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Sample records for cation channels inhibit

  1. Aluminium and hydrogen ions inhibit a mechanosensory calcium-selective cation channel

    Science.gov (United States)

    Ding, J. P.; Pickard, B. G.

    1993-01-01

    The tension-dependent activity of mechanosensory calcium-selective cation channels in excised plasmalemmal patches from onion bulb scale epidermis is modulated by pH in the physiologically meaningful range between 4.5 and 7.2. It is rapidly lowered by lowering pH and rapidly raised by raising pH. Channel activity is effectively inhibited by low levels of aluminium ions and activity can be partially restored by washing for a few minutes. We suggest that under normal conditions the sensitivity of the mechanosensory channels to pH of the wall free space plays important roles in regulation of plant activities such as growth. We further suggest that, when levels of acid and aluminium ions in the soil solution are high, they might inhibit similar sensory channels in cells of the root tip, thus contributing critically to the acid soil syndrome.

  2. Englerin A Agonizes the TRPC4/C5 Cation Channels to Inhibit Tumor Cell Line Proliferation.

    Directory of Open Access Journals (Sweden)

    Cheryl Carson

    Full Text Available Englerin A is a structurally unique natural product reported to selectively inhibit growth of renal cell carcinoma cell lines. A large scale phenotypic cell profiling experiment (CLiP of englerin A on ¬over 500 well characterized cancer cell lines showed that englerin A inhibits growth of a subset of tumor cell lines from many lineages, not just renal cell carcinomas. Expression of the TRPC4 cation channel was the cell line feature that best correlated with sensitivity to englerin A, suggesting the hypothesis that TRPC4 is the efficacy target for englerin A. Genetic experiments demonstrate that TRPC4 expression is both necessary and sufficient for englerin A induced growth inhibition. Englerin A induces calcium influx and membrane depolarization in cells expressing high levels of TRPC4 or its close ortholog TRPC5. Electrophysiology experiments confirmed that englerin A is a TRPC4 agonist. Both the englerin A induced current and the englerin A induced growth inhibition can be blocked by the TRPC4/C5 inhibitor ML204. These experiments confirm that activation of TRPC4/C5 channels inhibits tumor cell line proliferation and confirms the TRPC4 target hypothesis generated by the cell line profiling. In selectivity assays englerin A weakly inhibits TRPA1, TRPV3/V4, and TRPM8 which suggests that englerin A may bind a common feature of TRP ion channels. In vivo experiments show that englerin A is lethal in rodents near doses needed to activate the TRPC4 channel. This toxicity suggests that englerin A itself is probably unsuitable for further drug development. However, since englerin A can be synthesized in the laboratory, it may be a useful chemical starting point to identify novel modulators of other TRP family channels.

  3. Cell volume-regulated cation channels.

    Science.gov (United States)

    Wehner, Frank

    2006-01-01

    Considering the enormous turnover rates of ion channels when compared to carriers it is quite obvious that channel-mediated ion transport may serve as a rapid and efficient mechanism of cell volume regulation. Whenever studied in a quantitative fashion the hypertonic activation of non-selective cation channels is found to be the main mechanism of regulatory volume increase (RVI). Some channels are inhibited by amiloride (and may be related to the ENaC), others are blocked by Gd(3) and flufenamate (and possibly linked to the group of transient receptor potential (TRP) channels). Nevertheless, the actual architecture of hypertonicity-induced cation channels remains to be defined. In some preparations, hypertonic stress decreases K(+) channel activity so reducing the continuous K(+) leak out of the cell; this is equivalent to a net gain of cell osmolytes facilitating RVI. The hypotonic activation of K(+) selective channels appears to be one of the most common principles of regulatory volume decrease (RVD) and, in most instances, the actual channels involved could be identified on the molecular level. These are BKCa (or maxi K(+)) channels, IK(Ca) and SK(Ca) channels (of intermediate and small conductance, respectively), the group of voltage-gated (Kv) channels including their Beta (or Kv ancilliary) subunits, two-pore K(2P) channels, as well as inwardly rectifying K(+) (Kir) channels (also contributing to K(ATP) channels). In some cells, hypotonicity activates non-selective cation channels. This is surprising, at first sight, because of the inside negative membrane voltage and the sum of driving forces for Na(+) and K(+) diffusion across the cell membrane rather favouring net cation uptake. Some of these channels, however, exhibit a P(K)/P(Na) significantly higher than 1, whereas others are Ca(++) permeable linking hypotonic stress to the activation of Ca(++) dependent ion channels. In particular, the latter holds for the group of TRPs which are specialised in the

  4. Inhibition of the cation channel TRPV4 improves bladder function in mice and rats with cyclophosphamide-induced cystitis.

    Science.gov (United States)

    Everaerts, Wouter; Zhen, Xiaoguang; Ghosh, Debapriya; Vriens, Joris; Gevaert, Thomas; Gilbert, James P; Hayward, Neil J; McNamara, Colleen R; Xue, Fenqin; Moran, Magdalene M; Strassmaier, Timothy; Uykal, Eda; Owsianik, Grzegorz; Vennekens, Rudi; De Ridder, Dirk; Nilius, Bernd; Fanger, Christopher M; Voets, Thomas

    2010-11-02

    Reduced functional bladder capacity and concomitant increased micturition frequency (pollakisuria) are common lower urinary tract symptoms associated with conditions such as cystitis, prostatic hyperplasia, neurological disease, and overactive bladder syndrome. These symptoms can profoundly affect the quality of life of afflicted individuals, but available pharmacological treatments are often unsatisfactory. Recent work has demonstrated that the cation channel TRPV4 is highly expressed in urothelial cells and plays a role in sensing the normal filling state of the bladder. In this article, we show that the development of cystitis-induced bladder dysfunction is strongly impaired in Trpv4(-/-) mice. Moreover, we describe HC-067047, a previously uncharacterized, potent, and selective TRPV4 antagonist that increases functional bladder capacity and reduces micturition frequency in WT mice and rats with cystitis. HC-067047 did not affect bladder function in Trpv4(-/-) mice, demonstrating that its in vivo effects are on target. These results indicate that TRPV4 antagonists may provide a promising means of treating bladder dysfunction.

  5. Heavy metal cations permeate the TRPV6 epithelial cation channel.

    Science.gov (United States)

    Kovacs, Gergely; Danko, Tamas; Bergeron, Marc J; Balazs, Bernadett; Suzuki, Yoshiro; Zsembery, Akos; Hediger, Matthias A

    2011-01-01

    TRPV6 belongs to the vanilloid family of the transient receptor potential channel (TRP) superfamily. This calcium-selective channel is highly expressed in the duodenum and the placenta, being responsible for calcium absorption in the body and fetus. Previous observations have suggested that TRPV6 is not only permeable to calcium but also to other divalent cations in epithelial tissues. In this study, we tested whether TRPV6 is indeed also permeable to cations such as zinc and cadmium. We found that the basal intracellular calcium concentration was higher in HEK293 cells transfected with hTRPV6 than in non-transfected cells, and that this difference almost disappeared in nominally calcium-free solution. Live cell imaging experiments with Fura-2 and NewPort Green DCF showed that overexpression of human TRPV6 increased the permeability for Ca(2+), Ba(2+), Sr(2+), Mn(2+), Zn(2+), Cd(2+), and interestingly also for La(3+) and Gd(3+). These results were confirmed using the patch clamp technique. (45)Ca uptake experiments showed that cadmium, lanthanum and gadolinium were also highly efficient inhibitors of TRPV6-mediated calcium influx at higher micromolar concentrations. Our results suggest that TRPV6 is not only involved in calcium transport but also in the transport of other divalent cations, including heavy metal ions, which may have toxicological implications.

  6. P/Q-type and T-type calcium channels, but not type 3 transient receptor potential cation channels, are involved in inhibition of dendritic growth after chronic metabotropic glutamate receptor type 1 and protein kinase C activation in cerebellar Purkinje cells.

    Science.gov (United States)

    Gugger, Olivia S; Hartmann, Jana; Birnbaumer, Lutz; Kapfhammer, Josef P

    2012-01-01

    The development of a neuronal dendritic tree is modulated both by signals from afferent fibers and by an intrinsic program. We have previously shown that chronic activation of either type 1 metabotropic glutamate receptors (mGluR1s) or protein kinase C (PKC) in organotypic cerebellar slice cultures of mice and rats severely inhibits the growth and development of the Purkinje cell dendritic tree. The signaling events linking receptor activation to the regulation of dendritic growth remain largely unknown. We have studied whether channels allowing the entry of Ca(2+) into Purkinje cells, in particular the type 3 transient receptor potential cation channels (TRPC3s), P/Q-type Ca(2+) channels, and T-type Ca(2+) channels, might be involved in signaling after mGluR1 or PKC stimulation. We show that the inhibition of dendritic growth seen after mGluR1 or PKC stimulation is partially rescued by pharmacological blockade of P/Q-type and T-type Ca(2+) channels, indicating that activation of these channels mediating Ca(2+) influx contributes to the inhibition of dendritic growth. In contrast, the absence of Ca(2+) -permeable TRPC3s in TRPC3-deficient mice or pharmacological blockade had no effect on mGluR1-mediated and PKC-mediated inhibition of Purkinje cell dendritic growth. Similarly, blockade of Ca(2+) influx through glutamate receptor δ2 or R-type Ca(2+) channels or inhibition of release from intracellular stores did not influence mGluR1-mediated and PKC-mediated inhibition of Purkinje cell dendritic growth. These findings suggest that both T-type and P/Q-type Ca(2+) channels, but not TRPC3 or other Ca(2+) -permeable channels, are involved in mGluR1 and PKC signaling leading to the inhibition of dendritic growth in cerebellar Purkinje cells.

  7. Cation Selectivity in Biological Cation Channels Using Experimental Structural Information and Statistical Mechanical Simulation.

    Science.gov (United States)

    Finnerty, Justin John; Peyser, Alexander; Carloni, Paolo

    2015-01-01

    Cation selective channels constitute the gate for ion currents through the cell membrane. Here we present an improved statistical mechanical model based on atomistic structural information, cation hydration state and without tuned parameters that reproduces the selectivity of biological Na+ and Ca2+ ion channels. The importance of the inclusion of step-wise cation hydration in these results confirms the essential role partial dehydration plays in the bacterial Na+ channels. The model, proven reliable against experimental data, could be straightforwardly used for designing Na+ and Ca2+ selective nanopores.

  8. Pharmacology of the human cell voltage-dependent cation channel. Part II: inactivation and blocking

    DEFF Research Database (Denmark)

    Bennekou, Poul; Barksmann, Trine L.; Kristensen, Berit I.

    2004-01-01

    Human red cells; Nonselective voltage-dependent cation channel; NSVDC channel; Thiol group reagents......Human red cells; Nonselective voltage-dependent cation channel; NSVDC channel; Thiol group reagents...

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

    DEFF Research Database (Denmark)

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

    2004-01-01

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

  10. Modulation of fast synaptic transmission by presynaptic ligand-gated cation channels.

    Science.gov (United States)

    Khakh, B S; Henderson, G

    2000-07-01

    There is now considerable evidence demonstrating that ligand-gated cation channels (i.e., P2X, nicotinic, kainate, NMDA, AMPA and 5-HT(3) receptors), in addition to mediating fast excitatory neurotransmission, may be located presynaptically on nerve terminals in the peripheral and central nervous systems where they function to modulate neurotransmitter release. This modulation can be facilitation, inhibition or both. In this article, we first outline the multiple mechanisms by which activation of presynaptic ligand-gated cation channels can modulate spontaneous and evoked neurotransmitter release, before reviewing in detail published electrophysiological studies of presynaptic P2X, nicotinic, kainate, NMDA, AMPA and 5-HT(3) receptors.

  11. Cloning and first functional characterization of a plant cyclic nucleotide-gated cation channel

    Energy Technology Data Exchange (ETDEWEB)

    Leng, Q.; Mercier, R.W.; Yao, W.; Berkowitz, G.A.

    1999-11-01

    Cyclic nucleotide-gated (cng) non-selective cation channels have been cloned from a number of animal systems. These channels are characterized by direct gating upon cAMO or cGMO binding to the intracellular portion of the channel protein, which leads to an increase in channel conductance. Animal cng channels are involved in signal transduction systems; they translate stimulus-induced changes in cytosolic cyclic nucleotide into altered cell membrane potential and/or cation flux as part of a signal cascade pathway. Putative plant homologs of animal cng channels have been identified. However, functional characterization (i.e., demonstration of cyclic-nucleotide-dependent ion currents) of a plant cng channel has not yet been accomplished. The authors report the cloning and first functional characterization of a plant member of this family of ion channels. The Arabidopsis cDNA AtCNGC2 encodes a polypeptide with deduced homology to the {alpha}-subunit of animal channels, and facilitates cyclic nucleotide-dependent cation currents upon expression in a number of heterologous systems. AtCNGC2 expression in a yeast mutant lacking a low-affinity K{sup +} uptake system complements growth inhibition only when lipophilic nucleotides are present in the culture medium. Voltage clamp analysis indicates that Xenopus lawvis oocytes injected with AtCNGC2 cRNA demonstrate cyclic-nucleotide-dependent, inward-rectifying K{sup +} currents. Human embryonic kidney cells (HEK293) transfected with AtCNGC2 cDNA demonstrate increased permeability to Ca{sup 2+} only in the presence of lipophilic cyclic nucleotides. The evidence presented here supports the functional classification of AtCNGC2 as a cyclic-nucleotide-gated cation channel, and presents the first direct evidence identifying a plant member of this ion channel family.

  12. Divalent cation shrinks DNA but inhibits its compaction with trivalent cation

    Science.gov (United States)

    Tongu, Chika; Kenmotsu, Takahiro; Yoshikawa, Yuko; Zinchenko, Anatoly; Chen, Ning; Yoshikawa, Kenichi

    2016-05-01

    Our observation reveals the effects of divalent and trivalent cations on the higher-order structure of giant DNA (T4 DNA 166 kbp) by fluorescence microscopy. It was found that divalent cations, Mg(2+) and Ca(2+), inhibit DNA compaction induced by a trivalent cation, spermidine (SPD(3+)). On the other hand, in the absence of SPD(3+), divalent cations cause the shrinkage of DNA. As the control experiment, we have confirmed the minimum effect of monovalent cation, Na(+) on the DNA higher-order structure. We interpret the competition between 2+ and 3+ cations in terms of the change in the translational entropy of the counterions. For the compaction with SPD(3+), we consider the increase in translational entropy due to the ion-exchange of the intrinsic monovalent cations condensing on a highly charged polyelectrolyte, double-stranded DNA, by the 3+ cations. In contrast, the presence of 2+ cation decreases the gain of entropy contribution by the ion-exchange between monovalent and 3+ ions.

  13. Maitotoxin activates a nonselective cation channel and stimulates Ca2+ entry in MDCK renal epithelial cells.

    Science.gov (United States)

    Dietl, P; Völkl, H

    1994-02-01

    We examined the mechanisms of maitotoxin (MTX), a water-soluble polyether from the marine dinoflagellate Gambierdiscus toxicus, in stimulation of Ca2+ entry into Mardin-Darby canine kidney cells. In the presence of bath Ca2+, MTX (3 nM) caused an elevation of the intracellular calcium concentration ([Ca2+]i), which was partially inhibited by SK&F 96365 (25 microM) or La3+ (100 microM). A stimulation of Ca(2+)-dependent K+ channels in cell-attached membrane patches coincided with this rise in [Ca2+]i and was also partially inhibited by SK&F 96365. Before the rise in [Ca2+]i, a nonselective cation current (Ins), studied by the whole-cell patch-clamp technique, was irreversibly activated. Ins poorly discriminated between Na+, K+, and Cs+, was unaffected by replacement of Cl- with gluconate-, and was not voltage gated. MTX-induced Ins was partially blocked by La3+ ions (100 microM) but not by SK&F 96365 (25 microM) or nifedipine (10 microM). SK&F 96365 by itself induced a small but significant stimulation of Ins and a rise in [Ca2+]i. The activation of Ins by MTX was instantaneous and depended on the presence of extracellular Ca2+ ions. In the absence of other cations, the inward current of Ins was dependent on the bath Ca2+ concentration. Cell-attached and excised single-channel measurements revealed that MTX activated a SK&F 96365-insensitive, approximately 40-pS, nonselective cation channel from the outside. We conclude that the initial action of MTX is the stimulation of a nonselective cation channel, which requires the presence of extracellular Ca2+ ions. The subsequent rise in [Ca2+]i is at least in part caused by another, SK&F 96365-sensitive, Ca2+ entry pathway, which may be activated as a result of or independently of Ins.

  14. Stretch-activated cation channel from larval bullfrog skin

    DEFF Research Database (Denmark)

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

    2010-01-01

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

  15. Non-selective cation channels mediate chloroquine-induced relaxation in precontracted mouse airway smooth muscle.

    Directory of Open Access Journals (Sweden)

    Ting Zhang

    Full Text Available Bitter tastants can induce relaxation in precontracted airway smooth muscle by activating big-conductance potassium channels (BKs or by inactivating voltage-dependent L-type Ca2+ channels (VDLCCs. In this study, a new pathway for bitter tastant-induced relaxation was defined and investigated. We found nifedipine-insensitive and bitter tastant chloroquine-sensitive relaxation in epithelium-denuded mouse tracheal rings (TRs precontracted with acetylcholine (ACH. In the presence of nifedipine (10 µM, ACH induced cytosolic Ca2+ elevation and cell shortening in single airway smooth muscle cells (ASMCs, and these changes were inhibited by chloroquine. In TRs, ACH triggered a transient contraction under Ca2+-free conditions, and, following a restoration of Ca2+, a strong contraction occurred, which was inhibited by chloroquine. Moreover, the ACH-activated whole-cell and single channel currents of non-selective cation channels (NSCCs were blocked by chloroquine. Pyrazole 3 (Pyr3, an inhibitor of transient receptor potential C3 (TRPC3 channels, partially inhibited ACH-induced contraction, intracellular Ca2+ elevation, and NSCC currents. These results demonstrate that NSCCs play a role in bitter tastant-induced relaxation in precontracted airway smooth muscle.

  16. Transient receptor potential (TRP gene superfamily encoding cation channels

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    Pan Zan

    2011-01-01

    Full Text Available Abstract Transient receptor potential (TRP non-selective cation channels constitute a superfamily, which contains 28 different genes. In mammals, this superfamily is divided into six subfamilies based on differences in amino acid sequence homology between the different gene products. Proteins within a subfamily aggregate to form heteromeric or homomeric tetrameric configurations. These different groupings have very variable permeability ratios for calcium versus sodium ions. TRP expression is widely distributed in neuronal tissues, as well as a host of other tissues, including epithelial and endothelial cells. They are activated by environmental stresses that include tissue injury, changes in temperature, pH and osmolarity, as well as volatile chemicals, cytokines and plant compounds. Their activation induces, via intracellular calcium signalling, a host of responses, including stimulation of cell proliferation, migration, regulatory volume behaviour and the release of a host of cytokines. Their activation is greatly potentiated by phospholipase C (PLC activation mediated by coupled GTP-binding proteins and tyrosine receptors. In addition to their importance in maintaining tissue homeostasis, some of these responses may involve various underlying diseases. Given the wealth of literature describing the multiple roles of TRP in physiology in a very wide range of different mammalian tissues, this review limits itself to the literature describing the multiple roles of TRP channels in different ocular tissues. Accordingly, their importance to the corneal, trabecular meshwork, lens, ciliary muscle, retinal, microglial and retinal pigment epithelial physiology and pathology is reviewed.

  17. Nicotine activates the chemosensory cation channel TRPA1.

    Science.gov (United States)

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

    2009-10-01

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

  18. Epidermal growth factor regulation in adult rat alveolar type II cells of amiloride-sensitive cation channels.

    Science.gov (United States)

    Kemp, P J; Borok, Z; Kim, K J; Lubman, R L; Danto, S I; Crandall, E D

    1999-12-01

    Using the patch-clamp technique, we studied the effects of epidermal growth factor (EGF) on whole cell and single channel currents in adult rat alveolar epithelial type II cells in primary culture in the presence or absence of EGF for 48 h. In symmetrical sodium isethionate solutions, EGF exposure caused a significant increase in the type II cell whole cell conductance. Amiloride (10 microM) produced approximately 20-30% inhibition of the whole cell conductance in both the presence and absence of EGF, such that EGF caused the magnitude of the amiloride-sensitive component to more than double. Northern analysis showed that alpha-, beta- and gamma-subunits of rat epithelial Na(+) channel (rENaC) steady-state mRNA levels were all significantly decreased by EGF. At the single channel level, all active inside-out patches demonstrated only 25-pS channels that were amiloride sensitive and relatively nonselective for cations (P(Na(+))/P(K(+)) approximately 1.0:0.48). Although the biophysical characteristics (conductance, open-state probability, and selectivity) of the channels from EGF-treated and untreated cells were essentially identical, channel density was increased by EGF; the modal channel per patch was increased from 1 to 2. These findings indicate that EGF increases expression of nonselective, amiloride-sensitive cation channels in adult alveolar epithelial type II cells. The contribution of rENaC to the total EGF-dependent cation current under these conditions is quantitatively less important than that of the nonselective cation channels in these cells.

  19. The effect of external divalent cations on spontaneous non-selective cation channel currents in rabbit portal vein myocytes.

    Science.gov (United States)

    Albert, A P; Large, W A

    2001-10-15

    1. The effects of external divalent cations on spontaneous single non-selective cation channel currents were studied in outside-out patches from rabbit portal vein smooth muscle cells in K+-free conditions. 2. In an external medium containing 1.5 mM Ca2+ (Ca2+o) the majority of spontaneous channel currents had a unitary conductance of 23 pS, reversal potential (Vr) of +10 mV and a low open probability (Po) at negative patch potentials. Some channels opened to a lower conductance state of about 13 pS suggesting that the cation channels have two conductance states. Open time and burst duration distributions could both be described by two exponentials with time constants of about of 1 ms and 7 ms for open times and 3 ms and 16 ms for burst durations. 3. In 0 Ca2+o the majority of spontaneous cation channels had a unitary conductance of 13 pS and Vr was shifted to +4 mV. Moreover the longer open time and longer burst duration time constants were both reduced to approximately half the values in 1.5 mM Ca2+o. 4. Compared to 0 Ca2+o the single channel currents in 3 microM and 100 microM Ca2+o had a 5- to 6-fold increase in Po which was accompanied by increases in both open times and burst durations. In 3 microM and 100 microM Ca2+o the unitary conductance of the single channel currents was between 22 and 26 pS. 5. At positive membrane potentials the single channel currents had an increased Po compared to negative potentials which was associated with increased open times and burst durations but these values were similar in 3 microM, 100 microM and 1.5 mM Ca2+o. 6. In 1.5 mM Sr2+o and 1.5 mM Ba2+o channels opened to the higher conductance state of about 22-25 pS and had a 3- to 7-fold greater Po than in 0 Ca2+o. 7. In conclusion, external divalent cations have marked effects on the unitary conductance and kinetic behaviour of non-selective cation channels in rabbit portal vein smooth muscle cells.

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

    Science.gov (United States)

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

    2015-01-01

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

  1. The non-selective voltage-activated cation channel in the human red blood cell membrane: reconciliation between two conflicting reports and further characterisation

    DEFF Research Database (Denmark)

    Kaestner, Lars; Christophersen, Palle; Bernhardt, Ingolf;

    2000-01-01

    Erythrocyte; Patch-clamp; Non-specific; cation channel; Voltage dependence; Acetylcholin receptor......Erythrocyte; Patch-clamp; Non-specific; cation channel; Voltage dependence; Acetylcholin receptor...

  2. Inhibiting bacterial toxins by channel blockage.

    Science.gov (United States)

    Bezrukov, Sergey M; Nestorovich, Ekaterina M

    2016-03-01

    Emergent rational drug design techniques explore individual properties of target biomolecules, small and macromolecule drug candidates, and the physical forces governing their interactions. In this minireview, we focus on the single-molecule biophysical studies of channel-forming bacterial toxins that suggest new approaches for their inhibition. We discuss several examples of blockage of bacterial pore-forming and AB-type toxins by the tailor-made compounds. In the concluding remarks, the most effective rationally designed pore-blocking antitoxins are compared with the small-molecule inhibitors of ion-selective channels of neurophysiology.

  3. The Ca2+-activated cation channel TRPM4 is regulated by phosphatidylinositol 4,5-biphosphate.

    Science.gov (United States)

    Nilius, Bernd; Mahieu, Frank; Prenen, Jean; Janssens, Annelies; Owsianik, Grzegorz; Vennekens, Rudi; Voets, Thomas

    2006-02-08

    Transient receptor potential (TRP) channel, melastatin subfamily (TRPM)4 is a Ca2+-activated monovalent cation channel that depolarizes the plasma membrane and thereby modulates Ca2+ influx through Ca2+-permeable pathways. A typical feature of TRPM4 is its rapid desensitization to intracellular Ca2+ ([Ca2+]i). Here we show that phosphatidylinositol 4,5-biphosphate (PIP2) counteracts desensitization to [Ca2+]i in inside-out patches and rundown of TRPM4 currents in whole-cell patch-clamp experiments. PIP2 shifted the voltage dependence of TRPM4 activation towards negative potentials and increased the channel's Ca2+ sensitivity 100-fold. Conversely, activation of the phospholipase C (PLC)-coupled M1 muscarinic receptor or pharmacological depletion of cellular PIP2 potently inhibited currents through TRPM4. Neutralization of basic residues in a C-terminal pleckstrin homology (PH) domain accelerated TRPM4 current desensitization and strongly attenuated the effect of PIP2, whereas mutations to the C-terminal TRP box and TRP domain had no effect on the PIP2 sensitivity. Our data demonstrate that PIP2 is a strong positive modulator of TRPM4, and implicate the C-terminal PH domain in PIP2 action. PLC-mediated PIP2 breakdown may constitute a physiologically important brake on TRPM4 activity.

  4. Antiarrhythmic Mechanisms of SK Channel Inhibition in the Rat Atrium

    DEFF Research Database (Denmark)

    Skibsbye, Lasse; Wang, Xiaodong; Axelsen, Lene Nygaard

    2015-01-01

    remains unclear. OBJECTIVES: We speculated that together with a direct inhibition of repolarizing SK current, the previously observed depolarization of the atrial resting membrane potential (RMP) after SK channel inhibition reduces sodium channel availability thereby prolonging the effective refractory...

  5. Poorly selective cation channels in the apical membrane of A6 cells.

    Science.gov (United States)

    Van Driessche, W; De Smet, P; de Smedt, H

    1994-03-01

    This paper describes a Ca(2+)-blockable, poorly selective cation pathway in the apical membrane of A6 epithelia. This pathway has properties that resemble the cation-selective channels in the toad urinary bladder and frog skin. Transepithelial short circuit currents (Isc) and power density spectra (PDS) of the fluctuations in current were recorded. The basolateral surface of the tissues was exposed to Cl- or SO4(2-) solutions with Na+ as the major cation. Ca(2+)-blockable inward oriented currents and Lorentzian noise were recorded with isotonic (215 mosmol/kg) mucosal Cl- and hypotonic (144 mos-mol/kg serosal SO4(2-) solution with Na+, K+, Rb+ or Cs+ as the major mucosal cation. Experiments with mucosal K+ demonstrated that the cation-selective channel was markedly activated by serosal hypotonicity. Effects of an increased electrical driving force were excluded on the basis of the results obtained with microelectrode experiments and transepithelial voltage clamping. Cell volume expansion induced by isotonic replacements of serosal sucrose by glycerol or urea also activated the cation-selective pathway. Furthermore, the presence of Cl- in the mucosal solution was a prerequisite for a sustained response to hypotonicity or replacements of the organic compounds. Moreover, we found that the cation-selective channels are mainly expressed in the cells during the early period of epithelial growth.

  6. Evolutionary and structural perspectives of plant cyclic nucleotide-gated cation channels

    KAUST Repository

    Zelman, Alice K.

    2012-05-29

    Ligand-gated cation channels are a frequent component of signaling cascades in eukaryotes. Eukaryotes contain numerous diverse gene families encoding ion channels, some of which are shared and some of which are unique to particular kingdoms. Among the many different types are cyclic nucleotide-gated channels (CNGCs). CNGCs are cation channels with varying degrees of ion conduction selectivity. They are implicated in numerous signaling pathways and permit diffusion of divalent and monovalent cations, including Ca2+ and K+. CNGCs are present in both plant and animal cells, typically in the plasma membrane; recent studies have also documented their presence in prokaryotes. All eukaryote CNGC polypeptides have a cyclic nucleotide-binding domain and a calmodulin binding domain as well as a six transmembrane/one pore tertiary structure. This review summarizes existing knowledge about the functional domains present in these cation-conducting channels, and considers the evidence indicating that plant and animal CNGCs evolved separately. Additionally, an amino acid motif that is only found in the phosphate binding cassette and hinge regions of plant CNGCs, and is present in all experimentally confirmed CNGCs but no other channels was identified. This CNGC-specific amino acid motif provides an additional diagnostic tool to identify plant CNGCs, and can increase confidence in the annotation of open reading frames in newly sequenced genomes as putative CNGCs. Conversely, the absence of the motif in some plant sequences currently identified as probable CNGCs may suggest that they are misannotated or protein fragments. 2012 Zelman, Dawe, Gehring and Berkowitz.

  7. Role of TRPML and two-pore channels in endolysosomal cation homeostasis.

    Science.gov (United States)

    Grimm, Christian; Hassan, Sami; Wahl-Schott, Christian; Biel, Martin

    2012-08-01

    The transient receptor potential (TRP) channels TRPML1, TRPML2, and TRPML3 (also called mucolipins 1-3 or MCOLN1-3) are nonselective cation channels. Mutations in the Trpml1 gene cause mucolipidosis type IV in humans with clinical features including psychomotor retardation, corneal clouding, and retinal degeneration, whereas mutations in the Trpml3 gene cause deafness, circling behavior, and coat color dilution in mice. No disease-causing mutations are reported for the Trpml2 gene. Like TRPML channels, which are expressed in the endolysosomal pathway, two-pore channels (TPCs), namely TPC1, TPC2, and TPC3, are found in intracellular organelles, in particular in endosomes and lysosomes. Both TRPML channels and TPCs may function as calcium/cation release channels in endosomes, lysosomes, and lysosome-related organelles with TRPMLs being activated by phosphatidylinositol 3,5-bisphosphate and regulated by pH and TPCs being activated by nicotinic acid adenine dinucleotide phosphate in a calcium- and pH-dependent manner. They may also be involved in endolysosomal transport and fusion processes, e.g., as intracellular calcium sources. Currently, however, the exact physiological roles of TRPML channels and TPCs remain quite elusive, and whether TRPML channels are purely endolysosomal ion channels or whether they may also be functionally active at the plasma membrane in vivo remains to be determined.

  8. The antimalarial drug mefloquine inhibits cardiac inward rectifier K+ channels: evidence for interference in PIP2-channel interaction.

    Science.gov (United States)

    López-Izquierdo, Angélica; Ponce-Balbuena, Daniela; Moreno-Galindo, Eloy G; Aréchiga-Figueroa, Iván A; Rodríguez-Martínez, Martín; Ferrer, Tania; Rodríguez-Menchaca, Aldo A; Sánchez-Chapula, José A

    2011-04-01

    The antimalarial drug mefloquine was found to inhibit the KATP channel by an unknown mechanism. Because mefloquine is a Cationic amphiphilic drug and is known to insert into lipid bilayers, we postulate that mefloquine interferes with the interaction between PIP2 and Kir channels resulting in channel inhibition. We studied the inhibitory effects of mefloquine on Kir2.1, Kir2.3, Kir2.3(I213L), and Kir6.2/SUR2A channels expressed in HEK-293 cells, and on IK1 and IKATP from feline cardiac myocytes. The order of mefloquine inhibition was Kir6.2/SUR2A ≈ Kir2.3 (IC50 ≈ 2 μM) > Kir2.1 (IC50 > 30 μM). Similar results were obtained in cardiac myocytes. The Kir2.3(I213L) mutant, which enhances the strength of interaction with PIP2 (compared to WT), was significantly less sensitive (IC50 = 9 μM). In inside-out patches, continuous application of PIP2 strikingly prevented the mefloquine inhibition. Our results support the idea that mefloquine interferes with PIP2-Kir channels interactions.

  9. Similar cation channels mediate protection from cerebellar exitotoxicity by exercise and inheritance.

    Science.gov (United States)

    Ben-Ari, Shani; Ofek, Keren; Barbash, Shahar; Meiri, Hanoch; Kovalev, Eugenia; Greenberg, David Samuel; Soreq, Hermona; Shoham, Shai

    2012-03-01

    Exercise and inherited factors both affect recovery from stroke and head injury, but the underlying mechanisms and interconnections between them are yet unknown. Here, we report that similar cation channels mediate the protective effect of exercise and specific genetic background in a kainate injection model of cerebellar stroke. Microinjection to the cerebellum of the glutamatergic agonist, kainate, creates glutamatergic excito\\xE2\\x80\\x90toxicity characteristic of focal stroke, head injury or alcoholism. Inherited protection and prior exercise were both accompanied by higher cerebellar expression levels of the Kir6.1 ATP-dependent potassium channel in adjacent Bergmann glia, and voltage-gated KVbeta2 and cyclic nucleotide-gated cation HCN1 channels in basket cells. Sedentary FVB/N and exercised C57BL/6 mice both expressed higher levels of these cation channels compared to sedentary C57BL/6 mice, and were both found to be less sensitive to glutamate toxicity. Moreover, blocking ATP-dependent potassium channels with Glibenclamide enhanced kainate-induced cell death in cerebellar slices from the resilient sedentary FVB/N mice. Furthermore, exercise increased the number of acetylcholinesterase-positive fibres in the molecular layer, reduced cerebellar cytokine levels and suppressed serum acetylcholinesterase activity, suggesting anti-inflammatory protection by enhanced cholinergic signalling. Our findings demonstrate for the first time that routine exercise and specific genetic backgrounds confer protection from cerebellar glutamatergic damages by similar molecular mechanisms, including elevated expression of cation channels. In addition, our findings highlight the involvement of the cholinergic anti-inflammatory pathway in insult-inducible cerebellar processes. These mechanisms are likely to play similar roles in other brain regions and injuries as well, opening new venues for targeted research efforts.

  10. SOC and now also SIC: store-operated and store-inhibited channels.

    Science.gov (United States)

    Moreno, Claudia; Vaca, Luis

    2011-10-01

    There is a specialized form of calcium influx that involves a close communication between endoplasmic reticulum and the channels at the plasma membrane. In one side store depletion activates channels known as store-operated channels (SOC), which are responsible of the well-studied store-operated calcium entry (SOCE). SOC comprises two different types of channels. Orai, which is exclusively activated by store depletion being the channel responsible of the calcium release-activated calcium current, and transient receptor potential canonical channel, which in contrast, is activated by store depletion only under specific conditions and carries nonselective cationic currents. On the other hand, it has been recently shown that store depletion also inhibits calcium channels. The first member identified, of what we named as store-inhibited channels (SIC), is the L-type voltage-gated calcium channel. Stores control both SOC and SIC by means of the multifunctional protein STIM1. The identification of SOC and SIC opens a new scenario for the role of store depletion in the modulation of different calcium entry pathways, which may satisfy different cellular processes.

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

    Science.gov (United States)

    Zemkova, Hana; Tomić, Melanija; Kucka, Marek; Aguilera, Greti; Stojilkovic, Stanko S

    2016-04-01

    Transgenic mice expressing the tdimer2(12) form of Discosoma red fluorescent protein under control of the proopiomelanocortin gene's regulatory elements are a useful model for studying corticotrophs. Using these mice, we studied the ion channels and mechanisms controlling corticotroph excitability. Corticotrophs were either quiescent or electrically active, with a 22-mV difference in the resting membrane potential (RMP) between the 2 groups. In quiescent cells, CRH depolarized the membrane, leading to initial single spiking and sustained bursting; in active cells, CRH further facilitated or inhibited electrical activity and calcium spiking, depending on the initial activity pattern and CRH concentration. The stimulatory but not inhibitory action of CRH on electrical activity was mimicked by cAMP independently of the presence or absence of arachidonic acid. Removal of bath sodium silenced spiking and hyperpolarized the majority of cells; in contrast, the removal of bath calcium did not affect RMP but reduced CRH-induced depolarization, which abolished bursting electrical activity and decreased the spiking frequency but not the amplitude of single spikes. Corticotrophs with inhibited voltage-gated sodium channels fired calcium-dependent action potentials, whereas cells with inhibited L-type calcium channels fired sodium-dependent spikes; blockade of both channels abolished spiking without affecting the RMP. These results indicate that the background voltage-insensitive sodium conductance influences RMP, the CRH-depolarization current is driven by a cationic conductance, and the interplay between voltage-gated sodium and calcium channels plays a critical role in determining the status and pattern of electrical activity and calcium signaling.

  12. Effects of Octylphenol and Bisphenol A on the Metal Cation Transporter Channels of Mouse Placentas

    Science.gov (United States)

    Lee, Jae-Hwan; Ahn, Changhwan; Kang, Hee Young; Hong, Eui-Ju; Hyun, Sang-Hwan; Choi, Kyung-Chul; Jeung, Eui-Bae

    2016-01-01

    Octylphenol (OP) and bisphenol A (BPA) are known as endocrine-disrupting chemicals (EDCs). During pregnancy, the expression of steroid hormone receptors is controlled by maternal and fetal nutrition. To evaluate the impact of EDCs during pregnancy, ethinyl estradiol (EE, 0.2 mg/kg/day), OP (50 mg/kg/day), and BPA (50 mg/kg/day) were administered to pregnant mice. The mRNA levels of TRPV6 (transient receptor potential cation channels in subfamily V, member 6) decreased significantly by EE and OP. The PMCA1 (ATPase, Ca++ transporting, plasma membrane 1) mRNA and protein levels decreased significantly by EE, OP, and BPA. CTR1 (solute carrier family 31, member 1) and ATP7A (ATPase, Cu++ transporting, alpha polypeptide) expression decreased significantly by EE, OP, and BPA. The mRNA levels of IREG1 (iron-regulated transporter, member 1) decreased significantly by EE. Hephaestin (HEPH) mRNA levels decreased significantly by EE, OP, and BPA, and protein levels decreased significantly by BPA. As a result of immunohistochemistry analysis, all cation transporter proteins were found in labyrinth of placenta. To confirm the cytosolic level of cations, levels of cation level in fetal serum were measured. EE, OP, and BPA significantly reduced serum calcium and copper levels, and iron levels were reduced by BPA. Taken together, some EDCs, such as OP and BPA, could modulate the calcium, copper, and iron ion-transporting channels during pregnancy. The fetus relies on the mother for ionic transportation, and, therefore, pregnant women should avoid exposure to cation-channel-disrupting chemicals. PMID:27690074

  13. Adsorption and uptake of cadmium by Azolla pinnata: kinetics of inhibition by cations.

    Science.gov (United States)

    Gaur, J P; Noraho, N

    1995-06-01

    A. pinnata showed a remarkable ability of taking up Cd from the external medium. Of the total Cd taken up by the test plant, surface adsorption was about 90%. Cd adsorption did not occur at a constant rate, however, an equilibrium was reached in 2 h. The uptake of Cd occurred at a constant rate. Test cations, including heavy metals (Ca, Mg, K, Na, Ni, Fe, Cu and Zn), inhibited adsorption as well as uptake of Cd. Cd adsorption was competitively inhibited by Cu, Fe and Zn, whereas Ca, Mg, K, Na and Ni caused non-competitive inhibition. Similarly, Cd uptake was competitively (Ca, Mg, Fe) or non-competitively (Na, K, Ni, Zn) inhibited by cations. Inhibition of Cd uptake by Cu was not wholly competitive.

  14. Adsorption and Uptake of Cadmium by Azolla pinnata:Kinetics of Inhibition by Cations

    Institute of Scientific and Technical Information of China (English)

    J.P.GAUR; N.NORAHO

    1995-01-01

    A.pinnata showed a remarkable ability of taking up Cd from the external medium.Of the total Cd taken up by the test plant,surface adsorption was about 90%.Cd adsorption did not occur at a constant rate,however,an equilibrium was reached in 2h.The uptake of Cd occurred at a constant rate.Test cations.Including heavy metals(Ca,Mg,K,Na,Ni,F,Cu and Zn),inhibited adsorption as well as uptake of Cd.Cd adsorption was competitively inhibited by Cu,Fe and Zn,whereas Ca,Mg,K,Na and Ni caused non-competitive inhibition,Similarly,Cd uptake was competitively(Ca,Mg,Fe)or non-competitively(Na,K,Ni,Zn) inhibited by cations.Inhibition of Cd uptake by Cu was not wholly competitive.

  15. Identification and analysis of cation channel homologues in human pathogenic fungi.

    Directory of Open Access Journals (Sweden)

    David L Prole

    Full Text Available Fungi are major causes of human, animal and plant disease. Human fungal infections can be fatal, but there are limited options for therapy, and resistance to commonly used anti-fungal drugs is widespread. The genomes of many fungi have recently been sequenced, allowing identification of proteins that may become targets for novel therapies. We examined the genomes of human fungal pathogens for genes encoding homologues of cation channels, which are prominent drug targets. Many of the fungal genomes examined contain genes encoding homologues of potassium (K(+, calcium (Ca(2+ and transient receptor potential (Trp channels, but not sodium (Na(+ channels or ligand-gated channels. Some fungal genomes contain multiple genes encoding homologues of K(+ and Trp channel subunits, and genes encoding novel homologues of voltage-gated K(v channel subunits are found in Cryptococcus spp. Only a single gene encoding a homologue of a plasma membrane Ca(2+ channel was identified in the genome of each pathogenic fungus examined. These homologues are similar to the Cch1 Ca(2+ channel of Saccharomyces cerevisiae. The genomes of Aspergillus spp. and Cryptococcus spp., but not those of S. cerevisiae or the other pathogenic fungi examined, also encode homologues of the mitochondrial Ca(2+ uniporter (MCU. In contrast to humans, which express many K(+, Ca(2+ and Trp channels, the genomes of pathogenic fungi encode only very small numbers of K(+, Ca(2+ and Trp channel homologues. Furthermore, the sequences of fungal K(+, Ca(2+, Trp and MCU channels differ from those of human channels in regions that suggest differences in regulation and susceptibility to drugs.

  16. The human red cell voltage-dependent cation channel. Part III: Distribution homogeneity and pH dependence

    DEFF Research Database (Denmark)

    Bennekou, P.; Barksmann, T. L.; Christophersen, P.

    2006-01-01

    The homogeneity of the distribution of the non-selective voltage-dependent cation channel (the NSVDC channel) in the human erythrocyte, and the pH dependence was investigated. Activation of this channel caused a uniform cellular dehydration, which was characterized by the changes in the erythrocyte...

  17. Structural Insights into Divalent Cation Modulations of ATP-Gated P2X Receptor Channels

    Directory of Open Access Journals (Sweden)

    Go Kasuya

    2016-02-01

    Full Text Available P2X receptors are trimeric ATP-gated cation channels involved in physiological processes ranging widely from neurotransmission to pain and taste signal transduction. The modulation of the channel gating, including that by divalent cations, contributes to these diverse physiological functions of P2X receptors. Here, we report the crystal structure of an invertebrate P2X receptor from the Gulf Coast tick Amblyomma maculatum in the presence of ATP and Zn2+ ion, together with electrophysiological and computational analyses. The structure revealed two distinct metal binding sites, M1 and M2, in the extracellular region. The M1 site, located at the trimer interface, is responsible for Zn2+ potentiation by facilitating the structural change of the extracellular domain for pore opening. In contrast, the M2 site, coupled with the ATP binding site, might contribute to regulation by Mg2+. Overall, our work provides structural insights into the divalent cation modulations of P2X receptors.

  18. In vitro inhibition of rat small intestinal absorption by lipophilic organic cations.

    Science.gov (United States)

    Elsenhans, B; Blume, R; Lembcke, B; Caspary, W F

    1985-02-28

    Cationic, lipid-soluble organic compounds may interfere with cation-mediated membrane transport processes. Thus, small intestinal absorption may be influenced by lipophilic organic cations. Therefore a series of arylalkylamines was studied in the concentration range from 0.5 to 20 mmol/l for their effect on the transport of various monosaccharides and leucine in the rat small intestine in vitro by means of the tissue accumulation technique. Whereas the monophenyl substituted monoamines (e.g. benzylamine, 2-phenylethylamine, 3-phenylpropylamine) did not show a significant effect on the active transport, the corresponding omega,omega-diphenyl derivatives exhibited a strong inhibition of the active transport of the sugars and the amino acid. These monoamines and drugs of similar structure (e.g. benzoctamine, diphenydramine) exhibited a mixed or non-competitive type of inhibition which correlated quite well with their octanol-water partition coefficients. In contrast, di- or triamines (e.g. harmaline, imipramine, pyrilamine) revealed a rather pure competitive type of inhibition. These findings tentatively suggest a different mode of action on the active transport by lipid-soluble organic amines according to the molecular charge distribution. In addition, membrane vesicles were used to examine the effect of the different amines on the sucrase activity. Regarding the cation-dependent hydrolysis of sucrose, however, no distinct pattern developed.

  19. Modeling hysteresis observed in the human erythrocyte voltage-dependent cation channel

    DEFF Research Database (Denmark)

    Flyvbjerg, Henrik; Gudowska-Nowak, Ewa; Christophersen, Palle

    2012-01-01

    The non-selective voltage-activated cation channel from human red cells, which is activated at depolarizing potentials, has been shown to exhibit counter-clockwise gating hysteresis. Here, we analyze this phenomenon with the simplest possible phenomenological models. Specifically, the hysteresis...... cycle, including its direction, is reproduced by a model with 2×2 discrete states: the normal open/closed states and two different states of "gate tension". Rates of transitions between the two branches of the hysteresis curve are modeled with single-barrier kinetics by introducing a real...

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

    Science.gov (United States)

    Borbiro, Istvan; Badheka, Doreen; Rohacs, Tibor

    2015-02-10

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

  1. Inhibition of HERG potassium channels by celecoxib and its mechanism.

    Directory of Open Access Journals (Sweden)

    Roman V Frolov

    Full Text Available BACKGROUND: Celecoxib (Celebrex, a widely prescribed selective inhibitor of cyclooxygenase-2, can modulate ion channels independently of cyclooxygenase inhibition. Clinically relevant concentrations of celecoxib can affect ionic currents and alter functioning of neurons and myocytes. In particular, inhibition of Kv2.1 channels by celecoxib leads to arrhythmic beating of Drosophila heart and of rat heart cells in culture. However, the spectrum of ion channels involved in human cardiac excitability differs from that in animal models, including mammalian models, making it difficult to evaluate the relevance of these observations to humans. Our aim was to examine the effects of celecoxib on hERG and other human channels critically involved in regulating human cardiac rhythm, and to explore the mechanisms of any observed effect on the hERG channels. METHODS AND RESULTS: Celecoxib inhibited the hERG, SCN5A, KCNQ1 and KCNQ1/MinK channels expressed in HEK-293 cells with IC(50s of 6.0 µM, 7.5 µM, 3.5 µM and 3.7 µM respectively, and the KCND3/KChiP2 channels expressed in CHO cells with an IC(50 of 10.6 µM. Analysis of celecoxib's effects on hERG channels suggested gating modification as the mechanism of drug action. CONCLUSIONS: The above channels play a significant role in drug-induced long QT syndrome (LQTS and short QT syndrome (SQTS. Regulatory guidelines require that all new drugs under development be tested for effects on the hERG channel prior to first administration in humans. Our observations raise the question of celecoxib's potential to induce cardiac arrhythmias or other channel related adverse effects, and make a case for examining such possibilities.

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

    Science.gov (United States)

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

    2014-02-14

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

  3. Inhibition of N-Type Calcium Channels by Fluorophenoxyanilide Derivatives

    Directory of Open Access Journals (Sweden)

    Ellen C. Gleeson

    2015-04-01

    Full Text Available A set of fluorophenoxyanilides, designed to be simplified analogues of previously reported ω-conotoxin GVIA mimetics, were prepared and tested for N-type calcium channel inhibition in a SH-SY5Y neuroblastoma FLIPR assay. N-type or Cav2.2 channel is a validated target for the treatment of refractory chronic pain. Despite being significantly less complex than the originally designed mimetics, up to a seven-fold improvement in activity was observed.

  4. Inhibition of the ATPase activity of the catalytic portion of ATP synthases by cationic amphiphiles.

    Science.gov (United States)

    Datiles, Manuel J; Johnson, Eric A; McCarty, Richard E

    2008-04-01

    Melittin, a cationic, amphiphilic polypeptide, has been reported to inhibit the ATPase activity of the catalytic portions of the mitochondrial (MF1) and chloroplast (CF1) ATP synthases. Gledhill and Walker [J.R. Gledhill, J.E. Walker. Inhibition sites in F1-ATPase from bovine heart mitochondria, Biochem. J. 386 (2005) 591-598.] suggested that melittin bound to the same site on MF1 as IF1, the endogenous inhibitor polypeptide. We have studied the inhibition of the ATPase activity of CF1 and of F1 from Escherichia coli (ECF1) by melittin and the cationic detergent, cetyltrimethylammonium bromide (CTAB). The Ca2+- and Mg2+-ATPase activities of CF1 deficient in its inhibitory epsilon subunit (CF1-epsilon) are sensitive to inhibition by melittin and by CTAB. The inhibition of Ca2+-ATPase activity by CTAB is irreversible. The Ca2+-ATPase activity of F1 from E. coli (ECF1) is inhibited by melittin and the detergent, but Mg2+-ATPase activity is much less sensitive to both reagents. The addition of CTAB or melittin to a solution of CF1-epsilon or ECF1 caused a large increase in the fluorescence of the hydrophobic probe, N-phenyl-1-naphthylamine, indicating that the detergent and melittin cause at least partial dissociation of the enzymes. ATP partially protects CF1-epsilon from inhibition by CTAB. We also show that ATP can cause the aggregation of melittin. This result complicates the interpretation of experiments in which ATP is shown to protect enzyme activity from inhibition by melittin. It is concluded that melittin and CTAB cause at least partial dissociation of the alpha/beta heterohexamer.

  5. Effect of Extra-Framework Cations of LTL Nanozeolites to Inhibit Oil Oxidation

    Science.gov (United States)

    Tan, Kok-Hou; Cham, Hooi-Ying; Awala, Hussein; Ling, Tau Chuan; Mukti, Rino R.; Wong, Ka-Lun; Mintova, Svetlana; Ng, Eng-Poh

    2015-06-01

    Lubricant oils take significant part in current health and environmental considerations since they are an integral and indispensable component of modern technology. Antioxidants are probably the most important additives used in oils because oxidative deterioration plays a major role in oil degradation. Zeolite nanoparticles (NPs) have been proven as another option as green antioxidants in oil formulation. The anti-oxidative behavior of zeolite NPs is obvious; however, the phenomenon is still under investigation. Herein, a study of the effect of extra-framework cations stabilized on Linde Type L (LTL) zeolite NPs (ca. 20 nm) on inhibition of oxidation in palm oil-based lubricant oil is reported. Hydrophilic LTL zeolites with a Si/Al ratio of 3.2 containing four different inorganic cations (Li+, Na+, K+, Ca2+) were applied. The oxidation of the lubricant oil was followed by visual observation, colorimetry, fourier transform infrared (FTIR) spectroscopy, 1H NMR spectroscopy, total acid number (TAN), and rheology analyses. The effect of extra-framework cations to slow down the rate of oil oxidation and to control the viscosity of oil is demonstrated. The degradation rate of the lubricant oil samples is decreased considerably as the polarizability of cation is increased with the presence of zeolite NPs. More importantly, the microporous zeolite NPs have a great influence in halting the steps that lead to the polymerization of the oils and thus increasing the lifetime of oils.

  6. Hyperpolarization-activated cyclic nucleotide-gated cation channel subtypes differentially modulate the excitability of murine small intestinal afferents

    Institute of Scientific and Technical Information of China (English)

    Ying-Ping Wang; Bi-Ying Sun; Qian Li; Li Dong; Guo-Hua Zhang; David Grundy; Wei-Fang Rong

    2012-01-01

    AIM: To assess the role of hyperpolarization-activated cyclic nucleotide-gated cation (HCN) channels in regulating the excitability of vagal and spinal gut afferents.METHODS: The mechanosensory response of mesen-teric afferent activity was measured in an ex vivo murine jejunum preparation. HCN channel activity was recorded through voltage and current clamp in acutely dissociated dorsal root ganglia (DRG) and nodose ganglia (NG) neurons retrogradely labeled from the small intestine through injection of a fluorescent marker (DiI). The isoforms of HCN channels expressed in DRG and NG neurons were examined by immunohistochemistry.RESULTS: Ramp distension of the small intestine evoked biphasic increases in the afferent nerve activity, reflecting the activation of low- and high-threshold fibers.HCN blocker CsCl (5 mmol/L) preferentially inhibited the responses of low-threshold fibers to distension and showed no significant effects on the high-threshold responses. The effect of CsCl was mimicked by the more selective HCN blocker ZD7288 (10 ?mol/L). In 71.4% of DiI labeled DRG neurons (n = 20) and 90.9% of DiI labeled NG neurons (n = 10), an inward current (Ih current) was evoked by hyperpolarization pulses which was fully eliminated by extracellular CsCl. In neurons expressing Ih current, a typical "sag" was observed upon injection of hyperpolarizing current pulses in current-clamp recordings. CsCl abolished the sag entirely. In some DiI labeled DRG neurons, the Ih current was potentiated by 8-Br-cAMP, which had no effect on the Ih current of DiI labeled NG neurons. Immunohistochemistry revealed differential expression of HCN isoforms in vagal and spinal afferents, and HCN2 and HCN3 seemed to be the dominant isoform in DRG and NG, respectively.CONCLUSION: HCNs differentially regulate the excitability of vagal and spinal afferent of murine small intestine.

  7. Mutation of the melastatin-related cation channel, TRPM3, underlies inherited cataract and glaucoma.

    Directory of Open Access Journals (Sweden)

    Thomas M Bennett

    Full Text Available Inherited forms of cataract are a clinically important and genetically heterogeneous cause of visual impairment that usually present at an early age with or without systemic and/or other ocular abnormalities. Here we have identified a new locus for inherited cataract and high-tension glaucoma with variable anterior segment defects, and characterized an underlying mutation in the gene coding for transient receptor potential cation channel, subfamily M, member-3 (TRPM3, melastatin-2. Genome-wide linkage analysis mapped the ocular disease locus to the pericentric region of human chromosome 9. Whole exome and custom-target next-generation sequencing detected a heterozygous A-to-G transition in exon-3 of TRPM3 that co-segregated with disease. As a consequence of alternative splicing this missense mutation was predicted to result in the substitution of isoleucine-to-methionine at codon 65 (c.195A>G; p.I65 M of TRPM3 transcript variant 9, and at codon 8 (c.24A>G; p.I8 M of a novel TRPM3 transcript variant expressed in human lens. In both transcript variants the I-to-M substitution was predicted in silico to exert damaging effects on protein function. Furthermore, transient expression studies of a recombinant TRPM3-GFP reporter product predicted that the I-to-M substitution introduced an alternative translation start-site located 89 codons upstream from the native initiator methionine found in eight other TRPM3 transcript variants (1-8. Collectively, these studies have provided the first evidence that TRPM3 is associated with inherited ocular disease in humans, and further provide support for the important role of this cation channel in normal eye development.

  8. OmpW of Caulobacter crescentus Functions as an Outer Membrane Channel for Cations.

    Science.gov (United States)

    Benz, Roland; Jones, Michael D; Younas, Farhan; Maier, Elke; Modi, Niraj; Mentele, Reinhard; Lottspeich, Friedrich; Kleinekathöfer, Ulrich; Smit, John

    2015-01-01

    Caulobacter crescentus is an oligotrophic bacterium that lives in dilute organic environments such as soil and freshwater. This bacterium represents an interesting model for cellular differentiation and regulation because daughter cells after division have different forms: one is motile while the other is non-motile and can adhere to surfaces. Interestingly, the known genome of C. crescentus does not contain genes predicted to code for outer membrane porins of the OmpF/C general diffusion type present in enteric bacteria or those coding for specific porins selective for classes of substrates. Instead, genes coding for 67 TonB-dependent outer membrane receptors have been identified, suggesting that active transport of specific nutrients may be the norm. Here, we report that high channel-forming activity was observed with crude outer membrane extracts of C. crescentus in lipid bilayer experiments, indicating that the outer membrane of C. crescentus contained an ion-permeable channel with a single-channel conductance of about 120 pS in 1M KCl. The channel-forming protein with an apparent molecular mass of about 20 kDa was purified to homogeneity. Partial protein sequencing of the protein indicated it was a member of the OmpW family of outer membrane proteins from Gram-negative bacteria. This channel was not observed in reconstitution experiments with crude outer membrane extracts of an OmpW deficient C. crescentus mutant. Biophysical analysis of the C. crescentus OmpW suggested that it has features that are special for general diffusion porins of Gram-negative outer membranes because it was not a wide aqueous channel. Furthermore, OmpW of C. crescentus seems to be different to known OmpW porins and has a preference for ions, in particular cations. A putative model for OmpW of C. crescentus was built on the basis of the known 3D-structures of OmpW of Escherichia coli and OprG of Pseudomonas aeruginosa using homology modeling. A comparison of the two known structures

  9. OmpW of Caulobacter crescentus Functions as an Outer Membrane Channel for Cations.

    Directory of Open Access Journals (Sweden)

    Roland Benz

    Full Text Available Caulobacter crescentus is an oligotrophic bacterium that lives in dilute organic environments such as soil and freshwater. This bacterium represents an interesting model for cellular differentiation and regulation because daughter cells after division have different forms: one is motile while the other is non-motile and can adhere to surfaces. Interestingly, the known genome of C. crescentus does not contain genes predicted to code for outer membrane porins of the OmpF/C general diffusion type present in enteric bacteria or those coding for specific porins selective for classes of substrates. Instead, genes coding for 67 TonB-dependent outer membrane receptors have been identified, suggesting that active transport of specific nutrients may be the norm. Here, we report that high channel-forming activity was observed with crude outer membrane extracts of C. crescentus in lipid bilayer experiments, indicating that the outer membrane of C. crescentus contained an ion-permeable channel with a single-channel conductance of about 120 pS in 1M KCl. The channel-forming protein with an apparent molecular mass of about 20 kDa was purified to homogeneity. Partial protein sequencing of the protein indicated it was a member of the OmpW family of outer membrane proteins from Gram-negative bacteria. This channel was not observed in reconstitution experiments with crude outer membrane extracts of an OmpW deficient C. crescentus mutant. Biophysical analysis of the C. crescentus OmpW suggested that it has features that are special for general diffusion porins of Gram-negative outer membranes because it was not a wide aqueous channel. Furthermore, OmpW of C. crescentus seems to be different to known OmpW porins and has a preference for ions, in particular cations. A putative model for OmpW of C. crescentus was built on the basis of the known 3D-structures of OmpW of Escherichia coli and OprG of Pseudomonas aeruginosa using homology modeling. A comparison of the two

  10. Corrosion inhibition by inorganic cationic inhibitors on the high strength alumunium alloy, 2024-T3

    Science.gov (United States)

    Chilukuri, Anusha

    The toxicity and carcinogenic nature of chromates has led to the investigation of environmentally friendly compounds that offer good corrosion resistance to AA 2024-T3. Among the candidate inhibitors are rare earth metal cationic (REM) and zinc compounds, which have received much of attention over the past two decades. A comparative study on the corrosion inhibition caused by rare earth metal cations, Ce3+, Pr3+, La3+ and Zn2+ cations on the alloy was done. Cathodic polarization showed that these inhibitor ions suppress the oxygen reduction reaction (ORR) to varying extents with Zn2+ providing the best inhibition. Pr3+ exhibited windows of concentration (100-300 ppm) in which the corrosion rate is minimum; similar to the Ce3+ cation. Scanning Electron Microscopy (SEM) studies showed that the mechanism of inhibition of the Pr3+ ion is also similar to that of the Ce3+ ion. Potentiodynamic polarization experiments after 30 min immersion time showed greatest suppression of oxygen reduction reaction in neutral chloride solutions (pH 7), which reached a maximum at a Zn2+ ion concentration of 5 mM. Anodic polarization experiments after 30 min immersion time, showed no anodic inhibition by the inhibitor in any concentration (0.1 mM - 10 mM) and at any pH. However, anodic polarization of samples immersed after longer immersion times (upto 4 days) in mildly acidic Zn2+ (pH 4) solutions showed significant reduction in anodic kinetics indicating that zinc also acts as a “slow anodic inhibitor”. In contrast to the polarization experiments, coupons exposed to inhibited acidic solutions at pH 4 showed complete suppression of dissolution of Al2CuMg particles compared to zinc-free solutions in the SEM studies. Samples exposed in pH 4 Zn2+-bearing solution exhibited highest polarization resistance which was also observed to increase with time. In deaerated solutions, the inhibition by Zn2+ at pH 4 is not observed as strongly. The ability to make the interfacial electrolyte

  11. Dual-channel capillary electrophoresis for simultaneous determination of cations and anions.

    Science.gov (United States)

    Opekar, František; Tůma, Petr

    2016-05-13

    An original electrophoresis apparatus for simultaneous rapid determination of cations and anions has been designed and tested. The separation part of the apparatus consists of two identical fused-silica capillaries, each with a length of 10.5cm and inner diameter of 25μm. The injection space is formed by the crossing of four channels in a plexiglass cross-piece. The capillaries pass through two opposing channels and their injection ends are located opposite one another at a distance of approx. 0.5mm in the centre of the crossing point. The exit ends of the capillaries are placed in vessels containing the background electrolyte in which are immersed the electrodes of a high-voltage source. Contactless conductivity detectors with semi-cylindrical electrodes are located 2cm from the exit ends of the capillaries. The injection part of the apparatus consists of two piezoelectric micro-pumps bringing the solution through another channel in the cross-piece to the injection ends of the capillary. During the injection, the sample is brought through one of them and is injected electrokinetically for a defined time. Then the sample zone is forced out of the injection space by a stream of background electrolyte from the second micro-pump. The timing of the injection process is computer-controlled. Thus the equipment can be considered to constitute electrophoresis in one capillary with injection into its centre. The use of short capillaries and miniature micro-pumps without other mechanical components enabled the construction of the apparatus on a board with dimensions of 20×25cm. The proposed equipment was used to test simultaneous separation of a mixture of cations and anions, NH4(+), K(+), Ca(2+), Mg(2+), Sr(2+), Ba(2+), Cl(-), NO3(-), SO4(2-), ClO3(-) and F(-), in BGE with composition 500mM HAc+20mM Tris+2mM 18-crown-6 (pH 3.3). Baseline separation of all the components was achieved in time less than 1min. Quantification of the content of nitrate nitrogen (determined as

  12. PACSINs bind to the TRPV4 cation channel. PACSIN 3 modulates the subcellular localization of TRPV4.

    NARCIS (Netherlands)

    Cuajungco, M.P.; Grimm, C.; Oshima, K.; D'hoedt, D.; Nilius, B.; Mensenkamp, A.R.; Bindels, R.J.M.; Plomann, M.; Heller, S.

    2006-01-01

    TRPV4 is a cation channel that responds to a variety of stimuli including mechanical forces, temperature, and ligand binding. We set out to identify TRPV4-interacting proteins by performing yeast two-hybrid screens, and we isolated with the avian TRPV4 amino terminus the chicken orthologues of mamma

  13. Single mechanically-gated cation channel currents can trigger action potentials in neocortical and hippocampal pyramidal neurons.

    Science.gov (United States)

    Nikolaev, Yury A; Dosen, Peter J; Laver, Derek R; van Helden, Dirk F; Hamill, Owen P

    2015-05-22

    The mammalian brain is a mechanosensitive organ that responds to different mechanical forces ranging from intrinsic forces implicated in brain morphogenesis to extrinsic forces that can cause concussion and traumatic brain injury. However, little is known of the mechanosensors that transduce these forces. In this study we use cell-attached patch recording to measure single mechanically-gated (MG) channel currents and their affects on spike activity in identified neurons in neonatal mouse brain slices. We demonstrate that both neocortical and hippocampal pyramidal neurons express stretch-activated MG cation channels that are activated by suctions of ~25mm Hg, have a single channel conductance for inward current of 50-70pS and show weak selectivity for alkali metal cations (i.e., Na(+)channel currents activated on the soma trigger spiking/action potentials in both neocortical and hippocampal pyramidal neurons. Not all neuron types studied here expressed MG channel currents. In particular, locus coeruleus and cerebellar Purkinje neurons showed no detectable MG channel activity. Moreover their robust rhythmic spike activity was resistant to mechanical modulation. Our observation that a single MG channel current can trigger spiking predicates the need for reassessment of the long held view that the impulse output of central neurons depends only upon their intrinsic voltage-gated channels and/or their integrated synaptic input.

  14. Stretch-activated nonselective cation, Cl- and K+ channels in apical membrane of epithelial cells of Reissner's membrane.

    Science.gov (United States)

    Yeh, T H; Tsai, M C; Lee, S Y; Hsu, M M; Tran Ba Huy, P

    1997-07-01

    Ion channels on the apical membrane of epithelial cells (the surface facing the endolymph) of acutely isolated Reissner's membrane from guinea-pig cochlea were investigated by using patch-clamp technique in cell-attached and inside-out configurations. Three types of ion channel were identified: namely, a stretch-activated nonselective cation, a chloride and a potassium channel. When the pipette was filled with high-K+ endolymph-like solution, the most significant channel activity was nonselective cation channels (85/110, 77% patches). The current versus voltage relationship was linear with a unitary conductance of 22.1 +/- 0.4 pS and reversal potential (Vr) of 2.3 +/- 0.8 mV (n = 18). The channel exhibited a lower conductance (14.0 +/- 0.6 pS, n = 8) to Ca2+. The open probability was low (NPo approximately 0.1) in cell-attached configuration under +60 mV pipette potential and increased when the membrane was stretched with negative pressure. The channel was blocked by 10 microM extracellular Gd3+. The two other types of channels were a small voltage-sensitive Cl- channel (6.0 +/- 0.3 pS; 91/99, 92% patches) and a K+ channel (approximately 30 pS; 29/191, 15% patches). These channels might play roles in the regulation of cell volume, in balancing the hydrostatic pressure across Reissner's membrane and in maintaining the electrochemical composition of endolymph.

  15. Transient Receptor Potential Melastatin 7 Cation Channel Kinase: New Player in Angiotensin II-Induced Hypertension.

    Science.gov (United States)

    Antunes, Tayze T; Callera, Glaucia E; He, Ying; Yogi, Alvaro; Ryazanov, Alexey G; Ryazanova, Lillia V; Zhai, Alexander; Stewart, Duncan J; Shrier, Alvin; Touyz, Rhian M

    2016-04-01

    Transient receptor potential melastatin 7 (TRPM7) is a bifunctional protein comprising a magnesium (Mg(2+))/cation channel and a kinase domain. We previously demonstrated that vasoactive agents regulate vascular TRPM7. Whether TRPM7 plays a role in the pathophysiology of hypertension and associated cardiovascular dysfunction is unknown. We studied TRPM7 kinase-deficient mice (TRPM7Δkinase; heterozygous for TRPM7 kinase) and wild-type (WT) mice infused with angiotensin II (Ang II; 400 ng/kg per minute, 4 weeks). TRPM7 kinase expression was lower in heart and aorta from TRPM7Δkinase versus WT mice, effects that were further reduced by Ang II infusion. Plasma Mg(2+) was lower in TRPM7Δkinase versus WT mice in basal and stimulated conditions. Ang II increased blood pressure in both strains with exaggerated responses in TRPM7Δkinase versus WT groups (Phypertension is exaggerated, cardiac remodeling and left ventricular dysfunction are amplified, and endothelial function is impaired. These processes are associated with hypomagnesemia, blunted TRPM7 kinase expression/signaling, endothelial nitric oxide synthase downregulation, and proinflammatory vascular responses. Our findings identify TRPM7 kinase as a novel player in Ang II-induced hypertension and associated vascular and target organ damage.

