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Sample records for forms active channels

  1. Piezo proteins are pore-forming subunits of mechanically activated channels.

    Science.gov (United States)

    Coste, Bertrand; Xiao, Bailong; Santos, Jose S; Syeda, Ruhma; Grandl, Jörg; Spencer, Kathryn S; Kim, Sung Eun; Schmidt, Manuela; Mathur, Jayanti; Dubin, Adrienne E; Montal, Mauricio; Patapoutian, Ardem

    2012-02-19

    Mechanotransduction has an important role in physiology. Biological processes including sensing touch and sound waves require as-yet-unidentified cation channels that detect pressure. Mouse Piezo1 (MmPiezo1) and MmPiezo2 (also called Fam38a and Fam38b, respectively) induce mechanically activated cationic currents in cells; however, it is unknown whether Piezo proteins are pore-forming ion channels or modulate ion channels. Here we show that Drosophila melanogaster Piezo (DmPiezo, also called CG8486) also induces mechanically activated currents in cells, but through channels with remarkably distinct pore properties including sensitivity to the pore blocker ruthenium red and single channel conductances. MmPiezo1 assembles as a ∼1.2-million-dalton homo-oligomer, with no evidence of other proteins in this complex. Purified MmPiezo1 reconstituted into asymmetric lipid bilayers and liposomes forms ruthenium-red-sensitive ion channels. These data demonstrate that Piezo proteins are an evolutionarily conserved ion channel family involved in mechanotransduction.

  2. Raised activity of L-type calcium channels renders neurons prone to form paroxysmal depolarization shifts.

    Science.gov (United States)

    Rubi, Lena; Schandl, Ulla; Lagler, Michael; Geier, Petra; Spies, Daniel; Gupta, Kuheli Das; Boehm, Stefan; Kubista, Helmut

    2013-09-01

    Neuronal L-type voltage-gated calcium channels (LTCCs) are involved in several physiological functions, but increased activity of LTCCs has been linked to pathology. Due to the coupling of LTCC-mediated Ca(2+) influx to Ca(2+)-dependent conductances, such as KCa or non-specific cation channels, LTCCs act as important regulators of neuronal excitability. Augmentation of after-hyperpolarizations may be one mechanism that shows how elevated LTCC activity can lead to neurological malfunctions. However, little is known about other impacts on electrical discharge activity. We used pharmacological up-regulation of LTCCs to address this issue on primary rat hippocampal neurons. Potentiation of LTCCs with Bay K8644 enhanced excitatory postsynaptic potentials to various degrees and eventually resulted in paroxysmal depolarization shifts (PDS). Under conditions of disturbed Ca(2+) homeostasis, PDS were evoked frequently upon LTCC potentiation. Exposing the neurons to oxidative stress using hydrogen peroxide also induced LTCC-dependent PDS. Hence, raising LTCC activity had unidirectional effects on brief electrical signals and increased the likeliness of epileptiform events. However, long-lasting seizure-like activity induced by various pharmacological means was affected by Bay K8644 in a bimodal manner, with increases in one group of neurons and decreases in another group. In each group, isradipine exerted the opposite effect. This suggests that therapeutic reduction in LTCC activity may have little beneficial or even adverse effects on long-lasting abnormal discharge activities. However, our data identify enhanced activity of LTCCs as one precipitating cause of PDS. Because evidence is continuously accumulating that PDS represent important elements in neuropathogenesis, LTCCs may provide valuable targets for neuroprophylactic therapy.

  3. Structure-function of proteins interacting with the alpha1 pore-forming subunit of high voltage-activated calcium channel

    Directory of Open Access Journals (Sweden)

    Alan eNeely

    2014-06-01

    Full Text Available Openings of high-voltage-activated calcium channels lead to a transient increase in calcium concentration that in turn activate a plethora of cellular functions, including muscle contraction, secretion and gene transcription. To coordinate all these responses calcium channels form supramolecular assemblies containing effectors and regulatory proteins that couple calcium influx to the downstream signal cascades and to feedback elements. According to the original biochemical characterization of skeletal muscle Dihydropyridine receptors, high-voltage-activated calcium channels are multi-subunit protein complexes consisting of a pore-forming subunit (α1 associated with four additional polypeptide chains β, α2, δ and γ, often referred to as accessory subunits. Twenty-five years after the first purification of a high-voltage calcium channel, the concept of a flexible stoichiometry to expand the repertoire of mechanisms that regulate calcium channel influx has emerged. Several other proteins have been identified that associate directly with the α1-subunit, including calmodulin and multiple members of the small and large GTPase family. Some of these proteins only interact with a subset of α1-subunits and during specific stages of biogenesis. More strikingly, most of the α1-subunit interacting proteins, such as the β-subunit and small GTPases, regulate both gating and trafficking through a variety of mechanisms. Modulation of channel activity covers almost all biophysical properties of the channel. Likewise, regulation of the number of channels in the plasma membrane is performed by altering the release of the α1-subunit from the endoplasmic reticulum, by reducing its degradation or enhancing its recycling back to the cell surface. In this review, we discuss the structural basis, interplay and functional role of selected proteins that interact with the central pore-forming subunit of high-voltage-activated calcium channels.

  4. Channel-forming activity of syringopeptin 25A in mercury-supported phospholipid monolayers and negatively charged bilayers.

    Science.gov (United States)

    Becucci, Lucia; Toppi, Arianna; Fiore, Alberto; Scaloni, Andrea; Guidelli, Rolando

    2016-10-01

    Interactions of the cationic lipodepsipeptide syringopeptin 25A (SP25A) with mercury-supported dioleoylphosphatidylcholine (DOPC), dioleoylphosphatidylserine (DOPS) and dioeleoylphosphatidic acid (DOPA) self-assembled monolayers (SAMs) were investigated by AC voltammetry in 0.1M KCl at pH3, 5.4 and 6.8. SP25A targets and penetrates the DOPS SAM much more effectively than the other SAMs not only at pH6.8, where the DOPS SAM is negatively charged, but also at pH3, where it is positively charged just as SP25A. Similar investigations at tethered bilayer lipid membranes (tBLMs) consisting of a thiolipid called DPTL anchored to mercury, with a DOPS, DOPA or DOPC distal monolayer on top of it, showed that, at physiological transmembrane potentials, SP25A forms ion channels spanning the tBLM only if DOPS is the distal monolayer. The distinguishing chemical feature of the DOPS SAM is the ionic interaction between the protonated amino group of a DOPS molecule and the carboxylate group of an adjacent phospholipid molecule. Under the reasonable assumption that SP25A preferentially interacts with this ion pair, the selective lipodepsipeptide antimicrobial activity against Gram-positive bacteria may be tentatively explained by its affinity for similar protonated amino-carboxylate pairs, which are expected to be present in the peptide moieties of peptidoglycan strands.

  5. Structure-function of proteins interacting with the α1 pore-forming subunit of high-voltage-activated calcium channels

    Science.gov (United States)

    Neely, Alan; Hidalgo, Patricia

    2014-01-01

    Openings of high-voltage-activated (HVA) calcium channels lead to a transient increase in calcium concentration that in turn activate a plethora of cellular functions, including muscle contraction, secretion and gene transcription. To coordinate all these responses calcium channels form supramolecular assemblies containing effectors and regulatory proteins that couple calcium influx to the downstream signal cascades and to feedback elements. According to the original biochemical characterization of skeletal muscle Dihydropyridine receptors, HVA calcium channels are multi-subunit protein complexes consisting of a pore-forming subunit (α1) associated with four additional polypeptide chains β, α2, δ, and γ, often referred to as accessory subunits. Twenty-five years after the first purification of a high-voltage calcium channel, the concept of a flexible stoichiometry to expand the repertoire of mechanisms that regulate calcium channel influx has emerged. Several other proteins have been identified that associate directly with the α1-subunit, including calmodulin and multiple members of the small and large GTPase family. Some of these proteins only interact with a subset of α1-subunits and during specific stages of biogenesis. More strikingly, most of the α1-subunit interacting proteins, such as the β-subunit and small GTPases, regulate both gating and trafficking through a variety of mechanisms. Modulation of channel activity covers almost all biophysical properties of the channel. Likewise, regulation of the number of channels in the plasma membrane is performed by altering the release of the α1-subunit from the endoplasmic reticulum, by reducing its degradation or enhancing its recycling back to the cell surface. In this review, we discuss the structural basis, interplay and functional role of selected proteins that interact with the central pore-forming subunit of HVA calcium channels. PMID:24917826

  6. Tanshinone II-A sodium sulfonate (DS-201) enhances human BKCa channel activity by selectively targeting the pore-forming α subunit.

    Science.gov (United States)

    Tan, Xiao-qiu; Cheng, Xiu-li; Yang, Yan; Yan, Li; Gu, Jing-li; Li, Hui; Zeng, Xiao-rong; Cao, Ji-min

    2014-11-01

    Tanshinone II-A sodium sulfonate (DS-201), a water-soluble derivative of Tanshinone II-A, has been found to induce vascular relaxation and activate BKCa channels. The aim of this study was to explore the mechanisms underlying the action of DS-201 on BKCa channels. Human BKCa channels containing α subunit alone or α plus β1 subunits were expressed in HEK293 cells. BKCa currents were recorded from the cells using patch-clamp technique. The expression and trafficking of BKCa subunits in HEK293 cells or vascular smooth muscle cells (VSMCs) were detected by Western blotting, flow cytometry and confocal microscopy. DS-201 (40-160 μmol/L) concentration-dependently increased the total open probability of BKCa channels in HEK293 cells, associated with enhancements of Ca(2+) and voltage dependence as well as a delay in deactivation. Coexpression of β1 subunit did not affect the action of DS-201: the values of EC50 for BKCa channels containing α subunit alone and α plus β1 subunit were 66.6±1.5 and 62.0±1.1 μmol/L, respectively. In both HEK293 cells and VSMCs, DS-201 (80 μmol/L) markedly increased the expression of α subunit without affecting β1 subunit. In HEK293 cells, DS-201 enriched the membranous level of α subunit, likely by accelerating the trafficking and suppressing the internalization of α subunit. In both HEK293 cells and VSMCs, DS-201 (≥320 μmol/L) induced significant cytotoxicity. DS-201 selectively targets the pore-forming α subunit of human BKCa channels, thus enhancing the channel activities and increasing the subunit expression and trafficking, whereas the β1 subunit does not contribute to the action of DS-201.

  7. Drosophila TRPML forms PI(3,5)P2-activated cation channels in both endolysosomes and plasma membrane.

    Science.gov (United States)

    Feng, Xinghua; Huang, Yu; Lu, Yungang; Xiong, Jian; Wong, Ching-On; Yang, Pu; Xia, Jintang; Chen, De; Du, Guangwei; Venkatachalam, Kartik; Xia, Xuefeng; Zhu, Michael X

    2014-02-14

    Transient Receptor Potential mucolipin (TRPML) channels are implicated in endolysosomal trafficking, lysosomal Ca(2+) and Fe(2+) release, lysosomal biogenesis, and autophagy. Mutations in human TRPML1 cause the lysosome storage disease, mucolipidosis type IV (MLIV). Unlike vertebrates, which express three TRPML genes, TRPML1-3, the Drosophila genome encodes a single trpml gene. Although the trpml-deficient flies exhibit cellular defects similar to those in mammalian TRPML1 mutants, the biophysical properties of Drosophila TRPML channel remained uncharacterized. Here, we show that transgenic expression of human TRPML1 in the neurons of Drosophila trpml mutants partially suppressed the pupal lethality phenotype. When expressed in HEK293 cells, Drosophila TRPML was localized in both endolysosomes and plasma membrane and was activated by phosphatidylinositol 3,5-bisphosphate (PI(3,5)P2) applied to the cytoplasmic side in whole lysosomes and inside-out patches excised from plasma membrane. The PI(3,5)P2-evoked currents were blocked by phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2), but not other phosphoinositides. Using TRPML A487P, which mimics the varitint-waddler (Va) mutant of mouse TRPML3 with constitutive whole-cell currents, we show that TRPML is biphasically regulated by extracytosolic pH, with an optimal pH about 0.6 pH unit higher than that of human TRPML1. In addition to monovalent cations, TRPML exhibits high permeability to Ca(2+), Mn(2+), and Fe(2+), but not Fe(3+). The TRPML currents were inhibited by trivalent cations Fe(3+), La(3+), and Gd(3+). These features resemble more closely to mammalian TRPML1 than TRPML2 and TRPML3, but with some obvious differences. Together, our data support the use of Drosophila for assessing functional significance of TRPML1 in cell physiology.

  8. Controls on plan-form evolution of submarine channels

    Science.gov (United States)

    Imran, J.; Mohrig, D. C.

    2014-12-01

    Vertically aggrading sinuous channels constitute a basic building block of modern submarine fans and the greater continental slope. Interpretation of seismically imaged channels reveals a significant diversity in internal architecture, as well as important similarities and differences in the evolution of submarine channels relative to better studied rivers. Many submarine channel cross sections possess a 'gull wing' shape. Successive stacking of such channels demonstrates that systematic bank erosion is not required in order for lateral migration to occur. The lateral shift of such aggrading channels, however, is expected to be much less dynamic than in the case of terrestrial rivers. Recent high-resolution 3D seismic data from offshore Angola and an upstream segment of the Bengal Submarine Fan show intensely meandering channels that experience considerable lateral shifting during periods of active migration within submarine valleys. The cross sections of the actively migrating channels are similar to meandering river channels characterized by an outer cut-bank and inner-bank accretion. In submarine channels, the orientation of the secondary flow can be river-like or river-reverse depending on the channel gradient, cross sectional shape, and the adaptation length of the channel bend. In river channels, a single circulation cell commonly occupies the entire channel relief, redistributing the bed-load sediment across the channel, and influencing the thread of high velocity and thus the plan-form evolution of the channel. In submarine environments, the height of the circulation cell will be significantly smaller than channel relief, thus leading to development of lower relief point bars from bed-load transport. Nevertheless these "underfit" bars may play an important role in plan-form evolution of submarine channels. In rivers and submarine channels, the inclined surface accretion can be constructed via pure bed-load, suspended-load, or a combination of both transport

  9. Orai1 mediates the interaction between STIM1 and hTRPC1 and regulates the mode of activation of hTRPC1-forming Ca2+ channels.

    Science.gov (United States)

    Jardin, Isaac; Lopez, José J; Salido, Gines M; Rosado, Juan A

    2008-09-12

    Orai1 and hTRPC1 have been presented as essential components of store-operated channels mediating highly Ca(2+) selective I(CRAC) and relatively Ca(2+) selective I(SOC), respectively. STIM1 has been proposed to communicate the Ca(2+) content of the intracellular Ca(2+) stores to the plasma membrane store-operated Ca(2+) channels. Here we present evidence for the dynamic interaction between endogenously expressed Orai1 and both STIM1 and hTRPC1 regulated by depletion of the intracellular Ca(2+) stores, using the pharmacological tools thapsigargin plus ionomycin, or by the physiological agonist thrombin, independently of extracellular Ca(2+). In addition we report that Orai1 mediates the communication between STIM1 and hTRPC1, which is essential for the mode of activation of hTRPC1-forming Ca(2+) permeable channels. Electrotransjection of cells with anti-Orai1 antibody, directed toward the C-terminal region that mediates the interaction with STIM1, and stabilization of an actin cortical barrier with jasplakinolide prevented the interaction between STIM1 and hTRPC1. Under these conditions hTRPC1 was no longer involved in store-operated calcium entry but in diacylglycerol-activated non-capacitative Ca(2+) entry. These findings support the functional role of the STIM1-Orai1-hTRPC1 complex in the activation of store-operated Ca(2+) entry.

  10. THE FORMING CONDITIONS OF ALLUVIAL RIVER CHANNEL PATTERNS

    Institute of Scientific and Technical Information of China (English)

    Pu QI; Gouting LIANG; Zangying SUN; Honghai QI

    2002-01-01

    In normal fluvial processes the river channel is determined by river flows while the movement of river flows is contained by river channels. The relationship between the river morphology and its bend curvature shows that rivers with large bend curvatures always have narrow and deep channels and those with shallow and wide channels are always straight. The plan form of a river reaches is determined by the cross-sectional morphology. A meandering river reach may be developed under various water-sediment conditions as long as the narrow and deep channels are formed.

  11. Lubiprostone: a chloride channel activator.

    Science.gov (United States)

    Lacy, Brian E; Levy, L Campbell

    2007-04-01

    In January 2006 the Food and Drug Administration approved lubiprostone for the treatment of chronic constipation in men and women aged 18 and over. Lubiprostone is categorized as a prostone, a bicyclic fatty acid metabolite of prostaglandin E1. Lubiprostone activates a specific chloride channel (ClC-2) in the gastrointestinal (GI) tract to enhance intestinal fluid secretion, which increases GI transit and improves symptoms of constipation. This article reviews the role of chloride channels in the GI tract, describes the structure, function, and pharmacokinetics of lubiprostone, and discusses clinically important data on this new medication.

  12. Ion channel stability and hydrogen bonding. Molecular modelling of channels formed by synthetic alamethicin analogues.

    Science.gov (United States)

    Breed, J; Kerr, I D; Molle, G; Duclohier, H; Sansom, M S

    1997-12-04

    Several analogues of the channel-forming peptaibol alamethicin have been demonstrated to exhibit faster switching between channel substates than does unmodified alamethicin. Molecular modelling studies are used to explore the possible molecular basis of these differences. Models of channels formed by alamethicin analogues were generated by restrained molecular dynamics in vacuo and refined by short molecular dynamics simulations with water molecules within and at either mouth of the channel. A decrease in backbone solvation was found to correlate with a decrease in open channel stability between alamethicin and an analogue in which all alpha-amino-isobutyric acid residues of alamethicin were replaced by leucine. A decrease in the extent of hydrogen-bonding at residue 7 correlates with lower open channel stabilities of analogues in which the glutamine at position 7 was replaced by smaller polar sidechains. These two observations indicate the importance of alamethicin/water H-bonds in stabilizing the open channel.

  13. Pre-formed plasma channels for ion beam fusion

    Science.gov (United States)

    Peterson, R. R.; Olson, C. L.

    1997-04-01

    The transport of driver ions to the target in an IFE power plant is an important consideration in IFE target chamber design. Pre-formed laser-guided plasma discharge channels have been considered for light ions because they reduce the beam microdivergence constraints, allow long transport lengths, and require a target chamber fill gas that can help protect the target chamber from the target explosion. Here, pre-formed plasma discharge channels are considered for heavy ion transport. The channel formation parameters are similar to those for light ions. The allowable ion power per channel is limited by the onset of plasma instabilities and energy loss due to a reverse emf from the rapid channel expansion driven by the ion beam.

  14. Activation of ATP-sensitive potassium channels facilitates the function of human endothelial colony-forming cells via Ca(2+) /Akt/eNOS pathway.

    Science.gov (United States)

    Wu, Yan; He, Meng-Yu; Ye, Jian-Kui; Ma, Shu-Ying; Huang, Wen; Wei, Yong-Yue; Kong, Hui; Wang, Hong; Zeng, Xiao-Ning; Xie, Wei-Ping

    2017-03-01

    Accumulating data, including those from our laboratory, have shown that the opening of ATP-sensitive potassium channels (KATP ) plays a protective role in pulmonary vascular diseases (PVD). As maintainers of the endothelial framework, endothelial colony-forming cells (ECFCs) are considered excellent candidates for vascular regeneration in cases of PVD. Although KATP openers (KCOs) have been demonstrated to have beneficial effects on endothelial cells, the impact of KATP on ECFC function remains unclear. Herein, this study investigated whether there is a distribution of KATP in ECFCs and what role KATP play in ECFC modulation. By human ECFCs isolated from adult peripheral blood, KATP were confirmed for the first time to express in ECFCs, comprised subunits of Kir (Kir6.1, Kir6.2) and SUR2b. KCOs such as the classical agent nicorandil (Nico) and the novel agent iptakalim (Ipt) notably improved the function of ECFCs, promoting cell proliferation, migration and angiogenesis, which were abolished by a non-selective KATP blocker glibenclamide (Gli). To determine the underlying mechanisms, we investigated the impacts of KCOs on CaMKII, Akt and endothelial nitric oxide synthase pathways. Enhanced levels were detected by western blotting, which were abrogated by Gli. This suggested an involvement of Ca(2+) signalling in the regulation of ECFCs by KATP . Our findings demonstrated for the first time that there is a distribution of KATP in ECFCs and KATP play a vital role in ECFC function. The present work highlighted a novel profile of KATP as a potential target for ECFC modulation, which may hold the key to the treatment of PVD. © 2016 The Authors. Journal of Cellular and Molecular Medicine published by John Wiley & Sons Ltd and Foundation for Cellular and Molecular Medicine.

  15. Synthetic Channel-forming Peptides and Ion Selectivity

    Institute of Scientific and Technical Information of China (English)

    2006-01-01

    @@ Introduction Peptides made up of alternating L- and D- amino acids can form β-helices as in gramicidin A or cyclic peptides that aggregate to form tubes[1]. In both cases the structures are hollow with all the side chains projecting outwards. Kennedy et al. [2] postulated that peptides having the (LLLD)n configuration could form helices with every fourth side chain projecting inward.It is a fact that synthetic N-formyl-( LeuSerLeuGly)6-OH, when added to a lipid bilayer, dimerizes, to form ion channels having conductances greater than that of gramicidin.

  16. The SecY complex forms a channel capable of ionic discrimination.

    Science.gov (United States)

    Dalal, Kush; Duong, Franck

    2009-07-01

    Protein translocation across the bacterial membrane occurs at the SecY complex or channel. The resting SecY channel is impermeable to small molecules owing to a plug domain that creates a seal. Here, we report that a channel loosely sealed, or with a plug locked open, does not, however, lead to general membrane permeability. Instead, strong selectivity towards small monovalent anions, especially chloride, is observed. Mutations in the pore ring-structure increase both the translocation activity of the channel and its ionic conductance, however the selectivity is maintained. The same ionic specificity also occurs at the onset of protein translocation and across the archaeal SecY complex. Thus, the ion-conducting characteristic of the channel seems to be conserved as a normal consequence of protein translocation. We propose that the pore ring-structure forms a selectivity filter, allowing cells to tolerate channels with imperfect plugs.

  17. Experimental Study on CFRP Strengthened Cold Formed Channel Columns

    Directory of Open Access Journals (Sweden)

    Sreedhar Kalavagunta

    2013-09-01

    Full Text Available Cold-formed steel members usually display local-global buckling interaction which strongly effects the structural strength of columns. Through strengthening web of the members this buckling can be controlled to some extent. In this investigation, Carbon Fibre Reinforced Polymers (CFRP is used for strengthening cold formed steel channel member. This paper presents compression tests of cold-formed plain and CFRP strengthened steel channel section columns. This paper also proposes a design method based on Direct Strength Method provisions specified in American Iron and Steel Institute (AISI, for determining the axial compression strength. Results obtained from the proposed design method are compared with experimental test data and are found to be in good agreement.

  18. Three homologous subunits form a high affinity peptide-gated ion channel in Hydra

    DEFF Research Database (Denmark)

    Dürrnagel, Stefan; Kuhn, Anne; Tsiairis, Charisios D

    2010-01-01

    properties, like a low Na(+) selectivity and a low amiloride affinity, that are different from other channels of the DEG/ENaC gene family, suggesting that a component of the native Hydra channel might still be lacking. Here, we report the cloning of a new ion channel subunit from Hydra, HyNaC5. The new......Recently, three ion channel subunits of the degenerin (DEG)/epithelial Na(+) channel (ENaC) gene family have been cloned from the freshwater polyp Hydra magnipapillata, the Hydra Na(+) channels (HyNaCs) 2-4. Two of them, HyNaC2 and HyNaC3, co-assemble to form an ion channel that is gated...... by the neuropeptides Hydra-RFamides I and II. The HyNaC2/3 channel is so far the only cloned ionotropic receptor from cnidarians and, together with the related ionotropic receptor FMRFamide-activated Na(+) channel (FaNaC) from snails, the only known peptide-gated ionotropic receptor. The HyNaC2/3 channel has pore...

  19. Multivalent Inhibitors of Channel-Forming Bacterial Toxins.

    Science.gov (United States)

    Yamini, Goli; Nestorovich, Ekaterina M

    2016-07-29

    Rational design of multivalent molecules represents a remarkable modern tool to transform weak non-covalent interactions into strong binding by creating multiple finely-tuned points of contact between multivalent ligands and their supposed multivalent targets. Here, we describe several prominent examples where the multivalent blockers were investigated for their ability to directly obstruct oligomeric channel-forming bacterial exotoxins, such as the pore-forming bacterial toxins and B component of the binary bacterial toxins. We address problems related to the blocker/target symmetry match and nature of the functional groups, as well as chemistry and length of the linkers connecting the functional groups to their multivalent scaffolds. Using the anthrax toxin and AB5 toxin case studies, we briefly review how the oligomeric toxin components can be successfully disabled by the multivalent non-channel-blocking inhibitors, which are based on a variety of multivalent scaffolds.

  20. Channel-Forming Bacterial Toxins in Biosensing and Macromolecule Delivery

    Directory of Open Access Journals (Sweden)

    Philip A. Gurnev

    2014-08-01

    Full Text Available To intoxicate cells, pore-forming bacterial toxins are evolved to allow for the transmembrane traffic of different substrates, ranging from small inorganic ions to cell-specific polypeptides. Recent developments in single-channel electrical recordings, X-ray crystallography, protein engineering, and computational methods have generated a large body of knowledge about the basic principles of channel-mediated molecular transport. These discoveries provide a robust framework for expansion of the described principles and methods toward use of biological nanopores in the growing field of nanobiotechnology. This article, written for a special volume on “Intracellular Traffic and Transport of Bacterial Protein Toxins”, reviews the current state of applications of pore-forming bacterial toxins in small- and macromolecule-sensing, targeted cancer therapy, and drug delivery. We discuss the electrophysiological studies that explore molecular details of channel-facilitated protein and polymer transport across cellular membranes using both natural and foreign substrates. The review focuses on the structurally and functionally different bacterial toxins: gramicidin A of Bacillus brevis, α-hemolysin of Staphylococcus aureus, and binary toxin of Bacillus anthracis, which have found their “second life” in a variety of developing medical and technological applications.

  1. Channel-forming bacterial toxins in biosensing and macromolecule delivery.

    Science.gov (United States)

    Gurnev, Philip A; Nestorovich, Ekaterina M

    2014-08-21

    To intoxicate cells, pore-forming bacterial toxins are evolved to allow for the transmembrane traffic of different substrates, ranging from small inorganic ions to cell-specific polypeptides. Recent developments in single-channel electrical recordings, X-ray crystallography, protein engineering, and computational methods have generated a large body of knowledge about the basic principles of channel-mediated molecular transport. These discoveries provide a robust framework for expansion of the described principles and methods toward use of biological nanopores in the growing field of nanobiotechnology. This article, written for a special volume on "Intracellular Traffic and Transport of Bacterial Protein Toxins", reviews the current state of applications of pore-forming bacterial toxins in small- and macromolecule-sensing, targeted cancer therapy, and drug delivery. We discuss the electrophysiological studies that explore molecular details of channel-facilitated protein and polymer transport across cellular membranes using both natural and foreign substrates. The review focuses on the structurally and functionally different bacterial toxins: gramicidin A of Bacillus brevis, α-hemolysin of Staphylococcus aureus, and binary toxin of Bacillus anthracis, which have found their "second life" in a variety of developing medical and technological applications.

  2. BK channel activators and their therapeutic perspectives

    DEFF Research Database (Denmark)

    Bentzen, Bo Hjorth; Olesen, Søren-Peter; Rønn, Lars C B;

    2014-01-01

    The large conductance calcium- and voltage-activated K(+) channel (KCa1.1, BK, MaxiK) is ubiquitously expressed in the body, and holds the ability to integrate changes in intracellular calcium and membrane potential. This makes the BK channel an important negative feedback system linking increases...

  3. Channel properties of the splicing isoforms of the olfactory calcium-activated chloride channel Anoctamin 2.

    Science.gov (United States)

    Ponissery Saidu, Samsudeen; Stephan, Aaron B; Talaga, Anna K; Zhao, Haiqing; Reisert, Johannes

    2013-06-01

    Anoctamin (ANO)2 (or TMEM16B) forms a cell membrane Ca(2+)-activated Cl(-) channel that is present in cilia of olfactory receptor neurons, vomeronasal microvilli, and photoreceptor synaptic terminals. Alternative splicing of Ano2 transcripts generates multiple variants with the olfactory variants skipping exon 14 and having alternative splicing of exon 4. In the present study, 5' rapid amplification of cDNA ends analysis was conducted to characterize the 5' end of olfactory Ano2 transcripts, which showed that the most abundant Ano2 transcripts in the olfactory epithelium contain a novel starting exon that encodes a translation initiation site, whereas transcripts of the publically available sequence variant, which has an alternative and longer 5' end, were present in lower abundance. With two alternative starting exons and alternative splicing of exon 4, four olfactory ANO2 isoforms are thus possible. Patch-clamp experiments in transfected HEK293T cells expressing these isoforms showed that N-terminal sequences affect Ca(2+) sensitivity and that the exon 4-encoded sequence is required to form functional channels. Coexpression of the two predominant isoforms, one with and one without the exon 4 sequence, as well as coexpression of the two rarer isoforms showed alterations in channel properties, indicating that different isoforms interact with each other. Furthermore, channel properties observed from the coexpression of the predominant isoforms better recapitulated the native channel properties, suggesting that the native channel may be composed of two or more splicing isoforms acting as subunits that together shape the channel properties.

  4. Heart failure drug digitoxin induces calcium uptake into cells by forming transmembrane calcium channels.

    Science.gov (United States)

    Arispe, Nelson; Diaz, Juan Carlos; Simakova, Olga; Pollard, Harvey B

    2008-02-19

    Digitoxin and other cardiac glycosides are important, centuries-old drugs for treating congestive heart failure. However, the mechanism of action of these compounds is still being elucidated. Calcium is known to potentiate the toxicity of these drugs, and we have hypothesized that digitoxin might mediate calcium entry into cells. We report here that digitoxin molecules mediate calcium entry into intact cells. Multimers of digitoxin molecules also are able to form calcium channels in pure planar phospholipid bilayers. These digitoxin channels are blocked by Al(3+) and La(3+) but not by Mg(2+) or the classical l-type calcium channel blocker, nitrendipine. In bilayers, we find that the chemistry of the lipid affects the kinetics of the digitoxin channel activity, but not the cation selectivity. Antibodies against digitoxin promptly neutralize digitoxin channels in both cells and bilayers. We propose that these digitoxin calcium channels may be part of the mechanism by which digitoxin and other active cardiac glycosides, such as digoxin, exert system-wide actions at and above the therapeutic concentration range.

  5. Activation of TRPV1 channels inhibits mechanosensitive Piezo channel activity by depleting membrane phosphoinositides.

    Science.gov (United States)

    Borbiro, Istvan; Badheka, Doreen; Rohacs, Tibor

    2015-02-10

    Capsaicin is an activator of the heat-sensitive TRPV1 (transient receptor potential vanilloid 1) ion channels and has been used as a local analgesic. We found that activation of TRPV1 channels with capsaicin either in dorsal root ganglion neurons or in a heterologous expression system inhibited the mechanosensitive Piezo1 and Piezo2 channels by depleting phosphatidylinositol 4,5-bisphosphate [PI(4,5)P2] and its precursor phosphatidylinositol 4-phosphate [PI(4)P] from the plasma membrane through Ca(2+)-induced phospholipase Cδ (PLCδ) activation. Experiments with chemically inducible phosphoinositide phosphatases and receptor-induced activation of PLCβ indicated that inhibition of Piezo channels required depletion of both PI(4)P and PI(4,5)P2. The mechanically activated current amplitudes decreased substantially in the excised inside-out configuration, where the membrane patch containing Piezo1 channels is removed from the cell. PI(4,5)P2 and PI(4)P applied to these excised patches inhibited this decrease. Thus, we concluded that Piezo channel activity requires the presence of phosphoinositides, and the combined depletion of PI(4,5)P2 and PI(4)P reduces channel activity. In addition to revealing a role for distinct membrane lipids in mechanosensitive ion channel regulation, these data suggest that inhibition of Piezo2 channels may contribute to the analgesic effect of capsaicin.

  6. Channel protein engineering: Synthetic 22-mer peptide from the primary structure of the voltage-sensitive sodium channel forms ionic channels in lipid bilayers

    OpenAIRE

    1988-01-01

    A synthetic 22-mer peptide that mimics the sequence of a putative pore segment of the voltage-dependent sodium channel forms transmembrane ionic channels in lipid bilayers. Several features of the authentic sodium channel are exhibited by the synthetic peptide: (i) The single channel conductance of the most frequent event is 20 pS in 0.5 M NaCl. (ii) The single channel open and closed lifetimes are in the ms time range. (iii) The synthetic channel discriminates cations over anions but is nons...

  7. [Potential-dependent Cation Selective Ion Channels Formed by Peroxiredoxin 6 in the Lipid Bilayer].

    Science.gov (United States)

    Grigoriev, P A; Sharapov, M G; Novoselov, V I

    2015-01-01

    The antioxidant enzyme peroxiredoxin 6 forms cation selective ion cluster-type channels in the lipid bilayer. Channel clustering as oligomeric structure consists of three or more subunits--channels with conductance of about 350 pS in the 200 mM KCl. Mean dwell time of the channel's open states decreases with increasing membrane voltage. A possible molecular mechanism of the observed potential-dependent inactivation of the channel cluster is discussed.

  8. The unusual stoichiometry of ADP activation of the KATP channel

    Directory of Open Access Journals (Sweden)

    Eric eHosy

    2014-01-01

    Full Text Available KATP channels, oligomers of 4 pore-forming Kir6.2 proteins and 4 sulfonylurea receptors (SUR, sense metabolism by monitoring both cytosolic ATP, which closes the channel by interacting with Kir6.2, and ADP, which opens it via SUR. SUR mutations that alter activation by ADP are a major cause of KATP channelopathies. We examined the mechanism of ADP activation by analysis of single-channel and macropatch recordings from Xenopus oocytes expressing various mixtures of wild-type SUR2A and an ADP-activation-defective mutant. Evaluation of the data by a binomial distribution model suggests that wild-type and mutant SURs freely co-assemble and that channel activation results from interaction of ADP with only 2 of 4 SURs. This finding explains the heterozygous nature of most KATP channelopathies linked to mutations altering ADP activation. It also suggests that the channel deviates from circular symmetry and could function as a dimer-of-dimers.

  9. Turbine component having surface cooling channels and method of forming same

    Energy Technology Data Exchange (ETDEWEB)

    Miranda, Carlos Miguel; Trimmer, Andrew Lee; Kottilingam, Srikanth Chandrudu

    2017-09-05

    A component for a turbine engine includes a substrate that includes a first surface, and an insert coupled to the substrate proximate the substrate first surface. The component also includes a channel. The channel is defined by a first channel wall formed in the substrate and a second channel wall formed by at least one coating disposed on the substrate first surface. The component further includes an inlet opening defined in flow communication with the channel. The inlet opening is defined by a first inlet wall formed in the substrate and a second inlet wall defined by the insert.

  10. How voltage-gated calcium channels gate forms of homeostatic synaptic plasticity

    Directory of Open Access Journals (Sweden)

    C. Andrew eFrank

    2014-02-01

    Full Text Available Throughout life, animals face a variety of challenges such as developmental growth, the presence of toxins, or changes in temperature. Neuronal circuits and synapses respond to challenges by executing an array of neuroplasticity paradigms. Some paradigms allow neurons to up- or downregulate activity outputs, while countervailing ones ensure that outputs remain within appropriate physiological ranges. A growing body of evidence suggests that homeostatic synaptic plasticity (HSP is critical in the latter case. Voltage-gated calcium channels gate forms of HSP. Presynaptically, the aggregate data show that when synapse activity is weakened, homeostatic signaling systems can act to correct impairments, in part by increasing calcium influx through presynaptic CaV2-type channels. Increased calcium influx is often accompanied by parallel increases in the size of active zones and the size of the readily releasable pool of presynaptic vesicles. These changes coincide with homeostatic enhancements of neurotransmitter release. Postsynaptically, there is a great deal of evidence that reduced network activity and loss of calcium influx through CaV1-type calcium channels also results in adaptive homeostatic signaling. Some adaptations drive presynaptic enhancements of vesicle pool size and turnover rate via retrograde signaling, as well as de novo insertion of postsynaptic neurotransmitter receptors. Enhanced calcium influx through CaV1 after network activation or single cell stimulation can elicit the opposite response – homeostatic depression via removal of excitatory receptors.There exist intriguing links between HSP and calcium channelopathies – such as forms of epilepsy, migraine, ataxia, and myasthenia. The episodic nature of some of these disorders suggests alternating periods of stable and unstable function. Uncovering information about how calcium channels are regulated in the context of HSP could be relevant toward understanding these and other

  11. Did the martian outflow channels mostly form during the Amazonian Period?

    Science.gov (United States)

    Rodriguez, J. Alexis P.; Platz, Thomas; Gulick, Virginia; Baker, Victor R.; Fairén, Alberto G.; Kargel, Jeffrey; Yan, Jianguo; Miyamoto, Hideaki; Glines, Natalie

    2015-09-01

    Simud, Tiu, and Ares Valles comprise some of the largest outflow channels on Mars. Their excavation has been attributed variously to (or a combination of) erosion by catastrophic floods, glaciers, and debris flows. Numerous investigations indicate that they formed largely during the Late Hesperian (3.61-3.37 Ga). However, these studies mostly equate the ages of the outflow channel floors to those of the flows that generated mesoscale (several hundred meters to a few kilometers) bedforms within them. To improve the statistical accuracy in the age determinations of these flow events, we have used recently acquired high-resolution image and topographic data to map and date portions of Simud, Tiu and Ares Valles, which are extensively marked by these bedforms. Our results, which remove the statistical effects of older and younger outflow channel floor surfaces on the generation of modeled ages, reveal evidence for major outflow channel discharges occurring during the Early (3.37-1.23 Ga) and Middle (1.23-0.328 Ga) Amazonian, with activity significantly peaking during the Middle Amazonian stages. We also find that during the documented stages of Middle Amazonian discharges, the floor of Tiu Valles underwent widespread collapse, resulting in chaotic terrain formation. In addition, we present evidence showing that following the outflow channel discharges, collapse within northern Simud Valles generated another chaotic terrain. This younger chaos region likely represents the latest stage of large-scale outflow channel resurfacing within the study area. Our findings imply that in southern circum-Chryse the martian hydrosphere experienced large-scale drainage during the Amazonian, which likely led to periodic inundation and sedimentation within the northern plains.

  12. Circumnuclear Star Forming Activity in NGC 3982

    Institute of Scientific and Technical Information of China (English)

    Shui-Nai Zhang; Qiu-Sheng Gu; Yi-Peng Wang

    2008-01-01

    We present a study of the nearby Seyfert galaxy NGC 3982 using optical,infrared and X-ray data acquired by SDSS,Spitzer and Chandra.Our main results are as follows:(1) A simple stellar population synthesis on the nuclear and circumnuclear SDSS spectra gives unambiguous evidence of young stellar components in both the nuclear and circumnuclear regions.(2) The Spitzer Infrared Spectrograph (IRS) spectrum of the central region (~3") shows a power-law continuum,a silicate emission feature at 9.7 μm,and significant PAH emission features at 7.7,8.6,11.3 and 12.7/zm,suggesting the coexistence of AGN and starburst activities in the central region of NGC 3982.(3) We estimate the star formation rate (SFR) of the circumnuclear (~5"-20") region from the Ha luminosity to be for the active nucleus of NGC 3982 from radio to X-ray,and obtain a bolometric luminosity of Lbol=4.5×1042 erg s-1,corresponding to an Eddington ratio (Lbol/LEdd) of 0.014.The HST image of NGC 3982 shows a nuclear mini-spiral between the circumnuclear starforming region and the nucleus,which could be the channel through which gas is transported to the supermassive black hole from the circumnuclear star-forming region.

  13. Epithelial sodium channel modulates platelet collagen activation.

    Science.gov (United States)

    Cerecedo, Doris; Martínez-Vieyra, Ivette; Alonso-Rangel, Lea; Benítez-Cardoza, Claudia; Ortega, Arturo

    2014-03-01

    Activated platelets adhere to the exposed subendothelial extracellular matrix and undergo a rapid cytoskeletal rearrangement resulting in shape change and release of their intracellular dense and alpha granule contents to avoid hemorrhage. A central step in this process is the elevation of the intracellular Ca(2+) concentration through its release from intracellular stores and on throughout its influx from the extracellular space. The Epithelial sodium channel (ENaC) is a highly selective Na(+) channel involved in mechanosensation, nociception, fluid volume homeostasis, and control of arterial blood pressure. The present study describes the expression, distribution, and participation of ENaC in platelet migration and granule secretion using pharmacological inhibition with amiloride. Our biochemical and confocal analysis in suspended and adhered platelets suggests that ENaC is associated with Intermediate filaments (IF) and with Dystrophin-associated proteins (DAP) via α-syntrophin and β-dystroglycan. Migration assays, quantification of soluble P-selectin, and serotonin release suggest that ENaC is dispensable for migration and alpha and dense granule secretion, whereas Na(+) influx through this channel is fundamental for platelet collagen activation.

  14. Impact of Volcanic Activity on AMC Channel Operations

    Science.gov (United States)

    2014-06-13

    IMPACT OF VOLCANIC ACTIVITY ON AMC CHANNEL OPERATIONS GRADUATE RESEARCH PROJECT Matthew D... VOLCANIC ACTIVITY ON AMC CHANNEL OPERATIONS GRADUATE RESEARCH PROJECT Presented to the Faculty Department of Operational Sciences...AFIT-ENS-GRP-14-J-11 IMPACT OF VOLCANIC ACTIVITY ON AMC CHANNEL OPERATIONS Matthew D. Meshanko, BS, MA Major, USAF

  15. The Blurred Line between Form and Process: A Comparison of Stream Channel Classification Frameworks.

    Science.gov (United States)

    Kasprak, Alan; Hough-Snee, Nate; Beechie, Tim; Bouwes, Nicolaas; Brierley, Gary; Camp, Reid; Fryirs, Kirstie; Imaki, Hiroo; Jensen, Martha; O'Brien, Gary; Rosgen, David; Wheaton, Joseph

    2016-01-01

    Stream classification provides a means to understand the diversity and distribution of channels and floodplains that occur across a landscape while identifying links between geomorphic form and process. Accordingly, stream classification is frequently employed as a watershed planning, management, and restoration tool. At the same time, there has been intense debate and criticism of particular frameworks, on the grounds that these frameworks classify stream reaches based largely on their physical form, rather than direct measurements of their component hydrogeomorphic processes. Despite this debate surrounding stream classifications, and their ongoing use in watershed management, direct comparisons of channel classification frameworks are rare. Here we implement four stream classification frameworks and explore the degree to which each make inferences about hydrogeomorphic process from channel form within the Middle Fork John Day Basin, a watershed of high conservation interest within the Columbia River Basin, U.S.A. We compare the results of the River Styles Framework, Natural Channel Classification, Rosgen Classification System, and a channel form-based statistical classification at 33 field-monitored sites. We found that the four frameworks consistently classified reach types into similar groups based on each reach or segment's dominant hydrogeomorphic elements. Where classified channel types diverged, differences could be attributed to the (a) spatial scale of input data used, (b) the requisite metrics and their order in completing a framework's decision tree and/or, (c) whether the framework attempts to classify current or historic channel form. Divergence in framework agreement was also observed at reaches where channel planform was decoupled from valley setting. Overall, the relative agreement between frameworks indicates that criticism of individual classifications for their use of form in grouping stream channels may be overstated. These form

  16. The Blurred Line between Form and Process: A Comparison of Stream Channel Classification Frameworks.

    Directory of Open Access Journals (Sweden)

    Alan Kasprak

    Full Text Available Stream classification provides a means to understand the diversity and distribution of channels and floodplains that occur across a landscape while identifying links between geomorphic form and process. Accordingly, stream classification is frequently employed as a watershed planning, management, and restoration tool. At the same time, there has been intense debate and criticism of particular frameworks, on the grounds that these frameworks classify stream reaches based largely on their physical form, rather than direct measurements of their component hydrogeomorphic processes. Despite this debate surrounding stream classifications, and their ongoing use in watershed management, direct comparisons of channel classification frameworks are rare. Here we implement four stream classification frameworks and explore the degree to which each make inferences about hydrogeomorphic process from channel form within the Middle Fork John Day Basin, a watershed of high conservation interest within the Columbia River Basin, U.S.A. We compare the results of the River Styles Framework, Natural Channel Classification, Rosgen Classification System, and a channel form-based statistical classification at 33 field-monitored sites. We found that the four frameworks consistently classified reach types into similar groups based on each reach or segment's dominant hydrogeomorphic elements. Where classified channel types diverged, differences could be attributed to the (a spatial scale of input data used, (b the requisite metrics and their order in completing a framework's decision tree and/or, (c whether the framework attempts to classify current or historic channel form. Divergence in framework agreement was also observed at reaches where channel planform was decoupled from valley setting. Overall, the relative agreement between frameworks indicates that criticism of individual classifications for their use of form in grouping stream channels may be overstated. These

  17. Pharmacology of the human red cell voltage-dependent cation channel Part I. Activation by clotrimazole and analogues

    DEFF Research Database (Denmark)

    Barksmann, Trine Lyberth; Kristensen, Berit I.; Christophersen, Palle.

    2004-01-01

    Human red cells, Nonselective voltage dependent cation channel, NSVDC channel, Gárdos channel blockers, NSVDC channel activators......Human red cells, Nonselective voltage dependent cation channel, NSVDC channel, Gárdos channel blockers, NSVDC channel activators...

  18. KCNQ4 channel activation by BMS-204352 and retigabine

    DEFF Research Database (Denmark)

    Schrøder, Rikke Louise K.; Jespersen, Thomas; Christophersen, P

    2001-01-01

    Activation of potassium channels generally reduces cellular excitability, making potassium channel openers potential drug candidates for the treatment of diseases related to hyperexcitabilty such as epilepsy, neuropathic pain, and neurodegeneration. Two compounds, BMS-204352 and retigabine, prese...

  19. Functional reconstitution and channel activity measurements of purified wildtype and mutant CFTR protein.

    Science.gov (United States)

    Eckford, Paul D W; Li, Canhui; Bear, Christine E

    2015-03-09

    The Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) is a unique channel-forming member of the ATP Binding Cassette (ABC) superfamily of transporters. The phosphorylation and nucleotide dependent chloride channel activity of CFTR has been frequently studied in whole cell systems and as single channels in excised membrane patches. Many Cystic Fibrosis-causing mutations have been shown to alter this activity. While a small number of purification protocols have been published, a fast reconstitution method that retains channel activity and a suitable method for studying population channel activity in a purified system have been lacking. Here rapid methods are described for purification and functional reconstitution of the full-length CFTR protein into proteoliposomes of defined lipid composition that retains activity as a regulated halide channel. This reconstitution method together with a novel flux-based assay of channel activity is a suitable system for studying the population channel properties of wild type CFTR and the disease-causing mutants F508del- and G551D-CFTR. Specifically, the method has utility in studying the direct effects of phosphorylation, nucleotides and small molecules such as potentiators and inhibitors on CFTR channel activity. The methods are also amenable to the study of other membrane channels/transporters for anionic substrates.

  20. Epilepsy-Related Slack Channel Mutants Lead to Channel Over-Activity by Two Different Mechanisms

    Directory of Open Access Journals (Sweden)

    Qiong-Yao Tang

    2016-01-01

    Full Text Available Twelve sodium-activated potassium channel (KCNT1, Slack genetic mutants have been identified from severe early-onset epilepsy patients. The changes in biophysical properties of these mutants and the underlying mechanisms causing disease remain elusive. Here, we report that seven of the 12 mutations increase, whereas one mutation decreases, the channel’s sodium sensitivity. Two of the mutants exhibit channel over-activity only when the intracellular Na+ ([Na+]i concentration is ∼80 mM. In contrast, single-channel data reveal that all 12 mutants increase the maximal open probability (Po. We conclude that these mutant channels lead to channel over-activity predominantly by increasing the ability of sodium binding to activate the channel, which is indicated by its maximal Po. The sodium sensitivity of these epilepsy causing mutants probably determines the [Na+]i concentration at which these mutants exert their pathological effects.

  1. Bulge formed cooling channels with a variable lead helix on a hollow body of revolution

    Science.gov (United States)

    McAninch, Michael D. (Inventor); Holbrook, Richard L. (Inventor); Lacount, Dale F. (Inventor); Kawashige, Chester M. (Inventor); Crapuchettes, John M. (Inventor); Scala, James (Inventor)

    1993-01-01

    A method of constructing a nozzle having cooling channels comprises a shell and a liner which are formed into a body of revolution having an axis of revolution. Helical welds are formed to hold the liner and shell to each other with a channel position being defined between each pair of helical welds. Pressurized fluid which may be a gas or a liquid, is introduced between the weld pairs to outwardly bulge the material of at least one of the liner and shell to define the channels.

  2. Channel Networks on Large Fans: Refining Analogs for the Ridge-forming Unit, Sinus Meridiani

    Science.gov (United States)

    Wilkinson, Justin

    2009-01-01

    Stream channels are generally thought of as forming within confined valley settings, separated by interfluves. Sinuous ridges on Mars and Earth are often interpreted as stream channels inverted by subsequent erosion of valley sides. In the case of the ridge-forming unit (RFU), this interpretation fails to explain the (i) close spacing of the ridges, which are (ii) organized in networks, and which (iii) cover large areas (approximately 175,000 km (exp 2)). Channel networks on terrestrial fans develop unconfined by valley slopes. Large fans (100s km long) are low-angle, fluvial features, documented worldwide, with characteristics that address these aspects of the RFU. Ridge patterns Channels on large fans provide an analog for the sinuous and elongated morphology of RFU ridges, but more especially for other patterns such as subparallel, branching and crossing networks. Branches are related to splays (delta-like distributaries are rare), whose channels can rejoin the main channel. Crossing patterns can be caused by even slight sinuosity splay-related side channels often intersect. An avulsion node distant from the fan apex, gives rise to channels with slightly different, and hence intersecting, orientations. Channels on neighboring fans intersect along the common fan margin. 2. Network density Channels are the dominant feature on large terrestrial fans (lakes and dune fields are minor). Inverted landscapes on subsequently eroded fans thus display indurated channels as networks of significantly close-spaced ridges. 3. Channel networks covering large areas Areas of individual large terrestrial fans can reach >200,000 km 2 (105-6 km 2 with nested fans), providing an analog for the wide area distribution of the RFU.

  3. Forming of single-thread channels and multiple channel rivers on Titan and Earth

    Science.gov (United States)

    Misiura, Katarzyna; Czechowski, Leszek

    2016-10-01

    In our research we use numerical model of the river to determine the limits of different fluvial parameters that play important roles in evolution of the rivers on Titan and on Earth. We have found that transport of sediments as suspended load is the main way of transport for Titan [1]. We also determined the range of the river's parameters for which multiple channel rivers are developed rather than single channel. This work is aimed to investigate the similarity and differences between these processes on Titan and the Earth.Numerical modelThe dynamical analysis of the considered rivers is performed using the package CCHE modified for the specific conditions on Titan. The package is based on the Navier-Stokes equations for depth-integrated two dimensional, turbulent flow and three dimensional convection-diffusion equation of sediment transport. We use the same numerical package that in our previous work [1] and [2], i.e. CCHE2D package.Parameters of the modelFor Titan we consider liquid corresponding to a Titan's rain (75% methane, 25% nitrogen) and water ice as material transported in rivers, for Earth the water and the quartz. We model evolution of the river for at least 100-200 days.Results and ConclusionsOur preliminary results indicate that suspended load is the main way of transport in simulated Titan's conditions. We also indicate that multiple channel rivers appears for larger range of slope on Titan (e.g. S=0.01-0.04) than on Earth (e.g. S=0.004-0.009). Also, for the same type of river, the grain size on Titan is at least 10 times larger than on Earth (1 cm for Titan versus 1 mm for the Earth). It is very interesting that on Titan multiple channel rivers appear even for very little discharge (e.g. Q=30m3/s) and for very large grain size (e.g. 10 cm). In the future we plan the experimental modelling in sediment basin to confirm results from computer modelling.References[1] Misiura, K., Czechowski, L., 2015. Numerical modelling of sedimentary structures in

  4. MAJOR CHANNELS OF FORMING THE IMAGE OF A REGION ON THE INTERNET

    Directory of Open Access Journals (Sweden)

    Svetlana Lashova

    2015-01-01

    Full Text Available The article highlights the major channels of forming the image of a region on the Internet. The authors suppose that today the image of a region is formed as a certain communicative stream, with participation of both official and unofficial subjects of communication. The article analyzes such major channels of forming the image of a region on the Internet as blogs and social network. As stated by the authors, the abovementioned channels shape, firstly, the image which is expected by the people; secondly, the Internet forms the real but not the ideal (official image of the territory. In conclusion it is pointed out that the official image should be formed considering the positioning of a region in spontaneous communicative streams.

  5. [Isolation and purification of human blood plasma proteins able to form potassium channels in artificial bilayer lipid membrane].

    Science.gov (United States)

    Venediktova, N I; Kuznetsov, K V; Gritsenko, E N; Gulidova, G P; Mironova, G D

    2012-01-01

    Protein fraction able to induce K(+)-selective transport across bilayer lipid membrane was isolated from human blood plasma with the use of the detergent and proteolytic enzyme-free method developed at our laboratory. After addition of the studied sample to the artificial membrane in the presence of 100 mM KCl, a discrete current change was observed. No channel activity was recorded in the presence of calcium and sodium ions. Channel forming activity of fraction was observed only in the presence of K+. Using a threefold gradient of KCl in the presence of studied proteins the potassium-selective potential balanced by voltage of -29 mV was registered. This value is very close to the theoretical Nernst potential in this case. This means that the examined ion channel is cation-selective. According to data obtained with MS-MALDI-TOF/TOF and database NCBI three protein components were identified in isolated researched sample.

  6. Running out of time: the decline of channel activity and nucleotide activation in adenosine triphosphate-sensitive K-channels

    Science.gov (United States)

    Proks, Peter; Puljung, Michael C.; Vedovato, Natascia; Sachse, Gregor; Mulvaney, Rachel; Ashcroft, Frances M.

    2016-01-01

    KATP channels act as key regulators of electrical excitability by coupling metabolic cues—mainly intracellular adenine nucleotide concentrations—to cellular potassium ion efflux. However, their study has been hindered by their rapid loss of activity in excised membrane patches (rundown), and by a second phenomenon, the decline of activation by Mg-nucleotides (DAMN). Degradation of PI(4,5)P2 and other phosphoinositides is the strongest candidate for the molecular cause of rundown. Broad evidence indicates that most other determinants of rundown (e.g. phosphorylation, intracellular calcium, channel mutations that affect rundown) also act by influencing KATP channel regulation by phosphoinositides. Unfortunately, experimental conditions that reproducibly prevent rundown have remained elusive, necessitating post hoc data compensation. Rundown is clearly distinct from DAMN. While the former is associated with pore-forming Kir6.2 subunits, DAMN is generally a slower process involving the regulatory sulfonylurea receptor (SUR) subunits. We speculate that it arises when SUR subunits enter non-physiological conformational states associated with the loss of SUR nucleotide-binding domain dimerization following prolonged exposure to nucleotide-free conditions. This review presents new information on both rundown and DAMN, summarizes our current understanding of these processes and considers their physiological roles. This article is part of the themed issue ‘Evolution brings Ca2+ and ATP together to control life and death’. PMID:27377720

  7. Numerical Simulation of the Roll Forming Process of Aluminum Folded Micro-channel Tube

    Science.gov (United States)

    Zou, Tianxia; Zhou, Ning; Peng, Yinghong; Tang, Ding; Li, Dayong

    2016-08-01

    Micro-channel tube is the most important component of flat tube heat exchangers. The folded microchannel tube is made of clad aluminum sheet through roll forming process, and has great advantage in the aspect of corrosion resistance over extruded tube. The folded tube's sub-millimeter channel size as well as tight dimensional precision requirement brings great challenge to roll forming process design. In this paper, the finite element model of the whole roll forming process of a ten-channel tube is established by using ABAQUS/Explicit. The deformation at different forming stands are investigated and compared with experiment. The hydraulic pressure test is carried out on the developed tube and its pressure bearing capacity is evaluated.

  8. Wavelength-selective fluorescence in ion channels formed by gramicidin A in membranes

    Indian Academy of Sciences (India)

    Amitabha Chattopadhyay; Satinder S Rawat

    2007-03-01

    Gramicidins are linear peptides that form ion channels that are specific for monovalent cations in membranes. The tryptophan residues in the gramicidin channel play a crucial role in the organization and function of the channel. The natural mixture of gramicidins, denoted as gramicidin A', consists of mostly gramicidin A, but also contains gramicidins B, C and D as minor components. We have previously shown that the tryptophan residues in ion channels formed by the naturally occurring peptide, gramicidin A', display wavelength-dependent fluorescence characteristics due to the motionally restricted environment in which they are localized. In order to check the influence of ground-state heterogeneity in the observed wavelength-selective fluorescence of gramicidin A' in membranes, we performed similar experiments with pure gramicidin A in model membranes. Our results show that the observed wavelength-selective fluorescence characteristics of naturally occurring gramicidin A' are not due to groundstate heterogeneity.

  9. Plasma channel formed by ultraviolet laser pulses at 193 nm in air

    Institute of Scientific and Technical Information of China (English)

    Yuanyuan Ma; Xin Lu; Tingting Xi; Qihuang Gong; Jie Zhang

    2009-01-01

    The propagation of picosecond deep ultraviolet laser pulse at wavelength of 193 nm in air is numerically investigated.Long plasma channel can be formed due to the competition between Kerr self-focusing and ionization induced defocusing.The plasma channel with electron density of above 1013/cma can be formed over 70 m by 50-ps,20-mJ laser pulses.The fluctuation of laser intensity and electron density inside ultraviolet(UV)plasma channel is significantly lower than that of infrared pulse.The linear absorption of UV laser by air is considered in the simulation and it is shown that the linear absorption is important for the limit of the length of plasma channel.

  10. Hybrid onboard and ground based digital channelizer beam-forming for SATCOM interference mitigation and protection

    Science.gov (United States)

    Xiong, Wenhao; Wang, Gang; Tian, Xin; Pham, Khanh; Blasch, Erik; Chen, Genshe

    2016-05-01

    In this work, we propose a novel beam-forming power allocation method for a satellite communication (SATCOM) multiple-input multiple-output (MIMO) system to mitigate the co-channel interference (CCI) as well as limiting the signal leakage to the adversary users. In SATCOM systems, the beam-forming technique is a conventional way of avoiding interference, controlling the antenna beams, and mitigating undesired signals. We propose to use an advanced beam-forming technique which considers the number of independent channels used and transmitting power deployed to reduce and mitigate the unintentional interference effect. With certain quality of service (QoS) for the SATCOM system, independent channels components will be selected. It is desired to use less and stronger channel components when possible. On the other hand, considering that SATCOM systems often face the problem that adversary receiver detects the signal, a proposed power allocation method can efficiently reduce the received power at the adversary receiver. To reduce the computational burden on the transponder in order to minimize the size, mass, power consumption and delay for the satellite, we apply a hybrid onboard and ground based beam-forming design to distribute the calculation between the transponder and ground terminals. Also the digital channelizer beam-forming (DCB) technique is employed to achieve dynamic spatial control.

  11. Closed Form Secrecy Capacity of MIMO Wiretap Channels with Two Transmit Antennas

    CERN Document Server

    Li, Jiangyuan

    2011-01-01

    A Gaussian multiple-input multiple-output (MIMO) wiretap channel model is considered. The input is a two-antenna transmitter, while the outputs are the legitimate receiver and an eavesdropper, both equipped with multiple antennas. All channels are assumed to be known. The problem of obtaining the optimal input covariance matrix that achieves secrecy capacity subject to a power constraint is addressed, and a closed-form expression for the secrecy capacity is obtained.

  12. Probing the structure-function relationship of alpha-latrotoxin-formed channels with antibodies and pronase.

    Science.gov (United States)

    Chanturiya, A N; Nikolaenko, A N; Shatursky OYa; Lishko, V K

    1996-10-01

    The major toxic component of black widow spider (Latrodectus mactans tredecimguttatus) venom, alpha-latrotoxin, is known to form ionic channels in different membranes. In order to probe the extramembrane domains of alpha-latrotoxin molecule, alpha-latrotoxin channels in planar lipid membrane were treated with antibodies to latrotoxin or with pronase added to different sides of the membrane. It was found that antibody addition to the same side as the toxin (cis) decreased channel conductance only at positive potentials across the membrane. In contrast, trans side addition of antibodies changed the channel conductance at both positive and negative potentials: at positive potential conductance first slightly increased then decreased by more then 50%; at negative potential it decreased much more quickly, to only about 20% of the initial value. No dependence on membrane potential was found for pronase treatment of incorporated channels. For both cis and trans application of pronase, channel selectivity for Ca2+, Mg2+, Ba2+ and K+, Na+, Li+ ions did not change significantly but Cd2+ block was decreased. Trans pronase treatment also resulted in some rectification of I/V curves and an increase in channel conductance. We interpret these findings as evidence that alpha-latrotoxin channel has protruding parts on both sides of the membrane and that its conformation in the membrane depends on membrane potential.

  13. Activation of Slo2.1 channels by niflumic acid

    OpenAIRE

    Dai, Li; Garg, Vivek; Sanguinetti, Michael C.

    2010-01-01

    Slo2.1 channels conduct an outwardly rectifying K+ current when activated by high [Na+]i. Here, we show that gating of these channels can also be activated by fenamates such as niflumic acid (NFA), even in the absence of intracellular Na+. In Xenopus oocytes injected with

  14. Selective disruption of high sensitivity heat activation but not capsaicin activation of TRPV1 channels by pore turret mutations.

    Science.gov (United States)

    Cui, Yuanyuan; Yang, Fan; Cao, Xu; Yarov-Yarovoy, Vladimir; Wang, KeWei; Zheng, Jie

    2012-04-01

    The capsaicin receptor transient receptor potential vanilloid (TRPV)1 is a highly heat-sensitive ion channel. Although chemical activation and heat activation of TRPV1 elicit similar pungent, painful sensation, the molecular mechanism underlying synergistic activation remains mysterious. In particular, where the temperature sensor is located and whether heat and capsaicin share a common activation pathway are debated. To address these fundamental issues, we searched for channel mutations that selectively affected one form of activation. We found that deletion of the first 10 amino acids of the pore turret significantly reduced the heat response amplitude and shifted the heat activation threshold, whereas capsaicin activation remained unchanged. Removing larger portions of the turret disrupted channel function. Introducing an artificial sequence to replace the deleted region restored sensitive capsaicin activation in these nonfunctional channels. The heat activation, however, remained significantly impaired, with the current exhibiting diminishing heat sensitivity to a level indistinguishable from that of a voltage-gated potassium channel, Kv7.4. Our results demonstrate that heat and capsaicin activation of TRPV1 are structurally and mechanistically distinct processes, and the pore turret is an indispensible channel structure involved in the heat activation process but is not part of the capsaicin activation pathway. Synergistic effect of heat and capsaicin on TRPV1 activation may originate from convergence of the two pathways on a common activation gate.

  15. A structural view of ligand-dependent activation in thermoTRP channels

    Directory of Open Access Journals (Sweden)

    Ximena eSteinberg

    2014-05-01

    Full Text Available Transient Receptor Potential (TRP proteins are a large family of ion channels, grouped intoseven sub-families. Although great advances have been made regarding the activation andmodulation of TRP channel activity, detailed molecular mechanisms governing TRPchannel gating are still needed. Sensitive to electric, chemical, mechanical, and thermalcues, TRP channels are tightly associated with the detection and integration of sensoryinput, emerging as a model to study the polymodal activation of ion channel proteins.Among TRP channels, the temperature-activated kind constitute a subgroup by itself,formed by Vanilloid receptors 1-4, Melastatin receptors 2, 4, 5 and 8, TRPC5, and TRPA1.Some of the so-called thermoTRP channels participate in the detection of noxious stimulimaking them an interesting pharmacological target for the treatment of pain. However, thepoor specificity of the compounds available in the market represents an important obstacleto overcome. Understanding the molecular mechanics underlying ligand-dependentmodulation of TRP channels may help with the rational design of novel syntheticanalgesics. The present review focuses on the structural basis of ligand-dependentactivation of TRPV1 and TRPM8 channels. Special attention is drawn to the dissection ofligand-binding sites within TRPV1, PIP 2 -dependent modulation of TRP channels, and thestructure of natural and synthetic ligands.

  16. Alpha-helical hydrophobic polypeptides form proton-selective channels in lipid bilayers

    Science.gov (United States)

    Oliver, A. E.; Deamer, D. W.

    1994-01-01

    Proton translocation is important in membrane-mediated processes such as ATP-dependent proton pumps, ATP synthesis, bacteriorhodopsin, and cytochrome oxidase function. The fundamental mechanism, however, is poorly understood. To test the theoretical possibility that bundles of hydrophobic alpha-helices could provide a low energy pathway for ion translocation through the lipid bilayer, polyamino acids were incorporated into extruded liposomes and planar lipid membranes, and proton translocation was measured. Liposomes with incorporated long-chain poly-L-alanine or poly-L-leucine were found to have proton permeability coefficients 5 to 7 times greater than control liposomes, whereas short-chain polyamino acids had relatively little effect. Potassium permeability was not increased markedly by any of the polyamino acids tested. Analytical thin layer chromatography measurements of lipid content and a fluorescamine assay for amino acids showed that there were approximately 135 polyleucine or 65 polyalanine molecules associated with each liposome. Fourier transform infrared spectroscopy indicated that a major fraction of the long-chain hydrophobic peptides existed in an alpha-helical conformation. Single-channel recording in both 0.1 N HCl and 0.1 M KCl was also used to determine whether proton-conducting channels formed in planar lipid membranes (phosphatidylcholine/phosphatidylethanolamine, 1:1). Poly-L-leucine and poly-L-alanine in HCl caused a 10- to 30-fold increase in frequency of conductive events compared to that seen in KCl or by the other polyamino acids in either solution. This finding correlates well with the liposome observations in which these two polyamino acids caused the largest increase in membrane proton permeability but had little effect on potassium permeability. Poly-L-leucine was considerably more conductive than poly-L-alanine due primarily to larger event amplitudes and, to a lesser extent, a higher event frequency. Poly-L-leucine caused two

  17. Corynebacterium jeikeium jk0268 constitutes for the 40 amino acid long PorACj, which forms a homooligomeric and anion-selective cell wall channel.

    Directory of Open Access Journals (Sweden)

    Narges Abdali

    Full Text Available Corynebacterium jeikeium, a resident of human skin, is often associated with multidrug resistant nosocomial infections in immunodepressed patients. C. jeikeium K411 belongs to mycolic acid-containing actinomycetes, the mycolata and contains a channel-forming protein as judged from reconstitution experiments with artificial lipid bilayer experiments. The channel-forming protein was present in detergent treated cell walls and in extracts of whole cells using organic solvents. A gene coding for a 40 amino acid long polypeptide possibly responsible for the pore-forming activity was identified in the known genome of C. jeikeium by its similar chromosomal localization to known porH and porA genes of other Corynebacterium strains. The gene jk0268 was expressed in a porin deficient Corynebacterium glutamicum strain. For purification temporarily histidine-tailed or with a GST-tag at the N-terminus, the homogeneous protein caused channel-forming activity with an average conductance of 1.25 nS in 1M KCl identical to the channels formed by the detergent extracts. Zero-current membrane potential measurements of the voltage dependent channel implied selectivity for anions. This preference is according to single-channel analysis caused by some excess of cationic charges located in the channel lumen formed by oligomeric alpha-helical wheels. The channel has a suggested diameter of 1.4 nm as judged from the permeability of different sized hydrated anions using the Renkin correction factor. Surprisingly, the genome of C. jeikeium contained only one gene coding for a cell wall channel of the PorA/PorH type found in other Corynebacterium species. The possible evolutionary relationship between the heterooligomeric channels formed by certain Corynebacterium strains and the homooligomeric pore of C. jeikeium is discussed.

  18. Morphology of melt-rich channels formed during reaction infiltration experiments on partially molten mantle rocks

    Science.gov (United States)

    Pec, Matej; Holtzman, Benjamin; Zimmerman, Mark; Kohlstedt, David

    2016-04-01

    Geochemical, geophysical and geological observations suggest that melt extraction from the partially molten mantle occurs by some sort of channelized flow. Melt-solid reactions can lead to melt channelization due to a positive feedback between melt flow and reaction. If a melt-solid reaction increases local permeability, subsequent flow is increased as well and promotes further reaction. This process can lead to the development of high-permeability channels which emerge from background flow. In nature, anastomozing tabular dunite bodies within peridotitic massifs are thought to represent fossilized channels that formed by reactive flow. The conditions under which such channels can emerge are treated by the reaction infiltration instability (RII) theory (e.g. Szymczak and Ladd 2014). In this contribution, we report the results of a series of Darcy type experiments designed to study the development of channels due to RII in mantle lithologies (Pec et al. 2015). We sandwiched a partially molten rock between a melt source and a porous sink and annealed it at high-pressures (P = 300 MPa) and high-temperatures (T = 1200° or 1250° C) under a controlled pressure gradient (∇P = 0-100 MPa/mm) for up to 5 hours. The partially molten rock is formed by 50:50 mixtures of San Carlos olivine (Ol, Fo ˜ 88) and clinopyroxene (Cpx) with either 4, 10 or 20 vol% of alkali basalt added. The source and sink are disks of alkali basalt and porous alumina, respectively. During the experiments, silica undersaturated melt from the melt source dissolves Cpx and precipitates an iron rich Ol (Fo ˜ 82) thereby forming a Cpx-free reaction layer at the melt source - partially molten rock interface. The melt fraction in the reaction layer increases significantly (40% melt) compared to the protolith, confirming that the reaction increases the permeability of the partially molten rock. In experiments annealed under a low pressure gradient (and hence slow melt flow velocity) the reaction layer is

  19. NLOS UV Channel Modeling Using Numerical Integration and an Approximate Closed-Form Path Loss Model

    CERN Document Server

    Gupta, Ankit; Brandt-Pearce, Maïté

    2012-01-01

    In this paper we propose a simulation method using numerical integration, and develop a closed-form link loss model for physical layer channel characterization for non-line of sight (NLOS) ultraviolet (UV) communication systems. The impulse response of the channel is calculated by assuming both uniform and Gaussian profiles for transmitted beams and different geometries. The results are compared with previously published results. The accuracy of the integration approach is compared to the Monte Carlo simulation. Then the path loss using the simulation method and the suggested closed-form expression are presented for different link geometries. The accuracies are evaluated and compared to the results obtained using other methods.

  20. Dynamic changes of mechanically activated channels and K~+ channels at injury site of peripheral nerve in rat

    Institute of Scientific and Technical Information of China (English)

    凌琰; 谢益宽

    1996-01-01

    Ectopic ion channels developed locally at the injury site after nerve damage by light ligation around common sciatic nerve of the rats. Different channel types have different processes of formation, accumulation and degeneration. During the first three days after injury, mechanically activated channels that are modulated by Ca++ channel activities first appeared. As the nerve fibers begin to be excited by TEA, a blocker of K+ channels, suggesting that the accumulation of K+ channels, the responsibility of mechanically activated channels was declining. Onset of K+ channels was from the 3rd postoperative day and lasted up to the fiftieth day. This time course of K+ channel development was closely related to allodynia and hyperalgesia of neuropathic animal behaviour. The results suggest that chronic contraction injury induces a dynamic change in the ectopic mechanically activated channels and K+ channels at the injury site of nerve and there is an interchange in the development time courses of the mechanic

  1. Synchronized network activity in developing rat hippocampus involves regional hyperpolarization-activated cyclic nucleotide-gated (HCN) channel function

    OpenAIRE

    Bender, Roland A.; Galindo, Rafael; Mameli, Manuel; Gonzalez-Vega, Rebeca; Valenzuela, C. Fernando; Tallie Z. Baram

    2005-01-01

    The principal form of synchronized network activity in neonatal hippocampus consists of low frequency ‘giant depolarizing potentials’ (GDPs). Whereas contribution of both GABA and glutamate to their generation has been demonstrated, full understanding of the mechanisms underlying these synchronized activity bursts remains incomplete. A contribution of the h-current, conducted by HCN channels, to GDPs has been a topic of substantial interest. Here we focus on HCN1, the prevalent HCN channel is...

  2. A Relativistic Coupled-Channel Formalism for the Pion Form Factor

    Directory of Open Access Journals (Sweden)

    Klink W.H.

    2010-04-01

    Full Text Available The electromagnetic form factor of a confined quark-antiquark pair is calculated within the framework of point-form relativistic quantum mechanics. The dynamics of the exchanged photon is explicitly taken into account by treating the electromagnetic scattering of an electron by a meson as a relativistic two-channel problem for a Bakamjian-Thomas type mass operator. This approach guarantees Poincare invariance. Using a Feshbach reduction the coupled-channel problem can be converted into a one-channel problem for the elastic electron-meson channel. By comparing the one-photon-exchange optical potential at the constituent and hadronic levels, we are able to unambiguously identify the electromagnetic meson form factor. Violations of cluster-separability properties, which are inherent in the Bakamjian-Thomas approach, become negligible for su?ciently large invariant mass of the electron-meson system. In the limit of an in?nitely large invariant mass, an equivalence with form-factor calculations done in front-form relativistic quantum mechanics is established analytically.

  3. The deletion of several amino acid stretches of Escherichia coli alpha-hemolysin (HlyA suggests that the channel-forming domain contains beta-strands.

