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Sample records for affect channel activity

  1. Mechanically Activated Ion Channels.

    PubMed

    Ranade, Sanjeev S; Syeda, Ruhma; Patapoutian, Ardem

    2015-09-23

    Mechanotransduction, the conversion of physical forces into biochemical signals, is essential for various physiological processes such as the conscious sensations of touch and hearing, and the unconscious sensation of blood flow. Mechanically activated (MA) ion channels have been proposed as sensors of physical force, but the identity of these channels and an understanding of how mechanical force is transduced has remained elusive. A number of recent studies on previously known ion channels along with the identification of novel MA ion channels have greatly transformed our understanding of touch and hearing in both vertebrates and invertebrates. Here, we present an updated review of eukaryotic ion channel families that have been implicated in mechanotransduction processes and evaluate the qualifications of the candidate genes according to specified criteria. We then discuss the proposed gating models for MA ion channels and highlight recent structural studies of mechanosensitive potassium channels. PMID:26402601

  2. Sea Anemone Toxins Affecting Potassium Channels

    NASA Astrophysics Data System (ADS)

    Diochot, Sylvie; Lazdunski, Michel

    The great diversity of K+ channels and their wide distribution in many tissues are associated with important functions in cardiac and neuronal excitability that are now better understood thanks to the discovery of animal toxins. During the past few decades, sea anemones have provided a variety of toxins acting on voltage-sensitive sodium and, more recently, potassium channels. Currently there are three major structural groups of sea anemone K+ channel (SAK) toxins that have been characterized. Radioligand binding and electrophysiological experiments revealed that each group contains peptides displaying selective activities for different subfamilies of K+ channels. Short (35-37 amino acids) peptides in the group I display pore blocking effects on Kv1 channels. Molecular interactions of SAK-I toxins, important for activity and binding on Kv1 channels, implicate a spot of three conserved amino acid residues (Ser, Lys, Tyr) surrounded by other less conserved residues. Long (58-59 amino acids) SAK-II peptides display both enzymatic and K+ channel inhibitory activities. Medium size (42-43 amino acid) SAK-III peptides are gating modifiers which interact either with cardiac HERG or Kv3 channels by altering their voltage-dependent properties. SAK-III toxins bind to the S3C region in the outer vestibule of Kv channels. Sea anemones have proven to be a rich source of pharmacological tools, and some of the SAK toxins are now useful drugs for the diagnosis and treatment of autoimmune diseases.

  3. A mutation in TRPC6 channels abolishes their activation by hypoosmotic stretch but does not affect activation by diacylglycerol or G protein signaling cascades.

    PubMed

    Wilson, Cory; Dryer, Stuart E

    2014-05-01

    Canonical transient receptor potential-6 (TRPC6) channels have been implicated in the pathogenesis of kidney disease and in the regulation of vascular smooth muscle tone, podocyte function, and a variety of processes in other cell types. The question of whether their gating is intrinsically mechanosensitive has been controversial. In this study we have examined activation of two alleles of TRPC6 transiently expressed in CHO-K1 cells: the wild-type human TRPC6 channel, and TRPC6-N143S, an allele originally identified in a family with autosomal dominant familial focal and segmental glomerulosclerosis (FSGS). We observed that both channel variants carried robust cationic currents that could be evoked by application of membrane-permeable analogs of diacylglycerol (DAG) or by the P2Y receptor agonist ATP. The amplitudes and characteristics of currents evoked by the DAG analog or ATP were indistinguishable in cells expressing the two TRPC6 alleles. By contrast, hypoosmotic stretch evoked robust currents in wild-type TRPC6 channels but had no discernible effect on currents in cells expressing TRPC6-N143S, indicating that the mutant form lacks mechanosensitivity. Coexpression of TRPC6-N143S with wild-type TRPC6 or TRPC3 channels did not alter stretch-evoked responses compared with when TRPC3 channels were expressed by themselves, indicating that TRPC6-N143S does not function as a dominant-negative. These data indicate that mechanical activation and activation evoked by DAG or ATP occur through fundamentally distinct biophysical mechanisms, and they provide support for the hypothesis that protein complexes containing wild-type TRPC6 subunits can be intrinsically mechanosensitive. PMID:24598806

  4. Fe2O3 nanoparticles suppress Kv1.3 channels via affecting the redox activity of Kvβ2 subunit in Jurkat T cells

    NASA Astrophysics Data System (ADS)

    Yan, Li; Liu, Xiao; Liu, Wei-Xia; Tan, Xiao-Qiu; Xiong, Fei; Gu, Ning; Hao, Wei; Gao, Xue; Cao, Ji-Min

    2015-12-01

    Superparamagnetic iron oxide nanoparticles (SPIONs) are promising nanomaterials in medical practice due to their special magnetic characteristics and nanoscale size. However, their potential impacts on immune cells are not well documented. This study aims to investigate the effects of Fe2O3 nanoparticles (Fe2O3-NPs) on the electrophysiology of Kv1.3 channels in Jurkat T cells. Using the whole-cell patch-clamp technique, we demonstrate that incubation of Jurkat cells with Fe2O3-NPs dose- and time-dependently decreased the current density and shifted the steady-state inactivation curve and the recovery curve of Kv1.3 channels to a rightward direction. Fe2O3-NPs increased the NADP level but decreased the NADPH level of Jurkat cells. Direct induction of NADPH into the cytosole of Jurkat cells via the pipette abolished the rightward shift of the inactivation curve. In addition, transmission electron microscopy showed that Fe2O3-NPs could be endocytosed by Jurkat cells with relatively low speed and capacity. Fe2O3-NPs did not significantly affect the viability of Jurkat cells, but suppressed the expressions of certain cytokines (TNFα, IFNγ and IL-2) and interferon responsive genes (IRF-1 and PIM-1), and the time courses of Fe2O3-NPs endocytosis and effects on the expressions of cytokines and interferon responsive genes were compatible. We conclude that Fe2O3-NPs can be endocytosed by Jurkat cells and act intracellularly. Fe2O3-NPs decrease the current density and delay the inactivation and recovery kinetics of Kv1.3 channels in Jurkat cells by oxidizing NADPH and therefore disrupting the redox activity of the Kvβ2 auxiliary subunit, and as a result, lead to changes of the Kv1.3 channel function. These results suggest that iron oxide nanoparticles may affect T cell function by disturbing the activity of Kv1.3 channels. Further, the suppressing effects of Fe2O3-NPs on the expressions of certain inflammatory cytokines and interferon responsive genes suggest that iron

  5. REST levels affect the functional expression of voltage dependent calcium channels and the migratory activity in immortalized GnRH neurons.

    PubMed

    Antoniotti, Susanna; Ruffinatti, Federico Alessandro; Torriano, Simona; Luganini, Anna; D'Alessandro, Rosalba; Lovisolo, Davide

    2016-08-26

    The repressor element-1 silencing transcription factor (REST) has emerged as a key controller of neuronal differentiation and has been shown to play a critical role in the expression of the neuronal phenotype; however, much has still to be learned about its role at specific developmental stages and about the functional targets affected. Among these targets, calcium signaling mechanisms are critically dependent on the developmental stage and their full expression is a hallmark of the mature, functional neuron. We have analyzed the role played by REST in GN11 cells, an immortalized cell line derived from gonadotropin hormone releasing hormone (GnRH) neurons at an early developmental stage, electrically non-excitable and with a strong migratory activity. We show for the first time that functional voltage-dependent calcium channels are expressed in wild type GN11 cells; down-regulation of REST by a silencing approach shifts these cells towards a more differentiated phenotype, increasing the functional expression of P/Q-type channels and reducing their migratory potential. PMID:27349310

  6. Is microrheometry affected by channel deformation?

    PubMed

    Del Giudice, Francesco; Greco, Francesco; Netti, Paolo Antonio; Maffettone, Pier Luca

    2016-07-01

    Microrheometry is very important for exploring rheological behaviours of several systems when conventional techniques fail. Microrheometrical measurements are usually carried out in microfluidic devices made of Poly(dimethylsiloxane) (PDMS). Although PDMS is a very cheap material, it is also very easy to deform. In particular, a liquid flowing in a PDMS device, in some circumstances, can effectively deform the microchannel, thus altering the flow conditions. The measure of the fluid relaxation time might be performed through viscoelasticity induced particle migration in microfluidics devices. If the channel walls are deformed by the flow, the resulting measured value of the relaxation time could be not reliable. In this work, we study the effect of channel deformation on particle migration in square-shaped microchannel. Experiments are carried out in several PolyEthylene Oxyde solutions flowing in two devices made of PDMS and Poly(methylmethacrylate) (PMMA). The relevance of wall rigidity on particle migration is investigated, and the corresponding importance of wall rigidity on the determination of the relaxation time of the suspending liquid is examined. PMID:27098237

  7. Exogenous acetate ion reaches the type II copper centre in CueO through the water-excretion channel and potentially affects the enzymatic activity.

    PubMed

    Komori, Hirofumi; Kataoka, Kunishige; Tanaka, Sakiko; Matsuda, Nana; Higuchi, Yoshiki; Sakurai, Takeshi

    2016-07-01

    The acetate-bound form of the type II copper was found in the X-ray structure of the multicopper oxidase CueO crystallized in acetate buffer in addition to the conventional OH(-)-bound form as the major resting form. The acetate ion was retained bound to the type II copper even after prolonged exposure of a CueO crystal to X-ray radiation, which led to the stepwise reduction of the Cu centres. However, in this study, when CueO was crystallized in citrate buffer the OH(-)-bound form was present exclusively. This fact shows that an exogenous acetate ion reaches the type II Cu centre through the water channel constructed between domains 1 and 3 in the CueO molecule. It was also found that the enzymatic activity of CueO is enhanced in the presence of acetate ions in the solvent water. PMID:27380373

  8. Thermally activated TRPV3 channels.

    PubMed

    Luo, Jialie; Hu, Hongzhen

    2014-01-01

    TRPV3 is a temperature-sensitive transient receptor potential (TRP) ion channel. The TRPV3 protein functions as a Ca(2+)-permeable nonselective cation channel with six transmembrane domains forming a tetrameric complex. TRPV3 is known to be activated by warm temperatures, synthetic small-molecule chemicals, and natural compounds from plants. Its function is regulated by a variety of physiological factors including extracellular divalent cations and acidic pH, intracellular adenosine triphosphate, membrane voltage, and arachidonic acid. TRPV3 shows a broad expression pattern in both neuronal and non-neuronal tissues including epidermal keratinocytes, epithelial cells in the gut, endothelial cells in blood vessels, and neurons in dorsal root ganglia and CNS. TRPV3 null mice exhibit abnormal hair morphogenesis and compromised skin barrier function. Recent advances suggest that TRPV3 may play critical roles in inflammatory skin disorders, itch, and pain sensation. Thus, identification of selective TRPV3 activators and inhibitors could potentially lead to beneficial pharmacological interventions in several diseases. The intent of this review is to summarize our current knowledge of the tissue expression, structure, function, and mechanisms of activation of TRPV3. PMID:25366242

  9. Scorpion venom components that affect ion-channels function

    PubMed Central

    Quintero-Hernández, V.; Jiménez-Vargas, J.M.; Gurrola, G.B.; Valdivia, H.H.F.; Possani, L.D.

    2014-01-01

    SUMMARY The number and types of venom components that affect ion-channel function are reviewed. These are the most important venom components responsible for human intoxication, deserving medical attention, often requiring the use of specific anti-venoms. Special emphasis is given to peptides that recognize Na+-, K+- and Ca++-channels of excitable cells. Knowledge generated by direct isolation of peptides from venom and components deduced from cloned genes, whose amino acid sequences are deposited into databanks are now adays in the order of 1.5 thousands, out of an estimate biodiversity closed to 300,000. Here the diversity of components is briefly reviewed with mention to specific references. Structural characteristic are discussed with examples taken from published work. The principal mechanisms of action of the three different types of peptides are also reviewed. Na+-channel specific venom components usually are modifier of the open and closing kinetic mechanisms of the ion-channels, whereas peptides affecting K+-channels are normally pore blocking agents. The Ryanodine Ca++-channel specific peptides are known for causing sub-conducting stages of the channels conductance and some were shown to be able to internalize penetrating inside the muscle cells. PMID:23891887

  10. Target Promiscuity and Heterogeneous Effects of Tarantula Venom Peptides Affecting Na+ and K+ Ion Channels*

    PubMed Central

    Redaelli, Elisa; Cassulini, Rita Restano; Silva, Deyanira Fuentes; Clement, Herlinda; Schiavon, Emanuele; Zamudio, Fernando Z.; Odell, George; Arcangeli, Annarosa; Clare, Jeffrey J.; Alagón, Alejandro; de la Vega, Ricardo C. Rodríguez; Possani, Lourival D.; Wanke, Enzo

    2010-01-01

    Venom-derived peptide modulators of ion channel gating are regarded as essential tools for understanding the molecular motions that occur during the opening and closing of ion channels. In this study, we present the characterization of five spider toxins on 12 human voltage-gated ion channels, following observations about the target promiscuity of some spider toxins and the ongoing revision of their “canonical” gating-modifying mode of action. The peptides were purified de novo from the venom of Grammostola rosea tarantulas, and their sequences were confirmed by Edman degradation and mass spectrometry analysis. Their effects on seven tetrodotoxin-sensitive Na+ channels, the three human ether-à-go-go (hERG)-related K+ channels, and two human Shaker-related K+ channels were extensively characterized by electrophysiological techniques. All the peptides inhibited ion conduction through all the Na+ channels tested, although with distinctive patterns. The peptides also affected the three pharmaceutically relevant hERG isoforms differently. At higher concentrations, all peptides also modified the gating of the Na+ channels by shifting the activation to more positive potentials, whereas more complex effects were recorded on hERG channels. No effects were evident on the two Shaker-related K+ channels at concentrations well above the IC50 value for the affected channels. Given the sequence diversity of the tested peptides, we propose that tarantula toxins should be considered both as multimode and target-promiscuous ion channel modulators; both features should not be ignored when extracting mechanistic interpretations about ion channel gating. Our observations could also aid in future structure-function studies and might help the development of novel ion channel-specific drugs. PMID:19955179

  11. Halothane shortens acetylcholine receptor channel kinetics without affecting conductance.

    PubMed Central

    Lechleiter, J; Gruener, R

    1984-01-01

    The extracellular patch-clamp technique was used to examine how halothane, a general anesthetic, affects the properties of single nicotinic acetylcholine receptor channels of embryonic Xenopus skeletal muscle cells grown in culture. Under control conditions, single-channel events showed a bimodal distribution on the basis of current amplitudes. This distribution was maintained during exposure to halothane and its washout. In addition, the mean current value of the low-and high-amplitude channels was unaffected by the presence of the anesthetic at clinically relevant concentrations. In contrast, halothane shortened the burst durations of both channel types in a concentration-dependent manner. This shortening of burst durations may be an expression of the more rapid relaxation of the channel protein to the nonconducting state, possibly due to the disordering effect of the anesthetic on membrane lipids in which the receptor protein is embedded. This functional change, in the behavior of the synaptic receptor, provides further direct information on the mode of action of general anesthetics. Images PMID:6326154

  12. BK channel activation: structural and functional insights

    PubMed Central

    Lee, Urvi S.; Cui, Jianmin

    2010-01-01

    The voltage and Ca2+ activated K+ (BK) channels are involved in the regulation of neurotransmitter release and neuronal excitability. Structurally, BK channels are homologous to voltage- and ligand-gated K+ channels, having a voltage sensor and pore as the membrane-spanning domain and a cytosolic domain containing metal binding sites. Recently published electron cryomicroscopy (cryo-EM) and X-ray crystallographic structures of the BK channel provided the first look into the assembly of these domains, corroborating the close interactions among these domains during channel gating that have been suggested by functional studies. This review discusses these latest findings and an emerging new understanding about BK channel gating and implications for diseases such as epilepsy, in which mutations in BK channel genes have been associated. PMID:20663573

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

    PubMed Central

    Hermann, Anton; Sitdikova, Guzel F.; Weiger, Thomas M.

    2015-01-01

    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. PMID:26287261

  14. BK channels: multiple sensors, one activation gate.

    PubMed

    Yang, Huanghe; Zhang, Guohui; Cui, Jianmin

    2015-01-01

    Ion transport across cell membranes is essential to cell communication and signaling. Passive ion transport is mediated by ion channels, membrane proteins that create ion conducting pores across cell membrane to allow ion flux down electrochemical gradient. Under physiological conditions, majority of ion channel pores are not constitutively open. Instead, structural region(s) within these pores breaks the continuity of the aqueous ion pathway, thereby serves as activation gate(s) to control ions flow in and out. To achieve spatially and temporally regulated ion flux in cells, many ion channels have evolved sensors to detect various environmental stimuli or the metabolic states of the cell and trigger global conformational changes, thereby dynamically operate the opening and closing of their activation gate. The sensors of ion channels can be broadly categorized as chemical sensors and physical sensors to respond to chemical (such as neural transmitters, nucleotides and ions) and physical (such as voltage, mechanical force and temperature) signals, respectively. With the rapidly growing structural and functional information of different types of ion channels, it is now critical to understand how ion channel sensors dynamically control their gates at molecular and atomic level. The voltage and Ca(2+) activated BK channels, a K(+) channel with an electrical sensor and multiple chemical sensors, provide a unique model system for us to understand how physical and chemical energy synergistically operate its activation gate. PMID:25705194

  15. BK channels: multiple sensors, one activation gate

    PubMed Central

    Yang, Huanghe; Zhang, Guohui; Cui, Jianmin

    2015-01-01

    Ion transport across cell membranes is essential to cell communication and signaling. Passive ion transport is mediated by ion channels, membrane proteins that create ion conducting pores across cell membrane to allow ion flux down electrochemical gradient. Under physiological conditions, majority of ion channel pores are not constitutively open. Instead, structural region(s) within these pores breaks the continuity of the aqueous ion pathway, thereby serves as activation gate(s) to control ions flow in and out. To achieve spatially and temporally regulated ion flux in cells, many ion channels have evolved sensors to detect various environmental stimuli or the metabolic states of the cell and trigger global conformational changes, thereby dynamically operate the opening and closing of their activation gate. The sensors of ion channels can be broadly categorized as chemical sensors and physical sensors to respond to chemical (such as neural transmitters, nucleotides and ions) and physical (such as voltage, mechanical force and temperature) signals, respectively. With the rapidly growing structural and functional information of different types of ion channels, it is now critical to understand how ion channel sensors dynamically control their gates at molecular and atomic level. The voltage and Ca2+ activated BK channels, a K+ channel with an electrical sensor and multiple chemical sensors, provide a unique model system for us to understand how physical and chemical energy synergistically operate its activation gate. PMID:25705194

  16. Putative pore-loops of TMEM16/anoctamin channels affect channel density in cell membranes.

    PubMed

    Adomaviciene, Aiste; Smith, Keith J; Garnett, Hannah; Tammaro, Paolo

    2013-07-15

    The recently identified TMEM16/anoctamin protein family includes Ca(2+)-activated anion channels (TMEM16A, TMEM16B), a cation channel (TMEM16F) and proteins with unclear function. TMEM16 channels consist of eight putative transmembrane domains (TMs) with TM5-TM6 flanking a re-entrant loop thought to form the pore. In TMEM16A this region has also been suggested to contain residues involved in Ca(2+) binding. The role of the putative pore-loop of TMEM16 channels was investigated using a chimeric approach. Heterologous expression of either TMEM16A or TMEM16B resulted in whole-cell anion currents with very similar conduction properties but distinct kinetics and degrees of sensitivity to Ca(2+). Furthermore, whole-cell currents mediated by TMEM16A channels were ∼six times larger than TMEM16B-mediated currents. Replacement of the putative pore-loop of TMEM16A with that of TMEM16B (TMEM16A-B channels) reduced the currents by ∼six-fold, while the opposite modification (TMEM16B-A channels) produced a ∼six-fold increase in the currents. Unexpectedly, these changes were not secondary to variations in channel gating by Ca(2+) or voltage, nor were they due to changes in single-channel conductance. Instead, they depended on the number of functional channels present on the plasma membrane. Generation of additional, smaller chimeras within the putative pore-loop of TMEM16A and TMEM16B led to the identification of a region containing a non-canonical trafficking motif. Chimeras composed of the putative pore-loop of TMEM16F transplanted into the TMEM16A protein scaffold did not conduct anions or cations. These data suggest that the putative pore-loop does not form a complete, transferable pore domain. Furthermore, our data reveal an unexpected role for the putative pore-loop of TMEM16A and TMEM16B channels in the control of the whole-cell Ca(2+)-activated Cl(-) conductance. PMID:23613533

  17. Structure of Thermally Activated TRP Channels

    PubMed Central

    Cohen, Matthew R.; Moiseenkova-Bell, Vera Y.

    2015-01-01

    Temperature sensation is important for adaptation and survival of organisms. While temperature has the potential to affect all biological macromolecules, organisms have evolved specific thermosensitive molecular detectors that are able to transduce temperature changes into physiologically relevant signals. Among these thermosensors are ion channels from the transient receptor potential (TRP) family. Prime candidates include TRPV1–4, TRPA1, and TRPM8 (the so-called “thermoTRP” channels), which are expressed in sensory neurons and gated at specific temperatures. Electrophysiological and thermodynamic approaches have been employed to determine the nature by which thermoTRPs detect temperature and couple temperature changes to channel gating. To further understand how thermoTRPs sense temperature, high-resolution structures of full-length thermoTRPs channels will be required. Here, we will discuss current progress in unraveling the structures of thermoTRP channels. PMID:25366237

  18. Curcumin stimulates cystic fibrosis transmembrane conductance regulator Cl- channel activity.

    PubMed

    Berger, Allan L; Randak, Christoph O; Ostedgaard, Lynda S; Karp, Philip H; Vermeer, Daniel W; Welsh, Michael J

    2005-02-18

    Compounds that enhance either the function or biosynthetic processing of the cystic fibrosis transmembrane conductance regulator (CFTR) Cl(-) channel may be of value in developing new treatments for cystic fibrosis (CF). Previous studies suggested that the herbal extract curcumin might affect the processing of a common CF mutant, CFTR-DeltaF508. Here, we tested the hypothesis that curcumin influences channel function. Curcumin increased CFTR channel activity in excised, inside-out membrane patches by reducing channel closed time and prolonging the time channels remained open. Stimulation was dose-dependent, reversible, and greater than that observed with genistein, another compound that stimulates CFTR. Curcumin-dependent stimulation required phosphorylated channels and the presence of ATP. We found that curcumin increased the activity of both wild-type and DeltaF508 channels. Adding curcumin also increased Cl(-) transport in differentiated non-CF airway epithelia but not in CF epithelia. These results suggest that curcumin may directly stimulate CFTR Cl(-) channels. PMID:15582996

  19. Peptide Neurotoxins that Affect Voltage-Gated Calcium Channels: A Close-Up on ω-Agatoxins

    PubMed Central

    Pringos, Emilie; Vignes, Michel; Martinez, Jean; Rolland, Valerie

    2011-01-01

    Peptide neurotoxins found in animal venoms have gained great interest in the field of neurotransmission. As they are high affinity ligands for calcium, potassium and sodium channels, they have become useful tools for studying channel structure and activity. Peptide neurotoxins represent the clinical potential of ion-channel modulators across several therapeutic fields, especially in developing new strategies for treatment of ion channel-related diseases. The aim of this review is to overview the latest updates in the domain of peptide neurotoxins that affect voltage-gated calcium channels, with a special focus on ω-agatoxins. PMID:22069688

  20. Active channel for Fanno Creek, Oregon

    USGS Publications Warehouse

    Sobieszczyk, Steven

    2011-01-01

    Fanno Creek is a tributary to the Tualatin River and flows though parts of the southwest Portland metropolitan area. The stream is heavily influenced by urban runoff and shows characteristic flashy streamflow and poor water quality commonly associated with urban streams. This data set represents the active, wetted channel as derived from light detection and ranging (LiDAR) data and aerial photographic imagery. The wetted channel boundary is equivalent to the extent of water observed during a 2-yr high flow event.

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

    PubMed

    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. PMID:20435829

  2. Activation and inhibition of TMEM16A calcium-activated chloride channels.

    PubMed

    Ni, Yu-Li; Kuan, Ai-Seon; Chen, Tsung-Yu

    2014-01-01

    Calcium-activated chloride channels (CaCC) encoded by family members of transmembrane proteins of unknown function 16 (TMEM16) have recently been intensely studied for functional properties as well as their physiological roles as chloride channels in various tissues. One technical hurdle in studying these channels is the well-known channel rundown that frequently impairs the precision of electrophysiological measurements for the channels. Using experimental protocols that employ fast-solution exchange, we circumvented the problem of channel rundown by normalizing the Ca(2+)-induced current to the maximally-activated current obtained within a time period in which the channel rundown was negligible. We characterized the activation of the TMEM16A-encoded CaCC (also called ANO1) by Ca(2+), Sr(2+), and Ba(2+), and discovered that Mg(2+) competes with Ca(2+) in binding to the divalent-cation binding site without activating the channel. We also studied the permeability of the ANO1 pore for various anions and found that the anion occupancy in the pore-as revealed by the permeability ratios of these anions-appeared to be inversely correlated with the apparent affinity of the ANO1 inhibition by niflumic acid (NFA). On the other hand, the NFA inhibition was neither affected by the degree of the channel activation nor influenced by the types of divalent cations used for the channel activation. These results suggest that the NFA inhibition of ANO1 is likely mediated by altering the pore function but not through changing the channel gating. Our study provides a precise characterization of ANO1 and documents factors that can affect divalent cation activation and NFA inhibition of ANO1. PMID:24489780

  3. Hydrostatic and osmotic pressure activated channel in plant vacuole

    PubMed Central

    Alexandre, Joel; Lassalles, Jean-Paul

    1991-01-01

    The vacuolar membrane of red beet vacuoles contains a channel which was not gated by voltage or Ca2+ ions. Its unit conductance was 20 pS in 200 mM symmetrical KCl solutions. It was stretch activated: the conductance remained constant but the probability of opening was increased by suction or pressure applied to a membrane patch. A 1.5-kNm-2 suction applied to isolated patches or a 0.08-kNm-2 pressure applied to a 45-μm diameter vacuole induced an e-fold change in the mean current. A 75% inhibition of the channel current was obtained with 10 μM Gd3+ on the cytoplasmic side. The channel was more permeable for K+ than for Cl- (PK/PCl ∼ 3). A possible clustering for this channel was suggested by the recordings of the patch current. The channel properties were not significantly affected by a change in sorbitol osmolality in the solutions under isoosmotic conditions, between 0.6 and 1 mol/kg sorbitol. However, the channel was very sensitive to an osmotic gradient. A 0.2-mol/kg sorbitol gradient induced a two-fold increase in unit conductance and a thirty-fold increase in the mean patch current of the channel. A current was measured, when the osmotic gradient was the only driving force applied to the vacuolar membrane. The hydrostatic and osmotic pressure (HOP) activated channel described in this paper could be gated in vivo condition by a change in osmolality, without the need of a change in the turgor pressure in the cell. The HOP channel represents a possible example of an osmoreceptor for plant cells. PMID:19431814

  4. Mutations Affecting Internal TEA Blockade Identify the Probable Pore-Forming Region of a K^+ Channel

    NASA Astrophysics Data System (ADS)

    Yellen, Gary; Jurman, Mark E.; Abramson, Tatiana; MacKinnon, Roderick

    1991-02-01

    The active site of voltage-activated potassium channels is a transmembrane aqueous pore that permits ions to permeate the cell membrane in a rapid yet highly selective manner. A useful probe for the pore of potassium-selective channels is the organic ion tetraethylammonium (TEA), which binds with millimolar affinity to the intracellular opening of the pore and blocks potassium current. In the potassium channel encoded by the Drosophila Shaker gene, an amino acid residue that specifically affects the affinity for intracellular TEA has now been identified by site-directed mutagenesis. This residue is in the middle of a conserved stretch of 18 amino acids that separates two locations that are both near the external opening of the pore. These findings suggest that this conserved region is intimately involved in the formation of the ion conduction pore of voltage-activated potassium channels. Further, a stretch of only eight amino acid residues must traverse 80 percent of the transmembrane electric potential difference.

  5. Pretreating channel catfish with copper sulfate affects susceptibility to columnaris

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Columnaris disease is a costly bacterial disease of commercially grown channel catfish. Three experiments were conducted to evaluate the effects of pre-exposing channel catfish fingerlings to 0, 1/2, 1, or 2 times the recommended copper sulfate rate for 24 hours on their susceptibility to columnaris...

  6. Hydrogen peroxide affects ion channels in lily pollen grain protoplasts.

    PubMed

    Breygina, M A; Abramochkin, D V; Maksimov, N M; Yermakov, I P

    2016-09-01

    Ion homeostasis plays a central role in polarisation and polar growth. In several cell types ion channels are controlled by reactive oxygen species (ROS). One of the most important cells in the plant life cycle is the male gametophyte, which grows under the tight control of both ion fluxes and ROS balance. The precise relationship between these two factors in pollen tubes has not been completely elucidated, and in pollen grains it has never been studied to date. In the present study we used a simple model - protoplasts obtained from lily pollen grains at the early germination stage - to reveal the effect of H2 O2 on cation fluxes crucial for pollen germination. Here we present direct evidence for two ROS-sensitive currents on the pollen grain plasma membrane: the hyperpolarisation-activated calcium current, which is strongly enhanced by H2 O2 , and the outward potassium current, which is modestly enhanced by H2 O2 . We used low concentrations of H2 O2 that do not cause an intracellular oxidative burst and do not damage cells, as demonstrated with fluorescent staining. PMID:27115728

  7. Sodium channel activation mechanisms. Insights from deuterium oxide substitution

    SciTech Connect

    Alicata, D.A.; Rayner, M.D.; Starkus, J.G. )

    1990-04-01

    Schauf and Bullock, using Myxicola giant axons, demonstrated that solvent substitution with deuterium oxide (D2O) significantly affects both sodium channel activation and inactivation kinetics without corresponding changes in gating current or tail current rates. They concluded that (a) no significant component of gating current derives from the final channel opening step, and (b) channels must deactivate (during tail currents) by a different pathway from that used in channel opening. By contrast, Oxford found in squid axons that when a depolarizing pulse is interrupted by a brief (approximately 100 microseconds) return to holding potential, subsequent reactivation (secondary activation) is very rapid and shows almost monoexponential kinetics. Increasing the interpulse interval resulted in secondary activation rate returning towards control, sigmoid (primary activation) kinetics. He concluded that channels open and close (deactivate) via the same pathway. We have repeated both sets of observations in crayfish axons, confirming the results obtained in both previous studies, despite the apparently contradictory conclusions reached by these authors. On the other hand, we find that secondary activation after a brief interpulse interval (50 microseconds) is insensitive to D2O, although reactivation after longer interpulse intervals (approximately 400 microseconds) returns towards a D2O sensitivity similar to that of primary activation. We conclude that D2O-sensitive primary activation and D2O-insensitive tail current deactivation involve separate pathways. However, D2O-insensitive secondary activation involves reversal of the D2O-insensitive deactivation step. These conclusions are consistent with parallel gate models, provided that one gating particle has a substantially reduced effective valence.

  8. Iron status of channel catfish Ictalurus punctatus affected by channel catfish anemia and response to parenteral iron

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Originally reported in 1983, channel catfish anemia (CCA), also ‘white lip’ or ‘no blood,’ is a major idiopathic disease affecting commercial production in the Mississippi Delta region of the USA. Affected individuals are characterized by lethargy, anorexia, extreme pallor, and packed cell volumes o...

  9. Epithelial sodium channel modulates platelet collagen activation.

    PubMed

    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. PMID:24679405

  10. Golgi Anti-apoptotic Proteins Are Highly Conserved Ion Channels That Affect Apoptosis and Cell Migration*

    PubMed Central

    Carrara, Guia; Saraiva, Nuno; Parsons, Maddy; Byrne, Bernadette; Prole, David L.; Taylor, Colin W.; Smith, Geoffrey L.

    2015-01-01

    Golgi anti-apoptotic proteins (GAAPs) are multitransmembrane proteins that are expressed in the Golgi apparatus and are able to homo-oligomerize. They are highly conserved throughout eukaryotes and are present in some prokaryotes and orthopoxviruses. Within eukaryotes, GAAPs regulate the Ca2+ content of intracellular stores, inhibit apoptosis, and promote cell adhesion and migration. Data presented here demonstrate that purified viral GAAPs (vGAAPs) and human Bax inhibitor 1 form ion channels and that vGAAP from camelpox virus is selective for cations. Mutagenesis of vGAAP, including some residues conserved in the recently solved structure of a related bacterial protein, BsYetJ, altered the conductance (E207Q and D219N) and ion selectivity (E207Q) of the channel. Mutation of residue Glu-207 or -178 reduced the effects of GAAP on cell migration and adhesion without affecting protection from apoptosis. In contrast, mutation of Asp-219 abrogated the anti-apoptotic activity of GAAP but not its effects on cell migration and adhesion. These results demonstrate that GAAPs are ion channels and define residues that contribute to the ion-conducting pore and affect apoptosis, cell adhesion, and migration independently. PMID:25713081

  11. Calcium-Activated Potassium Channels: Potential Target for Cardiovascular Diseases.

    PubMed

    Dong, De-Li; Bai, Yun-Long; Cai, Ben-Zhi

    2016-01-01

    Ca(2+)-activated K(+) channels (KCa) are classified into three subtypes: big conductance (BKCa), intermediate conductance (IKCa), and small conductance (SKCa) KCa channels. The three types of KCa channels have distinct physiological or pathological functions in cardiovascular system. BKCa channels are mainly expressed in vascular smooth muscle cells (VSMCs) and inner mitochondrial membrane of cardiomyocytes, activation of BKCa channels in these locations results in vasodilation and cardioprotection against cardiac ischemia. IKCa channels are expressed in VSMCs, endothelial cells, and cardiac fibroblasts and involved in vascular smooth muscle proliferation, migration, vessel dilation, and cardiac fibrosis. SKCa channels are widely expressed in nervous and cardiovascular system, and activation of SKCa channels mainly contributes membrane hyperpolarization. In this chapter, we summarize the physiological and pathological roles of the three types of KCa channels in cardiovascular system and put forward the possibility of KCa channels as potential target for cardiovascular diseases. PMID:27038376

  12. Chemical activation of the mechanotransduction channel Piezo1

    PubMed Central

    Syeda, Ruhma; Xu, Jie; Dubin, Adrienne E; Coste, Bertrand; Mathur, Jayanti; Huynh, Truc; Matzen, Jason; Lao, Jianmin; Tully, David C; Engels, Ingo H; Petrassi, H Michael; Schumacher, Andrew M; Montal, Mauricio; Bandell, Michael; Patapoutian, Ardem

    2015-01-01

    Piezo ion channels are activated by various types of mechanical stimuli and function as biological pressure sensors in both vertebrates and invertebrates. To date, mechanical stimuli are the only means to activate Piezo ion channels and whether other modes of activation exist is not known. In this study, we screened ∼3.25 million compounds using a cell-based fluorescence assay and identified a synthetic small molecule we termed Yoda1 that acts as an agonist for both human and mouse Piezo1. Functional studies in cells revealed that Yoda1 affects the sensitivity and the inactivation kinetics of mechanically induced responses. Characterization of Yoda1 in artificial droplet lipid bilayers showed that Yoda1 activates purified Piezo1 channels in the absence of other cellular components. Our studies demonstrate that Piezo1 is amenable to chemical activation and raise the possibility that endogenous Piezo1 agonists might exist. Yoda1 will serve as a key tool compound to study Piezo1 regulation and function. DOI: http://dx.doi.org/10.7554/eLife.07369.001 PMID:26001275

  13. Mechanisms of caffeine activation of single calcium-release channels of sheep cardiac sarcoplasmic reticulum.

    PubMed Central

    Sitsapesan, R; Williams, A J

    1990-01-01

    1. Calcium-release channels of sheep cardiac junctional sarcoplasmic reticulum were incorporated into planar phospholipid bilayers. Single-channel current fluctuations were recorded under voltage clamp conditions. 2. Channels incorporate into the bilayer with a fixed orientation and channel open probability is regulated by the calcium concentration at the cytosolic face of the membrane. 3. Addition of caffeine (0.5-2.0 mM) to the cytosolic side of the membrane increased the open probability of the calcium-activated calcium-release channel by increasing the frequency of opening without significant alteration to the durations of open events. This effect was observed at both 0.1 and 10 microM-activating cytosolic calcium. 4. Caffeine (0.5-2.0 mM) did not activate the channel at a subactivating cytosolic calcium concentration (80 pM). 5. At subactivating calcium concentrations, channels could be activated by higher concentrations of caffeine (greater than 5.0 mM) revealing a second, calcium-independent, mechanism for channel activation. Channel openings induced by these high concentrations of caffeine at subactivating calcium concentrations displayed different kinetics from those observed with calcium as the sole activating ligand or with combinations of calcium and low concentrations of caffeine. 6. Activation of channel opening by caffeine in the presence of calcium did not affect single-channel conductance. Channel openings produced by caffeine at subactivating cytosolic calcium concentrations had identical conductance and relative permeability to those seen on calcium activation. 7. Channels activated by caffeine at both activating and subactivating calcium concentrations were characteristically modified by ryanodine, Ruthenium Red, ATP and magnesium, implying that the same channel is involved under both conditions. PMID:2167363

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

    PubMed

    Tang, Qiong-Yao; Zhang, Fei-Fei; Xu, Jie; Wang, Ran; Chen, Jian; Logothetis, Diomedes E; Zhang, Zhe

    2016-01-01

    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. PMID:26725113

  15. The activity of the TRP-like channel depends on its expression system

    PubMed Central

    Lev, Shaya; Katz, Ben; Minke, Baruch

    2012-01-01

    The Drosophila light activated TRP and TRPL channels have been a model for TRPC channel gating. Several gating mechanisms have been proposed following experiments conducted on photoreceptor and tissue cultured cells. However, conclusive evidence for any mechanism is still lacking. Here, we show that the Drosophila TRPL channel expressed in tissue cultured cells is constitutively active in S2 cells but is silent in HEK cells. Modulations of TRPL channel activity in different expression system by pharmacology or specific enzymes, which change the lipid content of the plasma membrane, resulted in conflicting effects. These findings demonstrate the difficulty in elucidating TRPC gating, as channel behavior is expression system dependent. However, clues on the gating mechanism may arise from understanding how different expression systems affect TRPC channel activation. PMID:22627924

  16. Dendritic NMDA receptors activate axonal calcium channels

    PubMed Central

    Christie, Jason M.; Jahr, Craig E.

    2008-01-01

    Summary NMDA receptor (NMDAR) activation can alter synaptic strength by regulating transmitter release from a variety of neurons in the CNS. As NMDARs are permeable to Ca2+ and monovalent cations, they could alter release directly by increasing presynaptic Ca2+ or indirectly by axonal depolarization sufficient to activate voltage-sensitive Ca2+ channels (VSCCs). Using two-photon microscopy to measure Ca2+ excursions, we found that somatic depolarization or focal activation of dendritic NMDARs elicited small Ca2+ transients in axon varicosities of cerebellar stellate cell interneurons. These axonal transients resulted from Ca2+ entry through VSCCs that were opened by the electrotonic spread of the NMDAR-mediated depolarization elicited in the dendrites. In contrast, we were unable to detect direct activation of NMDARs on axons indicating an exclusive somatodendritic expression of functional NMDARs. In cerebellar stellate cells, dendritic NMDAR activation masquerades as a presynaptic phenomenon and may influence Ca2+-dependent forms of presynaptic plasticity and release. PMID:18957221

  17. Proteolytic cleavage in the S1-S2 linker of the Kv1.5 channel does not affect channel function.

    PubMed

    Hogan-Cann, Andrew; Li, Wentao; Guo, Jun; Yang, Tonghua; Zhang, Shetuan

    2016-06-01

    Kv1.5 channels mediate the ultra-rapidly activating delayed rectifier potassium current (IKur), which is important for atrial repolarization. It has been shown that cell-surface Kv1.5 channels are sensitive to cleavage by the extracellular serine protease, proteinase K (PK). Here, we investigated the effects of extracellular proteolytic digestion on the function of Kv1.5 channels stably expressed in HEK 293 cells. Our data demonstrate that PK treatment cleaved mature membrane-bound (75kDa) Kv1.5 channels at a single locus in the S1-S2 linker, producing 42-kDa N-terminal fragments and 33-kDa C-terminal fragments. Interestingly, such PK treatment did not affect the Kv1.5 current (IKv1.5) recorded using the whole-cell patch clamp technique. Analysis of cell-surface proteins isolated using biotinylation indicated that the PK-generated N- and C-terminal fragments were both present in the plasma membrane. Co-immunoprecipitation (co-IP) experiments indicated that the N- and C-terminal fragments are no longer associated after cleavage. Furthermore, following PK digestion, the N- and C-fragments degraded at different rates. PK is frequently used as a tool to analyze cell-surface localization of membrane proteins, and cleavage of cell-surface channels has been shown to abolish channel function (e.g. hERG). Our data, for the first time, demonstrate that cleavage of cell-surface channels assessed by Western blot analysis does not necessarily correlate with an elimination of the channel activities. PMID:26874203

  18. Ion channels activated by light in Limulus ventral photoreceptors

    PubMed Central

    1986-01-01

    The light-activated conductance of Limulus ventral photoreceptors was studied using the patch-clamp technique. Channels (40 pS) were observed whose probability of opening was greatly increased by light. In some cells the latency of channel activation was nearly the same as that of the macroscopic response, while in other cells the channel latency was much greater. Like the macroscopic conductance, channel activity was reduced by light adaptation but enhanced by the intracellular injection of the calcium chelator EGTA. The latter observation indicates that channel activation was not a secondary result of the light-induced rise in intracellular calcium. A two-microelectrode voltage-clamp method was used to measure the voltage dependence of the light-activated macroscopic conductance. It was found that this conductance is constant over a wide voltage range more negative than zero, but it increases markedly at positive voltages. The single channel currents measured over this same voltage range show that the single channel conductance is independent of voltage, but that channel gating properties are dependent on voltage. Both the mean channel open time and the opening rate increase at positive voltages. These properties change in a manner consistent with the voltage dependence of the macroscopic conductance. The broad range of similarities between the macroscopic and single channel currents supports the conclusion that the 40-pS channel that we have observed is the principal channel underlying the response to light in these photoreceptors. PMID:2419481

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

    PubMed Central

    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

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

    PubMed

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

    2016-08-01

    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 Ca(2+) and ATP together to control life and death'. PMID:27377720

  1. γ-Band deficiency and abnormal thalamocortical activity in P/Q-type channel mutant mice

    PubMed Central

    Llinás, Rodolfo R.; Choi, Soonwook; Urbano, Francisco J.; Shin, Hee-Sup

    2007-01-01

    Thalamocortical in vivo and in vitro function was studied in mice lacking P/Q-type calcium channels (CaV2.1), in which N-type calcium channels (CaV2.2) supported central synaptic transmission. Unexpectedly, in vitro patch recordings from thalamic neurons demonstrated no γ-band subthreshold oscillation, and voltage-sensitive dye imaging demonstrated an absence of cortical γ-band-dependent columnar activation involving cortical inhibitory interneuron activity. In vivo electroencephalogram recordings showed persistent absence status and a dramatic reduction of γ-band activity. Pharmacological block of T-type calcium channels (CaV3), although not noticeably affecting normal control animals, left the knockout mice in a coma-like state. Hence, although N-type calcium channels can rescue P/Q-dependent synaptic transmission, P/Q calcium channels are essential in the generation of γ-band activity and resultant cognitive function. PMID:17968008

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

    PubMed Central

    Cui, Yuanyuan; Yang, Fan; Cao, Xu; Yarov-Yarovoy, Vladimir

    2012-01-01

    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. PMID:22412190

  3. Cumulative Activation of Voltage-Dependent KVS-1 Potassium Channels

    PubMed Central

    Rojas, Patricio; Garst-Orozco, Jonathan; Baban, Beravan; de Santiago-Castillo, Jose Antonio; Covarrubias, Manuel; Salkoff, Lawrence

    2008-01-01

    In this study, we reveal the existence of a novel use-dependent phenomenon in potassium channels, which we refer to as cumulative activation (CA). CA consists of an increase in current amplitude in response to repetitive depolarizing step pulses to the same potential. CA persists for up to 20 s and is similar to a phenomenon called “voltage-dependent facilitation” observed in some calcium channels. The KVS-1 K+ channel, which exhibits CA, is a rapidly activating and inactivating voltage-dependent potassium channel expressed in chemosensory and other neurons of Caenorhabditis elegans. It is unusual in being most closely related to the Shab (Kv2) family of potassium channels, which typically behave like delayed rectifier K+ channels in other species. The magnitude of CA depends on the frequency, voltage, and duration of the depolarizing step pulse. CA also radically changes the activation and inactivation kinetics of the channel, suggesting that the channel may undergo a physical modification in a use-dependent manner; thus, a model that closely simulates the behavior of the channel postulates the existence of two populations of channels, unmodified and modified. Use-dependent changes in the behavior of potassium channels, such as CA observed in KVS-1, could be involved in functional mechanisms of cellular plasticity such as synaptic depression that represent the cellular basis of learning and memory. PMID:18199775

  4. Sensory activity affects sensory axon development in C. elegans.

    PubMed

    Peckol, E L; Zallen, J A; Yarrow, J C; Bargmann, C I

    1999-05-01

    The simple nervous system of the nematode C. elegans consists of 302 neurons with highly reproducible morphologies, suggesting a hard-wired program of axon guidance. Surprisingly, we show here that sensory activity shapes sensory axon morphology in C. elegans. A class of mutants with deformed sensory cilia at their dendrite endings have extra axon branches, suggesting that sensory deprivation disrupts axon outgrowth. Mutations that alter calcium channels or membrane potential cause similar defects. Cell-specific perturbations of sensory activity can cause cell-autonomous changes in axon morphology. Although the sensory axons initially reach their targets in the embryo, the mutations that alter sensory activity cause extra axon growth late in development. Thus, perturbations of activity affect the maintenance of sensory axon morphology after an initial pattern of innervation is established. This system provides a genetically tractable model for identifying molecular mechanisms linking neuronal activity to nervous system structure. PMID:10101123

  5. Dual actions of procainamide on batrachotoxin-activated sodium channels: open channel block and prevention of inactivation.

    PubMed Central

    Zamponi, G W; Sui, X; Codding, P W; French, R J

    1993-01-01

    We have investigated the action of procainamide on batrachotoxin (BTX)-activated sodium channels from bovine heart and rat skeletal muscle. When applied to the intracellular side, procainamide induced rapid, open-channel block. We estimated rate constants using amplitude distribution analysis (Yellen, G. 1984. J. Gen. Physiol. 84:157). Membrane depolarization increased the blocking rate and slowed unblock. The rate constants were similar in both magnitude and voltage dependence for cardiac and skeletal muscle channels. Qualitatively, this block resembled the fast open-channel block by lidocaine (Zamponi, G. W., D. D. Doyle, and R. J. French. 1993. Biophys. J. 65:80), but procainamide was about sevenfold less potent. Molecular modeling suggests that the difference in potency between procainamide and lidocaine might arise from the relative orientation of their aromatic rings, or from differences in the structure of the aryl-amine link. For the cardiac channels, procainamide reduced the frequency of transitions to a long-lived closed state which shows features characteristic of inactivation (Zamponi, G. W., D. D. Doyle, and R. J. French. 1993. Biophys J. 65:91). Mean durations of kinetically identified closed states were not affected. The degree of fast block and of inhibition of the slow closures were correlated. Internally applied QX-314, a lidocaine derivative and also a fast blocker, produced a similar effect. Thus, drug binding to the fast blocking site appears to inhibit inactivation in BTX-activated cardiac channels. Images FIGURE 6 PMID:8312472

  6. 28 CFR 55.15 - Affected activities.

    Code of Federal Regulations, 2013 CFR

    2013-07-01

    ... RIGHTS ACT REGARDING LANGUAGE MINORITY GROUPS Minority Language Materials and Assistance § 55.15 Affected... of applicable language minority groups to be effectively informed of and participate effectively in voting-connected activities. Accordingly, the quoted language should be broadly construed to apply to...

  7. 28 CFR 55.15 - Affected activities.

    Code of Federal Regulations, 2011 CFR

    2011-07-01

    ... RIGHTS ACT REGARDING LANGUAGE MINORITY GROUPS Minority Language Materials and Assistance § 55.15 Affected... of applicable language minority groups to be effectively informed of and participate effectively in voting-connected activities. Accordingly, the quoted language should be broadly construed to apply to...

  8. 28 CFR 55.15 - Affected activities.

    Code of Federal Regulations, 2012 CFR

    2012-07-01

    ... RIGHTS ACT REGARDING LANGUAGE MINORITY GROUPS Minority Language Materials and Assistance § 55.15 Affected... of applicable language minority groups to be effectively informed of and participate effectively in voting-connected activities. Accordingly, the quoted language should be broadly construed to apply to...

  9. 28 CFR 55.15 - Affected activities.

    Code of Federal Regulations, 2014 CFR

    2014-07-01

    ... RIGHTS ACT REGARDING LANGUAGE MINORITY GROUPS Minority Language Materials and Assistance § 55.15 Affected... of applicable language minority groups to be effectively informed of and participate effectively in voting-connected activities. Accordingly, the quoted language should be broadly construed to apply to...

  10. 28 CFR 55.15 - Affected activities.

    Code of Federal Regulations, 2010 CFR

    2010-07-01

    ... RIGHTS ACT REGARDING LANGUAGE MINORITY GROUPS Minority Language Materials and Assistance § 55.15 Affected... of applicable language minority groups to be effectively informed of and participate effectively in voting-connected activities. Accordingly, the quoted language should be broadly construed to apply to...

  11. Allosterism and Structure in Thermally Activated Transient Receptor Potential Channels.

    PubMed

    Diaz-Franulic, Ignacio; Poblete, Horacio; Miño-Galaz, Germán; González, Carlos; Latorre, Ramón

    2016-07-01

    The molecular sensors that mediate temperature changes in living organisms are a large family of proteins known as thermosensitive transient receptor potential (TRP) ion channels. These membrane proteins are polymodal receptors that can be activated by cold or hot temperatures, depending on the channel subtype, voltage, and ligands. The stimuli sensors are allosterically coupled to a pore domain, increasing the probability of finding the channel in its ion conductive conformation. In this review we first discuss the allosteric coupling between the temperature and voltage sensor modules and the pore domain, and then discuss the thermodynamic foundations of thermo-TRP channel activation. We provide a structural overview of the molecular determinants of temperature sensing. We also posit an anisotropic thermal diffusion model that may explain the large temperature sensitivity of TRP channels. Additionally, we examine the effect of several ligands on TRP channel function and the evidence regarding their mechanisms of action. PMID:27297398

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

    PubMed

    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 < 0.05). CNTF-ACM produced a significant increase in BKα1 and BKβ3 expression (p < 0.05) but had no significant effect upon SK2 or SK3 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. PMID:27097551

  13. Do recreational activities affect coastal biodiversity?

    NASA Astrophysics Data System (ADS)

    Riera, Rodrigo; Menci, Cristiano; Sanabria-Fernández, José Antonio; Becerro, Mikel A.

    2016-09-01

    Human activities are largely affecting coastal communities worldwide. Recreational perturbations have been overlooked in comparison to other perturbations, yet they are potential threats to marine biodiversity. They affect coastal communities in different ways, underpinning consistent shifts in fish and invertebrates assemblages. Several sites were sampled subjected to varying effects by recreational fishermen (low and high pressure) and scuba divers (low and high) in an overpopulated Atlantic island. Non-consistent differences in ecological, trophic and functional diversity were found in coastal communities, considering both factors ("diving" and "fishing"). Multivariate analyses only showed significant differences in benthic invertebrates between intensively-dived and non-dived sites. The lack of clear trends may be explained by the depletion of coastal resources in the study area, an extensively-affected island by overfishing.

  14. Urinary Bladder-Relaxant Effect of Kurarinone Depending on Potentiation of Large-Conductance Ca2+-Activated K+ Channels.

    PubMed

    Lee, Sojung; Chae, Mee Ree; Lee, Byoung-Cheol; Kim, Yong-Chul; Choi, Jae Sue; Lee, Sung Won; Cheong, Jae Hoon; Park, Chul-Seung

    2016-08-01

    The large-conductance calcium-activated potassium channel (BKCa channel) plays critical roles in smooth muscle relaxation. In urinary bladder smooth muscle, BKCa channel activity underlies the maintenance of the resting membrane potential and repolarization of the spontaneous action potential triggering the phasic contraction. To identify novel BKCa channel activators, we screened a library of natural compounds using a cell-based fluorescence assay and a hyperactive mutant BKCa channel (Lee et al., 2013). From 794 natural compounds, kurarinone, a flavanone from Sophora flavescens, strongly potentiated BKCa channels. When treated from the extracellular side, this compound progressively shifted the conductance-voltage relationship of BKCa channels to more negative voltages and increased the maximum conductance in a dose-dependent manner. Whereas kurarinone strongly potentiated the homomeric BKCa channel composed of only the α subunit, its effects were much smaller on heteromeric channels coassembled with auxiliary β subunits. Although the activation kinetics was not altered significantly, the deactivation of BKCa channels was dramatically slowed by kurarinone treatment. At the single-channel level, kurarinone increased the open probability of the BKCa channel without affecting its single-channel conductance. Kurarinone potently relaxed acetylcholine-induced contraction of rat bladder smooth muscle and thus decreased the micturition frequency of rats with overactive bladder symptoms. These results indicate that kurarinone can directly potentiate BKCa channels and demonstrate the therapeutic potentials of kurarinone and its derivatives for developing antioveractive bladder medications and supplements. PMID:27251362

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

    PubMed

    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. PMID:7514038

  16. Slack, Slick, and Sodium-Activated Potassium Channels

    PubMed Central

    Kaczmarek, Leonard K.

    2013-01-01

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

  17. Calcium-mediated agonists activate an inwardly rectified K+ channel in colonic secretory cells.

    PubMed

    Devor, D C; Frizzell, R A

    1993-11-01

    Single-channel recording techniques were used to identify and characterize the K+ channel activated by Ca(2+)-mediated secretory agonists in T84 cells. Carbachol (CCh; 100 microM) and taurodeoxycholate (TDC; 0.75 mM) stimulated oscillatory outward K+ currents. With K gluconate in bath and pipette, cell-attached single-channel K+ currents stimulated by CCh and ionomycin (2 microM) were inwardly rectified and reversed at 0 mV. The single-channel chord conductance was 32 pS at -90 mV and 14 pS at +90 mV. Similar properties were observed in excised inside-out patches in symmetric K+, permitting further characterization of channel properties. Partial substitution of bath or pipette K+ with Na+ gave a K(+)-to-Na+ selectivity ratio of 5.5:1. Channel activity increased with increasing bath Ca2+ concentration in the physiological range of 50-800 nM. Maximal channel activity occurred at intracellular pH 7.2 and decreased at more acidic or alkaline pH values. Extracellular charybdotoxin (CTX; 50 nM) blocked inward but not outward currents. Extracellular tetraethylammonium (TEA; 10 mM) reduced single-channel amplitude at all voltages. No apparent block of the channel was observed with extracellular Ba2+ (1 mM), apamin (1 microM), 4-aminopyridine (4-AP; 4 mM), quinine (500 microM), or glyburide (10 microM). Cytosolic quinine and 4-AP blocked both inward and outward currents, whereas Ba2+ blocked only outward currents. Apamin, CTX, TEA, and glyburide did not affect channel activity. The agonist activation and pharmacological profile of this inwardly rectified K+ channel indicate that it is responsible for the increase in basolateral K+ conductance stimulated by Ca(2+)-mediated agonists in T84 cells. PMID:7694492

  18. Natural Bile Acids and Synthetic Analogues Modulate Large Conductance Ca2+-activated K+ (BKCa) Channel Activity in Smooth Muscle Cells

    PubMed Central

    Dopico, Alejandro M.; Walsh, John V.; Singer, Joshua J.

    2002-01-01

    Bile acids have been reported to produce relaxation of smooth muscle both in vitro and in vivo. The cellular mechanisms underlying bile acid–induced relaxation are largely unknown. Here we demonstrate, using patch-clamp techniques, that natural bile acids and synthetic analogues reversibly increase BKCa channel activity in rabbit mesenteric artery smooth muscle cells. In excised inside-out patches bile acid–induced increases in channel activity are characterized by a parallel leftward shift in the activity-voltage relationship. This increase in BKCa channel activity is not due to Ca2+-dependent mechanism(s) or changes in freely diffusible messengers, but to a direct action of the bile acid on the channel protein itself or some closely associated component in the cell membrane. For naturally occurring bile acids, the magnitude of bile acid–induced increase in BKCa channel activity is inversely related to the number of hydroxyl groups in the bile acid molecule. By using synthetic analogues, we demonstrate that such increase in activity is not affected by several chemical modifications in the lateral chain of the molecule, but is markedly favored by polar groups in the side of the steroid rings opposite to the side where the methyl groups are located, which stresses the importance of the planar polarity of the molecule. Bile acid–induced increases in BKCa channel activity are also observed in smooth muscle cells freshly dissociated from rabbit main pulmonary artery and gallbladder, raising the possibility that a direct activation of BKCa channels by these planar steroids is a widespread phenomenon in many smooth muscle cell types. Bile acid concentrations that increase BKCa channel activity in mesenteric artery smooth muscle cells are found in the systemic circulation under a variety of human pathophysiological conditions, and their ability to enhance BKCa channel activity may explain their relaxing effect on smooth muscle. PMID:11865021

  19. The regulation of BK channel activity by pre- and post-translational modifications

    PubMed Central

    Kyle, Barry D.; Braun, Andrew P.

    2014-01-01

    Large conductance, Ca2+-activated K+ (BK) channels represent an important pathway for the outward flux of K+ ions from the intracellular compartment in response to membrane depolarization, and/or an elevation in cytosolic free [Ca2+]. They are functionally expressed in a range of mammalian tissues (e.g., nerve and smooth muscles), where they can either enhance or dampen membrane excitability. The diversity of BK channel activity results from the considerable alternative mRNA splicing and post-translational modification (e.g., phosphorylation) of key domains within the pore-forming α subunit of the channel complex. Most of these modifications are regulated by distinct upstream cell signaling pathways that influence the structure and/or gating properties of the holo-channel and ultimately, cellular function. The channel complex may also contain auxiliary subunits that further affect channel gating and behavior, often in a tissue-specific manner. Recent studies in human and animal models have provided strong evidence that abnormal BK channel expression/function contributes to a range of pathologies in nerve and smooth muscle. By targeting the upstream regulatory events modulating BK channel behavior, it may be possible to therapeutically intervene and alter BK channel expression/function in a beneficial manner. PMID:25202279

  20. SLO2 Channels Are Inhibited by All Divalent Cations That Activate SLO1 K+ Channels.

    PubMed

    Budelli, Gonzalo; Sun, Qi; Ferreira, Juan; Butler, Alice; Santi, Celia M; Salkoff, Lawrence

    2016-04-01

    Two members of the family of high conductance K(+)channels SLO1 and SLO2 are both activated by intracellular cations. However, SLO1 is activated by Ca(2+)and other divalent cations, while SLO2 (Slack or SLO2.2 from rat) is activated by Na(+) Curiously though, we found that SLO2.2 is inhibited by all divalent cations that activate SLO1, with Zn(2+)being the most effective inhibitor with an IC50of ∼8 μmin contrast to Mg(2+), the least effective, with an IC50of ∼ 1.5 mm Our results suggest that divalent cations are not SLO2 pore blockers, but rather inhibit channel activity by an allosteric modification of channel gating. By site-directed mutagenesis we show that a histidine residue (His-347) downstream of S6 reduces inhibition by divalent cations. An analogous His residue present in some CNG channels is an inhibitory cation binding site. To investigate whether inhibition by divalent cations is conserved in an invertebrate SLO2 channel we cloned the SLO2 channel fromDrosophila(dSLO2) and compared its properties to those of rat SLO2.2. We found that, like rat SLO2.2, dSLO2 was also activated by Na(+)and inhibited by divalent cations. Inhibition of SLO2 channels in mammals andDrosophilaby divalent cations that have second messenger functions may reflect the physiological regulation of these channels by one or more of these ions. PMID:26823461

  1. Tonic PKA Activity Regulates SK Channel Nanoclustering and Somatodendritic Distribution.

    PubMed

    Abiraman, Krithika; Sah, Megha; Walikonis, Randall S; Lykotrafitis, George; Tzingounis, Anastasios V

    2016-06-01

    Small-conductance calcium-activated potassium (SK) channels mediate a potassium conductance in the brain and are involved in synaptic plasticity, learning, and memory. SK channels show a distinct subcellular localization that is crucial for their neuronal functions. However, the mechanisms that control this spatial distribution are unknown. We imaged SK channels labeled with fluorophore-tagged apamin and monitored SK channel nanoclustering at the single molecule level by combining atomic force microscopy and toxin (i.e., apamin) pharmacology. Using these two complementary approaches, we found that native SK channel distribution in pyramidal neurons, across the somatodendritic domain, depends on ongoing cyclic adenosine monophosphate (cAMP)-protein kinase A (PKA) levels, strongly limiting SK channel expression at the pyramidal neuron soma. Furthermore, tonic cAMP-PKA levels also controlled whether SK channels were expressed in nanodomains as single entities or as a group of multiple channels. Our study reveals a new level of regulation of SK channels by cAMP-PKA and suggests that ion channel topography and nanoclustering might be under the control of second messenger cascades. PMID:27107637

  2. Shikonin Inhibits Intestinal Calcium-Activated Chloride Channels and Prevents Rotaviral Diarrhea.

    PubMed

    Jiang, Yu; Yu, Bo; Yang, Hong; Ma, Tonghui

    2016-01-01

    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 Ca(2+) concentration as well as the other major enterocyte chloride channel conductance regulator. 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 calcium-activated chloride channels, the inhibitory effect was partially through inhbition of basolateral K(+) channel activity, and shikonin could be a lead compound in the treatment of rotaviral secretory diarrhea. PMID:27601995

  3. Shikonin Inhibits Intestinal Calcium-Activated Chloride Channels and Prevents Rotaviral Diarrhea

    PubMed Central

    Jiang, Yu; Yu, Bo; Yang, Hong; Ma, Tonghui

    2016-01-01

    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 conductance regulator. 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 calcium-activated chloride channels, the inhibitory effect was partially through inhbition of basolateral K+ channel activity, and shikonin could be a lead compound in the treatment of rotaviral secretory diarrhea. PMID:27601995

  4. Electrodermal activity analysis during affective haptic elicitation.

    PubMed

    Greco, Alberto; Valenza, Gaetano; Nardelli, Mimma; Bianchi, Matteo; Lanata, Antonio; Scilingo, Enzo Pasquale

    2015-08-01

    This paper investigates how the autonomic nervous system dynamics, quantified through the analysis of the electrodermal activity (EDA), is modulated according to affective haptic stimuli. Specifically, a haptic display able to convey caress-like stimuli is presented to 32 healthy subjects (16 female). Each stimulus is changed according to six combinations of three velocities and two forces levels of two motors stretching a strip of fabric. Subjects were also asked to score each stimulus in terms of arousal (high/low activation) and valence (pleasant/unpleasant), in agreement with the circumplex model of affect. EDA was processed using a deconvolutive method, separating tonic and phasic components. A statistical analysis was performed in order to identify significant differences in EDA features among force and velocity levels, as well as in their valence and arousal scores. Results show that the simulated caress induced by the haptic display significantly affects the EDA. In detail, the phasic component seems to be inversely related to the valence score. This finding is new and promising, since it can be used, e.g., as an additional cue for haptics design. PMID:26737605

  5. Voltage is a partial activator of rat thermosensitive TRP channels

    PubMed Central

    Matta, José A; Ahern, Gerard P

    2007-01-01

    TRPV1 and TRPM8 are sensory nerve ion channels activated by heating and cooling, respectively. A variety of physical and chemical stimuli activate these receptors in a synergistic manner but the underlying mechanisms are unclear. Both channels are voltage sensitive, and temperature and ligands modulate this voltage dependence. Thus, a voltage-sensing mechanism has become an attractive model to explain the generalized gating of these and other thermo-sensitive TRP channels. We show here using whole-cell and single channel measurements that voltage produces only a partial activation of TRPV1 and TRPM8. At room temperature (20–25°C) membrane depolarization evokes responses that saturate at ∼50–60% of the maximum open probability. Furthermore, high concentrations of capsaicin (10 μm), resiniferatoxin (5 μm) and menthol (6 mm) reveal voltage-independent gating. Similarly, other modes of TRPV1 regulation including heat, protein kinase C-dependent phosphorylation, and protons enhance both the efficacy and sensitivity of voltage activation. In contrast, the TRPV1 antagonist capsazepine produces the opposite effects. These data can be explained by an allosteric model in which voltage, temperature, agonists and inverse agonists are independently coupled, either positively or negatively, to channel gating. Thus, voltage acts separately but in concert with other stimuli to regulate channel activation, and, therefore, a voltage-sensitive mechanism is unlikely to represent a final, gating mechanism for these channels. PMID:17932142

  6. Human monocytes kill M-CSF-expressing glioma cells by BK channel activation.

    PubMed

    Hoa, Neil T; Zhang, Jian Gang; Delgado, Christina L; Myers, Michael P; Callahan, Linda L; Vandeusen, Gerald; Schiltz, Patric M; Wepsic, H Terry; Jadus, Martin R

    2007-02-01

    In this study, human monocytes/macrophages were observed to kill human U251 glioma cells expressing membrane macrophage colony-stimulating factor (mM-CSF) via a swelling and vacuolization process called paraptosis. Human monocytes responded to the mM-CSF-transduced U251 glioma cells, but not to viral vector control U251 glioma cells (U251-VV), by producing a respiratory burst within 20 min. Using patch clamp techniques, functional big potassium (BK) channels were observed on the membrane of the U251 glioma cell. It has been previously reported that oxygen indirectly regulates BK channel function. In this study, it was demonstrated that prolonged BK channel activation in response to the respiratory burst induced by monocytes initiates paraptosis in selected glioma cells. Forced BK channel opening within the glioma cells by BK channel activators (phloretin or pimaric acid) induced U251 glioma cell swelling and vacuolization occurred within 30 min. U251 glioma cell cytotoxicity, induced by using BK channel activators, required between 8 and 12 h. Swelling and vacuolization induced by phloretin and pimaric acid was prevented by iberiotoxin, a specific BK channel inhibitor. Confocal fluorescence microscopy demonstrated BK channels co-localized with the endoplasmic reticulum and mitochondria, the two targeted organelles affected in paraptosis. Iberiotoxin prevented monocytes from producing death in mM-CSF-expressing U251glioma cells in a 24 h assay. This study demonstrates a novel mechanism whereby monocytes can induce paraptosis via the disruption of internal potassium ion homeostasis. PMID:17318194

  7. Chloride dependence of hyperpolarization-activated chloride channel gates.

    PubMed

    Pusch, M; Jordt, S E; Stein, V; Jentsch, T J

    1999-03-01

    1. ClC proteins are a class of voltage-dependent Cl- channels with several members mutated in human diseases. The prototype ClC-0 Torpedo channel is a dimeric protein; each subunit forms a pore that can gate independently from the other one. A common slower gating mechanism acts on both pores simultaneously; slow gating activates ClC-0 at hyperpolarized voltages. The ClC-2 Cl- channel is also activated by hyperpolarization, as are some ClC-1 mutants (e.g. D136G) and wild-type (WT) ClC-1 at certain pH values. 2. We studied the dependence on internal Cl- ([Cl-]i) of the hyperpolarization-activated gates of several ClC channels (WT ClC-0, ClC-0 mutant P522G, ClC-1 mutant D136G and an N-terminal deletion mutant of ClC-2), by patch clamping channels expressed in Xenopus oocytes. 3. With all these channels, reducing [Cl-]i shifted activation to more negative voltages and reduced the maximal activation at most negative voltages. 4. We also investigated the external halide dependence of WT ClC-2 using two-electrode voltage-clamp recording. Reducing external Cl- ([Cl-]o) activated ClC-2 currents. Replacing [Cl-]o by the less permeant Br- reduced channel activity and accelerated deactivation. 5. Gating of the ClC-2 mutant K566Q in normal [Cl-]o resembled that of WT ClC-2 in low [Cl-]o, i.e. channels had a considerable open probability (Po) at resting membrane potential. Substituting external Cl- by Br- or I- led to a decrease in Po. 6. The [Cl-]i dependence of the hyperpolarization-activated gates of various ClC channels suggests a similar gating mechanism, and raises the possibility that the gating charge for the hyperpolarization-activated gate is provided by Cl-. 7. The external halide dependence of hyperpolarization-activated gating of ClC-2 suggests that it is mediated or modulated by anions as in other ClC channels. In contrast to the depolarization-activated fast gates of ClC-0 and ClC-1, the absence of Cl- favours channel opening. Lysine 556 may be important for the

  8. Oxidative Regulation of Large Conductance Calcium-Activated Potassium Channels

    PubMed Central

    Tang, Xiang D.; Daggett, Heather; Hanner, Markus; Garcia, Maria L.; McManus, Owen B.; Brot, Nathan; Weissbach, Herbert; Heinemann, Stefan H.; Hoshi, Toshinori

    2001-01-01

    Reactive oxygen/nitrogen species are readily generated in vivo, playing roles in many physiological and pathological conditions, such as Alzheimer's disease and Parkinson's disease, by oxidatively modifying various proteins. Previous studies indicate that large conductance Ca2+-activated K+ channels (BKCa or Slo) are subject to redox regulation. However, conflicting results exist whether oxidation increases or decreases the channel activity. We used chloramine-T, which preferentially oxidizes methionine, to examine the functional consequences of methionine oxidation in the cloned human Slo (hSlo) channel expressed in mammalian cells. In the virtual absence of Ca2+, the oxidant shifted the steady-state macroscopic conductance to a more negative direction and slowed deactivation. The results obtained suggest that oxidation enhances specific voltage-dependent opening transitions and slows the rate-limiting closing transition. Enhancement of the hSlo activity was partially reversed by the enzyme peptide methionine sulfoxide reductase, suggesting that the upregulation is mediated by methionine oxidation. In contrast, hydrogen peroxide and cysteine-specific reagents, DTNB, MTSEA, and PCMB, decreased the channel activity. Chloramine-T was much less effective when concurrently applied with the K+ channel blocker TEA, which is consistent with the possibility that the target methionine lies within the channel pore. Regulation of the Slo channel by methionine oxidation may represent an important link between cellular electrical excitability and metabolism. PMID:11222629

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

    PubMed Central

    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 channels. Voltage– and patch–clamp measurements, however, showed neither stimulation nor inhibition of ENaC-mediated conductance by activated CFTR. We conclude that the observed modulation of 22Na+ uptake by activated CFTR is due to the effect of CFTR-mediated chloride conductance on the membrane potential. These findings argue against the notion of a specific influence of CFTR on ENaC and emphasize the chloride channel function of CFTR. PMID:11266369

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

    PubMed

    Nagel, G; Szellas, T; Riordan, J R; Friedrich, T; Hartung, K

    2001-03-01

    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 (22)Na(+) 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 channels. Voltage- and patch-clamp measurements, however, showed neither stimulation nor inhibition of ENaC-mediated conductance by activated CFTR. We conclude that the observed modulation of (22)Na(+) uptake by activated CFTR is due to the effect of CFTR-mediated chloride conductance on the membrane potential. These findings argue against the notion of a specific influence of CFTR on ENaC and emphasize the chloride channel function of CFTR. PMID:11266369

  11. An anion channel in Arabidopsis hypocotyls activated by blue light.

    PubMed Central

    Cho, M H; Spalding, E P

    1996-01-01

    A rapid, transient depolarization of the plasma membrane in seedling stems is one of the earliest effects of blue light detected in plants. It appears to play a role in transducing blue light into inhibition of hypocotyl (stem) elongation, and perhaps other responses. The possibility that activation of a Cl- conductance is part of the depolarization mechanism was raised previously and addressed here. By patch clamping hypocotyl cells isolated from dark-grown (etiolated) Arabidopsis seedlings, blue light was found to activate an anion channel residing at the plasma membrane. An anion-channel blocker commonly known as NPPB 15-nitro-2-(3-phenylpropylamino)-benzoic acid] potently and reversibly blocked this anion channel. NPPB also blocked the blue-light-induced depolarization in vivo and decreased the inhibitory effect of blue light on hypocotyl elongation. These results indicate that activation of this anion channel plays a role in transducing blue light into growth inhibition. PMID:8755616

  12. An anion channel in Arabidopsis hypocotyls activated by blue light

    NASA Technical Reports Server (NTRS)

    Cho, M. H.; Spalding, E. P.; Evans, M. L. (Principal Investigator)

    1996-01-01

    A rapid, transient depolarization of the plasma membrane in seedling stems is one of the earliest effects of blue light detected in plants. It appears to play a role in transducing blue light into inhibition of hypocotyl (stem) elongation, and perhaps other responses. The possibility that activation of a Cl- conductance is part of the depolarization mechanism was raised previously and addressed here. By patch clamping hypocotyl cells isolated from dark-grown (etiolated) Arabidopsis seedlings, blue light was found to activate an anion channel residing at the plasma membrane. An anion-channel blocker commonly known as NPPB 15-nitro-2-(3-phenylpropylamino)-benzoic acid] potently and reversibly blocked this anion channel. NPPB also blocked the blue-light-induced depolarization in vivo and decreased the inhibitory effect of blue light on hypocotyl elongation. These results indicate that activation of this anion channel plays a role in transducing blue light into growth inhibition.

