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.

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

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

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.

[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.

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

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.

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

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.

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.

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

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.

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.

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

Structural model of the open–closed–inactivated cycle of prokaryotic voltage-gated sodium channels

PubMed Central

Bagnéris, Claire; Naylor, Claire E.; McCusker, Emily C.

2015-01-01

In excitable cells, the initiation of the action potential results from the opening of voltage-gated sodium channels. These channels undergo a series of conformational changes between open, closed, and inactivated states. Many models have been proposed for the structural transitions that result in these different functional states. Here, we compare the crystal structures of prokaryotic sodium channels captured in the different conformational forms and use them as the basis for examining molecular models for the activation, slow inactivation, and recovery processes. We compare structural similarities and differences in the pore domains, specifically in the transmembrane helices, the constrictions within the pore cavity, the activation gate at the cytoplasmic end of the last transmembrane helix, the C-terminal domain, and the selectivity filter. We discuss the observed differences in the context of previous models for opening, closing, and inactivation, and present a new structure-based model for the functional transitions. Our proposed prokaryotic channel activation mechanism is then compared with the activation transition in eukaryotic sodium channels. PMID:25512599

Gating modifier toxins isolated from spider venom: modulation of voltage-gated sodium channels and the role of lipid membranes.

PubMed

Agwa, Akello J; Peigneur, Steve; Chow, Chun Yuen; Lawrence, Nicole; Craik, David J; Tytgat, Jan; King, Glenn F; Henriques, Sonia Troeira; Schroeder, Christina I

2018-04-27

Gating modifier toxins (GMTs) are venom-derived peptides isolated from spiders and other venomous creatures that modulate activity of disease-relevant voltage-gated ion channels and are therefore being pursued as therapeutic leads. The amphipathic surface profile of GMTs has prompted the proposal that some GMTs simultaneously bind to the cell membrane and voltage-gated ion channels in a trimolecular complex. Here we examined whether there is a relationship among spider GMT amphipathicity, membrane binding and potency or selectivity for voltage-gated sodium (NaV) channels. We used NMR spectroscopy and in silico calculations to examine the structures and physicochemical properties of a panel of nine GMTs and deployed surface plasmon resonance to measure GMT affinity for lipids putatively found in proximity to NaV channels. Electrophysiology was used to quantify GMT activity on NaV1.7, an ion channel linked to chronic pain. Selectivity of the peptides was further examined against a panel of NaV channel subtypes. We show that GMTs adsorb to the outer leaflet of anionic lipid bilayers through electrostatic interactions. We did not observe a direct correlation between GMT amphipathicity and affinity for lipid bilayers. Furthermore, GMT-lipid bilayer interactions did not correlate with potency or selectivity for NaVs. We therefore propose that increased membrane binding is unlikely to improve subtype selectivity and that the conserved amphipathic GMT surface profile is an adaptation that facilitates simultaneous modulation of multiple NaVs. Published under license by The American Society for Biochemistry and Molecular Biology, Inc.

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.

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.

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

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.

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

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

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.

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

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.

Indoxacarb, Metaflumizone, and Other Sodium Channel Inhibitor Insecticides: Mechanism and Site of Action on Mammalian Voltage-Gated Sodium Channels

PubMed Central

von Stein, Richard T.; Silver, Kristopher S.; Soderlund, David M.

2013-01-01

Sodium channel inhibitor (SCI) insecticides were discovered almost four decades ago but have only recently yielded important commercial products (eg., indoxacarb and metaflumizone). SCI insecticides inhibit sodium channel function by binding selectively to slow-inactivated (non-conducting) sodium channel states. Characterization of the action of SCI insecticides on mammalian sodium channels using both biochemical and electrophysiological approaches demonstrates that they bind at or near a drug receptor site, the "local anesthetic (LA) receptor." This mechanism and site of action on sodium channels differentiates SCI insecticides from other insecticidal agents that act on sodium channels. However, SCI insecticides share a common mode of action with drugs currently under investigation as anticonvulsants and treatments for neuropathic pain. In this paper we summarize the development of the SCI insecticide class and the evidence that this structurally diverse group of compounds have a common mode of action on sodium channels. We then review research that has used site-directed mutagenesis and heterologous expression of cloned mammalian sodium channels in Xenopus laevis oocytes to further elucidate the site and mechanism of action of SCI insecticides. The results of these studies provide new insight into the mechanism of action of SCI insecticides on voltage-gated sodium channels, the location of the SCI insecticide receptor, and its relationship to the LA receptor that binds therapeutic SCI agents. PMID:24072940

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

Voltage-gated proton (H(v)1) channels, a singular voltage sensing domain.

PubMed

Castillo, Karen; Pupo, Amaury; Baez-Nieto, David; Contreras, Gustavo F; Morera, Francisco J; Neely, Alan; Latorre, Ramon; Gonzalez, Carlos

2015-11-14

The main role of voltage-gated proton channels (Hv1) is to extrude protons from the intracellular milieu when, mediated by different cellular processes, the H(+) concentration increases. Hv1 are exquisitely selective for protons and their structure is homologous to the voltage sensing domain (VSD) of other voltage-gated ion channels like sodium, potassium, and calcium channels. In clear contrast to the classical voltage-dependent channels, Hv1 lacks a pore domain and thus permeation necessarily occurs through the voltage sensing domain. Hv1 channels are activated by depolarizing voltages, and increases in internal proton concentration. It has been proposed that local conformational changes of the transmembrane segment S4, driven by depolarization, trigger the molecular rearrangements that open Hv1. However, it is still unclear how the electromechanical coupling is achieved between the VSD and the potential pore, allowing the proton flux from the intracellular to the extracellular side. Here we provide a revised view of voltage activation in Hv1 channels, offering a comparative scenario with other voltage sensing channels domains. Copyright © 2015 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

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.

[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.

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.

Transient sodium current at subthreshold voltages: activation by EPSP waveforms

PubMed Central

Carter, Brett C.; Giessel, Andrew J.; Sabatini, Bernardo L.; Bean, Bruce P.

2012-01-01

Summary Tetrodotoxin (TTX)-sensitive sodium channels carry large transient currents during action potentials and also “persistent” sodium current, a non-inactivating TTX-sensitive current present at subthreshold voltages. We examined gating of subthreshold sodium current in dissociated cerebellar Purkinje neurons and hippocampal CA1 neurons, studied at 37 °C with near-physiological ionic conditions. Unexpectedly, in both cell types small voltage steps at subthreshold voltages activated a substantial component of transient sodium current as well as persistent current. Subthreshold EPSP-like waveforms also activated a large component of transient sodium current, but IPSP-like waveforms engaged primarily persistent sodium current with only a small additional transient component. Activation of transient as well as persistent sodium current at subthreshold voltages produces amplification of EPSPs that is sensitive to the rate of depolarization and can help account for the dependence of spike threshold on depolarization rate, as previously observed in vivo. PMID:22998875

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.

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

Voltage-sensing domain of voltage-gated proton channel Hv1 shares mechanism of block with pore domains.

PubMed

Hong, Liang; Pathak, Medha M; Kim, Iris H; Ta, Dennis; Tombola, Francesco

2013-01-23

Voltage-gated sodium, potassium, and calcium channels are made of a pore domain (PD) controlled by four voltage-sensing domains (VSDs). The PD contains the ion permeation pathway and the activation gate located on the intracellular side of the membrane. A large number of small molecules are known to inhibit the PD by acting as open channel blockers. The voltage-gated proton channel Hv1 is made of two VSDs and lacks the PD. The location of the activation gate in the VSD is unknown and open channel blockers for VSDs have not yet been identified. Here, we describe a class of small molecules which act as open channel blockers on the Hv1 VSD and find that a highly conserved phenylalanine in the charge transfer center of the VSD plays a key role in blocker binding. We then use one of the blockers to show that Hv1 contains two intracellular and allosterically coupled gates. Copyright © 2013 Elsevier Inc. All rights reserved.

Carvacrol modulates voltage-gated sodium channels kinetics in dorsal root ganglia.

PubMed

Joca, Humberto Cavalcante; Vieira, Daiana Cardoso Oliveira; Vasconcelos, Aliny Perreira; Araújo, Demetrius Antônio Machado; Cruz, Jader Santos

2015-06-05

Recent studies have shown that many of plant-derived compounds interact with specific ion channels and thereby modulate many sensing mechanisms, such as nociception. The monoterpenoid carvacrol (5-isopropyl-2-methylphenol) has an anti-nociceptive effect related to a reduction in neuronal excitability and voltage-gated Na(+) channels (NaV) inhibition in peripheral neurons. However, the detailed mechanisms of carvacrol-induced inhibition of neuronal NaV remain elusive. This study explores the interaction between carvacrol and NaV in isolated dorsal root ganglia neurons. Carvacrol reduced the total voltage-gated Na(+) current and tetrodotoxin-resistant (TTX-R) Na(+) current component in a concentration-dependent manner. Carvacrol accelerates current inactivation and induced a negative-shift in voltage-dependence of steady-state fast inactivation in total and TTX-R Na(+) current. Furthermore, carvacrol slowed the recovery from inactivation. Carvacrol provoked a leftward shift in both the voltage-dependence of steady-state inactivation and activation of the TTX-R Na(+) current component. In addition, carvacrol-induced inhibition of TTX-R Na(+) current was enhanced by an increase in stimulation frequency and when neurons were pre-conditioned with long depolarization pulse (5s at -50 mV). Taken all results together, we herein demonstrated that carvacrol affects NaV gating properties. The present findings would help to explain the mechanisms underlying the analgesic activity of carvacrol. Copyright © 2015 Elsevier B.V. All rights reserved.

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.

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.

Hysteresis in voltage-gated channels.

PubMed

Villalba-Galea, Carlos A

2017-03-04

Ion channels constitute a superfamily of membrane proteins found in all living creatures. Their activity allows fast translocation of ions across the plasma membrane down the ion's transmembrane electrochemical gradient, resulting in a difference in electrical potential across the plasma membrane, known as the membrane potential. A group within this superfamily, namely voltage-gated channels, displays activity that is sensitive to the membrane potential. The activity of voltage-gated channels is controlled by the membrane potential, while the membrane potential is changed by these channels' activity. This interplay produces variations in the membrane potential that have evolved into electrical signals in many organisms. These signals are essential for numerous biological processes, including neuronal activity, insulin release, muscle contraction, fertilization and many others. In recent years, the activity of the voltage-gated channels has been observed not to follow a simple relationship with the membrane potential. Instead, it has been shown that the activity of voltage-gated channel displays hysteresis. In fact, a growing number of evidence have demonstrated that the voltage dependence of channel activity is dynamically modulated by activity itself. In spite of the great impact that this property can have on electrical signaling, hysteresis in voltage-gated channels is often overlooked. Addressing this issue, this review provides examples of voltage-gated ion channels displaying hysteretic behavior. Further, this review will discuss how Dynamic Voltage Dependence in voltage-gated channels can have a physiological role in electrical signaling. Furthermore, this review will elaborate on the current thoughts on the mechanism underlying hysteresis in voltage-gated channels.

Hysteresis in voltage-gated channels

PubMed Central

2017-01-01

ABSTRACT Ion channels constitute a superfamily of membrane proteins found in all living creatures. Their activity allows fast translocation of ions across the plasma membrane down the ion's transmembrane electrochemical gradient, resulting in a difference in electrical potential across the plasma membrane, known as the membrane potential. A group within this superfamily, namely voltage-gated channels, displays activity that is sensitive to the membrane potential. The activity of voltage-gated channels is controlled by the membrane potential, while the membrane potential is changed by these channels' activity. This interplay produces variations in the membrane potential that have evolved into electrical signals in many organisms. These signals are essential for numerous biological processes, including neuronal activity, insulin release, muscle contraction, fertilization and many others. In recent years, the activity of the voltage-gated channels has been observed not to follow a simple relationship with the membrane potential. Instead, it has been shown that the activity of voltage-gated channel displays hysteresis. In fact, a growing number of evidence have demonstrated that the voltage dependence of channel activity is dynamically modulated by activity itself. In spite of the great impact that this property can have on electrical signaling, hysteresis in voltage-gated channels is often overlooked. Addressing this issue, this review provides examples of voltage-gated ion channels displaying hysteretic behavior. Further, this review will discuss how Dynamic Voltage Dependence in voltage-gated channels can have a physiological role in electrical signaling. Furthermore, this review will elaborate on the current thoughts on the mechanism underlying hysteresis in voltage-gated channels. PMID:27689426

Genistein inhibits voltage-gated sodium currents in SCG neurons through protein tyrosine kinase-dependent and kinase-independent mechanisms.

PubMed

Jia, Zhanfeng; Jia, Yueqin; Liu, Boyi; Zhao, Zhiying; Jia, Qingzhong; Liang, Huiling; Zhang, Hailin

2008-08-01

Voltage-gated sodium channels play a crucial role in the initiation and propagation of neuronal action potentials. Genistein, an isoflavone phytoestrogen, has long been used as a broad-spectrum inhibitor of protein tyrosine kinases (PTK). In addition, genistein-induced modulation of ion channels has been described previously in the literature. In this study, we investigated the effect of genistein on voltage-gated sodium channels in rat superior cervical ganglia (SCG) neurons. The results show that genistein inhibits Na(+) currents in a concentration-dependent manner, with a concentration of half-maximal effect (IC(50)) at 9.1 +/- 0.9 microM. Genistein positively shifted the voltage dependence of activation but did not affect inactivation of the Na(+) current. The inactive genistein analog daidzein also inhibited Na(+) currents, but was less effective than genistein. The IC(50) for daidzein-induced inhibition was 20.7 +/- 0.1 microM. Vanadate, an inhibitor of protein tyrosine phosphatases, partially but significantly reversed genistein-induced inhibition of Na(+) currents. Other protein tyrosine kinase antagonists such as tyrphostin 23, an erbstatin analog, and PP2 all had small but significant inhibitory effects on Na(+) currents. Among all active and inactive tyrosine kinase inhibitors tested, genistein was the most potent inhibitor of Na(+) currents. These results suggest that genistein inhibits Na(+) currents in rat SCG neurons through two distinct mechanisms: protein tyrosine kinase-independent, and protein tyrosine kinase-dependent mechanisms. Furthermore, the Src kinase family may be involved in the basal phosphorylation of the Na(+) channel.

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.

Mechanism of Electromechanical Coupling in Voltage-Gated Potassium Channels

PubMed Central

Blunck, Rikard; Batulan, Zarah

2012-01-01

Voltage-gated ion channels play a central role in the generation of action potentials in the nervous system. They are selective for one type of ion – sodium, calcium, or potassium. Voltage-gated ion channels are composed of a central pore that allows ions to pass through the membrane and four peripheral voltage sensing domains that respond to changes in the membrane potential. Upon depolarization, voltage sensors in voltage-gated potassium channels (Kv) undergo conformational changes driven by positive charges in the S4 segment and aided by pairwise electrostatic interactions with the surrounding voltage sensor. Structure-function relations of Kv channels have been investigated in detail, and the resulting models on the movement of the voltage sensors now converge to a consensus; the S4 segment undergoes a combined movement of rotation, tilt, and vertical displacement in order to bring 3–4e+ each through the electric field focused in this region. Nevertheless, the mechanism by which the voltage sensor movement leads to pore opening, the electromechanical coupling, is still not fully understood. Thus, recently, electromechanical coupling in different Kv channels has been investigated with a multitude of techniques including electrophysiology, 3D crystal structures, fluorescence spectroscopy, and molecular dynamics simulations. Evidently, the S4–S5 linker, the covalent link between the voltage sensor and pore, plays a crucial role. The linker transfers the energy from the voltage sensor movement to the pore domain via an interaction with the S6 C-termini, which are pulled open during gating. In addition, other contact regions have been proposed. This review aims to provide (i) an in-depth comparison of the molecular mechanisms of electromechanical coupling in different Kv channels; (ii) insight as to how the voltage sensor and pore domain influence one another; and (iii) theoretical predictions on the movement of the cytosolic face of the Kv channels during

Recent Developments Regarding Voltage-Gated Sodium Channel Blockers for the Treatment of Inherited and Acquired Neuropathic Pain Syndromes

PubMed Central

Theile, Jonathan W.; Cummins, Theodore R.

2011-01-01

Chronic and neuropathic pain constitute significant health problems affecting millions of individuals each year. Pain sensations typically originate in sensory neurons of the peripheral nervous system which relay information to the central nervous system (CNS). Pathological pain sensations can arise as result of changes in excitability of these peripheral sensory neurons. Voltage-gated sodium channels are key determinants regulating action potential generation and propagation; thus, changes in sodium channel function can have profound effects on neuronal excitability and pain signaling. At present, most of the clinically available sodium channel blockers used to treat pain are non-selective across sodium channel isoforms and can contribute to cardio-toxicity, motor impairments, and CNS side effects. Numerous strides have been made over the last decade in an effort to develop more selective and efficacious sodium channel blockers to treat pain. The purpose of this review is to highlight some of the more recent developments put forth by research universities and pharmaceutical companies alike in the pursuit of developing more targeted sodium channel therapies for the treatment of a variety of neuropathic pain conditions. PMID:22007172

Attenuated effect of tungsten carbide nanoparticles on voltage-gated sodium current of hippocampal CA1 pyramidal neurons.

PubMed

Shan, Dehong; Xie, Yongling; Ren, Guogang; Yang, Zhuo

2013-02-01

Nanomaterials and relevant products are now being widely used in the world, and their safety becomes a great concern for the general public. Tungsten carbide nanoparticles (nano-WC) are widely used in metallurgy, aeronautics and astronautics, however our knowledge regarding the influence of nano-WC on neurons is still lacking. The aim of this study was to investigate the impact of nano-WC on tetrodotoxin (TTX)-sensitive voltage-activated sodium current (I(Na)) of hippocampal CA1 pyramidal neurons. Results showed that acute exposure of nano-WC attenuated the peak amplitudes of I(Na) in a concentration-dependent manner. The minimal effective concentration was 10(-5)g/ml. The exposure of nano-WC significantly decreased current amplitudes of the current-voltage curves of I(Na) from -50 to+50 mV, shifted the steady-state activation and inactivation curves of I(Na) negatively and delayed the recovery of I(Na) from inactivation state. After exposure to nano-WC, the peak amplitudes, overshoots and the V-thresholds of action potentials (APs) were markedly reduced. These results suggested that exposure of nano-WC could influence some characteristics of APs evoked from the hippocampal CA1 neurons by modifying the kinetics of voltage-gated sodium channels (VGSCs). Copyright © 2012 Elsevier Ltd. All rights reserved.

Outward stabilization of the voltage sensor in domain II but not domain I speeds inactivation of voltage-gated sodium channels.

PubMed

Sheets, Michael F; Chen, Tiehua; Hanck, Dorothy A

2013-10-15

To determine the roles of the individual S4 segments in domains I and II to activation and inactivation kinetics of sodium current (INa) in NaV1.5, we used a tethered biotin and avidin approach after a site-directed cysteine substitution was made in the second outermost Arg in each S4 (DI-R2C and DII-R2C). We first determined the fraction of gating charge contributed by the individual S4's to maximal gating current (Qmax), and found that the outermost Arg residue in each S4 contributed ∼19% to Qmax with minimal contributions by other arginines. Stabilization of the S4's in DI-R2C and DII-R2C was confirmed by measuring the expected reduction in Qmax. In DI-R2C, stabilization resulted in a decrease in peak INa of ∼45%, while its peak current-voltage (I-V) and voltage-dependent Na channel availability (SSI) curves were nearly unchanged from wild type (WT). In contrast, stabilization of the DII-R2C enhanced activation with a negative shift in the peak I-V relationship by -7 mV and a larger -17 mV shift in the voltage-dependent SSI curve. Furthermore, its INa decay time constants and time-to-peak INa became more rapid than WT. An explanation for these results is that the depolarized conformation of DII-S4, but not DI-S4, affects the receptor for the inactivation particle formed by the interdomain linker between DIII and IV. In addition, the leftward shifts of both activation and inactivation and the decrease in Gmax after stabilization of the DII-S4 support previous studies that showed β-scorpion toxins trap the voltage sensor of DII in an activated conformation.

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

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.

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

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.

Photocontrol of Voltage-Gated Ion Channel Activity by Azobenzene Trimethylammonium Bromide in Neonatal Rat Cardiomyocytes

PubMed Central

Frolova, Sheyda R.; Gaiko, Olga; Tsvelaya, Valeriya A.; Pimenov, Oleg Y.; Agladze, Konstantin I.

2016-01-01

The ability of azobenzene trimethylammonium bromide (azoTAB) to sensitize cardiac tissue excitability to light was recently reported. The dark, thermally relaxed trans- isomer of azoTAB suppressed spontaneous activity and excitation propagation speed, whereas the cis- isomer had no detectable effect on the electrical properties of cardiomyocyte monolayers. As the membrane potential of cardiac cells is mainly controlled by activity of voltage-gated ion channels, this study examined whether the sensitization effect of azoTAB was exerted primarily via the modulation of voltage-gated ion channel activity. The effects of trans- and cis- isomers of azoTAB on voltage-dependent sodium (INav), calcium (ICav), and potassium (IKv) currents in isolated neonatal rat cardiomyocytes were investigated using the whole-cell patch-clamp technique. The experiments showed that azoTAB modulated ion currents, causing suppression of sodium (Na+) and calcium (Ca2+) currents and potentiation of net potassium (K+) currents. This finding confirms that azoTAB-effect on cardiac tissue excitability do indeed result from modulation of voltage-gated ion channels responsible for action potential. PMID:27015602

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.

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.

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.

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.

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.

Gating of the designed trimeric/tetrameric voltage-gated H+ channel

PubMed Central

Fujiwara, Yuichiro; Kurokawa, Tatsuki; Takeshita, Kohei; Nakagawa, Atsushi; Larsson, H Peter; Okamura, Yasushi

2013-01-01

The voltage-gated H+ channel functions as a dimer, a configuration that is different from standard tetrameric voltage-gated channels. Each channel protomer has its own permeation pathway. The C-terminal coiled-coil domain has been shown to be necessary for both dimerization and cooperative gating in the two channel protomers. Here we report the gating cooperativity in trimeric and tetrameric Hv channels engineered by altering the hydrophobic core sequence of the coiled-coil assembly domain. Trimeric and tetrameric channels exhibited more rapid and less sigmoidal kinetics of activation of H+ permeation than dimeric channels, suggesting that some channel protomers in trimers and tetramers failed to produce gating cooperativity observed in wild-type dimers. Multimerization of trimer and tetramer channels were confirmed by the biochemical analysis of proteins, including crystallography. These findings indicate that the voltage-gated H+ channel is optimally designed as a dimeric channel on a solid foundation of the sequence pattern of the coiled-coil core, with efficient cooperative gating that ensures sustained and steep voltage-dependent H+ conductance in blood cells. PMID:23165764

Differential effect of brief electrical stimulation on voltage-gated potassium channels.

PubMed

Cameron, Morven A; Al Abed, Amr; Buskila, Yossi; Dokos, Socrates; Lovell, Nigel H; Morley, John W

2017-05-01

Electrical stimulation of neuronal tissue is a promising strategy to treat a variety of neurological disorders. The mechanism of neuronal activation by external electrical stimulation is governed by voltage-gated ion channels. This stimulus, typically brief in nature, leads to membrane potential depolarization, which increases ion flow across the membrane by increasing the open probability of these voltage-gated channels. In spiking neurons, it is activation of voltage-gated sodium channels (Na V channels) that leads to action potential generation. However, several other types of voltage-gated channels are expressed that also respond to electrical stimulation. In this study, we examine the response of voltage-gated potassium channels (K V channels) to brief electrical stimulation by whole cell patch-clamp electrophysiology and computational modeling. We show that nonspiking amacrine neurons of the retina exhibit a large variety of responses to stimulation, driven by different K V -channel subtypes. Computational modeling reveals substantial differences in the response of specific K V -channel subtypes that is dependent on channel kinetics. This suggests that the expression levels of different K V -channel subtypes in retinal neurons are a crucial predictor of the response that can be obtained. These data expand our knowledge of the mechanisms of neuronal activation and suggest that K V -channel expression is an important determinant of the sensitivity of neurons to electrical stimulation. NEW & NOTEWORTHY This paper describes the response of various voltage-gated potassium channels (K V channels) to brief electrical stimulation, such as is applied during prosthetic electrical stimulation. We show that the pattern of response greatly varies between K V channel subtypes depending on activation and inactivation kinetics of each channel. Our data suggest that problems encountered when artificially stimulating neurons such as cessation in firing at high frequencies, or

Differential effect of brief electrical stimulation on voltage-gated potassium channels

PubMed Central

Al Abed, Amr; Buskila, Yossi; Dokos, Socrates; Lovell, Nigel H.; Morley, John W.

2017-01-01

Electrical stimulation of neuronal tissue is a promising strategy to treat a variety of neurological disorders. The mechanism of neuronal activation by external electrical stimulation is governed by voltage-gated ion channels. This stimulus, typically brief in nature, leads to membrane potential depolarization, which increases ion flow across the membrane by increasing the open probability of these voltage-gated channels. In spiking neurons, it is activation of voltage-gated sodium channels (NaV channels) that leads to action potential generation. However, several other types of voltage-gated channels are expressed that also respond to electrical stimulation. In this study, we examine the response of voltage-gated potassium channels (KV channels) to brief electrical stimulation by whole cell patch-clamp electrophysiology and computational modeling. We show that nonspiking amacrine neurons of the retina exhibit a large variety of responses to stimulation, driven by different KV-channel subtypes. Computational modeling reveals substantial differences in the response of specific KV-channel subtypes that is dependent on channel kinetics. This suggests that the expression levels of different KV-channel subtypes in retinal neurons are a crucial predictor of the response that can be obtained. These data expand our knowledge of the mechanisms of neuronal activation and suggest that KV-channel expression is an important determinant of the sensitivity of neurons to electrical stimulation. NEW & NOTEWORTHY This paper describes the response of various voltage-gated potassium channels (KV channels) to brief electrical stimulation, such as is applied during prosthetic electrical stimulation. We show that the pattern of response greatly varies between KV channel subtypes depending on activation and inactivation kinetics of each channel. Our data suggest that problems encountered when artificially stimulating neurons such as cessation in firing at high frequencies, or �

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

Eslicarbazepine and the enhancement of slow inactivation of voltage-gated sodium channels: a comparison with carbamazepine, oxcarbazepine and lacosamide.

PubMed

Hebeisen, Simon; Pires, Nuno; Loureiro, Ana I; Bonifácio, Maria João; Palma, Nuno; Whyment, Andrew; Spanswick, David; Soares-da-Silva, Patrício

2015-02-01

This study aimed at evaluating the effects of eslicarbazepine, carbamazepine (CBZ), oxcarbazepine (OXC) and lacosamide (LCM) on the fast and slow inactivated states of voltage-gated sodium channels (VGSC). The anti-epileptiform activity was evaluated in mouse isolated hippocampal slices. The anticonvulsant effects were evaluated in MES and the 6-Hz psychomotor tests. The whole-cell patch-clamp technique was used to investigate the effects of eslicarbazepine, CBZ, OXC and LCM on sodium channels endogenously expressed in N1E-115 mouse neuroblastoma cells. CBZ and eslicarbazepine exhibit similar concentration dependent suppression of epileptiform activity in hippocampal slices. In N1E-115 mouse neuroblastoma cells, at a concentration of 250 μM, the voltage dependence of the fast inactivation was not influenced by eslicarbazepine, whereas LCM, CBZ and OXC shifted the V0.5 value (mV) by -4.8, -12.0 and -16.6, respectively. Eslicarbazepine- and LCM-treated fast-inactivated channels recovered similarly to control conditions, whereas CBZ- and OXC-treated channels required longer pulses to recover. CBZ, eslicarbazepine and LCM shifted the voltage dependence of the slow inactivation (V0.5, mV) by -4.6, -31.2 and -53.3, respectively. For eslicarbazepine, LCM, CBZ and OXC, the affinity to the slow inactivated state was 5.9, 10.4, 1.7 and 1.8 times higher than to the channels in the resting state, respectively. In conclusion, eslicarbazepine did not share with CBZ and OXC the ability to alter fast inactivation of VGSC. Both eslicarbazepine and LCM reduce VGSC availability through enhancement of slow inactivation, but LCM demonstrated higher interaction with VGSC in the resting state and with fast inactivation gating. Copyright © 2014 Elsevier Ltd. All rights reserved.

Pyrethroid resistance in Sitophilus zeamais is associated with a mutation (T929I) in the voltage-gated sodium channel.

PubMed

Araújo, Rúbia A; Williamson, Martin S; Bass, Christopher; Field, Linda M; Duce, Ian R

2011-08-01

The maize weevil, Sitophilus zeamais, is the most important pest affecting stored grain in Brazil and its control relies heavily on the use of insecticides. The intensive use of compounds such as the pyrethroids has led to the emergence of resistance, and previous studies have suggested that resistance to both pyrethroids and 1,1,1-trichloro-2,2-bis(p-chlorophenyl)ethane (DDT) may result from reduced sensitivity of the insecticide target, the voltage-gated sodium channel. To identify the molecular mechanisms underlying pyrethroid resistance in S. zeamais, the domain II region of the voltage-gated sodium channel (para-orthologue) gene was amplified by PCR and sequenced from susceptible and resistant laboratory S. zeamais strains that were selected with a discriminating dose of DDT. A single point mutation, T929I, was found in the para gene of the resistant S. zeamais populations and its presence in individual weevils was strongly associated with survival after DDT exposure. This is the first identification of a target-site resistance mutation in S. zeamais and unusually it is a super-kdr type mutation occurring in the absence of the more common kdr (L1014F) substitution. A high-throughput assay based on TaqMan single nucleotide polymorphism genotyping was developed for sensitive detection of the mutation and used to screen field-collected strains of S. zeamais. This showed that the mutation is present at low frequency in field populations and is a useful tool for informing control strategies. © 2011 The Authors. Insect Molecular Biology © 2011 The Royal Entomological Society.

Voltage gating of mechanosensitive PIEZO channels.

PubMed

Moroni, Mirko; Servin-Vences, M Rocio; Fleischer, Raluca; Sánchez-Carranza, Oscar; Lewin, Gary R

2018-03-15

Mechanosensitive PIEZO ion channels are evolutionarily conserved proteins whose presence is critical for normal physiology in multicellular organisms. Here we show that, in addition to mechanical stimuli, PIEZO channels are also powerfully modulated by voltage and can even switch to a purely voltage-gated mode. Mutations that cause human diseases, such as xerocytosis, profoundly shift voltage sensitivity of PIEZO1 channels toward the resting membrane potential and strongly promote voltage gating. Voltage modulation may be explained by the presence of an inactivation gate in the pore, the opening of which is promoted by outward permeation. Older invertebrate (fly) and vertebrate (fish) PIEZO proteins are also voltage sensitive, but voltage gating is a much more prominent feature of these older channels. We propose that the voltage sensitivity of PIEZO channels is a deep property co-opted to add a regulatory mechanism for PIEZO activation in widely different cellular contexts.

Beyond voltage-gated ion channels: Voltage-operated membrane proteins and cellular processes.

PubMed

Zhang, Jianping; Chen, Xingjuan; Xue, Yucong; Gamper, Nikita; Zhang, Xuan

2018-04-18

Voltage-gated ion channels were believed to be the only voltage-sensitive proteins in excitable (and some non-excitable) cells for a long time. Emerging evidence indicates that the voltage-operated model is shared by some other transmembrane proteins expressed in both excitable and non-excitable cells. In this review, we summarize current knowledge about voltage-operated proteins, which are not classic voltage-gated ion channels as well as the voltage-dependent processes in cells for which single voltage-sensitive proteins have yet to be identified. Particularly, we will focus on the following. (1) Voltage-sensitive phosphoinositide phosphatases (VSP) with four transmembrane segments homologous to the voltage sensor domain (VSD) of voltage-gated ion channels; VSPs are the first family of proteins, other than the voltage-gated ion channels, for which there is sufficient evidence for the existence of the VSD domain; (2) Voltage-gated proton channels comprising of a single voltage-sensing domain and lacking an identified pore domain; (3) G protein coupled receptors (GPCRs) that mediate the depolarization-evoked potentiation of Ca 2+ mobilization; (4) Plasma membrane (PM) depolarization-induced but Ca 2+ -independent exocytosis in neurons. (5) Voltage-dependent metabolism of phosphatidylinositol 4,5-bisphosphate (PtdIns[4,5]P 2 , PIP 2 ) in the PM. These recent discoveries expand our understanding of voltage-operated processes within cellular membranes. © 2018 Wiley Periodicals, Inc.

Pyrethroid Insecticides Directly Activate Microglia Through Interaction With Voltage-Gated Sodium Channels.

PubMed

Hossain, Muhammad M; Liu, Jason; Richardson, Jason R

2017-01-01

Microglia are considered to be the resident immune cells of the central nervous system and contribute significantly to ongoing neuroinflammation in a variety of neurodegenerative diseases. Recently, we and others identified that voltage-gated sodium channels (VGSC) are present on microglia cells and contribute to excessive accumulation of intracellular Na +  and release of major pro-inflammatory cytokine tumor necrosis factor alpha (TNF-α). Based on this finding and the fact that pyrethroid pesticides act on VGSC, we hypothesized that exposure of microglia to the pyrethroid pesticides, permethrin and deltamethrin, would activate microglia and increase the release of TNF-α. BV2 cells or primary microglia were treated with 0-5 µM deltamethrin or permethrin in the presence or absence of tetrodotoxin (TTX), a VGSC blocker for 24-48 h. Both pyrethroids caused a rapid Na +  influx and increased accumulation of intracellular sodium [(Na + )i] in the microglia in a dose- and time-dependent manner, which was significantly reduced by TTX. Furthermore, deltamethrin and permethrin increased the release of TNF-α in a dose- and time-dependent manner, which was significantly reduced by pre-treatment of cells with TTX. These results demonstrate that pyrethroid pesticides may directly activate microglial cells through their interaction with microglial VGSC. Because neuroinflammation plays a key role in many neurodegenerative diseases, these data provide an additional mechanism by which exposure to pyrethroid insecticides may contribute to neurodegeneration. © The Author 2016. Published by Oxford University Press on behalf of the Society of Toxicology. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.

Molecular basis of the interaction between gating modifier spider toxins and the voltage sensor of voltage-gated ion channels

NASA Astrophysics Data System (ADS)

Lau, Carus H. Y.; King, Glenn F.; Mobli, Mehdi

2016-09-01

Voltage-sensor domains (VSDs) are modular transmembrane domains of voltage-gated ion channels that respond to changes in membrane potential by undergoing conformational changes that are coupled to gating of the ion-conducting pore. Most spider-venom peptides function as gating modifiers by binding to the VSDs of voltage-gated channels and trapping them in a closed or open state. To understand the molecular basis underlying this mode of action, we used nuclear magnetic resonance to delineate the atomic details of the interaction between the VSD of the voltage-gated potassium channel KvAP and the spider-venom peptide VSTx1. Our data reveal that the toxin interacts with residues in an aqueous cleft formed between the extracellular S1-S2 and S3-S4 loops of the VSD whilst maintaining lipid interactions in the gaps formed between the S1-S4 and S2-S3 helices. The resulting network of interactions increases the energetic barrier to the conformational changes required for channel gating, and we propose that this is the mechanism by which gating modifier toxins inhibit voltage-gated ion channels.

Voltage-Dependent Gating of hERG Potassium Channels

PubMed Central

Cheng, Yen May; Claydon, Tom W.

2012-01-01

The mechanisms by which voltage-gated channels sense changes in membrane voltage and energetically couple this with opening of the ion conducting pore has been the source of significant interest. In voltage-gated potassium (Kv) channels, much of our knowledge in this area comes from Shaker-type channels, for which voltage-dependent gating is quite rapid. In these channels, activation and deactivation are associated with rapid reconfiguration of the voltage-sensing domain unit that is electromechanically coupled, via the S4–S5 linker helix, to the rate-limiting opening of an intracellular pore gate. However, fast voltage-dependent gating kinetics are not typical of all Kv channels, such as Kv11.1 (human ether-à-go-go related gene, hERG), which activates and deactivates very slowly. Compared to Shaker channels, our understanding of the mechanisms underlying slow hERG gating is much poorer. Here, we present a comparative review of the structure–function relationships underlying activation and deactivation gating in Shaker and hERG channels, with a focus on the roles of the voltage-sensing domain and the S4–S5 linker that couples voltage sensor movements to the pore. Measurements of gating current kinetics and fluorimetric analysis of voltage sensor movement are consistent with models suggesting that the hERG activation pathway contains a voltage independent step, which limits voltage sensor transitions. Constraints upon hERG voltage sensor movement may result from loose packing of the S4 helices and additional intra-voltage sensor counter-charge interactions. More recent data suggest that key amino acid differences in the hERG voltage-sensing unit and S4–S5 linker, relative to fast activating Shaker-type Kv channels, may also contribute to the increased stability of the resting state of the voltage sensor. PMID:22586397

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.

Actions and Mechanisms of Polyunsaturated Fatty Acids on Voltage-Gated Ion Channels.

PubMed

Elinder, Fredrik; Liin, Sara I

2017-01-01

Polyunsaturated fatty acids (PUFAs) act on most ion channels, thereby having significant physiological and pharmacological effects. In this review we summarize data from numerous PUFAs on voltage-gated ion channels containing one or several voltage-sensor domains, such as voltage-gated sodium (Na V ), potassium (K V ), calcium (Ca V ), and proton (H V ) channels, as well as calcium-activated potassium (K Ca ), and transient receptor potential (TRP) channels. Some effects of fatty acids appear to be channel specific, whereas others seem to be more general. Common features for the fatty acids to act on the ion channels are at least two double bonds in cis geometry and a charged carboxyl group. In total we identify and label five different sites for the PUFAs. PUFA site 1 : The intracellular cavity. Binding of PUFA reduces the current, sometimes as a time-dependent block, inducing an apparent inactivation. PUFA site 2 : The extracellular entrance to the pore. Binding leads to a block of the channel. PUFA site 3 : The intracellular gate. Binding to this site can bend the gate open and increase the current. PUFA site 4 : The interface between the extracellular leaflet of the lipid bilayer and the voltage-sensor domain. Binding to this site leads to an opening of the channel via an electrostatic attraction between the negatively charged PUFA and the positively charged voltage sensor. PUFA site 5 : The interface between the extracellular leaflet of the lipid bilayer and the pore domain. Binding to this site affects slow inactivation. This mapping of functional PUFA sites can form the basis for physiological and pharmacological modifications of voltage-gated ion channels.

Actions and Mechanisms of Polyunsaturated Fatty Acids on Voltage-Gated Ion Channels

PubMed Central

Elinder, Fredrik; Liin, Sara I.

2017-01-01

Polyunsaturated fatty acids (PUFAs) act on most ion channels, thereby having significant physiological and pharmacological effects. In this review we summarize data from numerous PUFAs on voltage-gated ion channels containing one or several voltage-sensor domains, such as voltage-gated sodium (NaV), potassium (KV), calcium (CaV), and proton (HV) channels, as well as calcium-activated potassium (KCa), and transient receptor potential (TRP) channels. Some effects of fatty acids appear to be channel specific, whereas others seem to be more general. Common features for the fatty acids to act on the ion channels are at least two double bonds in cis geometry and a charged carboxyl group. In total we identify and label five different sites for the PUFAs. PUFA site 1: The intracellular cavity. Binding of PUFA reduces the current, sometimes as a time-dependent block, inducing an apparent inactivation. PUFA site 2: The extracellular entrance to the pore. Binding leads to a block of the channel. PUFA site 3: The intracellular gate. Binding to this site can bend the gate open and increase the current. PUFA site 4: The interface between the extracellular leaflet of the lipid bilayer and the voltage-sensor domain. Binding to this site leads to an opening of the channel via an electrostatic attraction between the negatively charged PUFA and the positively charged voltage sensor. PUFA site 5: The interface between the extracellular leaflet of the lipid bilayer and the pore domain. Binding to this site affects slow inactivation. This mapping of functional PUFA sites can form the basis for physiological and pharmacological modifications of voltage-gated ion channels. PMID:28220076

Deletion of cytosolic gating ring decreases gate and voltage sensor coupling in BK channels.

PubMed

Zhang, Guohui; Geng, Yanyan; Jin, Yakang; Shi, Jingyi; McFarland, Kelli; Magleby, Karl L; Salkoff, Lawrence; Cui, Jianmin

2017-03-06

Large conductance Ca 2+ -activated K + channels (BK channels) gate open in response to both membrane voltage and intracellular Ca 2+ The channel is formed by a central pore-gate domain (PGD), which spans the membrane, plus transmembrane voltage sensors and a cytoplasmic gating ring that acts as a Ca 2+ sensor. How these voltage and Ca 2+ sensors influence the common activation gate, and interact with each other, is unclear. A previous study showed that a BK channel core lacking the entire cytoplasmic gating ring (Core-MT) was devoid of Ca 2+ activation but retained voltage sensitivity (Budelli et al. 2013. Proc. Natl. Acad. Sci. USA http://dx.doi.org/10.1073/pnas.1313433110). In this study, we measure voltage sensor activation and pore opening in this Core-MT channel over a wide range of voltages. We record gating currents and find that voltage sensor activation in this truncated channel is similar to WT but that the coupling between voltage sensor activation and gating of the pore is reduced. These results suggest that the gating ring, in addition to being the Ca 2+ sensor, enhances the effective coupling between voltage sensors and the PGD. We also find that removal of the gating ring alters modulation of the channels by the BK channel's β1 and β2 subunits. © 2017 Zhang et al.

Deletion of cytosolic gating ring decreases gate and voltage sensor coupling in BK channels

PubMed Central

Zhang, Guohui; Shi, Jingyi; McFarland, Kelli; Magleby, Karl L.; Salkoff, Lawrence

2017-01-01

Large conductance Ca2+-activated K+ channels (BK channels) gate open in response to both membrane voltage and intracellular Ca2+. The channel is formed by a central pore-gate domain (PGD), which spans the membrane, plus transmembrane voltage sensors and a cytoplasmic gating ring that acts as a Ca2+ sensor. How these voltage and Ca2+ sensors influence the common activation gate, and interact with each other, is unclear. A previous study showed that a BK channel core lacking the entire cytoplasmic gating ring (Core-MT) was devoid of Ca2+ activation but retained voltage sensitivity (Budelli et al. 2013. Proc. Natl. Acad. Sci. USA. http://dx.doi.org/10.1073/pnas.1313433110). In this study, we measure voltage sensor activation and pore opening in this Core-MT channel over a wide range of voltages. We record gating currents and find that voltage sensor activation in this truncated channel is similar to WT but that the coupling between voltage sensor activation and gating of the pore is reduced. These results suggest that the gating ring, in addition to being the Ca2+ sensor, enhances the effective coupling between voltage sensors and the PGD. We also find that removal of the gating ring alters modulation of the channels by the BK channel’s β1 and β2 subunits. PMID:28196879

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

Multi-country Survey Revealed Prevalent and Novel F1534S Mutation in Voltage-Gated Sodium Channel (VGSC) Gene in Aedes albopictus.

PubMed

Xu, Jiabao; Bonizzoni, Mariangela; Zhong, Daibin; Zhou, Guofa; Cai, Songwu; Li, Yiji; Wang, Xiaoming; Lo, Eugenia; Lee, Rebecca; Sheen, Roger; Duan, Jinhua; Yan, Guiyun; Chen, Xiao-Guang

2016-05-01

Aedes albopictus is an important dengue vector because of its aggressive biting behavior and rapid spread out of its native home range in Southeast Asia. Pyrethroids are widely used for adult mosquito control, and resistance to pyrethroids should be carefully monitored because vector control is the only effective method currently available to prevent dengue transmission. The voltage-gated sodium channel gene is the target site of pyrethroids, and mutations in this gene cause knockdown resistance (kdr). Previous studies reported various mutations in the voltage-gated sodium channel (VGSC) gene, but the spatial distribution of kdr mutations in Ae. albopictus has not been systematically examined, and the association between kdr mutation and phenotypic resistance has not been established. A total of 597 Ae. albopictus individuals from 12 populations across Asia, Africa, America and Europe were examined for mutations in the voltage-gated sodium channel gene. Three domains for a total of 1,107 bp were sequenced for every individual. Two populations from southern China were examined for pyrethroid resistance using the World Health Organization standard tube bioassay, and the association between kdr mutations and phenotypic resistance was tested. A total of 29 synonymous mutations were found across domain II, III and IV of the VGSC gene. Non-synonymous mutations in two codons of the VGSC gene were detected in 5 populations from 4 countries. A novel mutation at 1532 codon (I1532T) was found in Rome, Italy with a frequency of 19.7%. The second novel mutation at codon 1534 (F1534S) was detected in southern China and Florida, USA with a frequency ranging from 9.5-22.6%. The WHO insecticide susceptibility bioassay found 90.1% and 96.1% mortality in the two populations from southern China, suggesting resistance and probable resistance. Positive association between kdr mutations with deltamethrin resistance was established in these two populations. Two novel kdr mutations, I1532T

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.

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

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.

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

PubMed

Groome, James R; Winston, Vern

2013-05-01

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

Voltage-gated Proton Channels

PubMed Central

DeCoursey, Thomas E.

2014-01-01

Voltage-gated proton channels, HV1, have vaulted from the realm of the esoteric into the forefront of a central question facing ion channel biophysicists, namely the mechanism by which voltage-dependent gating occurs. This transformation is the result of several factors. Identification of the gene in 2006 revealed that proton channels are homologues of the voltage-sensing domain of most other voltage-gated ion channels. Unique, or at least eccentric, properties of proton channels include dimeric architecture with dual conduction pathways, perfect proton selectivity, a single-channel conductance ~103 smaller than most ion channels, voltage-dependent gating that is strongly modulated by the pH gradient, ΔpH, and potent inhibition by Zn2+ (in many species) but an absence of other potent inhibitors. The recent identification of HV1 in three unicellular marine plankton species has dramatically expanded the phylogenetic family tree. Interest in proton channels in their own right has increased as important physiological roles have been identified in many cells. Proton channels trigger the bioluminescent flash of dinoflagellates, facilitate calcification by coccolithophores, regulate pH-dependent processes in eggs and sperm during fertilization, secrete acid to control the pH of airway fluids, facilitate histamine secretion by basophils, and play a signaling role in facilitating B-cell receptor mediated responses in B lymphocytes. The most elaborate and best-established functions occur in phagocytes, where proton channels optimize the activity of NADPH oxidase, an important producer of reactive oxygen species. Proton efflux mediated by HV1 balances the charge translocated across the membrane by electrons through NADPH oxidase, minimizes changes in cytoplasmic and phagosomal pH, limits osmotic swelling of the phagosome, and provides substrate H+ for the production of H2O2 and HOCl, reactive oxygen species crucial to killing pathogens. PMID:23798303

Consequences of Phosphate-Arginine Complexes in Voltage-Gated Ion Channels

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

Green, Michael E.

2008-11-01

There are two reasons for suspecting that phosphate complexes of arginine make it very difficult to derive gating charge in voltage gated potassium (and presumably sodium) channels from the motion of charged arginines. For one thing, the arginines should be complexed with phosphate, thereby neutralizing the charge, at least partially. Second, Li et al.(1) have shown that there is a large energy penalty for putting a charged arginine into a membrane. on channel gating current is generally attributed to S4 motion, in that the S4 segment of the voltage sensing domain (VSD) of these channels contains arginines, some of whichmore » are not (or at least not obviously) salt bridged, or otherwise charge compensated. There is, however, good reason to expect that there should be a complex of these arginines with phosphate, very probably from lipid headgroups. This has consequences for gating current; the complexed arginines, if they moved, would carry too much of the membrane along. This leads to the suggestion that an alternative to S4 physical motion, H+ transport, should be considered as a possible resolution of the apparent paradox. The consequences for a gating model that was proposed in our earlier work are discussed; there is one major difference in the model in the present form (a conformational change), but the proton cascade as gating current and the role of water in the closed state are reinforced.« less

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.

Voltage-gated sodium channel polymorphism and metabolic resistance in pyrethroid-resistant Aedes aegypti from Brazil.