    Directory of Open Access Journals (Sweden)

    Roland Benz

    Full Text Available Escherichia coli α-hemolysin (HlyA is a pore-forming protein of 110 kDa belonging to the family of RTX toxins. A hydrophobic region between the amino acid residues 238 and 410 in the N-terminal half of HlyA has previously been suggested to form hydrophobic and/or amphipathic α-helices and has been shown to be important for hemolytic activity and pore formation in biological and artificial membranes. The structure of the HlyA transmembrane channel is, however, largely unknown. For further investigation of the channel structure, we deleted in HlyA different stretches of amino acids that could form amphipathic β-strands according to secondary structure predictions (residues 71-110, 158-167, 180-203, and 264-286. These deletions resulted in HlyA mutants with strongly reduced hemolytic activity. Lipid bilayer measurements demonstrated that HlyAΔ71-110 and HlyAΔ264-286 formed channels with much smaller single-channel conductance than wildtype HlyA, whereas their channel-forming activity was virtually as high as that of the wildtype toxin. HlyAΔ158-167 and HlyAΔ180-203 were unable to form defined channels in lipid bilayers. Calculations based on the single-channel data indicated that the channels generated by HlyAΔ71-110 and HlyAΔ264-286 had a smaller size (diameter about 1.4 to 1.8 nm than wildtype HlyA channels (diameter about 2.0 to 2.6 nm, suggesting that in these mutants part of the channel-forming domain was removed. Osmotic protection experiments with erythrocytes confirmed that HlyA, HlyAΔ71-110, and HlyAΔ264-286 form defined transmembrane pores and suggested channel diameters that largely agreed with those estimated from the single-channel data. Taken together, these results suggest that the channel-forming domain of HlyA might contain β-strands, possibly in addition to α-helical structures.

  4. Large-conductance calcium-activated potassium channels facilitate transmitter release in salamander rod synapse.

    Science.gov (United States)

    Xu, Jian Wei; Slaughter, Malcolm M

    2005-08-17

    Large-conductance calcium-activated potassium (BK) channels are colocalized with calcium channels at sites of exocytosis at the presynaptic terminals throughout the nervous system. It is expected that their activation would provide negative feedback to transmitter release, but the opposite is sometimes observed. Attempts to resolve this apparent paradox based on alterations in action potential waveform have been ambiguous. In an alternative approach, we investigated the influence of this channel on neurotransmitter release in a nonspiking neuron, the salamander rod photoreceptors. Surprisingly, the BK channel facilitates calcium-mediated transmitter release from rods. The two presynaptic channels form a positive coupled loop. Calcium influx activates the BK channel current, leading to potassium efflux that increases the calcium current. The normal physiological voltage range of the rod is well matched to the dynamics of this positive loop. When the rod is further depolarized, then the hyperpolarizing BK channel current exceeds its facilitatory effect, causing truncation of transmitter release. Thus, the calcium channel-BK channel linkage performs two functions at the synapse: nonlinear potentiator and safety brake.

  5. Adaptive beam forming for time-variant channels with a multi-beam antenna

    Institute of Scientific and Technical Information of China (English)

    JIANG Ze; MEI Yuan-yuan; RUAN Wei

    2008-01-01

    A multi-beam adaptive antenna is investigated by considering the adaptive beam forming performance for time-variant channels on the focus of mobile to base station or reverse link. This antenna is constructed by Fresnel zone phase-correcting plane (FZP) focusing element and uniformly spaced feeds array. An estimator for the beamforming weight vector in the presence of angle spreads is derived using a code filtering approach. Owing to the time-varying nature of the channels, a recursive method for computing and tracking the above optimal weight vector solution that is easy to implement is applied. The effects of angle spread, maximum Doppler frequency, and forgetting factor are studied. Simulation results showed that this type of adaptive antenna is capable of performing adaptive beam forming for time-variant channels effectively.

  6. Cell volume and membrane stretch independently control K+ channel activity

    DEFF Research Database (Denmark)

    Bomholtz, Sofia Hammami; Willumsen, Niels J; Olsen, Hervør L

    2009-01-01

    A number of potassium channels including members of the KCNQ family and the Ca(2+) activated IK and SK, but not BK, are strongly and reversibly regulated by small changes in cell volume. It has been argued that this general regulation is mediated through sensitivity to changes in membrane stretch...... was not affected by membrane stretch. The results indicate that (1) activation of BK channels by local membrane stretch is not mimicked by membrane stress induced by cell swelling, and (2) activation of KCNQ1 channels by cell volume increase is not mediated by local tension in the cell membrane. We conclude....... To test this hypothesis we have studied the regulation of KCNQ1 and BK channels after expression in Xenopus oocytes. Results from cell-attached patch clamp studies (approximately 50 microm(2) macropatches) in oocytes expressing BK channels demonstrate that the macroscopic volume-insensitive BK current...

  7. Phosphatase inhibitors activate normal and defective CFTR chloride channels

    OpenAIRE

    Becq, F; Jensen, T J; Chang, X B; Savoia, A.; Rommens, J M; Tsui, L C; Buchwald, M; Riordan, J R; Hanrahan, J W

    1994-01-01

    The cystic fibrosis transmembrane conductance regulator (CFTR) chloride channel is regulated by phosphorylation and dephosphorylation at multiple sites. Although activation by protein kinases has been studied in some detail, the dephosphorylation step has received little attention. This report examines the mechanisms responsible for the dephosphorylation and spontaneous deactivation ("rundown") of CFTR chloride channels excised from transfected Chinese hamster ovary (CHO) and human airway epi...

  8. Oxidative Stress and Maxi Calcium-Activated Potassium (BK Channels

    Directory of Open Access Journals (Sweden)

    Anton Hermann

    2015-08-01

    Full Text Available All cells contain ion channels in their outer (plasma and inner (organelle membranes. Ion channels, similar to other proteins, are targets of oxidative impact, which modulates ion fluxes across membranes. Subsequently, these ion currents affect electrical excitability, such as action potential discharge (in neurons, muscle, and receptor cells, alteration of the membrane resting potential, synaptic transmission, hormone secretion, muscle contraction or coordination of the cell cycle. In this chapter we summarize effects of oxidative stress and redox mechanisms on some ion channels, in particular on maxi calcium-activated potassium (BK channels which play an outstanding role in a plethora of physiological and pathophysiological functions in almost all cells and tissues. We first elaborate on some general features of ion channel structure and function and then summarize effects of oxidative alterations of ion channels and their functional consequences.

  9. ANTISTAPHYLOCOCCAL ACTIVITY OF LIPOSOMAL FORMS OF LINCOMYCIN

    Directory of Open Access Journals (Sweden)

    Derkach SA

    2015-04-01

    Full Text Available Nowadays the vital problem of modern medicine is a tendency to emerging of both nosocomial and community-acquired strains before antibiotic resistance forming. The complexity of antibiotic therapy of diseases caused by methicillin resistant staphylococci having high poly resistance almost to every classes of antibacterial agents is of prime importance. One of the ways to improve antibacterial preparations still remains the development of their liposomal forms. This work studies antistaphylococcal activity (according to MIC of the liposomal form of lincomycin developed in the Institute of Dermatology and Venereology of Ukraine by Ivanova N. N., the Candidate of Сhemical Sciences.The purpose of this research work was to study liposomal inhibiting concentration of the liposomalny form of lincomycin and a commercial preparation lincomycin (produced by CJSC “Pharmaceutical firm "Darnitsa". Determination of the minimum inhibiting concentration was carried out by a tablet micromethod by consecutive cultivations of the samples under study.It is shown that MIC of liposomal lincomycin is eight times as low as usual lincomycin (0,23mkg/ml to 1,87 mkg/ml. Antibacterial activity of the liposomal form of lincomycin is studied concerning the patients selected from the different biotopes with pyo inflammatory diseases of staphylococcus strains (15 strains – methicillin sensitive, 12 strains - methicillin resistant.It is shown authentically the higher sensitivity of S. aureus strains to the liposomal form of lincomycin in comparison with usual lincomycin . Also 50.0% of MRSA strains were sensitive to the liposomalny form of lincomycin that shows the perspective for the development of the liposomal forms of antibiotics to cure staphylococcal infections.

  10. The Topographic Design of River Channels for Form-Process Linkages

    Science.gov (United States)

    Brown, Rocko A.; Pasternack, Gregory B.; Lin, Tin

    2016-04-01

    Scientists and engineers design river topography for a wide variety of uses, such as experimentation, site remediation, dam mitigation, flood management, and river restoration. A recent advancement has been the notion of topographical design to yield specific fluvial mechanisms in conjunction with natural or environmental flow releases. For example, the flow convergence routing mechanism, whereby shear stress and spatially convergent flow migrate or jump from the topographic high (riffle) to the low point (pool) from low to high discharge, is thought to be a key process able to maintain undular relief in gravel bedded rivers. This paper develops an approach to creating riffle-pool topography with a form-process linkage to the flow convergence routing mechanism using an adjustable, quasi equilibrium synthetic channel model. The link from form to process is made through conceptualizing form-process relationships for riffle-pool couplets into geomorphic covariance structures (GCSs) that are then quantitatively embedded in a synthetic channel model. Herein, GCSs were used to parameterize a geometric model to create five straight, synthetic river channels with varying combinations of bed and width undulations. Shear stress and flow direction predictions from 2D hydrodynamic modeling were used to determine if scenarios recreated aspects of the flow convergence routing mechanism. Results show that the creation of riffle-pool couplets that experience flow convergence in straight channels requires GCSs with covarying bed and width undulations in their topography as supported in the literature. This shows that GCSs are a useful way to translate conceptualizations of form-process linkages into quantitative models of channel form.

  11. Effect of pyrrolidinium based ionic liquid on the channel form of gramicidin in lipid vesicles.

    Science.gov (United States)

    Singh, Upendra Kumar; Dohare, Neeraj; Mishra, Prabhash; Singh, Prashant; Bohidar, Himadri B; Patel, Rajan

    2015-08-01

    The present work is focused on the interaction between membrane bound gramicidin and 1-butyl-1-methyl-2-oxopyrrolidinium bromide (BMOP) ionic liquid. Ionic liquids (ILs) are solvents that are often liquid at room temperature and composed of organic cation and appropriate anion. The gramicidin peptide forms prototypical ion channels for cations, which have been extensively used to study the organization, dynamics, and function of membrane spanning channels. The interaction was studied by circular dichroism, steady state, time-resolved fluorescence spectroscopy in combination with dynamic surface tension and field emission scanning electron microscopic methods (FESEM). The results obtained from circular dichroism shows that the BMOP interacts with the channel form of gramicidin in lipid vesicle without any considerable effect on its conformation. The Red-edge excitation shift (REES) also supported the above findings. In addition, the fluorescence studies suggested that BMOP makes ground state complex with ion channel, which was further supported by time resolved measurements. Furthermore, dynamic surface tension analysis shows the faster adsorption of BMOP with membrane bound gramicidin at the air-water interface. Additionally, FESEM results indicated that BMOP forms a film around the membrane bound gramicidin at higher concentration. These results are potentially useful to analyze the effect of ionic liquids on the behaviour of membrane proteins.

  12. Protein kinase CK2 is coassembled with small conductance Ca(2+)-activated K+ channels and regulates channel gating

    DEFF Research Database (Denmark)

    Bildl, Wolfgang; Strassmaier, Tim; Thurm, Henrike

    2004-01-01

    Small conductance Ca(2+)-activated K+ channels (SK channels) couple the membrane potential to fluctuations in intracellular Ca2+ concentration in many types of cells. SK channels are gated by Ca2+ ions via calmodulin that is constitutively bound to the intracellular C terminus of the channels and...

  13. Punching Holes in Membranes: How Oligomeric Pore-Forming Proteins and Lipids Cooperate to Form Aqueous Channels in Membranes

    Science.gov (United States)

    Fradin, Cécile; Satsoura, Dmitri; Andrews, David W.

    Many important biological processes are carried out by a small number of proteins working together as a team to accomplish a specific task. Cooperation between the different proteins is often accomplished through the formation of a supramolecular complex, comprised of either identical or different subunits. Although the formation of protein assemblies is a favored mechanism throughout the cell, it becomes especially important in lipid membranes, as evidenced by the numerous cellular events that are either triggered by or result in the formation of protein complexes in membranes. However, due to the difficulties associated with the study of membrane proteins, the formation of oligomers in lipid membranes is perhaps one of the least understood cellular processes. In this chapter we focus our attention on a subset of membrane complexes — namely, those formed by proteins that are able to pass from a water-soluble to a transmembrane form in order to create a water-filled channel through the lipid membrane. These pore-forming proteins (PFPs) are found in many organisms throughout different kingdoms of life, from bacteria to human. They are often involved in cell death mechanisms through their capacity to break membrane permeability barriers, which can lead to dissipation of the membrane potential as well as introduction or leakage of enzymatic proteins. In fact, a large subset of the PFPs are toxins, and referred to in the literature as pore-forming toxins (PFTs). The association of several monomers into an oligomer is almost always an important aspect of the modus operandi of these proteins. Oligomerization can be useful in several ways: it results in structures large enough to delineate nanometer-size water-filled channels in lipid bilayers, it ensures the presence of large hydrophobic surfaces that can support insertion in the membrane, and it permits cooperative formation and insertion mechanisms.

  14. DESDynI Quad First Stage Processor - A Four Channel Digitizer and Digital Beam Forming Processor

    Science.gov (United States)

    Chuang, Chung-Lun; Shaffer, Scott; Smythe, Robert; Niamsuwan, Noppasin; Li, Samuel; Liao, Eric; Lim, Chester; Morfopolous, Arin; Veilleux, Louise

    2013-01-01

    The proposed Deformation, Eco-Systems, and Dynamics of Ice Radar (DESDynI-R) L-band SAR instrument employs multiple digital channels to optimize resolution while keeping a large swath on a single pass. High-speed digitization with very fine synchronization and digital beam forming are necessary in order to facilitate this new technique. The Quad First Stage Processor (qFSP) was developed to achieve both the processing performance as well as the digitizing fidelity in order to accomplish this sweeping SAR technique. The qFSP utilizes high precision and high-speed analog to digital converters (ADCs), each with a finely adjustable clock distribution network to digitize the channels at the fidelity necessary to allow for digital beam forming. The Xilinx produced FX130T Virtex 5 part handles the processing to digitally calibrate each channel as well as filter and beam form the receive signals. Demonstrating the digital processing required for digital beam forming and digital calibration is instrumental to the viability of the proposed DESDynI instrument. The qFSP development brings this implementation to Technology Readiness Level (TRL) 6. This paper will detail the design and development of the prototype qFSP as well as the preliminary results from hardware tests.

  15. DESDynI Quad First Stage Processor - A Four Channel Digitizer and Digital Beam Forming Processor

    Science.gov (United States)

    Chuang, Chung-Lun; Shaffer, Scott; Smythe, Robert; Niamsuwan, Noppasin; Li, Samuel; Liao, Eric; Lim, Chester; Morfopolous, Arin; Veilleux, Louise

    2013-01-01

    The proposed Deformation, Eco-Systems, and Dynamics of Ice Radar (DESDynI-R) L-band SAR instrument employs multiple digital channels to optimize resolution while keeping a large swath on a single pass. High-speed digitization with very fine synchronization and digital beam forming are necessary in order to facilitate this new technique. The Quad First Stage Processor (qFSP) was developed to achieve both the processing performance as well as the digitizing fidelity in order to accomplish this sweeping SAR technique. The qFSP utilizes high precision and high-speed analog to digital converters (ADCs), each with a finely adjustable clock distribution network to digitize the channels at the fidelity necessary to allow for digital beam forming. The Xilinx produced FX130T Virtex 5 part handles the processing to digitally calibrate each channel as well as filter and beam form the receive signals. Demonstrating the digital processing required for digital beam forming and digital calibration is instrumental to the viability of the proposed DESDynI instrument. The qFSP development brings this implementation to Technology Readiness Level (TRL) 6. This paper will detail the design and development of the prototype qFSP as well as the preliminary results from hardware tests.

  16. Plasma membrane mechanical stress activates TRPC5 channels.

    Directory of Open Access Journals (Sweden)

    Bing Shen

    Full Text Available Mechanical forces exerted on cells impose stress on the plasma membrane. Cells sense this stress and elicit a mechanoelectric transduction cascade that initiates compensatory mechanisms. Mechanosensitive ion channels in the plasma membrane are responsible for transducing the mechanical signals to electrical signals. However, the mechanisms underlying channel activation in response to mechanical stress remain incompletely understood. Transient Receptor Potential (TRP channels serve essential functions in several sensory modalities. These channels can also participate in mechanotransduction by either being autonomously sensitive to mechanical perturbation or by coupling to other mechanosensory components of the cell. Here, we investigated the response of a TRP family member, TRPC5, to mechanical stress. Hypoosmolarity triggers Ca2+ influx and cationic conductance through TRPC5. Importantly, for the first time we were able to record the stretch-activated TRPC5 current at single-channel level. The activation threshold for TRPC5 was found to be 240 mOsm for hypoosmotic stress and between -20 and -40 mmHg for pressure applied to membrane patch. In addition, we found that disruption of actin filaments suppresses TRPC5 response to hypoosmotic stress and patch pipette pressure, but does not prevent the activation of TRPC5 by stretch-independent mechanisms, indicating that actin cytoskeleton is an essential transduction component that confers mechanosensitivity to TRPC5. In summary, our findings establish that TRPC5 can be activated at the single-channel level when mechanical stress on the cell reaches a certain threshold.

  17. Structural basis of slow activation gating in the cardiac IKs channel complex

    DEFF Research Database (Denmark)

    Strutz-Seebohm, Nathalie; Pusch, Michael; Wolf, Steffen

    2011-01-01

    of the voltage sensor domain S4 of KCNQ1 in a putative pre-open channel state. Formation of this state may induce slow activation gating, the pivotal characteristic of native cardiac I(Ks) channels. This new KCNQ1-KCNE1 model may become useful for dynamic modeling of disease-associated mutant I(Ks) channels.......Accessory ß-subunits of the KCNE gene family modulate the function of various cation channel a-subunits by the formation of heteromultimers. Among the most dramatic changes of biophysical properties of a voltage-gated channel by KCNEs are the effects of KCNE1 on KCNQ1 channels. KCNQ1 and KCNE1...... are believed to form nativeI(Ks) channels. Here, we characterize molecular determinants of KCNE1 interaction with KCNQ1 channels by scanning mutagenesis, double mutant cycle analysis, and molecular dynamics simulations. Our findings suggest that KCNE1 binds to the outer face of the KCNQ1 channel pore domain...

  18. Effect of Diaminopropionic acid (Dap) on the Biophysical Properties of a Modified Synthetic Channel-Forming Peptide

    OpenAIRE

    Bukovnik, Urska; Sala-Rabanal, Monica; Francis, Simonne; Frazier, Shawnalea J.; Schultz, Bruce D.; Nichols, Colin G.; John M Tomich

    2013-01-01

    Channel replacement therapy, based on synthetic channel-forming peptides (CFPs) with the ability to supersede defective endogenous ion channels, is a novel treatment modality that may augment existing interventions against multiple diseases. Previously, we derived CFPs from the second transmembrane segment of the α-subunit of the glycine receptor, M2GlyR, which forms chloride-selective channels in its native form. The best candidate, NK4-M2GlyR T19R, S22W (p22-T19R, S22W), was water-soluble, ...

  19. Heart failure drug digitoxin induces calcium uptake into cells by forming transmembrane calcium channels

    OpenAIRE

    2008-01-01

    Digitoxin and other cardiac glycosides are important, centuries-old drugs for treating congestive heart failure. However, the mechanism of action of these compounds is still being elucidated. Calcium is known to potentiate the toxicity of these drugs, and we have hypothesized that digitoxin might mediate calcium entry into cells. We report here that digitoxin molecules mediate calcium entry into intact cells. Multimers of digitoxin molecules also are able to form calcium channels in pure plan...

  20. Membrane bioelectrogenesis and ionic channel activity simulation under drug action.

    Science.gov (United States)

    Pennec, J P; Iacobas, A D; Iacobas, S

    1995-01-01

    The concept of the ionic channel, as a protein with more discrete ionic conductance levels, is analyzed and a computer program, aiming to simulate its dynamics under the action of a pharmacological agent, is presented. The binding of a pharmacological agent modifies the set of the possible configurations and the channel energy spectrum. Therefore it modifies the transition probabilities and the dwelling times in the excited (generally conductive) states. The program creates a library of simulations of the electric activity of some user designated hypothetical models. The simulations are then statistically compared to the recorded single channel events to select the most suitable model.

  1. ROMK1 channel activity is regulated by monoubiquitination.

    Science.gov (United States)

    Lin, Dao-Hong; Sterling, Hyacinth; Wang, Zhijian; Babilonia, Elisa; Yang, Baofeng; Dong, Ke; Hebert, Steven C; Giebisch, Gerhard; Wang, Wen-Hui

    2005-03-22

    The ubiquitination of proteins can signal their degradation, modify their activity or target them to specific membranes or cellular organelles. Here, we show that monoubiquitination regulates the plasma membrane abundance and function of the potassium channel, ROMK. Immunoprecipitation of proteins obtained from renal cortex and outer medulla with ROMK antibody revealed that this channel was monoubiquitinated. To determine the ubiquitin binding site on ROMK1, all intracellular lysine (Lys) residues of ROMK1 were individually mutated to arginine (Arg), and a two-electrode voltage clamp was used to measure the ROMK1 channel activity in Xenopus oocytes. ROMK1 channel activity increased from 8.1 to 27.2 microA only when Lys-22 was mutated to Arg. Furthermore, Western blotting failed to detect the ubiquitinated ROMK1 in oocytes injected with R1K22R. Patch-clamp experiments showed that biophysical properties of R1K22R were identical to those of wild-type ROMK1. Although total protein expression levels of GFP-ROMK1 and GFP-R1K22R in oocytes were similar, confocal microscopy showed that the surface fluorescence intensity in oocytes injected with GFP-R1K22R was higher than that of GFP-ROMK1. In addition, biotin labeling of ROMK1 and R1K22R proteins expressed in HEK293 cells showed increased surface expression of the Lys-22 mutant channel. Finally, expression of R1K22R in COS7 cells significantly stimulated the surface expression of ROMK1. We conclude that ROMK1 can be monoubiquitinated and that Lys-22 is an ubiquitin-binding site. Thus, monoubiquitination of ROMK1 regulates channel activity by reducing the surface expression of channel protein. This finding implicates the linking of a single ubiquitin molecule to channels as an important posttranslational regulatory signal.

  2. Cell volume and membrane stretch independently control K+ channel activity.

    Science.gov (United States)

    Hammami, Sofia; Willumsen, Niels J; Olsen, Hervør L; Morera, Francisco J; Latorre, Ramón; Klaerke, Dan A

    2009-05-15

    A number of potassium channels including members of the KCNQ family and the Ca(2+) activated IK and SK, but not BK, are strongly and reversibly regulated by small changes in cell volume. It has been argued that this general regulation is mediated through sensitivity to changes in membrane stretch. To test this hypothesis we have studied the regulation of KCNQ1 and BK channels after expression in Xenopus oocytes. Results from cell-attached patch clamp studies (approximately 50 microm(2) macropatches) in oocytes expressing BK channels demonstrate that the macroscopic volume-insensitive BK current increases with increasing negative hydrostatic pressure (suction) applied to the pipette. Thus, at a pipette pressure of -5.0 +/- 0.1 mmHg the increase amounted to 381 +/- 146% (mean +/- S.E.M., n = 6, P < 0.025). In contrast, in oocytes expressing the strongly volume-sensitive KCNQ1 channel, the current was not affected by membrane stretch. The results indicate that (1) activation of BK channels by local membrane stretch is not mimicked by membrane stress induced by cell swelling, and (2) activation of KCNQ1 channels by cell volume increase is not mediated by local tension in the cell membrane. We conclude that stretch and volume sensitivity can be considered two independent regulatory mechanisms.

  3. Stretch-activated cation channel from larval bullfrog skin.

    Science.gov (United States)

    Hillyard, Stanley D; Willumsen, Niels J; Marrero, Mario B

    2010-05-01

    Cell-attached patches from isolated epithelial cells from larval bullfrog skin revealed a cation channel that was activated by applying suction (-1 kPa to -4.5 kPa) to the pipette. Activation was characterized by an initial large current spike that rapidly attenuated to a stable value and showed a variable pattern of opening and closing with continuing suction. Current-voltage plots demonstrated linear or inward rectification and single channel conductances of 44-56 pS with NaCl or KCl Ringer's solution as the pipette solution, and a reversal potential (-V(p)) of 20-40 mV. The conductance was markedly reduced with N-methyl-D-glucamide (NMDG)-Cl Ringer's solution in the pipette. Neither amiloride nor ATP, which are known to stimulate an apical cation channel in Ussing chamber preparations of larval frog skin, produced channel activation nor did these compounds affect the response to suction. Stretch activation was not affected by varying the pipette concentrations of Ca(2+) between 0 mmol l(-1) and 4 mmol l(-1) or by varying pH between 6.8 and 8.0. However, conductance was reduced with 4 mmol l(-1) Ca(2+). Western blot analysis of membrane homogenates from larval bullfrog and larval toad skin identified proteins that were immunoreactive with mammalian TRPC1 and TRPC5 (TRPC, canonical transient receptor potential channel) antibodies while homogenates of skin from newly metamorphosed bullfrogs were positive for TRPC1 and TRPC3/6/7 antibodies. The electrophysiological response of larval bullfrog skin resembles that of a stretch-activated cation channel characterized in Xenopus oocytes and proposed to be TRPC1. These results indicate this channel persists in all life stages of anurans and that TRP isoforms may be important for sensory functions of their skin.

  4. Variability in form and growth of sediment waves on turbidite channel levees

    Science.gov (United States)

    Normark, W.R.; Piper, D.J.W.; Posamentier, H.; Pirmez, C.; Migeon, S.

    2002-01-01

    Fine-grained sediment waves have been observed in many modern turbidite systems, generally restricted to the overbank depositional element. Sediment waves developed on six submarine fan systems are compared using high-resolution seismic-reflection profiles, sediment core samples (including ODP drilling), multibeam bathymetry, 3D seismic-reflection imaging (including examples of burried features), and direct measurements of turbidity currents that overflow their channels. These submarine fan examples extend over more than three orders of magnitude in physical scale. The presence or absence of sediment waves is not simply a matter of either the size of the turbidite channel-levee systems or the dominant initiation process for the turbidity currents that overflow the channels to form the wave fields. Both sediment-core data and seismic-reflection profiles document the upslope migration of the wave forms, with thicker and coarser beds deposited on the up-current flank of the waves. Some wave fields are orthogonal to channel trend and were initiated by large flows whose direction was controlled by upflow morphology, whereas fields subparallel to channel levees resulted from local spillover. In highly meandering systems, sediment waves may mimic meander planform. Larger sediment waves form on channel-levee systems with thicker overflow of turbidity currents, but available data indicate that sediment waves can be maintaned during conditions of relatively thin overflow. Coarser-grained units in sediment waves are typically laminated and thin-bedded sand as much as several centimetres thick, but sand beds as thick as several tens of centimetres have been documented from both modern and buried systems. Current production of hydrocarbons from sediment-wave deposits suggests that it is important to develop criteria for recognising this overbank element in outcrop exposures and borehole data, where the wavelength of typical waves (several kilometres) generally exceeds outcrop

  5. Block of a Ca(2+)-activated potassium channel by cocaine.

    Science.gov (United States)

    Premkumar, L S

    2005-04-01

    The primary target for cocaine is believed to be monoamine transporters because of cocaine's high-affinity binding that prevents re-uptake of released neurotransmitter. However, direct interaction with ion channels has been shown to be important for certain pharmacological/toxicological effects of cocaine. Here I show that cocaine selectively blocks a calcium-dependent K(+) channel in hippocampal neurons grown in culture (IC(50)=approximately 30 microM). Single-channel recordings show that in the presence of cocaine, the channel openings are interrupted with brief closures (flicker block). As the concentration of cocaine is increased the open-time is reduced, whereas the duration of brief closures is independent of concentration. The association and dissociation rate constants of cocaine for the neuronal Ca(2+)-activated K(+ )channels are 261+/-37 microM: (-1)s(-1) and 11451+/-1467 s(-1). The equilibrium dissociation constant (K(B)) for cocaine, determined from single-channel parameters, is 43 microM. The lack of voltage dependence of block suggests that cocaine probably binds to a site at the mouth of the pore. Block of Ca(2+)-dependent K(+) channels by cocaine may be involved in functions that include broadening of the action potential, which would facilitate transmitter release, enhancement of smooth muscle contraction particularly in blood vessels, and modulation of repetitive neuronal firing by altering the repolarization and afterhyperpolarization phases of the action potential.

  6. Selective activation of mechanosensitive ion channels using magnetic particles.

    Science.gov (United States)

    Hughes, Steven; McBain, Stuart; Dobson, Jon; El Haj, Alicia J

    2008-08-01

    This study reports the preliminary development of a novel magnetic particle-based technique that permits the application of highly localized mechanical forces directly to specific regions of an ion-channel structure. We demonstrate that this approach can be used to directly and selectively activate a mechanosensitive ion channel of interest, namely TREK-1. It is shown that manipulation of particles targeted against the extended extracellular loop region of TREK-1 leads to changes in whole-cell currents consistent with changes in TREK-1 activity. Responses were absent when particles were coated with RGD (Arg-Gly-Asp) peptide or when magnetic fields were applied in the absence of magnetic particles. It is concluded that changes in whole-cell current are the result of direct force application to the extracellular loop region of TREK-1 and thus these results implicate this region of the channel structure in mechano-gating. It is hypothesized that the extended loop region of TREK-1 may act as a tension spring that acts to regulate sensitivity to mechanical forces, in a nature similar to that described for MscL. The development of a technique that permits the direct manipulation of mechanosensitive ion channels in real time without the need for pharmacological drugs has huge potential benefits not only for basic biological research of ion-channel gating mechanisms, but also potentially as a tool for the treatment of human diseases caused by ion-channel dysfunction.

  7. [Polymethoxylated flavonoids activate cystic fibrosis transmembrane conductance regulator chloride channel].

    Science.gov (United States)

    Cao, Huan-Huan; Fang, Fang; Yu, Bo; Luan, Jian; Jiang, Yu; Yang, Hong

    2015-04-25

    Cystic fibrosis transmembrane conductance regulator (CFTR), a cAMP-dependent chloride channel, plays key roles in fluid secretion in serous epithelial cells. Previously, we identified two polymethoxylated flavonoids, 3',4',5,5',6,7-hexamethoxyflavone (HMF) and 5-hydroxy-6,7,3',4'-tetramethoxyflavone (HTF) which could potentiate CFTR chloride channel activities. The present study was aimed to investigate the potentiation effects of HMF and HTF on CFTR Cl(-) channel activities by using a cell-based fluorescence assay and the short circuit Ussing chamber assay. The results of cell-based fluorescence assay showed that both HMF and HTF could dose-dependently potentiate CFTR Cl(-) channel activities in rapid and reversible ways, and the activations could be reversed by the CFTR blocker CFTRinh-172. Notably, HMF showed the highest affinity (EC50 = 2 μmol/L) to CFTR protein among the flavonoid CFTR activators identified so far. The activation of CFTR by HMF or HTF was forskolin (FSK) dependent. Both compounds showed additive effect with FSK and 3-Isobutyl-1-methylx (IBMX) in the activation of CFTR, while had no additive effect with genistein (GEN). In ex vivo studies, HMF and HTF could stimulate transepithelial Cl(-) secretion in rat colonic mucosa and enhance fluid secretion in mouse trachea submucosal glands. These results suggest that HMF and HTF may potentiate CFTR Cl(-) channel activities through both elevation of cAMP level and binding to CFTR protein pathways. The results provide new clues in elucidating structure and activity relationship of flavonoid CFTR activators. HMF might be developed as a new drug in the therapy of CFTR-related diseases such as bronchiectasis and habitual constipation.

  8. Heterogeneity of Calcium Channel/cAMP-Dependent Transcriptional Activation.

    Science.gov (United States)

    Kobrinsky, Evgeny

    2015-01-01

    The major function of the voltage-gated calcium channels is to provide the Ca(2+) flux into the cell. L-type voltage-gated calcium channels (Cav1) serve as voltage sensors that couple membrane depolarization to many intracellular processes. Electrical activity in excitable cells affects gene expression through signaling pathways involved in the excitation-transcription (E-T) coupling. E-T coupling starts with activation of the Cav1 channel and results in initiation of the cAMP-response element binding protein (CREB)-dependent transcription. In this review we discuss the new quantitative approaches to measuring E-T signaling events. We describe the use of wavelet transform to detect heterogeneity of transcriptional activation in nuclei. Furthermore, we discuss the properties of discovered microdomains of nuclear signaling associated with the E-T coupling and the basis of the frequency-dependent transcriptional regulation.

  9. Light-Activated Ion Channels for Remote Control of Neural Activity

    OpenAIRE

    Chambers, James J.; Richard H Kramer

    2008-01-01

    Light-activated ion channels provide a new opportunity to precisely and remotely control neuronal activity for experimental applications in neurobiology. In the past few years, several strategies have arisen that allow light to control ion channels and therefore neuronal function. Light-based triggers for ion channel control include caged compounds, which release active neurotransmitters when photolyzed with light, and natural photoreceptive proteins, which can be expressed exogenously in neu...

  10. Non-specific activation of the epithelial sodium channel by the CFTR chloride channel

    OpenAIRE

    Nagel, Georg; Szellas, Tanjef; Riordan, John R.; Friedrich, Thomas; Hartung, Klaus

    2001-01-01

    The genetic disease cystic fibrosis is caused by mutation of the gene coding for the cystic fibrosis transmembrane conductance regulator (CFTR). Controversial studies reported regulation of the epithelial sodium channel (ENaC) by CFTR. We found that uptake of 22Na+ through ENaC is modulated by activation of CFTR in oocytes, coexpressing CFTR and ENaC, depending on extracellular chloride concentration. Furthermore we found that the effect of CFTR activation could be mimicked by other chloride ...

  11. Activation of Slo2.1 channels by niflumic acid.

    Science.gov (United States)

    Dai, Li; Garg, Vivek; Sanguinetti, Michael C

    2010-03-01

    Slo2.1 channels conduct an outwardly rectifying K(+) current when activated by high [Na(+)](i). Here, we show that gating of these channels can also be activated by fenamates such as niflumic acid (NFA), even in the absence of intracellular Na(+). In Xenopus oocytes injected with <10 ng cRNA, heterologously expressed human Slo2.1 current was negligible, but rapidly activated by extracellular application of NFA (EC(50) = 2.1 mM) or flufenamic acid (EC(50) = 1.4 mM). Slo2.1 channels activated by 1 mM NFA exhibited weak voltage dependence. In high [K(+)](e), the conductance-voltage (G-V) relationship had a V(1/2) of +95 mV and an effective valence, z, of 0.48 e. Higher concentrations of NFA shifted V(1/2) to more negative potentials (EC(50) = 2.1 mM) and increased the minimum value of G/G(max) (EC(50) = 2.4 mM); at 6 mM NFA, Slo2.1 channel activation was voltage independent. In contrast, V(1/2) of the G-V relationship was shifted to more positive potentials when [K(+)](e) was elevated from 1 to 300 mM (EC(50) = 21.2 mM). The slope conductance measured at the reversal potential exhibited the same [K(+)](e) dependency (EC(50) = 23.5 mM). Conductance was also [Na(+)](e) dependent. Outward currents were reduced when Na(+) was replaced with choline or mannitol, but unaffected by substitution with Rb(+) or Li(+). Neutralization of charged residues in the S1-S4 domains did not appreciably alter the voltage dependence of Slo2.1 activation. Thus, the weak voltage dependence of Slo2.1 channel activation is independent of charged residues in the S1-S4 segments. In contrast, mutation of R190 located in the adjacent S4-S5 linker to a neutral (Ala or Gln) or acidic (Glu) residue induced constitutive channel activity that was reduced by high [K(+)](e). Collectively, these findings indicate that Slo2.1 channel gating is modulated by [K(+)](e) and [Na(+)](e), and that NFA uncouples channel activation from its modulation by transmembrane voltage and intracellular Na(+).

  12. Anxiolytic effects of Maxipost (BMS-204352) and retigabine via activation of neuronal Kv7 channels.

    Science.gov (United States)

    Korsgaard, M P G; Hartz, B P; Brown, W D; Ahring, P K; Strøbaek, D; Mirza, N R

    2005-07-01

    Neuronal Kv7 channels are recognized as potential drug targets for treating hyperexcitability disorders such as pain, epilepsy, and mania. Hyperactivity of the amygdala has been described in clinical and preclinical studies of anxiety, and therefore, neuronal Kv7 channels may be a relevant target for this indication. In patch-clamp electrophysiology on cell lines expressing Kv7 channel subtypes, Maxipost (BMS-204352) exerted positive modulation of all neuronal Kv7 channels, whereas its R-enantiomer was a negative modulator. By contrast, at the Kv7.1 and the large conductance Ca2+-activated potassium channels, the two enantiomers showed the same effect, namely, negative and positive modulation at the two channels, respectively. At GABA(A) receptors (alpha1beta2gamma2s and alpha2beta2gamma2s) expressed in Xenopus oocytes, BMS-204352 was a negative modulator, and the R-enantiomer was a positive modulator. The observation that the S- and R-forms exhibited opposing effects on neuronal Kv7 channel subtypes allowed us to assess the potential role of Kv7 channels in anxiety. In vivo, BMS-204352 (3-30 mg/kg) was anxiolytic in the mouse zero maze and marble burying models of anxiety, with the effect in the burying model antagonized by the R-enantiomer (3 mg/kg). Likewise, the positive Kv7 channel modulator retigabine was anxiolytic in both models, and its effect in the burying model was blocked by the Kv7 channel inhibitor 10,10-bis-pyridin-4-ylmethyl-10H-anthracen-9-one (XE-991) (1 mg/kg). Doses at which BMS-204352 and retigabine induce anxiolysis could be dissociated from effects on sedation or memory impairment. In conclusion, these in vitro and in vivo studies provide compelling evidence that neuronal Kv7 channels are a target for developing novel anxiolytics.

  13. K sup + channel openers activate brain sulfonylurea-sensitive K sup + channels and block neurosecretion

    Energy Technology Data Exchange (ETDEWEB)

    Schmid-Antomarchi, H.; Amoroso, S.; Fosset, M.; Lazdunski, M. (Centre National de la Recherche Scientifique, Valbonne (France))

    1990-05-01

    Vascular K{sup +} channel openers such as cromakalim, nicorandil, and pinacidil potently stimulate {sup 86}Rb{sup +} efflux from slices of substantia nigra. This {sup 86}Rb{sup +} efflux is blocked by antidiabetic sulfonylureas, which are known to be potent and specific blockers of ATP-regulated K{sup +} channels in pancreatic beta cells, cardiac cells, and smooth muscle cells. K{sub 0.5}, the half-maximal effect of the enantiomer ({minus})-cromakalim, is as low as 10 nM, whereas K{sub 0.5} for nicorandil is 100 nM. These two compounds appear to have a much higher affinity for nerve cells than for smooth muscle cells. Openers of sulfonylurea-sensitive K{sup +} channels lead to inhibition of {gamma}-aminobutyric acid release. There is an excellent relationship between potency to activate {sup 86}Rb{sup +} efflux and potency to inhibit neurotransmitter release.

  14. Phosphoinositide isoforms determine compartment-specific ion channel activity.

    Science.gov (United States)

    Zhang, Xiaoli; Li, Xinran; Xu, Haoxing

    2012-07-10

    Phosphoinositides serve as address labels for recruiting peripheral cytoplasmic proteins to specific subcellular compartments, and as endogenous factors for modulating the activity of integral membrane proteins. Phosphatidylinositol 4,5-bisphosphate (PI(4,5)P(2)) is a plasma-membrane (PM)-specific phosphoinositide and a positive cofactor required for the activity of most PM channels and transporters. This requirement for phosphoinositide cofactors has been proposed to prevent PM channel/transporter activity during passage through the biosynthetic/secretory and endocytic pathways. To determine whether intracellularly localized channels are similarly "inactivated" at the PM, we studied PIP(2) modulation of intracellular TRPML1 channels. TRPML1 channels are primarily localized in lysosomes, but can also be detected temporarily in the PM upon lysosomal exocytosis. By directly patch-clamping isolated lysosomes, we previously found that lysosomal, but not PM-localized, TRPML1 is active with PI(3,5)P(2), a lysosome-specific PIP(2), as the underlying positive cofactor. Here we found that "silent" PM-localized TRPML1 could be activated by depleting PI(4,5)P(2) levels and/or by adding PI(3,5)P(2) to inside-out membrane patches. Unlike PM channels, surface-expressed TRPML1 underwent a unique and characteristic run-up upon patch excision, and was potently inhibited by a low micromolar concentration of PI(4,5)P(2). Conversely, depletion of PI(4,5)P(2) by either depolarization-induced activation or chemically induced translocation of 5'-phosphatase potentiated whole-cell TRPML1 currents. PI(3,5)P(2) activation and PI(4,5)P(2) inhibition of TRPML1 were mediated by distinct basic amino acid residues in a common PIP(2)-interacting domain. Thus, PI(4,5)P(2) may serve as a negative cofactor for intracellular channels such as TRPML1. Based on these results, we propose that phosphoinositide regulation sets compartment-specific activity codes for membrane channels and transporters.

  15. Radar Waveform Design in Active Communications Channel

    OpenAIRE

    Ric A. Romero; Shepherd, Kevin D.

    2013-01-01

    In this paper, we investigate spectrally adaptive radar transmit waveform design and its effects on an active communication system. We specifically look at waveform design for point targets. The transmit waveform is optimized by accounting for the modulation spectrum of the communication system while trying to efficiently use the remaining spectrum. With the use of spectrally-matched radar waveform, we show that the SER detection performance of the communication system ...

  16. Plan form changes of Gumara River channel over 50 years (Upper Blue Nile basin, Ethiopia)

    Science.gov (United States)

    Abate, Mengiste; Nyssen, Jan; Mehari, Michael

    2014-05-01

    Channel plan form changes were investigated along the 65 km long Gumara River in Lake Tana basin (Ethiopia) by overlaying information from aerial photographs and SPOT imagery. Two sets of aerial photographs (1957 and 1980) were scanned, and then orthorectified in ENVI 4.2 environment. Recent channel plan form information was extracted from SPOT images of 2006. ERDAS 2010 and ArcGIS 10.1 tools were used for the data preparation and analysis. The information on river plan form changes spans from 1957 to 2006 (49 years), during which time the Gumara catchment has been subjected to changes in land use/cover and increasing water abstraction, which may have affected its hydrogeomorphology. The results indicated that the lower reach of Gumara at its mouth has undergone major plan form changes. A delta of 1.12 km² was created between 1957 and 1980 and additional 1.00 km² land has been created between 1980 and 2006. The sinuosity of the plan form changed only slightly through the study period: 1.78 in 1957, 1.76 in 1980, and 1.81 in 2006. Comparison of cross sections at the hydrological gauging station showed that the river bed aggraded in the order of 1.5 m to 2.5 m for the period 1963-2009. The trend analysis of stream flow of Gumara River versus rainfall in the catchment also indicated that the bed level of the Gumara river at its gauging station has risen. From field observations, the impact of direct human interventions was identified. The building of artificial levees along the river banks has contributed to huge deposition in the river bed. At locations where intensive irrigation takes place in the floodplain, seepage water through the banks created river bank failure and modifications in plan form. The unstable segments of the river reach were identified and will be further analysed.

  17. Ligand-Gated Ion Channels: Permeation and Activation1

    Science.gov (United States)

    Lynch, Joseph W.; Barry, Peter H.

    Ligand-gated ion channels (LGICs) are fast-responding channels in which the receptor, which binds the activating molecule (the ligand), and the ion channel are part of the same nanomolecular protein complex. This chapter will describe the properties and functions of the nicotinic acetylcholine LGIC superfamily, which play a critical role in the fast chemical transmission of electrical signals between nerve cells at synapses and between nerve and muscle cells at endplates. All the processing functions of the brain and the resulting behavioral output depend on chemical transmission across such neuronal interconnections. To describe the properties of the channels of this LGIC superfamily,we will mainly use two examples of this family of channels: the excitatory nicotinic acetylcholine receptor (nAChR) and the inhibitory glycine receptor (GlyR) channels. In the chemical transmission of electrical signals, the arrival of an electrical signal at the synaptic terminal of a nerve causes the release of a chemical signal—a neurotransmitter molecule (the ligand, also referred to as the agonist). The neurotransmitter rapidly diffuses across the very narrow 20-40 nm synaptic gap between the cells and binds to the LGIC receptors in the membrane of the target (postsynaptic) cell and generates a new electrical signal in that cell (e.g., Kandel et al., 2000). How this chemical signal is converted into an electrical one depends on the fundamental properties of LGICs and the ionic composition of the postsynaptic cell and its external solution.

  18. STIM1 activates CRAC channels through rotation of the pore helix to open a hydrophobic gate

    Science.gov (United States)

    Yamashita, Megumi; Yeung, Priscilla S.-W.; Ing, Christopher E.; McNally, Beth A.; Pomès, Régis; Prakriya, Murali

    2017-02-01

    Store-operated Ca2+ release-activated Ca2+ (CRAC) channels constitute a major pathway for Ca2+ influx and mediate many essential signalling functions in animal cells, yet how they open remains elusive. Here, we investigate the gating mechanism of the human CRAC channel Orai1 by its activator, stromal interacting molecule 1 (STIM1). We find that two rings of pore-lining residues, V102 and F99, work together to form a hydrophobic gate. Mutations of these residues to polar amino acids produce channels with leaky gates that conduct ions in the resting state. STIM1-mediated channel activation occurs through rotation of the pore helix, which displaces the F99 residues away from the pore axis to increase pore hydration, allowing ions to flow through the V102-F99 hydrophobic band. Pore helix rotation by STIM1 also explains the dynamic coupling between CRAC channel gating and ion selectivity. This hydrophobic gating mechanism has implications for CRAC channel function, pharmacology and disease-causing mutations.

  19. How to Switch a Beam-Forming Network with Minimum Disturbance to Existing Communication Channels.

    Science.gov (United States)

    1976-04-02

    new EIRP, whichever is smaller. The temporary 3 dB drop in power is to take place over a short time and allows for the use of latching ferrite phase...all of the VPD’sif of the latch - ing ferrite varietymust go through the reset- set cycle (even if their power divisions are not to be changed) in order...LABORATORY 7F HOW TO SWITCH A BEAM -FORMING .NETWORK WITH MINIMUM PISTU1RBANCE TC EXISTING COMMUNICATION CHANNELS i ( J •: "h.’DeSIZE,/9 Group 6

  20. Fragmented inositol 1,4,5-trisphosphate receptors retain tetrameric architecture and form functional Ca2+ release channels.

    Science.gov (United States)

    Alzayady, Kamil J; Chandrasekhar, Rahul; Yule, David I

    2013-04-19

    Inositol 1,4,5-trisphosphate receptor isoforms are a family of ubiquitously expressed ligand-gated channels encoded by three individual genes. The proteins are localized to membranes of intracellular Ca(2+) stores and play pivotal roles in Ca(2+) homeostasis. Previous studies have demonstrated that IP3R1 is cleaved by the intracellular proteases calpain and caspase both in vivo and in vitro. However, the resultant cleavage products are poorly defined, and the functional consequences of these proteolytic events are not fully understood. We demonstrate that IP3R1 is cleaved during staurosporine-induced apoptosis, yielding N-terminal fragments encompassing the ligand-binding domain and the majority of the central modulatory domain together with a C-terminal fragment containing the channel domain and cytosolic tail. Notably, these fragments remain associated with the membrane after initiation of apoptotic cleavage. Furthermore, when recombinant IP3R1 fragments, corresponding to those predicted to be generated by caspase or calpain cleavage, are stably coexpressed in cells, they physically associate and form functional channels. These data provide novel insights regarding the regulation of IP3R1 during proteolysis and provide direct evidence that polypeptide continuity is not required for IP3R activation and Ca(2+) release.

  1. KCNQ4 channel activation by BMS-204352 and retigabine

    DEFF Research Database (Denmark)

    Schrøder, Rikke Louise K.; Jespersen, Thomas; Christophersen, P

    2001-01-01

    and concentration-dependent manner in the concentration range 0.1-10 microM. Both compounds shifted the KCNQ4 channel activation curves towards more negative potentials by about 10 mV. Further, the maximal current obtainable at large positive voltages was also increased concentration-dependently by both compounds...

  2. 75 FR 26782 - Agency Information Collection Activities: Form I-864, Form I-864A, Form I-864EZ, and Form I-864W...

    Science.gov (United States)

    2010-05-12

    ... Agency Information Collection Activities: Form I-864, Form I- 864A, Form I-864EZ, and Form I-864W... Collection Under Review; Form I- 864, Affidavit of Support Under Section 213A of the Act; Form I-864A, Contract Between Sponsor and Household Member, Form I-864EZ, Affidavit of Support Under Section 213A of...

  3. Combined single channel and single molecule detection identifies subunit composition of STIM1-activated transient receptor potential canonical (TRPC) channels.

    Science.gov (United States)

    Asanov, Alexander; Sampieri, Alicia; Moreno, Claudia; Pacheco, Jonathan; Salgado, Alfonso; Sherry, Ryan; Vaca, Luis

    2015-01-01

    Depletion of intracellular calcium ion stores initiates a rapid cascade of events culminating with the activation of the so-called Store-Operated Channels (SOC) at the plasma membrane. Calcium influx via SOC is essential in the initiation of calcium-dependent intracellular signaling and for the refilling of internal calcium stores, ensuring the regeneration of the signaling cascade. In spite of the significance of this evolutionary conserved mechanism, the molecular identity of SOC has been the center of a heated controversy spanning over the last 20 years. Initial studies positioned some members of the transient receptor potential canonical (TRPC) channel superfamily of channels (with the more robust evidence pointing to TRPC1) as a putative SOC. Recent evidence indicates that Stromal Interacting Molecule 1 (STIM1) activates some members from the TRPC family of channels. However, the exact subunit composition of TRPC channels remains undetermined to this date. To identify the subunit composition of STIM1-activated TRPC channels, we developed novel method, which combines single channel electrophysiological measurements based on the patch clamp technique with single molecule fluorescence imaging. We termed this method Single ion Channel Single Molecule Detection technique (SC-SMD). Using SC-SMD method, we have obtained direct evidence of the subunit composition of TRPC channels activated by STIM1. Furthermore, our electrophysiological-imaging SC-SMD method provides evidence at the molecular level of the mechanism by which STIM1 and calmodulin antagonize to modulate TRPC channel activity. Copyright © 2014 Elsevier Ltd. All rights reserved.

  4. Forming the organizational structure for activities

    Directory of Open Access Journals (Sweden)

    U. S. Barash

    2013-04-01

    Full Text Available Purpose. Development of theoretical and methodological foundations of efficiency of freight cars operating companies in railway reform through improved management structure them. Methodology. A theoretical and methodological approach for building effective management structure of freight wagons operating companies of different ownership forms is proposed, its introduction will significantly reduce detention of cars on technical stations under loading operations and maintenance, and thereby to improve the quality parameters of rolling stock usage in reform conditions of Ukraine railway transport. Findings. An improved control mechanism of cargo transportation is developed, it is different from the existing by its adaptation to the conditions of the reformed sector and the organization of management companies which together with the Ukrainian Transport and Logistics Center (UTLC centralize management of all freight cars of domestic and foreign operating companies. Originality. It is proposed for management of cargo transportation in wagons operating companies of different ownership to organize a series of management companies that would have the right to dispose of universal cars of other domestic operating companies, being on leasehold basis, and to direct them to current and scheduled repairs by themselves; to organize the cargo transportation in wagons of domestic and foreign operating companies on a contractual terms, depending on the type and content of the contract, on the basis of additional contracts for a separate fee to perform current and scheduled repair of freight cars; the management company organizational structure is developed, it includes simultaneously two directions of activity: commercial and repair, it will reduce the stay time of rolling stock on the engineering stations during loading and in a non-operating park as far as this company will manage a significant part of the production cycle of the transportation process

  5. [A comparative study of antiarrhythmic and antihypoxic effects of magnesium sulfate, its prolonged form and blockers of calcium channels].

    Science.gov (United States)

    Samsonia, M D; Kandelaki, M A

    2013-01-01

    The aim of the study is the comparative study of treatment of heart and brain damages during the hypoxia with magnesium sulfate, verapamil, diltiazem. As a result of the experiment carried out on rats it was proved that magnesium sulfate and its prolonged form are not less active than the blockers of calcium channels, such as verapamil and diltiazem. It is possible to avoid lethal fibrillations caused by calcium chloride with the help of 25% magnesium sulfate solution (after intraperitoneal administration with the dose of 1000 mg/kg) in case we make arrythmogenic injection 5 minutes after inputting magnesium sulfate solution. During the arrhythmia induced by calcium chloride prolonged form of magnesium sulfate is also effective only if we inject the drug subcutaneous 30 minutes before the arrythmogenic injection. If the interval is 5 minutes lethal fibrillations cant be avoided as the release of magnesium ions from the drug form is slowed down. The drugs containing magnesium ions also displayed cytoprotective activity on the model of normobaric hypoxia. This was resulted in the increase of protective index. Neuroprotective action of magnesium ions (in the condition of hypoxia) is caused by maintaining homeostasis of calcium ions and by inhibition of exocytosis of neuromediators in the synaptic cleft. Thus, magnesium sulfate and its prolonged form can be used with the purpose of pharmacocorrection of heart and brain injuries during hypoxic conditions.

  6. Cell swelling activates K+ and Cl- channels as well as nonselective, stretch-activated cation channels in ehrlich ascites tumor cells

    DEFF Research Database (Denmark)

    Christensen, Ove; Hoffmann, Else Kay

    1992-01-01

    Cell-attached patch-clamp recordings from Ehrlich ascites tumor cells reveal nonselective cation channels which are activated by mechanical deformation of the membrane. These channels are seen when suction is applied to the patch pipette or after osmotic cell swelling. The channel activation does...

  7. Design of a specific activator for skeletal muscle sodium channels uncovers channel architecture.

    Science.gov (United States)

    Cohen, Lior; Ilan, Nitza; Gur, Maya; Stühmer, Walter; Gordon, Dalia; Gurevitz, Michael

    2007-10-05

    Gating modifiers of voltage-gated sodium channels (Na(v)s) are important tools in neuroscience research and may have therapeutic potential in medicinal disorders. Analysis of the bioactive surface of the scorpion beta-toxin Css4 (from Centruroides suffusus suffusus) toward rat brain (rNa(v)1.2a) and skeletal muscle (rNa(v)1.4) channels using binding studies revealed commonality but also substantial differences, which were used to design a specific activator, Css4(F14A/E15A/E28R), of rNa(v)1.4 expressed in Xenopus oocytes. The therapeutic potential of Css4(F14A/E15A/E28R) was tested using an rNa(v)1.4 mutant carrying the same mutation present in the genetic disorder hypokalemic periodic paralysis. The activator restored the impaired gating properties of the mutant channel expressed in oocytes, thus offering a tentative new means for treatment of neuromuscular disorders with reduced muscle excitability. Mutant double cycle analysis employing toxin residues involved in the construction of Css4(F14A/E15A/E28R) and residues whose equivalents in the rat brain channel rNa(v)1.2a were shown to affect Css4 binding revealed significant coupling energy (>1.3 kcal/mol) between F14A and E592A at Domain-2/voltage sensor segments 1-2 (D2/S1-S2), R27Q and E1251N at D3/SS2-S6, and E28R with both E650A at D2/S3-S4 and E1251N at D3/SS2-S6. These results show that despite the differences in interactions with the rat brain and skeletal muscle Na(v)s, Css4 recognizes a similar region on both channel subtypes. Moreover, our data indicate that the S3-S4 loop of the voltage sensor module in Domain-2 is in very close proximity to the SS2-S6 segment of the pore module of Domain-3 in rNa(v)1.4. This is the first experimental evidence that the inter-domain spatial organization of mammalian Na(v)s resembles that of voltage-gated potassium channels.

  8. Plasmin in nephrotic urine activates the epithelial sodium channel

    DEFF Research Database (Denmark)

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

    2008-01-01

    Proteinuria and increased renal reabsorption of NaCl characterize the nephrotic syndrome. Here, we show that protein-rich urine from nephrotic rats and from patients with nephrotic syndrome activate the epithelial sodium channel (ENaC) in cultured M-1 mouse collecting duct cells and in Xenopus...... laevis oocytes heterologously expressing ENaC. The activation depended on urinary serine protease activity. We identified plasmin as a urinary serine protease by matrix-assisted laser desorption/ionization time of-flight mass spectrometry. Purified plasmin activated ENaC currents, and inhibitors...... of plasmin abolished urinary protease activity and the ability to activate ENaC. In nephrotic syndrome, tubular urokinase-type plasminogen activator likely converts filtered plasminogen to plasmin. Consistent with this, the combined application of urokinase-type plasminogen activator and plasminogen...

  9. The complete structure of an activated open sodium channel

    Science.gov (United States)

    Sula, Altin; Booker, Jennifer; Ng, Leo C. T.; Naylor, Claire E.; DeCaen, Paul G.; Wallace, B. A.

    2017-01-01

    Voltage-gated sodium channels (Navs) play essential roles in excitable tissues, with their activation and opening resulting in the initial phase of the action potential. The cycling of Navs through open, closed and inactivated states, and their closely choreographed relationships with the activities of other ion channels lead to exquisite control of intracellular ion concentrations in both prokaryotes and eukaryotes. Here we present the 2.45 Å resolution crystal structure of the complete NavMs prokaryotic sodium channel in a fully open conformation. A canonical activated conformation of the voltage sensor S4 helix, an open selectivity filter leading to an open activation gate at the intracellular membrane surface and the intracellular C-terminal domain are visible in the structure. It includes a heretofore unseen interaction motif between W77 of S3, the S4–S5 interdomain linker, and the C-terminus, which is associated with regulation of opening and closing of the intracellular gate. PMID:28205548

  10. Stretch-activated cation channel from larval bullfrog skin

    DEFF Research Database (Denmark)

    Hillyard, Stanley D; Willumsen, Niels J; Marrero, Mario B

    2010-01-01

    Cell-attached patches from isolated epithelial cells from larval bullfrog skin revealed a cation channel that was activated by applying suction (-1 kPa to -4.5 kPa) to the pipette. Activation was characterized by an initial large current spike that rapidly attenuated to a stable value and showed...... was markedly reduced with N-methyl-D-glucamide (NMDG)-Cl Ringer's solution in the pipette. Neither amiloride nor ATP, which are known to stimulate an apical cation channel in Ussing chamber preparations of larval frog skin, produced channel activation nor did these compounds affect the response to suction....... Stretch activation was not affected by varying the pipette concentrations of Ca(2+) between 0 mmol l(-1) and 4 mmol l(-1) or by varying pH between 6.8 and 8.0. However, conductance was reduced with 4 mmol l(-1) Ca(2+). Western blot analysis of membrane homogenates from larval bullfrog and larval toad skin...

  11. S-acylation dependent post-translational cross-talk regulates large conductance calcium- and voltage- activated potassium (BK channels

    Directory of Open Access Journals (Sweden)

    Michael J Shipston

    2014-08-01

    Full Text Available Mechanisms that control surface expression and/or activity of large conductance calcium-activated potassium (BK channels are important determinants of their (pathophysiological function. Indeed, BK channel dysfunction is associated with major human disorders ranging from epilepsy to hypertension and obesity. S-acylation (S-palmitoylation represents a major reversible, post-translational modification controlling the properties and function of many proteins including ion channels. Recent evidence reveals that both pore-forming and regulatory subunits of BK channels are S-acylated and control channel trafficking and regulation by AGC-family protein kinases. The pore-forming α-subunit is S-acylated at two distinct sites within the N- and C-terminus, each site being regulated by different palmitoyl acyl transferases (zDHHCs and acyl thioesterases. (APTs. S-acylation of the N-terminus controls channel trafficking and surface expression whereas S-acylation of the C-terminal domain determines regulation of channel activity by AGC-family protein kinases. S-acylation of the regulatory β4-subunit controls ER exit and surface expression of BK channels but does not affect ion channel kinetics at the plasma membrane. Furthermore, a significant number of previously identified BK-channel interacting proteins have been shown, or are predicted to be, S-acylated. Thus, the BK channel multi-molecular signalling complex may be dynamically regulated by this fundamental post-translational modification and thus S-acylation likely represents an important determinant of BK channel physiology in health and disease.

  12. Atomic basis for therapeutic activation of neuronal potassium channels

    DEFF Research Database (Denmark)

    Kim, Robin Y; Yau, Michael C; Galpin, Jason D

    2015-01-01

    chemical interactions required for retigabine action. Introduction of a non-natural isosteric H-bond-deficient Trp analogue abolishes channel potentiation, indicating that retigabine effects rely strongly on formation of a H-bond with the conserved pore Trp. Supporting this model, substitution...... with fluorinated Trp analogues, with increased H-bonding propensity, strengthens retigabine potency. In addition, potency of numerous retigabine analogues correlates with the negative electrostatic surface potential of a carbonyl/carbamate oxygen atom present in most KCNQ activators. These findings functionally...... pinpoint an atomic-scale interaction essential for effects of retigabine and provide stringent constraints that may guide rational improvement of the emerging drug class of KCNQ channel activators....

  13. NUMERICAL SIMULATION OF ROLL FORMING FOR CHANNEL SECTION WITH OUTER EDGE

    Institute of Scientific and Technical Information of China (English)

    韩志武; 刘才; 陆卫平

    2002-01-01

    The finite strip method in structural analysis has been extended, and elasticplastic large deformation spline finite strip method based on the Updated- Lagrange method ( U. L. method) was established to simulate roll forming process of channel section with outer edge. The deformation characteristics of strip was analyzed, and the threedimensional displacement field, strain field and stress field of deformed strip were got. The calculation example proves that the peak transverse pressing membrane strain is on the corner part of the deformed strip, and the peak longitudinal stretching strain is on the outer edge part of the deformed strip in front of rolls. In addition, the transverse deformation of the deformed strip is principal, and the longitudinal deformation is small.

  14. Active compounds release from semisolid dosage forms.

    Science.gov (United States)

    Olejnik, Anna; Goscianska, Joanna; Nowak, Izabela

    2012-11-01

    The aim of this paper is to review all the aspects of the in vitro release testing (IVRT) from semisolid dosage forms. Although none of the official dissolution methods has been specified for use with semisolid dosage forms, their utility for assessing release rates of drugs from semisolid dosage forms has become a topic of considerable interest. One can expect to overcome such complexity in the future, when the official "Topical and Transdermal Drug Products-Product Performance Tests" will be published in an issue of the Pharmacopeial Forum. Many factors such as type of the dissolution medium, membrane, temperature, and speed have an influence on the mechanism and kinetics of the release testing from gels, creams, and ointments; therefore, those parameters have been widely discussed.

  15. A conserved pre-block interaction motif regulates potassium channel activation and N-type inactivation.

    Directory of Open Access Journals (Sweden)

    Paul J Pfaffinger

    Full Text Available N-type inactivation occurs when the N-terminus of a potassium channel binds into the open pore of the channel. This study examined the relationship between activation and steady state inactivation for mutations affecting the N-type inactivation properties of the Aplysia potassium channel AKv1 expressed in Xenopus oocytes. The results show that the traditional single-step model for N-type inactivation fails to properly account for the observed relationship between steady state channel activation and inactivation curves. We find that the midpoint of the steady state inactivation curve depends in part on a secondary interaction between the channel core and a region of the N-terminus just proximal to the pore blocking peptide that we call the Inactivation Proximal (IP region. The IP interaction with the channel core produces a negative shift in the activation and inactivation curves, without blocking the pore. A tripeptide motif in the IP region was identified in a large number of different N-type inactivation domains most likely reflecting convergent evolution in addition to direct descent. Point mutating a conserved hydrophobic residue in this motif eliminates the gating voltage shift, accelerates recovery from inactivation and decreases the amount of pore block produced during inactivation. The IP interaction we have identified likely stabilizes the open state and positions the pore blocking region of the N-terminus at the internal opening to the transmembrane pore by forming a Pre-Block (P state interaction with residues lining the side window vestibule of the channel.

  16. Relevance of Viroporin Ion Channel Activity on Viral Replication and Pathogenesis

    Directory of Open Access Journals (Sweden)

    Jose L. Nieto-Torres

    2015-07-01

    Full Text Available Modification of host-cell ionic content is a significant issue for viruses, as several viral proteins displaying ion channel activity, named viroporins, have been identified. Viroporins interact with different cellular membranes and self-assemble forming ion conductive pores. In general, these channels display mild ion selectivity, and, eventually, membrane lipids play key structural and functional roles in the pore. Viroporins stimulate virus production through different mechanisms, and ion channel conductivity has been proved particularly relevant in several cases. Key stages of the viral cycle such as virus uncoating, transport and maturation are ion-influenced processes in many viral species. Besides boosting virus propagation, viroporins have also been associated with pathogenesis. Linking pathogenesis either to the ion conductivity or to other functions of viroporins has been elusive for a long time. This article summarizes novel pathways leading to disease stimulated by viroporin ion conduction, such as inflammasome driven immunopathology.

  17. Relevance of Viroporin Ion Channel Activity on Viral Replication and Pathogenesis.

    Science.gov (United States)

    Nieto-Torres, Jose L; Verdiá-Báguena, Carmina; Castaño-Rodriguez, Carlos; Aguilella, Vicente M; Enjuanes, Luis

    2015-07-03

    Modification of host-cell ionic content is a significant issue for viruses, as several viral proteins displaying ion channel activity, named viroporins, have been identified. Viroporins interact with different cellular membranes and self-assemble forming ion conductive pores. In general, these channels display mild ion selectivity, and, eventually, membrane lipids play key structural and functional roles in the pore. Viroporins stimulate virus production through different mechanisms, and ion channel conductivity has been proved particularly relevant in several cases. Key stages of the viral cycle such as virus uncoating, transport and maturation are ion-influenced processes in many viral species. Besides boosting virus propagation, viroporins have also been associated with pathogenesis. Linking pathogenesis either to the ion conductivity or to other functions of viroporins has been elusive for a long time. This article summarizes novel pathways leading to disease stimulated by viroporin ion conduction, such as inflammasome driven immunopathology.

  18. Taxation and forms of organizing business activities

    Directory of Open Access Journals (Sweden)

    Đinđić Srđan

    2013-01-01

    Full Text Available This paper takes sample tax regimes and tendencies from the developed countries in the EU-15 and the USA, and uses them to analyse the influence of taxation on the choice of organizational form of profit-oriented entities in Serbia. In order to understand how the procedure of taxation affects the sphere of business decision-making it is necessary to focus on the tax status of business losses and valorization and the effects of the double taxation of dividends. The rule of successive deduction of losses ensures the fiscally transparent entity receives a tax saving in the form of a reduction of the present value of the total paid tax. Meanwhile the corporation is handicapped because it postpones loss deductions, that is, it postpones tax saving, which directly influences the level of the present value of saved tax. The global trend of gradually moving from the classical system towards shareholder relief provision, above all in the form of a reduced withholding tax rate on dividends, has two opposing features: it simplifies the tax procedure while neglecting the distributional aims (consequences of taxation. The analysis of a particular practical example from the Serbian tax context enables us to draw a conclusion in relation to the relative taxes paid by entrepreneurs versus enterprises. The developed countries favour fiscally transparent entities, whereas Serbia allocates tax privileges to enterprises.

  19. Leptin excites POMC neurons via activation of TRPC channels

    Science.gov (United States)

    Qiu, Jian; Fang, Yuan; Rønnekleiv, Oline K.; Kelly, Martin J.

    2010-01-01

    Leptin can exert its potent appetite-suppressing effects via activation of hypothalamic proopiomelanocortin (POMC) neurons. It depolarizes POMC neurons via activation of a yet unidentified non-selective cation current. Therefore, we sought to identify the conductance activated by leptin using whole cell recording in EGFP-POMC neurons from transgenic mice. The TRPC channel blockers SKF96365, FFA and 2-APB potently inhibited the leptin-induced current. Also, lanthanum (La3+) and intracellular Ca2+ potentiated the effects of leptin. Moreover, the DAG permeable analog OAG failed to activate any TRPC current. Using a Cs+-gluconate-based internal solution, leptin-activated current reversed near -20 mV. After replacement of external Na+ and K+ with Cs+, the reversal shifted to near 0 mV, and the I/V curve exhibited a negative slope conductance at voltages more negative than –40 mV. Based on scRT-PCR, TRPC1 and TRPC4-7 mRNA were expressed in POMC neurons with TRPC5 being the most prevalent. The leptin-induced current was blocked by Jak2 inhibitor AG490, the PI3 Kinase inhibitor wortmannin and the phospholipase C inhibitors, U73122 and ET-18-OCH3. Notably, we identified PLCγ1 transcripts in the majority of POMC neurons. Therefore, leptin through a Jak2-PI3 kinase-PLCγ pathway activates TRPC channels, and TRPC1, 4 and 5 appear to be the key channels mediating the depolarizing effects of leptin in POMC neurons. PMID:20107083

  20. Hydrologically active palaeofluvial and subglacial channel networks beneath Humboldt Glacier, Greenland

    Science.gov (United States)

    Ely, Jeremy; Livingstone, Stephen; Chu, Winnie; Kingslake, Jonathan

    2017-04-01

    Subglacial drainage systems influence both the flow of overlying ice and the evolution of subglacial landscapes. Yet, the persistence, pattern, origin and spatio-temporal evolution of subglacial drainage remains poorly understood. Whilst the beds of former ice sheets record numerous examples of channelized subglacial drainage systems, any influence these may have had upon ice sheet dynamics is difficult to decipher without contemporary analogues. Therefore, in order to understand the fates of past, present and future ice sheets, further study of contemporary subglacial hydraulic systems is required. Here, we present evidence from satellite imagery, digital elevation models and radio-echo sounding data for previously unknown channelized networks beneath Humboldt Glacier, northern Greenland. We find that two major channel networks exist beneath Humboldt Glacier: (i) a dendritic channel network to the north of the catchment, which extends for over >250 km beneath the ice sheet; and (ii) a series of linear channels in the south of the catchment, which are up to 80 km in length, 2.5 km wide and 400 m deep. These two morphologically contrasting systems likely have separate origins. We interpret the dendritic channel network to be of palaeofluvial origin, whilst the linear channels are likely to be subglacially formed tunnel valleys - analogous to those observed on former ice sheet beds. Radio-echo sounding indicates that basal meltwater is actively being routed along both systems. The dichotomy in subglacial drainage system origin corresponds to a division in ice flow regime, with faster flowing ice occurring over the palaeo-fluvial system. We therefore hypothesise that the large-scale bed channelization by subglacial meltwater erosion, which occurs beneath the slower flowing southern portion of Humboldt, results in a long-term reduction in basal water pressures and ice flow velocities.

  1. 76 FR 41279 - Agency Information Collection Activities; Form I-864, Form I-864A, Form I-864EZ, and Form I-864W...

    Science.gov (United States)

    2011-07-13

    ... SECURITY U.S. Citizenship and Immigration Services Agency Information Collection Activities; Form I-864, Form I- 864A, Form I-864EZ, and Form I-864W; Extension of an Existing Information Collection; Comment Request ACTION: 30-Day Notice of Information Collection Under Review: Form I- 864, Affidavit of...

  2. 75 FR 51093 - Agency Information Collection Activities: Form I-864, Form I-864A, Form I-864EZ, and Form I-864W...

    Science.gov (United States)

    2010-08-18

    ... SECURITY U.S. Citizenship and Immigration Services Agency Information Collection Activities: Form I-864, Form I- 864A, Form I-864EZ, and Form I-864W; Extension of a Currently Approved Information Collection; Comment Request ACTION: 30-Day Notice of Information Collection Under Review: Form I- 864, Affidavit...

  3. Experience with ActiveX control for simple channel access

    Energy Technology Data Exchange (ETDEWEB)

    Timossi, C.; Nishimura, H.; McDonald, J.

    2003-05-15

    Accelerator control system applications at Berkeley Lab's Advanced Light Source (ALS) are typically deployed on operator consoles running Microsoft Windows 2000 and utilize EPICS[2]channel access for data access. In an effort to accommodate the wide variety of Windows based development tools and developers with little experience in network programming, ActiveX controls have been deployed on the operator stations. Use of ActiveX controls for use in the accelerator control environment has been presented previously[1]. Here we report on some of our experiences with the use and development of these controls.

  4. The S4-S5 linker of KCNQ1 channels forms a structural scaffold with the S6 segment controlling gate closure.

    Science.gov (United States)

    Labro, Alain J; Boulet, Inge R; Choveau, Frank S; Mayeur, Evy; Bruyns, Tine; Loussouarn, Gildas; Raes, Adam L; Snyders, Dirk J

    2011-01-07

    In vivo, KCNQ1 α-subunits associate with the β-subunit KCNE1 to generate the slowly activating cardiac potassium current (I(Ks)). Structurally, they share their topology with other Kv channels and consist out of six transmembrane helices (S1-S6) with the S1-S4 segments forming the voltage-sensing domain (VSD). The opening or closure of the intracellular channel gate, which localizes at the bottom of the S6 segment, is directly controlled by the movement of the VSD via an electromechanical coupling. In other Kv channels, this electromechanical coupling is realized by an interaction between the S4-S5 linker (S4S5(L)) and the C-terminal end of S6 (S6(T)). Previously we reported that substitutions for Leu(353) in S6(T) resulted in channels that failed to close completely. Closure could be incomplete because Leu(353) itself is the pore-occluding residue of the channel gate or because of a distorted electromechanical coupling. To resolve this and to address the role of S4S5(L) in KCNQ1 channel gating, we performed an alanine/tryptophan substitution scan of S4S5(L). The residues with a "high impact" on channel gating (when mutated) clustered on one side of the S4S5(L) α-helix. Hence, this side of S4S5(L) most likely contributes to the electromechanical coupling and finds its residue counterparts in S6(T). Accordingly, substitutions for Val(254) resulted in channels that were partially constitutively open and the ability to close completely was rescued by combination with substitutions for Leu(353) in S6(T). Double mutant cycle analysis supported this cross-talk indicating that both residues come in close contact and stabilize the closed state of the channel.