  13. Phosphatase inhibitors activate normal and defective CFTR chloride channels.

    PubMed Central

    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 epithelial cells. We report that the alkaline phosphatase inhibitors bromotetramisole, 3-isobutyl-1-methylxanthine, theophylline, and vanadate slow the rundown of CFTR channel activity in excised membrane patches and reduce dephosphorylation of CFTR protein in isolated membranes. It was also found that in unstimulated cells, CFTR channels can be activated by exposure to phosphatase inhibitors alone. Most importantly, exposure of mammalian cells to phosphatase inhibitors alone activates CFTR channels that have disease-causing mutations, provided the mutant channels are present in the plasma membrane (R117H, G551D, and delta F508 after cooling). These results suggest that CFTR dephosphorylation is dynamic and that membrane-associated phosphatase activity may be a potential therapeutic target for the treatment of cystic fibrosis. Images PMID:7522329

  14. Phosphatase inhibitors activate normal and defective CFTR chloride channels.

    PubMed

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

    1994-09-13

    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 epithelial cells. We report that the alkaline phosphatase inhibitors bromotetramisole, 3-isobutyl-1-methylxanthine, theophylline, and vanadate slow the rundown of CFTR channel activity in excised membrane patches and reduce dephosphorylation of CFTR protein in isolated membranes. It was also found that in unstimulated cells, CFTR channels can be activated by exposure to phosphatase inhibitors alone. Most importantly, exposure of mammalian cells to phosphatase inhibitors alone activates CFTR channels that have disease-causing mutations, provided the mutant channels are present in the plasma membrane (R117H, G551D, and delta F508 after cooling). These results suggest that CFTR dephosphorylation is dynamic and that membrane-associated phosphatase activity may be a potential therapeutic target for the treatment of cystic fibrosis. PMID:7522329

  15. Ion permeation of AQP6 water channel protein. Single channel recordings after Hg2+ activation.

    PubMed

    Hazama, Akihiro; Kozono, David; Guggino, William B; Agre, Peter; Yasui, Masato

    2002-08-01

    Aquaporin-6 (AQP6) has recently been identified as an intracellular vesicle water channel with anion permeability that is activated by low pH or HgCl2. Here we present direct evidence of AQP6 channel gating using patch clamp techniques. Cell-attached patch recordings of AQP6 expressed in Xenopus laevis oocytes indicated that AQP6 is a gated channel with intermediate conductance (49 picosiemens in 100 mm NaCl) induced by 10 microm HgCl2. Current-voltage relationships were linear, and open probability was fairly constant at any given voltage, indicating that Hg2+-induced AQP6 conductance is voltage-independent. The excised outside-out patch recording revealed rapid activation of AQP6 channels immediately after application of 10 microm HgCl2. Reduction of both Na+ and Cl- concentrations from 100 to 30 mm did not shift the reversal potential of the Hg2+-induced AQP6 current, suggesting that Na+ is as permeable as Cl-. The Na+ permeability of Hg2+-induced AQP6 current was further demonstrated by 22Na+ influx measurements. Site-directed mutagenesis identified Cys-155 and Cys-190 residues as the sites of Hg2+ activation both for water permeability and ion conductance. The Hill coefficient from the concentration-response curve for Hg2+-induced conductance was 1.1 +/- 0.3. These data provide the first evidence of AQP6 channel gating at a single-channel level and suggest that each monomer contains the pore region for ions based on the number of Hg2+-binding sites and the kinetics of Hg2+-activation of the channel. PMID:12034750

  16. Cytogenetic and molecular localization of tipE: A gene affecting sodium channels in Drosophila melanogaster

    SciTech Connect

    Feng, G.; Deak, P.; Hall, L.M.

    1995-04-01

    Voltage-sensitive sodium channels play a key role in nerve cells where they are responsible for the increase in sodium permeability during the rising phase of action potentials. In Drosophila melanogaster a subset of temperature-sensitive paralytic mutations affect sodium channel function. One such mutation is temperature-induced paralysis locus E (tipE), which has been shown by electrophysiology and ligand binding studies to reduce sodium channel numbers. Three new {gamma}-ray-induced tipE alleles associated with either visible deletions in 64AB or a translocation breakpoint within 64B2 provide landmarks for positional cloning of tipE. Beginning with the flanking cloned gene Ras2, a 140-kb walk across the translocation breakpoint was completed. Germline transformation using a 42-kb cosmid clone and successively smaller subclones localized the tipE gene within a 7.4-kb genomic DNA segment. Although this chromosome region is rich in transcripts, only three overlapping mRNAs (5.4, 4.4, and 1.7 kb) lie completely within the smallest rescuing construct. The small sizes of the rescuing construct and transcripts suggests that tipE does not encode a standard sodium channel {alpha}-subunit with four homologous repeats. Sequencing these transcripts will elucidate the role of the tipE gene product in sodium channel functional regulation. 55 refs., 4 figs., 2 tabs.

  17. Smooth muscle BK channel activity influences blood pressure independent of vascular tone in mice

    PubMed Central

    Sachse, Gregor; Faulhaber, Jörg; Seniuk, Anika; Ehmke, Heimo; Pongs, Olaf

    2014-01-01

    The large conductance voltage- and Ca2+-activated K+ (BK) channel is an important determinant of vascular tone and contributes to blood pressure regulation. Both activities depend on the ancillary BKβ1 subunit. To determine the significance of smooth muscle BK channel activity for blood pressure regulation, we investigated the potential link between changes in arterial tone and altered blood pressure in BKβ1 knockout (BKβ1−/−) mice from three different genetically defined strains. While vascular tone was consistently increased in all BKβ1−/− mice independent of genetic background, BKβ1−/− strains exhibited increased (strain A), unaltered (strain B) or decreased (strain C) mean arterial blood pressures compared to their corresponding BKβ1+/+ controls. In agreement with previous data on aldosterone regulation by renal/adrenal BK channel function, BKβ1−/− strain A mice have increased plasma aldosterone and increased blood pressure. Consistently, blockade of mineralocorticoid receptors by spironolactone treatment reversibly restored the elevated blood pressure to the BKβ1+/+ strain A level. In contrast, loss of BKβ1 did not affect plasma aldosterone in strain C mice. Smooth muscle-restricted restoration of BKβ1 expression increased blood pressure in BKβ1−/− strain C mice, implying that impaired smooth muscle BK channel activity lowers blood pressure in these animals. We conclude that BK channel activity directly affects vascular tone but influences blood pressure independent of this effect via different pathways. PMID:24687584

  18. Flow structures in submarine channels affected by Coriolis forces: Experimental observations

    NASA Astrophysics Data System (ADS)

    Cossu, R.; Wells, M. G.

    2011-12-01

    In this talk we will show how Coriolis forces can control the flow dynamics of turbidity currents flowing in sinuous channels at high latitudes. We describe how the internal velocity structure changes with latitude, based on observations from rotating laboratory experiments. When these results are combined with existing conceptual facies and depositional models we can now describe the changes in sedimentation patterns that are observed at different latitudes. The experiments were conducted in a sinuous channel model placed in a tank that was rotated at various rates (reflected by the Coriolis parameters f) ranging from f = 0 (at the equator) to ± 0.5 rad s-1 (at higher latitudes). The dependence of the density interface of gravity currents on rotation is shown in Figure 1a. At the equator the interface slopes up to the outer bend due to the centrifugal forces. In the Northern Hemisphere (NH) the tilt of the interface increases as now the Coriolis forces reinforce the centrifugal acceleration. In contrast, in the Southern Hemisphere (SH) the current ramps up to the inner bend and Coriolis forces dominate over centrifugal forces. Figure 1b shows the corresponding position of the downstream velocity core in the bend apex. At the equator the core is predominantly close to the centerline, whilst in the NH the core is deflected to the inner bend and in the SH the velocity core is shifted to the outer bank. Based upon our experimental results, we hypothesize that Coriolis forces can affect the velocity structure and sedimentation regime. Lateral accretion packages (LAPs) are built only on one side in the channel and finer sediments will be deposited mainly on the levee bank to which the high velocity core is deflected. The Rossby number RoW = U/fW (where U is the mean downstream velocity and W the channel width) can be used to determine the influence of Coriolis forces. In channel systems at high-latitudes (with RoW << 1) we predict that channels exhibit a low sinuosity

  19. Multi-channel fiber photometry for population neuronal activity recording

    PubMed Central

    Guo, Qingchun; Zhou, Jingfeng; Feng, Qiru; Lin, Rui; Gong, Hui; Luo, Qingming; Zeng, Shaoqun; Luo, Minmin; Fu, Ling

    2015-01-01

    Fiber photometry has become increasingly popular among neuroscientists as a convenient tool for the recording of genetically defined neuronal population in behaving animals. Here, we report the development of the multi-channel fiber photometry system to simultaneously monitor neural activities in several brain areas of an animal or in different animals. In this system, a galvano-mirror modulates and cyclically couples the excitation light to individual multimode optical fiber bundles. A single photodetector collects excited light and the configuration of fiber bundle assembly and the scanner determines the total channel number. We demonstrated that the system exhibited negligible crosstalk between channels and optical signals could be sampled simultaneously with a sample rate of at least 100 Hz for each channel, which is sufficient for recording calcium signals. Using this system, we successfully recorded GCaMP6 fluorescent signals from the bilateral barrel cortices of a head-restrained mouse in a dual-channel mode, and the orbitofrontal cortices of multiple freely moving mice in a triple-channel mode. The multi-channel fiber photometry system would be a valuable tool for simultaneous recordings of population activities in different brain areas of a given animal and different interacting individuals. PMID:26504642

  20. Active Integrated Filters for RF-Photonic Channelizers

    PubMed Central

    Nagdi, Amr El; Liu, Ke; LaFave, Tim P.; Hunt, Louis R.; Ramakrishna, Viswanath; Dabkowski, Mieczyslaw; MacFarlane, Duncan L.; Christensen, Marc P.

    2011-01-01

    A theoretical study of RF-photonic channelizers using four architectures formed by active integrated filters with tunable gains is presented. The integrated filters are enabled by two- and four-port nano-photonic couplers (NPCs). Lossless and three individual manufacturing cases with high transmission, high reflection, and symmetric couplers are assumed in the work. NPCs behavior is dependent upon the phenomenon of frustrated total internal reflection. Experimentally, photonic channelizers are fabricated in one single semiconductor chip on multi-quantum well epitaxial InP wafers using conventional microelectronics processing techniques. A state space modeling approach is used to derive the transfer functions and analyze the stability of these filters. The ability of adapting using the gains is demonstrated. Our simulation results indicate that the characteristic bandpass and notch filter responses of each structure are the basis of channelizer architectures, and optical gain may be used to adjust filter parameters to obtain a desired frequency magnitude response, especially in the range of 1–5 GHz for the chip with a coupler separation of ∼9 mm. Preliminarily, the measurement of spectral response shows enhancement of quality factor by using higher optical gains. The present compact active filters on an InP-based integrated photonic circuit hold the potential for a variety of channelizer applications. Compared to a pure RF channelizer, photonic channelizers may perform both channelization and down-conversion in an optical domain. PMID:22319352

  1. Designing Effective Persuasive Systems Utilizing the Power of Entanglement: Communication Channel, Strategy and Affect

    NASA Astrophysics Data System (ADS)

    Li, Haiqing; Chatterjee, Samir

    With rapid advances in information and communication technology, computer-mediated communication (CMC) technologies are utilizing multiple IT platforms such as email, websites, cell-phones/PDAs, social networking sites, and gaming environments. However, no studies have compared the effectiveness of a persuasive system using such alternative channels and various persuasive techniques. Moreover, how affective computing impacts the effectiveness of persuasive systems is not clear. This study proposes (1) persuasive technology channels in combination with persuasive strategies will have different persuasive effectiveness; (2) Adding positive emotion to a message that leads to a better overall user experience could increase persuasive effectiveness. The affective computing or emotion information was added to the experiment using emoticons. The initial results of a pilot study show that computer-mediated communication channels along with various persuasive strategies can affect the persuasive effectiveness to varying degrees. These results also shows that adding a positive emoticon to a message leads to a better user experience which increases the overall persuasive effectiveness of a system.

  2. LE135, a retinoid acid receptor antagonist, produces pain through direct activation of TRP channels

    PubMed Central

    Yin, Shijin; Luo, Jialie; Qian, Aihua; Yu, Weihua; Hu, Hongzhen

    2014-01-01

    Background and PurposeRetinoids, through their activation of retinoic acid receptors (RARs) and retinoid X receptors, regulate diverse cellular processes, and pharmacological intervention in their actions has been successful in the treatment of skin disorders and cancers. Despite the many beneficial effects, administration of retinoids causes irritating side effects with unknown mechanisms. Here, we demonstrate that LE135 [4-(7,8,9,10-tetrahydro-5,7,7,10,10-pentamethyl-5H-benzo[e]naphtho[2,3-b][1,4]diazepin-13-yl)benzoic acid], a selective antagonist of RARβ, is a potent activator of the capsaicin (TRPV1) and wasabi (TRPA1) receptors, two critical pain-initiating cation channels. Experimental ApproachWe performed to investigate the excitatory effects of LE135 on TRPV1 and TRPA1 channels expressed in HEK293T cells and in dorsal root ganglia neurons with calcium imaging and patch-clamp recordings. We also used site-directed mutagenesis of the channels to determine the structural basis of LE135-induced activation of TRPV1 and TRPA1 channels and behavioural testing to examine if pharmacological inhibition and genetic deletion of the channels affected LE135-evoked pain-related behaviours. Key ResultsLE135 activated both the capsaicin receptor (TRPV1) and the allyl isothiocyanate receptor (TRPA1) heterologously expressed in HEK293T cells and endogenously expressed by sensory nociceptors. Mutations disrupting the capsaicin-binding site attenuated LE135 activation of TRPV1 channels and a single mutation (K170R) eliminated TRPA1 activity evoked by LE135. Intraplantar injection of LE135 evoked pain-related behaviours. Both TRPV1 and TRPA1 channels were involved in LE135-elicited pain-related responses, as shown by pharmacological and genetic ablation studies. Conclusions and ImplicationsThis blocker of retinoid acid signalling also exerted non-genomic effects through activating the pain-initiating TRPV1 and TRPA1 channels. PMID:24308840

  3. Brain Activity, Personality Traits and Affect: Electrocortical Activity in Reaction to Affective Film Stimuli

    NASA Astrophysics Data System (ADS)

    Makvand Hosseini, Sh.; Azad Fallah, P.; Rasoolzadeh Tabatabaei, S. K.; Ghannadyan Ladani, S. H.; Heise, C.

    We studied the patterns of activation over the cerebral cortex in reaction to affective film stimuli in four groups of extroverts, introverts, neurotics and emotionally stables. Measures of extraversion and neuroticism were collected and resting EEG was recorded from 40 right handed undergraduate female students (19-23) on one occasion for five 30s periods in baseline condition and in affective states. Mean log-transformed absolute alpha power was extracted from 12 electrode sites and analyzed. Patterns of activation were different in personality groups. Different patterns of asymmetries were observed in personality groups in reaction to affective stimuli. Results were partly consistent with approach and withdrawal model and provided supportive evidence for the role of right frontal asymmetry in negative affects in two groups (introverts and emotionally stables) as well as the role of right central asymmetry (increase on right and decrease on left) in active affective states (anxiety and happiness) in all personality groups. Results were also emphasized on the role of decrease activity relative to baseline in cortical regions (bilaterally in frontal and unilaterally in left parietal and temporal regions) in moderating of positive and negative emotion.

  4. Cyclic nucleotide-activated channels in carp olfactory receptor cells.

    PubMed

    Kolesnikov, S S; Kosolapov, A V

    1993-07-25

    When applied from the cytoplasmic side, cyclic 3',5'-adenosine and guanosine monophosphates reversibly increased the ion permeability of inside-out patches of carp olfactory neuron plasma membrane. The cAMP (cGMP)-induced permeability via cAMP (cGMP) concentration was fitted by Hill's equation with the exponents of 1.07 +/- 0.15 (1.12 +/- 0.05) and EC50 = 1.3 +/- 0.6 microM (0.9 +/- 0.3 microM). Substitution of NaCl in the bathing solution by chlorides of other alkali metals resulted in a slight shift of reversal potential of the cyclic nucleotide-dependent (CN) current, which indicates a weak selectivity of the channels. Permeability coefficients calculated by Goldman-Hodgkin-Katz's equation corresponded to the following relation: PNa/PK/PLi/PRb/PCs = 1:0.98:0.94:0.70:0.61. Ca2+ and Mg2+ in physiological concentrations blocked the channels activated by cyclic nucleotides (CN-channels). In the absence of divalent cations the conductance of single CN-channels was equal to 51 +/- 9 pS in 100 mM NaCl solution. Channel density did not exceed 1 micron-2. The maximal open state probability of the channel (Po) tended towards 1.0 at a high concentration of cAMP or cGMP. Dichlorobenzamil decreased Po without changing the single CN-channel' conductance. CN-channels exhibited burst activity. Mean open and closed times as well as the burst duration depended on agonist concentration. A kinetic model with four states (an inactivated, a closed and two open ones) is suggested to explain the regularities of CN-channel gating and dose-response relations. PMID:8334139

  5. Novel Activation of Voltage-gated K+ Channels by Sevoflurane*

    PubMed Central

    Barber, Annika F.; Liang, Qiansheng; Covarrubias, Manuel

    2012-01-01

    Voltage-gated ion channels are modulated by halogenated inhaled general anesthetics, but the underlying molecular mechanisms are not understood. Alkanols and halogenated inhaled anesthetics such as halothane and isoflurane inhibit the archetypical voltage-gated Kv3 channel homolog K-Shaw2 by stabilizing the resting/closed states. By contrast, sevoflurane, a more heavily fluorinated ether commonly used in general anesthesia, specifically activates K-Shaw2 currents at relevant concentrations (0.05–1 mm) in a rapid and reversible manner. The concentration dependence of this modulation is consistent with the presence of high and low affinity interactions (KD = 0.06 and 4 mm, respectively). Sevoflurane (<1 mm) induces a negative shift in the conductance-voltage relation and increases the maximum conductance. Furthermore, suggesting possible roles in general anesthesia, mammalian Kv1.2 and Kv1.5 channels display similar changes. Quantitative description of the observations by an economical allosteric model indicates that sevoflurane binding favors activation gating and eliminates an unstable inactivated state outside the activation pathway. This study casts light on the mechanism of the novel sevoflurane-dependent activation of Kv channels, which helps explain how closely related inhaled anesthetics achieve specific actions and suggests strategies to develop novel Kv channel activators. PMID:23038249

  6. Activation of TRPC cationic channels by mercurial compounds confers the cytotoxicity of mercury exposure.

    PubMed

    Xu, Shang-Zhong; Zeng, Bo; Daskoulidou, Nikoleta; Chen, Gui-Lan; Atkin, Stephen L; Lukhele, Bhekithemba

    2012-01-01

    Mercury is an established worldwide environmental pollutant with well-known toxicity affecting neurodevelopment in humans, but the molecular basis of cytotoxicity and the detoxification procedure are still unclear. Here we examined the involvement of the canonical transient receptor potential (TRPC) channel in the mercury-induced cytotoxicity and the potential detoxification strategy. Whole-cell and excised patches, Ca(2+) imaging, and site-directed mutagenesis were used to determine the mechanism of action of mercurial compounds on TRPC channels overexpressed in HEK293 cells, and cytotoxicity and preventive effect were investigated in cell culture models using small interfering RNA and pharmacological blockers. Mercury potently activates TRPC4 and TRPC5 channels. The extracellular cysteine residues (C(553) and C(558)) near the channel pore region of TRPC5 are the molecular targets for channel activation by mercury. The sensitivity of mercury to TRPC5 is presumed to be specific because other divalent heavy metal pollutants, such as Cd(2+), Ni(2+), and Zn(2+), had no stimulating effect, and TRPC3, TRPC6, TRPV1, and TRPM2 were resistant to mercurial compounds. The channel activity of TRPC5, as well as TRPC4, induced by mercury, was prevented by 2-aminoethoxydiphenyl borate and modified by a reducing environment. The inhibition of TRPC5 channels by specific TRPC5 pore-blocking antibody or by SKF-96365 alleviated the cytotoxicity, whereas the mercury chelator, meso-2,3-dimercaptosuccinic acid, showed nonselective prevention of cell survival. Silencing of the TRPC5 gene reduced the mercury-induced neuronal damage. These results indicate that mercurial compounds are activators for TRPC5 and TRPC4 channels. Blockade of TRPC channels could be a novel strategy for preventing mercury-induced cytotoxicity and neurodevelopment impairment. PMID:21984481

  7. Kv1.3 potassium channel mediates macrophage migration in atherosclerosis by regulating ERK activity.

    PubMed

    Kan, Xiao-Hong; Gao, Hai-Qing; Ma, Zhi-Yong; Liu, Lin; Ling, Ming-Ying; Wang, Yuan-Yuan

    2016-02-01

    Ion channels expressed in macrophages have been tightly related to atherosclerosis by coupling cellular function. How the voltage-gated potassium channels (Kv) affect macrophage migration remain unknown. The aim of our study is to investigate whether Kv1.3-ERK signaling pathway plays an important role in the process. We explored the expression of Kv1.3 in coronary atherosclerotic heart disease and found Kv1.3 channel was increased in acute coronary syndrome patients. Treatment of RAW264.7 cells with Kv1.3 small interfering RNA, suppressed cell migration. The expression of phosphorylated ERK1/2 also decreased after knockdown of Kv1.3. On the other hand, overexpression of Kv1.3 channel promoted cell migration and ERK1/2 phosphorylation. U-0126, the mitogen-activated protein kinase inhibitors, could reverse macrophage migration induced by Kv1.3 channel overexpression. Downregulation of Kv1.3 channel by siRNA could not further inhibit cell migration when cells were treated with U-0126. It means that ERK is downstream signal of Kv1.3 channel. We concluded that Kv1.3 may stimulate macrophage migration through the activation of ERK. PMID:26748289

  8. Calcium-activated potassium channels and endothelial dysfunction: therapeutic options?

    PubMed Central

    Félétou, Michel

    2009-01-01

    The three subtypes of calcium-activated potassium channels (KCa) of large, intermediate and small conductance (BKCa, IKCa and SKCa) are present in the vascular wall. In healthy arteries, BKCa channels are preferentially expressed in vascular smooth muscle cells, while IKCa and SKCa are preferentially located in endothelial cells. The activation of endothelial IKCa and SKCa contributes to nitric oxide (NO) generation and is required to elicit endothelium-dependent hyperpolarizations. In the latter responses, the hyperpolarization of the smooth muscle cells is evoked either via electrical coupling through myo-endothelial gap junctions or by potassium ions, which by accumulating in the intercellular space activate the inwardly rectifying potassium channel Kir2.1 and/or the Na+/K+-ATPase. Additionally, endothelium-derived factors such as cytochrome P450-derived epoxyeicosatrienoic acids and under some circumstances NO, prostacyclin, lipoxygenase products and hydrogen peroxide (H2O2) hyperpolarize and relax the underlying smooth muscle cells by activating BKCa. In contrast, cytochrome P450-derived 20-hydroxyeicosatetraenoic acid and various endothelium-derived contracting factors inhibit BKCa. Aging and cardiovascular diseases are associated with endothelial dysfunctions that can involve a decrease in NO bioavailability, alterations of EDHF-mediated responses and/or enhanced production of endothelium-derived contracting factors. Because potassium channels are involved in these endothelium-dependent responses, activation of endothelial and/or smooth muscle KCa could prevent the occurrence of endothelial dysfunction. Therefore, direct activators of these potassium channels or compounds that regulate their activity or their expression may be of some therapeutic interest. Conversely, blockers of IKCa may prevent restenosis and that of BKCa channels sepsis-dependent hypotension. PMID:19187341

  9. Trypsin increases availability and open probability of cardiac L-type Ca2+ channels without affecting inactivation induced by Ca2+.

    PubMed Central

    Schmid, R; Seydl, K; Baumgartner, W; Groschner, K; Romanin, C

    1995-01-01

    The patch-clamp technique was employed to investigate the response of single L-type Ca2+ channels to the protease trypsin applied to the intracellular face of excised membrane patches from guinea pig ventricular myocytes. Calpastatin and ATP were used to prevent run-down of Ca2+ channel activity monitored with 96 mM Ba2+ as charge carrier in the presence of 2.5 microM (-)-BAYK 8644. Upon application of trypsin (100 micrograms/ml) channel activity was enhanced fourfold and remained elevated upon removal of trypsin, as expected of a proteolytic, irreversible modification. The trypsin effect was not mediated by a proteolytic activation of protein kinases, as evidenced by the insensitivity of this effect to protein kinase inhibitors. Trypsin-modified Ca2+ channels exhibited the usual run-down phanomenon upon removal of calpastatin and ATP. In ensemble average currents trypsin-induced changes of channel function are apparent as a threefold increase in peak current and a reduction in current inactivation. At the single channel level these effects were based on about a twofold increase in both Ca2+ channels' availability and open probability. Neither the actual number of channels in the patch nor their unitary conductance as well as reversal potential was changed by trypsin. The Ca(2+)-induced inactivation was not impaired, as judged by a comparable sensitivity of trypsin-modified Ca2+ channels to intracellular Ca2+. Similarly, trypsin treatment did not affect the sensitivity of Ca2+ channels to phenylalkylmine inhibition. The observed alterations in channel function are discussed in terms of possible structural correlates. PMID:8580328

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

    PubMed

    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. PMID:25896054

  11. Activation of peripheral KCNQ channels relieves gout pain

    PubMed Central

    Zheng, Yueming; Xu, Haiyan; Zhan, Li; Zhou, Xindi; Chen, Xueqin; Gao, Zhaobing

    2015-01-01

    Abstract Intense inflammatory pain caused by urate crystals in joints and other tissues is a major symptom of gout. Among therapy drugs that lower urate, benzbromarone (BBR), an inhibitor of urate transporters, is widely used because it is well tolerated and highly effective. We demonstrate that BBR is also an activator of voltage-gated KCNQ potassium channels. In cultured recombinant cells, BBR exhibited significant potentiation effects on KCNQ channels comparable to previously reported classical activators. In native dorsal root ganglion neurons, BBR effectively overcame the suppression of KCNQ currents, and the resultant neuronal hyperexcitability caused by inflammatory mediators, such as bradykinin (BK). Benzbromarone consistently attenuates BK-, formalin-, or monosodium urate–induced inflammatory pain in rat and mouse models. Notably, the analgesic effects of BBR are largely mediated through peripheral and not through central KCNQ channels, an observation supported both by pharmacokinetic studies and in vivo experiments. Moreover, multiple residues in the superficial part of the voltage sensing domain of KCNQ channels were identified critical for the potentiation activity of BBR by a molecular determinant investigation. Our data indicate that activation of peripheral KCNQ channels mediates the pain relief effects of BBR, potentially providing a new strategy for the development of more effective therapies for gout. PMID:25735002

  12. Na(+) -Activated K(+) Channels in Rat Supraoptic Neurones.

    PubMed

    Bansal, V; Fisher, T E

    2016-06-01

    The magnocellular neurosecretory cells (MNCs) of the hypothalamus secrete the neurohormones vasopressin and oxytocin. The systemic release of these hormones depends on the rate and pattern of MNC firing and it is therefore important to identify the ion channels that contribute to the electrical behaviour of MNCs. In the present study, we report evidence for the presence of Na(+) -activated K(+) (KN a ) channels in rat MNCs. KN a channels mediate outwardly rectifying K(+) currents activated by the increases in intracellular Na(+) that occur during electrical activity. Although the molecular identity of native KN a channels is unclear, their biophysical properties are consistent with those of expressed Slick (slo 2.1) and Slack (slo 2.2) proteins. Using immunocytochemistry and Western blot experiments, we found that both Slick and Slack proteins are expressed in rat MNCs. Using whole cell voltage clamp techniques on acutely isolated rat MNCs, we found that inhibiting Na(+) influx by the addition of the Na(+) channel blocker tetrodotoxin or the replacement of Na(+) in the external solution with Li(+) caused a significant decrease in sustained outward currents. Furthermore, the evoked outward current density was significantly higher in rat MNCs using patch pipettes containing 60 mm Na(+) than it was when patch pipettes containing 0 mm Na(+) were used. Our data show that functional KN a channels are expressed in rat MNCs. These channels could contribute to the activity-dependent afterhyperpolarisations that have been identified in the MNCs and thereby play a role in the regulation of their electrical behaviour. PMID:27091544

  13. Use of Small Fluorescent Molecules to Monitor Channel Activity

    NASA Astrophysics Data System (ADS)

    Jones, Sharon; Stringer, Sarah; Naik, Rajesh; Stone, Morley

    2001-03-01

    The Mechanosensitive channel of Large conductance (MscL) allows bacteria to rapidly adapt to changing environmental conditions such as osmolarity. The MscL channel opens in response to increases in membrane tension, which allows for the efflux of cytoplasmic constituents. Here we describe the cloning and expression of Salmonella typhimurium MscL (St-MscL). Using a fluorescence efflux assay, we demonstrate that efflux through the MscL channel during hypoosmotic shock can be monitored using endogenously produced fluorophores. In addition, we observe that thermal stimulation, i.e., heat shock, can also induce efflux through MscL. We present the first evidence of thermal activation of MscL efflux by heat shocking cells expressing the S. typhimurium protein variant. This finding has significant biosensor implications, especially for investigators exploring the use of channel proteins in biosensor applications. Thermal biosensors are relatively unexplored, but would have considerable commercial and military utility.

  14. Neuronal modulation of calcium channel activity in cultured rat astrocytes

    SciTech Connect

    Corvalan, V.; Cole, R.; De Vellis, J.; Hagiwara, Susumu )

    1990-06-01

    The patch-clamp technique was used to study whether cocultivation of neurons and astrocytes modulates the expression of calcium channel activity in astrocytes. Whole-cell patch-clamp recordings from rat brain astrocytes cocultured with rat embryonic neurons revealed two types of voltage-dependent inward currents carried by Ca{sup 2+} and blocked by either Cd{sup 2+} or Co{sup 2+} that otherwise were not detected in purified astrocytes. This expression of calcium channel activity in astrocytes was neuron dependent and was not observed when astrocytes were cocultured with purified oligodendrocytes.

  15. Science communication on YouTube: Factors that affect channel and video popularity.

    PubMed

    Welbourne, Dustin J; Grant, Will J

    2016-08-01

    YouTube has become one of the largest websites on the Internet. Among its many genres, both professional and amateur science communicators compete for audience attention. This article provides the first overview of science communication on YouTube and examines content factors that affect the popularity of science communication videos on the site. A content analysis of 390 videos from 39 YouTube channels was conducted. Although professionally generated content is superior in number, user-generated content was significantly more popular. Furthermore, videos that had consistent science communicators were more popular than those without a regular communicator. This study represents an important first step to understand content factors, which increases the channel and video popularity of science communication on YouTube. PMID:25698225

  16. Afferent Arteriolar Dilation to 11,12-EET Analogs Involves PP2A Activity and Ca2+-Activated K+ Channels

    PubMed Central

    Imig, John D.; Dimitropoulou, Christiana; Reddy, D. Sudarshan; White, Richard E.; Falck, John R.

    2008-01-01

    The epoxygenase metabolite, 11,12-epoxyeicosatrienoic acid (11,12-EET), has renal vascular actions. 11,12-EET analogs have been developed to determine the structure activity relationship for 11,12-EET and as a tool to investigate signaling mechanisms responsible for afferent arteriolar dilation. We hypothesized that 11,12-EET mediated afferent arteriolar dilation involves increased phosphoprotein phosphatase 2A (PP2A) and large-conductance calcium activated K+ (KCa) channels. We evaluated the chemically and/or metabolically stable 11,12-EET analogs: 11,12-EET-N-methylsulfonimide (11,12-EET-SI), 11-nonyloxy-undec-8(Z)-enoic acid (11,12-ether-EET-8-ZE), and 11,12-trans-oxidoeicosa-8(Z)-eonoic acid (11,12-tetra-EET-8-ZE). Afferent arteriolar responses were assessed. Activation of KCa channels by 11,12-EET analogs were established by single cell channel recordings in renal myocytes. Assessment of renal vascular responses revealed that 11,12-EET analogs increased afferent arteriolar diameter. Vasodilator responses to 11,12-EET analogs were abolished by K+ channel or PP2A inhibition. 11,12-EET analogs activated renal myocyte large-conductance KCa channels. 11,12-EET analogs increased cAMP by 2-fold and PP2A activity increased 3-8 fold in renal myocytes. PP2A inhibition did not significantly affect the 11,12-EET analog mediated increase in cAMP and PP2A increased renal myocyte KCa channel activity to a much greater extent than PKA. These data support the concept that 11,12-EET utilizes PP2A dependent pathways to activate large-conductance KCa channels and dilate the afferent arteriole. PMID:18260004

  17. Evidence for glucagon-like peptide-1 receptor signaling to activate ATP-sensitive potassium channels in pancreatic beta cells.

    PubMed

    Kwon, Hye-Jung; Park, Hyun-Sun; Park, Sung-Hee; Park, Jae-Hyung; Shin, Su-Kyung; Song, Seung Eun; Hwang, Meeyul; Cho, Ho-Chan; Song, Dae-Kyu

    2016-01-01

    Glucagon-like peptide-1 (GLP-1) is a gut peptide that promotes insulin release from pancreatic beta cells. GLP-1 has been shown to confer glucose-insensitive beta cells with glucose sensitivity by modulation of the activity of the ATP-sensitive potassium (KATP) channel. The channel closing effect of GLP-1, interacting with corresponding G-protein-coupled receptors, has been well established; however, to our knowledge, no study has shown whether GLP-1 directly induces activation of beta-cell KATP channels. Here, we aimed to evaluate whether the activation of beta-cell KATP channels by GLP-1 exists and affects intracellular Ca(2+) levels ([Ca(2+)]i). KATP channel activity was measured in isolated rat pancreatic beta cells by whole-cell perforated patch-clamp recordings with a diazoxide-containing pipette solution. Changes in [Ca(2+)]i and the subcellular localization of KATP channels were observed using the calcium-sensitive dye fura-4/AM and anti-Kir6.2 antibodies in INS-1 beta cells, respectively. To eliminate the well-known inhibitory effects of GLP-1 on KATP channel activity, channels were fully inhibited by pretreatment with methyl pyruvate and epigallocatechin-3-gallate. In the pretreated beta cells, GLP-1 and exendin-4 promptly activated the channels, reducing [Ca(2+)]i. The phosphoinositide 3-kinase (PI3K) inhibitor LY294002 blocked the effects of GLP-1 on channel activity. Moreover, phosphatidylinositol-3,4,5-trisphosphate mimicked the effects of GLP-1. These results suggested that beta-cell GLP-1 receptor signaling involved activation of KATP channels via a PI3K-dependent pathway. This alternative mechanism of GLP-1 function may act as a negative feedback pathway, modulating the glucose-dependent GLP-1 inhibition on KATP channel activity. PMID:26655814

  18. Detection of single ion channel activity with carbon nanotubes

    PubMed Central

    Zhou, Weiwei; Wang, Yung Yu; Lim, Tae-Sun; Pham, Ted; Jain, Dheeraj; Burke, Peter J.

    2015-01-01

    Many processes in life are based on ion currents and membrane voltages controlled by a sophisticated and diverse family of membrane proteins (ion channels), which are comparable in size to the most advanced nanoelectronic components currently under development. Here we demonstrate an electrical assay of individual ion channel activity by measuring the dynamic opening and closing of the ion channel nanopores using single-walled carbon nanotubes (SWNTs). Two canonical dynamic ion channels (gramicidin A (gA) and alamethicin) and one static biological nanopore (α-hemolysin (α-HL)) were successfully incorporated into supported lipid bilayers (SLBs, an artificial cell membrane), which in turn were interfaced to the carbon nanotubes through a variety of polymer-cushion surface functionalization schemes. The ion channel current directly charges the quantum capacitance of a single nanotube in a network of purified semiconducting nanotubes. This work forms the foundation for a scalable, massively parallel architecture of 1d nanoelectronic devices interrogating electrophysiology at the single ion channel level. PMID:25778101

  19. Detection of single ion channel activity with carbon nanotubes

    NASA Astrophysics Data System (ADS)

    Zhou, Weiwei; Wang, Yung Yu; Lim, Tae-Sun; Pham, Ted; Jain, Dheeraj; Burke, Peter J.

    2015-03-01

    Many processes in life are based on ion currents and membrane voltages controlled by a sophisticated and diverse family of membrane proteins (ion channels), which are comparable in size to the most advanced nanoelectronic components currently under development. Here we demonstrate an electrical assay of individual ion channel activity by measuring the dynamic opening and closing of the ion channel nanopores using single-walled carbon nanotubes (SWNTs). Two canonical dynamic ion channels (gramicidin A (gA) and alamethicin) and one static biological nanopore (α-hemolysin (α-HL)) were successfully incorporated into supported lipid bilayers (SLBs, an artificial cell membrane), which in turn were interfaced to the carbon nanotubes through a variety of polymer-cushion surface functionalization schemes. The ion channel current directly charges the quantum capacitance of a single nanotube in a network of purified semiconducting nanotubes. This work forms the foundation for a scalable, massively parallel architecture of 1d nanoelectronic devices interrogating electrophysiology at the single ion channel level.

  20. Detection of single ion channel activity with carbon nanotubes.

    PubMed

    Zhou, Weiwei; Wang, Yung Yu; Lim, Tae-Sun; Pham, Ted; Jain, Dheeraj; Burke, Peter J

    2015-01-01

    Many processes in life are based on ion currents and membrane voltages controlled by a sophisticated and diverse family of membrane proteins (ion channels), which are comparable in size to the most advanced nanoelectronic components currently under development. Here we demonstrate an electrical assay of individual ion channel activity by measuring the dynamic opening and closing of the ion channel nanopores using single-walled carbon nanotubes (SWNTs). Two canonical dynamic ion channels (gramicidin A (gA) and alamethicin) and one static biological nanopore (α-hemolysin (α-HL)) were successfully incorporated into supported lipid bilayers (SLBs, an artificial cell membrane), which in turn were interfaced to the carbon nanotubes through a variety of polymer-cushion surface functionalization schemes. The ion channel current directly charges the quantum capacitance of a single nanotube in a network of purified semiconducting nanotubes. This work forms the foundation for a scalable, massively parallel architecture of 1d nanoelectronic devices interrogating electrophysiology at the single ion channel level. PMID:25778101

  1. Phosphatidylinositol-3-kinase regulates mast cell ion channel activity.

    PubMed

    Lam, Rebecca S; Shumilina, Ekaterina; Matzner, Nicole; Zemtsova, Irina M; Sobiesiak, Malgorzata; Lang, Camelia; Felder, Edward; Dietl, Paul; Huber, Stephan M; Lang, Florian

    2008-01-01

    Stimulation of the mast cell IgE-receptor (FcepsilonRI) by antigen leads to stimulation of Ca(2+) entry with subsequent mast cell degranulation and release of inflammatory mediators. Ca(2+) further activates Ca(2+)-activated K(+) channels, which in turn provide the electrical driving force for Ca(2+) entry. Since phosphatidylinositol (PI)-3-kinase has previously been shown to be required for mast cell activation and degranulation, we explored, whether mast cell Ca(2+) and Ca(2+)-activated K(+) channels may be sensitive to PI3-kinase activity. Whole-cell patch clamp experiments and Fura-2 fluorescence measurements for determination of cytosolic Ca(2+) concentration were performed in mouse bone marrow-derived mast cells either treated or untreated with the PI3-kinase inhibitors LY-294002 (10 muM) and wortmannin (100 nM). Antigen-stimulated Ca(2+) entry but not Ca(2+) release from the intracellular stores was dramatically reduced upon PI3-kinase inhibition. Ca(2+) entry was further inhibited by TRPV blocker ruthenium red (10 muM). Ca(2+) entry following readdition after Ca(+)-store depletion with thapsigargin was again decreased by LY-294002, pointing to inhibition of store-operated channels (SOCs). Moreover, inhibition of PI3-kinase abrogated IgE-stimulated, but not ionomycin-induced stimulation of Ca(2+)-activated K(+) channels. These observations disclose PI3-kinase-dependent regulation of Ca(2+) entry and Ca(2+)-activated K(+)-channels, which in turn participate in triggering mast cell degranulation. PMID:18769043

  2. TRPV3 channels mediate strontium-induced mouse egg activation

    PubMed Central

    Carvacho, Ingrid; Lee, Hoi Chang; Fissore, Rafael A.; Clapham, David E.

    2014-01-01

    SUMMARY 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, as TrpV3−/− eggs failed to permeate Sr2+ or undergo strontium-induced activation. We propose that TRPV3 is the major mediator of calcium influx in mouse eggs and is a putative target for artificial egg activation. PMID:24316078

  3. Physiological mechanisms for the modulation of pannexin 1 channel activity

    PubMed Central

    Sandilos, Joanna K; Bayliss, Douglas A

    2012-01-01

    It is widely recognized that ATP, along with other nucleotides, subserves important intercellular signalling processes. Among various nucleotide release mechanisms, the relatively recently identified pannexin 1 (Panx1) channel is gaining prominence by virtue of its ability to support nucleotide permeation and release in a variety of different tissues. Here, we review recent advances in our understanding of the factors that control Panx1 channel activity. By using electrophysiological and biochemical approaches, diverse mechanisms that dynamically regulate Panx1 channel function have been identified in various settings; these include, among others, activation by caspase-mediated channel cleavage in apoptotic immune cells, by G protein-coupled receptors in vascular smooth muscle, by low oxygen tension in erythrocytes and neurons, by high extracellular K+ in various cell types and by stretch/strain in airway epithelia. Delineating the distinct mechanisms of Panx1 modulation that prevail in different physiological contexts provides the possibility that these channels, and ATP release, could ultimately be targeted in a context-dependent manner. PMID:23070703

  4. Molecular Mechanisms of Large-Conductance Ca2+-Activated Potassium Channel Activation by Ginseng Gintonin

    PubMed Central

    Choi, S. H.; Lee, B. H.; Hwang, S. H.; Kim, H. J.; Lee, S. M.; Kim, H. C.; Rhim, H. W.; Nah, S. Y.

    2013-01-01

    Gintonin is a unique lysophosphatidic acid (LPA) receptor ligand found in Panax ginseng. Gintonin induces transient [Ca2+]i through G protein-coupled LPA receptors. Large-conductance Ca2+-activated K+ (BKCa) channels are expressed in blood vessels and neurons and play important roles in blood vessel relaxation and attenuation of neuronal excitability. BKCa channels are activated by transient [Ca2+]i and are regulated by various Ca2+-dependent kinases. We investigated the molecular mechanisms of BKCa channel activation by gintonin. BKCa channels are heterologously expressed in Xenopus oocytes. Gintonin treatment induced BKCa channel activation in oocytes expressing the BKCa channel α subunit in a concentration-dependent manner (EC50 = 0.71 ± 0.08 µg/mL). Gintonin-mediated BKCa channel activation was blocked by a PKC inhibitor, calphostin, and by the calmodulin inhibitor, calmidazolium. Site-directed mutations in BKCa channels targeting CaM kinase II or PKC phosphorylation sites but not PKA phosphorylation sites attenuated gintonin action. Mutations in the Ca2+ bowl and the regulator of K+ conductance (RCK) site also blocked gintonin action. These results indicate that gintonin-mediated BKCa channel activations are achieved through LPA1 receptor-phospholipase C-IP3-Ca2+-PKC-calmodulin-CaM kinase II pathways and calcium binding to the Ca2+ bowl and RCK domain. Gintonin could be a novel contributor against blood vessel constriction and over-excitation of neurons. PMID:23662129

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

    PubMed Central

    Chambers, James J.; Kramer, Richard H.

    2009-01-01

    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 neurons. More recently, a third type of light trigger has been introduced: a photoisomerizable tethered ligand that directly controls ion channel activity in a light-dependent manner. Beyond the experimental applications for light-gated ion channels, there may be clinical applications in which these light-sensitive ion channels could prove advantageous over traditional methods. Electrodes for neural stimulation to control disease symptoms are invasive and often difficult to reposition between cells in tissue. Stimulation by chemical agents is difficult to constrain to individual cells and has limited temporal accuracy in tissue due to diffusional limitations. In contrast, ion channels that can be directly activated with light allow control with unparalleled spatial and temporal precision. The goal of this chapter is to describe light-regulated ion channels and how they have been tailored to control different aspects of neural activity, and how to use these channels to manipulate and better understand development, function, and plasticity of neurons and neural circuits. PMID:19195553

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

    PubMed

    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; Kremsner, Peter G; Hübner, Christian A; Rust, Marco B; Dedek, Karin; Jentsch, Thomas J; Lang, Florian

    2004-10-01

    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 dependence, cell volume sensitivity, and activation by oxidation are identical to ClC-2. Moreover, Western blots and FACS analysis showed protein and functional ClC-2 expression in human erythrocytes and erythrocytes from wild type (Clcn2(+/+)) but not from Clcn2(-/-) mice. Finally, patch clamp recording revealed activation of volume-sensitive inwardly rectifying channels in Plasmodium berghei-infected Clcn2(+/+) but not Clcn2(-/-) erythrocytes. Erythrocytes from infected mice of both genotypes differed in cell volume and inhibition of ClC-2 by ZnCl(2) (1 mm) induced an increase of cell volume only in parasitized Clcn2(+/+) erythrocytes. Lack of ClC-2 did not inhibit P. berghei development in vivo nor substantially affect the mortality of infected mice. In conclusion, activation of host ClC-2 channels participates in the altered permeability of Plasmodium-infected erythrocytes but is not required for intraerythrocytic parasite survival. PMID:15272009

  7. Lipid bilayer array for simultaneous recording of ion channel activities

    NASA Astrophysics Data System (ADS)

    Hirano-Iwata, Ayumi; Nasu, Tomohiro; Oshima, Azusa; Kimura, Yasuo; Niwano, Michio

    2012-07-01

    This paper describes an array of stable and reduced-solvent bilayer lipid membranes (BLMs) formed in microfabricated silicon chips. BLMs were first vertically formed simultaneously and then turned 90° in order to realize a horizontal BLM array. Since the present BLMs are mechanically stable and robust, the BLMs survive this relatively tough process. Typically, a ˜60% yield in simultaneous BLM formation over 9 sites was obtained. Parallel recordings of gramicidin channel activities from different BLMs were demonstrated. The present system has great potential as a platform of BLM-based high throughput drug screening for ion channel proteins.

  8. Amphetamine activates calcium channels through dopamine transporter-mediated depolarization.

    PubMed

    Cameron, Krasnodara N; Solis, Ernesto; Ruchala, Iwona; De Felice, Louis J; Eltit, Jose M

    2015-11-01

    Amphetamine (AMPH) and its more potent enantiomer S(+)AMPH are psychostimulants used therapeutically to treat attention deficit hyperactivity disorder and have significant abuse liability. AMPH is a dopamine transporter (DAT) substrate that inhibits dopamine (DA) uptake and is implicated in DA release. Furthermore, AMPH activates ionic currents through DAT that modify cell excitability presumably by modulating voltage-gated channel activity. Indeed, several studies suggest that monoamine transporter-induced depolarization opens voltage-gated Ca(2+) channels (CaV), which would constitute an additional AMPH mechanism of action. In this study we co-express human DAT (hDAT) with Ca(2+) channels that have decreasing sensitivity to membrane depolarization (CaV1.3, CaV1.2 or CaV2.2). Although S(+)AMPH is more potent than DA in transport-competition assays and inward-current generation, at saturating concentrations both substrates indirectly activate voltage-gated L-type Ca(2+) channels (CaV1.3 and CaV1.2) but not the N-type Ca(2+) channel (CaV2.2). Furthermore, the potency to achieve hDAT-CaV electrical coupling is dominated by the substrate affinity on hDAT, with negligible influence of L-type channel voltage sensitivity. In contrast, the maximal coupling-strength (defined as Ca(2+) signal change per unit hDAT current) is influenced by CaV voltage sensitivity, which is greater in CaV1.3- than in CaV1.2-expressing cells. Moreover, relative to DA, S(+)AMPH showed greater coupling-strength at concentrations that induced relatively small hDAT-mediated currents. Therefore S(+)AMPH is not only more potent than DA at inducing hDAT-mediated L-type Ca(2+) channel currents but is a better depolarizing agent since it produces tighter electrical coupling between hDAT-mediated depolarization and L-type Ca(2+) channel activation. PMID:26162812

  9. G-protein–gated TRP-like Cationic Channel Activated by Muscarinic Receptors

    PubMed Central

    Zholos, Alexander V.; Zholos, Andrey A.; Bolton, Thomas B.

    2004-01-01

    There is little information about the mechanisms by which G-protein–coupled receptors gate ion channels although many ionotropic receptors are well studied. We have investigated gating of the muscarinic cationic channel, which mediates the excitatory effect of acetylcholine in smooth muscles, and proposed a scheme consisting of four pairs of closed and open states. Channel kinetics appeared to be the same in cell-attached or outside-out patches whether the channel was activated by carbachol application or by intracellular dialysis with GTPγS. Since in the latter case G-proteins are permanently active, it is concluded that the cationic channel is the major determinant of its own gating, similarly to the KACh channel (Ivanova-Nikolova, T.T., and G.E. Breitwieser. 1997. J. Gen. Physiol. 109:245–253). Analysis of adjacent-state dwell times revealed connections between the states that showed features conserved among many other ligand-gated ion channels (e.g., nAChR, BKCa channel). Open probability (PO) of the cationic channel was increased by membrane depolarization consistent with the prominent U-shaped I-V relationship of the muscarinic whole-cell current at negative potentials. Membrane potential affected transitions within each closed-open state pair but had little effect on transitions between pairs; thus, the latter are likely to be caused by interactions of the channel with its ligands, e.g., Ca2+ and Gαo-GTP. Channel activity was highly heterogeneous, as was evident from the prominent cycling behavior when PO was measured over 5-s intervals. This was related to the variable frequency of openings (as in the KACh channel) and, especially, to the number of long openings between consecutive long shuttings. Analysis of the underlying Markov chain in terms of probabilities allowed us to evaluate the contribution of each open state to the integral current (from shortest to longest open state: 0.1, 3, 24, and 73%) as PO increased 525-fold in three stages. PMID

  10. Activation of peripheral KCNQ channels attenuates inflammatory pain

    PubMed Central

    2014-01-01

    Background Refractory chronic pain dramatically reduces the quality of life of patients. Existing drugs cannot fully achieve effective chronic pain control because of their lower efficacy and/or accompanying side effects. Voltage-gated potassium channels (KCNQ) openers have demonstrated their analgesic effect in preclinical and clinical studies, and are thus considered to be a potential therapeutic target as analgesics. However, these drugs exhibit a narrow therapeutic window due to their imposed central nerve system (CNS) side effects. To clarify the analgesic effect by peripheral KCNQ channel activation, we investigated whether the analgesic effect of the KCNQ channel opener, retigabine, is inhibited by intracerebroventricular (i.c.v.) administration of the KCNQ channel blocker, 10, 10-bis (4-Pyridinylmethyl)-9(10H) -anthracenone dihydrochloride (XE-991) in rats. Results Oral administration (p.o.) of retigabine showed an anticonvulsant effect on maximal electronic seizures and an analgesic effect on complete Freund’s adjuvant-induced thermal hyperalgesia. However, impaired motor coordination and reduced exploratory behavior were also observed at the analgesic doses of retigabine. Administration (i.c.v.) of XE-991 reversed the retigabine-induced anticonvulsant effect, impaired motor coordination and reduced exploratory behavior but not the analgesic effect. Moreover, intraplantar administration of retigabine or an additional KCNQ channel opener, N-(6-Chloro-pyridin-3-yl)-3,4-difluoro-benzamide (ICA-27243), inhibited formalin-induced nociceptive behavior. Conclusions Our findings suggest that the peripheral sensory neuron is the main target for KCNQ channel openers to induce analgesia. Therefore, peripheral KCNQ channel openers that do not penetrate the CNS may be suitable analgesic drugs as they would prevent CNS side effects. PMID:24555569

  11. Computational study of a calcium release-activated calcium channel

    NASA Astrophysics Data System (ADS)

    Talukdar, Keka; Shantappa, Anil

    2016-05-01

    The naturally occurring proteins that form hole in membrane are commonly known as ion channels. They play multiple roles in many important biological processes. Deletion or alteration of these channels often leads to serious problems in the physiological processes as it controls the flow of ions through it. The proper maintenance of the flow of ions, in turn, is required for normal health. Here we have investigated the behavior of a calcium release-activated calcium ion channel with pdb entry 4HKR in Drosophila Melanogaster. The equilibrium energy as well as molecular dynamics simulation is performed first. The protein is subjected to molecular dynamics simulation to find their energy minimized value. Simulation of the protein in the environment of water and ions has given us important results too. The solvation energy is also found using Charmm potential.

  12. Variomics Screen Identifies the Re-entrant Loop of the Calcium-activated Chloride Channel ANO1 That Facilitates Channel Activation*

    PubMed Central

    Bill, Anke; Popa, M. Oana; van Diepen, Michiel T.; Gutierrez, Abraham; Lilley, Sarah; Velkova, Maria; Acheson, Kathryn; Choudhury, Hedaythul; Renaud, Nicole A.; Auld, Douglas S.; Gosling, Martin; Groot-Kormelink, Paul J.; Gaither, L. Alex

    2015-01-01

    The calcium-activated chloride channel ANO1 regulates multiple physiological processes. However, little is known about the mechanism of channel gating and regulation of ANO1 activity. Using a high-throughput, random mutagenesis-based variomics screen, we generated and functionally characterized ∼6000 ANO1 mutants and identified novel mutations that affected channel activity, intracellular trafficking, or localization of ANO1. Mutations such as S741T increased ANO1 calcium sensitivity and rendered ANO1 calcium gating voltage-independent, demonstrating a critical role of the re-entrant loop in coupling calcium and voltage sensitivity of ANO1 and hence in regulating ANO1 activation. Our data present the first unbiased and comprehensive study of the structure-function relationship of ANO1. The novel ANO1 mutants reported have diverse functional characteristics, providing new tools to study ANO1 function in biological systems, paving the path for a better understanding of the function of ANO1 and its role in health and diseases. PMID:25425649

  13. Low affinity block of native and cloned hyperpolarization-activated Ih channels by Ba2+ ions.

    PubMed

    van Welie, Ingrid; Wadman, Wytse J; van Hooft, Johannes A

    2005-01-10

    Ba2+ is commonly used to discriminate two classes of ion currents. The classical inward-rectifying K+ current, I(Kir), is blocked by low millimolar concentrations of Ba2+, whereas the hyperpolarization-activated cation current, I(h), is assumed not to be sensitive to Ba2+. Here we investigated the effects of Ba2+ on I(h) currents recorded from rat hippocampal CA1 pyramidal neurons, and on cloned I(h) channels composed of either HCN1 or HCN2 subunits transiently expressed in Human Embryonic Kidney (HEK) 293 cells. The results show that low millimolar concentrations of Ba2+ reduce the maximal I(h) conductance (IC50 approximately 3-5 mM) in both CA1 pyramidal neurons and in HEK 293 cells without specificity for HCN1 or HCN2 subunits. In addition, Ba2+ decreases the rate of activation and increases the rate of deactivation of I(h) currents. Neither the half-maximal voltage of activation, V(h), nor the reversal potential of the I(h) channels were affected by Ba2+. The combined results suggest that B2+, at concentrations commonly used to block I(Kir) currents, also reduces the conductance of I(h) channels without subunit specificity, and affects the kinetics of I(h) channel gating. PMID:15659289

  14. Transmembrane helix straightening and buckling underlies activation of mechanosensitive and thermosensitive K(2P) channels.

    PubMed

    Lolicato, Marco; Riegelhaupt, Paul M; Arrigoni, Cristina; Clark, Kimberly A; Minor, Daniel L

    2014-12-17

    Mechanical and thermal activation of ion channels is central to touch, thermosensation, and pain. The TRAAK/TREK K(2P) potassium channel subfamily produces background currents that alter neuronal excitability in response to pressure, temperature, signaling lipids, and anesthetics. How such diverse stimuli control channel function is unclear. Here we report structures of K(2P)4.1 (TRAAK) bearing C-type gate-activating mutations that reveal a tilting and straightening of the M4 inner transmembrane helix and a buckling of the M2 transmembrane helix. These conformational changes move M4 in a direction opposite to that in classical potassium channel activation mechanisms and open a passage lateral to the pore that faces the lipid bilayer inner leaflet. Together, our findings uncover a unique aspect of K(2P) modulation, indicate a means for how the K(2P) C-terminal cytoplasmic domain affects the C-type gate which lies ∼40Å away, and suggest how lipids and bilayer inner leaflet deformations may gate the channel. PMID:25500157

  15. Zinc activates damage-sensing TRPA1 ion channels

    PubMed Central

    Hu, Hongzhen; Bandell, Michael; Petrus, Matt J.; Zhu, Michael X.; Patapoutian, Ardem

    2009-01-01

    Zinc is an essential biological trace element. It is required for the structure or function of over 300 proteins, and is increasingly recognized for its role in cell signaling. However, high concentrations of zinc have cytotoxic effects, and overexposure to zinc can cause pain and inflammation through unknown mechanisms. Here we show that zinc excites nociceptive somatosensory neurons and causes nociception in mice through TRPA1, a cation channel previously shown to mediate the pungency of wasabi and cinnamon through cysteine-modification. Zinc activates TRPA1 through a novel mechanism that requires zinc influx through TRPA1 channels and subsequent activation via specific intracellular cysteine and histidine residues. TRPA1 is highly sensitive to intracellular zinc, as low nanomolar concentrations activate TRPA1 and modulate its sensitivity. These findings identify TRPA1 as a major target for the sensory effects of zinc, and support an emerging role for zinc as a signaling molecule that can modulate sensory transmission. PMID:19202543

  16. Multiple types of voltage-dependent Ca2+-activated K+ channels of large conductance in rat brain synaptosomal membranes.

    PubMed Central

    Farley, J.; Rudy, B.

    1988-01-01

    K+-selective ion channels from a mammalian brain synaptosomal membrane preparation were inserted into planar phospholipid bilayers on the tips of patch-clamp pipettes, and single-channel currents were measured. Multiple distinct classes of K+ channels were observed. We have characterized and described the properties of several types of voltage-dependent, Ca2+-activated K+ channels of large single-channel conductance (greater than 50 pS in symmetrical KCl solutions). One class of channels (Type I) has a 200-250-pS single-channel conductance. It is activated by internal calcium concentrations greater than 10(-7) M, and its probability of opening is increased by membrane depolarization. This channel is blocked by 1-3 mM internal concentrations of tetraethylammonium (TEA). These channels are similar to the BK channel described in a variety of tissues. A second novel group of voltage-dependent, Ca2+-activated K+ channels was also studied. These channels were more sensitive to internal calcium, but less sensitive to voltage than the large (Type I) channel. These channels were minimally affected by internal TEA concentrations of 10 mM, but were blocked by a 50 mM concentration. In this class of channels we found a wide range of relatively large unitary channel conductances (65-140 pS). Within this group we have characterized two types (75-80 pS and 120-125 pS) that also differ in gating kinetics. The various types of voltage-dependent, Ca2+-activated K+ channels described here were blocked by charybdotoxin added to the external side of the channel. The activity of these channels was increased by exposure to nanomolar concentrations of the catalytic subunit of cAMP-dependent protein kinase. These results indicate that voltage-dependent, charybdotoxin-sensitive Ca2+-activated K+ channels comprise a class of related, but distinguishable channel types. Although the Ca2+-activated (Type I and II) K+ channels can be distinguished by their single-channel properties, both could

  17. Stretch-activated single ion channel currents in tissue-cultured embryonic chick skeletal muscle.

    PubMed Central

    Guharay, F; Sachs, F

    1984-01-01

    The membrane of tissue-cultured chick pectoral muscle contains an ionic channel which is activated by membrane stretch. Nicotinic channels and Ca2+-activated K+ channels are not affected by stretch. In 150 mM-external K+ and 150 mM-internal Na+ the channel has a conductance of 70 pS, linear current-voltage relationship between -50 and -140 mV and a reversal potential of +30 mV. Kinetic analysis of single-channel records indicates that there are one open (O) and three closed (C) states. The data can be fitted by the reaction scheme: C1-C2-C3-O. Only the rate constant that governs the C1-C2 transition (k1,2) is stretch-sensitive. None of the rates are voltage-sensitive. The rate constant k1,2 varies with the square of the tension as k1, 2 = k0 X e alpha T2, where alpha is a constant describing the sensitivity to stretch and T is the tension. A typical value of alpha is 0.08 (dyn cm-1)-2. Following exposure to cytochalasin B the channel becomes more sensitive to stretch. The stretch-sensitivity constant, alpha, increases from 0.08 to 2.4 (dyn cm-1)-2. The probability of the channel being open is strongly dependent upon the extracellular K+ concentration. With a suction of 2 cmHg the probability increases from 0.004 in normal saline (5 mM-K+) to 0.26 in 150 mM-K+. The channel appears to gather force from a large area of membrane (greater than 3 X 10(5) A2), probably by a cytochalasin-resistant cytoskeletal network. PMID:6086918

  18. Structural aspects of calcium-release activated calcium channel function

    PubMed Central

    Stathopulos, Peter B; Ikura, Mitsuhiko

    2013-01-01

    Store-operated calcium (Ca2+) entry is the process by which molecules located on the endo/sarcoplasmic reticulum (ER/SR) respond to decreased luminal Ca2+ levels by signaling Ca2+ release activated Ca2+ channels (CRAC) channels to open on the plasma membrane (PM). This activation of PM CRAC channels provides a sustained cytosolic Ca2+ elevation associated with myriad physiological processes. The identities of the molecules which mediate SOCE include stromal interaction molecules (STIMs), functioning as the ER/SR luminal Ca2+ sensors, and Orai proteins, forming the PM CRAC channels. This review examines the current available high-resolution structural information on these CRAC molecular components with particular focus on the solution structures of the luminal STIM Ca2+ sensing domains, the crystal structures of cytosolic STIM fragments, a closed Orai hexameric crystal structure and a structure of an Orai1 N-terminal fragment in complex with calmodulin. The accessible structural data are discussed in terms of potential mechanisms of action and cohesiveness with functional observations. PMID:24213636

  19. Atomic basis for therapeutic activation of neuronal potassium channels

    NASA Astrophysics Data System (ADS)

    Kim, Robin Y.; Yau, Michael C.; Galpin, Jason D.; Seebohm, Guiscard; Ahern, Christopher A.; Pless, Stephan A.; Kurata, Harley T.

    2015-09-01

    Retigabine is a recently approved anticonvulsant that acts by potentiating neuronal M-current generated by KCNQ2-5 channels, interacting with a conserved Trp residue in the channel pore domain. Using unnatural amino-acid mutagenesis, we subtly altered the properties of this Trp to reveal specific 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.

  20. Atomic basis for therapeutic activation of neuronal potassium channels

    PubMed Central

    Kim, Robin Y.; Yau, Michael C.; Galpin, Jason D.; Seebohm, Guiscard; Ahern, Christopher A.; Pless, Stephan A.; Kurata, Harley T.

    2015-01-01

    Retigabine is a recently approved anticonvulsant that acts by potentiating neuronal M-current generated by KCNQ2–5 channels, interacting with a conserved Trp residue in the channel pore domain. Using unnatural amino-acid mutagenesis, we subtly altered the properties of this Trp to reveal specific 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. PMID:26333338

  1. Local postsynaptic voltage-gated sodium channel activation in dendritic spines of olfactory bulb granule cells.

    PubMed

    Bywalez, Wolfgang G; Patirniche, Dinu; Rupprecht, Vanessa; Stemmler, Martin; Herz, Andreas V M; Pálfi, Dénes; Rózsa, Balázs; Egger, Veronica

    2015-02-01

    Neuronal dendritic spines have been speculated to function as independent computational units, yet evidence for active electrical computation in spines is scarce. Here we show that strictly local voltage-gated sodium channel (Nav) activation can occur during excitatory postsynaptic potentials in the spines of olfactory bulb granule cells, which we mimic and detect via combined two-photon uncaging of glutamate and calcium imaging in conjunction with whole-cell recordings. We find that local Nav activation boosts calcium entry into spines through high-voltage-activated calcium channels and accelerates postsynaptic somatic depolarization, without affecting NMDA receptor-mediated signaling. Hence, Nav-mediated boosting promotes rapid output from the reciprocal granule cell spine onto the lateral mitral cell dendrite and thus can speed up recurrent inhibition. This striking example of electrical compartmentalization both adds to the understanding of olfactory network processing and broadens the general view of spine function. PMID:25619656

  2. Inhibition of parathyroid hormone release by maitotoxin, a calcium channel activator

    SciTech Connect

    Fitzpatrick, L.A.; Yasumoto, T.; Aurbach, G.D.