PubMed

Martins, Ademir Jesus; Lins, Rachel Mazzei Moura de Andrade; Linss, Jutta Gerlinde Birgitt; Peixoto, Alexandre Afranio; Valle, Denise

2009-07-01

The nature of pyrethroid resistance in Aedes aegypti Brazilian populations was investigated. Quantification of enzymes related to metabolic resistance in two distinct populations, located in the Northeast and Southeast regions, revealed increases in Glutathione-S-transferase (GST) and Esterase levels. Additionally, polymorphism was found in the IIS6 region of Ae. aegypti voltage-gated sodium channel (AaNa(V)), the pyrethroid target site. Sequences were classified in two haplotype groups, A and B, according to the size of the intron in that region. Rockefeller, a susceptible control lineage, contains only B sequences. In field populations, some A sequences present a substitution in the 1011 site (Ile/Met). When resistant and susceptible individuals were compared, the frequency of both A (with the Met mutation) and B sequences were slightly increased in resistant specimens. The involvement of the AaNa(V) polymorphism in pyrethroid resistance and the metabolic mechanisms that lead to potential cross-resistance between organophosphate and pyrethroids are discussed.

Coupling between the Voltage-sensing and Pore Domains in a Voltage-gated Potassium Channel

PubMed Central

Schow, Eric V.; Freites, J. Alfredo; Nizkorodov, Alex; White, Stephen H.; Tobias, Douglas J.

2012-01-01

Voltage-dependent potassium (Kv), sodium (Nav), and calcium channels open and close in response to changes in transmembrane (TM) potential, thus regulating cell excitability by controlling ion flow across the membrane. An outstanding question concerning voltage gating is how voltage-induced conformational changes of the channel voltage-sensing domains (VSDs) are coupled through the S4-S5 interfacial linking helices to the opening and closing of the pore domain (PD). To investigate the coupling between the VSDs and the PD, we generated a closed Kv channel configuration from Aeropyrum pernix (KvAP) using atomistic simulations with experiment-based restraints on the VSDs. Full closure of the channel required, in addition to the experimentally determined TM displacement, that the VSDs be displaced both inwardly and laterally around the PD. This twisting motion generates a tight hydrophobic interface between the S4-S5 linkers and the C-terminal ends of the pore domain S6 helices in agreement with available experimental evidence. PMID:22425907

Coupling between the voltage-sensing and pore domains in a voltage-gated potassium channel.

PubMed

Schow, Eric V; Freites, J Alfredo; Nizkorodov, Alex; White, Stephen H; Tobias, Douglas J

2012-07-01

Voltage-dependent potassium (Kv), sodium (Nav), and calcium channels open and close in response to changes in transmembrane (TM) potential, thus regulating cell excitability by controlling ion flow across the membrane. An outstanding question concerning voltage gating is how voltage-induced conformational changes of the channel voltage-sensing domains (VSDs) are coupled through the S4-S5 interfacial linking helices to the opening and closing of the pore domain (PD). To investigate the coupling between the VSDs and the PD, we generated a closed Kv channel configuration from Aeropyrum pernix (KvAP) using atomistic simulations with experiment-based restraints on the VSDs. Full closure of the channel required, in addition to the experimentally determined TM displacement, that the VSDs be displaced both inwardly and laterally around the PD. This twisting motion generates a tight hydrophobic interface between the S4-S5 linkers and the C-terminal ends of the pore domain S6 helices in agreement with available experimental evidence.

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.

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

Voltage-Dependent Gating: Novel Insights from KCNQ1 Channels

PubMed Central

Cui, Jianmin

2016-01-01

Gating of voltage-dependent cation channels involves three general molecular processes: voltage sensor activation, sensor-pore coupling, and pore opening. KCNQ1 is a voltage-gated potassium (Kv) channel whose distinctive properties have provided novel insights on fundamental principles of voltage-dependent gating. 1) Similar to other Kv channels, KCNQ1 voltage sensor activation undergoes two resolvable steps; but, unique to KCNQ1, the pore opens at both the intermediate and activated state of voltage sensor activation. The voltage sensor-pore coupling differs in the intermediate-open and the activated-open states, resulting in changes of open pore properties during voltage sensor activation. 2) The voltage sensor-pore coupling and pore opening require the membrane lipid PIP2 and intracellular ATP, respectively, as cofactors, thus voltage-dependent gating is dependent on multiple stimuli, including the binding of intracellular signaling molecules. These mechanisms underlie the extraordinary KCNE1 subunit modification of the KCNQ1 channel and have significant physiological implications. PMID:26745405

Signaling complexes of voltage-gated calcium channels

PubMed Central

Turner, Ray W; Anderson, Dustin

2011-01-01

Voltage-gated calcium channels are key mediators of depolarization induced calcium entry into electrically excitable cells. There is increasing evidence that voltage-gated calcium channels, like many other types of ionic channels, do not operate in isolation, but instead form complexes with signaling molecules, G protein coupled receptors, and other types of ion channels. Furthermore, there appears to be bidirectional signaling within these protein complexes, thus allowing not only for efficient translation of calcium signals into cellular responses, but also for tight control of calcium entry per se. In this review, we will focus predominantly on signaling complexes between G protein-coupled receptors and high voltage activated calcium channels, and on complexes of voltage-gated calcium channels and members of the potassium channel superfamily. PMID:21832880

In vivo potency of different ligands on voltage-gated sodium channels.

PubMed

Safrany-Fark, Arpad; Petrovszki, Zita; Kekesi, Gabriella; Liszli, Peter; Benedek, Gyorgy; Keresztes, Csilla; Horvath, Gyongyi

2015-09-05

The Ranvier nodes of thick myelinated nerve fibers contain almost exclusively voltage-gated sodium channels (Navs), while the unmyelinated fibers have several receptors (e.g., cannabinoid, transient receptor potential vanilloid receptor 1), too. Therefore, a nerve which contains only motor fibers can be an appropriate in vivo model for selective influence of Navs. The goals were to evaluate the potency of local anesthetic drugs on such a nerve in vivo; furthermore, to investigate the effects of ligands with different structures (arachidonic acid, anandamide, capsaicin and nisoxetine) that were proved to inhibit Navs in vitro with antinociceptive properties. The marginal mandibular branch of the facial nerve was explored in anesthetized Wistar rats; after its stimulation, the electrical activity of the vibrissae muscles was registered following the perineural injection of different drugs. Lidocaine, bupivacaine and ropivacaine evoked dose-dependent decrease in electromyographic activity, i.e., lidocaine had lower potency than bupivacaine or ropivacaine. QX-314 did not cause any effect by itself, but its co-application with lidocaine produced a prolonged inhibition. Nisoxetine had a very low potency. While anandamide and capsaicin in high doses caused about 50% decrease in the amplitude of action potential, arachidonic acid did not influence the responses. We proved that the classical local anesthetics have high potency on motor nerves, suggesting that this method might be a reliable model for selective targeting of Navs in vivo circumstances. It is proposed that the effects of these endogenous lipids and capsaicin on sensory fibers are not primarily mediated by Navs. Copyright © 2015 Elsevier B.V. All rights reserved.

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.

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...

Oxidative Modulation of Voltage-Gated Potassium Channels

PubMed Central

Sahoo, Nirakar; Hoshi, Toshinori

2014-01-01

Abstract Significance: Voltage-gated K+ channels are a large family of K+-selective ion channel protein complexes that open on membrane depolarization. These K+ channels are expressed in diverse tissues and their function is vital for numerous physiological processes, in particular of neurons and muscle cells. Potentially reversible oxidative regulation of voltage-gated K+ channels by reactive species such as reactive oxygen species (ROS) represents a contributing mechanism of normal cellular plasticity and may play important roles in diverse pathologies including neurodegenerative diseases. Recent Advances: Studies using various protocols of oxidative modification, site-directed mutagenesis, and structural and kinetic modeling provide a broader phenomenology and emerging mechanistic insights. Critical Issues: Physicochemical mechanisms of the functional consequences of oxidative modifications of voltage-gated K+ channels are only beginning to be revealed. In vivo documentation of oxidative modifications of specific amino-acid residues of various voltage-gated K+ channel proteins, including the target specificity issue, is largely absent. Future Directions: High-resolution chemical and proteomic analysis of ion channel proteins with respect to oxidative modification combined with ongoing studies on channel structure and function will provide a better understanding of how the function of voltage-gated K+ channels is tuned by ROS and the corresponding reducing enzymes to meet cellular needs. Antioxid. Redox Signal. 21, 933–952. PMID:24040918

Dopaminergic modulation of the voltage-gated sodium current in the cochlear afferent neurons of the rat.

PubMed

Valdés-Baizabal, Catalina; Soto, Enrique; Vega, Rosario

2015-01-01

The cochlear inner hair cells synapse onto type I afferent terminal dendrites, constituting the main afferent pathway for auditory information flow. This pathway receives central control input from the lateral olivocochlear efferent neurons that release various neurotransmitters, among which dopamine (DA) plays a salient role. DA receptors activation exert a protective role in the over activation of the afferent glutamatergic synapses, which occurs when an animal is exposed to intense sound stimuli or during hypoxic events. However, the mechanism of action of DA at the cellular level is still not completely understood. In this work, we studied the actions of DA and its receptor agonists and antagonists on the voltage-gated sodium current (INa) in isolated cochlear afferent neurons of the rat to define the mechanisms of dopaminergic control of the afferent input in the cochlear pathway. Experiments were performed using the voltage and current clamp techniques in the whole-cell configuration in primary cultures of cochlear spiral ganglion neurons (SGNs). Recordings of the INa showed that DA receptor activation induced a significant inhibition of the peak current amplitude, leading to a significant decrease in cell excitability. Inhibition of the INa was produced by a phosphorylation of the sodium channels as shown by the use of phosphatase inhibitor that produced an inhibition analogous to that caused by DA receptor activation. Use of specific agonists and antagonists showed that inhibitory action of DA was mediated both by activation of D1- and D2-like DA receptors. The action of the D1- and D2-like receptors was shown to be mediated by a Gαs/AC/cAMP/PKA and Gαq/PLC/PKC pathways respectively. These results showed that DA receptor activation constitutes a significant modulatory input to SGNs, effectively modulating their excitability and information flow in the auditory pathway.

Voltage-dependent gating of KCNH potassium channels lacking a covalent link between voltage-sensing and pore domains

PubMed Central

Lörinczi, Éva; Gómez-Posada, Juan Camilo; de la Peña, Pilar; Tomczak, Adam P.; Fernández-Trillo, Jorge; Leipscher, Ulrike; Stühmer, Walter; Barros, Francisco; Pardo, Luis A.

2015-01-01

Voltage-gated channels open paths for ion permeation upon changes in membrane potential, but how voltage changes are coupled to gating is not entirely understood. Two modules can be recognized in voltage-gated potassium channels, one responsible for voltage sensing (transmembrane segments S1 to S4), the other for permeation (S5 and S6). It is generally assumed that the conversion of a conformational change in the voltage sensor into channel gating occurs through the intracellular S4–S5 linker that provides physical continuity between the two regions. Using the pathophysiologically relevant KCNH family, we show that truncated proteins interrupted at, or lacking the S4–S5 linker produce voltage-gated channels in a heterologous model that recapitulate both the voltage-sensing and permeation properties of the complete protein. These observations indicate that voltage sensing by the S4 segment is transduced to the channel gate in the absence of physical continuity between the modules. PMID:25818916

Voltage-dependent gating of KCNH potassium channels lacking a covalent link between voltage-sensing and pore domains.

PubMed

Lörinczi, Éva; Gómez-Posada, Juan Camilo; de la Peña, Pilar; Tomczak, Adam P; Fernández-Trillo, Jorge; Leipscher, Ulrike; Stühmer, Walter; Barros, Francisco; Pardo, Luis A

2015-03-30

Voltage-gated channels open paths for ion permeation upon changes in membrane potential, but how voltage changes are coupled to gating is not entirely understood. Two modules can be recognized in voltage-gated potassium channels, one responsible for voltage sensing (transmembrane segments S1 to S4), the other for permeation (S5 and S6). It is generally assumed that the conversion of a conformational change in the voltage sensor into channel gating occurs through the intracellular S4-S5 linker that provides physical continuity between the two regions. Using the pathophysiologically relevant KCNH family, we show that truncated proteins interrupted at, or lacking the S4-S5 linker produce voltage-gated channels in a heterologous model that recapitulate both the voltage-sensing and permeation properties of the complete protein. These observations indicate that voltage sensing by the S4 segment is transduced to the channel gate in the absence of physical continuity between the modules.

Voltage-dependent gating of KCNH potassium channels lacking a covalent link between voltage-sensing and pore domains

NASA Astrophysics Data System (ADS)

Lörinczi, Éva; Gómez-Posada, Juan Camilo; de La Peña, Pilar; Tomczak, Adam P.; Fernández-Trillo, Jorge; Leipscher, Ulrike; Stühmer, Walter; Barros, Francisco; Pardo, Luis A.

2015-03-01

Voltage-gated channels open paths for ion permeation upon changes in membrane potential, but how voltage changes are coupled to gating is not entirely understood. Two modules can be recognized in voltage-gated potassium channels, one responsible for voltage sensing (transmembrane segments S1 to S4), the other for permeation (S5 and S6). It is generally assumed that the conversion of a conformational change in the voltage sensor into channel gating occurs through the intracellular S4-S5 linker that provides physical continuity between the two regions. Using the pathophysiologically relevant KCNH family, we show that truncated proteins interrupted at, or lacking the S4-S5 linker produce voltage-gated channels in a heterologous model that recapitulate both the voltage-sensing and permeation properties of the complete protein. These observations indicate that voltage sensing by the S4 segment is transduced to the channel gate in the absence of physical continuity between the modules.

Characterization of the voltage-gated sodium channel of the Asian citrus psyllid, Diaphorina citri.

PubMed

Liu, Bin; Coy, Monique R; Wang, Jin-Jun; Stelinski, Lukasz L

2017-02-01

The Asian citrus psyllid, Diaphorina citri Kuwayama (Hemiptera: Liviidae), is an important insect pest of citrus. It is the vector of 'Candidatus' Liberibacter asiaticus, a phloem-limited bacterium that infects citrus, resulting in the disease Huanglongbing (HLB). Disease management relies heavily on suppression of D. citri populations with insecticides, including pyrethroids. In recent annual surveys to monitor insecticide resistance, reduced susceptibility to fenpropathrin was identified in several field populations of D. citri. The primary target of pyrethroids is the voltage-gated sodium channel (VGSC). The VGSC is prone to target-site insensitivity because of mutations that either reduce pyrethroid binding and/or alter gating kinetics. These mutations, known as knockdown resistance or kdr, have been reported in a wide diversity of arthropod species. Alternative splicing, in combination with kdr mutations, has been also associated with reduced pyrethroid efficacy. Here we report the molecular characterization of the VGSC in D. citri along with a survey of alternative splicing across developmental stages of this species. Previous studies demonstrated that D. citri has an exquisite enzymatic arsenal to detoxify insecticides resulting in reduced efficacy. The results from the current investigation demonstrate that target-site insensitivity is also a potential basis for insecticide resistance to pyrethroids in D. citri. The VGSC sequence and its molecular characterization should facilitate early elucidation of the underlying cause of an established case of resistance to pyrethroids. This is the first characterization of a VGSC from a hemipteran to this level of detail, with the majority of the previous studies on dipterans and lepidopterans. © 2015 Institute of Zoology, Chinese Academy of Sciences.

Cytoplasmic Domains and Voltage-Dependent Potassium Channel Gating

PubMed Central

Barros, Francisco; Domínguez, Pedro; de la Peña, Pilar

2012-01-01

The basic architecture of the voltage-dependent K+ channels (Kv channels) corresponds to a transmembrane protein core in which the permeation pore, the voltage-sensing components and the gating machinery (cytoplasmic facing gate and sensor–gate coupler) reside. Usually, large protein tails are attached to this core, hanging toward the inside of the cell. These cytoplasmic regions are essential for normal channel function and, due to their accessibility to the cytoplasmic environment, constitute obvious targets for cell-physiological control of channel behavior. Here we review the present knowledge about the molecular organization of these intracellular channel regions and their role in both setting and controlling Kv voltage-dependent gating properties. This includes the influence that they exert on Kv rapid/N-type inactivation and on activation/deactivation gating of Shaker-like and eag-type Kv channels. Some illustrative examples about the relevance of these cytoplasmic domains determining the possibilities for modulation of Kv channel gating by cellular components are also considered. PMID:22470342

Voltage-gated sodium channel expression and action potential generation in differentiated NG108-15 cells.

PubMed

Liu, Jinxu; Tu, Huiyin; Zhang, Dongze; Zheng, Hong; Li, Yu-Long

2012-10-25

The generation of action potential is required for stimulus-evoked neurotransmitter release in most neurons. Although various voltage-gated ion channels are involved in action potential production, the initiation of the action potential is mainly mediated by voltage-gated Na+ channels. In the present study, differentiation-induced changes of mRNA and protein expression of Na+ channels, Na+ currents, and cell membrane excitability were investigated in NG108-15 cells. Whole-cell patch-clamp results showed that differentiation (9 days) didn't change cell membrane excitability, compared to undifferentiated state. But differentiation (21 days) induced the action potential generation in 45.5% of NG108-15 cells (25/55 cells). In 9-day-differentiated cells, Na+ currents were mildly increased, which was also found in 21-day differentiated cells without action potential. In 21-day differentiated cells with action potential, Na+ currents were significantly enhanced. Western blot data showed that the expression of Na+ channels was increased with differentiated-time dependent manner. Single-cell real-time PCR data demonstrated that the expression of Na+ channel mRNA was increased by 21 days of differentiation in NG108-15 cells. More importantly, the mRNA level of Na+ channels in cells with action potential was higher than that in cells without action potential. Differentiation induces expression of voltage-gated Na+ channels and action potential generation in NG108-15 cells. A high level of the Na+ channel density is required for differentiation-triggered action potential generation.

Effects of Volatile Aromatic Anesthetics on Voltage-Gated Na+ Channels Expressed in Xenopus Oocytes

PubMed Central

Horishita, Takafumi; Eger, Edmond I; Harris, R. Adron

2008-01-01

Background Many inhaled anesthetics inhibit voltage-gated sodium channels at clinically relevant concentrations, and suppression of neurotransmitter release by these agents results, at least partly, from decreased presynaptic sodium channel activity. Volatile aromatic anesthetics can inhibit N-methyl-D-aspartate (NMDA) receptor function and enhance γ-amino butyric acid A (GABAA) receptor function, but these effects depend strongly on the chemical properties of the aromatic ompounds. The present study tested whether diverse aromatic anesthetics consistently inhibit sodium channel function. Methods We studied the effect of eight aromatic anesthetics on Nav1.2 sodium channels with β1 subunits, using whole-cell, two-electrode voltage-clamp techniques in Xenopus oocytes. Results All aromatic anesthetics inhibited INa (sodium currents) at a holding potential which produce half-maximal current (V1/2) (partial depolarization); inhibition was modest with 1,3,5-trifluorobenzene (8 ± 2%), pentafluorobenzene (13 ± 2%), and hexafluorobenzene (13 ± 2%), but greater with benzene (37 ± 2%), fluorobenzene (39 ± 2%), 1,2-difluorobenzene (48 ± 2%), 1,4-difluorobenzene (31 ± 3%), and 1,2,4-trifluorobenzene (33 ± 1%). Such dichotomous effects were noted by others for NMDA and GABAA receptors. Parallel, but much smaller inhibition, was found for INa at a holding potential which produced near maximal current (−90 mV) (VH-90), and hexafluorobenzene caused small (6 ± 1%) potentiation of this current. These changes in sodium channel function were correlated with effectiveness for inhibiting NMDA receptors, with lipid solubility of the compounds, with molecular volume, and with cation-π interactions. Conclusion Aromatic compounds vary in their actions on the kinetics of sodium channel gating and this may underlie their variable inhibition. The range of inhibition produced by MAC concentrations of inhaled anesthetics indicates that sodium channel inhibition may underlie the

Effect of gate bias sweep rate on the threshold voltage of in-plane gate nanowire transistor

NASA Astrophysics Data System (ADS)

Liu, H. X.; Li, J.; Tan, R. R.

2018-01-01

In2O3 nanowire electric-double-layer (EDL) transistors with in-plane gate gated by SiO2 solid-electrolyte are fabricated on transparent glass substrates. The gate voltage sweep rates can effectively modulate the threshold voltage (Vth) of nanowire device. Both depletion mode and enhancement mode are realized, and the Vth shift of the nanowire transistors is estimated to be 0.73V (without light). This phenomenon is due to increased adsorption of oxygen on the nanowire surface by the slower gate voltage sweep rates. Adsorbed oxygens capture electrons and cause a surface of nanowire channel was depleted. The operation voltage of transistor was 1.0 V, because the EDL gate dielectric can lead to high gate dielectric capacitance. These transparent in-plane gate nanowire transistors are promising for “see-through” nanoscale sensors.

Voltage-Gated Potassium Channels: A Structural Examination of Selectivity and Gating

PubMed Central

Kim, Dorothy M.; Nimigean, Crina M.

2016-01-01

Voltage-gated potassium channels play a fundamental role in the generation and propagation of the action potential. The discovery of these channels began with predictions made by early pioneers, and has culminated in their extensive functional and structural characterization by electrophysiological, spectroscopic, and crystallographic studies. With the aid of a variety of crystal structures of these channels, a highly detailed picture emerges of how the voltage-sensing domain reports changes in the membrane electric field and couples this to conformational changes in the activation gate. In addition, high-resolution structural and functional studies of K+ channel pores, such as KcsA and MthK, offer a comprehensive picture on how selectivity is achieved in K+ channels. Here, we illustrate the remarkable features of voltage-gated potassium channels and explain the mechanisms used by these machines with experimental data. PMID:27141052

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.

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

Redox regulation of neuronal voltage-gated calcium channels.

PubMed

Todorovic, Slobodan M; Jevtovic-Todorovic, Vesna

2014-08-20

Voltage-gated calcium channels are ubiquitously expressed in neurons and are key regulators of cellular excitability and synaptic transmitter release. There is accumulating evidence that multiple subtypes of voltage-gated calcium channels may be regulated by oxidation and reduction. However, the redox mechanisms involved in the regulation of channel function are not well understood. Several studies have established that both T-type and high-voltage-activated subtypes of voltage-gated calcium channel can be redox-regulated. This article reviews different mechanisms that can be involved in redox regulation of calcium channel function and their implication in neuronal function, particularly in pain pathways and thalamic oscillation. A current critical issue in the field is to decipher precise mechanisms of calcium channel modulation via redox reactions. In this review we discuss covalent post-translational modification via oxidation of cysteine molecules and chelation of trace metals, and reactions involving nitric oxide-related molecules and free radicals. Improved understanding of the roles of redox-based reactions in regulation of voltage-gated calcium channels may lead to improved understanding of novel redox mechanisms in physiological and pathological processes. Identification of redox mechanisms and sites on voltage-gated calcium channel may allow development of novel and specific ion channel therapies for unmet medical needs. Thus, it may be possible to regulate the redox state of these channels in treatment of pathological process such as epilepsy and neuropathic pain.

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.

Charge movement in gating-locked HCN channels reveals weak coupling of voltage sensors and gate.

PubMed

Ryu, Sujung; Yellen, Gary

2012-11-01

HCN (hyperpolarization-activated cyclic nucleotide gated) pacemaker channels have an architecture similar to that of voltage-gated K(+) channels, but they open with the opposite voltage dependence. HCN channels use essentially the same positively charged voltage sensors and intracellular activation gates as K(+) channels, but apparently these two components are coupled differently. In this study, we examine the energetics of coupling between the voltage sensor and the pore by using cysteine mutant channels for which low concentrations of Cd(2+) ions freeze the open-closed gating machinery but still allow the sensors to move. We were able to lock mutant channels either into open or into closed states by the application of Cd(2+) and measure the effect on voltage sensor movement. Cd(2+) did not immobilize the gating charge, as expected for strict coupling, but rather it produced shifts in the voltage dependence of voltage sensor charge movement, consistent with its effect of confining transitions to either closed or open states. From the magnitude of the Cd(2+)-induced shifts, we estimate that each voltage sensor produces a roughly three- to sevenfold effect on the open-closed equilibrium, corresponding to a coupling energy of ∼1.3-2 kT per sensor. Such coupling is not only opposite in sign to the coupling in K(+) channels, but also much weaker.

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.

Molecular mechanism of voltage sensing in voltage-gated proton channels

PubMed Central

Rebolledo, Santiago; Perez, Marta E.

2013-01-01

Voltage-gated proton (Hv) channels play an essential role in phagocytic cells by generating a hyperpolarizing proton current that electrically compensates for the depolarizing current generated by the NADPH oxidase during the respiratory burst, thereby ensuring a sustained production of reactive oxygen species by the NADPH oxidase in phagocytes to neutralize engulfed bacteria. Despite the importance of the voltage-dependent Hv current, it is at present unclear which residues in Hv channels are responsible for the voltage activation. Here we show that individual neutralizations of three charged residues in the fourth transmembrane domain, S4, all reduce the voltage dependence of activation. In addition, we show that the middle S4 charged residue moves from a position accessible from the cytosolic solution to a position accessible from the extracellular solution, suggesting that this residue moves across most of the membrane electric field during voltage activation of Hv channels. Our results show for the first time that the charge movement of these three S4 charges accounts for almost all of the measured gating charge in Hv channels. PMID:23401575

Redox Regulation of Neuronal Voltage-Gated Calcium Channels

PubMed Central

Jevtovic-Todorovic, Vesna

2014-01-01

Abstract Significance: Voltage-gated calcium channels are ubiquitously expressed in neurons and are key regulators of cellular excitability and synaptic transmitter release. There is accumulating evidence that multiple subtypes of voltage-gated calcium channels may be regulated by oxidation and reduction. However, the redox mechanisms involved in the regulation of channel function are not well understood. Recent Advances: Several studies have established that both T-type and high-voltage-activated subtypes of voltage-gated calcium channel can be redox-regulated. This article reviews different mechanisms that can be involved in redox regulation of calcium channel function and their implication in neuronal function, particularly in pain pathways and thalamic oscillation. Critical Issues: A current critical issue in the field is to decipher precise mechanisms of calcium channel modulation via redox reactions. In this review we discuss covalent post-translational modification via oxidation of cysteine molecules and chelation of trace metals, and reactions involving nitric oxide-related molecules and free radicals. Improved understanding of the roles of redox-based reactions in regulation of voltage-gated calcium channels may lead to improved understanding of novel redox mechanisms in physiological and pathological processes. Future Directions: Identification of redox mechanisms and sites on voltage-gated calcium channel may allow development of novel and specific ion channel therapies for unmet medical needs. Thus, it may be possible to regulate the redox state of these channels in treatment of pathological process such as epilepsy and neuropathic pain. Antioxid. Redox Signal. 21, 880–891. PMID:24161125

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.

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.

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

In silico insight into voltage-gated sodium channel 1.7 inhibition for anti-pain drug discovery.

PubMed

Wang, Mingxing; Li, Wei; Wang, Ying; Song, Yongbo; Wang, Jian; Cheng, Maosheng

2018-05-18

Studies on human genetics have implicated the voltage-gated sodium channel Nav1.7 as an appealing target for the treatment of pain. In this study, we put forward a ligand-based pharmacophore for the first time, which was generated by a set of multiple chemical scaffolds including sulfonamide and non-sulfonamide derivatives and consisted of four chemical features: an aromatic ring, a hydrophobic group and two hydrogen acceptors. The active cavity was divided into three regions according to the properties of the amino acids surrounded and was used for the docking of 16 known active inhibitors. Four accurate docking methods were employed to analyze the ligand-protein interactions in our molecular simulation study. Combining pharmacophore model with docking results, an interaction model was obtained with four features that were consistent with one another, which was more powerful in illuminating the binding site. The research elucidated a valuable relationship between structure and activity, at the same time it proposed an accurate binding model that was instructive in the development of novel and potent Nav1.7 inhibitors in the future. Copyright © 2018 Elsevier Inc. All rights reserved.

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

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

Structure of the voltage-gated K⁺ channel Eag1 reveals an alternative voltage sensing mechanism.

PubMed

Whicher, Jonathan R; MacKinnon, Roderick

2016-08-12

Voltage-gated potassium (K(v)) channels are gated by the movement of the transmembrane voltage sensor, which is coupled, through the helical S4-S5 linker, to the potassium pore. We determined the single-particle cryo-electron microscopy structure of mammalian K(v)10.1, or Eag1, bound to the channel inhibitor calmodulin, at 3.78 angstrom resolution. Unlike previous K(v) structures, the S4-S5 linker of Eag1 is a five-residue loop and the transmembrane segments are not domain swapped, which suggest an alternative mechanism of voltage-dependent gating. Additionally, the structure and position of the S4-S5 linker allow calmodulin to bind to the intracellular domains and to close the potassium pore, independent of voltage-sensor position. The structure reveals an alternative gating mechanism for K(v) channels and provides a template to further understand the gating properties of Eag1 and related channels. Copyright © 2016, American Association for the Advancement of Science.

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.

Modulation of voltage-gated channel currents by harmaline and harmane.

PubMed

Splettstoesser, Frank; Bonnet, Udo; Wiemann, Martin; Bingmann, Dieter; Büsselberg, Dietrich

2005-01-01

Harmala alkaloids are endogenous substances, which are involved in neurodegenerative disorders such as M. Parkinson, but some of them also have neuroprotective effects in the nervous system. While several sites of action at the cellular level (e.g. benzodiazepine receptors, 5-HT and GABA(A) receptors) have been identified, there is no report on how harmala alkaloids interact with voltage-gated membrane channels. The aim of this study was to investigate the effects of harmaline and harmane on voltage-activated calcium- (I(Ca(V))), sodium- (I(Na(V))) and potassium (I(K(V)))-channel currents, using the whole-cell patch-clamp method with cultured dorsal root ganglion neurones of 3-week-old rats. Currents were elicited by voltage steps from the holding potential to different command potentials. Harmaline and harmane reduced I(Ca(V)), I(Na(V)) and I(K(V)) concentration-dependent (10-500 microM) over the voltage range tested. I(Ca(V)) was reduced with an IC(50) of 100.6 microM for harmaline and by a significantly lower concentration of 75.8 microM (P<0.001, t-test) for harmane. The Hill coefficient was close to 1. Threshold concentration was around 10 microM for both substances. The steady state of inhibition of I(Ca(V)) by harmaline or harmane was reached within several minutes. The action was not use-dependent and at least partly reversible. It was mainly due to a reduction in the sustained calcium channel current (I(Ca(L+N))), while the transient voltage-gated calcium channel current (I(Ca(T))) was only partially affected. We conclude that harmaline and harmane are modulators of I(Ca(V)) in vitro. This might be related to their neuroprotective effects.

Modulation of voltage-gated channel currents by harmaline and harmane

PubMed Central

Splettstoesser, Frank; Bonnet, Udo; Wiemann, Martin; Bingmann, Dieter; Büsselberg, Dietrich

2004-01-01

Harmala alkaloids are endogenous substances, which are involved in neurodegenerative disorders such as M. Parkinson, but some of them also have neuroprotective effects in the nervous system. While several sites of action at the cellular level (e.g. benzodiazepine receptors, 5-HT and GABAA receptors) have been identified, there is no report on how harmala alkaloids interact with voltage-gated membrane channels. The aim of this study was to investigate the effects of harmaline and harmane on voltage-activated calcium- (ICa(V)), sodium- (INa(V)) and potassium (IK(V))-channel currents, using the whole-cell patch-clamp method with cultured dorsal root ganglion neurones of 3-week-old rats. Currents were elicited by voltage steps from the holding potential to different command potentials. Harmaline and harmane reduced ICa(V), INa(V) and IK(V) concentration-dependent (10–500 μM) over the voltage range tested. ICa(V) was reduced with an IC50 of 100.6 μM for harmaline and by a significantly lower concentration of 75.8 μM (P<0.001, t-test) for harmane. The Hill coefficient was close to 1. Threshold concentration was around 10 μM for both substances. The steady state of inhibition of ICa(V) by harmaline or harmane was reached within several minutes. The action was not use dependent and at least partly reversible. It was mainly due to a reduction in the sustained calcium channel current (ICa(L+N)), while the transient voltage-gated calcium channel current (ICa(T)) was only partially affected. We conclude that harmaline and harmane are modulators of ICa(V) in vitro. This might be related to their neuroprotective effects. PMID:15644868

Structural Mechanism of Voltage-Dependent Gating in an Isolated Voltage-Sensing Domain

PubMed Central

Li, Qufei; Wanderling, Sherry; Paduch, Marcin; Medovoy, David; Singharoy, Abhishek; McGreevy, Ryan; Villalba-Galea, Carlos; Hulse, Raymond E.; Roux, Benoit; Schulten, Klaus; Kossiakoff, Anthony; Perozo, Eduardo

2014-01-01

SUMMARY The transduction of transmembrane electric fields into protein motion plays an essential role in the generation and propagation of cellular signals. Voltage-sensing domains (VSD) carry out these functions through reorientations of S4 helix with discrete gating charges. Here, crystal structures of the VSD from Ci-VSP were determined in both, active (Up) and resting (Down) conformations. The S4 undergoes a ~5 Å displacement along its main axis accompanied by a ~60o rotation, consistent with the helix-screw gating mechanism. This movement is stabilized by a change in countercharge partners in helices S1 and S3, generating an estimated net charge transfer of ~1 eo. Gating charges move relative to a “hydrophobic gasket” that electrically divides intra and extracellular compartments. EPR spectroscopy confirms the limited nature of S4 movement in a membrane environment. These results provide an explicit mechanism for voltage sensing and set the basis for electromechanical coupling in voltage-dependent cellular activities. PMID:24487958

Voltage-gated proton channels: what' next?

PubMed Central

DeCoursey, Thomas E

2008-01-01

This review is an attempt to identify and place in context some of the many questions about voltage-gated proton channels that remain unsolved. As the gene was identified only 2 years ago, the situation is very different than in fields where the gene has been known for decades. For the proton channel, most of the obvious and less obvious structure–function questions are still wide open. Remarkably, the proton channel protein strongly resembles the voltage-sensing domain of many voltage-gated ion channels, and thus offers a novel approach to study gating mechanisms. Another surprise is that the proton channel appears to function as a dimer, with two separate conduction pathways. A number of significant biological questions remain in dispute, unanswered, or in some cases, not yet asked. This latter deficit is ascribable to the intrinsic difficulty in evaluating the importance of one component in a complex system, and in addition, to the lack, until recently, of a means of performing an unambiguous lesion experiment, that is, of selectively eliminating the molecule in question. We still lack a potent, selective pharmacological inhibitor, but the identification of the gene has allowed the development of powerful new tools including proton channel antibodies, siRNA and knockout mice. PMID:18801839

Resurgent current of voltage-gated Na+ channels

PubMed Central

Lewis, Amanda H; Raman, Indira M

2014-01-01

Resurgent Na+ current results from a distinctive form of Na+ channel gating, originally identified in cerebellar Purkinje neurons. In these neurons, the tetrodotoxin-sensitive voltage-gated Na+ channels responsible for action potential firing have specialized mechanisms that reduce the likelihood that they accumulate in fast inactivated states, thereby shortening refractory periods and permitting rapid, repetitive, and/or burst firing. Under voltage clamp, step depolarizations evoke transient Na+ currents that rapidly activate and quickly decay, and step repolarizations elicit slower channel reopening, or a ‘resurgent’ current. The generation of resurgent current depends on a factor in the Na+ channel complex, probably a subunit such as NaVβ4 (Scn4b), which blocks open Na+ channels at positive voltages, competing with the fast inactivation gate, and unblocks at negative voltages, permitting recovery from an open channel block along with a flow of current. Following its initial discovery, resurgent Na+ current has been found in nearly 20 types of neurons. Emerging research suggests that resurgent current is preferentially increased in a variety of clinical conditions associated with altered cellular excitability. Here we review the biophysical, molecular and structural mechanisms of resurgent current and their relation to the normal functions of excitable cells as well as pathophysiology. PMID:25172941

Voltage-dependent gating and gating charge measurements in the Kv1.2 potassium channel

PubMed Central

Ishida, Itzel G.; Rangel-Yescas, Gisela E.; Carrasco-Zanini, Julia

2015-01-01

Much has been learned about the voltage sensors of ion channels since the x-ray structure of the mammalian voltage-gated potassium channel Kv1.2 was published in 2005. High resolution structural data of a Kv channel enabled the structural interpretation of numerous electrophysiological findings collected in various ion channels, most notably Shaker, and permitted the development of meticulous computational simulations of the activation mechanism. The fundamental premise for the structural interpretation of functional measurements from Shaker is that this channel and Kv1.2 have the same characteristics, such that correlation of data from both channels would be a trivial task. We tested these assumptions by measuring Kv1.2 voltage-dependent gating and charge per channel. We found that the Kv1.2 gating charge is near 10 elementary charges (eo), ∼25% less than the well-established 13–14 eo in Shaker. Next, we neutralized positive residues in the Kv1.2 S4 transmembrane segment to investigate the cause of the reduction of the gating charge and found that, whereas replacing R1 with glutamine decreased voltage sensitivity to ∼50% of the wild-type channel value, mutation of the subsequent arginines had a much smaller effect. These data are in marked contrast to the effects of charge neutralization in Shaker, where removal of the first four basic residues reduces the gating charge by roughly the same amount. In light of these differences, we propose that the voltage-sensing domains (VSDs) of Kv1.2 and Shaker might undergo the same physical movement, but the septum that separates the aqueous crevices in the VSD of Kv1.2 might be thicker than Shaker’s, accounting for the smaller Kv1.2 gating charge. PMID:25779871

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

PubMed

Li, Yang; Liu, Huihui; Xia, Mengdie; Gong, Haipeng

2016-01-01

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

Direct Interaction between the Voltage Sensors Produces Cooperative Sustained Deactivation in Voltage-gated H+ Channel Dimers*

PubMed Central

Okuda, Hiroko; Yonezawa, Yasushige; Takano, Yu; Okamura, Yasushi; Fujiwara, Yuichiro

2016-01-01

The voltage-gated H+ channel (Hv) is a voltage sensor domain-like protein consisting of four transmembrane segments (S1–S4). The native Hv structure is a homodimer, with the two channel subunits functioning cooperatively. Here we show that the two voltage sensor S4 helices within the dimer directly cooperate via a π-stacking interaction between Trp residues at the middle of each segment. Scanning mutagenesis showed that Trp situated around the original position provides the slow gating kinetics characteristic of the dimer's cooperativity. Analyses of the Trp mutation on the dimeric and monomeric channel backgrounds and analyses with tandem channel constructs suggested that the two Trp residues within the dimer are functionally coupled during Hv deactivation but are less so during activation. Molecular dynamics simulation also showed direct π-stacking of the two Trp residues. These results provide new insight into the cooperative function of voltage-gated channels, where adjacent voltage sensor helices make direct physical contact and work as a single unit according to the gating process. PMID:26755722

Voltage-Gated Lipid Ion Channels

PubMed Central

Blicher, Andreas; Heimburg, Thomas

2013-01-01

Synthetic lipid membranes can display channel-like ion conduction events even in the absence of proteins. We show here that these events are voltage-gated with a quadratic voltage dependence as expected from electrostatic theory of capacitors. To this end, we recorded channel traces and current histograms in patch-experiments on lipid membranes. We derived a theoretical current-voltage relationship for pores in lipid membranes that describes the experimental data very well when assuming an asymmetric membrane. We determined the equilibrium constant between closed and open state and the open probability as a function of voltage. The voltage-dependence of the lipid pores is found comparable to that of protein channels. Lifetime distributions of open and closed events indicate that the channel open distribution does not follow exponential statistics but rather power law behavior for long open times. PMID:23823188

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

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.

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.

Exploring the Structure of the Voltage-gated Na+ Channel by an Engineered Drug Access Pathway to the Receptor Site for Local Anesthetics*

PubMed Central

Lukacs, Peter; Gawali, Vaibhavkumar S.; Cervenka, Rene; Ke, Song; Koenig, Xaver; Rubi, Lena; Zarrabi, Touran; Hilber, Karlheinz; Stary-Weinzinger, Anna; Todt, Hannes

2014-01-01

Despite the availability of several crystal structures of bacterial voltage-gated Na+ channels, the structure of eukaryotic Na+ channels is still undefined. We used predictions from available homology models and crystal structures to modulate an external access pathway for the membrane-impermeant local anesthetic derivative QX-222 into the internal vestibule of the mammalian rNaV1.4 channel. Potassium channel-based homology models predict amino acid Ile-1575 in domain IV segment 6 to be in close proximity to Lys-1237 of the domain III pore-loop selectivity filter. The mutation K1237E has been shown previously to increase the diameter of the selectivity filter. We found that an access pathway for external QX-222 created by mutations of Ile-1575 was abolished by the additional mutation K1237E, supporting the notion of a close spatial relationship between sites 1237 and 1575. Crystal structures of bacterial voltage-gated Na+ channels predict that the side chain of rNaV1.4 Trp-1531 of the domain IV pore-loop projects into the space between domain IV segment 6 and domain III pore-loop and, therefore, should obstruct the putative external access pathway. Indeed, mutations W1531A and W1531G allowed for exceptionally rapid access of QX-222. In addition, W1531G created a second non-selective ion-conducting pore, bypassing the outer vestibule but probably merging into the internal vestibule, allowing for control by the activation gate. These data suggest a strong structural similarity between bacterial and eukaryotic voltage-gated Na+ channels. PMID:24947510

Mapping the interaction site for the tarantula toxin hainantoxin-IV (β-TRTX-Hn2a) in the voltage sensor module of domain II of voltage-gated sodium channels.

PubMed

Cai, Tianfu; Luo, Ji; Meng, Er; Ding, Jiuping; Liang, Songping; Wang, Sheng; Liu, Zhonghua

2015-06-01

Peptide toxins often have pharmacological applications and are powerful tools for investigating the structure-function relationships of voltage-gated sodium channels (VGSCs). Although a group of potential VGSC inhibitors have been reported from tarantula venoms, little is known about the mechanism of their interaction with VGSCs. In this study, we showed that hainantoxin-IV (β-TRTX-Hn2a, HNTX-IV in brief), a 35-residue peptide from Ornithoctonus hainana venom, preferentially inhibited rNav1.2, rNav1.3 and hNav1.7 compared with rNav1.4 and hNav1.5. hNav1.7 was the most sensitive to HNTX-IV (IC50∼21nM). In contrast to many other tarantula toxins that affect VGSCs, HNTX-IV at subsaturating concentrations did not alter activation and inactivation kinetics in the physiological range of voltages, while very large depolarization above +70mV could partially activate toxin-bound hNav1.7 channel, indicating that HNTX-IV acts as a gating modifier rather than a pore blocker. Site-directed mutagenesis indicated that the toxin bound to site 4, which was located on the extracellular S3-S4 linker of hNav1.7 domain II. Mutants E753Q, D816N and E818Q of hNav1.7 decreased toxin affinity for hNav1.7 by 2.0-, 3.3- and 130-fold, respectively. In silico docking indicated that a three-toed claw substructure formed by residues with close contacts in the interface between HNTX-IV and hNav1.7 domain II stabilized the toxin-channel complex, impeding movement of the domain II voltage sensor and inhibiting hNav1.7 activation. Our data provide structural details for structure-based drug design and a useful template for the design of highly selective inhibitors of a specific subtype of VGSCs. Copyright © 2014 Elsevier Inc. All rights reserved.

Widespread Distribution of a Newly Found Point Mutation in Voltage-Gated Sodium Channel in Pyrethroid-Resistant Aedes aegypti Populations in Vietnam

PubMed Central

Kawada, Hitoshi; Higa, Yukiko; Komagata, Osamu; Kasai, Shinji; Tomita, Takashi; Thi Yen, Nguyen; Loan, Luu Lee; Sánchez, Rodrigo A. P.; Takagi, Masahiro

2009-01-01

Background Resistance of Aedes aegypti to photostable pyrethroid insecticides is a major problem for disease-vector control programs. Pyrethroids target the voltage-gated sodium channel on the insects' neurons. Single amino acid substitutions in this channel associated with pyrethroid resistance are one of the main factors that cause knockdown resistance in insects. Although kdr has been observed in several mosquito species, point mutations in the para gene have not been fully characterized in Ae. aegypti populations in Vietnam. The aim of this study was to determine the types and frequencies of mutations in the para gene in Ae. aegypti collected from used tires in Vietnam. Methods and Findings Several point mutations were examined that cause insensitivity of the voltage-gated sodium channel in the insect nervous system due to the replacement of the amino acids L1014F, the most commonly found point mutation in several mosquitoes; I1011M (or V) and V1016G (or I), which have been reported to be associated to knockdown resistance in Ae. aegypti located in segment 6, domain II; and a recently found amino acid replacement in F1269 in Ae. aegypti, located in segment 6, domain III. Among 756 larvae from 70 locations, no I1011M or I1011V nor L1014F mutations were found, and only two heterozygous V1016G mosquitoes were detected. However, F1269C mutations on domain III were distributed widely and with high frequency in 269 individuals among 757 larvae (53 collection sites among 70 locations surveyed). F1269C frequencies were low in the middle to north part of Vietnam but were high in the areas neighboring big cities and in the south of Vietnam, with the exception of the southern mountainous areas located at an elevation of 500–1000 m. Conclusions The overall percentage of homozygous F1269C seems to remain low (7.4%) in the present situation. However, extensive and uncontrolled frequent use of photostable pyrethroids might be a strong selection pressure for this mutation to

Sodium efflux from voltage clamped squid giant axons.

PubMed Central

Landowne, D

1977-01-01

1. The efflux of radioactive sodium was measured from squid axons during simultaneous voltage clamp experiments such that it was possible to determine the efflux of sodium associated with a measured voltage clamp current. 2. The extra efflux of sodium associated with voltage clamp pulses increased linearly with the magnitude of the depolarization above 40 mV. A 100 mV pulse of sufficient duration to produce all of the sodium current increased the rate constant of efflux by about 10(-6). 3. Application of 100 nM tetrodotoxin eliminated the sodium current and the extra efflux of radioactive sodium. 4. Cooling the axon increased the extra efflux/voltage clamp pulse slightly with a Q10 of 1/1-1. On the same axons cooling increased the integral of the sodium current with a Q10 of 1/1-4. 5. Replacing external sodium with Tris, dextrose or Mg-mannitol reduced the extra efflux of sodium by about 50%. The inward sodium current was replaced with an outward current as expected. 6. Replacing external sodium with lithium also reduced the extra efflux by about 50% but the currents seen in lithium were slightly larger than those in sodium. 7. The effect of replacing external sodium was not voltage dependent. Cooling reduced the effect so that there was less reduction of efflux on switching to Tris ASW in the cold than in the warm. 8. The extra efflux of sodium into sodium-free ASW is approximately the same as the integral of the sodium current. Adding external sodium produces a deviation from the independence principle such that there is more exchange of sodium than predicted. Such a deviation from prediction was noted by Hodgkin & Huxley (1952c). 9. Using the equations of Hodgkin & Huxley (1952c) modified to include the deviation from independence reported in this paper and its temperature dependence, one can predict the temperature dependence of the sodium efflux associated with action potentials and obtain much better agreement than is possibly without these phenomena. 10

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

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.

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.

Liquid-Solid Dual-Gate Organic Transistors with Tunable Threshold Voltage for Cell Sensing.

PubMed

Zhang, Yu; Li, Jun; Li, Rui; Sbircea, Dan-Tiberiu; Giovannitti, Alexander; Xu, Junling; Xu, Huihua; Zhou, Guodong; Bian, Liming; McCulloch, Iain; Zhao, Ni

2017-11-08

Liquid electrolyte-gated organic field effect transistors and organic electrochemical transistors have recently emerged as powerful technology platforms for sensing and simulation of living cells and organisms. For such applications, the transistors are operated at a gate voltage around or below 0.3 V because prolonged application of a higher voltage bias can lead to membrane rupturing and cell death. This constraint often prevents the operation of the transistors at their maximum transconductance or most sensitive regime. Here, we exploit a solid-liquid dual-gate organic transistor structure, where the threshold voltage of the liquid-gated conduction channel is controlled by an additional gate that is separated from the channel by a metal-oxide gate dielectric. With this design, the threshold voltage of the "sensing channel" can be linearly tuned in a voltage window exceeding 0.4 V. We have demonstrated that the dual-gate structure enables a much better sensor response to the detachment of human mesenchymal stem cells. In general, the capability of tuning the optimal sensing bias will not only improve the device performance but also broaden the material selection for cell-based organic bioelectronics.