  5. Nitric oxide activates ATP-sensitive potassium channels in mammalian sensory neurons: action by direct S-nitrosylation

    Directory of Open Access Journals (Sweden)

    Kwok Wai-Meng

    2009-03-01

    Full Text Available Abstract Background ATP-sensitive potassium (KATP channels in neurons regulate excitability, neurotransmitter release and mediate protection from cell-death. Furthermore, activation of KATP channels is suppressed in DRG neurons after painful-like nerve injury. NO-dependent mechanisms modulate both KATP channels and participate in the pathophysiology and pharmacology of neuropathic pain. Therefore, we investigated NO modulation of KATP channels in control and axotomized DRG neurons. Results Cell-attached and cell-free recordings of KATP currents in large DRG neurons from control rats (sham surgery, SS revealed activation of KATP channels by NO exogenously released by the NO donor SNAP, through decreased sensitivity to [ATP]i. This NO-induced KATP channel activation was not altered in ganglia from animals that demonstrated sustained hyperalgesia-type response to nociceptive stimulation following spinal nerve ligation. However, baseline opening of KATP channels and their activation induced by metabolic inhibition was suppressed by axotomy. Failure to block the NO-mediated amplification of KATP currents with specific inhibitors of sGC and PKG indicated that the classical sGC/cGMP/PKG signaling pathway was not involved in the activation by SNAP. NO-induced activation of KATP channels remained intact in cell-free patches, was reversed by DTT, a thiol-reducing agent, and prevented by NEM, a thiol-alkylating agent. Other findings indicated that the mechanisms by which NO activates KATP channels involve direct S-nitrosylation of cysteine residues in the SUR1 subunit. Specifically, current through recombinant wild-type SUR1/Kir6.2 channels expressed in COS7 cells was activated by NO, but channels formed only from truncated isoform Kir6.2 subunits without SUR1 subunits were insensitive to NO. Further, mutagenesis of SUR1 indicated that NO-induced KATP channel activation involves interaction of NO with residues in the NBD1 of the SUR1 subunit. Conclusion NO

  6. Activation of stretch-activated channels and maxi-K+ channels by membrane stress of human lamina cribrosa cells.

    LENUS (Irish Health Repository)

    Irnaten, Mustapha

    2009-01-01

    The lamina cribrosa (LC) region of the optic nerve head is considered the primary site of damage in glaucomatous optic neuropathy. Resident LC cells have a profibrotic potential when exposed to cyclical stretch. However, the mechanosensitive mechanisms of these cells remain unknown. Here the authors investigated the effects of membrane stretch on cell volume change and ion channel activity and examined the associated changes in intracellular calcium ([Ca(2+)](i)).

  7. UAV Measurement of the 2015 Large Flood Impact in Kinugawa River on Riverine Vegetation and Channel Form Changes

    Science.gov (United States)

    Miyamoto, Hitoshi; Inoue, Toshiya; Chigasaki, Yuka

    2016-04-01

    This presentation gives the results of field observation for a flood impact on riverine environment measured by using an unmanned aerial vehicle (UAV). The flood we examined occurred on September 9-10, 2015 in Kinugawa River, Japan, owing to the heavy rainfall that brought tremendous volume of water on the Kanto and Tohoku regions of Japan. In Kinugawa River, the largest record flood occurred in this time, resulting in the levee failure and the corresponding flood disaster in Joso City located in the downstream part of Kinugawa River, as well as the large flood impact on the riverine environment in the Kinugawa channel network. In order to investigate the very initial state of the after-flood-impact throughout the channel network, 13 channel sections with 2 km in longitudinal length were chosen and observed in October 2015. Orthochromatic images of the river channel sections obtained by the UAV measurement with a geographic information system (GIS) were used for analyzing the changes in riverine vegetation distributions and channel form profiles. The results show that there exist three characteristic river segments having different impact-response states in vegetation and channel form changes. The river sections in the most upstream segment indicated severe damage of trees and herbs as well as large movement of gravel bed material, while those in the most downstream segment showed relatively small damage in vegetation distribution and small change in channel forms. Furthermore, relationships between the vegetation damage, channel deformation, channel slopes, and bed shear stresses calculated by a numerical simulation model were discussed in detail along the river network.

  8. Assessment on Ultimate Load of Cold-formed Steel Channel (CFSC Stub Column

    Directory of Open Access Journals (Sweden)

    Mohd Sani Mohd Syahrul Hisyam

    2015-01-01

    Full Text Available Cold-formed steel is used as the non-structural and structural material in civil engineering work and building. Cold-formed steel channel is selected and cut into 100 mm, 200 mm, 300 mm, 400 mm and 500 mm. The slenderness ratio is calculated and noted as a stub or short column because below 40. The column is tested by using Universal Testing Machine to determine the ultimate load of the stub column. Besides, the CFSC is determined the material properties of CFSC for checking it’s the originality of steel based material. The experimental data are tested and compared with the Direct Strength Method (DSM. It showed that the CFSC1 with a height of 100 mm is reported to have a higher value of ultimate load when compared with other samples. When the height of the stub column increased, the ultimate load of the sample is decreased. Then, the CFSC1 also showed a higher in initial stiffness when compared with other samples. All samples are shown having a higher data in ultimate load when compared with the Direct Strength Method prediction. The ultimate load of experimental and DSM all gave a ratio below 1.03. Finally, all samples can further recommend determining the relation between the ultimate loads with variations of height of the column.

  9. Extraction of Electromagnetic Transition Form Factors for Nucleon Resonances within a Dynamical Coupled-Channels Model

    Energy Technology Data Exchange (ETDEWEB)

    N. Suzuki, T. Sato, T.-S. H. Lee

    2010-10-01

    We explain the application of a recently developed analytic continuation method to extract the electromagnetic transition form factors for the nucleon resonances ($N^*$) within a dynamical coupled-channel model of meson-baryon reactions.Illustrative results of the obtained $N^*\\rightarrow \\gamma N$ transition form factors, defined at the resonance pole positions on the complex energy plane, for the well isolated $P_{33}$ and $D_{13}$, and the complicated $P_{11}$ resonances are presented. A formula has been developed to give an unified representation of the effects due to the first two $P_{11}$ poles, which are near the $\\pi\\Delta$ threshold, but are on different Riemann sheets. We also find that a simple formula, with its parameters determined in the Laurent expansions of $\\pi N \\rightarrow \\pi N$ and $\\gamma N \\rightarrow\\pi N$ amplitudes, can reproduce to a very large extent the exact solutions of the considered model at energies near the real parts of the extracted resonance positions. We indicate the differences between our results and those extracted from the approaches using the Breit-Wigner parametrization of resonant amplitudes to fit the data.

  10. Study of the Ion Channel Behavior of Didodecyldimethylammonium Bromide Formed Bilayer Lipid Membrane Stimulated by PF-6

    Institute of Scientific and Technical Information of China (English)

    TONG,Yue-Hong; HAN,Xiao-Jun; WANG,Er-Kang

    2003-01-01

    Bilayer lipid membranes ( BLM ) formed from didodecyldimethylammonium bromide were made on the freshly exposed surface ofa glassy carbon (GC) ani were demonstrated by the ac impedance spectroscopy. The ion channels of membrane properties induced by PF6- were studied by the cyclic voltammetric methods.Experimental results indicated that the ion channel of BLM was open in the presence of the PF6- due to the interaction of PF6- with the BLM, while it was switched offin the absence of PF6-. Because the ion channel behavior was affected by the concentration of PF6-,a sensor for PF6- can be developed.

  11. TRPC1 protein forms only one type of native store-operated channels in HEK293 cells.

    Science.gov (United States)

    Skopin, Anton; Shalygin, Alexey; Vigont, Vladimir; Zimina, Olga; Glushankova, Lyubov; Mozhayeva, Galina N; Kaznacheyeva, Elena

    2013-02-01

    TRPC1 is a major component of store-operated calcium entry in many cell types. In our previous studies, three types of endogenous store-operated calcium channels have been described in HEK293 cells, but it remained unknown which of these channels are composed of TRPC1 proteins. Here, this issue has been addressed by performing single-channel analysis in HEK293 cells transfected with anti-TRPC1 siRNA (siTPRC1) or a TPRC1-encoding plasmid. The results show that thapsigargin-or agonist-induced calcium influx is significantly attenuated in siTRPC1-transfected HEK293 cells. TRPC1 knockdown by siRNA results in the disappearance of store-operated I(max) channels, while the properties of I(min) and I(NS) channels are unaffected. In HEK293 cells with overexpressed TRPC1 protein, the unitary current-voltage relationship of exogenous TRPC1 channels is almost linear, with a slope conductance of about 17 pS. The extrapolated reversal potential of expressed TRPC1 channels is +30 mV. Therefore, the main electrophysiological and regulatory properties of expressed TRPC1 and native I(max) channels are identical. Moreover, TRPC1 overexpression in HEK293 cells results in an increased number of store-operated I(max) channels. All these data allow us to conclude that TRPC1 protein forms native store-operated I(max) channels but is not an essential subunit for other store-operated channel types in HEK293 cells.

  12. Calcium-activated potassium channels sustain calcium signaling in T lymphocytes. Selective blockers and manipulated channel expression levels.

    Science.gov (United States)

    Fanger, C M; Rauer, H; Neben, A L; Miller, M J; Rauer, H; Wulff, H; Rosa, J C; Ganellin, C R; Chandy, K G; Cahalan, M D

    2001-04-13

    To maintain Ca(2+) entry during T lymphocyte activation, a balancing efflux of cations is necessary. Using three approaches, we demonstrate that this cation efflux is mediated by Ca(2+)-activated K(+) (K(Ca)) channels, hSKCa2 in the human leukemic T cell line Jurkat and hIKCa1 in mitogen-activated human T cells. First, several recently developed, selective and potent pharmacological inhibitors of K(Ca) channels but not K(V) channels reduce Ca(2+) entry in Jurkat and in mitogen-activated human T cells. Second, dominant-negative suppression of the native K(Ca) channel in Jurkat T cells by overexpression of a truncated fragment of the cloned hSKCa2 channel decreases Ca(2+) influx. Finally, introduction of the hIKCa1 channel into Jurkat T cells maintains rapid Ca(2+) entry despite pharmacological inhibition of the native small conductance K(Ca) channel. Thus, K(Ca) channels play a vital role in T cell Ca(2+) signaling.

  13. Modulation of KCNQ4 channel activity by changes in cell volume

    DEFF Research Database (Denmark)

    Hougaard, Charlotte; Klaerke, Dan A; Hoffmann, Else K;

    2004-01-01

    KCNQ4 channels expressed in HEK 293 cells are sensitive to cell volume changes, being activated by swelling and inhibited by shrinkage, respectively. The KCNQ4 channels contribute significantly to the regulatory volume decrease (RVD) process following cell swelling. Under isoosmotic conditions......, the KCNQ4 channel activity is modulated by protein kinases A and C, G protein activation, and a reduction in the intracellular Ca2+ concentration, but these signalling pathways are not responsible for the increased channel activity during cell swelling....

  14. Purinergic regulation of CFTR and Ca2+ -activated Cl- channels and K+ channels in human pancreatic duct epithelium

    DEFF Research Database (Denmark)

    Wang, Jing; Haanes, Kristian A; Novak, Ivana

    2013-01-01

    dependent on intracellular Ca(2+). Apically applied ATP/UTP stimulated CF transmembrane conductance regulator (CFTR) and Ca(2+)-activated Cl(-) (CaCC) channels, which were inhibited by CFTRinh-172 and niflumic acid, respectively. The basolaterally applied ATP stimulated CFTR. In CFPAC-1 cells, which have...... mutated CFTR, basolateral ATP and UTP had negligible effects. In addition to Cl(-) transport in Capan-1 cells, the effects of 5,6-dichloro-1-ethyl-1,3-dihydro-2H-benzimidazol-2-one (DC-EBIO) and clotrimazole indicated functional expression of the intermediate conductance K(+) channels (IK, KCa3...... receptors both Cl(-) channels (TMEM16A/ANO1 and CFTR) and K(+) channels (IK). The K(+) channels provide the driving force for Cl(-)-channel-dependent secretion, and luminal ATP provided locally or secreted from acini may potentiate secretory processes. Future strategies in augmenting pancreatic duct...

  15. Stretch-activated channels in stretch-induced muscle damage: role in muscular dystrophy.

    Science.gov (United States)

    Yeung, Ella W; Allen, David G

    2004-08-01

    1. Stretch-induced muscle injury results in the damage that causes reduced force and increased membrane permeability. This muscle damage is caused, in part, by ionic entry through stretch-activated channels and blocking these channels with Gd3+ or streptomycin reduces the force deficit associated with damage. 2. Dystrophin-deficient muscles are more susceptible to stretch-induced muscle injury and the recovery from injury can be incomplete. We have found that Na+ entry associated with stretch-induced injury is enhanced in dystrophin-deficient muscles and that blockers of stretch-activated channels are capable of preventing ionic entry and reducing muscle damage. 3. A model is presented that proposes links between stretch-induced injury, opening of stretch-activated channels, increased levels of intracellular ions and various forms of muscle damage. Although changes in Na+ accompany stretch-induced muscle injury, we believe that changes in Ca2+ probably have a more central role in the damage process.

  16. Channel Response to Gravel Mining Activities in Mountain Rivers

    Institute of Scientific and Technical Information of China (English)

    José Luis López S.

    2004-01-01

    The removal of bed material from active river channels usually affects the bed profile of the streambed, causing progressive degradation upstream and downstream of the extraction site. These effects can extend for kilometers affecting hydraulic structures located in the vicinity of the river reach. In this paper, the geomorphic effects of gravel mining are reviewed and summarized. Some cases in Venezuelan streams are presented to illustrate the problem. To describe the processes of erosion and sedimentation in a gravel extraction pit, a recent developed mathematical model for the simulation of flow and sediment transport in gravel-cobble bed streams is applied to a hypothetical case of gravel mining in a river channel. A simple rectangular dredge pit is imposed as initial condition in the channel bed, and changes in bed elevations and grain size distribution of bed material are calculated by using the numerical model. The process of deposition within the pit, and the downstream and upstream migration of the erosion wave are well simulated by the model and closely resemble the phenomena observed in laboratory experiments. The response of the friction coefficient to the changes in flow and bed elevations shows the importance in modeling adequately flow resistance and sediment transport in gravel-cobble bed streams.

  17. Endogenous Piezo1 Can Confound Mechanically Activated Channel Identification and Characterization.

    Science.gov (United States)

    Dubin, Adrienne E; Murthy, Swetha; Lewis, Amanda H; Brosse, Lucie; Cahalan, Stuart M; Grandl, Jörg; Coste, Bertrand; Patapoutian, Ardem

    2017-04-19

    A gold standard for characterizing mechanically activated (MA) currents is via heterologous expression of candidate channels in naive cells. Two recent studies described MA channels using this paradigm. TMEM150c was proposed to be a component of an MA channel partly based on a heterologous expression approach (Hong et al., 2016). In another study, Piezo1's N-terminal "propeller" domain was proposed to constitute an intrinsic mechanosensitive module based on expression of a chimera between a pore-forming domain of the mechanically insensitive ASIC1 channel and Piezo1 (Zhao et al., 2016). When we attempted to replicate these results, we found each construct conferred modest MA currents in a small fraction of naive HEK cells similar to the published work. Strikingly, these MA currents were not detected in cells in which endogenous Piezo1 was CRISPR/Cas9 inactivated. These results highlight the importance of choosing cells lacking endogenous MA channels to assay the mechanotransduction properties of various proteins. This Matters Arising paper is in response to Hong et al. (2016) and Zhao et al. (2016) in Neuron. See also the response papers by Hong et al. (2017) and Zhao et al. (2017) published concurrently with this Matters Arising. Copyright © 2017 Elsevier Inc. All rights reserved.

  18. Structural insights into Ca(2+)-activated long-range allosteric channel gating of RyR1.

    Science.gov (United States)

    Wei, Risheng; Wang, Xue; Zhang, Yan; Mukherjee, Saptarshi; Zhang, Lei; Chen, Qiang; Huang, Xinrui; Jing, Shan; Liu, Congcong; Li, Shuang; Wang, Guangyu; Xu, Yaofang; Zhu, Sujie; Williams, Alan J; Sun, Fei; Yin, Chang-Cheng

    2016-09-01

    Ryanodine receptors (RyRs) are a class of giant ion channels with molecular mass over 2.2 mega-Daltons. These channels mediate calcium signaling in a variety of cells. Since more than 80% of the RyR protein is folded into the cytoplasmic assembly and the remaining residues form the transmembrane domain, it has been hypothesized that the activation and regulation of RyR channels occur through an as yet uncharacterized long-range allosteric mechanism. Here we report the characterization of a Ca(2+)-activated open-state RyR1 structure by cryo-electron microscopy. The structure has an overall resolution of 4.9 Å and a resolution of 4.2 Å for the core region. In comparison with the previously determined apo/closed-state structure, we observed long-range allosteric gating of the channel upon Ca(2+) activation. In-depth structural analyses elucidated a novel channel-gating mechanism and a novel ion selectivity mechanism of RyR1. Our work not only provides structural insights into the molecular mechanisms of channel gating and regulation of RyRs, but also sheds light on structural basis for channel-gating and ion selectivity mechanisms for the six-transmembrane-helix cation channel family.

  19. Curcumin inhibits activation of TRPM2 channels in rat hepatocytes

    Directory of Open Access Journals (Sweden)

    E. Kheradpezhouh

    2016-04-01

    Full Text Available Oxidative stress is a hallmark of many liver diseases including viral and drug-induced hepatitis, ischemia-reperfusion injury, and non-alcoholic steatohepatitis. One of the consequences of oxidative stress in the liver is deregulation of Ca2+ homeostasis, resulting in a sustained elevation of the free cytosolic Ca2+ concentration ([Ca2+]c in hepatocytes, which leads to irreversible cellular damage. Recently it has been shown that liver damage induced by paracetamol and subsequent oxidative stress is, in large part, mediated by Ca2+ entry through Transient Receptor Potential Melastatin 2 (TRPM2 channels. Involvement of TRPM2 channels in hepatocellular damage induced by oxidative stress makes TRPM2 a potential therapeutic target for treatment of a range of oxidative stress-related liver diseases. We report here the identification of curcumin ((1E,6E-1,7-bis(4-hydroxy-3-methoxyphenyl-1,6-heptadiene-3,5-dione, a natural plant-derived polyphenol in turmeric spice, as a novel inhibitor of TRPM2 channel. Presence of 5 µM curcumin in the incubation medium prevented the H2O2- and paracetamol-induced [Ca2+]c rise in rat hepatocytes. Furthermore, in patch clamping experiments incubation of hepatocytes with curcumin inhibited activation of TRPM2 current by intracellular ADPR with IC50 of approximately 50 nM. These findings enhance understanding of the actions of curcumin and suggest that the known hepatoprotective properties of curcumin are, at least in part, mediated through inhibition of TRPM2 channels.

  20. Proteolytic fragmentation of inositol 1,4,5-trisphosphate receptors: a novel mechanism regulating channel activity?

    Science.gov (United States)

    Wang, Liwei; Alzayady, Kamil J; Yule, David I

    2016-06-01

    Inositol 1,4,5-trisphosphate receptors (IP3 Rs) are a family of ubiquitously expressed intracellular Ca(2+) release channels. Regulation of channel activity by Ca(2+) , nucleotides, phosphorylation, protein binding partners and other cellular factors is thought to play a major role in defining the specific spatiotemporal characteristics of intracellular Ca(2+) signals. These properties are, in turn, believed pivotal for the selective and specific physiological activation of Ca(2+) -dependent effectors. IP3 Rs are also substrates for the intracellular cysteine proteases, calpain and caspase. Cleavage of the IP3 R has been proposed to play a role in apoptotic cell death by uncoupling regions important for IP3 binding from the channel domain, leaving an unregulated leaky Ca(2+) pore. Contrary to this hypothesis, we demonstrate following proteolysis that N- and C-termini of IP3 R1 remain associated, presumably through non-covalent interactions. Further, we show that complementary fragments of IP3 R1 assemble into tetrameric structures and retain their ability to be regulated robustly by IP3 . While peptide continuity is clearly not necessary for IP3 -gating of the channel, we propose that cleavage of the IP3 R peptide chain may alter other important regulatory events to modulate channel activity. In this scenario, stimulation of the cleaved IP3 R may support distinct spatiotemporal Ca(2+) signals and activation of specific effectors. Notably, in many adaptive physiological events, the non-apoptotic activities of caspase and calpain are demonstrated to be important, but the substrates of the proteases are poorly defined. We speculate that proteolytic fragmentation may represent a novel form of IP3 R regulation, which plays a role in varied adaptive physiological processes.

  1. BAX channel activity mediates lysosomal disruption linked to Parkinson disease.

    Science.gov (United States)

    Bové, Jordi; Martínez-Vicente, Marta; Dehay, Benjamin; Perier, Celine; Recasens, Ariadna; Bombrun, Agnes; Antonsson, Bruno; Vila, Miquel

    2014-05-01

    Lysosomal disruption is increasingly regarded as a major pathogenic event in Parkinson disease (PD). A reduced number of intraneuronal lysosomes, decreased levels of lysosomal-associated proteins and accumulation of undegraded autophagosomes (AP) are observed in PD-derived samples, including fibroblasts, induced pluripotent stem cell-derived dopaminergic neurons, and post-mortem brain tissue. Mechanistic studies in toxic and genetic rodent PD models attribute PD-related lysosomal breakdown to abnormal lysosomal membrane permeabilization (LMP). However, the molecular mechanisms underlying PD-linked LMP and subsequent lysosomal defects remain virtually unknown, thereby precluding their potential therapeutic targeting. Here we show that the pro-apoptotic protein BAX (BCL2-associated X protein), which permeabilizes mitochondrial membranes in PD models and is activated in PD patients, translocates and internalizes into lysosomal membranes early following treatment with the parkinsonian neurotoxin MPTP, both in vitro and in vivo, within a time-frame correlating with LMP, lysosomal disruption, and autophagosome accumulation and preceding mitochondrial permeabilization and dopaminergic neurodegeneration. Supporting a direct permeabilizing effect of BAX on lysosomal membranes, recombinant BAX is able to induce LMP in purified mouse brain lysosomes and the latter can be prevented by pharmacological blockade of BAX channel activity. Furthermore, pharmacological BAX channel inhibition is able to prevent LMP, restore lysosomal levels, reverse AP accumulation, and attenuate mitochondrial permeabilization and overall nigrostriatal degeneration caused by MPTP, both in vitro and in vivo. Overall, our results reveal that PD-linked lysosomal impairment relies on BAX-induced LMP, and point to small molecules able to block BAX channel activity as potentially beneficial to attenuate both lysosomal defects and neurodegeneration occurring in PD.

  2. Flow, form, and function: Distinguishing eco-hydraulic controls with relevance beyond the stream reach using synthetic channel morphologies

    Science.gov (United States)

    Lane, Belize; Pasternack, Gregory; Sandoval-Solis, Samuel

    2017-04-01

    Rivers are highly complex, dynamic systems that support numerous ecosystem functions including transporting sediment, modulating biogeochemical processes, and regulating habitat availability for native species. The extent and timing of these functions is largely controlled by the interplay of hydrologic dynamics (i.e., flow) and the shape and structure of the river channel (i.e., form). In spite of this, the majority of river restoration studies are limited to the influence of flow on ecosystem function without regard for the role of channel form in modulating eco-hydraulic response. The few studies that have effectively examined the flow-form interface highlight the scientific and management value of such analyses, but are highly resource intensive. This study represents a first attempt to apply synthetic channel design to the evaluation of river flow-form-function linkages, with the aim of improving basic understanding of how the interplay between flow and form affects ecosystem functions across a range of regionally-significant flows and forms with minimal resource requirements. Archetypal Mediterranean-montane channel types were used to guide the design of 3D synthetic morphologies. These morphologies were then used to quantify 2D eco-hydraulic response to different channel configurations under select hydrologic scenarios (distinguished by alteration and water year type). The eco-hydraulic performance of alternative flow-form settings, based on spatiotemporal patterns of depth and velocity, was evaluated with respect to a suite of river ecosystem functions related to geomorphic diversity, aquatic habitat, and riparian habitat. The methods described herein provide a potential design and inventory tool for quantifying river ecosystem functions and management trade-offs of alternative flow-form combinations with minimal resource and data requirements. While addressing specific scientific questions of interest for Mediterranean-montane rivers, the general framework

  3. Profound defects in pupillary responses to light in TRPM-channel null mice: a role for TRPM channels in non-image-forming photoreception.

    Science.gov (United States)

    Hughes, Steven; Pothecary, Carina A; Jagannath, Aarti; Foster, Russell G; Hankins, Mark W; Peirson, Stuart N

    2012-01-01

    TRPM1 is a spontaneously active non-selective cation channel that has recently been shown to play an important role in the depolarizing light responses of ON bipolar cells. Consistent with this role, mutations in the TRPM1 gene have been identified as a principal cause of congenital stationary night blindness. However, previous microarray studies have shown that Trpm1 and Trpm3 are acutely regulated by light in the eyes of mice lacking rods and cones (rd/rd cl), a finding consistent with a role in non-image-forming photoreception. In this study we show that pupillary light responses are significantly attenuated in both Trpm1(-/-) and Trpm3(-/-) animals. Trpm1(-/-) mice exhibit a profound deficit in the pupillary response that is far in excess of that observed in mice lacking rods and cones (rd/rd cl) or melanopsin, and cannot be explained by defects in bipolar cell function alone. Immunolocalization studies suggest that TRPM1 is expressed in ON bipolar cells and also a subset of cells in the ganglion cell layer, including melanopsin-expressing photosensitive retinal ganglion cells (pRGCs). We conclude that, in addition to its role in bipolar cell signalling, TRPM1 is involved in non-image-forming responses to light and may perform a functional role within pRGCs. By contrast, TRPM3(-/-) mice display a more subtle pupillary phenotype with attenuated responses under bright light and dim light conditions. Expression of TRPM3 is detected in Muller cells and the ciliary body but is absent from pRGCs, and thus our data support an indirect role for TRPM3 in pupillary light responses.

  4. On the estimation of cooperativity in ion channel kinetics: activation free energy and kinetic mechanism of Shaker K+ channel.

    Science.gov (United States)

    Banerjee, Kinshuk; Das, Biswajit; Gangopadhyay, Gautam

    2013-04-28

    In this paper, we have explored generic criteria of cooperative behavior in ion channel kinetics treating it on the same footing with multistate receptor-ligand binding in a compact theoretical framework. We have shown that the characterization of cooperativity of ion channels in terms of the Hill coefficient violates the standard Hill criteria defined for allosteric cooperativity of ligand binding. To resolve the issue, an alternative measure of cooperativity is proposed here in terms of the cooperativity index that sets a unified criteria for both the systems. More importantly, for ion channel this index can be very useful to describe the cooperative kinetics as it can be readily determined from the experimentally measured ionic current combined with theoretical modelling. We have analyzed the correlation between the voltage value and slope of the voltage-activation curve at the half-activation point and consequently determined the standard free energy of activation of the ion channel using two well-established mechanisms of cooperativity, namely, Koshland-Nemethy-Filmer (KNF) and Monod-Wyman-Changeux (MWC) models. Comparison of the theoretical results for both the models with appropriate experimental data of mutational perturbation of Shaker K(+) channel supports the experimental fact that the KNF model is more suitable to describe the cooperative behavior of this class of ion channels, whereas the performance of the MWC model is unsatisfactory. We have also estimated the mechanistic performance through standard free energy of channel activation for both the models and proposed a possible functional disadvantage in the MWC scheme.

  5. An amino acid outside the pore region influences apamin sensitivity in small conductance Ca2+-activated K+ channels.

    Science.gov (United States)

    Nolting, Andreas; Ferraro, Teresa; D'hoedt, Dieter; Stocker, Martin

    2007-02-01

    Small conductance calcium-activated potassium channels (SK, K(Ca)) are a family of voltage-independent K+ channels with a distinct physiology and pharmacology. The bee venom toxin apamin inhibits exclusively the three cloned SK channel subtypes (SK1, SK2, and SK3) with different affinity, highest for SK2, lowest for SK1, and intermediate for SK3 channels. The high selectivity of apamin made it a valuable tool to study the molecular makeup and function of native SK channels. Three amino acids located in the outer vestibule of the pore are of particular importance for the different apamin sensitivities of SK channels. Chimeric SK1 channels, enabling the homomeric expression of the rat SK1 (rSK1) subunit and containing the core domain (S1-S6) of rSK1, are apamin-insensitive. By contrast, channels formed by the human orthologue human SK1 (hSK1) are sensitive to apamin. This finding hinted at the involvement of regions beyond the pore as determinants of apamin sensitivity, because hSK1 and rSK1 have an identical amino acid sequence in the pore region. Here we investigated which parts of the channels outside the pore region are important for apamin sensitivity by constructing chimeras between apamin-insensitive and -sensitive SK channel subunits and by introducing point mutations. We demonstrate that a single amino acid situated in the extracellular loop between the transmembrane segments S3 and S4 has a major impact on apamin sensitivity. Our findings enabled us to convert the hSK1 channel into a channel that was as sensitive for apamin as SK2, the SK channel with the highest sensitivity.

  6. Copper and protons directly activate the zinc-activated channel

    DEFF Research Database (Denmark)

    Trattnig, Sarah Maria; Gasiorek, Agnes; Deeb, Tarek Z

    2016-01-01

    further. We demonstrate that not only zinc (Zn(2+)) but also copper (Cu(2+)) and protons (H(+)) are agonists of ZAC, displaying potencies and efficacies in the rank orders of H(+)>Cu(2+)>Zn(2+) and H(+)>Zn(2+)>Cu(2+), respectively. The responses elicited by Zn(2+), Cu(2+) and H(+) through ZAC are all...... characterized by low degrees of desensitization. In contrast, currents evoked by high concentrations of the three agonists comprise distinctly different activation and decay components, with transitions to and from an open state being significantly faster for H(+) than for the two metal ions. The permeabilities...

  7. Adaptive vector quantization in SVD MIMO system backward link with limited number of active sub channels

    Directory of Open Access Journals (Sweden)

    Ivaniš Predrag

    2004-01-01

    Full Text Available This paper presents combination of Channel Optimized Vector Quantization based on LBG algorithm and sub channel power allocation for MIMO systems with Singular Value Decomposition and limited number of active sub channels. Proposed algorithm is designed to enable maximal throughput with bit error rate bellow some tar- get level in case of backward channel capacity limitation. Presence of errors effect in backward channel is also considered.

  8. Decay of the excited compound system *56Ni formed through various channels using deformed Coulomb and deformed nuclear proximity potentials

    Science.gov (United States)

    Santhosh, K. P.; Subha, P. V.

    2017-06-01

    The total cross section, the intermediate mass fragment (IMF) production cross section, and the cross section for the formation of light particles (LPs) for the decay of compound system *56Ni formed through the entrance channel 32S+24Mg have been evaluated by taking the scattering potential as the sum of deformed Coulomb and deformed nuclear proximity potentials, for various Ec .m . values. The computed results have been compared with the available experimental data of total cross section corresponding to Ec .m .=60.5 and 51.6 MeV for the entrance channel 32S+24Mg , which were found to be in good agreement. The experimental values for the LP production cross section and IMF cross section for the channel 32S+24Mg were also found to agree with our calculations. Hence we have extended our studies and have thus computed the total cross section, IMF cross section, and LP cross section for the decay of *56Ni formed through the other three entrance channels 36Ar+20Ne,40Ca+16O , and 28Si+28Si with different Ec .m . values. Hence, we hope that our predictions on the evaluations of the IMF cross sections and the LP cross sections for the decay of *56Ni formed through these three channels can be used for further experimental studies.

  9. 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.

  10. Inhibition of T cell proliferation by selective block of Ca(2+)-activated K(+) channels

    DEFF Research Database (Denmark)

    Jensen, B S; Odum, Niels; Jorgensen, N K;

    1999-01-01

    T lymphocytes express a plethora of distinct ion channels that participate in the control of calcium homeostasis and signal transduction. Potassium channels play a critical role in the modulation of T cell calcium signaling, and the significance of the voltage-dependent K channel, Kv1.3, is well...... established. The recent cloning of the Ca(2+)-activated, intermediate-conductance K(+) channel (IK channel) has enabled a detailed investigation of the role of this highly Ca(2+)-sensitive K(+) channel in the calcium signaling and subsequent regulation of T cell proliferation. The role IK channels play in T...

  11. High basal melting forming a channel at the grounding line of Ross Ice Shelf, Antarctica

    Science.gov (United States)

    Marsh, Oliver J.; Fricker, Helen A.; Siegfried, Matthew R.; Christianson, Knut; Nicholls, Keith W.; Corr, Hugh F. J.; Catania, Ginny

    2016-01-01

    Antarctica's ice shelves are thinning at an increasing rate, affecting their buttressing ability. Channels in the ice shelf base unevenly distribute melting, and their evolution provides insight into changing subglacial and oceanic conditions. Here we used phase-sensitive radar measurements to estimate basal melt rates in a channel beneath the currently stable Ross Ice Shelf. Melt rates of 22.2 ± 0.2 m a-1 (>2500% the overall background rate) were observed 1.7 km seaward of Mercer/Whillans Ice Stream grounding line, close to where subglacial water discharge is expected. Laser altimetry shows a corresponding, steadily deepening surface channel. Two relict channels to the north suggest recent subglacial drainage reorganization beneath Whillans Ice Stream approximately coincident with the shutdown of Kamb Ice Stream. This rapid channel formation implies that shifts in subglacial hydrology may impact ice shelf stability.

  12. Nicotine activates the chemosensory cation channel TRPA1.

    Science.gov (United States)

    Talavera, Karel; Gees, Maarten; Karashima, Yuji; Meseguer, Víctor M; Vanoirbeek, Jeroen A J; Damann, Nils; Everaerts, Wouter; Benoit, Melissa; Janssens, Annelies; Vennekens, Rudi; Viana, Félix; Nemery, Benoit; Nilius, Bernd; Voets, Thomas

    2009-10-01

    Topical application of nicotine, as used in nicotine replacement therapies, causes irritation of the mucosa and skin. This reaction has been attributed to activation of nicotinic acetylcholine receptors (nAChRs) in chemosensory neurons. In contrast with this view, we found that the chemosensory cation channel transient receptor potential A1 (TRPA1) is crucially involved in nicotine-induced irritation. We found that micromolar concentrations of nicotine activated heterologously expressed mouse and human TRPA1. Nicotine acted in a membrane-delimited manner, stabilizing the open state(s) and destabilizing the closed state(s) of the channel. In the presence of the general nAChR blocker hexamethonium, nociceptive neurons showed nicotine-induced responses that were strongly reduced in TRPA1-deficient mice. Finally, TRPA1 mediated the mouse airway constriction reflex to nasal instillation of nicotine. The identification of TRPA1 as a nicotine target suggests that existing models of nicotine-induced irritation should be revised and may facilitate the development of smoking cessation therapies with less adverse effects.

  13. Synchronization of active atomic clocks via quantum and classical channels

    Science.gov (United States)

    Roth, Alexander; Hammerer, Klemens

    2016-10-01

    Superradiant lasers based on atomic ensembles exhibiting ultranarrow optical transitions can emit light of unprecedented spectral purity and may serve as active atomic clocks. We consider two frequency-detuned active atomic clocks, which are coupled in a cascaded setup, i.e., as master and slave lasers, and study the synchronization of the slave to the master clock. In a setup where both atomic ensembles are coupled to a common cavity mode, such synchronization phenomena have been predicted by Xu et al. [M. Xu, D. A. Tieri, E. C. Fine, J. K. Thompson, and M. J. Holland, Phys. Rev. Lett. 113, 154101 (2014)., 10.1103/PhysRevLett.113.154101] and experimentally observed by Weiner et al. (J. M. Weiner et al., arXiv:1503.06464). Here we demonstrate that synchronization still occurs in cascaded setups but exhibits distinctly different phase diagrams. We study the characteristics of synchronization in comparison to the case of coupling through a common cavity. We also consider synchronization through a classical channel where light of the master laser is measured phase sensitively and the slave laser is injection locked by feedback and compare to the results achievable by coupling through quantum channels.

  14. Endogenous chloride channels of insect sf9 cells. Evidence for coordinated activity of small elementary channel units

    DEFF Research Database (Denmark)

    Larsen, Erik Hviid; Gabriel, S. E.; Stutts, M. J.

    1996-01-01

    ) openings interrupted by similar long closures. In the open state, channels exhibited fast burst-like closures. Since the patches normally contained more than a single channel, it was not possible to measure open and closed dwell-time distributions for comparing single-Cl- channel activity with the kinetic....../150) of approximately 3.5 pS and approximately 35 pS, respectively. All states reversed near the same membrane potential, and they exhibited similar halide ion selectivity, P1 > PCl approximately PBr. Accordingly, Cl- current amplitudes larger than current flow through the smallest channel unit resolved seem to result...... from simultaneous open/shut events of two or more channel units....

  15. Structure of a potentially open state of a proton-activated pentameric ligand-gated ion channel.

    Science.gov (United States)

    Hilf, Ricarda J C; Dutzler, Raimund

    2009-01-01

    The X-ray structure of a pentameric ligand-gated ion channel from Erwinia chrysanthemi (ELIC) has recently provided structural insight into this family of ion channels at high resolution. The structure shows a homo-pentameric protein with a barrel-stave architecture that defines an ion-conduction pore located on the fivefold axis of symmetry. In this structure, the wide aqueous vestibule that is encircled by the extracellular ligand-binding domains of the five subunits narrows to a discontinuous pore that spans the lipid bilayer. The pore is constricted by bulky hydrophobic residues towards the extracellular side, which probably serve as barriers that prevent the diffusion of ions. This interrupted pore architecture in ELIC thus depicts a non-conducting conformation of a pentameric ligand-gated ion channel, the thermodynamically stable state in the absence of bound ligand. As ligand binding promotes pore opening in these ion channels and the specific ligand for ELIC has not yet been identified, we have turned our attention towards a homologous protein from the cyanobacterium Gloebacter violaceus (GLIC). GLIC was shown to form proton-gated channels that are activated by a pH decrease on the extracellular side and that do not desensitize after activation. Both prokaryotic proteins, ELIC and GLIC form ion channels that are selective for cations over anions with poor discrimination among monovalent cations, characteristics that resemble the conduction properties of the cation-selective branch of the family that includes acetylcholine and serotonin receptors. Here we present the X-ray structure of GLIC at 3.1 A resolution. The structure reveals a conformation of the channel that is distinct from ELIC and that probably resembles the open state. In combination, both structures suggest a novel gating mechanism for pentameric ligand-gated ion channels where channel opening proceeds by a change in the tilt of the pore-forming helices.

  16. Closed-Form Performance Analysis of Dual Polarization Based MIMO System in Shadowed-Rician Fading LMS Channels

    Directory of Open Access Journals (Sweden)

    Suresh Kumar Jindal

    2015-04-01

    Full Text Available In this paper, the problem of dual polarization based MIMO Processing in Shadowed-Rician (SR fading land mobile satellite (LMS channels is studied. It is shown in literature that polarization is used as a interference rejection method; and, most of the existing analytical results are not in closed-form. In this paper, we derive the closed-form expressions of the moment generating (MGF function of the received signal-to-noise ratio of the MRC based receiver in SR fading LMS channels. Then we provide closed-form expressions of the symbol error rate (SER by using MGF approach. The analytical diversity order and capacity of the considered scheme is also derived. It is shown by by derived closed-form capacity expression that the capacity of the considered dual polarization based scheme is improved; and it is found very useful in practical satellite communication systems.

  17. Vanilloid Receptor–Related Osmotically Activated Channel (VR-OAC), a Candidate Vertebrate Osmoreceptor

    National Research Council Canada - National Science Library

    Liedtke, Wolfgang; Choe, Yong; Martí-Renom, Marc A; Bell, Andrea M; Denis, Charlotte S; AndrejŠali; Hudspeth, A.J; Friedman, Jeffrey M; Heller, Stefan

    2000-01-01

    .... By employing a candidate-gene approach based on genes encoding members of the TRP superfamily of ion channels, we cloned cDNAs encoding the vanilloid receptor-related osmotically activated channel (VR-OAC...

  18. Observations of the Behavior and Distribution of Fish in Relation to the Columbia River Navigation Channel and Channel Maintenance Activities

    Energy Technology Data Exchange (ETDEWEB)

    Carlson, Thomas J.; Ploskey, Gene R.; Johnson, R. L.; Mueller, Robert P.; Weiland, Mark A.; Johnson, P. N.

    2001-10-19

    This report is a compilation of 7 studies conducted for the U.S. Army Corps of Engineers between 1995 and 1998 which used hydroacoustic methods to study the behavior of migrating salmon in response to navigation channel maintenance activities in the lower Columbia River near river mile 45. Differences between daytime and nighttime behavior and fish densities were noted. Comparisons were made of fish distribution across the river (in the channel, channel margin or near shore) and fish depth upstream and downstream of dikes, dredges, and pile driving areas.

  19. New insights into the activation mechanism of store-operated calcium channels:roles of STIM and Orai

    Institute of Scientific and Technical Information of China (English)

    Rui-wei GUO; Lan HUANG

    2008-01-01

    The activation of Ca2+ entry through store-operated channels by agonists that deplete Ca2+ from the endoplasmic reticulum (ER)is a ubiquitous signaling mechanism,the molecular basis of which has remained elusive for the past two decades.Store-operated Ca2+-release-activated Ca2+(CRAC)channels constitute the sole pathway for Ca2+ entry following antigen-receptor engagement.In a set of breakthrough studies over the past two years,stromal interaction molecule l(STIM1,tbe ER Ca2+ sensor) and Orail(a pore-forming subunit of the CRAC channel)have been identified.Here we review these recent studies and the insights they provide into the mechanism of store-operated Ca2+ channels(SOCCs).

  20. Cardioprotective effects of mitochondrial KATP channels activated at different time

    Institute of Scientific and Technical Information of China (English)

    魏珂; 闵苏; 龙村

    2004-01-01

    Backgroud Recent studies in adult hearts have indicated that KATP channels in the inner mitochondrial membrance are responsible for the protection. And we investigated whether opening of mitochondrial KATP channels (mKATP) could provide myocardial protection for immature rabbits and determined its role in cardioprotection.Methods Thirty-four 3-4-week-old rabbits, weighing 300-350 g, were divided randomly into five groups: Group Ⅰ (control group, n=8); Group Ⅱ [diazoxide preconditioning group; n=8; the hearts were pretreated with 100 μmol/L diazoxide for 5 minutes followed by 10-minute wash out with Krebs-Henseleit buffer (KHB)]; Group Ⅲ [diazoxide+5-hydroxydeconate (5-HD) preconditioning group; n=5; the hearts were pretreated with 100 μmol/L diazoxide and 100 μmol/L 5-HD); Group Ⅳ (diazoxide+cardioplegia group; n=8; cardioplegia containing 100 μmol/L diazoxide perfused the hearts for 5 minutes before ischemia); Group Ⅴ (diazoxide+5-HD+cardioplegia group; n=5; the cardioplegia contained 100 μmol/L diazoxide and 100 μmol/L 5-HD). All hearts were excised and connected to langend ?Zrff perfusion system and passively perfused with KHB at 38℃ under a pressure of 70 cmH2O. After reperfusion, the recovery rate of left ventricular diastolic pressure (LVDP), ±dp/dtmax, coronary flow (CF), the creatinine kinase (CK), lactate dehydrogenase (LDH), aspartate aminotransferase (AST) in coronary sinus venous effluent and the tissue ATP were measured. Mitochondria were evaluated semiquantitatively by morphology.Results After ischemia and reperfusion (I/R), the two groups that were treated by diazoxide only (Groups Ⅱ and Ⅳ) had a significant improvement in LVDP, ±dp/dtmax, and CF recovery. AST, LDH, and CK were decreased, and the levels of tissue ATP in the two groups were higher. Mitochondria was protected better in Group Ⅳ than in other groups. Conclusions Activating mKATP channels before and during ischemia can similarly protect immature rabbit hearts

  1. Interaction between 2 extracellular loops influences the activity of the cystic fibrosis transmembrane conductance regulator chloride channel.

    Science.gov (United States)

    Broadbent, Steven D; Wang, Wuyang; Linsdell, Paul

    2014-10-01

    Activity of the cystic fibrosis transmembrane conductance regulator (CFTR) chloride channel is thought to be controlled by cytoplasmic factors. However, recent evidence has shown that overall channel activity is also influenced by extracellular anions that interact directly with the extracellular loops (ECLs) of the CFTR protein. Very little is known about the structure of the ECLs or how substances interacting with these ECLs might affect CFTR function. We used patch-clamp recording to investigate the accessibility of cysteine-reactive reagents to cysteines introduced throughout ECL1 and 2 key sites in ECL4. Furthermore, interactions between ECL1 and ECL4 were investigated by the formation of disulfide crosslinks between cysteines introduced into these 2 regions. Crosslinks could be formed between R899C (in ECL4) and a number of sites in ECL1 in a manner that was dependent on channel activity, suggesting that the relative orientation of these 2 loops changes on activation. Formation of these crosslinks inhibited channel function, suggesting that relative movement of these ECLs is important to normal channel function. Implications of these findings for the effects of mutations in the ECLs that are associated with cystic fibrosis and interactions with extracellular substances that influence channel activity are discussed.

  2. Numerical Simulation and Sensitivity Analysis of Parameters for Multistand Roll Forming of Channel Section With Outer Edge

    Institute of Scientific and Technical Information of China (English)

    ZENG Guo; LAI Xin-min; YU Zhong-qi; LIN Zhong-qin

    2009-01-01

    Cold roll forming is a high production but complex metal forming process under the conditions of coupled effects with multi-factor.A new booting finite element method (FEM) model using the updated Lagrangian (UL) method for multistand roll forming process is developed and validated.Compared with most of the literatures related to roll forming simulation,the new model can take the roll rotation into account and is well suited for simulating multistand roll forming.Based on the model,the process of a channel section with outer edge formed with twelve passes is simulated and the sensitivity analysis of parameters is conducted with orthogonal design combined FEM model.It is found that the multiatand roll forming process can he efficiently analyzed by the new booting model,and sensitivity analysis shows that the yield strength plays an important role in controlling the quality of the products.

  3. Detection of TRPV4 channel current-like activity in Fawn Hooded hypertensive (FHH) rat cerebral arterial muscle cells

    National Research Council Canada - National Science Library

    Debebe Gebremedhin; David X Zhang; Dorothee Weihrauch; Nnamdi N Uche; David R Harder

    2017-01-01

    ...) rat, known to display exaggerated KCa channel current activity and impaired myogenic tone, express TRPV4 channels at the transcript and protein level and exhibit TRPV4-like single-channel cationic current activity...

  4. Human myoblast differentiation: Ca(2+) channels are activated by K(+) channels.

    Science.gov (United States)

    Bernheim, Laurent; Bader, Charles R

    2002-02-01

    In a paradigm of cellular differentiation, human myoblast fusion, we investigated how a Ca(2+) influx, indispensable for fusion, is triggered. We show how newly expressed Kir2.1 K(+) channels, via their hyperpolarizing effect on the membrane potential, generate a window Ca(2+) current (mediated by alpha 1H T-type Ca(2+) channels), which causes intracellular Ca(2+) to rise.

  5. Simulations of the pore structures for a M2G1yR derived channel forming peptide in membrane

    Science.gov (United States)

    Al-Rawi, Ahlam N.; Al-Rawi, Asma; Chen, Jianhan; Herrera, Alvaro; Tomich, John; Rahman, Talat S.

    2008-03-01

    In an effort to develop a peptide-based compound suitable for clinical use as a channel replacement therapeutic for treating channelopathies such as cystic fibrosis, we present a reductionist model that appears to capture many of the biophysical properties of an intact ion channel using short channel-forming peptides. We have developed two anion selective channel-forming peptides with near native and altered properties from the peptides derived from the glycine receptor: NK4-M2GlyR-p22 WT (KKKKPAR-VGLGITTVLTMTTQS) and NK4-M2GlyR-p22 S22W (KKKKPARVGLGITTVLTMTTQW), respectively. Starting with the two structures determined by solution multidimensional NMR (800 MHz) in SDS, we used CHARMM and NAMD to perform molecular dynamics simulations on the monomers. Using the existing experimental data, we then built an initial 5- helix assembly by altering the tilted angle, rotational angle and pore radius. We investigated the impact of the single mutation at position 22 on the structure and dynamics of the pore formed in a membrane build in a hydrated POPC lipid bilayer. Probable structures for both assemblies are presented.

  6. Residues involved in the pore-forming activity of the Clostridium perfringens iota toxin.

    Science.gov (United States)

    Knapp, Oliver; Maier, Elke; Waltenberger, Eva; Mazuet, Christelle; Benz, Roland; Popoff, Michel R

    2015-02-01

    Clostridium perfringens iota toxin is a binary toxin that is organized into enzyme (Ia) and binding (Ib) components. Ib forms channels in lipid bilayers and mediates the transport of Ia into the target cells. Here we show that Ib residues 334-359 contain a conserved pattern of alternating hydrophobic and hydrophilic residues forming two amphipathic β-strands involved in membrane insertion and channel formation. This stretch of amino acids shows remarkable structural and functional analogies with the β-pore-forming domain of C. perfringens epsilon toxin. Several mutations within the two amphipathic β-strands affected pore formation, single-channel conductance and ion selectivity (S339E-S341E, Q345H N346E) confirming their involvement in channel formation. F454 of Ib corresponds to the Φ-clamp F427 of anthrax protective antigen and F428 of C2II binary toxins. The mutation F454A resulted in a loss of cytotoxicity and strong increase in single-channel conductance (500 pS as compared with 85 pS in 1 M KCl) with a slight decrease in cation selectivity, indicating that the Φ-clamp is highly conserved and crucial for binary toxin activity. In contrast, the mutants Q367D, N430D, L443E had no or only minor effects on Ib properties, while T360I, T360A and T360W caused a dramatic effect on ion selectivity and single-channel conductance, indicating gross disturbance of the oligomer structure. This suggests that, at least in the iota toxin family, T360 has a structural role in the pore organization. Moreover, introduction of charged residues within the channel (S339E-S341E) or in the vestibule (Q367D, N430D and L443E) had virtually no effect on chloroquine or Ia binding, whereas F454A, T360I, T360A and T360W strongly decreased the chloroquine and Ia affinity to Ib. These results support that distinct residues within the vestibule interact with chloroquine and Ia or are responsible for channel structure, while the channel lining amino acids play a less important role.

  7. Water Aerobics as a Form of Health Activities

    Directory of Open Access Journals (Sweden)

    Anna S. Batrak

    2013-09-01

    Full Text Available The offered literature review considers water aerobics as a form of health activities. Water aerobics is wide spread and popular, especially among women, because it is also the form of adaptive and health activities. It enlarges general physiological effect of physical exercises on the human body. Regular exercises improve physical fitness and physical development, health, mood, sleep, intensify activities and working efficiency.

  8. Diacylglycerols Activate Mitochondrial Cationic Channel(s) and Release Sequestered Ca2+

    Science.gov (United States)

    Chinopoulos, Christos; Starkov, Anatoly A.; Grigoriev, Sergey; Dejean, Laurent M.; Kinnally, Kathleen W.; Liu, Xibao; Ambudkar, Indu S.; Fiskum, Gary

    2008-01-01

    Mitochondria contribute to cytosolic Ca2+ homeostasis through several uptake and release pathways. Here we report that 1,2-sn-diacylglycerols (DAGs) induce Ca2+ release from Ca2+-loaded mammalian mitochondria. Release is not mediated by the uniporter or the Na+/Ca2+ exchanger, nor is it attributed to putative catabolites. DAGs-induced Ca2+ efflux is biphasic. Initial release is rapid and transient, insensitive to permeability transition inhibitors, and not accompanied by mitochondrial swelling. Following initial rapid release of Ca2+ and relatively slowreuptake, a secondary progressive release of Ca2+ occurs, associated with swelling, and mitigated by permeability transition inhibitors. The initial peak of DAGs-induced Ca2+ efflux is abolished by La3+ (1mM) and potentiated by protein kinase C inhibitors. Phorbol esters, 1,3-diacylglycerols and 1-monoacylglycerols do not induce mitochondrial Ca2+ efflux. Ca2+-loaded mitoplasts devoid of outer mitochondrial membrane also exhibit DAGsinduced Ca2+ release, indicating that this mechanism resides at the inner mitochondrial membrane. Patch clamping brainmitoplasts reveal DAGs-induced slightly cation-selective channel activity that is insensitive to bongkrekic acid and abolished by La3+. The presence of a second messenger-sensitive Ca2+ release mechanism in mitochondria could have an important impact on intracellular Ca2+ homeostasis. PMID:16167179

  9. A novel method to form conducting channels in SiOx(Si ) films for field emission application

    Science.gov (United States)

    Semenenko, M.; Evtukh, A.; Yilmazoglu, O.; Hartnagel, H. L.; Pavlidis, D.

    2010-01-01

    The electrical and field emission properties of SiOx(Si ) films are studied. SiOx(Si ) films of 40-100nm thick are obtained by plasma-enhanced chemical vapor deposition and thermal evaporation of Si powder onto Si substrates. Nanosized electrical conducting channels are formed in SiOx(Si ) films by electrical conditioning at high current densities. The structures with conducting channels demonstrate increased field emission current and decreased threshold voltage compared to as-deposited SiOx(Si ) films. The decrease in threshold voltage for electron field emission is explained by local enhancement of electric field. The diameters of conducting channels are estimated from the effective emission area to be in the range of 1-2nm.

  10. Increasing lifetime of the plasma channel formed in air using picosecond and nanosecond laser pulses

    Science.gov (United States)

    Narayanan, V.; Singh, V.; Pandey, Pramod K.; Shukla, Neeraj; Thareja, R. K.

    2007-04-01

    We report experiments on a pump-probe configuration to elucidate the formation of a plasma channel by the hydrodynamic evolution of air breakdown in laser focus. A stable air breakdown was produced by focusing a picosecond laser pulse to create a shock driven plasma channel in the laser focus for propagating a nanosecond pulse. A four fold increase in the lifetime of the channel estimated by monitoring the temporal evolution of the fluorescence of a spectral line at 504.5nm of N+ transition 3pS3-3sP03 is reported. Assuming plasma in local thermal equilibrium plasma temperature of ˜8.2eV and an electron density of ˜1.4×1018cm-3 were determined using a Stark broadening of 649.2nm line of NII transition 3dD03-4pD3 in the channel. An enhancement in the electron density of the plasma channel was observed at the 7ns delay of the nanosecond laser pulse relative to the picosecond laser pulse.

  11. Swelling-Activated Anion Channels Are Essential for Volume Regulation of Mouse Thymocytes

    Directory of Open Access Journals (Sweden)

    Ravshan Z. Sabirov

    2011-12-01

    Full Text Available Channel-mediated trans-membrane chloride movement is a key process in the active cell volume regulation under osmotic stress in most cells. However, thymocytes were hypothesized to regulate their volume by activating a coupled K-Cl cotransport mechanism. Under the patch-clamp, we found that osmotic swelling activates two types of macroscopic anion conductance with different voltage-dependence and pharmacology. At the single-channel level, we identified two types of events: one corresponded to the maxi-anion channel, and the other one had characteristics of the volume-sensitive outwardly rectifying (VSOR chloride channel of intermediate conductance. A VSOR inhibitor, phloretin, significantly suppressed both macroscopic VSOR-type conductance and single-channel activity of intermediate amplitude. The maxi-anion channel activity was largely suppressed by Gd3+ ions but not by phloretin. Surprisingly, [(dihydroindenyloxy] alkanoic acid (DIOA, a known antagonist of K-Cl cotransporter, was found to significantly suppress the activity of the VSOR-type single-channel events with no effect on the maxi-anion channels at 10 μM. The regulatory volume decrease (RVD phase of cellular response to hypotonicity was mildly suppressed by Gd3+ ions and was completely abolished by phloretin suggesting a major impact of the VSOR chloride channel and modulatory role of the maxi-anion channel. The inhibitory effect of DIOA was also strong, and, most likely, it occurred via blocking the VSOR Cl− channels.

  12. Localization of Ca2+ -activated big-conductance K+ channels in rabbit distal colon

    DEFF Research Database (Denmark)

    Hay-Schmidt, Anders; Grunnet, Morten; Abrahamse, Salomon L

    2003-01-01

    Big-conductance Ca(2+)-activated K(+) channels (BK channels) may play an important role in the regulation of epithelial salt and water transport, but little is known about the expression level and the precise localization of BK channels in epithelia. The aim of the present study was to quantify...

  13. 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 mRNA...

  14. 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…

  15. Ca(2+)-activated chloride channel activity during Ca(2+) alternans in ventricular myocytes.

    Science.gov (United States)

    Kanaporis, Giedrius; Blatter, Lothar A

    2016-11-01

    Cardiac alternans, defined beat-to-beat alternations in contraction, action potential (AP) morphology or cytosolic Ca transient (CaT) amplitude, is a high risk indicator for cardiac arrhythmias. We investigated mechanisms of cardiac alternans in single rabbit ventricular myocytes. CaTs were monitored simultaneously with membrane currents or APs recorded with the patch clamp technique. A strong correlation between beat-to-beat alternations of AP morphology and CaT alternans was observed. During CaT alternans application of voltage clamp protocols in form of pre-recorded APs revealed a prominent Ca(2+)-dependent membrane current consisting of a large outward component coinciding with AP phases 1 and 2, followed by an inward current during AP repolarization. Approximately 85% of the initial outward current was blocked by Cl(-) channel blocker DIDS or lowering external Cl(-) concentration identifying it as a Ca(2+)-activated Cl(-) current (ICaCC). The data suggest that ICaCC plays a critical role in shaping beat-to-beat alternations in AP morphology during alternans.

  16. Simulations of the Pore Structures for a M2GlyR Derived Channel Forming Peptide in Different Membrane Environments

    Science.gov (United States)

    Al-Rawi, A.; Herrera, A.; Tomich, J.; Rahman, T.

    2007-03-01

    As part of an effort to develop a peptide-based compound suitable for clinical use as a channel replacement therapeutic for treating channelopathies such as cystic fibrosis, we present a reductionist model that appears to grasp the characteristics of ion channeling peptides. In particular we present the observed changes in the functional characteristics of NK4-M2GlyR p22 (KKKKPARVGLGITTVLTMTTQS), a M2 GlyR derived channel forming peptide. Starting with a structure determined by multidimensional NMR (800 MHz) in SDS, a potential from CHARMM force-field was used to relax the structure of NK4-M2GlyR p22. Following the relaxation, numerous pore structures were generated for the symmetric five-helix assembly with geometries varying from cylindrical to conical. As it is difficult a priori to assign accurately the orientation of the hydrophilic portion of M2GlyR derived amphipath towards the inside of the pore, we tilted and rotated the helical structure by five different angles about the backbone axis before forming the pore. Energy minimization of the channel was performed in vacuum, in phosphotidylcholine (POPC) membrane, and 60% POPC 30% phosphotidylethanolamine (POPE) in order to determine the effect of the environment surrounding on the structure on its energy minimization. We will present the various pore assemblies, in the different membrane environments, used to predict the most probably membrane bound structure.

  17. A novel BK channel-targeted peptide suppresses sound evoked activity in the mouse inferior colliculus

    Science.gov (United States)

    Scott, L. L.; Brecht, E. J.; Philpo, A.; Iyer, S.; Wu, N. S.; Mihic, S. J.; Aldrich, R. W.; Pierce, J.; Walton, J. P.

    2017-01-01

    Large conductance calcium-activated (BK) channels are broadly expressed in neurons and muscle where they modulate cellular activity. Decades of research support an interest in pharmaceutical applications for modulating BK channel function. Here we report a novel BK channel-targeted peptide with functional activity in vitro and in vivo. This 9-amino acid peptide, LS3, has a unique action, suppressing channel gating rather than blocking the pore of heterologously expressed human BK channels. With an IC50 in the high picomolar range, the apparent affinity is higher than known high affinity BK channel toxins. LS3 suppresses locomotor activity via a BK channel-specific mechanism in wild-type or BK channel-humanized Caenorhabditis elegans. Topical application on the dural surface of the auditory midbrain in mouse suppresses sound evoked neural activity, similar to a well-characterized pore blocker of the BK channel. Moreover, this novel ion channel-targeted peptide rapidly crosses the BBB after systemic delivery to modulate auditory processing. Thus, a potent BK channel peptide modulator is open to neurological applications, such as preventing audiogenic seizures that originate in the auditory midbrain. PMID:28195225

  18. An improved ivermectin-activated chloride channel receptor for inhibiting electrical activity in defined neuronal populations

    DEFF Research Database (Denmark)

    Lynagh, Timothy Peter; Lynch, Joseph W

    2010-01-01

    for surgically implanted stimulus delivery methods and their use of nonhuman receptors. A third silencing method, an invertebrate glutamate-gated chloride channel receptor (GluClR) activated by ivermectin, solves the stimulus delivery problem as ivermectin is a safe, well tolerated drug that reaches the brain...

  19. A leucine zipper motif essential for gating of hyperpolarization-activated channels.

    Science.gov (United States)

    Wemhöner, Konstantin; Silbernagel, Nicole; Marzian, Stefanie; Netter, Michael F; Rinné, Susanne; Stansfeld, Phillip J; Decher, Niels

    2012-11-23

    It is poorly understood how hyperpolarization-activated cyclic nucleotide-gated channels (HCNs) function. We have identified a leucine zipper in the S5 segment of HCNs, regulating hyperpolarization-activated and instantaneous current components. The leucine zipper is essential for HCN channel gating. The identification and functional characterization of the leucine zipper is an important step toward the understanding of HCN channel function. Hyperpolarization-activated cyclic nucleotide-gated (HCN) channels are pacemakers in cardiac myocytes and neurons. Although their membrane topology closely resembles that of voltage-gated K(+) channels, the mechanism of their unique gating behavior in response to hyperpolarization is still poorly understood. We have identified a highly conserved leucine zipper motif in the S5 segment of HCN family members. In order to study the role of this motif for channel function, the leucine residues of the zipper were individually mutated to alanine, arginine, or glutamine residues. Leucine zipper mutants traffic to the plasma membrane, but the channels lose their sensitivity to open upon hyperpolarization. Thus, our data indicate that the leucine zipper is an important molecular determinant for hyperpolarization-activated channel gating. Residues of the leucine zipper interact with the adjacent S6 segment of the channel. This interaction is essential for voltage-dependent gating of the channel. The lower part of the leucine zipper, at the intracellular mouth of the channel, is important for stabilizing the closed state. Mutations at these sites increase current amplitudes or result in channels with deficient closing and increased min-P(o). Our data are further supported by homology models of the open and closed state of the HCN2 channel pore. Thus, we conclude that the leucine zipper of HCN channels is a major determinant for hyperpolarization-activated channel gating.

  20. A Leucine Zipper Motif Essential for Gating of Hyperpolarization-activated Channels*

    Science.gov (United States)

    Wemhöner, Konstantin; Silbernagel, Nicole; Marzian, Stefanie; Netter, Michael F.; Rinné, Susanne; Stansfeld, Phillip J.; Decher, Niels

    2012-01-01

    Hyperpolarization-activated cyclic nucleotide-gated (HCN) channels are pacemakers in cardiac myocytes and neurons. Although their membrane topology closely resembles that of voltage-gated K+ channels, the mechanism of their unique gating behavior in response to hyperpolarization is still poorly understood. We have identified a highly conserved leucine zipper motif in the S5 segment of HCN family members. In order to study the role of this motif for channel function, the leucine residues of the zipper were individually mutated to alanine, arginine, or glutamine residues. Leucine zipper mutants traffic to the plasma membrane, but the channels lose their sensitivity to open upon hyperpolarization. Thus, our data indicate that the leucine zipper is an important molecular determinant for hyperpolarization-activated channel gating. Residues of the leucine zipper interact with the adjacent S6 segment of the channel. This interaction is essential for voltage-dependent gating of the channel. The lower part of the leucine zipper, at the intracellular mouth of the channel, is important for stabilizing the closed state. Mutations at these sites increase current amplitudes or result in channels with deficient closing and increased min-Po. Our data are further supported by homology models of the open and closed state of the HCN2 channel pore. Thus, we conclude that the leucine zipper of HCN channels is a major determinant for hyperpolarization-activated channel gating. PMID:23048023

  1. Synchronization of Active Atomic Clocks via Quantum and Classical Channels

    CERN Document Server

    Roth, Alexander

    2016-01-01

    Superradiant lasers based on atomic ensembles exhibiting ultra-narrow optical transitions can emit light of unprecedented spectral purity and may serve as active atomic clocks. We consider two frequency-detuned active atomic clocks, which are coupled in a cascaded setup, i.e. as master & slave lasers, and study the synchronization of the slave to the master clock. In a setup where both atomic ensembles are coupled to a common cavity mode such synchronization phenomena have been predicted by Xu et al. [Phys. Rev. Lett. 113, 154101 (2014)] and experimentally observed by Weiner et al. [arXiv:1503.06464 (2015)]. Here we demonstrate that synchronization still occurs in cascaded setups but exhibits distinctly different phase diagrams. We study the characteristics of synchronization in comparison to the case of coupling through a common cavity. We also consider synchronization through a classical channel where light of the master laser is measured phase sensitively and the slave laser is injection locked by feed...

  2. Activation and deactivation of vibronic channels in intact phycocyanin rods

    Science.gov (United States)

    Nganou, C.; David, L.; Meinke, R.; Adir, N.; Maultzsch, J.; Mkandawire, M.; Pouhè, D.; Thomsen, C.

    2014-02-01

    We investigated the excitation modes of the light-harvesting protein phycocyanin (PC) from Thermosynechococcus vulcanus in the crystalline state using UV and near-infrared Raman spectroscopy. The spectra revealed the absence of a hydrogen out-of-plane wagging (HOOP) mode in the PC trimer, which suggests that the HOOP mode is activated in the intact PC rod, while it is not active in the PC trimer. Furthermore, in the PC trimer an intense mode at 984 cm-1 is assigned to the C-C stretching vibration while the mode at 454 cm-1 is likely due to ethyl group torsion. In contrast, in the similar chromophore phytochromobilin the C5,10,15-D wag mode at 622 cm-1 does not come from a downshift of the HOOP. Additionally, the absence of modes between 1200 and 1300 cm-1 rules out functional monomerization. A correlation between phycocyanobilin (PCB) and phycoerythrobilin (PEB) suggests that the PCB cofactors of the PC trimer appear in a conformation similar to that of PEB. The conformation of the PC rod is consistent with that of the allophycocyanin (APC) trimer, and thus excitonic flow is facilitated between these two independent light-harvesting compounds. This excitonic flow from the PC rod to APC appears to be modulated by the vibration channels during HOOP wagging, C = C stretching, and the N-H rocking in-plan vibration.

  3. Nitric oxide regulates neuronal activity via calcium-activated potassium channels.

    Directory of Open Access Journals (Sweden)

    Lei Ray Zhong

    Full Text Available Nitric oxide (NO is an unconventional membrane-permeable messenger molecule that has been shown to play various roles in the nervous system. How NO modulates ion channels to affect neuronal functions is not well understood. In gastropods, NO has been implicated in regulating the feeding motor program. The buccal motoneuron, B19, of the freshwater pond snail Helisoma trivolvis is active during the hyper-retraction phase of the feeding motor program and is located in the vicinity of NO-producing neurons in the buccal ganglion. Here, we asked whether B19 neurons might serve as direct targets of NO signaling. Previous work established NO as a key regulator of growth cone motility and neuronal excitability in another buccal neuron involved in feeding, the B5 neuron. This raised the question whether NO might modulate the electrical activity and neuronal excitability of B19 neurons as well, and if so whether NO acted on the same or a different set of ion channels in both neurons. To study specific responses of NO on B19 neurons and to eliminate indirect effects contributed by other cells, the majority of experiments were performed on single cultured B19 neurons. Addition of NO donors caused a prolonged depolarization of the membrane potential and an increase in neuronal excitability. The effects of NO could mainly be attributed to the inhibition of two types of calcium-activated potassium channels, apamin-sensitive and iberiotoxin-sensitive potassium channels. NO was found to also cause a depolarization in B19 neurons in situ, but only after NO synthase activity in buccal ganglia had been blocked. The results suggest that NO acts as a critical modulator of neuronal excitability in B19 neurons, and that calcium-activated potassium channels may serve as a common target of NO in neurons.

  4. Simulation of Multiple Cold Rolls Progressive Forming for Non-symmetrical Channel Section

    Institute of Scientific and Technical Information of China (English)

    2001-01-01

    In cold roll forming process, the sheet is progressively formed into a very complex three dimensional surface. The design procedure for the roll formed products, forming rolls, and roll pass sequences was considered more an art than a science. Good roll pass design was the key to successful roll forming. In order to reduce forming defects and trial production cost, computer simulation of cold roll forming was employed. Based on the Updated-Lagrange method in the deformation mechanics, a theoretical model of elastic-plastic large deformation spline finite strip method is proposed in this paper. The method is employed to analyze the progressive forming process of non-symmetrical section, and the displacement, the stress and the strain along rolling direction during the multiple cold rolls forming process are got. This program written in C Language can be used to analyze other simple cross sectional profiles also.

  5. Effects of Active Subsidence Vs. Existing Basin Geometry on Fluviodeltaic Channels and Stratal Architecture

    Science.gov (United States)

    Liang, M.; Kim, W.; Passalacqua, P.

    2015-12-01

    Tectonic subsidence and basin topography, both determining the accommodation, are fundamental controls on the basin filling processes. Their effects on the fluvial organization and the resultant subsurface patterns remain difficult to predict due to the lack of understanding about interaction between internal dynamics and external controls. Despite the intensive studies on tectonic steering effects on alluvial architecture, how the self-organization of deltaic channels, especially the distributary channel network, respond to tectonics and basin geometry is mostly unknown. Recently physical experiments and field studies have hinted dramatic differences in fluviodeltaic evolution between ones associated with active differential subsidence and existing basin depth. In this work we designed a series of numerical experiments using a reduced-complexity channel-resolving model for delta formation, and tested over a range of localized subsidence rates and topographic depression in basin geometry. We also used a set of robust delta metrics to analyze: i) shoreline planform asymmetry, ii) channel and lobe geometry, iii) channel network pattern, iv) autogenic timescales, and v) subsurface structure. The modeling results show that given a similar final thickness, active subsidence enhances channel branching with smaller channel sand bodies that are both laterally and vertically connected, whereas existing topographic depression causes more large-scale channel avulsions with larger channel sand bodies. In general, both subsidence and existing basin geometry could steer channels and/or lock channels in place but develop distinct channel patterns and thus stratal architecture.

  6. Forms and Methods of Agricultural Sector Innovative Activity Improvement

    Directory of Open Access Journals (Sweden)

    Aisha S. Ablyaeva

    2013-01-01

    Full Text Available The article is focused on basic forms and methods to improve the efficiency of innovative activity in the agricultural sector of Ukraine. It was determined that the development of agriculture in Ukraine is affected by a number of factors that must be considered to design innovative models of entrepreneurship development and ways to improve the efficiency of innovative entrepreneurship activity.

  7. The S4-S5 linker couples voltage sensing and activation of pacemaker channels.

    Science.gov (United States)

    Chen, J; Mitcheson, J S; Tristani-Firouzi, M; Lin, M; Sanguinetti, M C

    2001-09-25

    Voltage-gated channels are normally opened by depolarization and closed by repolarization of the membrane. Despite sharing significant sequence homology with voltage-gated K(+) channels, the gating of hyperpolarization-activated, cyclic-nucleotide-gated (HCN) pacemaker channels has the opposite dependence on membrane potential: hyperpolarization opens, whereas depolarization closes, these channels. The mechanism and structural basis of the process that couples voltage sensor movement to HCN channel opening and closing is not understood. On the basis of our previous studies of a mutant HERG (human ether-a-go-go-related gene) channel, we hypothesized that the intracellular linker that connects the fourth and fifth transmembrane domains (S4-S5 linker) of HCN channels might be important for channel gating. Here, we used alanine-scanning mutagenesis of the HCN2 S4-S5 linker to identify three residues, E324, Y331, and R339, that when mutated disrupted normal channel closing. Mutation of a basic residue in the S4 domain (R318Q) prevented channel opening, presumably by disrupting S4 movement. However, channels with R318Q and Y331S mutations were constitutively open, suggesting that these channels can open without a functioning S4 domain. We conclude that the S4-S5 linker mediates coupling between voltage sensing and HCN channel activation. Our findings also suggest that opening of HCN and related channels corresponds to activation of a gate located near the inner pore, rather than recovery of channels from a C-type inactivated state.

  8. Activity-dependent Phosphorylation of Neuronal Kv2.1 Potassium Channels by CDK5*

    OpenAIRE

    Cerda, Oscar; Trimmer, James S.

    2011-01-01

    Dynamic modulation of ion channel expression, localization, and/or function drives plasticity in intrinsic neuronal excitability. Voltage-gated Kv2.1 potassium channels are constitutively maintained in a highly phosphorylated state in neurons. Increased neuronal activity triggers rapid calcineurin-dependent dephosphorylation, loss of channel clustering, and hyperpolarizing shifts in voltage-dependent activation that homeostatically suppress neuronal excitability. These changes are reversible,...

  9. Hysteresis of KcsA potassium channel's activation- deactivation gating is caused by structural changes at the channel's selectivity filter.

