Expression, purification and functional reconstitution of slack sodium-activated potassium channels.

PubMed

Yan, Yangyang; Yang, Youshan; Bian, Shumin; Sigworth, Fred J

2012-11-01

The slack (slo2.2) gene codes for a potassium-channel α-subunit of the 6TM voltage-gated channel family. Expression of slack results in Na(+)-activated potassium channel activity in various cell types. We describe the purification and reconstitution of Slack protein and show that the Slack α-subunit alone is sufficient for potassium channel activity activated by sodium ions as assayed in planar bilayer membranes and in membrane vesicles.

International Union of Basic and Clinical Pharmacology. C. Nomenclature and Properties of Calcium-Activated and Sodium-Activated Potassium Channels.

PubMed

Kaczmarek, Leonard K; Aldrich, Richard W; Chandy, K George; Grissmer, Stephan; Wei, Aguan D; Wulff, Heike

2017-01-01

A subset of potassium channels is regulated primarily by changes in the cytoplasmic concentration of ions, including calcium, sodium, chloride, and protons. The eight members of this subfamily were originally all designated as calcium-activated channels. More recent studies have clarified the gating mechanisms for these channels and have documented that not all members are sensitive to calcium. This article describes the molecular relationships between these channels and provides an introduction to their functional properties. It also introduces a new nomenclature that differentiates between calcium- and sodium-activated potassium channels. Copyright © 2016 by The American Society for Pharmacology and Experimental Therapeutics.

Slack, Slick, and Sodium-Activated Potassium Channels

PubMed Central

Kaczmarek, Leonard K.

2013-01-01

The Slack and Slick genes encode potassium channels that are very widely expressed in the central nervous system. These channels are activated by elevations in intracellular sodium, such as those that occur during trains of one or more action potentials, or following activation of nonselective cationic neurotransmitter receptors such as AMPA receptors. This review covers the cellular and molecular properties of Slack and Slick channels and compares them with findings on the properties of sodium-activated potassium currents (termed KNa currents) in native neurons. Human mutations in Slack channels produce extremely severe defects in learning and development, suggesting that KNa channels play a central role in neuronal plasticity and intellectual function. PMID:24319675

Site-3 sea anemone toxins: molecular probes of gating mechanisms in voltage-dependent sodium channels.

PubMed

Smith, Jaime J; Blumenthal, Kenneth M

2007-02-01

Sea anemone toxins, whose biological function is the capture of marine prey, are invaluable tools for studying the structure and function of mammalian voltage-gated sodium channels. Their high degree of specificity and selectivity have allowed for detailed analysis of inactivation gating and assignment of molecular entities responsible for this process. Because of their ability to discriminate among channel isoforms, and their high degree of structural conservation, these toxins could serve as important lead compounds for future pharmaceutical design.

Alterations of sodium and potassium channels of RGCs in RCS rat with the development of retinal degeneration.

PubMed

Chen, Zhongshan; Song, Yanping; Yao, Junping; Weng, Chuanhuang; Yin, Zheng Qin

2013-11-01

All know that retinitis pigmentosa (RP) is a group of hereditary retinal degenerative diseases characterized by progressive dysfunction of photoreceptors and associated with progressive cells loss; nevertheless, little is known about how rods and cones loss affects the surviving inner retinal neurons and networks. Retinal ganglion cells (RGCs) process and convey visual information from retina to visual centers in the brain. The healthy various ion channels determine the normal reception and projection of visual signals from RGCs. Previous work on the Royal College of Surgeons (RCS) rat, as a kind of classical RP animal model, indicated that, at late stages of retinal degeneration in RCS rat, RGCs were also morphologically and functionally affected. Here, retrograde labeling for RGCs with Fluorogold was performed to investigate the distribution, density, and morphological changes of RGCs during retinal degeneration. Then, patch clamp recording, western blot, and immunofluorescence staining were performed to study the channels of sodium and potassium properties of RGCs, so as to explore the molecular and proteinic basis for understanding the alterations of RGCs membrane properties and firing functions. We found that the resting membrane potential, input resistance, and capacitance of RGCs changed significantly at the late stage of retinal degeneration. Action potential could not be evoked in a part of RGCs. Inward sodium current and outward potassium current recording showed that sodium current was impaired severely but only slightly in potassium current. Expressions of sodium channel protein were impaired dramatically at the late stage of retinal degeneration. The results suggested that the density of RGCs decreased, process ramification impaired, and sodium ion channel proteins destructed, which led to the impairment of electrophysiological functions of RGCs and eventually resulted in the loss of visual function.

A double tyrosine motif in the cardiac sodium channel domain III-IV linker couples calcium-dependent calmodulin binding to inactivation gating.

PubMed

Sarhan, Maen F; Van Petegem, Filip; Ahern, Christopher A

2009-11-27

Voltage-gated sodium channels maintain the electrical cadence and stability of neurons and muscle cells by selectively controlling the transmembrane passage of their namesake ion. The degree to which these channels contribute to cellular excitability can be managed therapeutically or fine-tuned by endogenous ligands. Intracellular calcium, for instance, modulates sodium channel inactivation, the process by which sodium conductance is negatively regulated. We explored the molecular basis for this effect by investigating the interaction between the ubiquitous calcium binding protein calmodulin (CaM) and the putative sodium channel inactivation gate composed of the cytosolic linker between homologous channel domains III and IV (DIII-IV). Experiments using isothermal titration calorimetry show that CaM binds to a novel double tyrosine motif in the center of the DIII-IV linker in a calcium-dependent manner, N-terminal to a region previously reported to be a CaM binding site. An alanine scan of aromatic residues in recombinant DIII-DIV linker peptides shows that whereas multiple side chains contribute to CaM binding, two tyrosines (Tyr(1494) and Tyr(1495)) play a crucial role in binding the CaM C-lobe. The functional relevance of these observations was then ascertained through electrophysiological measurement of sodium channel inactivation gating in the presence and absence of calcium. Experiments on patch-clamped transfected tsA201 cells show that only the Y1494A mutation of the five sites tested renders sodium channel steady-state inactivation insensitive to cytosolic calcium. The results demonstrate that calcium-dependent calmodulin binding to the sodium channel inactivation gate double tyrosine motif is required for calcium regulation of the cardiac sodium channel.

β1 subunit stabilises sodium channel Nav1.7 against mechanical stress.

PubMed

Körner, Jannis; Meents, Jannis; Machtens, Jan-Philipp; Lampert, Angelika

2018-06-01

The voltage-gated sodium channel Nav1.7 is a key player in neuronal excitability and pain signalling. In addition to voltage sensing, the channel is also modulated by mechanical stress. Using whole-cell patch-clamp experiments, we discovered that the sodium channel subunit β1 is able to prevent the impact of mechanical stress on Nav1.7. An intramolecular disulfide bond of β1 was identified to be essential for stabilisation of inactivation, but not activation, against mechanical stress using molecular dynamics simulations, homology modelling and site-directed mutagenesis. Our results highlight the role of segment 6 of domain IV in fast inactivation. We present a candidate mechanism for sodium channel stabilisation against mechanical stress, ensuring reliable channel functionality in living systems. Voltage-gated sodium channels are key players in neuronal excitability and pain signalling. Precise gating of these channels is crucial as even small functional alterations can lead to pathological phenotypes such as pain or heart failure. Mechanical stress has been shown to affect sodium channel activation and inactivation. This suggests that stabilising components are necessary to ensure precise channel gating in living organisms. Here, we show that mechanical shear stress affects voltage dependence of activation and fast inactivation of the Nav1.7 channel. Co-expression of the β1 subunit, however, protects both gating modes of Nav1.7 against mechanical shear stress. Using molecular dynamics simulation, homology modelling and site-directed mutagenesis, we identify an intramolecular disulfide bond of β1 (Cys21-Cys43) which is partially involved in this process: the β1-C43A mutant prevents mechanical modulation of voltage dependence of activation, but not of fast inactivation. Our data emphasise the unique role of segment 6 of domain IV for sodium channel fast inactivation and confirm previous reports that the intracellular process of fast inactivation can be modified by interfering with the extracellular end of segment 6 of domain IV. Thus, our data suggest that physiological gating of Nav1.7 may be protected against mechanical stress in a living organism by assembly with the β1 subunit. © 2018 The Authors. The Journal of Physiology © 2018 The Physiological Society.

Energetics of discrete selectivity bands and mutation-induced transitions in the calcium-sodium ion channels family.

PubMed

Kaufman, I; Luchinsky, D G; Tindjong, R; McClintock, P V E; Eisenberg, R S

2013-11-01

We use Brownian dynamics (BD) simulations to study the ionic conduction and valence selectivity of a generic electrostatic model of a biological ion channel as functions of the fixed charge Q(f) at its selectivity filter. We are thus able to reconcile the discrete calcium conduction bands recently revealed in our BD simulations, M0 (Q(f)=1e), M1 (3e), M2 (5e), with a set of sodium conduction bands L0 (0.5e), L1 (1.5e), thereby obtaining a completed pattern of conduction and selectivity bands vs Q(f) for the sodium-calcium channels family. An increase of Q(f) leads to an increase of calcium selectivity: L0 (sodium-selective, nonblocking channel) → M0 (nonselective channel) → L1 (sodium-selective channel with divalent block) → M1 (calcium-selective channel exhibiting the anomalous mole fraction effect). We create a consistent identification scheme where the L0 band is putatively identified with the eukaryotic sodium channel The scheme created is able to account for the experimentally observed mutation-induced transformations between nonselective channels, sodium-selective channels, and calcium-selective channels, which we interpret as transitions between different rows of the identification table. By considering the potential energy changes during permeation, we show explicitly that the multi-ion conduction bands of calcium and sodium channels arise as the result of resonant barrierless conduction. The pattern of periodic conduction bands is explained on the basis of sequential neutralization taking account of self-energy, as Q(f)(z,i)=ze(1/2+i), where i is the order of the band and z is the valence of the ion. Our results confirm the crucial influence of electrostatic interactions on conduction and on the Ca(2+)/Na(+) valence selectivity of calcium and sodium ion channels. The model and results could be also applicable to biomimetic nanopores with charged walls.

Energetics of discrete selectivity bands and mutation-induced transitions in the calcium-sodium ion channels family

NASA Astrophysics Data System (ADS)

Kaufman, I.; Luchinsky, D. G.; Tindjong, R.; McClintock, P. V. E.; Eisenberg, R. S.

2013-11-01

We use Brownian dynamics (BD) simulations to study the ionic conduction and valence selectivity of a generic electrostatic model of a biological ion channel as functions of the fixed charge Qf at its selectivity filter. We are thus able to reconcile the discrete calcium conduction bands recently revealed in our BD simulations, M0 (Qf=1e), M1 (3e), M2 (5e), with a set of sodium conduction bands L0 (0.5e), L1 (1.5e), thereby obtaining a completed pattern of conduction and selectivity bands vs Qf for the sodium-calcium channels family. An increase of Qf leads to an increase of calcium selectivity: L0 (sodium-selective, nonblocking channel) → M0 (nonselective channel) → L1 (sodium-selective channel with divalent block) → M1 (calcium-selective channel exhibiting the anomalous mole fraction effect). We create a consistent identification scheme where the L0 band is putatively identified with the eukaryotic sodium channel The scheme created is able to account for the experimentally observed mutation-induced transformations between nonselective channels, sodium-selective channels, and calcium-selective channels, which we interpret as transitions between different rows of the identification table. By considering the potential energy changes during permeation, we show explicitly that the multi-ion conduction bands of calcium and sodium channels arise as the result of resonant barrierless conduction. The pattern of periodic conduction bands is explained on the basis of sequential neutralization taking account of self-energy, as Qf(z,i)=ze(1/2+i), where i is the order of the band and z is the valence of the ion. Our results confirm the crucial influence of electrostatic interactions on conduction and on the Ca2+/Na+ valence selectivity of calcium and sodium ion channels. The model and results could be also applicable to biomimetic nanopores with charged walls.

Relative potencies of Type I and Type II pyrethroids for inhibition of spontaneous firing in neuronal networks.

EPA Science Inventory

Pyrethroids insecticides commonly used in pest control disrupt the normal function of voltage-sensitive sodium channels. We have previously demonstrated that permethrin (a Type I pyrethroid) and deltamethrin (a Type II pyrethroid) inhibit sodium channel-dependent spontaneous netw...

Sodium channel blockers for cystic fibrosis.

PubMed

Burrows, Elinor F; Southern, Kevin W; Noone, Peadar G

2014-04-09

People with cystic fibrosis (CF) have increased transport of the salt, sodium across their airway lining. Over-absorption of sodium results in the dehydration of the liquid that lines the airway surface and (along with defective chloride secretion) is a primary defect in people with CF. To determine whether the topical administration of drugs that block sodium transport improves the respiratory condition of people with CF. We searched the Cochrane Cystic Fibrosis and Genetic Disorders Group Trials Register comprising references identified from comprehensive electronic database searches, handsearching relevant journals and abstract books of conference proceedings. We contacted principal investigators known to work in the field, previous authors and pharmaceutical companies who manufacture ion transport agents for unpublished or follow-up data.Most recent search of the Group's register: 19 December 2013. Published or unpublished randomised controlled trials (RCTs) or quasi-randomised controlled trials of sodium channel blockers compared to placebo or another sodium channel blocker or the same sodium channel blocker at a different dosing regimen. Two authors independently extracted data. Meta-analysis was limited due to differing study designs. Five RCTs, with a total of 226 participants, examining the topical administration of the short-acting sodium channel blocker, amiloride, compared to placebo were identified as eligible for inclusion in the review. In three studies over six months, there was a significant difference found in the difference in relative change in FVC in favour of placebo (weighted mean difference 1.51% (95% confidence interval -2.77 to -0.25), although heterogeneity was evident. A two-week study demonstrated that hypertonic saline with amiloride pre-treatment did not result in a significant improvement in respiratory function or mucus clearance, in contrast to pre-treatment with placebo. There were no significant differences identified in other clinically relevant outcomes. We found no evidence that the topical administration of a short-acting sodium channel blocker improves respiratory condition in people with cystic fibrosis and some limited evidence of deterioration in lung function.

Sodium channel blockers for cystic fibrosis.

PubMed

Burrows, Elinor F; Southern, Kevin W; Noone, Peadar G

2012-03-14

People with cystic fibrosis (CF) have increased transport of the salt, sodium across their airway lining. Over-absorption of sodium results in the dehydration of the liquid that lines the airway surface and is a primary defect in people with CF. To determine whether the topical administration of drugs that block sodium transport improves the respiratory condition of people with CF. We searched the Cochrane Cystic Fibrosis and Genetic Disorders Group Trials Register comprising references identified from comprehensive electronic database searches, handsearching relevant journals and abstract books of conference proceedings. We contacted principal investigators known to work in the field, previous authors and pharmaceutical companies who manufacture ion transport agents for unpublished or follow-up data.Most recent search of the Group's register: 22nd August 2011. Published or unpublished randomised controlled trials (RCTs) or quasi-randomised controlled trials of sodium channel blockers compared to placebo or another sodium channel blocker or the same sodium channel blocker at a different dosing regimen. Two authors independently extracted data. Meta-analysis was limited due to differing study designs. Five RCTs, with a total of 226 participants, examining the topical administration of the short-acting sodium channel blocker, amiloride, compared to placebo were identified as eligible for inclusion in the review. In three studies over six months, there was a significant difference found in the difference in relative change in FVC in favour of placebo (weighted mean difference 1.51% (95% confidence interval -2.77 to -0.25), although heterogeneity was evident. A two-week study demonstrated that hypertonic saline with amiloride pre-treatment did not result in a significant improvement in respiratory function or mucus clearance, in contrast to pre-treatment with placebo. There were no significant differences identified in other clinically relevant outcomes. We found no evidence that the topical administration of a short-acting sodium channel blocker improves respiratory condition in people with cystic fibrosis and some limited evidence of deterioration in lung function.

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

PubMed Central

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. PMID:28484374

Sodium channel blockers as therapeutic target for treating epilepsy: recent updates.

PubMed

Zuliani, Valentina; Fantini, Marco; Rivara, Mirko

2012-01-01

The voltage-gated sodium channels (VGSCs) are a family of membrane proteins forming a pore, through which they selectively conduct sodium ions inward and outward cell's plasma membranes in response to variations of membrane potentials, playing a fundamental role in controlling cellular excitability. Growing evidences suggest that abnormal VGSCs are involved in the pathophysiology of both acquired and inherited epilepsy. Approximately two dozen drugs are currently marketed for the treatment of epilepsy and most of them act as sodium channel blockers, preventing the return of the channels to the active state by stabilizing the inactive form. Despite the many drugs on the market, 30% of patients continue to experience seizures even in the presence of optimal doses of AEDs, while others continue to suffer from medication induced side effects. Thus, there is a great need to continue the search for new AEDs that are not only more effective, but also have a better side effects profile. For this reason, many efforts have been made in the recent years to identify new sodium channel blockers for the treatment of epilepsy. These studies have led to different classes of compounds, characterized by a great structural diversity. The aim of this review is to provide an introduction on the structure and function of the sodium channels, followed by a brief historical perspective on the sodium channel blockers in use as anticonvulsant drugs. Moreover, it will focus on the medicinal chemistry of the sodium channel blockers recently published (2008-2011) and the drug design/molecular modeling studies related to the receptor.

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

PubMed Central

Payandeh, Jian; Minor, Daniel L.

2014-01-01

Voltage-gated sodium channels (NaVs) provide the initial electrical signal that drives action potential generation in many excitable cells of the brain, heart, and nervous system. For more than 60 years, functional studies of NaVs 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 (BacNaVs) prevalent in soil, marine, and salt lake environments that bear many of the core features of eukaryotic NaVs have reframed ideas for voltage-gated channel function, ion selectivity, and pharmacology. Here, we analyze the recent advances, unanswered questions, and potential of BacNaVs as templates for drug development efforts. PMID:25158094

β1-Adrenergic blocker bisoprolol reverses down-regulated ion channels in sinoatrial node of heart failure rats.

PubMed

Du, Yuan; Zhang, Junbo; Xi, Yutao; Wu, Geru; Han, Ke; Huang, Xin; Ma, Aiqun; Wang, Tingzhong

2016-06-01

Bisoprolol, an antagonist of β1-adrenergic receptors, is effective in reducing the morbidity and mortality in patients with heart failure (HF). It has been found that HF is accompanied with dysfunction of the sinoatrial node (SAN). However, whether bisoprolol reverses the decreased SAN function in HF and how the relevant ion channels in SAN change were relatively less studied. SAN function and messenger RNA (mRNA) expression of sodium channels and hyperpolarization-activated cyclic nucleotide-gated (HCN) channel subunits were assessed in sham-operated rats, abdominal arterio-venous shunt (volume overload)-induced HF rats, and bisoprolol- treated HF rats. SAN cells of rats were isolated by laser capture microdissection. Quantitative real-time PCR analysis was used to quantify mRNA expression of sodium channels and HCN channel subunits in SAN. Intrinsic heart rate declined and sinus node recovery time prolonged in HF rats, indicating the suppressed SAN function, which could be improved by bisoprolol treatment. Nav1.1, Nav1.6, and HCN4 mRNA expressions were reduced in SAN in HF rats compared with that in control rats. Treatment with bisoprolol could reverse both the SAN function and the Nav1.1, Nav1.6, and HCN4 mRNA expression partially. These data indicated that bisoprolol is effective in HF treatment partially due to improved SAN function by reversing the down-regulation of sodium channels (Nav1.1 and Nav1.6) and HCN channel (HCN4) subunits in SAN in failing hearts.

Chimeric Agents Derived from the Functionalized Amino Acid, Lacosamide, and the α-Aminoamide, Safinamide: Evaluation of Their Inhibitory Actions on Voltage-Gated Sodium Channels, and Antiseizure and Antinociception Activities and Comparison with Lacosamide and Safinamide

PubMed Central

2015-01-01

The functionalized amino acid, lacosamide ((R)-2), and the α-aminoamide, safinamide ((S)-3), are neurological agents that have been extensively investigated and have displayed potent anticonvulsant activities in seizure models. Both compounds have been reported to modulate voltage-gated sodium channel activity. We have prepared a series of chimeric compounds, (R)-7–(R)-10, by merging key structural units in these two clinical agents, and then compared their activities with (R)-2 and (S)-3. Compounds were assessed for their ability to alter sodium channel kinetics for inactivation, frequency (use)-dependence, and steady-state activation and fast inactivation. We report that chimeric compounds (R)-7–(R)-10 in catecholamine A-differentiated (CAD) cells and embryonic rat cortical neurons robustly enhanced sodium channel inactivation at concentrations far lower than those required for (R)-2 and (S)-3, and that (R)-9 and (R)-10, unlike (R)-2 and (S)-3, produce sodium channel frequency (use)-dependence at low micromolar concentrations. We further show that (R)-7–(R)-10 displayed excellent anticonvulsant activities and pain-attenuating properties in the animal formalin model. Of these compounds, only (R)-7 reversed mechanical hypersensitivity in the tibial-nerve injury model for neuropathic pain in rats. PMID:25418676

Chimeric agents derived from the functionalized amino acid, lacosamide, and the α-aminoamide, safinamide: evaluation of their inhibitory actions on voltage-gated sodium channels, and antiseizure and antinociception activities and comparison with lacosamide and safinamide.

PubMed

Park, Ki Duk; Yang, Xiao-Fang; Dustrude, Erik T; Wang, Yuying; Ripsch, Matthew S; White, Fletcher A; Khanna, Rajesh; Kohn, Harold

2015-02-18

The functionalized amino acid, lacosamide ((R)-2), and the α-aminoamide, safinamide ((S)-3), are neurological agents that have been extensively investigated and have displayed potent anticonvulsant activities in seizure models. Both compounds have been reported to modulate voltage-gated sodium channel activity. We have prepared a series of chimeric compounds, (R)-7-(R)-10, by merging key structural units in these two clinical agents, and then compared their activities with (R)-2 and (S)-3. Compounds were assessed for their ability to alter sodium channel kinetics for inactivation, frequency (use)-dependence, and steady-state activation and fast inactivation. We report that chimeric compounds (R)-7-(R)-10 in catecholamine A-differentiated (CAD) cells and embryonic rat cortical neurons robustly enhanced sodium channel inactivation at concentrations far lower than those required for (R)-2 and (S)-3, and that (R)-9 and (R)-10, unlike (R)-2 and (S)-3, produce sodium channel frequency (use)-dependence at low micromolar concentrations. We further show that (R)-7-(R)-10 displayed excellent anticonvulsant activities and pain-attenuating properties in the animal formalin model. Of these compounds, only (R)-7 reversed mechanical hypersensitivity in the tibial-nerve injury model for neuropathic pain in rats.

First report on an inotropic peptide activating tetrodotoxin-sensitive, "neuronal" sodium currents in the heart.

PubMed

Kirchhof, Paulus; Tal, Tzachy; Fabritz, Larissa; Klimas, Jan; Nesher, Nir; Schulte, Jan S; Ehling, Petra; Kanyshkova, Tatayana; Budde, Thomas; Nikol, Sigrid; Fortmueller, Lisa; Stallmeyer, Birgit; Müller, Frank U; Schulze-Bahr, Eric; Schmitz, Wilhelm; Zlotkin, Eliahu; Kirchhefer, Uwe

2015-01-01

New therapeutic approaches to improve cardiac contractility without severe risk would improve the management of acute heart failure. Increasing systolic sodium influx can increase cardiac contractility, but most sodium channel activators have proarrhythmic effects that limit their clinical use. Here, we report the cardiac effects of a novel positive inotropic peptide isolated from the toxin of the Black Judean scorpion that activates neuronal tetrodotoxin-sensitive sodium channels. All venoms and peptides were isolated from Black Judean Scorpions (Buthotus Hottentotta) caught in the Judean Desert. The full scorpion venom increased left ventricular function in sedated mice in vivo, prolonged ventricular repolarization, and provoked ventricular arrhythmias. An inotropic peptide (BjIP) isolated from the full venom by chromatography increased cardiac contractility but did neither provoke ventricular arrhythmias nor prolong cardiac repolarization. BjIP increased intracellular calcium in ventricular cardiomyocytes and prolonged inactivation of the cardiac sodium current. Low concentrations of tetrodotoxin (200 nmol/L) abolished the effect of BjIP on calcium transients and sodium current. BjIP did not alter the function of Nav1.5, but selectively activated the brain-type sodium channels Nav1.6 or Nav1.3 in cellular electrophysiological recordings obtained from rodent thalamic slices. Nav1.3 (SCN3A) mRNA was detected in human and mouse heart tissue. Our pilot experiments suggest that selective activation of tetrodotoxin-sensitive neuronal sodium channels can safely increase cardiac contractility. As such, the peptide described here may become a lead compound for a new class of positive inotropic agents. © 2014 American Heart Association, Inc.

Alanine to valine substitutions in the pore helix IIIP1 and linker-helix IIIL45 confer cockroach sodium channel resistance to DDT and pyrethroids.

PubMed

Chen, Mengli; Du, Yuzhe; Nomura, Yoshiko; Zhu, Guonian; Zhorov, Boris S; Dong, Ke

2017-05-01

Pyrethroid insecticides exert toxic effects by prolonging the opening of voltage-gated sodium channels. More than 20 sodium channel mutations from arthropod pests and disease vectors have been confirmed to confer pyrethroid resistance. These mutations have been valuable in elucidating the molecular interaction between pyrethroids and sodium channels, including identification of two pyrethroid receptor sites. Previously, two alanine to valine substitutions, one in the pore helix IIIP1 and the other in the linker-helix connecting S4 and S5 in domain III (IIIL45), were found in Drosophila melanogaster mutants that are resistant to DDT and deltamethrin (a type II pyrethroid with an α-cyano group at the phenylbenzyl alcohol position, which is lacking in type I pyrethroids), but their role in target-site-mediated insecticide resistance has not been functionally confirmed. In this study, we functionally examined the two mutations in cockroach sodium channels expressed in Xenopus laevis oocytes. Both mutations caused depolarizing shifts in the voltage dependence of activation, conferred DDT resistance and also resistance to two Type I pyrethroids by almost abolishing the tail currents induced by Type I pyrethroids. In contrast, neither mutation reduced the amplitude of tail currents induced by the Type II pyrethroids, deltamethrin or cypermethrin. However, both mutations accelerated the decay of Type II pyrethroid-induced tail currents, which normally decay extremely slowly. These results provided new insight into the molecular basis of different actions of Type I and Type II pyrethroids on sodium channels. Computer modeling predicts that both mutations may allosterically affect pyrethroid binding. Copyright © 2016 Elsevier B.V. All rights reserved.

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

PubMed

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.

Potential Roles of Amiloride-Sensitive Sodium Channels in Cancer Development.

PubMed

Xu, Siguang; Liu, Cui; Ma, Yana; Ji, Hong-Long; Li, Xiumin

2016-01-01

The ENaC/degenerin ion channel superfamily includes the amiloride-sensitive epithelial sodium channel (ENaC) and acid sensitive ionic channel (ASIC). ENaC is a multimeric ion channel formed by heteromultimeric membrane glycoproteins, which participate in a multitude of biological processes by mediating the transport of sodium (Na(+)) across epithelial tissues such as the kidney, lungs, bladder, and gut. Aberrant ENaC functions contribute to several human disease states including pseudohypoaldosteronism, Liddle syndrome, cystic fibrosis, and salt-sensitive hypertension. Increasing evidence suggests that ion channels not only regulate ion homeostasis and electric signaling in excitable cells but also play important roles in cancer cell behaviors such as proliferation, apoptosis, invasion, and migration. Indeed, ENaCs/ASICs had been reported to be associated with cancer characteristics. Given their cell surface localization and pharmacology, pharmacological strategies to target ENaC/ASIC family members may be promising cancer therapeutics.

Functional Studies of Sodium Channels: From Target to Compound Identification.

PubMed

Bertrand, Daniel; Biton, Bruno; Licher, Thomas; Chambard, Jean-Marie; Lanneau, Christophe; Partiseti, Michel; Lefevre, Isabel A

2016-12-13

Over the last six decades, voltage-gated sodium (Na v ) channels have attracted a great deal of scientific and pharmaceutical interest, driving fundamental advances in both biology and technology. The structure and physiological function of these channels have been extensively studied; clinical and genetic data have uncovered their implication in diseases such as epilepsy, arrhythmias, and pain, bringing them into focus as current and future drug targets. While different techniques have been established to record the activity of Na v channels, proper determination of their properties still presents serious challenges, depending upon the experimental conditions and the desired subtype of channel to be characterized. The aim of this unit is to review the characteristics of Na v channels, their properties, the cells in which they can be studied, and the currently available techniques. Topics covered include the determination of Na v -channel biophysical properties as well as the use of toxins to discriminate between subtypes using electrophysiological or optical methods. Perspectives on the development of high-throughput screening assays with their advantages and limitations are also discussed to allow a better understanding of the challenges encountered in voltage-gated sodium channel preclinical drug discovery. © 2016 by John Wiley & Sons, Inc. Copyright © 2016 John Wiley & Sons, Inc.

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

PubMed

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

2017-01-01

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.

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

PubMed Central

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.

2017-01-01

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. PMID:28202723

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

PubMed

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.

Sodium channels in the Cx43 gap junction perinexus may constitute a cardiac ephapse: an experimental and modeling study.

PubMed

Veeraraghavan, Rengasayee; Lin, Joyce; Hoeker, Gregory S; Keener, James P; Gourdie, Robert G; Poelzing, Steven

2015-10-01

It has long been held that electrical excitation spreads from cell-to-cell in the heart via low resistance gap junctions (GJ). However, it has also been proposed that myocytes could interact by non-GJ-mediated "ephaptic" mechanisms, facilitating propagation of action potentials in tandem with direct GJ-mediated coupling. We sought evidence that such mechanisms contribute to cardiac conduction. Using super-resolution microscopy, we demonstrate that Nav1.5 is localized within 200 nm of the GJ plaque (a region termed the perinexus). Electron microscopy revealed close apposition of adjacent cell membranes within perinexi suggesting that perinexal sodium channels could function as an ephapse, enabling ephaptic cell-to-cell transfer of electrical excitation. Acute interstitial edema (AIE) increased intermembrane distance at the perinexus and was associated with preferential transverse conduction slowing and increased spontaneous arrhythmia incidence. Inhibiting sodium channels with 0.5 μM flecainide uniformly slowed conduction, but sodium channel inhibition during AIE slowed conduction anisotropically and increased arrhythmia incidence more than AIE alone. Sodium channel inhibition during GJ uncoupling with 25 μM carbenoxolone slowed conduction anisotropically and was also highly proarrhythmic. A computational model of discretized extracellular microdomains (including ephaptic coupling) revealed that conduction trends associated with altered perinexal width, sodium channel conductance, and GJ coupling can be predicted when sodium channel density in the intercalated disk is relatively high. We provide evidence that cardiac conduction depends on a mathematically predicted ephaptic mode of coupling as well as GJ coupling. These data suggest opportunities for novel anti-arrhythmic therapies targeting noncanonical conduction pathways in the heart.

Amyloid precursor protein modulates Nav1.6 sodium channel currents through a Go-coupled JNK pathway.

PubMed

Li, Shao; Wang, Xi; Ma, Quan-Hong; Yang, Wu-Lin; Zhang, Xiao-Gang; Dawe, Gavin S; Xiao, Zhi-Cheng

2016-12-23

Amyloid precursor protein (APP), commonly associated with Alzheimer's disease, also marks axonal degeneration. In the recent studies, we demonstrated that APP aggregated at nodes of Ranvier (NORs) in myelinated central nervous system (CNS) axons and interacted with Nav1.6. However, the physiological function of APP remains unknown. In this study, we described reduced sodium current densities in APP knockout hippocampal neurons. Coexpression of APP or its intracellular domains containing a VTPEER motif with Na v 1.6 sodium channels in Xenopus oocytes resulted in an increase in peak sodium currents, which was enhanced by constitutively active Go mutant and blocked by a dominant negative mutant. JNK and CDK5 inhibitor attenuated increases in Nav1.6 sodium currents induced by overexpression of APP. Nav1.6 sodium currents were increased by APPT668E (mutant Thr to Glu) and decreased by T668A (mutant Thr to ALa) mutant, respectively. The cell surface expression of Nav1.6 sodium channels in the white matter of spinal cord and the spinal conduction velocity is decreased in APP, p35 and JNK3 knockout mice. Therefore, APP modulates Nav1.6 sodium channels through a Go-coupled JNK pathway, which is dependent on phosphorylation of APP at Thr668.

Amyloid precursor protein modulates Nav1.6 sodium channel currents through a Go-coupled JNK pathway

PubMed Central

Li, Shao; Wang, Xi; Ma, Quan-Hong; Yang, Wu-lin; Zhang, Xiao-Gang; Dawe, Gavin S.; Xiao, Zhi-Cheng

2016-01-01

Amyloid precursor protein (APP), commonly associated with Alzheimer’s disease, also marks axonal degeneration. In the recent studies, we demonstrated that APP aggregated at nodes of Ranvier (NORs) in myelinated central nervous system (CNS) axons and interacted with Nav1.6. However, the physiological function of APP remains unknown. In this study, we described reduced sodium current densities in APP knockout hippocampal neurons. Coexpression of APP or its intracellular domains containing a VTPEER motif with Nav1.6 sodium channels in Xenopus oocytes resulted in an increase in peak sodium currents, which was enhanced by constitutively active Go mutant and blocked by a dominant negative mutant. JNK and CDK5 inhibitor attenuated increases in Nav1.6 sodium currents induced by overexpression of APP. Nav1.6 sodium currents were increased by APPT668E (mutant Thr to Glu) and decreased by T668A (mutant Thr to ALa) mutant, respectively. The cell surface expression of Nav1.6 sodium channels in the white matter of spinal cord and the spinal conduction velocity is decreased in APP, p35 and JNK3 knockout mice. Therefore, APP modulates Nav1.6 sodium channels through a Go-coupled JNK pathway, which is dependent on phosphorylation of APP at Thr668. PMID:28008944

A uniquely adaptable pore is consistent with NALCN being an ion sensor

PubMed Central

Senatore, Adriano; Spafford, J. David

2013-01-01

NALCN is an intriguing, orphan ion channel among the 4x6TM family of related voltage-gated cation channels, sharing a common architecture of four homologous domains consisting of six transmembrane helices, separated by three cytoplasmic linkers and delimited by N and C-terminal ends. NALCN is one of the shortest 4x6TM family members, lacking much of the variation that provides the diverse palate of gating features, and tissue specific adaptations of sodium and calcium channels. NALCN’s most distinctive feature is that that it possesses a highly adaptable pore with a calcium-like EEEE selectivity filter in radially symmetrical animals and a more sodium-like EEKE or EKEE selectivity filter in bilaterally symmetrical animals including vertebrates. Two lineages of animals evolved alternative calcium-like EEEE and sodium-like EEKE / EKEE pores, spliced to regulate NALCN functions in differing cellular environments, such as muscle (heart and skeletal) and secretory tissue (brain and glands), respectively. A highly adaptable pore in an otherwise conserved ion channel in the 4x6TM channel family is not consistent with a role for NALCN in directly gating a significant ion conductance that can be either sodium ions or calcium ions. NALCN was proposed to be an expressible Gd3+-sensitive, NMDG+-impermeant, non-selective and ohmic leak conductance in HEK-293T cells, but we were unable to distinguish these reported currents from leaky patch currents (ILP) in control HEK-293T cells. We suggest that NALCN functions as a sensor for the much larger UNC80/UNC79 complex, in a manner consistent with the coupling mechanism known for other weakly or non-conducting 4x6TM channel sensor proteins such as Nax or Cav1.1. We propose that NALCN serves as a variable sensor that responds to calcium or sodium ion flux, depending on whether the total cellular current density is generated more from calcium-selective or sodium-selective channels. PMID:23442378

A uniquely adaptable pore is consistent with NALCN being an ion sensor.

PubMed

Senatore, Adriano; Spafford, J David

2013-01-01

NALCN is an intriguing, orphan ion channel among the 4x6TM family of related voltage-gated cation channels, sharing a common architecture of four homologous domains consisting of six transmembrane helices, separated by three cytoplasmic linkers and delimited by N and C-terminal ends. NALCN is one of the shortest 4x6TM family members, lacking much of the variation that provides the diverse palate of gating features, and tissue specific adaptations of sodium and calcium channels. NALCN's most distinctive feature is that that it possesses a highly adaptable pore with a calcium-like EEEE selectivity filter in radially symmetrical animals and a more sodium-like EEKE or EKEE selectivity filter in bilaterally symmetrical animals including vertebrates. Two lineages of animals evolved alternative calcium-like EEEE and sodium-like EEKE / EKEE pores, spliced to regulate NALCN functions in differing cellular environments, such as muscle (heart and skeletal) and secretory tissue (brain and glands), respectively. A highly adaptable pore in an otherwise conserved ion channel in the 4x6TM channel family is not consistent with a role for NALCN in directly gating a significant ion conductance that can be either sodium ions or calcium ions. NALCN was proposed to be an expressible Gd ( 3+) -sensitive, NMDG (+) -impermeant, non-selective and ohmic leak conductance in HEK-293T cells, but we were unable to distinguish these reported currents from leaky patch currents (ILP) in control HEK-293T cells. We suggest that NALCN functions as a sensor for the much larger UNC80/UNC79 complex, in a manner consistent with the coupling mechanism known for other weakly or non-conducting 4x6TM channel sensor proteins such as Nax or Cav 1.1. We propose that NALCN serves as a variable sensor that responds to calcium or sodium ion flux, depending on whether the total cellular current density is generated more from calcium-selective or sodium-selective channels.

Voltage-gated sodium channels as targets for pyrethroid insecticides.

PubMed

Field, Linda M; Emyr Davies, T G; O'Reilly, Andrias O; Williamson, Martin S; Wallace, B A

2017-10-01

The pyrethroid insecticides are a very successful group of compounds that have been used extensively for the control of arthropod pests of agricultural crops and vectors of animal and human disease. Unfortunately, this has led to the development of resistance to the compounds in many species. The mode of action of pyrethroids is known to be via interactions with the voltage-gated sodium channel. Understanding how binding to the channel is affected by amino acid substitutions that give rise to resistance has helped to elucidate the mode of action of the compounds and the molecular basis of their selectivity for insects vs mammals and between insects and other arthropods. Modelling of the channel/pyrethroid interactions, coupled with the ability to express mutant channels in oocytes and study function, has led to knowledge of both how the channels function and potentially how to design novel insecticides with greater species selectivity.

Controlling epithelial sodium channels with light using photoswitchable amilorides

NASA Astrophysics Data System (ADS)

Schönberger, Matthias; Althaus, Mike; Fronius, Martin; Clauss, Wolfgang; Trauner, Dirk

2014-08-01

Amiloride is a widely used diuretic that blocks epithelial sodium channels (ENaCs). These heterotrimeric transmembrane proteins, assembled from β, γ and α or δ subunits, effectively control water transport across epithelia and sodium influx into non-epithelial cells. The functional role of δβγENaC in various organs, including the human brain, is still poorly understood and no pharmacological tools are available for the functional differentiation between α- and δ-containing ENaCs. Here we report several photoswitchable versions of amiloride. One compound, termed PA1, enables the optical control of ENaC channels, in particular the δβγ isoform, by switching between blue and green light, or by turning on and off blue light. PA1 was used to modify functionally δβγENaC in amphibian and mammalian cells. We also show that PA1 can be used to differentiate between δβγENaC and αβγENaC in a model for the human lung epithelium.

Differences in sodium voltage-gated channel properties according to myosin heavy chain isoform expression in single muscle fibres.

PubMed

Rannou, F; Droguet, M; Giroux-Metges, M A; Pennec, Y; Gioux, M; Pennec, J P

2009-11-01

The myosin heavy chain (MHC) isoform determines the characteristics and shortening velocity of muscle fibres. The functional properties of the muscle fibre are also conditioned by its membrane excitability through the electrophysiological properties of sodium voltage-gated channels. Macropatch-clamp is used to study sodium channels in fibres from peroneus longus (PL) and soleus (Sol) muscles (Wistar rats, n = 8). After patch-clamp recordings, single fibres are identified by SDS-PAGE electrophoresis according to their myosin heavy chain isoform (slow type I and the three fast types IIa, IIx, IIb). Characteristics of sodium currents are compared (Student's t test) between fibres exhibiting only one MHC isoform. Four MHC isoforms are identified in PL and only type I in Sol single fibres. In PL, maximal sodium current (I(max)), maximal sodium conductance (g(Na,max)) and time constants of activation and inactivation ((m) and (h)) increase according to the scheme I-->IIa-->IIx-->IIb (P < 0.05). (m) values related to sodium channel type and/or function, are similar in Sol I and PL IIb fibres (P = 0.97) despite different contractile properties. The voltage dependence of activation (V(a,1/2)) shows a shift towards positive potentials from Sol type I to IIa, IIx and finally IIb fibres from PL (P < 0.05). These data are consistent with the earlier recruitment of slow fibres in a fast-mixed muscle like PL, while slow fibres of postural muscle such as soleus could be recruited in the same ways as IIb fibres in a fast muscle.

Structure-activity relationships for the action of 11 pyrethroid insecticides on rat Na{sub v}1.8 sodium channels expressed in Xenopus oocytes

DOE Office of Scientific and Technical Information (OSTI.GOV)

Choi, J.-S.; Soderlund, David M.

2006-03-15

Pyrethroid insecticides bind to voltage-sensitive sodium channels and modify their gating kinetics, thereby disrupting nerve function. This paper describes the action of 11 structurally diverse commercial pyrethroid insecticides on the rat Na{sub v}1.8 sodium channel isoform, the principal carrier of the tetrodotoxin-resistant, pyrethroid-sensitive sodium current of sensory neurons, expressed in Xenopus laevis oocytes. All 11 compounds produced characteristic sodium tail currents following a depolarizing pulse that ranged from rapidly-decaying monoexponential currents (allethrin, cismethrin and permethrin) to persistent biexponential currents (cyfluthrin, cyhalothrin, cypermethrin and deltamethrin). Tail currents for the remaining compounds (bifenthrin, fenpropathrin, fenvalerate and tefluthrin) were monoexponential and decayed withmore » kinetics intermediate between these extremes. Reconstruction of currents carried solely by the pyrethroid-modified subpopulation of channels revealed two types of pyrethroid-modified currents. The first type, found with cismethrin, allethrin, permethrin and tefluthrin, activated relatively rapidly and inactivated partially during a 40-ms depolarization. The second type, found with cypermethrin, cyfluthrin, cyhalothrin, deltamethrin, fenpropathrin and fenvalerate, activated more slowly and did not detectably inactivate during a 40-ms depolarization. Only bifenthrin did not produce modified currents that fit clearly into either of these categories. In all cases, the rate of activation of modified channels was strongly correlated with the rate of tail current decay following repolarization. Modification of Na{sub v}1.8 sodium channels by cyfluthrin, cyhalothrin, cypermethrin and deltamethrin was enhanced 2.3- to 3.4-fold by repetitive stimulation; this effect appeared to result from the accumulation of persistently open channels rather than preferential binding to open channel states. Fenpropathrin was the most effective compound against Na{sub v}1.8 sodium channels from the perspective of either resting or use-dependent modification. When use dependence is taken into account, cypermethrin, deltamethrin and tefluthrin approached the effectiveness of fenpropathrin. The selective expression of Na{sub v}1.8 sodium channels in nociceptive neurons suggests that these channels may be important targets for pyrethroids in the production of paresthesia following dermal expo0010su.« less

How do voltage-gated sodium channels enhance migration and invasiveness in cancer cells?

PubMed

Besson, Pierre; Driffort, Virginie; Bon, Émeline; Gradek, Frédéric; Chevalier, Stéphan; Roger, Sébastien

2015-10-01

Voltage-gated sodium channels are abnormally expressed in tumors, often as neonatal isoforms, while they are not expressed, or only at a low level, in the matching normal tissue. The level of their expression and their activity is related to the aggressiveness of the disease and to the formation of metastases. A vast knowledge on the regulation of their expression and functioning has been accumulated in normal excitable cells. This helped understand their regulation in cancer cells. However, how voltage-gated sodium channels impose a pro-metastatic behavior to cancer cells is much less documented. This aspect will be addressed in the review. This article is part of a Special Issue entitled: Membrane channels and transporters in cancers. Copyright © 2015 Elsevier B.V. All rights reserved.

Dysregulation of renal aquaporins and epithelial sodium channel in lithium-induced nephrogenic diabetes insipidus.

PubMed

Nielsen, Jakob; Kwon, Tae-Hwan; Christensen, Birgitte Mønster; Frøkiaer, Jørgen; Nielsen, Søren

2008-05-01

Lithium is used commonly to treat bipolar mood disorders. In addition to its primary therapeutic effects in the central nervous system lithium has a number of side effects in the kidney. The side effects include nephrogenic diabetes insipidus with polyuria, mild sodium wasting, and changes in acid/base balance. These functional changes are associated with marked structural changes in collecting duct cell composition and morphology, likely contributing to the functional changes. Over the past few years, investigations of lithium-induced renal changes have provided novel insight into the molecular mechanisms that are responsible for the disturbances in water, sodium, and acid/base metabolism. This includes dysregulation of renal aquaporins, epithelial sodium channel, and acid/base transporters. This review focuses on these issues with the aim to present this in context with clinically relevant features.

Cardiac sodium channel Markov model with temperature dependence and recovery from inactivation.

PubMed Central

Irvine, L A; Jafri, M S; Winslow, R L

1999-01-01

A Markov model of the cardiac sodium channel is presented. The model is similar to the CA1 hippocampal neuron sodium channel model developed by Kuo and Bean (1994. Neuron. 12:819-829) with the following modifications: 1) an additional open state is added; 2) open-inactivated transitions are made voltage-dependent; and 3) channel rate constants are exponential functions of enthalpy, entropy, and voltage and have explicit temperature dependence. Model parameters are determined using a simulated annealing algorithm to minimize the error between model responses and various experimental data sets. The model reproduces a wide range of experimental data including ionic currents, gating currents, tail currents, steady-state inactivation, recovery from inactivation, and open time distributions over a temperature range of 10 degrees C to 25 degrees C. The model also predicts measures of single channel activity such as first latency, probability of a null sweep, and probability of reopening. PMID:10096885

Mechanisms of pyrethroid insecticide-induced stimulation of calcium influx in neocortical neurons

EPA Science Inventory

Pyrethroid insecticides bind to voltage-gated sodium channels (VGSCs) and modify their gating kinetics, thereby disrupting neuronal function. Pyrethroids have also been reported to alter the function of other channel types, including activation of voltage-gated Ca2+ calcium chann...

Mining Protein Evolution for Insights into Mechanisms of Voltage-Dependent Sodium Channel Auxiliary Subunits.

PubMed

Molinarolo, Steven; Granata, Daniele; Carnevale, Vincenzo; Ahern, Christopher A

2018-02-21

Voltage-gated sodium channel (VGSC) beta (β) subunits have been called the "overachieving" auxiliary ion channel subunit. Indeed, these subunits regulate the trafficking of the sodium channel complex at the plasma membrane and simultaneously tune the voltage-dependent properties of the pore-forming alpha-subunit. It is now known that VGSC β-subunits are capable of similar modulation of multiple isoforms of related voltage-gated potassium channels, suggesting that their abilities extend into the broader voltage-gated channels. The gene family for these single transmembrane immunoglobulin beta-fold proteins extends well beyond the traditional VGSC β1-β4 subunit designation, with deep roots into the cell adhesion protein family and myelin-related proteins - where inherited mutations result in a myriad of electrical signaling disorders. Yet, very little is known about how VGSC β-subunits support protein trafficking pathways, the basis for their modulation of voltage-dependent gating, and, ultimately, their role in shaping neuronal excitability. An evolutionary approach can be useful in yielding new clues to such functions as it provides an unbiased assessment of protein residues, folds, and functions. An approach is described here which indicates the greater emergence of the modern β-subunits roughly 400 million years ago in the early neurons of Bilateria and bony fish, and the unexpected presence of distant homologues in bacteriophages. Recent structural breakthroughs containing α and β eukaryotic sodium channels containing subunits suggest a novel role for a highly conserved polar contact that occurs within the transmembrane segments. Overall, a mixture of approaches will ultimately advance our understanding of the mechanism for β-subunit interactions with voltage-sensor containing ion channels and membrane proteins.

Modulation of epithelial sodium channel trafficking and function by sodium 4-phenylbutyrate in human nasal epithelial cells.

PubMed

Prulière-Escabasse, Virginie; Planès, Carole; Escudier, Estelle; Fanen, Pascale; Coste, André; Clerici, Christine

2007-11-23

Sodium 4-phenylbutyrate (4-PBA) has been shown to correct the cellular trafficking of several mutant or nonmutant plasma membrane proteins such as cystic fibrosis transmembrane conductance regulator through the expression of 70-kDa heat shock proteins. The objective of the study was to determine whether 4-PBA may influence the functional expression of epithelial sodium channels (ENaC) in human nasal epithelial cells (HNEC). Using primary cultures of HNEC, we demonstrate that 4-PBA (5 mm for 6 h) markedly stimulated amiloride-sensitive sodium channel activity and that this was related to an increased abundance of alpha-, beta-, and gamma-ENaC subunits in the apical membrane. The increase in ENaC cell surface expression (i) was due to insertion of newly ENaC subunits as determined by brefeldin A experiments and (ii) was not associated with cell surface retention of ENaC subunits because endocytosis of ENaC subunits was unchanged. In addition, we find that ENaC co-immunoprecipitated with the heat shock protein constitutively expressed Hsc70, that has been reported to modulate ENaC trafficking, and that 4-PBA decreased Hsc70 protein level. Finally, we report that in cystic fibrosis HNEC obtained from two cystic fibrosis patients, 4-PBA increased functional expression of ENaC as demonstrated by the increase in amiloride-sensitive sodium transport and in alpha-, beta-, and gamma-ENaC subunit expression in the apical membrane. Our results suggest that in HNEC, 4-PBA increases the functional expression of ENaC through the insertion of new alpha-, beta-, and gamma-ENaC subunits into the apical membrane and also suggest that 4-PBA could modify ENaC trafficking by reducing Hsc70 protein expression.

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

DOE Office of Scientific and Technical Information (OSTI.GOV)

Nilsson, Mats F, E-mail: Mats.Nilsson@farmbio.uu.se; Sköld, Anna-Carin; Ericson, Ann-Christin

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 effectmore » 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.« less

Na+ influx via Orai1 inhibits intracellular ATP-induced mTORC2 signaling to disrupt CD4 T cell gene expression and differentiation.

PubMed

Miao, Yong; Bhushan, Jaya; Dani, Adish; Vig, Monika

2017-05-11

T cell effector functions require sustained calcium influx. However, the signaling and phenotypic consequences of non-specific sodium permeation via calcium channels remain unknown. α-SNAP is a crucial component of Orai1 channels, and its depletion disrupts the functional assembly of Orai1 multimers. Here we show that α-SNAP hypomorph, hydrocephalus with hopping gait, Napa hyh/hyh mice harbor significant defects in CD4 T cell gene expression and Foxp3 regulatory T cell (Treg) differentiation. Mechanistically, TCR stimulation induced rapid sodium influx in Napa hyh/hyh CD4 T cells, which reduced intracellular ATP, [ATP] i . Depletion of [ATP] i inhibited mTORC2 dependent NFκB activation in Napa hyh/hyh cells but ablation of Orai1 restored it. Remarkably, TCR stimulation in the presence of monensin phenocopied the defects in Napa hyh/hyh signaling and Treg differentiation, but not IL-2 expression. Thus, non-specific sodium influx via bonafide calcium channels disrupts unexpected signaling nodes and may provide mechanistic insights into some divergent phenotypes associated with Orai1 function.

Gating transitions in the selectivity filter region of a sodium channel are coupled to the domain IV voltage sensor.

PubMed

Capes, Deborah L; Arcisio-Miranda, Manoel; Jarecki, Brian W; French, Robert J; Chanda, Baron

2012-02-14

Voltage-dependent ion channels are crucial for generation and propagation of electrical activity in biological systems. The primary mechanism for voltage transduction in these proteins involves the movement of a voltage-sensing domain (D), which opens a gate located on the cytoplasmic side. A distinct conformational change in the selectivity filter near the extracellular side has been implicated in slow inactivation gating, which is important for spike frequency adaptation in neural circuits. However, it remains an open question whether gating transitions in the selectivity filter region are also actuated by voltage sensors. Here, we examine conformational coupling between each of the four voltage sensors and the outer pore of a eukaryotic voltage-dependent sodium channel. The voltage sensors of these sodium channels are not structurally symmetric and exhibit functional specialization. To track the conformational rearrangements of individual voltage-sensing domains, we recorded domain-specific gating pore currents. Our data show that, of the four voltage sensors, only the domain IV voltage sensor is coupled to the conformation of the selectivity filter region of the sodium channel. Trapping the outer pore in a particular conformation with a high-affinity toxin or disulphide crossbridge impedes the return of this voltage sensor to its resting conformation. Our findings directly establish that, in addition to the canonical electromechanical coupling between voltage sensor and inner pore gates of a sodium channel, gating transitions in the selectivity filter region are also coupled to the movement of a voltage sensor. Furthermore, our results also imply that the voltage sensor of domain IV is unique in this linkage and in the ability to initiate slow inactivation in sodium channels.

Optimized expression and purification of NavAb provide the structural insight into the voltage dependence.

PubMed

Irie, Katsumasa; Haga, Yukari; Shimomura, Takushi; Fujiyoshi, Yoshinori

2018-01-01

Voltage-gated sodium channels are crucial for electro-signalling in living systems. Analysis of the molecular mechanism requires both fine electrophysiological evaluation and high-resolution channel structures. Here, we optimized a dual expression system of NavAb, which is a well-established standard of prokaryotic voltage-gated sodium channels, for E. coli and insect cells using a single plasmid vector to analyse high-resolution protein structures and measure large ionic currents. Using this expression system, we evaluated the voltage dependence and determined the crystal structures of NavAb wild-type and two mutants, E32Q and N49K, whose voltage dependence were positively shifted and essential interactions were lost in voltage sensor domain. The structural and functional comparison elucidated the molecular mechanisms of the voltage dependence of prokaryotic voltage-gated sodium channels. © 2017 Federation of European Biochemical Societies.

Gain-of-Function Mutation W493R in the Epithelial Sodium Channel Allosterically Reconfigures Intersubunit Coupling*♦

PubMed Central

Shobair, Mahmoud; Dagliyan, Onur; Kota, Pradeep; Dang, Yan L.; He, Hong; Stutts, M. Jackson; Dokholyan, Nikolay V.

2016-01-01

Sodium absorption in epithelial cells is rate-limited by the epithelial sodium channel (ENaC) activity in lung, kidney, and the distal colon. Pathophysiological conditions, such as cystic fibrosis and Liddle syndrome, result from water-electrolyte imbalance partly due to malfunction of ENaC regulation. Because the quaternary structure of ENaC is yet undetermined, the bases of pathologically linked mutations in ENaC subunits α, β, and γ are largely unknown. Here, we present a structural model of heterotetrameric ENaC α1βα2γ that is consistent with previous cross-linking results and site-directed mutagenesis experiments. By using this model, we show that the disease-causing mutation αW493R rewires structural dynamics of the intersubunit interfaces α1β and α2γ. Changes in dynamics can allosterically propagate to the channel gate. We demonstrate that cleavage of the γ-subunit, which is critical for full channel activation, does not mediate activation of ENaC by αW493R. Our molecular dynamics simulations led us to identify a channel-activating electrostatic interaction between α2Arg-493 and γGlu-348 at the α2γ interface. By neutralizing a sodium-binding acidic patch at the α1β interface, we reduced ENaC activation of αW493R by more than 2-fold. By combining homology modeling, molecular dynamics, cysteine cross-linking, and voltage clamp experiments, we propose a dynamics-driven model for the gain-of-function in ENaC by αW493R. Our integrated computational and experimental approach advances our understanding of structure, dynamics, and function of ENaC in its disease-causing state. PMID:26668308

Effects of channel blocking on information transmission and energy efficiency in squid giant axons.

PubMed

Liu, Yujiang; Yue, Yuan; Yu, Yuguo; Liu, Liwei; Yu, Lianchun

2018-04-01

Action potentials are the information carriers of neural systems. The generation of action potentials involves the cooperative opening and closing of sodium and potassium channels. This process is metabolically expensive because the ions flowing through open channels need to be restored to maintain concentration gradients of these ions. Toxins like tetraethylammonium can block working ion channels, thus affecting the function and energy cost of neurons. In this paper, by computer simulation of the Hodgkin-Huxley neuron model, we studied the effects of channel blocking with toxins on the information transmission and energy efficiency in squid giant axons. We found that gradually blocking sodium channels will sequentially maximize the information transmission and energy efficiency of the axons, whereas moderate blocking of potassium channels will have little impact on the information transmission and will decrease the energy efficiency. Heavy blocking of potassium channels will cause self-sustained oscillation of membrane potentials. Simultaneously blocking sodium and potassium channels with the same ratio increases both information transmission and energy efficiency. Our results are in line with previous studies suggesting that information processing capacity and energy efficiency can be maximized by regulating the number of active ion channels, and this indicates a viable avenue for future experimentation.

Sodium channel slow inactivation as a therapeutic target for myotonia congenita

PubMed Central

Novak, Kevin R; Norman, Jennifer; Mitchell, Jacob R; Pinter, Martin J; Rich, Mark M

2014-01-01

Objective Patients with myotonia congenita have muscle hyperexcitability due to loss-of-function mutations in the chloride channel in skeletal muscle, which causes spontaneous firing of muscle action potentials (myotonia), producing muscle stiffness. In patients, muscle stiffness lessens with exercise, a change known as the warm-up phenomenon. Our goal was to identify the mechanism underlying warm up and to use this information to guide development of novel therapy. Methods To determine the mechanism underlying warm-up, we used a recently discovered drug to eliminate muscle contraction, thus allowing prolonged intracellular recording from individual muscle fibers during induction of warm-up in a mouse model of myotonia congenita. Results Changes in action potentials suggested slow inactivation of sodium channels as an important contributor to warm-up. These data suggested enhancing slow inactivation of sodium channels might offer effective therapy for myotonia. Lacosamide and ranolazine enhance slow inactivation of sodium channels and are FDA-approved for other uses in patients. We compared the efficacy of both drugs to mexiletine, a sodium channel blocker currently used to treat myotonia. In vitro studies suggested both lacosamide and ranolazine were superior to mexiletine. However, in vivo studies in a mouse model of myotonia congenita suggested side effects could limit the efficacy of lacosamide. Ranolazine produced fewer side effects and was as effective as mexiletine at a dose that produced none of mexiletine’s hypoexcitability side effects. Interpretation We conclude ranolazine has excellent therapeutic potential for treatment of patients with myotonia congenita. PMID:25515836

Calcium-Activated Potassium Channels at Nodes of Ranvier Secure Axonal Spike Propagation

PubMed Central

Gründemann, Jan; Clark, Beverley A.

2015-01-01

Summary Functional connectivity between brain regions relies on long-range signaling by myelinated axons. This is secured by saltatory action potential propagation that depends fundamentally on sodium channel availability at nodes of Ranvier. Although various potassium channel types have been anatomically localized to myelinated axons in the brain, direct evidence for their functional recruitment in maintaining node excitability is scarce. Cerebellar Purkinje cells provide continuous input to their targets in the cerebellar nuclei, reliably transmitting axonal spikes over a wide range of rates, requiring a constantly available pool of nodal sodium channels. We show that the recruitment of calcium-activated potassium channels (IK, KCa3.1) by local, activity-dependent calcium (Ca2+) influx at nodes of Ranvier via a T-type voltage-gated Ca2+ current provides a powerful mechanism that likely opposes depolarizing block at the nodes and is thus pivotal to securing continuous axonal spike propagation in spontaneously firing Purkinje cells. PMID:26344775

Influence of Pyrethroid Insecticides on Sodium and Calcium Influx in Neocortical Neurons

EPA Science Inventory

Pyrethroid insecticides bind to voltage-gated sodium channels and modify their gating kinetics, thereby disrupting neuronal function. Using murine neocortical neurons in primary culture, we have compared the ability of 11 structurally diverse pyrethroid insecticides to evoke Na+ ...

Additivity of Pyrethroid Actions on Sodium Influx in Cortical Neuronsin vitro

EPA Science Inventory

Background: Pyrethroid insecticides bind to voltage-gated sodium channels (VGSCs) and modify their gating kinetics, thereby disrupting neuronal function. While previous work has tested the additivity of pyrethroids in vivo, the additivity of these compounds at the major target si...

The sodium-activated potassium channel Slack is required for optimal cognitive flexibility in mice.

PubMed

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

2015-07-01

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 development. In particular, recent findings have shown that human Slack mutations produce very severe intellectual disability and that Slack channels interact directly with the Fragile X mental retardation protein (FMRP), a protein that when missing or mutated results in Fragile X syndrome (FXS), the most common form of inherited intellectual disability and autism in humans. We have now analyzed a recently developed Kcnt1 null mouse model in several behavioral tasks to assess which aspects of memory and learning are dependent on Slack. We demonstrate that Slack deficiency results in mildly altered general locomotor activity, but normal working memory, reference memory, as well as cerebellar control of motor functions. In contrast, we find that Slack channels are required for cognitive flexibility, including reversal learning processes and the ability to adapt quickly to unfamiliar situations and environments. Our data reveal that hippocampal-dependent spatial learning capabilities require the proper function of Slack channels. © 2015 Bausch et al.; Published by Cold Spring Harbor Laboratory Press.

The sodium-activated potassium channel Slack is required for optimal cognitive flexibility in mice

PubMed Central

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

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 development. In particular, recent findings have shown that human Slack mutations produce very severe intellectual disability and that Slack channels interact directly with the Fragile X mental retardation protein (FMRP), a protein that when missing or mutated results in Fragile X syndrome (FXS), the most common form of inherited intellectual disability and autism in humans. We have now analyzed a recently developed Kcnt1 null mouse model in several behavioral tasks to assess which aspects of memory and learning are dependent on Slack. We demonstrate that Slack deficiency results in mildly altered general locomotor activity, but normal working memory, reference memory, as well as cerebellar control of motor functions. In contrast, we find that Slack channels are required for cognitive flexibility, including reversal learning processes and the ability to adapt quickly to unfamiliar situations and environments. Our data reveal that hippocampal-dependent spatial learning capabilities require the proper function of Slack channels. PMID:26077685

Na+ influx via Orai1 inhibits intracellular ATP-induced mTORC2 signaling to disrupt CD4 T cell gene expression and differentiation

PubMed Central

Miao, Yong; Bhushan, Jaya; Dani, Adish; Vig, Monika

2017-01-01

T cell effector functions require sustained calcium influx. However, the signaling and phenotypic consequences of non-specific sodium permeation via calcium channels remain unknown. α-SNAP is a crucial component of Orai1 channels, and its depletion disrupts the functional assembly of Orai1 multimers. Here we show that α-SNAP hypomorph, hydrocephalus with hopping gait, Napahyh/hyh mice harbor significant defects in CD4 T cell gene expression and Foxp3 regulatory T cell (Treg) differentiation. Mechanistically, TCR stimulation induced rapid sodium influx in Napahyh/hyh CD4 T cells, which reduced intracellular ATP, [ATP]i. Depletion of [ATP]i inhibited mTORC2 dependent NFκB activation in Napahyh/hyh cells but ablation of Orai1 restored it. Remarkably, TCR stimulation in the presence of monensin phenocopied the defects in Napahyh/hyh signaling and Treg differentiation, but not IL-2 expression. Thus, non-specific sodium influx via bonafide calcium channels disrupts unexpected signaling nodes and may provide mechanistic insights into some divergent phenotypes associated with Orai1 function. DOI: http://dx.doi.org/10.7554/eLife.25155.001 PMID:28492364

Metaflumizone is a novel sodium channel blocker insecticide.

PubMed

Salgado, V L; Hayashi, J H

2007-12-15

Metaflumizone is a novel semicarbazone insecticide, derived chemically from the pyrazoline sodium channel blocker insecticides (SCBIs) discovered at Philips-Duphar in the early 1970s, but with greatly improved mammalian safety. This paper describes studies confirming that the insecticidal action of metaflumizone is due to the state-dependent blockage of sodium channels. Larvae of the moth Spodoptera eridania injected with metaflumizone became paralyzed, concomitant with blockage of all nerve activity. Furthermore, tonic firing of abdominal stretch receptor organs from Spodoptera frugiperda was blocked by metaflumizone applied in the bath, consistent with the block of voltage-dependent sodium channels. Studies on native sodium channels, in primary-cultured neurons isolated from the CNS of the larvae of the moth Manduca sexta and on Para/TipE sodium channels heterologously expressed in Xenopus (African clawed frog) oocytes, confirmed that metaflumizone blocks sodium channels by binding selectively to the slow-inactivated state, which is characteristic of the SCBIs. The results confirm that metaflumizone is a novel sodium channel blocker insecticide.

Sodium channel from rat brain. Reconstitution of voltage-dependent scorpion toxin binding in vesicles of defined lipid composition

DOE Office of Scientific and Technical Information (OSTI.GOV)

Feller, D.J.; Talvenheimo, J.A.; Catterall, W.A.

1985-09-25

Purified sodium channels incorporated into phosphatidylcholine (PC) vesicles mediate neurotoxin-activated SSNa influx but do not bind the alpha-scorpion toxin from Leiurus quinquestriatus (LqTx) with high affinity. Addition of phosphatidylethanolamine (PE) or phosphatidylserine to the reconstitution mixture restores high affinity LqTx binding with KD = 1.9 nM for PC/PE vesicles at -90 mV and 36 degrees C in sucrose-substituted medium. Other lipids tested were markedly less effective. The binding of LqTx in vesicles of PC/PE (65:35) is sensitive to both the membrane potential formed by sodium gradients across the reconstituted vesicle membrane and the cation concentration in the extravesicular medium. Bindingmore » of LqTx is reduced 3- to 4-fold upon depolarization to 0 mV from -50 to -60 mV in experiments in which (Na+)out/(Na+)in is varied by changing (Na+)in or (Na+)out at constant extravesicular ionic strength. It is concluded that the purified sodium channel contains the receptor site for LqTx in functional form and that restoration of high affinity, voltage-dependent binding of LqTx by the purified sodium channel requires an appropriate ratio of PC to PE and/or phosphatidylserine in the vesicle membrane.« less

[Mechanisms of action of voltage-gated sodium channel ligands].

PubMed

Tikhonov, D B

2007-05-01

The voltage-gated sodium channels play a key role in the generation of action potential in excitable cells. Sodium channels are targeted by a number of modulating ligands. Despite numerous studies, the mechanisms of action of many ligands are still unknown. The main cause of the problem is the absence of the channel structure. Sodium channels belong to the superfamily of P-loop channels that also the data abowt includes potassium and calcium channels and the channels of ionotropic glutamate receptors. Crystallization of several potassium channels has opened a possibility to analyze the structure of other members of the superfamily using the homology modeling approach. The present study summarizes the results of several recent modelling studies of such sodium channel ligands as tetrodotoxin, batrachotoxin and local anesthetics. Comparison of available experimental data with X-ray structures of potassium channels has provided a new level of understanding of the mechanisms of action of sodium channel ligands and has allowed proposing several testable hypotheses.

Additivity of Pyrethroid Actions on Sodium Influx in Cortical Neurons in Cerebrocortical Neurons in Primary Culture

EPA Science Inventory

BACKGROUND: Pyrethroid insecticides bind to voltage-gated sodium channels and modify their gating kinetics, thereby disrupting neuronal function. Although previous work has tested the additivity of pyrethroids in vivo, this has not been assessed directly at the primary molecular ...

The human Nav1.5 F1486 deletion associated with long QT syndrome leads to impaired sodium channel inactivation and reduced lidocaine sensitivity

PubMed Central

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

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

PubMed Central

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

Epithelial Sodium and Acid-Sensing Ion Channels

NASA Astrophysics Data System (ADS)

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.

The C-terminal coiled-coil of the bacterial voltage-gated sodium channel NaChBac is not essential for tetramer formation, but stabilizes subunit-to-subunit interactions.

PubMed

Mio, Kazuhiro; Mio, Muneyo; Arisaka, Fumio; Sato, Masahiko; Sato, Chikara

2010-09-01

The NaChBac is a prokaryotic homologue of the voltage-gated sodium channel found in the genome of the alkalophilic bacterium Bacillus halodurans C-125. Like a repeating cassette of mammalian sodium channel, the NaChBac possesses hydrophobic domains corresponding to six putative transmembrane segments and a pore loop, and exerts channel function by forming a tetramer although detailed mechanisms of subunit assembly remain unclear. We generated truncated mutants from NaChBac, and investigated their ability to form tetramers in relation to their channel functions. A mutant that deletes almost all of the C-terminal coiled-coil structure lost its voltage-dependent ion permeability, although it was properly translocated to the cell surface. The mutant protein was purified as a tetramer using a reduced concentration of detergent, but the association between the subunits was shown to be much weaker than the wild type. The chemical cross-linking, blue native PAGE, sedimentation velocity experiments, size exclusion chromatography, immunoprecipitation, and electron microscopy all supported its tetrameric assembly. We further purified the C-terminal cytoplasmic domain alone and confirmed its self-oligomerization. These data suggest that the C-terminal coiled-coil structure stabilizes subunit-to-subunit interactions of NaChBac, but is not critical for their tetramer formation. 2010 Elsevier Ltd. All rights reserved.

Chimeric Derivatives of Functionalized Amino Acids and α-Aminoamides: Compounds with Anticonvulsant Activity in Seizure Models and Inhibitory Actions on Central, Peripheral, and Cardiac Isoforms of Voltage-gated Sodium Channels

PubMed Central

Torregrosa, Robert; Yang, Xiao-Fang; Dustrude, Erik T.; Cummins, Theodore R.

2015-01-01

Six novel 3″-substituted (R)-N-(phenoxybenzyl) 2-N-acetamido-3-methoxypropionamides were prepared and then assessed using whole-cell, patch-clamp electrophysiology for their anticonvulsant activities in animal seizure models and for their sodium channel activities. We found compounds with various substituents at the terminal aromatic ring that had excellent anticonvulsant activity. Of these compounds, (R)-N-4′-((3″-chloro)phenoxy)benzyl 2-N-acetamido-3-methoxypropionamide ((R)-5) and (R)-N-4′-((3″-trifluoromethoxy)phenoxy)benzyl 2-N-acetamido-3-methoxypropionamide ((R)-9) exhibited high protective indices (PI = TD50/ED50) comparable with many antiseizure drugs when tested in the maximal electroshock seizure test to mice (intraperitoneally) and rats (intraperitoneally, orally). Most compounds potently transitioned sodium channels to the slow-inactivated state when evaluated in rat embryonic cortical neurons. Treating HEK293 recombinant cells that expressed hNav1.1, rNav1.3, hNav1.5, or hNav1.7 with (R)-9 recapitulated the high levels of sodium channel slow inactivation. PMID:25922183

Sodium channel selectivity and conduction: Prokaryotes have devised their own molecular strategy

PubMed Central

Finol-Urdaneta, Rocio K.; Wang, Yibo; Al-Sabi, Ahmed; Zhao, Chunfeng

2014-01-01

Striking structural differences between voltage-gated sodium (Nav) channels from prokaryotes (homotetramers) and eukaryotes (asymmetric, four-domain proteins) suggest the likelihood of different molecular mechanisms for common functions. For these two channel families, our data show similar selectivity sequences among alkali cations (relative permeability, Pion/PNa) and asymmetric, bi-ionic reversal potentials when the Na/K gradient is reversed. We performed coordinated experimental and computational studies, respectively, on the prokaryotic Nav channels NaChBac and NavAb. NaChBac shows an “anomalous,” nonmonotonic mole-fraction dependence in the presence of certain sodium–potassium mixtures; to our knowledge, no comparable observation has been reported for eukaryotic Nav channels. NaChBac’s preferential selectivity for sodium is reduced either by partial titration of its highly charged selectivity filter, when extracellular pH is lowered from 7.4 to 5.8, or by perturbation—likely steric—associated with a nominally electro-neutral substitution in the selectivity filter (E191D). Although no single molecular feature or energetic parameter appears to dominate, our atomistic simulations, based on the published NavAb crystal structure, revealed factors that may contribute to the normally observed selectivity for Na over K. These include: (a) a thermodynamic penalty to exchange one K+ for one Na+ in the wild-type (WT) channel, increasing the relative likelihood of Na+ occupying the binding site; (b) a small tendency toward weaker ion binding to the selectivity filter in Na–K mixtures, consistent with the higher conductance observed with both sodium and potassium present; and (c) integrated 1-D potentials of mean force for sodium or potassium movement that show less separation for the less selective E/D mutant than for WT. Overall, tight binding of a single favored ion to the selectivity filter, together with crucial inter-ion interactions within the pore, suggests that prokaryotic Nav channels use a selective strategy more akin to those of eukaryotic calcium and potassium channels than that of eukaryotic Nav channels. PMID:24420772

Pyrethroids and Nectar Toxins Have Subtle Effects on the Motor Function, Grooming and Wing Fanning Behaviour of Honeybees (Apis mellifera).

PubMed

Oliver, Caitlin J; Softley, Samantha; Williamson, Sally M; Stevenson, Philip C; Wright, Geraldine A

2015-01-01

Sodium channels, found ubiquitously in animal muscle cells and neurons, are one of the main target sites of many naturally-occurring, insecticidal plant compounds and agricultural pesticides. Pyrethroids, derived from compounds found only in the Asteraceae, are particularly toxic to insects and have been successfully used as pesticides including on flowering crops that are visited by pollinators. Pyrethrins, from which they were derived, occur naturally in the nectar of some flowering plant species. We know relatively little about how such compounds--i.e., compounds that target sodium channels--influence pollinators at low or sub-lethal doses. Here, we exposed individual adult forager honeybees to several compounds that bind to sodium channels to identify whether these compounds affect motor function. Using an assay previously developed to identify the effect of drugs and toxins on individual bees, we investigated how acute exposure to 10 ng doses (1 ppm) of the pyrethroid insecticides (cyfluthrin, tau-fluvalinate, allethrin and permethrin) and the nectar toxins (aconitine and grayanotoxin I) affected honeybee locomotion, grooming and wing fanning behaviour. Bees exposed to these compounds spent more time upside down and fanning their wings. They also had longer bouts of standing still. Bees exposed to the nectar toxin, aconitine, and the pyrethroid, allethrin, also spent less time grooming their antennae. We also found that the concentration of the nectar toxin, grayanotoxin I (GTX), fed to bees affected the time spent upside down (i.e., failure to perform the righting reflex). Our data show that low doses of pyrethroids and other nectar toxins that target sodium channels mainly influence motor function through their effect on the righting reflex of adult worker honeybees.

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

PubMed

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.

Dopamine negatively modulates the NCA ion channels in C. elegans

PubMed Central

Topalidou, Irini; Pereira, Laura

2017-01-01

The NALCN/NCA ion channel is a cation channel related to voltage-gated sodium and calcium channels. NALCN has been reported to be a sodium leak channel with a conserved role in establishing neuronal resting membrane potential, but its precise cellular role and regulation are unclear. The Caenorhabditis elegans orthologs of NALCN, NCA-1 and NCA-2, act in premotor interneurons to regulate motor circuit activity that sustains locomotion. Recently we found that NCA-1 and NCA-2 are activated by a signal transduction pathway acting downstream of the heterotrimeric G protein Gq and the small GTPase Rho. Through a forward genetic screen, here we identify the GPCR kinase GRK-2 as a new player affecting signaling through the Gq-Rho-NCA pathway. Using structure-function analysis, we find that the GPCR phosphorylation and membrane association domains of GRK-2 are required for its function. Genetic epistasis experiments suggest that GRK-2 acts on the D2-like dopamine receptor DOP-3 to inhibit Go signaling and positively modulate NCA-1 and NCA-2 activity. Through cell-specific rescuing experiments, we find that GRK-2 and DOP-3 act in premotor interneurons to modulate NCA channel function. Finally, we demonstrate that dopamine, through DOP-3, negatively regulates NCA activity. Thus, this study identifies a pathway by which dopamine modulates the activity of the NCA channels. PMID:28968387

Dopamine negatively modulates the NCA ion channels in C. elegans.

PubMed

Topalidou, Irini; Cooper, Kirsten; Pereira, Laura; Ailion, Michael

2017-10-01

The NALCN/NCA ion channel is a cation channel related to voltage-gated sodium and calcium channels. NALCN has been reported to be a sodium leak channel with a conserved role in establishing neuronal resting membrane potential, but its precise cellular role and regulation are unclear. The Caenorhabditis elegans orthologs of NALCN, NCA-1 and NCA-2, act in premotor interneurons to regulate motor circuit activity that sustains locomotion. Recently we found that NCA-1 and NCA-2 are activated by a signal transduction pathway acting downstream of the heterotrimeric G protein Gq and the small GTPase Rho. Through a forward genetic screen, here we identify the GPCR kinase GRK-2 as a new player affecting signaling through the Gq-Rho-NCA pathway. Using structure-function analysis, we find that the GPCR phosphorylation and membrane association domains of GRK-2 are required for its function. Genetic epistasis experiments suggest that GRK-2 acts on the D2-like dopamine receptor DOP-3 to inhibit Go signaling and positively modulate NCA-1 and NCA-2 activity. Through cell-specific rescuing experiments, we find that GRK-2 and DOP-3 act in premotor interneurons to modulate NCA channel function. Finally, we demonstrate that dopamine, through DOP-3, negatively regulates NCA activity. Thus, this study identifies a pathway by which dopamine modulates the activity of the NCA channels.

Analysis of the structural and molecular basis of voltage-sensitive sodium channel inhibition by the spider toxin huwentoxin-IV (μ-TRTX-Hh2a).

PubMed

Minassian, Natali A; Gibbs, Alan; Shih, Amy Y; Liu, Yi; Neff, Robert A; Sutton, Steven W; Mirzadegan, Tara; Connor, Judith; Fellows, Ross; Husovsky, Matthew; Nelson, Serena; Hunter, Michael J; Flinspach, Mack; Wickenden, Alan D

2013-08-02

Voltage-gated sodium channels (VGSCs) are essential to the normal function of the vertebrate nervous system. Aberrant function of VGSCs underlies a variety of disorders, including epilepsy, arrhythmia, and pain. A large number of animal toxins target these ion channels and may have significant therapeutic potential. Most of these toxins, however, have not been characterized in detail. Here, by combining patch clamp electrophysiology and radioligand binding studies with peptide mutagenesis, NMR structure determination, and molecular modeling, we have revealed key molecular determinants of the interaction between the tarantula toxin huwentoxin-IV and two VGSC isoforms, Nav1.7 and Nav1.2. Nine huwentoxin-IV residues (F6A, P11A, D14A, L22A, S25A, W30A, K32A, Y33A, and I35A) were important for block of Nav1.7 and Nav1.2. Importantly, molecular dynamics simulations and NMR studies indicated that folding was normal for several key mutants, suggesting that these amino acids probably make specific interactions with sodium channel residues. Additionally, we identified several amino acids (F6A, K18A, R26A, and K27A) that are involved in isoform-specific VGSC interactions. Our structural and functional data were used to model the docking of huwentoxin-IV into the domain II voltage sensor of Nav1.7. The model predicts that a hydrophobic patch composed of Trp-30 and Phe-6, along with the basic Lys-32 residue, docks into a groove formed by the Nav1.7 S1-S2 and S3-S4 loops. These results provide new insight into the structural and molecular basis of sodium channel block by huwentoxin-IV and may provide a basis for the rational design of toxin-based peptides with improved VGSC potency and/or selectivity.