Polysilicon Gate Enhancement of the Random Dopant Induced Threshold Voltage Fluctuations in Sub-100 nm MOSFET's with Ultrathin Gate Oxide

NASA Technical Reports Server (NTRS)

Asenov, Asen; Saini, Subhash

2000-01-01

In this paper, we investigate various aspects of the polysilicon gate influence on the random dopant induced threshold voltage fluctuations in sub-100 nm MOSFET's with ultrathin gate oxides. The study is done by using an efficient statistical three-dimensional (3-D) "atomistic" simulation technique described else-where. MOSFET's with uniform channel doping and with low doped epitaxial channels have been investigated. The simulations reveal that even in devices with a single crystal gate the gate depletion and the random dopants in it are responsible for a substantial fraction of the threshold voltage fluctuations when the gate oxide is scaled-in the range of 1-2 nm. Simulation experiments have been used in order to separate the enhancement in the threshold voltage fluctuations due to an effective increase in the oxide thickness associated with the gate depletion from the direct influence of the random dopants in the gate depletion layer. The results of the experiments show that the both factors contribute to the enhancement of the threshold voltage fluctuations, but the effective increase in the oxide-thickness has a dominant effect in the investigated range of devices. Simulations illustrating the effect or the polysilicon grain boundaries on the threshold voltage variation are also presented.

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

Effect of gate voltage polarity on the ionic liquid gating behavior of NdNiO 3/NdGaO 3 heterostructures

DOE PAGES

Dong, Yongqi; Xu, Haoran; Luo, Zhenlin; ...

2017-05-16

The effect of gate voltage polarity on the behavior of NdNiO 3 epitaxial thin films during ionic liquid gating is studied using in situ synchrotron X-ray techniques. We show that while negative biases have no discernible effect on the structure or composition of the films, large positive gate voltages result in the injection of a large concentration of oxygen vacancies (similar to 3%) and pronounced lattice expansion (0.17%) in addition to a 1000-fold increase in sheet resistance at room temperature. Despite the creation of large defect densities, the heterostructures exhibit a largely reversible switching behavior when sufficient time is providedmore » for the vacancies to migrate in and out of the thin film surface. The results confirm that electrostatic gating takes place at negative gate voltages for p-type complex oxides while positive voltages favor the electrochemical reduction of Ni 3+. Switching between positive and negative gate voltages therefore involves a combination of electronic and ionic doping processes that may be utilized in future electrochemical transistors.« less

Modulation of BK channel voltage gating by different auxiliary β subunits

PubMed Central

Contreras, Gustavo F.; Neely, Alan; Alvarez, Osvaldo; Gonzalez, Carlos; Latorre, Ramon

2012-01-01

Calcium- and voltage-activated potassium channels (BK) are regulated by a multiplicity of signals. The prevailing view is that different BK gating mechanisms converge to determine channel opening and that these gating mechanisms are allosterically coupled. In most instances the pore forming α subunit of BK is associated with one of four alternative β subunits that appear to target specific gating mechanisms to regulate the channel activity. In particular, β1 stabilizes the active configuration of the BK voltage sensor having a large effect on BK Ca2+ sensitivity. To determine the extent to which β subunits regulate the BK voltage sensor, we measured gating currents induced by the pore-forming BK α subunit alone and with the different β subunits expressed in Xenopus oocytes (β1, β2IR, β3b, and β4). We found that β1, β2, and β4 stabilize the BK voltage sensor in the active conformation. β3 has no effect on voltage sensor equilibrium. In addition, β4 decreases the apparent number of charges per voltage sensor. The decrease in the charge associated with the voltage sensor in α β4 channels explains most of their biophysical properties. For channels composed of the α subunit alone, gating charge increases slowly with pulse duration as expected if a significant fraction of this charge develops with a time course comparable to that of K+ current activation. In the presence of β1, β2, and β4 this slow component develops in advance of and much more rapidly than ion current activation, suggesting that BK channel opening proceeds in two steps. PMID:23112204

Mechanism of voltage-gated channel formation in lipid membranes.

PubMed

Guidelli, Rolando; Becucci, Lucia

2016-04-01

Although several molecular models for voltage-gated ion channels in lipid membranes have been proposed, a detailed mechanism accounting for the salient features of experimental data is lacking. A general treatment accounting for peptide dipole orientation in the electric field and their nucleation and growth kinetics with ion channel formation is provided. This is the first treatment that explains all the main features of the experimental current-voltage curves of peptides forming voltage-gated channels available in the literature. It predicts a regime of weakly voltage-dependent conductance, followed by one of strong voltage-dependent conductance at higher voltages. It also predicts values of the parameters expressing the exponential dependence of conductance upon voltage and peptide bulk concentration for both regimes, in good agreement with those reported in the literature. Most importantly, the only two adjustable parameters involved in the kinetics of nucleation and growth of ion channels can be varied over broad ranges without affecting the above predictions to a significant extent. Thus, the fitting of experimental current-voltage curves stems naturally from the treatment and depends only slightly upon the choice of the kinetic parameters. Copyright © 2015 Elsevier B.V. All rights reserved.

Voltage Gating of Shaker K+ Channels

PubMed Central

Rodríguez, Beatriz M.; Sigg, Daniel; Bezanilla, Francisco

1998-01-01

Ionic (Ii) and gating currents (Ig) from noninactivating Shaker H4 K+ channels were recorded with the cut-open oocyte voltage clamp and macropatch techniques. Steady state and kinetic properties were studied in the temperature range 2–22°C. The time course of Ii elicited by large depolarizations consists of an initial delay followed by an exponential rise with two kinetic components. The main Ii component is highly temperature dependent (Q10 > 4) and mildly voltage dependent, having a valence times the fraction of electric field (z) of 0.2–0.3 eo. The Ig On response obtained between −60 and 20 mV consists of a rising phase followed by a decay with fast and slow kinetic components. The main Ig component of decay is highly temperature dependent (Q10 > 4) and has a z between 1.6 and 2.8 eo in the voltage range from −60 to −10 mV, and ∼0.45 eo at more depolarized potentials. After a pulse to 0 mV, a variable recovery period at −50 mV reactivates the gating charge with a high temperature dependence (Q10 > 4). In contrast, the reactivation occurring between −90 and −50 mV has a Q10 = 1.2. Fluctuation analysis of ionic currents reveals that the open probability decreases 20% between 18 and 8°C and the unitary conductance has a low temperature dependence with a Q10 of 1.44. Plots of conductance and gating charge displacement are displaced to the left along the voltage axis when the temperature is decreased. The temperature data suggests that activation consists of a series of early steps with low enthalpic and negative entropic changes, followed by at least one step with high enthalpic and positive entropic changes, leading to final transition to the open state, which has a negative entropic change. PMID:9689029

Benchmarking the stability of human detergent-solubilised voltage-gated sodium channels for structural studies using eel as a reference

PubMed Central

Slowik, Daria; Henderson, Richard

2015-01-01

With the ultimate goal of detailed structural analysis of mammalian and particularly human voltage-gated sodium channels (VGSCs), we have investigated the relative stability of human and rat VGSCs and compared them with electric eel VGSC. We found that NaV1.3 from rat was the most stable after detergent solubilisation. The order of stability was rNaV1.3 > hNaV1.2 > hNaV1.1 > hNaV1.6 > hNaV1.3 > hNaV1.4. However, a comparison with the VGSC from Electrophorus electricus, which is most similar to NaV1.4, shows that the eel VGSC is considerably more stable in detergent than the human VGSCs examined. We conclude that current methods of structural analysis, such as single particle electron cryomicroscopy (cryoEM), may be most usefully targeted to eel VGSC or rNaV1.3, but that structural analysis on the full spectrum of VGSCs, by methods that require greater stability such as crystallisation and X-ray crystallography, will require further stabilisation of the channel. PMID:25838126

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.

The cooperative voltage sensor motion that gates a potassium channel.

PubMed

Pathak, Medha; Kurtz, Lisa; Tombola, Francesco; Isacoff, Ehud

2005-01-01

The four arginine-rich S4 helices of a voltage-gated channel move outward through the membrane in response to depolarization, opening and closing gates to generate a transient ionic current. Coupling of voltage sensing to gating was originally thought to operate with the S4s moving independently from an inward/resting to an outward/activated conformation, so that when all four S4s are activated, the gates are driven to open or closed. However, S4 has also been found to influence the cooperative opening step (Smith-Maxwell et al., 1998a), suggesting a more complex mechanism of coupling. Using fluorescence to monitor structural rearrangements in a Shaker channel mutant, the ILT channel (Ledwell and Aldrich, 1999), that energetically isolates the steps of activation from the cooperative opening step, we find that opening is accompanied by a previously unknown and cooperative movement of S4. This gating motion of S4 appears to be coupled to the internal S6 gate and to two forms of slow inactivation. Our results suggest that S4 plays a direct role in gating. While large transmembrane rearrangements of S4 may be required to unlock the gating machinery, as proposed before, it appears to be the gating motion of S4 that drives the gates to open and close.

The Cooperative Voltage Sensor Motion that Gates a Potassium Channel

PubMed Central

Pathak, Medha; Kurtz, Lisa; Tombola, Francesco; Isacoff, Ehud

2005-01-01

The four arginine-rich S4 helices of a voltage-gated channel move outward through the membrane in response to depolarization, opening and closing gates to generate a transient ionic current. Coupling of voltage sensing to gating was originally thought to operate with the S4s moving independently from an inward/resting to an outward/activated conformation, so that when all four S4s are activated, the gates are driven to open or closed. However, S4 has also been found to influence the cooperative opening step (Smith-Maxwell et al., 1998a), suggesting a more complex mechanism of coupling. Using fluorescence to monitor structural rearrangements in a Shaker channel mutant, the ILT channel (Ledwell and Aldrich, 1999), that energetically isolates the steps of activation from the cooperative opening step, we find that opening is accompanied by a previously unknown and cooperative movement of S4. This gating motion of S4 appears to be coupled to the internal S6 gate and to two forms of slow inactivation. Our results suggest that S4 plays a direct role in gating. While large transmembrane rearrangements of S4 may be required to unlock the gating machinery, as proposed before, it appears to be the gating motion of S4 that drives the gates to open and close. PMID:15623895

Free energy dissipation of the spontaneous gating of a single voltage-gated potassium channel.

PubMed

Wang, Jia-Zeng; Wang, Rui-Zhen

2018-02-01

Potassium channels mainly contribute to the resting potential and re-polarizations, with the potassium electrochemical gradient being maintained by the pump Na + /K + -ATPase. In this paper, we construct a stochastic model mimicking the kinetics of a potassium channel, which integrates temporal evolving of the membrane voltage and the spontaneous gating of the channel. Its stationary probability density functions (PDFs) are found to be singular at the boundaries, which result from the fact that the evolving rates of voltage are greater than the gating rates of the channel. We apply PDFs to calculate the power dissipations of the potassium current, the leakage, and the gating currents. On a physical perspective, the essential role of the system is the K + -battery charging the leakage (L-)battery. A part of power will inevitably be dissipated among the process. So, the efficiency of energy transference is calculated.

Free energy dissipation of the spontaneous gating of a single voltage-gated potassium channel

NASA Astrophysics Data System (ADS)

Wang, Jia-Zeng; Wang, Rui-Zhen

2018-02-01

Potassium channels mainly contribute to the resting potential and re-polarizations, with the potassium electrochemical gradient being maintained by the pump Na+/K+-ATPase. In this paper, we construct a stochastic model mimicking the kinetics of a potassium channel, which integrates temporal evolving of the membrane voltage and the spontaneous gating of the channel. Its stationary probability density functions (PDFs) are found to be singular at the boundaries, which result from the fact that the evolving rates of voltage are greater than the gating rates of the channel. We apply PDFs to calculate the power dissipations of the potassium current, the leakage, and the gating currents. On a physical perspective, the essential role of the system is the K+-battery charging the leakage (L-)battery. A part of power will inevitably be dissipated among the process. So, the efficiency of energy transference is calculated.

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.

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

The isolated voltage sensing domain of the Shaker potassium channel forms a voltage-gated cation channel.

PubMed

Zhao, Juan; Blunck, Rikard

2016-10-06

Domains in macromolecular complexes are often considered structurally and functionally conserved while energetically coupled to each other. In the modular voltage-gated ion channels the central ion-conducting pore is surrounded by four voltage sensing domains (VSDs). Here, the energetic coupling is mediated by interactions between the S4-S5 linker, covalently linking the domains, and the proximal C-terminus. In order to characterize the intrinsic gating of the voltage sensing domain in the absence of the pore domain, the Shaker Kv channel was truncated after the fourth transmembrane helix S4 (Shaker-iVSD). Shaker-iVSD showed significantly altered gating kinetics and formed a cation-selective ion channel with a strong preference for protons. Ion conduction in Shaker-iVSD developed despite identical primary sequence, indicating an allosteric influence of the pore domain. Shaker-iVSD also displays pronounced 'relaxation'. Closing of the pore correlates with entry into relaxation suggesting that the two processes are energetically related.

Elevated Neuronal Excitability Due to Modulation of the Voltage-Gated Sodium Channel Nav1.6 by Aβ1−42

PubMed Central

Wang, Xi; Zhang, Xiao-Gang; Zhou, Ting-Ting; Li, Na; Jang, Chun-Yan; Xiao, Zhi-Cheng; Ma, Quan-Hong; Li, Shao

2016-01-01

Aberrant increases in neuronal network excitability may contribute to the cognitive deficits in Alzheimer's disease (AD). However, the mechanisms underlying hyperexcitability are not fully understood. Such overexcitation of neuronal networks has been detected in the brains of APP/PS1 mice. In the present study, using current-clamp recording techniques, we observed that 12 days in vitro (DIV) primary cultured pyramidal neurons from P0 APP/PS1 mice exhibited a more prominent action potential burst and a lower threshold than WT littermates. Moreover, after treatment with Aβ1−42 peptide, 12 DIV primary cultured neurons showed similar changes, to a greater degree than in controls. Voltage-clamp recordings revealed that the voltage-dependent sodium current density of neurons incubated with Aβ1−42 was significantly increased, without change in the voltage-dependent sodium channel kinetic characteristics. Immunohistochemistry and western blot results showed that, after treatment with Aβ1−42, expressions of Nav and Nav1.6 subtype increased in cultured neurons or APP/PS1 brains compared to control groups. The intrinsic neuronal hyperexcitability of APP/PS1 mice might thus be due to an increased expression of voltage-dependent sodium channels induced by Aβ1−42. These results may illuminate the mechanism of aberrant neuronal networks in AD. PMID:27013956

Identification of an HV 1 voltage-gated proton channel in insects.

PubMed

Chaves, Gustavo; Derst, Christian; Franzen, Arne; Mashimo, Yuta; Machida, Ryuichiro; Musset, Boris

2016-04-01

The voltage-gated proton channel 1 (HV 1) is an important component of the cellular proton extrusion machinery and is essential for charge compensation during the respiratory burst of phagocytes. HV 1 has been identified in a wide range of eukaryotes throughout the animal kingdom, with the exception of insects. Therefore, it has been proposed that insects do not possess an HV 1 channel. In the present study, we report the existence of an HV 1-type proton channel in insects. We searched insect transcriptome shotgun assembly (TSA) sequence databases and found putative HV 1 orthologues in various polyneopteran insects. To confirm that these putative HV 1 orthologues were functional channels, we studied the HV 1 channel of Nicoletia phytophila (NpHV 1), an insect of the Zygentoma order, in more detail. NpHV 1 comprises 239 amino acids and is 33% identical to the human voltage-gated proton channel 1. Patch clamp measurements in a heterologous expression system showed proton selectivity, as well as pH- and voltage-dependent gating. Interestingly, NpHV 1 shows slightly enhanced pH-dependent gating compared to the human channel. Mutations in the first transmembrane segment at position 66 (Asp66), the presumed selectivity filter, lead to a loss of proton-selective conduction, confirming the importance of this aspartate residue in voltage-gated proton channels. Nucleotide sequence data have been deposited in the GenBank database under accession number KT780722. © 2016 Federation of European Biochemical Societies.

The voltage-sensor quartet

PubMed Central

Bankston, J. R.; Kass, R. S.

2009-01-01

Decoding the workings of voltage-gated sodium channels is crucial because their mutation leads to severe disease and their activity is modulated by toxins and drugs. An innovative approach now allows such investigations. PMID:19005542

The natural scorpion peptide, BmK NT1 activates voltage-gated sodium channels and produces neurotoxicity in primary cultured cerebellar granule cells.

PubMed

Zou, Xiaohan; He, Yuwei; Qiao, Jinping; Zhang, Chunlei; Cao, Zhengyu

2016-01-01

The scorpion Buthus martensii Karsch has been used in Traditional Chinese Medicine to treat neuronal diseases such as neuropathic pain, paralysis and epilepsy for thousands of years. Studies have demonstrated that scorpion venom is the primary active component. Although scorpion venom can effectively attenuate pain in the clinic, it also produces neurotoxic response. In this study, toxicity guided purification led to identify a mammalian toxin termed BmK NT1 comprising of 65 amino acid residues and an amidated C-terminus, a mature peptide encoded by the nucleotide sequence (GenBank No. AF464898). In contract to the recombinant product of the same nucleotide sequence, BmK AGAP, which displayed analgesic and anti-tumor effect, intravenous injection (i.v.) of BmK NT1 produced acute toxicity in mice with an LD50 value of 1.36 mg/kg. In primary cultured cerebellar granule cells, BmK NT1 produced a concentration-dependent cell death with an IC50 value of 0.65 μM (0.41-1.03 μM, 95% Confidence Intervals, 95% CI) which was abolished by TTX, a voltage-gated sodium channel (VGSC) blocker. We also demonstrated that BmK NT1 produced modest sodium influx in cerebellar granule cell cultures with an EC50 value of 2.19 μM (0.76-6.40 μM, 95% CI), an effect similar to VGSC agonist, veratridine. The sodium influx response was abolished by TTX suggesting that BmK NT1-induced sodium influx is solely through activation of VGSC. Considered these data together, we demonstrated that BmK NT1 activated VGSC and produced neurotoxicity in cerebellar granule cell cultures. Copyright © 2015 Elsevier Ltd. All rights reserved.

Gating currents from Kv7 channels carrying neuronal hyperexcitability mutations in the voltage-sensing domain.

PubMed

Miceli, Francesco; Vargas, Ernesto; Bezanilla, Francisco; Taglialatela, Maurizio

2012-03-21

Changes in voltage-dependent gating represent a common pathogenetic mechanism for genetically inherited channelopathies, such as benign familial neonatal seizures or peripheral nerve hyperexcitability caused by mutations in neuronal K(v)7.2 channels. Mutation-induced changes in channel voltage dependence are most often inferred from macroscopic current measurements, a technique unable to provide a detailed assessment of the structural rearrangements underlying channel gating behavior; by contrast, gating currents directly measure voltage-sensor displacement during voltage-dependent gating. In this work, we describe macroscopic and gating current measurements, together with molecular modeling and molecular-dynamics simulations, from channels carrying mutations responsible for benign familial neonatal seizures and/or peripheral nerve hyperexcitability; K(v)7.4 channels, highly related to K(v)7.2 channels both functionally and structurally, were used for these experiments. The data obtained showed that mutations affecting charged residues located in the more distal portion of S(4) decrease the stability of the open state and the active voltage-sensing domain configuration but do not directly participate in voltage sensing, whereas mutations affecting a residue (R4) located more proximally in S(4) caused activation of gating-pore currents at depolarized potentials. These results reveal that distinct molecular mechanisms underlie the altered gating behavior of channels carrying disease-causing mutations at different voltage-sensing domain locations, thereby expanding our current view of the pathogenesis of neuronal hyperexcitability diseases. Copyright © 2012 Biophysical Society. Published by Elsevier Inc. All rights reserved.

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

Molecular modelling studies of kdr mutations in voltage gated sodium channel revealed significant conformational variations contributing to insecticide resistance.

PubMed

Yellapu, Nanda Kumar; Gopal, Jeyakodi; Kasinathan, Gunasekaran; Purushothaman, Jambulingam

2018-06-01

Voltage gated sodium channels (VGSC) of mosquito vectors are the primary targets of dichlorodiphenyltrichloroethane (DDT) and other synthetic pyrethroids used in public health programmes. The knockdown resistant (kdr) mutations in VGSC are associated with the insecticide resistance especially in Anophelines. The present study is aimed to emphasize and demarcate the impact of three kdr-mutations such as L1014S, L1014F and L1014H on insecticide resistance. The membrane model of sodium transport domain of VGSC (STD-VGSC) was constructed using de novo approach based on domain and trans-membrane predictions. The comparative molecular modelling studies of wild type and mutant models of STD-VGSC revealed that L1014F mutant was observed to be near native to the wild type model in all the respects, but, L1014S and L1014H mutations showed drastic variations in the energy levels, root mean square fluctuations (RMSF) that resulted in conformational variations. The predicted binding sites also showed variable cavity volumes and RMSF in L1014S and L1014H mutants. Further, DDT also found be bound in near native manner to wild type in L1014F mutant and with variable orientation and affinities in L1014S and L1014H mutants. The variations and fluctuations observed in mutant structures explained that each mutation has its specific impact on the conformation of VGSC and its binding with DDT. The study provides new insights into the structure-function-correlations of mutant STD-VGSC structures and demonstrates the role and effects of kdr mutations on insecticide resistance in mosquito vectors.

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

Evaluation of a 125I-labelled benzazepinone derived voltage-gated sodium channel blocker for imaging with SPECT.

PubMed

Pérez-Medina, Carlos; Patel, Niral; Robson, Mathew; Badar, Adam; Lythgoe, Mark F; Årstad, Erik

2012-12-21

Voltage-gated sodium channels (VGSCs) are a family of transmembrane proteins that mediate fast neurotransmission, and are integral to sustain physiological conditions and higher cognitive functions. Imaging of VGSCs in vivo holds promise as a tool to elucidate operational functions in the brain and to aid the treatment of a wide range of neurological diseases. To assess the suitability of 1-benzazepin-2-one derived VGSC blockers for imaging, we have prepared a (125)I-labelled analogue of BNZA and evaluated the tracer in vivo. In an automated patch-clamp assay, a diastereomeric mixture of the non-radioactive compound blocked the Na(v)1.2 and Na(v)1.7 VGSC isoforms with IC(50) values of 4.1 ± 1.5 μM and 0.25 ± 0.07 μM, respectively. [(3)H]BTX displacement studies revealed a three-fold difference in affinity between the two diastereomers. Iodo-destannylation of a tin precursor with iodine-125 afforded the two diastereomerically pure tracers, which were used to assess binding to VGSCs in vivo by comparing their tissue distributions in mice. Whilst the results point to a lack of VGSC binding in vivo, SPECT imaging revealed highly localized uptake in the interscapular region, an area typically associated with brown adipose tissue, which in addition to high metabolic stability of the iodinated tracer, demonstrate the potential of 1-benzazepin-2-ones for in vivo imaging.

The isolated voltage sensing domain of the Shaker potassium channel forms a voltage-gated cation channel

PubMed Central

Zhao, Juan; Blunck, Rikard

2016-01-01

Domains in macromolecular complexes are often considered structurally and functionally conserved while energetically coupled to each other. In the modular voltage-gated ion channels the central ion-conducting pore is surrounded by four voltage sensing domains (VSDs). Here, the energetic coupling is mediated by interactions between the S4-S5 linker, covalently linking the domains, and the proximal C-terminus. In order to characterize the intrinsic gating of the voltage sensing domain in the absence of the pore domain, the Shaker Kv channel was truncated after the fourth transmembrane helix S4 (Shaker-iVSD). Shaker-iVSD showed significantly altered gating kinetics and formed a cation-selective ion channel with a strong preference for protons. Ion conduction in Shaker-iVSD developed despite identical primary sequence, indicating an allosteric influence of the pore domain. Shaker-iVSD also displays pronounced 'relaxation'. Closing of the pore correlates with entry into relaxation suggesting that the two processes are energetically related. DOI: http://dx.doi.org/10.7554/eLife.18130.001 PMID:27710769

Novel Mutations in the Voltage-Gated Sodium Channel of Pyrethroid-Resistant Varroa destructor Populations from the Southeastern USA

PubMed Central

González-Cabrera, Joel; Rodríguez-Vargas, Sonia; Davies, T. G. Emyr; Field, Linda M.; Schmehl, Daniel; Ellis, James D.; Krieger, Klemens; Williamson, Martin S.

2016-01-01

The parasitic mite Varroa destructor has a significant worldwide impact on bee colony health. In the absence of control measures, parasitized colonies invariably collapse within 3 years. The synthetic pyrethroids tau-fluvalinate and flumethrin have proven very effective at managing this mite within apiaries, but intensive control programs based mainly on one active ingredient have led to many reports of pyrethroid resistance. In Europe, a modification of leucine to valine at position 925 (L925V) of the V. destructor voltage-gated sodium channel was correlated with resistance, the mutation being found at high frequency exclusively in hives with a recent history of pyrethroid treatment. Here, we identify two novel mutations, L925M and L925I, in tau-fluvalinate resistant V. destructor collected at seven sites across Florida and Georgia in the Southeastern region of the USA. Using a multiplexed TaqMan® allelic discrimination assay, these mutations were found to be present in 98% of the mites surviving tau-fluvalinate treatment. The mutations were also found in 45% of the non-treated mites, suggesting a high potential for resistance evolution if selection pressure is applied. The results from a more extensive monitoring programme, using the Taqman® assay described here, would clearly help beekeepers with their decision making as to when to include or exclude pyrethroid control products and thereby facilitate more effective mite management programmes. PMID:27191597

Novel Mutations in the Voltage-Gated Sodium Channel of Pyrethroid-Resistant Varroa destructor Populations from the Southeastern USA.

PubMed

González-Cabrera, Joel; Rodríguez-Vargas, Sonia; Davies, T G Emyr; Field, Linda M; Schmehl, Daniel; Ellis, James D; Krieger, Klemens; Williamson, Martin S

2016-01-01

The parasitic mite Varroa destructor has a significant worldwide impact on bee colony health. In the absence of control measures, parasitized colonies invariably collapse within 3 years. The synthetic pyrethroids tau-fluvalinate and flumethrin have proven very effective at managing this mite within apiaries, but intensive control programs based mainly on one active ingredient have led to many reports of pyrethroid resistance. In Europe, a modification of leucine to valine at position 925 (L925V) of the V. destructor voltage-gated sodium channel was correlated with resistance, the mutation being found at high frequency exclusively in hives with a recent history of pyrethroid treatment. Here, we identify two novel mutations, L925M and L925I, in tau-fluvalinate resistant V. destructor collected at seven sites across Florida and Georgia in the Southeastern region of the USA. Using a multiplexed TaqMan® allelic discrimination assay, these mutations were found to be present in 98% of the mites surviving tau-fluvalinate treatment. The mutations were also found in 45% of the non-treated mites, suggesting a high potential for resistance evolution if selection pressure is applied. The results from a more extensive monitoring programme, using the Taqman® assay described here, would clearly help beekeepers with their decision making as to when to include or exclude pyrethroid control products and thereby facilitate more effective mite management programmes.

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.

Continuous adjustment of threshold voltage in carbon nanotube field-effect transistors through gate engineering

NASA Astrophysics Data System (ADS)

Zhong, Donglai; Zhao, Chenyi; Liu, Lijun; Zhang, Zhiyong; Peng, Lian-Mao

2018-04-01

In this letter, we report a gate engineering method to adjust threshold voltage of carbon nanotube (CNT) based field-effect transistors (FETs) continuously in a wide range, which makes the application of CNT FETs especially in digital integrated circuits (ICs) easier. Top-gated FETs are fabricated using solution-processed CNT network films with stacking Pd and Sc films as gate electrodes. By decreasing the thickness of the lower layer metal (Pd) from 20 nm to zero, the effective work function of the gate decreases, thus tuning the threshold voltage (Vt) of CNT FETs from -1.0 V to 0.2 V. The continuous adjustment of threshold voltage through gate engineering lays a solid foundation for multi-threshold technology in CNT based ICs, which then can simultaneously provide high performance and low power circuit modules on one chip.

Frequency of V1016I and F1534C mutations in the voltage-gated sodium channel gene in Aedes aegypti in Venezuela.

PubMed

Alvarez, Leslie C; Ponce, Gustavo; Saavedra-Rodriguez, Karla; Lopez, Beatriz; Flores, Adriana E

2015-06-01

The V1016I and F1534C mutations in the voltage-gated sodium channel gene have been associated with resistance to pyrethroids and DDT in Aedes aegypti mosquitoes. A study was carried out to determine the frequency of I1016 and C1534 by real-time PCR in five natural populations of Ae. aegypti in Venezuela during 2008, 2010 and 2012, as well as in a strain selected with 0.14 µg of deltamethrin for 15 generations. In natural populations, frequencies of I1016 varied between 0.01 and 0.37, and frequencies of C1534 between 0.35 and 1.0. In the Pampanito strain, the frequency of I1016 increased from 0.02 in F1 up to 0.5 in F15 and from 0.35 up to fixation for C1534 after selection with deltamethrin. The results showed that C1534 frequencies are higher than I1016 frequencies in natural populations of Ae. aegypti in Venezuela, and that deltamethrin selected the C1534 more rapidly than I1016. © 2014 Society of Chemical Industry.

A new mechanism of voltage-dependent gating exposed by KV10.1 channels interrupted between voltage sensor and pore.

PubMed

Tomczak, Adam P; Fernández-Trillo, Jorge; Bharill, Shashank; Papp, Ferenc; Panyi, Gyorgy; Stühmer, Walter; Isacoff, Ehud Y; Pardo, Luis A

2017-05-01

Voltage-gated ion channels couple transmembrane potential changes to ion flow. Conformational changes in the voltage-sensing domain (VSD) of the channel are thought to be transmitted to the pore domain (PD) through an α-helical linker between them (S4-S5 linker). However, our recent work on channels disrupted in the S4-S5 linker has challenged this interpretation for the KCNH family. Furthermore, a recent single-particle cryo-electron microscopy structure of K V 10.1 revealed that the S4-S5 linker is a short loop in this KCNH family member, confirming the need for an alternative gating model. Here we use "split" channels made by expression of VSD and PD as separate fragments to investigate the mechanism of gating in K V 10.1. We find that disruption of the covalent connection within the S4 helix compromises the ability of channels to close at negative voltage, whereas disconnecting the S4-S5 linker from S5 slows down activation and deactivation kinetics. Surprisingly, voltage-clamp fluorometry and MTS accessibility assays show that the motion of the S4 voltage sensor is virtually unaffected when VSD and PD are not covalently bound. Finally, experiments using constitutively open PD mutants suggest that the presence of the VSD is structurally important for the conducting conformation of the pore. Collectively, our observations offer partial support to the gating model that assumes that an inward motion of the C-terminal S4 helix, rather than the S4-S5 linker, closes the channel gate, while also suggesting that control of the pore by the voltage sensor involves more than one mechanism. © 2017 Tomczak et al.

A new mechanism of voltage-dependent gating exposed by KV10.1 channels interrupted between voltage sensor and pore

PubMed Central

Fernández-Trillo, Jorge; Bharill, Shashank; Panyi, Gyorgy; Stühmer, Walter; Isacoff, Ehud Y.

2017-01-01

Voltage-gated ion channels couple transmembrane potential changes to ion flow. Conformational changes in the voltage-sensing domain (VSD) of the channel are thought to be transmitted to the pore domain (PD) through an α-helical linker between them (S4–S5 linker). However, our recent work on channels disrupted in the S4–S5 linker has challenged this interpretation for the KCNH family. Furthermore, a recent single-particle cryo-electron microscopy structure of KV10.1 revealed that the S4–S5 linker is a short loop in this KCNH family member, confirming the need for an alternative gating model. Here we use “split” channels made by expression of VSD and PD as separate fragments to investigate the mechanism of gating in KV10.1. We find that disruption of the covalent connection within the S4 helix compromises the ability of channels to close at negative voltage, whereas disconnecting the S4–S5 linker from S5 slows down activation and deactivation kinetics. Surprisingly, voltage-clamp fluorometry and MTS accessibility assays show that the motion of the S4 voltage sensor is virtually unaffected when VSD and PD are not covalently bound. Finally, experiments using constitutively open PD mutants suggest that the presence of the VSD is structurally important for the conducting conformation of the pore. Collectively, our observations offer partial support to the gating model that assumes that an inward motion of the C-terminal S4 helix, rather than the S4–S5 linker, closes the channel gate, while also suggesting that control of the pore by the voltage sensor involves more than one mechanism. PMID:28360219

Voltage-Gated Calcium Channels

NASA Astrophysics Data System (ADS)

Zamponi, Gerald Werner

Voltage Gated Calcium Channels is the first comprehensive book in the calcium channel field, encompassing over thirty years of progress towards our understanding of calcium channel structure, function, regulation, physiology, pharmacology, and genetics. This book balances contributions from many of the leading authorities in the calcium channel field with fresh perspectives from risings stars in the area, taking into account the most recent literature and concepts. This is the only all-encompassing calcium channel book currently available, and is an essential resource for academic researchers at all levels in the areas neuroscience, biophysics, and cardiovascular sciences, as well as to researchers in the drug discovery area.

Method for voltage-gated protein fractionation

DOEpatents

Hatch, Anson [Tracy, CA; Singh, Anup K [Danville, CA

2012-04-24

We report unique findings on the voltage dependence of protein exclusion from the pores of nanoporous polymer exclusion membranes. The pores are small enough that proteins are excluded from passage with low applied electric fields, but increasing the field enables proteins to pass through. The requisite field necessary for a change in exclusion is protein-specific with a correlation to protein size. The field-dependence of exclusion is important to consider for preconcentration applications. The ability to selectively gate proteins at exclusion membranes is also a promising means for manipulating and characterizing proteins. We show that field-gated exclusion can be used to selectively remove proteins from a mixture, or to selectively trap protein at one exclusion membrane in a series.

Voltage-dependent K+ channel gating and voltage sensor toxin sensitivity depend on the mechanical state of the lipid membrane.

PubMed

Schmidt, Daniel; MacKinnon, Roderick

2008-12-09

Voltage-dependent K(+) (Kv) channels underlie action potentials through gating conformational changes that are driven by membrane voltage. In this study of the paddle chimera Kv channel, we demonstrate that the rate of channel opening, the voltage dependence of the open probability, and the maximum achievable open probability depend on the lipid membrane environment. The activity of the voltage sensor toxin VsTx1, which interferes with voltage-dependent gating by partitioning into the membrane and binding to the channel, also depends on the membrane. Membrane environmental factors that influence channel function are divisible into two general categories: lipid compositional and mechanical state. The mechanical state can have a surprisingly large effect on the function of a voltage-dependent K(+) channel, including its pharmacological interaction with voltage sensor toxins. The dependence of VSTx1 activity on the mechanical state of the membrane leads us to hypothesize that voltage sensor toxins exert their effect by perturbing the interaction forces that exist between the channel and the membrane.

Non-scaling behavior of electroosmotic flow in voltage-gated nanopores

DOE PAGES

Lian, Cheng; Gallegos, Alejandro; Liu, Honglai; ...

2016-11-17

Ionic transport through nanopores is of fundamental importance for the design and development of nanofiltration membranes and novel electrochemical devices including supercapacitors, fuel cells and batteries. Recent experiments have shown an unusual variation of electrical conductance with the pore size and the electrolyte parameters that defies conventional scaling relations. Here ionic transport through voltage-gated nanopores was studied by using the classical density functional theory for ion distributions in combination with the Navier–Stokes equation for the electroosmotic flow. We also identified a significant influence of the gating potential on the scaling behavior of the conductance with changes in the pore sizemore » and the salt concentration. Finally, for ion transport in narrow pores with a high gating voltage, the conductivity shows an oscillatory dependence on the pore size owing to the strong overlap of electric double layers.« less

Physical implication of transition voltage in organic nano-floating-gate nonvolatile memories

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

Wang, Shun; Gao, Xu, E-mail: wangsd@suda.edu.cn, E-mail: gaoxu@suda.edu.cn; Zhong, Ya-Nan

High-performance pentacene-based organic field-effect transistor nonvolatile memories, using polystyrene as a tunneling dielectric and Au nanoparticles as a nano-floating-gate, show parallelogram-like transfer characteristics with a featured transition point. The transition voltage at the transition point corresponds to a threshold electric field in the tunneling dielectric, over which stored electrons in the nano-floating-gate will start to leak out. The transition voltage can be modulated depending on the bias configuration and device structure. For p-type active layers, optimized transition voltage should be on the negative side of but close to the reading voltage, which can simultaneously achieve a high ON/OFF ratio andmore » good memory retention.« less

C-terminus-mediated voltage gating of Arabidopsis guard cell anion channel QUAC1.

PubMed

Mumm, Patrick; Imes, Dennis; Martinoia, Enrico; Al-Rasheid, Khaled A S; Geiger, Dietmar; Marten, Irene; Hedrich, Rainer

2013-09-01

Anion transporters in plants play a fundamental role in volume regulation and signaling. Currently, two plasma membrane-located anion channel families—SLAC/SLAH and ALMT—are known. Among the ALMT family, the root-expressed ALuminium-activated Malate Transporter 1 was identified by comparison of aluminum-tolerant and Al(3+)-sensitive wheat cultivars and was subsequently shown to mediate voltage-independent malate currents. In contrast, ALMT12/QUAC1 (QUickly activating Anion Channel1) is expressed in guard cells transporting malate in an Al(3+)-insensitive and highly voltage-dependent manner. So far, no information is available about the structure and mechanism of voltage-dependent gating with the QUAC1 channel protein. Here, we analyzed gating of QUAC1-type currents in the plasma membrane of guard cells and QUAC1-expressing oocytes revealing similar voltage dependencies and activation–deactivation kinetics. In the heterologous expression system, QUAC1 was electrophysiologically characterized at increasing extra- and intracellular malate concentrations. Thereby, malate additively stimulated the voltage-dependent QUAC1 activity. In search of structural determinants of the gating process, we could not identify transmembrane domains common for voltage-sensitive channels. However, site-directed mutations and deletions at the C-terminus of QUAC1 resulted in altered voltage-dependent channel activity. Interestingly, the replacement of a single glutamate residue, which is conserved in ALMT channels from different clades, by an alanine disrupted QUAC1 activity. Together with C- and N-terminal tagging, these results indicate that the cytosolic C-terminus is involved in the voltage-dependent gating mechanism of QUAC1.

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

High-throughput electrophysiological assays for voltage gated ion channels using SyncroPatch 768PE.

PubMed

Li, Tianbo; Lu, Gang; Chiang, Eugene Y; Chernov-Rogan, Tania; Grogan, Jane L; Chen, Jun

2017-01-01

Ion channels regulate a variety of physiological processes and represent an important class of drug target. Among the many methods of studying ion channel function, patch clamp electrophysiology is considered the gold standard by providing the ultimate precision and flexibility. However, its utility in ion channel drug discovery is impeded by low throughput. Additionally, characterization of endogenous ion channels in primary cells remains technical challenging. In recent years, many automated patch clamp (APC) platforms have been developed to overcome these challenges, albeit with varying throughput, data quality and success rate. In this study, we utilized SyncroPatch 768PE, one of the latest generation APC platforms which conducts parallel recording from two-384 modules with giga-seal data quality, to push these 2 boundaries. By optimizing various cell patching parameters and a two-step voltage protocol, we developed a high throughput APC assay for the voltage-gated sodium channel Nav1.7. By testing a group of Nav1.7 reference compounds' IC50, this assay was proved to be highly consistent with manual patch clamp (R > 0.9). In a pilot screening of 10,000 compounds, the success rate, defined by > 500 MΩ seal resistance and >500 pA peak current, was 79%. The assay was robust with daily throughput ~ 6,000 data points and Z' factor 0.72. Using the same platform, we also successfully recorded endogenous voltage-gated potassium channel Kv1.3 in primary T cells. Together, our data suggest that SyncroPatch 768PE provides a powerful platform for ion channel research and drug discovery.

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.

Voltage-gated currents in identified rat olfactory receptor neurons.

PubMed

Trombley, P Q; Westbrook, G L

1991-02-01

Whole-cell recording techniques were used to characterize voltage-gated membrane currents in neonatal rat olfactory receptor neurons (ORNs) in cell culture. Mature ORNs were identified in culture by their characteristic bipolar morphology, by retrograde labeling techniques, and by olfactory marker protein (OMP) immunoreactivity. ORNs did not have spontaneous activity, but fired action potentials to depolarizing current pulses. Action potentials were blocked by tetrodotoxin (TTX), which contrasts with the TTX-resistant action potentials in salamander olfactory receptor cells (e.g., Firestein and Werblin, 1987). Prolonged, suprathreshold current pulses evoked only a single action potential; however, repetitive firing up to 35 Hz could be elicited by a series of brief depolarizing pulses. Under voltage clamp, the TTX-sensitive sodium current had activation and inactivation properties similar to other excitable cells. In TTX and 20 mM barium, sustained inward current were evoked by voltage steps positive to -30 mV. This current was blocked by Cd (100 microM) and by nifedipine (IC50 = 368 nM) consistent with L-type calcium channels in other neurons. No T-type calcium current was observed. Voltage steps positive to -20 mV also evoked an outward current that did not inactivate during 100-msec depolarizations. Tail current analysis of this current was consistent with a selective potassium conductance. The outward current was blocked by external tetraethylammonium but was unaffected by Cd or 4-aminopyridine (4-AP) or by removal of external calcium. A transient outward current was not observed. The 3 voltage-dependent conductances in cultured rat ORNs appear to be sufficient for 2 essential functions: action potential generation and transmitter release. As a single odorant-activated channel can trigger an action potential (e.g., Lynch and Barry, 1989), the repetitive firing seen with brief depolarizing pulses suggests that ORNs do not integrate sensory input, but rather act

Charged Residues at the First Transmembrane Region Contribute to the Voltage Dependence of the Slow Gate of Connexins*

PubMed Central

Pinto, Bernardo I.; García, Isaac E.; Pupo, Amaury; Retamal, Mauricio A.; Martínez, Agustín D.; Latorre, Ramón; González, Carlos

2016-01-01

Connexins (Cxs) are a family of membrane-spanning proteins that form gap junction channels and hemichannels. Connexin-based channels exhibit two distinct voltage-dependent gating mechanisms termed slow and fast gating. Residues located at the C terminus of the first transmembrane segment (TM-1) are important structural components of the slow gate. Here, we determined the role of the charged residues at the end of TM-1 in voltage sensing in Cx26, Cx46, and Cx50. Conductance/voltage curves obtained from tail currents together with kinetics analysis reveal that the fast and slow gates of Cx26 involves the movement of two and four charges across the electric field, respectively. Primary sequence alignment of different Cxs shows the presence of well conserved glutamate residues in the C terminus of TM-1; only Cx26 contains a lysine in that position (lysine 41). Neutralization of lysine 41 in Cx26 increases the voltage dependence of the slow gate. Swapping of lysine 41 with glutamate 42 maintains the voltage dependence. In Cx46, neutralization of negative charges or addition of a positive charge in the Cx26 equivalent region reduced the slow gate voltage dependence. In Cx50, the addition of a glutamate in the same region decreased the voltage dependence, and the neutralization of a negative charge increased it. These results indicate that the charges at the end of TM-1 are part of the slow gate voltage sensor in Cxs. The fact that Cx42, which has no charge in this region, still presents voltage-dependent slow gating suggests that charges still unidentified also contribute to the slow gate voltage sensitivity. PMID:27143357

Dynamical characterization of inactivation path in voltage-gated Na+ ion channel by non-equilibrium response spectroscopy

PubMed Central

Pal, Krishnendu; Gangopadhyay, Gautam

2016-01-01

ABSTRACT Inactivation path of voltage gated sodium channel has been studied here under various voltage protocols as it is the main governing factor for the periodic occurrence and shape of the action potential. These voltage protocols actually serve as non-equilibrium response spectroscopic tools to study the ion channel in non-equilibrium environment. In contrast to a lot of effort in finding the crystal structure based molecular mechanism of closed-state(CSI) and open-state inactivation(OSI); here our approach is to understand the dynamical characterization of inactivation. The kinetic flux as well as energetic contribution of the closed and open- state inactivation path is compared here for voltage protocols, namely constant, pulsed and oscillating. The non-equilibrium thermodynamic quantities used in response to these voltage protocols serve as improved characterization tools for theoretical understanding which not only agrees with the previously known kinetic measurements but also predict the energetically optimum processes to sustain the auto-regulatory mechanism of action potential and the consequent inactivation steps needed. The time dependent voltage pattern governs the population of the conformational states which when couple with characteristic rate parameters, the CSI and OSI selectivity arise dynamically to control the inactivation path. Using constant, pulsed and continuous oscillating voltage protocols we have shown that during depolarization the OSI path is more favored path of inactivation however, in the hyper-polarized situation the CSI is favored. It is also shown that the re-factorisation of inactivated sodium channel to resting state occurs via CSI path. Here we have shown how the subtle energetic and entropic cost due to the change in the depolarization magnitude determines the optimum path of inactivation. It is shown that an efficient CSI and OSI dynamical profile in principle can characterize the open-state drug blocking phenomena. PMID

TPEN, a Specific Zn2+ Chelator, Inhibits Sodium Dithionite and Glucose Deprivation (SDGD)-Induced Neuronal Death by Modulating Apoptosis, Glutamate Signaling, and Voltage-Gated K+ and Na+ Channels.

PubMed

Zhang, Feng; Ma, Xue-Ling; Wang, Yu-Xiang; He, Cong-Cong; Tian, Kun; Wang, Hong-Gang; An, Di; Heng, Bin; Xie, Lai-Hua; Liu, Yan-Qiang

2017-03-01

Hypoxia-ischemia-induced neuronal death is an important pathophysiological process that accompanies ischemic stroke and represents a major challenge in preventing ischemic stroke. To elucidate factors related to and a potential preventative mechanism of hypoxia-ischemia-induced neuronal death, primary neurons were exposed to sodium dithionite and glucose deprivation (SDGD) to mimic hypoxic-ischemic conditions. The effects of N,N,N',N'-tetrakis (2-pyridylmethyl) ethylenediamine (TPEN), a specific Zn 2+ -chelating agent, on SDGD-induced neuronal death, glutamate signaling (including the free glutamate concentration and expression of α-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA) receptor (GluR2) and N-methyl-D-aspartate (NMDA) receptor subunits (NR2B), and voltage-dependent K + and Na + channel currents were also investigated. Our results demonstrated that TPEN significantly suppressed increases in cell death, apoptosis, neuronal glutamate release into the culture medium, NR2B protein expression, and I K as well as decreased GluR2 protein expression and Na + channel activity in primary cultured neurons exposed to SDGD. These results suggest that TPEN could inhibit SDGD-induced neuronal death by modulating apoptosis, glutamate signaling (via ligand-gated channels such as AMPA and NMDA receptors), and voltage-gated K + and Na + channels in neurons. Hence, Zn 2+ chelation might be a promising approach for counteracting the neuronal loss caused by transient global ischemia. Moreover, TPEN could represent a potential cell-targeted therapy.

Voltage-gated proton channel in a dinoflagellate

PubMed Central

Smith, Susan M. E.; Morgan, Deri; Musset, Boris; Cherny, Vladimir V.; Place, Allen R.; Hastings, J. Woodland; DeCoursey, Thomas E.

2011-01-01

Fogel and Hastings first hypothesized the existence of voltage-gated proton channels in 1972 in bioluminescent dinoflagellates, where they were thought to trigger the flash by activating luciferase. Proton channel genes were subsequently identified in human, mouse, and Ciona intestinalis, but their existence in dinoflagellates remained unconfirmed. We identified a candidate proton channel gene from a Karlodinium veneficum cDNA library based on homology with known proton channel genes. K. veneficum is a predatory, nonbioluminescent dinoflagellate that produces toxins responsible for fish kills worldwide. Patch clamp studies on the heterologously expressed gene confirm that it codes for a genuine voltage-gated proton channel, kHV1: it is proton-specific and activated by depolarization, its gH–V relationship shifts with changes in external or internal pH, and mutation of the selectivity filter (which we identify as Asp51) results in loss of proton-specific conduction. Indirect evidence suggests that kHV1 is monomeric, unlike other proton channels. Furthermore, kHV1 differs from all known proton channels in activating well negative to the Nernst potential for protons, EH. This unique voltage dependence makes the dinoflagellate proton channel ideally suited to mediate the proton influx postulated to trigger bioluminescence. In contrast to vertebrate proton channels, whose main function is acid extrusion, we propose that proton channels in dinoflagellates have fundamentally different functions of signaling and excitability. PMID:22006335

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.

Charged Residues at the First Transmembrane Region Contribute to the Voltage Dependence of the Slow Gate of Connexins.