    Science.gov (United States)

    Tilegenova, Cholpon; Cortes, D Marien; Cuello, Luis G

    2017-03-21

    Mode-shift or hysteresis has been reported in ion channels. Voltage-shift for gating currents is well documented for voltage-gated cation channels (VGCC), and it is considered a voltage-sensing domain's (VSD) intrinsic property. However, uncoupling the Shaker K(+) channel's pore domain (PD) from the VSD prevented the mode-shift of the gating currents. Consequently, it was proposed that an open-state stabilization of the PD imposes a mechanical load on the VSD, which causes its mode-shift. Furthermore, the mode-shift displayed by hyperpolarization-gated cation channels is likely caused by structural changes at the channel's PD similar to those underlying C-type inactivation. To demonstrate that the PD of VGCC undergoes hysteresis, it is imperative to study its gating process in the absence of the VSD. A back-door strategy is to use KcsA (a K(+) channel from the bacteria Streptomyces lividans) as a surrogate because it lacks a VSD and exhibits an activation coupled to C-type inactivation. By directly measuring KcsA's activation gate opening and closing in conditions that promote or halt C-type inactivation, we have found (i) that KcsA undergoes mode-shift of gating when having K(+) as the permeant ion; (ii) that Cs(+) or Rb(+), known to halt C-inactivation, prevented mode-shift of gating; and (iii) that, in the total absence of C-type inactivation, KcsA's mode-shift was prevented. Finally, our results demonstrate that an allosteric communication causes KcsA's activation gate to "remember" the conformation of the selectivity filter, and hence KcsA requires a different amount of energy for opening than for closing.

  10. Arecoline inhibits intermediate-conductance calcium-activated potassium channels in human glioblastoma cell lines.

    Science.gov (United States)

    So, Edmund Cheung; Huang, Yan-Ming; Hsing, Chung-Hsi; Liao, Yu-Kai; Wu, Sheng-Nan

    2015-07-05

    Arecoline (ARE) is an alkaloid-type natural product from areca nut. This compound has numerous pharmacological and toxicological effects. Whether this agent interacts with ion channels to perturb functional activity of cells remains unknown. The effects of ARE on ionic currents were studied in glioma cell lines (U373 and U87MG) using patch-clamp technique. Like TRAM-34(1-[(2-chlorophenyl)-diphenylmethyl]pyrazole), ARE suppressed the amplitude of whole-cell voltage-gated K(+) currents in U373 cells elicited by a ramp voltage clamp. In cell-attached configuration, ARE did not modify the single-channel conductance of intermediate-conductance Ca(2+)-activated K(+) (IKCa) channels; however, it did reduce channel activity. Its inhibition of IKCa channels was accompanied by a significant lengthening in the slow component of mean closed time of IKCa channels. Based on minimal kinetic scheme, the dissociation constant (KD) required for ARE-mediated prolongation of mean closed time was 11.2µM. ARE-induced inhibition of IKCa channels was voltage-dependent. Inability of ARE to perturb the activity of large-conductance Ca(2+)-activated K(+) (BKCa) channels was seen. Under current-clamp recordings, ARE depolarized the membrane of U373 cells and DCEBIO reversed ARE-induced depolarization. Similarly, ARE suppressed IKCa-channel activities in oral keratinocytes. This study provides the evidence that ARE block IKCa channels in a concentration, voltage and state-dependent manner. ARE-induced block of IKCa channels is unrelated to the binding of muscarinic receptors. The effects of ARE on these channels may partially be responsible for the underlying cellular mechanisms by which it influences the functional activities of glioma cells or oral keratinocytes, if similar findings occur in vivo. Copyright © 2015 Elsevier B.V. All rights reserved.

  11. Complex role of STIM1 in the activation of store-independent Orai1/3 channels

    Science.gov (United States)

    Zhang, Wei; González-Cobos, José C.; Jardin, Isaac; Romanin, Christoph; Matrougui, Khalid

    2014-01-01

    Orai proteins contribute to Ca2+ entry into cells through both store-dependent, Ca2+ release–activated Ca2+ (CRAC) channels (Orai1) and store-independent, arachidonic acid (AA)-regulated Ca2+ (ARC) and leukotriene C4 (LTC4)-regulated Ca2+ (LRC) channels (Orai1/3 heteromultimers). Although activated by fundamentally different mechanisms, CRAC channels, like ARC and LRC channels, require stromal interacting molecule 1 (STIM1). The role of endoplasmic reticulum–resident STIM1 (ER-STIM1) in CRAC channel activation is widely accepted. Although ER-STIM1 is necessary and sufficient for LRC channel activation in vascular smooth muscle cells (VSMCs), the minor pool of STIM1 located at the plasma membrane (PM-STIM1) is necessary for ARC channel activation in HEK293 cells. To determine whether ARC and LRC conductances are mediated by the same or different populations of STIM1, Orai1, and Orai3 proteins, we used whole-cell and perforated patch-clamp recording to compare AA- and LTC4-activated currents in VSMCs and HEK293 cells. We found that both cell types show indistinguishable nonadditive LTC4- and AA-activated currents that require both Orai1 and Orai3, suggesting that both conductances are mediated by the same channel. Experiments using a nonmetabolizable form of AA or an inhibitor of 5-lipooxygenase suggested that ARC and LRC currents in both cell types could be activated by either LTC4 or AA, with LTC4 being more potent. Although PM-STIM1 was required for current activation by LTC4 and AA under whole-cell patch-clamp recordings in both cell types, ER-STIM1 was sufficient with perforated patch recordings. These results demonstrate that ARC and LRC currents are mediated by the same cellular populations of STIM1, Orai1, and Orai3, and suggest a complex role for both ER-STIM1 and PM-STIM1 in regulating these store-independent Orai1/3 channels. PMID:24567509

  12. 5-HT1A receptors modulate small-conductance Ca2+-activated K+ channels

    DEFF Research Database (Denmark)

    Grunnet, Morten; Jespersen, Thomas; Perrier, Jean-François

    2004-01-01

    Small-conductance calcium-activated potassium channels (SK) are responsible for the medium afterhyperpolarisation (mAHP) following action potentials in neurons. Here we tested the ability of serotonin (5-HT) to modulate the activity of SK channels by coexpressing 5-HT1A receptors with different...

  13. Soft Matter-Regulated Active Nanovalves Locally Self-Assembled in Femtoliter Nanofluidic Channels.

    Science.gov (United States)

    Xu, Yan; Shinomiya, Misato; Harada, Atsushi

    2016-03-16

    Well-tailored thermoresponsive polymer brushes locally self-assembled in tiny nanofluidic channels enable the active regulation of femtoliter-scale fluids. Such soft-matter-regulated active nanovalves within nanofluidic channels can be extended to build well-controlled functional nanofluidic systems, allowing complex fluidic processes to be performed at the nanometer scales.

  14. Constitutive activity of the human TRPML2 channel induces cell degeneration.

    Science.gov (United States)

    Lev, Shaya; Zeevi, David A; Frumkin, Ayala; Offen-Glasner, Vered; Bach, Gideon; Minke, Baruch

    2010-01-22

    The mucolipin (TRPML) ion channel proteins represent a distinct subfamily of channel proteins within the transient receptor potential (TRP) superfamily of cation channels. Mucolipin 1, 2, and 3 (TRPML1, -2, and -3, respectively) are channel proteins that share high sequence homology with each other and homology in the transmembrane domain with other TRPs. Mutations in the TRPML1 protein are implicated in mucolipidosis type IV, whereas mutations in TRPML3 are found in the varitint-waddler mouse. The properties of the wild type TRPML2 channel are not well known. Here we show functional expression of the wild type human TRPML2 channel (h-TRPML2). The channel is functional at the plasma membrane and characterized by a significant inward rectification similar to other constitutively active TRPML mutant isoforms. The h-TRPML2 channel displays nonselective cation permeability, which is Ca(2+)-permeable and inhibited by low extracytosolic pH but not Ca(2+) regulated. In addition, constitutively active h-TRPML2 leads to cell death by causing Ca(2+) overload. Furthermore, we demonstrate by functional mutation analysis that h-TRPML2 shares similar characteristics and structural similarities with other TRPML channels that regulate the channel in a similar manner. Hence, in addition to overall structure, all three TRPML channels also share common modes of regulation.

  15. Application of the normal forms to analyze the interactions among the multi-control channels of UPFC

    Energy Technology Data Exchange (ETDEWEB)

    Zou, Z.Y.; Jiang, Q.Y.; Cao, Y.J. [College of Electrical Engineering and National Laboratory of Industrial Control Technology, Zhejiang University, Hangzhou, Zhejiang 310027 (China); Wang, H.F. [Department of Electrical and Electronics Engineering, University of Bath, BA2, 7AY (United Kingdom)

    2005-10-01

    One of the most important features of the unified power flow controller (UPFC) is its multiple control functions, which are implemented by multiple controllers. However, recent simulation studies have demonstrated the existence of the dynamic interactions among different controller channels of UPFC, i.e. power flow controller, AC voltage controller and DC voltage controller. This paper is concerned with the application of the normal forms to analyze the interactions among the multi-controller channels of UPFC in power systems. Moreover, a non-linear interaction index is developed to investigate the interactions among these UPFC controllers. The employed heat exchanger operators to plan cleaning schedules predictions can assisbus power system (SMIB) and the New England test power system (NETPS). The simulation results validate the proposed approach. (author)

  16. Carbon monoxide stimulates the Ca2(+)-activated big conductance k channels in cultured human endothelial cells.

    Science.gov (United States)

    Dong, De-Li; Zhang, Yan; Lin, Dao-Hong; Chen, Jun; Patschan, Susann; Goligorsky, Michael S; Nasjletti, Alberto; Yang, Bao-Feng; Wang, Wen-Hui

    2007-10-01

    We used the whole-cell patch-clamp technique to study K channels in the human umbilical vein endothelial cells and identified a 201 pS K channel, which was blocked by tetraethylammonium and iberiotoxin but not by TRAM34 and apamin. This suggests that the Ca(2+)-activated big-conductance K channel (BK) is expressed in endothelial cells. Application of carbon monoxide (CO) or tricarbonylchloro(glycinato)ruthenium(II), a water soluble CO donor, stimulated the BK channels. Moreover, application of hemin, a substrate of heme oxygenase, mimicked the effect of CO and increased the BK channel activity. The stimulatory effect of hemin was significantly diminished by tin mesoporphyrin, an inhibitor of heme oxygenase. To determine whether the stimulatory effect of CO on the BK channel was mediated by NO and the cGMP-dependent pathway, we examined the effect of CO on BK channels in cells treated with, N(G)-nitro-l-arginine methyl ester, 1H(1,2,4)oxadiazolo(4,3-a)quinoxalin-1-one, an inhibitor of soluble guanylate cyclase, or KT5823, an inhibitor of protein kinase G. Addition of either diethylamine NONOate or sodium nitroprusside significantly increased BK channel activity. Inhibition of endogenous NO synthesis with N(G)-nitro-l-arginine methyl ester, blocking soluble guanylate cyclase or protein kinase G, delayed but did not prevent the CO-induced activation of BK channels. Finally, application of an antioxidant agent, ebselen, had no effect on CO-mediated stimulation of BK channels in human umbilical vein endothelial cells. We conclude that BK channels are expressed in human umbilical vein endothelial cells and that they are activated by both CO and NO. CO activates BK channels directly, as well as via a mechanism involving NO or the cGMP-dependent pathway.

  17. BARP suppresses voltage-gated calcium channel activity and Ca2+-evoked exocytosis.

    Science.gov (United States)

    Béguin, Pascal; Nagashima, Kazuaki; Mahalakshmi, Ramasubbu N; Vigot, Réjan; Matsunaga, Atsuko; Miki, Takafumi; Ng, Mei Yong; Ng, Yu Jin Alvin; Lim, Chiaw Hwee; Tay, Hock Soon; Hwang, Le-Ann; Firsov, Dmitri; Tang, Bor Luen; Inagaki, Nobuya; Mori, Yasuo; Seino, Susumu; Launey, Thomas; Hunziker, Walter

    2014-04-28

    Voltage-gated calcium channels (VGCCs) are key regulators of cell signaling and Ca(2+)-dependent release of neurotransmitters and hormones. Understanding the mechanisms that inactivate VGCCs to prevent intracellular Ca(2+) overload and govern their specific subcellular localization is of critical importance. We report the identification and functional characterization of VGCC β-anchoring and -regulatory protein (BARP), a previously uncharacterized integral membrane glycoprotein expressed in neuroendocrine cells and neurons. BARP interacts via two cytosolic domains (I and II) with all Cavβ subunit isoforms, affecting their subcellular localization and suppressing VGCC activity. Domain I interacts at the α1 interaction domain-binding pocket in Cavβ and interferes with the association between Cavβ and Cavα1. In the absence of domain I binding, BARP can form a ternary complex with Cavα1 and Cavβ via domain II. BARP does not affect cell surface expression of Cavα1 but inhibits Ca(2+) channel activity at the plasma membrane, resulting in the inhibition of Ca(2+)-evoked exocytosis. Thus, BARP can modulate the localization of Cavβ and its association with the Cavα1 subunit to negatively regulate VGCC activity.

  18. A plasma membrane-targeted cytosolic domain of STIM1 selectively activates ARC channels, an arachidonate-regulated store-independent Orai channel.

    Science.gov (United States)

    Thompson, Jill L; Shuttleworth, Trevor J

    2012-01-01

    The Orai family of calcium channels includes the store-operated CRAC channels and store-independent, arachidonic acid (AA)-regulated ARC channels. Both depend on STIM1 for their activation but, whereas CRAC channel activation involves sensing the depletion of intracellular calcium stores via a luminal N terminal EF-hand of STIM1 in the endoplasmic reticulum (ER) membrane, ARC channels are exclusively activated by the pool of STIM1 that constitutively resides in the plasma membrane (PM). Here, the EF-hand is extracellular and unlikely to ever lose its bound calcium, suggesting that STIM1-dependent activation of ARC channels is very different from that of CRAC channels. We now show that attachment of the cytosolic portion of STIM1 to the inner face of the PM via an N terminal Lck-domain sequence is sufficient to enable normal AA-dependent activation of ARC channels, while failing to allow activation of store-operated CRAC channels. Introduction of a point mutation within the Lck-domain resulted in the loss of both PM localization and ARC channel activation. Reversing the orientation of the PM-anchored STIM1 C terminus via a C-terminal CAAX-box fails to support either CRAC or ARC channel activation. Finally, the Lck-anchored STIM1 C-terminal domain also enabled the exclusive activation of the ARC channels following physiological agonist addition. These data demonstrate that simple tethering of the cytosolic C-terminal domain of STIM1 to the inner face of the PM is sufficient to allow the full, normal and exclusive activation of ARC channels, and that the N-terminal regions of STIM1 (including the EF-hand domain) play no significant role in this activation.

  19. The pore-forming toxin proaerolysin is activated by furin.

    Science.gov (United States)

    Abrami, L; Fivaz, M; Decroly, E; Seidah, N G; Jean, F; Thomas, G; Leppla, S H; Buckley, J T; van der Goot, F G

    1998-12-04

    Aerolysin is secreted as an inactive dimeric precursor by the bacterium Aeromonas hydrophila. Proteolytic cleavage within a mobile loop near the C terminus of the protoxin is required for oligomerization and channel formation. This loop contains the sequence KVRRAR432, which should be recognized by mammalian proprotein convertases such as furin, PACE4, and PC5/6A. Here we show that these three proteases cleave proaerolysin after Arg-432 in vitro, yielding active toxin. We also investigated the potential role of these enzymes in the in vivo activation of the protoxin. We found that Chinese hamster ovary cells were able to convert the protoxin to aerolysin in the absence of exogenous proteases and that activation did not require internalization of the toxin. The furin inhibitor alpha1-antitrypsin Portland reduced the rate of proaerolysin activation in vivo, and proaerolysin processing was even further reduced in furin-deficient FD11 Chinese hamster ovary cells. The cells were also less sensitive to proaerolysin than wild type cells; however, transient transfection of FD11 cells with the cDNA encoding furin conferred normal sensitivity to the protoxin. Together these findings argue that furin catalyzes the cell-surface activation of proaerolysin in vivo.

  20. 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...

  1. Activation of big conductance Ca(2+)-activated K (+) channels (BK) protects the heart against ischemia-reperfusion injury

    DEFF Research Database (Denmark)

    Bentzen, Bo Hjorth; Osadchii, Oleg; Jespersen, Thomas;

    2009-01-01

    complexes, while producing no effect on cardiac K(ATP) channels. The cardioprotective effects of NS11021-induced BK channel activation were studied in isolated, perfused rat hearts subjected to 35 min of global ischemia followed by 120 min of reperfusion. 3 microM NS11021 applied prior to ischemia...... (3 microM) antagonized the protective effect. These findings suggest that tissue damage induced by ischemia and reperfusion can be reduced by activation of cardiac BK channels.......Activation of the large-conductance Ca(2+)-activated K(+) channel (BK) in the cardiac inner mitochondrial membrane has been suggested to protect the heart against ischemic injury. However, these findings are limited by the low selectivity profile and potency of the BK channel activator (NS1619...

  2. Effect of trimethyllead chloride on slowly activating (SV) channels in red beet (Beta vulgaris L.) taproots.

    Science.gov (United States)

    Trela, Zenon; Burdach, Zbigniew; Przestalski, Stanisław; Karcz, Waldemar

    2012-12-01

    The patch-clamp technique was used to examine the effect of trimethyllead chloride (Met(3)PbCl) on SV channel activity in red beet (Beta vulgaris L.) taproot vacuoles. It was found that in the control bath the macroscopic currents showed the typical slow activation and a strong outward rectification of the steady-state currents. An addition of Met(3)PbCl to the bath solution blocked, in a concentration-dependent manner, SV currents in red beet vacuoles. The time constant τ increased several times in the presence of 100 μM trimethyllead chloride at all voltages tested. When single channel properties were analyzed, only little channel activity could be recorded in the presence of 100 μM Met(3)PbCl. Trimethyllead chloride decreased significantly (by about one order of magnitude) the open probability of single channels. The recordings of single channel activity obtained in the presence and absence of Met(3)PbCl showed that organolead only slightly (by ca. 10%) decreased the unitary conductance of single channels. It was also found that Met(3)PbCl diminished significantly the number of SV channel openings, whereas it did not change the opening times of the channels. Taken together, these results suggest that Met(3)PbCl binding site is located outside the channel selectivity filter.

  3. Regulation of cloned, Ca2+-activated K+ channels by cell volume changes

    DEFF Research Database (Denmark)

    Grunnet, Morten; MacAulay, Nanna; Jorgensen, Nanna K;

    2002-01-01

    Ca2+-activated K+ channels of big (hBK), intermediate (hIK) or small (rSK3) conductance were co-expressed with aquaporin 1 (AQP1) in Xenopus laevis oocytes. hBK channels were activated by depolarization, whereas hIK and rSK3 channels were activated by direct injection of Ca2+ or Cd2+ into the ooc......Ca2+-activated K+ channels of big (hBK), intermediate (hIK) or small (rSK3) conductance were co-expressed with aquaporin 1 (AQP1) in Xenopus laevis oocytes. hBK channels were activated by depolarization, whereas hIK and rSK3 channels were activated by direct injection of Ca2+ or Cd2......+ into the oocyte cytoplasm, before the oocytes were subjected to hyperosmolar or hypoosmolar (+/-50 mOsm mannitol) challenges. In all cases, the oocytes responded rapidly to the osmotic changes with shrinkage or swelling and the effects on the K+ currents were measured. hIK and rSK3 currents were highly sensitive......IK/rSK3 and hBK channels suggest that the significant stimulation of hIK and rSK3 channels during swelling is not mediated by changes in intracellular Ca2+, but rather through interactions with the cytoskeleton, provided that a sufficient basal concentration of intracellular Ca2+ or Cd2+ is present....

  4. Two P2X1 receptor transcripts able to form functional channels are present in most human monocytes.

    Science.gov (United States)

    López-López, Cintya; Jaramillo-Polanco, Josue; Portales-Pérez, Diana P; Gómez-Coronado, Karen S; Rodríguez-Meléndez, Jessica G; Cortés-García, Juan D; Espinosa-Luna, Rosa; Montaño, Luis M; Barajas-López, Carlos

    2016-12-15

    To characterize the presence and general properties of P2X1 receptors in single human monocytes we used RT-PCR, flow cytometry, and the patch-clamp and the two-electrode voltage-clamp techniques. Most human monocytes expressed the canonical P2X1 (90%) and its splicing variant P2X1del (88%) mRNAs. P2X1 receptor immunoreactivity was also observed in 70% of these cells. Currents mediated by P2X1 (EC50=1.9±0.8µm) and P2X1del (EC50 >1000µm) channels, expressed in Xenopus leavis oocytes, have different ATP sensitivity and kinetics. Both currents mediated by P2X1 and P2X1del channels kept increasing during the continuous presence of high ATP concentrations. Currents mediated by the native P2X1 receptors in human monocytes showed an EC50=6.3±0.2µm. Currents have kinetics that resemble those observed for P2X1 and P2X1del receptors in oocytes. Our study is the first to demonstrate the expression of P2X1 transcript and its splicing variant P2X1del in most human monocytes. We also, for the first time, described functional homomeric P2X1del channels and demonstrated that currents mediated by P2X1 or P2X1del receptors, during heterologous expression, increased in amplitude when activated with high ATP concentrations in a similar fashion to those channels that increase their conductance under similar conditions, such as P2X7, P2X2, and P2X4 channels.

  5. Cell swelling activates separate taurine and chloride channels in Ehrlich mouse ascites tumor cells

    DEFF Research Database (Denmark)

    Lambert, Ian Henry; Hoffmann, Else Kay

    1994-01-01

    The taurine efflux from Ehrlich ascites tumor cells is stimulated by hypotonic cell swelling. The swelling-activated taurine efflux is unaffected by substitution of gluconate for extracellular Cl– but inhibited by addition of MK196 (anion channel blocker) and 4,4 -diisothiocyanostilbene-2......,2 -disulfonic acid (DIDS; anion channel and anion exchange blocker) and by depolarization of the cell membrane. This is taken to indicate that taurine does not leave the osmotically swollen Ehrlich cells in exchange for extracellular Cl–, i.e., via the anion exchanger but via a MK196- and DIDS-sensitive channel...... that is potential dependent. An additional stimulation of the swelling-activated taurine efflux is seen after addition of arachidonic acid and oleic acid. Cell swelling also activates a Mini Cl– channel. The Cl– efflux via this Cl– channel, in contrast to the swelling-activated taurine efflux, is unaffected by DIDS...

  6. Pore-forming activity of clostridial binary toxins.

    Science.gov (United States)

    Knapp, O; Benz, R; Popoff, M R

    2016-03-01

    Clostridial binary toxins (Clostridium perfringens Iota toxin, Clostridium difficile transferase, Clostridium spiroforme toxin, Clostridium botulinum C2 toxin) as Bacillus binary toxins, including Bacillus anthracis toxins consist of two independent proteins, one being the binding component which mediates the internalization into cell of the intracellularly active component. Clostridial binary toxins induce actin cytoskeleton disorganization through mono-ADP-ribosylation of globular actin and are responsible for enteric diseases. Clostridial and Bacillus binary toxins share structurally and functionally related binding components which recognize specific cell receptors, oligomerize, form pores in endocytic vesicle membrane, and mediate the transport of the enzymatic component into the cytosol. Binding components retain the global structure of pore-forming toxins (PFTs) from the cholesterol-dependent cytotoxin family such as perfringolysin. However, their pore-forming activity notably that of clostridial binding components is more related to that of heptameric PFT family including aerolysin and C. perfringens epsilon toxin. This review focuses upon pore-forming activity of clostridial binary toxins compared to other related PFTs. This article is part of a Special Issue entitled: Pore-Forming Toxins edited by Mauro Dalla Serra and Franco Gambale.

  7. Exploratory Topology Modelling of Form-Active Hybrid Structures

    DEFF Research Database (Denmark)

    Holden Deleuran, Anders; Pauly, Mark; Tamke, Martin;

    2016-01-01

    The development of novel form-active hybrid structures (FAHS) is impeded by a lack of modelling tools that allow for exploratory topology modelling of shaped assemblies. We present a flexible and real-time computational design modelling pipeline developed for the exploratory modelling of FAHS tha...

  8. Folding of Aggregated Proteins to Functionally Active Form

    Science.gov (United States)

    2006-06-01

    in Cn5 were reformed with a total protein yield of 87% and 100% recovery of activity [34]. Artificial chaperone-assisted refolding This method...typically formed using a mixture of reduced/oxidizedCurrent Opinion in Biotechnology 2006, 17:367–372 370 Protein technologiesglutathione, and glycerol

  9. Plasmodium induces swelling-activated ClC-2 anion channels in the host erythrocyte

    OpenAIRE

    Huber, Stephan M.; Duranton, Christophe; Henke, Guido; Van De Sand, Claudia; Heussler, Volker; Shumilina, Ekaterina; Sandu, Ciprian D.; Tanneur, Valerie; Brand, Verena; Kasinathan, Ravi S.; Lang, Karl S; Peter G Kremsner; Hübner, Christian A; Marco B Rust; Dedek, Karin

    2004-01-01

    Intraerythrocytic growth of the human malaria parasite Plasmodium falciparum depends on delivery of nutrients. Moreover, infection challenges cell volume constancy of the host erythrocyte requiring enhanced activity of cell volume regulatory mechanisms. Patch clamp recording demonstrated inwardly and outwardly rectifying anion channels in infected but not in control erythrocytes. The molecular identity of those channels remained elusive. We show here for one channel type that voltage dependen...

  10. Tremorgenic indole alkaloids potently inhibit smooth muscle high-conductance calcium-activated potassium channels.

    Science.gov (United States)

    Knaus, H G; McManus, O B; Lee, S H; Schmalhofer, W A; Garcia-Calvo, M; Helms, L M; Sanchez, M; Giangiacomo, K; Reuben, J P; Smith, A B

    1994-05-17

    Tremorgenic indole alkaloids produce neurological disorders (e.g., staggers syndromes) in ruminants. The mode of action of these fungal mycotoxins is not understood but may be related to their known effects on neurotransmitter release. To determine whether these effects could be due to inhibition of K+ channels, the interaction of various indole diterpenes with high-conductance Ca(2+)-activated K+ (maxi-K) channels was examined. Paspalitrem A, paspalitrem C, aflatrem, penitrem A, and paspalinine inhibit binding of [125I]charybdotoxin (ChTX) to maxi-K channels in bovine aortic smooth muscle sarcolemmal membranes. In contrast, three structurally related compounds, paxilline, verruculogen, and paspalicine, enhanced toxin binding. As predicted from the binding studies, covalent incorporation of [125I]ChTX into the 31-kDa subunit of the maxi-K channel was blocked by compounds that inhibit [125I]ChTX binding and enhanced by compounds that stimulate [125I]ChTX binding. Modulation of [125I]ChTX binding was due to allosteric mechanisms. Despite their different effects on binding of [125I]ChTX to maxi-K channels, all compounds potently inhibited maxi-K channels in electrophysiological experiments. Other types of voltage-dependent or Ca(2+)-activated K+ channels examined were not affected. Chemical modifications of paxilline indicate a defined structure-activity relationship for channel inhibition. Paspalicine, a deshydroxy analog of paspalinine lacking tremorgenic activity, also potently blocked maxi-K channels. Taken together, these data suggest that indole diterpenes are the most potent nonpeptidyl inhibitors of maxi-K channels identified to date. Some of their pharmacological properties could be explained by inhibition of maxi-K channels, although tremorgenicity may be unrelated to channel block.

  11. Plasmodium induces swelling-activated ClC-2 anion channels in the host erythrocyte

    OpenAIRE

    Huber, Stephan M.; Duranton, Christophe; Henke, Guido; Van De Sand, Claudia; Heussler, Volker; Shumilina, Ekaterina; Sandu, Ciprian D.; Tanneur, Valerie; Brand, Verena; Kasinathan, Ravi S.; Lang, Karl S; Peter G Kremsner; Christian A. Hübner; Rust, Marco B.; Dedek, Karin

    2004-01-01

    Intraerythrocytic growth of the human malaria parasite Plasmodium falciparum depends on delivery of nutrients. Moreover, infection challenges cell volume constancy of the host erythrocyte requiring enhanced activity of cell volume regulatory mechanisms. Patch clamp recording demonstrated inwardly and outwardly rectifying anion channels in infected but not in control erythrocytes. The molecular identity of those channels remained elusive. We show here for one channel type that voltage dependen...

  12. Ferroelectric active models of ion channels in biomembranes.

    Science.gov (United States)

    Bystrov, V S; Lakhno, V D; Molchanov, M

    1994-06-21

    Ferroactive models of ion channels in the theory of biological membranes are presented. The main equations are derived and their possible solutions are shown. The estimates of some experimentally measured parameters are given. Possible physical consequences of the suggested models are listed and the possibility of their experimental finding is discussed. The functioning of the biomembrane's ion channel is qualitatively described on the basis of the suggested ferroactive models. The main directions and prospects for development of the ferroactive approach to the theory of biological membranes and their structures are indicated.

  13. Thermodynamic view of activation energies of proton transfer in various gramicidin A channels.

    Science.gov (United States)

    Chernyshev, Anatoly; Cukierman, Samuel

    2002-01-01

    The temperature dependencies (range: 5-45 degrees C) of single-channel proton conductances (g(H)) in native gramicidin A (gA) and in two diastereoisomers (SS and RR) of the dioxolane-linked gA channels were measured in glycerylmonooleate/decane (GMO) and diphytanoylphosphatidylcholine/decane (DiPhPC) bilayers. Linear Arrhenius plots (ln (g(H)) versus K(-1)) were obtained for the native gA and RR channels in both types of bilayers, and for the SS channel in GMO bilayers only. The Arrhenius plot for proton transfer in the SS channel in DiPhPC bilayers had a break in linearity around 20 degrees C. This break seems to occur only when protons are the permeating cations in the SS channel. The activation energies (E(a)) for proton transfer in various gA channels (approximately 15 kJ/mol) are consistent with the rate-limiting step being in the channel and/or at the membrane-channel/solution interface, and not in bulk solution. E(a) values for proton transfer in gA channels are considerably smaller than for the permeation of nonproton currents in gA as well as in various other ion channels. The E(a) values for proton transfer in native gA channels are nearly the same in both GMO and DiPhPC bilayers. In contrast, for the dioxolane linked gA dimers, E(a) values were strongly modulated by the lipid environment. The Gibbs activation free energies (Delta G(#)(o)) for protons in various gA channels are within the range of 27-29 kJ/mol in GMO bilayers and of 20-22 kJ/mol in DiPhPC bilayers. The largest difference between Delta G(#)(o) for proton currents occurs between native gA (or SS channels) and the RR channel. In general, the activation entropy (Delta S) is mostly responsible for the differences between g(H) values in various gA channels, and also in distinct bilayers. However, significant differences between the activation enthalpies (Delta H(#)(o)) for proton transfer in the SS and RR channels occur in distinct membranes.

  14. CNTF-Treated Astrocyte Conditioned Medium Enhances Large-Conductance Calcium-Activated Potassium Channel Activity in Rat Cortical Neurons.

    Science.gov (United States)

    Sun, Meiqun; Liu, Hongli; Xu, Huanbai; Wang, Hongtao; Wang, Xiaojing

    2016-08-01

    Seizure activity is linked to astrocyte activation as well as dysfunctional cortical neuron excitability produced from changes in calcium-activated potassium (KCa) channel function. Ciliary neurotrophic factor-treated astrocyte conditioned medium (CNTF-ACM) can be used to investigate the peripheral effects of activated astrocytes upon cortical neurons. However, CNTF-ACM's effect upon KCa channel activity in cultured cortical neurons has not yet been investigated. Whole-cell patch clamp recordings were performed in rat cortical neurons to evaluate CNTF-ACM's effects upon charybdotoxin-sensitive large-conductance KCa (BK) channel currents and apamin-sensitive small-conductance KCa (SK) channel current. Biotinylation and RT-PCR were applied to assess CNTF-ACM's effects upon the protein and mRNA expression, respectively, of the SK channel subunits SK2 and SK3 and the BK channel subunits BKα1 and BKβ3. An anti-fibroblast growth factor-2 (FGF-2) monoclonal neutralizing antibody was used to assess the effects of the FGF-2 component of CNTF-ACM. CNTF-ACM significantly increased KCa channel current density, which was predominantly attributable to gains in BK channel activity (p ACM produced a significant increase in BKα1 and BKβ3 expression (p  0.05). Blocking FGF-2 produced significant reductions in KCa channel current density (p > 0.05) as well as BKα1 and BKβ3 expression in CNTF-ACM-treated neurons (p > 0.05). CNTF-ACM significantly enhances BK channel activity in rat cortical neurons and that FGF-2 is partially responsible for these effects. CNTF-induced astrocyte activation results in secretion of neuroactive factors which may affect neuronal excitability and resultant seizure activity in mammalian cortical neurons.

  15. CRL4A(CRBN) E3 ubiquitin ligase restricts BK channel activity and prevents epileptogenesis.

    Science.gov (United States)

    Liu, Jiye; Ye, Jia; Zou, Xiaolong; Xu, Zhenghao; Feng, Yan; Zou, Xianxian; Chen, Zhong; Li, Yuezhou; Cang, Yong

    2014-05-21

    Ion channels regulate membrane excitation, and mutations of ion channels often cause serious neurological disorders including epilepsy. Compared with extensive analyses of channel protein structure and function, much less is known about the fine tuning of channel activity by post-translational modification. Here we report that the large conductance, Ca(2+)- and voltage-activated K(+) (BK) channels are targeted by the E3 ubiquitin ligase CRL4A(CRBN) for polyubiquitination and retained in the endoplasmic reticulum (ER). Inactivation of CRL4A(CRBN) releases deubiquitinated BK channels from the ER to the plasma membrane, leading to markedly enhanced channel activity. Mice with CRL4A(CRBN) mutation in the brain or treated with a CRL4A(CRBN) inhibitor are very sensitive to seizure induction, which can be attenuated by blocking BK channels. Finally, the mutant mice develop spontaneous epilepsy when aged. Therefore, ubiquitination of BK channels before their cell surface expression is an important step to prevent systemic neuronal excitability and epileptogenesis.

  16. Cell swelling activates cloned Ca(2+)-activated K(+) channels: a role for the F-actin cytoskeleton

    DEFF Research Database (Denmark)

    Jorgensen, Nanna K; Pedersen, Stine F; Rasmussen, Hanne B;

    2003-01-01

    Cloned Ca(2+)-activated K(+) channels of intermediate (hIK) or small (rSK3) conductance were expressed in HEK 293 cells, and channel activity was monitored using whole-cell patch clamp. hIK and rSK3 currents already activated by intracellular calcium were further increased by 95% and 125......%, respectively, upon exposure of the cells to a 33% decrease in extracellular osmolarity. hIK and rSK3 currents were inhibited by 46% and 32%, respectively, by a 50% increase in extracellular osmolarity. Cell swelling and channel activation were not associated with detectable increases in [Ca(2+)](i), evidenced...... by population and single-cell measurements. In addition, inhibitors of IK and SK channels significantly reduced the rate of regulatory volume decrease (RVD) in cells expressing these channels. Cell swelling induced a decrease, and cell shrinkage an increase, in net cellular F-actin content. The swelling...

  17. WNK1 Activates Large-Conductance Ca2+-Activated K+ Channels through Modulation of ERK1/2 Signaling

    OpenAIRE

    Liu, Yingli; Song, Xiang; Shi, Yanling; Shi, Zhen; Niu, Weihui; Feng, Xiuyan; Gu, Dingying; Bao, Hui-Fang; Ma, He-Ping; Eaton, Douglas C.; Zhuang, Jieqiu; Cai, Hui

    2014-01-01

    With no lysine (WNK) kinases are members of the serine/threonine kinase family. We previously showed that WNK4 inhibits renal large-conductance Ca2+-activated K+ (BK) channel activity by enhancing its degradation through a lysosomal pathway. In this study, we investigated the effect of WNK1 on BK channel activity. In HEK293 cells stably expressing the α subunit of BK (HEK-BKα cells), siRNA-mediated knockdown of WNK1 expression significantly inhibited both BKα channel activity and open probabi...

  18. Calcium-calmodulin does not alter the anion permeability of the mouse TMEM16A calcium-activated chloride channel.

    Science.gov (United States)

    Yu, Yawei; Kuan, Ai-Seon; Chen, Tsung-Yu

    2014-07-01

    The transmembrane protein TMEM16A forms a Ca(2+)-activated Cl(-) channel that is permeable to many anions, including SCN(-), I(-), Br(-), Cl(-), and HCO3 (-), and has been implicated in various physiological functions. Indeed, controlling anion permeation through the TMEM16A channel pore may be critical in regulating the pH of exocrine fluids such as the pancreatic juice. The anion permeability of the TMEM16A channel pore has recently been reported to be modulated by Ca(2+)-calmodulin (CaCaM), such that the pore of the CaCaM-bound channel shows a reduced ability to discriminate between anions as measured by a shift of the reversal potential under bi-ionic conditions. Here, using a mouse TMEM16A clone that contains the two previously identified putative CaM-binding motifs, we were unable to demonstrate such CaCaM-dependent changes in the bi-ionic potential. We confirmed the activity of CaCaM used in our study by showing CaCaM modulation of the olfactory cyclic nucleotide-gated channel. We suspect that the different bi-ionic potentials that were obtained previously from whole-cell recordings in low and high intracellular [Ca(2+)] may result from different degrees of bi-ionic potential shift secondary to a series resistance problem, an ion accumulation effect, or both.

  19. Coassembly of big conductance Ca2+-activated K+ channels and L-type voltage-gated Ca2+ channels in rat brain

    DEFF Research Database (Denmark)

    Grunnet, Morten; Kaufmann, Walter A

    2004-01-01

    . The nature of the apparent coupling is not known. In the present study we report a direct coassembly of big conductance Ca(2+)-activated K(+) channels (BK) and L-type voltage-gated Ca(2+) channels in rat brain. Saturation immunoprecipitation studies were performed on membranes labeled for BK channels...... to separate ion channel complexes. Finally, immunochemical studies showed a distinct but overlapping expression pattern of the two types of ion channels investigated. BK and L-type Ca(2+) channels were colocalized in various compartments throughout the rat brain. Taken together, these results demonstrate...... a direct coassembly of BK channels and L-type Ca(2+) channels in certain areas of the brain....

  20. Effects of familial hemiplegic migraine type 1 mutations on neuronal P/Q-type Ca2+ channel activity and inhibitory synaptic transmission.

    Science.gov (United States)

    Cao, Yu-Qing; Tsien, Richard W

    2005-02-15

    Inhibitory synapses play key roles in the modulatory circuitry that regulates pain signaling and generation of migraine headache. A rare, dominant form of this common disease, familial hemiplegic migraine type 1 (FHM1), arises from missense mutations in the pore-forming alpha1A subunit of P/Q-type Ca2+ channels. These channels are normally vital for presynaptic Ca2+ entry and neurotransmitter release at many central synapses, raising questions about effects of FHM1 mutations on neuronal Ca2+ influx and inhibitory and excitatory neurotransmission. We have expressed the four original FHM1 mutant channels in hippocampal neurons from alpha1A knockout mice. Whole-cell recordings indicated that FHM1 mutant channels were less effective than wild-type channels in their ability to conduct P/Q-type current, but not generally different from wild type in voltage-dependent channel gating. Ca2+ influx triggered by action potential waveforms was also diminished. In keeping with decreased channel activity, FHM1 mutant channels were correspondingly impaired in supporting the P/Q-type component of inhibitory neurotransmission. When expressed in wild-type inhibitory neurons, FHM1 mutant channels reduced the contribution of P/Q-type channels to GABAergic synaptic currents, consistent with a competition of mutant and endogenous channels for P/Q-specific slots. In all cases, N-type channels took up the burden of supporting transmission and homeostatic mechanisms maintained overall synaptic strength. The shift to reliance on N-type channels greatly increased the susceptibility to G protein-coupled modulation of neurotransmission, studied with the GABAB agonist baclofen. Thus, mutant-expressing synapses might be weakened in a heightened state of neuromodulation like that provoked by triggers of migraine such as stress.

  1. BK channel activation by NS11021 decreases excitability and contractility of urinary bladder smooth muscle

    DEFF Research Database (Denmark)

    Layne, Jeffrey J; Nausch, Bernhard; Olesen, Søren-Peter

    2009-01-01

    Large-conductance Ca(2+)-activated potassium (BK) channels play an important role in regulating the function and activity of urinary bladder smooth muscle (UBSM), and the loss of BK channel function has been shown to increase UBSM excitability and contractility. However, it is not known whether......(o)) and whole cell BK channel currents. The frequency of spontaneous action potentials in UBSM strips was reduced by NS11021 from a control value of 20.9 + or - 5.9 to 10.9 + or - 3.7 per minute. NS11021 also reduced the force of UBSM spontaneous phasic contractions by approximately 50%, and this force...... reduction was blocked by pretreatment with the BK channel blocker iberiotoxin. NS11021 (3 microM) had no effect on contractions evoked by nerve stimulation. These findings indicate that activating BK channels reduces the force of UBSM spontaneous phasic contractions, principally through decreasing...

  2. Role of calcium activated potassium channels in atrial fibrillation pathophysiology and therapy

    DEFF Research Database (Denmark)

    Diness, Jonas G.; Bentzen, Bo H.; S. Sørensen, Ulrik

    2015-01-01

    Small-conductance Ca2+-activated potassium (SK) channels are relative newcomers within the field of cardiac electrophysiology. In recent years, an increased focus has been given to these channels since they might constitute a relatively atrial selective target. The present review will give...

  3. Inhibition of small-conductance Ca2+-activated K+ channels terminates and protects against atrial fibrillation

    DEFF Research Database (Denmark)

    Diness, Jonas Goldin; Sørensen, Ulrik S; Nissen, Jakob Dahl

    2010-01-01

    Recently, evidence has emerged that small-conductance Ca(2+)-activated K(+) (SK) channels are predominantly expressed in the atria in a number of species including human. In rat, guinea pig, and rabbit ex vivo and in vivo models of atrial fibrillation (AF), we used 3 different SK channel inhibito...

  4. Upregulation of voltage-activated potassium channels in hippocampus of Aβ25.35-treated rats

    Institute of Scientific and Technical Information of China (English)

    Xiao-liangWANG; Ya-pingPAN

    2004-01-01

    AIM: Potassium channels dysfunction has been indicated in Alzheimer disease. In the present study, the mRNA and protein expression alterations and the functional changes ot VOltage- activated potassium channels were studied in rat hippocampus after a single intracerebro- ventricular injection of β-amyloid peptide 25-35 (Aβ25.35). METHODS: The expressions of mRNA

  5. Quantification and distribution of big conductance Ca2+-activated K+ channels in kidney epithelia

    DEFF Research Database (Denmark)

    Grunnet, Morten; Hay-Schmidt, Anders; Klaerke, Dan A

    2005-01-01

    channels were determined by a isotope flux assay where up to 44% of the total K+ channel activity could be inhibited by iberiotoxin indicating that BK channels are widely present in kidney epithelia. Consistent with these functional studies, 125I-IbTX-D19Y/Y36F binds to membrane vesicles from outer cortex......, outer medulla and inner medulla with Bmax values (in fmol/mg protein) of 6.8, 2.6 and 21.4, respectively. These studies were performed applying rabbit kidney epithelia tissue. The distinct distribution of BK channels in both rabbit and rat kidney epithelia was confirmed by autoradiography...

  6. Stimulatory actions of a novel thiourea derivative on large-conductance, calcium-activated potassium channels.

    Science.gov (United States)

    Wu, Sheng-Nan; Chern, Jyh-Haur; Shen, Santai; Chen, Hwei-Hisen; Hsu, Ying-Ting; Lee, Chih-Chin; Chan, Ming-Huan; Lai, Ming-Chi; Shie, Feng-Shiun

    2017-12-01

    In this study, we examine whether an anti-inflammatory thiourea derivative, compound #326, actions on ion channels. The effects of compound #326 on Ca(2+) -activated K(+) channels were evaluated by patch-clamp recordings obtained in cell-attached, inside-out or whole-cell configuration. In pituitary GH3 cells, compound #326 increased the amplitude of Ca(2+) -activated K(+) currents (IK(Ca) ) with an EC50 value of 11.6 μM, which was reversed by verruculogen, but not tolbutamide or TRAM-34. Under inside-out configuration, a bath application of compound #326 raised the probability of large-conductance Ca(2+) -activated K(+) (BKCa ) channels. The activation curve of BKCa channels was shifted to less depolarised potential with no modification of the gating charge of the curve; consequently, the difference of free energy was reduced in the presence of this compound. Compound #326-stimulated activity of BKCa channels is explained by a shortening of mean closed time, despite its inability to alter single-channel conductance. Neither delayed-rectifier nor erg-mediated K(+) currents was modified. Compound #326 decreased the peak amplitude of voltage-gated Na(+) current with no clear change in the overall current-voltage relationship of this current. In HEK293T cells expressing α-hSlo, compound #326 enhanced BKCa channels effectively. Intriguingly, the inhibitory actions of compound #326 on interleukin 1β in lipopolysaccharide-activated microglia were significantly reversed by verruculogen, whereas BKCa channel inhibitors suppressed the expressions of inducible nitric oxide synthase. The BKCa channels could be an important target for compound #326 if similar in vivo results occur, and the multi-functionality of BKCa channels in modulating microglial immunity merit further investigation. © 2017 Wiley Periodicals, Inc.

  7. Small-conductance Ca2+ -activated K+ channels and cardiac arrhythmias.

    Science.gov (United States)

    Zhang, Xiao-Dong; Lieu, Deborah K; Chiamvimonvat, Nipavan

    2015-08-01

    Small-conductance Ca2+ -activated K+ (SK, KCa2) channels are unique in that they are gated solely by changes in intracellular Ca2+ and, hence, function to integrate intracellular Ca2+ and membrane potentials on a beat-to-beat basis. Recent studies have provided evidence for the existence and functional significance of SK channels in the heart. Indeed, our knowledge of cardiac SK channels has been greatly expanded over the past decade. Interests in cardiac SK channels are further driven by recent studies suggesting the critical roles of SK channels in human atrial fibrillation, the SK channel as a possible novel therapeutic target in atrial arrhythmias, and upregulation of SK channels in heart failure in animal models and in human heart failure. However, there remain critical gaps in our knowledge. Specifically, blockade of SK channels in cardiac arrhythmias has been shown to be both antiarrhythmic and proarrhythmic. This contemporary review provides an overview of the literature on the role of cardiac SK channels in cardiac arrhythmias and serves as a discussion platform for the current clinical perspectives. At the translational level, development of SK channel blockers as a new therapeutic strategy in the treatment of atrial fibrillation and the possible proarrhythmic effects merit further considerations and investigations.

  8. Activation of α7 nicotinic receptors by orthosteric and allosteric agonists: influence on single-channel kinetics and conductance.

    Science.gov (United States)

    Pałczyńska, Magda M; Jindrichova, Marie; Gibb, Alasdair J; Millar, Neil S

    2012-11-01

    Nicotinic acetylcholine receptors (nAChRs) are oligomeric transmembrane proteins in which five subunits coassemble to form a central ion channel pore. Conventional agonists, such as acetylcholine (ACh), bind to an orthosteric site, located at subunit interfaces in the extracellular domain. More recently, it has been demonstrated that nAChRs can also be activated by ligands binding to an allosteric transmembrane site. In the case of α7 nAChRs, ACh causes rapid activation and almost complete desensitization. In contrast, allosteric agonists such as 4-(4-bromophenyl)-3a,4,5,9b-tetrahydro-3H-cyclopenta[c] quin oline-8-sulfonamide (4BP-TQS) activate α7 nAChRs more slowly and cause only low levels of apparent desensitization. In the present study, single-channel patch-clamp recording has been used to investigate differences in the mechanism of activation of α7 nAChRs by ACh and 4BP-TQS. The most striking difference between activation by ACh and 4BP-TQS is in single-channel kinetics. In comparison with activation by ACh, single-channel open times and burst lengths are substantially longer (~160-800-fold, respectively), and shut times are shorter (~8-fold) when activated by 4BP-TQS. In addition, coapplication of ACh and 4BP-TQS results in a further increase in single-channel burst lengths. Mean burst lengths seen when the two agonists are coapplied (3099 ± 754 ms) are ~2.5-fold longer than with 4BP-TQS alone and ∼370-fold longer than with ACh alone. Intriguingly, the main single-channel conductance of α7 nAChRs, was significantly larger when activated by 4BP-TQS (100.3 ± 2.4 pS) than when activated by ACh (90.0 ± 2.7 pS), providing evidence that activation by allosteric and orthosteric agonists results in different α7 nAChRs open-channel conformations.

  9. Kv Channel S1-S2 Linker Working as a Binding Site of Human β-Defensin 2 for Channel Activation Modulation.

    Science.gov (United States)

    Feng, Jing; Yang, Weishan; Xie, Zili; Xiang, Fang; Cao, Zhijian; Li, Wenxin; Hu, Hongzhen; Chen, Zongyun; Wu, Yingliang

    2015-06-19

    Among the three extracellular domains of the tetrameric voltage-gated K(+) (Kv) channels consisting of six membrane-spanning helical segments named S1-S6, the functional role of the S1-S2 linker still remains unclear because of the lack of a peptide ligand. In this study, the Kv1.3 channel S1-S2 linker was reported as a novel receptor site for human β-defensin 2 (hBD2). hBD2 shifts the conductance-voltage relationship curve of the human Kv1.3 channel in a positive direction by nearly 10.5 mV and increases the activation time constant for the channel. Unlike classical gating modifiers of toxin peptides from animal venoms, which generally bind to the Kv channel S3-S4 linker, hBD2 only targets residues in both the N and C termini of the S1-S2 linker to influence channel gating and inhibit channel currents. The increment and decrement of the basic residue number in a positively charged S4 sensor of Kv1.3 channel yields conductance-voltage relationship curves in the positive direction by ∼31.2 mV and 2-4 mV, which suggests that positively charged hBD2 is anchored in the channel S1-S2 linker and is modulating channel activation through electrostatic repulsion with an adjacent S4 helix. Together, these findings reveal a novel peptide ligand that binds with the Kv channel S1-S2 linker to modulate channel activation. These findings also highlight the functional importance of the Kv channel S1-S2 linker in ligand recognition and modification of channel activation.

  10. Two distinct pools of large-conductance calcium-activated potassium channels in the somatic plasma membrane of central principal neurons

    Science.gov (United States)

    Kaufmann, W.A.; Kasugai, Y.; Ferraguti, F.; Storm, J.F.

    2010-01-01

    Although nerve cell membranes are often assumed to be uniform with respect to electrical properties, there is increasing evidence for compartmentalization into subdomains with heterogeneous impacts on the overall cell function. Such microdomains are characterized by specific sets of proteins determining their functional properties. Recently, clustering of large-conductance calcium-activated potassium (BKCa) channels was shown at sites of subsurface membrane cisterns in cerebellar Purkinje cells (PC), where they likely participate in building a subcellular signaling unit, the 'PLasmERosome'. By applying SDS-digested freeze-fracture replica labeling (SDS-FRL) and postembedding immunogold electron microscopy, we have now studied the spatial organization of somatic BKCa channels in neocortical layer 5 pyramidal neurons, principal neurons of the central and basolateral amygdaloid nuclei, hippocampal pyramidal neurons and dentate gyrus (DG) granule cells to establish whether there is a common organizational principle in the distribution of BKCa channels in central principal neurons. In all cell types analyzed, somatic BKCa channels were found to be non-homogenously distributed in the plasma membrane, forming two pools of channels with one pool consisting of clustered channels and the other of scattered channels in the extrasynaptic membrane. Quantitative analysis by means of SDS-FRL revealed that about two-thirds of BKCa channels belong to the scattered pool and about one-third to the clustered pool in principal cell somata. Overall densities of channels in both pools differed in the different cell types analyzed, although being considerably lower compared to cerebellar PC. Postembedding immunogold labeling revealed association of clustered channels with subsurface membrane cisterns and confirmed extrasynaptic localization of scattered channels. This study indicates a common organizational principle for somatic BKCa channels in central principal neurons with the

  11. Characterization of ryanodine receptor type 1 single channel activity using "on-nucleus" patch clamp.

    Science.gov (United States)

    Wagner, Larry E; Groom, Linda A; Dirksen, Robert T; Yule, David I

    2014-08-01

    In this study, we provide the first description of the biophysical and pharmacological properties of ryanodine receptor type 1 (RyR1) expressed in a native membrane using the on-nucleus configuration of the patch clamp technique. A stable cell line expressing rabbit RyR1 was established (HEK-RyR1) using the FLP-in 293 cell system. In contrast to untransfected cells, RyR1 expression was readily demonstrated by immunoblotting and immunocytochemistry in HEK-RyR1 cells. In addition, the RyR1 agonists 4-CMC and caffeine activated Ca(2+) release that was inhibited by high concentrations of ryanodine. On nucleus patch clamp was performed in nuclei prepared from HEK-RyR1 cells. Raising the [Ca(2+)] in the patch pipette resulted in the appearance of a large conductance cation channel with well resolved kinetics and the absence of prominent subconductance states. Current versus voltage relationships were ohmic and revealed a chord conductance of ∼750pS or 450pS in symmetrical 250mM KCl or CsCl, respectively. The channel activity was markedly enhanced by caffeine and exposure to ryanodine resulted in the appearance of a subconductance state with a conductance ∼40% of the full channel opening with a Po near unity. In total, these properties are entirely consistent with RyR1 channel activity. Exposure of RyR1 channels to cyclic ADP ribose (cADPr), nicotinic acid adenine dinucleotide phosphate (NAADP) or dantrolene did not alter the single channel activity stimulated by Ca(2+), and thus, it is unlikely these molecules directly modulate RyR1 channel activity. In summary, we describe an experimental platform to monitor the single channel properties of RyR channels. We envision that this system will be influential in characterizing disease-associated RyR mutations and the molecular determinants of RyR channel modulation.

  12. Antagonism of ligand-gated ion channel receptors: two domains of the glycine receptor alpha subunit form the strychnine-binding site.

    Science.gov (United States)

    Vandenberg, R J; French, C R; Barry, P H; Shine, J; Schofield, P R

    1992-01-01

    The inhibitory glycine receptor (GlyR) is a member of the ligand-gated ion channel receptor superfamily. Glycine activation of the receptor is antagonized by the convulsant alkaloid strychnine. Using in vitro mutagenesis and functional analysis of the cDNA encoding the alpha 1 subunit of the human GlyR, we have identified several amino acid residues that form the strychnine-binding site. These residues were identified by transient expression of mutated cDNAs in mammalian (293) cells and examination of resultant [3H]strychnine binding, glycine displacement of [3H]strychnine, and electrophysiological responses to the application of glycine and strychnine. This mutational analysis revealed that residues from two separate domains within the alpha 1 subunit form the binding site for the antagonist strychnine. The first domain includes the amino acid residues Gly-160 and Tyr-161, and the second domain includes the residues Lys-200 and Tyr-202. These results, combined with analyses of other ligand-gated ion channel receptors, suggest a conserved tertiary structure and a common mechanism for antagonism in this receptor superfamily. PMID:1311851

  13. Channel formation by RTX-toxins of pathogenic bacteria: Basis of their biological activity.

    Science.gov (United States)

    Benz, Roland

    2016-03-01

    The pore-forming cytolysins of the RTX-toxin (Repeats in ToXin) family are a relatively small fraction of a steadily increasing family of proteins that contain several functionally important glycine-rich and aspartate containing nonapeptide repeats. These cytolysins produced by a variety of Gram-negative bacteria form ion-permeable channels in erythrocytes and other eukaryotic cells. Hemolytic and cytolytic RTX-toxins represent pathogenicity factors of the toxin-producing bacteria and are very often important key factors in pathogenesis of the bacteria. Channel formation by RTX-toxins lead to the dissipation of ionic gradients and membrane potential across the cytoplasmic membrane of target cells, which results in cell death. Here we discuss channel formation and channel properties of some of the best known RTX-toxins, such as α-hemolysin (HlyA) of Escherichia coli and the uropathogenic EHEC strains, the adenylate cyclase toxin (ACT, CyaA) of Bordetella pertussis and the RTX-toxins (ApxI, ApxII and ApxIII) produced by different strains of Actinobacillus pleuropneumoniae. The channels formed by these RTX-toxins in lipid bilayers share some common properties such as cation selectivity and voltage-dependence. Furthermore the channels are transient and show frequent switching between different ion-conducting states. This article is part of a Special Issue entitled: Pore-Forming Toxins edited by Mauro Dalla Serra and Franco Gambale.

  14. UCP3 Regulates Single-Channel Activity of the Cardiac mCa1.

    Science.gov (United States)

    Motloch, Lukas J; Gebing, Tina; Reda, Sara; Schwaiger, Astrid; Wolny, Martin; Hoppe, Uta C

    2016-08-01

    Mitochondrial Ca(2+) uptake (mCa(2+) uptake) is thought to be mediated by the mitochondrial Ca(2+) uniporter (MCU). UCP2 and UCP3 belong to a superfamily of mitochondrial ion transporters. Both proteins are expressed in the inner mitochondrial membrane of the heart. Recently, UCP2 was reported to modulate the function of the cardiac MCU related channel mCa1. However, the possible role of UCP3 in modulating cardiac mCa(2+) uptake via the MCU remains inconclusive. To understand the role of UCP3, we analyzed cardiac mCa1 single-channel activity in mitoplast-attached single-channel recordings from isolated murine cardiac mitoplasts, from adult wild-type controls (WT), and from UCP3 knockout mice (UCP3(-/-)). Single-channel registrations in UCP3(-/-) confirmed a murine voltage-gated Ca(2+) channel, i.e., mCa1, which was inhibited by Ru360. Compared to WT, mCa1 in UCP3(-/-) revealed similar single-channel characteristics. However, in UCP3(-/-) the channel exhibited decreased single-channel activity, which was insensitive to adenosine triphosphate (ATP) inhibition. Our results suggest that beyond UCP2, UCP3 also exhibits regulatory effects on cardiac mCa1/MCU function. Furthermore, we speculate that UCP3 might modulate previously described inhibitory effects of ATP on mCa1/MCU activity as well.

  15. Control of anterior pituitary cell excitability by calcium-activated potassium channels.

    Science.gov (United States)

    Shipston, Michael J

    2017-06-05

    In anterior pituitary endocrine cells, large (BK), small (SK) and intermediate (IK) conductance calcium activated potassium channels are key determinants in shaping cellular excitability in a cell type- and context-specific manner. Indeed, these channels are targeted by multiple signaling pathways that stimulate or inhibit cellular excitability. BK channels can, paradoxically, both promote electrical bursting as well as terminate bursting and spiking dependent upon intrinsic BK channel properties and proximity to voltage gated calcium channels in somatotrophs, lactotrophs and corticotrophs. In contrast, SK channels are predominantly activated by calcium released from intracellular IP3-sensitive calcium stores and mediate membrane hyperpolarization in cells including gonadotrophs and corticotrophs. IK channels are predominantly expressed in corticotrophs where they limit membrane excitability. A major challenge for the future is to determine the cell-type specific molecular composition of calcium-activated potassium channels and how they control anterior pituitary hormone secretion as well as other calcium-dependent processes. Copyright © 2017. Published by Elsevier B.V.

  16. Calibrated and Interactive Modelling of Form-Active Hybrid Structures

    DEFF Research Database (Denmark)

    Quinn, Gregory; Holden Deleuran, Anders; Piker, Daniel

    2016-01-01

    Form-active hybrid structures (FAHS) couple two or more different structural elements of low self weight and low or negligible bending flexural stiffness (such as slender beams, cables and membranes) into one structural assembly of high global stiffness. They offer high load-bearing capacity...... materially-informed sketching. Making use of a projection-based dynamic relaxation solver for structural analysis, explorative design has proven to be highly effective....

  17. OSR1 and SPAK Sensitivity of Large-Conductance Ca2+ Activated K+ Channel

    Directory of Open Access Journals (Sweden)

    Bernat Elvira

    2016-04-01

    Full Text Available Background/Aims: The oxidative stress-responsive kinase 1 (OSR1 and the serine/threonine kinases SPAK (SPS1-related proline/alanine-rich kinase are under the control of WNK (with-no-K [Lys] kinases. OSR1 and SPAK participate in diverse functions including cell volume regulation and neuronal excitability. Cell volume and neuronal excitation are further modified by the large conductance Ca2+-activated K+ channels (maxi K+ channel or BK channels. An influence of OSR1 and/or SPAK on BK channel activity has, however, never been shown. The present study thus explored whether OSR1 and/or SPAK modify the activity of BK channels. Methods: cRNA encoding the Ca2+ insensitive BK channel mutant BKM513I+Δ899-903 was injected into Xenopus laevis oocytes without or with additional injection of cRNA encoding wild-type OSR1 or wild-type SPAK, constitutively active T185EOSR1, catalytically inactive D164AOSR1, constitutively active T233ESPAK or catalytically inactive D212ASPAK. K+ channel activity was measured utilizing dual electrode voltage clamp. Results: BK channel activity in BKM513I+Δ899-903 expressing oocytes was significantly decreased by co-expression of OSR1 or SPAK. The effect of wild-type OSR1/SPAK was mimicked by T185EOSR1 and T233ESPAK, but not by D164AOSR1 or D212ASPAK. Conclusions: OSR1 and SPAK suppress BK channels, an effect possibly contributing to cell volume regulation and neuroexcitability.

  18. Cardiac mechanosensitivity and stretch-activated ion channels.

    Science.gov (United States)

    Bett, G C; Sachs, F

    1997-01-01

    Mechanosensitivity is a ubiquitous property of cells, and mechanosensitive ion channels (MSCs) are hypothesized to be the transducers. In the heart, MSCs are likely to account for changes in beating rate as a function of filling and for initiating stretch-induced arrhythmias (for example, following a myocardial infarction). Pharmacological agents that affect MSCs may provide a new class of antiarrhythmic drugs. © 1997, Elsevier Science Inc. (Trends Cardiovasc Med 1997;7:4-8).

  19. Regulation of Voltage-Activated K(+) Channel Gating by Transmembrane β Subunits.

    Science.gov (United States)

    Sun, Xiaohui; Zaydman, Mark A; Cui, Jianmin

    2012-01-01

    Voltage-activated K(+) (K(V)) channels are important for shaping action potentials and maintaining resting membrane potential in excitable cells. K(V) channels contain a central pore-gate domain (PGD) surrounded by four voltage-sensing domains (VSDs). The VSDs will change conformation in response to alterations of the membrane potential thereby inducing the opening of the PGD. Many K(V) channels are heteromeric protein complexes containing auxiliary β subunits. These β subunits modulate channel expression and activity to increase functional diversity and render tissue specific phenotypes. This review focuses on the K(V) β subunits that contain transmembrane (TM) segments including the KCNE family and the β subunits of large conductance, Ca(2+)- and voltage-activated K(+) (BK) channels. These TM β subunits affect the voltage-dependent activation of K(V) α subunits. Experimental and computational studies have described the structural location of these β subunits in the channel complexes and the biophysical effects on VSD activation, PGD opening, and VSD-PGD coupling. These results reveal some common characteristics and mechanistic insights into K(V) channel modulation by TM β subunits.

  20. Regulation of KV channel voltage-dependent activation by transmembrane β subunits

    Directory of Open Access Journals (Sweden)

    Xiaohui eSun

    2012-04-01

    Full Text Available Voltage-activated K+ (KV channels are important for shaping action potentials and maintaining resting membrane potential in excitable cells. KV channels contain a central pore-gate domain (PGD surrounded by four voltage-sensing domains (VSD. The VSDs will change conformation in response to alterations of the membrane potential thereby inducing the opening of the PGD. Many KV channels are heteromeric protein complexes containing auxiliary β subunits. These β subunits modulate channel expression and activity to increase functional diversity and render tissue specific phenotypes. This review focuses on the KV β subunits that contain transmembrane (TM segments including the KCNE family and the β subunits of large conductance, Ca2+- and voltage-activated K+ (BK channels. These TM β subunits affect the voltage-dependent activation of KV α subunits. Experimental and computational studies have described the structural location of these β subunits in the channel complexes and the biophysical effects on VSD activation, PGD opening and VSD-PGD coupling. These results reveal some common characteristics and mechanistic insights into KV channel modulation by TM β subunits.

  1. Structure of the active form of human origin recognition complex and its ATPase motor module

    Energy Technology Data Exchange (ETDEWEB)

    Tocilj, Ante; On, Kin Fan; Yuan, Zuanning; Sun, Jingchuan; Elkayam, Elad; Li, Huilin; Stillman, Bruce; Joshua-Tor, Leemor

    2017-01-23

    Binding of the Origin Recognition Complex (ORC) to origins of replication marks the first step in the initiation of replication of the genome in all eukaryotic cells. Here, we report the structure of the active form of human ORC determined by X-ray crystallography and cryo-electron microscopy. The complex is composed of an ORC1/4/5 motor module lobe in an organization reminiscent of the DNA polymerase clamp loader complexes. A second lobe contains the ORC2/3 subunits. The complex is organized as a double-layered shallow corkscrew, with the AAA+ and AAA+-like domains forming one layer, and the winged-helix domains (WHDs) forming a top layer. CDC6 fits easily between ORC1 and ORC2, completing the ring and the DNA-binding channel, forming an additional ATP hydrolysis site. Analysis of the ATPase activity of the complex provides a basis for understanding ORC activity as well as molecular defects observed in Meier-Gorlin Syndrome mutations.

  2. Structure of the active form of human origin recognition complex and its ATPase motor module

    Science.gov (United States)

    Tocilj, Ante; On, Kin Fan; Yuan, Zuanning; Sun, Jingchuan; Elkayam, Elad; Li, Huilin; Stillman, Bruce; Joshua-Tor, Leemor

    2017-01-01

    Binding of the Origin Recognition Complex (ORC) to origins of replication marks the first step in the initiation of replication of the genome in all eukaryotic cells. Here, we report the structure of the active form of human ORC determined by X-ray crystallography and cryo-electron microscopy. The complex is composed of an ORC1/4/5 motor module lobe in an organization reminiscent of the DNA polymerase clamp loader complexes. A second lobe contains the ORC2/3 subunits. The complex is organized as a double-layered shallow corkscrew, with the AAA+ and AAA+-like domains forming one layer, and the winged-helix domains (WHDs) forming a top layer. CDC6 fits easily between ORC1 and ORC2, completing the ring and the DNA-binding channel, forming an additional ATP hydrolysis site. Analysis of the ATPase activity of the complex provides a basis for understanding ORC activity as well as molecular defects observed in Meier-Gorlin Syndrome mutations. DOI: http://dx.doi.org/10.7554/eLife.20818.001 PMID:28112645

  3. Qualitative computational bioanalytics: assembly of viral channel-forming peptides around mono and divalent ions.

    Science.gov (United States)

    Li, Li-Hua; Hsu, Hao-Jen; Fischer, Wolfgang B

    2013-12-06

    A fine-grained docking protocol was used to generate a bundle-like structure of the bitopic membrane protein Vpu from HIV-1. Vpu is a type I membrane protein with 81 amino acids. It is proposed that Vpu forms ion- and substrate-conducting bundles, which are located at the plasma membrane in the infected cell. The Vpu1-32 peptide that includes the transmembrane domain (TMD) is assembled into homo-pentameric bundles around prepositioned Na, K, Ca or Cl ions. For bundles with the lowest energy, the TMDs generate a hydrophobic pore. Bundles in which Ser-24 faces the pore have higher energy. The tilt of the helices in the lowest energy bundles is larger than bundles with serines facing the pore. Left-handed bundles are lowest in energy where the ions are located at the serines.

  4. How Lipid Membranes Affect Pore Forming Toxin Activity.

    Science.gov (United States)

    Rojko, Nejc; Anderluh, Gregor

    2015-12-15

    Pore forming toxins (PFTs) evolved to permeate the plasma membrane of target cells. This is achieved in a multistep mechanism that usually involves binding of soluble protein monomer to the lipid membrane, oligomerization at the plane of the membrane, and insertion of part of the polypeptide chain across the lipid membrane to form a conductive channel. Introduced pores allow uncontrolled transport of solutes across the membrane, inflicting damage to the target cell. PFTs are usually studied from the perspective of structure-function relationships, often neglecting the important role of the bulk membrane properties on the PFT mechanism of action. In this Account, we discuss how membrane lateral heterogeneity, thickness, and fluidity influence the pore forming process of PFTs. In general, lipid molecules are more accessible for binding in fluid membranes due to steric reasons. When PFT specifically binds ordered domains, it usually recognizes a specific lipid distribution pattern, like sphingomyelin (SM) clusters or SM/cholesterol complexes, and not individual lipid species. Lipid domains were also suggested to act as an additional concentration platform facilitating PFT oligomerization, but this is yet to be shown. The last stage in PFT action is the insertion of the transmembrane segment across the membranes to build the transmembrane pore walls. Conformational changes are a spontaneous process, and sufficient free energy has to be available for efficient membrane penetration. Therefore, fluid bilayers are permeabilized more readily in comparison to highly ordered and thicker liquid ordered lipid phase (Lo). Energetically more costly insertion into the Lo phase can be driven by the hydrophobic mismatch between the thinner liquid disordered phase (Ld) and large protein complexes, which are unable to tilt like single transmembrane segments. In the case of proteolipid pores, membrane properties can directly modulate pore size, stability, and even selectivity. Finally

  5. Large-conductance Ca2+-activated potassium channels in secretory neurons.

    Science.gov (United States)

    Lara, J; Acevedo, J J; Onetti, C G

    1999-09-01

    Large-conductance Ca2+-activated K+ channels (BK) are believed to underlie interburst intervals and contribute to the control of hormone release in several secretory cells. In crustacean neurosecretory cells, Ca2+ entry associated with electrical activity could act as a modulator of membrane K+ conductance. Therefore we studied the contribution of BK channels to the macroscopic outward current in the X-organ of crayfish, and their participation in electrophysiological activity, as well as their sensitivity toward intracellular Ca2+, ATP, and voltage, by using the patch-clamp technique. The BK channels had a conductance of 223 pS and rectified inwardly in symmetrical K+. These channels were highly selective to K+ ions; potassium permeability (PK) value was 2.3 x 10(-13) cm(3) s(-1). The BK channels were sensitive to internal Ca2+ concentration, voltage dependent, and activated by intracellular MgATP. Voltage sensitivity (k) was approximately 13 mV, and the half-activation membrane potentials depended on the internal Ca2+ concentration. Calcium ions (0.3-3 microM) applied to the internal membrane surface caused an enhancement of the channel activity. This activation of BK channels by internal calcium had a KD(0) of 0.22 microM and was probably due to the binding of only one or two Ca2+ ions to the channel. Addition of MgATP (0.01-3 mM) to the internal solution increased steady state-open probability. The dissociation constant for MgATP (KD) was 119 microM, and the Hill coefficient (h) was 0.6, according to the Hill analysis. Ca2+-activated K+ currents recorded from whole cells were suppressed by either adding Cd2+ (0.4 mM) or removing Ca2+ ions from the external solution. TEA (1 mM) or charybdotoxin (100 nM) blocked these currents. Our results showed that both BK and K(ATP) channels are present in the same cell. Even when BK and K(ATP) channels were voltage dependent and modulated by internal Ca2+ and ATP, the profile of sensitivity was quite different for each kind

  6. A Calcium-Dependent Plasticity Rule for HCN Channels Maintains Activity Homeostasis and Stable Synaptic Learning

    Science.gov (United States)

    Honnuraiah, Suraj; Narayanan, Rishikesh

    2013-01-01

    Theoretical and computational frameworks for synaptic plasticity and learning have a long and cherished history, with few parallels within the well-established literature for plasticity of voltage-gated ion channels. In this study, we derive rules for plasticity in the hyperpolarization-activated cyclic nucleotide-gated (HCN) channels, and assess the synergy between synaptic and HCN channel plasticity in establishing stability during synaptic learning. To do this, we employ a conductance-based model for the hippocampal pyramidal neuron, and incorporate synaptic plasticity through the well-established Bienenstock-Cooper-Munro (BCM)-like rule for synaptic plasticity, wherein the direction and strength of the plasticity is dependent on the concentration of calcium influx. Under this framework, we derive a rule for HCN channel plasticity to establish homeostasis in synaptically-driven firing rate, and incorporate such plasticity into our model. In demonstrating that this rule for HCN channel plasticity helps maintain firing rate homeostasis after bidirectional synaptic plasticity, we observe a linear relationship between synaptic plasticity and HCN channel plasticity for maintaining firing rate homeostasis. Motivated by this linear relationship, we derive a calcium-dependent rule for HCN-channel plasticity, and demonstrate that firing rate homeostasis is maintained in the face of synaptic plasticity when moderate and high levels of cytosolic calcium influx induced depression and potentiation of the HCN-channel conductance, respectively. Additionally, we show that such synergy between synaptic and HCN-channel plasticity enhances the stability of synaptic learning through metaplasticity in the BCM-like synaptic plasticity profile. Finally, we demonstrate that the synergistic interaction between synaptic and HCN-channel plasticity preserves robustness of information transfer across the neuron under a rate-coding schema. Our results establish specific physiological roles

  7. Prolonged AT1R activation induces CaV1.2 channel internalization in rat cardiomyocytes.

    Science.gov (United States)

    Hermosilla, Tamara; Encina, Matías; Morales, Danna; Moreno, Cristian; Conejeros, Carolina; Alfaro-Valdés, Hilda M; Lagos-Meza, Felipe; Simon, Felipe; Altier, Christophe; Varela, Diego

    2017-08-31

    The cardiac L-type calcium channel is a multi-subunit complex that requires co-assembling of the pore-forming subunit CaV1.2 with auxiliary subunits CaVα2δ and CaVβ. Its traffic has been shown to be controlled by these subunits and by the activation of various G-protein coupled receptors (GPCR). Here, we explore the consequences of the prolonged activation of angiotensin receptor type 1 (AT1R) over CaV1.2 channel trafficking. Bioluminescence Resonance Energy Transfer (BRET) assay between β-arrestin and L-type channels in angiotensin II-stimulated cells was used to assess the functional consequence of AT1R activation, while immunofluorescence of adult rat cardiomyocytes revealed the effects of GPCR activation on CaV1.2 trafficking. Angiotensin II exposure results in β-arrestin1 recruitment to the channel complex and an apparent loss of CaV1.2 immunostaining at the T-tubules. Accordingly, angiotensin II stimulation causes a decrease in L-type current, Ca(2+) transients and myocyte contractility, together with a faster repolarization phase of action potentials. Our results demonstrate that prolonged AT1R activation induces β-arrestin1 recruitment and the subsequent internalization of CaV1.2 channels with a half-dose of AngII on the order of 100 nM, suggesting that this effect depends on local renin-angiotensin system. This novel AT1R-dependent CaV1.2-trafficking modulation likely contributes to angiotensin II-mediated cardiac remodeling.

