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Sample records for sodium channels expressed

  1. Stable expression and characterization of human PN1 and PN3 sodium channels.

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    Akiba, Isamu; Seki, Tetsuo; Mori, Masayuki; Iizuka, Masaki; Nishimura, Seiichiro; Sasaki, Sachie; Imoto, Keiji; Barsoumian, Edward L

    2003-01-01

    Nociceptive transduction in inflammatory and neuropathic pain involves peripherally expressed voltage-gated sodium channels, such as tetrodotoxin (TTX)-sensitive PN1 and TTX-resistant PN3. We generated recombinant cell lines stably expressing the human PN1 and PN3 sodium channels in Chinese hamster ovary (CHO) cells using inducible expression vectors. The PN1 and PN3 cDNAs were isolated from human adrenal gland and heart poly(A)+ RNAs, respectively. The recombinant human PN1 currents exhibited rapid activation and inactivation kinetics and were blocked by TTX with a half-maximal inhibitory concentration (IC50) of 32.6 nM. The human PN3 channel expressed in stable transfectants showed TTX-resistant inward currents with slow inactivation kinetics. The IC50 value for TTX was 73.3 microM. The voltage-dependence of activation of the PN3 channel was shifted to the depolarizing direction, compared to that of the PN1 channel. Lidocaine and mexiletine exhibited tonic and use-dependent block of PN1 and PN3 channels. The PN1 channel was more susceptible to inhibition by mexiletine than PN3. These results suggest that stable transfectants expressing the human PN1 and PN3 sodium channels will be useful tools to define subtype selectivity for sodium channel blockers.

  2. Regulation of T-type calcium channel expression by sodium butyrate in prostate cancer cells.

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    Weaver, Erika M; Zamora, Francis J; Puplampu-Dove, Yvonne A; Kiessu, Ezechielle; Hearne, Jennifer L; Martin-Caraballo, Miguel

    2015-02-15

    Several cellular mechanisms contribute to the neuroendocrine differentiation of prostate cancer cells, including exposure to sodium butyrate (NaBu), a naturally occurring salt of the short chain fatty acid n-butyric acid. NaBu belongs to a class of histone deacetylase inhibitors with potential anticancer function. T-type calcium channel expression constitutes an important route for calcium influx in tumor cells that may trigger changes in cell proliferation and differentiation. In this work we investigated the role NaBu on the differentiation of lymph node carcinoma of the prostate (LNCaP) cells and its effect on T-type Ca(2+) channel expression. NaBu stimulates the morphological and molecular differentiation of LNCaP cells. Stimulation of LNCaP cells with NaBu evokes a significant increase in the expression of the Cav3.2 T-type channel subunits. Furthermore, the increased Cav3.2 expression promotes membrane insertion of T-type Ca(2+) channels capable of generating fast inactivating Ca(2+) currents, sensitive to 100μM Ni(2+) ions. Inhibition of T-type Ca(2+) channel function reduces the outgrowth of neurite-like processes in LNCaP cells. NaBu-evoked expression of T-type Ca(2+) channels is also involved in the regulation of cell viability. Inhibition of T-type Ca(2+) channels causes a significant reduction in the viability of LNCaP cells treated with 1mM NaBu, suggesting that Ca(2+) influx via T-type channels can promote cell proliferation. However, increased expression of T-type Ca(2+) channels enhanced the cytotoxic effect of thapsigargin and paclitaxel on cell proliferation. These findings demonstrate that NaBu stimulates T-type Ca(2+) channel expression, thereby regulating both the morphological differentiation and growth of prostate cancer cells. Copyright © 2015 Elsevier B.V. All rights reserved.

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

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

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

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    Antonio Cejudo-Roman

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

  5. Polymorphisms in the cardiac sodium channel promoter displaying variant in vitro expression activity

    NARCIS (Netherlands)

    Yang, P.; Koopmann, T. T.; Pfeufer, A.; Jalilzadeh, S.; Schulze-Bahr, E.; Kääb, S.; Wilde, A. A.; Roden, D. M.; Bezzina, C. R.

    2008-01-01

    Variable transcription of the cardiac sodium channel gene is a candidate mechanism determining arrhythmia susceptibility. We have previously cloned and characterized the core promoter and flanking region of SCN5A, encoding the cardiac sodium channel. Loss-of-function mutations in this gene have been

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

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    Radzioch Danuta

    2005-01-01

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

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

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    Zhixin Lin

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

  8. Gene expression of the concentration-sensitive sodium channel is suppressed in lipopolysaccharide-induced acute lung injury in mice.

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    Hagiwara, Teruki; Yoshida, Shigeru; Hidaka, Yuji

    2017-04-01

    The concentration-sensitive sodium channel (Na C ) is expressed in alveolar type II epithelial cells and pulmonary microvascular endothelial cells in mouse lungs. We recently reported that Na C contributes to amiloride-insensitive sodium transport in mouse lungs (Respiratory Physiology & Neurobiology, 2016). However, details regarding its physiological role in the lung remain unknown. To examine whether Na C is involved in alveolar fluid clearance during an acute lung injury (ALI), we analyzed the relationship between Na C gene expression in the lung and the development of pulmonary edema in lipopolysaccharide (LPS)-induced ALI mice. LPS-induced ALI mice were prepared by the intratracheal administration of LPS. Bronchoalveolar lavage (BAL) neutrophils and lung water content (LWCs) were used as a marker of ALI and pulmonary edema, respectively. Na C protein production in the lung was detected by immunoblotting and immunofluorescence. The gene expressions of Na C and the epithelial sodium channel (ENaC) of LPS-induced ALI mice were examined by quantitative RT-PCR over a time course of 14 days. The BAL neutrophil count increased until day 2 after LPS administration and had nearly recovered by day 6. LWCs in LPS-induced mice gradually increased until day 8 and had recovered by day 14. The expression of the Na C protein in the lungs of LPS-induced mice dramatically decreased from day 2 to day 6, but recovered by day 8. The mRNA expression of Na C decreased in the lung, as well as those for α-, β-, and γ-ENaC during ALI. Thus, Na C expression is suppressed during the development stage of pulmonary edema and then recovers in the convalescent phase. Our results suggest that suppression of the gene expression of Na C is involved in the development of pulmonary edema in ALI.

  9. Targeting sodium channels in cardiac arrhythmia

    NARCIS (Netherlands)

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

    2014-01-01

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

  10. Intron retention in mRNA encoding ancillary subunit of insect voltage-gated sodium channel modulates channel expression, gating regulation and drug sensitivity.

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    Céline M Bourdin

    Full Text Available Insect voltage-gated sodium (Nav channels are formed by a well-known pore-forming α-subunit encoded by para-like gene and ancillary subunits related to TipE from the mutation "temperature-induced-paralysis locus E." The role of these ancillary subunits in the modulation of biophysical and pharmacological properties of Na(+ currents are not enough documented. The unique neuronal ancillary subunit TipE-homologous protein 1 of Drosophila melanogaster (DmTEH1 strongly enhances the expression of insect Nav channels when heterologously expressed in Xenopus oocytes. Here we report the cloning and functional expression of two neuronal DmTEH1-homologs of the cockroach, Periplaneta americana, PaTEH1A and PaTEH1B, encoded by a single bicistronic gene. In PaTEH1B, the second exon encoding the last 11-amino-acid residues of PaTEH1A is shifted to 3'UTR by the retention of a 96-bp intron-containing coding-message, thus generating a new C-terminal end. We investigated the gating and pharmacological properties of the Drosophila Nav channel variant (DmNav1-1 co-expressed with DmTEH1, PaTEH1A, PaTEH1B or a truncated mutant PaTEH1Δ(270-280 in Xenopus oocytes. PaTEH1B caused a 2.2-fold current density decrease, concomitant with an equivalent α-subunit incorporation decrease in the plasma membrane, compared to PaTEH1A and PaTEH1Δ(270-280. PaTEH1B positively shifted the voltage-dependences of activation and slow inactivation of DmNav1-1 channels to more positive potentials compared to PaTEH1A, suggesting that the C-terminal end of both proteins may influence the function of the voltage-sensor and the pore of Nav channel. Interestingly, our findings showed that the sensitivity of DmNav1-1 channels to lidocaine and to the pyrazoline-type insecticide metabolite DCJW depends on associated TEH1-like subunits. In conclusion, our work demonstrates for the first time that density, gating and pharmacological properties of Nav channels expressed in Xenopus oocytes can be

  11. Insect sodium channels and insecticide resistance

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    Dong, Ke

    2007-01-01

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

  12. Excitability constraints on voltage-gated sodium channels.

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    Elaine Angelino

    2007-09-01

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

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

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

    2017-01-01

    Nav1. 9 voltage-gated sodium channel is preferentially expressed in peripheral nociceptive neurons. Recent progresses have proved its role in pain sensation, but our understanding of Nav1.9, in general, has lagged behind because of limitations in heterologous expression in mammal cells. In this work, functional expression of human Nav1.9 (hNav1.9) was achieved by fusing GFP to the C-terminal of hNav1.9 in ND7/23 cells, which has been proved to be a reliable method to the electrophysiological and pharmacological studies of hNav1.9. By using the hNav1.9 expression system, we investigated the electrophysiological properties of four mutations of hNav1.9 (K419N, A582T, A842P, and F1689L), whose electrophysiological functions have not been determined yet. The four mutations significantly caused positive shift of the steady-state fast inactivation and therefore increased hNav1.9 activity, consistent with the phenotype of painful peripheral neuropathy. Meanwhile, the effects of inflammatory mediators on hNav1.9 were also investigated. Impressively, histamine was found for the first time to enhance hNav1.9 activity, indicating its vital role in hNav1.9 modulating inflammatory pain. Taken together, our research provided a useful platform for hNav1.9 studies and new insight into mechanism of hNav1.9 linking to pain. PMID:29213238

  14. Gene expression profile of sodium channel subunits in the anterior cingulate cortex during experimental paclitaxel-induced neuropathic pain in mice

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    Willias Masocha

    2016-11-01

    Full Text Available Paclitaxel, a chemotherapeutic agent, causes neuropathic pain whose supraspinal pathophysiology is not fully understood. Dysregulation of sodium channel expression, studied mainly in the periphery and spinal cord level, contributes to the pathogenesis of neuropathic pain. We examined gene expression of sodium channel (Nav subunits by real time polymerase chain reaction (PCR in the anterior cingulate cortex (ACC at day 7 post first administration of paclitaxel, when mice had developed paclitaxel-induced thermal hyperalgesia. The ACC was chosen because increased activity in the ACC has been observed during neuropathic pain. In the ACC of vehicle-treated animals the threshold cycle (Ct values for Nav1.4, Nav1.5, Nav1.7, Nav1.8 and Nav1.9 were above 30 and/or not detectable in some samples. Thus, comparison in mRNA expression between untreated control, vehicle-treated and paclitaxel treated animals was done for Nav1.1, Nav1.2, Nav1.3, Nav1.6, Nax as well as Navβ1–Navβ4. There were no differences in the transcript levels of Nav1.1–Nav1.3, Nav1.6, Nax, Navβ1–Navβ3 between untreated and vehicle-treated mice, however, vehicle treatment increased Navβ4 expression. Paclitaxel treatment significantly increased the mRNA expression of Nav1.1, Nav1.2, Nav1.6 and Nax, but not Nav1.3, sodium channel alpha subunits compared to vehicle-treated animals. Treatment with paclitaxel significantly increased the expression of Navβ1 and Navβ3, but not Navβ2 and Navβ4, sodium channel beta subunits compared to vehicle-treated animals. These findings suggest that during paclitaxel-induced neuropathic pain (PINP there is differential upregulation of sodium channels in the ACC, which might contribute to the increased neuronal activity observed in the area during neuropathic pain.

  15. Synthetic ciguatoxins selectively activate Nav1.8-derived chimeric sodium channels expressed in HEK293 cells.

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    Yamaoka, Kaoru; Inoue, Masayuki; Miyazaki, Keisuke; Hirama, Masahiro; Kondo, Chie; Kinoshita, Eiji; Miyoshi, Hiroshi; Seyama, Issei

    2009-03-20

    The synthetic ciguatoxin CTX3C has been shown to activate tetrodotoxin (TTX)-sensitive sodium channels (Na(v)1.2, Na(v)1.4, and Na(v)1.5) by accelerating activation kinetics and shifting the activation curve toward hyperpolarization (Yamaoka, K., Inoue, M., Miyahara, H., Miyazaki, K., and Hirama, M. (2004) Br. J. Pharmacol. 142, 879-889). In this study, we further explored the effects of CTX3C on the TTX-resistant sodium channel Na(v)1.8. TTX-resistant channels have been shown to be involved in transducing pain and related sensations (Akopian, A. N., Sivilotti, L., and Wood, J. N. (1996) Nature 379, 257-262). Thus, we hypothesized that ciguatoxin-induced activation of the Na(v)1.8 current would account for the neurological symptoms of ciguatera poisoning. We found that 0.1 mum CTX3C preferentially affected the activation process of the Na(v)1.8 channel compared with those of the Na(v)1.2 and Na(v)1.4 channels. Importantly, without stimulation, 0.1 mum CTX3C induced a large leakage current (I (L)). The conductance of the I (L) calculated relative to the maximum conductance (G (max)) was 10 times larger than that of Na(v)1.2 or Na(v)1.4. To determine the molecular domain of Na(v)1.8 responsible for conferring higher sensitivity to CTX3C, we made two chimeric constructs from Na(v)1.4 and Na(v)1.8. Chimeras containing the N-terminal half of Na(v)1.8 exhibited a large response similar to wild-type Na(v)1.8, indicating that the region conferring high sensitivity to ciguatoxin action is located in the D1 or D2 domains.

  16. Synthetic Ciguatoxins Selectively Activate Nav1.8-derived Chimeric Sodium Channels Expressed in HEK293 Cells*

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    Yamaoka, Kaoru; Inoue, Masayuki; Miyazaki, Keisuke; Hirama, Masahiro; Kondo, Chie; Kinoshita, Eiji; Miyoshi, Hiroshi; Seyama, Issei

    2009-01-01

    The synthetic ciguatoxin CTX3C has been shown to activate tetrodotoxin (TTX)-sensitive sodium channels (Nav1.2, Nav1.4, and Nav1.5) by accelerating activation kinetics and shifting the activation curve toward hyperpolarization (Yamaoka, K., Inoue, M., Miyahara, H., Miyazaki, K., and Hirama, M. (2004) Br. J. Pharmacol. 142, 879–889). In this study, we further explored the effects of CTX3C on the TTX-resistant sodium channel Nav1.8. TTX-resistant channels have been shown to be involved in transducing pain and related sensations (Akopian, A. N., Sivilotti, L., and Wood, J. N. (1996) Nature 379, 257–262). Thus, we hypothesized that ciguatoxin-induced activation of the Nav1.8 current would account for the neurological symptoms of ciguatera poisoning. We found that 0.1 μm CTX3C preferentially affected the activation process of the Nav1.8 channel compared with those of the Nav1.2 and Nav1.4 channels. Importantly, without stimulation, 0.1 μm CTX3C induced a large leakage current (IL). The conductance of the IL calculated relative to the maximum conductance (Gmax) was 10 times larger than that of Nav1.2 or Nav1.4. To determine the molecular domain of Nav1.8 responsible for conferring higher sensitivity to CTX3C, we made two chimeric constructs from Nav1.4 and Nav1.8. Chimeras containing the N-terminal half of Nav1.8 exhibited a large response similar to wild-type Nav1.8, indicating that the region conferring high sensitivity to ciguatoxin action is located in the D1 or D2 domains. PMID:19164297

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

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    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.

  18. Voltage-gated sodium channels: mutations, channelopathies and targets.

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    Andavan, G S B; Lemmens-Gruber, R

    2011-01-01

    Voltage-gated sodium channels produce fast depolarization, which is responsible for the rising phase of the action potential in neurons, muscles and heart. These channels are very large membrane proteins and are encoded by ten genes in mammals. Sodium channels are a crucial component of excitable tissues; hence, they are a target for various neurotoxins that are produced by plants and animals for defence and protection, such as tetrodotoxin, scorpion toxins and batrachotoxin. Several mutations in various sodium channel subtypes cause multiple inherited diseases known as channelopathies. When these mutated sodium channel subtypes are expressed in various tissues, channelopathies in brain, skeletal muscle and cardiac muscle develop as well as neuropathic pain. In this review, we discuss aspects of voltage-gated sodium channel genes with an emphasis on cardiac muscle sodium channels. In addition, we report novel mutations that underlie a spectrum of diseases, such as Brugada, long QT syndrome and inherited conduction disorders. Furthermore, this review explains commonalities and differences among the channel subtypes, the channelopathies caused by the sodium channel gene mutation and the specificity of toxins and blockers of the channel subtypes.

  19. Sodium Channel Mutations and Pyrethroid Resistance in Aedes aegypti

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    Yuzhe Du

    2016-10-01

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

  20. Sensory renal innervation: a kidney-specific firing activity due to a unique expression pattern of voltage-gated sodium channels?

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    Freisinger, Wolfgang; Schatz, Johannes; Ditting, Tilmann; Lampert, Angelika; Heinlein, Sonja; Lale, Nena; Schmieder, Roland; Veelken, Roland

    2013-03-01

    Sensory neurons with afferent axons from the kidney are extraordinary in their response to electrical stimulation. More than 50% exhibit a tonic firing pattern, i.e., sustained action potential firing throughout depolarizing, pointing to an increased excitability, whereas nonrenal neurons show mainly a phasic response, i.e., less than five action potentials. Here we investigated whether these peculiar firing characteristics of renal afferent neurons are due to differences in the expression of voltage-gated sodium channels (Navs). Dorsal root ganglion (DRG) neurons from rats (Th11-L2) were recorded by the current-clamp technique and distinguished as "tonic" or "phasic." In voltage-clamp recordings, Navs were characterized by their tetrodotoxoxin (TTX) sensitivity, and their molecular identity was revealed by RT-PCR. The firing pattern of 66 DRG neurons (41 renal and 25 nonrenal) was investigated. Renal neurons exhibited more often a tonic firing pattern (56.1 vs. 12%). Tonic neurons showed a more positive threshold (-21.75 ± 1.43 vs.-29.33 ± 1.63 mV; P sodium currents. Interestingly, mRNA expression of TTX-resistant sodium channels was significantly increased in renal, predominantly tonic, DRG neurons. Hence, under physiological conditions, renal sensory neurons exhibit predominantly a firing pattern associated with higher excitability. Our findings support that this is due to an increased expression and activation of TTX-resistant Navs.

  1. NALCN ion channels have alternative selectivity filters resembling calcium channels or sodium channels.

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    Adriano Senatore

    Full Text Available NALCN is a member of the family of ion channels with four homologous, repeat domains that include voltage-gated calcium and sodium channels. NALCN is a highly conserved gene from simple, extant multicellular organisms without nervous systems such as sponges and placozoans and mostly remains a single gene compared to the calcium and sodium channels which diversified into twenty genes in humans. The single NALCN gene has alternatively-spliced exons at exons 15 or exon 31 that splices in novel selectivity filter residues that resemble calcium channels (EEEE or sodium channels (EKEE or EEKE. NALCN channels with alternative calcium, (EEEE and sodium, (EKEE or EEKE -selective pores are conserved in simple bilaterally symmetrical animals like flatworms to non-chordate deuterostomes. The single NALCN gene is limited as a sodium channel with a lysine (K-containing pore in vertebrates, but originally NALCN was a calcium-like channel, and evolved to operate as both a calcium channel and sodium channel for different roles in many invertebrates. Expression patterns of NALCN-EKEE in pond snail, Lymnaea stagnalis suggest roles for NALCN in secretion, with an abundant expression in brain, and an up-regulation in secretory organs of sexually-mature adults such as albumen gland and prostate. NALCN-EEEE is equally abundant as NALCN-EKEE in snails, but is greater expressed in heart and other muscle tissue, and 50% less expressed in the brain than NALCN-EKEE. Transfected snail NALCN-EEEE and NALCN-EKEE channel isoforms express in HEK-293T cells. We were not able to distinguish potential NALCN currents from background, non-selective leak conductances in HEK293T cells. Native leak currents without expressing NALCN genes in HEK-293T cells are NMDG(+ impermeant and blockable with 10 µM Gd(3+ ions and are indistinguishable from the hallmark currents ascribed to mammalian NALCN currents expressed in vitro by Lu et al. in Cell. 2007 Apr 20;129(2:371-83.

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

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    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

  3. Voltage-gated sodium channels in taste bud cells.

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

    2009-03-12

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

  4. Antillatoxin is a sodium channel activator that displays unique efficacy in heterologously expressed rNav1.2, rNav1.4 and rNav1.5 alpha subunits

    Directory of Open Access Journals (Sweden)

    Gerwick William H

    2010-12-01

    Full Text Available Abstract Background Antillatoxin (ATX is a structurally unique lipopeptide produced by the marine cyanobacterium Lyngbya majuscula. ATX activates voltage-gated sodium channel α-subunits at an undefined recognition site and stimulates sodium influx in neurons. However, the pharmacological properties and selectivity of ATX on the sodium channel α-subunits were not fully characterized. Results In this study, we characterized the pharmacological properties and selectivity of ATX in cells heterologously expressing rNav1.2, rNav1.4 or rNav1.5 α-subunits by using the Na+ selective fluorescent dye, sodium-binding benzofuran isophthalate. ATX produced sodium influx in cells expressing each sodium channel α-subunit, whereas two other sodium channel activators, veratridine and brevetoxin-2, were without effect. The ATX potency at rNav1.2, rNav1.4 and rNav1.5 did not differ significantly. Similarly, there were no significant differences in the efficacy for ATX-induced sodium influx between rNav1.2, rNav1.4 and rNav1.5 α-subunits. ATX also produced robust Ca2+ influx relative to other sodium channel activators in the calcium-permeable DEAA mutant of rNav1.4 α-subunit. Finally, we demonstrated that the 8-demethyl-8,9-dihydro-antillatoxin analog was less efficacious and less potent in stimulating sodium influx. Conclusions ATX displayed a unique efficacy with respect to stimulation of sodium influx in cells expressing rNav1.2, rNav1.4 and rNav1.5 α-subunits. The efficacy of ATX was distinctive inasmuch as it was not shared by activators of neurotoxin sites 2 and 5 on VGSC α-subunits. Given the unique pharmacological properties of ATX interaction with sodium channel α-subunits, decoding the molecular determinants and mechanism of action of antillatoxin may provide further insight into sodium channel gating mechanisms.

  5. Antillatoxin is a sodium channel activator that displays unique efficacy in heterologously expressed rNav1.2, rNav1.4 and rNav1.5 α subunits.

    Science.gov (United States)

    Cao, Zhengyu; Gerwick, William H; Murray, Thomas F

    2010-12-14

    Antillatoxin (ATX) is a structurally unique lipopeptide produced by the marine cyanobacterium Lyngbya majuscula. ATX activates voltage-gated sodium channel α-subunits at an undefined recognition site and stimulates sodium influx in neurons. However, the pharmacological properties and selectivity of ATX on the sodium channel α-subunits were not fully characterized. In this study, we characterized the pharmacological properties and selectivity of ATX in cells heterologously expressing rNa(v)1.2, rNa(v)1.4 or rNa(v)1.5 α-subunits by using the Na(+) selective fluorescent dye, sodium-binding benzofuran isophthalate. ATX produced sodium influx in cells expressing each sodium channel α-subunit, whereas two other sodium channel activators, veratridine and brevetoxin-2, were without effect. The ATX potency at rNa(v)1.2, rNa(v)1.4 and rNa(v)1.5 did not differ significantly. Similarly, there were no significant differences in the efficacy for ATX-induced sodium influx between rNa(v)1.2, rNa(v)1.4 and rNa(v)1.5 α-subunits. ATX also produced robust Ca²(+) influx relative to other sodium channel activators in the calcium-permeable DEAA mutant of rNa(v)1.4 α-subunit. Finally, we demonstrated that the 8-demethyl-8,9-dihydro-antillatoxin analog was less efficacious and less potent in stimulating sodium influx. ATX displayed a unique efficacy with respect to stimulation of sodium influx in cells expressing rNa(v)1.2, rNa(v)1.4 and rNa(v)1.5 α-subunits. The efficacy of ATX was distinctive inasmuch as it was not shared by activators of neurotoxin sites 2 and 5 on VGSC α-subunits. Given the unique pharmacological properties of ATX interaction with sodium channel α-subunits, decoding the molecular determinants and mechanism of action of antillatoxin may provide further insight into sodium channel gating mechanisms.

  6. The sodium-activated sodium channel is expressed in the rat kidney thick ascending limb and collecting duct cells and is upregulated during high salt intake.

    Science.gov (United States)

    Lara, Lucienne S; Satou, Ryousuke; Bourgeois, Camille R T; Gonzalez, Alexis A; Zsombok, Andrea; Prieto, Minolfa C; Navar, L Gabriel

    2012-07-01

    Increased dietary salt triggers oxidative stress and kidney injury in salt-sensitive hypertension; however, the mechanism for sensing increased extracellular Na(+) concentration ([Na(+)]) remains unclear. A Na(+)-activated Na(+) channel (Na sensor) described in the brain operates as a sensor of extracellular fluid [Na(+)]; nonetheless, its presence in the kidney has not been established. In the present study, we demonstrated the gene expression of the Na sensor by RT-PCR and Western blotting in the Sprague-Dawley rat kidney. Using immunofluorescence, the Na sensor was localized to the luminal side in tubular epithelial cells of collecting ducts colocalizing with aquaporin-2, a marker of principal cells, and in thick ascending limb, colocalizing with the glycoprotein Tamm-Horsfall. To determine the effect of a high-salt diet (HSD) on Na sensor gene expression, we quantified its transcript and protein levels primarily in renal medullas from control rats and rats subjected to 8% NaCl for 7 days (n = 5). HSD increased Na sensor expression levels (mRNA: from 1.2 ± 0.2 to 5.1 ± 1.3 au; protein: from 0.98 ± 0.15 to 1.74 ± 0.28 au P < 0.05) in the kidney medulla, but not in the cortex. These data indicate that rat kidney epithelial cells of the thick ascending limb and principal cells of the collecting duct possess a Na sensor that is upregulated by HSD, suggesting an important role in monitoring changes in tubular fluid [Na(+)].

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

    OpenAIRE

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

    2010-01-01

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

  8. Abnormal Conduction in the Diseased Heart - Enhanced fibrosis due to reduced gap junction and sodium channel expression

    NARCIS (Netherlands)

    Jansen, J.A.

    2011-01-01

    The velocity with which the impulse is conducted is dependent on three different factors: excitability, cell-to-cell coupling and tissue characteristics. Excitability is mainly determined by Nav1.5 channels, determining the upstroke-velocity of the action potential, but also by Kir2.1 channels. Gap

  9. Sodium channels in planar lipid bilayers. Channel gating kinetics of purified sodium channels modified by batrachotoxin

    Science.gov (United States)

    1986-01-01

    Single channel currents of sodium channels purified from rat brain and reconstituted into planar lipid bilayers were recorded. The kinetics of channel gating were investigated in the presence of batrachotoxin to eliminate inactivation and an analysis was conducted on membranes with a single active channel at any given time. Channel opening is favored by depolarization and is strongly voltage dependent. Probability density analysis of dwell times in the closed and open states of the channel indicates the occurrence of one open state and several distinct closed states in the voltage (V) range-120 mV less than or equal to V less than or equal to +120 mV. For V less than or equal to 0, the transition rates between stages are exponentially dependent on the applied voltage, as described in mouse neuroblastoma cells (Huang, L. M., N. Moran, and G. Ehrenstein. 1984. Biophysical Journal. 45:313- 322). In contrast, for V greater than or equal to 0, the transition rates are virtually voltage independent. Autocorrelation analysis (Labarca, P., J. Rice, D. Fredkin, and M. Montal. 1985. Biophysical Journal. 47:469-478) shows that there is no correlation in the durations of successive open or closing events. Several kinetic schemes that are consistent with the experimental data are considered. This approach may provide information about the mechanism underlying the voltage dependence of channel activation. PMID:2426388

  10. Voltage-gated sodium channels in taste bud cells

    Directory of Open Access Journals (Sweden)

    Williams Mark E

    2009-03-01

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

  11. DDT, pyrethrins, pyrethroids and insect sodium channels.

    Science.gov (United States)

    Davies, T G E; Field, L M; Usherwood, P N R; Williamson, M S

    2007-03-01

    The long term use of many insecticides is continually threatened by the ability of insects to evolve resistance mechanisms that render the chemicals ineffective. Such resistance poses a serious threat to insect pest control both in the UK and worldwide. Resistance may result from either an increase in the ability of the insect to detoxify the insecticide or by changes in the target protein with which the insecticide interacts. DDT, the pyrethrins and the synthetic pyrethroids (the latter currently accounting for around 17% of the world insecticide market), act on the voltage-gated sodium channel proteins found in insect nerve cell membranes. The correct functioning of these channels is essential for normal transmission of nerve impulses and this process is disrupted by binding of the insecticides, leading to paralysis and eventual death. Some insect pest populations have evolved modifications of the sodium channel protein which prevent the binding of the insecticide and result in the insect developing resistance. Here we review some of the work (done at Rothamsted Research and elsewhere) that has led to the identification of specific residues on the sodium channel that may constitute the DDT and pyrethroid binding sites.

  12. Excitability Constraints on Voltage-Gated Sodium Channels

    OpenAIRE

    Brenner, Michael; Angelino, Elaine

    2007-01-01

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

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

    Science.gov (United States)

    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.

  14. Soluble adenylyl cyclase in vascular endothelium: gene expression control of epithelial sodium channel-α, Na+/K+-ATPase-α/β, and mineralocorticoid receptor.

    Science.gov (United States)

    Schmitz, Boris; Nedele, Johanna; Guske, Katrin; Maase, Martina; Lenders, Malte; Schelleckes, Michael; Kusche-Vihrog, Kristina; Brand, Stefan-Martin; Brand, Eva

    2014-04-01

    The Ca(2+)- and bicarbonate-activated soluble adenylyl cyclase (sAC) has been identified recently as an important mediator of aldosterone signaling in the kidney. Nuclear sAC has been reported to stimulate cAMP response element-binding protein 1 phosphorylation via protein kinase A, suggesting an alternative cAMP pathway in the nucleus. In this study, we analyzed the sAC as a potential modulator of endothelial stiffness in the vascular endothelium. We determined the contribution of sAC to cAMP response element-mediated transcriptional activation in vascular endothelial cells and kidney collecting duct cells. Inhibition of sAC by the specific inhibitor KH7 significantly reduced cAMP response element-mediated promoter activity and affected cAMP response element-binding protein 1 phosphorylation. Furthermore, KH7 and anti-sAC small interfering RNA significantly decreased mRNA and protein levels of epithelial sodium channel-α and Na(+)/K(+)-ATPase-α. Using atomic force microscopy, a nano-technique that measures stiffness and deformability of living cells, we detected significant endothelial cell softening after sAC inhibition. Our results suggest that the sAC is a regulator of gene expression involved in aldosterone signaling and an important regulator of endothelial stiffness. Additional studies are warranted to investigate the protective action of sAC inhibitors in humans for potential clinical use.

  15. Voltage-gated sodium channels as targets for pyrethroid insecticides.

    Science.gov (United States)

    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.

  16. Molecular Biology of Insect Sodium Channels and Pyrethroid Resistance

    Science.gov (United States)

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

    2015-01-01

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

  17. On conduction in a bacterial sodium channel.

    Directory of Open Access Journals (Sweden)

    Simone Furini

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

  18. Ototrauma induces sodium channel plasticity in auditory afferent neurons

    OpenAIRE

    Fryatt, Alistair G.; Mulheran, Mike; Egerton, Julie; Gunthorpe, Martin J.; Grubb, Blair D.

    2011-01-01

    Exposure to intense sound can cause damage to the delicate sensory and neuronal components of the cochlea leading to hearing loss. Such damage often causes the dendrites of the spiral ganglion neurons (SGN), the neurons that provide the afferent innervation of the hair cells, to swell and degenerate thus damaging the synapse. In models of neuropathic pain, axotomy, another form of afferent nerve damage, is accompanied by altered voltage-gated sodium channel (VGSC) expression, leading to neuro...

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

    Science.gov (United States)

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

    2010-01-01

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

  20. Sodium Channel (Dys)Function and Cardiac Arrhythmias

    NARCIS (Netherlands)

    Remme, Carol Ann; Bezzina, Connie R.

    2010-01-01

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

  1. A sodium channel mutation identified in Aedes aegypti selectively reduces cockroach sodium channel sensitivity to type I, but not type II pyrethroids

    Science.gov (United States)

    Hu, Zhaonong; Du, Yuzhe; Nomura, Yoshiko; Dong, Ke

    2010-01-01

    Voltage-gated sodium channels are the primary target of pyrethroid insecticides. Numerous point mutations in sodium channel genes have been identified in pyrethroid-resistant insect species, and many have been confirmed to reduce or abolish sensitivity of channels expressed in Xenopus oocytes to pyrethroids. Recently, several novel mutations were reported in sodium channel genes of pyrethroid-resistant Aedes mosquito populations. One of the mutations is a phenylalanine (F) to cysteine (C) change in segment 6 of domain III (IIIS6) of the Aedes mosquito sodium channel. Curiously, a previous study showed that alanine substitution of this F did not alter the action of deltamethrin, a type II pyrethroid, on a cockroach sodium channel. In this study, we changed this F to C in a pyrethroid-sensitive cockroach sodium channel and examined mutant channel sensitivity to permethrin as well as five other type I or type II pyrethroids in Xenopus oocytes. Interestingly, the F to C mutation drastically reduced channel sensitivity to three type I pyrethroids, permethrin, NRDC 157 (a deltamethrin analogue lacking the α-cyano group) and bioresemthrin, but not to three type II pyrethroids, cypermethrin, deltamethrin and cyhalothrin. These results confirm the involvement of the F to C mutation in permethrin resistance, and raise the possibility that rotation of type I and type II pyrethroids might be considered in the control of insect pest populations where this particular mutation is present. PMID:20869441

  2. Voltage-Gated Sodium Channels: Biophysics, Pharmacology, and Related Channelopathies

    Science.gov (United States)

    Savio-Galimberti, Eleonora; Gollob, Michael H.; Darbar, Dawood

    2012-01-01

    Voltage-gated sodium channels (VGSC) are multi-molecular protein complexes expressed in both excitable and non-excitable cells. They are primarily formed by a pore-forming multi-spanning integral membrane glycoprotein (α-subunit) that can be associated with one or more regulatory β-subunits. The latter are single-span integral membrane proteins that modulate the sodium current (INa) and can also function as cell adhesion molecules. In vitro some of the cell-adhesive functions of the β-subunits may play important physiological roles independently of the α-subunits. Other endogenous regulatory proteins named “channel partners” or “channel interacting proteins” (ChiPs) like caveolin-3 and calmodulin/calmodulin kinase II (CaMKII) can also interact and modulate the expression and/or function of VGSC. In addition to their physiological roles in cell excitability and cell adhesion, VGSC are the site of action of toxins (like tetrodotoxin and saxitoxin), and pharmacologic agents (like antiarrhythmic drugs, local anesthetics, antiepileptic drugs, and newly developed analgesics). Mutations in genes that encode α- and/or β-subunits as well as the ChiPs can affect the structure and biophysical properties of VGSC, leading to the development of diseases termed sodium “channelopathies”.  This review will outline the structure, function, and biophysical properties of VGSC as well as their pharmacology and associated channelopathies and highlight some of the recent advances in this field. PMID:22798951

  3. Voltage-gated sodium channels: biophysics, pharmacology, and related channelopathies

    Directory of Open Access Journals (Sweden)

    Eleonora eSavio Galimberti

    2012-07-01

    Full Text Available Voltage-gated sodium channels (VGSC are multi-molecular protein complexes expressed in both excitable and non-excitable cells. They are primarily formed by a pore-forming multi-spanning integral membrane glycoprotein (α-subunit that can be associated with one or more regulatory β-subunits. The latter are single-span integral membrane proteins that modulate the sodium current (INa and can also function as cell-adhesion molecules (CAMs. In-vitro some of the cell-adhesive functions of the β-subunits may play important physiological roles independently of the α-subunits. Other endogenous regulatory proteins named channel partners or channel interacting proteins (ChiPs like caveolin-3 and calmodulin/calmodulin kinase II (CaMKII can also interact and modulate the expression and/or function of VGSC. In addition to their physiological roles in cell excitability and cell adhesion, VGSC are the site of action of toxins (like tetrodotoxin and saxitoxin, and pharmacologic agents (like antiarrhythmic drugs, local anesthetics, antiepileptic drugs, and newly developed analgesics. Mutations in genes that encode α- and/or β-subunits as well as the ChiPs can affect the structure and biophysical properties of VGSC, leading to the development of diseases termed sodium channelopathies. This review will outline the structure, function and biophysical properties of VGSC as well as their pharmacology and associated channelopathies and highlight some of the recent advances in this field

  4. Resveratrol attenuates cortical neuron activity: roles of large conductance calcium-activated potassium channels and voltage-gated sodium channels.

    Science.gov (United States)

    Wang, Ya-Jean; Chan, Ming-Huan; Chen, Linyi; Wu, Sheng-Nan; Chen, Hwei-Hisen

    2016-05-21

    Resveratrol, a phytoalexin found in grapes and red wine, exhibits diverse pharmacological activities. However, relatively little is known about whether resveratrol modulates the ion channels in cortical neurons. The large-conductance calcium-activated potassium channels (BKCa) and voltage-gated sodium channels were expressed in cortical neurons and play important roles in regulation of neuronal excitability. The present study aimed to determine the effects of resveratrol on BKCa currents and voltage-gated sodium currents in cortical neurons. Resveratrol concentration-dependently increased the current amplitude and the opening activity of BKCa channels, but suppressed the amplitude of voltage-gated sodium currents. Similar to the BKCa channel opener NS1619, resveratrol decreased the firing rate of action potentials. In addition, the enhancing effects of BKCa channel blockers tetraethylammonium (TEA) and paxilline on action potential firing were sensitive to resveratrol. Our results indicated that the attenuation of action potential firing rate by resveratrol might be mediated through opening the BKCa channels and closing the voltage-gated sodium channels. As BKCa channels and sodium channels are critical molecular determinants for seizure generation, our findings suggest that regulation of these two channels in cortical neurons probably makes a considerable contribution to the antiseizure activity of resveratrol.

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

    Science.gov (United States)

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

    2015-01-01

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

  6. Novel Sodium Channel Inhibitor From Leeches

    Directory of Open Access Journals (Sweden)

    Gan Wang

    2018-03-01

    Full Text Available Considering blood-sucking habits of leeches from surviving strategy of view, it can be hypothesized that leech saliva has analgesia or anesthesia functions for leeches to stay undetected by the host. However, no specific substance with analgesic function has been reported from leech saliva although clinical applications strongly indicated that leech therapy produces a strong and long lasting pain-reducing effect. Herein, a novel family of small peptides (HSTXs including 11 members which show low similarity with known peptides was identified from salivary glands of the leech Haemadipsa sylvestris. A typical HSTX is composed of 22–25 amino acid residues including four half-cysteines, forming two intra-molecular disulfide bridges, and an amidated C-terminus. HSTX-I exerts significant analgesic function by specifically inhibiting voltage-gated sodium (NaV channels (NaV1.8 and NaV1.9 which contribute to action potential electrogenesis in neurons and potential targets to develop analgesics. This study reveals that sodium channel inhibitors are analgesic substances in the leech. HSTXs are excellent candidates or templates for development of analgesics.

  7. NALCN ion channels have alternative selectivity filters resembling calcium channels or sodium channels

    NARCIS (Netherlands)

    Senatore, A.; Monteil, A.; van Minnen, J.; Smit, A.B.; Spafford, J.D.

    2013-01-01

    NALCN is a member of the family of ion channels with four homologous, repeat domains that include voltage-gated calcium and sodium channels. NALCN is a highly conserved gene from simple, extant multicellular organisms without nervous systems such as sponges and placozoans and mostly remains a single

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

    Energy Technology Data Exchange (ETDEWEB)

    He, Bingjun [College of Life Sciences, Nankai University, Tianjin 300071 (China); Soderlund, David M., E-mail: dms6@cornell.edu [Department of Entomology, Cornell University, New York State Agricultural Experiment Station, Geneva, NY 14456 (United States)

    2011-12-15

    We expressed rat Na{sub v}1.6 sodium channels in combination with the rat {beta}1 and {beta}2 auxiliary subunits in human embryonic kidney (HEK293) cells and evaluated the effects of the pyrethroid insecticides tefluthrin and deltamethrin on expressed sodium currents using the whole-cell patch clamp technique. Both pyrethroids produced concentration-dependent, resting modification of Na{sub v}1.6 channels, prolonging the kinetics of channel inactivation and deactivation to produce persistent 'late' currents during depolarization and tail currents following repolarization. Both pyrethroids also produced concentration dependent hyperpolarizing shifts in the voltage dependence of channel activation and steady-state inactivation. Maximal shifts in activation, determined from the voltage dependence of the pyrethroid-induced late and tail currents, were {approx} 25 mV for tefluthrin and {approx} 20 mV for deltamethrin. The highest attainable concentrations of these compounds also caused shifts of {approx} 5-10 mV in the voltage dependence of steady-state inactivation. In addition to their effects on the voltage dependence of inactivation, both compounds caused concentration-dependent increases in the fraction of sodium current that was resistant to inactivation following strong depolarizing prepulses. We assessed the use-dependent effects of tefluthrin and deltamethrin on Na{sub v}1.6 channels by determining the effect of trains of 1 to 100 5-ms depolarizing prepulses at frequencies of 20 or 66.7 Hz on the extent of channel modification. Repetitive depolarization at either frequency increased modification by deltamethrin by {approx} 2.3-fold but had no effect on modification by tefluthrin. Tefluthrin and deltamethrin were equally potent as modifiers of Na{sub v}1.6 channels in HEK293 cells using the conditions producing maximal modification as the basis for comparison. These findings show that the actions of tefluthrin and deltamethrin of Na{sub v}1.6 channels

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

    Directory of Open Access Journals (Sweden)

    Smith Paula L

    2011-09-01

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

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

    NARCIS (Netherlands)

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

    2006-01-01

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

  11. Human cardiac sodium channels are affected by pentobarbital

    NARCIS (Netherlands)

    Wartenberg, H. C.; Wartenberg, J. P.; Urban, B. W.

    2001-01-01

    BACKGROUND: AND OBJECTIVE: To investigate the response to general anaesthetics of different sodium channel subtypes, we examined the effects of pentobarbital, a close thiopental analogue, on single sodium channels from human ventricular muscle and compared them with existing data from human brain

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

    Science.gov (United States)

    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.

  13. Cardiac sodium channels and inherited electrophysiologic disorders: a pharmacogenetic overview

    NARCIS (Netherlands)

    Smits, Jeroen P. P.; Blom, Marieke T.; Wilde, Arthur A. M.; Tan, Hanno L.

    2008-01-01

    Sodium (Na) channels are essential for cardiac electrical activity. Cardiac Na channel dysfunction, inherited or acquired, can induce life-threatening conduction and arrhythmia disorders. Inherited Na channel dysfunction may put affected patients at a greater risk for these complications when

  14. Morphine decreases enteric neuron excitability via inhibition of sodium channels.

    Directory of Open Access Journals (Sweden)

    Tricia H Smith

    Full Text Available Gastrointestinal peristalsis is significantly dependent on the enteric nervous system. Constipation due to reduced peristalsis is a major side-effect of morphine, which limits the chronic usefulness of this excellent pain reliever in man. The ionic basis for the inhibition of enteric neuron excitability by morphine is not well characterized as previous studies have mainly utilized microelectrode recordings from whole mount myenteric plexus preparations in guinea pigs. Here we have developed a Swiss-Webster mouse myenteric neuron culture and examined their electrophysiological properties by patch-clamp techniques and determined the mechanism for morphine-induced decrease in neuronal excitability. Isolated neurons in culture were confirmed by immunostaining with pan-neuronal marker, β-III tubulin and two populations were identified by calbindin and calretinin staining. Distinct neuronal populations were further identified based on the presence and absence of an afterhyperpolarization (AHP. Cells with AHP expressed greater density of sodium currents. Morphine (3 µM significantly reduced the amplitude of the action potential, increased the threshold for spike generation but did not alter the resting membrane potential. The decrease in excitability resulted from inhibition of sodium currents. In the presence of morphine, the steady-state voltage dependence of Na channels was shifted to the left with almost 50% of channels unavailable for activation from hyperpolarized potentials. During prolonged exposure to morphine (two hours, action potentials recovered, indicative of the development of tolerance in single enteric neurons. These results demonstrate the feasibility of isolating mouse myenteric neurons and establish sodium channel inhibition as a mechanism for morphine-induced decrease in neuronal excitability.

  15. Channel properties of Nax expressed in neurons.

    Directory of Open Access Journals (Sweden)

    Masahito Matsumoto

    Full Text Available Nax is a sodium-concentration ([Na+]-sensitive Na channel with a gating threshold of ~150 mM for extracellular [Na+] ([Na+]o in vitro. We previously reported that Nax was preferentially expressed in the glial cells of sensory circumventricular organs including the subfornical organ, and was involved in [Na+] sensing for the control of salt-intake behavior. Although Nax was also suggested to be expressed in the neurons of some brain regions including the amygdala and cerebral cortex, the channel properties of Nax have not yet been adequately characterized in neurons. We herein verified that Nax was expressed in neurons in the lateral amygdala of mice using an antibody that was newly generated against mouse Nax. To investigate the channel properties of Nax expressed in neurons, we established an inducible cell line of Nax using the mouse neuroblastoma cell line, Neuro-2a, which is endogenously devoid of the expression of Nax. Functional analyses of this cell line revealed that the [Na+]-sensitivity of Nax in neuronal cells was similar to that expressed in glial cells. The cation selectivity sequence of the Nax channel in cations was revealed to be Na+ ≈ Li+ > Rb+ > Cs+ for the first time. Furthermore, we demonstrated that Nax bound to postsynaptic density protein 95 (PSD95 through its PSD95/Disc-large/ZO-1 (PDZ-binding motif at the C-terminus in neurons. The interaction between Nax and PSD95 may be involved in promoting the surface expression of Nax channels because the depletion of endogenous PSD95 resulted in a decrease in Nax at the plasma membrane. These results indicated, for the first time, that Nax functions as a [Na+]-sensitive Na channel in neurons as well as in glial cells.

  16. Epithelial Sodium and Acid-Sensing Ion Channels

    Science.gov (United States)

    Kellenberger, Stephan

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

  17. High Salt Diet Affects Renal Sodium Excretion and ERRα Expression.

    Science.gov (United States)

    Wang, Dan; Wang, Yang; Liu, Fu-Qiang; Yuan, Zu-Yi; Mu, Jian-Jun

    2016-04-01

    Kidneys regulate the balance of water and sodium and therefore are related to blood pressure. It is unclear whether estrogen-related receptor α (ERRα), an orphan nuclear receptor and transcription factor highly expressed in kidneys, affects the reabsorption of water and sodium. The aim of this study was to determine whether changes in the expressions of ERRα, Na⁺/K⁺-ATPase and epithelial sodium channel (ENaC) proteins affected the reabsorption of water and sodium in kidneys of Dahl salt-sensitive (DS) rats. SS.13BN rats, 98% homologous to the DS rats, were used as a normotensive control group. The 24 h urinary sodium excretion of the DS and SS.13BN rats increased after the 6-week high salt diet intervention, while sodium excretion was increased in DS rats with daidzein (agonist of ERRα) treatment. ERRα expression was decreased, while β- and γ-ENaC mRNA expressions were increased upon high sodium diet treatment in the DS rats. In the chromatin immunoprecipitation (CHIP) assay, positive PCR signals were obtained in samples treated with anti-ERRα antibody. The transcriptional activity of ERRα was decreased upon high salt diet intervention. ERRα reduced the expressions of β- and γ-ENaC by binding to the ENaC promoter, thereby increased Na+ reabsorption. Therefore, ERRα might be one of the factors causing salt-sensitive hypertension.

  18. Rapid evolution of a voltage-gated sodium channel gene in a lineage of electric fish leads to a persistent sodium current.

    Science.gov (United States)

    Thompson, Ammon; Infield, Daniel T; Smith, Adam R; Smith, G Troy; Ahern, Christopher A; Zakon, Harold H

    2018-03-01

    Most weakly electric fish navigate and communicate by sensing electric signals generated by their muscle-derived electric organs. Adults of one lineage (Apteronotidae), which discharge their electric organs in excess of 1 kHz, instead have an electric organ derived from the axons of specialized spinal neurons (electromotorneurons [EMNs]). EMNs fire spontaneously and are the fastest-firing neurons known. This biophysically extreme phenotype depends upon a persistent sodium current, the molecular underpinnings of which remain unknown. We show that a skeletal muscle-specific sodium channel gene duplicated in this lineage and, within approximately 2 million years, began expressing in the spinal cord, a novel site of expression for this isoform. Concurrently, amino acid replacements that cause a persistent sodium current accumulated in the regions of the channel underlying inactivation. Therefore, a novel adaptation allowing extreme neuronal firing arose from the duplication, change in expression, and rapid sequence evolution of a muscle-expressing sodium channel gene.

  19. Physiological regulation of epithelial sodium channel by proteolysis

    DEFF Research Database (Denmark)

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

    2011-01-01

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

  20. Channelopathies from mutations in the cardiac sodium channel protein complex.

    Science.gov (United States)

    Adsit, Graham S; Vaidyanathan, Ravi; Galler, Carla M; Kyle, John W; Makielski, Jonathan C

    2013-08-01

    The cardiac sodium current underlies excitability in heart, and inherited abnormalities of the proteins regulating and conducting this current cause inherited arrhythmia syndromes. This review focuses on inherited mutations in non-pore forming proteins of sodium channel complexes that cause cardiac arrhythmia, and the deduced mechanisms by which they affect function and dysfunction of the cardiac sodium current. Defining the structure and function of these complexes and how they are regulated will contribute to understanding the possible roles for this complex in normal and abnormal physiology and homeostasis. This article is part of a Special Issue entitled "Na(+) Regulation in Cardiac Myocytes". Copyright © 2013 Elsevier Ltd. All rights reserved.

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

    NARCIS (Netherlands)

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

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

  2. Molecular evidence for dual pyrethroid-receptor sites on a mosquito sodium channel

    Science.gov (United States)

    Nomura, Yoshiko; Satar, Gul; Hu, Zhaonong; Nauen, Ralf; He, Sheng Yang; Zhorov, Boris S.; Dong, Ke

    2013-01-01

    Pyrethroid insecticides are widely used as one of the most effective control measures in the global fight against agricultural arthropod pests and mosquito-borne diseases, including malaria and dengue. They exert toxic effects by altering the function of voltage-gated sodium channels, which are essential for proper electrical signaling in the nervous system. A major threat to the sustained use of pyrethroids for vector control is the emergence of mosquito resistance to pyrethroids worldwide. Here, we report the successful expression of a sodium channel, AaNav1–1, from Aedes aegypti in Xenopus oocytes, and the functional examination of nine sodium channel mutations that are associated with pyrethroid resistance in various Ae. aegypti and Anopheles gambiae populations around the world. Our analysis shows that five of the nine mutations reduce AaNav1–1 sensitivity to pyrethroids. Computer modeling and further mutational analysis revealed a surprising finding: Although two of the five confirmed mutations map to a previously proposed pyrethroid-receptor site in the house fly sodium channel, the other three mutations are mapped to a second receptor site. Discovery of this second putative receptor site provides a dual-receptor paradigm that could explain much of the molecular mechanisms of pyrethroid action and resistance as well as the high selectivity of pyrethroids on insect vs. mammalian sodium channels. Results from this study could impact future prediction and monitoring of pyrethroid resistance in mosquitoes and other arthropod pests and disease vectors. PMID:23821746

  3. [Progress in sodium channelopathies and biological functions of voltage-gated sodium channel blockers].

    Science.gov (United States)

    Wang, Hongyan; Gou, Meng; Xiao, Rong; Li, Qingwei

    2014-06-01

    Voltage-gated sodium channels (VGSCs), which are widely distributed in the excitable cells, are the primary mediators of electrical signal amplification and propagation. They play important roles in the excitative conduction of the neurons and cardiac muscle cells. The abnormalities of the structures and functions of VGSCs can change the excitability of the cells, resulting in a variety of diseases such as neuropathic pain, epilepsy and arrhythmia. At present, some voltage-gated sodium channel blockers are used for treating those diseases. In the recent years, several neurotoxins have been purified from the venom of the animals, which could inhibit the current of the voltage-gated sodium channels. Usually, these neurotoxins are compounds or small peptides that have been further designed and modified for targeted drugs of sodium channelopathies in the clinical treatment. In addition, a novel cysteine-rich secretory protein (CRBGP) has been isolated and purified from the buccal gland of the lampreys (Lampetra japonica), and it could inhibit the Na+ current of the hippocampus and dorsal root neurons for the first time. In the present study, the progress of the sodium channelopathies and the biological functions of voltage-gated sodium channel blockers are analyzed and summarized.

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

    African Journals Online (AJOL)

    Modulation of epithelial sodium channel in human alveolar epithelial cells by lipoxin A4 through AhR-cAMP-dependent pathway. Bi-Huan Cheng, Li-Wei Pan, Sheng-Rong Zhang, Bin-Yu Ying, Ben-Ji Wang, Guo-Liang Lin, Shi-Fang Ding ...

  5. Differential lipid dependence of the function of bacterial sodium channels.

    Directory of Open Access Journals (Sweden)

    Nazzareno D'Avanzo

    Full Text Available The lipid bilayer is important for maintaining the integrity of cellular compartments and plays a vital role in providing the hydrophobic and charged interactions necessary for membrane protein structure, conformational flexibility and function. To directly assess the lipid dependence of activity for voltage-gated sodium channels, we compared the activity of three bacterial sodium channel homologues (NaChBac, NavMs, and NavSp by cumulative (22Na(+ uptake into proteoliposomes containing a 3∶1 ratio of 1-palmitoyl 2-oleoyl phosphatidylethanolamine and different "guest" glycerophospholipids. We observed a unique lipid profile for each channel tested. NavMs and NavSp showed strong preference for different negatively-charged lipids (phosphatidylinositol and phosphatidylglycerol, respectively, whilst NaChBac exhibited a more modest variation with lipid type. To investigate the molecular bases of these differences we used synchrotron radiation circular dichroism spectroscopy to compare structures in liposomes of different composition, and molecular modeling and electrostatics calculations to rationalize the functional differences seen. We then examined pore-only constructs (with voltage sensor subdomains removed and found that in these channels the lipid specificity was drastically reduced, suggesting that the specific lipid influences on voltage-gated sodium channels arise primarily from their abilities to interact with the voltage-sensing subdomains.

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

    Science.gov (United States)

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

    2014-04-25

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

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

    Science.gov (United States)

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

    2010-04-01

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

  8. The batrachotoxin receptor on the voltage-gated sodium channel is guarded by the channel activation gate.

    Science.gov (United States)

    Li, Hong-Ling; Hadid, David; Ragsdale, David S

    2002-04-01

    Batrachotoxin (BTX), from South American frogs of the genus Phyllobates, irreversibly activates voltage-gated sodium channels. Previous work demonstrated that a phenylalanine residue approximately halfway through pore-lining transmembrane segment IVS6 is a critical determinant of channel sensitivity to BTX. In this study, we introduced a series of mutations at this site in the Na(v)1.3 sodium channel, expressed wild-type and mutant channels in Xenopus laevis oocytes, and examined their sensitivity to BTX using voltage clamp recording. We found that substitution of either alanine or isoleucine strongly reduced channel sensitivity to toxin, whereas cysteine, tyrosine, or tryptophan decreased toxin action only modestly. These data suggest an electrostatic ligand-receptor interaction at this site, possibly involving a charged tertiary amine on BTX. We then used a mutant channel (mutant F1710C) with intermediate toxin sensitivity to examine the properties of the toxin-receptor reaction in more detail. In contrast to wild-type channels, which bind BTX almost irreversibly, toxin dissociation from mutant channels was rapid, but only when the channels were open, not when they were closed. These data suggest the closed activation gate trapped bound toxin. Although BTX dissociation required channel activation, it was, paradoxically, slowed by strong membrane depolarization, suggesting additional state-dependent and/or electrostatic influences on the toxin binding reaction. We propose that BTX moves to and from its receptor through the cytoplasmic end of the open ion-conducting pore, in a manner similar to that of quaternary local anesthetics like QX314.

  9. Molecular model of the action potential sodium channel.

    OpenAIRE

    Guy, H R; Seetharamulu, P

    1986-01-01

    Secondary and tertiary structural models of sodium channel transmembrane segments were developed from its recently determined primary sequence in Electrophorus electricus. The model has four homologous domains, and each domain has eight homologous transmembrane segments, S1 through S8. Each domain contains three relatively apolar segments (S1, S2 and S3) and two very apolar segments (S5 and S8), all postulated to be transmembrane alpha-helices. S4 segments have positively charged residues, ma...

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

    African Journals Online (AJOL)

    Modulation of epithelial sodium channel in human alveolar epithelial cells by lipoxin A4 through AhR-cAMP-dependent pathway. Bi-Huan Cheng1,2, Li-Wei Pan2, Sheng-Rong Zhang3, Bin-Yu Ying2, Ben-Ji. Wang2, Guo-Liang Lin2 and Shi-Fang Ding1*. 1Department of Critical Care Medicine, Qilu Hospital of Shandong ...

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

    Directory of Open Access Journals (Sweden)

    Danielle L Tomasello

    2017-09-01

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

  12. Estrogen, progesterone, and genistein differentially regulate levels of expression of α-, β-, and γ-epithelial sodium channel (ENaC) and α-sodium potassium pump (Na⁺/K⁺-ATPase) in the uteri of sex steroid-deficient rats.

    Science.gov (United States)

    Chinigarzadeh, Asma; Muniandy, Sekaran; Salleh, Naguib

    2015-10-01

    Estrogen, progesterone, and genistein could induce changes in uterine fluid volume and Na(+) concentration. Progesterone upregulates expression of epithelial sodium channel (ENaC) and Na(+)/K(+)-ATPase which contributed toward these changes. However, effects of estrogen and genistein were unknown. This study therefore investigated changes in expression of these proteins in the uterus under estrogen, progesterone, and genistein influences to further understand mechanisms underlying sex steroids and phytoestrogen effects on uterine fluid Na(+) regulation. In this study, uteri of ovariectomized female rats receiving 7-day treatment with genistein (25, 50, and 100 mg/kg/day), estrogen (0.8 × 10(-4) mg/kg/day), or progesterone (4 mg/kg/day) were harvested, and expression levels of α-, β-, and γ-ENaC proteins and messenger RNAs (mRNAs) and α-Na(+)/K(+)-ATPase protein were determined by Western blotting (proteins) and real-time polymerase chain reaction (mRNA). Meanwhile, distribution of α-, β-, and γ-ENaC and α-Na(+)/K(+)-ATPase proteins in the uterus was identified by immunohistochemistry. Our findings indicated that expression of α-, β-, and γ-ENaC proteins and mRNAs and α-Na(+)/K(+)-ATPase protein were enhanced under progesterone influence. Lower expressions were noted under estrogen and genistein influences compared to progesterone. Under estrogen, progesterone, and genistein influences, α- and β-ENaC were distributed at apical membrane and γ-ENaC was distributed at apical and basolateral membranes of uterine luminal epithelia. Under progesterone influence, α-Na(+)/K(+)-ATPase was highly expressed at basolateral membrane. In conclusion, high expression of α-, β-, and γ-ENaC and α-Na(+)/K(+)-ATPase under progesterone influence would contribute toward increased uterine fluid Na(+) reabsorption, whereas lesser expression of these proteins under estrogen and genistein influences would contribute toward lower reabsorption of uterine fluid Na

  13. Plasmin in Nephrotic Urine Activates the Epithelial Sodium Channel

    DEFF Research Database (Denmark)

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

    2009-01-01

    stimulated amiloride-sensitive transepithelial sodium transport in M-1 cells and increased amiloride-sensitive whole-cell currents in Xenopus laevis oocytes heterologously expressing ENaC. Activation of ENaC by plasmin involved cleavage and release of an inhibitory peptide from the ENaC gamma subunit...