Toxins That Affect Voltage-Gated Sodium Channels.

PubMed

Ji, Yonghua

2017-10-26

Voltage-gated sodium channels (VGSCs) are critical in generation and conduction of electrical signals in multiple excitable tissues. Natural toxins, produced by animal, plant, and microorganisms, target VGSCs through diverse strategies developed over millions of years of evolutions. Studying of the diverse interaction between VGSC and VGSC-targeting toxins has been contributing to the increasing understanding of molecular structure and function, pharmacology, and drug development potential of VGSCs. This chapter aims to summarize some of the current views on the VGSC-toxin interaction based on the established receptor sites of VGSC for natural toxins.

Function and solution structure of huwentoxin-IV, a potent neuronal tetrodotoxin (TTX)-sensitive sodium channel antagonist from Chinese bird spider Selenocosmia huwena.

PubMed

Peng, Kuan; Shu, Qin; Liu, Zhonghua; Liang, Songping

2002-12-06

We have isolated a highly potent neurotoxin from the venom of the Chinese bird spider, Selenocosmia huwena. This 4.1-kDa toxin, which has been named huwentoxin-IV, contains 35 residues with three disulfide bridges: Cys-2-Cys-17, Cys-9-Cys-24, and Cys-16-Cys-31, assigned by a chemical strategy including partial reduction of the toxin and sequence analysis of the modified intermediates. It specifically inhibits the neuronal tetrodotoxin-sensitive (TTX-S) voltage-gated sodium channel with the IC(50) value of 30 nm in adult rat dorsal root ganglion neurons, while having no significant effect on the tetrodotoxin-resistant (TTX-R) voltage-gated sodium channel. This toxin seems to be a site I toxin affecting the sodium channel through a mechanism quite similar to that of TTX: it suppresses the peak sodium current without altering the activation or inactivation kinetics. The three-dimensional structure of huwentoxin-IV has been determined by two-dimensional (1)H NMR combined with distant geometry and simulated annealing calculation by using 527 nuclear Overhauser effect constraints and 14 dihedral constraints. The resulting structure is composed of a double-stranded antiparallel beta-sheet (Leu-22-Ser-25 and Trp-30-Tyr-33) and four turns (Glu-4-Lys-7, Pro-11-Asp-14, Lys-18-Lys-21 and Arg-26-Arg-29) and belongs to the inhibitor cystine knot structural family. After comparison with other toxins purified from the same species, we are convinced that the positively charged residues of loop IV (residues 25-29), especially residue Arg-26, must be crucial to its binding to the neuronal tetrodotoxin-sensitive voltage-gated sodium channel.

Functional coupling between sodium-activated potassium channels and voltage-dependent persistent sodium currents in cricket Kenyon cells.

PubMed

Takahashi, Izumi; Yoshino, Masami

2015-10-01

In this study, we examined the functional coupling between Na(+)-activated potassium (KNa) channels and Na(+) influx through voltage-dependent Na(+) channels in Kenyon cells isolated from the mushroom body of the cricket Gryllus bimaculatus. Single-channel activity of KNa channels was recorded with the cell-attached patch configuration. The open probability (Po) of KNa channels increased with increasing Na(+) concentration in a bath solution, whereas it decreased by the substitution of Na(+) with an equimolar concentration of Li(+). The Po of KNa channels was also found to be reduced by bath application of a high concentration of TTX (1 μM) and riluzole (100 μM), which inhibits both fast (INaf) and persistent (INaP) Na(+) currents, whereas it was unaffected by a low concentration of TTX (10 nM), which selectively blocks INaf. Bath application of Cd(2+) at a low concentration (50 μM), as an inhibitor of INaP, also decreased the Po of KNa channels. Conversely, bath application of the inorganic Ca(2+)-channel blockers Co(2+) and Ni(2+) at high concentrations (500 μM) had little effect on the Po of KNa channels, although Cd(2+) (500 μM) reduced the Po of KNa channels. Perforated whole cell clamp analysis further indicated the presence of sustained outward currents for which amplitude was dependent on the amount of Na(+) influx. Taken together, these results indicate that KNa channels could be activated by Na(+) influx passing through voltage-dependent persistent Na(+) channels. The functional significance of this coupling mechanism was discussed in relation to the membrane excitability of Kenyon cells and its possible role in the formation of long-term memory. Copyright © 2015 the American Physiological Society.

A negative charge in transmembrane segment 1 of domain II of the cockroach sodium channel is critical for channel gating and action of pyrethroid insecticides

DOE Office of Scientific and Technical Information (OSTI.GOV)

Du Yuzhe; Song Weizhong; Groome, James R.

2010-08-15

Voltage-gated sodium channels are the primary target of pyrethroids, an important class of synthetic insecticides. Pyrethroids bind to a distinct receptor site on sodium channels and prolong the open state by inhibiting channel deactivation and inactivation. Recent studies have begun to reveal sodium channel residues important for pyrethroid binding. However, how pyrethroid binding leads to inhibition of sodium channel deactivation and inactivation remains elusive. In this study, we show that a negatively charged aspartic acid residue at position 802 (D802) located in the extracellular end of transmembrane segment 1 of domain II (IIS1) is critical for both the action ofmore » pyrethroids and the voltage dependence of channel activation. Charge-reversing or -neutralizing substitutions (K, G, or A) of D802 shifted the voltage dependence of activation in the depolarizing direction and reduced channel sensitivity to deltamethrin, a pyrethroid insecticide. The charge-reversing mutation D802K also accelerated open-state deactivation, which may have counteracted the inhibition of sodium channel deactivation by deltamethrin. In contrast, the D802G substitution slowed open-state deactivation, suggesting an additional mechanism for neutralizing the action of deltamethrin. Importantly, Schild analysis showed that D802 is not involved in pyrethroid binding. Thus, we have identified a sodium channel residue that is critical for regulating the action of pyrethroids on the sodium channel without affecting the receptor site of pyrethroids.« less

COMPLETE INHIBITION OF SPONTANEOUS ACTIVITY IN NEURONAL NETWORKS IN VITRO BY DELTAMETHRIN AND PERMETHRIN

EPA Science Inventory

Type I and II pyrethroid insecticides cause temporally distinct decreases in voltage-gated sodium channel (VGSC) inactivation rates that are proposed to underlie their characteristic differences in toxicity signs. How alterations in VGSC channel function give rise to the characte...

USE OF GENOMIC TECHNOLOGIES AND ISOTONIC DOSE-RESPONSE MODELING IN THE DEVELOPMENT OF A BIOCHEMICAL MARKER OF EFFECT FOR PYRETHROID INSECTICIDE.

EPA Science Inventory

Pyrethroids are pesticides that disrupt nervous system function by prolongation of sodium currents

through voltage-sensitive sodium channels present in nerve membranes. Pyrethroid usage has

increased as use of other pesticides has declined. A sensitive, dose-respons...

H2O2 Regulates Lung Epithelial Sodium Channel (ENaC) via Ubiquitin-like Protein Nedd8

PubMed Central

Downs, Charles A.; Kumar, Amrita; Kreiner, Lisa H.; Johnson, Nicholle M.; Helms, My N.

2013-01-01

Redundancies in both the ubiquitin and epithelial sodium transport pathways allude to their importance of proteolytic degradation and ion transport in maintaining normal cell function. The classical pathway implicated in ubiquitination of the epithelial sodium channel (ENaC) involves Nedd4-2 regulation of sodium channel subunit expression and has been studied extensively studied. However, less attention has been given to the role of the ubiquitin-like protein Nedd8. Here we show that Nedd8 plays an important role in the ubiquitination of ENaC in alveolar epithelial cells. We report that the Nedd8 pathway is redox-sensitive and that under oxidizing conditions Nedd8 conjugation to Cullin-1 is attenuated, resulting in greater surface expression of α-ENaC. This observation was confirmed in our electrophysiology studies in which we inhibited Nedd8-activating enzyme using MLN4924 (a specific Nedd8-activating enzyme inhibitor) and observed a marked increase in ENaC activity (measured as the product of the number of channels (N) and the open probability (Po) of a channel). These results suggest that ubiquitination of lung ENaC is redox-sensitive and may have significant implications for our understanding of the role of ENaC in pulmonary conditions where oxidative stress occurs, such as pulmonary edema and acute lung injury. PMID:23362276

Novel Target for Ameliorating Pain and Other Problems after SCI: Spontaneous Activityin Nociceptors

DTIC Science & Technology

2015-10-01

Funding support (other than DoD) Mission Connect-TIRR Foundation, "Neuroprotective Effect of Targeting KCNQ/ Kv7 Channels in Spinal Cord Injury...the function of a sodium ion channel , Nav1.8, that is selectively expressed in primary afferent neurons (especially nociceptors) ameliorate reflex...our finding that antisense knockdown of TRPV1 channels or pharmacological blockade of TRPV1 channels -- which are expressed most abundantly in

Hydration status regulates sodium flux and inflammatory pathways through epithelial sodium channel (ENaC) in the skin.

PubMed

Xu, Wei; Hong, Seok Jong; Zeitchek, Michael; Cooper, Garry; Jia, Shengxian; Xie, Ping; Qureshi, Hannan A; Zhong, Aimei; Porterfield, Marshall D; Galiano, Robert D; Surmeier, D James; Mustoe, Thomas A

2015-03-01

Although it is known that the inflammatory response that results from disruption of epithelial barrier function after injury results in excessive scarring, the upstream signals remain unknown. It has also been observed that epithelial disruption results in reduced hydration status and that the use of occlusive dressings that prevent water loss from wounds decreases scar formation. We hypothesized that hydration status changes sodium homeostasis and induces sodium flux in keratinocytes, which result in activation of pathways responsible for keratinocyte-fibroblast signaling and ultimately lead to activation of fibroblasts. Here, we demonstrate that perturbations in epithelial barrier function lead to increased sodium flux in keratinocytes. We identified that sodium flux in keratinocytes is mediated by epithelial sodium channels (ENaCs) and causes increased secretion of proinflammatory cytokines, which activate fibroblast via the cyclooxygenase 2 (COX-2)/prostaglandin E2 (PGE2) pathway. Similar changes in signal transduction and sodium flux occur by increased sodium concentration, which simulates reduced hydration, in the media in epithelial cultures or human ex vivo skin cultures. Blockade of ENaC, prostaglandin synthesis, or PGE2 receptors all reduce markers of fibroblast activation and collagen synthesis. In addition, employing a validated in vivo excessive scar model in the rabbit ear, we demonstrate that utilization of either an ENaC blocker or a COX-2 inhibitor results in a marked reduction in scarring. Other experiments demonstrate that the activation of COX-2 in response to increased sodium flux is mediated through the PIK3/Akt pathway. Our results indicate that ENaC responds to small changes in sodium concentration with inflammatory mediators and suggest that the ENaC pathway is a potential target for a strategy to prevent fibrosis.

Local anesthetic-induced inhibition of human neutrophil priming: the influence of structure, lipophilicity, and charge.

PubMed

Picardi, Susanne; Cartellieri, Sibylle; Groves, Danja; Hahnenkamp, Klaus; Hahnenekamp, Klaus; Gerner, Peter; Durieux, Marcel E; Stevens, Markus F; Lirk, Philipp; Hollmann, Markus W

2013-01-01

Local anesthetics (LAs) are widely known for inhibition of voltage-gated sodium channels underlying their antiarrhythmic and antinociceptive effects. However, LAs have significant immunomodulatory properties and were shown to affect human neutrophil functions independent of sodium-channel blockade. Previous studies suggest a highly selective interaction between LAs and the α-subunit of G protein-coupled receptors of the Gq/G11 family as underlying mechanism. Providing a detailed structure function analysis, this study aimed to determine the active parts within the LA molecule responsible for the effects on human neutrophil priming. Human neutrophils were incubated with structurally different LAs for 60 minutes, followed by priming and activation using either platelet-activating factor or lysophosphatidic acid and N-formyl-methionyl-L-leucyl-L-phenylalanine. Superoxide anion generation was determined, using the cytochrome c reduction assay. Differences in priming inhibition of human neutrophils between LAs were smaller than expected, although significant. Ester-linked LAs blocked priming responses more effectively than did amide LAs. Furthermore, the inhibitory potency of LAs on priming decreased with an increase of their respective octanol-buffer coefficient, and inhibition did not correlate with sodium-channel-blocking potency. Charge was not crucially required for priming inhibition, yet it played a role in effect size. Local anesthetics significantly attenuated Gαq-protein-mediated neutrophil priming. The most potent inhibition was achieved by ester compounds, inversely correlated with their octanol-buffer coefficient, and enhanced by permanent charges within the LA molecule. No correlation to their potency of blocking sodium channels was found.

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

PubMed Central

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

2001-01-01

Tetrapentylammonium (TPeA) block of rat brain type IIA sodium channel α 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. PMID:11309247

Sodium Channel Nav1.8 Underlies TTX-Resistant Axonal Action Potential Conduction in Somatosensory C-Fibers of Distal Cutaneous Nerves.

PubMed

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 fibers, but clarifying the role of sodium channel subtypes in different axonal segments may be useful for the development of novel analgesic strategies. Here, we provide evidence from mice and nonhuman primates that a substantial portion of the C-fiber compound action potential in distal peripheral nerves, but not proximal nerves or dorsal roots, is resistant to tetrodotoxin and that, in mice, this effect is mediated solely by voltage-gated sodium channel 1.8 (Na V 1.8). The functional prominence of Na V 1.8 within the axonal compartment immediately proximal to its termination may affect strategies targeting pain of peripheral origin. Copyright © 2017 the authors 0270-6474/17/375205-11$15.00/0.

Pharmacological reversal of a pain phenotype in iPSC-derived sensory neurons and patients with inherited erythromelalgia.

PubMed

Cao, Lishuang; McDonnell, Aoibhinn; Nitzsche, Anja; Alexandrou, Aristos; Saintot, Pierre-Philippe; Loucif, Alexandre J C; Brown, Adam R; Young, Gareth; Mis, Malgorzata; Randall, Andrew; Waxman, Stephen G; Stanley, Philip; Kirby, Simon; Tarabar, Sanela; Gutteridge, Alex; Butt, Richard; McKernan, Ruth M; Whiting, Paul; Ali, Zahid; Bilsland, James; Stevens, Edward B

2016-04-20

In common with other chronic pain conditions, there is an unmet clinical need in the treatment of inherited erythromelalgia (IEM). TheSCN9Agene encoding the sodium channel Nav1.7 expressed in the peripheral nervous system plays a critical role in IEM. A gain-of-function mutation in this sodium channel leads to aberrant sensory neuronal activity and extreme pain, particularly in response to heat. Five patients with IEM were treated with a new potent and selective compound that blocked the Nav1.7 sodium channel resulting in a decrease in heat-induced pain in most of the patients. We derived induced pluripotent stem cell (iPSC) lines from four of five subjects and produced sensory neurons that emulated the clinical phenotype of hyperexcitability and aberrant responses to heat stimuli. When we compared the severity of the clinical phenotype with the hyperexcitability of the iPSC-derived sensory neurons, we saw a trend toward a correlation for individual mutations. The in vitro IEM phenotype was sensitive to Nav1.7 blockers, including the clinical test agent. Given the importance of peripherally expressed sodium channels in many pain conditions, our approach may have broader utility for a wide range of pain and sensory conditions. Copyright © 2016, American Association for the Advancement of Science.

The Tetrodotoxin Receptor of Voltage-Gated Sodium Channels—Perspectives from Interactions with μ-Conotoxins

PubMed Central

French, Robert J.; Yoshikami, Doju; Sheets, Michael F.; Olivera, Baldomero M.

2010-01-01

Neurotoxin receptor site 1, in the outer vestibule of the conducting pore of voltage-gated sodium channels (VGSCs), was first functionally defined by its ability to bind the guanidinium-containing agents, tetrodotoxin (TTX) and saxitoxin (STX). Subsequent studies showed that peptide μ-conotoxins competed for binding at site 1. All of these natural inhibitors block single sodium channels in an all-or-none manner on binding. With the discovery of an increasing variety of μ-conotoxins, and the synthesis of numerous derivatives, observed interactions between the channel and these different ligands have become more complex. Certain μ-conotoxin derivatives block single-channel currents partially, rather than completely, thus enabling the demonstration of interactions between the bound toxin and the channel’s voltage sensor. Most recently, the relatively small μ-conotoxin KIIIA (16 amino acids) and its variants have been shown to bind simultaneously with TTX and exhibit both synergistic and antagonistic interactions with TTX. These interactions raise new pharmacological possibilities and place new constraints on the possible structures of the bound complexes of VGSCs with these toxins. PMID:20714429

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

PubMed

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.

Identification of PN1, a Predominant Voltage-Dependent Sodium Channel Expressed Principally in Peripheral Neurons

NASA Astrophysics Data System (ADS)

Toledo-Aral, Juan J.; Moss, Brenda L.; He, Zhi-Jun; Koszowski, Adam G.; Whisenand, Teri; Levinson, Simon R.; Wolf, John J.; Silos-Santiago, Inmaculada; Halegoua, Simon; Mandel, Gail

1997-02-01

Membrane excitability in different tissues is due, in large part, to the selective expression of distinct genes encoding the voltage-dependent sodium channel. Although the predominant sodium channels in brain, skeletal muscle, and cardiac muscle have been identified, the major sodium channel types responsible for excitability within the peripheral nervous system have remained elusive. We now describe the deduced primary structure of a sodium channel, peripheral nerve type 1 (PN1), which is expressed at high levels throughout the peripheral nervous system and is targeted to nerve terminals of cultured dorsal root ganglion neurons. Studies using cultured PC12 cells indicate that both expression and targeting of PN1 is induced by treatment of the cells with nerve growth factor. The preferential localization suggests that the PN1 sodium channel plays a specific role in nerve excitability.

Voltage-gated sodium channels: pharmaceutical targets via anticonvulsants to treat epileptic syndromes.

PubMed

Abdelsayed, Mena; Sokolov, Stanislav

2013-01-01

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

Action of insecticidal N-alkylamides at site 2 of the voltage-sensitive sodium channel

DOE Office of Scientific and Technical Information (OSTI.GOV)

Ottea, J.A.; Payne, G.T.; Soderlund, D.M.

1990-08-01

Nine synthetic N-alkylamides were examined as inhibitors of the specific binding of ({sup 3}H)batrachotoxinin A 20{alpha}-benzoate (({sup 3}H)BTX-B) to sodium channels and as activators of sodium uptake in mouse brain synaptoneurosomes. In the presence of scorpion (Leiurus quinquestriatus) venom, the six insecticidal analogues were active as both inhibitors of ({sup 3}H)BTX-B binding and stimulators of sodium uptake. These findings are consistent with an action of these compounds at the alkaloid activator recognition site (site 2) of the voltage-sensitive sodium channel. The three noninsecticidal N-alkylamides also inhibited ({sup 3}H)BTX-B binding but were ineffective as activators of sodium uptake. Concentration-response studies revealedmore » that some of the insecticidal amides also enhanced sodium uptake through a second, high-affinity interaction that does not involve site 2, but this secondary effect does not appear to be correlated with insecticidal activity. The activities of N-alkylamides as sodium channel activators were influenced by the length of the alkenyl chain and the location of unsaturation within the molecule. These results further define the actions of N-alkylamides on sodium channels and illustrate the significance of the multiple binding domains of the sodium channel as target sites for insect control agents.« less

[Effects of phoxim and fenvalerate on TTX-S and TTX-R sodium channels in the DRG neurons of adult rat].

PubMed

Wang, X; Xiao, H; Dai, X; Liu, X; Yu, X; Wu, J

2000-05-01

To study the joint neurotoxic effects of phoxim (Pho) and fenvalerate (Fen) on tetrodotoxin-sensitive (TTX-S) and tetrodotoxin-resistant (TTX-R) Na(+) currents in dorsal root ganglion (DRG) neurons of adult rat. Whole cell patch clamp technique was used to test the effects of Pho and Fen on TTX-S and TTX-R sodium currents in DRG neurons. The inactivation of TTX-R sodium channel was obviously slowed down by Fen. The tau(Na) of peak currents at doses of 10, 50 and 100 micromol/L Fen and control groups were (8.10 +/- 2.41) ms, (11.78 +/- 2.76) ms, P < 0.01, (8.76 +/-1.94) ms, P < 0.05 and (6.41 +/- 1.32) ms respectively. The inactivation of TTX-R sodium channel tail currents was also significantly delayed by Fen. The tau(Na) of the tail currents at doses of 10, 50, 100 micromol/L Fen and control groups were 6.11 +/- 0.52 (P < 0.05), 7.82 +/- 0.82 (P < 0.05), 7.23 +/- 1.09 (P < 0.05) and (4.91 +/- 0.97) ms separately. As compared with TTX-R sodium channel, the TTX-S sodium channel was less responsive to Fen exposure, which only led to slowly decay TTX-S sodium tail currents. There was no any effect of Pho on the TTX-S and TTX-R sodium channels. The mixed treatment of a Pho and Fen did not show joint effect on the sodium currents. Both the peak and tail currents are changed by Fen, however, Fen has more remarkable effects on TTX-R than on TTX-S sodium channel. The combined exposure to Pho and Fen shows no joint effect on the sodium channel.

PnPP-19, a Synthetic and Nontoxic Peptide Designed from a Phoneutria nigriventer Toxin, Potentiates Erectile Function via NO/cGMP.

PubMed

Silva, Carolina Nunes; Nunes, Kenia Pedrosa; Torres, Fernanda Silva; Cassoli, Juliana Silva; Santos, Daniel Moreira; Almeida, Flávia De Marco; Matavel, Alessandra; Cruz, Jader Santos; Santos-Miranda, Arthur; Nunes, Allancer Divino C; Castro, Carlos Henrique; Machado de Ávila, Ricardo Andrés; Chávez-Olórtegui, Carlos; Láuar, Stephanie Stransky; Felicori, Liza; Resende, Jarbas Magalhães; Camargos, Elizabeth Ribeiro da Silva; Borges, Márcia Helena; Cordeiro, Marta Nascimento; Peigneur, Steve; Tytgat, Jan; de Lima, Maria Elena

2015-11-01

We designed a peptide, PnPP-19, comprising the potential active core of the Phoneutria nigriventer native toxin PnTx2-6. We investigated its role on erectile function, and its toxicity and immunogenicity. Erectile function was evaluated by the intracavernous pressure-to-mean arterial pressure ratio during electrical field stimulation on rat pelvic ganglia. Cavernous strips were contracted with phenylephrine and relaxation was induced by electrical field stimulation with or without PnPP-19 (10(-8) M). Activity on sodium channels was evaluated by electrophysiological screening of transfected channels on Xenopus oocytes and dorsal root ganglion cells. Antibodies were detected by indirect enzyme-linked immunosorbent assay in mice previously treated with the peptide. Histopathological studies were performed with mouse organs treated with different doses of PnPP-19. PnPP-19 was able to potentiate erection at 4 and 8 Hz in vivo and ex vivo. It showed no toxicity and low immunogenicity in mice, and did not affect sodium channels or rat hearts. PnPP-19 increased cyclic guanosine monophosphate levels at 8 Hz. This effect was inhibited by L-NAME (10(-4) M). Erectile function was partially inhibited by 7-nitroindazole (10(-5) M), a selective inhibitor of neuronal nitric oxide synthase. PnPP-19 potentiates erection in vivo and ex vivo via the nitric oxide/cyclic guanosine monophosphate pathway. It does not affect sodium channels or rat hearts and shows no toxicity and low immunogenicity. These findings make it a promising candidate as a novel drug in the therapy of erectile dysfunction. Copyright © 2015 American Urological Association Education and Research, Inc. Published by Elsevier Inc. All rights reserved.

The Acetylcholine Receptor and Its Ionic Channel as Targets for Drugs and Toxins

DTIC Science & Technology

1981-12-10

mlecular target that can have any number of different binding sites and is able to generate levels of energy barriers which &re a direct function of the...Albuquerque, E.X. XEatrachotoxi.-A 20- a -benzoate: A new radioactive ligand for voltage- sensitive sodium channels. Cell. Mol. Neurobio .. 1: 19-40

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

PubMed

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.

Congenital Insensitivity to Pain: Novel SCN9A Missense and In-Frame Deletion Mutations

PubMed Central

Cox, James J; Sheynin, Jony; Shorer, Zamir; Reimann, Frank; Nicholas, Adeline K; Zubovic, Lorena; Baralle, Marco; Wraige, Elizabeth; Manor, Esther; Levy, Jacov; Woods, C Geoffery; Parvari, Ruti

2010-01-01

SCN9A encodes the voltage-gated sodium channel Nav1.7, a protein highly expressed in pain-sensing neurons. Mutations in SCN9A cause three human pain disorders: bi-allelic loss of function mutations result in Channelopathy-associated Insensitivity to Pain (CIP), whereas activating mutations cause severe episodic pain in Paroxysmal Extreme Pain Disorder (PEPD) and Primary Erythermalgia (PE). To date, all mutations in SCN9A that cause a complete inability to experience pain are protein truncating and presumably lead to no protein being produced. Here, we describe the identification and functional characterization of two novel non-truncating mutations in families with CIP: a homozygously-inherited missense mutation found in a consanguineous Israeli Bedouin family (Nav1.7-R896Q) and a five amino acid in-frame deletion found in a sporadic compound heterozygote (Nav1.7-ΔR1370-L1374). Both of these mutations map to the pore region of the Nav1.7 sodium channel. Using transient transfection of PC12 cells we found a significant reduction in membrane localization of the mutant protein compared to the wild type. Furthermore, voltage clamp experiments of mutant-transfected HEK293 cells show a complete loss of function of the sodium channel, consistent with the absence of pain phenotype. In summary, this study has identified critical amino acids needed for the normal subcellular localization and function of Nav1.7. © 2010 Wiley-Liss, Inc. PMID:20635406

Congenital insensitivity to pain: novel SCN9A missense and in-frame deletion mutations.

PubMed

Cox, James J; Sheynin, Jony; Shorer, Zamir; Reimann, Frank; Nicholas, Adeline K; Zubovic, Lorena; Baralle, Marco; Wraige, Elizabeth; Manor, Esther; Levy, Jacov; Woods, C Geoffery; Parvari, Ruti

2010-09-01

SCN9Aencodes the voltage-gated sodium channel Na(v)1.7, a protein highly expressed in pain-sensing neurons. Mutations in SCN9A cause three human pain disorders: bi-allelic loss of function mutations result in Channelopathy-associated Insensitivity to Pain (CIP), whereas activating mutations cause severe episodic pain in Paroxysmal Extreme Pain Disorder (PEPD) and Primary Erythermalgia (PE). To date, all mutations in SCN9A that cause a complete inability to experience pain are protein truncating and presumably lead to no protein being produced. Here, we describe the identification and functional characterization of two novel non-truncating mutations in families with CIP: a homozygously-inherited missense mutation found in a consanguineous Israeli Bedouin family (Na(v)1.7-R896Q) and a five amino acid in-frame deletion found in a sporadic compound heterozygote (Na(v)1.7-DeltaR1370-L1374). Both of these mutations map to the pore region of the Na(v)1.7 sodium channel. Using transient transfection of PC12 cells we found a significant reduction in membrane localization of the mutant protein compared to the wild type. Furthermore, voltage clamp experiments of mutant-transfected HEK293 cells show a complete loss of function of the sodium channel, consistent with the absence of pain phenotype. In summary, this study has identified critical amino acids needed for the normal subcellular localization and function of Na(v)1.7. Copyright 2010 Wiley-Liss, Inc.

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

PubMed

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.

CFTR and lung homeostasis

PubMed Central

Matalon, Sadis

2014-01-01

CFTR is a cAMP-activated chloride and bicarbonate channel that is critical for lung homeostasis. Decreases in CFTR expression have dire consequences in cystic fibrosis (CF) and have been suggested to be a component of the lung pathology in chronic obstructive pulmonary disease. Decreases or loss of channel function often lead to mucus stasis, chronic bacterial infections, and the accompanying chronic inflammatory responses that promote progressive lung destruction, and, eventually in CF, lung failure. Here we discuss CFTR's functional role airway surface liquid hydration and pH, in regulation of other channels such as the epithelial sodium channel, and in regulating inflammatory responses in the lung. PMID:25381027

Evolutionary diversification of TTX-resistant sodium channels in a predator-prey interaction.

PubMed

Geffeney, Shana L; Fujimoto, Esther; Brodie, Edmund D; Brodie, Edmund D; Ruben, Peter C

2005-04-07

Understanding the molecular genetic basis of adaptations provides incomparable insight into the genetic mechanisms by which evolutionary diversification takes place. Whether the evolution of common traits in different lineages proceeds by similar or unique mutations, and the degree to which phenotypic evolution is controlled by changes in gene regulation as opposed to gene function, are fundamental questions in evolutionary biology that require such an understanding of genetic mechanisms. Here we identify novel changes in the molecular structure of a sodium channel expressed in snake skeletal muscle, tsNa(V)1.4, that are responsible for differences in tetrodotoxin (TTX) resistance among garter snake populations coevolving with toxic newts. By the functional expression of tsNa(V)1.4, we show how differences in the amino-acid sequence of the channel affect TTX binding and impart different levels of resistance in four snake populations. These results indicate that the evolution of a physiological trait has occurred through a series of unique functional changes in a gene that is otherwise highly conserved among vertebrates.

The sodium chloride cotransporter (NCC) and epithelial sodium channel (ENaC) associate.

PubMed

Mistry, Abinash C; Wynne, Brandi M; Yu, Ling; Tomilin, Viktor; Yue, Qiang; Zhou, Yiqun; Al-Khalili, Otor; Mallick, Rickta; Cai, Hui; Alli, Abdel A; Ko, Benjamin; Mattheyses, Alexa; Bao, Hui-Fang; Pochynyuk, Oleh; Theilig, Franziska; Eaton, Douglas C; Hoover, Robert S

2016-10-01

The thiazide-sensitive sodium chloride cotransporter (NCC) and the epithelial sodium channel (ENaC) are two of the most important determinants of salt balance and thus systemic blood pressure. Abnormalities in either result in profound changes in blood pressure. There is one segment of the nephron where these two sodium transporters are coexpressed, the second part of the distal convoluted tubule. This is a key part of the aldosterone-sensitive distal nephron, the final regulator of salt handling in the kidney. Aldosterone is the key hormonal regulator for both of these proteins. Despite these shared regulators and coexpression in a key nephron segment, associations between these proteins have not been investigated. After confirming apical localization of these proteins, we demonstrated the presence of functional transport proteins and native association by blue native PAGE. Extensive coimmunoprecipitation experiments demonstrated a consistent interaction of NCC with α- and γ-ENaC. Mammalian two-hybrid studies demonstrated direct binding of NCC to ENaC subunits. Fluorescence resonance energy transfer and immunogold EM studies confirmed that these transport proteins are within appropriate proximity for direct binding. Additionally, we demonstrate that there are functional consequences of this interaction, with inhibition of NCC affecting the function of ENaC. This novel finding of an association between ENaC and NCC could alter our understanding of salt transport in the distal tubule. © 2016 The Author(s); published by Portland Press Limited on behalf of the Biochemical Society.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Bean, Bruce Palmer

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 themore » 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.« less

Structure of a eukaryotic voltage-gated sodium channel at near-atomic resolution.

PubMed

Shen, Huaizong; Zhou, Qiang; Pan, Xiaojing; Li, Zhangqiang; Wu, Jianping; Yan, Nieng

2017-03-03

Voltage-gated sodium (Na v ) channels are responsible for the initiation and propagation of action potentials. They are associated with a variety of channelopathies and are targeted by multiple pharmaceutical drugs and natural toxins. Here, we report the cryogenic electron microscopy structure of a putative Na v channel from American cockroach (designated Na v PaS) at 3.8 angstrom resolution. The voltage-sensing domains (VSDs) of the four repeats exhibit distinct conformations. The entrance to the asymmetric selectivity filter vestibule is guarded by heavily glycosylated and disulfide bond-stabilized extracellular loops. On the cytoplasmic side, a conserved amino-terminal domain is placed below VSD I , and a carboxy-terminal domain binds to the III-IV linker. The structure of Na v PaS establishes an important foundation for understanding function and disease mechanism of Na v and related voltage-gated calcium channels. Copyright © 2017, American Association for the Advancement of Science.

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

PubMed Central

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

Progress in Studying Scintillator Proportionality: Phenomenological Model

NASA Astrophysics Data System (ADS)

Bizarri, G.; Cherepy, N. J.; Choong, W. S.; Hull, G.; Moses, W. W.; Payne, S. A.; Singh, J.; Valentine, J. D.; Vasilev, A. N.; Williams, R. T.

2009-08-01

We present a model to describe the origin of non-proportional dependence of scintillator light yield on the energy of an ionizing particle. The non-proportionality is discussed in terms of energy relaxation channels and their linear and non-linear dependences on the deposited energy. In this approach, the scintillation response is described as a function of the deposited energy deposition and the kinetic rates of each relaxation channel. This mathematical framework allows both a qualitative interpretation and a quantitative fitting representation of scintillation non-proportionality response as function of kinetic rates. This method was successfully applied to thallium doped sodium iodide measured with SLYNCI, a new facility using the Compton coincidence technique. Finally, attention is given to the physical meaning of the dominant relaxation channels, and to the potential causes responsible for the scintillation non-proportionality. We find that thallium doped sodium iodide behaves as if non-proportionality is due to competition between radiative recombinations and non-radiative Auger processes.

Pickpocket1 Is an Ionotropic Molecular Sensory Transducer*

PubMed Central

Boiko, Nina; Kucher, Volodymyr; Stockand, James D.; Eaton, Benjamin A.

2012-01-01

The molecular transformation of an external stimulus into changes in sensory neuron activity is incompletely described. Although a number of molecules have been identified that can respond to stimuli, evidence that these molecules can transduce stimulation into useful neural activity is lacking. Here we demonstrate that pickpocket1 (ppk1), a Drosophila homolog of mammalian Degenerin/epithelial sodium channels, encodes an acid-sensing sodium channel that conducts a transient depolarizing current in multidendritic sensory neurons of Drosophila melanogaster. Stimulation of Ppk1 is sufficient to bring these sensory neurons to threshold, eliciting a burst of action potentials. The transient nature of the neural activity produced by Ppk1 activation is the result of Ppk1 channel gating properties. This model is supported by the observation of enhanced bursting activity in neurons expressing a gain of function ppk1 mutant harboring the degenerin mutation. These findings demonstrate that Ppk1 can function as an ionotropic molecular sensory transducer capable of transforming the perception of a stimulus into phasic neuronal activity in sensory neurons. PMID:23033486

Developmentally-regulated sodium channel subunits are differentially sensitive to {alpha}-cyano containing pyrethroids

DOE Office of Scientific and Technical Information (OSTI.GOV)

Meacham, Connie A.; Brodfuehrer, Peter D.; Watkins, Jennifer A.

2008-09-15

Juvenile rats have been reported to be more sensitive to the acute neurotoxic effects of the pyrethroid deltamethrin than adults. While toxicokinetic differences between juveniles and adults are documented, toxicodynamic differences have not been examined. Voltage-gated sodium channels, the primary targets of pyrethroids, are comprised of {alpha} and {beta} subunits, each of which have multiple isoforms that are expressed in a developmentally-regulated manner. To begin to test whether toxicodynamic differences could contribute to age-dependent deltamethrin toxicity, deltamethrin effects were examined on sodium currents in Xenopus laevis oocytes injected with different combinations of rat {alpha} (Na{sub v}1.2 or Na{sub v}1.3) andmore » {beta} ({beta}{sub 1} or {beta}{sub 3}) subunits. Deltamethrin induced tail currents in all isoform combinations and increased the percent of modified channels in a concentration-dependent manner. Effects of deltamethrin were dependent on subunit combination; Na{sub v}1.3-containing channels were modified to a greater extent than were Na{sub v}1.2-containing channels. In the presence of a {beta} subunit, deltamethrin effects were significantly greater, an effect most pronounced for Na{sub v}1.3 channels; Na{sub v}1.3/{beta}{sub 3} channels were more sensitive to deltamethrin than Na{sub v}1.2/{beta}{sub 1} channels. Na{sub v}1.3/{beta}{sub 3} channels are expressed embryonically, while the Na{sub v}1.2 and {beta}{sub 1} subunits predominate in adults, supporting the hypothesis for age-dependent toxicodynamic differences. Structure-activity relationships for sensitivity of these subunit combinations were examined for other pyrethroids. Permethrin and tetramethrin did not modify currents mediated by either subunit combination. Cypermethrin, {beta}-cyfluthrin, esfenvalerate and fenpropathrin all modified sodium channel function; effects were significantly greater on Na{sub v}1.3/{beta}{sub 3} than on Na{sub v}1.2/{beta}{sub 1} channels. These data demonstrate a greater sensitivity of Na{sub v}1.3 vs Na{sub v}1.2 channels to deltamethrin and other cyano-containing pyrethroids, particularly in the presence of a {beta} subunit.« less

RELATIVE POTENCIES FOR ACUTE EFFECTS OF PYRETHROIDS ON MOTOR FUNCTION IN RATS.

EPA Science Inventory

A proposed common mode-of-action for pyrethroid insecticides, includes alterations in sodium channel dynamics in nervous system tissues, consequent disturbance of neuronal membrane polarization, abnormal discharge in targeted neurons, and changes in nervous system function. The p...

Effects of strychnine on the sodium conductance of the frog node of Ranvier

PubMed Central

1977-01-01

Strychnine blocks sodium conductance in the frog node of Ranvier. This block was studied by reducing and slowing sodium inactivation with scorpion venom. The block is voltage and time dependent. The more positive the axoplasm the greater the block and the faster the approach to equilibrium. Some evidence is presented suggesting that only open channels can be blocked. The block is reduced by raising external sodium or lithium but not impermeant cations. A quaternary derivative of strychnine was synthesized and found to have the same action only when applied intracellularly. We conclude that strychnine blocks sodium channels by a mechanism analogous to that by which it blocks potassium channels. The potassium channel block had previously been found to be identical to that by tetraethylammonium ion derivatives. In addition, strychnine resembles procaine and its derivatives in both its structure and the mechanism of sodium channel block. PMID:302321

Exon 11 skipping of SCN10A coding for voltage-gated sodium channels in dorsal root ganglia

PubMed Central

Schirmeyer, Jana; Szafranski, Karol; Leipold, Enrico; Mawrin, Christian; Platzer, Matthias; Heinemann, Stefan H

2014-01-01

The voltage-gated sodium channel NaV1.8 (encoded by SCN10A) is predominantly expressed in dorsal root ganglia (DRG) and plays a critical role in pain perception. We analyzed SCN10A transcripts isolated from human DRGs using deep sequencing and found a novel splice variant lacking exon 11, which codes for 98 amino acids of the domain I/II linker. Quantitative PCR analysis revealed an abundance of this variant of up to 5–10% in human, while no such variants were detected in mouse or rat. Since no obvious functional differences between channels with and without the exon-11 sequence were detected, it is suggested that SCN10A exon 11 skipping in humans is a tolerated event. PMID:24763188

Role of the Local Anesthetic Receptor in the State-Dependent Inhibition of Voltage-Gated Sodium Channels by the Insecticide Metaflumizone

PubMed Central

von Stein, Richard T.

2012-01-01

Sodium channel inhibitor (SCI) insecticides selectively target voltage-gated sodium (Nav) channels in the slow-inactivated state by binding at or near the local anesthetic receptor within the sodium channel pore. Metaflumizone is a new insecticide for the treatment of fleas on domesticated pets and has recently been reported to block insect sodium channels in the slow-inactivated state, thereby implying that it is also a member of the SCI class. Using the two-electrode voltage-clamp technique, we examined metaflumizone inhibition of rat Nav1.4 sodium channels expressed in Xenopus laevis oocytes. Metaflumizone selectively inhibited Nav1.4 channels at potentials that promoted slow inactivation and shifted the voltage dependence of slow inactivation in the direction of hyperpolarization. Metaflumizone perfusion at a hyperpolarized holding potential also shifted the conductance-voltage curve for activation in the direction of depolarization and antagonized use-dependent lidocaine inhibition of fast-inactivated sodium channels, actions not previously observed with other SCI insecticides. We expressed mutated Nav1.4/F1579A and Nav1.4/Y1586A channels to investigate whether metaflumizone shares the domain IV segment S6 (DIV-S6) binding determinants identified for other SCI insecticides. Consistent with previous investigations of SCI insecticides on rat Nav1.4 channels, the F1579A mutation reduced sensitivity to block by metaflumizone, whereas the Y1586A mutation paradoxically increased the sensitivity to metaflumizone. We conclude that metaflumizone selectively inhibits slow-inactivated Nav1.4 channels and shares DIV-S6 binding determinants with other SCI insecticides and therapeutic drugs. However, our results suggest that metaflumizone interacts with resting and fast-inactivated channels in a manner that is distinct from other compounds in this insecticide class. PMID:22127519

Unusual Voltage-Gated Sodium Currents as Targets for Pain.

PubMed

Barbosa, C; Cummins, T R

2016-01-01

Pain is a serious health problem that impacts the lives of many individuals. Hyperexcitability of peripheral sensory neurons contributes to both acute and chronic pain syndromes. Because voltage-gated sodium currents are crucial to the transmission of electrical signals in peripheral sensory neurons, the channels that underlie these currents are attractive targets for pain therapeutics. Sodium currents and channels in peripheral sensory neurons are complex. Multiple-channel isoforms contribute to the macroscopic currents in nociceptive sensory neurons. These different isoforms exhibit substantial variations in their kinetics and pharmacology. Furthermore, sodium current complexity is enhanced by an array of interacting proteins that can substantially modify the properties of voltage-gated sodium channels. Resurgent sodium currents, atypical currents that can enhance recovery from inactivation and neuronal firing, are increasingly being recognized as playing potentially important roles in sensory neuron hyperexcitability and pain sensations. Here we discuss unusual sodium channels and currents that have been identified in nociceptive sensory neurons, describe what is known about the molecular determinants of the complex sodium currents in these neurons. Finally, we provide an overview of therapeutic strategies to target voltage-gated sodium currents in nociceptive neurons. Copyright © 2016 Elsevier Inc. All rights reserved.

Single-channel analysis of functional epithelial sodium channel (ENaC) stability at the apical membrane of A6 distal kidney cells

PubMed Central

Yu, Ling; Helms, My N.; Yue, Qiang; Eaton, Douglas C.

2008-01-01

Epithelial sodium channels (ENaC) play an essential role in maintaining total body fluid and electrolyte homeostasis. As such, abnormal expression of ENaC at the cell surface is linked to several important human diseases. Although the stability of ENaC subunits has been extensively studied by protein biochemical analysis, the half-life of the functional channel in the apical membrane remains controversial. Because the functional stability of the multisubunit channel may be more physiologically relevant than the stability of individual subunit proteins, we performed studies of functional ENaC channels using A6 epithelial cells, a Xenopus laevis distal nephron cell line. We recorded single-channel activity in over 400 cells with the translation blockers cycloheximide (CHX) or puromycin, as well as the intracellular protein trafficking inhibitors brefeldin A (BFA) or nocodazole. Our cell-attached, single-channel recordings allow us to quantify the channel density in the apical membrane, as well as to determine channel open probability (Po) from control (untreated) cells and from cells at different times of drug treatment. The data suggest that the half-life of ENaC channels is ∼3.5 h following puromycin, BFA, and nocodazole treatment. Furthermore, these three drugs had no significant effect on the Po of ENaC for at least 6 h after exposure. A decrease in apical channel number and Po was observed following 2 h of CHX inhibition of protein synthesis, and the apparent channel half-life was closer to 1.5 h following CHX treatment. Treatment of cells with the translation inhibitors does not alter the expression of the protease furin, and therefore changes in protease activity cannot explain changes in ENaC Po. Confocal images show that BFA and nocodazole both disrupt most of the Golgi apparatus after 1-h exposure. In cells with the Golgi totally disrupted by overnight exposure to BFA, 20% of apical ENaC channels remained functional. This result suggests that ENaC is delivered to the apical membrane via a pathway that might bypass the Golgi vesicular trafficking pathway, or that there might be two pools of channels with markedly different half-lives in the apical membrane. PMID:18784262

Single-channel analysis of functional epithelial sodium channel (ENaC) stability at the apical membrane of A6 distal kidney cells.

PubMed

Yu, Ling; Helms, My N; Yue, Qiang; Eaton, Douglas C

2008-11-01

Epithelial sodium channels (ENaC) play an essential role in maintaining total body fluid and electrolyte homeostasis. As such, abnormal expression of ENaC at the cell surface is linked to several important human diseases. Although the stability of ENaC subunits has been extensively studied by protein biochemical analysis, the half-life of the functional channel in the apical membrane remains controversial. Because the functional stability of the multisubunit channel may be more physiologically relevant than the stability of individual subunit proteins, we performed studies of functional ENaC channels using A6 epithelial cells, a Xenopus laevis distal nephron cell line. We recorded single-channel activity in over 400 cells with the translation blockers cycloheximide (CHX) or puromycin, as well as the intracellular protein trafficking inhibitors brefeldin A (BFA) or nocodazole. Our cell-attached, single-channel recordings allow us to quantify the channel density in the apical membrane, as well as to determine channel open probability (Po) from control (untreated) cells and from cells at different times of drug treatment. The data suggest that the half-life of ENaC channels is approximately 3.5 h following puromycin, BFA, and nocodazole treatment. Furthermore, these three drugs had no significant effect on the Po of ENaC for at least 6 h after exposure. A decrease in apical channel number and Po was observed following 2 h of CHX inhibition of protein synthesis, and the apparent channel half-life was closer to 1.5 h following CHX treatment. Treatment of cells with the translation inhibitors does not alter the expression of the protease furin, and therefore changes in protease activity cannot explain changes in ENaC Po. Confocal images show that BFA and nocodazole both disrupt most of the Golgi apparatus after 1-h exposure. In cells with the Golgi totally disrupted by overnight exposure to BFA, 20% of apical ENaC channels remained functional. This result suggests that ENaC is delivered to the apical membrane via a pathway that might bypass the Golgi vesicular trafficking pathway, or that there might be two pools of channels with markedly different half-lives in the apical membrane.

To4, the first Tityus obscurus β-toxin fully electrophysiologically characterized on human sodium channel isoforms.

PubMed

Duque, Harry Morales; Mourão, Caroline Barbosa Farias; Tibery, Diogo Vieira; Barbosa, Eder Alves; Campos, Leandro Ambrósio; Schwartz, Elisabeth Ferroni

2017-09-01

Many scorpion toxins that act on sodium channels (NaScTxs) have been characterized till date. These toxins may act modulating the inactivation or the activation of sodium channels and are named α- or β-types, respectively. Some venom toxins from Tityus obscurus (Buthidae), a scorpion widely distributed in the Brazilian Amazon, have been partially characterized in previous studies; however, little information about their electrophysiological role on sodium ion channels has been published. In the present study, we describe the purification, identification and electrophysiological characterization of a NaScTx, which was first described as Tc54 and further fully sequenced and renamed To4. This toxin shows a marked β-type effect on different sodium channel subtypes (hNa v 1.1-hNa v 1.7) at low concentrations, and has more pronounced activity on hNa v 1.1, hNa v 1.2 and hNa v 1.4. By comparing To4 primary structure with other Tityus β-toxins which have already been electrophysiologically tested, it is possible to establish some key amino acid residues for the sodium channel activity. Thus, To4 is the first toxin from T. obscurus fully electrophysiologically characterized on different human sodium channel isoforms. Copyright © 2017 Elsevier Inc. All rights reserved.

Update on the mechanism of action of antiepileptic drugs.

PubMed

Meldrum, B S

1996-01-01

Novel antiepileptic drugs (AEDs) are thought to act on voltage-sensitive ion channels, on inhibitory neurotransmission or on excitatory neurotransmission. Two successful examples of rational AED design that potentiate GABA-mediated inhibition are vigabatrin (VGB) by irreversible inhibition of GABA-transaminase, and tiagabine (TGB) by blocking GABA uptake. Lamotrigine (LTG) prolongs inactivation of voltage-dependent sodium channels. The anticonvulsant action of remacemide (RCM) is probably largely due to blockade of NMDA receptors and prolonged inactivation of sodium channels induced by its desglycinated metabolite. Felbamate (FBM) apparently blocks NMDA receptors, potentiates GABA-mediated responses, blocks L-type calcium channels, and possibly also prolongs sodium channel inactivation. Similarly, topiramate (TPM) has multiple probable sites of action, including sodium channels, GABA receptors, and glutamate (AMPA) receptors. Gabapentin (GBP) apparently has a completely novel type of action, probably involving potentiation of GABA-mediated inhibition and possibly also inactivation of sodium channels. The therapeutic advantages of the novel AEDs are as yet only partially explained by our present understanding of their mechanisms of action.

A 3T Sodium and Proton Composite Array Breast Coil

PubMed Central

Kaggie, Joshua D.; Hadley, J. Rock; Badal, James; Campbell, John R.; Park, Daniel J.; Parker, Dennis L.; Morrell, Glen; Newbould, Rexford D.; Wood, Ali F.; Bangerter, Neal K.

2013-01-01

Purpose The objective of this study was to determine whether a sodium phased array would improve sodium breast MRI at 3T. The secondary objective was to create acceptable proton images with the sodium phased array in place. Methods A novel composite array for combined proton/sodium 3T breast MRI is compared to a coil with a single proton and sodium channel. The composite array consists of a 7-channel sodium receive array, a larger sodium transmit coil, and a 4-channel proton transceive array. The new composite array design utilizes smaller sodium receive loops than typically used in sodium imaging, uses novel decoupling methods between the receive loops and transmit loops, and uses a novel multi-channel proton transceive coil. The proton transceive coil reduces coupling between proton and sodium elements by intersecting the constituent loops to reduce their mutual inductance. The coil used for comparison consists of a concentric sodium and proton loop with passive decoupling traps. Results The composite array coil demonstrates a 2–5x improvement in SNR for sodium imaging and similar SNR for proton imaging when compared to a simple single-loop dual resonant design. Conclusion The improved SNR of the composite array gives breast sodium images of unprecedented quality in reasonable scan times. PMID:24105740

Elucidation of pyrethroid and DDT receptor sites in the voltage-gated sodium channel.

PubMed

Zhorov, Boris S; Dong, Ke

2017-05-01

DDT and pyrethroid insecticides were among the earliest neurotoxins identified to act on voltage-gated sodium channels. In the 1960s, equipped with, at the time, new voltage-clamp techniques, Professor Narahashi and associates provided the initial evidence that DDT and allethrin (the first commercial pyrethroid insecticide) caused prolonged flow of sodium currents in lobster and squid giant axons. Over the next several decades, continued efforts by Prof. Narahashi's group as well as other laboratories led to a comprehensive understanding of the mechanism of action of DDT and pyrethroids on sodium channels. Fast forward to the 1990s, genetic, pharmacological and toxicological data all further confirmed voltage-gated sodium channels as the primary targets of DDT and pyrethroid insecticides. Modifications of the gating kinetics of sodium channels by these insecticides result in repetitive firing and/or membrane depolarization in the nervous system. This mini-review focuses on studies from Prof. Narahashi's pioneer work and more recent mutational and computational modeling analyses which collectively elucidated the elusive pyrethroid receptor sites as well as the molecular basis of differential sensitivities of insect and mammalian sodium channels to pyrethroids. Copyright © 2016 Elsevier B.V. All rights reserved.

A novel Ile1455Thr variant in the skeletal muscle sodium channel alpha-subunit in a patient with a severe adult-onset proximal myopathy with electrical myotonia and a patient with mild paramyotonia phenotype.

PubMed

Bednarz, Marcin; Stunnenberg, Bas C; Kusters, Benno; Kamsteeg, Erik-Jan; Saris, Christiaan G; Groome, James; Winston, Vern; Meola, Giovanni; Jurkat-Rott, Karin; Voermans, Nicol C

2017-02-01

In sodium channelopathies, a severe fixed myopathy caused by a dominant mutation is rare. We describe two unrelated patients with a novel variant, p.Ile1455Thr, with phenotypes of paramyotonia in one case and fixed proximal myopathy with latent myotonia in another. In-vitro whole cell patch-clamp studies show that the mutation slows inactivation and accelerates recovery, in line with other paramyotonia variants with destabilized fast inactivation as pathomechanism. Additionally, p.IleI1455 causes a loss-of-function by reduced membrane insertion, right-shift of activation, and slowed kinetics. Molecular dynamics simulations comparing wild type and mutant Nav1.4 showed that threonine substitution hindered D4S4 mobility in response to membrane depolarization, consistent with effects of the mutation on channel inactivation. The fixed myopathy is likely to be associated to gain-of-function leading to sodium accumulation, regional edema, T-tubular swelling and mitochondrial stress. A possible contribution of the loss-of-function features towards myotonia and myopathy is discussed. Copyright © 2016. Published by Elsevier B.V.

Mice with an NaV1.4 sodium channel null allele have latent myasthenia, without susceptibility to periodic paralysis

PubMed Central

Wu, Fenfen; Mi, Wentao; Fu, Yu; Struyk, Arie

2016-01-01

Over 60 mutations of SCN4A encoding the NaV1.4 sodium channel of skeletal muscle have been identified in patients with myotonia, periodic paralysis, myasthenia, or congenital myopathy. Most mutations are missense with gain-of-function defects that cause susceptibility to myotonia or periodic paralysis. Loss-of-function from enhanced inactivation or null alleles is rare and has been associated with myasthenia and congenital myopathy, while a mix of loss and gain of function changes has an uncertain relation to hypokalaemic periodic paralysis. To better define the functional consequences for a loss-of-function, we generated NaV1.4 null mice by deletion of exon 12. Heterozygous null mice have latent myasthenia and a right shift of the force-stimulus relation, without evidence of periodic paralysis. Sodium current density was half that of wild-type muscle and no compensation by retained expression of the foetal NaV1.5 isoform was detected. Mice null for NaV1.4 did not survive beyond the second postnatal day. This mouse model shows remarkable preservation of muscle function and viability for haploinsufficiency of NaV1.4, as has been reported in humans, with a propensity for pseudo-myasthenia caused by a marginal Na+ current density to support sustained high-frequency action potentials in muscle. PMID:27048647

Molecular and kinetic determinants of local anaesthetic action on sodium channels.

PubMed

French, R J; Zamponi, G W; Sierralta, I E

1998-11-23

(1) Local anaesthetics (LA) rely for their clinical actions on state-dependent inhibition of voltage-dependent sodium channels. (2) Single, batrachoxin-modified sodium channels in planar lipid bilayers allow direct observation of drug-channel interactions. Two modes of inhibition of single-channel current are observed: fast block of the open channels and prolongation of a long-lived closed state, some of whose properties resemble those of the inactivated state of unmodified channels. (3) Analogues of different parts of the LA molecule separately mimic each blocking mode: amines--fast block, and water-soluble aromatics--closed state prolongation. (4) Interaction between a mu-conotoxin derivative and diethylammonium indicate an intrapore site of fast, open-state block. (5) Site-directed mutagenesis studies suggest that hydrophobic residues in transmembrane segment 6 of repeat domain 4 of sodium channels are critical for both LA binding and stabilization of the inactivated state.

Current understanding of the mechanism of action of the antiepileptic drug lacosamide.

PubMed

Rogawski, Michael A; Tofighy, Azita; White, H Steve; Matagne, Alain; Wolff, Christian

2015-02-01

The antiepileptic drug lacosamide [(R)-2-acetamido-N-benzyl-3-methoxypropanamide], a chiral functionalized amino acid, was originally identified by virtue of activity in the mouse and rat maximal electroshock (MES) test. Attention was drawn to lacosamide because of its high oral potency and stereoselectivity. Lacosamide is also active in the 6 Hz seizure model but inactive against clonic seizures in rodents induced by subcutaneous pentylenetetrazol, bicuculline and picrotoxin. It is also ineffective in genetic models of absence epilepsy. At doses greater than those required to confer protection in the MES test, lacosamide inhibits behavioral and electrographic seizures in hippocampal kindled rats. It also effectively terminates seizures in the rat perforant path stimulation status epilepticus model when administered early after the onset of seizures. Lacosamide does not exhibit antiepileptogenic effects in kindling or post-status epilepticus models. The profile of lacosamide in animal seizure and epilepsy models is similar to that of sodium channel blocking antiepileptic drugs, such as phenytoin and carbamazepine. However, unlike these agents, lacosamide does not affect sustained repetitive firing (SRF) on a time scale of hundreds of milliseconds or affect fast inactivation of voltage-gated sodium channels; however, it terminates SRF on a time scale of seconds by an apparent effect on sodium channel slow inactivation. Lacosamide shifts the slow inactivation curve to more hyperpolarized potentials and enhances the maximal fraction of channels that are in the slow inactivated state. Currently, lacosamide is the only known antiepileptic drug in clinical practice that exerts its anticonvulsant activity predominantly by selectively enhancing slow sodium channel inactivation. Copyright © 2014 Elsevier B.V. All rights reserved.

Sodium entry through endothelial store-operated calcium entry channels: regulation by Orai1

PubMed Central

Xu, Ningyong; Cioffi, Donna L.; Alexeyev, Mikhail; Rich, Thomas C.

2014-01-01

Orai1 interacts with transient receptor potential protein of the canonical subfamily (TRPC4) and contributes to calcium selectivity of the endothelial cell store-operated calcium entry current (ISOC). Orai1 silencing increases sodium permeability and decreases membrane-associated calcium, although it is not known whether Orai1 is an important determinant of cytosolic sodium transitions. We test the hypothesis that, upon activation of store-operated calcium entry channels, Orai1 is a critical determinant of cytosolic sodium transitions. Activation of store-operated calcium entry channels transiently increased cytosolic calcium and sodium, characteristic of release from an intracellular store. The sodium response occurred more abruptly and returned to baseline more rapidly than did the transient calcium rise. Extracellular choline substitution for sodium did not inhibit the response, although 2-aminoethoxydiphenyl borate and YM-58483 reduced it by ∼50%. After this transient response, cytosolic sodium continued to increase due to influx through activated store-operated calcium entry channels. The magnitude of this sustained increase in cytosolic sodium was greater when experiments were conducted in low extracellular calcium and when Orai1 expression was silenced; these two interventions were not additive, suggesting a common mechanism. 2-Aminoethoxydiphenyl borate and YM-58483 inhibited the sustained increase in cytosolic sodium, only in the presence of Orai1. These studies demonstrate that sodium permeates activated store-operated calcium entry channels, resulting in an increase in cytosolic sodium; the magnitude of this response is determined by Orai1. PMID:25428882

The DEG/ENaC cation channel protein UNC-8 drives activity-dependent synapse removal in remodeling GABAergic neurons

PubMed Central

Miller-Fleming, Tyne W; Petersen, Sarah C; Manning, Laura; Matthewman, Cristina; Gornet, Megan; Beers, Allison; Hori, Sayaka; Mitani, Shohei; Bianchi, Laura; Richmond, Janet; Miller, David M

2016-01-01

Genetic programming and neural activity drive synaptic remodeling in developing neural circuits, but the molecular components that link these pathways are poorly understood. Here we show that the C. elegans Degenerin/Epithelial Sodium Channel (DEG/ENaC) protein, UNC-8, is transcriptionally controlled to function as a trigger in an activity-dependent mechanism that removes synapses in remodeling GABAergic neurons. UNC-8 cation channel activity promotes disassembly of presynaptic domains in DD type GABA neurons, but not in VD class GABA neurons where unc-8 expression is blocked by the COUP/TF transcription factor, UNC-55. We propose that the depolarizing effect of UNC-8-dependent sodium import elevates intracellular calcium in a positive feedback loop involving the voltage-gated calcium channel UNC-2 and the calcium-activated phosphatase TAX-6/calcineurin to initiate a caspase-dependent mechanism that disassembles the presynaptic apparatus. Thus, UNC-8 serves as a link between genetic and activity-dependent pathways that function together to promote the elimination of GABA synapses in remodeling neurons. DOI: http://dx.doi.org/10.7554/eLife.14599.001 PMID:27403890

Molecular Properties of neurotoxin receptors sites associated with sodium channels from mammalian brain.

PubMed

Catterall, W A; Hartshorne, R P; Beneski, D A

1982-01-01

Neurotoxins that act at specific receptor sites on voltage-sensitive sodium channels have been used as molecular probes to identify and purify protein components of sodium channels from mammalian brain. Photoreactive derivatives of scorpion toxin have been prepared and used to covalently label sodium channels in intact synaptosomes. Two polypeptides, alpha with Mr approximately 270,000 and beta with Mr approximately 38,000, are specifically labeled indicating that they are components of the scorpion toxin receptor site on the sodium channel. The sodium channel can be solubilized with retention of specific binding of [3H] saxitoxin using nonionic detergents such as Triton X-100. The solubilized saxitoxin receptor has molecular weight of 316,000 +/- 63,000 and binds 0.9 g of Triton X-100 and phospholipid per g of protein. The solubilized receptor can be purified 750-fold by ion exchange chromatography, wheat germ lectin/Sepharose chromatography and sucrose gradient sedimentation to a final specific activity of 1488 pmol/mg. Analysis of the polypeptide chain composition of the most highly purified fractions indicates that alpha and beta comprise 65% of the protein of these fractions and are only the polypeptides whose presence correlates with saxitoxin binding activity. These studies lead to a working hypothesis of sodium channel structure in which the intact channel is comprised of a complex with Mr of approximately 316,000 containing one mole of alpha (Mr approximately 270,000) and one to three moles of beta (Mr approximately 38,000).

Molecular determinants on the insect sodium channel for the specific action of type II pyrethroid insecticides

DOE Office of Scientific and Technical Information (OSTI.GOV)

Du Yuzhe; Nomura, Yoshiko; Luo Ningguang

2009-01-15

Pyrethroid insecticides are classified as type I or type II based on their distinct symptomology and effects on sodium channel gating. Structurally, type II pyrethroids possess an {alpha}-cyano group at the phenylbenzyl alcohol position, which is lacking in type I pyrethroids. Both type I and type II pyrethroids inhibit deactivation consequently prolonging the opening of sodium channels. However, type II pyrethroids inhibit the deactivation of sodium channels to a greater extent than type I pyrethroids inducing much slower decaying of tail currents upon repolarization. The molecular basis of a type II-specific action, however, is not known. Here we report themore » identification of a residue G{sup 1111} and two positively charged lysines immediately downstream of G{sup 1111} in the intracellular linker connecting domains II and III of the cockroach sodium channel that are specifically involved in the action of type II pyrethroids, but not in the action of type I pyrethroids. Deletion of G{sup 1111}, a consequence of alternative splicing, reduced the sodium channel sensitivity to type II pyrethroids, but had no effect on channel sensitivity to type I pyrethroids. Interestingly, charge neutralization or charge reversal of two positively charged lysines (Ks) downstream of G{sup 1111} had a similar effect. These results provide the molecular insight into the type II-specific interaction of pyrethroids with the sodium channel at the molecular level.« less

New Theoretical Model of Nerve Conduction in Unmyelinated Nerves

PubMed Central

Akaishi, Tetsuya

2017-01-01

Nerve conduction in unmyelinated fibers has long been described based on the equivalent circuit model and cable theory. However, without the change in ionic concentration gradient across the membrane, there would be no generation or propagation of the action potential. Based on this concept, we employ a new conductive model focusing on the distribution of voltage-gated sodium ion channels and Coulomb force between electrolytes. Based on this new model, the propagation of the nerve conduction was suggested to take place far before the generation of action potential at each channel. We theoretically showed that propagation of action potential, which is enabled by the increasing Coulomb force produced by inflowing sodium ions, from one sodium ion channel to the next sodium channel would be inversely proportionate to the density of sodium channels on the axon membrane. Because the longitudinal number of sodium ion channel would be proportionate to the square root of channel density, the conduction velocity of unmyelinated nerves is theoretically shown to be proportionate to the square root of channel density. Also, from a viewpoint of equilibrium state of channel importation and degeneration, channel density was suggested to be proportionate to axonal diameter. Based on these simple basis, conduction velocity in unmyelinated nerves was theoretically shown to be proportionate to the square root of axonal diameter. This new model would also enable us to acquire more accurate and understandable vision on the phenomena in unmyelinated nerves in addition to the conventional electric circuit model and cable theory. PMID:29081751

The heart and potassium: a banana republic.

PubMed

Khan, Ehsan; Spiers, Christine; Khan, Maria

2013-03-01

The importance of potassium in maintaining stable cardiac function is a clinically understood phenomenon. Physiologically the importance of potassium in cardiac function is described by the large number of different kinds of potassium ions channels found in the heart compared to channels and membrane transport mechanisms for other ions such as sodium and calcium. Potassium is important in physiological homeostatic control of cardiac function, but is also of relevance to the diseased state, as potassium-related effects may stabilize or destabilize cardiac function. This article aims to provide a detailed understanding of potassium-mediated cardiac function. This will help the clinical practitioner evaluate how modulation of potassium ion channels by disease and pharmacological manipulation affect the cardiac patient, thus aiding in decision making when faced with clinical problems related to potassium.

Rab27a regulates epithelial sodium channel (ENaC) activity through synaptotagmin-like protein (SLP-5) and Munc13-4 effector mechanism

DOE Office of Scientific and Technical Information (OSTI.GOV)

Saxena, Sunil K.; Horiuchi, Hisanori; Fukuda, Mitsunori

Liddle's syndrome (excessive absorption of sodium ions) and PHA-1 (pseudohypoaldosteronism type 1) with decreased sodium absorption are caused by the mutations in the amiloride-sensitive epithelial sodium channel ENaC. Rab proteins are small GTPases involved in vesicle transport, docking, and fusion. Earlier, we reported that Rab27a inhibits ENaC-mediated currents through protein-protein interaction in HT-29 cells. We hereby report that Rab27a-dependent inhibition is associated with the GTP/GDP status as constitutively active or GTPase-deficient mutant Q78L inhibits amiloride-sensitive currents whereas GDP-locked inactive mutant T23N showed no effect. In order to further explore the molecular mechanism of this regulation, we performed competitive assays withmore » two Rab27a-binding proteins: synaptotagmin-like protein (SLP-5) and Munc13-4 (a putative priming factor for exocytosis). Both proteins eliminate negative modulation of Rab27a on ENaC function. The SLP-5 reversal of Rab27a effect was restricted to C-terminal C2A/C2B domains assigned for putative phospholipids-binding function while the Rab27a-binding SHD motif imparted higher inhibition. The ENaC-mediated currents remain unaffected by Rab27a though SLP-5 appears to strongly bind it. The immunoprecipitation experiments suggest that in the presence of excessive Munc13-4 and SLP-5 proteins, Rab27a interaction with ENaC is diminished. Munc13-4 and SLP-5 limit the Rab27a availability to ENaC, thus minimizing its effect on channel function. These observations decisively prove that Rab27a inhibits ENaC function through a complex mechanism that involves GTP/GDP status, and protein-protein interactions involving Munc13-4 and SLP-5 effector proteins.« less

Insulation of the conduction pathway of muscle transverse tubule calcium channels from the surface charge of bilayer phospholipid

PubMed Central

1986-01-01

Functional calcium channels present in purified skeletal muscle transverse tubules were inserted into planar phospholipid bilayers composed of the neutral lipid phosphatidylethanolamine (PE), the negatively charged lipid phosphatidylserine (PS), and mixtures of both. The lengthening of the mean open time and stabilization of single channel fluctuations under constant holding potentials was accomplished by the use of the agonist Bay K8644. It was found that the barium current carried through the channel saturates as a function of the BaCl2 concentration at a maximum current of 0.6 pA (at a holding potential of 0 mV) and a half-saturation value of 40 mM. Under saturation, the slope conductance of the channel is 20 pS at voltages more negative than -50 mV and 13 pS at a holding potential of 0 mV. At barium concentrations above and below the half-saturation point, the open channel currents were independent of the bilayer mole fraction of PS from XPS = 0 (pure PE) to XPS = 1.0 (pure PS). It is shown that in the absence of barium, the calcium channel transports sodium or potassium ions (P Na/PK = 1.4) at saturating rates higher than those for barium alone. The sodium conductance in pure PE bilayers saturates as a function of NaCl concentration, following a curve that can be described as a rectangular hyperbola with a half-saturation value of 200 mM and a maximum conductance of 68 pS (slope conductance at a holding potential of 0 mV). In pure PS bilayers, the sodium conductance is about twice that measured in PE at concentrations below 100 mM NaCl. The maximum channel conductance at high ionic strength is unaffected by the lipid charge. This effect at low ionic strength was analyzed according to J. Bell and C. Miller (1984. Biophysical Journal. 45:279- 287) and interpreted as if the conduction pathway of the calcium channel were separated from the bilayer lipid by approximately 20 A. This distance thereby effectively insulates the ion entry to the channel from the bulk of the bilayer lipid surface charge. Current vs. voltage curves measured in NaCl in pure PE and pure PS show that similarly small surface charge effects are present in both inward and outward currents. This suggests that the same conduction insulation is present at both ends of the calcium channel. PMID:2425043

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

PubMed Central

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

2011-01-01

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. PMID:21881211

Voltage-gated sodium channel β subunits: The power outside the pore in brain development and disease.

PubMed

Hull, Jacob M; Isom, Lori L

2018-04-01

Voltage gated sodium channels (VGSCs) were first identified in terms of their role in the upstroke of the action potential. The underlying proteins were later identified as saxitoxin and scorpion toxin receptors consisting of α and β subunits. We now know that VGSCs are heterotrimeric complexes consisting of a single pore forming α subunit joined by two β subunits; a noncovalently linked β1 or β3 and a covalently linked β2 or β4 subunit. VGSC α subunits contain all the machinery necessary for channel cell surface expression, ion conduction, voltage sensing, gating, and inactivation, in one central, polytopic, transmembrane protein. VGSC β subunits are more than simple accessories to α subunits. In the more than two decades since the original cloning of β1, our knowledge of their roles in physiology and pathophysiology has expanded immensely. VGSC β subunits are multifunctional. They confer unique gating mechanisms, regulate cellular excitability, affect brain development, confer distinct channel pharmacology, and have functions that are independent of the α subunits. The vast array of functions of these proteins stems from their special station in the channelome: being the only known constituents that are cell adhesion and intra/extracellular signaling molecules in addition to being part of channel complexes. This functional trifecta and how it goes awry demonstrates the power outside the pore in ion channel signaling complexes, broadening the term channelopathy beyond defects in ion conduction. This article is part of the Special Issue entitled 'Channelopathies.' Copyright © 2017 Elsevier Ltd. All rights reserved.

Role of aquaporin and sodium channel in pleural water movement.

PubMed

Jiang, Jinjun; Hu, Jie; Bai, Chunxue

2003-12-16

The role of the ENaC sodium channel and aquaporin-1 (AQP1) water channel on pleural fluid dynamics in mice was investigated. 0.25 ml of hypertonic or isosmolar fluid was infused into the pleural space in anesthetized wildtype and AQP1 null mice. Pleural fluid was sampled at specified times to quantify the osmolality and volume. The sodium channel activator terbutaline increased isosmolar fluid clearance by 90% while the sodium channel inhibitor amiloride decreased it by 15%, but had no effect on osmotically driven water transport. AQP1 deletion significantly decreased osmotic water transport in pleural space by twofold, but it had no effect on isosmolar fluid clearance. Pretreatment with dexamethasone increased pleural osmotic fluid entry by 25%, while intravenous injection of HgCl2 decreased osmotic pleural water movement by 43%. These results provided evidence for a role of a sodium channel in pleural fluid absorption; AQP1 plays a major role in osmotic liquid transport but it does not affect isosmolar fluid clearance.

The novel sodium channel modulator GS-458967 (GS967) is an effective treatment in a mouse model of SCN8A encephalopathy.

PubMed

Baker, Erin M; Thompson, Christopher H; Hawkins, Nicole A; Wagnon, Jacy L; Wengert, Eric R; Patel, Manoj K; George, Alfred L; Meisler, Miriam H; Kearney, Jennifer A

2018-06-01

De novo mutations of SCN8A, encoding the voltage-gated sodium channel Na V 1.6, have been associated with a severe infant onset epileptic encephalopathy. Individuals with SCN8A encephalopathy have a mean age of seizure onset of 4-5 months, with multiple seizure types that are often refractory to treatment with available drugs. Anecdotal reports suggest that high-dose phenytoin is effective for some patients, but there are associated adverse effects and potential for toxicity. Functional characterization of several SCN8A encephalopathy variants has shown that elevated persistent sodium current is one of several common biophysical defects. Therefore, specifically targeting elevated persistent current may be a useful therapeutic strategy in some cases. The novel sodium channel modulator GS967 has greater preference for persistent as opposed to peak current and nearly 10-fold greater potency than phenytoin. We evaluated the therapeutic effect of GS967 in the Scn8a N1768D/+ mouse model carrying an SCN8A patient mutation that results in elevated persistent sodium current. We also performed patch clamp recordings to assess the effect of GS967 on peak and persistent sodium current and excitability in hippocampal neurons from Scn8a N1768D/+ mice. GS967 potently blocked persistent sodium current without affecting peak current, normalized action potential morphology, and attenuated excitability in neurons from heterozygous Scn8a N1768D/+ mice. Acute treatment with GS967 provided dose-dependent protection against maximal electroshock-induced seizures in Scn8a N1768D/+ and wild-type mice. Chronic treatment of Scn8a N1768D/+ mice with GS967 resulted in lower seizure burden and complete protection from seizure-associated lethality observed in untreated Scn8a N1768D/+ mice. Protection was achieved at a chronic dose that did not cause overt behavioral toxicity or sedation. Persistent sodium current modulators like GS967 may be an effective precision targeting strategy for SCN8A encephalopathy and other functionally similar channelopathies when elevated persistent sodium current is the primary dysfunction. Wiley Periodicals, Inc. © 2018 International League Against Epilepsy.

Structure and function of splice variants of the cardiac voltage-gated sodium channel Na(v)1.5.

PubMed

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.

Genetically engineered SCN5A mutant pig hearts exhibit conduction defects and arrhythmias

PubMed Central

Park, David S.; Cerrone, Marina; Morley, Gregory; Vasquez, Carolina; Fowler, Steven; Liu, Nian; Bernstein, Scott A.; Liu, Fang-Yu; Zhang, Jie; Rogers, Christopher S.; Priori, Silvia G.; Chinitz, Larry A.; Fishman, Glenn I.

2014-01-01

SCN5A encodes the α subunit of the major cardiac sodium channel NaV1.5. Mutations in SCN5A are associated with conduction disease and ventricular fibrillation (VF); however, the mechanisms that link loss of sodium channel function to arrhythmic instability remain unresolved. Here, we generated a large-animal model of a human cardiac sodium channelopathy in pigs, which have cardiac structure and function similar to humans, to better define the arrhythmic substrate. We introduced a nonsense mutation originally identified in a child with Brugada syndrome into the orthologous position (E558X) in the pig SCN5A gene. SCN5AE558X/+ pigs exhibited conduction abnormalities in the absence of cardiac structural defects. Sudden cardiac death was not observed in young pigs; however, Langendorff-perfused SCN5AE558X/+ hearts had an increased propensity for pacing-induced or spontaneous VF initiated by short-coupled ventricular premature beats. Optical mapping during VF showed that activity often began as an organized focal source or broad wavefront on the right ventricular (RV) free wall. Together, the results from this study demonstrate that the SCN5AE558X/+ pig model accurately phenocopies many aspects of human cardiac sodium channelopathy, including conduction slowing and increased susceptibility to ventricular arrhythmias. PMID:25500882

Modulation of neuronal sodium channels by the sea anemone peptide BDS-I

PubMed Central

Liu, Pin; Jo, Sooyeon

2012-01-01

Blood-depressing substance I (BDS-I), a 43 amino-acid peptide from sea anemone venom, is used as a specific inhibitor of Kv3-family potassium channels. We found that BDS-I acts with even higher potency to modulate specific types of voltage-dependent sodium channels. In rat dorsal root ganglion (DRG) neurons, 3 μM BDS-I strongly enhanced tetrodotoxin (TTX)-sensitive sodium current but weakly inhibited TTX-resistant sodium current. In rat superior cervical ganglion (SCG) neurons, which express only TTX-sensitive sodium current, BDS-I enhanced current elicited by small depolarizations and slowed decay of currents at all voltages (EC50 ∼ 300 nM). BDS-I acted with exceptionally high potency and efficacy on cloned human Nav1.7 channels, slowing inactivation by 6-fold, with an EC50 of approximately 3 nM. BDS-I also slowed inactivation of sodium currents in N1E-115 neuroblastoma cells (mainly from Nav1.3 channels), with an EC50 ∼ 600 nM. In hippocampal CA3 pyramidal neurons (mouse) and cerebellar Purkinje neurons (mouse and rat), BDS-I had only small effects on current decay (slowing inactivation by 20–50%), suggesting relatively weak sensitivity of Nav1.1 and Nav1.6 channels. The biggest effect of BDS-I in central neurons was to enhance resurgent current in Purkinje neurons, an effect reflected in enhancement of sodium current during the repolarization phase of Purkinje neuron action potentials. Overall, these results show that BDS-I acts to modulate sodium channel gating in a manner similar to previously known neurotoxin receptor site 3 anemone toxins but with different isoform sensitivity. Most notably, BDS-I acts with very high potency on human Nav1.7 channels. PMID:22442564

Can robots patch-clamp as well as humans? Characterization of a novel sodium channel mutation

PubMed Central

Estacion, M; Choi, J S; Eastman, E M; Lin, Z; Li, Y; Tyrrell, L; Yang, Y; Dib-Hajj, S D; Waxman, S G

2010-01-01

Ion channel missense mutations cause disorders of excitability by changing channel biophysical properties. As an increasing number of new naturally occurring mutations have been identified, and the number of other mutations produced by molecular approaches such as in situ mutagenesis has increased, the need for functional analysis by patch-clamp has become rate limiting. Here we compare a patch-clamp robot using planar-chip technology with human patch-clamp in a functional assessment of a previously undescribed Nav1.7 sodium channel mutation, S211P, which causes erythromelalgia. This robotic patch-clamp device can increase throughput (the number of cells analysed per day) by 3- to 10-fold. Both modes of analysis show that the mutation hyperpolarizes activation voltage dependence (−8 mV by manual profiling, −11 mV by robotic profiling), alters steady-state fast inactivation so that it requires an additional Boltzmann function for a second fraction of total current (∼20% manual, ∼40% robotic), and enhances slow inactivation (hyperpolarizing shift −15 mV by human, −13 mV robotic). Manual patch-clamping demonstrated slower deactivation and enhanced (∼2-fold) ramp response for the mutant channel while robotic recording did not, possibly due to increased temperature and reduced signal-to-noise ratio on the robotic platform. If robotic profiling is used to screen ion channel mutations, we recommend that each measurement or protocol be validated by initial comparison to manual recording. With this caveat, we suggest that, if results are interpreted cautiously, robotic patch-clamp can be used with supervision and subsequent confirmation from human physiologists to facilitate the initial profiling of a variety of electrophysiological parameters of ion channel mutations. PMID:20123784

Domain IV voltage-sensor movement is both sufficient and rate limiting for fast inactivation in sodium channels.