PubMed

Pinto, Bernardo I; García, Isaac E; Pupo, Amaury; Retamal, Mauricio A; Martínez, Agustín D; Latorre, Ramón; González, Carlos

2016-07-22

Connexins (Cxs) are a family of membrane-spanning proteins that form gap junction channels and hemichannels. Connexin-based channels exhibit two distinct voltage-dependent gating mechanisms termed slow and fast gating. Residues located at the C terminus of the first transmembrane segment (TM-1) are important structural components of the slow gate. Here, we determined the role of the charged residues at the end of TM-1 in voltage sensing in Cx26, Cx46, and Cx50. Conductance/voltage curves obtained from tail currents together with kinetics analysis reveal that the fast and slow gates of Cx26 involves the movement of two and four charges across the electric field, respectively. Primary sequence alignment of different Cxs shows the presence of well conserved glutamate residues in the C terminus of TM-1; only Cx26 contains a lysine in that position (lysine 41). Neutralization of lysine 41 in Cx26 increases the voltage dependence of the slow gate. Swapping of lysine 41 with glutamate 42 maintains the voltage dependence. In Cx46, neutralization of negative charges or addition of a positive charge in the Cx26 equivalent region reduced the slow gate voltage dependence. In Cx50, the addition of a glutamate in the same region decreased the voltage dependence, and the neutralization of a negative charge increased it. These results indicate that the charges at the end of TM-1 are part of the slow gate voltage sensor in Cxs. The fact that Cx42, which has no charge in this region, still presents voltage-dependent slow gating suggests that charges still unidentified also contribute to the slow gate voltage sensitivity. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

Voltage-dependent K+ channel gating and voltage sensor toxin sensitivity depend on the mechanical state of the lipid membrane

PubMed Central

Schmidt, Daniel; MacKinnon, Roderick

2008-01-01

Voltage-dependent K+ (Kv) channels underlie action potentials through gating conformational changes that are driven by membrane voltage. In this study of the paddle chimera Kv channel, we demonstrate that the rate of channel opening, the voltage dependence of the open probability, and the maximum achievable open probability depend on the lipid membrane environment. The activity of the voltage sensor toxin VsTx1, which interferes with voltage-dependent gating by partitioning into the membrane and binding to the channel, also depends on the membrane. Membrane environmental factors that influence channel function are divisible into two general categories: lipid compositional and mechanical state. The mechanical state can have a surprisingly large effect on the function of a voltage-dependent K+ channel, including its pharmacological interaction with voltage sensor toxins. The dependence of VSTx1 activity on the mechanical state of the membrane leads us to hypothesize that voltage sensor toxins exert their effect by perturbing the interaction forces that exist between the channel and the membrane. PMID:19050073

Microscopic origin of gating current fluctuations in a potassium channel voltage sensor.

PubMed

Freites, J Alfredo; Schow, Eric V; White, Stephen H; Tobias, Douglas J

2012-06-06

Voltage-dependent ion channels open and close in response to changes in membrane electrical potential due to the motion of their voltage-sensing domains (VSDs). VSD charge displacements within the membrane electric field are observed in electrophysiology experiments as gating currents preceding ionic conduction. The elementary charge motions that give rise to the gating current cannot be observed directly, but appear as discrete current pulses that generate fluctuations in gating current measurements. Here we report direct observation of gating-charge displacements in an atomistic molecular dynamics simulation of the isolated VSD from the KvAP channel in a hydrated lipid bilayer on the timescale (10-μs) expected for elementary gating charge transitions. The results reveal that gating-charge displacements are associated with the water-catalyzed rearrangement of salt bridges between the S4 arginines and a set of conserved acidic side chains on the S1-S3 transmembrane segments in the hydrated interior of the VSD. Copyright © 2012 Biophysical Society. Published by Elsevier Inc. All rights reserved.

A mutation (L1014F) in the voltage-gated sodium channel of the grain aphid, Sitobion avenae, is associated with resistance to pyrethroid insecticides.

PubMed

Foster, Stephen P; Paul, Verity L; Slater, Russell; Warren, Anne; Denholm, Ian; Field, Linda M; Williamson, Martin S

2014-08-01

The grain aphid, Sitobion avenae Fabricius (Hemiptera: Aphididae), is an important pest of cereal crops. Pesticides are the main method for control but carry the risk of selecting for resistance. In response to reports of reduced efficacy of pyrethroid sprays applied to S. avenae, field samples were collected and screened for mutations in the voltage-gated sodium channel, the primary target site for pyrethroids. Aphid mobility and mortality to lambda-cyhalothrin were measured in coated glass vial bioassays. A single amino acid substitution (L1014F) was identified in the domain IIS6 segment of the sodium channel from the S. avenae samples exhibiting reduced pyrethroid efficacy. Bioassays on aphids heterozygous for the kdr mutation (SR) or homozygous for the wild-type allele (SS) showed that those carrying the mutation had significantly lower susceptibility to lambda-cyhalothrin. The L1014F (kdr) mutation, known to confer pyrethroid resistance in many insect pests, has been identified for the first time in S. avenae. Clonal lines heterozygous for the mutation showed 35-40-fold resistance to lambda-cyhalothrin in laboratory bioassays, consistent with the reported effect of this mutation on pyrethroid sensitivity in other aphid species. © 2013 Society of Chemical Industry.

Gating of Connexin Channels by transjunctional-voltage: Conformations and models of open and closed states.

PubMed

Bargiello, Thaddeus A; Oh, Seunghoon; Tang, Qingxiu; Bargiello, Nicholas K; Dowd, Terry L; Kwon, Taekyung

2018-01-01

Voltage is an important physiologic regulator of channels formed by the connexin gene family. Connexins are unique among ion channels in that both plasma membrane inserted hemichannels (undocked hemichannels) and intercellular channels (aggregates of which form gap junctions) have important physiological roles. The hemichannel is the fundamental unit of gap junction voltage-gating. Each hemichannel displays two distinct voltage-gating mechanisms that are primarily sensitive to a voltage gradient formed along the length of the channel pore (the transjunctional voltage) rather than sensitivity to the absolute membrane potential (V m or V i-o ). These transjunctional voltage dependent processes have been termed V j - or fast-gating and loop- or slow-gating. Understanding the mechanism of voltage-gating, defined as the sequence of voltage-driven transitions that connect open and closed states, first and foremost requires atomic resolution models of the end states. Although ion channels formed by connexins were among the first to be characterized structurally by electron microscopy and x-ray diffraction in the early 1980's, subsequent progress has been slow. Much of the current understanding of the structure-function relations of connexin channels is based on two crystal structures of Cx26 gap junction channels. Refinement of crystal structure by all-atom molecular dynamics and incorporation of charge changing protein modifications has resulted in an atomic model of the open state that arguably corresponds to the physiologic open state. Obtaining validated atomic models of voltage-dependent closed states is more challenging, as there are currently no methods to solve protein structure while a stable voltage gradient is applied across the length of an oriented channel. It is widely believed that the best approach to solve the atomic structure of a voltage-gated closed ion channel is to apply different but complementary experimental and computational methods and to use

Philosophy of voltage-gated proton channels

PubMed Central

DeCoursey, Thomas E.; Hosler, Jonathan

2014-01-01

In this review, voltage-gated proton channels are considered from a mainly teleological perspective. Why do proton channels exist? What good are they? Why did they go to such lengths to develop several unique hallmark properties such as extreme selectivity and ΔpH-dependent gating? Why is their current so minuscule? How do they manage to be so selective? What is the basis for our belief that they conduct H+ and not OH–? Why do they exist in many species as dimers when the monomeric form seems to work quite well? It is hoped that pondering these questions will provide an introduction to these channels and a way to logically organize their peculiar properties as well as to understand how they are able to carry out some of their better-established biological functions. PMID:24352668

Direct Evidence of Conformational Changes Associated with Voltage Gating in a Voltage Sensor Protein by Time-Resolved X-ray/Neutron Interferometry

PubMed Central

2015-01-01

The voltage sensor domain (VSD) of voltage-gated cation (e.g., Na+, K+) channels central to neurological signal transmission can function as a distinct module. When linked to an otherwise voltage-insensitive, ion-selective membrane pore, the VSD imparts voltage sensitivity to the channel. Proteins homologous with the VSD have recently been found to function themselves as voltage-gated proton channels or to impart voltage sensitivity to enzymes. Determining the conformational changes associated with voltage gating in the VSD itself in the absence of a pore domain thereby gains importance. We report the direct measurement of changes in the scattering-length density (SLD) profile of the VSD protein, vectorially oriented within a reconstituted phospholipid bilayer membrane, as a function of the transmembrane electric potential by time-resolved X-ray and neutron interferometry. The changes in the experimental SLD profiles for both polarizing and depolarizing potentials with respect to zero potential were found to extend over the entire length of the isolated VSD’s profile structure. The characteristics of the changes observed were in qualitative agreement with molecular dynamics simulations of a related membrane system, suggesting an initial interpretation of these changes in terms of the VSD’s atomic-level 3-D structure. PMID:24697545

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.

Low-voltage organic transistors on plastic comprising high-dielectric constant gate insulators

PubMed

Dimitrakopoulos; Purushothaman; Kymissis; Callegari; Shaw

1999-02-05

The gate bias dependence of the field-effect mobility in pentacene-based insulated gate field-effect transistors (IGFETs) was interpreted on the basis of the interaction of charge carriers with localized trap levels in the band gap. This understanding was used to design and fabricate IGFETs with mobility of more than 0.3 square centimeter per volt per second and current modulation of 10(5), with the use of amorphous metal oxide gate insulators. These values were obtained at operating voltage ranges as low as 5 volts, which are much smaller than previously reported results. An all-room-temperature fabrication process sequence was used, which enabled the demonstration of high-performance organic IGFETs on transparent plastic substrates, at low operating voltages for organic devices.

The voltage-sensing domain of a phosphatase gates the pore of a potassium channel.

PubMed

Arrigoni, Cristina; Schroeder, Indra; Romani, Giulia; Van Etten, James L; Thiel, Gerhard; Moroni, Anna

2013-03-01

The modular architecture of voltage-gated K(+) (Kv) channels suggests that they resulted from the fusion of a voltage-sensing domain (VSD) to a pore module. Here, we show that the VSD of Ciona intestinalis phosphatase (Ci-VSP) fused to the viral channel Kcv creates Kv(Synth1), a functional voltage-gated, outwardly rectifying K(+) channel. Kv(Synth1) displays the summed features of its individual components: pore properties of Kcv (selectivity and filter gating) and voltage dependence of Ci-VSP (V(1/2) = +56 mV; z of ~1), including the depolarization-induced mode shift. The degree of outward rectification of the channel is critically dependent on the length of the linker more than on its amino acid composition. This highlights a mechanistic role of the linker in transmitting the movement of the sensor to the pore and shows that electromechanical coupling can occur without coevolution of the two domains.

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

Voltage-dependent and -independent titration of specific residues accounts for complex gating of a ClC chloride channel by extracellular protons

PubMed Central

Niemeyer, María Isabel; Cid, L Pablo; Yusef, Yamil R; Briones, Rodolfo; Sepúlveda, Francisco V

2009-01-01

The ClC transport protein family comprises both Cl− ion channel and H+/Cl− and H+/NO3− exchanger members. Structural studies on a bacterial ClC transporter reveal a pore obstructed at its external opening by a glutamate side-chain which acts as a gate for Cl− passage and in addition serves as a staging post for H+ exchange. This same conserved glutamate acts as a gate to regulate Cl− flow in ClC channels. The activity of ClC-2, a genuine Cl− channel, has a biphasic response to extracellular pH with activation by moderate acidification followed by abrupt channel closure at pH values lower than ∼7. We have now investigated the molecular basis of this complex gating behaviour. First, we identify a sensor that couples extracellular acidification to complete closure of the channel. This is extracellularly-facing histidine 532 at the N-terminus of transmembrane helix Q whose neutralisation leads to channel closure in a cooperative manner. We go on to show that acidification-dependent activation of ClC-2 is voltage dependent and probably mediated by protonation of pore gate glutamate 207. Intracellular Cl− acts as a voltage-independent modulator, as though regulating the pKa of the protonatable residue. Our results suggest that voltage dependence of ClC-2 is given by hyperpolarisation-dependent penetration of protons from the extracellular side to neutralise the glutamate gate deep within the channel, which allows Cl− efflux. This is reminiscent of a partial exchanger cycle, suggesting that the ClC-2 channel evolved from its transporter counterparts. PMID:19153159

Low-voltage electric-double-layer paper transistors gated by microporous SiO2 processed at room temperature

NASA Astrophysics Data System (ADS)

Sun, Jia; Wan, Qing; Lu, Aixia; Jiang, Jie

2009-11-01

Battery drivable low-voltage SnO2-based paper thin-film transistors with a near-zero threshold voltage (Vth=0.06 V) gated by microporous SiO2 dielectric with electric-double-layer (EDL) effect are fabricated at room temperature. The operating voltage is found to be as low as 1.5 V due to the huge gate specific capacitance (1.34 μF/cm2 at 40 Hz) related to EDL formation. The subthreshold gate voltage swing and current on/off ratio is found to be 82 mV/decade and 2.0×105, respectively. The electron field-effect mobility is estimated to be 47.3 cm2/V s based on the measured gate specific capacitance at 40 Hz.

Voltage Gated Ion Channel Function: Gating, Conduction, and the Role of Water and Protons

PubMed Central

Kariev, Alisher M.; Green, Michael E.

2012-01-01

Ion channels, which are found in every biological cell, regulate the concentration of electrolytes, and are responsible for multiple biological functions, including in particular the propagation of nerve impulses. The channels with the latter function are gated (opened) by a voltage signal, which allows Na+ into the cell and K+ out. These channels have several positively charged amino acids on a transmembrane domain of their voltage sensor, and it is generally considered, based primarily on two lines of experimental evidence, that these charges move with respect to the membrane to open the channel. At least three forms of motion, with greatly differing extents and mechanisms of motion, have been proposed. There is a “gating current”, a capacitative current preceding the channel opening, that corresponds to several charges (for one class of channel typically 12–13) crossing the membrane field, which may not require protein physically crossing a large fraction of the membrane. The coupling to the opening of the channel would in these models depend on the motion. The conduction itself is usually assumed to require the “gate” of the channel to be pulled apart to allow ions to enter as a section of the protein partially crosses the membrane, and a selectivity filter at the opposite end of the channel determines the ion which is allowed to pass through. We will here primarily consider K+ channels, although Na+ channels are similar. We propose that the mechanism of gating differs from that which is generally accepted, in that the positively charged residues need not move (there may be some motion, but not as gating current). Instead, protons may constitute the gating current, causing the gate to open; opening consists of only increasing the diameter at the gate from approximately 6 Å to approximately 12 Å. We propose in addition that the gate oscillates rather than simply opens, and the ion experiences a barrier to its motion across the channel that is tuned

A Non-canonical Voltage-Sensing Mechanism Controls Gating in K2P K(+) Channels.

PubMed

Schewe, Marcus; Nematian-Ardestani, Ehsan; Sun, Han; Musinszki, Marianne; Cordeiro, Sönke; Bucci, Giovanna; de Groot, Bert L; Tucker, Stephen J; Rapedius, Markus; Baukrowitz, Thomas

2016-02-25

Two-pore domain (K2P) K(+) channels are major regulators of excitability that endow cells with an outwardly rectifying background "leak" conductance. In some K2P channels, strong voltage-dependent activation has been observed, but the mechanism remains unresolved because they lack a canonical voltage-sensing domain. Here, we show voltage-dependent gating is common to most K2P channels and that this voltage sensitivity originates from the movement of three to four ions into the high electric field of an inactive selectivity filter. Overall, this ion-flux gating mechanism generates a one-way "check valve" within the filter because outward movement of K(+) induces filter opening, whereas inward movement promotes inactivation. Furthermore, many physiological stimuli switch off this flux gating mode to convert K2P channels into a leak conductance. These findings provide insight into the functional plasticity of a K(+)-selective filter and also refine our understanding of K2P channels and the mechanisms by which ion channels can sense voltage. Copyright © 2016 The Authors. Published by Elsevier Inc. All rights reserved.

Structure of Voltage-gated Two-pore Channel TPC1 from Arabidopsis thaliana

PubMed Central

Guo, Jiangtao; Zeng, Weizhong; Chen, Qingfeng; Lee, Changkeun; Chen, Liping; Yang, Yi; Cang, Chunlei; Ren, Dejian; Jiang, Youxing

2015-01-01

Two-pore channels (TPCs) contain two copies of a Shaker-like six-transmembrane (6-TM) domain in each subunit and are ubiquitously expressed in both animals and plants as organellar cation channels. Here, we present the first crystal structure of a vacuolar two-pore channel from Arabidopsis thaliana, AtTPC1, which functions as a homodimer. AtTPC1 activation requires both voltage and cytosolic Ca2+. Ca2+ binding to the cytosolic EF-hand domain triggers conformational changes coupled to the pair of pore-lining inner helices (IS6 helices) from the first 6-TM domains, whereas membrane potential only activates the second voltage-sensing domain (VSD2) whose conformational changes are coupled to the pair of inner helices (IIS6 helices) from the second 6-TM domains. Luminal Ca2+ or Ba2+ can modulate voltage activation by stabilizing VSD2 in the resting state and shifts voltage activation towards more positive potentials. Our Ba2+ bound AtTPC1 structure reveals a voltage sensor in the resting state, providing hitherto unseen structural insight into the general voltage-gating mechanism among voltage-gated channels. PMID:26689363

Outward Rectification of Voltage-Gated K+ Channels Evolved at Least Twice in Life History.

PubMed

Riedelsberger, Janin; Dreyer, Ingo; Gonzalez, Wendy

2015-01-01

Voltage-gated potassium (K+) channels are present in all living systems. Despite high structural similarities in the transmembrane domains (TMD), this K+ channel type segregates into at least two main functional categories-hyperpolarization-activated, inward-rectifying (Kin) and depolarization-activated, outward-rectifying (Kout) channels. Voltage-gated K+ channels sense the membrane voltage via a voltage-sensing domain that is connected to the conduction pathway of the channel. It has been shown that the voltage-sensing mechanism is the same in Kin and Kout channels, but its performance results in opposite pore conformations. It is not known how the different coupling of voltage-sensor and pore is implemented. Here, we studied sequence and structural data of voltage-gated K+ channels from animals and plants with emphasis on the property of opposite rectification. We identified structural hotspots that alone allow already the distinction between Kin and Kout channels. Among them is a loop between TMD S5 and the pore that is very short in animal Kout, longer in plant and animal Kin and the longest in plant Kout channels. In combination with further structural and phylogenetic analyses this finding suggests that outward-rectification evolved twice and independently in the animal and plant kingdom.

Outward Rectification of Voltage-Gated K+ Channels Evolved at Least Twice in Life History

PubMed Central

Riedelsberger, Janin; Dreyer, Ingo; Gonzalez, Wendy

2015-01-01

Voltage-gated potassium (K+) channels are present in all living systems. Despite high structural similarities in the transmembrane domains (TMD), this K+ channel type segregates into at least two main functional categories—hyperpolarization-activated, inward-rectifying (Kin) and depolarization-activated, outward-rectifying (Kout) channels. Voltage-gated K+ channels sense the membrane voltage via a voltage-sensing domain that is connected to the conduction pathway of the channel. It has been shown that the voltage-sensing mechanism is the same in Kin and Kout channels, but its performance results in opposite pore conformations. It is not known how the different coupling of voltage-sensor and pore is implemented. Here, we studied sequence and structural data of voltage-gated K+ channels from animals and plants with emphasis on the property of opposite rectification. We identified structural hotspots that alone allow already the distinction between Kin and Kout channels. Among them is a loop between TMD S5 and the pore that is very short in animal Kout, longer in plant and animal Kin and the longest in plant Kout channels. In combination with further structural and phylogenetic analyses this finding suggests that outward-rectification evolved twice and independently in the animal and plant kingdom. PMID:26356684

Gating by Cyclic Gmp and Voltage in the α Subunit of the Cyclic Gmp–Gated Channel from Rod Photoreceptors

PubMed Central

Benndorf, Klaus; Koopmann, Rolf; Eismann, Elisabeth; Kaupp, U. Benjamin

1999-01-01

Gating by cGMP and voltage of the α subunit of the cGMP-gated channel from rod photoreceptor was examined with a patch-clamp technique. The channels were expressed in Xenopus oocytes. At low [cGMP] (<20 μM), the current displayed strong outward rectification. At low and high (700 μM) [cGMP], the channel activity was dominated by only one conductance level. Therefore, the outward rectification at low [cGMP] results solely from an increase in the open probability, P o. Kinetic analysis of single-channel openings revealed two exponential distributions. At low [cGMP], the larger P o at positive voltages with respect to negative voltages is caused by an increased frequency of openings in both components of the open-time distribution. In macroscopic currents, depolarizing voltage steps, starting from −100 mV, generated a time-dependent current that increased with the step size (activation). At low [cGMP] (20 μM), the degree of activation was large and the time course was slow, whereas at saturating [cGMP] (7 mM) the respective changes were small and fast. The dose–response relation at −100 mV was shifted to the right and saturated at significantly lower P o values with respect to that at +100 mV (0.77 vs. 0.96). P o was determined as function of the [cGMP] (at +100 and −100 mV) and voltage (at 20, 70, and 700 μM, and 7 mM cGMP). Both relations could be fitted with an allosteric state model consisting of four independent cGMP-binding reactions and one voltage-dependent allosteric opening reaction. At saturating [cGMP] (7 mM), the activation time course was monoexponential, which allowed us to determine the individual rate constants for the allosteric reaction. For the rapid rate constants of cGMP binding and unbinding, lower limits are determined. It is concluded that an allosteric model consisting of four independent cGMP-binding reactions and one voltage-dependent allosteric reaction, describes the cGMP- and voltage-dependent gating of cGMP-gated channels

High temperature sensitivity is intrinsic to voltage-gated potassium channels

PubMed Central

Yang, Fan; Zheng, Jie

2014-01-01

Temperature-sensitive transient receptor potential (TRP) ion channels are members of the large tetrameric cation channels superfamily but are considered to be uniquely sensitive to heat, which has been presumed to be due to the existence of an unidentified temperature-sensing domain. Here we report that the homologous voltage-gated potassium (Kv) channels also exhibit high temperature sensitivity comparable to that of TRPV1, which is detectable under specific conditions when the voltage sensor is functionally decoupled from the activation gate through either intrinsic mechanisms or mutations. Interestingly, mutations could tune Shaker channel to be either heat-activated or heat-deactivated. Therefore, high temperature sensitivity is intrinsic to both TRP and Kv channels. Our findings suggest important physiological roles of heat-induced variation in Kv channel activities. Mechanistically our findings indicate that temperature-sensing TRP channels may not contain a specialized heat-sensor domain; instead, non-obligatory allosteric gating permits the intrinsic heat sensitivity to drive channel activation, allowing temperature-sensitive TRP channels to function as polymodal nociceptors. DOI: http://dx.doi.org/10.7554/eLife.03255.001 PMID:25030910

Significance of the gate voltage-dependent mobility in the electrical characterization of organic field effect transistors

NASA Astrophysics Data System (ADS)

Kim, Jong Beom; Lee, Dong Ryeol

2018-04-01

We studied the effect of the addition of free hole- and electron-rich organic molecules to organic semiconductors (OSCs) in organic field effect transistors (OFETs) on the gate voltage-dependent mobility. The drain current versus gate voltage characteristics were quantitatively analyzed using an OFET mobility model of power law behavior based on hopping transport in an OSC. This analysis distinguished the threshold voltage shifts, depending on the materials and structures of the OFET device, and properly estimated the hopping transport of the charge carriers induced by the gate bias within the OSC from the power law exponent parameter. The addition of pentacene or C60 molecules to a one-monolayer pentacene-based OFET shifted the threshold voltages negatively or positively, respectively, due to the structural changes that occurred in the OFET device. On the other hand, the power law parameters revealed that the addition of charge carriers of the same or opposite polarity enhanced or hindered hopping transport, respectively. This study revealed the need for a quantitative analysis of the gate voltage-dependent mobility while distinguishing this effect from the threshold voltage effect in order to understand OSC hopping transport in OFETs.

The voltage-sensing domain of a phosphatase gates the pore of a potassium channel

PubMed Central

Arrigoni, Cristina; Schroeder, Indra; Romani, Giulia; Van Etten, James L.; Thiel, Gerhard

2013-01-01

The modular architecture of voltage-gated K+ (Kv) channels suggests that they resulted from the fusion of a voltage-sensing domain (VSD) to a pore module. Here, we show that the VSD of Ciona intestinalis phosphatase (Ci-VSP) fused to the viral channel Kcv creates KvSynth1, a functional voltage-gated, outwardly rectifying K+ channel. KvSynth1 displays the summed features of its individual components: pore properties of Kcv (selectivity and filter gating) and voltage dependence of Ci-VSP (V1/2 = +56 mV; z of ∼1), including the depolarization-induced mode shift. The degree of outward rectification of the channel is critically dependent on the length of the linker more than on its amino acid composition. This highlights a mechanistic role of the linker in transmitting the movement of the sensor to the pore and shows that electromechanical coupling can occur without coevolution of the two domains. PMID:23440279

Mechanisms of Gain Control by Voltage-Gated Channels in Intrinsically-Firing Neurons

PubMed Central

Patel, Ameera X.; Burdakov, Denis

2015-01-01

Gain modulation is a key feature of neural information processing, but underlying mechanisms remain unclear. In single neurons, gain can be measured as the slope of the current-frequency (input-output) relationship over any given range of inputs. While much work has focused on the control of basal firing rates and spike rate adaptation, gain control has been relatively unstudied. Of the limited studies on gain control, some have examined the roles of synaptic noise and passive somatic currents, but the roles of voltage-gated channels present ubiquitously in neurons have been less explored. Here, we systematically examined the relationship between gain and voltage-gated ion channels in a conductance-based, tonically-active, model neuron. Changes in expression (conductance density) of voltage-gated channels increased (Ca2+ channel), reduced (K+ channels), or produced little effect (h-type channel) on gain. We found that the gain-controlling ability of channels increased exponentially with the steepness of their activation within the dynamic voltage window (voltage range associated with firing). For depolarization-activated channels, this produced a greater channel current per action potential at higher firing rates. This allowed these channels to modulate gain by contributing to firing preferentially at states of higher excitation. A finer analysis of the current-voltage relationship during tonic firing identified narrow voltage windows at which the gain-modulating channels exerted their effects. As a proof of concept, we show that h-type channels can be tuned to modulate gain by changing the steepness of their activation within the dynamic voltage window. These results show how the impact of an ion channel on gain can be predicted from the relationship between channel kinetics and the membrane potential during firing. This is potentially relevant to understanding input-output scaling in a wide class of neurons found throughout the brain and other nervous systems

Low-voltage operation of Si-based ferroelectric field effect transistors using organic ferroelectrics, poly(vinylidene fluoride-trifluoroethylene), as a gate dielectric

NASA Astrophysics Data System (ADS)

Miyata, Yusuke; Yoshimura, Takeshi; Ashida, Atsushi; Fujimura, Norifumi

2016-04-01

Si-based metal-ferroelectric-semiconductor (MFS) capacitors have been fabricated using poly(vinylidene fluoride-trifluoroethylene) [P(VDF-TrFE)] as a ferroelectric gate. The pinhole-free P(VDF-TrFE) thin films with high resistivity were able to be prepared by spin-coating directly onto hydrogen-terminated Si. The capacitance-voltage (C-V) characteristics of the ferroelectric gate field effect transistor (FeFET) using this MFS structure clearly show butterfly-shaped hysteresis originating from the ferroelectricity, indicating carrier modulation on the Si surface at gate voltages below 2 V. The drain current-gate voltage (I D-V G) characteristics also show counterclockwise hysteresis at gate voltages below 5 V. This is the first report on the low-voltage operation of a Si-based FeFET using P(VDF-TrFE) as a gate dielectric. This organic gate FeFET without any insulator layer at the ferroelectric/Si interface should be one of the promising devices for overcoming the critical issues of the FeFET, such as depolarization field and a decrease in the gate voltage.

Mapping the Interaction Site for a β-Scorpion Toxin in the Pore Module of Domain III of Voltage-gated Na+ Channels*

PubMed Central

Zhang, Joel Z.; Yarov-Yarovoy, Vladimir; Scheuer, Todd; Karbat, Izhar; Cohen, Lior; Gordon, Dalia; Gurevitz, Michael; Catterall, William A.

2012-01-01

Activation of voltage-gated sodium (Nav) channels initiates and propagates action potentials in electrically excitable cells. β-Scorpion toxins, including toxin IV from Centruroides suffusus suffusus (CssIV), enhance activation of NaV channels. CssIV stabilizes the voltage sensor in domain II in its activated state via a voltage-sensor trapping mechanism. Amino acid residues required for the action of CssIV have been identified in the S1-S2 and S3-S4 extracellular loops of domain II. The extracellular loops of domain III are also involved in toxin action, but individual amino acid residues have not been identified. We used site-directed mutagenesis and voltage clamp recording to investigate amino acid residues of domain III that are involved in CssIV action. In the IIISS2-S6 loop, five substitutions at four positions altered voltage-sensor trapping by CssIVE15A. Three substitutions (E1438A, D1445A, and D1445Y) markedly decreased voltage-sensor trapping, whereas the other two substitutions (N1436G and L1439A) increased voltage-sensor trapping. These bidirectional effects suggest that residues in IIISS2-S6 make both positive and negative interactions with CssIV. N1436G enhanced voltage-sensor trapping via increased binding affinity to the resting state, whereas L1439A increased voltage-sensor trapping efficacy. Based on these results, a three-dimensional model of the toxin-channel interaction was developed using the Rosetta modeling method. These data provide additional molecular insight into the voltage-sensor trapping mechanism of toxin action and define a three-point interaction site for β-scorpion toxins on NaV channels. Binding of α- and β-scorpion toxins to two distinct, pseudo-symmetrically organized receptor sites on NaV channels acts synergistically to modify channel gating and paralyze prey. PMID:22761417

Barbiturates Block Sodium and Potassium Conductance Increases in Voltage-Clamped Lobster Axons

PubMed Central

Blaustein, M. P.

1968-01-01

Sodium pentobarbital and sodium thiopental decrease both the peak initial (Na) and late steady-state (K) currents and reduce the maximum sodium and potassium conductance increases in voltage-clamped lobster giant axons. These barbiturates also slow the rate at which the sodium conductance turns on, and shift the normalized sodium conductance vs. voltage curves in the direction of depolarization along the voltage axis. Since pentobarbital (pKa = 8.0) blocks the action potential more effectively at pH 8.5 than at pH 6.7, the anionic form of the drug appears to be active. The data suggest that these drugs affect the axon membrane directly, rather than secondarily through effects on intermediary metabolism. It is suggested that penetration of the lipid layer of the membrane by the nonpolar portion of the barbiturate molecules may cause the decrease in membrane conductances, while electrostatic interactions involving the anionic group on the barbiturate, divalent cations, and "fixed charges" in the membrane could account for the slowing of the rate of sodium conductance turn-on and the shift of the normalized conductance curves along the voltage axis. PMID:5648829

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

Effect of thiopental sodium on N-methyl-D-aspartate-gated currents.

PubMed

Liu, Hongliang; Dai, Tijun; Yao, Shanglong

2006-05-01

N-methyl-D-aspartate (NMDA) receptors in the prefrontal cortex (PFC) are closely related with the excitability of pyramidal neurons and PFC function. As the effect of thiopental sodium on the central nervous system may partly result from the inhibition of PFC NMDA receptors, we investigated the effect of thiopental sodium with different concentrations on NMDA-gated currents in acutely dissociated rat PFC pyramidal neurons. We sought to determine whether thiopental sodium inhibits NMDA receptor function. Three to four week old male Sprague-Dawley rats were sacrificed and the PFC was dissected. Pyramidal neurons from the PFC were prepared and standard whole-cell patch clamp recordings were performed. Escalating concentrations from 3-1000 microM NMDA were applied 100 microm from the pyramidal cells, and the concentration in the effect compartment related to 50% effect (EC50) of NMDA was determined for the ensuing experiments. One hundred microM NMDA alone (control) or NMDA with different concentrations (10-1000 microM) of thiopental sodium were applied. After the inhibitory concentration, in 50% of NMDA effect (IC50) of thiopental sodium was established this IC50 and NMDA 3-1000 microM were applied 100 microm from the pyramidal cells. The EC50 value of NMDA under the effect of IC50 thiopental sodium was determined. N-methyl-D-aspartate induced inward currents in a concentration-dependent manner, which were completely antagonized by 50 microM AP5. The maximal amplitude of NMDA-induced current was 1.15 +/- 0.27 nA. The EC50 of NMDA was 53.6 +/- 12.4 microM. The NMDA (100 microM)-gated current was inhibited by thiopental sodium in a concentration-dependent manner, and the IC50 of thiopental sodium was 33.6 +/- 6.1 microM. Under the effect of 33.6 microM thiopental sodium, the maximal amplitude of NMDA-induced current was 0.87 +/- 0.17 nA. The concentration-response curve of NMDA was shifted rightwards. The EC50 of NMDA was 128 +/- 15 microM, which was greater than that

Structural mechanism of voltage-dependent gating in an isolated voltage-sensing domain.

PubMed

Li, Qufei; Wanderling, Sherry; Paduch, Marcin; Medovoy, David; Singharoy, Abhishek; McGreevy, Ryan; Villalba-Galea, Carlos A; Hulse, Raymond E; Roux, Benoît; Schulten, Klaus; Kossiakoff, Anthony; Perozo, Eduardo

2014-03-01

The transduction of transmembrane electric fields into protein motion has an essential role in the generation and propagation of cellular signals. Voltage-sensing domains (VSDs) carry out these functions through reorientations of positive charges in the S4 helix. Here, we determined crystal structures of the Ciona intestinalis VSD (Ci-VSD) in putatively active and resting conformations. S4 undergoes an ~5-Å displacement along its main axis, accompanied by an ~60° rotation. This movement is stabilized by an exchange in countercharge partners in helices S1 and S3 that generates an estimated net charge transfer of ~1 eo. Gating charges move relative to a ''hydrophobic gasket' that electrically divides intra- and extracellular compartments. EPR spectroscopy confirms the limited nature of S4 movement in a membrane environment. These results provide an explicit mechanism for voltage sensing and set the basis for electromechanical coupling in voltage-dependent enzymes and ion channels.

Threshold voltage control in TmSiO/HfO2 high-k/metal gate MOSFETs

NASA Astrophysics Data System (ADS)

Dentoni Litta, E.; Hellström, P.-E.; Östling, M.

2015-06-01

High-k interfacial layers have been proposed as a way to extend the scalability of Hf-based high-k/metal gate CMOS technology, which is currently limited by strong degradations in threshold voltage control, channel mobility and device reliability when the chemical oxide (SiOx) interfacial layer is scaled below 0.4 nm. We have previously demonstrated that thulium silicate (TmSiO) is a promising candidate as a high-k interfacial layer, providing competitive advantages in terms of EOT scalability and channel mobility. In this work, the effect of the TmSiO interfacial layer on threshold voltage control is evaluated, showing that the TmSiO/HfO2 dielectric stack is compatible with threshold voltage control techniques commonly used with SiOx/HfO2 stacks. Specifically, we show that the flatband voltage can be set in the range -1 V to +0.5 V by the choice of gate metal and that the effective workfunction of the stack is properly controlled by the metal workfunction in a gate-last process flow. Compatibility with a gate-first approach is also demonstrated, showing that integration of La2O3 and Al2O3 capping layers can induce a flatband voltage shift of at least 150 mV. Finally, the effect of the annealing conditions on flatband voltage is investigated, finding that the duration of the final forming gas anneal can be used as a further process knob to tune the threshold voltage. The evaluation performed on MOS capacitors is confirmed by the fabrication of TmSiO/HfO2/TiN MOSFETs achieving near-symmetric threshold voltages at sub-nm EOT.

Low-voltage organic strain sensor on plastic using polymer/high- K inorganic hybrid gate dielectrics

NASA Astrophysics Data System (ADS)

Jung, Soyoun; Ji, Taeksoo; Varadan, Vijay K.

2007-12-01

In this paper, gate-induced pentacene semiconductor strain sensors based on hybrid-gate dielectrics using poly-vinylphenol (PVP) and high-K inorganic, Ta IIO 5 are fabricated on flexible substrates, polyethylene naphthalate (PEN). The Ta IIO 5 gate dielectric layer is combined with a thin PVP layer to obtain very smooth and hydrophobic surfaces which improve the molecular structures of pentacene films. The PVP-Ta IIO 5 hybrid-gate dielectric films exhibit a high dielectric capacitance and low leakage current. The sensors adopting thin film transistor (TFT)-like structures show a significantly reduced operating voltage (~6V), and good device characteristics with a field-effect mobility of 1.89 cm2/V•s, a threshold voltage of -0.5 V, and an on/off ratio of 10 3. The strain sensor, one of the practical applications in large-area organic electronics, was characterized with different bending radii of 50, 40, 30, and 20 mm. The sensor output signals were significantly improved with low-operating voltages.

Expression and distribution of voltage-gated ion channels in ferret sinoatrial node.

PubMed

Brahmajothi, Mulugu V; Morales, Michael J; Campbell, Donald L; Steenbergen, Charles; Strauss, Harold C

2010-10-01

Spontaneous diastolic depolarization in the sinoatrial (SA) node enables it to serve as pacemaker of the heart. The variable cell morphology within the SA node predicts that ion channel expression would be heterogeneous and different from that in the atrium. To evaluate ion channel heterogeneity within the SA node, we used fluorescent in situ hybridization to examine ion channel expression in the ferret SA node region and atrial appendage. SA nodal cells were distinguished from surrounding cardiac myocytes by expression of the slow (SA node) and cardiac (surrounding tissue) forms of troponin I. Nerve cells in the sections were identified by detection of GAP-43 and cytoskeletal middle neurofilament. Transcript expression was characterized for the 4 hyperpolarization-activated cation channels, 6 voltage-gated Na(+) channels, 3 voltage-gated Ca(2+) channels, 24 voltage-gated K(+) channel α-subunits, and 3 ancillary subunits. To ensure that transcript expression was representative of protein expression, immunofluorescence was used to verify localization patterns of voltage-dependent K(+) channels. Colocalizations were performed to observe any preferential patterns. Some overlapping and nonoverlapping binding patterns were observed. Measurement of different cation channel transcripts showed heterogeneous expression with many different patterns of expression, attesting to the complexity of electrical activity in the SA node. This study provides insight into the possible role ion channel heterogeneity plays in SA node pacemaker activity.

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...

Different role of TTX-sensitive voltage-gated sodium channel (NaV 1) subtypes in action potential initiation and conduction in vagal airway nociceptors.

PubMed

Kollarik, M; Sun, H; Herbstsomer, R A; Ru, F; Kocmalova, M; Meeker, S N; Undem, B J

2018-04-15

The action potential initiation in the nerve terminals and its subsequent conduction along the axons of afferent nerves are not necessarily dependent on the same voltage-gated sodium channel (Na V 1) subunits. The action potential initiation in jugular C-fibres within airway tissues is not blocked by TTX; nonetheless, conduction of action potentials along the vagal axons of these nerves is often dependent on TTX-sensitive channels. This is not the case for nodose airway Aδ-fibres and C-fibres, where both action potential initiation and conduction is abolished by TTX or selective Na V 1.7 blockers. The difference between the initiation of action potentials within the airways vs. conduction along the axons should be considered when developing Na V 1 blocking drugs for topical application to the respiratory tract. The action potential (AP) initiation in the nerve terminals and its subsequent AP conduction along the axons do not necessarily depend on the same subtypes of voltage-gated sodium channels (Na V 1s). We evaluated the role of TTX-sensitive and TTX-resistant Na V 1s in vagal afferent nociceptor nerves derived from jugular and nodose ganglia innervating the respiratory system. Single cell RT-PCR was performed on vagal afferent neurons retrogradely labelled from the guinea pig trachea. Almost all of the jugular neurons expressed the TTX-sensitive channel Na V 1.7 along with TTX-resistant Na V 1.8 and Na V 1.9. Tracheal nodose neurons also expressed Na V 1.7 but, less frequently, Na V 1.8 and Na V 1.9. Na V 1.6 were expressed in ∼40% of the jugular and 25% of nodose tracheal neurons. Other Na V 1 α subunits were only rarely expressed. Single fibre recordings were made from the vagal nodose and jugular nerve fibres innervating the trachea or lung in the isolated perfused vagally-innervated preparations that allowed for selective drug delivery to the nerve terminal compartment (AP initiation) or to the desheathed vagus nerve (AP conduction). AP initiation in

Knockdown resistance (kdr) of the voltage-gated sodium channel gene of Aedes aegypti population in Denpasar, Bali, Indonesia.

PubMed

Hamid, Penny Humaidah; Prastowo, Joko; Widyasari, Anis; Taubert, Anja; Hermosilla, Carlos

2017-06-05

Aedes aegypti is the main vector of several arthropod-borne viral infections in the tropics profoundly affecting humans, such as dengue fever (DF), West Nile (WN), chikungunya and more recently Zika. Eradication of Aedes still largely depends on insecticides, which is the most cost-effective strategy, and often inefficient due to resistance development in exposed Aedes populations. We here conducted a study of Ae. aegypti resistance towards several insecticides regularly used in the city of Denpasar, Bali, Indonesia. Aedes aegypti egg samples were collected with ovitraps and thereafter hatched in the insectary of the Gadjah Mada University. The F0 generation was used for all bioassay-related experiments and knockdown resistance (kdr) assays. Results clearly showed resistance development of Ae. aegypti against tested insecticides. Mortalities of Ae. aegypti were less than 90% with highest resistance observed against 0.75% permethrin. Mosquitoes from the southern parts of Denpasar presented high level of resistance pattern in comparison to those from the western and northern parts of Denpasar. Kdr analysis of voltage-gated sodium channel (Vgsc) gene showed significant association to S989P and V1016G mutations linked to resistance phenotypes against 0.75% permethrin. Conversely, Ae. aegypti F1534C gene mutation did not result in any significant correlation to resistance development. Periodically surveillance of insecticide resistances in Ae. aegypti mosquitoes will help local public health authorities to set better goals and allow proper evaluation of on-going mosquito control strategies. Initial detection of insecticide resistance will contribute to conduct proper actions in delaying mosquito resistance development such as insecticide rotation or combination of compounds in order to prolong chemical efficacy in combating Ae. aegypti vectors in Indonesia.

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+ ...

Cellular defibrillation: interaction of micro-scale electric fields with voltage-gated ion channels.

PubMed

Kargol, Armin; Malkinski, Leszek; Eskandari, Rahmatollah; Carter, Maya; Livingston, Daniel

2015-09-01

We study the effect of micro-scale electric fields on voltage-gated ion channels in mammalian cell membranes. Such micro- and nano-scale electric fields mimic the effects of multiferroic nanoparticles that were recently proposed [1] as a novel way of controlling the function of voltage-sensing biomolecules such as ion channels. This article describes experimental procedures and initial results that reveal the effect of the electric field, in close proximity of cells, on the ion transport through voltage-gated ion channels. We present two configurations of the whole-cell patch-clamping apparatus that were used to detect the effect of external stimulation on ionic currents and discuss preliminary results that indicate modulation of the ionic currents consistent with the applied stimulus.

Sterol Regulation of Voltage-Gated K+ Channels.

PubMed

Balajthy, Andras; Hajdu, Peter; Panyi, Gyorgy; Varga, Zoltan

2017-01-01

Cholesterol is an essential lipid building block of the cellular plasma membrane. In addition to its structural role, it regulates the fluidity and raft structure of the membrane and influences the course of numerous membrane-linked signaling pathways and the function of transmembrane proteins, including ion channels. This is supported by a vast body of scientific data, which demonstrates the modulation of ion channels with a great variety of ion selectivity, gating, and tissue distribution by changes in membrane cholesterol. Here, we review what is currently known about the modulation of voltage-gated K + (Kv) channels by changes in membrane cholesterol content, considering raft association of the channels, the roles of cholesterol recognition sites, and those of adaptor proteins in cholesterol-Kv channel interactions. We specifically focus on Kv1.3, the dominant K + channel of human T cells. Effects of cholesterol depletion and enrichment and 7-dehydrocholesterol enrichment on Kv1.3 gating are discussed in the context of the immunological synapse and the comparison of the in vitro effects of sterol modifications on Kv1.3 function with ex vivo effects on cells from hypercholesterolemic and Smith-Lemli-Opitz patients. © 2017 Elsevier Inc. All rights reserved.

Expression of Voltage-Gated Sodium Channel Nav1.8 in Human Prostate Cancer is Associated with High Histological Grade

PubMed Central

Suy, Simeng; Hansen, Todd P.; Auto, Heather D.; Kallakury, Bhaskar V.S.; Dailey, Vernon; Danner, Malika; MacArthur, Linda; Zhang, Ying; Miessau, Matthew J.; Collins, Sean P.; Brown, Milton L.

2013-01-01

Voltage-gated sodium (Nav) channels are required for impulse conductance in excitable tissues. Navs have been linked to human cancers, including prostate. The expression and distribution of Nav isoforms (Nav1.1-Nav1.9) in human prostate cancer are not well established. Here, we evaluated the expression of these isoforms and investigated the expression of Nav1.8 in human prostate cancer tissues. Nav1.8 was highly expressed in all examined cells. Expression of Nav1.1, Nav1.2, and Nav1.9 were high in DU-145, PC-3 and PC-3M cells compared to LNCaP (hormone-dependent), C4-2, C4-2B, and CWR22Rv-1 cells. Nav1.5 and Nav1.6 were expressed in all cells examined. Nav1.7 expression was absent in PC-3M and CWR22Rv-1, but expressed in the other cells examined. Immunohistochemistry revealed intensive Nav1.8 staining correlated with more advanced pathologic stage of disease. Increased intensity of nuclear Nav1.8 correlated with increased Gleason grade. Our results revealed that Nav1.8 is universally expressed in human prostate cancer cells. Nav1.8 expression statistically correlated with pathologic stage (P=0.04) and Gleason score (P=0.01) of human prostate tissue specimens. The aberrant nuclear localization of Nav1.8 with advanced prostate cancer tissues warrant further investigation into use of Nav1.8 as a potential biomarker to differentiate between early and advanced disease. PMID:24163825

Lithium-Responsive Seizure-Like Hyperexcitability Is Caused by a Mutation in the Drosophila Voltage-Gated Sodium Channel Gene paralytic

PubMed Central

Kasuya, Junko; Ueda, Atsushi; Iyengar, Atulya; Wu, Chun-Fang

2016-01-01

Abstract Shudderer (Shu) is an X-linked dominant mutation in Drosophila melanogaster identified more than 40 years ago. A previous study showed that Shu caused spontaneous tremors and defects in reactive climbing behavior, and that these phenotypes were significantly suppressed when mutants were fed food containing lithium, a mood stabilizer used in the treatment of bipolar disorder (Williamson, 1982). This unique observation suggested that the Shu mutation affects genes involved in lithium-responsive neurobiological processes. In the present study, we identified Shu as a novel mutant allele of the voltage-gated sodium (Nav) channel gene paralytic (para). Given that hypomorphic para alleles and RNA interference–mediated para knockdown reduced the severity of Shu phenotypes, Shu was classified as a para hypermorphic allele. We also demonstrated that lithium could improve the behavioral abnormalities displayed by other Nav mutants, including a fly model of the human generalized epilepsy with febrile seizures plus. Our electrophysiological analysis of Shu showed that lithium treatment did not acutely suppress Nav channel activity, indicating that the rescue effect of lithium resulted from chronic physiological adjustments to this drug. Microarray analysis revealed that lithium significantly alters the expression of various genes in Shu, including those involved in innate immune responses, amino acid metabolism, and oxidation-reduction processes, raising the interesting possibility that lithium-induced modulation of these biological pathways may contribute to such adjustments. Overall, our findings demonstrate that Nav channel mutants in Drosophila are valuable genetic tools for elucidating the effects of lithium on the nervous system in the context of neurophysiology and behavior. PMID:27844061

Three-Function Logic Gate Controlled by Analog Voltage

NASA Technical Reports Server (NTRS)

Zebulum, Ricardo; Stoica, Adrian

2006-01-01

The figure is a schematic diagram of a complementary metal oxide/semiconductor (CMOS) electronic circuit that performs one of three different logic functions, depending on the level of an externally applied control voltage, V(sub sel). Specifically, the circuit acts as A NAND gate at V(sub sel) = 0.0 V, A wire (the output equals one of the inputs) at V(sub sel) = 1.0 V, or An AND gate at V(sub sel) = -1.8 V. [The nominal power-supply potential (VDD) and logic "1" potential of this circuit is 1.8 V.] Like other multifunctional circuits described in several prior NASA Tech Briefs articles, this circuit was synthesized following an automated evolutionary approach that is so named because it is modeled partly after the repetitive trial-and-error process of biological evolution. An evolved circuit can be tested by computational simulation and/or tested in real hardware, and the results of the test can provide guidance for refining the design through further iteration. The evolutionary synthesis of electronic circuits can now be implemented by means of a software package Genetic Algorithms for Circuit Synthesis (GACS) that was developed specifically for this purpose. GACS was used to synthesize the present trifunctional circuit. As in the cases of other multifunctional circuits described in several prior NASA Tech Briefs articles, the multiple functionality of this circuit, the use of a single control voltage to select the function, and the automated evolutionary approach to synthesis all contribute synergistically to a combination of features that are potentially advantageous for the further development of robust, multiple-function logic circuits, including, especially, field-programmable gate arrays (FPGAs). These advantages include the following: This circuit contains only 9 transistors about half the number of transistors that would be needed to obtain equivalent NAND/wire/AND functionality by use of components from a standard digital design library. If

Controlling the layer localization of gapless states in bilayer graphene with a gate voltage

NASA Astrophysics Data System (ADS)

Jaskólski, W.; Pelc, M.; Bryant, Garnett W.; Chico, Leonor; Ayuela, A.