    1989-01-01

    Maitotoxin, a toxin derived from a marine dinoflagellate, is a potent activator of voltage-sensitive calcium channels. To further test the hypothesis that inhibition of PTH secretion by calcium is mediated via a calcium channel we studied the effect of maitotoxin on dispersed bovine parathyroid cells. Maitotoxin inhibited PTH release in a dose-dependent fashion, and inhibition was maximal at 1 ng/ml. Chelation of extracellular calcium by EGTA blocked the inhibition of PTH by maitotoxin. Maitotoxin enhanced the effects of the dihydropyridine calcium channel agonist (+)202-791 and increased the rate of radiocalcium uptake in parathyroid cells. Pertussis toxin, which ADP-ribosylates and inactivates a guanine nucleotide regulatory protein that interacts with calcium channels in the parathyroid cell, did not affect the inhibition of PTH secretion by maitotoxin. Maitotoxin, by its action on calcium channels allows entry of extracellular calcium and inhibits PTH release. Our results suggest that calcium channels are involved in the release of PTH. Inhibition of PTH release by maitotoxin is not sensitive to pertussis toxin, suggesting that maitotoxin may act distal to the site interacting with a guanine nucleotide regulatory protein, or maitotoxin could interact with other ions or second messengers to inhibit PTH release.

  3. Structure and activity of the acid-sensing ion channels

    PubMed Central

    Sherwood, Thomas W.; Frey, Erin N.

    2012-01-01

    The acid-sensing ion channels (ASICs) are a family of proton-sensing channels expressed throughout the nervous system. Their activity is linked to a variety of complex behaviors including fear, anxiety, pain, depression, learning, and memory. ASICs have also been implicated in neuronal degeneration accompanying ischemia and multiple sclerosis. As a whole, ASICs represent novel therapeutic targets for several clinically important disorders. An understanding of the correlation between ASIC structure and function will help to elucidate their mechanism of action and identify potential therapeutics that specifically target these ion channels. Despite the seemingly simple nature of proton binding, multiple studies have shown that proton-dependent gating of ASICs is quite complex, leading to activation and desensitization through distinct structural components. This review will focus on the structural aspects of ASIC gating in response to both protons and the newly discovered activators GMQ and MitTx. ASIC modulatory compounds and their action on proton-dependent gating will also be discussed. This review is dedicated to the memory of Dale Benos, who made a substantial contribution to our understanding of ASIC activity. PMID:22843794

  4. Ca(2+)-activated anion channels and membrane depolarizations induced by blue light and cold in Arabidopsis seedlings

    NASA Technical Reports Server (NTRS)

    Lewis, B. D.; Karlin-Neumann, G.; Davis, R. W.; Spalding, E. P.; Evans, M. L. (Principal Investigator)

    1997-01-01

    The activation of an anion channel in the plasma membrane of Arabidopsis thaliana hypocotyls by blue light (BL) is believed to be a signal-transducing event leading to growth inhibition. Here we report that the open probability of this particular anion channel depends on cytoplasmic Ca2+ ([Ca2+]cyt) within the concentration range of 1 to 10 microM, raising the possibility that BL activates the anion channel by increasing [Ca2+]cyt. Arabidopsis seedlings cytoplasmically expressing aequorin were generated to test this possibility. Aequorin luminescence did not increase during or after BL, providing evidence that Ca2+ does not play a second-messenger role in the activation of anion channels. However, cold shock simultaneously triggered a large increase in [Ca2+]cyt and a 110-mV transient depolarization of the plasma membrane. A blocker of the anion channel, 5-nitro-2-(3-phenylpropylamino)-benzoic acid, blocked 61% of the cold-induced depolarization without affecting the increase in [Ca2+]cyt. These data led us to propose that cold shock opens Ca2+ channels at the plasma membrane, allowing an inward, depolarizing Ca2+ current. The resulting large increase in [Ca2+]cyt activates the anion channel, which further depolarizes the membrane. Although an increase in [Ca2+]cyt may activate anion channels in response to cold, it appears that BL does so via a Ca(2+)-independent pathway.

  5. Fluctuation driven active molecular transport in passive channel proteins

    NASA Astrophysics Data System (ADS)

    Kosztin, Ioan

    2006-03-01

    Living cells interact with their extracellular environment through the cell membrane, which acts as a protective permeability barrier for preserving the internal integrity of the cell. However, cell metabolism requires controlled molecular transport across the cell membrane, a function that is fulfilled by a wide variety of transmembrane proteins, acting as either passive or active transporters. In this talk it is argued that, contrary to the general belief, in active cell membranes passive and spatially asymmetric channel proteins can act as active transporters by consuming energy from nonequilibrium fluctuations fueled by cell metabolism. This assertion is demonstrated in the case of the E. coli aquaglyceroporin GlpF channel protein, whose high resolution crystal structure is manifestly asymmetric. By calculating the glycerol flux through GlpF within the framework of a stochastic model, it is found that, as a result of channel asymmetry, glycerol uptake driven by a concentration gradient is enhanced significantly in the presence of non-equilibrium fluctuations. Furthermore, the enhancement caused by a ratchet-like mechanism is larger for the outward, i.e., from the cytoplasm to the periplasm, flux than for the inward one, suggesting that the same non-equilibrium fluctuations also play an important role in protecting the interior of the cell against poisoning by excess uptake of glycerol. Preliminary data on water and sugar transport through aquaporin and maltoporin channels, respectively, are indicative of the universality of the proposed nonequilibrium-fluctuation-driven active transport mechanism. This work was supported by grants from the Univ. of Missouri Research Board, the Institute for Theoretical Sciences and the Department of Energy (DOE Contract W-7405-ENG-36), and the National Science Foundation (FIBR-0526854).

  6. An Investigation of How the Channel of Input and Access to Test Questions Affect L2 Listening Test Performance

    ERIC Educational Resources Information Center

    Wagner, Elvis

    2013-01-01

    The use of video technology has become widespread in the teaching and testing of second-language (L2) listening, yet research into how this technology affects the learning and testing process has lagged. The current study investigated how the channel of input (audiovisual vs. audio-only) used on an L2 listening test affected test-taker…

  7. Modulation of bone remodeling via mechanically activated ion channels

    NASA Technical Reports Server (NTRS)

    Duncan, Randall L. (Principal Investigator)

    1996-01-01

    A critical factor in the maintenance of bone mass is the physical forces imposed upon the skeleton. Removal of these forces, such as in a weightless environment, results in a rapid loss of bone, whereas application of exogenous mechanical strain has been shown to increase bone formation. Numerous flight and ground-based experiments indicate that the osteoblast is the key bone cell influenced by mechanical stimulation. Aside from early transient fluctuations in response to unloading, osteoclast number and activity seem unaffected by removal of strain. However, bone formation is drastically reduced in weightlessness and osteoblasts respond to mechanical strain with an increase in the activity of a number of second messenger pathways resulting in increased anabolic activity. Unfortunately, the mechanism by which the osteoblast converts physical stimuli into a biochemical message, a process we have termed biochemical coupling, remains elusive. Prior to the application of this grant, we had characterized a mechanosensitive, cation nonselective channel (SA-cat) in osteoblast-like osteosarcoma cells that we proposed is the initial signalling mechanism for mechanotransduction. During the execution of this grant, we have made considerable progress to further characterize this channel as well as to determine its role in the osteoblastic response to mechanical strain. To achieve these goals, we combined electrophysiologic techniques with cellular and molecular biology methods to examine the role of these channels in the normal function of the osteoblast in vitro.

  8. 17Beta-Estradiol Inhibits Calcium-Activated Potassium Channel Expressions in Rat Whole Bladder

    PubMed Central

    2016-01-01

    Purpose: To investigate the effect of estrogen on the expression of calcium-activated potassium (KCa) channels in an overactive bladder rat model. To this end, mRNA and protein levels of KCa channel subtypes in the bladder of ovariectomized rats were measured by reverse transcription polymerase chain reaction and western blotting, respectively. Methods: Ten-week-old female Sprague-Dawley rats were divided randomly into 3 groups: sham-operated control group (n=11), ovariectomy group (n=11), and the group treated with estrogen after ovariectomy (n=12). Rats in the last group were subcutaneously injected with 17β-estradiol (50 μg/kg) every other day for 2 weeks, whereas rats in the other 2 groups received vehicle (soybean oil) alone. Two weeks after treatment, the whole bladder was excised for mRNA and protein measurements. Results: Protein levels of the large-conductance KCa (BK) channels in the ovariectomy group were 1.5 folds higher than those in the sham-operated control group. However, the protein levels of the other KCa channel subtypes did not change significantly upon bilateral ovariectomy. Treatment with 17β-estradiol after ovariectomy restored BK channel protein levels to the control value. In contrast, BK channel mRNA levels were not significantly affected by either ovariectomy alone or 17β-estradiol treatment. The small-conductance KCa type 3 channel (SK3) mRNA and protein levels decreased to 75% of control levels upon 17β-estradiol treatment. Conclusions: These results suggest that 17β-estradiol may influence urinary bladder function by modulating BK and SK3 channel expression. PMID:27032553

  9. Ca(2+)-activated K+ channels in rat thymic lymphocytes: activation by concanavalin A.

    PubMed

    Mahaut-Smith, M P; Mason, M J

    1991-08-01

    1. The role of ion channels in the mitogenic response of rat thymic lymphocytes to concanavalin A (ConA) was studied using single-channel patch-clamp recordings and measurements of membrane potential with the fluorescent probe bis-oxonol. 2. ConA (20 micrograms ml-1) evoked a rapid membrane hyperpolarization; Indo-1 measurements indicated a concurrent increase in [Ca2+]i. The hyperpolarization was blocked by cytoplasmic loading with the Ca2+ buffer BAPTA (bis(O-amino-phenoxy)ethane-N,N,N',N'-tetraacetic acid), or charybdotoxin, a component of scorpion venom known to block K+ channels in lymphocytes. 3. Cell-attached patch-clamp recordings showed that both ConA and the Ca2+ ionophore ionomycin activated channels with high selectivity for K+. Two conductance levels were observed -6-7 pS and 17-18 pS-measured as inward chord conductance at 60 mV from reversal potential (Erev) with 140 mM-KCl in the pipette. The current-voltage relationship for the larger channel displayed inward rectification and channel open probability was weakly dependent upon membrane potential. 4. These experiments provide the first direct evidence for mitogen-activated Ca(2+)-gated K+ channels (IK(Ca)) in lymphocytes. This conductance is relatively inactive in unstimulated rat thymocytes but following the intracellular Ca2+ rises induced by ConA, IK(Ca) channels are activated and produce a significant hyperpolarization of the cell potential. PMID:1716678

  10. Anion Permeation in Ca2+-Activated Cl− Channels

    PubMed Central

    Qu, Zhiqiang; Hartzell, H. Criss

    2000-01-01

    Ca2+-activated Cl channels (ClCaCs) are an important class of anion channels that are opened by increases in cytosolic [Ca2+]. Here, we examine the mechanisms of anion permeation through ClCaCs from Xenopus oocytes in excised inside-out and outside-out patches. ClCaCs exhibited moderate selectivity for Cl over Na: PNa/PCl = 0.1. The apparent affinity of ClCaCs for Cl was low: Kd = 73 mM. The channel had an estimated pore diameter >0.6 nm. The relative permeabilities measured under bi-ionic conditions by changes in Erev were as follows: C(CN)3 > SCN > N(CN)2 > ClO4 > I > N3 > Br > Cl > formate > HCO3 > acetate = F > gluconate. The conductance sequence was as follows: N3 > Br > Cl > N(CN)2 > I > SCN > COOH > ClO4 > acetate > HCO3 = C(CN)3 > gluconate. Permeant anions block in a voltage-dependent manner with the following affinities: C(CN)3 > SCN = ClO4 > N(CN)2 > I > N3 > Br > HCO3 > Cl > gluconate > formate > acetate. Although these data suggest that anionic selectivity is determined by ionic hydration energy, other factors contribute, because the energy barrier for permeation is exponentially related to anion hydration energy. ClCaCs exhibit weak anomalous mole fraction behavior, implying that the channel may be a multi-ion pore, but that ions interact weakly in the pore. The affinity of the channel for Ca2+ depended on the permeant anion at low [Ca2+] (100–500 nM). Apparently, occupancy of the pore by a permeant anion increased the affinity of the channel for Ca2+. The current was strongly dependent on pH. Increasing pH on the cytoplasmic side decreased the inward current, whereas increasing pH on the external side decreased the outward current. In both cases, the apparent pKa was voltage-dependent with apparent pKa at 0 mV = ∼9.2. The channel may be blocked by OH− ions, or protons may titrate a site in the pore necessary for ion permeation. These data demonstrate that the permeation properties of ClCaCs are different from those of CFTR or ClC-1, and provide

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

    PubMed

    Shipston, Michael J

    2014-01-01

    Mechanisms that control surface expression and/or activity of large conductance calcium-activated potassium (BK) channels are important determinants of their (patho)physiological 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 signaling 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. PMID:25140154

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

    PubMed Central

    Shipston, Michael J.

    2014-01-01

    Mechanisms that control surface expression and/or activity of large conductance calcium-activated potassium (BK) channels are important determinants of their (patho)physiological 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 signaling 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. PMID:25140154

  13. Location of Release Sites and Calcium-Activated Chloride Channels Relative to Calcium Channels at the Photoreceptor Ribbon Synapse

    PubMed Central

    Mercer, A. J.; Rabl, K.; Riccardi, G. E.; Brecha, N. C.; Stella, S. L.

    2011-01-01

    Vesicle release from photoreceptor ribbon synapses is regulated by L-type Ca2+ channels, which are in turn regulated by Cl− moving through calcium-activated chloride [Cl(Ca)] channels. We assessed the proximity of Ca2+ channels to release sites and Cl(Ca) channels in synaptic terminals of salamander photoreceptors by comparing fast (BAPTA) and slow (EGTA) intracellular Ca2+ buffers. BAPTA did not fully block synaptic release, indicating some release sites are <100 nm from Ca2+ channels. Comparing Cl(Ca) currents with predicted Ca2+ diffusion profiles suggested that Cl(Ca) and Ca2+ channels average a few hundred nanometers apart, but the inability of BAPTA to block Cl(Ca) currents completely suggested some channels are much closer together. Diffuse immunolabeling of terminals with an antibody to the putative Cl(Ca) channel TMEM16A supports the idea that Cl(Ca) channels are dispersed throughout the presynaptic terminal, in contrast with clustering of Ca2+ channels near ribbons. Cl(Ca) currents evoked by intracellular calcium ion concentration ([Ca2+]i) elevation through flash photolysis of DM-nitrophen exhibited EC50 values of 556 and 377 nM with Hill slopes of 1.8 and 2.4 in rods and cones, respectively. These relationships were used to estimate average submembrane [Ca2+]i in photoreceptor terminals. Consistent with control of exocytosis by [Ca2+] nanodomains near Ca2+ channels, average submembrane [Ca2+]i remained below the vesicle release threshold (∼400 nM) over much of the physiological voltage range for cones. Positioning Ca2+ channels near release sites may improve fidelity in converting voltage changes to synaptic release. A diffuse distribution of Cl(Ca) channels may allow Ca2+ influx at one site to influence relatively distant Ca2+ channels. PMID:21084687

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

    PubMed

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

    2016-02-25

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

  15. Ca2+ and Mn2+ Influx Through Receptor-Mediated Activation of Nonspecific Cation Channels in Mast Cells

    NASA Astrophysics Data System (ADS)

    Fasolato, Cristina; Hoth, Markus; Matthews, Gary; Penner, Reinhold

    1993-04-01

    Whole-cell patch-clamp recordings of membrane currents and Fura-2 measurements of free intracellular calcium concentration ([Ca2+]_i) were used to study calcium influx through receptor-activated cation channels in rat peritoneal mast cells. Cation channels were activated by the secretagogue compound 48/80, whereas a possible concomitant Ca2+ entry through pathways activated by depletion of calcium stores was blocked by dialyzing cells with heparin. Heparin effectively suppressed the transient Ca2+ release induced by 48/80 and abrogated inositol 1,4,5-trisphosphate-induced calcium influx without affecting activation of 50-pS cation channels. There was a clear correlation between changes in [Ca2+]_i and the activity of 50-pS channels. The changes in [Ca2+]_i increased with elevation of extracellular Ca2+. At the same time, inward currents through 50-pS channels were diminished as more Ca2+ permeated. This effect was due to a decrease in slope conductance and a reduction in the open probability of the cation channels. In physiological solutions, 3.6% of the total current was carried by Ca2+. The cation channels were not only permeable to Ca2+ but also to Mn2+, as evidenced by the quench of Fura-2 fluorescence. Mn2+ current through 50-pS channels could not be resolved at the single-channel level. Our results suggest that 50-pS cation channels partially contribute to sustained increases of [Ca2+]_i in mast cells following receptor activation.

  16. Eucalyptol induces hyperexcitability and epileptiform activity in snail neurons by inhibiting potassium channels.

    PubMed

    Zeraatpisheh, Zahra; Vatanparast, Jafar

    2015-10-01

    The effects of eucalyptol (1,8-cineole) were studied on the activity of central neurons of land snail Caucasotachea atrolabiata. Eucalyptol (3 mM) depolarized the membrane potential and increased the frequency of spontaneous activity in a time dependent and reversible manner. These effects were associated with suppression of afterhyperpolarization and significant reduction of amplitude and slope of rising and falling phases of action potentials. While the eucalyptol-induced suppression of action potential amplitude and rising slope were essentially dependent on membrane depolarization, its actions on repolarization slope and afterhyperpolarization were not affected by resetting the membrane potential close to the control value. These findings suggest an inhibitory action on the potassium channels that underlie repolarization and afterhyperpolarization. Eucalyptol also increased the frequency of driven action potentials but suppressed the post stimulus inhibitory period, indicating an inhibitory action on calcium-activated potassium channels. A higher concentration of eucalyptol, 5mM, reversibly changed the pattern of activity to burst firing associated with paroxysmal depolarization shift (PDS). Low doses of eucalyptol and potassium channel blockers, tetraethylammonium and 4-aminopyridine, synergistically acted to induce burst firing. At high concentration (30 mM), tetraethylammonium was able to induce burst firing and PDS. The sodium currents and ion channel phosphorylation by protein kinases A and C were not required for the eucalyptol-induced epileptiform activity, but calcium currents were essential for this action. Our findings show the excitatory and epileptogenic action of eucalyptol, which is most likely mediated through direct inhibitory action on potassium channels. PMID:26134504

  17. Selective potentiation of 2-APB-induced activation of TRPV1–3 channels by acid

    PubMed Central

    Gao, Luna; Yang, Pu; Qin, Peizhong; Lu, Yungang; Li, Xinxin; Tian, Quan; Li, Yang; Xie, Chang; Tian, Jin-bin; Zhang, Chengwei; Tian, Changlin; Zhu, Michael X.; Yao, Jing

    2016-01-01

    Temperature-sensitive TRP channels are important for responses to pain and inflammation, to both of which tissue acidosis is a major contributing factor. However, except for TRPV1, acid-sensing by other ThermoTRP channels remains mysterious. We show here that unique among TRPV1–3 channels, TRPV3 is directly activated by protons from cytoplasmic side. This effect is very weak and involves key cytoplasmic residues L508, D512, S518, or A520. However, mutations of these residues did not affect a strong proton induced potentiation of TRPV3 currents elicited by the TRPV1–3 common agonist, 2-aminoethoxydiphenyl borate (2-APB), no matter if the ligand was applied from extracellular or cytoplasmic side. The acid potentiation was common among TRPV1–3 and only seen with 2-APB-related ligands. Using 1H-nuclear magnetic resonance to examine the solution structures of 2-APB and its analogs, we observed striking structural differences of the boron-containing compounds at neutral/basic as compared to acidic pH, suggesting that a pH-dependent configuration switch of 2-APB-based drugs may underlie their functionality. Supporting this notion, protons also enhanced the inhibitory action of 2-APB on TRPM8. Collectively, our findings reveal novel insights into 2-APB action on TRP channels, which should facilitate the design of new drugs for these channels. PMID:26876731

  18. Molecular candidates for cardiac stretch-activated ion channels

    PubMed Central

    Reed, Alistair; Kohl, Peter; Peyronnet, Rémi

    2014-01-01

    The heart is a mechanically-active organ that dynamically senses its own mechanical environment. This environment is constantly changing, on a beat-by-beat basis, with additional modulation by respiratory activity and changes in posture or physical activity, and further overlaid with more slowly occurring physiological (e.g. pregnancy, endurance training) or pathological challenges (e.g. pressure or volume overload). Far from being a simple pump, the heart detects changes in mechanical demand and adjusts its performance accordingly, both via heart rate and stroke volume alteration. Many of the underlying regulatory processes are encoded intracardially, and are thus maintained even in heart transplant recipients. Over the last three decades, molecular substrates of cardiac mechanosensitivity have gained increasing recognition in the scientific and clinical communities. Nonetheless, the processes underlying this phenomenon are still poorly understood. Stretch-activated ion channels (SAC) have been identified as one contributor to mechanosensitive autoregulation of the heartbeat. They also appear to play important roles in the development of cardiac pathologies – most notably stretch-induced arrhythmias. As recently discovered, some established cardiac drugs act, in part at least, via mechanotransduction pathways suggesting SAC as potential therapeutic targets. Clearly, identification of the molecular substrate of cardiac SAC is of clinical importance and a number of candidate proteins have been identified. At the same time, experimental studies have revealed variable–and at times contrasting–results regarding their function. Further complication arises from the fact that many ion channels that are not classically defined as SAC, including voltage and ligand-gated ion channels, can respond to mechanical stimulation. Here, we summarise what is known about the molecular substrate of the main candidates for cardiac SAC, before identifying potential further

  19. Biologically active extracts with kidney affections applications

    NASA Astrophysics Data System (ADS)

    Pascu (Neagu), Mihaela; Pascu, Daniela-Elena; Cozea, Andreea; Bunaciu, Andrei A.; Miron, Alexandra Raluca; Nechifor, Cristina Aurelia

    2015-12-01

    This paper is aimed to select plant materials rich in bioflavonoid compounds, made from herbs known for their application performances in the prevention and therapy of renal diseases, namely kidney stones and urinary infections (renal lithiasis, nephritis, urethritis, cystitis, etc.). This paper presents a comparative study of the medicinal plant extracts composition belonging to Ericaceae-Cranberry (fruit and leaves) - Vaccinium vitis-idaea L. and Bilberry (fruit) - Vaccinium myrtillus L. Concentrated extracts obtained from medicinal plants used in this work were analyzed from structural, morphological and compositional points of view using different techniques: chromatographic methods (HPLC), scanning electronic microscopy, infrared, and UV spectrophotometry, also by using kinetic model. Liquid chromatography was able to identify the specific compounds of the Ericaceae family, present in all three extracts, arbutosid, as well as specific components of each species, mostly from the class of polyphenols. The identification and quantitative determination of the active ingredients from these extracts can give information related to their therapeutic effects.

  20. Tissue kallikrein activation of the epithelial Na channel

    PubMed Central

    Patel, Ankit B.; Chao, Julie

    2012-01-01

    Epithelial Na Channels (ENaC) are responsible for the apical entry of Na+ in a number of different epithelia including the renal connecting tubule and cortical collecting duct. Proteolytic cleavage of γ-ENaC by serine proteases, including trypsin, furin, elastase, and prostasin, has been shown to increase channel activity. Here, we investigate the ability of another serine protease, tissue kallikrein, to regulate ENaC. We show that excretion of tissue kallikrein, which is secreted into the lumen of the connecting tubule, is stimulated following 5 days of a high-K+ or low-Na+ diet in rats. Urinary proteins reconstituted in a low-Na buffer activated amiloride-sensitive currents (INa) in ENaC-expressing oocytes, suggesting an endogenous urinary protease can activate ENaC. We next tested whether tissue kallikrein can directly cleave and activate ENaC. When rat ENaC-expressing oocytes were exposed to purified tissue kallikrein from rat urine (RTK), ENaC currents increased threefold in both the presence and absence of a soybean trypsin inhibitor (SBTI). RTK and trypsin both decreased the apparent molecular mass of cleaved cell-surface γ-ENaC, while immunodepleted RTK produced no shift in apparent molecular mass, demonstrating the specificity of the tissue kallikrein. A decreased effect of RTK on Xenopus ENaC, which has variations in the putative prostasin cleavage sites in γ-ENaC, suggests these sites are important in RTK activation of ENaC. Mutating the prostasin site in mouse γ-ENaC (γRKRK186QQQQ) abolished ENaC activation and cleavage by RTK while wild-type mouse ENaC was activated and cleaved similar to that of the rat. We conclude that tissue kallikrein can be a physiologically relevant regulator of ENaC activity. PMID:22622459

  1. Affective Response to Physical Activity: Testing for Measurement Invariance of the Physical Activity Affect Scale across Active and Non-Active Individuals

    ERIC Educational Resources Information Center

    Carpenter, Laura C.; Tompkins, Sara Anne; Schmiege, Sarah J.; Nilsson, Renea; Bryan, Angela

    2010-01-01

    Affective responses to physical activity are assumed to play a role in exercise initiation and maintenance. The Physical Activity Affect Scale measures four dimensions of an individual's affective response to exercise. Group differences in the interpretation of scale items can impact the interpretability of mean differences, underscoring the need…

  2. Mechanism of allosteric activation of TMEM16A/ANO1 channels by a commonly used chloride channel blocker

    PubMed Central

    Ta, Chau M; Adomaviciene, Aiste; Rorsman, Nils J G; Garnett, Hannah

    2016-01-01

    Background and Purpose Calcium‐activated chloride channels (CaCCs) play varied physiological roles and constitute potential therapeutic targets for conditions such as asthma and hypertension. TMEM16A encodes a CaCC. CaCC pharmacology is restricted to compounds with relatively low potency and poorly defined selectivity. Anthracene‐9‐carboxylic acid (A9C), an inhibitor of various chloride channel types, exhibits complex effects on native CaCCs and cloned TMEM16A channels providing both activation and inhibition. The mechanisms underlying these effects are not fully defined. Experimental Approach Patch‐clamp electrophysiology in conjunction with concentration jump experiments was employed to define the mode of interaction of A9C with TMEM16A channels. Key Results In the presence of high intracellular Ca2+, A9C inhibited TMEM16A currents in a voltage‐dependent manner by entering the channel from the outside. A9C activation, revealed in the presence of submaximal intracellular Ca2+ concentrations, was also voltage‐dependent. The electric distance of A9C inhibiting and activating binding site was ~0.6 in each case. Inhibition occurred according to an open‐channel block mechanism. Activation was due to a dramatic leftward shift in the steady‐state activation curve and slowed deactivation kinetics. Extracellular A9C competed with extracellular Cl−, suggesting that A9C binds deep in the channel's pore to exert both inhibiting and activating effects. Conclusions and Implications A9C is an open TMEM16A channel blocker and gating modifier. These effects require A9C to bind to a region within the pore that is accessible from the extracellular side of the membrane. These data will aid the future drug design of compounds that selectively activate or inhibit TMEM16A channels. PMID:26562072

  3. Relaxin stimulates myometrial calcium-activated potassium channel activity via protein kinase A.

    PubMed

    Meera, P; Anwer, K; Monga, M; Oberti, C; Stefani, E; Toro, L; Sanborn, B M

    1995-08-01

    Relaxin, a hormone that is elevated during pregnancy, can suppress myometrial contractile activity. Ca(2+)-activated K+ channels (KCa) play a role in the modulation of uterine contractions and myometrial Ca2+ homeostasis and have been implicated in the control of smooth muscle excitability. We now show that relaxin stimulates KCa channels in cell-attached patches in a cell line derived from term pregnant human myometrium. This effect was prevented by the protein kinase A (PKA) antagonist, the Rp diastereomer of adenosine 3',5'-cyclic monophosphothioate (Rp-cAMPS). After patch excision, the channel was activated by PKA and inhibited by alkaline phosphatase. These data suggest that relaxin may promote myometrial quiescence in part by stimulation of KCa channels via a PKA-mediated mechanism. PMID:7653512

  4. Phosphoinositide interacting regulator of TRP (Pirt) enhances TRPM8 channel activity in vitro via increasing channel conductance

    PubMed Central

    Tang, Min; Wu, Guang-yi; Dong, Xin-zhong; Tang, Zong-xiang

    2016-01-01

    Aim: Pirt is a two-transmembrane domain protein that regulates the function of a variety of ion channels. Our previous study indicated that Pirt acts as a positive endogenous regulator of the TRPM8 channel. The aim of this study was to investigate the mechanism underlying the regulation of TRPM8 channel by Pirt. Methods: HEK293 cells were transfected with TRPM8+Pirt or TRPM8 alone. Menthol (1 mmol/L) was applied through perfusion to induce TRPM8-mediated voltage-dependent currents, which were recorded using a whole-cell recording technique. PIP2 (10 μmol/L) was added into the electrode pipettes (PI was taken as a control). Additionally, cell-attached single-channel recordings were conducted in CHO cells transfected with TRPM8+Pirt or TRPM8 alone, and menthol (1 mmol/L) was added into the pipette solution. Results: Either co-transfection with Pirt or intracellular application of PIP2 (but not PI) significantly enhanced menthol-induced TRPM8 currents. Furthermore, Pirt and PIP2 synergistically modulated menthol-induced TRPM8 currents. Single-channel recordings revealed that co-transfection with Pirt significantly increased the single channel conductance. Conclusion: Pirt and PIP2 synergistically enhance TRPM8 channel activity, and Pirt regulates TRPM8 channel activity by increasing the single channel conductance. PMID:26657057

  5. Prefrontal Cortex Haemodynamics and Affective Responses during Exercise: A Multi-Channel Near Infrared Spectroscopy Study

    PubMed Central

    Tempest, Gavin D.; Eston, Roger G.; Parfitt, Gaynor

    2014-01-01

    The dose-response effects of the intensity of exercise upon the potential regulation (through top-down processes) of affective (pleasure-displeasure) responses in the prefrontal cortex during an incremental exercise protocol have not been explored. This study examined the functional capacity of the prefrontal cortex (reflected by haemodynamics using near infrared spectroscopy) and affective responses during exercise at different intensities. Participants completed an incremental cycling exercise test to exhaustion. Changes (Δ) in oxygenation (O2Hb), deoxygenation (HHb), blood volume (tHb) and haemoglobin difference (HbDiff) were measured from bilateral dorsal and ventral prefrontal areas. Affective responses were measured every minute during exercise. Data were extracted at intensities standardised to: below ventilatory threshold, at ventilatory threshold, respiratory compensation point and the end of exercise. During exercise at intensities from ventilatory threshold to respiratory compensation point, ΔO2Hb, ΔHbDiff and ΔtHb were greater in mostly ventral than dorsal regions. From the respiratory compensation point to the end of exercise, ΔO2Hb remained stable and ΔHbDiff declined in dorsal regions. As the intensity increased above the ventilatory threshold, inverse associations between affective responses and oxygenation in (a) all regions of the left hemisphere and (b) lateral (dorsal and ventral) regions followed by the midline (ventral) region in the right hemisphere were observed. Differential activation patterns occur within the prefrontal cortex and are associated with affective responses during cycling exercise. PMID:24788166

  6. Prefrontal cortex haemodynamics and affective responses during exercise: a multi-channel near infrared spectroscopy study.

    PubMed

    Tempest, Gavin D; Eston, Roger G; Parfitt, Gaynor

    2014-01-01

    The dose-response effects of the intensity of exercise upon the potential regulation (through top-down processes) of affective (pleasure-displeasure) responses in the prefrontal cortex during an incremental exercise protocol have not been explored. This study examined the functional capacity of the prefrontal cortex (reflected by haemodynamics using near infrared spectroscopy) and affective responses during exercise at different intensities. Participants completed an incremental cycling exercise test to exhaustion. Changes (Δ) in oxygenation (O2Hb), deoxygenation (HHb), blood volume (tHb) and haemoglobin difference (HbDiff) were measured from bilateral dorsal and ventral prefrontal areas. Affective responses were measured every minute during exercise. Data were extracted at intensities standardised to: below ventilatory threshold, at ventilatory threshold, respiratory compensation point and the end of exercise. During exercise at intensities from ventilatory threshold to respiratory compensation point, ΔO2Hb, ΔHbDiff and ΔtHb were greater in mostly ventral than dorsal regions. From the respiratory compensation point to the end of exercise, ΔO2Hb remained stable and ΔHbDiff declined in dorsal regions. As the intensity increased above the ventilatory threshold, inverse associations between affective responses and oxygenation in (a) all regions of the left hemisphere and (b) lateral (dorsal and ventral) regions followed by the midline (ventral) region in the right hemisphere were observed. Differential activation patterns occur within the prefrontal cortex and are associated with affective responses during cycling exercise. PMID:24788166

  7. Curcumin inhibits activation of TRPM2 channels in rat hepatocytes

    PubMed Central

    Kheradpezhouh, E.; Barritt, G.J.; Rychkov, G.Y.

    2015-01-01

    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. PMID:26609559

  8. Curcumin inhibits activation of TRPM2 channels in rat hepatocytes.

    PubMed

    Kheradpezhouh, E; Barritt, G J; Rychkov, G Y

    2016-04-01

    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 Ca(2+) homeostasis, resulting in a sustained elevation of the free cytosolic Ca(2+) concentration ([Ca(2+)]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 Ca(2+) 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 5µM curcumin in the incubation medium prevented the H2O2- and paracetamol-induced [Ca(2+)]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 50nM. 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. PMID:26609559

  9. AMPK Dilates Resistance Arteries via Activation of SERCA and BKCa Channels in Smooth Muscle.

    PubMed

    Schneider, Holger; Schubert, Kai Michael; Blodow, Stephanie; Kreutz, Claus-Peter; Erdogmus, Serap; Wiedenmann, Margarethe; Qiu, Jiehua; Fey, Theres; Ruth, Peter; Lubomirov, Lubomir T; Pfitzer, Gabriele; Mederos Y Schnitzler, Michael; Hardie, D Grahame; Gudermann, Thomas; Pohl, Ulrich

    2015-07-01

    The protective effects of 5'-AMP-activated protein kinase (AMPK) on the metabolic syndrome may include direct effects on resistance artery vasomotor function. However, the precise actions of AMPK on microvessels and their potential interaction are largely unknown. Thus, we set to determine the effects of AMPK activation on vascular smooth muscle tone and the underlying mechanisms. Resistance arteries isolated from hamster and mouse exhibited a pronounced endothelium-independent dilation on direct pharmacological AMPK activation by 2 structurally unrelated compounds (PT1 and A769662). The dilation was associated with a decrease of intracellular-free calcium [Ca(2+)]i in vascular smooth muscle cell. AMPK stimulation induced activation of BKCa channels as assessed by patch clamp studies in freshly isolated hamster vascular smooth muscle cell and confirmed by direct proof of membrane hyperpolarization in intact arteries. The BKCa channel blocker iberiotoxin abolished the hyperpolarization but only partially reduced the dilation and did not affect the decrease of [Ca(2+)]i. By contrast, the sarcoplasmic/endoplasmic Ca(2+)-ATPase (SERCA) inhibitor thapsigargin largely reduced these effects, whereas combined inhibition of SERCA and BKCa channels virtually abolished them. AMPK stimulation significantly increased the phosphorylation of the SERCA modulator phospholamban at the regulatory T17 site. Stimulation of smooth muscle AMPK represents a new, potent vasodilator mechanism in resistance vessels. AMPK directly relaxes vascular smooth muscle cell by a decrease of [Ca(2+)]i. This is achieved by calcium sequestration via SERCA activation, as well as activation of BKCa channels. There is in part a mutual compensation of both calcium-lowering mechanisms. However, SERCA activation which involves an AMPK-dependent phosphorylation of phospholamban is the predominant mechanism in resistance vessels. PMID:26034200

  10. Study of permeation and blocker binding in TMEM16A calcium-activated chloride channels.

    PubMed

    Reyes, J P; Huanosta-Gutiérrez, A; López-Rodríguez, A; Martínez-Torres, A

    2015-01-01

    We studied the effects of mutations of positively charged amino acid residues in the pore of X. tropicalis TMEM16A calcium-activated chloride channels: K613E, K628E, K630E; R646E and R761E. The activation and deactivation kinetics were not affected, and only K613E showed a lower current density. K628E and R761E affect anion selectivity without affecting Na(+) permeation, whereas K613E, R646E and the double mutant K613E + R646E affect anion selectivity and permeability to Na(+). Furthermore, altered blockade by the chloride channel blockers anthracene-9-carboxylic acid (A-9-C), 4, 4'-Diisothiocyano-2,2'-stilbenedisulfonic acid (DIDS) and T16inh-A01 was observed. These results suggest the existence of 2 binding sites for anions within the pore at electrical distances of 0.3 and 0.5. These sites are also relevant for anion permeation and blockade. PMID:25853341

  11. Photochemical activation of TRPA1 channels in neurons and animals

    PubMed Central

    Kokel, David; Cheung, Chung Yan J.; Mills, Robert; Coutinho-Budd, Jaeda; Huang, Liyi; Setola, Vincent; Sprague, Jared; Jin, Shan; Jin, Youngnam N.; Huang, Xi-Ping; Bruni, Giancarlo; Woolf, Clifford; Roth, Bryan L.; Hamblin, Michael R; Zylka, Mark J.; Milan, David J.; Peterson, Randall T.

    2013-01-01

    Optogenetics is a powerful research tool because it enables high-resolution optical control of neuronal activity. However, current optogenetic approaches are limited to transgenic systems expressing microbial opsins and other exogenous photoreceptors. Here, we identify optovin, a small molecule that enables repeated photoactivation of motor behaviors in wild type animals. Surprisingly, optovin's behavioral effects are not visually mediated. Rather, photodetection is performed by sensory neurons expressing the cation channel TRPA1. TRPA1 is both necessary and sufficient for the optovin response. Optovin activates human TRPA1 via structure-dependent photochemical reactions with redox-sensitive cysteine residues. In animals with severed spinal cords, optovin treatment enables control of motor activity in the paralyzed extremities by localized illumination. These studies identify a light-based strategy for controlling endogenous TRPA1 receptors in vivo, with potential clinical and research applications in non-transgenic animals, including humans. PMID:23396078

  12. Blockade of TRPM7 Channel Activity and Cell Death by Inhibitors of 5-Lipoxygenase

    PubMed Central

    Chen, Hsiang-Chin; Xie, Jia; Zhang, Zheng; Su, Li-Ting; Yue, Lixia; Runnels, Loren W.

    2010-01-01

    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 the TRPM7 channel

  13. Modulation and pharmacology of low voltage-activated ("T-Type") calcium channels.

    PubMed

    Yunker, Anne Marie R

    2003-12-01

    Although T-type calcium channel currents were observed almost 30 years ago, the genes that encode the pore-forming subunits have only been recently reported. When expressed in heterologous systems, three distinct alpha1 subunits (alpha1G (Cav3.1), alpha1H (Car3.2), and alpha1I (Cav3.3)) conduct T-type currents with insert similar but not identical electrophysiological characteristics that. Alpha 1G, alpha 1H, and alpha 1I transcripts are found throughout neural and nonneural tissues, suggesting multiple types of T-type channels (also called low voltage-activated calcium channels (LVAs)) are coexpressed by many tissues. The study of endogenous LVAs has been hampered by a lack of highly selective antagonists that differentiate between LVA subtypes. Furthermore, many pharmacological agents attenuate currents conducted by LVA and high voltage-activated calcium channels (HVAs). At least 15 classes of pharmacological agents affect T-type currents, and the therapeutic use of many of these drugs has implicated LVAs in the etiology of a variety of diseases. Comparison of the responses of recombinant and native LVAs to pharmacological agents and endogenous modulatory molecules will lead to a better understanding of LVAs in normal and diseased cells. PMID:15000521

  14. Large-Conductance Calcium-Activated Potassium Channels in Glomerulus: From Cell Signal Integration to Disease.

    PubMed

    Tao, Jie; Lan, Zhen; Wang, Yunman; Hei, Hongya; Tian, Lulu; Pan, Wanma; Zhang, Xuemei; Peng, Wen

    2016-01-01

    Large-conductance calcium-activated potassium (BK) channels are currently considered as vital players in a variety of renal physiological processes. In podocytes, BK channels become active in response to stimuli that increase local cytosolic Ca(2+), possibly secondary to activation of slit diaphragm TRPC6 channels by chemical or mechanical stimuli. Insulin increases filtration barrier permeability through mobilization of BK channels. In mesangial cells, BK channels co-expressed with β1 subunits act as a major component of the counteractive response to contraction in order to regulate glomerular filtration. This review aims to highlight recent discoveries on the localization, physiological and pathological roles of BK channels in glomerulus. PMID:27445840

  15. Large-Conductance Calcium-Activated Potassium Channels in Glomerulus: From Cell Signal Integration to Disease

    PubMed Central

    Tao, Jie; Lan, Zhen; Wang, Yunman; Hei, Hongya; Tian, Lulu; Pan, Wanma; Zhang, Xuemei; Peng, Wen

    2016-01-01

    Large-conductance calcium-activated potassium (BK) channels are currently considered as vital players in a variety of renal physiological processes. In podocytes, BK channels become active in response to stimuli that increase local cytosolic Ca2+, possibly secondary to activation of slit diaphragm TRPC6 channels by chemical or mechanical stimuli. Insulin increases filtration barrier permeability through mobilization of BK channels. In mesangial cells, BK channels co-expressed with β1 subunits act as a major component of the counteractive response to contraction in order to regulate glomerular filtration. This review aims to highlight recent discoveries on the localization, physiological and pathological roles of BK channels in glomerulus.

  16. NSAIDs attenuate hyperalgesia induced by TRP channel activation.

    PubMed

    Nozadze, Ivliane; Tsiklauri, Nana; Gurtskaia, Gulnaz; Tsagareli, Merab G

    2016-03-01

    Transient receptor potential (TRP) cation channels have been extensively investigated as targets for analgesic drug discovery. Because some non-steroidal anti-inflammatory drugs (NSAIDs) are structural analogs of prostaglandins (mediators of inflammation) and NSAIDs attenuate heat nociception and mechanical allodynia in models of inflammatory and neuropathic pain, we examined three widely used NSAIDs (diclofenac, ketorolac, and xefocam) on the activation of TRPA1 and TRPV1 channels using thermal paw withdrawal (Hargreaves) test and mechanical paw withdrawal (von Frey) test in male rats. Thermal withdrawal latencies and mechanical thresholds for both hind paws were obtained with 5, 15, 30, 45, 60, and 120 min intraplantar post-injection of TRPA1 agonizts, allyl isothiocyanate (AITC) (a natural compound of mustard oil) and cinnamaldehyde (CA), and TRPV1 agonist capsaicin or vehicle. Twenty minutes prior to the start of the experiment with TRP agonizts, diclofenac, ketorolac or xefocam were pre-injected in the same hindpaw and animals were examined by these two tests. After pretreatment of all three NSAIDs in the ipsilateral (injected) hindpaw that produced strong antinociceptive effects, AITC, CA, and capsaicin caused significant decreases in latency of the thermal withdrawal reflex compared with vehicle or the contralateral hindpaw. The same findings were observed for the paw withdrawal threshold. In approximately 30 min the effects of CA, AITC, and capsaicin returned to baseline. The data are different from our previous evidence, where TRPA1 agonizts AITC and CA and TRPV1 agonist capsaicin produced hyperalgesia for nearly 2 h and resulted in facilitation of these withdrawal reflexes (Tsagareli et al., 2010, 2013). Thus, our data showing that NSAIDs suppress thermal and mechanical hyperalgesia following TRP activation could presumably due to inactivation or desensitization of TRPA1 and TRPV1 channels by NSAIDs. PMID:26909384

  17. NSAIDs attenuate hyperalgesia induced by TRP channel activation

    PubMed Central

    Nozadze, Ivliane; Tsiklauri, Nana; Gurtskaia, Gulnaz; Tsagareli, Merab G.

    2016-01-01

    Transient receptor potential (TRP) cation channels have been extensively investigated as targets for analgesic drug discovery. Because some non-steroidal anti-inflammatory drugs (NSAIDs) are structural analogs of prostaglandins (mediators of inflammation) and NSAIDs attenuate heat nociception and mechanical allodynia in models of inflammatory and neuropathic pain, we examined three widely used NSAIDs (diclofenac, ketorolac, and xefocam) on the activation of TRPA1 and TRPV1 channels using thermal paw withdrawal (Hargreaves) test and mechanical paw withdrawal (von Frey) test in male rats. Thermal withdrawal latencies and mechanical thresholds for both hind paws were obtained with 5, 15, 30, 45, 60, and 120 min intraplantar post-injection of TRPA1 agonizts, allyl isothiocyanate (AITC) (a natural compound of mustard oil) and cinnamaldehyde (CA), and TRPV1 agonist capsaicin or vehicle. Twenty minutes prior to the start of the experiment with TRP agonizts, diclofenac, ketorolac or xefocam were pre-injected in the same hindpaw and animals were examined by these two tests. After pretreatment of all three NSAIDs in the ipsilateral (injected) hindpaw that produced strong antinociceptive effects, AITC, CA, and capsaicin caused significant decreases in latency of the thermal withdrawal reflex compared with vehicle or the contralateral hindpaw. The same findings were observed for the paw withdrawal threshold. In approximately 30 min the effects of CA, AITC, and capsaicin returned to baseline. The data are different from our previous evidence, where TRPA1 agonizts AITC and CA and TRPV1 agonist capsaicin produced hyperalgesia for nearly 2 h and resulted in facilitation of these withdrawal reflexes (Tsagareli et al., 2010, 2013). Thus, our data showing that NSAIDs suppress thermal and mechanical hyperalgesia following TRP activation could presumably due to inactivation or desensitization of TRPA1 and TRPV1 channels by NSAIDs. PMID:26909384

  18. Membrane stretch and cytoplasmic Ca2+ independently modulate stretch-activated BK channel activity.

    PubMed

    Zhao, Hu-Cheng; Agula, Hasi; Zhang, Wei; Wang, Fa; Sokabe, Masahiro; Li, Lu-Ming

    2010-11-16

    Large conductance Ca(2+)-activated K(+) (BK) channels are responsible for changes in chemical and physical signals such as Ca(2+), Mg(2+) and membrane potentials. Previously, we reported that a BK channel cloned from chick heart (SAKCaC) is activated by membrane stretch. Molecular cloning and subsequent functional characterization of SAKCaC have shown that both the membrane stretch and intracellular Ca(2+) signal allosterically regulate the channel activity via the linker of the gating ring complex. Here we investigate how these two gating principles interact with each other. We found that stretch force activated SAKCaC in the absence of cytoplasmic Ca(2+). Lack of Ca(2+) bowl (a calcium binding motif) in SAKCaC diminished the Ca(2+)-dependent activation, but the mechanosensitivity of channel was intact. We also found that the abrogation of STREX (a proposed mechanosensing apparatus) in SAKCaC abolished the mechanosensitivity without altering the Ca(2+) sensitivity of channels. These observations indicate that membrane stretch and intracellular Ca(2+) could independently modulate SAKCaC activity. PMID:20673577

  19. Channel bed adjustment along mine-affected rivers of northeast Tasmania

    NASA Astrophysics Data System (ADS)

    Knighton, A. D.

    1991-10-01

    Forty million m 3 and 1 million m 3 of mining waste are estimated to have been supplied respectively to the Ringarooma and George Rivers between 1875 and 1984. Given the volumes involved and the fact that much of the input was less than 5 mm in diameter, the size composition of the bed material changed from gravel to sand as the rivers progressively aggraded their beds downstream. Increases in bed height of over 10 m and 1 m are predicted respectively for the two rivers, with change becoming more gradual further downstream. With upstream supplies becoming depleted first, degradation followed the same pattern as aggradation — progressing downstream. Maximum rates of almost 0.5 m yr -1 were measured. Degradation has returned the George River to its pre-1875 level but has yet to reach the downstream reaches of the Ringarooma, where sediment waves continue to pass over a slightly aggrading bed. Where degradation has been occurring long enough, the bed material has again become gravelly through re-exposure of the original bed and/or lag concentration of coarser fractions within the introduced load. Such armouring should improve bed stability and slow the rate of degradation. However, at Herrick, which is representative of intermediate reaches with only a thin gravel veneer, the annual amount of degradation is reasonably well correlated with flow conditions, suggesting that high discharges can strip the surface armour and continue to degrade actively. Incision has not only changed the vertical position of the rivers but also their planimetric and cross-sectional geometry. In particular, non-uniform lowering of the bed has narrowed the channel by up to 60%, changes which can be predicted quite accurately by Chang's quantitative model of channel adjustment. At least another 50 years will be needed for degradation to cleanse the Ringarooma of mining debris.

  20. Membrane stretching triggers mechanosensitive Ca2+ channel activation in Chara.

    PubMed

    Kaneko, Toshiyuki; Takahashi, Naoya; Kikuyama, Munehiro

    2009-03-01

    In order to confirm that mechanosensitive Ca(2+) channels are activated by membrane stretching, we stretched or compressed the plasma membrane of Chara by applying osmotic shrinkage or swelling of the cell by varying the osmotic potential of the bathing medium. Aequorin studies revealed that treatments causing membrane stretching induced a transient but large increase in cytoplasmic concentration of Ca(2+) (Delta[Ca(2+)](c)). However, the observed Delta[Ca(2+)](c) decreased during the treatments, resulting in membrane compression. A second experiment was carried out to study the relationship between changes in membrane potential (DeltaE(m)) and stretching or compression of the plasma membrane. Significant DeltaE(m) values, often accompanied by an action potential, were observed during the initial exchange of the bathing medium from a hypotonic medium to a hypertonic one (plasmolysis). DeltaE(m) appears to be triggered by a partial stretching of the membrane as it was peeled from the cell wall. After plasmolysis, other exchanges from hypertonic to hypotonic media, with their accompanying membrane stretching, always induced large DeltaE(m) values and were often accompanied by an action potential. By contrast, action potentials were scarcely observed during other exchanges from hypotonic to hypertonic solutions (=membrane compression). Thus, we concluded that activation of the mechanosensitive channels is triggered by membrane stretching in Chara. PMID:19234734

  1. TMEM16F is a component of a Ca2+-activated Cl- channel but not a volume-sensitive outwardly rectifying Cl- channel.

    PubMed

    Shimizu, Takahiro; Iehara, Takahiro; Sato, Kaori; Fujii, Takuto; Sakai, Hideki; Okada, Yasunobu

    2013-04-15

    TMEM16 (transmembrane protein 16) proteins, which possess eight putative transmembrane domains with intracellular NH2- and COOH-terminal tails, are thought to comprise a Cl(-) channel family. The function of TMEM16F, a member of the TMEM16 family, has been greatly controversial. In the present study, we performed whole cell patch-clamp recordings to investigate the function of human TMEM16F. In TMEM16F-transfected HEK293T cells but not TMEM16K- and mock-transfected cells, activation of membrane currents with strong outward rectification was found to be induced by application of a Ca(2+) ionophore, ionomycin, or by an increase in the intracellular free Ca(2+) concentration. The free Ca(2+) concentration for half-maximal activation of TMEM16F currents was 9.6 μM, which is distinctly higher than that for TMEM16A/B currents. The outwardly rectifying current-voltage relationship for TMEM16F currents was not changed by an increase in the intracellular Ca(2+) level, in contrast to TMEM16A/B currents. The Ca(2+)-activated TMEM16F currents were anion selective, because replacing Cl(-) with aspartate(-) in the bathing solution without changing cation concentrations caused a positive shift of the reversal potential. The anion selectivity sequence of the TMEM16F channel was I(-) > Br(-) > Cl(-) > F(-) > aspartate(-). Niflumic acid, a Ca(2+)-activated Cl(-) channel blocker, inhibited the TMEM16F-dependent Cl(-) currents. Neither overexpression nor knockdown of TMEM16F affected volume-sensitive outwardly rectifying Cl(-) channel (VSOR) currents activated by osmotic swelling or apoptotic stimulation. These results demonstrate that human TMEM16F is an essential component of a Ca(2+)-activated Cl(-) channel with a Ca(2+) sensitivity that is distinct from that of TMEM16A/B and that it is not related to VSOR activity. PMID:23426967

  2. A cAMP-Regulated Chloride Channel in Lymphocytes that is Affected in Cystic Fibrosis

    NASA Astrophysics Data System (ADS)

    Chen, Jennifer H.; Schulman, Howard; Gardner, Phyllis

    1989-02-01

    A defect in regulation of a chloride channel appears to be the molecular basis for cystic fibrosis (CF), a common lethal genetic disease. It is shown here that a chloride channel with kinetic and regulatory properties similar to those described for secretory epithelial cells is present in both T and B lymphocyte cell lines. The regulation of the channels by adenosine 3',5'-monophosphate (cAMP)--dependent protein kinase in transformed B cells from CF patients is defective. Thus, lymphocytes may be an accessible source of CF tissue for study of this defect, for cloning of the chloride channel complex, and for diagnosis of the disease.

  3. Structure-Function Relation of Phospholamban: Modulation of Channel Activity as a Potential Regulator of SERCA Activity

    PubMed Central

    Smeazzetto, Serena; Saponaro, Andrea; Young, Howard S.; Moncelli, Maria Rosa; Thiel, Gerhard

    2013-01-01

    Phospholamban (PLN) is a small integral membrane protein, which binds and inhibits in a yet unknown fashion the Ca2+-ATPase (SERCA) in the sarcoplasmic reticulum. When reconstituted in planar lipid bilayers PLN exhibits ion channel activity with a low unitary conductance. From the effect of non-electrolyte polymers on this unitary conductance we estimate a narrow pore with a diameter of ca. 2.2 Å for this channel. This value is similar to that reported for the central pore in the structure of the PLN pentamer. Hence the PLN pentamer, which is in equilibrium with the monomer, is the most likely channel forming structure. Reconstituted PLN mutants, which either stabilize (K27A and R9C) or destabilize (I47A) the PLN pentamer and also phosphorylated PLN still generate the same unitary conductance of the wt/non-phosphorylated PLN. However the open probability of the phosphorylated PLN and of the R9C mutant is significantly lower than that of the respective wt/non-phosphorylated control. In the context of data on PLN/SERCA interaction and on Ca2+ accumulation in the sarcoplasmic reticulum the present results are consistent with the view that PLN channel activity could participate in the balancing of charge during Ca2+ uptake. A reduced total conductance of the K+ transporting PLN by phosphorylation or by the R9C mutation may stimulate Ca2+ uptake in the same way as an inhibition of K+ channels in the SR membrane. The R9C-PLN mutation, a putative cause of dilated cardiomyopathy, might hence affect SERCA activity also via its inherent low open probability. PMID:23308118

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

    PubMed

    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. PMID:25253636

  5. Antisense oligodeoxynucleotide inhibition of a swelling-activated cation channel in osteoblast-like osteosarcoma cells.

    PubMed Central

    Duncan, R L; Kizer, N; Barry, E L; Friedman, P A; Hruska, K A

    1996-01-01

    By patch-clamp analysis, we have shown that chronic, intermittent mechanical strain (CMS) increases the activity of stretch-activated cation channels of osteoblast-like UMR-106.01 cells. CMS also produces a swelling-activated whole-cell conductance (Gm) regulated by varying strain levels. We questioned whether the swelling-activated conductance was produced by stretch-activated cation channel activity. We have identified a gene involved in the increase in conductance by using antisense oligodeoxynucleotides (ODN) derived from the alpha 1-subunit genes of calcium channels found in UMR-106.01 cells (alpha1S, alpha1C, and alpha1D). We demonstrate that alpha 1C antisense ODNs abolish the increase in Gm in response to hypotonic swelling following CMS. Antisense ODNs to alpha1S and alpha1D, sense ODNs to alpha1C, and sham permeabilization had no effect on the conductance increase. In addition, during cell-attached patch-clamp studies, antisense ODNs to alpha1c completely blocked the swelling-activated and stretch-activated nonselective cation channel response to strain. Antisense ODNs to alpha1S treatment produced no effect on either swelling-activated or stretch-activated cation channel activity. There were differences in the stretch-activated and swelling-activated cation channel activity, but whether they represent different channels could not be determined from our data. Our data indicate that the alpha1C gene product is involved in the Gm and the activation of the swelling-activated cation channels induced by CMS. The possibility that swelling-activated cation channel genes are members of the calcium channel superfamily exists, but if alpha1c is not the swelling-activated cation channel itself, then its expression is required for induction of swelling-activated cation channel activity by CMS. PMID:8700850

  6. Antisense oligodeoxynucleotide inhibition of a swelling-activated cation channel in osteoblast-like osteosarcoma cells

    NASA Technical Reports Server (NTRS)

    Duncan, R. L.; Kizer, N.; Barry, E. L.; Friedman, P. A.; Hruska, K. A.

    1996-01-01

    By patch-clamp analysis, we have shown that chronic, intermittent mechanical strain (CMS) increases the activity of stretch-activated cation channels of osteoblast-like UMR-106.01 cells. CMS also produces a swelling-activated whole-cell conductance (Gm) regulated by varying strain levels. We questioned whether the swelling-activated conductance was produced by stretch-activated cation channel activity. We have identified a gene involved in the increase in conductance by using antisense oligodeoxynucleotides (ODN) derived from the alpha 1-subunit genes of calcium channels found in UMR-106.01 cells (alpha1S, alpha1C, and alpha1D). We demonstrate that alpha 1C antisense ODNs abolish the increase in Gm in response to hypotonic swelling following CMS. Antisense ODNs to alpha1S and alpha1D, sense ODNs to alpha1C, and sham permeabilization had no effect on the conductance increase. In addition, during cell-attached patch-clamp studies, antisense ODNs to alpha1c completely blocked the swelling-activated and stretch-activated nonselective cation channel response to strain. Antisense ODNs to alpha1S treatment produced no effect on either swelling-activated or stretch-activated cation channel activity. There were differences in the stretch-activated and swelling-activated cation channel activity, but whether they represent different channels could not be determined from our data. Our data indicate that the alpha1C gene product is involved in the Gm and the activation of the swelling-activated cation channels induced by CMS. The possibility that swelling-activated cation channel genes are members of the calcium channel superfamily exists, but if alpha1c is not the swelling-activated cation channel itself, then its expression is required for induction of swelling-activated cation channel activity by CMS.

  7. Threshold bedrock channels in tectonically active mountains with frequent mass wasting

    NASA Astrophysics Data System (ADS)

    Korup, O.; Hayakawa, Y. S.; Codilean, A.; Oguchi, T.

    2013-12-01

    determining the fraction of landslide-affected terrain throughout Japan. We infer that neither average nor extreme bedrock channel steepness is a sufficiently sensitive measure for gauging landslide activity in terms of volumes mobilized as opposed to bedrock river sinuosity. This new and largely underexplored channel planform metric may reflect spacing of discontinuities in rock mass that constrains the susceptibility to large rock-slope failures, and may contain valuable compound information on both fluvial and hillslope erosion dynamics in tectonically active landscapes.

  8. Activation and Regulation of Purinergic P2X Receptor Channels

    PubMed Central

    Coddou, Claudio; Yan, Zonghe; Obsil, Tomas; Huidobro-Toro, J. Pablo

    2011-01-01

    Mammalian ATP-gated nonselective cation channels (P2XRs) can be composed of seven possible subunits, denoted P2X1 to P2X7. Each subunit contains a large ectodomain, two transmembrane domains, and intracellular N and C termini. Functional P2XRs are organized as homomeric and heteromeric trimers. This review focuses on the binding sites involved in the activation (orthosteric) and regulation (allosteric) of P2XRs. The ectodomains contain three ATP binding sites, presumably located between neighboring subunits and formed by highly conserved residues. The detection and coordination of three ATP phosphate residues by positively charged amino acids are likely to play a dominant role in determining agonist potency, whereas an AsnPheArg motif may contribute to binding by coordinating the adenine ring. Nonconserved ectodomain histidines provide the binding sites for trace metals, divalent cations, and protons. The transmembrane domains account not only for the formation of the channel pore but also for the binding of ivermectin (a specific P2X4R allosteric regulator) and alcohols. The N- and C- domains provide the structures that determine the kinetics of receptor desensitization and/or pore dilation and are critical for the regulation of receptor functions by intracellular messengers, kinases, reactive oxygen species and mercury. The recent publication of the crystal structure of the zebrafish P2X4.1R in a closed state provides a major advance in the understanding of this family of receptor channels. We will discuss data obtained from numerous site-directed mutagenesis experiments accumulated during the last 15 years with reference to the crystal structure, allowing a structural interpretation of the molecular basis of orthosteric and allosteric ligand actions. PMID:21737531

  9. ATP-Sensitive K+ Channels Regulate the Concentrative Adenosine Transporter CNT2 following Activation by A1 Adenosine Receptors

    PubMed Central

    Duflot, Sylvie; Riera, Bárbara; Fernández-Veledo, Sonia; Casadó, Vicent; Norman, Robert I.; Casado, F. Javier; Lluís, Carme; Franco, Rafael; Pastor-Anglada, Marçal

    2004-01-01

    This study describes a novel mechanism of regulation of the high-affinity Na+-dependent adenosine transporter (CNT2) via the activation of A1 adenosine receptors (A1R). This regulation is mediated by the activation of ATP-sensitive K+ (KATP) channels. The high-affinity Na+-dependent adenosine transporter CNT2 and A1R are coexpressed in the basolateral domain of the rat hepatocyte plasma membrane and are colocalized in the rat hepatoma cell line FAO. The transient increase in CNT2-mediated transport activity triggered by (−)-N6-(2-phenylisopropyl)adenosine is fully inhibited by KATP channel blockers and mimicked by a KATP channel opener. A1R agonist activation of CNT2 occurs in both hepatocytes and FAO cells, which express Kir6.1, Kir6.2, SUR1, SUR2A, and SUR2B mRNA channel subunits. With the available antibodies against Kir6.X, SUR2A, and SUR2B, it is shown that all of these proteins colocalize with CNT2 and A1R in defined plasma membrane domains of FAO cells. The extent of the purinergic modulation of CNT2 is affected by the glucose concentration, a finding which indicates that glycemia and glucose metabolism may affect this cross-regulation among A1R, CNT2, and KATP channels. These results also suggest that the activation of KATP channels under metabolic stress can be mediated by the activation of A1R. Cell protection under these circumstances may be achieved by potentiation of the uptake of adenosine and its further metabolization to ATP. Mediation of purinergic responses and a connection between the intracellular energy status and the need for an exogenous adenosine supply are novel roles for KATP channels. PMID:15024061

  10. Activation of the epithelial Na+ channel in the collecting duct by vasopressin contributes to water reabsorption.

    PubMed

    Bugaj, Vladislav; Pochynyuk, Oleh; Stockand, James D

    2009-11-01

    We used patch-clamp electrophysiology on isolated, split-open murine collecting ducts (CD) to test the hypothesis that regulation of epithelial sodium channel (ENaC) activity is a physiologically important effect of vasopressin. Surprisingly, this has not been tested directly before. We ask whether vasopressin affects ENaC activity distinguishing between acute and chronic effects, as well as, parsing the cellular signaling pathway and molecular mechanism of regulation. In addition, we quantified possible synergistic regulation of ENaC by vasopressin and aldosterone associating this with a requirement for distal nephron Na+ reabsorption during water conservation vs. maintenance of Na+ balance. We find that vasopressin significantly increases ENaC activity within 2-3 min by increasing open probability (P(o)). This activation was dependent on adenylyl cyclase (AC) and PKA. Water restriction (18-24 h) and pretreatment of isolated CD with vasopressin (approximately 30 min) resulted in a similar increase in P(o). In addition, this also increased the number (N) of active ENaC in the apical membrane. Similar to P(o), increases in N were sensitive to inhibitors of AC. Stressing animals with water and salt restriction separately and jointly revealed an important effect of vasopressin: conservation of water and Na+ each independently increased ENaC activity and jointly had a synergistic effect on channel activity. These results demonstrate a quantitatively important action of vasopressin on ENaC suggesting that distal nephron Na+ reabsorption mediated by this channel contributes to maintenance of water reabsorption. In addition, our results support that the combined actions of vasopressin and aldosterone are required to achieve maximally activated ENaC. PMID:19692483

  11. Effects of microgravity on liposome-reconstituted cardiac gap junction channeling activity

    NASA Technical Reports Server (NTRS)

    Claassen, D. E.; Spooner, B. S.

    1989-01-01

    Effects of microgravity on cardiac gap junction channeling activity were investigated aboard NASA zero-gravity aircraft. Liposome-reconstituted gap junctions were assayed for channel function during free-fall, and the data were compared with channeling at 1 g. Control experiments tested for 0 g effects on the structural stability of liposomes, and on the enzyme-substrate signalling system of the assay. The results demonstrate that short periods of microgravity do not perturb reconstituted cardiac gap junction channeling activity.

  12. Trypsin-Sensitive, Rapid Inactivation of a Calcium-Activated Potassium Channel

    NASA Astrophysics Data System (ADS)

    Solaro, Christopher R.; Lingle, Christopher J.

    1992-09-01

    Most calcium-activated potassium channels couple changes in intracellular calcium to membrane excitability by conducting a current with a probability that depends directly on submembrane calcium concentration. In rat adrenal chromaffin cells, however, a large conductance, voltage- and calcium-activated potassium channel (BK) undergoes rapid inactivation, suggesting that this channel has a physiological role different than that of other BK channels. The inactivation of the BK channel, like that of the voltage-gated Shaker B potassium channel, is removed by trypsin digestion and channels are blocked by the Shaker B amino-terminal inactivating domain. Thus, this BK channel shares functional and possibly structural homologies with other inactivating voltage-gated potassium channels.

  13. Mechanosensitive channels of Escherichia coli: the MscL gene, protein, and activities

    NASA Technical Reports Server (NTRS)

    Sukharev, S. I.; Blount, P.; Martinac, B.; Kung, C.

    1997-01-01

    Although mechanosensory responses are ubiquitous and diverse, the molecular bases of mechanosensation in most cases remain mysterious MscL, a mechanosensitive channel of large conductance of Escherichia coli and its bacterial homologues are the first and currently only channel molecules shown to directly sense mechanical stretch of the membrane. In response to the tension conveyed via the lipid bilayer, MscL increases its open probability by several orders of magnitude. In the present review we describe the identification, cloning, and first sets of biophysical and structural data on this simplest mechanosensory molecule. We discovered a 2.5-ns mechanosensitive conductance in giant E. coli spheroplasts. Using chromatographies to enrich the target and patch clamp to assay the channel activity in liposome-reconstituted fractions, we identified the MscL protein and cloned the mscL gene. MscL comprises 136 amino acid residues (15 kDa), with two highly hydrophobic regions, and resides in the inner membrane of the bacterium. PhoA-fusion experiments indicate that the protein spans the membrane twice with both termini in the cytoplasm. Spectroscopic techniques show that it is highly helical. Expression of MscL tandems and covalent cross-linking suggest that the active channel complex is a homo-hexamer. We have identified several residues, which when deleted or substituted, affect channel kinetics or mechanosensitivity. Although unique when discovered, highly conserved MscL homologues in both gram-negative and gram-positive bacteria have been found, suggesting their ubiquitous importance among bacteria.

  14. Proteolytic regulation of epithelial sodium channels by urokinase plasminogen activator: cutting edge and cleavage sites.

    PubMed

    Ji, Hong-Long; Zhao, Runzhen; Komissarov, Andrey A; Chang, Yongchang; Liu, Yongfeng; Matthay, Michael A

    2015-02-27

    Plasminogen activator inhibitor 1 (PAI-1) level is extremely elevated in the edematous fluid of acutely injured lungs and pleurae. Elevated PAI-1 specifically inactivates pulmonary urokinase-type (uPA) and tissue-type plasminogen activators (tPA). We hypothesized that plasminogen activation and fibrinolysis may alter epithelial sodium channel (ENaC) activity, a key player in clearing edematous fluid. Two-chain urokinase (tcuPA) has been found to strongly stimulate heterologous human αβγ ENaC activity in a dose- and time-dependent manner. This activity of tcuPA was completely ablated by PAI-1. Furthermore, a mutation (S195A) of the active site of the enzyme also prevented ENaC activation. By comparison, three truncation mutants of the amino-terminal fragment of tcuPA still activated ENaC. uPA enzymatic activity was positively correlated with ENaC current amplitude prior to reaching the maximal level. In sharp contrast to uPA, neither single-chain tPA nor derivatives, including two-chain tPA and tenecteplase, affected ENaC activity. Furthermore, γ but not α subunit of ENaC was proteolytically cleaved at ((177)GR↓KR(180)) by tcuPA. In summary, the underlying mechanisms of urokinase-mediated activation of ENaC include release of self-inhibition, proteolysis of γ ENaC, incremental increase in opening rate, and activation of closed (electrically "silent") channels. This study for the first time demonstrates multifaceted mechanisms for uPA-mediated up-regulation of ENaC, which form the cellular and molecular rationale for the beneficial effects of urokinase in mitigating mortal pulmonary edema and pleural effusions. PMID:25555911

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

    SciTech Connect

    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.