PubMed

Capes, Deborah L; Goldschen-Ohm, Marcel P; Arcisio-Miranda, Manoel; Bezanilla, Francisco; Chanda, Baron

2013-08-01

Voltage-gated sodium channels are critical for the generation and propagation of electrical signals in most excitable cells. Activation of Na(+) channels initiates an action potential, and fast inactivation facilitates repolarization of the membrane by the outward K(+) current. Fast inactivation is also the main determinant of the refractory period between successive electrical impulses. Although the voltage sensor of domain IV (DIV) has been implicated in fast inactivation, it remains unclear whether the activation of DIV alone is sufficient for fast inactivation to occur. Here, we functionally neutralize each specific voltage sensor by mutating several critical arginines in the S4 segment to glutamines. We assess the individual role of each voltage-sensing domain in the voltage dependence and kinetics of fast inactivation upon its specific inhibition. We show that movement of the DIV voltage sensor is the rate-limiting step for both development and recovery from fast inactivation. Our data suggest that activation of the DIV voltage sensor alone is sufficient for fast inactivation to occur, and that activation of DIV before channel opening is the molecular mechanism for closed-state inactivation. We propose a kinetic model of sodium channel gating that can account for our major findings over a wide voltage range by postulating that DIV movement is both necessary and sufficient for fast inactivation.

Sodium-dependent potassium channels of a Slack-like subtype contribute to the slow afterhyperpolarization in lamprey spinal neurons

PubMed Central

Wallén, Peter; Robertson, Brita; Cangiano, Lorenzo; Löw, Peter; Bhattacharjee, Arin; Kaczmarek, Leonard K; Grillner, Sten

2007-01-01

The slow afterhyperpolarization (sAHP) following the action potential is the main determinant of spike frequency regulation. The sAHP after single action potentials in neurons of the lamprey locomotor network is largely due to calcium-dependent K+ channels (80%), activated by calcium entering the cell during the spike. The residual (20%) component becomes prominent during high level activity (50% of the sAHP). It is not Ca2+ dependent, has a reversal potential like that of potassium, and is not affected by chloride injection. It is not due to rapid activation of Na+/K+-ATPase. This non-KCa-sAHP is reduced markedly in amplitude when sodium ions are replaced by lithium ions, and is thus sodium dependent. Quinidine also blocks this sAHP component, further indicating an involvement of sodium-dependent potassium channels (KNa). Modulators tested do not influence the KNa-sAHP amplitude. Immunofluorescence labelling with an anti-Slack antibody revealed distinct immunoreactivity of medium-sized and large neurons in the grey matter of the lamprey spinal cord, suggesting the presence of a Slack-like subtype of KNa channel. The results strongly indicate that a KNa potassium current contributes importantly to the sAHP and thereby to neuronal frequency regulation during high level burst activity as during locomotion. This is, to our knowledge, the first demonstration of a functional role for the Slack gene in contributing to the slow AHP. PMID:17884929

The Perceptual Characteristics of Sodium Chloride to Sodium-Depleted Rats

PubMed Central

2017-01-01

Three experiments assessed potential changes in the rat’s perception of sodium chloride (NaCl) during a state of sodium appetite. In Experiment 1, sodium-sufficient rats licking a range of NaCl concentrations (0.028–0.89M) in 15s trials showed an inverted U-shaped concentration response function peaking at 0.281M. Depleted rats (furosemide) showed an identical function, merely elevated, suggesting altered qualitative or hedonic perception but no change in perceived intensity. In Experiment 2, sodium-depleted rats were tested with NaCl, sodium gluconate, and potassium chloride (KCl; 0.028–0.89M) similar to Experiment 1. KCl was licked at the same rate as water except for a slight elevation at 0.158; sodium gluconate and NaCl were treated similarly, but rats showed more licking for hypertonic sodium gluconate than hypertonic NaCl. Sodium-depleted rats were also tested with NaCl mixed in amiloride (10–300 μM). Amiloride reduced licking but did not alter the shape of the concentration–response function. Collectively, these results suggest that transduction of sodium by epithelial sodium channels (which are blocked by amiloride and are more dominant in sodium gluconate than NaCl transduction) is crucial for the perception of sodium during physiological sodium depletion. In Experiment 3, sodium-deplete rats were tested with NaCl as in Experiment 1 but after taste aversion conditioning to 0.3M NaCl or sucrose. Rats conditioned to avoid NaCl but not sucrose failed to express a sodium appetite, strongly suggesting that NaCl does not undergo a change in taste quality during sodium appetite—rats show no confusion between sucrose and NaCl in this paradigm. PMID:27660150

The Perceptual Characteristics of Sodium Chloride to Sodium-Depleted Rats.

PubMed

St John, Steven J

2017-02-01

Three experiments assessed potential changes in the rat's perception of sodium chloride (NaCl) during a state of sodium appetite. In Experiment 1, sodium-sufficient rats licking a range of NaCl concentrations (0.028-0.89M) in 15s trials showed an inverted U-shaped concentration response function peaking at 0.281M. Depleted rats (furosemide) showed an identical function, merely elevated, suggesting altered qualitative or hedonic perception but no change in perceived intensity. In Experiment 2, sodium-depleted rats were tested with NaCl, sodium gluconate, and potassium chloride (KCl; 0.028-0.89M) similar to Experiment 1. KCl was licked at the same rate as water except for a slight elevation at 0.158; sodium gluconate and NaCl were treated similarly, but rats showed more licking for hypertonic sodium gluconate than hypertonic NaCl. Sodium-depleted rats were also tested with NaCl mixed in amiloride (10-300 μM). Amiloride reduced licking but did not alter the shape of the concentration-response function. Collectively, these results suggest that transduction of sodium by epithelial sodium channels (which are blocked by amiloride and are more dominant in sodium gluconate than NaCl transduction) is crucial for the perception of sodium during physiological sodium depletion. In Experiment 3, sodium-deplete rats were tested with NaCl as in Experiment 1 but after taste aversion conditioning to 0.3M NaCl or sucrose. Rats conditioned to avoid NaCl but not sucrose failed to express a sodium appetite, strongly suggesting that NaCl does not undergo a change in taste quality during sodium appetite-rats show no confusion between sucrose and NaCl in this paradigm. Published by Oxford University Press on behalf of US Government 2016.

Local anesthetic inhibition of a bacterial sodium channel

PubMed Central

Lee, Sora; Goodchild, Samuel J.

2012-01-01

Recent structural breakthroughs with the voltage-gated sodium channel from Arcobacter butzleri suggest that such bacterial channels may provide a structural platform to advance the understanding of eukaryotic sodium channel gating and pharmacology. We therefore set out to determine whether compounds known to interact with eukaryotic NaVs could also inhibit the bacterial channel from Bacillus halodurans and NaChBac and whether they did so through similar mechanisms as in their eukaryotic homologues. The data show that the archetypal local anesthetic (LA) lidocaine inhibits resting NaChBac channels with a dissociation constant (Kd) of 260 µM, and channels displayed a left-shifted steady-state inactivation gating relationship in the presence of the drug. Extracellular application of QX-314 to expressed NaChBac channels had no effect on sodium current, whereas internal exposure via injection of a bolus of the quaternary derivative rapidly reduced sodium conductance, consistent with a hydrophilic cytoplasmic access pathway to an internal binding site. However, the neutral derivative benzocaine applied externally inhibited NaChBac channels, suggesting that hydrophobic pathways can also provide drug access to inhibit channels. Alternatively, ranolazine, a putative preopen state blocker of eukaryotic NaVs, displayed a Kd of 60 µM and left-shifted the NaChBac activation-voltage relationship. In each case, block enhanced entry into the inactivated state of the channel, an effect that is well described by a simple kinetic scheme. The data suggest that although significant differences exist, LA block of eukaryotic NaVs also occurs in bacterial sodium channels and that NaChBac shares pharmacological homology to the resting state of vertebrate NaV homologues. PMID:22641643

Structure and function of μ-conotoxins, peptide-based sodium channel blockers with analgesic activity

PubMed Central

Green, Brad R; Bulaj, Grzegorz; Norton, Raymond S

2015-01-01

μ-Conotoxins block voltage-gated sodium channels (VGSCs) and compete with tetrodotoxin for binding to the sodium conductance pore. Early efforts identified μ-conotoxins that preferentially blocked the skeletal muscle subtype (NaV1.4). However, the last decade witnessed a significant increase in the number of μ-conotoxins and the range of VGSC subtypes inhibited (NaV1.2, NaV1.3 or NaV1.7). Twenty μ-conotoxin sequences have been identified to date and structure–activity relationship studies of several of these identified key residues responsible for interactions with VGSC subtypes. Efforts to engineer-in subtype specificity are driven by in vivo analgesic and neuromuscular blocking activities. This review summarizes structural and pharmacological studies of μ-conotoxins, which show promise for development of selective blockers of NaV1.2, and perhaps also NaV1.1,1.3 or 1.7. PMID:25406007

Feedforward activation of endothelial ENaC by high sodium

PubMed Central

Korte, Stefanie; Sträter, Alexandra S.; Drüppel, Verena; Oberleithner, Hans; Jeggle, Pia; Grossmann, Claudia; Fobker, Manfred; Nofer, Jerzy-Roch; Brand, Eva; Kusche-Vihrog, Kristina

2014-01-01

Kidney epithelial sodium channels (ENaCs) are known to be inactivated by high sodium concentrations (feedback inhibition). Recently, the endothelial sodium channel (EnNaC) was identified to control the nanomechanical properties of the endothelium. EnNaC-dependent endothelial stiffening reduces the release of nitric oxide, the hallmark of endothelial dysfunction. To study the regulatory impact of sodium on EnNaC, endothelial cells (EA.hy926 and ex vivo mouse endothelium) were incubated in aldosterone-free solutions containing either low (130 mM) or high (150 mM) sodium concentrations. By applying atomic force microscopy-based nanoindentation, an unexpected positive correlation between increasing sodium concentrations and cortical endothelial stiffness was observed, which can be attributed to functional EnNaC. In particular, an acute rise in sodium concentration (+20 mM) was sufficient to increase EnNaC membrane abundance by 90% and stiffening of the endothelial cortex by 18%. Despite the absence of exogenous aldosterone, these effects were prevented by the aldosterone synthase inhibitor FAD286 (100 nM) or the mineralocorticoid receptor (MR)-antagonist spironolactone (100 nM), indicating endogenous aldosterone synthesis and MR-dependent signaling. Interestingly, in the presence of high-sodium concentrations, FAD286 increased the transcription of the MR by 69%. Taken together, a novel feedforward activation of EnNaC by sodium is proposed that contrasts ENaC feedback inhibition in kidney.—Korte, S., Sträter, A. S., Drüppel, V., Oberleithner, H., Jeggle, P., Grossmann, C., Fobker, M., Nofer, J.-R., Brand, E., Kusche-Vihrog, K. Feedforward activation of endothelial ENaC by high sodium. PMID:24868010

Voltage-gated sodium channels

PubMed Central

Abdelsayed, Mena; Sokolov, Stanislav

2013-01-01

Epilepsy is a brain disorder characterized by seizures and convulsions. The basis of epilepsy is an increase in neuronal excitability that, in some cases, may be caused by functional defects in neuronal voltage gated sodium channels, Nav1.1 and Nav1.2. The effects of antiepileptic drugs (AEDs) as effective therapies for epilepsy have been characterized by extensive research. Most of the classic AEDs targeting Nav share a common mechanism of action by stabilizing the channel’s fast-inactivated state. In contrast, novel AEDs, such as lacosamide, stabilize the slow-inactivated state in neuronal Nav1.1 and Nav1.7 isoforms. This paper reviews the different mechanisms by which this stabilization occurs to determine new methods for treatment. PMID:23531742

The role of slow and persistent TTX-resistant sodium currents in acute tumor necrosis factor-α-mediated increase in nociceptors excitability

PubMed Central

Gudes, Sagi; Barkai, Omer; Caspi, Yaki; Katz, Ben; Lev, Shaya

2014-01-01

Tetrodotoxin-resistant (TTX-r) sodium channels are key players in determining the input-output properties of peripheral nociceptive neurons. Changes in gating kinetics or in expression levels of these channels by proinflammatory mediators are likely to cause the hyperexcitability of nociceptive neurons and pain hypersensitivity observed during inflammation. Proinflammatory mediator, tumor necrosis factor-α (TNF-α), is secreted during inflammation and is associated with the early onset, as well as long-lasting, inflammation-mediated increase in excitability of peripheral nociceptive neurons. Here we studied the underlying mechanisms of the rapid component of TNF-α-mediated nociceptive hyperexcitability and acute pain hypersensitivity. We showed that TNF-α leads to rapid onset, cyclooxygenase-independent pain hypersensitivity in adult rats. Furthermore, TNF-α rapidly and substantially increases nociceptive excitability in vitro, by decreasing action potential threshold, increasing neuronal gain and decreasing accommodation. We extended on previous studies entailing p38 MAPK-dependent increase in TTX-r sodium currents by showing that TNF-α via p38 MAPK leads to increased availability of TTX-r sodium channels by partial relief of voltage dependence of their slow inactivation, thereby contributing to increase in neuronal gain. Moreover, we showed that TNF-α also in a p38 MAPK-dependent manner increases persistent TTX-r current by shifting the voltage dependence of activation to a hyperpolarized direction, thus producing an increase in inward current at functionally critical subthreshold voltages. Our results suggest that rapid modulation of the gating of TTX-r sodium channels plays a major role in the mediated nociceptive hyperexcitability of TNF-α during acute inflammation and may lead to development of effective treatments for inflammatory pain, without modulating the inflammation-induced healing processes. PMID:25355965

Visualizing individual sodium channels on the move.

PubMed

Heinemann, Stefan H

2012-07-27

Visualization of voltage-gated sodium channels at work is an important requirement for the understanding of rapid electrical signaling in nerve cells. In this issue of Chemistry & Biology, Ondrus and colleagues have mastered this challenge by chemical synthesis of a fluorescent antagonist and by monitoring single sodium channels in living cells with unprecedented optical resolution. Copyright © 2012 Elsevier Ltd. All rights reserved.

Pinostrobin from Cajanus cajan (L.) Millsp. inhibits sodium channel-activated depolarization of mouse brain synaptoneurosomes.

PubMed

Nicholson, Russell A; David, Laurence S; Pan, Rui Le; Liu, Xin Min

2010-10-01

This investigation focuses on the in vitro neuroactive properties of pinostrobin, a substituted flavanone from Cajanus cajan (L.) Millsp. of the Fabaceae family. We demonstrate that pinostrobin inhibits voltage-gated sodium channels of mammalian brain (IC(50)=23 µM) based on the ability of this substance to suppress the depolarizing effects of the sodium channel-selective activator veratridine in a synaptoneurosomal preparation from mouse brain. The resting membrane potential of synaptoneurosomes was unaffected by pinostrobin. The pharmacological profile of pinostrobin resembles that of depressant drugs that block sodium channels. Copyright © 2010 Elsevier B.V. All rights reserved.

Inhibition of Neuronal Voltage-Gated Sodium Channels by Brilliant Blue G

PubMed Central

Jo, Sooyeon

2011-01-01

Brilliant blue G (BBG), best known as an antagonist of P2X7 receptors, was found to inhibit voltage-gated sodium currents in N1E-115 neuroblastoma cells. Sodium currents elicited from a holding potential of −60 mV were blocked with an IC50 of 2 μM. Block was enhanced in a use-dependent manner at higher stimulation rates. The voltage-dependence of inactivation was shifted in the hyperpolarizing direction, and recovery from inactivation was slowed by BBG. The most dramatic effect of BBG was to slow recovery from inactivation after long depolarizations, with 3 μM BBG increasing half-time for recovery (measured at −120 mV) from 24 to 854 ms after a 10-s step to 0 mV. These results were mimicked by a kinetic model in which BBG binds weakly to resting channels (Kd = 170 μM) but tightly to fast-inactivated channels (Kd = 5 μM) and even more tightly (Kd = 0.2 μM) to slow-inactivated channels. In contrast to BBG, the structurally related food-coloring dye Brilliant Blue FCF had very little effect at concentrations up to 30 μM. These results show that BBG inhibits voltage-gated sodium channels at micromolar concentrations. Although BBG inhibition of sodium channels is less potent than inhibition of P2X7 receptors, there may be significant inhibition of sodium channels at BBG concentrations achieved in spinal cord or brain during experimental treatment of spinal cord injury or Huntington's disease. Considered as a sodium channel blocker, BBG is remarkably potent, acting with more than 10-fold greater potency than lacosamide, another blocker thought to bind to slow-inactivated channels. PMID:21536754

From The Cover: Microtransplantation of functional receptors and channels from the Alzheimer's brain to frog oocytes

NASA Astrophysics Data System (ADS)

Miledi, R.; Dueñas, Z.; Martinez-Torres, A.; Kawas, C. H.; Eusebi, F.

2004-02-01

About a decade ago, cell membranes from the electric organ of Torpedo and from the rat brain were transplanted to frog oocytes, which thus acquired functional Torpedo and rat neurotransmitter receptors. Nevertheless, the great potential that this method has for studying human diseases has remained virtually untapped. Here, we show that cell membranes from the postmortem brains of humans that suffered Alzheimer's disease can be microtransplanted to the plasma membrane of Xenopus oocytes. We show also that these postmortem membranes carry neurotransmitter receptors and voltage-operated channels that are still functional, even after they have been kept frozen for many years. This method provides a new and powerful approach to study directly the functional characteristics and structure of receptors, channels, and other membrane proteins of the Alzheimer's brain. This knowledge may help in understanding the basis of Alzheimer's disease and also help in developing new treatments. -aminobutyric acid receptors | sodium channels | calcium channels | postmortem brain

Sidedness of Carbamazepine Accessibility to Voltage-Gated Sodium Channels

PubMed Central

Jo, Sooyeon

2014-01-01

Voltage-gated sodium channels are inhibited by many local anesthetics, antiarrhythmics, and antiepileptic drugs. The local anesthetic lidocaine appears to be able to access its binding site in the sodium channel only from the membrane phase or from the internal face of the channel. In contrast, the antiepileptic drug carbamazepine was found to inhibit voltage-gated sodium channels only with external, but not internal, application, implying a major difference. We investigated this point using both whole-cell and inside-out patch recordings from human Nav1.7 channels in a stable cell line. In the whole-cell configuration, carbamazepine inhibited sodium current within seconds when applied externally, but had little or no effect when applied internally for up to 15 minutes, confirming previous results. However, carbamazepine inhibited sodium channels effectively and rapidly when applied to the internal face of the membrane using inside-out patch recording. We found that lidocaine also has little or no effect when applied intracellularly in whole-cell recording, but blocks effectively and rapidly when applied to the internal surface using inside-out patches. In contrast, the cationic lidocaine derivative QX-314 (N-ethyl-lidocaine) blocks effectively when applied internally with whole-cell dialysis, as well as when applied to inside-out patches. We conclude that carbamazepine and lidocaine access the sodium channel in similar ways and hypothesize that their lack of effect with internal dialysis in whole-cell recording reflects rapid exit through membrane near the pipette recording site. This effect likely limits the ability of any compound with significant membrane permeability to be applied intracellularly by whole-cell dialysis. PMID:24319110

Sodium channel dysfunction in intractable childhood epilepsy with generalized tonic–clonic seizures

PubMed Central

Rhodes, Thomas H; Vanoye, Carlos G; Ohmori, Iori; Ogiwara, Ikuo; Yamakawa, Kazuhiro; George, Alfred L

2005-01-01

Mutations in SCN1A, the gene encoding the brain voltage-gated sodium channel α1 subunit (NaV1.1), are associated with genetic forms of epilepsy, including generalized epilepsy with febrile seizures plus (GEFS+ type 2), severe myoclonic epilepsy of infancy (SMEI) and related conditions. Several missense SCN1A mutations have been identified in probands affected by the syndrome of intractable childhood epilepsy with generalized tonic–clonic seizures (ICEGTC), which bears similarity to SMEI. To test whether ICEGTC arises from molecular mechanisms similar to those involved in SMEI, we characterized eight ICEGTC missense mutations by whole-cell patch clamp recording of recombinant human SCN1A heterologously expressed in cultured mammalian cells. Two mutations (G979R and T1709I) were non-functional. The remaining alleles (T808S, V983A, N1011I, V1611F, P1632S and F1808L) exhibited measurable sodium current, but had heterogeneous biophysical phenotypes. Mutant channels exhibited lower (V983A, N1011I and F1808L), greater (T808S) or similar (V1611F and P1632S) peak sodium current densities compared with wild-type (WT) SCN1A. Three mutations (V1611F, P1632S and F1808L) displayed hyperpolarized conductance–voltage relationships, while V983A exhibited a strong depolarizing shift in the voltage dependence of activation. All mutants except T808S had hyperpolarized shifts in the voltage dependence of steady-state channel availability. Three mutants (V1611F, P1632S and F1808L) exhibited persistent sodium current ranging from ∼1–3% of peak current amplitude that was significantly greater than WT-SCN1A. Several mutants had impaired slow inactivation, with V983A showing the most prominent effect. Finally, all of the functional alleles exhibited reduced use-dependent channel inhibition. In summary, SCN1A mutations associated with ICEGTC result in a wide spectrum of biophysical defects, including mild-to-moderate gating impairments, shifted voltage dependence and reduced use dependence. The constellation of biophysical abnormalities for some mutants is distinct from those previously observed for GEFS+ and SMEI, suggesting possible, but complex, genotype–phenotype correlations. PMID:16210358

Determining the Advantages, Costs, and Trade-Offs of a Novel Sodium Channel Mutation in the Copepod Acartia hudsonica to Paralytic Shellfish Toxins (PST)

PubMed Central

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

Spider toxin inhibits gating pore currents underlying periodic paralysis.

PubMed

Männikkö, Roope; Shenkarev, Zakhar O; Thor, Michael G; Berkut, Antonina A; Myshkin, Mikhail Yu; Paramonov, Alexander S; Kulbatskii, Dmitrii S; Kuzmin, Dmitry A; Sampedro Castañeda, Marisol; King, Louise; Wilson, Emma R; Lyukmanova, Ekaterina N; Kirpichnikov, Mikhail P; Schorge, Stephanie; Bosmans, Frank; Hanna, Michael G; Kullmann, Dimitri M; Vassilevski, Alexander A

2018-04-24

Gating pore currents through the voltage-sensing domains (VSDs) of the skeletal muscle voltage-gated sodium channel Na V 1.4 underlie hypokalemic periodic paralysis (HypoPP) type 2. Gating modifier toxins target ion channels by modifying the function of the VSDs. We tested the hypothesis that these toxins could function as blockers of the pathogenic gating pore currents. We report that a crab spider toxin Hm-3 from Heriaeus melloteei can inhibit gating pore currents due to mutations affecting the second arginine residue in the S4 helix of VSD-I that we have found in patients with HypoPP and describe here. NMR studies show that Hm-3 partitions into micelles through a hydrophobic cluster formed by aromatic residues and reveal complex formation with VSD-I through electrostatic and hydrophobic interactions with the S3b helix and the S3-S4 extracellular loop. Our data identify VSD-I as a specific binding site for neurotoxins on sodium channels. Gating modifier toxins may constitute useful hits for the treatment of HypoPP. Copyright © 2018 the Author(s). Published by PNAS.

Effects of channel noise on firing coherence of small-world Hodgkin-Huxley neuronal networks

NASA Astrophysics Data System (ADS)

Sun, X. J.; Lei, J. Z.; Perc, M.; Lu, Q. S.; Lv, S. J.

2011-01-01

We investigate the effects of channel noise on firing coherence of Watts-Strogatz small-world networks consisting of biophysically realistic HH neurons having a fraction of blocked voltage-gated sodium and potassium ion channels embedded in their neuronal membranes. The intensity of channel noise is determined by the number of non-blocked ion channels, which depends on the fraction of working ion channels and the membrane patch size with the assumption of homogeneous ion channel density. We find that firing coherence of the neuronal network can be either enhanced or reduced depending on the source of channel noise. As shown in this paper, sodium channel noise reduces firing coherence of neuronal networks; in contrast, potassium channel noise enhances it. Furthermore, compared with potassium channel noise, sodium channel noise plays a dominant role in affecting firing coherence of the neuronal network. Moreover, we declare that the observed phenomena are independent of the rewiring probability.

Propylparaben reduces the excitability of hippocampal neurons by blocking sodium channels.

PubMed

Lara-Valderrábano, Leonardo; Rocha, Luisa; Galván, Emilio J

2016-12-01

Propylparaben (PPB) is an antimicrobial preservative widely used in food, cosmetics, and pharmaceutics. Virtual screening methodologies predicted anticonvulsant activity of PPB that was confirmed in vivo. Thus, we explored the effects of PPB on the excitability of hippocampal neurons by using standard patch clamp techniques. Bath perfusion of PPB reduced the fast-inactivating sodium current (I Na ) amplitude, causing a hyperpolarizing shift in the inactivation curve of the I Na, and markedly delayed the sodium channel recovery from the inactivation state. Also, PPB effectively suppressed the riluzole-sensitive, persistent sodium current (I NaP ). PPB perfusion also modified the action potential kinetics, and higher concentrations of PPB suppressed the spike activity. Nevertheless, the modulatory effects of PPB did not occur when PPB was internally applied by whole-cell dialysis. These results indicate that PPB reduces the excitability of CA1 pyramidal neurons by modulating voltage-dependent sodium channels. The mechanistic basis of this effect is a marked delay in the recovery from inactivation state of the voltage-sensitive sodium channels. Our results indicate that similar to local anesthetics and anticonvulsant drugs that act on sodium channels, PPB acts in a use-dependent manner. Copyright © 2016 Elsevier B.V. All rights reserved.

The antiepileptic medications carbamazepine and phenytoin inhibit native sodium currents in murine osteoblasts.

PubMed

Petty, Sandra J; Milligan, Carol J; Todaro, Marian; Richards, Kay L; Kularathna, Pamuditha K; Pagel, Charles N; French, Chris R; Hill-Yardin, Elisa L; O'Brien, Terence J; Wark, John D; Mackie, Eleanor J; Petrou, Steven

2016-09-01

Fracture risk is a serious comorbidity in epilepsy and may relate to the use of antiepileptic drugs (AEDs). Many AEDs inhibit ion channel function, and the expression of these channels in osteoblasts raises the question of whether altered bone signaling increases bone fragility. We aimed to confirm the expression of voltage-gated sodium (NaV ) channels in mouse osteoblasts, and to investigate the action of carbamazepine and phenytoin on NaV channels. Immunocytochemistry was performed on primary calvarial osteoblasts extracted from neonatal C57BL/6J mice and additional RNA sequencing (RNASeq) was included to confirm expression of NaV . Whole-cell patch-clamp recordings were made to identify the native currents expressed and to assess the actions of carbamazepine (50 μm) or phenytoin (50 μm). NaV expression was demonstrated with immunocytochemistry, RNA sequencing, and functionally, with demonstration of robust tetrodotoxin-sensitive and voltage-activated inward currents. Application of carbamazepine or phenytoin resulted in significant inhibition of current amplitude for carbamazepine (31.6 ± 5.9%, n = 9; p < 0.001), and for phenytoin (35.5 ± 6.9%, n = 7; p < 0.001). Mouse osteoblasts express NaV , and native NaV currents are blocked by carbamazepine and phenytoin, supporting our hypothesis that AEDs can directly influence osteoblast function and potentially affect bone strength. Wiley Periodicals, Inc. © 2016 International League Against Epilepsy.

Frequency-dependent reliability of spike propagation is function of axonal voltage-gated sodium channels in cerebellar Purkinje cells.

PubMed

Yang, Zhilai; Wang, Jin-Hui

2013-12-01

The spike propagation on nerve axons, like synaptic transmission, is essential to ensure neuronal communication. The secure propagation of sequential spikes toward axonal terminals has been challenged in the neurons with a high firing rate, such as cerebellar Purkinje cells. The shortfall of spike propagation makes some digital spikes disappearing at axonal terminals, such that the elucidation of the mechanisms underlying spike propagation reliability is crucial to find the strategy of preventing loss of neuronal codes. As the spike propagation failure is influenced by the membrane potentials, this process is likely caused by altering the functional status of voltage-gated sodium channels (VGSC). We examined this hypothesis in Purkinje cells by using pair-recordings at their somata and axonal blebs in cerebellar slices. The reliability of spike propagation was deteriorated by elevating spike frequency. The frequency-dependent reliability of spike propagation was attenuated by inactivating VGSCs and improved by removing their inactivation. Thus, the functional status of axonal VGSCs influences the reliability of spike propagation.

Effects of stochastic sodium channels on extracellular excitation of myelinated nerve fibers.

PubMed

Mino, Hiroyuki; Grill, Warren M

2002-06-01

The effects of the stochastic gating properties of sodium channels on the extracellular excitation properties of mammalian nerve fibers was determined by computer simulation. To reduce computation time, a hybrid multicompartment cable model including five central nodes of Ranvier containing stochastic sodium channels and 16 flanking nodes containing detenninistic membrane dynamics was developed. The excitation properties of the hybrid cable model were comparable with those of a full stochastic cable model including 21 nodes of Ranvier containing stochastic sodium channels, indicating the validity of the hybrid cable model. The hybrid cable model was used to investigate whether or not the excitation properties of extracellularly activated fibers were influenced by the stochastic gating of sodium channels, including spike latencies, strength-duration (SD), current-distance (IX), and recruitment properties. The stochastic properties of the sodium channels in the hybrid cable model had the greatest impact when considering the temporal dynamics of nerve fibers, i.e., a large variability in latencies, while they did not influence the SD, IX, or recruitment properties as compared with those of the conventional deterministic cable model. These findings suggest that inclusion of stochastic nodes is not important for model-based design of stimulus waveforms for activation of motor nerve fibers. However, in cases where temporal fine structure is important, for example in sensory neural prostheses in the auditory and visual systems, the stochastic properties of the sodium channels may play a key role in the design of stimulus waveforms.

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

PubMed Central

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

Novel ion channel targets in atrial fibrillation.

PubMed

Hancox, Jules C; James, Andrew F; Marrion, Neil V; Zhang, Henggui; Thomas, Dierk

2016-08-01

Atrial fibrillation (AF) is the most common arrhythmia in humans. It is progressive and the development of electrical and structural remodeling makes early intervention desirable. Existing antiarrhythmic pharmacological approaches are not always effective and can produce unwanted side effects. Additional atrial-selective antiarrhythmic strategies are therefore desirable. Evidence for three novel ion channel atrial-selective therapeutic targets is evaluated: atrial-selective fast sodium channel current (INa) inhibition; small conductance calcium-activated potassium (SK) channels; and two-pore (K2P) potassium channels. Data from animal models support atrial-ventricular differences in INa kinetics and also suggest atrial-ventricular differences in sodium channel β subunit expression. Further work is required to determine whether intrinsic atrial-ventricular differences in human INa exist or whether functional differences occur due to distinct atrial and ventricular action and resting potentials. SK and K2P channels (particularly K2P 3.1) offer potentially attractive atrial-selective targets. Work is needed to identify the underlying basis of SK current that contributes to (patho)physiological atrial repolarization and settings in which SK inhibition is anti- versus pro-arrhythmic. Although K2P3.1 appears to be a promising target with comparatively selective drugs for experimental use, a lack of selective pharmacology hinders evaluation of other K2P channels as potential atrial-selective targets.

Biophysical Adaptations of Prokaryotic Voltage-Gated Sodium Channels.

PubMed

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.

Effects of carbamazepine and lamotrigine on functional magnetic resonance imaging cognitive networks.

PubMed

Xiao, Fenglai; Caciagli, Lorenzo; Wandschneider, Britta; Sander, Josemir W; Sidhu, Meneka; Winston, Gavin; Burdett, Jane; Trimmel, Karin; Hill, Andrea; Vollmar, Christian; Vos, Sjoerd B; Ourselin, Sebastien; Thompson, Pamela J; Zhou, Dong; Duncan, John S; Koepp, Matthias J

2018-06-13

To investigate the effects of sodium channel-blocking antiepileptic drugs (AEDs) on functional magnetic resonance imaging (fMRI) language network activations in patients with focal epilepsy. In a retrospective study, we identified patients who were treated at the time of language fMRI scanning with either carbamazepine (CBZ; n = 42) or lamotrigine (LTG; n = 42), but not another sodium channel-blocking AED. We propensity-matched 42 patients taking levetiracetam (LEV) as "patient-controls" and included further 42 age- and gender-matched healthy controls. After controlling for age, age at onset of epilepsy, gender, and antiepileptic comedications, we compared verbal fluency fMRI activations between groups and out-of-scanner psychometric measures of verbal fluency. Patients on CBZ performed less well on a verbal fluency tests than those taking LTG or LEV. Compared to either LEV-treated patients or controls, patients taking CBZ showed decreased activations in left inferior frontal gyrus and patients on LTG showed abnormal deactivations in frontal and parietal default mode areas. All patient groups showed fewer activations in the putamen bilaterally compared to controls. In a post hoc analysis, out-of-scanner fluency scores correlated positively with left putamen activation. Our study provides evidence of AED effects on the functional neuroanatomy of language, which might explain subtle language deficits in patients taking otherwise well-tolerated sodium channel-blocking agents. Patients on CBZ showed dysfunctional frontal activation and more pronounced impairment of performance than patients taking LTG, which was associated only with failure to deactivate task-negative networks. As previously shown for working memory, LEV treatment did not affect functional language networks. © 2018 The Authors. Epilepsia published by Wiley Periodicals, Inc. on behalf of International League Against Epilepsy.

Evolutionarily conserved intracellular gate of voltage-dependent sodium channels

NASA Astrophysics Data System (ADS)

Oelstrom, Kevin; Goldschen-Ohm, Marcel P.; Holmgren, Miguel; Chanda, Baron

2014-03-01

Members of the voltage-gated ion channel superfamily (VGIC) regulate ion flux and generate electrical signals in excitable cells by opening and closing pore gates. The location of the gate in voltage-gated sodium channels, a founding member of this superfamily, remains unresolved. Here we explore the chemical modification rates of introduced cysteines along the S6 helix of domain IV in an inactivation-removed background. We find that state-dependent accessibility is demarcated by an S6 hydrophobic residue; substituted cysteines above this site are not modified by charged thiol reagents when the channel is closed. These accessibilities are consistent with those inferred from open- and closed-state structures of prokaryotic sodium channels. Our findings suggest that an intracellular gate composed of a ring of hydrophobic residues is not only responsible for regulating access to the pore of sodium channels, but is also a conserved feature within canonical members of the VGIC superfamily.

β1-C121W Is Down But Not Out: Epilepsy-Associated Scn1b-C121W Results in a Deleterious Gain-of-Function

PubMed Central

Kruger, Larisa C.; O'Malley, Heather A.; Hull, Jacob M.; Kleeman, Amanda; Patino, Gustavo A.

2016-01-01

Voltage-gated sodium channel (VGSC) β subunits signal through multiple pathways on multiple time scales. In addition to modulating sodium and potassium currents, β subunits play nonconducting roles as cell adhesion molecules, which allow them to function in cell–cell communication, neuronal migration, neurite outgrowth, neuronal pathfinding, and axonal fasciculation. Mutations in SCN1B, encoding VGSC β1 and β1B, are associated with epilepsy. Autosomal-dominant SCN1B-C121W, the first epilepsy-associated VGSC mutation identified, results in genetic epilepsy with febrile seizures plus (GEFS+). This mutation has been shown to disrupt both the sodium-current-modulatory and cell-adhesive functions of β1 subunits expressed in heterologous systems. The goal of this study was to compare mice heterozygous for Scn1b-C121W (Scn1b+/W) with mice heterozygous for the Scn1b-null allele (Scn1b+/−) to determine whether the C121W mutation results in loss-of-function in vivo. We found that Scn1b+/W mice were more susceptible than Scn1b+/− and Scn1b+/+ mice to hyperthermia-induced convulsions, a model of pediatric febrile seizures. β1-C121W subunits are expressed at the neuronal cell surface in vivo. However, despite this, β1-C121W polypeptides are incompletely glycosylated and do not associate with VGSC α subunits in the brain. β1-C121W subcellular localization is restricted to neuronal cell bodies and is not detected at axon initial segments in the cortex or cerebellum or at optic nerve nodes of Ranvier of Scn1bW/W mice. These data, together with our previous results showing that β1-C121W cannot participate in trans-homophilic cell adhesion, lead to the hypothesis that SCN1B-C121W confers a deleterious gain-of-function in human GEFS+ patients. SIGNIFICANCE STATEMENT The mechanisms underlying genetic epilepsy syndromes are poorly understood. Closing this gap in knowledge is essential to the development of new medicines to treat epilepsy. We have used mouse models to understand the mechanism of a mutation in the sodium channel gene SCN1B linked to genetic epilepsy with febrile seizures plus. We report that sodium channel β1 subunit proteins encoded by this mutant gene are expressed at the surface of neuronal cell bodies; however, they do not associate with the ion channel complex nor are they transported to areas of the axon that are critical for proper neuronal firing. We conclude that this disease-causing mutation is not simply a loss-of-function, but instead results in a deleterious gain-of-function in the brain. PMID:27277800

Non-dystrophic myotonia: prospective study of objective and patient reported outcomes.

PubMed

Trivedi, Jaya R; Bundy, Brian; Statland, Jeffrey; Salajegheh, Mohammad; Rayan, Dipa Raja; Venance, Shannon L; Wang, Yunxia; Fialho, Doreen; Matthews, Emma; Cleland, James; Gorham, Nina; Herbelin, Laura; Cannon, Stephen; Amato, Anthony; Griggs, Robert C; Hanna, Michael G; Barohn, Richard J

2013-07-01

Non-dystrophic myotonias are rare diseases caused by mutations in skeletal muscle chloride and sodium ion channels with considerable phenotypic overlap between diseases. Few prospective studies have evaluated the sensitivity of symptoms and signs of myotonia in a large cohort of patients. We performed a prospective observational study of 95 participants with definite or clinically suspected non-dystrophic myotonia recruited from six sites in the USA, UK and Canada between March 2006 and March 2009. We used the common infrastructure and data elements provided by the NIH-funded Rare Disease Clinical Research Network. Outcomes included a standardized symptom interview and physical exam; the Short Form-36 and the Individualized Neuromuscular Quality of Life instruments; electrophysiological short and prolonged exercise tests; manual muscle testing; and a modified get-up-and-go test. Thirty-two participants had chloride channel mutations, 34 had sodium channel mutations, nine had myotonic dystrophy type 2, one had myotonic dystrophy type 1, and 17 had no identified mutation. Phenotype comparisons were restricted to those with sodium channel mutations, chloride channel mutations, and myotonic dystrophy type 2. Muscle stiffness was the most prominent symptom overall, seen in 66.7% to 100% of participants. In comparison with chloride channel mutations, participants with sodium mutations had an earlier age of onset of stiffness (5 years versus 10 years), frequent eye closure myotonia (73.5% versus 25%), more impairment on the Individualized Neuromuscular Quality of Life summary score (20.0 versus 9.44), and paradoxical eye closure myotonia (50% versus 0%). Handgrip myotonia was seen in three-quarters of participants, with warm up of myotonia in 75% chloride channel mutations, but also 35.3% of sodium channel mutations. The short exercise test showed ≥10% decrement in the compound muscle action potential amplitude in 59.3% of chloride channel participants compared with 27.6% of sodium channel participants, which increased post-cooling to 57.6% in sodium channel mutations. In evaluation of patients with clinical and electrical myotonia, despite considerable phenotypic overlap, the presence of eye closure myotonia, paradoxical myotonia, and an increase in short exercise test sensitivity post-cooling suggest sodium channel mutations. Outcomes designed to measure stiffness or the electrophysiological correlates of stiffness may prove useful for future clinical trials, regardless of underlying mutation, and include patient-reported stiffness, bedside manoeuvres to evaluate myotonia, muscle specific quality of life instruments and short exercise testing.

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

PubMed

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.

n-Alcohols Inhibit Voltage-Gated Na+ Channels Expressed in Xenopus Oocytes

PubMed Central

Horishita, Takafumi; Harris, R. Adron

2008-01-01

Voltage-gated sodium channels are essential for the initiation and propagation of action potentials in excitable cells and are known as a target of local anesthetics. In addition, inhibition of sodium channels by volatile anesthetics has been proposed as a mechanism of general anesthesia. The n-alcohols produce anesthesia, and their potency increases with carbon number until a “cut-off” is reached. In this study, we examined effects of a range of n-alcohols on Nav1.2 subunits to determine the alcohol cut-off for this channel. We also studied the effect of a short-chain alcohol (ethanol) and a long-chain alcohol (octanol) on Nav1.2, Nav1.4, Nav1.6, and Nav1.8 subunits, and we investigated the effects of alcohol on channel kinetics. Ethanol and octanol inhibited sodium currents of all subunits, and the inhibition of the Nav1.2 channel by n-alcohols indicated a cut-off at nonanol. Ethanol and octanol produced open-channel block, which was more pronounced for Nav1.8 than for the other sodium channels. Inhibition of Nav1.2 was due to decreased activation and increased inactivation. These results suggest that sodium channels may have a hydrophobic binding site for n-alcohols and demonstrate the differences in the kinetic mechanisms of inhibition for n-alcohols and inhaled anesthetics. PMID:18434586

The nonproton ligand of acid-sensing ion channel 3 activates mollusk-specific FaNaC channels via a mechanism independent of the native FMRFamide peptide.

PubMed

Yang, Xiao-Na; Niu, You-Ya; Liu, Yan; Yang, Yang; Wang, Jin; Cheng, Xiao-Yang; Liang, Hong; Wang, Heng-Shan; Hu, You-Min; Lu, Xiang-Yang; Zhu, Michael X; Xu, Tian-Le; Tian, Yun; Yu, Ye

2017-12-29

The degenerin/epithelial sodium channel (DEG/ENaC) superfamily of ion channels contains subfamilies with diverse functions that are fundamental to many physiological and pathological processes, ranging from synaptic transmission to epileptogenesis. The absence in mammals of some DEG/ENaCs subfamily orthologues such as FMRFamide peptide-activated sodium channels (FaNaCs), which have been identified only in mollusks, indicates that the various subfamilies diverged early in evolution. We recently reported that the nonproton agonist 2-guanidine-4-methylquinazoline (GMQ) activates acid-sensing ion channels (ASICs), a DEG/ENaC subfamily mainly in mammals, in the absence of acidosis. Here, we show that GMQ also could directly activate the mollusk-specific FaNaCs. Differences in ion selectivity and unitary conductance and effects of substitutions at key residues revealed that GMQ and FMRFamide activate FaNaCs via distinct mechanisms. The presence of two activation mechanisms in the FaNaC subfamily diverging early in the evolution of DEG/ENaCs suggested that dual gating is an ancient feature in this superfamily. Notably, the GMQ-gating mode is still preserved in the mammalian ASIC subfamily, whereas FMRFamide-mediated channel gating was lost during evolution. This implied that GMQ activation may be essential for the functions of mammalian DEG/ENaCs. Our findings provide new insights into the evolution of DEG/ENaCs and may facilitate the discovery and characterization of their endogenous agonists. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

Characterization of Rat Meibomian Gland Ion and Fluid Transport

PubMed Central

Yu, Dongfang; Davis, Richard M.; Aita, Megumi; Burns, Kimberlie A.; Clapp, Phillip W.; Gilmore, Rodney C.; Chua, Michael; O'Neal, Wanda K.; Schlegel, Richard; Randell, Scott H.; C. Boucher, Richard

2016-01-01

Purpose We establish novel primary rat meibomian gland (MG) cell culture systems and explore the ion transport activities of the rat MG. Methods Freshly excised rat MG tissues were characterized as follows: (1) mRNA expression of selected epithelial ion channels/transporters were measured by RT-PCR, (2) localization of epithelial sodium channel (ENaC) mRNAs was performed by in situ hybridization, and (3) protein expression and localization of βENaC, the Na+/K+/Cl− cotransporter (NKCC), and the Na+/K+ ATPase were evaluated by immunofluorescence. Primary isolated rat MG cells were cocultured with 3T3 feeder cells and a Rho-associated kinase (ROCK) inhibitor (Y-27632) for expansion. Passaged rat MG cells were cultured as planar sheets under air-liquid interface (ALI) conditions for gene expression and electrophysiologic studies. Passaged rat MG cells also were cultured in matrigel matrices to form spheroids, which were examined ultrastructurally by transmission electron microscopy (TEM) and functionally using swelling assays. Results Expression of multiple ion channel/transporter genes was detected in rat MG tissues. β-ENaC mRNA and protein were localized more to MG peripheral acinar cells than central acinar cells or ductular epithelial cells. Electrophysiologic studies of rat MG cell planar cultures demonstrated functional sodium, chloride, and potassium channels, and cotransporters activities. Transmission electron microscopic analyses of rat MG spheroids revealed highly differentiated MG cells with abundant lysosomal lamellar bodies. Rat MG spheroids culture-based measurements demonstrated active volume regulation by ion channels. Conclusions This study demonstrates the presence and function of ion channels and volume transport by rat MG. Two novel primary MG cell culture models that may be useful for MG research were established. PMID:27127933

β-Liddle mutation of the epithelial sodium channel increases alveolar fluid clearance and reduces the severity of hydrostatic pulmonary oedema in mice

PubMed Central

Randrianarison, Nadia; Escoubet, Brigitte; Ferreira, Chrystophe; Fontayne, Alexandre; Fowler-Jaeger, Nicole; Clerici, Christine; Hummler, Edith; Rossier, Bernard C; Planès, Carole

2007-01-01

Transepithelial sodium transport via alveolar epithelial Na+ channels and Na+,K+-ATPase constitutes the driving force for removal of alveolar oedema fluid. Decreased activity of the amiloride-sensitive epithelial Na+ channel (ENaC) in the apical membrane of alveolar epithelial cells impairs sodium-driven alveolar fluid clearance (AFC) and predisposes to pulmonary oedema. We hypothesized that hyperactivity of ENaC in the distal lung could improve AFC and facilitate the resolution of pulmonary oedema. AFC and lung fluid balance were studied at baseline and under conditions of hydrostatic pulmonary oedema in the β-Liddle (L) mouse strain harbouring a gain-of-function mutation (R566stop) within the Scnn1b gene. As compared with wild-type (+/+), baseline AFC was increased by 2- and 3-fold in heterozygous (+/L) and homozygous mutated (L/L) mice, respectively, mainly due to increased amiloride-sensitive AFC. The β2-agonist terbutaline stimulated AFC in +/+ and +/L mice, but not in L/L mice. Acute volume overload induced by saline infusion (40% of body weight over 2 h) significantly increased extravascular (i.e. interstitial and alveolar) lung water as assessed by the bloodless wet-to-dry lung weight ratio in +/+ and L/L mice, as compared with baseline. However, the increase was significantly larger in +/+ than in L/L groups (P= 0.01). Volume overload also increased the volume of the alveolar epithelial lining fluid in +/+ mice, indicating the presence of alveolar oedema, but not in L/L mice. Cardiac function as evaluated by echocardiography was comparable in both groups. These data show that constitutive ENaC activation improved sodium-driven AFC in the mouse lung, and attenuated the severity of hydrostatic pulmonary oedema. PMID:17430990

Serum and Glucocorticoid Regulated Kinase 1 in Sodium Homeostasis

PubMed Central

Lou, Yiyun; Zhang, Fan; Luo, Yuqin; Wang, Liya; Huang, Shisi; Jin, Fan

2016-01-01

The ubiquitously expressed serum and glucocorticoid regulated kinase 1 (SGK1) is tightly regulated by osmotic and hormonal signals, including glucocorticoids and mineralocorticoids. Recently, SGK1 has been implicated as a signal hub for the regulation of sodium transport. SGK1 modulates the activities of multiple ion channels and carriers, such as epithelial sodium channel (ENaC), voltage-gated sodium channel (Nav1.5), sodium hydrogen exchangers 1 and 3 (NHE1 and NHE3), sodium-chloride symporter (NCC), and sodium-potassium-chloride cotransporter 2 (NKCC2); as well as the sodium-potassium adenosine triphosphatase (Na+/K+-ATPase) and type A natriuretic peptide receptor (NPR-A). Accordingly, SGK1 is implicated in the physiology and pathophysiology of Na+ homeostasis. Here, we focus particularly on recent findings of SGK1’s involvement in Na+ transport in renal sodium reabsorption, hormone-stimulated salt appetite and fluid balance and discuss the abnormal SGK1-mediated Na+ reabsorption in hypertension, heart disease, edema with diabetes, and embryo implantation failure. PMID:27517916

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

PubMed

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 < 1 and < 0 for the ROR and IC, respectively, signified inverse associations. Furthermore, using a claims database, which contains 3 million insured persons, an event sequence symmetry analysis (ESSA) was performed to identify an inverse association between AEDs and cancer over the period of January 2005 to May 2014. The upper limit of the 95% CI of adjusted sequence ratio (ASR) < 1 signified an inverse association. In the FAERS database analyses, significant inverse associations were found between sodium channel-blocking AEDs and individual 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.

Lidocaine attenuation testing: An in vivo investigation of putative LQT3-associated variants in the SCN5A-encoded sodium channel.

PubMed

Anderson, Heather N; Bos, J Martijn; Kapplinger, Jamie D; Meskill, Jana M; Ye, Dan; Ackerman, Michael J

2017-08-01

Long QT syndrome type 3 (LQT3) accounts for 5%-10% of long QT syndrome and results from gain-of-function mutations in the SCN5A-encoded sodium channel. Approximately 2% of healthy individuals host rare SCN5A variants of uncertain significance (VUS). Distinction of true LQT3-causative mutations from background genetic noise is essential. The purpose of this study was to assess the use of the lidocaine attenuation test (LAT) in evaluating patients with possible LQT3. We reviewed the LAT results and medical records for 25 patients with a possible LQT3-associated SCN5A variant. The LAT involved a loading dose of 1 mg/kg of intravenous lidocaine followed by continuous infusion at 50 μg/(kg⋅min) for 20 minutes. If the corrected QT interval shortened by ≥30 ms, the LAT was defined as positive. Sixteen patients (64%) had a positive LAT, 6 of which demonstrated the E1784K variant. A positive LAT correlated in 86% of cases with abnormal in vitro channel function (mean corrected QT interval attenuation 43 ± 3 ms vs 25 ± 5 ms for wild-type variants; P = .03). Four of 5 patients (80%) with a VUS had a positive LAT (T1304M [2 patients], L1786P, and R800L). The T1304M variant demonstrated abnormal in vitro function and a positive LAT, opening the door for a potential variant promotion from VUS to likely pathogenic. The LAT may help distinguish true LQT3-causative mutations from an otherwise noncontributory VUS. Given that lidocaine acts as a late sodium current blocker, a positive LAT may enable the early identification of a pathological accentuation of the late sodium current that could be targeted therapeutically. Copyright © 2017 Heart Rhythm Society. Published by Elsevier Inc. All rights reserved.

Binding modes and functional surface of anti-mammalian scorpion α-toxins to sodium channels.

PubMed

Chen, Rong; Chung, Shin-Ho

2012-10-02

Scorpion α-toxins bind to the voltage-sensing domains of voltage-gated sodium (Na(V)) channels and interfere with the inactivation mechanisms. The functional surface of α-toxins has been shown to contain an NC-domain consisting of the five-residue turn (positions 8-12) and the C-terminus (positions 56-64) and a core-domain centered on the residue 18. The NC- and core-domains are interconnected by the linker-domain (positions 8-18). Here with atomistic molecular dynamics simulations, we examine the binding modes between two α-toxins, the anti-mammalian AahII and the anti-insect LqhαIT, and the voltage-sensing domain of rat Na(V)1.2, a subtype of Na(V) channels expressed in nerve cells. Both toxins are docked to the extracellular side of the voltage-sensing domain of Na(V)1.2 using molecular dynamics simulations, with the linker-domain assumed to wedge into the binding pocket. Several salt bridges and hydrophobic clusters are observed to form between the NC- and core-domains of the toxins and Na(V)1.2 and stabilize the toxin-channel complexes. The binding modes predicted are consistent with available mutagenesis data and can readily explain the relative affinities of AahII and LqhαIT for Na(V)1.2. The dissociation constants for the two toxin-channel complexes are derived, which compare favorably with experiment. Our models demonstrate that the functional surface of anti-mammalian scorpion α-toxins is centered on the linker-domain, similar to that of β-toxins.

Short-Term Depression of Axonal Spikes at the Mouse Hippocampal Mossy Fibers and Sodium Channel-Dependent Modulation

PubMed Central

Ohura, Shunsuke

2018-01-01

Axonal spike is an important upstream process of transmitter release, which directly impacts on release probability from the presynaptic terminals. Despite the functional significance, possible activity-dependent modulation of axonal spikes has not been studied extensively, partly due to inaccessibility of the small structures of axons for electrophysiological recordings. In this study, we tested the possibility of use-dependent changes in axonal spikes at the hippocampal mossy fibers, where direct recordings from the axon terminals are readily feasible. Hippocampal slices were made from mice of either sex, and loose-patch clamp recordings were obtained from the visually identified giant mossy fiber boutons located in the stratum lucidum of the CA3 region. Stimulation of the granule cell layer of the dentate gyrus elicited axonal spikes at the single bouton which occurred in all or none fashion. Unexpected from the digital nature of spike signaling, the peak amplitude of the second spikes in response to paired stimuli at a 50-ms interval was slightly but reproducibly smaller than the first spikes. Repetitive stimuli at 20 or 100 Hz also caused progressive use-dependent depression during the train. Notably, veratridine, an inhibitor of inactivation of sodium channels, significantly accelerated the depression with minimal effect on the initial spikes. These results suggest that sodium channels contribute to use-dependent depression of axonal spikes at the hippocampal mossy fibers, possibly by shaping the afterdepolarization (ADP) following axonal spikes. Prolonged depolarization during ADP may inactivate a fraction of sodium channels and thereby suppresses the subsequent spikes at the hippocampal mossy fibers. PMID:29468192

Calcium Homeostasis Modulator 1-Like Currents in Rat Fungiform Taste Cells Expressing Amiloride-Sensitive Sodium Currents.

PubMed

Bigiani, Albertino

2017-05-01

Salt reception by taste cells is still the less understood transduction process occurring in taste buds, the peripheral sensory organs for the detection of food chemicals. Although there is evidence suggesting that the epithelial sodium channel (ENaC) works as sodium receptor, yet it is not clear how salt-detecting cells signal the relevant information to nerve endings. Taste cells responding to sweet, bitter, and umami substances release ATP as neurotransmitter through a nonvesicular mechanism. Three different channel proteins have been proposed as conduit for ATP secretion: pannexin channels, connexin hemichannels, and calcium homeostasis modulator 1 (CALHM1) channels. In heterologous expression systems, these channels mediate outwardly rectifying membrane currents with distinct biophysical and pharmacological properties. I therefore tested whether also salt-detecting taste cells were endowed with these currents. To this aim, I applied the patch-clamp techniques to single cells in isolated taste buds from rat fungiform papillae. Salt-detecting cells were functionally identified by exploiting the effect of amiloride, which induces a current response by shutting down ENaCs. I looked for the presence of outwardly rectifying currents by using appropriate voltage-clamp protocols and specific pharmacological tools. I found that indeed salt-detecting cells possessed these currents with properties consistent with the presence, at least in part, of CALHM1 channels. Unexpectedly, CALHM1-like currents in taste cells were potentiated by known blockers of pannexin, suggesting a possible inhibitory action of this protein on CALMH1. These findings indicate that communication between salt-detecting cells and nerve endings might involve ATP release by CALMH1 channels. © The Author 2017. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

A Conserved Bicycle Model for Circadian Clock Control of Membrane Excitability

PubMed Central

Flourakis, Matthieu; Kula-Eversole, Elzbieta; Hutchison, Alan L.; Han, Tae Hee; Aranda, Kimberly; Moose, Devon L.; White, Kevin P.; Dinner, Aaron R.; Lear, Bridget C.; Ren, Dejian; Diekman, Casey O.; Raman, Indira M.; Allada, Ravi

2015-01-01

Summary Circadian clocks regulate membrane excitability in master pacemaker neurons to control daily rhythms of sleep and wake. Here we find that two distinctly timed electrical drives collaborate to impose rhythmicity on Drosophila clock neurons. In the morning, a voltage-independent sodium conductance via the NA/NALCN ion channel depolarizes these neurons. This current is driven by the rhythmic expression of NCA localization factor-1, linking the molecular clock to ion channel function. In the evening, basal potassium currents peak to silence clock neurons. Remarkably, daily antiphase cycles of sodium and potassium currents also drive mouse clock neuron rhythms. Thus, we reveal an evolutionarily ancient strategy for the neural mechanisms that govern daily sleep and wake. PMID:26276633

Compound-Specific Effects of Mutations at Val787 in DII-S6 of Nav1.4 Sodium Channels on the Action of Sodium Channel Inhibitor Insecticides

PubMed Central

von Stein, Richard T.; Soderlund, David M.

2012-01-01

Sodium channel inhibitor (SCI) insecticides are hypothesized to inhibit voltage-gated sodium channels by binding selectively to the slow-inactivated state. Replacement of valine at position 787 in the S6 segment of homology domain II of the rat Nav1.4 sodium channel by lysine (V787K) enchances slow inactivation of this channel whereas replacement by alanine or cysteine (V787A, V787C) inhibits slow inactivation. To test the hypothesis that SCI insecticides bind selectively to the slow-inactivated state, we constructed mutated Nav1.4/V787A, Nav1.4/V787C, and Nav1.4/V787K cDNAs, expressed wildtype and mutated channels with the auxiliary β1 subunit in Xenopus oocytes, and used the two-electrode voltage clamp technique to examine the effects of these mutations on channel inhibition by four SCI insecticides (indoxacarb, its bioactivated metabolite DCJW, metaflumizone, and RH3421). Mutations at Val787 affected SCI insecticide sensitivity in a manner that was independent of mutation-induced changes in slow inactivation gating. Sensitivity to inhibition by 10 μM indoxacarb was significantly increased in all three mutated channels, whereas sensitivity to inhibition by 10 μM metaflumizone was significantly reduced in Nav1.4/V787A channels and completely abolished in Nav1.4/V787K channels. The effects of Val787 mutations on metaflumizone were correlated with the hydrophobicity of the substituted amino acid rather than the extent of slow inactivation. None of the mutations at Val787 significantly affected the sensitivity to inhibition by DCJW or RH3421. These results demonstrate that the impact of mutations at Val787 on sodium channel inhibition by SCI insecticides depends on the specific insecticide examined and is independent of mutation-induced changes in slow inactivation gating. We propose that Val787 may be a unique determinant of metaflumizone binding. PMID:22983119

n-Alkanols potentiate sodium channel inactivation in squid giant axons.

PubMed Central

Oxford, G S; Swenson, R P

1979-01-01

The effects of n-octanol and n-decanol on nerve membrane sodium channels were examined in internally perfused, voltage-clamped squid giant axons. Both n-octanol and n-decanol almost completely eliminated the residual sodium conductance at the end of 8-ms voltage steps. In contrast, peak sodium conductance was only partially reduced. This block of peak and residual sodium conductance was very reversible and seen with both internal and external alkanol application. The differential sensitivity of peak and residual conductance to alkanol treatment was eliminated after internal pronase treatment, suggesting that n-octanol and n-decanol enhance the normal inactivation mechanism rather than directly blocking channels in a time-dependent manner. PMID:233577

Analgesic ineffectiveness of lacosamide after spinal nerve ligation and its sodium channel activity in injured neurons.

PubMed

Hagenacker, T; Schäfer, N; Büsselberg, D; Schäfers, M

2013-07-01

Lacosamide is a novel anti-epileptic drug that enhances the slow- and not fast-inactivating state of voltage-gated sodium channels. Lacosamide has demonstrated analgesic efficacy in several animal studies but preclinical studies on neuropathic pain models are rare, and recent clinical trials showed no superior analgesic effects. Here, we examine whether an acute or chronic administration of lacosamide (3-60 mg/kg, i.p.) attenuates pain behaviour induced by spinal nerve ligation (SNL). To validate the inhibitory efficacy of lacosamide on voltage-gated sodium channels, sodium currents in naïve and SNL-injured dorsal root ganglion (DRG) neurons were recorded using whole-cell patch clamping. Lacosamide only marginally attenuated thermal hyperalgesia, but not tactile allodynia when applied once 7 or 14 days after SNL and showed no analgesic effect when applied daily for 19 days. In naïve neurons, 100 μmol/L lacosamide inhibited sodium channel currents by 58% and enhanced the slow inactivation (87% for lacosamide vs. 47% for control). In contrast, lacosamide inhibited sodium currents in injured DRG neurons by only 15%, while the effects on slow inactivation were diminished. Isolated currents from the NaV 1.8 channel subtype were only marginally changed by lacosamide. The reduced effectiveness of lacosamide on voltage-gated sodium channel currents in injured DRG neurons may contribute to the reduced analgesic effect observed for the SNL model. © 2012 European Federation of International Association for the Study of Pain Chapters.

Alpha 1-acid glycoprotein reverses cocaine-induced sodium channel blockade in cardiac myocytes.

PubMed

Ma, Yu-Ling; Peters, Nicholas S; Henry, John A

2006-03-01

Alpha 1-acid glycoprotein (AAG) is an acute phase protein capable of binding basic drugs. This action explains its reversal of sodium channel blockade by drugs such as amitriptyline and quinidine. We report here the reversal of cocaine-induced sodium channel blockade by AAG. The sodium channel blocking property of cocaine is a major mechanism behind cocaine-induced sudden cardiac death, since sodium channels play a key role in the initiation and regulation of the heart beat. Voltage-gated sodium current (I(Na)) was recorded using whole-cell patch-clamp techniques. Guinea-pig cardiac ventricular myocytes were isolated and continuously perfused at room temperature with physiological solutions. At concentrations ranging from 5 to 320 microM cocaine showed a dose-dependent and reversible blockade of I(Na) with an IC50 of 45.9 microM. The addition of equimolar amounts of AAG to cocaine produced almost complete reversal of cocaine's effects, suggesting a single binding site for cocaine on the AAG molecule. With changes of peak I(Na) normalized against control as 1, cocaine at 20 and 40 microM reduced I(Na) to 0.62+/-0.042 (n = 6) and 0.57+/-0.052 (n = 9), respectively, and the addition of an equimolar concentration of AAG reversed I(Na) to 0.86+/-0.022 and 0.91+/-0.060, respectively. AAG reverses cocaine-induced sodium channel blockade in a dose-dependent manner, indicating a therapeutic potential to reverse acute cocaine cardiac toxicity.

Constraint shapes convergence in tetrodotoxin-resistant sodium channels of snakes.

PubMed

Feldman, Chris R; Brodie, Edmund D; Brodie, Edmund D; Pfrender, Michael E

2012-03-20

Natural selection often produces convergent changes in unrelated lineages, but the degree to which such adaptations occur via predictable genetic paths is unknown. If only a limited subset of possible mutations is fixed in independent lineages, then it is clear that constraint in the production or function of molecular variants is an important determinant of adaptation. We demonstrate remarkably constrained convergence during the evolution of resistance to the lethal poison, tetrodotoxin, in six snake species representing three distinct lineages from around the globe. Resistance-conferring amino acid substitutions in a voltage-gated sodium channel, Na(v)1.4, are clustered in only two regions of the protein, and a majority of the replacements are confined to the same three positions. The observed changes represent only a small fraction of the experimentally validated mutations known to increase Na(v)1.4 resistance to tetrodotoxin. These results suggest that constraints resulting from functional tradeoffs between ion channel function and toxin resistance led to predictable patterns of evolutionary convergence at the molecular level. Our data are consistent with theoretical predictions and recent microcosm work that suggest a predictable path is followed during an adaptive walk along a mutational landscape, and that natural selection may be frequently constrained to produce similar genetic outcomes even when operating on independent lineages.

Steady-state kinetics of solitary batrachotoxin-treated sodium channels. Kinetics on a bounded continuum of polymer conformations.

PubMed Central

Rubinson, K A

1992-01-01

The underlying principles of the kinetics and equilibrium of a solitary sodium channel in the steady state are examined. Both the open and closed kinetics are postulated to result from round-trip excursions from a transition region that separates the openable and closed forms. Exponential behavior of the kinetics can have origins different from small-molecule systems. These differences suggest that the probability density functions (PDFs) that describe the time dependences of the open and closed forms arise from a distribution of rate constants. The distribution is likely to arise from a thermal modulation of the channel structure, and this provides a physical basis for the following three-variable equation: [formula; see text] Here, A0 is a scaling term, k is the mean rate constant, and sigma quantifies the Gaussian spread for the contributions of a range of effective rate constants. The maximum contribution is made by k, with rates faster and slower contributing less. (When sigma, the standard deviation of the spread, goes to zero, then p(f) = A0 e-kt.) The equation is applied to the single-channel steady-state probability density functions for batrachotoxin-treated sodium channels (1986. Keller et al. J. Gen. Physiol. 88: 1-23). The following characteristics are found: (a) The data for both open and closed forms of the channel are fit well with the above equation, which represents a Gaussian distribution of first-order rate processes. (b) The simple relationship [formula; see text] holds for the mean effective rat constants. Or, equivalently stated, the values of P open calculated from the k values closely agree with the P open values found directly from the PDF data. (c) In agreement with the known behavior of voltage-dependent rate constants, the voltage dependences of the mean effective rate constants for the opening and closing of the channel are equal and opposite over the voltage range studied. That is, [formula; see text] "Bursts" are related to the well-known cage effect of solution chemistry. PMID:1312365

Fast- or Slow-inactivated State Preference of Na+ Channel Inhibitors: A Simulation and Experimental Study

PubMed Central

Karoly, Robert; Lenkey, Nora; Juhasz, Andras O.; Vizi, E. Sylvester; Mike, Arpad

2010-01-01

Sodium channels are one of the most intensively studied drug targets. Sodium channel inhibitors (e.g., local anesthetics, anticonvulsants, antiarrhythmics and analgesics) exert their effect by stabilizing an inactivated conformation of the channels. Besides the fast-inactivated conformation, sodium channels have several distinct slow-inactivated conformational states. Stabilization of a slow-inactivated state has been proposed to be advantageous for certain therapeutic applications. Special voltage protocols are used to evoke slow inactivation of sodium channels. It is assumed that efficacy of a drug in these protocols indicates slow-inactivated state preference. We tested this assumption in simulations using four prototypical drug inhibitory mechanisms (fast or slow-inactivated state preference, with either fast or slow binding kinetics) and a kinetic model for sodium channels. Unexpectedly, we found that efficacy in these protocols (e.g., a shift of the “steady-state slow inactivation curve”), was not a reliable indicator of slow-inactivated state preference. Slowly associating fast-inactivated state-preferring drugs were indistinguishable from slow-inactivated state-preferring drugs. On the other hand, fast- and slow-inactivated state-preferring drugs tended to preferentially affect onset and recovery, respectively. The robustness of these observations was verified: i) by performing a Monte Carlo study on the effects of randomly modifying model parameters, ii) by testing the same drugs in a fundamentally different model and iii) by an analysis of the effect of systematically changing drug-specific parameters. In patch clamp electrophysiology experiments we tested five sodium channel inhibitor drugs on native sodium channels of cultured hippocampal neurons. For lidocaine, phenytoin and carbamazepine our data indicate a preference for the fast-inactivated state, while the results for fluoxetine and desipramine are inconclusive. We suggest that conclusions based on voltage protocols that are used to detect slow-inactivated state preference are unreliable and should be re-evaluated. PMID:20585544

Unfolding of a Temperature-Sensitive Domain Controls Voltage-Gated Channel Activation.

PubMed

Arrigoni, Cristina; Rohaim, Ahmed; Shaya, David; Findeisen, Felix; Stein, Richard A; Nurva, Shailika Reddy; Mishra, Smriti; Mchaourab, Hassane S; Minor, Daniel L

2016-02-25

Voltage-gated ion channels (VGICs) are outfitted with diverse cytoplasmic domains that impact function. To examine how such elements may affect VGIC behavior, we addressed how the bacterial voltage-gated sodium channel (BacNa(V)) C-terminal cytoplasmic domain (CTD) affects function. Our studies show that the BacNa(V) CTD exerts a profound influence on gating through a temperature-dependent unfolding transition in a discrete cytoplasmic domain, the neck domain, proximal to the pore. Structural and functional studies establish that the BacNa(V) CTD comprises a bi-partite four-helix bundle that bears an unusual hydrophilic core whose integrity is central to the unfolding mechanism and that couples directly to the channel activation gate. Together, our findings define a general principle for how the widespread four-helix bundle cytoplasmic domain architecture can control VGIC responses, uncover a mechanism underlying the diverse BacNa(V) voltage dependencies, and demonstrate that a discrete domain can encode the temperature-dependent response of a channel. Copyright © 2016 Elsevier Inc. All rights reserved.

Unfolding of a temperature-sensitive domain controls voltage-gated channel activation

PubMed Central

Arrigoni, Cristina; Rohaim, Ahmed; Shaya, David; Findeisen, Felix; Stein, Richard A.; Nurva, Shailika Reddy; Mishra, Smriti; Mchaourab, Hassane S.; Minor, Daniel L.

2016-01-01

Voltage-gated ion channels (VGICs) are outfitted with diverse cytoplasmic domains that impact function. To examine how such elements may affect VGIC behavior, we addressed how the bacterial voltage-gated sodium channel (BacNaV) C-terminal cytoplasmic domain (CTD) affects function. Our studies show that the BacNaV CTD exerts a profound influence on gating through a temperature-dependent unfolding transition in a discrete cytoplasmic domain, the neck domain, proximal to the pore. Structural and functional studies establish that the BacNaV CTD comprises a bi-partite four-helix bundle that bears an unusual hydrophilic core whose integrity is central to the unfolding mechanism and that couples directly to the channel activation gate. Together, our findings define a general principle for how the widespread four-helix bundle cytoplasmic domain architecture can control VGIC responses, uncover a mechanism underlying the diverse BacNaV voltage dependencies, and demonstrate that a discrete domain can encode the temperature dependent response of a channel. PMID:26919429

Benzonatate inhibition of voltage-gated sodium currents.

PubMed

Evans, M Steven; Maglinger, G Benton; Fletcher, Anita M; Johnson, Stephen R

2016-02-01

Benzonatate was FDA-approved in 1958 as an antitussive. Its mechanism of action is thought to be anesthesia of vagal sensory nerve fibers that mediate cough. Vagal sensory neurons highly express the Nav1.7 subtype of voltage-gated sodium channels, and inhibition of this channel inhibits the cough reflex. Local anesthetics inhibit voltage-gated sodium channels, but there are no reports of whether benzonatate affects these channels. Our hypothesis is that benzonatate inhibits Nav1.7 voltage-gated sodium channels. We used whole cell voltage clamp recording to test the effects of benzonatate on voltage-gated sodium (Na(+)) currents in two murine cell lines, catecholamine A differentiated (CAD) cells, which express primarily Nav1.7, and N1E-115, which express primarily Nav1.3. We found that, like local anesthetics, benzonatate strongly and reversibly inhibits voltage-gated Na(+) channels. Benzonatate causes both tonic and phasic inhibition. It has greater effects on channel inactivation than on activation, and its potency is much greater at depolarized potentials, indicating inactivated-state-specific effects. Na(+) currents in CAD cells and N1E-115 cells are similarly affected, indicating that benzonatate is not Na(+) channel subtype-specific. Benzonatate is a mixture of polyethoxy esters of 4-(butylamino) benzoic acid having varying degrees of hydrophobicity. We found that Na(+) currents are inhibited most potently by a benzonatate fraction containing the 9-ethoxy component. Detectable effects of benzonatate occur at concentrations as low as 0.3 μM, which has been reported in humans. We conclude that benzonatate has local anesthetic-like effects on voltage-gated sodium channels, including Nav1.7, which is a possible mechanism for cough suppression by the drug. Copyright © 2015 Elsevier Ltd. All rights reserved.

Validation of a patch clamp screening protocol that simultaneously measures compound activity in multiple states of the voltage-gated sodium channel Nav1.2.

PubMed

Liu, Yi; Beck, Edward J; Flores, Christopher M

2011-12-01

Hyperactivity of voltage-gated sodium channels underlies, at least in part, a range of pathological states, including pain and epilepsy. Selective blockers of these channels may offer effective treatment of such disorders. Currently employed methods to screen for sodium channel blockers, however, are inadequate to rationally identify mechanistically diverse blockers, limiting the potential range of indications that may be treated by such agents. Here, we describe an improved patch clamp screening assay that increases the mechanistic diversity of sodium channel blockers being identified. Using QPatch HT, a medium-throughput, automated patch clamp system, we tested three common sodium channel blockers (phenytoin, lidocaine, and tetrodotoxin) with distinct mechanistic profiles at Nav1.2. The single-voltage protocol employed in this assay simultaneously measured the compound activity in multiple states, including the slow inactivated state, of the channel. A long compound incubation period (10 s) was introduced during channel inactivation to increase the probability of identifying "slow binders." As such, phenytoin, which preferentially binds with slow kinetics to the fast inactivated state, exhibited significantly higher potency than that obtained from a brief exposure (100 ms) used in typical assays. This assay also successfully detected the use-dependent block of tetrodotoxin, a well-documented property of this molecule yet unobserved in typical patch clamp protocols. These results indicate that the assay described here can increase the likelihood of identification and mechanistic diversity of sodium channel blockers from a primary screen. It can also be used to efficiently guide the in vitro optimization of leads that retain the desired mechanistic properties. © MARY ANN LIEBERT, INC.

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

PubMed Central

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

Reduced expression of Na(v)1.6 sodium channels and compensation by Na(v)1.2 channels in mice heterozygous for a null mutation in Scn8a.

PubMed

Vega, Ana V; Henry, Diane L; Matthews, Gary

2008-09-05

The voltage-gated sodium channel alpha subunit Na(v)1.6, encoded by the Scn8a gene, accumulates at high density at mature nodes of Ranvier of myelinated axons, replacing the Na(v)1.2 channels found at nodes earlier in development. To investigate this preferential expression of Na(v)1.6 at adult nodes, we examined isoform-specific expression of sodium channels in mice heterozygous for a null mutation in Scn8a. Immunoblots from these +/- mice had 50% of the wild-type level of Na(v)1.6 protein, and their optic-nerve nodes of Ranvier had correspondingly less anti-Na(v)1.6 immunofluorescence. Protein level and nodal immunofluorescence of the Na(v)1.2 alpha subunit increased in Scn8a(+/-) mice, keeping total sodium channel expression approximately constant despite partial loss of Na(v)1.6 channels. The results are consistent with a model in which Na(v)1.6 and Na(v)1.2 compete for binding partners at sites of high channel density, such as nodes of Ranvier. We suggest that Na(v)1.6 channels normally occupy most of the molecular machinery responsible for channel clustering because they have higher binding affinity, and not because they are exclusively recognized by mechanisms for transport and insertion of sodium channels in myelinated axons. The reduced amount of Na(v)1.6 protein in Scn8a(+/-) mice is apparently insufficient to saturate the nodal binding sites, allowing Na(v)1.2 channels to compete more successfully.

RNAi-mediated knockdown of the voltage gated sodium ion channel TcNav causes mortality in Tribolium castaneum.

PubMed

Abd El Halim, Hesham M; Alshukri, Baida M H; Ahmad, Munawar S; Nakasu, Erich Y T; Awwad, Mohammed H; Salama, Elham M; Gatehouse, Angharad M R; Edwards, Martin G

2016-07-14

The voltage-gated sodium ion channel (VGSC) belongs to the largest superfamily of ion channels. Since VGSCs play key roles in physiological processes they are major targets for effective insecticides. RNA interference (RNAi) is widely used to analyse gene function, but recently, it has shown potential to contribute to novel strategies for selectively controlling agricultural insect pests. The current study evaluates the delivery of dsRNA targeted to the sodium ion channel paralytic A (TcNav) gene in Tribolium castaneum as a viable means of controlling this insect pest. Delivery of TcNav dsRNA caused severe developmental arrest with larval mortalities up to 73% post injection of dsRNA. Injected larvae showed significant (p < 0.05) knockdown in gene expression between 30-60%. Expression was also significantly (p < 0.05) reduced in pupae following injection causing 30% and 42% knockdown for early and late pupal stages, respectively. Oral delivery of dsRNA caused dose-dependant mortalities of between 19 and 51.34%; this was accompanied by significant (p < 0.05) knockdown in gene expression following 3 days of continuous feeding. The majority of larvae injected with, or fed, dsRNA died during the final larval stage prior to pupation. This work provides evidence of a viable RNAi-based strategy for insect control.

Modulation of renal CNG-A3 sodium channel in rats subjected to low- and high-sodium diets.

PubMed

Novaira, Horacio J; Botelho, Bruno F; Goldenberg, Regina C; Guggino, Sandra E; Morales, Marcelo M

2004-10-11

In this work, we studied the mRNA distribution of CNG-A3, an amiloride-sensitive sodium channel that belongs to the cyclic nucleotide-gated (CNG) family of channels, along the rat nephron. The possible involvement of aldosterone in this process was also studied. We also evaluated its expression in rats subjected to diets with different concentrations of sodium or to alterations in aldosterone plasma levels. Total RNA isolated from whole kidney and/or dissected nephron segments of Wistar rats subjected to low- and high-sodium diets, furosemide treatment, adrenalectomy, and adrenalectomy with replacement by aldosterone were analyzed by the use of Western blot, ribonuclease protection assay (RPA) and/or reverse transcription followed by semi-quantitative polymerase chain reaction (RT-PCR). CNG-A3 sodium channel mRNA and protein expression, in whole kidneys of rats subjected to high-Na+ diet, were lower than those in animals given a low-salt diet. Renal CNG-A3 mRNA expression was also decreased in adrenalectomized rats, and was normalized by aldosterone replacement. Moreover, a CNG-A3 mRNA expression study in different nephron segments revealed that aldosterone modulation is present in the cortical thick ascending loop (cTAL) and cortical collecting duct (CCD). This result suggests that CNG-A3 is responsive to the same hormone signaling as the amiloride sensitive sodium channel ENaC and suggests the CNG-A3 may have a physiological role in sodium reabsorption.

Local anesthetics disrupt energetic coupling between the voltage-sensing segments of a sodium channel.

PubMed

Muroi, Yukiko; Chanda, Baron

2009-01-01

Local anesthetics block sodium channels in a state-dependent fashion, binding with higher affinity to open and/or inactivated states. Gating current measurements show that local anesthetics immobilize a fraction of the gating charge, suggesting that the movement of voltage sensors is modified when a local anesthetic binds to the pore of the sodium channel. Here, using voltage clamp fluorescence measurements, we provide a quantitative description of the effect of local anesthetics on the steady-state behavior of the voltage-sensing segments of a sodium channel. Lidocaine and QX-314 shifted the midpoints of the fluorescence-voltage (F-V) curves of S4 domain III in the hyperpolarizing direction by 57 and 65 mV, respectively. A single mutation in the S6 of domain IV (F1579A), a site critical for local anesthetic block, abolished the effect of QX-314 on the voltage sensor of domain III. Both local anesthetics modestly shifted the F-V relationships of S4 domain IV toward hyperpolarized potentials. In contrast, the F-V curve of the S4 domain I was shifted by 11 mV in the depolarizing direction upon QX-314 binding. These antagonistic effects of the local anesthetic indicate that the drug modifies the coupling between the voltage-sensing domains of the sodium channel. Our findings suggest a novel role of local anesthetics in modulating the gating apparatus of the sodium channel.