  16. TRPML cation channels regulate the specialized lysosomal compartment of vertebrate B-lymphocytes.

    Science.gov (United States)

    Song, Yumei; Dayalu, Rashmi; Matthews, Sharon A; Scharenberg, Andrew M

    2006-12-01

    B-lymphocytes possess a specialized lysosomal compartment, the regulated transformation of which has been implicated in B-cell antigen presentation. Members of the mucolipin (TRPML) family of cation channels have been implicated in regulated vesicular transport in several tissues, but a role for TRPML function in lymphocyte vesicular transport physiology has not been previously described. To address the role of TRPML proteins in lymphocyte vesicular transport, we analyzed the lysosomal compartment in cultured B-lymphocytes engineered to lack TRPML1 or after expression of N- or C-terminal GFP fusion proteins of TRPML1 or TRPML2. Consistent with previous analyses of lymphocytes derived from human patients with mutations in TRPML1, we were not able to detect abnormalities in the lysosomes of TRPML1-deficient DT40 B-lymphocytes. However, while N-terminal GFP fusions of TRPML2 localized to normal appearing lysosomes, C-terminal GFP fusions of either TRPML1 or TRPML2 acted to antagonize endogenous TRPML function, localizing to large vesicular structures, the histological properties of which were indistinguishable from the enlarged lysosomes observed in affected tissues of TRPML1-deficient humans. Endocytosed B-cell receptors were delivered to these enlarged lysosomes, demonstrating that a TRPML-dependent process is required for normal regulation of the specialized lysosome compartment of vertebrate B-lymphocytes.

  17. Inhibition of biofouling by modification of forward osmosis membrane using quaternary ammonium cation.

    Science.gov (United States)

    Park, Kang-Hee; Yu, Sang-Hyun; Kim, Han-Shin; Park, Hee-Deung

    2015-01-01

    In the operation of the forward osmosis (FO) process, biofouling of the membrane is a potentially serious problem. Development of an FO membrane with antibacterial properties could contribute to a reduction in biofouling. In this study, quaternary ammonium cation (QAC), a widely used biocidal material, was conjugated with a silane coupling agent (3-(trimethoxysilyl)-propyldimethyloctadecyl ammonium chloride) and used to modify an FO membrane to confer antibacterial properties. Fourier transform infrared spectroscopy (FT-IR) demonstrated that the conjugated QAC was successfully immobilized on the FO membrane via covalent bonding. Bacterial viability on the QAC-modified membrane was confirmed via colony count method and visualized via bacterial viability assay. The QAC membrane decreased the viability of Escherichia coli to 62% and Staphylococcus aureus to 77% versus the control membrane. Inhibition of biofilm formation on the QAC modified membrane was confirmed via anti-biofilm tests using the drip-flow reactor and FO unit, resulting in 64% and 68% inhibition in the QAC-modified membrane against the control membrane, respectively. The results demonstrate the effectiveness of the modified membrane in reducing bacterial viability and inhibiting biofilm formation, indicating the potential of QAC-modified membranes to decrease operation costs incurred by biofouling.

  18. Cation-modulated electron-transfer channel: H-atom transfer vs proton-coupled electron transfer with a variable electron-transfer channel in acylamide units.

    Science.gov (United States)

    Chen, Xiaohua; Bu, Yuxiang

    2007-08-08

    The mechanism of proton transfer (PT)/electron transfer (ET) in acylamide units was explored theoretically using density functional theory in a representative model (a cyclic coupling mode between formamide and the N-dehydrogenated formamidic radical, FF). In FF, PT/ET normally occurs via a seven-center cyclic proton-coupled electron transfer (PCET) mechanism with a N-->N PT and an O-->O ET. However, when different hydrated metal ions are bound to the two oxygen sites of FF, the PT/ET mechanism may significantly change. In addition to their inhibition of PT/ET rate, the hydrated metal ions can effectively regulate the FF PT/ET cooperative mechanism to produce a single pathway hydrogen atom transfer (HAT) or a flexible proton coupled electron transfer (PCET) mechanism by changing the ET channel. The regulation essentially originates from the change in the O...O bond strength in the transition state, subject to the binding ability of the hydrated metal ions. In general, the high valent metal ions and those with large binding energies can promote HAT, and the low valent metal ions and those with small binding energies favor PCET. Hydration may reduce the Lewis acidity of cations, and thus favor PCET. Good correlations among the binding energies, barrier heights, spin density distributions, O...O contacts, and hydrated metal ion properties have been found, which can be used to interpret the transition in the PT/ET mechanism. These findings regarding the modulation of the PT/ET pathway via hydrated metal ions may provide useful information for a greater understanding of PT/ET cooperative mechanisms, and a possible method for switching conductance in nanoelectronic devices.

  19. Influence of dexamethasone on the expression and distribution of transient receptor potential cation channel 6 in glomerular podocytes

    Institute of Scientific and Technical Information of China (English)

    王辉阳

    2014-01-01

    Objective To observe the changes of foot processes,expression and distribution of transient receptor potential cation channel 6(TRPC6)in podocytes by puromycin aminonucleoside(PAN)and dexamethasone(DEX)intervention,then to investigate the function of TRPC6 in podocytes and its relation to proteinuria in kidney diseases.Methods Podocytes cultured in vitro were divided into three group:control group,PAN stimulation group and DEX intervention group.Mouse podocyte cell line

  20. Curcumin inhibits activation of TRPM2 channels in rat hepatocytes

    Directory of Open Access Journals (Sweden)

    E. Kheradpezhouh

    2016-04-01

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

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

    Directory of Open Access Journals (Sweden)

    Tricia H Smith

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

  2. Inhibition of human organic cation transporters by the alkaloids matrine and oxymatrine.

    Science.gov (United States)

    Pan, Xiaolei; Wang, Li; Gründemann, Dirk; Sweet, Douglas H

    2014-01-01

    Human organic cation transporters (hOCTs; SLC22) are expressed in many organs, including intestine, liver, kidney, heart and brain, where they contribute to the absorption, distribution, and elimination of endogenous and exogenous substances. The alkaloids matrine and oxymatrine are widely used in herbal medicine for the treatment of cancer, as well as viral, and cardiac diseases. Their physicochemical properties indicated that they are potential inhibitors for hOCTs, leading to drug-drug interactions in vivo. Therefore, we assessed the inhibitory effects of matrine and oxymatrine on the function of hOCT1 (SLC22A1), hOCT2 (SLC22A2) and hOCT3 (SLC22A3) using stably transfected transporter-expressing cells. At 100-fold excess, oxymatrine exhibited marked inhibition of hOCT1-mediated substrate uptake (pmatrine failed to produce significant inhibition on hOCT1. The IC50 value for oxymatrine on hOCT1 was estimated as 513±132 μM. While there was no significant inhibition of hOCT2 or hOCT3 at 100-fold excess, oxymatrine and matrine showed 42% and 88% inhibition of hOCT3-mediated substrate uptake at 3 and 6mM, respectively. Considering the potential intestinal lumen and reported plasma concentrations of matrine and oxymatrine, these data suggest that drug-drug interactions may occur during hOCT1-mediated hepatic and renal uptake and during hOCT3-mediated intestinal absorption.

  3. Inhibition by acrolein of light-induced stomatal opening through inhibition of inward-rectifying potassium channels in Arabidopsis thaliana.

    Science.gov (United States)

    Islam, Md Moshiul; Ye, Wenxiu; Matsushima, Daiki; Khokon, Md Atiqur Rahman; Munemasa, Shintaro; Nakamura, Yoshimasa; Murata, Yoshiyuki

    2015-01-01

    Acrolein is a reactive α,β-unsaturated aldehyde derived from lipid peroxides, which are produced in plants under a variety of stress. We investigated effects of acrolein on light-induced stomatal opening using Arabidopsis thaliana. Acrolein inhibited light-induced stomatal opening in a dose-dependent manner. Acrolein at 100 μM inhibited plasma membrane inward-rectifying potassium (Kin) channels in guard cells. Acrolein at 100 μM inhibited Kin channel KAT1 expressed in a heterologous system using Xenopus leaves oocytes. These results suggest that acrolein inhibits light-induced stomatal opening through inhibition of Kin channels in guard cells.

  4. Concerted action of two cation filters in the aquaporin water channel

    DEFF Research Database (Denmark)

    Wu, Binghua; Steinbronn, Christina; Alsterfjord, Magnus;

    2009-01-01

    Aquaporin (AQP) facilitated water transport is common to virtually all cell membranes and is marked by almost perfect specificity and high flux rates. Simultaneously, protons and cations are strictly excluded to maintain ionic transmembrane gradients. Yet, the AQP cation filters have not been ide...

  5. Influence of some growth regulators and cations on inhibition of chlorophyll biosynthesis by lead in maize

    Energy Technology Data Exchange (ETDEWEB)

    Sinha, S.K. (Council of Science Technology, Lucknow (India)); Srivastava, H.S. (Rohilkhand Univ., Bareilly (India)); Tripathi, R.D. (National Botanical Research Institute, Lucknow (India))

    1993-08-01

    Phytotoxic effects of Pb pollution are well established. In order to analyse the physiological basis of toxic symptoms and of reduced plant productivity, its effect on chlorophyll content has been examined in some plants. Thus, a decrease in total chlorophyll content during Pb supply has been observed in oats, mung beam, pea, etc. The activity of delta aminolevulinic acid dehydratase, an important enzyme in the biosynthesis of heme pigments, is inhibited by Pb in mung bean and several other species. This observation may perhaps indicate that a reduction in chlorophyll content in the presence of lead is due to an inhibition of pigment synthesis. The effect of Pb on greening maize leaf segments in the presence of various precursors of chlorophyll has been studied in the present investigation to evaluate this hypothesis. The effect of some growth regulators and cations, which could otherwise modify chlorophyll biosynthesis, has been examined to see whether the toxic effects of Pb on photosynthetic pigments could also be modified by these effectors. 16 refs., 4 tabs.

  6. Proton pump inhibitors inhibit metformin uptake by organic cation transporters (OCTs.

    Directory of Open Access Journals (Sweden)

    Anne T Nies

    Full Text Available Metformin, an oral insulin-sensitizing drug, is actively transported into cells by organic cation transporters (OCT 1, 2, and 3 (encoded by SLC22A1, SLC22A2, or SLC22A3, which are tissue specifically expressed at significant levels in various organs such as liver, muscle, and kidney. Because metformin does not undergo hepatic metabolism, drug-drug interaction by inhibition of OCT transporters may be important. So far, comprehensive data on the interaction of proton pump inhibitors (PPIs with OCTs are missing although PPIs are frequently used in metformin-treated patients. Using in silico modeling and computational analyses, we derived pharmacophore models indicating that PPIs (i.e. omeprazole, pantoprazole, lansoprazole, rabeprazole, and tenatoprazole are potent OCT inhibitors. We then established stably transfected cell lines expressing the human uptake transporters OCT1, OCT2, or OCT3 and tested whether these PPIs inhibit OCT-mediated metformin uptake in vitro. All tested PPIs significantly inhibited metformin uptake by OCT1, OCT2, and OCT3 in a concentration-dependent manner. Half-maximal inhibitory concentration values (IC(50 were in the low micromolar range (3-36 µM and thereby in the range of IC(50 values of other potent OCT drug inhibitors. Finally, we tested whether the PPIs are also transported by OCTs, but did not identify PPIs as OCT substrates. In conclusion, PPIs are potent inhibitors of the OCT-mediated metformin transport in vitro. Further studies are needed to elucidate the clinical relevance of this drug-drug interaction with potential consequences on metformin disposition and/or efficacy.

  7. Gap-junction channels inhibit transverse propagation in cardiac muscle

    Directory of Open Access Journals (Sweden)

    Ramasamy Lakshminarayanan

    2005-01-01

    Full Text Available Abstract The effect of adding many gap-junctions (g-j channels between contiguous cells in a linear chain on transverse propagation between parallel chains was examined in a 5 × 5 model (5 parallel chains of 5 cells each for cardiac muscle. The action potential upstrokes were simulated using the PSpice program for circuit analysis. Either a single cell was stimulated (cell A1 or the entire chain was stimulated simultaneously (A-chain. Transverse velocity was calculated from the total propagation time (TPT from when the first AP crossed a Vm of -20 mV and the last AP crossed -20 mV. The number of g-j channels per junction was varied from zero to 100, 1,000 and 10,000 (Rgj of ∞, 100 MΩ, 10 MΩ, 1.0 MΩ, respectively. The longitudinal resistance of the interstitial fluid (ISF space between the parallel chains (Rol2 was varied between 200 KΩ (standard value and 1.0, 5.0, and 10 MΩ. The higher the Rol2 value, the tighter the packing of the chains. It was found that adding many g-j channels inhibited transverse propagation by blocking activation of all 5 chains, unless Rol2 was greatly increased above the standard value of 200 KΩ. This was true for either method of stimulation. This was explained by, when there is strong longitudinal coupling between all 5 cells of a chain awaiting excitation, there must be more transfer energy (i.e., more current to simultaneously excite all 5 cells of a chain.

  8. A genome-wide study of panic disorder suggests the amiloride-sensitive cation channel 1 as a candidate gene

    DEFF Research Database (Denmark)

    Gregersen, Noomi; Dahl, Hans A.; Buttenschön, Henriette N.;

    2012-01-01

    Panic disorder (PD) is a mental disorder with recurrent panic attacks that occur spontaneously and are not associated to any particular object or situation. There is no consensus on what causes PD. However, it is recognized that PD is influenced by environmental factors, as well as genetic factors...... of the Faroe Islands. Subsequently, we conducted a fine mapping, which revealed the amiloride-sensitive cation channel 1 (ACCN1) located on chromosome 17q11.2-q12 as a potential candidate gene for PD. The further analyses of the ACCN1 gene using single-nucleotide polymorphisms (SNPs) revealed significant...

  9. Triple-channel portable capillary electrophoresis instrument with individual background electrolytes for the concurrent separations of anionic and cationic species.

    Science.gov (United States)

    Mai, Thanh Duc; Le, Minh Duc; Sáiz, Jorge; Duong, Hong Anh; Koenka, Israel Joel; Pham, Hung Viet; Hauser, Peter C

    2016-03-10

    The portable capillary electrophoresis instrument is automated and features three independent channels with different background electrolytes to allow the concurrent optimized determination of three different categories of charged analytes. The fluidic system is based on a miniature manifold which is based on mechanically milled channels for injection of samples and buffers. The planar manifold pattern was designed to minimize the number of electronic valves required for each channel. The system utilizes pneumatic pressurization to transport solutions at the grounded as well as the high voltage side of the separation capillaries. The instrument has a compact design, with all components arranged in a briefcase with dimensions of 45 (w) × 35 (d) × 15 cm (h) and a weight of about 15 kg. It can operate continuously for 8 h in the battery-powered mode if only one electrophoresis channel is in use, or for about 2.5 h in the case of simultaneous employment of all three channels. The different operations, i.e. capillary flushing, rinsing of the interfaces at both capillary ends, sample injection and electrophoretic separation, are activated automatically with a control program featuring a graphical user interface. For demonstration, the system was employed successfully for the concurrent separation of different inorganic cations and anions, organic preservatives, additives and artificial sweeteners in various beverage and food matrices.

  10. Multiple cationic amphiphiles induce a Niemann-Pick C phenotype and inhibit Ebola virus entry and infection.

    Directory of Open Access Journals (Sweden)

    Charles J Shoemaker

    Full Text Available Ebola virus (EBOV is an enveloped RNA virus that causes hemorrhagic fever in humans and non-human primates. Infection requires internalization from the cell surface and trafficking to a late endocytic compartment, where viral fusion occurs, providing a conduit for the viral genome to enter the cytoplasm and initiate replication. In a concurrent study, we identified clomiphene as a potent inhibitor of EBOV entry. Here, we screened eleven inhibitors that target the same biosynthetic pathway as clomiphene. From this screen we identified six compounds, including U18666A, that block EBOV infection (IC(50 1.6 to 8.0 µM at a late stage of entry. Intriguingly, all six are cationic amphiphiles that share additional chemical features. U18666A induces phenotypes, including cholesterol accumulation in endosomes, associated with defects in Niemann-Pick C1 protein (NPC1, a late endosomal and lysosomal protein required for EBOV entry. We tested and found that all six EBOV entry inhibitors from our screen induced cholesterol accumulation. We further showed that higher concentrations of cationic amphiphiles are required to inhibit EBOV entry into cells that overexpress NPC1 than parental cells, supporting the contention that they inhibit EBOV entry in an NPC1-dependent manner. A previously reported inhibitor, compound 3.47, inhibits EBOV entry by blocking binding of the EBOV glycoprotein to NPC1. None of the cationic amphiphiles tested had this effect. Hence, multiple cationic amphiphiles (including several FDA approved agents inhibit EBOV entry in an NPC1-dependent fashion, but by a mechanism distinct from that of compound 3.47. Our findings suggest that there are minimally two ways of perturbing NPC1-dependent pathways that can block EBOV entry, increasing the attractiveness of NPC1 as an anti-filoviral therapeutic target.

  11. Multiple cationic amphiphiles induce a Niemann-Pick C phenotype and inhibit Ebola virus entry and infection.

    Science.gov (United States)

    Shoemaker, Charles J; Schornberg, Kathryn L; Delos, Sue E; Scully, Corinne; Pajouhesh, Hassan; Olinger, Gene G; Johansen, Lisa M; White, Judith M

    2013-01-01

    Ebola virus (EBOV) is an enveloped RNA virus that causes hemorrhagic fever in humans and non-human primates. Infection requires internalization from the cell surface and trafficking to a late endocytic compartment, where viral fusion occurs, providing a conduit for the viral genome to enter the cytoplasm and initiate replication. In a concurrent study, we identified clomiphene as a potent inhibitor of EBOV entry. Here, we screened eleven inhibitors that target the same biosynthetic pathway as clomiphene. From this screen we identified six compounds, including U18666A, that block EBOV infection (IC(50) 1.6 to 8.0 µM) at a late stage of entry. Intriguingly, all six are cationic amphiphiles that share additional chemical features. U18666A induces phenotypes, including cholesterol accumulation in endosomes, associated with defects in Niemann-Pick C1 protein (NPC1), a late endosomal and lysosomal protein required for EBOV entry. We tested and found that all six EBOV entry inhibitors from our screen induced cholesterol accumulation. We further showed that higher concentrations of cationic amphiphiles are required to inhibit EBOV entry into cells that overexpress NPC1 than parental cells, supporting the contention that they inhibit EBOV entry in an NPC1-dependent manner. A previously reported inhibitor, compound 3.47, inhibits EBOV entry by blocking binding of the EBOV glycoprotein to NPC1. None of the cationic amphiphiles tested had this effect. Hence, multiple cationic amphiphiles (including several FDA approved agents) inhibit EBOV entry in an NPC1-dependent fashion, but by a mechanism distinct from that of compound 3.47. Our findings suggest that there are minimally two ways of perturbing NPC1-dependent pathways that can block EBOV entry, increasing the attractiveness of NPC1 as an anti-filoviral therapeutic target.

  12. Divalent cation tolerance protein binds to β-secretase and inhibits the processing of amyloid precursor protein

    Institute of Scientific and Technical Information of China (English)

    Runzhong Liu; Haibo Hou; Xuelian Yi; Shanwen Wu; Huan Zeng

    2013-01-01

    The deposition of amyloid-beta is a pathological hallmark of Alzheimer's disease. Amyloid-beta is derived from amyloid precursor protein through sequential proteolytic cleavages by β-secretase (beta-site amyloid precursor protein-cleaving enzyme 1) and γ-secretase. To further elucidate the roles of beta-site amyloid precursor protein-cleaving enzyme 1 in the development of Alzheimer's disease, a yeast two-hybrid system was used to screen a human embryonic brain cDNA library for proteins directly interacting with the intracellular domain of beta-site amyloid precursor protein-cleaving enzyme 1. A potential beta-site amyloid precursor protein-cleaving enzyme 1- interacting protein identified from the positive clones was divalent cation tolerance protein. Immunoprecipitation studies in the neuroblastoma cell line N2a showed that exogenous divalent cation tolerance protein interacts with endogenous beta-site amyloid precursor protein-cleaving enzyme 1. The overexpression of divalent cation tolerance protein did not affect beta-site amyloid precursor protein-cleaving enzyme 1 protein levels, but led to increased amyloid precursor protein levels in N2a/APP695 cells, with a concomitant reduction in the processing product amyloid precursor protein C-terminal fragment, indicating that divalent cation tolerance protein inhibits the processing of amyloid precursor protein. Our experimental findings suggest that divalent cation tolerance protein negatively regulates the function of beta-site amyloid precursor protein-cleaving enzyme 1. Thus, divalent cation tolerance protein could play a protective role in Alzheimer's disease.

  13. Triple-channel portable capillary electrophoresis instrument with individual background electrolytes for the concurrent separations of anionic and cationic species

    Energy Technology Data Exchange (ETDEWEB)

    Mai, Thanh Duc; Le, Minh Duc [Centre for Environmental Technology and Sustainable Development (CETASD), Hanoi University of Science, Nguyen Trai Street 334, Hanoi (Viet Nam); Sáiz, Jorge [Department of Analytical Chemistry, Physical Chemistry and Chemical Engineering, University of Alcalá, Ctra. Madrid-Barcelona Km 33.6, Alcalá de Henares, Madrid (Spain); Duong, Hong Anh [Centre for Environmental Technology and Sustainable Development (CETASD), Hanoi University of Science, Nguyen Trai Street 334, Hanoi (Viet Nam); Koenka, Israel Joel [University of Basel, Department of Chemistry, Spitalstrasse 51, 4056 Basel (Switzerland); Pham, Hung Viet, E-mail: phamhungviet@hus.edu.vn [Centre for Environmental Technology and Sustainable Development (CETASD), Hanoi University of Science, Nguyen Trai Street 334, Hanoi (Viet Nam); Hauser, Peter C., E-mail: Peter.Hauser@unibas.ch [University of Basel, Department of Chemistry, Spitalstrasse 51, 4056 Basel (Switzerland)

    2016-03-10

    The portable capillary electrophoresis instrument is automated and features three independent channels with different background electrolytes to allow the concurrent optimized determination of three different categories of charged analytes. The fluidic system is based on a miniature manifold which is based on mechanically milled channels for injection of samples and buffers. The planar manifold pattern was designed to minimize the number of electronic valves required for each channel. The system utilizes pneumatic pressurization to transport solutions at the grounded as well as the high voltage side of the separation capillaries. The instrument has a compact design, with all components arranged in a briefcase with dimensions of 45 (w) × 35 (d) × 15 cm (h) and a weight of about 15 kg. It can operate continuously for 8 h in the battery-powered mode if only one electrophoresis channel is in use, or for about 2.5 h in the case of simultaneous employment of all three channels. The different operations, i.e. capillary flushing, rinsing of the interfaces at both capillary ends, sample injection and electrophoretic separation, are activated automatically with a control program featuring a graphical user interface. For demonstration, the system was employed successfully for the concurrent separation of different inorganic cations and anions, organic preservatives, additives and artificial sweeteners in various beverage and food matrices. - Highlights: • The use of parallel channels allows the concurrent separation of different classes of analytes. • Separate background electrolytes allow individual optimization. • The instrument is compact and field portable.

  14. Ancient association between cation leak channels and Mid1 proteins is conserved in fungi and animals

    Directory of Open Access Journals (Sweden)

    Alfredo eGhezzi

    2014-03-01

    Full Text Available Neuronal resting potential can tune the excitability of neural networks, affecting downstream behavior. Sodium leak channels (NALCN play a key role in rhythmic behaviors by helping set, or subtly changing neuronal resting potential. The full complexity of these newly described channels is just beginning to be appreciated, however. NALCN channels can associate with numerous subunits in different tissues and can be activated by several different peptides and second messengers. We recently showed that NALCN channels are closely related to fungal calcium channels, which they functionally resemble. Here, we use this relationship to predict a family of NALCN-associated proteins in animals on the basis of homology with the yeast protein Mid1, the subunit of the yeast calcium channel. These proteins all share a cysteine-rich region that is necessary for Mid1 function in yeast. We validate this predicted association by showing that the Mid1 homolog in Drosophila, encoded by the CG33988 gene, is coordinately expressed with NALCN, and that knockdown of either protein creates identical phenotypes in several behaviors associated with NALCN function. The relationship between Mid1 and leak channels has therefore persisted over a billion years of evolution, despite drastic changes to both proteins and the organisms in which they exist.

  15. δ-opioid Receptor Induced Inhibition of Sodium Channel Function

    Institute of Scientific and Technical Information of China (English)

    康学智; 顾全保; 丁光宏; 晁东满; 王英伟; G Balboni; LH Lazarus; 夏萤

    2008-01-01

    Objective: To study the precise role of DOR in the regulation of sodium channels at present. Methods: With Xenopus oocytes co-expressing sodium channel subtype 2 (Nav1.2) and DOR. Results: 1) Nav1.2 expression induced tetrodotoxin-sensitive inward currents; 2) DOR expression reduced the inward currents; 3) activation of DOR reduced the amplitude of the current and rightly shifted the activation curve of the current in the oocytes with both Nav1.2 and DOR, but not in ones with Nav1.2 alone; 4) the DOR agonist-induced inhibition of Nav1.2 currents was in a dose-dependent manner and saturable; 5) the DOR agonist had no effect on naive oocytes. Conclusion: These data represent the first demonstration that activation of DOR inhibits Na+ channel function by decreasing the amplitude of sodium currents and increasing its threshold of activation. This novel finding has far-reaching impacts on novel solutions of certain neurological disorders such as hypoxic/ischemic injury, epilepsy and pain. Also, our data may improve the understanding of the mechanisms underlying acupuncture since acupuncture is known to activate the brain opioid system.%目的:研究δ-阿片受体表达和激活对钠通道1.2亚型的电流特性的影响.方法:用双电极电压钳技术,在δ-阿片受体和钠通道亚型1.2共表达的非洲爪蟾第V期卵母细胞上,观察δ-阿片受体表达和/或激活后,钠通道1.2亚型电流特性的变化.结果:1)钠通道1.2亚型的表达产生河豚毒素(tetrodotoxin,TTX)敏感的内向电流;2)δ-阿片受体的表达减少钠通道激活电流的幅度;3)δ-阿片受体和钠通道1.2亚型共表达的卵母细胞中,δ-阿片受体激动剂可以抑制钠通道激活电流的幅度和电导,而只有钠通道1.2亚型表达的卵母细胞则无此现象;4)δ-阿片受体激动剂抑制钠电流的作用具有剂量依赖关系,并能达到饱和状态;5)δ-阿片受体激动剂对未表达外派陛蛋白的卵母细胞无影响.结论:本结

  16. Anion-Channel Blockers Inhibit S-Type Anion Channels and Abscisic Acid Responses in Guard Cells.

    Science.gov (United States)

    Schwartz, A.; Ilan, N.; Schwarz, M.; Scheaffer, J.; Assmann, S. M.; Schroeder, J. I.

    1995-10-01

    The effects of anion-channel blockers on light-mediated stomatal opening, on the potassium dependence of stomatal opening, on stomatal responses to abscisic acid (ABA), and on current through slow anion channels in the plasma membrane of guard cells were investigated. The anion-channel blockers anthracene-9-carboxylic acid (9-AC) and niflumic acid blocked current through slow anion channels of Vicia faba L. guard cells. Both 9-AC and niflumic acid reversed ABA inhibition of stomatal opening in V. faba L. and Commelina communis L. The anion-channel blocker probenecid also abolished ABA inhibition of stomatal opening in both species. Additional tests of 9-AC effects on stomatal aperture in Commelina revealed that application of this anion-channel blocker allowed wide stomatal opening under low (1 mM) KCI conditions and increased the rate of stomatal opening under both low and high (100 mM) KCI conditions. These results indicate that anion channels can function as a negative regulator of stomatal opening, presumably by allowing anion efflux and depolarization, which prohibits ion up-take in guard cells. Furthermore, 9-AC prevented ABA induction of stomatal closure. A model in which ABA activation of anion channels contributes a rate-limiting mechanism during ABA-induced stomatal closure and inhibition of stomatal opening is discussed.

  17. Data in support of the negative influence of divalent cations on (-)-epigallocatechin-3-gallate (EGCG)-mediated inhibition of matrix metalloproteinase-2 (MMP-2).

    Science.gov (United States)

    Deb, Gauri; Batra, Sahil; Limaye, Anil M

    2016-03-01

    In this data article we have provided evidence for the negative influence of divalent cations on (-)-epigallocatechin-3-gallate (EGCG)-mediated inhibition of matrix metalloproteinase-2 (MMP-2) activity in cell-free experiments. Chelating agents, such as EDTA and sodium citrate alone, did not affect MMP-2 activity. While EDTA enhanced, excess of divalent cations interfered with EGCG-mediated inhibition of MMP-2.

  18. Differential contribution of TRPM4 and TRPM5 nonselective cation channels to the slow afterdepolarization in mouse prefrontal cortex neurons

    Directory of Open Access Journals (Sweden)

    Ya-Ting eLei

    2014-09-01

    Full Text Available In certain neurons from different brain regions, a brief burst of action potentials can activate a slow afterdepolarization (sADP in the presence of muscarinic acetylcholine receptor agonists. The sADP, if suprathreshold, can contribute to persistent non-accommodating firing in some of these neurons. Previous studies have characterized a Ca2+-activated non-selective cation (CAN current (ICAN that is thought to underlie the sADP. ICAN depends on muscarinic receptor stimulation and exhibits a dependence on neuronal activity, membrane depolarization and Ca2+-influx similar to that observed for the sADP. Despite the widespread occurrence of sADPs in neurons throughout the brain, the molecular identity of the ion channels underlying these events, as well as ICAN, remains uncertain. Here we used a combination of genetic, pharmacological and electrophysiological approaches to characterize the molecular mechanisms underlying the muscarinic receptor-dependent sADP in layer 5 pyramidal neurons of mouse prefrontal cortex. First, we confirmed that in the presence of the cholinergic agonist carbachol a brief burst of action potentials triggers a prominent sADP in these neurons. Second, we confirmed that this sADP requires activation of a PLC signaling cascade and intracellular calcium signaling. Third, we obtained direct evidence that the transient receptor potential melastatin 5 channel (TRPM5, which is thought to function as a CAN channel in non-neural cells, contributes importantly to the sADP in the layer 5 neurons. In contrast, the closely related TRPM4 channel may play only a minor role in the sADP.

  19. A novel triazole-based cationic gemini surfactant: synthesis and effect on corrosion inhibition of carbon steel in hydrochloric acid

    Energy Technology Data Exchange (ETDEWEB)

    Qiu Lingguang [School of Chemistry and Chemical Engineering, Anhui University, Hefei 230039 (China)]. E-mail: lgahu@163.com; Xie Anjian [School of Chemistry and Chemical Engineering, Anhui University, Hefei 230039 (China); Shen Yuhua [School of Chemistry and Chemical Engineering, Anhui University, Hefei 230039 (China)

    2005-06-15

    A triazole-based cationic gemini surfactant, 3,5-bis(methylene octadecyl dimethylammonium chloride)-1,2,4-triazole (18-triazole-18) has been synthesized, and its effect on corrosion inhibition of A{sub 3} steel in 1 M HCl has been studied using the weight-loss method. The result showed that 18-triazole-18 acted as an excellent inhibitor in 1 M HCl. It was found that the adsorption mechanism of 18-triazole-18 on the steel surface in acid medium was quite different from that of cationic gemini surfactants containing dimethylene as a spacer, as well as that of conventional cationic single-chained surfactants, which is due to unique molecular structure of 18-triazole-18. 18-Triazole-18 may be adsorbed on the steel surface in acid medium through a maximum of four atoms or groups, i.e., the two nitrogen atoms of triazole ring and two quaternary ammonium head groups. Four regions of surfactant concentration could be divided to illustrate the adsorption of 18-triazole-18 on the steel surface, and four different adsorption mechanisms may take place in different regions of surfactant concentration.

  20. Emission channeling studies on transition-metal doped GaN and ZnO: Cation versus anion substitution

    CERN Document Server

    AUTHOR|(CDS)2070176; Wahl, Ulrich; Martins Correia, Joao; Amorim, Lígia; Silva, Daniel; Decoster, Stefan; Castro Ribeiro Da Silva, Manuel; Temst, Kristiaan; Vantomme, André

    2014-01-01

    The magnetic and electric properties of impurities in semiconductors are strongly dependent on the lattice sites which they occupy. While the majority site can often be predicted based on chemical similarities with the host elements and is usually simple to confirm experimentally, minority sites are far more complicated to predict, detect and identify. We have carried out extensive beta− emission channeling studies on the lattice location of transition metal impurities in wide-gap dilute magnetic semiconductors, namely Co and Mn in GaN and ZnO, making use of radioactive 61Co and 56Mn implanted at the ISOLDE facility at CERN. In addition to the majority occupation of cation (Ga, Zn) sites, we located significant fractions (of the order of 20%) of the Co and Mn impurities in anion (N, O) sites, which are virtually unaffected by thermal annealing up to 900 °C. Here, we present the beta− emission channeling experiments on 61Co-implanted GaN. We discuss these results in the context of our recent reports of mi...

  1. Stimulation of TRPC5 cationic channels by low micromolar concentrations of lead ions (Pb{sup 2+})

    Energy Technology Data Exchange (ETDEWEB)

    Sukumar, Piruthivi [Multidisciplinary Cardiovascular Research Centre and Institute of Membrane and Systems Biology, Faculty of Biological Sciences, University of Leeds, Leeds LS2 9JT (United Kingdom); Beech, David J., E-mail: d.j.beech@leeds.ac.uk [Multidisciplinary Cardiovascular Research Centre and Institute of Membrane and Systems Biology, Faculty of Biological Sciences, University of Leeds, Leeds LS2 9JT (United Kingdom)

    2010-02-26

    Lead toxicity is long-recognised but continues to be a major public health problem. Its effects are wide-ranging and include induction of hyper-anxiety states. In general it is thought to act by interfering with Ca{sup 2+} signalling but specific targets are not clearly identified. Transient receptor potential canonical 5 (TRPC5) is a Ca{sup 2+}-permeable ion channel that is linked positively to innate fear responses and unusual amongst ion channels in being stimulated by trivalent lanthanides, which include gadolinium. Here we show investigation of the effect of lead, which is a divalent ion (Pb{sup 2+}). Intracellular Ca{sup 2+} and whole-cell patch-clamp recordings were performed on HEK 293 cells conditionally over-expressing TRPC5 or other TRP channels. Extracellular application of Pb{sup 2+} stimulated TRPC5 at concentrations greater than 1 {mu}M. Control cells without TRPC5 showed little or no response to Pb{sup 2+} and expression of other TRP channels (TRPM2 or TRPM3) revealed partial inhibition by 10 {mu}M Pb{sup 2+}. The stimulatory effect on TRPC5 depended on an extracellular residue (E543) near the ion pore: similar to gadolinium action, E543Q TRPC5 was resistant to Pb{sup 2+} but showed normal stimulation by the receptor agonist sphingosine-1-phosphate. The study shows that Pb{sup 2+} is a relatively potent stimulator of the TRPC5 channel, generating the hypothesis that a function of the channel is to sense metal ion poisoning.

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

    DEFF Research Database (Denmark)

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

    2011-01-01

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

  3. Corrosion inhibition efficiency and surface activity of benzothiazol-3-ium cationic Schiff base derivatives in hydrochloric acid

    Energy Technology Data Exchange (ETDEWEB)

    Negm, N.A., E-mail: nabelnegm@hotmail.co [Petrochemicals Department, Egyptian Petroleum Research Institute, Nasr City, Cairo (Egypt); Elkholy, Y.M.; Zahran, M.K. [Chemistry Department, Faculty of Science, Helwan University, Helwan (Egypt); Tawfik, S.M. [Petrochemicals Department, Egyptian Petroleum Research Institute, Nasr City, Cairo (Egypt)

    2010-10-15

    Research highlights: {yields} Benzothiazolium inhibitors show high corrosion inhibition for carbon steel. {yields} Weight loss and corrosion rates decrease by increasing inhibitor dose. {yields} Inhibition mechanism suggested as physisorption mechanism supported by adsorption free energy values. {yields} The adsorption of the inhibitors obeys Langmuir isotherm. {yields} Side chains have ascending order on the corrosion inhibition of the different inhibitors. - Abstract: Two series of cationic Schiff base surfactants namely: 2-(benzylideneamino)-3-(2-oxo-2-alkoxyethyl)-1,3-benzothiazol-3-ium bromide and 2-[(4-methoxybenzylidene)amino]-3-(2-oxo-2-alkoxyethyl)-1, 3-benzothiazol-3-ium bromide were prepared and confirmed using elemental analysis, FTIR, and {sup 1}H NMR spectra. The surface activity of the synthesized Schiff bases showed their tendency towards adsorption at the interfaces. The prepared compounds were evaluated as corrosion inhibitors for carbon steel in 0.5 M HCl solution using gravimetric and polarization measurements. By fitting the gravimetric data, some thermodynamic and kinetic parameters were estimated. The adsorption of the inhibitors on the carbon steel surface obeyed Langmuir adsorption isotherm and had a physical mechanism. Polarization measurements showed that the synthesized inhibitors act as mixed inhibitors for carbon steel in the acidic media. The results of the corrosion inhibition using two different methods showed narrow differences in the obtained values between the two methods within 5%.

  4. Sigma-1 receptor agonists directly inhibit Nav1.2/1.4 channels.

    Directory of Open Access Journals (Sweden)

    Xiao-Fei Gao

    Full Text Available (+-SKF 10047 (N-allyl-normetazocine is a prototypic and specific sigma-1 receptor agonist that has been used extensively to study the function of sigma-1 receptors. (+-SKF 10047 inhibits K(+, Na(+ and Ca2+ channels via sigma-1 receptor activation. We found that (+-SKF 10047 inhibited Na(V1.2 and Na(V1.4 channels independently of sigma-1 receptor activation. (+-SKF 10047 equally inhibited Na(V1.2/1.4 channel currents in HEK293T cells with abundant sigma-1 receptor expression and in COS-7 cells, which barely express sigma-1 receptors. The sigma-1 receptor antagonists BD 1063,BD 1047 and NE-100 did not block the inhibitory effects of (+-SKF-10047. Blocking of the PKA, PKC and G-protein pathways did not affect (+-SKF 10047 inhibition of Na(V1.2 channel currents. The sigma-1 receptor agonists Dextromethorphan (DM and 1,3-di-o-tolyl-guanidine (DTG also inhibited Na(V1.2 currents through a sigma-1 receptor-independent pathway. The (+-SKF 10047 inhibition of Na(V1.2 currents was use- and frequency-dependent. Point mutations demonstrated the importance of Phe(1764 and Tyr(1771 in the IV-segment 6 domain of the Na(V1.2 channel and Phe(1579 in the Na(V1.4 channel for (+-SKF 10047 inhibition. In conclusion, our results suggest that sigma-1 receptor agonists directly inhibit Na(V1.2/1.4 channels and that these interactions should be given special attention for future sigma-1 receptor function studies.

  5. Mammalian target of rapamycin complex 2 signaling pathway regulates transient receptor potential cation channel 6 in podocytes.

    Directory of Open Access Journals (Sweden)

    Fangrui Ding

    Full Text Available Transient receptor potential cation channel 6 (TRPC6 is a nonselective cation channel, and abnormal expression and gain of function of TRPC6 are involved in the pathogenesis of hereditary and nonhereditary forms of renal disease. Although the molecular mechanisms underlying these diseases remain poorly understood, recent investigations revealed that many signaling pathways are involved in regulating TRPC6. We aimed to examine the effect of the mammalian target of rapamycin (mTOR complex (mTOR complex 1 [mTORC1] or mTOR complex 2 [mTORC2] signaling pathways on TRPC6 in podocytes, which are highly terminally differentiated renal epithelial cells that are critically required for the maintenance of the glomerular filtration barrier. We applied both pharmacological inhibitors of mTOR and specific siRNAs against mTOR components to explore which mTOR signaling pathway is involved in the regulation of TRPC6 in podocytes. The podocytes were exposed to rapamycin, an inhibitor of mTORC1, and ku0063794, a dual inhibitor of mTORC1 and mTORC2. In addition, specific siRNA-mediated knockdown of the mTORC1 component raptor and the mTORC2 component rictor was employed. The TRPC6 mRNA and protein expression levels were examined via real-time quantitative PCR and Western blot, respectively. Additionally, fluorescence calcium imaging was performed to evaluate the function of TRPC6 in podocytes. Rapamycin displayed no effect on the TRPC6 mRNA or protein expression levels or TRPC6-dependent calcium influx in podocytes. However, ku0063794 down-regulated the TRPC6 mRNA and protein levels and suppressed TRPC6-dependent calcium influx in podocytes. Furthermore, knockdown of raptor did not affect TRPC6 expression or function, whereas rictor knockdown suppressed TRPC6 protein expression and TRPC6-dependent calcium influx in podocytes. These findings indicate that the mTORC2 signaling pathway regulates TRPC6 in podocytes but that the mTORC1 signaling pathway does not appear

  6. Inhibition of Kv1.3 Channels in Human Jurkat T Cells by Xanthohumol and Isoxanthohumol.

    Science.gov (United States)

    Gąsiorowska, Justyna; Teisseyre, Andrzej; Uryga, Anna; Michalak, Krystyna

    2015-08-01

    Using whole-cell patch-clamp technique, we investigated influence of selected compounds from groups of prenylated chalcones and flavonoids: xanthohumol and isoxanthohumol on the activity of Kv1.3 channels in human leukemic Jurkat T cells. Obtained results provide evidence that both examined compounds were inhibitors of Kv1.3 channels in these cells. The inhibitory effects occurred in a concentration-dependent manner. The estimated value of the half-blocking concentration (EC50) was about 3 μM for xanthohumol and about 7.8 μM for isoxanthohumol. The inhibition of Kv1.3 channels by examined compounds was not complete. Upon an application of the compounds at the maximal concentrations equal to 30 μM, the activity of Kv1.3 channels was inhibited to about 0.13 of the control value. The inhibitory effect was reversible. The application of xanthohumol and isoxanthohumol did not change the currents' activation and inactivation rate. These results may confirm our earlier hypothesis that the presence of a prenyl group in a molecule is a factor that facilitates the inhibition of Kv1.3 channels by compounds from the groups of flavonoids and chalcones. The inhibition of Kv1.3 channels might be involved in antiproliferative and proapoptotic effects of the compounds observed in cancer cell lines expressing these channels.

  7. Biochemical requirements for inhibition of Connexin26-containing channels by natural and synthetic taurine analogs.

    Science.gov (United States)

    Tao, Liang; Harris, Andrew L

    2004-09-10

    Previous work has shown that protonated taurine and aminosulfonate pH buffers, including HEPES, can directly and reversibly inhibit connexin channels that contain connexin26 (Cx26) (Bevans, C. G., and Harris, A. L. (1999) J. Biol. Chem. 274, 3711-3719). The structural requirements for this inhibition were explored by studies of the effects of structural analogs of taurine on the activity of Cx26-containing reconstituted hemichannels from native tissue. Several analogs inhibited the channels, with a range of relative affinities and efficacies. Each active compound contains a protonated amine separated from an ionized sulfonate or sulfinate moiety by several methylene groups. The inhibition is eliminated if the sulfonate/sulfinate moiety or the amine is not present. Compounds that contain a protonated amine but lack a sulfonate/sulfinate moiety do not inhibit but do competitively block the effect of the active compounds. Compounds that lack the protonated amine do not significantly inhibit or antagonize inhibition. The results suggest involvement of the protonated amine in binding and of the ionized sulfur-containing moiety in effecting the inhibition. The maximal effect of the inhibitory compounds is enhanced when a carboxyl group is linked to the alpha-carbon. Inhibition but not binding is stereospecific, with l-isomers being inhibitory and the corresponding d-isomers being inactive but able to antagonize inhibition by the l-isomers. Whereas not all connexins are sensitive to aminosulfonates, the well defined structural requirements described here argue strongly for a highly specific regulatory interaction with some connexins. The finding that cytoplasmic aminosulfonates inhibit connexin channels whereas other cytoplasmic compounds antagonize the inhibition suggests that gap junction channels are regulated by a complex interplay of cytoplasmic ligands.

  8. Blockade of transient receptor potential cation channel subfamily V member 1 promotes regeneration after sciatic nerve injury

    Institute of Scientific and Technical Information of China (English)

    Fei Ren; Hong Zhang; Chao Qi; Mei-ling Gao; Hong Wang; Xia-qing Li

    2015-01-01

    The transient receptor potential cation channel subfamily V member 1 (TRPV1) provides the sensation of pain (nociception). However, it remains unknown whether TRPV1 is activated after peripheral nerve injury, or whether activation of TRPV1 affects neural regeneration. In the present study, we established rat models of unilateral sciatic nerve crush injury, with or without pretreatment with AMG517 (300 mg/kg), a TRPV1 antagonist, injected subcutaneously into the ipsilateral paw 60 minutes before injury. At 1 and 2 weeks after injury, we performed immuno-lfuorescence staining of the sciatic nerve at the center of injury, at 0.3 cm proximal and distal to the injury site, and in the dorsal root ganglia. Our results showed that Wallerian degeneration occurred distal to the injury site, and neurite outgrowth and Schwann cell regeneration occurred proximal to the injury. The number of regenerating myelinated and unmyelinated nerve clus-ters was greater in the AMG517-pretreated rats than in the vehicle-treated group, most notably 2 weeks after injury. TRPV1 expression in the injured sciatic nerve and ipsilateral dorsal root ganglia was markedly greater than on the contralateral side. Pretreatment with AMG517 blocked this effect. These data indicate that TRPV1 is activated or overexpressed after sciatic nerve crush injury, and that blockade of TRPV1 may accelerate regeneration of the injured sciatic nerve.

  9. GARP2 accelerates retinal degeneration in rod cGMP-gated cation channel β-subunit knockout mice

    Science.gov (United States)

    DeRamus, Marci L.; Stacks, Delores A.; Zhang, Youwen; Huisingh, Carrie E.; McGwin, Gerald; Pittler, Steven J.

    2017-01-01

    The Cngb1 locus-encoded β-subunit of rod cGMP-gated cation channel and associated glutamic acid rich proteins (GARPs) are required for phototransduction, disk morphogenesis, and rod structural integrity. To probe individual protein structure/function of the GARPs, we have characterized several transgenic mouse lines selectively restoring GARPs on a Cngb1 knockout (X1−/−) mouse background. Optical coherence tomography (OCT), light and transmission electron microscopy (TEM), and electroretinography (ERG) were used to analyze 6 genotypes including WT at three and ten weeks postnatal. Comparison of aligned histology/OCT images demonstrated that GARP2 accelerates the rate of degeneration. ERG results are consistent with the structural analyses showing the greatest attenuation of function when GARP2 is present. Even 100-fold or more overexpression of GARP1 could not accelerate degeneration as rapidly as GARP2, and when co-expressed GARP1 attenuated the structural and functional deficits elicited by GARP2. These results indicate that the GARPs are not fully interchangeable and thus, likely have separate and distinct functions in the photoreceptor. We also present a uniform murine OCT layer naming nomenclature system that is consistent with human retina layer designations to standardize murine OCT, which will facilitate data evaluation across different laboratories. PMID:28198469

  10. Efficient inhibition of C-26 colon carcinoma by VSVMP gene delivered by biodegradable cationic nanogel derived from polyethyleneimine.

    Science.gov (United States)

    Gou, MaLing; Men, Ke; Zhang, Juan; Li, YuHua; Song, Jia; Luo, Shan; Shi, HuaShan; Wen, YanJun; Guo, Gang; Huang, MeiJuan; Zhao, Xia; Qian, ZhiYong; Wei, YuQuan

    2010-10-26

    Biodegradable cationic nanoparticles have promising application as a gene delivery system. In this article, heparin-polyethyleneimine (HPEI) nanogels were prepared, and these nanogels were developed as a nonviral gene vector. The transfection efficiency of HPEI nanogels was comparable with that of PEI25K, while the cytotoxicity was lower than that of PEI2K and much lower than that of PEI25K in vitro. These HPEI nanogels also had better blood compatibility than PEI25K. After intravenous administration, HPEI nanogels degraded, and the degradation products were excreted through urine. The plasmid expressing vesicular stomatitis virus matrix protein (pVSVMP) could be efficiently transfected into C-26 colon carcinoma cells by HPEI nanogels in vitro, inhibiting the cell proliferation through apoptosis induction. Intraperitoneal injection of pVSVMP/HPEI complexes efficiently inhibited the abdominal metastases of C-26 colon carcinoma through apoptosis induction (mean tumor weight in mice treated with pVSVMP/HPEI complex = 0.93 g and in control mice = 3.28 g, difference = 2.35 g, 95% confidence interval [CI] = 1.75-2.95 g, P < 0.001) and prolonged the survival of treated mice. Moreover, intravenous application of pVSVMP/HPEI complexes also inhibited the growth of pulmonary metastases of C-26 colon carcinoma through apoptosis induction. The HPEI nanogels delivering pVSVMP have promising application in treating colon carcinoma.

  11. Evaluation of organic cation transporter 3 (SLC22A3) inhibition as a potential mechanism of antidepressant action.

    Science.gov (United States)

    Zhu, Hao-Jie; Appel, David I; Gründemann, Dirk; Richelson, Elliott; Markowitz, John S

    2012-04-01

    Organic cation transporter 3 (OCT3, SLC22A3) is a low-affinity, high-capacity transporter widely expressed in the central nervous system (CNS) and other major organs in both humans and rodents. It is postulated that OCT3 has a role in the overall regulation of neurotransmission and maintenance of homeostasis within the CNS. It is generally believed that all antidepressant drugs in current clinical use exert their primary therapeutic effects through inhibition of one or more of the high-affinity neuronal plasma membrane monoamine transporters, such as the norepinephrine transporter and the serotonin transporter. In the present study, we investigated the inhibitory effects of selected antidepressants on OCT3 activity in OCT3-transfected cells to evaluate whether OCT3 inhibition may at least in part contribute to the pharmacological effects of tested antidepressants. The studies demonstrated that all examined antidepressants inhibited OCT3-mediated uptake of the established OCT3 substrate 4-(4-(dimethylamino)styryl)-N-methylpyridinium iodide (4-Di-1-ASP) in a concentration-dependent manner. The IC(50) values were determined to be 4.7 μM, 7.4 μM, 12.0 μM, 18.6 μM, 11.2 μM, and 21.9 μM for desipramine, sertraline, paroxetine, amitriptyline, imipramine, and fluoxetine, respectively. Additionally, desipramine had an IC(50) value of 0.7 μM for the uptake of NE by OCT3, while the IC(50) value of sertraline was 2.3 μM for 5-HT uptake. Both desipramine and sertraline appeared to inhibit OCT3 activity via a non-competitive mechanism. In vivo studies are warranted to determine whether such effects on OCT3 inhibition are of sufficient magnitude to contribute to the overall therapeutic effects of antidepressants.

  12. Inhibition of hERG Potassium Channels by Celecoxib and Its Mechanism

    Science.gov (United States)

    Frolov, Roman V.; Ignatova, Irina I.; Singh, Satpal

    2011-01-01

    Background Celecoxib (Celebrex), a widely prescribed selective inhibitor of cyclooxygenase-2, can modulate ion channels independently of cyclooxygenase inhibition. Clinically relevant concentrations of celecoxib can affect ionic currents and alter functioning of neurons and myocytes. In particular, inhibition of Kv2.1 channels by celecoxib leads to arrhythmic beating of Drosophila heart and of rat heart cells in culture. However, the spectrum of ion channels involved in human cardiac excitability differs from that in animal models, including mammalian models, making it difficult to evaluate the relevance of these observations to humans. Our aim was to examine the effects of celecoxib on hERG and other human channels critically involved in regulating human cardiac rhythm, and to explore the mechanisms of any observed effect on the hERG channels. Methods and Results Celecoxib inhibited the hERG, SCN5A, KCNQ1 and KCNQ1/MinK channels expressed in HEK-293 cells with IC50s of 6.0 µM, 7.5 µM, 3.5 µM and 3.7 µM respectively, and the KCND3/KChiP2 channels expressed in CHO cells with an IC50 of 10.6 µM. Analysis of celecoxib's effects on hERG channels suggested gating modification as the mechanism of drug action. Conclusions The above channels play a significant role in drug-induced long QT syndrome (LQTS) and short QT syndrome (SQTS). Regulatory guidelines require that all new drugs under development be tested for effects on the hERG channel prior to first administration in humans. Our observations raise the question of celecoxib's potential to induce cardiac arrhythmias or other channel related adverse effects, and make a case for examining such possibilities. PMID:22039467

  13. Eosinophil cationic protein stimulates and major basic protein inhibits airway mucus secretion

    DEFF Research Database (Denmark)

    Lundgren, J D; Davey, R T; Lundgren, B

    1991-01-01

    Possible roles of eosinophil (EO) products in modulating the release of mucus from airway explants were investigated. Cell- and membrane-free lysates from purified human EOs (1 to 20 x 10(5)) caused a dose-dependent release of respiratory glycoconjugates (RGC) from cultured feline tracheal explants...... chromatography. ECP (0.025 to 25 micrograms/ml) caused a dose-dependent increase in RGC release from both feline and human airway explants and also stimulated the release of the serous cell-marker, lactoferrin, from human bronchial explants. EO-derived neurotoxin (0.025 to 50 micrograms/ml) failed to affect RGC...... release, whereas MBP (50 micrograms/ml) significantly inhibited RGC release from feline explants. Thus, ECP stimulates RGC and lactoferrin release from airway explants, whereas MBP inhibits RGC release....

  14. Characterization of inward-rectifier K+ channel inhibition by antiarrhythmic piperazine.

    Science.gov (United States)

    Xu, Yanping; Lu, Zhe

    2004-12-14

    Strong inward-rectifier K(+) (Kir) channels play a significant role in shaping the cardiac action potential: they help produce its long plateau and accelerate its rate of repolarization. Consequently, genetic deletion of the gene encoding the strongly rectifying K(+) channel IRK1 (Kir2.1) prolongs the cardiac action potential in mice. In principle, broadening the action potential lengthens the refractory period, which may in turn be antiarrhythmogenic. Interestingly, previous studies showed that piperazine, an inexpensive and safe anthelmintic, both inhibits IRK1 channels and is antiarrhythmic in some animal preparations. This potential pharmacological benefit motivated us to further characterize the energetic, kinetic, and molecular properties of IRK1 inhibition by piperazine. We show how its blocking characteristics, in particular, its shallow voltage dependence, allow piperazine to be effective even in the presence of high-affinity polyamine blockers. We also examine the channel selectivity of piperazine and its molecular determinants.

  15. Nitric oxide suppresses stomatal opening by inhibiting inward-rectifying Kin channels in Arabidopsis guard cells

    Institute of Scientific and Technical Information of China (English)

    XUE ShaoWu; YANG Pin; HE YiKun

    2008-01-01

    We explore nitric oxide (NO) effect on K+in channels in Arabidopsis guard cells. We observed NO inhib-ited K+in currents when Ca2+ chelator EGTA (Ethylene glycol-bis(2-aminoethylether)-N,N,N',N'tetraacetic acid) was not added in the pipette solution; K+in currents were not sensitive to NO when cytosolic Ca2+ was chelated by EGTA. NO inhibited the Arabidopsis stomatal opening, but when EGTA was added in the bath solution, inhibition effect of NO on stomatal opening vanished. Thus, it implies that NO ele-vates cytosolic Ca2+ by activating plasma membrane Ca2+ channels firstly, then inactivates K+in chan-nels, resulting in stomatal opening suppressed subsequently.

  16. A membrane-access mechanism of ion channel inhibition by voltage sensor toxins from spider venom

    Science.gov (United States)

    Lee, Seok-Yong; MacKinnon, Roderick

    2004-07-01

    Venomous animals produce small protein toxins that inhibit ion channels with high affinity. In several well-studied cases the inhibitory proteins are water-soluble and bind at a channel's aqueous-exposed extracellular surface. Here we show that a voltage-sensor toxin (VSTX1) from the Chilean Rose Tarantula (Grammostola spatulata) reaches its target by partitioning into the lipid membrane. Lipid membrane partitioning serves two purposes: to localize the toxin in the membrane where the voltage sensor resides and to exploit the free energy of partitioning to achieve apparent high-affinity inhibition. VSTX1, small hydrophobic poisons and anaesthetic molecules reveal a common theme of voltage sensor inhibition through lipid membrane access. The apparent requirement for such access is consistent with the recent proposal that the sensor in voltage-dependent K+ channels is located at the membrane-protein interface.

  17. Latent Ice Recrystallization Inhibition Activity in Nonantifreeze Proteins: Ca2+-Activated Plant Lectins and Cation-Activated Antimicrobial Peptides.

    Science.gov (United States)

    Mitchell, Daniel E; Gibson, Matthew I

    2015-10-12

    Organisms living in polar regions have evolved a series of antifreeze (glyco) proteins (AFGPs) to enable them to survive by modulating the structure of ice. These proteins have huge potential for use in cellular cryopreservation, ice-resistant surfaces, frozen food, and cryosurgery, but they are limited by their relatively low availability and questions regarding their mode of action. This has triggered the search for biomimetic materials capable of reproducing this function. The identification of new structures and sequences capable of inhibiting ice growth is crucial to aid our understanding of these proteins. Here, we show that plant c-type lectins, which have similar biological function to human c-type lectins (glycan recognition) but no sequence homology to AFPs, display calcium-dependent ice recrystallization inhibition (IRI) activity. This IRI activity can be switched on/off by changing the Ca2+ concentration. To show that more (nonantifreeze) proteins may exist with the potential to display IRI, a second motif was considered, amphipathicity. All known AFPs have defined hydrophobic/hydrophilic domains, rationalizing this choice. The cheap, and widely used, antimicrobial Nisin was found to have cation-dependent IRI activity, controlled by either acid or addition of histidine-binding ions such as zinc or nickel, which promote its amphipathic structure. These results demonstrate a new approach in the identification of antifreeze protein mimetic macromolecules and may help in the development of synthetic mimics of AFPs.

  18. Phentolamine and yohimbine inhibit ATP-sensitive K+ channels in mouse pancreatic beta-cells.

    OpenAIRE

    Plant, T D; Henquin, J C

    1990-01-01

    1. The effects of phentolamine and yohimbine on adenosine 5'-triphosphate (ATP)-sensitive K+ channels were studied in normal mouse beta-cells. 2. In the presence of 3 mM glucose, many ATP-sensitive K+ channels are open in the beta-cell membrane. Under these conditions, phentolamine inhibited 86Rb efflux from the islets. This inhibition was faster with 100 than with 20 microM phentolamine but its steady-state magnitude was similar with both concentrations. Yohimbine (20-100 microM) also inhibi...

  19. SLO BK Potassium Channels Couple Gap Junctions to Inhibition of Calcium Signaling in Olfactory Neuron Diversification.

    Science.gov (United States)

    Alqadah, Amel; Hsieh, Yi-Wen; Schumacher, Jennifer A; Wang, Xiaohong; Merrill, Sean A; Millington, Grethel; Bayne, Brittany; Jorgensen, Erik M; Chuang, Chiou-Fen

    2016-01-01

    The C. elegans AWC olfactory neuron pair communicates to specify asymmetric subtypes AWCOFF and AWCON in a stochastic manner. Intercellular communication between AWC and other neurons in a transient NSY-5 gap junction network antagonizes voltage-activated calcium channels, UNC-2 (CaV2) and EGL-19 (CaV1), in the AWCON cell, but how calcium signaling is downregulated by NSY-5 is only partly understood. Here, we show that voltage- and calcium-activated SLO BK potassium channels mediate gap junction signaling to inhibit calcium pathways for asymmetric AWC differentiation. Activation of vertebrate SLO-1 channels causes transient membrane hyperpolarization, which makes it an important negative feedback system for calcium entry through voltage-activated calcium channels. Consistent with the physiological roles of SLO-1, our genetic results suggest that slo-1 BK channels act downstream of NSY-5 gap junctions to inhibit calcium channel-mediated signaling in the specification of AWCON. We also show for the first time that slo-2 BK channels are important for AWC asymmetry and act redundantly with slo-1 to inhibit calcium signaling. In addition, nsy-5-dependent asymmetric expression of slo-1 and slo-2 in the AWCON neuron is necessary and sufficient for AWC asymmetry. SLO-1 and SLO-2 localize close to UNC-2 and EGL-19 in AWC, suggesting a role of possible functional coupling between SLO BK channels and voltage-activated calcium channels in AWC asymmetry. Furthermore, slo-1 and slo-2 regulate the localization of synaptic markers, UNC-2 and RAB-3, in AWC neurons to control AWC asymmetry. We also identify the requirement of bkip-1, which encodes a previously identified auxiliary subunit of SLO-1, for slo-1 and slo-2 function in AWC asymmetry. Together, these results provide an unprecedented molecular link between gap junctions and calcium pathways for terminal differentiation of olfactory neurons.

  20. Membrane coordination of receptors and channels mediating the inhibition of neuronal ion currents by ADP.

    Science.gov (United States)

    Gafar, Hend; Dominguez Rodriguez, Manuel; Chandaka, Giri K; Salzer, Isabella; Boehm, Stefan; Schicker, Klaus

    2016-09-01

    ADP and other nucleotides control ion currents in the nervous system via various P2Y receptors. In this respect, Cav2 and Kv7 channels have been investigated most frequently. The fine tuning of neuronal ion channel gating via G protein coupled receptors frequently relies on the formation of higher order protein complexes that are organized by scaffolding proteins and harbor receptors and channels together with interposed signaling components. However, ion channel complexes containing P2Y receptors have not been described. Therefore, the regulation of Cav2.2 and Kv7.2/7.3 channels via P2Y1 and P2Y12 receptors and the coordination of these ion channels and receptors in the plasma membranes of tsA 201 cells have been investigated here. ADP inhibited currents through Cav2.2 channels via both P2Y1 and P2Y12 receptors with phospholipase C and pertussis toxin-sensitive G proteins being involved, respectively. The nucleotide controlled the gating of Kv7 channels only via P2Y1 and phospholipase C. In fluorescence energy transfer assays using conventional as well as total internal reflection (TIRF) microscopy, both P2Y1 and P2Y12 receptors were found juxtaposed to Cav2.2 channels, but only P2Y1, and not P2Y12, was in close proximity to Kv7 channels. Using fluorescence recovery after photobleaching in TIRF microscopy, evidence for a physical interaction was obtained for the pair P2Y12/Cav2.2, but not for any other receptor/channel combination. These results reveal a membrane juxtaposition of P2Y receptors and ion channels in parallel with the control of neuronal ion currents by ADP. This juxtaposition may even result in apparent physical interactions between receptors and channels.

  1. Polyamine triggering of exocytosis in Paramecium involves an extracellular Ca(2+)/(polyvalent cation)-sensing receptor, subplasmalemmal Ca-store mobilization and store-operated Ca(2+)-influx via unspecific cation channels.

    Science.gov (United States)

    Klauke, N; Blanchard, M; Plattner, H

    2000-03-15

    The polyamine secretagogue, aminoethyldextran (AED), causes a cortical [Ca(2+)] transient in Paramecium cells, as analyzed by fluorochrome imaging. Our most essential findings are: (i) Cortical Ca(2+) signals also occur when AED is applied in presence of the fast Ca(2+) chelator, BAPTA. (ii) Extracellular La(3+) application causes within seconds a rapid, reversible fluorescence signal whose reversibility can be attributed to a physiological [Ca(2+)](i) transient (while injected La(3+) causes a sustained fluorescence signal). (iii) Simply increasing [Ca(2+)](o) causes a similar rapid, short-lived [Ca(2+)](i) transient. All these phenomena, (i-iii), are compatible with activation of an extracellular "Ca(2+)/(polyvalent cation)-sensing receptor" known from some higher eukaryotic systems, where this sensor (responding to Ca(2+), La(3+) and some multiply charged cations) is linked to cortical calcium stores which, thus, are activated. In Paramecium, such subplasmalemmal stores ("alveolar sacs") are physically linked to the cell membrane and they can also be activated by the Ca(2+) releasing agent, 4-chloro-m-cresol, just like in Sarcoplasmic Reticulum. Since this drug causes a cortical Ca(2+) signal also in absence of Ca(2+)(o) we largely exclude a "Ca(2+)-induced Ca(2+) release" (CICR) mechanism. Our finding of increased cortical Ca(2+) signals after store depletion and re-addition of extracellular Ca(2+) can be explained by a "store-operated Ca(2+) influx" (SOC), i.e., a Ca(2+) influx superimposing store activation. AED stimulation in presence of Mn(2+)(o) causes fluorescence quenching in Fura-2 loaded cells, indicating involvement of unspecific cation channels. Such channels, known to occur in Paramecium, share some general characteristics of SOC-type Ca(2+) influx channels. In conclusion, we assume the following sequence of events during AED stimulated exocytosis: (i) activation of an extracellular Ca(2+)/polyamine-sensing receptor, (ii) release of Ca(2+) from

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

    Science.gov (United States)

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

    2014-09-19

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

  3. Stereoselective inhibition of the hERG1 potassium channel

    Directory of Open Access Journals (Sweden)

    Liliana eSintra Grilo

    2010-11-01

    Full Text Available A growing number of drugs have been shown to prolong cardiac repolarization, predisposing individuals to life-threatening ventricular arrhythmias known as Torsades de Pointes. Most of these drugs are known to interfere with the human ether à-gogo related gene 1 (hERG1 channel, whose current is one of the main determinants of action potential duration. Prolonged repolarization is reflected by lengthening of the QT interval of the electrocardiogram, as seen in the suitably named drug-induced long QT syndrome. Chirality (presence of an asymmetric atom is a common feature of marketed drugs, which can therefore exist in at least two enantiomers with distinct three-dimensional structures and possibly distinct biological fates. Both the pharmacokinetic and pharmacodynamic properties can differ between enantiomers, as well as also between individuals who take the drug due to metabolic polymorphisms. Despite the large number of reports about drugs reducing the hERG1 current, potential stereoselective contributions have only been scarcely investigated. In this review, we present a non-exhaustive list of clinically important molecules which display chiral toxicity that may be related to hERG1-blocking properties. We particularly focus on methadone cardiotoxicity, which illustrates the importance of the stereoselective effect of drug chirality as well as individual variations resulting from pharmacogenetics. Furthermore, it seems likely that, during drug development, consideration of chirality in lead optimization and systematic assessment of the hERG1 current block with all enantiomers could contribute to the reduction of the risk of drug-induced LQTS.

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

    DEFF Research Database (Denmark)

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

    2011-01-01

    We have shown previously that inhibition of small conductance Ca(2+)-activated K(+) (SK) channels is antiarrhythmic in models of acutely induced atrial fibrillation (AF). These models, however, do not take into account that AF derives from a wide range of predisposing factors, the most prevalent ...

  5. Inhibition of G protein-activated inwardly rectifying K+ channels by different classes of antidepressants.

    Directory of Open Access Journals (Sweden)

    Toru Kobayashi

    Full Text Available Various antidepressants are commonly used for the treatment of depression and several other neuropsychiatric disorders. In addition to their primary effects on serotonergic or noradrenergic neurotransmitter systems, antidepressants have been shown to interact with several receptors and ion channels. However, the molecular mechanisms that underlie the effects of antidepressants have not yet been sufficiently clarified. G protein-activated inwardly rectifying K(+ (GIRK, Kir3 channels play an important role in regulating neuronal excitability and heart rate, and GIRK channel modulation has been suggested to have therapeutic potential for several neuropsychiatric disorders and cardiac arrhythmias. In the present study, we investigated the effects of various classes of antidepressants on GIRK channels using the Xenopus oocyte expression assay. In oocytes injected with mRNA for GIRK1/GIRK2 or GIRK1/GIRK4 subunits, extracellular application of sertraline, duloxetine, and amoxapine effectively reduced GIRK currents, whereas nefazodone, venlafaxine, mianserin, and mirtazapine weakly inhibited GIRK currents even at toxic levels. The inhibitory effects were concentration-dependent, with various degrees of potency and effectiveness. Furthermore, the effects of sertraline were voltage-independent and time-independent during each voltage pulse, whereas the effects of duloxetine were voltage-dependent with weaker inhibition with negative membrane potentials and time-dependent with a gradual decrease in each voltage pulse. However, Kir2.1 channels were insensitive to all of the drugs. Moreover, the GIRK currents induced by ethanol were inhibited by sertraline but not by intracellularly applied sertraline. The present results suggest that GIRK channel inhibition may reveal a novel characteristic of the commonly used antidepressants, particularly sertraline, and contributes to some of the therapeutic effects and adverse effects.

  6. Immunization with cationized BSA inhibits progression of disease in ApoBec-1/LDL receptor deficient mice with manifest atherosclerosis.