  14. Gating kinetics of batrachotoxin-modified sodium channels in neuroblastoma cells determined from single-channel measurements

    OpenAIRE

    Huang, L.Y.; Moran, N.; Ehrenstein, G.

    1984-01-01

    We have observed the opening and closing of single batrachotoxin (BTX)-modified sodium channels in neuroblastoma cells using the patch-clamp method. The conductance of a single BTX-modified channel is approximately 10 pS. At a given membrane potential, the channels are open longer than are normal sodium channels. As is the case for normal sodium channels, the open dwell times become longer as the membrane is depolarized. For membrane potentials more negative than about -70 mV, histograms of b...

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

    Directory of Open Access Journals (Sweden)

    Maryam Iman

    2013-09-01

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

  16. Adaptive evolution of the vertebrate skeletal muscle sodium channel

    Directory of Open Access Journals (Sweden)

    Jian Lu

    2011-01-01

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

  17. Marine Toxins That Target Voltage-gated Sodium Channels

    Directory of Open Access Journals (Sweden)

    Robert J. French

    2006-04-01

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

  18. Sodium channel β subunits: emerging targets in channelopathies.

    Science.gov (United States)

    O'Malley, Heather A; Isom, Lori L

    2015-01-01

    Voltage-gated sodium channels (VGSCs) are responsible for the initiation and propagation of action potentials in excitable cells. VGSCs in mammalian brain are heterotrimeric complexes of α and β subunits. Although β subunits were originally termed auxiliary, we now know that they are multifunctional signaling molecules that play roles in both excitable and nonexcitable cell types and with or without the pore-forming α subunit present. β subunits function in VGSC and potassium channel modulation, cell adhesion, and gene regulation, with particularly important roles in brain development. Mutations in the genes encoding β subunits are linked to a number of diseases, including epilepsy, sudden death syndromes like SUDEP and SIDS, and cardiac arrhythmia. Although VGSC β subunit-specific drugs have not yet been developed, this protein family is an emerging therapeutic target.

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

    Directory of Open Access Journals (Sweden)

    Mohamed-Yassine eAMAROUCH

    2015-02-01

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

  20. Single sodium channels from human ventricular muscle in planar lipid bilayers

    NARCIS (Netherlands)

    Wartenberg, H. C.; Wartenberg, J. P.; Urban, B. W.

    2001-01-01

    Sodium channels from human ventricular muscle membrane vesicles were incorporated into planar lipid bilayers and the steady-state behavior of single sodium channels were examined in the presence of batrachotoxin. In symmetrical 500 mM NaCl the averaged single channel conductance was 24.7 +/- 1.3 pS

  1. Sodium channel activators: model of binding inside the pore and a possible mechanism of action.

    Science.gov (United States)

    Tikhonov, Denis B; Zhorov, Boris S

    2005-08-15

    Sodium channel activators, batrachotoxin and veratridine, cause sodium channels to activate easier and stay open longer than normal channels. Traditionally, this was explained by an allosteric mechanism. However, increasing evidence suggests that activators can bind inside the pore. Here, we model the open sodium channel with activators and propose a novel mechanism of their action. The activator-bound channel retains a hydrophilic pathway for ions between the ligand and conserved asparagine in segment S6 of repeat II. One end of the activator approaches the selectivity filter, decreasing the channel conductance and selectivity. The opposite end reaches the gate stabilizing it in the open state.

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

    Science.gov (United States)

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

    2016-07-01

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

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

    Science.gov (United States)

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

    2013-01-01

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

  4. Two-dimensional probability density analysis of single channel currents from reconstituted acetylcholine receptors and sodium channels.

    Science.gov (United States)

    Keller, B U; Montal, M S; Hartshorne, R P; Montal, M

    1990-01-01

    Two-dimensional probability density analysis of single channel current recordings was applied to two purified channel proteins reconstituted in planar lipid bilayers: Torpedo acetylcholine receptors and voltage-sensitive sodium channels from rat brain. The information contained in the dynamic history of the gating process, i.e., the time sequence of opening and closing events was extracted from two-dimensional distributions of transitions between identifiable states. This approach allows one to identify kinetic models consistent with the observables. Gating of acetylcholine receptors expresses "memory" of the transition history: the receptor has two channel open (O) states; the residence time in each of them strongly depends on both the preceding open time and the intervening closed interval. Correspondingly, the residence time in the closed (C) states depends on both the preceding open time and the preceding closed time. This result confirms the scheme that considers, at least, two transition pathways between the open and closed states and extends the details of the model in that it defines that the short-lived open state is primarily entered from long-lived closed states while the long-lived open state is accessed mainly through short-lived closed states. Since ligand binding to the acetylcholine-binding sites is a reaction with channel closed states, we infer that the longest closed state (approximately 19 ms) is unliganded, the intermediate closed state (approximately 2 ms) is singly liganded and makes transitions to the short open state (approximately 0.5 ms) and the shortest closed state (approximately 0.4 ms) is doubly liganded and isomerizes to long open states (approximately 5 ms). This is the simplest interpretation consistent with available data. In contrast, sodium channels modified with batrachotoxin to eliminate inactivation show no correlation in the sequence of channel opening and closing events, i.e., have no memory of the transition history. This

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

    Science.gov (United States)

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

    2017-11-07

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

  6. [Steroidal alkaloid batrachotoxin--instrument for studying voltage-regulated sodium channels].

    Science.gov (United States)

    Khodorov, B I

    1985-01-01

    Results of recent studies on the batrachotoxin (BTX) effect on the properties of voltage-operated sodium channels in excitable membranes are summarized in the review. The following problems are considered: allosteric interaction of the BTX receptor with structural entities of the sodium channel responsible for its activation, inactivation, ion selectivity, binding of polypeptide (scorpion and anemone) toxins, local anesthetics and many blocking drugs; relationship between BTX-induced changes in the sodium conductance and intramembrane charge movement; relationship between ion selectivity and effective pK of the selectivity filter acid group of sodium channels modified by BTX or aconitine; effects of BTX on the behaviour and conductance (gamma) of single sodium channels. The problem of the BTX receptor location and possible mechanism of the sodium channel modification by BTX are discussed.

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

    Directory of Open Access Journals (Sweden)

    Jean-François eDesaphy

    2012-02-01

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

  8. Batrachotoxin modifies the gating kinetics of sodium channels in internally perfused neuroblastoma cells.

    Science.gov (United States)

    Huang, L Y; Moran, N; Ehrenstein, G

    1982-01-01

    We have studied the effects of batrachotoxin (BTX) on sodium channels in hybrid mouse neuroblastoma cells NG108-15 by using the suction pipet voltage clamp method. BTX-modified sodium channels activate with first-order kinetics and, over most of the potential range, activate more slowly than normal sodium channels. The peak conductance-voltage curve and the time constant of activation-versus-voltage curve for BTX-modified sodium channels are shifted about 50 mV in the hyperpolarizing direction compared to the corresponding curves for normal sodium channels. There is no change in the slope of the conductance-voltage curve. These results suggest that BTX slows down one of the steps leading to channel opening, which consequently becomes rate-limiting. In addition, BTX eliminates both fast and slow inactivation. PMID:6281796

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

    Science.gov (United States)

    Vien, T N; DeCaen, P G

    2016-01-01

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

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

    Directory of Open Access Journals (Sweden)

    Andrias O O'Reilly

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

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

    Science.gov (United States)

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

    2016-10-01

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

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

    Science.gov (United States)

    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

  13. Divergent Actions of the Pyrethroid Insecticides S-Bioallethrin, Tefluthrin and Deltamethrin on Rat Nav1.6 Sodium Channels

    Science.gov (United States)

    Tan, Jianguo; Soderlund, David M.

    2010-01-01

    We expressed rat Nav1.6 sodium channels in combination with the rat β1 and β2 auxiliary subunits in Xenopus laevis oocytes and evaluated the effects of the pyrethroid insecticides S-bioallethrin, deltamethrin and tefluthrin on expressed sodium currents using the two-electrode voltage clamp technique. S-Bioallethrin, a Type I structure, produced transient modification evident in the induction of rapidly-decaying sodium tail currents, weak resting modification (5.7% modification at 100 μM), and no further enhancement of modification upon repetitive activation by high-frequency trains of depolarizing pulses. By contrast deltamethrin, a Type II structure, produced sodium tail currents that were ~9-fold more persistent than those caused by S-bioallethrin, barely detectable resting modification (2.5% modification at 100 μM), and 3.7-fold enhancement of modification upon repetitive activation. Tefluthrin, a Type I structure with high mammalian toxicity, exhibited properties intermediate between S-bioallethrin and deltamethrin: intermediate tail current decay kinetics, much greater resting modification (14.1% at 100 μM), and 2.8-fold enhancement of resting modification upon repetitive activation. Comparison of concentration–effect data showed that repetitive depolarization increased the potency of tefluthrin ~15-fold and that tefluthrin was ~10-fold more potent than deltamethrin as a use-dependent modifier of Nav1.6 sodium channels. Concentration–effect data from parallel experiments with the rat Nav1.2 sodium channel co-expressed with the rat β1 and β2 subunits in oocytes showed that the Nav1.6 isoform was at least 15-fold more sensitive to tefluthrin and deltamethrin than the Nav1.2 isoform. These results implicate sodium channels containing the Nav1.6 isoform as potential targets for the central neurotoxic effects of pyrethroids. PMID:20624410

  14. Amiloride-insensitive sodium channels are directly regulated by actin cytoskeleton dynamics in human lymphoma cells.

    Science.gov (United States)

    Sudarikova, Anastasia V; Tsaplina, Olga A; Chubinskiy-Nadezhdin, Vladislav I; Morachevskaya, Elena A; Negulyaev, Yuri A

    2015-05-22

    Sodium influx mediated by ion channels of plasma membrane underlies fundamental physiological processes in cells of blood origin. However, little is known about the single channel activity and regulatory mechanisms of sodium-specific channels in native cells. In the present work, we used different modes of patch clamp technique to examine ion channels involved in Na-transporting pathway in U937 human lymphoma cells. The activity of native non-voltage-gated sodium (NVGS) channels with unitary conductance of 10 pS was revealed in cell-attached, inside-out and whole-cell configurations. NVGS channel activity is directly controlled by submembranous actin cytoskeleton. Specifically, an activation of sodium channels in U937 cells in response to microfilament disassembly was demonstrated on single-channel and integral current level. Inside-out experiments showed that filament assembly on cytoplasmic membrane surface caused fast inactivation of the channels. Biophysical characteristics of NVGS channels in U937 cells were similar to that of epithelial sodium channels (ENaCs). However, we found that amiloride, a known inhibitor of DEG/ENaC, did not block NVGS channels in U937 cells. Whole-cell current measurements revealed no amiloride-sensitive component of membrane current. Our data show that cortical actin structures represent the main factor that controls the activity of amiloride-insensitive ENaC-like channels in human lymphoma cells. Copyright © 2015 Elsevier Inc. All rights reserved.

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

    NARCIS (Netherlands)

    Wartenberg, Hans C.; Urban, Bernd W.

    2004-01-01

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

  16. An important role of a pyrethroid-sensing residue F1519 in the action of the N-alkylamide insecticide BTG 502 on the cockroach sodium channel.

    Science.gov (United States)

    Du, Yuzhe; Khambay, Bhupinder; Dong, Ke

    2011-07-01

    Deltamethrin, a pyrethroid insecticide, and BTG 502, an alkylamide insecticide, target voltage-gated sodium channels. Deltamethrin binds to a unique receptor site and causes prolonged opening of sodium channels by inhibiting deactivation and inactivation. Previous (22)Na(+) influx and receptor binding assays using mouse brain synaptoneurosomes showed that BTG 502 antagonized the binding and action of batrachotoxin (BTX), a site 2 sodium channel neurotoxin. However, the effect of BTG 502 has not been examined directly on sodium channels expressed in Xenopus oocytes. In this study, we examined the effect of BTG 502 on wild-type and mutant cockroach sodium channels expressed in Xenopus oocytes. Toxin competition experiments confirmed that BTG 502 antagonizes the action of BTX and possibly shares a common receptor site with BTX. However, unlike BTX which causes persistent activation of sodium channels, BTG 502 reduces the amplitude of peak sodium current. A previous study showed that BTG 502 was more toxic to pyrethroid-resistant house flies possessing a super-kdr (knockdown resistance) mechanism than to pyrethroid-susceptible house flies. However, we found that the cockroach sodium channels carrying the equivalent super-kdr mutations (M918T and L1014F) were not more sensitive to BTG 502 than the wild-type channel. Instead, a kdr mutation, F1519I, which reduces pyrethroid binding, abolished the action of BTG 502. These results provide evidence the actions of alkylamide and pyrethroid insecticides require a common sodium channel residue. Copyright © 2011 Elsevier Ltd. All rights reserved.

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

    Science.gov (United States)

    Wartenberg, Hans C; Urban, Bernd W

    2004-01-01

    To investigate the response to general anesthetics of different sodium-channel subtypes, we examined the effects of pentobarbital, a close thiopental analogue, on single sodium channels from human skeletal muscle and compared them to existing data from human brain and human ventricular muscle channels. Sodium channels from a preparation of human skeletal muscle were incorporated into planar lipid bilayers, and the steady-state behavior of single sodium channels and their response to pentobarbital was examined in the presence of batrachotoxin, a sodium-channel activator. Single-channel currents were recorded before and after the addition of pentobarbital (0.34-1.34 mM). In symmetrical 500 mM NaCl, human skeletal muscle sodium channels had an averaged single-channel conductance of 21.0 +/- 0.6 pS, and the channel fractional open time was 0.96 +/- 0.04. The activation midpoint potential was -96.2 +/- 1.6 mV. Extracellular tetrodotoxin blocked the channel with a half-maximal concentration (k1/2) of 60 nM at 0 mV. Pentobarbital reduced the time-averaged conductance of single skeletal muscle sodium channels in a concentration-dependent manner (inhibitory concentration 50% [IC50] = 0.66 mM). The steady-state activation was shifted to more hyperpolarized potentials (-16.7 mV at 0.67 mM pentobarbital). In the planar lipid bilayer system, skeletal muscle sodium channels have some electrophysiological properties that are significantly different compared with those of sodium channels from cardiac or from central nervous tissue. In contrast to the control data, these different human sodium channel subtypes showed the same qualitative and quantitative response to the general anesthetic pentobarbital. The implication of these effects for overall anesthesia will depend on the role the individual channels play within their neuronal networks, but suppression of both central nervous system and peripheral sodium channels may add to general anesthetic effects.

  18. Cation gating and selectivity in a purified, reconstituted, voltage-dependent sodium channel

    International Nuclear Information System (INIS)

    Barchi, R.L.; Tanaka, J.C.

    1984-01-01

    In excitable membranes, the voltage-dependent sodium channel controls the primary membrane conductance change necessary for the generation of an action potential. Over the past four decades, the time- and voltage-dependent sodium currents gated by this channel have been thoroughly documented with increasingly sophisticated voltage-clamp techniques. Recent advances in the biochemistry of membrane proteins have led to the solubilization and purification of this channel protein from nerve (6) and from muscle (4) or muscle-derived (1) membranes, and have provided an approach to the correlation of the channel's molecular structure with its functional properties. Each of these sodium channel preparations appears to contain a large glycoprotein either as its sole component (2) or in association with several small subunits (6, 3). Evidence that these purified proteins represent the excitable membrane sodium channel is presented. 8 refs., 1 fig., 1 tab

  19. The Receptor Site and Mechanism of Action of Sodium Channel Blocker Insecticides.

    Science.gov (United States)

    Zhang, Yongqiang; Du, Yuzhe; Jiang, Dingxin; Behnke, Caitlyn; Nomura, Yoshiko; Zhorov, Boris S; Dong, Ke

    2016-09-16

    Sodium channels are excellent targets of both natural and synthetic insecticides with high insect selectivity. Indoxacarb, its active metabolite DCJW, and metaflumizone (MFZ) belong to a relatively new class of sodium channel blocker insecticides (SCBIs) with a mode of action distinct from all other sodium channel-targeting insecticides, including pyrethroids. Electroneutral SCBIs preferably bind to and trap sodium channels in the inactivated state, a mechanism similar to that of cationic local anesthetics. Previous studies identified several SCBI-sensing residues that face the inner pore of sodium channels. However, the receptor site of SCBIs, their atomic mechanisms, and the cause of selective toxicity of MFZ remain elusive. Here, we have built a homology model of the open-state cockroach sodium channel BgNav1-1a. Our computations predicted that SCBIs bind in the inner pore, interact with a sodium ion at the focus of P1 helices, and extend their aromatic moiety into the III/IV domain interface (fenestration). Using model-driven mutagenesis and electrophysiology, we identified five new SCBI-sensing residues, including insect-specific residues. Our study proposes the first three-dimensional models of channel-bound SCBIs, sheds light on the molecular basis of MFZ selective toxicity, and suggests that a sodium ion located in the inner pore contributes to the receptor site for electroneutral SCBIs. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

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

    Science.gov (United States)

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

    2007-01-01

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

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

    Science.gov (United States)

    Tikhonov, D B

    2008-06-01

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

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

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    Olivier eTheriault

    2014-09-01

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

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

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

    2015-02-01

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

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

    NARCIS (Netherlands)

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

    1999-01-01

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

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

    Science.gov (United States)

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

    2018-02-15

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

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

    Energy Technology Data Exchange (ETDEWEB)

    Ottea, J.A.; Payne, G.T.; Soderlund, D.M. (Cornell Univ., Geneva, NY (USA))

    1990-08-01

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

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

    Science.gov (United States)

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

    2011-01-01

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

  8. Voltage-dependent activation in purified reconstituted sodium channels from rabbit T-tubular membranes

    Energy Technology Data Exchange (ETDEWEB)

    Furman, R.E.; Tanaka, J.C.; Mueller, P.; Barchi, R.L.

    1986-01-01

    The authors have examined the voltage-dependent gating of batrachotoxin-modified sodium channels purified from rabbit T-tubular membranes in two ways. First, purified channels were reconstituted into planar bilayers and single-channel properties were measured. Batrachotoxin-activated channels showed steep voltage-dependent activation with half-maximal opening probabilities at potentials between -95 and -116 mV. The single-channel conductance averaged 20 pS and was independent of membrane potential. A second approach was used to establish that this voltage dependence was a characteristic of the entire population of purified channels and not just those few channels observed in planar bilayers. Channels reconstituted into egg phosphophatidyl-choline vesicles were functionally oriented by inclusion of internal saxitoxin; vesicle membrane potentials were then generated by K/sup +/ gradients in the presence of valinomycin. All of the specific /sup 22/Na/sup +/ influx activated by batrachotoxin and blocked by saxitoxin was found to be voltage sensitive, activating between predicted membrane potentials of -100 and -50 mV. The single-channel properties of the purified T-tubular sodium channel correspond closely to those seen with native sodium channels from rat sarcolemma. The voltage-dependent activation of the bactrachotoxin-modified reconstituted channel is the same as that seen with native channels in situ or in bilayers after exposure this toxin.

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

    Science.gov (United States)

    Du, Yuzhe; Nomura, Yoshiko; Luo, Ningguang; Liu, Zhiqi; Lee, Jung-Eun; Khambay, Bhupinder; Dong, Ke

    2011-01-01

    Pyrethroid insecticides are classified as type I or type II based on their distinct symptomology and effects on sodium channel gating. Structurally, type II pyrethroids possess an α-cyano group at the phenylbenzyl alcohol position, which is lacking in type I pyrethroids. Both type I and type II pyrethroids inhibit deactivation consequently prolonging the opening of sodium channels. However, type II pyrethroids inhibit the deactivation of sodium channels to a greater extent than type I pyrethroids inducing much slower decaying of tail currents upon repolarization. The molecular basis of type II-specific action, however, is not known. Here we report the identification of a residue G1111 and two positively charged lysines immediately downstream of G1111 in the intracellular linker connecting domains II and III of the cockroach sodium channel that are specifically involved in the action of type II pyrethroids, but not in the action of type I pyrethroids. Deletion of G1111, a consequence of alternative splicing, reduced the sodium channel sensitivity to type II pyrethroids, but had no effect on channel sensitivity to type I pyrethroids. Interestingly, charge neutralization or charge reversal of two positively charged lysines (Ks) downstream of G1111 had a similar effect. These results provide the molecular insight into the type II-specific interaction of pyrethroids with the sodium channel at the molecular level. PMID:19022275

  10. Inhibition of Sodium Ion Channel Function with Truncated Forms of Batrachotoxin.

    Science.gov (United States)

    Toma, Tatsuya; Logan, Matthew M; Menard, Frederic; Devlin, A Sloan; Du Bois, J

    2016-10-19

    A novel family of small molecule inhibitors of voltage-gated sodium channels (Na V s) based on the structure of batrachotoxin (BTX), a well-known channel agonist, is described. Protein mutagenesis and electrophysiology experiments reveal the binding site as the inner pore region of the channel, analogous to BTX, alkaloid toxins, and local anesthetics. Homology modeling of the eukaryotic channel based on recent crystallographic analyses of bacterial Na V s suggests a mechanism of action for ion conduction block.

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

    Science.gov (United States)

    1985-10-14

    by veratridine, batrachotoxin , and other neurc.oxins acting at receptor site 2 on the sodium channel through an allosteric mechanism (9). Since GPT...activation of sodium channels by neurotoxins acting at neurotoxin receptor site 2 such as veratridine and batrachotoxin . The value of KO.5 for activation by...of 70 nM, and is completely blocked by appropriate concentrations of unlabeled batrachotoxin , veratridine, and aconitine (19). Our ion flux experiments

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

    NARCIS (Netherlands)

    Qiao, X.

    2013-01-01

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

  13. Effect of gamma radiation on sodium channels in different conformations in neuroblastoma cells

    Energy Technology Data Exchange (ETDEWEB)

    Freschi, J.E.; Moran

    1986-01-01

    The dose-response relationship between gamma radiation and batrachotoxin-stimulated sodium influx in neuroblastoma cells in tissue culture was studied. Also tested was the hypothesis that changes in sodium-channel conformation may alter the radiosensitivity of the channel. It was found that gamma radiation inhibited toxin-stimulated /sup 22/Na uptake at doses beyond a threshold of 200-300 Gy. No effects were seen following doses below 100 Gy. This inhibition of sodium permeability was seen when the cells were irradiated with sodium channels in the closed or inactivated, nonconducting states. However, when the channels were in the toxin-opened, conducting state, gamma radiation had no effect at doses up to 2000 Gy. Results support earlier electrophysiological studies that showed that high doses of ionizing radiation are required to produce a measureable decrease in sodium permeability. In addition, the data suggest that by changing the sodium-channel conformation, batrachotoxin appears to alter radiosensitive chemical bonds in the gating or ion-conducting portion of the channel.

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

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    Li Jia

    2011-11-01

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

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

    Directory of Open Access Journals (Sweden)

    Yiangou Yiangos

    2010-06-01

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

  16. Sodium ion channel mutations in glioblastoma patients correlate with shorter survival

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    Velculescu Victor E

    2011-02-01

    Full Text Available Abstract Background Glioblastoma Multiforme (GBM is the most common and invasive astrocytic tumor associated with dismal prognosis. Treatment for GBM patients has advanced, but the median survival remains a meager 15 months. In a recent study, 20,000 genes from 21 GBM patients were sequenced that identified frequent mutations in ion channel genes. The goal of this study was to determine whether ion channel mutations have a role in disease progression and whether molecular targeting of ion channels is a promising therapeutic strategy for GBM patients. Therefore, we compared GBM patient survival on the basis of presence or absence of mutations in calcium, potassium and sodium ion transport genes. Cardiac glycosides, known sodium channel inhibitors, were then tested for their ability to inhibit GBM cell proliferation. Results Nearly 90% of patients showed at least one mutation in ion transport genes. GBM patients with mutations in sodium channels showed a significantly shorter survival compared to patients with no sodium channel mutations, whereas a similar comparison based on mutational status of calcium or potassium ion channel mutations showed no survival differences. Experimentally, targeting GBM cells with cardiac glycosides such as digoxin and ouabain demonstrated preferential cytotoxicity against U-87 and D54 GBM cells compared to non-tumor astrocytes (NTAs. Conclusions These pilot studies of GBM patients with sodium channel mutations indicate an association with a more aggressive disease and significantly shorter survival. Moreover, inhibition of GBM cells by ion channel inhibitors such as cardiac glycosides suggest a therapeutic strategy with relatively safe drugs for targeting GBM ion channel mutations. Key Words: glioblastoma multiforme, ion channels, mutations, small molecule inhibitors, cardiac glycosides.

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

    Energy Technology Data Exchange (ETDEWEB)

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

    1979-01-01

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

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

    Science.gov (United States)

    Bao, Lan

    2015-09-30

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

  19. Alternative splicing of the cardiac sodium channel creates multiple variants of mutant T1620K channels.

    Directory of Open Access Journals (Sweden)

    Stefan Walzik

    2011-04-01

    Full Text Available Alternative splicing creates several Na(v1.5 transcripts in the mammalian myocardium and in various other tissues including brain, dorsal root ganglia, breast cancer cells as well as neuronal stem cell lines. In total nine Na(v1.5 splice variants have been discovered. Four of them, namely Na(v1.5a, Na(v1.5c, Na(v1.5d, and Na(v1.5e, generate functional channels in heterologous expression systems. The significance of alternatively spliced transcripts for cardiac excitation, in particular their role in SCN5A channelopathies, is less well understood. In the present study, we systematically investigated electrophysiological properties of mutant T1620K channels in the background of all known functional Na(v1.5 splice variants in HEK293 cells. This mutation has been previously associated with two distinct cardiac excitation disorders: with long QT syndrome type 3 (LQT3 and isolated cardiac conduction disease (CCD. When investigating the effect of the T1620K mutation, we noticed similar channel defects in the background of hNa(v1.5, hNa(v1.5a, and hNa(v1.5c. In contrast, the hNa(v1.5d background produced differential effects: In the mutant channel, some gain-of-function features did not emerge, whereas loss-of-function became more pronounced. In case of hNa(v1.5e, the neonatal variant of hNa(v1.5, both the splice variant itself as well as the corresponding mutant channel showed electrophysiological properties that were distinct from the wild-type and mutant reference channels, hNa(v1.5 and T1620K, respectively. In conclusion, our data show that alternative splicing is a mechanism capable of generating a variety of functionally distinct wild-type and mutant hNa(v1.5 channels. Thus, the cellular splicing machinery is a potential player affecting genotype-phenotype correlations in SCN5A channelopathies.

  20. State-dependent access to the batrachotoxin receptor on the sodium channel.

    Science.gov (United States)

    De Leon, Lyn; Ragsdale, David S

    2003-07-18

    Batrachotoxin causes sustained opening of voltage-gated sodium channels. Toxin binds irreversibly to wild type channels; however, it dissociates rapidly from channels with mutation F1710C in transmembrane segment IVS6. This dissociation requires channel activation, suggesting that the activation gate guards the toxin-binding site. Here we show that activity-dependent toxin dissociation was not affected by external sodium, arguing against a binding site within the pore, and demonstrate that dissociation occurred only during the first few milliseconds after membrane depolarization, as if the toxin leaves its binding site during closed states that precede the final open state in the activation pathway. Toxin interaction with preopen states may facilitate subsequent channel opening, thus accounting for the batrachotoxin-induced negative shift in channel activation.

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

    Science.gov (United States)

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

    2017-11-02

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

  2. The voltage-dependent action of pentobarbital on batrachotoxin-modified human brain sodium channels.

    Science.gov (United States)

    Rehberg, B; Duch, D S; Urban, B W

    1994-09-14

    The voltage-dependent action of the intravenous anesthetic pentobarbital on human brain sodium channels activated by batrachotoxin was examined using planar lipid bilayer methods. Fractional open time-data were fitted by Boltzmann functions to yield simple parameters characterizing the voltage-dependence of the fractional open time. Pentobarbital caused a dose-dependent reduction of the maximum fractional open time of the sodium channel and a shift of the potential of half-maximal open time towards hyperpolarized potentials, whereas the slope parameter of the Boltzmann-fits was unaffected. A statistically significant increase of the variability of these parameters was found only in the case of the maximum fractional open time, indicating a random fluctuation of pentobarbital-induced suppression of the sodium channels over time. The voltage-dependent action of pentobarbital probably results from either a pentobarbital-modification of channel activation gating and/or a modification of the pentobarbital action by the gating process itself.

  3. Identification of new batrachotoxin-sensing residues in segment IIIS6 of the sodium channel.

    Science.gov (United States)

    Du, Yuzhe; Garden, Daniel P; Wang, Lingxin; Zhorov, Boris S; Dong, Ke

    2011-04-15

    Ion permeation through voltage-gated sodium channels is modulated by various drugs and toxins. The atomistic mechanisms of action of many toxins are poorly understood. A steroidal alkaloid batrachotoxin (BTX) causes persistent channel activation by inhibiting inactivation and shifting the voltage dependence of activation to more negative potentials. Traditionally, BTX is considered to bind at the channel-lipid interface and allosterically modulate the ion permeation. However, amino acid residues critical for BTX action are found in the inner helices of all four repeats, suggesting that BTX binds in the pore. In the octapeptide segment IFGSFFTL in IIIS6 of a cockroach sodium channel BgNa(V), besides Ser_3i15 and Leu_3i19, which correspond to known BTX-sensing residues of mammalian sodium channels, we found that Gly_3i14 and Phe_3i16 are critical for BTX action. Using these data along with published data as distance constraints, we docked BTX in the Kv1.2-based homology model of the open BgNa(V) channel. We arrived at a model in which BTX adopts a horseshoe conformation with the horseshoe plane normal to the pore axis. The BTX ammonium group is engaged in cation-π interactions with Phe_3i16 and BTX moieties interact with known BTX-sensing residues in all four repeats. Oxygen atoms at the horseshoe inner surface constitute a transient binding site for permeating cations, whereas the bulky BTX molecule would resist the pore closure, thus causing persistent channel activation. Our study reinforces the concept that steroidal sodium channel agonists bind in the inner pore of sodium channels and elaborates the atomistic mechanism of BTX action.

  4. Chronic ciguatoxin treatment induces synaptic scaling through voltage gated sodium channels in cortical neurons.

    Science.gov (United States)

    Martín, Víctor; Vale, Carmen; Rubiolo, Juan A; Roel, Maria; Hirama, Masahiro; Yamashita, Shuji; Vieytes, Mercedes R; Botana, Luís M

    2015-06-15

    Ciguatoxins are sodium channels activators that cause ciguatera, one of the most widespread nonbacterial forms of food poisoning, which presents with long-term neurological alterations. In central neurons, chronic perturbations in activity induce homeostatic synaptic mechanisms that adjust the strength of excitatory synapses and modulate glutamate receptor expression in order to stabilize the overall activity. Immediate early genes, such as Arc and Egr1, are induced in response to activity changes and underlie the trafficking of glutamate receptors during neuronal homeostasis. To better understand the long lasting neurological consequences of ciguatera, it is important to establish the role that chronic changes in activity produced by ciguatoxins represent to central neurons. Here, the effect of a 30 min exposure of 10-13 days in vitro (DIV) cortical neurons to the synthetic ciguatoxin CTX 3C on Arc and Egr1 expression was evaluated using real-time polymerase chain reaction approaches. Since the toxin increased the mRNA levels of both Arc and Egr1, the effect of CTX 3C in NaV channels, membrane potential, firing activity, miniature excitatory postsynaptic currents (mEPSCs), and glutamate receptors expression in cortical neurons after a 24 h exposure was evaluated using electrophysiological and western blot approaches. The data presented here show that CTX 3C induced an upregulation of Arc and Egr1 that was prevented by previous coincubation of the neurons with the NaV channel blocker tetrodotoxin. In addition, chronic CTX 3C caused a concentration-dependent shift in the activation voltage of NaV channels to more negative potentials and produced membrane potential depolarization. Moreover, 24 h treatment of cortical neurons with 5 nM CTX 3C decreased neuronal firing and induced synaptic scaling mechanisms, as evidenced by a decrease in the amplitude of mEPSCs and downregulation in the protein level of glutamate receptors that was also prevented by tetrodotoxin

  5. Mouse brain synaptosomal sodium channels: activation by aconitine, batrachotoxin, and veratridine, and inhibition by tetrodotoxin.

    Science.gov (United States)

    Ghiasuddin, S M; Soderlund, D M

    1984-01-01

    Batrachotoxin, veratridine and aconitine, activators of the voltage-dependent sodium channel in excitable cell membranes, increase the rate of 22Na+ uptake by mouse brain synaptosomes. Batrachotoxin was both the most potent (K0.5, 0.49 microM) and most effective activator of specific 22Na+ uptake. Veratridine (K0.5, 34.5 microM) and aconitine (K0.5, 19.6 microM) produced maximal stimulations of 22Na+ uptake that were 73% and 46%, respectively, of that produced by batrachotoxin. Activation of 22Na+ uptake by veratridine was completely inhibited by tetrodotoxin (I50, 6 nM ), a specific blocker of nerve membrane sodium channels. These results identify appropriate conditions for measuring sodium channel-dependent 22Na+ flux in mouse brain synaptosomes. The pharmacological properties of mouse brain synaptosomal sodium channels described here are distinct from those previously described for sodium channels in rat brain synaptosomes and mouse neuroblastoma cells.

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

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    Michael B Butterworth

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

  7. Characterization of two Bunodosoma granulifera toxins active on cardiac sodium channels

    Science.gov (United States)

    Goudet, Cyril; Ferrer, Tania; Galàn, Loipa; Artiles, Adriana; Batista, Cesar F V; Possani, Lourival D; Alvarez, Julio; Aneiros, Abel; Tytgat, Jan

    2001-01-01

    Two sodium channel toxins, BgII and BgIII, have been isolated and purified from the sea anemone Bunodosoma granulifera. Combining different techniques, we have investigated the electrophysiological properties of these toxins. We examined the effect of BgII and BgIII on rat ventricular strips. These toxins prolong action potentials with EC50 values of 60 and 660 nM and modify the resting potentials. The effect on Na+ currents in rat cardiomyocytes was studied using the patch-clamp technique. BgII and BgIII slow the rapid inactivation process and increase the current density with EC50 values of 58 and 78 nM, respectively. On the cloned hH1 cardiac Na+ channel expressed in Xenopus laevis oocytes, BgII and BgIII slow the inactivation process of Na+ currents (respective EC50 values of 0.38 and 7.8 μM), shift the steady-state activation and inactivation parameters to more positive potentials and the reversal potential to more negative potentials. The amino acid sequences of these toxins are almost identical except for an asparagine at position 16 in BgII which is replaced by an aspartic acid in BgIII. In all experiments, BgII was more potent than BgIII suggesting that this conservative residue is important for the toxicity of sea anemone toxins. We conclude that BgII and BgIII, generally known as neurotoxins, are also cardiotoxic and combine the classical effects of sea anemone Na+ channels toxins (slowing of inactivation kinetics, shift of steady-state activation and inactivation parameters) with a striking decrease on the ionic selectivity of Na+ channels. PMID:11704639

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

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    Siguang Xu

    2016-01-01

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

  9. Molecular cloning and analysis of zebrafish voltage-gated sodium channel beta subunit genes: implications for the evolution of electrical signaling in vertebrates

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    Zhong Tao P

    2007-07-01

    Full Text Available Abstract Background Action potential generation in excitable cells such as myocytes and neurons critically depends on voltage-gated sodium channels. In mammals, sodium channels exist as macromolecular complexes that include a pore-forming alpha subunit and 1 or more modulatory beta subunits. Although alpha subunit genes have been cloned from diverse metazoans including flies, jellyfish, and humans, beta subunits have not previously been identified in any non-mammalian species. To gain further insight into the evolution of electrical signaling in vertebrates, we investigated beta subunit genes in the teleost Danio rerio (zebrafish. Results We identified and cloned single zebrafish gene homologs for beta1-beta3 (zbeta1-zbeta3 and duplicate genes for beta4 (zbeta4.1, zbeta4.2. Sodium channel beta subunit loci are similarly organized in fish and mammalian genomes. Unlike their mammalian counterparts, zbeta1 and zbeta2 subunit genes display extensive alternative splicing. Zebrafish beta subunit genes and their splice variants are differentially-expressed in excitable tissues, indicating tissue-specific regulation of zbeta1-4 expression and splicing. Co-expression of the genes encoding zbeta1 and the zebrafish sodium channel alpha subunit Nav1.5 in Chinese Hamster Ovary cells increased sodium current and altered channel gating, demonstrating functional interactions between zebrafish alpha and beta subunits. Analysis of the synteny and phylogeny of mammalian, teleost, amphibian, and avian beta subunit and related genes indicated that all extant vertebrate beta subunits are orthologous, that beta2/beta4 and beta1/beta3 share common ancestry, and that beta subunits are closely related to other proteins sharing the V-type immunoglobulin domain structure. Vertebrate sodium channel beta subunit genes were not identified in the genomes of invertebrate chordates and are unrelated to known subunits of the para sodium channel in Drosophila. Conclusion The

  10. Genetic and environmental factors in cardiac sodium channel disease

    NARCIS (Netherlands)

    Mizusawa, Y.

    2016-01-01

    Cardiac sodium channelopathies, such as long QT syndrome type3 (LQT3), Brugada syndrome (BrS) and cardiac conduction disease (CCD), are heritable diseases associated with mutations in the SCN5A gene and sudden cardiac death. They were classically thought to be a monogenic disease. However, while

  11. Hydrogen Sulfide Prevents Advanced Glycation End-Products Induced Activation of the Epithelial Sodium Channel

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    Qiushi Wang

    2015-01-01

    Full Text Available Advanced glycation end-products (AGEs are complex and heterogeneous compounds implicated in diabetes. Sodium reabsorption through the epithelial sodium channel (ENaC at the distal nephron plays an important role in diabetic hypertension. Here, we report that H2S antagonizes AGEs-induced ENaC activation in A6 cells. ENaC open probability (PO in A6 cells was significantly increased by exogenous AGEs and that this AGEs-induced ENaC activity was abolished by NaHS (a donor of H2S and TEMPOL. Incubating A6 cells with the catalase inhibitor 3-aminotriazole (3-AT mimicked the effects of AGEs on ENaC activity, but did not induce any additive effect. We found that the expression levels of catalase were significantly reduced by AGEs and both AGEs and 3-AT facilitated ROS uptake in A6 cells, which were significantly inhibited by NaHS. The specific PTEN and PI3K inhibitors, BPV(pic  and LY294002, influence ENaC activity in AGEs-pretreated A6 cells. Moreover, after removal of AGEs from AGEs-pretreated A6 cells for 72 hours, ENaC PO remained at a high level, suggesting that an AGEs-related “metabolic memory” may be involved in sodium homeostasis. Our data, for the first time, show that H2S prevents AGEs-induced ENaC activation by targeting the ROS/PI3K/PTEN pathway.

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

    Energy Technology Data Exchange (ETDEWEB)

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

    1990-08-01

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

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

    International Nuclear Information System (INIS)

    Dargent, B.; Couraud, F.

    1990-01-01

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

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

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    Jeffrey R. McArthur

    2012-11-01

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

  15. Antillatoxin is a marine cyanobacterial toxin that potently activates voltage-gated sodium channels.

    Science.gov (United States)

    Li, W I; Berman, F W; Okino, T; Yokokawa, F; Shioiri, T; Gerwick, W H; Murray, T F

    2001-06-19

    Antillatoxin (ATX) is a lipopeptide derived from the pantropical marine cyanobacterium Lyngbya majuscula. ATX is neurotoxic in primary cultures of rat cerebellar granule cells, and this neuronal death is prevented by either N-methyl-d-aspartate (NMDA) receptor antagonists or tetrodotoxin. To further explore the potential interaction of ATX with voltage-gated sodium channels, we assessed the influence of tetrodotoxin on ATX-induced Ca2+ influx in cerebellar granule cells. The rapid increase in intracellular Ca2+ produced by ATX (100 nM) was antagonized in a concentration-dependent manner by tetrodotoxin. Additional, more direct, evidence for an interaction with voltage-gated sodium channels was derived from the ATX-induced allosteric enhancement of [3H]batrachotoxin binding to neurotoxin site 2 of the alpha subunit of the sodium channel. ATX, moreover, produced a strong synergistic stimulation of [3H]batrachotoxin binding in combination with brevetoxin, which is a ligand for neurotoxin site 5 on the voltage-gated sodium channel. Positive allosteric interactions were not observed between ATX and either alpha-scorpion toxin or the pyrethroid deltamethrin. That ATX interaction with voltage-gated sodium channels produces a gain of function was demonstrated by the concentration-dependent and tetrodotoxin-sensitive stimulation of 22Na+ influx in cerebellar granule cells exposed to ATX. Together these results demonstrate that the lipopeptide ATX is an activator of voltage-gated sodium channels. The neurotoxic actions of ATX therefore resemble those of brevetoxins that produce neural insult through depolarization-evoked Na+ load, glutamate release, relief of Mg2+ block of NMDA receptors, and Ca2+ influx.

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

    Energy Technology Data Exchange (ETDEWEB)

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

    2013-10-15

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

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

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    Huiqiong He

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

  18. MTSET modification of D4S6 cysteines stabilize the fast inactivated state of Nav1.5 sodium channels

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    Michael E O'leary

    2015-06-01

    Full Text Available The transmembrane S6 segments of Na+ sodium channels form the cytoplasmic entrance of the channel and line the internal aspects of the aqueous pore. This region of the channel has been implicated in Na+ channel permeation, gating and pharmacology. In this study we utilized cysteine substitutions and methanethiosulfonate reagent (MTSET to investigate the role of the S6 segment of homologous domain 4 (D4S6 in the gating of the cardiac (Nav1.5 channel. D4S6 cysteine mutants were heterologously expressed in tsA201 cells and currents recorded using whole-cell patch clamp. Internal MTSET reduced the peak Na+ currents, induced hyperpolarizing shifts in steady-state inactivation and slowed the recovery of mutant channels with cysteines inserted near the middle (F1760C, V1763C and C-terminus (Y1767C of the D4S6. These findings suggested a link between the MTSET inhibition and fast inactivation. This was confirmed by expressing the V1763C and Y1767C mutations in non-inactivating Nav1.5 channels. Removing inactivation abolished the MTSET inhibition of the V1763C and Y1767C mutants. The data indicate that the MTSET-induced reduction in current primarily results from slower recovery from inactivation that produces hyperpolarizing shifts in fast inactivation and decreases the steady-state availability of the channels. This contrasted with a cysteine inserted near the C-terminus of the D4S6 (I1770C where MTSET increased the persistent Na+ current at depolarized voltages consistent with impaired fast inactivation. Covalent modification of D4S6 cysteines with MTSET adduct appears to reduce the mobility of the D4S6 segment and stabilize the channels in the fast inactivated state. These findings indicate that residues located near the middle and C-terminus of the D4S6 play an important role in fast inactivation.

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

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    Debora eBaroni

    2015-05-01

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

  20. No evidence for specific opioid effects on batrachotoxin-modified sodium channels from human brain synaptosomes.

    Science.gov (United States)

    Frenkel, C; Gerhard, A; Wartenberg, H C; Rehberg, B; Urban, B W

    1997-06-20

    Human central nervous system (CNS) sodium channels modified by batrachotoxin and incorporated inter voltage-clamped lipid bilayers, were exposed to various concentrations of the opioid alfentanil (0.2-8.0 mM). Alfentanil caused a concentration-dependent and membrane potential independent reduction of the single channel amplitude and the fractional channel open-time. The weighted computer fit of the dose-response curve yielded a maximal conductance block of 50% with an EC50 of 1.3 mM. These effects occurred at levels beyond clinically relevant human serum/brain levels (0.003 mM) but within the predicted concentration range using the Meyer-Overton (lipid solubility/anaesthetic potency) correlation. Thus, human CNS sodium channels are probably not a main target site for the clinical effects of alfentanil but they provide a model system to estimate the proportion of the lipophilic interactions contributing to its overall effect.

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

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    Kunichika Tsumoto

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

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

    Energy Technology Data Exchange (ETDEWEB)

    Gaudioso, Christelle; Carlier, Edmond; Youssouf, Fahamoe [INSERM U641, Institut Jean Roche, Marseille F-13344 (France); Universite de la Mediterranee, Faculte de Medecine Secteur Nord, IFR 11, Marseille F-13344 (France); Clare, Jeffrey J. [Eaton Pharma Consulting, Eaton Socon, Cambridgeshire PE19 8EF (United Kingdom); Debanne, Dominique [INSERM U641, Institut Jean Roche, Marseille F-13344 (France); Universite de la Mediterranee, Faculte de Medecine Secteur Nord, IFR 11, Marseille F-13344 (France); Alcaraz, Gisele, E-mail: gisele.alcaraz@univmed.fr [INSERM U641, Institut Jean Roche, Marseille F-13344 (France); Universite de la Mediterranee, Faculte de Medecine Secteur Nord, IFR 11, Marseille F-13344 (France)

    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 channel 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.

  3. Inhibition of cardiac voltage-gated sodium channels by grape polyphenols.

    Science.gov (United States)

    Wallace, C H R; Baczkó, I; Jones, L; Fercho, M; Light, P E

    2006-11-01

    The cardiovascular benefits of red wine consumption are often attributed to the antioxidant effects of its polyphenolic constituents, including quercetin, catechin and resveratrol. Inhibition of cardiac voltage-gated sodium channels (VGSCs) is antiarrhythmic and cardioprotective. As polyphenols may also modulate ion channels, and possess structural similarities to several antiarrhythmic VGSC inhibitors, we hypothesised that VGSC inhibition may contribute to cardioprotection by these polyphenols. The whole-cell voltage-clamp technique was used to record peak and late VGSC currents (INa) from recombinant human heart NaV1.5 channels expressed in tsA201 cells. Right ventricular myocytes from rat heart were isolated and single myocytes were field-stimulated. Either calcium transients or contractility were measured using the calcium-sensitive dye Calcium-Green 1AM or video edge detection, respectively. The red grape polyphenols quercetin, catechin and resveratrol blocked peak INa with IC50s of 19.4 microM, 76.8 microM and 77.3 microM, respectively. In contrast to lidocaine, resveratrol did not exhibit any frequency-dependence of peak INa block. Late INa induced by the VGSC long QT mutant R1623Q was reduced by resveratrol and quercetin. Resveratrol and quercetin also blocked late INa induced by the toxin, ATX II, with IC50s of 26.1 microM and 24.9 microM, respectively. In field-stimulated myocytes, ATXII-induced increases in diastolic calcium were prevented and reversed by resveratrol. ATXII-induced contractile dysfunction was delayed and reduced by resveratrol. Our results indicate that several red grape polyphenols inhibit cardiac VGSCs and that this effect may contribute to the documented cardioprotective efficacy of red grape products.

  4. High Salt Intake Down-Regulates Colonic Mineralocorticoid Receptors, Epithelial Sodium Channels and 11β-Hydroxysteroid Dehydrogenase Type 2

    Science.gov (United States)

    Lienhard, Daniel; Lauterburg, Meret; Escher, Geneviève; Frey, Felix J.; Frey, Brigitte M.

    2012-01-01

    Besides the kidneys, the gastrointestinal tract is the principal organ responsible for sodium homeostasis. For sodium transport across the cell membranes the epithelial sodium channel (ENaC) is of pivotal relevance. The ENaC is mainly regulated by mineralocorticoid receptor mediated actions. The MR activation by endogenous 11β-hydroxy-glucocorticoids is modulated by the 11β-hydroxysteroid dehydrogenase type 2 (11β-HSD2). Here we present evidence for intestinal segment specific 11β-HSD2 expression and hypothesize that a high salt intake and/or uninephrectomy (UNX) affects colonic 11β-HSD2, MR and ENaC expression. The 11β-HSD2 activity was measured by means of 3H-corticosterone conversion into 3H-11-dehydrocorticosterone in Sprague Dawley rats on a normal and high salt diet. The activity increased steadily from the ileum to the distal colon by a factor of about 3, an observation in line with the relevance of the distal colon for sodium handling. High salt intake diminished mRNA and protein of 11β-HSD2 by about 50% (phigh salt intake (psalt overload, a mechanism not modulated by UNX. PMID:22693583

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

    Science.gov (United States)

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

    2013-09-01

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

  6. Four-mode gating model of fast inactivation of sodium channel Nav1.2a.

    Science.gov (United States)

    Huth, Tobias; Schmidtmayer, Johann; Alzheimer, Christian; Hansen, Ulf-Peter

    2008-10-01

    Basic principles of the gating mechanisms of neuronal sodium channels, especially the fast inactivation process, were revealed by a quantitative analysis of the effects of the chemically irreversible modifying agent chloramine T. The compound is known to enhance the open probability of sodium channels by interfering with the inactivation process. The key for the deduction of structure-function relationships was obtained from the analysis of single-channel patch-clamp data, especially the finding that chloramine T-induced modification of inactivation occurred in four steps. These steps were termed modes 1-4 (four-mode gating model), and their temporal sequence was always the same. The kinetic analysis of single-channel traces with an improved two-dimensional dwell-time fit revealed the possible mechanism related to each mode. Similarities to the kinetics of the sodium channel mutant F1489Q led to the assignment of modes 1 and 2 to transient defects in the locking of the inactivation particle (hinged lid). In the third mode, the hinged lid was unable to lock permanently. Finally, in mode 4, the apparent single-channel current was reduced, which could be explained by fast gating, presumably related to the selectivity filter.

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

    Science.gov (United States)

    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.

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

    OpenAIRE

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

    2016-01-01

    Resurgent sodium currents arise from channel reopening during repolarisation, and are predicted to increase neuronal excitability. Patel et al. show that epilepsy-associated mutations in the voltage-gated sodium channel Nav1.6, but not Nav1.1, upregulate resurgent currents. Cannabidiol preferentially targets these currents, suggesting a strategy for reducing neuronal hyperexcitability associated with epilepsy.

  9. Epoxyeicosatrienoic acid analogue lowers blood pressure through vasodilation and sodium channel inhibition

    Czech Academy of Sciences Publication Activity Database

    Khan, M. A. H.; Pavlov, T. S.; Christain, S. V.; Neckář, Jan; Staruschenko, A.; Gauthier, K. M.; Capdevila, J. H.; Falck, J. R.; Campbell, W. B.; Imig, J. D.

    2014-01-01

    Roč. 127, č. 7 (2014), s. 463-474 ISSN 0143-5221 Institutional support: RVO:67985823 Keywords : angiotensin II * epithelial sodium channel (ENaC) * epoxyeicosatrienoic acid analogue * hypertension Subject RIV: FA - Cardiovascular Diseases incl. Cardiotharic Surgery Impact factor: 5.598, year: 2014

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

    NARCIS (Netherlands)

    Chockalingam, Priya; Wilde, Arthur A. M.

    2012-01-01

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

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

    DEFF Research Database (Denmark)

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

    2011-01-01

    Cardiac sodium channels are established therapeutic targets for the management of inherited and acquired arrhythmias by class I anti-arrhythmic drugs (AADs). These drugs share a common target receptor bearing two highly conserved aromatic side chains, and are subdivided by the Vaughan-Williams...

  12. Ionizing Radiation Alters the Properties of Sodium Channels in Rat Brain Synaptosomes

    Science.gov (United States)

    1986-01-01

    in the order of membrane lipids. Key greatest. Batrachotoxin -stimulated 22Na’ uptake was Words: Ionizing radiation-Sodium channels-Mem- less sensitive...action potential, Received October 18, 1985; accepted February 14, 1986. Abbreviations used: BTX, batrachotoxin DPH, 1.6-diphenyl- Address correspondence

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

    Science.gov (United States)

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

    2010-11-01

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

  14. Acute stages of batrachotoxin-induced neuropathy: a morphologic study of a sodium-channel toxin.

    Science.gov (United States)

    Moore, G R; Boegman, R J; Robertson, D M; Raine, C S

    1986-10-01

    The acute effects of batrachotoxin, a steroidal neurotoxin which opens the membrane sodium channel, were observed morphologically at various time points up to 3 h after injection into rat peroneal nerve. Three changes were found. First, there was massive swelling of the axon at the node of Ranvier accompanied by retraction of paranodal myelin. Second, a similar swelling of unmyelinated axons was seen. Third, extracellular fluid accumulated along the internode in the adaxonal space, the intraperiod line of myelin and, rarely, the external mesaxon, with concomitant shrinkage of the axon. The first two changes might be explained on the basis of massive shift of sodium through the batrachotoxin-modified sodium channel into the axon and subsequent osmotic shift of fluid. The reason for the third change is not clear but probably also has a ionic basis.

  15. Old Friends With New Faces: Are Sodium Channel Blockers the Future of Adjunct Pain Medication Management?

    Science.gov (United States)

    Thomas, Ashley M; Atkinson, Timothy J

    2018-01-01

    Providers are being asked to decrease the emphasis and overutilization of long-term opioid therapy, but many are left without proper guidance on appropriate utilization of nonopioid therapies. Furthermore, therapeutic options are quite limited and many providers lack confidence in distinguishing available alternatives. When first-line therapy has failed in a patient, there is an apparent lack of knowledge on how to proceed with choosing subsequent therapy. To choose among alternative agents, an understanding of pharmacology, pharmacokinetics, and efficacy in targeting various pain conditions is necessary. This article focuses on the use of the carboxamide class of sodium channel blockers (carbamazepine, oxcarbazepine, eslicarbazepine) for adjunct pain medication management including research updates in pharmacology, pharmacokinetics, and current evidence for pain along with promising areas of research. It is an evidence update for clinical use of sodium channel blockers, clarifies misconceptions regarding their use, and highlights emerging research for improved pain targets that justifies additional study. We performed a complete review of the literature using the search terms, "oxcarbazepine," "carbamazepine," and "eslicarbazepine" in conjunction with "pharmacokinetics," "adverse effects," "pharmacology," "voltage-gated sodium channel subtype," "neuropathic pain," "inflammatory pain," "metabolism," "epoxide metabolite formation," "drug interactions," "CYP450 interactions," "pain phenotype," and "chronic pain management." Databases searched included PubMed and Google Scholar. Package inserts were used for drug structure illustration, adverse reactions, and bioavailability. Pharmacology and pharmacokinetic data were taken from randomized controlled trials evaluating this area as well as in vitro published results. For validity, only peer-reviewed literature was included. Evidence for sodium channel blockers in chronic pain management was limited. This review focuses

  16. Polyunsaturated fatty acids are potent openers of human M-channels expressed in Xenopus laevis oocytes

    DEFF Research Database (Denmark)

    Liin, Sara I; Karlsson, Urban; Bentzen, Bo Hjorth

    2016-01-01

    the threshold current to evoke action potentials in dorsal root ganglion neurons. The polyunsaturated fatty acids docosahexaenoic acid, α-linolenic acid, and eicosapentaenoic acid facilitated opening of the human M-channel, comprised of the heteromeric human KV 7.2/3 channel expressed in Xenopus oocytes......AIM: Polyunsaturated fatty acids have been reported to reduce neuronal excitability, in part by promoting inactivation of voltage-gated sodium and calcium channels. Effects on neuronal potassium channels are less explored and experimental data ambiguous. The aim of this study was to investigate...... anti-excitable effects of polyunsaturated fatty acids on the neuronal M-channel, important for setting the resting membrane potential in hippocampal and dorsal root ganglion neurons. METHODS: Effects of fatty acids and fatty-acid analogues on mouse dorsal root ganglion neurons and on the human KV 7...

  17. Computational Structural Pharmacology and Toxicology of Voltage-Gated Sodium Channels.

    Science.gov (United States)

    Zhorov, B S; Tikhonov, D B

    2016-01-01

    Voltage-gated sodium channels are targets for many toxins and medically important drugs. Despite decades of intensive studies in industry and academia, atomic mechanisms of action are still not completely understood. The major cause is a lack of high-resolution structures of eukaryotic channels and their complexes with ligands. In these circumstances a useful approach is homology modeling that employs as templates X-ray structures of potassium channels and prokaryotic sodium channels. On one hand, due to inherent limitations of this approach, results should be treated with caution. In particular, models should be tested against relevant experimental data. On the other hand, docking of drugs and toxins in homology models provides a unique possibility to integrate diverse experimental data provided by mutational analysis, electrophysiology, and studies of structure-activity relations. Here we describe how homology modeling advanced our understanding of mechanisms of several classes of ligands. These include tetrodotoxins and mu-conotoxins that block the outer pore, local anesthetics that block of the inner pore, batrachotoxin that binds in the inner pore but, paradoxically, activates the channel, pyrethroid insecticides that activate the channel by binding at lipid-exposed repeat interfaces, and scorpion alpha and beta-toxins, which bind between the pore and voltage-sensing domains and modify the channel gating. We emphasize importance of experimental data for elaborating the models. Copyright © 2016 Elsevier Inc. All rights reserved.