2018-04-01

Experiments in gated bilayer graphene with stacking domain walls present topological gapless states protected by no-valley mixing. Here we research these states under gate voltages using atomistic models, which allow us to elucidate their origin. We find that the gate potential controls the layer localization of the two states, which switches non-trivially between layers depending on the applied gate voltage magnitude. We also show how these bilayer gapless states arise from bands of single-layer graphene by analyzing the formation of carbon bonds between layers. Based on this analysis we provide a model Hamiltonian with analytical solutions, which explains the layer localization as a function of the ratio between the applied potential and interlayer hopping. Our results open a route for the manipulation of gapless states in electronic devices, analogous to the proposed writing and reading memories in topological insulators.

Voltage-Gated Proton Channels: Molecular Biology, Physiology, and Pathophysiology of the HV Family

PubMed Central

2013-01-01

Voltage-gated proton channels (HV) are unique, in part because the ion they conduct is unique. HV channels are perfectly selective for protons and have a very small unitary conductance, both arguably manifestations of the extremely low H+ concentration in physiological solutions. They open with membrane depolarization, but their voltage dependence is strongly regulated by the pH gradient across the membrane (ΔpH), with the result that in most species they normally conduct only outward current. The HV channel protein is strikingly similar to the voltage-sensing domain (VSD, the first four membrane-spanning segments) of voltage-gated K+ and Na+ channels. In higher species, HV channels exist as dimers in which each protomer has its own conduction pathway, yet gating is cooperative. HV channels are phylogenetically diverse, distributed from humans to unicellular marine life, and perhaps even plants. Correspondingly, HV functions vary widely as well, from promoting calcification in coccolithophores and triggering bioluminescent flashes in dinoflagellates to facilitating killing bacteria, airway pH regulation, basophil histamine release, sperm maturation, and B lymphocyte responses in humans. Recent evidence that hHV1 may exacerbate breast cancer metastasis and cerebral damage from ischemic stroke highlights the rapidly expanding recognition of the clinical importance of hHV1. PMID:23589829

Shaping charge excitations in chiral edge states with a time-dependent gate voltage

NASA Astrophysics Data System (ADS)

Misiorny, Maciej; Fève, Gwendal; Splettstoesser, Janine

2018-02-01

We study a coherent conductor supporting a single edge channel in which alternating current pulses are created by local time-dependent gating and sent on a beam-splitter realized by a quantum point contact. The current response to the gate voltage in this setup is intrinsically linear. Based on a fully self-consistent treatment employing a Floquet scattering theory, we analyze the effect of different voltage shapes and frequencies, as well as the role of the gate geometry on the injected signal. In particular, we highlight the impact of frequency-dependent screening on the process of shaping the current signal. The feasibility of creating true single-particle excitations with this method is confirmed by investigating the suppression of excess noise, which is otherwise created by additional electron-hole pair excitations in the current signal.

Controllable Hysteresis and Threshold Voltage of Single-Walled Carbon Nano-tube Transistors with Ferroelectric Polymer Top-Gate Insulators

PubMed Central

Sun, Yi-Lin; Xie, Dan; Xu, Jian-Long; Zhang, Cheng; Dai, Rui-Xuan; Li, Xian; Meng, Xiang-Jian; Zhu, Hong-Wei

2016-01-01

Double-gated field effect transistors have been fabricated using the SWCNT networks as channel layer and the organic ferroelectric P(VDF-TrFE) film spin-coated as top gate insulators. Standard photolithography process has been adopted to achieve the patterning of organic P(VDF-TrFE) films and top-gate electrodes, which is compatible with conventional CMOS process technology. An effective way for modulating the threshold voltage in the channel of P(VDF-TrFE) top-gate transistors under polarization has been reported. The introduction of functional P(VDF-TrFE) gate dielectric also provides us an alternative method to suppress the initial hysteresis of SWCNT networks and obtain a controllable ferroelectric hysteresis behavior. Applied bottom gate voltage has been found to be another effective way to highly control the threshold voltage of the networked SWCNTs based FETs by electrostatic doping effect. PMID:26980284

Electric organ discharge diversification in mormyrid weakly electric fish is associated with differential expression of voltage-gated ion channel genes.

PubMed

Nagel, Rebecca; Kirschbaum, Frank; Tiedemann, Ralph

2017-03-01

In mormyrid weakly electric fish, the electric organ discharge (EOD) is used for species recognition, orientation and prey localization. Produced in the muscle-derived adult electric organ, the EOD exhibits a wide diversity across species in both waveform and duration. While certain defining EOD characteristics can be linked to anatomical features of the electric organ, many factors underlying EOD differentiation are yet unknown. Here, we report the differential expression of 13 Kv1 voltage-gated potassium channel genes, two inwardly rectifying potassium channel genes, two previously studied sodium channel genes and an ATPase pump in two sympatric species of the genus Campylomormyrus in both the adult electric organ and skeletal muscle. Campylomormyrus compressirostris displays a basal EOD, largely unchanged during development, while C. tshokwe has an elongated, putatively derived discharge. We report an upregulation in all Kv1 genes in the electric organ of Campylomormyrus tshokwe when compared to both skeletal muscle and C. compressirostris electric organ. This pattern of upregulation in a species with a derived EOD form suggests that voltage-gated potassium channels are potentially involved in the diversification of the EOD signal among mormyrid weakly electric fish.

Non-scaling behavior of electroosmotic flow in voltage-gated nanopores

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

Lian, Cheng; Gallegos, Alejandro; Liu, Honglai

2017-01-01

Ionic size effects and electrostatic correlations result in the non-monotonic dependence of the electrical conductivity on the pore size. For ion transport at a high gating voltage, the conductivity oscillates with the pore size due to a significant overlap of the electric double layers.

A localized interaction surface for voltage-sensing domains on the pore domain of a K+ channel.

PubMed

Li-Smerin, Y; Hackos, D H; Swartz, K J

2000-02-01

Voltage-gated K+ channels contain a central pore domain and four surrounding voltage-sensing domains. How and where changes in the structure of the voltage-sensing domains couple to the pore domain so as to gate ion conduction is not understood. The crystal structure of KcsA, a bacterial K+ channel homologous to the pore domain of voltage-gated K+ channels, provides a starting point for addressing this question. Guided by this structure, we used tryptophan-scanning mutagenesis on the transmembrane shell of the pore domain in the Shaker voltage-gated K+ channel to localize potential protein-protein and protein-lipid interfaces. Some mutants cause only minor changes in gating and when mapped onto the KcsA structure cluster away from the interface between pore domain subunits. In contrast, mutants producing large changes in gating tend to cluster near this interface. These results imply that voltage-sensing domains interact with localized regions near the interface between adjacent pore domain subunits.

The NH2 terminus regulates voltage-dependent gating of CALHM ion channels.

PubMed

Tanis, Jessica E; Ma, Zhongming; Foskett, J Kevin

2017-08-01

Calcium homeostasis modulator protein-1 (CALHM1) and its Caenorhabditis elegans (ce) homolog, CLHM-1, belong to a new family of physiologically important ion channels that are regulated by voltage and extracellular Ca 2+ (Ca 2+ o ) but lack a canonical voltage-sensing domain. Consequently, the intrinsic voltage-dependent gating mechanisms for CALHM channels are unknown. Here, we performed voltage-clamp experiments on ceCLHM-1 chimeric, deletion, insertion, and point mutants to assess the role of the NH 2 terminus (NT) in CALHM channel gating. Analyses of chimeric channels in which the ceCLHM-1 and human (h)CALHM1 NH 2 termini were interchanged showed that the hCALHM1 NT destabilized channel-closed states, whereas the ceCLHM-1 NT had a stabilizing effect. In the absence of Ca 2+ o , deletion of up to eight amino acids from the ceCLHM-1 NT caused a hyperpolarizing shift in the conductance-voltage relationship with little effect on voltage-dependent slope. However, deletion of nine or more amino acids decreased voltage dependence and induced a residual conductance at hyperpolarized voltages. Insertion of amino acids into the NH 2 -terminal helix also decreased voltage dependence but did not prevent channel closure. Mutation of ceCLHM-1 valine 9 and glutamine 13 altered half-maximal activation and voltage dependence, respectively, in 0 Ca 2+ In 2 mM Ca 2+ o , ceCLHM-1 NH 2 -terminal deletion and point mutant channels closed completely at hyperpolarized voltages with apparent affinity for Ca 2+ o indistinguishable from wild-type ceCLHM-1, although the ceCLHM-1 valine 9 mutant exhibited an altered conductance-voltage relationship and kinetics. We conclude that the NT plays critical roles modulating voltage dependence and stabilizing the closed states of CALHM channels. Copyright © 2017 the American Physiological Society.

Breathing of voltage dependent anion channel as revealed by the fractal property of its gating

NASA Astrophysics Data System (ADS)

Manna, Smarajit; Banerjee, Jyotirmoy; Ghosh, Subhendu

2007-12-01

The gating of voltage dependent anion channel (VDAC) depends on the movement of voltage sensors in the transmembrane region, but the actual mechanism is still not well understood. With a view to understand the phenomenon we have analyzed the current recordings of VDAC in lipid bilayer membrane (BLM) and found that the data show self-similarity and fractal characteristics. We look for the microscopic and molecular basis of fractal behavior of gating of VDAC. A model describing the oscillatory dynamics of voltage sensors of VDAC in the transmembrane region under applied potential has been proposed which gives rise to the aforesaid fractal behavior.

Micro-mechanical resonators for dynamically reconfigurable reduced voltage logic gates

NASA Astrophysics Data System (ADS)

Chappanda, K. N.; Ilyas, S.; Younis, M. I.

2018-05-01

Due to the limitations of transistor-based logic devices such as their poor performance at elevated temperature, alternative computing methods are being actively investigated. In this work, we present electromechanical logic gates using electrostatically coupled in-plane micro-cantilever resonators operated at modest vacuum conditions of 5 Torr. Operating in the first resonant mode, we demonstrate 2-bit XOR, 2- and 3-bit AND, 2- and 3-bit NOR, and 1-bit NOT gates; all condensed in the same device. Through the designed electrostatic coupling, the required voltage for the logic gates is reduced by 80%, along with the reduction in the number of electrical interconnects and devices per logic operation (contrary to transistors). The device is dynamically reconfigurable between any logic gates in real time without the need for any change in the electrical interconnects and the drive circuit. By operating in the first two resonant vibration modes, we demonstrate mechanical logic gates consisting of two 2-bit AND and two 2-bit XOR gates. The device is tested at elevated temperatures and is shown to be functional as a logic gate up to 150 °C. Also, the device has high reliability with demonstrated lifetime greater than 5  ×  1012 oscillations.

Protonic/electronic hybrid oxide transistor gated by chitosan and its full-swing low voltage inverter applications

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

Chao, Jin Yu; Ningbo Institute of Material Technology and Engineering, Chinese Academy of Sciences, Ningbo 315201; Zhu, Li Qiang, E-mail: lqzhu@nimte.ac.cn

Modulation of charge carrier density in condensed materials based on ionic/electronic interaction has attracted much attention. Here, protonic/electronic hybrid indium-zinc-oxide (IZO) transistors gated by chitosan based electrolyte were obtained. The chitosan-based electrolyte illustrates a high proton conductivity and an extremely strong proton gating behavior. The transistor illustrates good electrical performances at a low operating voltage of ∼1.0 V such as on/off ratio of ∼3 × 10{sup 7}, subthreshold swing of ∼65 mV/dec, threshold voltage of ∼0.3 V, and mobility of ∼7 cm{sup 2}/V s. Good positive gate bias stress stabilities are obtained. Furthermore, a low voltage driven resistor-loaded inverter was built by using an IZO transistor inmore » series with a load resistor, exhibiting a linear relationship between the voltage gain and the supplied voltage. The inverter is also used for decreasing noises of input signals. The protonic/electronic hybrid IZO transistors have potential applications in biochemical sensors and portable electronics.« less

Highly tunable local gate controlled complementary graphene device performing as inverter and voltage controlled resistor.

PubMed

Kim, Wonjae; Riikonen, Juha; Li, Changfeng; Chen, Ya; Lipsanen, Harri

2013-10-04

Using single-layer CVD graphene, a complementary field effect transistor (FET) device is fabricated on the top of separated back-gates. The local back-gate control of the transistors, which operate with low bias at room temperature, enables highly tunable device characteristics due to separate control over electrostatic doping of the channels. Local back-gating allows control of the doping level independently of the supply voltage, which enables device operation with very low VDD. Controllable characteristics also allow the compensation of variation in the unintentional doping typically observed in CVD graphene. Moreover, both p-n and n-p configurations of FETs can be achieved by electrostatic doping using the local back-gate. Therefore, the device operation can also be switched from inverter to voltage controlled resistor, opening new possibilities in using graphene in logic circuitry.

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.

Reduction of voltage gated sodium channel protein in DRG by vector mediated miRNA reduces pain in rats with painful diabetic neuropathy.

PubMed

Chattopadhyay, Munmun; Zhou, Zhigang; Hao, Shuanglin; Mata, Marina; Fink, David J

2012-03-22

Painful neuropathy is a common complication of diabetes. Previous studies have identified significant increases in the amount of voltage gated sodium channel isoforms Na(V)1.7 and Na(V)1.3 protein in the dorsal root ganglia (DRG) of rats with streptozotocin (STZ)-induced diabetes. We found that gene transfer-mediated release of the inhibitory neurotransmitters enkephalin or gamma amino butyric acid (GABA) from DRG neurons in diabetic animals reduced pain-related behaviors coincident with a reduction in Na(V)1.7 protein levels in DRG in vivo. To further evaluate the role of Na(V)α subunit levels in DRG in the pathogenesis of pain in diabetic neuropathy, we constructed a non-replicating herpes simplex virus (HSV)-based vector expressing a microRNA (miRNA) against Na(V)α subunits. Subcutaneous inoculation of the miRNA-expressing HSV vector into the feet of diabetic rats to transduce DRG resulted in a reduction in Na(V)α subunit levels in DRG neurons, coincident with a reduction in cold allodynia, thermal hyperalgesia and mechanical hyperalgesia. These data support the role of increased Na(V)α protein in DRG in the pathogenesis of pain in diabetic neuropathy, and provide a proof-of-principle demonstration for the development of a novel therapy that could be used to treat intractable pain in patients with diabetic neuropathy.

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.

Adaptive evolution of voltage-gated sodium channels: The first 800 million years

PubMed Central

Zakon, Harold H.

2012-01-01

Voltage-gated Na+-permeable (Nav) channels form the basis for electrical excitability in animals. Nav channels evolved from Ca2+ channels and were present in the common ancestor of choanoflagellates and animals, although this channel was likely permeable to both Na+ and Ca2+. Thus, like many other neuronal channels and receptors, Nav channels predated neurons. Invertebrates possess two Nav channels (Nav1 and Nav2), whereas vertebrate Nav channels are of the Nav1 family. Approximately 500 Mya in early chordates Nav channels evolved a motif that allowed them to cluster at axon initial segments, 50 million years later with the evolution of myelin, Nav channels “capitalized” on this property and clustered at nodes of Ranvier. The enhancement of conduction velocity along with the evolution of jaws likely made early gnathostomes fierce predators and the dominant vertebrates in the ocean. Later in vertebrate evolution, the Nav channel gene family expanded in parallel in tetrapods and teleosts (∼9 to 10 genes in amniotes, 8 in teleosts). This expansion occurred during or after the late Devonian extinction, when teleosts and tetrapods each diversified in their respective habitats, and coincided with an increase in the number of telencephalic nuclei in both groups. The expansion of Nav channels may have allowed for more sophisticated neural computation and tailoring of Nav channel kinetics with potassium channel kinetics to enhance energy savings. Nav channels show adaptive sequence evolution for increasing diversity in communication signals (electric fish), in protection against lethal Nav channel toxins (snakes, newts, pufferfish, insects), and in specialized habitats (naked mole rats). PMID:22723361

A novel amino acid substitution in a voltage-gated sodium channel is associated with knockdown resistance to permethrin in Aedes aegypti.

PubMed

Chang, Cheng; Shen, Wen-Kai; Wang, Tzu-Ting; Lin, Ying-Hsi; Hsu, Err-Lieh; Dai, Shu-Mei

2009-04-01

To identify pertinent mutations associated with knockdown resistance to permethrin, the entire coding sequence of the voltage-gated sodium channel gene Aa-para was sequenced and analyzed from a Per-R strain with 190-fold resistance to permethrin and two susceptible strains of Aedes aegypti. The longest transcript, a 6441bp open reading frame, encodes 2147 amino acid residues with an estimated molecular mass of 241kDa. A total of 33 exons were found in the Aa-para gene over 293kb of genomic DNA. Three previously unreported optional exons were identified. The first two exons, m and n, were located within the intracellular domain I/II, and the third, f', was found within the II/III linkers. The two mutually exclusive exons, d and l, were the only alternative exons in all the cDNA clones sequenced in this study. The most distinct finding was a novel amino acid substitution mutation, D1794Y, located within the extracellular linker between IVS5 and IVS6, which is concurrent with the known V1023G mutation in Aa-para of the Per-R strain. The high frequency and coexistence of the two mutations in the Per-R strain suggest that they might exert a synergistic effect to provide the knockdown resistance to permethrin. Furthermore, both cDNA and genomic DNA data from the same individual mosquitoes have demonstrated that RNA editing was not involved in amino acid substitutions of the Per-R strain.

Docking Simulation of the Binding Interactions of Saxitoxin Analogs Produced by the Marine Dinoflagellate Gymnodinium catenatum to the Voltage-Gated Sodium Channel Nav1.4.

PubMed

Durán-Riveroll, Lorena M; Cembella, Allan D; Band-Schmidt, Christine J; Bustillos-Guzmán, José J; Correa-Basurto, José

2016-05-06

Saxitoxin (STX) and its analogs are paralytic alkaloid neurotoxins that block the voltage-gated sodium channel pore (Nav), impeding passage of Na⁺ ions into the intracellular space, and thereby preventing the action potential in the peripheral nervous system and skeletal muscle. The marine dinoflagellate Gymnodinium catenatum produces an array of such toxins, including the recently discovered benzoyl analogs, for which the mammalian toxicities are essentially unknown. We subjected STX and its analogs to a theoretical docking simulation based upon two alternative tri-dimensional models of the Nav1.4 to find a relationship between the binding properties and the known mammalian toxicity of selected STX analogs. We inferred hypothetical toxicities for the benzoyl analogs from the modeled values. We demonstrate that these toxins exhibit different binding modes with similar free binding energies and that these alternative binding modes are equally probable. We propose that the principal binding that governs ligand recognition is mediated by electrostatic interactions. Our simulation constitutes the first in silico modeling study on benzoyl-type paralytic toxins and provides an approach towards a better understanding of the mode of action of STX and its analogs.

Docking Simulation of the Binding Interactions of Saxitoxin Analogs Produced by the Marine Dinoflagellate Gymnodinium catenatum to the Voltage-Gated Sodium Channel Nav1.4

PubMed Central

Durán-Riveroll, Lorena M.; Cembella, Allan D.; Band-Schmidt, Christine J.; Bustillos-Guzmán, José J.; Correa-Basurto, José

2016-01-01

Saxitoxin (STX) and its analogs are paralytic alkaloid neurotoxins that block the voltage-gated sodium channel pore (Nav), impeding passage of Na+ ions into the intracellular space, and thereby preventing the action potential in the peripheral nervous system and skeletal muscle. The marine dinoflagellate Gymnodinium catenatum produces an array of such toxins, including the recently discovered benzoyl analogs, for which the mammalian toxicities are essentially unknown. We subjected STX and its analogs to a theoretical docking simulation based upon two alternative tri-dimensional models of the Nav1.4 to find a relationship between the binding properties and the known mammalian toxicity of selected STX analogs. We inferred hypothetical toxicities for the benzoyl analogs from the modeled values. We demonstrate that these toxins exhibit different binding modes with similar free binding energies and that these alternative binding modes are equally probable. We propose that the principal binding that governs ligand recognition is mediated by electrostatic interactions. Our simulation constitutes the first in silico modeling study on benzoyl-type paralytic toxins and provides an approach towards a better understanding of the mode of action of STX and its analogs. PMID:27164145

L1014F-kdr Mutation in Indian Anopheles subpictus (Diptera: Culicidae) Arising From Two Alternative Transversions in the Voltage-Gated Sodium Channel and a Single PIRA-PCR for Their Detection

PubMed Central

Singh, O. P.; Dykes, C. L.; Sharma, G.; Das, M. K.

2015-01-01

Leucine-to-phenylalanine substitution at residue L1014 in the voltage-gated sodium channel, target site of action for dichlorodiphenyltrichloroethane (DDT) and pyrethroids, is the most common knockdown resistance (kdr) mutation reported in several insects conferring resistance against DDT and pyrethroids. Here, we report presence of two coexisting alternative transversions, A>T and A>C, on the third codon position of L1014 residue in malaria vector Anopheles subpictus Grassi (species A) from Jamshedpur (India), both leading to the same amino acid substitution of Leu-to-Phe with allelic frequencies of 19 and 67%, respectively. A single primer-introduced restriction analysis–polymerase chain reaction (PIRA-PCR) was devised for the identification of L1014F-kdr mutation in An. subpictus resulting from either type of point mutation. Genotyping of samples with PIRA-PCR revealed high frequency (82%) of L1014F-kdr mutation in the study area. PMID:26336276

Aconitum and Delphinium sp. Alkaloids as Antagonist Modulators of Voltage-Gated Na+ Channels. AM1/DFT Electronic Structure Investigations and QSAR Studies

PubMed Central

Turabekova, Malakhat A.; Rasulev, Bakhtiyor F.; Levkovich, Mikhail G.; Abdullaev, Nasrulla D.; Leszczynski, Jerzy

2015-01-01

Early pharmacological studies of Aconitum and Delphinium sp. alkaloids suggested that these neurotoxins act at site 2 of voltage-gated Na+ channel and allosterically modulate its function. Understanding structural requirements for these compounds to exhibit binding activity at voltage-gated Na+ channel has been important in various fields. This paper reports quantum-chemical studies and quantitative structure-activity relationships (QSARs) based on a total of 65 natural alkaloids from two plant species, which includes both blockers and openers of sodium ion channel. A series of 18 antagonist alkaloids (9 blockers and 9 openers) have been studied using AM1 and DFT computational methods in order to reveal their structure-activity (structure-toxicity) relationship at electronic level. An examination of frontier orbitals obtained for ground and protonated forms of the compounds revealed that HOMOs and LUMOs were mainly represented by nitrogen atom and benzyl/benzoylester orbitals with –OH and –OCOCH3 contributions. The results obtained from this research have confirmed the experimental findings suggesting that neurotoxins acting at type 2 receptor site of voltage-dependent sodium channel are activators and blockers with common structural features and differ only in efficacy. The energetic tendency of HOMO-LUMO energy gap can probably distinguish activators and blockers that have been observed. Genetic Algorithm with Multiple Linear Regression Analysis (GA-MLRA) technique was also applied for the generation of two-descriptor QSAR models for the set of 65 blockers. Additionally to the computational studies, the HOMO-LUMO gap descriptor in each obtained QSAR model has confirmed the crucial role of charge transfer in receptor-ligand interactions. A number of other descriptors such as logP, IBEG, nNH2, nHDon, nCO have been selected as complementary ones to LUMO and their role in activity alteration has also been discussed. PMID:18201930

A comparison of 15 Hz sine on-line and off-line magnetic stimulation affecting the voltage-gated sodium channel currents of prefrontal cortex pyramidal neurons

NASA Astrophysics Data System (ADS)

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

2016-10-01

Combined with the use of patch-clamp techniques, repetitive transcranial magnetic stimulation (rTMS) has proven to be a noninvasive neuromodulation tool that can inhibit or facilitate excitability of neurons after extensive research. The studies generally focused on the method: the neurons are first stimulated in an external standard magnetic exposure device, and then moved to the patch-clamp to record electrophysiological characteristics (off-line magnetic exposure). Despite its universality, real-time observation of the effects of magnetic stimulation on the neurons is more effective (on-line magnetic stimulation). In this study, we selected a standard exposure device for magnetic fields acting on mouse prefrontal cortex pyramidal neurons, and described a new method that a patch-clamp setup was modified to allow on-line magnetic stimulation. By comparing the off-line exposure and on-line stimulation of the same magnetic field intensity and frequency affecting the voltage-gated sodium channel currents, we succeeded in proving the feasibility of the new on-line stimulation device. We also demonstrated that the sodium channel currents of prefrontal cortex pyramidal neurons increased significantly under the 15 Hz sine 1 mT, and 2 mT off-line magnetic field exposure and under the 1 mT and 2 mT on-line magnetic stimulation, and the rate of acceleration was most significant on 2 mT on-line magnetic stimulation. This study described the development of a new on-line magnetic stimulator and successfully demonstrated its practicability for scientific stimulation of neurons.

[Human calcium channelopathies. Voltage-gated Ca(2+) channels in etiology, pathogenesis, and pharmacotherapy of neurologic disorders].

PubMed

Weiergräber, M; Hescheler, J; Schneider, T

2008-04-01

Voltage-gated calcium channels are key components in a variety of physiological processes. Within the last decade an increasing number of voltage-gated Ca(2+) channelopathies in both humans and animal models has been described, most of which are related to the neurologic and muscular system. In humans, mutations were found in L-type Ca(v)1.2 and Ca(v)1.4 Ca(2+) channels as well as the non-L-type Ca(v)2.1 and T-type Ca(v)3.2 channels, resulting in altered electrophysiologic properties. Based on their widespread distribution within the CNS, voltage-gated calcium channels are of particular importance in the etiology and pathogenesis of various forms of epilepsy and neuropsychiatric disorders. In this review we characterise the different human Ca(2+) channelopathies known so far, further illuminating basic pathophysiologic mechanisms and clinical aspects.

Voltage-Gated Channel Mechanosensitivity: Fact or Friction?

PubMed Central

Morris, Catherine E.

2011-01-01

The heart is a continually active pulsatile fluid pump. It generates appropriate forces by precisely timed and spaced engagement of its contractile machinery. Largely, it makes its own control signals, the most crucial of which are precisely timed and spaced fluxes of ions across the sarcolemma, achieved by the timely opening and closing of diverse voltage-gated channels (VGC). VGCs have four voltage sensors around a central ion-selective pore that opens and closes under the influence of membrane voltage. Operation of any VGC is secondarily tuned by the mechanical state (i.e., structure) of the bilayer in which it is embedded. Rates of opening and closing, in other words, vary with bilayer structure. Thus, in the intensely mechanical environment of the myocardium and its vasculature, VGCs kinetics might be routinely modulated by reversible and irreversible nano-scale changes in bilayer structure. If subtle bilayer deformations are routine in the pumping heart, VGCs could be subtly transducing bilayer mechanical signals, thereby tuning cardiac rhythmicity, collectively contributing to mechano-electric feedback. Reversible bilayer deformations would be expected with changing shear flows and tissue distension, while irreversible bilayer restructuring occurs with ischemia, inflammation, membrane remodeling, etc. I suggest that tools now available could be deployed to help probe whether/how the inherent mechanosensitivity of VGCs – an attribute substantially reflecting the dependence of voltage sensor stability on bilayer structure – contributes to cardiac rhythmicity. Chief among these tools are voltage sensor toxins (whose inhibitory efficacy varies with the mechanical state of bilayer) and arrhythmia-inducing VGC mutants with distinctive mechano-phenotypes. PMID:21660289

Gating mechanism of Kv11.1 (hERG) K+ channels without covalent connection between voltage sensor and pore domains.

PubMed

de la Peña, Pilar; Domínguez, Pedro; Barros, Francisco

2018-03-01

Kv11.1 (hERG, KCNH2) is a voltage-gated potassium channel crucial in setting the cardiac rhythm and the electrical behaviour of several non-cardiac cell types. Voltage-dependent gating of Kv11.1 can be reconstructed from non-covalently linked voltage sensing and pore modules (split channels), challenging classical views of voltage-dependent channel activation based on a S4-S5 linker acting as a rigid mechanical lever to open the gate. Progressive displacement of the split position from the end to the beginning of the S4-S5 linker induces an increasing negative shift in activation voltage dependence, a reduced z g value and a more negative ΔG 0 for current activation, an almost complete abolition of the activation time course sigmoid shape and a slowing of the voltage-dependent deactivation. Channels disconnected at the S4-S5 linker near the S4 helix show a destabilization of the closed state(s). Furthermore, the isochronal ion current mode shift magnitude is clearly reduced in the different splits. Interestingly, the progressive modifications of voltage dependence activation gating by changing the split position are accompanied by a shift in the voltage-dependent availability to a methanethiosulfonate reagent of a Cys introduced at the upper S4 helix. Our data demonstrate for the first time that alterations in the covalent connection between the voltage sensor and the pore domains impact on the structural reorganizations of the voltage sensor domain. Also, they support the hypothesis that the S4-S5 linker integrates signals coming from other cytoplasmic domains that constitute either an important component or a crucial regulator of the gating machinery in Kv11.1 and other KCNH channels.

Pentacene-based low voltage organic field-effect transistors with anodized Ta2O5 gate dielectric

NASA Astrophysics Data System (ADS)

Jeong, Yeon Taek; Dodabalapur, Ananth

2007-11-01

Pentacene-based low voltage organic field-effect transistors were realized using an anodized Ta2O5 gate dielectric. The Ta2O5 gate dielectric layer with a surface roughness of 1.3Å was obtained by anodizing an e-beam evaporated Ta film. The device exhibited values of saturation mobility, threshold voltage, and Ion/Ioff ratio of 0.45cm2/Vs, 0.56V, and 7.5×101, respectively. The gate leakage current was reduced by more than 70% with a hexamethyldisilazane (HMDS) treatment on the Ta2O5 layer. The HMDS treatment also resulted in enhanced mobility values and a larger pentacene grain size.

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

Block of voltage-gated potassium channels by Pacific ciguatoxin-1 contributes to increased neuronal excitability in rat sensory neurons

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

Birinyi-Strachan, Liesl C.; Gunning, Simon J.; Lewis, Richard J.

2005-04-15

The present study investigated the actions of the polyether marine toxin Pacific ciguatoxin-1 (P-CTX-1) on neuronal excitability in rat dorsal root ganglion (DRG) neurons using patch-clamp recording techniques. Under current-clamp conditions, bath application of 2-20 nM P-CTX-1 caused a rapid, concentration-dependent depolarization of the resting membrane potential in neurons expressing tetrodotoxin (TTX)-sensitive voltage-gated sodium (Na{sub v}) channels. This action was completely suppressed by the addition of 200 nM TTX to the external solution, indicating that this effect was mediated through TTX-sensitive Na{sub v} channels. In addition, P-CTX-1 also prolonged action potential and afterhyperpolarization (AHP) duration. In a subpopulation of neurons,more » P-CTX-1 also produced tonic action potential firing, an effect that was not accompanied by significant oscillation of the resting membrane potential. Conversely, in neurons expressing TTX-resistant Na{sub v} currents, P-CTX-1 failed to alter any parameter of neuronal excitability examined in this study. Under voltage-clamp conditions in rat DRG neurons, P-CTX-1 inhibited both delayed-rectifier and 'A-type' potassium currents in a dose-dependent manner, actions that occurred in the absence of alterations to the voltage dependence of activation. These actions appear to underlie the prolongation of the action potential and AHP, and contribute to repetitive firing. These data indicate that a block of potassium channels contributes to the increase in neuronal excitability, associated with a modulation of Na{sub v} channel gating, observed clinically in response to ciguatera poisoning.« less

Comparison between mouse and sea urchin orthologs of voltage-gated proton channel suggests role of S3 segment in activation gating.

PubMed

Sakata, Souhei; Miyawaki, Nana; McCormack, Thomas J; Arima, Hiroki; Kawanabe, Akira; Özkucur, Nurdan; Kurokawa, Tatsuki; Jinno, Yuka; Fujiwara, Yuichiro; Okamura, Yasushi

2016-12-01

The voltage-gated proton channel, Hv1, is expressed in blood cells, airway epithelium, sperm and microglia, playing important roles in diverse biological contexts including phagocytosis or sperm maturation through its regulation of membrane potential and pH. The gene encoding Hv1, HVCN1, is widely found across many species and is also conserved in unicellular organisms such as algae or dinoflagellates where Hv1 plays role in calcification or bioluminescence. Voltage-gated proton channels exhibit a large variation of activation rate among different species. Here we identify an Hv1 ortholog from sea urchin, Strongylocentrotus purpuratus, SpHv1. SpHv1 retains most of key properties of Hv1 but exhibits 20-60 times more rapid activation kinetics than mammalian orthologs upon heterologous expression in HEK293T cells. Comparison between SpHv1 and mHv1 highlights novel roles of the third transmembrane segment S3 in activation gating of Hv1. Copyright © 2016 Elsevier B.V. All rights reserved.

Large scale rearrangement of protein domains is associated with voltage gating of the VDAC channel.

PubMed Central

Peng, S; Blachly-Dyson, E; Forte, M; Colombini, M

1992-01-01

The VDAC channel of the mitochondrial outer membrane is voltage-gated like the larger, more complex voltage-gated channels of the plasma membrane. However, VDAC is a low molecular weight (30 kDa), abundant protein, which is readily purified and reconstituted, making it an ideal system for analyzing the molecular basis for ion selectivity and voltage-gating. We have probed the VDAC channel by subjecting the cloned yeast (S. cerevisiae) VDAC gene to site-directed mutagenesis and introducing the resulting mutant channels into planar bilayers to detect the effects of specific sequence changes on channel properties. This approach has allowed us to formulate and test a model of the open state structure of the VDAC channel. Now we have applied the same approach to analyzing the structure of the channel's low-conducting "closed state" (essentially closed to important metabolites). We have identified protein domains forming the wall of the closed conformation and domains that seem to be removed from the wall of the pore during channel closure. The latter can explain the reduction in pore diameter and volume and the dramatically altered channel selectivity resulting from the channel closure. This process would make a natural coupling between motion of the sensor and channel gating. PMID:1376163

Inhibitory effects of magnolol on voltage-gated Na+ and K+ channels of NG108-15 cells.

PubMed

Gong, Chi-Li; Wong, Kar-Lok; Cheng, Ka-Shun; Kuo, Chang-Shin; Chao, Chia-Chia; Tsai, Min-Fan; Leung, Yuk-Man

2012-05-05

Magnolol, a polyphenolic compound isolated from Houpu, a Chinese herb from the bark of Magnolia officinalis, has been reported to have in vitro and in vivo neuroprotective effects. In spite of these reported beneficial effects, studies on the direct impact of magnolol on neuronal ion channels have been scarce. Whether magnolol affects voltage-gated Na(+) channels (VGSC) and voltage-gated K(+) (Kv) channels is unknown. Using the whole-cell voltage-clamp method, we studied the effects of magnolol on voltage-gated ion channels in neuronal NG108-15 cells. Magnolol inhibited VGSC channels with mild state-dependence (IC(50) of 15 and 30 μM, at holding potentials of -70 and -100 mV, respectively). No frequency-dependence was observed in magnolol block. Magnolol caused a left-shift of 18 mV in the steady-state inactivation curve but did not affect the voltage-dependence of activation. Magnolol inhibited Kv channels with an IC(50) of 21 μM, and it caused a 20-mV left-shift in the steady-state inactivation curve without affecting the voltage-dependence of activation. In conclusion, magnolol is an inhibitor of both VGSC and Kv channels and these inhibitory effects may in part contribute to some of the reported neuroprotective effects of magnolol. Copyright © 2012 Elsevier B.V. All rights reserved.

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 ...

Co-occurrence of Point Mutations in the Voltage-Gated Sodium Channel of Pyrethroid-Resistant Aedes aegypti Populations in Myanmar

PubMed Central

Kawada, Hitoshi; Oo, Sai Zaw Min; Thaung, Sein; Kawashima, Emiko; Maung, Yan Naung Maung; Thu, Hlaing Myat; Thant, Kyaw Zin; Minakawa, Noboru

2014-01-01

Background Single amino acid substitutions in the voltage-gated sodium channel associated with pyrethroid resistance constitute one of the main causative factors of knockdown resistance in insects. The kdr gene has been observed in several mosquito species; however, point mutations in the para gene of Aedes aegypti populations in Myanmar have not been fully characterized. The aim of the present study was to determine the types and frequencies of mutations in the para gene of Aedes aegypti collected from used tires in Yangon City, Myanmar. Methodology/Principal Findings We determined high pyrethroid resistance in Aedes aegypti larvae at all collection sites in Yangon City, by using a simplified knockdown bioassay. We showed that V1016G and S989P mutations were widely distributed, with high frequencies (84.4% and 78.8%, respectively). By contrast, we were unable to detect I1011M (or I1011V) or L1014F mutations. F1534C mutations were also widely distributed, but with a lower frequency than the V1016G mutation (21.2%). High percentage of co-occurrence of the homozygous V1016G/S989P mutations was detected (65.7%). Additionally, co-occurrence of homozygous V1016G/F1534C mutations (2.9%) and homozygous V1016G/F1534C/S989P mutations (0.98%) were detected in the present study. Conclusions/Significance Pyrethroid insecticides were first used for malaria control in 1992, and have since been constantly used in Myanmar. This intensive use may explain the strong selection pressure toward Aedes aegypti, because this mosquito is generally a domestic and endophagic species with a preference for indoor breeding. Extensive use of DDT for malaria control before the use of this chemical was banned may also explain the development of pyrethroid resistance in Aedes aegypti. PMID:25077956

Co-occurrence of point mutations in the voltage-gated sodium channel of pyrethroid-resistant Aedes aegypti populations in Myanmar.

PubMed

Kawada, Hitoshi; Oo, Sai Zaw Min; Thaung, Sein; Kawashima, Emiko; Maung, Yan Naung Maung; Thu, Hlaing Myat; Thant, Kyaw Zin; Minakawa, Noboru

2014-01-01

Single amino acid substitutions in the voltage-gated sodium channel associated with pyrethroid resistance constitute one of the main causative factors of knockdown resistance in insects. The kdr gene has been observed in several mosquito species; however, point mutations in the para gene of Aedes aegypti populations in Myanmar have not been fully characterized. The aim of the present study was to determine the types and frequencies of mutations in the para gene of Aedes aegypti collected from used tires in Yangon City, Myanmar. We determined high pyrethroid resistance in Aedes aegypti larvae at all collection sites in Yangon City, by using a simplified knockdown bioassay. We showed that V1016G and S989P mutations were widely distributed, with high frequencies (84.4% and 78.8%, respectively). By contrast, we were unable to detect I1011M (or I1011V) or L1014F mutations. F1534C mutations were also widely distributed, but with a lower frequency than the V1016G mutation (21.2%). High percentage of co-occurrence of the homozygous V1016G/S989P mutations was detected (65.7%). Additionally, co-occurrence of homozygous V1016G/F1534C mutations (2.9%) and homozygous V1016G/F1534C/S989P mutations (0.98%) were detected in the present study. Pyrethroid insecticides were first used for malaria control in 1992, and have since been constantly used in Myanmar. This intensive use may explain the strong selection pressure toward Aedes aegypti, because this mosquito is generally a domestic and endophagic species with a preference for indoor breeding. Extensive use of DDT for malaria control before the use of this chemical was banned may also explain the development of pyrethroid resistance in Aedes aegypti.

mRNAs coding for neurotransmitter receptors and voltage-gated sodium channels in the adult rabbit visual cortex after monocular deafferentiation

PubMed Central

Nguyen, Quoc-Thang; Matute, Carlos; Miledi, Ricardo

1998-01-01

It has been postulated that, in the adult visual cortex, visual inputs modulate levels of mRNAs coding for neurotransmitter receptors in an activity-dependent manner. To investigate this possibility, we performed a monocular enucleation in adult rabbits and, 15 days later, collected their left and right visual cortices. Levels of mRNAs coding for voltage-activated sodium channels, and for receptors for kainate/α-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA), N-methyl-d-aspartate (NMDA), γ-aminobutyric acid (GABA), and glycine were semiquantitatively estimated in the visual cortices ipsilateral and contralateral to the lesion by the Xenopus oocyte/voltage-clamp expression system. This technique also allowed us to study some of the pharmacological and physiological properties of the channels and receptors expressed in the oocytes. In cells injected with mRNA from left or right cortices of monocularly enucleated and control animals, the amplitudes of currents elicited by kainate or AMPA, which reflect the abundance of mRNAs coding for kainate and AMPA receptors, were similar. There was no difference in the sensitivity to kainate and in the voltage dependence of the kainate response. Responses mediated by NMDA, GABA, and glycine were unaffected by monocular enucleation. Sodium channel peak currents, activation, steady-state inactivation, and sensitivity to tetrodotoxin also remained unchanged after the enucleation. Our data show that mRNAs for major neurotransmitter receptors and ion channels in the adult rabbit visual cortex are not obviously modified by monocular deafferentiation. Thus, our results do not support the idea of a widespread dynamic modulation of mRNAs coding for receptors and ion channels by visual activity in the rabbit visual system. PMID:9501250

Structural basis for the inhibition of voltage-dependent K+ channel by gating modifier toxin

PubMed Central

Ozawa, Shin-ichiro; Kimura, Tomomi; Nozaki, Tomohiro; Harada, Hitomi; Shimada, Ichio; Osawa, Masanori

2015-01-01

Voltage-dependent K+ (Kv) channels play crucial roles in nerve and muscle action potentials. Voltage-sensing domains (VSDs) of Kv channels sense changes in the transmembrane potential, regulating the K+-permeability across the membrane. Gating modifier toxins, which have been used for the functional analyses of Kv channels, inhibit Kv channels by binding to VSD. However, the structural basis for the inhibition remains elusive. Here, fluorescence and NMR analyses of the interaction between VSD derived from KvAP channel and its gating modifier toxin, VSTx1, indicate that VSTx1 recognizes VSD under depolarized condition. We identified the VSD-binding residues of VSTx1 and their proximal residues of VSD by the cross-saturation (CS) and amino acid selective CS experiments, which enabled to build a docking model of the complex. These results provide structural basis for the specific binding and inhibition of Kv channels by gating modifier toxins. PMID:26382304

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.

Calmodulin and calcium differentially regulate the neuronal Nav1.1 voltage-dependent sodium channel

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

Gaudioso, Christelle; Carlier, Edmond; Youssouf, Fahamoe

2011-07-29

Highlights: {yields} Both Ca{sup ++}-Calmodulin (CaM) and Ca{sup ++}-free CaM bind to the C-terminal region of Nav1.1. {yields} Ca{sup ++} and CaM have both opposite and convergent effects on I{sub Nav1.1}. {yields} Ca{sup ++}-CaM modulates I{sub Nav1.1} amplitude. {yields} CaM hyperpolarizes the voltage-dependence of activation, and increases the inactivation rate. {yields} Ca{sup ++} alone antagonizes CaM for both effects, and depolarizes the voltage-dependence of inactivation. -- Abstract: Mutations in the neuronal Nav1.1 voltage-gated sodium channel are responsible for mild to severe epileptic syndromes. The ubiquitous calcium sensor calmodulin (CaM) bound to rat brain Nav1.1 and to the human Nav1.1 channelmore » expressed by a stably transfected HEK-293 cell line. The C-terminal region of the channel, as a fusion protein or in the yeast two-hybrid system, interacted with CaM via a consensus C-terminal motif, the IQ domain. Patch clamp experiments on HEK1.1 cells showed that CaM overexpression increased peak current in a calcium-dependent way. CaM had no effect on the voltage-dependence of fast inactivation, and accelerated the inactivation kinetics. Elevating Ca{sup ++} depolarized the voltage-dependence of fast inactivation and slowed down the fast inactivation kinetics, and for high concentrations this effect competed with the acceleration induced by CaM alone. Similarly, the depolarizing action of calcium antagonized the hyperpolarizing shift of the voltage-dependence of activation due to CaM overexpression. Fluorescence spectroscopy measurements suggested that Ca{sup ++} could bind the Nav1.1 C-terminal region with micromolar affinity.« less

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

A magnesium–sodium hybrid battery with high operating voltage

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

Dong, Hui; Li, Yifei; Liang, Yanliang

2016-06-10

We report a high performance magnesium-sodium hybrid battery utilizing a magnesium-sodium dual-salt electrolyte, a magnesium anode, and a Berlin green cathode. The cell delivers an average discharge voltage of 2.2 V and a reversible capacity of 143 mAh g -1. We also demonstrate the cell with an energy density of 135 Wh kg -1 and a high power density of up to 1.67 kW kg -1.

Modulation of voltage-gated Na+ and K+ channels by pumiliotoxin 251D: a "joint venture" alkaloid from arthropods and amphibians.

PubMed

Vandendriessche, Thomas; Abdel-Mottaleb, Yousra; Maertens, Chantal; Cuypers, Eva; Sudau, Alexander; Nubbemeyer, Udo; Mebs, Dietrich; Tytgat, Jan

2008-03-01

Certain amphibians provide themselves with a chemical defense by accumulating lipophilic alkaloids into skin glands from dietary arthropods. Examples of such alkaloids are pumiliotoxins (PTXs). In general, PTXs are known as positive modulators of voltage-gated sodium channels (VGSCs). Unlike other PTXs, PTX 251D does not share this characteristic. However, mice and insect studies showed that PTX 251D is highly toxic and to date the basis of its toxicity remains unknown. In this work, we searched for the possible target of PTX 251D. The toxin was therefore made synthetically and tested on four VGSCs (mammalian rNa(v)1.2/beta(1), rNa(v)1.4/beta(1), hNa(v)1.5/beta(1) and insect Para/tipE) and five voltage-gated potassium channels (VGPCs) (mammalian rK(v)1.1-1.2, hK(v)1.3, hK(v)11.1 (hERG) and insect Shaker IR) expressed heterologously in Xenopus laevis oocytes, using the two-electrode voltage clamp technique. PTX 251D not only inhibited the Na(+) influx through the mammalian VGSCs but also affected the steady-state activation and inactivation. Interestingly, in the insect ortholog, the inactivation process was dramatically affected. Additionally, PTX 251D inhibited the K(+) efflux through all five tested VGPCs and slowed down the deactivation kinetics of the mammalian VGPCs. hK(v)1.3 was the most sensitive channel, with an IC(50) value 10.8+/-0.5 microM. To the best of our knowledge this is the first report of a PTX affecting VGPCs.

Further characterization of the effect of ethanol on voltage-gated Ca(2+) channel function in developing CA3 hippocampal pyramidal neurons.

PubMed

Morton, Russell A; Valenzuela, C Fernando

2016-02-15

Developmental ethanol exposure damages the hippocampus, a brain region involved in learning and memory. Alterations in synaptic transmission and plasticity may play a role in this effect of ethanol. We previously reported that acute and repeated exposure to ethanol during the third trimester-equivalent inhibits long-term potentiation of GABAA receptor-dependent synaptic currents in CA3 pyramidal neurons through a mechanism that depends on retrograde release of brain-derived neurotrophic factor driven by activation of voltage-gated Ca(2+) channels (Zucca and Valenzuela, 2010). We found evidence indicating that voltage-gated Ca(2+) channels are inhibited in the presence of ethanol, an effect that may play a role in its mechanism of action. Here, we further investigated the acute effect of ethanol on the function of voltage-gated Ca(2+) channels in CA3 pyramidal neurons using Ca(2+) imaging techniques. These experiments revealed that acute ethanol exposure inhibits voltage-gated Ca(2+) channels both in somatic and proximal dendritic compartments. To investigate the long-term consequences of ethanol on voltage-gated Ca(2+) channels, we used patch-clamp electrophysiological techniques to assess the function of L-type voltage-gated Ca(2+) channels during and following ten days of vapor ethanol exposure. During ethanol withdrawal periods, the function of these channels was not significantly affected by vapor chamber exposure. Taken together with our previous findings, our results suggest that 3(rd) trimester-equivalent ethanol exposure transiently inhibits L-type voltage-gated Ca(2+) channel function in CA3 pyramidal neurons and that compensatory mechanisms restore their function during ethanol withdrawal. Transient inhibition of these channels by ethanol may be, in part, responsible for the hippocampal abnormalities associated with developmental exposure to this agent. Copyright © 2015 Elsevier B.V. All rights reserved.