    Science.gov (United States)

    Kolbus, Daniel; Wigren, Maria; Ljungcrantz, Irena; Söderberg, Ingrid; Alm, Ragnar; Björkbacka, Harry; Nilsson, Jan; Fredrikson, Gunilla N

    2011-06-01

    Immune responses against modified self-antigens generated by hypercholesterolemia play an important role in atherosclerosis identifying the immune system as a possible novel target for prevention and treatment of cardiovascular disease. It has recently been shown that these immune responses can be modulated by subcutaneous injection of adjuvant. In the present study we immunized 25-week old ApoBec-1/LDL receptor deficient mice with manifest atherosclerosis with adjuvant and two different concentrations of the carrier molecule cationized BSA (cBSA). Plasma levels of Th2-induced apolipoprotein B (apoB)/IgG1 immune complexes were increased in the cBSA immunized groups verifying induction of immunity against a self-antigen. Mice were sacrificed at 36 weeks of age and atherosclerosis was monitored by en face Oil red O staining of the aorta. Immunization with 100 μg cBSA inhibited plaque progression, whereas the lower dose (50 μg) did not. In addition, the higher dose induced a more stable plaque phenotype, indicated by a higher content of collagen and less macrophages and T cells in the plaques. Moreover, there was an increased ratio of Foxp3+/Foxp3⁻ T cells in the circulation suggesting activation of a regulatory T cell response. In conclusion, we show that immunization with cBSA induces an immune response against apoB as well as an activation of Treg cells. This was associated with development of a more stable plaque phenotype and reduced atherosclerosis progression.

  7. Inhibition of Na(+) -K+ pump activity by divalent cations in intact peritoneal mast cells of the rat

    DEFF Research Database (Denmark)

    Knudsen, T; Berthelsen, Carsten; Johansen, Torben

    1990-01-01

    1. The inhibition by the divalent cations magnesium, barium and strontium and the trivalent ion lanthanum of the Na(+) -K+ pump in the plasma membrane of rat peritoneal mast cells was studied in pure mast cell populations by measurement of the ouabain-sensitive uptake of the radioactive potassium......-resistant uptake was not changed. Half maximum decrease in the ouabain-sensitive K+(86Rb+)-uptake was observed with 1.8 mM magnesium, 1.2mM barium and 0.7 mM strontium. 4. The trivalent ion lanthanum blocked almost completely the ouabain-sensitive K+(86Rb+)-uptake at a concentration of 1 microM as does 1 m......M calcium. Combining either of these ions with magnesium had no further inhibitory effect on the ouabain-sensitive uptake. 5. In conclusion, in addition to the previously suggested modulation by calcium of the activity of the Na+ (-)K+ pump, evidence is provided in this investigation that the modulation may...

  8. Cell-penetrating peptide derived from human eosinophil cationic protein inhibits mite allergen Der p 2 induced inflammasome activation.

    Directory of Open Access Journals (Sweden)

    Sheng-Jie Yu

    Full Text Available Newly discovered cell penetration peptides derived from human eosinophil cationic proteins (CPPecp have the characteristic of cell internalization, but the effect of CPPecp on immunomodulation has not been clarified. House dust mite (HDM major allergen, Der p 2, can induce proinflammatory cytokine production which contributes to airway inflammation and allergic asthma. However, the mechanism of Der p 2 on NLRP3 inflammasome activation remains unclear. The aim of this study was to investigate the immunomodulatory effect of CPPecp on inhibition of Der p 2 induced inflammasome activation. We showed that proinflammatory cytokines IL-1β, IL-6 and IL-8 were significantly upregulated in peripheral blood mononuclear cells (PBMCs derived from HDM allergic patients after Der p 2 stimulation. Expression of NLRP3, ASC, Caspase-1, IL-1β and Caspase-1 activity was upregulated in THP-1 cells after Der p 2 stimulation. Proinflammatory cytokine production, NLRP3 inflammasome activation and caspase-1 activity were downregulated in THP-1 cells and CD14+ cells co-cultured with Der p 2 and CPPecp. The immunomodulatory effect of CPPecp was through upregulation of IFN-α production but not induction of autophagy. These results suggested Der p 2 plays an important role in NLRP3 inflammasome activation and CPPecp has the potential to be a novel anti-inflammatory agent for allergic inflammation treatment in the future.

  9. Gating of the two-pore cation channel AtTPC1 in the plant vacuole is based on a single voltage-sensing domain.

    Science.gov (United States)

    Jaślan, D; Mueller, T D; Becker, D; Schultz, J; Cuin, T A; Marten, I; Dreyer, I; Schönknecht, G; Hedrich, R

    2016-09-01

    The two-pore cation channel TPC1 operates as a dimeric channel in animal and plant endomembranes. Each subunit consists of two homologous Shaker-like halves, with 12 transmembrane domains in total (S1-S6, S7-S12). In plants, TPC1 channels reside in the vacuolar membrane, and upon voltage stimulation, give rise to the well-known slow-activating SV currents. Here, we combined bioinformatics, structure modelling, site-directed mutagenesis, and in planta patch clamp studies to elucidate the molecular mechanisms of voltage-dependent channel gating in TPC1 in its native plant background. Structure-function analysis of the Arabidopsis TPC1 channel in planta confirmed that helix S10 operates as the major voltage-sensing site, with Glu450 and Glu478 identified as possible ion-pair partners for voltage-sensing Arg537. The contribution of helix S4 to voltage sensing was found to be negligible. Several conserved negative residues on the luminal site contribute to calcium binding, stabilizing the closed channel. During evolution of plant TPC1s from two separate Shaker-like domains, the voltage-sensing function in the N-terminal Shaker-unit (S1-S4) vanished.

  10. An RNA aptamer possessing a novel monovalent cation-mediated fold inhibits lysozyme catalysis by inhibiting the binding of long natural substrates.

    Science.gov (United States)

    Padlan, Camille S; Malashkevich, Vladimir N; Almo, Steve C; Levy, Matthew; Brenowitz, Michael; Girvin, Mark E

    2014-04-01

    RNA aptamers are being developed as inhibitors of macromolecular and cellular function, diagnostic tools, and potential therapeutics. Our understanding of the physical nature of this emerging class of nucleic acid-protein complexes is limited; few atomic resolution structures have been reported for aptamers bound to their protein target. Guided by chemical mapping, we systematically minimized an RNA aptamer (Lys1) selected against hen egg white lysozyme. The resultant 59-nucleotide compact aptamer (Lys1.2minE) retains nanomolar binding affinity and the ability to inhibit lysozyme's catalytic activity. Our 2.0-Å crystal structure of the aptamer-protein complex reveals a helical stem stabilizing two loops to form a protein binding platform that binds lysozyme distal to the catalytic cleft. This structure along with complementary solution analyses illuminate a novel protein-nucleic acid interface; (1) only 410 Å(2) of solvent accessible surface are buried by aptamer binding; (2) an unusually small fraction (∼18%) of the RNA-protein interaction is electrostatic, consistent with the limited protein phosphate backbone contacts observed in the structure; (3) a single Na(+) stabilizes the loops that constitute the protein-binding platform, and consistent with this observation, Lys1.2minE-lysozyme complex formation takes up rather than displaces cations at low ionic strength; (4) Lys1.2minE inhibits catalysis of large cell wall substrates but not catalysis of small model substrates; and (5) the helical stem of Lys1.2minE can be shortened to four base pairs (Lys1.2minF) without compromising binding affinity, yielding a 45-nucleotide aptamer whose structure may be an adaptable protein binding platform.

  11. Shikonin inhibits intestinal calcium-activated chloride channels and prevents rotaviral diarrhea

    Directory of Open Access Journals (Sweden)

    Yu Jiang

    2016-08-01

    Full Text Available Secretory diarrhea remains a global health burden and causes major mortality in children. There have been some focuses on antidiarrheal therapies that may reduce fluid losses and intestinal motility in diarrheal diseases. In the present study, we identified shikonin as an inhibitor of TMEM16A chloride channel activity using cell-based fluorescent-quenching assay. The IC50 value of shikonin was 6.5 μM. Short-circuit current measurements demonstrated that shikonin inhibited Eact-induced Cl current in a dose-dependent manner, with IC50 value of 1.5 μM. Short-circuit current measurement showed that shikonin exhibited inhibitory effect against CCh-induced Cl currents in mouse colonic epithelia but did not affect cytoplasmic Ca2+ concentration as well as the other major enterocyte chloride channel CFTR. Characterization study found that shikonin inhibited basolateral K+ channel activity without affecting Na+/K+-ATPase activities. In-vivo studies revealed that shikonin significantly delayed intestinal motility in mice and reduced stool water content in a neonatal mice model of rotaviral diarrhea without affecting the viral infection process in-vivo. Taken together, the results suggested that shikonin inhibited enterocyte CaCCs, the inhibitory effect was partially through inhbition of basolateral K+ channel acitivty, and shikonin could be a lead compound in the treatment of rotaviral secretory diarrhea.

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

    DEFF Research Database (Denmark)

    Christensen, Ove; Hoffmann, Else Kay

    1992-01-01

    not occur instantaneously but within a time delay of 1/2 to 1 min. The channel is permeable to Ba2+ and hence presumably to Ca2+. It seems likely that the function of the nonselective, stretch-activated channels is correlated with their inferred Ca2+ permeability, as part of the volume-activated signal...... external K+ is estimated at about 7 pS. A K+ channel with similar properties can be activated in the cellattached mode by addition of Ca2+ plus ionophore A23187. The channel is also activated by cell swelling, within 1 min following hypotonic exposure. No evidence was found of channel activation...... types of Cl– channels were regularly recorded in excised inside-out patches: a voltage-activated 400-pS channel and a 34-pS Cl– channel which show properties similar to the Cl– channel in the apical membrane in human airway epithelial cells. There is no evidence for a role in RVD for either of these two...

  13. Subtype-specific, bi-component inhibition of SK channels by low internal pH

    DEFF Research Database (Denmark)

    Peitersen, Torben; Jespersen, Thomas; Jorgensen, Nanna K;

    2006-01-01

    The effects of low intracellular pH (pH(i) 6.4) on cloned small-conductance Ca2+-activated K+ channel currents of all three subtypes (SK1, SK2, and SK3) were investigated in HEK293 cells using the patch-clamp technique. In 400 nM internal Ca2+ [Ca2+]i, all subtypes were inhibited by pH(i) 6...

  14. Extracellular potassium inhibits Kv7.1 potassium channels by stabilizing an inactivated state

    DEFF Research Database (Denmark)

    Larsen, Anders Peter; Steffensen, Annette Buur; Grunnet, Morten;

    2011-01-01

    Kv7.1 (KCNQ1) channels are regulators of several physiological processes including vasodilatation, repolarization of cardiomyocytes, and control of secretory processes. A number of Kv7.1 pore mutants are sensitive to extracellular potassium. We hypothesized that extracellular potassium also...... modulates wild-type Kv7.1 channels. The Kv7.1 currents were measured in Xenopus laevis oocytes at different concentrations of extracellular potassium (1-50 mM). As extracellular potassium was elevated, Kv7.1 currents were reduced significantly more than expected from theoretical calculations based...... on the Goldman-Hodgkin-Katz flux equation. Potassium inhibited the steady-state current with an IC(50) of 6.0 ± 0.2 mM. Analysis of tail-currents showed that potassium increased the fraction of channels in the inactivated state. Similarly, the recovery from inactivation was slowed by potassium, suggesting...

  15. Mechanosensitive ion channel Piezo2 is inhibited by D-GsMTx4.

    Science.gov (United States)

    Alcaino, Constanza; Knutson, Kaitlyn; Gottlieb, Philip A; Farrugia, Gianrico; Beyder, Arthur

    2017-01-13

    Enterochromaffin (EC) cells are the primary mechanosensors of the gastrointestinal (GI) epithelium. In response to mechanical stimuli EC cells release serotonin (5-hydroxytryptamine; 5-HT). The molecular details of EC cell mechanosensitivity are poorly understood. Recently, our group found that human and mouse EC cells express the mechanosensitive ion channel Piezo2. The mechanosensitive currents in a human EC cell model QGP-1 were blocked by the mechanosensitive channel blocker D-GsMTx4. In the present study we aimed to characterize the effects of the mechanosensitive ion channel inhibitor spider peptide D-GsMTx4 on the mechanically stimulated currents from both QGP-1 and human Piezo2 transfected HEK-293 cells. We found co-localization of 5-HT and Piezo2 in QGP-1 cells by immunohistochemistry. QGP-1 mechanosensitive currents had biophysical properties similar to dose-dependently Piezo2 and were inhibited by D-GsMTx4. In response to direct displacement of cell membranes, human Piezo2 transiently expressed in HEK-293 cells produced robust rapidly activating and inactivating inward currents. D-GsMTx4 reversibly and dose-dependently inhibited both the potency and efficacy of Piezo2 currents in response to mechanical force. Our data demonstrate an effective inhibition of Piezo2 mechanosensitive currents by the spider peptide D-GsMTx4.

  16. Structure and inhibition of the SARS coronavirus envelope protein ion channel.

    Directory of Open Access Journals (Sweden)

    Konstantin Pervushin

    2009-07-01

    Full Text Available The envelope (E protein from coronaviruses is a small polypeptide that contains at least one alpha-helical transmembrane domain. Absence, or inactivation, of E protein results in attenuated viruses, due to alterations in either virion morphology or tropism. Apart from its morphogenetic properties, protein E has been reported to have membrane permeabilizing activity. Further, the drug hexamethylene amiloride (HMA, but not amiloride, inhibited in vitro ion channel activity of some synthetic coronavirus E proteins, and also viral replication. We have previously shown for the coronavirus species responsible for severe acute respiratory syndrome (SARS-CoV that the transmembrane domain of E protein (ETM forms pentameric alpha-helical bundles that are likely responsible for the observed channel activity. Herein, using solution NMR in dodecylphosphatidylcholine micelles and energy minimization, we have obtained a model of this channel which features regular alpha-helices that form a pentameric left-handed parallel bundle. The drug HMA was found to bind inside the lumen of the channel, at both the C-terminal and the N-terminal openings, and, in contrast to amiloride, induced additional chemical shifts in ETM. Full length SARS-CoV E displayed channel activity when transiently expressed in human embryonic kidney 293 (HEK-293 cells in a whole-cell patch clamp set-up. This activity was significantly reduced by hexamethylene amiloride (HMA, but not by amiloride. The channel structure presented herein provides a possible rationale for inhibition, and a platform for future structure-based drug design of this potential pharmacological target.

  17. Fluoxetine-induced inhibition of synaptosomal ( sup 3 H)5-HT release: Possible Ca sup 2+ -channel inhibition

    Energy Technology Data Exchange (ETDEWEB)

    Stauderman, K.A. (Marion Merrell Dow Research Inst., Cincinnati, OH (United States)); Gandhi, V.C.; Jones, D.J. (Univ. of Texas Health Science Center, San Antonio, TX (United States))

    1992-01-01

    Fluoxetine, a selective 5-Ht uptake inhibitor, inhibited 15 mM K{sup +}-induced ({sup 3}H)5-HT release from rat spinal cord and cortical synaptosomes at concentrations > 0.5 uM. This effect reflected a property shared by another selective 5-HT uptake inhibitor paroxetine but not by less selective uptake inhibitors such as amitriptyline, desipramine, imipramine or nortriptyline. Inhibition of release by fluoxetine was inversely related to both the concentration of K{sup +} used to depolarize the synaptosomes and the concentration of external Ca{sup 2+}. Experiments aimed at determining a mechanism of action revealed that fluoxetine did not inhibit voltage-independent release of ({sup 3}H)5-HT release induced by the Ca{sup 2+}-ionophore A 23187 or Ca{sup 2+}-independent release induced by fenfluramine. Moreover the 5-HT autoreceptor antagonist methiothepin did not reverse the inhibitory actions of fluoxetine on K{sup +}-induced release. Further studies examined the effects of fluoxetine on voltage-dependent Ca{sup 2+} channels and Ca{sup 2+} entry.

  18. Estradiol inhibits Ca2+ and K+ channels in smooth muscle cells from pregnant rat myometrium.

    Science.gov (United States)

    Okabe, K; Inoue, Y; Soeda, H

    1999-07-02

    The purpose of this study was to investigate the actions of 17beta-estradiol on the electrical activity of pregnant rat myometrium. The longitudinal layer of the myometrium was dissected from pregnant rats (17 to 19 days of gestation), and single cells were isolated by enzymatic digestion. Calcium currents and potassium currents were recorded by the whole-cell voltage-clamp method, and the single calcium-dependent potassium current was recorded by the outside-out patch-clamp method. The effects of 17beta-estradiol on these currents were investigated. When a myometrial cell was held at -50 mV, depolarization to a potential more positive than -30 mV produced an inward current followed by a slowly developing outward current. Application of tetraethylammonium inhibited the outward current while the inward current was completely abolished in a calcium-free solution. Estradiol at high concentrations (> 3 microM) inhibited both inward and outward currents in a voltage-dependent manner. Removal of estradiol restored the amplitude of the outward but not of the inward current. Estradiol (30 microM) also inhibited the activity of single calcium-dependent potassium channels without changing single channel conductance. In conclusion, estradiol at high concentrations inhibited: (1) voltage-dependent calcium, (2) calcium-dependent potassium and (3) voltage-dependent potassium currents. These actions of estradiol would prevent action potential generation and after-hyperpolarizations. Suppression of the after-hyperpolarization might further prevent spike generation due to slowing of the calcium channel's recovery from the inactivated state.

  19. Parthenolide inhibits nociception and neurogenic vasodilatation in the trigeminovascular system by targeting the TRPA1 channel.

    Science.gov (United States)

    Materazzi, Serena; Benemei, Silvia; Fusi, Camilla; Gualdani, Roberta; De Siena, Gaetano; Vastani, Nisha; Andersson, David A; Trevisan, Gabriela; Moncelli, Maria Rosa; Wei, Xiaomei; Dussor, Gregory; Pollastro, Federica; Patacchini, Riccardo; Appendino, Giovanni; Geppetti, Pierangelo; Nassini, Romina

    2013-12-01

    Although feverfew has been used for centuries to treat pain and headaches and is recommended for migraine treatment, the mechanism for its protective action remains unknown. Migraine is triggered by calcitonin gene-related peptide (CGRP) release from trigeminal neurons. Peptidergic sensory neurons express a series of transient receptor potential (TRP) channels, including the ankyrin 1 (TRPA1) channel. Recent findings have identified agents either inhaled from the environment or produced endogenously that are known to trigger migraine or cluster headache attacks, such as TRPA1 simulants. A major constituent of feverfew, parthenolide, may interact with TRPA1 nucleophilic sites, suggesting that feverfew's antimigraine effect derives from its ability to target TRPA1. We found that parthenolide stimulates recombinant (transfected cells) or natively expressed (rat/mouse trigeminal neurons) TRPA1, where it, however, behaves as a partial agonist. Furthermore, in rodents, after initial stimulation, parthenolide desensitizes the TRPA1 channel and renders peptidergic TRPA1-expressing nerve terminals unresponsive to any stimulus. This effect of parthenolide abrogates nociceptive responses evoked by stimulation of peripheral trigeminal endings. TRPA1 targeting and neuronal desensitization by parthenolide inhibits CGRP release from trigeminal neurons and CGRP-mediated meningeal vasodilatation, evoked by either TRPA1 agonists or other unspecific stimuli. TRPA1 partial agonism, together with desensitization and nociceptor defunctionalization, ultimately resulting in inhibition of CGRP release within the trigeminovascular system, may contribute to the antimigraine effect of parthenolide.

  20. Parthenolide inhibits nociception and neurogenic vasodilatation in the trigeminovascular system by targeting TRPA1 channel

    Science.gov (United States)

    Materazzi, Serena; Benemei, Silvia; Fusi, Camilla; Gualdani, Roberta; De Siena, Gaetano; Vastani, Nisha; Andersson, David A.; Trevisan, Gabriela; Moncelli, Maria Rosa; Wei, Xiaomei; Dussor, Gregory; Pollastro, Federica; Patacchini, Riccardo; Appendino, Giovanni; Geppetti, Pierangelo; Nassini, Romina

    2013-01-01

    While feverfew has been used for centuries to treat pain and headaches and is recommended for migraine treatment, the mechanism for its protective action remains unknown. Migraine is triggered by calcitonin gene-related peptide (CGRP) release from trigeminal neurons. Peptidergic sensory neurons, express a series of transient receptor potential (TRP) channels, including the ankyrin 1 (TRPA1) channel. Recent findings have identified agents either inhaled from the environment or produced endogenously, which are known to trigger migraine or cluster headache attacks, as TRPA1 simulants. A major constituent of feverfew, parthenolide, may interact with TRPA1 nucleophilic sites, suggesting that feverfew antimigraine effect derives from its ability to target TRPA1. We found that parthenolide stimulates recombinant (transfected cells) or natively expressed (rat/mouse trigeminal neurons) TRPA1, where it, however, behaves as a partial agonist. Furthermore, in rodents, after initial stimulation, parthenolide desensitizes the TRPA1 channel, and renders peptidergic, TRPA1-expressing nerve terminals unresponsive to any stimulus. This effect of parthenolide abrogates nociceptive responses evoked by stimulation of peripheral trigeminal endings. TRPA1 targeting and neuronal desensitization by parthenolide inhibits CGRP release from trigeminal neurons and CGRP-mediated meningeal vasodilatation, evoked by either TRPA1 agonists or other unspecific stimuli. TRPA1 partial agonism, together with desensitization and nociceptor defunctionalization, ultimately resulting in inhibition of CGRP release within the trigeminovascular system, may contribute to the antimigraine effect of parthenolide. PMID:23933184

  1. CRAC channel is inhibited by neomycin in a Ptdlns(4,5)P2-independent manner.

    Science.gov (United States)

    Huang, Kun; Wang, Xuemei; Liu, Yanjun; Zhao, Yi

    2015-03-01

    Depletion of intracellular Ca(2+) stores evokes store-operated Ca(2+) entry through the Ca(2+) release-activated Ca(2+) (CRAC) channels. In this study, we found that the store-operated Ca(2+) entry was inhibited by neomycin, an aminoglycoside that strongly binds phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P2). Patch clamp recordings revealed that neomycin blocked the CRAC currents reconstituted by co-expression of Orai1 and Stim1 in HEK293 cells. Using a rapamycin-inducible PtdIns(4,5)P2-specific phosphatase (Inp54p) system to manipulate the PtdIns(4,5)P2 in the plasma membrane, we found that the CRAC current was not altered by PtdIns(4,5)P2 depletion. This result suggests that PtdIns(4,5)P2 is not required for CRAC channel activity, and thereby, neomycin inhibits CRAC channels in a manner that is independent of neomycin-PtdIns(4,5)P2 binding.

  2. The cyclic nucleotide gated cation channel AtCNGC10 traffics from the ER via Golgi vesicles to the plasma membrane of Arabidopsis root and leaf cells

    Directory of Open Access Journals (Sweden)

    Andres Marilou A

    2007-09-01

    Full Text Available Abstract Background The cyclic nucleotide-gated ion channels (CNGCs maintain cation homeostasis essential for a wide range of physiological processes in plant cells. However, the precise subcellular locations and trafficking of these membrane proteins are poorly understood. This is further complicated by a general deficiency of information about targeting pathways of membrane proteins in plants. To investigate CNGC trafficking and localization, we have measured Atcngc5 and Atcngc10 expression in roots and leaves, analyzed AtCNGC10-GFP fusions transiently expressed in protoplasts, and conducted immunofluorescence labeling of protoplasts and immunoelectron microscopic analysis of high pressure frozen leaves and roots. Results AtCNGC10 mRNA and protein levels were 2.5-fold higher in roots than leaves, while AtCNGC5 mRNA and protein levels were nearly equal in these tissues. The AtCNGC10-EGFP fusion was targeted to the plasma membrane in leaf protoplasts, and lightly labeled several intracellular structures. Immunofluorescence microscopy with affinity purified CNGC-specific antisera indicated that AtCNGC5 and AtCNGC10 are present in the plasma membrane of protoplasts. Immunoelectron microscopy demonstrated that AtCNGC10 was associated with the plasma membrane of mesophyll, palisade parenchyma and epidermal cells of leaves, and the meristem, columella and cap cells of roots. AtCNCG10 was also observed in the endoplasmic reticulum and Golgi cisternae and vesicles of 50–150 nm in size. Patch clamp assays of an AtCNGC10-GFP fusion expressed in HEK293 cells measured significant cation currents. Conclusion AtCNGC5 and AtCNGC10 are plasma membrane proteins. We postulate that AtCNGC10 traffics from the endoplasmic reticulum via the Golgi apparatus and associated vesicles to the plasma membrane. The presence of the cation channel, AtCNGC10, in root cap meristem cells, cell plate, and gravity-sensing columella cells, combined with the previously reported

  3. Ca2+ channel inhibition by endomorphins via the cloned mu-opioid receptor expressed in NG108-15 cells.

    Science.gov (United States)

    Mima, H; Morikawa, H; Fukuda, K; Kato, S; Shoda, T; Mori, K

    1997-12-11

    Endomorphin-1 and -2, recently isolated endogenous peptides specific for the mu-opioid receptor, inhibited Ca2+ channel currents with EC50 of 6 and 9 nM, respectively, in NG108-15 cells transformed to express the cloned rat mu-opioid receptor. On the other hand, they elicited no response in nontransfected NG108-15 cells. It is concluded that endomorphin-1 and -2 induce Ca2+ channel inhibition by selectively activating the mu-opioid receptor.

  4. Contribution of the S5-pore-S6 domain to the gating characteristics of the cation channels TRPM2 and TRPM8.

    Science.gov (United States)

    Kühn, Frank J P; Witschas, Katja; Kühn, Cornelia; Lückhoff, Andreas

    2010-08-27

    The closely related cation channels TRPM2 and TRPM8 show completely different requirements for stimulation and are regulated by Ca(2+) in an opposite manner. TRPM8 is basically gated in a voltage-dependent process enhanced by cold temperatures and cooling compounds such as menthol and icilin. The putative S4 voltage sensor of TRPM8 is closely similar to that of TRPM2, which, however, is mostly devoid of voltage sensitivity. To gain insight into principal interactions of critical channel domains during the gating process, we created chimeras in which the entire S5-pore-S6 domains were reciprocally exchanged. The chimera M2-M8P (i.e. TRPM2 with the pore of TRPM8) responded to ADP-ribose and hydrogen peroxide and was regulated by extracellular and intracellular Ca(2+) as was wild-type TRPM2. Single-channel recordings revealed the characteristic pattern of TRPM2 with extremely long open times. Only at far-negative membrane potentials (-120 to -140 mV) did differences become apparent because currents were reduced by hyperpolarization in M2-M8P but not in TRPM2. The reciprocal chimera, M8-M2P, showed currents after stimulation with high concentrations of menthol and icilin, but these currents were only slightly larger than in controls. The transfer of the NUDT9 domain to the C terminus of TRPM8 produced a channel sensitive to cold, menthol, or icilin but insensitive to ADP-ribose or hydrogen peroxide. We conclude that the gating processes in TRPM2 and TRPM8 differ in their requirements for specific structures within the pore. Moreover, the regulation by extracellular and intracellular Ca(2+) and the single-channel properties in TRPM2 are not determined by the S5-pore-S6 region.

  5. Inhibition of hepatic fibrosis with artificial microRNA using ultrasound and cationic liposome-bearing microbubbles.

    Science.gov (United States)

    Yang, D; Gao, Y-H; Tan, K-B; Zuo, Z-X; Yang, W-X; Hua, X; Li, P-J; Zhang, Y; Wang, G

    2013-12-01

    We sought to investigate the antifibrotic effects of an artificial microRNA (miRNA) targeting connective tissue growth factor (CTGF) using the ultrasound-targeted cationic liposome-bearing microbubble destruction gene delivery system. Cationic liposomes were conjugated with microbubbles using a biotin-avidin system. Plasmids carrying the most effective artificial miRNA sequences were delivered by ultrasound-targeted cationic liposome-bearing microbubble destruction gene delivery system to rats with hepatic fibrosis. The results show that this method of gene delivery effectively transported the plasmids to the rat liver. The artificial miRNA reduced hepatic fibrosis pathological alterations as well as the protein and mRNA expressions of CTGF and transforming growth factor β1. Furthermore, the CTGF gene silencing decreased the levels of type I collagen and α-smooth muscle actin (Pliposome-bearing microbubble destruction may be an efficacious therapeutic method to ameliorate hepatic fibrosis.

  6. Inhibition of hepatitis C virus p7 membrane channels in a liposome-based assay system.

    Science.gov (United States)

    StGelais, Corine; Tuthill, Tobias J; Clarke, Dean S; Rowlands, David J; Harris, Mark; Griffin, Stephen

    2007-10-01

    Chemotherapy for patients chronically infected with hepatitis C virus (HCV) is ineffective in over 50% of cases, generating a high demand for new drug targets. The p7 protein of HCV displays membrane channel activity in vitro and is essential for replication in vivo though its precise role in the virus life cycle is unknown. p7 channel activity can be specifically inhibited by several classes of compounds, making this protein an attractive candidate for drug development, though techniques used to date in characterising this protein are unsuited to compound library screening. Here we describe an assay for the channel forming ability of p7 based on the release of a fluorescent indicator from liposomes. We show that recombinant p7 from genotype 1b HCV causes a dose-dependent release of dye when mixed with liposomes and that this property is enhanced at acidic pH. We demonstrate that this activity is due to the formation of a size-selective pore rather than non-specific disruption of liposomes and that activity can be blocked by amantadine and several other compounds, validating it as a measure of p7 channel function. This system provides the first convenient in vitro assay for exploiting p7 as a therapeutic target.

  7. Blockade of chloride channels by DIDS stimulates renin release and inhibits contraction of afferent arterioles

    DEFF Research Database (Denmark)

    Jensen, B L; Skøtt, O

    1996-01-01

    arterioles with the chloride channel blocker 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS). Renin secretion was equally enhanced by omission of extracellular calcium and by addition of 0.5 mM DIDS. The inhibitory effect of calcium was blocked by DIDS. The stimulatory effects of low calcium [with....... Norepinephrine (5 x 10(-7)-1 x 10(-6) M) and angiotensin II (1 x 10(-8)-10(-6) M) evoked reversible and dose-dependent contractions of microperfused rabbit afferent arterioles. DIDS (0.5 mM) did not affect the basal diameter of the arterioles but strongly inhibited the response to angiotensin II and attenuated...... the duration of the contractile response to norepinephrine. The results support the hypothesis that DIDS-sensitive calcium-activated chloride channels are involved in regulation of renin release and in the afferent arteriolar contraction after angiotensin II but do not play a pivotal role in the response...

  8. Acid-sensitive channel inhibition prevents fetal alcohol spectrum disorders cerebellar Purkinje cell loss.

    Science.gov (United States)

    Ramadoss, Jayanth; Lunde, Emilie R; Ouyang, Nengtai; Chen, Wei-Jung A; Cudd, Timothy A

    2008-08-01

    Ethanol is now considered the most common human teratogen. Educational campaigns have not reduced the incidence of ethanol-mediated teratogenesis, leading to a growing interest in the development of therapeutic prevention or mitigation strategies. On the basis of the observation that maternal ethanol consumption reduces maternal and fetal pH, we hypothesized that a pH-sensitive pathway involving the TWIK-related acid-sensitive potassium channels (TASKs) is implicated in ethanol-induced injury to the fetal cerebellum, one of the most sensitive targets of prenatal ethanol exposure. Pregnant ewes were intravenously infused with ethanol (258+/-10 mg/dl peak blood ethanol concentration) or saline in a "3 days/wk binge" pattern throughout the third trimester. Quantitative stereological analysis demonstrated that ethanol resulted in a 45% reduction in the total number of fetal cerebellar Purkinje cells, the cell type most sensitive to developmental ethanol exposure. Extracellular pH manipulation to create the same degree and pattern of pH fall caused by ethanol (manipulations large enough to inhibit TASK 1 channels), resulted in a 24% decrease in Purkinje cell number. We determined immunohistochemically that TASK 1 channels are expressed in Purkinje cells and that the TASK 3 isoform is expressed in granule cells of the ovine fetal cerebellum. Pharmacological blockade of both TASK 1 and TASK 3 channels simultaneous with ethanol effectively prevented any reduction in fetal cerebellar Purkinje cell number. These results demonstrate for the first time functional significance of fetal cerebellar two-pore domain pH-sensitive channels and establishes them as a potential therapeutic target for prevention of ethanol teratogenesis.

  9. Transient receptor potential cation channel subfamily V member 1 expressing corneal sensory neurons can be subdivided into at least three subpopulations

    Directory of Open Access Journals (Sweden)

    Abdulhakeem eAlamri

    2015-06-01

    Full Text Available The cornea is innervated by 3 main functional classes of sensory neurons: polymodal nociceptors, pure mechano-nociceptors and cold-sensing neurons. Here we explored transient receptor potential cation channel subfamily V member 1 (TRPV1 expression in guinea pig corneal sensory neurons, a widely used molecular marker of polymodal nociceptors. We used retrograde tracing to identify corneal afferent neurons in the trigeminal ganglion and double label in situ hybridization and/or immunohistochemistry to determine their molecular profile. In addition, we used immunohistochemistry to reveal the neurochemistry and structure of TRPV1 expressing nerve endings in the corneal epithelium. Approximately 45% of corneal afferent neurons expressed TRPV1, 28% expressed Piezo2 (a marker of putative pure mechano-nociceptors and 8% expressed the transient receptor potential cation channel subfamily M member 8 (TRPM8; a marker of cold-sensing neurons. There was no co-expression of TRPV1 and Piezo2 in corneal afferent neurons, but 6% of TRPV1 neurons co-expressed TRPM8. The TRPV1 expressing corneal afferent neurons could be divided into 3 subpopulations on the basis of calcitonin gene-related peptide (CGRP and/or or glial cell line-derived neurotrophic factor family receptor alpha3 (GFRα3 co-expression. In the corneal epithelium, the TRPV1 axons that co-expressed CGRP and GFRα3 ended as simple unbranched endings in the wing cell layer. In contrast, those that only co-expressed GFRα3 had ramifying endings that branched and terminated in the squamous cell layer, whereas those that only co-expressed CGRP had simple endings in the basal epithelium. This study shows that the majority of TRPV1 expressing corneal afferent neurons (>90% are likely to be polymodal nociceptors. Furthermore, TRPV1 expressing corneal afferent neurons can be subdivided into specific subpopulations based on their molecular phenotype, nerve terminal morphology and distribution in the corneal

  10. Prevention of secretory diarrhea by ethanol extract of Bistortae rhizoma through inhibition of chloride channel

    Directory of Open Access Journals (Sweden)

    Bo Yu

    2015-08-01

    Full Text Available Inhibition of cystic fibrosis transmembrane conductance regulator (CFTR and Ca2+-activated Cl- channel (CaCC represents an attractive approach for the treatment of secretory diarrhea. The aim of the study is to investigate the molecular basis of the anti-diarrheal effect of traditional Chinese herbal anti-diarrheal medicine Bistortae rhizoma. Fluorescence quenching assay indicated that the 40% methanol /water fraction (D5 dose-dependently inhibited both CFTR and CaCC function in transfected Fischer rat thyroid (FRT cells. Ex vivo studies indicated that D5 inhibited both forskolin (FSK-activated CFTR current and CCh-induced CaCC current in rat colonic mucosa. In the mouse closed-loop model, intraluminal application of D5 (200 µg/mL significantly reduced cholera toxin-stimulated fluid secretion. In the intestinal motility model, D5 significantly delayed intestinal peristalsis in mice. Our research suggests that CFTR and CaCC-mediated intestinal epithelial Cl- secretion inhibiting and gastrointestinal motility delaying may account for the anti-diarrheal activity of B. rhizoma.

  11. Mutant connexin 50 (S276F) inhibits channel and hemichannel functions inducing cataract

    Indian Academy of Sciences (India)

    Yuanyuan Liu; Chen Qiao; Tanwei Wei; Fang Zheng; Shuren Guo; Qiang Chen; Ming Yan; Xin Zhou

    2015-06-01

    This study was designed to detect the expression, detergent resistance, subcellular localization, and channel and hemichannel functions of mutant Cx50 to understand the forming mechanism for inducing congenital cataract by a novel mutation p.S276F in connexin 50 (Cx50) reported previously by us. HeLa and human lens epithelial (HLE) cells were transfected with wild-type Cx50 and mutant Cx50 (S276F). We examined the functional characteristics of mutant Cx50 (S276F) in comparison with those of wild-type Cx50 using immunoblot, confocal fluorescence microscopy, dye transfer analysis and dye uptake assay. The mutant and wild-type Cx50 were expressed in equal levels and could efficiently localize to the plasma membrane without transportation and assembly problems. Scrape loading dye transfer was significantly evident in cells transfected with wild-type Cx50 compared to those in cells transfected with mutant Cx50 and cotransfected with wild-type and mutant Cx50. The dye uptake was found to be significantly lower in cells transfected with mutant Cx50 than in cells transfected with wild-type Cx50 and cells cotransfected with wild-type and mutant Cx50. The transfected HeLa and HLE cell lines showed similar performance in all the experiments. These results indicated that the mutant Cx50 (S276F) might inhibit the function of gap junction channel in a dominant negative manner, but inhibit the hemichannel function in a recessive negative manner.

  12. Myricetin inhibits Kv1.5 channels in HEK293 cells.

    Science.gov (United States)

    Ou, Xianhong; Bin, Xiaohong; Wang, Luzhen; Li, Miaoling; Yang, Yan; Fan, Xinrong; Zeng, Xiaorong

    2016-02-01

    Myricetin (Myr) is a flavonoid that exerts anti-arrhythmic effects. However, its potential effects on ion channels have remained elusive. The aim of the present study was to investigate the effects of Myr on Kv1.5 channels in HEK293 cells. The current of Kv1.5 channels (Ikur) in HEK293 cells was recorded using the whole-cell patch-clamp technique and the expression of the Kv1.5 protein was measured using western blot analysis 24 h after treatment with Myr. The results showed that 5 µM Myr significantly reduced Ikur from 215.04 ± 40.59 to 77.72 ± 17.94 pA/pF (PHEK293 cells treated with 10 µM Myr for 5 min. Furthermore, Myr reduced hKv1.5 protein expression in a dose-dependent manner. These results demonstrated that Myr inhibited Ikur and the expression of hKv1.5 in HEK293 cells in a dose-, time- and frequency-dependent manner. These observations partly explained the mechanisms by which Myr exerts anti-arrhythmic effect.

  13. Lipid storage disorders block lysosomal trafficking by inhibiting a TRP channel and lysosomal calcium release.

    Science.gov (United States)

    Shen, Dongbiao; Wang, Xiang; Li, Xinran; Zhang, Xiaoli; Yao, Zepeng; Dibble, Shannon; Dong, Xian-ping; Yu, Ting; Lieberman, Andrew P; Showalter, Hollis D; Xu, Haoxing

    2012-03-13

    Lysosomal lipid accumulation, defects in membrane trafficking and altered Ca(2+) homoeostasis are common features in many lysosomal storage diseases. Mucolipin transient receptor potential channel 1 (TRPML1) is the principle Ca(2+) channel in the lysosome. Here we show that TRPML1-mediated lysosomal Ca(2+) release, measured using a genetically encoded Ca(2+) indicator (GCaMP3) attached directly to TRPML1 and elicited by a potent membrane-permeable synthetic agonist, is dramatically reduced in Niemann-Pick (NP) disease cells. Sphingomyelins (SMs) are plasma membrane lipids that undergo sphingomyelinase (SMase)-mediated hydrolysis in the lysosomes of normal cells, but accumulate distinctively in lysosomes of NP cells. Patch-clamp analyses revealed that TRPML1 channel activity is inhibited by SMs, but potentiated by SMases. In NP-type C cells, increasing TRPML1's expression or activity was sufficient to correct the trafficking defects and reduce lysosome storage and cholesterol accumulation. We propose that abnormal accumulation of luminal lipids causes secondary lysosome storage by blocking TRPML1- and Ca(2+)-dependent lysosomal trafficking.

  14. Inhibition of herpes simplex virus type 1 entry by chloride channel inhibitors tamoxifen and NPPB

    Energy Technology Data Exchange (ETDEWEB)

    Zheng, Kai [Guangzhou Jinan Biomedicine Research and Development Center, National Engineering Research Center of Genetic Medicine, Jinan University, Guangzhou (China); College of Life Science and Technology, Jinan University, Guangzhou (China); Chen, Maoyun [Guangzhou Jinan Biomedicine Research and Development Center, National Engineering Research Center of Genetic Medicine, Jinan University, Guangzhou (China); College of pharmacy, Jinan University, Guangzhou (China); Xiang, Yangfei; Ma, Kaiqi [Guangzhou Jinan Biomedicine Research and Development Center, National Engineering Research Center of Genetic Medicine, Jinan University, Guangzhou (China); Jin, Fujun [Guangzhou Jinan Biomedicine Research and Development Center, National Engineering Research Center of Genetic Medicine, Jinan University, Guangzhou (China); College of pharmacy, Jinan University, Guangzhou (China); Wang, Xiao [School of Pharmaceutical Sciences, Sun Yat-sen University, Guangzhou 510006 (China); Wang, Xiaoyan; Wang, Shaoxiang [Guangzhou Jinan Biomedicine Research and Development Center, National Engineering Research Center of Genetic Medicine, Jinan University, Guangzhou (China); Wang, Yifei, E-mail: twang-yf@163.com [Guangzhou Jinan Biomedicine Research and Development Center, National Engineering Research Center of Genetic Medicine, Jinan University, Guangzhou (China)

    2014-04-18

    Highlights: • We analyze the anti-HSV potential of chloride channel inhibitors. • Tamoxifen and NPPB show anti-HSV-1 and anti-ACV-resistant HSV-1 activities. • HSV-1 infection induces intracellular chloride concentration increasing. • Tamoxifen and NPPB inhibit HSV-1 early infection. • Tamoxifen and NPPB prevent the fusion process of HSV-1. - Abstract: Herpes simplex virus type 1 (HSV-1) infection is very common worldwide and can cause significant health problems from periodic skin and corneal lesions to encephalitis. Appearance of drug-resistant viruses in clinical therapy has made exploring novel antiviral agents emergent. Here we show that chloride channel inhibitors, including tamoxifen and 5-nitro-2-(3-phenyl-propylamino) benzoic acid (NPPB), exhibited extensive antiviral activities toward HSV-1 and ACV-resistant HSV viruses. HSV-1 infection induced chloride ion influx while treatment with inhibitors reduced the increase of intracellular chloride ion concentration. Pretreatment or treatment of inhibitors at different time points during HSV-1 infection all suppressed viral RNA synthesis, protein expression and virus production. More detailed studies demonstrated that tamoxifen and NPPB acted as potent inhibitors of HSV-1 early entry step by preventing viral binding, penetration and nuclear translocation. Specifically the compounds appeared to affect viral fusion process by inhibiting virus binding to lipid rafts and interrupting calcium homeostasis. Taken together, the observation that tamoxifen and NPPB can block viral entry suggests a stronger potential for these compounds as well as other ion channel inhibitors in antiviral therapy against HSV-1, especially the compound tamoxifen is an immediately actionable drug that can be reused for treatment of HSV-1 infections.

  15. The open-close mechanism of M2 channel protein in influenza A virus: A computational study on the hydrogen bonds and cation-π interactions among His37 and Trp41

    Institute of Scientific and Technical Information of China (English)

    CHENG JiaGao; ZHU WeiLiang; WANG YanLi; YAN XiuHua; LI Zhong; TANG Yun; JIANG HuaLiang

    2008-01-01

    The M2 protein from influenza A virus is a tetrameric ion channel. It was reported that the permeation of the ion channel is correlated with the hydrogen bond network among His37 residues and the cation-π interactions between His37 and Trp41. In the present study, the hydrogen bonding network of 4-methyl-imidazoles was built to mimic the hydrogen bonds between His37 residues, and the cation-π interactions between 4-methyl-imidazolium and indole systems were selected to represent the interac-tions between His37 and Trp41. Then, quantum chemistry calculations at the MP2/6-311G** level were carried out to explore the properties of the hydrogen bonds and the cation-π interactions. The calculation results indicate that the binding strength of the N--H…N hydrogen bond between imidazole rings is up to -6.22 kcal·mol-1, and the binding strength of the strongest cation-π interaction is up to -18.8 kcal·mol-1 (T-shaped interaction) or -12.3 kcal·mol-1 (parallel stacking interaction). Thus, the calculated binding energies indicate that it is possible to control the permeation of the M2 ion channel through the hydrogen bond network and the cation-π interactions by altering the pH values.

  16. The open-close mechanism of M2 channel protein in influenza A virus:A computational study on the hydrogen bonds and cation-π interactions among His37 and Trp41

    Institute of Scientific and Technical Information of China (English)

    2008-01-01

    The M2 protein from influenza A virus is a tetrameric ion channel. It was reported that the permeation of the ion channel is correlated with the hydrogen bond network among His37 residues and the cation-π interactions between His37 and Trp41. In the present study,the hydrogen bonding network of 4-methyl-imidazoles was built to mimic the hydrogen bonds between His37 residues,and the cation-π interactions between 4-methyl-imidazolium and indole systems were selected to represent the interac-tions between His37 and Trp41. Then,quantum chemistry calculations at the MP2/6-311G level were carried out to explore the properties of the hydrogen bonds and the cation-π interactions. The calcula-tion results indicate that the binding strength of the N-H···N hydrogen bond between imidazole rings is up to -6.22 kcal·mol-1,and the binding strength of the strongest cation-π interaction is up to -18.8 kcal·mol-1(T-shaped interaction) or -12.3 kcal·mol-1(parallel stacking interaction). Thus,the calcu-lated binding energies indicate that it is possible to control the permeation of the M2 ion channel through the hydrogen bond network and the cation-π interactions by altering the pH values.

  17. Anesthetic drug midazolam inhibits cardiac human ether-à-go-go-related gene channels: mode of action.

    Science.gov (United States)

    Vonderlin, Nadine; Fischer, Fathima; Zitron, Edgar; Seyler, Claudia; Scherer, Daniel; Thomas, Dierk; Katus, Hugo A; Scholz, Eberhard P

    2015-01-01

    Midazolam is a short-acting benzodiazepine that is in wide clinical use as an anxiolytic, sedative, hypnotic, and anticonvulsant. Midazolam has been shown to inhibit ion channels, including calcium and potassium channels. So far, the effects of midazolam on cardiac human ether-à-go-go-related gene (hERG) channels have not been analyzed. The inhibitory effects of midazolam on heterologously expressed hERG channels were analyzed in Xenopus oocytes using the double-electrode voltage clamp technique. We found that midazolam inhibits hERG channels in a concentration-dependent manner, yielding an IC50 of 170 μM in Xenopus oocytes. When analyzed in a HEK 293 cell line using the patch-clamp technique, the IC50 was 13.6 μM. Midazolam resulted in a small negative shift of the activation curve of hERG channels. However, steady-state inactivation was not significantly affected. We further show that inhibition is state-dependent, occurring within the open and inactivated but not in the closed state. There was no frequency dependence of block. Using the hERG pore mutants F656A and Y652A we provide evidence that midazolam uses a classical binding site within the channel pore. Analyzing the subacute effects of midazolam on hERG channel trafficking, we further found that midazolam does not affect channel surface expression. Taken together, we show that the anesthetic midazolam is a low-affinity inhibitor of cardiac hERG channels without additional effects on channel surface expression. These data add to the current understanding of the pharmacological profile of the anesthetic midazolam.

  18. Cannabinoids inhibit acid-sensing ion channel currents in rat dorsal root ganglion neurons.

    Directory of Open Access Journals (Sweden)

    Yu-Qiang Liu

    Full Text Available Local acidosis has been found in various pain-generating conditions such as inflammation and tissue injury. Cannabinoids exert a powerful inhibitory control over pain initiation via peripheral cognate receptors. However, the peripheral molecular targets responsible for the antinociceptive effects of cannabinoids are still poorly understood. Here, we have found that WIN55,212-2, a cannabinoid receptor agonist, inhibits the activity of native acid-sensing ion channels (ASICs in rat dorsal root ganglion (DRG neurons. WIN55,212-2 dose-dependently inhibited proton-gated currents mediated by ASICs. WIN55,212-2 shifted the proton concentration-response curve downwards, with an decrease of 48.6±3.7% in the maximum current response but with no significant change in the EC(50 value. The inhibition of proton-gated current induced by WIN55,212-2 was almost completely blocked by the selective CB1 receptor antagonist AM 281, but not by the CB2 receptor antagonist AM630. Pretreatment of forskolin, an AC activator, and the addition of cAMP also reversed the inhibition of WIN55,212-2. Moreover, WIN55,212-2 altered acid-evoked excitability of rat DRG neurons and decreased the number of action potentials induced by acid stimuli. Finally, WIN55,212-2 attenuated nociceptive responses to injection of acetic acid in rats. These results suggest that WIN55,212-2 inhibits the activity of ASICs via CB1 receptor and cAMP dependent pathway in rat primary sensory neurons. Thus, cannabinoids can exert their analgesic action by interaction with ASICs in the primary afferent neurons, which was novel analgesic mechanism of cannabinoids.

  19. Eugenol dilates rat cerebral arteries by inhibiting smooth muscle cell voltage-dependent calcium channels.

    Science.gov (United States)

    Peixoto-Neves, Dieniffer; Leal-Cardoso, Jose Henrique; Jaggar, Jonathan H

    2014-11-01

    Plants high in eugenol, a phenylpropanoid compound, are used as folk medicines to alleviate diseases including hypertension. Eugenol has been demonstrated to relax conduit and ear arteries and reduce systemic blood pressure, but mechanisms involved are unclear. Here, we studied eugenol regulation of resistance-size cerebral arteries that control regional brain blood pressure and flow and investigated mechanisms involved. We demonstrate that eugenol dilates arteries constricted by either pressure or membrane depolarization (60 mM K) in a concentration-dependent manner. Experiments performed using patch-clamp electrophysiology demonstrated that eugenol inhibited voltage-dependent calcium (Ca) currents, when using Ba as a charge carrier, in isolated cerebral artery smooth muscle cells. Eugenol inhibition of voltage-dependent Ca currents involved pore block, a hyperpolarizing shift (∼-10 mV) in voltage-dependent inactivation, an increase in the proportion of steady-state inactivating current, and acceleration of inactivation rate. In summary, our data indicate that eugenol dilates cerebral arteries by means of multimodal inhibition of voltage-dependent Ca channels.

  20. K-channels inhibited by hydrogen peroxide mediate abscisic acid signaling in Vicia guard cells

    Institute of Scientific and Technical Information of China (English)

    2001-01-01

    A number of studies show that environmental stress conditions increase abscisic acid (ABA) and hydrogen peroxide (H2O2) levels in plant cells. Despite this central role of ABA in altering stomatal aperture by regulating guard cell ion transport, little is known concerning the relationship between ABA and H2O2 in signal transduction leading to stomatal movement. Epidermal strip bioassay illustrated that ABA-inhibited stomatal opening and ABA-induced stomatal closure were abolished partly by externally added catalase (CAT) or diphenylene iodonium (DPI), which are a H2O2 scavenger and a NADPH oxidase inhibitor respectively. In contrast, internally added CAT or DPI nearly completely or partly reversed ABA-induced closure in half-stoma. Consistent with these results, whole-cell patch-clamp analysis showed that intracellular application of CAT or DPI partly abolished ABA-inhibited inward K+ current across the plasma membrane of guard cells. H2O2 mimicked ABA to inhibit inward K+ current, an effect which was reversed by the addition of ascorbic acid (Vc) in patch clamping micropipettes. These results suggested that H2O2 mediated ABA-induced stomatal movement by targeting inward K+ channels at plasma membrane.

  1. A novel CaV2.2 channel inhibition by piracetam in peripheral and central neurons.

    Science.gov (United States)

    Bravo-Martínez, Jorge; Arenas, Isabel; Vivas, Oscar; Rebolledo-Antúnez, Santiago; Vázquez-García, Mario; Larrazolo, Arturo; García, David E

    2012-10-01

    No mechanistic actions for piracetam have been documented to support its nootropic effects. Voltage-gated calcium channels have been proposed as a promising pharmacological target of nootropic drugs. In this study, we investigated the effect of piracetam on Ca(V)2.2 channels in peripheral neurons, using patch-clamp recordings from cultured superior cervical ganglion neurons. In addition, we tested if Ca(V)2.2 channel inhibition could be related with the effects of piracetam on central neurons. We found that piracetam inhibited native Ca(V)2.2 channels in superior cervical ganglion neurons in a dose-dependent manner, with an IC(50) of 3.4 μmol/L and a Hill coefficient of 1.1. GDPβS dialysis did not prevent piracetam-induced inhibition of Ca(V)2.2 channels and G-protein-coupled receptor activation by noradrenaline did not occlude the piracetam effect. Piracetam altered the biophysical characteristics of Ca(V)2.2 channel such as facilitation ratio. In hippocampal slices, piracetam and ω-conotoxin GVIA diminished the frequency of excitatory postsynaptic potentials and action potentials. Our results provide evidence of piracetam's actions on Ca(V)2.2 channels in peripheral neurons, which might explain some of its nootropic effects in central neurons.

  2. Cardamonin, a Novel Antagonist of hTRPA1 Cation Channel, Reveals Therapeutic Mechanism of Pathological Pain

    Directory of Open Access Journals (Sweden)

    Shifeng Wang

    2016-08-01

    Full Text Available The increasing demand for safe and effective treatments of chronic pain has promoted the investigation of novel analgesic drugs. Some herbals have been known to be able to relieve pain, while the chemical basis and target involved in this process remained to be clarified. The current study aimed to find anti-nociceptive candidates targeting transient receptor potential ankyrin 1 (TRPA1, a receptor that implicates in hyperalgesia and neurogenic inflammation. In the current study, 156 chemicals were tested for blocking HEK293/TRPA1 ion channel by calcium-influx assay. Docking study was conducted to predict the binding modes of hit compound with TRPA1 using Discovery Studio. Cytotoxicity in HEK293 was conducted by Cell Titer-Glo assay. Additionally, cardiotoxicity was assessed via xCELLigence RTCA system. We uncovered that cardamonin selectively blocked TRPA1 activation while did not interact with TRPV1 nor TRPV4 channel. A concentration-dependent inhibitory effect was observed with IC50 of 454 nM. Docking analysis of cardamonin demonstrated a compatible interaction with A-967079-binding site of TRPA1. Meanwhile, cardamonin did not significantly reduce HEK293 cell viability, nor did it impair cardiomyocyte constriction. Our data suggest that cardamonin is a selective TRPA1 antagonist, providing novel insight into the target of its anti-nociceptive activity.

  3. Prostaglandin metabolite induces inhibition of TRPA1 and channel-dependent nociception

    Directory of Open Access Journals (Sweden)

    Weng Yingqi

    2012-09-01

    Full Text Available Abstract Background The Transient Receptor Potential (TRP ion channel TRPA1 is a key player in pain pathways. Irritant chemicals activate ion channel TRPA1 via covalent modification of N-terminal cysteines. We and others have shown that 15-Deoxy-Δ12, 14-prostaglandin J2 (15d-PGJ2 similarly activates TRPA1 and causes channel-dependent nociception. Paradoxically, 15d-PGJ2 can also be anti-nociceptive in several pain models. Here we hypothesized that activation and subsequent desensitization of TRPA1 in dorsal root ganglion (DRG neurons underlies the anti-nociceptive property of 15d-PGJ2. To investigate this, we utilized a battery of behavioral assays and intracellular Ca2+ imaging in DRG neurons to test if pre-treatment with 15d-PGJ2 inhibited TRPA1 to subsequent stimulation. Results Intraplantar pre-injection of 15d-PGJ2, in contrast to mustard oil (AITC, attenuated acute nocifensive responses to subsequent injections of 15d-PGJ2 and AITC, but not capsaicin (CAP. Intraplantar 15d-PGJ2—administered after the induction of inflammation—reduced mechanical hypersensitivity in the Complete Freund’s Adjuvant (CFA model for up to 2 h post-injection. The 15d-PGJ2-mediated reduction in mechanical hypersensitivity is dependent on TRPA1, as this effect was absent in TRPA1 knockout mice. Ca2+ imaging studies of DRG neurons demonstrated that 15d-PGJ2 pre-exposure reduced the magnitude and number of neuronal responses to AITC, but not CAP. AITC responses were not reduced when neurons were pre-exposed to 15d-PGJ2 combined with HC-030031 (TRPA1 antagonist, demonstrating that inhibitory effects of 15d-PGJ2 depend on TRPA1 activation. Single daily doses of 15d-PGJ2, administered during the course of 4 days in the CFA model, effectively reversed mechanical hypersensitivity without apparent tolerance or toxicity. Conclusions Taken together, our data support the hypothesis that 15d-PGJ2 induces activation followed by persistent inhibition of TRPA1 channels

  4. Synergistic antiarrhythmic effect of combining inhibition of Ca(2+)-activated K(+) (SK) channels and voltage-gated Na(+) channels in an isolated heart model of atrial fibrillation

    DEFF Research Database (Denmark)

    Kirchhoff, Jeppe Egedal; Goldin Diness, Jonas; Sheykhzade, Majid;

    2015-01-01

    of the adverse effect profile could be an additional advantage if compound concentrations could be reduced. OBJECTIVE: The purpose of this study was to test the hypothesis that combined inhibition of Ca(2+)-activated K(+) channels (SK channels) and voltage-gated Na(+) channels, in concentrations that would......)thiazol-2-amine (ICA) in a Langendorff-perfused guinea pig heart model in which AF was induced after acetylcholine application and burst pacing. RESULTS: AF duration was reduced when both flecainide and ranolazine were combined with ICA in doses that did not reduce AF as monotherapy. At higher...... concentrations, both flecainide and ranolazine revealed proarrhythmic properties. CONCLUSION: A synergistic effect in AF treatment was obtained by combining low concentrations of SK and Na(+) channel blockers....

  5. Inhibition of Gene Expression of Organic Cation/Carnitine Transporter and Antioxidant Enzymes in Oxazaphosphorines-Induced Acute Cardiomyopathic Rat Models

    Directory of Open Access Journals (Sweden)

    Mohamed M. Sayed-Ahmed

    2012-01-01

    Full Text Available It is well documented that high therapeutic doses of oxazaphosphorines, cyclophosphamide (CP and ifosfamide (IFO, are associated with cardiomyopathy. This study investigated whether oxazaphosphorines alter the expression of organic cation/carnitine transporter (OCTN2 and antioxidant genes and if so, whether these alterations contribute to CP and IFO-induced cardiotoxicity. Adult male Wistar albino rats were assigned to one of six treatment groups namely, control, L carnitine, CP, IFO, CP plus L carnitine and IFO plus L carnitine. In cardiac and kidney tissues, CP and IFO significantly decreased mRNA and protein expression of OCTN2. Oxazaphosphorines significantly increased serum acyl-carnitine/free carnitine ratio and urinary carnitine excretion and significantly decreased total carnitine in cardiac tissues. Interestingly, carnitine supplementation completely reversed the biochemical and gene expression changes-induced by oxazaphosphorines to the control values, except OCTN2 expression remained inhibited by IFO. Data from this study suggest that: (1 Oxazaphosphorines decreased myocardial carnitine content following the inhibition of OCTN2 mRNA and protein expression in cardiac tissues. (2 Oxazaphosphorine therapy increased urinary loss of carnitine secondary to the inhibition of OCTN2 mRNA and protein expression in proximal tubules of the kidney. (3 Carnitine supplementation attenuates CP but not IFO-induced inhibition of OCTN2 mRNA and protein expression in heart and kidney tissues.

  6. Like Extinction, Latent Inhibition of Conditioned Fear in Mice Is Blocked by Systemic Inhibition of L-Type Voltage-Gated Calcium Channels

    Science.gov (United States)

    Blouin, Ashley M.; Cain, Chris K.; Barad, Mike

    2004-01-01

    Having recently shown that extinction of conditioned fear depends on L-type voltage-gated calcium channels (LVGCCs), we have been seeking other protocols that require this unusual induction mechanism. We tested latent inhibition (LI) of fear, because LI resembles extinction except that cue exposures precede, rather than follow, cue-shock pairing.…

  7. Transient Receptor Potential Channel and Interleukin-17A Involvement in LTTL Gel Inhibition of Bone Cancer Pain in a Rat Model.

    Science.gov (United States)

    Wang, Juyong; Zhang, Ruixin; Dong, Changsheng; Jiao, Lijing; Xu, Ling; Liu, Jiyong; Wang, Zhengtao; Lao, Lixing

    2015-07-01

    Cancer pain management is a challenge for which Chinese herbal medicine might be useful. To study the spinal mechanisms of the Chinese medicated gel Long-Teng-Tong-Luo (LTTL), a 7-herb compound, on bone cancer pain, a bone cancer pain model was made by inoculating the tibias of female rats with Walker 256 cells. LTTL gel or inert gel, 0.5 g/cm(2)/d, was applied to the skin of tumor-bearing tibias for 21 days beginning a day after the inoculation. Mechanical threshold and paw withdrawal latency to thermal stimulation was measured. Transient receptor potential (TRP) cation channels in lumbar dorsal root ganglia (DRG) were immunostained and counted, and lumbar spinal cord interleukin-17A (IL-17A) was measured with real-time polymerase chain reaction and enzyme-linked immunosorbent assay. TRP antagonists and interleukin (IL)-17A antibodies were intrathecally administered to determine their effects on bone cancer pain. The gel significantly (P gel inhibits cancer pain, and this might be accounted for by the decrease in expression of DRG TRP channels and spinal astrocyte IL-17A.

  8. The cation channel mucolipin-1 is a bifunctional protein that facilitates membrane remodeling via its serine lipase domain.

    Science.gov (United States)

    LaPlante, Janice M; Falardeau, John L; Brown, Edward M; Slaugenhaupt, Susan A; Vassilev, Peter M

    2011-04-01

    Phospholipase modulators have been shown to affect the topology of lipid bilayers and the formation of tubulo-vesicular structures, but the specific endogenous phospholipases involved have yet to be identified. Here we show that TRPML1 (MLN1), a Ca(2+)-permeable channel, contributes to membrane remodeling through a serine lipase consensus domain, and thus represents a novel type of bifunctional protein. Remarkably, this serine lipase active site determines the ability of MLN1 to generate tubulo-vesicular extensions in mucolipin-1-expressing oocytes, human fibroblasts and model membrane vesicles. Our demonstration that MLN1 is involved in membrane remodeling and the formation of extensions suggests that it may play a role in the formation of cellular processes linked to the late endosome/lysosome (LE/L) pathway. MLN1 is absent or mutated in patients with mucolipidosis IV (MLIV), a lysosomal disorder with devastating neurological and other consequences. This study provides potential insight into the pathophysiology of MLIV.

  9. K channel activation by nucleotide diphosphates and its inhibition by glibenclamide in vascular smooth muscle cells.

    Science.gov (United States)

    Beech, D J; Zhang, H; Nakao, K; Bolton, T B

    1993-10-01

    1. Whole-cell and inside-out patch recordings were made from single smooth muscle cells that had been isolated enzymatically and mechanically from the rabbit portal vein. 2. In whole-cells the inclusion in the recording pipette solution of nucleotide diphosphates (NDPs), but not tri- or monophosphates, induced a K-current that developed gradually over 5 to 15 min. Intracellular 1 mM guanosine 5'-diphosphate (GDP) induced a slowly developing outward K-current at -37 mV that reached a maximum on average of 72 +/- 4 pA (n = 40). Half maximal effect was estimated to occur with about 0.2 mM GDP. Except for ADP, other NDPs had comparable effects. At 0.1 mM, ADP was equivalent to GDP but at higher concentration ADP was less effective. ADP induced its maximum effect at 1 mM but had almost no effect at 10 mM. 3. In 14% of inside-out patches exposed to 1 mM GDP at the intracellular surface, characteristic K channel activity was observed which showed long (> 1 s) bursts of openings separated by longer closed periods. The current-voltage relationship for the channel was linear in a 60 mM:130 mM K-gradient and the unitary conductance was 24 pS. 4. Glibenclamide applied via the extracellular solution was found to be a potent inhibitor of GDP-induced K-current (IK(GDP)) in the whole-cell. The Kd was 25 nM and the inhibition was fully reversible on wash-out. 5. IK(GDP) was not evoked if Mg ions were absent from the pipette solution. In contrast the omission of extracellular Mg ions had no effect on outward or inward IK(GDP). 6. Inclusion of 1 mM ATP in the recording pipette solution reduced IK(GDP) and also attenuated its decline during long (25 min) recordings. 7. When perforated-patch whole-cell recording was used, metabolic poisoning with cyanide and 2-deoxy-D-glucose induced a glibenclamide-sensitive K-current. This current was not observed when conventional whole-cell recording was used. Possible reasons for this difference are discussed. 8. These K channels appear similar to

  10. Novel cationic surfactants from fatty acids and their corrosion inhibition efficiency for carbon steel pipelines in 1 M HCl

    Directory of Open Access Journals (Sweden)

    A.M. Al-Sabagh

    2011-06-01

    Full Text Available Four fatty acids were used as a source of alkyl halides. Untraditionally tertiary amines were prepared by ethoxylation of aromatic and aliphatic fatty amines. These alkyl halide and tertiary amines were used to prepare 20 cationic quaternary ammonium surfactants (QASS. Their chemical structures were characterized and they tested as corrosion inhibitors for carbon steel in 1 M HCl solution. The corrosion inhibition efficiency was measured using, weight loss and potentiodynamic polarization methods. The inhibition efficiencies obtained from the two employed methods are nearly closed. From the obtained data it was found that, the inhibition efficiency increases with increasing the inhibitor concentration until the optimum one. Also, it was found that the inhibition efficiency of QASs which based on ethoxylated aromatic tertiary amine is greater than the obtained efficiencies by the QASs which based on ethoxylated aliphatic tertiary amines. The QASs based on alkyl halide C16 exhibited the maximum inhibition efficiency 98.8%. Adsorption of the inhibitors on the carbon steel surface was found to obey Langmuir’s adsorption isotherm. The quantum chemical calculations were done for some selected quaternary ammonium compounds based on their chemical structures QL1,4,5–QP3,4,5. The following quantum chemical indices such as the bond length, bond angle, charge density distribution, highest occupied molecular orbital (HOMO, lowest unoccupied molecular orbital (LUMO, energy gap ΔE = HOMO − LUMO, and dipole moment (u were considered. The relation between these parameters and the inhibition efficiencies was explained on the light of the chemical structure of the used inhibitors.

  11. Functional and molecular effects of mercury compounds on the human OCTN1 cation transporter: C50 and C136 are the targets for potent inhibition.

    Science.gov (United States)

    Galluccio, Michele; Pochini, Lorena; Peta, Valentina; Iannì, Maria; Scalise, Mariafrancesca; Indiveri, Cesare

    2015-03-01

    The effect of mercury compounds has been tested on the organic cation transporter, hOCTN1. MeHg(+), Hg(2+), or Cd(2+) caused strong inhibition of transport. 1,4-Dithioerythritol (DTE), cysteine (Cys), and N-acetyl-l-cysteine reversed (NAC) the inhibition at different extents. 2-Aminoethyl methanethiosulfonate hydrobromide (MTSEA), a prototype SH reagent, exerted inhibition of transport similar to that observed for the mercurial agents. To investigate the mechanism of action of mercurials, mutants of hOCTN1 in which each of the Cys residues was substituted by Ala have been constructed, over-expressed in Escherichia coli, and purified. Tetraethylammonium chloride (TEA) uptake mediated by each mutant in proteoliposomes was comparable to that of wild type (WT). IC50 values of the WT and mutants for the mercury compounds were derived from dose-response analyses. The mutants C50A and C136A showed significant increase of IC50 indicating that the 2 Cys residues were involved in the interaction with the mercury compounds and inhibition of the transporter. The double mutant C50A/C136A was constructed; the lack of inhibition confirmed that the 2 Cys residues are the targets of mercury compounds. MTSEA showed similar behavior with respect to the mercurial reagents with the difference that increased IC50 was observed also in the C81A mutant. Similar results were obtained when transport was measured as acetylcholine uptake. Ethyl mercury (Thimerosal) inhibited hOCTN1 as well. C50A, C50A/C136A and, at very lower extent, C136A showed increased IC50 indicating that C50 was the major target of this mercury compound. The homology model of hOCTN1 was built using as template PiPT and validated by the experimental data on mutant proteins.

  12. Inhibition of nitrite-induced toxicity in channel catfish by calcium chloride and sodium chloride

    Science.gov (United States)

    Tommasso J.R., Wright; Simco, B.A.; Davis, K.B.