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

    Science.gov (United States)

    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

  19. Annotation of functional impact of voltage‐gated sodium channel mutations

    Science.gov (United States)

    Hinard, Valérie; Britan, Aurore; Schaeffer, Mathieu; Zahn‐Zabal, Monique; Thomet, Urs; Rougier, Jean‐Sébastien; Bairoch, Amos; Abriel, Hugues

    2017-01-01

    Abstract Voltage‐gated sodium channels are pore‐forming transmembrane proteins that selectively allow sodium ions to flow across the plasma membrane according to the electro‐chemical gradient thus mediating the rising phase of action potentials in excitable cells and playing key roles in physiological processes such as neurotransmission, skeletal muscle contraction, heart rhythm, and pain sensation. Genetic variations in the nine human genes encoding these channels are known to cause a large range of diseases affecting the nervous and cardiac systems. Understanding the molecular effect of genetic variations is critical for elucidating the pathologic mechanisms of known variations and in predicting the effect of newly discovered ones. To this end, we have created a Web‐based tool, the Ion Channels Variants Portal, which compiles all variants characterized functionally in the human sodium channel genes. This portal describes 672 variants each associated with at least one molecular or clinical phenotypic impact, for a total of 4,658 observations extracted from 264 different research articles. These data were captured as structured annotations using standardized vocabularies and ontologies, such as the Gene Ontology and the Ion Channel ElectroPhysiology Ontology. All these data are available to the scientific community via neXtProt at https://www.nextprot.org/portals/navmut. PMID:28168870

  20. Annotation of functional impact of voltage-gated sodium channel mutations.

    Science.gov (United States)

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

    2017-05-01

    Voltage-gated sodium channels are pore-forming transmembrane proteins that selectively allow sodium ions to flow across the plasma membrane according to the electro-chemical gradient thus mediating the rising phase of action potentials in excitable cells and playing key roles in physiological processes such as neurotransmission, skeletal muscle contraction, heart rhythm, and pain sensation. Genetic variations in the nine human genes encoding these channels are known to cause a large range of diseases affecting the nervous and cardiac systems. Understanding the molecular effect of genetic variations is critical for elucidating the pathologic mechanisms of known variations and in predicting the effect of newly discovered ones. To this end, we have created a Web-based tool, the Ion Channels Variants Portal, which compiles all variants characterized functionally in the human sodium channel genes. This portal describes 672 variants each associated with at least one molecular or clinical phenotypic impact, for a total of 4,658 observations extracted from 264 different research articles. These data were captured as structured annotations using standardized vocabularies and ontologies, such as the Gene Ontology and the Ion Channel ElectroPhysiology Ontology. All these data are available to the scientific community via neXtProt at https://www.nextprot.org/portals/navmut. © 2017 The Authors. **Human Mutation published by Wiley Periodicals, Inc.

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

    Science.gov (United States)

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

    2006-12-13

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

  2. Molecular identity of dendritic voltage-gated sodium channels.

    Science.gov (United States)

    Lorincz, Andrea; Nusser, Zoltan

    2010-05-14

    Active invasion of the dendritic tree by action potentials (APs) generated in the axon is essential for associative synaptic plasticity and neuronal ensemble formation. In cortical pyramidal cells (PCs), this AP back-propagation is supported by dendritic voltage-gated Na+ (Nav) channels, whose molecular identity is unknown. Using a highly sensitive electron microscopic immunogold technique, we revealed the presence of the Nav1.6 subunit in hippocampal CA1 PC proximal and distal dendrites. Here, the subunit density is lower by a factor of 35 to 80 than that found in axon initial segments. A gradual decrease in Nav1.6 density along the proximodistal axis of the dendritic tree was also detected without any labeling in dendritic spines. Our results reveal the characteristic subcellular distribution of the Nav1.6 subunit, identifying this molecule as a key substrate enabling dendritic excitability.

  3. Batrachotoxin-modified sodium channels from squid optic nerve in planar bilayers. Ion conduction and gating properties

    Science.gov (United States)

    1989-01-01

    Squid optic nerve sodium channels were characterized in planar bilayers in the presence of batrachotoxin (BTX). The channel exhibits a conductance of 20 pS in symmetrical 200 mM NaCl and behaves as a sodium electrode. The single-channel conductance saturates with increasing the concentration of sodium and the channel conductance vs. sodium concentration relation is well described by a simple rectangular hyperbola. The apparent dissociation constant of the channel for sodium is 11 mM and the maximal conductance is 23 pS. The selectivity determined from reversal potentials obtained in mixed ionic conditions is Na+ approximately Li+ greater than K+ greater than Rb+ greater than Cs+. Calcium blocks the channel in a voltage-dependent manner. Analysis of single-channel membranes showed that the probability of being open (Po) vs. voltage relation is sigmoidal with a value of 0.5 between -90 and -100 mV. The fitting of Po requires at least two closed and one open state. The apparent gating charge required to move through the whole transmembrane voltage during the closed-open transition is four to five electronic charges per channel. Distribution of open and closed times are well described by single exponentials in most of the voltage range tested and mean open and mean closed times are voltage dependent. The number of charges associated with channel closing is 1.6 electronic charges per channel. Tetrodotoxin blocked the BTX-modified channel being the blockade favored by negative voltages. The apparent dissociation constant at zero potential is 16 nM. We concluded that sodium channels from the squid optic nerve are similar to other BTX- modified channels reconstituted in bilayers and to the BTX-modified sodium channel detected in the squid giant axon. PMID:2536797

  4. Neuronal sodium leak channel is responsible for the detection of sodium in the rat median preoptic nucleus.

    Science.gov (United States)

    Tremblay, Christina; Berret, Emmanuelle; Henry, Mélaine; Nehmé, Benjamin; Nadeau, Louis; Mouginot, Didier

    2011-02-01

    Sodium (Na(+)) ions are of primary importance for hydromineral and cardiovascular homeostasis, and the level of Na(+) in the body fluid compartments [plasma and cerebrospinal fluid (CSF)] is precisely monitored in the hypothalamus. Glial cells seem to play a critical role in the mechanism of Na(+) detection. However, the precise role of neurons in the detection of extracellular Na(+) concentration ([Na(+)](out)) remains unclear. Here we demonstrate that neurons of the median preoptic nucleus (MnPO), a structure in close contact with the CSF, are specific Na(+) sensors. Electrophysiological recordings were performed on dissociated rat MnPO neurons under isotonic [Na(+)] (100 mM NaCl) with local application of hypernatriuric (150, 180 mM NaCl) or hyponatriuric (50 mM NaCl) external solution. The hyper- and hyponatriuric conditions triggered an in- and an outward current, respectively. The reversal potential of the current matched the equilibrium potential of Na(+), indicating that a change in [Na(+)](out) modified the influx of Na(+) in the MnPO neurons. The conductance of the Na(+) current was not affected by either the membrane potential or the [Na(+)](out). Moreover, the channel was highly selective for lithium over guanidinium. Together, these data identified the channel as a Na(+) leak channel. A high correlation between the electrophysiological recordings and immunofluorescent labeling for the Na(X) channel in dissociated MnPO neurons strongly supports this channel as a candidate for the Na(+) leak channel responsible for the Na(+)-sensing ability of rat MnPO neurons. The absence of Na(X) labeling and of a specific current evoked by a change in [Na(+)](out) in mouse MnPO neurons suggests species specificity in the hypothalamus structures participating in central Na(+) detection.

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

    Directory of Open Access Journals (Sweden)

    Weiyun Huang

    2017-02-01

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

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

    Science.gov (United States)

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

    2017-01-01

    Human intoxication with the seafood poison ciguatoxin, a dinoflagellate polyether that activates voltage-gated sodium channels (NaV), causes ciguatera, a disease characterised by gastrointestinal and neurological disturbances. We assessed the activity of the most potent congener, Pacific ciguatoxin-1 (P-CTX-1), on NaV1.1–1.9 using imaging and electrophysiological approaches. Although P-CTX-1 is essentially a non-selective NaV toxin and shifted the voltage-dependence of activation to more hyperpolarising potentials at all NaV subtypes, an increase in the inactivation time constant was observed only at NaV1.8, while the slope factor of the conductance-voltage curves was significantly increased for NaV1.7 and peak current was significantly increased for NaV1.6. Accordingly, P-CTX-1-induced visceral and cutaneous pain behaviours were significantly decreased after pharmacological inhibition of NaV1.8 and the tetrodotoxin-sensitive isoforms NaV1.7 and NaV1.6, respectively. The contribution of these isoforms to excitability of peripheral C- and A-fibre sensory neurons, confirmed using murine skin and visceral single-fibre recordings, reflects the expression pattern of NaV isoforms in peripheral sensory neurons and their contribution to membrane depolarisation, action potential initiation and propagation. PMID:28225079

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

    Science.gov (United States)

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

    2016-10-25

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

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

    Directory of Open Access Journals (Sweden)

    Michael Finiguerra

    Full Text Available The marine copepod Acartia hudsonica was shown to be adapted to dinoflagellate prey, Alexandrium fundyense, which produce paralytic shellfish toxins (PST. Adaptation to PSTs in other organisms is caused by a mutation in the sodium channel. Recently, a mutation in the sodium channel in A. hudsonica was found. In this study, we rigorously tested for advantages, costs, and trade-offs associated with the mutant isoform of A. hudsonica under toxic and non-toxic conditions. We combined fitness with wild-type: mutant isoform ratio measurements on the same individual copepod to test our hypotheses. All A. hudsonica copepods express both the wild-type and mutant sodium channel isoforms, but in different proportions; some individuals express predominantly mutant (PMI or wild-type isoforms (PWI, while most individuals express relatively equal amounts of each (EI. There was no consistent pattern of improved performance as a function of toxin dose for egg production rate (EPR, ingestion rate (I, and gross growth efficiency (GGE for individuals in the PMI group relative to individuals in the PWI expression group. Neither was there any evidence to indicate a fitness benefit to the mutant isoform at intermediate toxin doses. No clear advantage under toxic conditions was associated with the mutation. Using a mixed-diet approach, there was also no observed relationship between individual wild-type: mutant isoform ratios and among expression groups, on both toxic and non-toxic diets, for eggs produced over three days. Lastly, expression of the mutant isoform did not mitigate the negative effects of the toxin. That is, the reductions in EPR from a toxic to non-toxic diet for copepods were independent of expression groups. Overall, the results did not support our hypotheses; the mutant sodium channel isoform does not appear to be related to adaptation to PST in A. hudsonica. Other potential mechanisms responsible for the adaptation are discussed.

  9. Tetrodotoxin-resistant sodium channels contribute to directional responses in starburst amacrine cells.

    Science.gov (United States)

    Oesch, Nicholas W; Taylor, W Rowland

    2010-08-27

    The biophysical mechanisms that give rise to direction selectivity in the retina remain uncertain. Current evidence suggests that the directional signal first arises within the dendrites of starburst amacrine cells (SBACs). Two models have been proposed to explain this phenomenon, one based on mutual inhibitory interactions between SBACs, and the other positing an intrinsic dendritic mechanism requiring a voltage-gradient depolarizing towards the dendritic tips. We tested these models by recording current and voltage responses to visual stimuli in SBACs. In agreement with previous work, we found that the excitatory currents in the SBACs were directional, and remained directional when GABA receptors were blocked. Contrary to the mutual-inhibitory model, stimuli that produce strong directional signals in ganglion cells failed to reveal a significant inhibitory input to SBACs. Suppression of the tonic excitatory conductance, proposed to generate the dendritic voltage-gradient required for the dendrite autonomous model, failed to eliminate the directional signal in SBACs. However, selective block of tetrodotoxin-resistant sodium channels did reduce the strength of the directional excitatory signal in the SBACs. These results indicate that current models of direction-selectivity in the SBACs are inadequate, and suggest that voltage-gated excitatory channels, specifically tetrodotoxin-resistant sodium channels, are important elements in directional signaling. This is the first physiological evidence that tetrodotoxin-resistant sodium channels play a role in retinal information processing.

  10. Tetrodotoxin-resistant sodium channels contribute to directional responses in starburst amacrine cells.

    Directory of Open Access Journals (Sweden)

    Nicholas W Oesch

    2010-08-01

    Full Text Available The biophysical mechanisms that give rise to direction selectivity in the retina remain uncertain. Current evidence suggests that the directional signal first arises within the dendrites of starburst amacrine cells (SBACs. Two models have been proposed to explain this phenomenon, one based on mutual inhibitory interactions between SBACs, and the other positing an intrinsic dendritic mechanism requiring a voltage-gradient depolarizing towards the dendritic tips. We tested these models by recording current and voltage responses to visual stimuli in SBACs. In agreement with previous work, we found that the excitatory currents in the SBACs were directional, and remained directional when GABA receptors were blocked. Contrary to the mutual-inhibitory model, stimuli that produce strong directional signals in ganglion cells failed to reveal a significant inhibitory input to SBACs. Suppression of the tonic excitatory conductance, proposed to generate the dendritic voltage-gradient required for the dendrite autonomous model, failed to eliminate the directional signal in SBACs. However, selective block of tetrodotoxin-resistant sodium channels did reduce the strength of the directional excitatory signal in the SBACs. These results indicate that current models of direction-selectivity in the SBACs are inadequate, and suggest that voltage-gated excitatory channels, specifically tetrodotoxin-resistant sodium channels, are important elements in directional signaling. This is the first physiological evidence that tetrodotoxin-resistant sodium channels play a role in retinal information processing.

  11. Pyrethroids differentially alter voltage-gated sodium channels from the honeybee central olfactory neurons.

    Science.gov (United States)

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

    2014-01-01

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

  12. Pacemaker rate and depolarization block in nigral dopamine neurons: a somatic sodium channel balancing act

    Science.gov (United States)

    Tucker, Kristal R.; Huertas, Marco A.; Horn, John P.; Canavier, Carmen C.; Levitan, Edwin S.

    2012-01-01

    Midbrain dopamine (DA) neurons are slow intrinsic pacemakers that undergo depolarization (DP) block upon moderate stimulation. Understanding DP block is important because it has been correlated with the clinical efficacy of chronic antipsychotic drug treatment. Here we describe how voltage-gated sodium (NaV) channels regulate DP block and pacemaker activity in DA neurons of the substantia nigra using rat brain slices. The distribution, density and gating of NaV currents were manipulated by blocking native channels with tetrodotoxin and by creating virtual channels and anti-channels with dynamic clamp. Although action potentials initiate in the axon initial segment (AIS) and NaV channels are distributed in multiple dendrites, selective reduction of NaV channel activity in the soma was sufficient to decrease pacemaker frequency and increase susceptibility to DP block. Conversely, increasing somatic NaV current density raised pacemaker frequency and lowered susceptibility to DP block. Finally, when NaV currents were restricted to the soma, pacemaker activity occurred at abnormally high rates due to excessive local subthreshold NaV current. Together with computational simulations, these data show that both the slow pacemaker rate and the sensitivity to DP block that characterizes DA neurons result from the low density of somatic NaV channels. More generally, we conclude that the somatodendritic distribution of NaV channels is a major determinant of repetitive spiking frequency. PMID:23077037

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

    Science.gov (United States)

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

    2012-01-01

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

  14. Salt-induced hypertension in a mouse model of Liddle syndrome is mediated by epithelial sodium channels in the brain.

    Science.gov (United States)

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

    2012-09-01

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

  15. Individual variation and hormonal modulation of a sodium channel beta subunit in the electric organ correlate with variation in a social signal.

    Science.gov (United States)

    Liu, He; Wu, Ming-Ming; Zakon, Harold H

    2007-09-01

    The sodium channel beta1 subunit affects sodium channel gating and surface density, but little is known about the factors that regulate beta1 expression or its participation in the fine control of cellular excitability. In this study we examined whether graded expression of the beta1 subunit contributes to the gradient in sodium current inactivation, which is tightly controlled and directly related to a social behavior, the electric organ discharge (EOD), in a weakly electric fish Sternopygus macrurus. We found the mRNA and protein levels of beta1 in the electric organ both correlate with EOD frequency. We identified a novel mRNA splice form of this gene and found the splicing preference for this novel splice form also correlates with EOD frequency. Androgen implants lowered EOD frequency and decreased the beta1 mRNA level but did not affect splicing. Coexpression of each splice form in Xenopus oocytes with either the human muscle sodium channel gene, hNav1.4, or a Sternopygus ortholog, smNav1.4b, sped the rate of inactivation of the sodium current and shifted the steady-state inactivation toward less negative membrane potentials. The translational product of the novel mRNA splice form lacks a previously identified important tyrosine residue but still functions normally. The properties of the fish alpha and coexpressed beta1 subunits in the oocyte replicate those of the electric organ's endogenous sodium current. These data highlight the role of ion channel beta subunits in regulating cellular excitability.

  16. Management of untreatable ventricular arrhythmias during pharmacologic challenges with sodium channel blockers for suspected Brugada syndrome.

    Science.gov (United States)

    Poli, Stefano; Toniolo, Mauro; Maiani, Massimo; Zanuttini, Davide; Rebellato, Luca; Vendramin, Igor; Dametto, Ermanno; Bernardi, Guglielmo; Bassi, Flavio; Napolitano, Carlo; Livi, Ugolino; Proclemer, Alessandro

    2018-02-01

    Pharmacologic challenge with sodium channel blockers is part of the diagnostic workout in patients with suspected Brugada syndrome. The test is overall considered safe but both ajmaline and flecainide detain well known pro-arrhythmic properties. Moreover, the treatment of patients with life-threatening arrhythmias during these diagnostic procedures is not well defined. Current consensus guidelines suggest to adopt cautious protocols interrupting the sodium channel blockers as soon as any ECG alteration appears. Nevertheless, the risk of life-threatening arrhythmias persists, even adopting a safe and cautious protocol and in absence of major arrhythmic risk factors. The authors revise the main published case studies of sodium channel blockers challenge in adults and in children, and summarize three cases of untreatable ventricular arrhythmias discussing their management. In particular, the role of advanced cardiopulmonary resuscitation with extra-corporeal membrane oxygenation is stressed as it can reveal to be the only reliable lifesaving facility in prolonged cardiac arrest. Published on behalf of the European Society of Cardiology. All rights reserved. © The Author 2017. For permissions, please email: journals.permissions@oup.com.

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

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

    1987-05-01

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

  18. Biophysical characterization of theVarroa destructorNaV1 sodium channel and its affinity for τ-fluvalinate insecticide.

    Science.gov (United States)

    Gosselin-Badaroudine, Pascal; Chahine, Mohamed

    2017-07-01

    The decline of the western honeybee ( Apis mellifera ) has been reported to be due to parasitism by Varroa destructor mites and to colony collapse disorder in which these mites may be involved. In-hive chemicals such as τ-fluvalinate are being used to control V destructor populations. This approach may lead to the chronic exposure of bees to this liposoluble chemical, which tends to accumulate in hives. We cloned a variant of the V. destructor voltage-dependent sodium (VdNa V 1) channel and studied its biophysical characteristics and sensitivity to τ-fluvalinate using the Xenopus oocyte expression system and the 2-microelectrode voltage-clamp technique. We compared the affinity of VdNa V 1 for τ-fluvalinate with the honeybee voltage-dependent sodium ortholog. Our results showed that the honeybee sodium channel is more sensitive to τ-fluvalinate than the V. destructor channel, suggesting that care must be taken when treating hives with this chemical.-Gosselin-Badaroudine, P., Chahine, M. Biophysical characterization of the Varroa destructor Na V 1 sodium channel and its affinity for τ-fluvalinate insecticide. © FASEB.

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

    Directory of Open Access Journals (Sweden)

    Vargas-Alarcon Gilberto

    2012-02-01

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

  20. The sodium channel isoform transition at developing nodes of Ranvier in the peripheral nervous system: dependence on a Genetic program and myelination-induced cluster formation.

    Science.gov (United States)

    Luo, Songjiang; Jaegle, Martine; Li, Roy; Ehring, George R; Meijer, Dies; Levinson, Simon R

    2014-12-15

    Among sodium channel isoforms, Nav 1.6 is selectively expressed at nodes of Ranvier in both the CNS and the PNS. However, non-Nav 1.6 isoforms such as Nav 1.2 are also present at the CNS nodes in early development but gradually diminish later. It has been proposed that myelination is part of a glia-neuron signaling mechanism that produces this change in nodal isoform expression. The present study used isoform-specific antibodies to demonstrate that, in the PNS, four other neuronal sodium channel isoforms were also clustered at nodes in early development but eventually disappeared during maturation. To study possible roles of myelination in such transitions, we investigated the nodal expression of selected isoforms in the sciatic nerve of the transgenic mouse Oct6(ΔSCE/βgeo) , whose PNS myelination is delayed in the first postnatal week but eventually resumes. We found that delayed myelination retarded the formation of nodal channel clusters and altered the expression-elimination patterns of sodium channel isoforms, resulting in significantly reduced expression levels of non-Nav 1.6 isoforms in such delayed nodes. However, delayed myelination did not significantly affect the gene expression, protein synthesis, or axonal trafficking of any isoform studied. Rather, we found evidence for a developmentally programmed increase in neuronal Nav 1.6 expression with constant or decreasing neuronal expression of other isoforms that were unaffected by delayed myelination. Thus our results suggest that, in the developmental isoform switch of the PNS, myelination does not play a signaling role as that proposed for the CNS but rather serves only to form nodal clusters from existing isoform pools. © 2014 Wiley Periodicals, Inc.

  1. TWIK-1 two-pore domain potassium channels change ion selectivity and conduct inward leak sodium currents in hypokalemia.

    Science.gov (United States)

    Ma, Liqun; Zhang, Xuexin; Chen, Haijun

    2011-06-07

    Background potassium (K+) channels, which are normally selectively permeable to K+, maintain the cardiac resting membrane potential at around -80 mV. In subphysiological extracellular K+ concentrations ([K+]o), which occur in pathological hypokalemia, the resting membrane potential of human cardiomyocytes can depolarize to around -50 mV, whereas rat and mouse cardiomyocytes become hyperpolarized, consistent with the Nernst equation for K+. This paradoxical depolarization of cardiomyocytes in subphysiological [K+]o, which may contribute to cardiac arrhythmias, is thought to involve an inward leak sodium (Na+) current. Here, we show that human cardiac TWIK-1 (also known as K2P1) two-pore domain K+ channels change ion selectivity, becoming permeable to external Na+, and conduct inward leak Na+ currents in subphysiological [K+]o. A specific threonine residue (Thr118) within the pore selectivity sequence TxGYG was required for this altered ion selectivity. Mouse cardiomyocyte-derived HL-1 cells exhibited paradoxical depolarization with ectopic expression of TWIK-1 channels, whereas TWIK-1 knockdown in human spherical primary cardiac myocytes eliminated paradoxical depolarization. These findings indicate that ion selectivity of TWIK-1 K+ channels changes during pathological hypokalemia, elucidate a molecular basis for inward leak Na+ currents that could trigger or contribute to cardiac paradoxical depolarization in lowered [K+]o, and identify a mechanism for regulating cardiac excitability.

  2. Malignant perinatal variant of long-QT syndrome caused by a profoundly dysfunctional cardiac sodium channel.

    Science.gov (United States)

    Wang, Dao W; Crotti, Lia; Shimizu, Wataru; Pedrazzini, Matteo; Cantu, Francesco; De Filippo, Paolo; Kishiki, Kanako; Miyazaki, Aya; Ikeda, Tomoaki; Schwartz, Peter J; George, Alfred L

    2008-12-01

    Inherited cardiac arrhythmia susceptibility contributes to sudden death during infancy and may contribute to perinatal and neonatal mortality, but the molecular basis of this risk and the relationship to genetic disorders presenting later in life is unclear. We studied the functional and pharmacological properties of a novel de novo cardiac sodium channel gene (SCN5A) mutation associated with an extremely severe perinatal presentation of long-QT syndrome in unrelated probands of different ethnicity. Two subjects exhibiting severe fetal and perinatal ventricular arrhythmias were screened for SCN5A mutations, and the functional properties of a novel missense mutation (G1631D) were determined by whole-cell patch clamp recording. In vitro electrophysiological studies revealed a profound defect in sodium channel function characterized by approximately 10-fold slowing of inactivation, increased persistent current, slowing of recovery from inactivation, and depolarized voltage dependence of activation and inactivation. Single-channel recordings demonstrated increased frequency of late openings, prolonged mean open time, and increased latency to first opening for the mutant. Subjects carrying this mutation responded clinically to the combination of mexiletine with propranolol and survived. Pharmacologically, the mutant exhibited 2-fold greater tonic and use-dependent mexiletine block than wild-type channels. The mutant also exhibited enhanced tonic (2.4-fold) and use-dependent block ( approximately 5-fold) by propranolol, and we observed additive effects of the 2 drugs on the mutant. Our study demonstrates the molecular basis for a malignant perinatal presentation of long-QT syndrome, illustrates novel functional and pharmacological properties of SCN5A-G1631D, which caused the disorder, and reveals therapeutic benefits of propranolol block of mutant sodium channels in this setting.

  3. Asymmetric functional contributions of acidic and aromatic side chains in sodium channel voltage-sensor domains

    DEFF Research Database (Denmark)

    Pless, Stephan Alexander; Elstone, Fisal D; Niciforovic, Ana P

    2014-01-01

    functional phenotypes that are different from those observed previously in Kv VSDs. In contrast, and similar to results obtained with Kv channels, individually neutralizing acidic side chains with synthetic derivatives and with natural amino acid substitutions in the INC had little or no effect......Voltage-gated sodium (NaV) channels mediate electrical excitability in animals. Despite strong sequence conservation among the voltage-sensor domains (VSDs) of closely related voltage-gated potassium (KV) and NaV channels, the functional contributions of individual side chains in Nav VSDs remain.......4). The results show that the highly conserved aromatic side chain constituting the S2 HC makes distinct functional contributions in each of the four NaV domains. No obvious cation-pi interaction exists with nearby S4 charges in any domain, and natural and unnatural mutations at these aromatic sites produce...

  4. Rotational Symmetry of Two Pyrethroid Receptor Sites in the Mosquito Sodium Channel

    Science.gov (United States)

    Du, Yuzhe; Nomura, Yoshiko; Zhorov, Boris S.

    2015-01-01

    Voltage-gated sodium channels are the primary target of pyrethroid insecticides. Although it is well known that specific mutations in insect sodium channels confer knockdown resistance (kdr) to pyrethroids, the atomic mechanisms of pyrethroid-sodium channel interactions are not clearly understood. Previously, computer modeling and mutational analysis predicted two pyrethroid receptors, pyrethroid receptor site 1 (PyR1) (initial) and pyrethroid receptor site 2 (PyR2), located in the domain interfaces II/III and I/II, respectively. The models differ in ligand orientation and the number of transmembrane helices involved. In this study, we elaborated a revised PyR1 model of the mosquito sodium channel. Computational docking in the Kv1.2-based open channel model yielded a complex in which a pyrethroid (deltamethrin) binds between the linker helix IIL45 and transmembrane helices IIS5, IIS6, and IIIS6 with its dibromoethenyl and diphenylether moieties oriented in the intra- and extracellular directions, respectively. The PyR2 and revised PyR1 models explained recently discovered kdr mutations and predicted new deltamethrin-channel contacts. Further model-driven mutagenesis identified seven new pyrethroid-sensing residues, three in the revised PyR1 and four in PyR2. Our data support the following conclusions: 1) each pyrethroid receptor is formed by a linker-helix L45 and three transmembrane helices (S5 and two S6s); 2) IIS6 contains four residues that contribute to PyR1 and another four to PyR2; 3) seven pairs of pyrethroid-sensing residues are located in symmetric positions within PyR1 and PyR2; and 4) pyrethroids bind to PyR1 and PyR2 in similar orientations, penetrating deeply into the respective domain interfaces. Our study elaborates the dual pyrethroid-receptor sites concept and provides a structural background for rational development of new insecticides. PMID:25972447

  5. Batrachotoxinin-A-ortho-azidobenzoate: a photoaffinity probe of the batrachotoxin binding site of voltage-sensitive sodium channels.

    Science.gov (United States)

    Casebolt, T L; Brown, G B

    1993-09-01

    Batrachotoxin (BTX) is one of a group of potent lipid-soluble neurotoxins which binds voltage-sensitive sodium channels. Here we show that [3H]batrachotoxinin-A-ortho-azidobenzoate ([3H]BTX-OAB), a photolabile derivative of BTX, binds covalently upon irradiation to the BTX sodium channel site of rat cerebral cortical synaptoneurosomes. Another ligand specific for the BTX sodium channel receptor, batrachotoxinin-A 20-alpha-benzoate (BTX-B), competitively inhibited the specific binding of [3H]BTX-OAB. The specific binding of [3H]BTX-OAB was increased by the addition of Leiurus quinquestriatus quinquestriatus scorpion venom (ScTx) and inhibited by veratridine, a member of the same class of sodium channel activators. Examination of the [3H]BTX-OAB-labeled components revealed that over 90% of the specifically incorporated [3H]BTX-OAB was recovered in lipid extracts of photolabeled synaptoneurosomes. Addition of tetrodotoxin (TTX) to the binding mixture increased the specific incorporation of [3H]BTX-OAB into protein components as much as 15-fold. Increasing the incubation temperature from 25 degree C to 37 degrees C had a similar but less marked effect. We conclude that the BTX binding site lies at a lipid-protein interface and that treatments which induce conformational changes in the sodium channel protein (i.e. addition of TTX) can result in a reorientation of BTX at its binding site relative to the protein and lipid domains of voltage-sensitive sodium channels.

  6. Voltage gated sodium and calcium channel blockers for the treatment of chronic inflammatory pain.

    Science.gov (United States)

    Rahman, Wahida; Dickenson, Anthony H

    2013-12-17

    The inflammatory response is a natural response of the body that occurs immediately following tissue damage, which may be due to injury, infection or disease. The acute inflammatory response is an essential mechanism that promotes healing and a key aspect is the ensuing pain, which warns the subject to protect the site of injury. Thus, it is common to see a zone of primary sensitization as well as consequential central sensitization that generally, is maintained by a peripheral drive from the zone of tissue injury. Inflammation associated with chronic pain states, such as rheumatoid and osteoarthritis, cancer and migraine etc. is deleterious to health and often debilitating for the patient. Thus there is a large unmet clinical need. The mechanisms underlying both acute and chronic inflammatory pain are extensive and complex, involving a diversity of cell types, receptors and proteins. Among these the contribution of voltage gated sodium and calcium channels on peripheral nociceptors is critical for nociceptive transmission beyond the peripheral transducers and changes in their distribution, accumulation, clustering and functional activities have been linked to both inflammatory and neuropathic pain. The latter has been the main area for trials and use of drugs that modulate ion channels such as carbamazepine and gabapentin, but given the large peripheral drive that follows tissue damage, there is a clear rationale for blocking voltage gated sodium and calcium channels in these pain states. It has been hypothesized that pain of inflammatory origin may evolve into a condition that resembles neuropathic pain, but mixed pains such as low back pain and cancer pain often include elements of both pain states. This review considers the therapeutic potential for sodium and calcium channel blockers for the treatment of chronic inflammatory pain states. Copyright © 2013 Elsevier Ireland Ltd. All rights reserved.

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

    Science.gov (United States)

    Chanda, Baron; Bezanilla, Francisco

    2002-11-01

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

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

    Science.gov (United States)

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

    2017-05-01

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

  9. Regions of KCNQ K+ Channels Controlling Functional Expression

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    Frank eChoveau

    2012-10-01

    Full Text Available KCNQ1-5 α-subunits assemble to form K+ channels that play critical roles in the function of numerous tissues. The channels are tetramers of subunits containing six transmembrane domains. Each subunit consists of a pore region (S5-pore-S6 and a voltage sensor domain (S1-S4. Despite similar structures, KCNQ2 and KCNQ3 homomers yield small current amplitudes compared to other KCNQ homomers and KCNQ2/3 heteromers. Two major mechanisms have been suggested as governing functional expression. The first involves control of channel trafficking to the plasma membrane by the distal part of the C-terminus, containing two coiled-coiled domains, required for channel trafficking and assembly. The proximal half of the C-terminus is the crucial region for channel modulation by signaling molecules such as calmodulin, which may mediate C- and N-terminal interactions. The N-terminus of KCNQ channels has also been postulated as critical for channel surface expression. The second mechanism suggests networks of interactions between the pore helix and the selectivity filter, and between the pore helix and the S6 domain that govern KCNQ current amplitudes. Here, we summarize the role of these different regions in expression of functional KCNQ channels.

  10. Fragile X mental retardation protein controls ion channel expression and activity.

    Science.gov (United States)

    Ferron, Laurent

    2016-10-15

    Fragile X-associated disorders are a family of genetic conditions resulting from the partial or complete loss of fragile X mental retardation protein (FMRP). Among these disorders is fragile X syndrome, the most common cause of inherited intellectual disability and autism. FMRP is an RNA-binding protein involved in the control of local translation, which has pleiotropic effects, in particular on synaptic function. Analysis of the brain FMRP transcriptome has revealed hundreds of potential mRNA targets encoding postsynaptic and presynaptic proteins, including a number of ion channels. FMRP has been confirmed to bind voltage-gated potassium channels (K v 3.1 and K v 4.2) mRNAs and regulates their expression in somatodendritic compartments of neurons. Recent studies have uncovered a number of additional roles for FMRP besides RNA regulation. FMRP was shown to directly interact with, and modulate, a number of ion channel complexes. The sodium-activated potassium (Slack) channel was the first ion channel shown to directly interact with FMRP; this interaction alters the single-channel properties of the Slack channel. FMRP was also shown to interact with the auxiliary β4 subunit of the calcium-activated potassium (BK) channel; this interaction increases calcium-dependent activation of the BK channel. More recently, FMRP was shown to directly interact with the voltage-gated calcium channel, Ca v 2.2, and reduce its trafficking to the plasma membrane. Studies performed on animal models of fragile X syndrome have revealed links between modifications of ion channel activity and changes in neuronal excitability, suggesting that these modifications could contribute to the phenotypes observed in patients with fragile X-associated disorders. © 2016 The Authors. The Journal of Physiology © 2016 The Physiological Society.

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

    DEFF Research Database (Denmark)

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

    2011-01-01

    AIMS: Atrial fibrillation (AF) is the most frequent arrhythmia. Screening of SCN5A-the gene encoding the a-subunit of the cardiac sodium channel-has indicated that disturbances of the sodium current may play a central role in the mechanism of lone AF. We tested the hypothesis that lone AF in young...... across species. Electrophysiological studies on the SCN3B mutation were carried out and all three SCN3B mutations caused a functionally reduced sodium channel current. One synonymous variant was found in SCN4B. CONCLUSION: In 192 young lone AF patients, we found three patients with suspected disease...

  12. Biological activity of the functional epitope of ciguatoxin fragment AB on the neuroblastoma sodium channel in tissue culture.

    Science.gov (United States)

    Hokama, Y; Chun, K E; Campora, C E; Higa, N; Suma, C; Hamajima, A; Isobe, M

    2006-01-01

    It is well established that the targeted receptor for ciguatoxin (CTX) in mammalian tissues is the sodium channel, affecting the influx of sodium into cells and altering the action potential and function of the cell. Since the syntheses of fragments of CTX has become available, our focus has been on the receptor functions of the west sphere AB and east sphere JKLM fragments using the neuroblastoma cell assay, guinea pig atrium assay, and the membrane immunobead assay (MIA). The data presented here suggest that the west sphere AB of the ciguatoxin molecule is the active portion and is responsible for the activation of the sodium channels. (c) 2006 Wiley-Liss, Inc.

  13. Ionizing radiation alters the properties of sodium channels in rat brain synaptosomes

    Energy Technology Data Exchange (ETDEWEB)

    Mullin, M.J.; Hunt, W.A.; Harris, R.A.

    1986-08-01

    The effect of ionizing radiation on neuronal membrane function was assessed by measurement of neurotoxin-stimulated /sup 22/Na/sup +/ uptake by rat brain synaptosomes. High-energy electrons and gamma photons were equally effective in reducing the maximal uptake of /sup 22/Na/sup +/ with no significant change in the affinity of veratridine for its binding site in the channel. Ionizing radiation reduced the veratridine-stimulated uptake at the earliest times measured (3 and 5 s), when the rate of uptake was greatest. Batrachotoxin-stimulated /sup 22/Na/sup +/ uptake was less sensitive to inhibition by radiation. The binding of (/sup 3/H)saxitoxin to its receptor in the sodium channel was unaffected by exposure to ionizing radiation. The effect of ionizing radiation on the lipid order of rat brain synaptic plasma membranes was measured by the fluorescence polarization of the molecular probes 1,6-diphenyl-1,3,5-hexatriene and 1-(4-(trimethylammonium)phenyl)-6-phenyl-1,3,5-hexatriene. A dose of radiation that reduced the veratridine-stimulated uptake of /sup 22/Na/sup +/ had no effect on the fluorescence polarization of either probe. These results demonstrate an inhibitory effect of ionizing radiation on the voltage-sensitive sodium channels in rat brain synaptosomes. This effect of radiation is not dependent on changes in the order of membrane lipids.

  14. Differential expression of the Kv1 voltage-gated potassium channel family in the rat nephron.

    Science.gov (United States)

    Carrisoza-Gaytán, Rolando; Salvador, Carolina; Diaz-Bello, Beatriz; Escobar, Laura I

    2014-10-01

    Several potassium (K(+)) channels contribute to maintaining the resting membrane potential of renal epithelial cells. Apart from buffering the cell membrane potential and cell volume, K(+) channels allow sodium reabsorption in the proximal tubule (PT), K(+) recycling and K(+) reabsorption in the thick ascending limb (TAL) and K(+) secretion and K(+) reabsorption in the distal convoluted tubule (DCT), connecting tubule (CNT) and collecting duct. Previously, we identified Kv.1.1, Kv1.3 and Kv1.6 channels in collecting ducts of the rat inner medulla. We also detected intracellular Kv1.3 channel in the acid secretory intercalated cells, which is trafficked to the apical membrane in response to dietary K(+) to function as a secretory K(+) channel. In this work we sought to characterize the expression of all members of the Kv1 family in the rat nephron. mRNA and protein expression were detected for all Kv1 channels. Immunoblots identified differential expression of each Kv1 in the cortex, outer and inner medulla. Immunofluorescence labeling detected Kv1.5 in Bowman´s capsule and endothelial cells and Kv1.7 in podocytes, endothelial cells and macula densa in glomeruli; Kv1.4, Kv1.5 and Kv1.7 in PT; Kv1.2, Kv1.4 and Kv1.6 in TAL; Kv1.1, Kv1.4 and Kv1.6 in DCT and CNT and Kv1.3 in DCT, and all the Kv1 family in the cortical and medullary collecting ducts. Recently, some hereditary renal syndromes have been attributed to mutations in K(+) channels. Our results expand the repertoire of K(+) channels that contribute to K(+) homeostasis to include the Kv1 family.

  15. Physical model of voltage sensing in sodium channels based on the sliding helix complex

    Science.gov (United States)

    Chancey, C. C.; George, S. A.

    1996-05-01

    We have modeled voltage sensing in the sodium channel by evaluating forces on the S4 α-helix portion of the channel molecule, which we assume moves outward during activation. The interaction between the S4 α-helix segment and its environment was modeled by (i) nearest-neighbor Coulombic forces, (ii) the electric force due to an external electric field, and (iii) static mechanical and electrostatic forces. These terms collectively describe a depolarization-dependent effective potential within which the S4 segment moves. Thermal transitions between center-of-mass energy states of the segment were modeled starting from the Boltzmann distribution, and the time evolution of the segment's position relative to the membrane was simulated. Combining the histories of four such processes models the activation history of the channel molecule. The model simulation is in good qualitative agreement with batrachotoxin-modified single channel open and closed dwell time distributions and with such a channel's open probability as a function of depolarization. The model also qualitatively agrees with site-specific mutagenesis experiments, which show the different effects of eliminating positive charges on the cytoplasmic and extracellular ends of the S4 segment.

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

    Science.gov (United States)

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

    2017-04-01

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

  17. Batrachotoxin, Pyrethroids, and BTG 502 Share Overlapping Binding Sites on Insect Sodium ChannelsS⃞

    Science.gov (United States)

    Du, Yuzhe; Garden, Daniel; Khambay, Bhupinder; Zhorov, Boris S.

    2011-01-01

    Batrachotoxin (BTX), a steroidal alkaloid, and pyrethroid insecticides bind to distinct but allosterically coupled receptor sites on voltage-gated sodium channels and cause persistent channel activation. BTX presumably binds in the inner pore, whereas pyrethroids are predicted to bind at the lipid-exposed cavity formed by the short intracellular linker-helix IIS4-S5 and transmembrane helices IIS5 and IIIS6. The alkylamide insecticide (2E,4E)-N-(1,2-dimethylpropyl)-6-(5-bromo-2-naphthalenyl)-2,4-hexadienamide (BTG 502) reduces sodium currents and antagonizes the action of BTX on cockroach sodium channels, suggesting that it also binds inside the pore. However, a pyrethroid-sensing residue, Phe3i17 in IIIS6, which does not face the pore, is essential for the activity of BTG 502 but not for BTX. In this study, we found that three additional deltamethrin-sensing residues in IIIS6, Ile3i12, Gly3i14, and Phe3i16 (the latter two are also BTX-sensing), and three BTX-sensing residues, Ser3i15 and Leu3i19 in IIIS6 and Phe4i15 in IVS6, are all critical for BTG 502 action on cockroach sodium channels. Using these data as constraints, we constructed a BTG 502 binding model in which BTG 502 wraps around IIIS6, probably making direct contacts with all of the above residues on the opposite faces of the IIIS6 helix, except for the putative gating hinge Gly3i14. BTG 502 and its inactive analog DAP 1855 antagonize the action of deltamethrin. The antagonism was eliminated by mutations of Ser3i15, Phe3i17, Leu3i19, and Phe4i15 but not by mutations of Ile3i12, Gly3i14, and Phe3i16. Our analysis revealed a unique mode of action of BTG 502, its receptor site overlapping with those of both BTX and deltamethrin. PMID:21680776

  18. Batrachotoxin, pyrethroids, and BTG 502 share overlapping binding sites on insect sodium channels.

    Science.gov (United States)

    Du, Yuzhe; Garden, Daniel; Khambay, Bhupinder; Zhorov, Boris S; Dong, Ke

    2011-09-01

    Batrachotoxin (BTX), a steroidal alkaloid, and pyrethroid insecticides bind to distinct but allosterically coupled receptor sites on voltage-gated sodium channels and cause persistent channel activation. BTX presumably binds in the inner pore, whereas pyrethroids are predicted to bind at the lipid-exposed cavity formed by the short intracellular linker-helix IIS4-S5 and transmembrane helices IIS5 and IIIS6. The alkylamide insecticide (2E,4E)-N-(1,2-dimethylpropyl)-6-(5-bromo-2-naphthalenyl)-2,4-hexadienamide (BTG 502) reduces sodium currents and antagonizes the action of BTX on cockroach sodium channels, suggesting that it also binds inside the pore. However, a pyrethroid-sensing residue, Phe(3i17) in IIIS6, which does not face the pore, is essential for the activity of BTG 502 but not for BTX. In this study, we found that three additional deltamethrin-sensing residues in IIIS6, Ile(3i12), Gly(3i14), and Phe(3i16) (the latter two are also BTX-sensing), and three BTX-sensing residues, Ser(3i15) and Leu(3i19) in IIIS6 and Phe(4i15) in IVS6, are all critical for BTG 502 action on cockroach sodium channels. Using these data as constraints, we constructed a BTG 502 binding model in which BTG 502 wraps around IIIS6, probably making direct contacts with all of the above residues on the opposite faces of the IIIS6 helix, except for the putative gating hinge Gly(3i14). BTG 502 and its inactive analog DAP 1855 antagonize the action of deltamethrin. The antagonism was eliminated by mutations of Ser(3i15), Phe(3i17), Leu(3i19), and Phe(4i15) but not by mutations of Ile(3i12), Gly(3i14), and Phe(3i16). Our analysis revealed a unique mode of action of BTG 502, its receptor site overlapping with those of both BTX and deltamethrin.

  19. Endogenous opioids contribute to insensitivity to pain in humans and mice lacking sodium channel Nav1.7.

    Science.gov (United States)

    Minett, Michael S; Pereira, Vanessa; Sikandar, Shafaq; Matsuyama, Ayako; Lolignier, Stéphane; Kanellopoulos, Alexandros H; Mancini, Flavia; Iannetti, Gian D; Bogdanov, Yury D; Santana-Varela, Sonia; Millet, Queensta; Baskozos, Giorgios; MacAllister, Raymond; Cox, James J; Zhao, Jing; Wood, John N

    2015-12-04

    Loss-of-function mutations in the SCN9A gene encoding voltage-gated sodium channel Nav1.7 cause congenital insensitivity to pain in humans and mice. Surprisingly, many potent selective antagonists of Nav1.7 are weak analgesics. We investigated whether Nav1.7, as well as contributing to electrical signalling, may have additional functions. Here we report that Nav1.7 deletion has profound effects on gene expression, leading to an upregulation of enkephalin precursor Penk mRNA and met-enkephalin protein in sensory neurons. In contrast, Nav1.8-null mutant sensory neurons show no upregulated Penk mRNA expression. Application of the opioid antagonist naloxone potentiates noxious peripheral input into the spinal cord and dramatically reduces analgesia in both female and male Nav1.7-null mutant mice, as well as in a human Nav1.7-null mutant. These data suggest that Nav1.7 channel blockers alone may not replicate the analgesic phenotype of null mutant humans and mice, but may be potentiated with exogenous opioids.

  20. [Potential-dependent changes in the ionic selectivity of batrachotoxin-modified sodium channels of a frog nerve fiber].

    Science.gov (United States)

    Mozhaeva, G N; Naumov, A P; Khodorov, B I

    1983-01-01

    Currents through batrachotoxin-modified sodium channels in frog myelinated fibres were measured under voltage-clamp conditions. Reversal potential (Erev) of steady-state currents is shown to be about 5 mV less positive than Erev of initial (peak) currents. Control experiments with procaine and tetrodotoxin in external solutions showed that this shift of Erev during depolarizing pulse cannot be accounted for by the presence of unmodified sodium channels, unblocked potassium channels, nonlinearity of the leakage or any changes in transmembrane gradients of current-carrying cations. "Instantaneous" current measurements showed that Erev becomes less positive as amplitude and duration of preliminary depolarization increase. The results obtained are consistent with assumption that sodium-potassium selectivity of the batrachotoxin-modified channels depends on potential.

  1. [Selectivity and sensitivity to blocking by hydrogen ions of batrachotoxin-modified sodium channels in nerve fiber membranes].

    Science.gov (United States)

    Mozhaeva, G N; Naumov, A P; Khodorov, B I

    1983-01-01

    Currents through normal and batrachotoxin-modified sodium channels in frog nerve were measured under voltage clamp conditions. Measured reversal potentials and the Goldman equation were used to calculate relative permeabilities. The permeability ratios were: PNa: PNH4: PK = 1: 0.47: 0.19. Hydrogen-to-sodium permeability ratio was estimated from reversal potential measurements in Na-free acid (pH 3.7-3.8) solutions. It was 528 +/- 46 for batrachotoxin-modified sodium channels. Modified channels were less sensitive to hydrogen block as compared with normal ones. The difference in apparent pKa for acid group between normal and modified channels was about 0.40.

  2. SK2 channel expression and function in cerebellar Purkinje cells

    Science.gov (United States)

    Hosy, Eric; Piochon, Claire; Teuling, Eva; Rinaldo, Lorenzo; Hansel, Christian

    2011-01-01

    Abstract Small-conductance calcium-activated K+ channels (SK channels) regulate the excitability of neurons and their responsiveness to synaptic input patterns. SK channels contribute to the afterhyperpolarization (AHP) following action potential bursts, and curtail excitatory postsynaptic potentials (EPSPs) in neuronal dendrites. Here we review evidence that SK2 channels are expressed in rat cerebellar Purkinje cells during development and throughout adulthood, and play a key role in diverse cellular processes such as the regulation of the spike firing frequency and the modulation of calcium transients in dendritic spines. In Purkinje cells as well as in other types of neurons, SK2 channel plasticity seems to provide an important mechanism allowing these cells to adjust their intrinsic excitability and to alter the probabilities for the induction of synaptic learning correlates, such as long-term potentiation (LTP). PMID:21521760

  3. Angiotensin II stimulates renin in inner medullary collecting duct cells via protein kinase C and independent of epithelial sodium channel and mineralocorticoid receptor activity.

    Science.gov (United States)

    Gonzalez, Alexis A; Liu, Liu; Lara, Lucienne S; Seth, Dale M; Navar, L Gabriel; Prieto, Minolfa C

    2011-03-01

    Collecting duct (CD) renin is stimulated by angiotensin (Ang) II, providing a pathway for Ang I generation and further conversion to Ang II. Ang II stimulates the epithelial sodium channel via the Ang II type 1 receptor and increases mineralocorticoid receptor activity attributed to increased aldosterone release. Our objective was to determine whether CD renin augmentation is mediated directly by Ang II type 1 receptor or via the epithelial sodium channel and mineralocorticoid receptor. In vivo studies examined the effects of epithelial sodium channel blockade (amiloride; 5 mg/kg per day) on CD renin expression and urinary renin content in Ang II-infused rats (80 ng/min, 2 weeks). Ang II infusion increased systolic blood pressure, medullary renin mRNA, urinary renin content, and intrarenal Ang II levels. Amiloride cotreatment did not alter these responses despite a reduction in the rate of progression of systolic blood pressure. In primary cultures of inner medullary CD cells, renin mRNA and (pro)renin protein levels increased with Ang II (100 nmol/L), and candesartan (Ang II type 1 receptor antagonist) prevented this effect. Aldosterone (10(-10) to 10(-7) mol/L) with or without amiloride did not modify the upregulation of renin mRNA in Ang II-treated cells. However, inhibition of protein kinase C with calphostin C prevented the Ang II-mediated increases in renin mRNA and (pro)renin protein levels. Furthermore, protein kinase C activation with phorbol 12-myristate 13-acetate increased renin expression to the same extent as Ang II. These data indicate that an Ang II type 1 receptor-mediated increase in CD renin is induced directly by Ang II via the protein kinase C pathway and that this regulation is independent of mineralocorticoid receptor activation or epithelial sodium channel activity.

  4. Partial Block by Riluzole of Muscle Sodium Channels in Myotubes from Amyotrophic Lateral Sclerosis Patients

    Directory of Open Access Journals (Sweden)

    Cristina Deflorio

    2014-01-01

    Full Text Available Denervated muscles undergo fibrillations due to spontaneous activation of voltage-gated sodium (Na+ channels generating action potentials. Fibrillations also occur in patients with amyotrophic lateral sclerosis (ALS. Riluzole, the only approved drug for ALS treatment, blocks voltage-gated Na+ channels, but its effects on muscle Na+ channels and fibrillations are yet poorly characterized. Using patch-clamp technique, we studied riluzole effect on Na+ channels in cultured myotubes from ALS patients. Needle electromyography was used to study fibrillation potentials (Fibs in ALS patients during riluzole treatment and after one week of suspension. Patients were clinically characterized in all recording sessions. In myotubes, riluzole (1 μM, a therapeutic concentration reduced Na+ current by 20%. The rate of rise and amplitude of spikes evoked by depolarizing stimuli were also reduced. Fibs were detected in all patients tested during riluzole treatment and riluzole washout had no univocal effect. Our study indicates that, in human myotubes, riluzole partially blocks Na+ currents and affects action potentials but does not prevent firing. In line with this in vitro finding, muscle Fibs in ALS patients appear to be largely unaffected by riluzole.

  5. RING finger protein 121 facilitates the degradation and membrane localization of voltage-gated sodium channels

    Science.gov (United States)

    Ogino, Kazutoyo; Low, Sean E.; Yamada, Kenta; Saint-Amant, Louis; Zhou, Weibin; Muto, Akira; Asakawa, Kazuhide; Nakai, Junichi; Kawakami, Koichi; Kuwada, John Y.; Hirata, Hiromi

    2015-01-01

    Following their synthesis in the endoplasmic reticulum (ER), voltage-gated sodium channels (NaV) are transported to the membranes of excitable cells, where they often cluster, such as at the axon initial segment of neurons. Although the mechanisms by which NaV channels form and maintain clusters have been extensively examined, the processes that govern their transport and degradation have received less attention. Our entry into the study of these processes began with the isolation of a new allele of the zebrafish mutant alligator, which we found to be caused by mutations in the gene encoding really interesting new gene (RING) finger protein 121 (RNF121), an E3-ubiquitin ligase present in the ER and cis-Golgi compartments. Here we demonstrate that RNF121 facilitates two opposing fates of NaV channels: (i) ubiquitin-mediated proteasome degradation and (ii) membrane localization when coexpressed with auxiliary NaVβ subunits. Collectively, these results indicate that RNF121 participates in the quality control of NaV channels during their synthesis and subsequent transport to the membrane. PMID:25691753

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

    Directory of Open Access Journals (Sweden)

    Mohamed eChahine

    2011-11-01

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

  7. Actions of Ethanol on Voltage-Sensitive Sodium Channels. Effects on Neurotoxin-Stimulated Sodium Uptake in Synaptosomes

    Science.gov (United States)

    1985-01-01

    scorpion venom-induced enhancement of batrachotoxin - Seffect of ethanol on rotoxin-stimulated sodium uptake was stimulated sodium uptake. The inhibitory...Ethanol reduced rate of batrachotoxin -stimulated sodium ethanol has an inhibitory effect on neurotoxin-stimulated sodium CC uptake whenq sjjred at 3, 5 and...potential were due to a transient increased ) . AUUBROREVTIONS CNS, central nervous system; TTX, tetrodotoxin; BTX, batrachotoxin ; VER, veratitdne; ScV

  8. Adaptive evolution of insect selective excitatory β-type sodium channel neurotoxins from scorpion venom.

    Science.gov (United States)

    Wu, Wenlan; Li, Zhongjie; Ma, Yibao

    2017-06-01

    Insect selective excitatory β-type sodium channel neurotoxins from scorpion venom (β-NaScTxs) are composed of about 70-76 amino acid residues and share a common scaffold stabilized by four unique disulfide bonds. The phylogenetic analysis of these toxins was hindered by limited sequence data. In our recent study, two new insect selective excitatory β-NaScTxs, LmIT and ImIT, were isolated from Lychas mucronatus and Isometrus maculatus, respectively. With the sequences previously reported, we examined the adaptive molecular evolution of insect selective excitatory β-NaScTxs by estimating the nonsynonymous-to-synonymous rate ratio (ω=d N /d S ). The results revealed 12 positively selected sites in the genes of insect selective excitatory β-NaScTxs. Moreover, these positively selected sites match well with the sites important for interacting with sodium channels, as demonstrated in previous mutagenesis study. These results reveal that adaptive evolution after gene duplication is one of the most important genetic mechanisms of scorpion neurotoxin diversification. Copyright © 2017 Elsevier Inc. All rights reserved.

  9. Voltage-Gated Sodium Channel β1/β1B Subunits Regulate Cardiac Physiology and Pathophysiology

    Directory of Open Access Journals (Sweden)

    Nnamdi Edokobi

    2018-04-01

    Full Text Available Cardiac myocyte contraction is initiated by a set of intricately orchestrated electrical impulses, collectively known as action potentials (APs. Voltage-gated sodium channels (NaVs are responsible for the upstroke and propagation of APs in excitable cells, including cardiomyocytes. NaVs consist of a single, pore-forming α subunit and two different β subunits. The β subunits are multifunctional cell adhesion molecules and channel modulators that have cell type and subcellular domain specific functional effects. Variants in SCN1B, the gene encoding the Nav-β1 and -β1B subunits, are linked to atrial and ventricular arrhythmias, e.g., Brugada syndrome, as well as to the early infantile epileptic encephalopathy Dravet syndrome, all of which put patients at risk for sudden death. Evidence over the past two decades has demonstrated that Nav-β1/β1B subunits play critical roles in cardiac myocyte physiology, in which they regulate tetrodotoxin-resistant and -sensitive sodium currents, potassium currents, and calcium handling, and that Nav-β1/β1B subunit dysfunction generates substrates for arrhythmias. This review will highlight the role of Nav-β1/β1B subunits in cardiac physiology and pathophysiology.

  10. Both Laminin and Schwann cell Dystroglycan are necessary for proper clustering of Sodium Channels at Nodes of Ranvier.

    Science.gov (United States)

    Occhi, S.; Zambroni, D.; Del Carro, U.; Amadio, S.; Sirkowski, E. E.; Scherer, SS.; Campbell, K.; Moore, S.A.; Chen, Z-L.; Strickland, S.; Di Muzio, A.; Uncini, A.; Wrabetz, L.; Feltri, ML.

    2005-01-01

    Nodes of Ranvier are specialized axonal domains, where voltage-gated sodium channels cluster. How axons cluster molecules in discrete domains is largely unknown. Both axons and glia probably provide constraining mechanisms that contribute to domain formation. Proper sodium channel clustering in peripheral nerves depends on contact from Schwann cell microvilli, where at least one molecule, gliomedin, binds the sodium channel complex and induces its clustering. Furthermore, mice lacking Schwann cell dystroglycan have aberrant microvilli and poorly clustered sodium channels. Dystroglycan could interact at the basal lamina or at the axon-glial surface. Since dystroglycan is a laminin receptor, and laminin-2 mutations (merosin-deficient-congenital-muscular-dystrophy, MDC1A) cause reduced nerve conduction velocity, we asked whether laminins are involved. Here we show that the composition of both laminins and the dystroglycan complex at nodes differs from that of internodes. Mice defective in laminin-2 have poorly formed microvilli and abnormal sodium clusters. These abnormalities are similar, albeit less severe, than those of mice lacking dystroglycan. However, mice lacking all Schwann cell laminins show severe nodal abnormalities, suggesting other laminins compensate for the lack of laminin 2. Thus, even though laminins are located at a distance from the axo-glial junction, they are required for proper clustering of sodium channels. Laminins, through their specific nodal receptors and cytoskeletal linkages, may participate in the formation of mechanisms that constrain clusters at nodes. Finally, abnormal sodium channel clusters are present in a patient with MDC1A, providing a molecular basis for the reduced nerve conduction velocity in this disorder. PMID:16221851

  11. Pharmacological modification of sodium channels from the human heart atrium in planar lipid bilayers: electrophysiological characterization of responses to batrachotoxin and pentobarbital.

    Science.gov (United States)

    Wartenberg, H C; Wartenberg, J P; Urban, B W

    2003-05-01

    To investigate the effects of barbiturates on batrachotoxin-modified sodium channels from different regions of the human heart. Single sodium channels from human atria were studied and compared with existing data from the human ventricle and from the central nervous system. Sodium channels from preparations of human atrial muscle were incorporated into planar lipid bilayers in the presence of batrachotoxin, a sodium channel activator. The steady-state behaviour of single sodium channels was recorded in symmetrical 500 mmol NaCl before and after the addition of pentobarbital 0.34-1.34 mmol. The batrachotoxin-treated human atrial sodium channel had an average single-channel conductance of 23.8 +/- 1.6 pS in symmetrical 500 mmol NaCl and a channel fractional open time of 0.83 +/- 0.06. The activation mid-point potential was -98.0 +/- 2.3 mV. Extracellular tetrodotoxin (a specific sodium channel blocking agent) blocked these channels with a k(1/2) = 0.53 micromol at 0 mV. Pentobarbital reduced the time average conductance of single atrial sodium channels in a concentration-dependent manner (ID50 = 0.71 mmol). In the same way, the steady-state activation was shifted to more hyperpolarized potentials (-10.6 mV at 0.67 mmol pentobarbital). The properties of batrachotoxin-modified sodium channels from human atrial tissue did not differ greatly from those described for ventricular sodium channels in the literature. Our data yielded no explanation for the observed functional diversity. However, cardiac sodium channels differ from those found in the central nervous system.