  8. Effect of channel block on the collective spiking activity of coupled stochastic Hodgkin-Huxley neurons

    Institute of Scientific and Technical Information of China (English)

    2008-01-01

    Toxins, such as tetraethylammonium (TEA) and tetrodotoxin (TTX), can make potassium or sodium ion channels poisoned, respectively, and hence reduce the number of working ion channels and lead to the diminishment of conductance. In this paper, we have studied by numerical simulations the effects of sodium and potassium ion channel poisoning on the collective spiking activity of an array of coupled stochastic Hodgkin-Huxley (HH) neurons. It is found for a given number of neurons sodium or potas- sium ion channel block can either enhance or reduce the collective spiking regularity, depending on the membrane patch size. For a given smaller or larger patch size, potassium and sodium ion channel block can reduce or enhance the collective spiking regularity, but they have different patch size ranges for the transformation. This result shows that sodium or potassium ion channel block might have dif- ferent effects on the collective spiking activity in coupled HH neurons from the effects for a single neuron, which represents the interplay among the diminishment of maximal conductance and the in- crease of channel noise strength due to the channel blocks, as well as the bi-directional coupling be- tween the neurons.

  9. Dendrotoxins: structure-activity relationships and effects on potassium ion channels.

    Science.gov (United States)

    Harvey, A L; Robertson, B

    2004-12-01

    Dendrotoxins are small proteins isolated from mamba (Dendroaspis) snakes. The original dendrotoxin was found in venom of the Eastern green mamba, Dendroaspis angusticeps, and related proteins were subsequently found in other mamba venoms. The dendrotoxins contain 57-60 amino acid residues cross-linked by three disulphide bridges, and they are homologous to Kunitz-type serine protease inhibitors, such as aprotinin (BPTI). The dendrotoxins have little or no anti-protease activity, but they block particular subtypes of voltage-dependent potassium channels of the Kv1 subfamily in neurones. Alpha-dendrotoxin from green mamba Dendroaspis angusticeps and toxin I from the black mamba Dendroaspis polylepis block cloned Kv1.1, Kv1.2 and Kv1.6 channels in the low nanomolar range; toxin K, also from the black mamba Dendroaspis polylepis, preferentially blocks Kv1.1 channels and is active at picomolar concentrations. Structural modifications and mutations to dendrotoxins have helped to define the molecular recognition properties of different types of K+ channels, although more work is needed to characterise the chemical features of the toxins that underlie their selectivity and potency at particular subtypes of channels. Dendrotoxins have been useful markers of subtypes of K+ channels in vivo, and dendrotoxins have become widely used as probes for studying the function of K+ channels in physiology and pathophysiology. With some pathological conditions being associated with voltage-gated K+ channels, analogues of dendrotoxins might have therapeutic potential.

  10. Effect of channel block on the collective spiking activity of coupled stochastic Hodgkin-Huxley neurons

    Institute of Scientific and Technical Information of China (English)

    GONG YuBing; XU Bo; MA XiaoGuang; HAN JiQu

    2008-01-01

    Toxins, such as tetraethylammonium (TEA) and tetrodotoxin (TTX), can make potassium or sodium ion channels poisoned, respectively, and hence reduce the number of working ion channels and lead to the diminishment of conductance. In this paper, we have studied by numerical simulations the effects of sodium and potassium ion channel poisoning on the collective spiking activity of an array of coupled stochastic Hodgkin-Huxley (HH) neurons. It is found for a given number of neurons sodium or potas-sium ion channel block can either enhance or reduce the collective spiking regularity, depending on the membrane patch size. For a given smaller or larger patch size, potassium and sodium ion channel block can reduce or enhance the collective spiking regularity, but they have different patch size ranges for the transformation. This result shows that sodium or potassium ion channel block might have dif-ferent effects on the collective spiking activity in coupled HH neurons from the effects for a single neuron, which represents the interplay among the diminishment of maximal conductance and the in-crease of channel noise strength due to the channel blocks, as well as the bi-directional coupling be-tween the neurons.

  11. Involvement of Ca2+ Activated Cl- Channel Ano6 in Platelet Activation and Apoptosis

    Directory of Open Access Journals (Sweden)

    Guoxing Liu

    2015-11-01

    Full Text Available Background/Aims: The ubiquitously expressed Ca2+ Activated Cl- Channel Ano6 participates in the stimulation of cell membrane scrambling. Defective Ano6 underlies the Scott syndrome, an inherited bleeding disorder with impaired scrambling of plasma membrane phospholipids. At least in theory, the bleeding disorder of Scott syndrome may result from impaired platelet function. Activators of platelets include thrombin and collagen related peptide (CRP, which trigger increase of cytosolic Ca2+-activity ([Ca2+]i, production of reactive oxygen species (ROS, degranulation, integrin activation, as well as cell shrinkage and phospholipid scrambling of the cell membrane. The present study thus explored whether Ano6 modifies activation-induced alterations of cytosolic Ca2+-activity ([Ca2+]i, degranulation (P-selectin exposure, integrin activation, phosphatidylserine exposure on the platelet surface and platelet volume. Methods: Platelets from mice lacking Ano6 (ano6-/- were compared to platelets from corresponding wild-type mice (ano6+/+. [Ca2+]i was estimated from Fluo-3 fluorescence, ROS from DCFDA fluorescence, degranulation from P-selectin abundance, integrin activation from αIIbβ3-integrin abundance, phosphatidylserine abundance from annexin-V-binding, and cell volume from forward scatter. Results: Platelet number in blood was slightly higher in ano6-/- mice than in ano6+/+ mice. Without activation [Ca2+]i and volume were similar in ano6-/- and ano6+/+ platelets as well as ROS abundance, P-selectin abundance, αIIbβ3 integrin activation, and phosphatidylserine exposure were negligible in both genotypes. Thrombin (0.01 U/ml and CRP (2 or 5 µg/ml increased [Ca2+]i, ROS abundance, platelet degranulation, αIIbβ3 integrin activation, and triggered annexin-V-binding as well as cell shrinkage, all effects less pronounced in ano6-/- than in ano6+/+ platelets. Conclusions: Genetic knockout of Ano6 blunts thrombin- and CRP-induced activation and apoptosis

  12. Control of Ca2+ Influx and Calmodulin Activation by SK-Channels in Dendritic Spines.

    Directory of Open Access Journals (Sweden)

    Thom Griffith

    2016-05-01

    Full Text Available The key trigger for Hebbian synaptic plasticity is influx of Ca2+ into postsynaptic dendritic spines. The magnitude of [Ca2+] increase caused by NMDA-receptor (NMDAR and voltage-gated Ca2+ -channel (VGCC activation is thought to determine both the amplitude and direction of synaptic plasticity by differential activation of Ca2+ -sensitive enzymes such as calmodulin. Ca2+ influx is negatively regulated by Ca2+ -activated K+ channels (SK-channels which are in turn inhibited by neuromodulators such as acetylcholine. However, the precise mechanisms by which SK-channels control the induction of synaptic plasticity remain unclear. Using a 3-dimensional model of Ca2+ and calmodulin dynamics within an idealised, but biophysically-plausible, dendritic spine, we show that SK-channels regulate calmodulin activation specifically during neuron-firing patterns associated with induction of spike timing-dependent plasticity. SK-channel activation and the subsequent reduction in Ca2+ influx through NMDARs and L-type VGCCs results in an order of magnitude decrease in calmodulin (CaM activation, providing a mechanism for the effective gating of synaptic plasticity induction. This provides a common mechanism for the regulation of synaptic plasticity by neuromodulators.

  13. Enzymatic activity in the surface microlayer and subsurface water in the harbour channel

    Science.gov (United States)

    Perliński, Piotr; Mudryk, Zbigniew J.; Antonowicz, Józef

    2017-09-01

    Hydrolytic activity of eight extracellular enzymes was determined spectrofluorimetric method in the surface microlayer and subsurface water in the harbour channel in Ustka. The ranking order of the potential enzyme activity rates in the studied water layers was as follows: lipase > phosphatase > aminopeptidase > β-glucosidase > α-glucosidase > xylanase > cellulase > chitinase. The level of activity of all studied hydrolases was higher in the surface microlayer than subsurface water. No clear gradients in the level of enzymatic activity were determined along the horizontal profile of the studied channel. Activity of extracellular enzymes was strongly influenced by the season.

  14. Coupling Optical and Electrical Measurements in Artificial Membranes: Lateral Diffusion of Lipids and Channel Forming Peptides in Planar Bilayers

    Directory of Open Access Journals (Sweden)

    Duclohier H

    1998-01-01

    Full Text Available Planar lipid bilayers (PLB were prepared by the Montal-Mueller technique in a FRAP system designed to simultaneously measure conductivity across, and lateral diffusion of, the bilayer. In the first stage of the project the FRAP system was used to characterise the lateral dynamics of bilayer lipids with regards to phospholipid composition (headgroup, chain unsaturation etc., presence of cholesterol and the effect of divalent cations on negatively-charged bilayers. In the second stage of the project, lateral diffusion of two fluorescently-labelled voltage-dependent pore-forming peptides (alamethicin and S4s from Shaker K+ channel was determined at rest and in the conducting state. This study demonstrates the feasibility of such experiments with PLBs, amenable to physical constraints, and thus offers new opportunities for systematic studies of structure-function relationships in membrane-associating molecules.

  15. 76 FR 25364 - Agency Information Collection Activities: Form I-864, Form I-864A, Form I-864EZ, and From I-864W...

    Science.gov (United States)

    2011-05-04

    ... SECURITY U.S. Citizenship and Immigration Services Agency Information Collection Activities: Form I-864, Form I- 864A, Form I-864EZ, and From I-864W; Extension of an Existing Information Collection; Comment Request. ACTION: 60-Day Notice of Information Collection Under Review; Form I- 864, Affidavit of...

  16. Exact closed form expressions for outage probability of GSC receivers over Rayleigh fading channel subject to self-interference

    KAUST Repository

    Nam, Sungsik

    2010-11-01

    Previous work on performance analyses of generalized selection combining (GSC) RAKE receivers based on the signal to noise ratio focused on the development of methodologies to derive exact closed-form expressions for various performance measures. However, some open problems related to the performance evaluation of GSC RAKE receivers still remain to be solved such that an assessment of the impact of self-interference on the performance of GSC RAKE receivers. To have a full and exact understanding of the performance of GSC RAKE receivers, the outage probability of GSC RAKE receivers needs to be analyzed as closed-form expressions. The major difficulty in this problem is to derive some joint statistics of ordered exponential variates. With this motivation in mind, we capitalize in this paper on some new order statistics results to derive exact closed-form expressions for outage probability of GSC RAKE receivers subject to self-interference over independent and identically distributed Rayleigh fading channels. © 2010 IEEE.

  17. The Fungal Sexual Pheromone Sirenin Activates the Human CatSper Channel Complex.

    Science.gov (United States)

    Syeda, Shameem Sultana; Carlson, Erick J; Miller, Melissa R; Francis, Rawle; Clapham, David E; Lishko, Polina V; Hawkinson, Jon E; Hook, Derek; Georg, Gunda I

    2016-02-19

    The basal fungus Allomyces macrogynus (A. macrogynus) produces motile male gametes displaying well-studied chemotaxis toward their female counterparts. This chemotaxis is driven by sirenin, a sexual pheromone released by the female gametes. The pheromone evokes a large calcium influx in the motile gametes, which could proceed through the cation channel of sperm (CatSper) complex. Herein, we report the total synthesis of sirenin in 10 steps and 8% overall yield and show that the synthetic pheromone activates the CatSper channel complex, indicated by a concentration-dependent increase in intracellular calcium in human sperm. Sirenin activation of the CatSper channel was confirmed using whole-cell patch clamp electrophysiology with human sperm. Based on this proficient synthetic route and confirmed activation of CatSper, analogues of sirenin can be designed as blockers of the CatSper channel that could provide male contraceptive agents.

  18. Dysfunctional Hyperpolarization-Activated Cyclic Nucleotide-gated Ion Channels in Cardiac Diseases

    Directory of Open Access Journals (Sweden)

    Xiaoqi Zhao

    Full Text Available Abstract Hyperpolarization-activated cyclic nucleotide-gated (HCN channels are reverse voltage-dependent, and their activation depends on the hyperpolarization of the membrane and may be directly or indirectly regulated by the cyclic adenosine monophosphate (cAMP or other signal-transduction cascades. The distribution, quantity and activation states of HCN channels differ in tissues throughout the body. Evidence exhibits that HCN channels play critical roles in the generation and conduction of the electrical impulse and the physiopathological process of some cardiac diseases. They may constitute promising drug targets in the treatment of these cardiac diseases. Pharmacological treatment targeting HCN channels is of benefit to these cardiac conditions.

  19. Activity-dependent regulation of T-type calcium channels by submembrane calcium ions.

    Science.gov (United States)

    Cazade, Magali; Bidaud, Isabelle; Lory, Philippe; Chemin, Jean

    2017-01-21

    Voltage-gated Ca(2+) channels are involved in numerous physiological functions and various mechanisms finely tune their activity, including the Ca(2+) ion itself. This is well exemplified by the Ca(2+)-dependent inactivation of L-type Ca(2+) channels, whose alteration contributes to the dramatic disease Timothy Syndrome. For T-type Ca(2+) channels, a long-held view is that they are not regulated by intracellular Ca(2+). Here we challenge this notion by using dedicated electrophysiological protocols on both native and expressed T-type Ca(2+) channels. We demonstrate that a rise in submembrane Ca(2+) induces a large decrease in T-type current amplitude due to a hyperpolarizing shift in the steady-state inactivation. Activation of most representative Ca(2+)-permeable ionotropic receptors similarly regulate T-type current properties. Altogether, our data clearly establish that Ca(2+) entry exerts a feedback control on T-type channel activity, by modulating the channel availability, a mechanism that critically links cellular properties of T-type Ca(2+) channels to their physiological roles.

  20. Calcium-Activated Potassium Channels in Ischemia Reperfusion: A Brief Update

    Directory of Open Access Journals (Sweden)

    Jean-Yves eTano

    2014-10-01

    Full Text Available Ischemia and reperfusion (IR injury constitutes one of the major causes of cardiovascular morbidity and mortality. The discovery of new therapies to block/mediate the effects of IR is therefore an important goal in the biomedical sciences. Dysfunction associated with IR involves modification of calcium-activated potassium channels (KCa through different mechanisms, which are still under study. Respectively, the KCa family, major contributors to plasma membrane calcium influx in cells and essential players in the regulation of the vascular tone are interesting candidates. This family is divided into two groups including the large conductance (BKCa and the small/intermediate conductance (SKCa/IKCa K+ channels. In the heart and brain, these channels have been described to offer protection against IR injury. BKCa and SKCa channels deserve special attention since new data demonstrate that these channels are also expressed in mitochondria. More studies are however needed to fully determine their potential use as therapeutic targets.

  1. Kv3 channel assembly, trafficking and activity are regulated by zinc through different binding sites.

    Science.gov (United States)

    Gu, Yuanzheng; Barry, Joshua; Gu, Chen

    2013-05-15

    Zinc, a divalent heavy metal ion and an essential mineral for life, regulates synaptic transmission and neuronal excitability via ion channels. However, its binding sites and regulatory mechanisms are poorly understood. Here, we report that Kv3 channel assembly, localization and activity are regulated by zinc through different binding sites. Local perfusion of zinc reversibly reduced spiking frequency of cultured neurons most likely by suppressing Kv3 channels. Indeed, zinc inhibited Kv3.1 channel activity and slowed activation kinetics, independent of its site in the N-terminal T1 domain. Biochemical assays surprisingly identified a novel zinc-binding site in the Kv3.1 C-terminus, critical for channel activity and axonal targeting, but not for the zinc inhibition. Finally, mutagenesis revealed an important role of the junction between the first transmembrane (TM) segment and the first extracellular loop in sensing zinc. Its mutant enabled fast spiking with relative resistance to the zinc inhibition. Therefore, our studies provide novel mechanistic insights into the multifaceted regulation of Kv3 channel activity and localization by divalent heavy metal ions.

  2. Identification and characterization of Ca2+-activated K+ channels in granulosa cells of the human ovary

    Directory of Open Access Journals (Sweden)

    Berg Ulrike

    2009-04-01

    Full Text Available Abstract Background Granulosa cells (GCs represent a major endocrine compartment of the ovary producing sex steroid hormones. Recently, we identified in human GCs a Ca2+-activated K+ channel (KCa of big conductance (BKCa, which is involved in steroidogenesis. This channel is activated by intraovarian signalling molecules (e.g. acetylcholine via raised intracellular Ca2+ levels. In this study, we aimed at characterizing 1. expression and functions of KCa channels (including BKCa beta-subunits, and 2. biophysical properties of BKCa channels. Methods GCs were obtained from in vitro-fertilization patients and cultured. Expression of mRNA was determined by standard RT-PCR and protein expression in human ovarian slices was detected by immunohistochemistry. Progesterone production was measured in cell culture supernatants using ELISAs. Single channels were recorded in the inside-out configuration of the patch-clamp technique. Results We identified two KCa types in human GCs, the intermediate- (IK and the small-conductance KCa (SK. Their functionality was concluded from attenuation of human chorionic gonadotropin-stimulated progesterone production by KCa blockers (TRAM-34, apamin. Functional IK channels were also demonstrated by electrophysiological recording of single KCa channels with distinctive features. Both, IK and BKCa channels were found to be simultaneously active in individual GCs. In agreement with functional data, we identified mRNAs encoding IK, SK1, SK2 and SK3 in human GCs and proteins of IK and SK2 in corresponding human ovarian cells. Molecular characterization of the BKCa channel revealed the presence of mRNAs encoding several BKCa beta-subunits (beta2, beta3, beta4 in human GCs. The multitude of beta-subunits detected might contribute to variations in Ca2+ dependence of individual BKCa channels which we observed in electrophysiological recordings. Conclusion Functional and molecular studies indicate the presence of active IK and SK

  3. Activation of TRPV2 and BKCa channels by the LL-37 enantiomers stimulates calcium entry and migration of cancer cells

    Science.gov (United States)

    Guéguinou, Maxime; Chourpa, Igor; Fromont, Gaëlle; Bouchet, Ana Maria; Burlaud-Gaillard, Julien; Potier-Cartereau, Marie; Roger, Sébastien; Aucagne, Vincent; Chevalier, Stéphan; Vandier, Christophe

    2016-01-01

    Expression of the antimicrobial peptide hCAP18/LL-37 is associated to malignancy in various cancer forms, stimulating cell migration and metastasis. We report that LL-37 induces migration of three cancer cell lines by activating the TRPV2 calcium-permeable channel and recruiting it to pseudopodia through activation of the PI3K/AKT pathway. Ca2+ entry through TRPV2 cooperated with a K+ efflux through the BKCa channel. In a panel of human breast tumors, the expression of TRPV2 and LL-37 was found to be positively correlated. The D-enantiomer of LL-37 showed identical effects as the L-peptide, suggesting that no binding to a specific receptor was involved. LL-37 attached to caveolae and pseudopodia membranes and decreased membrane fluidity, suggesting that a modification of the physical properties of the lipid membrane bilayer was the underlying mechanism of its effects. PMID:26993604

  4. Synthesis of Rh/Macro-Porous Alumina Over Micro-Channel Plate and Its Catalytic Activity Tests for Diesel Reforming.

    Science.gov (United States)

    Seong, Yeon Baek; Kim, Yong Sul; Park, No-Kuk; Lee, Tae Jin

    2015-11-01

    Macro-porous Al2O3 as the catalytic support material was synthesized using colloidal polystyrene spheres over a micro-channel plate. The colloidal polystyrene spheres were used as a template for the production of an ordered macro porous material using an alumina nitrate solution as the precursor for Al2O3. The close-packed colloidal crystal array template method was applied to the formulation of ordered macro-porous Al2O3 used as a catalytic support material over a micro-channel plate. The solvent in the mixture solution, which also contained the colloidal polystyrene solution, aluminum nitrate solution and the precursor of the catalytic active materials (Rh), was evaporated in a vacuum oven at 50 degrees C. The ordered polystyrene spheres and aluminum salt of the solid state were deposited over a micro channel plate, and macro-porous Al2O3 was formed after calcination at 600 degrees C to remove the polystyrene spheres. The catalytic activity of the Rh/macro-porous alumina supported over the micro-channel plate was tested for diesel reforming.

  5. Graded activation of CRAC channel by binding of different numbers of STIM1 to Orai1 subunits

    Institute of Scientific and Technical Information of China (English)

    Zhengzheng Li; Lin Liu; Yongqiang Deng; Wei Ji; Wen Du; Pingyong Xu; Liangyi Chen; Tao Xu

    2011-01-01

    The Ca2+release-activated Ca2+(CRAC)channel pore is formed by Orail and gated by STIM1 after intracellular Ca2+store depletion.To resolve how many STIM1 molecules are required to open a CRAC channel,we fused different numbers of Orail subunits with functional two-tandem cytoplasmic domains of STIM1(residues 336-485,designated as S domain).Whole-cell patch clamp recordings of these chimeric molecules revealed that CRAC current reached maximum at a stoichiometry of four Orail and eight S domains.Further experiments indicate that two-tandem S domains specifically interact with the C-terminus of one Orail subunit,and CRAC current can be gradually increased as more Orail subunits can interact with S domains or STIM1 proteins.Our data suggest that maximal opening of one CRAC channel requires eight STIM1 molecules,and support a model that the CRAC channel activation is not in an"all-or-none"fashion but undergoes a graded process via binding of different numbers of STIM1.

  6. CaV2.1 voltage activated calcium channels and synaptic transmission in familial hemiplegic migraine pathogenesis.

    Science.gov (United States)

    Uchitel, Osvaldo D; Inchauspe, Carlota González; Urbano, Francisco J; Di Guilmi, Mariano N

    2012-01-01

    Studies on the genetic forms of epilepsy, chronic pain, and migraine caused by mutations in ion channels have given crucial insights into the molecular mechanisms, pathogenesis, and therapeutic approaches to complex neurological disorders. In this review we focus on the role of mutated CaV2.1 (i.e., P/Q-type) voltage-activated Ca2+ channels, and on the ultimate consequences that mutations causing familial hemiplegic migraine type-1 (FHM1) have in neurotransmitter release. Transgenic mice harboring the human pathogenic FHM1 mutation R192Q or S218L (KI) have been used as models to study neurotransmission at several central and peripheral synapses. FHM1 KI mice are a powerful tool to explore presynaptic regulation associated with expression of CaV2.1 channels. Mutated CaV2.1 channels activate at more hyperpolarizing potentials and lead to a gain-of-function in synaptic transmission. This gain-of-function might underlie alterations in the excitatory/ inhibitory balance of synaptic transmission, favoring a persistent state of hyperexcitability in cortical neurons that would increase the susceptibility for cortical spreading depression (CSD), a mechanism believed to initiate the attacks of migraine with aura. Copyright © 2011 Elsevier Ltd. All rights reserved.

  7. Up-Regulatory Effects of Curcumin on Large Conductance Ca2+-Activated K+ Channels.

    Directory of Open Access Journals (Sweden)

    Qijing Chen

    Full Text Available Large conductance Ca2+-activated potassium channels (BK are targets for research that explores therapeutic means to various diseases, owing to the roles of the channels in mediating multiple physiological processes in various cells and tissues. We investigated the pharmacological effects of curcumin, a compound isolated from the herb Curcuma longa, on BK channels. As recorded by whole-cell patch-clamp, curcumin increased BK (α and BK (α+β1 currents in transfected HEK293 cells as well as the current density of BK in A7r5 smooth muscle cells in a dose-dependent manner. By incubating with curcumin for 24 hours, the current density of exogenous BK (α in HEK293 cells and the endogenous BK in A7r5 cells were both enhanced notably, though the steady-state activation of the channels did not shift significantly, except for BK (α+β1. Curcumin up-regulated the BK protein expression without changing its mRNA level in A7r5 cells. The surface expression and the half-life of BK channels were also increased by curcumin in HEK293 cells. These effects of curcumin were abolished by MG-132, a proteasome inhibitor. Curcumin also increased ERK 1/2 phosphorylation, while inhibiting ERK by U0126 attenuated the curcumin-induced up-regulation of BK protein expression. We also observed that the curcumin-induced relaxation in the isolated rat aortic rings was significantly attenuated by paxilline, a BK channel specific blocker. These results show that curcumin enhances the activity of the BK channels by interacting with BK directly as well as enhancing BK protein expression through inhibiting proteasomal degradation and activating ERK signaling pathway. The findings suggest that curcumin is a potential BK channel activator and provide novel insight into its complicated pharmacological effects and the underlying mechanisms.

  8. Digoxin activates sarcoplasmic reticulum Ca(2+)-release channels: a possible role in cardiac inotropy.

    Science.gov (United States)

    McGarry, S J; Williams, A J

    1993-04-01

    1. The effect of digoxin on rapid 45Ca2+ efflux from cardiac and skeletal sarcoplasmic reticulum (SR) vesicles was investigated. Additionally the interaction of digoxin with single cardiac and skeletal muscle SR Ca(2+)-release channels incorporated into planar phospholipid bilayers and held under voltage clamp was determined. 2. Digoxin (1 nM) increased the initial rate and amount of Ca(2+)-induced release of 45Ca2+ from cardiac SR vesicles, passively loaded with 45CaCl2, at an extravesicular [Ca2+] of 0.1 microM. The efflux in the presence and absence of digoxin was inhibited at pM extravesicular Ca2+ and blocked by 5 mM Mg2+. 3. To elucidate the mechanism of action of digoxin, single-channel recording was used. Digoxin (1-20 nM) increased single-channel open probability (Po) when added to the cytosolic but not the luminal face of the cardiac channel in the presence of sub-maximally activating Ca2+ (0.1 microM-10 microM) with an EC50 of 0.91 nM at 10 microM Ca2+. The mechanisms underlying the action of digoxin appear to be concentration-dependent. The activation observed at 1 nM digoxin appears to be consistent with the sensitization of the channel to the effects of Ca2+. At higher concentrations the drug appears to interact synergistically with Ca2+ to produce values of Po considerably greater than those seen with Ca2+ as the sole activating ligand. 4. Digoxin had no effect on single-channel conductance or the Ca2+/Tris permeability ratio. In channels activated by digoxin the Po was decreased by Mg2+. Single-channels were characteristically modified to along lasting open, but reduced, conductance state when 100 nM ryanodine was added to the cytosolic side of the channel.5. Activation of the cardiac SR Ca2+-release channel was observed with similar concentrations of digitoxin, however, higher concentrations of ouabain were required to increase PO. In contrast, a steroid which is not positively inotropic, chlormadinone acetate, had no effect on either cardiac or

  9. The role of Hv1 and CatSper channels in sperm activation.

    Science.gov (United States)

    Lishko, Polina V; Kirichok, Yuriy

    2010-12-01

    Elevations of sperm intracellular pH and Ca(2+) regulate sperm motility, chemotaxis, capacitation and the acrosome reaction, and play a vital role in the ability of the sperm cell to reach and fertilise the egg. In human spermatozoa, the flagellar voltage-gated proton channel Hv1 is the main H(+) extrusion pathway that controls sperm intracellular pH, and the pH-dependent flagellar Ca²(+) channel CatSper is the main pathway for Ca²(+) entry as measured by the whole-cell patch clamp technique. Hv1 and CatSper channels are co-localized within the principal piece of the sperm flagellum. Hv1 is dedicated to proton extrusion from flagellum and is activated by membrane depolarisation, an alkaline extracellular environment, the endocannabinoid anandamide, and removal of extracellular zinc, a potent Hv1 blocker. The CatSper channel is strongly potentiated by intracellular alkalinisation. Since Hv1 and CatSper channels are located in the same subcellular domain, proton extrusion via Hv1 channels should induce intraflagellar alkalinisation and activate CatSper ion channels. Therefore the combined action of Hv1 and CatSper channels in human spermatozoa can induce elevation of both intracellular pH and Ca²(+) required for sperm activation in the female reproductive tract. Here, we discuss how Hv1 and CatSper channels regulate human sperm physiology and the differences in control of sperm intracellular pH and Ca²(+) between species.

  10. Hyperkalemic periodic paralysis M1592V mutation modifies activation in human skeletal muscle Na+ channel.

    Science.gov (United States)

    Rojas, C V; Neely, A; Velasco-Loyden, G; Palma, V; Kukuljan, M

    1999-01-01

    Mutations in the human skeletal muscle Na+ channel underlie the autosomal dominant disease hyperkalemic periodic paralysis (HPP). Muscle fibers from affected individuals exhibit sustained Na+ currents thought to depolarize the sarcolemma and thus inactivate normal Na+ channels. We expressed human wild-type or M1592V mutant alpha-subunits with the beta1-subunit in Xenopus laevis oocytes and recorded Na+ currents using two-electrode and cut-open oocyte voltage-clamp techniques. The most prominent functional difference between M1592V mutant and wild-type channels is a 5- to 10-mV shift in the hyperpolarized direction of the steady-state activation curve. The shift in the activation curve for the mutant results in a larger overlap with the inactivation curve than that observed for wild-type channels. Accordingly, the current through M1592V channels displays a larger noninactivating component than does that through wild-type channels at membrane potentials near -40 mV. The functional properties of the M1592V mutant resemble those of the previously characterized HPP T704M mutant. Both clinically similar phenotypes arise from mutations located at a distance from the putative voltage sensor of the channel.

  11. The Natural Plant Product Rottlerin Activates Kv7.1/KCNE1 Channels

    Directory of Open Access Journals (Sweden)

    Veronika Matschke

    2016-12-01

    Full Text Available Background/Aims: Acquired as well as inherited channelopathies are disorders that are caused by altered ion channel function. A family of channels whose malfunction is associated with different channelopathies is the Kv7 K+ channel family; and restoration of normal Kv7 channel function by small molecule modulators is a promising approach for treatment of these often fatal diseases. Methods: Here, we show the modulation of Kv7 channels by the natural compound Rottlerin heterologously expressed in Xenopus laevis oocytes and on iPSC cardiomyocytes overexpressing Kv7.1 channels. Results: We show that currents carried by Kv7.1 (EC50 = 1.48 μM, Kv7.1/KCNE1 (EC50 = 4.9 μM, and Kv7.4 (EC50 = 0.148 μM are strongly enhanced by the compound, whereas Kv7.2, Kv7.2/Kv7.3, and Kv7.5 are not sensitive to Rottlerin. Studies on Kv7.1/KCNE1 mutants and in silico modelling indicate that Rottlerin binds to the R-L3-activator site. Rottlerin mediated activation of Kv7.1/KCNE1 channels might be a promising approach in long QT syndrome. As a proof of concept, we show that Rottlerin shortens cardiac repolarisation in iPSC-derived cardiomyocytes expressing Kv7.1.Conclusion: Rottlerin or an optimized derivative holds a potential as QT interval correcting drug.

  12. Molecular mechanisms of diabetic coronary dysfunction due to large conductance Ca2+-activated K+ channel impairment

    Institute of Scientific and Technical Information of China (English)

    WANG Ru-xing; ZHENG Jie; GUO Su-xia; LI Xiao-rong; LU Tong; SHI Hai-feng; CHAI Qiang; WU Ying; SUN Wei; JI Yuan; YAO Yong; LI Ku-lin; ZHANG Chang-ying

    2012-01-01

    Background Diabetes mellitus is associated with coronary dysfunction,contributing to a 2- to 4-fold increase in the risk of coronary heart diseases.The mechanisms by which diabetes induces vasculopathy involve endothelial-dependent and -independent vascular dysfunction in both type 1 and type 2 diabetes mellitus.The purpose of this study is to determine the role of vascular large conductance Ca2+-activated K+ (BK) channel activities in coronary dysfunction in streptozotocin-induced diabetic rats.Methods Using videomicroscopy,immunoblotting,fluorescent assay and patch clamp techniques,we investigated the coronary BK channel activities and BK channel-mediated coronary vasoreactivity in streptozotocin-induced diabetic rats.Results BK currents (defined as the iberiotoxin-sensitive K+ component) contribute (65±4)% of the total K+ currents in freshly isolated coronary smooth muscle cells and >50% of the contraction of the inner diameter of coronary arteries from normal rats.However,BK current density is remarkably reduced in coronary smooth muscle cells of streptozotocin-induced diabetic rats,leading to an increase in coronary artery tension.BK channel activity in response to free Ca2+ is impaired in diabetic rats.Moreover,cytoplasmic application of DHS-1 (a specific BK channel β1 subunit activator) robustly enhanced the open probability of BK channels in coronary smooth muscle cells of normal rats.In diabetic rats,the DHS-1 effect was diminished in the presence of 200 nmol/L Ca2+ and was significantly attenuated in the presence of high free calcium concentration,i.e.,1 μmol/L Ca2+.Immunoblotting experiments confirmed that there was a 2-fold decrease in BK-β1 protein expression in diabetic vessels,without altering the BK channel α-subunit expression.Although the cytosolic Ca2+ concentration of coronary arterial smooth muscle cells was increased from (103±23)nmol/L (n=5) of control rats to (193±22) nmol/L (n=6,P<0.05) of STZ-induced diabetic rats,reduced BK

  13. Actively evolving subglacial conduits and eskers initiate ice shelf channels at an Antarctic grounding line.

    Science.gov (United States)

    Drews, R; Pattyn, F; Hewitt, I J; Ng, F S L; Berger, S; Matsuoka, K; Helm, V; Bergeot, N; Favier, L; Neckel, N

    2017-05-09

    Ice-shelf channels are long curvilinear tracts of thin ice found on Antarctic ice shelves. Many of them originate near the grounding line, but their formation mechanisms remain poorly understood. Here we use ice-penetrating radar data from Roi Baudouin Ice Shelf, East Antarctica, to infer that the morphology of several ice-shelf channels is seeded upstream of the grounding line by large basal obstacles indenting the ice from below. We interpret each obstacle as an esker ridge formed from sediments deposited by subglacial water conduits, and calculate that the eskers' size grows towards the grounding line where deposition rates are maximum. Relict features on the shelf indicate that these linked systems of subglacial conduits and ice-shelf channels have been changing over the past few centuries. Because ice-shelf channels are loci where intense melting occurs to thin an ice shelf, these findings expose a novel link between subglacial drainage, sedimentation and ice-shelf stability.

  14. Effects on atrial fibrillation in aged hypertensive rats by Ca(2+)-activated K(+) channel inhibition

    DEFF Research Database (Denmark)

    Diness, Jonas Goldin; Skibsbye, Lasse; Jespersen, Thomas

    2011-01-01

    We have shown previously that inhibition of small conductance Ca(2+)-activated K(+) (SK) channels is antiarrhythmic in models of acutely induced atrial fibrillation (AF). These models, however, do not take into account that AF derives from a wide range of predisposing factors, the most prevalent ...... the notion that SK channels may offer a promising new therapeutic target in the treatment of AF.......We have shown previously that inhibition of small conductance Ca(2+)-activated K(+) (SK) channels is antiarrhythmic in models of acutely induced atrial fibrillation (AF). These models, however, do not take into account that AF derives from a wide range of predisposing factors, the most prevalent...... being hypertension. In this study we assessed the effects of two different SK channel inhibitors, NS8593 and UCL1684, in aging, spontaneously hypertensive rats to examine their antiarrhythmic properties in a setting of hypertension-induced atrial remodeling. Male spontaneously hypertensive rats...

  15. Effects of large conductance Ca(2+)-activated K(+) channels on nitroglycerin-mediated vasorelaxation in humans

    DEFF Research Database (Denmark)

    Gruhn, Nicolai; Boesgaard, Søren; Eiberg, Jonas

    2002-01-01

    Nitric oxide (NO)-induced vasorelaxation and the regulation of endothelial superoxide anion levels is partly mediated by vascular large conductance Ca(2+)-activated K(+) (BK(Ca)) channels. Nitroglycerin acts through the release of NO and its effect is modulated by changes in endothelial superoxide...... levels. This study examines the effect of BK(Ca) channel blockade on nitroglycerin-induced vasorelaxation in human arterial and venous vascular segments and whether responses to BK(Ca) channel blockade are influenced by the development of venous nitroglycerin tolerance. Dose-relaxation curves...... suggest that primarily arterial effects of nitroglycerin are significantly inhibited by changes in the activity of the endothelial BK(Ca) channels. Although endothelial BK(Ca) are likely regulators of mechanisms underlying arterial tolerance development to nitroglycerin, they do not appear to play a role...

  16. P2X(7) receptor activation enhances SK3 channels- and cystein cathepsin-dependent cancer cells invasiveness.

    Science.gov (United States)

    Jelassi, B; Chantôme, A; Alcaraz-Pérez, F; Baroja-Mazo, A; Cayuela, M L; Pelegrin, P; Surprenant, A; Roger, S

    2011-05-05

    ATP-gated P2X(7) receptors (P2X(7)R) are unusual plasma membrane ion channels that have been extensively studied in immune cells. More recently, P2X(7)R have been described as potential cancer cell biomarkers. However, mechanistic links between P2X(7)R and cancer cell processes are unknown. Here, we show, in the highly aggressive human breast cancer cell line MDA-MB-435s, that P2X(7) receptor is highly expressed and fully functional. Its activation is responsible for the extension of neurite-like cellular prolongations, of the increase in cell migration by 35% and in cell invasion through extracellular matrix by 150%. The change in cancer cell morphology and the increased migration appeared to be due to the activation of Ca(2+)-activated SK3 potassium channels. The enhanced invasion through the extracellular matrix was related to the increase of mature forms of cysteine cathepsins in the extracellular medium, which was independent of SK3 channel activity and not associated with cell death. Pharmacological targeting of P2X(7)R in vivo was crucial for cell invasiveness in a zebrafish model of metastases. Our results demonstrate a novel mechanistic link between P2X(7)R functionality in cancer cells and invasiveness, a key parameter in tumour growth and in the development of metastases. They also suggest a potential therapeutic role for the newly developed P2X(7)R antagonists.

  17. Anoctamin 1 induces calcium-activated chloride secretion and proliferation of renal cyst-forming epithelial cells.

    Science.gov (United States)

    Buchholz, Bjoern; Faria, Diana; Schley, Gunnar; Schreiber, Rainer; Eckardt, Kai-Uwe; Kunzelmann, Karl

    2014-05-01

    Polycystic kidney diseases are characterized by multiple bilateral renal cysts that gradually enlarge and lead to a decline in renal function. Cyst enlargement is driven by transepithelial chloride secretion, stimulated by enhanced levels of cyclic adenosine monophosphate, which activates apical cystic fibrosis transmembrane conductance regulator chloride channels. However, chloride secretion by calcium-dependent chloride channels, activated through stimulation of purinergic receptors, also has a major impact. To identify the molecular basis of calcium-dependent chloride secretion in cyst expansion, we determined the role of anoctamin 1 and 6, two recently discovered calcium-activated chloride channels both of which are expressed in epithelial cells. We found that anoctamin 1, which plays a role in epithelial fluid secretion and proliferation, is strongly expressed in principal-like MDCK cells (PLCs) forming cysts within a collagen matrix, in an embryonic kidney cyst model, and in human autosomal dominant polycystic kidney disease tissue. Knockdown of anoctamin 1 but not anoctamin 6 strongly diminished the calcium-dependent chloride secretion of PLCs. Moreover, two inhibitors of anoctamin ion channels, tannic acid and a more selective inhibitor of anoctamin 1, significantly inhibited PLC cyst growth and cyst enlargement in an embryonic kidney cyst model. Knockdown of ANO1 by morpholino analogs also attenuated embryonic cyst growth. Thus, calcium-activated chloride secretion by anoctamin 1 appears to be a crucial component of renal cyst growth.

  18. STIM1 dimers undergo unimolecular coupling to activate Orai1 channels

    Science.gov (United States)

    Zhou, Yandong; Wang, Xizhuo; Wang, Xianming; Loktionova, Natalia A.; Cai, Xiangyu; Nwokonko, Robert M.; Vrana, Erin; Wang, Youjun; Rothberg, Brad S.; Gill, Donald L.

    2015-09-01

    The endoplasmic reticulum (ER) Ca2+ sensor, STIM1, becomes activated when ER-stored Ca2+ is depleted and translocates into ER-plasma membrane junctions where it tethers and activates Orai1 Ca2+ entry channels. The dimeric STIM1 protein contains a small STIM-Orai-activating region (SOAR)--the minimal sequence sufficient to activate Orai1 channels. Since SOAR itself is a dimer, we constructed SOAR concatemer-dimers and introduced mutations at F394, which is critical for Orai1 coupling and activation. The F394H mutation in both SOAR monomers completely blocks dimer function, but F394H introduced in only one of the dimeric SOAR monomers has no effect on Orai1 binding or activation. This reveals an unexpected unimolecular coupling between STIM1 and Orai1 and argues against recent evidence suggesting dimeric interaction between STIM1 and two adjacent Orai1 channel subunits. The model predicts that STIM1 dimers may be involved in crosslinking between Orai1 channels with implications for the kinetics and localization of Orai1 channel opening.

  19. Numerical study on the thermal and flow characteristics of periodically formed inner wavy structures in a cooling channel

    Energy Technology Data Exchange (ETDEWEB)

    Lee, Ju Chul; Park, Sang Hu; Son, Chang Min; Min, June Kee; Ha, Man Yeong [Pusan National University, Busan (Korea, Republic of); Cho, Jong Rae [Korea Maritime University, Busan (Korea, Republic of)

    2015-09-15

    In industrial fields of machine and aerospace, cooling systems consisting of channels are widely used to increase energy efficiency and prevent system overheat. In cooling channels, a reduced pressure drop, an enhanced heat transfer, and a short channel length are considered key design requirements for optimizing the total volume and weight of a system. In this work, we improved heat transfer efficiency by using milli-scale wavy structures inside the channel. By optimizing the inner structures through computational fluid dynamics analysis and Taguchi method, the Nusselt number increased by approximately 11.7% with a similar pressure drop compared with that of a normal channel for a Reynolds number of 1000.

  20. Voltage-gated Na+ Channel Activity Increases Colon Cancer Transcriptional Activity and Invasion Via Persistent MAPK Signaling

    Science.gov (United States)

    House, Carrie D.; Wang, Bi-Dar; Ceniccola, Kristin; Williams, Russell; Simaan, May; Olender, Jacqueline; Patel, Vyomesh; Baptista-Hon, Daniel T.; Annunziata, Christina M.; Silvio Gutkind, J.; Hales, Tim G.; Lee, Norman H.

    2015-06-01

    Functional expression of voltage-gated Na+ channels (VGSCs) has been demonstrated in multiple cancer cell types where channel activity induces invasive activity. The signaling mechanisms by which VGSCs promote oncogenesis remain poorly understood. We explored the signal transduction process critical to VGSC-mediated invasion on the basis of reports linking channel activity to gene expression changes in excitable cells. Coincidentally, many genes transcriptionally regulated by the SCN5A isoform in colon cancer have an over-representation of cis-acting sites for transcription factors phosphorylated by ERK1/2 MAPK. We hypothesized that VGSC activity promotes MAPK activation to induce transcriptional changes in invasion-related genes. Using pharmacological inhibitors/activators and siRNA-mediated gene knockdowns, we correlated channel activity with Rap1-dependent persistent MAPK activation in the SW620 human colon cancer cell line. We further demonstrated that VGSC activity induces downstream changes in invasion-related gene expression via a PKA/ERK/c-JUN/ELK-1/ETS-1 transcriptional pathway. This is the first study illustrating a molecular mechanism linking functional activity of VGSCs to transcriptional activation of invasion-related genes.

  1. Cholesterol regulates HERG K+ channel activation by increasing phospholipase C β1 expression

    Science.gov (United States)

    Chun, Yoon Sun; Oh, Hyun Geun; Park, Myoung Kyu; Cho, Hana; Chung, Sungkwon

    2013-01-01

    Human ether-a-go-go-related gene (HERG) K+ channel underlies the rapidly activating delayed rectifier K+ conductance (IKr) during normal cardiac repolarization. Also, it may regulate excitability in many neuronal cells. Recently, we showed that enrichment of cell membrane with cholesterol inhibits HERG channels by reducing the levels of phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P2] due to the activation of phospholipase C (PLC). In this study, we further explored the effect of cholesterol enrichment on HERG channel kinetics. When membrane cholesterol level was mildly increased in human embryonic kidney (HEK) 293 cells expressing HERG channel, the inactivation and deactivation kinetics of HERG current were not affected, but the activation rate was significantly decelerated at all voltages tested. The application of PtdIns(4,5)P2 or inhibitor for PLC prevented the effect of cholesterol enrichment, while the presence of antibody against PtdIns(4,5)P2 in pipette solution mimicked the effect of cholesterol enrichment. These results indicate that the effect of cholesterol enrichment on HERG channel is due to the depletion of PtdIns(4,5)P2. We also found that cholesterol enrichment significantly increases the expression of β1 and β3 isoforms of PLC (PLCβ1, PLCβ3) in the membrane. Since the effects of cholesterol enrichment on HERG channel were prevented by inhibiting transcription or by inhibiting PLCβ1 expression, we conclude that increased PLCβ1 expression leads to the deceleration of HERG channel activation rate via downregulation of PtdIns(4,5)P2. These results confirm a crosstalk between two plasma membrane-enriched lipids, cholesterol and PtdIns(4,5)P2, in the regulation of HERG channels. PMID:23793622

  2. Glucose deprivation activates diversity of potassium channels in cultured rat hippocampal neurons.

    Science.gov (United States)

    Velasco, Myrian; García, Esperanza; Onetti, Carlos G

    2006-05-01

    1. Glucose is one of the most important substrates for generating metabolic energy required for the maintenance of cellular functions. Glucose-mediated changes in neuronal firing pattern have been observed in the central nervous system of mammals. K(+) channels directly regulated by intracellular ATP have been postulated as a linkage between cellular energetic metabolism and excitability; the functional roles ascribed to these channels include glucose-sensing to regulate energy homeostasis and neuroprotection under energy depletion conditions. The hippocampus is highly sensitive to metabolic insults and is the brain region most sensitive to ischemic damage. Because the identity of metabolically regulated potassium channels present in hippocampal neurons is obscure, we decided to study the biophysical properties of glucose-sensitive potassium channels in hippocampal neurons. 2. The dependence of membrane potential and the sensitivity of potassium channels to glucose and ATP in rat hippocampal neurons were studied in cell-attached and excised inside-out membrane patches. 3. We found that under hypoglycemic conditions, at least three types of potassium channels were activated; their unitary conductance values were 37, 147, and 241 pS in symmetrical K(+), and they were sensitive to ATP. For K(+) channels with unitary conductance of 37 and 241, when the membrane potential was depolarized the longer closed time constant diminished and this produced an increase in the open-state probability; nevertheless, the 147-pS channels were not voltage-dependent. 4. We propose that neuronal glucose-sensitive K(+) channels in rat hippocampus include subtypes of ATP-sensitive channels with a potential role in neuroprotection during short-term or prolonged metabolic stress.

  3. Stochastic ion channel gating in dendritic neurons: morphology dependence and probabilistic synaptic activation of dendritic spikes.

    Directory of Open Access Journals (Sweden)

    Robert C Cannon

    Full Text Available Neuronal activity is mediated through changes in the probability of stochastic transitions between open and closed states of ion channels. While differences in morphology define neuronal cell types and may underlie neurological disorders, very little is known about influences of stochastic ion channel gating in neurons with complex morphology. We introduce and validate new computational tools that enable efficient generation and simulation of models containing stochastic ion channels distributed across dendritic and axonal membranes. Comparison of five morphologically distinct neuronal cell types reveals that when all simulated neurons contain identical densities of stochastic ion channels, the amplitude of stochastic membrane potential fluctuations differs between cell types and depends on sub-cellular location. For typical neurons, the amplitude of membrane potential fluctuations depends on channel kinetics as well as open probability. Using a detailed model of a hippocampal CA1 pyramidal neuron, we show that when intrinsic ion channels gate stochastically, the probability of initiation of dendritic or somatic spikes by dendritic synaptic input varies continuously between zero and one, whereas when ion channels gate deterministically, the probability is either zero or one. At physiological firing rates, stochastic gating of dendritic ion channels almost completely accounts for probabilistic somatic and dendritic spikes generated by the fully stochastic model. These results suggest that the consequences of stochastic ion channel gating differ globally between neuronal cell-types and locally between neuronal compartments. Whereas dendritic neurons are often assumed to behave deterministically, our simulations suggest that a direct consequence of stochastic gating of intrinsic ion channels is that spike output may instead be a probabilistic function of patterns of synaptic input to dendrites.

  4. Calcium regulation of HCN channels supports persistent activity in a multiscale model of neocortex.

    Science.gov (United States)

    Neymotin, S A; McDougal, R A; Bulanova, A S; Zeki, M; Lakatos, P; Terman, D; Hines, M L; Lytton, W W

    2016-03-01

    Neuronal persistent activity has been primarily assessed in terms of electrical mechanisms, without attention to the complex array of molecular events that also control cell excitability. We developed a multiscale neocortical model proceeding from the molecular to the network level to assess the contributions of calcium (Ca(2+)) regulation of hyperpolarization-activated cyclic nucleotide-gated (HCN) channels in providing additional and complementary support of continuing activation in the network. The network contained 776 compartmental neurons arranged in the cortical layers, connected using synapses containing AMPA/NMDA/GABAA/GABAB receptors. Metabotropic glutamate receptors (mGluR) produced inositol triphosphate (IP3) which caused the release of Ca(2+) from endoplasmic reticulum (ER) stores, with reuptake by sarco/ER Ca(2+)-ATP-ase pumps (SERCA), and influence on HCN channels. Stimulus-induced depolarization led to Ca(2+) influx via NMDA and voltage-gated Ca(2+) channels (VGCCs). After a delay, mGluR activation led to ER Ca(2+) release via IP3 receptors. These factors increased HCN channel conductance and produced firing lasting for ∼1min. The model displayed inter-scale synergies among synaptic weights, excitation/inhibition balance, firing rates, membrane depolarization, Ca(2+) levels, regulation of HCN channels, and induction of persistent activity. The interaction between inhibition and Ca(2+) at the HCN channel nexus determined a limited range of inhibition strengths for which intracellular Ca(2+) could prepare population-specific persistent activity. Interactions between metabotropic and ionotropic inputs to the neuron demonstrated how multiple pathways could contribute in a complementary manner to persistent activity. Such redundancy and complementarity via multiple pathways is a critical feature of biological systems. Mediation of activation at different time scales, and through different pathways, would be expected to protect against disruption, in

  5. Apical Ca2+-activated potassium channels in mouse parotid acinar cells.

    Science.gov (United States)

    Almassy, Janos; Won, Jong Hak; Begenisich, Ted B; Yule, David I

    2012-02-01

    Ca(2+) activation of Cl and K channels is a key event underlying stimulated fluid secretion from parotid salivary glands. Cl channels are exclusively present on the apical plasma membrane (PM), whereas the localization of K channels has not been established. Mathematical models have suggested that localization of some K channels to the apical PM is optimum for fluid secretion. A combination of whole cell electrophysiology and temporally resolved digital imaging with local manipulation of intracellular [Ca(2+)] was used to investigate if Ca(2+)-activated K channels are present in the apical PM of parotid acinar cells. Initial experiments established Ca(2+)-buffering conditions that produced brief, localized increases in [Ca(2+)] after focal laser photolysis of caged Ca(2+). Conditions were used to isolate K(+) and Cl(-) conductances. Photolysis at the apical PM resulted in a robust increase in K(+) and Cl(-) currents. A localized reduction in [Ca(2+)] at the apical PM after photolysis of Diazo-2, a caged Ca(2+) chelator, resulted in a decrease in both K(+) and Cl(-) currents. The K(+) currents evoked by apical photolysis were partially blocked by both paxilline and TRAM-34, specific blockers of large-conductance "maxi-K" (BK) and intermediate K (IK), respectively, and almost abolished by incubation with both antagonists. Apical TRAM-34-sensitive K(+) currents were also observed in BK-null parotid acini. In contrast, when the [Ca(2+)] was increased at the basal or lateral PM, no increase in either K(+) or Cl(-) currents was evoked. These data provide strong evidence that K and Cl channels are similarly distributed in the apical PM. Furthermore, both IK and BK channels are present in this domain, and the density of these channels appears higher in the apical versus basolateral PM. Collectively, this study provides support for a model in which fluid secretion is optimized after expression of K channels specifically in the apical PM.

  6. Neuronal Ca2+-activated K+ channels limit brain infarction and promote survival.

    Directory of Open Access Journals (Sweden)

    Yiliu Liao

    Full Text Available Neuronal calcium-activated potassium channels of the BK type are activated by membrane depolarization and intracellular Ca(2+ ions. It has been suggested that these channels may play a key neuroprotective role during and after brain ischemia, but this hypothesis has so far not been tested by selective BK-channel manipulations in vivo. To elucidate the in vivo contribution of neuronal BK channels in acute focal cerebral ischemia, we performed middle cerebral artery occlusion (MCAO in mice lacking BK channels (homozygous mice lacking the BK channel alpha subunit, BK(-/-. MCAO was performed in BK(-/- and WT mice for 90 minutes followed by a 7-hour-reperfusion period. Coronal 1 mm thick sections were stained with 2,3,5-triphenyltetrazolium chloride to reveal the infarction area. We found that transient focal cerebral ischemia by MCAO produced larger infarct volume, more severe neurological deficits, and higher post-ischemic mortality in BK(-/- mice compared to WT littermates. However, the regional cerebral blood flow was not significantly different between genotypes as measured by Laser Doppler (LD flowmetry pre-ischemically, intra-ischemically, and post-ischemically, suggesting that the different impact of MCAO in BK(-/- vs. WT was not due to vascular BK channels. Furthermore, when NMDA was injected intracerebrally in non-ischemic mice, NMDA-induced neurotoxicity was found to be larger in BK(-/- mice compared to WT. Whole-cell patch clamp recordings from CA1 pyramidal cells in organotypic hippocampal slice cultures revealed that BK channels contribute to rapid action potential repolarization, as previously found in acute slices. When these cultures were exposed to ischemia-like conditions this induced significantly more neuronal death in BK(-/- than in WT cultures. These results indicate that neuronal BK channels are important for protection against ischemic brain damage.

  7. Neuronal Ca2+-activated K+ channels limit brain infarction and promote survival.

    Science.gov (United States)

    Liao, Yiliu; Kristiansen, Ase-Marit; Oksvold, Cecilie P; Tuvnes, Frode A; Gu, Ning; Rundén-Pran, Elise; Ruth, Peter; Sausbier, Matthias; Storm, Johan F

    2010-12-30

    Neuronal calcium-activated potassium channels of the BK type are activated by membrane depolarization and intracellular Ca(2+) ions. It has been suggested that these channels may play a key neuroprotective role during and after brain ischemia, but this hypothesis has so far not been tested by selective BK-channel manipulations in vivo. To elucidate the in vivo contribution of neuronal BK channels in acute focal cerebral ischemia, we performed middle cerebral artery occlusion (MCAO) in mice lacking BK channels (homozygous mice lacking the BK channel alpha subunit, BK(-/-)). MCAO was performed in BK(-/-) and WT mice for 90 minutes followed by a 7-hour-reperfusion period. Coronal 1 mm thick sections were stained with 2,3,5-triphenyltetrazolium chloride to reveal the infarction area. We found that transient focal cerebral ischemia by MCAO produced larger infarct volume, more severe neurological deficits, and higher post-ischemic mortality in BK(-/-) mice compared to WT littermates. However, the regional cerebral blood flow was not significantly different between genotypes as measured by Laser Doppler (LD) flowmetry pre-ischemically, intra-ischemically, and post-ischemically, suggesting that the different impact of MCAO in BK(-/-) vs. WT was not due to vascular BK channels. Furthermore, when NMDA was injected intracerebrally in non-ischemic mice, NMDA-induced neurotoxicity was found to be larger in BK(-/-) mice compared to WT. Whole-cell patch clamp recordings from CA1 pyramidal cells in organotypic hippocampal slice cultures revealed that BK channels contribute to rapid action potential repolarization, as previously found in acute slices. When these cultures were exposed to ischemia-like conditions this induced significantly more neuronal death in BK(-/-) than in WT cultures. These results indicate that neuronal BK channels are important for protection against ischemic brain damage.

  8. Differential antifungal activity of isomeric forms of nystatin.

    Science.gov (United States)

    Ostrosky-Zeichner, L; Bazemore, S; Paetznick, V L; Rodriguez, J R; Chen, E; Wallace, T; Cossum, P; Rex, J H

    2001-10-01

    When nystatin is placed in RPMI and other biological fluids, there is loss of pure nystatin, with the development of two distinguishable chromatographic peaks, 1 and 2. Peak 1 appears identical to commercially prepared nystatin. By nuclear magnetic resonance (NMR) and mass spectral analysis, peak 2 appears to be an isomer of peak 1. The isomers are quantitatively and fully interconvertible. Formation of peak 2 is accelerated at a pH of >7.0 and ultimately reaches a near 55:45 (peak 1/peak 2 ratio) mixture. We sought to determine the relative activities of peaks 1 and 2 against Candida spp. Peak 2 consistently showed higher MICs when it was the predominant form during the experiment. Time-kill analyses showed that peak 2 required > or =8 x the concentration of peak 1 to produce a modest and delayed killing effect, which was never of the same magnitude as that produced by peak 1. In both types of assays, the activity of peak 2 corresponded with intra-assay formation of peak 1. Both MIC measurements and time-kill analysis suggest that peak 2 has considerably less activity, if any at all, against Candida spp. Peak 2 may serve as a reservoir for peak 1.

  9. Synthesis, QSAR and calcium channel modulator activity of new hexahydroquinoline derivatives containing nitroimidazole.

    Science.gov (United States)

    Miri, Ramin; Javidnia, Katayoun; Mirkhani, Hossein; Hemmateenejad, Bahram; Sepeher, Zahra; Zalpour, Masomeh; Behzad, Taherh; Khoshneviszadeh, Mehdi; Edraki, Najmeh; Mehdipour, Ahmad R

    2007-10-01

    The discovery that 1,4-dihydropyridine class of calcium channel antagonists inhibit Ca2+ influx represented a major therapeutic advance in the treatment of cardiovascular disease. In contrast to the effects of known calcium channel blockers of the Nifedipine-type, the so-called calcium channel agonists, such as Bay K8644 and CGP 28392, increase calcium influx by binding at the same receptor regions. Our goal was to discover a dual cardioselective Ca2+-channel agonist/vascular selective smooth muscle Ca2+ channel antagonist third-generation 1,4-dihydropyridine drug which would have a suitable therapeutic profile for treating congestive heart failure (CHF) patients. A series of unsymmetrical alkyl, cycloalkyl and aryl ester analogues of 2-methyl-4-(1-methyl)-5-nitro-2-imidazolyl-5-oxo-1,4,5,6,7, 8-hexahydroquinolin-3-arboxylate were synthesized using modified Hantzsch reaction. All compounds show calcium antagonist activity on guinea-pig ileum longitudinal smooth muscle and some of them show agonist effect activity on guinea-pig auricle. Effect of structural parameters on the Ca2+ channel agonist/antagonist was evaluated by quantitative structure-activity relationship analysis. These compounds could be considered as a synthon for developing a suitable drug for treating CHF patients.

  10. Acid activation mechanism of the influenza A M2 proton channel.

    Science.gov (United States)

    Liang, Ruibin; Swanson, Jessica M J; Madsen, Jesper J; Hong, Mei; DeGrado, William F; Voth, Gregory A

    2016-10-24

    The homotetrameric influenza A M2 channel (AM2) is an acid-activated proton channel responsible for the acidification of the influenza virus interior, an important step in the viral lifecycle. Four histidine residues (His37) in the center of the channel act as a pH sensor and proton selectivity filter. Despite intense study, the pH-dependent activation mechanism of the AM2 channel has to date not been completely understood at a molecular level. Herein we have used multiscale computer simulations to characterize (with explicit proton transport free energy profiles and their associated calculated conductances) the activation mechanism of AM2. All proton transfer steps involved in proton diffusion through the channel, including the protonation/deprotonation of His37, are explicitly considered using classical, quantum, and reactive molecular dynamics methods. The asymmetry of the proton transport free energy profile under high-pH conditions qualitatively explains the rectification behavior of AM2 (i.e., why the inward proton flux is allowed when the pH is low in viral exterior and high in viral interior, but outward proton flux is prohibited when the pH gradient is reversed). Also, in agreement with electrophysiological results, our simulations indicate that the C-terminal amphipathic helix does not significantly change the proton conduction mechanism in the AM2 transmembrane domain; the four transmembrane helices flanking the channel lumen alone seem to determine the proton conduction mechanism.

  11. A new homogeneous high-throughput screening assay for profiling compound activity on the human ether-a-go-go-related gene channel.

    Science.gov (United States)

    Titus, Steven A; Beacham, Daniel; Shahane, Sampada A; Southall, Noel; Xia, Menghang; Huang, Ruili; Hooten, Elizabeth; Zhao, Yong; Shou, Louie; Austin, Christopher P; Zheng, Wei

    2009-11-01

    Long QT syndrome, either inherited or acquired from drug treatments, can result in ventricular arrhythmia (torsade de pointes) and sudden death. Human ether-a-go-go-related gene (hERG) channel inhibition by drugs is now recognized as a common reason for the acquired form of long QT syndrome. It has been reported that more than 100 known drugs inhibit the activity of the hERG channel. Since 1997, several drugs have been withdrawn from the market due to the long QT syndrome caused by hERG inhibition. Food and Drug Administration regulations now require safety data on hERG channels for investigative new drug (IND) applications. The assessment of compound activity on the hERG channel has now become an important part of the safety evaluation in the process of drug discovery. During the past decade, several in vitro assay methods have been developed and significant resources have been used to characterize hERG channel activities. However, evaluation of compound activities on hERG have not been performed for large compound collections due to technical difficulty, lack of throughput, and/or lack of biological relevance to function. Here we report a modified form of the FluxOR thallium flux assay, capable of measuring hERG activity in a homogeneous 1536-well plate format. To validate the assay, we screened a 7-point dilution series of the LOPAC 1280 library collection and reported rank order potencies of ten common hERG inhibitors. A correlation was also observed for the hERG channel activities of 10 known hERG inhibitors determined in this thallium flux assay and in the patch clamp experiment. Our findings indicate that this thallium flux assay can be used as an alternative method to profile large-volume compound libraries for compound activity on the hERG channel.

  12. Coronatin-1 isolated from entomopathogenic fungus Conidiobolus coronatus kills Galleria mellonella hemocytes in vitro and forms potassium channels in planar lipid membrane.

    Science.gov (United States)

    Wieloch, Wioletta; Boguś, Mieczysława I; Ligęza, Marta; Koszela-Piotrowska, Izabela; Szewczyk, Adam

    2011-09-15

    Entomopathogenic fungi are important natural regulatory factors of insect populations and have potential as biological control agents of insect pests. The cosmopolitan soil fungus Conidiobolus coronatus (Entomopthorales) easily attacks Galleria mellonella (Lepidoptera) larvae. Prompt death of invaded insects is attributed to the action of toxic metabolites released by the invader. Effect of fungal metabolites on hemocytes, insect blood cells involved in innate defense response, remains underexplored to date. C. coronatus isolate 3491 inducing 100% mortality of G. mellonella last instar larvae exposed to sporulating colonies, was cultivated at 20 °C in minimal medium. Post-incubation filtrates were used as a source of fungal metabolites. A two-step HPLC (1 step: Shodex KW-803 column eluted with 50 mM KH(2)PO(4) supplemented with 0.1 M KCl, pH 6.5; 2 step: ProteinPak™ CM 8HR column equilibrated with 5 mM KH(2)PO(4), pH 6.5, proteins eluted with a linear gradient of 0.5 M KCl) allowed the isolation of coronatin-1, an insecticidal 36 kDa protein showing both elastolytic and chitinolytic activities. Addition of coronatin-1 into primary in vitro cultures of G. mellonella hemocytes resulted in rapid disintegration of spherulocytes freely floating in culture medium and shrinkage of plasmatocytes adhering to the bottom of culture well. Coronatin-1 stimulated pseudopodia atrophy and, in consequence, disintegration of nets formed by cultured hemocytes. After incorporation of coronatin-1 into planar lipid membrane (PLM) ion channels selective for K(+) ions in 50/450 mM KCl solutions were observed. Potassium current flows were recorded in nearly 70% of experiments with conductance from 300 pS up to 1 nS. All observed channels were active at both positive and negative membrane potentials. Under experimental conditions incorporated coronatin-1 exhibited a zero current potential (E(rev)) of 47.7 mV, which indicates K(+)-selectivity of this protein. The success of the

  13. Magnesium excretion in C. elegans requires the activity of the GTL-2 TRPM channel.

    Directory of Open Access Journals (Sweden)

    Takayuki Teramoto

    Full Text Available Systemic magnesium homeostasis in mammals is primarily governed by the activities of the TRPM6 and TRPM7 cation channels, which mediate both uptake by the intestinal epithelial cells and reabsorption by the distal convoluted tubule cells in the kidney. In the nematode, C. elegans, intestinal magnesium uptake is dependent on the activities of the TRPM channel proteins, GON-2 and GTL-1. In this paper we provide evidence that another member of the TRPM protein family, GTL-2, acts within the C. elegans excretory cell to mediate the excretion of excess magnesium. Thus, the activity of GTL-2 balances the activities of the paralogous TRPM channel proteins, GON-2 and GTL-1.

  14. ALTERNATIVE EQUATIONS FOR DYNAMIC BEHAVIOR OF IONIC CHANNEL ACTIVATION AND INACTIVATION GATES

    Directory of Open Access Journals (Sweden)

    Mahmut ÖZER

    2003-03-01

    Full Text Available In this paper, alternative equations for dynamics of ionic channel activation and inactivation gates are proposed based on the path probability method. Dynamic behavior of a voltage-gated ionic channel is modeled by the conventional Hodgkin-Huxley (H-H mathematical formalism. In that model, conductance of the channel is defined in terms of activation and inactivation gates. Dynamics of the activation and inactivation gates is modeled by first-order differential equations dependent on the gate variable and the membrane potential. In the new approach proposed in this study, dynamic behavior of activation and inactivation gates is modeled by a firstorder differential equation dependent on internal energy and membrane potential by using the path probability method which is widely used in statistical physics. The new model doesn't require the time constant and steadystate values which are used explicitly in the H-H model. The numerical results show validity of the proposed method.

  15. Activation Effect of Cathartic Natural Compound Rhein to CFTR Chloride Channel

    Institute of Scientific and Technical Information of China (English)

    2006-01-01

    The cystic fibrosis transmembrane conductance regulator (CFTR) is a cAMP-activated chloride channel expressed in intestinal exocrine glands, which plays a key role in intestinal fluid secretion. A natural anthraquinone activator of CFTR Cl- channel, rhein, was identified by screening 217 single compounds from Chinese herbs via a cellbased halide-sensitive fluorescent assay. Rhein activates CFTR Cl- transportation in a dose-dependent manner in the presence of cAMP with a physiological concentration. This study provides a novel molecular pharmacological mechanism for the laxative drugs in Traditional Chinese Medicine such as aloe, cascara and senna.

  16. Analysis of G-protein-activated inward rectifying K(+) (GIRK) channel currents upon GABAB receptor activation in rat supraoptic neurons.

    Science.gov (United States)

    Harayama, Nobuya; Kayano, Tomohiko; Moriya, Taiki; Kitamura, Naoki; Shibuya, Izumi; Tanaka-Yamamoto, Keiko; Uezono, Yasuhito; Ueta, Yoichi; Sata, Takeyoshi

    2014-12-03

    While magnocellular neurons in the supraoptic nucleus (SON) possess rich Gi/o-mediated mechanisms, molecular and cellular properties of G-protein-activated inwardly rectifying K(+) (GIRK) channels have been controversial. Here, properties of GIRK channels are examined by RT-PCR and whole-cell patch-clamp techniques in rat SON neurons. Patch clamp experiments showed that the selective GABAB agonist, baclofen, enhanced currents in a high K(+) condition. The baclofen-enhanced currents exhibited evident inward rectification and were blocked by the selective GABAB antagonist, CGP55845A, the IRK channel blocker, Ba(2+), and the selective GIRK channel blocker, tertiapin, indicating that baclofen activates GIRK channels via GABAB receptors. The GIRK currents were abolished by N-ethylmaleimide pretreatment, and prolonged by GTPγS inclusion in the patch pipette, suggesting that Gi/o proteins are involved. RT-PCR analysis revealed mRNAs for all four GIRK 1-4 channels and for both GABABR1 and GABABR2 receptors in rat SON. However, the concentration-dependency of the baclofen-induced activation of GIRK currents had an EC50 of 110 µM, which is about 100 times higher than that of baclofen-induced inhibition of voltage-dependent Ca(2+) channels. Moreover, baclofen caused no significant changes in the membrane potential and the firing rate. These results suggest that although GIRK channels can be activated by GABAB receptors via the Gi/o pathway, this occurs at high agonist concentrations, and thus may not be a physiological mechanism regulating the function of SON neurons. This property that the membrane potential receives little influence from GIRK currents seems to be uncommon for CNS neurons possessing rich Gi/o-coupled receptors, and could be a special feature of rat SON neurons. Copyright © 2014 Elsevier B.V. All rights reserved.

  17. Mechanosensitive channel activity and F-actin organization in cholesterol-depleted human leukaemia cells.

    Science.gov (United States)

    Morachevskaya, Elena; Sudarikova, Anastasiya; Negulyaev, Yuri

    2007-04-01

    This study focuses on the functional role of cellular cholesterol in the regulation of mechanosensitive cation channels activated by stretch in human leukaemia K562 cells. The patch-clamp method was employed to examine the effect of methyl-beta-cyclodextrin (MbetaCD), a synthetic cholesterol-sequestering agent, on stretch-activated single currents. We found that cholesterol-depleting treatment with MbetaCD resulted in a suppression of the activity of mechanosensitive channels without a change in the unitary conductance. The probability that the channel was open significantly decreased after treatment with MbetaCD. Fluorescent microscopy revealed F-actin reorganization, possibly involving actin assembly, after incubation of the cells with MbetaCD. We suggest that suppression of mechanosensitive channel activation in cholesterol-depleted leukaemia cells is due to F-actin rearrangement, presumably induced by lipid raft destruction. Our observations are consistent with the notion that stretch-activated cation channels in eukaryotic cells are regulated by the membrane-cytoskeleton complex rather than by tension developed purely in the lipid bilayer.

  18. Ca2+ Channel Re-localization to Plasma-Membrane Microdomains Strengthens Activation of Ca2+-Dependent Nuclear Gene Expression

    Directory of Open Access Journals (Sweden)

    Krishna Samanta

    2015-07-01

    Full Text Available In polarized cells or cells with complex geometry, clustering of plasma-membrane (PM ion channels is an effective mechanism for eliciting spatially restricted signals. However, channel clustering is also seen in cells with relatively simple topology, suggesting it fulfills a more fundamental role in cell biology than simply orchestrating compartmentalized responses. Here, we have compared the ability of store-operated Ca2+ release-activated Ca2+ (CRAC channels confined to PM microdomains with a similar number of dispersed CRAC channels to activate transcription factors, which subsequently increase nuclear gene expression. For similar levels of channel activity, we find that channel confinement is considerably more effective in stimulating gene expression. Our results identify a long-range signaling advantage to the tight evolutionary conservation of channel clustering and reveal that CRAC channel aggregation increases the strength, fidelity, and reliability of the general process of excitation-transcription coupling.

  19. Radiometric microbiologic assay for the biologically active forms of niacin

    Energy Technology Data Exchange (ETDEWEB)

    Kertcher, J.A.; Guilarte, T.R.; Chen, M.F.; Rider, A.A.; McIntyre, P.A.

    1979-05-01

    A radiometric microbiologic assay has been developed for the determination of niacin in biologic fluids. Lactobacillus plantarum produced /sup 14/CO/sub 2/ from L-(U-/sup 14/C) malic acid in quantities proportional to the amount of niacin present. The assay is specific for the biologically active forms of niacin in humans. Thirty normal hemolysates were analyzed and the values ranged from 13.0 to 17.8 ..mu..g niacin/ml RBC (mean = 15.27 +- 1.33 s.d.). Good recovery and reproducibility studies were obtained with this assay. On thirty blood samples, correlation was excellent between the radiometric and the conventional turbidimetric assays.

  20. The Circumnuclear Star-forming Activities along the Hubble Sequence

    CERN Document Server

    Shi, L; Peng, Z; Shi, Lei; Gu, Qiusheng; Peng, Zhixin

    2005-01-01

    In order to study the circumnuclear star-forming activity along the Hubble sequence, we cross-correlate the Sloan Digital Sky Survey Data Release 2 (SDSS DR2) with the Third Reference Catalog of Bright Galaxies (RC3) to derive a large sample of 1015 galaxies with both morphological and spectral information. Among these, 385 sources are classified as star-forming galaxies and the SDSS fibre covered the circumnuclear regions (0.2 $-$ 2.0 kpc). By using the spectral synthesis method to remove the contribution from the underlying old stellar population, we measure the emission lines fluxes accurately which are then used to estimate the star formation rates(SFRs). Our main findings are that: (1) Early-type spirals show much larger H$\\alpha$ luminosities and hence higher SFRs, they also suffer more extinctions than late-type ones. The equivalent widths (EWs) of H$\\alpha$ emission lines show the similar trend, however, the very late types (Sdm $\\sim$ Irr) do have large fractions of high EWs. (2) We confirm that D$_n...