Genistein inhibition of OGD-induced brain neuron death correlates with its modulation of apoptosis, voltage-gated potassium and sodium currents and glutamate signal pathway.

PubMed

Ma, Xue-Ling; Zhang, Feng; Wang, Yu-Xiang; He, Cong-Cong; Tian, Kun; Wang, Hong-Gang; An, Di; Heng, Bin; Liu, Yan-Qiang

2016-07-25

In the present study, we established an in vitro model of hypoxic-ischemia via exposing primary neurons of newborn rats to oxygen-glucose deprivation (OGD) and observing the effects of genistein, a soybean isoflavone, on hypoxic-ischemic neuron viability, apoptosis, voltage-activated potassium (Kv) and sodium (Nav) currents, and glutamate receptor subunits. The results indicated that OGD exposure reduced the viability and increased the apoptosis of brain neurons. Meanwhile, OGD exposure caused changes in the current-voltage curves and current amplitude values of voltage-activated potassium and sodium currents; OGD exposure also decreased GluR2 expression and increased NR2 expression. However, genistein at least partially reversed the effects caused by OGD. The results suggest that hypoxic-ischemia-caused neuronal apoptosis/death is related to an increase in K(+) efflux, a decrease in Na(+) influx, a down-regulation of GluR2, and an up-regulation of NR2. Genistein may exert some neuroprotective effects via the modulation of Kv and Nav currents and the glutamate signal pathway, mediated by GluR2 and NR2. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

Dextromethorphan inhibition of voltage-gated proton currents in BV2 microglial cells.

PubMed

Song, Jin-Ho; Yeh, Jay Z

2012-05-10

Dextromethorphan, an antitussive drug, has a neuroprotective property as evidenced by its inhibition of microglial production of pro-inflammatory cytokines and reactive oxygen species. The microglial activation requires NADPH oxidase activity, which is sustained by voltage-gated proton channels in microglia as they dissipate an intracellular acid buildup. In the present study, we examined the effect of dextromethorphan on proton currents in microglial BV2 cells. Dextromethorphan reversibly inhibited proton currents with an IC(50) value of 51.7 μM at an intracellular/extracellular pH gradient of 5.5/7.3. Dextromethorphan did not change the reversal potential or the voltage dependence of the gating. Dextrorphan and 3-hydroxymorphinan, major metabolites of dextromethorphan, and dextromethorphan methiodide were ineffective in inhibiting proton currents. The results indicate that dextromethorphan inhibition of proton currents would suppress NADPH oxidase activity and, eventually, microglial activation. Copyright © 2012 Elsevier Ireland Ltd. All rights reserved.

Epidermal growth factor upregulates motility of Mat-LyLu rat prostate cancer cells partially via voltage-gated Na+ channel activity

PubMed Central

Ding, Yanning; Brackenbury, William J.; Onganer, Pinar U.; Montano, Ximena; Porter, Louise M.; Bates, Lucy F.; Djamgoz, Mustafa B. A.

2014-01-01

The main aim of this investigation was to determine whether a functional relationship existed between epidermal growth factor (EGF) and voltage-gated sodium channel (VGSC) upregulation, both associated with strongly metastatic prostate cancer cells. Incubation with EGF for 24 h more than doubled VGSC current density. Similar treatment with EGF significantly and dose-dependently enhanced the cells’ migration through Transwell filters. Both the patch clamp recordings and the migration assay suggested that endogenous EGF played a similar role. Importantly, co-application of EGF and tetrodotoxin, a highly selective VGSC blocker, abolished 65% of the potentiating effect of EGF. It is suggested that a significant portion of the EGF-induced enhancement of migration occurred via VGSC activity. PMID:17960590

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 Natural and Unnatural History of the Voltage-Gated Na(+) Channel.

PubMed

Moczydlowski, E G

2016-01-01

This review glances at the voltage-gated sodium (Na(+)) channel (NaV) from the skewed perspective of natural history and the history of ideas. Beginning with the earliest natural philosophers, the objective of biological science and physiology was to understand the basis of life and discover its intimate secrets. The idea that the living state of matter differs from inanimate matter by an incorporeal spirit or mystical force was central to vitalism, a doctrine based on ancient beliefs that persisted until the last century. Experimental electrophysiology played a major role in the abandonment of vitalism by elucidating physiochemical mechanisms that explained the electrical excitability of muscle and nerve. Indeed, as a principal biomolecule underlying membrane excitability, the NaV channel may be considered as the physical analog or surrogate for the vital spirit once presumed to animate higher forms of life. NaV also epitomizes the "other secret of life" and functions as a quantal transistor element of biological intelligence. Subplots of this incredible but true story run the gamut from electric fish to electromagnetism, invention of the battery, venomous animals, neurotoxins, channelopathies, arrhythmia, anesthesia, astrobiology, etc. Copyright © 2016 Elsevier Inc. All rights reserved.

A magnesium–sodium hybrid battery with high operating voltage

DOE PAGES

Dong, Hui; Li, Yifei; Liang, Yanliang; ...

2016-06-10

Here, we report a high performance magnesium-sodium hybrid battery utilizing a magnesium-sodium dual-salt electrolyte, a magnesium anode, and a Berlin green cathode. The cell delivers an average discharge voltage of 2.2 V and a reversible capacity of 143 mA h g –1. We also demonstrate the cell with an energy density of 135 W h kg –1 and a high power density of up to 1.67 kW kg –1.

Voltage-sensing domain mode shift is coupled to the activation gate by the N-terminal tail of hERG channels.

PubMed

Tan, Peter S; Perry, Matthew D; Ng, Chai Ann; Vandenberg, Jamie I; Hill, Adam P

2012-09-01

Human ether-a-go-go-related gene (hERG) potassium channels exhibit unique gating kinetics characterized by unusually slow activation and deactivation. The N terminus of the channel, which contains an amphipathic helix and an unstructured tail, has been shown to be involved in regulation of this slow deactivation. However, the mechanism of how this occurs and the connection between voltage-sensing domain (VSD) return and closing of the gate are unclear. To examine this relationship, we have used voltage-clamp fluorometry to simultaneously measure VSD motion and gate closure in N-terminally truncated constructs. We report that mode shifting of the hERG VSD results in a corresponding shift in the voltage-dependent equilibrium of channel closing and that at negative potentials, coupling of the mode-shifted VSD to the gate defines the rate of channel closure. Deletion of the first 25 aa from the N terminus of hERG does not alter mode shifting of the VSD but uncouples the shift from closure of the cytoplasmic gate. Based on these observations, we propose the N-terminal tail as an adaptor that couples voltage sensor return to gate closure to define slow deactivation gating in hERG channels. Furthermore, because the mode shift occurs on a time scale relevant to the cardiac action potential, we suggest a physiological role for this phenomenon in maximizing current flow through hERG channels during repolarization.

Molecule desorption induced by gate-voltage application in MOS structure

NASA Astrophysics Data System (ADS)

Hirota, Nozomu; Hattori, Ken; Daimon, Hiroshi; Hattori, Azusa N.; Tanaka, Hidekazu

2016-04-01

For the first time, we demonstrate desorption from a MOS surface by applying gate voltages (V G). We observed CH4, CO, and CO2 desorption from a MOS (Fe nanofilm/a-SiO2/Si) surface in vacuum only when applying negative V G, suggesting the occurrence of electronic excitation by hot-hole injection. This demonstration is the first step in the application of MOSs to electrically controlled catalysts.

Voltage-gated calcium flux mediates Escherichia coli mechanosensation.

PubMed

Bruni, Giancarlo N; Weekley, R Andrew; Dodd, Benjamin J T; Kralj, Joel M

2017-08-29

Electrically excitable cells harness voltage-coupled calcium influx to transmit intracellular signals, typically studied in neurons and cardiomyocytes. Despite intense study in higher organisms, investigations of voltage and calcium signaling in bacteria have lagged due to their small size and a lack of sensitive tools. Only recently were bacteria shown to modulate their membrane potential on the timescale of seconds, and little is known about the downstream effects from this modulation. In this paper, we report on the effects of electrophysiology in individual bacteria. A genetically encoded calcium sensor expressed in Escherichia coli revealed calcium transients in single cells. A fusion sensor that simultaneously reports voltage and calcium indicated that calcium influx is induced by voltage depolarizations, similar to metazoan action potentials. Cytoplasmic calcium levels and transients increased upon mechanical stimulation with a hydrogel, and single cells altered protein concentrations dependent on the mechanical environment. Blocking voltage and calcium flux altered mechanically induced changes in protein concentration, while inducing calcium flux reproduced these changes. Thus, voltage and calcium relay a bacterial sense of touch and alter cellular lifestyle. Although the calcium effectors remain unknown, these data open a host of new questions about E. coli , including the identity of the underlying molecular players, as well as other signals conveyed by voltage and calcium. These data also provide evidence that dynamic voltage and calcium exists as a signaling modality in the oldest domain of life, and therefore studying electrophysiology beyond canonical electrically excitable cells could yield exciting new findings.

Voltage-gated calcium flux mediates Escherichia coli mechanosensation

PubMed Central

Weekley, R. Andrew; Dodd, Benjamin J. T.

2017-01-01

Electrically excitable cells harness voltage-coupled calcium influx to transmit intracellular signals, typically studied in neurons and cardiomyocytes. Despite intense study in higher organisms, investigations of voltage and calcium signaling in bacteria have lagged due to their small size and a lack of sensitive tools. Only recently were bacteria shown to modulate their membrane potential on the timescale of seconds, and little is known about the downstream effects from this modulation. In this paper, we report on the effects of electrophysiology in individual bacteria. A genetically encoded calcium sensor expressed in Escherichia coli revealed calcium transients in single cells. A fusion sensor that simultaneously reports voltage and calcium indicated that calcium influx is induced by voltage depolarizations, similar to metazoan action potentials. Cytoplasmic calcium levels and transients increased upon mechanical stimulation with a hydrogel, and single cells altered protein concentrations dependent on the mechanical environment. Blocking voltage and calcium flux altered mechanically induced changes in protein concentration, while inducing calcium flux reproduced these changes. Thus, voltage and calcium relay a bacterial sense of touch and alter cellular lifestyle. Although the calcium effectors remain unknown, these data open a host of new questions about E. coli, including the identity of the underlying molecular players, as well as other signals conveyed by voltage and calcium. These data also provide evidence that dynamic voltage and calcium exists as a signaling modality in the oldest domain of life, and therefore studying electrophysiology beyond canonical electrically excitable cells could yield exciting new findings. PMID:28808010

Effect of angiotension II on voltage-gated sodium currents in aortic baroreceptor neurons and arterial baroreflex sensitivity in heart failure rats

PubMed Central

Zhang, Dongze; Liu, Jinxu; Zheng, Hong; Tu, Huiyin; Muelleman, Robert L.; Li, Yu-Long

2016-01-01

Impairment of arterial baroreflex sensitivity is associated with mortality in patients with chronic heart failure (CHF). Elevation of plasma angiotension II (Ang II) contributes to arterial baroreflex dysfunction in CHF. A reduced number of voltage-gated sodium (Nav) channels in aortic baroreceptor neurons are involved in CHF-blunted arterial baroreflex. In this study, we investigated acute effect of Ang II on Nav currents in the aortic baroreceptor neuron and on arterial baroreflex in sham and coronary artery ligation-induced CHF rats. Using Ang II 125I radioimmunoassay, real-time RT-PCR and western blot, we found that Ang II levels, and mRNA and protein expression of angiotension II type 1 receptor (AT1R) in nodose ganglia (NG) from CHF rats were higher than that from sham rats. Local microinjection of Ang II (0.2 nmol) into the NG decreased the arterial baroreflex sensitivity in sham rats, whereas losartan (1 nmol, an AT1R antagonist) improved the arterial baroreflex sensitivity in CHF rats. Data from patch-clamp recording showed that Ang II (100 nM) acutely inhibited Nav currents in the aortic baroreceptor neurons from sham and CHF rats. In particular, inhibitory effect of Ang II on Nav currents in the aortic baroreceptor neurons was larger in CHF rats than that in sham rats. Losartan (1 μM) totally abolished the inhibitory effect of Ang II on Nav currents in sham and CHF aortic baroreceptor neurons. These results suggest that elevation of endogenous Ang II in the NG contributes to impairment of the arterial baroreflex function in CHF rats through inhibiting Nav channels. PMID:25827427

Structural characterization of the voltage sensor domain and voltage-gated K+- channel proteins vectorially-oriented within a single bilayer membrane at the solid/vapor and solid/liquid interfaces via neutron interferometry

PubMed Central

Gupta, S.; Dura, J.A.; Freites, J.A.; Tobias, D.J.; Blasie, J. K.

2012-01-01

The voltage-sensor domain (VSD) is a modular 4-helix bundle component that confers voltage sensitivity to voltage-gated cation channels in biological membranes. Despite extensive biophysical studies and the recent availability of x-ray crystal structures for a few voltage-gated potassium (Kv-) channels and a voltage-gate sodium (Nav-) channel, a complete understanding of the cooperative mechanism of electromechanical coupling, interconverting the closed-to-open states (i.e. non-conducting to cation conducting) remains undetermined. Moreover, the function of these domains is highly dependent on the physical-chemical properties of the surrounding lipid membrane environment. The basis for this work was provided by a recent structural study of the VSD from a prokaryotic Kv-channel vectorially-oriented within a single phospholipid (POPC; 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine) membrane investigated by x-ray interferometry at the solid/moist He (or solid/vapor) and solid/liquid interfaces thus achieving partial to full hydration, respectively (Gupta et. al. Phys. Rev E. 2011, 84). Here, we utilize neutron interferometry to characterize this system in substantially greater structural detail at the sub-molecular level, due to its inherent advantages arising from solvent contrast variation coupled with the deuteration of selected sub-molecular membrane components, especially important for the membrane at the solid/liquid interface. We demonstrate the unique vectorial orientation of the VSD and the retention of its molecular conformation manifest in the asymmetric profile structure of the protein within the profile structure of this single bilayer membrane system. We definitively characterize the asymmetric phospholipid bilayer solvating the lateral surfaces of the VSD protein within the membrane. The profile structures of both the VSD protein and phospholipid bilayer depend upon the hydration state of the membrane. We also determine the distribution of water and

L1014F-kdr Mutation in Indian Anopheles subpictus (Diptera: Culicidae) Arising From Two Alternative Transversions in the Voltage-Gated Sodium Channel and a Single PIRA-PCR for Their Detection.

PubMed

Singh, O P; Dykes, C L; Sharma, G; Das, M K

2015-01-01

Leucine-to-phenylalanine substitution at residue L1014 in the voltage-gated sodium channel, target site of action for dichlorodiphenyltrichloroethane (DDT) and pyrethroids, is the most common knockdown resistance (kdr) mutation reported in several insects conferring resistance against DDT and pyrethroids. Here, we report presence of two coexisting alternative transversions, A>T and A>C, on the third codon position of L1014 residue in malaria vector Anopheles subpictus Grassi (species A) from Jamshedpur (India), both leading to the same amino acid substitution of Leu-to-Phe with allelic frequencies of 19 and 67%, respectively. A single primer-introduced restriction analysis-polymerase chain reaction (PIRA-PCR) was devised for the identification of L1014F-kdr mutation in An. subpictus resulting from either type of point mutation. Genotyping of samples with PIRA-PCR revealed high frequency (82%) of L1014F-kdr mutation in the study area. © The Authors 2015. Published by Oxford University Press on behalf of Entomological Society of America. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

Reversible control of doping in graphene-on-SiO2 by cooling under gate-voltage

NASA Astrophysics Data System (ADS)

Singh, Anil Kumar; Gupta, Anjan Kumar

2017-11-01

The electronic properties of graphene can be modulated by various doping techniques other than back-gate, but most such methods are not easily reversible and also lead to mobility reduction. Here, we report on the reversible control of doping in graphene by cooling under back-gate-voltage. The observed variation in hysteresis in our devices with the temperature and interface preparation method is attributed to the variation in the density of redox species, namely, H2O and O2, at the graphene/SiO2 interface, and their diffusion. With careful interface preparation, we have been able to make devices with negligible hysteresis at room temperature and by exploiting hysteresis at high temperatures, we get a wide, but reversible tunability of interface charge density and graphene doping, by cooling to room temperature under gate-voltage. Such reversible control of graphene doping by manipulating the interface defect charge density can help in making new data storage devices using graphene.

Mechanism of the Association between Na+ Binding and Conformations at the Intracellular Gate in Neurotransmitter:Sodium Symporters*

PubMed Central

Stolzenberg, Sebastian; Quick, Matthias; Zhao, Chunfeng; Gotfryd, Kamil; Khelashvili, George; Gether, Ulrik; Loland, Claus J.; Javitch, Jonathan A.; Noskov, Sergei; Weinstein, Harel; Shi, Lei

2015-01-01

Neurotransmitter:sodium symporters (NSSs) terminate neurotransmission by Na+-dependent reuptake of released neurotransmitters. Previous studies suggested that Na+-binding reconfigures dynamically coupled structural elements in an allosteric interaction network (AIN) responsible for function-related conformational changes, but the intramolecular pathway of this mechanism has remained uncharted. We describe a new approach for the modeling and analysis of intramolecular dynamics in the bacterial NSS homolog LeuT. From microsecond-scale molecular dynamics simulations and cognate experimental verifications in both LeuT and human dopamine transporter (hDAT), we apply the novel method to identify the composition and the dynamic properties of their conserved AIN. In LeuT, two different perturbations disrupting Na+ binding and transport (i.e. replacing Na+ with Li+ or the Y268A mutation at the intracellular gate) affect the AIN in strikingly similar ways. In contrast, other mutations that affect the intracellular gate (i.e. R5A and D369A) do not significantly impair Na+ cooperativity and transport. Our analysis shows these perturbations to have much lesser effects on the AIN, underscoring the sensitivity of this novel method to the mechanistic nature of the perturbation. Notably, this set of observations holds as well for hDAT, where the aligned Y335A, R60A, and D436A mutations also produce different impacts on Na+ dependence. Thus, the detailed AIN generated from our method is shown to connect Na+ binding with global conformational changes that are critical for the transport mechanism. That the AIN between the Na+ binding sites and the intracellular gate in bacterial LeuT resembles that in eukaryotic hDAT highlights the conservation of allosteric pathways underlying NSS function. PMID:25869126

Mechanism of the association between Na + binding and conformations at the intracellular gate in neurotransmitter:sodium symporters

DOE PAGES

Stolzenberg, Sebastian; Quick, Matthias; Zhao, Chunfeng; ...

2015-04-13

Neurotransmitter:sodium symporters (NSSs) terminate neurotransmission by Na +-dependent reuptake of released neurotransmitters. Previous studies suggested that Na +-binding reconfigures dynamically coupled structural elements in an allosteric interaction network (AIN) responsible for function-related conformational changes, but the intramolecular pathway of this mechanism has remained uncharted. Here we describe a new approach for the modeling and analysis of intramolecular dynamics in the bacterial NSS homolog LeuT. From microsecond-scale molecular dynamics simulations and cognate experimental verifications in both LeuT and human dopamine transporter (hDAT), we apply the novel method to identify the composition and the dynamic properties of their conserved AIN. In LeuT,more » two different perturbations disrupting Na+ binding and transport ( i.e. replacing Na + with Li + or the Y268A mutation at the intracellular gate) affect the AIN in strikingly similar ways. In contrast, other mutations that affect the intracellular gate (i.e. R5A and D369A) do not significantly impair Na + cooperativity and transport. Our analysis shows these perturbations to have much lesser effects on the AIN, underscoring the sensitivity of this novel method to the mechanistic nature of the perturbation. Notably, this set of observations holds as well for hDAT, where the aligned Y335A, R60A, and D436A mutations also produce different impacts on Na + dependence. Furthermore, the detailed AIN generated from our method is shown to connect Na + binding with global conformational changes that are critical for the transport mechanism. Lastly, that the AIN between the Na + binding sites and the intracellular gate in bacterial LeuT resembles that in eukaryotic hDAT highlights the conservation of allosteric pathways underlying NSS function.« less

Aptiom (eslicarbazepine acetate) as a dual inhibitor of β-secretase and voltage-gated sodium channel: advancement in Alzheimer's disease-epilepsy linkage via an enzoinformatics study.

PubMed

Shaikh, Sibhghatulla; Rizvi, Syed M D; Hameed, Nida; Biswas, Deboshree; Khan, Mahiuddin; Shakil, Shazi; Kamal, Mohammad A

2014-01-01

Neurodegenerative disorders are increasingly identified as one of the major causes of epilepsy. The relationship of epileptic activity to Alzheimer's disease (AD) is of clinical importance. Voltage-gated sodium channel (VSC) is one of the best targets in the treatment of epilepsy while β-secretase (BACE) has long been observed as a curative target for AD. To explore a possible link between the treatment of AD and epilepsy, the molecular interactions of recently Food and Drug Administration approved antiepileptic drug Aptiom (Eslicarbazepine acetate) with BACE and VSC were studied. Docking study was performed using 'Autodock4.2'. Hydrophobic and pi-pi interactions play critical role in the correct positioning of Eslicarbazepine acetate within the catalytic site of VSC and BACE enzyme to permit docking. Free energy of binding (ΔG) of 'Eslicarbazepine acetate-VSC' interaction and 'Eslicarbazepine acetate-CAS domain of BACE' interaction was found to be -5.97 and -7.19 kcal/mol, respectively. Hence, Eslicarbazepine acetate might act as a potent dual inhibitor of BACE and VSC. However, scope still remains in the determination of the three-dimensional structure of BACE-Eslicarbazepine acetate and VSC-Eslicarbazepine acetate complexes by X-ray crystallography to validate the described data. Further, Aptiom (Eslicarbazepine acetate) could be expected to form the basis of future dual therapy against epilepsy associated neurological disorders.

Ephaptic coupling rescues conduction failure in weakly coupled cardiac tissue with voltage-gated gap junctions

NASA Astrophysics Data System (ADS)

Weinberg, S. H.

2017-09-01

Electrical conduction in cardiac tissue is usually considered to be primarily facilitated by gap junctions, providing a pathway between the intracellular spaces of neighboring cells. However, recent studies have highlighted the role of coupling via extracellular electric fields, also known as ephaptic coupling, particularly in the setting of reduced gap junction expression. Further, in the setting of reduced gap junctional coupling, voltage-dependent gating of gap junctions, an oft-neglected biophysical property in computational studies, produces a positive feedback that promotes conduction failure. We hypothesized that ephaptic coupling can break the positive feedback loop and rescue conduction failure in weakly coupled cardiac tissue. In a computational tissue model incorporating voltage-gated gap junctions and ephaptic coupling, we demonstrate that ephaptic coupling can rescue conduction failure in weakly coupled tissue. Further, ephaptic coupling increased conduction velocity in weakly coupled tissue, and importantly, reduced the minimum gap junctional coupling necessary for conduction, most prominently at fast pacing rates. Finally, we find that, although neglecting gap junction voltage-gating results in negligible differences in well coupled tissue, more significant differences occur in weakly coupled tissue, greatly underestimating the minimal gap junctional coupling that can maintain conduction. Our study suggests that ephaptic coupling plays a conduction-preserving role, particularly at rapid heart rates.

Characterization of high-dose and high-energy implanted gate and source diode and analysis of lateral spreading of p gate profile in high voltage SiC static induction transistors

NASA Astrophysics Data System (ADS)

Onose, Hidekatsu; Kobayashi, Yutaka; Onuki, Jin

2017-03-01

The effect of the p gate dose on the characteristics of the gate-source diode in SiC static induction transistors (SIT) was investigated. It was found that a dose of 1.5 × 1014 cm-2 yields a pn junction breakdown voltage higher than 60 V and good forward characteristics. A normally on SiC SIT was fabricated and demonstrated. A blocking voltage higher than 2.0 kV at a gate-source voltage of -50 V and on-resistance of 70 mΩ cm2 were obtained. Device simulations were performed to investigate the effect of the lateral spreading. By comparing the measured I-V curves with simulation results, the lateral spreading factor was estimated to be about 0.5. The lateral spreading detrimentally affected the electrical properties of the SIT made using implantations at energies higher than 1 MeV.

Beta-Estradiol Regulates Voltage-Gated Calcium Channels and Estrogen Receptors in Telocytes from Human Myometrium.

PubMed

Banciu, Adela; Banciu, Daniel Dumitru; Mustaciosu, Cosmin Catalin; Radu, Mihai; Cretoiu, Dragos; Xiao, Junjie; Cretoiu, Sanda Maria; Suciu, Nicolae; Radu, Beatrice Mihaela

2018-05-09

Voltage-gated calcium channels and estrogen receptors are essential players in uterine physiology, and their association with different calcium signaling pathways contributes to healthy and pathological conditions of the uterine myometrium. Among the properties of the various cell subtypes present in human uterine myometrium, there is increasing evidence that calcium oscillations in telocytes (TCs) contribute to contractile activity and pregnancy. Our study aimed to evaluate the effects of beta-estradiol on voltage-gated calcium channels and estrogen receptors in TCs from human uterine myometrium and to understand their role in pregnancy. For this purpose, we employed patch-clamp recordings, ratiometric Fura-2-based calcium imaging analysis, and qRT-PCR techniques for the analysis of cultured human myometrial TCs derived from pregnant and non-pregnant uterine samples. In human myometrial TCs from both non-pregnant and pregnant uterus, we evidenced by qRT-PCR the presence of genes encoding for voltage-gated calcium channels (Cav3.1, Ca3.2, Cav3.3, Cav2.1), estrogen receptors (ESR1, ESR2, GPR30), and nuclear receptor coactivator 3 (NCOA3). Pregnancy significantly upregulated Cav3.1 and downregulated Cav3.2, Cav3.3, ESR1, ESR2, and NCOA3, compared to the non-pregnant condition. Beta-estradiol treatment (24 h, 10, 100, 1000 nM) downregulated Cav3.2, Cav3.3, Cav1.2, ESR1, ESR2, GRP30, and NCOA3 in TCs from human pregnant uterine myometrium. We also confirmed the functional expression of voltage-gated calcium channels by patch-clamp recordings and calcium imaging analysis of TCs from pregnant human myometrium by perfusing with BAY K8644, which induced calcium influx through these channels. Additionally, we demonstrated that beta-estradiol (1000 nM) antagonized the effect of BAY K8644 (2.5 or 5 µM) in the same preparations. In conclusion, we evidenced the presence of voltage-gated calcium channels and estrogen receptors in TCs from non-pregnant and pregnant human

Sodium influxes in internally perfused squid giant axon during voltage clamp.

PubMed

Atwater, I; Bezanilla, F; Rojas, E

1969-05-01

1. An experimental method for measuring ionic influxes during voltage clamp in the giant axon of Dosidicus is described; the technique combines intracellular perfusion with a method for controlling membrane potential.2. Sodium influx determinations were carried out while applying rectangular pulses of membrane depolarization. The ratio ;measured sodium influx/computed ionic flux during the early current' is 0.92 +/- 0.12.3. Plots of measured sodium influx and computed ionic flux during the early current against membrane potential are very similar. There was evidence that the membrane potential at which the sodium influx vanishes is the potential at which the early current reverses.

Sodium influxes in internally perfused squid giant axon during voltage clamp

PubMed Central

Atwater, I.; Bezanilla, F.; Rojas, E.

1969-01-01

1. An experimental method for measuring ionic influxes during voltage clamp in the giant axon of Dosidicus is described; the technique combines intracellular perfusion with a method for controlling membrane potential. 2. Sodium influx determinations were carried out while applying rectangular pulses of membrane depolarization. The ratio `measured sodium influx/computed ionic flux during the early current' is 0·92 ± 0·12. 3. Plots of measured sodium influx and computed ionic flux during the early current against membrane potential are very similar. There was evidence that the membrane potential at which the sodium influx vanishes is the potential at which the early current reverses. PMID:5767887

The voltage-gated proton channel: a riddle, wrapped in a mystery, inside an enigma

PubMed Central

DeCoursey, Thomas E.

2016-01-01

The main properties of voltage gated proton channels are described, along with what is known about how the channel protein structure accomplishes these functions. Just as protons are unique among ions, proton channels are unique among ion channels. Their four transmembrane helices sense voltage, the pH gradient, and conduct protons exclusively. Selectivity is achieved by the unique ability of H3O+ to protonate an Asp-Arg salt bridge. Pathognomonic sensitivity of gating to the pH gradient ensures channel opening only when acid extrusion will result, which is crucial to most biological functions. An exception occurs in dinoflagellates in which H+ influx through HV1 triggers the bioluminescent flash. Pharmacological interventions that promise to ameliorate cancer, asthma, brain damage in ischemic stroke, Alzheimer’s disease, autoimmune diseases, and numerous other conditions, await future progress. PMID:25964989

Histidine168 is crucial for ΔpH-dependent gating of the human voltage-gated proton channel, hHV1.

PubMed

Cherny, Vladimir V; Morgan, Deri; Thomas, Sarah; Smith, Susan M E; DeCoursey, Thomas E

2018-05-09

We recently identified a voltage-gated proton channel gene in the snail Helisoma trivolvis , HtH V 1, and determined its electrophysiological properties. Consistent with early studies of proton currents in snail neurons, HtH V 1 opens rapidly, but it unexpectedly exhibits uniquely defective sensitivity to intracellular pH (pH i ). The H + conductance ( g H )- V relationship in the voltage-gated proton channel (H V 1) from other species shifts 40 mV when either pH i or pH o (extracellular pH) is changed by 1 unit. This property, called ΔpH-dependent gating, is crucial to the functions of H V 1 in many species and in numerous human tissues. The HtH V 1 channel exhibits normal pH o dependence but anomalously weak pH i dependence. In this study, we show that a single point mutation in human hH V 1-changing His 168 to Gln 168 , the corresponding residue in HtH V 1-compromises the pH i dependence of gating in the human channel so that it recapitulates the HtH V 1 response. This location was previously identified as a contributor to the rapid gating kinetics of H V 1 in Strongylocentrotus purpuratus His 168 mutation in human H V 1 accelerates activation but accounts for only a fraction of the species difference. H168Q, H168S, or H168T mutants exhibit normal pH o dependence, but changing pH i shifts the g H - V relationship on average by <20 mV/unit. Thus, His 168 is critical to pH i sensing in hH V 1. His 168 , located at the inner end of the pore on the S3 transmembrane helix, is the first residue identified in H V 1 that significantly impairs pH sensing when mutated. Because pH o dependence remains intact, the selective erosion of pH i dependence supports the idea that there are distinct internal and external pH sensors. Although His 168 may itself be a pH i sensor, the converse mutation, Q229H, does not normalize the pH i sensitivity of the HtH V 1 channel. We hypothesize that the imidazole group of His 168 interacts with nearby Phe 165 or other parts of hH V 1 to

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.

Modulation of voltage-gated conductances of retinal horizontal cells by UV-excited TiO2 nanoparticles.

PubMed

Meshik, Xenia; Choi, Min; Baker, Adam; Malchow, R Paul; Covnot, Leigha; Doan, Samuel; Mukherjee, Souvik; Farid, Sidra; Dutta, Mitra; Stroscio, Michael A

2017-04-01

This study examines the ability of optically-excited titanium dioxide nanoparticles to influence voltage-gated ion channels in retinal horizontal cells. Voltage clamp recordings were obtained in the presence and absence of TiO 2 and ultraviolet laser excitation. Significant current changes were observed in response to UV light, particularly in the -40 mV to +40 mV region where voltage-gated Na + and K + channels have the highest conductance. Cells in proximity to UV-excited TiO 2 exhibited a left-shift in the current-voltage relation of around 10 mV in the activation of Na + currents. These trends were not observed in control experiments where cells were excited with UV light without being exposed to TiO 2 . Electrostatic force microscopy confirmed that electric fields can be induced in TiO 2 with UV light. Simulations using the Hodgkin-Huxley model yielded results which agreed with the experimental data and showed the I-V characteristics of individual ion channels in the presence of UV-excited TiO 2 . Copyright © 2016 Elsevier Inc. All rights reserved.

Extended-gate-type IGZO electric-double-layer TFT immunosensor with high sensitivity and low operation voltage

NASA Astrophysics Data System (ADS)

Liang, Lingyan; Zhang, Shengnan; Wu, Weihua; Zhu, Liqiang; Xiao, Hui; Liu, Yanghui; Zhang, Hongliang; Javaid, Kashif; Cao, Hongtao

2016-10-01

An immunosensor is proposed based on the indium-gallium-zinc-oxide (IGZO) electric-double-layer thin-film transistor (EDL TFT) with a separating extended gate. The IGZO EDL TFT has a field-effect mobility of 24.5 cm2 V-1 s-1 and an operation voltage less than 1.5 V. The sensors exhibit the linear current response to label-free target immune molecule in the concentrations ranging from 1.6 to 368 × 10-15 g/ml with a detection limit of 1.6 × 10-15 g/ml (0.01 fM) under an ultralow operation voltage of 0.5 V. The IGZO TFT component demonstrates a consecutive assay stability and recyclability due to the unique structure with the separating extended gate. With the excellent electrical properties and the potential for plug-in-card-type multifunctional sensing, extended-gate-type IGZO EDL TFTs can be promising candidates for the development of a label-free biosensor for public health applications.

[Voltage-gated potassium channels and human neurological diseases].

PubMed

Jin, Hong-Wei; Wang, Xiao-Liang

2002-01-01

Voltage-gated potassium channels (Kv) is the largest, most complex in potassium channel superfamily. It can be divided into Kv alpha subunit and auxiliary two groups. The roles of some Kv channels types, e.g. rapidly inactivating (A-Type channel) and muscarine sensitive channels (M-type channel) are beginning to be understood. They are prominent in nervous system, acting in delicate and accurate ways to control or modify many physiological and pathological functions including membrane excitability, neurotransmitter release, cell proliferation or degeneration, signal transduction in neuronal network. Many human neurological disease pathogenesis are found to be related to mutant of Kv-channels subunit or subtype, such as, learning and memory impairing, ataxia, epilepsy, deafness, etc.

Single-molecule fluorimetry and gating currents inspire an improved optical voltage indicator

PubMed Central

Treger, Jeremy S; Priest, Michael F; Bezanilla, Francisco

2015-01-01

Voltage-sensing domains (VSDs) underlie the movement of voltage-gated ion channels, as well as the voltage-sensitive fluorescent responses observed from a common class of genetically encoded voltage indicators (GEVIs). Despite the widespread use and potential utility of these GEVIs, the biophysical underpinnings of the relationship between VSD movement and fluorophore response remain unclear. We investigated the recently developed GEVI ArcLight, and its close variant Arclight', at both the single-molecule and macroscopic levels to better understand their characteristics and mechanisms of activity. These studies revealed a number of previously unobserved features of ArcLight's behavior, including millisecond-scale fluorescence fluctuations in single molecules as well as a previously unreported delay prior to macroscopic fluorescence onset. Finally, these mechanistic insights allowed us to improve the optical response of ArcLight to fast or repetitive pulses with the development of ArcLightning, a novel GEVI with improved kinetics. DOI: http://dx.doi.org/10.7554/eLife.10482.001 PMID:26599732

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.

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

PubMed

Hovey, Liam; Fowler, C Andrew; Mahling, Ryan; Lin, Zesen; Miller, Mark Stephen; Marx, Dagan C; Yoder, Jesse B; Kim, Elaine H; Tefft, Kristin M; Waite, Brett C; Feldkamp, Michael D; Yu, Liping; Shea, Madeline A

2017-05-01

Several members of the voltage-gated sodium channel family are regulated by calmodulin (CaM) and ionic calcium. The neuronal voltage-gated sodium channel Na V 1.2 contains binding sites for both apo (calcium-depleted) and calcium-saturated CaM. We have determined equilibrium dissociation constants for rat Na V 1.2 IQ motif [IQRAYRRYLLK] binding to apo CaM (~3nM) and (Ca 2+ ) 4 -CaM (~85nM), showing that apo CaM binding is favored by 30-fold. For both apo and (Ca 2+ ) 4 -CaM, NMR demonstrated that Na V 1.2 IQ motif peptide (Na V 1.2 IQp ) exclusively made contacts with C-domain residues of CaM (CaM C ). To understand how calcium triggers conformational change at the CaM-IQ interface, we determined a solution structure (2M5E.pdb) of (Ca 2+ ) 2 -CaM C bound to Na V 1.2 IQp . The polarity of (Ca 2+ ) 2 -CaM C relative to the IQ motif was opposite to that seen in apo CaM C -Na v 1.2 IQp (2KXW), revealing that CaM C recognizes nested, anti-parallel sites in Na v 1.2 IQp . Reversal of CaM may require transient release from the IQ motif during calcium binding, and facilitate a re-orientation of CaM N allowing interactions with non-IQ Na V 1.2 residues or auxiliary regulatory proteins interacting in the vicinity of the IQ motif. Copyright © 2017 Elsevier B.V. All rights reserved.

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…

A fully on-chip fast-transient NMOS low dropout voltage regulator with quasi floating gate pass element

NASA Astrophysics Data System (ADS)

Wang, Han; Gou, Chao; Luo, Kai

2017-04-01

This paper presents a fully on-chip NMOS low-dropout regulator (LDO) for portable applications with quasi floating gate pass element and fast transient response. The quasi floating gate structure makes the gate of the NMOS transistor only periodically charged or refreshed by the charge pump, which allows the charge pump to be a small economical circuit with small silicon area. In addition, a variable reference circuit is introduced enlarging the dynamic range of error amplifier during load transient. The proposed LDO has been implemented in a 0.35 μm BCD process. From experimental results, the regulator can operate with a minimum dropout voltage of 250 mV at a maximum 1 A load and {I}{{Q}} of 395 μA. Under full-range load current step, the voltage undershoot and overshoot of the proposed LDO are reduced to 50 and 26 mV, respectively.

An inherent curvature-compensated voltage reference using non-linearity of gate coupling coefficient

NASA Astrophysics Data System (ADS)

Hande, Vinayak; Shojaei Baghini, Maryam

2015-08-01

A novel current-mode voltage reference circuit which is capable of generating sub-1 V output voltage is presented. The proposed architecture exhibits the inherent curvature compensation ability. The curvature compensation is achieved by utilizing the non-linear behavior of gate coupling coefficient to compensate non-linear temperature dependence of base-emitter voltage. We have also utilized the developments in CMOS process to reduce power and area consumption. The proposed voltage reference is analyzed theoretically and compared with other existing methods. The circuit is designed and simulated in 180 nm mixed-mode CMOS UMC technology which gives a reference level of 246 mV. The minimum required supply voltage is 1 V with maximum current drawn of 9.24 μA. A temperature coefficient of 9 ppm/°C is achieved over -25 to 125 °C temperature range. The reference voltage varies by ±11 mV across process corners. The reference circuit shows the line sensitivity of 0.9 mV/V with area consumption of 100 × 110 μm2

Comparative study of the distribution of the alpha-subunits of voltage-gated sodium channels in normal and axotomized rat dorsal root ganglion neurons.

PubMed

Fukuoka, Tetsuo; Kobayashi, Kimiko; Yamanaka, Hiroki; Obata, Koichi; Dai, Yi; Noguchi, Koichi

2008-09-10

We compared the distribution of the alpha-subunit mRNAs of voltage-gated sodium channels Nav1.1-1.3 and Nav1.6-1.9 and a related channel, Nax, in histochemically identified neuronal subpopulations of the rat dorsal root ganglia (DRG). In the naïve DRG, the expression of Nav1.1 and Nav1.6 was restricted to A-fiber neurons, and they were preferentially expressed by TrkC neurons, suggesting that proprioceptive neurons possess these channels. Nav1.7, -1.8, and -1.9 mRNAs were more abundant in C-fiber neurons compared with A-fiber ones. Nax was evenly expressed in both populations. Although Nav1.8 and -1.9 were preferentially expressed by TrkA neurons, other alpha-subunits were expressed independently of TrkA expression. Actually, all IB4(+) neurons expressed both Nav1.8 and -1.9, and relatively limited subpopulations of IB4(+) neurons (3% and 12%, respectively) expressed Nav1.1 and/or Nav1.6. These findings provide useful information in interpreting the electrophysiological characteristics of some neuronal subpopulations of naïve DRG. After L5 spinal nerve ligation, Nav1.3 mRNA was up-regulated mainly in A-fiber neurons in the ipsilateral L5 DRG. Although previous studies demonstrated that nerve growth factor (NGF) and glial cell-derived neurotrophic factor (GDNF) reversed this up-regulation, the Nav1.3 induction was independent of either TrkA or GFRalpha1 expression, suggesting that the induction of Nav1.3 may be one of the common responses of axotomized DRG neurons without a direct relationship to NGF/GDNF supply. (c) 2008 Wiley-Liss, Inc.

Multidrug resistance in epilepsy and polymorphisms in the voltage-gated sodium channel genes SCN1A, SCN2A, and SCN3A: correlation among phenotype, genotype, and mRNA expression.

PubMed

Kwan, Patrick; Poon, Wai Sang; Ng, Ho-Keung; Kang, David E; Wong, Virginia; Ng, Ping Wing; Lui, Colin H T; Sin, Ngai Chuen; Wong, Ka S; Baum, Larry

2008-11-01

Many antiepileptic drugs (AEDs) prevent seizures by blocking voltage-gated brain sodium channels. However, treatment is ineffective in 30% of epilepsy patients, which might, at least in part, result from polymorphisms of the sodium channel genes. We investigated the association of AED responsiveness with genetic polymorphisms and correlated any association with mRNA expression of the neuronal sodium channels. We performed genotyping of tagging and candidate single nucleotide polymorphisms (SNPs) of SCN1A, 2A, and 3A in 471 Chinese epilepsy patients (272 drug responsive and 199 drug resistant). A total of 27 SNPs were selected based on the HapMap database. Genotype distributions in drug-responsive and drug-resistant patients were compared. SCN2A mRNA was quantified by real-time PCR in 24 brain and 57 blood samples. Its level was compared between patients with different genotypes of an SCN2A SNP found to be associated with drug responsiveness. SCN2A IVS7-32A>G (rs2304016) A alleles were associated with drug resistance (odds ratio = 2.1, 95% confidence interval: 1.2-3.7, P=0.007). Haplotypes containing the IVS7-32A>G allele A were also associated with drug resistance. IVS7-32A>G is located within the putative splicing branch site for splicing exons 7 and 9. PCR of reverse-transcribed RNA from blood or brain of patients with different IVS7-32A>G genotypes using primers in exons 7 and 9 showed no skipping of exon 8, and real-time PCR showed no difference in SCN2A mRNA levels among genotypes. Results of this study suggest an association between SCN2A IVS7-32A>G and AED responsiveness, without evidence of an effect on splicing or mRNA expression.

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...

Low voltage operation of IGZO thin film transistors enabled by ultrathin Al2O3 gate dielectric

NASA Astrophysics Data System (ADS)

Ma, Pengfei; Du, Lulu; Wang, Yiming; Jiang, Ran; Xin, Qian; Li, Yuxiang; Song, Aimin

2018-01-01

An ultrathin, 5 nm, Al2O3 film grown by atomic-layer deposition was used as a gate dielectric for amorphous indium-gallium-zinc oxide (a-IGZO) thin-film transistors (TFTs). The Al2O3 layer showed a low surface roughness of 0.15 nm, a low leakage current, and a high breakdown voltage of 6 V. In particular, a very high gate capacitance of 720 nF/cm2 was achieved, making it possible for the a-IGZO TFTs to not only operate at a low voltage of 1 V but also exhibit desirable properties including a low threshold voltage of 0.3 V, a small subthreshold swing of 100 mV/decade, and a high on/off current ratio of 1.2 × 107. Furthermore, even under an ultralow operation voltage of 0.6 V, well-behaved transistor characteristics were still observed with an on/off ratio as high as 3 × 106. The electron transport through the Al2O3 layer has also been analyzed, indicating the Fowler-Nordheim tunneling mechanism.

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

Trafficking Mechanisms Underlying Neuronal Voltage-gated Ion Channel Localization at the Axon Initial Segment

PubMed Central

Vacher, Helene; Trimmer, James S.

2012-01-01

Summary Voltage-gated ion channels are diverse and fundamental determinants of neuronal intrinsic excitability. Voltage-gated K+ (Kv) and Na+ (Nav) channels play complex yet fundamentally important roles in determining intrinsic excitability. The Kv and Nav channels located at the axon initial segment (AIS) play a unique and especially important role in generating neuronal output in the form of anterograde axonal and backpropagating action potentials, Aberrant intrinsic excitability in individual neurons within networks contributes to synchronous neuronal activity leading to seizures. Mutations in ion channel genes gives rise to a variety of seizure-related “Channelopathies”, and many of the ion channel subunits associated with epilepsy mutations are localized at the AIS, making this a hotspot for epileptogenesis. Here we review the cellular mechanisms that underlie the trafficking of Kv and Nav channels found at the AIS, and how Kv and Nav channel mutations associated with epilepsy can alter these processes. PMID:23216576

Mutations in the voltage-gated sodium channel gene of anophelines and their association with resistance to pyrethroids - a review.

PubMed

Silva, Ana Paula B; Santos, Joselita Maria M; Martins, Ademir J

2014-10-07

Constant and extensive use of chemical insecticides has created a selection pressure and favored resistance development in many insect species worldwide. One of the most important pyrethroid resistance mechanisms is classified as target site insensitivity, due to conformational changes in the target site that impair a proper binding of the insecticide molecule. The voltage-gated sodium channel (NaV) is the target of pyrethroids and DDT insecticides, used to control insects of medical, agricultural and veterinary importance, such as anophelines. It has been reported that the presence of a few non-silent point mutations in the NaV gene are associated with pyrethroid resistance, termed as 'kdr' (knockdown resistance) for preventing the knockdown effect of these insecticides. The presence of these mutations, as well as their effects, has been thoroughly studied in Anopheles mosquitoes. So far, kdr mutations have already been detected in at least 13 species (Anopheles gambiae, Anopheles arabiensis, Anopheles sinensis, Anopheles stephensi, Anopheles subpictus, Anopheles sacharovi, Anopheles culicifacies, Anopheles sundaicus, Anopheles aconitus, Anopheles vagus, Anopheles paraliae, Anopheles peditaeniatus and Anopheles albimanus) from populations of African, Asian and, more recently, American continents. Seven mutational variants (L1014F, L1014S, L1014C, L1014W, N1013S, N1575Y and V1010L) were described, with the highest prevalence of L1014F, which occurs at the 1014 site in NaV IIS6 domain. The increase of frequency and distribution of kdr mutations clearly shows the importance of this mechanism in the process of pyrethroid resistance. In this sense, several species-specific and highly sensitive methods have been designed in order to genotype individual mosquitoes for kdr in large scale, which may serve as important tolls for monitoring the dynamics of pyrethroid resistance in natural populations. We also briefly discuss investigations concerning the course of Plasmodium

Voltage Sensor Inactivation in Potassium Channels

PubMed Central

Bähring, Robert; Barghaan, Jan; Westermeier, Regina; Wollberg, Jessica

2012-01-01

In voltage-gated potassium (Kv) channels membrane depolarization causes movement of a voltage sensor domain. This conformational change of the protein is transmitted to the pore domain and eventually leads to pore opening. However, the voltage sensor domain may interact with two distinct gates in the pore domain: the activation gate (A-gate), involving the cytoplasmic S6 bundle crossing, and the pore gate (P-gate), located externally in the selectivity filter. How the voltage sensor moves and how tightly it interacts with these two gates on its way to adopt a relaxed conformation when the membrane is depolarized may critically determine the mode of Kv channel inactivation. In certain Kv channels, voltage sensor movement leads to a tight interaction with the P-gate, which may cause conformational changes that render the selectivity filter non-conductive (“P/C-type inactivation”). Other Kv channels may preferably undergo inactivation from pre-open closed-states during voltage sensor movement, because the voltage sensor temporarily uncouples from the A-gate. For this behavior, known as “preferential” closed-state inactivation, we introduce the term “A/C-type inactivation”. Mechanistically, P/C- and A/C-type inactivation represent two forms of “voltage sensor inactivation.” PMID:22654758

Neutralisation of a single voltage sensor affects gating determinants in all four pore-forming S6 segments of Ca(V)1.2: a cooperative gating model.