Pharmacological rescue of trafficking-impaired ATP-sensitive potassium channels

PubMed Central

Martin, Gregory M.; Chen, Pei-Chun; Devaraneni, Prasanna; Shyng, Show-Ling

2013-01-01

ATP-sensitive potassium (KATP) channels link cell metabolism to membrane excitability and are involved in a wide range of physiological processes including hormone secretion, control of vascular tone, and protection of cardiac and neuronal cells against ischemic injuries. In pancreatic β-cells, KATP channels play a key role in glucose-stimulated insulin secretion, and gain or loss of channel function results in neonatal diabetes or congenital hyperinsulinism, respectively. The β-cell KATP channel is formed by co-assembly of four Kir6.2 inwardly rectifying potassium channel subunits encoded by KCNJ11 and four sulfonylurea receptor 1 subunits encoded by ABCC8. Many mutations in ABCC8 or KCNJ11 cause loss of channel function, thus, congenital hyperinsulinism by hampering channel biogenesis and hence trafficking to the cell surface. The trafficking defects caused by a subset of these mutations can be corrected by sulfonylureas, KATP channel antagonists that have long been used to treat type 2 diabetes. More recently, carbamazepine, an anticonvulsant that is thought to target primarily voltage-gated sodium channels has been shown to correct KATP channel trafficking defects. This article reviews studies to date aimed at understanding the mechanisms by which mutations impair channel biogenesis and trafficking and the mechanisms by which pharmacological ligands overcome channel trafficking defects. Insight into channel structure-function relationships and therapeutic implications from these studies are discussed. PMID:24399968

USING ARRAY TECHNOLOGY TO IDENTIFY POTENTIAL BIOMARKERS FOR PYRETHROID INSECTICIDES.

EPA Science Inventory

Pyrethroid insecticides affect nervous system function by disruption of sodium channels in nerve membranes. FQPA requirements for assessing cumulative risk have increased the need for rapid and sensitive biomarkers of effect. This project aims to develop biochemical markers of n...

Anaesthetic Tricaine Acts Preferentially on Neural Voltage-Gated Sodium Channels and Fails to Block Directly Evoked Muscle Contraction

PubMed Central

Attili, Seetharamaiah; Hughes, Simon M.

2014-01-01

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. PMID:25090007

Transportation behavior of alkali ions through a cell membrane ion channel. A quantum chemical description of a simplified isolated model.

PubMed

Billes, Ferenc; Mohammed-Ziegler, Ildikó; Mikosch, Hans

2012-08-01

Quantum chemical model calculations were carried out for modeling the ion transport through an isolated ion channel of a cell membrane. An isolated part of a natural ion channel was modeled. The model channel was a calixarene derivative, hydrated sodium and potassium ions were the models of the transported ion. The electrostatic potential of the channel and the energy of the channel-ion system were calculated as a function of the alkali ion position. Both attractive and repulsive ion-channel interactions were found. The calculations - namely the dependence of the system energy and the atomic charges of the water molecules with respect to the position of the alkali ion in the channel - revealed the molecular-structural background of the potassium selectivity of this artificial ion channel. It was concluded that the studied ion channel mimics real biological ion channel quite well.

Functional Interaction between the Scaffold Protein Kidins220/ARMS and Neuronal Voltage-Gated Na+ Channels.

PubMed

Cesca, Fabrizia; Satapathy, Annyesha; Ferrea, Enrico; Nieus, Thierry; Benfenati, Fabio; Scholz-Starke, Joachim

2015-07-17

Kidins220 (kinase D-interacting substrate of 220 kDa)/ankyrin repeat-rich membrane spanning (ARMS) acts as a signaling platform at the plasma membrane and is implicated in a multitude of neuronal functions, including the control of neuronal activity. Here, we used the Kidins220(-/-) mouse model to study the effects of Kidins220 ablation on neuronal excitability. Multielectrode array recordings showed reduced evoked spiking activity in Kidins220(-/-) hippocampal networks, which was compatible with the increased excitability of GABAergic neurons determined by current-clamp recordings. Spike waveform analysis further indicated an increased sodium conductance in this neuronal subpopulation. Kidins220 association with brain voltage-gated sodium channels was shown by co-immunoprecipitation experiments and Na(+) current recordings in transfected HEK293 cells, which revealed dramatic alterations of kinetics and voltage dependence. Finally, an in silico interneuronal model incorporating the Kidins220-induced Na(+) current alterations reproduced the firing phenotype observed in Kidins220(-/-) neurons. These results identify Kidins220 as a novel modulator of Nav channel activity, broadening our understanding of the molecular mechanisms regulating network excitability. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

Inhibition of Protease-Epithelial Sodium Channel Signaling Improves Mucociliary Function in Cystic Fibrosis Airways.

PubMed

Reihill, James A; Walker, Brian; Hamilton, Robert A; Ferguson, Timothy E G; Elborn, J Stuart; Stutts, M Jackson; Harvey, Brian J; Saint-Criq, Vinciane; Hendrick, Siobhan M; Martin, S Lorraine

2016-09-15

In cystic fibrosis (CF) a reduction in airway surface liquid (ASL) height compromises mucociliary clearance, favoring mucus plugging and chronic bacterial infection. Inhibitors of the epithelial sodium channel (ENaC) have therapeutic potential in CF airways to reduce hyperstimulated sodium and fluid absorption to levels that can restore airway hydration. To determine whether a novel compound (QUB-TL1) designed to inhibit protease/ENaC signaling in CF airways restores ASL volume and mucociliary function. Protease activity was measured using fluorogenic activity assays. Differentiated primary airway epithelial cell cultures (F508del homozygotes) were used to determined ENaC activity (Ussing chamber recordings), ASL height (confocal microscopy), and mucociliary function (by tracking the surface flow of apically applied microbeads). Cell toxicity was measured using a lactate dehydrogenase assay. QUB-TL1 inhibits extracellularly located channel activating proteases (CAPs), including prostasin, matriptase, and furin, the activities of which are observed at excessive levels at the apical surface of CF airway epithelial cells. QUB-TL1-mediated CAP inhibition results in diminished ENaC-mediated Na(+) absorption in CF airway epithelial cells caused by internalization of a prominent pool of cleaved (active) ENaCγ from the cell surface. Importantly, diminished ENaC activity correlates with improved airway hydration status and mucociliary clearance. We further demonstrate QUB-TL1-mediated furin inhibition, which is in contrast to other serine protease inhibitors (camostat mesylate and aprotinin), affords protection against neutrophil elastase-mediated ENaC activation and Pseudomonas aeruginosa exotoxin A-induced cell death. QUB-TL1 corrects aberrant CAP activities, providing a mechanism to delay or prevent the development of CF lung disease in a manner independent of CF transmembrane conductance regulator mutation.

Defective trafficking of cone photoreceptor CNG channels induces the unfolded protein response and ER-stress-associated cell death.

PubMed

Duricka, Deborah L; Brown, R Lane; Varnum, Michael D

2012-01-15

Mutations that perturb the function of photoreceptor CNG (cyclic nucleotide-gated) channels are associated with several human retinal disorders, but the molecular and cellular mechanisms leading to photoreceptor dysfunction and degeneration remain unclear. Many loss-of-function mutations result in intracellular accumulation of CNG channel subunits. Accumulation of proteins in the ER (endoplasmic reticulum) is known to cause ER stress and trigger the UPR (unfolded protein response), an evolutionarily conserved cellular programme that results in either adaptation via increased protein processing capacity or apoptotic cell death. We hypothesize that defective trafficking of cone photoreceptor CNG channels can induce UPR-mediated cell death. To test this idea, CNGA3 subunits bearing the R563H and Q655X mutations were expressed in photoreceptor-derived 661W cells with CNGB3 subunits. Compared with wild-type, R563H and Q655X subunits displayed altered degradation rates and/or were retained in the ER. ER retention was associated with increased expression of UPR-related markers of ER stress and with decreased cell viability. Chemical and pharmacological chaperones {TUDCA (tauroursodeoxycholate sodium salt), 4-PBA (sodium 4-phenylbutyrate) and the cGMP analogue CPT-cGMP [8-(4-chlorophenylthio)-cGMP]} differentially reduced degradation and/or promoted plasma-membrane localization of defective subunits. Improved subunit maturation was concordant with reduced expression of ER-stress markers and improved viability of cells expressing localization-defective channels. These results indicate that ER stress can arise from expression of localization-defective CNG channels, and may represent a contributing factor for photoreceptor degeneration.

SODIUM CHANNELS (NAV1.2/B1) EXPRESSED IN XENOPUS OOCYTES DEMONSTRATE SENSITIVITY TO PYRETHROIDS.

EPA Science Inventory

Voltage-sensitive sodium channels (VSSCs) are hypothesized to be a primary target of pyrethroid insecticides. However, multiple isoforms of VSSCs exist and the sensitivity of different isoforms to pyrethroids has not been well characterized. The Nav1.2/1 channel predominates in a...

Blood pressure and risk of cancer progression - A possible connection with salt and voltage-gated sodium channel.

PubMed

Djamgoz, Mustafa B A

2015-11-01

Although it is well known that high blood pressure promotes cancer, the underlying cause(s) is not well understood. Here, we advance the hypothesis that the extracellular sodium level could be a contributing factor. The hypothesis is based upon emerging evidence showing (i) that voltage-gated sodium channels are expressed de novo in cancer cells and tissues, and (ii) that the influx of sodium from the extracellular medium into cancer cells, mediated by the channel activity, promotes their metastatic potential. Clinical and lifestyle implications of the hypothesis are discussed. Copyright © 2015 Elsevier Ltd. All rights reserved.

Direct evidence for high affinity blockade of NaV1.6 channel subtype by huwentoxin-IV spider peptide, using multiscale functional approaches.

PubMed

Gonçalves, Tânia C; Boukaiba, Rachid; Molgó, Jordi; Amar, Muriel; Partiseti, Michel; Servent, Denis; Benoit, Evelyne

2018-05-01

The Chinese bird spider huwentoxin-IV (HwTx-IV) is well-known to be a highly potent blocker of Na V 1.7 subtype of voltage-gated sodium (Na V ) channels, a genetically validated analgesic target, and thus promising as a potential lead molecule for the development of novel pain therapeutics. In the present study, the interaction between HwTx-IV and Na V 1.6 channel subtype was investigated using multiscale (from in vivo to individual cell) functional approaches. HwTx-IV was approximatively 2 times more efficient than tetrodotoxin (TTX) to inhibit the compound muscle action potential recorded from the mouse skeletal neuromuscular system in vivo, and 30 times more effective to inhibit nerve-evoked than directly-elicited muscle contractile force of isolated mouse hemidiaphragms. These results strongly suggest that the inhibition of nerve-evoked skeletal muscle functioning, produced by HwTx-IV, resulted from a toxin-induced preferential blockade of Na V 1.6, compared to Na V 1.4, channel subtype. This was confirmed by whole-cell automated patch-clamp experiments performed on human embryonic kidney (HEK)-293 cells overexpressing hNa V 1.1-1.8 channel subtypes. HwTx-IV was also approximatively 850 times more efficient to inhibit TTX-sensitive than TTX-resistant sodium currents recorded from mouse dorsal root ganglia neurons. Finally, based on our data, we predict that blockade of the Na V 1.6 channel subtype was involved in the in vivo toxicity of HwTx-IV, although this toxicity was more than 2 times lower than that of TTX. In conclusion, our results provide detailed information regarding the effects of HwTx-IV and allow a better understanding of the side-effect mechanisms involved in vivo and of channel subtype interactions resulting from the toxin activity. Copyright © 2018 Elsevier Ltd. All rights reserved.

Rat epileptic seizures evoked by BmK {alpha}IV and its possible mechanisms involved in sodium channels

DOE Office of Scientific and Technical Information (OSTI.GOV)

Chai Zhifang; Bai Zhantao; Zhang Xuying

2007-05-01

This study showed that rat unilateral intracerebroventricular injection of BmK {alpha}IV, a sodium channel modulator derived from scorpion Buthus martensi Karsch, induced clusters of spikes, epileptic discharges and convulsion-related behavioral changes. BmK {alpha}IV potently promoted the release of endogenous glutamate from rat cerebrocortical synaptosomes. In vitro examination of the effect of BmK {alpha}IV on intrasynaptosomal free calcium concentration [Ca{sup 2+}]{sub i} and sodium concentration [Na{sup +}]{sub i} revealed that BmK {alpha}IV-evoked glutamate release from synaptosomes was associated with an increase in Ca{sup 2+} and Na{sup +} influx. Moreover, BmK {alpha}IV-mediated glutamate release and ion influx was completely blocked by tetrodotoxin,more » a blocker of sodium channel. Together, these results suggest that the induction of BmK {alpha}IV-evoked epileptic seizures may be involved in the modulation of BmK {alpha}IV on tetrodotoxin-sensitive sodium channels located on the nerve terminal, which subsequently enhances the Ca{sup 2+} influx to cause an increase of glutamate release. These findings may provide some insight regarding the mechanism of neuronal action of BmK {alpha}IV in the central nervous system for understanding epileptogenesis involved in sodium channels.« less

Autoradiographic localization of voltage-dependent sodium channels on the mouse neuromuscular junction using /sup 125/I-alpha scorpion toxin. I. Preferential labeling of glial cells on the presynaptic side

DOE Office of Scientific and Technical Information (OSTI.GOV)

Boudier, J.L.; Jover, E.; Cau, P.

1988-05-01

Alpha-scorpion toxins bind specifically to the voltage-sensitive sodium channel in excitable membranes, and binding is potential-dependent. The radioiodinated toxin II from the scorpion Androctonus australis Hector (alpha ScTx) was used to localize voltage-sensitive sodium channels on the presynaptic side of mouse neuromuscular junctions (NMJ) by autoradiography using both light and electron microscopy. Silver grain localization was analyzed by the cross-fire method. At the light-microscopic level, grain density over NMJ appeared 6-8x higher than over nonjunctional muscle membrane. The specificity of labeling was verified by competition/displacement with an excess of native alpha ScTx. Labeling was also inhibited by incubation in depolarizingmore » conditions, showing its potential-dependence. At the electron-microscopic level, analysis showed that voltage-sensitive sodium channels labeled with alpha ScTx were almost exclusively localized on membranes, as expected. Due to washout after incubation, appreciable numbers of binding sites were not found on the postsynaptic membranes. However, on the presynaptic side, alpha ScTx-labeled voltage-sensitive sodium channels were localized on the membrane of non-myelin-forming Schwann cells covering NMJ. The axonal presynaptic membrane was not labeled. These results show that voltage-sensitive sodium channels are present on glial cells in vivo, as already demonstrated in vitro. It is proposed that these glial channels could be indirectly involved in the ionic homeostasis of the axonal environment.« less

Effects of an Environmentally-relevant Mixture of Pyrethroid Insecticides on Spontaneous Activity in Primary Cortical Networks on Microelectrode Arrays

EPA Science Inventory

Pyrethroid insecticides exert their insecticidal and toxicological effects primarily by disrupting voltage-gated sodium channel (VGSC) function, resulting in altered neuronal excitability. Numerous studies of individual pyrethroids have characterized effects on mammalian VGSC fun...

Parallel evolution of tetrodotoxin resistance in three voltage-gated sodium channel genes in the garter snake Thamnophis sirtalis.

PubMed

McGlothlin, Joel W; Chuckalovcak, John P; Janes, Daniel E; Edwards, Scott V; Feldman, Chris R; Brodie, Edmund D; Pfrender, Michael E; Brodie, Edmund D

2014-11-01

Members of a gene family expressed in a single species often experience common selection pressures. Consequently, the molecular basis of complex adaptations may be expected to involve parallel evolutionary changes in multiple paralogs. Here, we use bacterial artificial chromosome library scans to investigate the evolution of the voltage-gated sodium channel (Nav) family in the garter snake Thamnophis sirtalis, a predator of highly toxic Taricha newts. Newts possess tetrodotoxin (TTX), which blocks Nav's, arresting action potentials in nerves and muscle. Some Thamnophis populations have evolved resistance to extremely high levels of TTX. Previous work has identified amino acid sites in the skeletal muscle sodium channel Nav1.4 that confer resistance to TTX and vary across populations. We identify parallel evolution of TTX resistance in two additional Nav paralogs, Nav1.6 and 1.7, which are known to be expressed in the peripheral nervous system and should thus be exposed to ingested TTX. Each paralog contains at least one TTX-resistant substitution identical to a substitution previously identified in Nav1.4. These sites are fixed across populations, suggesting that the resistant peripheral nerves antedate resistant muscle. In contrast, three sodium channels expressed solely in the central nervous system (Nav1.1-1.3) showed no evidence of TTX resistance, consistent with protection from toxins by the blood-brain barrier. We also report the exon-intron structure of six Nav paralogs, the first such analysis for snake genes. Our results demonstrate that the molecular basis of adaptation may be both repeatable across members of a gene family and predictable based on functional considerations. © The Author 2014. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution.

[Effect of nitric oxide on the somatic membrane of rat DRG neurons].

PubMed

Cheng, Hong-Ju; Ma, Ke-Tao; Zhao, Lei; Li, Li; Cao, Ying-Ying; Si, Jun-Qiang

2009-11-01

To observe the role of nitric oxide in dorsal root ganglion (DRG) neurons and its related ionic mechanisms, and explore the function of NO in pain transmission process. In freshly isolated rat DRG samples, using intracellular recording technique, we perfused sodium nitroprusside (NO donor) to observe the role of NO in DRG neurons. In 77.45% of the bath cells, application of sodium nitroprusside (10 -100 mmol/L) induced concentration-dependent membrane hyperpolarization (79/102), and remaining neurons had no response. The membrane conductance increased from control value of (21.06 +/- 1.94) nS to (23.08 +/- 0.92) nS during sodium nitroprusside induced hyperpolarization. L-NAME (1 mmol/L), CdCl2 (0.1 mmol/L) and non-sodium BSS failed to change the amplitude of sodium nitroprusside induced hyperpolarization. When BSS containing 10 mmol/L TEA was used, sodium nitroprusside induced hyperpolarization was obviously inhibited. Sodium nitroprusside could cause concentration-dependent hyperpolarization in DRG neurons by activating K+ channels.

Aldosterone Modulates the Association between NCC and ENaC.

PubMed

Wynne, Brandi M; Mistry, Abinash C; Al-Khalili, Otor; Mallick, Rickta; Theilig, Franziska; Eaton, Douglas C; Hoover, Robert S

2017-06-23

Distal sodium transport is a final step in the regulation of blood pressure. As such, understanding how the two main sodium transport proteins, the thiazide-sensitive sodium chloride cotransporter (NCC) and the epithelial sodium channel (ENaC), are regulated is paramount. Both are expressed in the late distal nephron; however, no evidence has suggested that these two sodium transport proteins interact. Recently, we established that these two sodium transport proteins functionally interact in the second part of the distal nephron (DCT2). Given their co-localization within the DCT2, we hypothesized that NCC and ENaC interactions might be modulated by aldosterone (Aldo). Aldo treatment increased NCC and αENaC colocalization (electron microscopy) and interaction (coimmunoprecipitation). Finally, with co-expression of the Aldo-induced protein serum- and glucocorticoid-inducible kinase 1 (SGK1), NCC and αENaC interactions were increased. These data demonstrate that Aldo promotes increased interaction of NCC and ENaC, within the DCT2 revealing a novel method of regulation for distal sodium reabsorption.

Strong G-Protein-Mediated Inhibition of Sodium Channels.

PubMed

Mattheisen, Glynis B; Tsintsadze, Timur; Smith, Stephen M

2018-05-29

Voltage-gated sodium channels (VGSCs) are strategically positioned to mediate neuronal plasticity because of their influence on action potential waveform. VGSC function may be strongly inhibited by local anesthetic and antiepileptic drugs and modestly modulated via second messenger pathways. Here, we report that the allosteric modulators of the calcium-sensing receptor (CaSR) cinacalcet, calindol, calhex, and NPS 2143 completely inhibit VGSC current in the vast majority of cultured mouse neocortical neurons. This form of VGSC current block persisted in CaSR-deficient neurons, indicating a CaSR-independent mechanism. Cinacalcet-mediated blockade of VGSCs was prevented by the guanosine diphosphate (GDP) analog GDPβs, indicating that G-proteins mediated this effect. Cinacalcet inhibited VGSCs by increasing channel inactivation, and block was reversed by prolonged hyperpolarization. Strong cinacalcet inhibition of VGSC currents was also present in acutely isolated mouse cortical neurons. These data identify a dynamic signaling pathway by which G-proteins regulate VGSC current to indirectly modulate central neuronal excitability. Published by Elsevier Inc.

Channel-transporter complexes: an emerging theme in cell signaling.

PubMed

Abbott, Geoffrey W

2016-11-01

In a recent edition of Biochemical Journal, Mistry et al. described the discovery of a novel protein complex, formed from the epithelial sodium channel (ENaC) and the sodium chloride cotransporter (NCC) [Mistry et al. (2016) Biochem. J. 473, 3237–3252]. The importance of these two proteins in the regulation of salt balance and blood pressure has long been known, as has their overlapping expression in the distal convoluted tubule of the kidney. The new study by Mistry et al. now demonstrates their physical interaction in the kidney and when heterologously co-expressed. Furthermore, the authors demonstrate some degree of functional co-dependence between ENaC and NCC, with pharmacological inhibition of the latter diminishing activity of the former when the two are co-assembled. This novel and potentially important interaction adds to a growing number of recently identified channel-transporter ('chansporter') complexes, which together constitute an emerging theme in cell signaling. © 2016 The Author(s); published by Portland Press Limited on behalf of the Biochemical Society.

Study of diffusion and local structure of sodium-silicate liquid: the molecular dynamic simulation

NASA Astrophysics Data System (ADS)

Hung, Pham Khac; Noritake, Fumiya; San, Luyen Thi; Van, To Ba; Vinh, Le The

2017-10-01

A systematic analysis on sodium-silicate melt with various silica contents was carried out. The simulation revealed two diffusion mechanisms occurred in the melt: the bond-breaking and hopping between sites. The local structure was analyzed through T-simplexes. It was revealed that T-clusters have a non-spherical shape and represent the diffusion channel, in which Na atoms are dominant, but no any O atoms are located. The SiO2-poor melt acquires a long channel. In contrast, the SiO2-rich melt consists of unconnected short channels. The simulation also revealed the immobile and mobile regions which differ in local structure and constituent composition. We propose a new CL-function to characterizing the spatial distribution of different atom component. The spatial distribution of mobile and immobile atoms is found quite different. In particular, the immobile atoms are concentrated in high-density regions possessing very large density of immobile atoms. The spatial distribution of mobile atoms in contrast is more homogeneous.

Molecular Mechanism of Action and Selectivity of Sodium Channel Blocker Insecticides

PubMed Central

Silver, Kristopher; Dong, Ke; Zhorov, Boris S.

2017-01-01

Sodium channel blocker insecticides (SCBIs) are a relatively new class of insecticides that are represented by two commercially registered compounds, indoxacarb and metaflumizone. SCBIs, like pyrethroids and DDT, target voltage-gated sodium channels (VGSCs) to intoxicate insects. In contrast to pyrethroids, however, SCBIs inhibit VGSCs at a distinct receptor site that overlaps those of therapeutic inhibitors of sodium channels, such as local anesthetics, anticonvulsants and antiarrhythmics. This review will recount the development of the SCBI insecticide class from its roots as chitin synthesis inhibitors, discuss the symptoms of poisoning and evidence supporting inhibition of VGSCs as their mechanism of action, describe the current model for SCBI-induced inhibition of VGSCs, present a model for the receptor for SCBIs on VGSCs, and highlight differences between data collected from mammalian and insect experimental models. PMID:27993108

Coupling interactions between voltage sensors of the sodium channel as revealed by site-specific measurements.

PubMed

Chanda, Baron; Asamoah, Osei Kwame; Bezanilla, Francisco

2004-03-01

The voltage-sensing S4 segments in the sodium channel undergo conformational rearrangements in response to changes in the electric field. However, it remains unclear whether these structures move independently or in a coordinated manner. Previously, site-directed fluorescence measurements were shown to track S4 transitions in each of the four domains. Here, using a similar technique, we provide direct evidence of coupling interactions between voltage sensors in the sodium channel. Pairwise interactions between S4s were evaluated by comparing site-specific conformational changes in the presence and absence of a gating perturbation in a distal domain. Reciprocity of effect, a fundamental property of thermodynamically coupled systems, was measured by generating converse mutants. The magnitude of a local gating perturbation induced by a remote S4 mutation depends on the coupling strength and the relative equilibrium positions of the two voltage sensors. In general, our data indicates that the movement of all four voltage sensors in the sodium channel are coupled to a varying extent. Moreover, a gating perturbation in S4-DI has the largest effect on the activation of S4-DIV and vice versa, demonstrating an energetic linkage between S4-DI and S4-DIV. This result suggests a physical mechanism by which the activation and inactivation process may be coupled in voltage-gated sodium channels. In addition, we propose that cooperative interactions between voltage sensors may be the mechanistic basis for the fast activation kinetics of the sodium channel.

Crystal Structure of a Fibroblast Growth Factor Homologous Factor (FHF) Defines a Conserved Surface on FHFs for Binding and Modulation of Voltage-gated Sodium Channels

DOE Office of Scientific and Technical Information (OSTI.GOV)

Goetz, R.; Dover, K; Laezza, F

2009-01-01

Voltage-gated sodium channels (Nav) produce sodium currents that underlie the initiation and propagation of action potentials in nerve and muscle cells. Fibroblast growth factor homologous factors (FHFs) bind to the intracellular C-terminal region of the Nav alpha subunit to modulate fast inactivation of the channel. In this study we solved the crystal structure of a 149-residue-long fragment of human FHF2A which unveils the structural features of the homology core domain of all 10 human FHF isoforms. Through analysis of crystal packing contacts and site-directed mutagenesis experiments we identified a conserved surface on the FHF core domain that mediates channel bindingmore » in vitro and in vivo. Mutations at this channel binding surface impaired the ability of FHFs to co-localize with Navs at the axon initial segment of hippocampal neurons. The mutations also disabled FHF modulation of voltage-dependent fast inactivation of sodium channels in neuronal cells. Based on our data, we propose that FHFs constitute auxiliary subunits for Navs.« less

Arizona bark scorpion venom resistance in the pallid bat, Antrozous pallidus

PubMed Central

Hopp, Bradley H.; Arvidson, Ryan S.; Adams, Michael E.; Razak, Khaleel A.

2017-01-01

The pallid bat (Antrozous pallidus), a gleaning bat found in the western United States and Mexico, hunts a wide variety of ground-dwelling prey, including scorpions. Anecdotal evidence suggests that the pallid bat is resistant to scorpion venom, but no systematic study has been performed. Here we show with behavioral measures and direct injection of venom that the pallid bat is resistant to venom of the Arizona bark scorpion, Centruroides sculpturatus. Our results show that the pallid bat is stung multiple times during a hunt without any noticeable effect on behavior. In addition, direct injection of venom at mouse LD50 concentrations (1.5 mg/kg) has no effect on bat behavior. At the highest concentration tested (10 mg/kg), three out of four bats showed no effects. One of the four bats showed a transient effect suggesting that additional studies are required to identify potential regional variation in venom tolerance. Scorpion venom is a cocktail of toxins, some of which activate voltage-gated sodium ion channels, causing intense pain. Dorsal root ganglia (DRG) contain nociceptive neurons and are principal targets of scorpion venom toxins. To understand if mutations in specific ion channels contribute to venom resistance, a pallid bat DRG transcriptome was generated. As sodium channels are a major target of scorpion venom, we identified amino acid substitutions present in the pallid bat that may lead to venom resistance. Some of these substitutions are similar to corresponding amino acids in sodium channel isoforms responsible for reduced venom binding activity. The substitution found previously in the grasshopper mouse providing venom resistance to the bark scorpion is not present in the pallid bat, indicating a potentially novel mechanism for venom resistance in the bat that remains to be identified. Taken together, these results indicate that the pallid bat is resistant to venom of the bark scorpion and altered sodium ion channel function may partly underlie such resistance. PMID:28854259

Arizona bark scorpion venom resistance in the pallid bat, Antrozous pallidus.

PubMed

Hopp, Bradley H; Arvidson, Ryan S; Adams, Michael E; Razak, Khaleel A

2017-01-01

The pallid bat (Antrozous pallidus), a gleaning bat found in the western United States and Mexico, hunts a wide variety of ground-dwelling prey, including scorpions. Anecdotal evidence suggests that the pallid bat is resistant to scorpion venom, but no systematic study has been performed. Here we show with behavioral measures and direct injection of venom that the pallid bat is resistant to venom of the Arizona bark scorpion, Centruroides sculpturatus. Our results show that the pallid bat is stung multiple times during a hunt without any noticeable effect on behavior. In addition, direct injection of venom at mouse LD50 concentrations (1.5 mg/kg) has no effect on bat behavior. At the highest concentration tested (10 mg/kg), three out of four bats showed no effects. One of the four bats showed a transient effect suggesting that additional studies are required to identify potential regional variation in venom tolerance. Scorpion venom is a cocktail of toxins, some of which activate voltage-gated sodium ion channels, causing intense pain. Dorsal root ganglia (DRG) contain nociceptive neurons and are principal targets of scorpion venom toxins. To understand if mutations in specific ion channels contribute to venom resistance, a pallid bat DRG transcriptome was generated. As sodium channels are a major target of scorpion venom, we identified amino acid substitutions present in the pallid bat that may lead to venom resistance. Some of these substitutions are similar to corresponding amino acids in sodium channel isoforms responsible for reduced venom binding activity. The substitution found previously in the grasshopper mouse providing venom resistance to the bark scorpion is not present in the pallid bat, indicating a potentially novel mechanism for venom resistance in the bat that remains to be identified. Taken together, these results indicate that the pallid bat is resistant to venom of the bark scorpion and altered sodium ion channel function may partly underlie such resistance.

Local anesthetic and antiepileptic drug access and binding to a bacterial voltage-gated sodium channel

PubMed Central

Boiteux, Céline; Vorobyov, Igor; French, Robert J.; French, Christopher; Yarov-Yarovoy, Vladimir; Allen, Toby W.

2014-01-01

Voltage-gated sodium (Nav) channels are important targets in the treatment of a range of pathologies. Bacterial channels, for which crystal structures have been solved, exhibit modulation by local anesthetic and anti-epileptic agents, allowing molecular-level investigations into sodium channel-drug interactions. These structures reveal no basis for the “hinged lid”-based fast inactivation, seen in eukaryotic Nav channels. Thus, they enable examination of potential mechanisms of use- or state-dependent drug action based on activation gating, or slower pore-based inactivation processes. Multimicrosecond simulations of NavAb reveal high-affinity binding of benzocaine to F203 that is a surrogate for FS6, conserved in helix S6 of Domain IV of mammalian sodium channels, as well as low-affinity sites suggested to stabilize different states of the channel. Phenytoin exhibits a different binding distribution owing to preferential interactions at the membrane and water–protein interfaces. Two drug-access pathways into the pore are observed: via lateral fenestrations connecting to the membrane lipid phase, as well as via an aqueous pathway through the intracellular activation gate, despite being closed. These observations provide insight into drug modulation that will guide further developments of Nav inhibitors. PMID:25136136

Properties of an intermediate-duration inactivation process of the voltage-gated sodium conductance in rat hippocampal CA1 neurons.

PubMed

French, Christopher R; Zeng, Zhen; Williams, David A; Hill-Yardin, Elisa L; O'Brien, Terence J

2016-02-01

Rapid transmembrane flow of sodium ions produces the depolarizing phase of action potentials (APs) in most excitable tissue through voltage-gated sodium channels (NaV). Macroscopic currents display rapid activation followed by fast inactivation (IF) within milliseconds. Slow inactivation (IS) has been subsequently observed in several preparations including neuronal tissues. IS serves important physiological functions, but the kinetic properties are incompletely characterized, especially the operative timescales. Here we present evidence for an "intermediate inactivation" (II) process in rat hippocampal CA1 neurons with time constants of the order of 100 ms. The half-inactivation potentials (V0.5) of steady-state inactivation curves were hyperpolarized by increasing conditioning pulse duration from 50 to 500 ms and could be described by a sum of Boltzmann relations. II state transitions were observed after opening as well as subthreshold potentials. Entry into II after opening was relatively insensitive to membrane potential, and recovery of II became more rapid at hyperpolarized potentials. Removal of fast inactivation with cytoplasmic papaine revealed time constants of INa decay corresponding to II and IS with long depolarizations. Dynamic clamp revealed attenuation of trains of APs over the 10(2)-ms timescale, suggesting a functional role of II in repetitive firing accommodation. These experimental findings could be reproduced with a five-state Markov model. It is likely that II affects important aspects of hippocampal neuron response and may provide a drug target for sodium channel modulation. Copyright © 2016 the American Physiological Society.

Robustness, Death of Spiral Wave in the Network of Neurons under Partial Ion Channel Block

NASA Astrophysics Data System (ADS)

Ma, Jun; Huang, Long; Wang, Chun-Ni; Pu, Zhong-Sheng

2013-02-01

The development of spiral wave in a two-dimensional square array due to partial ion channel block (Potassium, Sodium) is investigated, the dynamics of the node is described by Hodgkin—Huxley neuron and these neurons are coupled with nearest neighbor connection. The parameter ratio xNa (and xK), which defines the ratio of working ion channel number of sodium (potassium) to the total ion channel number of sodium (and potassium), is used to measure the shift conductance induced by channel block. The distribution of statistical variable R in the two-parameter phase space (parameter ratio vs. poisoning area) is extensively calculated to mark the parameter region for transition of spiral wave induced by partial ion channel block, the area with smaller factors of synchronization R is associated the parameter region that spiral wave keeps alive and robust to the channel poisoning. Spiral wave keeps alive when the poisoned area (potassium or sodium) and degree of intoxication are small, distinct transition (death, several spiral waves coexist or multi-arm spiral wave emergence) occurs under moderate ratio xNa (and xK) when the size of blocked area exceeds certain thresholds. Breakup of spiral wave occurs and multi-arm of spiral waves are observed when the channel noise is considered.

Transcriptomic Analysis of Ciguatoxin-Induced Changes in Gene Expression in Primary Cultures of Mice Cortical Neurons.

PubMed

Rubiolo, Juan Andrés; Vale, Carmen; Boente-Juncal, Andrea; Hirama, Masahiro; Yamashita, Shuji; Camiña, Mercedes; Vieytes, Mercedes R; Botana, Luis M

2018-05-10

Ciguatoxins are polyether marine toxins that act as sodium channel activators. These toxins cause ciguatera, one of the most widespread nonbacterial forms of food poisoning, which presents several symptoms in humans including long-term neurological alterations. Earlier work has shown that both acute and chronic exposure of primary cortical neurons to synthetic ciguatoxin CTX3C have profound impacts on neuronal function. Thus, the present work aimed to identify relevant neuronal genes and metabolic pathways that could be altered by ciguatoxin exposure. To study the effect of ciguatoxins in primary neurons in culture, we performed a transcriptomic analysis using whole mouse genome microarrays, for primary cortical neurons exposed during 6, 24, or 72 h in culture to CTX3C. Here, we have shown that the effects of the toxin on gene expression differ with the exposure time. The results presented here have identified several relevant genes and pathways related to the effect of ciguatoxins on neurons and may assist in future research or even treatment of ciguatera. Moreover, we demonstrated that the effects of the toxin on gene expression were exclusively consequential of its action as a voltage-gated sodium channel activator, since all the effects of CTX3C were avoided by preincubation of the neurons with the sodium channel blocker tetrodotoxin.

Hyperforin--a key constituent of St. John's wort specifically activates TRPC6 channels.

PubMed

Leuner, Kristina; Kazanski, Victor; Müller, Margarethe; Essin, Kirill; Henke, Bettina; Gollasch, Maik; Harteneck, Christian; Müller, Walter E

2007-12-01

Hyperforin, a bicyclic polyprenylated acylphloroglucinol derivative, is the main active principle of St. John's wort extract responsible for its antidepressive profile. Hyperforin inhibits the neuronal serotonin and norepinephrine uptake comparable to synthetic antidepressants. In contrast to synthetic antidepressants directly blocking neuronal amine uptake, hyperforin increases synaptic serotonin and norepinephrine concentrations by an indirect and yet unknown mechanism. Our attempts to identify the molecular target of hyperforin resulted in the identification of TRPC6. Hyperforin induced sodium and calcium entry as well as currents in TRPC6-expressing cells. Sodium currents and the subsequent breakdown of the membrane sodium gradients may be the rationale for the inhibition of neuronal amine uptake. The hyperforin-induced cation entry was highly specific and related to TRPC6 and was suppressed in cells expressing a dominant negative mutant of TRPC6, whereas phylogenetically related channels, i.e., TRPC3 remained unaffected. Furthermore, hyperforin induces neuronal axonal sprouting like nerve growth factor in a TRPC6-dependent manner. These findings support the role of TRPC channels in neurite extension and identify hyperforin as the first selective pharmacological tool to study TRPC6 function. Hyperforin integrates inhibition of neurotransmitter uptake and neurotrophic property by specific activation of TRPC6 and represents an interesting lead-structure for a new class of antidepressants.

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

PubMed

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

2016-07-21

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.

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

NASA Astrophysics Data System (ADS)

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.

A remarkably stable TipE gene cluster: evolution of insect Para sodium channel auxiliary subunits

PubMed Central

2011-01-01

Background First identified in fruit flies with temperature-sensitive paralysis phenotypes, the Drosophila melanogaster TipE locus encodes four voltage-gated sodium (NaV) channel auxiliary subunits. This cluster of TipE-like genes on chromosome 3L, and a fifth family member on chromosome 3R, are important for the optional expression and functionality of the Para NaV channel but appear quite distinct from auxiliary subunits in vertebrates. Here, we exploited available arthropod genomic resources to trace the origin of TipE-like genes by mapping their evolutionary histories and examining their genomic architectures. Results We identified a remarkably conserved synteny block of TipE-like orthologues with well-maintained local gene arrangements from 21 insect species. Homologues in the water flea, Daphnia pulex, suggest an ancestral pancrustacean repertoire of four TipE-like genes; a subsequent gene duplication may have generated functional redundancy allowing gene losses in the silk moth and mosquitoes. Intronic nesting of the insect TipE gene cluster probably occurred following the divergence from crustaceans, but in the flour beetle and silk moth genomes the clusters apparently escaped from nesting. Across Pancrustacea, TipE gene family members have experienced intronic nesting, escape from nesting, retrotransposition, translocation, and gene loss events while generally maintaining their local gene neighbourhoods. D. melanogaster TipE-like genes exhibit coordinated spatial and temporal regulation of expression distinct from their host gene but well-correlated with their regulatory target, the Para NaV channel, suggesting that functional constraints may preserve the TipE gene cluster. We identified homology between TipE-like NaV channel regulators and vertebrate Slo-beta auxiliary subunits of big-conductance calcium-activated potassium (BKCa) channels, which suggests that ion channel regulatory partners have evolved distinct lineage-specific characteristics. Conclusions TipE-like genes form a remarkably conserved genomic cluster across all examined insect genomes. This study reveals likely structural and functional constraints on the genomic evolution of insect TipE gene family members maintained in synteny over hundreds of millions of years of evolution. The likely common origin of these NaV channel regulators with BKCa auxiliary subunits highlights the evolutionary plasticity of ion channel regulatory mechanisms. PMID:22098672

Vitex negundo induces an anticonvulsant effect by inhibiting voltage gated sodium channels in murine Neuro 2A cell line.

PubMed

Khan, Faisal; Saify, Zafar Saeed; Jamali, Khawar Saeed; Naz, Saima; Hassan, Sohail; Siddiqui, Sonia

2018-01-01

Vitex negundo (Vn) extract is famous for the treatment of neurological diseases such as migraine and epilepsy. These neurological diseases have been associated with abnormally increased influx of sodium ions into the neurons. Drugs that inhibit voltage gated sodium channels can be used as potent anti-epileptics. Till now, the effects of Vn on sodium channels have not been investigated. Therefore, we have investigated the effects of methalonic fraction of Vn extract in Murine Neuro 2A cell line. Cells were cultured in a defined medium with or without the Vn extract (100 μg/ml). Sodium currents were recorded using whole-cell patch clamp method. The data show that methanolic extract of Vn inhibited sodium currents in a dose dependent manner (IC50 =161μg/ml). Vn (100 μg/ml) shifted the steady-state inactivation curve to the left or towards the hyper polarization state. However, Vn did not show any effects on outward rectifying potassium currents. Moreover, Vn (100 μg/ml) significantly reduced the sustained repetitive (48±4.8%, P<0.01) firing from neonatal hippocampal neurons at 12 DIV. Hence, our data suggested that inhibition of sodium channels by Vn may exert pharmacological effects in reducing pain and convulsions.

Multiple pore conformations driven by asynchronous movements of voltage sensors in a eukaryotic sodium channel

PubMed Central

Goldschen-Ohm, Marcel P.; Capes, Deborah L.; Oelstrom, Kevin M.; Chanda, Baron

2013-01-01

Voltage-dependent Na+ channels are crucial for electrical signalling in excitable cells. Membrane depolarization initiates asynchronous movements in four non-identical voltage-sensing domains of the Na+ channel. It remains unclear to what extent this structural asymmetry influences pore gating as compared with outwardly rectifying K+ channels, where channel opening results from a final concerted transition of symmetric pore gates. Here we combine single channel recordings, cysteine accessibility and voltage clamp fluorimetry to probe the relationships between voltage sensors and pore conformations in an inactivation deficient Nav1.4 channel. We observe three distinct conductance levels such that DI-III voltage sensor activation is kinetically correlated with formation of a fully open pore, whereas DIV voltage sensor movement underlies formation of a distinct subconducting pore conformation preceding inactivation in wild-type channels. Our experiments reveal that pore gating in sodium channels involves multiple transitions driven by asynchronous movements of voltage sensors. These findings shed new light on the mechanism of coupling between activation and fast inactivation in voltage-gated sodium channels. PMID:23322038

Modeling and Validation of Sodium Plugging for Heat Exchangers in Sodium-cooled Fast Reactor Systems

DOE Office of Scientific and Technical Information (OSTI.GOV)

Ferroni, Paolo; Tatli, Emre; Czerniak, Luke

The project “Modeling and Validation of Sodium Plugging for Heat Exchangers in Sodium-cooled Fast Reactor Systems” was conducted jointly by Westinghouse Electric Company (Westinghouse) and Argonne National Laboratory (ANL), over the period October 1, 2013- March 31, 2016. The project’s motivation was the need to provide designers of Sodium Fast Reactors (SFRs) with a validated, state-of-the-art computational tool for the prediction of sodium oxide (Na 2O) deposition in small-diameter sodium heat exchanger (HX) channels, such as those in the diffusion bonded HXs proposed for SFRs coupled with a supercritical CO 2 (sCO 2) Brayton cycle power conversion system. In SFRs,more » Na 2O deposition can potentially occur following accidental air ingress in the intermediate heat transport system (IHTS) sodium and simultaneous failure of the IHTS sodium cold trap. In this scenario, oxygen can travel through the IHTS loop and reach the coldest regions, represented by the cold end of the sodium channels of the HXs, where Na 2O precipitation may initiate and continue. In addition to deteriorating HX heat transfer and pressure drop performance, Na 2O deposition can lead to channel plugging especially when the size of the sodium channels is small, which is the case for diffusion bonded HXs whose sodium channel hydraulic diameter is generally below 5 mm. Sodium oxide melts at a high temperature well above the sodium melting temperature such that removal of a solid plug such as through dissolution by pure sodium could take a lengthy time. The Sodium Plugging Phenomena Loop (SPPL) was developed at ANL, prior to this project, for investigating Na 2O deposition phenomena within sodium channels that are prototypical of the diffusion bonded HX channels envisioned for SFR-sCO 2 systems. In this project, a Computational Fluid Dynamic (CFD) model capable of simulating the thermal-hydraulics of the SPPL test section and provided with Na 2O deposition prediction capabilities, was developed. This state-of-the-art computational tool incorporates a first-principles Na 2O deposition model developed by ANL, and combines it with predictive capabilities for the spatial and temporal variation of temperature, velocity, dissolved oxygen concentration, and wall temperature under flowing sodium conditions. The CFD model was validated under no-deposition conditions using experimental data collected with the SPPL, demonstrating the model’s capability to predict the thermal-hydraulics of the SPPL test section within the measurement uncertainty characterizing the SPPL instrumentation. The model’s deposition prediction capability was not, however, validated as the SPPL could not be operated under plugging conditions during the project, resulting in the lack of deposition data with adequate pedigree for a CFD model validation. Two novel diagnostic techniques to detect and characterize Na 2O deposits, i.e. Ultrasonic Time Domain Reflectometry (UTDR) and Potential Drop (PD) techniques, were developed to ultimately assist in the validation effort under plugging conditions, which can be performed once the SPPL becomes operational. This development effort consisted first in demonstrating, analytically and/or computationally, the capability of these techniques to diagnose Na 2O deposits inside of small channels (particularly the deposit’s thickness), and subsequently in the fabrication and testing of prototypical UTDR and PD instrumentation. The testing, performed on mockups of the SPPL test section, demonstrated the capability of these techniques to detect and characterize material discontinuities like those induced by sodium oxide deposition on stainless steel channel walls. Because of the mentioned impossibility to run the SPPL in a plugging mode, the developed instrumentation could not be tested in-situ, i.e. at the SPPL while deposits are being formed inside of the SPPL test section. Recommended future work includes a possible enhancement in the CFD modeling technique and installation of the developed UTDR and PD instrumentation on the test section, followed by plugging tests to be conducted with the SPPL. The installation of the UTDR and PD diagnostic instrumentation on the SPPL test section will allow collection of Na 2O deposition data after the onset of deposition to nearly complete channel plugging, which can ultimately be used for the validation of the CFD model.« less

Comparison of electrophysiological data from human-induced pluripotent stem cell-derived cardiomyocytes to functional preclinical safety assays.

PubMed

Harris, Kate; Aylott, Mike; Cui, Yi; Louttit, James B; McMahon, Nicholas C; Sridhar, Arun

2013-08-01

Human-induced pluripotent stem cell cardiomyocytes (hiPSC-CMs) are a potential source to develop assays for predictive electrophysiological safety screening. Published studies show that the relevant physiology and pharmacology exist but does not show the translation between stem cell cardiomyocyte assays and other preclinical safety screening assays, which is crucial for drug discovery and safety scientists and the regulators. Our studies are the first to show the pharmacology of ion channel blockade and compare them with existing functional cardiac electrophysiology studies. Ten compounds (a mixture of pure hERG [E-4031 and Cisapride], hERG and sodium [Flecainide, Mexiletine, Quinidine, and Terfenadine], calcium channel blockers [Nifedipine and Verapamil], and two proprietary compounds [GSK A and B]) were tested, and results from hiPSC-CMs studied on multielectrode arrays (MEA) were compared with other preclincial models and clinical drug concentrations and effects using integrated risk assessment plots. All ion channel blockers produced (1) functional effects on repolarization and depolarization around the IC25 and IC50 values and (2) excessive blockade of hERG and/or blockade of sodium current precipitated arrhythmias. Our MEA data show that hiPSC-CMs demonstrate relevant pharmacology and show excellent correlations to current functional cardiac electrophysiological studies. Based on these results, MEA assays using iPSC-CMs offer a reliable, cost effective, and surrogate to preclinical in vitro testing, in addition to the 3Rs (refine, reduce, and replace animals in research) benefit.

Ion channels in inflammation.

PubMed

Eisenhut, Michael; Wallace, Helen

2011-04-01

Most physical illness in vertebrates involves inflammation. Inflammation causes disease by fluid shifts across cell membranes and cell layers, changes in muscle function and generation of pain. These disease processes can be explained by changes in numbers or function of ion channels. Changes in ion channels have been detected in diarrhoeal illnesses, pyelonephritis, allergy, acute lung injury and systemic inflammatory response syndromes involving septic shock. The key role played by changes in ion transport is directly evident in inflammation-induced pain. Expression or function of all major categories of ion channels like sodium, chloride, calcium, potassium, transient receptor potential, purinergic receptor and acid-sensing ion channels can be influenced by cyto- and chemokines, prostaglandins, leukotrienes, histamine, ATP, reactive oxygen species and protons released in inflammation. Key pathways in this interaction are cyclic nucleotide, phosphoinositide and mitogen-activated protein kinase-mediated signalling, direct modification by reactive oxygen species like nitric oxide, ATP or protons and disruption of the cytoskeleton. Therapeutic interventions to modulate the adverse and overlapping effects of the numerous different inflammatory mediators on each ion transport system need to target adversely affected ion transport systems directly and locally.

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

PubMed Central

Tikhonov, Denis B.

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, 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. PMID:28258204

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

PubMed

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.

Voltage-gated sodium channels in taste bud cells.

PubMed

Gao, Na; Lu, Min; Echeverri, Fernando; Laita, Bianca; Kalabat, Dalia; Williams, Mark E; Hevezi, Peter; Zlotnik, Albert; Moyer, Bryan D

2009-03-12

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. 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. 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.

Design, synthesis and structure-activity relationship of indoxacarb analogs as voltage-gated sodium channel blocker.

PubMed

Hao, Wenbo; Fu, Chunling; Yu, Huijuan; Chen, Jian; Xu, Hanhong; Shao, Guang; Jiang, Dingxin

2015-10-15

Indoxacarb, the first commercialized pyrazoline-type sodium-channel blocker, is a commonly used insecticide because of high selectivity. To discover sodium-channel blocker with high insecticidal activity, a series of novel indoxacarb analogs were designed and synthesized by judicious structural modifications of the substituent group of C5, C6 in indenone and C'4 in benzene ring. Some analogs exhibited significant insecticidal activities against Spodoptera litura F. and excellent BgNav1-1a channel inhibitory activity. The structure-activity analysis indicated that the presence of strong electron-withdrawing group and decreased steric hindrance of indenone ring (R(1), R(2)) in 5- and 6-position could enhance larvicidal activity and BgNav1-1a channel inhibitory activity. Copyright © 2015 Elsevier Ltd. All rights reserved.

Molecular Basis of Paraltyic Neurotoxin Action on Voltage-Sensitive Sodium Channels

DTIC Science & Technology

1985-10-14

of 9,700 daltons isolated from the coral Goni2oora gy. (1). The toxin enhances neurally mediated contraction of blood vessels and taenia coli of the...sites on the solium channel and to identify the site of GPT action within the structure of the sodium channel protein. 2. Site of Action of Brvyetoxin

Locating the route of entry and binding sites of benzocaine and phenytoin in a bacterial voltage gated sodium channel.

PubMed

Martin, Lewis J; Corry, Ben

2014-07-01

Sodium channel blockers are used to control electrical excitability in cells as a treatment for epileptic seizures and cardiac arrhythmia, and to provide short term control of pain. Development of the next generation of drugs that can selectively target one of the nine types of voltage-gated sodium channel expressed in the body requires a much better understanding of how current channel blockers work. Here we make use of the recently determined crystal structure of the bacterial voltage gated sodium channel NavAb in molecular dynamics simulations to elucidate the position at which the sodium channel blocking drugs benzocaine and phenytoin bind to the protein as well as to understand how these drugs find their way into resting channels. We show that both drugs have two likely binding sites in the pore characterised by nonspecific, hydrophobic interactions: one just above the activation gate, and one at the entrance to the the lateral lipid filled fenestrations. Three independent methods find the same sites and all suggest that binding to the activation gate is slightly more favourable than at the fenestration. Both drugs are found to be able to pass through the fenestrations into the lipid with only small energy barriers, suggesting that this can represent the long posited hydrophobic entrance route for neutral drugs. Our simulations highlight the importance of a number of residues in directing drugs into and through the fenestration, and in forming the drug binding sites.

Locating the Route of Entry and Binding Sites of Benzocaine and Phenytoin in a Bacterial Voltage Gated Sodium Channel

PubMed Central

Martin, Lewis J.; Corry, Ben

2014-01-01

Sodium channel blockers are used to control electrical excitability in cells as a treatment for epileptic seizures and cardiac arrhythmia, and to provide short term control of pain. Development of the next generation of drugs that can selectively target one of the nine types of voltage-gated sodium channel expressed in the body requires a much better understanding of how current channel blockers work. Here we make use of the recently determined crystal structure of the bacterial voltage gated sodium channel NavAb in molecular dynamics simulations to elucidate the position at which the sodium channel blocking drugs benzocaine and phenytoin bind to the protein as well as to understand how these drugs find their way into resting channels. We show that both drugs have two likely binding sites in the pore characterised by nonspecific, hydrophobic interactions: one just above the activation gate, and one at the entrance to the the lateral lipid filled fenestrations. Three independent methods find the same sites and all suggest that binding to the activation gate is slightly more favourable than at the fenestration. Both drugs are found to be able to pass through the fenestrations into the lipid with only small energy barriers, suggesting that this can represent the long posited hydrophobic entrance route for neutral drugs. Our simulations highlight the importance of a number of residues in directing drugs into and through the fenestration, and in forming the drug binding sites. PMID:24992293

Structure of a prokaryotic sodium channel pore reveals essential gating elements and an outer ion binding site common to eukaryotic channels

PubMed Central

Shaya, David; Findeisen, Felix; Abderemane-Ali, Fayal; Arrigoni, Cristina; Wong, Stephanie; Nurva, Shailika Reddy; Loussouarn, Gildas; Minor, Daniel L.

2013-01-01

Voltage-gated sodium channels (NaVs) are central elements of cellular excitation. Notwithstanding advances from recent bacterial NaV (BacNaV) structures, key questions about gating and ion selectivity remain. Here, we present a closed conformation of NaVAe1p, a pore-only BacNaV derived from NaVAe1, a BacNaV from the arsenite oxidizer Alkalilimnicola ehrlichei found in Mono Lake, California, that provides insight into both fundamental properties. The structure reveals a pore domain in which the pore-lining S6 helix connects to a helical cytoplasmic tail. Electrophysiological studies of full-length BacNaVs show that two elements defined by the NaVAe1p structure, an S6 activation gate position and the cytoplasmic tail ‘neck’, are central to BacNaV gating. The structure also reveals the selectivity filter ion entry site, termed the ‘outer ion’ site. Comparison with mammalian voltage-gated calcium channel (CaV) selectivity filters, together with functional studies shows that this site forms a previously unknown determinant of CaV high affinity calcium binding. Our findings underscore commonalities between BacNaVs and eukaryotic voltage-gated channels and provide a framework for understanding gating and ion permeation in this superfamily. PMID:24120938

Effects of 4-phenyl butyric acid on high glucose-induced alterations in dorsal root ganglion neurons.

PubMed

Sharma, Dilip; Singh, Jitendra Narain; Sharma, Shyam S

2016-12-02

Mechanisms and pathways involving in diabetic neuropathy are still not fully understood but can be unified by the process of overproduction of reactive oxygen species (ROS) such as superoxide, endoplasmic reticulum (ER) stress, downstream intracellular signaling pathways and their modulation. Susceptibility of dorsal root ganglion (DRG) to internal/external hyperglycemic environment stress contributes to the pathogenesis and progression of diabetic neuropathy. ER stress leads to abnormal ion channel function, gene expression, transcriptional regulation, metabolism and protein folding. 4-phenyl butyric acid (4-PBA) is a potent and selective chemical chaperone; which may inhibit ER stress. It may be hypothesized that 4-PBA could attenuate via channels in DRG in diabetic neuropathy. Effects of 4-PBA were determined by applying different parameters of oxidative stress, cell viability, apoptosis assays and channel expression in cultured DRG neurons. Hyperglycemia-induced apoptosis in the DRG neuron was inhibited by 4-PBA. Cell viability of DRG neurons was not altered by 4-PBA. Oxidative stress was significantly blocked by the 4-PBA. Sodium channel expression was not altered by the 4-PBA. Our data provide evidence that the hyperglycemia-induced alteration may be reduced by the 4-PBA without altering the sodium channel expression. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

Folding similarity of the outer pore region in prokaryotic and eukaryotic sodium channels revealed by docking of conotoxins GIIIA, PIIIA, and KIIIA in a NavAb-based model of Nav1.4

PubMed Central

Korkosh, Viacheslav S.; Zhorov, Boris S.

2014-01-01

Voltage-gated sodium channels are targets for many drugs and toxins. However, the rational design of medically relevant channel modulators is hampered by the lack of x-ray structures of eukaryotic channels. Here, we used a homology model based on the x-ray structure of the NavAb prokaryotic sodium channel together with published experimental data to analyze interactions of the μ-conotoxins GIIIA, PIIIA, and KIIIA with the Nav1.4 eukaryotic channel. Using Monte Carlo energy minimizations and published experimentally defined pairwise contacts as distance constraints, we developed a model in which specific contacts between GIIIA and Nav1.4 were readily reproduced without deformation of the channel or toxin backbones. Computed energies of specific interactions between individual residues of GIIIA and the channel correlated with experimental estimates. The predicted complexes of PIIIA and KIIIA with Nav1.4 are consistent with a large body of experimental data. In particular, a model of Nav1.4 interactions with KIIIA and tetrodotoxin (TTX) indicated that TTX can pass between Nav1.4 and channel-bound KIIIA to reach its binding site at the selectivity filter. Our models also allowed us to explain experimental data that currently lack structural interpretations. For instance, consistent with the incomplete block observed with KIIIA and some GIIIA and PIIIA mutants, our computations predict an uninterrupted pathway for sodium ions between the extracellular space and the selectivity filter if at least one of the four outer carboxylates is not bound to the toxin. We found a good correlation between computational and experimental data on complete and incomplete channel block by native and mutant toxins. Thus, our study suggests similar folding of the outer pore region in eukaryotic and prokaryotic sodium channels. PMID:25156117

Dendritic sodium channels promote active decorrelation and reduce phase locking to parkinsonian input oscillations in model globus pallidus neurons

PubMed Central

Edgerton, Jeremy R.; Jaeger, Dieter

2011-01-01

Correlated firing among populations of neurons is present throughout the brain and is often rhythmic in nature, observable as an oscillatory fluctuation in the local field potential. Although rhythmic population activity is believed to be critical for normal function in many brain areas, synchronized neural oscillations are associated with disease states in other cases. In the globus pallidus (GP in rodents, homolog of the primate GPe), pairs of neurons generally have uncorrelated firing in normal animals despite an anatomical organization suggesting that they should receive substantial common input. By contrast, correlated and rhythmic GP firing is observed in animal models of Parkinson's disease (PD). Based in part on these findings it has been proposed that an important part of basal ganglia function is active decorrelation, whereby redundant information is compressed. Mechanisms that implement active decorrelation, and changes that cause it to fail in PD, are subjects of great interest. Rat GP neurons express fast, transient voltage-dependent sodium channels (NaF channels) in their dendrites, with the expression level being highest near asymmetric synapses. We recently showed that the dendritic NaF density strongly influences the responsiveness of model GP neurons to synchronous excitatory inputs. In the present study we use rat GP neuron models to show that dendritic NaF channel expression is a potential cellular mechanism of active decorrelation. We further show that model neurons with lower dendritic NaF channel expression have a greater tendency to phase lock with oscillatory synaptic input patterns like those observed in PD. PMID:21795543

Glial cells have heart: rH1 Na+ channel mRNA and protein in spinal cord astrocytes.

PubMed

Black, J A; Dib-Hajj, S; Cohen, S; Hinson, A W; Waxman, S G

1998-07-01

Astrocytes in vitro express several distinct voltage-sensitive sodium currents, including tetrodotoxin (TTX)-resistant in non-stellate astrocytes and TTX-sensitive currents in stellate astrocytes. However, the molecular identity of the underlying channels, and the mechanisms that regulate their expression, have yet to be identified. Since spinal cord astrocytes in vitro express sodium currents that are nearly ten-fold greater that those of astrocytes derived from other regions, we used reverse transcription polymerase chain reaction (RT-PCR), in situ hybridization, and immunocytochemistry to search for a sodium channel mRNA and protein corresponding to a TTX-resistant channel in these cells. RT-PCR did not detect transcripts for SNS, which is known to encode a TTX-resistant current in dorsal root ganglion neurons. However, RT-PCR demonstrated the presence of rH1 mRNA in cultured spinal cord astrocytes derived from postnatal day 0 (P0) Sprague Dawley rats at 7 days in vitro and in also intact spinal cords of P0 and P7 rats. Hybridization signal for rH1 mRNA was detected by in situ hybridization cytochemistry in most non-stellate and, at varying levels, in stellate astrocytes in these cultures. Immunocytochemical studies, utilizing a polyclonal antibody (R-12) generated against a conserved polypeptide sequence of sodium channels, demonstrated sodium channel immunoreactivity in non-stellate and stellate astrocytes in these cultures. Spinal cord cultures reacted with a rH1-specific polyclonal antibody also showed rH1 immunostaining in non-stellate and stellate astrocytes, although the intensity of the rH1 immunoreactivity in both astrocyte morphologies was attenuated compared to that observed with the R-12 generic sodium channel antibody. The presence of rH1 mRNA and protein in non-stellate astrocytes in vitro provides a possible correlate for the TTX-resistant current that has been recorded in these cells. Since TTX-resistant current is not present in stellate astrocytes, the presence of rH1 mRNA and protein in these cells suggests, in addition, that post-translational mechanisms participate in the control of sodium channel expression in these cells.

Profiling neuronal ion channelopathies with non-invasive brain imaging and dynamic causal models: Case studies of single gene mutations

PubMed Central

Gilbert, Jessica R.; Symmonds, Mkael; Hanna, Michael G.; Dolan, Raymond J.; Friston, Karl J.; Moran, Rosalyn J.

2016-01-01

Clinical assessments of brain function rely upon visual inspection of electroencephalographic waveform abnormalities in tandem with functional magnetic resonance imaging. However, no current technology proffers in vivo assessments of activity at synapses, receptors and ion-channels, the basis of neuronal communication. Using dynamic causal modeling we compared electrophysiological responses from two patients with distinct monogenic ion channelopathies and a large cohort of healthy controls to demonstrate the feasibility of assaying synaptic-level channel communication non-invasively. Synaptic channel abnormality was identified in both patients (100% sensitivity) with assay specificity above 89%, furnishing estimates of neurotransmitter and voltage-gated ion throughput of sodium, calcium, chloride and potassium. This performance indicates a potential novel application as an adjunct for clinical assessments in neurological and psychiatric settings. More broadly, these findings indicate that biophysical models of synaptic channels can be estimated non-invasively, having important implications for advancing human neuroimaging to the level of non-invasive ion channel assays. PMID:26342528

Two barriers for sodium in vascular endothelium?

PubMed Central

Oberleithner, Hans

2012-01-01

Vascular endothelium plays a key role in blood pressure regulation. Recently, it has been shown that a 5% increase of plasma sodium concentration (sodium excess) stiffens endothelial cells by about 25%, leading to cellular dysfunction. Surface measurements demonstrated that the endothelial glycocalyx (eGC), an anionic biopolymer, deteriorates when sodium is elevated. In view of these results, a two-barrier model for sodium exiting the circulation across the endothelium is suggested. The first sodium barrier is the eGC which selectively buffers sodium ions with its negatively charged prote-oglycans.The second sodium barrier is the endothelial plasma membrane which contains sodium channels. Sodium excess, in the presence of aldosterone, leads to eGC break-down and, in parallel, to an up-regulation of plasma membrane sodium channels. The following hypothesis is postulated: Sodium excess increases vascular sodium permeability. Under such con-ditions (e.g. high-sodium diet), day-by-day ingested sodium, instead of being readily buffered by the eGC and then rapidly excreted by the kidneys, is distributed in the whole body before being finally excreted. Gradually, the sodium overload damages the organism. PMID:22471931

Co-Localization of Sodium Channel Na[v]1.6 and the Sodium--Calcium Exchanger at Sites of Axonal Injury in the Spinal Cord in EAE

ERIC Educational Resources Information Center

Craner, Matthew J.; Hains, Bryan C.; Lo, Albert C.; Black, Joel A.; Waxman, Stephen G.

2004-01-01

Axonal degeneration contributes to the development of non-remitting neurological deficits and disability in multiple sclerosis, but the molecular mechanisms that underlie axonal loss in multiple sclerosis are not clearly understood. Studies of white matter axonal injury have demonstrated that voltage-gated sodium channels can provide a route for…

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

PubMed

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.

Surface dynamics of voltage-gated ion channels.

PubMed

Heine, Martin; Ciuraszkiewicz, Anna; Voigt, Andreas; Heck, Jennifer; Bikbaev, Arthur

2016-07-03

Neurons encode information in fast changes of the membrane potential, and thus electrical membrane properties are critically important for the integration and processing of synaptic inputs by a neuron. These electrical properties are largely determined by ion channels embedded in the membrane. The distribution of most ion channels in the membrane is not spatially uniform: they undergo activity-driven changes in the range of minutes to days. Even in the range of milliseconds, the composition and topology of ion channels are not static but engage in highly dynamic processes including stochastic or activity-dependent transient association of the pore-forming and auxiliary subunits, lateral diffusion, as well as clustering of different channels. In this review we briefly discuss the potential impact of mobile sodium, calcium and potassium ion channels and the functional significance of this for individual neurons and neuronal networks.

Surface dynamics of voltage-gated ion channels

PubMed Central

Heine, Martin; Ciuraszkiewicz, Anna; Voigt, Andreas; Heck, Jennifer; Bikbaev, Arthur

2016-01-01

ABSTRACT Neurons encode information in fast changes of the membrane potential, and thus electrical membrane properties are critically important for the integration and processing of synaptic inputs by a neuron. These electrical properties are largely determined by ion channels embedded in the membrane. The distribution of most ion channels in the membrane is not spatially uniform: they undergo activity-driven changes in the range of minutes to days. Even in the range of milliseconds, the composition and topology of ion channels are not static but engage in highly dynamic processes including stochastic or activity-dependent transient association of the pore-forming and auxiliary subunits, lateral diffusion, as well as clustering of different channels. In this review we briefly discuss the potential impact of mobile sodium, calcium and potassium ion channels and the functional significance of this for individual neurons and neuronal networks. PMID:26891382

CFTR fails to inhibit the epithelial sodium channel ENaC expressed in Xenopus laevis oocytes

PubMed Central

Nagel, G; Barbry, P; Chabot, H; Brochiero, E; Hartung, K; Grygorczyk, R

2005-01-01

The cystic fibrosis transmembrane conductance regulator (CFTR) plays a crucial role in regulating fluid secretion by the airways, intestines, sweat glands and other epithelial tissues. It is well established that the CFTR is a cAMP-activated, nucleotide-dependent anion channel, but additional functions are often attributed to it, including regulation of the epithelial sodium channel (ENaC). The absence of CFTR-dependent ENaC inhibition and the resulting sodium hyperabsorption were postulated to be a major electrolyte transport abnormality in cystic fibrosis (CF)-affected epithelia. Several ex vivo studies, including those that used the Xenopus oocyte expression system, have reported ENaC inhibition by activated CFTR, but contradictory results have also been obtained. Because CFTR–ENaC interactions have important implications in the pathogenesis of CF, the present investigation was undertaken by our three independent laboratories to resolve whether CFTR regulates ENaC in oocytes and to clarify potential sources of previously reported dissimilar observations. Using different experimental protocols and a wide range of channel expression levels, we found no evidence that activated CFTR regulates ENaC when oocyte membrane potential was carefully clamped. We determined that an apparent CFTR-dependent ENaC inhibition could be observed when resistance in series with the oocyte membrane was not low enough or the feedback voltage gain was not high enough. We suggest that the inhibitory effect of CFTR on ENaC reported in some earlier oocyte studies could be attributed to problems arising from high levels of channel expression and suboptimal recording conditions, that is, large series resistance and/or insufficient feedback voltage gain. PMID:15746174

Inhibitors of voltage-gated sodium channel Nav1.7: patent applications since 2010.

PubMed

Sun, Shaoyi; Cohen, Charles J; Dehnhardt, Christoph M

2014-09-01

There has been intense interest in developing inhibitors of the sodium channel Nav1.7 because genetic studies have established very strong validation for the efficacy to alleviate both inflammatory and neuropathic pain. This review summarizes patent applications targeting Nav1.7 since 2010 until May, 2014. We have classified the patents into three categories as follows: small molecules with well-defined molecular selectivity among sodium channel isoforms; biologicals with well-defined molecular selectivity; and, small molecules that inhibit Nav1.7 with unknown molecular selectivity. Most of the review is dedicated to small molecule selective compounds.

Mechanosensitive Piezo Channels in the Gastrointestinal Tract

PubMed Central

Alcaino, C.; Farrugia, G.; Beyder, A.

2017-01-01

Sensation of mechanical forces is critical for normal function of the gastrointestinal (GI) tract and abnormalities in mechanosensation are linked to GI pathologies. In the GI tract there are several mechanosensitive cell types—epithelial enterochromaffin cells, intrinsic and extrinsic enteric neurons, smooth muscle cells and interstitial cells of Cajal. These cells use mechanosensitive ion channels that respond to mechanical forces by altering transmembrane ionic currents in a process called mechanoelectrical coupling. Several mechanosensitive ionic conductances have been identified in the mechano-sensory GI cells, ranging from mechanosensitive voltage-gated sodium and calcium channels to the mechanogated ion channels, such as the two-pore domain potassium channels K2P (TREK-1) and nonselective cation channels from the transient receptor potential family. The recently discovered Piezo channels are increasingly recognized as significant contributors to cellular mechanosensitivity. Piezo1 and Piezo2 are nonselective cationic ion channels that are directly activated by mechanical forces and have well-defined biophysical and pharmacologic properties. The role of Piezo channels in the GI epithelium is currently under investigation and their role in the smooth muscle syncytium and enteric neurons is still not known. In this review, we outline the current state of knowledge on mechanosensitive ion channels in the GI tract, with a focus on the known and potential functions of the Piezo channels. PMID:28728818

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

PubMed Central

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

2011-01-01

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 (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. 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 results confirm our former hypothesis by showing that the presence of hydroxyl groups to the aryloxy moiety of mexiletine greatly reduced sodium channel block, and provide molecular insights into the intimate interaction of local anesthetics with their receptor. PMID:22403541

Loss-of-function mutations in sodium channel Nav1.7 cause anosmia

PubMed Central

Weiss, Jan; Pyrski, Martina; Jacobi, Eric; Bufe, Bernd; Willnecker, Vivienne; Schick, Bernhard; Zizzari, Philippe; Gossage, Samuel J.; Greer, Charles A.; Leinders-Zufall, Trese; Woods, C. Geoffrey; Wood, John N.; Zufall, Frank

2013-01-01

Loss of function of the gene SCN9A, encoding the voltage-gated sodium channel Nav1.7, causes a congenital inability to experience pain in humans. Here we show that Nav1.7 is not only necessary for pain sensation but is also an essential requirement for odour perception in both mice and humans. We examined human patients with loss-of-function mutations in SCN9A and show that they are unable to sense odours. To establish the essential role of Nav1.7 in odour perception, we generated conditional null mice in which Nav1.7 was removed from all olfactory sensory neurons. In the absence of Nav1.7, these neurons still produce odour-evoked action potentials but fail to initiate synaptic signalling from their axon terminals at the first synapse in the olfactory system. The mutant mice no longer display vital, odour-guided behaviours such as innate odour recognition and avoidance, short-term odour learning, and maternal pup retrieval. Our study creates a mouse model of congenital general anosmia and provides new strategies to explore the genetic basis of the human sense of smell. PMID:21441906

Loss-of-function mutations in sodium channel Nav1.7 cause anosmia.

PubMed

Weiss, Jan; Pyrski, Martina; Jacobi, Eric; Bufe, Bernd; Willnecker, Vivienne; Schick, Bernhard; Zizzari, Philippe; Gossage, Samuel J; Greer, Charles A; Leinders-Zufall, Trese; Woods, C Geoffrey; Wood, John N; Zufall, Frank

2011-04-14

Loss of function of the gene SCN9A, encoding the voltage-gated sodium channel Na(v)1.7, causes a congenital inability to experience pain in humans. Here we show that Na(v)1.7 is not only necessary for pain sensation but is also an essential requirement for odour perception in both mice and humans. We examined human patients with loss-of-function mutations in SCN9A and show that they are unable to sense odours. To establish the essential role of Na(v)1.7 in odour perception, we generated conditional null mice in which Na(v)1.7 was removed from all olfactory sensory neurons. In the absence of Na(v)1.7, these neurons still produce odour-evoked action potentials but fail to initiate synaptic signalling from their axon terminals at the first synapse in the olfactory system. The mutant mice no longer display vital, odour-guided behaviours such as innate odour recognition and avoidance, short-term odour learning, and maternal pup retrieval. Our study creates a mouse model of congenital general anosmia and provides new strategies to explore the genetic basis of the human sense of smell.

Voltage gating by molecular subunits of Na+ and K+ ion channels: higher-dimensional cubic kinetics, rate constants, and temperature

PubMed Central

2015-01-01

The structural similarity between the primary molecules of voltage-gated Na and K channels (alpha subunits) and activation gating in the Hodgkin-Huxley model is brought into full agreement by increasing the model's sodium kinetics to fourth order (m3 → m4). Both structures then virtually imply activation gating by four independent subprocesses acting in parallel. The kinetics coalesce in four-dimensional (4D) cubic diagrams (16 states, 32 reversible transitions) that show the structure to be highly failure resistant against significant partial loss of gating function. Rate constants, as fitted in phase plot data of retinal ganglion cell excitation, reflect the molecular nature of the gating transitions. Additional dimensions (6D cubic diagrams) accommodate kinetically coupled sodium inactivation and gating processes associated with beta subunits. The gating transitions of coupled sodium inactivation appear to be thermodynamically irreversible; response to dielectric surface charges (capacitive displacement) provides a potential energy source for those transitions and yields highly energy-efficient excitation. A comparison of temperature responses of the squid giant axon (apparently Arrhenius) and mammalian channel gating yields kinetic Q10 = 2.2 for alpha unit gating, whose transitions are rate-limiting at mammalian temperatures; beta unit kinetic Q10 = 14 reproduces the observed non-Arrhenius deviation of mammalian gating at low temperatures; the Q10 of sodium inactivation gating matches the rate-limiting component of activation gating at all temperatures. The model kinetics reproduce the physiologically large frequency range for repetitive firing in ganglion cells and the physiologically observed strong temperature dependence of recovery from inactivation. PMID:25867741

Nav1.7-A1632G Mutation from a Family with Inherited Erythromelalgia: Enhanced Firing of Dorsal Root Ganglia Neurons Evoked by Thermal Stimuli.

PubMed

Yang, Yang; Huang, Jianying; Mis, Malgorzata A; Estacion, Mark; Macala, Lawrence; Shah, Palak; Schulman, Betsy R; Horton, Daniel B; Dib-Hajj, Sulayman D; Waxman, Stephen G

2016-07-13

Voltage-gated sodium channel Nav1.7 is a central player in human pain. Mutations in Nav1.7 produce several pain syndromes, including inherited erythromelalgia (IEM), a disorder in which gain-of-function mutations render dorsal root ganglia (DRG) neurons hyperexcitable. Although patients with IEM suffer from episodes of intense burning pain triggered by warmth, the effects of increased temperature on DRG neurons expressing mutant Nav1.7 channels have not been well documented. Here, using structural modeling, voltage-clamp, current-clamp, and multielectrode array recordings, we have studied a newly identified Nav1.7 mutation, Ala1632Gly, from a multigeneration family with IEM. Structural modeling suggests that Ala1632 is a molecular hinge and that the Ala1632Gly mutation may affect channel gating. Voltage-clamp recordings revealed that the Nav1.7-A1632G mutation hyperpolarizes activation and depolarizes fast-inactivation, both gain-of-function attributes at the channel level. Whole-cell current-clamp recordings demonstrated increased spontaneous firing, lower current threshold, and enhanced evoked firing in rat DRG neurons expressing Nav1.7-A1632G mutant channels. Multielectrode array recordings further revealed that intact rat DRG neurons expressing Nav1.7-A1632G mutant channels are more active than those expressing Nav1.7 WT channels. We also showed that physiologically relevant thermal stimuli markedly increase the mean firing frequencies and the number of active rat DRG neurons expressing Nav1.7-A1632G mutant channels, whereas the same thermal stimuli only increase these parameters slightly in rat DRG neurons expressing Nav1.7 WT channels. The response of DRG neurons expressing Nav1.7-A1632G mutant channels upon increase in temperature suggests a cellular basis for warmth-triggered pain in IEM. Inherited erythromelalgia (IEM), a severe pain syndrome characterized by episodes of intense burning pain triggered by warmth, is caused by mutations in sodium channel Nav1.7, which are preferentially expressed in sensory and sympathetic neurons. More than 20 gain-of-function Nav1.7 mutations have been identified from IEM patients, but the question of how warmth triggers episodes of pain in IEM has not been well addressed. Combining multielectrode array, voltage-clamp, and current-clamp recordings, we assessed a newly identified IEM mutation (Nav1.7-A1632G) from a multigeneration family. Our data demonstrate gain-of-function attributes at the channel level and differential effects of physiologically relevant thermal stimuli on the excitability of DRG neurons expressing mutant and WT Nav1.7 channels, suggesting a cellular mechanism for warmth-triggered pain episodes in IEM patients. Copyright © 2016 the authors 0270-6474/16/367512-12$15.00/0.

Selectivity filters and cysteine-rich extracellular loops in voltage-gated sodium, calcium, and NALCN channels

PubMed Central

Stephens, Robert F.; Guan, W.; Zhorov, Boris S.; Spafford, J. David

2015-01-01

How nature discriminates sodium from calcium ions in eukaryotic channels has been difficult to resolve because they contain four homologous, but markedly different repeat domains. We glean clues from analyzing the changing pore region in sodium, calcium and NALCN channels, from single-cell eukaryotes to mammals. Alternative splicing in invertebrate homologs provides insights into different structural features underlying calcium and sodium selectivity. NALCN generates alternative ion selectivity with splicing that changes the high field strength (HFS) site at the narrowest level of the hourglass shaped pore where the selectivity filter is located. Alternative splicing creates NALCN isoforms, in which the HFS site has a ring of glutamates contributed by all four repeat domains (EEEE), or three glutamates and a lysine residue in the third (EEKE) or second (EKEE) position. Alternative splicing provides sodium and/or calcium selectivity in T-type channels with extracellular loops between S5 and P-helices (S5P) of different lengths that contain three or five cysteines. All eukaryotic channels have a set of eight core cysteines in extracellular regions, but the T-type channels have an infusion of 4–12 extra cysteines in extracellular regions. The pattern of conservation suggests a possible pairing of long loops in Domains I and III, which are bridged with core cysteines in NALCN, Cav, and Nav channels, and pairing of shorter loops in Domains II and IV in T-type channel through disulfide bonds involving T-type specific cysteines. Extracellular turrets of increasing lengths in potassium channels (Kir2.2, hERG, and K2P1) contribute to a changing landscape above the pore selectivity filter that can limit drug access and serve as an ion pre-filter before ions reach the pore selectivity filter below. Pairing of extended loops likely contributes to the large extracellular appendage as seen in single particle electron cryo-microscopy images of the eel Nav1 channel. PMID:26042044

Evans Blue is not a suitable inhibitor of the epithelial sodium channel δ-subunit.