  1. Distinct regions that control ion selectivity and calcium-dependent activation in the bestrophin ion channel.

    Science.gov (United States)

    Vaisey, George; Miller, Alexandria N; Long, Stephen B

    2016-11-22

    Cytoplasmic calcium (Ca(2+)) activates the bestrophin anion channel, allowing chloride ions to flow down their electrochemical gradient. Mutations in bestrophin 1 (BEST1) cause macular degenerative disorders. Previously, we determined an X-ray structure of chicken BEST1 that revealed the architecture of the channel. Here, we present electrophysiological studies of purified wild-type and mutant BEST1 channels and an X-ray structure of a Ca(2+)-independent mutant. From these experiments, we identify regions of BEST1 responsible for Ca(2+) activation and ion selectivity. A "Ca(2+) clasp" within the channel's intracellular region acts as a sensor of cytoplasmic Ca(2+). Alanine substitutions within a hydrophobic "neck" of the pore, which widen it, cause the channel to be constitutively active, irrespective of Ca(2+). We conclude that the primary function of the neck is as a "gate" that controls chloride permeation in a Ca(2+)-dependent manner. In contrast to what others have proposed, we find that the neck is not a major contributor to the channel's ion selectivity. We find that mutation of a cytosolic "aperture" of the pore does not perturb the Ca(2+) dependence of the channel or its preference for anions over cations, but its mutation dramatically alters relative permeabilities among anions. The data suggest that the aperture functions as a size-selective filter that permits the passage of small entities such as partially dehydrated chloride ions while excluding larger molecules such as amino acids. Thus, unlike ion channels that have a single "selectivity filter," in bestrophin, distinct regions of the pore govern anion-vs.-cation selectivity and the relative permeabilities among anions.

  2. Molecular mechanism of ATP binding and ion channel activation in P2X receptors

    Energy Technology Data Exchange (ETDEWEB)

    Hattori, Motoyuki; Gouaux, Eric (Oregon HSU)

    2012-10-24

    P2X receptors are trimeric ATP-activated ion channels permeable to Na{sup +}, K{sup +} and Ca{sup 2+}. The seven P2X receptor subtypes are implicated in physiological processes that include modulation of synaptic transmission, contraction of smooth muscle, secretion of chemical transmitters and regulation of immune responses. Despite the importance of P2X receptors in cellular physiology, the three-dimensional composition of the ATP-binding site, the structural mechanism of ATP-dependent ion channel gating and the architecture of the open ion channel pore are unknown. Here we report the crystal structure of the zebrafish P2X4 receptor in complex with ATP and a new structure of the apo receptor. The agonist-bound structure reveals a previously unseen ATP-binding motif and an open ion channel pore. ATP binding induces cleft closure of the nucleotide-binding pocket, flexing of the lower body {beta}-sheet and a radial expansion of the extracellular vestibule. The structural widening of the extracellular vestibule is directly coupled to the opening of the ion channel pore by way of an iris-like expansion of the transmembrane helices. The structural delineation of the ATP-binding site and the ion channel pore, together with the conformational changes associated with ion channel gating, will stimulate development of new pharmacological agents.

  3. The Activation Effect of Hainantoxin-I, a Peptide Toxin from the Chinese Spider, Ornithoctonus hainana, on Intermediate-Conductance Ca2+-Activated K+ Channels

    Directory of Open Access Journals (Sweden)

    Pengfei Huang

    2014-08-01

    Full Text Available Intermediate-conductance Ca2+-activated K+ (IK channels are calcium/calmodulin-regulated voltage-independent K+ channels. Activation of IK currents is important in vessel and respiratory tissues, rendering the channels potential drug targets. A variety of small organic molecules have been synthesized and found to be potent activators of IK channels. However, the poor selectivity of these molecules limits their therapeutic value. Venom-derived peptides usually block their targets with high specificity. Therefore, we searched for novel peptide activators of IK channels by testing a series of toxins from spiders. Using electrophysiological experiments, we identified hainantoxin-I (HNTX-I as an IK-channel activator. HNTX-I has little effect on voltage-gated Na+ and Ca2+ channels from rat dorsal root ganglion neurons and on the heterologous expression of voltage-gated rapidly activating delayed rectifier K+ channels (human ether-à-go-go-related gene; human ERG in HEK293T cells. Only 35.2% ± 0.4% of the currents were activated in SK channels, and there was no effect on BK channels. We demonstrated that HNTX-I was not a phrenic nerve conduction blocker or acutely toxic. This is believed to be the first report of a peptide activator effect on IK channels. Our study suggests that the activity and selectivity of HNTX-I on IK channels make HNTX-I a promising template for designing new drugs for cardiovascular diseases.

  4. Regulation of the membrane insertion and conductance activity of the metamorphic chloride intracellular channel protein CLIC1 by cholesterol.

    Directory of Open Access Journals (Sweden)

    Stella M Valenzuela

    Full Text Available The Chloride Intracellular ion channel protein CLIC1 has the ability to spontaneously insert into lipid membranes from a soluble, globular state. The precise mechanism of how this occurs and what regulates this insertion is still largely unknown, although factors such as pH and redox environment are known contributors. In the current study, we demonstrate that the presence and concentration of cholesterol in the membrane regulates the spontaneous insertion of CLIC1 into the membrane as well as its ion channel activity. The study employed pressure versus area change measurements of Langmuir lipid monolayer films; and impedance spectroscopy measurements using tethered bilayer membranes to monitor membrane conductance during and following the addition of CLIC1 protein. The observed cholesterol dependent behaviour of CLIC1 is highly reminiscent of the cholesterol-dependent-cytolysin family of bacterial pore-forming proteins, suggesting common regulatory mechanisms for spontaneous protein insertion into the membrane bilayer.

  5. Regulation of the membrane insertion and conductance activity of the metamorphic chloride intracellular channel protein CLIC1 by cholesterol.

    Science.gov (United States)

    Valenzuela, Stella M; Alkhamici, Heba; Brown, Louise J; Almond, Oscar C; Goodchild, Sophia C; Carne, Sonia; Curmi, Paul M G; Holt, Stephen A; Cornell, Bruce A

    2013-01-01

    The Chloride Intracellular ion channel protein CLIC1 has the ability to spontaneously insert into lipid membranes from a soluble, globular state. The precise mechanism of how this occurs and what regulates this insertion is still largely unknown, although factors such as pH and redox environment are known contributors. In the current study, we demonstrate that the presence and concentration of cholesterol in the membrane regulates the spontaneous insertion of CLIC1 into the membrane as well as its ion channel activity. The study employed pressure versus area change measurements of Langmuir lipid monolayer films; and impedance spectroscopy measurements using tethered bilayer membranes to monitor membrane conductance during and following the addition of CLIC1 protein. The observed cholesterol dependent behaviour of CLIC1 is highly reminiscent of the cholesterol-dependent-cytolysin family of bacterial pore-forming proteins, suggesting common regulatory mechanisms for spontaneous protein insertion into the membrane bilayer.

  6. Incensole acetate, an incense component, elicits psychoactivity by activating TRPV3 channels in the brain.

    Science.gov (United States)

    Moussaieff, Arieh; Rimmerman, Neta; Bregman, Tatiana; Straiker, Alex; Felder, Christian C; Shoham, Shai; Kashman, Yoel; Huang, Susan M; Lee, Hyosang; Shohami, Esther; Mackie, Ken; Caterina, Michael J; Walker, J Michael; Fride, Ester; Mechoulam, Raphael

    2008-08-01

    Burning of Boswellia resin as incense has been part of religious and cultural ceremonies for millennia and is believed to contribute to the spiritual exaltation associated with such events. Transient receptor potential vanilloid (TRPV) 3 is an ion channel implicated in the perception of warmth in the skin. TRPV3 mRNA has also been found in neurons throughout the brain; however, the role of TRPV3 channels there remains unknown. Here we show that incensole acetate (IA), a Boswellia resin constituent, is a potent TRPV3 agonist that causes anxiolytic-like and antidepressive-like behavioral effects in wild-type (WT) mice with concomitant changes in c-Fos activation in the brain. These behavioral effects were not noted in TRPV3(-/-) mice, suggesting that they are mediated via TRPV3 channels. IA activated TRPV3 channels stably expressed in HEK293 cells and in keratinocytes from TRPV3(+/+) mice. It had no effect on keratinocytes from TRPV3(-/-) mice and showed modest or no effect on TRPV1, TRPV2, and TRPV4, as well as on 24 other receptors, ion channels, and transport proteins. Our results imply that TRPV3 channels in the brain may play a role in emotional regulation. Furthermore, the biochemical and pharmacological effects of IA may provide a biological basis for deeply rooted cultural and religious traditions.

  7. Identification of a cation-specific channel (TipA) in the cell wall of the gram-positive mycolata Tsukamurella inchonensis: the gene of the channel-forming protein is identical to mspA of Mycobacterium smegmatis and mppA of Mycobacterium phlei.

    Science.gov (United States)

    Dörner, Ursula; Maier, Elke; Benz, Roland

    2004-11-17

    Detergent extracts of whole cells of the Gram-positive bacterium Tsukamurella inchonensis ATCC 700082, which belongs to the mycolata, were studied for the presence of ion-permeable channels using lipid bilayer experiments. One channel with a conductance of about 4.5 nS in 1 M KCl was identified in the extracts. The channel-forming protein was purified to homogeneity by preparative SDS-PAGE. The protein responsible for channel-forming activity had an apparent molecular mass of about 33 kDa as judged by SDS-PAGE. Interestingly, the protein showed cross-reactivity with polyclonal antibodies raised against a polypeptide derived from MspA of Mycobacterium smegmatis similarly as the cell wall channel of Mycobacterium phlei. Primers derived from mspA were used to clone and sequence the gene of the cell wall channels of T. inchonensis (named tipA for T. inchonensis porin A) and M. phlei (named mppA for M. phlei porin A). Surprisingly, both genes, tipA and mppA, were found to be identical to mspA of M. smegmatis, indicating that the genomes of T. inchonensis, M. phlei and M. smegmatis contain the same genes for the major cell wall channel. RT-PCR revealed that tipA is transcribed in T. inchonensis and mppA in M. phlei. The results suggest that despite a certain distance between the three organisms, their genomes contain the same gene coding for the major cell wall channel, with a molecular mass of 22 kDa for the monomer.

  8. Intermolecular Interactions in the TMEM16A Dimer Controlling Channel Activity

    Science.gov (United States)

    Scudieri, Paolo; Musante, Ilaria; Gianotti, Ambra; Moran, Oscar; Galietta, Luis J. V.

    2016-12-01

    TMEM16A and TMEM16B are plasma membrane proteins with Ca2+-dependent Cl- channel function. By replacing the carboxy-terminus of TMEM16A with the equivalent region of TMEM16B, we obtained channels with potentiation of channel activity. Progressive shortening of the chimeric region restricted the “activating domain” to a short sequence close to the last transmembrane domain and led to TMEM16A channels with high activity at very low intracellular Ca2+ concentrations. To elucidate the molecular mechanism underlying this effect, we carried out experiments based on double chimeras, Forster resonance energy transfer, and intermolecular cross-linking. We also modeled TMEM16A structure using the Nectria haematococca TMEM16 protein as template. Our results indicate that the enhanced activity in chimeric channels is due to altered interaction between the carboxy-terminus and the first intracellular loop in the TMEM16A homo-dimer. Mimicking this perturbation with a small molecule could be the basis for a pharmacological stimulation of TMEM16A-dependent Cl- transport.

  9. Calmodulin mediates calcium-dependent activation of the intermediate conductance KCa channel, IKCa1.

    Science.gov (United States)

    Fanger, C M; Ghanshani, S; Logsdon, N J; Rauer, H; Kalman, K; Zhou, J; Beckingham, K; Chandy, K G; Cahalan, M D; Aiyar, J

    1999-02-26

    Small and intermediate conductance Ca2+-activated K+ channels play a crucial role in hyperpolarizing the membrane potential of excitable and nonexcitable cells. These channels are exquisitely sensitive to cytoplasmic Ca2+, yet their protein-coding regions do not contain consensus Ca2+-binding motifs. We investigated the involvement of an accessory protein in the Ca2+-dependent gating of hIKCa1, a human intermediate conductance channel expressed in peripheral tissues. Cal- modulin was found to interact strongly with the cytoplasmic carboxyl (C)-tail of hIKCa1 in a yeast two-hybrid system. Deletion analyses defined a requirement for the first 62 amino acids of the C-tail, and the binding of calmodulin to this region did not require Ca2+. The C-tail of hSKCa3, a human neuronal small conductance channel, also bound calmodulin, whereas that of a voltage-gated K+ channel, mKv1.3, did not. Calmodulin co-precipitated with the channel in cell lines transfected with hIKCa1, but not with mKv1. 3-transfected lines. A mutant calmodulin, defective in Ca2+ sensing but retaining binding to the channel, dramatically reduced current amplitudes when co-expressed with hIKCa1 in mammalian cells. Co-expression with varying amounts of wild-type and mutant calmodulin resulted in a dominant-negative suppression of current, consistent with four calmodulin molecules being associated with the channel. Taken together, our results suggest that Ca2+-calmodulin-induced conformational changes in all four subunits are necessary for the channel to open.

  10. The secret life of CFTR as a calcium-activated chloride channel.

    Science.gov (United States)

    Billet, Arnaud; Hanrahan, John W

    2013-11-01

    cAMP-stimulated anion conductance is defective in cystic fibrosis (CF). The regulatory domain of CFTR, the anion channel protein encoded by the CF gene, possesses an unusually high density of consensus sequences for phosphorylation by protein kinase A (14 in a stretch of CFTR is viewed primarily as a cAMP-stimulated anion channel, and most studies have focused on this mode of activation. However, there is growing evidence that CFTR also responds to Ca(2+)-mobilizing secretagogues and contributes substantially to cholinergic and purinergic responses in native tissues. G protein-coupled receptors that signal through Gαq can stimulate CFTR channels by activating Ca(2+)-dependent adenylyl cyclase and tyrosine kinases, and also by inhibiting protein phosphatase type 2A. Here we review evidence for these novel mechanisms of CFTR activation and discuss how they may help explain previous observations.

  11. Involvement of Ca2+-activated K+ Channels in Receptor-Regulated Sperm Motility in Rats

    Institute of Scientific and Technical Information of China (English)

    2002-01-01

    Previous voltage-clamp studies have demonstrated the modulation of sperm Ca2+-activated K+ (KCa) channels expressed in Xenopus oocytes by angiotensin Ⅱ (Ang Ⅱ)and extracellular ATP via AT1 receptor and P2U receptor, respectively. In the presentstudy, we investigated the involvement of KCa channels in receptor-regulated spermmotility of the rat using a computer-aided sperm analysis system, HTM-IVOS, in con-junction with Ca2+-mobilizing agents, receptor agonists/antagonists and KCa channelsblockers.The percentage of motile sperm was increased by ionomycin (0. 5 μmol/L), whichcould be inhibited by K+ channel blockers, tetraethylammonium (TEA 1 μmol/L ) orcharybdotoxin (ChTX, 300 nmol/L) indicating the presence of KCa channels. AngⅡ, at low concentration, 10 nmol/L, was found to increase motility, however, athigher concentration, 1 μmol/L, percentage of motility was found to be suppressed.Both stimulatory and inhibitory effects of Ang Ⅱ could be reversed by losartan, aspecific antagonist of AT 1 receptors, but not AT 2 antagonist PD123177, indicating theinvolvement of AT1 but not AT2 receptor in mediating both effects. ChTX also abol-ished both stimulatory and inhibitory effects of Ang H, suggesting the involvement ofKCa channels. The percentage of motility was also enhanced by extracellular ATP, afactor known to be involved in sperm activation. The ATP-enhanced sperm motilitywas mimicked by UTP , and inhibited by ChTX and reactive blue, an antagonist of P2receptor, indicating the involvement of both P2U and KCa channels. RT-PCR studywas also conducted to confirm the expression of KCa channels, AT1 receptors and P2Ureceptor, but not AT2 receptor, in rat caudal epididymal sperm. The present findingssuggest an important role of KCa channels in the regulation of sperm motility by AT1and P 2U receptors.

  12. Activation, permeability, and inhibition of astrocytic and neuronal large pore (hemi)channels.

    Science.gov (United States)

    Hansen, Daniel Bloch; Ye, Zu-Cheng; Calloe, Kirstine; Braunstein, Thomas Hartig; Hofgaard, Johannes Pauli; Ransom, Bruce R; Nielsen, Morten Schak; MacAulay, Nanna

    2014-09-19

    Astrocytes and neurons express several large pore (hemi)channels that may open in response to various stimuli, allowing fluorescent dyes, ions, and cytoplasmic molecules such as ATP and glutamate to permeate. Several of these large pore (hemi)channels have similar characteristics with regard to activation, permeability, and inhibitor sensitivity. Consequently, their behaviors and roles in astrocytic and neuronal (patho)physiology remain undefined. We took advantage of the Xenopus laevis expression system to determine the individual characteristics of several large pore channels in isolation. Expression of connexins Cx26, Cx30, Cx36, or Cx43, the pannexins Px1 or Px2, or the purinergic receptor P2X7 yielded functional (hemi)channels with isoform-specific characteristics. Connexin hemichannels had distinct sensitivity to alterations of extracellular Ca(2+) and their permeability to dyes and small atomic ions (conductance) were not proportional. Px1 and Px2 exhibited conductance at positive membrane potentials, but only Px1 displayed detectable fluorescent dye uptake. P2X7, in the absence of Px1, was permeable to fluorescent dyes in an agonist-dependent manner. The large pore channels displayed overlapping sensitivity to the inhibitors Brilliant Blue, gadolinium, and carbenoxolone. These results demonstrated isoform-specific characteristics among the large pore membrane channels; an open (hemi)channel is not a nonselective channel. With these isoform-specific properties in mind, we characterized the divalent cation-sensitive permeation pathway in primary cultured astrocytes. We observed no activation of membrane conductance or Cx43-mediated dye uptake in astrocytes nor in Cx43-expressing C6 cells. Our data underscore that although Cx43-mediated transport is observed in overexpressing cell systems, such transport may not be detectable in native cells under comparable experimental conditions.

  13. Can water sensitive urban design systems help to preserve natural channel-forming flow regimes in an urbanised catchment?

    Science.gov (United States)

    Wella-Hewage, Chathurika Subhashini; Alankarage Hewa, Guna; Pezzaniti, David

    2016-01-01

    Increased stormwater runoff and pollutant loads due to catchment urbanisation bring inevitable impacts on the physical and ecological conditions of environmentally sensitive urban streams. Water sensitive urban design (WSUD) has been recognised as a possible means to minimise these negative impacts. This paper reports on a study that investigated the ability of infiltration-based WSUD systems to replicate the predevelopment channel-forming flow (CFF) regime in urban catchments. Catchment models were developed for the 'pre-urban', 'urban' and 'managed' conditions of a case study catchment and the hydrological effect on CFF regime was investigated using a number of flow indices. The results clearly show that changes to flow regime are apparent under urban catchment conditions and are even more severe under highly urbanised conditions. The use of WSUD systems was found to result in the replication of predevelopment flow regimes, particularly at low levels of urbanisation. Under highly urbanised conditions (of managed catchments) overcontrol of the CFF indices was observed as indicated by flow statistics below their pre-urban values. The overall results suggest that WSUD systems are highly effective in replicating the predevelopment CFF regime in urban streams and could be used as a means to protect environmentally sensitive urban streams.

  14. Multi-channel Kalman filters for active noise control

    NARCIS (Netherlands)

    Ophem, S. van; Berkhoff, A.P.

    2013-01-01

    By formulating the feed-forward broadband active noise control problem as a state estimation problem it is possible to achieve a faster rate of convergence than the filtered reference least mean squares algorithm and possibly also a better tracking performance. A multiple input/multiple output

  15. Multi-channel Kalman filters for active noise control

    NARCIS (Netherlands)

    van Ophem, S.; Berkhoff, Arthur P.

    By formulating the feed-forward broadband active noise control problem as a state estimation problem it is possible to achieve a faster rate of convergence than the filtered reference least mean squares algorithm and possibly also a better tracking performance. A multiple input/multiple output

  16. Intermediate-Conductance-Ca2-Activated K Channel Intermediate-Conductance Calcium-Activated Potassium Channel (IKCa1) is Upregulated and Promotes Cell Proliferation in Cervical Cancer

    Science.gov (United States)

    Liu, Ling; Zhan, Ping; Nie, Dan; Fan, Lingye; Lin, Hairui; Gao, Lanyang; Mao, Xiguang

    2017-01-01

    Background Accumulating data point to intermediate-conductance calcium-activated potassium channel (IKCa1) as a key player in controlling cell cycle progression and proliferation of human cancer cells. However, the role that IKCa1 plays in the growth of human cervical cancer cells is largely unexplored. Material/Methods In this study, Western blot analysis, immunohistochemical staining, and RT-PCR were first used for IKCa1protein and gene expression assays in cervical cancer tissues and HeLa cells. Then, IKCa1 channel blocker and siRNA were employed to inhibit the functionality of IKCa1 and downregulate gene expression in HeLa cells, respectively. After these treatments, we examined the level of cell proliferation by MTT method and measured IKCa1 currents by conventional whole-cell patch clamp technique. Cell apoptosis was assessed using the Annexin V-FITC/Propidium Iodide (PI) double-staining apoptosis detection kit. Results We demonstrated that IKCa1 mRNA and protein are preferentially expressed in cervical cancer tissues and HeLa cells. We also showed that the IKCa1 channel blocker, clotrimazole, and IKCa1 channel siRNA can be used to suppress cervical cancer cell proliferation and decrease IKCa1 channel current. IKCa1 downregulation by specific siRNAs induced a significant increase in the proportion of apoptotic cells in HeLa cells. Conclusions IKCa1 is overexpressed in cervical cancer tissues, and IKCa1 upregulation in cervical cancer cell linea enhances cell proliferation, partly by reducing the proportion of apoptotic cells. PMID:28280257

  17. Differential regulation of voltage- and calcium-activated potassium channels in human B lymphocytes.

    Science.gov (United States)

    Partiseti, M; Choquet, D; Diu, A; Korn, H

    1992-06-01

    The expression and characteristics of K+ channels of human B lymphocytes were studied by using single and whole-cell patch-clamp recordings. They were gated by depolarization (voltage-gated potassium current, IKv, 11-20 pS) and by an increase in intracellular Ca2+ concentration (calcium-activated potassium current, IKCa, 26 pS), respectively. The level of expression of these channels was correlated with the activational status of the cell. Both conductances are blocked by tetraethylammonium, verapamil, and charybdotoxin, and are insensitive to apamin; 4-aminopyridine blocks IK, preferentially. We used a protein kinase C activator (PMA) or antibodies to membrane Ig (anti-mu) to activate resting splenocytes in culture. Although IKv was recorded in the majority of the resting lymphocytic population, less than 20% of the activated cells expressed this conductance. However, in this subset the magnitude of IKv was 20-fold larger than in resting cells. On the other hand, IKCa was detected in nearly one half of the resting cells, whereas all activated cells expressed this current. The magnitude of IKCa was, on average, 30 times larger in activated than in nonactivated cells. These results probably reflect that during the course of activation 1) the number of voltage-dependent K+ channels per cell decreases and increases in a small subset and 2) the number of Ca(2+)-dependent K+ channels per cell increases in all cells. We suggest that the expression of functional Ca(2+)- and voltage-activated K+ channels are under the control of different regulatory signals.

  18. CLOSED-FORM SPACE-TIME CHANNEL BLIND ESTIMATION FOR SPACE-TIME CODED MC-CDMA SYTEMS WITH UNIFORM LINEAR ARRAY

    Institute of Scientific and Technical Information of China (English)

    2006-01-01

    This paper proposes a closed-form joint space-time channel and Direction Of Arrival (DOA) blind estimation algorithm for space-time coded Multi-Carrier Code Division Multiple Access (MC-CDMA) systems equipped with a Uniform Linear Array (ULA) at the base station in frequency-selective fading environments. The algorithm uses an ESPRIT-like method to separate multiple co-channel users with different impinging DOAs. As a result, the DOAs for multiple users are obtained. In particular, a set of signal subspaces,every one of which is spanned by the space-time vector channels of an individual user, are also obtained. From these signal subspaces, the space-time channels of multiple users are estimated using the subspace method.Computer simulations illustrate both the validity and the overall performance of the proposed scheme.

  19. Active membrane having uniform physico-chemically functionalized ion channels

    Science.gov (United States)

    Gerald, II, Rex E; Ruscic, Katarina J; Sears, Devin N; Smith, Luis J; Klingler, Robert J; Rathke, Jerome W

    2012-09-24

    The present invention relates to a physicochemically-active porous membrane for electrochemical cells that purports dual functions: an electronic insulator (separator) and a unidirectional ion-transporter (electrolyte). The electrochemical cell membrane is activated for the transport of ions by contiguous ion coordination sites on the interior two-dimensional surfaces of the trans-membrane unidirectional pores. One dimension of the pore surface has a macroscopic length (1 nm-1000 .mu.m) and is directed parallel to the direction of an electric field, which is produced between the cathode and the anode electrodes of an electrochemical cell. The membrane material is designed to have physicochemical interaction with ions. Control of the extent of the interactions between the ions and the interior pore walls of the membrane and other materials, chemicals, or structures contained within the pores provides adjustability of the ionic conductivity of the membrane.

  20. Intracellular long-chain acyl CoAs activate TRPV1 channels.

    Directory of Open Access Journals (Sweden)

    Yi Yu

    Full Text Available TRPV1 channels are an important class of membrane proteins that play an integral role in the regulation of intracellular cations such as calcium in many different tissue types. The anionic phospholipid phosphatidylinositol 4,5-bisphosphate (PIP2 is a known positive modulator of TRPV1 channels and the negatively charged phosphate groups interact with several basic amino acid residues in the proximal C-terminal TRP domain of the TRPV1 channel. We and other groups have shown that physiological sub-micromolar levels of long-chain acyl CoAs (LC-CoAs, another ubiquitous anionic lipid, can also act as positive modulators of ion channels and exchangers. Therefore, we investigated whether TRPV1 channel activity is similarly regulated by LC-CoAs. Our results show that LC-CoAs are potent activators of the TRPV1 channel and interact with the same PIP2-binding residues in TRPV1. In contrast to PIP2, LC-CoA modulation of TRPV1 is independent of Ca2+i, acting in an acyl side-chain saturation and chain-length dependent manner. Elevation of LC-CoAs in intact Jurkat T-cells leads to significant increases in agonist-induced Ca2+i levels. Our novel findings indicate that LC-CoAs represent a new fundamental mechanism for regulation of TRPV1 channel activity that may play a role in diverse cell types under physiological and pathophysiological conditions that alter fatty acid transport and metabolism such as obesity and diabetes.

  1. Critical role of gap junction coupled KATP channel activity for regulated insulin secretion.

    Directory of Open Access Journals (Sweden)

    Jonathan V Rocheleau

    2006-02-01

    Full Text Available Pancreatic beta-cells secrete insulin in response to closure of ATP-sensitive K+ (KATP channels, which causes membrane depolarization and a concomitant rise in intracellular Ca2+ (Cai. In intact islets, beta-cells are coupled by gap junctions, which are proposed to synchronize electrical activity and Cai oscillations after exposure to stimulatory glucose (>7 mM. To determine the significance of this coupling in regulating insulin secretion, we examined islets and beta-cells from transgenic mice that express zero functional KATP channels in approximately 70% of their beta-cells, but normal KATP channel density in the remainder. We found that KATP channel activity from approximately 30% of the beta-cells is sufficient to maintain strong glucose dependence of metabolism, Cai, membrane potential, and insulin secretion from intact islets, but that glucose dependence is lost in isolated transgenic cells. Further, inhibition of gap junctions caused loss of glucose sensitivity specifically in transgenic islets. These data demonstrate a critical role of gap junctional coupling of KATP channel activity in control of membrane potential across the islet. Control via coupling lessens the effects of cell-cell variation and provides resistance to defects in excitability that would otherwise lead to a profound diabetic state, such as occurs in persistent neonatal diabetes mellitus.

  2. Critical role of gap junction coupled KATP channel activity for regulated insulin secretion.

    Science.gov (United States)

    Rocheleau, Jonathan V; Remedi, Maria S; Granada, Butch; Head, W Steven; Koster, Joseph C; Nichols, Colin G; Piston, David W

    2006-02-01

    Pancreatic beta-cells secrete insulin in response to closure of ATP-sensitive K+ (KATP) channels, which causes membrane depolarization and a concomitant rise in intracellular Ca2+ (Cai). In intact islets, beta-cells are coupled by gap junctions, which are proposed to synchronize electrical activity and Cai oscillations after exposure to stimulatory glucose (>7 mM). To determine the significance of this coupling in regulating insulin secretion, we examined islets and beta-cells from transgenic mice that express zero functional KATP channels in approximately 70% of their beta-cells, but normal KATP channel density in the remainder. We found that KATP channel activity from approximately 30% of the beta-cells is sufficient to maintain strong glucose dependence of metabolism, Cai, membrane potential, and insulin secretion from intact islets, but that glucose dependence is lost in isolated transgenic cells. Further, inhibition of gap junctions caused loss of glucose sensitivity specifically in transgenic islets. These data demonstrate a critical role of gap junctional coupling of KATP channel activity in control of membrane potential across the islet. Control via coupling lessens the effects of cell-cell variation and provides resistance to defects in excitability that would otherwise lead to a profound diabetic state, such as occurs in persistent neonatal diabetes mellitus.

  3. SENSITIVE EFFECTS OF POTASSIUM AND CALCIUM CHANNEL BLOCKING AND ATP-SENSITIVE POTASSIUM CHANNEL ACTIVATORS ON SEMINAL VESICLE SMOOTH MUSCLE CONTRACTIONS

    Directory of Open Access Journals (Sweden)

    H SADRAEI

    2000-12-01

    Full Text Available Background. Seminal vesicle smooth muscle contraction is mediated through sympathetic and parasympathetic neurons activity. Although seminal vesicle plays an important role in male fertility, but little attention is given to mechanism involved in contraction of this organ.
    Methods. In this study effects of drugs which activate ATP - sensitive K channels and blockers of K and Ca channels were examined on contraction of guinea - pig isolated seminal vesicle due to electrical filled stimulation (EFS, noradrenaline, carbachol and KCI.
    Results. The K channel blocker tetraethyl ammonium potentate the EFS responses at all frequencies, while, the ATP - sensitive K channel inhibitor glibenclamide and the K channel opener levcromakalim, diazoxide, minoxidil and Ca channel blocker nifedipine all had relaxant effect on guinea - pig seminal vesicle.
    Discussion. This study indicate that activities of K and Ca channels is important in regulation of seminal vesicle contraction due to nerve stimulation, noradrenaline or carbachol.

  4. A putatively novel form of spontaneous coordination in neural activity.

    Science.gov (United States)

    Hermer-Vazquez, Raymond; Hermer-Vazquez, Linda; Srinivasan, Sridhar

    2009-04-06

    We simultaneously recorded local field potentials from three sites along the olfactory-entorhinal axis in rats lightly anesthetized with isoflurane, as part of another experiment. While analyzing the initial data from that experiment with spectrograms, we discovered a potentially novel form of correlated neural activity, with near-simultaneous occurrence across the three widely separated brain sites. After validating their existence further, we named these events Synchronous Frequency Bursts (SFBs). Here we report our initial investigations into their properties and their potential functional significance. In Experiment 1, we found that SFBs have highly regular properties, consisting of brief (approximately 250 ms), high amplitude bursts of LFP energy spanning frequency ranges from the delta band (1-4 Hz) to at least the low gamma band (30-50 Hz). SFBs occurred almost simultaneously across recording sites, usually with onsets sites. While the SFBs had fairly typical, exponentially decaying power spectral density plots, their coherence structure was unusual, with high peaks in several narrow frequency ranges and little coherence in other bands. In Experiment 2, we found that SFBs occurred far more often under light anesthesia than deeper anesthetic states, and were especially prevalent as the animals regained consciousness. Finally, in Experiment 3 we showed that SFBs occur simultaneously at a significant rate across brain sites from putatively different functional subsystems--olfactory versus motor pathways. We suggest that SFBs do not carry information per se, but rather, play a role in coordinating activity in different frequency bands, potentially brain-wide, as animals progress from sleep or anesthesia toward full consciousness.

  5. Mutations of the S4-S5 linker alter activation properties of HERG potassium channels expressed in Xenopus oocytes.

    Science.gov (United States)

    Sanguinetti, M C; Xu, Q P

    1999-02-01

    1. The structural basis for the activation gate of voltage-dependent K+ channels is not known, but indirect evidence has implicated the S4-S5 linker, the cytoplasmic region between the fourth and fifth transmembrane domains of the channel subunit. We have studied the effects of mutations in the S4-S5 linker of HERG (human ether-á-go-go-related gene), a human delayed rectifier K+ channel, in Xenopus oocytes. 2. Mutation of acidic residues (D540, E544) in the S4-S5 linker of HERG channels to neutral (Ala) or basic (Lys) residues accelerated the rate of channel deactivation. Most mutations greatly accelerated the rate of activation. However, E544K HERG channels activated more slowly than wild-type HERG channels. 3. Mutation of residues in the S4-S5 linker had little or no effect on fast inactivation, consistent with independence of HERG channel activation and inactivation 4. In response to large hyperpolarizations, D540K HERG channels can reopen into a state that is distinct from the normal depolarization-induced open state. It is proposed that substitution of a negatively charged Asp with the positively charged Lys disrupts a subunit interaction that normally stabilizes the channel in a closed state at negative transmembrane potentials. 5. The results indicate that the S4-S5 linker is a crucial component of the activation gate of HERG channels.

  6. Partial Agonist and Antagonist Activities of a Mutant Scorpion β-Toxin on Sodium Channels*

    OpenAIRE

    Karbat, Izhar; Ilan, Nitza; Zhang, Joel Z.; Cohen, Lior; Kahn, Roy; Benveniste, Morris; Scheuer, Todd; Catterall, William A.; Gordon, Dalia; Gurevitz, Michael

    2010-01-01

    Scorpion β-toxin 4 from Centruroides suffusus suffusus (Css4) enhances the activation of voltage-gated sodium channels through a voltage sensor trapping mechanism by binding the activated state of the voltage sensor in domain II and stabilizing it in its activated conformation. Here we describe the antagonist and partial agonist properties of a mutant derivative of this toxin. Substitution of seven different amino acid residues for Glu15 in Css4 yielded toxin derivatives with both increased a...

  7. Presynaptic calcium channels and α3-integrins are complexed with synaptic cleft laminins, cytoskeletal elements and active zone components.

    Science.gov (United States)

    Carlson, Steven S; Valdez, Gregorio; Sanes, Joshua R

    2010-11-01

    At chemical synapses, synaptic cleft components interact with elements of the nerve terminal membrane to promote differentiation and regulate function. Laminins containing the β2 subunit are key cleft components, and they act in part by binding the pore-forming subunit of a pre-synaptic voltage-gated calcium channel (Ca(v)α) (Nishimune et al. 2004). In this study, we identify Ca(v)α-associated intracellular proteins that may couple channel-anchoring to assembly or stabilization of neurotransmitter release sites called active zones. Using Ca(v)α-antibodies, we isolated a protein complex from Torpedo electric organ synapses, which resemble neuromuscular junctions but are easier to isolate in bulk. We identified 10 components of the complex: six cytoskeletal proteins (α2/β2 spectrins, plectin 1, AHNAK/desmoyokin, dystrophin, and myosin 1), two active zone components (bassoon and piccolo), synaptic laminin, and a calcium channel β subunit. Immunocytochemistry confirmed these proteins in electric organ synapses, and PCR analysis revealed their expression by developing mammalian motor neurons. Finally, we show that synaptic laminins also interact with pre-synaptic integrins containing the α3 subunit. Together with our previous finding that a distinct synaptic laminin interacts with SV2 on nerve terminals (Son et al. 2000), our results identify three paths by which synaptic cleft laminins can send developmentally important signals to nerve terminals.

  8. Anoctamin Calcium-Activated Chloride Channels May Modulate Inhibitory Transmission in the Cerebellar Cortex.

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    Weiping Zhang

    Full Text Available Calcium-activated chloride channels of the anoctamin (alias TMEM16 protein family fulfill critical functions in epithelial fluid transport, smooth muscle contraction and sensory signal processing. Little is known, however, about their contribution to information processing in the central nervous system. Here we examined the recent finding that a calcium-dependent chloride conductance impacts on GABAergic synaptic inhibition in Purkinje cells of the cerebellum. We asked whether anoctamin channels may underlie this chloride conductance. We identified two anoctamin channel proteins, ANO1 and ANO2, in the cerebellar cortex. ANO1 was expressed in inhibitory interneurons of the molecular layer and the granule cell layer. Both channels were expressed in Purkinje cells but, while ANO1 appeared to be retained in the cell body, ANO2 was targeted to the dendritic tree. Functional studies confirmed that ANO2 was involved in a calcium-dependent mode of ionic plasticity that reduces the efficacy of GABAergic synapses. ANO2 channels attenuated GABAergic transmission by increasing the postsynaptic chloride concentration, hence reducing the driving force for chloride influx. Our data suggest that ANO2 channels are involved in a Ca2+-dependent regulation of synaptic weight in GABAergic inhibition. Thus, in balance with the chloride extrusion mechanism via the co-transporter KCC2, ANO2 appears to regulate ionic plasticity in the cerebellum.

  9. P2Y2 and P2Y4 receptors regulate pancreatic Ca²+-activated K+ channels differently

    DEFF Research Database (Denmark)

    Klærke, Susanne Edeling Hede; Amstrup, Jan; Klærke, Dan Arne;

    2005-01-01

    Extracellular ATP is an important regulator of transepithelial transport in a number of tissues. In pancreatic ducts, we have shown that ATP modulates epithelial K+ channels via purinergic receptors, most likely the P2Y2 and P2Y4 receptors, but the identity of the involved K+ channels was not clear....... In this study, we show by RT-PCR analysis that rat pancreatic ducts express Ca(2+)-activated K+ channels of intermediate conductance (IK) and big conductance (BK), but not small conductance (SK). Possible interactions between P2Y receptors and these Ca(2+)-activated K+ channels were examined in co......-expression experiments in Xenopus laevis oocytes. K+ channel activity was measured electrophysiologically in oocytes stimulated with UTP (0.1 mM). UTP stimulation of oocytes expressing P2Y4 receptors and BK channels resulted in a 30% increase in the current through the expressed channels. In contrast, stimulation of P2Y...

  10. Activation of Na+ channels in cell membrane by capacitive stimulation with silicon chip

    Science.gov (United States)

    Schoen, Ingmar; Fromherz, Peter

    2005-11-01

    Sodium channels are the crucial electrical elements of neuronal excitation. As a step toward hybrid neuron-semiconductor devices, we studied the activation of recombinant NaV1.4 sodium channels in human embryonic kidney (HEK293) cells by stimulation from an electrolyte/oxide/silicon (EOS) capacitor. HfO2 was used as an insulator to attain a high capacitance. An effective activation was achieved by decaying voltage ramps at constant intracellular voltage at a depleted NaCl concentration in the bath to enhance the resistance of the cell-chip contact. We were also able to open sodium channels at a NaCl concentration close to physiological conditions. This experiment provides a basis for noninvasive capacitive stimulation of nerve cells with semiconductor chips.

  11. Ethanol affects network activity in cultured rat hippocampus: mediation by potassium channels.

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    Eduard Korkotian

    Full Text Available The effects of ethanol on neuronal network activity were studied in dissociated cultures of rat hippocampus. Exposure to low (0.25-0.5% ethanol concentrations caused an increase in synchronized network spikes, and a decrease in the duration of individual spikes. Ethanol also caused an increase in rate of miniature spontaneous excitatory postsynaptic currents. Higher concentrations of ethanol eliminated network spikes. These effects were reversible upon wash. The effects of the high, but not the low ethanol were blocked by the GABA antagonist bicuculline. The enhancing action of low ethanol was blocked by apamin, an SK potassium channel antagonist, and mimicked by 1-EBIO, an SK channel opener. It is proposed that in cultured hippocampal networks low concentration of ethanol is associated with SK channel activity, rather than the GABAergic receptor.

  12. Differential activation of vascular smooth muscle Kv7.4, Kv7.5, and Kv7.4/7.5 channels by ML213 and ICA-069673.

    Science.gov (United States)

    Brueggemann, Lyubov I; Haick, Jennifer M; Cribbs, Leanne L; Byron, Kenneth L

    2014-09-01

    Recent research suggests that smooth muscle cells express Kv7.4 and Kv7.5 voltage-activated potassium channels, which contribute to maintenance of their resting membrane voltage. New pharmacologic activators of Kv7 channels, ML213 (N-mesitybicyclo[2.2.1]heptane-2-carboxamide) and ICA-069673 N-(6-chloropyridin-3-yl)-3,4-difluorobenzamide), have been reported to discriminate among channels formed from different Kv7 subtypes. We compared the effects of ML213 and ICA-069673 on homomeric human Kv7.4, Kv7.5, and heteromeric Kv7.4/7.5 channels exogenously expressed in A7r5 vascular smooth muscle cells. We found that, despite its previous description as a selective activator of Kv7.2 and Kv7.4, ML213 significantly increased the maximum conductance of homomeric Kv7.4 and Kv7.5, as well as heteromeric Kv7.4/7.5 channels, and induced a negative shift of their activation curves. Current deactivation rates decreased in the presence of the ML213 (10 μM) for all three channel combinations. Mutants of Kv7.4 (W242L) and Kv7.5 (W235L), previously found to be insensitive to another Kv7 channel activator, retigabine, were also insensitive to ML213 (10 μM). In contrast to ML213, ICA-069673 robustly activated Kv7.4 channels but was significantly less effective on homomeric Kv7.5 channels. Heteromeric Kv7.4/7.5 channels displayed intermediate responses to ICA-069673. In each case, ICA-069673 induced a negative shift of the activation curves without significantly increasing maximal conductance. Current deactivation rates decreased in the presence of ICA-069673 in a subunit-specific manner. Kv7.4 W242L responded to ICA-069673-like wild-type Kv7.4, but a Kv7.4 F143A mutant was much less sensitive to ICA-069673. Based on these results, ML213 and ICA-069673 likely bind to different sites and are differentially selective among Kv7.4, Kv7.5, and Kv7.4/7.5 channel subtypes.

  13. Being flexible: the voltage-controllable activation gate of Kv channels

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    Alain J. Labro

    2012-09-01

    Full Text Available Kv channels form voltage-dependent potassium selective pores in the outer cell membrane and are composed out of four -subunits, each having six membrane-spanning -helices (S1-S6. The -subunits tetramerize such that the S5-S6 pore domains co-assemble into a centrally located K+ pore which is surrounded by four operational voltage sensing domains (VSD that are each formed by the S1-S4 segments. Consequently, each subunit is capable of responding to changes in membrane potential and dictates whether the pore should be conductive or not. K+ permeation through the pore can be sealed off by two separate gates in series: (a at the inner S6 bundle crossing (BC gate and (b at the level of the selectivity-filter (SF gate located at the extracellular entrance of the pore. Within the last years a general consensus emerged that a direct communication between the S4S5-linker and the bottom part of S6 (S6c constitutes the coupling with the VSD thus making the BC gate the main voltage-controllable activation gate. While the BC gate listens to the VSD, the SF changes its conformation depending on the status of the BC gate. Through the eyes of an entering K+ ion, the operation of the BC gate apparatus can be compared with the iris-like motion of the diaphragm from a camera whereby its diameter widens. Two main gating motions have been proposed to create this BC gate widening: (1 tilting of the helix whereby the S6 converts from a straight -helix to a tilted one or (2 swiveling of the S6c whereby the S6 remains bent. Such motions require a flexible hinge that decouples the pre- and post-hinge segment. Roughly at the middle of the S6 there exists a highly conserved glycine residue and a tandem proline motif that seem to fulfill the role of a gating hinge which allows for tilting/swiveling/rotations of the post-hinge S6 segment. In this review we delineate our current view on the operation of the BC gate for controlling K+ permeation in Kv channels.

  14. Role of low voltage activated calcium channels in neuritogenesis and active migration of embryonic neural progenitor cells.

    Science.gov (United States)

    Louhivuori, Lauri M; Louhivuori, Verna; Wigren, Henna-Kaisa; Hakala, Elina; Jansson, Linda C; Nordström, Tommy; Castrén, Maija L; Akerman, Karl E

    2013-04-15

    The central role of calcium influx and electrical activity in embryonic development raises important questions about the role and regulation of voltage-dependent calcium influx. Using cultured neural progenitor cell (NPC) preparations, we recorded barium currents through voltage-activated channels using the whole-cell configuration of the patch-clamp technique and monitored intracellular free calcium concentrations with Fura-2 digital imaging. We found that NPCs as well as expressing high-voltage-activated (HVA) calcium channels express functional low-threshold voltage-dependent calcium channels in the very early stages of differentiation (5 h to 1 day). The size of the currents recorded at -50 versus -20 mV after 1 day in differentiation was dependent on the nature of the charge carrier. Peak currents measured at -20 mV in the presence 10 mM Ca2+ instead of 10 mM Ba2+ had a tendency to be smaller, whereas the nature of the divalent species did not influence the amplitude measured at -50 mV. The T-type channel blockers mibefradil and NNC 55-0396 significantly reduced the calcium responses elicited by depolarizing with extracellular potassium, while the overall effect of the HVA calcium channel blockers was small at differentiation day 1. At differentiation day 20, the calcium responses were effectively blocked by nifedipine. Time-lapse imaging of differentiating neurospheres cultured in the presence of low-voltage-activated (LVA) blockers showed a significant decrease in the number of active migrating neuron-like cells and neurite extensions. Together, these data provide evidence that LVA calcium channels are involved in the physiology of differentiating and migrating NPCs.

  15. Levamisole-activated single-channel currents from muscle of the nematode parasite Ascaris suum.

    Science.gov (United States)

    Robertson, S. J.; Martin, R. J.

    1993-01-01

    1. The patch-clamp technique was used to examine levamisole-activated channels in muscle vesicles from Ascaris suum. Cell-attached and isolated inside-out patches were used. 2. Levamisole (1-90 microM), applied to the extracellular surface, activated channels which had apparent mean open-times in the range 0.80-2.85 ms and linear I/V relationships with conductances in the range 19-46 pS. Ion-replacement experiments showed the channels to be cation selective. 3. The kinetics of the channels were analysed. Generally open- and closed-time distributions were best fitted by two, and three exponentials respectively, indicating the presence of at least two open states and at least three closed states. The distributions of burst-times were best-fitted by two exponentials. 4. Channel open- and burst-times were voltage-sensitive: at low levamisole concentrations (1-10 microM), they increased with hyperpolarization. At higher concentrations of levamisole (30 microM and 90 microM) flickering channel-block was observed at hyperpolarized potentials. Using a simple channel-block model, values for the blocking dissociation constant, KB were determined as 123 microM at -50 mV, 46 microM at -75 mV and 9.4 microM at -100 mV. 5. At the higher concentration of levamisole (30 microM and 90 microM) long closed-times separating 'clusters' of bursts were observed, at both hyperpolarized and depolarized membrane potentials and this was interpreted as desensitization. PMID:7679027

  16. Stochastically gating ion channels enable patterned spike firing through activity-dependent modulation of spike probability.

    Directory of Open Access Journals (Sweden)

    Joshua T Dudman

    2009-02-01

    Full Text Available The transformation of synaptic input into patterns of spike output is a fundamental operation that is determined by the particular complement of ion channels that a neuron expresses. Although it is well established that individual ion channel proteins make stochastic transitions between conducting and non-conducting states, most models of synaptic integration are deterministic, and relatively little is known about the functional consequences of interactions between stochastically gating ion channels. Here, we show that a model of stellate neurons from layer II of the medial entorhinal cortex implemented with either stochastic or deterministically gating ion channels can reproduce the resting membrane properties of stellate neurons, but only the stochastic version of the model can fully account for perithreshold membrane potential fluctuations and clustered patterns of spike output that are recorded from stellate neurons during depolarized states. We demonstrate that the stochastic model implements an example of a general mechanism for patterning of neuronal output through activity-dependent changes in the probability of spike firing. Unlike deterministic mechanisms that generate spike patterns through slow changes in the state of model parameters, this general stochastic mechanism does not require retention of information beyond the duration of a single spike and its associated afterhyperpolarization. Instead, clustered patterns of spikes emerge in the stochastic model of stellate neurons as a result of a transient increase in firing probability driven by activation of HCN channels during recovery from the spike afterhyperpolarization. Using this model, we infer conditions in which stochastic ion channel gating may influence firing patterns in vivo and predict consequences of modifications of HCN channel function for in vivo firing patterns.

  17. TRPV3 Channels Mediate Strontium-Induced Mouse-Egg Activation

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    Ingrid Carvacho

    2013-12-01

    Full Text Available In mammals, calcium influx is required for oocyte maturation and egg activation. The molecular identities of the calcium-permeant channels that underlie the initiation of embryonic development are not established. Here, we describe a transient receptor potential (TRP ion channel current activated by TRP agonists that is absent in TrpV3−/− eggs. TRPV3 current is differentially expressed during oocyte maturation, reaching a peak of maximum density and activity at metaphase of meiosis II (MII, the stage of fertilization. Selective activation of TRPV3 channels provokes egg activation by mediating massive calcium entry. Widely used to activate eggs, strontium application is known to yield normal offspring in combination with somatic cell nuclear transfer. We show that TRPV3 is required for strontium influx, because TrpV3−/− eggs failed to conduct Sr2+ or undergo strontium-induced activation. We propose that TRPV3 is a major mediator of calcium influx in mouse eggs and is a putative target for artificial egg activation.

  18. Smoking Discriminately Changes the Serum Active and Non-Active Forms of Vitamin B12.

    Science.gov (United States)

    Shekoohi, Niloofar; Javanbakht, Mohammad Hassan; Sohrabi, Marjan; Zarei, Mahnaz; Mohammadi, Hamed; Djalali, Mahmoud

    2017-06-01

    Smoking may modify the appetite, and consequently affect nutrient intake and serum micronutrients. The effect of smoking on vitamin B12 status has been considered in several studies. The research proposed that organic nitrites, nitro oxide, cyanides, and isocyanides of cigarette smoke interfere with vitamin B12 metabolism, and convert it to inactive forms. This research was carried out to determine the serum level of active and inactive forms of vitamin B12 in male smokers in comparison with male nonsmokers. This is a case-control study, in which the participants were 85 male smokers and 85 male nonsmokers. The serum levels of total and active form of vitamin B12 were measured. Dietary intake was recorded by a quantitative food frequency questionnaire and one-day 24-hour dietary recall method. Independent two sample T test was used to compare quantitative variables between the case and control groups. The serum level of total vitamin B12 was not significantly different between two groups, but serum level of active form of vitamin B12 in the smoking group was significantly lower than non-smoking group (Pvitamin B12 in smokers in the Iranian community. The results of this study identified that serum level of total vitamin B12 might be not different between smoking and non-smoking people, but the function of this vitamin is disturbed in the body of smokers through the reduction of serum level of active form of vitamin B12.

  19. TRPC1 regulates calcium-activated chloride channels in salivary gland cells.

    Science.gov (United States)

    Sun, Yuyang; Birnbaumer, Lutz; Singh, Brij B

    2015-11-01

    Calcium-activated chloride channel (CaCC) plays an important role in modulating epithelial secretion. It has been suggested that in salivary tissues, sustained fluid secretion is dependent on Ca(2+) influx that activates ion channels such as CaCC to initiate Cl(-) efflux. However direct evidence as well as the molecular identity of the Ca(2+) channel responsible for activating CaCC in salivary tissues is not yet identified. Here we provide evidence that in human salivary cells, an outward rectifying Cl(-) current was activated by increasing [Ca(2+)]i, which was inhibited by the addition of pharmacological agents niflumic acid (NFA), an antagonist of CaCC, or T16Ainh-A01, a specific TMEM16a inhibitor. Addition of thapsigargin (Tg), that induces store-depletion and activates TRPC1-mediated Ca(2+) entry, potentiated the Cl(-) current, which was inhibited by the addition of a non-specific TRPC channel blocker SKF96365 or removal of external Ca(2+). Stimulation with Tg also increased plasma membrane expression of TMEM16a protein, which was also dependent on Ca(2+) entry. Importantly, in salivary cells, TRPC1 silencing, but not that of TRPC3, inhibited CaCC especially upon store depletion. Moreover, primary acinar cells isolated from submandibular gland also showed outward rectifying Cl(-) currents upon increasing [Ca(2+)]i. These Cl(-) currents were again potentiated with the addition of Tg, but inhibited in the presence of T16Ainh-A01. Finally, acinar cells isolated from the submandibular glands of TRPC1 knockout mice showed significant inhibition of the outward Cl(-) currents without decreasing TMEM16a expression. Together the data suggests that Ca(2+) entry via the TRPC1 channels is essential for the activation of CaCC.

  20. Mitochondrial ATP-sensitive potassium channel activity and hypoxic preconditioning are independent of an inwardly rectifying potassium channel subunit in Caenorhabditis elegans.

    Science.gov (United States)

    Wojtovich, Andrew P; DiStefano, Peter; Sherman, Teresa; Brookes, Paul S; Nehrke, Keith

    2012-02-17

    Hypoxic preconditioning (HP) is an evolutionarily-conserved mechanism that protects an organism against stress. The mitochondrial ATP-sensitive K(+) channel (mK(ATP)) plays an essential role in the protective signaling, but remains molecularly undefined. Several lines of evidence suggest that mK(ATP) may arise from an inward rectifying K(+) channel (Kir). The genetic model organism Caenorhabditis elegans exhibits HP and displays mK(ATP) activity. Here, we investigate the tissue expression profile of the three C. elegans Kir genes and demonstrate that mutant strains where the irk genes have been deleted either individually or in combination can be protected by HP and exhibit robust mK(ATP) channel activity in purified mitochondria. These data suggest that the mK(ATP) in C. elegans does not arise from a Kir derived channel.

  1. Mitochondrial ATP-sensitive potassium channel activity and hypoxic preconditioning are independent of an inwardly rectifying potassium channel subunit in C. elegans

    Science.gov (United States)

    Wojtovich, Andrew P.; DiStefano, Peter; Sherman, Teresa; Brookes, Paul S.; Nehrke, Keith

    2012-01-01

    Hypoxic preconditioning (HP) is an evolutionarily-conserved mechanism that protects an organism against stress. The mitochondrial ATP-sensitive K+ channel (mKATP) plays an essential role in the protective signaling, but remains molecularly undefined. Several lines of evidence suggest that mKATP may arise from an inward rectifying K+ channel (Kir). The genetic model organism C. elegans exhibits HP and displays mKATP activity. Here, we investigate the tissue expression profile of the three C. elegans Kir genes and demonstrate that mutant strains where the irk genes have been deleted either individually or in combination can be protected by HP and exhibit robust mKATP channel activity in purified mitochondria. These data suggest that the mKATP in C. elegans does not arise from a Kir derived channel. PMID:22281198

  2. Functional insights into modulation of BKCa channel activity to alter myometrial contractility

    Directory of Open Access Journals (Sweden)

    Ramón A Lorca

    2014-07-01

    Full Text Available The large-conductance voltage- and Ca2+-activated K+ channel (BKCa is an important regulator of membrane excitability in a wide variety of cells and tissues. In myometrial smooth muscle, activation of BKCa plays essential roles in buffering contractility to maintain uterine quiescence during pregnancy and in the transition to a more contractile state at the onset of labor. Multiple mechanisms of modulation have been described to alter BKCa channel activity, expression, and cellular localization. In the myometrium, BKCa is regulated by alternative splicing, protein targeting to the plasma membrane, compartmentation in membrane microdomains, and posttranslational modifications. In addition, interaction with auxiliary proteins (i.e., β1- and β2-subunits, association with G-protein coupled receptor signaling pathways, such as those activated by adrenergic and oxytocin receptors, and hormonal regulation provide further mechanisms of variable modulation of BKCa channel function in myometrial smooth muscle. Here, we provide an overview of these mechanisms of BKCa channel modulation and provide a context for them in relation to myometrial function.

  3. The immunophilin FKBP52 inhibits the activity of the epithelial Ca2+ channel TRPV5

    NARCIS (Netherlands)

    Gkika, D.; Topala, C.N.; Hoenderop, J.G.J.; Bindels, R.J.M.

    2006-01-01

    In the kidney, the epithelial Ca(2+) channel TRPV5 constitutes the apical entry pathway in the process of active Ca(2+) reabsorption. The regulation of Ca(2+) influx through TRPV5 is of crucial importance, because it determines the final amount of Ca(2+) excreted in the urine. The present study iden

  4. "Slow" Voltage-Dependent Inactivation of CaV2.2 Calcium Channels Is Modulated by the PKC Activator Phorbol 12-Myristate 13-Acetate (PMA.

    Directory of Open Access Journals (Sweden)

    Lei Zhu

    Full Text Available CaV2.2 (N-type voltage-gated calcium channels (Ca2+ channels play key roles in neurons and neuroendocrine cells including the control of cellular excitability, neurotransmitter / hormone secretion, and gene expression. Calcium entry is precisely controlled by channel gating properties including multiple forms of inactivation. "Fast" voltage-dependent inactivation is relatively well-characterized and occurs over the tens-to- hundreds of milliseconds timeframe. Superimposed on this is the molecularly distinct, but poorly understood process of "slow" voltage-dependent inactivation, which develops / recovers over seconds-to-minutes. Protein kinases can modulate "slow" inactivation of sodium channels, but little is known about if/how second messengers control "slow" inactivation of Ca2+ channels. We investigated this using recombinant CaV2.2 channels expressed in HEK293 cells and native CaV2 channels endogenously expressed in adrenal chromaffin cells. The PKC activator phorbol 12-myristate 13-acetate (PMA dramatically prolonged recovery from "slow" inactivation, but an inactive control (4α-PMA had no effect. This effect of PMA was prevented by calphostin C, which targets the C1-domain on PKC, but only partially reduced by inhibitors that target the catalytic domain of PKC. The subtype of the channel β-subunit altered the kinetics of inactivation but not the magnitude of slowing produced by PMA. Intracellular GDP-β-S reduced the effect of PMA suggesting a role for G proteins in modulating "slow" inactivation. We postulate that the kinetics of recovery from "slow" inactivation could provide a molecular memory of recent cellular activity and help control CaV2 channel availability, electrical excitability, and neurotransmission in the seconds-to-minutes timeframe.

  5. Increased anion channel activity is an unavoidable event in ozone-induced programmed cell death.

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    Takashi Kadono

    Full Text Available BACKGROUND: Ozone is a major secondary air pollutant often reaching high concentrations in urban areas under strong daylight, high temperature and stagnant high-pressure systems. Ozone in the troposphere is a pollutant that is harmful to the plant. PRINCIPAL FINDINGS: By exposing cells to a strong pulse of ozonized air, an acute cell death was observed in suspension cells of Arabidopsis thaliana used as a model. We demonstrated that O(3 treatment induced the activation of a plasma membrane anion channel that is an early prerequisite of O(3-induced cell death in A. thaliana. Our data further suggest interplay of anion channel activation with well known plant responses to O(3, Ca(2+ influx and NADPH-oxidase generated reactive oxygen species (ROS in mediating the oxidative cell death. This interplay might be fuelled by several mechanisms in addition to the direct ROS generation by O(3; namely, H(2O(2 generation by salicylic and abscisic acids. Anion channel activation was also shown to promote the accumulation of transcripts encoding vacuolar processing enzymes, a family of proteases previously reported to contribute to the disruption of vacuole integrity observed during programmed cell death. SIGNIFICANCE: Collectively, our data indicate that anion efflux is an early key component of morphological and biochemical events leading to O(3-induced programmed cell death. Because ion channels and more specifically anion channels assume a crucial position in cells, an understanding about the underlying role(s for ion channels in the signalling pathway leading to programmed cell death is a subject that warrants future investigation.

  6. Molecular Basis of Regulating High Voltage-Activated Calcium Channels by S-Nitrosylation.

    Science.gov (United States)

    Zhou, Meng-Hua; Bavencoffe, Alexis; Pan, Hui-Lin

    2015-12-18

    Nitric oxide (NO) is involved in a variety of physiological processes, such as vasoregulation and neurotransmission, and has a complex role in the regulation of pain transduction and synaptic transmission. We have shown previously that NO inhibits high voltage-activated Ca(2+) channels in primary sensory neurons and excitatory synaptic transmission in the spinal dorsal horn. However, the molecular mechanism involved in this inhibitory action remains unclear. In this study, we investigated the role of S-nitrosylation in the NO regulation of high voltage-activated Ca(2+) channels. The NO donor S-nitroso-N-acetyl-DL-penicillamine (SNAP) rapidly reduced N-type currents when Cav2.2 was coexpressed with the Cavβ1 or Cavβ3 subunits in HEK293 cells. In contrast, SNAP only slightly inhibited P/Q-type and L-type currents reconstituted with various Cavβ subunits. SNAP caused a depolarizing shift in voltage-dependent N-type channel activation, but it had no effect on Cav2.2 protein levels on the membrane surface. The inhibitory effect of SNAP on N-type currents was blocked by the sulfhydryl-specific modifying reagent methanethiosulfonate ethylammonium. Furthermore, the consensus motifs of S-nitrosylation were much more abundant in Cav2.2 than in Cav1.2 and Cav2.1. Site-directed mutagenesis studies showed that Cys-805, Cys-930, and Cys-1045 in the II-III intracellular loop, Cys-1835 and Cys-2145 in the C terminus of Cav2.2, and Cys-346 in the Cavβ3 subunit were nitrosylation sites mediating NO sensitivity of N-type channels. Our findings demonstrate that the consensus motifs of S-nitrosylation in cytoplasmically accessible sites are critically involved in post-translational regulation of N-type Ca(2+) channels by NO. S-Nitrosylation mediates the feedback regulation of N-type channels by NO.

  7. Reduction of Streptolysin O (SLO Pore-Forming Activity Enhances Inflammasome Activation

    Directory of Open Access Journals (Sweden)

    Peter A. Keyel

    2013-06-01

    Full Text Available Pore-forming toxins are utilized by bacterial and mammalian cells to exert pathogenic effects and induce cell lysis. In addition to rapid plasma membrane repair, macrophages respond to pore-forming toxins through activation of the NLRP3 inflammasome, leading to IL-1β secretion and pyroptosis. The structural determinants of pore-forming toxins required for NLRP3 activation remain unknown. Here, we demonstrate using streptolysin O (SLO that pore-formation controls IL-1β secretion and direct toxicity. An SLO mutant incapable of pore-formation did not promote direct killing, pyroptosis or IL-1β production. This indicated that pore formation is necessary for inflammasome activation. However, a partially active mutant (SLO N402C that was less toxic to macrophages than wild-type SLO, even at concentrations that directly lysed an equivalent number of red blood cells, enhanced IL-1β production but did not alter pyroptosis. This suggests that direct lysis may attenuate immune responses by preventing macrophages from successfully repairing their plasma membrane and elaborating more robust cytokine production. We suggest that mutagenesis of pore-forming toxins represents a strategy to enhance adjuvant activity.

  8. Sodium channel cleavage is associated with aberrant neuronal activity and cognitive deficits in a mouse model of Alzheimer's disease.

    Science.gov (United States)

    Corbett, Brian F; Leiser, Steven C; Ling, Huai-Ping; Nagy, Reka; Breysse, Nathalie; Zhang, Xiaohong; Hazra, Anupam; Brown, Jon T; Randall, Andrew D; Wood, Andrew; Pangalos, Menelas N; Reinhart, Peter H; Chin, Jeannie

    2013-04-17

    BACE1 is the rate-limiting enzyme that cleaves amyloid precursor protein (APP) to produce the amyloid β peptides that accumulate in Alzheimer's disease (AD). BACE1, which is elevated in AD patients and APP transgenic mice, also cleaves the β2-subunit of voltage-gated sodium channels (Navβ2). Although increased BACE1 levels are associated with Navβ2 cleavage in AD patients, whether Navβ2 cleavage occurs in APP mice had not yet been examined. Such a finding would be of interest because of its potential impact on neuronal activity: previous studies demonstrated that BACE1-overexpressing mice exhibit excessive cleavage of Navβ2 and reduced sodium current density, but the phenotype associated with loss of function mutations in either Navβ-subunits or pore-forming α-subunits is epilepsy. Because mounting evidence suggests that epileptiform activity may play an important role in the development of AD-related cognitive deficits, we examined whether enhanced cleavage of Navβ2 occurs in APP transgenic mice, and whether it is associated with aberrant neuronal activity and cognitive deficits. We found increased levels of BACE1 expression and Navβ2 cleavage fragments in cortical lysates from APP transgenic mice, as well as associated alterations in Nav1.1α expression and localization. Both pyramidal neurons and inhibitory interneurons exhibited evidence of increased Navβ2 cleavage. Moreover, the magnitude of alterations in sodium channel subunits was associated with aberrant EEG activity and impairments in the Morris water maze. Together, these results suggest that altered processing of voltage-gated sodium channels may contribute to aberrant neuronal activity and cognitive deficits in AD.

  9. Pharmacological investigation of the bioluminescence signaling pathway of the dinoflagellate Lingulodinium polyedrum: evidence for the role of stretch-activated ion channels.

    Science.gov (United States)

    Jin, Kelly; Klima, Jason C; Deane, Grant; Dale Stokes, Malcolm; Latz, Michael I

    2013-08-01

    Dinoflagellate bioluminescence serves as a whole-cell reporter of mechanical stress, which activates a signaling pathway that appears to involve the opening of voltage-sensitive ion channels and release of calcium from intracellular stores. However, little else is known about the initial signaling events that facilitate the transduction of mechanical stimuli. In the present study using the red tide dinoflagellate Lingulodinium polyedrum (Stein) Dodge, two forms of dinoflagellate bioluminescence, mechanically stimulated and spontaneous flashes, were used as reporter systems to pharmacological treatments that targeted various predicted signaling events at the plasma membrane level of the signaling pathway. Pretreatment with 200 μM Gadolinium III (Gd(3+) ), a nonspecific blocker of stretch-activated and some voltage-gated ion channels, resulted in strong inhibition of both forms of bioluminescence. Pretreatment with 50 μM nifedipine, an inhibitor of L-type voltage-gated Ca(2+) channels that inhibits mechanically stimulated bioluminescence, did not inhibit spontaneous bioluminescence. Treatment with 1 mM benzyl alcohol, a membrane fluidizer, was very effective in stimulating bioluminescence. Benzyl alcohol-stimulated bioluminescence was inhibited by Gd(3+) but not by nifedipine, suggesting that its role is through stretch activation via a change in plasma membrane fluidity. These results are consistent with the presence of stretch-activated and voltage-gated ion channels in the bioluminescence mechanotransduction signaling pathway, with spontaneous flashing associated with a stretch-activated component at the plasma membrane.

  10. Active star-forming galaxies in the X ray foreground

    Science.gov (United States)

    Griffiths, R. E.; Padovani, P.

    1989-01-01

    Star forming galaxies were discovered as a component of the X-ray background (XRB) in the Einstein deep surveys. Such star forming galaxies may be largely powered by superluminous Population 1 massive X-ray binaries (MXRB), formed in the wake of star formation in regions of low metallicity. The star forming galaxies with moderate numbers of MXRB may evolve into the infrared starburst galaxies found at low redshifts using IRAS (Infrared Astronomy Satellite), and may also be related to those galaxies identified with sub-mJy radio sources. A conservative contribution to the XRB of at least approximately 15 percent, without evolution is estimated. It is shown that moderate evolution leads to a contribution at least equalling that of quasars. Above 3 keV, star forming galaxies may dominate the XRB.

  11. Overexpression of the Large-Conductance, Ca2+-Activated K+ (BK) Channel Shortens Action Potential Duration in HL-1 Cardiomyocytes.

    Science.gov (United States)

    Stimers, Joseph R; Song, Li; Rusch, Nancy J; Rhee, Sung W

    2015-01-01

    Long QT syndrome is characterized by a prolongation of the interval between the Q wave and the T wave on the electrocardiogram. This abnormality reflects a prolongation of the ventricular action potential caused by a number of genetic mutations or a variety of drugs. Since effective treatments are unavailable, we explored the possibility of using cardiac expression of the large-conductance, Ca2+-activated K+ (BK) channel to shorten action potential duration (APD). We hypothesized that expression of the pore-forming α subunit of human BK channels (hBKα) in HL-1 cells would shorten action potential duration in this mouse atrial cell line. Expression of hBKα had minimal effects on expression levels of other ion channels with the exception of a small but significant reduction in Kv11.1. Patch-clamped hBKα expressing HL-1 cells exhibited an outward voltage- and Ca2+-sensitive K+ current, which was inhibited by the BK channel blocker iberiotoxin (100 nM). This BK current phenotype was not detected in untransfected HL-1 cells or in HL-1 null cells sham-transfected with an empty vector. Importantly, APD in hBKα-expressing HL-1 cells averaged 14.3 ± 2.8 ms (n = 10), which represented a 53% reduction in APD compared to HL-1 null cells lacking BKα expression. APD in the latter cells averaged 31.0 ± 5.1 ms (n = 13). The shortened APD in hBKα-expressing cells was restored to normal duration by 100 nM iberiotoxin, suggesting that a repolarizing K+ current attributed to BK channels accounted for action potential shortening. These findings provide initial proof-of-concept that the introduction of hBKα channels into a cardiac cell line can shorten APD, and raise the possibility that gene-based interventions to increase hBKα channels in cardiac cells may hold promise as a therapeutic strategy for long QT syndrome.

  12. Blockade of TRPM7 channel activity and cell death by inhibitors of 5-lipoxygenase.

    Directory of Open Access Journals (Sweden)

    Hsiang-Chin Chen

    Full Text Available TRPM7 is a ubiquitous divalent-selective ion channel with its own kinase domain. Recent studies have shown that suppression of TRPM7 protein expression by RNA interference increases resistance to ischemia-induced neuronal cell death in vivo and in vitro, making the channel a potentially attractive pharmacological target for molecular intervention. Here, we report the identification of the 5-lipoxygenase inhibitors, NDGA, AA861, and MK886, as potent blockers of the TRPM7 channel. Using a cell-based assay, application of these compounds prevented cell rounding caused by overexpression of TRPM7 in HEK-293 cells, whereas inhibitors of 12-lipoxygenase and 15-lipoxygenase did not prevent the change in cell morphology. Application of the 5-lipoxygenase inhibitors blocked heterologously expressed TRPM7 whole-cell currents without affecting the protein's expression level or its cell surface concentration. All three inhibitors were also effective in blocking the native TRPM7 current in HEK-293 cells. However, two other 5-lipoxygenase specific inhibitors, 5,6-dehydro-arachidonic acid and zileuton, were ineffective in suppressing TRPM7 channel activity. Targeted knockdown of 5-lipoxygenase did not reduce TRPM7 whole-cell currents. In addition, application of 5-hydroperoxyeicosatetraenoic acid (5-HPETE, the product of 5-lipoxygenase, or 5-HPETE's downstream metabolites, leukotriene B4 and leukotriene D4, did not stimulate TRPM7 channel activity. These data suggested that NDGA, AA861, and MK886 reduced the TRPM7 channel activity independent of their effect on 5-lipoxygenase activity. Application of AA861 and NDGA reduced cell death for cells overexpressing TRPM7 cultured in low extracellular divalent cations. Moreover, treatment of HEK-293 cells with AA861 increased cell resistance to apoptotic stimuli to a level similar to that obtained for cells in which TRPM7 was knocked down by RNA interference. In conclusion, NDGA, AA861, and MK886 are potent blockers of

  13. Contribution of KV7 Channels to Basal Coronary Flow and Active Response to Ischemia

    DEFF Research Database (Denmark)

    Khanamiri, Saereh; Soltysinska, Ewa; Jepps, Thomas A

    2013-01-01

    .5 activators produced relaxations, which were considerably less in arteries from hypertensive rats and were not mimicked by the KV7.1-specific activator R-L3. In isolated, perfused heart preparations, coronary flow rate increased in response to the KV7.2 to 7.5 activator (S)-1 and was diminished......The goal of the present study was to determine the role of KCNQ-encoded KV channels (KV7 channels) in the passive and active regulation of coronary flow in normotensive and hypertensive rats. In left anterior descending coronary arteries from normotensive rats, structurally different KV7.2 to 7....... Application of adenosine or A2A receptor agonist CGS-21680 produced concentration-dependent relaxations of coronary arteries from normotensive rats, which were attenuated by application of KV7 inhibitors. KV7 blockers also attenuated the ischemia-induced increase in coronary perfusion in Langendorff studies...

  14. Derivation of Hodgkin-Huxley equations for a Na+ channel from a master equation for coupled activation and inactivation

    Science.gov (United States)

    Vaccaro, S. R.

    2016-11-01

    The Na+ current in nerve and muscle membranes may be described in terms of the activation variable m (t ) and the inactivation variable h (t ) , which are dependent on the transitions of S4 sensors of each of the Na+ channel domains DI to DIV. The time-dependence of the Na+ current and the rate equations satisfied by m (t ) and h (t ) may be derived from the solution to a master equation that describes the coupling between two or three activation sensors regulating the Na+ channel conductance and a two-stage inactivation process. If the inactivation rate from the closed or open states increases as the S4 sensors activate, a more general form of the Hodgkin-Huxley expression for the open-state probability may be derived where m (t ) is dependent on both activation and inactivation processes. The voltage dependence of the rate functions for inactivation and recovery from inactivation are consistent with the empirically determined expressions and exhibit saturation for both depolarized and hyperpolarized clamp potentials.

  15. Derivation of Hodgkin-Huxley equations for a Na^{+} channel from a master equation for coupled activation and inactivation.