  12. Sodium

    Science.gov (United States)

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

  13. Distinct functional defect of three novel Brugada syndrome related cardiac sodium channel mutations

    Directory of Open Access Journals (Sweden)

    Juang Jyh-Ming

    2009-02-01

    Full Text Available Abstract The Brugada syndrome is characterized by ST segment elevation in the right precodial leads V1-V3 on surface ECG accompanied by episodes of ventricular fibrillation causing syncope or even sudden death. The molecular and cellular mechanisms that lead to Brugada syndrome are not yet completely understood. However, SCN5A is the most well known responsible gene that causes Brugada syndrome. Until now, more than a hundred mutations in SCN5A responsible for Brugada syndrome have been described. Functional studies of some of the mutations have been performed and show that a reduction of human cardiac sodium current accounts for the pathogenesis of Brugada syndrome. Here we reported three novel SCN5A mutations identified in patients with Brugada syndrome in Taiwan (p.I848fs, p.R965C, and p.1876insM. Their electrophysiological properties were altered by patch clamp analysis. The p.I848fs mutant generated no sodium current. The p.R965C and p.1876insM mutants produced channels with steady state inactivation shifted to a more negative potential (9.4 mV and 8.5 mV respectively, and slower recovery from inactivation. Besides, the steady state activation of p.1876insM was altered and was shifted to a more positive potential (7.69 mV. In conclusion, the SCN5A channel defect related to Brugada syndrome might be diverse but all resulted in a decrease of sodium current.

  14. Functional and molecular characterization of voltage gated sodium channel Nav1.8 in bull spermatozoa.

    Science.gov (United States)

    Chauhan, Dharmendra Singh; Swain, Dilip Kumar; Shah, Nadeem; Yadav, Hanuman Prasad; Nakade, Udayraj P; Singh, Vijay Kumar; Nigam, Rajesh; Yadav, Sarvajeet; Garg, Satish Kumar

    2017-03-01

    The aim of our study was to characterize the voltage gated sodium channel Na v 1.8 in bull spermatozoa. Forty ejaculates were collected from four Hariana bulls and semen samples were pooled in view of the nonsignificant variations between different ejaculates. Functional characterization was undertaken using A-803467, a selective blocker of Na v 1.8, and veratridine as an opener of the voltage gated sodium channels while molecular characterization was done using western blotting and indirect immunofluorescence assays. In vitro capacitation was induced using heparin, and to study the functional involvement of Na v 1.8 in regulation of capacitation induced hyper sperm motility, A-803467 was used. Selective blocking of Na V 1.8 by A-803467 at 6 and 8 μM concentration significantly (P spermatozoa after 2 h of incubation and it was observed up to 3 h. Treatment of sperm cells with veratridine (6, 8, 10, 15, 20 μM) resulted in concentration- and time-dependent increase in forward progressive sperm motility and it persisted up to 4 h. However, hyperactive motility was induced by veratridine at higher concentrations (10 and 15 μM) after 2 h of incubation. In vitro capacitated spermatozoa treated with A-803467 revealed significant (P spermatozoa showing high mitochondrial transmembrane potential in concentration- and time-dependent manner. High concentrations (10 and 15 μM) of A-803467 and veratridine resulted in bent neck condition in spermatozoa along with significant (P spermatozoa. Strongest IR was observed in the neck and middle piece whereas weak IR was observed in tail and acrosomal region of the spermatozoa. Results of our present study evidently revealed the presence of voltage gated sodium channel Na v 1.8 in bull spermatozoa and its functional involvement in regulation of spermatozoa dynamics in terms of motility, membrane integrity, acrosome integrity, capacitation and mitochondrial transmembrane potential. Further studies are warranted to

  15. Molecular Surface of JZTX-V (β-Theraphotoxin-Cj2a Interacting with Voltage-Gated Sodium Channel Subtype NaV1.4

    Directory of Open Access Journals (Sweden)

    Ji Luo

    2014-07-01

    Full Text Available Voltage-gated sodium channels (VGSCs; NaV1.1–NaV1.9 have been proven to be critical in controlling the function of excitable cells, and human genetic evidence shows that aberrant function of these channels causes channelopathies, including epilepsy, arrhythmia, paralytic myotonia, and pain. The effects of peptide toxins, especially those isolated from spider venom, have shed light on the structure–function relationship of these channels. However, most of these toxins have not been analyzed in detail. In particular, the bioactive faces of these toxins have not been determined. Jingzhaotoxin (JZTX-V (also known as β-theraphotoxin-Cj2a is a 29-amino acid peptide toxin isolated from the venom of the spider Chilobrachys jingzhao. JZTX-V adopts an inhibitory cysteine knot (ICK motif and has an inhibitory effect on voltage-gated sodium and potassium channels. Previous experiments have shown that JZTX-V has an inhibitory effect on TTX-S and TTX-R sodium currents on rat DRG cells with IC50 values of 27.6 and 30.2 nM, respectively, and is able to shift the activation and inactivation curves to the depolarizing and the hyperpolarizing direction, respectively. Here, we show that JZTX-V has a much stronger inhibitory effect on NaV1.4, the isoform of voltage-gated sodium channels predominantly expressed in skeletal muscle cells, with an IC50 value of 5.12 nM, compared with IC50 values of 61.7–2700 nM for other heterologously expressed NaV1 subtypes. Furthermore, we investigated the bioactive surface of JZTX-V by alanine-scanning the effect of toxin on NaV1.4 and demonstrate that the bioactive face of JZTX-V is composed of three hydrophobic (W5, M6, and W7 and two cationic (R20 and K22 residues. Our results establish that, consistent with previous assumptions, JZTX-V is a Janus-faced toxin which may be a useful tool for the further investigation of the structure and function of sodium channels.

  16. The sodium channel activator Lu AE98134 normalizes the altered firing properties of fast spiking interneurons in Dlx5/6+/- mice

    DEFF Research Database (Denmark)

    von Schoubye, Nadia Lybøl; Frederiksen, Kristen; Kristiansen, Uffe

    2018-01-01

    Mental disorders such as schizophrenia are associated with impaired firing properties of fast spiking inhibitory interneurons (FSINs) causing reduced task-evoked gamma-oscillation in prefrontal cortex. The voltage-gated sodium channel NaV1.1 is highly expressed in PV-positive interneurons, but only...... facilitated the sodium current mediated by NaV1.1 expressed in HEK cells by shifting its activation to more negative values, decreasing its inactivation kinetics and promoting a persistent inward current. In a slice preparation from the brain of adult mice, Lu AE98134 promoted the excitability of fast spiking...... interneurons by decreasing the threshold for action potentials. We then tested if Lu AE98134 could normalize the altered firing properties of FSINs in Dlx5/6+/- mutant mice. FSINs of this model for schizophrenia are characterized by broader action potentials and higher spike threshold. We found...

  17. Voltage dependence of intramembrane charge movement and conductance activation of batrachotoxin-modified sodium channels in frog node of Ranvier

    Science.gov (United States)

    1983-01-01

    Sodium current and sodium channel intramembrane gating charge movement (Q) were monitored in voltage-clamped frog node of Ranvier after modification of all sodium channels by batrachotoxin (BTX). BTX caused an approximately threefold increase in steepness of the Q vs. voltage relationship and a 50-mV negative shift in its midpoint. The maximum amount of intramembrane charge was virtually identical before and after BTX treatment. BTX treatment eliminated the charge immobilization observed in untreated nodes after relatively long depolarizing pulses and slowed the rate of OFF charge movement after a pulse. After BTX treatment, the voltage dependence of charge movement was the same as the steady-state voltage dependence of sodium conductance activation. The observations are consistent with the hypothesis that BTX induces an aggregation of the charged gating particles associated with each channel and causes them to move as a unit having approximately three times the average valence of the individual particles. Movement of this single aggregated unit would open the BTX-modified sodium channel. PMID:6308127

  18. Tumor necrosis factor α modulates sodium-activated potassium channel SLICK in rat dorsal horn neurons via p38 MAPK activation pathway

    Directory of Open Access Journals (Sweden)

    Wang K

    2017-05-01

    Full Text Available Kun Wang,1 Feng Wang,1 Jun-Ping Bao,2 Zhi-Yang Xie,1 Lu Chen,1 Bao-Yi Zhou,1 Xin-Hui Xie,2 Xiao-Tao Wu1,2 1Medical School of Southeast University, 2Department of Orthopaedics, Zhongda Hospital, Southeast University, Nanjing, People’s Republic of China Abstract: The dorsal horn (DH of the spinal cord is the integrative center that processes and transmits pain sensation. Abnormal changes in ion channel expression can enhance the excitability of pain-related DH neurons. Sodium-activated potassium (KNa channels are highly expressed particularly in the central nervous system; however, information about whether rat DH neurons express the SLICK channel protein is lacking, and the direct effects on SLICK in response to inflammation and the potential signaling pathway mediating such effects are yet to be elucidated. Here, using cultured DH neurons, we have shown that tumor necrosis factor-α inhibits the total outward potassium current IK and the KNa current predominantly as well as induces a progressive loss of firing accommodation. However, we found that this change in channel activity is offset by the p38 inhibitor SB202190, thereby suggesting the modulation of SLICK channel activity via the p38 MAPK pathway. Furthermore, we have demonstrated that the tumor necrosis factor-α modulation of KNa channels does not occur at the level of SLICK channel gating but arises from possible posttranslational modification. Keywords: p38 MAPK, SLICK channel, neuropathic pain, dorsal horn, TNF-α

  19. Safety and efficacy of a Nav1.7 selective sodium channel blocker in patients with trigeminal neuralgia

    DEFF Research Database (Denmark)

    Zakrzewska, Joanna M; Palmer, Joanne; Morisset, Valerie

    2017-01-01

    , can be administered at therapeutic doses without titration, and has shown good tolerability in healthy individuals in phase 1 studies. We therefore assessed the safety and efficacy of BIIB074 in patients with trigeminal neuralgia in a phase 2a study. METHODS: We did a double-blind, multicentre......BACKGROUND: Current standard of care for trigeminal neuralgia is treatment with the sodium channel blockers carbamazepine and oxcarbazepine, which although effective are associated with poor tolerability and the need for titration. BIIB074, a Nav1.7-selective, state-dependent sodium-channel blocker...

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

    Science.gov (United States)

    Groome, James R; Winston, Vern

    2013-05-01

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

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

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    Lorena M. Durán-Riveroll

    2017-10-01

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

  2. A residue in the transmembrane segment 6 of domain I in insect and mammalian sodium channels regulate differential sensitivities to pyrethroid insecticides

    Science.gov (United States)

    Oliveira, Eugênio E.; Du, Yuzhe; Nomura, Yoshiko; Dong, Ke

    2013-01-01

    Voltage-gated sodium channels are critical for electrical signaling in the nervous system. Pyrethroid insecticides exert their toxic action by modifying the gating of sodium channels. A valine to methionine mutation in the transmembrane segment 6 of domain I (IS6) of sodium channels from tobacco budworms (Heliothis virescens) has been shown to alter channel gating and reduce insect sodium channel sensitivity to pyrethroids. A valine to leucine substitution was subsequently reported in pyrethroid-resistant bedbug populations. Intriguingly, pyrethroid-resistant mammalian sodium channels possess an isoleucine at the corresponding position. To determine whether different substitutions at this position alter channel gating and confer pyrethroid resistance, we made valine to methionine, isoleucine or leucine substitutions at the corresponding position, V409, in a cockroach sodium channel and examined the gating properties and pyrethroid sensitivity of the three mutants in Xenopus oocytes. All three mutations reduced the channel sensitivity to three pyrethroids (permethrin, cismethrin and deltamethrin). V409M, but not V409I or V409L, caused 6-7 mV depolarizing shifts in the voltage dependences of both activation and inactivation. V409M and V409L slowed channel activation kinetics and accelerated open-state deactivation kinetics, but V409I did not. Furthermore, the substitution of isoleucine with valine, but not with methionine nor leucine, at the corresponding position in a rat skeletal muscle sodium channel, rNav1.4, enhanced channel sensitivity to deltamethrin. Collectively, our study highlights an important role of residues at 409 in regulating not only sodium channel gating, but also the differential sensitivities of insect and mammalian sodium channels to pyrethroids. PMID:23764339

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

    DEFF Research Database (Denmark)

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

    2015-01-01

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

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

    Energy Technology Data Exchange (ETDEWEB)

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

    1994-09-01

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

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

    Science.gov (United States)

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

    2012-10-01

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

  6. The epithelial sodium channel γ-subunit is processed proteolytically in human kidney

    DEFF Research Database (Denmark)

    Langkilde, Rikke Zachar; Skjødt, Karsten; Marcussen, Niels

    2015-01-01

    The epithelial sodium channel (ENaC) of the kidney is necessary for extracellular volume homeostasis and normal arterial BP. Activity of ENaC is enhanced by proteolytic cleavage of the gamma-subunit and putative release of a 43-amino acid inhibitory tract from the gamma-subunit ectodomain. We...... hypothesized that proteolytic processing of gammaENaC occurs in the human kidney under physiologic conditions and that proteinuria contributes to aberrant proteolytic activation. Here, we used monoclonal antibodies (mAbs) with specificity to the human 43-mer inhibitory tract (N and C termini, mAbinhibit, and m......Ab4C11) and the neoepitope generated after proteolytic cleavage at the prostasin/kallikrein cleavage site (K181-V182 and mAbprostasin) to examine human nephrectomy specimens. By immunoblotting, kidney cortex homogenate from patients treated with angiotensin II type 1 receptor antagonists (n=6...

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

    Directory of Open Access Journals (Sweden)

    Chen J

    2012-11-01

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

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

    Science.gov (United States)

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

    2016-08-01

    Mutations in brain isoforms of voltage-gated sodium channels have been identified in patients with distinct epileptic phenotypes. Clinically, these patients often do not respond well to classic anti-epileptics and many remain refractory to treatment. Exogenous as well as endogenous cannabinoids have been shown to target voltage-gated sodium channels and cannabidiol has recently received attention for its potential efficacy in the treatment of childhood epilepsies. In this study, we further investigated the ability of cannabinoids to modulate sodium currents from wild-type and epilepsy-associated mutant voltage-gated sodium channels. We first determined the biophysical consequences of epilepsy-associated missense mutations in both Nav1.1 (arginine 1648 to histidine and asparagine 1788 to lysine) and Nav1.6 (asparagine 1768 to aspartic acid and leucine 1331 to valine) by obtaining whole-cell patch clamp recordings in human embryonic kidney 293T cells with 200 μM Navβ4 peptide in the pipette solution to induce resurgent sodium currents. Resurgent sodium current is an atypical near threshold current predicted to increase neuronal excitability and has been implicated in multiple disorders of excitability. We found that both mutations in Nav1.6 dramatically increased resurgent currents while mutations in Nav1.1 did not. We then examined the effects of anandamide and cannabidiol on peak transient and resurgent currents from wild-type and mutant channels. Interestingly, we found that cannabidiol can preferentially target resurgent sodium currents over peak transient currents generated by wild-type Nav1.6 as well as the aberrant resurgent and persistent current generated by Nav1.6 mutant channels. To further validate our findings, we examined the effects of cannabidiol on endogenous sodium currents from striatal neurons, and similarly we found an inhibition of resurgent and persistent current by cannabidiol. Moreover, current clamp recordings show that cannabidiol reduces

  9. FGF14 N-Terminal Splice Variants Differentially Modulate Nav1.2 and Nav1.6-Encoded Sodium Channels

    Science.gov (United States)

    Laezza, Fernanda; Lampert, Angelika; Kozel, Marie A.; Gerber, Benjamin R.; Rush, Anthony M.; Nerbonne, Jeanne M.; Waxman, Stephen G.; Dib-Hajj, Sulayman D.; Ornitz, David M.

    2009-01-01

    The Intracellular Fibroblast Growth Factor (iFGF) subfamily includes four members (FGFs 11–14) of the structurally related FGF superfamily. Previous studies showed that the iFGFs interact directly with the pore-forming (α) subunits of voltage-gated sodium (Nav) channels and regulate the functional properties of sodium channel currents. Sequence heterogeneity among the iFGFs is thought to confer specificity to this regulation. Here, we demonstrate that the two N-terminal alternatively spliced FGF14 variants, FGF14-1a and FGF14-1b, differentially regulate currents produced by Nav1.2-and Nav1.6 channels. FGF14-1b, but not FGF14-1a, attenuates both Nav1.2 and Nav1.6 current densities. In contrast, co-expression of an FGF14 mutant, lacking the N-terminus, increased Nav1.6 current densities. In neurons, both FGF14-1a and FGF14-1b localized at the axonal initial segment, and deletion of the N-terminus abolished this localization. Thus, the FGF14 N-terminus is required for targeting and functional regulation of Nav channels, suggesting an important function for FGF14 alternative splicing in regulating neuronal excitability. PMID:19465131

  10. The Epithelial Sodium Channel α subunit (α ENaC alternatively spliced form "b" in Dahl rats: What's next?

    Directory of Open Access Journals (Sweden)

    Shehata Marlene F

    2010-07-01

    Full Text Available Abstract Background The amiloride-sensitive Epithelial Sodium Channel (ENaC is critical in maintaining Na+ balance, extracellular fluid volume and long term blood pressure control. ENaC is composed of three main subunits α, β, & γ. While α ENaC is critical for channel functionality, β & γ ENaC maximize channel function. To date, there are four alternatively spliced forms of the α subunit of ENaC (α ENaC-a, -b, -c, & -d that have been published in rats, in addition to the major α ENaC transcript. While α ENaC-a, -c & -d transcripts are low abundance transcripts compared to full-length α ENaC, α ENaC-b is a higher abundance and salt-sensitive transcript compared to full-length α ENaC. Presentation of the hypothesis α ENaC-b protein, which is preferentially produced in Dahl R rats, to a greater extent on high salt diet, exerts a dominant negative effect on full-length α ENaC subunit by physically binding to and trapping full-length α ENaC subunit in the endoplasmic reticulum, and finally accelerating full-length α ENaC proteolytic degradation in a dose-dependent manner. Testing the hypothesis 1 To examine the mRNA and protein abundance of α ENaC-b relative to α ENaC full-length in kidney, lung, and taste tissues of Dahl rats. 2 To compare the expression (mRNA and protein of α ENaC-b in kidneys of Dahl S and R rats on regular and high salt diet. 3 To examine the putative binding of α ENaC-b proteins to full-length α ENaC in vitro and to determine the impact of such binding on full-length α ENaC expression in vitro. Implications of the hypothesis Our studies will be the first to demonstrate the over-expression of salt-sensitive α ENaC-b spliced form in kidney tissues of Dahl R rats at the expense of full-length α ENaC. The current proposal will provide highly novel insights into the putative mechanisms leading to ENaC hypoactivity in high-salt-fed Dahl R rats. Finally, findings from the present proposal will uncover a new

  11. Prostaglandin E2 Upregulated Trigeminal Ganglionic Sodium Channel 1.7 Involving Temporomandibular Joint Inflammatory Pain in Rats.

    Science.gov (United States)

    Zhang, Peng; Gan, Ye-Hua

    2017-06-01

    Prostaglandin E 2 (PGE 2 ) is a key proinflammatory mediator that contributes to inflammatory hyperalgesia. Voltage-gated sodium channel 1.7 (Na v 1.7) plays an important role in inflammatory pain. However, the modulation of Na v 1.7 in inflammatory pain remains poorly understood. We hypothesized that PGE 2 might regulate Na v 1.7 expression in inflammatory pain. We here showed that treatment of rat trigeminal ganglion (TG) explants with PGE 2 significantly upregulated the mRNA and protein expressions of Na v 1.7 through PGE 2 receptor EP2. This finding was confirmed by studies on EP2-selective antagonist PF-04418948. We also demonstrated that Na v 1.7 and COX-2 expressions, as well as PGE 2 levels, were upregulated in the TG after induction of rats' temporomandibular joint (TMJ) inflammation. Correspondingly, hyperalgesia, as indicated by head withdrawal threshold, was observed. Moreover, TMJ inflammation-induced upregulation of Na v 1.7 expression and PGE 2 levels in the TG could be reversed by COX-2-selective inhibitor meloxicam given by oral gavage, and meanwhile, the hyperalgesia of inflamed TMJ was also mitigated. So we concluded that PGE 2 upregulated trigeminal ganglionic Na v 1.7 expression to contribute to TMJ inflammatory pain in rats. Our finding suggests that PGE 2 was an important regulator of Na v 1.7 in TMJ inflammatory pain, which may help increase understanding on the hyperalgesia of peripheral inflammation and develop a new strategy to address inflammatory pain.

  12. A transient receptor potential channel expressed in taste receptor cells.

    Science.gov (United States)

    Pérez, Cristian A; Huang, Liquan; Rong, Minqing; Kozak, J Ashot; Preuss, Axel K; Zhang, Hailin; Max, Marianna; Margolskee, Robert F

    2002-11-01

    We used differential screening of cDNAs from individual taste receptor cells to identify candidate taste transduction elements in mice. Among the differentially expressed clones, one encoded Trpm5, a member of the mammalian family of transient receptor potential (TRP) channels. We found Trpm5 to be expressed in a restricted manner, with particularly high levels in taste tissue. In taste cells, Trpm5 was coexpressed with taste-signaling molecules such as alpha-gustducin, Ggamma13, phospholipase C-beta2 (PLC-beta2) and inositol 1,4,5-trisphosphate receptor type III (IP3R3). Our heterologous expression studies of Trpm5 indicate that it functions as a cationic channel that is gated when internal calcium stores are depleted. Trpm5 may be responsible for capacitative calcium entry in taste receptor cells that respond to bitter and/or sweet compounds.

  13. The plant-derived glucocorticoid receptor agonist Endiandrin A acts as co-stimulator of colonic epithelial sodium channels (ENaC via SGK-1 and MAPKs.

    Directory of Open Access Journals (Sweden)

    Dana Kuntzsch

    Full Text Available In a search for secondary plant compounds that bind to the glucocorticoid receptor (GR, the cyclobutane lignan endiandrin A was discovered from the rainforest tree Endiandra anthropophagorum Domin. Our present study aims to characterize the effect of endiandrin A on GR-dependent induction of colonic sodium transport. The effect of endiandrin A was analyzed in GR-expressing colonic HT-29/B6 cells (HT-29/B6-GR. GR transactivation and subcellular localization were investigated by reporter gene assay and immunofluorescence. Epithelial sodium channel (ENaC was analyzed by qRT-PCR and by measuring amiloride-sensitive short-circuit current (I(sc in Ussing chambers. Endiandrin A (End A has been identified as GR receptor binder. However, it did not cause significant GR transactivation as pGRE-luciferase activity was only 7% of that of the maximum effect of dexamethasone. Interestingly, endiandrin A had a significant impact on dexamethasone-dependent sodium absorption in cells co-exposed to tumor necrosis factor (TNF-α. This was in part due to up-regulation of β- and γ-ENaC subunit expression. Endiandrin A potentiated GR-mediated transcription by increasing GR protein expression and phosphorylation. It inhibited c-Jun N-terminal kinase (JNK activation induced by dexamethasone and/or TNF-α and increased levels of GR localized to the nucleus. Additionally, endiandrin A increased the serum- and glucocorticoid-induced kinase (sgk-1 via activation of p38. Finally, the regulation of ENaC function by endiandrin A was confirmed in rat native colon. In conclusion, endiandrin A potentiates glucocorticoid-driven activation of colonic epithelial sodium channels via JNK inhibition and p38 activation due to transcriptional up-regulation of β- and γ-ENaC-subunits along with induction of sgk-1.

  14. How batrachotoxin modifies the sodium channel permeation pathway: computer modeling and site-directed mutagenesis.

    Science.gov (United States)

    Wang, Sho-Ya; Mitchell, Jane; Tikhonov, Denis B; Zhorov, Boris S; Wang, Ging Kuo

    2006-03-01

    A structural model of the rNav1.4 Na+ channel with batrachotoxin (BTX) bound within the inner cavity suggested that the BTX pyrrole moiety is located between a lysine residue at the DEKA selectivity filter (Lys1237) and an adjacent phenylalanine residue (Phe1236). We tested this pyrrole-binding model by site-directed mutagenesis of Phe1236 at D3/P-loop with 11 amino acids. Mutants F1236D and F1236E expressed poorly, whereas nine other mutants either expressed robust Na+ currents, like the wild-type (F1236Y/Q/K), or somewhat reduced current (F1236G/A/C/N/W/R). Gating properties were altered modestly in most mutant channels, with F1236G displaying the greatest shift in activation and steady-state fast inactivation (-10.1 and -7.5 mV, respectively). Mutants F1236K and F1236R were severely resistant to BTX after 1000 repetitive pulses (+50 mV/20 ms at 2 Hz), whereas seven other mutants were sensitive but with reduced magnitudes compared with the wild type. It is noteworthy that rNav1.4-F1236K mutant Na+ channels remained highly sensitive to block by the local anesthetic bupivacaine, unlike several other BTX-resistant mutant channels. Our data thus support a model in which BTX, when bound within the inner cavity, interacts with the D3/P-loop directly. Such a direct interaction provides clues on how BTX alters the Na+ channel selectivity and conductance.

  15. cAMP-dependent kinase does not modulate the Slack sodium-activated potassium channel.

    Science.gov (United States)

    Nuwer, Megan O; Picchione, Kelly E; Bhattacharjee, Arin

    2009-09-01

    The Slack gene encodes a Na(+)-activated K(+) channel and is expressed in many different types of neurons. Like the prokaryotic Ca(2+)-gated K(+) channel MthK, Slack contains two 'regulator of K(+) conductance' (RCK) domains within its carboxy terminal, domains likely involved in Na(+) binding and channel gating. It also contains multiple consensus protein kinase C (PKC) and protein kinase A (PKA) phosphorylation sites and although regulated by protein kinase C (PKC) phosphorylation, modulation by PKA has not been determined. To test if PKA directly regulates Slack, nystatin-perforated patch whole-cell currents were recorded from a human embryonic kidney (HEK-293) cell line stably expressing Slack. Bath application of forskolin, an adenylate cyclase activator, caused a rapid and complete inhibition of Slack currents however, the inactive homolog of forskolin, 1,9-dideoxyforskolin caused a similar effect. In contrast, bath application of 8-bromo-cAMP did not affect the amplitude nor the activation kinetics of Slack currents. In excised inside-out patch recordings, direct application of the PKA catalytic subunit to patches did not affect the open probability of Slack channels nor was open probability affected by direct application of protein phosphatase 2B. Preincubation of cells with the protein kinase A inhibitor KT5720 also did not change current density. Finally, mutating the consensus phosphorylation site located between RCK domain 1 and domain 2 from serine to glutamate did not affect current activation kinetics. We conclude that unlike PKC, phosphorylation by PKA does not acutely modulate the function and gating activation kinetics of Slack channels.

  16. Identification of cyclic nucleotide gated channels using regular expressions

    KAUST Repository

    Zelman, Alice K.

    2013-09-03

    Cyclic nucleotide-gated channels (CNGCs) are nonselective cation channels found in plants, animals, and some bacteria. They have a six-transmembrane/one- pore structure, a cytosolic cyclic nucleotide-binding domain, and a cytosolic calmodulin-binding domain. Despite their functional similarities, the plant CNGC family members appear to have different conserved amino acid motifs within corresponding functional domains than animal and bacterial CNGCs do. Here we describe the development and application of methods employing plant CNGC-specific sequence motifs as diagnostic tools to identify novel candidate channels in different plants. These methods are used to evaluate the validity of annotations of putative orthologs of CNGCs from plant genomes. The methods detail how to employ regular expressions of conserved amino acids in functional domains of annotated CNGCs and together with Web tools such as PHI-BLAST and ScanProsite to identify novel candidate CNGCs in species including Physcomitrella patens. © Springer Science+Business Media New York 2013.

  17. Pharmacological modification of sodium channels from the human heart atrium in planar lipid bilayers: electrophysiological characterization of responses to batrachotoxin and pentobarbital

    NARCIS (Netherlands)

    Wartenberg, H. C.; Wartenberg, J. P.; Urban, B. W.

    2003-01-01

    BACKGROUND AND OBJECTIVE: To investigate the effects of barbiturates on batrachotoxin-modified sodium channels from different regions of the human heart. Single sodium channels from human atria were studied and compared with existing data from the human ventricle and from the central nervous system.

  18. Decreased expression of Kv7 channels in Hirchsprung's disease.

    Science.gov (United States)

    O'Donnell, Anne-Marie; Coyle, David; Puri, Prem

    2017-07-01

    Voltage-dependent K + channels (Kv channels) participate in electrical rhythmicity and smooth muscle responses and are regulated by excitatory and inhibitory neurotransmitters. Kv channels also participate in the interstitial cell of Cajal (ICC) and smooth muscle cell (SMC) responses to neural inputs. The Kv family consists of 12 subfamilies, Kv1-Kv12, with five members of the Kv7 family identified to date: Kv7.1-Kv7.5. A recent study identified the potassium channel Kv7.5 as having a role in the excitability of ICC-IM in the mouse colon. We therefore designed this study to test the hypothesis that Kv7 channels are present in the normal human colon and are reduced in Hirschprung's disease (HSCR). HSCR tissue specimens were collected at the time of pull-through surgery (n=10), while normal control tissue specimens were obtained at the time of colostomy closure in patients with imperforate anus (n=10). Kv7.3-Kv7.5 immunohistochemistry was performed and visualized using confocal microscopy to assess their distribution. Western blot analysis was undertaken to determine Kv7.3-Kv7.5 protein quantification. Kv7.3 and Kv7.4-immunoreactivity was co-localized with neuron and ICC markers, while Kv7.5 was found to be expressed on both ICCs and SMCs. Western blot analysis revealed similar levels of Kv7.3 and Kv7.5 expression in the normal colon and HSCR colon, while Kv7.4 proteins were found to be markedly decreased in ganglionic specimens and decreased further in aganglionic specimens. A deficiency of Kv7.4 channels in the ganglionic and aganglionic bowel may place a role in colonic dysmotility in HSCR. Copyright © 2017 Elsevier Inc. All rights reserved.

  19. Hoiamide a, a sodium channel activator of unusual architecture from a consortium of two papua new Guinea cyanobacteria.

    Science.gov (United States)

    Pereira, Alban; Cao, Zhengyu; Murray, Thomas F; Gerwick, William H

    2009-08-28

    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 alpha-methylated thiazolines and one thiazole, and a highly oxygenated and methylated C15-polyketide substructure. Pure hoiamide A potently inhibited [(3)H]batrachotoxin binding to voltage-gated sodium channels (IC(50) = 92.8 nM), activated sodium influx (EC(50) = 2.31 microM) in mouse neocortical neurons, and 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.

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

    Science.gov (United States)

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

    2017-01-01

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

  1. Interplay between R513 methylation and S516 phosphorylation of the cardiac voltage-gated sodium channel.

    Science.gov (United States)

    Beltran-Alvarez, Pedro; Feixas, Ferran; Osuna, Sílvia; Díaz-Hernández, Rubí; Brugada, Ramon; Pagans, Sara

    2015-02-01

    Arginine methylation is a novel post-translational modification within the voltage-gated ion channel superfamily, including the cardiac sodium channel, NaV1.5. We show that NaV1.5 R513 methylation decreases S516 phosphorylation rate by 4 orders of magnitude, the first evidence of protein kinase A inhibition by arginine methylation. Reciprocally, S516 phosphorylation blocks R513 methylation. NaV1.5 p.G514C, associated to cardiac conduction disease, abrogates R513 methylation, while leaving S516 phosphorylation rate unchanged. This is the first report of methylation-phosphorylation cross-talk of a cardiac ion channel.

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

    Science.gov (United States)

    Takahashi, Izumi; Yoshino, Masami

    2015-10-01

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

  3. Trimethyloxonium modification of single batrachotoxin-activated sodium channels in planar bilayers. Changes in unit conductance and in block by saxitoxin and calcium

    Science.gov (United States)

    Worley JF; French, RJ; Krueger, BK

    1986-01-01

    Single batrachotoxin-activated sodium channels from rat brain were modified by trimethyloxonium (TMO) after incorporation in planar lipid bilayers. TMO modification eliminated saxitoxin (STX) sensitivity, reduced the single channel conductance by 37%, and reduced calcium block of inward sodium currents. These effects always occurred concomitantly, in an all-or-none fashion. Calcium and STX protected sodium channels from TMO modification with potencies similar to their affinities for block. Calcium inhibited STX binding to rat brain membrane vesicles and relieved toxin block of channels in bilayers, apparently by competing with STX for the toxin binding site. These results suggest that toxins, permeant cations, and blocking cations can interact with a common site on the sodium channel near the extracellular surface. It is likely that permeant cations transiently bind to this superficial site, as the first of several steps in passing inward through the channel. PMID:2419487

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

    Science.gov (United States)

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

    2017-09-27

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

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

    Science.gov (United States)

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

    2012-04-01

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

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

    Science.gov (United States)

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

    2008-09-10

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

  7. An increase in [Ca2+]i activates basolateral chloride channels and inhibits apical sodium channels in frog skin epithelium

    DEFF Research Database (Denmark)

    Brodin, Birger; Rytved, K A; Nielsen, R

    1996-01-01

    The aim of this study was to investigate the mechanisms by which increases in free cytosolic calcium ([Ca2+]i) cause a decrease in macroscopic sodium absorption across principal cells of the frog skin epithelium. [Ca2+]i was measured with fura-2 in an epifluorescence microscope set-up, sodium...

  8. Ethanol alters alveolar fluid balance via Nadph oxidase (NOX signaling to epithelial sodium channels (ENaC in the lung.

    Directory of Open Access Journals (Sweden)

    Charles A Downs

    Full Text Available Chronic alcohol consumption is associated with increased incidence of ICU-related morbidity and mortality, primarily from acute respiratory distress syndrome (ARDS. However, the mechanisms involved are unknown. One explanation is that alcohol regulates epithelial sodium channels (ENaC via oxidant signaling to promote a pro- injury environment. We used small rodent models to mimic acute and chronic alcohol consumption and tested the hypothesis that ethanol (EtOH would affect lung fluid clearance by up-regulating ENaC activity in the lung. Fluorescence labeling of rat lung slices and in vivo mouse lung revealed an increase in ROS production in response to acute EtOH exposure. Using western blots and fluorescein-5-maleimide labeling, we conclude that EtOH exposure modifies cysteines of α-ENaC while data from single channel patch clamp analysis confirm that 0.16% EtOH increased ENaC activity in rat alveolar cells. In vivo lung fluid clearance demonstrated a latent increase in fluid clearance in mice receiving EtOH diet. Ethanol mice given a tracheal instillation of LPS demonstrated early lung fluid clearance compared to caloric control mice and C57Bl/6 mice. Standard biochemical techniques reveal that chronic EtOH consumption resulted in greater protein expression of the catalytic gp91(phox subunit and the obligate Rac1 protein. Collectively these data suggest that chronic EtOH consumption may lead to altered regulation of ENaC, contributing to a 'pro-injury' environment in the alcohol lung.

  9. Epidermal growth factor potentiates in vitro metastatic behaviour of human prostate cancer PC-3M cells: involvement of voltage-gated sodium channel

    Directory of Open Access Journals (Sweden)

    Uysal-Onganer Pinar

    2007-11-01

    Full Text Available Abstract Background Although a high level of functional voltage-gated sodium channel (VGSC expression has been found in strongly metastatic human and rat prostate cancer (PCa cells, the mechanism(s responsible for the upregulation is unknown. The concentration of epidermal growth factor (EGF, a modulator of ion channels, in the body is highest in prostatic fluid. Thus, EGF could be involved in the VGSC upregulation in PCa. The effects of EGF on VGSC expression in the highly metastatic human PCa PC-3M cell line, which was shown previously to express both functional VGSCs and EGF receptors, were investigated. A quantitative approach, from gene level to cell behaviour, was used. mRNA levels were determined by real-time PCR. Protein expression was studied by Western blots and immunocytochemistry and digital image analysis. Functional assays involved measurements of transverse migration, endocytic membrane activity and Matrigel invasion. Results Exogenous EGF enhanced the cells' in vitro metastatic behaviours (migration, endocytosis and invasion. Endogenous EGF had a similar involvement. EGF increased VGSC Nav1.7 (predominant isoform in PCa mRNA and protein expressions. Co-application of the highly specific VGSC blocker tetrodotoxin (TTX suppressed the effect of EGF on all three metastatic cell behaviours studied. Conclusion 1 EGF has a major involvement in the upregulation of functional VGSC expression in human PCa PC-3M cells. (2 VGSC activity has a significant intermediary role in potentiating effect of EGF in human PCa.

  10. Kinetics of intramembrane charge movement and conductance activation of batrachotoxin-modified sodium channels in frog node of Ranvier.

    Science.gov (United States)

    Dubois, J M; Schneider, M F

    1985-09-01

    Sodium current and intramembrane gating charge movement (Q) were monitored in voltage-clamped frog node of Ranvier after modification of all sodium channels by batrachotoxin (BTX). Sodium current activation followed a single-exponential time course, provided a delay was interposed between the onset of the step ON depolarization and that of the current change. The delay decreased with increased ON depolarization and, for a constant ON depolarization, increased with prehyperpolarization. ON charge movement followed a single-exponential time course with time constants tau Q,ON slightly larger than tau Na, ON. For pulses between -70 and -50 mV, tau Q,ON/tau Na,ON = 1.14 +/- 0.08. The OFF charge movement and OFF sodium current tails after a depolarizing pulse followed single-exponential time courses, with tau Q, OFF larger than tau Na, OFF. tau Q,OFF/tau Na,OFF increased with OFF voltage from 1 near -100 mV to 2 near -160 mV. At a set OFF potential (-120 mV), both tau Q,OFF and tau Na,OFF increased with ON pulse duration. The delay in INa activation and the effect of ON pulse duration on tau Q,OFF and tau Na,OFF are inconsistent with a simple two-state, single-transition model for the gating of batrachotoxin-modified sodium channels.

  11. Sculpting ion channel functional expression with engineered ubiquitin ligases

    Science.gov (United States)

    Kanner, Scott A; Morgenstern, Travis

    2017-01-01

    The functional repertoire of surface ion channels is sustained by dynamic processes of trafficking, sorting, and degradation. Dysregulation of these processes underlies diverse ion channelopathies including cardiac arrhythmias and cystic fibrosis. Ubiquitination powerfully regulates multiple steps in the channel lifecycle, yet basic mechanistic understanding is confounded by promiscuity among E3 ligase/substrate interactions and ubiquitin code complexity. Here we targeted the catalytic domain of E3 ligase, CHIP, to YFP-tagged KCNQ1 ± KCNE1 subunits with a GFP-nanobody to selectively manipulate this channel complex in heterologous cells and adult rat cardiomyocytes. Engineered CHIP enhanced KCNQ1 ubiquitination, eliminated KCNQ1 surface-density, and abolished reconstituted K+ currents without affecting protein expression. A chemo-genetic variation enabling chemical control of ubiquitination revealed KCNQ1 surface-density declined with a ~ 3.5 hr t1/2 by impaired forward trafficking. The results illustrate utility of engineered E3 ligases to elucidate mechanisms underlying ubiquitin regulation of membrane proteins, and to achieve effective post-translational functional knockdown of ion channels. PMID:29256394

  12. Convergent Substitutions in a Sodium Channel Suggest Multiple Origins of Toxin Resistance in Poison Frogs.

    Science.gov (United States)

    Tarvin, Rebecca D; Santos, Juan C; O'Connell, Lauren A; Zakon, Harold H; Cannatella, David C

    2016-04-01

    Complex phenotypes typically have a correspondingly multifaceted genetic component. However, the genotype-phenotype association between chemical defense and resistance is often simple: genetic changes in the binding site of a toxin alter how it affects its target. Some toxic organisms, such as poison frogs (Anura: Dendrobatidae), have defensive alkaloids that disrupt the function of ion channels, proteins that are crucial for nerve and muscle activity. Using protein-docking models, we predict that three major classes of poison frog alkaloids (histrionicotoxins, pumiliotoxins, and batrachotoxins) bind to similar sites in the highly conserved inner pore of the muscle voltage-gated sodium channel, Nav1.4. We predict that poison frogs are somewhat resistant to these compounds because they have six types of amino acid replacements in the Nav1.4 inner pore that are absent in all other frogs except for a distantly related alkaloid-defended frog from Madagascar, Mantella aurantiaca. Protein-docking models and comparative phylogenetics support the role of these replacements in alkaloid resistance. Taking into account the four independent origins of chemical defense in Dendrobatidae, phylogenetic patterns of the amino acid replacements suggest that 1) alkaloid resistance in Nav1.4 evolved independently at least seven times in these frogs, 2) variation in resistance-conferring replacements is likely a result of differences in alkaloid exposure across species, and 3) functional constraint shapes the evolution of the Nav1.4 inner pore. Our study is the first to demonstrate the genetic basis of autoresistance in frogs with alkaloid defenses. © The Author 2016. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

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

    Science.gov (United States)

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

    2011-08-01

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

  14. Efficacy of sodium channel blockers in SCN2A early infantile epileptic encephalopathy.

    Science.gov (United States)

    Dilena, Robertino; Striano, Pasquale; Gennaro, Elena; Bassi, Laura; Olivotto, Sara; Tadini, Laura; Mosca, Fabio; Barbieri, Sergio; Zara, Federico; Fumagalli, Monica

    2017-04-01

    Recent clinical evidence supports a targeted therapeutic approach for genetic epileptic encephalopathies based on the molecular dysfunction. A 2-day-old male infant presented with epileptic encephalopathy characterized by burst-suppression EEG background and tonic-clonic migrating partial seizures. The condition was refractory to phenobarbital, pyridoxine, pyridoxal phosphate and levetiracetam, but a dramatic response to an intravenous loading dose of phenytoin was documented by video-EEG monitoring. Over weeks phenytoin was successfully switched to carbamazepine to prevent seizure relapses associated with difficulty in maintaining proper blood levels of phenytoin. Genetic analysis identified a novel de novo heterozygous mutation (c.[4633A>G]p.[Met1545Val]) in SCN2A. At two years and three months of age the patient is still seizure-free on carbamazepine, although a developmental delay is evident. Sodium channel blockers represent the first-line treatment for confirmed or suspected SCN2A-related epileptic encephalopathies. In severe cases with compatible electro-clinical features we propose a treatment algorithm based on a test trial with high dose intravenous phenytoin followed in case of a positive response by carbamazepine, more suitable for long-term maintenance treatment. Because of their rarity, collaborative studies are needed to delineate shared therapeutic protocols for EIEE based on the electro-clinical features and the presumed underlying genetic substrate. Copyright © 2016 The Japanese Society of Child Neurology. Published by Elsevier B.V. All rights reserved.

  15. Effect of ionizing radiation on voltage-sensitive sodium channels in the brain

    International Nuclear Information System (INIS)

    Hunt, W.A.; Mullin, M.J.

    1984-01-01

    It has generally been assumed that the mature central nervous system is not sensitive to ionizing radiation because cell division of neurons no longer occurs. In general, rather large doses have been required in order to observe changes in morphological, physiological, biochemical, and behavioral endpoints. In order to focus on one of the most fundamental mechanisms in the generation and propagation of action potentials, experiments were undertaken to determine whether ionizing radiation could alter the ability of the neurotoxins, veratridine and batrachotoxin, to stimulate /sup 22/Na uptake into synaptosomes. Synaptosomal preparations were irradiated with varying doses of high-energy electrons or gamma photons. Both qualities of radiation reduced /sup 22/Na uptake stimulated by either toxin after doses as low as 100 rad in a concentration-dependent manner. When concentration-response curves for the neurotoxins were derived at increasing doses of radiation, the maximum effectiveness of the toxins was progressively diminished. The data suggest that the central nervous system may be more sensitive to the effects of ionizing radiation than previously believed and that the effects observed may reflect a loss of viable sodium channels. Such effects may have biological significance

  16. Calcium Channel Expression and Applicability as Targeted Therapies in Melanoma

    Directory of Open Access Journals (Sweden)

    A. Macià

    2015-01-01

    Full Text Available The remodeling of Ca2+ signaling is a common finding in cancer pathophysiology serving the purpose of facilitating proliferation, migration, or survival of cancer cells subjected to stressful conditions. One particular facet of these adaptive changes is the alteration of Ca2+ fluxes through the plasma membrane, as described in several studies. In this review, we summarize the current knowledge about the expression of different Ca2+ channels in the plasma membrane of melanoma cells and its impact on oncogenic Ca2+ signaling. In the last few years, new molecular components of Ca2+ influx pathways have been identified in melanoma cells. In addition, new links between Ca2+ homeostasis and specific cell processes important in melanoma tumor progression have been unveiled. Thus, not only do Ca2+ channels appear to have a potential as prognostic markers, but their pharmacological blockade or gene silencing is hinted as interesting therapeutic approaches.

  17. [Ion currents through batrachotoxin-modified sodium channels of node of Ranvier membranes at high positive and negative potentials].

    Science.gov (United States)

    Mozhaeva, G N; Naumova, A P; Khodorov, B I

    1983-01-01

    Ionic currents through batrachotoxin-modified sodium channels in frog nerve fibres were measured over a wide range of membrane potentials. At potentials above +80 mV currents decay in time and their steady-state level decreased as potentials increased. "Instantaneous" current measurements have shown that this phenomenon was due to the decrease in net channel conductance. Scorpion toxin affected current kinetics only slightly at these potentials, which suggested that these decays were not caused by usual inactivation process. Externally applied procaine induced slow (tens of ms) potential-dependent block of batrachotoxin-modified channels at large positive potentials. At large negative potentials (above -100 mV) "instantaneus" currents decreased due to fast voltage-dependent block of the channels by calcium ions.

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

    Science.gov (United States)

    Song, Weihua; Xiao, Yucheng; Chen, Hanying; Ashpole, Nicole M; Piekarz, Andrew D; Ma, Peilin; Hudmon, Andy; Cummins, Theodore R; Shou, Weinian

    2012-01-01

    The deletion of phenylalanine 1486 (F1486del) in the human cardiac voltage-gated sodium channel (hNav1.5) is associated with fatal long QT (LQT) syndrome. In this study we determined how F1486del impairs the functional properties of hNav1.5 and alters action potential firing in heterologous expression systems (human embryonic kidney (HEK) 293 cells) and their native cardiomyocyte background. Cells expressing hNav1.5-F1486del exhibited a loss-of-function alteration, reflected by an 80% reduction of peak current density, and several gain-of-function alterations, including reduced channel inactivation, enlarged window current, substantial augmentation of persistent late sodium current and an increase in ramp current. We also observed substantial action potential duration (APD) prolongation and prominent early afterdepolarizations (EADs) in neonatal cardiomyocytes expressing the F1486del channels, as well as in computer simulations of myocyte activity. In addition, lidocaine sensitivity was dramatically reduced, which probably contributed to the poor therapeutic outcome observed in the patient carrying the hNav1.5-F1486del mutation. Therefore, despite the significant reduction in peak current density, the F1486del mutation also leads to substantial gain-of-function alterations that are sufficient to cause APD prolongation and EADs, the predominant characteristic of LQTs. These data demonstrate that hNav1.5 mutations can have complex functional consequences and highlight the importance of identifying the specific molecular defect when evaluating potential treatments for individuals with prolonged QT intervals. PMID:22826127

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

    Science.gov (United States)

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

    2014-01-16

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

  20. sodium

    International Development Research Centre (IDRC) Digital Library (Canada)

    Les initiatives de réduction de la consommation de sel qui visent l'ensemble de la population et qui ciblent la teneur en sodium des aliments et sensibilisent les consommateurs sont susceptibles de réduire la consommation de sel dans toutes les couches de la population et d'améliorer la santé cardiovasculaire. Ce projet a ...

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

    Science.gov (United States)

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

    2014-03-01

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

  2. Characterization of the binding of the Ptychodiscus brevis neurotoxin T17 to sodium channels in rat brain synaptosomes

    International Nuclear Information System (INIS)

    Poli, M.A.

    1985-01-01

    The lipid-soluble polyether neurotoxins isolated from the marine dinoflagellate Ptychodiscus brevis (formerly Gymnodinium breve) have been determined to bind to a unique receptor site associated with the voltage-sensitive sodium channel in rat brain synaptosomes. Reduction of the C 42 aldehyde function of T34 to the alcohol function of T17 using NaB 3 H 4 yielded 3 H-T17 with a specific activity of 15 Ci;/mmol. Using this specific probe, binding to sodium channels was measured at 4 0 CC, 22 0 C, and 37 0 C. Rosenthal analysis of the binding data yielded a K/sub d/ of 2.9 nM and B/sub max/ of 6.8 pmoles 3 H-T17 per mg of synaptosomal protein at 4 0 C. Both K/sub d/ and B/sub max/ were found to be temperature dependent. Depolarization of the synaptosomes by osmotic lysis resulted in the loss of 34% of the available receptor sites, with no decrease in binding affinity. Unlabeled T17, T34, and synthetic T17 (reduced T34) were equipotent in their ability to displace 3 H-T17 from its specific receptor site. Competition experiments using natural toxin probes specific for sites I-IV on the voltage-sensitive sodium channel demonstrate that 3 H-T17 does not bind to any of the previously-described neurotoxin receptor sites. A fifth site is proposed

  3. Affinity purification of the voltage-sensitive sodium channel from electroplax with resins selective for sialic acid

    Energy Technology Data Exchange (ETDEWEB)

    James, W.M.; Emerick, M.C.; Agnew, W.S. (Yale Univ. School of medicine, New Haven, CT (USA))

    1989-07-11

    The voltage-sensitive sodium channel present in the eel (Electrophorus electricus) has an unusually high content of sialic acid, including {alpha}-(2{yields}8)-linked polysialic acid, not found in other electroplax membrane glycopeptides. Lectins from Limax flavus (LFA) and wheat germ (WGA) proved the most effective of 11 lectin resins tried. The most selective resin was prepared from IgM antibodies against Neisseria meningitidis {alpha}-(2{yields}8)-polysialic acid which were affinity purified and coupled to Sepharose 4B. The sodium channel was found to bind to WGA, LFA, and IgM resins and was readily eluted with the appropriate soluble carbohydrates. Experiments with LFA and IgM resins demonstrated binding and unbinding rates and displacement kinetics, which suggest highly specific binding at multiple sites on the sodium channel protein. In preparative-scale purification of protein previously fractionated by anion-exchange chromatography, without stabilizing TTX, high yields were reproducibly obtained. Further, when detergent extracts were prepared from electroplax membranes fractionated by low-speed sedimentation, a single step over the IgM resin provided a 70-fold purification, yielding specific activities of 3,200 pmol of ({sup 3}H)TTX-binding sites/mg of protein and a single polypeptide of {approximately}285,000 Da on SDS-acrylamide gels. No small peptides were observed after this 5-h isolation. The authors describe a cation-dependent stabilization with millimolar levels of monovalent and micromolar levels of divalent species.

  4. Effects of a novel sodium channel blocker, GSK2339345, in patients with refractory chronic cough
.

    Science.gov (United States)

    Smith, Jaclyn A; McGarvey, Lorcan P A; Badri, Huda; Satia, Imran; Warren, Francis; Siederer, Sarah; Liefaard, Lia; Murdoch, Robert D; Povey, Kathryn; Marks-Konczalik, Joanna

    2017-09-01

    Voltage-gated sodium channels (VGSC) are important in the initiation and propagation of action potentials in afferent sensory nerve fibers responsible for evoking cough. This study investigated the efficacy of GSK2339345, a VGSC inhibitor, in the treatment of refractory chronic cough (RCC). A three-part randomized, double-blind, placebo-controlled, cross-over study was conducted in the UK. In part A, patients with RCC received two inhaled doses of either GSK2339345 or placebo, 4 hours apart during three study periods. Patients were monitored for cough for 8 hours post-first dose using the VitaloJAK, ambulatory cough monitor. In parts B and C, patients underwent full dose-response cough challenges with capsaicin and citric acid respectively following single doses of randomly assigned GSK2339345 or placebo (4 study days). Part A was analyzed using a mixed effects model and parts B and C using population non-linear mixed effects models. Of 16 enrolled patients, 11 completed the study. 8-hour cough counts increased following GSK2339345 treatment compared with placebo (GSK2339345/placebo ratio of adjusted geometric means: 1.26 (90% credible interval 1.10, 1.44), associated with GSK2339345-evoked coughing, recorded during the 2 minutes post-dose. This was not observed with placebo. The effect of GSK2339345 on cough responses during cough challenges was inconclusive. GSK2339345 was well tolerated. While these data could not determine if GSK2339345 reached the target VGSC, they strongly suggest that GSK2339345 has no anti-tussive effect despite reaching airway sensory nerves as evidenced by the evoked transient cough.
.

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

    Science.gov (United States)

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

    2014-06-01

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

  6. Actions of Ethanol on Voltage-Sensitive Sodium Channels: Effects on Neurotoxin-Stimulated Sodium Uptake in Synaptosomes

    Science.gov (United States)

    1985-01-01

    e o t .brane. Using the squid giant axon, Hodgkin and Huxley (1952)R ceived f r publication April 20, 19P4. ’ Supported in part by a Postdoctoral...to 100 mM was required to produce a HODGKIN , A. L. AND HUXLEY , A. F.: A quantitative description of membrane significant inhibitory effect. Previous...scorpion venom; HEPES, 4-(2- 01hydroxyethyl)-l -piporazineethanesulfonic acid. 41" -’--1K_ 414 Mullin and Hunt Vol. 232 perneabilit y to sodium

  7. SK2 channel expression and function in cerebellar Purkinje cells

    NARCIS (Netherlands)

    Hosy, Eric; Piochon, Claire; Teuling, Eva; Rinaldo, Lorenzo; Hansel, Christian

    2011-01-01

    Small-conductance calcium-activated K(+) channels (SK channels) regulate the excitability of neurons and their responsiveness to synaptic input patterns. SK channels contribute to the afterhyperpolarization (AHP) following action potential bursts, and curtail excitatory postsynaptic potentials

  8. Binding of the anticonvulsant drug lamotrigine and the neurotoxin batrachotoxin to voltage-gated sodium channels induces conformational changes associated with block and steady-state activation.

    Science.gov (United States)

    Cronin, Nora B; O'Reilly, Andrias; Duclohier, Hervé; Wallace, B A

    2003-03-21

    Voltage-gated sodium channels are dynamic membrane proteins characterized by rapid conformational changes that switch the molecule between closed resting, activated, and inactivated states. Sodium channels are specifically blocked by the anticonvulsant drug lamotrigine, which preferentially binds to the channel pore in the inactivated open state. Batrachotoxin is a lipid-soluble alkaloid that causes steady-state activation and binds in the inner pore of the sodium channel with overlapping but distinct molecular determinants from those of lamotrigine. Using circular dichroism spectroscopy on purified voltage-gated sodium channels from Electrophorus electricus, the secondary structures associated with the mixture of states present at equilibrium in the absence of these ligands were compared with specific stabilized states in their presence. As the channel shifts to open states, there appears to be a significant change in secondary structure to a more alpha-helical conformation. The observed changes are consistent with increased order involving the S6 segments that form the pore, the domain III-IV linker, and the P-loops that form the outer pore and selectivity filter. A molecular model has been constructed for the sodium channel based on its homology with the pore-forming regions of bacterial potassium channels, and automated docking of the crystal structure of lamotrigine with this model produces a structure in which the close contacts of the drug are with the residues previously identified by mutational studies as forming the binding site for this drug.

  9. Skeletal muscle sodium channelopathies.

    Science.gov (United States)

    Nicole, Sophie; Fontaine, Bertrand

    2015-10-01

    This is an update on skeletal muscle sodium channelopathies since knowledge in the field have dramatically increased in the past years. The relationship between two phenotypes and SCN4A has been confirmed with additional cases that remain extremely rare: severe neonatal episodic laryngospasm mimicking encephalopathy, which should be actively searched for since patients respond well to sodium channel blockers; congenital myasthenic syndromes, which have the particularity to be the first recessive Nav1.4 channelopathy. Deep DNA sequencing suggests the contribution of other ion channels in the clinical expressivity of sodium channelopathies, which may be one of the factors modulating the latter. The increased knowledge of channel molecular structure, the quantity of sodium channel blockers, and the availability of preclinical models would permit a most personalized choice of medication for patients suffering from these debilitating neuromuscular diseases. Advances in the understanding of the molecular structure of voltage-gated sodium channels, as well as availability of preclinical models, would lead to improved medical care of patients suffering from skeletal muscle, as well as other sodium channelopathies.

  10. ASIC and ENaC type sodium channels: conformational states and the structures of the ion selectivity filters.

    Science.gov (United States)

    Hanukoglu, Israel

    2017-02-01

    The acid-sensing ion channels (ASICs) and epithelial sodium channels (ENaC) are members of a superfamily of channels that play critical roles in mechanosensation, chemosensation, nociception, and regulation of blood volume and pressure. These channels look and function like a tripartite funnel that directs the flow of Na + ions into the cytoplasm via the channel pore in the membrane. The subunits that form these channels share a common structure with two transmembrane segments (TM1 and TM2) and a large extracellular part. In most vertebrates, there are five paralogous genes that code for ASICs (ASIC1-ASIC5), and four for ENaC subunits alpha, beta, gamma, and delta (α, β, γ, and δ). While ASICs can form functional channels as a homo- or heterotrimer, ENaC functions as an obligate heterotrimer composed of α-β-γ or β-γ-δ subunits. The structure of ASIC has been determined in several conformations, including desensitized and open states. This review presents a comparison of the structures of these states using easy-to-understand molecular models of the full complex, the central tunnel that includes an outer vestibule, the channel pore, and ion selectivity filter. The differences in the secondary, tertiary, and quaternary structures of the states are summarized to pinpoint the conformational changes responsible for channel opening. Results of site-directed mutagenesis studies of ENaC subunits are examined in light of ASIC1 models. Based on these comparisons, a molecular model for the selectivity filter of ENaC is built by in silico mutagenesis of an ASIC1 structure. These models suggest that Na + ions pass through the filter in a hydrated state. © 2016 Federation of European Biochemical Societies.

  11. Spontaneous excitation patterns computed for axons with injury-like impairments of sodium channels and Na/K pumps.

    Directory of Open Access Journals (Sweden)

    Na Yu

    Full Text Available In injured neurons, "leaky" voltage-gated sodium channels (Nav underlie dysfunctional excitability that ranges from spontaneous subthreshold oscillations (STO, to ectopic (sometimes paroxysmal excitation, to depolarizing block. In recombinant systems, mechanical injury to Nav1.6-rich membranes causes cytoplasmic Na(+-loading and "Nav-CLS", i.e., coupled left-(hyperpolarizing-shift of Nav activation and availability. Metabolic injury of hippocampal neurons (epileptic discharge results in comparable impairment: left-shifted activation and availability and hence left-shifted I(Na-window. A recent computation study revealed that CLS-based I(Na-window left-shift dissipates ion gradients and impairs excitability. Here, via dynamical analyses, we focus on sustained excitability patterns in mildly damaged nodes, in particular with more realistic Gaussian-distributed Nav-CLS to mimic "smeared" injury intensity. Since our interest is axons that might survive injury, pumps (sine qua non for live axons are included. In some simulations, pump efficacy and system volumes are varied. Impacts of current noise inputs are also characterized. The diverse modes of spontaneous rhythmic activity evident in these scenarios are studied using bifurcation analysis. For "mild CLS injury", a prominent feature is slow pump/leak-mediated E(Ion oscillations. These slow oscillations yield dynamic firing thresholds that underlie complex voltage STO and bursting behaviors. Thus, Nav-CLS, a biophysically justified mode of injury, in parallel with functioning pumps, robustly engenders an emergent slow process that triggers a plethora of pathological excitability patterns. This minimalist "device" could have physiological analogs. At first nodes of Ranvier and at nociceptors, e.g., localized lipid-tuning that modulated Nav midpoints could produce Nav-CLS, as could co-expression of appropriately differing Nav isoforms.