PubMed

Perniss, Alexander; Wolf, Annemarie; Wichmann, Lukas; Schönberger, Matthias; Althaus, Mike

2015-10-23

The Epithelial Sodium Channel (ENaC) is a heterotrimeric ion channel which can be either formed by assembly of its α-, β- and γ-subunits or, alternatively, its δ-, β- and γ-subunits. The physiological function of αβγ-ENaC is well established, but the function of δβγ-ENaC remains elusive. The azo-dye Evans Blue (EvB) has been routinely used to discriminate between the two channel isoforms by decreasing transmembrane currents and amiloride-sensitive current fractions of δβγ-ENaC expressing Xenopus oocytes. Even though these results could be reproduced, it was found by precipitation experiments and spectroscopic methods that the cationic amiloride and the anionic EvB directly interact in solution, forming a strong complex. Thereby a large amount of pharmacologically available amiloride is removed from physiological buffer solutions and the effective amiloride concentration is reduced. This interaction did not occur in the presence of albumin. In microelectrode recordings, EvB was able to abrogate the block of δβγ-ENaC by amiloride or its derivative benzamil. In sum, EvB reduces amiloride-sensitive ion current fractions in electrophysiological experiments. This is not a result of a specific inhibition of δβγ-ENaC but rather represents a pharmacological artefact. EvB should therefore not be used as an inhibitor of δ-ENaC. Copyright © 2015 Elsevier Inc. All rights reserved.

A novel frequency analysis method for assessing K(ir)2.1 and Na (v)1.5 currents.

PubMed

Rigby, J R; Poelzing, S

2012-04-01

Voltage clamping is an important tool for measuring individual currents from an electrically active cell. However, it is difficult to isolate individual currents without pharmacological or voltage inhibition. Herein, we present a technique that involves inserting a noise function into a standard voltage step protocol, which allows one to characterize the unique frequency response of an ion channel at different step potentials. Specifically, we compute the fast Fourier transform for a family of current traces at different step potentials for the inward rectifying potassium channel, K(ir)2.1, and the channel encoding the cardiac fast sodium current, Na(v)1.5. Each individual frequency magnitude, as a function of voltage step, is correlated to the peak current produced by each channel. The correlation coefficient vs. frequency relationship reveals that these two channels are associated with some unique frequencies with high absolute correlation. The individual IV relationship can then be recreated using only the unique frequencies with magnitudes of high absolute correlation. Thus, this study demonstrates that ion channels may exhibit unique frequency responses.

Tarantula huwentoxin-IV inhibits neuronal sodium channels by binding to receptor site 4 and trapping the domain ii voltage sensor in the closed configuration.

PubMed

Xiao, Yucheng; Bingham, Jon-Paul; Zhu, Weiguo; Moczydlowski, Edward; Liang, Songping; Cummins, Theodore R

2008-10-03

Peptide toxins with high affinity, divergent pharmacological functions, and isoform-specific selectivity are powerful tools for investigating the structure-function relationships of voltage-gated sodium channels (VGSCs). Although a number of interesting inhibitors have been reported from tarantula venoms, little is known about the mechanism for their interaction with VGSCs. We show that huwentoxin-IV (HWTX-IV), a 35-residue peptide from tarantula Ornithoctonus huwena venom, preferentially inhibits neuronal VGSC subtypes rNav1.2, rNav1.3, and hNav1.7 compared with muscle subtypes rNav1.4 and hNav1.5. Of the five VGSCs examined, hNav1.7 was most sensitive to HWTX-IV (IC(50) approximately 26 nM). Following application of 1 microm HWTX-IV, hNav1.7 currents could only be elicited with extreme depolarizations (>+100 mV). Recovery of hNav1.7 channels from HWTX-IV inhibition could be induced by extreme depolarizations or moderate depolarizations lasting several minutes. Site-directed mutagenesis analysis indicated that the toxin docked at neurotoxin receptor site 4 located at the extracellular S3-S4 linker of domain II. Mutations E818Q and D816N in hNav1.7 decreased toxin affinity for hNav1.7 by approximately 300-fold, whereas the reverse mutations in rNav1.4 (N655D/Q657E) and the corresponding mutations in hNav1.5 (R812D/S814E) greatly increased the sensitivity of the muscle VGSCs to HWTX-IV. Our data identify a novel mechanism for sodium channel inhibition by tarantula toxins involving binding to neurotoxin receptor site 4. In contrast to scorpion beta-toxins that trap the IIS4 voltage sensor in an outward configuration, we propose that HWTX-IV traps the voltage sensor of domain II in the inward, closed configuration.

Development of sodium channel protein during chemically induced differentiation of neuroblastoma cells.

PubMed

Baumgold, J; Spector, I

1987-04-01

We have previously shown that the [3H]saxitoxin binding site of the sodium channel is expressed independently of the [125I]scorpion toxin binding site in chick muscle cultures and in rat brain. In the present work, we studied the development of the sodium channel protein during chemically induced differentiation of N1E-115 neuroblastoma cells, using [3H]saxitoxin binding, [125I]scorpion toxin binding, and 22Na uptake techniques. When grown in their normal culture medium, these cells are mostly undifferentiated, bind 90 +/- 10 fmol of [3H]saxitoxin/mg of protein and 112 +/- 14 fmol of [125I]scorpion toxin/mg protein, and, when stimulated with scorpion toxin and batrachotoxin, take up 70 +/- 5 nmol of 22Na/min/mg of protein. Cells treated with dimethyl sulfoxide (DMSO) or hexamethylene-bis-acetamide (HMBA) differentiate morphologically within 3 days. At this time, the [3H]saxitoxin binding, the [125I]scorpion toxin binding, and the 22Na uptake values are not very different from those of undifferentiated cells. With subsequent time in DMSO or HMBA, these values continue to increase, a result indicating that the main period of sodium channel expression occurs well after the cells have assumed the morphologically differentiated state. The data indicate that the expression of sodium channels and morphological differentiation are independently regulated neuronal properties, that the attainment of morphological differentiation is necessary but not in itself sufficient for full expression of the sodium channel proteins, and that, in contrast to the chick muscle cultures and rat brain, the [3H]saxitoxin site and [125I]scorpion toxin site appear to be coregulated in N1E-115 cells.

Native pyroglutamation of huwentoxin-IV: a post-translational modification that increases the trapping ability to the sodium channel.

PubMed

Rong, Mingqiang; Duan, Zhigui; Chen, Juliang; Li, Jianglin; Xiao, Yuchen; Liang, Songping

2013-01-01

Huwentoxin-IV (HWTX-IV), a tetrodotoxin-sensitive (TTX-s) sodium channel antagonist, is found in the venom of the Chinese spider Ornithoctonus huwena. A naturally modified HWTX-IV (mHWTX-IV), having a molecular mass 18 Da lower than HWTX-IV, has also been isolated from the venom of the same spider. By a combination of enzymatic fragmentation and MS/MS de novo sequencing, mHWTX-IV has been shown to have the same amino acid sequence as that of HWTX-IV, except that the N-terminal glutamic acid replaced by pyroglutamic acid. mHWTX-IV inhibited tetrodotoxin-sensitive voltage-gated sodium channels of dorsal root ganglion neurons with an IC50 nearly equal to native HWTX-IV. mHWTX-IV showed the same activation and inactivation kinetics seen for native HWTX-IV. In contrast with HWTX-IV, which dissociates at moderate voltage depolarization voltages (+50 mV, 180000 ms), mHWTX-IV inhibition of TTX-sensitive sodium channels is not reversed by strong depolarization voltages (+200 mV, 500 ms). Recovery of Nav1.7current was voltage-dependent and was induced by extreme depolarization in the presence of HWTX-IV, but no obvious current was elicited after application of mHWTX-IV. Our data indicate that the N-terminal modification of HWTX-IV gives the peptide toxin a greater ability to trap the voltage sensor in the sodium channel. Loss of a negative charge, caused by cyclization at the N-terminus, is a possible reason why the modified toxin binds much stronger. To our knowledge, this is the first report of a pyroglutamic acid residue in a spider toxin; this modification seems to increase the trapping ability of the voltage sensor in the sodium channel.

Native Pyroglutamation of Huwentoxin-IV: A Post-Translational Modification that Increases the Trapping Ability to the Sodium Channel

PubMed Central

Rong, Mingqiang; Duan, Zhigui; Chen, Juliang; Li, Jianglin; Xiao, Yuchen; Liang, Songping

2013-01-01

Huwentoxin-IV (HWTX-IV), a tetrodotoxin-sensitive (TTX-s) sodium channel antagonist, is found in the venom of the Chinese spider Ornithoctonus huwena. A naturally modified HWTX-IV (mHWTX-IV), having a molecular mass 18 Da lower than HWTX-IV, has also been isolated from the venom of the same spider. By a combination of enzymatic fragmentation and MS/MS de novo sequencing, mHWTX-IV has been shown to have the same amino acid sequence as that of HWTX-IV, except that the N-terminal glutamic acid replaced by pyroglutamic acid. mHWTX-IV inhibited tetrodotoxin-sensitive voltage-gated sodium channels of dorsal root ganglion neurons with an IC50 nearly equal to native HWTX-IV. mHWTX-IV showed the same activation and inactivation kinetics seen for native HWTX-IV. In contrast with HWTX-IV, which dissociates at moderate voltage depolarization voltages (+50 mV, 180000 ms), mHWTX-IV inhibition of TTX-sensitive sodium channels is not reversed by strong depolarization voltages (+200 mV, 500 ms). Recovery of Nav1.7current was voltage-dependent and was induced by extreme depolarization in the presence of HWTX-IV, but no obvious current was elicited after application of mHWTX-IV. Our data indicate that the N-terminal modification of HWTX-IV gives the peptide toxin a greater ability to trap the voltage sensor in the sodium channel. Loss of a negative charge, caused by cyclization at the N-terminus, is a possible reason why the modified toxin binds much stronger. To our knowledge, this is the first report of a pyroglutamic acid residue in a spider toxin; this modification seems to increase the trapping ability of the voltage sensor in the sodium channel. PMID:23826086

Topographical localization of the C-terminal region of the voltage-dependent sodium channel from Electrophorus electricus using antibodies raised against a synthetic peptide

DOE Office of Scientific and Technical Information (OSTI.GOV)

Gordon, R.D.; Fieles, W.E.; Schotland, D.L.

1987-01-01

A peptide corresponding to amino acid residues 1783-1794 near the C terminus of the electric eel sodium channel primary sequence of the eel (Electrophorus electricus) sodium channel has been synthesized and used to raise an antiserum in rabbits. This antiserum specifically recognized the peptide in a solid-phase radioimmunoassay. Specificity of the antiserum for the native channel protein was shown by its specific binding to a 280-kDa protein in immunoblots of eel electroplax membrane proteins. The antiserum also specifically labeled the innervated membrane of the eel electroplax in immunofluorescent studies. The membrane topology of the peptide recognized by this antiserum wasmore » proved in binding studies using oriented electroplax membrane vesicles. These vesicles were 98% right-side-out as determined by (/sup 3/H)saxitoxin binding. Binding of the antipeptide antiserum to this fraction was measured before and after permeabilization with 0.01% saponin. Specific binding to intact vesicles was low, but this binding increased 10-fold after permeabilization, implying a cytoplasmic orientation for the peptide. Confirmation for this orientation was then sought by localizing the antibody bound to intact electroplax cells with immunogold electron microscopy. The data imply that the region of the sodium channel primary sequence near the C terminus that is recognized by the anitserum is localized on the cytoplasmic side of the membrane; this localization provides some further constraints on models of sodium channel tertiary structure.« less

Regulation of Sodium Transport in the Inner Ear

PubMed Central

Kim, Sung Huhn; Marcus, Daniel C.

2011-01-01

Na+ concentrations in endolymph must be controlled to maintain hair cell function since the transduction channels of hair cells are cation-permeable, but not K+-selective. Flooding or fluctuations of the hair cell cytosol with Na+ would be expected to lead to cellular dysfunction, hearing loss and vertigo. This review briefly describes cellular mechanisms known to be responsible for Na+homeostasis in each compartment of the inner ear, including the cochlea, saccule, semicircular canals and endolymphatic sac. The influx of Na+into endolymph of each of the organs is likely via passive diffusion, but these pathways have not yet been identified or characterized. Na+ absorption is controlled by gate -keeper channels in the apical (endolymphatic) membrane of the transporting cells. Highly Na+-selective epithelial sodium channels (ENaC) control absorption by Reissner’s membrane, saccular extramacular epithelium, semicircular canal duct epithelium and endolymphatic sac. ENaC activity is controlled by a number of signal pathways, but most notably by genomic regulation of channel numbers in the membrane via glucocorticoid signaling. Nonselective cation channels in the apical membrane of outer sulcus epithelial cells and vestibular transitional cells mediate Na+ and parasensory K+ absorption. The K+-mediated transduction current in hair cells is also accompanied by a Na+ flux since the transduction channels are nonselective cation channels. Cation absorption by all of these cells is regulated by extracellular ATP via apical nonselective cation channels (P2X receptors). The heterogeneous population of epithelial cells in the endolymphatic sac is thought to have multiple absorptive pathways for Na+ with regulatory pathways that include glucocorticoids and purinergic agonists. PMID:21620939

Faster voltage-dependent activation of Na+ channels in growth cones versus somata of neuroblastoma N1E-115 cells.

PubMed Central

Zhang, J; Loew, L M; Davidson, R M

1996-01-01

Kinetics of voltage-gated ionic channels fundamentally reflect the response of the channels to local electric fields. In this report cell-attached patch-clamp studies reveal that the voltage-dependent activation rate of sodium channels residing in the growth cone membrane differs from that of soma sodium channels in differentiating N1E-115 neuroblastoma cells. Because other electrophysiological properties of these channels do not differ, this finding may be a reflection of the difference in intramembrane electric field in these two regions of the cell. This represents a new mechanism for channels to attain a range of activities both within and between cells. PMID:8913589

Faster voltage-dependent activation of Na+ channels in growth cones versus somata of neuroblastoma N1E-115 cells.

PubMed

Zhang, J; Loew, L M; Davidson, R M

1996-11-01

Kinetics of voltage-gated ionic channels fundamentally reflect the response of the channels to local electric fields. In this report cell-attached patch-clamp studies reveal that the voltage-dependent activation rate of sodium channels residing in the growth cone membrane differs from that of soma sodium channels in differentiating N1E-115 neuroblastoma cells. Because other electrophysiological properties of these channels do not differ, this finding may be a reflection of the difference in intramembrane electric field in these two regions of the cell. This represents a new mechanism for channels to attain a range of activities both within and between cells.

Collecting Duct Nitric Oxide Synthase 1ß Activation Maintains Sodium Homeostasis During High Sodium Intake Through Suppression of Aldosterone and Renal Angiotensin II Pathways.

PubMed

Hyndman, Kelly A; Mironova, Elena V; Giani, Jorge F; Dugas, Courtney; Collins, Jessika; McDonough, Alicia A; Stockand, James D; Pollock, Jennifer S

2017-10-24

During high sodium intake, the renin-angiotensin-aldosterone system is downregulated and nitric oxide signaling is upregulated in order to remain in sodium balance. Recently, we showed that collecting duct nitric oxide synthase 1β is critical for fluid-electrolyte balance and subsequently blood pressure regulation during high sodium feeding. The current study tested the hypothesis that high sodium activation of the collecting duct nitric oxide synthase 1β pathway is critical for maintaining sodium homeostasis and for the downregulation of the renin-angiotensin-aldosterone system-epithelial sodium channel axis. Male control and collecting duct nitric oxide synthase 1β knockout (CDNOS1KO) mice were placed on low, normal, and high sodium diets for 1 week. In response to the high sodium diet, plasma sodium was significantly increased in control mice and to a significantly greater level in CDNOS1KO mice. CDNOS1KO mice did not suppress plasma aldosterone in response to the high sodium diet, which may be partially explained by increased adrenal AT1R expression. Plasma renin concentration was appropriately suppressed in both genotypes. Furthermore, CDNOS1KO mice had significantly higher intrarenal angiotensin II with high sodium diet, although intrarenal angiotensinogen levels and angiotensin-converting enzyme activity were similar between knockout mice and controls. In agreement with inappropriate renin-angiotensin-aldosterone system activation in the CDNOS1KO mice on a high sodium diet, epithelial sodium channel activity and sodium transporter abundance were significantly higher compared with controls. These data demonstrate that high sodium activation of collecting duct nitric oxide synthase 1β signaling induces suppression of systemic and intrarenal renin-angiotensin-aldosterone system, thereby modulating epithelial sodium channel and other sodium transporter abundance and activity to maintain sodium homeostasis. © 2017 The Authors. Published on behalf of the American Heart Association, Inc., by Wiley.

Transcriptomic Analysis of Ciguatoxin-Induced Changes in Gene Expression in Primary Cultures of Mice Cortical Neurons

PubMed Central

Rubiolo, Juan Andrés; Boente-Juncal, Andrea; Hirama, Masahiro; Yamashita, Shuji; Camiña, Mercedes; Vieytes, Mercedes R.

2018-01-01

Ciguatoxins are polyether marine toxins that act as sodium channel activators. These toxins cause ciguatera, one of the most widespread nonbacterial forms of food poisoning, which presents several symptoms in humans including long-term neurological alterations. Earlier work has shown that both acute and chronic exposure of primary cortical neurons to synthetic ciguatoxin CTX3C have profound impacts on neuronal function. Thus, the present work aimed to identify relevant neuronal genes and metabolic pathways that could be altered by ciguatoxin exposure. To study the effect of ciguatoxins in primary neurons in culture, we performed a transcriptomic analysis using whole mouse genome microarrays, for primary cortical neurons exposed during 6, 24, or 72 h in culture to CTX3C. Here, we have shown that the effects of the toxin on gene expression differ with the exposure time. The results presented here have identified several relevant genes and pathways related to the effect of ciguatoxins on neurons and may assist in future research or even treatment of ciguatera. Moreover, we demonstrated that the effects of the toxin on gene expression were exclusively consequential of its action as a voltage-gated sodium channel activator, since all the effects of CTX3C were avoided by preincubation of the neurons with the sodium channel blocker tetrodotoxin. PMID:29748486

Loss of Sodium-Activated Potassium Channel Slack and FMRP Differentially Affect Social Behavior in Mice.

PubMed

Bausch, Anne E; Ehinger, Rebekka; Straubinger, Julia; Zerfass, Patrick; Nann, Yvette; Lukowski, Robert

2018-05-31

The sodium-activated potassium channel Slack (Slo2.2) is widely expressed in central and peripheral neurons where it is supposed to shape firing properties important for neuronal excitability. Slack activity is enhanced by interaction with the Fragile-X-Mental-Retardation-Protein (FMRP) and loss of FMRP leads to decreased sodium-activated potassium currents in medial nucleus of the trapezoid body neurons of the Fmr1-knockout (KO) mouse representing a mouse model of the human Fragile-X-Syndrome (FXS) and autism. Autism is a frequent comorbidity of FXS, but it is unclear whether Slack is involved in autistic or related conditions of FXS in vivo. By applying a wide range of behavioral tests, we compared social and autism-related behaviors in Slack- and FMRP-deficient mice. In our hands, as expected, FMRP-deficiency causes autism-related behavioral changes in nesting and in a marble-burying test. In contrast, Slack-deficient males exhibited specific abnormalities in sociability in direct and indirect social interaction tests. Hence, we show for the first time that a proper Slack channel function is mandatory for normal social behavior in mice. Nevertheless, as deficits in social behaviors seem to occur independently from each other in FMRP and Slack null mutants, we conclude that Slack is not involved in the autistic phenotype of FMRP KO mice. Copyright © 2018 IBRO. Published by Elsevier Ltd. All rights reserved.

Development of lacosamide for the treatment of partial-onset seizures

PubMed Central

Doty, Pamela; Hebert, David; Mathy, Francois-Xavier; Byrnes, William; Zackheim, James; Simontacchi, Kelly

2013-01-01

Lacosamide is an antiepileptic drug (AED) available in multiple formulations that was first approved in 2008 as adjunctive therapy for partial-onset seizures (POS) in adults. Unlike traditional sodium channel blockers affecting fast inactivation, lacosamide selectively enhances sodium channel slow inactivation. This mechanism of action results in stabilization of hyperexcitable neuronal membranes, inhibition of neuronal firing, and reduction in long-term channel availability without affecting physiological function. Lacosamide has a well-characterized and favorable pharmacokinetic profile, including a fast absorption rate, minimal or no interaction with cytochrome P-450 izoenzymes, and a low potential for drug–drug interactions. Lacosamide clinical development included three placebo-controlled, double-blind, randomized trials conducted in more than 1300 patients, each demonstrating safety and efficacy of lacosamide compared to placebo as adjunctive therapy for adults with POS. The clinical use of lacosamide may broaden, pending results of trials evaluating its use as monotherapy for POS in adults, as treatment for epilepsy in pediatric subjects, and as adjunctive treatment for uncontrolled primary generalized tonic–clonic seizures in those with idiopathic generalized epilepsy. PMID:23859801

α-SNAP regulates dynamic, on-site assembly and calcium selectivity of Orai1 channels

PubMed Central

Li, Peiyao; Miao, Yong; Dani, Adish; Vig, Monika

2016-01-01

Orai1 forms a highly calcium-selective pore of the calcium release activated channel, and α-SNAP is necessary for its function. Here we show that α-SNAP regulates on-site assembly of Orai1 dimers into calcium-selective multimers. We find that Orai1 is a dimer in resting primary mouse embryonic fibroblasts but displays variable stoichiometry in the plasma membrane of store-depleted cells. Remarkably, α-SNAP depletion induces formation of higher-order Orai1 oligomers, which permeate significant levels of sodium via Orai1 channels. Sodium permeation in α-SNAP–deficient cells cannot be corrected by tethering multiple Stim1 domains to Orai1 C-terminal tail, demonstrating that α-SNAP regulates functional assembly and calcium selectivity of Orai1 multimers independently of Stim1 levels. Fluorescence nanoscopy reveals sustained coassociation of α-SNAP with Stim1 and Orai1, and α-SNAP–depleted cells show faster and less constrained mobility of Orai1 within ER-PM junctions, suggesting Orai1 and Stim1 coentrapment without stable contacts. Furthermore, α-SNAP depletion significantly reduces fluorescence resonance energy transfer between Stim1 and Orai1 N-terminus but not C-terminus. Taken together, these data reveal a unique role of α-SNAP in the on-site functional assembly of Orai1 subunits and suggest that this process may, in part, involve enabling crucial low-affinity interactions between Orai1 N-terminus and Stim1. PMID:27335124

Coexistence of CLCN1 and SCN4A mutations in one family suffering from myotonia.

PubMed

Maggi, Lorenzo; Ravaglia, Sabrina; Farinato, Alessandro; Brugnoni, Raffaella; Altamura, Concetta; Imbrici, Paola; Camerino, Diana Conte; Padovani, Alessandro; Mantegazza, Renato; Bernasconi, Pia; Desaphy, Jean-François; Filosto, Massimiliano

2017-12-01

Non-dystrophic myotonias are characterized by clinical overlap making it challenging to establish genotype-phenotype correlations. We report clinical and electrophysiological findings in a girl and her father concomitantly harbouring single heterozygous mutations in SCN4A and CLCN1 genes. Functional characterization of N1297S hNav1.4 mutant was performed by patch clamp. The patients displayed a mild phenotype, mostly resembling a sodium channel myotonia. The CLCN1 c.501C>G (p.F167L) mutation has been already described in recessive pedigrees, whereas the SCN4A c.3890A>G (p.N1297S) variation is novel. Patch clamp experiments showed impairment of fast and slow inactivation of the mutated Nav1.4 sodium channel. The present findings suggest that analysis of both SCN4A and CLCN1 genes should be considered in myotonic patients with atypical clinical and neurophysiological features.

Pathophysiology and Treatment of Resistant Hypertension: The Role of Aldosterone and Amiloride-Sensitive Sodium Channels

PubMed Central

Judd, Eric K.; Calhoun, David A.; Warnock, David G.

2015-01-01

Summary Resistant hypertension is a clinically distinct subgroup of hypertension defined by the failure to achieve blood pressure control on optimal dosing of at least 3 antihypertensive medications of different classes, including a diuretic. The pathophysiology of hypertension can be attributed to aldosterone excess in more than 20% of patients with resistant hypertension. Existing dogma attributes the increase in blood pressure seen with increases in aldosterone to its antinatriuretic effects in the distal nephron. However, emerging research, which has identified and has begun to define the function of amiloride-sensitive sodium channels and mineralocorticoid receptors in the systemic vasculature, challenges impaired natriuresis as the sole cause of aldosterone-mediated resistant hypertension. This review integrates these findings to better define the role of the vasculature and aldosterone in the pathophysiology of resistant hypertension. In addition, a brief guide to the treatment of resistant hypertension is presented. PMID:25416662

Pathophysiology and treatment of resistant hypertension: the role of aldosterone and amiloride-sensitive sodium channels.

PubMed

Judd, Eric K; Calhoun, David A; Warnock, David G

2014-01-01

Resistant hypertension is a clinically distinct subgroup of hypertension defined by the failure to achieve blood pressure control on optimal dosing of at least 3 antihypertensive medications of different classes, including a diuretic. The pathophysiology of hypertension can be attributed to aldosterone excess in more than 20% of patients with resistant hypertension. Existing dogma attributes the increase in blood pressure seen with increases in aldosterone to its antinatriuretic effects in the distal nephron. However, emerging research, which has identified and has begun to define the function of amiloride-sensitive sodium channels and mineralocorticoid receptors in the systemic vasculature, challenges impaired natriuresis as the sole cause of aldosterone-mediated resistant hypertension. This review integrates these findings to better define the role of the vasculature and aldosterone in the pathophysiology of resistant hypertension. In addition, a brief guide to the treatment of resistant hypertension is presented.

Dysfunction of NaV1.4, a skeletal muscle voltage-gated sodium channel, in sudden infant death syndrome: a case-control study.

PubMed

Männikkö, Roope; Wong, Leonie; Tester, David J; Thor, Michael G; Sud, Richa; Kullmann, Dimitri M; Sweeney, Mary G; Leu, Costin; Sisodiya, Sanjay M; FitzPatrick, David R; Evans, Margaret J; Jeffrey, Iona J M; Tfelt-Hansen, Jacob; Cohen, Marta C; Fleming, Peter J; Jaye, Amie; Simpson, Michael A; Ackerman, Michael J; Hanna, Michael G; Behr, Elijah R; Matthews, Emma

2018-04-14

Sudden infant death syndrome (SIDS) is the leading cause of post-neonatal infant death in high-income countries. Central respiratory system dysfunction seems to contribute to these deaths. Excitation that drives contraction of skeletal respiratory muscles is controlled by the sodium channel NaV1.4, which is encoded by the gene SCN4A. Variants in NaV1.4 that directly alter skeletal muscle excitability can cause myotonia, periodic paralysis, congenital myopathy, and myasthenic syndrome. SCN4A variants have also been found in infants with life-threatening apnoea and laryngospasm. We therefore hypothesised that rare, functionally disruptive SCN4A variants might be over-represented in infants who died from SIDS. We did a case-control study, including two consecutive cohorts that included 278 SIDS cases of European ancestry and 729 ethnically matched controls without a history of cardiovascular, respiratory, or neurological disease. We compared the frequency of rare variants in SCN4A between groups (minor allele frequency <0·00005 in the Exome Aggregation Consortium). We assessed biophysical characterisation of the variant channels using a heterologous expression system. Four (1·4%) of the 278 infants in the SIDS cohort had a rare functionally disruptive SCN4A variant compared with none (0%) of 729 ethnically matched controls (p=0·0057). Rare SCN4A variants that directly alter NaV1.4 function occur in infants who had died from SIDS. These variants are predicted to significantly alter muscle membrane excitability and compromise respiratory and laryngeal function. These findings indicate that dysfunction of muscle sodium channels is a potentially modifiable risk factor in a subset of infant sudden deaths. UK Medical Research Council, the Wellcome Trust, National Institute for Health Research, the British Heart Foundation, Biotronik, Cardiac Risk in the Young, Higher Education Funding Council for England, Dravet Syndrome UK, the Epilepsy Society, the Eunice Kennedy Shriver National Institute of Child Health & Human Development of the National Institutes of Health, and the Mayo Clinic Windland Smith Rice Comprehensive Sudden Cardiac Death Program. Copyright © 2018 The Author(s). Published by Elsevier Ltd. This is an Open Access article under the CC BY 4.0 license. Published by Elsevier Ltd.. All rights reserved.

Electrocardiographic Biomarkers for Detection of Drug-Induced Late Sodium Current Block

DOE Office of Scientific and Technical Information (OSTI.GOV)

Vicente, Jose; Johannesen, Lars; Hosseini, Meisam

Drugs that prolong the heart rate corrected QT interval (QTc) on the electrocardiogram (ECG) by blocking the hERG potassium channel and also block inward currents (late sodium or L-type calcium) are not associated with torsade de pointes (e.g. ranolazine and verapamil). Furthermore, identifying ECG signs of late sodium current block could aid in the determination of proarrhythmic risk for new drugs. A new cardiac safety paradigm for drug development (the "CiPA" initiative) will involve the preclinical assessment of multiple human cardiac ion channels and ECG biomarkers are needed to determine if there are unexpected ion channel effects in humans.

Electrocardiographic Biomarkers for Detection of Drug-Induced Late Sodium Current Block

DOE PAGES

Vicente, Jose; Johannesen, Lars; Hosseini, Meisam; ...

2016-12-30

Drugs that prolong the heart rate corrected QT interval (QTc) on the electrocardiogram (ECG) by blocking the hERG potassium channel and also block inward currents (late sodium or L-type calcium) are not associated with torsade de pointes (e.g. ranolazine and verapamil). Furthermore, identifying ECG signs of late sodium current block could aid in the determination of proarrhythmic risk for new drugs. A new cardiac safety paradigm for drug development (the "CiPA" initiative) will involve the preclinical assessment of multiple human cardiac ion channels and ECG biomarkers are needed to determine if there are unexpected ion channel effects in humans.

Evidence for a separate mechanism of toxicity for the Type I and the Type II pyrethroid insecticides.

PubMed

Breckenridge, Charles B; Holden, Larry; Sturgess, Nicholas; Weiner, Myra; Sheets, Larry; Sargent, Dana; Soderlund, David M; Choi, Jin-Sung; Symington, Steve; Clark, J Marshall; Burr, Steve; Ray, David

2009-11-01

Neurotoxicity and mechanistic data were collected for six alpha-cyano pyrethroids (beta-cyfluthrin, cypermethrin, deltamethrin, esfenvalerate, fenpropathrin and lambda-cyhalothrin) and up to six non-cyano containing pyrethroids (bifenthrin, S-bioallethrin [or allethrin], permethrin, pyrethrins, resmethrin [or its cis-isomer, cismethrin] and tefluthrin under standard conditions. Factor analysis and multivariate dissimilarity analysis were employed to evaluate four independent data sets comprised of (1) fifty-six behavioral and physiological parameters from an acute neurotoxicity functional observatory battery (FOB), (2) eight electrophysiological parameters from voltage clamp experiments conducted on the Na(v)1.8 sodium channel expressed in Xenopus oocytes, (3) indices of efficacy, potency and binding calculated for calcium ion influx across neuronal membranes, membrane depolarization and glutamate released from rat brain synaptosomes and (4) changes in chloride channel open state probability using a patch voltage clamp technique for membranes isolated from mouse neuroblastoma cells. The pyrethroids segregated into Type I (T--syndrome-tremors) and Type II (CS syndrome--choreoathetosis with salivation) groups based on FOB data. Of the alpha-cyano pyrethroids, deltamethrin, lambda-cyhalothrin, cyfluthrin and cypermethrin arrayed themselves strongly in a dose-dependent manner along two factors that characterize the CS syndrome. Esfenvalerate and fenpropathrin displayed weaker response profiles compared to the non-cyano pyrethroids. Visual clustering on multidimensional scaling (MDS) maps based upon sodium ion channel and calcium influx and glutamate release dissimilarities gave similar groupings. The non-cyano containing pyrethroids were arrayed in a dose-dependent manner along two different factors that characterize the T-syndrome. Bifenthrin was an outlier when MDS maps of the non-cyano pyrethroids were based on sodium ion channel characteristics and permethrin was an outlier when the MDS maps were based on calcium influx/glutamate release potency. Four of six alpha-cyano pyrethroids (lambda-cyfluthrin, cypermethrin, deltamethrin and fenpropathrin) reduced open chloride channel probability. The R-isomers of lambda-l-cyhalothrin reduced open channel probability whereas the S-isomers, antagonized the action of the R-isomers. None of the non-cyano pyrethroids reduced open channel probability, except bioallethrin, which gave a weak response. Overall, based upon neurotoxicity data and the effect of pyrethroids on sodium, calcium and chloride ion channels, it is proposed that bioallethrin, cismethrin, tefluthrin, bifenthrin and permethrin belong to one common mechanism group and deltamethrin, lambda-cyhalothrin, cyfluthrin and cypermethrin belong to a second. Fenpropathrin and esfenvalerate occupy an intermediate position between these two groups.

[Channels: a new way to revisit pathology].

PubMed

Fournier, Emmanuel

2014-02-01

Many "essential" diseases that manifest themselves in the form of crises or fits (epilepsies, episodic ataxia, periodic paralyses, myotonia, heart rhythm disorders, etc.) are due to ionic channel dysfunction and are thus referred to as "channelopathies". Some of these disorders are congenital, due to mutations of genes encoding channel subunits, while others result from toxic, immune or hormonal disturbances affecting channelfunction. Channelopathies take on a wide variety of clinical forms, depending on the type of channel (sodium, potassium, calcium, chloride...) and the type of dysfunction (loss or gain of function). Some apparently unrelated diseases affecting distinct organs are due to a similar dysfunction of the same channel, revealing unsuspected relationships between organs and between medical specialties. In addition, a given syndrome can be caused by distinct channel dysfunctions. This provides new opportunities for diferential diagnosis and specific correction of the causal defects, although some treatments find applications across multiple medical specialties.

Ion Channels in Innate and Adaptive Immunity

PubMed Central

Feske, Stefan; Wulff, Heike; Skolnik, Edward Y.

2016-01-01

Ion channels and transporters mediate the transport of charged ions across hydrophobic lipid membranes. In immune cells, divalent cations such as calcium, magnesium, and zinc have important roles as second messengers to regulate intracellular signaling pathways. By contrast, monovalent cations such as sodium and potassium mainly regulate the membrane potential, which indirectly controls the influx of calcium and immune cell signaling. Studies investigating human patients with mutations in ion channels and transporters, analysis of gene-targeted mice, or pharmacological experiments with ion channel inhibitors have revealed important roles of ionic signals in lymphocyte development and in innate and adaptive immune responses. We here review the mechanisms underlying the function of ion channels and transporters in lymphocytes and innate immune cells and discuss their roles in lymphocyte development, adaptive and innate immune responses, and autoimmunity, as well as recent efforts to develop pharmacological inhibitors of ion channels for immunomodulatory therapy. PMID:25861976

Extracellular zinc and ATP-gated P2X receptor calcium entry channels: New zinc receptors as physiological sensors and therapeutic targets.

PubMed

Schwiebert, Erik M; Liang, Lihua; Cheng, Nai-Lin; Williams, Clintoria Richards; Olteanu, Dragos; Welty, Elisabeth A; Zsembery, Akos

2005-12-01

In this review, we focus on two attributes of P2X receptor channel function, one essential and one novel. First, we propose that P2X receptors are extracellular sensors as well as receptors and ion channels. In particular, the large extracellular domain (that comprises 70% of the molecular mass of the receptor channel protein) lends itself to be a cellular sensor. Moreover, its exquisite sensitivity to extracellular pH, ionic strength, and multiple ligands evokes the function of a sensor. Second, we propose that P2X receptors are extracellular zinc receptors as well as receptors for nucleotides. We provide novel data in multiple publications and illustrative data in this invited review to suggest that zinc triggers ATP-independent activation of P2X receptor channel function. In this light, P2X receptors are the cellular site of integration between autocrine and paracrine zinc signaling and autocrine and paracrine purinergic signaling. P2X receptors may sense changes in these ligands as well as in extracellular pH and ionic strength and transduce these sensations via calcium and/or sodium entry and changes in membrane potential.

Divergent actions of the pyrethroid insecticides S-bioallethrin, tefluthrin, and deltamethrin on rat Na{sub v}1.6 sodium channels

DOE Office of Scientific and Technical Information (OSTI.GOV)

Tan Jianguo; Soderlund, David M., E-mail: dms6@cornell.ed

2010-09-15

We expressed rat Na{sub v}1.6 sodium channels in combination with the rat {beta}{sub 1} and {beta}{sub 2} auxiliary subunits in Xenopus laevis oocytes and evaluated the effects of the pyrethroid insecticides S-bioallethrin, deltamethrin, and tefluthrin on expressed sodium currents using the two-electrode voltage clamp technique. S-Bioallethrin, a type I structure, produced transient modification evident in the induction of rapidly decaying sodium tail currents, weak resting modification (5.7% modification at 100 {mu}M), and no further enhancement of modification upon repetitive activation by high-frequency trains of depolarizing pulses. By contrast deltamethrin, a type II structure, produced sodium tail currents that were {approx}more » 9-fold more persistent than those caused by S-bioallethrin, barely detectable resting modification (2.5% modification at 100 {mu}M), and 3.7-fold enhancement of modification upon repetitive activation. Tefluthrin, a type I structure with high mammalian toxicity, exhibited properties intermediate between S-bioallethrin and deltamethrin: intermediate tail current decay kinetics, much greater resting modification (14.1% at 100 {mu}M), and 2.8-fold enhancement of resting modification upon repetitive activation. Comparison of concentration-effect data showed that repetitive depolarization increased the potency of tefluthrin {approx} 15-fold and that tefluthrin was {approx} 10-fold more potent than deltamethrin as a use-dependent modifier of Na{sub v}1.6 sodium channels. Concentration-effect data from parallel experiments with the rat Na{sub v}1.2 sodium channel coexpressed with the rat {beta}{sub 1} and {beta}{sub 2} subunits in oocytes showed that the Na{sub v}1.6 isoform was at least 15-fold more sensitive to tefluthrin and deltamethrin than the Na{sub v}1.2 isoform. These results implicate sodium channels containing the Na{sub v}1.6 isoform as potential targets for the central neurotoxic effects of pyrethroids.« less

µ-Conotoxins Modulating Sodium Currents in Pain Perception and Transmission: A Therapeutic Potential

PubMed Central

Tosti, Elisabetta; Boni, Raffaele

2017-01-01

The Conus genus includes around 500 species of marine mollusks with a peculiar production of venomous peptides known as conotoxins (CTX). Each species is able to produce up to 200 different biological active peptides. Common structure of CTX is the low number of amino acids stabilized by disulfide bridges and post-translational modifications that give rise to different isoforms. µ and µO-CTX are two isoforms that specifically target voltage-gated sodium channels. These, by inducing the entrance of sodium ions in the cell, modulate the neuronal excitability by depolarizing plasma membrane and propagating the action potential. Hyperexcitability and mutations of sodium channels are responsible for perception and transmission of inflammatory and neuropathic pain states. In this review, we describe the current knowledge of µ-CTX interacting with the different sodium channels subtypes, the mechanism of action and their potential therapeutic use as analgesic compounds in the clinical management of pain conditions. PMID:28937587

Structure of a prokaryotic sodium channel pore reveals essential gating elements and an outer ion binding site common to eukaryotic channels.

PubMed

Shaya, David; Findeisen, Felix; Abderemane-Ali, Fayal; Arrigoni, Cristina; Wong, Stephanie; Nurva, Shailika Reddy; Loussouarn, Gildas; Minor, Daniel L

2014-01-23

Voltage-gated sodium channels (NaVs) are central elements of cellular excitation. Notwithstanding advances from recent bacterial NaV (BacNaV) structures, key questions about gating and ion selectivity remain. Here, we present a closed conformation of NaVAe1p, a pore-only BacNaV derived from NaVAe1, a BacNaV from the arsenite oxidizer Alkalilimnicola ehrlichei found in Mono Lake, California, that provides insight into both fundamental properties. The structure reveals a pore domain in which the pore-lining S6 helix connects to a helical cytoplasmic tail. Electrophysiological studies of full-length BacNaVs show that two elements defined by the NaVAe1p structure, an S6 activation gate position and the cytoplasmic tail "neck", are central to BacNaV gating. The structure also reveals the selectivity filter ion entry site, termed the "outer ion" site. Comparison with mammalian voltage-gated calcium channel (CaV) selectivity filters, together with functional studies, shows that this site forms a previously unknown determinant of CaV high-affinity calcium binding. Our findings underscore commonalities between BacNaVs and eukaryotic voltage-gated channels and provide a framework for understanding gating and ion permeation in this superfamily. © 2013. Published by Elsevier Ltd. All rights reserved.

Modeling the human Nav1.5 sodium channel: structural and mechanistic insights of ion permeation and drug blockade

PubMed Central

Ahmed, Marawan; Jalily Hasani, Horia; Ganesan, Aravindhan; Houghton, Michael; Barakat, Khaled

2017-01-01

Abnormalities in the human Nav1.5 (hNav1.5) voltage-gated sodium ion channel (VGSC) are associated with a wide range of cardiac problems and diseases in humans. Current structural models of hNav1.5 are still far from complete and, consequently, their ability to study atomistic interactions of this channel is very limited. Here, we report a comprehensive atomistic model of the hNav1.5 ion channel, constructed using homology modeling technique and refined through long molecular dynamics simulations (680 ns) in the lipid membrane bilayer. Our model was comprehensively validated by using reported mutagenesis data, comparisons with previous models, and binding to a panel of known hNav1.5 blockers. The relatively long classical MD simulation was sufficient to observe a natural sodium permeation event across the channel’s selectivity filters to reach the channel’s central cavity, together with the identification of a unique role of the lysine residue. Electrostatic potential calculations revealed the existence of two potential binding sites for the sodium ion at the outer selectivity filters. To obtain further mechanistic insight into the permeation event from the central cavity to the intracellular region of the channel, we further employed “state-of-the-art” steered molecular dynamics (SMD) simulations. Our SMD simulations revealed two different pathways through which a sodium ion can be expelled from the channel. Further, the SMD simulations identified the key residues that are likely to control these processes. Finally, we discuss the potential binding modes of a panel of known hNav1.5 blockers to our structural model of hNav1.5. We believe that the data presented here will enhance our understanding of the structure–property relationships of the hNav1.5 ion channel and the underlying molecular mechanisms in sodium ion permeation and drug interactions. The results presented here could be useful for designing safer drugs that do not block the hNav1.5 channel. PMID:28831242

Ion Channels in Brain Metastasis

PubMed Central

Klumpp, Lukas; Sezgin, Efe C.; Eckert, Franziska; Huber, Stephan M.

2016-01-01

Breast cancer, lung cancer and melanoma exhibit a high metastatic tropism to the brain. Development of brain metastases severely worsens the prognosis of cancer patients and constrains curative treatment options. Metastasizing to the brain by cancer cells can be dissected in consecutive processes including epithelial–mesenchymal transition, evasion from the primary tumor, intravasation and circulation in the blood, extravasation across the blood–brain barrier, formation of metastatic niches, and colonization in the brain. Ion channels have been demonstrated to be aberrantly expressed in tumor cells where they regulate neoplastic transformation, malignant progression or therapy resistance. Moreover, many ion channel modulators are FDA-approved drugs and in clinical use proposing ion channels as druggable targets for future anti-cancer therapy. The present review article aims to summarize the current knowledge on the function of ion channels in the different processes of brain metastasis. The data suggest that certain channel types involving voltage-gated sodium channels, ATP-release channels, ionotropic neurotransmitter receptors and gap junction-generating connexins interfere with distinct processes of brain metastazation. PMID:27618016

CONCENTRATION DEPENDENT ACCUMULATION OF [3H]-DELTAMETHRIN IN SODIUM CHANNEL N AV1.2 EXPRESSING XENOPUS LAEVIS OOCYTES.

EPA Science Inventory

Disruption of neuronal voltage-sensitive sodium channels (VSSCs) by pyrethroid insecticides such as deltamethrin (DLT) has been widely studied using Xenopus laevis oocytes transfected with VSSC. However, the extent of pyrethroid accumulation in VSSC-expressing oocytes is unknown....

Molecular Basis of Paralytic Neurotoxin Action on Voltage-Sensitive Sodium Channels

DTIC Science & Technology

1989-10-20

acting at neurotoxin receptor site 2, and markedly potentiated and prolonged by polypeptide a-scorpion toxins and sea anemone toxins acting at neurotoxin...to block the actions of scorpion toxins and sea anemone toxins on the sodium channel. Transmembrane organization of amino acid sequences between

Prevention of Paralytic Neurotoxin Action on Voltage-Sensitive Sodium Channels.

DTIC Science & Technology

1992-02-10

sodium channel from mammalian brain. Neurotoxin receptor site 3, which binds scorpion and sea anemone toxins, has been located to an extracellular...against them, and use these reagents to develop an effective treatment to prevent neurotoxicity of a-o toxins and sea anemone toxins; 2. Covalently

Mechanosensitive Piezo Channels in the Gastrointestinal Tract.

PubMed

Alcaino, C; Farrugia, G; Beyder, A

2017-01-01

Sensation of mechanical forces is critical for normal function of the gastrointestinal (GI) tract and abnormalities in mechanosensation are linked to GI pathologies. In the GI tract there are several mechanosensitive cell types-epithelial enterochromaffin cells, intrinsic and extrinsic enteric neurons, smooth muscle cells and interstitial cells of Cajal. These cells use mechanosensitive ion channels that respond to mechanical forces by altering transmembrane ionic currents in a process called mechanoelectrical coupling. Several mechanosensitive ionic conductances have been identified in the mechanosensory GI cells, ranging from mechanosensitive voltage-gated sodium and calcium channels to the mechanogated ion channels, such as the two-pore domain potassium channels K2P (TREK-1) and nonselective cation channels from the transient receptor potential family. The recently discovered Piezo channels are increasingly recognized as significant contributors to cellular mechanosensitivity. Piezo1 and Piezo2 are nonselective cationic ion channels that are directly activated by mechanical forces and have well-defined biophysical and pharmacologic properties. The role of Piezo channels in the GI epithelium is currently under investigation and their role in the smooth muscle syncytium and enteric neurons is still not known. In this review, we outline the current state of knowledge on mechanosensitive ion channels in the GI tract, with a focus on the known and potential functions of the Piezo channels. Copyright © 2017 Elsevier Inc. All rights reserved.

EFFECTS OF SUBSCUTE EXPOSURE TO NANOMOLAR CONCENTRATIONS OF METHYLMERCURY ON VOLTAGE-GATES SODIUM AND CALCIUM CURRENTS IN PC12 CELLS.

EPA Science Inventory

Methylmercury (CH3Hg+) alters the function of voltage-gated Na+ and Ca2+ channels in neuronal preparations following acute, in vitro, exposure. Because the developing nervous system is particularly sensitive to CH3Hg+ neurotoxicity, effects on voltage-gated Na+ (INa) and Ca2+ (IC...

Uncompacted Myelin Lamellae and Nodal Ion Channel Disruption in POEMS Syndrome.

PubMed

Hashimoto, Rina; Koike, Haruki; Takahashi, Mie; Ohyama, Ken; Kawagashira, Yuichi; Iijima, Masahiro; Sobue, Gen

2015-12-01

To elucidate the significance of uncompacted myelin lamellae (UML) and ion channel disruption at the nodes of Ranvier in the polyneuropathy, organomegaly, endocrinopathy, monoclonal gammopathy, and skin changes (POEMS) syndrome, we evaluated sural nerve biopsy specimens from 33 patients with POEMS syndrome and from 7 control patients. Uncompacted myelin lamellae distribution was assessed by electron microscopy and immunofluorescence microscopy. In the POEMS patient biopsies, UML were seen more frequently in small versus large myelinated fibers. Paranodes and Schmidt-Lanterman incisures, where normal physiologic UM is located, were frequently associated with UM. Widening of the nodes of Ranvier (i.e. segmental demyelination) was not associated with UML. There was axonal hollowing with neurofilament condensation at Schmidt-Lanterman incisures with abnormal UML, suggesting axonal damage at those sites in the POEMS patient biopsies. Myelin sheath irregularity was conspicuous in large myelinated fibers and was associated with abnormally widened bizarrely shaped Schmidt-Lanterman incisures. Indirect immunofluorescent studies revealed abnormalities of sodium (pan sodium) and potassium (KCNQ2) channels, even at nonwidened nodes of Ranvier. Thus, UML was not apparently associated with segmental demyelination but seemed to be associated with axonal damage. These observations suggest that nodal ion channel disruption may be associated with functional deficits in POEMS syndrome patient nerves.

Voltage gating by molecular subunits of Na+ and K+ ion channels: higher-dimensional cubic kinetics, rate constants, and temperature.

PubMed

Fohlmeister, Jürgen F

2015-06-01

The structural similarity between the primary molecules of voltage-gated Na and K channels (alpha subunits) and activation gating in the Hodgkin-Huxley model is brought into full agreement by increasing the model's sodium kinetics to fourth order (m(3) → m(4)). Both structures then virtually imply activation gating by four independent subprocesses acting in parallel. The kinetics coalesce in four-dimensional (4D) cubic diagrams (16 states, 32 reversible transitions) that show the structure to be highly failure resistant against significant partial loss of gating function. Rate constants, as fitted in phase plot data of retinal ganglion cell excitation, reflect the molecular nature of the gating transitions. Additional dimensions (6D cubic diagrams) accommodate kinetically coupled sodium inactivation and gating processes associated with beta subunits. The gating transitions of coupled sodium inactivation appear to be thermodynamically irreversible; response to dielectric surface charges (capacitive displacement) provides a potential energy source for those transitions and yields highly energy-efficient excitation. A comparison of temperature responses of the squid giant axon (apparently Arrhenius) and mammalian channel gating yields kinetic Q10 = 2.2 for alpha unit gating, whose transitions are rate-limiting at mammalian temperatures; beta unit kinetic Q10 = 14 reproduces the observed non-Arrhenius deviation of mammalian gating at low temperatures; the Q10 of sodium inactivation gating matches the rate-limiting component of activation gating at all temperatures. The model kinetics reproduce the physiologically large frequency range for repetitive firing in ganglion cells and the physiologically observed strong temperature dependence of recovery from inactivation. Copyright © 2015 the American Physiological Society.

Effect of phenytoin on sodium conductances in rat hippocampal CA1 pyramidal neurons

PubMed Central

Zeng, Zhen; Hill-Yardin, Elisa L.; Williams, David; O'Brien, Terence; Serelis, Andris

2016-01-01

The antiepileptic drug phenytoin (PHT) is thought to reduce the excitability of neural tissue by stabilizing sodium channels (NaV) in inactivated states. It has been suggested the fast-inactivated state (IF) is the main target, although slow inactivation (IS) has also been implicated. Other studies on local anesthetics with similar effects on sodium channels have implicated the NaV voltage sensor interactions. In this study, we reexamined the effect of PHT in both equilibrium and dynamic transitions between fast and slower forms of inactivation in rat hippocampal CA1 pyramidal neurons. The effects of PHT were observed on fast and slow inactivation processes, as well as on another identified “intermediate” inactivation process. The effect of enzymatic removal of IF was also studied, as well as effects on the residual persistent sodium current (INaP). A computational model based on a gating charge interaction was derived that reproduced a range of PHT effects on NaV equilibrium and state transitions. No effect of PHT on IF was observed; rather, PHT appeared to facilitate the occupancy of other closed states, either through enhancement of slow inactivation or through formation of analogous drug-bound states. The overall significance of these observations is that our data are inconsistent with the commonly held view that the archetypal NaV channel inhibitor PHT stabilizes fast inactivation states, and we demonstrate that conventional slow activation “IS” and the more recently identified intermediate-duration inactivation process “II” are the primary functional targets of PHT. In addition, we show that the traditional explanatory frameworks based on the “modulated receptor hypothesis” can be substituted by simple, physiologically plausible interactions with voltage sensors. Additionally, INaP was not preferentially inhibited compared with peak INa at short latencies (50 ms) by PHT. PMID:27489371

Functional Na+ Channels in Cell Adhesion probed by Transistor Recording

PubMed Central

Schmidtner, Markus; Fromherz, Peter

2006-01-01

Cell membranes in a tissue are in close contact to each other, embedded in the extracellular matrix. Standard electrophysiological methods are not able to characterize ion channels under these conditions. Here we consider the area of cell adhesion on a solid substrate as a model system. We used HEK 293 cells cultured on fibronectin and studied the activation of NaV1.4 sodium channels in the adherent membrane with field-effect transistors in a silicon substrate. Under voltage clamp, we compared the transistor response with the whole-cell current. We observed that the extracellular voltage in the cell-chip contact was proportional to the total membrane current. The relation was calibrated by alternating-current stimulation. We found that Na+ channels are present in the area of cell adhesion on fibronectin with a functionality and a density that is indistinguishable from the free membrane. The experiment provides a basis for studying selective accumulation and depletion of ion channels in cell adhesion and also for a development of cell-based biosensoric devices and neuroelectronic systems. PMID:16227504

Thermal Mechanisms of Millimeter Wave Stimulation of Excitable Cells

PubMed Central

Shapiro, Mikhail G.; Priest, Michael F.; Siegel, Peter H.; Bezanilla, Francisco

2013-01-01

Interactions between millimeter waves (MMWs) and biological systems have received increasing attention due to the growing use of MMW radiation in technologies ranging from experimental medical devices to telecommunications and airport security. Studies have shown that MMW exposure alters cellular function, especially in neurons and muscles. However, the biophysical mechanisms underlying such effects are still poorly understood. Due to the high aqueous absorbance of MMW, thermal mechanisms are likely. However, nonthermal mechanisms based on resonance effects have also been postulated. We studied MMW stimulation in a simplified preparation comprising Xenopus laevis oocytes expressing proteins that underlie membrane excitability. Using electrophysiological recordings simultaneously with 60 GHz stimulation, we observed changes in the kinetics and activity levels of voltage-gated potassium and sodium channels and a sodium-potassium pump that are consistent with a thermal mechanism. Furthermore, we showed that MMW stimulation significantly increased the action potential firing rate in oocytes coexpressing voltage-gated sodium and potassium channels, as predicted by thermal terms in the Hodgkin-Huxley model of neurons. Our results suggest that MMW stimulation produces significant thermally mediated effects on excitable cells via basic thermodynamic mechanisms that must be taken into account in the study and use of MMW radiation in biological systems. PMID:23790370

Addition of verapamil in the treatment of severe myoclonic epilepsy in infancy.

PubMed

Iannetti, Paola; Parisi, Pasquale; Spalice, Alberto; Ruggieri, Martino; Zara, Federico

2009-07-01

We report on the use of the voltage-gated calcium channel blocker (Vg-CCB), verapamil, as an add-on anticonvulsant medication in two girls, 4 and 14 years of age, who were affected by severe myoclonic epilepsy in infancy (SMEI) or Dravet syndrome, a channelopathy caused by abnormalities in the voltage-gated sodium channel neuronal type alpha1 subunit (SCN1A) gene at 2q24. Both girls had pharmacoresistant epilepsy and developmental delay. Mutation analysis for the SCN1A gene revealed a missense mutation in exon 2 in the 4-year-old girl. Verapamil was co-administered in both children with a prompt response in controlling status epilepticus, myoclonic jerks, and partial and generalized seizures. The therapeutic effect lasted 13 months in the 14-year-old girl, while it is still present after a 20-month follow-up period in the 4-year-old girl who, in addition, has experienced improvement in motor and language development. The verapamil vVg-CCB, which crosses the blood-brain barrier (BBB): (a) inhibits the P-glycoprotein, an active efflux transporter protein expressed in normal tissue, including the brain, which is believed to contribute to the in situ phenomenon of multidrug resistance; and (b) may regulate membrane depolarization induced by abnormal sodium channels functions by modulating the abnormal Ca++ influxes into neurons with subsequent cell resting. This is the first report on long-lasting verapamil therapy in SMEI. The functional consequences of such in vivo modulating effects on Ca++ channels could contribute to rational targeting for future molecular therapeutic approaches in pharmacoresistant epileptic channelopathies.

Serine protease activity in m-1 cortical collecting duct cells.

PubMed

Liu, Lian; Hering-Smith, Kathleen S; Schiro, Faith R; Hamm, L Lee

2002-04-01

An apical serine protease, channel-activating protease 1 (CAP1), augments sodium transport in A6 cells. Prostasin, a novel serine protease originally purified from seminal fluid, has been proposed to be the mammalian ortholog of CAP1. We have recently found functional evidence for a similar protease activity in the M-1 cortical collecting duct cell line. The purposes of the present studies were to determine whether prostasin (or CAP1) is present in collecting duct cells by use of mouse M-1 cells, to sequence mouse prostasin, and to further characterize the identity of the serine protease activity and additional functional features in M-1 cells. Using mouse expressed sequence tag sequences that are highly homologous to the published human prostasin sequence as templates, reverse transcription-polymerase chain reaction and RACE (rapid amplification of cDNA ends) were used to sequence mouse prostasin mRNA, which shows 99% identical to published mouse CAP1 sequence. A single 1800-bp transcript was found by Northern analysis, and this was not altered by aldosterone. Equivalent short-circuit current (I(eq)), which represents sodium transport in these cells, dropped to 59+/-3% of control value within 1 hour of incubation with aprotinin, a serine protease inhibitor. Trypsin increased the I(eq) in aprotinin-treated cells to the value of the control group within 5 minutes. Application of aprotinin not only inhibited amiloride sensitive I(eq) but also reduced transepithelial resistance (R(te)) to 43+/-2%, an effect not expected with simple inhibition of sodium channels. Trypsin partially reversed the effect of aprotinin on R(te). Another serine protease inhibitor, soybean trypsin inhibitor (STI), decreased I(eq) in M-1 cells. STI inhibited I(eq) gradually over 6 hours, and the inhibition of I(eq) by 2 inhibitors was additive. STI decreased transepithelial resistance much less than did aprotinin. Neither aldosterone nor dexamethasone significantly augmented protease activity or prostasin mRNA levels, and in fact, dexamethasone decreased prostasin mRNA expression. In conclusion, although prostasin is present in M-1 cells and probably augments sodium transport in these cells, serine proteases probably have other effects (eg, resistance) in the collecting duct in addition to effects on sodium channels. Steroids do not alter these effects in M-1 cells. Additional proteases are likely also present in mouse collecting duct cells.

Therapeutic Approaches to Genetic Ion Channelopathies and Perspectives in Drug Discovery

PubMed Central

Imbrici, Paola; Liantonio, Antonella; Camerino, Giulia M.; De Bellis, Michela; Camerino, Claudia; Mele, Antonietta; Giustino, Arcangela; Pierno, Sabata; De Luca, Annamaria; Tricarico, Domenico; Desaphy, Jean-Francois; Conte, Diana

2016-01-01

In the human genome more than 400 genes encode ion channels, which are transmembrane proteins mediating ion fluxes across membranes. Being expressed in all cell types, they are involved in almost all physiological processes, including sense perception, neurotransmission, muscle contraction, secretion, immune response, cell proliferation, and differentiation. Due to the widespread tissue distribution of ion channels and their physiological functions, mutations in genes encoding ion channel subunits, or their interacting proteins, are responsible for inherited ion channelopathies. These diseases can range from common to very rare disorders and their severity can be mild, disabling, or life-threatening. In spite of this, ion channels are the primary target of only about 5% of the marketed drugs suggesting their potential in drug discovery. The current review summarizes the therapeutic management of the principal ion channelopathies of central and peripheral nervous system, heart, kidney, bone, skeletal muscle and pancreas, resulting from mutations in calcium, sodium, potassium, and chloride ion channels. For most channelopathies the therapy is mainly empirical and symptomatic, often limited by lack of efficacy and tolerability for a significant number of patients. Other channelopathies can exploit ion channel targeted drugs, such as marketed sodium channel blockers. Developing new and more specific therapeutic approaches is therefore required. To this aim, a major advancement in the pharmacotherapy of channelopathies has been the discovery that ion channel mutations lead to change in biophysics that can in turn specifically modify the sensitivity to drugs: this opens the way to a pharmacogenetics strategy, allowing the development of a personalized therapy with increased efficacy and reduced side effects. In addition, the identification of disease modifiers in ion channelopathies appears an alternative strategy to discover novel druggable targets. PMID:27242528

Therapeutic Approaches to Genetic Ion Channelopathies and Perspectives in Drug Discovery.

PubMed

Imbrici, Paola; Liantonio, Antonella; Camerino, Giulia M; De Bellis, Michela; Camerino, Claudia; Mele, Antonietta; Giustino, Arcangela; Pierno, Sabata; De Luca, Annamaria; Tricarico, Domenico; Desaphy, Jean-Francois; Conte, Diana

2016-01-01

In the human genome more than 400 genes encode ion channels, which are transmembrane proteins mediating ion fluxes across membranes. Being expressed in all cell types, they are involved in almost all physiological processes, including sense perception, neurotransmission, muscle contraction, secretion, immune response, cell proliferation, and differentiation. Due to the widespread tissue distribution of ion channels and their physiological functions, mutations in genes encoding ion channel subunits, or their interacting proteins, are responsible for inherited ion channelopathies. These diseases can range from common to very rare disorders and their severity can be mild, disabling, or life-threatening. In spite of this, ion channels are the primary target of only about 5% of the marketed drugs suggesting their potential in drug discovery. The current review summarizes the therapeutic management of the principal ion channelopathies of central and peripheral nervous system, heart, kidney, bone, skeletal muscle and pancreas, resulting from mutations in calcium, sodium, potassium, and chloride ion channels. For most channelopathies the therapy is mainly empirical and symptomatic, often limited by lack of efficacy and tolerability for a significant number of patients. Other channelopathies can exploit ion channel targeted drugs, such as marketed sodium channel blockers. Developing new and more specific therapeutic approaches is therefore required. To this aim, a major advancement in the pharmacotherapy of channelopathies has been the discovery that ion channel mutations lead to change in biophysics that can in turn specifically modify the sensitivity to drugs: this opens the way to a pharmacogenetics strategy, allowing the development of a personalized therapy with increased efficacy and reduced side effects. In addition, the identification of disease modifiers in ion channelopathies appears an alternative strategy to discover novel druggable targets.

ADULT AND JUVENILE RAT SODIUM CHANNEL (NAV1.2 AND NAV1.3) SENSITIVITY TO THE PYRETHROID INSECTICIDE DELTAMETHRIN.

EPA Science Inventory

Adult rats are less sensitive than juveniles to the acute neurotoxicity of the Type II pyrethroid insecticide deltamethrin (DLT). Voltage-sensitive sodium channels (VSSCs) are the primary target of DLT and are differentially expressed during development, with expression of Nav1.2...

CK2 activity is required for the interaction of FGF14 with voltage-gated sodium channels and neuronal excitability

PubMed Central

Hsu, Wei-Chun J.; Scala, Federico; Nenov, Miroslav N.; Wildburger, Norelle C.; Elferink, Hannah; Singh, Aditya K.; Chesson, Charles B.; Buzhdygan, Tetyana; Sohail, Maveen; Shavkunov, Alexander S.; Panova, Neli I.; Nilsson, Carol L.; Rudra, Jai S.; Lichti, Cheryl F.; Laezza, Fernanda

2016-01-01

Recent data shows that fibroblast growth factor 14 (FGF14) binds to and controls the function of the voltage-gated sodium (Nav) channel with phenotypic outcomes on neuronal excitability. Mutations in the FGF14 gene in humans have been associated with brain disorders that are partially recapitulated in Fgf14−/− mice. Thus, signaling pathways that modulate the FGF14:Nav channel interaction may be important therapeutic targets. Bioluminescence-based screening of small molecule modulators of the FGF14:Nav1.6 complex identified 4,5,6,7-tetrabromobenzotriazole (TBB), a potent casein kinase 2 (CK2) inhibitor, as a strong suppressor of FGF14:Nav1.6 interaction. Inhibition of CK2 through TBB reduces the interaction of FGF14 with Nav1.6 and Nav1.2 channels. Mass spectrometry confirmed direct phosphorylation of FGF14 by CK2 at S228 and S230, and mutation to alanine at these sites modified FGF14 modulation of Nav1.6-mediated currents. In 1 d in vitro hippocampal neurons, TBB induced a reduction in FGF14 expression, a decrease in transient Na+ current amplitude, and a hyperpolarizing shift in the voltage dependence of Nav channel steady-state inactivation. In mature neurons, TBB reduces the axodendritic polarity of FGF14. In cornu ammonis area 1 hippocampal slices from wild-type mice, TBB impairs neuronal excitability by increasing action potential threshold and lowering firing frequency. Importantly, these changes in excitability are recapitulated in Fgf14−/− mice, and deletion of Fgf14 occludes TBB-dependent phenotypes observed in wild-type mice. These results suggest that a CK2-FGF14 axis may regulate Nav channels and neuronal excitability.—Hsu, W.-C. J., Scala, F., Nenov, M. N., Wildburger, N. C., Elferink, H., Singh, A. K., Chesson, C. B., Buzhdygan, T., Sohail, M., Shavkunov, A. S., Panova, N. I., Nilsson, C. L., Rudra, J. S., Lichti, C. F., Laezza, F. CK2 activity is required for the interaction of FGF14 with voltage-gated sodium channels and neuronal excitability. PMID:26917740

An external sodium ion binding site controls allosteric gating in TRPV1 channels

PubMed Central

Jara-Oseguera, Andres; Bae, Chanhyung; Swartz, Kenton J

2016-01-01

TRPV1 channels in sensory neurons are integrators of painful stimuli and heat, yet how they integrate diverse stimuli and sense temperature remains elusive. Here, we show that external sodium ions stabilize the TRPV1 channel in a closed state, such that removing the external ion leads to channel activation. In studying the underlying mechanism, we find that the temperature sensors in TRPV1 activate in two steps to favor opening, and that the binding of sodium to an extracellular site exerts allosteric control over temperature-sensor activation and opening of the pore. The binding of a tarantula toxin to the external pore also exerts control over temperature-sensor activation, whereas binding of vanilloids influences temperature-sensitivity by largely affecting the open/closed equilibrium. Our results reveal a fundamental role of the external pore in the allosteric control of TRPV1 channel gating and provide essential constraints for understanding how these channels can be tuned by diverse stimuli. DOI: http://dx.doi.org/10.7554/eLife.13356.001 PMID:26882503

The Phe932Ile mutation in KCNT1 channels associated with severe epilepsy, delayed myelination and leukoencephalopathy produces a loss-of-function channel phenotype.

PubMed

Evely, Katherine M; Pryce, Kerri D; Bhattacharjee, Arin

2017-05-20

Sodium-activated potassium (K Na ) channels contribute to firing frequency adaptation and slow after hyperpolarization. The KCNT1 gene (also known as SLACK) encodes a K Na subunit that is expressed throughout the central and peripheral nervous systems. Missense mutations of the SLACK C-terminus have been reported in several patients with rare forms of early onset epilepsy and in some cases severely delayed myelination. To date, such mutations identified in patients with autosomal dominant nocturnal frontal lobe epilepsy (ADNFLE), epilepsy of infancy with migrating focal seizures (EIMFS) and Ohtahara syndrome (OS) have been reported to be gain-of-function mutations (Villa and Combi, 2016). An exome sequencing study identified a p.Phe932Ile KCNT1 mutation as the disease-causing change in a child with severe early infantile epileptic encephalopathy and abnormal myelination (Vanderver et al., 2014). We characterized an analogous mutation in the rat Slack channel and unexpectedly found this mutation to produce a loss-of-function phenotype. In an effort to restore current, we tested the known Slack channel opener loxapine. Loxapine exhibited no effect, indicating that this mutation either caused the channel to be insensitive to this established opener or proper translation and trafficking to the membrane was disrupted. Protein analysis confirmed that while total mutant protein did not differ from wild type, membrane expression of the mutant channel was substantially reduced. Although gain-of-function mutations to the Slack channel are linked to epileptic phenotypes, this is the first reported loss-of-function mutation linked to severe epilepsy and delayed myelination. Copyright © 2017 IBRO. Published by Elsevier Ltd. All rights reserved.

Specific binding of lacosamide to collapsin response mediator protein 2 (CRMP2) and direct impairment of its canonical function: implications for the therapeutic potential of lacosamide.

PubMed

Wilson, Sarah M; Khanna, Rajesh

2015-04-01

The novel antiepileptic drug lacosamide (LCM; SPM927, Vimpat®) has been heralded as having a dual-mode of action through interactions with both the voltage-gated sodium channel and the neurite outgrowth-promoting collapsin response mediator protein 2 (CRMP2). Lacosamide's ability to dampen neuronal excitability through the voltage-gated sodium channel likely underlies its efficacy in attenuating the symptoms of epilepsy (i.e., seizures). While the role of CRMP2 in epilepsy has not been well studied, given the proposed involvement of circuit reorganization in epileptogenesis, the ability of lacosamide to alter CRMP2 function may prove disease modifying. Recently, however, the validity of lacosamide's interaction with CRMP2 has come under scrutiny. In this review, we address the contradictory reports concerning the binding of lacosamide to CRMP2 as well as the ability of lacosamide to directly impact CRMP2 function. Additionally, we address similarly the contradicting reports regarding the potential disease-modifying effect of lacosamide on the development and progression of epilepsy. As the vast majority of antiepileptic drugs influences only the symptoms of epilepsy, the ability to hinder disease progression would be a major breakthrough in efforts to cure or prevent this debilitating syndrome.

Specific binding of lacosamide to collapsin response mediator protein 2 (CRMP2) and direct impairment of its canonical function: implications for the therapeutic potential of lacosamide

PubMed Central

Wilson, Sarah M.; Khanna, Rajesh

2014-01-01

The novel anti-epileptic drug lacosamide (LCM; SPM927, Vimpat®) has been heralded as having a dual-mode of action through interactions with both the voltage-gated sodium channel and the neurite outgrowth-promoting collapsin response mediator protein 2 (CRMP2). Lacosamide’s ability to dampen neuronal excitability through the voltage-gated sodium channel likely underlies its efficacy in attenuating the symptoms of epilepsy (i.e. seizures). While the role of CRMP2 in epilepsy has not been well-studied, given the proposed involvement of circuit reorganization in epileptogenesis, the ability of lacosamide to alter CRMP2 function may prove disease-modifying. Recently, however, the validity of lacosamide’s interaction with CRMP2 has come under scrutiny. In this review, we address the contradictory reports concerning the binding of lacosamide to CRMP2, as well as, the ability of lacosamide to directly impact CRMP2 function. Additionally, we address similarly contradicting reports regarding the potential disease-modifying effect of lacosamide on the development and progression of epilepsy. As the vast majority of anti-epileptic drugs influence only the symptoms of epilepsy, the ability to hinder disease progression would be a major breakthrough in efforts to cure or prevent this debilitating syndrome. PMID:24944082

Bupivacaine inhibits large conductance, voltage- and Ca2+- activated K+ channels in human umbilical artery smooth muscle cells

PubMed Central

Martín, Pedro; Enrique, Nicolás; Palomo, Ana R. Roldán; Rebolledo, Alejandro; Milesi, Veronica

2012-01-01

Bupivacaine is a local anesthetic compound belonging to the amino amide group. Its anesthetic effect is commonly related to its inhibitory effect on voltage-gated sodium channels. However, several studies have shown that this drug can also inhibit voltage-operated K+ channels by a different blocking mechanism. This could explain the observed contractile effects of bupivacaine on blood vessels. Up to now, there were no previous reports in the literature about bupivacaine effects on large conductance voltage- and Ca2+-activated K+ channels (BKCa). Using the patch-clamp technique, it is shown that bupivacaine inhibits single-channel and whole-cell K+ currents carried by BKCa channels in smooth muscle cells isolated from human umbilical artery (HUA). At the single-channel level bupivacaine produced, in a concentration- and voltage-dependent manner (IC50 324 µM at +80 mV), a reduction of single-channel current amplitude and induced a flickery mode of the open channel state. Bupivacaine (300 µM) can also block whole-cell K+ currents (~45% blockage) in which, under our working conditions, BKCa is the main component. This study presents a new inhibitory effect of bupivacaine on an ion channel involved in different cell functions. Hence, the inhibitory effect of bupivacaine on BKCa channel activity could affect different physiological functions where these channels are involved. Since bupivacaine is commonly used during labor and delivery, its effects on umbilical arteries, where this channel is highly expressed, should be taken into account. PMID:22688134

Cardiac safety of lacosamide: the non-clinical perspective.

PubMed

Delaunois, A; Colomar, A; Depelchin, B O; Cornet, M

2015-11-01

Lacosamide is indicated for the adjunctive treatment of partial-onset seizures in adult patients. Unlike other sodium channel-blocking antiepileptic drugs, lacosamide selectively enhances sodium channel slow inactivation. Potential effects of lacosamide on cardiac sodium channels and their cardiovascular consequences were comprehensively assessed. This manuscript presents the non-clinical cardiac safety profile of lacosamide. Lacosamide was tested in vitro on sodium and L-type calcium currents from isolated human atrial myocytes and on hERG-mediated potassium currents from stably transfected HEK293 cells. Cardiac action potentials were recorded in guinea pig ventricular myocytes. In vivo, hemodynamic and ECG parameters were evaluated in anesthetized dogs and monkeys receiving acute cumulative intravenous doses of lacosamide. Following intravenous dosing with lacosamide, dose-dependent PR and QRS prolongation and ECG abnormalities (loss of P waves, atrioventricular and intraventricular blocks, junctional premature contractions) were observed in anesthetized dogs and monkeys. In vitro, lacosamide reduced human cardiac sodium currents in a concentration-, voltage- and state-dependent manner. Lacosamide reductions in Vmax in guinea pig myocytes were similar to lamotrigine and carbamazepine. Lacosamide showed no relevant inhibitory effects on hERG and L-type calcium channels and did not prolong QTc in vivo. ECG findings in anesthetized animals correlate well with in vitro sodium channel-related effects and are also consistent with those (PR prolongation, first-degree atrioventricular block) reported in healthy volunteers and patients with epilepsy. Both in vivo and in vitro effects were detected from exposure levels 1.5- to 2-fold above those achieved with the maximum-recommended human lacosamide dose (400 mg/day). © 2015 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

Cardiac Repolarization Abnormalities and Potential Evidence for Loss of Cardiac Sodium Currents on ECGs of Patients with Chagas' Heart Disease

NASA Technical Reports Server (NTRS)

Schlegel, T. T.; Medina, R.; Jugo, D.; Nunez, T. J.; Borrego, A.; Arellano, E.; Arenare, B.; DePalma, J. L.; Greco, E. C.; Starc, V.

2007-01-01

Some individuals with Chagas disease develop right precordial lead ST segment elevation in response to an ajmaline challenge test, and the prevalence of right bundle branch block (RBBB) is also high in Chagas disease. Because these same electrocardiographic abnormalities occur in the Brugada syndrome, which involves genetically defective cardiac sodium channels, acquired damage to cardiac sodium channels may also occur in Chagas disease. We studied several conventional and advanced resting 12-lead/derived Frank-lead ECG parameters in 34 patients with Chagas -related heart disease (mean age 39 14 years) and in 34 age-/gender-matched healthy controls. All ECG recordings were of 5-10 min duration, obtained in the supine position using high fidelity hardware/software (CardioSoft, Houston, TX). Even after excluding those Chagas patients who had resting BBBs, tachycardia and/or pathologic arrhythmia (n=8), significant differences remained in multiple conventional and advanced ECG parameters between the Chagas and control groups (n=26/group), especially in their respective QT interval variability indices, maximal spatial QRS-T angles and low frequency HRV powers (p=0.0006, p=0.0015 and p=0.0314 respectively). In relation to the issue of potential damage to cardiac sodium channels, the Chagas patients had: 1) greater than or equal to twice the incidence of resting ST segment elevation in leads V1-V3 (n=10/26 vs. n=5/26) and of both leftward (n=5/26 versus n=0/26) and rightward (n=7/26 versus n=3/26) QRS axis deviation than controls; 2) significantly increased filtered (40-250 Hz) QRS interval durations (92.1 8.5 versus 85.3 plus or minus 9.0 ms, p=0.022) versus controls; and 3) significantly decreased QT and especially JT interval durations versus controls (QT interval: 387.5 plus or minus 26.4 versus 408.9 plus or minus 34.6 ms, p=0.013; JT interval: 290.5 plus or minus 26.3 versus 314.8 plus or minus 31.3 ms; p=0.0029). Heart rates and Bazett-corrected QTc/JTc intervals were not significantly different between groups. Patients with Chagas heart disease have increased cardiac repolarization abnormalities, especially by advanced ECG. Moreover, as a group, they have decreased uncorrected JT and QT interval durations and increased filtered QRS interval durations (versus age/gender-matched controls), all suggesting a potential loss of cardiac sodium channel function that might be mediated, in part, by cardiac autonomic damage. Overall findings support Brugada et al's recent hypothesis that the pathway leading to sudden death may often be similar in Chagas' disease and Brugada syndrome i.e., damage to the sodium channel (infectious/immunologic/autonomic in Chagas' genetic in Brugada) with consequent loss of sodium currents may facilitate a phase II-reentry based arrhythmic substrate for ventricular fibrillation in both conditions. In general, JT interval-related results have been underreported in the Chagas literature.

Analysis of inter-residue contacts reveals folding stabilizers in P-loops of potassium, sodium, and TRPV channels.

PubMed

Korkosh, V S; Zhorov, B S; Tikhonov, D B

2016-05-01

The family of P-loop channels includes potassium, sodium, calcium, cyclic nucleotide-gated and TRPV channels, as well as ionotropic glutamate receptors. Despite vastly different physiological and pharmacological properties, the channels have structurally conserved folding of the pore domain. Furthermore, crystallographic data demonstrate surprisingly similar mutual disposition of transmembrane and membrane-diving helices. To understand determinants of this conservation, here we have compared available high-resolution structures of sodium, potassium, and TRPV1 channels. We found that some residues, which are in matching positions of the sequence alignment, occur in different positions in the 3D alignment. Surprisingly, we found 3D mismatches in well-packed P-helices. Analysis of energetics of individual residues in Monte Carlo minimized structures revealed cyclic patterns of energetically favorable inter- and intra-subunit contacts of P-helices with S6 helices. The inter-subunit contacts are rather conserved in all the channels, whereas the intra-subunit contacts are specific for particular types of the channels. Our results suggest that these residue-residue contacts contribute to the folding stabilization. Analysis of such contacts is important for structural and phylogenetic studies of homologous proteins.