    Science.gov (United States)

    Vaccaro, S R

    2016-11-01

    The Na^{+} current in nerve and muscle membranes may be described in terms of the activation variable m(t) and the inactivation variable h(t), which are dependent on the transitions of S4 sensors of each of the Na^{+} channel domains DI to DIV. The time-dependence of the Na^{+} current and the rate equations satisfied by m(t) and h(t) may be derived from the solution to a master equation that describes the coupling between two or three activation sensors regulating the Na^{+} channel conductance and a two-stage inactivation process. If the inactivation rate from the closed or open states increases as the S4 sensors activate, a more general form of the Hodgkin-Huxley expression for the open-state probability may be derived where m(t) is dependent on both activation and inactivation processes. The voltage dependence of the rate functions for inactivation and recovery from inactivation are consistent with the empirically determined expressions and exhibit saturation for both depolarized and hyperpolarized clamp potentials.

  16. [Determination of the antioxidant properties of activators of mitochondrial ATP-dependent potassium channels with the Amplex Red fluorescent indicator].

    Science.gov (United States)

    Murzaeva, S V; Belova, S P; Mironova, G D

    2013-01-01

    The effect of adaptogens-antihypoxants that participate in the activation of mitochondrial ATP-dependent potassium channels (mitoK(ATP)) at the oxidation of the Amplex Red (AR) fluorescent indicator in a peroxidase system was tested. It was shown that Extralife, Hypoxen, taurine, and synthetic antioxidant ionol can be arranged in the following row, according to the fluorescence inhibition activity: Extralife > Hypoxen > > ionol > taurine; their effect was shown to be concentration-dependent. The calculated K(i) value of fluorescence indicators demonstrate fast and slow phases of inhibition of the AR oxidation by Extralife and Hypoxen. The fast phase occurs in the presence of microdoses (0.05-3 microg/mL) of adaptogens and is related to the competition for H2O2, which is in agreement with our previous data on the mitoK(ATP) activation by doses of adaptogens related to the H2O2 consumption. The slow phase is characteristic of high adaptogen and ionol concentrations and is related to the competition for phenoxyl radicals of resorufin formed during AR oxidation. The obtained results allow one to suggest the application of a highly sensitive model peroxidase system with AR for the preliminary testing of compounds activating mitoK(ATP) channels.

  17. [Biochemical mechanisms of the cardioprotective effect of the K(ATP) channels opener flocalin (medicinal form) in ischemia-reperfusion of myocardium].

    Science.gov (United States)

    Strutyns'kyĭ, R B; Kotsiuruba, A V; Rovenets', R A; Strutyns'ka, N A; Iagupols'kyĭ, Iu L; Sagach, V F; Moĭbenko, O O

    2013-01-01

    In experiments on the anaesthetized dogs with modeling of experimental ischemia (90 min) and reperfusion (180 min) of myocardium it was investigated changes of biochemical processes in arterial blood at intragastric introduction of medicinal form (tablets) of flocalin (the fluorine-containing opener of ATP-sensitive potassium channels) in a dose 2,2 mg/kg. The data analysis allowed to define a few possible mechanisms of cardioprotective action offlocalin, which prevented the opening of a mitochondrial permeability transition pore (MPTP) and inhibition of apoptosis induced by it. They consist, from one side, in activating of the constitutive de novo biosynthesis of nitric oxide by cNOS, from other side, in suppression of inducible nitric oxide de novo synthesis by iNOS in such way to prevent the formation of toxic peroxynitrite by co-operation of surplus nitric oxide with superoxide anion, thereby limits the generation of toxic active forms of nitrogen (*NO2) and oxygen (*OH). The first effect of flocalin takes place due to limitation the degradation of L-arginine by arginase which keeps substrat for cNOS, second--due to the inhibition of superoxide generation, in particular, by xanthine oxidase (marker uric acid), lipoxigenase (marker LTC4) and cyclooxygenase (marker TxB2). Because LTC4 have coronaroconstrictory, arrhythmogenic and chemoattractory properties in the conditions of myocardial ischemia, inhibition of its production both with superoxide generation (markers H2O2 and diene conjugates) may be the another mechanisms of flocalin's cardioprotection. Powerful antiischemic action of flocalin (marker nitrite anion) as the mechanisms of cardioprotection is possible as well as inhibition of ATP and GTP degradation (marker hypoxanthine+xanthine+inosine levels in the blood) and, possibly, stimulation ofhaem degradation by haem oxygenase (markers total bilirubin and Fe in the blood). Diminishing content of free arachidonic acid in arterial blood can testify

  18. Molecular mechanism for H(2)S-induced activation of K(ATP) channels.

    Science.gov (United States)

    Jiang, Bo; Tang, Guanghua; Cao, Kun; Wu, Lingyun; Wang, Rui

    2010-05-15

    Hydrogen sulfide (H(2)S) is an endogenous opener of K(ATP) channels in many different types of cells. However, the molecular mechanism for an interaction between H(2)S and K(ATP) channel proteins remains unclear. The whole-cell patch-clamp technique and mutagenesis approach were used to examine the effects of H(2)S on different K(ATP) channel subunits, rvKir6.1 and rvSUR1, heterologously expressed in HEK-293 cells. H(2)S stimulated coexpressed rvKir6.1/rvSUR1 K(ATP) channels, but had no effect on K(ATP) currents generated by rvKir6.1 expression alone. Intracellularly applied sulfhydryl alkylating agent (N-ethylmaleimide, NEM), oxidizing agent (chloramine T, CLT), and a disulfide bond-oxidizing enzyme (protein disulfide isomerase) did not alter H(2)S effects on this recombinant channels. CLT, but not NEM, inhibited basal rvKir6.1/rvSUR1 currents, and both abolished the stimulatory effects of H(2)S on K(ATP) currents, when applied extracellularly. After selective cysteine residues (C6S and C26S but not C1051S and C1057S) in the extracellular loop of rvSUR1 subunits were point-mutated, H(2)S lost its stimulatory effects on rvKir6.1/rvSUR1 currents. Our results demonstrate that H(2)S interacts with Cys6 and Cys26 residues of the extracellular N terminal of rvSUR1 subunit of K(ATP) channel complex. Direct chemical modification of rvSUR1 subunit protein constitutes a molecular mechanism for the activation of K(ATP) channels by H(2)S.

  19. Relationship between Customer Perception about CSR activities and Purchase Intention: The Role of CSR Communication Channels

    OpenAIRE

    SONG, JINWEN; Fang, Qi; Wang, Jieru

    2015-01-01

    With the incorporation of businesses as major players in a country’s economy and society, Corporate Social Responsibility is becoming a very important aspect of corporate activity. This field is greatly understudied and only limited research has been done on the consequences of conducting CSR activities through different channels of communication. This thesis therefore contributes to a better understanding of the relationship between customer perception and purchase intention in the setting o...

  20. Taurine activates delayed rectifier KV channels via a metabotropic pathway in retinal neurons

    Science.gov (United States)

    Bulley, Simon; Liu, Yufei; Ripps, Harris; Shen, Wen

    2013-01-01

    Taurine is one of the most abundant amino acids in the retina, throughout the CNS, and in heart and muscle cells. In keeping with its broad tissue distribution, taurine serves as a modulator of numerous basic processes, such as enzyme activity, cell development, myocardial function and cytoprotection. Despite this multitude of functional roles, the precise mechanism underlying taurine's actions has not yet been identified. In this study we report findings that indicate a novel role for taurine in the regulation of voltage-gated delayed rectifier potassium (KV) channels in retinal neurons by means of a metabotropic receptor pathway. The metabotropic taurine response was insensitive to the Cl− channel blockers, picrotoxin and strychnine, but it was inhibited by a specific serotonin 5-HT2A receptor antagonist, MDL11939. Moreover, we found that taurine enhanced KV channels via intracellular protein kinase C-mediated pathways. When 5-HT2A receptors were expressed in human embryonic kidney cells, taurine and AL34662, a non-specific 5-HT2 receptor activator, produced a similar regulation of KIR channels. In sum, this study provides new evidence that taurine activates a serotonin system, apparently via 5-HT2A receptors and related intracellular pathways. PMID:23045337

  1. Role of the S4-S5 linker in CNG channel activation.

    Science.gov (United States)

    Kusch, Jana; Zimmer, Thomas; Holschuh, Jascha; Biskup, Christoph; Schulz, Eckhard; Nache, Vasilica; Benndorf, Klaus

    2010-10-20

    Cyclic nucleotide-gated (CNG) channels mediate sensory signal transduction in retinal and olfactory cells. The channels are activated by the binding of cyclic nucleotides to a cyclic nucleotide-binding domain (CNBD) in the C-terminus that is located at the intracellular side. The molecular events translating the ligand binding to the pore opening are still unknown. We investigated the role of the S4-S5 linker in the activation process by quantifying its interaction with other intracellular regions. To this end, we constructed chimeric channels in which the N-terminus, the S4-S5 linker, the C-linker, and the CNBD of the retinal CNGA1 subunit were systematically replaced by the respective regions of the olfactory CNGA2 subunit. Macroscopic concentration-response relations were analyzed, yielding the apparent affinity to cGMP and the Hill coefficient. The degree of functional coupling of intracellular regions in the activation gating was determined by thermodynamic double-mutant cycle analysis. We observed that all four intracellular regions, including the relatively short S4-S5 linker, are involved in controlling the apparent affinity of the channel to cGMP and, moreover, in determining the degree of cooperativity between the subunits, as derived from the Hill coefficient. The interaction energies reveal an interaction of the S4-S5 linker with both the N-terminus and the C-linker, but no interaction with the CNBD.

  2. Shikonin inhibits intestinal calcium-activated chloride channels and prevents rotaviral diarrhea

    Directory of Open Access Journals (Sweden)

    Yu Jiang

    2016-08-01

    Full Text Available Secretory diarrhea remains a global health burden and causes major mortality in children. There have been some focuses on antidiarrheal therapies that may reduce fluid losses and intestinal motility in diarrheal diseases. In the present study, we identified shikonin as an inhibitor of TMEM16A chloride channel activity using cell-based fluorescent-quenching assay. The IC50 value of shikonin was 6.5 μM. Short-circuit current measurements demonstrated that shikonin inhibited Eact-induced Cl current in a dose-dependent manner, with IC50 value of 1.5 μM. Short-circuit current measurement showed that shikonin exhibited inhibitory effect against CCh-induced Cl currents in mouse colonic epithelia but did not affect cytoplasmic Ca2+ concentration as well as the other major enterocyte chloride channel CFTR. Characterization study found that shikonin inhibited basolateral K+ channel activity without affecting Na+/K+-ATPase activities. In-vivo studies revealed that shikonin significantly delayed intestinal motility in mice and reduced stool water content in a neonatal mice model of rotaviral diarrhea without affecting the viral infection process in-vivo. Taken together, the results suggested that shikonin inhibited enterocyte CaCCs, the inhibitory effect was partially through inhbition of basolateral K+ channel acitivty, and shikonin could be a lead compound in the treatment of rotaviral secretory diarrhea.

  3. A multichannel integrated circuit for electrical recording of neural activity, with independent channel programmability.

    Science.gov (United States)

    Mora Lopez, Carolina; Prodanov, Dimiter; Braeken, Dries; Gligorijevic, Ivan; Eberle, Wolfgang; Bartic, Carmen; Puers, Robert; Gielen, Georges

    2012-04-01

    Since a few decades, micro-fabricated neural probes are being used, together with microelectronic interfaces, to get more insight in the activity of neuronal networks. The need for higher temporal and spatial recording resolutions imposes new challenges on the design of integrated neural interfaces with respect to power consumption, data handling and versatility. In this paper, we present an integrated acquisition system for in vitro and in vivo recording of neural activity. The ASIC consists of 16 low-noise, fully-differential input channels with independent programmability of its amplification (from 100 to 6000 V/V) and filtering (1-6000 Hz range) capabilities. Each channel is AC-coupled and implements a fourth-order band-pass filter in order to steeply attenuate out-of-band noise and DC input offsets. The system achieves an input-referred noise density of 37 nV/√Hz, a NEF of 5.1, a CMRR > 60 dB, a THD < 1% and a sampling rate of 30 kS/s per channel, while consuming a maximum of 70 μA per channel from a single 3.3 V. The ASIC was implemented in a 0.35 μm CMOS technology and has a total area of 5.6 × 4.5 mm². The recording system was successfully validated in in vitro and in vivo experiments, achieving simultaneous multichannel recordings of cell activity with satisfactory signal-to-noise ratios.

  4. TMEM16 proteins: the long awaited calcium-activated chloride channels?

    Directory of Open Access Journals (Sweden)

    C.A. Flores

    2009-11-01

    Full Text Available Currents mediated by calcium-activated chloride channels (CaCCs, observed for the first time in Xenopus oocytes, have been recorded in many cells and tissues ranging from different types of neurons to epithelial and muscle cells. CaCCs play a role in the regulation of excitability in neurons including sensory receptors. In addition, they are crucial mediators of chloride movements in epithelial cells where their activity regulates electrolyte and fluid transport. The roles of CaCCs, particularly in epithelia, are briefly reviewed with emphasis on their function in secretory epithelia. The recent identification by three independent groups, using different strategies, of TMEM16A as the molecular counterpart of the CaCC is discussed. TMEM16A is part of a family that has 10 other members in mice. The discovery of the potential TMEM16 anion channel activity opens the way for the molecular investigation of the role of these anion channels in specific cells and in organ physiology and pathophysiology. The identification of TMEM16A protein as a CaCC chloride channel molecule represents a great triumph of scientific perseverance and ingenuity. The varied approaches used by the three independent research groups also augur well for the solidity of the discovery.

  5. Molecular mechanism underlying β1 regulation in voltage- and calcium-activated potassium (BK) channels.

    Science.gov (United States)

    Castillo, Karen; Contreras, Gustavo F; Pupo, Amaury; Torres, Yolima P; Neely, Alan; González, Carlos; Latorre, Ramon

    2015-04-14

    Being activated by depolarizing voltages and increases in cytoplasmic Ca(2+), voltage- and calcium-activated potassium (BK) channels and their modulatory β-subunits are able to dampen or stop excitatory stimuli in a wide range of cellular types, including both neuronal and nonneuronal tissues. Minimal alterations in BK channel function may contribute to the pathophysiology of several diseases, including hypertension, asthma, cancer, epilepsy, and diabetes. Several gating processes, allosterically coupled to each other, control BK channel activity and are potential targets for regulation by auxiliary β-subunits that are expressed together with the α (BK)-subunit in almost every tissue type where they are found. By measuring gating currents in BK channels coexpressed with chimeras between β1 and β3 or β2 auxiliary subunits, we were able to identify that the cytoplasmic regions of β1 are responsible for the modulation of the voltage sensors. In addition, we narrowed down the structural determinants to the N terminus of β1, which contains two lysine residues (i.e., K3 and K4), which upon substitution virtually abolished the effects of β1 on charge movement. The mechanism by which K3 and K4 stabilize the voltage sensor is not electrostatic but specific, and the α (BK)-residues involved remain to be identified. This is the first report, to our knowledge, where the regulatory effects of the β1-subunit have been clearly assigned to a particular segment, with two pivotal amino acids being responsible for this modulation.

  6. Calmodulin and calcium interplay in the modulation of TRPC5 channel activity. Identification of a novel C-terminal domain for calcium/calmodulin-mediated facilitation.

    Science.gov (United States)

    Ordaz, Benito; Tang, Jisen; Xiao, Rui; Salgado, Alfonso; Sampieri, Alicia; Zhu, Michael X; Vaca, Luis

    2005-09-02

    TRPC5 forms Ca2+-permeable nonselective cation channels important for neurite outgrowth and growth cone morphology of hippocampal neurons. Here we studied the activation of mouse TRPC5 expressed in Chinese hamster ovary and human embryonic kidney 293 cells by agonist stimulation of several receptors that couple to the phosphoinositide signaling cascade and the role of calmodulin (CaM) on the activation. We showed that exogenous application of 10 microM CaM through patch pipette accelerated the agonist-induced channel activation by 2.8-fold, with the time constant for half-activation reduced from 4.25 +/- 0.4 to 1.56 +/- 0.85 min. We identified a novel CaM-binding site located at the C terminus of TRPC5, 95 amino acids downstream from the previously determined common CaM/IP3R-binding (CIRB) domain for all TRPC proteins. Deletion of the novel CaM-binding site attenuated the acceleration in channel activation induced by CaM. However, disruption of the CIRB domain from TRPC5 rendered the channel irresponsive to agonist stimulation without affecting the cell surface expression of the channel protein. Furthermore, we showed that high (>5 microM) intracellular free Ca2+ inhibited the current density without affecting the time course of TRPC5 activation by receptor agonists. These results demonstrated that intracellular Ca2+ has dual and opposite effects on the activation of TRPC5. The novel CaM-binding site is important for the Ca2+/CaM-mediated facilitation, whereas the CIRB domain is critical for the overall response of receptor-induced TRPC5 channel activation.

  7. Robust Stimulation of W1282X-CFTR Channel Activity by a Combination of Allosteric Modulators.

    Directory of Open Access Journals (Sweden)

    Wei Wang

    Full Text Available W1282X is a common nonsense mutation among cystic fibrosis patients that results in the production of a truncated Cystic Fibrosis Transmembrane Conductance Regulator (CFTR channel. Here we show that the channel activity of the W1282X-CFTR polypeptide is exceptionally low in excised membrane patches at normally saturating doses of ATP and PKA (single channel open probability (PO 0.9 when treated with both modulators. VX-770 and curcumin also additively stimulated W1282X-CFTR mediated currents in polarized FRT epithelial monolayers. In this setting, however, the stimulated W1282X-CFTR currents were smaller than those mediated by wild type CFTR (3-5% due presumably to lower expression levels or cell surface targeting of the truncated protein. Combining allosteric modulators of different mechanistic classes is worth considering as a treatment option for W1282X CF patients perhaps when coupled with maneuvers to increase expression of the truncated protein.

  8. A conserved threonine in the S1-S2 loop of KV7.2 and K V7.3 channels regulates voltage-dependent activation.

    Science.gov (United States)

    Füll, Yvonne; Seebohm, Guiscard; Lerche, Holger; Maljevic, Snezana

    2013-06-01

    The voltage-gated potassium channels KV7.2 and KV7.3 (KCNQ2/3 genes) play an important role in regulating neuronal excitability. More than 50 KCNQ2/3 mutations have been identified to cause an inherited form of epilepsy in newborns. For two of those (E119G and S122L) found in the S1-S2 region of KV7.2, we previously showed a decreased channel availability mainly at action potential subthreshold voltages caused by a slight depolarizing shift of the activation curve. Interestingly, recent studies revealed that a threonine residue within the S1-S2 loop, highly conserved among different classes of KV channels, is crucial for both their function and surface expression. To investigate the functional role of the homologous threonine residues in KV7.2 (T114) and KV7.3 (T144) channels, we replaced them with alanine and examined the electrophysiological properties using heterologous expression in CHO cells and whole cell patch clamping. Channels comprising mutant subunits yielded decreased potassium currents with slowed activation and accelerated deactivation kinetics. However, the most striking effect was a depolarizing shift in the voltage dependence of activation reaching +30 mV upon co-expression of both mutant subunits. Potential interactions of T114 within the channel were analyzed by creating a 3D homology model of KV7.2 in an open state suggesting that this residue plays a central role in the formation of a stable interface between the S1-S2 and the S5 segment helices. This could be the explanation why substitution of the conserved threonine in KV7.2 and KV7.3 channels destabilizes the open and favors the closed state of these channels.

  9. Antischistosomal activity of a calcium channel antagonist on schistosomula and adult Schistosoma mansoni worms

    Directory of Open Access Journals (Sweden)

    Vanessa Silva-Moraes

    2013-08-01

    Full Text Available Current schistosomiasis control strategies are largely based on chemotherapeutic agents and a limited number of drugs are available today. Praziquantel (PZQ is the only drug currently used in schistosomiasis control programs. Unfortunately, this drug shows poor efficacy in patients during the earliest infection phases. The effects of PZQ appear to operate on the voltage-operated Ca2+channels, which are located on the external Schistosoma mansoni membrane. Because some Ca2+channels have dihydropyridine drug class (a class that includes nifedipine sensitivity, an in vitro analysis using a calcium channel antagonist (clinically used for cardiovascular hypertension was performed to determine the antischistosomal effects of nifedipine on schistosomula and adult worm cultures. Nifedipine demonstrated antischistosomal activity against schistosomula and significantly reduced viability at all of the concentrations used alone or in combination with PZQ. In contrast, PZQ did not show significant efficacy when used alone. Adult worms were also affected by nifedipine after a 24 h incubation and exhibited impaired motility, several lesions on the tegument and intense contractility. These data support the idea of Ca2+channels subunits as drug targets and favour alternative therapeutic schemes when drug resistance has been reported. In this paper, strong arguments encouraging drug research are presented, with a focus on exploring schistosomal Ca2+channels.

  10. Roscovitine binds to novel L-channel (CaV1.2) sites that separately affect activation and inactivation.

    Science.gov (United States)

    Yarotskyy, Viktor; Gao, Guofeng; Du, Lei; Ganapathi, Sindura B; Peterson, Blaise Z; Elmslie, Keith S

    2010-01-01

    L-type (Ca(V)1.2) calcium channel antagonists play an important role in the treatment of cardiovascular disease. (R)-Roscovitine, a trisubstituted purine, has been shown to inhibit L-currents by slowing activation and enhancing inactivation. This study utilized molecular and pharmacological approaches to determine whether these effects result from (R)-roscovitine binding to a single site. Using the S enantiomer, we find that (S)-roscovitine enhances inactivation without affecting activation, which suggests multiple sites. This was further supported in studies using chimeric channels comprised of N- and L-channel domains. Those chimeras containing L-channel domains I and IV showed (R)-roscovitine-induced slowed activation like that of wild type L-channels, whereas chimeric channels containing L-channel domain I responded to (R)-roscovitine with enhanced inactivation. We conclude that (R)-roscovitine binds to distinct sites on L-type channels to slow activation and enhance inactivation. These sites appear to be unique from other calcium channel antagonist sites that reside within domains III and IV and are thus novel sites that could be exploited for future drug development. Trisubstituted purines could become a new class of drugs for the treatment of diseases related to hyperfunction of L-type channels, such as Torsades de Pointes.

  11. Phospholipase C not protein kinase C is required for the activation of TRPC5 channels by cholecystokinin.

    Science.gov (United States)

    Grisanti, Laurel A; Kurada, Lalitha; Cilz, Nicholas I; Porter, James E; Lei, Saobo

    2012-08-15

    Cholecystokinin (CCK) is one of the most abundant neuropeptides in the brain where it interacts with two G protein-coupled receptors (CCK1 and CCK2). Both types of CCK receptors are coupled to G(q/11) proteins resulting in increased function of phospholipase C (PLC) pathway. Whereas CCK has been suggested to increase neuronal excitability in the brain via activation of cationic channels, the types of cationic channels have not yet been identified. Here, we co-expressed CCK2 receptors and TRPC5 channels in human embryonic kidney (HEK) 293 cells and studied the effects of CCK on TRPC5 channels using patch-clamp techniques. Our results demonstrate that activation of CCK2 receptors robustly potentiates the function of TRPC5 channels. CCK-induced activation of TRPC5 channels requires the functions of G-proteins and PLC and depends on extracellular Ca(2+). The activation of TRPC5 channels mediated by CCK2 receptors is independent of IP(3) receptors and protein kinase C. CCK-induced opening of TRPC5 channels is not store-operated because application of thapsigargin to deplete intracellular Ca(2+) stores failed to alter CCK-induced TRPC5 channel currents significantly. Bath application of CCK also significantly increased the open probability of TRPC5 single channel currents in cell-attached patches. Because CCK exerts extensive effects in the brain, our results may provide a novel mechanism to explain its roles in modulating neuronal excitability.

  12. Activating mutations of the TRPML1 channel revealed by proline-scanning mutagenesis.

    Science.gov (United States)

    Dong, Xian-ping; Wang, Xiang; Shen, Dongbiao; Chen, Su; Liu, Meiling; Wang, Yanbin; Mills, Eric; Cheng, Xiping; Delling, Markus; Xu, Haoxing

    2009-11-13

    The mucolipin TRP (TRPML) proteins are a family of endolysosomal cation channels with genetically established importance in humans and rodent. Mutations of human TRPML1 cause type IV mucolipidosis, a devastating pediatric neurodegenerative disease. Our recent electrophysiological studies revealed that, although a TRPML1-mediated current can only be recorded in late endosome and lysosome (LEL) using the lysosome patch clamp technique, a proline substitution in TRPML1 (TRPML1(V432P)) results in a large whole cell current. Thus, it remains unknown whether the large TRPML1(V432P)-mediated current results from an increased surface expression (trafficking), elevated channel activity (gating), or both. Here we performed systemic Pro substitutions in a region previously implicated in the gating of various 6 transmembrane cation channels. We found that several Pro substitutions displayed gain-of-function (GOF) constitutive activities at both the plasma membrane (PM) and endolysosomal membranes. Although wild-type TRPML1 and non-GOF Pro substitutions localized exclusively in LEL and were barely detectable in the PM, the GOF mutations with high constitutive activities were not restricted to LEL compartments, and most significantly, exhibited significant surface expression. Because lysosomal exocytosis is Ca(2+)-dependent, constitutive Ca(2+) permeability due to Pro substitutions may have resulted in stimulus-independent intralysosomal Ca(2+) release, hence the surface expression and whole cell current of TRPML1. Indeed, surface staining of lysosome-associated membrane protein-1 (Lamp-1) was dramatically increased in cells expressing GOF TRPML1 channels. We conclude that TRPML1 is an inwardly rectifying, proton-impermeable, Ca(2+) and Fe(2+)/Mn(2+) dually permeable cation channel that may be gated by unidentified cellular mechanisms through a conformational change in the cytoplasmic face of the transmembrane 5 (TM5). Furthermore, activation of TRPML1 in LEL may lead to the

  13. Ion Channel Blockers as Antimicrobial Agents, Efflux Inhibitors, and Enhancers of Macrophage Killing Activity against Drug Resistant Mycobacterium tuberculosis.

    Directory of Open Access Journals (Sweden)

    Diana Machado

    irrespective of their resistance pattern. This work highlights the potential value ion channel blockers as adjuvants of tuberculosis chemotherapy, in particular for the development of new therapeutic strategies, with strong potential for treatment shortening against drug susceptible and resistant forms of tuberculosis. Medicinal chemistry studies are now needed to improve the properties of these compounds, increasing their M. tuberculosis efflux-inhibition and killing-enhancement activity and reduce their toxicity for humans, therefore optimizing their potential for clinical usage.

  14. 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...... and morphometric analysis on the intact tissue. BK(Ca) channels appeared to be localized along all the plasma membrane of the enterocytes; the apical part of the villi showed the most intense immunostaining. These channels were silent in basal condition, but were activated on both membranes (apical and basolateral......) by increasing intracellular Ca(2+) concentration with the Ca(2+) ionophore ionomycin (1 microM). BK(Ca) channels were also activated on both membranes by hypotonic swelling of the epithelium and their inhibition by 100 nM iberiotoxin (specific BK(Ca) inhibitor) abolished the Regulatory Volume Decrease (RVD...

  15. Influenza matrix protein 2 alters CFTR expression and function through its ion channel activity.

    Science.gov (United States)

    Londino, James D; Lazrak, Ahmed; Jurkuvenaite, Asta; Collawn, James F; Noah, James W; Matalon, Sadis

    2013-05-01

    The human cystic fibrosis transmembrane conductance regulator (CFTR) is a cyclic AMP-activated chloride (Cl(-)) channel in the lung epithelium that helps regulate the thickness and composition of the lung epithelial lining fluid. We investigated whether influenza M2 protein, a pH-activated proton (H(+)) channel that traffics to the plasma membrane of infected cells, altered CFTR expression and function. M2 decreased CFTR activity in 1) Xenopus oocytes injected with human CFTR, 2) epithelial cells (HEK-293) stably transfected with CFTR, and 3) human bronchial epithelial cells (16HBE14o-) expressing native CFTR. This inhibition was partially reversed by an inhibitor of the ubiquitin-activating enzyme E1. Next we investigated whether the M2 inhibition of CFTR activity was due to an increase of secretory organelle pH by M2. Incubation of Xenopus oocytes expressing CFTR with ammonium chloride or concanamycin A, two agents that alkalinize the secretory pathway, inhibited CFTR activity in a dose-dependent manner. Treatment of M2- and CFTR-expressing oocytes with the M2 ion channel inhibitor amantadine prevented the loss in CFTR expression and activity; in addition, M2 mutants, lacking the ability to transport H(+), did not alter CFTR activity in Xenopus oocytes and HEK cells. Expression of an M2 mutant retained in the endoplasmic reticulum also failed to alter CFTR activity. In summary, our data show that M2 decreases CFTR activity by increasing secretory organelle pH, which targets CFTR for destruction by the ubiquitin system. Alteration of CFTR activity has important consequences for fluid regulation and may potentially modify the immune response to viral infection.

  16. Synergistic antiarrhythmic effect of combining inhibition of Ca(2+)-activated K(+) (SK) channels and voltage-gated Na(+) channels in an isolated heart model of atrial fibrillation

    DEFF Research Database (Denmark)

    Kirchhoff, Jeppe Egedal; Goldin Diness, Jonas; Sheykhzade, Majid

    2015-01-01

    be subefficacious as monotherapy, may prevent atrial fibrillation (AF) and have reduced proarrhythmic potential in the ventricles. METHODS: Subefficacious concentrations of ranolazine, flecainide, and lidocaine were tested alone or in combination with the SK channel blocker N-(pyridin-2-yl)-4-(pyridin-2-yl...... of the adverse effect profile could be an additional advantage if compound concentrations could be reduced. OBJECTIVE: The purpose of this study was to test the hypothesis that combined inhibition of Ca(2+)-activated K(+) channels (SK channels) and voltage-gated Na(+) channels, in concentrations that would......)thiazol-2-amine (ICA) in a Langendorff-perfused guinea pig heart model in which AF was induced after acetylcholine application and burst pacing. RESULTS: AF duration was reduced when both flecainide and ranolazine were combined with ICA in doses that did not reduce AF as monotherapy. At higher...

  17. Insulin Excites Anorexigenic Proopiomelanocortin Neurons via Activation of Canonical Transient Receptor Potential Channels

    Science.gov (United States)

    Qiu, Jian; Zhang, Chunguang; Borgquist, Amanda; Nestor, Casey C; Smith, Arik W.; Bosch, Martha A.; Ku, Stephen; Wagner, Edward J.; Rønnekleiv, Oline K.; Kelly, Martin J.

    2014-01-01

    SUMMARY Proopiomelanocortin (POMC) neurons within the hypothalamic arcuate nucleus are vital anorexigenic neurons. Although both the leptin receptor and insulin receptor are coupled to activation of phosphatidylinositide3-kinase (PI3K) in POMC neurons, they are thought to have disparate actions on POMC excitability. Using whole-cell recording and selective pharmacological tools, we have found that similar to leptin, purified insulin depolarized POMC, and adjacent kisspeptin neurons via activation of TRPC5 channels, which are highly expressed in these neurons. In contrast, insulin hyperpolarized and inhibited NPY/AgRP neurons via activation of KATP channels. Moreover, Zn2+, which is found in insulin formulations at nanomolar concentrations, inhibited POMC neurons via activation of KATP channels. Finally as predicted, insulin given intracerebroventrically robustly inhibited food intake and activated c-fos expression in arcuate POMC neurons. Our results show that purified insulin excites POMC neurons in the arcuate nucleus, which we propose is a major mechanism by which insulin regulates energy homeostasis. PMID:24703699

  18. Mapping ECoG channel contributions to trajectory and muscle activity prediction in human sensorimotor cortex

    Science.gov (United States)

    Nakanishi, Yasuhiko; Yanagisawa, Takufumi; Shin, Duk; Kambara, Hiroyuki; Yoshimura, Natsue; Tanaka, Masataka; Fukuma, Ryohei; Kishima, Haruhiko; Hirata, Masayuki; Koike, Yasuharu

    2017-01-01

    Studies on brain-machine interface techniques have shown that electrocorticography (ECoG) is an effective modality for predicting limb trajectories and muscle activity in humans. Motor control studies have also identified distributions of “extrinsic-like” and “intrinsic-like” neurons in the premotor (PM) and primary motor (M1) cortices. Here, we investigated whether trajectories and muscle activity predicted from ECoG were obtained based on signals derived from extrinsic-like or intrinsic-like neurons. Three participants carried objects of three different masses along the same counterclockwise path on a table. Trajectories of the object and upper arm muscle activity were predicted using a sparse linear regression. Weight matrices for the predictors were then compared to determine if the ECoG channels contributed more information about trajectory or muscle activity. We found that channels over both PM and M1 contributed highly to trajectory prediction, while a channel over M1 was the highest contributor for muscle activity prediction. PMID:28361947

  19. Biophysical characterization of KV3.1 potassium channel activating compounds

    DEFF Research Database (Denmark)

    Taskin, Bahar; von Schoubye, Nadia Lybøl; Sheykhzade, Majid

    2015-01-01

    The effect of two positive modulators, RE1 and EX15, on the voltage-gated K+ channel Kv3.1 was investigated using the whole-cell patch-clamp technique on HEK293 cells expressing Kv3.1a. RE1 and EX15 increased the Kv3.1 currents in a concentration-dependent manner with an EC50 value of 4.5 and 1.3...... the first detailed biophysical characterization of two new Kv3.1 channel modifying compounds with different modulating properties.......The effect of two positive modulators, RE1 and EX15, on the voltage-gated K+ channel Kv3.1 was investigated using the whole-cell patch-clamp technique on HEK293 cells expressing Kv3.1a. RE1 and EX15 increased the Kv3.1 currents in a concentration-dependent manner with an EC50 value of 4.5 and 1.3µ......M, respectively. However, high compound concentrations caused an inhibition of the Kv3.1 current. The compound-induced activation of Kv3.1 channels showed a profound hyperpolarized shift in activation kinetics. 30µM RE1 shifted V½ from 5.63±0.31mV to -9.71±1.00mV and 10µM EX15 induced a shift from 10.77±0.32m...

  20. Rate-dependent activation failure in isolated cardiac cells and tissue due to Na+ channel block.

    Science.gov (United States)

    Varghese, Anthony; Spindler, Anthony J; Paterson, David; Noble, Denis

    2015-11-15

    While it is well established that class-I antiarrhythmics block cardiac sodium channels, the mechanism of action of therapeutic levels of these drugs is not well understood. Using a combination of mathematical modeling and in vitro experiments, we studied the failure of activation of action potentials in single ventricular cells and in tissue caused by Na(+) channel block. Our computations of block and unblock of sodium channels by a theoretical class-Ib antiarrhythmic agent predict differences in the concentrations required to cause activation failure in single cells as opposed to multicellular preparations. We tested and confirmed these in silico predictions with in vitro experiments on isolated guinea-pig ventricular cells and papillary muscles stimulated at various rates (2-6.67 Hz) and exposed to various concentrations (5 × 10(-6) to 500 × 10(-6) mol/l) of lidocaine. The most salient result was that whereas large doses (5 × 10(-4) mol/l or higher) of lidocaine were required to inhibit action potentials temporarily in single cells, much lower doses (5 × 10(-6) mol/l), i.e., therapeutic levels, were sufficient to have the same effect in papillary muscles: a hundredfold difference. Our experimental results and mathematical analysis indicate that the syncytial nature of cardiac tissue explains the effects of clinically relevant doses of Na(+) channel blockers.

  1. KV1 channels identified in rodent myelinated axons, linked to Cx29 in innermost myelin: support for electrically active myelin in mammalian saltatory conduction.

    Science.gov (United States)

    Rash, John E; Vanderpool, Kimberly G; Yasumura, Thomas; Hickman, Jordan; Beatty, Jonathan T; Nagy, James I

    2016-04-01

    Saltatory conduction in mammalian myelinated axons was thought to be well understood before recent discoveries revealed unexpected subcellular distributions and molecular identities of the K(+)-conductance pathways that provide for rapid axonal repolarization. In this study, we visualize, identify, localize, quantify, and ultrastructurally characterize axonal KV1.1/KV1.2 channels in sciatic nerves of rodents. With the use of light microscopic immunocytochemistry and freeze-fracture replica immunogold labeling electron microscopy, KV1.1/KV1.2 channels are localized to three anatomically and compositionally distinct domains in the internodal axolemmas of large myelinated axons, where they form densely packed "rosettes" of 9-nm intramembrane particles. These axolemmal KV1.1/KV1.2 rosettes are precisely aligned with and ultrastructurally coupled to connexin29 (Cx29) channels, also in matching rosettes, in the surrounding juxtaparanodal myelin collars and along the inner mesaxon. As >98% of transmembrane proteins large enough to represent ion channels in these specialized domains, ∼500,000 KV1.1/KV1.2 channels define the paired juxtaparanodal regions as exclusive membrane domains for the voltage-gated K(+)conductance that underlies rapid axonal repolarization in mammals. The 1:1 molecular linkage of KV1 channels to Cx29 channels in the apposed juxtaparanodal collars, plus their linkage to an additional 250,000-400,000 Cx29 channels along each inner mesaxon in every large-diameter myelinated axon examined, supports previously proposed K(+)conductance directly from juxtaparanodal axoplasm into juxtaparanodal myeloplasm in mammalian axons. With neither Cx29 protein nor myelin rosettes detectable in frog myelinated axons, these data showing axon-to-myelin linkage by abundant KV1/Cx29 channels in rodent axons support renewed consideration of an electrically active role for myelin in increasing both saltatory conduction velocity and maximum propagation frequency in

  2. Structural basis for the coupling between activation and inactivation gates in K[superscript +] channels

    Energy Technology Data Exchange (ETDEWEB)

    Cuello, Luis G.; Jogini, Vishwanath; Cortes, D. Marien; Pan, Albert C.; Gagnon, Dominique G.; Dalmas, Olivier; Cordero-Morales, Julio F.; Chakrapani, Sudha; Roux, Benoît; Perozo, Eduardo (UC)

    2010-08-30

    The coupled interplay between activation and inactivation gating is a functional hallmark of K{sup +} channels. This coupling has been experimentally demonstrated through ion interaction effects and cysteine accessibility, and is associated with a well defined boundary of energetically coupled residues. The structure of the K{sup +} channel KcsA in its fully open conformation, in addition to four other partial channel openings, richly illustrates the structural basis of activation-inactivation gating. Here, we identify the mechanistic principles by which movements on the inner bundle gate trigger conformational changes at the selectivity filter, leading to the non-conductive C-type inactivated state. Analysis of a series of KcsA open structures suggests that, as a consequence of the hinge-bending and rotation of the TM2 helix, the aromatic ring of Phe103 tilts towards residues Thr74 and Thr75 in the pore-helix and towards Ile100 in the neighbouring subunit. This allows the network of hydrogen bonds among residues Trp67, Glu71 and Asp80 to destabilize the selectivity filter, allowing entry to its non-conductive conformation. Mutations at position 103 have a size-dependent effect on gating kinetics: small side-chain substitutions F103A and F103C severely impair inactivation kinetics, whereas larger side chains such as F103W have more subtle effects. This suggests that the allosteric coupling between the inner helical bundle and the selectivity filter might rely on straightforward mechanical deformation propagated through a network of steric contacts. Average interactions calculated from molecular dynamics simulations show favourable open-state interaction-energies between Phe103 and the surrounding residues. We probed similar interactions in the Shaker K{sup +} channel where inactivation was impaired in the mutant I470A. We propose that side-chain rearrangements at position 103 mechanically couple activation and inactivation in KcsA and a variety of other K

  3. Structural basis for the coupling between activation and inactivation gates in K(+) channels.

    Science.gov (United States)

    Cuello, Luis G; Jogini, Vishwanath; Cortes, D Marien; Pan, Albert C; Gagnon, Dominique G; Dalmas, Olivier; Cordero-Morales, Julio F; Chakrapani, Sudha; Roux, Benoît; Perozo, Eduardo

    2010-07-08

    The coupled interplay between activation and inactivation gating is a functional hallmark of K(+) channels. This coupling has been experimentally demonstrated through ion interaction effects and cysteine accessibility, and is associated with a well defined boundary of energetically coupled residues. The structure of the K(+) channel KcsA in its fully open conformation, in addition to four other partial channel openings, richly illustrates the structural basis of activation-inactivation gating. Here, we identify the mechanistic principles by which movements on the inner bundle gate trigger conformational changes at the selectivity filter, leading to the non-conductive C-type inactivated state. Analysis of a series of KcsA open structures suggests that, as a consequence of the hinge-bending and rotation of the TM2 helix, the aromatic ring of Phe 103 tilts towards residues Thr 74 and Thr 75 in the pore-helix and towards Ile 100 in the neighbouring subunit. This allows the network of hydrogen bonds among residues Trp 67, Glu 71 and Asp 80 to destabilize the selectivity filter, allowing entry to its non-conductive conformation. Mutations at position 103 have a size-dependent effect on gating kinetics: small side-chain substitutions F103A and F103C severely impair inactivation kinetics, whereas larger side chains such as F103W have more subtle effects. This suggests that the allosteric coupling between the inner helical bundle and the selectivity filter might rely on straightforward mechanical deformation propagated through a network of steric contacts. Average interactions calculated from molecular dynamics simulations show favourable open-state interaction-energies between Phe 103 and the surrounding residues. We probed similar interactions in the Shaker K(+) channel where inactivation was impaired in the mutant I470A. We propose that side-chain rearrangements at position 103 mechanically couple activation and inactivation in KcsA and a variety of other K(+) channels.

  4. Partial Agonist and Antagonist Activities of a Mutant Scorpion β-Toxin on Sodium Channels*

    Science.gov (United States)

    Karbat, Izhar; Ilan, Nitza; Zhang, Joel Z.; Cohen, Lior; Kahn, Roy; Benveniste, Morris; Scheuer, Todd; Catterall, William A.; Gordon, Dalia; Gurevitz, Michael

    2010-01-01

    Scorpion β-toxin 4 from Centruroides suffusus suffusus (Css4) enhances the activation of voltage-gated sodium channels through a voltage sensor trapping mechanism by binding the activated state of the voltage sensor in domain II and stabilizing it in its activated conformation. Here we describe the antagonist and partial agonist properties of a mutant derivative of this toxin. Substitution of seven different amino acid residues for Glu15 in Css4 yielded toxin derivatives with both increased and decreased affinities for binding to neurotoxin receptor site 4 on sodium channels. Css4E15R is unique among this set of mutants in that it retained nearly normal binding affinity but lost its functional activity for modification of sodium channel gating in our standard electrophysiological assay for voltage sensor trapping. More detailed analysis of the functional effects of Css4E15R revealed weak voltage sensor trapping activity, which was very rapidly reversed upon repolarization and therefore was not observed in our standard assay of toxin effects. This partial agonist activity of Css4E15R is observed clearly in voltage sensor trapping assays with brief (5 ms) repolarization between the conditioning prepulse and the test pulse. The effects of Css4E15R are fit well by a three-step model of toxin action involving concentration-dependent toxin binding to its receptor site followed by depolarization-dependent activation of the voltage sensor and subsequent voltage sensor trapping. Because it is a partial agonist with much reduced efficacy for voltage sensor trapping, Css4E15R can antagonize the effects of wild-type Css4 on sodium channel activation and can prevent paralysis by Css4 when injected into mice. Our results define the first partial agonist and antagonist activities for scorpion toxins and open new avenues of research toward better understanding of the structure-function relationships for toxin action on sodium channel voltage sensors and toward potential toxin

  5. Partial agonist and antagonist activities of a mutant scorpion beta-toxin on sodium channels.

    Science.gov (United States)

    Karbat, Izhar; Ilan, Nitza; Zhang, Joel Z; Cohen, Lior; Kahn, Roy; Benveniste, Morris; Scheuer, Todd; Catterall, William A; Gordon, Dalia; Gurevitz, Michael

    2010-10-01

    Scorpion β-toxin 4 from Centruroides suffusus suffusus (Css4) enhances the activation of voltage-gated sodium channels through a voltage sensor trapping mechanism by binding the activated state of the voltage sensor in domain II and stabilizing it in its activated conformation. Here we describe the antagonist and partial agonist properties of a mutant derivative of this toxin. Substitution of seven different amino acid residues for Glu(15) in Css4 yielded toxin derivatives with both increased and decreased affinities for binding to neurotoxin receptor site 4 on sodium channels. Css4(E15R) is unique among this set of mutants in that it retained nearly normal binding affinity but lost its functional activity for modification of sodium channel gating in our standard electrophysiological assay for voltage sensor trapping. More detailed analysis of the functional effects of Css4(E15R) revealed weak voltage sensor trapping activity, which was very rapidly reversed upon repolarization and therefore was not observed in our standard assay of toxin effects. This partial agonist activity of Css4(E15R) is observed clearly in voltage sensor trapping assays with brief (5 ms) repolarization between the conditioning prepulse and the test pulse. The effects of Css4(E15R) are fit well by a three-step model of toxin action involving concentration-dependent toxin binding to its receptor site followed by depolarization-dependent activation of the voltage sensor and subsequent voltage sensor trapping. Because it is a partial agonist with much reduced efficacy for voltage sensor trapping, Css4(E15R) can antagonize the effects of wild-type Css4 on sodium channel activation and can prevent paralysis by Css4 when injected into mice. Our results define the first partial agonist and antagonist activities for scorpion toxins and open new avenues of research toward better understanding of the structure-function relationships for toxin action on sodium channel voltage sensors and toward

  6. Transparent form-active system with structural glass

    NARCIS (Netherlands)

    Nikolaou, M.S.N.; Veer, F.A.; Eigenraam, P.

    2015-01-01

    Free-form transparent wide-span spatial structures which have being constructed so far, are based on the concept of three sets of components, the structural components, usually steel elements to ensure both compressive and tensional capacity; the glass cladding elements for expressing transparency;

  7. Transparent form-active system with structural glass

    NARCIS (Netherlands)

    Nikolaou, M.S.N.; Veer, F.A.; Eigenraam, P.

    2015-01-01

    Free-form transparent wide-span spatial structures which have being constructed so far, are based on the concept of three sets of components, the structural components, usually steel elements to ensure both compressive and tensional capacity; the glass cladding elements for expressing transparency;

  8. Inhibition of G protein-activated inwardly rectifying K+ channels by different classes of antidepressants.

    Directory of Open Access Journals (Sweden)

    Toru Kobayashi

    Full Text Available Various antidepressants are commonly used for the treatment of depression and several other neuropsychiatric disorders. In addition to their primary effects on serotonergic or noradrenergic neurotransmitter systems, antidepressants have been shown to interact with several receptors and ion channels. However, the molecular mechanisms that underlie the effects of antidepressants have not yet been sufficiently clarified. G protein-activated inwardly rectifying K(+ (GIRK, Kir3 channels play an important role in regulating neuronal excitability and heart rate, and GIRK channel modulation has been suggested to have therapeutic potential for several neuropsychiatric disorders and cardiac arrhythmias. In the present study, we investigated the effects of various classes of antidepressants on GIRK channels using the Xenopus oocyte expression assay. In oocytes injected with mRNA for GIRK1/GIRK2 or GIRK1/GIRK4 subunits, extracellular application of sertraline, duloxetine, and amoxapine effectively reduced GIRK currents, whereas nefazodone, venlafaxine, mianserin, and mirtazapine weakly inhibited GIRK currents even at toxic levels. The inhibitory effects were concentration-dependent, with various degrees of potency and effectiveness. Furthermore, the effects of sertraline were voltage-independent and time-independent during each voltage pulse, whereas the effects of duloxetine were voltage-dependent with weaker inhibition with negative membrane potentials and time-dependent with a gradual decrease in each voltage pulse. However, Kir2.1 channels were insensitive to all of the drugs. Moreover, the GIRK currents induced by ethanol were inhibited by sertraline but not by intracellularly applied sertraline. The present results suggest that GIRK channel inhibition may reveal a novel characteristic of the commonly used antidepressants, particularly sertraline, and contributes to some of the therapeutic effects and adverse effects.

  9. A cytosolic activator of DNA replication is tyrosine phosphorylated in its active form.

    Science.gov (United States)

    Fresa, K L; Autieri, M V; Coffman, F D; Georgoff, I; Cohen, S

    1993-04-01

    Cytosolic extracts from actively dividing lymphoid cells have been shown to induce DNA synthesis in isolated, quiescent nuclei. An initiating factor in such extracts (activator of DNA replication; ADR) is a > 90-kDa aprotinin-binding protein whose activity is inhibitable not only by aprotinin, but also by several other protease inhibitors as well. Although cytosol from non-proliferating lymphocytes is devoid of ADR activity, we have shown that these preparations can be induced to express ADR activity by brief exposure to a membrane-enriched fraction of spontaneously proliferating MOLT-4 cells via a kinase-dependent mechanism. In the present study, we examine the role of tyrosine kinases in this process. Three inhibitors of tyrosine kinases (genistein, kaempferol, and quercetin) can inhibit the in vitro generation of ADR activity. In vitro generation of ADR activity is associated with the de novo phosphorylation of several proteins, many of which are detectable using anti-phosphotyrosine monoclonal antibodies. ADR itself may be tyrosine phosphorylated in active form as immunoprecipitation using such monoclonal antibodies leads to the depletion of its activity. Moreover, immunoprecipitation results in the removal of several de novo tyrosine-phosphorylated proteins, including species at approximately 122, 105, 93, 86, 79, and 65 kDa. A subset of de novo-phosphorylated proteins, migrating at approximately 105, 93, and 70 kDa, also bound to aprotinin, suggesting that at least one of these proteins may represent ADR itself.

  10. Phentolamine inhibits the pacemaker activity of mouse interstitial cells of Cajal by activating ATP-sensitive K+ channels.

    Science.gov (United States)

    Ahn, Seung Whan; Kim, Sang Hun; Kim, Jin Ho; Choi, Seok; Yeum, Cheol Ho; Wie, Hee Wook; Sun, Jae Myeong; So, Insuk; Jun, Jae Yeoul

    2010-03-01

    The aim of this study was to clarify if phentolamine has proven effects on the pacemaker activities of interstitial cells of Cajal (ICC) from the mouse small intestine involving the ATPsensitive K(+) channels and adrenergic receptor. The actions of phentolamine on pacemaker activities were investigated using whole-cell patch-clamp technique and intracellular Ca(2+) analysis at 30 degrees C in cultured mouse intestinal ICC. ICC generated spontaneous pacemaker currents at a holding potential of -70 mV. Treatment with phentolamine reduced the frequency and amplitude of the pacemaker currents and increased the resting outward currents. Moreover, under current clamping (I = 0), phentolamine hyperpolarized the ICC membrane and decreased the amplitude of the pacemaker potentials. We also observed that phentolamine inhibited spontaneous [Ca(2+)](i) oscillations in ICC. The alpha-adrenergic drugs prazosin, yohimbine, methoxamine, and clonidine had no effect on ICC intestinal pacemaker activity and did not block phentolamine-induced effects. Phentolamine-induced effects on the pacemaker currents and the pacemaker potentials were significantly inhibited by ATP sensitive K(+) channel blocker glibenclamide, but not by TEA, apamin, or 4-aminopyridine. In addition, the NO synthase inhibitor, L-NAME and the guanylate cyclase inhibitor, ODQ were incapable of blocking the phentolamine-induced effects. These results demonstrate that phentolamine regulates the pacemaker activity of ICC via ATP-sensitive K(+) channel activation. Phentolamine could act through an adrenergic receptor- and also through NO-independent mechanism that involves intracellular Ca(2+) signaling.

  11. Cholecystokinin facilitates neuronal excitability in the entorhinal cortex via activation of TRPC-like channels.

    Science.gov (United States)

    Wang, Shouping; Zhang, An-Ping; Kurada, Lalitha; Matsui, Toshimitsu; Lei, Saobo

    2011-09-01

    Cholecystokinin (CCK) is one of the most abundant neuropeptides in the brain, where it interacts with two G protein-coupled receptors (CCK-1 and CCK-2). Activation of both CCK receptors increases the activity of PLC, resulting in increases in intracellular calcium ion (Ca(2+)) release and activation of PKC. Whereas high density of CCK receptors has been detected in the superficial layers of the entorhinal cortex (EC), the functions of CCK in this brain region have not been determined. Here, we studied the effects of CCK on neuronal excitability of layer III pyramidal neurons in the EC. Our results showed that CCK remarkably increased the firing frequency of action potentials (APs). The effects of CCK on neuronal excitability were mediated via activation of CCK-2 receptors and required the functions of G proteins and PLC. However, CCK-mediated facilitation of neuronal excitability was independent of inositol trisphosphate receptors and PKC. CCK facilitated neuronal excitability by activating a cationic channel to generate membrane depolarization. The effects of CCK were suppressed by the generic, nonselective cationic channel blockers, 2-aminoethyldiphenyl borate and flufenamic acid, but potentiated by gadolinium ion and lanthanum ion at 100 μM. Depletion of extracellular Ca(2+) also counteracted CCK-induced increases in AC firing frequency. Moreover, CCK-induced enhancement of neuronal excitability was inhibited significantly by intracellular application of the antibody to transient receptor potential channel 5 (TRPC5), suggesting the involvement of TRPC5 channels. Our results provide a cellular and molecular mechanism to help explain the functions of CCK in vivo.

  12. The Ca2+-activated cation channel TRPM4 is regulated by phosphatidylinositol 4,5-biphosphate.

    Science.gov (United States)

    Nilius, Bernd; Mahieu, Frank; Prenen, Jean; Janssens, Annelies; Owsianik, Grzegorz; Vennekens, Rudi; Voets, Thomas

    2006-02-08

    Transient receptor potential (TRP) channel, melastatin subfamily (TRPM)4 is a Ca2+-activated monovalent cation channel that depolarizes the plasma membrane and thereby modulates Ca2+ influx through Ca2+-permeable pathways. A typical feature of TRPM4 is its rapid desensitization to intracellular Ca2+ ([Ca2+]i). Here we show that phosphatidylinositol 4,5-biphosphate (PIP2) counteracts desensitization to [Ca2+]i in inside-out patches and rundown of TRPM4 currents in whole-cell patch-clamp experiments. PIP2 shifted the voltage dependence of TRPM4 activation towards negative potentials and increased the channel's Ca2+ sensitivity 100-fold. Conversely, activation of the phospholipase C (PLC)-coupled M1 muscarinic receptor or pharmacological depletion of cellular PIP2 potently inhibited currents through TRPM4. Neutralization of basic residues in a C-terminal pleckstrin homology (PH) domain accelerated TRPM4 current desensitization and strongly attenuated the effect of PIP2, whereas mutations to the C-terminal TRP box and TRP domain had no effect on the PIP2 sensitivity. Our data demonstrate that PIP2 is a strong positive modulator of TRPM4, and implicate the C-terminal PH domain in PIP2 action. PLC-mediated PIP2 breakdown may constitute a physiologically important brake on TRPM4 activity.

  13. Activation of volume-regulated Cl− channels by ACh and ATP in Xenopus follicles

    Science.gov (United States)

    Pérez-Samartín, Alberto L; Miledi, Ricardo; Arellano, Rogelio O

    2000-01-01

    Osmolarity-dependent ionic currents from follicle-enclosed Xenopus oocytes (follicles) were studied using electrophysiological techniques. Whole follicle currents were monitored using a two-electrode voltage clamp and single-channel activity was measured using the patch-clamp technique.In follicles held at -60 mV two chloride currents were activated in external hyposmotic solutions. One was the habitual volume-regulated current elicited by external hyposmolarity (ICl,swell), and the second was a slow and smooth current (Sin) generated by ACh or ATP application.In follicles, the permeability ratios for different anions with respect to Cl− were similar for both ICl,swell and Sin, with a sequence of: SCN− > I− > Br−≥ NO3−≥ Cl− > gluconate ≥ cyclamate > acetate > SO42−.Extracellular ATP blocked the outward component of Sin. Also, extracellular pH modulated the inactivation kinetics of Sin elicited by ACh; e.g. inactivation at +80 mV was ∼100% slower at pH 8.0 compared with that at pH 6.0.Lanthanides inhibited ICl,swell and Sin. La3+ completely inhibited ICl,swell with a half-maximal inhibitory concentration (IC50) of 17 ± 1.9 μm, while Sin was blocked up to 55% with an apparent IC50 of 36 ± 2.6 μm.Patch-clamp recordings in follicular cells showed that hyposmotic challenge opened inward single-channel currents. The single channel conductance (4.7 ± 0.4 pS) had a linear current-voltage relationship with a reversal membrane potential close to −20 mV. This single-channel activity was increased by application of ACh or ATP.The ICl,swell generation was not affected by pirenzepine or metoctramine, and did not affect the purinergic activation of the chloride current named Fin. Thus, ICl,swell was not generated via neurotransmitters released during cellular swelling.All together, equal discrimination for different anions, similar modulatory effects by extracellular pH, the blocking effects by ATP and La3+, and the same single-channel activity

  14. Putative calcium-binding domains of the Caenorhabditis elegans BK channel are dispensable for intoxication and ethanol activation.

    Science.gov (United States)

    Davis, S J; Scott, L L; Ordemann, G; Philpo, A; Cohn, J; Pierce-Shimomura, J T

    2015-07-01

    Alcohol modulates the highly conserved, voltage- and calcium-activated potassium (BK) channel, which contributes to alcohol-mediated behaviors in species from worms to humans. Previous studies have shown that the calcium-sensitive domains, RCK1 and the Ca(2+) bowl, are required for ethanol activation of the mammalian BK channel in vitro. In the nematode Caenorhabditis elegans, ethanol activates the BK channel in vivo, and deletion of the worm BK channel, SLO-1, confers strong resistance to intoxication. To determine if the conserved RCK1 and calcium bowl domains were also critical for intoxication and basal BK channel-dependent behaviors in C. elegans, we generated transgenic worms that express mutated SLO-1 channels predicted to have the RCK1, Ca(2+) bowl or both domains rendered insensitive to calcium. As expected, mutating these domains inhibited basal function of SLO-1 in vivo as neck and body curvature of these mutants mimicked that of the BK null mutant. Unexpectedly, however, mutating these domains singly or together in SLO-1 had no effect on intoxication in C. elegans. Consistent with these behavioral results, we found that ethanol activated the SLO-1 channel in vitro with or without these domains. By contrast, in agreement with previous in vitro findings, C. elegans harboring a human BK channel with mutated calcium-sensing domains displayed resistance to intoxication. Thus, for the worm SLO-1 channel, the putative calcium-sensitive domains are critical for basal in vivo function but unnecessary for in vivo ethanol action.

  15. Activity of Palythoa caribaeorum Venom on Voltage-Gated Ion Channels in Mammalian Superior Cervical Ganglion Neurons

    Directory of Open Access Journals (Sweden)

    Fernando Lazcano-Pérez

    2016-05-01

    Full Text Available The Zoanthids are an order of cnidarians whose venoms and toxins have been poorly studied. Palythoa caribaeorum is a zoanthid commonly found around the Mexican coastline. In this study, we tested the activity of P. caribaeorum venom on voltage-gated sodium channel (NaV1.7, voltage-gated calcium channel (CaV2.2, the A-type transient outward (IA and delayed rectifier (IDR currents of KV channels of the superior cervical ganglion (SCG neurons of the rat. These results showed that the venom reversibly delays the inactivation process of voltage-gated sodium channels and inhibits voltage-gated calcium and potassium channels in this mammalian model. The compounds responsible for these effects seem to be low molecular weight peptides. Together, these results provide evidence for the potential use of zoanthids as a novel source of cnidarian toxins active on voltage-gated ion channels.

  16. Functional differences in pore properties between wild-type and cysteine-less forms of the CFTR chloride channel.

    Science.gov (United States)

    Holstead, Ryan G; Li, Man-Song; Linsdell, Paul

    2011-10-01

    Studies of the structure and function of the cystic fibrosis transmembrane conductance regulator (CFTR) Cl(-) channel have been advanced by the development of functional channel variants in which all 18 endogenous cysteine residues have been mutated ("cys-less" CFTR). However, cys-less CFTR has a slightly higher single-channel conductance than wild-type CFTR, raising questions as to the suitability of cys-less as a model of the wild-type CFTR pore. We used site-directed mutagenesis and patch-clamp recording to investigate the origin of this conductance difference and to determine the extent of functional differences between wild-type and cys-less CFTR channel permeation properties. Our results suggest that the conductance difference is the result of a single substitution, of C343: the point mutant C343S has a conductance similar to cys-less, whereas the reverse mutation, S343C in a cys-less background, restores wild-type conductance levels. Other cysteine substitutions (C128S, C225S, C376S, C866S) were without effect. Substitution of other residues for C343 suggested that conductance is dependent on amino acid side chain volume at this position. A range of other functional pore properties, including interactions with channel blockers (Au[CN] (2) (-) , 5-nitro-2-[3-phenylpropylamino]benzoic acid, suramin) and anion permeability, were not significantly different between wild-type and cys-less CFTR. Our results suggest that functional differences between these two CFTR constructs are of limited scale and scope and result from a small change in side chain volume at position 343. These results therefore support the use of cys-less as a model of the CFTR pore region.

  17. Effects of heat and mass transfer on peristaltic flow of a Bingham fluid in the presence of inclined magnetic field and channel with different wave forms

    Energy Technology Data Exchange (ETDEWEB)

    Akram, Safia, E-mail: safia_akram@yahoo.com [Department of Basic Sciences, MCS, National University of Sciences and Technology, Rawalpindi 46000 (Pakistan); Nadeem, S.; Hussain, Anwar [Department of Mathematics, Quaid-i-Azam University, Islamabad 45320 (Pakistan)

    2014-08-01

    In the present analysis we discussed the influence of heat and mass transfer on the peristaltic flow of a Bingham in an inclined magnetic field and channel with different wave forms. The governing two dimensional equations of momentum, heat and mass transfer are simplified under the assumptions of long wavelength and low Reynolds number approximation. The exact solutions of momentum, heat and mass transfer are calculated. Finally, graphical behaviors of various physical parameters are also discussed through the graphical behavior of pressure rise, pressure gradient, temperature concentration and stream functions. - Highlights: • Combine effects of heat and mass transfer on peristaltic flow problem is discussed. • Effects of inclined magnetic field and channel on new fluid model are discussed. • Effects of different wave forms are also discussed in the present flow problem.

  18. Activation of transient receptor potential vanilloid type-1 channel prevents adipogenesis and obesity

    DEFF Research Database (Denmark)

    Zhang, Li Li; Yan Liu, Dao; Ma, Li Qun

    2007-01-01

    in visceral adipose tissue from obese humans was accompanied by reduced capsaicin-induced calcium influx. The oral administration of capsaicin for 120 days prevented obesity in male wild type mice but not in TRPV1 knockout mice assigned to high fat diet. We conclude that the activation of TRPV1 channels......We tested the hypothesis that activation of transient receptor potential vanilloid type-1 (TRPV1) by capsaicin prevents adipogenesis. TRPV1 channels in 3T3-L1-preadipocytes and visceral adipose tissue from mice and humans were detected by immunoblotting and quantitative real-time RT-PCR. The effect...... of TRPV1 on cytosolic calcium was determined fluorometrically in 3T3-L1-preadipocytes and in human visceral fat tissue. Adipogenesis in stimulated 3T3-L1-preadipocytes was determined by oil red O-staining of intracellular lipid droplets, triglyceride levels, expression of peroxisome proliferator...

  19. Effect of a chloride channel activator, lubiprostone, on colonic sensory and motor functions in healthy subjects

    OpenAIRE

    Sweetser, Seth; Busciglio, Irene A.; Camilleri, Michael; Bharucha, Adil E.; Szarka, Lawrence A.; Papathanasopoulos, Athanasios; Burton, Duane D.; Eckert, Deborah J.; Zinsmeister, Alan R.

    2008-01-01

    Lubiprostone, a bicyclic fatty acid chloride channel activator, is efficacious in treatment of chronic constipation and constipation-predominant irritable bowel syndrome. The study aim was to compare effects of lubiprostone and placebo on colonic sensory and motor functions in humans. In double-blind, randomized fashion, 60 healthy adults received three oral doses of placebo or 24 μg lubiprostone per day in a parallel-group, placebo-controlled trial. A barostat-manometry tube was placed in th...

  20. Anti-Convulsant Activity of Boerhaavia diffusa: Plausible Role of Calcium Channel Antagonism

    OpenAIRE

    Mandeep Kaur; Rajesh Kumar Goel

    2011-01-01

    “Ethnopharmacological” use of roots of Boerhaavia diffusa (B. diffusa) in the treatment of epilepsy in Nigerian folk medicine and reports showing the presence of a calcium channel antagonistic compound “liriodendrin” in its roots, led us to undertake the present study. The study was designed to investigate the methanolic root extract of B. diffusa and its different fractions including liriodendrin-rich fraction for exploring the possible role of liriodendrin in its anti-convulsant activity. A...

  1. Maitotoxin activates a nonselective cation channel and stimulates Ca2+ entry in MDCK renal epithelial cells.

    Science.gov (United States)

    Dietl, P; Völkl, H

    1994-02-01

    We examined the mechanisms of maitotoxin (MTX), a water-soluble polyether from the marine dinoflagellate Gambierdiscus toxicus, in stimulation of Ca2+ entry into Mardin-Darby canine kidney cells. In the presence of bath Ca2+, MTX (3 nM) caused an elevation of the intracellular calcium concentration ([Ca2+]i), which was partially inhibited by SK&F 96365 (25 microM) or La3+ (100 microM). A stimulation of Ca(2+)-dependent K+ channels in cell-attached membrane patches coincided with this rise in [Ca2+]i and was also partially inhibited by SK&F 96365. Before the rise in [Ca2+]i, a nonselective cation current (Ins), studied by the whole-cell patch-clamp technique, was irreversibly activated. Ins poorly discriminated between Na+, K+, and Cs+, was unaffected by replacement of Cl- with gluconate-, and was not voltage gated. MTX-induced Ins was partially blocked by La3+ ions (100 microM) but not by SK&F 96365 (25 microM) or nifedipine (10 microM). SK&F 96365 by itself induced a small but significant stimulation of Ins and a rise in [Ca2+]i. The activation of Ins by MTX was instantaneous and depended on the presence of extracellular Ca2+ ions. In the absence of other cations, the inward current of Ins was dependent on the bath Ca2+ concentration. Cell-attached and excised single-channel measurements revealed that MTX activated a SK&F 96365-insensitive, approximately 40-pS, nonselective cation channel from the outside. We conclude that the initial action of MTX is the stimulation of a nonselective cation channel, which requires the presence of extracellular Ca2+ ions. The subsequent rise in [Ca2+]i is at least in part caused by another, SK&F 96365-sensitive, Ca2+ entry pathway, which may be activated as a result of or independently of Ins.

  2. Direct tests of micro channel plates as the active element of a new shower maximum detector

    Energy Technology Data Exchange (ETDEWEB)

    Ronzhin, A., E-mail: ronzhin@fnal.gov [Fermilab, Batavia, IL 60510 (United States); Los, S.; Ramberg, E. [Fermilab, Batavia, IL 60510 (United States); Apresyan, A.; Xie, S.; Spiropulu, M. [California Institute of Technology, Pasadena, CA (United States); Kim, H. [University of Chicago, Chicago, IL 60637 (United States)

    2015-09-21

    We continue the study of micro channel plates (MCP) as the active element of a shower maximum (SM) detector. We present below test beam results obtained with MCPs detecting directly secondary particles of an electromagnetic shower. The MCP efficiency to shower particles is close to 100%. The time resolution obtained for this new type of the SM detector is at the level of 40 ps.

  3. Modulation of voltage-gated sodium channels hyperpolarizes the voltage threshold for activation in spinal motoneurones.

    Science.gov (United States)

    Power, Kevin E; Carlin, Kevin P; Fedirchuk, Brent

    2012-03-01

    Previous work has shown that motoneurone excitability is enhanced by a hyperpolarization of the membrane potential at which an action potential is initiated (V(th)) at the onset, and throughout brainstem-evoked fictive locomotion in the adult decerebrate cat and neonatal rat. Modeling work has suggested the modulation of Na(+) conductance as a putative mechanism underlying this state-dependent change in excitability. This study sought to determine whether modulation of voltage-gated sodium channels could induce V(th) hyperpolarization. Whole-cell patch-clamp recordings were made from antidromically identified lumbar spinal motoneurones in an isolated neonatal rat spinal cord preparation. Recordings were made with and without the bath application of veratridine, a plant alkaloid neurotoxin that acts as a sodium channel modulator. As seen in HEK 293 cells expressing Nav1.2 channels, veratridine-modified channels demonstrated a hyperpolarizing shift in their voltage-dependence of activation and a slowing of inactivation that resulted in an enhanced inward current in response to voltage ramp stimulations. In the native rat motoneurones, veratridine-modified sodium channels induced a hyperpolarization of V(th) in all 29 neonatal rat motoneurones examined (mean hyperpolarization: -6.6 ± 4.3 mV). V(th) hyperpolarization was not due to the effects on Ca(2+) and/or K(+) channels as blockade of these currents did not alter V(th). Veratridine also significantly increased the amplitude of persistent inward currents (PICs; mean increase: 72.5 ± 98.5 pA) evoked in response to slow depolarizing current ramps. However, the enhancement of the PIC amplitude had a slower time course than the hyperpolarization of V(th), and the PIC onset voltage could be either depolarized or hyperpolarized, suggesting that PIC facilitation did not mediate the V(th) hyperpolarization. We therefore suggest that central neuronal circuitry in mammals could affect V(th) in a mechanism similar to that of

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

    Directory of Open Access Journals (Sweden)

    Sheyda R Frolova

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

  5. Hydraphiles: A Rigorously Studied Class of Synthetic Channel Compounds with In Vivo Activity

    Directory of Open Access Journals (Sweden)

    Saeedeh Negin

    2013-01-01

    Full Text Available Hydraphiles are a class of synthetic ion channels that now have a twenty-year history of analysis and success. In early studies, these compounds were rigorously validated in a wide range of in vitro assays including liposomal ion flow detected by NMR or ion-selective electrodes, as well as biophysical experiments in planar bilayers. During the past decade, biological activity was observed for these compounds including toxicity to bacteria, yeast, and mammalian cells due to stress caused by the disruption of ion homeostasis. The channel mechanism was verified in cells using membrane polarity sensitive dyes, as well as patch clamping studies. This body of work has provided a solid foundation with which hydraphiles have recently demonstrated acute biological toxicity in the muscle tissue of living mice, as measured by whole animal fluorescence imaging and histological studies. Here we review the critical structure-activity relationships in the hydraphile family of compounds and the in vitro and in cellulo experiments that have validated their channel behavior. This report culminates with a description of recently reported efforts in which these molecules have demonstrated activity in living mice.

  6. The Alternatively Spliced Form “b” of the Epithelial Sodium Channel α Subunit (α ENaC: Any Prior Evidence of its Existence?

    Directory of Open Access Journals (Sweden)

    Marlene F. Shehata

    2010-08-01

    Full Text Available The epithelial sodium channel (ENaC is critical in maintaining sodium balance across aldosterone-responsive epithelia. ENaC is a combined channel formed of three subunits (αβγ with α ENaC subunit being the most critical for channel functionality. In a previous report, we have demonstrated the existence and mRNA expression levels of four alternatively spliced forms of the α ENaC subunit denoted by -a, -b, -c and -d in kidney cortex of Dahl S and R rats. Of the four alternatively spliced forms presently identified, α ENaC-b is considered the most interesting for the following reasons: Aside from being a salt-sensitive transcript, α ENaC-b mRNA expression is ∼32 fold higher than α ENaC wildtype in kidney cortex of Dahl rats. Additionally, the splice site used to generate α ENaC-b is conserved across species. Finally, α ENaC-b mRNA expression is significantly higher in salt-resistant Dahl R rats versus salt-sensitive Dahl S rats. As such, this commentary aims to highlight some of the previously published research articles that described the existence of an additional protein band on α ENaC western blots that could account for α ENaC-b in other rat species.

  7. Shaping of action potentials by type I and type II large-conductance Ca²+-activated K+ channels.

    Science.gov (United States)

    Jaffe, D B; Wang, B; Brenner, R

    2011-09-29

    The BK channel is a Ca(2+) and voltage-gated conductance responsible for shaping action potential waveforms in many types of neurons. Type II BK channels are differentiated from type I channels by their pharmacology and slow gating kinetics. The β4 accessory subunit confers type II properties on BK α subunits. Empirically derived properties of BK channels, with and without the β4 accessory subunit, were obtained using a heterologous expression system under physiological ionic conditions. These data were then used to study how BK channels alone (type I) and with the accessory β4 subunit (type II) modulate action potential properties in biophysical neuron models. Overall, the models support the hypothesis that it is the slower kinetics provided by the β4 subunit that endows the BK channel with type II properties, which leads to broadening of action potentials and, secondarily, to greater recruitment of SK channels reducing neuronal excitability. Two regions of parameter space distinguished type II and type I effects; one where the range of BK-activating Ca(2+) was high (>20 μM) and the other where BK-activating Ca(2+) was low (∼0.4-1.2 μM). The latter required an elevated BK channel density, possibly beyond a likely physiological range. BK-mediated sharpening of the spike waveform associated with the lack of the β4 subunit was sensitive to the properties of voltage-gated Ca(2+) channels due to electrogenic effects on spike duration. We also found that depending on Ca(2+) dynamics, type II BK channels may have the ability to contribute to the medium AHP, a property not generally ascribed to BK channels, influencing the frequency-current relationship. Finally, we show how the broadening of action potentials conferred by type II BK channels can also indirectly increase the recruitment of SK-type channels decreasing the excitability of the neuron.