    1980-01-01

    Environmental chloride has been shown to inhibit methemoglobin formation in fish, thereby offering a protective effect against nitrite toxicity. Channel catfish (Ictalurus punctatus) were simultaneously exposed to various environmental nitrite and chloride levels (as either CaCl2 or NaCl) in dechlorinated tap water (40 mg/L total hardness, 47 mg/L alkalinity, 4 mg/L chloride, pH = 6.9-7.1, and temperature 21-24°C). Methemoglobin levels in fish simultaneously exposed to 2.5 mg/L nitrite and up to 30 mg/L chloride as either CaCl2 or NaCl were similar but significantly lower than in unprotected fish. Exposure to 10 mg/L nitrite and 60 mg/L chloride resulted in methemoglobin levels similar to those of the controls; most unprotected fish died. Fish exposed to 10 mg/L nitrite had significantly lower methemoglobin levels when protected with 15.0 mg/L chloride as CaCl2 than with NaCl. Fish exposed to nitrite in the presence of 60 mg/L chloride (as either CaCl2 or NaCl) had similar 24-h LC50 values that were significantly elevated above those obtained in the absence of chloride. Calcium had little effect on tolerance to nitrite toxicity in channel catfish in contrast to its large effect reported in steelhead trout (Salmo gairdneri).

  13. 1,3-propanediol binds deep inside the channel to inhibit water permeation through aquaporins.

    Science.gov (United States)

    Yu, Lili; Rodriguez, Roberto A; Chen, L Laurie; Chen, Liao Y; Perry, George; McHardy, Stanton F; Yeh, Chih-Ko

    2016-02-01

    Aquaporins and aquaglyceroporins (AQPs) are membrane channel proteins responsible for transport of water and for transport of glycerol in addition to water across the cell membrane, respectively. They are expressed throughout the human body and also in other forms of life. Inhibitors of human AQPs have been sought for therapeutic treatment for various medical conditions including hypertension, refractory edema, neurotoxic brain edema, and so forth. Conducting all-atom molecular dynamics simulations, we computed the binding affinity of acetazolamide to human AQP4 that agrees closely with in vitro experiments. Using this validated computational method, we found that 1,3-propanediol (PDO) binds deep inside the AQP4 channel to inhibit that particular aquaporin efficaciously. Furthermore, we used the same method to compute the affinities of PDO binding to four other AQPs and one aquaglyceroporin whose atomic coordinates are available from the protein data bank (PDB). For bovine AQP1, human AQP2, AQP4, AQP5, and Plasmodium falciparum PfAQP whose structures were resolved with high resolution, we obtained definitive predictions on the PDO dissociation constant. For human AQP1 whose PDB coordinates are less accurate, we estimated the dissociation constant with a rather large error bar. Taking into account the fact that PDO is generally recognized as safe by the US FDA, we predict that PDO can be an effective diuretic which directly modulates water flow through the protein channels. It should be free from the serious side effects associated with other diuretics that change the hydro-homeostasis indirectly by altering the osmotic gradients.

  14. Changes in cationic selectivity of the nicotinic channel at the rat ganglionic synapse: a role for chloride ions?

    Directory of Open Access Journals (Sweden)

    Oscar Sacchi

    Full Text Available The permeability of the nicotinic channel (nAChR at the ganglionic synapse has been examined, in the intact rat superior cervical ganglion in vitro, by fitting the Goldman current equation to the synaptic current (EPSC I-V relationship. Subsynaptic nAChRs, activated by neurally-released acetylcholine (ACh, were thus analyzed in an intact environment as natively expressed by the mature sympathetic neuron. Postsynaptic neuron hyperpolarization (from -40 to -90 mV resulted in a change of the synaptic potassium/sodium permeability ratio (P(K/P(Na from 1.40 to 0.92, corresponding to a reversible shift of the apparent acetylcholine equilibrium potential, E(ACh, by about +10 mV. The effect was accompanied by a decrease of the peak synaptic conductance (g(syn and of the EPSC decay time constant. Reduction of [Cl(-](o to 18 mM resulted in a change of P(K/P(Na from 1.57 (control to 2.26, associated with a reversible shift of E(ACh by about -10 mV. Application of 200 nM αBgTx evoked P(K/P(Na and g(syn modifications similar to those observed in reduced [Cl(-](o. The two treatments were overlapping and complementary, as if the same site/mechanism were involved. The difference current before and after chloride reduction or toxin application exhibited a strongly positive equilibrium potential, which could not be explained by the block of a calcium component of the EPSC. Observations under current-clamp conditions suggest that the driving force modification of the EPSC due to P(K/P(Na changes represent an additional powerful integrative mechanism of neuron behavior. A possible role for chloride ions is suggested: the nAChR selectivity was actually reduced by increased chloride gradient (membrane hyperpolarization, while it was increased, moving towards a channel preferentially permeable for potassium, when the chloride gradient was reduced.

  15. Acute simvastatin inhibits K ATP channels of porcine coronary artery myocytes.

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    Sai Wang Seto

    Full Text Available BACKGROUND: Statins (3-hydroxy-3-methyl-glutaryl coenzyme A (HMG-CoA reductase inhibitors consumption provides beneficial effects on cardiovascular systems. However, effects of statins on vascular KATP channel gatings are unknown. METHODS: Pig left anterior descending coronary artery and human left internal mammary artery were isolated and endothelium-denuded for tension measurements and Western immunoblots. Enzymatically-dissociated/cultured arterial myocytes were used for patch-clamp electrophysiological studies and for [Ca(2+]i, [ATP]i and [glucose]o uptake measurements. RESULTS: The cromakalim (10 nM to 10 µM- and pinacidil (10 nM to 10 µM-induced concentration-dependent relaxation of porcine coronary artery was inhibited by simvastatin (3 and 10 µM. Simvastatin (1, 3 and 10 µM suppressed (in okadaic acid (10 nM-sensitive manner cromakalim (10 µM- and pinacidil (10 µM-mediated opening of whole-cell KATP channels of arterial myocytes. Simvastatin (10 µM and AICAR (1 mM elicited a time-dependent, compound C (1 µM-sensitive [(3H]-2-deoxy-glucose uptake and an increase in [ATP]i levels. A time (2-30 min- and concentration (0.1-10 µM-dependent increase by simvastatin of p-AMPKα-Thr(172 and p-PP2A-Tyr(307 expression was observed. The enhanced p-AMPKα-Thr(172 expression was inhibited by compound C, ryanodine (100 µM and KN93 (10 µM. Simvastatin-induced p-PP2A-Tyr(307 expression was suppressed by okadaic acid, compound C, ryanodine, KN93, phloridzin (1 mM, ouabain (10 µM, and in [glucose]o-free or [Na(+]o-free conditions. CONCLUSIONS: Simvastatin causes ryanodine-sensitive Ca(2+ release which is important for AMPKα-Thr(172 phosphorylation via Ca(2+/CaMK II. AMPKα-Thr(172 phosphorylation causes [glucose]o uptake (and an [ATP]i increase, closure of KATP channels, and phosphorylation of AMPKα-Thr(172 and PP2A-Tyr(307 resulted. Phosphorylation of PP2A-Tyr(307 occurs at a site downstream of AMPKα-Thr(172 phosphorylation.

  16. Autoantibody against transient receptor potential M1 cation channels of retinal ON bipolar cells in paraneoplastic vitelliform retinopathy

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    Wang Yujuan

    2012-11-01

    degenerated and replaced with empty vacuoles and disintegrated organelles. Conclusion This case provides a convincing histological evidence of melanoma-associated autoantibodies directly against transient receptor potential M1 channels that target the ON bipolar cell structures in the inner nuclear and outer plexiform layers in paraneoplastic vitelliform retinopathy.

  17. Endostatin is protective against monocrotaline-induced right heart disease through the inhibition of T-type Ca(2+) channel.

    Science.gov (United States)

    Imoto, Keisuke; Kumatani, Sayaka; Okada, Muneyoshi; Yamawaki, Hideyuki

    2016-07-01

    Endostatin (ES), a C-terminal fragment of collagen XVIIIα1, has a potent anti-angiogenic effect. ES prevents tumor proliferation through inhibiting T-type Ca(2+) channel. T-type Ca(2+) channel is re-expressed during heart diseases including monocrotaline (MCT)-induced right heart failure. The present study aimed to clarify the effects of ES on T-type Ca(2+) channel and pathogenesis of MCT-induced right ventricular disease. MCT or saline was injected intraperitoneally to rats. After cardiomyocytes were isolated from right ventricles (RVs), T-type Ca(2+) channel current (I CaT) was measured by a patch-clamp method. After ES small interfering RNA (siRNA) or control siRNA (20 μg) was administrated for 1 week via the right jugular vein 1 week after MCT injection, echocardiography and histological analysis were done. I CaT was significantly increased in RV from MCT-injected rats, and ES significantly inhibited it. The survival rate of ES siRNA-administrated MCT rats (MCT ES si group) was decreased. In echocardiography, although ES siRNA did not affect pulmonary arterial pressure, RV systolic function was impaired in MCT ES si group compared with control siRNA-administrated MCT rats (MCT cont si group). In the histological analysis of RV, ES expression was increased in MCT cont si group, and ES siRNA inhibited it. Furthermore, although MCT cont si group showed only cardiomyocyte hypertrophy, MCT ES si group showed notable enlargement of intercellular spaces. The present study for the first time revealed that ES inhibits T-type Ca(2+) channel activity in RV from MCT-injected rats. ES gene knockdown deteriorates MCT-induced right heart disease. ES is thus cardioprotective possibly through inhibiting T-type Ca(2+) channel activity.

  18. TRESK background K(+ channel is inhibited by PAR-1/MARK microtubule affinity-regulating kinases in Xenopus oocytes.

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    Gabriella Braun

    Full Text Available TRESK (TWIK-related spinal cord K(+ channel, KCNK18 is a major background K(+ channel of sensory neurons. Dominant-negative mutation of TRESK is linked to familial migraine. This important two-pore domain K(+ channel is uniquely activated by calcineurin. The calcium/calmodulin-dependent protein phosphatase directly binds to the channel and activates TRESK current several-fold in Xenopus oocytes and HEK293 cells. We have recently shown that the kinase, which is responsible for the basal inhibition of the K(+ current, is sensitive to the adaptor protein 14-3-3. Therefore we have examined the effect of the 14-3-3-inhibited PAR-1/MARK, microtubule-associated-protein/microtubule affinity-regulating kinase on TRESK in the Xenopus oocyte expression system. MARK1, MARK2 and MARK3 accelerated the return of TRESK current to the resting state after the calcium-dependent activation. Several other serine-threonine kinase types, generally involved in the modulation of other ion channels, failed to influence TRESK current recovery. MARK2 phosphorylated the primary determinant of regulation, the cluster of three adjacent serine residues (S274, 276 and 279 in the intracellular loop of mouse TRESK. In contrast, serine 264, the 14-3-3-binding site of TRESK, was not phosphorylated by the kinase. Thus MARK2 selectively inhibits TRESK activity via the S274/276/279 cluster, but does not affect the direct recruitment of 14-3-3 to the channel. TRESK is the first example of an ion channel phosphorylated by the dynamically membrane-localized MARK kinases, also known as general determinants of cellular polarity. These results raise the possibility that microtubule dynamics is coupled to the regulation of excitability in the neurons, which express TRESK background potassium channel.

  19. Drug-induced Inhibition and Trafficking Disruption of ion Channels: Pathogenesis of QT Abnormalities and Drug-induced Fatal Arrhythmias.

    Science.gov (United States)

    Cubeddu, Luigi X

    2016-01-01

    Risk of severe and fatal ventricular arrhythmias, presenting as Torsade de Pointes (TdP), is increased in congenital and acquired forms of long QT syndromes (LQTS). Drug-induced inhibition of K+ currents, IKs, IKr, IK1, and/or Ito, delay repolarization, prolong QT, and increase the risk of TdP. Drug-induced interference with IKr is the most common cause of acquired LQTS/TdP. Multiple drugs bind to KNCH2-hERG-K+ channels affecting IKr, including antiarrythmics, antibiotics, antivirals, azole-antifungals, antimalarials, anticancer, antiemetics, prokinetics, antipsychotics, and antidepressants. Azithromycin has been recently added to this list. In addition to direct channel inhibition, some drugs interfere with the traffic of channels from the endoplasmic reticulum to the cell membrane, decreasing mature channel membrane density; e.g., pentamidine, geldalamicin, arsenic trioxide, digoxin, and probucol. Other drugs, such as ketoconazole, fluoxetine, norfluoxetine, citalopram, escitalopram, donepezil, tamoxifen, endoxifen, atazanavir, and roxitromycin, induce both direct channel inhibition and impaired channel trafficking. Although many drugs prolong the QT interval, TdP is a rare event. The following conditions increase the risk of drug-induced TdP: a) Disease states/electrolyte levels (heart failure, structural cardiac disease, bradycardia, hypokalemia); b) Pharmacogenomic variables (presence of congenital LQTS, subclinical ion-channel mutations, history of or having a relative with history of drug-induced long QT/TdP); c) Pharmacodynamic and kinetic factors (high doses, women, elderly, metabolism inhibitors, combining two or more QT prolonging drugs, drugs that prolong the QT and increase QT dispersion, and drugs with multiple actions on ion channels). Because most of these conditions are preventable, careful evaluation of risk factors and increased knowledge of drug use associated with repolarization abnormalities are strongly recommended.

  20. Transient receptor potential vanilloid 1 activation by dietary capsaicin promotes urinary sodium excretion by inhibiting epithelial sodium channel α subunit-mediated sodium reabsorption.

    Science.gov (United States)

    Li, Li; Wang, Fei; Wei, Xing; Liang, Yi; Cui, Yuanting; Gao, Feng; Zhong, Jian; Pu, Yunfei; Zhao, Yu; Yan, Zhencheng; Arendshorst, William J; Nilius, Bernd; Chen, Jing; Liu, Daoyan; Zhu, Zhiming

    2014-08-01

    High salt (HS) intake contributes to the development of hypertension. Epithelial sodium channels play crucial roles in regulating renal sodium reabsorption and blood pressure. The renal transient receptor potential vanilloid 1 (TRPV1) cation channel can be activated by its agonist capsaicin. However, it is unknown whether dietary factors can act on urinary sodium excretion and renal epithelial sodium channel (ENaC) function. Here, we report that TRPV1 activation by dietary capsaicin increased urinary sodium excretion through reducing sodium reabsorption in wild-type (WT) mice on a HS diet but not in TRPV1(-/-) mice. The effect of capsaicin on urinary sodium excretion was involved in inhibiting αENaC and its related with-no-lysine kinase 1/serum- and glucocorticoid-inducible protein kinase 1 pathway in renal cortical collecting ducts of WT mice. Dietary capsaicin further reduced the increased αENaC activity in WT mice attributed to the HS diet. In contrast, this capsaicin effect was absent in TRPV1(-/-) mice. Immunoprecipitation study indicated αENaC specifically coexpressed and functionally interact with TRPV1 in renal cortical collecting ducts of WT mice. Additionally, ENaC activity and expression were suppressed by capsaicin-mediated TRPV1 activation in cultured M1-cortical collecting duct cells. Long-term dietary capsaicin prevented the development of high blood pressure in WT mice on a HS diet. It concludes that TRPV1 activation in the cortical collecting ducts by capsaicin increases urinary sodium excretion and avoids HS diet-induced hypertension through antagonizing αENaC-mediated urinary sodium reabsorption. Dietary capsaicin may represent a promising lifestyle intervention in populations exposed to a high dietary salt intake.

  1. Inhibition of mitochondrial permeability transition pore contributes to the neuroprotection induced by activation of mitochondrial ATP-sensitive potassium channel

    Institute of Scientific and Technical Information of China (English)

    Li-pingWU; FangSHEN; QiangXIA

    2004-01-01

    AIM: To investigate whether the neuroprotection via activating mitochondrial ATP-sensitive potassium channel (mitoKTP) is mediated by the inhibition of mitochondrial permeability transition pore (MPTP). METHODS: Adult male Sprague-Dawleyrats were undergoing 90 min of middle cerebral artery occlusion(MCAO) by introducing a nylon monofilament through the external

  2. Isolation of bovine platelet cationic proteins which inhibit the surface-mediated activation of factor XII and prekallikrein.

    Science.gov (United States)

    Kodama, K; Kato, H; Iwanaga, S

    1985-01-01

    A possible role of bovine platelets in the surface-mediated activation of Factor XII and prekallikrein was studied, using the contact system reconstituted with the purified proteins from bovine plasma. The washed platelets before and after aggregation by ADP, thrombin or collagen did not show any ability to trigger or accelerate the activation of Factor XII and prekallikrein. On the contrary, these aggregates showed a potent inhibitory activity on the activation of those zymogens triggered by kaolin, amylose sulfate and sulfatide. The inhibitory substances from the supernatant of the thrombin-induced aggregates were separated into two major fractions, a low affinity fraction and a high affinity fraction, on a heparin-Sepharose column. The high affinity protein was identified as platelet factor 4, based on the amino acid composition. From the low affinity fraction, a beta-thromboglobulin (beta-TG)-like substance and three kinds of unknown proteins, named LA1, LA2, and LA3, were isolated by gel-filtration on a column of Sephadex G-100 or Sephadex G-75 followed by chromatography on a column of Mono S. The molecular weights of LA1, LA2, and LA3 were estimated to be 35,000, 26,000, and 11,000, respectively, on SDS-PAGE. LA2 was identified as a carbohydrate-less LA1, as judged from the amino acid composition and carbohydrate content. The inhibitory activities of these five cationic proteins on the activation of Factor XII and prekallikrein mediated with amylose sulfate, sulfatide and kaolin were different from each other. In the case of kaolin-mediated activation, LA3 was the most potent inhibitor, while platelet factor 4 and beta-TG-like substance did not show any significant inhibitory activity. Moreover, the inhibitory activities of all the cationic proteins were not correlated with their anti-heparin activities. Since these proteins were rapidly liberated from platelets by the action of the stimulants, the present results demonstrate a negative role of platelets in

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

    DEFF Research Database (Denmark)

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

    1999-01-01

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

  4. Calcium influx through L-type channels attenuates skeletal muscle contraction via inhibition of adenylyl cyclases.

    Science.gov (United States)

    Menezes-Rodrigues, Francisco Sandro; Pires-Oliveira, Marcelo; Duarte, Thiago; Paredes-Gamero, Edgar Julian; Chiavegatti, Tiago; Godinho, Rosely Oliveira

    2013-11-15

    Skeletal muscle contraction is triggered by acetylcholine induced release of Ca(2+) from sarcoplasmic reticulum. Although this signaling pathway is independent of extracellular Ca(2+), L-type voltage-gated calcium channel (Cav) blockers have inotropic effects on frog skeletal muscles which occur by an unknown mechanism. Taking into account that skeletal muscle fiber expresses Ca(+2)-sensitive adenylyl cyclase (AC) isoforms and that cAMP is able to increase skeletal muscle contraction force, we investigated the role of Ca(2+) influx on mouse skeletal muscle contraction and the putative crosstalk between extracellular Ca(2+) and intracellular cAMP signaling pathways. The effects of Cav blockers (verapamil and nifedipine) and extracellular Ca(2+) chelator EGTA were evaluated on isometric contractility of mouse diaphragm muscle under direct electrical stimulus (supramaximal voltage, 2 ms, 0.1 Hz). Production of cAMP was evaluated by radiometric assay while Ca(2+) transients were assessed by confocal microscopy using L6 cells loaded with fluo-4/AM. Ca(2+) channel blockers verapamil and nifedipine had positive inotropic effect, which was mimicked by removal of extracellular Ca(+2) with EGTA or Ca(2+)-free Tyrode. While phosphodiesterase inhibitor IBMX potentiates verapamil positive inotropic effect, it was abolished by AC inhibitors SQ22536 and NYK80. Finally, the inotropic effect of verapamil was associated with increased intracellular cAMP content and mobilization of intracellular Ca(2+), indicating that positive inotropic effects of Ca(2+) blockers depend on cAMP formation. Together, our results show that extracellular Ca(2+) modulates skeletal muscle contraction, through inhibition of Ca(2+)-sensitive AC. The cross-talk between extracellular calcium and cAMP-dependent signaling pathways appears to regulate the extent of skeletal muscle contraction responses.

  5. A tale of switched functions: from cyclooxygenase inhibition to M-channel modulation in new diphenylamine derivatives.

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    Asher Peretz

    Full Text Available Cyclooxygenase (COX enzymes are molecular targets of nonsteroidal anti-inflammatory drugs (NSAIDs, the most used medication worldwide. However, the COX enzymes are not the sole molecular targets of NSAIDs. Recently, we showed that two NSAIDs, diclofenac and meclofenamate, also act as openers of Kv7.2/3 K(+ channels underlying the neuronal M-current. Here we designed new derivatives of diphenylamine carboxylate to dissociate the M-channel opener property from COX inhibition. The carboxylate moiety was derivatized into amides or esters and linked to various alkyl and ether chains. Powerful M-channel openers were generated, provided that the diphenylamine moiety and a terminal hydroxyl group are preserved. In transfected CHO cells, they activated recombinant Kv7.2/3 K(+ channels, causing a hyperpolarizing shift of current activation as measured by whole-cell patch-clamp recording. In sensory dorsal root ganglion and hippocampal neurons, the openers hyperpolarized the membrane potential and robustly depressed evoked spike discharges. They also decreased hippocampal glutamate and GABA release by reducing the frequency of spontaneous excitatory and inhibitory post-synaptic currents. In vivo, the openers exhibited anti-convulsant activity, as measured in mice by the maximal electroshock seizure model. Conversion of the carboxylate function into amide abolished COX inhibition but preserved M-channel modulation. Remarkably, the very same template let us generating potent M-channel blockers. Our results reveal a new and crucial determinant of NSAID-mediated COX inhibition. They also provide a structural framework for designing novel M-channel modulators, including openers and blockers.

  6. Inhibition of phosphatidylinositol 3-kinase stimulates activity of the small-conductance K channel in the CCD.

    Science.gov (United States)

    Li, Dimin; Wei, Yuan; Babilonia, Elisa; Wang, Zhijian; Wang, Wen-Hui

    2006-04-01

    We used Western blotting to examine the expression of phosphatidylinositol 3-kinase (PI3K) in the renal cortex and outer medulla and employed the patch-clamp technique to study the effect of PI3K on the ROMK-like small-conductance K (SK) channels in the cortical collecting duct (CCD). Low K intake increased the expression of the 110-kDa alpha-subunit (p110alpha) of PI3K compared with rats on a normal-K diet. Because low K intake increases superoxide levels (2), the possibility that increases in superoxide anions may be responsible for the effect of low K intake on the expression of PI3K is supported by finding that addition of H(2)O(2) stimulates the expression of p110alpha in M1 cells. Inhibition of PI3K with either wortmannin or LY-294002 significantly increased channel activity in the CCD from rats on a K-deficient (KD) diet or on a normal-K diet. The stimulatory effect of wortmannin on ROMK channel activity cannot be mimicked by inhibition of phospholipase C with U-73122. This suggests that the effect of inhibiting PI3K was not the result of increasing the phosphatidylinositol 4,5-bisphosphate level. Moreover, application of the exogenous phosphatidylinositol 3,4,5-trisphosphate analog had no effect on channel activity in excised patches. Because low K intake has been shown to increase the activity of protein tyrosine kinase (PTK), we explored the role of the interaction between PTK and PI3K in the regulation of the SK channel activity. Inhibition of PTK increased SK channel activity in the CCD from rats on a KD diet. However, addition of wortmannin did not further increase ROMK channel activity. Also, the effect of wortmannin was abolished by treatment of CCD with phalloidin. We conclude that PI3K is involved in mediating the effect of low K intake on ROMK channel activity in the CCD and that the effect of PI3K on SK channels requires the involvement of PTK and the cytoskeleton.

  7. Pungent agents from Szechuan peppers excite sensory neurons by inhibiting two-pore potassium channels.

    Science.gov (United States)

    Bautista, Diana M; Sigal, Yaron M; Milstein, Aaron D; Garrison, Jennifer L; Zorn, Julie A; Tsuruda, Pamela R; Nicoll, Roger A; Julius, David

    2008-07-01

    In traditional folk medicine, Xanthoxylum plants are referred to as 'toothache trees' because their anesthetic or counter-irritant properties render them useful in the treatment of pain. Psychophysical studies have identified hydroxy-alpha-sanshool as the compound most responsible for the unique tingling and buzzing sensations produced by Szechuan peppercorns or other Xanthoxylum preparations. Although it is generally agreed that sanshool elicits its effects by activating somatosensory neurons, the underlying cellular and molecular mechanisms remain a matter of debate. Here we show that hydroxy-alpha-sanshool excites two types of sensory neurons, including small-diameter unmyelinated cells that respond to capsaicin (but not mustard oil) as well as large-diameter myelinated neurons that express the neurotrophin receptor TrkC. We found that hydroxy-alpha-sanshool excites neurons through a unique mechanism involving inhibition of pH- and anesthetic-sensitive two-pore potassium channels (KCNK3, KCNK9 and KCNK18), providing a framework for understanding the unique and complex psychophysical sensations associated with the Szechuan pepper experience.

  8. Mercury toxicity in the shark (Squalus acanthias) rectal gland: apical CFTR chloride channels are inhibited by mercuric chloride.

    Science.gov (United States)

    Ratner, Martha A; Decker, Sarah E; Aller, Stephen G; Weber, Gerhard; Forrest, John N

    2006-03-01

    In the shark rectal gland, basolateral membrane proteins have been suggested as targets for mercury. To examine the membrane polarity of mercury toxicity, we performed experiments in three preparations: isolated perfused rectal glands, primary monolayer cultures of rectal gland epithelial cells, and Xenopus oocytes expressing the shark cystic fibrosis transmembrane conductance regulator (CFTR) chloride channel. In perfused rectal glands we observed: (1) a dose-dependent inhibition by mercury of forskolin/3-isobutyl-1-methylxanthine (IBMX)-stimulated chloride secretion; (2) inhibition was maximal when mercury was added before stimulation with forskolin/IBMX; (3) dithiothrietol (DTT) and glutathione (GSH) completely prevented inhibition of chloride secretion. Short-circuit current (Isc) measurements in monolayers of rectal gland epithelial cells were performed to examine the membrane polarity of this effect. Mercuric chloride inhibited Isc more potently when applied to the solution bathing the apical vs. the basolateral membrane (23 +/- 5% and 68 +/- 5% inhibition at 1 and 10 microM HgCl2 in the apical solution vs. 2 +/- 0.9% and 14 +/- 5% in the basolateral solution). This inhibition was prevented by pre-treatment with apical DTT or GSH; however, only the permeant reducing agent DTT reversed mercury inhibition when added after exposure. When the shark rectal gland CFTR channel was expressed in Xenopus oocytes and chloride conductance was measured by two-electrode voltage clamping, we found that 1 microM HgCl2 inhibited forskolin/IBMX conductance by 69.2 +/- 2.0%. We conclude that in the shark rectal gland, mercury inhibits chloride secretion by interacting with the apical membrane and that CFTR is the likely site of this action.

  9. Absence of the ER Cation Channel TMEM38B/TRIC-B Disrupts Intracellular Calcium Homeostasis and Dysregulates Collagen Synthesis in Recessive Osteogenesis Imperfecta.

    Directory of Open Access Journals (Sweden)

    Wayne A Cabral

    2016-07-01

    Full Text Available Recessive osteogenesis imperfecta (OI is caused by defects in proteins involved in post-translational interactions with type I collagen. Recently, a novel form of moderately severe OI caused by null mutations in TMEM38B was identified. TMEM38B encodes the ER membrane monovalent cation channel, TRIC-B, proposed to counterbalance IP3R-mediated Ca2+ release from intracellular stores. The molecular mechanisms by which TMEM38B mutations cause OI are unknown. We identified 3 probands with recessive defects in TMEM38B. TRIC-B protein is undetectable in proband fibroblasts and osteoblasts, although reduced TMEM38B transcripts are present. TRIC-B deficiency causes impaired release of ER luminal Ca2+, associated with deficient store-operated calcium entry, although SERCA and IP3R have normal stability. Notably, steady state ER Ca2+ is unchanged in TRIC-B deficiency, supporting a role for TRIC-B in the kinetics of ER calcium depletion and recovery. The disturbed Ca2+ flux causes ER stress and increased BiP, and dysregulates synthesis of proband type I collagen at multiple steps. Collagen helical lysine hydroxylation is reduced, while telopeptide hydroxylation is increased, despite increased LH1 and decreased Ca2+-dependent FKBP65, respectively. Although PDI levels are maintained, procollagen chain assembly is delayed in proband cells. The resulting misfolded collagen is substantially retained in TRIC-B null cells, consistent with a 50-70% reduction in secreted collagen. Lower-stability forms of collagen that elude proteasomal degradation are not incorporated into extracellular matrix, which contains only normal stability collagen, resulting in matrix insufficiency. These data support a role for TRIC-B in intracellular Ca2+ homeostasis, and demonstrate that absence of TMEM38B causes OI by dysregulation of calcium flux kinetics in the ER, impacting multiple collagen-specific chaperones and modifying enzymes.

  10. Benzopyrimido-pyrrolo-oxazine-dione (R)-BPO-27 Inhibits CFTR Chloride Channel Gating by Competition with ATP.

    Science.gov (United States)

    Kim, Yonjung; Anderson, Marc O; Park, Jinhong; Lee, Min Goo; Namkung, Wan; Verkman, A S

    2015-10-01

    We previously reported that benzopyrimido-pyrrolo-oxazinedione BPO-27 [6-(5-bromofuran-2-yl)-7,9-dimethyl-8,10-dioxo-11-phenyl-7,8,9,10-tetrahydro-6H-benzo[b]pyrimido [4',5':3,4]pyrrolo [1,2-d][1,4]oxazine-2-carboxylic acid] inhibits the cystic fibrosis transmembrane conductance regulator (CFTR) chloride channel with low nanomolar potency and reduces cystogenesis in a model of polycystic kidney disease. We used computational chemistry and patch-clamp to show that enantiomerically pure (R)-BPO-27 inhibits CFTR by competition with ATP, whereas (S)-BPO-27 is inactive. Docking computations using a homology model of CFTR structure suggested that (R)-BPO-27 binds near the canonical ATP binding site, and these findings were supported by molecular dynamics simulations showing a lower binding energy for the (R) versus (S) stereoisomers. Three additional lower-potency BPO-27 analogs were modeled in a similar fashion, with the binding energies predicted in the correct order. Whole-cell patch-clamp studies showed linear CFTR currents with a voltage-independent (R)-BPO-27 block mechanism. Single-channel recordings in inside-out patches showed reduced CFTR channel open probability and increased channel closed time by (R)-BPO-27 without altered unitary channel conductance. At a concentration of (R)-BPO-27 that inhibited CFTR chloride current by ∼50%, the EC50 for ATP activation of CFTR increased from 0.27 to 1.77 mM but was not changed by CFTRinh-172 [4-[[4-oxo-2-thioxo-3-[3-trifluoromethyl)phenyl]-5-thiazolidinylidene]methyl]benzoic acid], a thiazolidinone CFTR inhibitor that acts at a site distinct from the ATP binding site. Our results suggest that (R)-BPO-27 inhibition of CFTR involves competition with ATP.

  11. Squalamine, a novel cationic steroid, specifically inhibits the brush-border Na+/H+ exchanger isoform NHE3.

    Science.gov (United States)

    Akhter, S; Nath, S K; Tse, C M; Williams, J; Zasloff, M; Donowitz, M

    1999-01-01

    Squalamine, an endogenous molecule found in the liver and other tissues of Squalus acanthias, has antibiotic properties and causes changes in endothelial cell shape. The latter suggested that its potential targets might include transport proteins that control cell volume or cell shape. The effect of purified squalamine was examined on cloned Na+/H+ exchanger isoforms NHE1, NHE2, and NHE3 stably transfected in PS120 fibroblasts. Squalamine (1-h pretreatment) decreased the maximal velocity of rabbit NHE3 in a concentration-dependent manner (13, 47, and 57% inhibition with 3, 5, and 7 micrograms/ml, respectively) and also increased K'[H+]i. Squalamine did not affect rabbit NHE1 or NHE2 function. The inhibitory effect of squalamine was 1) time dependent, with no effect of immediate addition and maximum effect with 1 h of exposure, and 2) fully reversible. Squalamine pretreatment of the ileum for 60 min inhibited brush-border membrane vesicle Na+/H+ activity by 51%. Further investigation into the mechanism of squalamine's effects showed that squalamine required the COOH-terminal 76 amino acids of NHE3. Squalamine had no cytotoxic effect at the concentrations studied, as indicated by monitoring lactate dehydrogenase release. These results indicate that squalamine 1) is a specific inhibitor of the brush-border NHE isoform NHE3 and not NHE1 or NHE2, 2) acts in a nontoxic and fully reversible manner, and 3) has a delayed effect, indicating that it may influence brush-border Na+/H+ exchanger function indirectly, through an intracellular signaling pathway or by acting as an intracellular modulator.

  12. 5,6-EET potently inhibits T-type calcium channels

    DEFF Research Database (Denmark)

    Cazade, M.; Bidaud, I.; Hansen, Pernille B. Lærkegaard;

    2014-01-01

    T-type calcium channels (T-channels) are important actors in neuronal pacemaking, in heart rhythm, and in the control of the vascular tone. T-channels are regulated by several endogenous lipids including the primary eicosanoid arachidonic acid (AA), which display an important role in vasodilation...

  13. Inhibition of the corrosion process of alloy AA5083 (Al-Mg) in seawater by cerium cations. An EIS study

    Energy Technology Data Exchange (ETDEWEB)

    Aballe, A.; Bethencourt, M.; Botana, F.J.; Cano, M.J. [Cadiz Univ. (Spain). Dept. de Ciencia de los Materiales e Ingenieria Metalurgica y Quimica Inorganica; Marcos, M. [Cadiz Univ. (Spain). Dept. de Ingenieria Mecanica y Diseno Industrial

    2001-05-01

    The principal corrosion process of the alloy AA5083 in aerated NaCl solutions mainly affects the zones occupied by Al(Mn,Fe,Cr) cathodic precipitates, over which the oxygen reduction reaction takes place. Thus, the design of an effective protection system could be based on the use of cathodic inhibitors. In previous papers, the inhibition efficiency of various lanthanide chlorides, particularly CeCl{sub 3}, has been proved. These compounds act as cathodic inhibitors, blocking these cathodic sites by means of the precipitation of a lanthanide oxide or hydroxide film. In this paper, EIS has been used to detect the blocking effect of the cathodic sites. This fact is reflected in the electrical response of the system through the minimisation of the values of those elements of the electrical loop that are related to the response of the cathodic intermetallics. In addition, the dielectric properties of the anodic film have been evaluated by measuring the value of the associated capacitance. From these values, it can be concluded that the thickness of the film produced over the matrix when the inhibitor is added is much lower than that produced in the bare solution. This finding is in good agreement with the results obtained with other techniques and may be related to the minimisation of the anodic reaction as a consequence of the decrease in the intensity of the cathodic process by the action of CeCl{sub 3}. (orig.)

  14. Gynura procumbens Merr. decreases blood pressure in rats by vasodilatation via inhibition of calcium channels

    Directory of Open Access Journals (Sweden)

    See-Ziau Hoe

    2011-01-01

    Full Text Available INTRODUCTION: Gynura procumbens has been shown to decrease blood pressure via inhibition of the angiotensinconverting enzyme. However, other mechanisms that may contribute to the hypotensive effect have not been studied. OBJECTIVES: To investigate the cardiovascular effects of a butanolic fraction of Gynura procumbens in rats. METHODS: Anaesthetized rats were given intravenous bolus injections of butanolic fraction at doses of 2.5-20 mg/kg in vivo. The effect of butanolic fraction on vascular reactivity was recorded in isolated rat aortic rings in vitro. RESULTS: Intravenous administrations of butanolic fraction elicited significant (p<0.001 and dose-dependent decreases in the mean arterial pressure. However, a significant (p<0.05 decrease in the heart rate was observed only at the higher doses (10 and 20 mg/kg. In isolated preparations of rat aortic rings, phenylephrine (1×10-6 M- or potassium chloride (8×10-2 M-precontracted endothelium-intact and -denuded tissue; butanolic fraction (1×10-6-1×10-1 g/ml induced similar concentration-dependent relaxation of the vessels. In the presence of 2.5×10-3 and 5.0×10-3 g/ml butanolic fraction, the contractions induced by phenylephrine (1×10-9-3×10-5 M and potassium chloride (1×10-2-8×10-2 M were significantly antagonized. The calcium-induced vasocontractions (1×10-4-1×10-2 M were antagonized by butanolic fraction concentration-dependently in calcium-free and high potassium (6×10-2 M medium, as well as in calcium- and potassium-free medium containing 1×10-6 M phenylephrine. However, the contractions induced by noradrenaline (1×10-6 M and caffeine (4.5×10-2 M were not affected by butanolic fraction. CONCLUSION: Butanolic fraction contains putative hypotensive compounds that appear to inhibit calcium influx via receptor-operated and/or voltage-dependent calcium channels to cause vasodilation and a consequent fall in blood pressure.

  15. Selective serotonin reuptake inhibitor sertraline inhibits voltage-dependent K+ channels in rabbit coronary arterial smooth muscle cells.

    Science.gov (United States)

    Kim, Han Sol; Li, Hongliang; Kim, Hye Won; Shin, Sung Eun; Choi, Il-Whan; Firth, Amy L; Bang, Hyoweon; Bae, Young Min; Park, Won Sun

    2016-12-01

    We examined the effects of the selective serotonin reuptake inhibitor (SSRI) sertraline on voltage-dependent K+ (Kv) channels in freshly isolated rabbit coronary arterial smooth muscle cells using the voltage-clamp technique. Sertraline decreased the Kv channel current in a dose-dependent manner, with an IC50 value of 0.18 mu M and a slope value (Hill coefficient) of 0.61. Although the application of 1 mu M sertraline did not affect the steady-state activation curves, sertraline caused a significant, negative shift in the inactivation curves. Pretreatment with another SSRI, paroxetine, had no significant effect on Kv currents and did not alter the inhibitory effects of sertraline on Kv currents. From these results, we concluded that sertraline dose-dependently inhibited Kv currents independently of serotonin reuptake inhibition by shifting inactivation curves to a more negative potential.

  16. Exposure to extremely low-frequency electromagnetic fields inhibits T-type calcium channels via AA/LTE4 signaling pathway.

    Science.gov (United States)

    Cui, Yujie; Liu, Xiaoyu; Yang, Tingting; Mei, Yan-Ai; Hu, Changlong

    2014-01-01

    Extremely low-frequency electromagnetic fields (ELF-EMF) causes various biological effects through altering intracellular calcium homeostasis. The role of high voltage-gated (HVA) calcium channels in ELF-EMF induced effects has been extensively studied. However, the effect of ELF-EMF on low-voltage-gated (LVA) T-type calcium channels has not been reported. In this study, we test the effect of ELF-EMF (50Hz) on human T-type calcium channels transfected in HEK293 cells. Conversely to its stimulant effects on HVA channels, ELF-EMF exposure inhibited all T-type (Cav3.1, Cav3.2 and Cav3.3) channels. Neither the protein expression nor the steady-state activation and inactivation kinetics of Cav3.2 channels were altered by ELF-EMF (50Hz, 0.2mT) exposure. Exposure to ELF-EMF increased both arachidonic acid (AA) and leukotriene E4 (LTE4) levels in HEK293 cells. CAY10502 and bestatin, which block the increase of AA and LTE4 respectively, abrogated the ELF-EMF inhibitory effect on Cav3.2 channels. Exogenous LTE4 mimicked the ELF-EMF inhibition of T-type calcium channels. ELF-EMF (50Hz) inhibits native T-type calcium channels in primary cultured mouse cortical neurons via LTE4. We conclude that 50Hz ELF-EMF inhibits T-type calcium channels through AA/LTE4 signaling pathway.

  17. L—type calcium channel blockers inhibit the development but not the expression of sensitization to morphine in mice

    Institute of Scientific and Technical Information of China (English)

    ZhanQ; ZhenJW

    2002-01-01

    The relationship between opioid actions and L-type calcium channel blockers has been well documented.However,there is no report relevant to L-type calcium channel blockers and morphinesensitization,which is suggested to be an analog of behaviors that are the characteristics of drug addiction.Here the effects of three L-type calcium channel blockers,nimodipine,nifedipine and verapamil,on morphine-induced locomotor activity,the development and the expression of sensitization to morphine were studied systematically.The results showed that both nimodipine and verapamil attenuated,while nifedipine had only a tendency to decrease morphine-induced locomotor activity.All the three drugs inhibited the development of sensitization to morphine.However,none of them showed any effects on the expression of morphine sensitization.These results indicate that blocking L-tpye calcium channel attenuates the locomotor stimulating effects of morphine and inhibits the development but not the expression of morphine-sensitization.

  18. A cationic-independent mannose 6-phosphate receptor inhibitor (PXS64 ameliorates kidney fibrosis by inhibiting activation of transforming growth factor-β1.

    Directory of Open Access Journals (Sweden)

    Jie Zhang

    Full Text Available The activity of transforming growth factor-β1 (TGF-β1 is regulated by its conversion from the latent to the active form. We have previously shown that the conversion is at least in part mediated by the cationic-independent mannose 6-phosphate receptor (CI-M6PR, as the CI-M6PR inhibitor, PXS-25 has anti-fibrotic properties in human kidney tubular (HK-2 cells under high glucose conditions. However, its clinical use is limited by low bioavailability. Our aim was to determine the effects of PXS64, a pro-drug of PXS25, in in vitro and in vivo models of renal fibrosis. HK-2 cells were exposed to latent TGFβ1+/- PXS64 for 48 hours. The mRNA and protein levels of pro-fibrotic and pro-inflammatory markers were determined. A 7 day unilateral ureteric obstruction (UUO model was used and the following experimental groups were studied: (i Sham operated, (ii UUO, (iii UUO + telmisartan (iv UUO + PSX64. HK-2 cells exposed to PXS64 reduced TGFβ mediated effects on collagen IV, fibronectin, macrophage chemotactic protein-1 (MCP-1 and phospho-smad2 protein expression, consistent with inhibition of the conversion of latent to active TGF-β1. PXS 64 treated UUO mice had a lower tubulointerstitial fibrosis index, collagen IV and fibronectin protein and mRNA expression when compared to untreated UUO mice. In addition, these animals had lower MCP-1 mRNA expression, reduced inflammarory cell infiltrate, as indicated by fewer CD45, F4/80 positive cells, and reduced phospho-Smad2 protein expression when compared to untreated UUO animals. Our data demonstrates that PSX64 is an effective anti-fibrotic agent by inhibiting the activation of latent TGF-β1.

  19. Cortical neurons immunoreactive for the potassium channel Kv3.1b subunit are predominantly surrounded by perineuronal nets presumed as a buffering system for cations.

    Science.gov (United States)

    Härtig, W; Derouiche, A; Welt, K; Brauer, K; Grosche, J; Mäder, M; Reichenbach, A; Brückner, G

    1999-09-18

    Perineuronal nets (PNs) are known as chondroitin sulphate-rich, lattice-like coatings of the extracellular matrix. In the cortex of mammalian species investigated so far, they were mainly found around GABAergic neurons, but to a lesser degree also around pyramidal cells. Previous investigations in the rat revealed similar distribution patterns of fast-firing neurons expressing both the Kv3.1b subunit of voltage-gated potassium channels and the calcium-binding protein parvalbumin. In the present study, triple fluorescence labelling was applied for the simultaneous demonstration of PNs with the N-acetylgalactosamine-specific Wisteria floribunda agglutinin (WFA), parvalbumin-immunoreactivity (ir) with a monoclonal antibody and of Kv3.1b-ir with several rabbit antibodies. Subsets of non-pyramidal neurons - enwrapped by PNs and expressing parvalbumin and Kv3.1b - were detected in the rat and monkey neocortex and hippocampus. In the rat, faintly stained PNs were additionally found around several layer II/III and V pyramidal cells immunonegative for Kv3.1b, but contacted by Kv3.1b-containing boutons. In the monkey, more intensely labelled PNs frequently occurred around pyramidal cells which themselves appeared to be Kv3. 1b-immunopositive. We also observed minor Kv3.1b-ir and parvalbumin-ir cortical cell populations which were devoid of PNs; occasionally, nets were detected around neurons lacking both immunoreactivities. By confocal laser scanning microscopy, Kv3.1b-ir and WFA-binding sites were found adjoining at the soma and proximal dendritic surface, while lectin-binding sites usually extended on more distal dendritic segments and the axon initial segments which failed to express detectable Kv3.1b-ir. This spatial relationship of both markers was also confirmed by combined WFA-gold labelling and Kv3.1b-immunoperoxidase staining at the electron microscopic level. The data are used for a critical examination of current hypotheses concerning the functional role of PNs

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

    DEFF Research Database (Denmark)

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

    2010-01-01

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

  1. Steviol reduces MDCK Cyst formation and growth by inhibiting CFTR channel activity and promoting proteasome-mediated CFTR degradation.

    Directory of Open Access Journals (Sweden)

    Chaowalit Yuajit

    Full Text Available Cyst enlargement in polycystic kidney disease (PKD involves cAMP-activated proliferation of cyst-lining epithelial cells and transepithelial fluid secretion into the cyst lumen via cystic fibrosis transmembrane conductance regulator (CFTR chloride channel. This study aimed to investigate an inhibitory effect and detailed mechanisms of steviol and its derivatives on cyst growth using a cyst model in Madin-Darby canine kidney (MDCK cells. Among 4 steviol-related compounds tested, steviol was found to be the most potent at inhibiting MDCK cyst growth. Steviol inhibition of cyst growth was dose-dependent; steviol (100 microM reversibly inhibited cyst formation and cyst growth by 72.53.6% and 38.2±8.5%, respectively. Steviol at doses up to 200 microM had no effect on MDCK cell viability, proliferation and apoptosis. However, steviol acutely inhibited forskolin-stimulated apical chloride current in MDCK epithelia, measured with the Ussing chamber technique, in a dose-dependent manner. Prolonged treatment (24 h with steviol (100 microM also strongly inhibited forskolin-stimulated apical chloride current, in part by reducing CFTR protein expression in MDCK cells. Interestingly, proteasome inhibitor, MG-132, abolished the effect of steviol on CFTR protein expression. Immunofluorescence studies demonstrated that prolonged treatment (24 h with steviol (100 microM markedly reduced CFTR expression at the plasma membrane. Taken together, the data suggest that steviol retards MDCK cyst progression in two ways: first by directly inhibiting CFTR chloride channel activity and second by reducing CFTR expression, in part, by promoting proteasomal degradation of CFTR. Steviol and related compounds therefore represent drug candidates for treatment of polycystic kidney disease.

  2. Lacosamide Inhibition of Nav1.7 Voltage-Gated Sodium Channels: Slow Binding to Fast-Inactivated States.

    Science.gov (United States)

    Jo, Sooyeon; Bean, Bruce P

    2017-04-01

    Lacosamide is an antiseizure agent that targets voltage-dependent sodium channels. Previous experiments have suggested that lacosamide is unusual in binding selectively to the slow-inactivated state of sodium channels, in contrast to drugs like carbamazepine and phenytoin, which bind tightly to fast-inactivated states. Using heterologously expressed human Nav1.7 sodium channels, we examined the state-dependent effects of lacosamide. Lacosamide induced a reversible shift in the voltage dependence of fast inactivation studied with 100-millisecond prepulses, suggesting binding to fast-inactivated states. Using steady holding potentials, lacosamide block was very weak at -120 mV (3% inhibition by 100 µM lacosamide) but greatly enhanced at -80 mV (43% inhibition by 100 µM lacosamide), where there is partial fast inactivation but little or no slow inactivation. During long depolarizations, lacosamide slowly (over seconds) put channels into states that recovered availability slowly (hundreds of milliseconds) at -120 mV. This resembles enhancement of slow inactivation, but the effect was much more pronounced at -40 mV, where fast inactivation is complete, but slow inactivation is not, than at 0 mV, where slow inactivation is maximal, more consistent with slow binding to fast-inactivated states than selective binding to slow-inactivated states. Furthermore, inhibition by lacosamide was greatly reduced by pretreatment with 300 µM lidocaine or 300 µM carbamazepine, suggesting that lacosamide, lidocaine, and carbamazepine all bind to the same site. The results suggest that lacosamide binds to fast-inactivated states in a manner similar to other antiseizure agents but with slower kinetics of binding and unbinding.

  3. Rock Tea extract (Jasonia glutinosa) relaxes rat aortic smooth muscle by inhibition of L-type Ca(2+) channels.

    Science.gov (United States)

    Valero, Marta Sofía; Oliván-Viguera, Aida; Garrido, Irene; Langa, Elisa; Berzosa, César; López, Víctor; Gómez-Rincón, Carlota; Murillo, María Divina; Köhler, Ralf

    2015-12-01

    In traditional herbal medicine, Rock Tea (Jasonia glutinosa) is known for its prophylactic and therapeutic value in various disorders including arterial hypertension. However, the mechanism by which Rock Tea exerts blood pressure-lowering actions has not been elucidated yet. Our aim was to demonstrate vasorelaxing effects of Rock Tea extract and to reveal its possible action mechanism. Isometric myography was conducted on high-K+-precontracted rings from rat thoracic aorta and tested extracts at concentrations of 0.5-5 mg/ml. Whole-cell patch-clamp experiments were performed in rat aortic vascular smooth muscle cells (line A7r5) to determine blocking effects on L-type Ca(2+) channels. Rock Tea extract relaxed the aorta contracted by high [K+] concentration dependently with an EC50 of ≈2.4 mg/ml and produced ≈75 % relaxation at the highest concentration tested. The L-type Ca(2+) channel blocker, verapamil (10(-6) M), had similar effects. Rock Tea extract had no effect in nominally Ca(2+)-free high-K(+) buffer but significantly inhibited contractions to re-addition of Ca(2+). Rock Tea extract inhibited the contractions induced by the L-type Ca(2+) channel activator Bay K 8644 (10(-5) M) and by phenylephrine (10(-6) M). Rock Tea extract and Y-27632 (10(-6) M), Rho-kinase inhibitor, had similar effects and the respective effects were not additive. Patch-clamp experiments demonstrated that Rock Tea extract (2.5 mg/ml) virtually abolished L-type Ca(2+) currents in A7r5. We conclude that Rock Tea extract produced vasorelaxation of rat aorta and that this relaxant effect is mediated by inhibition of L-type Ca(2+) channels. Rock Tea extracts may be of phytomedicinal value for prevention and adjuvant treatment of hypertension and other cardiovascular diseases.

  4. Molecular physiology of the insect K-activated amino acid transporter 1 (KAAT1) and cation-anion activated amino acid transporter/channel 1 (CAATCH1) in the light of the structure of the homologous protein LeuT.

    Science.gov (United States)

    Castagna, M; Bossi, E; Sacchi, V F

    2009-06-01

    K-activated amino acid transporter 1 (KAAT1) and cation-anion-activated amino acid transporter/channel 1 (CAATCH1) are amino acid cotransporters, belonging to the Na/Cl-dependent neurotransmitter transporter family (also called SLC6/NSS), that have been cloned from Manduca sexta midgut. They have been thoroughly studied by expression in Xenopus laevis oocytes, and structure/function analyses have made it possible to identify the structural determinants of their cation and amino acid selectivity. About 40 mutants of these proteins have been studied by measuring amino acid uptake and current/voltage relationships. The results obtained since the cloning of KAAT1 and CAATCH1 are here discussed in the light of the 3D model of the first crystallized member of the family, the leucine transporter LeuT.

  5. High glucose inhibits ClC-2 chloride channels and attenuates cell migration of rat keratinocytes

    Directory of Open Access Journals (Sweden)

    Pan F

    2015-08-01

    Full Text Available Fuqiang Pan, Rui Guo, Wenguang Cheng, Linlin Chai, Wenping Wang, Chuan Cao, Shirong LiDepartment of Plastic and Reconstructive Surgery, Southwestern Hospital, Third Military Medical University, Chongqing, People’s Republic of China Background: Accumulating evidence has demonstrated that migration of keratinocytes is critical to wound epithelialization, and defects of this function result in chronic delayed-healing wounds in diabetes mellitus patients, and the migration has been proved to be associated with volume-activated chloride channels. The aim of the study is to investigate the effects of high glucose (HG, 25 mM on ClC-2 chloride channels and cell migration of keratinocytes.Methods: Newborn Sprague Dawley rats were used to isolate and culture the keratinocyte in this study. Immunofluorescence assay, real-time polymerase chain reaction, and Western blot assay were used to examine the expression of ClC-2 protein or mRNA. Scratch wound assay was used to measure the migratory ability of keratinocytes. Transwell cell migration assay was used to measure the invasion and migration of keratinocytes. Recombinant lentivirus vectors were established and transducted to keratinocytes. Whole-cell patch clamp was used to perform the electrophysiological studies.Results: We found that the expression of ClC-2 was significantly inhibited when keratinocytes were exposed to a HG (25 mM medium, accompanied by the decline of volume-activated Cl- current (ICl,vol, migration potential, and phosphorylated PI3K as compared to control group. When knockdown of ClC-2 by RNAi or pretreatment with wortmannin, similar results were observed, including ICl,vol and migration keratinocytes were inhibited.Conclusion: Our study proved that HG inhibited ClC-2 chloride channels and attenuated cell migration of rat keratinocytes via inhibiting PI3K signaling.Keywords: high glucose, keratinocytes, ClC-2, cell migration, PI3K

  6. Carbenoxolone inhibits Pannexin1 channels through interactions in the first extracellular loop.

    Science.gov (United States)

    Michalski, Kevin; Kawate, Toshimitsu

    2016-02-01

    Pannexin1 (Panx1) is an ATP release channel important for controlling immune responses and synaptic strength. Various stimuli including C-terminal cleavage, a high concentration of extracellular potassium, and voltage have been demonstrated to activate Panx1. However, it remains unclear how Panx1 senses and integrates such diverse stimuli to form an open channel. To provide a clue on the mechanism underlying Panx1 channel gating, we investigated the action mechanism of carbenoxolone (CBX), the most commonly used small molecule for attenuating Panx1 function triggered by a wide range of stimuli. Using a chimeric approach, we discovered that CBX reverses its action polarity and potentiates the voltage-gated channel activity of Panx1 when W74 in the first extracellular loop is mutated to a nonaromatic residue. A systematic mutagenesis study revealed that conserved residues in this loop also play important roles in CBX function, potentially by mediating CBX binding. We extended our experiments to other Panx1 inhibitors such as probenecid and ATP, which also potentiate the voltage-gated channel activity of a Panx1 mutant at position 74. Notably, probenecid alone can activate this mutant at a resting membrane potential. These data suggest that CBX and other inhibitors, including probenecid, attenuate Panx1 channel activity through modulation of the first extracellular loop. Our experiments are the first step toward identifying a previously unknown mode of CBX action, which provide insight into the role of the first extracellular loop in Panx1 channel gating.

  7. Voltage-independent inhibition of Cav2.2 channels is delimited to a specific region of the membrane potential in rat SCG neurons

    Institute of Scientific and Technical Information of China (English)

    Oscar Vivas; Isabel Arenas; David E.García

    2012-01-01

    Neurotransmitters and hormones regulate Cav2.2 channels through a voltage-independent pathway which is not well understood.It has been suggested that this voltageindependent inhibition is constant at all membrane voltages.However,changes in the percent of voltageindependent inhibition of Cav2.2 have not been tested within a physiological voltage range.Here,we used a double-pulse protocol to isolate the voltage-independent inhibition of Cav2.2 channels induced by noradrenaline in rat superior cervical ganglion neurons.To assess changes in the percent of the voltage-independent inhibition,the activation voltage of the channels was tested between -40 and +40 mV.We found that the percent of voltage-independent inhibition induced by noradrenaline changed with the activation voltage used.In addition,voltage-independent inhibition induced by oxo-M,a muscarinic agonist,exhibited thesame dependence on activation voltage,which supports that this pattern is not exclusive for adrenergic activation.Our results suggested that voltage-independent inhibition of Cav2.2 channels depends on the activation voltage of the channel in a physiological voltage range.This may have relevant implications in the understanding of the mechanism involved in voltage-independent inhibition.

  8. Phentolamine inhibits the pacemaker activity of mouse interstitial cells of Cajal by activating ATP-sensitive K+ channels.

    Science.gov (United States)

    Ahn, Seung Whan; Kim, Sang Hun; Kim, Jin Ho; Choi, Seok; Yeum, Cheol Ho; Wie, Hee Wook; Sun, Jae Myeong; So, Insuk; Jun, Jae Yeoul

    2010-03-01

    The aim of this study was to clarify if phentolamine has proven effects on the pacemaker activities of interstitial cells of Cajal (ICC) from the mouse small intestine involving the ATPsensitive K(+) channels and adrenergic receptor. The actions of phentolamine on pacemaker activities were investigated using whole-cell patch-clamp technique and intracellular Ca(2+) analysis at 30 degrees C in cultured mouse intestinal ICC. ICC generated spontaneous pacemaker currents at a holding potential of -70 mV. Treatment with phentolamine reduced the frequency and amplitude of the pacemaker currents and increased the resting outward currents. Moreover, under current clamping (I = 0), phentolamine hyperpolarized the ICC membrane and decreased the amplitude of the pacemaker potentials. We also observed that phentolamine inhibited spontaneous [Ca(2+)](i) oscillations in ICC. The alpha-adrenergic drugs prazosin, yohimbine, methoxamine, and clonidine had no effect on ICC intestinal pacemaker activity and did not block phentolamine-induced effects. Phentolamine-induced effects on the pacemaker currents and the pacemaker potentials were significantly inhibited by ATP sensitive K(+) channel blocker glibenclamide, but not by TEA, apamin, or 4-aminopyridine. In addition, the NO synthase inhibitor, L-NAME and the guanylate cyclase inhibitor, ODQ were incapable of blocking the phentolamine-induced effects. These results demonstrate that phentolamine regulates the pacemaker activity of ICC via ATP-sensitive K(+) channel activation. Phentolamine could act through an adrenergic receptor- and also through NO-independent mechanism that involves intracellular Ca(2+) signaling.

  9. Agmatine suppresses peripheral sympathetic tone by inhibiting N-type Ca(2+) channel activity via imidazoline I2 receptor activation.

    Science.gov (United States)

    Kim, Young-Hwan; Jeong, Ji-Hyun; Ahn, Duck-Sun; Chung, Seungsoo

    2016-08-26

    Agmatine, a putative endogenous ligand of imidazoline receptors, suppresses cardiovascular function by inhibiting peripheral sympathetic tone. However, the molecular identity of imidazoline receptor subtypes and its cellular mechanism underlying the agmatine-induced sympathetic suppression remains unknown. Meanwhile, N-type Ca(2+) channels are important for the regulation of NA release in the peripheral sympathetic nervous system. Therefore, it is possible that agmatine suppresses NA release in peripheral sympathetic nerve terminals by inhibiting Ca(2+) influx through N-type Ca(2+) channels. We tested this hypothesis by investigating agmatine effect on electrical field stimulation (EFS)-evoked contraction and NA release in endothelium-denuded rat superior mesenteric arterial strips. We also investigated the effect of agmatine on the N-type Ca(2+) current in superior cervical ganglion (SCG) neurons in rats. Our study demonstrates that agmatine suppresses peripheral sympathetic outflow via the imidazoline I2 receptor in rat mesenteric arteries. In addition, the agmatine-induced suppression of peripheral vascular sympathetic tone is mediated by modulating voltage-dependent N-type Ca(2+) channels in sympathetic nerve terminals. These results suggest a potential cellular mechanism for the agmatine-induced suppression of peripheral sympathetic tone. Furthermore, they provide basic and theoretical information regarding the development of new agents to treat hypertension.

  10. INVESTIGATION OF SEIZURE ACTIVITY AFTER CYCLIC NUCLEOTIDE PHOSPHODIESTERASE INHIBITION WITH SECOND MESSENGER AND CALCIUM ION CHANNEL INHIBITION IN MICE

    Directory of Open Access Journals (Sweden)

    J Nandhakumar

    2012-03-01

    Full Text Available The role of PDE-4 inhibitor etazolate, was evaluated in the presence of PDE-7 inhibitor, BRL-50481, in animal models of epilepsy. Seizures were induced in the animals by subjecting them to injection of chemical convulsants, Pilocarpine, Kainic acid (KA and maximal electroshock (MES. The combination of etazolate and BRL50481 treated mice showed a significant (P<0.001 quick onset of action, jerky movements and convulsion when compared to gabapentin. The combination of etazolate and sGC inhibitor, methylene blue (MB treated mice showed a significant (P<0.001 delay in onset of action, jerky movements and convulsion when compare to gabapentin as well as against the combination of etazolate with BRL 50481.The present study mainly highlights the individual effects of etazolate and combination with BRL-50481 potentiates (P<0.001 the onset of seizure activity against all models of convulsion. The study mainly comprises the onset of seizures, mortality/recovery, percentage of prevention of seizures (anticonvulsant and total duration of convulsive time. The total convulsive time was prolonged significantly (P<0.05 and P<0.01 in combination of methylene blue with etazolate treated (28.59% and 35.15 % groups, compared to DMSO received group (100% in the MES model. In the same way, the combination of calcium channel modulator (CCM and calcium channel blocker (CCB amiodarone and nifedipine respectively, with etazolate showed a significant (P<0.001 delay in onset of seizures, compared to DMSO and etazolate treated groups in all models of epilepsy. This confirms that both CCM and CCB possess anticonvulsant activity. Finally, the study reveals that identification of new cAMP mediated phosphodiesterases family members offers a potential new therapy for epilepsy management in future.

  11. Involvement of presynaptic voltage-dependent Kv3 channel in endothelin-1-induced inhibition of noradrenaline release from rat gastric sympathetic nerves.

    Science.gov (United States)

    Nakamura, Kumiko; Shimizu, Takahiro; Tanaka, Kenjiro; Taniuchi, Keisuke; Yokotani, Kunihiko

    2012-11-05

    We previously reported that two types of K(+) channels, the BK type Ca(2+)-activated K(+) channel coupled with phospholipase C (PLC) and the voltage-dependent K(+) channel (Kv channel), are, respectively, involved in the prostanoid TP receptor- and muscarinic M(2) receptor-mediated inhibition of noradrenaline (NA) release from rat gastric sympathetic nerves. In the present study, therefore, we examined whether these K(+) channels are involved in endothelin-1-induced inhibition of NA release, using an isolated, vascularly perfused rat stomach. The gastric sympathetic postganglionic nerves around the left gastric artery were electrically stimulated twice at 2.5 Hz for 1 min, and endothelin-1 was added during the second stimulation. Endothelin-1 (1, 2 and 10 nM) dose-dependently inhibited gastric NA release. Endothelin-1 (2 nM)-induced inhibition of NA release was neither attenuated by PLC inhibitors [U-73122 (3 μM) and ET-18-OCH(3) (3 μM)] nor by Ca(2+)-activated K(+) channel blockers [charybdotoxin (0.1 μM) (a blocker of BK type K(+) channel) and apamin (0.3 μM) (a blocker of SK type K(+) channel)]. The endothelin-1-induced inhibitory response was also not attenuated by α-dendrotoxin (0.1 μM) (a selective inhibitor of Kv1 channel), but abolished by 4-aminopyridine (20 μM) (a selectively inhibitory dose for Kv3 channel). These results suggest the involvement of a voltage-dependent Kv3 channel in the endothelin-1-induced inhibition of NA release from the gastric sympathetic nerves in rats.

  12. Mice with deficient BK channel function show impaired prepulse inhibition and spatial learning, but normal working and spatial reference memory.

    Directory of Open Access Journals (Sweden)

    Marei Typlt

    Full Text Available Genetic variations in the large-conductance, voltage- and calcium activated potassium channels (BK channels have been recently implicated in mental retardation, autism and schizophrenia which all come along with severe cognitive impairments. In the present study we investigate the effects of functional BK channel deletion on cognition using a genetic mouse model with a knock-out of the gene for the pore forming α-subunit of the channel. We tested the F1 generation of a hybrid SV129/C57BL6 mouse line in which the slo1 gene was deleted in both parent strains. We first evaluated hearing and motor function to establish the suitability of this model for cognitive testing. Auditory brain stem responses to click stimuli showed no threshold differences between knockout mice and their wild-type littermates. Despite of muscular tremor, reduced grip force, and impaired gait, knockout mice exhibited normal locomotion. These findings allowed for testing of sensorimotor gating using the acoustic startle reflex, as well as of working memory, spatial learning and memory in the Y-maze and the Morris water maze, respectively. Prepulse inhibition on the first day of testing was normal, but the knockout mice did not improve over the days of testing as their wild-type littermates did. Spontaneous alternation in the y-maze was normal as well, suggesting that the BK channel knock-out does not impair working memory. In the Morris water maze knock-out mice showed significantly slower acquisition of the task, but normal memory once the task was learned. Thus, we propose a crucial role of the BK channels in learning, but not in memory storage or recollection.

  13. Marked increases in mucociliary clearance produced by synergistic secretory agonists or inhibition of the epithelial sodium channel

    Science.gov (United States)

    Joo, Nam Soo; Jeong, Jin Hyeok; Cho, Hyung-Ju; Wine, Jeffrey J.

    2016-01-01

    Mucociliary clearance (MCC) is a critical host innate defense mechanism in airways, and it is impaired in cystic fibrosis (CF) and other obstructive lung diseases. Epithelial fluid secretion and absorption modify MCC velocity (MCCV). We tested the hypotheses that inhibiting fluid absorption accelerates MCCV, whereas inhibiting fluid secretion decelerates it. In airways, ENaC is mainly responsible for fluid absorption, while anion channels, including CFTR and Ca2+-activated chloride channels mediate anion/fluid secretion. MCCV was increased by the cAMP-elevating agonists, forskolin or isoproterenol (10 μM) and by the Ca2+-elevating agonist, carbachol (0.3 μM). The CFTR-selective inhibitor, CFTRinh-172, modestly reduced MCCV-increases induced by forskolin or isoproterenol but not increases induced by carbachol. The ENaC inhibitor benzamil increased basal MCCV as well as MCCV increases produced by forskolin or carbachol. MCC velocity was most dramatically accelerated by the synergistic combination of forskolin and carbachol, which produced near-maximal clearance rates regardless of prior treatment with CFTR or ENaC inhibitors. In CF airways, where CFTR-mediated secretion (and possibly synergistic MCC) is lost, ENaC inhibition via exogenous agents may provide therapeutic benefit, as has long been proposed. PMID:27830759

  14. Selective serotonin reuptake inhibitor sertraline inhibits voltage-dependent K+ channels in rabbit coronary arterial smooth muscle cells

    Indian Academy of Sciences (India)

    HAN SOL KIM; HONGLIANG LI; HYE WON KIM; SUNG EUN SHIN; IL-WHAN CHOI; AMY L FIRTH; HYOWEON BANG; YOUNG MIN BAE; WON SUN PARK

    2016-12-01

    We examined the effects of the selective serotonin reuptake inhibitor (SSRI) sertraline on voltage-dependent K+ (Kv)channels in freshly isolated rabbit coronary arterial smooth muscle cells using the voltage-clamp technique. Sertralinedecreased the Kv channel current in a dose-dependent manner, with an IC50 value of 0.18 μM and a slope value (Hillcoefficient) of 0.61. Although the application of 1 μM sertraline did not affect the steady-state activation curves,sertraline caused a significant, negative shift in the inactivation curves. Pretreatment with another SSRI, paroxetine,had no significant effect on Kv currents and did not alter the inhibitory effects of sertraline on Kv currents. From theseresults, we concluded that sertraline dose-dependently inhibited Kv currents independently of serotonin reuptakeinhibition by shifting inactivation curves to a more negative potential.