  12. Developmental regulation of voltage-sensitive sodium channels in rat skeletal muscle

    International Nuclear Information System (INIS)

    Sherman, S.J.

    1985-01-01

    The developmental regulation of the voltage-sensitive Na + channel in rat skeletal muscle was studied in vivo and in vitro. In triceps surae muscle developing in vivo the development of TTX-sensitive Na + channel occurred primarily during the first three postnatal weeks as determined by the specific binding of [ 3 H]saxitoxin. This development proceeded in two separate phases. The first phase occurs independently of continuing motor neuron innervation and accounts for 60% of the adult density of TTX-sensitive Na + channels. The second phase, which begins about day 11, requires innervation. Muscle cells in primary culture were found to have both TTX-sensitive and insensitive Na + channels. The development of the TTX-sensitive channel, in vitro, paralleled the initial innervation-independent phase of development observed in vivo. The density of TTX-sensitive Na + channels in cultured muscle cells was regulated by electrical activity and cytosolic Ca ++ levels. Pharmacological blockade of the spontaneous electrical activity present in these cells lead to a nearly 2-fold increase in the surface density of TTX-sensitive channels. The turnover time of the TTX-sensitive Na + channel was measured by blocking the incorporation of newly synthesized channels with tunicamycin, an inhibitor of N-linked protein glycosylation. The regulation of channel density by electrical activity, cytosolic Ca ++ levels, and agents affecting cyclic neucleotide levels had no effect on the turnover time of the TTX-sensitive Na + channel, indicating that these regulatory agents instead affect the synthesis of the channel

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

    Science.gov (United States)

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

    2017-04-01

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

  14. Does the Altered Expression of Ion Channels Give Rise to the Enhanced Excitability of Neurons Isolated from Nf1 +/- Mice?

    Science.gov (United States)

    2011-09-01

    complications of neurofibromatosis type 1 in adulthood. Brain 122:473-481. Cregg R, Momin A, Rugiero F, Wood JN, Zhao J (2010) Pain channelopathies . J...TR (2010) Human voltage-gated sodium channel mutations that cause inherited neuronal and muscle channelopathies increase resurgent sodium currents...Jackson JO 2nd, Cummins TR (2010) Hu- man voltage-gated sodium channel mutations that cause inherited neuronal and muscle channelopathies increase

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

    Science.gov (United States)

    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.

  16. Tetraethylammonium block of water flux in Aquaporin-1 channels expressed in kidney thin limbs of Henle's loop and a kidney-derived cell line.

    Directory of Open Access Journals (Sweden)

    Pannabecker Thomas L

    2002-03-01

    Full Text Available Abstract Background Aquaporin-1 (AQP1 channels are constitutively active water channels that allow rapid transmembrane osmotic water flux, and also serve as cyclic-GMP-gated ion channels. Tetraethylammonium chloride (TEA; 0.05 to 10 mM was shown previously to inhibit the osmotic water permeability of human AQP1 channels expressed in Xenopus oocytes. The purpose of the present study was to determine if TEA blocks osmotic water flux of native AQP1 channels in kidney, and recombinant AQP1 channels expressed in a kidney derived MDCK cell line. We also demonstrate that TEA does not inhibit the cGMP-dependent ionic conductance of AQP1 expressed in oocytes, supporting the idea that water and ion fluxes involve pharmacologically distinct pathways in the AQP1 tetrameric complex. Results TEA blocked water permeability of AQP1 channels in kidney and kidney-derived cells, demonstrating this effect is not limited to the oocyte expression system. Equivalent inhibition is seen in MDCK cells with viral-mediated AQP1 expression, and in rat renal descending thin limbs of Henle's loops which abundantly express native AQP1, but not in ascending thin limbs which do not express AQP1. External TEA (10 mM does not block the cGMP-dependent AQP1 ionic conductance, measured by two-electrode voltage clamp after pre-incubation of oocytes in 8Br-cGMP (10–50 mM or during application of the nitric oxide donor, sodium nitroprusside (2–4 mM. Conclusions TEA selectively inhibits osmotic water permeability through native and heterologously expressed AQP1 channels. The pathways for water and ions in AQP1 differ in pharmacological sensitivity to TEA, and are consistent with the idea of independent solute pathways within the channel structure. The results confirm the usefulness of TEA as a pharmacological tool for the analysis of AQP1 function.

  17. Regulation of sodium channel function by bilayer elasticity: the importance of hydrophobic coupling. Effects of Micelle-forming amphiphiles and cholesterol

    DEFF Research Database (Denmark)

    Lundbæk, Jens August; Birn, Pia; Hansen, Anker J

    2004-01-01

    , Triton X-100, and reduced Triton X-100) that make lipid bilayers less "stiff", as measured using gA channels, shift the voltage dependence of sodium channel inactivation toward more hyperpolarized potentials. At low amphiphile concentration, the magnitude of the shift is linearly correlated to the change...

  18. The jellyfish green fluorescent protein: a new tool for studying ion channel expression and function.

    Science.gov (United States)

    Marshall, J; Molloy, R; Moss, G W; Howe, J R; Hughes, T E

    1995-02-01

    Two methods are described for using the jellyfish green fluorescent protein (GFP) as a reporter gene for ion channel expression. GFP fluorescence can be used to identify the transfected cells, and to estimate the relative levels of ion channel expression, in cotransfection experiments. A GFP-NMDAR1 chimera can be constructed that produces a functional, fluorescent receptor subunit. These methods should facilitate studies of ion channel expression, localization, and processing.

  19. Developmentally-regulated sodium channel subunits are differentially sensitive to α–cyano containing pyrethroids

    Science.gov (United States)

    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...

  20. Characterization of the binding of the Ptychodiscus brevis neurotoxin T17 to sodium channels in rat brain synaptosomes

    Energy Technology Data Exchange (ETDEWEB)

    Poli, M.A.

    1985-01-01

    The lipid-soluble polyether neurotoxins isolated from the marine dinoflagellate Ptychodiscus brevis (formerly Gymnodinium breve) have been determined to bind to a unique receptor site associated with the voltage-sensitive sodium channel in rat brain synaptosomes. Reduction of the C/sub 42/ aldehyde function of T34 to the alcohol function of T17 using NaB/sup 3/H/sub 4/ yielded /sup 3/H-T17 with a specific activity of 15 Ci;/mmol. Using this specific probe, binding to sodium channels was measured at 4/sup 0/CC, 22/sup 0/C, and 37/sup 0/C. Rosenthal analysis of the binding data yielded a K/sub d/ of 2.9 nM and B/sub max/ of 6.8 pmoles /sup 3/H-T17 per mg of synaptosomal protein at 4/sup 0/C. Both K/sub d/ and B/sub max/ were found to be temperature dependent. Depolarization of the synaptosomes by osmotic lysis resulted in the loss of 34% of the available receptor sites, with no decrease in binding affinity. Unlabeled T17, T34, and synthetic T17 (reduced T34) were equipotent in their ability to displace /sup 3/H-T17 from its specific receptor site. Competition experiments using natural toxin probes specific for sites I-IV on the voltage-sensitive sodium channel demonstrate that /sup 3/H-T17 does not bind to any of the previously-described neurotoxin receptor sites. A fifth site is proposed.

  1. Cardiac sodium channelopathies

    NARCIS (Netherlands)

    Amin, A.S.; Asghari-Roodsari, A.; Tan, H.L.

    2010-01-01

    Cardiac sodium channel are protein complexes that are expressed in the sarcolemma of cardiomyocytes to carry a large inward depolarizing current (I-Na) during phase 0 of the cardiac action potential. The importance of I-Na for normal cardiac electrical activity is reflected by the high incidence of

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

    Czech Academy of Sciences Publication Activity Database

    Zemková, Hana; Tomič, M.; Kučka, M.; Aguilera, G.; Stojilkovic, S. S.

    2016-01-01

    Roč. 157, č. 4 (2016), s. 1576-1589 ISSN 0013-7227 R&D Projects: GA ČR(CZ) GBP304/12/G069; GA MŠk(CZ) LQ1604; GA MŠk(CZ) ED1.1.00/02.0109 Institutional support: RVO:67985823 Keywords : action potential * background sodium conductance * bursting activity * cation-conducting channels * cytosolic calcium concentration * resting membrane potential Subject RIV: FB - Endocrinology, Diabetology, Metabolism, Nutrition Impact factor: 4.286, year: 2016

  3. Local anesthetics QX 572 and benzocaine act at separate sites on the batrachotoxin-activated sodium channel

    OpenAIRE

    1981-01-01

    We have studied the effect of local anesthetics QX 572, which is permanently charged, and benzocaine, which is neutral, on batrachotoxin- activated sodium channels in mouse neuroblastoma N18 cells. The dose- response curves for each drug suggest that QX 752 and benzocaine each act on a single class of binding sites. The dissociation constants are 3.15 X 10(-5) M for QX 572 and 2.65 X 10(-4) M for benzocaine. Equilibrium and kinetic experiments indicate that both drugs are competitive inhibito...

  4. Spin-dependent observables in electron-sodium scattering calculated using the coupled-channel optical method

    International Nuclear Information System (INIS)

    Bray, I.; McCarthy, I.E.

    1992-06-01

    The calculations of 3 2 S-3 S and 3 2 S-3 2 P electron sodium scattering at 1 to 40 eV is presented. An excellent agreement with measurements of the spin asymmetries and P'/P for both channels, and L the angular momentum for singlet, triplet, and summed spin states at all energies was found. This may only be achieved at energies above the ionization threshold by including the coupling of the low-lying target discrete states to the target continuum. 25 refs., 6 figs

  5. Cloning and expression analysis of potassium channel gene NKT3 ...

    African Journals Online (AJOL)

    Potassium (K+) is the predominant inorganic ion of plant cells. K+ channels in higher plant cells play an important role in regulating the influx and efflux of K+ from cells, and activity of these channels might be involved in plant stress resistance. A completely new K+ channel gene of Nicotiana tabacum was obtained through ...

  6. Prolongation of action potential duration and QT interval during epilepsy linked to increased contribution of neuronal sodium channels to cardiac late Na+ current: potential mechanism for sudden death in epilepsy.

    Science.gov (United States)

    Biet, Michael; Morin, Nathalie; Lessard-Beaudoin, Melissa; Graham, Rona K; Duss, Sandra; Gagné, Jonathan; Sanon, Nathalie T; Carmant, Lionel; Dumaine, Robert

    2015-08-01

    Arrhythmias associated with QT prolongation on the ECG often lead to sudden unexpected death in epilepsy. The mechanism causing a prolongation of the QT interval during epilepsy remains unknown. Based on observations showing an upregulation of neuronal sodium channels in the brain during epilepsy, we tested the hypothesis that a similar phenomenon occurs in the heart and contributes to QT prolongation by altering cardiac sodium current properties (INa). We used the patch clamp technique to assess the effects of epilepsy on the cardiac action potential and INa in rat ventricular myocytes. Consistent with QT prolongation, epileptic rats had longer ventricular action potential durations attributable to a sustained component of INa (INaL). The increase in INaL was because of a larger contribution of neuronal Na channels characterized by their high sensitivity to tetrodotoxin. As in the brain, epilepsy was associated with an enhanced expression of the neuronal isoform NaV1.1 in cardiomyocyte. Epilepsy was also associated with a lower INa activation threshold resulting in increased cell excitability. This is the first study correlating increased expression of neuronal sodium channels within the heart to epilepsy-related cardiac arrhythmias. This represents a new paradigm in our understanding of cardiac complications related to epilepsy. © 2015 American Heart Association, Inc.

  7. A digital atlas of ion channel expression patterns in the two-week-old rat brain.

    Science.gov (United States)

    Shcherbatyy, Volodymyr; Carson, James; Yaylaoglu, Murat; Jäckle, Katharina; Grabbe, Frauke; Brockmeyer, Maren; Yavuz, Halenur; Eichele, Gregor

    2015-01-01

    The approximately 350 ion channels encoded by the mammalian genome are a main pillar of the nervous system. We have determined the expression pattern of 320 channels in the two-week-old (P14) rat brain by means of non-radioactive robotic in situ hybridization. Optimized methods were developed and implemented to generate stringently coronal brain sections. The use of standardized methods permits a direct comparison of expression patterns across the entire ion channel expression pattern data set and facilitates recognizing ion channel co-expression. All expression data are made publically available at the Genepaint.org database. Inwardly rectifying potassium channels (Kir, encoded by the Kcnj genes) regulate a broad spectrum of physiological processes. Kcnj channel expression patterns generated in the present study were fitted with a deformable subdivision mesh atlas produced for the P14 rat brain. This co-registration, when combined with numerical quantification of expression strengths, allowed for semi-quantitative automated annotation of expression patterns as well as comparisons among and between Kcnj subfamilies. The expression patterns of Kcnj channel were also cross validated against previously published expression patterns of Kcnj channel genes.

  8. TRPA1 expression levels and excitability brake by KV channels influence cold sensitivity of TRPA1-expressing neurons.

    Science.gov (United States)

    Memon, Tosifa; Chase, Kevin; Leavitt, Lee S; Olivera, Baldomero M; Teichert, Russell W

    2017-06-14

    The molecular sensor of innocuous (painless) cold sensation is well-established to be transient receptor potential cation channel, subfamily M, member 8 (TRPM8). However, the role of transient receptor potential cation channel, subfamily A, member 1 (TRPA1) in noxious (painful) cold sensation has been controversial. We find that TRPA1 channels contribute to the noxious cold sensitivity of mouse somatosensory neurons, independent of TRPM8 channels, and that TRPA1-expressing neurons are largely non-overlapping with TRPM8-expressing neurons in mouse dorsal-root ganglia (DRG). However, relatively few TRPA1-expressing neurons (e.g., responsive to allyl isothiocyanate or AITC, a selective TRPA1 agonist) respond overtly to cold temperature in vitro, unlike TRPM8-expressing neurons, which almost all respond to cold. Using somatosensory neurons from TRPM8-/- mice and subtype-selective blockers of TRPM8 and TRPA1 channels, we demonstrate that responses to cold temperatures from TRPA1-expressing neurons are mediated by TRPA1 channels. We also identify two factors that affect the cold-sensitivity of TRPA1-expressing neurons: (1) cold-sensitive AITC-sensitive neurons express relatively more TRPA1 transcripts than cold-insensitive AITC-sensitive neurons and (2) voltage-gated potassium (K V ) channels attenuate the cold-sensitivity of some TRPA1-expressing neurons. The combination of these two factors, combined with the relatively weak agonist-like activity of cold temperature on TRPA1 channels, partially explains why few TRPA1-expressing neurons respond to cold. Blocking K V channels also reveals another subclass of noxious cold-sensitive DRG neurons that do not express TRPM8 or TRPA1 channels. Altogether, the results of this study provide novel insights into the cold-sensitivity of different subclasses of somatosensory neurons. Copyright © 2017 IBRO. Published by Elsevier Ltd. All rights reserved.

  9. Age-dependent expression of Nav1.9 channels in medial prefrontal cortex pyramidal neurons in rats.

    Science.gov (United States)

    Gawlak, Maciej; Szulczyk, Bartłomiej; Berłowski, Adam; Grzelka, Katarzyna; Stachurska, Anna; Pełka, Justyna; Czarzasta, Katarzyna; Małecki, Maciej; Kurowski, Przemysław; Nurowska, Ewa; Szulczyk, Paweł

    2017-12-01

    Developmental changes that occur in the prefrontal cortex during adolescence alter behavior. These behavioral alterations likely stem from changes in prefrontal cortex neuronal activity, which may depend on the properties and expression of ion channels. Nav1.9 sodium channels conduct a Na + current that is TTX resistant with a low threshold and noninactivating over time. The purpose of this study was to assess the presence of Nav1.9 channels in medial prefrontal cortex (mPFC) layer II and V pyramidal neurons in young (20-day old), late adolescent (60-day old), and adult (6- to 7-month old) rats. First, we demonstrated that layer II and V mPFC pyramidal neurons in slices obtained from young rats exhibited a TTX-resistant, low-threshold, noninactivating, and voltage-dependent Na + current. The mRNA expression of the SCN11a gene (which encodes the Nav1.9 channel) in mPFC tissue was significantly higher in young rats than in late adolescent and adult rats. Nav1.9 protein was immunofluorescently labeled in mPFC cells in slices and analyzed via confocal microscopy. Nav1.9 immunolabeling was present in layer II and V mPFC pyramidal neurons and was more prominent in the neurons of young rats than in the neurons of late adolescent and adult rats. We conclude that Nav1.9 channels are expressed in layer II and V mPFC pyramidal neurons and that Nav1.9 protein expression in the mPFC pyramidal neurons of late adolescent and adult rats is lower than that in the neurons of young rats. © 2017 Wiley Periodicals, Inc. Develop Neurobiol 77: 1371-1384, 2017. © 2017 Wiley Periodicals, Inc.

  10. Glioblastoma cancer stem cell lines express functional acid sensing ion channels ASIC1a and ASIC3

    DEFF Research Database (Denmark)

    Tian, Yuemin; Bresenitz, Pia; Reska, Anna

    2017-01-01

    Acidic microenvironment is commonly observed in tumour tissues, including glioblastoma (GBM), the most aggressive and lethal brain tumour in adults. Acid sensing ion channels (ASICs) are neuronal voltage-insensitive sodium channels, which are sensors of extracellular protons. Here we studied...... and functionally characterized ASICs in two primary glioblastoma stem cell lines as cell culture models. We detected transcripts of the ACCN2 and ACCN3 genes, coding for ASIC1 and ASIC3, respectively, but not transcripts of ACCN1 (coding for ASIC2). Available microarray data confirmed that ACCN1 is downregulated...... in glioma. Western blotting confirmed expression of ASIC1 and ASIC3, the most proton-sensitive ASICs, in both GBM cell lines. We characterized ASICs functionally using whole-cell patch clamp and detected different types of acid-sensitive currents. Some of these currents had kinetics typical for ASICs...

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

    Directory of Open Access Journals (Sweden)

    Janelle Elizabeth O'Brien

    2013-10-01

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

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

    Science.gov (United States)

    O'Leary, Michael E; Hancox, Jules C

    2010-01-01

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

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

    Directory of Open Access Journals (Sweden)

    Somayeh Mahdavi

    2014-12-01

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

  14. A De Novo Mutation in the Sodium-Activated Potassium Channel KCNT2 Alters Ion Selectivity and Causes Epileptic Encephalopathy

    Directory of Open Access Journals (Sweden)

    Sushmitha Gururaj

    2017-10-01

    Full Text Available Early infantile epileptic encephalopathies (EOEE are a debilitating spectrum of disorders associated with cognitive impairments. We present a clinical report of a KCNT2 mutation in an EOEE patient. The de novo heterozygous variant Phe240Leu SLICK was identified by exome sequencing and confirmed by Sanger sequencing. Phe240Leu rSlick and hSLICK channels were electrophysiologically, heterologously characterized to reveal three significant alterations to channel function. First, [Cl−]i sensitivity was reversed in Phe240Leu channels. Second, predominantly K+-selective WT channels were made to favor Na+ over K+ by Phe240Leu. Third, and consequent to altered ion selectivity, Phe240Leu channels had larger inward conductance. Further, rSlick channels induced membrane hyperexcitability when expressed in primary neurons, resembling the cellular seizure phenotype. Taken together, our results confirm that Phe240Leu is a “change-of-function” KCNT2 mutation, demonstrating unusual altered selectivity in KNa channels. These findings establish pathogenicity of the Phe240Leu KCNT2 mutation in the reported EOEE patient.

  15. Biophysical costs associated with tetrodotoxin resistance in the sodium channel pore of the garter snake, Thamnophis sirtalis.

    Science.gov (United States)

    Lee, Chong Hyun; Jones, David K; Ahern, Christopher; Sarhan, Maen F; Ruben, Peter C

    2011-01-01

    Tetrodotoxin (TTX) is a potent toxin that specifically binds to voltage-gated sodium channels (NaV). TTX binding physically blocks the flow of sodium ions through NaV, thereby preventing action potential generation and propagation. TTX has different binding affinities for different NaV isoforms. These differences are imparted by amino acid substitutions in positions within, or proximal to, the TTX-binding site in the channel pore. These substitutions confer TTX-resistance to a variety of species. The garter snake Thamnophis sirtalis has evolved TTX-resistance over the course of an arms race, allowing some populations of snakes to feed on tetrodotoxic newts, including Taricha granulosa. Different populations of the garter snake have different degrees of TTX-resistance, which is closely related to the number of amino acid substitutions. We tested the biophysical properties and ion selectivity of NaV of three garter snake populations from Bear Lake, Idaho; Warrenton, Oregon; and Willow Creek, California. We observed changes in gating properties of TTX-resistant (TTXr) NaV. In addition, ion selectivity of TTXr NaV was significantly different from that of TTX-sensitive NaV. These results suggest TTX-resistance comes at a cost to performance caused by changes in the biophysical properties and ion selectivity of TTXr NaV.

  16. Mutations at opposite ends of the DIII/S4-S5 linker of sodium channel NaV1.7 produce distinct pain disorders

    Directory of Open Access Journals (Sweden)

    Tyrrell Lynda

    2010-04-01

    Full Text Available Abstract Background Two groups of gain-of-function mutations in sodium channel NaV1.7, which are expressed in dorsal root ganglion (DRG neurons, produce two clinically-distinct pain syndromes - inherited erythromelalgia (IEM and paroxysmal extreme pain disorder (PEPD. IEM is characterized by intermittent burning pain and skin redness in the feet or hands, triggered by warmth or mild exercise, while PEPD is characterized by episodes of rectal, ocular and mandibular pain accompanied with skin flushing, triggered by bowel movement and perianal stimulation. Most of the IEM mutations are located within channel domains I and II, while most of the PEPD mutations are located within domains III and IV. The structural dichotomy parallels the biophysical effects of the two types of mutations, with IEM mutations shifting voltage-dependence of NaV1.7 activation in a hyperpolarized direction, and PEPD mutations shifting fast-inactivation of NaV1.7 in a depolarized direction. While four IEM and four PEPD mutations are located within cytoplasmic linkers joining segments 4 and 5 (S4-S5 linkers in the different domains (IEM: domains I and II; PEPD: domains III and IV, no S4-S5 linker has been reported to house both IEM and PEPD mutations thus far. Results We have identified a new IEM mutation P1308L within the C-terminus of the DIII/S4-S5 linker of NaV1.7, ten amino acids from a known PEPD mutation V1298F which is located within the N-terminus of this linker. We used voltage-clamp to compare the biophysical properties of the two mutant channels and current-clamp to study their effects on DRG neuron excitability. We confirm that P1308L and V1298F behave as prototypical IEM and PEPD mutations, respectively. We also show that DRG neurons expressing either P1308L or V1298F become hyperexcitable, compared to DRG neurons expressing wild-type channels. Conclusions Our results provide evidence for differential roles of the DIII/S4-S5 linker N- and C-termini in channel

  17. Functional reconstitution of the voltage-regulated sodium channel purified from electroplax of Electrophorus electricus

    Energy Technology Data Exchange (ETDEWEB)

    Rosenberg, R.L.

    1985-01-01

    The voltage-regulated NA channel is responsible for the depolarization of the excitable cell membrane during the normal action potential. This research has focused on the functional properties of the Na channel, purified from detergent extracts of electroplax membranes of the electric eel, and reconstituted into vesicles of defined phospholipid. These properties were assessed by measuring neurotoxin-modulated ion flux into the reconstituted membrane vesicles and by recording the single-channel currents of the purified channel by the patch-clamp method. The binding of tritiated tetrodotoxin (TTX) was employed as a marker for the purification of the channel. Two high-resolution fractionation steps, based on molecular charge and protein size, were used to obtain a preparation that is 80% homogeneous for a large peptide of 270,000 daltons. Radiotracer /sup 22/Na/sup +/ influx into the vesicles was stimulated by veratridine and by batrachotoxin (BTX) at concentrations of 100 ..mu..M and 5 ..mu..M, respectively. The stimulation by BTX was greater than that by veratridine, and can be as much as 16-fold over control influx levels. The stimulated influx is blocked by TTX with a K/sub i/ of 35 nM, and by local anesthetics in the normal pharmacological range. Large multilamellar vesicles prepared with a freeze-thaw step are suitable for single-channel recording techniques. When excised patches of the reconstituted membranes were voltage-clamped in the absence of activating neurotoxins, voltage-dependent single-channel currents were recorded. These displayed properties similar to those from native membranes of nerve and muscle. These results indicate that the protein purified on the basis of TTX binding is a functional Na channel possessing these functional domains: the ion-selective channel, the voltage sensors controlling activation and inactivation, and the sites of action of TTX, alkaloid neurotoxins, and local anesthetics.

  18. Functional reconstitution of the voltage-regulated sodium channel purified from electroplax of Electrophorus electricus

    International Nuclear Information System (INIS)

    Rosenberg, R.L.

    1985-01-01

    The voltage-regulated NA channel is responsible for the depolarization of the excitable cell membrane during the normal action potential. This research has focused on the functional properties of the Na channel, purified from detergent extracts of electroplax membranes of the electric eel, and reconstituted into vesicles of defined phospholipid. These properties were assessed by measuring neurotoxin-modulated ion flux into the reconstituted membrane vesicles and by recording the single-channel currents of the purified channel by the patch-clamp method. The binding of tritiated tetrodotoxin (TTX) was employed as a marker for the purification of the channel. Two high-resolution fractionation steps, based on molecular charge and protein size, were used to obtain a preparation that is 80% homogeneous for a large peptide of 270,000 daltons. Radiotracer 22 Na + influx into the vesicles was stimulated by veratridine and by batrachotoxin (BTX) at concentrations of 100 μM and 5 μM, respectively. The stimulation by BTX was greater than that by veratridine, and can be as much as 16-fold over control influx levels. The stimulated influx is blocked by TTX with a K/sub i/ of 35 nM, and by local anesthetics in the normal pharmacological range. Large multilamellar vesicles prepared with a freeze-thaw step are suitable for single-channel recording techniques. When excised patches of the reconstituted membranes were voltage-clamped in the absence of activating neurotoxins, voltage-dependent single-channel currents were recorded. These displayed properties similar to those from native membranes of nerve and muscle. These results indicate that the protein purified on the basis of TTX binding is a functional Na channel possessing these functional domains: the ion-selective channel, the voltage sensors controlling activation and inactivation, and the sites of action of TTX, alkaloid neurotoxins, and local anesthetics

  19. Trans-channel interactions in batrachotoxin-modified rat skeletal muscle sodium channels: kinetic analysis of mutual inhibition between mu-conotoxin GIIIA derivatives and amine blockers.

    Science.gov (United States)

    Ma, Quanli; Pavlov, Evgeny; Britvina, Tatiana; Zamponi, Gerald W; French, Robert J

    2008-11-01

    R13X derivatives of mu-conotoxin GIIIA bind externally to single sodium channels and block current incompletely with mean "blocked" durations of several seconds. We studied interactions between two classes of blockers (mu-conotoxins and amines) by steady state, kinetic analysis of block of BTX-modified Na channels in planar bilayers. The amines cause all-or-none block at a site internal to the selectivity filter. TPrA and DEA block single Na channels with very different kinetics. TPrA induces discrete, all-or-none, blocked events (mean blocked durations, approximately 100 ms), whereas DEA produces a concentration-dependent reduction of the apparent single channel amplitude ("fast" block). These distinct modes of action allow simultaneous evaluation of block by TPrA and DEA, showing a classical, competitive interaction between them. The apparent affinity of TPrA decreases with increasing [DEA], based on a decrease in the association rate for TPrA. When an R13X mu-conotoxin derivative and one of the amines are applied simultaneously on opposite sides of the membrane, a mutually inhibitory interaction is observed. Dissociation constants, at +50 mV, for TPrA ( approximately 4 mM) and DEA ( approximately 30 mM) increase by approximately 20%-50% when R13E (nominal net charge, +4) or R13Q (+5) is bound. Analysis of the slow blocking kinetics for the two toxin derivatives showed comparable decreases in affinity of the mu-conotoxins in the presence of an amine. Although this mutual inhibition seems to be qualitatively consistent with an electrostatic interaction across the selectivity filter, quantitative considerations raise questions about the mechanistic details of the interaction.

  20. A Mutation in the Intracellular Loop III/IV of Mosquito Sodium Channel Synergizes the Effect of Mutations in Helix IIS6 on Pyrethroid Resistance

    Science.gov (United States)

    Wang, Lingxin; Nomura, Yoshiko; Du, Yuzhe; Liu, Nannan; Zhorov, Boris S.

    2015-01-01

    Activation and inactivation of voltage-gated sodium channels are critical for proper electrical signaling in excitable cells. Pyrethroid insecticides promote activation and inhibit inactivation of sodium channels, resulting in prolonged opening of sodium channels. They preferably bind to the open state of the sodium channel by interacting with two distinct receptor sites, pyrethroid receptor sites PyR1 and PyR2, formed by the interfaces of domains II/III and I/II, respectively. Specific mutations in PyR1 or PyR2 confer pyrethroid resistance in various arthropod pests and disease vectors. Recently, a unique mutation, N1575Y, in the cytoplasmic loop linking domains III and IV (LIII/IV) was found to coexist with a PyR2 mutation, L1014F in IIS6, in pyrethroid-resistant populations of Anopheles gambiae. To examine the role of this mutation in pyrethroid resistance, N1575Y alone or N1575Y + L1014F were introduced into an Aedes aegypti sodium channel, AaNav1-1, and the mutants were functionally examined in Xenopus oocytes. N1575Y did not alter AaNav1-1 sensitivity to pyrethroids. However, the N1575Y + L1014F double mutant was more resistant to pyrethroids than the L1014F mutant channel. Further mutational analysis showed that N1575Y could also synergize the effect of L1014S/W, but not L1014G or other pyrethroid-resistant mutations in IS6 or IIS6. Computer modeling predicts that N1575Y allosterically alters PyR2 via a small shift of IIS6. Our findings provide the molecular basis for the coexistence of N1575Y with L1014F in pyrethroid resistance, and suggest an allosteric interaction between IIS6 and LIII/IV in the sodium channel. PMID:25523031

  1. Quantitative modelling of interaction of propafenone with sodium channels in cardiac cells

    Czech Academy of Sciences Publication Activity Database

    Pásek, Michal; Šimurda, J.

    2004-01-01

    Roč. 42, č. 2 (2004), s. 151-157 ISSN 0140-0118 R&D Projects: GA ČR GP204/02/D129 Institutional research plan: CEZ:AV0Z2076919 Keywords : cardiac cell * sodium current block * quantitative modelling Subject RIV: BO - Biophysics Impact factor: 1.070, year: 2004

  2. Inhibition of human Na(v)1.5 sodium channels by strychnine and its analogs.

    Science.gov (United States)

    Yuan, Chunhua; Sun, Lirong; Zhang, Meng; Li, Shuji; Wang, Xuemin; Gao, Tianming; Zhu, Xinhong

    2011-08-15

    Strychnine and brucine from the seeds of the plant Strychnos nux vomica have been shown to have interesting pharmacological effects on several neurotransmitter receptors. In this study, we have characterized the pharmacological properties of strychnine and its analogs on human Na(v)1.5 channels to assess their potential therapeutic advantage in certain arrhythmias. Among the eight alkaloids, only strychnine and icajine exhibited inhibition potency on the Na(v)1.5 channel with the half-maximum inhibition (IC(50)) values of 83.1μM and 104.6μM, respectively. Structure-function analysis indicated that the increased bulky methoxy groups on the phenyl ring or the negatively charged oxygen atom may account for this lack of inhibition on the Na(v)1.5 channel. Strychnine and icajine may bind to the channel by cation-π interactions. The substitution with a large side chain on the phenyl ring or the increased molecular volume may alter the optimized position for the compound close to the binding sites of the channel. Strychnine and icajine bind to the Na(v)1.5 channel with a new mechanism that is different from TTX and local anesthetics. They bind to the outer vestibule of the channel pore with fast association and dissociation rates at resting state. Strychnine and icajine had little effect on steady-state fast inactivation but markedly shifted the slow inactivation of Na(v)1.5 currents toward more hyperpolarized potentials. The property of icajine influencing slow-inactivated state of Na(v)1.5 channel would be potential therapeutic advantages in certain arrhythmias. Copyright © 2011 Elsevier Inc. All rights reserved.

  3. Effect of batrachotoxin on the electroplax of electric eel: evidence for voltage-dependent interaction with sodium channels.

    Science.gov (United States)

    Bartels-Bernal, E; Rosenberry, T L; Daly, J W

    1977-03-01

    Batrachotoxin under certain conditions has a strong depolarizing effect on the innervated membrane of the monocellular electroplax preparation from the electric eel, El-ectrophorus electricus. No effect is observed when the toxin (50-200 nM) is applied to the resting membrane for periods up to 1 hr. However, if the membrane is exposed to batrachotoxin and the cell is subjected to stimulation at a stimulus voltage slightly above the threshold for action potential firing, a progressive prolongation of the action potential and concomitant progressive depolarization of the innervated membrane is observed. When the membrane is depolarized by 15-20 mV, a further abrupt all-or-none depolarization occurs, and the potential attains a steady-state value between 0 and -10 mV. Brief stimulation of a cell in the presence of batrachotoxin is sufficient to define a batrachotoxin-treated cell, even though negligible depolarization occurs. If depolarizing agents such as carbamoylcholine or potassium chloride are introduced to such a cell in concentrations that normally produce a 20-30 mV depolarization, the abrupt all-or-none depolarization immediately occurs. All-or-none depolarizations arising from either electrical stimulation or depolarizing agents are unaffected by d-tubocurarine but are completely reversed by tetrodotoxin. Batrachotoxin thus appears to activate only the action potential sodium channels. In the batrachotoxin-treated membrane, these channels can attain stable steady states in either a closed configuration at the normal resting potential or in an open configuration after complete depolarization. A striking hysteresis cycle thus can be generated, which is strongly indicative of a voltage-dependent interaction of the toxin with the action potential sodium channels.

  4. Sodium butyrate increases expression of the coxsackie and adenovirus receptor in colon cancer cells.

    Science.gov (United States)

    Küster, Katrin; Grötzinger, Carsten; Koschel, Annika; Fischer, Andreas; Wiedenmann, Bertram; Anders, Mario

    2010-03-01

    Histone deacetylase inhibitors (HDACI), e.g., sodium butyrate (NaB), have been suggested to upregulate the coxsackie and adenovirus receptor (CAR). Its impact on CAR in colon carcinomas, however, is poorly understood. NaB treatment of colon cancer cells increased CAR expression preferentially in cell lines with low basic CAR levels. These findings suggest that downregulation of CAR gene expression is mediated by transcriptional regulation and that activation of the CAR gene promoter is modulated by histone acetylation. The employment of HDACI may, therefore, represent a promising approach for improving adenovirus-based therapies of colon cancers with low CAR expression levels.

  5. TNF Lectin-Like Domain Restores Epithelial Sodium Channel Function in Frameshift Mutants Associated with Pseudohypoaldosteronism Type 1B

    Directory of Open Access Journals (Sweden)

    Anita Willam

    2017-05-01

    Full Text Available Previous in vitro studies have indicated that tumor necrosis factor (TNF activates amiloride-sensitive epithelial sodium channel (ENaC current through its lectin-like (TIP domain, since cyclic peptides mimicking the TIP domain (e.g., solnatide, showed ENaC-activating properties. In the current study, the effects of TNF and solnatide on individual ENaC subunits or ENaC carrying mutated glycosylation sites in the α-ENaC subunit were compared, revealing a similar mode of action for TNF and solnatide and corroborating the previous assumption that the lectin-like domain of TNF is the relevant molecular structure for ENaC activation. Accordingly, TNF enhanced ENaC current by increasing open probability of the glycosylated channel, position N511 in the α-ENaC subunit being identified as the most important glycosylation site. TNF significantly increased Na+ current through ENaC comprising only the pore forming subunits α or δ, was less active in ENaC comprising only β-subunits, and showed no effect on ENaC comprising γ-subunits. TNF did not increase the membrane abundance of ENaC subunits to the extent observed with solnatide. Since the α-subunit is believed to play a prominent role in the ENaC current activating effect of TNF and TIP, we investigated whether TNF and solnatide can enhance αβγ-ENaC current in α-ENaC loss-of-function frameshift mutants. The efficacy of solnatide has been already proven in pathological conditions involving ENaC in phase II clinical trials. The frameshift mutations αI68fs, αT169fs, αP197fs, αE272fs, αF435fs, αR438fs, αY447fs, αR448fs, αS452fs, and αT482fs have been reported to cause pseudohypoaldosteronism type 1B (PHA1B, a rare, life-threatening, salt-wasting disease, which hitherto has been treated only symptomatically. In a heterologous expression system, all frameshift mutants showed significantly reduced amiloride-sensitive whole-cell current compared to wild type αβγ-ENaC, whereas membrane

  6. Molecular characterization and expression analysis of sodium pump genes in the marine red alga Porphyra yezoensis.

    Science.gov (United States)

    Uji, Toshiki; Hirata, Ryo; Mikami, Koji; Mizuta, Hiroyuki; Saga, Naotsune

    2012-08-01

    Sodium pumps (EC 3.6.3.9, Na(+)-ATPase), which mediate excretion of Na(+) from the cell, play a crucial role in Na(+) homeostasis in eukaryotic cells. The objective of this study is to understand the Na(+) efflux system in a marine red alga. We identified a novel sodium pump gene, PyKPA2, from the marine red alga Porphyra yezoensis. The amino acid sequence of PyKPA2 shares 65 % identity with PyKPA1, a previously identified P. yezoensis sodium pump. Similar to PyKPA1, PyKPA2 contains conserved sequences for functions such as phosphorylation, ATP binding, and cation binding. Phylogenetic analysis revealed that the two genes cluster with sodium pumps from algae. Reverse-transcription polymerase chain reaction (RT-PCR) analysis showed that PyKPA1 is expressed preferentially in sporophytes, whereas PyKPA2 is expressed specifically in gametophytes. RT-PCR and quantitative real-time PCR analysis revealed that PyKPA1 and PyKPA2 transcripts were upregulated and downregulated, respectively, in gametophytes during exposure to alkali stress. In addition, transcription of both genes in gametophytes was also induced by cold stress. These results suggest that PyKPA1 and PyKPA2 play an important role in alkali and cold stress tolerance.

  7. Genetic strategies to analyze primary TRP channel-expressing cells in mice.

    Science.gov (United States)

    Wyatt, Amanda; Wartenberg, Philipp; Candlish, Michael; Krasteva-Christ, Gabriela; Flockerzi, Veit; Boehm, Ulrich

    2017-11-01

    Transient receptor potential (TRP) ion channels regulate fundamental biological processes throughout the body. TRP channel dysfunction has been causally linked to a number of disease states and thus establishes these channels as promising therapeutic targets. In order to dissect the physiological role of individual TRP channels in specific tissues, a detailed understanding of the expression pattern of the different TRP channels throughout the organism is essential. We provide an overview of recent efforts to generate novel TRP channel reporter mouse strains for all 28 TRP channels encoded in the mouse genome to understand expression of these channels with a single-cell resolution in an organism-wide manner. The reporter mice will enable both the visualization and manipulation of all primary TRP channel-expressing cells allowing an unprecedented wealth in variety to investigate TRP channel function in vivo. As proof of principle, we provide preliminary results documenting TRPM5 expression throughout the entire body of juvenile and adult mice. Copyright © 2017 Elsevier Ltd. All rights reserved.

  8. Expression of transient receptor potential channels in the ependymal cells of the developing rat brain.

    Science.gov (United States)

    Jo, Kwang Deog; Lee, Kyu-Seok; Lee, Won Taek; Hur, Mi-Sun; Kim, Ho-Jeong

    2013-03-01

    Cerebrospinal fluid (CSF) plays an important role in providing brain tissue with a stable internal environment as well as in absorbing mechanical and thermal stresses. From its initial composition, derived from the amniotic fluid trapped by the closure of neuropores, CSF is modified by developing and differentiating ependymal cells lining the ventricular surface or forming the choroid plexus. Its osmolarity and ionic composition brings about a change through the action of many channels expressed on the ependymal cells. Some newly discovered transient receptor potential (TRP) channels are known to be expressed in the choroid plexus ependyma. To detect additional TRP channel expression, immunohistochemical screening was performed at the choroid plexus of 13-, 15-, 17-, and 19-day embryos, using antibodies against TRPV1, TRPV3, and TRPA1, and the expression was compared with those in the adult TRP channels. The level of TRP channel expression was higher in the choroid plexus which suggests more active functioning of TRP channels in the developing choroid plexus than the ventricular lining ependyma in the 15- and 17-day embryos. All the expression of TRP channels decreased at the 19th day of gestation. TRPA1 was expressed at a higher level than TRPV1 and TRPV3 in almost all stages in both the choroid plexus and ventricular lining epithelium. The highest level of TRPV1 and TRPV3 expression was observed in association with the glycogen deposits in the cytoplasm of the choroid plexus ependymal cells of the 15- and 17-day embryos.

  9. Cardiac sodium channelopathies.

    Science.gov (United States)

    Amin, Ahmad S; Asghari-Roodsari, Alaleh; Tan, Hanno L

    2010-07-01

    Cardiac sodium channel are protein complexes that are expressed in the sarcolemma of cardiomyocytes to carry a large inward depolarizing current (INa) during phase 0 of the cardiac action potential. The importance of INa for normal cardiac electrical activity is reflected by the high incidence of arrhythmias in cardiac sodium channelopathies, i.e., arrhythmogenic diseases in patients with mutations in SCN5A, the gene responsible for the pore-forming ion-conducting alpha-subunit, or in genes that encode the ancillary beta-subunits or regulatory proteins of the cardiac sodium channel. While clinical and genetic studies have laid the foundation for our understanding of cardiac sodium channelopathies by establishing links between arrhythmogenic diseases and mutations in genes that encode various subunits of the cardiac sodium channel, biophysical studies (particularly in heterologous expression systems and transgenic mouse models) have provided insights into the mechanisms by which INa dysfunction causes disease in such channelopathies. It is now recognized that mutations that increase INa delay cardiac repolarization, prolong action potential duration, and cause long QT syndrome, while mutations that reduce INa decrease cardiac excitability, reduce electrical conduction velocity, and induce Brugada syndrome, progressive cardiac conduction disease, sick sinus syndrome, or combinations thereof. Recently, mutation-induced INa dysfunction was also linked to dilated cardiomyopathy, atrial fibrillation, and sudden infant death syndrome. This review describes the structure and function of the cardiac sodium channel and its various subunits, summarizes major cardiac sodium channelopathies and the current knowledge concerning their genetic background and underlying molecular mechanisms, and discusses recent advances in the discovery of mutation-specific therapies in the management of these channelopathies.

  10. Expression of factor VIII-related antigen in human aqueous drainage channels.

    Science.gov (United States)

    Krohn, J

    1999-02-01

    Previous studies have indicated that there are preformed channels at the inner aspect of the anterior sclera, capable of draining fluid from the suprachoroidal space into scleral veins. The aim of this study was to characterize the cellular lining of these channels, and to compare it with the endothelium of Schlemm's canal, the collector channels and the scleral blood vessels. Histological sections from the angular region of human eyes were prepared by an immunoperoxidase method to evaluate the expression of factor VIII-related antigen in different aqueous drainage channels. The cellular lining of the scleral channels showed a weak immunostaining to factor VIII-related antigen. Factor VIII-related antigen was also detected in the endothelium of the collector channels and Schlemm's canal. The positive immunoreaction to factor VIII-related antigen indicates that the previously described scleral channels, the collector channels and Schlemm's canal are all lined by an endothelium derived from a vascular origin.

  11. Comparison and optimization of in silico algorithms for predicting the pathogenicity of sodium channel variants in epilepsy.

    Science.gov (United States)

    Holland, Katherine D; Bouley, Thomas M; Horn, Paul S

    2017-07-01

    Variants in neuronal voltage-gated sodium channel α-subunits genes SCN1A, SCN2A, and SCN8A are common in early onset epileptic encephalopathies and other autosomal dominant childhood epilepsy syndromes. However, in clinical practice, missense variants are often classified as variants of uncertain significance when missense variants are identified but heritability cannot be determined. Genetic testing reports often include results of computational tests to estimate pathogenicity and the frequency of that variant in population-based databases. The objective of this work was to enhance clinicians' understanding of results by (1) determining how effectively computational algorithms predict epileptogenicity of sodium channel (SCN) missense variants; (2) optimizing their predictive capabilities; and (3) determining if epilepsy-associated SCN variants are present in population-based databases. This will help clinicians better understand the results of indeterminate SCN test results in people with epilepsy. Pathogenic, likely pathogenic, and benign variants in SCNs were identified using databases of sodium channel variants. Benign variants were also identified from population-based databases. Eight algorithms commonly used to predict pathogenicity were compared. In addition, logistic regression was used to determine if a combination of algorithms could better predict pathogenicity. Based on American College of Medical Genetic Criteria, 440 variants were classified as pathogenic or likely pathogenic and 84 were classified as benign or likely benign. Twenty-eight variants previously associated with epilepsy were present in population-based gene databases. The output provided by most computational algorithms had a high sensitivity but low specificity with an accuracy of 0.52-0.77. Accuracy could be improved by adjusting the threshold for pathogenicity. Using this adjustment, the Mendelian Clinically Applicable Pathogenicity (M-CAP) algorithm had an accuracy of 0.90 and a

  12. Investigations of the Navβ1b sodium channel subunit in human ventricle; functional characterization of the H162P Brugada Syndrome mutant

    DEFF Research Database (Denmark)

    Yuan, Lei; Koivumaki, Jussi; Liang, Bo

    2014-01-01

    Brugada Syndrome (BrS) is a rare inherited disease which can give rise to ventricular arrhythmia and ultimately sudden cardiac death. Numerous loss-of-function mutations in the cardiac sodium channel Nav1.5 have been associated with BrS. However, few mutations in the auxiliary Navβ1-4 subunits have...... to support the notion that BrS can be linked to the function of Navβ1b and is associated with loss-of-function of the cardiac sodium channel....

  13. Alpha-lipoic acid induces sodium iodide symporter expression in TPC-1 thyroid cancer cell line

    International Nuclear Information System (INIS)

    Choi, Hyun-Jeung; Kim, Tae Yong; Ruiz-Llorente, Sergio; Jeon, Min Ji; Han, Ji Min; Kim, Won Gu; Shong, Young Kee; Kim, Won Bae

    2012-01-01

    Introduction: Patients with metastatic thyroid cancers that do not uptake iodine need effective therapeutic option. Differentiation-inducing agents have been tried to restore functional expression of sodium iodide symporter (NIS) without success. Our objective was to assess the effect of alpha-lipoic acid (ALA), known as potential antioxidant, on expression of sodium iodide symporter in thyroid cancer cells. Methods: Human thyroid cancer-derived cell lines, TPC-1, were treated with ALA, and changes in NIS mRNA and protein expression were measured. ALA's effect on NIS gene promoter was evaluated, and functional NIS expression was assessed by iodide uptake assay. Results: Treatment with ALA increased NIS mRNA expression up to ten folds of control dose-dependently after 24 h of exposure. ALA increased NIS promoter activity, and increased iodide uptake by 1.6 fold. ALA induced expression of NIS protein, but had no significant effect on the plasma membrane trafficking. ALA increased phosphorylation of CREB and nuclear translocation of pCREB, and co-treatment of ALA and trichostatin A increased iodide uptake by three folds in TPC-1 cells. Conclusions: ALA is a potential agent to increase NIS transcription in TPC-1. It could be used as an adjunctive agent to increase efficacy of radioiodine therapy if combined with a strategy to increase NIS protein trafficking to cell membrane.

  14. Novel molecular determinants in the pore region of sodium channels regulate local anesthetic binding.

    Science.gov (United States)

    Yamagishi, Toshio; Xiong, Wei; Kondratiev, Andre; Vélez, Patricio; Méndez-Fitzwilliam, Ailsa; Balser, Jeffrey R; Marbán, Eduardo; Tomaselli, Gordon F

    2009-10-01

    The pore of the Na+ channel is lined by asymmetric loops formed by the linkers between the fifth and sixth transmembrane segments (S5-S6). We investigated the role of the N-terminal portion (SS1) of the S5-S6 linkers in channel gating and local anesthetic (LA) block using site-directed cysteine mutagenesis of the rat skeletal muscle (Na(V)1.4) channel. The mutants examined have variable effects on voltage dependence and kinetics of fast inactivation. Of the cysteine mutants immediately N-terminal to the putative DEKA selectivity filter in four domains, only Q399C in domain I and F1236C in domain III exhibit reduced use-dependent block. These two mutations also markedly accelerated the recovery from use-dependent block. Moreover, F1236C and Q399C significantly decreased the affinity of QX-314 for binding to its channel receptor by 8.5- and 3.3-fold, respectively. Oddly enough, F1236C enhanced stabilization of slow inactivation by both hastening entry into and delaying recovery from slow inactivation states. It is noteworthy that symmetric applications of QX-314 on both external and internal sides of F1236C mutant channels reduced recovery from use-dependent block, indicating an allosteric effect of external QX-314 binding on the recovery of availability of F1236C. These observations suggest that cysteine mutation in the SS1 region, particularly immediate adjacent to the DEKA ring, may lead to a structural rearrangement that alters binding of permanently charged QX-314 to its receptor. The results lend further support for a role for the selectivity filter region as a structural determinant for local anesthetic block.

  15. Local anesthetics QX 572 and benzocaine act at separate sites on the batrachotoxin-activated sodium channel.

    Science.gov (United States)

    Huang, L M; Ehrenstein, G

    1981-02-01

    We have studied the effect of local anesthetics QX 572, which is permanently charged, and benzocaine, which is neutral, on batrachotoxin-activated sodium channels in mouse neuroblastoma N18 cells. The dose-response curves for each drug suggest that QX 752 and benzocaine each act on a single class of binding sites. The dissociation constants are 3.15 X 10(-5) M for QX 572 and 2.65 X 10(-4) M for benzocaine. Equilibrium and kinetic experiments indicate that both drugs are competitive inhibitors of batrachotoxin. When benzocaine and QX 572 are present with batrachotoxin, they are much more effective at inhibiting Na+ flux than would be predicted by a one-site model. Our results indicate that QX 572 and benzocaine bind to separate sites, each of which interacts competitively with batrachotoxin.

  16. Update on the frequency of Ile1016 mutation in voltage-gated sodium channel gene of Aedes aegypti in Mexico.

    Science.gov (United States)

    Siller, Quetzaly; Ponce, Gustavo; Lozano, Saul; Flores, Adriana E

    2011-12-01

    We analyzed 790 Aedes aegypti from 14 localities of Mexico in 2009 to update information on the frequency of the Ile1016 allele in the voltage-gated sodium channel gene that confers resistance to pyrethroids and DDT. The Ile1016 mutation was present in all 17 collections, and was close to fixation in Acapulco (frequency = 0.97), Iguala (0.93), and San Nicolas (0.90). Genotypes at the 1016 locus were not in Hardy-Weinberg proportions in collections from Panuco, Veracruz, Cosoleacaque, Coatzacoalcos, Tantoyuca, and Monterrey due in every case to an excess of homozygotes. The high frequencies of this mutation in Ae. aegypti are probably due to selection pressure from pyrethroid insecticides, particularly permethrin, which has been used in mosquito control programs for >10 years in Mexico.

  17. High affinity for the rat brain sodium channel of newly discovered hydroxybenzoate saxitoxin analogues from the dinoflagellate Gymnodinium catenatum.

    Science.gov (United States)

    Llewellyn, Lyndon; Negri, Andrew; Quilliam, Michael

    2004-01-01

    The paralytic shellfish poison family has been recently extended by the discovery of several analogues possessing a hydoxybenzoate moiety instead of the carbamoyl group one finds in saxitoxin, the parent molecule of this toxin family. We have investigated the potency of these new analogues on a representative isoform of the pharmacological target of these toxins, the voltage gated sodium channel. These toxins were found to have K1's in the low nanomolar range, only slightly less potent than saxitoxin. The hydroxybenzoate group may increase the lipophilicity of these toxins and improve their ability to pass through epithelia and therefore its uptake and elimination in both intoxication victims and animals that bioaccumulate paralytic shellfish toxins.

  18. Sodium channel point mutations associated with pyrethroid resistance in Chinese strains of Culex pipiens quinquefasciatus (Diptera: Culicidae).

    Science.gov (United States)

    Zhao, Minghui; Dong, Yande; Ran, Xin; Guo, Xiaoxia; Xing, Dan; Zhang, Yingmei; Yan, Ting; Zhu, Xiaojuan; Su, Jianxin; Zhang, Hengduan; Wang, Gang; Hou, Wenjun; Wu, Zhiming; Li, Chunxiao; Zhao, Tongyan

    2014-08-15

    Pesticide resistance due to sodium channel point mutations has been well documented in many mosquito species. We tested the resistance of six, wild, Chinese populations of the mosquito Culex pipiens quinquefasciatus to deltamethrin and cyhalothrin. The full length of the sodium channel gene was cloned and sequenced from a pooled sample of mosquitoes from each population. Seven amino acid alterations were found (V250M, R436K, M943V, I973T, L1035F, L1035S and E1901D). Correlation between the frequencies of these mutations and the level of pesticide resistance (LC50) associated with them indicates that those at position L1035 (corresponding to position L1014F in the house fly, Musca domestica; GenBank Accession No.: X96668) are associated with resistance to deltamethrin and cyhalothrin. The frequency of the L1035F mutation was significantly correlated with resistance to deltamethrin (R2 = 0.536, P = 0.049) and cyhalothrin (R2 = 0.626, P = 0.030), and the combined frequency of the L1035F and L1035S mutations was significantly correlated with resistance to both deltamethrin (R2 = 0.661, P = 0.025), and cyhalothrin (R2 = 0.803, P = 0.008). None of the other mutations were correlated with either deltamethrin or cyhalothrin resistance. Interestingly, an HWE test indicated significant linkage between the M943V and I973T mutations (P mutations may be of practical benefit to the development of pesticide resistance management programs.

  19. Dilazep inhibits binding of batrachotoxin to sodium channels in canine sarcolemmal vesicles.

    Science.gov (United States)

    Chiba, K; Hashizume, H; Inagaki, S I; Abiko, Y

    1995-01-01

    We studied the effect of dilazep on the binding of [3H]- batrachotoxinin A 20 alpha-benzoate ([3H]BTXB), which binds to and stabilizes the activated state of the Na+ channel, and compared it with that of lidocaine in canine cardiac sarcolemmal vesicles. Dilazep inhibited the specific [3H]BTXB binding in a dose-dependent manner with an IC50 value of 0.37 microM, while lidocaine inhibited it with an IC50 value of 92 microM. Scatchard analysis of [3H]BTXB binding demonstrated that both dilazep and lidocaine reduced the amax without a marked effect on the K(D). The inhibition of [3H]BTXB induced by dilazep was reversible. Both dilazep (4 microM) and lidocaine (100 microM) increased the dissociation rate constant of [3H]BTXB only in concentrations which are about a 10-fold greater than their IC50, indicating the low affinity of both drugs for the [3H]BTXB-bound Na+ channel. However, dilazep (0.5 microM) and lidocaine (100 microM) decreased significantly the association rate constant of the [3H]BTXB binding at concentrations near their IC50, indicating that the affinity of both drugs for the [3H]BTXB-unbound Na+ channel is relatively high. These results suggest that, in canine cardiac membrane vesicles, the effect of dilazep in inhibiting the binding of [3H]BTXB and stabilizing the Na+ channel is similar to that of lidocaine, but the potency of dilazep is greater than that of lidocaine.

  20. Functional interaction between S1 and S4 segments in voltage-gated sodium channels revealed by human channelopathies.

    Science.gov (United States)

    Amarouch, Mohamed-Yassine; Kasimova, Marina A; Tarek, Mounir; Abriel, Hugues

    2014-01-01

    The p.I141V mutation of the voltage-gated sodium channel is associated with several clinical hyper-excitability phenotypes. To understand the structural bases of the p.I141V biophysical alterations, molecular dynamics simulations were performed. These simulations predicted that the p.I141V substitution induces the formation of a hydrogen bond between the Y168 residue of the S2 segment and the R225 residue of the S4 segment. We generated a p.I141V-Y168F double mutant for both the Nav1.4 and Nav1.5 channels. The double mutants demonstrated the abolition of the functional effects of the p.I141V mutation, consistent with the formation of a specific interaction between Y168-S2 and R225-S4. The single p.Y168F mutation, however, positively shifted the activation curve, suggesting a compensatory role of these residues on the stability of the voltage-sensing domain.

  1. Patch clamp and perfusion techniques for studying ion channels expressed in Xenopus oocytes.

    Science.gov (United States)

    Yang, Junqiu; Delaloye, Kelli; Lee, Urvi S; Cui, Jianmin

    2011-01-10

    The protocol presented here is designed to study the activation of the large conductance, voltage- and Ca(2+)-activated K(+) (BK) channels. The protocol may also be used to study the structure-function relationship for other ion channels and neurotransmitter receptors. BK channels are widely expressed in different tissues and have been implicated in many physiological functions, including regulation of smooth muscle contraction, frequency tuning of inner hair cells and regulation of neurotransmitter release. BK channels are activated by membrane depolarization and by intracellular Ca(2+) and Mg(2+). Therefore, the protocol is designed to control both the membrane voltage and the intracellular solution. In this protocol, messenger RNA of BK channels is injected into Xenopus laevis oocytes (stage V-VI) followed by 2-5 days of incubation at 18°C. Membrane patches that contain single or multiple BK channels are excised with the inside-out configuration using patch clamp techniques. The intracellular side of the patch is perfused with desired solutions during recording so that the channel activation under different conditions can be examined. To summarize, the mRNA of BK channels is injected into Xenopus laevis oocytes to express channel proteins on the oocyte membrane; patch clamp techniques are used to record currents flowing through the channels under controlled voltage and intracellular solutions.

  2. Reserpine: interactions with batrachotoxin and brevetoxin sites on voltage-dependent sodium channels.

    Science.gov (United States)

    Flowers, Andrew; Onwueme, Kenolisa; Creveling, Cyrus R; Daly, John W

    2002-02-01

    Reserpine inhibited batrachotoxin-elicited sodium influx in guinea pig brain synaptoneurosomes with an IC50 of about 1 microM. In the presence of brevetoxin the IC50 increased to about 80 microM. Reserpine inhibited binding of batrachotoxinin-A [3H]benzoate ([3H]BTX-B) binding in a complex manner causing a partial inhibition from 0.001 to 0.08 microM, then a rebound stimulation from 0.1 to 0.8 microM, followed by complete inhibition by 80 microM. The stimulation was prevented by the presence of brevetoxin; reserpine then smoothly inhibited binding with an IC50 of about 1 microM. Reserpine at 1 microM slightly reduced the off-rate of [3H]BTX-B binding measured in the presence of veratridine, while at a concentration of 50 microM it enhanced the off-rate, presumably by an allosteric mechanism. Reserpine at 0.3-10 microM elicited a partial inhibition of the binding of [3H]brevetoxin-3. The local anesthetic dibucaine had effects similar to reserpine: It partially inhibited binding of [3H]brevetoxin. The presence of brevetoxin reduced the potency of dibucaine as an inhibitor of batrachotoxin-elicited sodium influx from an IC50 of about 2 microM to an IC50 of about 50 microM. The results suggest that reserpine binds at both a local anesthetic site to cause allosteric inhibition of batrachotoxin-binding and action, but that it also binds to another site causing, like brevetoxin, an enhancement of batrachotoxin-binding and action. Local anesthetics also may bind to the brevetoxin site.