In Silico Docking and Electrophysiological Characterization of Lacosamide Binding Sites on Collapsin Response Mediator Protein-2 Identifies a Pocket Important in Modulating Sodium Channel Slow Inactivation*

PubMed Central

Wang, Yuying; Brittain, Joel M.; Jarecki, Brian W.; Park, Ki Duk; Wilson, Sarah M.; Wang, Bo; Hale, Rachel; Meroueh, Samy O.; Cummins, Theodore R.; Khanna, Rajesh

2010-01-01

The anti-epileptic drug (R)-lacosamide ((2R)-2-(acetylamino)-N-benzyl-3-methoxypropanamide (LCM)) modulates voltage-gated sodium channels (VGSCs) by preferentially interacting with slow inactivated sodium channels, but the observation that LCM binds to collapsin response mediator protein 2 (CRMP-2) suggests additional mechanisms of action for LCM. We postulated that CRMP-2 levels affects the actions of LCM on VGSCs. CRMP-2 labeling by LCM analogs was competitively displaced by excess LCM in rat brain lysates. Manipulation of CRMP-2 levels in the neuronal model system CAD cells affected slow inactivation of VGSCs without any effects on other voltage-dependent properties. In silico docking was performed to identify putative binding sites in CRMP-2 that may modulate the effects of LCM on VGSCs. These studies identified five cavities in CRMP-2 that can accommodate LCM. CRMP-2 alanine mutants of key residues within these cavities were functionally similar to wild-type CRMP-2 as assessed by similar levels of enhancement in dendritic complexity of cortical neurons. Next, we examined the effects of expression of wild-type and mutant CRMP-2 constructs on voltage-sensitive properties of VGSCs in CAD cells: 1) steady-state voltage-dependent activation and fast-inactivation properties were not affected by LCM, 2) CRMP-2 single alanine mutants reduced the LCM-mediated effects on the ability of endogenous Na+ channels to transition to a slow inactivated state, and 3) a quintuplicate CRMP-2 alanine mutant further decreased this slow inactivated fraction. Collectively, these results identify key CRMP-2 residues that can coordinate LCM binding thus making it more effective on its primary clinical target. PMID:20538611

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

NASA Astrophysics Data System (ADS)

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.

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

PubMed

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

2008-02-15

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 microm) and carbamazepine (30-500 microm) 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.

Resistance Status and Resistance Mechanisms in a Strain of Aedes aegypti (Diptera: Culicidae) From Puerto Rico.

PubMed

Estep, Alden S; Sanscrainte, Neil D; Waits, Christy M; Louton, Jessica E; Becnel, James J

2017-11-07

Puerto Rico (PR) has a long history of vector-borne disease and insecticide-resistant Aedes aegypti (L.). Defining contributing mechanisms behind phenotypic resistance is critical for effective vector control intervention. However, previous studies from PR have each focused on only one mechanism of pyrethroid resistance. This study examines the contribution of P450-mediated enzymatic detoxification and sodium channel target site changes to the overall resistance phenotype of Ae. aegypti collected from San Juan, PR, in 2012. Screening of a panel of toxicants found broad resistance relative to the lab susceptible Orlando (ORL1952) strain. We identified significant resistance to representative Type I, Type II, and nonester pyrethroids, a sodium channel blocker, and a sodium channel blocking inhibitor, all of which interact with the sodium channel. Testing of fipronil, a chloride channel agonist, also showed low but significant levels of resistance. In contrast, the PR and ORL1952 strains were equally susceptible to chlorfenapyr, which has been suggested as an alternative public health insecticide. Molecular characterization of the strain indicated that two common sodium channel mutations were fixed in the population. Topical bioassay with piperonyl butoxide (PBO) indicated cytochrome P450-mediated detoxification accounts for approximately half of the resistance profile. Transcript expression screening of cytochrome P450s and glutathione-S-transferases identified the presence of overexpressed transcripts. This study of Puerto Rican Ae. aegypti with significant contributions from both genetic changes and enzymatic detoxification highlights the necessity of monitoring for resistance but also defining the multiple resistance mechanisms to inform effective mosquito control. Published by Oxford University Press on behalf of Entomological Society of America 2017. This work is written by US Government employees and is in the public domain in the US.

Activity of the anticonvulsant lacosamide in experimental and human epilepsy via selective effects on slow Na+ channel inactivation.

PubMed

Holtkamp, Dominik; Opitz, Thoralf; Niespodziany, Isabelle; Wolff, Christian; Beck, Heinz

2017-01-01

In human epilepsy, pharmacoresistance to antiepileptic drug therapy is a major problem affecting ~30% of patients with epilepsy. Many classical antiepileptic drugs target voltage-gated sodium channels, and their potent activity in inhibiting high-frequency firing has been attributed to their strong use-dependent blocking action. In chronic epilepsy, a loss of use-dependent block has emerged as a potential cellular mechanism of pharmacoresistance for anticonvulsants acting on voltage-gated sodium channels. The anticonvulsant drug lacosamide (LCM) also targets sodium channels, but has been shown to preferentially affect sodium channel slow inactivation processes, in contrast to most other anticonvulsants. We used whole-cell voltage clamp recordings in acutely isolated cells to investigate the effects of LCM on transient Na + currents. Furthermore, we used whole-cell current clamp recordings to assess effects on repetitive action potential firing in hippocampal slices. We show here that LCM exerts its effects primarily via shifting the slow inactivation voltage dependence to more hyperpolarized potentials in hippocampal dentate granule cells from control and epileptic rats, and from patients with epilepsy. It is important to note that this activity of LCM was maintained in chronic experimental and human epilepsy. Furthermore, we demonstrate that the efficacy of LCM in inhibiting high-frequency firing is undiminished in chronic experimental and human epilepsy. Taken together, these results show that LCM exhibits maintained efficacy in chronic epilepsy, in contrast to conventional use-dependent sodium channel blockers such as carbamazepine. They also establish that targeting slow inactivation may be a promising strategy for overcoming target mechanisms of pharmacoresistance. Wiley Periodicals, Inc. © 2016 International League Against Epilepsy.

Pyrethroids Differentially Alter Voltage-Gated Sodium Channels from the Honeybee Central Olfactory Neurons

PubMed Central

Kadala, Aklesso; Charreton, Mercedes; Jakob, Ingrid; Cens, Thierry; Rousset, Matthieu; Chahine, Mohamed; Le Conte, Yves; Charnet, Pierre; Collet, Claude

2014-01-01

The sensitivity of neurons from the honey bee olfactory system to pyrethroid insecticides was studied using the patch-clamp technique on central ‘antennal lobe neurons’ (ALNs) in cell culture. In these neurons, the voltage-dependent sodium currents are characterized by negative potential for activation, fast kinetics of activation and inactivation, and the presence of cumulative inactivation during train of depolarizations. Perfusion of pyrethroids on these ALN neurons submitted to repetitive stimulations induced (1) an acceleration of cumulative inactivation, and (2) a marked slowing of the tail current recorded upon repolarization. Cypermethrin and permethrin accelerated cumulative inactivation of the sodium current peak in a similar manner and tetramethrin was even more effective. The slow-down of channel deactivation was markedly dependent on the type of pyrethroid. With cypermethrin, a progressive increase of the tail current amplitude along with successive stimulations reveals a traditionally described use-dependent recruitment of modified sodium channels. However, an unexpected decrease in this tail current was revealed with tetramethrin. If one considers the calculated percentage of modified channels as an index of pyrethroids effects, ALNs are significantly more susceptible to tetramethrin than to permethrin or cypermethrin for a single depolarization, but this difference attenuates with repetitive activity. Further comparison with peripheral neurons from antennae suggest that these modifications are neuron type specific. Modeling the sodium channel as a multi-state channel with fast and slow inactivation allows to underline the effects of pyrethroids on a set of rate constants connecting open and inactivated conformations, and give some insights to their specificity. Altogether, our results revealed a differential sensitivity of central olfactory neurons to pyrethroids that emphasize the ability for these compounds to impair detection and processing of information at several levels of the bees olfactory pathway. PMID:25390654

Bromodomain-containing Protein 4 Activates Voltage-gated Sodium Channel 1.7 Transcription in Dorsal Root Ganglia Neurons to Mediate Thermal Hyperalgesia in Rats.

PubMed

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 likely mediated by the enhanced expression of voltage-gated sodium channel 1.7.

Changes in ion transport in inflammatory disease.

PubMed

Eisenhut, Michael

2006-03-29

Ion transport is essential for maintenance of transmembranous and transcellular electric potential, fluid transport and cellular volume. Disturbance of ion transport has been associated with cellular dysfunction, intra and extracellular edema and abnormalities of epithelial surface liquid volume. There is increasing evidence that conditions characterized by an intense local or systemic inflammatory response are associated with abnormal ion transport. This abnormal ion transport has been involved in the pathogenesis of conditions like hypovolemia due to fluid losses, hyponatremia and hypokalemia in diarrhoeal diseases, electrolyte abnormalities in pyelonephritis of early infancy, septicemia induced pulmonary edema, and in hypersecretion and edema induced by inflammatory reactions of the mucosa of the upper respiratory tract. Components of membranous ion transport systems, which have been shown to undergo a change in function during an inflammatory response include the sodium potassium ATPase, the epithelial sodium channel, the Cystic Fibrosis Transmembrane Conductance Regulator and calcium activated chloride channels and the sodium potassium chloride co-transporter. Inflammatory mediators, which influence ion transport are tumor necrosis factor, gamma interferon, interleukins, transforming growth factor, leukotrienes and bradykinin. They trigger the release of specific messengers like prostaglandins, nitric oxide and histamine which alter ion transport system function through specific receptors, intracellular second messengers and protein kinases. This review summarizes data on in vivo measurements of changes in ion transport in acute inflammatory conditions and in vitro studies, which have explored the underlying mechanisms. Potential interventions directed at a correction of the observed abnormalities are discussed.

Changes in ion transport in inflammatory disease

PubMed Central

Eisenhut, Michael

2006-01-01

Ion transport is essential for maintenance of transmembranous and transcellular electric potential, fluid transport and cellular volume. Disturbance of ion transport has been associated with cellular dysfunction, intra and extracellular edema and abnormalities of epithelial surface liquid volume. There is increasing evidence that conditions characterized by an intense local or systemic inflammatory response are associated with abnormal ion transport. This abnormal ion transport has been involved in the pathogenesis of conditions like hypovolemia due to fluid losses, hyponatremia and hypokalemia in diarrhoeal diseases, electrolyte abnormalites in pyelonephritis of early infancy, septicemia induced pulmonary edema, and in hypersecretion and edema induced by inflammatory reactions of the mucosa of the upper respiratory tract. Components of membranous ion transport systems, which have been shown to undergo a change in function during an inflammatory response include the sodium potassium ATPase, the epithelial sodium channel, the Cystic Fibrosis Transmembrane Conductance Regulator and calcium activated chloride channels and the sodium potassium chloride co-transporter. Inflammatory mediators, which influence ion transport are tumor necrosis factor, gamma interferon, interleukins, transforming growth factor, leukotrienes and bradykinin. They trigger the release of specific messengers like prostaglandins, nitric oxide and histamine which alter ion transport system function through specific receptors, intracellular second messengers and protein kinases. This review summarizes data on in vivo measurements of changes in ion transport in acute inflammatory conditions and in vitro studies, which have explored the underlying mechanisms. Potential interventions directed at a correction of the observed abnormalities are discussed. PMID:16571116

Development and Testing of an In Vitro Assay for Screening of Potential Therapeutic Agents against Na Channel Neurotoxins

DTIC Science & Technology

1987-02-03

or by synthesis , the search almost certainly would have failed (3). Fortunately, the accumulated knowledge of sodium channel pharmacology allows the...distinct sodium channel domains. These studies were made possible by the synthesis of a tritiated analog of BTX, batrachotoxinin-A benzoate (BTX-B), of...elaborated upon. Creveling et al. (11) found that the local anesthetic lidocaine ethiodide was 10 times more potent at displacing specifically bound [3H]BTX-B

THE PRESENCE OF A B SUBUNIT INCREASES SENSITIVITY OF SODIUM CHANNEL NAV1.3, BUT NOT NAV1.2, TO TYPE II PYRETHROIDS.

EPA Science Inventory

Voltage-sensitive sodium channels (VSSCs) are a primary target of pyrethroid insecticides. VSSCs are comprised of a pore-forming ¿ and auxillary ß subunits, and multiple isoforms of both subunit types exist. The sensitivity of different isoform combinations to pyrethroids has not...

Slack sodium-activated potassium channel membrane expression requires p38 mitogen-activated protein kinase phosphorylation.

PubMed

Gururaj, Sushmitha; Fleites, John; Bhattacharjee, Arin

2016-04-01

p38 MAPK has long been understood as an inducible kinase under conditions of cellular stress, but there is now increasing evidence to support its role in the regulation of neuronal function. Several phosphorylation targets have been identified, an appreciable number of which are ion channels, implicating the possible involvement of p38 MAPK in neuronal excitability. The KNa channel Slack is an important protein to be studied as it is highly and ubiquitously expressed in DRG neurons and is important in the maintenance of their firing accommodation. We sought to examine if the Slack channel could be a substrate of p38 MAPK activity. First, we found that the Slack C-terminus contains two putative p38 MAPK phosphorylation sites that are highly conserved across species. Second, we show via electrophysiology experiments that KNa currents and further, Slack currents, are subject to tonic modulation by p38 MAPK. Third, biochemical approaches revealed that Slack channel regulation by p38 MAPK occurs through direct phosphorylation at the two putative sites of interaction, and mutating both sites prevented surface expression of Slack channels. Based on these results, we conclude that p38 MAPK is an obligate regulator of Slack channel function via the trafficking of channels into the membrane. The present study identifies Slack KNa channels as p38 MAPK substrates. Copyright © 2015 Elsevier Ltd. All rights reserved.

Slack Sodium-activated Potassium Channel Membrane Expression Requires p38 Mitogen-Activated Protein Kinase Phosphorylation

PubMed Central

Gururaj, Sushmitha; Fleites, John; Bhattacharjee, Arin

2016-01-01

p38 MAPK has long been understood as an inducible kinase under conditions of cellular stress, but there is now increasing evidence to support its role in the regulation of neuronal function. Several phosphorylation targets have been identified, an appreciable number of which are ion channels, implicating the possible involvement of p38 MAPK in neuronal excitability. The KNa channel Slack is an important protein to be studied as it is highly and ubiquitously expressed in DRG neurons and is important in the maintenance of their firing accommodation. We sought to examine if the Slack channel could be a substrate of p38 MAPK activity. First, we found that the Slack C-terminus contains two putative p38 MAPK phosphorylation sites that are highly conserved across species. Second, we show via electrophysiology experiments that KNa currents and further, Slack currents, are subject to tonic modulation by p38 MAPK. Third, biochemical approaches revealed that Slack channel regulation by p38 MAPK occurs through direct phosphorylation at the two putative sites of interaction, and mutating both sites prevented surface expression of Slack channels. Based on these results, we conclude that p38 MAPK is an obligate regulator of Slack channel function via the trafficking of channels into the membrane. The present study identifies Slack KNa channels as p38 MAPK substrates. PMID:26721627

Transepithelial nasal potential difference (NPD) measurements in cystic fibrosis (CF).

PubMed

Sands, Dorota

2013-01-01

The main underlying physiologic abnormality in cystic fibrosis (CF) is dysfunction of the CF transmembrane conductance regulator (CFTR), which results in abnormal transport of sodium and chloride across epithelial surfaces. CFTR function could be tested in vivo using measurements of nasal transepithelial potential difference (PD). Nasal measurements show characteristic features of CF epithelia, including hyperpolarized baseline readings (basal PD), excessive depolarization in response to sodium channel inhibitors, such as amiloride (ΔAmiloride), and little or no chloride (Cl-) secretion in response to isoproterenol in a chloride-free solution (ΔCl- free-isoproterenol). PD test is applied for CF diagnosis and monitoring of new therapeutic modulations and corrections.

Ciguatoxins activate specific cold pain pathways to elicit burning pain from cooling

PubMed Central

Vetter, Irina; Touska, Filip; Hess, Andreas; Hinsbey, Rachel; Sattler, Simon; Lampert, Angelika; Sergejeva, Marina; Sharov, Anastasia; Collins, Lindon S; Eberhardt, Mirjam; Engel, Matthias; Cabot, Peter J; Wood, John N; Vlachová, Viktorie; Reeh, Peter W; Lewis, Richard J; Zimmermann, Katharina

2012-01-01

Ciguatoxins are sodium channel activator toxins that cause ciguatera, the most common form of ichthyosarcotoxism, which presents with peripheral sensory disturbances, including the pathognomonic symptom of cold allodynia which is characterized by intense stabbing and burning pain in response to mild cooling. We show that intraplantar injection of P-CTX-1 elicits cold allodynia in mice by targeting specific unmyelinated and myelinated primary sensory neurons. These include both tetrodotoxin-resistant, TRPA1-expressing peptidergic C-fibres and tetrodotoxin-sensitive A-fibres. P-CTX-1 does not directly open heterologously expressed TRPA1, but when co-expressed with Nav channels, sodium channel activation by P-CTX-1 is sufficient to drive TRPA1-dependent calcium influx that is responsible for the development of cold allodynia, as evidenced by a large reduction of excitatory effect of P-CTX-1 on TRPA1-deficient nociceptive C-fibres and of ciguatoxin-induced cold allodynia in TRPA1-null mutant mice. Functional MRI studies revealed that ciguatoxin-induced cold allodynia enhanced the BOLD (Blood Oxygenation Level Dependent) signal, an effect that was blunted in TRPA1-deficient mice, confirming an important role for TRPA1 in the pathogenesis of cold allodynia. PMID:22850668

Development of lacosamide for the treatment of partial-onset seizures.

PubMed

Doty, Pamela; Hebert, David; Mathy, Francois-Xavier; Byrnes, William; Zackheim, James; Simontacchi, Kelly

2013-07-01

Lacosamide is an antiepileptic drug (AED) available in multiple formulations that was first approved in 2008 as adjunctive therapy for partial-onset seizures (POS) in adults. Unlike traditional sodium channel blockers affecting fast inactivation, lacosamide selectively enhances sodium channel slow inactivation. This mechanism of action results in stabilization of hyperexcitable neuronal membranes, inhibition of neuronal firing, and reduction in long-term channel availability without affecting physiological function. Lacosamide has a well-characterized and favorable pharmacokinetic profile, including a fast absorption rate, minimal or no interaction with cytochrome P-450 izoenzymes, and a low potential for drug-drug interactions. Lacosamide clinical development included three placebo-controlled, double-blind, randomized trials conducted in more than 1300 patients, each demonstrating safety and efficacy of lacosamide compared to placebo as adjunctive therapy for adults with POS. The clinical use of lacosamide may broaden, pending results of trials evaluating its use as monotherapy for POS in adults, as treatment for epilepsy in pediatric subjects, and as adjunctive treatment for uncontrolled primary generalized tonic-clonic seizures in those with idiopathic generalized epilepsy. © 2013 The Authors. Annals of the New York Academy of Sciences published by Wiley Periodicals Inc. on behalf of The New York Academy of Sciences.

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

PubMed

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

2018-02-15

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 (I Na ) 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 Na v 1.5 channels, we examined how restricting the extracellular space modulates I Na 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 I Na at step potentials near the threshold of I Na activation and decreased peak I Na at step potentials far above threshold (7 cells, P = 0.0156, Wilcoxon signed rank test). These effects were consistent with corresponding control simulations with a uniform Na + channel distribution. In the intercalated disc computer model, redistributing the Na + channels into a central cluster of the disc potentiated ephaptic effects. Moreover, ephaptic impulse transmission from one cell to another was facilitated by clusters of Na + channels facing each other across the intercellular cleft when gap junctional coupling was reduced. In conclusion, our proof-of-principle experiments demonstrate that confining the extracellular space modulates cardiac I Na , and our simulations reveal the functional role of the aggregation of Na + channels in the perinexus. These findings highlight novel concepts in the physiology of cardiac excitation. © 2017 The Authors. The Journal of Physiology © 2017 The Physiological Society.

Evolutionary diversification of Mesobuthus α-scorpion toxins affecting sodium channels.

PubMed

Zhu, Shunyi; Peigneur, Steve; Gao, Bin; Lu, Xiuxiu; Cao, Chunyang; Tytgat, Jan

2012-01-01

α-Scorpion toxins constitute a family of peptide modulators that induce a prolongation of the action potential of excitable cells by inhibiting voltage-gated sodium channel inactivation. Although they all adopt a conserved structural scaffold, the potency and phylogentic preference of these toxins largely vary, which render them an intriguing model for studying evolutionary diversification among family members. Here, we report molecular characterization of a new multigene family of α-toxins comprising 13 members (named MeuNaTxα-1 to MeuNaTxα-13) from the scorpion Mesobuthus eupeus. Of them, five native toxins (MeuNaTxα-1 to -5) were purified to homogeneity from the venom and the solution structure of MeuNaTxα-5 was solved by nuclear magnetic resonance. A systematic functional evaluation of MeuNaTxα-1, -2, -4, and -5 was conducted by two-electrode voltage-clamp recordings on seven cloned mammalian voltage-gated sodium channels (Na(v)1.2 to Na(v)1.8) and the insect counterpart DmNa(v)1 expressed in Xenopus oocytes. Results show that all these four peptides slow inactivation of DmNa(v)1 and are inactive on Na(v)1.8 at micromolar concentrations. However, they exhibit differential specificity for the other six channel isoforms (Na(v)1.2 to Na(v)1.7), in which MeuNaTxα-4 shows no activity on these isoforms and thus represents the first Mesobuthus-derived insect-selective α-toxin identified so far with a half maximal effective concentration of 130 ± 2 nm on DmNa(v)1 and a half maximal lethal dose of about 200 pmol g(-1) on the insect Musca domestica; MeuNaTxα-2 only affects Na(v)1.4; MeuNaTxα-1 and MeuNaTxα-5 have a wider range of channel spectrum, the former active on Na(v)1.2, Na(v)1.3, Na(v)1.6, and Na(v)1.7, whereas the latter acting on Na(v)1.3-Na(v)1.7. Remarkably, MeuNaTxα-4 and MeuNaTxα-5 are two nearly identical peptides differing by only one point mutation at site 50 (A50V) but exhibit rather different channel subtype selectivity, highlighting a switch role of this site in altering the target specificity. By the maximum likelihood models of codon substitution, we detected nine positively selected sites (PSSs) that could be involved in functional diversification of Mesobuthus α-toxins. The PSSs include site 50 and other seven sites located in functional surfaces of α-toxins. This work represents the first thorough investigation of evolutionary diversification of α-toxins derived from a specific scorpion lineage from the perspectives of sequence, structure, function, and evolution.

The Nav1.2 channel is regulated by GSK3

PubMed Central

James, Thomas F.; Nenov, Miroslav N.; Wildburger, Norelle C.; Lichti, Cheryl; Luisi, Jonathan; Vergara, Fernanda; Panova-Electronova, Neli I.; Nilsson, Carol L.; Rudra, Jai; Green, Thomas A.; Labate, Demetrio; Laezza, Fernanda

2015-01-01

Background Phosphorylation plays an essential role in regulating the voltage-gated sodium (Nav) channels and excitability. Yet, a surprisingly limited number of kinases have been identified as regulators of Nav channels. Herein, we posited that glycogen synthase kinase 3 (GSK3), a critical kinase found associated with numerous brain disorders, might directly regulate neuronal Nav channels. Methods We used patch-clamp electrophysiology to record sodium currents from Nav1.2 channels stably expressed in HEK-293 cells. mRNA and protein levels were quantified with RT-PCR, Western blot, or confocal microscopy, and in vitro phosphorylation and mass spectrometry to identify phosphorylated residues. Results We found that exposure of cells to GSK3 inhibitor XIII significantly potentiates the peak current density of Nav1.2, a phenotype reproduced by silencing GSK3 with siRNA. Contrarily, overexpression of GSK3β suppressed Nav1.2-encoded currents. Neither mRNA nor total protein expression were changed upon GSK3 inhibition. Cell surface labeling of CD4-chimeric constructs expressing intracellular domains of the Nav1.2 channel indicates that cell surface expression of CD4-Nav1.2-Ctail was up-regulated upon pharmacological inhibition of GSK3, resulting in an increase of surface puncta at the plasma membrane. Finally, using in vitro phosphorylation in combination with high resolution mass spectrometry, we further demonstrate that GSK3β phosphorylates T1966 at the C-terminal tail of Nav1.2. Conclusion These findings provide evidence for a new mechanism by which GSK3 modulate Nav channel function via its C-terminal tail. General Significance These findings provide fundamental knowledge in understanding signaling dysfunction common in several neuropsychiatric disorders. PMID:25615535

Regulation of voltage-gated sodium channel expression in cancer: hormones, growth factors and auto-regulation

PubMed Central

Fraser, Scott P.; Ozerlat-Gunduz, Iley; Brackenbury, William J.; Fitzgerald, Elizabeth M.; Campbell, Thomas M.; Coombes, R. Charles; Djamgoz, Mustafa B. A.

2014-01-01

Although ion channels are increasingly being discovered in cancer cells in vitro and in vivo, and shown to contribute to different aspects and stages of the cancer process, much less is known about the mechanisms controlling their expression. Here, we focus on voltage-gated Na+ channels (VGSCs) which are upregulated in many types of carcinomas where their activity potentiates cell behaviours integral to the metastatic cascade. Regulation of VGSCs occurs at a hierarchy of levels from transcription to post-translation. Importantly, mainstream cancer mechanisms, especially hormones and growth factors, play a significant role in the regulation. On the whole, in major hormone-sensitive cancers, such as breast and prostate cancer, there is a negative association between genomic steroid hormone sensitivity and functional VGSC expression. Activity-dependent regulation by positive feedback has been demonstrated in strongly metastatic cells whereby the VGSC is self-sustaining, with its activity promoting further functional channel expression. Such auto-regulation is unlike normal cells in which activity-dependent regulation occurs mostly via negative feedback. Throughout, we highlight the possible clinical implications of functional VGSC expression and regulation in cancer. PMID:24493753

DRG Voltage-Gated Sodium Channel 1.7 Is Upregulated in Paclitaxel-Induced Neuropathy in Rats and in Humans with Neuropathic Pain.

PubMed

Li, Yan; North, Robert Y; Rhines, Laurence D; Tatsui, Claudio Esteves; Rao, Ganesh; Edwards, Denaya D; Cassidy, Ryan M; Harrison, Daniel S; Johansson, Caj A; Zhang, Hongmei; Dougherty, Patrick M

2018-01-31

Chemotherapy-induced peripheral neuropathy (CIPN) is a common adverse effect experienced by cancer patients receiving treatment with paclitaxel. The voltage-gated sodium channel 1.7 (Na v 1.7) plays an important role in multiple preclinical models of neuropathic pain and in inherited human pain phenotypes, and its gene expression is increased in dorsal root ganglia (DRGs) of paclitaxel-treated rats. Hence, the potential of change in the expression and function of Na v 1.7 protein in DRGs from male rats with paclitaxel-related CIPN and from male and female humans with cancer-related neuropathic pain was tested here. Double immunofluorescence in CIPN rats showed that Na v 1.7 was upregulated in small DRG neuron somata, especially those also expressing calcitonin gene-related peptide (CGRP), and in central processes of these cells in the superficial spinal dorsal horn. Whole-cell patch-clamp recordings in rat DRG neurons revealed that paclitaxel induced an enhancement of ProTx II (a selective Na v 1.7 channel blocker)-sensitive sodium currents. Bath-applied ProTx II suppressed spontaneous action potentials in DRG neurons occurring in rats with CIPN, while intrathecal injection of ProTx II significantly attenuated behavioral signs of CIPN. Complementarily, DRG neurons isolated from segments where patients had a history of neuropathic pain also showed electrophysiological and immunofluorescence results indicating an increased expression of Na v 1.7 associated with spontaneous activity. Na v 1.7 was also colocalized in human cells expressing transient receptor potential vanilloid 1 and CGRP. Furthermore, ProTx II decreased firing frequency in human DRGs with spontaneous action potentials. This study suggests that Na v 1.7 may provide a potential new target for the treatment of neuropathic pain, including chemotherapy (paclitaxel)-induced neuropathic pain. SIGNIFICANCE STATEMENT This work demonstrates that the expression and function of the voltage-gated sodium channel Na v 1.7 are increased in a preclinical model of chemotherapy-induced peripheral neuropathy (CIPN), the most common treatment-limiting side effect of all the most common anticancer therapies. This is key as gain-of-function mutations in human Na v 1.7 recapitulate both the distribution and pain percept as shown by CIPN patients. This work also shows that Na v 1.7 is increased in human DRG neurons only in dermatomes where patients are experiencing acquired neuropathic pain symptoms. This work therefore has major translational impact, indicating an important novel therapeutic avenue for neuropathic pain as a class. Copyright © 2018 the authors 0270-6474/18/381124-13$15.00/0.

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

PubMed

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.

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).

PubMed

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 venomous species including cone snails, scorpions, spiders, and anemones. Enhanced activation or delayed inactivation of sodium channels by toxins is associated with the extremely rapid onset of tetanic/excitatory paralysis in envenomed prey animals. A strong selection pressure exists for the evolution of such toxins where there is a high chance of prey escape. However, despite their prevalence in other venomous species, toxins causing delay of sodium channel inhibition have never previously been described in vertebrate venoms. Here we show that Na V modulators, convergent with those of invertebrates, have evolved in the venom of the long-glanded coral snake. Calliotoxin represents a functionally novel class of 3FTx and a structurally novel class of Na V toxins that will provide significant insights into the pharmacology and physiology of Na V . The toxin represents a remarkable case of functional convergence between invertebrate and vertebrate venom systems in response to similar selection pressures. These results underscore the dynamic evolution of the Toxicofera reptile system and reinforces the value of using evolution as a roadmap for biodiscovery.

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)

PubMed Central

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.

2016-01-01

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 NaV1.4 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 (NaV) 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 NaV function provide a key defensive and predatory advantage to a range of invertebrate venomous species including cone snails, scorpions, spiders, and anemones. Enhanced activation or delayed inactivation of sodium channels by toxins is associated with the extremely rapid onset of tetanic/excitatory paralysis in envenomed prey animals. A strong selection pressure exists for the evolution of such toxins where there is a high chance of prey escape. However, despite their prevalence in other venomous species, toxins causing delay of sodium channel inhibition have never previously been described in vertebrate venoms. Here we show that NaV modulators, convergent with those of invertebrates, have evolved in the venom of the long-glanded coral snake. Calliotoxin represents a functionally novel class of 3FTx and a structurally novel class of NaV toxins that will provide significant insights into the pharmacology and physiology of NaV. The toxin represents a remarkable case of functional convergence between invertebrate and vertebrate venom systems in response to similar selection pressures. These results underscore the dynamic evolution of the Toxicofera reptile system and reinforces the value of using evolution as a roadmap for biodiscovery. PMID:27763551

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

PubMed Central

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

Differential state-dependent modification of rat Na{sub v}1.6 sodium channels expressed in human embryonic kidney (HEK293) cells by the pyrethroid insecticides tefluthrin and deltamethrin

DOE Office of Scientific and Technical Information (OSTI.GOV)

He, Bingjun; Soderlund, David M., E-mail: dms6@cornell.edu

2011-12-15

We expressed rat Na{sub v}1.6 sodium channels in combination with the rat {beta}1 and {beta}2 auxiliary subunits in human embryonic kidney (HEK293) cells and evaluated the effects of the pyrethroid insecticides tefluthrin and deltamethrin on expressed sodium currents using the whole-cell patch clamp technique. Both pyrethroids produced concentration-dependent, resting modification of Na{sub v}1.6 channels, prolonging the kinetics of channel inactivation and deactivation to produce persistent 'late' currents during depolarization and tail currents following repolarization. Both pyrethroids also produced concentration dependent hyperpolarizing shifts in the voltage dependence of channel activation and steady-state inactivation. Maximal shifts in activation, determined from the voltagemore » dependence of the pyrethroid-induced late and tail currents, were {approx} 25 mV for tefluthrin and {approx} 20 mV for deltamethrin. The highest attainable concentrations of these compounds also caused shifts of {approx} 5-10 mV in the voltage dependence of steady-state inactivation. In addition to their effects on the voltage dependence of inactivation, both compounds caused concentration-dependent increases in the fraction of sodium current that was resistant to inactivation following strong depolarizing prepulses. We assessed the use-dependent effects of tefluthrin and deltamethrin on Na{sub v}1.6 channels by determining the effect of trains of 1 to 100 5-ms depolarizing prepulses at frequencies of 20 or 66.7 Hz on the extent of channel modification. Repetitive depolarization at either frequency increased modification by deltamethrin by {approx} 2.3-fold but had no effect on modification by tefluthrin. Tefluthrin and deltamethrin were equally potent as modifiers of Na{sub v}1.6 channels in HEK293 cells using the conditions producing maximal modification as the basis for comparison. These findings show that the actions of tefluthrin and deltamethrin of Na{sub v}1.6 channels in HEK293 cells differ from the effects of these compounds on Na{sub v}1.6 channels in Xenopus oocytes and more closely reflect the actions of pyrethroids on channels in their native neuronal environment. -- Highlights: Black-Right-Pointing-Pointer We expressed rat Na{sub v}1.6 voltage-gated sodium channels in HEK293 cells. Black-Right-Pointing-Pointer Tefluthrin and deltamethrin caused resting modification of Na{sub v}1.6 channels. Black-Right-Pointing-Pointer Only deltamethrin exhibited use-dependent enhancement of modification. Black-Right-Pointing-Pointer State-dependent effects of pyrethroids are influenced by the cellular context. Black-Right-Pointing-Pointer Channels in HEK293 cells exhibit properties similar to native neuronal channels.« less

Distribution Patterns of Three Sodium Channel Mutations Associated with Pyrethroid Resistance in Rhipicephalus (Boophilus) Microplus Populations from North and South America, South Africa and Australia

USDA-ARS?s Scientific Manuscript database

Resistance to synthetic pyrethroids (SP) in the cattle tick Rhipicephalus (Boophilus) microplus is widespread throughout its distribution area. Three single nucleotide substitutions identified in the Domains II and III of the sodium channel gene of R. (B.) microplus are known to be associated with t...

Voltage-gated sodium channel as a target for metastatic risk reduction with re-purposed drugs

PubMed Central

Koltai, Tomas

2015-01-01

Objective: To determine the exact role of sodium channel proteins in migration, invasion and metastasis and understand the possible anti-invasion and anti-metastatic activity of repurposed drugs with voltage gated sodium channel blocking properties. Material and methods: A review of the published medical literature was performed searching for pharmaceuticals used in daily practice, with inhibitory activity on voltage gated sodium channels. For every drug found, the literature was reviewed in order to define if it may act against cancer cells as an anti-invasion and anti-metastatic agent and if it was tested with this purpose in the experimental and clinical settings. Results: The following pharmaceuticals that fulfill the above mentioned effects, were found: phenytoin, carbamazepine, valproate, lamotrigine, ranolazine, resveratrol, ropivacaine, lidocaine, mexiletine, flunarizine, and riluzole. Each of them are independently described and analyzed. Conclusions: The above mentioned pharmaceuticals have shown anti-metastatic and anti-invasion activity and many of them deserve to be tested in well-planned clinical trials as adjunct therapies for solid tumors and as anti-metastatic agents. Antiepileptic drugs like phenytoin, carbamazepine and valproate and the vasodilator flunarizine emerged as particularly useful for anti-metastatic purposes. PMID:27408684

The Permeability of the Sodium Channel to Metal Cations in Myelinated Nerve

PubMed Central

Hille, Bertil

1972-01-01

The relative permeability of sodium channels to eight metal cations is studied in myelinated nerve fibers. Ionic currents under voltage-clamp conditions are measured in Na-free solutions containing the test ion. Measured reversal potentials and the Goldman equation are used to calculate the permeability sequence: Na+ ≈ Li+ > Tl+ > K+. The ratio P K/P Na is 1/12. The permeabilities to Rb+, Cs+, Ca++, and Mg++ are too small to measure. The permeability ratios agree with observations on the squid giant axon and show that the reversal potential E Na differs significantly from the Nernst potential for Na+ in normal axons. Opening and closing rates for sodium channels are relatively insensitive to the ionic composition of the bathing medium, implying that gating is a structural property of the channel rather than a result of the movement or accumulation of particular ions around the channel. A previously proposed pore model of the channel accommodates the permeant metal cations in a partly hydrated form. The observed sequence of permeabilities follows the order expected for binding to a high field strength anion in Eisenman's theory of ion exchange equilibria. PMID:5025743

Hoiamide A, a Sodium Channel Activator of Unusual Architecture from a Consortium of Two Papua New Guinea Cyanobacteria

PubMed Central

Pereira, Alban; Cao, Zhengyu; Murray, Thomas F.; Gerwick, William H.

2009-01-01

Summary Hoiamide A, a novel bioactive cyclic depsipeptide, was isolated from an environmental assemblage of the marine cyanobacteria Lyngbya majuscula and Phormidium gracile collected in Papua New Guinea. This stereochemically complex metabolite possesses a highly unusual structure which likely derives from a mixed peptide-polyketide biogenetic origin, and includes a peptidic section featuring an acetate extended and S-adenosyl methionine modified isoleucine moiety, a triheterocyclic fragment bearing two a-methylated thiazolines and one thiazole, as well as a highly oxygenated and methylated C15-polyketide substructure. Pure hoiamide A potently inhibited [3H]batrachotoxin binding to voltage-gated sodium channels (IC50 = 92.8 nM) and activated sodium influx (EC50 = 1.73 μM) in mouse neocortical neurons, as well as exhibited modest cytotoxicity to cancer cells. Further investigation revealed that hoiamide A is a partial agonist of site 2 on the voltage gated sodium channel. PMID:19716479

Predicting a double mutant in the twilight zone of low homology modeling for the skeletal muscle voltage-gated sodium channel subunit beta-1 (Nav1.4 β1)

PubMed Central

Scior, Thomas; Paiz-Candia, Bertin; Islas, Ángel A.; Sánchez-Solano, Alfredo; Millan-Perez Peña, Lourdes; Mancilla-Simbro, Claudia; Salinas-Stefanon, Eduardo M.

2015-01-01

The molecular structure modeling of the β1 subunit of the skeletal muscle voltage-gated sodium channel (Nav1.4) was carried out in the twilight zone of very low homology. Structural significance can per se be confounded with random sequence similarities. Hence, we combined (i) not automated computational modeling of weakly homologous 3D templates, some with interfaces to analogous structures to the pore-bearing Nav1.4 α subunit with (ii) site-directed mutagenesis (SDM), as well as (iii) electrophysiological experiments to study the structure and function of the β1 subunit. Despite the distant phylogenic relationships, we found a 3D-template to identify two adjacent amino acids leading to the long-awaited loss of function (inactivation) of Nav1.4 channels. This mutant type (T109A, N110A, herein called TANA) was expressed and tested on cells of hamster ovary (CHO). The present electrophysiological results showed that the double alanine substitution TANA disrupted channel inactivation as if the β1 subunit would not be in complex with the α subunit. Exhaustive and unbiased sampling of “all β proteins” (Ig-like, Ig) resulted in a plethora of 3D templates which were compared to the target secondary structure prediction. The location of TANA was made possible thanks to another “all β protein” structure in complex with an irreversible bound protein as well as a reversible protein–protein interface (our “Rosetta Stone” effect). This finding coincides with our electrophysiological data (disrupted β1-like voltage dependence) and it is safe to utter that the Nav1.4 α/β1 interface is likely to be of reversible nature. PMID:25904995

Predicting a double mutant in the twilight zone of low homology modeling for the skeletal muscle voltage-gated sodium channel subunit beta-1 (Nav1.4 β1).

PubMed

Scior, Thomas; Paiz-Candia, Bertin; Islas, Ángel A; Sánchez-Solano, Alfredo; Millan-Perez Peña, Lourdes; Mancilla-Simbro, Claudia; Salinas-Stefanon, Eduardo M

2015-01-01

The molecular structure modeling of the β1 subunit of the skeletal muscle voltage-gated sodium channel (Nav1.4) was carried out in the twilight zone of very low homology. Structural significance can per se be confounded with random sequence similarities. Hence, we combined (i) not automated computational modeling of weakly homologous 3D templates, some with interfaces to analogous structures to the pore-bearing Nav1.4 α subunit with (ii) site-directed mutagenesis (SDM), as well as (iii) electrophysiological experiments to study the structure and function of the β1 subunit. Despite the distant phylogenic relationships, we found a 3D-template to identify two adjacent amino acids leading to the long-awaited loss of function (inactivation) of Nav1.4 channels. This mutant type (T109A, N110A, herein called TANA) was expressed and tested on cells of hamster ovary (CHO). The present electrophysiological results showed that the double alanine substitution TANA disrupted channel inactivation as if the β1 subunit would not be in complex with the α subunit. Exhaustive and unbiased sampling of "all β proteins" (Ig-like, Ig) resulted in a plethora of 3D templates which were compared to the target secondary structure prediction. The location of TANA was made possible thanks to another "all β protein" structure in complex with an irreversible bound protein as well as a reversible protein-protein interface (our "Rosetta Stone" effect). This finding coincides with our electrophysiological data (disrupted β1-like voltage dependence) and it is safe to utter that the Nav1.4 α/β1 interface is likely to be of reversible nature.

Sexy DEG/ENaC channels involved in gustatory detection of fruit fly pheromones.

PubMed

Pikielny, Claudio W

2012-11-06

Hydrocarbon pheromones on the cuticle of Drosophila melanogaster modulate the complex courtship behavior of males. Recently, three members of the degenerin/epithelial Na+ channel (DEG/ENaC) family of sodium channel subunits, Ppk25, Ppk23, and Ppk29 (also known as Nope), have been shown to function in gustatory perception of courtship-modulating contact pheromones. All three proteins are required for the activation of male courtship by female pheromones. Specific interactions between two of them have been demonstrated in cultured cells, suggesting that, in a subset of cells where they are coexpressed, these three subunits function within a common heterotrimeric DEG/ENaC channel. Such a DEG/ENaC channel may be gated by pheromones, either directly or indirectly, or alternatively may control the excitability of pheromone-sensing cells. In addition, these studies identify taste neurons that respond specifically to courtship-modulating pheromones and mediate their effects on male behavior. Two types of pheromone-sensing taste neurons, F and M cells, have been defined on the basis of their specific response to either female or male pheromones. These reports set the stage for the dissection of the molecular and cellular mechanisms that mediate gustatory detection of contact pheromones.

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

PubMed Central

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

Zn2+ reduction induces neuronal death with changes in voltage-gated potassium and sodium channel currents.

PubMed

Tian, Kun; He, Cong-Cong; Xu, Hui-Nan; Wang, Yu-Xiang; Wang, Hong-Gang; An, Di; Heng, Bin; Pang, Wei; Jiang, Yu-Gang; Liu, Yan-Qiang

2017-05-01

In the present study, cultured rat primary neurons were exposed to a medium containing N,N,N',N'-tetrakis(2-pyridylmethyl)ethylenediamine (TPEN), a specific cell membrane-permeant Zn 2+ chelator, to establish a model of free Zn 2+ deficiency in neurons. The effects of TPEN-mediated free Zn 2+ ion reduction on neuronal viability and on the performance of voltage-gated sodium channels (VGSCs) and potassium channels (Kvs) were assessed. Free Zn 2+ deficiency 1) markedly reduced the neuronal survival rate, 2) reduced the peak amplitude of I Na , 3) shifted the I Na activation curve towards depolarization, 4) modulated the sensitivity of sodium channel voltage-dependent inactivation to a depolarization voltage, and 5) increased the time course of recovery from sodium channel inactivation. In addition, free Zn 2+ deficiency by TPEN notably enhanced the peak amplitude of transient outward K + currents (I A ) and delayed rectifier K + currents (I K ), as well as caused hyperpolarization and depolarization directional shifts in their steady-state activation curves, respectively. Zn 2+ supplementation reversed the effects induced by TPEN. Our results indicate that free Zn 2+ deficiency causes neuronal damage and alters the dynamic characteristics of VGSC and Kv currents. Thus, neuronal injury caused by free Zn 2+ deficiency may correlate with its modulation of the electrophysiological properties of VGSCs and Kvs. Copyright © 2017 Elsevier GmbH. All rights reserved.

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

PubMed

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 involved in transduction or encoding of muscle stretch. We propose that NaVs contribute to multiple steps in sensory signaling by muscle spindles as it does in other types of slowly adapting sensory neurons. Copyright © 2017 the American Physiological Society.

Pharmacology of the Nav1.1 domain IV voltage sensor reveals coupling between inactivation gating processes.

PubMed

Osteen, Jeremiah D; Sampson, Kevin; Iyer, Vivek; Julius, David; Bosmans, Frank

2017-06-27

The Na v 1.1 voltage-gated sodium channel is a critical contributor to excitability in the brain, where pathological loss of function leads to such disorders as epilepsy, Alzheimer's disease, and autism. This voltage-gated sodium (Na v ) channel subtype also plays an important role in mechanical pain signaling by primary afferent somatosensory neurons. Therefore, pharmacologic modulation of Na v 1.1 represents a potential strategy for treating excitability disorders of the brain and periphery. Inactivation is a complex aspect of Na v channel gating and consists of fast and slow components, each of which may involve a contribution from one or more voltage-sensing domains. Here, we exploit the Hm1a spider toxin, a Na v 1.1-selective modulator, to better understand the relationship between these temporally distinct modes of inactivation and ask whether they can be distinguished pharmacologically. We show that Hm1a inhibits the gating movement of the domain IV voltage sensor (VSDIV), hindering both fast and slow inactivation and leading to an increase in Na v 1.1 availability during high-frequency stimulation. In contrast, ICA-121431, a small-molecule Na v 1.1 inhibitor, accelerates a subsequent VSDIV gating transition to accelerate entry into the slow inactivated state, resulting in use-dependent block. Further evidence for functional coupling between fast and slow inactivation is provided by a Na v 1.1 mutant in which fast inactivation removal has complex effects on slow inactivation. Taken together, our data substantiate the key role of VSDIV in Na v channel fast and slow inactivation and demonstrate that these gating processes are sequential and coupled through VSDIV. These findings provide insight into a pharmacophore on VSDIV through which modulation of inactivation gating can inhibit or facilitate Na v 1.1 function.

Activity of Palythoa caribaeorum Venom on Voltage-Gated Ion Channels in Mammalian Superior Cervical Ganglion Neurons.

PubMed

Lazcano-Pérez, Fernando; Castro, Héctor; Arenas, Isabel; García, David E; González-Muñoz, Ricardo; Arreguín-Espinosa, Roberto

2016-05-05

The Zoanthids are an order of cnidarians whose venoms and toxins have been poorly studied. Palythoa caribaeorum is a zoanthid commonly found around the Mexican coastline. In this study, we tested the activity of P. caribaeorum venom on voltage-gated sodium channel (NaV1.7), voltage-gated calcium channel (CaV2.2), the A-type transient outward (IA) and delayed rectifier (IDR) currents of KV channels of the superior cervical ganglion (SCG) neurons of the rat. These results showed that the venom reversibly delays the inactivation process of voltage-gated sodium channels and inhibits voltage-gated calcium and potassium channels in this mammalian model. The compounds responsible for these effects seem to be low molecular weight peptides. Together, these results provide evidence for the potential use of zoanthids as a novel source of cnidarian toxins active on voltage-gated ion channels.

Activity of Palythoa caribaeorum Venom on Voltage-Gated Ion Channels in Mammalian Superior Cervical Ganglion Neurons

PubMed Central

Lazcano-Pérez, Fernando; Castro, Héctor; Arenas, Isabel; García, David E.; González-Muñoz, Ricardo; Arreguín-Espinosa, Roberto

2016-01-01

The Zoanthids are an order of cnidarians whose venoms and toxins have been poorly studied. Palythoa caribaeorum is a zoanthid commonly found around the Mexican coastline. In this study, we tested the activity of P. caribaeorum venom on voltage-gated sodium channel (NaV1.7), voltage-gated calcium channel (CaV2.2), the A-type transient outward (IA) and delayed rectifier (IDR) currents of KV channels of the superior cervical ganglion (SCG) neurons of the rat. These results showed that the venom reversibly delays the inactivation process of voltage-gated sodium channels and inhibits voltage-gated calcium and potassium channels in this mammalian model. The compounds responsible for these effects seem to be low molecular weight peptides. Together, these results provide evidence for the potential use of zoanthids as a novel source of cnidarian toxins active on voltage-gated ion channels. PMID:27164140

Ion Channels in Obesity: Pathophysiology and Potential Therapeutic Targets

PubMed Central

Vasconcelos, Luiz H. C.; Souza, Iara L. L.; Pinheiro, Lílian S.; Silva, Bagnólia A.

2016-01-01

Obesity is a multifactorial disease related to metabolic disorders and associated with genetic determinants. Currently, ion channels activity has been linked to many of these disorders, in addition to the central regulation of food intake, energetic balance, hormone release and response, as well as the adipocyte cell proliferation. Therefore, the objective of this work is to review the current knowledge about the influence of ion channels in obesity development. This review used different sources of literature (Google Scholar, PubMed, Scopus, and Web of Science) to assess the role of ion channels in the pathophysiology of obesity. Ion channels present diverse key functions, such as the maintenance of physiological homeostasis and cell proliferation. Cell biology and pharmacological experimental evidences demonstrate that proliferating cells exhibit ion channel expression, conductance, and electrical properties different from the resting cells. Thereby, a large variety of ion channels has been identified in the pathogenesis of obesity such as potassium, sodium, calcium and chloride channels, nicotinic acetylcholine receptor and transient receptor potential channels. The fundamental involvement of these channels on the generation of obesity leads to the progress in the knowledge about the mechanisms responsible for the obesity pathophysiology, consequently emerging as new targets for pharmacological modulation. PMID:27065858

Ion Selectivity Mechanism in a Bacterial Pentameric Ligand-Gated Ion Channel

DOE Office of Scientific and Technical Information (OSTI.GOV)

Fritsch, Sebastian; Ivanov, Ivaylo; Wang, Hailong

2010-01-01

The proton-gated ion channel from Gloeobacter violaceus (GLIC) is a prokaryotic homolog of the eukaryotic nicotinic acetylcholine receptor that responds to the binding of neurotransmitter acetylcholine and mediates fast signal transmission. Recent emergence of a high-resolution crystal structure of GLIC captured in a potentially open state allowed detailed, atomic-level insight into ion conduction and selectivity mechanisms in these channels. Herein, we have examined the barriers to ion conduction and origins of ion selectivity in the GLIC channel by the construction of potential-of-mean-force profiles for sodium and chloride ions inside the transmembrane region. Our calculations reveal that the GLIC channel ismore » open for a sodium ion to transport, but presents a 11 kcal/mol free energy barrier for a chloride ion. Our collective findings identify three distinct contributions to the observed preference for the permeant ions. First, there is a substantial contribution due to a ring of negatively charged glutamate residues (E-2 ) at the narrow intracellular end of the channel. The negative electrostatics of this region and the ability of the glutamate side chains to directly bind cations would strongly favor the passage of sodium ions while hindering translocation of chloride ions. Second, our results imply a significant hydrophobic contribution to selectivity linked to differences in the desolvation penalty for the sodium versus chloride ions in the central hydrophobic region of the pore. This hydrophobic contribution is evidenced by the large free energy barriers experienced by Cl in the middle of the pore for both GLIC and the E-2 A mutant. Finally, there is a distinct contribution arising from the overall negative electrostatics of the channel.« less

Simultaneous mapping of membrane voltage and calcium in zebrafish heart in vivo reveals chamber-specific developmental transitions in ionic currents

PubMed Central

Hou, Jennifer H.; Kralj, Joel M.; Douglass, Adam D.; Engert, Florian; Cohen, Adam E.

2014-01-01

The cardiac action potential (AP) and the consequent cytosolic Ca2+ transient are key indicators of cardiac function. Natural developmental processes, as well as many drugs and pathologies change the waveform, propagation, or variability (between cells or over time) of these parameters. Here we apply a genetically encoded dual-function calcium and voltage reporter (CaViar) to study the development of the zebrafish heart in vivo between 1.5 and 4 days post fertilization (dpf). We developed a high-sensitivity spinning disk confocal microscope and associated software for simultaneous three-dimensional optical mapping of voltage and calcium. We produced a transgenic zebrafish line expressing CaViar under control of the heart-specific cmlc2 promoter, and applied ion channel blockers at a series of developmental stages to map the maturation of the action potential in vivo. Early in development, the AP initiated via a calcium current through L-type calcium channels. Between 90 and 102 h post fertilization (hpf), the ventricular AP switched to a sodium-driven upswing, while the atrial AP remained calcium driven. In the adult zebrafish heart, a sodium current drives the AP in both the atrium and ventricle. Simultaneous voltage and calcium imaging with genetically encoded reporters provides a new approach for monitoring cardiac development, and the effects of drugs on cardiac function. PMID:25309445

Simultaneous mapping of membrane voltage and calcium in zebrafish heart in vivo reveals chamber-specific developmental transitions in ionic currents.

PubMed

Hou, Jennifer H; Kralj, Joel M; Douglass, Adam D; Engert, Florian; Cohen, Adam E

2014-01-01

The cardiac action potential (AP) and the consequent cytosolic Ca(2+) transient are key indicators of cardiac function. Natural developmental processes, as well as many drugs and pathologies change the waveform, propagation, or variability (between cells or over time) of these parameters. Here we apply a genetically encoded dual-function calcium and voltage reporter (CaViar) to study the development of the zebrafish heart in vivo between 1.5 and 4 days post fertilization (dpf). We developed a high-sensitivity spinning disk confocal microscope and associated software for simultaneous three-dimensional optical mapping of voltage and calcium. We produced a transgenic zebrafish line expressing CaViar under control of the heart-specific cmlc2 promoter, and applied ion channel blockers at a series of developmental stages to map the maturation of the action potential in vivo. Early in development, the AP initiated via a calcium current through L-type calcium channels. Between 90 and 102 h post fertilization (hpf), the ventricular AP switched to a sodium-driven upswing, while the atrial AP remained calcium driven. In the adult zebrafish heart, a sodium current drives the AP in both the atrium and ventricle. Simultaneous voltage and calcium imaging with genetically encoded reporters provides a new approach for monitoring cardiac development, and the effects of drugs on cardiac function.

Identification of the Sensory Neuron Specific Regulatory Region for the Mouse Gene Encoding the Voltage Gated Sodium Channel Nav1.8

PubMed Central

Puhl, Henry L.; Ikeda, Stephen R.

2008-01-01

Voltage-gated sodium channels (VGSC) are critical membrane components that participate in the electrical activity of excitable cells. The type one VGSC family includes the tetrodotoxin insensitive sodium channel, Nav1.8, encoded by the Scn10a gene. Nav1.8 expression is restricted to small and medium diameter nociceptive sensory neurons of the dorsal root (DRG) and cranial sensory ganglia. In order to understand the stringent transcriptional regulation of the Scn10a gene, the sensory neuron specific promoter was functionally identified. While identifying the mRNA 5’ end, alternative splicing within the 5’ UTR was observed to create heterogeneity in the RNA transcript. Four kilobases of upstream genomic DNA was cloned and the presence of tissue specific promoter activity was tested by microinjection and adenoviral infection of fluorescent protein reporter constructs into primary mouse and rat neurons, and cell lines. The region contained many putative transcription factor binding sites and strong homology with the predicted rat ortholog. Homology to the predicted human ortholog was limited to the proximal end and several conserved cis elements were noted. Two regulatory modules were identified by microinjection of reporter constructs into DRG and superior cervical ganglia neurons: a neuron specific proximal promoter region between −1.6 and −0.2kb of the transcription start site cluster, and a distal sensory neuron switch region beyond −1.6kb that restricted fluorescent protein expression to a subset of primary sensory neurons. PMID:18466327

Bothriurus bonariensis scorpion venom activates voltage-dependent sodium channels in insect and mammalian nervous systems.

PubMed

Dos Santos, Douglas Silva; Carvalho, Evelise Leis; de Lima, Jeferson Camargo; Breda, Ricardo Vaz; Oliveira, Raquel Soares; de Freitas, Thiago Carrazoni; Salamoni, Simone Denise; Domingues, Michelle Flores; Piovesan, Angela Regina; Boldo, Juliano Tomazzoni; de Assis, Dênis Reis; da Costa, Jaderson Costa; Dal Belo, Cháriston André; Pinto, Paulo Marcos

2016-10-25

Animal venoms have been widely recognized as a major source of biologically active molecules. Bothriurus bonariensis, popularly known as black scorpion, is the arthropod responsible for the highest number of accidents involving scorpion sting in Southern Brazil. Here we reported the first attempt to investigate the neurobiology of B. bonariensis venom (BBV) in the insect and mammalian nervous system. BBV (32 μg/g) induced a slow neuromuscular blockade in the in vivo cockroach nerve-muscle preparations (70 ± 4%, n = 6, p < 0.001), provoking repetitive twitches and significantly decreasing the frequency of spontaneous leg action potentials (SNCAPs) from 82 ± 3 min(-1) to 36 ± 1.3 min(-1) (n = 6, p < 0.05), without affecting the amplitude. When tested in primary cultures of rat hippocampal cells, BBV induced a massive increase of Ca(2+) influx (250 ± 1% peak increase, n = 3, p < 0.0001). The disturbance of calcium homeostasis induced by BBV on the mammalian central nervous system was not accompanied by cellular death and was prevented by the co-treatment of the hippocampal cells with tetrodotoxin, a selective sodium channel blocker. The results suggest that the biological activity of BBV is mostly related to a modulation of sodium channels function. Our biological activity survey suggests that BBV may have a promising insecticidal and therapeutic potential. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

Cardiac sodium channel blockade after an intentional ingestion of lacosamide, cyclobenzaprine, and levetiracetam: Case report.

PubMed

Chua-Tuan, Jenny L; Cao, Dazhe; Iwanicki, Janetta L; Hoyte, Christopher O

2015-07-01

Lacosamide treats partial seizures by enhancing slow inactivation of voltage-gated sodium channels. The described cardiac toxicity of lacosamide in the literature to date includes atrioventricular blockade (PR prolongation), atrial flutter, atrial fibrillation, sinus pauses, ventricular tachycardia and a single cardiac arrest. We report a second case of cardiac arrest following an intentional lacosamide overdose. A 16 year-old female with a seizure disorder was found unresponsive in pulseless ventricular tachycardia after intentionally ingesting 4.5 g (76 mg/kg) lacosamide, 120 mg (2 mg/kg) cyclobenzaprine and an unknown amount of levetiracetam. Exact time of ingestion was unknown. Her initial electrocardiogram (ECG) demonstrated sinus tachycardia at 139 beats per minute, QRS duration 112 ms, and terminal R-wave in lead aVR > 3 mm. Despite treatment with 150 mEq of sodium bicarbonate, she had persistent EKG findings eight hours after presentation. Her serum lacosamide concentration nine hours after presentation was elevated at 22.8 μg/mL, while serum cyclobenzaprine concentration was 16 ng/mL (therapeutic: 10-30 ng/mL), and serum levetiracetam concentration was 22.7 μg/mL (therapeutic: 12-46 μg/mL). On hospital day three, ECG demonstrated resolution of the terminal R-wave with QRS of 78 ms. The patient recovered without physical or neurologic sequelae. The patient's lacosamide, cyclobenzaprine and levetiracetam overdose was associated with QRS prolongation and terminal right axis deviation--suggesting sodium channel blockade as a likely etiology for her cardiac arrest. Cyclobenzaprine has potential for sodium channel blockade and ventricular dysrhythmias although cardiac toxicity due to cyclobenzaprine alone is rare. The combination of cyclobenzaprine with lacosamide may have resulted in cardiovascular collapse. In conclusion, overdose of lacosamide combined with therapeutic concentrations of sodium channel blocking xenobiotics may cause cardiac conduction delays and cardiac arrest.

α-SNAP regulates dynamic, on-site assembly and calcium selectivity of Orai1 channels.

PubMed

Li, Peiyao; Miao, Yong; Dani, Adish; Vig, Monika

2016-08-15

Orai1 forms a highly calcium-selective pore of the calcium release activated channel, and α-SNAP is necessary for its function. Here we show that α-SNAP regulates on-site assembly of Orai1 dimers into calcium-selective multimers. We find that Orai1 is a dimer in resting primary mouse embryonic fibroblasts but displays variable stoichiometry in the plasma membrane of store-depleted cells. Remarkably, α-SNAP depletion induces formation of higher-order Orai1 oligomers, which permeate significant levels of sodium via Orai1 channels. Sodium permeation in α-SNAP-deficient cells cannot be corrected by tethering multiple Stim1 domains to Orai1 C-terminal tail, demonstrating that α-SNAP regulates functional assembly and calcium selectivity of Orai1 multimers independently of Stim1 levels. Fluorescence nanoscopy reveals sustained coassociation of α-SNAP with Stim1 and Orai1, and α-SNAP-depleted cells show faster and less constrained mobility of Orai1 within ER-PM junctions, suggesting Orai1 and Stim1 coentrapment without stable contacts. Furthermore, α-SNAP depletion significantly reduces fluorescence resonance energy transfer between Stim1 and Orai1 N-terminus but not C-terminus. Taken together, these data reveal a unique role of α-SNAP in the on-site functional assembly of Orai1 subunits and suggest that this process may, in part, involve enabling crucial low-affinity interactions between Orai1 N-terminus and Stim1. © 2016 Li, Miao, et al. This article is distributed by The American Society for Cell Biology under license from the author(s). Two months after publication it is available to the public under an Attribution–Noncommercial–Share Alike 3.0 Unported Creative Commons License (http://creativecommons.org/licenses/by-nc-sa/3.0).

Chloride channels in myotonia congenita assessed by velocity recovery cycles.

PubMed

Tan, S Veronica; Z'Graggen, Werner J; Boërio, Delphine; Rayan, Dipa Raja; Norwood, Fiona; Ruddy, Deborah; Howard, R; Hanna, Michael G; Bostock, Hugh

2014-06-01

Myotonia congenita (MC) is caused by congenital defects in the muscle chloride channel CLC-1. This study used muscle velocity recovery cycles (MVRCs) to investigate how membrane function is affected. MVRCs and responses to repetitive stimulation were compared between 18 patients with genetically confirmed MC (13 recessive, 7 dominant) and 30 age-matched, normal controls. MC patients exhibited increased early supernormality, but this was prevented by treatment with sodium channel blockers. After multiple conditioning stimuli, late supernormality was enhanced in all MC patients, indicating delayed repolarization. These abnormalities were similar between the MC subtypes, but recessive patients showed a greater drop in amplitude during repetitive stimulation. MVRCs indicate that chloride conductance only becomes important when muscle fibers are depolarized. The differential responses to repetitive stimulation suggest that, in dominant MC, the affected chloride channels are activated by strong depolarization, consistent with a positive shift of the CLC-1 activation curve. Copyright © 2013 Wiley Periodicals, Inc.

Ciguatoxins: Cyclic Polyether Modulators of Voltage-gated Iion Channel Function

PubMed Central

Nicholson, Graham M.; Lewis, Richard J.

2006-01-01

Ciguatoxins are cyclic polyether toxins, derived from marine dinoflagellates, which are responsible for the symptoms of ciguatera poisoning. Ingestion of tropical and subtropical fin fish contaminated by ciguatoxins results in an illness characterised by neurological, cardiovascular and gastrointestinal disorders. The pharmacology of ciguatoxins is characterised by their ability to cause persistent activation of voltage-gated sodium channels, to increase neuronal excitability and neurotransmitter release, to impair synaptic vesicle recycling, and to cause cell swelling. It is these effects, in combination with an action to block voltage-gated potassium channels at high doses, which are believed to underlie the complex of symptoms associated with ciguatera. This review examines the sources, structures and pharmacology of ciguatoxins. In particular, attention is placed on their cellular modes of actions to modulate voltage-gated ion channels and other Na+-dependent mechanisms in numerous cell types and to current approaches for detection and treatment of ciguatera.

Salt, chloride, bleach, and innate host defense

PubMed Central

Wang, Guoshun; Nauseef, William M.

2015-01-01

Salt provides 2 life-essential elements: sodium and chlorine. Chloride, the ionic form of chlorine, derived exclusively from dietary absorption and constituting the most abundant anion in the human body, plays critical roles in many vital physiologic functions, from fluid retention and secretion to osmotic maintenance and pH balance. However, an often overlooked role of chloride is its function in innate host defense against infection. Chloride serves as a substrate for the generation of the potent microbicide chlorine bleach by stimulated neutrophils and also contributes to regulation of ionic homeostasis for optimal antimicrobial activity within phagosomes. An inadequate supply of chloride to phagocytes and their phagosomes, such as in CF disease and other chloride channel disorders, severely compromises host defense against infection. We provide an overview of the roles that chloride plays in normal innate immunity, highlighting specific links between defective chloride channel function and failures in host defense. PMID:26048979

Salt, chloride, bleach, and innate host defense.

PubMed

Wang, Guoshun; Nauseef, William M

2015-08-01

Salt provides 2 life-essential elements: sodium and chlorine. Chloride, the ionic form of chlorine, derived exclusively from dietary absorption and constituting the most abundant anion in the human body, plays critical roles in many vital physiologic functions, from fluid retention and secretion to osmotic maintenance and pH balance. However, an often overlooked role of chloride is its function in innate host defense against infection. Chloride serves as a substrate for the generation of the potent microbicide chlorine bleach by stimulated neutrophils and also contributes to regulation of ionic homeostasis for optimal antimicrobial activity within phagosomes. An inadequate supply of chloride to phagocytes and their phagosomes, such as in CF disease and other chloride channel disorders, severely compromises host defense against infection. We provide an overview of the roles that chloride plays in normal innate immunity, highlighting specific links between defective chloride channel function and failures in host defense. © Society for Leukocyte Biology.

The pH dependence of cocaine interaction with cardiac sodium channels.

PubMed

Crumb, W J; Clarkson, C W

1995-09-01

Previous in vitro and in vivo studies have provided evidence implicating cocaine block of cardiac sodium channels as a putative mechanism for cocaine-induced arrhythmias and sudden death. Cocaine also has been shown to cause seizures which can result in respiratory and/or metabolic acidosis. In this study we investigated how changes in both internal pH (pHi) and external pH (pHo) over the range of 6.6 to 9.2 modify the sodium channel blocking properties of cocaine in isolated guinea pig ventricular myocytes by using the whole-cell variant of the patch clamp technique. Use-dependent block produced by a train of 1-sec pulses to -20 mV was not affected by changes in pHi, but both the amplitude and time constant for approaching steady-state block were significantly affected by changes in pHo. Characterization of the time course of cocaine binding during a depolarizing pulse indicated that the kinetics of drug interaction with inactivated channels were independent of pHi, but were significantly affected by changes in pHo. The rate of recovery from channel block at a holding potential of -140 mV also was independent of pHi, but strongly dependent on pHo, with the unblocking time constant decreasing exponentially as pHo was increased. The results of this study indicate that cocaine's effect on cardiac sodium channels can be modulated significantly by changes in pHo, and provide further support for previously poorly tested assumptions of the modulated receptor hypothesis.

Functional reconstitution of the voltage-regulated sodium channel purified from electroplax of Electrophorus electricus

DOE Office of Scientific and Technical Information (OSTI.GOV)

Rosenberg, R.L.

1985-01-01

The voltage-regulated NA channel is responsible for the depolarization of the excitable cell membrane during the normal action potential. This research has focused on the functional properties of the Na channel, purified from detergent extracts of electroplax membranes of the electric eel, and reconstituted into vesicles of defined phospholipid. These properties were assessed by measuring neurotoxin-modulated ion flux into the reconstituted membrane vesicles and by recording the single-channel currents of the purified channel by the patch-clamp method. The binding of tritiated tetrodotoxin (TTX) was employed as a marker for the purification of the channel. Two high-resolution fractionation steps, based onmore » molecular charge and protein size, were used to obtain a preparation that is 80% homogeneous for a large peptide of 270,000 daltons. Radiotracer /sup 22/Na/sup +/ influx into the vesicles was stimulated by veratridine and by batrachotoxin (BTX) at concentrations of 100 ..mu..M and 5 ..mu..M, respectively. The stimulation by BTX was greater than that by veratridine, and can be as much as 16-fold over control influx levels. The stimulated influx is blocked by TTX with a K/sub i/ of 35 nM, and by local anesthetics in the normal pharmacological range. Large multilamellar vesicles prepared with a freeze-thaw step are suitable for single-channel recording techniques. When excised patches of the reconstituted membranes were voltage-clamped in the absence of activating neurotoxins, voltage-dependent single-channel currents were recorded. These displayed properties similar to those from native membranes of nerve and muscle. These results indicate that the protein purified on the basis of TTX binding is a functional Na channel possessing these functional domains: the ion-selective channel, the voltage sensors controlling activation and inactivation, and the sites of action of TTX, alkaloid neurotoxins, and local anesthetics.« less

Evidence for Dual Binding Sites for 1,1,1-Trichloro-2,2-bis(p-chlorophenyl)ethane (DDT) in Insect Sodium Channels*

PubMed Central

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

Evidence for Dual Binding Sites for 1,1,1-Trichloro-2,2-bis(p-chlorophenyl)ethane (DDT) in Insect Sodium Channels.

PubMed

Du, Yuzhe; Nomura, Yoshiko; Zhorov, Boris S; Dong, Ke

2016-02-26

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. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

Two novel sodium channel mutations associated with resistance to indoxacarb and metaflumizone in the diamondback moth, Plutella xylostella

PubMed Central

Wang, Xing-Liang; Su, Wen; Zhang, Jian-Heng; Yang, Yi-Hua; Dong, Ke; Wu, Yi-Dong

2015-01-01

Indoxacarb and metaflumizone belong to a relatively new class of sodium channel blocker insecticides (SCBIs). Due to intensive use of indoxacarb, field-evolved indoxacarb resistance has been reported in several lepidopteran pests including the diamondback moth, Plutella xylostella, a serious pest of cruciferous crops. In particular, the BY12 population of P. xylostella, collected from Baiyun, Guangdong province of China in 2012, was 750-fold more resistant to indoxacarb and 70-fold more resistant to metaflumizone compared with the susceptible Roth strain. Comparison of cDNA sequences encoding the sodium channel genes of Roth and BY12 revealed two point mutations (F1845Y and V1848I) in the 6th segment of domain IV of the PxNav protein in the BY population. Both mutations are located within a highly conserved sequence region that is predicted to be involved in the binding sites of local anesthetics and SCBIs based on mammalian sodium channels. A significant correlation was observed among ten field-collected populations between the mutant allele (Y1845 or I1848) frequencies (1.7% to 52.5%) and resistance levels to both indoxacarb (34- to 870-fold) and metaflumizone (1- to 70-fold). The two mutant alleles were never found to co-exist in the same allele of PxNav, suggesting that they arose independently. This is the first time that sodium channel mutations have been associated with high levels of resistance to SCBIs. F1845Y and V1848I are molecular markers for resistance monitoring in the diamondback moth and possibly other insect pest species. PMID:25850422

Two novel sodium channel mutations associated with resistance to indoxacarb and metaflumizone in the diamondback moth, Plutella xylostella.

PubMed

Wang, Xing-Liang; Su, Wen; Zhang, Jian-Heng; Yang, Yi-Hua; Dong, Ke; Wu, Yi-Dong

2016-02-01

Indoxacarb and metaflumizone belong to a relatively new class of sodium channel blocker insecticides (SCBIs). Due to intensive use of indoxacarb, field-evolved indoxacarb resistance has been reported in several lepidopteran pests, including the diamondback moth Plutella xylostella, a serious pest of cruciferous crops. In particular, the BY12 population of P. xylostella, collected from Baiyun, Guangdong Province of China in 2012, was 750-fold more resistant to indoxacarb and 70-fold more resistant to metaflumizone compared with the susceptible Roth strain. Comparison of complementary DNA sequences encoding the sodium channel genes of Roth and BY12 revealed two point mutations (F1845Y and V1848I) in the sixth segment of domain IV of the PxNav protein in the BY population. Both mutations are located within a highly conserved sequence region that is predicted to be involved in the binding sites of local anesthetics and SCBIs based on mammalian sodium channels. A significant correlation was observed among 10 field-collected populations between the mutant allele (Y1845 or I1848) frequencies (1.7% to 52.5%) and resistance levels to both indoxacarb (34- to 870-fold) and metaflumizone (1- to 70-fold). The two mutations were never found to co-exist in the same allele of PxNav , suggesting that they arose independently. This is the first time that sodium channel mutations have been associated with high levels of resistance to SCBIs. F1845Y and V1848I are molecular markers for resistance monitoring in the diamondback moth and possibly other insect pest species. © 2015 Institute of Zoology, Chinese Academy of Sciences.

Lacosamide

PubMed Central

Curia, Giulia; Biagini, Giuseppe; Perucca, Emilio; Avoli, Massimo

2016-01-01

The mechanism of action of several antiepileptic drugs (AEDs) rests on their ability to modulate the activity of voltage-gated sodium currents that are responsible for fast action potential generation. Recent data indicate that lacosamide (a compound with analgesic and anticonvulsant effects in animal models) shares a similar mechanism. When compared with other AEDs, lacosamide has the unique ability to interact with sodium channel slow inactivation without affecting fast inactivation. This article reviews these findings and discusses their relevance within the context of neuronal activity seen during epileptiform discharges generated by limbic neuronal networks in the presence of chemical convulsants. These seizure-like events are characterized by sustained discharges of sodium-dependent action potentials supported by robust depolarizations, thus providing synchronization within neuronal networks. Generally, AEDs such as phenytoin, carbamazepine and lamotrigine block sodium channels when activated. In contrast, lacosamide facilitates slow inactivation of sodium channels both in terms of kinetics and voltage dependency. This effect may be relatively selective for repeatedly depolarized neurons, such as those participating in seizure activity in which the persistence of sodium currents is more pronounced and promotes neuronal excitation. The clinical effectiveness of lacosamide has been demonstrated in randomized, double-blind, parallel-group, placebo-controlled, adjunctive-therapy trials in patients with refractory partial seizures. Further studies should determine whether the effects of lacosamide in animal models and in clinical settings are fully explained by its selective action on sodium current slow inactivation or whether other effects (e.g. interactions with the collapsin-response mediator protein-2) play a contributory role. PMID:19552484

Polyamine FTX-3.3 and polyamine amide sFTX-3.3 inhibit presynaptic calcium currents and acetylcholine release at mouse motor nerve terminals.

PubMed

Fatehi, M; Rowan, E G; Harvey, A L; Moya, E; Blagbrough, I S

1997-02-01

FTX-3.3 is the proposed structure of a calcium-channel blocking toxin that has been isolated from the funnel web spider (Agelenopsis aperta). The effects of FTX-3.3 and one of its analogues, sFTX-3.3, on acetylcholine release, on presynaptic currents at mouse motor nerve terminals and on whole-cell sodium currents in SK.N.SH cells (a human neuroblastoma cell line) have been studied. FTX-3.3 (10-30 microM) and sFTX-3.3 (100-300 microM) reversibly reduced release of acetylcholine by approximately 70-90% and 40-60%, respectively. FTX-3.3 (10 microM) blocked the fast component of presynaptic calcium currents by approximately 60%. sFTX-3.3 (100 microM) reduced the duration of the slow component of presynaptic calcium currents by about 50% of the control and also reduced presynaptic sodium current by approximately 20% of the control. sFTX-3.3 (100 microM) reduced whole-cell sodium current recorded from SK.N.SH cells by approximately 15%, whereas FTX-3.3, even at 200 microM, did not affect this current. Since the only difference in chemical structures of these toxins is that sFTX-3.3 has an amide function which is absent in FTX-3.3, the amide function may be responsible for the reduced potency and selectivity of sFTX-3.3. This study also provides further support for the existence of P-type calcium channels at mouse motor nerve terminals.

Targeting of sodium channel blockers into nociceptors to produce long-duration analgesia: a systematic study and review

PubMed Central

Roberson, DP; Binshtok, AM; Blasl, F; Bean, BP; Woolf, CJ

2011-01-01

BACKGROUND AND PURPOSE We have developed a strategy to target the permanently charged lidocaine derivative lidocaine N-ethyl bromide (QX-314) selectively into nociceptive sensory neurons through the large-pore transient receptor potential cation channel subfamily V (TRPV1) noxious heat detector channel. This involves co-administration of QX-314 and a TRPV1 agonist to produce a long-lasting local analgesia. For potential clinical use we propose using lidocaine as the TRPV1 agonist, because it activates TRPV1 at clinical doses. EXPERIMENTAL APPROACH We conducted experiments in rats to determine optimal concentrations and ratios of lidocaine and QX-314 that produce the greatest degree and duration of pain-selective block when administered nearby the sciatic nerve: reduction in the response to noxious mechanical (pinch) and to radiant heat stimuli, with minimal disruption in motor function (grip strength). KEY RESULTS A combination of 0.5% QX-314 and 2% lidocaine produced 1 h of non-selective sensory and motor block followed by >9 h of pain-selective block, where grip strength was unimpaired. QX-314 at this concentration had no effect by itself, while 2% lidocaine by itself produced 1 h of non-selective block. The combination of 0.5% QX-314 and 2% lidocaine was the best of the many tested, in terms of the duration and selectivity of local analgesia. CONCLUSIONS AND IMPLICATIONS Targeting charged sodium channel blockers into specific sets of axons via activation of differentially expressed large-pore channels provides an opportunity to produce prolonged local analgesia, and represents an example of how exploiting ion channels as a drug delivery port can be used to increase the specificity and efficacy of therapeutics. PMID:21457220

Lens intracellular hydrostatic pressure is generated by the circulation of sodium and modulated by gap junction coupling

PubMed Central

Gao, Junyuan; Sun, Xiurong; Moore, Leon C.; White, Thomas W.; Brink, Peter R.

2011-01-01

We recently modeled fluid flow through gap junction channels coupling the pigmented and nonpigmented layers of the ciliary body. The model suggested the channels could transport the secretion of aqueous humor, but flow would be driven by hydrostatic pressure rather than osmosis. The pressure required to drive fluid through a single layer of gap junctions might be just a few mmHg and difficult to measure. In the lens, however, there is a circulation of Na+ that may be coupled to intracellular fluid flow. Based on this hypothesis, the fluid would cross hundreds of layers of gap junctions, and this might require a large hydrostatic gradient. Therefore, we measured hydrostatic pressure as a function of distance from the center of the lens using an intracellular microelectrode-based pressure-sensing system. In wild-type mouse lenses, intracellular pressure varied from ∼330 mmHg at the center to zero at the surface. We have several knockout/knock-in mouse models with differing levels of expression of gap junction channels coupling lens fiber cells. Intracellular hydrostatic pressure in lenses from these mouse models varied inversely with the number of channels. When the lens’ circulation of Na+ was either blocked or reduced, intracellular hydrostatic pressure in central fiber cells was either eliminated or reduced proportionally. These data are consistent with our hypotheses: fluid circulates through the lens; the intracellular leg of fluid circulation is through gap junction channels and is driven by hydrostatic pressure; and the fluid flow is generated by membrane transport of sodium. PMID:21624945

Redox regulation of epithelial sodium channels examined in alveolar type 1 and 2 cells patch-clamped in lung slice tissue.