  16. Impact of calcium-activated potassium channels on NMDA spikes in cortical layer 5 pyramidal neurons.

    PubMed

    Bock, Tobias; Stuart, Greg J

    2016-03-01

    Active electrical events play an important role in shaping signal processing in dendrites. As these events are usually associated with an increase in intracellular calcium, they are likely to be under the control of calcium-activated potassium channels. Here, we investigate the impact of calcium-activated potassium channels onN-methyl-d-aspartate (NMDA) receptor-dependent spikes, or NMDA spikes, evoked by glutamate iontophoresis onto basal dendrites of cortical layer 5 pyramidal neurons. We found that small-conductance calcium-activated potassium channels (SK channels) act to reduce NMDA spike amplitude but at the same time, also decrease the iontophoretic current required for their generation. This SK-mediated decrease in NMDA spike threshold was dependent on R-type voltage-gated calcium channels and indicates a counterintuitive, excitatory effect of SK channels on NMDA spike generation, whereas the capacity of SK channels to suppress NMDA spike amplitude is in line with the expected inhibitory action of potassium channels on dendritic excitability. Large-conductance calcium-activated potassium channels had no significant impact on NMDA spikes, indicating that these channels are either absent from basal dendrites or not activated by NMDA spikes. These experiments reveal complex and opposing interactions among NMDA receptors, SK channels, and voltage-gated calcium channels in basal dendrites of cortical layer 5 pyramidal neurons during NMDA spike generation, which are likely to play an important role in regulating the way these neurons integrate the thousands of synaptic inputs they receive. PMID:26936985

  17. Alternatively Spliced Isoforms of KV10.1 Potassium Channels Modulate Channel Properties and Can Activate Cyclin-dependent Kinase in Xenopus Oocytes*

    PubMed Central

    Ramos Gomes, Fernanda; Romaniello, Vincenzo; Sánchez, Araceli; Weber, Claudia; Narayanan, Pratibha; Psol, Maryna; Pardo, Luis A.

    2015-01-01

    KV10.1 is a voltage-gated potassium channel expressed selectively in the mammalian brain but also aberrantly in cancer cells. In this study we identified short splice variants of KV10.1 resulting from exon-skipping events (E65 and E70) in human brain and cancer cell lines. The presence of the variants was confirmed by Northern blot and RNase protection assays. Both variants completely lacked the transmembrane domains of the channel and produced cytoplasmic proteins without channel function. In a reconstituted system, both variants co-precipitated with the full-length channel and induced a robust down-regulation of KV10.1 current when co-expressed with the full-length form, but their effect was mechanistically different. E65 required a tetramerization domain and induced a reduction in the overall expression of full-length KV10.1, whereas E70 mainly affected its glycosylation pattern. E65 triggered the activation of cyclin-dependent kinases in Xenopus laevis oocytes, suggesting a role in cell cycle control. Our observations highlight the relevance of noncanonical functions for the oncogenicity of KV10.1, which need to be considered when ion channels are targeted for cancer therapy. PMID:26518875

  18. Alternatively Spliced Isoforms of KV10.1 Potassium Channels Modulate Channel Properties and Can Activate Cyclin-dependent Kinase in Xenopus Oocytes.

    PubMed

    Ramos Gomes, Fernanda; Romaniello, Vincenzo; Sánchez, Araceli; Weber, Claudia; Narayanan, Pratibha; Psol, Maryna; Pardo, Luis A

    2015-12-18

    KV10.1 is a voltage-gated potassium channel expressed selectively in the mammalian brain but also aberrantly in cancer cells. In this study we identified short splice variants of KV10.1 resulting from exon-skipping events (E65 and E70) in human brain and cancer cell lines. The presence of the variants was confirmed by Northern blot and RNase protection assays. Both variants completely lacked the transmembrane domains of the channel and produced cytoplasmic proteins without channel function. In a reconstituted system, both variants co-precipitated with the full-length channel and induced a robust down-regulation of KV10.1 current when co-expressed with the full-length form, but their effect was mechanistically different. E65 required a tetramerization domain and induced a reduction in the overall expression of full-length KV10.1, whereas E70 mainly affected its glycosylation pattern. E65 triggered the activation of cyclin-dependent kinases in Xenopus laevis oocytes, suggesting a role in cell cycle control. Our observations highlight the relevance of noncanonical functions for the oncogenicity of KV10.1, which need to be considered when ion channels are targeted for cancer therapy. PMID:26518875

  19. Calcium-activated potassium channels mask vascular dysfunction associated with oxidized LDL exposure in rabbit aorta.

    PubMed

    Bocker, J M; Miller, F J; Oltman, C L; Chappell, D A; Gutterman, D D

    2001-05-01

    Endothelium-dependent vasodilation is impaired in atherosclerosis. Oxidized low density lipoprotein (ox-LDL) plays an important role, possibly through alterations in G-protein activation. We examined the effect of acute exposure to ox-LDL on the dilator responses of isolated rabbit aorta segments. We sought also to evaluate the specificity of this dysfunction for dilator stimuli that traditionally operate through a Gi-protein mechanism. Aortic segments were prepared for measurement of isometric tension. After contraction with prostaglandin F2alpha, relaxation to thrombin, adenosine diphosphate (ADP), or the endothelium-independent agonists, sodium nitroprusside (SNP) or papaverine was examined. Maximal relaxation to thrombin was impaired in the presence of ox-LDL (17.7+/-3.7% p<0.05) compared to control (no LDL) (52.6+/-4.0%). Ox-LDL did not affect maximal relaxation to ADP or SNP. However, in the presence of charybdotoxin (CHTX: calcium-activated potassium channel inhibitor) ox-LDL impaired relaxation to ADP (17.4+/-3.2%). CHTX did not affect control (no LDL) responses to ADP (69.6+/-5.0%) or relaxation to thrombin or papaverine. In conclusion, ox-LDL impairs relaxation to thrombin, but in the case of ADP, calcium-activated potassium channels compensate to maintain this relaxation. PMID:11605770

  20. Calcium ions open a selectivity filter gate during activation of the MthK potassium channel

    NASA Astrophysics Data System (ADS)

    Posson, David J.; Rusinova, Radda; Andersen, Olaf S.; Nimigean, Crina M.

    2015-09-01

    Ion channel opening and closing are fundamental to cellular signalling and homeostasis. Gates that control K+ channel activity were found both at an intracellular pore constriction and within the selectivity filter near the extracellular side but the specific location of the gate that opens Ca2+-activated K+ channels has remained elusive. Using the Methanobacterium thermoautotrophicum homologue (MthK) and a stopped-flow fluorometric assay for fast channel activation, we show that intracellular quaternary ammonium blockers bind to closed MthK channels. Since the blockers are known to bind inside a central channel cavity, past the intracellular entryway, the gate must be within the selectivity filter. Furthermore, the blockers access the closed channel slower than the open channel, suggesting that the intracellular entryway narrows upon pore closure, without preventing access of either the blockers or the smaller K+. Thus, Ca2+-dependent gating in MthK occurs at the selectivity filter with coupled movement of the intracellular helices.

  1. Calcineurin enhances L-type Ca(2+) channel activity in hippocampal neurons: increased effect with age in culture.

    PubMed

    Norris, C M; Blalock, E M; Chen, K-C; Porter, N M; Landfield, P W

    2002-01-01

    The Ca(2+)/calmodulin-dependent protein phosphatase, calcineurin, modulates a number of key Ca(2+) signaling pathways in neurons, and has been implicated in Ca(2+)-dependent negative feedback inactivation of N-methyl-D-aspartate receptors and voltage-sensitive Ca(2+) channels. In contrast, we report here that three mechanistically disparate calcineurin inhibitors, FK-506, cyclosporin A, and the calcineurin autoinhibitory peptide, inhibited high-voltage-activated Ca(2+) channel currents by up to 40% in cultured hippocampal neurons, suggesting that calcineurin acts to enhance Ca(2+) currents. This effect occurred with Ba(2+) or Ca(2+) as charge carrier, and with or without intracellular Ca(2+) buffered by EGTA. Ca(2+)-dependent inactivation of Ca(2+) channels was not affected by FK-506. The immunosuppressant, rapamycin, and the protein phosphatase 1/2A inhibitor, okadaic acid, did not decrease Ca(2+) channel current, showing specificity for effects on calcineurin. Blockade of L-type Ca(2+) channels with nimodipine fully negated the effect of FK-506 on Ca(2+) channel current, while blockade of N-, and P-/Q-type Ca(2+) channels enhanced FK-506-mediated inhibition of the remaining L-type-enriched current. FK-506 also inhibited substantially more Ca(2+) channel current in 4-week-old vs. 2-week-old cultures, an effect paralleled by an increase in calcineurin A mRNA levels. These studies provide the first evidence that calcineurin selectively enhances L-type Ca(2+) channel activity in neurons. Moreover, this action appears to be increased concomitantly with the well-characterized increase in L-type Ca(2+) channel availability in hippocampal neurons with age-in-culture. PMID:11958864

  2. Keeping active channels in their place: membrane phosphoinositides regulate TRPM channel activity in a compartment-selective manner.

    PubMed

    Braun, Andrew P

    2012-01-01

    We have long appreciated that the controlled movement of ions and solutes across the cell surface or plasma membrane affects every aspect of cell function, ranging from membrane excitability to metabolism to secretion, and is also critical for the long-term maintenance of cell viability. Studies examining these physiological transport processes have revealed a vast array of ion channels, transporters and ATPase-driven pumps that underlie these transmembrane ionic movements and how acquired or genetic disruption of these processes are linked to disease. More recently, it has become evident that the ongoing function of intracellular organelles and subcellular compartments also depends heavily on the controlled movement of ions to establish distinct pH or ionic environments. However, limited experimental access to these subcellular domains/structures has hampered scientific progress in this area, due in large part to the difficulty of applying proven functional assays, such as patch clamp and radiotracer methodologies, to these specialized membrane locations. Using both functional and immune-labeling assays, we now know that the types and complement of channels, transporters and pumps located within intracellular membranes and organelles often differ from those present on the plasma membrane. Moreover, it appears that this differential distribution is due to the presence of discrete tags/signals present within these transport proteins that dictate their sorting/trafficking to spatially discrete membrane compartments, where they may also interact with scaffolding proteins that help maintain their localization. Such targeting signals may thus operate in a manner analogous to the way a postal code is used to direct the delivery of a letter. PMID:23151432

  3. Severe acute respiratory syndrome coronavirus envelope protein ion channel activity promotes virus fitness and pathogenesis.

    PubMed

    Nieto-Torres, Jose L; DeDiego, Marta L; Verdiá-Báguena, Carmina; Jimenez-Guardeño, Jose M; Regla-Nava, Jose A; Fernandez-Delgado, Raul; Castaño-Rodriguez, Carlos; Alcaraz, Antonio; Torres, Jaume; Aguilella, Vicente M; Enjuanes, Luis

    2014-05-01

    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-CoV virulence. PMID:24788150

  4. Severe Acute Respiratory Syndrome Coronavirus Envelope Protein Ion Channel Activity Promotes Virus Fitness and Pathogenesis

    PubMed Central

    Nieto-Torres, Jose L.; DeDiego, Marta L.; Verdiá-Báguena, Carmina; Jimenez-Guardeño, Jose M.; Regla-Nava, Jose A.; Fernandez-Delgado, Raul; Castaño-Rodriguez, Carlos; Alcaraz, Antonio; Torres, Jaume; Aguilella, Vicente M.; Enjuanes, Luis

    2014-01-01

    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-CoV virulence. PMID:24788150

  5. Pitch underlies activation of the vocal system during affective vocalization.

    PubMed

    Belyk, Michel; Brown, Steven

    2016-07-01

    Affective prosody is that aspect of speech that conveys a speaker's emotional state through modulations in various vocal parameters, most prominently pitch. While a large body of research implicates the cingulate vocalization area in controlling affective vocalizations in monkeys, no systematic test of functional homology for this area has yet been reported in humans. In this study, we used functional magnetic resonance imaging to compare brain activations when subjects produced affective vocalizations in the form of exclamations vs non-affective vocalizations with similar pitch contours. We also examined the perception of affective vocalizations by having participants make judgments about either the emotions being conveyed by recorded affective vocalizations or the pitch contours of the same vocalizations. Production of affective vocalizations and matched pitch contours activated a highly overlapping set of brain areas, including the larynx-phonation area of the primary motor cortex and a region of the anterior cingulate cortex that is consistent with the macro-anatomical position of the cingulate vocalization area. This overlap contradicts the dominant view that these areas form two distinct vocal pathways with dissociable functions. Instead, we propose that these brain areas are nodes in a single vocal network, with an emphasis on pitch modulation as a vehicle for affective expression. PMID:26078385

  6. Regulation of epithelial sodium channels in urokinase plasminogen activator deficiency

    PubMed Central

    Chen, Zaixing; Zhao, Runzhen; Zhao, Meimi; Liang, Xinrong; Bhattarai, Deepa; Dhiman, Rohan; Shetty, Sreerama; Idell, Steven

    2014-01-01

    Epithelial sodium channels (ENaC) govern transepithelial salt and fluid homeostasis. ENaC contributes to polarization, apoptosis, epithelial-mesenchymal transformation, etc. Fibrinolytic proteases play a crucial role in virtually all of these processes and are elaborated by the airway epithelium. We hypothesized that urokinase-like plasminogen activator (uPA) regulates ENaC function in airway epithelial cells and tested that possibility in primary murine tracheal epithelial cells (MTE). Both basal and cAMP-activated Na+ flow through ENaC were significantly reduced in monolayers of uPA-deficient cells. The reduction in ENaC activity was further confirmed in basolateral membrane-permeabilized cells. A decrease in the Na+-K+-ATPase activity in the basolateral membrane could contribute to the attenuation of ENaC function in intact monolayer cells. Dysfunctional fluid resolution was seen in uPA-disrupted cells. Administration of uPA and plasmin partially restores ENaC activity and fluid reabsorption by MTEs. ERK1/2, but not Akt, phosphorylation was observed in the cells and lungs of uPA-deficient mice. On the other hand, cleavage of γ ENaC is significantly depressed in the lungs of uPA knockout mice vs. those of wild-type controls. Expression of caspase 8, however, did not differ between wild-type and uPA−/− mice. In addition, uPA deficiency did not alter transepithelial resistance. Taken together, the mechanisms for the regulation of ENaC by uPA in MTEs include augmentation of Na+-K+-ATPase, proteolysis, and restriction of ERK1/2 phosphorylation. We demonstrate for the first time that ENaC may serve as a downstream signaling target by which uPA controls the biophysical profiles of airway fluid and epithelial function. PMID:25172911

  7. MEG brain activities reflecting affection for visual food stimuli.

    PubMed

    Kuriki, Shinya; Miyamura, Takahiro; Uchikawa, Yoshinori

    2010-01-01

    This study aimed to explore the modulation of alpha rhythm in response to food pictures with distinct affection values. We examined the method to discriminate subject's state, i.e., whether he/she liked the article of food or not, from MEG signals detected over the head. Pictures of familiar foods were used as affective stimuli, while those pictures with complementary color phase were used as non-affective stimuli. Alpha band signals in a narrow frequency window around the spectral peak of individual subjects were wavelet analyzed and phase-locked component to the stimulus onset was obtained as a complex number. The amplitude of the phase-locked component was averaged during 0-1 s after stimulus onset for 30 epochs in a measurement session and across 76 channels of MEG sensor. In statistical test of individual subjects, significant difference was found in the real part of the averaged phase-locked amplitude between the normal-color and reverse-color pictures. These results suggest that affective information processing of food pictures is reflected in the synchronized component of narrow band alpha rhythm. PMID:21096510

  8. Channel

    NASA Technical Reports Server (NTRS)

    2006-01-01

    [figure removed for brevity, see original site] Context image for PIA03693 Channel

    This channel is located south of Iani Chaos.

    Image information: VIS instrument. Latitude -10.9N, Longitude 345.5E. 17 meter/pixel resolution.

    Note: this THEMIS visual image has not been radiometrically nor geometrically calibrated for this preliminary release. An empirical correction has been performed to remove instrumental effects. A linear shift has been applied in the cross-track and down-track direction to approximate spacecraft and planetary motion. Fully calibrated and geometrically projected images will be released through the Planetary Data System in accordance with Project policies at a later time.

    NASA's Jet Propulsion Laboratory manages the 2001 Mars Odyssey mission for NASA's Office of Space Science, Washington, D.C. The Thermal Emission Imaging System (THEMIS) was developed by Arizona State University, Tempe, in collaboration with Raytheon Santa Barbara Remote Sensing. The THEMIS investigation is led by Dr. Philip Christensen at Arizona State University. Lockheed Martin Astronautics, Denver, is the prime contractor for the Odyssey project, and developed and built the orbiter. Mission operations are conducted jointly from Lockheed Martin and from JPL, a division of the California Institute of Technology in Pasadena.

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

    ERIC Educational Resources Information Center

    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…

  10. Activation of raphe nuclei triggers rapid and distinct effects on parallel olfactory bulb output channels.

    PubMed

    Kapoor, Vikrant; Provost, Allison C; Agarwal, Prateek; Murthy, Venkatesh N

    2016-02-01

    The serotonergic raphe nuclei are involved in regulating brain states over timescales of minutes and hours. We examined more rapid effects of raphe activation on two classes of principal neurons in the mouse olfactory bulb, mitral and tufted cells, which send olfactory information to distinct targets. Brief stimulation of the raphe nuclei led to excitation of tufted cells at rest and potentiation of their odor responses. While mitral cells at rest were also excited by raphe activation, their odor responses were bidirectionally modulated, leading to improved pattern separation of odors. In vitro whole-cell recordings revealed that specific optogenetic activation of raphe axons affected bulbar neurons through dual release of serotonin and glutamate. Therefore, the raphe nuclei, in addition to their role in neuromodulation of brain states, are also involved in fast, sub-second top-down modulation similar to cortical feedback. This modulation can selectively and differentially sensitize or decorrelate distinct output channels. PMID:26752161

  11. Activation of raphe nuclei triggers rapid and distinct effects on parallel olfactory bulb output channels

    PubMed Central

    Kapoor, Vikrant; Provost, Allison; Agarwal, Prateek; Murthy, Venkatesh N.

    2015-01-01

    The serotonergic raphe nuclei are involved in regulating brain states over time-scales of minutes and hours. We examined more rapid effects of serotonergic activation on two classes of principal neurons in the mouse olfactory bulb, mitral and tufted cells, which send olfactory information to distinct targets. Brief stimulation of the raphe nuclei led to excitation of tufted cells at rest and potentiation of their odor responses. While mitral cells at rest were also excited by raphe activation, their odor responses were bidirectionally modulated, leading to improved pattern separation of odors. In vitro whole-cell recordings revealed that specific optogenetic activation of raphe axons affected bulbar neurons through dual release of serotonin and glutamate. Therefore, the raphe nuclei, in addition to their role in neuromodulation of brain states, are also involved in fast, sub-second top-down modulation, similar to cortical feedback. This modulation can selectively and differentially sensitize or decorrelate distinct output channels. PMID:26752161

  12. Arachidonic acid activation of a new family of K+ channels in cultured rat neuronal cells.

    PubMed Central

    Kim, D; Sladek, C D; Aguado-Velasco, C; Mathiasen, J R

    1995-01-01

    1. The presence and properties of K+ channels activated by arachidonic acid were studied in neuronal cells cultured from the mesencephalic and hypothalamic areas of rat brain. 2. Arachidonic acid produced a concentration-dependent (5-50 microM) and reversible activation of whole-cell currents. 3. In excised membrane patches, arachidonic acid applied to the cytoplasmic or extracellular side of the membrane caused opening of three types of channels whose current-voltage relationships were slightly outwardly rectifying, inwardly rectifying and linear, and whose single channel slope conductances at +60 mV were 143, 45 and 52 pS, respectively. 4. All three currents were K+ selective and blocked by 2 mM Ba2+ but not by other K+ channel blockers such as tetraethylammonium chloride, 4-aminopyridine and quinidine. The outwardly and inwardly rectifying currents were slightly voltage dependent with higher channel activity at more depolarized potentials. 5. Arachidonic acid activated the K+ channels in cells treated with cyclo-oxygenase and lipoxygenase inhibitors (indomethacin and nordihydroguaiaretic acid), indicating that arachidonic acid itself can directly activate the channels. Alcohol and methyl ester derivatives of arachidonic acid failed to activate the K+ channels, indicating that the charged carboxyl group is important for activation. 6. Certain unsaturated fatty acids (linoleic, linolenic and docosahexaenoic acids), but not saturated fatty acids (myristic, palmitic, stearic acids), also reversibly activated all three types of K+ channel. 7. All three K+ channels were activated by pressure applied to the membrane (i.e. channels were stretch sensitive) with a half-maximal pressure of approximately 18 mmHg. The K+ channels were not blocked by 100 microM GdCl3. 8. A decrease in intracellular pH (over the range 5.6-7.2) caused a reversible, pH-dependent increase in channel activity whether the channel was initially activated by arachidonic acid or stretch. 9. Glutamate

  13. In vitro detection of cardiotoxins or neurotoxins affecting ion channels or pumps using beating cardiomyocytes as alternative for animal testing.

    PubMed

    Nicolas, Jonathan; Hendriksen, Peter J M; de Haan, Laura H J; Koning, Rosella; Rietjens, Ivonne M C M; Bovee, Toine F H

    2015-03-01

    The present study investigated if and to what extent murine stem cell-derived beating cardiomyocytes within embryoid bodies can be used as a broad screening in vitro assay for neurotoxicity testing, replacing for example in vivo tests for marine neurotoxins. Effect of nine model compounds, acting on either the Na(+), K(+), or Ca(2+) channels or the Na(+)/K(+) ATP-ase pump, on the beating was assessed. Diphenhydramine, veratridine, isradipine, verapamil and ouabain induced specific beating arrests that were reversible and none of the concentrations tested induced cytotoxicity. Three K(+) channel blockers, amiodarone, clofilium and sematilide, and the Na(+)/K(+) ATPase pump inhibitor digoxin had no specific effect on the beating. In addition, two marine neurotoxins i.e. saxitoxin and tetrodotoxin elicited specific beating arrests in cardiomyocytes. Comparison of the results obtained with cardiomyocytes to those obtained with the neuroblastoma neuro-2a assay revealed that the cardiomyocytes were generally somewhat more sensitive for the model compounds affecting Na(+) and Ca(2+) channels, but less sensitive for the compounds affecting K(+) channels. The stem cell-derived cardiomyocytes were not as sensitive as the neuroblastoma neuro-2a assay for saxitoxin and tetrodotoxin. It is concluded that the murine stem cell-derived beating cardiomyocytes provide a sensitive model for detection of specific neurotoxins and that the neuroblastoma neuro-2a assay may be a more promising cell-based assay for the screening of marine biotoxins. PMID:25479353

  14. GABA/sub B/ receptor activation inhibits Ca/sup 2 +/-activated potassium channels in synaptosomes: involvement of G-proteins

    SciTech Connect

    Ticku, M.K.; Delgado, A.

    1989-01-01

    /sup 86/Rb-efflux assay from preloaded synaptosomes of rat cerebral cortex was developed to study the effect of GABA/sub B/ receptor agonist baclofen on Ca/sup 2 +/-activated K/sup +/-channels. Depolarization of /sup 86/Rb-loaded synaptosomes in physiological buffer increased Ca/sup 2 +/-activated /sup 86/Rb-efflux by 400%. The /sup 86/Rb-efflux was blocked by quinine sulfate, tetraethylammonium, and La/sup 3 +/ indicating the involvement of Ca/sup 2 +/-activated K/sup +/-channels. (-)Baclofen inhibited Ca/sup 2 +/-activated /sup 86/Rb-efflux in a stereospecific manner. The inhibitory effect of (-)baclofen was mediated by GABA/sub B/ receptor activation, since it was blocked by GABA/sub B/ antagonist phaclofen, but not by bicuculline. Further, pertussis toxin also blocked the ability of baclofen or depolarizing action to affect Ca/sup 2 +/-activated K/sup +/-channels. These results suggest that baclofen inhibits Ca/sup 2 +/-activated K/sup +/-channels in synaptosomes and these channels are regulated by G-proteins. This assay may provide an ideal in vitro model to study GABA/sub B/ receptor pharmacology.

  15. Point mutations in the pore region directly or indirectly affect glibenclamide block of the CFTR chloride channel.

    PubMed

    Gupta, Jyoti; Linsdell, Paul

    2002-03-01

    The sulfonylurea glibenclamide is a relatively potent inhibitor of the CFTR Cl(-) channel. This inhibition is thought to be via an open channel block mechanism. However, nothing is known about the physical nature of the glibenclamide-binding site on CFTR. Here we show that mutations in the pore-forming 6th and 12th transmembrane regions of CFTR affect block by intracellular glibenclamide, confirming previous suggestions that glibenclamide enters the pore in order to block the channel. Two mutations in the 6th transmembrane region, F337A and T338A, significantly weakened glibenclamide block, consistent with a direct interaction between glibenclamide and this region of the pore. Interestingly, two mutations in the 12th transmembrane region (N1138A and T1142A) significantly strengthened block. These two mutations also abolished the dependence of block on the extracellular Cl(-) concentration, which in wild-type CFTR suggests an interaction between Cl(-) and glibenclamide within the channel pore that limits block. We suggest that mutations in the 12th transmembrane region strengthen glibenclamide block not by directly altering interactions between glibenclamide and the pore walls, but indirectly by reducing interactions between Cl(-) ions and glibenclamide within the pore. This work demonstrates that glibenclamide binds within the CFTR channel pore and begins to define its intrapore binding site. PMID:11889571

  16. Pupil Size Changes as an Active Information Channel for Biofeedback Applications.

    PubMed

    Ehlers, Jan; Strauch, Christoph; Georgi, Juliane; Huckauf, Anke

    2016-09-01

    Pupil size is usually regarded as a passive information channel that provides insight into cognitive and affective states but defies any further control. However, in a recent study (Ehlers et al. 2015) we demonstrate that sympathetic activity indexed by pupil dynamics allows strategic interference by means of simple cognitive techniques. Utilizing positive/negative imaginings, subjects were able to expand pupil diameter beyond baseline variations; albeit with varying degrees of success and only over brief periods. The current study provides a comprehensive replication on the basis of considerable changes to the experimental set-up. Results show that stricter methodological conditions (controlled baseline settings and specified user instructions) strengthen the reported effect, whereas overall performance increases by one standard deviation. Effects are thereby not restricted to pupillary level. Parallel recordings of skin conductance changes prove a general enhancement of induced autonomic arousal. Considering the stability of the results across studies, we conclude that pupil size information exceeds affective monitoring and may constitute an active input channel in human-computer interaction. Furthermore, since variations in pupil diameter reliably display self-induced changes in sympathetic arousal, the relevance of this parameter is strongly indicated for future approaches in clinical biofeedback. PMID:27113096

  17. How Will Climate Change Affect Channel Morphology and Salmonid Habitat in Mountain Basins?

    NASA Astrophysics Data System (ADS)

    Buffington, J. M.; Goode, J.

    2010-12-01

    Riverine habitat for salmonids is intimately linked to channel morphology and fluvial processes (channel hydraulics, sediment transport and scour regime) which are, in turn, controlled by watershed hydrology and erosional processes that input sediment to the fluvial system. Climate change has the potential to alter the timing, magnitude, and style of sediment and water inputs to mountain rivers. Channel response to these changes may range from small-scale adjustments of channel characteristics (e.g., width, depth, grain size, scour depth) to larger-scale changes in channel type (e.g., metamorphosis from a pool-riffle channel to a plane-bed morphology). Identifying which parts of the river network will remain relatively stable in response to climate change, and which are likely to cross critical morphologic and scour thresholds is important for predicting effects on salmonid populations. Toward this end, a regime framework is presented for predicting the relative degree of morphologic stability and scour potential in different physiographic settings (different water and sediment regimes). Digital elevation models are used to explore the spatial distribution of these conditions and potential consequences for salmonid habitat across the landscape. Results suggest that the potential for scour and morphologic variability are strongly influenced by hydroclimate; snowmelt channels are relatively stable across floods of different magnitude, while rainfall-dominated channels are more variable and less stable. Transitional changes in hydrologic regime (mixed rain and snow) have the greatest potential for altering geomorphic conditions and salmonid habitat. However, the vulnerability of salmonids to climate-driven changes in scour regime depend on the species and its life history (i.e., depth to which eggs are buried and timing of incubation relative to scouring flows). Overall, the regime approach provides a useful first-order assessment of channel condition and response

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

    NASA Astrophysics Data System (ADS)

    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.

  19. Corrugator activity confirms immediate negative affect in surprise.

    PubMed

    Topolinski, Sascha; Strack, Fritz

    2015-01-01

    The emotion of surprise entails a complex of immediate responses, such as cognitive interruption, attention allocation to, and more systematic processing of the surprising stimulus. All these processes serve the ultimate function to increase processing depth and thus cognitively master the surprising stimulus. The present account introduces phasic negative affect as the underlying mechanism responsible for this switch in operating mode. Surprising stimuli are schema-discrepant and thus entail cognitive disfluency, which elicits immediate negative affect. This affect in turn works like a phasic cognitive tuning switching the current processing mode from more automatic and heuristic to more systematic and reflective processing. Directly testing the initial elicitation of negative affect by surprising events, the present experiment presented high and low surprising neutral trivia statements to N = 28 participants while assessing their spontaneous facial expressions via facial electromyography. High compared to low surprising trivia elicited higher corrugator activity, indicative of negative affect and mental effort, while leaving zygomaticus (positive affect) and frontalis (cultural surprise expression) activity unaffected. Future research shall investigate the mediating role of negative affect in eliciting surprise-related outcomes. PMID:25762956

  20. Corrugator activity confirms immediate negative affect in surprise

    PubMed Central

    Topolinski, Sascha; Strack, Fritz

    2015-01-01

    The emotion of surprise entails a complex of immediate responses, such as cognitive interruption, attention allocation to, and more systematic processing of the surprising stimulus. All these processes serve the ultimate function to increase processing depth and thus cognitively master the surprising stimulus. The present account introduces phasic negative affect as the underlying mechanism responsible for this switch in operating mode. Surprising stimuli are schema-discrepant and thus entail cognitive disfluency, which elicits immediate negative affect. This affect in turn works like a phasic cognitive tuning switching the current processing mode from more automatic and heuristic to more systematic and reflective processing. Directly testing the initial elicitation of negative affect by surprising events, the present experiment presented high and low surprising neutral trivia statements to N = 28 participants while assessing their spontaneous facial expressions via facial electromyography. High compared to low surprising trivia elicited higher corrugator activity, indicative of negative affect and mental effort, while leaving zygomaticus (positive affect) and frontalis (cultural surprise expression) activity unaffected. Future research shall investigate the mediating role of negative affect in eliciting surprise-related outcomes. PMID:25762956

  1. GlialCAM, a CLC-2 Cl(-) channel subunit, activates the slow gate of CLC chloride channels.

    PubMed

    Jeworutzki, Elena; Lagostena, Laura; Elorza-Vidal, Xabier; López-Hernández, Tania; Estévez, Raúl; Pusch, Michael

    2014-09-01

    GlialCAM, a glial cell adhesion molecule mutated in megalencephalic leukoencephalopathy with subcortical cysts, targets the CLC-2 Cl(-) channel to cell contacts in glia and activates CLC-2 currents in vitro and in vivo. We found that GlialCAM clusters all CLC channels at cell contacts in vitro and thus studied GlialCAM interaction with CLC channels to investigate the mechanism of functional activation. GlialCAM slowed deactivation kinetics of CLC-Ka/barttin channels and increased CLC-0 currents opening the common gate and slowing its deactivation. No functional effect was seen for common gate deficient CLC-0 mutants. Similarly, GlialCAM targets the common gate deficient CLC-2 mutant E211V/H816A to cell contacts, without altering its function. Thus, GlialCAM is able to interact with all CLC channels tested, targeting them to cell junctions and activating them by stabilizing the open configuration of the common gate. These results are important to better understand the physiological role of GlialCAM/CLC-2 interaction. PMID:25185546

  2. GlialCAM, a CLC-2 Cl- Channel Subunit, Activates the Slow Gate of CLC Chloride Channels

    PubMed Central

    Jeworutzki, Elena; Lagostena, Laura; Elorza-Vidal, Xabier; López-Hernández, Tania; Estévez, Raúl; Pusch, Michael

    2014-01-01

    GlialCAM, a glial cell adhesion molecule mutated in megalencephalic leukoencephalopathy with subcortical cysts, targets the CLC-2 Cl- channel to cell contacts in glia and activates CLC-2 currents in vitro and in vivo. We found that GlialCAM clusters all CLC channels at cell contacts in vitro and thus studied GlialCAM interaction with CLC channels to investigate the mechanism of functional activation. GlialCAM slowed deactivation kinetics of CLC-Ka/barttin channels and increased CLC-0 currents opening the common gate and slowing its deactivation. No functional effect was seen for common gate deficient CLC-0 mutants. Similarly, GlialCAM targets the common gate deficient CLC-2 mutant E211V/H816A to cell contacts, without altering its function. Thus, GlialCAM is able to interact with all CLC channels tested, targeting them to cell junctions and activating them by stabilizing the open configuration of the common gate. These results are important to better understand the physiological role of GlialCAM/CLC-2 interaction. PMID:25185546

  3. Abnormal activation of potassium channels in aortic smooth muscle of rats with peritonitis-induced septic shock.

    PubMed

    Kuo, Jiunn-Horng; Chen, Shiu-Jen; Shih, Chih-Chin; Lue, Wei-Ming; Wu, Chin-Chen

    2009-07-01

    This study was conducted to examine the role of membrane hyperpolarization in mediating vascular hyporeactivity induced by cecal ligation and puncture (CLP) in endothelial-denuded strips of rat thoracic aorta ex vivo. The CLP for 18 h elicited a significant fall of blood pressure and a severe vascular hyporeactivity to norepinephrine as seen in severe sepsis. At the end of the in vivo experiments, thoracic aortas were removed from both CLP-treated and control rats. After removal of the endothelium, aortic segments were mounted in myographs for the recording of isometric tension and smooth muscle membrane potential. The membrane potential recording showed that a hyperpolarization was observed in the CLP-treated rats when compared with the control rats. This hyperpolarization was reversed by iberiotoxin (a large-conductance Ca2+-activated K+ channel blocker), 4-aminopyridine (a voltage-dependent K+ channel blocker), barium (an inward rectifier K+ channels blocker), N-(1-adamantyl)-N'-cyclohexyl-4-morpholinecarboxamidine hydrochloride (a pore-forming blocker of adenosine triphosphate (ATP)-sensitive K+ channels [KATP]), or methylene blue (a nonspecific guanylyl cyclase [GC] inhibitor). However, this hyperpolarization was not significantly affected by apamin (a small-conductance Ca2+-activated K+ channel blocker), glibenclamide (a sulfonylurea blocker of KATP), N(omega)-nitro-L-arginine methyl ester (a NOS inhibitor), or 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (an NO-sensitive GC inhibitor). In addition, the basal tension of the tissues obtained from CLP rats was increased simultaneously, whereas membrane potential was reversed. In contrast, none of these inhibitors had significant effects on the membrane potential or the basal tension in control tissues. Thus, we provide electrophysiological and functional evidence demonstrating that an abnormal activation of K+ channels in vascular smooth muscle in animals with septic shock induced by CLP. Our observations

  4. Long-range forces affecting equilibrium inertial focusing behavior in straight high aspect ratio microfluidic channels

    NASA Astrophysics Data System (ADS)

    Reece, Amy E.; Oakey, John

    2016-04-01

    The controlled and directed focusing of particles within flowing fluids is a problem of fundamental and technological significance. Microfluidic inertial focusing provides passive and precise lateral and longitudinal alignment of small particles without the need for external actuation or sheath fluid. The benefits of inertial focusing have quickly enabled the development of miniaturized flow cytometers, size-selective sorting devices, and other high-throughput particle screening tools. Straight channel inertial focusing device design requires knowledge of fluid properties and particle-channel size ratio. Equilibrium behavior of inertially focused particles has been extensively characterized and the constitutive phenomena described by scaling relationships for straight channels of square and rectangular cross section. In concentrated particle suspensions, however, long-range hydrodynamic repulsions give rise to complex particle ordering that, while interesting and potentially useful, can also dramatically diminish the technique's effectiveness for high-throughput particle handling applications. We have empirically investigated particle focusing behavior within channels of increasing aspect ratio and have identified three scaling regimes that produce varying degrees of geometrical ordering between focused particles. To explore the limits of inertial particle focusing and identify the origins of these long-range interparticle forces, we have explored equilibrium focusing behavior as a function of channel geometry and particle concentration. Experimental results for highly concentrated particle solutions identify equilibrium thresholds for focusing that scale weakly with concentration and strongly with channel geometry. Balancing geometry mediated inertial forces with estimates for interparticle repulsive forces now provide a complete picture of pattern formation among concentrated inertially focused particles and enhance our understanding of the fundamental limits of

  5. The effects of the putative potassium channel activator WAY-120,491 on 86Rb efflux from the rabbit aorta

    SciTech Connect

    Lodge, N.J.; Cohen, R.B.; Havens, C.N.; Colatsky, T.J. )

    1991-02-01

    WAY-120,491 ((-)-(3S-trans)-2-(3,4-dihydro-3-hydroxy-2,2-dimethyl-6-(trifluoromet hox y)- 2H-1-benzopyran-4-yl)-2,3-dihydro-1H-isoindol-1-one) is a novel antihypertensive agent. We have investigated the effects of this compound on contractile force and 86Rb efflux, using the rabbit aorta, in order to assess its K channel activator properties. K channel blockers and ionic conditions thought to modulate specific K channel types have been used to provide insight into the K channel(s) affected by this compound. WAY-120,491 evoked relaxation of precontracted rabbit aortic rings and increased the rate of 86Rb efflux from strips of rabbit aorta; both effects occurring in a concentration-dependent manner. The WAY-120,491 (1 microM)-induced 86Rb efflux was inhibited by tetraethylammonium (IC50 = 0.38 mM), indicating that the increased efflux was mediated by K channels. Glyburide completely blocked the WAY-120,491 (1 microM)-evoked 86Rb efflux with 50% block occurring at a concentration of 0.48 microM. Glyburide also antagonized the WAY-120,491-induced relaxation of aortic rings. Omission of Ca from the solution bathing the aorta did not inhibit the WAY-120,491 induced 86Rb efflux but rather caused an augmentation of the response. It is concluded that WAY-120,491 may be classified as a K channel opener. Furthermore, the K channel upon which WAY-120,491 acts exhibits some characteristics normally associated with the ATP regulated K channel although the involvement of other K channel types has not been ruled out.

  6. Ca(2+) influx through L-type Ca(2+) channels and transient receptor potential channels activates pathological hypertrophy signaling.

    PubMed

    Gao, Hui; Wang, Fang; Wang, Wei; Makarewich, Catherine A; Zhang, Hongyu; Kubo, Hajime; Berretta, Remus M; Barr, Larry A; Molkentin, Jeffery D; Houser, Steven R

    2012-11-01

    Common cardiovascular diseases such as hypertension and myocardial infarction require that myocytes develop greater than normal force to maintain cardiac pump function. This requires increases in [Ca(2+)]. These diseases induce cardiac hypertrophy and increases in [Ca(2+)] are known to be an essential proximal signal for activation of hypertrophic genes. However, the source of "hypertrophic" [Ca(2+)] is not known and is the topic of this study. The role of Ca(2+) influx through L-type Ca(2+) channels (LTCC), T-type Ca(2+) channels (TTCC) and transient receptor potential (TRP) channels on the activation of calcineurin (Cn)-nuclear factor of activated T cells (NFAT) signaling and myocyte hypertrophy was studied. Neonatal rat ventricular myocytes (NRVMs) and adult feline ventricular myocytes (AFVMs) were infected with an adenovirus containing NFAT-GFP, to determine factors that could induce NFAT nuclear translocation. Four millimolar Ca(2+) or pacing induced NFAT nuclear translocation. This effect was blocked by Cn inhibitors. In NRVMs Nifedipine (Nif, LTCC antagonist) blocked high Ca(2+)-induced NFAT nuclear translocation while SKF-96365 (TRP channel antagonist) and Nickel (Ni, TTCC antagonist) were less effective. The relative potency of these antagonists against Ca(2+) induced NFAT nuclear translocation (Nif>SKF-96365>Ni) was similar to their effects on Ca(2+) transients and the LTCC current. Infection of NRVM with viruses containing TRP channels also activated NFAT-GFP nuclear translocation and caused myocyte hypertrophy. TRP effects were reduced by SKF-96365, but were more effectively antagonized by Nif. These experiments suggest that Ca(2+) influx through LTCCs is the primary source of Ca(2+) to activate Cn-NFAT signaling in NRVMs and AFVMs. While TRP channels cause hypertrophy, they appear to do so through a mechanism involving Ca(2+) entry via LTCCs. PMID:22921230

  7. Inhibitory effects of Tyrphostin AG-related compounds on oxidative stress-sensitive transient receptor potential channel activation.

    PubMed

    Toda, Takahiro; Yamamoto, Shinichiro; Yonezawa, Ryo; Mori, Yasuo; Shimizu, Shunichi

    2016-09-01

    Some transient receptor potential (TRP) proteins including TRPA1, TPRM2 and TRPV1 are oxidative stress-sensitive Ca(2+)-permeable channels. Ca(2+) signaling via these TRP channels activated by oxidative stress has been implicated in the aggravation of various inflammatory diseases and pain sensation. We recently reported that Tyrphostin AG490 exerted inhibitory effects on H2O2-induced TRPM2 activation by scavenging the hydroxyl radical. In order to identify stronger inhibitors of oxidative stress-sensitive TRP channels than AG490, we examined the inhibitory effects of Tyrphostin AG-related compounds on H2O2-induced TRP channel activation in human embryonic kidney 293 cells expressing TRP channels. AG555 and AG556 blocked the activation of TRPM2 by H2O2 more strongly than AG490. Regarding TRPV1 and TRPA1, none of the three compounds tested affected H2O2-induced TRPV1 activation; however, AG555 and AG556 reduced H2O2-induced TRPA1 activation more than AG490. Thus, we herein identified AG555 and AG556 as new compounds that exert stronger inhibitory effects on H2O2-induced TRPM2 and TRPA1 activation than AG490. Edaravone, a hydroxyl radical scavenger used in the treatment of cerebral hemorrhage and cerebral infarction, did not affect H2O2-induced TRPM2 or TRPA1 activation. AG555 and AG556 may be useful seed compounds as therapeutic agents for several TRP-related diseases associated with oxidative stress. PMID:27238971

  8. Membrane stretch affects gating modes of a skeletal muscle sodium channel.

    PubMed

    Tabarean, I V; Juranka, P; Morris, C E

    1999-08-01

    The alpha subunit of the human skeletal muscle Na(+) channel recorded from cell-attached patches yielded, as expected for Xenopus oocytes, two current components that were stable for tens of minutes during 0.2 Hz stimulation. Within seconds of applying sustained stretch, however, the slower component began decreasing and, depending on stretch intensity, disappeared in 1-3 min. Simultaneously, the faster current increased. The resulting fast current kinetics and voltage sensitivity were indistinguishable from the fast components 1) left after 10 Hz depolarizations, and 2) that dominated when alpha subunit was co-expressed with human beta1 subunit. Although high frequency depolarization-induced loss of slow current was reversible, the stretch-induced slow-to-fast conversion was irreversible. The conclusion that stretch converted a single population of alpha subunits from an abnormal slow to a bona fide fast gating mode was confirmed by using gigaohm seals formed without suction, in which fast gating was originally absent. For brain Na(+) channels, co-expressing G proteins with the channel alpha subunit yields slow gating. Because both stretch and beta1 subunits induced the fast gating mode, perhaps they do so by minimizing alpha subunit interactions with G proteins or with other regulatory molecules available in oocyte membrane. Because of the possible involvement of oocyte molecules, it remains to be determined whether the Na(+) channel alpha subunit was directly or secondarily susceptible to bilayer tension. PMID:10423424

  9. Membrane stretch affects gating modes of a skeletal muscle sodium channel.

    PubMed Central

    Tabarean, I V; Juranka, P; Morris, C E

    1999-01-01

    The alpha subunit of the human skeletal muscle Na(+) channel recorded from cell-attached patches yielded, as expected for Xenopus oocytes, two current components that were stable for tens of minutes during 0.2 Hz stimulation. Within seconds of applying sustained stretch, however, the slower component began decreasing and, depending on stretch intensity, disappeared in 1-3 min. Simultaneously, the faster current increased. The resulting fast current kinetics and voltage sensitivity were indistinguishable from the fast components 1) left after 10 Hz depolarizations, and 2) that dominated when alpha subunit was co-expressed with human beta1 subunit. Although high frequency depolarization-induced loss of slow current was reversible, the stretch-induced slow-to-fast conversion was irreversible. The conclusion that stretch converted a single population of alpha subunits from an abnormal slow to a bona fide fast gating mode was confirmed by using gigaohm seals formed without suction, in which fast gating was originally absent. For brain Na(+) channels, co-expressing G proteins with the channel alpha subunit yields slow gating. Because both stretch and beta1 subunits induced the fast gating mode, perhaps they do so by minimizing alpha subunit interactions with G proteins or with other regulatory molecules available in oocyte membrane. Because of the possible involvement of oocyte molecules, it remains to be determined whether the Na(+) channel alpha subunit was directly or secondarily susceptible to bilayer tension. PMID:10423424

  10. Breeding strategy of US farm-raised channel catfish, Ictalurus punctatus affects progeny production and performance

    Technology Transfer Automated Retrieval System (TEKTRAN)

    catfish propagation for decades has been dependent on random mating of male and female channel catfish in ponds. It is simple and has been fairly successful in fulfilling the needs of the US farm-raised catfish industry. However, natural pond spawning is unreliable, unpredictable, and incurs 30 t...

  11. Designing Effective Persuasive Systems Utilizing the Power of Entanglement: Communication Channel, Strategy & Affect

    ERIC Educational Resources Information Center

    Li, Haiqing

    2010-01-01

    With rapid advancements in information and communication technologies, computer-mediated communication channels such as email, web, mobile smart-phones with SMS, social networking websites (Facebook), multimedia websites, and OEM devices provide users with multiple technology choices to seek information. However, no study has compared the…

  12. Pre-treating channel catfish with copper sulfate affects susceptibility to columnaris disease

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Columnaris disease is one of the most important bacterial diseases of channel catfish, Ictalurus punctatus, commercially grown in the US. This disease can greatly diminish the profitability of aquaculture operations by large-scale mortality events, particularly in the fingerling production phase. ...

  13. Role of calcium-activated potassium channels in acetylcholine-induced vasodilation of rat retinal arterioles in vivo.

    PubMed

    Mori, Asami; Suzuki, Sachi; Sakamoto, Kenji; Nakahara, Tsutomu; Ishii, Kunio

    2011-01-01

    The vascular endothelium plays an important role in regulating retinal blood flow via actions of several vasodilators, including nitric oxide (NO), prostaglandin I₂, and an endothelium-derived hyperpolarizing factor (EDHF). Our previous in vivo studies demonstrated that acetylcholine (ACh) dilates the rat retinal arteriole partly through NO- and prostaglandin-independent pathway, possibly the EDHF-mediated pathway, but the underlying mechanism(s) remains to be elucidated. It has been suggested that activation of Ca²+-activated K+ (K(Ca)) channels contributes to the EDHF-mediated responses; therefore, the roles of K(Ca) channels in ACh-induced vasodilation of retinal arterioles were examined in rats. The retinal vascular responses were assessed by determining changes in diameters of retinal arterioles in ocular fundus images that were captured with an original fundus camera system. Intravitreal injection of charybdotoxin, an inhibitor of intermediate- and large-conductance K(Ca) (I/BK(Ca)) channels, or iberiotoxin, an inhibitor of large-conductance K(Ca) (BK(Ca)) channels, significantly reduced the ACh-induced vasodilation of retinal arterioles, whereas neither apamin, an inhibitor of small-conductance K(Ca) (SK(Ca)) channels, nor TRAM-34, an inhibitor of intermediate-conductance K(Ca) (IK(Ca)) channels, altered the response. The vasodilator response to ACh observed under the combined blockade of NO synthase and cyclooxygenase with N(G)-nitro-L-arginine methyl ester plus indomethacin was also diminished by iberiotoxin. Iberiotoxin did not affect the NO donor NOR3-induced vasodilation of retinal arterioles, whereas it significantly reduced the BK(Ca) channel opener BMS-191011-induced responses. These results suggest that activation of BK(Ca) channels is involved in the EDHF-mediated component of the vasodilator response to ACh in the rat retinal arterioles in vivo. PMID:20978884

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

    PubMed Central

    Steinberg, Ximena; Lespay-Rebolledo, Carolyne; Brauchi, Sebastian

    2014-01-01

    Transient Receptor Potential (TRP) proteins are a large family of ion channels, grouped into seven sub-families. Although great advances have been made regarding the activation and modulation of TRP channel activity, detailed molecular mechanisms governing TRP channel gating are still needed. Sensitive to electric, chemical, mechanical, and thermal cues, TRP channels are tightly associated with the detection and integration of sensory input, 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 stimuli making them an interesting pharmacological target for the treatment of pain. However, the poor specificity of the compounds available in the market represents an important obstacle to overcome. Understanding the molecular mechanics underlying ligand-dependent modulation of TRP channels may help with the rational design of novel synthetic analgesics. The present review focuses on the structural basis of ligand-dependent activation of TRPV1 and TRPM8 channels. Special attention is drawn to the dissection of ligand-binding sites within TRPV1, PIP2-dependent modulation of TRP channels, and the structure of natural and synthetic ligands. PMID:24847275

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

    NASA Astrophysics Data System (ADS)

    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.

  16. X-ray irradiation activates K+ channels via H2O2 signaling.

    PubMed

    Gibhardt, Christine S; Roth, Bastian; Schroeder, Indra; Fuck, Sebastian; Becker, Patrick; Jakob, Burkhard; Fournier, Claudia; Moroni, Anna; Thiel, Gerhard

    2015-01-01

    Ionizing radiation is a universal tool in tumor therapy but may also cause secondary cancers or cell invasiveness. These negative side effects could be causally related to the human-intermediate-conductance Ca2+-activated-K+-channel (hIK), which is activated by X-ray irradiation and affects cell proliferation and migration. To analyze the signaling cascade downstream of ionizing radiation we use genetically encoded reporters for H2O2 (HyPer) and for the dominant redox-buffer glutathione (Grx1-roGFP2) to monitor with high spatial and temporal resolution, radiation-triggered excursions of H2O2 in A549 and HEK293 cells. The data show that challenging cells with ≥1 Gy X-rays or with UV-A laser micro-irradiation causes a rapid rise of H2O2 in the nucleus and in the cytosol. This rise, which is determined by the rate of H2O2 production and glutathione-buffering, is sufficient for triggering a signaling cascade that involves an elevation of cytosolic Ca2+ and eventually an activation of hIK channels. PMID:26350345

  17. X-ray irradiation activates K+ channels via H2O2 signaling

    PubMed Central

    Gibhardt, Christine S.; Roth, Bastian; Schroeder, Indra; Fuck, Sebastian; Becker, Patrick; Jakob, Burkhard; Fournier, Claudia; Moroni, Anna; Thiel, Gerhard

    2015-01-01

    Ionizing radiation is a universal tool in tumor therapy but may also cause secondary cancers or cell invasiveness. These negative side effects could be causally related to the human-intermediate-conductance Ca2+-activated-K+-channel (hIK), which is activated by X-ray irradiation and affects cell proliferation and migration. To analyze the signaling cascade downstream of ionizing radiation we use genetically encoded reporters for H2O2 (HyPer) and for the dominant redox-buffer glutathione (Grx1-roGFP2) to monitor with high spatial and temporal resolution, radiation-triggered excursions of H2O2 in A549 and HEK293 cells. The data show that challenging cells with ≥1 Gy X-rays or with UV-A laser micro-irradiation causes a rapid rise of H2O2 in the nucleus and in the cytosol. This rise, which is determined by the rate of H2O2 production and glutathione-buffering, is sufficient for triggering a signaling cascade that involves an elevation of cytosolic Ca2+ and eventually an activation of hIK channels. PMID:26350345

  18. Monitoring Affect States during Effortful Problem Solving Activities

    ERIC Educational Resources Information Center

    D'Mello, Sidney K.; Lehman, Blair; Person, Natalie

    2010-01-01

    We explored the affective states that students experienced during effortful problem solving activities. We conducted a study where 41 students solved difficult analytical reasoning problems from the Law School Admission Test. Students viewed videos of their faces and screen captures and judged their emotions from a set of 14 states (basic…

  19. Tityus bahiensis toxin IV-5b selectively affects Na channel inactivation in chick dorsal root ganglion neurons.

    PubMed

    Trequattrini, C; Zamudio, F Z; Petris, A; Prestipino, G; Possani, L D; Franciolini, F

    1995-09-01

    A novel toxin was isolated from the venom of the Brazilian scorpion Tityus (T.) bahiensis. The N-terminal amino acid sequence of this toxin was shown to be 80% identical to the corresponding segment of T. serrulatus toxin IV-5. The new toxin was thus named toxin IV-5b. Toxin IV-5b was found to markedly slow inactivation of Na channel in dorsal root ganglion neurons from chick embryo. By contrast, Na channel activation was only negligibly delayed, and deactivation completely unaffected. Similarly unaffected by the toxin were K and Ca currents. The slowing effect of the toxin starts to appear at concentrations of c. 80 nM, and shows a KD of 143 nM. With a toxin concentration of 2.4 microM, the Na channel inactivation time constant was increased c. 3-fold with respect to the control. The slowing of inactivation was voltage dependent, and increased with depolarization. PMID:7553331

  20. Structural elements in the Girk1 subunit that potentiate G protein-gated potassium channel activity.

    PubMed

    Wydeven, Nicole; Young, Daniele; Mirkovic, Kelsey; Wickman, Kevin

    2012-12-26

    G protein-gated inwardly rectifying K(+) (Girk/K(IR)3) channels mediate the inhibitory effect of many neurotransmitters on excitable cells. Girk channels are tetramers consisting of various combinations of four mammalian Girk subunits (Girk1 to -4). Although Girk1 is unable to form functional homomeric channels, its presence in cardiac and neuronal channel complexes correlates with robust channel activity. This study sought to better understand the potentiating influence of Girk1, using the GABA(B) receptor and Girk1/Girk2 heteromer as a model system. Girk1 did not increase the protein levels or alter the trafficking of Girk2-containing channels to the cell surface in transfected cells or hippocampal neurons, indicating that its potentiating influence involves enhancement of channel activity. Structural elements in both the distal carboxyl-terminal domain and channel core were identified as key determinants of robust channel activity. In the distal carboxyl-terminal domain, residue Q404 was identified as a key determinant of receptor-induced channel activity. In the Girk1 core, three unique residues in the pore (P) loop (F137, A142, Y150) were identified as a collective potentiating influence on both receptor-dependent and receptor-independent channel activity, exerting their influence, at least in part, by enhancing mean open time and single-channel conductance. Interestingly, the potentiating influence of the Girk1 P-loop is tempered by residue F162 in the second membrane-spanning domain. Thus, discontinuous and sometime opposing elements in Girk1 underlie the Girk1-dependent potentiation of receptor-dependent and receptor-independent heteromeric channel activity. PMID:23236146

  1. Classical-quantum arbitrarily varying wiretap channel: Ahlswede dichotomy, positivity, resources, super-activation

    NASA Astrophysics Data System (ADS)

    Boche, Holger; Cai, Minglai; Deppe, Christian; Nötzel, Janis

    2016-08-01

    We establish the Ahlswede dichotomy for arbitrarily varying classical-quantum wiretap channels, i.e., either the deterministic secrecy capacity of the channel is zero, or it equals its randomness-assisted secrecy capacity. We analyze the secrecy capacity of these channels when the sender and the receiver use various resources. It turns out that randomness, common randomness, and correlation as resources are very helpful for achieving a positive secrecy capacity. We prove the phenomenon "super-activation" for arbitrarily varying classical-quantum wiretap channels, i.e., two channels, both with zero deterministic secrecy capacity, if used together allow perfect secure transmission.

  2. Rare Mutations of CACNB2 Found in Autism Spectrum Disease-Affected Families Alter Calcium Channel Function

    PubMed Central

    Breitenkamp, Alexandra F. S.; Matthes, Jan; Nass, Robert Daniel; Sinzig, Judith; Lehmkuhl, Gerd; Nürnberg, Peter; Herzig, Stefan

    2014-01-01

    Autism Spectrum Disorders (ASD) are complex neurodevelopmental diseases clinically defined by dysfunction of social interaction. Dysregulation of cellular calcium homeostasis might be involved in ASD pathogenesis, and genes coding for the L-type calcium channel subunits CaV1.2 (CACNA1C) and CaVβ2 (CACNB2) were recently identified as risk loci for psychiatric diseases. Here, we present three rare missense mutations of CACNB2 (G167S, S197F, and F240L) found in ASD-affected families, two of them described here for the first time (G167S and F240L). All these mutations affect highly conserved regions while being absent in a sample of ethnically matched controls. We suggest the mutations to be of physiological relevance since they modulate whole-cell Ba2+ currents through calcium channels when expressed in a recombinant system (HEK-293 cells). Two mutations displayed significantly decelerated time-dependent inactivation as well as increased sensitivity of voltage-dependent inactivation. In contrast, the third mutation (F240L) showed significantly accelerated time-dependent inactivation. By altering the kinetic parameters, the mutations are reminiscent of the CACNA1C mutation causing Timothy Syndrome, a Mendelian disease presenting with ASD. In conclusion, the results of our first-time biophysical characterization of these three rare CACNB2 missense mutations identified in ASD patients support the hypothesis that calcium channel dysfunction may contribute to autism. PMID:24752249

  3. A Ca2+-activated channel from Xenopus laevis oocyte membranes reconstituted into planar bilayers.

    PubMed Central

    Young, G P; Young, J D; Deshpande, A K; Goldstein, M; Koide, S S; Cohn, Z A

    1984-01-01

    Plasma membrane fractions from Xenopus laevis oocytes were incorporated into planar lipid bilayers. We show the existence of numerous Ca2+-activated nonspecific channels that are more permeable to anions. These channels are activated by Ca2+ at micromolar concentration but not by Mg2+, Zn2+, or Mn2+, even at millimolar concentrations. Decreasing Ca2+ concentration to less than 1 microM decreases the time of channel opening until channels close completely in the absence of Ca2+ and in the presence of EGTA. I- and Br- are more permeable through this channel than Cl-. The time during which the channels remain open is also voltage-dependent, with the channels switching off at higher voltages in both polarities. Single-channel activity shows a conductance of 380 pS in 1 M NaCl and 1 mM CaCl2, with an average open lifetime of 1.5 s at 40 mV. Similar channels are found in different stages of oocyte maturation. These observations support the hypothesis that an increase in oocyte-free Ca2+ activates directly these channels, and the resultant Cl- efflux forms the ionic basis for the fertilization potential in X. laevis. PMID:6089180

  4. The 4′lysine in the putative channel lining domain affects desensitization but not the single-channel conductance of recombinant homomeric 5-HT3A receptors

    PubMed Central

    Gunthorpe, Martin J; Peters, John A; Gill, Catherine H; Lambert, Jeremy J; Lummis, Sarah C R

    2000-01-01

    The 5-HT3 receptor is a transmitter-gated ion channel of the Cys-loop superfamily. Uniquely, 5-HT3 receptor subunits (5-HT3A and 5-HT3B) possess a positively charged lysine residue within the putative channel lining M2 domain (4′ position). Using whole cell recording techniques, we examined the role of this residue in receptor function using wild-type (WT) and mutant 5-HT3A receptor subunits of murine origin transiently expressed in human embryonic kidney (HEK 293) cells. WT 5-HT3A receptors mediated rapidly activating currents in response to 5-HT (10–90 % rise time, 103 ms; EC50, 2.34 μm; Hill coefficient, nH, 2.87). The currents rectified inwardly, reversed in sign at a potential of −9 mV and desensitized in the continuous presence of agonist (half-time of desensitization, t1/2, 2.13 s). 5-HT3A receptor subunits in which the 4′lysine was mutated to arginine, glutamine, serine or glycine formed functional receptors. 5-HT EC50 values were approximately 2-fold lower than for WT 5-HT3A receptors, but Hill coefficients, kinetics of current activation, rectification, and reversal potentials were unaltered. Each of the mutants desensitized more slowly than the WT 5-HT3A receptor, with the arginine and glycine mutations exhibiting the greatest effect (5-fold reduction). The rank order of effect was arginine > glycine > serine > glutamine. The single-channel conductance of the WT 5-HT3A receptor, as assessed by fluctuation analysis of macroscopic currents, was 390 fS. A similar value was obtained for the 4′lysine mutant receptors. Thus it appears unlikely that 4′lysine is exposed to the channel lumen. Mutation of residues immediately adjacent to 4′lysine to glutamate or lysine resulted in lack of receptor expression or function. We conclude that 4′lysine does not form part of the channel lining, but may play an important role in 5-HT3 receptor desensitization. PMID:10639097

  5. Investigating neuronal activity by SPYCODE multi-channel data analyzer.

    PubMed

    Bologna, Luca Leonardo; Pasquale, Valentina; Garofalo, Matteo; Gandolfo, Mauro; Baljon, Pieter Laurens; Maccione, Alessandro; Martinoia, Sergio; Chiappalone, Michela

    2010-08-01

    Multi-channel acquisition from neuronal networks, either in vivo or in vitro, is becoming a standard in modern neuroscience in order to infer how cell assemblies communicate. In spite of the large diffusion of micro-electrode-array-based systems, researchers usually find it difficult to manage the huge quantity of data routinely recorded during the experimental sessions. In fact, many of the available open-source toolboxes still lack two fundamental requirements for treating multi-channel recordings: (i) a rich repertoire of algorithms for extracting information both at a single channel and at the whole network level; (ii) the capability of autonomously repeating the same set of computational operations to 'multiple' recording streams (also from different experiments) and without a manual intervention. The software package we are proposing, named SPYCODE, was mainly developed to respond to the above constraints and generally to offer the scientific community a 'smart' tool for multi-channel data processing. PMID:20554151

  6. Voltage-induced membrane displacement in patch pipettes activates mechanosensitive channels

    PubMed Central

    Gil, Ziv; Silberberg, Shai D.; Magleby, Karl L.

    1999-01-01

    The patch-clamp technique allows currents to be recorded through single ion channels in patches of cell membrane in the tips of glass pipettes. When recording, voltage is typically applied across the membrane patch to drive ions through open channels and to probe the voltage-sensitivity of channel activity. In this study, we used video microscopy and single-channel recording to show that prolonged depolarization of a membrane patch in borosilicate pipettes results in delayed slow displacement of the membrane into the pipette and that this displacement is associated with the activation of mechanosensitive (MS) channels in the same patch. The membrane displacement, ≈1 μm with each prolonged depolarization, occurs after variable delays ranging from tens of milliseconds to many seconds and is correlated in time with activation of MS channels. Increasing the voltage step shortens both the delay to membrane displacement and the delay to activation. Preventing depolarization-induced membrane displacement by applying positive pressure to the shank of the pipette or by coating the tips of the borosilicate pipettes with soft glass prevents the depolarization-induced activation of MS channels. The correlation between depolarization-induced membrane displacement and activation of MS channels indicates that the membrane displacement is associated with sufficient membrane tension to activate MS channels. Because membrane tension can modulate the activity of various ligand and voltage-activated ion channels as well as some transporters, an apparent voltage dependence of a channel or transporter in a membrane patch in a borosilicate pipette may result from voltage-induced tension rather than from direct modulation by voltage. PMID:10588750

  7. The Clinically Tested Gardos Channel Inhibitor Senicapoc Exhibits Antimalarial Activity

    PubMed Central

    Tubman, Venée N.; Mejia, Pedro; Shmukler, Boris E.; Bei, Amy K.; Alper, Seth L.; Mitchell, James R.

    2015-01-01

    Senicapoc, a Gardos channel inhibitor, prevented erythrocyte dehydration in clinical trials of patients with sickle cell disease. We tested the hypothesis that senicapoc-induced blockade of the Gardos channel inhibits Plasmodium growth. Senicapoc inhibited in vitro growth of human and primate plasmodia during the clinical blood stage. Senicapoc treatment suppressed P. yoelii parasitemia in vivo in C57BL/6 mice. The reassuring safety and biochemical profile of senicapoc encourage its use in antimalarial development. PMID:26459896

  8. Peptide fragments of the dihydropyridine receptor can modulate cardiac ryanodine receptor channel activity and sarcoplasmic reticulum Ca2+ release.

    PubMed Central

    Dulhunty, Angela F; Curtis, Suzanne M; Cengia, Louise; Sakowska, Magdalena; Casarotto, Marco G

    2004-01-01

    We show that peptide fragments of the dihydropyridine receptor II-III loop alter cardiac RyR (ryanodine receptor) channel activity in a cytoplasmic Ca2+-dependent manner. The peptides were AC (Thr-793-Ala-812 of the cardiac dihydropyridine receptor), AS (Thr-671-Leu-690 of the skeletal dihydropyridine receptor), and a modified AS peptide [AS(D-R18)], with an extended helical structure. The peptides added to the cytoplasmic side of channels in lipid bilayers at > or = 10 nM activated channels when the cytoplasmic [Ca2+] was 100 nM, but either inhibited or did not affect channel activity when the cytoplasmic [Ca2+] was 10 or 100 microM. Both activation and inhibition were independent of bilayer potential. Activation by AS, but not by AC or AS(D-R18), was reduced at peptide concentrations >1 mM in a voltage-dependent manner (at +40 mV). In control experiments, channels were not activated by the scrambled AS sequence (ASS) or skeletal II-III loop peptide (NB). Resting Ca2+ release from cardiac sarcoplasmic reticulum was not altered by peptide AC, but Ca2+-induced Ca2+ release was depressed. Resting and Ca2+-induced Ca2+ release were enhanced by both the native and modified AS peptides. NMR revealed (i) that the structure of peptide AS(D-R18) is not influenced by [Ca2+] and (ii) that peptide AC adopts a helical structure, particularly in the region containing positively charged residues. This is the first report of specific functional interactions between dihydropyridine receptor A region peptides and cardiac RyR ion channels in lipid bilayers. PMID:14678014

  9. Transcainide causes two modes of open-channel block with different voltage sensitivities in batrachotoxin-activated sodium channels.

    PubMed Central

    Zamponi, G W; French, R J

    1994-01-01

    Transcainide, a complex derivative of lidocaine, blocks the open state of BTX-activated sodium channels from bovine heart and rat skeletal muscle in two distinct ways. When applied to either side of the membrane, transcainide caused discrete blocking events a few hundred milliseconds in duration (slow block), and a concomitant reduction in apparent single-channel amplitude, presumably because of rapid block beyond the temporal resolution of our recordings (fast block). We quantitatively analyzed block from the cytoplasmic side. Both modes of block occurred via binding of the drug to the open channel, approximately followed 1:1 stoichiometry, and were similar for both channel subtypes. For slow block, the blocking rate increased, and the unblocking rate decreased with depolarization, yielding an overall enhancement of block at positive potentials, and suggesting a blocking site at an apparent electrical distance about 45% of the way from the cytoplasmic end of the channel (z delta approximately 0.45). In contrast, the fast blocking mode was only slightly enhanced by depolarization (z delta approximately 0.15). Phenomenologically, the bulky and complex transcainide molecule combines the almost voltage-insensitive blocking action of phenylhydrazine (Zamponi and French, 1994a (companion paper)) with a slow open-channel blocking action that shows a voltage dependence typical of simpler amines. Only the slower blocking mode was sensitive to the removal of external sodium ions, suggesting that the two types of block occur at distinct sites. Dose-response relations were also consistent with independent binding of transcainide to two separate sites on the channel. PMID:7811913

  10. Oscillating activity of a calcium-activated K+ channel in normal and cancerous mammary cells in culture.

    PubMed

    Enomoto, K; Furuya, K; Maeno, T; Edwards, C; Oka, T

    1991-01-01

    Calcium-activated potassium channels were the channels most frequently observed in primary cultured normal mammary cell and in the established mammary tumor cell, MMT060562. In both cells, single-channel and whole-cell clamp recordings sometimes showed slow oscillations of the Ca2(+)-gated K+ current. The characteristics of the Ca2(+)-activated K+ channels in normal and cancerous mammary cells were quite similar. The slope conductances changed from 8 to 70 pS depending on the mode of recording and the ionic composition in the patch electrode. The open probability of this channel increased between 0.1 to 1 microM of the intracellular Ca2+, but it was independent of the membrane potential. Charybdotoxin reduced the activity of the Ca2(+)-activated K+ channel and the oscillation of the membrane current, but apamin had no apparent effect. The application of tetraethylammonium (TEA) from outside and BaCl2 from inside of the cell diminished the activity of the channel. The properties of this channel were different from those of both the large conductance (BK or MAXI K) and small conductance (SK) type Ca2(+)-activated K+ channels. PMID:1710671

  11. Regulation of Arterial Tone by Activation of Calcium-Dependent Potassium Channels

    NASA Astrophysics Data System (ADS)

    Brayden, Joseph E.; Nelson, Mark T.