  3. Loss-of-function of the voltage-gated sodium channel NaV1.5 (channelopathies) in patients with irritable bowel syndrome.

    Science.gov (United States)

    Beyder, Arthur; Mazzone, Amelia; Strege, Peter R; Tester, David J; Saito, Yuri A; Bernard, Cheryl E; Enders, Felicity T; Ek, Weronica E; Schmidt, Peter T; Dlugosz, Aldona; Lindberg, Greger; Karling, Pontus; Ohlsson, Bodil; Gazouli, Maria; Nardone, Gerardo; Cuomo, Rosario; Usai-Satta, Paolo; Galeazzi, Francesca; Neri, Matteo; Portincasa, Piero; Bellini, Massimo; Barbara, Giovanni; Camilleri, Michael; Locke, G Richard; Talley, Nicholas J; D'Amato, Mauro; Ackerman, Michael J; Farrugia, Gianrico

    2014-06-01

    SCN5A encodes the α-subunit of the voltage-gated sodium channel NaV1.5. Many patients with cardiac arrhythmias caused by mutations in SCN5A also have symptoms of irritable bowel syndrome (IBS). We investigated whether patients with IBS have SCN5A variants that affect the function of NaV1.5. We performed genotype analysis of SCN5A in 584 persons with IBS and 1380 without IBS (controls). Mutant forms of SCN5A were expressed in human embryonic kidney-293 cells, and functions were assessed by voltage clamp analysis. A genome-wide association study was analyzed for an association signal for the SCN5A gene, and replicated in 1745 patients in 4 independent cohorts of IBS patients and controls. Missense mutations were found in SCN5A in 13 of 584 patients (2.2%, probands). Diarrhea-predominant IBS was the most prevalent form of IBS in the overall study population (25%). However, a greater percentage of individuals with SCN5A mutations had constipation-predominant IBS (31%) than diarrhea-predominant IBS (10%; P < .05). Electrophysiologic analysis showed that 10 of 13 detected mutations disrupted NaV1.5 function (9 loss-of-function and 1 gain-of-function function). The p. A997T-NaV1.5 had the greatest effect in reducing NaV1.5 function. Incubation of cells that expressed this variant with mexiletine restored their sodium current and administration of mexiletine to 1 carrier of this mutation (who had constipation-predominant IBS) normalized their bowel habits. In the genome-wide association study and 4 replicated studies, the SCN5A locus was strongly associated with IBS. About 2% of patients with IBS carry mutations in SCN5A. Most of these are loss-of-function mutations that disrupt NaV1.5 channel function. These findings provide a new pathogenic mechanism for IBS and possible treatment options. Copyright © 2014 AGA Institute. Published by Elsevier Inc. All rights reserved.

  4. Coordinated role of voltage-gated sodium channels and the Na{sup +}/H{sup +} exchanger in sustaining microglial activation during inflammation

    Energy Technology Data Exchange (ETDEWEB)

    Hossain, Muhammad M. [Department of Environmental and Occupational Medicine and Environmental and Occupational Health Sciences Institute, Rutgers Robert Wood Johnson Medical School, Piscataway, NJ (United States); Sonsalla, Patricia K. [Department of Neurology, Rutgers Robert Wood Johnson Medical School, Piscataway, NJ (United States); Richardson, Jason R., E-mail: jricha3@eohsi.rutgers.edu [Department of Environmental and Occupational Medicine and Environmental and Occupational Health Sciences Institute, Rutgers Robert Wood Johnson Medical School, Piscataway, NJ (United States)

    2013-12-01

    Persistent neuroinflammation and microglial activation play an integral role in the pathogenesis of many neurological disorders. We investigated the role of voltage-gated sodium channels (VGSC) and Na{sup +}/H{sup +} exchangers (NHE) in the activation of immortalized microglial cells (BV-2) after lipopolysaccharide (LPS) exposure. LPS (10 and 100 ng/ml) caused a dose- and time-dependent accumulation of intracellular sodium [(Na{sup +}){sub i}] in BV-2 cells. Pre-treatment of cells with the VGSC antagonist tetrodotoxin (TTX, 1 μM) abolished short-term Na{sup +} influx, but was unable to prevent the accumulation of (Na{sup +}){sub i} observed at 6 and 24 h after LPS exposure. The NHE inhibitor cariporide (1 μM) significantly reduced accumulation of (Na{sup +}){sub i} 6 and 24 h after LPS exposure. Furthermore, LPS increased the mRNA expression and protein level of NHE-1 in a dose- and time-dependent manner, which was significantly reduced after co-treatment with TTX and/or cariporide. LPS increased production of TNF-α, ROS, and H{sub 2}O{sub 2} and expression of gp91{sup phox}, an active subunit of NADPH oxidase, in a dose- and time-dependent manner, which was significantly reduced by TTX or TTX + cariporide. Collectively, these data demonstrate a closely-linked temporal relationship between VGSC and NHE-1 in regulating function in activated microglia, which may provide avenues for therapeutic interventions aimed at reducing neuroinflammation. - Highlights: • LPS causes immediate increase in sodium through VGSC and subsequently through the NHE-1. • Inhibition of VGSC reduces increases in NHE-1 and gp91{sup phox}. • Inhibition of VGSC and NHE-1 reduces NADPH oxidase-mediated Tnf-α, ROS, and H{sub 2}O{sub 2} production. • NHE-1 and Na{sub v}1.6 may be viable targets for therapeutic interventions to reduce neuroinflammation in neurodegenerative disease.

  5. Functional suppression of Kcnq1 leads to early sodium channel remodelling and cardiac conduction system dysmorphogenesis

    Czech Academy of Sciences Publication Activity Database

    De la Rosa, A. J.; Domínguez, J. N.; Sedmera, D.; Šaňková, Barbora; Hove-Madsen, L.; Franco, D.; Aránega, A. E.

    2013-01-01

    Roč. 98, č. 3 (2013), s. 504-514 ISSN 0008-6363 R&D Projects: GA ČR(CZ) GA304/08/0615; GA ČR(CZ) GAP302/11/1308; GA ČR(CZ) GD204/09/H084; GA ČR(CZ) GA13-12412S Institutional research plan: CEZ:AV0Z50110509 Institutional support: RVO:67985823 Keywords : ion channels * Long-QT syndrome * sudden death * cardiac hypertrophy Subject RIV: FA - Cardiovascular Diseases incl. Cardiotharic Surgery Impact factor: 5.808, year: 2013

  6. A Novel Mechanism in Regulating the Alpha-Subunit of the Epithelial Sodium Channel (α ENaC by the Alternatively Spliced Form α ENaC-b

    Directory of Open Access Journals (Sweden)

    Marlene F. Shehata

    2009-01-01

    Full Text Available Introduction: In Dahl rats’ kidney cortex, the alternatively spliced form of the epithelial sodium channel α subunit (α ENaC-b is the most abundant mRNA transcript (32+/-3 fold α ENaC-wt as was investigated by quantitative RT-PCR analysis. α ENaC-b mRNA levels were significantly higher in Dahl R versus S rats, and were further augmented by high salt diet.Objectives: In the present study, we described the molecular cloning and searched for a possible role of α ENaC-b by testing its potential expression in COS7 cells as well as its impact on α ENaC-wt expression levels when co-expressed in COS7 cells in a dose-dependent manner.Methods: Using RT-PCR strategy, the full-length wildtype α ENaC transcript and the alternatively spliced form α ENaC-b were amplified, sequenced, cloned, subcloned into PCMV-sport6 expression vector, expressed and co-expressed into COS7 cells in a dose-dependent manner. A combination of denaturing and native western blotting techniques was employed to examine the expression of α ENaC-b in vitro, and to determine if an interaction between α ENaC-b and α ENaC-wt occurs in vitro, and finally to demonstrate if degradation of α ENaC-wt protein does occur.Results: α ENaC-b is translated in COS7 cells. Co-expression of α ENaC-b together with α ENaC-wt reduced α ENaC-wt levels in a dose-dependent manner. α ENaC-wt and α ENaC-b appear to form a complex that enhances the degradation of α ENaC-wt.Conclusions: Western blots suggest a novel mechanism in α ENaC regulation whereby α ENaC-b exerts a dominant negative effect on α ENaC-wt expression. This is potentially by sequestering α ENaC-wt, enhancing its proteolytic degradation, and possibly explaining the mechanism of salt-resistance in Dahl R rats.

  7. Corticolimbic expression of TRPC4 and TRPC5 channels in the rodent brain.

    Directory of Open Access Journals (Sweden)

    Melissa A Fowler

    2007-06-01

    Full Text Available The canonical transient receptor potential (TRPC channels are a family of non-selective cation channels that are activated by increases in intracellular Ca(2+ and G(q/phospholipase C-coupled receptors. We used quantitative real-time PCR, in situ hybridization, immunoblots and patch-clamp recording from several brain regions to examine the expression of the predominant TRPC channels in the rodent brain. Quantitative real-time PCR of the seven TRPC channels in the rodent brain revealed that TRPC4 and TRPC5 channels were the predominant TRPC subtypes in the adult rat brain. In situ hybridization histochemistry and immunoblotting further resolved a dense corticolimbic expression of the TRPC4 and TRPC5 channels. Total protein expression of HIP TRPC4 and 5 proteins increased throughout development and peaked late in adulthood (6-9 weeks. In adults, TRPC4 expression was high throughout the frontal cortex, lateral septum (LS, pyramidal cell layer of the hippocampus (HIP, dentate gyrus (DG, and ventral subiculum (vSUB. TRPC5 was highly expressed in the frontal cortex, pyramidal cell layer of the HIP, DG, and hypothalamus. Detailed examination of frontal cortical layer mRNA expression indicated TRPC4 mRNA is distributed throughout layers 2-6 of the prefrontal cortex (PFC, motor cortex (MCx, and somatosensory cortex (SCx. TRPC5 mRNA expression was concentrated specifically in the deep layers 5/6 and superficial layers 2/3 of the PFC and anterior cingulate. Patch-clamp recording indicated a strong metabotropic glutamate-activated cation current-mediated depolarization that was dependent on intracellular Ca(2+and inhibited by protein kinase C in brain regions associated with dense TRPC4 or 5 expression and absent in regions lacking TRPC4 and 5 expression. Overall, the dense corticolimbic expression pattern suggests that these Gq/PLC coupled nonselective cation channels may be involved in learning, memory, and goal-directed behaviors.

  8. Intracellular calcium oscillations in strongly metastatic human breast and prostate cancer cells: control by voltage-gated sodium channel activity.

    Science.gov (United States)

    Rizaner, Nahit; Onkal, Rustem; Fraser, Scott P; Pristerá, Alessandro; Okuse, Kenji; Djamgoz, Mustafa B A

    2016-10-01

    The possible association of intracellular Ca 2+ with metastasis in human cancer cells is poorly understood. We have studied Ca 2+ signaling in human prostate and breast cancer cell lines of strongly versus weakly metastatic potential in a comparative approach. Intracellular free Ca 2+ was measured using a membrane-permeant fluorescent Ca 2+ -indicator dye (Fluo-4 AM) and confocal microscopy. Spontaneous Ca 2+ oscillations were observed in a proportion of strongly metastatic human prostate and breast cancer cells (PC-3M and MDA-MB-231, respectively). In contrast, no such oscillations were observed in weakly/non metastatic LNCaP and MCF-7 cells, although a rise in the resting Ca 2+ level could be induced by applying a high-K + solution. Various parameters of the oscillations depended on extracellular Ca 2+ and voltage-gated Na + channel activity. Treatment with either tetrodotoxin (a general blocker of voltage-gated Na + channels) or ranolazine (a blocker of the persistent component of the channel current) suppressed the Ca 2+ oscillations. It is concluded that the functional voltage-gated Na + channel expression in strongly metastatic cancer cells makes a significant contribution to generation of oscillatory intracellular Ca 2+ activity. Possible mechanisms and consequences of the Ca 2+ oscillations are discussed.

  9. Isolation and identification of a sodium channel-inhibiting protein from eggs of black widow spiders.

    Science.gov (United States)

    Li, Jianjun; Yan, Yizhong; Yu, Hai; Peng, Xiaozhen; Zhang, Yiya; Hu, Weijun; Duan, Zhigui; Wang, Xianchun; Liang, Songping

    2014-04-01

    The eggs of black widow spider (L. tredecimguttatus) have been demonstrated to be rich in biologically active components that exhibit great research value and application foreground. In the present study, a protein toxin, named Latroeggtoxin-II, was isolated from the eggs using the combination of gel filtration, ion exchange chromatography and reversed-phase high performance liquid chromatography. Electrospray mass spectrometric analysis indicated that the molecular weight of the protein was 28.69 kDa, and Edman degradation revealed that its N-terminal sequence was ESIQT STYVP NTPNQ KFDYE VGKDY-. After being abdominally injected into mice and P. americana, the protein could make the animals especially P. americana display a series of poisoning symptoms. Electrophysiological experiments demonstrated that the protein could selectively inhibit tetrodotoxin-resistant Na(+) channel currents in rat dorsal root ganglion neurons, without significant effect on the tetrodotoxin-sensitive Na(+) channel currents. Using multiple proteomic strategies, the purified protein was shown to have only a few similarities to the existing proteins in the databases, suggesting that it was a novel protein isolated from the eggs of black widow spiders. Copyright © 2014. Published by Elsevier B.V.

  10. Acid-sensing ion channel (ASIC) 4 predominantly localizes to an early endosome-related organelle upon heterologous expression.

    Science.gov (United States)

    Schwartz, Verena; Friedrich, Katharina; Polleichtner, Georg; Gründer, Stefan

    2015-12-15

    Acid-sensing ion channels (ASICs) are voltage-independent proton-gated amiloride sensitive sodium channels, belonging to the DEG/ENaC gene family. Six different ASICs have been identified (ASIC1a, ASIC1b, ASIC2a, ASIC2b, ASIC3, ASIC4) that are activated by a drop in extracellular pH, either as homo- or heteromers. An exception is ASIC4, which is not activated by protons as a homomer and which does not contribute to functional heteromeric ASICs. Insensitivity of ASIC4 to protons and its comparatively low sequence identity to other ASICs (45%) raises the question whether ASIC4 may have different functions than other ASICs. In this study, we therefore investigated the subcellular localization of ASIC4 in heterologous cell lines, which revealed a surprising accumulation of the channel in early endosome-related vacuoles. Moreover, we identified an unique amino-terminal motif as important for forward-trafficking from the ER/Golgi to the early endosome-related compartment. Collectively, our results show that heterologously expressed ASIC4 predominantly resides in an intracellular endosomal compartment.

  11. The brain-specific Beta4 subunit downregulates BK channel cell surface expression.

    Directory of Open Access Journals (Sweden)

    Sonal Shruti

    Full Text Available The large-conductance K(+ channel (BK channel can control neural excitability, and enhanced channel currents facilitate high firing rates in cortical neurons. The brain-specific auxiliary subunit β4 alters channel Ca(++- and voltage-sensitivity, and β4 knock-out animals exhibit spontaneous seizures. Here we investigate β4's effect on BK channel trafficking to the plasma membrane. Using a novel genetic tag to track the cellular location of the pore-forming BKα subunit in living cells, we find that β4 expression profoundly reduces surface localization of BK channels via a C-terminal ER retention sequence. In hippocampal CA3 neurons from C57BL/6 mice with endogenously high β4 expression, whole-cell BK channel currents display none of the characteristic properties of BKα+β4 channels observed in heterologous cells. Finally, β4 knock-out animals exhibit a 2.5-fold increase in whole-cell BK channel current, indicating that β4 also regulates current magnitude in vivo. Thus, we propose that a major function of the brain-specific β4 subunit in CA3 neurons is control of surface trafficking.

  12. Inhibitory effects and mechanism of dihydroberberine on hERG channels expressed in HEK293 cells.

    Directory of Open Access Journals (Sweden)

    Dahai Yu

    Full Text Available The human ether-a-go-go-related gene (hERG potassium channel conducts rapid delayed rectifier potassium currents (IKr and contributes to phase III cardiac action potential repolarization. Drugs inhibit hERG channels by binding to aromatic residues in hERG helixes. Berberine (BBR has multiple actions, and its hydrogenated derivative dihydroberberine (DHB is a potential candidate for developing new drugs. Previous studies have demonstrated that BBR blocks hERG channels and prolongs action potential duration (APD. Our present study aimed to investigate the effects and mechanism of DHB on hERG channels. Protein expression and the hERG current were analyzed using western blotting and patch-clamp, respectively. DHB inhibited the hERG current concentration-dependently after instantaneous perfusion, accelerated channel inactivation by directly binding tyrosine (Tyr652 and phenylalanine (Phe656, and decreased mature (155-kDa and simultaneously increased immature (135-kDa hERG expression, respectively. This suggests disruption of forward trafficking of hERG channels. Besides, DHB remarkably reduced heat shock protein 90 (Hsp90 expression and its interaction with hERG, indicating that DHB disrupted hERG trafficking by impairing channel folding. Meanwhie, DHB enhanced the expression of cleaved activating transcription factor-6 (ATF-6, a biomarker of unfolded protein response (UPR. Expression of calnexin and calreticulin, chaperones activated by ATF-6 to facilitate channel folding, were also increased, which indicating UPR activation. Additionally, the degradation rate of mature 155-kDa hERG increased following DHB exposure. In conclusion, we demonstrated that DHB acutely blocked hERG channels by binding the aromatic Tyr652 and Phe656. DHB may decrease hERG plasma membrane expression through two pathways involving disruption of forward trafficking of immature hERG channels and enhanced degradation of mature hERG channels. Furthermore, forward trafficking was

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

    NARCIS (Netherlands)

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

    2016-01-01

    Antiarrhythmic drugs are widely used to treat patients with atrial fibrillation (AF), but the mechanisms conveying their variable effectiveness are not known. Recent data suggested that paired like homeodomain-2 transcription factor (PITX2) might play an important role in regulating gene expression

  14. High Glucose Represses hERG K+ Channel Expression through Trafficking Inhibition

    Directory of Open Access Journals (Sweden)

    Yuan-Qi Shi

    2015-08-01

    Full Text Available Background/Aims: Abnormal QT prolongation is the most prominent cardiac electrical disturbance in patients with diabetes mellitus (DM. It is well known that the human ether-ago-go-related gene (hERG controls the rapid delayed rectifier K+ current (IKr in cardiac cells. The expression of the hERG channel is severely down-regulated in diabetic hearts, and this down-regulation is a critical contributor to the slowing of repolarization and QT prolongation. However, the intracellular mechanisms underlying the diabetes-induced hERG deficiency remain unknown. Methods: The expression of the hERG channel was assessed via western blot analysis, and the hERG current was detected with a patch-clamp technique. Results: The results of our study revealed that the expression of the hERG protein and the hERG current were substantially decreased in high-glucose-treated hERG-HEK cells. Moreover, we demonstrated that the high-glucose-mediated damage to the hERG channel depended on the down-regulation of protein levels but not the alteration of channel kinetics. These discoveries indicated that high glucose likely disrupted hERG channel trafficking. From the western blot and immunoprecipitation analyses, we found that high glucose induced trafficking inhibition through an effect on the expression of Hsp90 and its interaction with hERG. Furthermore, the high-glucose-induced inhibition of hERG channel trafficking could activate the unfolded protein response (UPR by up-regulating the expression levels of activating transcription factor-6 (ATF-6 and the ER chaperone protein calnexin. In addition, we demonstrated that 100 nM insulin up-regulated the expression of the hERG channel and rescued the hERG channel repression caused by high glucose. Conclusion: The results of our study provide the first evidence of a high-glucose-induced hERG channel deficiency resulting from the inhibition of channel trafficking. Furthermore, insulin promotes the expression of the hERG channel

  15. Effects of an epilepsy-causing mutation in the SCN1A sodium channel gene on cocaine-induced seizure susceptibility in mice.

    Science.gov (United States)

    Purcell, Ryan H; Papale, Ligia A; Makinson, Christopher D; Sawyer, Nikki T; Schroeder, Jason P; Escayg, Andrew; Weinshenker, David

    2013-07-01

    High doses of cocaine can elicit seizures in humans and in laboratory animals. Several mechanisms have been proposed for the induction of seizures by cocaine, including enhanced monoaminergic signaling, blockade of ion channels, and alterations in GABA and glutamate transmission. Mutations in the SCN1A gene, which encodes the central nervous system (CNS) voltage-gated sodium channel (VGSC) Nav1.1, are responsible for several human epilepsy disorders including Dravet syndrome and genetic (generalized) epilepsy with febrile seizures plus (GEFS+). Mice heterozygous for the R1648H GEFS+ mutation (RH mice) exhibit reduced interneuron excitability, spontaneous seizures, and lower thresholds to flurothyl- and hyperthermia-induced seizures. However, it is unknown whether impaired CNS VGSC function or a genetic predisposition to epilepsy increases susceptibility to cocaine-induced seizures. Our primary goal was to determine whether Scn1a dysfunction caused by the RH mutation alters sensitivity to cocaine-induced behavioral and electrographic (EEG) seizures. We also tested novelty- and cocaine-induced locomotor activity and assessed the expression of Nav1.1 in midbrain dopaminergic neurons. We found that RH mice had a profound increase in cocaine-induced behavioral seizure susceptibility compared to wild-type (WT) controls, which was confirmed with cortical EEG recordings. By contrast, although the RH mice were hyperactive in novel environments, cocaine-induced locomotor activity was comparable between the mutants and WT littermates. Finally, immunofluorescence experiments revealed a lack of Nav1.1 immunoreactivity in dopaminergic neurons. These data indicate that a disease-causing CNS VGSC mutation confers susceptibility to the proconvulsant, but not motoric, effects of cocaine.

  16. Species selective resistance of cardiac muscle voltage gated sodium channels: characterization of brevetoxin and ciguatoxin binding sites in rats and fish.

    Science.gov (United States)

    Dechraoui, Marie-Yasmine Bottein; Wacksman, Jeremy J; Ramsdell, John S

    2006-11-01

    Brevetoxins (PbTxs) and ciguatoxins (CTXs) are two suites of dinoflagellate derived marine polyether neurotoxins that target the voltage gated sodium channel (VGSC). PbTxs are commonly responsible for massive fish kills and unusual mortalities in marine mammals. CTXs, more often noted for human intoxication, are suspected causes of fish and marine mammal intoxication, although this has never been reported in the field. VGSCs, present in the membrane of all excitable cells including those found in skeletal muscle, nervous and heart tissues, are found as isoforms with differential expression within species and tissues. To investigate the tissue and species susceptibility to these biotoxins, we determined the relative affinity of PbTx-2 and -3 and P-CTX-1 to native VGSCs in the brain, heart, and skeletal muscle of rat and the marine teleost fish Centropristis striata by competitive binding in the presence of [(3)H]PbTx-3. No differences between rat and fish were observed in the binding of PbTxs and CTX to either brain or skeletal muscle. However, [(3)H]PbTx-3 showed substantial lower affinity to rat heart tissue while in the fish it bound with the same affinity to heart than to brain or skeletal muscle. These new insights into PbTxs and CTXs binding in fish and mammalian excitable tissues indicate a species related resistance of heart VGSC in the rat; yet, with comparable sensitivity between the species for brain and skeletal muscle.

  17. A recombinant fusion protein containing a spider toxin specific for the insect voltage-gated sodium ion channel shows oral toxicity towards insects of different orders

    Science.gov (United States)

    Yang, Sheng; Pyati, Prashant; Fitches, Elaine; Gatehouse, John A.

    2014-01-01

    Recombinant fusion protein technology allows specific insecticidal protein and peptide toxins to display activity in orally-delivered biopesticides. The spider venom peptide δ-amaurobitoxin-PI1a, which targets insect voltage-gated sodium channels, was fused to the “carrier” snowdrop lectin (GNA) to confer oral toxicity. The toxin itself (PI1a) and an amaurobitoxin/GNA fusion protein (PI1a/GNA) were produced using the yeast Pichia pastoris as expression host. Although both proteins caused mortality when injected into cabbage moth (Mamestra brassicae) larvae, the PI1a/GNA fusion was approximately 6 times as effective as recombinant PI1a on a molar basis. PI1a alone was not orally active against cabbage moth larvae, but a single 30 μg dose of the PI1a/GNA fusion protein caused 100% larval mortality within 6 days when fed to 3rd instar larvae, and caused significant reductions in survival, growth and feeding in 4th – 6th instar larvae. Transport of fusion protein from gut contents to the haemolymph of cabbage moth larvae, and binding to the nerve chord, was shown by Western blotting. The PI1a/GNA fusion protein also caused mortality when delivered orally to dipteran (Musca domestica; housefly) and hemipteran (Acyrthosiphon pisum; pea aphid) insects, making it a promising candidate for development as a biopesticide. PMID:24486516

  18. Radiostatine and radioiodine uptake characterization in sodium iodine symporter-expressing cell lines

    International Nuclear Information System (INIS)

    Petrich, T.; Helmeke, H.J.; Meyer, G.J.; Knapp, W.H.; Poetter, E.

    2002-01-01

    Full text: The sodium iodide symporter (NIS) has been recognized as an attractive target for cancer gene therapy. Here we investigated NIS-mediated transport of the high LET α-emitter astatine, 211 At, in comparison to radioiodine. A constitutive expression vector harbouring the human NIS cDNA was used in combination with reporter gene vectors for transient transfection of 13 different human cancer cell lines. Radioiodine uptake was measured as well as transfection efficiencies. Six stable NIS-expressing cell lines (3 derived from thyroid carcinomas, 2 colon carcinoma, 1 glioblastoma) were generated by antibiotic selection. NIS expression was monitored by immunohistochemistry and RT-PCR. Subsequently the radioastatine and radioiodine uptake characteristics of genetically modified cells were studied in comparison to the respective control cells. After xenotransplantation in nude mice in vivo tumor imaging by scintigraphy and biodistribution studies following organ removal were performed. Transient transfection of NIS cDNA led to high specific sodium perchlorate-sensitive radioiodine uptake in NIS-expressing cells that roughly correlates to transfection efficiencies. Similarly, stable NIS-expressing cell lines were able to concentrate high levels of radioiodine and in addition showed comparable transport capacity for radioastatine. Accumulation of 211 At was inhibited by sodium perchlorate like iodide uptake and displayed dependency an extracellular Na + - and I - -ions as well. Compared to wash-out experiments in cell culture the effective half life of radioiodine and radioastatine in vivo was significantly prolonged. Preliminary dose calculations by MIRD concepts indicated higher tumor radiation doses for 211 At compared to 131 I. Tumor cells of different origins transfected with the NIS-expression vector specifically and significantly take-up radioiodine and radioastatine in vitro and in vivo. The data provide direct evidence that the NIS efficiently transports

  19. Effect of insecticidal fusion proteins containing spider toxins targeting sodium and calcium ion channels on pyrethroid-resistant strains of peach-potato aphid (Myzus persicae).

    Science.gov (United States)

    Yang, Sheng; Fitches, Elaine; Pyati, Prashant; Gatehouse, John A

    2015-07-01

    The recombinant fusion proteins Pl1a/GNA and Hv1a/GNA contain the spider venom peptides δ-amaurobitoxin-PI1a or ω-hexatoxin-Hv1a respectively, linked to snowdrop lectin (GNA). Pl1a targets receptor site 4 of insect voltage-gated sodium channels (NaCh), while Hv1a targets voltage-gated calcium channels. Insecticide-resistant strains of peach-potato aphid (Myzus persicae) contain mutations in NaCh. The pyrethroid-resistant kdr (794J) and super-kdr (UKO) strains contain mutations at residues L1014 and M918 in the channel α-subunit respectively, while the kdr + super-kdr strain (4824J), insensitive to pyrethroids, contains mutations at both L1014 and M918. Pl1a/GNA and Hv1a/GNA fusion proteins have estimated LC50 values of 0.35 and 0.19 mg mL(-1) when fed to wild-type M. persicae. For insecticide-resistant aphids, LC50 for the Pl1a/GNA fusion protein increased by 2-6-fold, correlating with pyrethroid resistance (wild type < kdr < super-kdr < kdr + super-kdr strains). In contrast, LC50 for the Hv1a/GNA fusion protein showed limited correlation with pyrethroid resistance. Mutations in the sodium channel in pyrethroid-resistant aphids also protect against a fusion protein containing a sodium-channel-specific toxin, in spite of differences in ligand-channel interactions, but do not confer resistance to a fusion protein targeting calcium channels. The use of fusion proteins with differing targets could play a role in managing pesticide resistance. © 2014 Society of Chemical Industry.

  20. On the Mechanism of Human Red Blood Cell Longevity: Roles of Calcium, the Sodium Pump, PIEZO1, and Gardos Channels

    Directory of Open Access Journals (Sweden)

    Virgilio L. Lew

    2017-12-01

    Full Text Available In a healthy adult, the transport of O2 and CO2 between lungs and tissues is performed by about 2 · 1013 red blood cells, of which around 1.7 · 1011 are renewed every day, a turnover resulting from an average circulatory lifespan of about 120 days. Cellular lifespan is the result of an evolutionary balance between the energy costs of maintaining cells in a fit functional state versus cell renewal. In this Review we examine how the set of passive and active membrane transporters of the mature red blood cells interact to maximize their circulatory longevity thus minimizing costs on expensive cell turnover. Red blood cell deformability is critical for optimal rheology and gas exchange functionality during capillary flow, best fulfilled when the volume of each human red blood cell is kept at a fraction of about 0.55–0.60 of the maximal spherical volume allowed by its membrane area, the optimal-volume-ratio range. The extent to which red blood cell volumes can be preserved within or near these narrow optimal-volume-ratio margins determines the potential for circulatory longevity. We show that the low cation permeability of red blood cells allows volume stability to be achieved with extraordinary cost-efficiency, favouring cell longevity over cell turnover. We suggest a mechanism by which the interplay of a declining sodium pump and two passive membrane transporters, the mechanosensitive PIEZO1 channel, a candidate mediator of Psickle in sickle cells, and the Ca2+-sensitive, K+-selective Gardos channel, can implement red blood cell volume stability around the optimal-volume-ratio range, as required for extended circulatory longevity.

  1. Expression of inwardly rectifying K+ channels in the carotid body of rat

    OpenAIRE

    Yamamoto, Y.; Ishikawa, R.; Omoe, K.; Taniguchi, K.

    2008-01-01

    The inwardly rectifying K+ channels, Kir1.1, Kir2.3, Kir4.1-Kir5.1, and Kir4.2-Kir5.1, are candidate chemosensory molecules for CO2/H+. Here, we determined the mRNA expression and immunohistochemical localization of these channels in the carotid body (CB) and petrosal ganglion (PG) of the rat. RT-PCR analysis revealed mRNA expression of Kir4.1 and Kir5.1 in CB, and Kir1.1, Kir4.1, and Kir5.1 in PG. Immunohistochemistry identified the glomus cells in CB to express bo...

  2. Do cysteine residues regulate transient receptor potential canonical type 6 (TRPC6) channel protein expression?

    DEFF Research Database (Denmark)

    Thilo, Florian; Liu, Ying; Krueger, Katharina

    2012-01-01

    The regulation of calcium influx through transient receptor potential canonical type 6 channel is mandatory for the activity of human monocytes. We submit the first evidence that cysteine residues of homocysteine or acetylcysteine affect TRPC6 expression in human monocytes. We observed that patie......The regulation of calcium influx through transient receptor potential canonical type 6 channel is mandatory for the activity of human monocytes. We submit the first evidence that cysteine residues of homocysteine or acetylcysteine affect TRPC6 expression in human monocytes. We observed...... that patients with chronic renal failure had significantly elevated homocysteine levels and TRPC6 mRNA expression levels in monocytes compared to control subjects. We further observed that administration of homocysteine or acetylcysteine significantly increased TRPC6 channel protein expression compared...

  3. Expression of TRPC6 channels in human epithelial breast cancer cells

    Directory of Open Access Journals (Sweden)

    Ahidouch Ahmed

    2008-05-01

    Full Text Available Abstract Background TRP channels have been shown to be involved in tumour generation and malignant growth. However, the expression of these channels in breast cancer remains unclear. Here we studied the expression and function of endogenous TRPC6 channels in a breast cancer cell line (MCF-7, a human breast cancer epithelial primary culture (hBCE and in normal and tumour breast tissues. Methods Molecular (Western blot and RT-PCR, and immunohistochemical techniques were used to investigate TRPC6 expression. To investigate the channel activity in both MCF-7 cells and hBCE we used electrophysiological technique (whole cell patch clamp configuration. Results A non selective cationic current was activated by the oleoyl-2-acetyl-sn-glycerol (OAG in both hBCE and MCF-7 cells. OAG-inward current was inhibited by 2-APB, SK&F 96365 and La3+. TRPC6, but not TRPC7, was expressed both in hBCE and in MCF-7 cells. TRPC3 was only expressed in hBCE. Clinically, TRPC6 mRNA and protein were elevated in breast carcinoma specimens in comparison to normal breast tissue. Furthermore, we found that the overexpression of TRPC6 protein levels were not correlated with tumour grades, estrogen receptor expression or lymph node positive tumours. Conclusion Our results indicate that TRPC6 channels are strongly expressed and functional in breast cancer epithelial cells. Moreover, the overexpression of these channels appears without any correlation with tumour grade, ER expression and lymph node metastasis. Our findings support the idea that TRPC6 may have a role in breast carcinogenesis.

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

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    Joel González-Cabrera

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

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

    Science.gov (United States)

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

    2016-01-01

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

  6. Immunolocalization and expression of small-conductance calcium-activated potassium channels in human myometrium

    DEFF Research Database (Denmark)

    Rosenbaum, Sofia T; Svalø, Julie; Nielsen, Karsten

    2012-01-01

    Small-conductance calcium-activated potassium (SK3) channels have been detected in human myometrium and we have previously shown a functional role of SK channels in human myometrium in vitro. The aims of this study were to identify the precise localization of SK3 channels and to quantify SK3 m...... muscle cells, and that the molecular expression of SK3 channels is higher in non-pregnant compared to pregnant myometrium. On the basis of our previous study and the present findings, we propose that SK3 activators reduce contractility in human myometrium by modulating telocyte function....... This is the first report to provide evidence for a possible role of SK3 channels in human uterine telocytes....

  7. Ion channel gene expressions in infertile men: A case-control study

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    Serkan Carkci

    2017-12-01

    Full Text Available Background: Infertility is described as not receiving pregnancy despite unprotected and regular sexual intercourse in a 1 yr period. It is detected by 15% of the couples. Male and female factor in the etiology may be detected in similar rates. Objective: The present study aims to investigate ion channel gene expression in semen samples of infertile male compared with fertile men. Materials and Methods: A total of 150 men who applied to the urology clinic due to infertility were divided into five equal groups: asthenozoospermia, oligozoospermia, oligoasthenoteratozoospermia, teratozoospermia, and normozoospermia (control. All paticipants were evaluated with Cation Channel Spermia (CatSper 1, 2, 3, 4, Proton Voltage Gated Ion Channel1 (Hv1, Potassium Channel Subfamily U1 (KCNU1, and transmembrane protein (TMEM16A gene expression in semen samples. Results: “CatSper1, 4, HV1, KCNU1, and TMEM16A gene expression were detected higher in the oligozoospermia group compared to the controls. CatSper1, 2, 3, 4, KCNU1, and TMEM16A gene expression in the asthenozoospermia group and CatSper1, 2, 3, 4, KCNU1, and TMEM16A gene expression in the teratozoospermia group were detected lower compared to the controls. CatSper1, 4, HV1, and TMEM16A gen expression were higher in the oligoasthenoteratozoospermia men than the controls while CatSper3 gen expression was detected as lower.” Conclusion: It was detected that these ion channels have an effect on sperm progressive motility and morphology. It may be considered that mutations in these ion channels may result in infertility

  8. Development of a Rapid Throughput Assay for Identification of hNav1.7 Antagonist Using Unique Efficacious Sodium Channel Agonist, Antillatoxin

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    Fang Zhao

    2016-02-01

    Full Text Available Voltage-gated sodium channels (VGSCs are responsible for the generation of the action potential. Among nine classified VGSC subtypes (Nav1.1–Nav1.9, Nav1.7 is primarily expressed in the sensory neurons, contributing to the nociception transmission. Therefore Nav1.7 becomes a promising target for analgesic drug development. In this study, we compared the influence of an array of VGSC agonists including veratridine, BmK NT1, brevetoxin-2, deltamethrin and antillatoxin (ATX on membrane depolarization which was detected by Fluorescence Imaging Plate Reader (FLIPR membrane potential (FMP blue dye. In HEK-293 cells heterologously expressing hNav1.7 α-subunit, ATX produced a robust membrane depolarization with an EC50 value of 7.8 ± 2.9 nM whereas veratridine, BmK NT1, and deltamethrin produced marginal response. Brevetoxin-2 was without effect on membrane potential change. The ATX response was completely inhibited by tetrodotoxin suggesting that the ATX response was solely derived from hNav1.7 activation, which was consistent with the results where ATX produced a negligible response in null HEK-293 cells. Six VGSC antagonists including lidocaine, lamotrigine, phenytoin, carbamazepine, riluzole, and 2-amino-6-trifluoromethylthiobenzothiazole all concentration-dependently inhibited ATX response with IC50 values comparable to that reported from patch-clamp experiments. Considered together, we demonstrate that ATX is a unique efficacious hNav1.7 activator which offers a useful probe to develop a rapid throughput screening assay to identify hNav1.7 antagonists.

  9. Development of a Rapid Throughput Assay for Identification of hNav1.7 Antagonist Using Unique Efficacious Sodium Channel Agonist, Antillatoxin.

    Science.gov (United States)

    Zhao, Fang; Li, Xichun; Jin, Liang; Zhang, Fan; Inoue, Masayuki; Yu, Boyang; Cao, Zhengyu

    2016-02-16

    Voltage-gated sodium channels (VGSCs) are responsible for the generation of the action potential. Among nine classified VGSC subtypes (Nav1.1-Nav1.9), Nav1.7 is primarily expressed in the sensory neurons, contributing to the nociception transmission. Therefore Nav1.7 becomes a promising target for analgesic drug development. In this study, we compared the influence of an array of VGSC agonists including veratridine, BmK NT1, brevetoxin-2, deltamethrin and antillatoxin (ATX) on membrane depolarization which was detected by Fluorescence Imaging Plate Reader (FLIPR) membrane potential (FMP) blue dye. In HEK-293 cells heterologously expressing hNav1.7 α-subunit, ATX produced a robust membrane depolarization with an EC50 value of 7.8 ± 2.9 nM whereas veratridine, BmK NT1, and deltamethrin produced marginal response. Brevetoxin-2 was without effect on membrane potential change. The ATX response was completely inhibited by tetrodotoxin suggesting that the ATX response was solely derived from hNav1.7 activation, which was consistent with the results where ATX produced a negligible response in null HEK-293 cells. Six VGSC antagonists including lidocaine, lamotrigine, phenytoin, carbamazepine, riluzole, and 2-amino-6-trifluoromethylthiobenzothiazole all concentration-dependently inhibited ATX response with IC50 values comparable to that reported from patch-clamp experiments. Considered together, we demonstrate that ATX is a unique efficacious hNav1.7 activator which offers a useful probe to develop a rapid throughput screening assay to identify hNav1.7 antagonists.

  10. The voltage-gated sodium channel EF-hands form an interaction with the III-IV linker that is disturbed by disease-causing mutations.

    Science.gov (United States)

    Gardill, Bernd R; Rivera-Acevedo, Ricardo E; Tung, Ching-Chieh; Okon, Mark; McIntosh, Lawrence P; Van Petegem, Filip

    2018-03-14

    Voltage-gated sodium channels (Na V ) are responsible for the rapid depolarization of many excitable cells. They readily inactivate, a process where currents diminish after milliseconds of channel opening. They are also targets for a multitude of disease-causing mutations, many of which have been shown to affect inactivation. A cluster of disease mutations, linked to Long-QT and Brugada syndromes, is located in a C-terminal EF-hand like domain of Na V 1.5, the predominant cardiac sodium channel isoform. Previous studies have suggested interactions with the III-IV linker, a cytosolic element directly involved in inactivation. Here we validate and map the interaction interface using isothermal titration calorimetry (ITC) and NMR spectroscopy. We investigated the impact of various disease mutations on the stability of the domain, and found that mutations that cause misfolding of the EF-hand domain result in hyperpolarizing shifts in the steady-state inactivation curve. Conversely, mutations in the III-IV linker that disrupt the interaction with the EF-hand domain also result in large hyperpolarization shifts, supporting the interaction between both elements in intact channels. Disrupting the interaction also causes large late currents, pointing to a dual role of the interaction in reducing the population of channels entering inactivation and in stabilizing the inactivated state.

  11. Streptomycin potency is dependent on MscL channel expression

    Science.gov (United States)

    Iscla, Irene; Wray, Robin; Wei, Shuguang; Posner, Bruce; Blount, Paul

    2014-09-01

    The antibiotic streptomycin is widely used in the treatment of microbial infections. The primary mechanism of action is inhibition of translation by binding to the ribosome, but how it enters the bacterial cell is unclear. Early in the study of this antibiotic, a mysterious streptomycin-induced potassium efflux preceding any decrease in viability was observed; it was speculated that this changed the electrochemical gradient such that streptomycin better accessed the cytoplasm. Here we use a high-throughput screen to search for compounds targeting the mechanosensitive channel of large conductance (MscL) and find dihydrostreptomycin among the ‘hits’. Furthermore, we find that MscL is not only necessary for the previously described streptomycin-induced potassium efflux, but also directly increases MscL activity in electrophysiological studies. The data suggest that gating MscL is a novel mode of action of dihydrostreptomycin, and that MscL’s large pore may provide a mechanism for cell entry.

  12. K-ATP channel expression and pharmacological in vivo and in vitro studies of the K-ATP channel blocker PNU-37883A in rat middle meningeal arteries

    DEFF Research Database (Denmark)

    Ploug, K.B.; Boni, L.J.; Baun, M.

    2008-01-01

    Background and purpose: Dilatation of cerebral and dural arteries causes a throbbing, migraine-like pain, indicating that these structures are involved in migraine. Clinical trials suggest that adenosine 5'-triphosphate-sensitive K+ (K-ATP) channel opening may cause migraine by dilatating...... intracranial arteries, including the middle meningeal artery (MMA). We studied the K-ATP channel expression profile in rat MMA and examined the potential inhibitory effects of the K-ATP channel blocker PNU-37883A on K-ATP channel opener-induced relaxation of the rat MMA, using the three K-ATP channel openers...

  13. MiR-30b Attenuates Neuropathic Pain by Regulating Voltage-Gated Sodium Channel Nav1.3 in Rats

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    Songxue Su

    2017-05-01

    Full Text Available Nav1.3 is a tetrodotoxin-sensitive isoform among voltage-gated sodium channels that are closely associated with neuropathic pain. It can be up-regulated following nerve injury, but its biological function remains uncertain. MicroRNAs (miRNAs are endogenous non-coding RNAs that can regulate post-transcriptional gene expression by binding with their target mRNAs. Using Target Scan software, we discovered that SCN3A is the major target of miR-30b, and we then determined whether miR-30b regulated the expression of Nav1.3 by transfecting miR-30b agomir through the stimulation of TNF-α or by transfecting miR-30b antagomir in primary dorsal root ganglion (DRG neurons. The spinal nerve ligation (SNL model was used to determine the contribution of miR-30b to neuropathic pain, to evaluate changes in Nav1.3 mRNA and protein expression, and to understand the sensitivity of rats to mechanical and thermal stimuli. Our results showed that miR-30b agomir transfection down-regulated Nav1.3 mRNA stimulated with TNF-α in primary DRG neurons. Moreover, miR-30b overexpression significantly attenuated neuropathic pain induced by SNL, with decreases in the expression of Nav1.3 mRNA and protein both in DRG neurons and spinal cord. Activation of Nav1.3 caused by miR-30b antagomir was identified. These data suggest that miR-30b is involved in the development of neuropathic pain, probably by regulating the expression of Nav1.3, and might be a novel therapeutic target for neuropathic pain.Perspective: This study is the first to explore the important role of miR-30b and Nav1.3 in spinal nerve ligation-induced neuropathic pain, and our evidence may provide new insight for improving therapeutic approaches to pain.

  14. Role of voltage-gated sodium channel blockers in the treatment of chronic pain: Potential uses in clinical practice based on available evidence.

    Science.gov (United States)

    Alcántara Montero, A; Sánchez Carnerero, C I

    2018-02-26

    Once patients have failed first line therapy, there is an apparent lack of knowledge on how to proceed with choosing subsequent therapy. To choose amongst alternative agents, an understanding of pharmacology, pharmacokinetics, and available evidence in targeting various pain conditions is necessary. This article focuses on the use of the carboxamide class of voltage-gated sodium channel blockers (carbamazepine, oxcarbazepine, eslicarbazepine acetate) for adjunct pain medication management; including research updates in pharmacology, pharmacokinetics, and evidence for pain along on this therapeutic group with promising future areas of research. Although evidence for voltage-gated sodium channel blockers in chronic pain management is limited, emerging research has identified this area as promising for additional clinical trials to better guide clinical practice. Copyright © 2018 Sociedad Española de Anestesiología, Reanimación y Terapéutica del Dolor. Publicado por Elsevier España, S.L.U. All rights reserved.

  15. Subcloning, localization, and expression of the rat intestinal sodium-hydrogen exchanger isoform 8.

    Science.gov (United States)

    Xu, Hua; Chen, Rongji; Ghishan, Fayez K

    2005-07-01

    Apically expressed intestinal and renal sodium-hydrogen exchangers (NHEs) play a major role in Na(+) absorption. Our previous studies on NHE ontogeny have shown that NHE-2 and NHE-3 are expressed at very low levels in young animals. Furthermore, single and/or double NHE-2 and NHE-3 knockout mice display no obvious abnormalities before weaning. These observations suggest that other transporter(s) may be involved in intestinal Na+ absorption during early life. The present studies were designed to clone the novel rat intestinal NHE-8 cDNA and to decipher the NHE-8 protein localization and gene expression pattern during different developmental stages. The rat NHE-8 cDNA has 2,160 bp and encodes a 575-amino acid protein. An antibody against NHE-8 protein was developed. Immunohistochemistry staining indicated apical localization of NHE-8 protein in rat intestinal epithelial cells. The apical localization of NHE-8 was also confirmed by its presence in brush-border membrane and its absence in basolateral membrane preparations. Northern blotting utilizing a NHE-8-specific probe demonstrated higher NHE-8 mRNA expression in young animals compared with adult animals. Western blot analysis revealed a similar pattern. Tissue distribution with multiple human tissue RNA blot showed that NHE-8 was expressed in multiple tissues including the gastrointestinal tract. In conclusion, we have cloned the full-length NHE-8 cDNA from rat intestine and further showed its apical localization in intestinal epithelial cells. We have also shown that NHE-8 gene expression and protein expression were regulated during ontogeny. Our data suggests that NHE-8 may play an important role in intestinal Na+ absorption during early life.

  16. Beraprost sodium, a stable prostacyclin analogue, elicits dilation of isolated porcine retinal arterioles: roles of eNOS and potassium channels.

    Science.gov (United States)

    Ono, Shinji; Nagaoka, Taiji; Omae, Tsuneaki; Tanano, Ichiro; Kamiya, Takayuki; Otani, Shinichi; Ishibazawa, Akihiro; Yoshida, Akitoshi

    2014-07-31

    Prostacyclin (PGI2) is usually described as an endoEDRFsthelium-derived relaxing factor, but the vasoreactivity to PGI2 in the retinal arterioles and the underlying mechanisms are not fully understood. We examined the effects of PGI2 on the retinal microcirculation using beraprost sodium (BPS), a stable PGI2 analogue, and the signaling mechanisms involved in this vasomotor activity. Porcine retinal arterioles were isolated, cannulated, and pressurized without flow in vitro. Video microscopic techniques recorded the diametric responses to BPS. Beraprost sodium elicited dose-dependent (0.1 pM-0.1 μM) vasodilation of the retinal arterioles that was abolished by the PGI2 receptor (IP) antagonist CAY10441. Beraprost sodium-induced vasodilation decreased by 50% after the endothelium was removed and was inhibited by the nitric oxide (NO) synthase inhibitor N(G)-nitro-L-arginine methyl ester (L-NAME) comparable with denudation. Inhibition of soluble guanylyl cyclase by 1H-1,2,4-oxadiazolo[4,3-a]quinoxalin-1-one (ODQ) and blockage of protein kinase A (PKA) by Rp-8-Br-cAMPS were comparable to L-NAME. Beraprost sodium-induced vasodilation was also inhibited by the nonselective potassium channel inhibitor, tetraethylammonium, and the adenosine triphosphate-sensitive potassium (KATP) channel blocker, glibenclamide. Residual vasodilation in the presence of glibenclamide decreased further with subsequent application of ODQ. Beraprost sodium, a stable PGI2 analogue, causes vasodilation of the retinal arterioles mediated via the IP receptor. The current findings suggest that BPS elicits endothelium-dependent and -independent dilation of the retinal arterioles mediated by NO induced by activation of PKA in the endothelium and the KATP channel activation in the vascular smooth muscle, respectively. Copyright 2014 The Association for Research in Vision and Ophthalmology, Inc.

  17. Endoglin Selectively Modulates Transient Receptor Potential Channel Expression in Left and Right Heart Failure

    Science.gov (United States)

    Morine, Kevin J.; Paruchuri, Vikram; Qiao, Xiaoying; Aronovitz, Mark; Pham, Duc Thinh; Huggins, Gordon S.; DeNofrio, David; Kiernan, Michael S.; Karas, Richard H.; Kapur, Navin K.

    2017-01-01

    Introduction Transient receptor potential (TRP) channels are broadly expressed cation channels that mediate diverse physiological stimuli and include canonical (TRPC), melastatin (TRPM) and vanilloid (TRPV) subtypes. Recent studies have implicated a role for TRPC6 channels as an important component of signaling via the cytokine, transforming growth factor beta 1 (TGFb1) in right (RV) or left ventricular (LV) failure. Endoglin is a transmembrane glycoprotein that promotes TRPC6 expression and TGFb1 activity. No studies have defined biventricular expression of all TRP channel family members in heart failure. Hypothesis We hypothesized that heart failure is associated with distinct patterns of TRP channel expression in the LV and RV. Methods Paired viable left (LV) and right (RV) ventricular free wall tissue was obtained from human subjects with end-stage heart failure (n=12) referred for cardiac transplantation or biventricular assist device implantation. Paired LV and RV samples from human subjects without heart failure served as controls (n=3). To explore a functional role for endoglin (Eng) as a regulator of TRP expression in response to RV or LV pressure overload, wild-type (Eng+/+) and Eng haploinsufficient (Eng+/−) mice were exposed to thoracic aortic (TAC) or pulmonary arterial (PAC) constriction for 8 weeks. Biventricular tissue was analyzed by real-time polymerase chain reaction. Results Compared to non-failing human LV and RV samples, mRNA levels of TRPC1, 3, 4, 6 and TRPV-2 were increased and TRPM2, 3, and 8 were decreased in failing LV and RV samples. TRPC1 and 6 levels were higher in failing RV compared to failing LV samples. After TAC, murine LV levels of TPRC1 and 6 were increased in both Eng +/+ and Eng +/− mice compared to sham controls. LV levels of TRPC4; TRPM3 and 7; TRPV2 and 4 were increased in Eng +/+, not Eng +/− mice after TAC. After PAC, all TRP channel family members were increased in the RV, but not LV, of Eng +/+ compared to sham

  18. Dysfunction of the Scn8a Voltage-gated Sodium Channel Alters Sleep Architecture, Reduces Diurnal Corticosterone Levels, and Enhances Spatial Memory

    OpenAIRE

    Papale, Ligia Assumpção [UNIFESP; Paul, Ketema N.; Sawyer, Nikki T.; Manns, Joseph R.; Tufik, Sergio [UNIFESP; Escayg, Andrew

    2010-01-01

    Voltage-gated sodium channels (VGSCs) are responsible for the initiation and propagation of transient depolarizing currents and play a critical role in the electrical signaling between neurons. A null mutation in the VGSC gene SCN8A, which encodes the transmembrane protein Na(v)1.6, was identified previously in a human family. Heterozygous mutation carriers displayed a range of phenotypes, including ataxia, cognitive deficits, and emotional instability. A possible role for SCN8A was also prop...

  19. Developmental expression of Kv1 voltage-gated potassium channels in the avian hypothalamus.

    Science.gov (United States)

    Doczi, Megan A; Vitzthum, Carl M; Forehand, Cynthia J

    2016-03-11

    Specialized hypothalamic neurons integrate the homeostatic balance between food intake and energy expenditure, processes that may become dysregulated during the development of diabetes, obesity, and other metabolic disorders. Shaker family voltage-gated potassium channels (Kv1) contribute to the maintenance of resting membrane potential, action potential characteristics, and neurotransmitter release in many populations of neurons, although hypothalamic Kv1 channel expression has been largely unexplored. Whole-cell patch clamp recordings from avian hypothalamic brain slices demonstrate a developmental shift in the electrophysiological properties of avian arcuate nucleus neurons, identifying an increase in outward ionic current that corresponds with action potential maturation. Additionally, RT-PCR experiments identified the early expression of Kv1.2, Kv1.3, and Kv1.5 mRNA in the embryonic avian hypothalamus, suggesting that these channels may underlie the electrophysiological changes observed in these neurons. Real-time quantitative PCR analysis on intact microdissections of embryonic hypothalamic tissue revealed a concomitant increase in Kv1.2 and Kv1.5 gene expression at key electrophysiological time points during development. This study is the first to demonstrate hypothalamic mRNA expression of Kv1 channels in developing avian embryos and may suggest a role for voltage-gated ion channel regulation in the physiological patterning of embryonic hypothalamic circuits governing energy homeostasis. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

  20. Understanding evolutionary paradigm of knockdown resistance in mosquitoes by analyzing DNA sequence polymorphisms in voltage-gated sodium channel in Culex quinquefasciatus

    Directory of Open Access Journals (Sweden)

    Manas Sarkar

    2011-09-01

    Full Text Available The Voltage Gated Sodium Channel (VGSC is critical for binding of different insecticides and plays a key role in insecticide resistance. The insect sodium channel consists of four homologous domains (I to IV, each containing six transmembrane segments (S1 to S6. An important mechanism of resistance to DDT and pyrethroids is termed knockdown resistance (kdr, caused by a single nucleotide polymorphism in IIS6 domain of sodium channels. We analyzed the polymorphisms, nucleotide diversity, and phylogenies in VGSC-IIS6 gene in Culex quinquefasciatus from India. We analyzed the neutral model / hypothesis to infer if natural selection is acting upon the analyzed vgsc gene. Tajima’s D, Fu and Li’s D* and F* and Fu’s Fs test were performed to determine whether the distribution of nucleotide variation within the samples was consistent with neutral model. We theorized that the evolutionary pattern of intra-population distribution of variability in vgsc gene is consistent with the neutral expectation.

  1. Activation of Recombinantly Expressed l-Amino Acid Oxidase from Rhizoctonia solani by Sodium Dodecyl Sulfate

    Directory of Open Access Journals (Sweden)

    Katharina Hahn

    2017-12-01

    Full Text Available l-Amino acid oxidases (l-AAO catalyze the oxidative deamination of l-amino acids to the corresponding α-keto acids. The non-covalently bound cofactor FAD is reoxidized by oxygen under formation of hydrogen peroxide. We expressed an active l-AAO from the fungus Rhizoctonia solani as a fusion protein in E. coli. Treatment with small amounts of the detergent sodium dodecyl sulfate (SDS stimulated the activity of the enzyme strongly. Here, we investigated whether other detergents and amphiphilic molecules activate 9His-rsLAAO1. We found that 9His-rsLAAO1 was also activated by sodium tetradecyl sulfate. Other detergents and fatty acids were not effective. Moreover, effects of SDS on the oligomerization state and the protein structure were analyzed. Native and SDS-activated 9His-rsLAAO1 behaved as dimers by size-exclusion chromatography. SDS treatment induced an increase in hydrodynamic radius as observed by size-exclusion chromatography and dynamic light scattering. The activated enzyme showed accelerated thermal inactivation and an exposure of additional protease sites. Changes in tryptophan fluorescence point to a more hydrophilic environment. Moreover, FAD fluorescence increased and a lower concentration of sulfites was sufficient to form adducts with FAD. Taken together, these data point towards a more open conformation of SDS-activated l-amino acid oxidase facilitating access to the active site.

  2. Expression of Inwardly Rectifying Potassium Channel Subunits in Native Human Retinal Pigment Epithelium

    OpenAIRE

    Yang, Dongli; Zhang, Xiaoming; Hughes, Bret A.

    2008-01-01

    Previously, we demonstrated that the inwardly rectifying K+ (Kir) channel subunit Kir7.1 is highly expressed in bovine and human retinal pigment epithelium (RPE). The purpose of this study was to determine whether any of the 14 other members of the Kir gene family are expressed in native human RPE. Conventional reverse transcription-polymerase chain reaction (RT-PCR) analysis indicated that in addition to Kir7.1, 7 other Kir channel subunits (Kir1.1, Kir2.1, Kir2.2, Kir3.1, Kir3.4, Kir4.2 and...

  3. Mutation in the Sodium Channel Gene Corresponds With Phenotypic Resistance of Rhipicephalus sanguineus sensu lato (Acari: Ixodidae) to Pyrethroids.

    Science.gov (United States)

    Klafke, G M; Miller, R J; Tidwell, J; Barreto, R; Guerrero, F D; Kaufman, P E; Pérez de León, A A

    2017-11-07

    The brown dog tick, Rhipicephalus sanguineus sensu lato (Latreille), is a cosmopolitan ectoparasite and vector of pathogens that kill humans and animals. Pyrethroids represent a class of synthetic acaricides that have been used intensely to try to control the brown dog tick and mitigate the risk of tick-borne disease transmission. However, acaricide resistance is an emerging problem in the management of the brown dog tick. Understanding the mechanism of resistance to acaricides, including pyrethroids, is important to adapt brown dog tick control strategies. The main objective of this study was to determine if target-site mutations associated with pyrethroid resistance in other pests could be associated with phenotypic resistance detected in a brown dog tick population from Florida. We amplified segment 6 of the domain III of the voltage-sensitive sodium channel protein, using cDNAs synthesized from pyrethroid-susceptible and pyrethroid-resistant tick strains. A single nucleotide point mutation (SNP) identified in a highly conserved region of domain III S6 in the resistant ticks resulted in an amino acid change from phenylalanine to leucine. This mutation is characteristic of resistance phenotypes in other tick species, and is the first report of this mutation in R. sanguineus. Molecular assays based on this knowledge could be developed to diagnose the risk for pyrethroid resistance, and to inform decisions on integrated brown dog tick management practices. © The Authors 2017. Published by Oxford University Press on behalf of Entomological Society of America. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

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

    Science.gov (United States)

    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.