PubMed

Helms, My N; Jain, Lucky; Self, Julie L; Eaton, Douglas C

2008-08-15

The alveolar surface of the lung is lined by alveolar type 1 (AT1) and type 2 (AT2) cells. Using single channel patch clamp analysis in lung slice preparations, we are able to uniquely study AT1 and AT2 cells separately from intact lung. We report for the first time the Na+ transport properties of type 2 cells accessed in live lung tissue (as we have done in type 1 cells). Type 2 cells in lung tissue slices express both highly selective cation and nonselective cation channels with average conductances of 8.8 +/- 3.2 and 22.5 +/- 6.3 picosiemens, respectively. Anion channels with 10-picosiemen conductance are also present in the apical membrane of type 2 cells. Our lung slice studies importantly verify the use of cultured cell model systems commonly used in lung epithelial sodium channel (ENaC) studies. Furthermore, we identify novel functional differences between the cells that make up the alveolar epithelium. One important difference is that exposure to the nitric oxide (NO) donor, PAPA-NONOate (1.5 microm), significantly decreases average ENaC NPo in type 2 cells (from 1.38 +/- 0.26 to 0.82 +/- 0.16; p < 0.05 and n = 18) but failed to alter ENaC activity in alveolar type 1 cells. Elevating endogenous superoxide (O2.) levels with Ethiolat, a superoxide dismutase inhibitor, prevented NO inhibition of ENaC activity in type 2 cells, supporting the novel hypothesis that O2. and NO signaling plays an important role in maintaining lung fluid balance.

Molecular Characterization of Voltage-Gated Sodium Channels and Their Relations with Paralytic Shellfish Toxin Bioaccumulation in the Pacific Oyster Crassostrea gigas

PubMed Central

Boullot, Floriane; Castrec, Justine; Bidault, Adeline; Dantas, Natanael; Payton, Laura; Perrigault, Mickael; Tran, Damien; Amzil, Zouher; Boudry, Pierre; Soudant, Philippe; Hégaret, Hélène; Fabioux, Caroline

2017-01-01

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. PMID:28106838

Distinct Roles of the DmNav and DSC1 Channels in the Action of DDT and Pyrethroids

PubMed Central

Rinkevich, Frank D.; Du, Yuzhe; Tolinski, Josh; Ueda, Atsushi; Wu, Chun-Fang; Zhorov, Boris S.; Dong, Ke

2015-01-01

Voltage-gated sodium channels (Nav channels) are critical for electrical signaling in the nervous system and are the primary targets of the insecticides DDT and pyrethroids. In Drosophila melanogaster, besides the canonical Nav channel, Para (also called DmNav), there is a sodium channel-like cation channel called DSC1 (Drosophila sodium channel 1). Temperature-sensitive paralytic mutations in DmNav (parats) confer resistance to DDT and pyrethroids, whereas DSC1 knockout flies exhibit enhanced sensitivity to pyrethroids. To further define the roles and interaction of DmNav and DSC1 channels in DDT and pyrethroid neurotoxicology, we generated a DmNav/DSC1 double mutant line by introducing a parats1 allele (carrying the I265N mutation) into a DSC1 knockout line. We confirmed that the I265N mutation reduced the sensitivity to two pyrethroids, permethrin and deltamethrin of a DmNav variant expressed in Xenopus oocytes. Computer modeling predicts that the I265N mutation confers pyrethroid resistance by allosterically altering the second pyrethroid receptor site on the DmNav channel. Furthermore, we found that I265N-mediated pyrethroid resistance in parats1 mutant flies was almost completely abolished in parats1;DSC1−/− double mutant flies. Unexpectedly, however, the DSC1 knockout flies were less sensitive to DDT, compared to the control flies (w1118A), and the parats1;DSC1−/− double mutant flies were even more resistant to DDT compared to the DSC1 knockout or parats1 mutant. Our findings revealed distinct roles of the DmNav and DSC1 channels in the neurotoxicology of DDT vs. pyrethroids and implicate the exciting possibility of using DSC1 channel blockers or modifiers in the management of pyrethroid resistance. PMID:25687544

Harnessing the Flow of Excitation: TRP, Voltage-Gated Na(+), and Voltage-Gated Ca(2+) Channels in Contemporary Medicine.

PubMed

Frolov, Roman V; Weckström, Matti

2016-01-01

Cellular signaling in both excitable and nonexcitable cells involves several classes of ion channels. Some of them are of minor importance, with very specialized roles in physiology, but here we concentrate on three major channel classes: TRP (transient receptor potential channels), voltage-gated sodium channels (Nav), and voltage-gated calcium channels (Cav). Here, we first propose a conceptual framework binding together all three classes of ion channels, a "flow-of-excitation model" that takes into account the inputs mediated by TRP and other similar channels, the outputs invariably provided by Cav channels, and the regenerative transmission of signals in the neural networks, for which Nav channels are responsible. We use this framework to examine the function, structure, and pharmacology of these channel classes both at cellular and also at whole-body physiological level. Building on that basis we go through the pathologies arising from the direct or indirect malfunction of the channels, utilizing ion channel defects, the channelopathies. The pharmacological interventions affecting these channels are numerous. Part of those are well-established treatments, like treatment of hypertension or some forms of epilepsy, but many other are deeply problematic due to poor drug specificity, ion channel diversity, and widespread expression of the channels in tissues other than those actually targeted. © 2016 Elsevier Inc. All rights reserved.

Antagonist action of progesterone at σ-receptors in the modulation of voltage-gated sodium channels

PubMed Central

Johannessen, Molly; Fontanilla, Dominique; Mavlyutov, Timur; Ruoho, Arnold E.

2011-01-01

σ-Receptors are integral membrane proteins that have been implicated in a number of biological functions, many of which involve the modulation of ion channels. A wide range of synthetic ligands activate σ-receptors, but endogenous σ-receptor ligands have proven elusive. One endogenous ligand, dimethyltryptamine (DMT), has been shown to act as a σ-receptor agonist. Progesterone and other steroids bind σ-receptors, but the functional consequences of these interactions are unclear. Here we investigated progesterone binding to σ1- and σ2-receptors and evaluated its effect on σ-receptor-mediated modulation of voltage-gated Na+ channels. Progesterone binds both σ-receptor subtypes in liver membranes with comparable affinities and blocks photolabeling of both subtypes in human embryonic kidney 293 cells that stably express the human cardiac Na+ channel Nav1.5. Patch-clamp recording in this cell line tested Na+ current modulation by the σ-receptor ligands ditolylguanidine, PB28, (+)SKF10047, and DMT. Progesterone inhibited the action of these ligands to varying degrees, and some of these actions were reduced by σ1-receptor knockdown with small interfering RNA. Progesterone inhibition of channel modulation by drugs was consistent with stronger antagonism of σ2-receptors. By contrast, progesterone inhibition of channel modulation by DMT was consistent with stronger antagonism of σ1-receptors. Progesterone binding to σ-receptors blocks σ-receptor-mediated modulation of a voltage-gated ion channel, and this novel membrane action of progesterone may be relevant to changes in brain and cardiovascular function during endocrine transitions. PMID:21084640

Antagonist action of progesterone at σ-receptors in the modulation of voltage-gated sodium channels.

PubMed

Johannessen, Molly; Fontanilla, Dominique; Mavlyutov, Timur; Ruoho, Arnold E; Jackson, Meyer B

2011-02-01

σ-Receptors are integral membrane proteins that have been implicated in a number of biological functions, many of which involve the modulation of ion channels. A wide range of synthetic ligands activate σ-receptors, but endogenous σ-receptor ligands have proven elusive. One endogenous ligand, dimethyltryptamine (DMT), has been shown to act as a σ-receptor agonist. Progesterone and other steroids bind σ-receptors, but the functional consequences of these interactions are unclear. Here we investigated progesterone binding to σ(1)- and σ(2)-receptors and evaluated its effect on σ-receptor-mediated modulation of voltage-gated Na(+) channels. Progesterone binds both σ-receptor subtypes in liver membranes with comparable affinities and blocks photolabeling of both subtypes in human embryonic kidney 293 cells that stably express the human cardiac Na(+) channel Na(v)1.5. Patch-clamp recording in this cell line tested Na(+) current modulation by the σ-receptor ligands ditolylguanidine, PB28, (+)SKF10047, and DMT. Progesterone inhibited the action of these ligands to varying degrees, and some of these actions were reduced by σ(1)-receptor knockdown with small interfering RNA. Progesterone inhibition of channel modulation by drugs was consistent with stronger antagonism of σ(2)-receptors. By contrast, progesterone inhibition of channel modulation by DMT was consistent with stronger antagonism of σ(1)-receptors. Progesterone binding to σ-receptors blocks σ-receptor-mediated modulation of a voltage-gated ion channel, and this novel membrane action of progesterone may be relevant to changes in brain and cardiovascular function during endocrine transitions.

The Helicoverpa zea (Boddie) (Lepidoptera: Noctuidae) voltage-gated sodium channel and mutations associated with pyrethroid resistance in field-collected adult males.

PubMed

Hopkins, B W; Pietrantonio, P V

2010-05-01

Helicoverpa zea is one of the most costly insect pests of food and fiber crops throughout the Americas. Pyrethroid insecticides are widely applied for its control as they are effective and relatively inexpensive; however, resistance to pyrethroids threatens agricultural systems sustainability because alternative insecticides are often more expensive or less effective. Although pyrethroid resistance has been identified in this pest since 1990, the mechanisms of resistance have not yet been elucidated at the molecular level. Pyrethroids exert their toxicity by prolonging the open state of the voltage-gated sodium channel. Here we report the cDNA sequence of the H. zea sodium channel alpha-subunit homologous to the para gene from Drosophila melanogaster. In field-collected males which were resistant to cypermethrin as determined by the adult vial test, we identify known resistance-conferring mutations L1029H and V421M, along with two novel mutations at the V421 residue, V421A and V421G. An additional mutation, I951V, may be the first example of a pyrethroid resistance mutation caused by RNA editing. Identification of the sodium channel cDNA sequence will allow for testing hypotheses on target-site resistance for insecticides acting on this channel through modeling and expression studies. Understanding the mechanisms responsible for resistance will greatly improve our ability to identify and predict resistance, as well as preserve susceptibility to pyrethroid insecticides. Copyright 2010 Elsevier Ltd. All rights reserved.

A distinct sodium channel voltage-sensor locus determines insect selectivity of the spider toxin Dc1a.

PubMed

Bende, Niraj S; Dziemborowicz, Sławomir; Mobli, Mehdi; Herzig, Volker; Gilchrist, John; Wagner, Jordan; Nicholson, Graham M; King, Glenn F; Bosmans, Frank

2014-07-11

β-Diguetoxin-Dc1a (Dc1a) is a toxin from the desert bush spider Diguetia canities that incapacitates insects at concentrations that are non-toxic to mammals. Dc1a promotes opening of German cockroach voltage-gated sodium (Nav) channels (BgNav1), whereas human Nav channels are insensitive. Here, by transplanting commonly targeted S3b-S4 paddle motifs within BgNav1 voltage sensors into Kv2.1, we find that Dc1a interacts with the domain II voltage sensor. In contrast, Dc1a has little effect on sodium currents mediated by PaNav1 channels from the American cockroach even though their domain II paddle motifs are identical. When exploring regions responsible for PaNav1 resistance to Dc1a, we identified two residues within the BgNav1 domain II S1-S2 loop that when mutated to their PaNav1 counterparts drastically reduce toxin susceptibility. Overall, our results reveal a distinct region within insect Nav channels that helps determine Dc1a sensitivity, a concept that will be valuable for the design of insect-selective insecticides.

Regulation of blood pressure and renal function by NCC and ENaC: lessons from genetically engineered mice.

PubMed

Verouti, Sophia N; Boscardin, Emilie; Hummler, Edith; Frateschi, Simona

2015-04-01

The activity of the thiazide-sensitive Na(+)/Cl(-) cotransporter (NCC) and of the amiloride-sensitive epithelial Na(+) channel (ENaC) is pivotal for blood pressure regulation. NCC is responsible for Na(+) reabsorption in the distal convoluted tubule (DCT) of the nephron, while ENaC reabsorbs the filtered Na(+) in the late DCT and in the cortical collecting ducts (CCD) providing the final renal adjustment to Na(+) balance. Here, we aim to highlight the recent advances made using transgenic mouse models towards the understanding of the regulation of NCC and ENaC function relevant to the control of sodium balance and blood pressure. We thus like to pave the way for common mechanisms regulating these two sodium-transporting proteins and their potential implication in structural remodeling of the nephron segments and Na(+) and Cl(-) reabsorption. Copyright © 2015 Elsevier Ltd. All rights reserved.

ProTx-II, a selective inhibitor of NaV1.7 sodium channels, blocks action potential propagation in nociceptors.

PubMed

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.

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

NASA Technical Reports Server (NTRS)

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.

Impaired natriuretic response to high-NaCl diet plus aldosterone infusion in mice overexpressing human CD39, an ectonucleotidase (NTPDase1).

PubMed

Zhang, Yue; Robson, Simon C; Morris, Kaiya L; Heiney, Kristina M; Dwyer, Karen M; Kishore, Bellamkonda K; Ecelbarger, Carolyn M

2015-06-15

Extracellular nucleotides acting through P2 receptors facilitate natriuresis. To define how purinergic mechanisms are involved in sodium homeostasis, we used transgenic (TG) mice that globally overexpress human CD39 (hCD39, NTPDase1), an ectonucleotidase that hydrolyzes extracellular ATP/ADP to AMP, resulting in an altered extracellular purine profile. On a high-sodium diet (HSD, 3.5% Na(+)), urine volume and serum sodium were significantly higher in TG mice but sodium excretion was unaltered. Furthermore, TG mice showed an attenuated fall in urine aldosterone with HSD. Western blot analysis revealed significantly lower densities (∼40%) of the β-subunit of the epithelial sodium channel (ENaC) in medulla, and the major band (85-kDa) of γ-ENaC in TG mice cortex. To evaluate aldosterone-independent differences, in a second experiment, aldosterone was clamped by osmotic minipump at 20 μg/day, and mice were fed either an HSD or a low-sodium diet (LSD, 0.03% Na(+)). Here, no differences in urine volume or osmolality, or serum aldosterone were found, but TG mice showed a modest, yet significant impairment in late natriuresis (days 3 and 4). Several major sodium transporters or channel subunits were differentially expressed between the genotypes. HSD caused a downregulation of Na-Cl cotransporter (NCC) in both genotypes; and had higher cortical levels of NCC, Na-K-ATPase (α-1 subunit), and α- and γ-ENaC. The Na-K-2Cl cotransporter (NKCC2) was downregulated by HSD in wild-type mice, but it increased in TG mice. In summary, our data support the concept that extracellular nucleotides facilitate natriuresis; they also reveal an aldosterone-independent downregulation of major renal sodium transporters and channel subunits by purinergic signaling.

The Origin and Early Evolution of Membrane Proteins

NASA Technical Reports Server (NTRS)

Pohorille, Andrew; Schweighofter, Karl; Wilson, Michael A.

2006-01-01

The origin and early evolution of membrane proteins, and in particular ion channels, are considered from the point of view that the transmembrane segments of membrane proteins are structurally quite simple and do not require specific sequences to fold. We argue that the transport of solute species, especially ions, required an early evolution of efficient transport mechanisms, and that the emergence of simple ion channels was protobiologically plausible. We also argue that, despite their simple structure, such channels could possess properties that, at the first sight, appear to require markedly larger complexity. These properties can be subtly modulated by local modifications to the sequence rather than global changes in molecular architecture. In order to address the evolution and development of ion channels, we focus on identifying those protein domains that are commonly associated with ion channel proteins and are conserved throughout the three main domains of life (Eukarya, Prokarya, and Archaea). We discuss the potassium-sodium-calcium superfamily of voltage-gated ion channels, mechanosensitive channels, porins, and ABC-transporters and argue that these families of membrane channels have sufficiently universal architectures that they can readily adapt to the diverse functional demands arising during evolution.

Activation of sodium channels by α-scorpion toxin, BmK NT1, produced neurotoxicity in cerebellar granule cells: an association with intracellular Ca2+ overloading.

PubMed

He, Yuwei; Zou, Xiaohan; Li, Xichun; Chen, Juan; Jin, Liang; Zhang, Fan; Yu, Boyang; Cao, Zhengyu

2017-02-01

Voltage-gated sodium channels (VGSCs) are responsible for the action potential generation in excitable cells including neurons and involved in many physiological and pathological processes. Scorpion toxins are invaluable tools to explore the structure and function of ion channels. BmK NT1, a scorpion toxin from Buthus martensii Karsch, stimulates sodium influx in cerebellar granule cells (CGCs). In this study, we characterized the mode of action of BmK NT1 on the VGSCs and explored the cellular response in CGC cultures. BmK NT1 delayed the fast inactivation of VGSCs, increased the Na + currents, and shifted the steady-state activation and inactivation to more hyperpolarized membrane potential, which was similar to the mode of action of α-scorpion toxins. BmK NT1 stimulated neuron death (EC 50  = 0.68 µM) and produced massive intracellular Ca 2+ overloading (EC 50  = 0.98 µM). TTX abrogated these responses, suggesting that both responses were subsequent to the activation of VGSCs. The Ca 2+ response of BmK NT1 was primary through extracellular Ca 2+ influx since reducing the extracellular Ca 2+ concentration suppressed the Ca 2+ response. Further pharmacological evaluation demonstrated that BmK NT1-induced Ca 2+ influx and neurotoxicity were partially blocked either by MK-801, an NMDA receptor blocker, or by KB-R7943, an inhibitor of Na + /Ca 2+ exchangers. Nifedipine, an L-type Ca 2+ channel inhibitor, slightly suppressed both Ca 2+ response and neurotoxicity. A combination of these three inhibitors abrogated both responses. Considered together, these data ambiguously demonstrated that activation of VGSCs by an α-scorpion toxin was sufficient to produce neurotoxicity which was associated with intracellular Ca 2+ overloading through both NMDA receptor- and Na + /Ca 2+ exchanger-mediated Ca 2+ influx.

Dynamic reciprocity of sodium and potassium channel expression in a macromolecular complex controls cardiac excitability and arrhythmia.

PubMed

Milstein, Michelle L; Musa, Hassan; Balbuena, Daniela Ponce; Anumonwo, Justus M B; Auerbach, David S; Furspan, Philip B; Hou, Luqia; Hu, Bin; Schumacher, Sarah M; Vaidyanathan, Ravi; Martens, Jeffrey R; Jalife, José

2012-07-31

The cardiac electrical impulse depends on an orchestrated interplay of transmembrane ionic currents in myocardial cells. Two critical ionic current mechanisms are the inwardly rectifying potassium current (I(K1)), which is important for maintenance of the cell resting membrane potential, and the sodium current (I(Na)), which provides a rapid depolarizing current during the upstroke of the action potential. By controlling the resting membrane potential, I(K1) modifies sodium channel availability and therefore, cell excitability, action potential duration, and velocity of impulse propagation. Additionally, I(K1)-I(Na) interactions are key determinants of electrical rotor frequency responsible for abnormal, often lethal, cardiac reentrant activity. Here, we have used a multidisciplinary approach based on molecular and biochemical techniques, acute gene transfer or silencing, and electrophysiology to show that I(K1)-I(Na) interactions involve a reciprocal modulation of expression of their respective channel proteins (Kir2.1 and Na(V)1.5) within a macromolecular complex. Thus, an increase in functional expression of one channel reciprocally modulates the other to enhance cardiac excitability. The modulation is model-independent; it is demonstrable in myocytes isolated from mouse and rat hearts and with transgenic and adenoviral-mediated overexpression/silencing. We also show that the post synaptic density, discs large, and zonula occludens-1 (PDZ) domain protein SAP97 is a component of this macromolecular complex. We show that the interplay between Na(v)1.5 and Kir2.1 has electrophysiological consequences on the myocardium and that SAP97 may affect the integrity of this complex or the nature of Na(v)1.5-Kir2.1 interactions. The reciprocal modulation between Na(v)1.5 and Kir2.1 and the respective ionic currents should be important in the ability of the heart to undergo self-sustaining cardiac rhythm disturbances.

Channelopathies of skeletal muscle excitability

PubMed Central

Cannon, Stephen C.

2016-01-01

Familial disorders of skeletal muscle excitability were initially described early in the last century and are now known to be caused by mutations of voltage-gated ion channels. The clinical manifestations are often striking, with an inability to relax after voluntary contraction (myotonia) or transient attacks of severe weakness (periodic paralysis). An essential feature of these disorders is fluctuation of symptoms that are strongly impacted by environmental triggers such as exercise, temperature, or serum K+ levels. These phenomena have intrigued physiologists for decades, and in the past 25 years the molecular lesions underlying these disorders have been identified and mechanistic studies are providing insights for therapeutic strategies of disease modification. These familial disorders of muscle fiber excitability are “channelopathies” caused by mutations of a chloride channel (ClC-1), sodium channel (NaV1.4), calcium channel (CaV1.1) and several potassium channels (Kir2.1, Kir2.6, Kir3.4). This review provides a synthesis of the mechanistic connections between functional defects of mutant ion channels, their impact on muscle excitability, how these changes cause clinical phenotypes, and approaches toward therapeutics. PMID:25880512

A Sodium Channel Myotonia Presenting with Intermittent Dysphagia as a Manifestation of a Rare SCN4A Variant.

PubMed

Benhammou, Jihane N; Phan, Jennifer; Lee, Hane; Ghassemi, Kevin; Parsons, William; Grody, Wayne W; Pisegna, Joseph R

2017-03-01

The voltage gated sodium channel SCN4A mutations account for non-dystrophic myotonia and include a heterogeneous group of conditions that include hyperkalemic periodic paralysis, paramyotonica congenita, potassium-aggravated myotonia, and hypokalemic periodic paralysis type 2. This case report proposes that a rare variant p.Pro1629Leu in SCN4A can cause a skeletal muscle deficit with intermittent dysphagia.

A Sodium Channel Myotonia Presenting with Intermittent Dysphagia as a Manifestation of a Rare SCN4A Variant

PubMed Central

Benhammou, Jihane N.; Phan, Jennifer; Lee, Hane; Ghassemi, Kevin; Parsons, William; Grody, Wayne W.; Pisegna, Joseph R.

2016-01-01

The voltage gated sodium channel SCN4A mutations account for non-dystrophic myotonia and include a heterogenous group of conditions that include hyperkalemic periodic paralysis, paramyotonica congenita, potassium-aggravated myotonia and hypokalemic periodic paralysis type 2. This case report proposes that a rare variant p.Pro1629Leu in SCN4A can cause skeletal muscle deficit with intermittent dysphagia. PMID:28012096

Molecular dynamics study of ion transport through an open model of voltage-gated sodium channel.

PubMed

Li, Yang; Sun, Ruining; Liu, Huihui; Gong, Haipeng

2017-05-01

Voltage-gated sodium (Na V ) channels are critical in the signal transduction of excitable cells. In this work, we modeled the open conformation for the pore domain of a prokaryotic Na V channel (Na V Rh), and used molecular dynamics simulations to track the translocation of dozens of Na + ions through the channel in the presence of a physiological transmembrane ion concentration gradient and a transmembrane electrical field that was closer to the physiological one than previous studies. Channel conductance was then estimated from simulations on the wide-type and DEKA mutant of Na V Rh. Interestingly, the conductivity predicted from the DEKA mutant agrees well with experimental measurement on eukaryotic Na V 1.4 channel. Moreover, the wide-type and DEKA mutant of Na V Rh exhibited markedly distinct ion permeation patterns, which thus implies the mechanistic difference between prokaryotic and eukaryotic Na V channels. Copyright © 2017 Elsevier B.V. All rights reserved.

The SCN8A encephalopathy mutation p.Ile1327Val displays elevated sensitivity to the anticonvulsant phenytoin.

PubMed

Barker, Bryan S; Ottolini, Matteo; Wagnon, Jacy L; Hollander, Rachel M; Meisler, Miriam H; Patel, Manoj K

2016-09-01

SCN8A encephalopathy (early infantile epileptic encephalopathy; EIEE13) is caused by gain-of-function mutations resulting in hyperactivity of the voltage-gated sodium channel Nav 1.6. The channel is concentrated at the axon initial segment (AIS) and is involved in establishing neuronal excitability. Clinical features of SCN8A encephalopathy include seizure onset between 0 and 18 months of age, intellectual disability, and developmental delay. Seizures are often refractory to treatment with standard antiepileptic drugs, and sudden unexpected death in epilepsy (SUDEP) has been reported in approximately 10% of patients. In a recent study, high doses of phenytoin were effective in four patients with SCN8A encephalopathy. In view of this observation, we have investigated the relationship between the functional effect of the SCN8A mutation p.Ile1327Val and its response to phenytoin. The mutation was introduced into the Scn8a cDNA by site-directed mutagenesis. Channel activity was characterized in transfected ND7/23 cells. The effects of phenytoin (100 μm) on mutant and wild-type (WT) channels were compared. Channel activation parameters were shifted in a hyperpolarizing direction in the mutant channel, whereas inactivation parameters were shifted in a depolarizing direction, increasing Na channel window current. Macroscopic current decay was slowed in I1327V channels, indicating an impairment in the transition from open state to inactivated state. Channel deactivation was also delayed, allowing more channels to remain in the open state. Phenytoin (100 μm) resulted in hyperpolarized activation and inactivation curves as well as greater tonic block and use-dependent block of I1327V mutant channels relative to WT. SCN8A - I1327V is a gain-of-function mutation with altered features that are predicted to increase neuronal excitability and seizure susceptibility. Phenytoin is an effective inhibitor of the mutant channel and may be of use in treating patients with gain-of-function mutations of SCN8A. Wiley Periodicals, Inc. © 2016 International League Against Epilepsy.

The SCN8A encephalopathy mutation p.Ile1327Val displays elevated sensitivity to the anticonvulsant phenytoin

PubMed Central

Barker, Bryan S.; Ottolini, Matteo; Wagnon, Jacy L.; Hollander, Rachel; Meisler, Miriam H.; Patel, Manoj K.

2016-01-01

Objective SCN8A encephalopathy (EIEE13) is caused by gain-of-function mutations resulting in hyperactivity of the voltage-gated sodium channel Nav1.6. The channel is concentrated at the axon initial segment (AIS) and is involved in establishing neuronal excitability. Clinical features of SCN8A encephalopathy include seizure onset between 0–18 months of age, intellectual disability, and developmental delay. Seizures are often refractory to treatment with standard anti-epileptic drugs, and sudden unexpected death in epilepsy (SUDEP) has been reported in approximately 10% of patients. In a recent study, high doses of phenytoin were effective in four patients with SCN8A encephalopathy. In view of this observation, we have investigated the relationship between the functional effect of the SCN8A mutation p.Ile1327Val and its response to phenytoin. Methods The mutation was introduced into the Scn8a cDNA by site-directed mutagenesis. Channel activity was characterized in transfected ND7/23 cells. The effects of phenytoin (100 μM) on mutant and wild type (WT) channels were compared. Results Channel activation parameters were shifted in a hyperpolarizing direction in the mutant channel, while inactivation parameters were shifted in a depolarizing direction, increasing Na channel window current. Macroscopic current decay was slowed in I1327V channels, indicating an impairment in the transition from open state to inactivated state. Channel deactivation was also delayed, allowing more channels to remain in the open state. Phenytoin (100 μM) resulted in hyperpolarized activation and inactivation curves as well as greater tonic block and use dependent block of I1327V mutant channels relative to WT. Significance SCN8A – I1327V is a gain-of-function mutation with altered features that are predicted to increase neuronal excitability and seizure susceptibility. Phenytoin is an effective inhibitor of the mutant channel and may be of use in treating patients with gain-of-function mutations of SCN8A. PMID:27375106

Permeation and block of TRPV1 channels by the cationic lidocaine derivative QX-314

PubMed Central

Puopolo, Michelino; Binshtok, Alexander M.; Yao, Gui-Lan; Oh, Seog Bae; Woolf, Clifford J.

2013-01-01

QX-314 (N-ethyl-lidocaine) is a cationic lidocaine derivative that blocks voltage-dependent sodium channels when applied internally to axons or neuronal cell bodies. Coapplication of external QX-314 with the transient receptor potential vanilloid 1 protein (TRPV1) agonist capsaicin produces long-lasting sodium channel inhibition in TRPV1-expressing neurons, suggestive of QX-314 entry into the neurons. We asked whether QX-314 entry occurs directly through TRPV1 channels or through a different pathway (e.g., pannexin channels) activated downstream of TRPV1 and whether QX-314 entry requires the phenomenon of “pore dilation” previously reported for TRPV1. With external solutions containing 10 or 20 mM QX-314 as the only cation, inward currents were activated by stimulation of both heterologously expressed and native TRPV1 channels in rat dorsal root ganglion neurons. QX-314-mediated inward current did not require pore dilation, as it activated within several seconds and in parallel with Cs-mediated outward current, with a reversal potential consistent with PQX-314/PCs = 0.12. QX-314-mediated current was no different when TRPV1 channels were expressed in C6 glioma cells, which lack expression of pannexin channels. Rapid addition of QX-314 to physiological external solutions produced instant partial inhibition of inward currents carried by sodium ions, suggesting that QX-314 is a permeant blocker. Maintained coapplication of QX-314 with capsaicin produced slowly developing reduction of outward currents carried by internal Cs, consistent with intracellular accumulation of QX-314 to concentrations of 50–100 μM. We conclude that QX-314 is directly permeant in the “standard” pore formed by TRPV1 channels and does not require either pore dilation or activation of additional downstream channels for entry. PMID:23303863

Sodium-potassium-adenosinetriphosphatase-dependent sodium transport in the kidney: hormonal control.

PubMed

Féraille, E; Doucet, A

2001-01-01

Tubular reabsorption of filtered sodium is quantitatively the main contribution of kidneys to salt and water homeostasis. The transcellular reabsorption of sodium proceeds by a two-step mechanism: Na(+)-K(+)-ATPase-energized basolateral active extrusion of sodium permits passive apical entry through various sodium transport systems. In the past 15 years, most of the renal sodium transport systems (Na(+)-K(+)-ATPase, channels, cotransporters, and exchangers) have been characterized at a molecular level. Coupled to the methods developed during the 1965-1985 decades to circumvent kidney heterogeneity and analyze sodium transport at the level of single nephron segments, cloning of the transporters allowed us to move our understanding of hormone regulation of sodium transport from a cellular to a molecular level. The main purpose of this review is to analyze how molecular events at the transporter level account for the physiological changes in tubular handling of sodium promoted by hormones. In recent years, it also became obvious that intracellular signaling pathways interacted with each other, leading to synergisms or antagonisms. A second aim of this review is therefore to analyze the integrated network of signaling pathways underlying hormone action. Given the central role of Na(+)-K(+)-ATPase in sodium reabsorption, the first part of this review focuses on its structural and functional properties, with a special mention of the specificity of Na(+)-K(+)-ATPase expressed in renal tubule. In a second part, the general mechanisms of hormone signaling are briefly introduced before a more detailed discussion of the nephron segment-specific expression of hormone receptors and signaling pathways. The three following parts integrate the molecular and physiological aspects of the hormonal regulation of sodium transport processes in three nephron segments: the proximal tubule, the thick ascending limb of Henle's loop, and the collecting duct.

Uptake, elimination, and relative distribution of perchlorate in various tissues of channel catfish

USGS Publications Warehouse

Park, J.-W.; Bradford, C.M.; Rinchard, J.; Liu, F.; Wages, M.; Waters, A.; Kendall, R.J.; Anderson, T.A.; Theodorakis, C.W.

2007-01-01

This study was undertaken to determine the kinetics of uptake and elimination of perchlorate in channel catfish, Ictalurus punctatus. Perchlorate - an oxidizer used in solid fuel rockets, fireworks, and illuminating munitions - has been shown to effect thyroid function, causing hormone disruption and potential perturbations of metabolic activities. For the uptake study, catfish were exposed to 100 mg/L sodium perchlorate for 12 h to 5 d in the laboratory. Perchlorate in tissues was analyzed using ion chromatography. The highest perchlorate concentrations were found in the head and fillet, indicating that these tissues are the most important tissues to analyze when determining perchlorate uptake into large fish. To calculate uptake and elimination rate constants for fillet, gills, G-I tract, liver, and head, fish were exposed to 100 ppm sodium perchlorate for 5 days, and allowed to depurate in clean water for up to 20 days. The animals rapidly eliminated the perchlorate accumulated showing the highest elimination in fillet (Ke = 1.67 day -1) and lowest elimination in liver (Ke = 0.79 day -1). ?? 2007 American Chemical Society.

The C. elegans ceh-36 gene encodes a putative homemodomain transcription factor involved in chemosensory functions of ASE and AWC neurons.

PubMed

Koga, Makoto; Ohshima, Yasumi

2004-02-20

Chemotaxis to water-soluble chemicals such as sodium ion is an important behavior of Caenorhabditis elegans for seeking food, and ASE chemosensory neurons have a major role in this behavior. We isolated mutants defective in chemotaxis to sodium acetate. We show here that among them ks86 had a mutation in the ceh-36 gene. ceh-36 :: gfp reporter constructs were expressed in ASE and AWC neurons. In a mutant of the che-1 gene, which encodes another transcription factor and is required for specification of ASE neurons, expression of the ceh-36 :: gfp reporter in ASE is lost. This indicates that the ceh-36 gene functions downstream of the che-1 gene in ASE. In the ceh-36(ks86) mutant, expression of the tax-2 gene encoding a cyclic nucleotide-gated channel was reduced in ASE and AWC. This affords an explanation for defects of the ceh-36 mutant in the chemotaxis mediated by ASE and AWC. When a ceh-36 cDNA was expressed in an adult ceh-36 mutant by a heat shock promoter, chemotaxis to sodium acetate was recovered. These results suggest that ceh-36 is required for functions, and not for development, of ASE.

Trans-channel interactions in batrachotoxin-modified skeletal muscle sodium channels: voltage-dependent block by cytoplasmic amines, and the influence of mu-conotoxin GIIIA derivatives and permeant ions.

PubMed

Pavlov, Evgeny; Britvina, Tatiana; McArthur, Jeff R; Ma, Quanli; Sierralta, Iván; Zamponi, Gerald W; French, Robert J

2008-11-01

External mu-conotoxins and internal amine blockers inhibit each other's block of voltage-gated sodium channels. We explore the basis of this interaction by measuring the shifts in voltage-dependence of channel inhibition by internal amines induced by two mu-conotoxin derivatives with different charge distributions and net charges. Charge changes on the toxin were made at residue 13, which is thought to penetrate most deeply into the channel, making it likely to have the strongest individual interaction with an internal charged ligand. When an R13Q or R13E molecule was bound to the channel, the voltage dependence of diethylammonium (DEA)-block shifted toward more depolarized potentials (23 mV for R13Q, and 16 mV for R13E). An electrostatic model of the repulsion between DEA and the toxin simulated these data, with a distance between residue 13 of the mu-conotoxin and the DEA-binding site of approximately 15 A. Surprisingly, for tetrapropylammonium, the shifts were only 9 mV for R13Q, and 7 mV for R13E. The smaller shifts associated with R13E, the toxin with a smaller net charge, are generally consistent with an electrostatic interaction. However, the smaller shifts observed for tetrapropylammonium than for DEA suggest that other factors must be involved. Two observations indicate that the coupling of permeant ion occupancy of the channel to blocker binding may contribute to the overall amine-toxin interaction: 1), R13Q binding decreases the apparent affinity of sodium for the conducting pore by approximately 4-fold; and 2), increasing external [Na(+)] decreases block by DEA at constant voltage. Thus, even though a number of studies suggest that sodium channels are occupied by no more than one ion most of the time, measurable coupling occurs between permeant ions and toxin or amine blockers. Such interactions likely determine, in part, the strength of trans-channel, amine-conotoxin interactions.

Trans-Channel Interactions in Batrachotoxin-Modified Skeletal Muscle Sodium Channels: Voltage-Dependent Block by Cytoplasmic Amines, and the Influence of μ-Conotoxin GIIIA Derivatives and Permeant Ions

PubMed Central

Pavlov, Evgeny; Britvina, Tatiana; McArthur, Jeff R.; Ma, Quanli; Sierralta, Iván; Zamponi, Gerald W.; French, Robert J.

2008-01-01

External μ-conotoxins and internal amine blockers inhibit each other's block of voltage-gated sodium channels. We explore the basis of this interaction by measuring the shifts in voltage-dependence of channel inhibition by internal amines induced by two μ-conotoxin derivatives with different charge distributions and net charges. Charge changes on the toxin were made at residue 13, which is thought to penetrate most deeply into the channel, making it likely to have the strongest individual interaction with an internal charged ligand. When an R13Q or R13E molecule was bound to the channel, the voltage dependence of diethylammonium (DEA)-block shifted toward more depolarized potentials (23 mV for R13Q, and 16 mV for R13E). An electrostatic model of the repulsion between DEA and the toxin simulated these data, with a distance between residue 13 of the μ-conotoxin and the DEA-binding site of ∼15 Å. Surprisingly, for tetrapropylammonium, the shifts were only 9 mV for R13Q, and 7 mV for R13E. The smaller shifts associated with R13E, the toxin with a smaller net charge, are generally consistent with an electrostatic interaction. However, the smaller shifts observed for tetrapropylammonium than for DEA suggest that other factors must be involved. Two observations indicate that the coupling of permeant ion occupancy of the channel to blocker binding may contribute to the overall amine-toxin interaction: 1), R13Q binding decreases the apparent affinity of sodium for the conducting pore by ∼4-fold; and 2), increasing external [Na+] decreases block by DEA at constant voltage. Thus, even though a number of studies suggest that sodium channels are occupied by no more than one ion most of the time, measurable coupling occurs between permeant ions and toxin or amine blockers. Such interactions likely determine, in part, the strength of trans-channel, amine-conotoxin interactions. PMID:18658222

Myotonia permanens with Nav1.4-G1306E displays varied phenotypes during course of life.

PubMed

Lehmann-Horn, Frank; D'Amico, Adele; Bertini, Enrico; Lomonaco, Mauro; Merlini, Luciano; Nelson, Kevin R; Philippi, Heike; Siciliano, Gabriele; Spaans, Frank; Jurkat-Rott, Karin

2017-09-01

Myotonia permanens due to Nav1.4-G1306E is a rare sodium channelopathy with potentially life-threatening respiratory complications. Our goal was to study phenotypic variability throughout life. Clinical neurophysiology and genetic analysis were performed. Using existing functional expression data we determined the sodium window by integration. In 10 unrelated patients who were believed to have epilepsy, respiratory disease or Schwartz-Jampel syndrome, we made the same prima facie diagnosis and detected the same heterologous Nav1.4-G1306E channel mutation as for our first myotonia permanens patient published in 1993. Eight mutations were de-novo, two were inherited from the affected parent each. Seven patients improved with age, one had a benign phenotype from birth, and two died of respiratory complications. The clinical features age-dependently varied with severe neonatal episodic laryngospasm in childhood and myotonia throughout life. Weakness of varying degrees was present. The responses to cold, exercise and warm-up were different for lower than for upper extremities. Spontaneous membrane depolarization increased frequency and decreased size of action potentials; self-generated repolarization did the opposite. The overlapping of steady-state activation and inactivation curves generated a 3.1-fold window area for G1306E vs. normal channels. Residue G1306 Neonatal laryngospasm and unusual distribution of myotonia, muscle hypertrophy, and weakness encourage direct search for the G1306E mutation, a hotspot for de-novo mutations. Successful therapy with the sodium channel blocker flecainide is due to stabilization of the inactivated state and special effectiveness for enlarged window currents. Our G1306E collection is the first genetically clarified case series from newborn period to adulthood and therefore helpful for counselling.

Myotonia permanens with Nav1.4-G1306E displays varied phenotypes during course of life

PubMed Central

LEHMANN-HORN, FRANK; D’AMICO, ADELE; BERTINI, ENRICO; LOMONACO, MAURO; MERLINI, LUCIANO; NELSON, KEVIN R.; PHILIPPI, HEIKE; SICILIANO, GABRIELE; SPAANS, FRANK; JURKAT-ROTT, KARIN

2017-01-01

Introduction Myotonia permanens due to Nav1.4-G1306E is a rare sodium channelopathy with potentially life-threatening respiratory complications. Our goal was to study phenotypic variability throughout life. Methods Clinical neurophysiology and genetic analysis were performed. Using existing functional expression data we determined the sodium window by integration. Results In 10 unrelated patients who were believed to have epilepsy, respiratory disease or Schwartz-Jampel syndrome, we made the same prima facie diagnosis and detected the same heterologous Nav1.4-G1306E channel mutation as for our first myotonia permanens patient published in 1993. Eight mutations were de-novo, two were inherited from the affected parent each. Seven patients improved with age, one had a benign phenotype from birth, and two died of respiratory complications. The clinical features age-dependently varied with severe neonatal episodic laryngospasm in childhood and myotonia throughout life. Weakness of varying degrees was present. The responses to cold, exercise and warm-up were different for lower than for upper extremities. Spontaneous membrane depolarization increased frequency and decreased size of action potentials; self-generated repolarization did the opposite. The overlapping of steady-state activation and inactivation curves generated a 3.1-fold window area for G1306E vs. normal channels. Discussion Residue G1306 Neonatal laryngospasm and unusual distribution of myotonia, muscle hypertrophy, and weakness encourage direct search for the G1306E mutation, a hotspot for de-novo mutations. Successful therapy with the sodium channel blocker flecainide is due to stabilization of the inactivated state and special effectiveness for enlarged window currents. Our G1306E collection is the first genetically clarified case series from newborn period to adulthood and therefore helpful for counselling. PMID:29774303

Sodium and calcium currents in neuroblastoma x glioma hybrid cells before and after morphological differentiation by dibutyryl cyclic AMP.

PubMed

Bodewei, R; Hering, S; Schubert, B; Wollenberger, A

1985-04-01

Sodium and calcium inward currents (INa and ICa) were measured in neuroblastoma X glioma hybrid cells of clones 108CC5 and 108CC15 by a single suction pipette method for internal perfusion and voltage clamp. Morphologically undifferentiated, exponentially growing cells were compared with cells differentiated by cultivation with 1 mmol/l dibutyryl cyclic AMP. Outward currents were eliminated by perfusing the cells with a K+-free solution. Voltage dependence and ion selectivity as well as steady state inactivation characteristics of INa and ICa resembled those of differentiated mouse neuroblastoma cells, clone N1E-115 (Moolenaar and Spector 1978, 1979). These parameters were identical in undifferentiated and differentiated cells of both clones. After differentiation the average density of the peak sodium and calcium currents was increased two and four-fold, respectively, in both cell lines. Our data indicate that exponentially growing, morphologically undifferentiated 108CC5 and 108CC15 neuroblastoma X glioma hybrid cells possess functional Na+ and Ca2+ channels undistinguishable from those of non-proliferating cells of these clones differentiated morphologically by treatment with dibutyryl cyclic AMP. That Na+ and Ca2+ spikes were not detected by other authors in these cells prior to morphological differentiation by dibutyryl cyclic AMP may be attributed to the fact that at the low resting membrane potential measured the Na+ and Ca2+ channels are inactivated.

Mechanisms of tubular sodium chloride transport.

PubMed

Venkatesh, S; Schrier, R W; Andreoli, T E

1998-11-01

Extracellular fluid volume is determined by sodium and its accompanying anions. There are control mechanisms which regulate sodium balance in the body. These include high and low pressure baroreceptors, intrarenal baroreceptors, renal autoregulation, tubuloglomerular feedback, aldosterone, and numerous other physical and hormonal factors. Sodium transport by the nephron involves active and passive processes which occur in several different nephron segments. Mechanisms of cotransport, Na(+)-H+ exchange, antiporters and ion-specific channels are all utilized by the nephron to maintain sodium balance. These regulatory factors and transport mechanisms for sodium in the kidney will he discussed in detail.

Roderick MacKinnon and Ion Channels - Potassium Channels and Sodium

Science.gov Websites

very first potassium channel structure, which revealed the way that positively charged potassium ions explain how a fundamental feedback loop worked. Now, with the structure of the voltage-dependent ion channel, based on research carried out at The Rockefeller University, the National Synchrotron Light

A single Markov-type kinetic model accounting for the macroscopic currents of all human voltage-gated sodium channel isoforms.

PubMed

Balbi, Pietro; Massobrio, Paolo; Hellgren Kotaleski, Jeanette

2017-09-01

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 kinetic model, developed in the NEURON simulation environment, appears to be the simplest and most parsimonious way for a detailed phenomenological description of the human VGSCs electrophysiological behaviour.

Initial post marketing experience with lacosamide in adult patients with epilepsy.

PubMed

Harden, Cynthia L; Cohn, Aaron; Lowe, Merredith; Serrano, Enrique

2012-02-01

The outcomes of adult epilepsy patients prescribed lacosamide for additional seizure control. Responders were defined as having at least a 50% decrease in seizure frequency Sixty-seven patients were evaluated. Forty-six out of 67 patients (69%) were responders. Twelve of 14 patients not taking sodium channel-acting AEDs were responders (86%) and 34/53 patients taking sodium channel-acting AEDs were responders (64%) (difference not significant). Copyright © 2011 Elsevier B.V. All rights reserved.

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

DOE Office of Scientific and Technical Information (OSTI.GOV)

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 localizationsmore » 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.« less

Molecular basis of the remarkable species selectivity of an insecticidal sodium channel toxin from the African spider Augacephalus ezendami

NASA Astrophysics Data System (ADS)

Herzig, Volker; Ikonomopoulou, Maria; Smith, Jennifer J.; Dziemborowicz, Sławomir; Gilchrist, John; Kuhn-Nentwig, Lucia; Rezende, Fernanda Oliveira; Moreira, Luciano Andrade; Nicholson, Graham M.; Bosmans, Frank; King, Glenn F.

2016-07-01

The inexorable decline in the armament of registered chemical insecticides has stimulated research into environmentally-friendly alternatives. Insecticidal spider-venom peptides are promising candidates for bioinsecticide development but it is challenging to find peptides that are specific for targeted pests. In the present study, we isolated an insecticidal peptide (Ae1a) from venom of the African spider Augacephalus ezendami (family Theraphosidae). Injection of Ae1a into sheep blowflies (Lucilia cuprina) induced rapid but reversible paralysis. In striking contrast, Ae1a was lethal to closely related fruit flies (Drosophila melanogaster) but induced no adverse effects in the recalcitrant lepidopteran pest Helicoverpa armigera. Electrophysiological experiments revealed that Ae1a potently inhibits the voltage-gated sodium channel BgNaV1 from the German cockroach Blattella germanica by shifting the threshold for channel activation to more depolarized potentials. In contrast, Ae1a failed to significantly affect sodium currents in dorsal unpaired median neurons from the American cockroach Periplaneta americana. We show that Ae1a interacts with the domain II voltage sensor and that sensitivity to the toxin is conferred by natural sequence variations in the S1-S2 loop of domain II. The phyletic specificity of Ae1a provides crucial information for development of sodium channel insecticides that target key insect pests without harming beneficial species.

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

PubMed

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.

Identification of an alternative knockdown resistance (kdr)-like mutation, M918L, and a novel mutation, V1010A, in the Thrips tabaci voltage-gated sodium channel gene.

PubMed

Wu, Meixiang; Gotoh, Hiroki; Waters, Timothy; Walsh, Douglas B; Lavine, Laura Corley

2014-06-01

Knockdown resistance (kdr) has been identified as a main mechanism against pyrethroid insecticides in many arthropod pests including in the onion thrips, Thrips tabaci. To characterize and identify pyrethroid-resistance in onion thrips in Washington state, we conducted insecticide bioassays and sequenced a region of the voltage gated sodium channel gene from several different T. tabaci populations. Field collected Thrips tabaci were found to have large variations in resistance to the pyrethroid insecticide lambda-cyhalothrin. We identified two single nucleotide substitutions in our analysis of a partial sequence of the T. tabaci voltage-gated sodium channel gene. One mutation resulted in the non-synonymous substitution of methionine with leucine (M918L), which is well known to be responsible for super knockdown resistance in some pest species. Another non-synonymous substitution, a valine (GTT) to alanine (GCT) replacement at amino acid 1010 (V1010A) was identified in our study and was associated with lambda-cyhalothrin resistance. We have characterized a known kdr mutation and identified a novel mutation in the voltage-gated sodium channel gene of Thrips tabaci associated with resistance to lambda-cyhalothrin. This gene region and these mutations are expected to be useful in the development of a diagnostic test to detect kdr resistance in many onion thrips populations. © 2013 Society of Chemical Industry.

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

PubMed Central

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 first effect was studied in crayfish axons. We found that the removal of the fast Na inactivation did not depend on the states of the channel since the channel could be modified by chloramine-T at holding potential (from -80 to -100 mV) or at depolarized potential of -30 mV. After removal of fast Na inactivation, the slow inactivation mechanism was still present, and more channels could undergo slow inactivation. This result indicates that in crayfish axons the transition through the fast inactivated state is not a prerequisite for the slow inactivation to occur. During chloramine-T treatment, a distinct blocking phase occurred, which recovered upon washing out the drug. This second effect of chloramine-T was studied in detail in squid axons. After 24 h, chloramine-T solution lost its ability to remove fast inactivation but retained its blocking action. After removal of the fast Na inactivation, both fresh and aged chloramine-T solutions blocked the Na currents with a similar potency and in a voltage-dependent manner, being more pronounced at lower depolarizing potentials. A similar voltage-dependent block was observed with aged chloramine-T solution in an axon with intact inactivation. In contrast to the action of the fresh solution, the aged chloramine-T solution was found to accelerate the decay of Na currents.These results suggest that chloramine-T solution contains at least two active molecular forms that act at different sites in the Na channel. PMID:2444276

A complicated complex: Ion channels, voltage sensing, cell membranes and peptide inhibitors.

PubMed

Zhang, Alan H; Sharma, Gagan; Undheim, Eivind A B; Jia, Xinying; Mobli, Mehdi

2018-04-21

Voltage-gated ion channels (VGICs) are specialised ion channels that have a voltage dependent mode of action, where ion conduction, or gating, is controlled by a voltage-sensing mechanism. VGICs are critical for electrical signalling and are therefore important pharmacological targets. Among these, voltage-gated sodium channels (Na V s) have attracted particular attention as potential analgesic targets. Na V s, however, comprise several structurally similar subtypes with unique localisations and distinct functions, ranging from amplification of action potentials in nociception (e.g. Na V 1.7) to controlling electrical signalling in cardiac function (Na V 1.5). Understanding the structural basis of Na V function is therefore of great significance, both to our knowledge of electrical signalling and in development of subtype and state selective drugs. An important tool in this pursuit has been the use of peptides from animal venoms as selective Na V modulators. In this review, we look at peptides, particularly from spider venoms, that inhibit Na V s by binding to the voltage sensing domain (VSD) of this channel, known as gating modifier toxins (GMT). In the first part of the review, we look at the structural determinants of voltage sensing in VGICs, the gating cycle and the conformational changes that accompany VSD movement. Next, the modulation of the analgesic target Na V 1.7 by GMTs is reviewed to develop bioinformatic tools that, based on sequence information alone, can identify toxins that are likely to inhibit this channel. The same approach is also used to define VSD sequences, other than that from Na V 1.7, which are likely to be sensitive to this class of toxins. The final section of the review focuses on the important role of the cellular membrane in channel modulation and also how the lipid composition affects measurements of peptide-channel interactions both in binding kinetics measurements in solution and in cell-based functional assays. Copyright © 2018 Elsevier B.V. All rights reserved.

Bicarbonate refractory QRS prolongation and left bundle-branch block following escitalopram and lamotrigine overdose: A case report and literature review of toxic left bundle-branch block.

PubMed

Farkas, A N; Marcott, M; Yanta, J H; Pizon, A F

2018-05-02

Toxic prolongation of the QRS interval most often results from blockade of cardiac voltage-gated sodium channels and manifests on electrocardiogram with a right bundle-branch block-like morphology. Rarely, a left bundle-branch block (LBBB) morphology has been reported. We report a case of transient LBBB resultant from ingestion of lamotrigine and citalopram which was refractory to sodium bicarbonate therapy and eventually resolved spontaneously. Cases of toxic LBBB are less likely to respond to bicarbonate therapy, suggesting that this finding is due to a mechanism other than sodium channel blockade. © 2018 John Wiley & Sons Ltd.

Simulating the Activation of Voltage Sensing Domain for a Voltage-Gated Sodium Channel Using Polarizable Force Field.

PubMed

Sun, Rui-Ning; Gong, Haipeng

2017-03-02

Voltage-gated sodium (Na V ) channels play vital roles in the signal transduction of excitable cells. Upon activation of a Na V channel, the change of transmembrane voltage triggers conformational change of the voltage sensing domain, which then elicits opening of the pore domain and thus allows an influx of Na + ions. Description of this process with atomistic details is in urgent demand. In this work, we simulated the partial activation process of the voltage sensing domain of a prokaryotic Na V channel using a polarizable force field. We not only observed the conformational change of the voltage sensing domain from resting to preactive state, but also rigorously estimated the free energy profile along the identified reaction pathway. Comparison with the control simulation using an additive force field indicates that voltage-gating thermodynamics of Na V channels may be inaccurately described without considering the electrostatic polarization effect.

Bioluminescence methodology for the detection of protein-protein interactions within the voltage-gated sodium channel macromolecular complex.

PubMed

Shavkunov, Alexander; Panova, Neli; Prasai, Anesh; Veselenak, Ron; Bourne, Nigel; Stoilova-McPhie, Svetla; Laezza, Fernanda

2012-04-01

Protein-protein interactions are critical molecular determinants of ion channel function and emerging targets for pharmacological interventions. Yet, current methodologies for the rapid detection of ion channel macromolecular complexes are still lacking. In this study we have adapted a split-luciferase complementation assay (LCA) for detecting the assembly of the voltage-gated Na+ (Nav) channel C-tail and the intracellular fibroblast growth factor 14 (FGF14), a functionally relevant component of the Nav channelosome that controls gating and targeting of Nav channels through direct interaction with the channel C-tail. In the LCA, two complementary N-terminus and C-terminus fragments of the firefly luciferase were fused, respectively, to a chimera of the CD4 transmembrane segment and the C-tail of Nav1.6 channel (CD4-Nav1.6-NLuc) or FGF14 (CLuc-FGF14). Co-expression of CLuc-FGF14 and CD4-Nav1.6-NLuc in live cells led to a robust assembly of the FGF14:Nav1.6 C-tail complex, which was attenuated by introducing single-point mutations at the predicted FGF14:Nav channel interface. To evaluate the dynamic regulation of the FGF14:Nav1.6 C-tail complex by signaling pathways, we investigated the effect of kinase inhibitors on the complex formation. Through a platform of counter screenings, we show that the p38/MAPK inhibitor, PD169316, and the IκB kinase inhibitor, BAY 11-7082, reduce the FGF14:Nav1.6 C-tail complementation, highlighting a potential role of the p38MAPK and the IκB/NFκB pathways in controlling neuronal excitability through protein-protein interactions. We envision the methodology presented here as a new valuable tool to allow functional evaluations of protein-channel complexes toward probe development and drug discovery targeting ion channels implicated in human disorders.

Defective Fast Inactivation Recovery of Nav1.4 in Congenital Myasthenic Syndrome

PubMed Central

Arnold, W. David; Feldman, Daniel H.; Ramirez, Sandra; He, Liuyuan; Kassar, Darine; Quick, Adam; Klassen, Tara L.; Lara, Marian; Nguyen, Joanna; Kissel, John T.; Lossin, Christoph; Maselli, Ricardo A.

2015-01-01

Objective To describe the unique phenotype and genetic findings in a 57-year-old female with a rare form of congenital myasthenic syndrome (CMS) associated with longstanding muscle fatigability, and to investigate the underlying pathophysiology. Methods We used whole-cell voltage clamping to compare the biophysical parameters of wild-type and Arg1457His-mutant Nav1.4. Results Clinical and neurophysiological evaluation revealed features consistent with CMS. Sequencing of candidate genes indicated no abnormalities. However, analysis of SCN4A, the gene encoding the skeletal muscle sodium channel Nav1.4, revealed a homozygous mutation predicting an arginine-to-histidine substitution at position 1457 (Arg1457His), which maps to the channel’s voltage sensor, specifically D4/S4. Whole-cell patch clamp studies revealed that the mutant required longer hyperpolarization to recover from fast inactivation, which produced a profound use-dependent current attenuation not seen in the wild type. The mutant channel also had a marked hyperpolarizing shift in its voltage dependence of inactivation as well as slowed inactivation kinetics. Interpretation We conclude that Arg1457His compromises muscle fiber excitability. The mutant fast-inactivates with significantly less depolarization, and it recovers only after extended hyperpolarization. The resulting enhancement in its use dependence reduces channel availability, which explains the patient’s muscle fatigability. Arg1457His offers molecular insight into a rare form of CMS precipitated by sodium channel inactivation defects. Given this channel’s involvement in other muscle disorders such as paramyotonia congenita and hyperkalemic periodic paralysis, our study exemplifies how variations within the same gene can give rise to multiple distinct dysfunctions and phenotypes, revealing residues important in basic channel function. PMID:25707578

PSD-95 interacts with NBCn1 and enhances channel-like activity without affecting Na/HCO(3) cotransport.

PubMed

Lee, Soojung; Yang, Han Soo; Kim, Eunjin; Ju, Eun Ji; Kwon, Min Hyung; Dudley, R Kyle; Smith, Yoland; Yun, C Chris; Choi, Inyeong

2012-01-01

The sodium/bicarbonate transporter NBCn1 plays an essential role in intracellular pH regulation and transepithelial HCO(3)(-) movement in the body. NBCn1 also has sodium channel-like activity uncoupled to Na/HCO(3) cotransport. We previously reported that NBCn1 interacts with the postsynaptic density protein PSD-95 in the brain. Here, we elucidated the structural determinant and functional consequence of NBCn1/PSD-95 interaction. In rat hippocampal CA3 neurons, NBCn1 was localized to the postsynaptic membranes of both dendritic shafts and spines and occasionally to the presynaptic membranes. A GST/NBCn1 fusion protein containing the C-terminal 131 amino acids of NBCn1 pulled down PSD-95 from rat brain lysates, whereas GST/NBCn1-ΔETSL (deletion of the last four amino acids) and GST/NBCn2 (NCBE) lacking the same ETSL did not. NBCn1 and PSD-95 were coimmunoprecipitated in HEK 293 cells, and their interaction did not affect the efficacy of PSD-95 to bind to the NMDA receptor NR2A. PSD-95 has negligible effects on intracellular pH changes mediated by NBCn1 in HEK 293 cells and Xenopus oocytes. However, PSD-95 increased an ionic conductance produced by NBCn1 channel-like activity. This increase was abolished by NBCn1-ΔETSL or by the peptide containing the last 15 amino acids of NBCn1. Our data suggest that PSD-95 interacts with NBCn1 and increases its channel-like activity while negligibly affecting Na/HCO(3) cotransport. The possibility that the channel-like activity occurs via an intermolecular cavity of multimeric NBCn1 proteins is discussed. Copyright © 2012 S. Karger AG, Basel.

PSD-95 Interacts with NBCn1 and Enhances Channel-like Activity without Affecting Na/HCO3 Cotransport

PubMed Central

Lee, Soojung; Yang, Han Soo; Kim, Eunjin; Ju, Eun Ji; Kwon, Min Hyung; Dudley, R. Kyle; Smith, Yoland; Yun, C. Chris; Choi, Inyeong

2013-01-01

Background/Aims The sodium/bicarbonate transporter NBCn1 plays an essential role in intracellular pH regulation and transepithelial HCO3− movement in the body. NBCn1 also has sodium channel-like activity uncoupled to Na/HCO3 cotransport. We previously reported that NBCn1 interacts with the postsynaptic density protein PSD-95 in the brain. Here, we elucidated the structural determinant and functional consequence of NBCn1/PSD-95 interaction. Methods: Results In rat hippocampal CA3 neurons, NBCn1 was localized to the postsynaptic membranes of both dendritic shafts and spines and occasionally to the presynaptic membranes. A GST/NBCn1 fusion protein containing the C-terminal 131 amino acids of NBCn1 pulled down PSD-95 from rat brain lysates, whereas GST/NBCn1-ΔETSL (deletion of the last four amino acids) and GST/NBCn2 (NCBE) lacking the same ETSL did not. NBCn1 and PSD-95 were coimmunoprecipitated in HEK 293 cells, and their interaction did not affect the efficacy of PSD-95 to bind to the NMDA receptor NR2A. PSD-95 has negligible effects on intracellular pH changes mediated by NBCn1 in HEK 293 cells and Xenopus oocytes. However, PSD-95 increased an ionic conductance produced by NBCn1 channel-like activity. This increase was abolished by NBCn1-ΔETSL or by the peptide containing the last 15 amino acids of NBCn1. Conclusion Our data suggest that PSD-95 interacts with NBCn1 and increases its channel-like activity while negligibly affecting Na/HCO3 cotransport. The possibility that the channel-like activity occurs via an intermolecular cavity of multimeric NBCn1 proteins is discussed. PMID:23183381

TPC Proteins Are Phosphoinositide-activated Sodium-selective Ion Channels in Endosomes and Lysosomes

PubMed Central

Wang, Xiang; Zhang, Xiaoli; Dong, Xian-ping; Samie, Mohammad; Li, Xinran; Cheng, Xiping; Goschka, Andrew; Shen, Dongbiao; Zhou, Yandong; Harlow, Janice; Zhu, Michael X.; Clapham, David E.; Ren, Dejian; Xu, Haoxing

2012-01-01

Summary Mammalian Two-Pore Channels (TPC1, 2; TPCN1, TPCN2) encode ion channels in intracellular endosomes and lysosomes and were proposed to mediate endolysosomal calcium release triggered by the second messenger, nicotinic acid adenine dinucleotide phosphate (NAADP). By directly recording TPCs in endolysosomes from wild-type and TPC double knockout mice, here we show that, in contrast to previous conclusions, TPCs are in fact sodium-selective channels activated by PI(3,5)P2, and are not activated by NAADP. Moreover, the primary endolysosomal ion is Na+, not K+, as had been previously assumed. These findings suggest that the organellar membrane potential may undergo large regulatory changes, and may explain the specificity of PI(3,5)P2 in regulating the fusogenic potential of intracellular organelles. PMID:23063126

[Membrane mechanisms of effects of antihypoxic agents bemethyl and almide on neurons of Mollusca].

PubMed

Vislobokov, A I; Marysheva, V V; Shabanov, P D

2003-01-01

Membranotropic effects of the antihypoxants bemithyl and almide, structural analogs of thiobenzimidazole, have been studied on the isolated neuronal preparations of Lymaea stagnalis branchycephalic mollusk. Both drugs in a concentration range of 100-1000 microM produced a reversible, dose-dependent nonselective single-phase blocking action upon the ion channels and completely blocked the channels at a concentration of 10 mM. Therefore, bemithyl and almide are active membranotropic compounds capable (in sufficiently high concentrations) of changing the conductivity of slow sodium, calcium, and potassium ion channels in excitable cells. The protective antihypoxant drug reactions on a systemic level of the organism are probably related to the fact that both drugs in small concentrations are capable of hyperpolarizing the cell membrane, activating the ion channel function, and stabilizing the action potential under hypoxia conditions; in greater concentrations, bemithyl and almide are capable of blocking ion currents, thus reducing the excitability of cells and protecting them from overstress.

Endothelin-1 mediates natriuresis but not polyuria during vitamin D-induced acute hypercalcaemia.

PubMed

Tokonami, Natsuko; Cheval, Lydie; Monnay, Isabelle; Meurice, Guillaume; Loffing, Johannes; Feraille, Eric; Houillier, Pascal

2017-04-15

Hypercalcaemia can occur under various pathological conditions, such as primary hyperparathyroidism, malignancy or granulomatosis, and it induces natriuresis and polyuria in various species via an unknown mechanism. A previous study demonstrated that hypercalcaemia induced by vitamin D in rats increased endothelin (ET)-1 expression in the distal nephron, which suggests the involvement of the ET system in hypercalcaemia-induced effects. In the present study, we demonstrate that, during vitamin D-induced hypercalcaemia, the activation of ET system by increased ET-1 is responsible for natriuresis but not for polyuria. Vitamin D-treated hypercalcaemic mice showed a blunted response to amiloride, suggesting that epithelial sodium channel function is inhibited. We have identified an original pathway that specifically mediates the effects of vitamin D-induced hypercalcaemia on sodium handling in the distal nephron without affecting water handling. Acute hypercalcaemia increases urinary sodium and water excretion; however, the underlying molecular mechanism remains unclear. Because vitamin D-induced hypercalcaemia increases the renal expression of endothelin (ET)-1, we hypothesized that ET-1 mediates the effects of hypercalcaemia on renal sodium and water handling. Hypercalcaemia was induced in 8-week-old, parathyroid hormone-supplemented, male mice by oral administration of dihydrotachysterol (DHT) for 3 days. DHT-treated mice became hypercalcaemic and displayed increased urinary water and sodium excretion compared to controls. mRNA levels of ET-1 and the transcription factors CCAAT-enhancer binding protein β and δ were specifically increased in the distal convoluted tubule and downstream segments in DHT-treated mice. To examine the role of the ET system in hypercalcaemia-induced natriuresis and polyuria, mice were treated with the ET-1 receptor antagonist macitentan, with or without DHT. Mice treated with both macitentan and DHT displayed hypercalcaemia and polyuria similar to that in mice treated with DHT alone; however, no increase in urinary sodium excretion was observed. To identify the affected sodium transport mechanism, we assessed the response to various diuretics in control and DHT-treated hypercalcaemic mice. Amiloride, an inhibitor of the epithelial sodium channel (ENaC), increased sodium excretion to a lesser extent in DHT-treated mice compared to control mice. Mice treated with either macitentan+DHT or macitentan alone had a similar response to amiloride. In summary, vitamin D-induced hypercalcaemia increases the renal production of ET-1 and decreases ENaC activity, which is probably responsible for the rise in urinary sodium excretion but not for polyuria. © 2017 The Authors. The Journal of Physiology © 2017 The Physiological Society.

Endothelin‐1 mediates natriuresis but not polyuria during vitamin D‐induced acute hypercalcaemia

PubMed Central

Tokonami, Natsuko; Cheval, Lydie; Monnay, Isabelle; Meurice, Guillaume; Loffing, Johannes; Feraille, Eric

2017-01-01

Key points Hypercalcaemia can occur under various pathological conditions, such as primary hyperparathyroidism, malignancy or granulomatosis, and it induces natriuresis and polyuria in various species via an unknown mechanism.A previous study demonstrated that hypercalcaemia induced by vitamin D in rats increased endothelin (ET)‐1 expression in the distal nephron, which suggests the involvement of the ET system in hypercalcaemia‐induced effects.In the present study, we demonstrate that, during vitamin D‐induced hypercalcaemia, the activation of ET system by increased ET‐1 is responsible for natriuresis but not for polyuria.Vitamin D‐treated hypercalcaemic mice showed a blunted response to amiloride, suggesting that epithelial sodium channel function is inhibited.We have identified an original pathway that specifically mediates the effects of vitamin D‐induced hypercalcaemia on sodium handling in the distal nephron without affecting water handling. Abstract Acute hypercalcaemia increases urinary sodium and water excretion; however, the underlying molecular mechanism remains unclear. Because vitamin D‐induced hypercalcaemia increases the renal expression of endothelin (ET)‐1, we hypothesized that ET‐1 mediates the effects of hypercalcaemia on renal sodium and water handling. Hypercalcaemia was induced in 8‐week‐old, parathyroid hormone‐supplemented, male mice by oral administration of dihydrotachysterol (DHT) for 3 days. DHT‐treated mice became hypercalcaemic and displayed increased urinary water and sodium excretion compared to controls. mRNA levels of ET‐1 and the transcription factors CCAAT‐enhancer binding protein β and δ were specifically increased in the distal convoluted tubule and downstream segments in DHT‐treated mice. To examine the role of the ET system in hypercalcaemia‐induced natriuresis and polyuria, mice were treated with the ET‐1 receptor antagonist macitentan, with or without DHT. Mice treated with both macitentan and DHT displayed hypercalcaemia and polyuria similar to that in mice treated with DHT alone; however, no increase in urinary sodium excretion was observed. To identify the affected sodium transport mechanism, we assessed the response to various diuretics in control and DHT‐treated hypercalcaemic mice. Amiloride, an inhibitor of the epithelial sodium channel (ENaC), increased sodium excretion to a lesser extent in DHT‐treated mice compared to control mice. Mice treated with either macitentan+DHT or macitentan alone had a similar response to amiloride. In summary, vitamin D‐induced hypercalcaemia increases the renal production of ET‐1 and decreases ENaC activity, which is probably responsible for the rise in urinary sodium excretion but not for polyuria. PMID:28120456

Identification of potential novel interaction partners of the sodium-activated potassium channels Slick and Slack in mouse brain.

PubMed

Rizzi, Sandra; Schwarzer, Christoph; Kremser, Leopold; Lindner, Herbert H; Knaus, Hans-Günther

2015-12-01

The sodium-activated potassium channels Slick (Slo2.1, KCNT2) and Slack (Slo2.2, KCNT1) are paralogous channels of the Slo family of high-conductance potassium channels. Slick and Slack channels are widely distributed in the mammalian CNS and they play a role in slow afterhyperpolarization, generation of depolarizing afterpotentials and in setting and stabilizing the resting potential. In the present study we used a combined approach of (co)-immunoprecipitation studies, Western blot analysis, double immunofluorescence and mass spectrometric sequencing in order to investigate protein-protein interactions of the Slick and Slack channels. The data strongly suggest that Slick and Slack channels co-assemble into identical cellular complexes. Double immunofluorescence experiments revealed that Slick and Slack channels co-localize in distinct mouse brain regions. Moreover, we identified the small cytoplasmic protein beta-synuclein and the transmembrane protein 263 (TMEM 263) as novel interaction partners of both, native Slick and Slack channels. In addition, the inactive dipeptidyl-peptidase (DPP 10) and the synapse associated protein 102 (SAP 102) were identified as constituents of the native Slick and Slack channel complexes in the mouse brain. This study presents new insights into protein-protein interactions of native Slick and Slack channels in the mouse brain.

Ion channels to inactivate neurons in Drosophila.

PubMed

Hodge, James J L

2009-01-01

Ion channels are the determinants of excitability; therefore, manipulation of their levels and properties provides an opportunity for the investigator to modulate neuronal and circuit function. There are a number of ways to suppress electrical activity in Drosophila neurons, for instance, over-expression of potassium channels (i.e. Shaker Kv1, Shaw Kv3, Kir2.1 and DORK) that are open at resting membrane potential. This will result in increased potassium efflux and membrane hyperpolarisation setting resting membrane potential below the threshold required to fire action potentials. Alternatively over-expression of other channels, pumps or co-transporters that result in a hyperpolarised membrane potential will also prevent firing. Lastly, neurons can be inactivated by, disrupting or reducing the level of functional voltage-gated sodium (Nav1 paralytic) or calcium (Cav2 cacophony) channels that mediate the depolarisation phase of action potentials. Similarly, strategies involving the opposite channel manipulation should allow net depolarisation and hyperexcitation in a given neuron. These changes in ion channel expression can be brought about by the versatile transgenic (i.e. Gal4/UAS based) systems available in Drosophila allowing fine temporal and spatial control of (channel) transgene expression. These systems are making it possible to electrically inactivate (or hyperexcite) any neuron or neural circuit in the fly brain, and much like an exquisite lesion experiment, potentially elucidate whatever interesting behaviour or phenotype each network mediates. These techniques are now being used in Drosophila to reprogram electrical activity of well-defined circuits and bring about robust and easily quantifiable changes in behaviour, allowing different models and hypotheses to be rapidly tested.

Distinct roles of the DmNav and DSC1 channels in the action of DDT and pyrethroids.

PubMed

Rinkevich, Frank D; Du, Yuzhe; Tolinski, Josh; Ueda, Atsushi; Wu, Chun-Fang; Zhorov, Boris S; Dong, Ke

2015-03-01

Voltage-gated sodium channels (Nav channels) are critical for electrical signaling in the nervous system and are the primary targets of the insecticides DDT and pyrethroids. In Drosophila melanogaster, besides the canonical Nav channel, Para (also called DmNav), there is a sodium channel-like cation channel called DSC1 (Drosophila sodium channel 1). Temperature-sensitive paralytic mutations in DmNav (para(ts)) confer resistance to DDT and pyrethroids, whereas DSC1 knockout flies exhibit enhanced sensitivity to pyrethroids. To further define the roles and interaction of DmNav and DSC1 channels in DDT and pyrethroid neurotoxicology, we generated a DmNav/DSC1 double mutant line by introducing a para(ts1) allele (carrying the I265N mutation) into a DSC1 knockout line. We confirmed that the I265N mutation reduced the sensitivity to two pyrethroids, permethrin and deltamethrin of a DmNav variant expressed in Xenopus oocytes. Computer modeling predicts that the I265N mutation confers pyrethroid resistance by allosterically altering the second pyrethroid receptor site on the DmNav channel. Furthermore, we found that I265N-mediated pyrethroid resistance in para(ts1) mutant flies was almost completely abolished in para(ts1);DSC1(-/-) double mutant flies. Unexpectedly, however, the DSC1 knockout flies were less sensitive to DDT, compared to the control flies (w(1118A)), and the para(ts1);DSC1(-/-) double mutant flies were even more resistant to DDT compared to the DSC1 knockout or para(ts1) mutant. Our findings revealed distinct roles of the DmNav and DSC1 channels in the neurotoxicology of DDT vs. pyrethroids and implicate the exciting possibility of using DSC1 channel blockers or modifiers in the management of pyrethroid resistance. Copyright © 2015 Elsevier Inc. All rights reserved.

Gain-of-function mutation of a voltage-gated sodium channel NaV1.7 associated with peripheral pain and impaired limb development.

PubMed

Tanaka, Brian S; Nguyen, Phuong T; Zhou, Eray Yihui; Yang, Yong; Yarov-Yarovoy, Vladimir; Dib-Hajj, Sulayman D; Waxman, Stephen G

2017-06-02

Dominant mutations in voltage-gated sodium channel Na V 1.7 cause inherited erythromelalgia, a debilitating pain disorder characterized by severe burning pain and redness of the distal extremities. Na V 1.7 is preferentially expressed within peripheral sensory and sympathetic neurons. Here, we describe a novel Na V 1.7 mutation in an 11-year-old male with underdevelopment of the limbs, recurrent attacks of burning pain with erythema, and swelling in his feet and hands. Frequency and duration of the episodes gradually increased with age, and relief by cooling became less effective. The patient's sister had short stature and reported similar complaints of erythema and burning pain, but with less intensity. Genetic analysis revealed a novel missense mutation in Na V 1.7 (2567G>C; p.Gly856Arg) in both siblings. The G856R mutation, located within the DII/S4-S5 linker of the channel, substitutes a highly conserved non-polar glycine by a positively charged arginine. Voltage-clamp analysis of G856R currents revealed that the mutation hyperpolarized (-11.2 mV) voltage dependence of activation and slowed deactivation but did not affect fast inactivation, compared with wild-type channels. A mutation of Gly-856 to aspartic acid was previously found in a family with limb pain and limb underdevelopment, and its functional assessment showed hyperpolarized activation, depolarized fast inactivation, and increased ramp current. Structural modeling using the Rosetta computational modeling suite provided structural clues to the divergent effects of the substitution of Gly-856 by arginine and aspartic acid. Although the proexcitatory changes in gating properties of G856R contribute to the pathophysiology of inherited erythromelalgia, the link to limb underdevelopment is not well understood. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

Evaluation of microtransplantation of rat brain neurolemma into Xenopus laevis oocytes as a technique to study the effect of neurotoxicants on endogenous voltage-sensitive ion channels.

PubMed

Murenzi, Edwin; Toltin, Abigail C; Symington, Steven B; Morgan, Molly M; Clark, John M

2017-05-01

Microtransplantation of mammalian brain neurolemma into the plasma membrane of Xenopus oocytes is used to study ion channels in their native form as they appear in the central nervous system. Use of microtransplanted neurolemma is advantageous for various reasons: tissue can be obtained from various sources and at different developmental stages; ion channels and receptors are present in their native configuration in their proper lipid environment along with appropriate auxiliary subunits; allowing the evaluation of numerous channelpathies caused by neurotoxicants in an ex vivo state. Here we show that Xenopus oocytes injected with post-natal day 90 (PND90) rat brain neurolemma fragments successfully express functional ion channels. Using a high throughput two electrode voltage clamp (TEVC) electrophysiological system, currents that were sensitive to tetrodotoxin, ω-conotoxin MVIIC, and tetraethylammonium were detected, indicating the presence of multiple voltage-sensitive ion channels (voltage-sensitive sodium (VSSC), calcium and potassium channels, respectively). The protein expression pattern for nine different VSSC isoforms (Na v 1.1-Na v 1.9) was determined in neurolemma using automated western blotting, with the predominant isoforms expressed being Na v 1.2 and Na v 1.6. VSSC were also successfully detected in the plasma membrane of Xenopus oocytes microtransplanted with neurolemma. Using this approach, a "proof-of-principle" experiment was conducted where a well-established structure-activity relationship between the neurotoxicant, 1,1,1-trichloro-2,2-di(4-chlorophenyl)ethane (DDT) and its non-neurotoxic metabolite, 1,1-bis-(4-chlorophenyl)-2,2-dichloroethene (DDE) was examined. A differential sensitivity of DDT and DDE on neurolemma-injected oocytes was determined where DDT elicited a concentration-dependent increase in TTX-sensitive inward sodium current upon pulse-depolarization whereas DDE resulted in no significant effect. Additionally, DDT resulted in a slowing of sodium channel inactivation kinetics whereas DDE was without effect. These results are consistent with the findings obtained using heterologous expression of single isoforms of rat brain VSSCs in Xenopus oocytes and with many other electrophysiological approaches, validating the use of the microtransplantation procedure as a toxicologically-relevant ex vivo assay. Once fully characterized, it is likely that this approach could be expanded to study the role of environmental toxicants and contaminants on various target tissues (e.g. neural, reproductive, developmental) from many species. Copyright © 2016 Elsevier B.V. All rights reserved.