  15. Tolperisone-type drugs inhibit spinal reflexes via blockade of voltage-gated sodium and calcium channels.

    Science.gov (United States)

    Kocsis, Pál; Farkas, Sándor; Fodor, László; Bielik, Norbert; Thán, Márta; Kolok, Sándor; Gere, Anikó; Csejtei, Mónika; Tarnawa, István

    2005-12-01

    The spinal reflex depressant mechanism of tolperisone and some of its structural analogs with central muscle relaxant action was investigated. Tolperisone (50-400 microM), eperisone, lanperisone, inaperisone, and silperisone (25-200 microM) dose dependently depressed the ventral root potential of isolated hemisected spinal cord of 6-day-old rats. The local anesthetic lidocaine (100-800 microM) produced qualitatively similar depression of spinal functions in the hemicord preparation, whereas its blocking effect on afferent nerve conduction was clearly stronger. In vivo, tolperisone and silperisone as well as lidocaine (10 mg/kg intravenously) depressed ventral root reflexes and excitability of motoneurons. However, in contrast with lidocaine, the muscle relaxant drugs seemed to have a more pronounced action on the synaptic responses than on the excitability of motoneurons. Whole-cell measurements in dorsal root ganglion cells revealed that tolperisone and silperisone depressed voltage-gated sodium channel conductance at concentrations that inhibited spinal reflexes. Results obtained with tolperisone and its analogs in the [3H]batrachotoxinin A 20-alpha-benzoate binding in cortical neurons and in a fluorimetric membrane potential assay in cerebellar neurons further supported the view that blockade of sodium channels may be a major component of the action of tolperisone-type centrally acting muscle relaxant drugs. Furthermore, tolperisone, eperisone, and especially silperisone had a marked effect on voltage-gated calcium channels, whereas calcium currents were hardly influenced by lidocaine. These data suggest that tolperisone-type muscle relaxants exert their spinal reflex inhibitory action predominantly via a presynaptic inhibition of the transmitter release from the primary afferent endings via a combined action on voltage-gated sodium and calcium channels.

  16. Ferritin ion channel disorder inhibits Fe(II)/O2 reactivity at distant sites.

    Science.gov (United States)

    Tosha, Takehiko; Behera, Rabindra K; Theil, Elizabeth C

    2012-11-05

    Ferritins, a complex, mineralized, protein nanocage family essential for life, provide iron concentrates and oxidant protection. Protein-based ion channels and Fe(II)/O(2) catalysis initiate conversion of thousands of Fe atoms to caged, ferritin Fe(2)O(3)·H(2)O minerals. The ion channels consist of six helical segments, contributed by 3 of 12 or 24 polypeptide subunits, around the 3-fold cage axes. The channel structure guides entering Fe(II) ions toward multiple, catalytic, diiron sites buried inside ferritin protein helices, ~20 Å away from channel internal exits. The catalytic product, Fe(III)-O(H)-Fe(III), is a mineral precursor; mineral nucleation begins inside the protein cage with mineral growth in the central protein cavity (5-8 nm diameter). Amino acid substitutions that changed ionic or hydrophobic channel interactions R72D, D122R, and L134P increased ion channel structural disorder (protein crystallographic analyses) and increased Fe(II) exit [chelated Fe(II) after ferric mineral reduction/dissolution]. Since substitutions of some channel carboxylate residues diminished ferritin catalysis with no effect on Fe(II) exit, such as E130A and D127A, we investigated catalysis in ferritins with altered Fe(II) exit, R72D, D122R and L134P. The results indicate that simply changing the ionic properties of the channels, as in the R72D variant, need not change the forward catalytic rate. However, both D122R and L134P, which had dramatic effects on ferritin catalysis, also caused larger effects on channel structure and order, contrasting with R72D. All three amino acid substitutions, however, decreased the stability of the catalytic intermediate, diferric peroxo, even though overall ferritin cage structure is very stable, resisting 80 °C and 6 M urea. The localized structural changes in ferritin subdomains that affect ferritin function over long distances illustrate new properties of the protein cage in natural ferritin function and for applied ferritin uses.

  17. The stress protein heat shock cognate 70 (Hsc70) inhibits the Transient Receptor Potential Vanilloid type 1 (TRPV1) channel

    Science.gov (United States)

    Iftinca, Mircea; Flynn, Robyn; Basso, Lilian; Melo, Helvira; Aboushousha, Reem; Taylor, Lauren

    2016-01-01

    Background Specialized cellular defense mechanisms prevent damage from chemical, biological, and physical hazards. The heat shock proteins have been recognized as key chaperones that maintain cell survival against a variety of exogenous and endogenous stress signals including noxious temperature. However, the role of heat shock proteins in nociception remains poorly understood. We carried out an expression analysis of the constitutively expressed 70 kDa heat-shock cognate protein, a member of the stress-induced HSP70 family in lumbar dorsal root ganglia from a mouse model of Complete Freund’s Adjuvant-induced chronic inflammatory pain. We used immunolabeling of dorsal root ganglion neurons, behavioral analysis and patch clamp electrophysiology in both dorsal root ganglion neurons and HEK cells transfected with Hsc70 and Transient Receptor Potential Channels to examine their functional interaction in heat shock stress condition. Results We report an increase in protein levels of Hsc70 in mouse dorsal root ganglia, 3 days post Complete Freund’s Adjuvant injection in the hind paw. Immunostaining of Hsc70 was observed in most of the dorsal root ganglion neurons, including the small size nociceptors immunoreactive to the TRPV1 channel. Standard whole-cell patch-clamp technique was used to record Transient Receptor Potential Vanilloid type 1 current after exposure to heat shock. We found that capsaicin-evoked currents are inhibited by heat shock in dorsal root ganglion neurons and transfected HEK cells expressing Hsc70 and TRPV1. Blocking Hsc70 with matrine or spergualin compounds prevented heat shock-induced inhibition of the channel. We also found that, in contrast to TRPV1, both the cold sensor channels TRPA1 and TRPM8 were unresponsive to heat shock stress. Finally, we show that inhibition of TRPV1 depends on the ATPase activity of Hsc70 and involves the rho-associated protein kinase. Conclusions Our work identified Hsc70 and its ATPase activity as a central

  18. Channel catfish reovirus (CRV) inhibits replication of channel catfish herpesvirus (CCV) by two distinct mechanisms: viral interference and induction of an anti-viral factor.

    Science.gov (United States)

    Chinchar, V G; Logue, O; Antao, A; Chinchar, G D

    1998-06-19

    Catfish reovirus (CRV), a double stranded RNA virus, inhibited channel catfish herpes-virus (CCV) replication by 2 different mechanisms: (1) directly as a consequence of its own replication, and (2) indirectly due to the induction of an anti-viral factor. In the former, prior infection with CRV significantly reduced subsequent CCV protein synthesis and virus yield. CRV mediated-interference was greatest when CRV infection preceded CCV infection by 16 h, and was least when cell cultures were simultaneously infected with both viruses. in the latter case, the infection of channel catfish ovary (CCO) cultures with UV-inactivated CRV resulted in the synthesis (or release) of an anti-viral factor. Cells producing the factor were protected from CCV infection, as were cells which had been treated with spent culture medium containing anti-viral activity. Interestingly an anti-viral activity was constitutively present in long-term cultures of catfish T-cells and macrophages. Whether this factor and the one induced by UV-inactivated CRV are identical is not known, but analogy to mammalian systems suggests that the former may be similar to type II interferon, whereas the latter may be the piscine equivalent of type I interferon. These results suggest that UV-inactivated CRV may prove useful in the induction and characterization of interferon-like anti-viral proteins in the channel catfish and that long-term cultures of catfish T-cells and monocytes may serve as a ready source of additional anti-viral factors.

  19. Inhibition of KV7 channels protects against myocardial ischemia and reperfusion injury

    DEFF Research Database (Denmark)

    Hedegaard, Elise Røge; Johnsen, Jacob; Povlsen, Jonas Agerlund;

    2015-01-01

    Aims: KV7 channel are activated by ischemia and mediate hypoxic vasodilatation. We investigated the effect of KV7 channel modulation on cardiac ischemia and reperfusion (IR) injury and the interaction with cardioprotection by ischemic preconditioning (IPC). Methods and Results: We investigated......-flow, global ischemia and reperfusion with and without IPC. Infarct size (IS) was quantified by 2,3,5-triphenyltetrazolium chloride (TTC) staining. Hemodynamics were measured using a catheter inserted in the left ventricle. Functional studies on isolated coronary arteries were performed in a wire myograph. KV7.......1, KV7.4 and KV7.5 were expressed in rat coronary arteries and all KV7 subtypes (KV7.1-5) in the left and right ventricles of the heart. KV7 channel blockade by XE991 and linopirdine reduced infarct size additive to infarct reduction by IPC. Flupirtine abolished infarct size reduction by IPC...

  20. Filter gate closure inhibits ion but not water transport through potassium channels.

    Science.gov (United States)

    Hoomann, Torben; Jahnke, Nadin; Horner, Andreas; Keller, Sandro; Pohl, Peter

    2013-06-25

    The selectivity filter of K(+) channels is conserved throughout all kingdoms of life. Carbonyl groups of highly conserved amino acids point toward the lumen to act as surrogates for the water molecules of K(+) hydration. Ion conductivity is abrogated if some of these carbonyl groups flip out of the lumen, which happens (i) in the process of C-type inactivation or (ii) during filter collapse in the absence of K(+). Here, we show that K(+) channels remain permeable to water, even after entering such an electrically silent conformation. We reconstituted fluorescently labeled and constitutively open mutants of the bacterial K(+) channel KcsA into lipid vesicles that were either C-type inactivating or noninactivating. Fluorescence correlation spectroscopy allowed us to count both the number of proteoliposomes and the number of protein-containing micelles after solubilization, providing the number of reconstituted channels per proteoliposome. Quantification of the per-channel increment in proteoliposome water permeability with the aid of stopped-flow experiments yielded a unitary water permeability pf of (6.9 ± 0.6) × 10(-13) cm(3)⋅s(-1) for both mutants. "Collapse" of the selectivity filter upon K(+) removal did not alter pf and was fully reversible, as demonstrated by current measurements through planar bilayers in a K(+)-containing medium to which K(+)-free proteoliposomes were fused. Water flow through KcsA is halved by 200 mM K(+) in the aqueous solution, which indicates an effective K(+) dissociation constant in that range for a singly occupied channel. This questions the widely accepted hypothesis that multiple K(+) ions in the selectivity filter act to mutually destabilize binding.

  1. The ryanodine receptor pore blocker neomycin also inhibits channel activity via a previously undescribed high-affinity Ca(2+) binding site.

    Science.gov (United States)

    Laver, Derek R; Hamada, Tomoyo; Fessenden, James D; Ikemoto, Noriaki

    2007-12-01

    In this study, we present evidence for the mechanism of neomycin inhibition of skeletal ryanodine receptors (RyRs). In single-channel recordings, neomycin produced monophasic inhibition of RyR open probability and biphasic inhibition of [(3)H]ryanodine binding. The half-maximal inhibitory concentration (IC(50)) for channel blockade by neomycin was dependent on membrane potential and cytoplasmic [Ca(2+)], suggesting that neomycin acts both as a pore plug and as a competitive antagonist at a cytoplasmic Ca(2+) binding site that causes allosteric inhibition. This novel Ca(2+)/neomycin binding site had a neomycin affinity of 100 nM: and a Ca(2+) affinity of 35 nM,: which is 30-fold higher than that of the well-described cytoplasmic Ca(2+) activation site. Therefore, a new high-affinity class of Ca(2+) binding site(s) on the RyR exists that mediates neomycin inhibition. Neomycin plugging of the channel pore induced brief (1-2 ms) conductance substates at 30% of the fully open conductance, whereas allosteric inhibition caused complete channel closure with durations that depended on the neomycin concentration. We quantitatively account for these results using a dual inhibition model for neomycin that incorporates voltage-dependent pore plugging and Ca(2+)-dependent allosteric inhibition.

  2. Lipid Storage Disorders Block Lysosomal Trafficking By Inhibiting TRP Channel and Calcium Release

    OpenAIRE

    2012-01-01

    Lysosomal lipid accumulation, defects in membrane trafficking, and altered Ca2+ homeostasis are common features in many lysosomal storage diseases. Mucolipin TRP channel 1 (TRPML1) is the principle Ca2+ channel in the lysosome. Here we show that TRPML1-mediated lysosomal Ca2+ release, measured using a genetically-encoded Ca2+ indicator (GCaMP3) attached directly to TRPML1 and elicited by a potent membrane-permeable synthetic agonist, is dramatically reduced in Niemann-Pick (NP) disease cells....

  3. Inhibition of collagen synthesis by select calcium and sodium channel blockers can be mitigated by ascorbic acid and ascorbyl palmitate.

    Science.gov (United States)

    Ivanov, Vadim; Ivanova, Svetlana; Kalinovsky, Tatiana; Niedzwiecki, Aleksandra; Rath, Matthias

    2016-01-01

    muscle cells, assayed by measuring intracellular collagen content. We observed increased intracellular levels of ascorbate under supplementation with elevated doses of ascorbic acid, as well as its lipid soluble derivative ascorbyl palmitate. Nifedipine reduced ascorbic acid intracellular influx in cultured aortic smooth muscle cells with nifedipine (50 µM) compared to control. Adverse effects of nifedipine were neutralized either by an increased level of cell supplementation with ascorbic acid or by substituting it with ascorbyl palmitate. These studies suggest that adverse effects of channel blockers could be caused by their weakening the arterial wall integrity by interfering with proper extracellular matrix formation. In conclusion, these studies confirm the adverse effects of channel blockers on collagen type l and lV deposition, the key ECM components essential for maintaining optimal structural integrity of the arterial walls. Ascorbate supplementation reversed channel blocker inhibition of these collagen types synthesis and deposition. The results of this study imply the benefits of ascorbate and ascorbate palmitate supplementation in medical management of cardiovascular disease in order to compensate for adverse effects of channel blockers.

  4. Competitive inhibition of the nondepolarizing muscle relaxant rocuronium on nicotinic acetylcholine receptor channels in the rat superior cervical ganglia.

    Science.gov (United States)

    Zhang, Chengmi; Wang, Zhenmeng; Zhang, Jinmin; Qiu, Haibo; Sun, Yuming; Yang, Liqun; Wu, Feixiang; Zheng, Jijian; Yu, Weifeng

    2014-05-01

    A number of case reports now indicate that rocuronium can induce a number of serious side effects. We hypothesized that these side effects might be mediated by the inhibition of nicotinic acetylcholine receptors (nAChRs) at superior cervical ganglion (SCG) neurons. Conventional patch clamp recordings were used to study the effects of rocuronium on nAChR currents from enzymatically dissociated rat SCG neurons. We found that ACh induced a peak transient inward current in rat SCG neurons. Additionally, rocuronium suppressed the peak ACh-evoked currents in rat SCG neurons in a concentration-dependent and competitive manner, and it increased the extent of desensitization of nAChRs. The inhibitory rate of rocuronium on nAChR currents did not change significantly at membrane potentials between -70 and -20 mV, suggesting that this inhibition was voltage independent. Lastly, rocuronium preapplication enhanced its inhibitory effect, indicating that this drug might prefer to act on the closed state of nAChR channels. In conclusion, rocuronium, at clinically relevant concentrations, directly inhibits nAChRs at the SCG by interacting with both opened and closed states. This inhibition is competitive, dose dependent, and voltage independent. Blockade of synaptic transmission in the sympathetic ganglia by rocuronium might have potentially inhibitory effects on the cardiovascular system.

  5. Inhibition of voltage-gated calcium channels by sequestration of beta subunits.

    Science.gov (United States)

    Cuchillo-Ibañez, Inmaculada; Aldea, Marcos; Brocard, Jacques; Albillos, Almudena; Weiss, Norbert; Garcia, Antonio G; De Waard, Michel

    2003-11-28

    The auxiliary Ca(v)beta subunit is essential for functional expression of high-voltage activated Ca(2+) channels. Here, we describe a lure sequence designed to sequester the Ca(v)beta subunits in transfected bovine chromaffin cells. This sequence is composed of the extracellular and transmembrane domains of the alpha chain of the human CD8, the I-II loop of Ca(v)2.1 subunit, and EGFP. We showed that expressing the CD8-I-II-EGFP sequence in chromaffin cells led to a >50% decrease in overall Ca(2+) current density. Although this decrease involved all the Ca(2+) channel types (L, N, P/Q, R), the proportion of each type supporting the remaining current was altered. A similar effect was observed after transfection when measuring the functional role of Ca(2+) channels in catecholamine release by chromaffin cells: global decrease of release and change of balance between the different channel types supporting it. Possible explanations for this apparent discrepancy are further discussed.

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

    DEFF Research Database (Denmark)

    Lynagh, Timothy Peter; Lynch, Joseph W

    2010-01-01

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

  7. Blockade of microglial KATP -channel abrogates suppression of inflammatory-mediated inhibition of neural precursor cells.

    Science.gov (United States)

    Ortega, Francisco J; Vukovic, Jana; Rodríguez, Manuel J; Bartlett, Perry F

    2014-02-01

    Microglia positively affect neural progenitor cell physiology through the release of inflammatory mediators or trophic factors. We demonstrated previously that reactive microglia foster K(ATP) -channel expression and that blocking this channel using glibenclamide administration enhances striatal neurogenesis after stroke. In this study, we investigated whether the microglial K(ATP) -channel directly influences the activation of neural precursor cells (NPCs) from the subventricular zone using transgenic Csf1r-GFP mice. In vitro exposure of NPCs to lipopolysaccharide and interferon-gamma resulted in a significant decrease in precursor cell number. The complete removal of microglia from the culture or exposure to enriched microglia culture also decreased the precursor cell number. The addition of glibenclamide rescued the negative effects of enriched microglia on neurosphere formation and promoted a ∼20% improvement in precursor cell number. Similar results were found using microglial-conditioned media from isolated microglia. Using primary mixed glial and pure microglial cultures, glibenclamide specifically targeted reactive microglia to restore neurogenesis and increased the microglial production of the chemokine monocyte chemoattractant protein-1 (MCP-1). These findings provide the first direct evidence that the microglial K(ATP) -channel is a regulator of the proliferation of NPCs under inflammatory conditions.

  8. The immunophilin FKBP52 inhibits the activity of the epithelial Ca2+ channel TRPV5

    NARCIS (Netherlands)

    Gkika, D.; Topala, C.N.; Hoenderop, J.G.J.; Bindels, R.J.M.

    2006-01-01

    In the kidney, the epithelial Ca(2+) channel TRPV5 constitutes the apical entry pathway in the process of active Ca(2+) reabsorption. The regulation of Ca(2+) influx through TRPV5 is of crucial importance, because it determines the final amount of Ca(2+) excreted in the urine. The present study iden

  9. The Inhibition by Oxaliplatin, a Platinum-Based Anti-Neoplastic Agent, of the Activity of Intermediate-Conductance Ca2+-Activated K+ Channels in Human Glioma Cells

    Directory of Open Access Journals (Sweden)

    Mei-Han Huang

    2015-10-01

    Full Text Available Oxaliplatin (OXAL is a third-generation organoplatinum which is effective against advanced cancer cells including glioma cells. How this agent and other related compounds interacts with ion channels in glioma cells is poorly understood. OXAL (100 µM suppressed the amplitude of whole-cell K+ currents (IK; and, either DCEBIO or ionomycin significantly reversed OXAL-mediated inhibition of IK in human 13-06-MG glioma cells. In OXAL-treated cells, TRAM-34 did not suppress IK amplitude in these cells. The intermediate-conductance Ca2+-activated K+ (IKCa channels subject to activation by DCEBIO and to inhibition by TRAM-34 or clotrimazole were functionally expressed in these cells. Unlike cisplatin, OXAL decreased the probability of IKCa-channel openings in a concentration-dependent manner with an IC50 value of 67 µM. No significant change in single-channel conductance of IKCa channels in the presence of OXAL was demonstrated. Neither large-conductance Ca2+-activated K+ channels nor inwardly rectifying K+ currents in these cells were affected in the presence of OXAL. OXAL also suppressed the proliferation and migration of 13-06-MG cells in a concentration- and time-dependent manner. OXAL reduced IKCa-channel activity in LoVo colorectal cancer cells. Taken together, the inhibition by OXAL of IKCa channels would conceivably be an important mechanism through which it acts on the functional activities of glioma cells occurring in vivo.

  10. Low concentrations of alcohol inhibit BDNF-dependent GABAergic plasticity via L-type Ca2+ channel inhibition in developing CA3 hippocampal pyramidal neurons.

    Science.gov (United States)

    Zucca, Stefano; Valenzuela, C Fernando

    2010-05-12

    Fetal alcohol spectrum disorder (FASD) is associated with learning and memory alterations that could be, in part, a consequence of hippocampal damage. The CA3 hippocampal subfield is one of the regions affected by ethanol (EtOH), including exposure during the third trimester-equivalent (i.e., neonatal period in rats). However, the mechanism of action of EtOH is poorly understood. In CA3 pyramidal neurons from neonatal rats, dendritic BDNF release causes long-term potentiation of the frequency of GABAA receptor-mediated spontaneous postsynaptic currents (LTP-GABAA) and this mechanism is thought to play a role in GABAergic synapse maturation. Here, we show that short- and long-term exposure of neonatal male rats to low EtOH concentrations abolishes LTP-GABAA by inhibiting L-type voltage-gated Ca2+ channels. These findings support the recommendation that even light drinking should be avoided during pregnancy.

  11. Inhibition of ANO1/TMEM16A Chloride Channel by Idebenone and Its Cytotoxicity to Cancer Cell Lines.

    Directory of Open Access Journals (Sweden)

    Yohan Seo

    Full Text Available The expression levels of anoctamin 1 (ANO1, TMEM16A, a calcium-activated chloride channel (CaCC, are significantly increased in several tumors, and inhibition of ANO1 is known to reduce cell proliferation and migration. Here, we performed cell-based screening of a collection of natural products and drug-like compounds to identify inhibitors of ANO1. As a result of the screening, idebenone, miconazole and plumbagin were identified as novel ANO1 inhibitors. Electrophysiological studies showed that idebenone, a synthetic analog of coenzyme Q10, completely blocked ANO1 activity in FRT cells expressing ANO1 without any effect on intracellular calcium signaling and CFTR, a cAMP-regulated chloride channel. The CaCC activities in PC-3 and CFPAC-1 cells expressing abundant endogenous ANO1 were strongly blocked by idebenone. Idebenone inhibited cell proliferation and induced apoptosis in PC-3 and CFPAC-1 cells, but not in A549 cells, which do not express ANO1. These data suggest that idebenone, a novel ANO1 inhibitor, has potential for use in cancer therapy.

  12. ZD7288, a selective hyperpolarization-activated cyclic nucleotide-gated channel blocker, inhibits hippocampal synaptic plasticity

    Institute of Scientific and Technical Information of China (English)

    Xiao-xue Zhang; Xiao-chun Min; Xu-lin Xu; Min Zheng; Lian-jun Guo

    2016-01-01

    The selective hyperpolarization-activated cyclic nucleotide-gated (HCN) channel blocker 4-(N-ethyl-N-phenylamino)-1,2-dimeth-yl-6-(methylamino) pyrimidinium chloride (ZD7288) blocks the induction of long-term potentiation in the perforant path–CA3 region in rat hippocampusin vivo. To explore the mechanisms underlying the action of ZD7288, we recorded excitatory postsynaptic potentials in perforant path–CA3 synapses in male Sprague-Dawley rats. We measured glutamate content in the hippocampus and in cultured hip-pocampal neurons using high performance liquid chromatography, and determined intracellular Ca2+ concentration ([Ca2+]i) using Fura-2. ZD7288 inhibited the induction and maintenance of long-term potentiation, and these effects were mirrored by the nonspeciifc HCN channel blocker cesium. ZD7288 also decreased glutamate release in hippocampal tissue and in cultured hippocampal neurons. Further-more, ZD7288 attenuated glutamate-induced rises in [Ca2+]i in a concentration-dependent manner and reversed 8-Br-cAMP-mediated facilitation of these glutamate-induced [Ca2+]i rises. Our results suggest that ZD7288 inhibits hippocampal synaptic plasticity both gluta-mate release and resultant [Ca2+]i increases in rat hippocampal neurons.

  13. Phenolic acids isolated from the fungus Schizophyllum commune exert analgesic activity by inhibiting voltage-gated sodium channels.

    Science.gov (United States)

    Yao, Hui-Min; Wang, Gan; Liu, Ya-Ping; Rong, Ming-Qiang; Shen, Chuan-Bin; Yan, Xiu-Wen; Luo, Xiao-Dong; Lai, Ren

    2016-09-01

    The present study was designed to search for compounds with analgesic activity from the Schizophyllum commune (SC), which is widely consumed as edible and medicinal mushroom world. Thin layer chromatography (TLC), tosilica gel column chromatography, sephadex LH 20, and reverse-phase high performance liquid chromatography (RP-HPLC) were used to isolate and purify compounds from SC. Structural analysis of the isolated compounds was based on nuclear magnetic resonance (NMR). The effects of these compounds on voltage-gated sodium (NaV) channels were evaluated using patch clamp. The analgesic activity of these compounds was tested in two types of mouse pain models induced by noxious chemicals. Five phenolic acids identified from SC extracts in the present study included vanillic acid, m-hydroxybenzoic acid, o-hydroxybenzeneacetic acid, 3-hydroxy-5-methybenzoic acid, and p-hydroxybenzoic acid. They inhibited the activity of both tetrodotoxin-resistant (TTX-r) and tetrodotoxin-sensitive (TTX-s) NaV channels. All the compounds showed low selectivity on NaV channel subtypes. After intraperitoneal injection, three compounds of these compounds exerted analgesic activity in mice. In conclusion, phenolic acids identified in SC demonstrated analgesic activity, facilitating the mechanistic studies of SC in the treatment of neurasthenia.

  14. Strong Inhibition of O-Atom Transfer Reactivity for Mn(IV)(O)(π-Radical-Cation)(Lewis Acid) versus Mn(V)(O) Porphyrinoid Complexes.

    Science.gov (United States)

    Zaragoza, Jan Paulo T; Baglia, Regina A; Siegler, Maxime A; Goldberg, David P

    2015-05-27

    The oxygen atom transfer (OAT) reactivity of two valence tautomers of a Mn(V)(O) porphyrinoid complex was compared. The OAT kinetics of Mn(V)(O)(TBP8Cz) (TBP8Cz = octakis(p-tert-butylphenyl)corrolazinato(3-)) reacting with a series of triarylphosphine (PAr3) substrates were monitored by stopped-flow UV-vis spectroscopy, and revealed second-order rate constants ranging from 16(1) to 1.43(6) × 10(4) M(-1) s(-1). Characterization of the OAT transition state analogues Mn(III)(OPPh3)(TBP8Cz) and Mn(III)(OP(o-tolyl)3)(TBP8Cz) was carried out by single-crystal X-ray diffraction (XRD). A valence tautomer of the closed-shell Mn(V)(O)(TBP8Cz) can be stabilized by the addition of Lewis and Brønsted acids, resulting in the open-shell Mn(IV)(O)(TBP8Cz(•+)):LA (LA = Zn(II), B(C6F5)3, H(+)) complexes. These Mn(IV)(O)(π-radical-cation) derivatives exhibit dramatically inhibited rates of OAT with the PAr3 substrates (k = 8.5(2) × 10(-3) - 8.7 M(-1) s(-1)), contrasting the previously observed rate increase of H-atom transfer (HAT) for Mn(IV)(O)(TBP8Cz(•+)):LA with phenols. A Hammett analysis showed that the OAT reactivity for Mn(IV)(O)(TBP8Cz(•+)):LA is influenced by the Lewis acid strength. Spectral redox titration of Mn(IV)(O)(TBP8Cz(•+)):Zn(II) gives Ered = 0.69 V vs SCE, which is nearly +700 mV above its valence tautomer Mn(V)(O)(TBP8Cz) (Ered = -0.05 V). These data suggest that the two-electron electrophilicity of the Mn(O) valence tautomers dominate OAT reactivity and do not follow the trend in one-electron redox potentials, which appear to dominate HAT reactivity. This study provides new fundamental insights regarding the relative OAT and HAT reactivity of valence tautomers such as M(V)(O)(porph) versus M(IV)(O)(porph(•+)) (M = Mn or Fe) found in heme enzymes.

  15. Synthetic cation-selective nanotube: Permeant cations chaperoned by anions

    Science.gov (United States)

    Hilder, Tamsyn A.; Gordon, Dan; Chung, Shin-Ho

    2011-01-01

    The ability to design ion-selective, synthetic nanotubes which mimic biological ion channels may have significant implications for the future treatment of bacteria, diseases, and as ultrasensitive biosensors. We present the design of a synthetic nanotube made from carbon atoms that selectively allows monovalent cations to move across and rejects all anions. The cation-selective nanotube mimics some of the salient properties of biological ion channels. Before practical nanodevices are successfully fabricated it is vital that proof-of-concept computational studies are performed. With this in mind we use molecular and stochastic dynamics simulations to characterize the dynamics of ion permeation across a single-walled (10, 10), 36 Å long, carbon nanotube terminated with carboxylic acid with an effective radius of 5.08 Å. Although cations encounter a high energy barrier of 7 kT, its height is drastically reduced by a chloride ion in the nanotube. The presence of a chloride ion near the pore entrance thus enables a cation to enter the pore and, once in the pore, it is chaperoned by the resident counterion across the narrow pore. The moment the chaperoned cation transits the pore, the counterion moves back to the entrance to ferry another ion. The synthetic nanotube has a high sodium conductance of 124 pS and shows linear current-voltage and current-concentration profiles. The cation-anion selectivity ratio ranges from 8 to 25, depending on the ionic concentrations in the reservoirs.

  16. Acid-sensitive channel inhibition prevents fetal alcohol spectrum disorders cerebellar Purkinje cell loss

    OpenAIRE

    Ramadoss, Jayanth; Lunde, Emilie R.; Ouyang, Nengtai; Chen, Wei-Jung A.; Cudd, Timothy A.

    2008-01-01

    Ethanol is now considered the most common human teratogen. Educational campaigns have not reduced the incidence of ethanol-mediated teratogenesis, leading to a growing interest in the development of therapeutic prevention or mitigation strategies. On the basis of the observation that maternal ethanol consumption reduces maternal and fetal pH, we hypothesized that a pH-sensitive pathway involving the TWIK-related acid-sensitive potassium channels (TASKs) is implicated in ethanol-induced injury...

  17. Structural Basis for the Function and Inhibition of an Influenze Virus Proton Channel

    Energy Technology Data Exchange (ETDEWEB)

    Stouffer,A.; Acharya, R.; Salom, D.; Levine, A.; Di Costanzo, L.; Soto, C.; Tershko, V.; Nanda, V.; Stayrook, S.; DeGrado, W.

    2008-01-01

    The M2 protein from influenza A virus is a pH-activated proton channel that mediates acidification of the interior of viral particles entrapped in endosomes. M2 is the target of the anti-influenza drugs amantadine and rimantadine; recently, resistance to these drugs in humans, birds and pigs has reached more than 90% (ref. 1). Here we describe the crystal structure of the transmembrane-spanning region of the homotetrameric protein in the presence and absence of the channel-blocking drug amantadine. pH-dependent structural changes occur near a set of conserved His and Trp residues that are involved in proton gating2. The drug-binding site is lined by residues that are mutated in amantadine-resistant viruses3, 4. Binding of amantadine physically occludes the pore, and might also perturb the pKa of the critical His residue. The structure provides a starting point for solving the problem of resistance to M2-channel blockers.

  18. Deltamethrin Inhibits the Human T-type Voltage-Sensitive Calcium Channel (Cav3.2

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    Steven B. Symington

    2009-01-01

    Full Text Available The goal of this study was to determine the effect of deltamethrin, a pyrethroid insecticide, on CaV3.2, a human T-type voltage-sensitive calcium channel expressed in Xenopus laevis (X.laevis oocytes. Cav3.2 cDNA was transcribed into cRNA; the cRNA was then injected into X.laevis oocytes and electrophysiologically characterized using the two-electrode voltage clamp technique with Ba2+ as a charge carrier. Deltamethrin (10-7 M reduced peak current in a nonreversible manner compared to the untreated control, but had no effect on the voltagedependent activation and inactivation kinetics. These findings confirm that human CaV3.2 is a target for deltamethrin and quite possibly other pyrethroid insecticides. These studies provide insight into the molecular mechanisms of the effect that pyrethroids have on voltage-sensitive calcium channels in general. This information will allow a more complete understanding of the molecular and cellular nature of pyrethroid-induced toxicity and expand our knowledge of the structure-activity relationships of pyrethroids with regard to their action on voltage-sensitive calcium channels.

  19. Spicing up the sensation of stretch: TRPV1 controls mechanosensitive Piezo channels.

    Science.gov (United States)

    Altier, Christophe

    2015-02-10

    Piezo proteins--a family of mammalian cation-selective ion channels that respond to mechanical stretch--are molecular mediators of biological processes, including vascular tone, hearing, touch, and pain. In this issue of Science Signaling, Rohacs and colleagues demonstrate that activation of the heat-sensitive transient receptor potential vanilloid 1 (TRPV1), another cation channel, inhibits Piezo channels through a calcium-induced depletion of phosphoinositides. This regulation could contribute to the cellular mechanisms by which the TRPV1 activator capsaicin mitigates mechanical hypersensitivity.

  20. Two pore channel 2 (TPC2) inhibits autophagosomal-lysosomal fusion by alkalinizing lysosomal pH.

    Science.gov (United States)

    Lu, Yingying; Hao, Bai-Xia; Graeff, Richard; Wong, Connie W M; Wu, Wu-Tian; Yue, Jianbo

    2013-08-16

    Autophagy is an evolutionarily conserved lysosomal degradation pathway, yet the underlying mechanisms remain poorly understood. Nicotinic acid adenine dinucleotide phosphate (NAADP), one of the most potent Ca(2+) mobilizing messengers, elicits Ca(2+) release from lysosomes via the two pore channel 2 (TPC2) in many cell types. Here we found that overexpression of TPC2 in HeLa or mouse embryonic stem cells inhibited autophagosomal-lysosomal fusion, thereby resulting in the accumulation of autophagosomes. Treatment of TPC2 expressing cells with a cell permeant-NAADP agonist, NAADP-AM, further induced autophagosome accumulation. On the other hand, TPC2 knockdown or treatment of cells with Ned-19, a NAADP antagonist, markedly decreased the accumulation of autophagosomes. TPC2-induced accumulation of autophagosomes was also markedly blocked by ATG5 knockdown. Interestingly, inhibiting mTOR activity failed to increase TPC2-induced autophagosome accumulation. Instead, we found that overexpression of TPC2 alkalinized lysosomal pH, and lysosomal re-acidification abolished TPC2-induced autophagosome accumulation. In addition, TPC2 overexpression had no effect on general endosomal-lysosomal degradation but prevented the recruitment of Rab-7 to autophagosomes. Taken together, our data demonstrate that TPC2/NAADP/Ca(2+) signaling alkalinizes lysosomal pH to specifically inhibit the later stage of basal autophagy progression.

  1. The pungent substances piperine, capsaicin, 6-gingerol and polygodial inhibit the human two-pore domain potassium channels TASK-1, TASK-3 and TRESK.

    Science.gov (United States)

    Beltrán, Leopoldo R; Dawid, Corinna; Beltrán, Madeline; Gisselmann, Guenter; Degenhardt, Katharina; Mathie, Klaus; Hofmann, Thomas; Hatt, Hanns

    2013-01-01

    For a long time, the focus of trigeminal chemoperception has rested almost exclusively on TRP channels. However, two-pore domain (K2P) potassium channels have recently been identified as targets for substances associated with typical trigeminal sensations, such as numbing and tingling. In addition, they have been shown to be modulated by several TRP agonists. We investigated whether the pungent substances piperine, capsaicin, 6-gingerol and polygodial have an effect on human K2P channels. For this purpose, we evaluated the effects of these pungent substances on both wild-type and mutant K2P channels by means of two-electrode voltage-clamp experiments using Xenopus laevis oocytes. All four pungent substances were found to inhibit the basal activity of TASK-1 (K2P 3.1), TASK-3 (K2P 9.1), and TRESK (K2P 18.1) channels. This inhibitory effect was dose-dependent and, with the exception of polygodial on TASK-1, fully reversible. However, only piperine exhibited an IC50 similar to its reported EC50 on TRP channels. Finally, we observed for TASK-3 that mutating H98 to E markedly decreased the inhibition induced by piperine, capsaicin, and 6-gingerol, but not by polygodial. Our data contribute to the relatively sparse knowledge concerning the pharmacology of K2P channels and also raise the question of whether K2P channels could be involved in the pungency perception of piperine.

  2. The pungent substances piperine, capsaicin, 6-gingerol and polygodial inhibit the human two-pore domain potassium channels TASK-1, TASK-3 and TRESK

    Directory of Open Access Journals (Sweden)

    Leopoldo Raul Beltran

    2013-11-01

    Full Text Available For a long time, the focus of trigeminal chemoperception has rested almost exclusively on TRP channels. However, two-pore domain (K2P potassium channels have recently been identified as targets for substances associated with typical trigeminal sensations, such as numbing and tingling. In addition, they have been shown to be modulated by several TRP agonists. We investigated whether the pungent substances piperine, capsaicin, 6-gingerol and polygodial have an effect on human K2P channels. For this purpose, we evaluated the effects of these pungent substances on both wild-type and mutant K2P channels by means of two-electrode voltage-clamp experiments using Xenopus laevis oocytes. All four pungent substances were found to inhibit the basal activity of TASK-1 (K2P 3.1, TASK-3 (K2P 9.1, and TRESK (K2P 18.1 channels. This inhibitory effect was dose-dependent and, with the exception of polygodial on TASK-1, fully reversible. However, only piperine exhibited an IC50 similar to its reported EC50 on TRP channels. Finally, we observed for TASK-3 that mutating H98 to E markedly decreases the inhibition induced by piperine, capsaicin, and 6-gingerol, but not by polygodial. Our data contribute to the relatively sparse knowledge concerning the pharmacology of K2P channels and also raise the question of whether K2P channels could be involved in the pungency perception of piperine.

  3. Elevated Expression of Acid-Sensing Ion Channel 3 Inhibits Epilepsy via Activation of Interneurons.

    Science.gov (United States)

    Cao, Qingqing; Wang, Wei; Gu, Juan; Jiang, Guohui; Bian, Xiling; Wang, Kewei; Xu, Zucai; Li, Jie; Chen, Guojun; Wang, Xuefeng

    2016-01-01

    Recent studies have indicated that acid-sensing ion channels may play a significant role in the termination of epilepsy. In particular, acid-sensing ion channel 3 (ASIC3) is expressed in the central nervous system and is most sensitive to extracellular pH. However, whether ASIC3 plays a role in epilepsy is unknown. In this study, qRT-PCR, Western blot, immunohistochemistry, double immunofluorescence labeling, and slice recordings were used. We first detected elevated ASIC3 expression patterns in the brains of temporal lobe epilepsy patients and epileptic rats. ASIC3 was expressed in neurons and glia in both humans and in an experimental model of epilepsy, and ASIC3 was colocalized with inhibitory GABAergic interneurons. By blocking ASIC3 with its antagonist APETx2, we observed that injected APETx2 shortened the latency to seizure and increased the incidence of generalized tonic clonic seizure compared to the control group in models of both pilocarpine- and pentylenetetrazole (PTZ)-induced seizures. Additionally, blocking ASIC3 significantly decreased the frequency of action potential (AP) firing in interneurons. Moreover, APETx2 significantly reduced the amplitudes and frequencies of miniature inhibitory postsynaptic currents (mIPSCs) while showed no differences with the APETx2 + bicuculline group and the bicuculline group. These findings suggest that elevated levels of ASIC3 may serve as an anti-epileptic mechanism via postsynaptic mechanisms in interneurons. It could represent a novel therapeutic strategy for epilepsy treatment.

  4. Mambalgin-1 Pain-relieving Peptide, Stepwise Solid-phase Synthesis, Crystal Structure, and Functional Domain for Acid-sensing Ion Channel 1a Inhibition.

    Science.gov (United States)

    Mourier, Gilles; Salinas, Miguel; Kessler, Pascal; Stura, Enrico A; Leblanc, Mathieu; Tepshi, Livia; Besson, Thomas; Diochot, Sylvie; Baron, Anne; Douguet, Dominique; Lingueglia, Eric; Servent, Denis

    2016-02-05

    Mambalgins are peptides isolated from mamba venom that specifically inhibit a set of acid-sensing ion channels (ASICs) to relieve pain. We show here the first full stepwise solid phase peptide synthesis of mambalgin-1 and confirm the biological activity of the synthetic toxin both in vitro and in vivo. We also report the determination of its three-dimensional crystal structure showing differences with previously described NMR structures. Finally, the functional domain by which the toxin inhibits ASIC1a channels was identified in its loop II and more precisely in the face containing Phe-27, Leu-32, and Leu-34 residues. Moreover, proximity between Leu-32 in mambalgin-1 and Phe-350 in rASIC1a was proposed from double mutant cycle analysis. These data provide information on the structure and on the pharmacophore for ASIC channel inhibition by mambalgins that could have therapeutic value against pain and probably other neurological disorders.

  5. Rem, a member of the RGK GTPases, inhibits recombinant CaV1.2 channels using multiple mechanisms that require distinct conformations of the GTPase.

    Science.gov (United States)

    Yang, Tingting; Xu, Xianghua; Kernan, Timothy; Wu, Vincent; Colecraft, Henry M

    2010-05-15

    Rad/Rem/Gem/Kir (RGK) GTPases potently inhibit Ca(V)1 and Ca(V)2 (Ca(V)1-2) channels, a paradigm of ion channel regulation by monomeric G-proteins with significant physiological ramifications and potential biotechnology applications. The mechanism(s) underlying how RGK proteins inhibit I(Ca) is unknown, and it is unclear how key structural and regulatory properties of these GTPases (such as the role of GTP binding to the nucleotide binding domain (NBD), and the C-terminus which contains a membrane-targeting motif) feature in this effect. Here, we show that Rem inhibits Ca(V)1.2 channels by three independent mechanisms that rely on distinct configurations of the GTPase: (1) a reduction in surface density of channels is accomplished by enhancing dynamin-dependent endocytosis, (2) a diminution of channel open probability (P(o)) that occurs without impacting on voltage sensor movement, and (3) an immobilization of Ca(V) channel voltage sensors. The presence of both the Rem NBD and C-terminus (whether membrane-targeted or not) in one molecule is sufficient to reconstitute all three mechanisms. However, membrane localization of the NBD by a generic membrane-targeting module reconstitutes only the decreased P(o) function (mechanism 2). A point mutation that prevents GTP binding to the NBD selectively eliminates the capacity to immobilize voltage sensors (mechanism 3). The results reveal an uncommon multiplicity in the mechanisms Rem uses to inhibit I(Ca), predict new physiological dimensions of the RGK GTPase-Ca(V) channel crosstalk, and suggest original approaches for developing novel Ca(V) channel blockers.

  6. Chick RGS2L demonstrates concentration-dependent selectivity for pertussis toxin-sensitive and -insensitive pathways that inhibit L-type Ca2+ channels.

    Science.gov (United States)

    Tosetti, Patrizia; Parente, Valeria; Taglietti, Vanni; Dunlap, Kathleen; Toselli, Mauro

    2003-05-15

    In neuronal cells, the influx of Ca2+ ions through voltage-dependent L-type calcium (L) channels couples excitation to multiple cellular functions. In addition to voltage, several neurotransmitters, hormones and cytokines regulate L channel gating via binding to G-protein-coupled receptors. Intracellular molecules that modify G-protein activity - such as regulator of G-protein-signalling (RGS) proteins - are therefore potential candidates for regulating Ca2+ influx through L channels. Here we show that a novel RGS2 splice variant from chick dorsal root ganglion (DRG) neurons, RGS2L, reduces bradykinin (BK)-mediated inhibition of neuronal L channels and accelerates recovery from inhibition. Chick RGS2 reduces the inhibition mediated by both the pertussis toxin (PTX)-sensitive (Gi/o-coupled) and the PTX-insensitive (presumably Gq/11-coupled) pathways. However, we demonstrate for the first time in a living cell that the extent of coupling to each pathway varies with RGS2L concentration. A low concentration of recombinant chick RGS2L (10 nM) preferentially reduces the inhibition mediated by the PTX-insensitive pathway, whereas a 100-fold higher concentration attenuates both PTX-sensitive- and PTX-insensitive-mediated components equally. Our data suggest that factors promoting RGS2L gene induction may regulate Ca2+ influx through L channels by recruiting low-affinity interactions with Gi/o that are absent at basal RGS2L levels.

  7. Pharmacologic inhibition of the renal outer medullary potassium channel causes diuresis and natriuresis in the absence of kaliuresis.

    Science.gov (United States)

    Garcia, Maria L; Priest, Birgit T; Alonso-Galicia, Magdalena; Zhou, Xiaoyan; Felix, John P; Brochu, Richard M; Bailey, Timothy; Thomas-Fowlkes, Brande; Liu, Jessica; Swensen, Andrew; Pai, Lee-Yuh; Xiao, Jianying; Hernandez, Melba; Hoagland, Kimberly; Owens, Karen; Tang, Haifeng; de Jesus, Reynalda K; Roy, Sophie; Kaczorowski, Gregory J; Pasternak, Alexander

    2014-01-01

    The renal outer medullary potassium (ROMK) channel, which is located at the apical membrane of epithelial cells lining the thick ascending loop of Henle and cortical collecting duct, plays an important role in kidney physiology by regulating salt reabsorption. Loss-of-function mutations in the human ROMK channel are associated with antenatal type II Bartter's syndrome, an autosomal recessive life-threatening salt-wasting disorder with mild hypokalemia. Similar observations have been reported from studies with ROMK knockout mice and rats. It is noteworthy that heterozygous carriers of Kir1.1 mutations associated with antenatal Bartter's syndrome have reduced blood pressure and a decreased risk of developing hypertension by age 60. Although selective ROMK inhibitors would be expected to represent a new class of diuretics, this hypothesis has not been pharmacologically tested. Compound A [5-(2-(4-(2-(4-(1H-tetrazol-1-yl)phenyl)acetyl)piperazin-1-yl)ethyl)isobenzofuran-1(3H)-one)], a potent ROMK inhibitor with appropriate selectivity and characteristics for in vivo testing, has been identified. Compound A accesses the channel through the cytoplasmic side and binds to residues lining the pore within the transmembrane region below the selectivity filter. In normotensive rats and dogs, short-term oral administration of compound A caused concentration-dependent diuresis and natriuresis that were comparable to hydrochlorothiazide. Unlike hydrochlorothiazide, however, compound A did not cause any significant urinary potassium losses or changes in plasma electrolyte levels. These data indicate that pharmacologic inhibition of ROMK has the potential for affording diuretic/natriuretic efficacy similar to that of clinically used diuretics but without the dose-limiting hypokalemia associated with the use of loop and thiazide-like diuretics.

  8. Nonthermal activation of transient receptor potential vanilloid-1 channels in abdominal viscera tonically inhibits autonomic cold-defense effectors.

    Science.gov (United States)

    Steiner, Alexandre A; Turek, Victoria F; Almeida, Maria C; Burmeister, Jeffrey J; Oliveira, Daniela L; Roberts, Jennifer L; Bannon, Anthony W; Norman, Mark H; Louis, Jean-Claude; Treanor, James J S; Gavva, Narender R; Romanovsky, Andrej A

    2007-07-11

    An involvement of the transient receptor potential vanilloid (TRPV) 1 channel in the regulation of body temperature (T(b)) has not been established decisively. To provide decisive evidence for such an involvement and determine its mechanisms were the aims of the present study. We synthesized a new TRPV1 antagonist, AMG0347 [(E)-N-(7-hydroxy-5,6,7,8-tetrahydronaphthalen-1-yl)-3-(2-(piperidin-1-yl)-6-(trifluoromethyl)pyridin-3-yl)acrylamide], and characterized it in vitro. We then found that this drug is the most potent TRPV1 antagonist known to increase T(b) of rats and mice and showed (by using knock-out mice) that the entire hyperthermic effect of AMG0347 is TRPV1 dependent. AMG0347-induced hyperthermia was brought about by one or both of the two major autonomic cold-defense effector mechanisms (tail-skin vasoconstriction and/or thermogenesis), but it did not involve warmth-seeking behavior. The magnitude of the hyperthermic response depended on neither T(b) nor tail-skin temperature at the time of AMG0347 administration, thus indicating that AMG0347-induced hyperthermia results from blockade of tonic TRPV1 activation by nonthermal factors. AMG0347 was no more effective in causing hyperthermia when administered into the brain (intracerebroventricularly) or spinal cord (intrathecally) than when given systemically (intravenously), which indicates a peripheral site of action. We then established that localized intra-abdominal desensitization of TRPV1 channels with intraperitoneal resiniferatoxin blocks the T(b) response to systemic AMG0347; the extent of desensitization was determined by using a comprehensive battery of functional tests. We conclude that tonic activation of TRPV1 channels in the abdominal viscera by yet unidentified nonthermal factors inhibits skin vasoconstriction and thermogenesis, thus having a suppressive effect on T(b).

  9. Lipopolysaccharide Inhibits the Channel Activity of the P2X7 Receptor

    Directory of Open Access Journals (Sweden)

    Elias Leiva-Salcedo

    2011-01-01

    Full Text Available The purinergic P2X7 receptor (P2X7R plays an important role during the immune response, participating in several events such as cytokine release, apoptosis, and necrosis. The bacterial endotoxin lipopolysaccharide (LPS is one of the strongest stimuli of the immune response, and it has been shown that P2X7R activation can modulate LPS-induced responses. Moreover, a C-terminal binding site for LPS has been proposed. In order to evaluate if LPS can directly modulate the activity of the P2X7R, we tested several signaling pathways associated with P2X7R activation in HEK293 cells that do not express the TLR-4 receptor. We found that LPS alone was unable to induce any P2X7R-related activity, suggesting that the P2X7R is not directly activated by the endotoxin. On the other hand, preapplication of LPS inhibited ATP-induced currents, intracellular calcium increase, and ethidium bromide uptake and had no effect on ERK activation in HEK293 cells. In splenocytes-derived T-regulatory cells, in which ATP-induced apoptosis is driven by the P2X7R, LPS inhibited ATP-induced apoptosis. Altogether, these results demonstrate that LPS modulates the activity of the P2X7R and suggest that this effect could be of physiological relevance.

  10. Lipopolysaccharide Inhibits the Channel Activity of the P2X7 Receptor

    Science.gov (United States)

    Leiva-Salcedo, Elias; Coddou, Claudio; Rodríguez, Felipe E.; Penna, Antonello; Lopez, Ximena; Neira, Tanya; Fernández, Ricardo; Imarai, Mónica; Rios, Miguel; Escobar, Jorge; Montoya, Margarita; Huidobro-Toro, J. Pablo; Escobar, Alejandro; Acuña-Castillo, Claudio

    2011-01-01

    The purinergic P2X7 receptor (P2X7R) plays an important role during the immune response, participating in several events such as cytokine release, apoptosis, and necrosis. The bacterial endotoxin lipopolysaccharide (LPS) is one of the strongest stimuli of the immune response, and it has been shown that P2X7R activation can modulate LPS-induced responses. Moreover, a C-terminal binding site for LPS has been proposed. In order to evaluate if LPS can directly modulate the activity of the P2X7R, we tested several signaling pathways associated with P2X7R activation in HEK293 cells that do not express the TLR-4 receptor. We found that LPS alone was unable to induce any P2X7R-related activity, suggesting that the P2X7R is not directly activated by the endotoxin. On the other hand, preapplication of LPS inhibited ATP-induced currents, intracellular calcium increase, and ethidium bromide uptake and had no effect on ERK activation in HEK293 cells. In splenocytes-derived T-regulatory cells, in which ATP-induced apoptosis is driven by the P2X7R, LPS inhibited ATP-induced apoptosis. Altogether, these results demonstrate that LPS modulates the activity of the P2X7R and suggest that this effect could be of physiological relevance. PMID:21941410

  11. Selective inhibition of caspases in skeletal muscle reverses the apoptotic synaptic degeneration in slow-channel myasthenic syndrome.

    Science.gov (United States)

    Zhu, Haipeng; Pytel, Peter; Gomez, Christopher M

    2014-01-01

    Slow-channel syndrome (SCS) is a congenital myasthenic disorder caused by point mutations in subunits of skeletal muscle acetylcholine receptor leading to Ca(2+) overload and degeneration of the postsynaptic membrane, nuclei and mitochondria of the neuromuscular junction (NMJ). In both SCS muscle biopsies and transgenic mouse models for SCS (mSCS), the endplate regions are shrunken, and there is evidence of DNA damage in the subsynaptic region. Activated caspase-9, -3 and -7 are intensely co-localized at the NMJ, and the Ca(2+)-activated protease, calpain, and the atypical cyclin-dependent kinase (Cdk5) are overactivated in mSCS. Thus, the true mediator(s) of the disease process is not clear. Here, we demonstrate that selective inhibition of effector caspases, caspase-3 and -7, or initiator caspase, caspase-9, in limb muscle in vivo by localized expression of recombinant inhibitor proteins dramatically decreases subsynaptic DNA damage, increases endplate area and improves ultrastructural abnormalities in SCS transgenic mice. Calpain and Cdk5 are not affected by this treatment. On the other hand, inhibition of Cdk5 by expression of a dominant-negative form of Cdk5 has no effect on the degeneration. Together with previous studies, these results indicate that focal activation of caspase activity at the NMJ is the principal pathological process responsible for the synaptic apoptosis in SCS. Thus, treatments that reduce muscle caspase activity are likely to be of benefit for SCS patients.

  12. Arctigenin, a Potential Anti-Arrhythmic Agent, Inhibits Aconitine-Induced Arrhythmia by Regulating Multi-Ion Channels

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    Zhenying Zhao

    2013-11-01

    Full Text Available Background/Aims: Arctigenin possesses biological activities, but its underlying mechanisms at the cellular and ion channel levels are not completely understood. Therefore, the present study was designed to identify the anti-arrhythmia effect of arctigenin in vivo, as well as its cellular targets and mechanisms. Methods: A rat arrhythmia model was established via continuous aconitine infusion, and the onset times of ventricular premature contraction, ventricular tachycardia and death were recorded. The Action Potential Duration (APD, sodium current (INa, L-type calcium current (ICa, L and transient outward potassium current (Ito were measured and analysed using a patch-clamp recording technique in normal rat cardiomyocytes and myocytes of arrhythmia aconitine-induced by. Results: Arctigenin significantly delayed the arrhythmia onset in the aconitine-induced rat model. The 50% and 90% repolarisations (APD50 and APD90 were shortened by 100 µM arctigenin; the arctigenin dose also inhibited the prolongation of APD50 and APD90 caused by 1 µM aconitine. Arctigenin inhibited INa and ICa,L and attenuated the aconitine-increased INa and ICa,L by accelerating the activation process and delaying the inactivation process. Arctigenin enhanced Ito by facilitating the activation process and delaying the inactivation process, and recoverd the decreased Ito induced by aconitine. Conclusions: Arctigenin has displayed anti-arrhythmia effects, both in vivo and in vitro. In the context of electrophysiology, INa, ICa, L, and Ito may be multiple targets of arctigenin, leading to its antiarrhythmic effect.

  13. Extracellular acid block and acid-enhanced inactivation of the Ca2+-activated cation channel TRPM5 involve residues in the S3-S4 and S5-S6 extracellular domains.

    Science.gov (United States)

    Liu, Dan; Zhang, Zheng; Liman, Emily R

    2005-05-27

    TRPM5, a member of the superfamily of transient receptor potential ion channels, is essential for the detection of bitter, sweet, and amino acid tastes. In heterologous cell types it forms a nonselective cation channel that is activated by intracellular Ca(2+). TRPM5 is likely to be part of the taste transduction cascade, and regulators of TRPM5 are likely to affect taste sensation. In this report we show that TRPM5, but not the related channel TRPM4b, is potently blocked by extracellular acidification. External acidification has two effects, a fast reversible block of the current (IC(50) pH = 6.2) and a slower irreversible enhancement of current inactivation. Mutation of a single Glu residue in the S3-S4 linker and a His residue in the pore region each reduced sensitivity of TRPM5 currents to fast acid block (IC(50) pH = 5.8 for both), and the double mutant was nearly insensitive to acidic pH (IC(50) pH = 5.0). Prolonged exposure to acidic pH enhanced inactivation of TRPM5 currents, and mutant channels that were less sensitive to acid block were also less sensitive to acid-enhanced inactivation, suggesting an intimate association between the two processes. These processes are, however, distinct because the pore mutant H896N, which has normal sensitivity to acid block, shows significant recovery from acid-enhanced inactivation. These data show that extracellular acidification acts through specific residues on TRPM5 to block conduction through two distinct but related mechanisms and suggest a possible interaction between extracellular pH and activation and adaptation of bitter, sweet, and amino acid taste transduction.

  14. Inhibition of voltage-gated calcium channels as common mode of action for (mixtures of) distinct classes of insecticides.

    Science.gov (United States)

    Meijer, Marieke; Dingemans, Milou M L; van den Berg, Martin; Westerink, Remco H S

    2014-09-01

    Humans are exposed to distinct structural classes of insecticides with different neurotoxic modes of action. Because calcium homeostasis is essential for proper neuronal function and development, we investigated the effects of insecticides from different classes (pyrethroid: (α-)cypermethrin; organophosphate: chlorpyrifos; organochlorine: endosulfan; neonicotinoid: imidacloprid) and mixtures thereof on the intracellular calcium concentration ([Ca(2+)]i). Effects of acute (20 min) exposure to (mixtures of) insecticides on basal and depolarization-evoked [Ca(2+)]i were studied in vitro with Fura-2-loaded PC12 cells and high resolution single-cell fluorescence microscopy. The data demonstrate that cypermethrin, α-cypermethrin, endosulfan, and chlorpyrifos concentration-dependently decreased depolarization-evoked [Ca(2+)]i, with 50% (IC50) at 78nM, 239nM, 250nM, and 899nM, respectively. Additionally, acute exposure to chlorpyrifos or endosulfan (10μM) induced a modest increase in basal [Ca(2+)]i, amounting to 68 ± 8nM and 53 ± 8nM, respectively. Imidacloprid did not disturb basal or depolarization-evoked [Ca(2+)]i at 10μM. Following exposure to binary mixtures, effects on depolarization-evoked [Ca(2+)]i were within the expected effect additivity range, whereas the effect of the tertiary mixture was less than this expected additivity effect range. These results demonstrate that different types of insecticides inhibit depolarization-evoked [Ca(2+)]i in PC12 cells by inhibiting voltage-gated calcium channels (VGCCs) in vitro at concentrations comparable with human occupational exposure levels. Moreover, the effective concentrations in this study are below those for earlier described modes of action. Because inhibition of VGCCs appears to be a common and potentially additive mode of action of several classes of insecticides, this target should be considered in neurotoxicity risk assessment studies.

  15. Inhibition of Hyperpolarization-Activated Cation Current in Medium-Sized DRG Neurons Contributed to the Antiallodynic Effect of Methylcobalamin in the Rat of a Chronic Compression of the DRG

    Directory of Open Access Journals (Sweden)

    Ming Zhang

    2015-01-01

    Full Text Available Recently several lines of evidence demonstrated that methylcobalamin (MeCbl might have potential analgesic effect in experimental and clinical studies. However, it was reported that MeCbl had no effect on treating lumbar spinal stenosis induced pain. Thus, the effects of short-term and long-term administration of MeCbl were examined in the chronic compression of dorsal root ganglion (CCD model. We found that mechanical allodynia was significantly inhibited by a continuous application of high dose and a single treatment of a super high dose of MeCbl. Little is known about mechanisms underlying the analgesia of MeCbl. We examined the effect of MeCbl on the spontaneous activity (SA, the excitability, and hyperpolarization-activated nonselective cation ion current in compressed medium-sized dorsal root ganglion (DRG neurons using extracellular single fiber recording in vivo and whole-cell patch clamp in vitro. We found that MeCbl significantly inhibited the SA of A-type sensory neurons in a dose-dependent manner and inhibited the excitability of medium-sized DRG neurons. In addition, MeCbl also decreased Ih current density in injured medium-sized DRG neurons. Our results proved that MeCbl might exert an analgesic effect through the inhibition Ih current and then might inhibit the hyperexcitability of primary sensory neurons under neuropathic pain state.

  16. Inhibition of Hyperpolarization-Activated Cation Current in Medium-Sized DRG Neurons Contributed to the Antiallodynic Effect of Methylcobalamin in the Rat of a Chronic Compression of the DRG.

    Science.gov (United States)

    Zhang, Ming; Han, Wenjuan; Zheng, Jianyong; Meng, Fancheng; Jiao, Xiying; Hu, Sanjue; Xu, Hui

    2015-01-01

    Recently several lines of evidence demonstrated that methylcobalamin (MeCbl) might have potential analgesic effect in experimental and clinical studies. However, it was reported that MeCbl had no effect on treating lumbar spinal stenosis induced pain. Thus, the effects of short-term and long-term administration of MeCbl were examined in the chronic compression of dorsal root ganglion (CCD) model. We found that mechanical allodynia was significantly inhibited by a continuous application of high dose and a single treatment of a super high dose of MeCbl. Little is known about mechanisms underlying the analgesia of MeCbl. We examined the effect of MeCbl on the spontaneous activity (SA), the excitability, and hyperpolarization-activated nonselective cation ion current in compressed medium-sized dorsal root ganglion (DRG) neurons using extracellular single fiber recording in vivo and whole-cell patch clamp in vitro. We found that MeCbl significantly inhibited the SA of A-type sensory neurons in a dose-dependent manner and inhibited the excitability of medium-sized DRG neurons. In addition, MeCbl also decreased I h current density in injured medium-sized DRG neurons. Our results proved that MeCbl might exert an analgesic effect through the inhibition I h current and then might inhibit the hyperexcitability of primary sensory neurons under neuropathic pain state.

  17. The calmodulin inhibitor CGS 9343B inhibits voltage-dependent K{sup +} channels in rabbit coronary arterial smooth muscle cells

    Energy Technology Data Exchange (ETDEWEB)

    Li, Hongliang; Hong, Da Hye; Kim, Han Sol; Kim, Hye Won [Institute of Medical Sciences, Department of Physiology, Kangwon National University School of Medicine, Chuncheon, 200-701 (Korea, Republic of); Jung, Won-Kyo [Department of Biomedical Engineering, Center for Marine-Integrated Biomedical Technology (BK21 Plus), Pukyong National University, Busan 608-737 (Korea, Republic of); Na, Sung Hun [Institute of Medical Sciences, Department of Obstetrics and Gynecology, Kangwon National University Hospital, School of Medicine, Kangwon National University, Chuncheon, 200-701 (Korea, Republic of); Jung, In Duk; Park, Yeong-Min [Department of Immunology, Lab of Dendritic Cell Differentiation and Regulation, College of Medicine, Konkuk University, Chungju 380-701 (Korea, Republic of); Choi, Il-Whan, E-mail: cihima@inje.ac.kr [Department of Microbiology, Inje University College of Medicine, Busan, 614-735 (Korea, Republic of); Park, Won Sun, E-mail: parkws@kangwon.ac.kr [Institute of Medical Sciences, Department of Physiology, Kangwon National University School of Medicine, Chuncheon, 200-701 (Korea, Republic of)

    2015-06-15

    We investigated the effects of the calmodulin inhibitor CGS 9343B on voltage-dependent K{sup +} (Kv) channels using whole-cell patch clamp technique in freshly isolated rabbit coronary arterial smooth muscle cells. CGS 9343B inhibited Kv currents in a concentration-dependent manner, with a half-maximal inhibitory concentration (IC{sub 50}) value of 0.81 μM. The decay rate of Kv channel inactivation was accelerated by CGS 9343B. The rate constants of association and dissociation for CGS 9343B were 2.77 ± 0.04 μM{sup −1} s{sup −1} and 2.55 ± 1.50 s{sup −1}, respectively. CGS 9343B did not affect the steady-state activation curve, but shifted the inactivation curve toward to a more negative potential. Train pulses (1 or 2 Hz) application progressively increased the CGS 9343B-induced Kv channel inhibition. In addition, the inactivation recovery time constant was increased in the presence of CGS 9343B, suggesting that CGS 9343B-induced inhibition of Kv channel was use-dependent. Another calmodulin inhibitor, W-13, did not affect Kv currents, and did not change the inhibitory effect of CGS 9343B on Kv current. Our results demonstrated that CGS 9343B inhibited Kv currents in a state-, time-, and use-dependent manner, independent of calmodulin inhibition. - Highlights: • We investigated the effects of CGS 9394B on Kv channels. • CGS 9394B inhibited Kv current in a state-, time-, and use-dependent manner. • Caution is required when using CGS 9394B in vascular function studies.

  18. Anesthetic drug midazolam inhibits cardiac human ether-à-go-go-related gene channels: mode of action

    Directory of Open Access Journals (Sweden)

    Vonderlin N

    2015-02-01

    Full Text Available Nadine Vonderlin,1 Fathima Fischer,1 Edgar Zitron,1,2 Claudia Seyler,1 Daniel Scherer,1 Dierk Thomas,1,2 Hugo A Katus,1,2 Eberhard P Scholz1 1Department of Internal Medicine III, University Hospital Heidelberg, 2German Centre for Cardiovascular Research, Partner Site Heidelberg/Mannheim, Heidelberg, Germany Abstract: Midazolam is a short-acting benzodiazepine that is in wide clinical use as an anxiolytic, sedative, hypnotic, and anticonvulsant. Midazolam has been shown to inhibit ion channels, including calcium and potassium channels. So far, the effects of midazolam on cardiac human ether-à-go-go-related gene (hERG channels have not been analyzed. The inhibitory effects of midazolam on heterologously expressed hERG channels were analyzed in Xenopus oocytes using the double-electrode voltage clamp technique. We found that midazolam inhibits hERG channels in a concentration-dependent manner, yielding an IC50 of 170 µM in Xenopus oocytes. When analyzed in a HEK 293 cell line using the patch-clamp technique, the IC50 was 13.6 µM. Midazolam resulted in a small negative shift of the activation curve of hERG channels. However, steady-state inactivation was not significantly affected. We further show that inhibition is state-dependent, occurring within the open and inactivated but not in the closed state. There was no frequency dependence of block. Using the hERG pore mutants F656A and Y652A we provide evidence that midazolam uses a classical binding site within the channel pore. Analyzing the subacute effects of midazolam on hERG channel trafficking, we further found that midazolam does not affect channel surface expression. Taken together, we show that the anesthetic midazolam is a low-affinity inhibitor of cardiac hERG channels without additional effects on channel surface expression. These data add to the current understanding of the pharmacological profile of the anesthetic midazolam. Keywords: midazolam, anesthetics, human ether

  19. Inhibition of voltage-gated calcium channels after subchronic and repeated exposure of PC12 cells to different classes of insecticides

    NARCIS (Netherlands)

    Meijer, Marieke; Brandsema, Joske A R; Nieuwenhuis, Desirée; Wijnolts, Fiona M J; Dingemans, Milou M L; Westerink, Remco H S

    2015-01-01

    We previously demonstrated that acute inhibition of voltage-gated calcium channels (VGCCs) is a common mode of action for (sub)micromolar concentrations of chemicals, including insecticides. However, since human exposure to chemicals is usually chronic and repeated, we investigated if selected insec

  20. Inhibition of SK4 Potassium Channels Suppresses Cell Proliferation, Migration and the Epithelial-Mesenchymal Transition in Triple-Negative Breast Cancer Cells.

    Science.gov (United States)

    Zhang, Panshi; Yang, Xiaowei; Yin, Qian; Yi, Jilin; Shen, Wenzhuang; Zhao, Lu; Zhu, Zhi; Liu, Jinwen

    2016-01-01

    Treatments for triple-negative breast cancer (TNBC) are limited; intermediate-conductance calcium-activated potassium (SK4) channels are closely involved in tumor progression, but little is known about these channels in TNBC. We aimed to investigate whether SK4 channels affect TNBC. First, by immunohistochemistry (IHC) and western blotting (WB), increased SK4 protein expression in breast tumor tissues was detected relative to that in non-tumor breast tissues, but there was no apparent expression difference between various subtypes of breast cancer (p>0.05). Next, functional SK4 channels were detected in the TNBC cell line MDA-MB-231 using WB, real-time PCR, immunofluorescence and patch-clamp recording. By employing SK4 specific siRNAs and blockers, including TRAM-34 and clotrimazole, in combination with an MTT assay, a colony-formation assay, flow cytometry and a cell motility assay, we found that the suppression of SK4 channels significantly inhibited cell proliferation and migration and promoted apoptosis in MDA-MB-231 cells (pMDA-MB-231 cells to undergo the epithelial-mesenchymal transition (EMT) and to show increased SK4 mRNA expression. In addition, the down-regulation of SK4 expression inhibited the EMT markers Vimentin and Snail1. Collectively, our findings suggest that SK4 channels are expressed in TNBC and are involved in the proliferation, apoptosis, migration and EMT processes of TNBC cells.