    1992-04-01

    Blood pressure and tissue perfusion are controlled in part by the level of intrinsic (myogenic) vascular tone. However, many of the molecular determinants of this response are unknown. Evidence is now presented that the degree of myogenic tone is regulated in part by the activation of large-conductance calcium-activated potassium channels in arterial smooth muscle. Tetraethylammonium ion (TEA^+) and charybdotoxin (CTX), at concentrations that block calcium-activated potassium channels in smooth muscle cells isolated from cerebral arteries, depolarized and constricted pressurized cerebral arteries with myogenic tone. Both TEA^+ and CTX had little effect on arteries when intracellular calcium was reduced by lowering intravascular pressure or by blocking calcium channels. Elevation of intravascular pressure through membrane depolarization and an increase in intracellular calcium may activate calcium-activated potassium channels. Thus, these channels may serve as a negative feedback pathway to control the degree of membrane depolarization and vasoconstriction.

  12. [Osmoregulatory reactions of frog erythrocytes under conditions of activation and blockade of Ca2+-channels].

    PubMed

    Skorkina, M Iu

    2012-01-01

    The kinetics of cell osmoregulatory reactions under conditions of activation and blockade of Ca2+-channels was studied on a model of frog polyfunctional nuclear erythrocyte. Both activation and blockade of Ca2+-channels has been established to promote swelling of nuclei and an increase of the nuclear-cytoplasmic ratios under conditions of hypotonic exposure. The osmoregulatory cell reactions after activation of Ca2+-channels are expressed as a decrease of the cell volume. The blockator of Ca2+-channels verapamil produces an alternated increase and decrease of the erythrocyte volume with time intervals of 30 and 60 s. The clearly expressed functional activity of the nuclear membrane in response to the hypotonic action under conditions of activation and blockade of Ca2+-channels indicates participation of Ca2+ ions in mechanisms of the nuclear-cytoplasmic transfer. PMID:22645976

  13. Structural determinants of the transient receptor potential 1 (TRPV1) channel activation by phospholipid analogs.

    PubMed

    Morales-Lázaro, Sara L; Serrano-Flores, Barbara; Llorente, Itzel; Hernández-García, Enrique; González-Ramírez, Ricardo; Banerjee, Souvik; Miller, Duane; Gududuru, Veeresh; Fells, James; Norman, Derek; Tigyi, Gabor; Escalante-Alcalde, Diana; Rosenbaum, Tamara

    2014-08-29

    The transient receptor potential vanilloid 1 (TRPV1) ion channel is a polymodal protein that responds to various stimuli, including capsaicin (the pungent compound found in chili peppers), extracellular acid, and basic intracellular pH, temperatures close to 42 °C, and several lipids. Lysophosphatidic acid (LPA), an endogenous lipid widely associated with neuropathic pain, is an agonist of the TRPV1 channel found in primary afferent nociceptors and is activated by other noxious stimuli. Agonists or antagonists of lipid and other chemical natures are known to possess specific structural requirements for producing functional effects on their targets. To better understand how LPA and other lipid analogs might interact and affect the function of TRPV1, we set out to determine the structural features of these lipids that result in the activation of TRPV1. By changing the acyl chain length, saturation, and headgroup of these LPA analogs, we established strict requirements for activation of TRPV1. Among the natural LPA analogs, we found that only LPA 18:1, alkylglycerophosphate 18:1, and cyclic phosphatidic acid 18:1, all with a monounsaturated C18 hydrocarbon chain activate TRPV1, whereas polyunsaturated and saturated analogs do not. Thus, TRPV1 shows a more restricted ligand specificity compared with LPA G-protein-coupled receptors. We synthesized fatty alcohol phosphates and thiophosphates and found that many of them with a single double bond in position Δ9, 10, or 11 and Δ9 cyclopropyl group can activate TRPV1 with efficacy similar to capsaicin. Finally, we developed a pharmacophore and proposed a mechanistic model for how these lipids could induce a conformational change that activates TRPV1. PMID:25035428

  14. Gain-of-function mutations in the mechanically activated ion channel PIEZO2 cause a subtype of Distal Arthrogryposis

    PubMed Central

    Coste, Bertrand; Houge, Gunnar; Murray, Michael F.; Stitziel, Nathan; Bandell, Michael; Giovanni, Monica A.; Philippakis, Anthony; Hoischen, Alexander; Riemer, Gunnar; Steen, Unni; Steen, Vidar Martin; Mathur, Jayanti; Cox, James; Lebo, Matthew; Rehm, Heidi; Weiss, Scott T.; Wood, John N.; Maas, Richard L.; Sunyaev, Shamil R.; Patapoutian, Ardem

    2013-01-01

    Mechanotransduction, the pathway by which mechanical forces are translated to biological signals, plays important but poorly characterized roles in physiology. PIEZOs are recently identified, widely expressed, mechanically activated ion channels that are hypothesized to play a role in mechanotransduction in mammals. Here, we describe two distinct PIEZO2 mutations in patients with a subtype of Distal Arthrogryposis Type 5 characterized by generalized autosomal dominant contractures with limited eye movements, restrictive lung disease, and variable absence of cruciate knee ligaments. Electrophysiological studies reveal that the two PIEZO2 mutations affect biophysical properties related to channel inactivation: both E2727del and I802F mutations cause the PIEZO2-dependent, mechanically activated currents to recover faster from inactivation, while E2727del also causes a slowing of inactivation. Both types of changes in kinetics result in increased channel activity in response to a given mechanical stimulus, suggesting that Distal Arthrogryposis Type 5 can be caused by gain-of-function mutations in PIEZO2. We further show that overexpression of mutated PIEZO2 cDNAs does not cause constitutive activity or toxicity to cells, indicating that the observed phenotype is likely due to a mechanotransduction defect. Our studies identify a type of channelopathy and link the dysfunction of mechanically activated ion channels to developmental malformations and joint contractures. PMID:23487782

  15. Chemoselective tarantula toxins report voltage activation of wild-type ion channels in live cells.

    PubMed

    Tilley, Drew C; Eum, Kenneth S; Fletcher-Taylor, Sebastian; Austin, Daniel C; Dupré, Christophe; Patrón, Lilian A; Garcia, Rita L; Lam, Kit; Yarov-Yarovoy, Vladimir; Cohen, Bruce E; Sack, Jon T

    2014-11-01

    Electrically excitable cells, such as neurons, exhibit tremendous diversity in their firing patterns, a consequence of the complex collection of ion channels present in any specific cell. Although numerous methods are capable of measuring cellular electrical signals, understanding which types of ion channels give rise to these signals remains a significant challenge. Here, we describe exogenous probes which use a novel mechanism to report activity of voltage-gated channels. We have synthesized chemoselective derivatives of the tarantula toxin guangxitoxin-1E (GxTX), an inhibitory cystine knot peptide that binds selectively to Kv2-type voltage gated potassium channels. We find that voltage activation of Kv2.1 channels triggers GxTX dissociation, and thus GxTX binding dynamically marks Kv2 activation. We identify GxTX residues that can be replaced by thiol- or alkyne-bearing amino acids, without disrupting toxin folding or activity, and chemoselectively ligate fluorophores or affinity probes to these sites. We find that GxTX-fluorophore conjugates colocalize with Kv2.1 clusters in live cells and are released from channels activated by voltage stimuli. Kv2.1 activation can be detected with concentrations of probe that have a trivial impact on cellular currents. Chemoselective GxTX mutants conjugated to dendrimeric beads likewise bind live cells expressing Kv2.1, and the beads are released by channel activation. These optical sensors of conformational change are prototype probes that can indicate when ion channels contribute to electrical signaling. PMID:25331865

  16. Low-dose photon irradiation alters cell differentiation via activation of hIK channels.

    PubMed

    Roth, Bastian; Gibhardt, Christine S; Becker, Patrick; Gebhardt, Manuela; Knoop, Jan; Fournier, Claudia; Moroni, Anna; Thiel, Gerhard

    2015-08-01

    To understand the impact of ionizing irradiation from diagnostics and radiotherapy on cells, we examined K(+) channel activity before and immediately after exposing cells to X-rays. Already, low dose in the cGy range caused in adenocarcinoma A549 cells within minutes a hyperpolarization following activation of the human intermediate-conductance Ca(2+)-activated K(+) channel (hIK). The response was specific for cells, which functionally expressed hIK channels and in which hIK activity was low before irradiation. HEK293 cells, which do not respond to X-ray irradiation, accordingly develop a sensitivity to this stress after heterologous expression of hIK channels. The data suggest that hIK activation involves a Ca(2+)-mediated signaling cascade because channel activation is suppressed by a strong cytosolic Ca(2+) buffer. The finding that an elevation of H2O2 causes an increase in the concentration of cytosolic Ca(2+) suggests that radicals, which emerge early in response to irradiation, trigger this Ca(2+) signaling cascade. Inhibition of hIK channels by specific blockers clotrimazole and TRAM-34 slowed cell proliferation and migration in "wound" scratch assays; ionizing irradiation, in turn, stimulated the latter process presumably via its activation of the hIK channels. These data stress an indirect radiosensitivity of hIK channels with an impact on cell differentiation. PMID:25277267

  17. The synaptic vesicle protein synaptophysin: purification and characterization of its channel activity.

    PubMed Central

    Gincel, Dan; Shoshan-Barmatz, Varda

    2002-01-01

    The synaptic vesicle protein synaptophysin was solubilized from rat brain synaptosomes with a relatively low concentration of Triton X-100 (0.2%) and was highly purified (above 95%) using a rapid single chromatography step on hydroxyapatite/celite resin. Purified synaptophysin was reconstituted into a planar lipid bilayer and the channel activity of synaptophysin was characterized. In asymmetric KCl solutions (cis 300 mM/trans 100 mM), synaptophysin formed a fast-fluctuating channel with a conductance of 414 +/- 13 pS at +60 mV. The open probability of synaptophysin channels was decreased upon depolarization, and channels were found to be cation-selective. Synaptophysin channels showed higher selectivity for K(+) over Cl(-) (P(K(+))/P(Cl(-)) > 8) and preferred K(+) over Li(+), Na(+), Rb(+), Cs(+), or choline(+). The synaptophysin channel is impermeable to Ca(2+), which has no effect on its channel activity. This study is the second demonstration of purified synaptophysin channel activity, but the first biophysical characterization of its channel properties. The availability of large amounts of purified synaptophysin and of its characteristic channel properties might help to establish the role of synaptophysin in synaptic transmission. PMID:12496091

  18. Allosteric interactions and the modular nature of the voltage- and Ca2+-activated (BK) channel

    PubMed Central

    Latorre, Ramon; Morera, Francisco J; Zaelzer, Cristian

    2010-01-01

    The high conductance voltage- and Ca2+-activated K+ channel is one of the most broadly expressed channels in mammals. This channel is named BK for ‘big K’ because of its single-channel conductance that can be as large as 250 pS in 100 mm symmetrical K+. BK channels increase their activity by membrane depolarization or an increase in cytosolic Ca2+. One of the key features that defines the behaviour of BK channels is that neither Ca2+ nor voltage is strictly necessary for channel activation. This and several other observations led to the idea that both Ca2+ and voltage increase the open probability by an allosteric mechanism. In this type of mechanism, the processes of voltage sensor displacement, Ca2+ binding and pore opening are independent equilibria that interact allosterically with each other. These allosteric interactions in BK channels reside in the structural characteristics of the BK channel in the sense that voltage and Ca2+ sensors and the pore need to be contained in different structures or ‘modules’. Through electrophysiological, mutagenesis, biochemical and fluorescence studies these modules have been identified and, more important, some of the interactions between them have been unveiled. In this review, we have covered the main advances achieved during the last few years in the elucidation of the structure of the BK channel and how this is related with its function as an allosteric protein. PMID:20603335

  19. Calcium-activated chloride channels in cultured embryonic Xenopus spinal neurons.

    PubMed

    Hussy, N

    1992-12-01

    1. Single-channel currents were recorded from Xenopus spinal neurons developing in vitro using the patch-clamp technique, to identify the channels underlying the large and small macroscopic Ca(2+)-activated Cl- currents (ICl(Ca)) present in these cells. 2. Channels of large (maxi-channels; 310 pS) and smaller conductance (mini-channels; 50-60 pS) are activated by elevation of cytoplasmic Ca2+ concentration. Channel activity is not altered by subsequent removal of Ca2+ from the bath, arguing against a direct ligand-type Ca2+ dependence. The much higher incidence of channel activation in cell-attached patches from cells permeabilized with the Ca2+ ionophore A23187 than in excised patches also suggests the involvement of some unidentified intracellular factor. 3. The reversal potential of maxi-Cl- channels is not altered by changes in Na+ concentration, but is shifted in the negative direction by the substitution of Cl- by methanesulfonate on the intracellular side of the patch, indicating their anionic selectivity. 4. Maxi-Cl- channels exhibited the presence of multiple probable subconductance states and showed marked voltage-dependent inactivation above and below +/- 20 mV. 5. Examination of maxi-Cl- channels at early times in culture (6-9 h) and 24 h later did not reveal any developmental change in the characteristics described above. However, the mean open duration of the channel was found to increase twofold during this period of time. 6. The simultaneous presence of maxi- and mini-Cl- channels prevented detailed characterization of the latter. The anionic selectivity of mini-Cl- channels is suggested by their reversal potential that lies close to the Cl- equilibrium potential.(ABSTRACT TRUNCATED AT 250 WORDS) PMID:1283407

  20. Role of Calcium-activated Potassium Channels in Atrial Fibrillation Pathophysiology and Therapy

    PubMed Central

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

    2015-01-01

    Abstract: 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 because they might constitute a relatively atrial-selective target. This review will give a general introduction to SK channels followed by their proposed function in the heart under normal and pathophysiological conditions. It is revealed how antiarrhythmic effects can be obtained by SK channel inhibition in a number of species in situations of atrial fibrillation. On the contrary, the beneficial effects of SK channel inhibition in situations of heart failure are questionable and still needs investigation. The understanding of cardiac SK channels is rapidly increasing these years, and it is hoped that this will clarify whether SK channel inhibition has potential as a new anti–atrial fibrillation principle. PMID:25830485

  1. Role of Calcium-activated Potassium Channels in Atrial Fibrillation Pathophysiology and Therapy.

    PubMed

    Diness, Jonas G; Bentzen, Bo H; Sørensen, Ulrik S; Grunnet, Morten

    2015-11-01

    Small-conductance Ca(2+)-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 because they might constitute a relatively atrial-selective target. This review will give a general introduction to SK channels followed by their proposed function in the heart under normal and pathophysiological conditions. It is revealed how antiarrhythmic effects can be obtained by SK channel inhibition in a number of species in situations of atrial fibrillation. On the contrary, the beneficial effects of SK channel inhibition in situations of heart failure are questionable and still needs investigation. The understanding of cardiac SK channels is rapidly increasing these years, and it is hoped that this will clarify whether SK channel inhibition has potential as a new anti-atrial fibrillation principle. PMID:25830485

  2. PYR/PYL/RCAR Abscisic Acid Receptors Regulate K+ and Cl− Channels through Reactive Oxygen Species-Mediated Activation of Ca2+ Channels at the Plasma Membrane of Intact Arabidopsis Guard Cells1[W][OPEN

    PubMed Central

    Wang, Yizhou; Chen, Zhong-Hua; Zhang, Ben; Hills, Adrian; Blatt, Michael R.

    2013-01-01

    The discovery of the START family of abscisic acid (ABA) receptors places these proteins at the front of a protein kinase/phosphatase signal cascade that promotes stomatal closure. The connection of these receptors to Ca2+ signals evoked by ABA has proven more difficult to resolve, although it has been implicated by studies of the pyrbactin-insensitive pyr1/pyl1/pyl2/pyl4 quadruple mutant. One difficulty is that flux through plasma membrane Ca2+ channels and Ca2+ release from endomembrane stores coordinately elevate cytosolic free Ca2+ concentration ([Ca2+]i) in guard cells, and both processes are facilitated by ABA. Here, we describe a method for recording Ca2+ channels at the plasma membrane of intact guard cells of Arabidopsis (Arabidopsis thaliana). We have used this method to resolve the loss of ABA-evoked Ca2+ channel activity at the plasma membrane in the pyr1/pyl1/pyl2/pyl4 mutant and show the consequent suppression of [Ca2+]i increases in vivo. The basal activity of Ca2+ channels was not affected in the mutant; raising the concentration of Ca2+ outside was sufficient to promote Ca2+ entry, to inactivate current carried by inward-rectifying K+ channels and to activate current carried by the anion channels, both of which are sensitive to [Ca2+]i elevations. However, the ABA-dependent increase in reactive oxygen species (ROS) was impaired. Adding the ROS hydrogen peroxide was sufficient to activate the Ca2+ channels and trigger stomatal closure in the mutant. These results offer direct evidence of PYR/PYL/RCAR receptor coupling to the activation by ABA of plasma membrane Ca2+ channels through ROS, thus affecting [Ca2+]i and its regulation of stomatal closure. PMID:23899646

  3. SK channel activation modulates mitochondrial respiration and attenuates neuronal HT-22 cell damage induced by H2O2.

    PubMed

    Richter, Maren; Nickel, Catharina; Apel, Lisa; Kaas, Alexander; Dodel, Richard; Culmsee, Carsten; Dolga, Amalia M

    2015-02-01

    Previous studies established an essential role for small conductance calcium-activated potassium (SK) channels in neuronal cell death pathways induced by glutamate excitotoxicity in cortical neurons in vitro and after cerebral ischemia in vivo. In addition to the intracellular calcium deregulation, glutamate-induced cell death also involves mechanisms of oxidative stress and mitochondrial dysfunction. Therefore, we sought to investigate whether SK channel activation might also affect mechanisms of intrinsic death pathways induced by reactive oxygen species (ROS) such as hydrogen peroxide (H2O2). Exposure of immortalized hippocampal HT-22 cells to H2O2 imposed activation of a cascade of intracellular toxic events resulting in intracellular ROS production, mitochondrial loss of function, and ultimately cell death. Using a pharmacological approach to activate SK channels with CyPPA, we demonstrated a reduction of H2O2-mediated intracellular ROS production and cell death. Interestingly, CyPPA mediated neuroprotection in conditions of extracellular calcium and/or pyruvate depletion, pointing to a neuroprotective role of mitochondrial SK channels. Moreover, CyPPA partially inhibited H2O2-induced mitochondrial superoxide production, but did not prevent mitochondrial membrane depolarization. CyPPA treatment resulted in slight ATP depletion and a reduction of mitochondrial respiration/oxygen consumption. These findings postulate that SK channels mediate a protective effect by preventing neuronal death from subsequent oxidative stress through an adaptive metabolic response at the level of mitochondria. Therefore, SK channel activation may serve as a therapeutic target, where mitochondrial dysfunction and related mechanisms of oxidative stress contribute to progressive degeneration and death of neurons. PMID:25576183

  4. Tubular Unimolecular Transmembrane Channels: Construction Strategy and Transport Activities.

    PubMed

    Si, Wen; Xin, Pengyang; Li, Zhan-Ting; Hou, Jun-Li

    2015-06-16

    Lipid bilayer membranes separate living cells from their environment. Membrane proteins are responsible for the processing of ion and molecular inputs and exports, sensing stimuli and signals across the bilayers, which may operate in a channel or carrier mechanism. Inspired by these wide-ranging functions of membrane proteins, chemists have made great efforts in constructing synthetic mimics in order to understand the transport mechanisms, create materials for separation, and develop therapeutic agents. Since the report of an alkylated cyclodextrin for transporting Cu(2+) and Co(2+) by Tabushi and co-workers in 1982, chemists have constructed a variety of artificial transmembrane channels by making use of either the multimolecular self-assembly or unimolecular strategy. In the context of the design of unimolecular channels, important advances have been made, including, among others, the tethering of natural gramicidin A or alamethicin and the modification of various macrocycles such as crown ethers, cyclodextrins, calixarenes, and cucurbiturils. Many of these unimolecular channels exhibit high transport ability for metal ions, particularly K(+) and Na(+). Concerning the development of artificial channels based on macrocyclic frameworks, one straightforward and efficient approach is to introduce discrete chains to reinforce their capability to insert into bilayers. Currently, this approach has found the widest applications in the systems of crown ethers and calixarenes. We envisioned that for macrocycle-based unimolecular channels, control of the arrangement of the appended chains in the upward and/or downward direction would favor the insertion of the molecular systems into bilayers, while the introduction of additional interactions among the chains would further stabilize a tubular conformation. Both factors should be helpful for the formation of new efficient channels. In this Account, we discuss our efforts in designing new unimolecular artificial channels from

  5. How does the anthropogenic activity affect the spring discharge?

    NASA Astrophysics Data System (ADS)

    Hao, Yonghong; Zhang, Juan; Wang, Jiaojiao; Li, Ruifang; Hao, Pengmei; Zhan, Hongbin

    2016-09-01

    Karst hydrological process has largely been altered by climate change and human activity. In many places throughout the world, human activity (e.g. groundwater pumping and dewatering from mining) has intensified and surpassed climate change, where human activity becomes the primary factor that affects groundwater system. But it is still largely unclear how the human activity affects spring discharge in magnitude and periodicity. This study investigates the effects of anthropogenic activity on spring discharge, using the Xin'an Springs of China as an example. The Xin'an Spring discharge were divided into two time periods: the pre-development period from 1956 to 1971 and the post-development period from 1972 to 2013. We confirm the dividing time (i.e. 1971) of these two periods using the Wilcoxon rank-sum test. Then the wavelet transform and wavelet coherence were used to analyze the karst hydrological processes for the two periods respectively. We analyze the correlations of precipitation and the Xin'an spring discharge with the monsoons including the Indian Summer Monsoon (ISM) and the West North Pacific Monsoon (WNPM) and the climate teleconnections including El Niño Southern Oscillation (ENSO) and Pacific Decadal Oscillation (PDO), respectively. The results indicated that the spring discharge was attenuated about 19.63% under the influence of human activity in the Xin'an Springs basin. However, human activity did not alter the size of the resonance frequencies between the spring discharge and the monsoons. In contrast, it reinforced the periodicities of the monsoons-driven spring discharge. It suggested that human has adapted to the major climate periodicities, and human activity had the same rhyme with the primary climate periodicity. In return, human activity enhances the correlation between the monsoons and the spring discharge.

  6. Brain-Derived Neurotrophic Factor Inhibits Calcium Channel Activation, Exocytosis, and Endocytosis at a Central Nerve Terminal

    PubMed Central

    Baydyuk, Maryna; Wu, Xin-Sheng; He, Liming

    2015-01-01

    Brain-derived neurotrophic factor (BDNF) is a neurotrophin that regulates synaptic function and plasticity and plays important roles in neuronal development, survival, and brain disorders. Despite such diverse and important roles, how BDNF, or more generally speaking, neurotrophins affect synapses, particularly nerve terminals, remains unclear. By measuring calcium currents and membrane capacitance during depolarization at a large mammalian central nerve terminal, the rat calyx of Held, we report for the first time that BDNF slows down calcium channel activation, including P/Q-type channels, and inhibits exocytosis induced by brief depolarization or single action potentials, inhibits slow and rapid endocytosis, and inhibits vesicle mobilization to the readily releasable pool. These presynaptic mechanisms may contribute to the important roles of BDNF in regulating synapses and neuronal circuits and suggest that regulation of presynaptic calcium channels, exocytosis, and endocytosis are potential mechanisms by which neurotrophins achieve diverse neuronal functions. PMID:25788684

  7. Lifetime and conductance of acetylcholine-activated channels in normal and denervated toad sartorius muscle.

    PubMed Central

    Gage, P W; Hamill, O P

    1980-01-01

    1. The average lifetime and conductance of acetylcholine-activated channels were measured in normal and denervated, voltage-clamped toad sartorius muscle fibres at 10 degrees C. 2. The null potential was -4 +/- 1 mV for subsynaptic channels in normal fibres and -6 +/- 3 mV for extrasynaptic channels in denervated fibres. 3. There was a linear relationship between variance of conductance fluctuations and mean conductance for acetylcholine-induced currents up to 50 nA, in denervated fibres clamped at -50 mV. The ratio gave a channel conductance of 14 pS. 4. At the same membrane potential, the average lifetime of extrasynaptic channels in denervated fibres was approximately double, whereas channel conductance was approximately half, that of subsynaptic channels in normal fibres: there was little difference in net charge transfer through the two types of channel under similar conditions. 5. Single channel conductance increased, whereas average channel lifetime decreased, as the membrane potential became more positive (depolarized). The effect of potential on channel lifetime and conductance was more pronounced in denervated than in normal fibres. PMID:6767026

  8. Regulation of Human Kv1.4 Channel Activity by the Antidepressant Metergoline.

    PubMed

    Yeom, Hye Duck; Lee, Jun-Ho

    2016-01-01

    Metergoline is an ergot-derived psychoactive drug that is a ligand for various serotonin and dopamine receptors. Little is known about the effect of metergoline on different types of receptors and ion channels. Potassium channels are the most diverse group of ion channels. Kv1.4, a shaker family K channel alpha subunit, is one of a family of voltage gated K channels that mediates transient and rapid inactivating A-type currents and N-type inactivation. We demonstrated previously that metergoline inhibited the activity of neuronal voltage-dependent Na(+) channels in Xenopus laevis oocytes (Acta Pharmacol. Sin., 35, 2014, Lee et al.). In this study, we sought to elucidate the regulatory effects underlying metergoline-induced human Kv1.4 channel inhibition. We used the two electrode voltage-clamp (TEVC) technique to investigate the effect of metergoline on human Kv1.4 channel currents in Xenopus laevis oocytes expressing human Kv1.4 alpha subunits. Interestingly, metergoline treatment also induced inhibition of peak currents in human Kv1.4 channels in a concentration-dependent manner. The IC50 of peak currents of hKv1.4 currents was 3.6±0.6 µM. These results indicate that metergoline might regulate the human Kv1.4 channel activity that is expressed in X. laevis oocytes. Further, this regulation of potassium currents by metergoline might be one of the pharmacological actions of metergoline-mediated psychoactivity. PMID:27251511

  9. Luminal acetylcholine does not affect the activity of the CFTR in tracheal epithelia of pigs.

    PubMed

    Dittrich, Nikolaus P; Kummer, Wolfgang; Clauss, Wolfgang G; Fronius, Martin

    2015-11-01

    Fluid homeostasis mediated by the airway epithelium is required for proper lung function, and the CFTR (cystic fibrosis transmembrane conductance regulator) Cl(-) channel is crucial for these processes. Luminal acetylcholine (ACh) acts as an auto-/paracrine mediator to activate Cl(-) channels in airway epithelia and evidence exists showing that nicotinic ACh receptors activate CFTR in murine airway epithelia. The present study investigated whether or not luminal ACh regulates CFTR activity in airway epithelia of pigs, an emerging model for investigations of human airway disease and cystic fibrosis (CF) in particular. Transepithelial ion currents of freshly dissected pig tracheal preparations were measured with Ussing chambers. Application of luminal ACh (100 μM) induced an increase of the short-circuit current (I(SC)). The ACh effect was mimicked by muscarine and pilocarpine (100 μM each) and was sensitive to muscarinic receptor antagonists (atropine, 4-DAMP, pirenzepine). No changes of the I(SC) were observed by nicotine (100 μM) and ACh responses were not affected by nicotine or mecamylamine (25 μM). Luminal application of IBMX (I, 100 μM) and forskolin (F, 10 μM), increase the I(SC) and the I/F-induced current were decreased by the CFTR inhibitor GlyH-101 (GlyH, 50 μM) indicating increased CFTR activity by I/F. In contrast, GlyH did not affect the ACh-induced current, indicating that the ACh response does not involve the activation of the CFTR. Results from this study suggest that luminal ACh does not regulate the activity of the CFTR in tracheal epithelia of pigs which opposes observation from studies using mice airway epithelium. PMID:26286842

  10. Factors affecting measurement of channel thickness in asymmetrical flow field-flow fractionation.

    PubMed

    Dou, Haiyang; Jung, Euo Chang; Lee, Seungho

    2015-05-01

    Asymmetrical flow field-flow fractionation (AF4) has been considered to be a useful tool for simultaneous separation and characterization of polydisperse macromolecules or colloidal nanoparticles. AF4 analysis requires the knowledge of the channel thickness (w), which is usually measured by injecting a standard with known diffusion coefficient (D) or hydrodynamic diameter (dh). An accurate w determination is a challenge due to its uncertainties arising from the membrane's compressibility, which may vary with experimental condition. In the present study, influence of factors including the size and type of the standard on the measurement of w was systematically investigated. The results revealed that steric effect and the particles-membrane interaction by van der Waals or electrostatic force may result in an error in w measurement. PMID:25817708

  11. The AQP-3 water channel is a pivotal modulator of glycerol-induced chloride channel activation in nasopharyngeal carcinoma cells.

    PubMed

    Zhang, Haifeng; Deng, Zhiqin; Yang, Lili; Luo, Hai; Liu, Shanwen; Li, Yuan; Wei, Yan; Peng, Shuang; Zhu, Linyan; Wang, Liwei; Chen, Lixin

    2016-03-01

    Aquaporin (AQP) and chloride channels are ubiquitous in virtually all living cells, playing pivotal roles in cell proliferation, migration and apoptosis. We previously reported that AQP-3 aquaglyceroporin and ClC-3 chloride channels could form complexes to regulate cell volume in nasopharyngeal carcinoma cells. In this study, the roles of AQP-3 in their hetero-complexes were further investigated. Glycerol entered the cells via AQP-3 and induced two different Cl(-) currents through cell swelling-dependent or -independent pathways. The swelling-dependent Cl(-) current was significantly inhibited by pretreatment with CuCl2 and AQP-3-siRNA. After siRNA-induced AQP-3 knock-down, the 140 mM glycerol isoosmotic solution swelled cells by 22% (45% in AQP-3-intact cells) and induced a smaller Cl(-) current; this current was smaller than that activated by 8% cell volume swelling, which induced by the 140 mM glycerol hyperosmotic solution in AQP-3-intact cells. This suggests that the interaction between AQP-3 and ClC-3 plays an important role in cell volume regulation and that AQP-3 may be a modulator that opens volume-regulated chloride channels. The swelling-independent Cl(-) current, which was activated by extracellular glycerol, was reduced by CuCl2 and AQP-3-siRNA pretreatment. Dialyzing glycerol into cells via the pipette directly induced the swelling-independent Cl(-) current; however this current was blocked by AQP-3 down-regulation, suggesting AQP-3 is essential for the opening of chloride channels. In conclusion, AQP-3 is the pathway for water, glycerol and other small solutes to enter cells, and it may be an essential modulator for the gating of chloride channels. PMID:26794461

  12. Control of Inward Rectifier K Channel Activity by Lipid Tethering of Cytoplasmic Domains

    PubMed Central

    Enkvetchakul, Decha; Jeliazkova, Iana; Bhattacharyya, Jaya; Nichols, Colin G.

    2007-01-01

    Interactions between nontransmembrane domains and the lipid membrane are proposed to modulate activity of many ion channels. In Kir channels, the so-called “slide-helix” is proposed to interact with the lipid headgroups and control channel gating. We examined this possibility directly in a cell-free system consisting of KirBac1.1 reconstituted into pure lipid vesicles. Cysteine substitution of positively charged slide-helix residues (R49C and K57C) leads to loss of channel activity that is rescued by in situ restoration of charge following modification by MTSET+ or MTSEA+, but not MTSES− or neutral MMTS. Strikingly, activity is also rescued by modification with long-chain alkyl-MTS reagents. Such reagents are expected to partition into, and hence tether the side chain to, the membrane. Systematic scanning reveals additional slide-helix residues that are activated or inhibited following alkyl-MTS modification. A pattern emerges whereby lipid tethering of the N terminus, or C terminus, of the slide-helix, respectively inhibits, or activates, channel activity. This study establishes a critical role of the slide-helix in Kir channel gating, and directly demonstrates that physical interaction of soluble domains with the membrane can control ion channel activity. PMID:17698595

  13. Cyclic GMP-mediated inhibition of L-type Ca2+ channel activity by human natriuretic peptide in rabbit heart cells.

    PubMed Central

    Tohse, N; Nakaya, H; Takeda, Y; Kanno, M

    1995-01-01

    1. Effects of atrial natriuretic peptide (ANP) on the L-type Ca2+ channels were examined in rabbit isolated ventricular cells by use of whole-cell and cell-attached configurations of the patch clamp methods. ANP produced a concentration-dependent decrease (10-100 nM) in amplitude of a basal Ca2+ channel current. 2. The inactive ANP (methionine-oxidized ANP, 30 nM) failed to decrease the current. 3. 8-Bromo-cyclic GMP (300 microM), a potent activator of cyclic GMP-dependent protein kinase (PKG), produced the same effects on the basal Ca2+ channel current as those produced by ANP. The cyclic GMP-induced inhibition of the Ca2+ channel current was still evoked in the presence of 1-isobutyl-3-methyl-xanthine, an inhibitor of phosphodiesterase. ANP failed to produce inhibition of the Ca2+ channel current in the presence of 8-bromo-cyclic GMP. 4. In the single channel recording, ANP and 8-bromo-cyclic GMP also inhibited the activities of the L-type Ca2+ channels. Both agents decreased the open probability (NPo) without affecting the unit amplitude. 5. The present results suggest that ANP inhibits the cardiac L-type Ca2+ channel activity through the intracellular production of cyclic GMP and then activation of PKG. PMID:7540093

  14. Identification of a Peptide Toxin from Grammostola spatulata Spider Venom That Blocks Cation-Selective Stretch-Activated Channels

    PubMed Central

    Suchyna, Thomas M.; Johnson, Janice H.; Hamer, Katherine; Leykam, Joseph F.; Gage, Douglas A.; Clemo, Henry F.; Baumgarten, Clive M.; Sachs, Frederick

    2000-01-01

    We have identified a 35 amino acid peptide toxin of the inhibitor cysteine knot family that blocks cationic stretch-activated ion channels. The toxin, denoted GsMTx-4, was isolated from the venom of the spider Grammostola spatulata and has <50% homology to other neuroactive peptides. It was isolated by fractionating whole venom using reverse phase HPLC, and then assaying fractions on stretch-activated channels (SACs) in outside-out patches from adult rat astrocytes. Although the channel gating kinetics were different between cell-attached and outside-out patches, the properties associated with the channel pore, such as selectivity for alkali cations, conductance (∼45 pS at −100 mV) and a mild rectification were unaffected by outside-out formation. GsMTx-4 produced a complete block of SACs in outside-out patches and appeared specific since it had no effect on whole-cell voltage-sensitive currents. The equilibrium dissociation constant of ∼630 nM was calculated from the ratio of association and dissociation rate constants. In hypotonically swollen astrocytes, GsMTx-4 produces ∼40% reduction in swelling-activated whole-cell current. Similarly, in isolated ventricular cells from a rabbit dilated cardiomyopathy model, GsMTx-4 produced a near complete block of the volume-sensitive cation-selective current, but did not affect the anion current. In the myopathic heart cells, where the swell-induced current is tonically active, GsMTx-4 also reduced the cell size. This is the first report of a peptide toxin that specifically blocks stretch-activated currents. The toxin affect on swelling-activated whole-cell currents implicates SACs in volume regulation. PMID:10779316

  15. A synthetic prostone activates apical chloride channels in A6 epithelial cells

    PubMed Central

    Bao, Hui Fang; Liu, Lian; Self, Julie; Duke, Billie Jeanne; Ueno, Ryuji; Eaton, Douglas C.

    2008-01-01

    The bicyclic fatty acid lubiprostone (formerly known as SPI-0211) activates two types of anion channels in A6 cells. Both channel types are rarely, if ever, observed in untreated cells. The first channel type was activated at low concentrations of lubiprostone (<100 nM) in >80% of cell-attached patches and had a unit conductance of ∼3–4 pS. The second channel type required higher concentrations (>100 nM) of lubiprostone to activate, was observed in ∼30% of patches, and had a unit conductance of 8–9 pS. The properties of the first type of channel were consistent with ClC-2 and the second with CFTR. ClC-2's unit current strongly inwardly rectified that could be best fit by models of the channel with multiple energy barrier and multiple anion binding sites in the conductance pore. The open probability and mean open time of ClC-2 was voltage dependent, decreasing dramatically as the patches were depolarized. The order of anion selectivity for ClC-2 was Cl > Br > NO3 > I > SCN, where SCN is thiocyanate. ClC-2 was a “double-barreled” channel favoring even numbers of levels over odd numbers as if the channel protein had two conductance pathways that opened independently of one another. The channel could be, at least, partially blocked by glibenclamide. The properties of the channel in A6 cells were indistinguishable from ClC-2 channels stably transfected in HEK293 cells. CFTR in the patches had a selectivity of Cl > Br ≫ NO3 ≅ SCN ≅ I. It outwardly rectified as expected for a single-site anion channel. Because of its properties, ClC-2 is uniquely suitable to promote anion secretion with little anion reabsorption. CFTR, on the other hand, could promote either reabsorption or secretion depending on the anion driving forces. PMID:18511742

  16. Metal Toxicity Affects Fungal and Bacterial Activities in Soil Differently

    PubMed Central

    Rajapaksha, R. M. C. P.; Tobor-Kapłon, M. A; Bååth, E.

    2004-01-01

    Although the toxic effect of heavy metals on soil microorganism activity is well known, little is known about the effects on different organism groups. The influence of heavy metal addition on total, bacterial, and fungal activities was therefore studied for up to 60 days in a laboratory experiment using forest soil contaminated with different concentrations of Zn or Cu. The effects of the metals differed between the different activity measurements. During the first week after metal addition, the total activity (respiration rate) decreased by 30% at the highest level of contamination and then remained stable during the 60 days of incubation. The bacterial activity (thymidine incorporation rate) decreased during the first days with the level of metal contamination, resulting in a 90% decrease at the highest level of contamination. Bacterial activity then slowly recovered to values similar to those of the control soil. The recovery was faster when soil pH, which had decreased due to metal addition, was restored to control values by liming. Fungal activity (acetate-in-ergosterol incorporation rate) initially increased with the level of metal contamination, being up to 3 and 7 times higher than that in the control samples during the first week at the highest levels of Zn and Cu addition, respectively. The positive effect of metal addition on fungal activity then decreased, but fungal activity was still higher in contaminated than in control soil after 35 days. This is the first direct evidence that fungal and bacterial activities in soil are differently affected by heavy metals. The different responses of bacteria and fungi to heavy metals were reflected in an increase in the relative fungal/bacterial ratio (estimated using phospholipid fatty acid analysis) with increased metal load. PMID:15128558

  17. Metal toxicity affects fungal and bacterial activities in soil differently.

    PubMed

    Rajapaksha, R M C P; Tobor-Kapłon, M A; Bååth, E

    2004-05-01

    Although the toxic effect of heavy metals on soil microorganism activity is well known, little is known about the effects on different organism groups. The influence of heavy metal addition on total, bacterial, and fungal activities was therefore studied for up to 60 days in a laboratory experiment using forest soil contaminated with different concentrations of Zn or Cu. The effects of the metals differed between the different activity measurements. During the first week after metal addition, the total activity (respiration rate) decreased by 30% at the highest level of contamination and then remained stable during the 60 days of incubation. The bacterial activity (thymidine incorporation rate) decreased during the first days with the level of metal contamination, resulting in a 90% decrease at the highest level of contamination. Bacterial activity then slowly recovered to values similar to those of the control soil. The recovery was faster when soil pH, which had decreased due to metal addition, was restored to control values by liming. Fungal activity (acetate-in-ergosterol incorporation rate) initially increased with the level of metal contamination, being up to 3 and 7 times higher than that in the control samples during the first week at the highest levels of Zn and Cu addition, respectively. The positive effect of metal addition on fungal activity then decreased, but fungal activity was still higher in contaminated than in control soil after 35 days. This is the first direct evidence that fungal and bacterial activities in soil are differently affected by heavy metals. The different responses of bacteria and fungi to heavy metals were reflected in an increase in the relative fungal/bacterial ratio (estimated using phospholipid fatty acid analysis) with increased metal load. PMID:15128558

  18. Let It Go and Open Up, an Ensemble of Ion Channel Active States.

    PubMed

    Minor, Daniel L

    2016-02-11

    Ligand binding usually moves the target protein from an ensemble of inactive states to a well-defined active conformation. Matthies et al. flip this scheme around, finding that, for the magnesium channel CorA, loss of ligand binding induces an ensemble of conformations that turn the channel on. PMID:26871624

  19. Endogenous KCNE Subunits Govern Kv2.1 K+ Channel Activation Kinetics in Xenopus Oocyte Studies

    PubMed Central

    Gordon, Earl; Roepke, Torsten K.; Abbott, Geoffrey W.

    2006-01-01

    Kv2.1 is a voltage-gated potassium (Kv) channel that generates delayed rectifier currents in mammalian heart and brain. The biophysical properties of Kv2.1 and other ion channels have been characterized by functional expression in heterologous systems, and most commonly in Xenopus laevis oocytes. A number of previous oocyte-based studies of mammalian potassium channels have revealed expression-level-dependent changes in channel properties, leading to the suggestion that endogenous oocyte factors regulate channel gating. Here, we show that endogenous oocyte potassium channel KCNE ancillary subunits xMinK and xMiRP2 slow the activation of oocyte-expressed mammalian Kv2.1 channels two-to-fourfold. This produces a sigmoidal relationship between Kv2.1 current density and activation rate in oocyte-based two-electrode voltage clamp studies. The effect of endogenous xMiRP2 and xMinK on Kv2.1 activation is diluted at high Kv2.1 expression levels, or by RNAi knockdown of either endogenous subunit. RNAi knockdown of both xMiRP2 and xMinK eliminates the correlation between Kv2.1 expression level and activation kinetics. The data demonstrate a molecular basis for expression-level-dependent changes in Kv channel gating observed in heterologous expression studies. PMID:16326911

  20. Human Immunodeficiency Syndromes Affecting Human Natural Killer Cell Cytolytic Activity

    PubMed Central

    Ham, Hyoungjun; Billadeau, Daniel D.

    2013-01-01

    Natural killer (NK) cells are lymphocytes of the innate immune system that secrete cytokines upon activation and mediate the killing of tumor cells and virus-infected cells, especially those that escape the adaptive T cell response caused by the down regulation of MHC-I. The induction of cytotoxicity requires that NK cells contact target cells through adhesion receptors, and initiate activation signaling leading to increased adhesion and accumulation of F-actin at the NK cell cytotoxic synapse. Concurrently, lytic granules undergo minus-end directed movement and accumulate at the microtubule-organizing center through the interaction with microtubule motor proteins, followed by polarization of the lethal cargo toward the target cell. Ultimately, myosin-dependent movement of the lytic granules toward the NK cell plasma membrane through F-actin channels, along with soluble N-ethylmaleimide-sensitive factor attachment protein receptor-dependent fusion, promotes the release of the lytic granule contents into the cleft between the NK cell and target cell resulting in target cell killing. Herein, we will discuss several disease-causing mutations in primary immunodeficiency syndromes and how they impact NK cell-mediated killing by disrupting distinct steps of this tightly regulated process. PMID:24478771

  1. Ion channel modifying agents influence the electrical activity generated by canine intrinsic cardiac neurons in situ.

    PubMed

    Thompson, G W; Horackova, M; Armour, J A

    2000-04-01

    This study was designed to establish whether agents known to modify neuronal ion channels influence the behavior of mammalian intrinsic cardiac neurons in situ and, if so, in a manner consistent with that found previously in vitro. The activity generated by right atrial neurons was recorded extracellularly in varying numbers of anesthetized dogs before and during continuous local arterial infusion of several neuronal ion channel modifying agents. Veratridine (7.5 microM), the specific modifier of Na+-selective channels, increased neuronal activity (95% above control) in 80% of dogs tested (n = 25). The membrane depolarizing agent potassium chloride (40 mM) reduced neuronal activity (43% below control) in 84% of dogs tested (n = 19). The inhibitor of voltage-sensitive K+ channels, tetraethylammonium (10 mM), decreased neuronal activity (42% below control) in 73% of dogs tested (n = 11). The nonspecific potassium channel inhibitor barium chloride (5 mM) excited neurons (47% above control) in 13 of 19 animals tested. Cadmium chloride (200 microM), which inhibits Ca2+-selective channels and Ca2+-dependent K+ channels, increased neuronal activity (65% above control) in 79% of dogs tested (n = 14). The specific L-type Ca2+ channel blocking agent nifedipine (5 microM) reduced neuronal activity (52% blow control in 72% of 11 dogs tested), as did the nonspecific inhibitor of L-type Ca2+ channels, nickel chloride (5 mM) (36% below control in 69% of 13 dogs tested). Each agent induced either excitatory or inhibitory responses, depending on the agent tested. It is concluded that specific ion channels (I(Na), I(CaL), I(Kv), and I(KCa)) that have been associated with intrinsic cardiac neurons in vitro are involved in their capacity to generate action potentials in situ. PMID:10772056

  2. Calmodulin-dependent activation and inactivation of anoctamin calcium-gated chloride channels

    PubMed Central

    Vocke, Kerstin; Dauner, Kristin; Hahn, Anne; Ulbrich, Anne; Broecker, Jana; Keller, Sandro; Frings, Stephan

    2013-01-01

    Calcium-dependent chloride channels serve critical functions in diverse biological systems. Driven by cellular calcium signals, the channels codetermine excitatory processes and promote solute transport. The anoctamin (ANO) family of membrane proteins encodes three calcium-activated chloride channels, named ANO 1 (also TMEM16A), ANO 2 (also TMEM16B), and ANO 6 (also TMEM16F). Here we examined how ANO 1 and ANO 2 interact with Ca2+/calmodulin using nonstationary current analysis during channel activation. We identified a putative calmodulin-binding domain in the N-terminal region of the channel proteins that is involved in channel activation. Binding studies with peptides indicated that this domain, a regulatory calmodulin-binding motif (RCBM), provides two distinct modes of interaction with Ca2+/calmodulin, one at submicromolar Ca2+ concentrations and one in the micromolar Ca2+ range. Functional, structural, and pharmacological data support the concept that calmodulin serves as a calcium sensor that is stably associated with the RCBM domain and regulates the activation of ANO 1 and ANO 2 channels. Moreover, the predominant splice variant of ANO 2 in the brain exhibits Ca2+/calmodulin-dependent inactivation, a loss of channel activity within 30 s. This property may curtail ANO 2 activity during persistent Ca2+ signals in neurons. Mutagenesis data indicated that the RCBM domain is also involved in ANO 2 inactivation, and that inactivation is suppressed in the retinal ANO 2 splice variant. These results advance the understanding of Ca2+ regulation in anoctamin Cl− channels and its significance for the physiological function that anoctamin channels subserve in neurons and other cell types. PMID:24081981

  3. Neutron field for activation experiments in horizontal channel of training reactor VR-1

    NASA Astrophysics Data System (ADS)

    Stefanik, Milan; Katovsky, Karel; Vins, Miroslav; Soltes, Jaroslav; Zavorka, Lukas

    2014-11-01

    The experimental channels of nuclear reactors often serve for nuclear data measurement and validation. The dosimetry-foils activation technique was employed to measure neutron field parameters in the horizontal radial channel of the training reactor VR-1, and to test the possibility of using the reactor for scientific purposes. The reaction rates, energy spectral indexes, and neutron spectrum at several irradiation positions of the experimental channel were determined. The experimental results show the feasibility of the radial channel for irradiating experiments and open new possibilities for data validation by using this nuclear facility.

  4. Serotonin and Dopamine: Unifying Affective, Activational, and Decision Functions

    PubMed Central

    Cools, Roshan; Nakamura, Kae; Daw, Nathaniel D

    2011-01-01

    Serotonin, like dopamine (DA), has long been implicated in adaptive behavior, including decision making and reinforcement learning. However, although the two neuromodulators are tightly related and have a similar degree of functional importance, compared with DA, we have a much less specific understanding about the mechanisms by which serotonin affects behavior. Here, we draw on recent work on computational models of dopaminergic function to suggest a framework by which many of the seemingly diverse functions associated with both DA and serotonin—comprising both affective and activational ones, as well as a number of other functions not overtly related to either—can be seen as consequences of a single root mechanism. PMID:20736991

  5. Single Mutations in the Transmembrane Domains of Maize Plasma Membrane Aquaporins Affect the Activity of Monomers within a Heterotetramer.

    PubMed

    Berny, Marie C; Gilis, Dimitri; Rooman, Marianne; Chaumont, François

    2016-07-01

    Aquaporins are channels facilitating the diffusion of water and/or small uncharged solutes across biological membranes. They assemble as homotetramers but some of them also form heterotetramers, especially in plants. In Zea mays, aquaporins belonging to the plasma membrane intrinsic protein (PIP) subfamily are clustered into two groups, PIP1 and PIP2, which exhibit different water-channel activities when expressed in Xenopus oocytes. When PIP1 and PIP2 isoforms are co-expressed, they physically interact to modulate their subcellular localization and channel activity. Here, we demonstrated by affinity chromatography purification that, when co-expressed in Xenopus oocytes, the maize PIP1;2 and PIP2;5 isoforms assemble as homo- and heterodimers within heterotetramers. We built the 3D structure of such heterotetramers by comparative modeling on the basis of the spinach SoPIP2;1 X-ray structure and identified amino acid residues in the transmembrane domains which putatively interact at the interfaces between monomers. Their roles in the water-channel activity, subcellular localization, protein abundance, and physical interaction were investigated by mutagenesis. We highlighted single-residue substitutions that either inactivated PIP2;5 or activated PIP1;2 without affecting their interaction. Interestingly, the Phe220Ala mutation in the transmembrane domain 5 of PIP1;2 activated its water-channel activity and, at the same time, inactivated PIP2;5 within a heterotetramer. Altogether, these data contribute to a better understanding of the interaction mechanisms between PIP isoforms and the role of heterotetramerization on their water-channel activity. PMID:27109604

  6. Fe(2+) substrate transport through ferritin protein cage ion channels influences enzyme activity and biomineralization.

    PubMed

    Behera, Rabindra K; Torres, Rodrigo; Tosha, Takehiko; Bradley, Justin M; Goulding, Celia W; Theil, Elizabeth C

    2015-09-01

    Ferritins, complex protein nanocages, form internal iron-oxy minerals (Fe2O3·H2O), by moving cytoplasmic Fe(2+) through intracage ion channels to cage-embedded enzyme (2Fe(2+)/O2 oxidoreductase) sites where ferritin biomineralization is initiated. The products of ferritin enzyme activity are diferric oxy complexes that are mineral precursors. Conserved, carboxylate amino acid side chains of D127 from each of three cage subunits project into ferritin ion channels near the interior ion channel exits and, thus, could direct Fe(2+) movement to the internal enzyme sites. Ferritin D127E was designed and analyzed to probe properties of ion channel size and carboxylate crowding near the internal ion channel opening. Glu side chains are chemically equivalent to, but longer by one -CH2 than Asp, side chains. Ferritin D127E assembled into normal protein cages, but diferric peroxo formation (enzyme activity) was not observed, when measured at 650 nm (DFP λ max). The caged biomineral formation, measured at 350 nm in the middle of the broad, nonspecific Fe(3+)-O absorption band, was slower. Structural differences (protein X-ray crystallography), between ion channels in wild type and ferritin D127E, which correlate with the inhibition of ferritin D127E enzyme activity include: (1) narrower interior ion channel openings/pores; (2) increased numbers of ion channel protein-metal binding sites, and (3) a change in ion channel electrostatics due to carboxylate crowding. The contributions of ion channel size and structure to ferritin activity reflect metal ion transport in ion channels are precisely regulated both in ferritin protein nanocages and membranes of living cells. PMID:26202907

  7. Ca2+-Activated K+ Channels in Gonadotropin-Releasing Hormone-Stimulated Mouse Gonadotrophs

    PubMed Central

    Waring, Dennis W.; Turgeon, Judith L.

    2009-01-01

    GnRH receptor activation elicits release of intracellular Ca2+, which leads to secretion and also activates Ca2+-activated ion channels underlying membrane voltage changes. The predominant Ca2+-activated ion channels in rat and mouse gonadotrophs are Ca2+-activated K+ channels. To establish the temporal relationship between GnRH-induced changes in intracellular [Ca2+] ([Ca2+]i) and membrane current (Im), and to identify specific Ca2+-activated K+ channels linking GnRH-induced increase in [Ca2+]i to changes in plasma membrane electrical activity, we used single female mouse gonadotrophs in the perforated patch configuration of the patch-clamp technique, which preserves signaling pathways. Simultaneous measurement of [Ca2+]i and Im in voltage-clamped gonadotrophs revealed that GnRH stimulates an increase in [Ca2+]i that precedes outward Im, and that activates two kinetically distinct currents identified, using specific toxin inhibitors, as small conductance Ca2+-activated K+ (SK) current (ISK) and large (big) conductance voltage- and Ca2+-activated K+ (BK) current (IBK). We show that the apamin-sensitive current has an IC50 of 69 pM, consistent with the SK2 channel subtype and confirmed by immunocytochemistry. The magnitude of the SK current response to GnRH was attenuated by 17β-estradiol (E2) pretreatment. Iberiotoxin, an inhibitor of BK channels, completely blocked the residual apamin-insensitive outward Im, substantiating that IBK is a component of the GnRH-induced outward Im. In contrast to its suppression of ISK, E2 pretreatment augmented peak IBK. SK or BK channel inhibition modulated GnRH-stimulated LH secretion, implicating a role for these channels in gonadotroph function. In summary, in mouse gonadotrophs the GnRH-stimulated increase in [Ca2+]i activates ISK and IBK, which are differentially regulated by E2 and which may be targets for E2 positive feedback in LH secretion. PMID:19106218

  8. Factors affecting the behavior of unburned carbon upon steam activation

    NASA Astrophysics Data System (ADS)

    Lu, Zhe

    The main objective of this study is to investigate the factors that could affect the behavior of unburned carbon samples upon steam activation. Through this work, the relationships among the factors that could influence the carbon-steam reaction with the surface area of the produced activated carbon were explored. Statistical analysis was used to relate the chemical and physical properties of the unburned carbon to the surface area of the activated carbon. Six unburned carbons were selected as feedstocks for activated carbon, and marked as UCA through UCF. The unburned carbons were activated using steam at 850°C for 90 minutes, and the surface areas of their activated counterparts were measured using N2 adsorption isotherms at 77K. The activated carbons produced from different unburned carbon precursors presented different surface areas at similar carbon burn-off levels. Moreover, in different carbon burn-off regions, the sequences for surface area of activated carbons from different unburned carbon samples were different. The factors that may affect the carbon-steam gasification reactions, including the concentration of carbon active sites, the crystallite size of the carbon, the intrinsic porous structure of carbon, and the inorganic impurities, were investigated. All unburned carbons investigated in this study were similar in that they showed the very broad (002) and (10 ) carbon peaks, which are characteristic of highly disordered carbonaceous materials. In this study, the unburned carbon samples contained about 17--48% of inorganic impurities. Compared to coals, the unburned carbon samples contain a larger amount of inorganic impurities as a result of the burn-off, or at lease part, of the carbon during the combustion process. These inorganic particles were divided into two groups in terms of the way they are associated with carbon particles: free single particles, and particles combined with carbon particles. As indicated from the present work, unburned

  9. Tamoxifen inhibits BK channels in chick cochlea without alterations in voltage-dependent activation.

    PubMed

    Tong, Mingjie; Duncan, R Keith

    2009-07-01

    Large-conductance, Ca(2+)-activated, and voltage-gated potassium channels (BK, BK(Ca), or Maxi-K) play an important role in electrical tuning in nonmammalian vertebrate hair cells. Systematic changes in tuning frequency along the tonotopic axis largely result from variations in BK channel kinetics, but the molecular changes underpinning these functional variations remain unknown. Auxiliary beta(1) have been implicated in low-frequency tuning at the cochlear apex because these subunits dramatically slow channel kinetics. Tamoxifen (Tx), a (xeno)estrogen compound known to activate BK channels through the beta-subunit, was used to test for the functional presence of beta(1). The hypotheses were that Tx would activate the majority of BK channels in hair cells from the cochlear apex due to the presence of beta(1) and that the level of activation would exhibit a tonotopic gradient following the expression profile of beta(1). Outside-out patches of BK channels were excised from tall hair cells along the apical half of the chicken basilar papilla. In low-density patches, single-channel conductance was reduced and the averaged open probability was unaffected by Tx. In high-density patches, the amplitude of ensemble-averaged BK current was inhibited, whereas half-activation potential and activation kinetics were unaffected by Tx. In both cases, no tonotopic Tx-dependent activation of channel activity was observed. Therefore, contrary to the hypotheses, electrophysiological assessment suggests that molecular mechanisms other than auxiliary beta-subunits are involved in generating a tonotopic distribution of BK channel kinetics and electric tuning in chick basilar papilla. PMID:19439526

  10. Deletion of phenylalanine 508 causes attenuated phosphorylation-dependent activation of CFTR chloride channels.

    PubMed

    Wang, F; Zeltwanger, S; Hu, S; Hwang, T C

    2000-05-01

    In cell-attached patches stimulated with cAMP agonists, the single-channel open probability (Po) of the phenylalanine 508-deleted cystic fibrosis transmembrane conductance regulator (DeltaF508-CFTR) channel, the most common disease-associated mutation in cystic fibrosis, was abnormally low (a functional defect). To investigate the mechanism for the poor response of DeltaF508-CFTR to cAMP stimulation, we examined, in excised inside-out patches, protein kinase A (PKA)-dependent phosphorylation activation and ATP-dependent gating of wild-type (WT) and DeltaF508-CFTR channels expressed in NIH3T3 mouse fibroblasts. For WT-CFTR, the activation time course of CFTR channel current upon addition of PKA and ATP followed a sigmoidal function with time constants that decreased as [PKA] was increased. The curvilinear relationship between [PKA] and the apparent activation rate suggests an incremental phosphorylation-dependent activation of CFTR at multiple phosphorylation sites. The time course of PKA-dependent activation of DeltaF508-CFTR channel current also followed a sigmoidal function, but the rate of activation was at least 7-fold slower than that with WT channels. This result suggests that deletion of phenylalanine 508 causes attenuated PKA-dependent phosphorylation of the CFTR chloride channel. Once DeltaF508-CFTR channels were maximally activated with PKA, the mutant channel and WT channel had indistinguishable steady-state Po values, ATP dose-response relationships and single-channel kinetics, indicating that DeltaF508-CFTR is not defective in ATP-dependent gating. By measuring whole-cell current density, we compared the number of functional channels in WT- and DeltaF508-CFTR cell membrane. Our data showed that the estimated channel density for DeltaF508-CFTR was approximately 10-fold lower than that for WT-CFTR, but the cAMP-dependent whole-cell current density differed by approximately 200-fold. We thus conclude that the functional defect (a decrease in Po) of Delta

  11. Deletion of phenylalanine 508 causes attenuated phosphorylation-dependent activation of CFTR chloride channels

    PubMed Central

    Wang, Fei; Zeltwanger, Shawn; Hu, Shenghui; Hwang, Tzyh-Chang

    2000-01-01

    In cell-attached patches stimulated with cAMP agonists, the single-channel open probability (Po) of the phenylalanine 508-deleted cystic fibrosis transmembrane conductance regulator (ΔF508-CFTR) channel, the most common disease-associated mutation in cystic fibrosis, was abnormally low (a functional defect). To investigate the mechanism for the poor response of ΔF508-CFTR to cAMP stimulation, we examined, in excised inside-out patches, protein kinase A (PKA)-dependent phosphorylation activation and ATP-dependent gating of wild-type (WT) and ΔF508-CFTR channels expressed in NIH3T3 mouse fibroblasts.For WT-CFTR, the activation time course of CFTR channel current upon addition of PKA and ATP followed a sigmoidal function with time constants that decreased as [PKA] was increased. The curvilinear relationship between [PKA] and the apparent activation rate suggests an incremental phosphorylation-dependent activation of CFTR at multiple phosphorylation sites.The time course of PKA-dependent activation of ΔF508-CFTR channel current also followed a sigmoidal function, but the rate of activation was at least 7-fold slower than that with WT channels. This result suggests that deletion of phenylalanine 508 causes attenuated PKA-dependent phosphorylation of the CFTR chloride channel.Once ΔF508-CFTR channels were maximally activated with PKA, the mutant channel and WT channel had indistinguishable steady-state Po values, ATP dose-response relationships and single-channel kinetics, indicating that ΔF508-CFTR is not defective in ATP-dependent gating.By measuring whole-cell current density, we compared the number of functional channels in WT- and ΔF508-CFTR cell membrane. Our data showed that the estimated channel density for ΔF508-CFTR was ∼10-fold lower than that for WT-CFTR, but the cAMP-dependent whole-cell current density differed by ∼200-fold. We thus conclude that the functional defect (a decrease in Po) of ΔF508-CFTR is as important as the trafficking defect (a

  12. Using fluvial channel morphology to obtain the neotectonic characteristics of the Liuchia fault, an important active structure in southwestern Taiwan

    NASA Astrophysics Data System (ADS)

    Shyu, J. H.; Du, K.

    2013-12-01

    The Liuchia fault in southwestern Taiwan has been considered as one of the major active faults in the active Taiwan orogen. It is identified by its clear geomorphic features, and forms a major geologic boundary of Taiwan's Western Foothills. No unanimous historical evidence for seismic activity of the Liuchia fault exists, thus the fault poses large earthquake hazard potentials for the populous southwestern Taiwan. Here we attempted to analyze the characteristics of the fault from fluvial channel morphology of the Kueichung River that flows across the fault. We also calculated actual river incision rates from the age of river terraces along the river to obtain the rock uplift rates of the hanging-wall block of the fault. We have obtained a detailed river long profile of the Kueichung River from surveys using RTK-GPS, and a channel width profile from actual field measurements using a Laser Rangefinder. The fluvial channel morphology of the Kueichung River appears to have been affected by active folding in the hanging-wall block of the Liuchia fault. Such active deformation pattern is also evident from river incision rate patterns. Combining these different datasets, we constructed a realistic model of the subsurface geometry of the Liuchia fault in southwestern Taiwan, and calculated the long-term slip rates of this important active structure in southwestern Taiwan.

  13. Can gramicidin ion channel affect the dipole potential of neighboring phospholipid headgroups?

    PubMed

    Becucci, Lucia; Guidelli, Rolando

    2015-12-01

    The cyclic voltammetry behavior of a mercury-supported tethered bilayer lipid membrane (tBLM) incorporating gramicidin A was investigated in aqueous 0.1 M KCl at pH 6.8, 5.4 and 3. The distal leaflet of the lipid bilayer consisted of dioleoylphosphatidylcholine (DOPC), dioleoylphosphatidylserine (DOPS), dioleoylphosphatidic acid or a DOPC/cholesterol mixture. In passing from pH 6.8 to pH 3, the midpoint potential between the negative current peak, due to K(+) inflow into the spacer, and the positive current peak, due to K(+) ejection into the aqueous solution, shifts toward more positive potentials, while the separation between these two peaks decreases. This behavior is interpreted quantitatively on the basis of a model relying on tBLM structural features estimated independently in previous works. The only adjustable parameter is the rate constant for cation translocation across a potential energy barrier located in the hydrocarbon tail region. The behavior is ascribed to a dragging of the lipid headgroups adjacent to the gramicidin channel mouth toward the hydrocarbon tail region, with a resulting decrease in the negative charge of the DOPC phosphate group, or of the DOPS carboxyl group, with decreasing pH. PMID:26190793

  14. Overexpression of the rice AKT1 potassium channel affects potassium nutrition and rice drought tolerance

    PubMed Central

    Ahmad, Izhar; Mian, Afaq; Maathuis, Frans J. M.

    2016-01-01

    Potassium (K+) is the most important cationic nutrient for all living organisms and has roles in most aspects of plant physiology. To assess the impact of one of the main K+ uptake components, the K+ inward rectifying channel AKT1, we characterized both loss of function and overexpression of OsAKT1 in rice. In many conditions, AKT1 expression correlated with K+ uptake and tissue K+ levels. No salinity-related growth phenotype was observed for either loss or gain of function mutants. However, a correlation between AKT1 expression and root Na+ when the external Na/K ratio was high suggests that there may be a role for AKT1 in Na+ uptake in such conditions. In contrast to findings with Arabidopsis thaliana, we did not detect any change in growth of AKT1 loss of function mutants in the presence of NH4 +. Nevertheless, NH4 +-dependent inhibition was detected during K+ uptake assays in loss of function and wild type plants, depending on pre-growth conditions. The most prominent result of OsAKT1 overexpression was a reduction in sensitivity to osmotic/drought stress in transgenic plants: the data suggest that AKT1 overexpression improved rice osmotic and drought stress tolerance by increasing tissue levels of K+, especially in the root. PMID:26969743

  15. Molecular characterization of a gene affecting potassium channels in Drosophila melanogaster

    SciTech Connect

    Drysdale, R.A.

    1988-01-01

    This study describes the molecular isolation and characterization of the ether-a-go-go (eag) gene of Drosophila melanogaster. Electrophysiological and genetic evidence suggest that the product of the eag locus is intimately involved in the normal functioning of voltage-gated potassium channels. A molecular analysis of eag was undertaken in order to elucidate the contribution of eag{sup +} to the proper operation of the nervous system. An inverted chromosome, In(1)sc{sup 29}, broken in the scute complex and in the eag locus, was used to isolate DNA from the eag region. 85kb of DNA around this starting point were isolated by chromosome waling. Analysis of the corresponding genomic DNA identified the molecular lesions associated with three additional eag allels: two dysgeneis-induced insertion mutations and a lambda-ray-induced insertional translocation. The molecular defects associated with these alleles are spread throughout 27kb within the chromosome walk. Several cDNAs have been isolated on the basis of homology to parts of the chromosome walk. One of these is multiply spliced over 32kb of genomic DNA in a pattern that strongly suggests that it represents at least part of the eag message.