  5. Effect of voltage-gated sodium channels blockers on motility and viability of human sperm in vitro

    Directory of Open Access Journals (Sweden)

    Hammad Ahmad Gakhar

    2018-03-01

    Full Text Available Objective: To test the effect of voltage-gated sodium channels (VGSCs blockers on the motility and viability of human sperm in-vitro and to evaluate the tested compounds as potential contact spermicidal. Methods: Sperm samples were obtained from healthy nonsmoking volunteers of age 25-30 years who had not taken any drug 3 months before and during the course of the study. The effect of VGSCs blockers evaluated from two pharmacological classes including antiarrhythmic (amiodarone, procainamide and disopyramide and antiepileptic (carbamazepine, oxcarbazepine, phenytoin, and lamotrigine drugs. They were tested on the in-vitro motility and viability of human sperm using Computer Assisted Semen Analyzer. Results: All tested drugs except oxcarbazepine showed dose dependent inhibition of total motility with significant reduction (P<0.05 at the maximum concentration of 200 μM when compared with the control. The concentrations of drugs that reduced total sperm motility to 50% of control (half maximal inhibitory concentration were 2.76, 14.16 and 20.29 μM for phenytoin, lamotrigine and carbamazepine, respectively; and 2.53, 5.32 and 0.37 μM for amiodarone, procainamide and disopyramide, respectively. The anti-motility effects were reversible to various degrees. There was statistically insignificant difference in the inhibition of sperm viability among amiodarone, procainamide and disopyramide. Phenytoin demonstrated the most potent spermicidal action. Conclusions: VGSCs blockers have significant adverse effects on in-vitro motility of human spermatozoa. So in-vivo studies are required to determine their potential toxicological effects on human semen quality, which is an important factor regarding fertility. Moreover, these drugs have the potential to be developed into contact spermicidal.

  6. Cholesterol Down-Regulates BK Channels Stably Expressed in HEK 293 Cells

    Science.gov (United States)

    Deng, Xiu-Ling; Sun, Hai-Ying; Li, Gui-Rong

    2013-01-01

    Cholesterol is one of the major lipid components of the plasma membrane in mammalian cells and is involved in the regulation of a number of ion channels. The present study investigates how large conductance Ca2+-activated K+ (BK) channels are regulated by membrane cholesterol in BK-HEK 293 cells expressing both the α-subunit hKCa1.1 and the auxiliary β1-subunit or in hKCa1.1-HEK 293 cells expressing only the α-subunit hKCa1.1 using approaches of electrophysiology, molecular biology, and immunocytochemistry. Membrane cholesterol was depleted in these cells with methyl-β-cyclodextrin (MβCD), and enriched with cholesterol-saturated MβCD (MβCD-cholesterol) or low-density lipoprotein (LDL). We found that BK current density was decreased by cholesterol enrichment in BK-HEK 293 cells, with a reduced expression of KCa1.1 protein, but not the β1-subunit protein. This effect was fully countered by the proteasome inhibitor lactacystin or the lysosome function inhibitor bafilomycin A1. Interestingly, in hKCa1.1-HEK 293 cells, the current density was not affected by cholesterol enrichment, but directly decreased by MβCD, suggesting that the down-regulation of BK channels by cholesterol depends on the auxiliary β1-subunit. The reduced KCa1.1 channel protein expression was also observed in cultured human coronary artery smooth muscle cells with cholesterol enrichment using MβCD-cholesterol or LDL. These results demonstrate the novel information that cholesterol down-regulates BK channels by reducing KCa1.1 protein expression via increasing the channel protein degradation, and the effect is dependent on the auxiliary β1-subunit. PMID:24260325

  7. Unidirectional photoreceptor-to-Müller glia coupling and unique K+ channel expression in Caiman retina.

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    Astrid Zayas-Santiago

    Full Text Available Müller cells, the principal glial cells of the vertebrate retina, are fundamental for the maintenance and function of neuronal cells. In most vertebrates, including humans, Müller cells abundantly express Kir4.1 inwardly rectifying potassium channels responsible for hyperpolarized membrane potential and for various vital functions such as potassium buffering and glutamate clearance; inter-species differences in Kir4.1 expression were, however, observed. Localization and function of potassium channels in Müller cells from the retina of crocodiles remain, hitherto, unknown.We studied retinae of the Spectacled caiman (Caiman crocodilus fuscus, endowed with both diurnal and nocturnal vision, by (i immunohistochemistry, (ii whole-cell voltage-clamp, and (iii fluorescent dye tracing to investigate K+ channel distribution and glia-to-neuron communications.Immunohistochemistry revealed that caiman Müller cells, similarly to other vertebrates, express vimentin, GFAP, S100β, and glutamine synthetase. In contrast, Kir4.1 channel protein was not found in Müller cells but was localized in photoreceptor cells. Instead, 2P-domain TASK-1 channels were expressed in Müller cells. Electrophysiological properties of enzymatically dissociated Müller cells without photoreceptors and isolated Müller cells with adhering photoreceptors were significantly different. This suggests ion coupling between Müller cells and photoreceptors in the caiman retina. Sulforhodamine-B injected into cones permeated to adhering Müller cells thus revealing a uni-directional dye coupling.Our data indicate that caiman Müller glial cells are unique among vertebrates studied so far by predominantly expressing TASK-1 rather than Kir4.1 K+ channels and by bi-directional ion and uni-directional dye coupling to photoreceptor cells. This coupling may play an important role in specific glia-neuron signaling pathways and in a new type of K+ buffering.

  8. Endurance Exercise Training Reduces Cardiac Sodium/Calcium Exchanger Expression in Animals Susceptible to Ventricular Fibrillation

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    Monica eKukielka

    2011-02-01

    Full Text Available Aim: Increased sodium/calcium exchanger activity (NCX1, an important regulator of cardiomyocyte cystolic calcium may provoke arrhythmias. Exercise training can decrease NCX1 expression in animals with heart failure improving cytosolic calcium regulation, and could thereby reduce the risk for ventricular fibrillation (VF. Methods: To test this hypothesis, a 2-min coronary occlusion was made during the last min. of exercise in dogs with healed myocardial infarctions; 23 had VF (S, susceptible and 13 did not (R, resistant. The animals were randomly assigned to either 10-wk exercise training (progressively increasing treadmill running (S n = 9; R n = 8 or 10-wk sedentary (S n = 14; R n = 5 groups. At the end of the 10-wk period, the exercise + ischemia test provoked VF in sedentary but not trained susceptible dogs. On a subsequent day, cardiac tissue was harvested and NCX1 protein expression was determined by Western blot. Results: In the sedentary group, NCX1 expression was significantly (ANOVA, P<0.05 higher in susceptible compared to resistant dogs. In contrast, NCX1 levels were similar in the exercise trained resistant and susceptible animals. Conclusion: These data suggest that exercise training can restore a more normal NCX1 level in dogs susceptible to ventricular fibrillation, improving cystolic calcium regulation and could thereby reduce the risk for sudden death following myocardial infarction.

  9. Modulation of Sodium/Iodide Symporter Expression in the Salivary Gland

    Science.gov (United States)

    La Perle, Krista M.D.; Kim, Dong Chul; Hall, Nathan C.; Bobbey, Adam; Shen, Daniel H.; Nagy, Rebecca S.; Wakely, Paul E.; Lehman, Amy; Jarjoura, David

    2013-01-01

    Background Physiologic iodide-uptake, mediated by the sodium/iodide symporter (NIS), in the salivary gland confers its susceptibility to radioactive iodine–induced damage following 131I treatment of thyroid cancer. Subsequent quality of life for thyroid cancer survivors can be decreased due to recurrent sialoadenitis and persistent xerostomia. NIS expression at the three principal salivary duct components in various pathological conditions was examined to better our understanding of NIS modulation in the salivary gland. Methods NIS expression was evaluated by immunohistochemistry in human salivary gland tissue microarrays constructed of normal, inflamed, and neoplastic salivary tissue cores. Cumulative 123I radioactivity reflecting the combination of NIS activity with clearance of saliva secretion in submandibular and parotid salivary glands was evaluated by single-photon emission computed tomography/computed tomography imaging 24 hours after 123I administration in 50 thyroid cancer patients. Results NIS is highly expressed in the basolateral membranes of the majority of striated ducts, yet weakly expressed in few intercalated and excretory duct cells. The ratio of 123I accumulation between parotid and submandibular glands is 2.38±0.19. However, the corresponding ratio of 123I accumulation normalized by volume of interest is 1.19±0.06. The percentage of NIS-positive striated duct cells in submandibular salivary glands was statistically greater than in parotid salivary glands, suggesting a higher clearance rate of saliva secretion in submandibular salivary glands. NIS expression in striated ducts was heterogeneously decreased or absent in sialoadenitis. Most ductal salivary gland tumors did not express NIS. However, Warthin's tumors of striated duct origin exhibited consistent and intense NIS staining, corresponding with radioactive iodine uptake. Conclusions NIS expression is tightly modulated during the transition of intercalated to striated ducts and striated

  10. Linking loss of sodium-iodide symporter expression to DNA damage

    Energy Technology Data Exchange (ETDEWEB)

    Lyckesvärd, Madeleine Nordén [Sahlgrenska Cancer Center, University of Gothenburg, Göteborg (Sweden); Department of Medical Chemistry and Cell Biology, University of Gothenburg, Göteborg (Sweden); Kapoor, Nirmal [Department of Medical Chemistry and Cell Biology, University of Gothenburg, Göteborg (Sweden); Ingeson-Carlsson, Camilla; Carlsson, Therese [Sahlgrenska Cancer Center, University of Gothenburg, Göteborg (Sweden); Department of Medical Chemistry and Cell Biology, University of Gothenburg, Göteborg (Sweden); Karlsson, Jan-Olof [Department of Medical Chemistry and Cell Biology, University of Gothenburg, Göteborg (Sweden); Postgård, Per; Himmelman, Jakob; Forssell-Aronsson, Eva [Department of Radiation Physics, University of Gothenburg, Göteborg (Sweden); Hammarsten, Ola [Department of Clinical Chemistry, University of Gothenburg, Göteborg (Sweden); Nilsson, Mikael, E-mail: mikael.nilsson@gu.se [Sahlgrenska Cancer Center, University of Gothenburg, Göteborg (Sweden); Department of Medical Chemistry and Cell Biology, University of Gothenburg, Göteborg (Sweden)

    2016-05-15

    Radiotherapy of thyroid cancer with I-131 is abrogated by inherent loss of radioiodine uptake due to loss of sodium iodide symporter (NIS) expression in poorly differentiated tumor cells. It is also known that ionizing radiation per se down-regulates NIS (the stunning effect), but the mechanism is unknown. Here we investigated whether loss of NIS-mediated iodide transport may be elicited by DNA damage. Calicheamicin, a fungal toxin that specifically cleaves double-stranded DNA, induced a full scale DNA damage response mediated by the ataxia-telangiectasia mutated (ATM) kinase in quiescent normal thyrocytes. At sublethal concentrations (<1 nM) calicheamicin blocked NIS mRNA expression and transepithelial iodide transport as stimulated by thyrotropin; loss of function occurred at a much faster rate than after I-131 irradiation. KU-55933, a selective ATM kinase inhibitor, partly rescued NIS expression and iodide transport in DNA-damaged cells. Prolonged ATM inhibition in healthy cells also repressed NIS-mediated iodide transport. ATM-dependent loss of iodide transport was counteracted by IGF-1. Together, these findings indicate that NIS, the major iodide transporter of the thyroid gland, is susceptible to DNA damage involving ATM-mediated mechanisms. This uncovers novel means of poor radioiodine uptake in thyroid cells subjected to extrinsic or intrinsic genotoxic stress. - Highlights: • DNA damage inhibits polarized iodide transport in normal thyroid cells. • Down-regulation of NIS expression is mediated by activation of the ATM kinase. • Long-term ATM inhibition also represses NIS-mediated iodide transport. • IGF-1 rescues NIS expression and iodide transport in DNA-damaged cells.

  11. N-linked glycans are required on epithelial Na+ channel subunits for maturation and surface expression.

    Science.gov (United States)

    Kashlan, Ossama B; Kinlough, Carol L; Myerburg, Michael M; Shi, Shujie; Chen, Jingxin; Blobner, Brandon M; Buck, Teresa M; Brodsky, Jeffrey L; Hughey, Rebecca P; Kleyman, Thomas R

    2018-03-01

    Epithelial Na + channel (ENaC) subunits undergo N-linked glycosylation in the endoplasmic reticulum where they assemble into an αβγ complex. Six, 13, and 5 consensus sites (Asn-X-Ser/Thr) for N-glycosylation reside in the extracellular domains of the mouse α-, β-, and γ-subunits, respectively. Because the importance of ENaC N-linked glycans has not been fully addressed, we examined the effect of preventing N-glycosylation of specific subunits on channel function, expression, maturation, and folding. Heterologous expression in Xenopus oocytes or Fischer rat thyroid cells with αβγ-ENaC lacking N-linked glycans on a single subunit reduced ENaC activity as well as the inhibitory response to extracellular Na + . The lack of N-linked glycans on the β-subunit also precluded channel activation by trypsin. However, channel activation by shear stress was N-linked glycan independent, regardless of which subunit was modified. We also discovered that the lack of N-linked glycans on any one subunit reduced the total and surface levels of cognate subunits. The lack of N-linked glycans on the β-subunit had the largest effect on total levels, with the lack of N-linked glycans on the γ- and α-subunits having intermediate and modest effects, respectively. Finally, channels with wild-type β-subunits were more sensitive to limited trypsin proteolysis than channels lacking N-linked glycans on the β-subunit. Our results indicate that N-linked glycans on each subunit are required for proper folding, maturation, surface expression, and function of the channel.

  12. High salt loading induces urinary storage dysfunction via upregulation of epithelial sodium channel alpha in the bladder epithelium in Dahl salt-sensitive rats

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    Seiji Yamamoto

    2017-11-01

    Full Text Available We aimed to investigate whether high salt intake affects bladder function via epithelial sodium channel (ENaC by using Dahl salt-resistant (DR and salt-sensitive (DS rats. Bladder weight of DR + high-salt diet (HS, 8% NaCl and DS + HS groups were significantly higher than those of DR + normal-salt diet (NS, 0.3% NaCl and DS + NS groups after one week treatment. We thereafter used only DR + HS and DS + HS group. Systolic and diastolic blood pressures were significantly higher in DS + HS group than in DR + HS group after the treatment period. Cystometrogram showed the intercontraction intervals (ICI were significantly shorter in DS + HS group than in DR + HS group during infusion of saline. Subsequent infusion of amiloride significantly prolonged ICI in DS + HS group, while no intra-group difference in ICI was observed in DR + HS group. No intra- or inter-group differences in maximum intravesical pressure were observed. Protein expression levels of ENaCα in the bladder were significantly higher in DS + HS group than in DR + HS group. ENaCα protein was localized at bladder epithelium in both groups. In conclusion, high salt intake is considered to cause urinary storage dysfunction via upregulation of ENaC in the bladder epithelium with salt-sensitive hypertension, suggesting that ENaC might be a candidate for therapeutic target for urinary storage dysfunction.

  13. High salt loading induces urinary storage dysfunction via upregulation of epithelial sodium channel alpha in the bladder epithelium in Dahl salt-sensitive rats.

    Science.gov (United States)

    Yamamoto, Seiji; Hotta, Yuji; Maeda, Kotomi; Kataoka, Tomoya; Maeda, Yasuhiro; Hamakawa, Takashi; Shibata, Yasuhiro; Sasaki, Shoichi; Ugawa, Shinya; Yasui, Takahiro; Kimura, Kazunori

    2017-11-01

    We aimed to investigate whether high salt intake affects bladder function via epithelial sodium channel (ENaC) by using Dahl salt-resistant (DR) and salt-sensitive (DS) rats. Bladder weight of DR + high-salt diet (HS, 8% NaCl) and DS + HS groups were significantly higher than those of DR + normal-salt diet (NS, 0.3% NaCl) and DS + NS groups after one week treatment. We thereafter used only DR + HS and DS + HS group. Systolic and diastolic blood pressures were significantly higher in DS + HS group than in DR + HS group after the treatment period. Cystometrogram showed the intercontraction intervals (ICI) were significantly shorter in DS + HS group than in DR + HS group during infusion of saline. Subsequent infusion of amiloride significantly prolonged ICI in DS + HS group, while no intra-group difference in ICI was observed in DR + HS group. No intra- or inter-group differences in maximum intravesical pressure were observed. Protein expression levels of ENaCα in the bladder were significantly higher in DS + HS group than in DR + HS group. ENaCα protein was localized at bladder epithelium in both groups. In conclusion, high salt intake is considered to cause urinary storage dysfunction via upregulation of ENaC in the bladder epithelium with salt-sensitive hypertension, suggesting that ENaC might be a candidate for therapeutic target for urinary storage dysfunction. Copyright © 2017 The Authors. Production and hosting by Elsevier B.V. All rights reserved.

  14. delta-Atracotoxins from australian funnel-web spiders compete with scorpion alpha-toxin binding but differentially modulate alkaloid toxin activation of voltage-gated sodium channels.

    Science.gov (United States)

    Little, M J; Zappia, C; Gilles, N; Connor, M; Tyler, M I; Martin-Eauclaire, M F; Gordon, D; Nicholson, G M

    1998-10-16

    delta-Atracotoxins from the venom of Australian funnel-web spiders are a unique group of peptide toxins that slow sodium current inactivation in a manner similar to scorpion alpha-toxins. To analyze their interaction with known sodium channel neurotoxin receptor sites, we studied their effect on [3H]batrachotoxin and 125I-Lqh II (where Lqh is alpha-toxin II from the venom of the scorpion Leiurus quinquestriatus hebraeus) binding and on alkaloid toxin-stimulated 22Na+ uptake in rat brain synaptosomes. delta-Atracotoxins significantly increased [3H]batrachotoxin binding yet decreased maximal batrachotoxin-activated 22Na+ uptake by 70-80%, the latter in marked contrast to the effect of scorpion alpha-toxins. Unlike the inhibition of batrachotoxin-activated 22Na+ uptake, delta-atracotoxins increased veratridine-stimulated 22Na+ uptake by converting veratridine from a partial to a full agonist, analogous to scorpion alpha-toxins. Hence, delta-atracotoxins are able to differentiate between the open state of the sodium channel stabilized by batrachotoxin and veratridine and suggest a distinct sub-conductance state stabilized by delta-atracotoxins. Despite these actions, low concentrations of delta-atracotoxins completely inhibited the binding of the scorpion alpha-toxin, 125I-Lqh II, indicating that they bind to similar, or partially overlapping, receptor sites. The apparent uncoupling between the increase in binding but inhibition of the effect of batrachotoxin induced by delta-atracotoxins suggests that the binding and action of certain alkaloid toxins may represent at least two distinguishable steps. These results further contribute to the understanding of the complex dynamic interactions between neurotoxin receptor site areas related to sodium channel gating.

  15. Heterologous expression and purification of an active human TRPV3 ion channel

    DEFF Research Database (Denmark)

    Kol, Stefan; Braun, Christian; Thiel, Gerhard

    2013-01-01

    and interactions with other proteins at the atomic level. This is mainly caused by difficulties in obtaining functionally active samples of high homogeneity. Here, we report on the high‐level Escherichia coli expression of the human TRPV3 channel, for which no structural information has been reported to date. We...

  16. Sequence genomic organization and expression of two channel catfish Ictalurus punctatus Ghrelin receptors

    Science.gov (United States)

    Two ghrelin receptor (GHS-R) genes were isolated from channel catfish tissue and a bacterial artificial chromosome (BAC) library. The two receptors were characterized by determining tissue distribution, ontogeny of receptor mRNA expression, and effects of exogenous homologous ghrelin administration ...

  17. Distribution and expression of non-neuronal transient receptor potential (TRPV) ion channels in rosacea.

    Science.gov (United States)

    Sulk, Mathias; Seeliger, Stephan; Aubert, Jerome; Schwab, Verena D; Cevikbas, Ferda; Rivier, Michel; Nowak, Pawel; Voegel, Johannes J; Buddenkotte, Jörg; Steinhoff, Martin

    2012-04-01

    Rosacea is a frequent chronic inflammatory skin disease of unknown etiology. Because early rosacea reveals all characteristics of neurogenic inflammation, a central role of sensory nerves in its pathophysiology has been discussed. Neuroinflammatory mediators and their receptors involved in rosacea are poorly defined. Good candidates may be transient receptor potential (TRP) ion channels of vanilloid type (TRPV), which can be activated by many trigger factors of rosacea. Interestingly, TRPV2, TRPV3, and TRPV4 are expressed by both neuronal and non-neuronal cells. Here, we analyzed the expression and distribution of TRPV receptors in the various subtypes of rosacea on non-neuronal cells using immunohistochemistry, morphometry, double immunoflourescence, and quantitative real-time PCR (qRT-PCR) as compared with healthy skin and lupus erythematosus. Our results show that dermal immunolabeling of TRPV2 and TRPV3 and gene expression of TRPV1 is significantly increased in erythematotelangiectatic rosacea (ETR). Papulopustular rosacea (PPR) displayed an enhanced immunoreactivity for TRPV2, TRPV4, and also of TRPV2 gene expression. In phymatous rosacea (PhR)-affected skin, dermal immunostaining of TRPV3 and TRPV4 and gene expression of TRPV1 and TRPV3 was enhanced, whereas epidermal TRPV2 staining was decreased. Thus, dysregulation of TRPV channels also expressed by non-neuronal cells may be critically involved in the initiation and/or development of rosacea. TRP ion channels may be targets for the treatment of rosacea.

  18. Recombinant Expression and Functional Characterization of Martentoxin: A Selective Inhibitor for BK Channel (α + β4

    Directory of Open Access Journals (Sweden)

    Jie Tao

    2014-04-01

    Full Text Available Martentoxin (MarTX, a 37-residue peptide purified from the venom of East-Asian scorpion (Buthus martensi Karsch, was capable of blocking large-conductance Ca2+-activated K+ (BK channels. Here, we report an effective expression and purification approach for this toxin. The cDNA encoding martentoxin was expressed by the prokaryotic expression system pGEX-4T-3 which was added an enterokinase cleavage site by PCR. The fusion protein (GST-rMarTX was digested by enterokinase to release hetero-expressed toxin and further purified via reverse-phase HPLC. The molecular weight of the hetero-expressed rMarTX was 4059.06 Da, which is identical to that of the natural peptide isolated from scorpion venom. Functional characterization through whole-cell patch clamp showed that rMarTX selectively and potently inhibited the currents of neuronal BK channels (α + β4 (IC50 = 186 nM, partly inhibited mKv1.3, but hardly having any significant effect on hKv4.2 and hKv3.1a even at 10 μM. Successful expression of martentoxin lays basis for further studies of structure-function relationship underlying martentoxin or other potassium-channel specific blockers.

  19. Distribution and Expression of Non-Neuronal Transient Receptor Potential (TRPV) Ion Channels in Rosacea

    Science.gov (United States)

    Sulk, Mathias; Seeliger, Stephan; Aubert, Jerome; Schwab, Verena D.; Cevikbas, Ferda; Rivier, Michel; Nowak, Pawel; Voegel, Johannes J.; Buddenkotte, Jörg; Steinhoff, Martin

    2011-01-01

    Rosacea is a frequent chronic inflammatory skin disease of unknown etiology. Because early rosacea reveals all characteristics of neurogenic inflammation, a central role of sensory nerves in its pathophysiology has been discussed. Neuroinflammatory mediators and their receptors involved in rosacea are poorly defined. Good candidates may be transient receptor potential (TRP) ion channels of vanilloid type (TRPV), which can be activated by many trigger factors of rosacea. Interestingly, TRPV2, TRPV3, and TRPV4 are expressed by both neuronal and non-neuronal cells. Here, we analyzed the expression and distribution of TRPV receptors in the various subtypes of rosacea on non-neuronal cells using immunohistochemistry, morphometry, double immunoflourescence, and quantitative real-time PCR (qRT-PCR) as compared with healthy skin and lupus erythematosus. Our results show that dermal immunolabeling of TRPV2 and TRPV3 and gene expression of TRPV1 is significantly increased in erythematotelangiectatic rosacea (ETR). Papulopustular rosacea (PPR) displayed an enhanced immunoreactivity for TRPV2, TRPV4, and also of TRPV2 gene expression. In phymatous rosacea (PhR)-affected skin, dermal immunostaining of TRPV3 and TRPV4 and gene expression of TRPV1 and TRPV3 was enhanced, whereas epidermal TRPV2 staining was decreased. Thus, dysregulation of TRPV channels also expressed by non-neuronal cells may be critically involved in the initiation and/or development of rosacea. TRP ion channels may be targets for the treatment of rosacea. PMID:22189789

  20. Multiple saxitoxin-binding sites in bullfrog muscle: tetrodotoxin-sensitive sodium channels and tetrodotoxin-insensitive sites of unknown function

    Energy Technology Data Exchange (ETDEWEB)

    Moczydlowski, E.; Mahar, J.; Ravindran, A.

    1988-02-01

    The possible presence of multiple sodium channel subtypes in bullfrog skeletal muscle was investigated in binding experiments with (/sup 3/H)saxitoxin and in single-channel studies using planar lipid bilayers. Two classes of (/sup 3/H)saxitoxin-binding sites were identified in membrane preparations. One class displayed a toxin specificity characteristic of voltage-dependent sodium channels: high affinity for saxitoxin (KD approximately equal to 0.5 nM), neosaxitoxin (KD approximately equal to 0.1 nM), and tetrodotoxin (KD approximately equal to 1.3 nM). A second class of membrane-associated binding sites exhibited high affinity for saxitoxin (KD approximately equal to 0.1 nM), lower affinity for neosaxitoxin (KD approximately equal to 25 nM), and complete insensitivity to tetrodotoxin at concentrations up to 32 microM. The first class corresponded to functional tetrodotoxin-sensitive sodium channels that could be incorporated and observed in planar bilayers in the presence of batrachotoxin. The unusual, tetrodotoxin-insensitive binding activity for (/sup 3/H)saxitoxin was also found at nM levels in the high speed supernatant of homogenized skeletal muscle without the addition of detergents. This soluble class of sites exhibited low affinity for neosaxitoxin (KD approximately equal to 60 nM) and a very slow dissociation rate of (/sup 3/H)saxitoxin (t0.5 approximately equal to 90 min), properties nearly identical to those of the tetrodotoxin-insensitive sites in membranes. The soluble saxitoxin-binding activity is also characterized by a more basic pH dependence and a complete lack of binding competition between saxitoxin and alkali cations. Bullfrog muscle appears to be a good tissue source for the purification of this soluble saxitoxin-binding protein.

  1. The sodium-bicarbonate cotransporter NBCe2 (slc4a5) expressed in human renal proximal tubules shows increased apical expression under high-salt conditions.

    Science.gov (United States)

    Gildea, John J; Xu, Peng; Carlson, Julia M; Gaglione, Robert T; Bigler Wang, Dora; Kemp, Brandon A; Reyes, Camellia M; McGrath, Helen E; Carey, Robert M; Jose, Pedro A; Felder, Robin A

    2015-12-01

    The electrogenic sodium bicarbonate cotransporter (NBCe2) is encoded by SLC4A5, variants of which have been associated with salt sensitivity of blood pressure, which affects 25% of the adult population. NBCe2 is thought to mediate sodium bicarbonate cotransport primarily in the renal collecting duct, but NBCe2 mRNA is also found in the rodent renal proximal tubule (RPT). The protein expression or function of NBCe2 has not been demonstrated in the human RPT. We validated an NBCe2 antibody by shRNA and Western blot analysis, as well as overexpression of an epitope-tagged NBCe2 construct in both RPT cells (RPTCs) and human embryonic kidney 293 (HEK293) cells. Using this validated NBCe2 antibody, we found NBCe2 protein expression in the RPT of fresh and frozen human kidney slices, RPTCs isolated from human urine, and isolated RPTC apical membrane. Under basal conditions, NBCe2 was primarily found in the Golgi, while NBCe1 was primarily found at the basolateral membrane. Following an acute short-term increase in intracellular sodium, NBCe2 expression was increased at the apical membrane in cultured slices of human kidney and polarized, immortalized RPTCs. Sodium bicarbonate transport was increased by monensin and overexpression of NBCe2, decreased by NBCe2 shRNA, but not by NBCe1 shRNA, and blocked by 2,2'-(1,2-ethenediyl)bis[5-isothiocyanato-benzenesulfonic acid]. NBCe2 could be important in apical sodium and bicarbonate cotransport under high-salt conditions; the implication of the ex vivo studies to the in vivo situation when salt intake is increased remains unclear. Therefore, future studies will examine the role of NBCe2 in mediating increased renal sodium transport in humans whose blood pressures are elevated by an increase in sodium intake. Copyright © 2015 the American Physiological Society.

  2. Mammary radioiodine accumulation due to functional sodium iodide symporter expression in a benign fibroadenoma

    International Nuclear Information System (INIS)

    Berger, F.; Unterholzner, S.; Diebold, J.; Knesewitsch, P.; Hahn, K.; Spitzweg, C.

    2006-01-01

    The sodium iodide symporter (NIS) has been characterized to mediate the active transport of iodide not only in the thyroid gland but also in various non-thyroidal tissues, including lactating mammary gland and the majority of breast cancers, thereby offering the possibility of diagnostic and therapeutic radioiodine application in breast cancer. In this report, we present a 57-year-old patient with multifocal papillary thyroid carcinoma, who showed focal radioiodine accumulation in a lesion in the right breast on a posttherapy 131 I scan following radioiodine therapy. CT and MR-mammography showed a focal solid lesion in the right breast suggestive of a fibroadenoma, which was confirmed by histological examination. Immunostaining of paraffin-embedded tumor tissue sections using a human NIS antibody demonstrated NIS-specific immunoreactivity confined to epithelial cells of mammary ducts. In conclusion, in a thyroid cancer patient we identified a benign fibroadenoma of the breast expressing high levels of functionally active NIS protein as underlying cause of focal mammary radioiodine accumulation on a posttherapy 131 I scan. These data show for the first time that functional NIS expression is not restricted to lactating mammary gland and malignant breast tissue, but can also be detected in benign breast lesions, such as fibroadenomata of the breast

  3. The effect of tanespimycin (17-AAG) on radioiodine accumulation in sodium iodide symporter expressing cells

    International Nuclear Information System (INIS)

    Yu, Kyoung Hyun; Youn, Hyewon; Song, Myung Geun; Lee, Dong Soo; Chung, June Key

    2012-01-01

    The heat shock protein 90 inhibitor, tanespimycin, is an anticancer agent known to increase iodine accumulation in normal and cancerous thyroid cells. Iodine accumulation is regulated by membrane proteins such as sodium iodide sym porter (NIS) and pendrin (PDS), and thus we attempted to characterize the effects of tanespimycin on those genes. Cells were incubated with tanespimycin in order to evaluate 125 I accumulation and efflux ability. Radioiodine uptake and efflux were measured by a gamma counter and normalized by protein amount. RT PCR were performed to measure the level of gene expression. After tanespimycin treatment, 125 uptake was in creased by ∼2.5 fold in FRTL 5, hNIS ARO. and hNIS MDA MB 231 cells, but no changes were detected in the hNIS HeLa cells. Tanespimycin significantly reduced the radioiodine efflux rate only in the FRTL 5 cell. in the FRTL 5 and hNIS ARO cells, PDS mRNA levels were markedly reduced; the only other observed alteration in the levels of NIS mRNA after tanespimtycin treatment was an observed increase in the h hNIS ARO cells. These results indicate that cellular responses against tanespimycin treatment differed between the normal rat thyroid cells and human cancer cells, and the reduction in the 125I efflux rate by tanespimycin in the normal rat thyroid cells might be attributable to reduced PDS gene expression

  4. Development and validation of a thallium flux-based functional assay for the sodium channel NaV1.7 and its utility for lead discovery and compound profiling.

    Science.gov (United States)

    Du, Yu; Days, Emily; Romaine, Ian; Abney, Kris K; Kaufmann, Kristian; Sulikowski, Gary; Stauffer, Shaun; Lindsley, Craig W; Weaver, C David

    2015-06-17

    Ion channels are critical for life, and they are targets of numerous drugs. The sequencing of the human genome has revealed the existence of hundreds of different ion channel subunits capable of forming thousands of ion channels. In the face of this diversity, we only have a few selective small-molecule tools to aid in our understanding of the role specific ion channels in physiology which may in turn help illuminate their therapeutic potential. Although the advent of automated electrophysiology has increased the rate at which we can screen for and characterize ion channel modulators, the technique's high per-measurement cost and moderate throughput compared to other high-throughput screening approaches limit its utility for large-scale high-throughput screening. Therefore, lower cost, more rapid techniques are needed. While ion channel types capable of fluxing calcium are well-served by low cost, very high-throughput fluorescence-based assays, other channel types such as sodium channels remain underserved by present functional assay techniques. In order to address this shortcoming, we have developed a thallium flux-based assay for sodium channels using the NaV1.7 channel as a model target. We show that the assay is able to rapidly and cost-effectively identify NaV1.7 inhibitors thus providing a new method useful for the discovery and profiling of sodium channel modulators.

  5. A quantitative and comparative study of the effects of a synthetic ciguatoxin CTX3C on the kinetic properties of voltage-dependent sodium channels

    Science.gov (United States)

    Yamaoka, Kaoru; Inoue, Masayuki; Miyahara, Hidemichi; Miyazaki, Keisuke; Hirama, Masahiro

    2004-01-01

    Ciguatoxins (CTXs) are known to bind to receptor site 5 of the voltage-dependent Na channel, but the toxin's physiological effects are poorly understood. In this study, we investigated the effects of a ciguatoxin congener (CTX3C) on three different Na-channel isoforms, rNav1.2, rNav1.4, and rNav1.5, which were transiently expressed in HEK293 cells. The toxin (1.0 μmol l−1) shifted the activation potential (V1/2 of activation curve) in the negative direction by 4–9 mV and increased the slope factor (k) from 8 mV to between 9 and 12 mV (indicative of decreased steepness of the activation curve), thereby resulting in a hyperpolarizing shift of the threshold potential by 30 mV for all Na channel isoforms. The toxin (1.0 μmol l−1) significantly accelerated the time-to-peak current from 0.62 to 0.52 ms in isoform rNav1.2. Higher doses of the toxin (3–10 μmol l−1) additionally decreased time-to-peak current in rNav1.4 and rNav1.5. A toxin effect on decay of INa at −20 mV was either absent or marginal even at relatively high doses of CTX3C. The toxin (1 μmol l−1) shifted the inactivation potential (V1/2 of inactivation curve) in the negative direction by 15–18 mV in all isoforms. INa maxima of the I–V curve (at −20 mV) were suppressed by application of 1.0 μmol l−1 CTX3C to a similar extent (80–85% of the control) in all the three isoforms. Higher doses of CTX3C up to 10 μmol l−1 further suppressed INa to 61–72% of the control. Recovery from slow inactivation induced by a depolarizing prepulse of intermediate duration (500 ms) was dramatically delayed in the presence of 1.0 μmol l−1 CTX3C, as time constants describing the monoexponential recovery were increased from 38±8 to 588±151 ms (n=5), 53±6 to 338±85 ms (n=4), and 23±3 to 232±117 ms (n=3) in rNav1.2, rNav1.4, and rNav1.5, respectively. CTX3C exerted multimodal effects on sodium channels, with simultaneous stimulatory and inhibitory aspects, probably due to the large

  6. Development of heart failure is independent of K+ channel-interacting protein 2 expression

    DEFF Research Database (Denmark)

    Speerschneider, Tobias; Grubb, Søren; Metoska, Artina

    2013-01-01

    Abstract  Abnormal ventricular repolarization in ion channelopathies and heart disease is a major cause of ventricular arrhythmias and sudden cardiac death. K(+) channel-interacting protein 2 (KChIP2) expression is significantly reduced in human heart failure (HF), contributing to a loss of the t......Abstract  Abnormal ventricular repolarization in ion channelopathies and heart disease is a major cause of ventricular arrhythmias and sudden cardiac death. K(+) channel-interacting protein 2 (KChIP2) expression is significantly reduced in human heart failure (HF), contributing to a loss...... of the transient outward K(+) current (Ito). We aim to investigate the possible significance of a changed KChIP2 expression on the development of HF and proarrhythmia. Transverse aortic constrictions (TAC) and sham operations were performed in wild-type (WT) and KChIP2(-/-) mice. Echocardiography was performed...

  7. Heterologous expression of tulip petal plasma membrane aquaporins in Pichia pastoris for water channel analysis.

    Science.gov (United States)

    Azad, Abul Kalam; Sawa, Yoshihiro; Ishikawa, Takahiro; Shibata, Hitoshi

    2009-05-01

    Water channels formed by aquaporins (AQPs) play an important role in the control of water homeostasis in individual cells and in multicellular organisms. Plasma membrane intrinsic proteins (PIPs) constitute a subclass of plant AQPs. TgPIP2;1 and TgPIP2;2 from tulip petals are members of the PIP family. In this study, we overexpressed TgPIP2;1 and TgPIP2;2 in Pichia pastoris and monitored their water channel activity (WCA) either by an in vivo spheroplast-bursting assay performed after hypo-osmotic shock or by growth assay. Osmolarity, pH, and inhibitors of AQPs, protein kinases (PKs), and protein phosphatases (PPs) affect the WCA of heterologous AQPs in this expression system. The WCA of TgPIP2;2-expressing spheroplasts was affected by inhibitors of PKs and PPs, which indicates that the water channel of this homologue is regulated by phosphorylation in P. pastoris. From the results reported herein, we suggest that P. pastoris can be employed as a heterologous expression system to assay the WCA of PIPs and to monitor the AQP-mediated channel gating mechanism, and it can be developed to screen inhibitors/effectors of PIPs.

  8. Heterologous Expression of Tulip Petal Plasma Membrane Aquaporins in Pichia pastoris for Water Channel Analysis▿

    Science.gov (United States)

    Azad, Abul Kalam; Sawa, Yoshihiro; Ishikawa, Takahiro; Shibata, Hitoshi

    2009-01-01

    Water channels formed by aquaporins (AQPs) play an important role in the control of water homeostasis in individual cells and in multicellular organisms. Plasma membrane intrinsic proteins (PIPs) constitute a subclass of plant AQPs. TgPIP2;1 and TgPIP2;2 from tulip petals are members of the PIP family. In this study, we overexpressed TgPIP2;1 and TgPIP2;2 in Pichia pastoris and monitored their water channel activity (WCA) either by an in vivo spheroplast-bursting assay performed after hypo-osmotic shock or by growth assay. Osmolarity, pH, and inhibitors of AQPs, protein kinases (PKs), and protein phosphatases (PPs) affect the WCA of heterologous AQPs in this expression system. The WCA of TgPIP2;2-expressing spheroplasts was affected by inhibitors of PKs and PPs, which indicates that the water channel of this homologue is regulated by phosphorylation in P. pastoris. From the results reported herein, we suggest that P. pastoris can be employed as a heterologous expression system to assay the WCA of PIPs and to monitor the AQP-mediated channel gating mechanism, and it can be developed to screen inhibitors/effectors of PIPs. PMID:19251885

  9. 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.

    Science.gov (United States)

    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.

  10. Gene expression of stretch-activated channels and mechanoelectric feedback in the heart.

    Science.gov (United States)

    Kelly, D; Mackenzie, L; Hunter, P; Smaill, B; Saint, D A

    2006-07-01

    1. Mechanoelectric feedback (MEF) in the heart is the process by which mechanical forces on the myocardium can change its electrical properties. Mechanoelectric feedback has been demonstrated in many animal models, ranging from isolated cells, through isolated hearts to whole animals. In humans, MEF has been demonstrated directly in both the atria and the ventricles. It seems likely that MEF provides either the trigger or the substrate for some types of clinically important arrhythmias. 2. Mechanoelectric feedback may arise because of the presence of stretch-sensitive (or mechano-sensitive) ion channels in the cell membrane of the cardiac myocytes. Two types have been demonstrated: (i) a non-specific cation channel (stretch-activated channel (SAC); conductance of approximately 25 pS); and (ii) a potassium channel with a conductance of approximately 100 pS. The gene coding for the SAC has not yet been identified. The gene for the potassium channel is likely to be TREK, a member of the tandem pore potassium channel gene family. We have recorded stretch-sensitive potassium channels in rat isolated myocytes that have the properties of TREK channels expressed in heterologous systems. 3. It has been shown that TREK mRNA is expressed heterogeneously in the rat ventricular wall, with 17-fold more expression in endocardial compared with epicardial cells. This difference is reflected in the TREK currents recorded from endocardial and epicardial cells using whole-cell patch-clamp techniques, although the difference in current density was less pronounced (approximately threefold). Consistent with this, we show here that when the ventricle is stretched by inflation of an intraventricular balloon in a Langendorff perfused rat isolated heart, action potential shortening was more pronounced in the endocardium (30% shortening at 40 mmHg) compared with that in the epicardium (10% shortening at the same pressure). 4. Computer models of the mechanics of the (pig) heart show pronounced

  11. Expression of calcium-activated chloride channels Ano1 and Ano2 in mouse taste cells.

    Science.gov (United States)

    Cherkashin, Alexander P; Kolesnikova, Alisa S; Tarasov, Michail V; Romanov, Roman A; Rogachevskaja, Olga A; Bystrova, Marina F; Kolesnikov, Stanislav S

    2016-02-01

    Specialized Ca(2+)-dependent ion channels ubiquitously couple intracellular Ca(2+) signals to a change in cell polarization. The existing physiological evidence suggests that Ca(2+)-activated Cl(-) channels (CaCCs) are functional in taste cells. Because Ano1 and Ano2 encode channel proteins that form CaCCs in a variety of cells, we analyzed their expression in mouse taste cells. Transcripts for Ano1 and Ano2 were detected in circumvallate (CV) papillae, and their expression in taste cells was confirmed using immunohistochemistry. When dialyzed with CsCl, taste cells of the type III exhibited no ion currents dependent on cytosolic Ca(2+). Large Ca(2+)-gated currents mediated by TRPM5 were elicited in type II cells by Ca(2+) uncaging. When TRPM5 was inhibited by triphenylphosphine oxide (TPPO), ionomycin stimulated a small but resolvable inward current that was eliminated by anion channel blockers, including T16Ainh-A01 (T16), a specific Ano1 antagonist. This suggests that CaCCs, including Ano1-like channels, are functional in type II cells. In type I cells, CaCCs were prominently active, blockable with the CaCC antagonist CaCCinh-A01 but insensitive to T16. By profiling Ano1 and Ano2 expressions in individual taste cells, we revealed Ano1 transcripts in type II cells only, while Ano2 transcripts were detected in both type I and type II cells. P2Y agonists stimulated Ca(2+)-gated Cl(-) currents in type I cells. Thus, CaCCs, possibly formed by Ano2, serve as effectors downstream of P2Y receptors in type I cells. While the role for TRPM5 in taste transduction is well established, the physiological significance of expression of CaCCs in type II cells remains to be elucidated.

  12. Fluorescence resonance energy transfer on the voltage-dependent sodium channel. Spatial relationship and site coupling between the batrachotoxin and Leiurus quinquestriatus quinquestriatus alpha-scorpion toxin receptors.

    Science.gov (United States)

    Angelides, K J; Brown, G B

    1984-05-25

    A fluorescent N- methylanthraniloyl derivative of the potent depolarizing agent batrachotoxin has been used to probe the structural and conformational properties of the neurotoxin receptor site on the voltage-dependent sodium channel. Batrachotoxin A 20-alpha-N- methylanthranilate (BTX-NMA) retains high affinity for its receptor site on the synaptosomal sodium channel with a Kd between 78 and 91 nM and an average site capacity of 2 pmol/mg of synaptosomal protein in the presence of Leiurus quinquestriatus quinquestriatus alpha-scorpion toxin. The fluorescence emission of BTX-NMA upon binding to synaptosomes indicates a hydrophobic environment. Toxin V from L. quinquestriatus, an allosteric activator, effects a 20-nm red shift in the spectrum of bound BTX-NMA and a 4-fold enhancement in the fluorescence quantum yield disclosing a conformational change into a hydrophilic environment. Fluorescence resonance energy transfer measurements show that the distance separating the receptor sites is 37 +/- 10 A. Thus, the binding of alpha-scorpion toxin must involve conformational changes that extend over large distances from the batrachotoxin-binding locus. This information together with the distance measurements between the tetrodotoxin and alpha-scorpion toxin receptors and the conformational transition associated with this distance upon batrachotoxin addition indicate a conformationally flexible channel with coupling of sites through the polyatomic framework of individual subunits or through extensive alterations in subunit/subunit interactions.

  13. Expression and distribution of transient receptor potential (TRP) channels in bladder epithelium.

    Science.gov (United States)

    Yu, Weiqun; Hill, Warren G; Apodaca, Gerard; Zeidel, Mark L

    2011-01-01

    The urothelium is proposed to be a sensory tissue that responds to mechanical stress by undergoing dynamic membrane trafficking and neurotransmitter release; however, the molecular basis of this function is poorly understood. Transient receptor potential (TRP) channels are ideal candidates to fulfill such a role as they can sense changes in temperature, osmolarity, and mechanical stimuli, and several are reported to be expressed in the bladder epithelium. However, their complete expression profile is unknown and their cellular localization is largely undefined. We analyzed expression of all 33 TRP family members in mouse bladder and urothelium by RT-PCR and found 22 specifically expressed in the urothelium. Of the latter, 10 were chosen for closer investigation based on their known mechanosensory or membrane trafficking functions in other cell types. Western blots confirmed urothelial expression of TRPC1, TRPC4, TRPV1, TRPV2, TRPV4, TRPM4, TRPM7, TRPML1, and polycystins 1 and 2 (PKD1 and PKD2) proteins. We further defined the cellular and subcellular localization of all 10 TRP channels. TRPV2 and TRPM4 were prominently localized to the umbrella cell apical membrane, while TRPC4 and TRPV4 were identified on their abluminal surfaces. TRPC1, TRPM7, and TRPML1 were localized to the cytoplasm, while PKD1 and PKD2 were expressed on the apical and basolateral membranes of umbrella cells as well as in the cytoplasm. The cellular location of TRPV1 in the bladder has been debated, but colocalization with neuronal marker calcitonin gene-related peptide indicated clearly that it is present on afferent neurons that extend into the urothelium, but may not be expressed by the urothelium itself. These findings are consistent with the hypothesis that the urothelium acts as a sentinel and by expressing multiple TRP channels it is likely it can detect and presumably respond to a diversity of external stimuli and suggest that it plays an important role in urothelial signal

  14. Novel insights into the distribution of cardiac HCN channels: an expression study in the mouse heart.

    Science.gov (United States)

    Herrmann, Stefan; Layh, Beate; Ludwig, Andreas

    2011-12-01

    HCN pacemaker channels (I(f) channels) are believed to contribute to important functions in the heart; thus these channels became an attractive target for generating transgenic mouse mutants to elucidate their role in physiological and pathophysiological cardiac conditions. A full understanding of cardiac I(f) and the interpretation of studies using HCN mouse mutants require detailed information about the expression profile of the individual HCN subunits. Here we investigate the cardiac expression pattern of the HCN isoforms at the mRNA as well as at the protein level. The specificity of antibodies used was strictly confirmed by the use of HCN1, HCN2 and HCN4 knockout animals. We find a low, but highly differential HCN expression profile outside the cardiac conduction pathway including left and right atria and ventricles. Additionally HCN distribution was investigated in tissue slices of the sinoatrial node, the atrioventricular node, the bundle of His and the bundle branches. The conduction system was marked by acetylcholine esterase staining. HCN4 was confirmed as the predominant isoform of the primary pacemaker followed by a distinct expression of HCN1. In contrast HCN2 shows only a confined expression to individual pacemaker cells. Immunolabeling of the AV-node reveals also a pronounced specificity for HCN1 and HCN4. Compared to the SN and AVN we found a low but selective expression of HCN4 as the only isoform in the atrioventricular bundle. However in the bundle branches HCN1, HCN4 and also HCN2 show a prominent and selective expression pattern. Our results display a characteristic distribution of individual HCN isoforms in several cardiac compartments and reveal that beside HCN4, HCN1 represents the isoform which is selectively expressed in most parts of the conduction system suggesting a substantial contribution of HCN1 to pacemaking. 2011 Elsevier Ltd. All rights reserved.

  15. Aberrant Sodium Channel Currents and Hyperexcitability of Medial Entorhinal Cortex Neurons in a Mouse Model of SCN8A Encephalopathy.

    Science.gov (United States)

    Ottolini, Matteo; Barker, Bryan S; Gaykema, Ronald P; Meisler, Miriam H; Patel, Manoj K

    2017-08-09

    Na v 1.6. In addition to seizures, patients suffer from cognitive and developmental delays and are at high risk for sudden unexpected death in epilepsy (SUDEP). A mouse knock-in model expressing the patient mutation N1768D reproduces several pathological phenotypes, including spontaneous seizures and sudden death. We demonstrate that medial entorhinal cortex (mEC) neurons from the mouse model exhibit proexcitatory alterations in Na channel activity, some of which were not seen in hippocampal or cortical neurons, and resulting in neuronal hyperexcitability. Because mEC neurons regulate the activity of the hippocampus, which plays an important role in seizure onset, we propose that these profound changes in mEC neuron excitability associated with the gain-of-function mutation of Na v 1.6 may increase excitatory drive into the hippocampus, culminating in seizure activity and SUDEP. Copyright © 2017 the authors 0270-6474/17/377643-13$15.00/0.

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

    Science.gov (United States)

    Klein, Amanda H; Vyshnevska, Alina; Hartke, Timothy V; De Col, Roberto; Mankowski, Joseph L; Turnquist, Brian; Bosmans, Frank; Reeh, Peter W; Schmelz, Martin; Carr, Richard W; Ringkamp, Matthias

    2017-05-17

    Voltage-gated sodium (Na V ) channels are responsible for the initiation and conduction of action potentials within primary afferents. The nine Na V channel isoforms recognized in mammals are often functionally divided into tetrodotoxin (TTX)-sensitive (TTX-s) channels (Na V 1.1-Na V 1.4, Na V 1.6-Na V 1.7) that are blocked by nanomolar concentrations and TTX-resistant (TTX-r) channels (Na V 1.8 and Na V 1.9) inhibited by millimolar concentrations, with Na V 1.5 having an intermediate toxin sensitivity. For small-diameter primary afferent neurons, it is unclear to what extent different Na V channel isoforms are distributed along the peripheral and central branches of their bifurcated axons. To determine the relative contribution of TTX-s and TTX-r channels to action potential conduction in different axonal compartments, we investigated the effects of TTX on C-fiber-mediated compound action potentials (C-CAPs) of proximal and distal peripheral nerve segments and dorsal roots from mice and pigtail monkeys ( Macaca nemestrina ). In the dorsal roots and proximal peripheral nerves of mice and nonhuman primates, TTX reduced the C-CAP amplitude to 16% of the baseline. In contrast, >30% of the C-CAP was resistant to TTX in distal peripheral branches of monkeys and WT and Na V 1.9 -/- mice. In nerves from Na V 1.8 -/- mice, TTX-r C-CAPs could not be detected. These data indicate that Na V 1.8 is the primary isoform underlying TTX-r conduction in distal axons of somatosensory C-fibers. Furthermore, there is a differential spatial distribution of Na V 1.8 within C-fiber axons, being functionally more prominent in the most distal axons and terminal regions. The enrichment of Na V 1.8 in distal axons may provide a useful target in the treatment of pain of peripheral origin. SIGNIFICANCE STATEMENT It is unclear whether individual sodium channel isoforms exert differential roles in action potential conduction along the axonal membrane of nociceptive, unmyelinated peripheral nerve

  17. Interaction of batrachotoxin with the local anesthetic receptor site in transmembrane segment IVS6 of the voltage-gated sodium channel.

    Science.gov (United States)

    Linford, N J; Cantrell, A R; Qu, Y; Scheuer, T; Catterall, W A

    1998-11-10

    The voltage-gated sodium channel is the site of action of more than six classes of neurotoxins and drugs that alter its function by interaction with distinct, allosterically coupled receptor sites. Batrachotoxin (BTX) is a steroidal alkaloid that binds to neurotoxin receptor site 2 and causes persistent activation. BTX binding is inhibited allosterically by local anesthetics. We have investigated the interaction of BTX with amino acid residues I1760, F1764, and Y1771, which form part of local anesthetic receptor site in transmembrane segment IVS6 of type IIA sodium channels. Alanine substitution for F1764 (mutant F1764A) reduces tritiated BTX-A-20-alpha-benzoate binding affinity, causing a 60-fold increase in Kd. Alanine substitution for I1760, which is adjacent to F1764 in the predicted IVS6 transmembrane alpha helix, causes only a 4-fold increase in Kd. In contrast, mutant Y1771A shows no change in BTX binding affinity. For wild-type and mutant Y1771A, BTX shifted the voltage for half-maximal activation approximately 40 mV in the hyperpolarizing direction and increased the percentage of noninactivating sodium current to approximately 60%. In contrast, these BTX effects were eliminated completely for the F1764A mutant and were reduced substantially for mutant I1760A. Our data suggest that the BTX receptor site shares overlapping but nonidentical molecular determinants with the local anesthetic receptor site in transmembrane segment IVS6 as well as having unique molecular determinants in transmembrane segment IS6, as demonstrated in previous work. Evidently, BTX conforms to a domain-interface allosteric model of ligand binding and action, as previously proposed for calcium agonist and antagonist drugs acting on L-type calcium channels.

  18. Molecular and functional expression of high conductance Ca 2+ activated K+ channels in the eel intestinal epithelium

    DEFF Research Database (Denmark)

    Lionetto, Maria G; Rizzello, Antonia; Giordano, Maria E

    2008-01-01

    Several types of K(+) channels have been identified in epithelial cells. Among them high conductance Ca(2+)-activated K(+) channels (BK channels) are of relevant importance for their involvement in regulatory volume decrease (RVD) response following hypotonic stress. The aim of the present work...... was to investigate the functional and molecular expression of BK in the eel intestine, which is a useful experimental model for cell volume regulation research. In the present paper using rat BK channel-specific primer, a RT-PCR signal of 696 pb cDNA was detected in eel intestine, whole nucleotide sequence showed...... high similarity (83%) to the alpha subunit of BK channel family. BK channel protein expression was verified by immunoblotting and confocal microscopy, while the functional role of BK channels in epithelial ion transport mechanisms and cell volume regulation was examined by electrophysiological...

  19. Caracterización del canal epitelial de sodio en sinciciotrofoblasto de placenta humana preeclamptica Characterization of the epithelial sodium channel in human pre-eclampsia syncytiotrophoblast

    Directory of Open Access Journals (Sweden)

    Silvana del Mónaco

    2006-02-01

    epithelial Na channel (ENaC in placental tissue from normal and pre-eclamptic women and in BeWo cell, a model of a human SCT. Changes in the expression of these proteins during sodium transport across the placenta may be related to the pathogeny of pre-eclampsia. The role that ENaC and Na+ transport deregulation play on human placental tissues still remains unknown although in aldosterone-responsive epithelial cells (kidney, colon, abnormalities upregulating its activity lead to increased Na+ uptake and hypertension (i.e. Liddle´s syndrome whereas a diminished channel activity can result in the pseudohypoaldosteronisn syndrome with salt loss and hypotension. Our results show that ENaC is expressed in the apical membrane of normal syncytiotrophoblast. The amplified fragment of a-ENaC was cloned and sequenced having a 100% identity with the sequence of a-ENaC obtained from GenBankTM (SCNN1A, accession number Z92981. We found that the transcription of the a-ENaC mRNA was not detectable in preeclamptic placentas and the protein was not observed with immunohistochemistry staining, probably indicating a low protein expression level. In BeWo cells ENac was found and its expression is regulated by aldosterone, vasopressin, progesterone and estradiol. With patch clamp techniques we studied the currents trough ENaC channels in Bewo cells. We observed currents that were blocked by 10 µM amiloride in cells incubated in 100 nM aldosterone for 12 hs. The amplitude of this current was 20-fold the basal current, a reversal potential of 3 mV and a conductance of 127 ± 26 pS/pF with pulses between -60 and -140 mV. These characteristics are similar to those reported in ENaC channels in several tissues. Although their roles in placenta are still poorly understood, the differences in the expression of ENaC in pre-eclamptic placentas may have consequences for ion transport and these data could lead to future studies concerning the mechanism involved in the pathophysiology of pre-eclampsia.

  20. Distribution, expression and functional effects of small conductance Ca-activated potassium (SK) channels in rat myometrium.

    Science.gov (United States)

    Noble, Karen; Floyd, Rachel; Shmygol, Andre; Shmygol, Anatoly; Mobasheri, A; Wray, Susan

    2010-01-01

    Calcium-activated potassium channels are important in a variety of smooth muscles, contributing to excitability and contractility. In the myometrium previous work has focussed on the large conductance channels (BK), and the role of small conductance channels (SK) has received scant attention, despite the finding that over-expression of an SK channel isoform (SK3) results in uterine dysfunction and delayed parturition. This study therefore characterises the expression of the three SK channel isoforms (SK1-3) in rat myometrium throughout pregnancy and investigates their effect on cytosolic [Ca] and force and compares this with that of BK channels. Consistent expression of all SK isoform transcripts and clear immunostaining of SK1-3 was found. Inhibition of SK1-3 channels (apamin, scyllatoxin) significantly inhibited outward current, caused membrane depolarisation and elicited action potentials in previously quiescent cells. Apamin or scyllatoxin increased the amplitude of [Ca] and force in spontaneously contracting myometrial strips throughout gestation. The functional effect of SK inhibition was larger than that of BK channel inhibition. Thus we show for the first time that SK1-3 channels are expressed and translated throughout pregnancy and contribute to outward current, regulate membrane potential and hence Ca signals in pregnant rat myometrium. They contribute more to quiescence that BK channels. 2009 Elsevier Ltd. All rights reserved.