A melanosomal two-pore sodium channel regulates pigmentation

PubMed Central

Bellono, Nicholas W.; Escobar, Iliana E.; Oancea, Elena

2016-01-01

Intracellular organelles mediate complex cellular functions that often require ion transport across their membranes. Melanosomes are organelles responsible for the synthesis of the major mammalian pigment melanin. Defects in melanin synthesis result in pigmentation defects, visual deficits, and increased susceptibility to skin and eye cancers. Although genes encoding putative melanosomal ion transporters have been identified as key regulators of melanin synthesis, melanosome ion transport and its contribution to pigmentation remain poorly understood. Here we identify two-pore channel 2 (TPC2) as the first reported melanosomal cation conductance by directly patch-clamping skin and eye melanosomes. TPC2 has been implicated in human pigmentation and melanoma, but the molecular mechanism mediating this function was entirely unknown. We demonstrate that the vesicular signaling lipid phosphatidylinositol bisphosphate PI(3,5)P2 modulates TPC2 activity to control melanosomal membrane potential, pH, and regulate pigmentation. PMID:27231233

Maintenance of Mouse Gustatory Terminal Field Organization Is Disrupted following Selective Removal of Peripheral Sodium Salt Taste Activity at Adulthood

PubMed Central

Sun, Chengsan

2017-01-01

Neural activity plays a critical role in the development of central circuits in sensory systems. However, the maintenance of these circuits at adulthood is usually not dependent on sensory-elicited neural activity. Recent work in the mouse gustatory system showed that selectively deleting the primary transduction channel for sodium taste, the epithelial sodium channel (ENaC), throughout development dramatically impacted the organization of the central terminal fields of three nerves that carry taste information to the nucleus of the solitary tract. More specifically, deleting ENaCs during development prevented the normal maturation of the fields. The present study was designed to extend these findings by testing the hypothesis that the loss of sodium taste activity impacts the maintenance of the normal adult terminal field organization in male and female mice. To do this, we used an inducible Cre-dependent genetic recombination strategy to delete ENaC function after terminal field maturation occurred. We found that removal of sodium taste neural activity at adulthood resulted in significant reorganization of mature gustatory afferent terminal fields in the nucleus of the solitary tract. Specifically, the chorda tympani and greater superficial petrosal nerve terminal fields were 1.4× and 1.6× larger than age-matched controls, respectively. By contrast, the glossopharyngeal nerve, which is not highly sensitive to sodium taste stimulation, did not undergo terminal field reorganization. These surprising results suggest that gustatory nerve terminal fields remain plastic well into adulthood, which likely impacts central coding of taste information and taste-related behaviors with altered taste experience. SIGNIFICANCE STATEMENT Neural activity plays a major role in the development of sensory circuits in the mammalian brain. However, the importance of sensory-driven activity in maintaining these circuits at adulthood, especially in subcortical structures, appears to be much less. Here, we tested whether the loss of sodium taste activity in adult mice impacts the maintenance of how taste nerves project to the first central relay. We found that specific loss of sodium-elicited taste activity at adulthood produced dramatic and selective reorganization of terminal fields in the brainstem. This demonstrates, for the first time, that taste-elicited activity is necessary for the normal maintenance of central gustatory circuits at adulthood and highlights a level of plasticity not seen in other sensory system subcortical circuits. PMID:28676575

Do defects in ion channel glycosylation set the stage for lethal cardiac arrhythmias?

PubMed

Fozzard, Harry A; Kyle, John W

2002-04-30

Many ion channels are modified by the addition of carbohydrate residues. Fozzard and Kyle discuss evidence that sialic acid residues on glycosylated cardiac sodium and potassium channels may be important for preventing early after-depolarizations that can result in cardiac arrhythmias.

New Molecular Targets for Antiepileptic Drugs: α2δ, SV2A, and Kv7/KCNQ/M Potassium Channels

PubMed Central

Rogawski, Michael A.; Bazil, Carl W.

2008-01-01

Many currently prescribed antiepileptic drugs (AEDs) act via voltage-gated sodium channels, through effects on γ-aminobutyric acid–mediated inhibition, or via voltage-gated calcium channels. Some newer AEDs do not act via these traditional mechanisms. The molecular targets for several of these nontraditional AEDs have been defined using cellular electrophysiology and molecular approaches. Here, we describe three of these targets: α2δ, auxiliary subunits of voltage-gated calcium channels through which the gabapentinoids gabapentin and pregabalin exert their anticonvulsant and analgesic actions; SV2A, a ubiquitous synaptic vesicle glycoprotein that may prepare vesicles for fusion and serves as the target for levetiracetam and its analog brivaracetam (which is currently in late-stage clinical development); and Kv7/KCNQ/M potassium channels that mediate the M-current, which acts a brake on repetitive firing and burst generation and serves as the target for the investigational AEDs retigabine and ICA-105665. Functionally, all of the new targets modulate neurotransmitter output at synapses, focusing attention on presynaptic terminals as critical sites of action for AEDs. PMID:18590620

New molecular targets for antiepileptic drugs: alpha(2)delta, SV2A, and K(v)7/KCNQ/M potassium channels.

PubMed

Rogawski, Michael A; Bazil, Carl W

2008-07-01

Many currently prescribed antiepileptic drugs (AEDs) act via voltage-gated sodium channels, through effects on gamma-aminobutyric acid-mediated inhibition, or via voltage-gated calcium channels. Some newer AEDs do not act via these traditional mechanisms. The molecular targets for several of these nontraditional AEDs have been defined using cellular electrophysiology and molecular approaches. Here, we describe three of these targets: alpha(2)delta, auxiliary subunits of voltage-gated calcium channels through which the gabapentinoids gabapentin and pregabalin exert their anticonvulsant and analgesic actions; SV2A, a ubiquitous synaptic vesicle glycoprotein that may prepare vesicles for fusion and serves as the target for levetiracetam and its analog brivaracetam (which is currently in late-stage clinical development); and K(v)7/KCNQ/M potassium channels that mediate the M-current, which acts a brake on repetitive firing and burst generation and serves as the target for the investigational AEDs retigabine and ICA-105665. Functionally, all of the new targets modulate neurotransmitter output at synapses, focusing attention on presynaptic terminals as critical sites of action for AEDs.

Scaffold Protein Connector Enhancer of Kinase Suppressor of Ras Isoform 3 (CNK3) Coordinates Assembly of a Multiprotein Epithelial Sodium Channel (ENaC)-regulatory Complex*

PubMed Central

Soundararajan, Rama; Ziera, Tim; Koo, Eric; Ling, Karen; Wang, Jian; Borden, Steffen A.; Pearce, David

2012-01-01

Hormone regulation of ion transport in the kidney tubules is essential for fluid and electrolyte homeostasis in vertebrates. A large body of evidence has suggested that transporters and channels exist in multiprotein regulatory complexes; however, relatively little is known about the composition of these complexes or their assembly. The epithelial sodium channel (ENaC) in particular is tightly regulated by the salt-regulatory hormone aldosterone, which acts at least in part by increasing expression of the serine-threonine kinase SGK1. Here we show that aldosterone induces the formation of a 1.0–1.2-MDa plasma membrane complex, which includes ENaC, SGK1, and the ENaC inhibitor Nedd4-2, a key target of SGK1. We further show that this complex contains the PDZ domain-containing protein connector enhancer of kinase suppressor of Ras isoform 3 (CNK3). CNK3 physically interacts with ENaC, Nedd4-2, and SGK1; enhances the interactions among them; and stimulates ENaC function in a PDZ domain-dependent, aldosterone-induced manner. These results strongly suggest that CNK3 is a molecular scaffold, which coordinates the assembly of a multiprotein ENaC-regulatory complex and hence plays a central role in Na+ homeostasis. PMID:22851176

Blood pressure regulation via the epithelial sodium channel: from gene to kidney and beyond.

PubMed

Büsst, Cara J

2013-08-01

The epithelial sodium channel (ENaC) has long been recognized as playing a vital role in blood pressure (BP) regulation due to its involvement in fluid balance. The genes encoding the three ENaC subunits are likewise important contributors to hypertension, both in rare monogenic diseases and in the general population. The unusually high numbers of genetic variants associated with complex traits, including BP, that are located in non-coding areas suggest an involvement of these variants in regulatory functions. This may involve differential regulation of expression in different tissues. Emerging evidence indicates that the ENaC plays an important role in BP determination not only via its actions in the kidney, but also in other tissues commonly involved in BP regulation. The ENaC in the central nervous system is proposed to regulate BP via sympathetic nervous system activity. Recent evidence suggests that the ENaC contributes to vascular function and the myogenic response. Additional roles potentially include initiation of the baroreceptor reflex via ENaC in the baroreceptors and driving high salt intake with a 'taste for salt' via ENaC in the tongue. The present review describes the involvement of the ENaC in the determination of BP at a genetic and physiological level, detailing recent evidence for its role in the kidney and in other pertinent tissues. © 2013 Wiley Publishing Asia Pty Ltd.

Molecular morphology of the tetrodotoxin-binding sodium channel protein from Electrophorus electricus in solubilized and reconstituted preparations

PubMed Central

1983-01-01

The appearance of detergent-solubilized voltage-regulated sodium channel protein was recently characterized by this laboratory. Negative- staining revealed rod-shaped particles measuring 40 X 170 A. Further studies have suggested that the actual configuration of this protein may be quite different from the rod-shaped structures. Freeze-fracture and freeze-etch images of the protein in reconstituted membranes indicated that the channel is cylindrical with a diameter of 100 A and a minimum length of 80 A. Experiments with two detergent systems (Lubrol-PX and sodium cholate) enabled us to explain the discrepancy between this structure and the rod-shaped particles visualized earlier. Negative staining in either detergent at low pH (4.5) produced rod- shaped structures. As the pH was increased, doughnut-shaped particles, consistent with the structure of the protein in freeze-etch, appeared in negative stain. The tendency of the protein to change shape under different pH conditions appears to be a peculiar property of this protein. PMID:6315745

Vocal Fold Surface Hydration: A review

PubMed Central

Leydon, Ciara; Sivasankar, Mahalakshmi; Falciglia, Danielle Lodewyck; Atkins, Christopher; Fisher, Kimberly V.

2009-01-01

Vocal fold surface liquid homeostasis contributes to optimal vocal physiology. In this paper we review emerging evidence that vocal fold surface liquid is maintained in part by salt and water fluxes across the epithelium. Based on recent immunolocalization and electrophysiological findings, we describe a transcellular pathway as one mechanism for regulating superficial vocal fold hydration. We propose that the pathway includes the sodium-potassium pump, sodium-potassium-chloride cotransporter, epithelial sodium channels, cystic fibrosis transmembrane regulator chloride channels, and aquaporin water channels. By integrating knowledge of the regulating mechanisms underlying ion and fluid transport with observations from hydration challenges and treatments using in vitro and in vivo studies, we provide a theoretical basis for understanding how environmental and behavioral challenges and clinical interventions may modify vocal fold surface liquid composition. We present converging evidence that clinical protocols directed at facilitating vocal fold epithelial ion and fluid transport may benefit healthy speakers, those with voice disorders, and those at risk for voice disorders. PMID:19111440

Pharmacological characterization of ionic currents that regulate high-frequency spontaneous activity of electromotor neurons in the weakly electric fish, Apteronotus leptorhynchus.

PubMed

Smith, G Troy

2006-01-01

The neural circuit that controls the electric organ discharge (EOD) of the brown ghost knifefish (Apteronotus leptorhynchus) contains two spontaneous oscillators. Both pacemaker neurons in the medulla and electromotor neurons (EMNs) in the spinal cord fire spontaneously at frequencies of 500-1,000 Hz to control the EOD. These neurons continue to fire in vitro at frequencies that are highly correlated with in vivo EOD frequency. Previous studies used channel blocking drugs to pharmacologically characterize ionic currents that control high-frequency firing in pacemaker neurons. The goal of the present study was to use similar techniques to investigate ionic currents in EMNs, the other type of spontaneously active neuron in the electromotor circuit. As in pacemaker neurons, high-frequency firing of EMNs was regulated primarily by tetrodotoxin-sensitive sodium currents and by potassium currents that were sensitive to 4-aminopyridine and kappaA-conotoxin SIVA, but resistant to tetraethylammonium. EMNs, however, differed from pacemaker neurons in their sensitivity to some channel blocking drugs. Alpha-dendrotoxin, which blocks a subset of Kv1 potassium channels, increased firing rates in EMNs, but not pacemaker neurons; and the sodium channel blocker muO-conotoxin MrVIA, which reduced firing rates of pacemaker neurons, had no effect on EMNs. These results suggest that similar, but not identical, ionic currents regulate high-frequency firing in EMNs and pacemaker neurons. The differences in the ionic currents expressed in pacemaker neurons and EMNs might be related to differences in the morphology, connectivity, or function of these two cell types.

Substitutions in the domain III voltage-sensing module enhance the sensitivity of an insect sodium channel to a scorpion beta-toxin.

PubMed

Song, Weizhong; Du, Yuzhe; Liu, Zhiqi; Luo, Ningguang; Turkov, Michael; Gordon, Dalia; Gurevitz, Michael; Goldin, Alan L; Dong, Ke

2011-05-06

Scorpion β-toxins bind to the extracellular regions of the voltage-sensing module of domain II and to the pore module of domain III in voltage-gated sodium channels and enhance channel activation by trapping and stabilizing the voltage sensor of domain II in its activated state. We investigated the interaction of a highly potent insect-selective scorpion depressant β-toxin, Lqh-dprIT(3), from Leiurus quinquestriatus hebraeus with insect sodium channels from Blattella germanica (BgNa(v)). Like other scorpion β-toxins, Lqh-dprIT(3) shifts the voltage dependence of activation of BgNa(v) channels expressed in Xenopus oocytes to more negative membrane potentials but only after strong depolarizing prepulses. Notably, among 10 BgNa(v) splice variants tested for their sensitivity to the toxin, only BgNa(v)1-1 was hypersensitive due to an L1285P substitution in IIIS1 resulting from a U-to-C RNA-editing event. Furthermore, charge reversal of a negatively charged residue (E1290K) at the extracellular end of IIIS1 and the two innermost positively charged residues (R4E and R5E) in IIIS4 also increased the channel sensitivity to Lqh-dprIT(3). Besides enhancement of toxin sensitivity, the R4E substitution caused an additional 20-mV negative shift in the voltage dependence of activation of toxin-modified channels, inducing a unique toxin-modified state. Our findings provide the first direct evidence for the involvement of the domain III voltage-sensing module in the action of scorpion β-toxins. This hypersensitivity most likely reflects an increase in IIS4 trapping via allosteric mechanisms, suggesting coupling between the voltage sensors in neighboring domains during channel activation.

On the Mechanism of Human Red Blood Cell Longevity: Roles of Calcium, the Sodium Pump, PIEZO1, and Gardos Channels.

PubMed

Lew, Virgilio L; Tiffert, Teresa

2017-01-01

In a healthy adult, the transport of O 2 and CO 2 between lungs and tissues is performed by about 2 · 10 13 red blood cells, of which around 1.7 · 10 11 are renewed every day, a turnover resulting from an average circulatory lifespan of about 120 days. Cellular lifespan is the result of an evolutionary balance between the energy costs of maintaining cells in a fit functional state versus cell renewal. In this Review we examine how the set of passive and active membrane transporters of the mature red blood cells interact to maximize their circulatory longevity thus minimizing costs on expensive cell turnover. Red blood cell deformability is critical for optimal rheology and gas exchange functionality during capillary flow, best fulfilled when the volume of each human red blood cell is kept at a fraction of about 0.55-0.60 of the maximal spherical volume allowed by its membrane area, the optimal-volume-ratio range. The extent to which red blood cell volumes can be preserved within or near these narrow optimal-volume-ratio margins determines the potential for circulatory longevity. We show that the low cation permeability of red blood cells allows volume stability to be achieved with extraordinary cost-efficiency, favouring cell longevity over cell turnover. We suggest a mechanism by which the interplay of a declining sodium pump and two passive membrane transporters, the mechanosensitive PIEZO1 channel, a candidate mediator of P sickle in sickle cells, and the Ca 2+ -sensitive, K + -selective Gardos channel, can implement red blood cell volume stability around the optimal-volume-ratio range, as required for extended circulatory longevity.

Convergent Substitutions in a Sodium Channel Suggest Multiple Origins of Toxin Resistance in Poison Frogs.

PubMed

Tarvin, Rebecca D; Santos, Juan C; O'Connell, Lauren A; Zakon, Harold H; Cannatella, David C

2016-04-01

Complex phenotypes typically have a correspondingly multifaceted genetic component. However, the genotype-phenotype association between chemical defense and resistance is often simple: genetic changes in the binding site of a toxin alter how it affects its target. Some toxic organisms, such as poison frogs (Anura: Dendrobatidae), have defensive alkaloids that disrupt the function of ion channels, proteins that are crucial for nerve and muscle activity. Using protein-docking models, we predict that three major classes of poison frog alkaloids (histrionicotoxins, pumiliotoxins, and batrachotoxins) bind to similar sites in the highly conserved inner pore of the muscle voltage-gated sodium channel, Nav1.4. We predict that poison frogs are somewhat resistant to these compounds because they have six types of amino acid replacements in the Nav1.4 inner pore that are absent in all other frogs except for a distantly related alkaloid-defended frog from Madagascar, Mantella aurantiaca. Protein-docking models and comparative phylogenetics support the role of these replacements in alkaloid resistance. Taking into account the four independent origins of chemical defense in Dendrobatidae, phylogenetic patterns of the amino acid replacements suggest that 1) alkaloid resistance in Nav1.4 evolved independently at least seven times in these frogs, 2) variation in resistance-conferring replacements is likely a result of differences in alkaloid exposure across species, and 3) functional constraint shapes the evolution of the Nav1.4 inner pore. Our study is the first to demonstrate the genetic basis of autoresistance in frogs with alkaloid defenses. © The Author 2016. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

Modular organization of α-toxins from scorpion venom mirrors domain structure of their targets, sodium channels.

PubMed

Chugunov, Anton O; Koromyslova, Anna D; Berkut, Antonina A; Peigneur, Steve; Tytgat, Jan; Polyansky, Anton A; Pentkovsky, Vladimir M; Vassilevski, Alexander A; Grishin, Eugene V; Efremov, Roman G

2013-06-28

To gain success in the evolutionary "arms race," venomous animals such as scorpions produce diverse neurotoxins selected to hit targets in the nervous system of prey. Scorpion α-toxins affect insect and/or mammalian voltage-gated sodium channels (Na(v)s) and thereby modify the excitability of muscle and nerve cells. Although more than 100 α-toxins are known and a number of them have been studied into detail, the molecular mechanism of their interaction with Na(v)s is still poorly understood. Here, we employ extensive molecular dynamics simulations and spatial mapping of hydrophobic/hydrophilic properties distributed over the molecular surface of α-toxins. It is revealed that despite the small size and relatively rigid structure, these toxins possess modular organization from structural, functional, and evolutionary perspectives. The more conserved and rigid "core module" is supplemented with the "specificity module" (SM) that is comparatively flexible and variable and determines the taxon (mammal versus insect) specificity of α-toxin activity. We further show that SMs in mammal toxins are more flexible and hydrophilic than in insect toxins. Concomitant sequence-based analysis of the extracellular loops of Na(v)s suggests that α-toxins recognize the channels using both modules. We propose that the core module binds to the voltage-sensing domain IV, whereas the more versatile SM interacts with the pore domain in repeat I of Na(v)s. These findings corroborate and expand the hypothesis on different functional epitopes of toxins that has been reported previously. In effect, we propose that the modular structure in toxins evolved to match the domain architecture of Na(v)s.

On the Mechanism of Human Red Blood Cell Longevity: Roles of Calcium, the Sodium Pump, PIEZO1, and Gardos Channels

PubMed Central

Lew, Virgilio L.; Tiffert, Teresa

2017-01-01

In a healthy adult, the transport of O2 and CO2 between lungs and tissues is performed by about 2 · 1013 red blood cells, of which around 1.7 · 1011 are renewed every day, a turnover resulting from an average circulatory lifespan of about 120 days. Cellular lifespan is the result of an evolutionary balance between the energy costs of maintaining cells in a fit functional state versus cell renewal. In this Review we examine how the set of passive and active membrane transporters of the mature red blood cells interact to maximize their circulatory longevity thus minimizing costs on expensive cell turnover. Red blood cell deformability is critical for optimal rheology and gas exchange functionality during capillary flow, best fulfilled when the volume of each human red blood cell is kept at a fraction of about 0.55–0.60 of the maximal spherical volume allowed by its membrane area, the optimal-volume-ratio range. The extent to which red blood cell volumes can be preserved within or near these narrow optimal-volume-ratio margins determines the potential for circulatory longevity. We show that the low cation permeability of red blood cells allows volume stability to be achieved with extraordinary cost-efficiency, favouring cell longevity over cell turnover. We suggest a mechanism by which the interplay of a declining sodium pump and two passive membrane transporters, the mechanosensitive PIEZO1 channel, a candidate mediator of Psickle in sickle cells, and the Ca2+-sensitive, K+-selective Gardos channel, can implement red blood cell volume stability around the optimal-volume-ratio range, as required for extended circulatory longevity. PMID:29311949

Caffeine intake antagonizes salt sensitive hypertension through improvement of renal sodium handling

PubMed Central

Yu, Hao; Yang, Tao; Gao, Peng; Wei, Xing; Zhang, Hexuan; Xiong, Shiqiang; Lu, Zongshi; Li, Li; Wei, Xiao; Chen, Jing; Zhao, Yu; Arendshorst, William J.; Shang, Qianhui; Liu, Daoyan; Zhu, Zhiming

2016-01-01

High salt intake is a major risk factor for hypertension. Although acute caffeine intake produces moderate diuresis and natriuresis, caffeine increases the blood pressure (BP) through activating sympathetic activity. However, the long-term effects of caffeine on urinary sodium excretion and blood pressure are rarely investigated. Here, we investigated whether chronic caffeine administration antagonizes salt sensitive hypertension by promoting urinary sodium excretion. Dahl salt-sensitive (Dahl-S) rats were fed with high salt diet with or without 0.1% caffeine in drinking water for 15 days. The BP, heart rate and locomotor activity of rats was analyzed and urinary sodium excretion was determined. The renal epithelial Na+ channel (ENaC) expression and function were measured by in vivo and in vitro experiments. Chronic consumption of caffeine attenuates hypertension induced by high salt without affecting sympathetic nerve activity in Dahl-S rats. The renal α-ENaC expression and ENaC activity of rats decreased after chronic caffeine administration. Caffeine increased phosphorylation of AMPK and decrease α-ENaC expression in cortical collecting duct cells. Inhibiting AMPK abolished the effect of caffeine on α-ENaC. Chronic caffeine intake prevented the development of salt-sensitive hypertension through promoting urinary sodium excretion, which was associated with activation of renal AMPK and inhibition of renal tubular ENaC. PMID:27173481

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

PubMed Central

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 muscle cells, assayed by measuring intracellular collagen content. We observed increased intracellular levels of ascorbate under supplementation with elevated doses of ascorbic acid, as well as its lipid soluble derivative ascorbyl palmitate. Nifedipine reduced ascorbic acid intracellular influx in cultured aortic smooth muscle cells with nifedipine (50 µM) compared to control. Adverse effects of nifedipine were neutralized either by an increased level of cell supplementation with ascorbic acid or by substituting it with ascorbyl palmitate. These studies suggest that adverse effects of channel blockers could be caused by their weakening the arterial wall integrity by interfering with proper extracellular matrix formation. In conclusion, these studies confirm the adverse effects of channel blockers on collagen type l and lV deposition, the key ECM components essential for maintaining optimal structural integrity of the arterial walls. Ascorbate supplementation reversed channel blocker inhibition of these collagen types synthesis and deposition. The results of this study imply the benefits of ascorbate and ascorbate palmitate supplementation in medical management of cardiovascular disease in order to compensate for adverse effects of channel blockers. PMID:27335688

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

PubMed

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 muscle cells, assayed by measuring intracellular collagen content. We observed increased intracellular levels of ascorbate under supplementation with elevated doses of ascorbic acid, as well as its lipid soluble derivative ascorbyl palmitate. Nifedipine reduced ascorbic acid intracellular influx in cultured aortic smooth muscle cells with nifedipine (50 µM) compared to control. Adverse effects of nifedipine were neutralized either by an increased level of cell supplementation with ascorbic acid or by substituting it with ascorbyl palmitate. These studies suggest that adverse effects of channel blockers could be caused by their weakening the arterial wall integrity by interfering with proper extracellular matrix formation. In conclusion, these studies confirm the adverse effects of channel blockers on collagen type l and lV deposition, the key ECM components essential for maintaining optimal structural integrity of the arterial walls. Ascorbate supplementation reversed channel blocker inhibition of these collagen types synthesis and deposition. The results of this study imply the benefits of ascorbate and ascorbate palmitate supplementation in medical management of cardiovascular disease in order to compensate for adverse effects of channel blockers.

Molecular Basis of Paralytic Neurotoxin Action on Voltage-Sensitive Sodium Channels

DTIC Science & Technology

1988-10-20

channels bind a scorpion toxins and sea anemone toxins, which act at an extracellular site and spccifically slow Na+ channel inactivation (Catterall, 1980...the molecule by antibodies as well as by polypeptide neurotoxins from scorpions, sea anemones , coral and snail (Catterall, 1980; Strichartz et al

Energy absorber for sodium-heated heat exchanger

DOEpatents

Essebaggers, J.

1975-12-01

A heat exchanger is described in which water-carrying tubes are heated by liquid sodium and in which the results of accidental contact between the water and the sodium caused by failure of one or more of the water tubes is minimized. An energy absorbing chamber contains a compressible gas and is connected to the body of flowing sodium by a channel so that, in the event of a sodium-water reaction, products of the reaction will partially fill the energy absorbing chamber to attenuate the rise in pressure within the heat exchanger.

Lacosamide Inhibition of Nav1.7 Voltage-Gated Sodium Channels: Slow Binding to Fast-Inactivated States

PubMed Central

Jo, Sooyeon

2017-01-01

Lacosamide is an antiseizure agent that targets voltage-dependent sodium channels. Previous experiments have suggested that lacosamide is unusual in binding selectively to the slow-inactivated state of sodium channels, in contrast to drugs like carbamazepine and phenytoin, which bind tightly to fast-inactivated states. Using heterologously expressed human Nav1.7 sodium channels, we examined the state-dependent effects of lacosamide. Lacosamide induced a reversible shift in the voltage dependence of fast inactivation studied with 100-millisecond prepulses, suggesting binding to fast-inactivated states. Using steady holding potentials, lacosamide block was very weak at −120 mV (3% inhibition by 100 µM lacosamide) but greatly enhanced at −80 mV (43% inhibition by 100 µM lacosamide), where there is partial fast inactivation but little or no slow inactivation. During long depolarizations, lacosamide slowly (over seconds) put channels into states that recovered availability slowly (hundreds of milliseconds) at −120 mV. This resembles enhancement of slow inactivation, but the effect was much more pronounced at −40 mV, where fast inactivation is complete, but slow inactivation is not, than at 0 mV, where slow inactivation is maximal, more consistent with slow binding to fast-inactivated states than selective binding to slow-inactivated states. Furthermore, inhibition by lacosamide was greatly reduced by pretreatment with 300 µM lidocaine or 300 µM carbamazepine, suggesting that lacosamide, lidocaine, and carbamazepine all bind to the same site. The results suggest that lacosamide binds to fast-inactivated states in a manner similar to other antiseizure agents but with slower kinetics of binding and unbinding. PMID:28119481

Lacosamide Inhibition of Nav1.7 Voltage-Gated Sodium Channels: Slow Binding to Fast-Inactivated States.

PubMed

Jo, Sooyeon; Bean, Bruce P

2017-04-01

Lacosamide is an antiseizure agent that targets voltage-dependent sodium channels. Previous experiments have suggested that lacosamide is unusual in binding selectively to the slow-inactivated state of sodium channels, in contrast to drugs like carbamazepine and phenytoin, which bind tightly to fast-inactivated states. Using heterologously expressed human Nav1.7 sodium channels, we examined the state-dependent effects of lacosamide. Lacosamide induced a reversible shift in the voltage dependence of fast inactivation studied with 100-millisecond prepulses, suggesting binding to fast-inactivated states. Using steady holding potentials, lacosamide block was very weak at -120 mV (3% inhibition by 100 µ M lacosamide) but greatly enhanced at -80 mV (43% inhibition by 100 µ M lacosamide), where there is partial fast inactivation but little or no slow inactivation. During long depolarizations, lacosamide slowly (over seconds) put channels into states that recovered availability slowly (hundreds of milliseconds) at -120 mV. This resembles enhancement of slow inactivation, but the effect was much more pronounced at -40 mV, where fast inactivation is complete, but slow inactivation is not, than at 0 mV, where slow inactivation is maximal, more consistent with slow binding to fast-inactivated states than selective binding to slow-inactivated states. Furthermore, inhibition by lacosamide was greatly reduced by pretreatment with 300 µ M lidocaine or 300 µ M carbamazepine, suggesting that lacosamide, lidocaine, and carbamazepine all bind to the same site. The results suggest that lacosamide binds to fast-inactivated states in a manner similar to other antiseizure agents but with slower kinetics of binding and unbinding. Copyright © 2017 by The American Society for Pharmacology and Experimental Therapeutics.

Affinity purification of the voltage-sensitive sodium channel from electroplax with resins selective for sialic acid

DOE Office of Scientific and Technical Information (OSTI.GOV)

James, W.M.; Emerick, M.C.; Agnew, W.S.

1989-07-11

The voltage-sensitive sodium channel present in the eel (Electrophorus electricus) has an unusually high content of sialic acid, including {alpha}-(2{yields}8)-linked polysialic acid, not found in other electroplax membrane glycopeptides. Lectins from Limax flavus (LFA) and wheat germ (WGA) proved the most effective of 11 lectin resins tried. The most selective resin was prepared from IgM antibodies against Neisseria meningitidis {alpha}-(2{yields}8)-polysialic acid which were affinity purified and coupled to Sepharose 4B. The sodium channel was found to bind to WGA, LFA, and IgM resins and was readily eluted with the appropriate soluble carbohydrates. Experiments with LFA and IgM resins demonstrated bindingmore » and unbinding rates and displacement kinetics, which suggest highly specific binding at multiple sites on the sodium channel protein. In preparative-scale purification of protein previously fractionated by anion-exchange chromatography, without stabilizing TTX, high yields were reproducibly obtained. Further, when detergent extracts were prepared from electroplax membranes fractionated by low-speed sedimentation, a single step over the IgM resin provided a 70-fold purification, yielding specific activities of 3,200 pmol of ({sup 3}H)TTX-binding sites/mg of protein and a single polypeptide of {approximately}285,000 Da on SDS-acrylamide gels. No small peptides were observed after this 5-h isolation. The authors describe a cation-dependent stabilization with millimolar levels of monovalent and micromolar levels of divalent species.« less

DOE Office of Scientific and Technical Information (OSTI.GOV)

Tatli, Emre; Ferroni, Paolo; Mazzoccoli, Jason

The possible use of compact heat exchangers (HXs) in sodium-cooled fast reactors (SFR) employing a Brayton cycle is promising due to their high power density and resulting small volume in comparison with conventional shell-and-tube HXs. However, the small diameter of their channels makes them more susceptible to plugging due to Na2O deposition during accident conditions. Although cold traps are designed to reduce oxygen impurity levels in the sodium coolant, their failure, in conjunction with accidental air ingress into the sodium boundary, could result in coolant oxygen levels that are above the saturation limit in the cooler parts of the HXmore » channels. This can result in Na2O crystallization and the formation of solid deposits on cooled channel surfaces, limiting or even blocking coolant flow. The development of analysis tools capable of modeling the formation of these deposits in the presence of sodium flow will allow designers of SFRs to properly size the HX channels so that, in the scenario mentioned above, the reactor operator has sufficient time to detect and react to the affected HX. Until now, analytical methodologies to predict the formation of these deposits have been developed, but never implemented in a high-fidelity computational tool suited to modern reactor design techniques. This paper summarizes the challenges and the current status in the development of a Computational Fluid Dynamics (CFD) methodology to predict deposit formation, with particular emphasis on sensitivity studies on some parameters affecting deposition.« less

Discovery of Lacosamide Affinity Bait Agents That Exhibit Potent Voltage-Gated Sodium Channel Blocking Properties

PubMed Central

2012-01-01

Lacosamide ((R)-1) is a recently marketed, first-in-class, antiepileptic drug. Patch-clamp electrophysiology studies are consistent with the notion that (R)-1 modulates voltage-gated Na+ channel function by increasing and stabilizing the slow inactivation state without affecting fast inactivation. The molecular pathway(s) that regulate slow inactivation are poorly understood. Affinity baits are chemical reactive units, which when appended to a ligand (drug) can lead to irreversible, covalent modification of the receptor thus permitting drug binding site identification including, possibly, the site of ligand function. We describe, herein, the synthesis of four (R)-1 affinity baits, (R)-N-(4″-isothiocyanatobiphenyl-4′-yl)methyl 2-acetamido-3-methoxypropionamide ((R)-8), (S)-N-(4″-isothiocyanatobiphenyl-4′-yl)methyl 2-acetamido-3-methoxypropionamide ((S)-8), (R)-N-(3″-isothiocyanatobiphenyl-4′-yl)methyl 2-acetamido-3-methoxypropionamide ((R)-9), and (R)-N-(3″-acrylamidobiphenyl-4′-yl)methyl 2-acetamido-3-methoxypropionamide ((R)-10). The affinity bait compounds were designed to interact with the receptor(s) responsible for (R)-1-mediated slow inactivation. We show that (R)-8 and (R)-9 are potent inhibitors of Na+ channel function and function by a pathway similar to that observed for (R)-1. We further demonstrate that (R)-8 function is stereospecific. The calculated IC50 values determined for Na+ channel slow inactivation for (R)-1, (R)-8, and (R)-9 were 85.1, 0.1, and 0.2 μM, respectively. Incubating (R)-9 with the neuronal-like CAD cells led to appreciable levels of Na+ channel slow inactivation after cellular wash, and the level of slow inactivation only modestly decreased with further incubation and washing. Collectively, these findings have identified a promising structural template to investigate the voltage-gated Na+ channel slow inactivation process. PMID:23509982

Evaluation of stochastic differential equation approximation of ion channel gating models.

PubMed

Bruce, Ian C

2009-04-01

Fox and Lu derived an algorithm based on stochastic differential equations for approximating the kinetics of ion channel gating that is simpler and faster than "exact" algorithms for simulating Markov process models of channel gating. However, the approximation may not be sufficiently accurate to predict statistics of action potential generation in some cases. The objective of this study was to develop a framework for analyzing the inaccuracies and determining their origin. Simulations of a patch of membrane with voltage-gated sodium and potassium channels were performed using an exact algorithm for the kinetics of channel gating and the approximate algorithm of Fox & Lu. The Fox & Lu algorithm assumes that channel gating particle dynamics have a stochastic term that is uncorrelated, zero-mean Gaussian noise, whereas the results of this study demonstrate that in many cases the stochastic term in the Fox & Lu algorithm should be correlated and non-Gaussian noise with a non-zero mean. The results indicate that: (i) the source of the inaccuracy is that the Fox & Lu algorithm does not adequately describe the combined behavior of the multiple activation particles in each sodium and potassium channel, and (ii) the accuracy does not improve with increasing numbers of channels.

Nonlinear effects of hyperpolarizing shifts in activation of mutant Nav1.7 channels on resting membrane potential

PubMed Central

Estacion, Mark

2017-01-01

The Nav1.7 sodium channel is preferentially expressed within dorsal root ganglion (DRG) and sympathetic ganglion neurons. Gain-of-function mutations that cause the painful disorder inherited erythromelalgia (IEM) shift channel activation in a hyperpolarizing direction. When expressed within DRG neurons, these mutations produce a depolarization of resting membrane potential (RMP). The biophysical basis for the depolarized RMP has to date not been established. To explore the effect on RMP of the shift in activation associated with a prototypical IEM mutation (L858H), we used dynamic-clamp models that represent graded shifts that fractionate the effect of the mutation on activation voltage dependence. Dynamic-clamp recording from DRG neurons using a before-and-after protocol for each cell made it possible, even in the presence of cell-to-cell variation in starting RMP, to assess the effects of these graded mutant models. Our results demonstrate a nonlinear, progressively larger effect on RMP as the shift in activation voltage dependence becomes more hyperpolarized. The observed differences in RMP were predicted by the “late” current of each mutant model. Since the depolarization of RMP imposed by IEM mutant channels is known, in itself, to produce hyperexcitability of DRG neurons, the development of pharmacological agents that normalize or partially normalize activation voltage dependence of IEM mutant channels merits further study. NEW & NOTEWORTHY Inherited erythromelalgia (IEM), the first human pain disorder linked to a sodium channel, is widely regarded as a genetic model of neuropathic pain. IEM is produced by Nav1.7 mutations that hyperpolarize activation. These mutations produce a depolarization of resting membrane potential (RMP) in dorsal root ganglion neurons. Using dynamic clamp to explore the effect on RMP of the shift in activation, we demonstrate a nonlinear effect on RMP as the shift in activation voltage dependence becomes more hyperpolarized. PMID:28148645

Mechanism of action of novel lung edema therapeutic AP301 by activation of the epithelial sodium channel.

PubMed

Shabbir, Waheed; Scherbaum-Hazemi, Parastoo; Tzotzos, Susan; Fischer, Bernhard; Fischer, Hendrik; Pietschmann, Helmut; Lucas, Rudolf; Lemmens-Gruber, Rosa

2013-12-01

AP301 [Cyclo(CGQRETPEGAEAKPWYC)], a cyclic peptide comprising the human tumor necrosis factor lectin-like domain (TIP domain) sequence, is currently being developed as a treatment for lung edema and has been shown to reduce extravascular lung water and improve lung function in mouse, rat, and pig models. The current paradigm for liquid homeostasis in the adult mammalian lung is that passive apical uptake of sodium via the amiloride-sensitive epithelial Na⁺ channel (ENaC) and nonselective cyclic-nucleotide-gated cation channels creates the major driving force for reabsorption of water through the alveolar epithelium in addition to other ion channels such as potassium and chloride channels. AP301 can increase amiloride-sensitive current in A549 cells as well as in freshly isolated type II alveolar epithelial cells from different species. ENaC is expressed endogenously in all of these cell types. Consequently, this study was undertaken to determine whether ENaC is the specific target of AP301. The effect of AP301 in A549 cells as well as in human embryonic kidney cells and Chinese hamster ovary cells heterologously expressing human ENaC subunits (α, β, γ, and δ) was measured in patch clamp experiments. The congener TIP peptide AP318 [Cyclo(4-aminobutanoic acid-GQRETPEGAEAKPWYD)] activated ENaC by increasing single-channel open probability. AP301 increased current in proteolytically activated (cleaved) but not near-silent (uncleaved) ENaC in a reversible manner. αβγ- or δβγ-ENaC coexpression was required for maximal activity. No increase in current was observed after deglycosylation of extracellular domains of ENaC. Thus, our data suggest that the specific interaction of AP301 with both endogenously and heterologously expressed ENaC requires precedent binding to glycosylated extracellular loop(s).

Membrane Assembly and Ion Transport Ability of a Fluorinated Nanopore

PubMed Central

Godbout, Raphaël; Légaré, Sébastien; Auger, Maud; Carpentier, Claudia; Otis, François; Auger, Michèle; Lagüe, Patrick; Voyer, Normand

2016-01-01

A novel 21-residue peptide incorporating six fluorinated amino acids was prepared. It was designed to fold into an amphiphilic alpha helical structure of nanoscale length with one hydrophobic face and one fluorinated face. The formation of a fluorous interface serves as the main vector for the formation of a superstructure in a bilayer membrane. Fluorescence assays showed this ion channel's ability to facilitate the translocation of alkali metal ions through a phospholipid membrane, with selectivity for sodium ions. Computational studies showed that a tetramer structure is the most probable and stable supramolecular assembly for the active ion channel structure. The results illustrate the possibility of exploiting multiple Fδ-:M+ interactions for ion transport and using fluorous interfaces to create functional nanostructures. PMID:27835700

Membrane Assembly and Ion Transport Ability of a Fluorinated Nanopore.

PubMed

Godbout, Raphaël; Légaré, Sébastien; Auger, Maud; Carpentier, Claudia; Otis, François; Auger, Michèle; Lagüe, Patrick; Voyer, Normand

2016-01-01

A novel 21-residue peptide incorporating six fluorinated amino acids was prepared. It was designed to fold into an amphiphilic alpha helical structure of nanoscale length with one hydrophobic face and one fluorinated face. The formation of a fluorous interface serves as the main vector for the formation of a superstructure in a bilayer membrane. Fluorescence assays showed this ion channel's ability to facilitate the translocation of alkali metal ions through a phospholipid membrane, with selectivity for sodium ions. Computational studies showed that a tetramer structure is the most probable and stable supramolecular assembly for the active ion channel structure. The results illustrate the possibility of exploiting multiple Fδ-:M+ interactions for ion transport and using fluorous interfaces to create functional nanostructures.

Activation of Ih and TTX-sensitive sodium current at subthreshold voltages during CA1 pyramidal neuron firing

PubMed Central

Yamada-Hanff, Jason

2015-01-01

We used dynamic clamp and action potential clamp techniques to explore how currents carried by tetrodotoxin-sensitive sodium channels and HCN channels (Ih) regulate the behavior of CA1 pyramidal neurons at resting and subthreshold voltages. Recording from rat CA1 pyramidal neurons in hippocampal slices, we found that the apparent input resistance and membrane time constant were strongly affected by both conductances, with Ih acting to decrease apparent input resistance and time constant and sodium current acting to increase both. We found that both Ih and sodium current were active during subthreshold summation of artificial excitatory postsynaptic potentials (EPSPs) generated by dynamic clamp, with Ih dominating at less depolarized voltages and sodium current at more depolarized voltages. Subthreshold sodium current—which amplifies EPSPs—was most effectively recruited by rapid voltage changes, while Ih—which blunts EPSPs—was maximal for slow voltage changes. The combined effect is to selectively amplify rapid EPSPs. We did similar experiments in mouse CA1 pyramidal neurons, doing voltage-clamp experiments using experimental records of action potential firing of CA1 neurons previously recorded in awake, behaving animals as command voltages to quantify flow of Ih and sodium current at subthreshold voltages. Subthreshold sodium current was larger and subthreshold Ih was smaller in mouse neurons than in rat neurons. Overall, the results show opposing effects of subthreshold sodium current and Ih in regulating subthreshold behavior of CA1 neurons, with subthreshold sodium current prominent in both rat and mouse CA1 pyramidal neurons and additional regulation by Ih in rat neurons. PMID:26289465

Collecting duct prorenin receptor knockout reduces renal function, increases sodium excretion, and mitigates renal responses in ANG II-induced hypertensive mice.

PubMed

Prieto, Minolfa C; Reverte, Virginia; Mamenko, Mykola; Kuczeriszka, Marta; Veiras, Luciana C; Rosales, Carla B; McLellan, Matthew; Gentile, Oliver; Jensen, V Behrana; Ichihara, Atsuhiro; McDonough, Alicia A; Pochynyuk, Oleh M; Gonzalez, Alexis A

2017-12-01

Augmented intratubular angiotensin (ANG) II is a key determinant of enhanced distal Na + reabsorption via activation of epithelial Na + channels (ENaC) and other transporters, which leads to the development of high blood pressure (BP). In ANG II-induced hypertension, there is increased expression of the prorenin receptor (PRR) in the collecting duct (CD), which has been implicated in the stimulation of the sodium transporters and resultant hypertension. The impact of PRR deletion along the nephron on BP regulation and Na + handling remains controversial. In the present study, we investigate the role of PRR in the regulation of renal function and BP by using a mouse model with specific deletion of PRR in the CD ( CD PRR-KO). At basal conditions, CD PRR-KO mice had decreased renal function and lower systolic BP associated with higher fractional Na + excretion and lower ANG II levels in urine. After 14 days of ANG II infusion (400 ng·kg -1 ·min -1 ), the increases in systolic BP and diastolic BP were mitigated in CD PRR-KO mice. CD PRR-KO mice had lower abundance of cleaved αENaC and γENaC, as well as lower ANG II and renin content in urine compared with wild-type mice. In isolated CD from CD PRR-KO mice, patch-clamp studies demonstrated that ANG II-dependent stimulation of ENaC activity was reduced because of fewer active channels and lower open probability. These data indicate that CD PRR contributes to renal function and BP responses during chronic ANG II infusion by enhancing renin activity, increasing ANG II, and activating ENaC in the distal nephron segments. Copyright © 2017 the American Physiological Society.

Merging Structural Motifs of Functionalized Amino Acids and α-Aminoamides Results in Novel Anticonvulsant Compounds with Significant Effects on Slow and Fast Inactivation of Voltage-Gated Sodium Channels and in the Treatment of Neuropathic Pain

PubMed Central

2011-01-01

We recently reported that merging key structural pharmacophores of the anticonvulsant drugs lacosamide (a functionalized amino acid) with safinamide (an α-aminoamide) resulted in novel compounds with anticonvulsant activities superior to that of either drug alone. Here, we examined the effects of six such chimeric compounds on Na+-channel function in central nervous system catecholaminergic (CAD) cells. Using whole-cell patch clamp electrophysiology, we demonstrated that these compounds affected Na+ channel fast and slow inactivation processes. Detailed electrophysiological characterization of two of these chimeric compounds that contained either an oxymethylene ((R)-7) or a chemical bond ((R)-11) between the two aromatic rings showed comparable effects on slow inactivation, use-dependence of block, development of slow inactivation, and recovery of Na+ channels from inactivation. Both compounds were equally effective at inducing slow inactivation; (R)-7 shifted the fast inactivation curve in the hyperpolarizing direction greater than (R)-11, suggesting that in the presence of (R)-7 a larger fraction of the channels are in an inactivated state. None of the chimeric compounds affected veratridine- or KCl-induced glutamate release in neonatal cortical neurons. There was modest inhibition of KCl-induced calcium influx in cortical neurons. Finally, a single intraperitoneal administration of (R)-7, but not (R)-11, completely reversed mechanical hypersensitivity in a tibial-nerve injury model of neuropathic pain. The strong effects of (R)-7 on slow and fast inactivation of Na+ channels may contribute to its efficacy and provide a promising novel therapy for neuropathic pain, in addition to its antiepileptic potential. PMID:21765969

Mechanisms of Pyrethroid Insecticide-Induced Stimulation of Calcium Influx in Neocortical Neurons

PubMed Central

Cao, Zhengyu; Shafer, Timothy J.

2011-01-01

Pyrethroid insecticides bind to voltage-gated sodium channels (VGSCs) and modify their gating kinetics, thereby disrupting neuronal function. Pyrethroids have also been reported to alter the function of other channel types, including activation of voltage-gated calcium channels. Therefore, the present study compared the ability of 11 structurally diverse pyrethroids to evoke Ca2+ influx in primary cultures of mouse neocortical neurons. Nine pyrethroids (tefluthrin, deltamethrin, λ-cyhalothrin, β-cyfluthrin, esfenvalerate, S-bioallethrin, fenpropathrin, cypermethrin, and bifenthrin) produced concentration-dependent elevations in intracellular calcium concentration ([Ca2+]i) in neocortical neurons. Permethrin and resmethrin were without effect on [Ca2+]i. These pyrethroids displayed a range of efficacies on Ca2+ influx; however, the EC50 values for active pyrethroids all were within one order of magnitude. Tetrodotoxin blocked increases in [Ca2+]i caused by all nine active pyrethroids, indicating that the effects depended on VGSC activation. The pathways for deltamethrin- and tefluthrin-induced Ca2+ influx include N-methyl-d-aspartic acid receptors, L-type Ca2+ channels, and reverse mode of operation of the Na+/Ca2+ exchanger inasmuch as antagonists of these sites blocked deltamethrin-induced Ca2+ influx. These data demonstrate that pyrethroids stimulate Ca2+ entry into neurons subsequent to their actions on VGSCs. PMID:20881019

Remarkable Phenytoin Sensitivity in 4 Children with SCN8A-related Epilepsy: A Molecular Neuropharmacological Approach.

PubMed

Boerma, Ragna S; Braun, Kees P; van den Broek, Marcel P H; van de Broek, Maarten P H; van Berkestijn, Frederique M C; Swinkels, Marielle E; Hagebeuk, Eveline O; Lindhout, Dick; van Kempen, Marjan; Boon, Maartje; Nicolai, Joost; de Kovel, Carolien G; Brilstra, Eva H; Koeleman, Bobby P C

2016-01-01

Mutations in SCN8A are associated with epilepsy and intellectual disability. SCN8A encodes for sodium channel Nav1.6, which is located in the brain. Gain-of-function missense mutations in SCN8A are thought to lead to increased firing of excitatory neurons containing Nav1.6, and therefore to lead to increased seizure susceptibility. We hypothesized that sodium channel blockers could have a beneficial effect in patients with SCN8A-related epilepsy by blocking the overactive Nav1.6 and thereby counteracting the effect of the mutation. Herein, we describe 4 patients with a missense SCN8A mutation and epilepsy who all show a remarkably good response on high doses of phenytoin and loss of seizure control when phenytoin medication was reduced, while side effects were relatively mild. In 2 patients, repeated withdrawal of phenytoin led to the reoccurrence of seizures. Based on the findings in these patients and the underlying molecular mechanism we consider treatment with (high-dose) phenytoin as a possible treatment option in patients with difficult-to-control seizures due to an SCN8A mutation.

ASIC3 channels in multimodal sensory perception.

PubMed

Li, Wei-Guang; Xu, Tian-Le

2011-01-19

Acid-sensing ion channels (ASICs), which are members of the sodium-selective cation channels belonging to the epithelial sodium channel/degenerin (ENaC/DEG) family, act as membrane-bound receptors for extracellular protons as well as nonproton ligands. At least five ASIC subunits have been identified in mammalian neurons, which form both homotrimeric and heterotrimeric channels. The highly proton sensitive ASIC3 channels are predominantly distributed in peripheral sensory neurons, correlating with their roles in multimodal sensory perception, including nociception, mechanosensation, and chemosensation. Different from other ASIC subunit composing ion channels, ASIC3 channels can mediate a sustained window current in response to mild extracellular acidosis (pH 7.3-6.7), which often occurs accompanied by many sensory stimuli. Furthermore, recent evidence indicates that the sustained component of ASIC3 currents can be enhanced by nonproton ligands including the endogenous metabolite agmatine. In this review, we first summarize the growing body of evidence for the involvement of ASIC3 channels in multimodal sensory perception and then discuss the potential mechanisms underlying ASIC3 activation and mediation of sensory perception, with a special emphasis on its role in nociception. We conclude that ASIC3 activation and modulation by diverse sensory stimuli represent a new avenue for understanding the role of ASIC3 channels in sensory perception. Furthermore, the emerging implications of ASIC3 channels in multiple sensory dysfunctions including nociception allow the development of new pharmacotherapy.

Inhibition of cardiac sodium currents by toluene exposure

PubMed Central

Cruz, Silvia L; Orta-Salazar, Gerardo; Gauthereau, Marcia Y; Millan-Perez Peña, Lourdes; Salinas-Stefanón, Eduardo M

2003-01-01

Toluene is an industrial solvent widely used as a drug of abuse, which can produce sudden sniffing death due to cardiac arrhythmias. In this paper, we tested the hypothesis that toluene inhibits cardiac sodium channels in Xenopus laevis oocytes transfected with Nav1.5 cDNA and in isolated rat ventricular myocytes. In oocytes, toluene inhibited sodium currents (INa+) in a concentration-dependent manner, with an IC50 of 274 μM (confidence limits: 141–407μM). The inhibition was complete, voltage-independent, and slowly reversible. Toluene had no effect on: (i) the shape of the I–V curves; (ii) the reversal potential of Na+; and (iii) the steady-state inactivation. The slow recovery time constant from inactivation of INa+ decreased with toluene exposure, while the fast recovery time constant remained unchanged. Block of INa+ by toluene was use- and frequency-dependent. In rat cardiac myocytes, 300 μM toluene inhibited the sodium current (INa+) by 62%; this inhibition was voltage independent. These results suggest that toluene binds to cardiac Na+ channels in the open state and unbinds either when channels move between inactivated states or from an inactivated to a closed state. The use- and frequency-dependent block of INa+ by toluene might be responsible, at least in part, for its arrhythmogenic effect. PMID:14534149

Detectability and medico-legal value of the gunshot residues in the intracorporeal channel.

PubMed

Migliorini, Arnaldo Stanislao; Andreola, Salvatore; Battistini, Alessio; Gentile, Guendalina; Muccino, Enrico; Vancheri, Giulia; Zoja, Riccardo

2015-11-01

The application of the histochemical stain of sodium rhodizonate to the entrance wound for the detection of the lead (Pb) residues coming from the gunshot may be affected by false positive cases due to the contamination of the environmental Pb. The aim of the Authors is to histochemically search the Pb of GSR in a region which should be more protected by the contamination: the intracorporeal channel. Two hundreds and eighteen serial histological specimens of the intracorporeal channels coming from 25 subjects (dead due to gunshots and being autopsied at the Section of Legal Medicine of the Milan University, in the years 2013-2014) were stained with the sodium rhodizonate and sodium rhodizonate in acid environment (HCl 5%), and then observed by the microscope. The sodium rhodizonate showed a positivity for the Pb residues in the intracorporeal channel, with the detection of the particles within the first 2 cm beyond the entrance wound in 6 cases over the total number of 25 (24%). Victims were characterized by common features: short-barreled weapon; contact shots or short-distance shots; involvement of regions that were not covered by clothing; preservation of the microscopic structure of organs interested by the intracorporeal channel. The searching of GSR in the intracorporeal channel, even in conditions securing a high sensitivity, could represent an important test for the discrimination between an environmental contamination of Pb and the presence of Pb residues by GSR: once confirmed the presence of GSR in the intracorporeal channel by the histochemical analysis, the diagnostic process should require the application of the SEM-EDX for the confirmation of the results. Although not yet studied, this combination could be applied to cadavers exposed to the environment, with advanced post-mortal phenomena permitting at least the suspects of the existence of gunshot wounds at the macroscopic autopsy evaluation. Indeed, in some cases, the putrefaction is so advanced that no suspect of gunshot injuries could be derived from the soft tissue. Copyright © 2015 Elsevier Ltd and Faculty of Forensic and Legal Medicine. All rights reserved.

The ladder-shaped polyether toxin gambierol anchors the gating machinery of Kv3.1 channels in the resting state

PubMed Central

Kopljar, Ivan; Labro, Alain J.; de Block, Tessa; Rainier, Jon D.; Tytgat, Jan

2013-01-01

Voltage-gated potassium (Kv) and sodium (Nav) channels are key determinants of cellular excitability and serve as targets of neurotoxins. Most marine ciguatoxins potentiate Nav channels and cause ciguatera seafood poisoning. Several ciguatoxins have also been shown to affect Kv channels, and we showed previously that the ladder-shaped polyether toxin gambierol is a potent Kv channel inhibitor. Most likely, gambierol acts via a lipid-exposed binding site, located outside the K+ permeation pathway. However, the mechanism by which gambierol inhibits Kv channels remained unknown. Using gating and ionic current analysis to investigate how gambierol affected S6 gate opening and voltage-sensing domain (VSD) movements, we show that the resting (closed) channel conformation forms the high-affinity state for gambierol. The voltage dependence of activation was shifted by >120 mV in the depolarizing direction, precluding channel opening in the physiological voltage range. The (early) transitions between the resting and the open state were monitored with gating currents, and provided evidence that strong depolarizations allowed VSD movement up to the activated-not-open state. However, for transition to the fully open (ion-conducting) state, the toxin first needed to dissociate. These dissociation kinetics were markedly accelerated in the activated-not-open state, presumably because this state displayed a much lower affinity for gambierol. A tetrameric concatemer with only one high-affinity binding site still displayed high toxin sensitivity, suggesting that interaction with a single binding site prevented the concerted step required for channel opening. We propose a mechanism whereby gambierol anchors the channel’s gating machinery in the resting state, requiring more work from the VSD to open the channel. This mechanism is quite different from the action of classical gating modifier peptides (e.g., hanatoxin). Therefore, polyether toxins open new opportunities in structure–function relationship studies in Kv channels and in drug design to modulate channel function. PMID:23401573

The Role of Nav1.9 Channel in the Development of Neuropathic Orofacial Pain Associated with Trigeminal Neuralgia.

PubMed

Lulz, Ana Paula; Kopach, Olga; Santana-Varela, Sonia; Wood, John N

2015-01-01

Trigeminal neuralgia is accompanied by severe mechanical, thermal and chemical hypersensitivity of the orofacial area innervated by neurons of trigeminal ganglion (TG). We examined the role of the voltage-gated sodium channel subtype Nav1.9 in the development of trigeminal neuralgia. We found that Nav1.9 is required for the development of both thermal and mechanical hypersensitivity induced by constriction of the infraorbital nerve (CION). The CION model does not induce change on Nav1.9 mRNA expression in the ipsilateral TG neurons when evaluated 9 days after surgery. These results demonstrate that Nav1.9 channels play a critical role in the development of orofacial neuropathic pain. New routes for the treatment of orofacial neuropathic pain focussing on regulation of the voltage-gated Nav1.9 sodium channel activity should be investigated. © 2015 Luiz et al.

Initiation and blocking of the action potential in an axon in weak ultrasonic or microwave fields

NASA Astrophysics Data System (ADS)

Shneider, M. N.; Pekker, M.

2014-05-01

In this paper, we analyze the effect of the redistribution of the transmembrane ion channels in an axon caused by longitudinal acoustic vibrations of the membrane. These oscillations can be excited by an external source of ultrasound and weak microwave radiation interacting with the charges sitting on the surface of the lipid membrane. It is shown, using the Hodgkin-Huxley model of the axon, that the density redistribution of transmembrane sodium channels may reduce the threshold of the action potential, up to its spontaneous initiation. At the significant redistribution of sodium channels in the membrane, the rarefaction zones of the transmembrane channel density are formed, blocking the propagation of the action potential. Blocking the action potential propagation along the axon is shown to cause anesthesia in the example case of a squid axon. Various approaches to experimental observation of the effects considered in this paper are discussed.

The alpha subunit of the epithelial sodium channel in the mouse: developmental regulation of its expression.

PubMed

Dagenais, A; Kothary, R; Berthiaume, Y

1997-09-01

Sodium reabsorption by the amiloride-sensitive sodium channel of epithelial cells plays a crucial role in the management of ionic composition and fluid volume in the body. In the respiratory system, sodium transport is involved in the clearance of pulmonary edema and of liquid secreted during fetal life at birth. We have cloned a partial cDNA of the alpha subunit of the mouse amiloride-sensitive sodium channel (alpha mENaC). In the region of comparison, the mouse alpha subunit shows 92% identity at the DNA level and 95% identity at the amino acid level with the rat sequence. The kidneys, lungs, and distal colon are major sites of expression of a 3.5-kb alpha mENaC mRNA. During mouse development, alpha mENaC transcripts appear late during gestation (d 17.5) and are expressed continuously thereafter. In the distal colon, a short 1.2-kb mRNA deleted of the 5' part of the transcript is detected during gestation and is replaced gradually by the mature 3.5-kb transcript after birth. Alpha mENaC and alpha1 Na+-K+-ATPase mRNAs have an expression profile that is modulated similarly during development for a given tissue. The expression of alpha mENaC transcripts increases transiently in the lungs at birth (2.5-fold), as for alpha1 Na+-K+-ATPase mRNAs (1.5-fold), suggesting that the expression of several components of the sodium transport system is modulated in the lungs at that time. In the kidney, there is no significant increase of alpha mENaC and alpha1 Na+-K+-ATPase mRNAs in newborns.

Mechanisms involved in cardiac sensitization by volatile anesthetics: general applicability to halogenated hydrocarbons?

PubMed

Himmel, Herbert M

2008-01-01

An increased sensitivity of the heart to catecholamines or cardiac sensitization is a recognized risk during acute human exposure to halogenated hydrocarbons used as solvents, foam-blowing or fire-extinguishing agents, refrigerants, and aerosol propellants. Although cardiac sensitization to such "industrial" halocarbons can result in serious arrhythmia and death, research into its mechanistic basis has been limited, whereas the literature on volatile anesthetics (e.g., halothane, chloroform) is comparably extensive. A review of the literature on halocarbons and related volatile anesthetics was conducted. The available experimental evidence suggests that volatile anesthetics at physiologically relevant concentrations interact predominantly with the main repolarizing cardiac potassium channels hERG and I(Ks), as well as with calcium and sodium channels at slightly higher concentrations. On the level of the heart, inhibition of these ion channels is prone to alter both action potential shape (triangulation) and electrical impulse conduction, which may facilitate arrhythmogenesis by volatile anesthetics per se and is potentiated by catecholamines. Action potential triangulation by regionally heterogeneous inhibition of calcium and potassium channels will facilitate catecholamine-induced afterdepolarizations, triggered activity, and enhanced automaticity. Inhibition of cardiac sodium channels will reduce conduction velocity and alter refractory period; this is potentiated by catecholamines and promotes reentry arrhythmias. Other cardiac and/or neuronal mechanisms might also contribute to arrhythmogenesis. The few scattered in vitro data available for halocarbons (e.g., FC-12, halon 1301, trichloroethylene) suggest inhibition of cardiac sodium (conduction), calcium and potassium channels (triangulation), extraneuronal catecholamine reuptake, and various neuronal ion channels. Therefore, it is hypothesized that halocarbons promote cardiac sensitization by similar mechanisms as volatile anesthetics. Experimental approaches for further investigation of these sensitization mechanisms by selected halocarbons are suggested.

Role of mixed ion channel effects in the cardiovascular safety assessment of the novel anti-MRSA fluoroquinolone JNJ-Q2.

PubMed

Eichenbaum, G; Pugsley, M K; Gallacher, D J; Towart, R; McIntyre, G; Shukla, U; Davenport, J M; Lu, H R; Rohrbacher, J; Hillsamer, V

2012-07-01

JNJ-Q2, a novel broad-spectrum fluoroquinolone with anti-methicillin-resistant Staphylococcus aureus activity, was evaluated in a comprehensive set of non-clinical and clinical cardiovascular safety studies. The effect of JNJ-Q2 on different cardiovascular parameters was compared with that of moxifloxacin, sparfloxacin and ofloxacin. Through comparisons with these well-known fluoroquinolones, the importance of effects on compensatory ion channels to the cardiovascular safety of JNJ-Q2 was investigated. JNJ-Q2 and comparator fluoroquinolones were evaluated in the following models/test systems: hERG-transfected HEK293 cells sodium channel-transfected CHO cells, guinea pig right atria, arterially perfused rabbit left ventricular wedge preparations and in vivo studies in anaesthetized guinea pigs, anaesthetized and conscious telemetered dogs, and a thorough QT study in humans. The trend for effects of JNJ-Q2 on Tp-Te, QT, QRS and PR intervals in the non-clinical models and the plateau in QTc with increasing plasma concentration in humans are consistent with offsetting sodium and calcium channel activities that were observed in the non-clinical studies. These mixed ion channel activities result in the less pronounced or comparable increase in QTc interval for JNJ-Q2 compared with moxifloxacin and sparfloxacin despite its greater in vitro inhibition of I(Kr). Based on the non-clinical and clinical cardiovascular safety assessment, JNJ-Q2 has a safe cardiovascular profile for administration in humans with comparable or reduced potential to prolong QT intervals, compared with moxifloxacin. The results demonstrate the importance of compensatory sodium and calcium channel activity in offsetting potassium channel activity for compounds with a fluoroquinolone core. © 2012 Janssen Pharmaceutical Companies of Johnson & Johnson. British Journal of Pharmacology © 2012 The British Pharmacological Society.

Functional diversity of secreted cestode Kunitz proteins: Inhibition of serine peptidases and blockade of cation channels

PubMed Central

Fló, Martín; Margenat, Mariana; Pellizza, Leonardo; Durán, Rosario; Salceda, Emilio; Alvarez, Beatriz

2017-01-01

We previously reported a multigene family of monodomain Kunitz proteins from Echinococcus granulosus (EgKU-1-EgKU-8), and provided evidence that some EgKUs are secreted by larval worms to the host interface. In addition, functional studies and homology modeling suggested that, similar to monodomain Kunitz families present in animal venoms, the E. granulosus family could include peptidase inhibitors as well as channel blockers. Using enzyme kinetics and whole-cell patch-clamp, we now demonstrate that the EgKUs are indeed functionally diverse. In fact, most of them behaved as high affinity inhibitors of either chymotrypsin (EgKU-2-EgKU-3) or trypsin (EgKU-5-EgKU-8). In contrast, the close paralogs EgKU-1 and EgKU-4 blocked voltage-dependent potassium channels (Kv); and also pH-dependent sodium channels (ASICs), while showing null (EgKU-1) or marginal (EgKU-4) peptidase inhibitory activity. We also confirmed the presence of EgKUs in secretions from other parasite stages, notably from adult worms and metacestodes. Interestingly, data from genome projects reveal that at least eight additional monodomain Kunitz proteins are encoded in the genome; that particular EgKUs are up-regulated in various stages; and that analogous Kunitz families exist in other medically important cestodes, but not in trematodes. Members of this expanded family of secreted cestode proteins thus have the potential to block, through high affinity interactions, the function of host counterparts (either peptidases or cation channels) and contribute to the establishment and persistence of infection. From a more general perspective, our results confirm that multigene families of Kunitz inhibitors from parasite secretions and animal venoms display a similar functional diversity and thus, that host-parasite co-evolution may also drive the emergence of a new function associated with the Kunitz scaffold. PMID:28192542

Sodium cyanate-induced opening of calcium-activated potassium currents in hippocampal neuron-derived H19-7 cells.

PubMed

Huang, Chin-Wei; Huang, Chao-Ching; Huang, Mei-Han; Wu, Sheng-Nan; Hsieh, Yi-Jung

2005-03-29

We investigated the chemical toxic agent sodium cyanate (NaOCN) on the large conductance calcium-activated potassium channels (BK(Ca)) on hippocampal neuron-derived H19-7 cells. The whole-cell and cell-attach configuration of patch-clamp technique were applied to investigate the BK(Ca) currents in H19-7 cells in the presence of NaOCN (0.3 mM). NaOCN activated BK(Ca) channels on H19-7 cells. The single-channel conductance of BK(Ca) channels was 138+/-7pS. The presence of NaOCN (0.3 mM) caused an obvious increase in open probability of BK(Ca) channels. NaOCN did not exert effect on the slope of the activation curve and stimulated the activity of BK(Ca) channels in a voltage-dependent fashion in H19-7 cells. The presence of paxilline or EGTA significantly reduced the BK(Ca) amplitude, in comparison with the presence of NaOCN. These findings suggest that during NaOCN exposure, the activation of BK(Ca) channels in neurons could be one of the ionic mechanisms underlying the decreased neuronal excitability and neurological disorders.

Pacsin 2 is required for the maintenance of a normal cardiac function in the developing mouse heart.

PubMed

Semmler, Judith; Kormann, Jan; Srinivasan, Sureshkumar Perumal; Köster, Annette; Sälzer, Daniel; Reppel, Michael; Hescheler, Jürgen; Plomann, Markus; Nguemo, Filomain

2018-02-01

The Pacsin proteins (Pacsin 1, 2 and 3) play an important role in intracellular trafficking and thereby signal transduction in many cells types. This study was designed to examine the role of Pacsin 2 in cardiac development and function. We investigated the development and electrophysiological properties of Pacsin 2 knockout (P2KO) hearts and single cardiomyocytes isolated from 11.5 and 15.5days old fetal mice. Immunofluorescence experiments confirmed the lack of Pacsin 2 protein expression in P2KO cardiac myocytes in comparison to wildtype (WT). Western blotting demonstrates low expression levels of connexin 43 and T-box 3 proteins in P2KO compared to wildtype (WT). Electrophysiology measurements including online Multi-Electrode Array (MEA) based field potential (FP) recordings on isolated whole heart of P2KO mice showed a prolonged AV-conduction time. Patch clamp measurements of P2KO cardiomyocytes revealed differences in action potential (AP) parameters and decreased pacemaker funny channel (I f ), as well as L-type Ca 2+ channel (I CaL ), and sodium channel (I Na ). These findings demonstrate that Pacsin 2 is necessary for cardiac development and function in mouse embryos, which will enhance our knowledge to better understand the genesis of cardiovascular diseases. Copyright © 2017 Elsevier Ltd. All rights reserved.

Developmentally-regulated sodium channel subunits are differentially sensitive to α–cyano containing pyrethroids

EPA Science Inventory

Juvenile rats have been reported to be more sensitive to the acute neurotoxic effects of the pyrethroid deltamethrin than adults. While toxicokinetic differences between juveniles and adults are documented, toxicodynamic differences have not been examined. Voltage-gated sodium ch...

Identification of an ovarian voltage-activated Na+-channel type: hints to involvement in luteolysis.

PubMed

Bulling, A; Berg, F D; Berg, U; Duffy, D M; Stouffer, R L; Ojeda, S R; Gratzl, M; Mayerhofer, A

2000-07-01

An endocrine type of voltage-activated sodium channel (eNaCh) was identified in the human ovary and human luteinized granulosa cells (GC). Whole-cell patch-clamp studies showed that the eNaCh in GC is functional and tetrodotoxin (TTX) sensitive. The luteotrophic hormone human CG (hCG) was found to decrease the peak amplitude of the sodium current within seconds. Treatment with hCG for 24-48 h suppressed not only eNaCh mRNA levels, but also mean Na+ peak currents and resting membrane potentials. An unexpected role for eNaChs in regulating cell morphology and function was indicated after pharmacological modulation of presumed eNaCh steady-state activity in GC cultures for 24-48 h using TTX (NaCh blocker) and veratridine (NaCh activator). TTX preserved a highly differentiated cellular phenotype. Veratridine not only increased the number of secondary lysosomes but also led to a significantly reduced progesterone production. Importantly, endocrine cells of the nonhuman primate corpus luteum (CL), which represent in vivo counterparts of luteinized GC, also contain eNaCh mRNA. Although the mechanism of channel activity under physiological conditions is not clear, it may include persistent Na+ currents. As observed in GC in culture, abundant secondary lysosomes were particularly evident in the regressing CL, suggesting a functional link between eNaCh activity and this form of cellular regression in vivo. Our results identify eNaCh in ovarian endocrine cells and demonstrate that their expression is under the inhibitory control of hCG. Activation of eNaChs in luteal cells, due to loss of gonadotropin support, may initiate a cascade of events leading to decreased CL function, a process that involves lysosomal activation and autophagy. These results imply that ovarian eNaChs are involved in the physiological demise of the temporary endocrine organ CL in the primate ovary during the menstrual cycle. Because commonly used drugs, including phenytoin, target NaChs, these results may be of clinical relevance.

Ethanol stimulates epithelial sodium channels by elevating reactive oxygen species

PubMed Central

Bao, Hui-Fang; Song, John Z.; Duke, Billie J.; Ma, He-Ping; Denson, Donald D.

2012-01-01

Alcohol affects total body sodium balance, but the molecular mechanism of its effect remains unclear. We used single-channel methods to examine how ethanol affects epithelial sodium channels (ENaC) in A6 distal nephron cells. The data showed that ethanol significantly increased both ENaC open probability (Po) and the number of active ENaC in patches (N). 1-Propanol and 1-butanol also increased ENaC activity, but iso-alcohols did not. The effects of ethanol were mimicked by acetaldehyde, the first metabolic product of ethanol, but not by acetone, the metabolic product of 2-propanol. Besides increasing open probability and apparent density of active channels, confocal microscopy and surface biotinylation showed that ethanol significantly increased α-ENaC protein in the apical membrane. The effects of ethanol on ENaC Po and N were abolished by a superoxide scavenger, 4-hydroxy-2,2,6,6-tetramethylpiperidinyloxy (TEMPOL) and blocked by the phosphatidylinositol 3-kinase inhibitor LY294002. Consistent with an effect of ethanol-induced reactive oxygen species (ROS) on ENaC, primary alcohols and acetaldehyde elevated intracellular ROS, but secondary alcohols did not. Taken together with our previous finding that ROS stimulate ENaC, the current results suggest that ethanol stimulates ENaC by elevating intracellular ROS probably via its metabolic product acetaldehyde. PMID:22895258

Contribution of Sialic Acid to the Voltage Dependence of Sodium Channel Gating

PubMed Central

Bennett, Eric; Urcan, Mary S.; Tinkle, Sally S.; Koszowski, Adam G.; Levinson, Simon R.

1997-01-01

A potential role for sialic acid in the voltage-dependent gating of rat skeletal muscle sodium channels (rSkM1) was investigated using Chinese hamster ovary (CHO) cells stably transfected with rSkM1. Changes in the voltage dependence of channel gating were observed after enzymatic (neuraminidase) removal of sialic acid from cells expressing rSkM1 and through the expression of rSkM1 in a sialylation-deficient cell line (lec2). The steady-state half-activation voltages (Va) of channels under each condition of reduced sialylation were ∼10 mV more depolarized than control channels. The voltage dependence of the time constants of channel activation and inactivation were also shifted in the same direction and by a similar magnitude. In addition, recombinant deletion of likely glycosylation sites from the rSkM1 sequence resulted in mutant channels that gated at voltages up to 10 mV more positive than wild-type channels. Thus three independent means of reducing channel sialylation show very similar effects on the voltage dependence of channel gating. Finally, steady-state activation voltages for channels subjected to reduced sialylation conditions were much less sensitive to the effects of external calcium than those measured under control conditions, indicating that sialic acid directly contributes to the negative surface potential. These results are consistent with an electrostatic mechanism by which external, negatively charged sialic acid residues on rSkM1 alter the electric field sensed by channel gating elements. PMID:9089440

Adaptive evolution of insect selective excitatory β-type sodium channel neurotoxins from scorpion venom.

PubMed

Wu, Wenlan; Li, Zhongjie; Ma, Yibao

2017-06-01

Insect selective excitatory β-type sodium channel neurotoxins from scorpion venom (β-NaScTxs) are composed of about 70-76 amino acid residues and share a common scaffold stabilized by four unique disulfide bonds. The phylogenetic analysis of these toxins was hindered by limited sequence data. In our recent study, two new insect selective excitatory β-NaScTxs, LmIT and ImIT, were isolated from Lychas mucronatus and Isometrus maculatus, respectively. With the sequences previously reported, we examined the adaptive molecular evolution of insect selective excitatory β-NaScTxs by estimating the nonsynonymous-to-synonymous rate ratio (ω=d N /d S ). The results revealed 12 positively selected sites in the genes of insect selective excitatory β-NaScTxs. Moreover, these positively selected sites match well with the sites important for interacting with sodium channels, as demonstrated in previous mutagenesis study. These results reveal that adaptive evolution after gene duplication is one of the most important genetic mechanisms of scorpion neurotoxin diversification. Copyright © 2017 Elsevier Inc. All rights reserved.

Comparative effects of sodium bicarbonate and sodium chloride on reversing cocaine-induced changes in the electrocardiogram.

PubMed

Parker, R B; Perry, G Y; Horan, L G; Flowers, N C

1999-12-01

Cocaine abuse is associated with a number of cardiovascular complications that include arrhythmias and sudden cardiac death. Although the mechanism(s) remain unclear, cocaine-induced block of sodium channels resulting in slowed cardiac conduction is thought to play an important role. Several reports suggest that the effects of cocaine effects on cardiac sodium channels can be reversed by administration of sodium bicarbonate. Whether the beneficial effects of sodium bicarbonate are due to sodium ions or an increase in blood pH is unknown. Therefore the purpose of this study was to compare the effects of sodium loading alone (by using sodium chloride) versus sodium loading with an associated increase in arterial pH (by using sodium bicarbonate) on reversing cocaine-induced effects on the electrocardiogram (ECG) in a canine model. Seventeen anesthetized dogs received three i.v. injections of cocaine, 5 mg/kg, with each dose separated by 15 min. Two minutes after the third cocaine dose, each dog was randomly assigned to receive 2 mEq/kg i.v. sodium bicarbonate (1 mEq/ml) or 2 mEq/kg i.v. sodium chloride (1 mEq/ml). ECG, electrophysiologic, and hemodynamic data were recorded at baseline, after each cocaine injection, and after administration of sodium bicarbonate or sodium chloride. In both groups of animals, the first cocaine injection significantly (p < 0.05) prolonged the PR, QTc, AH, and HV intervals, and QRS duration compared with baseline. All intervals continued to lengthen in a dose-dependent manner after the second and third cocaine doses. Sodium bicarbonate significantly (p < 0.05) reduced cocaine-induced prolongation of PR [(147 +/- 5-130 +/- 5 ms), AH (81 +/- 6 - 72 +/- 6 ms), and HV intervals (55 +/- 2 - 39 +/- 1 ms). and QRS duration (96 +/- 6 - 66 +/- 4 ms), peak effect after third cocaine dose versus after sodium bicarbonate, respectively]. Sodium chloride had no effect on reversing cocaine-induced effects on the ECG. Cocaine produces dose-dependent slowing of cardiac conduction that is effectively reversed by sodium bicarbonate. The lack of efficacy of sodium chloride suggests that the increase in arterial pH associated with sodium bicarbonate is responsible for reversal of the effects of cocaine on the ECG. Therefore sodium bicarbonate may be clinically useful in the treatment of cocaine-induced cardiac arrhythmias, primarily as a result of its effects on arterial pH.

Revelation of Brugada electrocardiographic pattern during a febrile state associated with acute myocardial infarction.

PubMed

Patanè, Salvatore; Marte, Filippo

2010-09-24

The prevalence of the Brugada-type ECG and its natural history are still unclear. The Brugada syndrome is usually identified by a characteristic Brugada-type ECG that consists of ST elevation of a coved type in the precordial leads V1 to V3 and ventricular fibrillation that can lead to sudden cardiac death, although affected individuals may have a normal ECG. Mutations in the cardiac sodium channel gene SCN5A, which encodes the alpha-subunit of the human cardiac voltage-dependent Na+ channel (Na(v)1.5), are identified in 15-30% of patients with Brugada syndrome. Most SCN5A mutations lead to a 'loss-of-function' phenotype, reducing the Na+ current during the early phases of the action potential. Several nongenetic factors have been mentioned in the literature as possible inductors of the ECG pattern resembling Brugada syndrome. As such, a Brugada-type ECG may appear in some patients during febrile states and in those who are under the influence of cocaine and pharmaceutical drugs that have a sodium channel-blocking effect. It has been also reported chest pain and ST elevation Brugada pattern during febrile states. We present a case of revelation of Brugada pattern in a 69-year-old Italian man during a febrile state associated with acute myocardial infarction. Also this report confirms that Brugada pattern should be considered as one of differential diagnoses when we examine the patients during a febrile state. Copyright © 2008 Elsevier Ireland Ltd. All rights reserved.

Convergent adaptation to dangerous prey proceeds through the same first-step mutation in the garter snake Thamnophis sirtalis.

PubMed

Hague, Michael T J; Feldman, Chris R; Brodie, Edmund D; Brodie, Edmund D

2017-06-01

Convergent phenotypes often result from similar underlying genetics, but recent work suggests convergence may also occur in the historical order of substitutions en route to an adaptive outcome. We characterized convergence in the mutational steps to two independent outcomes of tetrodotoxin (TTX) resistance in separate geographic lineages of the common garter snake (Thamnophis sirtalis) that coevolved with toxic newts. Resistance is largely conferred by amino acid changes in the skeletal muscle sodium channel (Na V 1.4) that interfere with TTX-binding. We sampled variation in Na V 1.4 throughout western North America and found clear evidence that TTX-resistant changes in both lineages began with the same isoleucine-valine mutation (I1561V) within the outer pore of Na V 1.4. Other point mutations in the pore, shown to confer much greater resistance, accumulate later in the evolutionary progression and always occur together with the initial I1561V change. A gene tree of Na V 1.4 suggests the I1561V mutations in each lineage are not identical-by-decent, but rather they arose independently. Convergence in the evolution of channel resistance is likely the result of shared biases in the two lineages of T. sirtalis-only a few mutational routes can confer TTX resistance while maintaining the conserved function of voltage-gated sodium channels. © 2017 The Author(s). Evolution © 2017 The Society for the Study of Evolution.