  16. Schizophrenia-Associated MIR204 Regulates Noncoding RNAs and Affects Neurotransmitter and Ion Channel Gene Sets

    PubMed Central

    Cammaerts, Sophia; Strazisar, Mojca; Smets, Bart; Weckhuysen, Sarah; Nordin, Annelie; De Jonghe, Peter; Adolfsson, Rolf; De Rijk, Peter; Del Favero, Jurgen

    2015-01-01

    As regulators of gene expression, microRNAs (miRNAs) are likely to play an important role in the development of disease. In this study we present a large-scale strategy to identify miRNAs with a role in the regulation of neuronal processes. Thereby we found variant rs7861254 located near the MIR204 gene to be significantly associated with schizophrenia. This variant resulted in reduced expression of miR-204 in neuronal-like SH-SY5Y cells. Analysis of the consequences of the altered miR-204 expression on the transcriptome of these cells uncovered a new mode of action for miR-204, being the regulation of noncoding RNAs (ncRNAs), including several miRNAs, such as MIR296. Furthermore, pathway analysis showed downstream effects of miR-204 on neurotransmitter and ion channel related gene sets, potentially mediated by miRNAs regulated through miR-204. PMID:26714269

  17. Transient potassium channels augment degeneracy in hippocampal active dendritic spectral tuning.

    PubMed

    Rathour, Rahul Kumar; Malik, Ruchi; Narayanan, Rishikesh

    2016-01-01

    Hippocampal pyramidal neurons express an intraneuronal map of spectral tuning mediated by hyperpolarization-activated cyclic-nucleotide-gated nonspecific-cation channels. Modeling studies have predicted a critical regulatory role for A-type potassium (KA) channels towards augmenting functional robustness of this map. To test this, we performed patch-clamp recordings from soma and dendrites of rat hippocampal pyramidal neurons, and measured spectral tuning before and after blocking KA channels using two structurally distinct pharmacological agents. Consistent with computational predictions, we found that blocking KA channels resulted in a significant reduction in resonance frequency and significant increases in input resistance, impedance amplitude and action-potential firing frequency across the somato-apical trunk. Furthermore, across all measured locations, blocking KA channels enhanced temporal summation of postsynaptic potentials and critically altered the impedance phase profile, resulting in a significant reduction in total inductive phase. Finally, pair-wise correlations between intraneuronal percentage changes (after blocking KA channels) in different measurements were mostly weak, suggesting differential regulation of different physiological properties by KA channels. Our results unveil a pivotal role for fast transient channels in regulating theta-frequency spectral tuning and intrinsic phase response, and suggest that degeneracy with reference to several coexisting functional maps is mediated by cross-channel interactions across the active dendritic arbor. PMID:27094086

  18. Transient potassium channels augment degeneracy in hippocampal active dendritic spectral tuning

    PubMed Central

    Rathour, Rahul Kumar; Malik, Ruchi; Narayanan, Rishikesh

    2016-01-01

    Hippocampal pyramidal neurons express an intraneuronal map of spectral tuning mediated by hyperpolarization-activated cyclic-nucleotide-gated nonspecific-cation channels. Modeling studies have predicted a critical regulatory role for A-type potassium (KA) channels towards augmenting functional robustness of this map. To test this, we performed patch-clamp recordings from soma and dendrites of rat hippocampal pyramidal neurons, and measured spectral tuning before and after blocking KA channels using two structurally distinct pharmacological agents. Consistent with computational predictions, we found that blocking KA channels resulted in a significant reduction in resonance frequency and significant increases in input resistance, impedance amplitude and action-potential firing frequency across the somato-apical trunk. Furthermore, across all measured locations, blocking KA channels enhanced temporal summation of postsynaptic potentials and critically altered the impedance phase profile, resulting in a significant reduction in total inductive phase. Finally, pair-wise correlations between intraneuronal percentage changes (after blocking KA channels) in different measurements were mostly weak, suggesting differential regulation of different physiological properties by KA channels. Our results unveil a pivotal role for fast transient channels in regulating theta-frequency spectral tuning and intrinsic phase response, and suggest that degeneracy with reference to several coexisting functional maps is mediated by cross-channel interactions across the active dendritic arbor. PMID:27094086

  19. Stretch-activated channels in single early distal tubule cells of the frog.

    PubMed Central

    Hurst, A M; Hunter, M

    1990-01-01

    1. Single stretch-activated channels have been studied in cell-attached and excised patches from single early distal tubule (diluting segment) cells of Rana temporaria. 2. The channels can be reversibly activated, in both cell-attached and excised patches, by the application of negative pressure to the pipette causing mechanical stretching of the cell membrane. In cell-attached patches, application of 14.8 cmH2O negative pressure to the patch pipette increased reversibly the open probably from 0.11 to 0.87. 3. The channel conductance in the cell-attached configuration with standard Ringer solution in the pipette is 21.3 pS. 4. The channel is non-specific. In excised inside-out patches ion substitution experiments show that the channel does not discriminate between sodium and potassium ions, nor does it appear to select for cations over anions. 5. The channel is voltage sensitive such that depolarizing the cell opens the channel. The open probability at the resting membrane potential, 0.89, was reduced to 0.26 at a hyperpolarizing potential of 100 mV (holding pressure of -20.1 cmH2O or -206 Pa). 6. The sensitivity of the channel to mechanical stretching suggests that the channel may be involved in cell volume regulation. PMID:1707964

  20. pH regulation of amphotericin B channels activity in the bilayer lipid membrane

    PubMed Central

    Shahmoradi, Tahereh; Sepehry, Hamid; Ashrafpour, Manuchehr

    2016-01-01

    Background: Amphotericin B (AmB) is a polyene antibiotic frequently applied in the treatment of systemic fungal infections in spite of its secondary effects. The pH plays a crucial role in modulating biophysical features of ion channels in the bilayer lipid membranes. Aim: In this study, the role of pH in the regulation of AmB channel was assessed by single channel recording of ion channel incorporated in the artificial membrane. Materials and Methods: Bilayer lipid membrane was formed by phosphatidylcholine in a 350 μm diameter aperture between two chambers, cis and trans contained 200/50 mMKCl solutions, respectively; then AmB was incorporated into the bilayer lipid membrane. Single channel recordings were used to indicate the effects of pH changes on AmB channels activity. The records were analyzed by Clamp fit 10 software. Results: A kinetic analysis of single channel currents indicated a cation ion channel with 500 pS conductance and voltage-dependence of the open probability of the AmB channel (Po). A reduction of cis pH to 6 decreased Po and conductance. This effect was also voltage-dependent, being greater at a more positive above −40. The pH changes in the range of 6-8 had no effect on the reversal potential and ion selectivity. Conclusion: Our data indicated that extracellular acidity can reduce AmB activity. PMID:27003977

  1. Environmental layout complexity affects neural activity during navigation in humans.

    PubMed

    Slone, Edward; Burles, Ford; Iaria, Giuseppe

    2016-05-01

    Navigating large-scale surroundings is a fundamental ability. In humans, it is commonly assumed that navigational performance is affected by individual differences, such as age, sex, and cognitive strategies adopted for orientation. We recently showed that the layout of the environment itself also influences how well people are able to find their way within it, yet it remains unclear whether differences in environmental complexity are associated with changes in brain activity during navigation. We used functional magnetic resonance imaging to investigate how the brain responds to a change in environmental complexity by asking participants to perform a navigation task in two large-scale virtual environments that differed solely in interconnection density, a measure of complexity defined as the average number of directional choices at decision points. The results showed that navigation in the simpler, less interconnected environment was faster and more accurate relative to the complex environment, and such performance was associated with increased activity in a number of brain areas (i.e. precuneus, retrosplenial cortex, and hippocampus) known to be involved in mental imagery, navigation, and memory. These findings provide novel evidence that environmental complexity not only affects navigational behaviour, but also modulates activity in brain regions that are important for successful orientation and navigation. PMID:26990572

  2. [Cooperative phenomena in the activity of single ion channels].

    PubMed

    Geletiuk, V I; Kazachenko, V N

    1989-01-01

    Using the patch-voltage-clamp method kinetics of the fast potential-dependent K+-channels in molluscan neurones was investigated. It was found that under given experimental conditions the amplitudes of single current impulses have a wide spectrum. The amplitudes are proportional to a number of the current substates involved. Averaged fronts of the current impulses are S-shaped, and have duration greater than 1 ms. Averaged duration of the current impulses increases (from 0.25 to 30-40 ms) with the impulse amplitude (or with the number of the substates involved). There is a sharp bend of the dependence at the impulse amplitude 0.6-0.7 of maximal value. The phenomena investigated reflect, probably, cooperativity of the channel transitions between the substates. The degree of the cooperativity depends on the membrane potential value. PMID:2804147

  3. Ion Channels in Regulation of Neuronal Regenerative Activities

    PubMed Central

    Chen, Dongdong; Yu, Shan Ping; Wei, Ling

    2014-01-01

    The regeneration of the nervous system is achieved by the regrowth of damaged neuronal axons, the restoration of damaged nerve cells, and the generation of new neurons to replace those that have been lost. In the central nervous system the regenerative ability is limited by various factors including damaged oligodendrocytes that are essential for neuronal axon myelination, an emerging glial scar, and secondary injury in the surrounding areas. Stem cell transplantation therapy has been shown to be a promising approach to treating neurodegenerative diseases because of the regenerative capability of stem cells that secrete neurotrophic factors and give rise to differentiated progeny. However, some issues of stem cell transplantation, such as survival, homing, and efficiency of neural differentiation after transplantation, still need to be improved. Ion channels allow for the exchange of ions between the intra- and extracellular spaces or between the cytoplasm and organelles. These ion channels maintain the ion homeostasis in the brain and play a key role in regulating the physiological function of the nervous system and allowing the processing of neuronal signals. In seeking a potential strategy to enhance the efficacy of stem cell therapy in neurological and neurodegenerative diseases, this review briefly summarizes the roles of ion channels in cell proliferation, differentiation, migration, chemotropic axon guidance of growth cones and axon outgrowth after injury. PMID:24399572

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

    PubMed Central

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

    2009-01-01

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

  5. Fast and slow activation kinetics of voltage-gated sodium channels in molluscan neurons.

    PubMed

    Gilly, W F; Gillette, R; McFarlane, M

    1997-05-01

    Whole cell patch-clamp recordings of Na current (I(Na)) were made under identical experimental conditions from isolated neurons from cephalopod (Loligo, Octopus) and gastropod (Aplysia, Pleurobranchaea, Doriopsilla) species to compare properties of activation gating. Voltage dependence of peak Na conductance (gNa) is very similar in all cases, but activation kinetics in the gastropod neurons studied are markedly slower. Kinetic differences are very pronounced only over the voltage range spanned by the gNa-voltage relation. At positive and negative extremes of voltage, activation and deactivation kinetics of I(Na) are practically indistinguishable in all species studied. Voltage-dependent rate constants underlying activation of the slow type of Na channel found in gastropods thus appear to be much more voltage dependent than are the equivalent rates in the universally fast type of channel that predominates in cephalopods. Voltage dependence of inactivation kinetics shows a similar pattern and is representative of activation kinetics for the two types of Na channels. Neurons with fast Na channels can thus make much more rapid adjustments in the number of open Na channels at physiologically relevant voltages than would be possible with only slow Na channels. This capability appears to be an adaptation that is highly evolved in cephalopods, which are well known for their high-speed swimming behaviors. Similarities in slow and fast Na channel subtypes in molluscan and mammalian neurons are discussed. PMID:9163364

  6. Regulation of large conductance calcium- and voltage-activated potassium (BK) channels by S-palmitoylation.

    PubMed

    Shipston, Michael J

    2013-02-01

    BK (large conductance calcium- and voltage-activated potassium) channels are important determinants of physiological control in the nervous, endocrine and vascular systems with channel dysfunction associated with major disorders ranging from epilepsy to hypertension and obesity. Thus the mechanisms that control channel surface expression and/or activity are important determinants of their (patho)physiological function. BK channels are S-acylated (palmitoylated) at two distinct sites within the N- and C-terminus of the pore-forming α-subunit. Palmitoylation of the N-terminus controls channel trafficking and surface expression whereas palmitoylation of the C-terminal domain determines regulation of channel activity by AGC-family protein kinases. Recent studies are beginning to reveal mechanistic insights into how palmitoylation controls channel trafficking and cross-talk with phosphorylation-dependent signalling pathways. Intriguingly, each site of palmitoylation is regulated by distinct zDHHCs (palmitoyl acyltransferases) and APTs (acyl thioesterases). This supports that different mechanisms may control substrate specificity by zDHHCs and APTs even within the same target protein. As palmitoylation is dynamically regulated, this fundamental post-translational modification represents an important determinant of BK channel physiology in health and disease. PMID:23356260

  7. Does the choice of the forcing term affect flow statistics in DNS of turbulent channel flow?

    NASA Astrophysics Data System (ADS)

    Quadrio, Maurizio; Frohnapfel, Bettina; Hasegawa, Yosuke

    2016-01-01

    We seek possible statistical consequences of the way a forcing term is added to the Navier--Stokes equations in the Direct Numerical Simulation (DNS) of incompressible channel flow. Simulations driven by constant flow rate, constant pressure gradient and constant power input are used to build large databases, and in particular to store the complete temporal trace of the wall-shear stress for later analysis. As these approaches correspond to different dynamical systems, it can in principle be envisaged that these differences are reflect by certain statistics of the turbulent flow field. The instantaneous realizations of the flow in the various simulations are obviously different, but, as expected, the usual one-point, one-time statistics do not show any appreciable difference. However, the PDF for the fluctuations of the streamwise component of wall friction reveals that the simulation with constant flow rate presents lower probabilities for extreme events of large positive friction. The low probability value of such events explains their negligible contribution to the commonly computed statistics; however, the very existence of a difference in the PDF demonstrates that the forcing term is not entirely uninfluential. Other statistics for wall-based quantities (the two components of friction and pressure) are examined; in particular spatio-temporal autocorrelations show small differences at large temporal separations, where unfortunately the residual statistical uncertainty is still of the same order of the observed difference. Hence we suggest that the specific choice of the forcing term does not produce important statistical consequences, unless one is interested in the strongest events of high wall friction, that are underestimated by a simulation run at constant flow rate.

  8. Potent KCNQ2/3-Specific Channel Activator Suppresses In Vivo Epileptic Activity and Prevents the Development of Tinnitus

    PubMed Central

    Kalappa, Bopanna I.; Soh, Heun; Duignan, Kevin M.; Furuya, Takeru; Edwards, Scott

    2015-01-01

    Voltage-gated Kv7 (KCNQ) channels are voltage-dependent potassium channels that are activated at resting membrane potentials and therefore provide a powerful brake on neuronal excitability. Genetic or experience-dependent reduction of KCNQ2/3 channel activity is linked with disorders that are characterized by neuronal hyperexcitability, such as epilepsy and tinnitus. Retigabine, a small molecule that activates KCNQ2–5 channels by shifting their voltage-dependent opening to more negative voltages, is an US Food and Drug Administration (FDA) approved anti-epileptic drug. However, recently identified side effects have limited its clinical use. As a result, the development of improved KCNQ2/3 channel activators is crucial for the treatment of hyperexcitability-related disorders. By incorporating a fluorine substituent in the 3-position of the tri-aminophenyl ring of retigabine, we synthesized a small-molecule activator (SF0034) with novel properties. Heterologous expression of KCNQ2/3 channels in HEK293T cells showed that SF0034 was five times more potent than retigabine at shifting the voltage dependence of KCNQ2/3 channels to more negative voltages. Moreover, unlike retigabine, SF0034 did not shift the voltage dependence of either KCNQ4 or KCNQ5 homomeric channels. Conditional deletion of Kcnq2 from cerebral cortical pyramidal neurons showed that SF0034 requires the expression of KCNQ2/3 channels for reducing the excitability of CA1 hippocampal neurons. Behavioral studies demonstrated that SF0034 was a more potent and less toxic anticonvulsant than retigabine in rodents. Furthermore, SF0034 prevented the development of tinnitus in mice. We propose that SF0034 provides, not only a powerful tool for investigating ion channel properties, but, most importantly, it provides a clinical candidate for treating epilepsy and preventing tinnitus. PMID:26063916

  9. Screening of Transient Receptor Potential Canonical Channel Activators Identifies Novel Neurotrophic Piperazine Compounds.

    PubMed

    Sawamura, Seishiro; Hatano, Masahiko; Takada, Yoshinori; Hino, Kyosuke; Kawamura, Tetsuya; Tanikawa, Jun; Nakagawa, Hiroshi; Hase, Hideharu; Nakao, Akito; Hirano, Mitsuru; Rotrattanadumrong, Rachapun; Kiyonaka, Shigeki; Mori, Masayuki X; Nishida, Motohiro; Hu, Yaopeng; Inoue, Ryuji; Nagata, Ryu; Mori, Yasuo

    2016-03-01

    Transient receptor potential canonical (TRPC) proteins form Ca(2+)-permeable cation channels activated upon stimulation of metabotropic receptors coupled to phospholipase C. Among the TRPC subfamily, TRPC3 and TRPC6 channels activated directly by diacylglycerol (DAG) play important roles in brain-derived neurotrophic factor (BDNF) signaling, promoting neuronal development and survival. In various disease models, BDNF restores neurologic deficits, but its therapeutic potential is limited by its poor pharmacokinetic profile. Elucidation of a framework for designing small molecules, which elicit BDNF-like activity via TRPC3 and TRPC6, establishes a solid basis to overcome this limitation. We discovered, through library screening, a group of piperazine-derived compounds that activate DAG-activated TRPC3/TRPC6/TRPC7 channels. The compounds [4-(5-chloro-2-methylphenyl)piperazin-1-yl](3-fluorophenyl)methanone (PPZ1) and 2-[4-(2,3-dimethylphenyl)piperazin-1-yl]-N-(2-ethoxyphenyl)acetamide (PPZ2) activated, in a dose-dependent manner, recombinant TRPC3/TRPC6/TRPC7 channels, but not other TRPCs, in human embryonic kidney cells. PPZ2 activated native TRPC6-like channels in smooth muscle cells isolated from rabbit portal vein. Also, PPZ2 evoked cation currents and Ca(2+) influx in rat cultured central neurons. Strikingly, both compounds induced BDNF-like neurite growth and neuroprotection, which were abolished by a knockdown or inhibition of TRPC3/TRPC6/TRPC7 in cultured neurons. Inhibitors of Ca(2+) signaling pathways, except calcineurin, impaired neurite outgrowth promotion induced by PPZ compounds. PPZ2 increased activation of the Ca(2+)-dependent transcription factor, cAMP response element-binding protein. These findings suggest that Ca(2+) signaling mediated by activation of DAG-activated TRPC channels underlies neurotrophic effects of PPZ compounds. Thus, piperazine-derived activators of DAG-activated TRPC channels provide important insights for future development of a

  10. Evaluation of macrophage antiviral activity in patients affected by neoplasia.

    PubMed

    Merendino, R A; Iannello, D; Arena, A; Bonina, L; Greco, V; Mesiti, M; Chillemi, S; Mastroeni, P

    1988-01-01

    The intrinsic antiviral activity of macrophages has been studied in healthy donors and in patients affected by breast cancer and melanoma. In vitro differentiated macrophages from blood-derived monocytes were infected with measles virus, herpes simplex virus type 2 and adenovirus 17. The challenge was carried out with different multiplicities of infection and the synthesis of virus was tested by evaluating the single cycle growth curve in 24 h. The results obtained show that the restriction of virus infectivity by macrophages is strongly influenced by the multiplicity of infection. This was particularly evident with the adenovirus 17. Moreover, macrophages from patients with melanoma and breast cancer showed an impairment of the intrinsic antiviral activity in comparison with normal subjects. PMID:2842553

  11. Activities affecting surface water resources: A general overview

    SciTech Connect

    Not Available

    1990-01-01

    In November 1987, P.E.I. signed a federal/provincial work-sharing arrangement on water resource management focusing on groundwater pollution, surface water degradation and estuarine eutrophication. The surface water program was designed to identify current surface water uses and users within 12 major watersheds across the Island containing 26 individual rivers, as well as problems arising due to practices that degrade the quality of surface water and restricts its value to other user groups. This report presents a general overview of the program, covering the general characteristics of the Island; operations in agriculture, fish and wildlife, forestry, recreation, fisheries, and industry; alterations of natural features of waterways; wetlands; additional watershed activities such as hydrometric stations and subdivision development; and activities affecting surface water resources such as sedimentation sources, pollution point sources and instream obstructions.

  12. Allelic variation in the vacuolar TPK1 channel affects its calcium dependence and may impact on stomatal conductance.

    PubMed

    Hartley, Tom N; Maathuis, Frans J M

    2016-01-01

    Natural variation can be exploited to identify allelic variants of proteins. In this study, patch clamp was used to determine transport properties of two AtTPK1 alleles from Landsberg and Kas-2 ecotypes. No difference in conductance or ion selectivity was observed but the Kas version of TPK1 showed different Ca(2+) dependence in its open probability compared to Ler. Leaves from Kas showed lower rates of water loss than those of Ler, in either the absence or presence of ABA, an observation that is consistent with higher TPK1 channel activity at comparable cytoplasmic Ca(2+) concentrations. A model that explains the results is presented. PMID:26765783

  13. BK channel activation by tungstate requires the β1 subunit extracellular loop residues essential to modulate voltage sensor function and channel gating.

    PubMed

    Fernández-Mariño, Ana I; Valverde, Miguel A; Fernández-Fernández, José M

    2014-07-01

    Tungstate, a compound with antidiabetic, antiobesity, and antihypertensive properties, activates the large-conductance voltage- and Ca(2+)-dependent K(+) (BK) channel containing either β1 or β4 subunits. The BK activation by tungstate is Mg(2+)-dependent and promotes arterial vasodilation, but only in precontracted mouse arteries expressing β1. In this study, we further explored how the β1 subunit participates in tungstate activation of BK channels. Activation of heterologously expressed human BKαβ1 channels in inside-out patches is fully dependent on the Mg(2+) sensitivity of the BK α channel subunit even at high (10 μM) cytosolic Ca(2+) concentration. Alanine mutagenesis of β1 extracellular residues Y74 or S104, which destabilize the active voltage sensor, greatly decreased the tungstate-induced left-shift of the BKαβ1 G-V curves in either the absence or presence of physiologically relevant cytosolic Ca(2+) levels (10 μM). The weakened tungstate activation of the BKαβ1Y74A and BKαβ1S104A mutant channels was not related to decreased Mg(2+) sensitivity. These results, together with previously published reports, support the idea that the putative binding site for tungstate-mediated BK channel activation is located in the pore-forming α channel subunit, around the Mg(2+) binding site. The role of β1 in tungstate-induced channel activation seems to rely on its interaction with the BK α subunit to modulate channel activity. Loop residues that are essential for the regulation of voltage sensor activation and gating of the BK channel are also relevant for BK activation by tungstate. PMID:24158430

  14. Methyl p-hydroxybenzoate causes pain sensation through activation of TRPA1 channels

    PubMed Central

    Fujita, F; Moriyama, T; Higashi, T; Shima, A; Tominaga, M

    2007-01-01

    Background and purpose: Parabens are commonly added in pharmaceutical, cosmetic and food products because of their wide antibacterial properties, low toxicity, inertness and chemical stability, although the molecular mechanism of their antibacterial effect is not fully understood. Some agonists of the transient receptor potential (TRP) A1 channels are known to have strong antibacterial activities. Therefore, a series of experiments was conducted to find out the effects of parabens on TRP channels expressed in sensory neurons, particularly the TRPA1 channels. Experimental approach: Effects of parabens, especially of methyl p-hydroxybenzoate (methyl paraben) on TRP channel activities were examined using Ca2+-imaging and patch-clamp methods. In addition, an involvement of methyl paraben in the development of pain-related behavior in mice was investigated. Key results: Methyl paraben specifically activated TRPA1 in both HEK293 cells expressing TRPA1 and in mouse sensory neurons with an EC50 value of 4.4 mM, an attainable concentration in methyl paraben-containing products. Methyl paraben caused pain-related behavior in mice similar to that caused by allyl isothiocyanate, which was blocked by the TRP channel blocker, ruthenium red. Conclusions and implications: Our data indicate that methyl paraben is able to activate TRPA1 channels and can cause pain sensation. As such, methyl paraben provides a useful tool for investigating TRPA1 function and development of antinociceptive agents acting on TRPA1 channels. PMID:17351650

  15. Toward the rational design of constitutively active KCa3.1 mutant channels.

    PubMed

    Garneau, Line; Klein, Hélène; Parent, Lucie; Sauvé, Rémy

    2010-01-01

    The Ca²+ activated potassium channel of intermediate conductance KCa3.1 is now emerging as a therapeutic target for a large variety of health disorders. KCa3.1 is a tetrameric membrane protein with each subunit formed of six transmembrane helices (S1-S6). Ca²+ sensitivity is conferred by the Ca²+ binding protein calmodulin (CaM), with the CaM C-lobe constitutively bound to an intracellular domain of the channel C-terminus, located proximal to the membrane and connected to the S6 transmembrane segment. Patch clamp single channel recordings have demonstrated that binding of Ca²+ to CaM allows the channel to transit dose dependently from a nonconducting to an ion-conducting configuration. Here we present a general strategy to generate KCa3.1 mutant channels that remain in an ion-conducting state in the absence of Ca²+. Our strategy is first based on the production of a 3D model of the channel pore region, followed by SCAM experiments to confirm that residues along each of the channel S6 transmembrane helix form the channel pore lumen as predicted. In a simple model, constitutive activity can be obtained by removing the steric hindrances inside the channel pore susceptible to prevent ion flow when the channel is in the closed configuration. Using charged MTS reagents and Ag+ ions as probes acting on Cys residues engineered in the pore lumen, we found that the S6 transmembrane helices of KCa3.1 cannot form a pore constriction tight enough to prevent ion flow for channels in the closed state. These observations ruled out experimental strategies where constitutive activity would be generated by producing a "leaky" closed channel. A more successful approach consisted however in perturbing the channel open/closed state equilibrium free energy. In particular, we found that substituting the hydrophobic residue V282 in S6 by hydrophilic amino acids could lock the channel in an open-like state, resulting in channels that were ion conducting in the absence of Ca²+. PMID

  16. Sequential acquisition of cacophony calcium currents, sodium channels and voltage-dependent potassium currents affects spike shape and dendrite growth during postembryonic maturation of an identified Drosophila motoneuron

    PubMed Central

    Ryglewski, Stefanie; Kilo, Lukas; Duch, Carsten

    2015-01-01

    During metamorphosis the CNS undergoes profound changes to accommodate the switch from larval to adult behaviors. In Drosophila and other holometabolous insects, adult neurons differentiate either from respecified larval neurons, newly born neurons, or are born embryonically but remain developmentally arrested until differentiation during pupal life. This study addresses the latter in the identified Drosophila flight motoneuron 5. In situ patch-clamp recordings, intracellular dye fills and immunocytochemistry address the interplay between dendritic shape, excitability and ionic current development. During pupal life, changes in excitability and spike shape correspond to a stereotyped, progressive appearance of voltage-gated ion channels. High-voltage-activated calcium current is the first current to appear at pupal stage P4, prior to the onset of dendrite growth. This is followed by voltage-gated sodium as well as transient potassium channel expression, when first dendrites grow, and sodium-dependent action potentials can be evoked by somatic current injection. Sustained potassium current appears later than transient potassium current. During the early stages of rapid dendritic growth, sodium-dependent action potentials are broadened by a calcium component. Narrowing of spike shape coincides with sequential increases in transient and sustained potassium currents during stages when dendritic growth ceases. Targeted RNAi knockdown of pupal calcium current significantly reduces dendritic growth. These data indicate that the stereotyped sequential acquisition of different voltage-gated ion channels affects spike shape and excitability such that activity-dependent calcium influx serves as a partner of genetic programs during critical stages of motoneuron dendrite growth. PMID:24620836

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

    PubMed

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

    2016-07-01

    The sodium-activated potassium channels Slick (Slo2.1, KCNT2) and Slack (Slo2.2, KCNT1) are high-conductance potassium channels of the Slo family. In neurons, Slick and Slack channels are involved in the generation of slow afterhyperpolarization, in the regulation of firing patterns, and in setting and stabilizing the resting membrane potential. The distribution and subcellular localization of Slick and Slack channels in the mouse brain have not yet been established in detail. The present study addresses this issue through in situ hybridization and immunohistochemistry. Both channels were widely distributed and exhibited distinct distribution patterns. However, in some brain regions, their expression overlapped. Intense Slick channel immunoreactivity was observed in processes, varicosities, and neuronal cell bodies of the olfactory bulb, granular zones of cortical regions, hippocampus, amygdala, lateral septal nuclei, certain hypothalamic and midbrain nuclei, and several regions of the brainstem. The Slack channel showed primarily a diffuse immunostaining pattern, and labeling of cell somata and processes was observed only occasionally. The highest Slack channel expression was detected in the olfactory bulb, lateral septal nuclei, basal ganglia, and distinct areas of the midbrain, brainstem, and cerebellar cortex. In addition, comparing our data obtained from mouse brain with a previously published study on rat brain revealed some differences in the expression and distribution of Slick and Slack channels in these species. J. Comp. Neurol. 524:2093-2116, 2016. © 2015 The Authors The Journal of Comparative Neurology Published by Wiley Periodicals, Inc. PMID:26587966

  18. Functional Expression of a Ca(2+)-activated Cl(-) Channel Modulator Involved in Ion Transport and Epithelial Cell Differentiation.

    PubMed

    Yamazaki, Jun

    2016-01-01

      Cl(-)-permeable channels and transporters expressed on the cell membranes of various mammalian cell types play pivotal roles in the transport of electrolytes and water, pH regulation, cell volume and membrane excitability, and are therefore expected to be useful molecular targets for drug discovery. Both TMEM16A (a possible candidate for Ca(2+)-regulated Cl(-) channels recently identified) and cystic fibrosis transmembrane conductance regulator (CFTR) (or cAMP-regulated Cl(-) channels) have been known to be involved in Cl(-) secretion and reabsorption in the rat salivary gland. Crosstalk between two types of regulatory pathways through these two types of channels has also been described. Previously, we demonstrated that CLCA, a Ca(2+)-activated Cl(-) channel modulator, was involved in Cl(-) absorption in rat salivary ducts. In addition to Ca(2+), basal NF-κB activity in a mouse keratinocyte line was shown to be involved in the transcriptional regulation of CLCA. Conversely, a truncated isoform of CLCA was found in undifferentiated epithelial cells present in the rat epidermal basal layers. Under regulation by Ca(2+) and PKC, the surface expression of β1-integrin and cell adhesion were decreased in the CLCA-overexpressing cells. Knockdown of this isoform elevated the expression of β1-integrin in rat epidermis in vivo. These results indicate that the specific differentiation-dependent localization of CLCA, and transcriptional regulation through Ca(2+), are likely to affect ion permeability and the adhesive potential of epithelial cells. In summary, these types of Cl(-) channels and their modulators may function in a coordinated manner in regulating the functions of epithelial cells under different physiological conditions. PMID:26935091

  19. Paradoxical Contribution of SK3 and GIRK Channels to the Activation of Mouse Vomeronasal Organ

    PubMed Central

    Kim, SangSeong; Ma, Limei; Jensen, Kristi L.; Kim, Michelle M.; Bond, Chris T.; Adelman, John P.; Yu, C. Ron

    2012-01-01

    The vomeronasal organ (VNO) plays an essential role in intraspecies communication for terrestrial vertebrates. The ionic mechanisms of VNO activation remain unclear. We find that the calcium–activated potassium channel SK3 and G–protein activated potassium channel GIRK are part of an independent pathway for VNO activation. In slice preparations, the potassium channels attenuate inward currents carried by TRPC2 and calcium–activated chloride channels (CACCs). In intact tissue preparations, paradoxically, the potassium channels enhance urine–evoked inward currents. This discrepancy results from the loss of a high concentration of lumenal potassium, which enables the influx of potassium ions to depolarize the VNO neurons in vivo. SK3−/− and GIRK1−/− mice show deficits in both mating and aggressive behaviors and deficiency in SK3−/− is exacerbated by TRPC2 knockout. Our results suggest a model of VNO activation that is mediated by TRPC2, CACCs and two potassium channels, all contributing to the in vivo depolarization of VNO neurons. PMID:22842147

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

    PubMed Central

    Griffith, Thom; Tsaneva-Atanasova, Krasimira; Mellor, Jack R.

    2016-01-01

    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. PMID:27232631

  1. Paradoxical contribution of SK3 and GIRK channels to the activation of mouse vomeronasal organ.

    PubMed

    Kim, SangSeong; Ma, Limei; Jensen, Kristi L; Kim, Michelle M; Bond, Chris T; Adelman, John P; Yu, C Ron

    2012-09-01

    The vomeronasal organ (VNO) is essential for intraspecies communication in many terrestrial vertebrates. The ionic mechanisms of VNO activation remain unclear. We found that the calcium-activated potassium channel SK3 and the G protein-activated potassium channel GIRK are part of an independent pathway for VNO activation. In slice preparations, the potassium channels attenuated inward currents carried by TRPC2 and calcium-activated chloride channels (CACCs). In intact tissue preparations, paradoxically, the potassium channels enhanced urine-evoked inward currents. This discrepancy resulted from the loss of a high concentration of lumenal potassium, which enabled the influx of potassium ions to depolarize the VNO neurons in vivo. Both Sk3 (also known as Kcnn3) and Girk1 (also known as Kcnj3) homozygous null mice showed deficits in mating and aggressive behaviors, and the deficiencies in Sk3(-/-) mice were exacerbated by Trpc2 knockout. Our results suggest that VNO activation is mediated by TRPC2, CACCs and two potassium channels, all of which contributed to the in vivo depolarization of VNO neurons. PMID:22842147

  2. Cryo-EM structure of the Slo2.2 Na+-activated K+ channel

    PubMed Central

    Hite, Richard; Yuan, Peng; Li, Zongli; Hsuing, Yichun; Walz, Thomas; MacKinnon, Roderick

    2015-01-01

    Na+-activated K+ channels are members of the Slo family of large conductance K+ channels that are widely expressed in the brain, where their opening regulates neuronal excitability. These channels are fascinating for the biological roles they fulfill as well as for their intriguing biophysical properties, including conductance levels ten times most other K+ channels and gating sensitivity to intracellular Na+. Here we present the structure a complete Na+-activated K+ channel, Slo2.2, in the Na+-free state, determined by cryo-electron microscopy at a nominal resolution of 4.5 Å. The channel is composed of a large cytoplasmic gating ring within which resides the Na+-binding site and a transmembrane domain that closely resembles voltage-gated K+ channels. In the structure, the cytoplasmic domain adopts a closed conformation and the ion conduction pore is also closed. The structure provides a first view of a member of the Slo K+ channel family, which reveals features explaining their high conductance and gating mechanism. PMID:26436452

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

    PubMed

    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. PMID:15579000

  4. Direct Activation of β-Cell KATP Channels with a Novel Xanthine Derivative

    PubMed Central

    Raphemot, Rene; Swale, Daniel R.; Dadi, Prasanna K.; Jacobson, David A.; Cooper, Paige; Wojtovich, Andrew P.; Banerjee, Sreedatta; Nichols, Colin G.

    2014-01-01

    ATP-regulated potassium (KATP) channel complexes of inward rectifier potassium channel (Kir) 6.2 and sulfonylurea receptor (SUR) 1 critically regulate pancreatic islet β-cell membrane potential, calcium influx, and insulin secretion, and consequently, represent important drug targets for metabolic disorders of glucose homeostasis. The KATP channel opener diazoxide is used clinically to treat intractable hypoglycemia caused by excessive insulin secretion, but its use is limited by off-target effects due to lack of potency and selectivity. Some progress has been made in developing improved Kir6.2/SUR1 agonists from existing chemical scaffolds and compound screening, but there are surprisingly few distinct chemotypes that are specific for SUR1-containing KATP channels. Here we report the serendipitous discovery in a high-throughput screen of a novel activator of Kir6.2/SUR1: VU0071063 [7-(4-(tert-butyl)benzyl)-1,3-dimethyl-1H-purine-2,6(3H,7H)-dione]. The xanthine derivative rapidly and dose-dependently activates Kir6.2/SUR1 with a half-effective concentration (EC50) of approximately 7 μM, is more efficacious than diazoxide at low micromolar concentrations, directly activates the channel in excised membrane patches, and is selective for SUR1- over SUR2A-containing Kir6.1 or Kir6.2 channels, as well as Kir2.1, Kir2.2, Kir2.3, Kir3.1/3.2, and voltage-gated potassium channel 2.1. Finally, we show that VU0071063 activates native Kir6.2/SUR1 channels, thereby inhibiting glucose-stimulated calcium entry in isolated mouse pancreatic β cells. VU0071063 represents a novel tool/compound for investigating β-cell physiology, KATP channel gating, and a new chemical scaffold for developing improved activators with medicinal chemistry. PMID:24646456

  5. Mechanisms of Rose Bengal inhibition on SecA ATPase and ion channel activities.

    PubMed

    Hsieh, Ying-Hsin; Huang, Ying-Ju; Jin, Jin-Shan; Yu, Liyan; Yang, Hsiuchin; Jiang, Chun; Wang, Binghe; Tai, Phang C

    2014-11-14

    SecA is an essential protein possessing ATPase activity in bacterial protein translocation for which Rose Bengal (RB) is the first reported sub-micromolar inhibitor in ATPase activity and protein translocation. Here, we examined the mechanisms of inhibition on various forms of SecA ATPase by conventional enzymatic assays, and by monitoring the SecA-dependent channel activity in the semi-physiological system in cells. We build on the previous observation that SecA with liposomes form active protein-conducting channels in the oocytes. Such ion channel activity is enhanced by purified Escherichia coli SecYEG-SecDF·YajC liposome complexes. Inhibition by RB could be monitored, providing correlation of in vitro activity and intact cell functionality. In this work, we found the intrinsic SecA ATPase is inhibited by RB competitively at low ATP concentration, and non-competitively at high ATP concentrations while the translocation ATPase with precursors and SecYEG is inhibited non-competitively by RB. The Inhibition by RB on SecA channel activity in the oocytes with exogenous ATP-Mg(2+), mimicking translocation ATPase activity, is also non-competitive. The non-competitive inhibition on channel activity has also been observed with SecA from other bacteria which otherwise would be difficult to examine without the cognate precursors and membranes. PMID:25450394

  6. Active Urbanization and Channel Adjustment in Apple Creek, Appleton, WI

    NASA Astrophysics Data System (ADS)

    Clark, J. J.

    2002-12-01

    Headwaters of the Apple Creek watershed have been and continue to be rapidly developed as part of the City of Appleton's long-term growth plan. Concurrent with early development, and prior to development over the past 4 years, two regional stormwater management facilities were constructed. Cross-sectional surveys and core transects were used to determine channel response to urbanization mitigated by stormwater management. The reach immediately downstream of the first pond complex has a narrow, but well established, wooded riparian zone and has not changed in size or shape over the past two years. An engineered reach approximately one mile downstream, however has exhibited widespread bed aggradation. Cross-sectional area decreased an average of 51% over the past four years. Despite the use of sediment and erosion control BMPs, sediment concentrations exceeding 1000 mg/L during base flow are not uncommon downstream of construction sites adjacent to the stream. The artificially widened channel, a reduction in stream gradient, and the backwater effect from downstream ponds caused much of this sediment to remain within the engineered reach. It is estimated that approximately 21,000 Mg of sediment is stored in this mile-long reach. As this sediment migrates downstream, the forebay of the second set of stormwater ponds will begin to fill, reducing storage capacity and thereby limiting its effectiveness in mitigating peak discharges and sequestering nutrients.

  7. Exploring the biophysical evidence that mammalian two-pore channels are NAADP-activated calcium-permeable channels.

    PubMed

    Pitt, Samantha J; Reilly-O'Donnell, Benedict; Sitsapesan, Rebecca

    2016-08-01

    Nicotinic acid adenine dinucleotide phosphate (NAADP) potently releases Ca(2+) from acidic intracellular endolysosomal Ca(2+) stores. It is widely accepted that two types of two-pore channels, termed TPC1 and TPC2, are responsible for the NAADP-mediated Ca(2+) release but the underlying mechanisms regulating their gating appear to be different. For example, although both TPC1 and TPC2 are activated by NAADP, TPC1 appears to be additionally regulated by cytosolic Ca(2+) . Ion conduction and permeability also differ markedly. TPC1 and TPC2 are permeable to a range of cations although biophysical experiments suggest that TPC2 is slightly more selective for Ca(2+) over K(+) than TPC1 and hence capable of releasing greater quantities of Ca(2+) from acidic stores. TPC1 is also permeable to H(+) and therefore may play a role in regulating lysosomal and cytosolic pH, possibly creating localised acidic domains. The significantly different gating and ion conducting properties of TPC1 and TPC2 suggest that these two ion channels may play complementary physiological roles as Ca(2+) -release channels of the endolysosomal system. PMID:26872338

  8. Preferential Phosphorylation of R-domain Serine 768 Dampens Activation of CFTR Channels by PKA

    PubMed Central

    Csanády, László; Seto-Young, Donna; Chan, Kim W.; Cenciarelli, Cristina; Angel, Benjamin B.; Qin, Jun; McLachlin, Derek T.; Krutchinsky, Andrew N.; Chait, Brian T.; Nairn, Angus C.; Gadsby, David C.

    2005-01-01

    CFTR (cystic fibrosis transmembrane conductance regulator), the protein whose dysfunction causes cystic fibrosis, is a chloride ion channel whose gating is controlled by interactions of MgATP with CFTR's two cytoplasmic nucleotide binding domains, but only after several serines in CFTR's regulatory (R) domain have been phosphorylated by cAMP-dependent protein kinase (PKA). Whereas eight R-domain serines have previously been shown to be phosphorylated in purified CFTR, it is not known how individual phosphoserines regulate channel gating, although two of them, at positions 737 and 768, have been suggested to be inhibitory. Here we show, using mass spectrometric analysis, that Ser 768 is the first site phosphorylated in purified R-domain protein, and that it and five other R-domain sites are already phosphorylated in resting Xenopus oocytes expressing wild-type (WT) human epithelial CFTR. The WT channels have lower activity than S768A channels (with Ser 768 mutated to Ala) in resting oocytes, confirming the inhibitory influence of phosphoserine 768. In excised patches exposed to a range of PKA concentrations, the open probability (Po) of mutant S768A channels exceeded that of WT CFTR channels at all [PKA], and the half-maximally activating [PKA] for WT channels was twice that for S768A channels. As the open burst duration of S768A CFTR channels was almost double that of WT channels, at both low (55 nM) and high (550 nM) [PKA], we conclude that the principal mechanism by which phosphoserine 768 inhibits WT CFTR is by hastening the termination of open channel bursts. The right-shifted Po-[PKA] curve of WT channels might explain their slower activation, compared with S768A channels, at low [PKA]. The finding that phosphorylation kinetics of WT or S768A R-domain peptides were similar provides no support for an alternative explanation, that early phosphorylation of Ser 768 in WT CFTR might also impair subsequent phosphorylation of stimulatory R-domain serines. The

  9. Attracting Views and Going Viral: How Message Features and News-Sharing Channels Affect Health News Diffusion

    PubMed Central

    Kim, Hyun Suk

    2015-01-01

    This study examined how intrinsic as well as perceived message features affect the extent to which online health news stories prompt audience selections and social retransmissions, and how news-sharing channels (e-mail vs. social media) shape what goes viral. The study analyzed actual behavioral data on audience viewing and sharing of New York Times health news articles, and associated article content and context data. News articles with high informational utility and positive sentiment invited more frequent selections and retransmissions. Articles were also more frequently selected when they presented controversial, emotionally evocative, and familiar content. Informational utility and novelty had stronger positive associations with e-mail-specific virality, while emotional evocativeness, content familiarity, and exemplification played a larger role in triggering social media-based retransmissions. PMID:26441472

  10. Discovery of novel tetrahydroisoquinoline derivatives as orally active N-type calcium channel blockers with high selectivity for hERG potassium channels.

    PubMed

    Ogiyama, Takashi; Inoue, Makoto; Honda, Shugo; Yamada, Hiroyoshi; Watanabe, Toshihiro; Gotoh, Takayasu; Kiso, Tetsuo; Koakutsu, Akiko; Kakimoto, Shuichiro; Shishikura, Jun-ichi

    2014-12-15

    N-type calcium channels represent a promising target for the treatment of neuropathic pain. The selective N-type calcium channel blocker ziconotide ameliorates severe chronic pain but has a narrow therapeutic window and requires intrathecal administration. We identified tetrahydroisoquinoline derivative 1a as a novel potent N-type calcium channel blocker. However, this compound also exhibited potent inhibitory activity against hERG channels. Structural optimizations led to identification of (1S)-(1-cyclohexyl-3,4-dihydroisoquinolin-2(1H)-yl)-2-{[(1-hydroxycyclohexyl)methyl]amino}ethanone ((S)-1h), which exhibited high selectivity for hERG channels while retaining potency for N-type calcium channel inhibition. (S)-1h went on to demonstrate in vivo efficacy as an orally available N-type calcium channel blocker in a rat spinal nerve ligation model of neuropathic pain. PMID:25456079

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

    PubMed

    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. PMID:26674547

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

    PubMed Central

    2015-01-01

    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. PMID:26674547

  13. On Application Of Langevin Dynamics In Logarithmic Potential To Model Ion Channel Gate Activity.

    PubMed

    Wawrzkiewicz-Jałowiecka, Agata; Borys, Przemysław; Grzywna, Zbigniew J

    2015-12-01

    We model the activity of an ion channel gate by Langevin dynamics in a logarithmic potential. This approach enables one to describe the power-law dwell-time distributions of the considered system, and the long-term correlations between the durations of the subsequent channel states, or fractal scaling of statistical characteristics of the gate's movement with time. Activity of an ion channel gate is described as an overdamped motion of the reaction coordinate in a confining logarithmic potential, which ensures great flexibility of the model. Depending on the chosen parameters, it allows one to reproduce many types of gate dynamics within the family of non-Markovian, anomalous conformational diffusion processes. In this study we apply the constructed model to largeconductance voltage and Ca2+-activated potassium channels (BKCa). The interpretation of model assumptions and parameters is provided in terms of this biological system. Our results show good agreement with the experimental data. PMID:26317442

  14. Block by ruthenium red of the ryanodine-activated calcium release channel of skeletal muscle

    PubMed Central

    1993-01-01

    The effects of ruthenium red and the related compounds tetraamine palladium (4APd) and tetraamine platinum (4APt) were studied on the ryanodine activated Ca2+ release channel reconstituted in planar bilayers with the immunoaffinity purified ryanodine receptor. Ruthenium red, applied at submicromolar concentrations to the myoplasmic side (cis), induced an all-or-none flickery block of the ryanodine activated channel. The blocking effect was strongly voltage dependent, as large positive potentials that favored the movement of ruthenium red into the channel conduction pore produced stronger block. The half dissociation constants (Kd) for ruthenium red block of the 500 pS channel were 0.22, 0.38, and 0.62 microM, at +100, +80, and +60 mV, respectively. Multiple ruthenium red molecules seemed to be involved in the inhibition, because a Hill coefficient of close to 2 was obtained from the dose response curve. The half dissociation constant of ruthenium red block of the lower conductance state of the ryanodine activated channel (250 pS) was higher (Kd = 0.82 microM at +100 mV), while the Hill coefficient remained approximately the same (nH = 2.7). Ruthenium red block of the channel was highly asymmetric, as trans ruthenium red produced a different blocking effect. The blocking and unblocking events (induced by cis ruthenium red) can be resolved at the single channel level at a cutoff frequency of 2 kHz. The closing rate of the channel in the presence of ruthenium red increased linearly with ruthenium red concentration, and the unblocking rate of the channel was independent of ruthenium red concentrations. This suggests that ruthenium red block of the channel occurred via a simple blocking mechanism. The on-rate of ruthenium red binding to the channel was 1.32 x 10(9) M-1 s-1, and the off-rate of ruthenium red binding was 0.75 x 10(3) s-1 at +60 mV, in the presence of 200 nM ryanodine. The two related compounds, 4APd and 4APt, blocked the channel in a similar way to that

  15. Ophiobolin A induces paraptosis-like cell death in human glioblastoma cells by decreasing BKCa channel activity.

    PubMed

    Bury, M; Girault, A; Mégalizzi, V; Spiegl-Kreinecker, S; Mathieu, V; Berger, W; Evidente, A; Kornienko, A; Gailly, P; Vandier, C; Kiss, R

    2013-01-01

    Glioblastoma multiforme (GBM) is the most lethal and common malignant human brain tumor. The intrinsic resistance of highly invasive GBM cells to radiation- and chemotherapy-induced apoptosis accounts for the generally dismal treatment outcomes. This study investigated ophiobolin A (OP-A), a fungal metabolite from Bipolaris species, for its promising anticancer activity against human GBM cells exhibiting varying degrees of resistance to proapoptotic stimuli. We found that OP-A induced marked changes in the dynamic organization of the F-actin cytoskeleton, and inhibited the proliferation and migration of GBM cells, likely by inhibiting big conductance Ca(2+)-activated K(+) channel (BKCa) channel activity. Moreover, our results indicated that OP-A induced paraptosis-like cell death in GBM cells, which correlated with the vacuolization, possibly brought about by the swelling and fusion of mitochondria and/or the endoplasmic reticulum (ER). In addition, the OP-A-induced cell death did not involve the activation of caspases. We also showed that the expression of BKCa channels colocalized with these two organelles (mitochondria and ER) was affected in this programmed cell death pathway. Thus, this study reveals a novel mechanism of action associated with the anticancer effects of OP-A, which involves the induction of paraptosis through the disruption of internal potassium ion homeostasis. Our findings offer a promising therapeutic strategy to overcome the intrinsic resistance of GBM cells to proapoptotic stimuli. PMID:23538442

  16. Activation and inhibition of thermosensitive TRP channels by voacangine, an alkaloid present in Voacanga africana, an African tree.

    PubMed

    Terada, Yuko; Horie, Syunji; Takayama, Hiromitsu; Uchida, Kunitoshi; Tominaga, Makoto; Watanabe, Tatsuo

    2014-02-28

    Voacangine (1) is an alkaloid found in the root bark of Voacanga africana. Our previous work has suggested that 1 is a novel transient receptor potential vanilloid type 1 (TRPV1) antagonist. In this study, the agonist and antagonist activities of 1 were examined against thermosensitive TRP channels. Channel activity was evaluated mainly using TRP channel-expressing HEK cells and calcium imaging. Herein, it was shown that 1 acts as an antagonist for TRPV1 and TRPM8 but as an agonist for TRPA1 (EC50, 8 μM). The compound competitively blocked capsaicin binding to TRPV1 (IC50, 50 μM). Voacangine (1) competitively inhibited the binding of menthol to TRPM8 (IC50, 9 μM), but it showed noncompetitive inhibition against icilin (IC50, 7 μM). Moreover, the compound selectively abrogated chemical agonist-induced TRPM8 activation and did not affect cold-induced activation. Among these effects, the TRPM8 inhibition profile is unique and noteworthy, because to date no studies have reported a menthol competitive inhibitor of TRPM8 derived from a natural source. Furthermore, this is the first report of a stimulus-selective TRPM8 antagonist. Accordingly, 1 may contribute to the development of a novel class of stimulus-selective TRPM8 blockers. PMID:24484240

  17. Single-Channel Kinetic Analysis for Activation and Desensitization of Homomeric 5-HT3A Receptors

    PubMed Central

    Corradi, Jeremías; Gumilar, Fernanda; Bouzat, Cecilia

    2009-01-01

    Abstract The 5-HT3A receptor is a member of the Cys-loop family of ligand-gated ion channels. To perform kinetic analysis, we mutated the 5-HT3A subunit to obtain a high-conductance form so that single-channel currents can be detected. At all 5-HT concentrations (>0.1 μM), channel activity appears as openings in quick succession that form bursts, which coalesce into clusters. By combining single-channel and macroscopic data, we generated a kinetic model that perfectly describes activation, deactivation, and desensitization. The model shows that full activation arises from receptors with three molecules of agonist bound. It reveals an earlier conformational change of the fully liganded receptor that occurs while the channel is still closed. From this pre-open closed state, the receptor enters into an open-closed cycle involving three open states, which form the cluster whose duration parallels the time constant of desensitization. A similar model lacking the pre-open closed state can describe the data only if the opening rates are fixed to account for the slow activation rate. The application of the model to M4 mutant receptors shows that position 10′ contributes to channel opening and closing rates. Thus, our kinetic model provides a foundation for understanding structural bases of activation and drug action. PMID:19720021

  18. Calcium-channel number critically influences synaptic strength and plasticity at the active zone

    PubMed Central

    Sheng, Jiansong; He, Liming; Zheng, Hongwei; Xue, Lei; Luo, Fujun; Shin, Wonchul; Sun, Tao; Kuner, Thomas; Yue, David T; Wu, Ling-Gang

    2016-01-01

    How synaptic-vesicle release is controlled at the basic release structure, the active zone, is poorly understood. By performing cell-attached current and capacitance recordings predominantly at single active zones in rat calyces, we found that single active zones contained 5-218 (mean, 42) calcium channels and 1–10 (mean, 5) readily releasable vesicles (RRVs) and released 0–5 vesicles during a 2-ms depolarization. Large variation in the number of calcium channels caused wide variation in release strength (measured during a 2-ms depolarization) by regulating the RRV release probability (PRRV) and the RRV number. Consequently, an action potential opened ~1–35 (mean, ~7) channels, resulting in different release probabilities at different active zones. As the number of calcium-channels determined PRRV, it critically influenced whether subsequent release would be facilitated or depressed. Regulating calcium channel density at active zones may thus be a major mechanism to yield synapses with different release properties and plasticity. These findings may explain large differences reported at synapses regarding release strength (release of 0, 1 or multiple vesicles), PRRV, short-term plasticity, calcium transients and the requisite calcium-channel number for triggering release. PMID:22683682

  19. Neural activities during affective processing in people with Alzheimer's disease.

    PubMed

    Lee, Tatia M C; Sun, Delin; Leung, Mei-Kei; Chu, Leung-Wing; Keysers, Christian

    2013-03-01

    This study examined brain activities in people with Alzheimer's disease when viewing happy, sad, and fearful facial expressions of others. A functional magnetic resonance imaging and a voxel-based morphometry methodology together with a passive viewing of emotional faces paradigm were employed to compare the affective processing in 12 people with mild Alzheimer's disease and 12 matched controls. The main finding was that the clinical participants showed reduced activations in regions associated with the motor simulation system (the ventral premotor cortex) and in regions associated with emotional simulation-empathy (the anterior insula and adjacent frontal operculum). This regional decline in blood oxygen level-dependent signals appeared to be lateralized in the left hemisphere and was not related to any structural degeneration in the clinical participants. Furthermore, the regions that showed changes in neural activity differed for the 3 emotional facial expressions studied. Findings of our study indicate that neural changes in regions associated with the motor and emotional simulation systems might play an important role in the development of Alzheimer's disease. PMID:22840336

  20. Language affects patterns of brain activation associated with perceptual decision.

    PubMed

    Tan, Li Hai; Chan, Alice H D; Kay, Paul; Khong, Pek-Lan; Yip, Lawrance K C; Luke, Kang-Kwong

    2008-03-11

    Well over half a century ago, Benjamin Lee Whorf [Carroll JB (1956) Language, Thought, and Reality: Selected Writings of Benjamin Lee Whorf (MIT Press, Cambridge, MA)] proposed that language affects perception and thought and is used to segment nature, a hypothesis that has since been tested by linguistic and behavioral studies. Although clear Whorfian effects have been found, it has not yet been demonstrated that language influences brain activity associated with perception and/or immediate postperceptual processes (referred hereafter as "perceptual decision"). Here, by using functional magnetic resonance imaging, we show that brain regions mediating language processes participate in neural networks activated by perceptual decision. When subjects performed a perceptual discrimination task on easy-to-name and hard-to-name colored squares, largely overlapping cortical regions were identified, which included areas of the occipital cortex critical for color vision and regions in the bilateral frontal gyrus. Crucially, however, in comparison with hard-to-name colored squares, perceptual discrimination of easy-to-name colors evoked stronger activation in the left posterior superior temporal gyrus and inferior parietal lobule, two regions responsible for word-finding processes, as demonstrated by a localizer experiment that uses an explicit color patch naming task. This finding suggests that the language-processing areas of the brain are directly involved in visual perceptual decision, thus providing neuroimaging support for the Whorf hypothesis. PMID:18316728

  1. Robust Stimulation of W1282X-CFTR Channel Activity by a Combination of Allosteric Modulators

    PubMed Central

    Wang, Wei; Hong, Jeong S.; Rab, Andras; Sorscher, Eric J.; Kirk, Kevin L.

    2016-01-01

    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) < 0.01). However, W1282X-CFTR channels were stimulated by two CFTR modulators, the FDA-approved VX-770 and the dietary compound curcumin. Each of these compounds is an allosteric modulator of CFTR gating that promotes channel activity in the absence of the native ligand, ATP. Although W1282X-CFTR channels were stimulated by VX-770 in the absence of ATP their activities remained dependent on PKA phosphorylation. Thus, activated W1282X-CFTR channels should remain under physiologic control by cyclic nucleotide signaling pathways in vivo. VX-770 and curcumin exerted additive effects on W1282X-CFTR channel gating (opening/closing) in excised patches such that the Po of the truncated channel approached unity (> 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. PMID:27007499

  2. Robust Stimulation of W1282X-CFTR Channel Activity by a Combination of Allosteric Modulators.

    PubMed

    Wang, Wei; Hong, Jeong S; Rab, Andras; Sorscher, Eric J; Kirk, Kevin L

    2016-01-01

    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) < 0.01). However, W1282X-CFTR channels were stimulated by two CFTR modulators, the FDA-approved VX-770 and the dietary compound curcumin. Each of these compounds is an allosteric modulator of CFTR gating that promotes channel activity in the absence of the native ligand, ATP. Although W1282X-CFTR channels were stimulated by VX-770 in the absence of ATP their activities remained dependent on PKA phosphorylation. Thus, activated W1282X-CFTR channels should remain under physiologic control by cyclic nucleotide signaling pathways in vivo. VX-770 and curcumin exerted additive effects on W1282X-CFTR channel gating (opening/closing) in excised patches such that the Po of the truncated channel approached unity (> 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. PMID:27007499

  3. Modulation of Ca2+ oscillation and melatonin secretion by BKCa channel activity in rat pinealocytes.

    PubMed

    Mizutani, Hiroya; Yamamura, Hisao; Muramatsu, Makoto; Hagihara, Yumiko; Suzuki, Yoshiaki; Imaizumi, Yuji

    2016-05-01

    The pineal glands regulate circadian rhythm through the synthesis and secretion of melatonin. The stimulation of nicotinic acetylcholine receptor due to parasympathetic nerve activity causes an increase in intracellular Ca(2+) concentration and eventually downregulates melatonin production. Our previous report shows that rat pinealocytes have spontaneous and nicotine-induced Ca(2+) oscillations that are evoked by membrane depolarization followed by Ca(2+) influx through voltage-dependent Ca(2+) channels (VDCCs). These Ca(2+) oscillations are supposed to contribute to the inhibitory mechanism of melatonin secretion. Here we examined the involvement of large-conductance Ca(2+)-activated K(+) (BKCa) channel conductance on the regulation of Ca(2+) oscillation and melatonin production in rat pinealocytes. Spontaneous Ca(2+) oscillations were markedly enhanced by BKCa channel blockers (1 μM paxilline or 100 nM iberiotoxin). Nicotine (100 μM)-induced Ca(2+) oscillations were also augmented by paxilline. In contrast, spontaneous Ca(2+) oscillations were abolished by BKCa channel opener [3 μM 12,14-dichlorodehydroabietic acid (diCl-DHAA)]. Under whole cell voltage-clamp configurations, depolarization-elicited outward currents were significantly activated by diCl-DHAA and blocked by paxilline. Expression analyses revealed that the α and β3 subunits of BKCa channel were highly expressed in rat pinealocytes. Importantly, the activity of BKCa channels modulated melatonin secretion from whole pineal gland of the rat. Taken together, BKCa channel activation attenuates these Ca(2+) oscillations due to depolarization-synchronized Ca(2+) influx through VDCCs and results in a recovery of reduced melatonin secretion during parasympathetic nerve activity. BKCa channels may play a physiological role for melatonin production via a negative-feedback mechanism. PMID:26791489

  4. Vanilloid receptor-related osmotically activated channel (VR-OAC), a candidate vertebrate osmoreceptor

    PubMed Central

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

    2008-01-01

    SUMMARY The detection of osmotic stimuli is essential for all organisms, yet few osmoreceptive proteins are known, none of them in vertebrates. 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) from the rat, mouse, human, and chicken. This novel cation-selective channel is gated by exposure to hypotonicity within the physiological range. In the central nevous system, the channel is expressed neurons of the circumventricular organs, neurosensory cells responsive to systemic osmotic pressure. The channel also occurs in other neurosensory cells, including inner-ear hair cells, sensory neurons, and Merkel cells. PMID:11081638

  5. How Lipid Membranes Affect Pore Forming Toxin Activity.

    PubMed

    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

  6. Mechanisms of STIM1 Activation of Store-Independent Leukotriene C4-Regulated Ca2+ Channels

    PubMed Central

    Zhang, Xuexin; González-Cobos, José C.; Schindl, Rainer; Muik, Martin; Ruhle, Brian; Motiani, Rajender K.; Bisaillon, Jonathan M.; Zhang, Wei; Fahrner, Marc; Barroso, Margarida; Matrougui, Khalid; Romanin, Christoph

    2013-01-01

    We recently showed, in primary vascular smooth muscle cells (VSMCs), that the platelet-derived growth factor activates canonical store-operated Ca2+ entry and Ca2+ release-activated Ca2+ currents encoded by Orai1 and STIM1 genes. However, thrombin activates store-independent Ca2+ selective channels contributed by both Orai3 and Orai1. These store-independent Orai3/Orai1 channels are gated by cytosolic leukotriene C4 (LTC4) and require STIM1 downstream LTC4 action. However, the source of LTC4 and the signaling mechanisms of STIM1 in the activation of this LTC4-regulated Ca2+ (LRC) channel are unknown. Here, we show that upon thrombin stimulation, LTC4 is produced through the sequential activities of phospholipase C, diacylglycerol lipase, 5-lipo-oxygenease, and leukotriene C4 synthase. We show that the endoplasmic reticulum-resident STIM1 is necessary and sufficient for LRC channel activation by thrombin. STIM1 does not form sustained puncta and does not colocalize with Orai1 either under basal conditions or in response to thrombin. However, STIM1 is precoupled to Orai3 and Orai3/Orai1 channels under basal conditions as shown using Forster resonance energy transfer (FRET) imaging. The second coiled-coil domain of STIM1 is required for coupling to either Orai3 or Orai3/Orai1 channels and for LRC channel activation. We conclude that STIM1 employs distinct mechanisms in the activation of store-dependent and store-independent Ca2+ entry pathways. PMID:23878392

  7. Gadolinium inhibits mechanoelectrical transduction in rabbit carotid baroreceptors. Implication of stretch-activated channels.

    PubMed Central

    Hajduczok, G; Chapleau, M W; Ferlic, R J; Mao, H Z; Abboud, F M

    1994-01-01

    Gadolinium (Gd3+) has been shown to prevent mechanoelectrical transduction believed to be mediated through stretch-activated channels. We investigated the possible role of Gd(3+)-sensitive channels in mediating baroreceptor activity in the carotid sinus of rabbits. Baroreceptor activity induced by a ramp increase of carotid sinus pressure was reduced significantly during exposure to Gd3+. The inhibition was dose-related and reversible, and was not associated with alteration of carotid sinus wall mechanics as the pressure-strain relationship was unaffected. Veratrine triggered action potentials from single- and multiple-baroreceptor fibers when their response to pressure was inhibited by Gd3+. This suggests that the effect of Gd3+ on baroreceptors in the isolated carotid sinus was specific to their mechanical activation. The results suggest that stretch-activated ion channels sensitive to Gd3+ may be the mechanoelectrical transducers of rabbit carotid sinus baroreceptors. PMID:7527431

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

    PubMed

    Chen, Qijing; Tao, Jie; Hei, Hongya; Li, Fangping; Wang, Yunman; Peng, Wen; Zhang, Xuemei

    2015-01-01

    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. PMID:26672753

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

    PubMed Central

    Hei, Hongya; Li, Fangping; Wang, Yunman; Peng, Wen; Zhang, Xuemei

    2015-01-01

    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. PMID:26672753

  10. Chaotic model and memory in single calcium-activated potassium channel kinetics

    NASA Astrophysics Data System (ADS)

    Bandeira, Heliovânio T.; Barbosa, Catão T. F.; Campos De Oliveira, Regina A.; Aguiar, José F.; Nogueira, Romildo A.

    2008-09-01

    Ion channels are pores formed by proteins and responsible for carrying ion fluxes through cellular membranes. The ion channels can assume conformational states thereby controlling ion flow. Physically, the conformational transitions from one state to another are associated with energy barriers between them and are dependent on stimulus, such as, electrical field, ligands, second messengers, etc. Several models have been proposed to describe the kinetics of ion channels. The classical Markovian model assumes that a future transition is independent of the time that the ion channel stayed in a previous state. Others models as the fractal and the chaotic assume that the rate of transitions between the states depend on the time that the ionic channel stayed in a previous state. For the calcium activated potassium channels of Leydig cells the R/S Hurst analysis has indicated that the channels are long-term correlated with a Hurst coefficient H around 0.7, showing a persistent memory in this kinetic. Here, we applied the R /S analysis to the opening and closing dwell time series obtained from simulated data from a chaotic model proposed by L. Liebovitch and T. Tóth [J. Theor. Biol. 148, 243 (1991)] and we show that this chaotic model or any model that treats the set of channel openings and closings as independent events is inadequate to describe the long-term correlation (memory) already described for the experimental data.

  11. The vacuolar Ca2+-activated channel TPC1 regulates germination and stomatal movement.

    PubMed

    Peiter, Edgar; Maathuis, Frans J M; Mills, Lewis N; Knight, Heather; Pelloux, Jérôme; Hetherington, Alistair M; Sanders, Dale

    2005-03-17

    Cytosolic free calcium ([Ca2+]cyt) is a ubiquitous signalling component in plant cells. Numerous stimuli trigger sustained or transient elevations of [Ca2+]cyt that evoke downstream stimulus-specific responses. Generation of [Ca2+]cyt signals is effected through stimulus-induced opening of Ca2+-permeable ion channels that catalyse a flux of Ca2+ into the cytosol from extracellular or intracellular stores. Many classes of Ca2+ current have been characterized electrophysiologically in plant membranes. However, the identity of the ion channels that underlie these currents has until now remained obscure. Here we show that the TPC1 ('two-pore channel 1') gene of Arabidopsis thaliana encodes a class of Ca2+-dependent Ca2+-release channel that is known from numerous electrophysiological studies as the slow vacuolar channel. Slow vacuolar channels are ubiquitous in plant vacuoles, where they form the dominant conductance at micromolar [Ca2+]cyt. We show that a tpc1 knockout mutant lacks functional slow vacuolar channel activity and is defective in both abscisic acid-induced repression of germination and in the response of stomata to extracellular calcium. These studies unequivocally demonstrate a critical role of intracellular Ca2+-release channels in the physiological processes of plants. PMID:15772667

  12. RIM Promotes Calcium Channel Accumulation at Active Zones of the Drosophila Neuromuscular Junction

    PubMed Central

    Graf, Ethan R.; Valakh, Vera; Wright, Christina M.; Wu, Chunlai; Liu, Zhihua; Zhang, Yong Q.; DiAntonio, Aaron

    2012-01-01

    Summary Synaptic communication requires the controlled release of synaptic vesicles from presynaptic axon terminals. Release efficacy is regulated by the many proteins that comprise the presynaptic release apparatus, including Ca2+ channels and proteins that influence Ca2+ channel accumulation at release sites. Here we identify Drosophila RIM and demonstrate that it localizes to active zones at the larval neuromuscular junction. In Drosophila RIM mutants, there is a large decrease in evoked synaptic transmission, due to a significant reduction in both the clustering of Ca2+ channels and the size of the readily releasable pool of synaptic vesicles at active zones. Hence, RIM plays an evolutionarily conserved role in regulating synaptic calcium channel localization and readily releasable pool size. Since RIM has traditionally been studied as an effector of Rab3 function, we investigate whether RIM is involved in the newly identified function of Rab3 in the distribution of presynaptic release machinery components across release sites. Bruchpilot (Brp), an essential component of the active zone cytomatrix T bar, is unaffected by RIM disruption, indicating that Brp localization and distribution across active zones does not require wild type RIM. In addition, larvae containing mutations in both RIM and rab3 have reduced Ca2+ channel levels and a Brp distribution that is very similar to that of the rab3 single mutant, indicating that RIM functions to regulate Ca2+ channel accumulation but is not a Rab3 effector for release machinery distribution across release sites. PMID:23175814

  13. Thermosensitive TRP channel pore turret is part of the temperature activation pathway

    PubMed Central

    Yang, Fan; Cui, Yuanyuan; Wang, KeWei; Zheng, Jie

    2010-01-01

    Temperature sensing is crucial for homeotherms, including human beings, to maintain a stable body core temperature and respond to the ambient environment. A group of exquisitely temperature-sensitive transient receptor potential channels, termed thermoTRPs, serve as cellular temperature sensors. How thermoTRPs convert thermal energy (heat) into protein conformational changes leading to channel opening remains unknown. Here we demonstrate that the pathway for temperature-dependent activation is distinct from those for ligand- and voltage-dependent activation and involves the pore turret. We found that mutant channels with an artificial pore turret sequence lose temperature sensitivity but maintain normal ligand responses. Using site-directed fluorescence recordings we observed that temperature change induces a significant rearrangement of TRPV1 pore turret that is coupled to channel opening. This movement is specifically associated to temperature-dependent activation and is not observed during ligand- and voltage-dependent channel activation. These observations suggest that the turret is part of the temperature-sensing apparatus in thermoTRP channels, and its conformational change may give rise to the large entropy that defines high temperature sensitivity. PMID:20351268

  14. Long-term correlation in single calcium-activated potassium channel kinetics

    NASA Astrophysics Data System (ADS)

    Campos de Oliveira, R. A.; Barbosa, C. T. F.; Consoni, L. H. A.; Rodrigues, A. R. A.; Varanda, W. A.; Nogueira, R. A.

    2006-05-01

    Ion channels are protein molecules found in biological membranes, which can assume distinct open and closed conformational states, a phenomenon called ion channel kinetics. The transitions from one state to another are dependent on the potential energy barrier that separates them and can be controlled by the electrical field, ions and/or drugs. Both Markovian and fractal models have been used for modeling the ion channel kinetics. Ion single channel records are characterized by successive openings and closings, which are correlated in time. Here the rescaled range analysis ( R/S Hurst analysis) is used to test for the occurrence of long-term correlation in the kinetics of a calcium-activated potassium channel of Leydig cells. A Hurst coefficient H=0.640±0.064 ( n=5) was found for the single calcium-activated potassium channel clamped at -80 mV and exposed to a free Ca 2+ concentration equal to 10 nM. This numerical value indicates the presence of long-term correlation (memory) in this kinetic process. However, when the R/ S analysis was applied to ion channel data simulated using Markovian and fractal models, it could not account for the long-term correlation previously found in the experimental data. In summary, in this work we show that: (i) opening and closing dwell times for the single calcium-activated potassium channel of Leydig cells present long-term correlation and (ii) Markovian and fractal models, which describe well the dwell time distributions, are not adequate to describe the memory found in the kinetics of this channel.

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

    NASA Astrophysics Data System (ADS)

    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.