  1. Competitive inhibition of the luminal efflux by multidrug and toxin extrusions, but not basolateral uptake by organic cation transporter 2, is the likely mechanism underlying the pharmacokinetic drug-drug interactions caused by cimetidine in the kidney.

    Science.gov (United States)

    Ito, Sumito; Kusuhara, Hiroyuki; Yokochi, Miyu; Toyoshima, Junko; Inoue, Katsuhisa; Yuasa, Hiroaki; Sugiyama, Yuichi

    2012-02-01

    Cimetidine, an H₂ receptor antagonist, has been used to investigate the tubular secretion of organic cations in human kidney. We report a systematic comprehensive analysis of the inhibition potency of cimetidine for the influx and efflux transporters of organic cations [human organic cation transporter 1 (hOCT1) and hOCT2 and human multidrug and toxin extrusion 1 (hMATE1) and hMATE2-K, respectively]. Inhibition constants (K(i)) of cimetidine were determined by using five substrates [tetraethylammonium (TEA), metformin, 1-methyl-4-phenylpyridinium, 4-(4-(dimethylamino)styryl)-N-methylpyridinium, and m-iodobenzylguanidine]. They were 95 to 146 μM for hOCT2, providing at most 10% inhibition based on its clinically reported plasma unbound concentrations (3.6-7.8 μM). In contrast, cimetidine is a potent inhibitor of MATE1 and MATE2-K with K(i) values (μM) of 1.1 to 3.8 and 2.1 to 6.9, respectively. The same tendency was observed for mouse Oct1 (mOct1), mOct2, and mouse Mate1. Cimetidine showed a negligible effect on the uptake of metformin by mouse kidney slices at 20 μM. Cimetidine was administered to mice by a constant infusion to achieve a plasma unbound concentration of 21.6 μM to examine its effect on the renal disposition of Mate1 probes (metformin, TEA, and cephalexin) in vivo. The kidney- and liver-to-plasma ratios of metformin both were increased 2.4-fold by cimetidine, whereas the renal clearance was not changed. Cimetidine also increased the kidney-to-plasma ratio of TEA and cephalexin 8.0- and 3.3-fold compared with a control and decreased the renal clearance from 49 to 23 and 11 to 6.6 ml/min/kg, respectively. These results suggest that the inhibition of MATEs, but not OCT2, is a likely mechanism underlying the drug-drug interactions with cimetidine in renal elimination.

  2. Tetrodotoxin-sensitive α-subunits of voltage-gated sodium channels are relevant for inhibition of cardiac sodium currents by local anesthetics.

    Science.gov (United States)

    Stoetzer, C; Doll, T; Stueber, T; Herzog, C; Echtermeyer, F; Greulich, F; Rudat, C; Kispert, A; Wegner, F; Leffler, A

    2016-06-01

    The sodium channel α-subunit (Nav) Nav1.5 is regarded as the most prevalent cardiac sodium channel required for generation of action potentials in cardiomyocytes. Accordingly, Nav1.5 seems to be the main target molecule for local anesthetic (LA)-induced cardiotoxicity. However, recent reports demonstrated functional expression of several "neuronal" Nav's in cardiomyocytes being involved in cardiac contractility and rhythmogenesis. In this study, we examined the relevance of neuronal tetrodotoxin (TTX)-sensitive Nav's for inhibition of cardiac sodium channels by the cardiotoxic LAs ropivacaine and bupivacaine. Effects of LAs on recombinant Nav1.2, 1.3, 1.4, and 1.5 expressed in human embryonic kidney cell line 293 (HEK-293) cells, and on sodium currents in murine, cardiomyocytes were investigated by whole-cell patch clamp recordings. Expression analyses were performed by reverse transcription PCR (RT-PCR). Cultured cardiomyocytes from neonatal mice express messenger RNA (mRNA) for Nav1.2, 1.3, 1.5, 1.8, and 1.9 and generate TTX-sensitive sodium currents. Tonic and use-dependent block of sodium currents in cardiomyocytes by ropivacaine and bupivacaine were enhanced by 200 nM TTX. Inhibition of recombinant Nav1.5 channels was similar to that of TTX-resistant currents in cardiomyocytes but stronger as compared to inhibition of total sodium current in cardiomyocytes. Recombinant Nav1.2, 1.3, 1.4, and 1.5 channels displayed significant differences in regard to use-dependent block by ropivacaine. Finally, bupivacaine blocked sodium currents in cardiomyocytes as well as recombinant Nav1.5 currents significantly stronger in comparison to ropivacaine. Our data demonstrate for the first time that cardiac TTX-sensitive sodium channels are relevant for inhibition of cardiac sodium currents by LAs.

  3. Retina derived relaxation is mediated by K(ir) channels and the inhibition of Ca(2+) sensitization in isolated bovine retinal arteries.

    Science.gov (United States)

    Takır, Selçuk; Uydeş-Doğan, B Sönmez; Özdemir, Osman

    2015-03-01

    Retinal relaxing factor (RRF) has recently been identified as a novel paracrine regulator of retinal circulation acting differently from well known mediators of the endothelium and the retina. Herein, we aimed to characterize the relaxing mechanism of the retina, i.e. RRF, by evaluating the role of Ca(+2)-dependent and -independent signaling mechanisms as well as inward rectifier K(+) (Kir) channels. Retinal relaxation was determined by placing a piece of retinal tissue just on top of the precontracted bovine retinal arteries mounted in a wire myograph. The retina produced a complete relaxation response, which display a biphasic character, in depolarized arteries contracted by L-type Ca(2+) channel agonist, Bay k 8644. Blockade of L-type Ca(2+) channel by nifedipine, inhibition of sarcoplasmic reticulum Ca(2+)-ATPase by cyclopiazonic acid or removal of extracellular Ca(2+) did not influence the prominent relaxation to the retina. Originally, retinal relaxation was found to be unaffected from the inhibition of myosin light chain kinase by ML7, whereas, completely abolished in the presence of myosin light chain phosphatase (MLCP) inhibitor, Calyculin A. Moreover, the inhibition of Rho kinase by its putative inhibitor, Y-27632 displayed comparable relaxant effects to RRF in retinal arteries precontracted either by prostaglandin F2α or K(+), and augmented the moderate response to the retina in K(+) precontracted arteries. In addition, retinal relaxation was significantly inhibited and lost its biphasic character in the presence of Kir channel blocker, Ba(2+). Our results suggested that inhibition of Ca(2+) sensitization through the activation of MLCP, possibly via interfering with Rho kinase, and the opening of Kir channels are likely to be involved in the inhibitory influence of RRF on the retinal arteries.

  4. Mitogen-activated protein kinases inhibit the ROMK (Kir 1.1)-like small conductance K channels in the cortical collecting duct.

    Science.gov (United States)

    Babilonia, Elisa; Li, Dimin; Wang, Zhijian; Sun, Peng; Lin, Dao-Hong; Jin, Yan; Wang, Wen-Hui

    2006-10-01

    It was demonstrated previously that low dietary potassium (K) intake stimulates Src family protein tyrosine kinase (PTK) expression via a superoxide-dependent signaling. This study explored the role of mitogen-activated protein kinase (MAPK) in mediating the effect of superoxide anions on PTK expression and ROMK (Kir 1.1) channel activity. Western blot analysis demonstrated that low K intake significantly increased the phosphorylation of P38 MAPK (P38) and extracellular signal-regulated kinase (ERK) but had no effect on phosphorylation of c-JUN N-terminus kinase in renal cortex and outer medulla. The stimulatory effect of low K intake on P38 and ERK was abolished by treatment of rats with tempol. The possibility that increases in superoxide and related products that are induced by low K intake were responsible for stimulating phosphorylation of P38 and ERK also was supported by the finding that application of H(2)O(2) increased the phosphorylation of ERK and P38 in the cultured mouse collecting duct cells. Simultaneous blocking of ERK and P38 completely abolished the effect of H(2)O(2) on c-Src expression in mouse collecting duct cells. For determination of the role of P38 and ERK in the regulation of ROMK-like small-conductance K (SK) channels, the patch-clamp technique was used to study the effect of inhibiting P38 and ERK on SK channels in the cortical collecting duct from rats that were on a control K diet (1.1%) and on a K-deficient diet for 1 d. Inhibition of ERK, c-JUN N-terminus kinase, or P38 alone had no effect on SK channels. In contrast, simultaneous inhibition of P38 and ERK significantly increased channel activity. The effect of inhibiting MAPK on SK channels was not affected in the presence of herbimycin A, a PTK inhibitor, and was larger in rats that were on a K-deficient diet than in rats that were on a normal-K diet. However, the stimulatory effect of inhibiting ERK and P38 on SK was absent in the cortical collecting duct that was treated with

  5. Transient receptor potential channels in essential hypertension

    DEFF Research Database (Denmark)

    Liu, Daoyan; Scholze, Alexandra; Zhu, Zhiming;

    2006-01-01

    The role of nonselective cation channels of the transient receptor potential channel (TRPC) family in essential hypertension has not yet been investigated.......The role of nonselective cation channels of the transient receptor potential channel (TRPC) family in essential hypertension has not yet been investigated....

  6. Tumor necrosis factor-alpha (TNF-α enhances functional thermal and chemical responses of TRP cation channels in human synoviocytes

    Directory of Open Access Journals (Sweden)

    Ma Fei

    2009-08-01

    Full Text Available Abstract Background We have shown functional expression of several TRP channels on human synovial cells, proposing significance in known calcium dependent proliferative and secretory responses in joint inflammation. The present study further characterizes synoviocyte TRP expression and activation responses to thermal and osmotic stimuli after pre-treatment with proinflammatory mediator tumor necrosis factor alpha (TNF-α, EC50 1.3221 × 10-10g/L. Results Fluorescent imaging of Fura-2 loaded human SW982 synoviocytes reveals immediate and delayed cytosolic calcium oscillations elicited by (1 TRPV1 agonists capsaicin and resiniferatoxin (20 – 40% of cells, (2 moderate and noxious temperature change, and (3 osmotic stress TRPV4 activation (11.5% of cells. TNF-alpha pre-treatment (1 ng/ml, 8 – 16 hr significantly increases (doubles capsaicin responsive cell numbers and [Ca2+]i spike frequency, as well as enhances average amplitude of temperature induced [Ca2+]i responses. With TNF-alpha pre-treatment for 8, 12, and 16 hr, activation with 36 or 45 degree bath solution induces bimodal [Ca2+]i increase (temperature controlled chamber. Initial temperature induced rapid transient spikes and subsequent slower rise reflect TRPV1 and TRPV4 channel activation, respectively. Only after prolonged TNF-alpha exposure (12 and 16 hr is recruitment of synoviocytes observed with sensitized TRPV4 responses to hypoosmolarity (3–4 fold increase. TNF-alpha increases TRPV1 (8 hr peak and TRPV4 (12 hr peak immunostaining, mRNA and protein expression, with a TRPV1 shift to membrane fractions. Conclusion TNF-α provides differentially enhanced synoviocyte TRPV1 and TRPV4 expression and [Ca2+]i response dependent on the TRP stimulus and time after exposure. Augmented relevance of TRPV1 and TRPV4 as inflammatory conditions persist would provide calcium mediated cell signaling required for pathophysiological responses of synoviocytes in inflammatory pain states.

  7. Inhibition of rat hippocampal excitability by the plant alkaloid 3-acetylaconitine mediated by interaction with voltage-dependent sodium channels.

    Science.gov (United States)

    Ameri, A

    1997-02-01

    The effects of the Aconitum alkaloid 3-acetylaconitine on neuronal activity were investigated in the slice preparation and on cultivated neurons of rat hippocampus by extracellular and patch-clamp recordings, respectively. 3-Acetylaconitine (0.01-1 microM) diminished the orthodromic and antidromic population spike in a concentration-dependent manner. The inhibitory action of the drug was preceded by a transiently enhanced excitability. The latency of onset of the inhibition was accelerated by increased stimulation frequency, whereas recovery during washout of the alkaloid was accelerated by decreased stimulation frequency. Moreover, the inhibitory effect of 3-acetylaconitine was evaluated in two different models of epileptiform activity induced either by blockade of GABA receptors by bicuculline (10 microM) or by a nominal Mg(2+)-free bathing medium. In accordance with the activity-dependent mode of action, this compound abolished the synaptically evoked population spikes in the presence of bicuculline or nominal Mg(2+)-free bathing medium, respectively. Whole-cell patch-clamp recordings revealed an interaction of 3-acetylaconitine with the voltage-dependent sodium channel. At a concentration of 1 microM, 3-acetylaconitine did not affect the peak amplitude of the sodium current, but shifted the current-voltage relationship in the hyperpolarized direction such that sodium currents were already activated at the resting potential.

  8. Calcitriol inhibits Ether-a go-go potassium channel expression and cell proliferation in human breast cancer cells

    Energy Technology Data Exchange (ETDEWEB)

    Garcia-Becerra, Rocio [Department of Reproductive Biology, Instituto Nacional de Ciencias Medicas y Nutricion Salvador Zubiran, Vasco de Quiroga No. 15, Tlalpan 14000 Mexico, D.F. (Mexico); Diaz, Lorenza, E-mail: lorenzadiaz@gmail.com [Department of Reproductive Biology, Instituto Nacional de Ciencias Medicas y Nutricion Salvador Zubiran, Vasco de Quiroga No. 15, Tlalpan 14000 Mexico, D.F. (Mexico); Camacho, Javier [Department of Pharmacology, Centro de Investigacion y de Estudios Avanzados, Instituto Politecnico Nacional, Av. Instituto Politecnico Nacional 2508, San Pedro Zacatenco 07360, Mexico, D.F. (Mexico); Barrera, David; Ordaz-Rosado, David; Morales, Angelica [Department of Reproductive Biology, Instituto Nacional de Ciencias Medicas y Nutricion Salvador Zubiran, Vasco de Quiroga No. 15, Tlalpan 14000 Mexico, D.F. (Mexico); Ortiz, Cindy Sharon [Department of Pathology, Instituto Nacional de Ciencias Medicas y Nutricion Salvador Zubiran, Vasco de Quiroga No. 15, Tlalpan 14000 Mexico, D.F. (Mexico); Avila, Euclides [Department of Reproductive Biology, Instituto Nacional de Ciencias Medicas y Nutricion Salvador Zubiran, Vasco de Quiroga No. 15, Tlalpan 14000 Mexico, D.F. (Mexico); Bargallo, Enrique [Department of Breast Tumors, Instituto Nacional de Cancerologia, Av. San Fernando No. 22, Tlalpan 14080, Mexico, D.F. (Mexico); Arrecillas, Myrna [Department of Pathology, Instituto Nacional de Cancerologia, Av. San Fernando No. 22, Tlalpan 14080, Mexico, D.F. (Mexico); Halhali, Ali; Larrea, Fernando [Department of Reproductive Biology, Instituto Nacional de Ciencias Medicas y Nutricion Salvador Zubiran, Vasco de Quiroga No. 15, Tlalpan 14000 Mexico, D.F. (Mexico)

    2010-02-01

    Antiproliferative actions of calcitriol have been shown to occur in many cell types; however, little is known regarding the molecular basis of this process in breast carcinoma. Ether-a-go-go (Eag1) potassium channels promote oncogenesis and are implicated in breast cancer cell proliferation. Since calcitriol displays antineoplastic effects while Eag1 promotes tumorigenesis, and both factors antagonically regulate cell cycle progression, we investigated a possible regulatory effect of calcitriol upon Eag1 as a mean to uncover new molecular events involved in the antiproliferative activity of this hormone in human breast tumor-derived cells. RT real-time PCR and immunocytochemistry showed that calcitriol suppressed Eag1 expression by a vitamin D receptor (VDR)-dependent mechanism. This effect was accompanied by inhibition of cell proliferation, which was potentiated by astemizole, a nonspecific Eag1 inhibitor. Immunohistochemistry and Western blot demonstrated that Eag1 and VDR abundance was higher in invasive-ductal carcinoma than in fibroadenoma, and immunoreactivity of both proteins was located in ductal epithelial cells. Our results provide evidence of a novel mechanism involved in the antiproliferative effects of calcitriol and highlight VDR as a cancer therapeutic target for breast cancer treatment and prevention.

  9. Anti-addiction drug ibogaine inhibits voltage-gated ionic currents: A study to assess the drug's cardiac ion channel profile

    Energy Technology Data Exchange (ETDEWEB)

    Koenig, Xaver; Kovar, Michael; Rubi, Lena; Mike, Agnes K.; Lukacs, Peter; Gawali, Vaibhavkumar S.; Todt, Hannes [Center for Physiology and Pharmacology, Department of Neurophysiology and -pharmacology, Medical University of Vienna, 1090 Vienna (Austria); Hilber, Karlheinz, E-mail: karlheinz.hilber@meduniwien.ac.at [Center for Physiology and Pharmacology, Department of Neurophysiology and -pharmacology, Medical University of Vienna, 1090 Vienna (Austria); Sandtner, Walter [Center for Physiology and Pharmacology, Institute of Pharmacology, Medical University of Vienna, 1090 Vienna (Austria)

    2013-12-01

    The plant alkaloid ibogaine has promising anti-addictive properties. Albeit not licenced as a therapeutic drug, and despite hints that ibogaine may perturb the heart rhythm, this alkaloid is used to treat drug addicts. We have recently reported that ibogaine inhibits human ERG (hERG) potassium channels at concentrations similar to the drugs affinity for several of its known brain targets. Thereby the drug may disturb the heart's electrophysiology. Here, to assess the drug's cardiac ion channel profile in more detail, we studied the effects of ibogaine and its congener 18-Methoxycoronaridine (18-MC) on various cardiac voltage-gated ion channels. We confirmed that heterologously expressed hERG currents are reduced by ibogaine in low micromolar concentrations. Moreover, at higher concentrations, the drug also reduced human Na{sub v}1.5 sodium and Ca{sub v}1.2 calcium currents. Ion currents were as well reduced by 18-MC, yet with diminished potency. Unexpectedly, although blocking hERG channels, ibogaine did not prolong the action potential (AP) in guinea pig cardiomyocytes at low micromolar concentrations. Higher concentrations (≥ 10 μM) even shortened the AP. These findings can be explained by the drug's calcium channel inhibition, which counteracts the AP-prolonging effect generated by hERG blockade. Implementation of ibogaine's inhibitory effects on human ion channels in a computer model of a ventricular cardiomyocyte, on the other hand, suggested that ibogaine does prolong the AP in the human heart. We conclude that therapeutic concentrations of ibogaine have the propensity to prolong the QT interval of the electrocardiogram in humans. In some cases this may lead to cardiac arrhythmias. - Highlights: • We study effects of anti-addiction drug ibogaine on ionic currents in cardiomyocytes. • We assess the cardiac ion channel profile of ibogaine. • Ibogaine inhibits hERG potassium, sodium and calcium channels. • Ibogaine’s effects on

  10. Sulfonation of 17{beta}-estradiol and inhibition of sulfotransferase activity by polychlorobiphenylols and celecoxib in channel catfish, Ictalurus punctatus

    Energy Technology Data Exchange (ETDEWEB)

    Wang Liquan [Department of Medicinal Chemistry, College of Pharmacy, University of Florida, Gainesville, FL 32610 (United States); James, Margaret O. [Department of Medicinal Chemistry, College of Pharmacy, University of Florida, Gainesville, FL 32610 (United States)]. E-mail: mojames@ufl.edu

    2007-03-10

    The sulfonation of 17{beta}-estradiol (E2) by human liver and recombinant sulfotransferases is influenced by environmental contaminants such as hydroxylated metabolites of polychlorinated biphenyls (OH-PCBs), which are potent inhibitors, and the therapeutic drug, celecoxib, which affects positional sulfonation of E2. In some locations, the aquatic environment is contaminated by PCBs, OH-PCBs and widely used therapeutic drugs. The objectives of this study were to investigate the sulfonation kinetics of E2 in liver cytosol from channel catfish (Ictalurus punctatus); to examine the effect of OH-PCBs on E2 sulfonation; and to determine if celecoxib altered the position of E2 sulfonation, as it does with human liver cytosol. E2 was converted to both 3- and 17-sulfates by catfish liver cytosol. At E2 concentrations below 1{mu}M, formation of E2-3-sulfate (E2-3-S) predominated, but substrate inhibition was observed at higher concentrations. Rates of E2-3-S formation at different E2 concentrations were fit to a substrate inhibition model, with K{sup '}{sub m} and V{sup '}{sub max} values of 0.40+/-0.10{mu}M and 91.0+/-4.7pmol/min/mg protein, respectively and K{sub i} of 1.08+/-0.09{mu}M. The formation of E2-17-S fit Michaelis-Menten kinetics over the concentration range 25nM to 2.5{mu}M, with K{sub m} and V{sub max} values of 1.07+/-0.23{mu}M and 25.7+/-4.43pmol/min/mg protein, respectively. The efficiency (V{sub max}/K{sub m}) of formation of E2-3-S was 9.8-fold higher than that of E2-17-S. Several OH-PCBs inhibited E2 3-sulfonation, measured at an E2 concentration of 1nM. Of those tested, the most potent inhibitor was 4'-OH-CB79, with two chlorine atoms flanking the OH group (IC{sub 50}: 94nM). The inhibition of estrogen sulfonation by OH-PCBs may disrupt the endocrine system and thus contribute to the known toxic effects of these compounds. Celecoxib did not stimulate E2-17-S formation, as is the case with human liver cytosol, but did inhibit the

  11. Inhibition of human two-pore domain K+ channel TREK1 by local anesthetic lidocaine: negative cooperativity and half-of-sites saturation kinetics.

    Science.gov (United States)

    Nayak, Tapan K; Harinath, S; Nama, S; Somasundaram, K; Sikdar, S K

    2009-10-01

    TWIK-related K+ channel TREK1, a background leak K+ channel, has been strongly implicated as the target of several general and local anesthetics. Here, using the whole-cell and single-channel patch-clamp technique, we investigated the effect of lidocaine, a local anesthetic, on the human (h)TREK1 channel heterologously expressed in human embryonic kidney 293 cells by an adenoviral-mediated expression system. Lidocaine, at clinical concentrations, produced reversible, concentration-dependent inhibition of hTREK1 current, with IC(50) value of 180 muM, by reducing the single-channel open probability and stabilizing the closed state. We have identified a strategically placed unique aromatic couplet (Tyr352 and Phe355) in the vicinity of the protein kinase A phosphorylation site, Ser348, in the C-terminal domain (CTD) of hTREK1, that is critical for the action of lidocaine. Furthermore, the phosphorylation state of Ser348 was found to have a regulatory role in lidocaine-mediated inhibition of hTREK1. It is interesting that we observed strong intersubunit negative cooperativity (Hill coefficient = 0.49) and half-of-sites saturation binding stoichiometry (half-reaction order) for the binding of lidocaine to hTREK1. Studies with the heterodimer of wild-type (wt)-hTREK1 and Delta119 C-terminal deletion mutant (hTREK1(wt)-Delta119) revealed that single CTD of hTREK1 was capable of mediating partial inhibition by lidocaine, but complete inhibition necessitates the cooperative interaction between both the CTDs upon binding of lidocaine. Based on our observations, we propose a model that explains the unique kinetics and provides a plausible paradigm for the inhibitory action of lidocaine on hTREK1.

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

    Energy Technology Data Exchange (ETDEWEB)

    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.

  13. Villin promoter-mediated transgenic expression of transient receptor potential cation channel, subfamily V, member 6 (TRPV6) increases intestinal calcium absorption in wild-type and vitamin D receptor knockout mice.

    Science.gov (United States)

    Cui, Min; Li, Qiang; Johnson, Robert; Fleet, James C

    2012-10-01

    Transient receptor potential cation channel, subfamily V, member 6 (TRPV6) is an apical membrane calcium (Ca) channel in the small intestine proposed to be essential for vitamin D-regulated intestinal Ca absorption. Recent studies have challenged the proposed role for TRPV6 in Ca absorption. We directly tested intestinal TRPV6 function in Ca and bone metabolism in wild-type (WT) and vitamin D receptor knockout (VDRKO) mice. TRPV6 transgenic mice (TG) were made with intestinal epithelium-specific expression of a 3X Flag-tagged human TRPV6 protein. TG and VDRKO mice were crossed to make TG-VDRKO mice. Ca and bone metabolism was examined in WT, TG, VDRKO, and TG-VDRKO mice. TG mice developed hypercalcemia and soft tissue calcification on a chow diet. In TG mice fed a 0.25% Ca diet, Ca absorption was more than three-fold higher and femur bone mineral density (BMD) was 26% higher than WT. Renal 1α hydroxylase (CYP27B1) mRNA and intestinal expression of the natural mouse TRPV6 gene were reduced to intestine calbindin-D(9k) expression was elevated >15 times in TG mice. TG-VDRKO mice had high Ca absorption that prevented the low serum Ca, high renal CYP27B1 mRNA, low BMD, and abnormal bone microarchitecture seen in VDRKO mice. In addition, small intestinal calbindin D(9K) mRNA and protein levels were elevated in TG-VDRKO. Transgenic TRPV6 expression in intestine is sufficient to increase Ca absorption and bone density, even in VDRKO mice. VDR-independent upregulation of intestinal calbindin D(9k) in TG-VDRKO suggests this protein may buffer intracellular Ca during Ca absorption. © 2012 American Society for Bone and Mineral Research.

  14. Inhibition by a novel anti-arrhythmic agent, NIP-142, of cloned human cardiac K+ channel Kv1.5 current.

    Science.gov (United States)

    Matsuda, T; Masumiya, H; Tanaka, N; Yamashita, T; Tsuruzoe, N; Tanaka, Y; Tanaka, H; Shigenoba, K

    2001-03-16

    NIP-142 was shown to prolong atrial effective refractory period and to terminate atrial fibrillation and flutter in in vivo canine models. To obtain information on its antiarrhythmic action, we examined the effect of NIP-142 on cloned human cardiac K+ channel Kv1.5 (hKv1.5) currents stably expressed in a human cell line using whole-cell voltage clamp methods. NIP-142 inhibited the hKv1.5 current in a concentration-dependent and voltage-independent manner. The inhibition was larger at the end of depolarizing pulse than at the outward current peak. The IC50 for inhibition of the steady-state phase was 4.75 microM. A cross-over phenomenon was observed when current traces in the absence and presence of NIP-142 were superimposed. Inhibition of hKv1.5 current by NIP-142 was frequency-independent; changing the depolarizing pulse frequencies (0.1, 0.2, 1 Hz) and little effect on the degree of inhibition. NIP-142 decreased the maximal peak amplitude of kHv1.5 current at the first command pulse after 3 min rest in the presence of the drug. These results suggest that NIP-142 has inhibitory effects on the hKv 1.5 current through interaction with both open and closed states of the channel, which may underlie its antiarrhythmic activity in the atria.

  15. Cilnidipine, but not amlodipine, ameliorates osteoporosis in ovariectomized hypertensive rats through inhibition of the N-type calcium channel.

    Science.gov (United States)

    Shimizu, Hideo; Nakagami, Hironori; Yasumasa, Natsuki; Mariana, Osako Kiomy; Kyutoku, Mariko; Koriyama, Hiroshi; Nakagami, Futoshi; Shimamura, Munehisa; Rakugi, Hiromi; Morishita, Ryuichi

    2012-01-01

    Both osteoporosis and high blood pressure are major diseases in aging populations. Recent studies demonstrated that some antihypertensive drugs reduced the risk of bone fracture in elderly patients. Although calcium channel blockers (CCB) are widely used as first-line antihypertensive agents, there is no evidence that they prevent osteoporosis. In this study, we investigated the effects of two types of CCB on bone metabolism: cilnidipine (L-/N-type CCB), which suppresses norepinephrine release from the sympathetic nerve, and amlodipine (L-type CCB). In ovariectomized female spontaneous hypertensive rats, administration of cilnidipine, but not amlodipine, resulted in a significant increase in the ratio of alkaline phosphatase to tartrate-resistant acid phosphatase (TRAP) and a decrease in the number of osteoclasts, as assessed by TRAP staining in the proximal tibia. Bone mineral density, moreover, was significantly higher in the cilnidipine group as compared with the amlodipine group and was associated with a significant decrease in a urinary collagen degradation product (deoxypyridinoline). The degree of prevention of osteoporosis by cilnidipine was similar to that of carvedilol (a β-blocker) because β-blockers reduce fracture risks though the inhibition of osteoclast activation. Interestingly, these effects cannot be attributed to the reduction of blood pressure because all three drugs significantly decreased blood pressure. In contrast, both cilnidipine and carvedilol, but not amlodipine, significantly decreased heart rate, indicating that both cilnidipine and carvedilol suppressed sympathetic nervous activity. Overall, our present data showed that cilnidipine (L-/N-type CCB) ameliorated osteoporosis in ovariectomized hypertensive rats. These pleiotropic effects of antihypertensive drugs such as cilnidipine and carvedilol might provide additional benefits in the treatment of hypertensive postmenopausal women.

  16. Ginsenoside Rb1 selectively inhibits the activity of L-type voltage-gated calcium channels in cultured rat hippocampal neurons

    Institute of Scientific and Technical Information of China (English)

    Zhi-ying LIN; Li-min CHEN; Jing ZHANG; Xiao-dong PAN; Yuan-gui ZHU; Qin-yong YE; Hua-pin HUANG; Xiao-chun CHEN

    2012-01-01

    Aim:To investigate the effect of ginsenoside Rb1 on voltage-gated calcium currents in cultured rat hippocampal neurons and the modulatory mechanism.Methods:Cultured hippocampal neurons were prepared from Sprague Dawley rat embryos.Whole-cell configuration of the patchclamp technique was used to record the voltage-gated calcium currents (VGCCs)from the hippocampal neurons,and the effect of Rb1 was examined.Results:Rb1 (2-100 μmol/L)inhibited VGCCs in a concentration-dependent manner,and the current was mostly recovered upon wash-out.The specific L-type Ca2+ channel inhibitor nifedipine (10 μmol/L)occluded Rb1-induced inhibition on VGCCs.Neither the selective N-type Ca2+ channel blocker ω-conotoxin-GVlA (1 μmoVL),nor the selective P/Q-type Ca2+ channel blocker ωo-agatoxin IVA (30 nmol/L)diminished Rb1-sensitive VGCCs.Rb1 induced a leftward shift of the steady-state inactivation curve of Ica to a negative potential without affecting its activation kinetics or reversal potential in the I-V curve.The inhibitory effect of Rb1 was neither abolished by the adenylyl cyclase activator forskolin (10 μmol/L),nor by the PKA inhibitor H-89 (10 μmol/L).Conclusion:Ginsenoside Rb1 selectively inhibits the activity of L-type voltage-gated calcium channels,without affecting the N-type or P/Q-type Ca2+ channels in hippocampal neurons,cAMP-PKA signaling pathway is not involved in this effect.

  17. Lys-[Leu8,des-Arg9]-bradykinin blocks lipopolysaccharide-induced SHR aorta hyperpolarization by inhibition of Ca(++)- and ATP-dependent K+ channels.

    Science.gov (United States)

    Farias, Nelson C; Feres, Teresa; Paiva, Antonio C M; Paiva, Therezinha B

    2004-09-13

    The mediators involved in the hyperpolarizing effects of lipopolysaccharide and of the bradykinin B1 receptor agonist des-Arg9-bradykinin on the rat aorta were investigated by comparing the responses of aortic rings of spontaneously hypertensive and normotensive Wistar rats. Endothelized rings from hypertensive rats were hyperpolarized by des-Arg9-bradykinin and lipopolysaccharide, whereas de-endothelized rings responded to lipopolysaccharide but not to des-Arg9-bradykinin. In endothelized preparations, the responses to des-Arg9-bradykinin were inhibited by Nomega-nitro-L-arginine and iberiotoxin. De-endothelized ring responses to lipopolysaccharide were inhibited by iberiotoxin, glibenclamide and B1 antagonist Lys-[Leu8,des-Arg9]-bradykinin. This antagonist also inhibited hyperpolarization by des-Arg9-bradykinin and by the á2-adrenoceptor agonist, brimonidine. Our results indicate that Ca(2+)-sensitive K+ channels are the final mediators of the responses to des-Arg9-bradykinin, whereas both Ca(2+)- and ATP-sensitive K+ channels mediate the responses to lipopolysaccharide. The inhibitory effects of Lys-[Leu8,des-Arg9]-bradykinin is due to a direct action on Ca(2+)- and ATP-sensitive potassium channels.

  18. The potential role of cobalt ions released from metal prosthesis on the inhibition of Hv1 proton channels and the decrease in Staphyloccocus epidermidis killing by human neutrophils.

    Science.gov (United States)

    Daou, Samira; El Chemaly, Antoun; Christofilopoulos, Panayiotis; Bernard, Louis; Hoffmeyer, Pierre; Demaurex, Nicolas

    2011-03-01

    Infection by Staphylococcus epidermidis is a devastating complication of metal-on-metal (MM) total hip arthroplasty (THA). Neutrophils are the first line of defense against infection, and these innate immune cells are potentially exposed to Co(2+) ions released in the peri-prosthetic tissue by the wear of MM THA. The toxicity of Co(2+) is still debated, but Co(2+) is a potential inhibitor of the Hv1 proton channel that sustains the production of superoxide by neutrophils. In this study, we show that the Co(2+) concentration in peri-prosthetic tissue from patients with MM THA averages 53 μM and that such high concentrations of Co(2+) alter the antibacterial activity of human neutrophils in vitro by inhibiting Hv1 proton channels. We show that submillimolar concentrations of Co(2+) inhibit proton currents, impair the extrusion of cytosolic acid, and decrease the production of superoxide in human neutrophils. As a result, Co(2+) reduces the ability of human neutrophils to kill two strains of Staphyloccocus epidermidis by up to 7-fold at the maximal concentration tested of 100 μM Co(2+). By inhibiting proton channels, the Co(2+) ions released by metal prostheses might therefore promote bacterial infections in patients with metal-on-metal total hip arthroplasty.

  19. Endomorphins inhibit high-threshold Ca2+ channel currents in rodent NG108-15 cells overexpressing mu-opioid receptors.

    Science.gov (United States)

    Higashida, H; Hoshi, N; Knijnik, R; Zadina, J E; Kastin, A J

    1998-02-15

    1. Extracellular application of the novel brain peptides endomorphin 1 (EM1) and endomorphin 2 (EM2) inhibited high-threshold Ca2+ channel currents in NGMO-251 cells, a daughter clone of NG108-15 mouse neuroblastoma x rat glioma hybrid cells, in which mu-opioid receptors are overexpressed. 2. In contrast, EM1 and EM2 did not induce this inhibition in the parental NG108-15 cells that predominantly express endogenous delta-receptors. 3. The IC50 for EM1 and EM2 was 7.7 and 23.1 nM, respectively. 4. EM-induced Ca2+ channel current inhibition was blocked by treatment or pretreatment of the cells with 100 microM N-methylmaleimide or 100 ng ml-1 pertussis toxin. 5. These results show that a decrease in conductance of Ca2+ channels results following interaction of EMs with cloned mu-receptors, which couple via Gi/Go-type G proteins, and that EMs fulfill one of the necessary synaptic conditions for them to be identified as neurotransmitters.

  20. Inhibition of SK4 Potassium Channels Suppresses Cell Proliferation, Migration and the Epithelial-Mesenchymal Transition in Triple-Negative Breast Cancer Cells.

    Directory of Open Access Journals (Sweden)

    Panshi Zhang

    Full Text Available Treatments for triple-negative breast cancer (TNBC are limited; intermediate-conductance calcium-activated potassium (SK4 channels are closely involved in tumor progression, but little is known about these channels in TNBC. We aimed to investigate whether SK4 channels affect TNBC. First, by immunohistochemistry (IHC and western blotting (WB, increased SK4 protein expression in breast tumor tissues was detected relative to that in non-tumor breast tissues, but there was no apparent expression difference between various subtypes of breast cancer (p>0.05. Next, functional SK4 channels were detected in the TNBC cell line MDA-MB-231 using WB, real-time PCR, immunofluorescence and patch-clamp recording. By employing SK4 specific siRNAs and blockers, including TRAM-34 and clotrimazole, in combination with an MTT assay, a colony-formation assay, flow cytometry and a cell motility assay, we found that the suppression of SK4 channels significantly inhibited cell proliferation and migration and promoted apoptosis in MDA-MB-231 cells (p<0.05. Further investigation revealed that treatment with epidermal growth factor (EGF/basic fibroblast growth factor (bFGF caused MDA-MB-231 cells to undergo the epithelial-mesenchymal transition (EMT and to show increased SK4 mRNA expression. In addition, the down-regulation of SK4 expression inhibited the EMT markers Vimentin and Snail1. Collectively, our findings suggest that SK4 channels are expressed in TNBC and are involved in the proliferation, apoptosis, migration and EMT processes of TNBC cells.

  1. Anti-addiction drug ibogaine inhibits voltage-gated ionic currents: a study to assess the drug's cardiac ion channel profile.

    Science.gov (United States)

    Koenig, Xaver; Kovar, Michael; Rubi, Lena; Mike, Agnes K; Lukacs, Peter; Gawali, Vaibhavkumar S; Todt, Hannes; Hilber, Karlheinz; Sandtner, Walter

    2013-12-01

    The plant alkaloid ibogaine has promising anti-addictive properties. Albeit not licensed as a therapeutic drug, and despite hints that ibogaine may perturb the heart rhythm, this alkaloid is used to treat drug addicts. We have recently reported that ibogaine inhibits human ERG (hERG) potassium channels at concentrations similar to the drugs affinity for several of its known brain targets. Thereby the drug may disturb the heart's electrophysiology. Here, to assess the drug's cardiac ion channel profile in more detail, we studied the effects of ibogaine and its congener 18-Methoxycoronaridine (18-MC) on various cardiac voltage-gated ion channels. We confirmed that heterologously expressed hERG currents are reduced by ibogaine in low micromolar concentrations. Moreover, at higher concentrations, the drug also reduced human Nav1.5 sodium and Cav1.2 calcium currents. Ion currents were as well reduced by 18-MC, yet with diminished potency. Unexpectedly, although blocking hERG channels, ibogaine did not prolong the action potential (AP) in guinea pig cardiomyocytes at low micromolar concentrations. Higher concentrations (≥ 10 μM) even shortened the AP. These findings can be explained by the drug's calcium channel inhibition, which counteracts the AP-prolonging effect generated by hERG blockade. Implementation of ibogaine's inhibitory effects on human ion channels in a computer model of a ventricular cardiomyocyte, on the other hand, suggested that ibogaine does prolong the AP in the human heart. We conclude that therapeutic concentrations of ibogaine have the propensity to prolong the QT interval of the electrocardiogram in humans. In some cases this may lead to cardiac arrhythmias.

  2. Inhibition of collagen synthesis by select calcium and sodium channel blockers can be mitigated by ascorbic acid and ascorbyl palmitate

    OpenAIRE

    Ivanov, Vadim; Ivanova, Svetlana; KALINOVSKY, TATIANA; NIEDZWIECKI, ALEKSANDRA; Rath, Matthias

    2016-01-01

    Calcium, sodium and potassium channel blockers are widely prescribed medications for a variety of health problems, most frequently for cardiac arrhythmias, hypertension, angina pectoris and other disorders. However, chronic application of channel blockers is associated with numerous side effects, including worsening cardiac pathology. For example, nifedipine, a calcium-channel blocker was found to be associated with increased mortality and increased risk for myocardial infarction. In addition...

  3. Berberine Reduces cAMP-Induced Chloride Secretion in T84 Human Colonic Carcinoma Cells through Inhibition of Basolateral KCNQ1 Channels.

    LENUS (Irish Health Repository)

    Alzamora, Rodrigo

    2011-01-01

    Berberine is a plant alkaloid with multiple pharmacological actions, including antidiarrhoeal activity and has been shown to inhibit Cl(-) secretion in distal colon. The aims of this study were to determine the molecular signaling mechanisms of action of berberine on Cl(-) secretion and the ion transporter targets. Monolayers of T84 human colonic carcinoma cells grown in permeable supports were placed in Ussing chambers and short-circuit current measured in response to secretagogues and berberine. Whole-cell current recordings were performed in T84 cells using the patch-clamp technique. Berberine decreased forskolin-induced short-circuit current in a concentration-dependent manner (IC(50) 80 ± 8 μM). In apically permeabilized monolayers and whole-cell current recordings, berberine inhibited a cAMP-dependent and chromanol 293B-sensitive basolateral membrane K(+) current by 88%, suggesting inhibition of KCNQ1 K(+) channels. Berberine did not affect either apical Cl(-) conductance or basolateral Na(+)-K(+)-ATPase activity. Berberine stimulated p38 MAPK, PKCα and PKA, but had no effect on p42\\/p44 MAPK and PKCδ. However, berberine pre-treatment prevented stimulation of p42\\/p44 MAPK by epidermal growth factor. The inhibitory effect of berberine on Cl(-) secretion was partially blocked by HBDDE (∼65%), an inhibitor of PKCα and to a smaller extent by inhibition of p38 MAPK with SB202190 (∼15%). Berberine treatment induced an increase in association between PKCα and PKA with KCNQ1 and produced phosphorylation of the channel. We conclude that berberine exerts its inhibitory effect on colonic Cl(-) secretion through inhibition of basolateral KCNQ1 channels responsible for K(+) recycling via a PKCα-dependent pathway.

  4. Berberine Reduces cAMP-Induced Chloride Secretion in T84 Human Colonic Carcinoma Cells through Inhibition of Basolateral KCNQ1 Channels.

    LENUS (Irish Health Repository)

    Alzamora, Rodrigo

    2012-02-01

    Berberine is a plant alkaloid with multiple pharmacological actions, including antidiarrhoeal activity and has been shown to inhibit Cl(-) secretion in distal colon. The aims of this study were to determine the molecular signaling mechanisms of action of berberine on Cl(-) secretion and the ion transporter targets. Monolayers of T84 human colonic carcinoma cells grown in permeable supports were placed in Ussing chambers and short-circuit current measured in response to secretagogues and berberine. Whole-cell current recordings were performed in T84 cells using the patch-clamp technique. Berberine decreased forskolin-induced short-circuit current in a concentration-dependent manner (IC(50) 80 +\\/- 8 muM). In apically permeabilized monolayers and whole-cell current recordings, berberine inhibited a cAMP-dependent and chromanol 293B-sensitive basolateral membrane K(+) current by 88%, suggesting inhibition of KCNQ1 K(+) channels. Berberine did not affect either apical Cl(-) conductance or basolateral Na(+)-K(+)-ATPase activity. Berberine stimulated p38 MAPK, PKCalpha and PKA, but had no effect on p42\\/p44 MAPK and PKCdelta. However, berberine pre-treatment prevented stimulation of p42\\/p44 MAPK by epidermal growth factor. The inhibitory effect of berberine on Cl(-) secretion was partially blocked by HBDDE ( approximately 65%), an inhibitor of PKCalpha and to a smaller extent by inhibition of p38 MAPK with SB202190 ( approximately 15%). Berberine treatment induced an increase in association between PKCalpha and PKA with KCNQ1 and produced phosphorylation of the channel. We conclude that berberine exerts its inhibitory effect on colonic Cl(-) secretion through inhibition of basolateral KCNQ1 channels responsible for K(+) recycling via a PKCalpha-dependent pathway.

  5. Berberine reduces cAMP-induced chloride secretion in T84 human colonic carcinoma cells through inhibition of basolateral KCNQ1 channels

    Directory of Open Access Journals (Sweden)

    Rodrigo eAlzamora

    2011-06-01

    Full Text Available Berberine is a plant alkaloid with multiple pharmacological actions, including antidiarrhoeal activity and has been shown to inhibit Cl- secretion in distal colon. The aims of this study were to determine the molecular signalling mechanisms of action of berberine on Cl- secretion and the ion transporter targets. Monolayers of T84 human colonic carcinoma cells grown in permeable supports were placed in Ussing chambers and short-circuit current measured in response to secretagogues and berberine. Whole-cell current recordings were performed in T84 cells using the patch-clamp technique. Berberine decreased forskolin-induced short-circuit current in a concentration-dependent manner (IC50 80  8 M. In apically permeabilized monolayers and whole-cell current recordings, berberine inhibited a cAMP-dependent and chromanol 293B-sensitive basolateral membrane K+ current by 88%, suggesting inhibition of KCNQ1 K+ channels. Berberine did not affect either apical Cl- conductance or basolateral Na+-K+-ATPase activity. Berberine stimulated p38 MAPK, PKC and PKA, but had no effect on p42/p44 MAPK and PKC. However, berberine pre-treatment prevented stimulation of p42/p44 MAPK by epidermal growth factor. The inhibitory effect of berberine on Cl- secretion was partially blocked by HBDDE (65 %, an inhibitor of PKC and to a smaller extent by inhibition of p38 MAPK with SB202190 (15 %. Berberine treatment induced an increase in association between PKC and PKA with KCNQ1 and produced phosphorylation of the channel. We conclude that berberine exerts its inhibitory effect on colonic Cl- secretion through inhibition of basolateral KCNQ1 channels responsible for K+ recycling via a PKC-dependent pathway.

  6. Functional diversity and evolutionary dynamics of thermoTRP channels.

    Science.gov (United States)

    Saito, Shigeru; Tominaga, Makoto

    2015-03-01

    Animals have evolved sophisticated physiological systems for sensing ambient temperature since changes in environmental temperatures affect various biological processes. Thermosensitive transient receptor potential (thermoTRP) channels serve as thermal sensors in diverse animal species. They are multimodal receptors that are activated by temperature as well as other physical and chemical stimuli. Since thermoTRP channels are calcium permeable non-selective cation channels, their activation leads to an influx of calcium and sodium ions into the cell and triggers downstream signal transduction. ThermoTRP channels have been characterized in diverse animal species over the past several years, illuminating the diversification of thermoTRP channels in the course of evolution. The gene repertoires of thermoTRP channels differ among animal species. Additionally, in some cases, the temperature and chemical sensitivities among orthologous thermoTRP channels vary among species. The evolutionary flexibility of thermoTRP channels enabled them to contribute to unique physiological systems such as infrared sensation in snakes and bats and seasonal adaptation in silk moth. On the other hand, the functional differences of thermoTRP channels among species have been utilized for understanding the molecular basis for their activation (or inhibition) mechanisms, and amino acid residues (or domains) responsible for the respective channel properties have been identified in various thermoTRP channels. Here we summarize the current understanding of the functional diversity and evolutionary dynamics of thermoTRP channels.

  7. Ghrelin inhibits proliferation and increases T-type Ca{sup 2+} channel expression in PC-3 human prostate carcinoma cells

    Energy Technology Data Exchange (ETDEWEB)

    Diaz-Lezama, Nundehui; Hernandez-Elvira, Mariana [Laboratory of Neuroendocrinology, Institute of Physiology, Autonomous University of Puebla (BUAP), Puebla (Mexico); Sandoval, Alejandro [School of Medicine FES Iztacala, National Autonomous University of Mexico (UNAM), Tlalnepantla (Mexico); Monroy, Alma; Felix, Ricardo [Department of Cell Biology, Center for Research and Advanced Studies of the National Polytechnic Institute (Cinvestav-IPN), Mexico City (Mexico); Monjaraz, Eduardo, E-mail: emguzman@siu.buap.mx [Laboratory of Neuroendocrinology, Institute of Physiology, Autonomous University of Puebla (BUAP), Puebla (Mexico)

    2010-12-03

    Research highlights: {yields} Ghrelin decreases prostate carcinoma PC-3 cells proliferation. {yields} Ghrelin favors apoptosis in PC-3 cells. {yields} Ghrelin increase in intracellular free Ca{sup 2+} levels in PC-3 cells. {yields} Grelin up-regulates expression of T-type Ca{sup 2+} channels in PC-3 cells. {yields} PC-3 cells express T-channels of the Ca{sub V}3.1 and Ca{sub V}3.2 subtype. -- Abstract: Ghrelin is a multifunctional peptide hormone with roles in growth hormone release, food intake and cell proliferation. With ghrelin now recognized as important in neoplastic processes, the aim of this report is to present findings from a series of in vitro studies evaluating the cellular mechanisms involved in ghrelin regulation of proliferation in the PC-3 human prostate carcinoma cells. The results showed that ghrelin significantly decreased proliferation and induced apoptosis. Consistent with a role in apoptosis, an increase in intracellular free Ca{sup 2+} levels was observed in the ghrelin-treated cells, which was accompanied by up-regulated expression of T-type voltage-gated Ca{sup 2+} channels. Interestingly, T-channel antagonists were able to prevent the effects of ghrelin on cell proliferation. These results suggest that ghrelin inhibits proliferation and may promote apoptosis by regulating T-type Ca{sup 2+} channel expression.

  8. Inhibitive effect of Ce(III) and La(III) cations for AA2219 aluminium alloy corrosion in sodium chloride medium

    Energy Technology Data Exchange (ETDEWEB)

    Venkatasubramanian, G., E-mail: venkatguru_1966@yahoo.co.in [Department of Chemistry, Sathyabama University, Jeppiaar Nagar, Chennai 600119 (India); Sheik Mideen, A. [Department of Chemistry, Sathyabama University, Jeppiaar Nagar, Chennai 600119 (India); Jha, Abhay K. [Materials Processing Division, Vikram Sarabhai Space Centre, Indian Space Research Organisation, Thiruvananthapuram 695022 (India); Kulandainathan, M. Anbu [Electro Organic Chemistry Division, Central Electrochemical Research Institute, Karaikudi 630006 (India)

    2014-11-14

    In this paper, the rare earth chlorides were used to mitigate the dealloying of Al{sub 2}Cu intermetallic particles in aluminium alloy 2219-T87 plate exposed to 0.6 M NaCl solution. The negative shift of cathodic branches of potentiodynamic polarisation curves revealed the cathodic inhibition towards intermetallic dealloying. The low and high frequency time constants obtained from electrochemical impedance spectra revealed an increase in the corrosion resistance due to precipitation of rare earth oxides/hydroxides on the porous surface of native oxide film of aluminium alloy. Scanning electron microscopy coupled with energy dispersive X-ray analysis and atomic force microscopy revealed that intermetallic inclusions are the sites for the formation of cerium and lanthanum deposits which suppress copper redeposition and minimise galvanic interactions between the matrix and Al{sub 2}Cu particles. - Graphical abstract: Scanning electron microscopy and atomic force microscopy reveal the intermetallic inclusions, the sites for the formation of cerium and lanthanum oxide/hydroxide precipitates which prevent intermetallic de-alloying, suppress copper redeposition and minimise galvanic interactions in aluminium alloy 2219-T87 plate. - Highlights: • Ce{sup 3+} and La{sup 3+} act as cathodic inhibitors for AA2219-T87 aluminium alloy in 0.6 M NaCl. • Al{sub 2}Cu intermetallic particles are the sites for the formation of Ce and La precipitates. • The decrease in surface potential by rare earth chlorides confirms their inhibition.

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

    Science.gov (United States)

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

    2001-04-13

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

  10. Regulation of epileptiform discharges in rat neocortex by HCN channels.

    Science.gov (United States)

    Albertson, Asher J; Williams, Sidney B; Hablitz, John J

    2013-10-01

    Hyperpolarization-activated, cyclic nucleotide-gated, nonspecific cation (HCN) channels have a well-characterized role in regulation of cellular excitability and network activity. The role of these channels in control of epileptiform discharges is less thoroughly understood. This is especially pertinent given the altered HCN channel expression in epilepsy. We hypothesized that inhibition of HCN channels would enhance bicuculline-induced epileptiform discharges. Whole cell recordings were obtained from layer (L)2/3 and L5 pyramidal neurons and L1 and L5 GABAergic interneurons. In the presence of bicuculline (10 μM), HCN channel inhibition with ZD 7288 (20 μM) significantly increased the magnitude (defined as area) of evoked epileptiform events in both L2/3 and L5 neurons. We recorded activity associated with epileptiform discharges in L1 and L5 interneurons to test the hypothesis that HCN channels regulate excitatory synaptic inputs differently in interneurons versus pyramidal neurons. HCN channel inhibition increased the magnitude of epileptiform events in both L1 and L5 interneurons. The increased magnitude of epileptiform events in both pyramidal cells and interneurons was due to an increase in network activity, since holding cells at depolarized potentials under voltage-clamp conditions to minimize HCN channel opening did not prevent enhancement in the presence of ZD 7288. In neurons recorded with ZD 7288-containing pipettes, bath application of the noninactivating inward cationic current (Ih) antagonist still produced increases in epileptiform responses. These results show that epileptiform discharges in disinhibited rat neocortex are modulated by HCN channels.

  11. A mechanism for the auto-inhibition of hyperpolarization-activated cyclic nucleotide-gated (HCN) channel opening and its relief by cAMP.

    Science.gov (United States)

    Akimoto, Madoka; Zhang, Zaiyong; Boulton, Stephen; Selvaratnam, Rajeevan; VanSchouwen, Bryan; Gloyd, Melanie; Accili, Eric A; Lange, Oliver F; Melacini, Giuseppe

    2014-08-01

    Hyperpolarization-activated cyclic nucleotide-gated (HCN) ion channels control neuronal and cardiac electrical rhythmicity. There are four homologous isoforms (HCN1-4) sharing a common multidomain architecture that includes an N-terminal transmembrane tetrameric ion channel followed by a cytoplasmic "C-linker," which connects a more distal cAMP-binding domain (CBD) to the inner pore. Channel opening is primarily stimulated by transmembrane elements that sense membrane hyperpolarization, although cAMP reduces the voltage required for HCN activation by promoting tetramerization of the intracellular C-linker, which in turn relieves auto-inhibition of the inner pore gate. Although binding of cAMP has been proposed to relieve auto-inhibition by affecting the structure of the C-linker and CBD, the nature and extent of these cAMP-dependent changes remain limitedly explored. Here, we used NMR to probe the changes caused by the binding of cAMP and of cCMP, a partial agonist, to the apo-CBD of HCN4. Our data indicate that the CBD exists in a dynamic two-state equilibrium, whose position as gauged by NMR chemical shifts correlates with the V½ voltage measured through electrophysiology. In the absence of cAMP, the most populated CBD state leads to steric clashes with the activated or "tetrameric" C-linker, which becomes energetically unfavored. The steric clashes of the apo tetramer are eliminated either by cAMP binding, which selects for a CBD state devoid of steric clashes with the tetrameric C-linker and facilitates channel opening, or by a transition of apo-HCN to monomers or dimer of dimers, in which the C-linker becomes less structured, and channel opening is not facilitated.

  12. Characterization of Kbot21 Reveals Novel Side Chain Interactions of Scorpion Toxins Inhibiting Voltage-Gated Potassium Channels.

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    Rym ElFessi-Magouri

    Full Text Available Scorpion toxins are important pharmacological tools for probing the physiological roles of ion channels which are involved in many physiological processes and as such have significant therapeutic potential. The discovery of new scorpion toxins with different specificities and affinities is needed to further characterize the physiology of ion channels. In this regard, a new short polypeptide called Kbot21 has been purified to homogeneity from the venom of Buthus occitanus tunetanus scorpion. Kbot21 is structurally related to BmBKTx1 from the venom of the Asian scorpion Buthus martensii Karsch. These two toxins differ by only two residues at position 13 (R /V and 24 (D/N.Despite their very similar sequences, Kbot21 and BmBKTx1 differ in their electrophysiological activities. Kbot21 targets KV channel subtypes whereas BmBKTx1 is active on both big conductance (BK and small conductance (SK Ca2+-activated K+ channel subtypes, but has no effects on Kv channel subtypes. The docking model of Kbot21 with the Kv1.2 channel shows that the D24 and R13 side-chain of Kbot21 are critical for its interaction with KV channels.

  13. Characterization of Kbot21 Reveals Novel Side Chain Interactions of Scorpion Toxins Inhibiting Voltage-Gated Potassium Channels.

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    ElFessi-Magouri, Rym; Peigneur, Steve; Othman, Houcemeddine; Srairi-Abid, Najet; ElAyeb, Mohamed; Tytgat, Jan; Kharrat, Riadh

    2015-01-01

    Scorpion toxins are important pharmacological tools for probing the physiological roles of ion channels which are involved in many physiological processes and as such have significant therapeutic potential. The discovery of new scorpion toxins with different specificities and affinities is needed to further characterize the physiology of ion channels. In this regard, a new short polypeptide called Kbot21 has been purified to homogeneity from the venom of Buthus occitanus tunetanus scorpion. Kbot21 is structurally related to BmBKTx1 from the venom of the Asian scorpion Buthus martensii Karsch. These two toxins differ by only two residues at position 13 (R /V) and 24 (D/N).Despite their very similar sequences, Kbot21 and BmBKTx1 differ in their electrophysiological activities. Kbot21 targets KV channel subtypes whereas BmBKTx1 is active on both big conductance (BK) and small conductance (SK) Ca2+-activated K+ channel subtypes, but has no effects on Kv channel subtypes. The docking model of Kbot21 with the Kv1.2 channel shows that the D24 and R13 side-chain of Kbot21 are critical for its interaction with KV channels.

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

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

    2010-01-22

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

  15. Upregulated Expression of Transient Receptor Potential Cation Channel Subfamily V Receptors in Mucosae of Patients with Oral Squamous Cell Carcinoma and Patients with a History of Alcohol Consumption or Smoking

    Science.gov (United States)

    Sakakibara, Akiko; Sakakibara, Shunsuke; Kusumoto, Junya; Takeda, Daisuke; Hasegawa, Takumi; Akashi, Masaya; Minamikawa, Tsutomu; Hashikawa, Kazunobu; Terashi, Hiroto; Komori, Takahide

    2017-01-01

    Objectives Transient receptor potential cation channel (subfamily V, members 1–4) (TRPV1–4) are expressed in skin and neurons and activated by external stimuli in normal mucosae of all oral cavity sites. The oral cavity is exposed to various stimuli, including temperature, mechanical stimuli, chemical substances, and changes in pH, and, notably, the risk factors for oncogenic transformation in oral squamous epithelium are the same as the external stimuli received by TRPV1–4 receptors. Hence, we examined the relationship between oral squamous cell carcinoma (SCC) and TRPV1–4 expression. Materials and Methods Oral SCC patients (n = 37) who underwent surgical resection were included in this study. We investigated the expression of TRPV1–4 by immunohistochemical staining and quantification of TRPV1–4 mRNA in human oral mucosa. In addition, we compared the TRPV1–4 levels in mucosa from patients with SCC to those in normal oral mucosa. Results The receptors were expressed in oral mucosa at all sites (tongue, buccal mucosa, gingiva, and oral floor) and the expression was stronger in epithelia from patients with SCC than in normal epithelia. Furthermore, alcohol consumption and tobacco use were strongly associated with the occurrence of oral cancer and were found to have a remarkable influence on TRPV1–4 receptor expression in normal oral mucosa. In particular, patients with a history of alcohol consumption demonstrated significantly higher expression levels. Conclusion Various external stimuli may influence the behavior of cancer cells. Overexpression of TRPV1-4 is likely to be a factor in enhanced sensitivity to external stimuli. These findings could contribute to the establishment of novel strategies for cancer therapy or prevention. PMID:28081185

  16. Selective inhibition of the Kir2 family of inward rectifier potassium channels by a small molecule probe: the discovery, SAR and pharmacological characterization of ML133

    Science.gov (United States)

    Wang, Hao-Ran; Wu, Meng; Yu, Haibo; Long, Shunyou; Stevens, Amy; Engers, Darren W.; Sackin, Henry; Daniels, J. Scott; Dawson, Eric S.; Hopkins, Corey R.; Lindsley, Craig W.; Li, Min; McManus, Owen B

    2011-01-01

    The Kir inward rectifying potassium channels have a broad tissue distribution and are implicated in a variety of functional roles. At least seven classes (Kir1 – Kir7) of structurally related inward rectifier potassium channels are known, and there are no selective small molecule tools to study their function. In an effort to develop selective Kir2.1 inhibitors, we performed a high-throughput screen (HTS) of more than 300,000 small molecules within the MLPCN for modulators of Kir2.1 function. Here we report one potent Kir2.1 inhibitor, ML133, which inhibits Kir2.1 with IC50 of 1.8 μM at pH 7.4 and 290 nM at pH 8.5, but exhibits little selectivity against other members of Kir2.x family channels. However, ML133 has no effect on Kir1.1 (IC50 > 300 μM), and displays weak activity for Kir4.1 (76 μM) and Kir7.1 (33 μM), making ML133 the most selective small molecule inhibitor of the Kir family reported to date. Due to the high homology within the Kir family, the channels share a common design of a pore region flanked by two transmembrane domains, identification of site(s) critical for isoform specificity would be an important basis for future development of more specific and potent Kir inhibitors. Using chimeric channels between Kir2.1 and Kir1.1 and site-directed mutagenesis, we have identified D172 and I176 within M2 segment of Kir2.1 as molecular determinants critical for the potency of ML133 mediated inhibition. Double mutation of the corresponding residues of Kir1.1 to those of Kir2.1 (N171D and C175I) transplants ML133 inhibition to Kir1.1. Together, the combination of a potent, Kir2 family selective inhibitor and identification of molecular determinants for the specificity provides both a tool and a model system to enable further mechanistic studies of modulation of Kir2 inward rectifier potassium channels. PMID:21615117

  17. Lutein inhibits the function of the transient receptor potential A1 ion channel in different in vitro and in vivo models.

    Science.gov (United States)

    Horváth, Györgyi; Szoke, Éva; Kemény, Ágnes; Bagoly, Teréz; Deli, József; Szente, Lajos; Pál, Szilárd; Sándor, Katalin; Szolcsányi, János; Helyes, Zsuzsanna

    2012-01-01

    Transient receptor potential (TRP) ion channels, such as TRP vanilloid 1 and ankyrin repeat domain 1 (TRPV1 and TRPA1), are expressed on primary sensory neurons. Lutein, a natural tetraterpene carotenoid, can be incorporated into membranes and might modulate TRP channels. Therefore, the effects of the water-soluble randomly methylated-β-cyclodextrin (RAMEB) complex of lutein were investigated on TRPV1 and TRPA1 activation. RAMEB-lutein (100 μM) significantly diminished Ca(2+) influx to cultured rat trigeminal neurons induced by TRPA1 activation with mustard oil, but not by TRPV1 stimulation with capsaicin, as determined with microfluorimetry. Calcitonin gene-related peptide release from afferents of isolated tracheae evoked by mustard oil, but not by capsaicin, was inhibited by RAMEB-lutein. Mustard oil-induced neurogenic mouse ear swelling was also significantly decreased by 100 μg/ml s.c. RAMEB-lutein pretreatment, while capsaicin-evoked edema was not altered. Myeloperoxidase activity indicating non-neurogenic granulocyte accumulation in the ear was not influenced by RAMEB-lutein in either case. It is concluded that lutein inhibits TRPA1, but not TRPV1 stimulation-induced responses on cell bodies and peripheral terminals of sensory neurons in vitro and in vivo. Based on these distinct actions and the carotenoid structure, the ability of lutein to modulate lipid rafts in the membrane around TRP channels can be suggested.

  18. Inhibition of Voltage-Gated Calcium Channels After Subchronic and Repeated Exposure of PC12 Cells to Different Classes of Insecticides.

    Science.gov (United States)

    Meijer, Marieke; Brandsema, Joske A R; Nieuwenhuis, Desirée; Wijnolts, Fiona M J; Dingemans, Milou M L; Westerink, Remco H S

    2015-10-01

    We previously demonstrated that acute inhibition of voltage-gated calcium channels (VGCCs) is a common mode of action for (sub)micromolar concentrations of chemicals, including insecticides. However, because human exposure to chemicals is usually chronic and repeated, we investigated if selected insecticides from different chemical classes (organochlorines, organophosphates, pyrethroids, carbamates, and neonicotinoids) also disturb calcium homeostasis after subchronic (24 h) exposure and after a subsequent (repeated) acute exposure. Effects on calcium homeostasis were investigated with single-cell fluorescence (Fura-2) imaging of PC12 cells. Cells were depolarized with high-K(+) saline to study effects of subchronic or repeated exposure on VGCC-mediated Ca(2+) influx. The results demonstrate that except for carbaryl and imidacloprid, all selected insecticides inhibited depolarization (K(+))-evoked Ca(2+) influx after subchronic exposure (IC50's: approximately 1-10 µM) in PC12 cells. These inhibitory effects were not or only slowly reversible. Moreover, repeated exposure augmented the inhibition of the K(+)-evoked increase in intracellular calcium concentration induced by subchronic exposure to cypermethrin, chlorpyrifos, chlorpyrifos-oxon, and endosulfan (IC50's: approximately 0.1-4 µM). In rat primary cortical cultures, acute and repeated chlorpyrifos exposure also augmented inhibition of VGCCs compared with subchronic exposure. In conclusion, compared with subchronic exposure, repeated exposure increases the potency of insecticides to inhibit VGCCs. However, the potency of insecticides to inhibit VGCCs upon repeated exposure was comparable with the inhibition previously observed following acute exposure, with the exception of chlorpyrifos. The data suggest that an acute exposure paradigm is sufficient for screening chemicals for effects on VGCCs and that PC12 cells are a sensitive model for detection of effects on VGCCs.

  19. The use of amlodipine, but not of P-glycoprotein inhibiting calcium channel blockers is associated with clopidogrel poor-response.

    Science.gov (United States)

    Harmsze, Ankie M; Robijns, Karen; van Werkum, Jochem W; Breet, Nicoline J; Hackeng, Christian M; Ten Berg, Jurrien M; Ruven, Hendrik J T; Klungel, Olaf H; de Boer, Anthonius; Deneer, Vera H M

    2010-05-01

    Clopidogrel is a prodrug that has to be converted in vivo to its active metabolite by cytochrome (CYP)P450 iso-enzymes. As calcium channel blockers (CCBs) are inhibitors of CYP3A4, concomitant use of these drugs might play a role in the wide inter-individual variability in the response to clopidogrel. However, some CCBs also have strong inhibitory effects on the drug transporter P-glycoprotein (Pgp), which mediates clopidogrel's intestinal absorption. It was the aim of this study to evaluate the effect of co-administration of Pgp-inhibiting and non-Pgp-inhibiting CCBs on on-clopidogrel platelet reactivity in patients on dual antiplatelet therapy undergoing elective percutaneous coronary intervention (PCI). In a total of 623 consecutive patients undergoing elective PCI treated with clopidogrel and aspirin, platelet reactivity to 5 and 20 muM adenosine diphospate (ADP) and clopidogrel poor-response (defined as > 70% platelet aggregation to 20 muM ADP) were evaluated by light transmittance aggregometry. A total of 222 patients (35.6%) were on CCB treatment, of which 98 used Pgp-inhibiting CCBs (verapamil, nifedipine, diltiazem, barnidipine) and 124 patients used the non-Pgp-inhibiting CCB amlodipine. Adjusted mean ADP-induced on-clopidogrel platelet reactivity was significantly higher in both users of Pgp-inhibiting CCBs and amlodipine as compared to CCB non-users (all p<0.05). However, only the use of amlodipine was significantly associated with a 2.3-fold increased risk of clopidogrel poor-response. This study demonstrates that concomitant use of Pgp-inhibiting CCBs and amlodipine increases on-clopidogrel platelet reactivity. Only amlodipine was associated with clopidogrel poor-response. The drug-drug interaction between clopidogrel and amlodipine might be more clinically relevant as compared to P-glycoprotein-inhibiting CCBs.

  20. Rebaudioside A directly stimulates insulin secretion from pancreatic beta cells: a glucose-dependent action via inhibition of ATP-sensitive K-channels.

    Science.gov (United States)

    Abudula, R; Matchkov, V V; Jeppesen, P B; Nilsson, H; Aalkjaer, C; Hermansen, K

    2008-11-01

    Recently, we showed that rebaudioside A potently stimulates the insulin secretion from isolated mouse islets in a dose-, glucose- and Ca(2+)-dependent manner. Little is known about the mechanisms underlying the insulinotropic action of rebaudioside A. The aim of this study was to define the signalling system by which, rebaudioside A acts. Isolated mouse islets were used in the cAMP[(125)I] scintillation proximity assay to measure total cAMP level, and in a luminometric method to measure intracellular ATP and ADP concentrations. Conventional and permeabilized whole-cell configuration of the patch-clamp technique was used to verify the effect of rebaudioside A on ATP-sensitive K(+)-channels from dispersed single beta cells from isolated mouse islets. Insulin was measured by radioimmunoassay from insulinoma MIN6 cells. In the presence of 16.7 mM glucose, the addition of the maximally effective concentration of rebaudioside A (10(-9) M) increased the ATP/ADP ratio significantly, while it did not change the intracellular cAMP level. Rebaudioside A (10(-9) M) and stevioside (10(-6) M) reduced the ATP-sensitive potassium channel (K(ATP)) conductance in a glucose-dependent manner. Moreover, rebaudioside A stimulated the insulin secretion from MIN6 cells in a dose- and glucose-dependent manner. In conclusion, the insulinotropic effect of rebaudioside A is mediated via inhibition of ATP-sensitive K(+)-channels and requires the presence of high glucose. The inhibition of ATP-sensitive K(+)-channels is probably induced by changes in the ATP/ADP ratio. The results indicate that rebaudioside A may offer a distinct therapeutic advantage over sulphonylureas because of less risk of causing hypoglycaemia.