PubMed

Beyl, Stanislav; Depil, Katrin; Hohaus, Annette; Stary-Weinzinger, Anna; Linder, Tobias; Timin, Eugen; Hering, Steffen

2012-10-01

Voltage sensors trigger the closed-open transitions in the pore of voltage-gated ion channels. To probe the transmission of voltage sensor signalling to the channel pore of Ca(V)1.2, we investigated how elimination of positive charges in the S4 segments (charged residues were replaced by neutral glutamine) modulates gating perturbations induced by mutations in pore-lining S6 segments. Neutralisation of all positively charged residues in IIS4 produced a functional channel (IIS4(N)), while replacement of the charged residues in IS4, IIIS4 and IVS4 segments resulted in nonfunctional channels. The IIS4(N) channel displayed activation kinetics similar to wild type. Mutations in a highly conserved structure motif on S6 segments ("GAGA ring": G432W in IS6, A780T in IIS6, G1193T in IIIS6 and A1503G in IVS6) induce strong left-shifted activation curves and decelerated channel deactivation kinetics. When IIS4(N) was combined with these mutations, the activation curves were shifted back towards wild type and current kinetics were accelerated. In contrast, 12 other mutations adjacent to the GAGA ring in IS6-IVS6, which also affect activation gating, were not rescued by IIS4(N). Thus, the rescue of gating distortions in segments IS6-IVS6 by IIS4(N) is highly position-specific. Thermodynamic cycle analysis supports the hypothesis that IIS4 is energetically coupled with the distantly located GAGA residues. We speculate that conformational changes caused by neutralisation of IIS4 are not restricted to domain II (IIS6) but are transmitted to gating structures in domains I, III and IV via the GAGA ring.

Analytical model of threshold voltage degradation due to localized charges in gate material engineered Schottky barrier cylindrical GAA MOSFETs

NASA Astrophysics Data System (ADS)

Kumar, Manoj; Haldar, Subhasis; Gupta, Mridula; Gupta, R. S.

2016-10-01

The threshold voltage degradation due to the hot carrier induced localized charges (LC) is a major reliability concern for nanoscale Schottky barrier (SB) cylindrical gate all around (GAA) metal-oxide-semiconductor field-effect transistors (MOSFETs). The degradation physics of gate material engineered (GME)-SB-GAA MOSFETs due to LC is still unexplored. An explicit threshold voltage degradation model for GME-SB-GAA-MOSFETs with the incorporation of localized charges (N it) is developed. To accurately model the threshold voltage the minimum channel carrier density has been taken into account. The model renders how +/- LC affects the device subthreshold performance. One-dimensional (1D) Poisson’s and 2D Laplace equations have been solved for two different regions (fresh and damaged) with two different gate metal work-functions. LCs are considered at the drain side with low gate metal work-function as N it is more vulnerable towards the drain. For the reduction of carrier mobility degradation, a lightly doped channel has been considered. The proposed model also includes the effect of barrier height lowering at the metal-semiconductor interface. The developed model results have been verified using numerical simulation data obtained by the ATLAS-3D device simulator and excellent agreement is observed between analytical and simulation results.

[Mechanism of action of neurotoxins acting on the inactivation of voltage-gated sodium channels].

PubMed

Benoit, E

1998-01-01

This review focuses on the mechanism(s) of action of neurotoxins acting on the inactivation of voltage-gated Na channels. Na channels are transmembrane proteins which are fundamental for cellular communication. These proteins form pores in the plasma membrane allowing passive ionic movements to occur. Their opening and closing are controlled by gating systems which depend on both membrane potential and time. Na channels have three functional properties, mainly studied using electrophysiological and biochemical techniques, to ensure their role in the generation and propagation of action potentials: 1) a highly selectivity for Na ions, 2) a rapid opening ("activation"), responsible for the depolarizing phase of the action potential, and 3) a late closing ("inactivation") involved in the repolarizing phase of the action potential. As an essential protein for membrane excitability, the Na channel is the specific target of a number of vegetal and animal toxins which, by binding to the channel, alter its activity by affecting one or more of its properties. At least six toxin receptor sites have been identified on the neuronal Na channel on the basis of binding studies. However, only toxins interacting with four of these sites (sites 2, 3, 5 et 6) produce alterations of channel inactivation. The maximal percentage of Na channels modified by the binding of neurotoxins to sites 2 (batrachotoxin and some alkaloids), 3 (alpha-scorpion and sea anemone toxins), 5 (brevetoxins and ciguatoxins) et 6 (delta-conotoxins) is different according to the site considered. However, in all cases, these channels do not inactivate. Moreover, Na channels modified by toxins which bind to sites 2, 5 and 6 activate at membrane potentials more negative than do unmodified channels. The physiological consequences of Na channel modifications, induced by the binding of neurotoxins to sites 2, 3, 5 and 6, are (i) an inhibition of cellular excitability due to an important membrane depolarization (site

Chloride concentration affects Kv channel voltage-gating kinetics: Importance of experimental anion concentrations.

PubMed

Bekar, L K; Loewen, M E; Forsyth, G W; Walz, W

2005-09-30

Chloride concentration has been shown to have a dramatic impact on protein folding and subsequent tertiary conformation [K.D. Collins, Ions from the Hofmeister series and osmolytes: effects on proteins in solution and in the crystallization process, Methods 34 (2004) 300-311; I. Jelesarov, E. Durr, R.M. Thomas, H.R. Bosshard, Salt effects on hydrophobic interaction and charge screening in the folding of a negatively charged peptide to a coiled coil (leucine zipper), Biochemistry 37 (1998) 7539-7550]. As it is known that Kv channel gating is linked to the stability of the cytoplasmic T1 multimerization domain conformation [D.L. Minor, Y.F. Lin, B.C. Mobley, A. Avelar, Y.N. Jan, L.Y. Jan, J.M. Berger, The polar T1 interface is linked to conformational changes that open the voltage-gated potassium channel, Cell 102 (2000) 657-670; B.A. Yi, D.L. Minor Jr., Y.F. Lin, Y.N. Jan, L.Y. Jan, Controlling potassium channel activities: interplay between the membrane and intracellular factors, Proc. Natl. Acad. Sci. U.S.A. 98 (2001) 11016-11023] and that intracellular chloride concentration has been linked to Kv channel kinetics [L.K. Bekar, W. Walz, Intracellular chloride modulates A-type potassium currents in astrocytes, Glia 39 (2002) 207-216; W.B. Thoreson, S.L. Stella, Anion modulation of calcium current voltage dependence and amplitude in salamander rods, Biochim. Biophys. Acta 1464 (2000) 142-150], the objective of the present study was to address how chloride concentration changes affect Kv channel kinetics more closely in an isolated expression system. Initially, no significant chloride concentration-dependent effects on channel steady-state gating kinetics were observed. Only after disruption of the cytoskeleton with cytochalasin-D did we see significant chloride concentration-dependent shifts in gating kinetics. This suggests that the shift in gating kinetics is mediated through effects of intracellular chloride concentration on cytoplasmic domain tertiary

Blockade of voltage-gated sodium channels inhibits invasion of endocrine-resistant breast cancer cells.

PubMed

Mohammed, Fatima H; Khajah, Maitham A; Yang, Ming; Brackenbury, William J; Luqmani, Yunus A

2016-01-01

Voltage-gated Na+ channels (VGSCs) are membrane proteins which are normally expressed in excitable cells but have also been detected in cancer cells, where they are thought to be involved in malignancy progression. In this study we examined the ion current and expression profile of VGSC (Nav1.5) in estrogen receptor (ER)-positive (MCF-7) and silenced (pII) breast cancer cells and its possible influence on their proliferation, motility and invasion. VGSC currents were analysed by whole cell patch clamp recording. Nav1.5 expression and localization, in response to EGF stimulation, was examined by western blotting and immunofluorescence respectively. Cell invasion (under-agarose and Matrigel assays), motility (wound healing assay) and proliferation (MTT assay) were assessed in pII cells in response to VGSC blockers, phenytoin (PHT) and tetrodotoxin (TTX), or by siRNA knockdown of Nav1.5. The effect of PHT and TTX on modulating EGF-induced phosphorylation of Akt and ERK1/2 was determined by western blotting. Total matrix metalloproteinase (MMP) was determined using a fluorometric-based activity assay. The level of various human proteases was detected by using proteome profiler array kit. VGSC currents were detected in pII cells, but were absent in MCF-7. Nav1.5 showed cytoplasmic and perinuclear expression in both MCF-7 and pII cells, with enhanced expression upon EGF stimulation. Treatment of pII cells with PHT, TTX or siRNA significantly reduced invasion towards serum components and EGF, in part through reduction of P-ERK1/2 and proteases such as cathepsin E, kallikrein-10 and MMP-7, as well as total MMP activity. At high concentrations, PHT inhibited motility while TTX reduced cell proliferation. Pharmacological or genetic blockade of Nav1.5 may serve as a potential anti-metastatic therapy for breast cancer.

A rugged 650 V SOI-based high-voltage half-bridge IGBT gate driver IC for motor drive applications

NASA Astrophysics Data System (ADS)

Hua, Qing; Li, Zehong; Zhang, Bo; Chen, Weizhong; Huang, Xiangjun; Feng, Yuxiang

2015-05-01

This paper proposes a rugged high-voltage N-channel insulated gate bipolar transistor (IGBT) gate driver integrated circuit. The device integrates a high-side and a low-side output stages on a single chip, which is designed specifically for motor drive applications. High-voltage level shift technology enables the high-side stage of this device to operate up to 650 V. The logic inputs are complementary metal oxide semiconductor (CMOS)/transistor transistor logic compatible down to 3.3 V. Undervoltage protection functionality with hysteresis characteristic has also been integrated to enhance the device reliability. The device is fabricated in a 1.0 μm, 650 V high-voltage bipolar CMOS double-diffused metal oxide semiconductor (BCD) on silicon-on-insulator (SOI) process. Deep trench dielectric isolation technology is employed to provide complete electrical isolation with advantages such as reduced parasitic effects, excellent noise immunity and low leakage current. Experimental results show that the isolation voltage of this device can be up to approximately 779 V at 25°C, and the leakage current is only 5 nA at 650 V, which is 15% higher and 67% lower than the conventional ones. In addition, it delivers an excellent thermal stability and needs very low quiescent current and offers a high gate driver capability which is needed to adequately drive IGBTs that have large input capacitances.

Tetrodotoxin-sensitive, voltage-dependent sodium currents in hair cells from the alligator cochlea.

PubMed

Evans, M G; Fuchs, P A

1987-10-01

We have used whole-cell patch clamp techniques to record from tall hair cells isolated from the apical half of the alligator cochlea. Some of these cells gave action potentials in response to depolarizing current injections. When the same cells were voltage clamped, large transient inward currents followed by smaller outward currents were seen in response to depolarizing steps. We studied the transient inward current after the outward current had been blocked by external tetraethylammonium (20 mM) or by replacing internal potassium with cesium. It was found to be a sodium current because it was abolished by either replacing external sodium with choline or by external application of tetrodotoxin (100 nM). The sodium current showed voltage-dependent activation and inactivation. Most of the spiking hair cells came from the apex of the cochlea, where they would be subject to low-frequency mechanical stimulation in vivo.

Interaction of a dinoflagellate neurotoxin with voltage-activated ion channels in a marine diatom.

PubMed

Kitchen, Sheila A; Bourdelais, Andrea J; Taylor, Alison R

2018-01-01

The potent neurotoxins produced by the harmful algal bloom species Karenia brevis are activators of sodium voltage-gated channels (VGC) in animals, resulting in altered channel kinetics and membrane hyperexcitability. Recent biophysical and genomic evidence supports widespread presence of homologous sodium (Na + ) and calcium (Ca 2+ ) permeable VGCs in unicellular algae, including marine phytoplankton. We therefore hypothesized that VGCs of these phytoplankton may be an allelopathic target for waterborne neurotoxins produced by K. brevis blooms that could lead to ion channel dysfunction and disruption of signaling in a similar manner to animal Na + VGCs. We examined the interaction of brevetoxin-3 (PbTx-3), a K. brevis neurotoxin, with the Na + /Ca 2+ VGC of the non-toxic diatom Odontella sinensi s using electrophysiology. Single electrode current- and voltage- clamp recordings from O. sinensis in the presence of PbTx-3 were used to examine the toxin's effect on voltage gated Na + /Ca 2+ currents. In silico analysis was used to identify the putative PbTx binding site in the diatoms. We identified Na + /Ca 2+ VCG homologs from the transcriptomes and genomes of 12 diatoms, including three transcripts from O. sinensis and aligned them with site-5 of Na + VGCs, previously identified as the PbTx binding site in animals. Up to 1 µM PbTx had no effect on diatom resting membrane potential or membrane excitability. The kinetics of fast inward Na + /Ca 2+ currents that underlie diatom action potentials were also unaffected. However, the peak inward current was inhibited by 33%, delayed outward current was inhibited by 25%, and reversal potential of the currents shifted positive, indicating a change in permeability of the underlying channels. Sequence analysis showed a lack of conservation of the PbTx binding site in diatom VGC homologs, many of which share molecular features more similar to single-domain bacterial Na + /Ca 2+ VGCs than the 4-domain eukaryote channels

Switches from pi- to sigma-bonding complexes controlled by gate voltages.

PubMed

Matsui, Eriko; Harnack, Oliver; Matsuzawa, Nobuyuki N; Yasuda, Akio

2005-10-01

A conjugated polymer/metal ion/liquid-crystal molecular system was set between source and drain electrodes with a 100 nm gap. When gate voltage (Vg) increases, the current between source and drain electrodes increases. Infrared spectra show this system to be composed of pi and sigma complexes. At Vg = 0, the pi complex dominates the sigma complex, whereas the sigma complex becomes dominant when Vg is switched on. Calculations found that the pi complex has lower conductivity than the sigma complex.

L-type Voltage-Gated Calcium Channels in Conditioned Fear: A Genetic and Pharmacological Analysis

ERIC Educational Resources Information Center

McKinney, Brandon C.; Sze, Wilson; White, Jessica A.; Murphy, Geoffrey G.

2008-01-01

Using pharmacological approaches, others have suggested that L-type voltage-gated calcium channels (L-VGCCs) mediate both consolidation and extinction of conditioned fear. In the absence of L-VGCC isoform-specific antagonists, we have begun to investigate the subtype-specific role of LVGCCs in consolidation and extinction of conditioned fear…

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.

Common molecular determinants of tarantula huwentoxin-IV inhibition of Na+ channel voltage sensors in domains II and IV.

PubMed

Xiao, Yucheng; Jackson, James O; Liang, Songping; Cummins, Theodore R

2011-08-05

The voltage sensors of domains II and IV of sodium channels are important determinants of activation and inactivation, respectively. Animal toxins that alter electrophysiological excitability of muscles and neurons often modify sodium channel activation by selectively interacting with domain II and inactivation by selectively interacting with domain IV. This suggests that there may be substantial differences between the toxin-binding sites in these two important domains. Here we explore the ability of the tarantula huwentoxin-IV (HWTX-IV) to inhibit the activity of the domain II and IV voltage sensors. HWTX-IV is specific for domain II, and we identify five residues in the S1-S2 (Glu-753) and S3-S4 (Glu-811, Leu-814, Asp-816, and Glu-818) regions of domain II that are crucial for inhibition of activation by HWTX-IV. These data indicate that a single residue in the S3-S4 linker (Glu-818 in hNav1.7) is crucial for allowing HWTX-IV to interact with the other key residues and trap the voltage sensor in the closed configuration. Mutagenesis analysis indicates that the five corresponding residues in domain IV are all critical for endowing HWTX-IV with the ability to inhibit fast inactivation. Our data suggest that the toxin-binding motif in domain II is conserved in domain IV. Increasing our understanding of the molecular determinants of toxin interactions with voltage-gated sodium channels may permit development of enhanced isoform-specific voltage-gating modifiers.

The Anion Paradox in Sodium Taste Reception: Resolution by Voltage-Clamp Studies

NASA Astrophysics Data System (ADS)

Ye, Qing; Heck, Gerard L.; Desimone, John A.

1991-11-01

Sodium salts are potent taste stimuli, but their effectiveness is markedly dependent on the anion, with chloride yielding the greatest response. The cellular mechanisms that mediate this phenomenon are not known. This "anion paradox" has been resolved by considering the field potential that is generated by restricted electrodiffusion of the anion through paracellular shunts between taste-bud cells. Neural responses to sodium chloride, sodium acetate, and sodium gluconate were studied while the field potential was voltage-clamped. Clamping at electronegative values eliminated the anion effect, whereas clamping at electropositive potentials exaggerated it. Thus, field potentials across the lingual epithelium modulate taste reception, indicating that the functional unit of taste reception includes the taste cell and its paracellular microenvironment.

Voltage-gated K+ channel modulators as neuroprotective agents.

PubMed

Leung, Yuk-Man

2010-05-22

A manifestation in neurodegeneration is apoptosis of neurons. Neurons undergoing apoptosis may lose a substantial amount of cytosolic K+ through a number of pathways including K+ efflux via voltage-gated K+ (Kv) channels. The consequent drop in cytosolic [K+] relieves inhibition of an array of pro-apoptotic enzymes such as caspases and nucleases. Blocking Kv channels has been known to prevent neuronal apoptosis by preventing K+ efflux. Some neural diseases such as epilepsy are caused by neuronal hyperexcitability, which eventually may lead to neuronal apoptosis. Reduction in activities of A-type Kv channels and Kv7 subfamily members is amongst the etiological causes of neuronal hyperexcitation; enhancing the opening of these channels may offer opportunities of remedy. This review discusses the potential uses of Kv channel modulators as neuroprotective drugs.

Discovery of Point Mutations in the Voltage-Gated Sodium Channel from African Aedes aegypti Populations: Potential Phylogenetic Reasons for Gene Introgression

PubMed Central

Muranami, Yuto; Kawashima, Emiko; Osei, Joseph H. N.; Sakyi, Kojo Yirenkyi; Dadzie, Samuel; de Souza, Dziedzom K.; Appawu, Maxwell; Ohta, Nobuo; Minakawa, Noboru

2016-01-01

Background Yellow fever is endemic in some countries in Africa, and Aedes aegpyti is one of the most important vectors implicated in the outbreak. The mapping of the nation-wide distribution and the detection of insecticide resistance of vector mosquitoes will provide the beneficial information for forecasting of dengue and yellow fever outbreaks and effective control measures. Methodology/Principal Findings High resistance to DDT was observed in all mosquito colonies collected in Ghana. The resistance and the possible existence of resistance or tolerance to permethrin were suspected in some colonies. High frequencies of point mutations at the voltage-gated sodium channel (F1534C) and one heterozygote of the other mutation (V1016I) were detected, and this is the first detection on the African continent. The frequency of F1534C allele and the ratio of F1534C homozygotes in Ae. aegypti aegypti (Aaa) were significantly higher than those in Ae. aegypti formosus (Aaf). We could detect the two types of introns between exon 20 and 21, and the F1534C mutations were strongly linked with one type of intron, which was commonly found in South East Asian and South and Central American countries, suggesting the possibility that this mutation was introduced from other continents or convergently selected after the introgression of Aaa genes from the above area. Conclusions/Significance The worldwide eradication programs in 1940s and 1950s might have caused high selection pressure on the mosquito populations and expanded the distribution of insecticide-resistant Ae. aegypti populations. Selection of the F1534C point mutation could be hypothesized to have taken place during this period. The selection of the resistant population of Ae. aegypti with the point mutation of F1534C, and the worldwide transportation of vector mosquitoes in accordance with human activity such as trading of used tires, might result in the widespread distribution of F1534C point mutation in tropical countries

Discovery of Point Mutations in the Voltage-Gated Sodium Channel from African Aedes aegypti Populations: Potential Phylogenetic Reasons for Gene Introgression.

PubMed

Kawada, Hitoshi; Higa, Yukiko; Futami, Kyoko; Muranami, Yuto; Kawashima, Emiko; Osei, Joseph H N; Sakyi, Kojo Yirenkyi; Dadzie, Samuel; de Souza, Dziedzom K; Appawu, Maxwell; Ohta, Nobuo; Suzuki, Takashi; Minakawa, Noboru

2016-06-01

Yellow fever is endemic in some countries in Africa, and Aedes aegpyti is one of the most important vectors implicated in the outbreak. The mapping of the nation-wide distribution and the detection of insecticide resistance of vector mosquitoes will provide the beneficial information for forecasting of dengue and yellow fever outbreaks and effective control measures. High resistance to DDT was observed in all mosquito colonies collected in Ghana. The resistance and the possible existence of resistance or tolerance to permethrin were suspected in some colonies. High frequencies of point mutations at the voltage-gated sodium channel (F1534C) and one heterozygote of the other mutation (V1016I) were detected, and this is the first detection on the African continent. The frequency of F1534C allele and the ratio of F1534C homozygotes in Ae. aegypti aegypti (Aaa) were significantly higher than those in Ae. aegypti formosus (Aaf). We could detect the two types of introns between exon 20 and 21, and the F1534C mutations were strongly linked with one type of intron, which was commonly found in South East Asian and South and Central American countries, suggesting the possibility that this mutation was introduced from other continents or convergently selected after the introgression of Aaa genes from the above area. The worldwide eradication programs in 1940s and 1950s might have caused high selection pressure on the mosquito populations and expanded the distribution of insecticide-resistant Ae. aegypti populations. Selection of the F1534C point mutation could be hypothesized to have taken place during this period. The selection of the resistant population of Ae. aegypti with the point mutation of F1534C, and the worldwide transportation of vector mosquitoes in accordance with human activity such as trading of used tires, might result in the widespread distribution of F1534C point mutation in tropical countries.

Common Gating of Both CLC Transporter Subunits Underlies Voltage-dependent Activation of the 2Cl−/1H+ Exchanger ClC-7/Ostm1*

PubMed Central

Ludwig, Carmen F.; Ullrich, Florian; Leisle, Lilia; Stauber, Tobias; Jentsch, Thomas J.

2013-01-01

CLC anion transporters form dimers that function either as Cl− channels or as electrogenic Cl−/H+ exchangers. CLC channels display two different types of “gates,” “protopore” gates that open and close the two pores of a CLC dimer independently of each other and common gates that act on both pores simultaneously. ClC-7/Ostm1 is a lysosomal 2Cl−/1H+ exchanger that is slowly activated by depolarization. This gating process is drastically accelerated by many CLCN7 mutations underlying human osteopetrosis. Making use of some of these mutants, we now investigate whether slow voltage activation of plasma membrane-targeted ClC-7/Ostm1 involves protopore or common gates. Voltage activation of wild-type ClC-7 subunits was accelerated by co-expressing an excess of ClC-7 subunits carrying an accelerating mutation together with a point mutation rendering these subunits transport-deficient. Conversely, voltage activation of a fast ClC-7 mutant could be slowed by co-expressing an excess of a transport-deficient mutant. These effects did not depend on whether the accelerating mutation localized to the transmembrane part or to cytoplasmic cystathionine-β-synthase (CBS) domains of ClC-7. Combining accelerating mutations in the same subunit did not speed up gating further. No currents were observed when ClC-7 was truncated after the last intramembrane helix. Currents and slow gating were restored when the C terminus was co-expressed by itself or fused to the C terminus of the β-subunit Ostm1. We conclude that common gating underlies the slow voltage activation of ClC-7. It depends on the CBS domain-containing C terminus that does not require covalent binding to the membrane domain of ClC-7. PMID:23983121

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.

Voltage-Boosting Driver For Switching Regulator

NASA Technical Reports Server (NTRS)

Trump, Ronald C.

1990-01-01

Driver circuit assures availability of 10- to 15-V gate-to-source voltage needed to turn on n-channel metal oxide/semiconductor field-effect transistor (MOSFET) acting as switch in switching voltage regulator. Includes voltage-boosting circuit efficiently providing gate voltage 10 to 15 V above supply voltage. Contains no exotic parts and does not require additional power supply. Consists of NAND gate and dual voltage booster operating in conjunction with pulse-width modulator part of regulator.

Components of gating charge movement and S4 voltage-sensor exposure during activation of hERG channels.

PubMed

Wang, Zhuren; Dou, Ying; Goodchild, Samuel J; Es-Salah-Lamoureux, Zeineb; Fedida, David

2013-04-01

The human ether-á-go-go-related gene (hERG) K(+) channel encodes the pore-forming α subunit of the rapid delayed rectifier current, IKr, and has unique activation gating kinetics, in that the α subunit of the channel activates and deactivates very slowly, which focuses the role of IKr current to a critical period during action potential repolarization in the heart. Despite its physiological importance, fundamental mechanistic properties of hERG channel activation gating remain unclear, including how voltage-sensor movement rate limits pore opening. Here, we study this directly by recording voltage-sensor domain currents in mammalian cells for the first time and measuring the rates of voltage-sensor modification by [2-(trimethylammonium)ethyl] methanethiosulfonate chloride (MTSET). Gating currents recorded from hERG channels expressed in mammalian tsA201 cells using low resistance pipettes show two charge systems, defined as Q(1) and Q(2), with V(1/2)'s of -55.7 (equivalent charge, z = 1.60) and -54.2 mV (z = 1.30), respectively, with the Q(2) charge system carrying approximately two thirds of the overall gating charge. The time constants for charge movement at 0 mV were 2.5 and 36.2 ms for Q(1) and Q(2), decreasing to 4.3 ms for Q(2) at +60 mV, an order of magnitude faster than the time constants of ionic current appearance at these potentials. The voltage and time dependence of Q2 movement closely correlated with the rate of MTSET modification of I521C in the outermost region of the S4 segment, which had a V(1/2) of -64 mV and time constants of 36 ± 8.5 ms and 11.6 ± 6.3 ms at 0 and +60 mV, respectively. Modeling of Q(1) and Q(2) charge systems showed that a minimal scheme of three transitions is sufficient to account for the experimental findings. These data point to activation steps further downstream of voltage-sensor movement that provide the major delays to pore opening in hERG channels.

Exotic properties of a voltage-gated proton channel from the snail Helisoma trivolvis.

PubMed

Thomas, Sarah; Cherny, Vladimir V; Morgan, Deri; Artinian, Liana R; Rehder, Vincent; Smith, Susan M E; DeCoursey, Thomas E

2018-06-04

Voltage-gated proton channels, H V 1, were first reported in Helix aspersa snail neurons. These H + channels open very rapidly, two to three orders of magnitude faster than mammalian H V 1. Here we identify an H V 1 gene in the snail Helisoma trivolvis and verify protein level expression by Western blotting of H. trivolvis brain lysate. Expressed in mammalian cells, HtH V 1 currents in most respects resemble those described in other snails, including rapid activation, 476 times faster than hH V 1 (human) at pH o 7, between 50 and 90 mV. In contrast to most H V 1, activation of HtH V 1 is exponential, suggesting first-order kinetics. However, the large gating charge of ∼5.5 e 0 suggests that HtH V 1 functions as a dimer, evidently with highly cooperative gating. HtH V 1 opening is exquisitely sensitive to pH o , whereas closing is nearly independent of pH o Zn 2+ and Cd 2+ inhibit HtH V 1 currents in the micromolar range, slowing activation, shifting the proton conductance-voltage ( g H - V ) relationship to more positive potentials, and lowering the maximum conductance. This is consistent with HtH V 1 possessing three of the four amino acids that coordinate Zn 2+ in mammalian H V 1. All known H V 1 exhibit ΔpH-dependent gating that results in a 40-mV shift of the g H - V relationship for a unit change in either pH o or pH i This property is crucial for all the functions of H V 1 in many species and numerous human cells. The HtH V 1 channel exhibits normal or supernormal pH o dependence, but weak pH i dependence. Under favorable conditions, this might result in the HtH V 1 channel conducting inward currents and perhaps mediating a proton action potential. The anomalous ΔpH-dependent gating of HtH V 1 channels suggests a structural basis for this important property, which is further explored in this issue (Cherny et al. 2018. J. Gen. Physiol. https://doi.org/10.1085/jgp.201711968). © 2018 Thomas et al.

Artificial phosphorylation sites modulate the activity of a voltage-gated potassium channel

NASA Astrophysics Data System (ADS)

Ariyaratne, Amila; Zocchi, Giovanni

2015-03-01

The KvAP potassium channel is representative of a family of voltage-gated ion channels where the membrane potential is sensed by a transmembrane helix containing several positively charged arginines. Previous work by Wang and Zocchi [A. Wang and G. Zocchi, PLoS ONE 6, e18598 (2011), 10.1371/journal.pone.0018598] showed how a negatively charged polyelectrolyte attached in proximity to the voltage sensing element can bias the opening probability of the channel. Here we introduce three phosphorylation sites at the same location and show that the response curve of the channel shifts by about 20 mV upon phosphorylation, while other characteristics such as the single-channel conductance are unaffected. In summary, we construct an artificial phosphorylation site which confers allosteric regulation to the channel.

On the Structural Basis for Size-selective Permeation of Organic Cations through the Voltage-gated Sodium Channel

PubMed Central

Sun, Ye-Ming; Favre, Isabelle; Schild, Laurent; Moczydlowski, Edward

1997-01-01

Recent evidence indicates that ionic selectivity in voltage-gated Na+ channels is mediated by a small number of residues in P-region segments that link transmembrane elements S5 and S6 in each of four homologous domains denoted I, II, III, and IV. Important determinants for this function appear to be a set of conserved charged residues in the first three homologous domains, Asp(I), Glu(II), and Lys(III), located in a region of the pore called the DEKA locus. In this study, we examined several Ala-substitution mutations of these residues for alterations in ionic selectivity, inhibition of macroscopic current by external Ca2+ and H+, and molecular sieving behavior using a series of organic cations ranging in size from ammonium to tetraethylammonium. Whole-cell recording of wild-type and mutant channels of the rat muscle μ1 Na+ channel stably expressed in HEK293 cells was used to compare macroscopic current–voltage behavior in the presence of various external cations and an intracellular reference solution containing Cs+ and very low Ca2+. In particular, we tested the hypothesis that the Lys residue in domain III of the DEKA locus is responsible for restricting the permeation of large organic cations. Mutation of Lys(III) to Ala largely eliminated selectivity among the group IA monovalent alkali cations (Li+, Na+, K+, Rb+, Cs+) and permitted inward current of group IIA divalent cations (Mg2+, Ca2+, Sr2+, Ba2+). This same mutation also resulted in the acquisition of permeability to many large organic cations such as methylammonium, tetramethylammonium, and tetraethylammonium, all of which are impermeant in the native channel. The results lead to the conclusion that charged residues of the DEKA locus play an important role in molecular sieving behavior of the Na+ channel pore, a function that has been previously attributed to a hypothetical region of the channel called the “selectivity filter.” A detailed examination of individual contributions of the Asp(I), Glu

Kcnh1 Voltage-gated Potassium Channels Are Essential for Early Zebrafish Development*

PubMed Central

Stengel, Rayk; Rivera-Milla, Eric; Sahoo, Nirakar; Ebert, Christina; Bollig, Frank; Heinemann, Stefan H.; Schönherr, Roland; Englert, Christoph

2012-01-01

The Kcnh1 gene encodes a voltage-gated potassium channel highly expressed in neurons and involved in tumor cell proliferation, yet its physiological roles remain unclear. We have used the zebrafish as a model to analyze Kcnh1 function in vitro and in vivo. We found that the kcnh1 gene is duplicated in teleost fish (i.e. kcnh1a and kcnh1b) and that both genes are maternally expressed during early development. In adult zebrafish, kcnh1a and kcnh1b have distinct expression patterns but share expression in brain and testis. Heterologous expression of both genes in Xenopus oocytes revealed a strong conservation of characteristic functional properties between human and fish channels, including a unique sensitivity to intracellular Ca2+/calmodulin and modulation of voltage-dependent gating by extracellular Mg2+. Using a morpholino antisense approach, we demonstrate a strong kcnh1 loss-of-function phenotype in developing zebrafish, characterized by growth retardation, delayed hindbrain formation, and embryonic lethality. This late phenotype was preceded by transcriptional up-regulation of known cell-cycle inhibitors (p21, p27, cdh2) and down-regulation of pro-proliferative factors, including cyclin D1, at 70% epiboly. These results reveal an unanticipated basic activity of kcnh1 that is crucial for early embryonic development and patterning. PMID:22927438

The orientation and molecular movement of a k(+) channel voltage-sensing domain.

PubMed

Gandhi, Chris S; Clark, Eliana; Loots, Eli; Pralle, Arnd; Isacoff, Ehud Y

2003-10-30

Voltage-gated channels operate through the action of a voltage-sensing domain (membrane segments S1-S4) that controls the conformation of gates located in the pore domain (membrane segments S5-S6). Recent structural studies on the bacterial K(v)AP potassium channel have led to a new model of voltage sensing in which S4 lies in the lipid at the channel periphery and moves through the membrane as a unit with a portion of S3. Here we describe accessibility probing and disulfide scanning experiments aimed at determining how well the K(v)AP model describes the Drosophila Shaker potassium channel. We find that the S1-S3 helices have one end that is externally exposed, S3 does not undergo a transmembrane motion, and S4 lies in close apposition to the pore domain in the resting and activated state.

Voltage-gated sodium channel expression in mouse DRG after SNI leads to re-evaluation of projections of injured fibers.

PubMed

Laedermann, Cédric J; Pertin, Marie; Suter, Marc R; Decosterd, Isabelle

2014-03-11

Dysregulation of voltage-gated sodium channels (Na(v)s) is believed to play a major role in nerve fiber hyperexcitability associated with neuropathic pain. A complete transcriptional characterization of the different isoforms of Na(v)s under normal and pathological conditions had never been performed on mice, despite their widespread use in pain research. Na(v)s mRNA levels in mouse dorsal root ganglia (DRG) were studied in the spared nerve injury (SNI) and spinal nerve ligation (SNL) models of neuropathic pain. In the SNI model, injured and non-injured neurons were intermingled in lumbar DRG, which were pooled to increase the tissue available for experiments. A strong downregulation was observed for every Na(v)s isoform expressed except for Na(v)1.2; even Na(v)1.3, known to be upregulated in rat neuropathic pain models, was lower in the SNI mouse model. This suggests differences between these two species. In the SNL model, where the cell bodies of injured and non-injured fibers are anatomically separated between different DRG, most Na(v)s were observed to be downregulated in the L5 DRG receiving axotomized fibers. Transcription was then investigated independently in the L3, L4 and L5 DRG in the SNI model, and an important downregulation of many Na(v)s isoforms was observed in the L3 DRG, suggesting the presence of numerous injured neurons there after SNI. Consequently, the proportion of axotomized neurons in the L3, L4 and L5 DRG after SNI was characterized by studying the expression of activating transcription factor 3 (ATF3). Using this marker of nerve injury confirmed that most injured fibers find their cell bodies in the L3 and L4 DRG after SNI in C57BL/6 J mice; this contrasts with their L4 and L5 DRG localization in rats. The spared sural nerve, through which pain hypersensitivity is measured in behavioral studies, mostly projects into the L4 and L5 DRG. The complex regulation of Na(v)s, together with the anatomical rostral shift of the DRG harboring injured

Voltage-gated sodium channel expression in mouse DRG after SNI leads to re-evaluation of projections of injured fibers

PubMed Central

2014-01-01

Background Dysregulation of voltage-gated sodium channels (Navs) is believed to play a major role in nerve fiber hyperexcitability associated with neuropathic pain. A complete transcriptional characterization of the different isoforms of Navs under normal and pathological conditions had never been performed on mice, despite their widespread use in pain research. Navs mRNA levels in mouse dorsal root ganglia (DRG) were studied in the spared nerve injury (SNI) and spinal nerve ligation (SNL) models of neuropathic pain. In the SNI model, injured and non-injured neurons were intermingled in lumbar DRG, which were pooled to increase the tissue available for experiments. Results A strong downregulation was observed for every Navs isoform expressed except for Nav1.2; even Nav1.3, known to be upregulated in rat neuropathic pain models, was lower in the SNI mouse model. This suggests differences between these two species. In the SNL model, where the cell bodies of injured and non-injured fibers are anatomically separated between different DRG, most Navs were observed to be downregulated in the L5 DRG receiving axotomized fibers. Transcription was then investigated independently in the L3, L4 and L5 DRG in the SNI model, and an important downregulation of many Navs isoforms was observed in the L3 DRG, suggesting the presence of numerous injured neurons there after SNI. Consequently, the proportion of axotomized neurons in the L3, L4 and L5 DRG after SNI was characterized by studying the expression of activating transcription factor 3 (ATF3). Using this marker of nerve injury confirmed that most injured fibers find their cell bodies in the L3 and L4 DRG after SNI in C57BL/6 J mice; this contrasts with their L4 and L5 DRG localization in rats. The spared sural nerve, through which pain hypersensitivity is measured in behavioral studies, mostly projects into the L4 and L5 DRG. Conclusions The complex regulation of Navs, together with the anatomical rostral shift of the DRG

Molecular Dynamics Simulations of Voltage-Gated Cation Channels: Insights on Voltage-Sensor Domain Function and Modulation

PubMed Central

Delemotte, Lucie; Klein, Michael L.; Tarek, Mounir

2012-01-01

Since their discovery in the 1950s, the structure and function of voltage-gated cation channels (VGCC) has been largely understood thanks to results stemming from electrophysiology, pharmacology, spectroscopy, and structural biology. Over the past decade, computational methods such as molecular dynamics (MD) simulations have also contributed, providing molecular level information that can be tested against experimental results, thereby allowing the validation of the models and protocols. Importantly, MD can shed light on elements of VGCC function that cannot be easily accessed through “classical” experiments. Here, we review the results of recent MD simulations addressing key questions that pertain to the function and modulation of the VGCC’s voltage-sensor domain (VSD) highlighting: (1) the movement of the S4-helix basic residues during channel activation, articulating how the electrical driving force acts upon them; (2) the nature of the VSD intermediate states on transitioning between open and closed states of the VGCC; and (3) the molecular level effects on the VSD arising from mutations of specific S4 positively charged residues involved in certain genetic diseases. PMID:22654756

The polar T1 interface is linked to conformational changes that open the voltage-gated potassium channel.

PubMed

Minor, D L; Lin, Y F; Mobley, B C; Avelar, A; Jan, Y N; Jan, L Y; Berger, J M

2000-09-01

Kv voltage-gated potassium channels share a cytoplasmic assembly domain, T1. Recent mutagenesis of two T1 C-terminal loop residues implicates T1 in channel gating. However, structural alterations of these mutants leave open the question concerning direct involvement of T1 in gating. We find in mammalian Kv1.2 that gating depends critically on residues at complementary T1 surfaces in an unusually polar interface. An isosteric mutation in this interface causes surprisingly little structural alteration while stabilizing the closed channel and increasing the stability of T1 tetramers. Replacing T1 with a tetrameric coiled-coil destabilizes the closed channel. Together, these data suggest that structural changes involving the buried polar T1 surfaces play a key role in the conformational changes leading to channel opening.

Caution Is Required in Interpretation of Mutations in the Voltage Sensing Domain of Voltage Gated Channels as Evidence for Gating Mechanisms

PubMed Central

Kariev, Alisher M.; Green, Michael E.

2015-01-01

The gating mechanism of voltage sensitive ion channels is generally considered to be the motion of the S4 transmembrane segment of the voltage sensing domains (VSD). The primary supporting evidence came from R→C mutations on the S4 transmembrane segment of the VSD, followed by reaction with a methanethiosulfonate (MTS) reagent. The cys side chain is –SH (reactive form –S−); the arginine side chain is much larger, leaving space big enough to accommodate the MTS sulfonate head group. The cavity created by the mutation has space for up to seven more water molecules than were present in wild type, which could be displaced irreversibly by the MTS reagent. Our quantum calculations show there is major reorientation of three aromatic residues that face into the cavity in response to proton displacement within the VSD. Two phenylalanines reorient sufficiently to shield/unshield the cysteine from the intracellular and extracellular ends, depending on the proton positions, and a tyrosine forms a hydrogen bond to the cysteine sulfur with its side chain –OH. These could produce the results of the experiments that have been interpreted as evidence for physical motion of the S4 segment, without physical motion of the S4 backbone. The computations strongly suggest that the interpretation of cysteine substitution reaction experiments be re-examined in the light of these considerations. PMID:25588216

Caution is required in interpretation of mutations in the voltage sensing domain of voltage gated channels as evidence for gating mechanisms.

PubMed

Kariev, Alisher M; Green, Michael E

2015-01-12

The gating mechanism of voltage sensitive ion channels is generally considered to be the motion of the S4 transmembrane segment of the voltage sensing domains (VSD). The primary supporting evidence came from R → C mutations on the S4 transmembrane segment of the VSD, followed by reaction with a methanethiosulfonate (MTS) reagent. The cys side chain is -SH (reactive form -S-); the arginine side chain is much larger, leaving space big enough to accommodate the MTS sulfonate head group. The cavity created by the mutation has space for up to seven more water molecules than were present in wild type, which could be displaced irreversibly by the MTS reagent. Our quantum calculations show there is major reorientation of three aromatic residues that face into the cavity in response to proton displacement within the VSD. Two phenylalanines reorient sufficiently to shield/unshield the cysteine from the intracellular and extracellular ends, depending on the proton positions, and a tyrosine forms a hydrogen bond to the cysteine sulfur with its side chain -OH. These could produce the results of the experiments that have been interpreted as evidence for physical motion of the S4 segment, without physical motion of the S4 backbone. The computations strongly suggest that the interpretation of cysteine substitution reaction experiments be re-examined in the light of these considerations.

Mapping of Residues Forming the Voltage Sensor of the Voltage-Dependent Anion-Selective Channel

NASA Astrophysics Data System (ADS)

Thomas, Lorie; Blachly-Dyson, Elizabeth; Colombini, Marco; Forte, Michael

1993-06-01

Voltage-gated ion-channel proteins contain "voltage-sensing" domains that drive the conformational transitions between open and closed states in response to changes in transmembrane voltage. We have used site-directed mutagenesis to identify residues affecting the voltage sensitivity of a mitochondrial channel, the voltage-dependent anion-selective channel (VDAC). Although charge changes at many sites had no effect, at other sites substitutions that increased positive charge also increased the steepness of voltage dependance and substitutions that decreased positive charge decreased voltage dependance by an appropriate amount. In contrast to the plasma membrane K^+ and Na^+ channels, these residues are distributed over large parts of the VDAC protein. These results have been used to define the conformational transitions that accompany voltage gating of an ion channel. This gating mechanism requires the movement of large portions of the VDAC protein through the membrane.

Niflumic acid alters gating of HCN2 pacemaker channels by interaction with the outer region of S4 voltage sensing domains.

PubMed

Cheng, Lan; Sanguinetti, Michael C

2009-05-01

Niflumic acid, 2-[[3-(trifluoromethyl)phenyl]amino]pyridine-3-carboxylic acid (NFA), is a nonsteroidal anti-inflammatory drug that also blocks or modifies the gating of many ion channels. Here, we investigated the effects of NFA on hyperpolarization-activated cyclic nucleotide-gated cation (HCN) pacemaker channels expressed in X. laevis oocytes using site-directed mutagenesis and the two-electrode voltage-clamp technique. Extracellular NFA acted rapidly and caused a slowing of activation and deactivation and a hyperpolarizing shift in the voltage dependence of HCN2 channel activation (-24.5 +/- 1.2 mV at 1 mM). Slowed channel gating and reduction of current magnitude was marked in oocytes treated with NFA, while clamped at 0 mV but minimal in oocytes clamped at -100 mV, indicating the drug preferentially interacts with channels in the closed state. NFA at 0.1 to 3 mM shifted the half-point for channel activation in a concentration-dependent manner, with an EC(50) of 0.54 +/- 0.068 mM and a predicted maximum shift of -38 mV. NFA at 1 mM also reduced maximum HCN2 conductance by approximately 20%, presumably by direct block of the pore. The rapid onset and state-dependence of NFA-induced changes in channel gating suggests an interaction with the extracellular region of the S4 transmembrane helix, the primary voltage-sensing domain of HCN2. Neutralization (by mutation to Gln) of any three of the outer four basic charged residues in S4, but not single mutations, abrogated the NFA-induced shift in channel activation. We conclude that NFA alters HCN2 gating by interacting with the extracellular end of the S4 voltage sensor domains.

ClC-7 is a slowly voltage-gated 2Cl−/1H+-exchanger and requires Ostm1 for transport activity

PubMed Central

Leisle, Lilia; Ludwig, Carmen F; Wagner, Florian A; Jentsch, Thomas J; Stauber, Tobias

2011-01-01

Mutations in the ClC-7/Ostm1 ion transporter lead to osteopetrosis and lysosomal storage disease. Its lysosomal localization hitherto precluded detailed functional characterization. Using a mutated ClC-7 that reaches the plasma membrane, we now show that both the aminoterminus and transmembrane span of the Ostm1 β-subunit are required for ClC-7 Cl−/H+-exchange, whereas the Ostm1 transmembrane domain suffices for its ClC-7-dependent trafficking to lysosomes. ClC-7/Ostm1 currents were strongly outwardly rectifying owing to slow gating of ion exchange, which itself displays an intrinsically almost linear voltage dependence. Reversal potentials of tail currents revealed a 2Cl−/1H+-exchange stoichiometry. Several disease-causing CLCN7 mutations accelerated gating. Such mutations cluster to the second cytosolic cystathionine-β-synthase domain and potential contact sites at the transmembrane segment. Our work suggests that gating underlies the rectification of all endosomal/lysosomal CLCs and extends the concept of voltage gating beyond channels to ion exchangers. PMID:21527911

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.

Transparently wrap-gated semiconductor nanowire arrays for studies of gate-controlled photoluminescence

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

Nylund, Gustav; Storm, Kristian; Torstensson, Henrik

2013-12-04

We present a technique to measure gate-controlled photoluminescence (PL) on arrays of semiconductor nanowire (NW) capacitors using a transparent film of Indium-Tin-Oxide (ITO) wrapping around the nanowires as the gate electrode. By tuning the wrap-gate voltage, it is possible to increase the PL peak intensity of an array of undoped InP NWs by more than an order of magnitude. The fine structure of the PL spectrum reveals three subpeaks whose relative peak intensities change with gate voltage. We interpret this as gate-controlled state-filling of luminescing quantum dot segments formed by zincblende stacking faults in the mainly wurtzite NW crystal structure.

Antibodies to voltage-gated potassium and calcium channels in epilepsy.

PubMed

Majoie, H J Marian; de Baets, Mark; Renier, Willy; Lang, Bethan; Vincent, Angela

2006-10-01

To determine the prevalence of antibodies to ion channels in patients with long standing epilepsy. Although the CNS is thought to be protected from circulating antibodies by the blood brain barrier, glutamate receptor antibodies have been reported in Rasmussen's encephalitis, glutamic acid decarboxylase (GAD) antibodies have been found in a few patients with epilepsy, and antibodies to voltage-gated potassium channels (VGKC) have been found in a non-paraneoplastic form of limbic encephalitis (with amnesia and seizures) that responds to immunosuppressive therapy. We retrospectively screened sera from female epilepsy patients (n=106) for autoantibodies to VGKC (Kv 1.1, 1.2 or 1.6), voltage-gated calcium channels (VGCC) (P/Q-type), and GAD. All positive results, based on the values of control data [McKnight, K., Jiang, Y., et al. (2005). Serum antibodies in epilepsy and seizure-associated disorders. Neurology 65, 1730-1735], were retested at lower serum concentrations, and results compared with previously published control data. Demographics, medical history, and epilepsy related information was gathered. The studied group consisted predominantly of patients with long standing drug resistant epilepsy. VGKC antibodies were raised (>100 pM) in six patients. VGCC antibodies (>45 pM) were slightly raised in only one patient. GAD antibodies were <3 U/ml in all patients. The clinical features of the patients with VGKC antibodies differed from previously described patients with limbic encephalitis-like syndrome, and were not different with respect to seizure type, age at first seizure, duration of epilepsy, or use of anti-epileptic drugs from the VGKC antibody negative patients. The results demonstrate that antibodies to VGKC are present in 6% of patients with typical long-standing epilepsy, but whether these antibodies are pathogenic or secondary to the primary disease process needs to be determined.

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

Engineering of a genetically encodable fluorescent voltage sensor exploiting fast Ci-VSP voltage-sensing movements.