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

    International Nuclear Information System (INIS)

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

    1988-01-01

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

  2. Differentially Expressed Potassium Channels Are Associated with Function of Human Effector Memory CD8+T Cells.

    Science.gov (United States)

    Sim, Ji Hyun; Kim, Kyung Soo; Park, Hyoungjun; Kim, Kyung-Jin; Lin, Haiyue; Kim, Tae-Joo; Shin, Hyun Mu; Kim, Gwanghun; Lee, Dong-Sup; Park, Chan-Wook; Lee, Dong Hun; Kang, Insoo; Kim, Sung Joon; Cho, Chung-Hyun; Doh, Junsang; Kim, Hang-Rae

    2017-01-01

    The voltage-gated potassium channel, Kv1.3, and the Ca 2+ -activated potassium channel, KCa3.1, regulate membrane potentials in T cells, thereby controlling T cell activation and cytokine production. However, little is known about the expression and function of potassium channels in human effector memory (EM) CD8 + T cells that can be further divided into functionally distinct subsets based on the expression of the interleukin (IL)-7 receptor alpha (IL-7Rα) chain. Herein, we investigated the functional expression and roles of Kv1.3 and KCa3.1 in EM CD8 + T cells that express high or low levels of the IL-7 receptor alpha chain (IL-7Rα high and IL-7Rα low , respectively). In contrast to the significant activity of Kv1.3 and KCa3.1 in IL-7Rα high EM CD8 + T cells, IL-7Rα low EM CD8 + T cells showed lower expression of Kv1.3 and insignificant expression of KCa3.1. Kv1.3 was involved in the modulation of cell proliferation and IL-2 production, whereas KCa3.1 affected the motility of EM CD8 + T cells. The lower motility of IL-7Rα low EM CD8 + T cells was demonstrated using transendothelial migration and motility assays with intercellular adhesion molecule 1- and/or chemokine stromal cell-derived factor-1α-coated surfaces. Consistent with the lower migration property, IL-7Rα low EM CD8 + T cells were found less frequently in human skin. Stimulating IL-7Rα low EM CD8 + T cells with IL-2 or IL-15 increased their motility and recovery of KCa3.1 activity. Our findings demonstrate that Kv1.3 and KCa3.1 are differentially involved in the functions of EM CD8 + T cells. The weak expression of potassium channels in IL-7Rα low EM CD8 + T cells can be revived by stimulation with IL-2 or IL-15, which restores the associated functions. This study suggests that IL-7Rα high EM CD8 + T cells with functional potassium channels may serve as a reservoir for effector CD8 + T cells during peripheral inflammation.

  3. Duodenal Sodium/Glucose Cotransporter 1 Expression Under Fasting Conditions Is Associated With Postload Hyperglycemia.

    Science.gov (United States)

    Fiorentino, Teresa Vanessa; Suraci, Evelina; Arcidiacono, Gaetano Paride; Cimellaro, Antonio; Mignogna, Chiara; Presta, Ivan; Andreozzi, Francesco; Hribal, Marta Letizia; Perticone, Francesco; Donato, Giuseppe; Luzza, Francesco; Sesti, Giorgio

    2017-11-01

    Type 2 diabetes (T2DM) is associated with a higher intestinal expression of the glucose transporters sodium/glucose cotransporter 1 (SGLT-1) and glucose transporter 2 (GLUT-2). It is currently unsettled whether prediabetes conditions characterized by postprandial hyperglycemia, such as impaired glucose tolerance (IGT) and normal glucose tolerance (NGT) with 1-hour postload glucose ≥155 mg/dL (8.6 mmol/L) (NGT-1h-high) are associated with increased expression of these glucose carriers in the intestine. We evaluated whether duodenal abundance of SGLT-1 and GLUT-2 is augmented in subjects with IGT and NGT-1h-high, in comparison with subjects with NGT and 1-hour postload glucose ˂155 mg/dL (NGT-1h-low). Cross-sectional. A total of 54 individuals underwent an upper gastrointestinal endoscopy. Duodenal SGLT-1 and GLUT-2 protein and messenger RNA levels were assessed by Western blot and reverse transcription polymerase chain reaction, respectively. Of the 54 subjects examined, 18 had NGT-1h-low, 12 had NGT-1h-high, 12 had IGT, and 12 had T2DM. Duodenal SGLT-1 protein and messenger RNA levels were significantly higher in individuals with NGT-1h-high, IGT, or T2DM in comparison with NGT-1h-low subjects. GLUT-2 abundance was higher in individuals with T2DM in comparison with NGT-1h-low subjects; no substantial increase in GLUT-2 expression was observed in NGT-1h-high or IGT individuals. Univariate correlations showed that duodenal SGLT-1 abundance was positively correlated with 1-hour postload plasma glucose levels (r = 0.44; P = 0.003) but not with fasting or 2-hour postload glucose levels. Duodenal SGLT-1 expression is increased in individuals with 1-hour postload hyperglycemia or IGT, as well as in subjects with T2DM, and it positively correlates with early postload glucose excursion. Copyright © 2017 Endocrine Society

  4. Expression of inward rectifier potassium channel subunits in optic nerve glia

    OpenAIRE

    Brasko, Csilla

    2013-01-01

    In glia inward rectifying potassium channels (Kir) are predominantly responsible for the high selective membrane permeability to K+, for the maintenance of the RMP close to the EK and for the clearance of excess K+ released during action potentials by the process of K+ buffering. In this study I have investigated the expression, subcellular localisation and heteromer forming ability of Kir4.1, Kir5.1 and Kir2.1 subunits in optic nerve glia. Immunocytochemistry results demonstrated the e...

  5. System of closing relations of a two-fluid model for the HYDRA-IBRAE/LM/V1 code for calculation of sodium boiling in channels of power equipment

    Science.gov (United States)

    Usov, E. V.; Butov, A. A.; Dugarov, G. A.; Kudasov, I. G.; Lezhnin, S. I.; Mosunova, N. A.; Pribaturin, N. A.

    2017-07-01

    The system of equations from a two-fluid model is widely used in modeling thermohydraulic processes during accidents in nuclear reactors. The model includes conservation equations governing the balance of mass, momentum, and energy in each phase of the coolant. The features of heat and mass transfer, as well as of mechanical interaction between phases or with the channel wall, are described by a system of closing relations. Properly verified foreign and Russian codes with a comprehensive system of closing relations are available to predict processes in water coolant. As to the sodium coolant, only a few open publications on this subject are known. A complete system of closing relations used in the HYDRA-IBRAE/LM/V1 thermohydraulic code for calculation of sodium boiling in channels of power equipment is presented. The selection of these relations is corroborated on the basis of results of analysis of available publications with an account taken of the processes occurring in liquid sodium. A comparison with approaches outlined in foreign publications is presented. Particular attention has been given to the calculation of the sodium two-phase flow boiling. The flow regime map and a procedure for the calculation of interfacial friction and heat transfer in a sodium flow with account taken of high conductivity of sodium are described in sufficient detail. Correlations are presented for calculation of heat transfer for a single-phase sodium flow, sodium flow boiling, and sodium flow boiling crisis. A method is proposed for prediction of flow boiling crisis initiation.

  6. TRPV3, a thermosensitive channel is expressed in mouse distal colon epithelium

    Energy Technology Data Exchange (ETDEWEB)

    Ueda, Takashi, E-mail: tueda@med.nagoya-cu.ac.jp [Department of Neurobiology and Anatomy, Graduate School of Medical Sciences, Nagoya City University, 1 Kawasumi, Mizuho-cho, Mizuho-ku, Nagoya, Aichi 467-8601 (Japan); Yamada, Takahiro, E-mail: yamada-taka@syd.odn.ne.jp [Department of Neurobiology and Anatomy, Graduate School of Medical Sciences, Nagoya City University, 1 Kawasumi, Mizuho-cho, Mizuho-ku, Nagoya, Aichi 467-8601 (Japan); Ugawa, Shinya, E-mail: ugawa@med.nagoya-cu.ac.jp [Department of Neurobiology and Anatomy, Graduate School of Medical Sciences, Nagoya City University, 1 Kawasumi, Mizuho-cho, Mizuho-ku, Nagoya, Aichi 467-8601 (Japan); Ishida, Yusuke, E-mail: y.ishida@med.nagoya-cu.ac.jp [Department of Neurobiology and Anatomy, Graduate School of Medical Sciences, Nagoya City University, 1 Kawasumi, Mizuho-cho, Mizuho-ku, Nagoya, Aichi 467-8601 (Japan); Shimada, Shoichi, E-mail: sshimada@med.nagoya-cu.ac.jp [Department of Neurobiology and Anatomy, Graduate School of Medical Sciences, Nagoya City University, 1 Kawasumi, Mizuho-cho, Mizuho-ku, Nagoya, Aichi 467-8601 (Japan)

    2009-05-22

    The thermo-transient receptor potential (thermoTRP) subfamily is composed of channels that are important in nociception and thermo-sensing. Here, we show a selective expression of TRPV3 channel in the distal colon throughout the gastrointestinal tract. Expression analyses clearly revealed that TRPV3 mRNA and proteins were expressed in the superficial epithelial cells of the distal colon, but not in those of the stomach, duodenum or proximal colon. In a subset of primary epithelial cells cultured from the distal colon, carvacrol, an agonist for TRPV3, elevated cytosolic Ca{sup 2+}concentration in a concentration-dependent manner. This response was inhibited by ruthenium red, a TRPV channel antagonist. Organotypic culture supported that the carvacrol-responsive cells were present in superficial epithelial cells. Moreover, application of carvacrol evoked ATP release in primary colonic epithelial cells. We conclude that TRPV3 is present in absorptive cells in the distal colon and may be involved in a variety of cellular functions.

  7. TRPV3, a thermosensitive channel is expressed in mouse distal colon epithelium

    International Nuclear Information System (INIS)

    Ueda, Takashi; Yamada, Takahiro; Ugawa, Shinya; Ishida, Yusuke; Shimada, Shoichi

    2009-01-01

    The thermo-transient receptor potential (thermoTRP) subfamily is composed of channels that are important in nociception and thermo-sensing. Here, we show a selective expression of TRPV3 channel in the distal colon throughout the gastrointestinal tract. Expression analyses clearly revealed that TRPV3 mRNA and proteins were expressed in the superficial epithelial cells of the distal colon, but not in those of the stomach, duodenum or proximal colon. In a subset of primary epithelial cells cultured from the distal colon, carvacrol, an agonist for TRPV3, elevated cytosolic Ca 2+ concentration in a concentration-dependent manner. This response was inhibited by ruthenium red, a TRPV channel antagonist. Organotypic culture supported that the carvacrol-responsive cells were present in superficial epithelial cells. Moreover, application of carvacrol evoked ATP release in primary colonic epithelial cells. We conclude that TRPV3 is present in absorptive cells in the distal colon and may be involved in a variety of cellular functions.

  8. Sodium Butyrate Prevents Memory Impairment by Re-establishing BDNF and GDNF Expression in Experimental Pneumococcal Meningitis.

    Science.gov (United States)

    Barichello, Tatiana; Generoso, Jaqueline S; Simões, Lutiana R; Faller, Cristiano Julio; Ceretta, Renan A; Petronilho, Fabricia; Lopes-Borges, Jéssica; Valvassori, Samira S; Quevedo, João

    2015-08-01

    Pneumococcal meningitis is a serious infection of the central nervous system (CNS) with high fatality rates that causes reduced psychomotor performance, slight mental slowness, impairments in attention executive functions and learning and memory deficiencies. Previously, we demonstrated a correlation between memory impairment and decreased levels of brain-derived neurotropic factor (BDNF) in the hippocampi of rats subjected to pneumococcal meningitis. Emerging evidence demonstrates that histone acetylation regulates neurotrophins; therefore, a potential molecular intervention against cognitive impairment in bacterial meningitis may be the histone deacetylase (HDAC) inhibitor, sodium butyrate, which stimulates the acetylation of histones and increases BDNF expression. In this study, animals received either artificial cerebrospinal fluid as a placebo or a Streptococcus pneumoniae suspension at a concentration of 5 × 10(9) colony-forming units (CFU/mL). The animals received antibiotic treatment as usual and received saline or sodium butyrate as an adjuvant treatment. Ten days after, meningitis was induced; the animals were subjected to open-field habituation and the step-down inhibitory avoidance task. Immediately after these behavioural tasks, the animals were killed, and their hippocampi were removed to evaluate the expression of BDNF, nerve growth factor (NGF) and glial cell line-derived neurotrophic factor (GDNF). In the meningitis group that received saline, the animals presented memory impairment in both behavioural tasks, and hippocampal BDNF and GDNF expression was decreased. Sodium butyrate was able to prevent memory impairment and re-establish hippocampal neurotrophin expression in experimental pneumococcal meningitis.

  9. Differential expression of BK channel isoforms and beta-subunits in rat neuro-vascular tissues

    DEFF Research Database (Denmark)

    Poulsen, Asser Nyander; Wulf, Helle; Hay-Schmidt, Anders

    2009-01-01

    We investigated the expression of splice variants and beta-subunits of the BK channel (big conductance Ca(2+)-activated K(+) channel, Slo1, MaxiK, K(Ca)1.1) in rat cerebral blood vessels, meninges, trigeminal ganglion among other tissues. An alpha-subunit splice variant X1(+24) was found expressed...... (RT-PCR) in nervous tissue only where also the SS4(+81) variant was dominating with little expression of the short form SS4(0). SS4(+81) was present in some cerebral vessels too. The SS2(+174) variant (STREX) was found in both blood vessels and in nervous tissue. In situ hybridization data supported...... the finding of SS4(+81) and SS2(+174) in vascular smooth muscle and trigeminal ganglion. beta-subunits beta2 and beta4 showed high expression in brain and trigeminal ganglion and some in cerebral vessels while beta1 showed highest expression in blood vessels. beta3 was found only in testis and possibly brain...

  10. Effects of fluoxetine on protein expression of potassium ion channels in the brain of chronic mild stress rats

    Directory of Open Access Journals (Sweden)

    Chunlin Chen

    2015-01-01

    Full Text Available The purpose of this study is to investigate the expression of major potassium channel subtypes in the brain of chronical mild stress (CMS rats and reveal the effects of fluoxetine on the expression of these channels. Rats were exposed to a variety of unpredictable stress for three weeks and induced anhedonia, lower sucrose preference, locomotor activity and lower body weight. The protein expressions were determined by Western blot. CMS significantly increased the expression of Kv2.1 channel in frontal cortex but not in hippocampus, and the expression level was normalized after fluoxetine treatment. The expression of TREK-1 channel was also obviously increased in frontal cortex in CMS rats. Fluoxetine treatment might prevent this increase. However, the expression of Kv3.1 and Kv4.2 channels was considerably decreased in hippocampus after CMS, and was not affected by fluoxetine. These results suggest that different subtypes of potassium channels are associated with the pathophysiology of depression and that the therapeutical effects of fluoxetine may relate to Kv2.1 and TREK-1 potassium channels.

  11. Effects of 2,3-butanedione monoxime (BDM) on calcium channels expressed in Xenopus oocytes.

    Science.gov (United States)

    Allen, T J; Mikala, G; Wu, X; Dolphin, A C

    1998-04-01

    1. We examine the actions of a chemical phosphatase, 2,3-butanedione monoxime (BDM), on endogenous and expressed Ca2+ channel currents in Xenopus oocytes. In previous studies on L-type Ca2+ channel currents in cardiomyocytes and dorsal root ganglia, the inhibitory effects of BDM were attenuated by activation of protein kinase A. 2. Ba2+ currents (IBa) through a human wild-type L-type Ca2+ channel complex (i.e. halpha1C, alpha2-deltaa and hbeta1b) are inhibited by BDM with an IC50 of 16 mM, with 10 mM producing a 36.1 +/- 2.2 % inhibition. IBa through endogenous oocyte N-type Ca2+ channels, upregulated by exogenous alpha2-deltaa and hbeta1b subunits, are inhibited to a similar degree by BDM. 3. To examine whether the action of BDM is dependent on PKA-dependent phosphorylation, a clone of halpha1C deficient in all five serine PKA consensus sites (halpha1C-SA5) was co-expressed with alpha2-deltaa and the human cardiac hbeta3 subunit, which naturally lacks PKA consensus sites. This complex exhibited a sensitivity to BDM that was similar to the wild-type complex, with 10 mM BDM producing 31.6 +/- 1.5 % inhibition. 4. As limited proteolysis upregulates Ca2+ channels in cardiomyocytes and renders them less sensitive to BDM, experiments were performed with a carboxyl terminus deletion mutant, halpha1C-Delta1633. IBa through this subunit showed a sensitivity to BDM that was similar to the wild-type complex, with 10 mM BDM producing 31.3 +/- 1.4 % inhibition. However, co-expression with alpha2-deltaa and hbeta3 subunits reduced potency, and is reflected by an increased IC50 of 22.7 mM. 5. The actions of BDM were examined on a rat brain rbA-1 Ca2+ channel clone, alpha1A, co-expressed with alpha2-deltab and beta1b subunit homologues from rat brain. BDM inhibited the current through this channel complex to a similar degree to that seen for cardiac wild-type channels, with 10 mM BDM causing a 33.1 +/- 3.5 % inhibition. 6. The effects of BDM were compared at two holding

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

    Science.gov (United States)

    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.

  13. Development and utilization of a fluorescence-based receptor-binding assay for the site 5 voltage-sensitive sodium channel ligands brevetoxin and ciguatoxin.

    Science.gov (United States)

    McCall, Jennifer R; Jacocks, Henry M; Niven, Susan C; Poli, Mark A; Baden, Daniel G; Bourdelais, Andrea J

    2014-01-01

    Brevetoxins are a family of ladder-frame polyether toxins produced during blooms of the marine dinoflagellate Karenia brevis. Consumption of fish exposed to K. brevis blooms can lead to the development of neurotoxic shellfish poisoning. The toxic effects of brevetoxins are due to activation of voltage-sensitive sodium channels (VSSCs) in cell membranes. Binding of toxins has historically been measured using a radioligand competition assay that is fraught with difficulty. In this study, we developed a novel fluorescence-based binding assay for the brevetoxin receptor. Several fluorophores were conjugated to polyether brevetoxin-2 and used as the labeled ligand. Brevetoxin analogs were able to compete for binding with the fluorescent ligands. This assay was qualified against the standard radioligand receptor assay for the brevetoxin receptor. Furthermore, the fluorescence-based assay was used to determine relative concentrations of toxins in raw extracts of K. brevis culture, and to determine ciguatoxin affinity to site 5 of VSSCs. The fluorescence-based assay was quicker, safer, and far less expensive. As such, this assay can be used to replace the current radioligand assay and will be a vital tool for future experiments examining the binding affinity of various ligands for site 5 on sodium channels.

  14. Identification of a mutation associated with permethrin resistance in the para-type sodium channel of the stable fly (Diptera: Muscidae).

    Science.gov (United States)

    Olafson, Pia U; Pitzer, Jimmy B; Kaufman, Phillip E

    2011-02-01

    The insect sodium channel is of particular interest for evaluating resistance to pyrethroids because it is the target molecule for this major class of neurotoxic insecticides. The stable fly, Stomoxys calcitrans (L.) (Diptera: Muscidae), sodium channel coding sequence representing domains IS6 through IVS6 was isolated, and the sequence encoding domain II was compared among individuals of a laboratory strain selected for resistance to permethrin and the unselected, parental generation. A point mutation resulting in a leucine-to-histidine amino acid change was identified (Leul014His), and its location corresponded with that observed for knockdown resistance (kdr) mutations in other insects. As a result, the allele was designated kdr-his. A molecular assay was developed to assess the frequency of this mutation in genomic DNA of individual stable flies from the laboratory selections, which provided further evidence that the kdr-his allele accounts for the observed level ofpermethrin resistance in the selected strain. The assay was then used to evaluate the frequency of the mutation from five field-collected populations originating from three horse farms near Ocala, FL; one horse farm near Gainesville, FL; and one dairy farm near Hague, FL. Frequency of the kdr-his allele ranged from 0.46 to 0.78, supporting further investigation of allele prevalence throughout the stable fly season and in response to field insecticide application.

  15. Common variants of the beta and gamma subunits of the epithelial sodium channel and their relation to plasma renin and aldosterone levels in essential hypertension

    Directory of Open Access Journals (Sweden)

    Krusius Tom

    2005-01-01

    Full Text Available Abstract Background Rare mutations of the epithelial sodium channel (ENaC result in the monogenic hypertension form of Liddle's syndrome. We decided to screen for common variants in the ENaC βand γ subunits in patients with essential hypertension and to relate their occurrence to the activity of circulating renin-angiotensin-aldosterone system. Methods Initially, DNA samples from 27 patients with low renin/low aldosterone hypertension were examined. The DNA variants were subsequently screened for in 347 patients with treatment-resistant hypertension, 175 male subjects with documented long-lasting normotension and 301 healthy Plasma renin and aldosterone levels were measured under baseline conditions and during postural and captopril challenge tests. Results Two commonly occurring βENaC variants (G589S and a novel intronic i12-17CT substitution and one novel γENaC variant (V546I were detected. One of these variants occurred in a heterozygous form in 32 patients, a prevalence (9.2% significantly higher than that in normotensive males (2.9%, p = 0.007 and blood donors (3.0%, p = 0.001. βENaC i12-17CT was significantly more prevalent in the hypertension group than in the two control groups combined (4.6% vs. 1.1%, p = 0.001. When expressed in Xenopus oocytes, neither of the two ENaC amino acid-changing variants showed a significant difference in activity compared with ENaC wild-type. No direct evidence for a mRNA splicing defect could be obtained for the βENaC intronic variant. The ratio of daily urinary potassium excretion to upright and mean (of supine and upright values plasma renin activity was higher in variant allele carriers than in non-carriers (p = 0.034 and p = 0.048. Conclusions At least 9% of Finnish patients with hypertension admitted to a specialized center carry genetic variants of β and γENaC, a three times higher prevalence than in the normotensive individuals or in random healthy controls. Patients with the variant alleles

  16. Cloning and expression of a Kv1.2 class delayed rectifier K+ channel from canine colonic smooth muscle.

    Science.gov (United States)

    Hart, P J; Overturf, K E; Russell, S N; Carl, A; Hume, J R; Sanders, K M; Horowitz, B

    1993-10-15

    A cDNA (CSMK1) encoding a delayed rectifier K+ channel of the Kv1.2 class was cloned from canine colonic circular smooth muscle and expressed in Xenopus oocytes. These channels appear to be uniquely expressed in gastrointestinal muscles and may participate in the electrical slow wave activity. Functional expression of CSMK1 in Xenopus oocytes demonstrated a K+ current that activated in a voltage-dependent manner upon depolarization. This current was highly sensitive to 4-aminopyridine (IC50, 74 microM). A low-conductance K+ channel was identified in inside-out patches from oocytes injected with CSMK1. This channel displayed a linear current-voltage relation with a slope conductance of 14 pS. The channels were blocked in a concentration-dependent manner by 4-aminopyridine. Northern blot analysis demonstrated that CSMK1 is expressed in a wide variety of gastrointestinal smooth muscles. Portal vein, renal artery, and uterus do not express CSMK1, suggesting that, among smooth muscles, expression of this K+ channel may be restricted to gastrointestinal smooth muscles. CSMK1 is 91% homologous to RAK, a delayed rectifier K+ channel cloned from rat heart, but displays unique pharmacological properties and tissue distribution.

  17. Expression and function of K(V)2-containing channels in human urinary bladder smooth muscle.

    Science.gov (United States)

    Hristov, Kiril L; Chen, Muyan; Afeli, Serge A Y; Cheng, Qiuping; Rovner, Eric S; Petkov, Georgi V

    2012-06-01

    The functional role of the voltage-gated K(+) (K(V)) channels in human detrusor smooth muscle (DSM) is largely unexplored. Here, we provide molecular, electrophysiological, and functional evidence for the expression of K(V)2.1, K(V)2.2, and the electrically silent K(V)9.3 subunits in human DSM. Stromatoxin-1 (ScTx1), a selective inhibitor of K(V)2.1, K(V)2.2, and K(V)4.2 homotetrameric channels and of K(V)2.1/9.3 heterotetrameric channels, was used to examine the role of these channels in human DSM function. Human DSM tissues were obtained during open bladder surgeries from patients without a history of overactive bladder. Freshly isolated human DSM cells were studied using RT-PCR, immunocytochemistry, live-cell Ca(2+) imaging, and the perforated whole cell patch-clamp technique. Isometric DSM tension recordings of human DSM isolated strips were conducted using tissue baths. RT-PCR experiments showed mRNA expression of K(V)2.1, K(V)2.2, and K(V)9.3 (but not K(V)4.2) channel subunits in human isolated DSM cells. K(V)2.1 and K(V)2.2 protein expression was confirmed by Western blot analysis and immunocytochemistry. Perforated whole cell patch-clamp experiments revealed that ScTx1 (100 nM) inhibited the amplitude of the voltage step-induced K(V) current in freshly isolated human DSM cells. ScTx1 (100 nM) significantly increased the intracellular Ca(2+) level in DSM cells. In human DSM isolated strips, ScTx1 (100 nM) increased the spontaneous phasic contraction amplitude and muscle force, and enhanced the amplitude of the electrical field stimulation-induced contractions within the range of 3.5-30 Hz stimulation frequencies. These findings reveal that ScTx1-sensitive K(V)2-containing channels are key regulators of human DSM excitability and contractility and may represent new targets for pharmacological or genetic intervention for bladder dysfunction.

  18. Dietary sodium deprivation evokes activation of brain regional neurons and down-regulation of angiotensin II type 1 receptor and angiotensin-convertion enzyme mRNA expression.

    Science.gov (United States)

    Lu, B; Yang, X J; Chen, K; Yang, D J; Yan, J Q

    2009-12-15

    Previous studies have indicated that the renin-angiotensin-aldosterone system (RAAS) is implicated in the induction of sodium appetite in rats and that different dietary sodium intakes influence the mRNA expression of central and peripheral RAAS components. To determine whether dietary sodium deprivation activates regional brain neurons related to sodium appetite, and changes their gene expression of RAAS components of rats, the present study examined the c-Fos expression after chronic exposure to low sodium diet, and determined the relationship between plasma and brain angiotensin I (ANG I), angiotensin II (ANG II) and aldosterone (ALD) levels and the sodium ingestive behavior variations, as well as the effects of prolonged dietary sodium deprivation on ANG II type 1 (AT1) and ANG II type 2 (AT2) receptors and angiotensin-convertion enzyme (ACE) mRNA levels in the involved brain regions using the method of real-time polymerase chain reaction (PCR). Results showed that the Fos immunoreactivity (Fos-ir) expression in forebrain areas such as subfornical organ (SFO), paraventricular hypothalamic nuclei (PVN), supraoptic nucleus (SON) and organum vasculosum laminae terminalis (OVLT) all increased significantly and that the levels of ANG I, ANG II and ALD also increased in plasma and forebrain in rats fed with low sodium diet. In contrast, AT1, ACE mRNA in PVN, SON and OVLT decreased significantly in dietary sodium depleted rats, while AT2 mRNA expression did not change in the examined areas. These results suggest that many brain areas are activated by increased levels of plasma and/or brain ANG II and ALD, which underlies the elevated preference for hypertonic salt solution after prolonged exposure to low sodium diet, and that the regional AT1 and ACE mRNA are down-regulated after dietary sodium deprivation, which may be mediated by increased ANG II in plasma and/or brain tissue.

  19. A novel K+ channel expressed in carrot roots with a low susceptibility toward metal ions.

    Science.gov (United States)

    Paganetto, A; Bregante, M; Downey, P; Lo Schiavo, F; Hoth, S; Hedrich, R; Gambale, F

    2001-02-01

    Kdc1 is a novel K+-channel gene cloned from carrot roots, and which is also present in cultured carrot cells. We investigated the characteristics of the ionic current elicited in Xenopus oocytes coinjected with KDC1 (K+-Daucus carota 1) and KAT1 (from Arabidopsis thaliana) RNA. Expressed heteromeric channels displayed inward-rectifying potassium currents whose kinetics, voltage characteristics, and inhibition by metal ions depended on KDC1:KAT1 ratios. At low KDC1:KAT1 ratios, Zn2+ inhibition of heteromeric K+ current was less pronounced compared to homomeric KAT1 channels, while at higher KDC1:KAT1 ratios, the addition of Zn2+ even produced an increase in current. Under the same conditions, the Ni2+ inhibition of the current was also reduced, but no current increase was observed. These effects might be explained by the unusual amino acid composition of the KDC1 protein in terms of histidine residues that are absent in the pore region, but abundant (four per subunit) in the proximity of the pore entrance. Channels like KDC1 could be at least partially responsible for the higher resistance of carrot cells in the presence of metals.

  20. Expression of Transient Receptor Potential Canonical Channel Proteins in Human Non-small Cell Lung Cancer

    Directory of Open Access Journals (Sweden)

    Qi ZHANG

    2010-06-01

    Full Text Available Background and objective Transient receptor potential canonical (TRPC proteins, a group of Ca2+ permeable nonselective cation channels, are thought to constitute store-operated calcium channels (SOCC and mediate store-operated calcium entry (SOCE in various cell types. Members of TRPC have been found to be involved in abnormal proliferation, differentiation, and growth of cancer cells. The aim of this study is to detect the mRNA and protein expression of TRPC in non-small cell lung cancer (NSCLC. Methods Real-time quantitative PCR was performed to screen the expression of TRPC mRNA in NSCLC tissue. Protein expression of TRPC was detected by Western blot. Results Among the seven family members of TRPC so far identified (TRPC1-7, we detected the expression of TRPC1, TRPC3, TRPC4, TRPC6 mRNA in 24 cases of NSCLC tissue; TRPC2, TRPC5 and TRPC7 mRNA were not detectable. The relative abundance of the expressed TRPC was TRPC1≈TRPC6>TRPC3>TRPC4. Western blot confirmed the protein expression of TRPC1, TRPC3, TRPC4 and TRPC6 in NSCLC tissue. Conclusion Out of the seven members of TRPC, we found TRPC1, TRPC3, TRPC4, TRPC6 mRNA and protein were selectively expressed in human NSCLC tissue. This study could provide a basis for future exploration of the individual role of these TRPC proteins in mediating SOCE and in the progression of lung cancer.

  1. Altered expression of genes for Kir ion channels in dilated cardiomyopathy.

    Science.gov (United States)

    Szuts, Viktoria; Ménesi, Dalma; Varga-Orvos, Zoltán; Zvara, Ágnes; Houshmand, Nazanin; Bitay, Miklós; Bogáts, Gábor; Virág, László; Baczkó, István; Szalontai, Balázs; Geramipoor, Amir; Cotella, Diego; Wettwer, Erich; Ravens, Ursula; Deák, Ferenc; Puskás, László G; Papp, Julius Gy; Kiss, Ibolya; Varró, András; Jost, Norbert

    2013-08-01

    Dilated cardiomyopathy (DCM) is a multifactorial disease characterized by left ventricular dilation that is associated with systolic dysfunction and increased action potential duration. The Kir2.x K⁺ channels (encoded by KCNJ genes) regulate the inward rectifier current (IK1) contributing to the final repolarization in cardiac muscle. Here, we describe the transitions in the gene expression profiles of 4 KCNJ genes from healthy or dilated cardiomyopathic human hearts. In the healthy adult ventricles, KCNJ2, KCNJ12, and KCNJ4 (Kir2.1-2.3, respectively) genes were expressed at high levels, while expression of the KCNJ14 (Kir2.4) gene was low. In DCM ventricles, the levels of Kir2.1 and Kir2.3 were upregulated, but those of Kir2.2 channels were downregulated. Additionally, the expression of the DLG1 gene coding for the synapse-associated protein 97 (SAP97) anchoring molecule exhibited a 2-fold decline with increasing age in normal hearts, and it was robustly downregulated in young DCM patients. These adaptations could offer a new aspect for the explanation of the generally observed physiological and molecular alterations found in DCM.

  2. Sodium nitroprusside may modulate Escherichia coli antioxidant enzyme expression by interacting with the ferric uptake regulator.

    Science.gov (United States)

    Bertrand, R; Danielson, D; Gong, V; Olynik, B; Eze, M O

    2012-01-01

    Efforts to explore possible relationships between nitric oxide (NO) and antioxidant enzymes in an Escherichia coli model have uncovered a possible interaction between sodium nitroprusside (SNP), a potent, NO-donating drug, and the ferric uptake regulator (Fur), an iron(II)--dependent regulator of antioxidant and iron acquisition proteins present in Gram-negative bacteria. The enzymatic profiles of superoxide dismutase and hydroperoxidase during logarithmic phase of growth were studied via non-denaturing polyacrylamide gel electrophoresis and activity staining specific to each enzyme. Though NO is known to induce transcription of the manganese-bearing isozyme of SOD (MnSOD), treatment with SNP paradoxically suppressed MnSOD expression and greatly enhanced the activity of the iron-containing equivalent (FeSOD). Fur, one of six global regulators of MnSOD transcription, is uniquely capable of suppressing MnSOD while enhancing FeSOD expression through distinct mechanisms. We thus hypothesize that Fur is complacent in causing this behaviour and that the iron(II) component of SNP is activating Fur. E. coli was also treated with the SNP structural analogues, potassium ferricyanide (PFi) and potassium ferrocyanide (PFo). Remarkably, the ferrous PFo was capable of mimicking the SNP-related pattern, whereas the ferric PFi was not. As Fur depends upon ferrous iron for activation, we submit this observation of redox-specificity as preliminary supporting evidence for the hypothesized Fur-SNP interaction. Iron is an essential metal that the human innate immune system sequesters to prevent its use by invading pathogens. As NO is known to inhibit iron-bound Fur, and as activated Fur regulates iron uptake through feedback inhibition, we speculate that the administration of this drug may disrupt this strategic management of iron in favour of residing Gram-negative species by providing a source of iron in an otherwise iron-scarce environment capable of encouraging its own uptake

  3. TREK-1 Channel Expression in Smooth Muscle as a Target for Regulating Murine Intestinal Contractility: Therapeutic Implications for Motility Disorders

    Directory of Open Access Journals (Sweden)

    Ruolin Ma

    2018-03-01

    Full Text Available Gastrointestinal (GI motility disorders such as irritable bowel syndrome (IBS can occur when coordinated smooth muscle contractility is disrupted. Potassium (K+ channels regulate GI smooth muscle tone and are key to GI tract relaxation, but their molecular and functional phenotypes are poorly described. Here we define the expression and functional roles of mechano-gated K2P channels in mouse ileum and colon. Expression and distribution of the K2P channel family were investigated using quantitative RT-PCR (qPCR, immunohistochemistry and confocal microscopy. The contribution of mechano-gated K2P channels to mouse intestinal muscle tension was studied pharmacologically using organ bath. Multiple K2P gene transcripts were detected in mouse ileum and colon whole tissue preparations. Immunohistochemistry confirmed TREK-1 expression was smooth muscle specific in both ileum and colon, whereas TREK-2 and TRAAK channels were detected in enteric neurons but not smooth muscle. In organ bath, mechano-gated K2P channel activators (Riluzole, BL-1249, flufenamic acid, and cinnamyl 1-3,4-dihydroxy-alpha-cyanocinnamate induced relaxation of KCl and CCh pre-contracted ileum and colon tissues and reduced the amplitude of spontaneous contractions. These data reveal the specific expression of mechano-gated K2P channels in mouse ileum and colon tissues and highlight TREK-1, a smooth muscle specific K2P channel in GI tract, as a potential therapeutic target for combating motility pathologies arising from hyper-contractility.

  4. β-Escin sodium inhibits inducible nitric oxide synthase expression via downregulation of the JAK/STAT pathway in A549 cells.

    Science.gov (United States)

    Ji, Deng Bo; Xu, Bo; Liu, Jing Tao; Ran, Fu Xiang; Cui, Jing Rong

    2011-12-01

    β-escin, a triterpene saponin, is one of the major active compounds extracted from horse chestnut (Aesculus hippocastanum) seed. Previous work has found that β-escin sodium has antiinflammatory and antitumor effects. In the present study, we investigated its effect on cell proliferation and inducible nitric-oxide synthase (iNOS) expression in human lung carcinoma A549 cells. β-escin sodium (5-40 µg/mL) inhibited cytokine mixture (CM)-induced nitric oxide (NO) production in A549 cells by reducing the expression of iNOS. β-escin sodium suppressed phosphorylation and nuclear translocation of STAT1 (Tyr701) and STAT3 (Tyr705) induced by CM but did not affect the activation of c-Jun and NF-κB. β-escin sodium inhibited the activation of protein tyrosine kinase JAK2. Pervanadate treatment reversed the β-escin sodium-induced downregulation of STAT3 and STAT1. β-escin sodium treatment enhanced an activating phosphorylation of the phosphatase SHP2. Small interfering RNA-mediated knockdown of SHP2 inhibited β-escin sodium-induced phospho-STAT dephosphorylation. Moreover β-escin sodium reduced the activation of p38 MAPK. Finally, β-escin sodium inhibited the proliferation of A549 cells, did not change the cell membrane's permeability, nuclear morphology and size and the mitochondria's transmembrane potential of A549 cells. Taken together, these results demonstrate that β-escin sodium could downregulate iNOS expression through inhibiting JAK/STAT signaling and p38 MAPK activation in A549 cells. β-escin sodium has a marked antiproliferative effect on A549 cells at least in part by inhibiting the JAK/STAT signaling pathway, but not by a cytotoxic effect. β-escin sodium would be useful as a chemopreventive agent or a therapeutic against inflammatory-associated tumor. © 2011 Wiley Periodicals, Inc. Copyright © 2011 Wiley Periodicals, Inc.

  5. Calcium-dependent expression of transient receptor potential canonical type 3 channels in patients with chronic kidney disease

    DEFF Research Database (Denmark)

    Liu, Ying; Krueger, Katharina; Hovsepian, Anahit

    2011-01-01

    It is unknown whether extracellular calcium may regulate the expression of transient receptor potential canonical type 3 (TRPC3) channels in patients with chronic kidney disease. Using quantitative in-cell Western assay we compared the expression of TRPC3 channel protein in monocytes from 20...... patients with chronic kidney disease and 19 age- and sex-matched healthy control subjects. TRPC3 channels were identified by immunoblotting using specific antibodies and TRPC3 protein was further confirmed by mass spectrometry. We observed a significant increase of TRPC3 channel protein expression...... in patients with chronic kidney disease compared to healthy control subjects (normalized expression, 0.42±0.06 vs. 0.19±0.03; p...

  6. Lyso-globotriaosylceramide downregulates KCa3.1 channel expression to inhibit collagen synthesis in fibroblasts.

    Science.gov (United States)

    Choi, Ju Yeon; Shin, Mee-Young; Suh, Suk Hyo; Park, Seonghee

    2015-12-25

    Fabry disease is an X-linked lysosomal storage disorder that is caused by a deficiency of α-galactosidase A. The disease ultimately manifests as multiple organ dysfunctions owing to excessive accumulation of globotriaosylceramide (Gb3). Among the several complications of Fabry disease, ascending thoracic aortic aneurysm is relatively common, which is classically associated with connective tissue disorders characterized by abnormal defects or deficiencies in structural proteins such as collagen and elastin. Although an elevated Gb3 level is regarded as a prerequisite for the manifestations of Fabry disease, only this excess accumulation cannot explain the pathophysiology of these complications. Recently, an increased plasma level of lyso-Gb3 was suggested as a new biomarker in Fabry disease. Therefore, the aim of this study was to assess the effects of lyso-Gb3 on the pathogenesis of thoracic ascending aortic aneurysms in Fabry disease, with a particular focus on the responses related to aortic remodeling by fibroblasts. We found that lyso-Gb3 inhibited the growth of fibroblasts, as well as their differentiation into myofibroblasts, and collagen expression. Moreover, all of these compromised responses could be attributed to the effects of lyso-Gb3 on downregulation of KCa3.1 channel expression, and these impairments could be rescued when activating the KCa3.1 channel or increasing intracellular Ca(2+) concentration. This study provides new evidence that lyso-Gb3 inhibits the differentiation into myofibroblasts and collagen synthesis of fibroblasts owing to decreased Ca(2+) levels by KCa3.1 channel dysfunction. These findings suggest that the KCa3.1 channel can serve as a new target to attenuate and prevent development of ascending thoracic aortic aneurysm in Fabry disease. Copyright © 2015 Elsevier Inc. All rights reserved.

  7. Sodium arsenite down-regulates the expression of X-linked inhibitor of apoptosis protein via translational and post-translational mechanisms in hepatocellular carcinoma

    International Nuclear Information System (INIS)

    Chen, Hong; Hao, Yuqing; Wang, Lijing; Jia, Dongwei; Ruan, Yuanyuan; Gu, Jianxin

    2012-01-01

    Highlights: ► Sodium arsenite down-regulates the protein expression level of XIAP in HCC. ► Sodium arsenite inhibits the de novo XIAP synthesis and its IRES activity. ► Sodium arsenite decreases XIAP stability and promotes its proteasomal degradation. ► Overexpression of XIAP attenuates the pro-apoptotic effect of sodium arsenite. -- Abstract: X-linked inhibitor of apoptosis protein (XIAP) is a member of the inhibitors of apoptosis protein (IAP) family, and has been reported to exhibit elevated expression levels in hepatocellular carcinoma (HCC) and promote cell survival, metastasis and tumor recurrence. Targeting XIAP has proven effective for the inhibition of cancer cell proliferation and restoration of cancer cell chemosensitivity. Arsenic (or sodium arsenite) is a potent anti-tumor agent used to treat patients with acute promyelocytic leukemia (APL). Additionally, arsenic induces cell growth inhibition, cell cycle arrest and apoptosis in human HCC cells. In this study, we identified XIAP as a target for sodium arsenite-induced cytotoxicity in HCC. The exposure of HCC cell lines to sodium arsenite resulted in inhibition of XIAP expression in both a dose- and time-dependent manner. Sodium arsenite blocked the de novo XIAP synthesis and the activity of its internal ribosome entry site (IRES) element. Moreover, treatment with sodium arsenite decreased the protein stability of XIAP and induced its ubiquitin–proteasomal degradation. Overexpression of XIAP attenuated the pro-apoptotic effect of sodium arsenite in HCC. Taken together, our data demonstrate that sodium arsenite suppresses XIAP expression via translational and post-translational mechanisms in HCC.

  8. Site of covalent labeling by a photoreactive batrachotoxin derivative near transmembrane segment IS6 of the sodium channel alpha subunit.

    Science.gov (United States)

    Trainer, V L; Brown, G B; Catterall, W A

    1996-05-10

    The binding site for batrachotoxin, a lipid-soluble neurotoxin acting at Na+ channel receptor site 2, was localized using a photoreactive radiolabeled batrachotoxin derivative to covalently label purified and reconstituted rat brain Na+ channels. In the presence of the brevetoxin 1 from Ptychodiscus brevis and the pyrethroid RU51049, positive allosteric enhancers of batrachotoxin binding, a protein with an apparent molecular mass of 240 kDa corresponding to the Na+ channel alpha subunit was specifically covalently labeled. The region of the alpha subunit specifically photolabeled by the photoreactive batrachotoxin derivative was identified by antibody mapping of proteolytic fragments. Even after extensive trypsinization, and anti-peptide antibody recognizing an amino acid sequence adjacent to Na+ channel transmembrane segment IS6 was able to immunoprecipitate up to 70% of the labeled peptides. Analysis of a more complete digestion with trypsin or V8 protease indicated that the batrachotoxin receptor site is formed in part by a portion of domain I. The identification of a specifically immunoprecipitated photolabeled 7.3-kDa peptide containing transmembrane segment S6 from domain I restricted the site of labeling to residues Asn-388 to Glu-429 if V8 protease digestion was complete or Leu-380 to Glu-429 if digestion was incomplete. These results implicate the S6 transmembrane region of domain I of the Na+ channel alpha subunit as an important component of the batrachotoxin receptor site.

  9. Crystal Structure of the Mammalian GIRK2 K+ Channel and Gating Regulation by G-Proteins, PIP2 and Sodium

    Science.gov (United States)

    Whorton, Matthew R.; MacKinnon, Roderick

    2011-01-01

    Summary G-protein-gated K+ channels (Kir3.1–Kir3.4) control electrical excitability in many different cells. Among their functions relevant to human physiology and disease, they regulate the heart rate and govern a wide range of neuronal activities. Here we present the first crystal structures of a G-protein-gated K+ channel. By comparing the wild-type structure to that of a constitutively active mutant, we identify a global conformational change through which G-proteins could open a G-loop gate in the cytoplasmic domain. The structures of both channels in the absence and presence of PIP2 show that G-proteins open only the G-loop gate in the absence of PIP2, but in the presence of PIP2 the G-loop gate and a second inner helix gate become coupled, so that both gates open. We also identify a strategically located Na+ ion-binding site, which would allow intracellular Na+ to modulate GIRK channel activity. These data provide a mechanistic description of multi-ligand regulation of GIRK channel gating. PMID:21962516

  10. A new member of the amiloride-sensitive sodium channel family in Drosophila melanogaster peripheral nervous system.

    Science.gov (United States)

    Darboux, I; Lingueglia, E; Pauron, D; Barbry, P; Lazdunski, M

    1998-05-08

    Amiloride sensitivity is a common characteristic of structurally related cationic channels that are associated with a wide range of physiological functions. In Caenorhabditis elegans, neuronal and muscular degenerins are involved in mechanoperception. In animal epithelia, a Na(+)-selective channel participates in vectorial Na+ transport. In the snail nervous system, an ionotropic receptor for the peptide FMRFamide forms a Na(+)-selective channel. In mammalian brain and/or in sensory neurons, ASIC channels form H(+)-activated cation channels involved in nociception linked to acidosis. We have now cloned a new member of this family from Drosophila melanogaster. The corresponding protein displays low sequence identity with the previously cloned members of the super-family but it has the same structural organization. Its mRNA was detected from late embryogenesis (14-17 hours) and was present in the dendritic arbor subtype of the Drosophila peripheral nervous system multiple dendritic (md) sensory neurons. While the origin and specification of md neurons are well documented, their roles are still poorly understood. They could function as stretch or touch receptors, raising the possibility that this Drosophila gene product, called dmdNaC1, could also be involved in mechanotransduction.

  11. Molecular cloning, characterization and expression of thyroid-stimulating hormone receptor in channel catfish.

    Science.gov (United States)

    Goto-Kazeto, Rie; Kazeto, Yukinori; Trant, John M

    2009-05-01

    Thyroid-stimulating hormone receptors (TSHRs) are primarily expressed in the thyroid of vertebrates, however recently, transcripts encoding TSHR have been found abundantly in the gonads in a variety of fish species. The purpose of this study is to characterize the channel catfish TSHR and to examine whether the transcript are translated into protein in the gonad or store the transcript as maternal RNA for later use. The cDNA encoding the TSHR was isolated from the channel catfish thyroid but the transcript was determined to be expressed in a number of tissues, including the gonads. In fact, the ovarian expression of TSHR changed significantly during the reproductive season and peaked after the vitellogenic growth phase. Furthermore, the TSHR transcript was also detected in unfertilized eggs but not in fertilized egg of catfish. LM-PAT analysis demonstrated that catfish TSHR transcripts were fully polyadenylated in thyroidal follicles, gonads and unfertilized eggs suggesting that they were translated into protein opposed to being "stored mRNA". Western blot analysis using polyclonal antibodies against the catfish TSHR verified this assumption by visualizing immunoreactive protein in the thyroid, testis, and the post-vitellogenic ovary in abundance. A functional assay clearly showed that the recombinant catfish TSHR was specifically activated by bovine TSH but not by recombinant catfish follicle-stimulating hormone (FSH) and luteinizing hormone (LH). As in other species, the heterologous gonadotropin, hCG, partially activated the receptor. These results suggested that TSHR plays important roles for gametogenesis rather than embryogenesis.

  12. Sequence, genomic organization and expression of two channel catfish, Ictalurus punctatus, ghrelin receptors.

    Science.gov (United States)

    Small, Brian C; Quiniou, Sylvie M A; Kaiya, Hiroyuki

    2009-12-01

    Two ghrelin receptor (GHS-R) genes were isolated from channel catfish tissue and a bacterial artificial chromosome (BAC) library. The two receptors were characterized by determining tissue distribution, ontogeny of receptor mRNA expression, and effects of exogenous homologous ghrelin administration on target tissue mRNA expression. Analysis of sequence similarities indicated two genes putatively encoding GHS-R1 and GHS-R2, respectively, which have been known to be present in zebrafish. Organization and tissue expression of the GHS-R1 gene was similar to that reported for other species, and likewise yielded two detectable mRNA products as a result of alternative splicing. Expression of both full-length, GHS-R1a, and splice variant, GHS-R1b, mRNA was highest in the pituitary. Gene organization of GHS-R2 was similar to GHS-R1, but no splice variant was identified. Expression of GHS-R2a mRNA was highest in the Brockmann bodies. GHS-R1a mRNA was detected in unfertilized eggs and throughout embryogenesis, whereas GHR-R2a mRNA was not expressed in unfertilized eggs or early developing embryos and was the highest at the time of hatching. Catfish intraperitoneally injected with catfish ghrelin-Gly had greater mRNA expression of GHS-R1a in pituitaries at 2 h and Brockmann bodies at 4 h, and of GHS-R2a in Brockmann bodies at 6 h post injection. Amidated catfish ghrelin (ghrelin-amide) had no observable effect on expression of either pituitary receptor; however, GHS-R1a and GHS-R2a mRNA expression levels were increased 4 h post injection of ghrelin-amide in Brockmann bodies. This is the first characterization of GHS-R2a and suggests regulatory and functional differences between the two catfish receptors.

  13. The interaction between the activator agents batrachotoxin and veratridine and the gating processes of neuronal sodium channels.

    Science.gov (United States)

    Rando, T A; Wang, G K; Strichartz, G R

    1986-05-01

    The depolarization of frog sciatic nerves by the Na channel-activating toxins, batrachotoxin and veratridine, was studied using the sucrose-gap technique. To study the interaction between the activators and the gating processes of Na channels, we measured the depolarizations of unstimulated nerves, of nerves during repetitive stimulation, and of nerves whose Na channel inactivation process had been pharmacologically modified. Stimulation enhanced the rates of depolarization by the activators but did not effect the steady state depolarization values. Of the three inhibitors of Na channel inactivation that were tested (Leiurus alpha-scorpion toxin, chloramine T, and Ni2+), only Leiurus toxin enhanced the potencies of the activators. Neither chloramine T nor Ni2+ had any effect on the steady state level of depolarization produced by either activator. Both chloramine T and Ni2+, however, enhanced the rate of batrachotoxin action, although neither affected the rate of veratridine action. Leiurus toxin also potentiated the effects of the activators in chloramine T-treated nerves. We tested the interaction between the Na channel activators and a class of agents, local anesthetics, that stabilize a non-conducting state of the Na channel. The presence of lidocaine inhibited the depolarization produced by addition of either activator, although the addition of lidocaine subsequent to the development of batrachotoxin-induced depolarization produced repolarization very weakly and slowly. We also found that the lidocaine homologue, RAC 109I, was about 3 times as potent as its stereoisomer, RAC 109II, in its ability both to reduce the compound action potential amplitude and to inhibit the veratridine-induced depolarization.

  14. Multiple mutations in the para-sodium channel gene are associated with pyrethroid resistance in Rhipicephalus microplus from the United States and Mexico.

    Science.gov (United States)

    Stone, Nathan E; Olafson, Pia U; Davey, Ronald B; Buckmeier, Greta; Bodine, Deanna; Sidak-Loftis, Lindsay C; Giles, John R; Duhaime, Roberta; Miller, Robert J; Mosqueda, Juan; Scoles, Glen A; Wagner, David M; Busch, Joseph D

    2014-10-01

    Acaricide resistant Rhipicephalus microplus populations have become a major problem for many cattle producing areas of the world. Pyrethroid resistance in arthropods is typically associated with mutations in domains I, II, III, and IV of voltage-gated sodium channel genes. In R. microplus, known resistance mutations include a domain II change (C190A) in populations from Australia, Africa, and South America and a domain III mutation (T2134A) that only occurs in Mexico and the U.S. We investigated pyrethroid resistance in cattle fever ticks from Texas and Mexico by estimating resistance levels in field-collected ticks using larval packet discriminating dose (DD) assays and identifying single nucleotide polymorphisms (SNPs) in the para-sodium channel gene that associated with resistance. We then developed qPCR assays for three SNPs and screened a larger set of 1,488 R. microplus ticks, representing 77 field collections and four laboratory strains, for SNP frequency. We detected resistance SNPs in 21 of 68 U.S. field collections and six of nine Mexico field collections. We expected to identify the domain III SNP (T2134A) at a high frequency; however, we only found it in three U.S. collections. A much more common SNP in the U.S. (detected in 19 of 21 field collections) was the C190A domain II mutation, which has never before been reported from North America. We also discovered a novel domain II SNP (T170C) in ten U.S. and two Mexico field collections. The T170C transition mutation has previously been associated with extreme levels of resistance (super-knockdown resistance) in insects. We found a significant correlation (r = 0.81) between the proportion of individuals in field collections that carried any two resistance SNPs and the percent survivorship of F1 larvae from these collections in DD assays. This relationship is accurately predicted by a simple linear regression model (R2 = 0.6635). These findings demonstrate that multiple mutations in the para-sodium channel

  15. Increased transient receptor potential vanilloid type 1 (TRPV1) channel expression in hypertrophic heart

    DEFF Research Database (Denmark)

    Thilo, Florian; Liu, Ying; Schulz, Nico

    2010-01-01

    The aim of this study was to compare the expression of transient receptor potential vanilloid type 1 (TRPV1) channels in hypertrophic hearts from transgenic mice showing overexpression of the catalytic subunit alpha of protein phosphatase 2A alpha (PP2Ac alpha) with wild-type mice and with TRPV1-...... alpha transgenic mice compared to wild-type mice and TRPV1-/- mice (8.6±1.3mg/g; 5.4±0.3mg/g; and 5.4±0.4mg/g; respectively; p...

  16. Differential protein expression of Caco-2 cells treated with selenium nanoparticles compared with sodium selenite and selenomethionine

    Science.gov (United States)

    Fu, Linglin; Yan, Xuxia; Ruan, Xinming; Lin, Junda; Wang, Yanbo

    2014-10-01

    The study was designed to determine the differential protein expression of Caco-2 cells treated with different forms of selenium including sodium selenite, selenomethionine (Se-Met), and selenium nanoparticles (nano-Se). Two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) and mass spectrometry (MS) were used to identify the differentially expressed proteins. The results indicated that seven protein spots, ubiquitin-conjugating enzyme E2 (E2), glutathione synthetases (GS), triosephosphate isomerase (TSP), T-complex protein 1 subunit zeta (TCPZ), lamin-B1, heterogeneous nuclear ribonucleoprotein F (hnRNP F), and superoxide dismutase [Cu-Zn] (Cu, Zn-SOD) were significantly different among all the groups. According to the order of control, sodium selenite, Se-Met, and Nano-Se, the expression levels of two proteins (E2 and GS) increased and the other differential proteins were reverse. Except for E2, there were no significant differences in other protein expressions between the groups treated with nano-Se and Se-Met.

  17. Oligosaccharide composition of the neurotoxin-responsive sodium channel of mouse neuroblastoma and requirement of sialic acid for biological activity

    NARCIS (Netherlands)

    Vliegenthart, J.F.G.; Negishi, M.; Kuik, J.A. van; Glick, M.C.

    1992-01-01

    A glycoprotein, Mr, 200000, which has the biological activity of the neurotoxin-responsive Na+ channel, was isolated from a clonal line of mouse neuroblastoma cells, N-18. The glycoprotein was purified to homogeneity in 18% yield by methods used to purify glycoproteins, which included metabolic

  18. ASIC3, an acid-sensing ion channel, is expressed in metaboreceptive sensory neurons

    Directory of Open Access Journals (Sweden)

    Fierro Leonardo

    2005-11-01

    Full Text Available Abstract Background ASIC3, the most sensitive of the acid-sensing ion channels, depolarizes certain rat sensory neurons when lactic acid appears in the extracellular medium. Two functions have been proposed for it: 1 ASIC3 might trigger ischemic pain in heart and muscle; 2 it might contribute to some forms of touch mechanosensation. Here, we used immunocytochemistry, retrograde labelling, and electrophysiology to ask whether the distribution of ASIC3 in rat sensory neurons is consistent with either of these hypotheses. Results Less than half (40% of dorsal root ganglion sensory neurons react with anti-ASIC3, and the population is heterogeneous. They vary widely in cell diameter and express different growth factor receptors: 68% express TrkA, the receptor for nerve growth factor, and 25% express TrkC, the NT3 growth factor receptor. Consistent with a role in muscle nociception, small ( Conclusion Our data indicates that: 1 ASIC3 is expressed in a restricted population of nociceptors and probably in some non-nociceptors; 2 co-expression of ASIC3 and CGRP, and the absence of P2X3, are distinguishing properties of a class of sensory neurons, some of which innervate blood vessels. We suggest that these latter afferents may be muscle metaboreceptors, neurons that sense the metabolic state of muscle and can trigger pain when there is insufficient oxygen.

  19. Aberrant Chloride Intracellular Channel 4 Expression Contributes to Endothelial Dysfunction in Pulmonary Arterial Hypertension

    Science.gov (United States)

    Wojciak-Stothard, Beata; Abdul-Salam, Vahitha B.; Lao, Ka Hou; Tsang, Hilda; Irwin, David C.; Lisk, Christina; Loomis, Zoe; Stenmark, Kurt R.; Edwards, John C; Yuspa, Stuart H.; Howard, Luke S.; Edwards, Robert J.; Rhodes, Christopher J.; Gibbs, J Simon R.; Wharton, John; Zhao, Lan; Wilkins, Martin R.

    2014-01-01

    Background Chloride intracellular channel 4 (CLIC4) is highly expressed in the endothelium of remodelled pulmonary vessels and plexiform lesions of patients with pulmonary arterial hypertension (PAH). CLIC4 regulates vasculogenesis through endothelial tube formation. Aberrant CLIC4 expression may contribute to the vascular pathology of PAH. Methods and Results CLIC4 protein expression was increased in plasma and blood-derived endothelial cells from patients with idiopathic PAH (IPAH) and in the pulmonary vascular endothelium of 3 rat models of pulmonary hypertension. CLIC4 gene deletion markedly attenuated the development of chronic hypoxia-induced pulmonary hypertension in mice. Adenoviral overexpression of CLIC4 in cultured human pulmonary artery endothelial cells compromised pulmonary endothelial barrier function and enhanced their survival and angiogenic capacity, while CLIC4 shRNA had an inhibitory effect. Similarly, inhibition of CLIC4 expression in blood-derived endothelial cells from patients with IPAH attenuated the abnormal angiogenic behaviour that characterises these cells. The mechanism of CLIC4 effects involves p65-mediated activation of nuclear factor-κB, followed by stabilisation of hypoxia-inducible factor-1α and increased downstream production of vascular endothelial growth factor and endothelin-1. Conclusions Increased CLIC4 expression is an early manifestation and mediator of endothelial dysfunction in pulmonary hypertension. PMID:24503951

  20. Rotenone induces KATP channel opening in PC12 cells in association with the expression of tyrosine hydroxylase.

    Science.gov (United States)

    Bai, Qunhua; He, Junlin; Qiu, Jingfu; Wang, Yang; Wang, Shibo; Xiu, Yun; Yu, Chao

    2012-10-01

    The activation of ATP-sensitive potassium (KATP) channels in PC12 cells play a pivotal role in protection against the neurotoxic effect of rotenone. However, it remains unclear why rotenone seems to preferentially affect activation of KATP channels and if this could affect its physiological activity. In this study, we sought to determine how the different energy states caused by various doses of rotenone affect the KATP opening state and whether the KATP opening state influences the expression of tyrosine hydroxylase (TH) which is related with DA synthesis. With patch clamp technology, results showed that treatment of PC12 cells with rotenone (0.2-1 µg/ml) for 15 min can cause KATP channel opening with significantly increased intracellular ROS production. Treatment with rotenone (2-16 ng/ml) for 24 h also caused the channels to open with gently increased ROS. In order to study if the rather long-term action on KATP channel opening states could affect the specified function of PC12 cells, the KATP channel opener pinacidil and the inhibito