  1. Inhibition of human ether-a-go-go-related gene potassium channels by alpha 1-adrenoceptor antagonists prazosin, doxazosin, and terazosin.

    Science.gov (United States)

    Thomas, Dierk; Wimmer, Anna-Britt; Wu, Kezhong; Hammerling, Bettina C; Ficker, Eckhard K; Kuryshev, Yuri A; Kiehn, Johann; Katus, Hugo A; Schoels, Wolfgang; Karle, Christoph A

    2004-05-01

    Human ether-a-go-go-related gene (HERG) potassium channels are expressed in multiple tissues including the heart and adenocarcinomas. In cardiomyocytes, HERG encodes the alpha-subunit underlying the rapid component of the delayed rectifier potassium current, I(Kr), and pharmacological reduction of HERG currents may cause acquired long QT syndrome. In addition, HERG currents have been shown to be involved in the regulation of cell proliferation and apoptosis. Selective alpha 1-adrenoceptor antagonists are commonly used in the treatment of hypertension and benign prostatic hyperplasia. Recently, doxazosin has been associated with an increased risk of heart failure. Moreover, quinazoline-derived alpha 1-inhibitors induce apoptosis in cardiomyocytes and prostate tumor cells independently of alpha1-adrenoceptor blockade. To assess the action of the effects of prazosin, doxazosin, and terazosin on HERG currents, we investigated their acute electrophysiological effects on cloned HERG potassium channels heterologously expressed in Xenopus oocytes and HEK 293 cells.Prazosin, doxazosin, and terazosin blocked HERG currents in Xenopus oocytes with IC(50) values of 10.1, 18.2, and 113.2 microM respectively, whereas the IC(50) values for HERG channel inhibition in human HEK 293 cells were 1.57 microM, 585.1 nM, and 17.7 microM. Detailed biophysical studies revealed that inhibition by the prototype alpha 1-blocker prazosin occurred in closed, open, and inactivated channels. Analysis of the voltage-dependence of block displayed a reduction of inhibition at positive membrane potentials. Frequency-dependence was not observed. Prazosin caused a negative shift in the voltage-dependence of both activation (-3.8 mV) and inactivation (-9.4 mV). The S6 mutations Y652A and F656A partially attenuated (Y652A) or abolished (F656A) HERG current blockade, indicating that prazosin binds to a common drug receptor within the pore-S6 region. In conclusion, this study demonstrates that HERG

  2. Apelin-13 inhibits large-conductance Ca2+-activated K+ channels in cerebral artery smooth muscle cells via a PI3-kinase dependent mechanism.

    Directory of Open Access Journals (Sweden)

    Amit Modgil

    Full Text Available Apelin-13 causes vasoconstriction by acting directly on APJ receptors in vascular smooth muscle (VSM cells; however, the ionic mechanisms underlying this action at the cellular level remain unclear. Large-conductance Ca(2+-activated K(+ (BKCa channels in VSM cells are critical regulators of membrane potential and vascular tone. In the present study, we examined the effect of apelin-13 on BK(Ca channel activity in VSM cells, freshly isolated from rat middle cerebral arteries. In whole-cell patch clamp mode, apelin-13 (0.001-1 μM caused concentration-dependent inhibition of BK(Ca in VSM cells. Apelin-13 (0.1 µM significantly decreased BK(Ca current density from 71.25 ± 8.14 pA/pF to 44.52 ± 7.10 pA/pF (n=14 cells, P<0.05. This inhibitory effect of apelin-13 was confirmed by single channel recording in cell-attached patches, in which extracellular application of apelin-13 (0.1 µM decreased the open-state probability (NPo of BK(Ca channels in freshly isolated VSM cells. However, in inside-out patches, extracellular application of apelin-13 (0.1 µM did not alter the NPo of BK(Ca channels, suggesting that the inhibitory effect of apelin-13 on BKCa is not mediated by a direct action on BK(Ca. In whole cell patches, pretreatment of VSM cells with LY-294002, a PI3-kinase inhibitor, markedly attenuated the apelin-13-induced decrease in BK(Ca current density. In addition, treatment of arteries with apelin-13 (0.1 µM significantly increased the ratio of phosphorylated-Akt/total Akt, indicating that apelin-13 significantly increases PI3-kinase activity. Taken together, the data suggest that apelin-13 inhibits BK(Ca channel via a PI3-kinase-dependent signaling pathway in cerebral artery VSM cells, which may contribute to its regulatory action in the control of vascular tone.

  3. Anti-metastatic Potential of Amide-linked Local Anesthetics: Inhibition of Lung Adenocarcinoma Cell Migration and Inflammatory Src Signaling Independent of Sodium Channel Blockade

    Science.gov (United States)

    Piegeler, Tobias; Votta-Velis, E. Gina; Liu, Guoquan; Place, Aaron T.; Schwartz, David E.; Beck-Schimmer, Beatrice; Minshall, Richard D.; Borgeat, Alain

    2012-01-01

    Background Retrospective analysis of patients undergoing cancer surgery suggests the use of regional anesthesia may reduce cancer recurrence and improve survival. Amide-linked local anesthetics have anti-inflammatory properties, although the mechanism of action in this regard is unclear. As inflammatory processes involving Src tyrosine protein kinase and intercellular adhesion molecule-1 are important in tumor growth and metastasis, we hypothesized that amide-linked local anesthetics may inhibit inflammatory Src-signaling involved in migration of adenocarcinoma cells. Methods NCI-H838 lung cancer cells were incubated with Tumor Necrosis Factor-α in absence/presence of ropivacaine, lidocaine, or chloroprocaine (1nM-100μM). Cell migration and total cell lysate Src-activation and Intercellular Adhesion Molecule-1 phosphorylation were assessed. The role of voltage-gated sodium-channels in the mechanism of local anesthetic effects was also evaluated. Results Ropivacaine treatment (100μM) of H838 cells for 20 minutes decreased basal Src activity by 62% (p=0.003), and both ropivacaine and lidocaine co-administered with Tumor Necrosis Factor-α statistically significantly decreased Src-activation and Intercellular Adhesion Molecule-1 phosphorylation, whereas chloroprocaine had no such effect. Migration of these cells at 4 hours was inhibited by 26% (p=0.005) in presence of 1μM ropivacaine and 21% by 1μM lidocaine (p=0.004). These effects of ropivacaine and lidocaine were independent of voltage-gated sodium-channel inhibition. Conclusions This study indicates that amide-, but not ester-linked local anesthetics may provide beneficial anti-metastatic effects. The observed inhibition of NCI-H838 cell migration by lidocaine and ropivacaine was associated with the inhibition of Tumor Necrosis Factor-α-induced Src-activation and Intercellular Adhesion Molecule-1 phosphorylation, providing the first evidence of a molecular mechanism which appears to be independent of their

  4. Inhibition of Intermediate-Conductance Calcium-Activated K Channel (KCa3.1) and Fibroblast Mitogenesis by α-Linolenic Acid and Alterations of Channel Expression in the Lysosomal Storage Disorders, Fabry Disease, and Niemann Pick C

    Science.gov (United States)

    Oliván-Viguera, Aida; Lozano-Gerona, Javier; López de Frutos, Laura; Cebolla, Jorge J.; Irún, Pilar; Abarca-Lachen, Edgar; García-Malinis, Ana J.; García-Otín, Ángel Luis; Gilaberte, Yolanda; Giraldo, Pilar; Köhler, Ralf

    2017-01-01

    The calcium/calmodulin-gated KCa3.1 channel regulates normal and abnormal mitogenesis by controlling K+-efflux, cell volume, and membrane hyperpolarization-driven calcium-entry. Recent studies suggest modulation of KCa3.1 by omega-3 fatty acids as negative modulators and impaired KCa3.1 functions in the inherited lysosomal storage disorder (LSD), Fabry disease (FD). In the first part of present study, we characterize KCa3.1 in murine and human fibroblasts and test the impact of omega-3 fatty acids on fibroblast proliferation. In the second, we study whether KCa3.1 is altered in the LSDs, FD, and Niemann-Pick disease type C (NPC). Our patch-clamp and mRNA-expression studies on murine and human fibroblasts show functional expression of KCa3.1. KCa currents display the typical pharmacological fingerprint of KCa3.1: Ca2+-activation, potentiation by the positive-gating modulators, SKA-31 and SKA-121, and inhibition by TRAM-34, Senicapoc (ICA-17043), and the negative-gating modulator, 13b. Considering modulation by omega-3 fatty acids we found that α-linolenic acid (α-LA) and docosahexanenoic acid (DHA) inhibit KCa3.1 currents and strongly reduce fibroblast growth. The α-LA-rich linseed oil and γ-LA-rich borage oil at 0.5% produce channel inhibition while α-LA/γ-LA-low oils has no anti-proliferative effect. Concerning KCa3.1 in LSD, mRNA expression studies, and patch-clamp on primary fibroblasts from FD and NPC patients reveal lower KCa3.1-gene expression and membrane expression than in control fibroblasts. In conclusion, the omega-3 fatty acid, α-LA, and α-LA/γ-LA-rich plant oils, inhibit fibroblast KCa3.1 channels and mitogenesis. Reduced fibroblast KCa3.1 functions are a feature and possible biomarker of cell dysfunction in FD and NPC and supports the concept that biased lipid metabolism is capable of negatively modulating KCa3.1 expression. PMID:28197106

  5. Magnolol inhibits colonic motility through down-regulation of voltage-sensitive L-type Ca2+ channels of colonic smooth muscle cells in rats.

    Science.gov (United States)

    Zhang, Man; Zang, Kai-Hong; Luo, Jia-Lie; Leung, Fung-Ping; Huang, Yu; Lin, Cheng-Yuan; Yang, Zhi-Jun; Lu, Ai-Ping; Tang, Xu-Dong; Xu, Hong-Xi; Sung, Joseph Jao-yiu; Bian, Zhao-Xiang

    2013-11-15

    This study aimed to investigate the effect of magnolol (5,5'-diallyl-2,2'-biphenyldiol) on contraction in distal colonic segments of rats and the underlying mechanisms. Colonic segments were mounted in organ baths for isometric force measurement. Whole-cell voltage-sensitive L-type Ca(2+) currents were recorded on isolated single colonic smooth muscle cells using patch-clamp technique. The spontaneous contractions and acetylcholine (ACh)- and Bay K 8644-induced contractions were inhibited by magnolol (3-100 μM). In the presence of Bay K8644 (100 nM), magnolol (10-100 μM) inhibited the contraction induced by 10 μM ACh. By contrast, tetrodotoxin (100 nM) and Nώ-nitro-L-arginine methyl ester (L-NAME 100 μM) did not change the inhibitory effect of magnolol (10 μM). In addition, magnolol (3-100 μM) inhibited the L-type Ca(2+) currents. The present results suggest that magnolol inhibits colonic smooth muscle contraction through downregulating L-type Ca(2+) channel activity.

  6. The Methanolic Extract from Murraya koenigii L. Inhibits Glutamate-Induced Pain and Involves ATP-Sensitive K+ Channel as Antinociceptive Mechanism

    Science.gov (United States)

    Sharmin Ani, Nushrat; Chakraborty, Sudip

    2016-01-01

    Murraya koenigii L. is a perennial shrub, belonging to the family Rutaceae. Traditionally, the leaves of this plant are extensively used in treatment of a wide range of diseases and disorders including pain and inflammation. Although researchers have revealed the antinociceptive effects of this plant's leaves during past few years, the mechanisms underlying these effects are still unknown. Therefore, the present study evaluated some antinociceptive mechanisms of the methanolic extract of M. koenigii (MEMK) leaves along with its antinociceptive potential using several animal models. The antinociceptive effects of MEMK were evaluated using formalin-induced licking and acetic acid-induced writhing tests at the doses of 50, 100, and 200 mg/kg. In addition, we also justified the possible participations of glutamatergic system and ATP-sensitive potassium channels in the observed activities. Our results demonstrated that MEMK significantly (p < 0.01) inhibited the pain thresholds induced by formalin and acetic acid in a dose-dependent manner. MEMK also significantly (p < 0.01) suppressed glutamate-induced pain. Moreover, pretreatment with glibenclamide (an ATP-sensitive potassium channel blocker) at 10 mg/kg significantly (p < 0.05) reversed the MEMK-mediated antinociception. These revealed that MEMK might have the potential to interact with glutamatergic system and the ATP-sensitive potassium channels to exhibit its antinociceptive activities. Therefore, our results strongly support the antinociceptive effects of M. koenigii leaves and provide scientific basis of their analgesic uses in the traditional medicine. PMID:27812367

  7. Inhibition by 5-N-(4-chlorobenzyl)-2`,4`-dimethylbenzamil of Na{sup +}/Ca{sup 2+} exchange and L-type Ca{sup 2+} channels in isolated cardiomyocytes

    Energy Technology Data Exchange (ETDEWEB)

    Sharikabad, M.N.; Broers, O. [Ullevaal Univ. Hospital, Clinical Chemistry dept., Div. of Clinical Pharmacology and Toxicology, Olso (Norway); Cragoe, E.J. Jr.

    1997-02-01

    The inhibitory effect of the amiloride derivative 5-N-(4-chlorobenzyl)-2`,4`-dimethylbenzamil (CBDMB) on calcium (Ca{sup 2+}) uptake via sarcolemmal sodium-calcium (Na{sup +}/Ca{sup 2+}) exchange and L-type Ca{sup 2+} channels was investigated in isolated adult rat ventricular cardiomyocytes under depolarizing conditions in cells preincubated with 1 mM ouabain or 137 mM lithium (Li{sup +}), respectively. Fifteen or 120 min. preincubation with CBDMB inhibited Ca{sup 2+} uptake via Na{sup +}/Ca{sup 2+} exchange in Na{sup +}-loaded depolarized cells completely at 100 {mu}M with an IC{sub 50} of 21 {mu}M. After 120 min. preincubation, CBDMB inhibited Ca{sup 2+} uptake via L-type Ca{sup 2+} channels by 75.1{+-}8.1% (mean and S.E.M.) and IC{sub 50} of 4 {mu}M, whereas no significant inhibition was observed after 15 min. preincubation. (+)-Isradipine (10 {mu}3M) inhibited high potassium (K{sup +}) induced Ca{sup 2+} uptake via L-type Ca{sup 2+} channels by 35% after 15 min. and by 70% after 120 min. preincubation. Inhibition by CBDMB of specific (+)-[{sup 3}]isradipine binding to L-type Ca{sup 2+} channels showed similar concentration dependency as inhibition of Ca{sup 2+} uptake via L-type Ca{sup 2+} channels. In conclusio, CBDMB inhibits sarcolemmal Na{sup +}/Ca{sup 2+} exchange in rat ventricular cardiomyocytes rapidly. However, after longer preincubation periods, L-type Ca{sup 2+} channels are inhibited as well and with higher potency than Na{sup +}/Ca{sup 2+} exchange. (au) 17 refs.

  8. Cationic PAMAM dendrimers as pore-blocking binary toxin inhibitors.

    Science.gov (United States)

    Förstner, Philip; Bayer, Fabienne; Kalu, Nnanya; Felsen, Susanne; Förtsch, Christina; Aloufi, Abrar; Ng, David Y W; Weil, Tanja; Nestorovich, Ekaterina M; Barth, Holger

    2014-07-14

    Dendrimers are unique highly branched macromolecules with numerous groundbreaking biomedical applications under development. Here we identified poly(amido amine) (PAMAM) dendrimers as novel blockers for the pore-forming B components of the binary anthrax toxin (PA63) and Clostridium botulinum C2 toxin (C2IIa). These pores are essential for delivery of the enzymatic A components of the internalized toxins from endosomes into the cytosol of target cells. We demonstrate that at low μM concentrations cationic PAMAM dendrimers block PA63 and C2IIa to inhibit channel-mediated transport of the A components, thereby protecting HeLa and Vero cells from intoxication. By channel reconstitution and high-resolution current recording, we show that the PAMAM dendrimers obstruct transmembrane PA63 and C2IIa pores in planar lipid bilayers at nM concentrations. These findings suggest a new potential role for the PAMAM dendrimers as effective polyvalent channel-blocking inhibitors, which can protect human target cells from intoxication with binary toxins from pathogenic bacteria.

  9. Inhibition of Nav1.7 channels by methyl eugenol as a mechanism underlying its antinociceptive and anesthetic actions

    OpenAIRE

    Wang, Ze-Jun; Tabakoff, Boris; Levinson, Simon R.; Heinbockel, Thomas

    2015-01-01

    Aim: Methyl eugenol is a major active component extracted from the Chinese herb Asari Radix et Rhizoma, which has been used to treat toothache and other pain. Previous in vivo studies have shown that methyl eugenol has anesthetic and antinociceptive effects. The aim of this study was to determine the possible mechanism underlying its effect on nervous system disorders. Methods: The direct interaction of methyl eugenol with Na+ channels was explored and characterized using electrophysiological...

  10. Lack of conventional ATPase properties in CFTR chloride channel gating.

    Science.gov (United States)

    Schultz, B D; Bridges, R J; Frizzell, R A

    1996-05-01

    CFTR shares structural homology with the ABC transporter superfamily of proteins which hydrolyze ATP to effect the transport of compounds across cell membranes. Some superfamily members are characterized as P-type ATPases because ATP-dependent transport is sensitive to the presence of vanadate. It has been widely postulated that CFTR hydrolyzes ATP to gate its chloride channel. However, direct evidence of CFTR hydrolytic activity in channel gating is lacking and existing circumstantial evidence is contradictory. Therefore, we evaluated CFTR chloride channel activity under conditions known to inhibit the activity of ATPases; i.e., in the absence of divalent cations and in the presence of a variety of ATPase inhibitors. Removal of the cytosolic cofactor, Mg2+, reduced both the opening and closing rates of CFTR suggesting that Mg2+ plays a modulatory role in channel gating. However, channels continued to both open and close showing that Mg2+ is not an absolute requirement for channel activity. The nonselective P-type ATPase inhibitor, vanadate, did not alter the gating of CFTR when used at concentrations which completely inhibit the activity of other ABC transporters (1 mM). Higher concentrations of vanadate (10 mM) blocked the closing of CFTR, but did not affect the opening of the channel. As expected, more selective P-type (Sch28080, ouabain), V-type (bafilomycin A1, SCN-) and F-type (oligomycin) ATPase inhibitors did not affect either the opening or closing of CFTR. Thus, CFTR does not share a pharmacological inhibition profile with other ATPases and channel gating occurs in the apparent absence of hydrolysis, although with altered kinetics. Vanadate inhibition of channel closure might suggest that a hydrolytic step is involved although the requirement for a high concentration raises the possibility of previously uncharacterized effects of this compound. Most conservatively, the requirement for high concentrations of vanadate demonstrates that the binding site for

  11. Downregulation of the Ca(2+)-activated K(+) channel KC a3.1 by histone deacetylase inhibition in human breast cancer cells.

    Science.gov (United States)

    Ohya, Susumu; Kanatsuka, Saki; Hatano, Noriyuki; Kito, Hiroaki; Matsui, Azusa; Fujimoto, Mayu; Matsuba, Sayo; Niwa, Satomi; Zhan, Peng; Suzuki, Takayoshi; Muraki, Katsuhiko

    2016-04-01

    The intermediate-conductance Ca(2+)-activated K(+) channel KC a3.1 is involved in the promotion of tumor growth and metastasis, and is a potential therapeutic target and biomarker for cancer. Histone deacetylase inhibitors (HDACis) have considerable potential for cancer therapy, however, the effects of HDACis on ion channel expression have not yet been investigated in detail. The results of this study showed a significant decrease in KC a3.1 transcription by HDAC inhibition in the human breast cancer cell line YMB-1, which functionally expresses KCa3.1. A treatment with the clinically available, class I, II, and IV HDAC inhibitor, vorinostat significantly downregulated KC a3.1 transcription in a concentration-dependent manner, and the plasmalemmal expression of the KC a3.1 protein and its functional activity were correspondingly decreased. Pharmacological and siRNA-based HDAC inhibition both revealed the involvement of HDAC2 and HDAC3 in KC a3.1 transcription through the same mechanism. The downregulation of KC a3.1 in YMB-1 was not due to the upregulation of the repressor element-1 silencing transcription factor, REST and the insulin-like growth factor-binding protein 5, IGFBP5. The significant decrease in KC a3.1 transcription by HDAC inhibition was also observed in the KC a3.1-expressing human prostate cancer cell line, PC-3. These results suggest that vorinostat and the selective HDACis for HDAC2 and/or HDAC3 are effective drug candidates for KC a3.1-overexpressing cancers.

  12. Inhibition of inward K+ channels and stomatal response by abscisic acid: an intracellular locus of phytohormone action.

    Science.gov (United States)

    Schwartz, A; Wu, W H; Tucker, E B; Assmann, S M

    1994-04-26

    Abscisic acid (ABA), a plant hormone whose production is stimulated by water stress, reduces the apertures of stomatal pores in the leaf surface, thereby lessening transpirational water loss. It has been thought that inhibition of stomatal opening and promotion of stomatal closure by ABA are initiated by the binding of extracellular ABA to a receptor located in the guard-cell plasma membrane. However, in the present research, we employ three distinct experimental approaches to demonstrate that ABA can act from within guard cells to regulate stomatal apertures. (i) The extent to which ABA inhibits stomatal opening and promotes stomatal closure in Commelina communis L. is proportional to the extent of ABA uptake, as assayed with [3H]ABA. (ii) Direct microinjection of ABA into the cytoplasm of Commelina guard cells precipitates stomatal closure. (iii) Application of ABA to the cytosol of Vicia faba L. guard-cell protoplasts via patch-clamp techniques inhibits inward K+ currents, an effect sufficient to inhibit stomatal opening. These results demonstrate an intracellular locus of phytohormone action and imply that the search for hormone receptor proteins should be extended to include intracellular compartments.

  13. Inhibition of the K+ channel K(Ca3.1 reduces TGF-β1-induced premature senescence, myofibroblast phenotype transition and proliferation of mesangial cells.

    Directory of Open Access Journals (Sweden)

    Rong-Guo Fu

    Full Text Available OBJECTIVE: K(Ca3.1 channel participates in many important cellular functions. This study planned to investigate the potential involvement of K(Ca3.1 channel in premature senescence, myofibroblast phenotype transition and proliferation of mesangial cells. METHODS & MATERIALS: Rat mesangial cells were cultured together with TGF-β1 (2 ng/ml and TGF-β1 (2 ng/ml + TRAM-34 (16 nM separately for specified times from 0 min to 60 min. The cells without treatment served as controls. The location of K(Ca3.1 channels in mesangial cells was determined with Confocal laser microscope, the cell cycle of mesangial cells was assessed with flow cytometry, the protein and mRNA expression of K(Ca3.1, α-smooth muscle actin (α-SMA and fibroblast-specific protein-1 (FSP-1 were detected with Western blot and RT-PCR. One-way analysis of variance (ANOVA and Student-Newman-Keuls-q test (SNK-q were used to do statistical analysis. Statistical significance was considered at P<0.05. RESULTS: Kca3.1 channels were located in the cell membranes and/or in the cytoplasm of mesangial cells. The percentage of cells in G0-G1 phase and the expression of K(ca3.1, α-SMA and FSP-1 were elevated under the induction of TGF-β1 when compared to the control and decreased under the induction of TGF-β1+TRAM-34 when compared to the TGF-β1 induced (P<0.05 or P<0.01. CONCLUSION: Targeted disruption of K(Ca3.1 inhibits TGF-β1-induced premature aging, myofibroblast-like phenotype transdifferentiation and proliferation of mesangial cells.

  14. Quercetin Inhibits Pacemaker Potentials via Nitric Oxide/cGMP-Dependent Activation and TRPM7/ANO1 Channels in Cultured Interstitial Cells of Cajal from Mouse Small Intestine

    Directory of Open Access Journals (Sweden)

    Huijin Gim

    2015-04-01

    Full Text Available Background: Quercetin regulates gastrointestinal (GI motor activity but the molecular mechanism involved has not been determined. The authors investigated the effects of quercetin, a flavonoid present in various foods, on the pacemaker activities of interstitial cells of Cajal (ICCs in murine small intestine in vitro and on GI motility in vivo. Materials and Methods: Enzymatic digestion was used to dissociate ICCs from mouse small intestines. The whole-cell patch-clamp configuration was used to record pacemaker potentials in cultured ICCs in the absence or presence of quercetin and to record membrane currents of transient receptor potential melastatin (TRPM 7 or transmembrane protein 16A (Tmem16A, anoctamin1 (ANO1 overexpressed in human embryonic kidney (HEK 293 cells. The in vivo effects of quercetin on GI motility were investigated by measuring the intestinal transit rates (ITRs of Evans blue in normal mice. Results: Quercetin (100-200 μM decreased the amplitudes and frequencies of pacemaker activity in a concentration-dependent manner in current clamp mode, but this action was blocked by naloxone (a pan-opioid receptor antagonist and by GDPβS (a GTP-binding protein inhibitor. However, potassium channels were not involved in these inhibitory effects of quercetin. To study the quercetin signaling pathway, we examined the effects of 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ, an inhibitor of guanylate cyclase, and of RP-8-CPT-cGMPS, an inhibitor of protein kinase G (PKG. These inhibitors blocked the inhibitory effects of quercetin on pacemaker activities. Also, L-NAME (100 μM, a non-selective NO synthase (NOS inhibitor, blocked the effects of quercetin on pacemaker activity and quercetin stimulated cGMP production. Furthermore, quercetin inhibited both Ca2+-activated Cl- channels (TMEM16A, ANO1 and TRPM7 channels. In vivo, quercetin (10-100 mg/kg, p.o. decreased ITRs in normal mice in a dose-dependent manner. Conclusions: Quercetin

  15. Inhibition of inward K+ channels and stomatal response by abscisic acid: an intracellular locus of phytohormone action.

    OpenAIRE

    Schwartz, A; Wu, W. H.; Tucker, E B; Assmann, S M

    1994-01-01

    Abscisic acid (ABA), a plant hormone whose production is stimulated by water stress, reduces the apertures of stomatal pores in the leaf surface, thereby lessening transpirational water loss. It has been thought that inhibition of stomatal opening and promotion of stomatal closure by ABA are initiated by the binding of extracellular ABA to a receptor located in the guard-cell plasma membrane. However, in the present research, we employ three distinct experimental approaches to demonstrate tha...

  16. NEUROSCIENCE. Natural light-gated anion channels: A family of microbial rhodopsins for advanced optogenetics.

    Science.gov (United States)

    Govorunova, Elena G; Sineshchekov, Oleg A; Janz, Roger; Liu, Xiaoqin; Spudich, John L

    2015-08-01

    Light-gated rhodopsin cation channels from chlorophyte algae have transformed neuroscience research through their use as membrane-depolarizing optogenetic tools for targeted photoactivation of neuron firing. Photosuppression of neuronal action potentials has been limited by the lack of equally efficient tools for membrane hyperpolarization. We describe anion channel rhodopsins (ACRs), a family of light-gated anion channels from cryptophyte algae that provide highly sensitive and efficient membrane hyperpolarization and neuronal silencing through light-gated chloride conduction. ACRs strictly conducted anions, completely excluding protons and larger cations, and hyperpolarized the membrane of cultured animal cells with much faster kinetics at less than one-thousandth of the light intensity required by the most efficient currently available optogenetic proteins. Natural ACRs provide optogenetic inhibition tools with unprecedented light sensitivity and temporal precision.

  17. IgG anti-GalNAc-GD1a antibody inhibits the voltage-dependent calcium channel currents in PC12 pheochromocytoma cells.

    Science.gov (United States)

    Nakatani, Yoshihiko; Nagaoka, Takumi; Hotta, Sayako; Utsunomiya, Iku; Yoshino, Hiide; Miyatake, Tadashi; Hoshi, Keiko; Taguchi, Kyoji

    2007-03-01

    We investigated the effects of IgG anti-GalNAc-GD1a antibodies, produced by immunizing rabbits with GalNAc-GD1a, on the voltage-dependent calcium channel (VDCCs) currents in nerve growth factor (NGF)-differentiated PC12 pheochromocytoma cells. VDCCs currents in NGF-differentiated PC12 cells were recorded using the whole-cell patch-clamp technique. Immunized rabbit serum that had a high titer of anti-GalNAc-GD1a antibodies inhibited the VDCCs currents in the NGF-differentiated PC12 cells (36.0+/-9.6% reduction). The inhibitory effect of this serum was reversed to some degree within 3-4 min by washing with bath solution. Similarly, application of purified IgG from rabbit serum immunized with GalNAc-GD1a significantly inhibited the VDCCs currents in PC12 cells (30.6+/-2.5% reduction), and this inhibition was recovered by washing with bath solution. Furthermore, the inhibitory effect was also observed in the GalNAc-GD1a affinity column binding fraction (reduction of 31.1+/-9.85%), while the GalNAc-GD1a affinity column pass-through fraction attenuated the inhibitory effect on VDCCs currents. Normal rabbit serum and normal rabbit IgG did not affect the VDCCs currents in the PC12 cells. In an immunocytochemical study using fluorescence staining, the PC12 cells were stained using GalNAc-GD1a binding fraction. These results indicate that anti-GalNAc-GD1a antibodies inhibit the VDCCs currents in NGF-differentiated PC12 cells.

  18. Inhibition of cancer cell growth by exposure to a specific time-varying electromagnetic field involves T-type calcium channels.

    Directory of Open Access Journals (Sweden)

    Carly A Buckner

    Full Text Available Electromagnetic field (EMF exposures affect many biological systems. The reproducibility of these effects is related to the intensity, duration, frequency, and pattern of the EMF. We have shown that exposure to a specific time-varying EMF can inhibit the growth of malignant cells. Thomas-EMF is a low-intensity, frequency-modulated (25-6 Hz EMF pattern. Daily, 1 h, exposures to Thomas-EMF inhibited the growth of malignant cell lines including B16-BL6, MDA-MB-231, MCF-7, and HeLa cells but did not affect the growth of non-malignant cells. Thomas-EMF also inhibited B16-BL6 cell proliferation in vivo. B16-BL6 cells implanted in syngeneic C57b mice and exposed daily to Thomas-EMF produced smaller tumours than in sham-treated controls. In vitro studies showed that exposure of malignant cells to Thomas-EMF for > 15 min promoted Ca(2+ influx which could be blocked by inhibitors of voltage-gated T-type Ca(2+ channels. Blocking Ca(2+ uptake also blocked Thomas-EMF-dependent inhibition of cell proliferation. Exposure to Thomas-EMF delayed cell cycle progression and altered cyclin expression consistent with the decrease in cell proliferation. Non-malignant cells did not show any EMF-dependent changes in Ca(2+ influx or cell growth. These data confirm that exposure to a specific EMF pattern can affect cellular processes and that exposure to Thomas-EMF may provide a potential anti-cancer therapy.

  19. Cystic fibrosis transmembrane regulator inhibitors CFTR(inh)-172 and GlyH-101 target mitochondrial functions, independently of chloride channel inhibition.

    Science.gov (United States)

    Kelly, Mairead; Trudel, Stephanie; Brouillard, Franck; Bouillaud, Frederick; Colas, Julien; Nguyen-Khoa, Thao; Ollero, Mario; Edelman, Aleksander; Fritsch, Janine

    2010-04-01

    Two highly potent and selective cystic fibrosis (CF) transmembrane regulator (CFTR) inhibitors have been identified by high-throughput screening: the thiazolidinone CFTR(inh)-172 [3-[(3-trifluoromethyl)phenyl]-5-[(4-carboxyphenyl)methylene]- 2-thioxo-4-thiazolidinone] and the glycine hydrazide GlyH-101 [N-(2-naphthalenyl)-((3,5-dibromo-2,4-dihydroxyphenyl)methylene)glycine hydrazide]. Inhibition of the CFTR chloride channel by these compounds has been suggested to be of pharmacological interest in the treatment of secretory diarrheas and polycystic kidney disease. In addition, functional inhibition of CFTR by CFTR(inh)-172 has been proposed to be sufficient to mimic the CF inflammatory profile. In the present study, we investigated the effects of the two compounds on reactive oxygen species (ROS) production and mitochondrial membrane potential in several cell lines: the CFTR-deficient human lung epithelial IB3-1 (expressing the heterozygous F508del/W1282X mutation), the isogenic CFTR-corrected C38, and HeLa and A549 as non-CFTR-expressing controls. Both inhibitors were able to induce a rapid increase in ROS levels and depolarize mitochondria in the four cell types, suggesting that these effects are independent of CFTR inhibition. In HeLa cells, these events were associated with a decrease in the rate of oxygen consumption, with GlyH-101 demonstrating a higher potency than CFTR(inh)-172. The impact of CFTR inhibitors on inflammatory parameters was also tested in HeLa cells. CFTR(inh)-172, but not GlyH-101, induced nuclear translocation of nuclear factor-kappaB (NF-kappaB). CFTR(inh)-172 slightly decreased interleukin-8 secretion, whereas GlyH-101 induced a slight increase. These results support the conclusion that CFTR inhibitors may exert nonspecific effects regarding ROS production, mitochondrial failure, and activation of the NF-kappaB signaling pathway, independently of CFTR inhibition.

  20. Inhibition of cancer cell growth by exposure to a specific time-varying electromagnetic field involves T-type calcium channels.

    Science.gov (United States)

    Buckner, Carly A; Buckner, Alison L; Koren, Stan A; Persinger, Michael A; Lafrenie, Robert M

    2015-01-01

    Electromagnetic field (EMF) exposures affect many biological systems. The reproducibility of these effects is related to the intensity, duration, frequency, and pattern of the EMF. We have shown that exposure to a specific time-varying EMF can inhibit the growth of malignant cells. Thomas-EMF is a low-intensity, frequency-modulated (25-6 Hz) EMF pattern. Daily, 1 h, exposures to Thomas-EMF inhibited the growth of malignant cell lines including B16-BL6, MDA-MB-231, MCF-7, and HeLa cells but did not affect the growth of non-malignant cells. Thomas-EMF also inhibited B16-BL6 cell proliferation in vivo. B16-BL6 cells implanted in syngeneic C57b mice and exposed daily to Thomas-EMF produced smaller tumours than in sham-treated controls. In vitro studies showed that exposure of malignant cells to Thomas-EMF for > 15 min promoted Ca(2+) influx which could be blocked by inhibitors of voltage-gated T-type Ca(2+) channels. Blocking Ca(2+) uptake also blocked Thomas-EMF-dependent inhibition of cell proliferation. Exposure to Thomas-EMF delayed cell cycle progression and altered cyclin expression consistent with the decrease in cell proliferation. Non-malignant cells did not show any EMF-dependent changes in Ca(2+) influx or cell growth. These data confirm that exposure to a specific EMF pattern can affect cellular processes and that exposure to Thomas-EMF may provide a potential anti-cancer therapy.

  1. Inhibition of TRPA1 channel activity in sensory neurons by the glial cell line-derived neurotrophic factor family member, artemin

    Directory of Open Access Journals (Sweden)

    Wang Shenglan

    2011-05-01

    Full Text Available Abstract Background The transient receptor potential (TRP channel subtype A1 (TRPA1 is known to be expressed on sensory neurons and respond to changes in temperature, pH and local application of certain noxious chemicals such as allyl isothiocyanate (AITC. Artemin is a neuronal survival and differentiation factor and belongs to the glial cell line-derived neurotrophic factor (GDNF family. Both TRPA1 and artemin have been reported to be involved in pathological pain initiation and maintenance. In the present study, using whole-cell patch clamp recording technique, in situ hybridization and behavioral analyses, we examined the functional interaction between TRPA1 and artemin. Results We found that 85.8 ± 1.9% of TRPA1-expressing neurons also expressed GDNF family receptor alpha 3 (GFR α3, and 87.5 ± 4.1% of GFRα3-expressing neurons were TRPA1-positive. In whole-cell patch clamp analysis, a short-term treatment of 100 ng/ml artemin significantly suppressed the AITC-induced TRPA1 currents. A concentration-response curve of AITC resulting from the effect of artemin showed that this inhibition did not change EC50 but did lower the AITC-induced maximum response. In addition, pre-treatment of artemin significantly suppressed the number of paw lifts induced by intraplantar injection of AITC, as well as the formalin-induced pain behaviors. Conclusions These findings that a short-term application of artemin inhibits the TRPA1 channel's activity and the sequential pain behaviors suggest a role of artemin in regulation of sensory neurons.

  2. Poly(ethylene glycol-cholesterol inhibits L-type Ca2+ channel currents and augments voltage-dependent inactivation in A7r5 cells.

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    Rikuo Ochi

    Full Text Available Cholesterol distributes at a high density in the membrane lipid raft and modulates ion channel currents. Poly(ethylene glycol cholesteryl ether (PEG-cholesterol is a nonionic amphipathic lipid consisting of lipophilic cholesterol and covalently bound hydrophilic PEG. PEG-cholesterol is used to formulate lipoplexes to transfect cultured cells, and liposomes for encapsulated drug delivery. PEG-cholesterol is dissolved in the external leaflet of the lipid bilayer, and expands it to flatten the caveolae and widen the gap between the two leaflets. We studied the effect of PEG-cholesterol on whole cell L-type Ca(2+ channel currents (I(Ca,L recorded from cultured A7r5 arterial smooth muscle cells. The pretreatment of cells with PEG-cholesterol decreased the density of ICa,L and augmented the voltage-dependent inactivation with acceleration of time course of inactivation and negative shift of steady-state inactivation curve. Methyl-β-cyclodextrin (MβCD is a cholesterol-binding oligosaccharide. The enrichment of cholesterol by the MβCD:cholesterol complex (cholesterol (MβCD caused inhibition of I(Ca,L but did not augment voltage-dependent inactivation. Incubation with MβCD increased I(Ca,L, slowed the time course of inactivation and shifted the inactivation curve to a positive direction. Additional pretreatment by a high concentration of MβCD of the cells initially pretreated with PEG-cholesterol, increased I(Ca,L to a greater level than the control, and removed the augmented voltage-dependent inactivation. Due to the enhancement of the voltage-dependent inactivation, PEG-cholesterol inhibited window I(Ca,L more strongly as compared with cholesterol (MβCD. Poly(ethylene glycol conferred to cholesterol the efficacy to induce sustained augmentation of voltage-dependent inactivation of I(Ca,L.

  3. Mg2+ ions reduce microglial and THP-1 cell neurotoxicity by inhibiting Ca2+ entry through purinergic channels.

    Science.gov (United States)

    Lee, Moonhee; Jantaratnotai, Nattinee; McGeer, Edith; McLarnon, James G; McGeer, Patrick L

    2011-01-19

    Mg(2+) is a known antagonist of some Ca(2+) ion channels. It may therefore be able to counteract the toxic consequences of excessive Ca(2+) entry into immune-type cells. Here we examined the effects of Mg(2+) on inflammation induced by Ca(2+) influx into microglia and THP-1 cells following activation of purinergic receptors. Using tissue culture, an inflammatory response was induced by treatment with either the P2X7 purinergic receptor agonist 2',3'-[benzoyl-4-benzoyl]-ATP (BzATP) or the P2Y2,4 receptor agonist uridine 5'-triphosphate (UTP). Both microglia and THP-1 cells expressed the mRNAs for these receptors. Treatment produced a rapid rise in intracellular Ca(2+) which was significantly reduced by Mg(2+) or the calcium chelator BAPTA-AM. Purinergic receptor stimulation activated the intracellular inflammatory pathway P38 MAP kinase and NFκB. This caused release of TNFα, IL-6, nitrite ions and other materials that are neurotoxic to SH-SY5Y cells. These effects were all ameliorated by Mg(2+). They were also partly ameliorated by the P2X7R antagonists, oxATP and KN-62, the P2YR antagonist MRS2179, and the store operated Ca(2+) channel blocker, SK96365. These results indicate that elevated Mg(2+) is a broad spectrum inhibitor of Ca(2+) entry into microglia or THP-1 cells. Mg(2+) administration may be a strategy for reducing the damaging consequences Ca(2+) induced neuroinflammation in degenerative neurological disorders such as Alzheimer disease and Parkinson disease.

  4. Transverse relaxation dispersion of the p7 membrane channel from hepatitis C virus reveals conformational breathing

    Energy Technology Data Exchange (ETDEWEB)

    Dev, Jyoti; Brüschweiler, Sven [Harvard Medical School, Department of Biological Chemistry and Molecular Pharmacology (United States); Ouyang, Bo [Chinese Academy of Sciences, State Key Laboratory of Molecular Biology, Shanghai Institute of Biochemistry and Cell Biology (China); Chou, James J., E-mail: james-chou@hms.harvard.edu [Harvard Medical School, Department of Biological Chemistry and Molecular Pharmacology (United States)

    2015-04-15

    The p7 membrane protein encoded by hepatitis C virus (HCV) assembles into a homo-hexamer that selectively conducts cations. An earlier solution NMR structure of the hexameric complex revealed a funnel-like architecture and suggests that a ring of conserved asparagines near the narrow end of the funnel are important for cation interaction. NMR based drug-binding experiments also suggest that rimantadine can allosterically inhibit ion conduction via a molecular wedge mechanism. These results suggest the presence of dilation and contraction of the funnel tip that are important for channel activity and that the action of the drug is attenuating this motion. Here, we determined the conformational dynamics and solvent accessibility of the p7 channel. The proton exchange measurements show that the cavity-lining residues are largely water accessible, consistent with the overall funnel shape of the channel. Our relaxation dispersion data show that residues Val7 and Leu8 near the asparagine ring are subject to large chemical exchange, suggesting significant intrinsic channel breathing at the tip of the funnel. Moreover, the hinge regions connecting the narrow and wide regions of the funnel show strong relaxation dispersion and these regions are the binding sites for rimantadine. Presence of rimantadine decreases the conformational dynamics near the asparagine ring and the hinge area. Our data provide direct observation of μs–ms dynamics of the p7 channel and support the molecular wedge mechanism of rimantadine inhibition of the HCV p7 channel.

  5. Metamizol acts as an ATP sensitive potassium channel opener to inhibit the contracting response induced by angiotensin II but not to norepinephrine in rat thoracic aorta smooth muscle.

    Science.gov (United States)

    Valenzuela, Fermín; García-Saisó, Sebastián; Lemini, Cristina; Ramírez-Solares, Rafael; Vidrio, Horacio; Mendoza-Fernández, Víctor

    2005-08-01

    Clinically metamizol (MZ) has been related to alteration on haemodynamic parameters and modifications on blood pressure in humans when administered intravenously. These effects have been observed at MZ therapeutic doses. Experimentally, MZ is able to induce relaxation on several types of vascular smooth muscles and modulates the contraction induced by phenylephrine. However, the mechanism underlying the MZ effects on vascular reactivity is not clear. Potassium channels (K) present on vascular smooth muscle cells closely regulate the vascular reactivity and membrane potential. There are four described types of K in vascular tissue: K voltage sensitive (K(V)), K calcium sensitive (K(Ca)2+), K ATP sensitive (K(ATP) and K inward rectification (K(IR), voltage sensitive). The aim of this work was to investigate MZ effects on angiotensin II (AT II) and noradrenaline (NA) induced contraction and to evaluate the K participation on MZ modulating effect on vascular smooth muscle contraction, using isometric and patch clamp techniques. MZ induces relaxation in a concentration dependent manner. Furthermore, MZ strongly inhibits in a concentration dependent fashion the contraction induced by AT II. However, MZ inhibition on NA induced contraction was moderated compared with that observed on AT II. MZ effects on AT II induced contraction was blocked by glybenclamide (a specific K(ATP) blocker, 3 microM, *p < 0.01). In patch clamp experiments, MZ (3 mM) induces an increase on potassium current (K+) mediated by K(ATP) in similar way as diazoxide (a specific K(ATP) opener, 3 microM). Our results suggest that MZ induces relaxation and inhibits contraction induced by AT II acting as a K(ATP) opener.

  6. Inhibition of cell proliferation by a selective inhibitor of the Ca{sup 2+}-activated Cl{sup -} channel, Ano1

    Energy Technology Data Exchange (ETDEWEB)

    Mazzone, Amelia; Eisenman, Seth T.; Strege, Peter R. [Enteric NeuroScience Program, Mayo Clinic, Rochester, MN (United States); Yao, Zhen [Laboratory of Molecular Genetics, UCSF, San Francisco, CA (United States); Ordog, Tamas; Gibbons, Simon J. [Enteric NeuroScience Program, Mayo Clinic, Rochester, MN (United States); Farrugia, Gianrico, E-mail: farrugia.gianrico@mayo.edu [Enteric NeuroScience Program, Mayo Clinic, Rochester, MN (United States)

    2012-10-19

    Highlights: Black-Right-Pointing-Pointer T16A{sub inh}-A01 blocked Ano1 currents in HEK cells expressing Ano1. Black-Right-Pointing-Pointer T16A{sub inh}-A01 reduced proliferation in ICC primary cultures and CFPAC-1 cell line. Black-Right-Pointing-Pointer T16A{sub inh}-A01 reduced proliferation of ICC in intact smooth muscle strips. -- Abstract: Background: Ion channels play important roles in regulation of cellular proliferation. Ano1 (TMEM16A) is a Ca{sup 2+}-activated Cl{sup -} channel expressed in several tumors and cell types. In the muscle layers of the gastrointestinal tract Ano1 is selectively expressed in interstitial cells of Cajal (ICC) and appears to be required for normal gastrointestinal slow wave electrical activity. However, Ano1 is expressed in all classes of ICC, including those that do not generate slow waves suggesting that Ano1 may have other functions. Indeed, a role for Ano1 in regulating proliferation of tumors and ICC has been recently suggested. Recently, a high-throughput screen identified a small molecule, T16A{sub inh}-A01 as a specific inhibitor of Ano1. Aim: To investigate the effect of the T16A{sub inh}-A01 inhibitor on proliferation in ICC and in the Ano1-expressing human pancreatic cancer cell line CFPAC-1. Methods: Inhibition of Ano1 was demonstrated by whole cell voltage clamp recordings of currents in cells transfected with full-length human Ano1. The effect of T16A{sub inh}-A01 on ICC proliferation was examined in situ in organotypic cultures of intact mouse small intestinal smooth muscle strips and in primary cell cultures prepared from these tissues. ICC were identified by Kit immunoreactivity. Proliferating ICC and CFPAC-1 cells were identified by immunoreactivity for the nuclear antigen Ki67 or EdU incorporation, respectively. Results: T16A{sub inh}-A01 inhibited Ca{sup 2+}-activated Cl{sup -} currents by 60% at 10 {mu}M in a voltage-independent fashion. Proliferation of ICC was significantly reduced in primary cultures

  7. Identification of TRPM7 channels in human intestinal interstitial cells of Cajal

    Institute of Scientific and Technical Information of China (English)

    Byung Joo Kim; Kyu Joo Park; Hyung Woo Kim; Seok Choi; Jae Yeoul Jun; In Youb Chang; Ju-Hong Jeon; Insuk So; Seon Jeong Kim

    2009-01-01

    AIM: To investigate the characteristics of slow electrical waves and the presence of transient receptor potential melastatin-type 7 (TRPM7) in the human gastrointestinal (GI) tract. METHODS: Conventional microelectrode techniques were used to record intracellular electrical responses from human GI smooth muscle tissue. Immunohistochemistry was used to identify TRPM7 channels in interstitial cells of Cajal (ICCs). RESULTS: The human GI tract generated slow electrical waves and had ICCs which functioned as pacemaker cells. Flufenamic acid, a nonselective cation channel blocker, and 2-APB (2-aminoethoxydiphenyl borate) and La3~+, TRPM7 channel blockers, inhibited the slow waves. Also, TRPM7 channels were expressed in ICCs in human tissue. CONCLUSION: These results suggest that the human GI tract generates slow waves and that TRPM7 channels expressed in the ICCs may be involved in the generation of the slow waves.

  8. Differential modulation of TWIK-related K(+) channel (TREK) and TWIK-related acid-sensitive K(+) channel 2 (TASK2) activity by pyrazole compounds.

    Science.gov (United States)

    Kim, Hyun Jong; Woo, Joohan; Nam, Yuran; Nam, Joo Hyun; Kim, Woo Kyung

    2016-11-15

    Pyrazole derivatives were originally suggested as selective blockers of the transient receptor potential cation 3 (TRPC3) and channel. In particular, pyr3 and 10 selectively inhibit TRPC3, whereas pyr2 (BTP2) and 6 inhibit ORAI1. However, their effects on background K(+) channel activity have not been elucidated. In this study, the effects of BTP2, pyr3, pyr6, and pyr10 were studied on cloned human TWIK-related K(+) channels (TREKs) and TWIK-related acid-sensitive K(+) channel 2 (TASK-2) channels, which modulate Ca(2+) signaling by controlling membrane potential, in HEK293T-overexpressing cells by using a whole-cell patch clamp technique. Pyr3 potently inhibited TREK-1 (ITREK1), TREK-2 (ITREK2), and TASK2 current (ITASK-2) with half-maximal inhibitory concentrations (IC50) of 0.89±0.27, 1.95±1.44, and 2.42±0.39µM, respectively. BTP2 slightly inhibited ITASK-2 (80.3±2.5% at 100μM). In contrast, pyr6 at 100µM potentiated ITREK1 and ITREK2 by approximately 2.6- and 3.6-fold compared to the control and inhibited ITASK2 (38.7±9.2%). Pyr10 showed a subtype-specific inhibition of ITREK1 but not ITREK2. It also inhibited ITASK2 (70.9±3.1% at 100μM). To the best of our knowledge, this study is the first to describe the differential modulation of TREKs and TASK2 channels by pyrazole derivatives, previously used as inhibitors of TRPC3 and ORAI1. Therefore, studies using these drugs should consider their modulation of other channels such as TREK and TASK-2.

  9. Amyloid-β-induced reactive oxygen species production and priming are differentially regulated by ion channels in microglia.

    Science.gov (United States)

    Schilling, Tom; Eder, Claudia

    2011-12-01

    Production of reactive oxygen species (ROS) by microglial cells and subsequent oxidative stress are strongly implicated in the pathogenesis of Alzheimer's disease. Although it is recognized that amyloid-β (Aβ) plays a major role in inducing and regulating microglial ROS production in Alzheimer's disease, to date little is known about cellular mechanisms underlying Aβ-stimulated ROS production. Here, we identified ion channels involved in Aβ-induced microglial ROS production and in Aβ-induced microglial priming. Acute stimulation of microglial cells with either fibrillar Aβ(1-42) (fAβ(1-42) ) or soluble Aβ(1-42) (sAβ(1-42) ) caused significant increases in microglial ROS production, which were abolished by inhibition of TRPV1 cation channels with 5-iodo-resiniferatoxin (I-RTX), but were unaffected by inhibition of K(+) channels with charybdotoxin (CTX). Furthermore, pretreatment with either fAβ(1-42) or sAβ(1-42) induced microglial priming, that is, increased ROS production upon secondary stimulation with the phorbol ester PMA. Microglial priming induced by fAβ(1-42) or sAβ(1-42) remained unaffected by TRPV1 channel inhibition with I-RTX. However, sAβ(1-42) -induced priming was inhibited by CTX and margatoxin, but not by TRAM-34 or paxilline, indicating a role of Kv1.3 voltage-gated K(+) channels, but not of Ca(2+) -activated K(+) channels, in the priming process. In summary, our data suggest that in microglia Aβ-induced ROS production and priming are differentially regulated by ion channels, and that TRPV1 cation channels and Kv1.3 K(+) channels may provide potential therapeutic targets to reduce microglia-induced oxidative stress in Alzheimer's disease.

  10. Computational modeling of voltage-gated Ca channels inhibition: identification of different effects on uterine and cardiac action potentials

    Directory of Open Access Journals (Sweden)

    Wing Chiu eTong

    2014-10-01

    Full Text Available The uterus and heart share the important physiological feature whereby contractile activation of the muscle tissue is regulated by the generation of periodic, spontaneous electrical action potentials (APs. Preterm birth arising from premature uterine contractions is a major complication of pregnancy and there remains a need to pursue avenues of research that facilitate the use of drugs, tocolytics, to limit these inappropriate contractions without deleterious actions on cardiac electrical excitation. A novel approach is to make use of mathematical models of uterine and cardiac APs, which incorporate many ionic currents contributing to the AP forms, and test the cell-specific responses to interventions. We have used three such models – of uterine smooth muscle cells (USMC, cardiac sinoatrial node cells (SAN and ventricular cells – to investigate the relative effects of reducing two important voltage-gated Ca currents – the L-type (ICaL and T-type (ICaT Ca currents. Reduction of ICaL (10% alone, or ICaT (40% alone, blunted USMC APs with little effect on ventricular APs and only mild effects on SAN activity. Larger reductions in either current further attenuated the USMC APs but with also greater effects on SAN APs. Encouragingly, a combination of ICaL and ICaT reduction did blunt USMC APs as intended with little detriment to APs of either cardiac cell type. Subsequent overlapping maps of ICaL and ICaT inhibition profiles from each model revealed a range of combined reductions of ICaL and ICaT over which an appreciable diminution of USMC APs could be achieved with no deleterious action on cardiac SAN or ventricular APs. This novel approach illustrates the potential for computational biology to inform us of possible uterine and cardiac cell-specific mechanisms. Incorporating such computational approaches in future studies directed at designing new, or repurposing existing, tocolytics will be beneficial for establishing a desired uterine

  11. Inhibition of TREK-2 K(+) channels by PI(4,5)P2: an intrinsic mode of regulation by intracellular ATP via phosphatidylinositol kinase.

    Science.gov (United States)

    Woo, Joohan; Shin, Dong Hoon; Kim, Hyun Jong; Yoo, Hae Young; Zhang, Yin-Hua; Nam, Joo Hyun; Kim, Woo Kyung; Kim, Sung Joon

    2016-08-01

    TWIK-related two-pore domain K(+) channels 1 and 2 (TREKs) are activated under various physicochemical conditions. However, the directions in which they are regulated by PI(4,5)P2 and intracellular ATP are not clearly presented yet. In this study, we investigated the effects of ATP and PI(4,5)P2 on overexpressed TREKs (HEK293T and COS-7) and endogenously expressed TREK-2 (mouse astrocytes and WEHI-231 B cells). In all of these cells, both TREK-1 and TREK-2 currents were spontaneously increased by dialysis with ATP-free pipette solution for whole-cell recording (ITREK-1,w-c and ITREK-2w-c) or by membrane excision for inside-out patch clamping without ATP (ITREK-1,i-o and ITREK-2,i-o). Steady state ITREK-2,i-o was reversibly decreased by 3 mM ATP applied to the cytoplasmic side, and this reduction was prevented by wortmannin, a PI-kinase inhibitor. An exogenous application of PI(4,5)P2 inhibited the spontaneously increased ITREKs,i-o, suggesting that intrinsic PI(4,5)P2 maintained by intracellular ATP and PI kinase may set the basal activity of TREKs in the intact cells. The inhibition of intrinsic TREK-2 by ATP was more prominent in WEHI-231 cells than astrocytes. Interestingly, unspecific screening of negative charges by poly-L-lysine also inhibited ITREK-2,i-o. Application of PI(4,5)P2 after the poly-L-lysine treatment showed dose-dependent dual effects, initial activation and subsequent inhibition of ITREK-2,i-o at low and high concentrations, respectively. In HEK293T cells coexpressing TREK-2 and a voltage-sensitive PI(4,5)P2 phosphatase, sustained depolarization increased ITREK-2,w-c initially (P2 suggests the existence of dual regulatory modes that depend on PI(4,5)P2 concentration.

  12. Inhibition of lysosomal degradation rescues pentamidine-mediated decreases of K(IR)2.1 ion channel expression but not that of K(v)11.1.

    Science.gov (United States)

    Nalos, Lukas; de Boer, Teun P; Houtman, Marien J C; Rook, Martin B; Vos, Marc A; van der Heyden, Marcel A G

    2011-02-10

    The antiprotozoal drug pentamidine inhibits two types of cardiac rectifier potassium currents, which can precipitate life-threatening arrhythmias. Here, we use pentamidine as a tool to investigate whether a single drug affects trafficking of two structurally different potassium channels by identical or different mechanisms, and whether the adverse drug effect can be suppressed in a channel specific fashion. Whole cell patch clamp, Western blot, real time PCR, and confocal laser scanning microscopy were used to determine potassium current density, ion channel protein levels, mRNA expression levels, and subcellular localization, respectively. We demonstrate that pentamidine inhibits delayed (I(Kr)) and inward (I(K1)) rectifier currents in cultured adult canine cardiomyocytes. In HEK293 cells, pentamidine inhibits functional K(v)11.1 channels, responsible for I(Kr), by interfering at the level of full glycosylation, yielding less mature form of K(v)11.1 at the plasma membrane. In contrast, total K(IR)2.1 expression levels, underlying I(K1), are strongly decreased, which cannot be explained from mRNA expression levels. No changes in molecular size of K(IR)2.1 protein were observed, excluding interference in overt glycosylation. Remaining K(IR)2.1 protein is mainly expressed at the plasma membrane. Inhibition of lysosomal protein degradation is able to partially rescue K(IR)2.1 levels, but not those of K(v)11.1. We conclude that 1) a single drug can interfere in cardiac potassium channel trafficking in a subtype specific mode and 2) adverse drug effects can be corrected in a channel specific manner.

  13. Inhibition of T-Type Voltage Sensitive Calcium Channel Reduces Load-Induced OA in Mice and Suppresses the Catabolic Effect of Bone Mechanical Stress on Chondrocytes.

    Directory of Open Access Journals (Sweden)

    Padma P Srinivasan

    Full Text Available Voltage-sensitive calcium channels (VSCC regulate cellular calcium influx, one of the earliest responses to mechanical stimulation in osteoblasts. Here, we postulate that T-type VSCCs play an essential role in bone mechanical response to load and participate in events leading to the pathology of load-induced OA. Repetitive mechanical insult was used to induce OA in Cav3.2 T-VSCC null and wild-type control mouse knees. Osteoblasts (MC3T3-E1 and chondrocytes were treated with a selective T-VSCC inhibitor and subjected to fluid shear stress to determine how blocking of T-VSCCs alters the expression profile of each cell type upon mechanical stimulation. Conditioned-media (CM obtained from static and sheared MC3T3-E1 was used to assess the effect of osteoblast-derived factors on the chondrocyte phenotype. T-VSCC null knees exhibited significantly lower focal articular cartilage damage than age-matched controls. In vitro inhibition of T-VSCC significantly reduced the expression of both early and late mechanoresponsive genes in osteoblasts but had no effect on gene expression in chondrocytes. Furthermore, treatment of chondrocytes with CM obtained from sheared osteoblasts induced expression of markers of hypertrophy in chondrocytes and this was nearly abolished when osteoblasts were pre-treated with the T-VSCC-specific inhibitor. These results indicate that T-VSCC plays a role in signaling events associated with induction of OA and is essential to the release of osteoblast-derived factors that promote an early OA phenotype in chondrocytes. Further, these findings suggest that local inhibition of T-VSCC may serve as a therapy for blocking load-induced bone formation that results in cartilage degeneration.

  14. Growth inhibition of fungus Phycomyces blakesleeanus by anion channel inhibitors anthracene-9-carboxylic and niflumic acid attained through decrease in cellular respiration and energy metabolites.

    Science.gov (United States)

    Stanić, Marina; Križak, Strahinja; Jovanović, Mirna; Pajić, Tanja; Ćirić, Ana; Žižić, Milan; Zakrzewska, Joanna; Cvetić Antić, Tijana; Todorović, Nataša; Živić, Miroslav

    2017-01-18

    Increasing resistance of fungal strains to known fungicides has prompted identification of new candidates for fungicides among substances previously used for other purposes. We have tested the effects of known anion channel inhibitors anthracene-9-carboxylic (A9C) and niflumic acid (NFA) on growth, energy metabolism and anionic current of mycelium of fungus Phycomyces blakesleeanus. Both inhibitors significantly decreased growth and respiration of mycelium, but complete inhibition was only achieved by 100 or 500 µM NFA, for growth and respiration, respectively. A9C had no effect on respiration of human NCI-H460 cell line, and very little effect on cucumber root sprout clippings, which nominates this inhibitor for further investigation as a potential new fungicide. Effects of A9C and NFA on respiration of isolated mitochondria of P. blakesleeanus were significantly smaller, which indicates that their inhibitory effect on respiration of mycelium is indirect. NMR spectroscopy showed that both A9C and NFA decrease the levels of ATP and polyphosphates in the mycelium of P. blakesleanus, but only A9C caused intracellular acidification. Outwardly rectifying, fast inactivating instantaneous anionic current (ORIC) was also reduced to 33±5% and 21±3% of its pre-treatment size by A9C and NFA, respectively, but only in the absence of ATP. It can be assumed from our results that the regulation of ORIC is tightly linked to cellular energy metabolism in P. blakesleeanus, and the decrease in ATP and polyphosphate levels could be a direct cause of growth inhibition.

  15. Antinociception produced by Thalassia testudinum extract BM-21 is mediated by the inhibition of acid sensing ionic channels by the phenolic compound thalassiolin B

    Directory of Open Access Journals (Sweden)

    Thomas Olivier P

    2011-01-01

    Full Text Available Abstract Background Acid-sensing ion channels (ASICs have a significant role in the sensation of pain and constitute an important target for the search of new antinociceptive drugs. In this work we studied the antinociceptive properties of the BM-21 extract, obtained from the sea grass Thalassia testudinum, in chemical and thermal models of nociception in mice. The action of the BM-21 extract and the major phenolic component isolated from this extract, a sulphated flavone glycoside named thalassiolin B, was studied in the chemical nociception test and in the ASIC currents of the dorsal root ganglion (DRG neurons obtained from Wistar rats. Results Behavioral antinociceptive experiments were made on male OF-1 mice. Single oral administration of BM-21 produced a significant inhibition of chemical nociception caused by acetic acid and formalin (specifically during its second phase, and increased the reaction time in the hot plate test. Thalassiolin B reduced the licking behavior during both the phasic and tonic phases in the formalin test. It was also found that BM-21 and thalassiolin B selectively inhibited the fast desensitizing (τ Conclusions To our knowledge, this is the first report of an ASIC-current inhibitor derived of a marine-plant extract, and in a phenolic compound. The antinociceptive effects of BM-21 and thalassiolin B may be partially because of this action on the ASICs. That the active components of the extract are able to cross the blood-brain barrier gives them an additional advantage for future uses as tools to study pain mechanisms with a potential therapeutic application.

  16. Current status of hyperpolarization-activated cyclic nucleotide-gated cation channel%超极化激活环核苷酸门控阳离子通道的研究现状

    Institute of Scientific and Technical Information of China (English)

    严慧; 黄裕新; 王景杰

    2009-01-01

    @@ 0 引言 超极化激活环核苷酸门控的超极化阳离子通道(hyperpolarization-activated cyclic nucleotide-gated cation chan-nel,HCN)的研究起源于Ih的发现,Noma和Irisawa已经在研究窦房结起搏活动时发现这一离子流并命名为Ih(hyperpolar-ization-activated current),20世纪80年代初Di Francesco和Irisawa等

  17. Voltage-Gated R-Type Calcium Channel Inhibition via Human μ-, δ-, and κ-opioid Receptors Is Voltage-Independently Mediated by Gβγ Protein Subunits.

    Science.gov (United States)

    Berecki, Géza; Motin, Leonid; Adams, David J

    2016-01-01

    Elucidating the mechanisms that modulate calcium channels via opioid receptor activation is fundamental to our understanding of both pain perception and how opioids modulate pain. Neuronal voltage-gated N-type calcium channels (Cav2.2) are inhibited by activation of G protein-coupled opioid receptors (ORs). However, inhibition of R-type (Cav2.3) channels by μ- or κ-ORs is poorly defined and has not been reported for δ-ORs. To investigate such interactions, we coexpressed human μ-, δ-, or κ-ORs with human Cav2.3 or Cav2.2 in human embryonic kidney 293 cells and measured depolarization-activated Ba(2+) currents (IBa). Selective agonists of μ-, δ-, and κ-ORs inhibited IBa through Cav2.3 channels by 35%. Cav2.2 channels were inhibited to a similar extent by κ-ORs, but more potently (60%) via μ- and δ-ORs. Antagonists of δ- and κ-ORs potentiated IBa amplitude mediated by Cav2.3 and Cav2.2 channels. Consistent with G protein βγ (Gβγ) interaction, modulation of Cav2.2 was primarily voltage-dependent and transiently relieved by depolarizing prepulses. In contrast, Cav2.3 modulation was voltage-independent and unaffected by depolarizing prepulses. However, Cav2.3 inhibition was sensitive to pertussis toxin and to intracellular application of guanosine 5'-[β-thio]diphosphate trilithium salt and guanosine 5'-[γ-thio]triphosphate tetralithium salt. Coexpression of Gβγ-specific scavengers-namely, the carboxyl terminus of the G protein-coupled receptor kinase 2 or membrane-targeted myristoylated-phosducin-attenuated or abolished Cav2.3 modulation. Our study reveals the diversity of OR-mediated signaling at Cav2 channels and identifies neuronal Cav2.3 channels as potential targets for opioid analgesics. Their novel modulation is dependent on pre-existing OR activity and mediated by membrane-delimited Gβγ subunits in a voltage-independent manner.

  18. Inhibition of cystathionine β-synthetase suppresses sodium channel activities of dorsal root ganglion neurons of rats with lumbar disc herniation

    Science.gov (United States)

    Yan, Jun; Hu, Shufen; Zou, Kang; Xu, Min; Wang, Qianliang; Miao, Xiuhua; Yu, Shan Ping; Xu, Guang-Yin

    2016-01-01

    The pathogenesis of pain in lumbar disc herniation (LDH) remains poorly understood. We have recently demonstrated that voltage-gated sodium channels (VGSCs) in dorsal root ganglion (DRG) neurons were sensitized in a rat model of LDH. However, the detailed molecular mechanism for sensitization of VGSCs remains largely unknown. This study was designed to examine roles of the endogenous hydrogen sulfide synthesizing enzyme cystathionine β-synthetase (CBS) in sensitization of VGSCs in a previously validated rat model of LDH. Here we showed that inhibition of CBS activity by O-(Carboxymethyl) hydroxylamine hemihydrochloride (AOAA) significantly attenuated pain hypersensitivity in LDH rats. Administration of AOAA also reduced neuronal hyperexcitability, suppressed the sodium current density, and right-shifted the V1/2 of the inactivation curve, of hindpaw innervating DRG neurons, which is retrogradely labeled by DiI. In vitro incubation of AOAA did not alter the excitability of acutely isolated DRG neurons. Furthermore, CBS was colocalized with NaV1.7 and NaV1.8 in hindpaw-innervating DRG neurons. Treatment of AOAA markedly suppressed expression of NaV1.7 and NaV1.8 in DRGs of LDH rats. These data suggest that targeting the CBS-H2S signaling at the DRG level might represent a novel therapeutic strategy for chronic pain relief in patients with LDH. PMID:27905525

  19. Effects of ginger and its pungent constituents on transient receptor potential channels.

    Science.gov (United States)

    Kim, Young-Soo; Hong, Chan Sik; Lee, Sang Weon; Nam, Joo Hyun; Kim, Byung Joo

    2016-12-01

    Ginger extract is used as an analeptic in herbal medicine and has been reported to exert antioxidant effects. Transient receptor potential (TRP) canonical 5 (TRPC5), TRP cation channel, subfamily M, member 7 (TRPM7; melastatin 7), and TRP cation channel, subfamily A, member 1 (TRPA1; ankyrin 1) are non-selective cation channels that are modulated by reactive oxygen/nitrogen species (ROS/RNS) and subsequently control various cellular processes. The aim of this study was to evaluate whether ginger and its pungent constituents modulate these channels and exert antioxidant effects. It was found that TRPC5 and TRPA1 currents were modulated by ginger extract and by its pungent constituents, [6]-gingerol, zingerone and [6]-shogaol. In particular, [6]-shogaol markedly and dose-dependently inhibited TRPC5 currents with an IC50 of value of ~18.3 µM. Furthermore, the strong dose-dependent activation of TRPA1 currents by [6]-shogaol was abolished by A‑967079 (a selective TRPA1 inhibitor). However, ginger extract and its pungent constituents had no effect on TRPM7 currents. These results suggest the antioxidant effects of ginger extract and its pungent constituents are mediated through TRPC5 and TRPA1, and that [6]-shogaol is predominantly responsible for the regulation of TRPC5 and TRPA1 currents by ginger extract.