  16. Dehydrated Hereditary Stomatocytosislinked to gain-of-function mutations in mechanically activated PIEZO1 ion channels

    PubMed Central

    Albuisson, Juliette; Murthy, Swetha E.; Bandell, Michael; Coste, Bertrand; Louis-dit-Picard, Hélène; Mathur, Jayanti; Fénéant-Thibault, Madeleine; Tertian, Gérard; de Jaureguiberry, Jean-Pierre; Syfuss, Pierre-Yves; Cahalan, Stuart; Garçon, Loic; Toutain, Fabienne; Rohrlich, Pierre Simon; Delaunay, Jean; Picard, Véronique; Jeunemaitre, Xavier; Patapoutian, Ardem

    2013-01-01

    Dehydrated hereditary stomatocytosis (DHS) is a genetic condition with defective red blood cell (RBC) membrane properties that causes an imbalance in intracellular cation concentrations. Recently, two missense mutations inthe mechanically activated PIEZO1(FAM38A) ion channel were associated with DHS. However, it is not known how these mutations affect PIEZO1 function. Here, by combining linkage analysis and whole-exome sequencing in a large pedigree and Sanger sequencing in two additional kindreds and 11 unrelated DHS cases, we identifythree novel missense mutations and one recurrent duplication in PIEZO1, demonstrating that it is the major gene for DHS. All the DHS-associated mutations locate at C-terminal half of PIEZO1. Remarkably, we find that all PIEZO1 mutations give rise to mechanically activated currents that inactivate more slowly than wild-type currents. This gain-of-function PIEZO1 phenotype provides insight that helps to explain the increased permeability of cations in RBCs of DHS patients. Our findings also suggest a new role for mechanotransduction in RBC biology and pathophysiology. PMID:23695678

  17. Divalent cations potentiate TRPV1 channel by lowering the heat activation threshold

    PubMed Central

    Cao, Xu; Ma, Linlin; Yang, Fan

    2014-01-01

    Transient receptor potential vanilloid type 1 (TRPV1) channel responds to a wide spectrum of physical and chemical stimuli. In doing so, it serves as a polymodal cellular sensor for temperature change and pain. Many chemicals are known to strongly potentiate TRPV1 activation, though how this is achieved remains unclear. In this study we investigated the molecular mechanism underlying the gating effects of divalent cations Mg2+ and Ba2+. Using a combination of fluorescence imaging and patch-clamp analysis, we found that these cations potentiate TRPV1 gating by most likely promoting the heat activation process. Mg2+ substantially lowers the activation threshold temperature; as a result, a significant fraction of channels are heat-activated at room temperature. Although Mg2+ also potentiates capsaicin- and voltage-dependent activation, these processes were found either to be not required (in the case of capsaicin) or insufficient (in the case of voltage) to mediate the activating effect. In support of a selective effect on heat activation, Mg2+ and Ba2+ cause a Ca2+-independent desensitization that specifically prevents heat-induced channel activation but does not prevent capsaicin-induced activation. These results can be satisfactorily explained within an allosteric gating framework in which divalent cations strongly promote the heat-dependent conformational change or its coupling to channel activation, which is further coupled to the voltage- and capsaicin-dependent processes. PMID:24344247

  18. Modulation of channel activity and gadolinium block of MscL by static magnetic fields.

    PubMed

    Petrov, Evgeny; Martinac, Boris

    2007-02-01

    The magnetic field of the Earth has for long been known to influence the behaviour and orientation of a variety of living organisms. Experimental studies of the magnetic sense have, however, been impaired by the lack of a plausible cellular and/or molecular mechanism providing meaningful explanation for detection of magnetic fields by these organisms. Recently, mechanosensitive (MS) ion channels have been implied to play a role in magnetoreception. In this study we have investigated the effect of static magnetic fields (SMFs) of moderate intensity on the activity and gadolinium block of MscL, the bacterial MS channel of large conductance, which has served as a model channel to study the basic physical principles of mechanosensory transduction in living cells. In addition to showing that direct application of the magnetic field decreased the activity of the MscL channel, our study demonstrates for the first time that SMFs can reverse the effect of gadolinium, a well-known blocker of MS channels. The results of our study are consistent with a notion that (1) the effects of SMFs on the MscL channels may result from changes in physical properties of the lipid bilayer due to diamagnetic anisotropy of phospholipid molecules and consequently (2) cooperative superdiamagnetism of phospholipid molecules under influence of SMFs could cause displacement of Gd(3+) ions from the membrane bilayer and thus remove the MscL channel block. PMID:17089151

  19. Glial potassium channels activated by neuronal firing or intracellular cyclic AMP in Helix.

    PubMed Central

    Gommerat, I; Gola, M

    1996-01-01

    1. Cell-attached and whole cell patch clamp experiments were performed on satellite glial cells adhering to the cell body of neurones in situ within the nervous system of the snail Helix pomatia. The underlying neurone was under current or voltage-clamp control. 2. Neuronal firing induced a delayed (20-30 s) persistent (3-4 min) increase in the opening probability of glial K+ channels. The channels were also activated by perfusing the ganglion with a depolarizing high-K+ saline, except when the underlying neurone was prevented from depolarizing under voltage-clamp conditions. 3. Two K(+)-selective channels were detected in the glial membrane. The channel responding to neuronal firing was present in 95% of the patches (n = 393). It had a unitary conductance of 56 pS, a Na+ :K+ permeability ratio < 0.02 and displayed slight inward rectification in symmetrical [K+] conditions. It was sensitive to TEA, Ba2+ and Cs+. The following results refer to this channel as studied in the cell-attached configuration. 4. The glial K+ channel was activated by bath application of the membrane-permeant cyclic AMP derivatives 8-bromo-cAMP and dibutyryl-cAMP, the adenylyl cyclase activator forskolin and the diesterase inhibitors IBMX, theophylline and caffeine. It was insensitive to cyclic GMP activators and to conditions that might alter the intracellular [Ca2+] (ionomycin, low-Ca2+ saline and Ca2+ channel blockers). 5. The forskolin-induced changes in channel behaviour (open and closed time distributions, burst duration, short and long gaps within bursts) could be accounted for by a four-state model (3 closed states, 1 open state) by simply changing one of the six rate parameters. 6. The present results suggest that the signal sent by an active neurone to satellite glial cells is confined to the glial cells round that neurone. The effect of this signal on the class of glial K+ channels studied can be mimicked by an increase in glial cAMP concentration. The subsequent delayed opening

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

    PubMed

    Gururaj, Sushmitha; Fleites, John; Bhattacharjee, Arin

    2016-04-01

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

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

    PubMed Central

    Gründemann, Jan; Clark, Beverley A.

    2015-01-01

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

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

    PubMed

    Gründemann, Jan; Clark, Beverley A

    2015-09-22

    Functional connectivity between brain regions relies on long-range signaling by myelinated axons. This is secured by saltatory action potential propagation that depends fundamentally on sodium channel availability at nodes of Ranvier. Although various potassium channel types have been anatomically localized to myelinated axons in the brain, direct evidence for their functional recruitment in maintaining node excitability is scarce. Cerebellar Purkinje cells provide continuous input to their targets in the cerebellar nuclei, reliably transmitting axonal spikes over a wide range of rates, requiring a constantly available pool of nodal sodium channels. We show that the recruitment of calcium-activated potassium channels (IK, K(Ca)3.1) by local, activity-dependent calcium (Ca(2+)) influx at nodes of Ranvier via a T-type voltage-gated Ca(2+) current provides a powerful mechanism that likely opposes depolarizing block at the nodes and is thus pivotal to securing continuous axonal spike propagation in spontaneously firing Purkinje cells. PMID:26344775

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

    PubMed

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

    2010-10-01

    This investigation focuses on the in vitro neuroactive properties of pinostrobin, a substituted flavanone from Cajanus cajan (L.) Millsp. of the Fabaceae family. We demonstrate that pinostrobin inhibits voltage-gated sodium channels of mammalian brain (IC(50)=23 µM) based on the ability of this substance to suppress the depolarizing effects of the sodium channel-selective activator veratridine in a synaptoneurosomal preparation from mouse brain. The resting membrane potential of synaptoneurosomes was unaffected by pinostrobin. The pharmacological profile of pinostrobin resembles that of depressant drugs that block sodium channels. PMID:20472040

  4. Inaudible high-frequency sounds affect brain activity: hypersonic effect.

    PubMed

    Oohashi, T; Nishina, E; Honda, M; Yonekura, Y; Fuwamoto, Y; Kawai, N; Maekawa, T; Nakamura, S; Fukuyama, H; Shibasaki, H

    2000-06-01

    Although it is generally accepted that humans cannot perceive sounds in the frequency range above 20 kHz, the question of whether the existence of such "inaudible" high-frequency components may affect the acoustic perception of audible sounds remains unanswered. In this study, we used noninvasive physiological measurements of brain responses to provide evidence that sounds containing high-frequency components (HFCs) above the audible range significantly affect the brain activity of listeners. We used the gamelan music of Bali, which is extremely rich in HFCs with a nonstationary structure, as a natural sound source, dividing it into two components: an audible low-frequency component (LFC) below 22 kHz and an HFC above 22 kHz. Brain electrical activity and regional cerebral blood flow (rCBF) were measured as markers of neuronal activity while subjects were exposed to sounds with various combinations of LFCs and HFCs. None of the subjects recognized the HFC as sound when it was presented alone. Nevertheless, the power spectra of the alpha frequency range of the spontaneous electroencephalogram (alpha-EEG) recorded from the occipital region increased with statistical significance when the subjects were exposed to sound containing both an HFC and an LFC, compared with an otherwise identical sound from which the HFC was removed (i.e., LFC alone). In contrast, compared with the baseline, no enhancement of alpha-EEG was evident when either an HFC or an LFC was presented separately. Positron emission tomography measurements revealed that, when an HFC and an LFC were presented together, the rCBF in the brain stem and the left thalamus increased significantly compared with a sound lacking the HFC above 22 kHz but that was otherwise identical. Simultaneous EEG measurements showed that the power of occipital alpha-EEGs correlated significantly with the rCBF in the left thalamus. Psychological evaluation indicated that the subjects felt the sound containing an HFC to be more

  5. STIM1 dimers undergo unimolecular coupling to activate Orai1 channels

    NASA Astrophysics Data System (ADS)

    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.

  6. Activity of Ca2+ -activated Cl- channels contributes to regulating receptor- and store-operated Ca2+ entry in human pulmonary artery smooth muscle cells

    PubMed Central

    Yamamura, Aya; Yamamura, Hisao; Zeifman, Amy; Yuan, Jason X.-J.

    2011-01-01

    Intracellular Ca2+ plays a fundamental role in regulating cell functions in pulmonary arterial smooth muscle cells (PASMCs). A rise in cytosolic Ca2+ concentration ([Ca2+]cyt) triggers pulmonary vasoconstriction and stimulates PASMC proliferation. [Ca2+]cyt is increased mainly by Ca2+ release from intracellular stores and Ca2+ influx through plasmalemmal Ca2+-permeable channels. Given the high concentration of intracellular Cl- in PASMCs, Ca2+-activated Cl-(ClCa) channels play an important role in regulating membrane potential and cell excitability of PASMCs. In this study, we examined whether activity of ClCa channels was involved in regulating [Ca2+]cyt in human PASMCs via regulating receptor- (ROCE) and store- (SOCE) operated Ca2+ entry. The data demonstrated that an angiotensin II (100 nM)-mediated increase in [Ca2+]cyt via ROCE was markedly attenuated by the ClCa channel inhibitors, niflumic acid (100 μM), flufenamic acid (100 μM), and 4,4’-diisothiocyanatostilbene-2,2’-disulfonic acid (100 μM). The inhibition of ClCa channels by niflumic acid and flufenamic acid significantly reduced both transient and plateau phases of SOCE that was induced by passive depletion of Ca2+ from the sarcoplasmic reticulum by 10 μM cyclopiazonic acid. In addition, ROCE and SOCE were abolished by SKF-96365 (50 μM) and 2-aminoethyl diphenylborinate (100 μM), and were slightly decreased in the presence of diltiazem (10 μM). The electrophysiological and immunocytochemical data indicate that ClCa currents were present and TMEM16A was functionally expressed in human PASMCs. The results from this study suggest that the function of ClCa channels, potentially formed by TMEM16A proteins, contributes to regulating [Ca2+]cyt by affecting ROCE and SOCE in human PASMCs. PMID:22034612

  7. Proton Channel Activity of Influenza A Virus Matrix Protein 2 Contributes to Autophagy Arrest

    PubMed Central

    Ren, Yizhong; Feng, Liqiang; Pan, Weiqi; Li, Liang; Wang, Qian; Li, Jiashun; Li, Na; Han, Ling; Zheng, Xuehua; Niu, Xuefeng; Sun, Caijun

    2015-01-01

    Influenza A virus infection can arrest autophagy, as evidenced by autophagosome accumulation in infected cells. Here, we report that this autophagosome accumulation can be inhibited by amantadine, an antiviral proton channel inhibitor, in amantadine-sensitive virus infected cells or cells expressing influenza A virus matrix protein 2 (M2). Thus, M2 proton channel activity plays a role in blocking the fusion of autophagosomes with lysosomes, which might be a key mechanism for arresting autophagy. PMID:26468520

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

    PubMed

    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

  9. Endoxifen, the active metabolite of tamoxifen, inhibits cloned hERG potassium channels.

    PubMed

    Chae, Yun Ju; Lee, Keon Jin; Lee, Hong Joon; Sung, Ki-Wug; Choi, Jin-Sung; Lee, Eun Hui; Hahn, Sang June

    2015-04-01

    The effects of tamoxifen, and its active metabolite endoxifen (4-hydroxy-N-desmethyl-tamoxifen), on hERG currents stably expressed in HEK cells were investigated using the whole-cell patch-clamp technique and an immunoblot assay. Tamoxifen and endoxifen inhibited hERG tail currents at -50mV in a concentration-dependent manner with IC50 values of 1.2 and 1.6μM, respectively. The steady-state activation curve of the hERG currents was shifted to the hyperpolarizing direction in the presence of endoxifen. The voltage-dependent inhibition of hERG currents by endoxifen increased steeply in the voltage range of channel activation. The inhibition by endoxifen displayed a shallow voltage dependence (δ=0.18) in the full activation voltage range. A fast application of endoxifen induced a reversible block of hERG tail currents during repolarization in a concentration-dependent manner, which suggested an interaction with the open state of the channel. Endoxifen also decreased the hERG current elicited by a 5s depolarizing pulse to +60mV to inactivate the hERG currents, suggesting an interaction with the activated (open and/or inactivated) states of the channels. Tamoxifen and endoxifen inhibited the hERG channel protein trafficking to the plasma membrane in a concentration-dependent manner with endoxifen being more potent than tamoxifen. These results indicated that tamoxifen and endoxifen inhibited the hERG current by direct channel blockage and by the disruption of channel trafficking to the plasma membrane in a concentration-dependent manner. A therapeutic concentration of endoxifen inhibited the hERG current by preferentially interacting with the activated (open and/or inactivated) states of the channel. PMID:25680947

  10. Hormone-regulated K+ channels in follicle-enclosed oocytes are activated by vasorelaxing K+ channel openers and blocked by antidiabetic sulfonylureas.

    PubMed Central

    Honoré, E; Lazdunski, M

    1991-01-01

    Follicular oocytes from Xenopus laevis contain K+ channels activated by members of the recently recognized class of vasorelaxants that include cromakalim and pinacidil and blocked by antidiabetic sulfonylureas, such as glibenclamide. These channels are situated on the adherent follicular cells and are not present in denuded oocytes. Cromakalim-activated K+ channels are also activated by increases in intracellular cAMP, and cAMP-activated K+ channels are blocked by glibenclamide. Although cromakalim and cAMP effects are synergistic, cromakalim activation of K+ channels is drastically reduced or abolished by treatments that stimulate protein kinase C (e.g., muscarinic effectors, phorbol esters). Gonadotropins, known to play an essential role in ovarian physiology, also activate cromakalim and sulfonylurea-sensitive K+ channels. Follicular oocytes constitute an excellent system for studying regulation of cromakalim-sensitive K+ channels that are important in relation to a variety of disease processes, such as cardiovascular dysfunction and asthma, as well as brain function. PMID:1647032

  11. Quantifying the transient response of bedrock channels to Active Normal Faulting: New Field Observations

    NASA Astrophysics Data System (ADS)

    Whittaker, A. C.; Cowie, P. A.; Tucker, G. E.; Attal, M.; Roberts, G.

    2005-12-01

    Understanding the morphological response of the fluvial system to transient tectonic forcing is one of the major challenges facing quantitative geomorphology. In theory, insight gained from studying channel adjustment to changing tectonic rates should provide clear diagnostic tests of the many competing `erosion laws' which aim to quantify stream incision. However, fluvial algorithms in current landscape models tend to be parameterised in terms of hydraulic scaling relationships, which only describe channel width and depth as power-law functions of river discharge or upstream drainage area. Unfortunately, these scaling relationships, which have been derived from channels in tectonically quiescent areas, are not appropriate for bedrock rivers in active settings. This problem is serious for understanding non-equilibrium systems because hydraulic adjustments are an important aspect of the morphodynamic response to tectonic and climatic forcing. Recent theoretical attempts to resolve this issue still rely fundamentally on assumptions of steady-state channel form. To devise an alternative approach we need to collect geometrical data for channels incising in areas where the boundary conditions are well-constrained independently. We address this challenge by providing new and detailed field measurements of valley and bankfull channel width, depth, slope and grain-size data for an out-of-equilibrium channel with a drainage area of 65km2 crossing an active extensional fault near Fiamignano, Italy, where there are excellent constraints on current rates of fault movement, and good evidence for an increase in throw-rate approximately 700 Kyr ago. We show that in this situation channel width becomes strongly decoupled from drainage area immediately upstream of the fault and that channel aspect ratio and median grain-size are correlated with channel slope. The ratio of total stream power to coarse-fraction grain size peaks in precisely the areas where channel width

  12. A Kv7.2 mutation associated with early onset epileptic encephalopathy with suppression-burst enhances Kv7/M channel activity.

    PubMed

    Devaux, Jérôme; Abidi, Affef; Roubertie, Agathe; Molinari, Florence; Becq, Hélène; Lacoste, Caroline; Villard, Laurent; Milh, Mathieu; Aniksztejn, Laurent

    2016-05-01

    Mutations in the KCNQ2 gene encoding the voltage-gated potassium channel subunit Kv7.2 cause early onset epileptic encephalopathy (EOEE). Most mutations have been shown to induce a loss of function or to affect the subcellular distribution of Kv7 channels in neurons. Herein, we investigated functional consequences and subcellular distribution of the p.V175L mutation of Kv7.2 (Kv7.2(V175L) ) found in a patient presenting EOEE. We observed that the mutation produced a 25-40 mV hyperpolarizing shift of the conductance-voltage relationship of both the homomeric Kv7.2(V175L) and heteromeric Kv7.2(V175L) /Kv7.3 channels compared to wild-type channels and a 10 mV hyperpolarizing shift of Kv7.2(V175L) /Kv7.2/Kv7.3 channels in a 1:1:2 ratio mimicking the patient situation. Mutant channels also displayed faster activation kinetics and an increased current density that was prevented by 1 μm linopirdine. The p.V175L mutation did not affect the protein expression of Kv7 channels and its localization at the axon initial segment. We conclude that p.V175L is a gain of function mutation. This confirms previous observations showing that mutations having opposite consequences on M channels can produce EOEE. These findings alert us that drugs aiming to increase Kv7 channel activity might have adverse effects in EOEE in the case of gain-of-function variants. PMID:27030113

  13. KCNQ channel openers reverse depressive symptoms via an active resilience mechanism

    PubMed Central

    Friedman, Allyson K.; Juarez, Barbara; Ku, Stacy M.; Zhang, Hongxing; Calizo, Rhodora C.; Walsh, Jessica J.; Chaudhury, Dipesh; Zhang, Song; Hawkins, Angel; Dietz, David M.; Murrough, James W.; Ribadeneira, Maria; Wong, Erik H.; Neve, Rachael L.; Han, Ming-Hu

    2016-01-01

    Less than half of patients suffering from major depressive disorder, a leading cause of disability worldwide, achieve remission with current antidepressants, making it imperative to develop more effective treatment. A new therapeutic direction is emerging from the increased understanding of natural resilience as an active stress-coping process. It is known that potassium (K+) channels in the ventral tegmental area (VTA) are an active mediator of resilience. However, no druggable targets have been identified to potentiate active resilience mechanisms. In the chronic social defeat stress model of depression, we report that KCNQ-type K+ channel openers, including FDA-approved drug retigabine (ezogabine), show antidepressant efficacy. We demonstrate that overexpression of KCNQ channels in the VTA dopaminergic neurons and either local infusion or systemic administration of retigabine normalized neuronal hyperactivity and depressive behaviours. These findings identify KCNQ as a target for conceptually novel antidepressants that function through the potentiation of active resilience mechanisms. PMID:27216573

  14. Role of CFTR's intrinsic adenylate kinase activity in gating of the Cl(-) channel.

    PubMed

    Randak, Christoph O; Welsh, Michael J

    2007-12-01

    The cystic fibrosis transmembrane conductance regulator (CFTR) is a Cl(-)channel in the ATP-binding cassette (ABC) transporter protein family. CFTR features the modular design characteristic of ABC transporters, which includes two membrane-spanning domains forming the channel pore, and two ABC nucleotide-binding domains that interact with ATP and contain the enzymatic activity coupled to normal gating. Like other ABC transporters CFTR is an ATPase (ATP + H(2)O --> ADP + Pi). Recent work has shown that CFTR also possesses intrinsic adenylate kinase activity (ATP + AMP left arrow over right arrow ADP + ADP). This finding raises important questions: How does AMP influence CFTR gating? Why does ADP inhibit CFTR current? Which enzymatic activity gates CFTR in vivo? Are there implications for other ABC transporters? This minireview attempts to shed light on these questions by summarizing recent advances in our understanding of the role of the CFTR adenylate kinase activity for channel gating. PMID:17965924

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

    PubMed Central

    Almassy, Janos; Won, Jong Hak; Begenisich, Ted B.

    2012-01-01

    Ca2+ 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 [Ca2+] was used to investigate if Ca2+-activated K channels are present in the apical PM of parotid acinar cells. Initial experiments established Ca2+-buffering conditions that produced brief, localized increases in [Ca2+] after focal laser photolysis of caged Ca2+. 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 [Ca2+] at the apical PM after photolysis of Diazo-2, a caged Ca2+ 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 [Ca2+] 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. PMID:22291145

  16. Mouse cortical collecting duct cells show nonselective cation channel activity and express a gene related to the cGMP-gated rod photoreceptor channel.

    PubMed Central

    Ahmad, I; Korbmacher, C; Segal, A S; Cheung, P; Boulpaep, E L; Barnstable, C J

    1992-01-01

    Apical nonselective cation channels with an average single-channel conductance of 34 +/- 2.3 pS were found in M-1 mouse cortical collecting duct cells. Channel activity is increased by depolarization and abolished by cytoplasmic calcium removal. Cytoplasmic application of 0.1 mM cGMP decreases channel open probability by 27%. cDNAs corresponding to approximately 40% of the coding region of the photoreceptor channel were isolated by the polymerase chain reaction from M-1 cells and a rat kidney cDNA library. The rat kidney-derived sequence differs by a single base, and the M-1-cell-derived sequence differs by only two bases, from the photoreceptor sequence. A second clone from M-1 cells differs by 20 out of 426 bases from the photoreceptor sequence. In all three clones, the deduced amino acid sequence is identical to that of the rat photoreceptor channel. Northern blot analysis of poly(A)+ RNA from M-1 cells reveals the presence of a 3.2-kilobase band hybridizing with a retinal cGMP-gated cation channel probe. The results suggest the expression in M-1 cells of more than one gene coding for nonselective cation channels or channel subunits, one of which is identical to the cGMP-gated cation channel gene of rod photoreceptors. Images PMID:1279673

  17. Transient Receptor Potential Canonical 7 (TRPC7): A Diacylglycerol-Activated Non-Selective Cation Channel

    PubMed Central

    Zhang, Xuexin

    2016-01-01

    Transient receptor potential canonical 7 (TRPC7) channel is the seventh member of the mammalian TRPC channel family. TRPC7 mRNA, protein and channel activity have been detected in many tissues and organs from mouse, rat and human. TRPC7 has high sequence homology with TRPC3 and TRPC6 and all three channels are activated by membrane receptors that couple to isoforms of phospholipase C (PLC) and mediate non-selective cation currents. TRPC7, along with TRPC3 and TRPC6 can be activated by direct exogenous application of diacylglycerol (DAG) analogs and by pharmacological maneuvers that increase endogenous DAG in cells. TRPC7 shows distinct properties of activation, such as constitutive activity, susceptibility to negative regulation by extracellular Ca2+ and by protein kinase C. TRPC7 can form heteromultimers with TRPC3 and TRPC6. Although TRPC7 remains one of the least studied TRPC channel, its role in various cell types and physiological and pathophysiological conditions is begining to emerge. PMID:24756707

  18. SecA Alone Can Promote Protein Translocation and Ion Channel Activity

    PubMed Central

    Hsieh, Ying-hsin; Zhang, Hao; Lin, Bor-ruei; Cui, Ningren; Na, Bing; Yang, Hsiuchin; Jiang, Chun; Sui, Sen-fang; Tai, Phang C.

    2011-01-01

    SecA is an essential component of the Sec-dependent protein translocation pathway across cytoplasmic membranes in bacteria. Escherichia coli SecA binds to cytoplasmic membranes at SecYEG high affinity sites and at phospholipid low affinity sites. It has been widely viewed that SecYEG functions as the essential protein-conducting channel through which precursors cross the membranes in bacterial Sec-dependent pathways, and that SecA functions as a motor to hydrolyze ATP in translocating precursors through SecYEG channels. We have now found that SecA alone can promote precursor translocation into phospholiposomes. Moreover, SecA-liposomes elicit ionic currents in Xenopus oocytes. Patch-clamp recordings further show that SecA alone promotes signal peptide- or precursor-dependent single channel activity. These activities were observed with the functional SecA at about 1–2 μm. The results show that SecA alone is sufficient to promote protein translocation into liposomes and to elicit ionic channel activity at the phospholipids low affinity binding sites, thus indicating that SecA is able to form the protein-conducting channels. Even so, such SecA-liposomes are less efficient than those with a full complement of Sec proteins, and lose the signal-peptide proofreading function, resembling the effects of PrlA mutations. Addition of purified SecYEG restores the signal peptide specificity and increases protein translocation and ion channel activities. These data show that SecA can promote protein translocation and ion channel activities both when it is bound to lipids at low affinity sites and when it is bound to SecYEG with high affinity. The latter of the two interactions confers high efficiency and specificity. PMID:22033925

  19. Vasodilation of retinal arterioles induced by activation of BKCa channels is attenuated in diabetic rats.

    PubMed

    Mori, Asami; Suzuki, Sachi; Sakamoto, Kenji; Nakahara, Tsutomu; Ishii, Kunio

    2011-11-01

    The large-conductance Ca(2+)-activated K(+) (BK(Ca)) channels modulate the retinal vascular tone, but question of whether the impairment of the channel function contributes to abnormalities of retinal circulation has not yet been completely elucidated. The purpose of this study was to examine effects of diabetes on the vasodilation induced by activation of BK(Ca) channels. Male Wistar rats were treated with streptozotocin and experiments were performed 2 weeks later. The streptozotocin-treated animals were given drinking water containing 5% d-glucose to shorten the term in the development of retinal vascular dysfunction. The retinal vascular responses were assessed by measuring diameter of retinal arterioles in the fundus images that were captured with an original fundus camera system. In non-diabetic rats, vasodilator effects of acetylcholine on retinal arterioles were significantly reduced by iberiotoxin, an inhibitor of BK(Ca) channels. However, the inhibitory effect of iberiotoxin was not observed in diabetic rats, and the responses to the BK(Ca) channel opener BMS-191011 were almost completely abolished. The retinal vasodilator response to acetylcholine, possibly an endothelium-derived hyperpolarizing factor-mediated response, observed after treatment with N(G)-nitro-l-arginine methyl ester and indomethacin was markedly reduced in diabetic rats. The responses to pinacidil, an opener of ATP-sensitive K(+) channels, were unchanged. These results suggest that the retinal vasodilator response mediated through mechanisms involving activation of BK(Ca) channels is diminished at the early stage of diabetes in rats. The impairment of BK(Ca) channel function may contribute to abnormal retinal hemodynamics in diabetes and consequently play an important role in the pathogenesis of diabetic retinopathy. PMID:21871885

  20. Odorant receptors activated by amino acids in sensory neurons of the channel catfish Ictalurus punctatus.

    PubMed

    Ivanova, T T; Caprio, J

    1993-12-01

    Odorant receptors activated by amino acids were investigated with patch-clamp techniques in olfactory receptor neurons of the channel catfish, Ictalurus punctatus. The L-isomers of alanine, norvaline, arginine, and glutamate, known to act predominantly on different olfactory receptor sites, activated nondesensitizing inward currents with amplitudes of -2.5 to -280 pA in olfactory neurons voltage-clamped at membrane potentials of -72 or -82 mV. Different amino acids were shown to induce responses in the same sensory neurons; however, the amplitude and the kinetics of the observed whole cell currents differed among the stimuli and may therefore reflect activation of different amino acid receptor types or combinations of receptor types in these cells. Amino acid-induced currents appeared to have diverse voltage dependence and could also be classified according to the amplitude of the spontaneous channel fluctuations underlying the macroscopic currents. A mean single-channel conductance (gamma) of 360 fS was estimated from small noise whole-cell currents evoked by arginine within the same olfactory neuron in which a mean gamma value of 23.6 pS was estimated from 'large noise' response to norvaline. Quiescent olfactory neurons fired bursts of action potentials in response to either amino acid stimulation or application of 8-Br-cyclic GMP (100 microM), and voltage-gated channels underlying generation of action potentials were similar in these neurons. However, in whole-cell voltage-clamp, 8-Br-cyclic GMP evoked large rectangular current pulses, and single-channel conductances of 275, 220, and 110 pS were obtained from the discrete current levels. These results suggest that in addition to the cyclic nucleotide-gated transduction channels, olfactory neurons of the channel catfish possess a variety of odor receptors coupled to different types of transduction channels. PMID:8133240

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

    PubMed Central

    Huang, Pengfei; Zhang, Yiya; Chen, Xinyi; Zhu, Li; Yin, Dazhong; Zeng, Xiongzhi; Liang, Songping

    2014-01-01

    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. PMID:25153257

  2. Channel morphodynamics and habitat recovery in a river reach affected by gravel-mining (River Ésera, Ebro basin)

    NASA Astrophysics Data System (ADS)

    Lopez-Tarazon, J. A.; Lobera, G.; Andrés-Doménech, I.; Martínez-Capel, F.; Muñoz-Mas, R.; Vallés, F.; Tena, A.; Vericat, D.; Batalla, R. J.

    2012-04-01

    Physical processes in rivers are the result of the interaction between flow regime and hydraulics, morphology, sedimentology and sediment transport. The frequency and magnitude of physical disturbance (i.e. bed stability) control habitat integrity and, consequently, ecological diversity of a particular fluvial system. Most rivers experience human-induced perturbations that alter such hydrosedimentary equilibrium, thus affecting the habitat of aquatic species. A dynamic balance may take long time to be newly attained. Within this context, gravel mining is well known to affect channel characteristics mostly at the local scale, but its effect may also propagate downstream and upstream. Sedimentary forms are modified during extraction and habitat features are reduced or even eliminated. Effects tend to be most acute in contrasted climatic environments, such as the Mediterranean areas, in which climatic and hydrological variability maximises effects of impacts and precludes short regeneration periods. Present research focuses on the evolution of a river reach, which has experienced an intense gravel extraction. The selected area is located in the River Ésera (Ebro basin), where interactions between morphodynamics and habitat recovery are examined. Emphasis is put on monitoring sedimentary, morphological and hydraulic variables to later compare pre (t0) and post (t1, t2... tn) extraction situations. Methodology for all time monitoring steps (i.e. ti) includes: i) characterization of grain size distribution at all of the different hydromorphological units within the reach; ii) description of channel morphology (together with changes before and after floods) by means of close-range aerial photographs, which are taken with a digital camera attached to a 1m3 helium balloon (i.e. BLIMP); and iii) determination of flow parameters from 2D hydraulic modelling that is based on detailed topographical data obtained from Leica® GNSS/GPS and robotic total station, and River

  3. TRPM8 Channel Activation Induced by Monoterpenoid Rotundifolone Underlies Mesenteric Artery Relaxation

    PubMed Central

    Silva, Darizy Flavia; de Almeida, Monica Moura; Chaves, Cinthia Guedes; Braz, Ana Letícia; Gomes, Maria Aparecida; Pinho-da-Silva, Leidiane; Pesquero, Jorge Luiz; Andrade, Viviane Aguiar; Leite, Maria de Fátima; de Albuquerque, José George Ferreira; Araujo, Islania Giselia Albuquerque; Nunes, Xirley Pereira; Barbosa-Filho, José Maria; Cruz, Jader dos Santos; Correia, Nadja de Azevedo; de Medeiros, Isac Almeida

    2015-01-01

    In this study, our aims were to investigate transient receptor potential melastatin-8 channels (TRPM8) involvement in rotundifolone induced relaxation in the mesenteric artery and to increase the understanding of the role of these thermosensitive TRP channels in vascular tissue. Thus, message and protein levels of TRPM8 were measured by semi-quantitative PCR and western blotting in superior mesenteric arteries from 12 week-old Spague-Dawley (SD) rats. Isometric tension recordings evaluated the relaxant response in mesenteric rings were also performed. Additionally, the intracellular Ca2+ changes in mesenteric artery myocytes were measured using confocal microscopy. Using PCR and western blotting, both TRPM8 channel mRNA and protein expression was measured in SD rat mesenteric artery. Rotundifolone and menthol induced relaxation in the isolated superior mesenteric artery from SD rats and improved the relaxant response induced by cool temperatures. Also, this monoterpene induced an increase in transient intracellular Ca2+. These responses were significantly attenuated by pretreatment with capsazepine or BCTC, both TRPM8 channels blockers. The response induced by rotundifolone was not significantly attenuated by ruthenium red, a non-selective TRP channels blocker, or following capsaicin-mediated desensitization of TRPV1. Our findings suggest that rotundifolone induces relaxation by activating TRPM8 channels in rat superior mesenteric artery, more selectively than menthol, the classic TRPM8 agonist, and TRPM8 channels participates in vasodilatory pathways in isolated rat mesenteric arteries. PMID:26599698

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

    SciTech Connect

    Hattori, Motoyuki; Gouaux, Eric

    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.

  5. Influence of proline position upon the ion channel activity of alamethicin.

    PubMed Central

    Kaduk, C; Duclohier, H; Dathe, M; Wenschuh, H; Beyermann, M; Molle, G; Bienert, M

    1997-01-01

    Alamethicin, a 20-residue peptaibol, induces voltage-dependent ion channels in lipid bilayers according to the barrel-stave model. To study relationships between the proline-14-induced kink region and the channel-forming behavior of the peptide, a set of alamethicin analogs with proline incorporated at positions 11, 12, 13, 14, 15, 16, and 17, respectively, as well as an analog with alanine instead of proline at position 14 were synthesized. Macroscopic conductance experiments show that the voltage dependence of the peptides is conserved although slightly influenced, but the apparent mean number of monomers forming the channels is significantly reduced when proline is not located at position 14. This is confirmed in single-channel experiments. The analogs with proline next to position 14 (i.e., 13, 15, 16) show stable conductance levels, but of reduced number, which follows the order Alam-P14 > Alam-P15 > Alam-P16 > Alam-P13. This reduction in the number of levels is connected with changes in the lifetime of the channels. Analogs with proline at position 11, 12, or 17 produce erratic, extremely short-lived current events that could not be resolved. The changes in functional properties are related to structural properties as probed by circular dichroism. The results indicate that proline at position 14 results in optimal channel activity, whereas channels formed by the analogs bearing proline at different positions are considerably less stable. PMID:9129817

  6. Relaxation training affects success and activation on a teaching test.

    PubMed

    Helin, P; Hänninen, O

    1987-12-01

    We studied the effects of an audiocassette-relaxation training period (ART) and its timing on success at a teaching test (lecture type), on observed tension and on a number of physiological responses. The electrical activity of the upper trapezius muscle (EMG), heart rate (HR) and blood pressure (BP), of female and male instructor candidates, were examined before, during and after the teaching test as well as during its critique. The relaxation period (18 min) was presented either on the preceding night (ARTnt) or immediately before the teaching test (ARTimm). The influence of personality (types A-B and extrovert-introvert) was also studied. ART improved success at the teaching test in both sexes. In males (but not in females), ARTimm decreased EMG level during the test, but ARTnt increased EMG at the test period as compared to the control group. In females, both ARTnt and ARTimm lowered HR more than in the control group. ARTimm lowered systolic BP in both sexes. Personality types affected the ART responses; ART was more beneficial for type A than B subjects. PMID:3325481

  7. Swelling-Activated Ca2+ Channels Trigger Ca2+ Signals in Merkel Cells

    PubMed Central

    Haeberle, Henry; Bryan, Leigh A.; Vadakkan, Tegy J.; Dickinson, Mary E.; Lumpkin, Ellen A.

    2008-01-01

    Merkel cell-neurite complexes are highly sensitive touch receptors comprising epidermal Merkel cells and sensory afferents. Based on morphological and molecular studies, Merkel cells are proposed to be mechanosensory cells that signal afferents via neurotransmission; however, functional studies testing this hypothesis in intact skin have produced conflicting results. To test this model in a simplified system, we asked whether purified Merkel cells are directly activated by mechanical stimulation. Cell shape was manipulated with anisotonic solution changes and responses were monitored by Ca2+ imaging with fura-2. We found that hypotonic-induced cell swelling, but not hypertonic solutions, triggered cytoplasmic Ca2+ transients. Several lines of evidence indicate that these signals arise from swelling-activated Ca2+-permeable ion channels. First, transients were reversibly abolished by chelating extracellular Ca2+, demonstrating a requirement for Ca2+ influx across the plasma membrane. Second, Ca2+ transients were initially observed near the plasma membrane in cytoplasmic processes. Third, voltage-activated Ca2+ channel (VACC) antagonists reduced transients by half, suggesting that swelling-activated channels depolarize plasma membranes to activate VACCs. Finally, emptying internal Ca2+ stores attenuated transients by 80%, suggesting Ca2+ release from stores augments swelling-activated Ca2+ signals. To identify candidate mechanotransduction channels, we used RT-PCR to amplify ion-channel transcripts whose pharmacological profiles matched those of hypotonic-evoked Ca2+ signals in Merkel cells. We found 11 amplicons, including PKD1, PKD2, and TRPC1, channels previously implicated in mechanotransduction in other cells. Collectively, these results directly demonstrate that Merkel cells are activated by hypotonic-evoked swelling, identify cellular signaling mechanisms that mediate these responses, and support the hypothesis that Merkel cells contribute to touch reception

  8. Up-Regulation of Pressure-activated Ca2+-permeable Cation Channel in Intact Vascular Endothelium of Hypertensive Rats

    NASA Astrophysics Data System (ADS)

    Hoyer, J.; Kohler, R.; Haase, W.; Distler, A.

    1996-10-01

    In endothelial cells, stretch-activated cation channels have been proposed to act as mechanosensors for changes in hemodynamic forces. We have identified a novel mechanosensitive pressure-activated channel in intact endothelium from rat aorta and mesenteric artery. The 18-pS cation channel responded with a multifold increase in channel activity when positive pressure was applied to the luminal cell surface with the patch pipette and inactivated at negative pipette pressure. Channel permeability ratio for K+, Na+, and Ca2+ ions was 1:0.98:0.23. Ca2+ influx through the channel was sufficient to activate a neighboring Ca2+-dependent K+ channel. Hemodynamic forces are chronically disturbed in arterial hypertension. Endothelial cell dysfunction has been implicated in the pathogenesis of arterial hypertension. In two comparative studies, density of the pressure-activated channel was found to be significantly higher in spontaneously hypertensive rats and renovascular hypertensive rats compared with their respective normotensive controls. Channel activity presumably leads to mechanosensitive Ca2+ influx and induces cell hyperpolarization by K+ channel activity. Both Ca2+ influx and hyperpolarization are known to induce a vasodilatory endothelial response by stimulating endothelial nitric oxide (NO) production. Up-regulation of channel density in hypertension could, therefore, represent a counterregulatory mechanism of vascular endothelium.

  9. Members of the Chloride Intracellular Ion Channel Protein Family Demonstrate Glutaredoxin-Like Enzymatic Activity

    PubMed Central

    Al Khamici, Heba; Brown, Louise J.; Hossain, Khondker R.; Hudson, Amanda L.; Sinclair-Burton, Alxcia A.; Ng, Jane Phui Mun; Daniel, Elizabeth L.; Hare, Joanna E.; Cornell, Bruce A.; Curmi, Paul M. G.; Davey, Mary W.; Valenzuela, Stella M.

    2015-01-01

    The Chloride Intracellular Ion Channel (CLIC) family consists of six evolutionarily conserved proteins in humans. Members of this family are unusual, existing as both monomeric soluble proteins and as integral membrane proteins where they function as chloride selective ion channels, however no function has previously been assigned to their soluble form. Structural studies have shown that in the soluble form, CLIC proteins adopt a glutathione S-transferase (GST) fold, however, they have an active site with a conserved glutaredoxin monothiol motif, similar to the omega class GSTs. We demonstrate that CLIC proteins have glutaredoxin-like glutathione-dependent oxidoreductase enzymatic activity. CLICs 1, 2 and 4 demonstrate typical glutaredoxin-like activity using 2-hydroxyethyl disulfide as a substrate. Mutagenesis experiments identify cysteine 24 as the catalytic cysteine residue in CLIC1, which is consistent with its structure. CLIC1 was shown to reduce sodium selenite and dehydroascorbate in a glutathione-dependent manner. Previous electrophysiological studies have shown that the drugs IAA-94 and A9C specifically block CLIC channel activity. These same compounds inhibit CLIC1 oxidoreductase activity. This work for the first time assigns a functional activity to the soluble form of the CLIC proteins. Our results demonstrate that the soluble form of the CLIC proteins has an enzymatic activity that is distinct from the channel activity of their integral membrane form. This CLIC enzymatic activity may be important for protecting the intracellular environment against oxidation. It is also likely that this enzymatic activity regulates the CLIC ion channel function. PMID:25581026

  10. Activation of Plant Plasma Membrane Ca2+-Permeable Channels by Race-Specific Fungal Elicitors.

    PubMed Central

    Gelli, A.; Higgins, V. J.; Blumwald, E.

    1997-01-01

    The response of plant cells to invading pathogens is regulated by fluctuations in cytosolic Ca2+ levels that are mediated by Ca2+-permeable channels located at the plasma membrane of the host cell. The mechanisms by which fungal elicitors can induce Ca2+ uptake by the host cell were examined by the application of conventional patch-clamp techniques. Whole-cell and single-channel experiments on tomato (Lycopersicon esculentum L.) protoplasts revealed a race-specific fungal elicitor-induced activation of a plasma membrane Ca2+-permeable channel. The presence of the fungal elicitor resulted in a greater probability of channel opening. Guanosine 5[prime]-[[beta]-thio]diphosphate, a GDP analog that locks heterotrimeric G-proteins into their inactivated state, abolished the channel activation induced by the fungal elicitor, whereas guanosine 5[prime][[gamma]-thio]triphosphate, a nonhydrolyzable GTP analog that locks heterotrimeric G-proteins into their activated state, produced an effect similar to that observed with the fungal elicitor. Mastoparan, which stimulates GTPase activity, mimicked the effect of GTP[[gamma

  11. Small-Conductance Ca2+-Activated Potassium Channels Negatively Regulate Aldosterone Secretion in Human Adrenocortical Cells.

    PubMed

    Yang, Tingting; Zhang, Hai-Liang; Liang, Qingnan; Shi, Yingtang; Mei, Yan-Ai; Barrett, Paula Q; Hu, Changlong

    2016-09-01

    Aldosterone, which plays a key role in maintaining water and electrolyte balance, is produced by zona glomerulosa cells of the adrenal cortex. Autonomous overproduction of aldosterone from zona glomerulosa cells causes primary hyperaldosteronism. Recent clinical studies have highlighted the pathological role of the KCNJ5 potassium channel in primary hyperaldosteronism. Our objective was to determine whether small-conductance Ca(2+)-activated potassium (SK) channels may also regulate aldosterone secretion in human adrenocortical cells. We found that apamin, the prototypic inhibitor of SK channels, decreased membrane voltage, raised intracellular Ca(2+) and dose dependently increased aldosterone secretion from human adrenocortical H295R cells. By contrast, 1-Ethyl-2-benzimidazolinone, an agonist of SK channels, antagonized apamin's action and decreased aldosterone secretion. Commensurate with an increase in aldosterone production, apamin increased mRNA expression of steroidogenic acute regulatory protein and aldosterone synthase that control the early and late rate-limiting steps in aldosterone biosynthesis, respectively. In addition, apamin increased angiotensin II-stimulated aldosterone secretion, whereas 1-Ethyl-2-benzimidazolinone suppressed both angiotensin II- and high K(+)-stimulated production of aldosterone in H295R cells. These findings were supported by apamin-modulation of basal and angiotensin II-stimulated aldosterone secretion from acutely prepared slices of human adrenals. We conclude that SK channel activity negatively regulates aldosterone secretion in human adrenocortical cells. Genetic association studies are necessary to determine whether mutations in SK channel subtype 2 genes may also drive aldosterone excess in primary hyperaldosteronism. PMID:27432863

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

    PubMed

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

    2015-07-01

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

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

    PubMed

    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 <200 amino acids). Thus it is not surprising that 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. PMID:23959675

  14. Ion conductance of the Ca(2+)-activated maxi-K+ channel from the embryonic rat brain.

    PubMed

    Mienville, J M; Clay, J R

    1997-01-01

    By using single-channel recording techniques, we measured the conductance (gK) of the Ca(2+)-activated Maxi-K+ channel from the embryonic rat brain, and examined its dependence on K+ ions present in equimolar concentrations on both sides of the membrane patch. With ionic strength maintained constant by substitution of N-methyl-D-glucamine for K+, gK has a sigmoidal dependence upon [K+]. This result has been obscured in previous work by variations in ionic strength, which has a marked effect on single-channel conductance, especially in the limit for which this variable approaches zero. The gK versus [K+] relationship is described, theoretically, by a three-barrier, two-binding-site model in which the barrier that an ion must cross to leave the channel is decreased as [K+] is increased. PMID:8994603

  15. Impedance spectroscopy of micro-Droplets reveals activation of Bacterial Mechanosensitive Channels in Hypotonic Solutions

    NASA Astrophysics Data System (ADS)

    Ebrahimi, Aida; Alam, Muhammad A.

    Rapid detection of bacterial pathogens is of great importance in healthcare, food safety, environmental monitoring, and homeland security. Most bacterial detection platforms rely on binary fission (i.e. cell growth) to reach a threshold cell population that can be resolved by the sensing method. Since cell division depends on the bacteria type, the detection time of such methods can vary from hours to days. In contrast, in this work, we show that bacteria cells can be detected within minutes by relying on activation of specific protein channels, i.e. mechanosensitive channels (MS channels). When cells are exposed to hypotonic solutions, MS channels allow efflux of solutes to the external solution which leads to release the excessive membrane tension. Release of the cytoplasmic solutes, in turn, results in increase of the electrical conductance measured by droplet-based impedance sensing. The approach can be an effective technique for fast, pre-screening of bacterial contamination at ultra-low concentration.

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

    DOEpatents

    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.

  17. Characterization of apamin-sensitive Ca(2+)-activated potassium channels in human leukaemic T lymphocytes.

    PubMed

    Hanselmann, C; Grissmer, S

    1996-11-01

    1. The whole-cell recording mode of the patch-clamp technique was used to study the effect of extracellularly applied ions, toxins and drugs on voltage-independent, apamin-sensitive Ca(2+)-activated K+ channels, K(Ca), expressed in the Jurkat human leukaemic T cell line. 2. Extracellular Ba2+ and Sr+ produced a voltage-dependent block. The equilibrium dissociation constant of the Ba2+/K(Ca) channel complex increased e-fold for a 20 mV change of potential. Ba2+ block of Jurkat K(Ca) channels is therefore as steep as expected from the movement of a single divalent cation about half-way into the electric field of the membrane from the outside. 3. We determined the ion selectivity as well as the conductance of these channels. Calculated permeability ratios, PX/PK, for these K(Ca) channels were 1.0, 0.96, 0.26 and 0.53 for K+, Rb+, Cs+ and NH4+, respectively. Conductance ratios, gX/gK, for the same ions were 1.0, 1.0, 0.67 and 0.11, respectively. Most strikingly this channel can also carry significant current with Cs+ as current carrier. 4. Scyllatoxin (ScTX), a thirty-one amino acid peptide toxin, reduced current through these K(Ca) channels with a half-blocking concentration of approximately 0.3 nM independent of the pH. Other drugs that were able to reduce current through these channels include the classical calcium antagonists diltiazem and verapamil. In contrast, nifedipine, clotrimazole and kaliotoxin (100 nM) were unable to block current through these channels in Jurkat T cells. PMID:8930831

  18. EMERGENCE OF HELICAL FLUX AND THE FORMATION OF AN ACTIVE REGION FILAMENT CHANNEL

    SciTech Connect

    Lites, B. W.; Kubo, M.; Berger, T.; Frank, Z.; Shine, R.; Tarbell, T.; Title, A.; Okamoto, T. J.; Otsuji, K.

    2010-07-20

    We present comprehensive observations of the formation and evolution of a filament channel within NOAA Active Region (AR) 10978 from Hinode/Solar Optical Telescope and TRACE. We employ sequences of Hinode spectro-polarimeter maps of the AR, accompanying Hinode Narrowband Filter Instrument magnetograms in the Na I D1 line, Hinode Broadband Filter Instrument filtergrams in the Ca II H line and G-band, Hinode X-ray telescope X-ray images, and TRACE Fe IX 171 A image sequences. The development of the channel resembles qualitatively that presented by Okamoto et al. in that many indicators point to the emergence of a pre-existing sub-surface magnetic flux rope. The consolidation of the filament channel into a coherent structure takes place rapidly during the course of a few hours, and the filament form then gradually shrinks in width over the following two days. Particular to this filament channel is the observation of a segment along its length of horizontal, weak (500 G) flux that, unlike the rest of the filament channel, is not immediately flanked by strong vertical plage fields of opposite polarity on each side of the filament. Because this isolated horizontal field is observed in photospheric lines, we infer that it is unlikely that the channel formed as a result of reconnection in the corona, but the low values of inferred magnetic fill fraction along the entire length of the filament channel suggest that the bulk of the field resides somewhat above the low photosphere. Correlation tracking of granulation in the G band presents no evidence for either systematic flows toward the channel or systematic shear flows along it. The absence of these flows, along with other indications of these data from multiple sources, reinforces (but does not conclusively demonstrate) the picture of an emerging flux rope as the origin of this AR filament channel.

  19. Burst activity and ultrafast activation kinetics of CaV1.3 Ca2+ channels support presynaptic activity in adult gerbil hair cell ribbon synapses

    PubMed Central

    Zampini, Valeria; Johnson, Stuart L; Franz, Christoph; Knipper, Marlies; Holley, Matthew C; Magistretti, Jacopo; Masetto, Sergio; Marcotti, Walter

    2013-01-01

    Auditory information transfer to afferent neurons relies on precise triggering of neurotransmitter release at the inner hair cell (IHC) ribbon synapses by Ca2+ entry through CaV1.3 Ca2+ channels. Despite the crucial role of CaV1.3 Ca2+ channels in governing synaptic vesicle fusion, their elementary properties in adult mammals remain unknown. Using near-physiological recording conditions we investigated Ca2+ channel activity in adult gerbil IHCs. We found that Ca2+ channels are partially active at the IHC resting membrane potential (−60 mV). At −20 mV, the large majority (>70%) of Ca2+ channel first openings occurred with an estimated delay of about 50 μs in physiological conditions, with a mean open time of 0.5 ms. Similar to other ribbon synapses, Ca2+ channels in IHCs showed a low mean open probability (0.21 at −20 mV), but this increased significantly (up to 0.91) when Ca2+ channel activity switched to a bursting modality. We propose that IHC Ca2+ channels are sufficiently rapid to transmit fast signals of sound onset and support phase-locking. Short-latency Ca2+ channel opening coupled to multivesicular release would ensure precise and reliable signal transmission at the IHC ribbon synapse. PMID:23713031

  20. Thalamic Kv7 channels: pharmacological properties and activity control during noxious signal processing

    PubMed Central

    Cerina, Manuela; Szkudlarek, Hanna J; Coulon, Philippe; Meuth, Patrick; Kanyshkova, Tatyana; Nguyen, Xuan Vinh; Göbel, Kerstin; Seidenbecher, Thomas; Meuth, Sven G; Pape, Hans-Christian; Budde, Thomas

    2015-01-01

    Background and Purpose The existence of functional Kv7 channels in thalamocortical (TC) relay neurons and the effects of the K+-current termed M-current (IM) on thalamic signal processing have long been debated. Immunocytochemical evidence suggests their presence in this brain region. Therefore, we aimed to verify their existence, pharmacological properties and function in regulating activity in neurons of the ventrobasal thalamus (VB). Experimental Approach Characterization of Kv7 channels was performed by combining in vitro, in vivo and in silico techniques with a pharmacological approach. Retigabine (30 μM) and XE991 (20 μM), a specific Kv7 channel enhancer and blocker, respectively, were applied in acute brain slices during electrophysiological recordings. The effects of intrathalamic injection of retigabine (3 mM, 300 nL) and/or XE991 (2 mM, 300 nL) were investigated in freely moving animals during hot-plate tests by recording behaviour and neuronal activity. Key Results Kv7.2 and Kv7.3 subunits were found to be abundantly expressed in TC neurons of mouse VB. A slow K+-current with properties of IM was activated by retigabine and inhibited by XE991. Kv7 channel activation evoked membrane hyperpolarization, a reduction in tonic action potential firing, and increased burst firing in vitro and in computational models. Single-unit recordings and pharmacological intervention demonstrated a specific burst-firing increase upon IM activation in vivo. A Kv7 channel-mediated increase in pain threshold was associated with fewer VB units responding to noxious stimuli, and increased burst firing in responsive neurons. Conclusions and Implications Kv7 channel enhancement alters somatosensory activity and may reflect an anti-nociceptive mechanism during acute pain processing. PMID:25684311

  1. Excessive activation of cyclic nucleotide-gated channels contributes to neuronal degeneration of photoreceptors.

    PubMed

    Vallazza-Deschamps, Géraldine; Cia, David; Gong, Jie; Jellali, Abdeljelil; Duboc, Agnès; Forster, Valérie; Sahel, Jose A; Tessier, Luc-Henri; Picaud, Serge

    2005-09-01

    In different animal models, photoreceptor degeneration was correlated to an abnormal increase in cGMP concentration. The cGMP-induced photoreceptor toxicity was demonstrated by applying the phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine on retinal explants. To assess the role of cGMP-gated channels in this cGMP toxicity, the Ca(2+) channel blockers verapamil and L- and D-diltiazem, which block cGMP-gated channels with different efficacies, were applied to in vitro animal models of photoreceptor degeneration. These models included: (i) adult rat retinal explants incubated with zaprinast, a more specific inhibitor of the rod phosphodiesterase than 3-isobutyl-1-methylxanthine and (ii) rd mouse retinal explants. Photoreceptor apoptosis was assessed by terminal dUTP nick end labelling and caspase 3 activation. Effects of the blockers on the synaptic rod Ca(2+) channels were measured by patch-clamp recording. In the zaprinast-induced photoreceptor degeneration model, both diltiazem isomers rescued photoreceptors whereas verapamil had no influence. Their neuroprotective efficacy was correlated to their inhibition of cGMP-gated channels (l-diltiazem>d-diltiazem>verapamil=0). In contrast, all three Ca(2+) channel blockers suppressed rod Ca(2+) channel currents similarly. This suppression of the currents by the diltiazem isomers was very weak (16.5%) at the neuroprotective concentration (10 microm). In rd retinal explants, both diltiazem isomers also slowed down rod degeneration in contrast to verapamil. L-diltiazem exhibited this effect at concentrations ranging from 1 to 20 microm. This study further supports the photoreceptor neuroprotection by diltiazem particularly in the rd mouse retina, whereas the absence of neuroprotection by verapamil further suggests the role of cGMP-gated channel activation in the induction of photoreceptor degeneration. PMID:16176343

  2. Reporting Sodium Channel Activity Using Calcium Flux: Pharmacological Promiscuity of Cardiac Nav1.5

    PubMed Central

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

    2015-01-01

    Voltage-gated sodium (Nav) channels are essential for membrane excitability and represent therapeutic targets for treating human diseases. Recent reports suggest that these channels, e.g., Nav1.3 and Nav1.5, are inhibited by multiple structurally distinctive small molecule drugs. These studies give reason to wonder whether these drugs collectively target a single site or multiple sites in manifesting such pharmacological promiscuity. We thus investigate the pharmacological profile of Nav1.5 through systemic analysis of its sensitivity to diverse compound collections. Here, we report a dual-color fluorescent method that exploits a customized Nav1.5 [calcium permeable Nav channel, subtype 5 (SoCal5)] with engineered-enhanced calcium permeability. SoCal5 retains wild-type (WT) Nav1.5 pharmacological profiles. WT SoCal5 and SoCal5 with the local anesthetics binding site mutated (F1760A) could be expressed in separate cells, each with a different-colored genetically encoded calcium sensor, which allows a simultaneous report of compound activity and site dependence. The pharmacological profile of SoCal5 reveals a hit rate (>50% inhibition) of around 13% at 10 μM, comparable to that of hERG. The channel activity is susceptible to blockage by known drugs and structurally diverse compounds. The broad inhibition profile is highly dependent on the F1760 residue in the inner cavity, which is a residue conserved among all nine subtypes of Nav channels. Both promiscuity and dependence on F1760 seen in Nav1.5 were replicated in Nav1.4. Our evidence of a broad inhibition profile of Nav channels suggests a need to consider off-target effects on Nav channels. The site-dependent promiscuity forms a foundation to better understand Nav channels and compound interactions. PMID:25422141

  3. Effects of the small molecule HERG activator NS1643 on Kv11.3 channels.

    PubMed

    Bilet, Arne; Bauer, Christiane K

    2012-01-01

    NS1643 is one of the small molecule HERG (Kv11.1) channel activators and has also been found to increase erg2 (Kv11.2) currents. We now investigated whether NS1643 is also able to act as an activator of Kv11.3 (erg3) channels expressed in CHO cells. Activation of rat Kv11.3 current occurred in a dose-dependent manner and maximal current increasing effects were obtained with 10 µM NS1643. At this concentration, steady-state outward current increased by about 80% and the current increase was associated with a significant shift in the voltage dependence of activation to more negative potentials by about 15 mV. In addition, activation kinetics were accelerated, whereas deactivation was slowed. There was no significant effect on the kinetics of inactivation and recovery from inactivation. The strong current-activating agonistic effect of NS1643 did not result from a shift in the voltage dependence of Kv11.3 channel inactivation and was independent from external Na(+) or Ca(2+). At the higher concentration of 20 µM, NS1643 induced clearly less current increase. The left shift in the voltage dependence of activation reversed and the voltage sensitivity of activation dramatically decreased along with a slowing of Kv11.3 channel activation. These data show that, in comparison to other Kv11 family members, NS1643 exerts distinct effects on Kv11.3 channels with especially pronounced partial antagonistic effects at higher concentration. PMID:23226420

  4. Acid-sensing ion channel 1a is a postsynaptic proton receptor that affects the density of dendritic spines

    PubMed Central

    Zha, Xiang-ming; Wemmie, John A.; Green, Steven H.; Welsh, Michael J.

    2006-01-01

    Extracellular proton concentrations in the brain may be an important signal for neuron function. Proton concentrations change both acutely when synaptic vesicles release their acidic contents into the synaptic cleft and chronically during ischemia and seizures. However, the brain receptors that detect protons and their physiologic importance remain uncertain. Using organotypic hippocampal slices and biolistic transfection, we found the acid-sensing ion channel 1a (ASIC1a), localized in dendritic spines where it functioned as a proton receptor. ASIC1a also affected the density of spines, the postsynaptic site of most excitatory synapses. Decreasing ASIC1a reduced the number of spines, whereas overexpressing ASIC1a had the opposite effect. Ca2+-mediated Ca2+/calmodulin-dependent protein kinase II (CaMKII) signaling was probably responsible, because acid evoked an ASIC1a-dependent elevation of spine intracellular Ca2+ concentration, and reducing or increasing ASIC1a levels caused parallel changes in CaMKII phosphorylation in vivo. Moreover, inhibiting CaMKII prevented ASIC1a from increasing spine density. These data indicate that ASIC1a functions as a postsynaptic proton receptor that influences intracellular Ca2+ concentration and CaMKII phosphorylation and thereby the density of dendritic spines. The results provide insight into how protons influence brain function and how they may contribute to pathophysiology. PMID:17060608

  5. Dissection of the components for PIP2 activation and thermosensation in TRP channels

    PubMed Central

    Brauchi, Sebastian; Orta, Gerardo; Mascayano, Carolina; Salazar, Marcelo; Raddatz, Natalia; Urbina, Hector; Rosenmann, Eduardo; Gonzalez-Nilo, Fernando; Latorre, Ramon

    2007-01-01

    Phosphatidylinositol 4,5-bisphosphate (PIP2) plays a central role in the activation of several transient receptor potential (TRP) channels. The role of PIP2 on temperature gating of thermoTRP channels has not been explored in detail, and the process of temperature activation is largely unexplained. In this work, we have exchanged different segments of the C-terminal region between cold-sensitive (TRPM8) and heat-sensitive (TRPV1) channels, trying to understand the role of the segment in PIP2 and temperature activation. A chimera in which the proximal part of the C-terminal of TRPV1 replaces an equivalent section of TRPM8 C-terminal is activated by PIP2 and confers the phenotype of heat activation. PIP2, but not temperature sensitivity, disappears when positively charged residues contained in the exchanged region are neutralized. Shortening the exchanged segment to a length of 11 aa produces voltage-dependent and temperature-insensitive channels. Our findings suggest the existence of different activation domains for temperature, PIP2, and voltage. We provide an interpretation for channel–PIP2 interaction using a full-atom molecular model of TRPV1 and PIP2 docking analysis. PMID:17548815

  6. Arabidopsis MSL10 Has a Regulated Cell Death Signaling Activity That Is Separable from Its Mechanosensitive Ion Channel Activity[C][W

    PubMed Central

    Veley, Kira M.; Maksaev, Grigory; Frick, Elizabeth M.; January, Emma; Kloepper, Sarah C.; Haswell, Elizabeth S.

    2014-01-01

    Members of the MscS superfamily of mechanosensitive ion channels function as osmotic safety valves, releasing osmolytes under increased membrane tension. MscS homologs exhibit diverse topology and domain structure, and it has been proposed that the more complex members of the family might have novel regulatory mechanisms or molecular functions. Here, we present a study of MscS-Like (MSL)10 from Arabidopsis thaliana that supports these ideas. High-level expression of MSL10-GFP in Arabidopsis induced small stature, hydrogen peroxide accumulation, ectopic cell death, and reactive oxygen species- and cell death-associated gene expression. Phosphomimetic mutations in the MSL10 N-terminal domain prevented these phenotypes. The phosphorylation state of MSL10 also regulated its ability to induce cell death when transiently expressed in Nicotiana benthamiana leaves but did not affect subcellular localization, assembly, or channel behavior. Finally, the N-terminal domain of MSL10 was sufficient to induce cell death in tobacco, independent of phosphorylation state. We conclude that the plant-specific N-terminal domain of MSL10 is capable of inducing cell death, this activity is regulated by phosphorylation, and MSL10 has two separable activities—one as an ion channel and one as an inducer of cell death. These findings further our understanding of the evolution and significance of mechanosensitive ion channels. PMID:25052715

  7. Cardiac sodium/calcium exchanger preconditioning promotes anti-arrhythmic and cardioprotective effects through mitochondrial calcium-activated potassium channel

    PubMed Central

    Zhang, Jian-Ying; Cheng, Kang; Lai, Dong; Kong, Ling-Heng; Shen, Min; Yi, Fu; Liu, Bing; Wu, Feng; Zhou, Jing-Jun

    2015-01-01

    Background: Reverse-mode of the Na+/Ca2+ exchanger (NCX) stimulation provides cardioprotective effects for the ischemic/reperfused heart during ischemic preconditioning (IP). This study was designed to test the hypothesis that pretreatment with an inhibitor of cardiac delayed-rectifying K+ channel (IKr), E4031, increases reverse-mode of NCX activity, and triggers preconditioning against infarct size (IS) and arrhythmias caused by ischemia/reperfusion injury through mitoKCa channels. Materials and methods: In the isolated perfused rat heart, myocardial ischemia/reperfusion injury was created by occlusion of the left anterior descending coronary artery for 30 min followed by 120 min reperfusion. Two cycles of coronary occlusion for 5 min and reperfusion were performed, or pretreatment with E4031 or sevoflurane (Sevo) before the 30 min occlusion with the reversed-mode of NCX inhibitor (KB-R7943) or not. Results: E4031 or Sevo preconditioning not only markedly decreased IS but also reduced arrhythmias, which was significantly blunted by KB-R7943. Furthermore, these effects of E4031 preconditioning on IS and arrhythmias were abolished by inhibition of the mitoKCa channels. Similarly, pretreatment with NS1619, an opener of the mitoKCa channels, for 10 min before occlusion reduced both the infarct size and arrhythmias caused by ischemia/reperfusion. However, these effects weren’t affected by blockade of the NCX with KB-R7943. Conclusion: Taken together, these preliminary results conclude that pretreatment with E4031 reduces infarct size and produces anti-arrhythmic effect via stimulating the reverse-mode NCX, and that the mitoKCa channels mediate the protective effects. PMID:26617732

  8. Fly DPP10 acts as a channel ancillary subunit and possesses peptidase activity.

    PubMed

    Shiina, Yohei; Muto, Tomohiro; Zhang, Zhili; Baihaqie, Ahmad; Yoshizawa, Takamasa; Lee, Hye-In J; Park, Eulsoon; Tsukiji, Shinya; Takimoto, Koichi

    2016-01-01

    Mammalian DPP6 (DPPX) and DPP10 (DPPY) belong to a family of dipeptidyl peptidases, but lack enzyme activity. Instead, these proteins form complexes with voltage-gated K(+) channels in Kv4 family to control their gating and other properties. Here, we find that the fly DPP10 ortholog acts as an ancillary subunit of Kv4 channels and digests peptides. Similarly to mammalian DPP10, the fly ortholog tightly binds to rat Kv4.3 protein. The association causes negative shifts in voltage dependence of channel activation and steady state inactivation. It also results in faster inactivation and recovery from inactivation. In addition to its channel regulatory role, fly DPP10 exhibits significant dipeptidyl peptidase activity with Gly-Pro-MCA (glycyl-L-proline 4-methylcoumaryl-7-amide) as a substrate. Heterologously expressed Flag-tagged fly DPP10 and human DPP4 show similar Km values towards this substrate. However, fly DPP10 exhibits approximately a 6-times-lower relative kcat value normalized with anti-Flag immunoreactivity than human DPP4. These results demonstrate that fly DPP10 is a dual functional protein, controlling Kv4 channel gating and removing bioactive peptides. PMID:27198182

  9. Formation of cellular projections in neural progenitor cells depends on SK3 channel activity.

    PubMed

    Liebau, Stefan; Vaida, Bianca; Proepper, Christian; Grissmer, Stephan; Storch, Alexander; Boeckers, Tobias M; Dietl, Paul; Wittekindt, Oliver H

    2007-06-01

    Ion channels are potent modulators for developmental processes in progenitor cells. In a screening approach for different ion channels in neural progenitor cells (NPCs) we observed a 1-ethyl-2-benzimidazolinone (1-EBIO) activated inward current, which could be blocked by scyllatoxin (ScTX, IC50=2+/- 0.3 nmol/L). This initial evidence for the expression of the small conductance Ca2+ activated K+-channel SK3 was confirmed by the detection of SK3 transcripts and protein in NPCs. Interestingly, SK3 proteins were highly expressed in non-differentiated NPCs with a focused localization in lamellipodia as well as filopodial structures. The activation of SK3 channels using 1-EBIO lead to an immediate filopodial sprouting and the translocation of the protein into these novel filopodial protrusions. Both effects could be prevented by the pre-incubation of NPCs with ScTX. Our study gives first evidence that the formation and prolongation of filopodia in NPCs is, at least in part, effectively induced and regulated by SK3 channels. PMID:17459146

  10. A chimeric prokaryotic pentameric ligand–gated channel reveals distinct pathways of activation

    PubMed Central

    Schmandt, Nicolaus; Velisetty, Phanindra; Chalamalasetti, Sreevatsa V.; Stein, Richard A.; Bonner, Ross; Talley, Lauren; Parker, Mark D.; Mchaourab, Hassane S.; Yee, Vivien C.; Lodowski, David T.