  8. Constitutive Activity in an Ancestral Form of Abl Tyrosine Kinase.

    Science.gov (United States)

    Aleem, Saadat U; Craddock, Barbara P; Miller, W Todd

    2015-01-01

    The c-abl proto-oncogene encodes a nonreceptor tyrosine kinase that is found in all metazoans, and is ubiquitously expressed in mammalian tissues. The Abl tyrosine kinase plays important roles in the regulation of mammalian cell physiology. Abl-like kinases have been identified in the genomes of unicellular choanoflagellates, the closest relatives to the Metazoa, and in related unicellular organisms. Here, we have carried out the first characterization of a premetazoan Abl kinase, MbAbl2, from the choanoflagellate Monosiga brevicollis. The enzyme possesses SH3, SH2, and kinase domains in a similar arrangement to its mammalian counterparts, and is an active tyrosine kinase. MbAbl2 lacks the N-terminal myristoylation and cap sequences that are critical regulators of mammalian Abl kinase activity, and we show that MbAbl2 is constitutively active. When expressed in mammalian cells, MbAbl2 strongly phosphorylates cellular proteins on tyrosine, and transforms cells much more potently than mammalian Abl kinase. Thus, MbAbl2 appears to lack the autoinhibitory mechanism that tightly constrains the activity of mammalian Abl kinases, suggesting that this regulatory apparatus arose more recently in metazoan evolution.

  9. Constitutive Activity in an Ancestral Form of Abl Tyrosine Kinase.

    Directory of Open Access Journals (Sweden)

    Saadat U Aleem

    Full Text Available The c-abl proto-oncogene encodes a nonreceptor tyrosine kinase that is found in all metazoans, and is ubiquitously expressed in mammalian tissues. The Abl tyrosine kinase plays important roles in the regulation of mammalian cell physiology. Abl-like kinases have been identified in the genomes of unicellular choanoflagellates, the closest relatives to the Metazoa, and in related unicellular organisms. Here, we have carried out the first characterization of a premetazoan Abl kinase, MbAbl2, from the choanoflagellate Monosiga brevicollis. The enzyme possesses SH3, SH2, and kinase domains in a similar arrangement to its mammalian counterparts, and is an active tyrosine kinase. MbAbl2 lacks the N-terminal myristoylation and cap sequences that are critical regulators of mammalian Abl kinase activity, and we show that MbAbl2 is constitutively active. When expressed in mammalian cells, MbAbl2 strongly phosphorylates cellular proteins on tyrosine, and transforms cells much more potently than mammalian Abl kinase. Thus, MbAbl2 appears to lack the autoinhibitory mechanism that tightly constrains the activity of mammalian Abl kinases, suggesting that this regulatory apparatus arose more recently in metazoan evolution.

  10. Activation of endothelial and epithelial KCa2.3 calcium-activated potassium channels by NS309 relaxes human small pulmonary arteries and bronchioles

    DEFF Research Database (Denmark)

    Kroigaard, Christel; Dalsgaard, Thomas; Nielsen, Gorm

    2012-01-01

    BACKGROUND AND PURPOSE: Small (K(Ca) 2) and intermediate (K(Ca) 3.1) conductance calcium-activated potassium channels (K(Ca) ) may contribute to both epithelium- and endothelium-dependent relaxations, but this has not been established in human pulmonary arteries and bronchioles. Therefore, we...... investigated the expression of K(Ca) 2.3 and K(Ca) 3.1 channels, and hypothesized that activation of these channels would produce relaxation of human bronchioles and pulmonary arteries. EXPERIMENTAL APPROACH: Channel expression and functional studies were conducted in human isolated small pulmonary arteries.......1 activator, NS309, induced concentration-dependent relaxations. NS309 was equally potent in relaxing pulmonary arteries, but less potent in bronchioles, than salbutamol. NS309 relaxations were blocked by the K(Ca) 2 channel blocker apamin, while the K(Ca) 3.1 channel blocker, charybdotoxin failed to reduce...

  11. Store-operated interactions between plasmalemmal STIM1 and TRPC1 proteins stimulate PLCβ1 to induce TRPC1 channel activation in vascular smooth muscle cells.

    Science.gov (United States)

    Shi, Jian; Miralles, Francesc; Birnbaumer, Lutz; Large, William A; Albert, Anthony P

    2017-02-15

    1 and STIM1 antibodies and STIM1 short hairpin RNA (shRNA) in wild-type VSMCs, and was absent in TRPC1(-/-) VSMCs. Store-operated PKC phosphorylation of TRPC1 was reduced by knockdown of STIM1. Moreover, store-operated PLCβ1 activity measured with the fluorescent phosphatidylinositol 4,5-bisphosphate/inositol 1,4,5-trisphosphate biosensor GFP-PLCδ1-PH was reduced by STIM1 shRNA and absent in TRPC1(-/-) cells. Immunocytochemistry, co-immunoprecipitation and proximity ligation assays revealed that store depletion activated STIM1 translocation from within the cell to the plasma membrane (PM) where it formed STIM1-TRPC1 complexes, which then associated with Gαq and PLCβ1. Noradrenaline also evoked TRPC1 channel activity and associations between TRPC1, STIM1, Gαq and PLCβ1, which were inhibited by STIM1 knockdown. Effects of N-terminal and C-terminal STIM1 antibodies on TRPC1-based SOCs and STIM1 staining suggest that channel activation may involve insertion of STIM1 into the PM. The findings of the present study identify a new activation mechanism of TRPC1-based SOCs in VSMCs, and a novel role for STIM1, in which store-operated STIM1-TRPC1 interactions stimulate PLCβ1 activity to induce PKC phosphorylation of TRPC1 and channel gating. © 2016 The Authors. The Journal of Physiology published by John Wiley & Sons Ltd on behalf of The Physiological Society.

  12. 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.

  13. Emerging roles of calcium-activated K channels and TRPV4 channels in lung oedema and pulmonary circulatory collapse

    DEFF Research Database (Denmark)

    Simonsen, Ulf; Wandall-Frostholm, Christine; Oliván-Viguera, Aida;

    2016-01-01

    endothelial/epithelial barrier functions and vascular integrity, while KCa3.1 channels provide the driving force required for Cl(-) and water transport in some cells and most secretory epithelia. The three conditions, increased pulmonary venous pressure caused by left heart disease, high inflation pressure......, fluid extravasation, hemorrhage, pulmonary circulatory collapse, and cardiac arrest in vivo. These data identify KCa3.1 channels as crucial molecular components in downstream TRPV4-signal transduction and as a potential target for the prevention of undesired fluid extravasation, vasodilatation...

  14. Novel Compound-Forming Technology Using Bioprinting and Electrospinning for Patterning a 3D Scaffold Construct with Multiscale Channels

    Directory of Open Access Journals (Sweden)

    Yuanshao Sun

    2016-12-01

    Full Text Available One of the biggest challenges for tissue engineering is to efficiently provide oxygen and nutrients to cells on a three-dimensional (3D engineered scaffold structure. Thus, achieving sufficient vascularization of the structure is a critical problem in tissue engineering. This facilitates the need to develop novel methods to enhance vascularization. Use of patterned hydrogel structures with multiscale channels can be used to achieve the required vascularization. Patterned structures need to be biocompatible and biodegradable. In this study, gelatin was used as the main part of a hydrogel to prepare a biological structure with 3D multiscale channels using bioprinting combined with selection of suitable materials and electrostatic spinning. Human umbilical vein endothelial cells (HUVECs were then used to confirm efficacy of the structure, inferred from cell viability on different engineered construct designs. HUVECs were seeded on the surface of channels and cultured in vitro. HUVECs showed high viability and diffusion within the construct. This method can be used as a practical platform for the fabrication of engineered construct for vascularization.

  15. Noxious cold ion channel TRPA1 is activated by pungent compounds and bradykinin.

    Science.gov (United States)

    Bandell, Michael; Story, Gina M; Hwang, Sun Wook; Viswanath, Veena; Eid, Samer R; Petrus, Matt J; Earley, Taryn J; Patapoutian, Ardem

    2004-03-25

    Six members of the mammalian transient receptor potential (TRP) ion channels respond to varied temperature thresholds. The natural compounds capsaicin and menthol activate noxious heat-sensitive TRPV1 and cold-sensitive TRPM8, respectively. The burning and cooling perception of capsaicin and menthol demonstrate that these ion channels mediate thermosensation. We show that, in addition to noxious cold, pungent natural compounds present in cinnamon oil, wintergreen oil, clove oil, mustard oil, and ginger all activate TRPA1 (ANKTM1). Bradykinin, an inflammatory peptide acting through its G protein-coupled receptor, also activates TRPA1. We further show that phospholipase C is an important signaling component for TRPA1 activation. Cinnamaldehyde, the most specific TRPA1 activator, excites a subset of sensory neurons highly enriched in cold-sensitive neurons and elicits nociceptive behavior in mice. Collectively, these data demonstrate that TRPA1 activation elicits a painful sensation and provide a potential molecular model for why noxious cold can paradoxically be perceived as burning pain.

  16. Multiple forms of endopeptidase activity from jojoba seeds.

    Science.gov (United States)

    Wolf, M J; Storey, R D

    1990-01-01

    The cotyledons of 27 day post-germination jojoba seedlings (Simmondsia chinensis) contained five distinct endopeptidase activities separable by DEAE Bio-Gel and CM-cellulose ion exchange chromatography. The endopeptidases were purified 108- to 266-fold and their individuality was confirmed by activity-specific assays in native acrylamide gels along with differences in their Mr and catalytic properties. The five endopeptidases, which showed activity on model substrates and protein, were named EP Ia, EP Ib, EP II, EP III and EP IV. EP Ia was a serine proteinase with a pH optimum of ca 8 and Mr of 58,000. EP Ib, II and III were discrete cysteine proteinases showing pH optima of ca 6.8, 6.0 and 5.4 and Mr of 41,000, 47,000 and 35,000 respectively. EP IV was an aspartic acid proteinase with a ca pH optimum of 3.5 and Mr of 33,000.

  17. Channel Power in Multi-Channel Environments

    NARCIS (Netherlands)

    M.G. Dekimpe (Marnik); B. Skiera (Bernd)

    2004-01-01

    textabstractIn the literature, little attention has been paid to instances where companies add an Internet channel to their direct channel portfolio. However, actively managing multiple sales channels requires knowing the customers’ channel preferences and the resulting channel power. Two key compon

  18. Channel Power in Multi-Channel Environments

    NARCIS (Netherlands)

    M.G. Dekimpe (Marnik); B. Skiera (Bernd)

    2004-01-01

    textabstractIn the literature, little attention has been paid to instances where companies add an Internet channel to their direct channel portfolio. However, actively managing multiple sales channels requires knowing the customers’ channel preferences and the resulting channel power. Two key

  19. Channel-forming activity of syringomycin E in two mercury-supported biomimetic membranes

    NARCIS (Netherlands)

    Becucci, L.; Tramonti, V.; Fiore, A.; Fogliano, V.; Scaloni, A.; Guidelli, R.

    2015-01-01

    The lipodepsipeptide syringomycin E (SR-E) interacts with two mercury-supported biomimetic membranes, which consist of a self-assembled phospholipid monolayer (SAM) and of a tethered bilayer lipid membrane (tBLM) separated from the mercury surface by a hydrophilic tetraethyleneoxy (TEO) spacer that

  20. Severe acute respiratory syndrome coronavirus envelope protein ion channel activity promotes virus fitness and pathogenesis.

    Directory of Open Access Journals (Sweden)

    Jose L Nieto-Torres

    2014-05-01

    Full Text Available Deletion of Severe Acute Respiratory Syndrome Coronavirus (SARS-CoV envelope (E gene attenuates the virus. E gene encodes a small multifunctional protein that possesses ion channel (IC activity, an important function in virus-host interaction. To test the contribution of E protein IC activity in virus pathogenesis, two recombinant mouse-adapted SARS-CoVs, each containing one single amino acid mutation that suppressed ion conductivity, were engineered. After serial infections, mutant viruses, in general, incorporated compensatory mutations within E gene that rendered active ion channels. Furthermore, IC activity conferred better fitness in competition assays, suggesting that ion conductivity represents an advantage for the virus. Interestingly, mice infected with viruses displaying E protein IC activity, either with the wild-type E protein sequence or with the revertants that restored ion transport, rapidly lost weight and died. In contrast, mice infected with mutants lacking IC activity, which did not incorporate mutations within E gene during the experiment, recovered from disease and most survived. Knocking down E protein IC activity did not significantly affect virus growth in infected mice but decreased edema accumulation, the major determinant of acute respiratory distress syndrome (ARDS leading to death. Reduced edema correlated with lung epithelia integrity and proper localization of Na+/K+ ATPase, which participates in edema resolution. Levels of inflammasome-activated IL-1β were reduced in the lung airways of the animals infected with viruses lacking E protein IC activity, indicating that E protein IC function is required for inflammasome activation. Reduction of IL-1β was accompanied by diminished amounts of TNF and IL-6 in the absence of E protein ion conductivity. All these key cytokines promote the progression of lung damage and ARDS pathology. In conclusion, E protein IC activity represents a new determinant for SARS

  1. Recombinant goose-type lysozyme in channel catfish: Lysozyme activity and efficacy as plasmid DNA immunostimulant against Aeromonas hydrophila infection

    Science.gov (United States)

    The objectives of this study were: 1) to investigate whether recombinant channel catfish lysozyme g (CC-Lys-g) produced in E. coli expression system possesses any lysozyme activity; and 2) to evaluate whether channel catfish lysozyme g plasmid DNA could be used as an immunostimulant to protect chann...

  2. Blockade of Ca2+-activated K+ channels in T cells: an option for the treatment of multiple sclerosis?

    DEFF Research Database (Denmark)

    Madsen, Lars Siim; Christophersen, Palle; Olesen, Søren-Peter

    2005-01-01

    Voltage- and Ca(2+)-dependent K(+) channels in the membrane of both T and B lymphocytes are important for the cellular immune response. In the current issue of the European Journal of Immunology, Reich et al. demonstrate that selective blockade of the intermediate-conductance Ca(2+)-activated K......(+) channel (the IK channel encoded by the KCNN4 gene) prevents cytokine production in the spinal chord and ameliorates the development of EAE caused by injection of myelin oligodendrocyte glycoprotein (MOG)(35-55) in mice. These data renew the focus on the IK channel as a potential target for the development...... of new immune-suppressant drugs for the treatment of autoimmune diseases....

  3. Repetitive transcranial magnetic stimulation regulates L-type Ca(2+) channel activity inhibited by early sevoflurane exposure.

    Science.gov (United States)

    Liu, Yang; Yang, Huiyun; Tang, Xiaohong; Bai, Wenwen; Wang, Guolin; Tian, Xin

    2016-09-01

    Sevoflurane might be harmful to the developing brain. Therefore, it is essential to reverse sevoflurane-induced brain injury. This study aimed to determine whether low-frequency repetitive transcranial magnetic stimulation (rTMS) can regulate L-type Ca(2+) channel activity, which is inhibited by early sevoflurane exposure. Rats were randomly divided into three groups: control, sevoflurane, and rTMS groups. A Whole-cell patch clamp technique was applied to record L-type Ca(2+) channel currents. The I-V curve, steady-state activation and inactivation curves were studied in rats of each group at different ages (1 week, 2 weeks, 3 weeks, 4 weeks and 5 weeks old). In the control group, L-type Ca(2+) channel current density significantly increased from week 2 to week 3. Compared with the control group, L-type Ca(2+) channel currents of rats in the sevoflurane group were significantly inhibited from week 1 to week 3. Activation curves of L-type Ca(2+) channel shifted significantly towards depolarization at week 1 and week 2. Moreover, steady-state inactivation curves shifted towards hyperpolarization from week 1 to week 3. Compared with the sevoflurane group, rTMS significantly increased L-type Ca(2+) channel currents at week 2 and week 3. Activation curves of L-type Ca(2+) channel significantly shifted towards hyperpolarization at week 2. Meanwhile, steady-state inactivation curves significantly shifted towards depolarization at week 2. The period between week 2 and week 3 is critical for the development of L-type Ca(2+) channels. Early sevoflurane exposure inhibits L-type Ca(2+) channel activity and rTMS can regulate L-type Ca(2+) channel activity inhibited by sevoflurane. Copyright © 2016 Elsevier B.V. All rights reserved.

  4. An orally active TRPV4 channel blocker prevents and resolves pulmonary edema induced by heart failure.

    Science.gov (United States)

    Thorneloe, Kevin S; Cheung, Mui; Bao, Weike; Alsaid, Hasan; Lenhard, Stephen; Jian, Ming-Yuan; Costell, Melissa; Maniscalco-Hauk, Kristeen; Krawiec, John A; Olzinski, Alan; Gordon, Earl; Lozinskaya, Irina; Elefante, Lou; Qin, Pu; Matasic, Daniel S; James, Chris; Tunstead, James; Donovan, Brian; Kallal, Lorena; Waszkiewicz, Anna; Vaidya, Kalindi; Davenport, Elizabeth A; Larkin, Jonathan; Burgert, Mark; Casillas, Linda N; Marquis, Robert W; Ye, Guosen; Eidam, Hilary S; Goodman, Krista B; Toomey, John R; Roethke, Theresa J; Jucker, Beat M; Schnackenberg, Christine G; Townsley, Mary I; Lepore, John J; Willette, Robert N

    2012-11-01

    Pulmonary edema resulting from high pulmonary venous pressure (PVP) is a major cause of morbidity and mortality in heart failure (HF) patients, but current treatment options demonstrate substantial limitations. Recent evidence from rodent lungs suggests that PVP-induced edema is driven by activation of pulmonary capillary endothelial transient receptor potential vanilloid 4 (TRPV4) channels. To examine the therapeutic potential of this mechanism, we evaluated TRPV4 expression in human congestive HF lungs and developed small-molecule TRPV4 channel blockers for testing in animal models of HF. TRPV4 immunolabeling of human lung sections demonstrated expression of TRPV4 in the pulmonary vasculature that was enhanced in sections from HF patients compared to controls. GSK2193874 was identified as a selective, orally active TRPV4 blocker that inhibits Ca(2+) influx through recombinant TRPV4 channels and native endothelial TRPV4 currents. In isolated rodent and canine lungs, TRPV4 blockade prevented the increased vascular permeability and resultant pulmonary edema associated with elevated PVP. Furthermore, in both acute and chronic HF models, GSK2193874 pretreatment inhibited the formation of pulmonary edema and enhanced arterial oxygenation. Finally, GSK2193874 treatment resolved pulmonary edema already established by myocardial infarction in mice. These findings identify a crucial role for TRPV4 in the formation of HF-induced pulmonary edema and suggest that TRPV4 blockade is a potential therapeutic strategy for HF patients.

  5. Active Galactic Videos: A YouTube Channel for Astronomy Education and Outreach

    Science.gov (United States)

    Austin, Carmen; Calahan, Jenny; Resi Baucco, Alexandria; Bullivant, Christopher William; Eckley, Ross; Ekstrom, W. Haydon; Fitzpatrick, M. Ryleigh; Genovese, Taylor Fay; Impey, Chris David; Libby, Kaitlin; McCaw, Galen; Olmedo, Alexander N.; Ritter, Joshua; Wenger, Matthew; Williams, Stephanie

    2017-01-01

    Active Galactic Videos is an astronomy-focused YouTube channel run by a team at the University of Arizona. The channel has two main purposes: to produce educational content for public audiences, and to learn about astronomy and to open a window into the world of professional astronomy by showcasing the work done at Steward Observatory and in Southern Arizona. Our team consists of faculty, staff, and students from a variety of backgrounds including: astronomy, education, film, music, english, and writing. In addition to providing educational content for public audiences, this project provides opportunities for undergraduate students to learn about astronomy content, educational practice, and science communication while developing the practical skills needed to write, film, score, direct, and edit videos that effectively engage and teach viewers about topics in astronomy. The team has produced various styles of video: presentational, interviews, musical/poetic, and documentaries. In addition to YouTube, the Active Galactic Videos team maintains a social media presence on Facebook, Twitter, and Instagram. These help to widely distribute the content as well as to publicize the main Youtube channel. In addition to providing an overview of our educational work, this poster will present a year's worth of online analytics that we are using to better understand our audience, to examine what videos have been popular and successful and how people are accessing our content. We will present our experience in order to help others learn about improving astronomy education online, and astronomy communication and outreach in general.

  6. Phentolamine relaxes human corpus cavernosum by a nonadrenergic mechanism activating ATP-sensitive K+ channel.

    Science.gov (United States)

    Silva, L F G; Nascimento, N R F; Fonteles, M C; de Nucci, G; Moraes, M E; Vasconcelos, P R L; Moraes, M O

    2005-01-01

    To investigate the pharmacodynamics of phentolamine in human corpus cavernosum (HCC) with special attention to the role of the K+ channels. Strips of HCC precontracted with nonadrenergic stimuli and kept in isometric organ bath immersed in a modified Krebs-Henseleit solution enriched with guanethidine and indomethacine were used in order to study the mechanism of the phentolamine-induced relaxation. Phentolamine caused relaxation (approximately 50%) in HCC strips precontracted with K+ 40 mM. This effect was not blocked by tetrodotoxin (1 microM) (54.6+/-4.6 vs 48.9+/-6.4%) or (atropine (10 microM) (52.7+/-6.5 vs 58.6+/-5.6%). However, this relaxation was significantly attenuated by L-NAME (100 microM) (59.7+/-5.8 vs 27.8+/-7.1%; Pphentolamine relaxations (54.6+/-4.6 vs 59.3+/-5.2%). Glibenclamide (100 microM), an inhibitor of K(ATP)-channel, caused a significant inhibition (56.7+/-6.3 vs 11.3+/-2.3%; Pphentolamine-induced relaxation. In addition, the association of glibenclamide and L-NAME almost abolished the phentolamine-mediated relaxation (54.6+/-5.6 vs 5.7+/-1.4%; Pphentolamine relaxes HCC by a nonadrenergic-noncholinergic mechanism dependent on nitric oxide synthase activity and activation of K(ATP)-channel.

  7. The large conductance calcium-activated K(+) channel interacts with the small GTPase Rab11b.

    Science.gov (United States)

    Sokolowski, Sophia; Harvey, Margaret; Sakai, Yoshihisa; Jordan, Amy; Sokolowski, Bernd

    2012-09-21

    The transduction of sound by the receptor or hair cells of the cochlea leads to the activation of ion channels found in the basal and lateral regions of these cells. Thus, the processing of these transduced signals to the central nervous system is tied to the regulation of baso-lateral ion channels. The large conductance calcium-activated potassium or BK channel was revealed to interact with the small GTPase, Rab11b, which is one of many Rabs found in various endosomal pathways. Immunoelectron microscopy showed the colocalization of these two proteins in receptor cells and auditory neurons. Using Chinese hamster ovary cells as a heterologous expression system, Rab11b increased or decreased BK expression, depending on the overexpression or RNAi knockdown of Rab, respectively. Additional mutation analyses, using a yeast two-hybrid assay, suggested that this GTPase moderately interacts within a region of BK exclusive of the N- or C-terminal tails. These data suggest that this small GTPase regulates BK in a slow recycling process through the endocytic compartment and to the plasmalemma.

  8. Voltage-dependent gating of hyperpolarization-activated, cyclic nucleotide-gated pacemaker channels: molecular coupling between the S4-S5 and C-linkers.

    Science.gov (United States)

    Decher, Niels; Chen, Jun; Sanguinetti, Michael C

    2004-04-02

    Hyperpolarization-activated, cyclic nucleotide-gated (HCN) channels have a transmembrane topology that is highly similar to voltage-gated K(+) channels, yet HCN channels open in response to membrane hyperpolarization instead of depolarization. The structural basis for the "inverted" voltage dependence of HCN gating and how voltage sensing by the S1-S4 domains is coupled to the opening of the intracellular gate formed by the S6 domain are unknown. Coupling could arise from interaction between specific residues or entire transmembrane domains. We previously reported that the mutation of specific residues in the S4-S5 linker of HCN2 (i.e. Tyr-331 and Arg-339) prevented normal channel closure presumably by disruption of a crucial interaction with the activation gate. Here we hypothesized that the C-linker, a carboxyl terminus segment that connects S6 to the cyclic nucleotide binding domain, interacts with specific residues of the S4-S5 linker to mediate coupling. The recently solved structure of the C-linker of HCN2 indicates that an alpha-helix (the A'-helix) is located near the end of each S6 domain, the presumed location of the activation gate. Ala-scanning mutagenesis of the end of S6 and the A'-helix identified five residues that were important for normal gating as mutations disrupted channel closure. However, partial deletion of the C-linker indicated that the presence of only two of these residues was required for normal coupling. Further mutation analyses suggested that a specific electrostatic interaction between Arg-339 of the S4-S5 linker and Asp-443 of the C-linker stabilizes the closed state and thus participates in the coupling of voltage sensing and activation gating in HCN channels.

  9. Recombinant goose-type lysozyme in channel catfish: lysozyme activity and efficacy as plasmid DNA immunostimulant against Aeromonas hydrophila infection.

    Science.gov (United States)

    Pridgeon, Julia W; Klesius, Phillip H; Dominowski, Paul J; Yancey, Robert J; Kievit, Michele S

    2013-10-01

    The objectives of this study were: 1) to investigate whether recombinant channel catfish lysozyme-g (CC-Lys-g) produced in Escherichia coli expression system possesses any lysozyme activity; and 2) to evaluate whether channel catfish lysozyme-g plasmid DNA could be used as an immunostimulant to protect channel catfish against Aeromonas hydrophila infection. Recombinant CC-Lys-g produced in E. coli expression system exhibited significant (P recombinant channel catfish lysozyme-g (pcDNA-Lys-g) was transfected in channel catfish gill cells G1B, the over-expression of pcDNA-Lys-g offered significant (P DNA injection. Macrophages of fish injected with pcDNA-Lys-g produced significantly (P DNA injection. Taken together, our results suggest that pcDNA-Lys-g could be used as a novel immunostimulant to offer immediate protection to channel catfish against A. hydrophila infection.

  10. Structural basis for ether-a-go-go-related gene K+ channel subtype-dependent activation by niflumic acid.

    Science.gov (United States)

    Fernandez, David; Sargent, John; Sachse, Frank B; Sanguinetti, Michael C

    2008-04-01

    Niflumic acid [2-((3-(trifluoromethyl)phenyl)amino)-3-pyridinecarboxylic acid, NFA] is a nonsteroidal anti-inflammatory drug that also blocks or modulates the gating of a wide spectrum of ion channels. Here we investigated the mechanism of channel activation by NFA on ether-a-go-go-related gene (ERG) K(+) channel subtypes expressed in Xenopus laevis oocytes using two-electrode voltage-clamp techniques. NFA acted from the extracellular side of the membrane to differentially enhance ERG channel currents independent of channel state. At 1 mM, NFA shifted the half-point for activation by -6, -18, and -11 mV for ERG1, ERG2, and ERG3 channels, respectively. The half-point for channel inactivation was shifted by +5 to +9 mV by NFA. The structural basis for the ERG subtype-specific response to NFA was explored with chimeric channels and site-directed mutagenesis. The molecular determinants of enhanced sensitivity of ERG2 channels to NFA were isolated to an Arg and a Thr triplet in the extracellular S3-S4 linker.

  11. Dual activity of quinolinate synthase: triose phosphate isomerase and dehydration activities play together to form quinolinate.

    Science.gov (United States)

    Reichmann, Debora; Couté, Yohann; Ollagnier de Choudens, Sandrine

    2015-10-27

    Quinolinate synthase (NadA) is an Fe4S4 cluster-containing dehydrating enzyme involved in the synthesis of quinolinic acid (QA), the universal precursor of the essential coenzyme nicotinamide adenine dinucleotide. The reaction catalyzed by NadA is not well understood, and two mechanisms have been proposed in the literature that differ in the nature of the molecule (DHAP or G-3P) that condenses with iminoaspartate (IA) to form QA. In this article, using biochemical approaches, we demonstrate that DHAP is the triose that condenses with IA to form QA. The capacity of NadA to use G-3P is due to its previously unknown triose phosphate isomerase activity.

  12. Mineralocorticoids decrease the activity of the apical small-conductance K channel in the cortical collecting duct.

    Science.gov (United States)

    Wei, Yuan; Babilonia, Elisa; Sterling, Hyacinth; Jin, Yan; Wang, Wen-Hui

    2005-11-01

    We used the patch-clamp technique to examine the effect of DOCA treatment (2 mg/kg) on the apical small-conductance K (SK) channels, epithelial Na channels (ENaC), and the basolateral 18-pS K channels in the cortical collecting duct (CCD). Treatment of rats with DOCA for 6 days significantly decreased the plasma K from 3.8 to 3.1 meq and reduced the activity of the SK channel, defined as NP(o), from 1.3 in the CCD of control rats to 0.6. In contrast, DOCA treatment significantly increased ENaC activity from 0.01 to 0.53 and the basolateral 18-pS K channel activity from 0.67 to 1.63. Moreover, Western blot analysis revealed that DOCA treatment significantly increased the expression of the nonreceptor type of protein tyrosine kinase (PTK), cSrc, and the tyrosine phosphorylation of ROMK in the renal cortex and outer medulla. The possibility that decreases in apical SK channel activity induced by DOCA treatment were the result of stimulation of PTK activity was further supported by experiments in which inhibition of PTK with herbimycin A significantly increased NP(o) from 0.6 to 2.1 in the CCD from rats receiving DOCA. Also, when rats were fed a high-K (10%) diet, DOCA treatment did not increase the expression of c-Src and decrease the activity of the SK channel in the CCD. We conclude that DOCA treatment decreased the apical SK channel activity in rats on a normal-K diet and that an increase in PTK expression may be responsible for decreased channel activity in the CCD from DOCA-treated rats.

  13. Tectonic Activity on Pluto After the Charon-Forming Impact

    CERN Document Server

    Barr, Amy C

    2014-01-01

    The Pluto-Charon system, likely formed from an impact, has reached the endpoint of its tidal evolution. During its evolution into the dual-synchronous state, the equilibrium tidal figures of Pluto and Charon would have also evolved as angular momentum was transferred from Pluto's spin to Charon's orbit. The rate of tidal evolution is controlled by Pluto's interior physical and thermal state. We examine three interior models for Pluto: an undifferentiated rock/ice mixture, differentiated with ice above rock, and differentiated with an ocean. For the undifferentiated case without an ocean, the Pluto-Charon binary does not evolve to its current state unless its internal temperature $T_i>200$ K, which would likely lead to strong tidal heating, melting, and differentiation. Without an ocean, Pluto's interior temperature must be higher than 240 K for Charon to evolve on a time scale less than the age of the solar system. Further tidal heating would likely create an ocean. If New Horizons finds evidence of ancient t...

  14. Differential modulation of TWIK-related K(+) channel (TREK) and TWIK-related acid-sensitive K(+) channel 2 (TASK2) activity by pyrazole compounds.

    Science.gov (United States)

    Kim, Hyun Jong; Woo, Joohan; Nam, Yuran; Nam, Joo Hyun; Kim, Woo Kyung

    2016-11-15

    Pyrazole derivatives were originally suggested as selective blockers of the transient receptor potential cation 3 (TRPC3) and channel. In particular, pyr3 and 10 selectively inhibit TRPC3, whereas pyr2 (BTP2) and 6 inhibit ORAI1. However, their effects on background K(+) channel activity have not been elucidated. In this study, the effects of BTP2, pyr3, pyr6, and pyr10 were studied on cloned human TWIK-related K(+) channels (TREKs) and TWIK-related acid-sensitive K(+) channel 2 (TASK-2) channels, which modulate Ca(2+) signaling by controlling membrane potential, in HEK293T-overexpressing cells by using a whole-cell patch clamp technique. Pyr3 potently inhibited TREK-1 (ITREK1), TREK-2 (ITREK2), and TASK2 current (ITASK-2) with half-maximal inhibitory concentrations (IC50) of 0.89±0.27, 1.95±1.44, and 2.42±0.39µM, respectively. BTP2 slightly inhibited ITASK-2 (80.3±2.5% at 100μM). In contrast, pyr6 at 100µM potentiated ITREK1 and ITREK2 by approximately 2.6- and 3.6-fold compared to the control and inhibited ITASK2 (38.7±9.2%). Pyr10 showed a subtype-specific inhibition of ITREK1 but not ITREK2. It also inhibited ITASK2 (70.9±3.1% at 100μM). To the best of our knowledge, this study is the first to describe the differential modulation of TREKs and TASK2 channels by pyrazole derivatives, previously used as inhibitors of TRPC3 and ORAI1. Therefore, studies using these drugs should consider their modulation of other channels such as TREK and TASK-2.

  15. Jamming Games in the MIMO Wiretap Channel With an Active Eavesdropper

    CERN Document Server

    Mukherjee, Amitav

    2010-01-01

    This paper investigates reliable and covert transmission strategies in a MIMO wiretap channel with a transmitter, receiver and an adversarial wiretapper, each equipped with multiple antennas. In a departure from existing work, the wiretapper possesses the dual capability to act either as a passive eavesdropper or as an active jammer, under a halfduplex constraint. The transmitter therefore faces a choice between allocating all of its power for data, or broadcasting artificial noise along with the information signal in order to selectively jam the eavesdropper (assuming its instantaneous channel state is unknown). To examine the resulting tradeoffs for both agents, we model the network as a two-person zero-sum game with the ergodic MIMO secrecy rate as the payoff function. We first quantify and rank the various possible MIMO secrecy rate outcomes of the actions available to each player, and derive asymptotic expressions for the same. We then examine conditions for the existence of pure and mixed Nash equilibri...

  16. Acacetin Blocks Kv1.3 Channels and Inhibits Human T Cell Activation

    Directory of Open Access Journals (Sweden)

    Ning Zhao

    2014-10-01

    Full Text Available Backgrounds/Aims: Acacetin, a natural flavonoid compound, has been proven to exert anti-inflammatory and immunomodulatory effects. Kv1.3 channels, highly expressed in human T cells, are attractive therapeutic targets to treat inflammatory and immunological disorders. The present study was designed to characterize the inhibition of Kv1.3 channels by Acacetin in human T cells and examine its role in T cell activation. Methods: Whole-cell patch-clamp was applied to record the Kv1.3 and KCa currents in human T cells; Western blot was used to detect Kv1.3 expression as well as NFAT1 and NF-κB activity; Fluo-4, CCK-8 and an ELISA kit were used to measure Ca2+ influx, proliferation, and IL-2 secretion, respectively. Results: Acacetin decreased the Kv1.3 current, accelerated the decay rate and negatively shifted the steady-state inactivation curves in a concentration-dependent manner. The IC50 values at +40 mV for peak and the current at end of pulse were 21.09 ± 2.75 and 3.63 ± 0.25 µmol/L, respectively. Treatment with Acacetin for 24 h significantly inhibited Kv1.3 protein expression. Additionally, paralleling Kv1.3 inhibition, Acacetin also inhibited Ca2+ influx, the Ca2+-activated transcription factors NFAT1, NF-κB p65/p50 activity, and proliferation as well as IL-2 production. Small interfering RNA against Kv1.3 reduced the inhibitory effect of Acacetin on IL-2 secretion. Conclusions: Acacetin blocks the Kv1.3 channel and inhibits human T cell activation. This action most likely contributes to its immunomodulatory and anti-inflammatory actions.

  17. A dual action of saturated fatty acids on electrical activity in rat pancreatic β-cells. Role of volume-regulated anion channel and KATP channel currents.

    Science.gov (United States)

    Best, L; Jarman, E; Brown, P D

    2011-03-15

    Free fatty acids (FFAs) exert complex actions on pancreatic β-cells. Typically, an initial potentiation of insulin release is followed by a gradual impairment of β-cell function, the latter effect being of possible relevance to hyperlipidaemia in type 2 diabetes mellitus. The molecular actions of FFAs are poorly understood. The present study investigated the acute effects of saturated FFAs on electrophysiological responses of rat pancreatic β-cells. Membrane potential and KATP channel activity were recorded using the perforated patch technique. Volume-regulated anion channel (VRAC) activity was assessed from conventional whole-cell recordings. Cell volume regulation was measured using a video-imaging technique. Addition of octanoate caused a transient potentiation of glucose-induced electrical activity, followed by a gradual hyper-polarisation and a prolonged inhibition of electrical activity. Octanoate caused an initial increase in VRAC activity followed by a secondary inhibition coinciding with increased KATP channel activity. Similar effects were observed with palmitate and 2-bromopalmitate whereas butyrate was virtually ineffective. Octanoate and palmitate also exerted a dual effect on electrical activity evoked by tolbutamide. Octanoate significantly attenuated cell volume regulation in hypotonic solutions, consistent with VRAC inhibition. It is concluded that medium and long chain FFAs have a dual action on glucose-induced electrical activity in rat pancreatic β-cells: an initial stimulatory effect followed by a secondary inhibition. These effects appear to be the result of reciprocal actions on VRAC and KATP channel currents, and could contribute towards the stimulatory and inhibitory actions of FFAs on pancreatic β-cell function.

  18. Social influence and adolescent health-related physical activity in structured and unstructured settings: role of channel and type.

    Science.gov (United States)

    Spink, Kevin S; Wilson, Kathleen S; Ulvick, Jocelyn

    2012-08-01

    Social influence channels (e.g., parents) and types (e.g., compliance) have each been related to physical activity independently, but little is known about how these two categories of influence may operate in combination. This study examined the relationships between various combinations of social influence and physical activity among youth across structured and unstructured settings. Adolescents (N=304), classified as high or low active, reported the social influence combinations they received for being active. Participants identified three channels and three types of influence associated with being active. For structured activity, compliance with peers and significant others predicted membership in the high active group (values of psetting, peer compliance (p= .009) and conformity (p= .019) were associated with active group membership. These findings reinforce considering both setting, as well as the channel/type combinations of social influence, when examining health-related physical activity.

  19. SUMOylation of the Hyperpolarization-Activated Cyclic Nucleotide-Gated Channel 2 Increases Surface Expression and the Maximal Conductance of the Hyperpolarization-Activated Current

    Science.gov (United States)

    Parker, Anna R.; Welch, Meghyn A.; Forster, Lori A.; Tasneem, Sarah M.; Dubhashi, Janhavi A.; Baro, Deborah J.

    2017-01-01

    Small Ubiquitin-like Modifier (SUMO) is a ∼10 kDa peptide that can be post-translationally added to a lysine (K) on a target protein to facilitate protein–protein interactions. Recent studies have found that SUMOylation can be regulated in an activity-dependent manner and that ion channel SUMOylation can alter the biophysical properties and surface expression of the channel. Hyperpolarization-activated cyclic nucleotide-gated (HCN) channel surface expression can be regulated in an activity-dependent manner through unknown processes. We hypothesized that SUMOylation might influence the surface expression of HCN2 channels. In this manuscript, we show that HCN2 channels are SUMOylated in the mouse brain. Baseline levels of SUMOylation were also observed for a GFP-tagged HCN2 channel stably expressed in Human embryonic kidney (Hek) cells. Elevating GFP-HCN2 channel SUMOylation above baseline in Hek cells led to an increase in surface expression that augmented the hyperpolarization-activated current (Ih) mediated by these channels. Increased SUMOylation did not alter Ih voltage-dependence or kinetics of activation. There are five predicted intracellular SUMOylation sites on HCN2. Site-directed mutagenesis indicated that more than one K on the GFP-HCN2 channel was SUMOylated. Enhancing SUMOylation at one of the five predicted sites, K669, led to the increase in surface expression and Ih Gmax. The role of SUMOylation at additional sites is currently unknown. The SUMOylation site at K669 is also conserved in HCN1 channels. Aberrant SUMOylation has been linked to neurological diseases that also display alterations in HCN1 and HCN2 channel expression, such as seizures and Parkinson’s disease. This work is the first report that HCN channels can be SUMOylated and that this can regulate surface expression and Ih. PMID:28127275

  20. The ethylene bis-dithiocarbamate fungicide Mancozeb activates voltage-gated KCNQ2 potassium channel.

    Science.gov (United States)

    Li, Ping; Zhu, Jin; Kong, Qingya; Jiang, Baifeng; Wan, Xia; Yue, Jinfeng; Li, Min; Jiang, Hualiang; Li, Jian; Gao, Zhaobing

    2013-06-07

    Mancozeb (manganese/zinc ethylene bis-dithiocarbamate) is an organometallic fungicide that has been associated with human neurotoxicity and neurodegeneration. In a high-throughput screen for modulators of KCNQ2 channel, a fundamental player modulating neuronal excitability, Mancozeb, was found to significantly potentiate KCNQ2 activity. Mancozeb was validated electrophysiologically as a KCNQ2 activator with an EC50 value of 0.92±0.23μM. Further examination showed that manganese but not zinc ethylene bis-dithiocarbamate is the active component for the positive modulation effects. In addition, the compounds are effective when the metal ions are substituted by iron but lack potentiation activity when the metal ions are substituted by sodium, signifying the importance of the metal ion. However, the iron (Fe(3+)) alone, organic ligands alone or the mixture of iron with the organic ligand did not show any potentiation effect, suggesting as the active ingredient is a specific complex rather than two separate additive or synergistic components. Our study suggests that potentiation on KCNQ2 potassium channels might be the possible mechanism of Mancozeb toxicity in the nervous system.

  1. Small-conductance Ca2+-activated potassium type 2 channels regulate the formation of contextual fear memory.

    Directory of Open Access Journals (Sweden)

    Saravana R K Murthy

    Full Text Available Small-conductance, Ca2+ activated K+ channels (SK channels are expressed at high levels in brain regions responsible for learning and memory. In the current study we characterized the contribution of SK2 channels to synaptic plasticity and to different phases of hippocampal memory formation. Selective SK2 antisense-treatment facilitated basal synaptic transmission and theta-burst induced LTP in hippocampal brain slices. Using the selective SK2 antagonist Lei-Dab7 or SK2 antisense probes, we found that hippocampal SK2 channels are critical during two different time windows: 1 blockade of SK2 channels before the training impaired fear memory, whereas, 2 blockade of SK2 channels immediately after the training enhanced contextual fear memory. We provided the evidence that the post-training cleavage of the SK2 channels was responsible for the observed bidirectional effect of SK2 channel blockade on memory consolidation. Thus, Lei-Dab7-injection before training impaired the C-terminal cleavage of SK2 channels, while Lei-Dab7 given immediately after training facilitated the C-terminal cleavage. Application of the synthetic peptide comprising a leucine-zipper domain of the C-terminal fragment to Jurkat cells impaired SK2 channel-mediated currents, indicating that the endogenously cleaved fragment might exert its effects on memory formation by blocking SK2 channel-mediated currents. Our present findings suggest that SK2 channel proteins contribute to synaptic plasticity and memory not only as ion channels but also by additionally generating a SK2 C-terminal fragment, involved in both processes. The modulation of fear memory by down-regulating SK2 C-terminal cleavage might have applicability in the treatment of anxiety disorders in which fear conditioning is enhanced.

  2. Single channel currents of different amplitude activated by glutamate in a tonic (slow) crayfish muscle.

    Science.gov (United States)

    Finger, W; Pareto, A

    1987-09-11

    Single channel currents were recorded by means of the patch-clamp technique from a tonic (slow) crayfish muscle in the presence of 5 mM glutamate. The experiments were carried out with 'Gigaohm-seals' in the 'cell-attached' mode at 15-17 degrees C. Five classes of single channel currents with different mean amplitudes were resolved: i1 = -0.75 +/- 0.43 (S.D.) pA, i2 = -1.4 +/- 0.4 pA, i3 = -3.5 +/- 0.63 pA, i4 = -8.5 +/- 0.92 pA and i5 approximately equal to 2 X i4, i2, i3 and i4 were recorded at resting membrane potential, Eo approximately equal to -80 mV (pipette potential Vp = 0), while i1 and i5 were recorded at 40 mV hyperpolarized to Eo (Vp = +40 mV). The current most frequently seen was i4 which is the excitatory glutamate-activated single channel current recorded previously by Franke et al. The membrane reversal potentials and channel conductances for i2 and i4 were estimated to be +60 mV (Eo + 140 mV), 13 pS for i2 and +40 mV (Eo + 120 mV), 80 pS for i4. It was assumed that up to 40 i1 currents could superpose in a single patch to generate a DC current of up to -30 pA with current fluctuations the intensity of which increased with the DC current amplitude. Often variable combinations of i1 to i4 currents could be recorded simultaneously in a single patch. In particular, simultaneous activity of i1, i4; i2, i4 and i3, i4 currents was observed in different single patches.

  3. Heat of Hydration of Low Activity Cementitious Waste Forms

    Energy Technology Data Exchange (ETDEWEB)

    Nasol, D. [Savannah River Site (SRS), Aiken, SC (United States). Savannah River National Lab. (SRNL)

    2015-07-23

    During the curing of secondary waste grout, the hydraulic materials in the dry mix react exothermally with the water in the secondary low-activity waste (LAW). The heat released, called the heat of hydration, can be measured using a TAM Air Isothermal Calorimeter. By holding temperature constant in the instrument, the heat of hydration during the curing process can be determined. This will provide information that can be used in the design of a waste solidification facility. At the Savannah River National Laboratory (SRNL), the heat of hydration and other physical properties are being collected on grout prepared using three simulants of liquid secondary waste generated at the Hanford Site. From this study it was found that both the simulant and dry mix each had an effect on the heat of hydration. It was also concluded that the higher the cement content in the dry materials mix, the greater the heat of hydration during the curing of grout.

  4. Surface active complexes formed between keratin polypeptides and ionic surfactants.

    Science.gov (United States)

    Pan, Fang; Lu, Zhiming; Tucker, Ian; Hosking, Sarah; Petkov, Jordan; Lu, Jian R

    2016-12-15

    Keratins are a group of important proteins in skin and hair and as biomaterials they can provide desirable properties such as strength, biocompatibility, and moisture regaining and retaining. The aim of this work is to develop water-soluble keratin polypeptides from sheep wool and then explore how their surface adsorption behaves with and without surfactants. Successful preparation of keratin samples was demonstrated by identification of the key components from gel electrophoresis and the reproducible production of gram scale samples with and without SDS (sodium dodecylsulphate) during wool fibre dissolution. SDS micelles could reduce the formation of disulphide bonds between keratins during extraction, reducing inter-molecular crosslinking and improving keratin polypeptide solubility. However, Zeta potential measurements of the two polypeptide batches demonstrated almost identical pH dependent surface charge distributions with isoelectric points around pH 3.5, showing complete removal of SDS during purification by dialysis. In spite of different solubility from the two batches of keratin samples prepared, very similar adsorption and aggregation behavior was revealed from surface tension measurements and dynamic light scattering. Mixing of keratin polypeptides with SDS and C12TAB (dodecyltrimethylammonium bromide) led to the formation of keratin-surfactant complexes that were substantially more effective at reducing surface tension than the polypeptides alone, showing great promise in the delivery of keratin polypeptides via the surface active complexes. Neutron reflection measurements revealed the coexistence of surfactant and keratin polypeptides at the interface, thus providing the structural support to the observed surface tension changes associated with the formation of the surface active complexes.

  5. Search after new agents for hyperpolarization-activated and cyclic nucleotide-gated ion channels; Suche nach neuen Wirkstoffen fuer Hyperpolarisationsaktivierte und zyklisch Nukleotid-gesteuerte Ionenkanaele

    Energy Technology Data Exchange (ETDEWEB)

    Struenker, T.

    2005-12-01

    Rhythmic activity of single cells or cellular networks is a common feature of most organisms. Cellular rhythms govern the beating of the heart, cycles of sleep and wakefulness, breathing, and the release of hormones. The endogenous rhythmic activity of many neurons and cardiac relies on a complex interplay between several distinct ion channels. In particular, one type of ion channel plays a prominent role in the control of rhythmic electrical activity because it determines the frequency of the oscillations. The activity of the channels is thus setting the ''pace'' of the activity; therefore, these channels are often referred to as ''pacemaker'' channels. Despite their obvious physiological importance it hasn't been until a few years ago that the genes encoding pacemaker channels have been identified. Because both hyperpolarization and cyclic nucleotides are key elements that control their activity, pacemaker channels have now been designated hyperpolarization-activated and cyclic nucleotide-gated (HCN) channels. From a scientific as well as medical point of view, HCN channels are interesting drug targets. Only a few substances are known that specifically affect HCN channels. In the present study, a microtiter plate-based high throughput screening assay for HCN1 and HCN4 channels was developed. With this assay, known drugs for HCN channels were characterized. Subsequently, venoms of snails, spiders, scorpions, and snakes were screened for toxins affecting HCN channel activity. A few venoms were identified that possibly contain drugs that act on HCN channels. (orig.)

  6. The small molecule NS11021 is a potent and specific activator of Ca2+-activated big-conductance K+ channels

    DEFF Research Database (Denmark)

    Bentzen, Bo Hjorth; Nardi, Antonio; Calloe, Kirstine

    2007-01-01

    -channel analysis revealed that NS11021 increased the open probability of the channel by altering gating kinetics without affecting the single-channel conductance. NS11021 (10 microM) influenced neither a number of cloned Kv channels nor endogenous Na(+) and Ca(2+) channels (L- and T-type) in guinea pig cardiac...

  7. Downregulation of transient K+ channels in dendrites of hippocampal CA1 pyramidal neurons by activation of PKA and PKC.

    Science.gov (United States)

    Hoffman, D A; Johnston, D

    1998-05-15

    We have reported recently a high density of transient A-type K+ channels located in the distal dendrites of CA1 hippocampal pyramidal neurons and shown that these channels shape EPSPs, limit the back-propagation of action potentials, and prevent dendritic action potential initiation (). Because of the importance of these channels in dendritic signal propagation, their modulation by protein kinases would be of significant interest. We investigated the effects of activators of cAMP-dependent protein kinase (PKA) and the Ca2+-dependent phospholipid-sensitive protein kinase (PKC) on K+ channels in cell-attached patches from the distal dendrites of hippocampal CA1 pyramidal neurons. Inclusion of the membrane-permeant PKA activators 8-bromo-cAMP (8-br-cAMP) or forskolin in the dendritic patch pipette resulted in a depolarizing shift in the activation curve for the transient channels of approximately 15 mV. Activation of PKC by either of two phorbol esters also resulted in a 15 mV depolarizing shift of the activation curve. Neither PKA nor PKC activation affected the sustained or slowly inactivating component of the total outward current. This downregulation of transient K+ channels in the distal dendrites may be responsible for some of the frequently reported increases in cell excitability found after PKA and PKC activation. In support of this hypothesis, we found that activation of either PKA or PKC significantly increased the amplitude of back-propagating action potentials in distal dendrites.

  8. H-ras transformation sensitizes volume-activated anion channels and increases migratory activity of NIH3T3 fibroblasts

    DEFF Research Database (Denmark)

    Schneider, Linda; Klausen, Thomas K; Stock, Christian;

    2008-01-01

    The expression of the H-ras oncogene increases the migratory activity of many cell types and thereby contributes to the metastatic behavior of tumor cells. Other studies point to an involvement of volume-activated anion channels (VRAC) in (tumor) cell migration. In this paper, we tested whether...... VRACs are required for the stimulation of cell migration upon expression of the H-ras oncogene. We compared VRAC activation and migration of wild-type and H-ras-transformed NIH3T3 fibroblasts by means of patch-clamp techniques and time-lapse video microscopy. Both cell types achieve the same degree...... of VRAC activation upon maximal stimulation, induced by reducing extracellular osmolarity from 300 to 190 mOsm/l. However, upon physiologically relevant reductions in extracellular osmolarity (275 mOsm/l), the level of VRAC activation is almost three times higher in H-ras-transformed compared to wild...

  9. PAD-MAC: primary user activity-aware distributed MAC for multi-channel cognitive radio networks.

    Science.gov (United States)

    Ali, Amjad; Piran, Md Jalil; Kim, Hansoo; Yun, Jihyeok; Suh, Doug Young

    2015-03-30

    Cognitive radio (CR) has emerged as a promising technology to solve problems related to spectrum scarcity and provides a ubiquitous wireless access environment. CR-enabled secondary users (SUs) exploit spectrum white spaces opportunistically and immediately vacate the acquired licensed channels as primary users (PUs) arrive. Accessing the licensed channels without the prior knowledge of PU traffic patterns causes severe throughput degradation due to excessive channel switching and PU-to-SU collisions. Therefore, it is significantly important to design a PU activity-aware medium access control (MAC) protocol for cognitive radio networks (CRNs). In this paper, we first propose a licensed channel usage pattern identification scheme, based on a two-state Markov model, and then estimate the future idle slots using previous observations of the channels. Furthermore, based on these past observations, we compute the rank of each available licensed channel that gives SU transmission success assessment during the estimated idle slot. Secondly, we propose a PU activity-aware distributed MAC (PAD-MAC) protocol for heterogeneous multi-channel CRNs that selects the best channel for each SU to enhance its throughput. PAD-MAC controls SU activities by allowing them to exploit the licensed channels only for the duration of estimated idle slots and enables predictive and fast channel switching. To evaluate the performance of the proposed PAD-MAC, we compare it with the distributed QoS-aware MAC (QC-MAC) and listen-before-talk MAC schemes. Extensive numerical results show the significant improvements of the PAD-MAC in terms of the SU throughput, SU channel switching rate and PU-to-SU collision rate.

  10. Pregnenolone sulfate activates basic region leucine zipper transcription factors in insulinoma cells: role of voltage-gated Ca2+ channels and transient receptor potential melastatin 3 channels.

    Science.gov (United States)

    Müller, Isabelle; Rössler, Oliver G; Thiel, Gerald

    2011-12-01

    The neurosteroid pregnenolone sulfate activates a signaling cascade in insulinoma cells involving activation of extracellular signal-regulated protein kinase and enhanced expression of the transcription factor Egr-1. Here, we show that pregnenolone sulfate stimulation leads to a significant elevation of activator protein-1 (AP-1) activity in insulinoma cells. Expression of the basic region leucine zipper (bZIP) transcription factors c-Jun and c-Fos is up-regulated in insulinoma cells and pancreatic β-cells in primary culture after pregnenolone sulfate stimulation. Up-regulation of a chromatin-embedded c-Jun promoter/luciferase reporter gene transcription in pregnenolone sulfate-stimulated insulinoma cells was impaired when the AP-1 binding sites were mutated, indicating that these motifs function as pregnenolone sulfate response elements. In addition, phosphorylation of cAMP response element (CRE)-binding protein is induced and transcription of a CRE-controlled reporter gene is stimulated after pregnenolone sulfate treatment, indicating that the CRE functions as a pregnenolone sulfate response element as well. Pharmacological and genetic experiments revealed that both L-type Ca(2+) channels and transient receptor potential melastatin 3 (TRPM3) channels are essential for connecting pregnenolone sulfate stimulation with enhanced AP-1 activity and bZIP-mediated transcription in insulinoma cells. In contrast, pregnenolone sulfate stimulation did not enhance AP-1 activity or c-Jun and c-Fos expression in pituitary corticotrophs that express functional L-type Ca(2+) channels but only trace amounts of TRPM3. We conclude that expression of L-type Ca(2+) channels is not sufficient to activate bZIP-mediated gene transcription by pregnenolone sulfate. Rather, additional expression of TRPM3 or depolarization of the cells is required to connect pregnenolone sulfate stimulation with enhanced gene transcription.

  11. Mono-Heteromeric Configurations of Gap Junction Channels Formed by Connexin43 and Connexin45 Reduce Unitary Conductance and Determine both Voltage Gating and Metabolic Flux Asymmetry

    Directory of Open Access Journals (Sweden)

    Guoqiang Zhong

    2017-05-01

    Full Text Available In cardiac tissues, the expression of multiple connexins (Cx40, Cx43, Cx45, and Cx30.2 is a requirement for proper development and function. Gap junctions formed by these connexins have distinct permeability and gating mechanisms. Since a single cell can express more than one connexin isoform, the formation of hetero-multimeric gap junction channels provides a tissue with an enormous repertoire of combinations to modulate intercellular communication. To study further the perm-selectivity and gating properties of channels containing Cx43 and Cx45, we studied two monoheteromeric combinations in which a HeLa cell co-transfected with Cx43 and Cx45 was paired with a cell expressing only one of these connexins. Macroscopic measurements of total conductance between cell pairs indicated a drastic reduction in total conductance for mono-heteromeric channels. In terms of Vj dependent gating, Cx43 homomeric connexons facing heteromeric connexons only responded weakly to voltage negativity. Cx45 homomeric connexons exhibited no change in Vj gating when facing heteromeric connexons. The distributions of unitary conductances (γj for both mono-heteromeric channels were smaller than predicted, and both showed low permeability to the fluorescent dyes Lucifer yellow and Rhodamine123. For both mono-heteromeric channels, we observed flux asymmetry regardless of dye charge: flux was higher in the direction of the heteromeric connexon for MhetCx45 and in the direction of the homomeric Cx43 connexon for MhetCx43. Thus, our data suggest that co-expression of Cx45 and Cx43 induces the formation of heteromeric connexons with greatly reduced permeability and unitary conductance. Furthermore, it increases the asymmetry for voltage gating for opposing connexons, and it favors asymmetric flux of molecules across the junction that depends primarily on the size (not the charge of the crossing molecules.

  12. Additional Observations of Actively Forming Lava Tubes and Associated Structures, Hawaii

    Science.gov (United States)

    Greeley, Ronald

    1972-01-01

    Extensive changes occurred after the initial observations (Greeley, 1971) of lava tube and channel formation associated with the eruption of Mauna Ulu. Individual vents, which apparently acted somewhat independently, merged by collapse of intervening sections to form an elongate trench. Lava erupted from the summit vent flowed down the trench to the lower end and drained through lava tubes into Alae lava lake. Alae lava lake is in turn drained occasionally by other lava tubes and lava tube networks.

  13. Wound Healing Activity of Topical Application Forms Based on Ayurveda

    Directory of Open Access Journals (Sweden)

    Hema Sharma Datta

    2011-01-01

    Full Text Available The traditional Indian medicine—Ayurveda, describes various herbs, fats, oils and minerals with anti-aging as well as wound healing properties. With aging, numerous changes occur in skin, including decrease in tissue cell regeneration, decrease in collagen content, loss of skin elasticity and mechanical strength. We prepared five topical anti-aging formulations using cow ghee, flax seed oil, Phyllanthus emblica fruits, Shorea robusta resin, Yashada bhasma as study materials. For preliminary efficacy evaluation of the anti-aging activity we chose excision and incision wound healing animal models and studied the parameters including wound contraction, collagen content and skin breaking strength which in turn is indicative of the tissue cell regeneration capacity, collagenation capacity and mechanical strength of skin. The group treated with the formulations containing Yashada bhasma along with Shorea robusta resin and flax seed oil showed significantly better wound contraction (P < .01, higher collagen content (P < .05 and better skin breaking strength (P < .01 as compared to control group; thus proposing them to be effective prospective anti-aging formulations.

  14. Biophysical characterization of KV3.1 potassium channel activating compounds.

    Science.gov (United States)

    Taskin, Bahar; von Schoubye, Nadia Lybøl; Sheykhzade, Majid; Bastlund, Jesper Frank; Grunnet, Morten; Jespersen, Thomas

    2015-07-05

    The effect of two positive modulators, RE1 and EX15, on the voltage-gated K(+) channel Kv3.1 was investigated using the whole-cell patch-clamp technique on HEK293 cells expressing Kv3.1a. RE1 and EX15 increased the Kv3.1 currents in a concentration-dependent manner with an EC50 value of 4.5 and 1.3µM, respectively. However, high compound concentrations caused an inhibition of the Kv3.1 current. The compound-induced activation of Kv3.1 channels showed a profound hyperpolarized shift in activation kinetics. 30µM RE1 shifted V1/2 from 5.63±0.31mV to -9.71±1.00mV and 10µM EX15 induced a shift from 10.77±0.32mV to -15.11±1.57mV. The activation time constant (Tauact) was reduced for both RE1 and EX15, with RE1 being the fastest activator. The deactivation time constant (Taudeact) was also markedly reduced for both RE1 and EX15, with EX15 inducing the most prominent effect. Furthermore, subjected to depolarizing pulses at 30Hz, both compounds were showing a use-dependent effect resulting in a reduction of the compound-mediated effect. However, during these conditions, RE1- and EX15-modified current amplitudes still exceeded the control condition amplitudes by up to 200%. In summary, the present study introduces the first detailed biophysical characterization of two new Kv3.1 channel modifying compounds with different modulating properties.

  15. Hydroclimatic signal and LBK cultural activity in the Upper and Lower Rhine, inferred from abandoned channel fill deposits

    Science.gov (United States)

    Berger, J. F.; Salvador, P. G.; Erkens, G.; Toonen, W. H. J.; Purdue, L.; Barra, A.; Houben, P.

    2012-04-01

    The Linear Band Ceramic (LBK) culture represents a major event in the spread of agriculture in Europe. Occupation particularly occurred in river valleys, with largest densities found along the rivers Danube, Elbe and Rhine. The interaction between the emergence of this culture and the dominant climatic and hydrological conditions is not yet fully established. As part of the ANR OBRESOC project, in which LBK activity is investigated in a transect from France (Marne river) to the catchment of the Danube river (Tisza), we studied palaeo-environmental changes in the Rhine valley between 7600-6600 cal. yrs. BP. Focus is on the Upper Rhine Graben and the Lower Rhine valley near the Rhine Delta apex, which is thought to be a peripheral region of LBK-activity. In these regions, a total of five cores from abandoned channels were analysed to reconstruct palaeo-environmental dynamics in vegetation and fluvial activity during the period of LBK development. Abandoned channel fills are excellent sites to perform detailed studies of palaeo-environmental dynamics, as they (i) form proximal locations to occupation sites of the LBK culture, (ii) act as efficient traps of sediments in which different environmental proxies are well preserved, (iii) contain well-datable material for the construction of detailed age-depth models, and (iv) provide a long proxy record, potentially over more than a millennium at a single site. On all cores, high resolution analysis of channel fill deposits (grain size and geophysical properties) and biotic proxies (micro-charcoal fluxes and pollen assemblages) were preformed to reconstruct palaeo-environmental signals, such as changes in fluvial activity, forest fires, and vegetation evolution, which may be related to agricultural activity, and climatic and hydrogeomorphic changes in the region. In this contribution we compare the results of the high-resolution core analyses (1,5 to 5m sequences for the studied timeframe) derived from the more densely

  16. NS19504: a novel BK channel activator with relaxing effect on bladder smooth muscle spontaneous phasic contractions.

    Science.gov (United States)

    Nausch, Bernhard; Rode, Frederik; Jørgensen, Susanne; Nardi, Antonio; Korsgaard, Mads P G; Hougaard, Charlotte; Bonev, Adrian D; Brown, William D; Dyhring, Tino; Strøbæk, Dorte; Olesen, Søren-Peter; Christophersen, Palle; Grunnet, Morten; Nelson, Mark T; Rønn, Lars C B

    2014-09-01

    Large-conductance Ca(2+)-activated K(+) channels (BK, KCa1.1, MaxiK) are important regulators of urinary bladder function and may be an attractive therapeutic target in bladder disorders. In this study, we established a high-throughput fluorometric imaging plate reader-based screening assay for BK channel activators and identified a small-molecule positive modulator, NS19504 (5-[(4-bromophenyl)methyl]-1,3-thiazol-2-amine), which activated the BK channel with an EC50 value of 11.0 ± 1.4 µM. Hit validation was performed using high-throughput electrophysiology (QPatch), and further characterization was achieved in manual whole-cell and inside-out patch-clamp studies in human embryonic kidney 293 cells expressing hBK channels: NS19504 caused distinct activation from a concentration of 0.3 and 10 µM NS19504 left-shifted the voltage activation curve by 60 mV. Furthermore, whole-cell recording showed that NS19504 activated BK channels in native smooth muscle cells from guinea pig urinary bladder. In guinea pig urinary bladder strips, NS19504 (1 µM) reduced spontaneous phasic contractions, an effect that was significantly inhibited by the specific BK channel blocker iberiotoxin. In contrast, NS19504 (1 µM) only modestly inhibited nerve-evoked contractions and had no effect on contractions induced by a high K(+) concentration consistent with a K(+) channel-mediated action. Collectively, these results show that NS19504 is a positive modulator of BK channels and provide support for the role of BK channels in urinary bladder function. The pharmacologic profile of NS19504 indicates that this compound may have the potential to reduce nonvoiding contractions associated with spontaneous bladder overactivity while having a minimal effect on normal voiding.

  17. The Inhibition by Oxaliplatin, a Platinum-Based Anti-Neoplastic Agent, of the Activity of Intermediate-Conductance Ca2+-Activated K+ Channels in Human Glioma Cells

    Directory of Open Access Journals (Sweden)

    Mei-Han Huang

    2015-10-01

    Full Text Available Oxaliplatin (OXAL is a third-generation organoplatinum which is effective against advanced cancer cells including glioma cells. How this agent and other related compounds interacts with ion channels in glioma cells is poorly understood. OXAL (100 µM suppressed the amplitude of whole-cell K+ currents (IK; and, either DCEBIO or ionomycin significantly reversed OXAL-mediated inhibition of IK in human 13-06-MG glioma cells. In OXAL-treated cells, TRAM-34 did not suppress IK amplitude in these cells. The intermediate-conductance Ca2+-activated K+ (IKCa channels subject to activation by DCEBIO and to inhibition by TRAM-34 or clotrimazole were functionally expressed in these cells. Unlike cisplatin, OXAL decreased the probability of IKCa-channel openings in a concentration-dependent manner with an IC50 value of 67 µM. No significant change in single-channel conductance of IKCa channels in the presence of OXAL was demonstrated. Neither large-conductance Ca2+-activated K+ channels nor inwardly rectifying K+ currents in these cells were affected in the presence of OXAL. OXAL also suppressed the proliferation and migration of 13-06-MG cells in a concentration- and time-dependent manner. OXAL reduced IKCa-channel activity in LoVo colorectal cancer cells. Taken together, the inhibition by OXAL of IKCa channels would conceivably be an important mechanism through which it acts on the functional activities of glioma cells occurring in vivo.

  18. Immature human dendritic cells enhance their migration through KCa3.1 channel activation.

    Science.gov (United States)

    Crottès, David; Félix, Romain; Meley, Daniel; Chadet, Stéphanie; Herr, Florence; Audiger, Cindy; Soriani, Olivier; Vandier, Christophe; Roger, Sébastien; Angoulvant, Denis; Velge-Roussel, Florence

    2016-04-01

    Migration capacity is essential for dendritic cells (DCs) to present antigen to T cells for the induction of immune response. The DC migration is supposed to be a calcium-dependent process, while not fully understood. Here, we report a role of the KCa3.1/IK1/SK4 channels in the migration capacity of both immature (iDC) and mature (mDC) human CD14(+)-derived DCs. KCa3.1 channels were shown to control the membrane potential of human DC and the Ca(2+) entry, which is directly related to migration capacities. The expression of migration marker such as CCR5 and CCR7 was modified in both types of DCs by TRAM-34 (100nM). But, only the migration of iDC was decreased by use of both TRAM-34 and KCa3.1 siRNA. Confocal analyses showed a close localization of CCR5 with KCa3.1 in the steady state of iDC. Finally, the implication of KCa3.1 seems to be limited to the migration capacities as T cell activation of DCs appeared unchanged. Altogether, these results demonstrated that KCa3.1 channels have a pro-migratory effect on iDC migration. Our findings suggest that KCa3.1 in human iDC play a major role in their migration and constitute an attractive target for the cell therapy optimization.

  19. Sustaining sleep spindles through enhanced SK2 channel activity consolidates sleep and elevates arousal threshold

    Science.gov (United States)

    Wimmer, Ralf D.; Astori, Simone; Bond, Chris T.; Rovó, Zita; Chatton, Jean-Yves; Adelman, John P.; Franken, Paul; Lüthi, Anita

    2013-01-01

    Sleep spindles are synchronized 11–15 Hz electroencephalographic (EEG) oscillations predominant during non-rapid-eye-movement sleep (NREMS). Rhythmic bursting in the reticular thalamic nucleus (nRt), arising from interplay between Cav3.3-type Ca2+ channels and Ca2+-dependent small-conductance-type 2 (SK2) K+ channels, underlies spindle generation. Correlative evidence indicates that spindles contribute to memory consolidation and protection against environmental noise in human NREMS. Here, we describe a molecular mechanism through which spindle power is selectively extended and we probed the actions of intensified spindling in the naturally sleeping mouse. Using electrophysiological recordings in acute brain slices from SK2 channel-over-expressing (SK2-OE) mice, we found that nRt bursting was potentiated and thalamic circuit oscillations were prolonged. Moreover, nRt cells showed greater resilience to transit from burst to tonic discharge in response to gradual depolarization, mimicking transitions out of NREMS. Compared to wild-type littermates, chronic EEG recordings of SK2-OE mice contained less fragmented NREMS, while the NREMS EEG power spectrum was conserved. Furthermore, EEG spindle activity was prolonged at NREMS exit. Finally, when exposed to white noise, SK2-OE mice needed stronger stimuli to arouse. Increased nRt bursting thus strengthens spindles and improves sleep quality through mechanisms independent of EEG slow-waves (< 4 Hz), suggesting SK2 signaling as a new potential therapeutic target for sleep disorders and for neuropsychiatric diseases accompanied by weakened sleep spindles. PMID:23035101

  20. Sustaining sleep spindles through enhanced SK2-channel activity consolidates sleep and elevates arousal threshold.

    Science.gov (United States)

    Wimmer, Ralf D; Astori, Simone; Bond, Chris T; Rovó, Zita; Chatton, Jean-Yves; Adelman, John P; Franken, Paul; Lüthi, Anita

    2012-10-03

    Sleep spindles are synchronized 11-15 Hz electroencephalographic (EEG) oscillations predominant during nonrapid-eye-movement sleep (NREMS). Rhythmic bursting in the reticular thalamic nucleus (nRt), arising from interplay between Ca(v)3.3-type Ca(2+) channels and Ca(2+)-dependent small-conductance-type 2 (SK2) K(+) channels, underlies spindle generation. Correlative evidence indicates that spindles contribute to memory consolidation and protection against environmental noise in human NREMS. Here, we describe a molecular mechanism through which spindle power is selectively extended and we probed the actions of intensified spindling in the naturally sleeping mouse. Using electrophysiological recordings in acute brain slices from SK2 channel-overexpressing (SK2-OE) mice, we found that nRt bursting was potentiated and thalamic circuit oscillations were prolonged. Moreover, nRt cells showed greater resilience to transit from burst to tonic discharge in response to gradual depolarization, mimicking transitions out of NREMS. Compared with wild-type littermates, chronic EEG recordings of SK2-OE mice contained less fragmented NREMS, while the NREMS EEG power spectrum was conserved. Furthermore, EEG spindle activity was prolonged at NREMS exit. Finally, when exposed to white noise, SK2-OE mice needed stronger stimuli to arouse. Increased nRt bursting thus strengthens spindles and improves sleep quality through mechanisms independent of EEG slow waves (<4 Hz), suggesting SK2 signaling as a new potential therapeutic target for sleep disorders and for neuropsychiatric diseases accompanied by weakened sleep spindles.

  1. Active zone scaffolds differentially accumulate Unc13 isoforms to tune Ca(2+) channel-vesicle coupling.

    Science.gov (United States)

    Böhme, Mathias A; Beis, Christina; Reddy-Alla, Suneel; Reynolds, Eric; Mampell, Malou M; Grasskamp, Andreas T; Lützkendorf, Janine; Bergeron, Dominique Dufour; Driller, Jan H; Babikir, Husam; Göttfert, Fabian; Robinson, Iain M; O'Kane, Cahir J; Hell, Stefan W; Wahl, Markus C; Stelzl, Ulrich; Loll, Bernhard; Walter, Alexander M; Sigrist, Stephan J

    2016-10-01

    Brain function relies on fast and precisely timed synaptic vesicle (SV) release at active zones (AZs). Efficacy of SV release depends on distance from SV to Ca(2+) channel, but molecular mechanisms controlling this are unknown. Here we found that distances can be defined by targeting two unc-13 (Unc13) isoforms to presynaptic AZ subdomains. Super-resolution and intravital imaging of developing Drosophila melanogaster glutamatergic synapses revealed that the Unc13B isoform was recruited to nascent AZs by the scaffolding proteins Syd-1 and Liprin-α, and Unc13A was positioned by Bruchpilot and Rim-binding protein complexes at maturing AZs. Unc13B localized 120 nm away from Ca(2+) channels, whereas Unc13A localized only 70 nm away and was responsible for docking SVs at this distance. Unc13A(null) mutants suffered from inefficient, delayed and EGTA-supersensitive release. Mathematical modeling suggested that synapses normally operate via two independent release pathways differentially positioned by either isoform. We identified isoform-specific Unc13-AZ scaffold interactions regulating SV-Ca(2+)-channel topology whose developmental tightening optimizes synaptic transmission.

  2. Light-activated control of protein channel assembly mediated by membrane mechanics

    Science.gov (United States)

    Miller, David M.; Findlay, Heather E.; Ces, Oscar; Templer, Richard H.; Booth, Paula J.

    2016-12-01

    Photochemical processes provide versatile triggers of chemical reactions. Here, we use a photoactivated lipid switch to modulate the folding and assembly of a protein channel within a model biological membrane. In contrast to the information rich field of water-soluble protein folding, there is only a limited understanding of the assembly of proteins that are integral to biological membranes. It is however possible to exploit the foreboding hydrophobic lipid environment and control membrane protein folding via lipid bilayer mechanics. Mechanical properties such as lipid chain lateral pressure influence the insertion and folding of proteins in membranes, with different stages of folding having contrasting sensitivities to the bilayer properties. Studies to date have relied on altering bilayer properties through lipid compositional changes made at equilibrium, and thus can only be made before or after folding. We show that light-activation of photoisomerisable di-(5-[[4-(4-butylphenyl)azo]phenoxy]pentyl)phosphate (4-Azo-5P) lipids influences the folding and assembly of the pentameric bacterial mechanosensitive channel MscL. The use of a photochemical reaction enables the bilayer properties to be altered during folding, which is unprecedented. This mechanical manipulation during folding, allows for optimisation of different stages of the component insertion, folding and assembly steps within the same lipid system. The photochemical approach offers the potential to control channel assembly when generating synthetic devices that exploit the mechanosensitive protein as a nanovalve.

  3. Transduction for pheromones in the main olfactory epithelium is mediated by the Ca2+ -activated channel TRPM5.

    Science.gov (United States)

    L