PubMed

Lundby, Alicia; Mutoh, Hiroki; Dimitrov, Dimitar; Akemann, Walther; Knöpfel, Thomas

2008-06-25

Ci-VSP contains a voltage-sensing domain (VSD) homologous to that of voltage-gated potassium channels. Using charge displacement ('gating' current) measurements we show that voltage-sensing movements of this VSD can occur within 1 ms in mammalian membranes. Our analysis lead to development of a genetically encodable fluorescent protein voltage sensor (VSFP) in which the fast, voltage-dependent conformational changes of the Ci-VSP voltage sensor are transduced to similarly fast fluorescence read-outs.

Depolarization of the conductance-voltage relationship in the NaV1.5 mutant, E1784K, is due to altered fast inactivation.

PubMed

Peters, Colin H; Yu, Alec; Zhu, Wandi; Silva, Jonathan R; Ruben, Peter C

2017-01-01

E1784K is the most common mixed long QT syndrome/Brugada syndrome mutant in the cardiac voltage-gated sodium channel NaV1.5. E1784K shifts the midpoint of the channel conductance-voltage relationship to more depolarized membrane potentials and accelerates the rate of channel fast inactivation. The depolarizing shift in the midpoint of the conductance curve in E1784K is exacerbated by low extracellular pH. We tested whether the E1784K mutant shifts the channel conductance curve to more depolarized membrane potentials by affecting the channel voltage-sensors. We measured ionic currents and gating currents at pH 7.4 and pH 6.0 in Xenopus laevis oocytes. Contrary to our expectation, the movement of gating charges is shifted to more hyperpolarized membrane potentials by E1784K. Voltage-clamp fluorimetry experiments show that this gating charge shift is due to the movement of the DIVS4 voltage-sensor being shifted to more hyperpolarized membrane potentials. Using a model and experiments on fast inactivation-deficient channels, we show that changes to the rate and voltage-dependence of fast inactivation are sufficient to shift the conductance curve in E1784K. Our results localize the effects of E1784K to DIVS4, and provide novel insight into the role of the DIV-VSD in regulating the voltage-dependencies of activation and fast inactivation.

A two-dimensional analytical modeling for channel potential and threshold voltage of short channel triple material symmetrical gate Stack (TMGS) DG-MOSFET

NASA Astrophysics Data System (ADS)

Tripathi, Shweta

2016-10-01

In the present work, a two-dimensional (2D) analytical framework of triple material symmetrical gate stack (TMGS) DG-MOSFET is presented in order to subdue the short channel effects. A lightly doped channel along with triple material gate having different work functions and symmetrical gate stack structure, showcases substantial betterment in quashing short channel effects to a good extent. The device functioning amends in terms of improved exemption to threshold voltage roll-off, thereby suppressing the short channel effects. The encroachments of respective device arguments on the threshold voltage of the proposed structure are examined in detail. The significant outcomes are compared with the numerical simulation data obtained by using 2D ATLAS™ device simulator to affirm and formalize the proposed device structure.

Interaction between the Pore and a Fast Gate of the Cardiac Sodium Channel

PubMed Central

Townsend, Claire; Horn, Richard

1999-01-01

Permeant ions affect a fast gating process observed in human cardiac sodium channels (Townsend, C., H.A. Hartmann, and R. Horn. 1997. J. Gen. Physiol. 110:11–21). Removal of extracellular permeant ions causes a reduction of open probability at positive membrane potentials. These results suggest an intimate relationship between the ion-conducting pore and the gates of the channel. We tested this hypothesis by three sets of manipulations designed to affect the binding of cations within the pore: application of intracellular pore blockers, mutagenesis of residues known to contribute to permeation, and chemical modification of a native cysteine residue (C373) near the extracellular mouth of the pore. The coupling between extracellular permeant ions and this fast gating process is abolished both by pore blockers and by a mutation that severely affects selectivity. A more superficial pore mutation or chemical modification of C373 reduces single channel conductance while preserving both selectivity of the pore and the modulatory effects of extracellular cations. Our results demonstrate a modulatory gating role for a region deep within the pore and suggest that the structure of the permeation pathway is largely preserved when a channel is closed. PMID:9925827

MEMS Gate Structures for Electric Propulsion Applications

DTIC Science & Technology

2006-07-12

distance between gates of dual gate system V = grid voltage Dsheath = sheath thickness Va = anode voltage E = electric field Vemitter = emitter voltage Es...minutes. A hot pressed boron nitride target (4N) in the hexagonal phase (h- BN) was sputtered in a RF magnetron sputtering gun. To promote the nucleation...and nanoFETs. This paper concludes with a discussion on using MEMS gates for dual -grid electron field emission applications. II. Gate Design I I

Post-translational cleavage of Hv1 in human sperm tunes pH- and voltage-dependent gating.

PubMed

Berger, Thomas K; Fußhöller, David M; Goodwin, Normann; Bönigk, Wolfgang; Müller, Astrid; Dokani Khesroshahi, Nasim; Brenker, Christoph; Wachten, Dagmar; Krause, Eberhard; Kaupp, U Benjamin; Strünker, Timo

2017-03-01

In human sperm, proton flux across the membrane is controlled by the voltage-gated proton channel Hv1. We show that sperm harbour both Hv1 and an N-terminally cleaved isoform termed Hv1Sper. The pH-control of Hv1Sper and Hv1 is distinctively different. Hv1Sper and Hv1 can form heterodimers that combine features of both constituents. Cleavage and heterodimerization of Hv1 might represent an adaptation to the specific requirements of pH control in sperm. In human sperm, the voltage-gated proton channel Hv1 controls the flux of protons across the flagellar membrane. Here, we show that sperm harbour Hv1 and a shorter isoform, termed Hv1Sper. Hv1Sper is generated from Hv1 by removal of 68 amino acids from the N-terminus by post-translational proteolytic cleavage. The pH-dependent gating of the channel isoforms is distinctly different. In both Hv1 and Hv1Sper, the conductance-voltage relationship is determined by the pH difference across the membrane (∆pH). However, simultaneous changes in intracellular and extracellular pH that leave ΔpH constant strongly shift the activation curve of Hv1Sper but not that of Hv1, demonstrating that cleavage of the N-terminus tunes pH sensing in Hv1. Moreover, we show that Hv1 and Hv1Sper assemble as heterodimers that combine features of both constituents. We suggest that cleavage and heterodimerization of Hv1 represents an adaptation to the specific requirements of pH control in sperm. © 2016 The Authors. The Journal of Physiology © 2016 The Physiological Society.

Modulation of mononuclear phagocyte inflammatory response by liposome-encapsulated voltage gated sodium channel inhibitor ameliorates myocardial ischemia/reperfusion injury in rats.

PubMed

Zhou, Xin; Luo, Yue-Chen; Ji, Wen-Jie; Zhang, Li; Dong, Yan; Ge, Lan; Lu, Rui-Yi; Sun, Hai-Ying; Guo, Zao-Zeng; Yang, Guo-Hong; Jiang, Tie-Min; Li, Yu-Ming

2013-01-01

Emerging evidence shows that anti-inflammatory strategies targeting inflammatory monocyte subset could reduce excessive inflammation and improve cardiovascular outcomes. Functional expression of voltage-gated sodium channels (VGSCs) have been demonstrated in monocytes and macrophages. We hypothesized that mononuclear phagocyte VGSCs are a target for monocyte/macrophage phenotypic switch, and liposome mediated inhibition of mononuclear phagocyte VGSC may attenuate myocardial ischemia/reperfusion (I/R) injury and improve post-infarction left ventricular remodeling. Thin film dispersion method was used to prepare phenytoin (PHT, a non-selective VGSC inhibitor) entrapped liposomes. Pharmacokinetic study revealed that the distribution and elimination half-life of PHT entrapped liposomes were shorter than those of free PHT, indicating a rapid uptake by mononuclear phagocytes after intravenous injection. In rat peritoneal macrophages, several VGSC α subunits (NaV1.1, NaV1.3, NaV1.4, NaV1.5, NaV1.6, NaV1.7, NaVX, Scn1b, Scn3b and Scn4b) and β subunits were expressed at mRNA level, and PHT could suppress lipopolysaccharide induced M1 polarization (decreased TNF-α and CCL5 expression) and facilitate interleukin-4 induced M2 polarization (increased Arg1 and TGF-β1 expression). In vivo study using rat model of myocardial I/R injury, demonstrated that PHT entrapped liposome could partially suppress I/R injury induced CD43+ inflammatory monocyte expansion, along with decreased infarct size and left ventricular fibrosis. Transthoracic echocardiography and invasive hemodynamic analysis revealed that PHT entrapped liposome treatment could attenuate left ventricular structural and functional remodeling, as shown by increased ejection fraction, reduced end-systolic and end-diastolic volume, as well as an amelioration of left ventricular systolic (+dP/dt max) and diastolic (-dP/dt min) functions. Our work for the first time demonstrates the therapeutic potential of VGSC antagonism

G protein modulation of CaV2 voltage-gated calcium channels.

PubMed

Currie, Kevin P M

2010-01-01

Voltage-gated Ca(2+) channels translate the electrical inputs of excitable cells into biochemical outputs by controlling influx of the ubiquitous second messenger Ca(2+) . As such the channels play pivotal roles in many cellular functions including the triggering of neurotransmitter and hormone release by CaV2.1 (P/Q-type) and CaV2.2 (N-type) channels. It is well established that G protein coupled receptors (GPCRs) orchestrate precise regulation neurotransmitter and hormone release through inhibition of CaV2 channels. Although the GPCRs recruit a number of different pathways, perhaps the most prominent, and certainly most studied among these is the so-called voltage-dependent inhibition mediated by direct binding of Gβγ to the α1 subunit of CaV2 channels. This article will review the basics of Ca(2+) -channels and G protein signaling, and the functional impact of this now classical inhibitory mechanism on channel function. It will also provide an update on more recent developments in the field, both related to functional effects and crosstalk with other signaling pathways, and advances made toward understanding the molecular interactions that underlie binding of Gβγ to the channel and the voltage-dependence that is a signature characteristic of this mechanism.

Gating kinetics of batrachotoxin-modified Na+ channels in the squid giant axon. Voltage and temperature effects.

PubMed Central

Correa, A M; Bezanilla, F; Latorre, R

1992-01-01

The gating kinetics of batrachotoxin-modified Na+ channels were studied in outside-out patches of axolemma from the squid giant axon by means of the cut-open axon technique. Single channel kinetics were characterized at different membrane voltages and temperatures. The probability of channel opening (Po) as a function of voltage was well described by a Boltzmann distribution with an equivalent number of gating particles of 3.58. The voltage at which the channel was open 50% of the time was a function of [Na+] and temperature. A decrease in the internal [Na+] induced a shift to the right of the Po vs. V curve, suggesting the presence of an integral negative fixed charge near the activation gate. An increase in temperature decreased Po, indicating a stabilization of the closed configuration of the channel and also a decrease in entropy upon channel opening. Probability density analysis of dwell times in the closed and open states of the channel at 0 degrees C revealed the presence of three closed and three open states. The slowest open kinetic component constituted only a small fraction of the total number of transitions and became negligible at voltages greater than -65 mV. Adjacent interval analysis showed that there is no correlation in the duration of successive open and closed events. Consistent with this analysis, maximum likelihood estimation of the rate constants for nine different single-channel models produced a preferred model (model 1) having a linear sequence of closed states and two open states emerging from the last closed state. The effect of temperature on the rate constants of model 1 was studied. An increase in temperature increased all rate constants; the shift in Po would be the result of an increase in the closing rates predominant over the change in the opening rates. The temperature study also provided the basis for building an energy diagram for the transitions between channel states. PMID:1318096

The GaN trench gate MOSFET with floating islands: High breakdown voltage and improved BFOM

NASA Astrophysics Data System (ADS)

Shen, Lingyan; Müller, Stephan; Cheng, Xinhong; Zhang, Dongliang; Zheng, Li; Xu, Dawei; Yu, Yuehui; Meissner, Elke; Erlbacher, Tobias

2018-02-01

A novel GaN trench gate (TG) MOSFET with P-type floating islands (FLI) in drift region, which can suppress the electric field peak at bottom of gate trench during the blocking state and prevent premature breakdown in gate oxide, is proposed and investigated by TCAD simulations. The influence of thickness, position, doping concentration and length of the FLI on breakdown voltage (BV) and specific on-resistance (Ron_sp) is studied, providing useful guidelines for design of this new type of device. Using optimized parameters for the FLI, GaN FLI TG-MOSFET obtains a BV as high as 2464 V with a Ron_sp of 3.0 mΩ cm2. Compared to the conventional GaN TG-MOSFET with the same structure parameters, the Baliga figure of merit (BFOM) is enhanced by 150%, getting closer to theoretical limit for GaN devices.

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.

Sodium Channel Voltage-Gated Beta 2 Plays a Vital Role in Brain Aging Associated with Synaptic Plasticity and Expression of COX5A and FGF-2.

PubMed

XiYang, Yan-Bin; Wang, You-Cui; Zhao, Ya; Ru, Jin; Lu, Bing-Tuan; Zhang, Yue-Ning; Wang, Nai-Chao; Hu, Wei-Yan; Liu, Jia; Yang, Jin-Wei; Wang, Zhao-Jun; Hao, Chun-Guang; Feng, Zhong-Tang; Xiao, Zhi-Cheng; Dong, Wei; Quan, Xiong-Zhi; Zhang, Lian-Feng; Wang, Ting-Hua

2016-03-01

The role of sodium channel voltage-gated beta 2 (SCN2B) in brain aging is largely unknown. The present study was therefore designed to determine the role of SCN2B in brain aging by using the senescence-accelerated mice prone 8 (SAMP8), a brain senescence-accelerated animal model, together with the SCN2B transgenic mice. The results showed that SAMP8 exhibited impaired learning and memory functions, assessed by the Morris water maze test, as early as 8 months of age. The messenger RNA (mRNA) and protein expressions of SCN2B were also upregulated in the prefrontal cortex at this age. Treatment with traditional Chinese anti-aging medicine Xueshuangtong (Panax notoginseng saponins, PNS) significantly reversed the SCN2B expressions in the prefrontal cortex, resulting in improved learning and memory. Moreover, SCN2B knockdown transgenic mice were generated and bred to determine the roles of SCN2B in brain senescence. A reduction in the SCN2B level by 60.68% resulted in improvement in the hippocampus-dependent spatial recognition memory and long-term potential (LTP) slope of field excitatory postsynaptic potential (fEPSP), followed by an upregulation of COX5A mRNA levels and downregulation of fibroblast growth factor-2 (FGF-2) mRNA expression. Together, the present findings indicated that SCN2B could play an important role in the aging-related cognitive deterioration, which is associated with the regulations of COX5A and FGF-2. These findings could provide the potential strategy of candidate target to develop antisenescence drugs for the treatment of brain aging.

Polymorphism of intron-1 in the voltage-gated sodium channel gene of Anopheles gambiae s.s. populations from Cameroon with emphasis on insecticide knockdown resistance mutations.

PubMed

Etang, Josiane; Vicente, Jose L; Nwane, Philippe; Chouaibou, Mouhamadou; Morlais, Isabelle; Do Rosario, Virgilio E; Simard, Frederic; Awono-Ambene, Parfait; Toto, Jean Claude; Pinto, Joao

2009-07-01

Sequence variation at the intron-1 of the voltage-gated sodium channel gene in Anopheles gambiae M- and S-forms from Cameroon was assessed to explore the number of mutational events originating knockdown resistance (kdr) alleles. Mosquitoes were sampled between December 2005 and June 2006 from three geographical areas: (i) Magba in the western region; (ii) Loum, Tiko, Douala, Kribi, and Campo along the Atlantic coast; and (iii) Bertoua, in the eastern continental plateau. Both 1014S and 1014F kdr alleles were found in the S-form with overall frequencies of 14% and 42% respectively. Only the 1014F allele was found in the M-form at lower frequency (11%). Analysis of a 455 bp region of intron-1 upstream the kdr locus revealed four independent mutation events originating kdr alleles, here named MS1 -1014F, S1-1014S and S2-1014S kdr-intron-1 haplotypes in S-form and MS3-1014F kdr-intron-1 haplotype in the M-form. Furthermore, there was evidence for mutual introgression of kdr 1014F allele between the two molecular forms, MS1 and MS3 being widely shared by them. Although no M/S hybrid was observed in analysed samples, this wide distribution of haplotypes MS1 and MS3 suggests inter-form hybridizing at significant level and emphasizes the rapid diffusion of the kdr alleles in Africa. The mosaic of genetic events found in Cameroon is representative of the situation in the West-Central African region and highlights the importance of evaluating the spatial and temporal evolution of kdr alleles for a better management of insecticide resistance.

Titanium-tungsten nanocrystals embedded in a SiO(2)/Al(2)O(3) gate dielectric stack for low-voltage operation in non-volatile memory.

PubMed

Yang, Shiqian; Wang, Qin; Zhang, Manhong; Long, Shibing; Liu, Jing; Liu, Ming

2010-06-18

Titanium-tungsten nanocrystals (NCs) were fabricated by a self-assembly rapid thermal annealing (RTA) process. Well isolated Ti(0.46)W(0.54) NCs were embedded in the gate dielectric stack of SiO(2)/Al(2)O(3). A metal-oxide-semiconductor (MOS) capacitor was fabricated to investigate its application in a non-volatile memory (NVM) device. It demonstrated a large memory window of 6.2 V in terms of flat-band voltage (V(FB)) shift under a dual-directional sweeping gate voltage of - 10 to 10 V. A 1.1 V V(FB) shift under a low dual-directional sweeping gate voltage of - 4 to 4 V was also observed. The retention characteristic of this MOS capacitor was demonstrated by a 0.5 V memory window after 10(4) s of elapsed time at room temperature. The endurance characteristic was demonstrated by a program/erase cycling test.

Knockdown resistance (kdr)-like mutations in the voltage-gated sodium channel of a malaria vector Anopheles stephensi and PCR assays for their detection.

PubMed

Singh, Om P; Dykes, Cherry L; Lather, Manila; Agrawal, Om P; Adak, Tridibes

2011-03-14

Knockdown resistance (kdr) in insects, resulting from mutation(s) in the voltage-gated sodium channel (vgsc) gene is one of the mechanisms of resistance against DDT and pyrethroid-group of insecticides. The most common mutation(s) associated with knockdown resistance in insects, including anophelines, has been reported to be present at residue Leu1014 in the IIS6 transmembrane segment of the vgsc gene. This study reports the presence of two alternative kdr-like mutations, L1014S and L1014F, at this residue in a major malaria vector Anopheles stephensi and describes new PCR assays for their detection. Part of the vgsc (IIS4-S5 linker-to-IIS6 transmembrane segment) of An. stephensi collected from Alwar (Rajasthan, India) was PCR-amplified from genomic DNA, sequenced and analysed for the presence of deduced amino acid substitution(s). Analysis of DNA sequences revealed the presence of two alternative non-synonymous point mutations at L1014 residue in the IIS6 transmembrane segment of vgsc, i.e., T>C mutation on the second position and A>T mutation on the third position of the codon, leading to Leu (TTA)-to-Ser (TCA) and -Phe (TTT) amino acid substitutions, respectively. Polymerase chain reaction (PCR) assays were developed for identification of each of these two point mutations. Genotyping of An. stephensi mosquitoes from Alwar by PCR assays revealed the presence of both mutations, with a high frequency of L1014S. The PCR assays developed for detection of the kdr mutations were specific as confirmed by DNA sequencing of PCR-genotyped samples. Two alternative kdr-like mutations, L1014S and L1014F, were detected in An. stephensi with a high allelic frequency of L1014S. The occurrence of L1014S is being reported for the first time in An. stephensi. Two specific PCR assays were developed for detection of two kdr-like mutations in An. stephensi.

Temperature-dependent degradation mechanisms of threshold voltage in La2O3-gated n-channel metal-oxide-semiconductor field-effect transistors

NASA Astrophysics Data System (ADS)

Wang, Ming-Tsong; Hsu, De-Cheng; Juan, Pi-Chun; Wang, Y. L.; Lee, Joseph Ya-min

2010-09-01

Metal-oxide-semiconductor capacitors and n-channel metal-oxide-semiconductor field-effect transistors with La2O3 gate dielectric were fabricated. The positive bias temperature instability was studied. The degradation of threshold voltage (ΔVT) showed an exponential dependence on the stress time in the temperature range from 25 to 75 °C. The degradation of subthreshold slope (ΔS) and gate leakage (IG) with stress voltage was also measured. The degradation of VT is attributed to the oxide trap charges Qot. The extracted activation energy of 0.2 eV is related to a degradation dominated by the release of atomic hydrogen in La2O3 thin films.

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

Tunable valley polarization by a gate voltage when an electron tunnels through multiple line defects in graphene.

PubMed

Liu, Zhe; Jiang, Liwei; Zheng, Yisong

2015-02-04

By means of an appropriate wave function connection condition, we study the electronic structure of a line defect superlattice of graphene with the Dirac equation method. We obtain the analytical dispersion relation, which can simulate well the tight-binding numerical result about the band structure of the superlattice. Then, we generalize this theoretical method to study the electronic transmission through a potential barrier where multiple line defects are periodically patterned. We find that there exists a critical incident angle which restricts the electronic transmission through multiple line defects within a specific incident angle range. The critical angle depends sensitively on the potential barrier height, which can be modulated by a gate voltage. As a result, non-trivial transmissions of K and K' valley electrons are restricted, respectively, in two distinct ranges of the incident angle. Our theoretical result demonstrates that a gate voltage can act as a feasible measure to tune the valley polarization when electrons tunnel through multiple line defects.

Silicon direct bonding approach to high voltage power device (insulated gate bipolar transistors)

NASA Astrophysics Data System (ADS)

Cha, Giho; Kim, Youngchul; Jang, Hyungwoo; Kang, Hyunsoon; Song, Changsub

2001-10-01

Silicon direct bonding technique was successfully applied for the fabrication of high voltage IGBT (Insulated Gate Bipolar Transistor). In this work, 5 inch, p-type CZ wafer for handle wafer and n-type FZ wafer for device wafer were used and bonding the two wafers was performed at reduced pressure (1mmTorr) using a modified vacuum bonding machine. Since the breakdown voltage in high voltage device has been determined by the remained thickness of device layer, grinding and CMP steps should be carefully designed in order to acquire better uniformity of device layer. In order to obtain the higher removal rate and the final better uniformity of device layer, the harmony of the two processes must be considered. We found that the concave type of grinding profile and the optimal thickness of ground wafer was able to reduce the process time of CMP step and also to enhance the final thickness uniformity of device layer up to +/- 1%. Finally, when compared epitaxy layer with SDB wafer, the SDB wafer was found to be more favorable in terms of cost and electrical characteristics.

Calmodulins from Schistosoma mansoni: Biochemical analysis and interaction with IQ-motifs from voltage-gated calcium channels.

PubMed

Thomas, Charlotte M; Timson, David J

2018-05-17

The trematode Schistosoma mansoni is a causative agent of schistosomiasis, the second most common parasitic disease of humans after malaria. Calcium homeostasis and calcium-mediated signalling pathways are of particular interest in this species. The drug of choice for treating schistosomiasis, praziquantel, disrupts the regulation of calcium uptake and there is interest in exploiting calcium-mediated processes for future drug discovery. Calmodulin is a calcium sensing protein, present in most eukaryotes. It is a critical regulator of processes as diverse as muscle contraction, cell division and, partly through interaction with voltage-gated calcium channels, intra-cellular calcium concentrations. S. mansoni expresses two highly similar calmodulins - SmCaM1 and SmCaM2. Both proteins interact with calcium, manganese, cadmium (II), iron (II) and lead ions in native gel electrophoresis. These ions also cause conformational changes in the proteins resulting in the exposure of a more hydrophobic surface (as demonstrated by anilinonaphthalene-8-sulfonate fluorescence assays). The proteins are primarily dimeric in the absence of calcium ions, but monomeric in the presence of this ion. Both SmCaM1 and SmCaM2 interact with a peptide corresponding to an IQ-motif derived from the α-subunit of the voltage-gated calcium channel SmCa v 1B (residues 1923-1945). Both proteins bound with slightly higher affinity in the presence of calcium ions. However, there was no difference between the affinities of the two proteins for the peptide. This interaction could be antagonised by chlorpromazine and trifluoperazine, but not praziquantel or thiamylal. Interestingly no interaction could be detected with the other three IQ-motifs identified in S. mansoni voltage-gated ion calcium channels. Copyright © 2018 Elsevier Ltd. All rights reserved.

The Molecular Basis of Polyunsaturated Fatty Acid Interactions with the Shaker Voltage-Gated Potassium Channel

PubMed Central

Yazdi, Samira; Stein, Matthias; Elinder, Fredrik; Andersson, Magnus; Lindahl, Erik

2016-01-01

Voltage-gated potassium (KV) channels are membrane proteins that respond to changes in membrane potential by enabling K+ ion flux across the membrane. Polyunsaturated fatty acids (PUFAs) induce channel opening by modulating the voltage-sensitivity, which can provide effective treatment against refractory epilepsy by means of a ketogenic diet. While PUFAs have been reported to influence the gating mechanism by electrostatic interactions to the voltage-sensor domain (VSD), the exact PUFA-protein interactions are still elusive. In this study, we report on the interactions between the Shaker KV channel in open and closed states and a PUFA-enriched lipid bilayer using microsecond molecular dynamics simulations. We determined a putative PUFA binding site in the open state of the channel located at the protein-lipid interface in the vicinity of the extracellular halves of the S3 and S4 helices of the VSD. In particular, the lipophilic PUFA tail covered a wide range of non-specific hydrophobic interactions in the hydrophobic central core of the protein-lipid interface, while the carboxylic head group displayed more specific interactions to polar/charged residues at the extracellular regions of the S3 and S4 helices, encompassing the S3-S4 linker. Moreover, by studying the interactions between saturated fatty acids (SFA) and the Shaker KV channel, our study confirmed an increased conformational flexibility in the polyunsaturated carbon tails compared to saturated carbon chains, which may explain the specificity of PUFA action on channel proteins. PMID:26751683

A common pathway for charge transport through voltage-sensing domains.

PubMed

Chanda, Baron; Bezanilla, Francisco

2008-02-07

Voltage-gated ion channels derive their voltage sensitivity from the movement of specific charged residues in response to a change in transmembrane potential. Several studies on mechanisms of voltage sensing in ion channels support the idea that these gating charges move through a well-defined permeation pathway. This gating pathway in a voltage-gated ion channel can also be mutated to transport free cations, including protons. The recent discovery of proton channels with sequence homology to the voltage-sensing domains suggests that evolution has perhaps exploited the same gating pathway to generate a bona fide voltage-dependent proton transporter. Here we will discuss implications of these findings on the mechanisms underlying charge (and ion) transport by voltage-sensing domains.

Depolarization of the conductance-voltage relationship in the NaV1.5 mutant, E1784K, is due to altered fast inactivation

PubMed Central

Yu, Alec; Zhu, Wandi; Silva, Jonathan R.; Ruben, Peter C.

2017-01-01

E1784K is the most common mixed long QT syndrome/Brugada syndrome mutant in the cardiac voltage-gated sodium channel NaV1.5. E1784K shifts the midpoint of the channel conductance-voltage relationship to more depolarized membrane potentials and accelerates the rate of channel fast inactivation. The depolarizing shift in the midpoint of the conductance curve in E1784K is exacerbated by low extracellular pH. We tested whether the E1784K mutant shifts the channel conductance curve to more depolarized membrane potentials by affecting the channel voltage-sensors. We measured ionic currents and gating currents at pH 7.4 and pH 6.0 in Xenopus laevis oocytes. Contrary to our expectation, the movement of gating charges is shifted to more hyperpolarized membrane potentials by E1784K. Voltage-clamp fluorimetry experiments show that this gating charge shift is due to the movement of the DIVS4 voltage-sensor being shifted to more hyperpolarized membrane potentials. Using a model and experiments on fast inactivation-deficient channels, we show that changes to the rate and voltage-dependence of fast inactivation are sufficient to shift the conductance curve in E1784K. Our results localize the effects of E1784K to DIVS4, and provide novel insight into the role of the DIV-VSD in regulating the voltage-dependencies of activation and fast inactivation. PMID:28898267

N-acetylcysteine-induced vasodilation involves voltage-gated potassium channels in rat aorta.

PubMed

Han, Wei-Qing; Zhu, Ding-Liang; Wu, Ling-Yun; Chen, Qi-Zhi; Guo, Shu-Jie; Gao, Ping-Jin

2009-05-22

N-acetylcysteine (NAC) has a protective effect against vascular dysfunction by decreasing the level of reactive oxygen species (ROS) in experimental and human hypertension. This study was designed to examine whether NAC would relax vascular rings in vitro via nitric oxide-cyclic guanosine monophosphate (NO-cGMP) pathway, extracellular Ca2+ and/or K+ channels. Rat aortic arteries were mounted in an organ bath, contracted with 0.1, 0.5 or 1 micromol/L phenylephrine to plateau, and the vasodilatory effect of NAC was examined in the absence or presence of ROS scavengers, inhibitors of NO-cGMP pathway or K+ channels. Vascular smooth muscle cells (VSMCs) were loaded with a calcium sensitive fluorescent dye fluo-3 AM, and [Ca2+](i) was determined with laser-scanning confocal microscopy. NAC (0.1-4 mmol/L) dose-dependently relaxed rat aorta pre-contracted with phenylephrine. Endothelium removal, endothelial nitric oxide synthase inhibitor N(omega)-Nitro-l-arginine (L-NNA) (100 micromol/L) or soluble guanylyl cyclase (sGC) inhibitor (ODQ) (10 micromol/L) did not affect NAC-induced vasodilation. In contrast, NAC-induced vasodilation was blunted after extracellular calcium was removed and calcium imaging showed that 4 mmol/L NAC quickly decreased [Ca2+](i) in fluo-3 AM loaded VSMCs. NAC-induced vasodilation was significantly reduced in the presence of voltage-gated K+ channels (Kv) inhibitor 4-aminopyridine (4-AP). The vasodilatory effect of NAC may be explained at least partly by activation of voltage-gated K+ channels.

Voltage controlled spintronic devices for logic applications

DOEpatents

You, Chun-Yeol; Bader, Samuel D.

2001-01-01

A reprogrammable logic gate comprising first and second voltage-controlled rotation transistors. Each transistor comprises three ferromagnetic layers with a spacer and insulating layer between the first and second ferromagnetic layers and an additional insulating layer between the second and third ferromagnetic layers. The third ferromagnetic layer of each transistor is connected to each other, and a constant external voltage source is applied to the second ferromagnetic layer of the first transistor. As input voltages are applied to the first ferromagnetic layer of each transistor, the relative directions of magnetization of the ferromagnetic layers and the magnitude of the external voltage determines the output voltage of the gate. By altering these parameters, the logic gate is capable of behaving as AND, OR, NAND, or NOR gates.

Camel Gate Field Effect Transistors.

DTIC Science & Technology

1983-01-01

CAMFETs can be designed to yield relatively voltage independent transconductances, large for- * ward turn-on voltages, and large gate-drain breakdown...doping. The FATFET area is 4.6 x 10- 4 cm2. I.- . - . . - , - 36 80 * Camel Gate U_-- Eperimental 60 * -Theoretical % Schottky Gate ~--Experimental CL 4...in the design of other devices. Finally, a comparative study of the reliabil- ities of CAMFETs, JFETs, and MESFETs should be attempted. 43 VII

A novel NaV1.5 voltage sensor mutation associated with severe atrial and ventricular arrhythmias.

PubMed

Wang, Hong-Gang; Zhu, Wandi; Kanter, Ronald J; Silva, Jonathan R; Honeywell, Christina; Gow, Robert M; Pitt, Geoffrey S

2016-03-01

Inherited autosomal dominant mutations in cardiac sodium channels (NaV1.5) cause various arrhythmias, such as long QT syndrome and Brugada syndrome. Although dozens of mutations throughout the protein have been reported, there are few reported mutations within a voltage sensor S4 transmembrane segment and few that are homozygous. Here we report analysis of a novel lidocaine-sensitive recessive mutation, p.R1309H, in the NaV1.5 DIII/S4 voltage sensor in a patient with a complex arrhythmia syndrome. We expressed the wild type or mutant NaV1.5 heterologously for analysis with the patch-clamp and voltage clamp fluorometry (VCF) techniques. p.R1309H depolarized the voltage-dependence of activation, hyperpolarized the voltage-dependence of inactivation, and slowed recovery from inactivation, thereby reducing the channel availability at physiologic membrane potentials. Additionally, p.R1309H increased the "late" Na(+) current. The location of the mutation in DIIIS4 prompted testing for a gating pore current. We observed an inward current at hyperpolarizing voltages that likely exacerbates the loss-of-function defects at resting membrane potentials. Lidocaine reduced the gating pore current. The p.R1309H homozygous NaV1.5 mutation conferred both gain-of-function and loss-of-function effects on NaV1.5 channel activity. Reduction of a mutation-induced gating pore current by lidocaine suggested a therapeutic mechanism. Copyright © 2016 Elsevier Ltd. All rights reserved.

Current-voltage characteristics and increase in the quantum efficiency of three-terminal gate and avalanche-based silicon LEDs.

PubMed

Xu, Kaikai

2013-09-20

In this paper, the emission of visible light by a monolithically integrated silicon p-n junction under reverse-bias is discussed. The modulation of light intensity is achieved using an insulated-gate terminal on the surface of the p-n junction. By varying the gate voltage, the breakdown voltage of the p-n junction will be adjustable so that the reverse current I(sub) flowing through the p-n junction at a fixed reverse-bias voltage is changed. It is observed that the light, which is emitted from the defects located at the p-n junction, depends closely on the reverse current I(sub). In regard to the phenomenon of electroluminescence, the relationship between the optical emission power and the reverse current I(sub) is linear. On the other hand, it is observed that both the quantum efficiency and the power conversion efficiency are able to have obvious enhancement, although the reverse-bias of the p-n junction is reduced and the corresponding reverse-current is much lower. Moreover, the successful fabrication on monolithic silicon light source on the bulk silicon by means of standard silicon complementary metal-oxide-semiconductor process technology is presented.

Wafer-scale solution-derived molecular gate dielectrics for low-voltage graphene electronics

NASA Astrophysics Data System (ADS)

Sangwan, Vinod K.; Jariwala, Deep; Everaerts, Ken; McMorrow, Julian J.; He, Jianting; Grayson, Matthew; Lauhon, Lincoln J.; Marks, Tobin J.; Hersam, Mark C.

2014-02-01

Graphene field-effect transistors are integrated with solution-processed multilayer hybrid organic-inorganic self-assembled nanodielectrics (SANDs). The resulting devices exhibit low-operating voltage (2 V), negligible hysteresis, current saturation with intrinsic gain >1.0 in vacuum (pressure < 2 × 10-5 Torr), and overall improved performance compared to control devices on conventional SiO2 gate dielectrics. Statistical analysis of the field-effect mobility and residual carrier concentration demonstrate high spatial uniformity of the dielectric interfacial properties and graphene transistor characteristics over full 3 in. wafers. This work thus establishes SANDs as an effective platform for large-area, high-performance graphene electronics.

Sleep disturbances in voltage-gated potassium channel antibody syndrome.

PubMed

Barone, Daniel A; Krieger, Ana C

2016-05-01

Voltage-gated potassium channels (VGKCs) are a family of membrane proteins responsible for controlling cell membrane potential. The presence of antibodies (Ab) against neuronal VGKC complexes aids in the diagnosis of idiopathic and paraneoplastic autoimmune neurologic disorders. The diagnosis of VGKC Ab-associated encephalopathy (VCKC Ab syndrome) should be suspected in patients with subacute onset of disorientation, confusion, and memory loss in the presence of seizures or a movement disorder. VGKC Ab syndrome may present with sleep-related symptoms, and the purpose of this communication is to alert sleep and neurology clinicians of this still-under-recognized condition. In this case, we are presenting the VGKC Ab syndrome which improved after treatment with solumedrol. The prompt recognition and treatment of this condition may prevent the morbidity associated with cerebral atrophy and the mortality associated with intractable seizures and electrolyte disturbances. Copyright © 2016. Published by Elsevier B.V.

Influence of gate width on gate-channel carrier mobility in AlGaN/GaN heterostructure field-effect transistors

NASA Astrophysics Data System (ADS)

Yang, Ming; Ji, Qizheng; Gao, Zhiliang; Zhang, Shufeng; Lin, Zhaojun; Yuan, Yafei; Song, Bo; Mei, Gaofeng; Lu, Ziwei; He, Jihao

2017-11-01

For the fabricated AlGaN/GaN heterostructure field-effect transistors (HFETs) with different gate widths, the gate-channel carrier mobility is experimentally obtained from the measured current-voltage and capacitance-voltage curves. Under each gate voltage, the mobility gets lower with gate width increasing. Analysis shows that the phenomenon results from the polarization Coulomb field (PCF) scattering, which originates from the irregularly distributed polarization charges at the AlGaN/GaN interface. The device with a larger gate width is with a larger PCF scattering potential and a stronger PCF scattering intensity. As a function of gate width, PCF scattering potential shows a same trend with the mobility variation. And the theoretically calculated mobility values fits well with the experimentally obtained values. Varying gate widths will be a new perspective for the improvement of device characteristics by modulating the gate-channel carrier mobility.

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.

Anomalous positive flatband voltage shifts in metal gate stacks containing rare-earth oxide capping layers

NASA Astrophysics Data System (ADS)

Caraveo-Frescas, J. A.; Hedhili, M. N.; Wang, H.; Schwingenschlögl, U.; Alshareef, H. N.

2012-03-01

It is shown that the well-known negative flatband voltage (VFB) shift, induced by rare-earth oxide capping in metal gate stacks, can be completely reversed in the absence of the silicon overlayer. Using TaN metal gates and Gd2O3-doped dielectric, we measure a ˜350 mV negative shift with the Si overlayer present and a ˜110 mV positive shift with the Si overlayer removed. This effect is correlated to a positive change in the average electrostatic potential at the TaN/dielectric interface which originates from an interfacial dipole. The dipole is created by the replacement of interfacial oxygen atoms in the HfO2 lattice with nitrogen atoms from TaN.

Effects of trap-assisted tunneling on gate-induced drain leakage in silicon-germanium channel p-type FET for scaled supply voltages

NASA Astrophysics Data System (ADS)

Tiwari, Vishal A.; Divakaruni, Rama; Hook, Terence B.; Nair, Deleep R.

2016-04-01

Silicon-germanium is considered as an alternative channel material to silicon p-type FET (pFET) for the development of energy efficient high performance transistors for 28 nm and beyond in a high-k metal gate technology because of its lower threshold voltage and higher mobility. However, gate-induced drain leakage (GIDL) is a concern for high threshold voltage device design because of tunneling at reduced bandgap. In this work, the trap-assisted tunneling and band-to-band tunneling (BTBT) effects on GIDL is analyzed and modeled for SiGe pFETs. Experimental results and Monte Carlo simulation results reveal that the pre-halo germanium pre-amorphization implant used to contain the short channel effects contribute to GIDL at the drain sidewall in addition to GIDL due to BTBT in SiGe devices. The results are validated by comparing the experimental observations with the numerical simulation and a set of calibrated models are used to describe the GIDL mechanisms for various drain and gate bias.

Identification of an evolutionarily conserved extracellular threonine residue critical for surface expression and its potential coupling of adjacent voltage-sensing and gating domains in voltage-gated potassium channels.

PubMed

Mckeown, Lynn; Burnham, Matthew P; Hodson, Charlotte; Jones, Owen T

2008-10-31

The dynamic expression of voltage-gated potassium channels (Kvs) at the cell surface is a fundamental factor controlling membrane excitability. In exploring possible mechanisms controlling Kv surface expression, we identified a region in the extracellular linker between the first and second of the six (S1-S6) transmembrane-spanning domains of the Kv1.4 channel, which we hypothesized to be critical for its biogenesis. Using immunofluorescence microscopy, flow cytometry, patch clamp electrophysiology, and mutagenesis, we identified a single threonine residue at position 330 within the Kv1.4 S1-S2 linker that is absolutely required for cell surface expression. Mutation of Thr-330 to an alanine, aspartate, or lysine prevented surface expression. However, surface expression occurred upon co-expression of mutant and wild type Kv1.4 subunits or mutation of Thr-330 to a serine. Mutation of the corresponding residue (Thr-211) in Kv3.1 to alanine also caused intracellular retention, suggesting that the conserved threonine plays a generalized role in surface expression. In support of this idea, sequence comparisons showed conservation of the critical threonine in all Kv families and in organisms across the evolutionary spectrum. Based upon the Kv1.2 crystal structure, further mutagenesis, and the partial restoration of surface expression in an electrostatic T330K bridging mutant, we suggest that Thr-330 hydrogen bonds to equally conserved outer pore residues, which may include a glutamate at position 502 that is also critical for surface expression. We propose that Thr-330 serves to interlock the voltage-sensing and gating domains of adjacent monomers, thereby yielding a structure competent for the surface expression of functional tetramers.

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.

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

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

Voltage-Gated Ion Channels in Cancer Cell Proliferation

PubMed Central

Rao, Vidhya R.; Perez-Neut, Mathew; Kaja, Simon; Gentile, Saverio

2015-01-01

Changes of the electrical charges across the surface cell membrane are absolutely necessary to maintain cellular homeostasis in physiological as well as in pathological conditions. The opening of ion channels alter the charge distribution across the surface membrane as they allow the diffusion of ions such as K+, Ca++, Cl−, Na+. Traditionally, voltage-gated ion channels (VGIC) are known to play fundamental roles in controlling rapid bioelectrical signaling including action potential and/or contraction. However, several investigations have revealed that these classes of proteins can also contribute significantly to cell mitotic biochemical signaling, cell cycle progression, as well as cell volume regulation. All these functions are critically important for cancer cell proliferation. Interestingly, a variety of distinct VGICs are expressed in different cancer cell types, including metastasis but not in the tissues from which these tumors were generated. Given the increasing evidence suggesting that VGIC play a major role in cancer cell biology, in this review we discuss the role of distinct VGIC in cancer cell proliferation and possible therapeutic potential of VIGC pharmacological manipulation. PMID:26010603

Probing the Energy Landscape of Activation Gating of the Bacterial Potassium Channel KcsA

PubMed Central

Linder, Tobias; de Groot, Bert L.; Stary-Weinzinger, Anna

2013-01-01

The bacterial potassium channel KcsA, which has been crystallized in several conformations, offers an ideal model to investigate activation gating of ion channels. In this study, essential dynamics simulations are applied to obtain insights into the transition pathways and the energy profile of KcsA pore gating. In agreement with previous hypotheses, our simulations reveal a two phasic activation gating process. In the first phase, local structural rearrangements in TM2 are observed leading to an intermediate channel conformation, followed by large structural rearrangements leading to full opening of KcsA. Conformational changes of a highly conserved phenylalanine, F114, at the bundle crossing region are crucial for the transition from a closed to an intermediate state. 3.9 µs umbrella sampling calculations reveal that there are two well-defined energy barriers dividing closed, intermediate, and open channel states. In agreement with mutational studies, the closed state was found to be energetically more favorable compared to the open state. Further, the simulations provide new insights into the dynamical coupling effects of F103 between the activation gate and the selectivity filter. Investigations on individual subunits support cooperativity of subunits during activation gating. PMID:23658510

Calmodulin regulates Cav3 T-type channels at their gating brake

PubMed Central

Taiakina, Valentina; Monteil, Arnaud; Piazza, Michael; Guan, Wendy; Stephens, Robert F.; Dieckmann, Thorsten; Guillemette, Joseph Guy; Spafford, J. David

2017-01-01

Calcium (Cav1 and Cav2) and sodium channels possess homologous CaM-binding motifs, known as IQ motifs in their C termini, which associate with calmodulin (CaM), a universal calcium sensor. Cav3 T-type channels, which serve as pacemakers of the mammalian brain and heart, lack a C-terminal IQ motif. We illustrate that T-type channels associate with CaM using co-immunoprecipitation experiments and single particle cryo-electron microscopy. We demonstrate that protostome invertebrate (LCav3) and human Cav3.1, Cav3.2, and Cav3.3 T-type channels specifically associate with CaM at helix 2 of the gating brake in the I–II linker of the channels. Isothermal titration calorimetry results revealed that the gating brake and CaM bind each other with high-nanomolar affinity. We show that the gating brake assumes a helical conformation upon binding CaM, with associated conformational changes to both CaM lobes as indicated by amide chemical shifts of the amino acids of CaM in 1H-15N HSQC NMR spectra. Intact Ca2+-binding sites on CaM and an intact gating brake sequence (first 39 amino acids of the I–II linker) were required in Cav3.2 channels to prevent the runaway gating phenotype, a hyperpolarizing shift in voltage sensitivities and faster gating kinetics. We conclude that the presence of high-nanomolar affinity binding sites for CaM at its universal gating brake and its unique form of regulation via the tuning of the voltage range of activity could influence the participation of Cav3 T-type channels in heart and brain rhythms. Our findings may have implications for arrhythmia disorders arising from mutations in the gating brake or CaM. PMID:28972185

Effects of osmotic swelling on voltage-gated calcium channel currents in rat anterior pituitary cells.

PubMed

Ben-Tabou De-Leon, Shlomo; Blotnick, Edna; Nussinovitch, Itzhak

2003-10-01

Decrease in extracellular osmolarity ([Os]e) results in stimulation of hormone secretion from pituitary cells. Different mechanisms can account for this stimulation of hormone secretion. In this study we examined the possibility that hyposmolarity directly modulates voltage-gated calcium influx in pituitary cells. The effects of hyposmolarity on L-type (IL) and T-type (IT) calcium currents in pituitary cells were investigated by using two hyposmotic stimuli, moderate (18-22% decrease in [Os]e) and strong (31-32% decrease in [Os]e). Exposure to moderate hyposmotic stimuli resulted in three response types in IL (a decrease, a biphasic effect, and an increase in IL) and in increase in IT. Exposure to strong hyposmotic stimuli resulted only in increases in both IL and IT. Similarly, in intact pituitary cells (perforated patch method), exposure to either moderate or strong hyposmotic stimuli resulted only in increases in both IL and IT. Thus it appears that the main effect of decrease in [Os]e is increase in calcium channel currents. This increase was differential (IL were more sensitive than IT) and voltage independent. In addition, we show that these hyposmotic effects cannot be explained by activation of an anionic conductance or by an increase in cell membrane surface area. In conclusion, this study shows that hyposmotic swelling of pituitary cells can directly modulate voltage-gated calcium influx. This hyposmotic modulation of IL and IT may contribute to the previously reported hyposmotic stimulation of hormone secretion. The mechanisms underlying these hyposmotic effects and their possible physiological relevance are discussed.

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

Ph-Dependent Inhibition of Voltage-Gated H+ Currents in Rat Alveolar Epithelial Cells by Zn2+ and Other Divalent Cations

PubMed Central

Cherny, Vladimir V.; DeCoursey, Thomas E.

1999-01-01

Inhibition by polyvalent cations is a defining characteristic of voltage-gated proton channels. The mechanism of this inhibition was studied in rat alveolar epithelial cells using tight-seal voltage clamp techniques. Metal concentrations were corrected for measured binding to buffers. Externally applied ZnCl2 reduced the H+ current, shifted the voltage-activation curve toward positive potentials, and slowed the turn-on of H+ current upon depolarization more than could be accounted for by a simple voltage shift, with minimal effects on the closing rate. The effects of Zn2+ were inconsistent with classical voltage-dependent block in which Zn2+ binds within the membrane voltage field. Instead, Zn2+ binds to superficial sites on the channel and modulates gating. The effects of extracellular Zn2+ were strongly pHo dependent but were insensitive to pHi, suggesting that protons and Zn2+ compete for external sites on H+ channels. The apparent potency of Zn2+ in slowing activation was ∼10× greater at pHo 7 than at pHo 6, and ∼100× greater at pHo 6 than at pHo 5. The pHo dependence suggests that Zn2+, not ZnOH+, is the active species. Evidently, the Zn2+ receptor is formed by multiple groups, protonation of any of which inhibits Zn2+ binding. The external receptor bound H+ and Zn2+ with pK a 6.2–6.6 and pK M 6.5, as described by several models. Zn2+ effects on the proton chord conductance–voltage (g H–V) relationship indicated higher affinities, pK a 7 and pK M 8. CdCl2 had similar effects as ZnCl2 and competed with H+, but had lower affinity. Zn2+ applied internally via the pipette solution or to inside-out patches had comparatively small effects, but at high concentrations reduced H+ currents and slowed channel closing. Thus, external and internal zinc-binding sites are different. The external Zn2+ receptor may be the same modulatory protonation site(s) at which pHo regulates H+ channel gating. PMID:10578017