    2015-01-01

    Recent high resolution structures of several pentameric ligand–gated ion channels have provided unprecedented details of their molecular architecture. However, the conformational dynamics and structural rearrangements that underlie gating and allosteric modulation remain poorly understood. We used a combination of electrophysiology, double electron–electron resonance (DEER) spectroscopy, and x-ray crystallography to investigate activation mechanisms in a novel functional chimera with the extracellular domain (ECD) of amine-gated Erwinia chrysanthemi ligand–gated ion channel, which is activated by primary amines, and the transmembrane domain of Gloeobacter violaceus ligand–gated ion channel, which is activated by protons. We found that the chimera was independently gated by primary amines and by protons. The crystal structure of the chimera in its resting state, at pH 7.0 and in the absence of primary amines, revealed a closed-pore conformation and an ECD that is twisted with respect to the transmembrane region. Amine- and pH-induced conformational changes measured by DEER spectroscopy showed that the chimera exhibits a dual mode of gating that preserves the distinct conformational changes of the parent channels. Collectively, our findings shed light on both conserved and divergent features of gating mechanisms in this class of channels, and will facilitate the design of better allosteric modulators. PMID:26415570

  11. Fly DPP10 acts as a channel ancillary subunit and possesses peptidase activity

    PubMed Central

    Shiina, Yohei; Muto, Tomohiro; Zhang, Zhili; Baihaqie, Ahmad; Yoshizawa, Takamasa; Lee, Hye-in J.; Park, Eulsoon; Tsukiji, Shinya; Takimoto, Koichi

    2016-01-01

    Mammalian DPP6 (DPPX) and DPP10 (DPPY) belong to a family of dipeptidyl peptidases, but lack enzyme activity. Instead, these proteins form complexes with voltage-gated K+ channels in Kv4 family to control their gating and other properties. Here, we find that the fly DPP10 ortholog acts as an ancillary subunit of Kv4 channels and digests peptides. Similarly to mammalian DPP10, the fly ortholog tightly binds to rat Kv4.3 protein. The association causes negative shifts in voltage dependence of channel activation and steady state inactivation. It also results in faster inactivation and recovery from inactivation. In addition to its channel regulatory role, fly DPP10 exhibits significant dipeptidyl peptidase activity with Gly-Pro-MCA (glycyl-L-proline 4-methylcoumaryl-7-amide) as a substrate. Heterologously expressed Flag-tagged fly DPP10 and human DPP4 show similar Km values towards this substrate. However, fly DPP10 exhibits approximately a 6-times-lower relative kcat value normalized with anti-Flag immunoreactivity than human DPP4. These results demonstrate that fly DPP10 is a dual functional protein, controlling Kv4 channel gating and removing bioactive peptides. PMID:27198182

  12. Channel-Forming Activities in the Glycosomal Fraction from the Bloodstream Form of Trypanosoma brucei

    PubMed Central

    Miinalainen, Ilkka J.; Hiltunen, J. Kalervo; Michels, Paul A. M.; Antonenkov, Vasily D.

    2012-01-01

    Background Glycosomes are a specialized form of peroxisomes (microbodies) present in unicellular eukaryotes that belong to the Kinetoplastea order, such as Trypanosoma and Leishmania species, parasitic protists causing severe diseases of livestock and humans in subtropical and tropical countries. The organelles harbour most enzymes of the glycolytic pathway that is responsible for substrate-level ATP production in the cell. Glycolysis is essential for bloodstream-form Trypanosoma brucei and enzymes comprising this pathway have been validated as drug targets. Glycosomes are surrounded by a single membrane. How glycolytic metabolites are transported across the glycosomal membrane is unclear. Methods/Principal Findings We hypothesized that glycosomal membrane, similarly to membranes of yeast and mammalian peroxisomes, contains channel-forming proteins involved in the selective transfer of metabolites. To verify this prediction, we isolated a glycosomal fraction from bloodstream-form T.brucei and reconstituted solubilized membrane proteins into planar lipid bilayers. The electrophysiological characteristics of the channels were studied using multiple channel recording and single channel analysis. Three main channel-forming activities were detected with current amplitudes 70–80 pA, 20–25 pA, and 8–11 pA, respectively (holding potential +10 mV and 3.0 M KCl as an electrolyte). All channels were in fully open state in a range of voltages ±150 mV and showed no sub-conductance transitions. The channel with current amplitude 20–25 pA is anion-selective (PK+/PCl−∼0.31), while the other two types of channels are slightly selective for cations (PK+/PCl− ratios ∼1.15 and ∼1.27 for the high- and low-conductance channels, respectively). The anion-selective channel showed an intrinsic current rectification that may suggest a functional asymmetry of the channel's pore. Conclusions/Significance These results indicate that the membrane of glycosomes apparently

  13. Involvement of transient receptor potential-like channels in responses to mGluR-I activation in midbrain dopamine neurons.

    PubMed

    Tozzi, Alessandro; Bengtson, C Peter; Longone, Patrizia; Carignani, Corrado; Fusco, Francesca R; Bernardi, Giorgio; Mercuri, Nicola B

    2003-10-01

    We investigated the involvement of store-operated channels (SOCs) and transient receptor potential (TRP) channels in the response to activation of the group I metabotropic glutamate receptor subtype 1 (mGluR1) with the agonist (S)-3,5-dihydroxyphenylglycine (DHPG, puff application) in dopamine neurons in rat brain slices. The mGluR1-induced conductance reversed polarity close to 0 mV and at more positive potentials when extracellular potassium concentrations were increased, indicating the involvement of a cationic channel. DHPG currents but not intracellular calcium responses were reduced by low extracellular sodium concentrations but were not affected by sodium channel blockers, tetrodotoxin and saxitoxin or by inhibition of the h-current with cesium. Abolition of calcium responses with intracellular BAPTA (1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid; 10 mm) did not affect current responses, indicating they were not calcium activated. Extracellular application of non-selective SOCs and TRP channel blockers 2-aminoethoxydiphenylborane (2-APB), SKF96365, ruthenium red and flufenamic acid (but not gadolinium) reduced DHPG current and calcium responses. Intracellular application of ruthenium red and 2-APB did not affect DHPG currents, indicating that IP3 and ryanodine receptors did not mediate their actions. Single-cell PCR revealed the presence of TRPC1 and 5 mRNA in most dopamine neurons and subtypes 3, 4 and 6 in some. Store depletion evoked calcium entry indicative of SOCs, providing the first functional observation of such channels in native central neurons. Store depletion with either cyclopiazonic acid or ryanodine abolished calcium but not current responses to DHPG. The electrophysiological and pharmacological properties of the mGluR1-induced inward current are consistent with the involvement of TRP channels whereas calcium responses are dependent on the function of SOCs in voltage clamp recordings. PMID:14622174

  14. A Semi-Synthetic Ion Channel Platform for Detection of Phosphatase and Protease Activity

    PubMed Central

    Macrae, Michael X.; Blake, Steven; Jiang, Xiayun; Capone, Ricardo; Estes, Daniel J.; Mayer, Michael; Yang, Jerry

    2009-01-01

    Sensitive methods to probe the activity of enzymes are important for clinical assays and for elucidating the role of these proteins in complex biochemical networks. This paper describes a semi-synthetic ion channel platform for detecting the activity of two different classes of enzymes with high sensitivity. In the first case, this method uses single ion channel conductance measurements to follow the enzyme-catalyzed hydrolysis of a phosphate group attached to the C-terminus of gramicidin A (gA, an ion channel-forming peptide) in the presence of alkaline phosphatase (AP). Enzymatic hydrolysis of this phosphate group removes negative charges from the entrance of the gA pore, resulting in a product with measurably reduced single ion channel conductance compared to the original gA-phosphate substrate. This technique employs a standard, commercial bilayer setup and takes advantage of the catalytic turnover of enzymes and the amplification characteristics of ion flux through individual gA pores to detect picomolar concentrations of active AP in solution. Furthermore, this technique makes it possible to study the kinetics of an enzyme and provides an estimate for the observed rate constant (kcat) and the Michaelis constant (KM) by following the conversion of the gA-phosphate substrate to product over time in the presence of different concentrations of AP. In the second case, modification of gA with a substrate for proteolytic cleavage by anthrax lethal factor (LF) afforded a sensitive method for detection of LF activity, illustrating the utility of ion channel-based sensing for detection of a potential biowarfare agent. This ion channel-based platform represents a powerful, novel approach to monitor the activity of femtomoles to picomoles of two different classes of enzymes in solution. Furthermore, this platform has the potential for realizing miniaturized, cost-effective bioanalytical assays that complement currently established assays. PMID:19860382

  15. A comprehensive search for calcium binding sites critical for TMEM16A calcium-activated chloride channel activity.

    PubMed

    Tien, Jason; Peters, Christian J; Wong, Xiu Ming; Cheng, Tong; Jan, Yuh Nung; Jan, Lily Yeh; Yang, Huanghe

    2014-01-01

    TMEM16A forms calcium-activated chloride channels (CaCCs) that regulate physiological processes such as the secretions of airway epithelia and exocrine glands, the contraction of smooth muscles, and the excitability of neurons. Notwithstanding intense interest in the mechanism behind TMEM16A-CaCC calcium-dependent gating, comprehensive surveys to identify and characterize potential calcium sensors of this channel are still lacking. By aligning distantly related calcium-activated ion channels in the TMEM16 family and conducting systematic mutagenesis of all conserved acidic residues thought to be exposed to the cytoplasm, we identify four acidic amino acids as putative calcium-binding residues. Alterations of the charge, polarity, and size of amino acid side chains at these sites alter the ability of different divalent cations to activate the channel. Furthermore, TMEM16A mutant channels containing double cysteine substitutions at these residues are sensitive to the redox potential of the internal solution, providing evidence for their physical proximity and solvent accessibility. PMID:24980701

  16. Increased TMEM16A-encoded calcium-activated chloride channel activity is associated with pulmonary hypertension.

    PubMed

    Forrest, Abigail S; Joyce, Talia C; Huebner, Marissa L; Ayon, Ramon J; Wiwchar, Michael; Joyce, John; Freitas, Natalie; Davis, Alison J; Ye, Linda; Duan, Dayue D; Singer, Cherie A; Valencik, Maria L; Greenwood, Iain A; Leblanc, Normand

    2012-12-15

    Pulmonary artery smooth muscle cells (PASMCs) are more depolarized and display higher Ca(2+) levels in pulmonary hypertension (PH). Whether the functional properties and expression of Ca(2+)-activated Cl- channels (Cl(Ca)), an important excitatory mechanism in PASMCs, are altered in PH is unknown. The potential role of Cl(Ca) channels in PH was investigated using the monocrotaline (MCT)-induced PH model in the rat. Three weeks postinjection with a single dose of MCT (50 mg/kg ip), the animals developed right ventricular hypertrophy (heart weight measurements) and changes in pulmonary arterial flow (pulse-waved Doppler imaging) that were consistent with increased pulmonary arterial pressure and PH. Whole cell patch experiments revealed an increase in niflumic acid (NFA)-sensitive Ca(2+)-activated Cl(-) current [I(Cl(Ca))] density in PASMCs from large conduit and small intralobar pulmonary arteries of MCT-treated rats vs. aged-matched saline-injected controls. Quantitative RT-PCR and Western blot analysis revealed that the alterations in I(Cl(Ca)) were accompanied by parallel changes in the expression of TMEM16A, a gene recently shown to encode for Cl(Ca) channels. The contraction to serotonin of conduit and intralobar pulmonary arteries from MCT-treated rats exhibited greater sensitivity to nifedipine (1 μM), an l-type Ca(2+) channel blocker, and NFA (30 or 100 μM, with or without 10 μM indomethacin to inhibit cyclooxygenases) or T16A(Inh)-A01 (10 μM), TMEM16A/Cl(Ca) channel inhibitors, than that of control animals. In conclusion, augmented Cl(Ca)/TMEM16A channel activity is a major contributor to the changes in electromechanical coupling of PA in this model of PH. TMEM16A-encoded channels may therefore represent a novel therapeutic target in this disease. PMID:23034390

  17. Calibration activities on the BepiColombo High-Resolution Channel (HRIC) of SIMBIO-SYS instrument

    NASA Astrophysics Data System (ADS)

    Della Corte, V.; Zusi, M.; Palumbo, P.; Baroni, M.; Ficai Veltroni, I.; Flamini, E.; Mugnuolo, R.

    2015-10-01

    HRIC (High Resolution Imaging Channel) is the high resolution channel of the SIMBIO-SYS instrument on- board the ESA BepiColombo Mission. Calibration activities were performed at SelexES premises in spring- summer 2014 in order to check for Channel performances (radiometric performances, quality image and geometrical performances) and to obtain data necessary to setup a calibration pipeline necessary to process the raw images acquired by the channel when in operative scenario.

  18. Role of phospholipase D and diacylglycerol in activating constitutive TRPC-like cation channels in rabbit ear artery myocytes.

    PubMed

    Albert, A P; Piper, A S; Large, W A

    2005-08-01

    Previously we have described a constitutively active Ca2+-permeable non-selective cation channel in freshly dispersed rabbit ear artery myocytes that has similar properties to canonical transient receptor potential (TRPC) channel proteins. In the present study we have investigated the transduction pathways responsible for stimulating constitutive channel activity in these myocytes. Application of the pharmacological inhibitors of phosphatidylcholine-phospholipase D (PC-PLD), butan-1-ol and C2 ceramide, produced marked inhibition of constitutive channel activity in cell-attached patches and also butan-1-ol produced pronounced suppression of resting membrane conductance measured with whole-cell recording whereas the inactive isomer butan-2-ol had no effect on constitutive whole-cell or channel activity. In addition butan-1-ol had no effect on channel activity evoked by the diacylglycerol (DAG) analogue 1-oleoyl-2-acetyl-sn-glycerol (OAG). Inhibitors of PC-phospholipase C (PC-PLC) and phospholipase A2 (PLA2) had no effect on constitutive channel activity. Application of a purified PC-PLD enzyme and its metabolite phosphatidic acid to inside-out patches markedly increased channel activity. The phosphatidic acid phosphohydrolase (PAP) inhibitor dl-propranolol also inhibited constitutive and phosphatidic acid-induced increases in channel activity but had no effect on OAG-evoked responses. The DAG lipase and DAG kinase inhibitors, RHC80267 and R59949 respectively, which inhibit DAG metabolism, produced transient increases in channel activity which were mimicked by relatively high concentrations (40 microm) of OAG. The protein kinase C (PKC) inhibitor chelerythrine did not prevent channel activation by OAG but blocked the secondary inhibitory response of OAG. It is proposed that endogenous DAG is involved in the activation of channel activity and that its effects on channel activity are concentration-dependent with higher concentrations of DAG also inhibiting channel

  19. Apamin-sensitive, small-conductance, calcium-activated potassium channels mediate cholinergic inhibition of chick auditory hair cells.

    PubMed

    Yuhas, W A; Fuchs, P A

    1999-11-01

    Acetylcholine released from efferent neurons in the cochlea causes inhibition of mechanosensory hair cells due to the activation of calcium-dependent potassium channels. Hair cells are known to have large-conductance, "BK"-type potassium channels associated with the afferent synapse, but these channels have different properties than those activated by acetylcholine. Whole-cell (tight-seal) and cell-attached patch-clamp recordings were made from short (outer) hair cells isolated from the chicken basilar papilla (cochlea equivalent). The peptides apamin and charybdotoxin were used to distinguish the calcium-activated potassium channels involved in the acetylcholine response from the BK-type channels associated with the afferent synapse. Differential toxin blockade of these potassium currents provides definitive evidence that ACh activates apamin-sensitive, "SK"-type potassium channels, but does not activate carybdotoxin-sensitive BK channels. This conclusion is supported by tentative identification of small-conductance, calcium-sensitive but voltage-insensitive potassium channels in cell-attached patches. The distinction between these channel types is important for understanding the segregation of opposing afferent and efferent synaptic activity in the hair cell, both of which depend on calcium influx. These different calcium-activated potassium channels serve as sensitive indicators for functionally significant calcium influx in the hair cell. PMID:10573868

  20. Synthesis and evaluation of 1,4-dihydropyridine derivatives with calcium channel blocking activity.

    PubMed

    Bladen, Chris; Gündüz, Miyase Gözde; Şimşek, Rahime; Şafak, Cihat; Zamponi, Gerald W

    2014-07-01

    1,4-Dihydropyridines (DHPs) are an important class of L-type calcium channel blockers that are used to treat conditions such as hypertension and angina. Their primary target in the cardiovascular system is the Cav1.2 L-type calcium channel isoform, however, a number of DHPs also block low-voltage-activated T-type calcium channels. Here, we describe the synthesis of a series of novel DHP derivatives that have a condensed 1,4-DHP ring system (hexahydroquinoline) and report on their abilities to block both L- and T-type calcium channels. Within this series of compounds, modification of a key ester moiety not only regulates the blocking affinity for both L- and T-type channels, but also allows for the development of DHPs with 30-fold selectivity for T-type channels over the L-type. Our data suggest that a condensed dihydropyridine-based scaffold may serve as a pharmacophore for a new class of T-type selective inhibitors. PMID:24149495

  1. Molecular Interactions between Tarantula Toxins and Low-Voltage-Activated Calcium Channels

    PubMed Central

    Salari, Autoosa; Vega, Benjamin S.; Milescu, Lorin S.; Milescu, Mirela

    2016-01-01

    Few gating-modifier toxins have been reported to target low-voltage-activated (LVA) calcium channels, and the structural basis of toxin sensitivity remains incompletely understood. Studies of voltage-gated potassium (Kv) channels have identified the S3b–S4 “paddle motif,” which moves at the protein-lipid interface to drive channel opening, as the target for these amphipathic neurotoxins. Voltage-gated calcium (Cav) channels contain four homologous voltage sensor domains, suggesting multiple toxin binding sites. We show here that the S3–S4 segments within Cav3.1 can be transplanted into Kv2.1 to examine their individual contributions to voltage sensing and pharmacology. With these results, we now have a more complete picture of the conserved nature of the paddle motif in all three major voltage-gated ion channel types (Kv, Nav, and Cav). When screened with tarantula toxins, the four paddle sequences display distinct toxin binding properties, demonstrating that gating-modifier toxins can bind to Cav channels in a domain specific fashion. Domain III was the most commonly and strongly targeted, and mutagenesis revealed an acidic residue that is important for toxin binding. We also measured the lipid partitioning strength of all toxins tested and observed a positive correlation with their inhibition of Cav3.1, suggesting a key role for membrane partitioning. PMID:27045173

  2. Molecular Interactions between Tarantula Toxins and Low-Voltage-Activated Calcium Channels.

    PubMed

    Salari, Autoosa; Vega, Benjamin S; Milescu, Lorin S; Milescu, Mirela

    2016-01-01

    Few gating-modifier toxins have been reported to target low-voltage-activated (LVA) calcium channels, and the structural basis of toxin sensitivity remains incompletely understood. Studies of voltage-gated potassium (Kv) channels have identified the S3b-S4 "paddle motif," which moves at the protein-lipid interface to drive channel opening, as the target for these amphipathic neurotoxins. Voltage-gated calcium (Cav) channels contain four homologous voltage sensor domains, suggesting multiple toxin binding sites. We show here that the S3-S4 segments within Cav3.1 can be transplanted into Kv2.1 to examine their individual contributions to voltage sensing and pharmacology. With these results, we now have a more complete picture of the conserved nature of the paddle motif in all three major voltage-gated ion channel types (Kv, Nav, and Cav). When screened with tarantula toxins, the four paddle sequences display distinct toxin binding properties, demonstrating that gating-modifier toxins can bind to Cav channels in a domain specific fashion. Domain III was the most commonly and strongly targeted, and mutagenesis revealed an acidic residue that is important for toxin binding. We also measured the lipid partitioning strength of all toxins tested and observed a positive correlation with their inhibition of Cav3.1, suggesting a key role for membrane partitioning. PMID:27045173

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

    PubMed

    Zhang, Weiping; Schmelzeisen, Steffen; Parthier, Daniel; Frings, Stephan; Möhrlen, Frank

    2015-01-01

    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. PMID:26558388

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

    PubMed Central

    Parthier, Daniel; Frings, Stephan; Möhrlen, Frank

    2015-01-01

    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. PMID:26558388

  5. Hyperpolarization-activated, cyclic nucleotide-gated cation channels in Aplysia: Contribution to classical conditioning

    PubMed Central

    Yang, Qizong; Kuzyk, Pavlo; Antonov, Igor; Bostwick, Caleb J.; Kohn, Andrea B.; Moroz, Leonid L.; Hawkins, Robert D.

    2015-01-01

    Hyperpolarization-activated, cyclic nucleotide-gated cation (HCN) channels are critical regulators of neuronal excitability, but less is known about their possible roles in synaptic plasticity and memory circuits. Here, we characterized the HCN gene organization, channel properties, distribution, and involvement in associative and nonassociative forms of learning in Aplysia californica. Aplysia has only one HCN gene, which codes for a channel that has many similarities to the mammalian HCN channel. The cloned acHCN gene was expressed in Xenopus oocytes, which displayed a hyperpolarization-induced inward current that was enhanced by cGMP as well as cAMP. Similarly to its homologs in other animals, acHCN is permeable to K+ and Na+ ions, and is selectively blocked by Cs+ and ZD7288. We found that acHCN is predominantly expressed in inter- and motor neurons, including LFS siphon motor neurons, and therefore tested whether HCN channels are involved in simple forms of learning of the siphon-withdrawal reflex in a semiintact preparation. ZD7288 (100 μM) significantly reduced an associative form of learning (classical conditioning) but had no effect on two nonassociative forms of learning (intermediate-term sensitization and unpaired training) or baseline responses. The HCN current is enhanced by nitric oxide (NO), which may explain the postsynaptic role of NO during conditioning. HCN current in turn enhances the NMDA-like current in the motor neurons, suggesting that HCN channels contribute to conditioning through this pathway. PMID:26668355

  6. Large Conductance, Ca2+-Activated K+ Channels (BKCa) and Arteriolar Myogenic Signaling

    PubMed Central

    Hill, Michael A.; Yang, Yan; Ella, Srikanth R.; Davis, Michael J.; Braun, Andrew P.

    2010-01-01

    Summary Myogenic, or pressure-induced, vasoconstriction is critical for local blood flow autoregulation. Underlying this VSM response are events including membrane depolarization, Ca2+ entry and mobilization, and activation of contractile proteins. BKCa has been implicated in several of these steps including, 1) channel closure causing membrane depolarization, and 2) channel opening causing hyperpolarization to oppose excessive pressure-induced vasoconstriction. As multiple mechanisms regulate BKCa activity, (subunit composition, Em and Ca2+ levels, post-translational modification) tissue level diversity is predicted. Importantly, heterogeneity may contribute to tissue-specific differences in regulation of myogenic vasoconstriction, allowing local hemodynamics to be matched to metabolic requirements. Knowledge of such variability will be important to exploiting the BKCa channel as a therapeutic target and understanding systemic effects of its pharmacological manipulation. PMID:20178789

  7. Multi-channel Kalman filters for active noise control.

    PubMed

    van Ophem, S; Berkhoff, A P

    2013-04-01

    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 Kalman algorithm is derived to perform this state estimation. To make the algorithm more suitable for real-time applications, the Kalman filter is written in a fast array form and the secondary path state matrices are implemented in output normal form. The resulting filter implementation is tested in simulations and in real-time experiments. It was found that for a constant primary path the filter has a fast rate of convergence and is able to track changes in the frequency spectrum. For a forgetting factor equal to unity the system is robust but the filter is unable to track rapid changes in the primary path. A forgetting factor lower than 1 gives a significantly improved tracking performance but leads to a numerical instability for the fast array form of the algorithm. PMID:23556580

  8. Functional Roles of a Ca2+-Activated K+ Channel (SK2) in Atrioventricular Nodes

    PubMed Central

    Zhang, Qian; Timofeyev, Valeriy; Lu, Ling; Li, Ning; Singapuri, Anil; Long, Melissa K.; Bond, Chris T.; Adelman, John P.; Chiamvimonvat, Nipavan

    2013-01-01

    Since the first description of the anatomical atrioventricular nodes (AVN), a large number of studies have provided insights into the heterogeneity of the structure as well as a repertoire of ion channel proteins which govern this complex conduction pathway between the atria and ventricles. These studies have revealed the intricate organization of multiple nodal and nodal-like myocytes contributing to the unique electrophysiology of the AVN in health and diseases. On the other hand, information regarding contribution of specific ion channels to the function of the AVN remains incomplete. We reason that the identification of AVN-specific ion channels may provide a more direct and rationale design of therapeutic target in the control of AVN conduction in atrial flutter/fibrillation, one of the most common arrhythmias seen clinically. In this study, we took advantage of two genetically altered mouse models with over-expression or null mutation of one of the small conductance Ca2+-activated K+ channel isoform, SK2 channel and demonstrated robust phenotypes of AVN dysfunction in these experimental models. Over-expression of SK2 channels results in the shortening of the spontaneous action potentials (APs) of the AVN cells and an increase in the firing frequency. On the other hand, ablation of the SK2 channel results in the opposite effects on the spontaneous APs of the AVN. Furthermore, we directly documented the expression of SK2 channel in mouse AVN using multiple techniques. The new insights may have important implications in providing novel drug targets for the modification of AVN conduction in the treatment of atrial arrhythmias. PMID:18096820

  9. Apparent intermediate K conductance channel hyposmotic activation in human lens epithelial cells.

    PubMed

    Lauf, Peter K; Misri, Sandeep; Chimote, Ameet A; Adragna, Norma C

    2008-03-01

    This study explores the nature of K fluxes in human lens epithelial cells (LECs) in hyposmotic solutions. Total ion fluxes, Na-K pump, Cl-dependent Na-K-2Cl (NKCC), K-Cl (KCC) cotransport, and K channels were determined by 85Rb uptake and cell K (Kc) by atomic absorption spectrophotometry, and cell water gravimetrically after exposure to ouabain +/- bumetanide (Na-K pump and NKCC inhibitors), and ion channel inhibitors in varying osmolalities with Na, K, or methyl-d-glucamine and Cl, sulfamate, or nitrate. Reverse transcriptase polymerase chain reaction (RT-PCR), Western blot analyses, and immunochemistry were also performed. In isosmotic (300 mosM) media approximately 90% of the total Rb influx occurred through the Na-K pump and NKCC and approximately 10% through KCC and a residual leak. Hyposmotic media (150 mosM) decreased K(c) by a 16-fold higher K permeability and cell water, but failed to inactivate NKCC and activate KCC. Sucrose replacement or extracellular K to >57 mM, but not Rb or Cs, in hyposmotic media prevented Kc and water loss. Rb influx equaled Kc loss, both blocked by clotrimazole (IC50 approximately 25 microM) and partially by 1-[(2-chlorophenyl) diphenylmethyl]-1H-pyrazole (TRAM-34) inhibitors of the IK channel KCa3.1 but not by other K channel or connexin hemichannel blockers. Of several anion channel blockers (dihydro-indenyl)oxy]alkanoic acid (DIOA), 4-2(butyl-6,7-dichloro-2-cyclopentylindan-1-on-5-yl)oxybutyric acid (DCPIB), and phloretin totally or partially inhibited Kc loss and Rb influx, respectively. RT-PCR and immunochemistry confirmed the presence of KCa3.1 channels, aside of the KCC1, KCC2, KCC3 and KCC4 isoforms. Apparently, IK channels, possibly in parallel with volume-sensitive outwardly rectifying Cl channels, effect regulatory volume decrease in LECs. PMID:18184876

  10. Stoichiometry of altered hERG1 channel gating by small molecule activators.

    PubMed

    Wu, Wei; Sachse, Frank B; Gardner, Alison; Sanguinetti, Michael C

    2014-04-01

    Voltage-gated K(+) channels are tetramers formed by coassembly of four identical or highly related subunits. All four subunits contribute to formation of the selectivity filter, the narrowest region of the channel pore which determines K(+) selective conductance. In some K(+) channels, the selectivity filter can undergo a conformational change to reduce K(+) flux by a mechanism called C-type inactivation. In human ether-a-go-go-related gene 1 (hERG1) K(+) channels, C-type inactivation is allosterically inhibited by ICA-105574, a substituted benzamide. PD-118057, a 2-(phenylamino) benzoic acid, alters selectivity filter gating to enhance open probability of channels. Both compounds bind to a hydrophobic pocket located between adjacent hERG1 subunits. Accordingly, a homotetrameric channel contains four identical activator binding sites. Here we determine the number of binding sites required for maximal drug effect and determine the role of subunit interactions in the modulation of hERG1 gating by these compounds. Concatenated tetramers were constructed to contain a variable number (zero to four) of wild-type and mutant hERG1 subunits, either L646E to inhibit PD-118057 binding or F557L to inhibit ICA-105574 binding. Enhancement of hERG1 channel current magnitude by PD-118057 and attenuated inactivation by ICA-105574 were mediated by cooperative subunit interactions. Maximal effects of the both compounds required the presence of all four binding sites. Understanding how hERG1 agonists allosterically modify channel gating may facilitate mechanism-based drug design of novel agents for treatment of long QT syndrome. PMID:24638994

  11. Stoichiometry of altered hERG1 channel gating by small molecule activators

    PubMed Central

    Wu, Wei; Sachse, Frank B.; Gardner, Alison

    2014-01-01

    Voltage-gated K+ channels are tetramers formed by coassembly of four identical or highly related subunits. All four subunits contribute to formation of the selectivity filter, the narrowest region of the channel pore which determines K+ selective conductance. In some K+ channels, the selectivity filter can undergo a conformational change to reduce K+ flux by a mechanism called C-type inactivation. In human ether-a-go-go–related gene 1 (hERG1) K+ channels, C-type inactivation is allosterically inhibited by ICA-105574, a substituted benzamide. PD-118057, a 2-(phenylamino) benzoic acid, alters selectivity filter gating to enhance open probability of channels. Both compounds bind to a hydrophobic pocket located between adjacent hERG1 subunits. Accordingly, a homotetrameric channel contains four identical activator binding sites. Here we determine the number of binding sites required for maximal drug effect and determine the role of subunit interactions in the modulation of hERG1 gating by these compounds. Concatenated tetramers were constructed to contain a variable number (zero to four) of wild-type and mutant hERG1 subunits, either L646E to inhibit PD-118057 binding or F557L to inhibit ICA-105574 binding. Enhancement of hERG1 channel current magnitude by PD-118057 and attenuated inactivation by ICA-105574 were mediated by cooperative subunit interactions. Maximal effects of the both compounds required the presence of all four binding sites. Understanding how hERG1 agonists allosterically modify channel gating may facilitate mechanism-based drug design of novel agents for treatment of long QT syndrome. PMID:24638994

  12. Role of thromboxane A₂-activated nonselective cation channels in hypoxic pulmonary vasoconstriction of rat.

    PubMed

    Yoo, Hae Young; Park, Su Jung; Seo, Eun-Young; Park, Kyung Sun; Han, Jung-A; Kim, Kyung Soo; Shin, Dong Hoon; Earm, Yung E; Zhang, Yin-Hua; Kim, Sung Joon

    2012-01-01

    Hypoxia-induced pulmonary vasoconstriction (HPV) is critical for matching of ventilation/perfusion in lungs. Although hypoxic inhibition of K(+) channels has been a leading hypothesis for depolarization of pulmonary arterial smooth muscle cells (PASMCs) under hypoxia, pharmacological inhibition of K(+) channels does not induce significant contraction in rat pulmonary arteries. Because a partial contraction by thromboxane A(2) (TXA(2)) is required for induction of HPV, we hypothesize that TXA(2) receptor (TP) stimulation might activate depolarizing nonselective cation channels (NSCs). Consistently, we found that 5-10 nM U46619, a stable agonist for TP, was indispensible for contraction of rat pulmonary arteries by 4-aminopyridine, a blocker of voltage-gated K(+) channel (K(v)). Whole cell voltage clamp with rat PASMC revealed that U46619 induced a NSC current (I(NSC,TXA2)) with weakly outward rectifying current-voltage relation. I(NSC,TXA2) was blocked by ruthenium red (RR), an antagonist of the transient receptor potential vanilloid-related channel (TRPV) subfamily. 2-Aminoethoxydiphenyl borate, an agonist for TRPV1-3, consistently activated NSC channels in PASMCs. In contrast, agonists for TRPV1 (capsaicin), TRPV3 (camphor), or TRPV4 (α-PDD) rarely induced an increase in the membrane conductance of PASMCs. RT-PCR analysis showed the expression of transcripts for TRPV2 and -4 in rat PASMCs. Finally, it was confirmed that pretreatment with RR largely inhibited HPV in the presence of U46619. The pretreatment with agonists for TRPV1 (capsaicin) and TRPV4 (α-PDD) was ineffective as pretone agents for HPV. Taken together, it is suggested that the concerted effects of I(NSC,TXA2) activation and K(v) inhibition under hypoxia induce membrane depolarization sufficient for HPV. TRPV2 is carefully suggested as the TXA(2)-activated NSC in rat PASMC. PMID:21998141

  13. Molecular and functional significance of Ca2+-activated Cl− channels in pulmonary arterial smooth muscle

    PubMed Central

    Forrest, Abigail S.; Ayon, Ramon J.; Wiwchar, Michael; Angermann, Jeff E.; Pritchard, Harry A. T.; Singer, Cherie A.; Valencik, Maria L.; Britton, Fiona; Greenwood, Iain A.

    2015-01-01

    Abstract Increased peripheral resistance of small distal pulmonary arteries is a hallmark signature of pulmonary hypertension (PH) and is believed to be the consequence of enhanced vasoconstriction to agonists, thickening of the arterial wall due to remodeling, and increased thrombosis. The elevation in arterial tone in PH is attributable, at least in part, to smooth muscle cells of PH patients being more depolarized and displaying higher intracellular Ca2+ levels than cells from normal subjects. It is now clear that downregulation of voltage-dependent K+ channels (e.g., Kv1.5) and increased expression and activity of voltage-dependent (Cav1.2) and voltage-independent (e.g., canonical and vanilloid transient receptor potential [TRPC and TRPV]) Ca2+ channels play an important role in the functional remodeling of pulmonary arteries in PH. This review focuses on an anion-permeable channel that is now considered a novel excitatory mechanism in the systemic and pulmonary circulations. It is permeable to Cl− and is activated by a rise in intracellular Ca2+ concentration (Ca2+-activated Cl− channel, or CaCC). The first section outlines the biophysical and pharmacological properties of the channel and ends with a description of the molecular candidate genes postulated to encode for CaCCs, with particular emphasis on the bestrophin and the newly discovered TMEM16 and anoctamin families of genes. The second section provides a review of the various sources of Ca2+ activating CaCCs, which include stimulation by mobilization from intracellular Ca2+ stores and Ca2+ entry through voltage-dependent and voltage-independent Ca2+ channels. The third and final section summarizes recent findings that suggest a potentially important role for CaCCs and the gene TMEM16A in PH. PMID:26064450

  14. Assessing water source and channel type as factors affecting benthic macroinvertebrate and periphyton assemblages in the highly urbanized Santa Ana River Basin, California

    USGS Publications Warehouse

    Burton, C.A.; Brown, L.R.; Belitz, K.

    2005-01-01

    The Santa Ana River basin is the largest stream system in Southern California and includes a densely populated coastal area. Extensive urbanization has altered the geomorphology and hydrology of the streams, adversely affecting aquatic communities. We studied macroinvertebrate and periphyton assemblages in relation to two categorical features of the highly engineered hydrologic system-water source and channel type. Four water sources were identified-natural, urban-impacted groundwater, urban runoff, and treated wastewater. Three channel types were identified-natural, channelized with natural bottom, and concrete-lined. Nineteen sites, covering the range of these two categorical features, were sampled in summer 2000. To minimize the effects of different substrate types among sites, artificial substrates were used for assessing macroinvertebrate and periphyton assemblages. Physical and chemical variables and metrics calculated from macroinvertebrate and periphyton assemblage data were compared among water sources and channel types using analysis of variance and multiple comparison tests. Macroinvertebrate metrics exhibiting significant (P < 0.05) differences between water sources included taxa and Ephemeroptera-Plecoptera-Trichoptera richness, relative richness and abundance of nonchironomid dipterans, orthoclads, oligochaetes, and some functional-feeding groups such as parasites and shredders. Periphyton metrics showing significant differences between water sources included blue-green algae biovolume and relative abundance of nitrogen heterotrophic, eutrophic, motile, and pollution-sensitive diatoms. The relative abundance of trichopterans, tanytarsini chironomids, noninsects, and filter feeders, as well as the relative richness and abundance of diatoms, were significantly different between channel types. Most physical variables were related to channel type, whereas chemical variables and some physical variables (e.g., discharge, velocity, and channel width) were

  15. Hexachlorophene Is a Potent KCNQ1/KCNE1 Potassium Channel Activator Which Rescues LQTs Mutants

    PubMed Central

    Zheng, Yueming; Zhu, Xuejing; Zhou, Pingzheng; Lan, Xi; Xu, Haiyan; Li, Min; Gao, Zhaobing

    2012-01-01

    The voltage-gated KCNQ1 potassium channel is expressed in cardiac tissues, and coassembly of KCNQ1 with an auxiliary KCNE1 subunit mediates a slowly activating current that accelerates the repolarization of action potential in cardiomyocytes. Mutations of KCNQ1 genes that result in reduction or loss of channel activity cause prolongation of repolarization during action potential, thereby causing long QT syndrome (LQTs). Small molecule activators of KCNQ1/KCNE1 are useful both for understanding the mechanism of the complex activity and for developing therapeutics for LQTs. In this study we report that hexachlorophene (HCP), the active component of the topical anti-infective prescription drug pHisoHex, is a KCNQ1/KCNE1 activator. HCP potently increases the current amplitude of KCNQ1/KCNE1 expressed by stabilizing the channel in an open state with an EC50 of 4.61±1.29 μM. Further studies in cardiomyocytes showed that HCP significantly shortens the action potential duration at 1 μM. In addition, HCP is capable of rescuing the loss of function of the LQTs mutants caused by either impaired activation gating or phosphatidylinositol-4,5-bisphosphate (PIP2) binding affinity. Our results indicate HCP is a novel KCNQ1/KCNE1 activator and may be a useful tool compound for the development of LQTs therapeutics. PMID:23251633

  16. Effects of urine composition on epithelial Na+ channel-targeted protease activity.

    PubMed

    Berman, Jonathan M; Awayda, Ryan G; Awayda, Mouhamed S

    2015-11-01

    We examined human urinary proteolytic activity toward the Epithelial Sodium Channel (ENaC). We focused on two sites in each of alpha and gamma ENaC that are targets of endogenous and exogenous proteases. We examined the effects of ionic strength, pH and urinary H(+)-buffers, metabolic intermediates, redox molecules, and large urinary proteins. Monoatomic cations caused the largest effect, with sodium inhibiting activity in the 15-515 mEq range. Multivalent cations zinc and copper inhibited urinary proteolytic activity at concentrations below 100 μmol/L. Similar to sodium, urea caused a 30% inhibition in the 0-500 mmol/L range. This was not observed with acetone and ethanol. Modulating urinary redox status modified activity with H2O2 stimulated and ascorbate inhibited activity. Minimal effects (<10%) were observed with caffeine, glucose, several TCA cycle intermediates, salicylic acid, inorganic phosphate, albumin, creatinine, and Tamm-Horsfall protein. The cumulative activity of ENaC-cleaving proteases was highest at neutral pH, however, alpha and gamma proteases exhibited an inverse dependence with alpha stimulated at acidic and gamma stimulated at alkaline pH. These data indicate that ENaC-targeting urinary proteolytic activity is sensitive to sodium, urea and pH and changes in these components can modify channel cleavage and activation status, and likely downstream sodium absorption unrelated to changes in protein or channel density. PMID:26564065

  17. Ethanol inhibits ligand-activated calcium channels in human B lymphocytes

    SciTech Connect

    Mazer, B.D.; Brodie, C.; Domenico, J.; Gelfand, E.W. )

    1991-03-11

    The authors examined the acute effect of ethanol on the Ca{sup 2+} signal induced by two ligands, anti-{mu} antibody and platelet activating factor (PAF), during activation of human EBV-transformed B-cell lines. Stimulation of cells with either ligand results in an increase in free cytosolic Ca{sup 2+} as a result of release of Ca{sup 2+} from internal stores and of Ca{sup 2+} entry across the plasma membrane. Cells pretreated with 0.5% ethanol displayed a significantly smaller Ca{sup 2+} response following binding of either anti-{mu} or PAF, while ethanol itself did not effect basal levels of Ca{sup 2+}. The inhibitory effect of ethanol was time-dependent, the maximal effect being reached after 30 min of pre-incubation. In an attempt to define which component of the ligand-induced Ca{sup 2+} signal was inhibited by ethanol, the authors performed experiments in Ca{sup 2+}-free medium to eliminate Ca{sup 2+} influx. Ethanol treatment did not affect ligand-induced Ca{sup 2+} release from internal stores, but decreased the Ca{sup 2+} influx upon reintroduction of 1 mM Ca{sup 2+}. Ethanol also inhibited the Ca{sup 2+}-influx dependent induction of c-fos, triggered by anti-{mu} or PAF. The inhibitory effects of ethanol on Ca{sup 2+} channels in B cells are similar to those already reported in the CNS and may provide a basis for the immunosuppressive effect of ethanol.

  18. Anoctamin 1 (Tmem16A) Ca2+-activated chloride channel stoichiometrically interacts with an ezrin-radixin-moesin network.

    PubMed

    Perez-Cornejo, Patricia; Gokhale, Avanti; Duran, Charity; Cui, Yuanyuan; Xiao, Qinghuan; Hartzell, H Criss; Faundez, Victor

    2012-06-26

    The newly discovered Ca(2+)-activated Cl(-) channel (CaCC), Anoctamin 1 (Ano1 or TMEM16A), has been implicated in vital physiological functions including epithelial fluid secretion, gut motility, and smooth muscle tone. Overexpression of Ano1 in HEK cells or Xenopus oocytes is sufficient to generate Ca(2+)-activated Cl(-) currents, but the details of channel composition and the regulatory factors that control channel biology are incompletely understood. We used a highly sensitive quantitative SILAC proteomics approach to obtain insights into stoichiometric protein networks associated with the Ano1 channel. These studies provide a comprehensive footprint of putative Ano1 regulatory networks. We find that Ano1 associates with the signaling/scaffolding proteins ezrin, radixin, moesin, and RhoA, which link the plasma membrane to the cytoskeleton with very high stoichiometry. Ano1, ezrin, and moesin/radixin colocalize apically in salivary gland epithelial cells, and overexpression of moesin and Ano1 in HEK cells alters the subcellular localization of both proteins. Moreover, interfering RNA for moesin modifies Ano1 current without affecting its surface expression level. Another network associated with Ano1 includes the SNARE and SM proteins VAMP3, syntaxins 2 and -4, and syntaxin-binding proteins munc18b and munc18c, which are integral to translocation of vesicles to the plasma membrane. A number of other regulatory proteins, including GTPases, Ca(2+)-binding proteins, kinases, and lipid-interacting proteins are enriched in the Ano1 complex. These data provide stoichiometrically prioritized information about mechanisms regulating Ano1 function and trafficking to polarized domains of the plasma membrane. PMID:22685202

  19. Anoctamin 1 (Tmem16A) Ca2+-activated chloride channel stoichiometrically interacts with an ezrin–radixin–moesin network

    PubMed Central

    Perez-Cornejo, Patricia; Gokhale, Avanti; Duran, Charity; Cui, Yuanyuan; Xiao, Qinghuan; Hartzell, H. Criss; Faundez, Victor

    2012-01-01

    The newly discovered Ca2+-activated Cl− channel (CaCC), Anoctamin 1 (Ano1 or TMEM16A), has been implicated in vital physiological functions including epithelial fluid secretion, gut motility, and smooth muscle tone. Overexpression of Ano1 in HEK cells or Xenopus oocytes is sufficient to generate Ca2+-activated Cl− currents, but the details of channel composition and the regulatory factors that control channel biology are incompletely understood. We used a highly sensitive quantitative SILAC proteomics approach to obtain insights into stoichiometric protein networks associated with the Ano1 channel. These studies provide a comprehensive footprint of putative Ano1 regulatory networks. We find that Ano1 associates with the signaling/scaffolding proteins ezrin, radixin, moesin, and RhoA, which link the plasma membrane to the cytoskeleton with very high stoichiometry. Ano1, ezrin, and moesin/radixin colocalize apically in salivary gland epithelial cells, and overexpression of moesin and Ano1 in HEK cells alters the subcellular localization of both proteins. Moreover, interfering RNA for moesin modifies Ano1 current without affecting its surface expression level. Another network associated with Ano1 includes the SNARE and SM proteins VAMP3, syntaxins 2 and -4, and syntaxin-binding proteins munc18b and munc18c, which are integral to translocation of vesicles to the plasma membrane. A number of other regulatory proteins, including GTPases, Ca2+-binding proteins, kinases, and lipid-interacting proteins are enriched in the Ano1 complex. These data provide stoichiometrically prioritized information about mechanisms regulating Ano1 function and trafficking to polarized domains of the plasma membrane. PMID:22685202

  20. Active Sites of Spinoxin, a Potassium Channel Scorpion Toxin, Elucidated by Systematic Alanine Scanning.

    PubMed

    Peigneur, Steve; Yamaguchi, Yoko; Kawano, Chihiro; Nose, Takeru; Nirthanan, Selvanayagam; Gopalakrishnakone, Ponnampalam; Tytgat, Jan; Sato, Kazuki

    2016-05-31

    Peptide toxins from scorpion venoms constitute the largest group of toxins that target the voltage-gated potassium channel (Kv). Spinoxin (SPX) isolated from the venom of scorpion Heterometrus spinifer is a 34-residue peptide neurotoxin cross-linked by four disulfide bridges. SPX is a potent inhibitor of Kv1.3 potassium channels (IC50 = 63 nM), which are considered to be valid molecular targets in the diagnostics and therapy of various autoimmune disorders and cancers. Here we synthesized 25 analogues of SPX and analyzed the role of each amino acid in SPX using alanine scanning to study its structure-function relationships. All synthetic analogues showed similar disulfide bond pairings and secondary structures as native SPX. Alanine replacements at Lys(23), Asn(26), and Lys(30) resulted in loss of activity against Kv1.3 potassium channels, whereas replacements at Arg(7), Met(14), Lys(27), and Tyr(32) also largely reduced inhibitory activity. These results suggest that the side chains of these amino acids in SPX play an important role in its interaction with Kv1.3 channels. In particular, Lys(23) appears to be a key residue that underpins Kv1.3 channel inhibition. Of these seven amino acid residues, four are basic amino acids, suggesting that the positive electrostatic potential on the surface of SPX is likely required for high affinity interaction with Kv1.3 channels. This study provides insight into the structure-function relationships of SPX with implications for the rational design of new lead compounds targeting potassium channels with high potency. PMID:27159046

  1. Stochastically Gating Ion Channels Enable Patterned Spike Firing through Activity-Dependent Modulation of Spike Probability

    PubMed Central

    Dudman, Joshua T.; Nolan, Matthew F.

    2009-01-01

    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. PMID:19214199

  2. Natural and synthetic modulators of SK (Kca2) potassium channels inhibit magnesium-dependent activity of the kinase-coupled cation channel TRPM7

    PubMed Central

    Chubanov, V; Mederos y Schnitzler, M; Meißner, M; Schäfer, S; Abstiens, K; Hofmann, T; Gudermann, T

    2012-01-01

    BACKGROUND AND PURPOSE Transient receptor potential cation channel subfamily M member 7 (TRPM7) is a bifunctional protein comprising a TRP ion channel segment linked to an α-type protein kinase domain. TRPM7 is essential for proliferation and cell growth. Up-regulation of TRPM7 function is involved in anoxic neuronal death, cardiac fibrosis and tumour cell proliferation. The goal of this work was to identify non-toxic inhibitors of the TRPM7 channel and to assess the effect of blocking endogenous TRPM7 currents on the phenotype of living cells. EXPERIMENTAL APPROACH We developed an aequorin bioluminescence-based assay of TRPM7 channel activity and performed a hypothesis-driven screen for inhibitors of the channel. The candidates identified were further assessed electrophysiologically and in cell biological experiments. KEY RESULTS TRPM7 currents were inhibited by modulators of small conductance Ca2+-activated K+ channels (KCa2.1–2.3; SK) channels, including the antimalarial plant alkaloid quinine, CyPPA, dequalinium, NS8593, SKA31 and UCL 1684. The most potent compound NS8593 (IC50 1.6 µM) specifically targeted TRPM7 as compared with other TRP channels, interfered with Mg2+-dependent regulation of TRPM7 channel and inhibited the motility of cultured cells. NS8593 exhibited full and reversible block of native TRPM7-like currents in HEK 293 cells, freshly isolated smooth muscle cells, primary podocytes and ventricular myocytes. CONCLUSIONS AND IMPLICATIONS This study reveals a tight overlap in the pharmacological profiles of TRPM7 and KCa2.1–2.3 channels. NS8593 acts as a negative gating modulator of TRPM7 and is well-suited to study functional features and cellular roles of endogenous TRPM7. PMID:22242975

  3. Quantifying the transition from fluvial- to wave-dominance for river deltas with multiple active channels

    NASA Astrophysics Data System (ADS)

    Nienhuis, J.; Ashton, A. D.; Giosan, L.

    2012-12-01

    The plan-view morphologies of fluvial- and wave-dominated deltas are clearly distinctive, but transitional forms are numerous. A quantitative, process-based description of this transition remains unexplored, particularly for river deltas with multiple active channels. Previous studies focused on general attributes of the fluvial and marine environment, such as the balance between wave energy and river discharge. Here, we propose that the transition between fluvial and wave dominance is directly related to the magnitude of the fluvial bedload flux to the nearshore region versus the alongshore sediment transport capacity of waves removing sediment away from the mouth. In the case of a single-channel delta, this balance can be computed for a given distribution of waves approaching shore. Fluvial dominance occurs when fluvial sediment input exceeds the wave-sustained maximum alongshore sediment transport for all potential shoreline orientations both up- and downdrift of the river mouth. However, deltaic channels have the tendency to bifurcate with increasing fluvial strength. Initial bifurcation splits the fluvial sediment flux among individual channels, while the potential sediment transport by waves remains constant for both river mouths. At higher bifurcation orders, multiple channels interact with each other alongshore, a situation more complicated than the single channel case and one that cannot be simple addressed analytically. We apply a model of plan-view shoreline evolution to simulate the evolution of a deltaic environment with multiple active channels. A highly simplified fluvial domain is represented by deposition of sediment where channels meet the coast. We investigate two scenarios of fluvial delivery. The first scenario deposits fluvial sediment alongshore on a self-similar predefined network of channels. We analyze the effects of different network geometrical parameters, such as bifurcation length, bifurcation angle, and sediment partitioning. In the

  4. Positive affect modulates activity in the visual cortex to images of high calorie foods.

    PubMed

    Killgore, William D S; Yurgelun-Todd, Deborah A

    2007-05-01

    Activity within the visual cortex can be influenced by the emotional salience of a stimulus, but it is not clear whether such cortical activity is modulated by the affective status of the individual. This study used functional magnetic resonance imaging (fMRI) to examine the relationship between affect ratings on the Positive and Negative Affect Schedule and activity within the occipital cortex of 13 normal-weight women while viewing images of high calorie and low calorie foods. Regression analyses revealed that when participants viewed high calorie foods, Positive Affect correlated significantly with activity within the lingual gyrus and calcarine cortex, whereas Negative Affect was unrelated to visual cortex activity. In contrast, during presentations of low calorie foods, affect ratings, regardless of valence, were unrelated to occipital cortex activity. These findings suggest a mechanism whereby positive affective state may affect the early stages of sensory processing, possibly influencing subsequent perceptual experience of a stimulus. PMID:17464782

  5. Affect and Subsequent Physical Activity: An Ambulatory Assessment Study Examining the Affect-Activity Association in a Real-Life Context.

    PubMed

    Niermann, Christina Y N; Herrmann, Christian; von Haaren, Birte; van Kann, Dave; Woll, Alexander

    2016-01-01

    Traditionally, cognitive, motivational, and volitional determinants have been used to explain and predict health behaviors such as physical activity. Recently, the role of affect in influencing and regulating health behaviors received more attention. Affects as internal cues may automatically activate unconscious processes of behavior regulation. The aim of our study was to examine the association between affect and physical activity in daily life. In addition, we studied the influence of the habit of being physically active on this relationship. An ambulatory assessment study in 89 persons (33.7% male, 25 to 65 years, M = 45.2, SD = 8.1) was conducted. Affect was assessed in the afternoon on 5 weekdays using smartphones. Physical activity was measured continuously objectively using accelerometers and subjectively using smartphones in the evening. Habit strength was assessed at the beginning of the diary period. The outcomes were objectively and subjectively measured moderate-to-vigorous physical activity (MVPA) performed after work. Multilevel regression models were used to analyze the association between affect and after work MVPA. In addition, the cross-level interaction of habit strength and affect on after work MVPA was tested. Positive affect was positively related to objectively measured and self-reported after work MVPA: the greater the positive affect the more time persons subsequently spent on MVPA. An inverse relationship was found for negative affect: the greater the negative affect the less time persons spent on MVPA. The cross-level interaction effect was significant only for objectively measured MVPA. A strong habit seems to strengthen both the positive influence of positive affect and the negative influence of negative affect. The results of this study confirm previous results and indicate that affect plays an important role for the regulation of physical activity behavior in daily life. The results for positive affect were consistent. However, in

  6. Affect and Subsequent Physical Activity: An Ambulatory Assessment Study Examining the Affect-Activity Association in a Real-Life Context

    PubMed Central

    Niermann, Christina Y. N.; Herrmann, Christian; von Haaren, Birte; van Kann, Dave; Woll, Alexander

    2016-01-01

    Traditionally, cognitive, motivational, and volitional determinants have been used to explain and predict health behaviors such as physical activity. Recently, the role of affect in influencing and regulating health behaviors received more attention. Affects as internal cues may automatically activate unconscious processes of behavior regulation. The aim of our study was to examine the association between affect and physical activity in daily life. In addition, we studied the influence of the habit of being physically active on this relationship. An ambulatory assessment study in 89 persons (33.7% male, 25 to 65 years, M = 45.2, SD = 8.1) was conducted. Affect was assessed in the afternoon on 5 weekdays using smartphones. Physical activity was measured continuously objectively using accelerometers and subjectively using smartphones in the evening. Habit strength was assessed at the beginning of the diary period. The outcomes were objectively and subjectively measured moderate-to-vigorous physical activity (MVPA) performed after work. Multilevel regression models were used to analyze the association between affect and after work MVPA. In addition, the cross-level interaction of habit strength and affect on after work MVPA was tested. Positive affect was positively related to objectively measured and self-reported after work MVPA: the greater the positive affect the more time persons subsequently spent on MVPA. An inverse relationship was found for negative affect: the greater the negative affect the less time persons spent on MVPA. The cross-level interaction effect was significant only for objectively measured MVPA. A strong habit seems to strengthen both the positive influence of positive affect and the negative influence of negative affect. The results of this study confirm previous results and indicate that affect plays an important role for the regulation of physical activity behavior in daily life. The results for positive affect were consistent. However, in

  7. Calcium-activated chloride channels in the apical region of mouse vomeronasal sensory neurons.

    PubMed

    Dibattista, Michele; Amjad, Asma; Maurya, Devendra Kumar; Sagheddu, Claudia; Montani, Giorgia; Tirindelli, Roberto; Menini, Anna

    2012-07-01

    The rodent vomeronasal organ plays a crucial role in several social behaviors. Detection of pheromones or other emitted signaling molecules occurs in the dendritic microvilli of vomeronasal sensory neurons, where the binding of molecules to vomeronasal receptors leads to the influx of sodium and calcium ions mainly through the transient receptor potential canonical 2 (TRPC2) channel. To investigate the physiological role played by the increase in intracellular calcium concentration in the apical region of these neurons, we produced localized, rapid, and reproducible increases in calcium concentration with flash photolysis of caged calcium and measured calcium-activated currents with the whole cell voltage-clamp technique. On average, a large inward calcium-activated current of -261 pA was measured at -50 mV, rising with a time constant of 13 ms. Ion substitution experiments showed that this current is anion selective. Moreover, the chloride channel blockers niflumic acid and 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid partially inhibited the calcium-activated current. These results directly demonstrate that a large chloride current can be activated by calcium in the apical region of mouse vomeronasal sensory neurons. Furthermore, we showed by immunohistochemistry that the calcium-activated chloride channels TMEM16A/anoctamin1 and TMEM16B/anoctamin2 are present in the apical layer of the vomeronasal epithelium, where they largely colocalize with the TRPC2 transduction channel. Immunocytochemistry on isolated vomeronasal sensory neurons showed that TMEM16A and TMEM16B coexpress in the neuronal microvilli. Therefore, we conclude that microvilli of mouse vomeronasal sensory neurons have a high density of calcium-activated chloride channels that may play an important role in vomeronasal transduction. PMID:22732308

  8. Hyperpolarization-activated ion channels as targets for nitric oxide signalling in deep cerebellar nuclei

    PubMed Central

    Wilson, Gary W; Garthwaite, John

    2010-01-01

    Most biological effects of nitric oxide (NO) in the brain are mediated by guanylyl cyclase-coupled NO receptors, whose activation results in increased intracellular cGMP levels. Apart from protein kinase activation little is known about subsequent cGMP signal transduction. In optic nerve axons, hyperpolarization-activated cyclic nucleotide-modulated cation (HCN) channels, which bind cGMP or cAMP directly, were recently suggested to be a target. The aim here was to test this possibility more directly. Neurones of the rat deep cerebellar nuclei were selected for this purpose, their suitability being attested by immunocytochemistry showing that the principal neurones expressed guanylyl cyclase protein and that NO synthase-containing fibres were abundant in the neuropil. Using whole-cell voltage-clamp recording, HCN channels in the neurones were activated in response to isoprenaline and exogenous cAMP but only occasionally did they respond to NO, although exogenous cGMP was routinely effective. With the less invasive sharp microelectrode recording technique, however, exogenous NO modulated the channels reproducibly, as measured by the size of the HCN channel-mediated voltage sag following hyperpolarization. Moreover, NO also blunted the subsequent rebound depolarizing potentials, consistent with it increasing the hyperpolarization-activated current. Optimizing the whole-cell solution to improve the functioning of NO-activated guanylyl cyclase failed to restore NO sensitivity. Minimizing cellular dialysis by using the perforated-patch technique, however, was successful. The results provide evidence that HCN channels are potential downstream mediators of NO signalling in deep cerebellar nuclei neurones and suggest that the more general importance of this transduction pathway may have been overlooked previously because of unsuitable recording methods. PMID:20529121

  9. Basolateral K channel activated by carbachol in the epithelial cell line T84.

    PubMed

    Tabcharani, J A; Harris, R A; Boucher, A; Eng, J W; Hanrahan, J W

    1994-11-01

    Cholinergic stimulation of chloride secretion involves the activation of a basolateral membrane potassium conductance, which maintains the electrical gradient favoring apical Cl efflux and allows K to recycle at the basolateral membrane. We have used transepithelial short-circuit current (Isc), fluorescence imaging, and patch clamp studies to identify and characterize the K channel that mediates this response in T84 cells. Carbachol had little effect on Isc when added alone but produced large, transient currents if added to monolayers prestimulated with cAMP. cAMP also enhanced the subsequent Isc response to calcium ionophores. Carbachol (100 microM) transiently elevated intracellular free calcium ([Ca2+]i) by approximately 3-fold in confluent cells cultured on glass coverslips with a time course resembling the Isc response of confluent monolayers that had been grown on porous supports. In parallel patch clamp experiments, carbachol activated an inwardly rectifying potassium channel on the basolateral aspect of polarized monolayers which had been dissected from porous culture supports. The same channel was transiently activated on the surface of subconfluent monolayers during stimulation by carbachol. Activation was more prolonged when cells were exposed to calcium ionophores. The conductance of the inward rectifier in cell-attached patches was 55 pS near the resting membrane potential (-54 mV) with pipette solution containing 150 mM KCl (37 degrees C). This rectification persisted when patches were bathed in symmetrical 150 mM KCl solutions. The selectivity sequence was 1 K > 0.88 Rb > 0.18 Na > Cs based on permeability ratios under bi-ionic conditions. The channel exhibited fast block by external sodium ions, was weakly inhibited by external TEA, was relatively insensitive to charybdotoxin, kaliotoxin, 4-aminopyridine and quinidine, and was unaffected by external 10 mM barium. It is referred to as the KBIC channel based on its most distinctive properties (Ba

  10. Activation of the melastatin-related cation channel TRPM3 by D-erythro-sphingosine [corrected].

    PubMed

    Grimm, Christian; Kraft, Robert; Schultz, Günter; Harteneck, Christian

    2005-03-01

    TRPM3, a member of the melastatin-like transient receptor potential channel subfamily (TRPM), is predominantly expressed in human kidney and brain. TRPM3 mediates spontaneous Ca2+ entry and nonselective cation currents in transiently transfected human embryonic kidney 293 cells. Using measurements with the Ca2+-sensitive fluorescent dye fura-2 and the whole-cell patch-clamp technique, we found that D-erythro-sphingosine, a metabolite arising during the de novo synthesis of cellular sphingolipids, activated TRPM3. Other transient receptor potential (TRP) channels tested [classic or canonical TRP (TRPC3, TRPC4, TRPC5), vanilloid-like TRP (TRPV4, TRPV5, TRPV6), and melastatin-like TRP (TRPM2)] did not significantly respond to application of sphingosine. Sphingosine-induced TRPM3 activation was not mediated by inhibition of protein kinase C, depletion of intracellular Ca2+ stores, and intracellular conversion of sphingosine to sphingosine-1-phosphate. Although sphingosine-1-phosphate and ceramides had no effect, two structural analogs of sphingosine, dihydro-D-erythro-sphingosine and N,N-dimethyl-D-erythro-sphingosine, also activated TRPM3. Sphingolipids, including sphingosine, are known to have inhibitory effects on a variety of ion channels. Thus, TRPM3 is the first ion channel activated by sphingolipids. PMID:15550678

  11. Emodin augments calcium activated chloride channel in colonic smooth muscle cells by Gi/Go protein.

    PubMed

    Xu, Long; Ting-Lou; Lv, Nonghua; Zhu, Xuan; Chen, Youxiang; Yang, Jing

    2009-08-01

    Emodin is a natural anthraquinone in rhubarb. It has been identified as a prokinetic drug for gastrointestinal motility in Chinese traditional medicine. Emodin contracts smooth muscle by increasing the concentration of intracellular Ca(2+). In many smooth muscles, increasing intracellular Ca(2+) activates Ca(2+)-activated Cl(-) channels (ClCA). The study was aimed to investigate the effects of emodin on ClCA channels in colonic smooth muscle. 4 channel physiology signal acquire system was used to measure isometric contraction of smooth muscle strips. ClCA currents were recorded by EPC10 with perforated whole cell model. Emodin contracted strips and cells in colonic smooth muscle and augmented ClCA currents. Niflumic acid (NFA) and 4', 4'-diisothiostilbene-2, 2-disulfonic acid (DIDS) blocked the effects. Gi/Go protein inhibits protein kinase A (PKA) and protein kinase C (PKC), and PKA and PKC reduced ClCA currents. Pertussis toxin (PTX, a special inhibitor of Gi/Go protein), 8-bromoadenosine 38, 58-cyclic monophosphate (8-BrcAMP, a membrane-permeant protein kinase A activator) and Phorbol-12-myristate-13-acetate (PMA, a membrane-permeant protein kinase C activator) inhibited the effects on ClCA currents significantly. Our findings suggest that emodin augments ClCA channels to contract smooth muscle in colon, and the effect is induced mostly by enhancement of membrane Gi/Go protein signal transducer pathway. PMID:19409890

  12. Functional insights into modulation of BKCa channel activity to alter myometrial contractility

    PubMed Central

    Lorca, Ramón A.; Prabagaran, Monali; England, Sarah K.

    2014-01-01

    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. PMID:25132821

  13. Synthesis and characterization of a Redox-active artificial ion channel.

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The synthesis and characterization of an artificial ion channel containing both fluorescent and redox-active centers is described. fluorescence spectroscopy was used to study qualitative and quantitative aspects of the coordination of alkali metal cations and black lipid membrane studies were used ...

  14. Ethylene activates a plasma membrane Ca(2+)-permeable channel in tobacco suspension cells.

    PubMed

    Zhao, Min-Gui; Tian, Qiu-Ying; Zhang, Wen-Hao

    2007-01-01

    Here, the effects of the ethylene-releasing compound, ethephon, and the ethylene precursor, 1-aminocyclopropane-1-carboxylic acid (ACC), on ionic currents across plasma membranes and on the cytosolic Ca(2+) activity ([Ca(2+)](c)) of tobacco (Nicotiana tabacum) suspension cells were characterized using a patch-clamp technique and confocal laser scanning microscopy. Exposure of tobacco protoplasts to ethephon and ACC led to activation of a plasma membrane cation channel that was permeable to Ba(2+), Mg(2+) and Ca(2+), and inhibited by La(3+), Gd(3+) and Al(3+). The ethephon- and ACC-induced Ca(2+)-permeable channel was abolished by the antagonist of ethylene perception (1-metycyclopropene) and by the inhibitor of ACC synthase (aminovinylglycin), indicating that activation of the Ca(2+)-permeable channels results from ethylene. Ethephon elicited an increase in the [Ca(2+)](c) of tobacco suspension cells, as visualized by the Ca(2+)-sensitive probe Fluo-3 and confocal microscopy. The ethephon-induced elevation of [Ca(2+)](c) was markedly inhibited by Gd(3+) and BAPTA, suggesting that an influx of Ca(2+) underlies the elevation of [Ca(2+)](c). These results indicate that an elevation of [Ca(2+)](c), resulting from activation of the plasma membrane Ca(2+)-permeable channels by ethylene, is an essential component in ethylene signaling in plants. PMID:17447907

  15. Tuning voltage-gated channel activity and cellular excitability with a sphingomyelinase

    PubMed Central

    Combs, David J.; Shin, Hyeon-Gyu; Xu, Yanping; Ramu, Yajamana

    2013-01-01

    Voltage-gated ion channels generate action potentials in excitable cells and help set the resting membrane potential in nonexcitable cells like lymphocytes. It has been difficult to investigate what kinds of phospholipids interact with these membrane proteins in their native environments and what functional impacts such interactions create. This problem might be circumvented if we could modify specific lipid types in situ. Using certain voltage-gated K+ (KV) channels heterologously expressed in Xenopus laevis oocytes as a model, our group has shown previously that sphingomyelinase (SMase) D may serve this purpose. SMase D is known to remove the choline group from sphingomyelin, a phospholipid primarily present in the outer leaflet of plasma membranes. This SMase D action lowers the energy required for voltage sensors of a KV channel to enter the activated state, causing a hyperpolarizing shift of the Q-V and G-V curves and thus activating them at more hyperpolarized potentials. Here, we find that this SMase D effect vanishes after removing most of the voltage-sensor paddle sequence, a finding supporting the notion that SMase D modification of sphingomyelin molecules alters these lipids’ interactions with voltage sensors. Then, using SMase D to probe lipid–channel interactions, we find that SMase D not only similarly stimulates voltage-gated Na+ (NaV) and Ca2+ channels but also markedly slows NaV channel inactivation. However, the latter effect is not observed in tested mammalian cells, an observation highlighting the profound impact of the membrane environment on channel function. Finally, we directly demonstrate that SMase D stimulates both native KV1.3 in nonexcitable human T lymphocytes at their typical resting membrane potential and native NaV channels in excitable cells, such that it shifts the action potential threshold in the hyperpolarized direction. These proof-of-concept studies illustrate that the voltage-gated channel activity in both excitable and

  16. Novel spider toxin slows down the activation kinetics of P-type Ca2+ channels in Purkinje neurons of rat.

    PubMed

    Fisyunov, Alexander; Pluzhnikov, Kirill; Molyavka, Anton; Grishin, Eugene; Lozovaya, Natalia; Krishtal, Oleg

    2005-02-01

    We have identified a novel polypeptide toxin (Lsp-1) from the venom of the spider Lycosa (LS). Its effect has been examined on the P-type calcium channels in Purkinje neurons, using whole-cell patch-clamp. This toxin (at saturating concentration 7 nM) produces prominent (four-fold) deceleration of the activation kinetics and partial (71+/-6%) decrease of the amplitude of P-current without affecting either deactivation or inactivation kinetics. These effects are not use-dependent. They are partially reversible within a minute upon the wash-out of the toxin. Intracellular perfusion of Purkinje neurons with 100 microM of GDP or 2 microM of GTPgammaS, as well as strong depolarising pre-pulses (+100 mV), do not eliminate the action of Lsp-1 on P-channels indicating that down-modulation via guanine nucleotide-binding proteins (G-proteins) is not involved in the observed phenomenon. In view of extremely high functional significance of P-channels, the toxin can be suggested as a useful pharmacological tool. PMID:15590128

  17. Network-scale dynamics of sediment mixtures: how do tectonics affect surface bed texture and channel slope?

    NASA Astrophysics Data System (ADS)

    Gasparini, N. M.; Bras, R. L.; Tucker, G. E.

    2003-04-01

    An alluvial channel's slope and bed texture are intimately linked. Along with fluvial discharge, these variables are the key players in setting alluvial transport rates. We know that both channel slope and mean grain size usually decrease downstream, but how sensitive are these variables to tectonic changes? Are basin concavity and downstream fining drastically disrupted during transitions from one tectonic regime to another? We explore these questions using the CHILD numerical landscape evolution model to generate alluvial networks composed of a sand and gravel mixture. The steady-state and transient patterns of both channel slope and sediment texture are investigated. The steady-state patterns in slope and sediment texture are verified independently by solving the erosion equations under equilibrium conditions, i.e. the case when the erosion rate is equal to the uplift rate across the entire landscape. The inclusion of surface texture as a free parameter (as opposed to just channel slope) leads to some surprising results. In all cases, an increase in uplift rate results in channel beds which are finer at equilibrium (for a given drainage area). Higher uplif