  20. Role of extracellular cations in cell motility, polarity, and chemotaxis

    Directory of Open Access Journals (Sweden)

    Soll D

    2011-04-01

    Full Text Available David R Soll1, Deborah Wessels1, Daniel F Lusche1, Spencer Kuhl1, Amanda Scherer1, Shawna Grimm1,21Monoclonal Antibody Research Institute, Developmental Studies, Hybridoma Bank, Department of Biology, University of Iowa, Iowa City; 2Mercy Medical Center, Surgical Residency Program, Des Moines, Iowa, USAAbstract: The concentration of cations in the aqueous environment of free living organisms and cells within the human body influence motility, shape, and chemotaxis. The role of extracellular cations is usually perceived to be the source for intracellular cations in the process of homeostasis. The role of surface molecules that interact with extracellular cations is believed to be that of channels, transporters, and exchangers. However, the role of Ca2+ as a signal and chemoattractant and the discovery of the Ca2+ receptor have demonstrated that extracellular cations can function as signals at the cell surface, and the plasma membrane molecules they interact with can function as bona fide receptors that activate coupled signal transduction pathways, associated molecules in the plasma membrane, or the cytoskeleton. With this perspective in mind, we have reviewed the cationic composition of aqueous environments of free living cells and cells that move in multicellular organisms, most notably humans, the range of molecules interacting with cations at the cell surface, the concept of a cell surface cation receptor, and the roles extracellular cations and plasma membrane proteins that interact with them play in the regulation of motility, shape, and chemotaxis. Hopefully, the perspective of this review will increase awareness of the roles extracellular cations play and the possibility that many of the plasma membrane proteins that interact with them could also play roles as receptors.Keywords: extracellular cations, chemotaxis, transporters, calcium, receptors

  1. TRPM7-like channels are functionally expressed in oocytes and modulate post-fertilization embryo development in mouse

    Science.gov (United States)

    Carvacho, Ingrid; Ardestani, Goli; Lee, Hoi Chang; McGarvey, Kaitlyn; Fissore, Rafael A.; Lykke-Hartmann, Karin

    2016-01-01

    The Transient Receptor Potential (TRP) channels are a family of cationic ion channels widely distributed in mammalian tissues. In general, the global genetic disruption of individual TRP channels result in phenotypes associated with impairment of a particular tissue and/or organ function. An exception is the genetic ablation of the TRP channel TRPM7, which results in early embryonic lethality. Nevertheless, the function of TRPM7 in oocytes, eggs and pre-implantation embryos remains unknown. Here, we described an outward rectifying non-selective current mediated by a TRP ion channel in immature oocytes (germinal vesicle stage), matured oocytes (metaphase II eggs) and 2-cell stage embryos. The current is activated by specific agonists and inhibited by distinct blockers consistent with the functional expression of TRPM7 channels. We demonstrated that the TRPM7-like channels are homo-tetramers and their activation mediates calcium influx in oocytes and eggs, which is fundamental to support fertilization and egg activation. Lastly, we showed that pharmacological inhibition of the channel function delays pre-implantation embryo development and reduces progression to the blastocyst stage. Our data demonstrate functional expression of TRPM7-like channels in mouse oocytes, eggs and embryos that may play an essential role in the initiation of embryo development. PMID:27681336

  2. Competition by meperidine for the organic cation renal excretory system.

    Science.gov (United States)

    Acara, M; Gessner, T; Trudnowski, R J

    1981-07-01

    Renal tubular excretory transport of meperidine was studied using the Sperber preparation in chickens. When urine samples from infused and uninfused kidneys were analyzed for meperidine by gas chromatography, meperidine was always present in greater amounts in the urine from the infused kidney, demonstrating active tubular excretion. Meperidine at an infusion rate of 1 mumole/min, also inhibited the excretion of the organic cations choline and acetylcholine, indicating occupation of the renal organic cation excretory system in the chicken.

  3. Evidence for a common pharmacological interaction site on K(Ca)2 channels providing both selective activation and selective inhibition of the human K(Ca)2.1 subtype

    DEFF Research Database (Denmark)

    Hougaard, Charlotte; Hammami, Sofia; Eriksen, Birgitte L;

    2012-01-01

    ]pyrimidines, act either as activators or as inhibitors of the human K(Ca)2.1 channel. Whereas (-)-CM-TPMF activates K(Ca)2.1 with an EC(50) value of 24 nM, (-)-B-TPMF inhibits the channel with an IC(50) value of 31 nM. In contrast, their (+)-enantiomers are 40 to 100 times less active. Both (-)-CM-TPMF and (-)-B......-TPMF are subtype-selective, with 10- to 20-fold discrimination toward other K(Ca)2 channels and the K(Ca)3 channel. Coapplication experiments reveal competitive-like functional interactions between the effects of (-)-CM-TPMF and (-)-B-TPMF. Despite belonging to a different chemical class than GW542573X, the K(Ca)2......-TPMF is 10 times more potent on K(Ca)2.1 than NS309 (6,7-dichloro-1H-indole-2,3-dione 3-oxime), an unselective but hitherto the most potent K(Ca)3/K(Ca)2 channel activator. (-)-B-TPMF is the first small-molecule inhibitor with significant selectivity among the K(Ca)2 channel subtypes. In contrast to peptide...

  4. Channel allo cation game algorithm based on lifetime mo del in wireless sensor network%基于生命期模型的无线传感器网络信道分配博弈算法∗

    Institute of Scientific and Technical Information of China (English)

    郝晓辰; 姚宁; 汝小月; 刘伟静; 辛敏洁

    2015-01-01

    针对无线传感器网络中节点因干扰过大导致重传能耗增加,进而节点过早失效、网络生命期缩短的问题,根据网络拓扑信息和路由信息设计节点的负载模型,从而构建了节点的生命期模型。然后利用博弈论将路径增益、交叉干扰和节点生命期等性能参数融入到效益函数中,构建信道分配博弈模型。理论分析证明该博弈模型存在纳什均衡。进而运用最佳回应策略,在所构建的信道分配博弈模型的基础上,设计了一种优化网络生命期的抗干扰信道分配算法。该算法使节点在选择信道时避免与网络中交叉干扰较大的节点和生命期较小的节点使用相同信道,实现干扰小、能耗低且均衡的信道选择。理论分析与仿真结果证明该算法最终能够快速地收敛到纳什均衡,且具有较小的信息复杂度,从而减小算法本身的通信能耗。同时,该算法具有良好的抗干扰性和信道均衡性,能够有效地延长网络生命期。%In wireless sensor network, the lager interference makes the data transmission failed, thus leading to data retrans-mission of nodes. This situation exacerbates the energy consumption of retransmission. As a result, some nodes will prematurely fail to work, thus reducing the network lifetime. In order to deal with the above issue, this paper takes full advantage of the topology and route information to design a novel load model of nodes. Then, a lifetime model of each node is constructed based on the load model. Subsequently, the path gain, intersecting interference and node lifetime are integrated into a utility function to construct a channel allocation game model called channel allocation based-game (CABG) by taking advantage of the game theory. The theoretical analysis proves the existence of the Nash Equilibrium of CABG. And then, using the best response dynamics, a channel allocation game algorithm for anti

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

    Directory of Open Access Journals (Sweden)

    Melissa A Fowler

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

  6. Pharmacologic inhibition of small-conductance calcium-activated potassium (SK) channels by NS8593 reveals atrial antiarrhythmic potential in horses

    DEFF Research Database (Denmark)

    Haugaard, Maria Mathilde; Hesselkilde, Eva Zander; Pehrson, Steen Michael

    2015-01-01

    BACKGROUND: Small-conductance calcium-activated potassium (SK) channels have been found to play an important role in atrial repolarization and atrial fibrillation (AF). OBJECTIVE: The purpose of this study was to investigate the existence and functional role of SK channels in the equine heart...

  7. Stimulation of large-conductance calcium-activated potassium channels inhibits neurogenic contraction of human bladder from patients with urinary symptoms and reverses acetic acid-induced bladder hyperactivity in rats.

    Science.gov (United States)

    La Fuente, José M; Fernández, Argentina; Cuevas, Pedro; González-Corrochano, Rocío; Chen, Mao Xiang; Angulo, Javier

    2014-07-15

    We have analysed the effects of large-conductance calcium-activated potassium channel (BK) stimulation on neurogenic and myogenic contraction of human bladder from healthy subjects and patients with urinary symptoms and evaluated the efficacy of activating BK to relief bladder hyperactivity in rats. Bladder specimens were obtained from organ donors and from men with benign prostatic hyperplasia (BPH). Contractions elicited by electrical field stimulation (EFS) and carbachol (CCh) were evaluated in isolated bladder strips. in vivo cystometric recordings were obtained in anesthetized rats under control and acetic acid-induced hyperactive conditions. Neurogenic contractions of human bladder were potentiated by blockade of BK and small-conductance calcium-activated potassium channels (SK) but were unaffected by the blockade of intermediate calcium-activated potassium channels (IK). EFS-induced contractions were inhibited by BK stimulation with NS-8 or NS1619 or by SK/IK stimulation with NS309 (3µM). CCh-induced contractions were not modified by blockade or stimulation of BK, IK or SK. The anti-cholinergic agent, oxybutynin (0.3µM) inhibited either neurogenic or CCh-induced contractions. Neurogenic contractions of bladders from BPH patients were less sensitive to BK inhibition and more sensitive to BK activation than healthy bladders. The BK activator, NS-8 (5mg/kg; i.v.), reversed bladder hyperactivity induced by acetic acid in rats, while oxybutynin was ineffective. NS-8 did not significantly impact blood pressure or heart rate. BK stimulation specifically inhibits neurogenic contractions in patients with urinary symptoms and relieves bladder hyperactivity in vivo without compromising bladder contractile capacity or cardiovascular safety, supporting its potential therapeutic use for relieving bladder overactivity.

  8. HCN Channels and Heart Rate

    Directory of Open Access Journals (Sweden)

    Ilaria Dentamaro

    2012-04-01

    Full Text Available Hyperpolarization and Cyclic Nucleotide (HCN -gated channels represent the molecular correlates of the “funny” pacemaker current (If, a current activated by hyperpolarization and considered able to influence the sinus node function in generating cardiac impulses. HCN channels are a family of six transmembrane domain, single pore-loop, hyperpolarization activated, non-selective cation channels. This channel family comprises four members: HCN1-4, but there is a general agreement to consider HCN4 as the main isoform able to control heart rate. This review aims to summarize advanced insights into the structure, function and cellular regulation of HCN channels in order to better understand the role of such channels in regulating heart rate and heart function in normal and pathological conditions. Therefore, we evaluated the possible therapeutic application of the selective HCN channels blockers in heart rate control.

  9. Lipopolysaccharide Neutralization by Cationic-Amphiphilic Polymers through Pseudoaggregate Formation.

    Science.gov (United States)

    Uppu, Divakara S S M; Haldar, Jayanta

    2016-03-14

    Synthetic polymers incorporating the cationic charge and hydrophobicity to mimic the function of antimicrobial peptides (AMPs) have been developed. These cationic-amphiphilic polymers bind to bacterial membranes that generally contain negatively charged phospholipids and cause membrane disintegration resulting in cell death; however, cationic-amphiphilic antibacterial polymers with endotoxin neutralization properties, to the best of our knowledge, have not been reported. Bacterial endotoxins such as lipopolysaccharide (LPS) cause sepsis that is responsible for a great amount of mortality worldwide. These cationic-amphiphilic polymers can also bind to negatively charged and hydrophobic LPS and cause detoxification. Hence, we envisaged that cationic-amphiphilic polymers can have both antibacterial as well as LPS binding properties. Here we report synthetic amphiphilic polymers with both antibacterial as well as endotoxin neutralizing properties. Levels of proinflammatory cytokines in human monocytes caused by LPS stimulation were inhibited by >80% when coincubated with these polymers. These reductions were found to be dependent on concentration and, more importantly, on the side-chain chemical structure due to variations in the hydrophobicity profiles of these polymers. These cationic-amphiphilic polymers bind and cause LPS neutralization and detoxification. Investigations of polymer interaction with LPS using fluorescence spectroscopy and dynamic light scattering (DLS) showed that these polymers bind but neither dissociate nor promote LPS aggregation. We show that polymer binding to LPS leads to sort of a pseudoaggregate formation resulting in LPS neutralization/detoxification. These findings provide an unusual mechanism of LPS neutralization using novel synthetic cationic-amphiphilic polymers.

  10. Competitive Effects of 2+ and 3+ Cations on DNA Compaction

    CERN Document Server

    Tongu, C; Yoshikawa, Y; Zinchenko, A A; Chen, N; Yoshikawa, K

    2016-01-01

    By using single-DNA observation with fluorescence microscopy, we observed the effects of divalent and trivalent cations on the higher-order structure of giant DNA (T4 DNA with 166 kbp). It was found that divalent cations, such as Mg(2+) and Ca(2+), inhibit DNA compaction induced by a trivalent cation, spermidine (SPD(3+)). On the other hand, in the absence of SPD(3+), divalent cations cause the shrinkage of DNA. These experimental observations are inconsistent with the well-established Debye-Huckel scheme regarding the shielding effect of counter ions, which is given as the additivity of contributions of cations with different valences. We interpreted the competition between 2+ and 3+ cations in terms of the change in the translational entropy of the counter ions before and after the folding transition of DNA. For the compaction with SPD(3+), we considered the increase in translational entropy due to the ion-exchange of the intrinsic monovalent cations condensing on a highly-charged polyelectrolyte, double-st...

  11. Mouse sperm patch-clamp recordings reveal single Cl- channels sensitive to niflumic acid, a blocker of the sperm acrosome reaction.

    Science.gov (United States)

    Espinosa, F; de la Vega-Beltrán, J L; López-González, I; Delgado, R; Labarca, P; Darszon, A

    1998-04-10

    Ion channels lie at the heart of gamete signaling. Understanding their regulation will improve our knowledge of sperm physiology, and may lead to novel contraceptive strategies. Sperm are tiny (approximately 3 microm diameter) and, until now, direct evidence of ion channel activity in these cells was lacking. Using patch-clamp recording we document here, for the first time, the presence of cationic and anionic channels in mouse sperm. Anion selective channels were blocked by niflumic acid (NA) (IC50 = 11 microM). The blocker was effective also in inhibiting the acrosome reaction induced by the zona pellucida, GABA or progesterone. These observations suggest that Cl- channels participate in the sperm acrosome reaction in mammals.

  12. Impaired fast-spiking, suppressed cortical inhibition and increased susceptibility to seizures in mice lacking Kv3.2 K+ channel proteins

    OpenAIRE

    Lau, David; Vega-Saenz de Miera, Eleazar; Contreras, Diego; Ozaita Mintegui, Andrés, 1969-; Harvey, Michael; Chow, Alan; Noebels, Jeffrey L; Paylor, Richard; Morgan, James I.; Leonard, Christopher S.; Rudy, Bernardo

    2000-01-01

    Voltage-gated K+ channels of the Kv3 subfamily have unusual electrophysiological properties, including activation at very depolarized voltages (positive to −10 mV) and very fast deactivation rates, suggesting special roles in neuronal excitability. In the brain, Kv3 channels are prominently expressed in select neuronal populations, which include fast-spiking (FS) GABAergic interneurons of the neocortex, hippocampus, and caudate, as well as other high-frequency firing neurons. Although evidenc...

  13. A Change in the Ion Selectivity of Ligand-Gated Ion Channels Provides a Mechanism to Switch Behavior.

    Directory of Open Access Journals (Sweden)

    Jennifer K Pirri

    Full Text Available Behavioral output of neural networks depends on a delicate balance between excitatory and inhibitory synaptic connections. However, it is not known whether network formation and stability is constrained by the sign of synaptic connections between neurons within the network. Here we show that switching the sign of a synapse within a neural circuit can reverse the behavioral output. The inhibitory tyramine-gated chloride channel, LGC-55, induces head relaxation and inhibits forward locomotion during the Caenorhabditis elegans escape response. We switched the ion selectivity of an inhibitory LGC-55 anion channel to an excitatory LGC-55 cation channel. The engineered cation channel is properly trafficked in the native neural circuit and results in behavioral responses that are opposite to those produced by activation of the LGC-55 anion channel. Our findings indicate that switches in ion selectivity of ligand-gated ion channels (LGICs do not affect network connectivity or stability and may provide an evolutionary and a synthetic mechanism to change behavior.

  14. Heteromultimeric TRPML channel assemblies play a crucial role in the regulation of cell viability models and starvation-induced autophagy.

    Science.gov (United States)

    Zeevi, David A; Lev, Shaya; Frumkin, Ayala; Minke, Baruch; Bach, Gideon

    2010-09-15

    The mucolipin (TRPML) subfamily of transient receptor potential (TRP) cation channels consists of three members that play various roles in the regulation of membrane and protein sorting along endo-lysosomal pathways. Loss-of-function mutations in TRPML1 cause the neurodegenerative lysosomal storage disorder, mucolipidosis type IV (MLIV), whereas a gain-of-function mutation in TRPML3 is principally implicated in the hearing-impaired and abnormally pigmented varitint-waddler mouse. Currently, TRPML2 is not implicated in any pathological disorder, but we have recently shown that it is a functional cation channel that physically interacts with TRPML1 and TRPML3 to potentially regulate lysosomal integrity. Here, we show that mutant TRPMLs heteromultimerize with other mutant and wild-type TRPMLs to regulate cell viability and starvation-induced autophagy, a process that mediates macromolecular and organellar turnover under cell starvation conditions. Heteromultimerization of dominant-negative TRPMLs with constitutively active TRPMLs rescues cells from the cytotoxic effects of TRPML constitutive activity. Moreover, dominant-negative TRPML1 channels, including a mutant channel directly implicated in MLIV pathology, also inhibit starvation-induced autophagy by interacting with and affecting native TRPML channel function. Collectively, our results indicate that heteromultimerization of TRPML channels plays a role in various TRPML-regulated mechanisms.

  15. Inhibition of Ca2+-activated large-conductance K+ channel activity alters synaptic AMPA receptor phenotype in mouse cerebellar stellate cells.

    Science.gov (United States)

    Liu, Yu; Savtchouk, Iaroslav; Acharjee, Shoana; Liu, Siqiong June

    2011-07-01

    Many fast-spiking inhibitory interneurons, including cerebellar stellate cells, fire brief action potentials and express α-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA)-type glutamate receptors (AMPAR) that are permeable to Ca(2+) and do not contain the GluR2 subunit. In a recent study, we found that increasing action potential duration promotes GluR2 gene transcription in stellate cells. We have now tested the prediction that activation of potassium channels that control the duration of action potentials can suppress the expression of GluR2-containing AMPARs at stellate cell synapses. We find that large-conductance Ca(2+)-activated potassium (BK) channels mediate a large proportion of the depolarization-evoked noninactivating potassium current in stellate cells. Pharmacological blockade of BK channels prolonged the action potential duration in postsynaptic stellate cells and altered synaptic AMPAR subtype from GluR2-lacking to GluR2-containing Ca(2+)-impermeable AMPARs. An L-type channel blocker abolished an increase in Ca(2+) entry that was associated with spike broadening and also prevented the BK channel blocker-induced switch in AMPAR phenotype. Thus blocking BK potassium channels prolongs the action potential duration and increases the expression of GluR2-containing receptors at the synapse by enhancing Ca(2+) entry in cerebellar stellate cells.

  16. Characterization of the slow calcium channel binding sites for ( sup 3 H)SR 33557 in rat heart sarcolemmal membranes

    Energy Technology Data Exchange (ETDEWEB)

    Chatelain, P.; Beaufort, P.; Meysmans, L.; Clinet, M. (Sanofi-Labaz Research Centre, Brussels (Belgium))

    1991-01-01

    SR 33557 represents a new class of compounds (indolizine sulfone) that inhibit L-type Ca2+ channels. ({sup 3}H)SR 33557 has been shown to bind with high affinity (Kd congruent to 0.36 nM, calculated from saturation isotherms and association/dissociation kinetics) to a single class of sites in a purified preparation of rat cardiac sarcolemmal membranes. The binding was found to be saturable and reversible. The maximal binding capacity was in approximately 1:1 stoichiometry with that of other Ca2+ channel antagonists. Various divalent cations (Mg2+, Mn2+, Ca2+, Ba2+, and Cd2+) were shown to inhibit specific ({sup 3}H)SR 33557 binding, with Cd2+ being the most potent. Among several receptor or channel ligands (including omega-conotoxin and Na+ and K+ channel modulators), only the L-type Ca2+ channel antagonists were found to displace ({sup 3}H)SR 33557. However, dihydropyridines, phenylalkylamines, benzothiazepines, and diphenylbutylpiperidines were found to inhibit ({sup 3}H)SR 33557 in a noncompetitive manner as demonstrated by displacement and saturation experiments in addition to dissociation kinetics. From these results, we suggest that SR 33557 binds with high affinity to a unique site on the L-type Ca2+ channel found in rat cardiac sarcolemmal membranes.

  17. TPEN, a Specific Zn(2+) Chelator, Inhibits Sodium Dithionite and Glucose Deprivation (SDGD)-Induced Neuronal Death by Modulating Apoptosis, Glutamate Signaling, and Voltage-Gated K(+) and Na(+) Channels.

    Science.gov (United States)

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

    2017-03-01

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

  18. 离子交换型缓蚀填料在防腐蚀涂层中的应用Ⅰ阳离子交换型填料%Application of Ion-exchange Compounds as Corrosion Inhibiting Pigments to Organic Anticorrosion Coatings Ⅰ Cation-exchange Pigments

    Institute of Scientific and Technical Information of China (English)

    吴俊升; 肖葵; 李欣荣; 董超芳; 李晓刚

    2011-01-01

    铬酸盐等重金属类缓蚀性颜填料会对环境造成严重的污染,未来该类有害物质在防腐蚀涂层中的应用将被禁止。新型的离子交换型填料因其具有可同时释放缓蚀性离子和吸附固定侵蚀性离子(H+、Cl-、SO24-等)的双重功效,被认为是替代传统重金属类颜填料的理想材料。本文对Zn2+、Ce3+、Ca2+等缓蚀性阳离子改性膨润土、氧化硅、分子筛等新型离子交换型缓蚀填料在有机防腐蚀涂层中的应用进行了综述。%It is well documented that chromate pigments and other heavy metal compounds must be eliminated from organic anticorrosion coatings due to their toxic nature and carcinogenic effects in the future. The novel ion-exchange pigments can play a double role of absorbing the harmful ions such as H+ ,C1- ,SO24- and releasing the inhibiting ions on contact with aggressive electrolyte invading the coating. The released inhibitors can provide active corrosion protection to the defects in the coating and substrate, on the other hand, the uptake of harmful ions decreases the aggressiveness of the corrosive medium, and thereby reduces the rate of ecorrosion processes. Therefore, such new inhibiting compounds have been developed as the potential alternate materials of the traditional toxic pigments, and have attracted a lot of attention. In this paper, the application of ion-exchange pigments, such as Ce3+ , Znz+ cation-exchanged bentonite, Ca2+ cation-exchanged silica and MoO22+ cation-exchanged zeolite, to organic anticorrosion coatings is reviewed.

  19. Effects of disodium cromoglycate on cationic exchange of deoxygenated sickle cells.

    Science.gov (United States)

    Bizumukama, Léonidas; Ferster, Alina; Gulbis, Béatrice; Kumps, Alain; Cotton, Frédéric

    2011-08-31

    In the present work, we explored the way in which cromoglycate, a drug used to treat allergies acts on ion movements in sickle cells. Cells were either slowly deoxygenated by overnight exposure to nitrogen or acutely deoxygenated by exposure to metabisulfite, a strong reducing agent which induces sickling of red blood sickle cells. Flushing the cells with nitrogen increased the intracellular concentration of Na(+) and decreased the intracellular concentration of K(+) and the sum of the concentrations of the two cations. One hundred nM cromoglycate inhibited the decrease of intracellular K(+) and the increase of intracellular Na(+) induced by deoxygenation (n=17). Metabisulfite (100mM) increased the intracellular concentration of Ca(2+) (measured by Fura Red) (n=15) and the shape of the cells (measured by light scattering) (n=9). One μM cromoglycate partially inhibited these two responses. In conclusion, cromoglycate partially inhibits abnormal K(+) loss, Ca(2+) entry pathways or Ca(2+) channels opened by cell deoxygenation and ensuing membrane modifications and prevents cell sickling.

  20. Kv1.5 blockers preferentially inhibit TASK-1 channels: TASK-1 as a target against atrial fibrillation and obstructive sleep apnea?

    Science.gov (United States)

    Kiper, Aytug K; Rinné, Susanne; Rolfes, Caroline; Ramírez, David; Seebohm, Guiscard; Netter, Michael F; González, Wendy; Decher, Niels

    2015-05-01

    Atrial fibrillation and obstructive sleep apnea are responsible for significant morbidity and mortality in the industrialized world. There is a high medical need for novel drugs against both diseases, and here, Kv1.5 channels have emerged as promising drug targets. In humans, TASK-1 has an atrium-specific expression and TASK-1 is also abundantly expressed in the hypoglossal motor nucleus. We asked whether known Kv1.5 channel blockers, effective against atrial fibrillation and/or obstructive sleep apnea, modulate TASK-1 channels. Therefore, we tested Kv1.5 blockers with different chemical structures for their TASK-1 affinity, utilizing two-electrode voltage clamp (TEVC) recordings in Xenopus oocytes. Despite the low structural conservation of Kv1.5 and TASK-1 channels, we found all Kv1.5 blockers analyzed to be even more effective on TASK-1 than on Kv1.5. For instance, the half-maximal inhibitory concentration (IC50) values of AVE0118 and AVE1231 (A293) were 10- and 43-fold lower on TASK-1. Also for MSD-D, ICAGEN-4, S20951 (A1899), and S9947, the IC50 values were 1.4- to 70-fold lower than for Kv1.5. To describe this phenomenon on a molecular level, we used in silico models and identified unexpected structural similarities between the two drug binding sites. Kv1.5 blockers, like AVE0118 and AVE1231, which are promising drugs against atrial fibrillation or obstructive sleep apnea, are in fact potent TASK-1 blockers. Accordingly, block of TASK-1 channels by these compounds might contribute to the clinical effectiveness of these drugs. The higher affinity of these blockers for TASK-1 channels suggests that TASK-1 might be an unrecognized molecular target of Kv1.5 blockers effective in atrial fibrillation or obstructive sleep apnea.

  1. Chemo-nociceptive signalling from the colon is enhanced by mild colitis and blocked by inhibition of transient receptor potential ankyrin 1 channels

    DEFF Research Database (Denmark)

    Mitrovic, Martina; Shahbazian, Anaid; Bock, Elisabeth;

    2010-01-01

    Transient receptor potential ankyrin 1 (TRPA1) channels are expressed by primary afferent neurones and activated by irritant chemicals including allyl isothiocyanate (AITC). Here we investigated whether intracolonic AITC causes afferent input to the spinal cord and whether this response is modified...

  2. Effects of acidic pH on voltage-gated ion channels in rat trigeminal mesencephalic nucleus neurons.

    Science.gov (United States)

    Han, Jin-Eon; Cho, Jin-Hwa; Choi, In-Sun; Kim, Do-Yeon; Jang, Il-Sung

    2017-03-01

    The effects of acidic pH on several voltage-dependent ion channels, such as voltage-dependent K(+) and Ca(2+) channels, and hyperpolarization-gated and cyclic nucleotide-activated cation (HCN) channels, were examined using a whole-cell patch clamp technique on mechanically isolated rat mesencephalic trigeminal nucleus neurons. The application of a pH 6.5 solution had no effect on the peak amplitude of voltage-dependent K(+) currents. A pH 6.0 solution slightly, but significantly inhibited the peak amplitude of voltage-dependent K(+) currents. The pH 6.0 also shifted both the current-voltage and conductance-voltage relationships to the depolarization range. The application of a pH 6.5 solution scarcely affected the peak amplitude of membrane currents mediated by HCN channels, which were profoundly inhibited by the general HCN channel blocker Cs(+) (1 mM). However, the pH 6.0 solution slightly, but significantly inhibited the peak amplitude of HCN-mediated currents. Although the pH 6.0 solution showed complex modulation of the current-voltage and conductance-voltage relationships, the midpoint voltages for the activation of HCN channels were not changed by acidic pH. On the other hand, voltage-dependent Ca(2+) channels were significantly inhibited by an acidic pH. The application of an acidic pH solution significantly shifted the current-voltage and conductance-voltage relationships to the depolarization range. The modulation of several voltage-dependent ion channels by an acidic pH might affect the excitability of mesencephalic trigeminal nucleus neurons, and thus physiological functions mediated by the mesencephalic trigeminal nucleus could be affected in acidic pH conditions.

  3. Impaired fast-spiking, suppressed cortical inhibition, and increased susceptibility to seizures in mice lacking Kv3.2 K+ channel proteins.

    Science.gov (United States)

    Lau, D; Vega-Saenz de Miera, E C; Contreras, D; Ozaita, A; Harvey, M; Chow, A; Noebels, J L; Paylor, R; Morgan, J I; Leonard, C S; Rudy, B

    2000-12-15

    Voltage-gated K(+) channels of the Kv3 subfamily have unusual electrophysiological properties, including activation at very depolarized voltages (positive to -10 mV) and very fast deactivation rates, suggesting special roles in neuronal excitability. In the brain, Kv3 channels are prominently expressed in select neuronal populations, which include fast-spiking (FS) GABAergic interneurons of the neocortex, hippocampus, and caudate, as well as other high-frequency firing neurons. Although evidence points to a key role in high-frequency firing, a definitive understanding of the function of these channels has been hampered by a lack of selective pharmacological tools. We therefore generated mouse lines in which one of the Kv3 genes, Kv3.2, was disrupted by gene-targeting methods. Whole-cell electrophysiological recording showed that the ability to fire spikes at high frequencies was impaired in immunocytochemically identified FS interneurons of deep cortical layers (5-6) in which Kv3.2 proteins are normally prominent. No such impairment was found for FS neurons of superficial layers (2-4) in which Kv3.2 proteins are normally only weakly expressed. These data directly support the hypothesis that Kv3 channels are necessary for high-frequency firing. Moreover, we found that Kv3.2 -/- mice showed specific alterations in their cortical EEG patterns and an increased susceptibility to epileptic seizures consistent with an impairment of cortical inhibitory mechanisms. This implies that, rather than producing hyperexcitability of the inhibitory interneurons, Kv3.2 channel elimination suppresses their activity. These data suggest that normal cortical operations depend on the ability of inhibitory interneurons to generate high-frequency firing.

  4. Effects of hydrochloric acid modification on the channel characteristics, composition, surface potential and cation exchange behavior of natural clinoptilolite%盐酸改性对天然斜发沸石孔道特征、成分、表面电位及阳离子交换性能的影响

    Institute of Scientific and Technical Information of China (English)

    霍汉鑫; 林海; 董颖博; 刘泉利; 曹丽霞; 程皝; 汪涵; 傅川

    2015-01-01

    采用不同浓度盐酸对天然斜发沸石进行改性,并系统地研究了改性沸石的孔道特征、化学成分、表面电位及阳离子交换性能的变化.盐酸改性后,沸石晶体结构破坏较小,表面变得疏松粗糙,K+、Na+、Ca2+和Mg2+元素含量均小幅下降;表面负电荷增加,阳离子交换容量减小;比表面积和总孔体积均有所提高,最高分别从原沸石的35.97 m2·g -1和0.0761 m3·g-1提高至64.46 m2·g-1和0.1156 m3·g-1.盐酸改性对沸石微孔、介孔和大孔的分布影响明显.从迟滞回线形状判断沸石孔道类型均为不均匀狭缝型孔道,盐酸改性不会改变沸石孔道类型.%ABSTRACT Natural clinoptilolite was modified by different concentrations of hydrochloric acid. Systemic investigations were performed on the channel characteristics, chemical composition, surface potential, and cation exchange behavior of the modified clinoptilolite. It is found that the surface of the modified clinoptilolite becomes loose and rough, the contents of K+, Na+, Ca2+, and Mg2+ slightly decrease, the surface negative charges increases, and the cation exchange capacity decreases to some extent. The specif-ic surface area and total pore volume are improved and the maximum values of them increase from 35.97 m2·g-1 and 0.0761 m3·g-1 to 64.46 m2·g-1 and 0.1156 m3·g-1 , respectively. The distribution of micropores, mesopores, and macropores is obviously impacted, and the type of zeolite cavities is determined to be uneven slits by the hysteresis loop shape, indicating that this acid modification cannot change zeolite cavities.

  5. Binding of the auxiliary subunit TRIP8b to HCN channels shifts the mode of action of cAMP.

    Science.gov (United States)

    Hu, Lei; Santoro, Bina; Saponaro, Andrea; Liu, Haiying; Moroni, Anna; Siegelbaum, Steven

    2013-12-01

    Hyperpolarization-activated cyclic nucleotide-regulated cation (HCN) channels generate the hyperpolarization-activated cation current Ih present in many neurons. These channels are directly regulated by the binding of cAMP, which both shifts the voltage dependence of HCN channel opening to more positive potentials and increases maximal Ih at extreme negative voltages where voltage gating is complete. Here we report that the HCN channel brain-specific auxiliary subunit TRIP8b produces opposing actions on these two effects of cAMP. In the first action, TRIP8b inhibits the effect of cAMP to shift voltage gating, decreasing both the sensitivity of the channel to cAMP (K1/2) and the efficacy of cAMP (maximal voltage shift); conversely, cAMP binding inhibits these actions of TRIP8b. These mutually antagonistic actions are well described by a cyclic allosteric mechanism in which TRIP8b binding reduces the affinity of the channel for cAMP, with the affinity of the open state for cAMP being reduced to a greater extent than the cAMP affinity of the closed state. In a second apparently independent action, TRIP8b enhances the action of cAMP to increase maximal Ih. This latter effect cannot be explained by the cyclic allosteric model but results from a previously uncharacterized action of TRIP8b to reduce maximal current through the channel in the absence of cAMP. Because the binding of cAMP also antagonizes this second effect of TRIP8b, application of cAMP produces a larger increase in maximal Ih in the presence of TRIP8b than in its absence. These findings may provide a mechanistic explanation for the wide variability in the effects of modulatory transmitters on the voltage gating and maximal amplitude of Ih reported for different neurons in the brain.

  6. TRP Channels in Skin Biology and Pathophysiology

    OpenAIRE

    2016-01-01

    Ion channels of the Transient Receptor Potential (TRP) family mediate the influx of monovalent and/or divalent cations into cells in response to a host of chemical or physical stimuli. In the skin, TRP channels are expressed in many cell types, including keratinocytes, sensory neurons, melanocytes, and immune/inflammatory cells. Within these diverse cell types, TRP channels participate in physiological processes ranging from sensation to skin homeostasis. In addition, there is a growing body ...

  7. Statins and selective inhibition of Rho kinase protect small conductance calcium-activated potassium channel function (K(Ca2.3 in cerebral arteries.

    Directory of Open Access Journals (Sweden)

    Alister J McNeish

    Full Text Available BACKGROUND: In rat middle cerebral and mesenteric arteries the K(Ca2.3 component of endothelium-dependent hyperpolarization (EDH is lost following stimulation of thromboxane (TP receptors, an effect that may contribute to the endothelial dysfunction associated with cardiovascular disease. In cerebral arteries, K(Ca2.3 loss is associated with NO synthase inhibition, but is restored if TP receptors are blocked. The Rho/Rho kinase pathway is central for TP signalling and statins indirectly inhibit this pathway. The possibility that Rho kinase inhibition and statins sustain K(Ca2.3 hyperpolarization was investigated in rat middle cerebral arteries (MCA. METHODS: MCAs were mounted in a wire myograph. The PAR2 agonist, SLIGRL was used to stimulate EDH responses, assessed by simultaneous measurement of smooth muscle membrane potential and tension. TP expression was assessed with rt-PCR and immunofluorescence. RESULTS: Immunofluorescence detected TP in the endothelial cell layer of MCA. Vasoconstriction to the TP agonist, U46619 was reduced by Rho kinase inhibition. TP receptor stimulation lead to loss of K(Ca2.3 mediated hyperpolarization, an effect that was reversed by Rho kinase inhibitors or simvastatin. K(Ca2.3 activity was lost in L-NAME-treated arteries, but was restored by Rho kinase inhibition or statin treatment. The restorative effect of simvastatin was blocked after incubation with geranylgeranyl-pyrophosphate to circumvent loss of isoprenylation. CONCLUSIONS: Rho/Rho kinase signalling following TP stimulation and L-NAME regulates endothelial cell K(Ca2.3 function. The ability of statins to prevent isoprenylation and perhaps inhibit of Rho restores/protects the input of K(Ca2.3 to EDH in the MCA, and represents a beneficial pleiotropic effect of statin treatment.

  8. Fusion Pore Diameter Regulation by Cations Modulating Local Membrane Anisotropy

    Directory of Open Access Journals (Sweden)

    Doron Kabaso

    2012-01-01

    Full Text Available The fusion pore is an aqueous channel that is formed upon the fusion of the vesicle membrane with the plasma membrane. Once the pore is open, it may close again (transient fusion or widen completely (full fusion to permit vesicle cargo discharge. While repetitive transient fusion pore openings of the vesicle with the plasma membrane have been observed in the absence of stimulation, their frequency can be further increased using a cAMP-increasing agent that drives the opening of nonspecific cation channels. Our model hypothesis is that the openings and closings of the fusion pore are driven by changes in the local concentration of cations in the connected vesicle. The proposed mechanism of fusion pore dynamics is considered as follows: when the fusion pore is closed or is extremely narrow, the accumulation of cations in the vesicle (increased cation concentration likely leads to lipid demixing at the fusion pore. This process may affect local membrane anisotropy, which reduces the spontaneous curvature and thus leads to the opening of the fusion pore. Based on the theory of membrane elasticity, we used a continuum model to explain the rhythmic opening and closing of the fusion pore.

  9. Tris(triazole) tripodal receptors as selective probes for citrate anion recognition and multichannel transition and heavy metal cation sensing.

    Science.gov (United States)

    González, María del Carmen; Otón, Francisco; Espinosa, Arturo; Tárraga, Alberto; Molina, Pedro

    2015-02-01

    The three-armed pyrenyl-triazole receptor 1 behaves as a highly selective fluorescent molecular sensor for citrate anions over similar carboxylates such as malate or tartrate. In addition, this receptor senses Cu(2+) cations through absorption and emission channels even in the presence of Hg(2+) metal cations. The related three-armed ferrocenyl-triazole receptor 2 behaves as a highly selective dual (redox and chromogenic) chemosensor molecule for Pb(2+) metal cations.

  10. Asymmetric cation-binding catalysis

    DEFF Research Database (Denmark)

    Oliveira, Maria Teresa; Lee, Jiwoong

    2017-01-01

    and KCN, are selectively bound to the catalyst, providing exceptionally high enantioselectivities for kinetic resolutions, elimination reactions (fluoride base), and Strecker synthesis (cyanide nucleophile). Asymmetric cation-binding catalysis was recently expanded to silicon-based reagents, enabling...... solvents, thus increasing their applicability in synthesis. The expansion of this concept to chiral polyethers led to the emergence of asymmetric cation-binding catalysis, where chiral counter anions are generated from metal salts, particularly using BINOL-based polyethers. Alkali metal salts, namely KF...

  11. Menthol inhibits detrusor contractility independently of TRPM8 activation.

    Directory of Open Access Journals (Sweden)

    Antonio Celso Saragossa Ramos-Filho

    Full Text Available Agonists such as icilin and menthol can activate the cool temperature-sensitive ion channel TRPM8. However, biological responses to menthol may occur independently of TRPM8 activation. In the rodent urinary bladder, menthol facilitates the micturition reflex but inhibits muscarinic contractions of the detrusor smooth muscle. The site(s of TRPM8 expression in the bladder are controversial. In this study we investigated the regulation of bladder contractility in vitro by menthol. Bladder strips from wild type and TRPM8 knockout male mice (25-30 g were dissected free and mounted in organ baths. Isometric contractions to carbachol (1 nM-30 µM, CaCl2 (1 µM to 100 mM and electrical field stimulation (EFS; 8, 16, 32 Hz were measured. Strips from both groups contracted similarly in response to both carbachol and EFS. Menthol (300 µM or nifedipine (1 µM inhibited carbachol and EFS-induced contractions in both wild type and TRPM8 knockout bladder strips. Incubation with the sodium channel blocker tetrodotoxin (1 µM, replacement of extracellular sodium with the impermeant cation N-Methyl-D-Glucamine, incubation with a cocktail of potassium channel inhibitors (100 nM charybdotoxin, 1 µM apamin, 10 µM glibenclamide and 1 µM tetraethylammonium or removal of the urothelium did not affect the inhibitory actions of menthol. Contraction to CaCl2 was markedly inhibited by either menthol or nifedipine. In cultured bladder smooth muscle cells, menthol or nifedipine abrogated the carbachol or KCl-induced increases in [Ca2+]i. Intravesical administration of menthol increased voiding frequency while decreasing peak voiding pressure. We conclude that menthol inhibits muscarinic bladder contractions through blockade of L-type calcium channels, independently of TRPM8 activation.

  12. Menthol inhibits detrusor contractility independently of TRPM8 activation.

    Science.gov (United States)

    Ramos-Filho, Antonio Celso Saragossa; Shah, Ajay; Augusto, Taize Machado; Barbosa, Guilherme Oliveira; Leiria, Luiz Osorio; de Carvalho, Hernandes Faustino; Antunes, Edson; Grant, Andrew Douglas

    2014-01-01

    Agonists such as icilin and menthol can activate the cool temperature-sensitive ion channel TRPM8. However, biological responses to menthol may occur independently of TRPM8 activation. In the rodent urinary bladder, menthol facilitates the micturition reflex but inhibits muscarinic contractions of the detrusor smooth muscle. The site(s) of TRPM8 expression in the bladder are controversial. In this study we investigated the regulation of bladder contractility in vitro by menthol. Bladder strips from wild type and TRPM8 knockout male mice (25-30 g) were dissected free and mounted in organ baths. Isometric contractions to carbachol (1 nM-30 µM), CaCl2 (1 µM to 100 mM) and electrical field stimulation (EFS; 8, 16, 32 Hz) were measured. Strips from both groups contracted similarly in response to both carbachol and EFS. Menthol (300 µM) or nifedipine (1 µM) inhibited carbachol and EFS-induced contractions in both wild type and TRPM8 knockout bladder strips. Incubation with the sodium channel blocker tetrodotoxin (1 µM), replacement of extracellular sodium with the impermeant cation N-Methyl-D-Glucamine, incubation with a cocktail of potassium channel inhibitors (100 nM charybdotoxin, 1 µM apamin, 10 µM glibenclamide and 1 µM tetraethylammonium) or removal of the urothelium did not affect the inhibitory actions of menthol. Contraction to CaCl2 was markedly inhibited by either menthol or nifedipine. In cultured bladder smooth muscle cells, menthol or nifedipine abrogated the carbachol or KCl-induced increases in [Ca2+]i. Intravesical administration of menthol increased voiding frequency while decreasing peak voiding pressure. We conclude that menthol inhibits muscarinic bladder contractions through blockade of L-type calcium channels, independently of TRPM8 activation.

  13. Differential role of the menthol-binding residue Y745 in the antagonism of thermally gated TRPM8 channels

    Directory of Open Access Journals (Sweden)

    Ferrer-Montiel Antonio

    2009-11-01

    Full Text Available Abstract Background TRPM8 is a non-selective cation channel that belongs to the melastatin subfamily of the transient receptor potential (TRP ion channels. TRPM8 is activated by voltage, cold and cooling compounds such as menthol. Despite its essential role for cold temperature sensing in mammals, the pharmacology of TRPM8 is still in its infancy. Recently, tyrosine 745 (Y745 was identified as a critical residue for menthol sensitivity of the channel. In this report, we study the effect of mutating this residue on the action of several known TRPM8 antagonists: BCTC, capsazepine, SKF96365, and clotrimazole as well as two new inhibitor candidates, econazole and imidazole. Results We show that Y745 at the menthol binding site is critical for inhibition mediated by SKF96365 of cold- and voltage-activated TRPM8 currents. In contrast, the inhibition by other antagonists was unaffected by the mutation (BCTC or only partially reduced (capsazepine, clotrimazole, econazole, suggesting that additional binding sites exist on the TRPM8 channel from where the inhibitors exert their negative modulation. Indeed, a molecular docking model implies that menthol and SKF96365 interact readily with Y745, while BCTC is unable to bind to this residue. Conclusion In summary, we identify structural elements on the TRPM8 channel that are critical for the action of channel antagonists, providing valuable information for the future design of new, specific modulator compounds.

  14. 1,25 (OH)2D3 enhances PTH-induced Ca2+ transients in preosteoblasts by activating L-type Ca2+ channels

    Science.gov (United States)

    Li, W.; Duncan, R. L.; Karin, N. J.; Farach-Carson, M. C.

    1997-01-01

    We previously demonstrated electrophysiologically that 1,25-dihydroxyvitamin D3 [1,25(OH)2D3] shifts the activation threshold of L-type Ca2+ channels in osteoblasts toward the resting potential and prolongs mean open time. Presently, we used single-cell Ca2+ imaging to study the combined effects of 1,25(OH)2D3 and parathyroid hormone (PTH) during generation of Ca2+ transients in fura 2-loaded MC3T3-E1 cells. Pretreatment with 1,25(OH)2D3 concentrations, which alone did not produce Ca2+ transients, consistently enhanced Ca2+ responses to PTH. Enhancement was dose dependent over the range of 1 to 10 nM and was blocked by pretreatment with 5 microM nitrendipine during pretreatment. A 1,25(OH)2D3 analog that activates L-type channels and shifts their activation threshold also enhanced PTH responses. In contrast, an analog devoid of membrane Ca2+ effects did not enhance PTH-induced Ca2+ transients. The PTH-induced Ca2+ transient involved activation of a dihydropyridine-insensitive cation channel that was inhibited by Gd3+. Together, these data suggest that 1,25(OH)2D3 increases osteoblast responsiveness to PTH through rapid modification of L-type Ca2+ channel gating properties, whose activation enhances Ca2+ entry through other channels such as the PTH-responsive, Gd(3+)-sensitive cation channel.

  15. Highly Selective Artificial K(+) Channels: An Example of Selectivity-Induced Transmembrane Potential.

    Science.gov (United States)

    Gilles, Arnaud; Barboiu, Mihail

    2016-01-13

    Natural KcsA K(+) channels conduct at high rates with an extraordinary selectivity for K(+) cations, excluding the Na(+) or other cations. Biomimetic artificial channels have been designed in order to mimick the ionic activity of KcSA channels, but simple artificial systems presenting high K(+)/Na(+) selectivity are rare. Here we report an artificial ion channel of H-bonded hexyl-benzoureido-15-crown-5-ether, where K(+) cations are highly preferred to Na(+) cations. The K(+)-channel conductance is interpreted as arising in the formation of oligomeric highly cooperative channels, resulting in the cation-induced membrane polarization and enhanced transport rates without or under pH-active gradient. These channels are selectively responsive to the presence of K(+) cations, even in the presence of a large excess of Na(+). From the conceptual point of view, these channels express a synergistic adaptive behavior: the addition of the K(+) cation drives the selection and the construction of constitutional polarized ion channels toward the selective conduction of the K(+) cation that promotes their generation in the first place.

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

    Science.gov (United States)

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

    2014-08-01

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

  17. CyPPA, a positive modulator of small-conductance Ca(2+)-activated K(+) channels, inhibits phasic uterine contractions and delays preterm birth in mice.

    Science.gov (United States)

    Skarra, Dana V; Cornwell, Trudy; Solodushko, Viktoriya; Brown, Amber; Taylor, Mark S

    2011-11-01

    Organized uterine contractions, including those necessary for parturition, are dependent on calcium entry through voltage-gated calcium channels in myometrial smooth muscle cells. Recent evidence suggests that small-conductance Ca(2+)-activated potassium channels (K(Ca)2), specifically isoforms K(Ca)2.2 and 2.3, may control these contractions through negative feedback regulation of Ca(2+) entry. We tested whether selective pharmacologic activation of K(Ca)2.2/2.3 channels might depress uterine contractions, providing a new strategy for preterm labor intervention. Western blot analysis and immunofluorescence microscopy revealed expression of both K(Ca)2.2 and K(Ca)2.3 in the myometrium of nonpregnant (NP) and pregnant (gestation day 10 and 16; D10 and D16, respectively) mice. Spontaneous phasic contractions of isolated NP, D10, and D16 uterine strips were all suppressed by the K(Ca)2.2/2.3-selective activator CyPPA in a concentration-dependent manner. This effect was antagonized by the selective K(Ca)2 inhibitor apamin. Whereas CyPPA sensitivity was reduced in D10 and D16 versus NP strips (pIC(50) 5.33 ± 0.09, 4.64 ± 0.03, 4.72 ± 0.10, respectively), all contractions were abolished between 30 and 60 μM. Blunted contractions were associated with CyPPA depression of spontaneous Ca(2+) events in myometrial smooth muscle bundles. Augmentation of uterine contractions with oxytocin or prostaglandin F(2α) did not reduce CyPPA sensitivity or efficacy. Finally, in an RU486-induced preterm labor model, CyPPA significantly delayed time to delivery by 3.4 h and caused a 2.5-fold increase in pup retention. These data indicate that pharmacologic stimulation of myometrial K(Ca)2.2/2.3 channels effectively suppresses Ca(2+)-mediated uterine contractions and delays preterm birth in mice, supporting the potential utility of this approach in tocolytic therapies.

  18. Inhibition of Ca2+-activated large-conductance K+ channel activity alters synaptic AMPA receptor phenotype in mouse cerebellar stellate cells

    OpenAIRE

    Yu LIU; Savtchouk, Iaroslav; Acharjee, Shoana; Liu, Siqiong June

    2011-01-01

    Many fast-spiking inhibitory interneurons, including cerebellar stellate cells, fire brief action potentials and express α-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA)-type glutamate receptors (AMPAR) that are permeable to Ca2+ and do not contain the GluR2 subunit. In a recent study, we found that increasing action potential duration promotes GluR2 gene transcription in stellate cells. We have now tested the prediction that activation of potassium channels that control the durati...

  19. TRPM8 ion channels differentially modulate proliferation and cell cycle distribution of normal and cancer prostate cells.

    Science.gov (United States)

    Valero, María Ll; Mello de Queiroz, Fernanda; Stühmer, Walter; Viana, Félix; Pardo, Luis A

    2012-01-01

    Overexpression of the cation-permeable channel TRPM8 in prostate cancers might represent a novel opportunity for their treatment. Inhibitors of TRPM8 reduce the growth of prostate cancer cells. We have used two recently described and highly specific blockers, AMTB and JNJ41876666, and RNAi to determine the relevance of TRPM8 expression in the proliferation of non-tumor and tumor cells. Inhibition of the expression or function of the channel reduces proliferation rates and proliferative fraction in all tumor cells tested, but not of non-tumor prostate cells. We observed no consistent acceleration of growth after stimulation of the channel with menthol or icilin, indicating that basal TRPM8 expression is enough to sustain growth of prostate cancer cells.

  20. TRPM8 ion channels differentially modulate proliferation and cell cycle distribution of normal and cancer prostate cells.

    Directory of Open Access Journals (Sweden)

    María Ll Valero

    Full Text Available Overexpression of the cation-permeable channel TRPM8 in prostate cancers might represent a novel opportunity for their treatment. Inhibitors of TRPM8 reduce the growth of prostate cancer cells. We have used two recently described and highly specific blockers, AMTB and JNJ41876666, and RNAi to determine the relevance of TRPM8 expression in the proliferation of non-tumor and tumor cells. Inhibition of the expression or function of the channel reduces proliferation rates and proliferative fraction in all tumor cells tested, but not of non-tumor prostate cells. We observed no consistent acceleration of growth after stimulation of the channel with menthol or icilin, indicating that basal TRPM8 expression is enough to sustain growth of prostate cancer cells.

  1. The selectivity of conantokin-G for ion channel inhibition of NR2B subunit-containing NMDA receptors is regulated by amino acid residues in the S2 region of NR2B.

    Science.gov (United States)

    Sheng, Zhenyu; Liang, Zhong; Geiger, James H; Prorok, Mary; Castellino, Francis J

    2009-08-01

    The conantokins are short, naturally occurring peptides that inhibit ion flow through N-methyl-d-aspartate receptor (NMDAR) channels. One member of this peptide family, conantokin-G (con-G), shows high selectivity for antagonism of NR2B-containing NMDAR channels, whereas other known conantokins are less selective inhibitors with regard to the nature of the NR2 subunit of the NMDAR complex. In order to define the molecular determinants of NR2B that govern con-G selectivity, we evaluated the ability of con-G to inhibit NMDAR ion channels expressed in human embryonic kidney (HEK)293 cells transfected with NR1, in combination with various NR2A/2B chimeras and point mutants, by electrophysiology using cells voltage-clamped in the whole-cell configuration. We found that a variant of the con-G-insensitive subunit, NR2A, in which the 158 residues comprising the S2 peptide segment (E(657)-I(814)) were replaced by the corresponding S2 region of NR2B (E(658)-I(815)), results in receptors that are highly sensitive to inhibition by con-G. Of the 22 amino acids that are different between the NR2A-S2 and the NR2B-S2 regions, exchange of one of these, M(739) of NR2B for the equivalent K(738) of NR2A, was sufficient to completely import the inhibitory activity of con-G into NR1b/NR2A-containing NMDARs. Some reinforcement of this effect was found by substitution of a second amino acid, K(755) of NR2B for Y(754) of NR2A. The discovery of the molecular determinants of NR2B selectivity with con-G has implications for the design of subunit-selective neurobiological probes and drug therapies, in addition to advancing our understanding of NR2B- versus NR2A-mediated neurological processes.

  2. Caffeine-mediated inhibition of calcium release channel inositol 1,4,5-trisphosphate receptor subtype 3 blocks glioblastoma invasion and extends survival.

    Science.gov (United States)

    Kang, Sang Soo; Han, Kyung-Seok; Ku, Bo Mi; Lee, Yeon Kyung; Hong, Jinpyo; Shin, Hye Young; Almonte, Antoine G; Woo, Dong Ho; Brat, Daniel J; Hwang, Eun Mi; Yoo, Seung Hyun; Chung, Chun Kee; Park, Sung-Hye; Paek, Sun Ha; Roh, Eun Joo; Lee, Sung Joong; Park, Jae-Yong; Traynelis, Stephen F; Lee, C Justin

    2010-02-01

    Calcium signaling is important in many signaling processes in cancer cell proliferation and motility including in deadly glioblastomas of the brain that aggressively invade neighboring tissue. We hypothesized that disturbing Ca(2+) signaling pathways might decrease the invasive behavior of giloblastoma, extending survival. Evaluating a panel of small-molecule modulators of Ca(2+) signaling, we identified caffeine as an inhibitor of glioblastoma cell motility. Caffeine, which is known to activate ryanodine receptors, paradoxically inhibits Ca(2+) increase by inositol 1,4,5-trisphospate receptor subtype 3 (IP(3)R3), the expression of which is increased in glioblastoma cells. Consequently, by inhibiting IP(3)R3-mediated Ca(2+) release, caffeine inhibited migration of glioblastoma cells in various in vitro assays. Consistent with these effects, caffeine greatly increased mean survival in a mouse xenograft model of glioblastoma. These findings suggest IP(3)R3 as a novel therapeutic target and identify caffeine as a possible adjunct therapy to slow invasive growth of glioblastoma.

  3. Chloride channels in stroke

    Institute of Scientific and Technical Information of China (English)

    Ya-ping ZHANG; Hao ZHANG; Dayue Darrel DUAN

    2013-01-01

    Vascular remodeling of cerebral arterioles,including proliferation,migration,and apoptosis of vascular smooth muscle cells (VSMCs),is the major cause of changes in the cross-sectional area and diameter of the arteries and sudden interruption of blood flow or hemorrhage in the brain,ie,stroke.Accumulating evidence strongly supports an important role for chloride (Clˉ) channels in vascular remodeling and stroke.At least three Clˉ channel genes are expressed in VSMCs:1) the TMEM16A (or Ano1),which may encode the calcium-activated Clˉ channels (CACCs); 2) the CLC-3 Clˉ channel and Clˉ/H+ antiporter,which is closely related to the volume-regulated Clˉ channels (VRCCs); and 3) the cystic fibrosis transmembrane conductance regulator (CFTR),which encodes the PKA-and PKC-activated Clˉ channels.Activation of the CACCs by agonist-induced increase in intracellular Ca2+ causes membrane depolarization,vasoconstriction,and inhibition of VSMC proliferation.Activation of VRCCs by cell volume increase or membrane stretch promotes the production of reactive oxygen species,induces proliferation and inhibits apoptosis of VSMCs.Activation of CFTR inhibits oxidative stress and may prevent the development of hypertension.In addition,Clˉ current mediated by gammaaminobutyric acid (GABA) receptor has also been implicated a role in ischemic neuron death.This review focuses on the functional roles of Clˉ channels in the development of stroke and provides a perspective on the future directions for research and the potential to develop Clˉ channels as new targets for the prevention and treatment of stroke.

  4. Antibacterial Activity of Geminized Amphiphilic Cationic Homopolymers.

    Science.gov (United States)

    Wang, Hui; Shi, Xuefeng; Yu, Danfeng; Zhang, Jian; Yang, Guang; Cui, Yingxian; Sun, Keji; Wang, Jinben; Yan, Haike

    2015-12-22

    The current study is aimed at investigating the effect of cationic charge density and hydrophobicity on the antibacterial and hemolytic activities. Two kinds of cationic surfmers, containing single or double hydrophobic tails (octyl chains or benzyl groups), and the corresponding homopolymers were synthesized. The antimicrobial activity of these candidate antibacterials was studied by microbial growth inhibition assays against Escherichia coli, and hemolysis activity was carried out using human red blood cells. It was interestingly found that the homopolymers were much more effective in antibacterial property than their corresponding monomers. Furthermore, the geminized homopolymers had significantly higher antibacterial activity than that of their counterparts but with single amphiphilic side chains in each repeated unit. Geminized homopolymers, with high positive charge density and moderate hydrophobicity (such as benzyl groups), combine both advantages of efficient antibacterial property and prominently high selectivity. To further explain the antibacterial performance of the novel polymer series, the molecular interaction mechanism is proposed according to experimental data which shows that these specimens are likely to kill microbes by disrupting bacterial membranes, leading them unlikely to induce resistance.

  5. Novel role for the transient potential receptor melastatin 4 channel in guinea pig detrusor smooth muscle physiology.

    Science.gov (United States)

    Smith, Amy C; Hristov, Kiril L; Cheng, Qiuping; Xin, Wenkuan; Parajuli, Shankar P; Earley, Scott; Malysz, John; Petkov, Georgi V

    2013-03-01

    Members of the transient receptor potential (TRP) channel superfamily, including the Ca(2+)-activated monovalent cation-selective TRP melastatin 4 (TRPM4) channel, have been recently identified in the urinary bladder. However, their expression and function at the level of detrusor smooth muscle (DSM) remain largely unexplored. In this study, for the first time we investigated the role of TRPM4 channels in guinea pig DSM excitation-contraction coupling using a multidisciplinary approach encompassing protein detection, electrophysiology, live-cell Ca(2+) imaging, DSM contractility, and 9-phenanthrol, a recently characterized selective inhibitor of the TRPM4 channel. Western blot and immunocytochemistry experiments demonstrated the expression of the TRPM4 channel in whole DSM tissue and freshly isolated DSM cells with specific localization on the plasma membrane. Perforated whole cell patch-clamp recordings and real-time Ca(2+) imaging experiments with fura 2-AM, both using freshly isolated DSM cells, revealed that 9-phenanthrol (30 μM) significantly reduced the cation current and decreased intracellular Ca(2+) levels. 9-Phenanthrol (0.1-30 μM) significantly inhibited spontaneous, 0.1 μM carbachol-induced, 20 mM KCl-induced, and nerve-evoked contractions in guinea pig DSM-isolated strips with IC50 values of 1-7 μM and 70-80% maximum inhibition. 9-Phenanthrol also reduced nerve-evoked contraction amplitude induced by continuous repetitive electrical field stimulation of 10-Hz frequency and shifted the frequency-response curve (0.5-50 Hz) relative to the control. Collectively, our data demonstrate the novel finding that TRPM4 channels are expressed in guinea pig DSM and reveal their critical role in the regulation of guinea pig DSM excitation-contraction coupling.

  6. Cation binding site of cytochrome c oxidase: progress report.

    Science.gov (United States)

    Vygodina, Tatiana V; Kirichenko, Anna; Konstantinov, Alexander A

    2014-07-01

    Cytochrome c oxidase from bovine heart binds Ca(2+) reversibly at a specific Cation Binding Site located near the outer face of the mitochondrial membrane. Ca(2+) shifts the absorption spectrum of heme a, which allowed earlier the determination of the kinetic and equilibrium characteristics of the binding, and, as shown recently, the binding of calcium to the site inhibits cytochrome oxidase activity at low turnover rates of the enzyme [Vygodina, Т., Kirichenko, A., Konstantinov, A.A (2013). Direct Regulation of Cytochrome c Oxidase by Calcium Ions. PloS ONE 8, e74436]. This paper summarizes further progress in the studies of the Cation Binding Site in this group presenting the results to be reported at 18th EBEC Meeting in Lisbon, 2014. The paper revises specificity of the bovine oxidase Cation Binding Site for different cations, describes dependence of the Ca(2+)-induced inhibition on turnover rate of the enzyme and reports very high affinity binding of calcium with the "slow" form of cytochrome oxidase. This article is part of a Special Issue entitled: 18th European Bioenergetic Conference. Guest Editors: Manuela Pereira and Miguel Teixeira.

  7. Role of hyperpolarization-activated cyclic nucleotide-gated cation nonselective channel in spontaneous contrac-tion of smooth muscles in human isolated ureters%HCN通道在人体输尿管自发性收缩中的作用

    Institute of Scientific and Technical Information of China (English)

    冯观贵; 刘骞; 孙碧韶; 朱景振; 龙洲; 李龙坤

    2015-01-01

    目的应用ZD7288特异性阻断超极化激活环核苷酸门控阳离子非选择性通道( HCN通道)后观察人输尿管离体平滑肌肌条自发性收缩活动的变化,从功能学上初步探讨HCN通道在输尿管平滑肌自发性收缩中的作用。方法收集临床上因肾肿瘤或囊肿等行肾切除手术患者的离体输尿管标本12例,经确认患者手术前均未接受放疗、化疗或免疫治疗等特殊处理,进行输尿管离体平滑肌肌条实验,观察在ZD7288作用下输尿管离体平滑肌肌条的收缩幅度和频率的变化。以人肠道组织为阳性对照,剩余标本用于检测HCN通道在人输尿管上的表达情况。结果经RT-PCR、WB法( Western blot)技术及免疫组化法检测,发现正常人输尿管存在HCN通道的表达,且HCN通道主要表达于人输尿管的黏膜层与平滑肌层;离体肌条实验中加入ZD7288后,肌条的收缩频率明显变慢,收缩幅度变化不明显,自身前后对比有统计学差异(P<0.01)。结论 HCN通道可能在人输尿管自发性收缩中发挥着重要作用。%Objective To investigate effects of hyperpolarization-activated cyclic nucleotide-gated cation nonselective channel in the hu-man ureter on the spontaneous contraction of smooth muscles. Methods Four HCN subtypes were detected in human ureteral tissue using reverse transcription polymerase chain reaction,Western blotting and immunohistochemical. ZD7288,the HCN blocker, was used to observe the changes of ureteral muscle contraction amplitude and frequency by applying the ureteral smooth muscle strip test in vitro. Results HCN1-4 isoforms were all identified in human ureter using reverse transcription-polymerase chain reaction and Western blotting. Through the immunohistochemical,HCN channel was found mostly in the urothelium layer and muscular layer of human ureteral wall. ZD7288 significantly decreased the bladder excitation. Conclusion All 4 HCN channel hypotypes exist

  8. Inhibition of Inactive States of Tetrodotoxin-Sensitive Sodium Channels Reduces Spontaneous Firing of C-Fiber Nociceptors and Produces Analgesia in Formalin and Complete Freund's Adjuvant Models of Pain.

    Directory of Open Access Journals (Sweden)

    David J Matson

    Full Text Available While genetic evidence shows that the Nav1.7 voltage-gated sodium ion channel is a key regulator of pain, it is unclear exactly how Nav1.7 governs neuronal firing and what biophysical, physiological, and distribution properties of a pharmacological Nav1.7 inhibitor are required to produce analgesia. Here we characterize a series of aminotriazine inhibitors of Nav1.7 in vitro and in rodent models of pain and test the effects of the previously reported "compound 52" aminotriazine inhibitor on the spiking properties of nociceptors in vivo. Multiple aminotriazines, including some with low terminal brain to plasma concentration ratios, showed analgesic efficacy in the formalin model of pain. Effective concentrations were consistent with the in vitro potency as measured on partially-inactivated Nav1.7 but were far below concentrations required to inhibit non-inactivated Nav1.7. Compound 52 also reversed thermal hyperalgesia in the complete Freund's adjuvant (CFA model of pain. To study neuronal mechanisms, electrophysiological recordings were made in vivo from single nociceptive fibers from the rat tibial nerve one day after CFA injection. Compound 52 reduced the spontaneous firing of C-fiber nociceptors from approximately 0.7 Hz to 0.2 Hz and decreased the number of action potentials evoked by suprathreshold tactile and heat stimuli. It did not, however, appreciably alter the C-fiber thresholds for response to tactile or thermal stimuli. Surprisingly, compound 52 did not affect spontaneous activity or evoked responses of Aδ-fiber nociceptors. Results suggest that inhibition of inactivated states of TTX-S channels, mostly likely Nav1.7, in the peripheral nervous system produces analgesia by regulating the spontaneous discharge of C-fiber nociceptors.

  9. Acute and Chronic Toxicity, Cytochrome P450 Enzyme Inhibition, and hERG Channel Blockade Studies with a Polyherbal, Ayurvedic Formulation for Inflammation

    Directory of Open Access Journals (Sweden)

    Debendranath Dey

    2015-01-01

    Full Text Available Ayurvedic plants are known for thousands of years to have anti-inflammatory and antiarthritic effect. We have recently shown that BV-9238, a proprietary formulation of Withania somnifera, Boswellia serrata, Zingiber officinale, and Curcuma longa, inhibits LPS-induced TNF-alpha and nitric oxide production from mouse macrophage and reduces inflammation in different animal models. To evaluate the safety parameters of BV-9238, we conducted a cytotoxicity study in RAW 264.7 cells (0.005–1 mg/mL by MTT/formazan method, an acute single dose (2–10 g/kg bodyweight toxicity study and a 180-day chronic study with 1 g and 2 g/kg bodyweight in Sprague Dawley rats. Some sedation, ptosis, and ataxia were observed for first 15–20 min in very high acute doses and hence not used for further chronic studies. At the end of 180 days, gross and histopathology, blood cell counts, liver and renal functions were all at normal levels. Further, a modest attempt was made to assess the effects of BV-9238 (0.5 µg/mL on six major human cytochrome P450 enzymes and 3H radioligand binding assay with human hERG receptors. BV-9238 did not show any significant inhibition of these enzymes at the tested dose. All these suggest that BV-9238 has potential as a safe and well tolerated anti-inflammatory formulation for future use.

  10. Vulnerability of the retinal microvasculature to oxidative stress: ion channel-dependent mechanisms.

    Science.gov (United States)

    Fukumoto, Masanori; Nakaizumi, Atsuko; Zhang, Ting; Lentz, Stephen I; Shibata, Maho; Puro, Donald G

    2012-05-01

    Although oxidative stress is a hallmark of important vascular disorders such as diabetic retinopathy, it remains unclear why the retinal microvasculature is particularly vulnerable to this pathophysiological condition. We postulated that redox-sensitive ion channels may play a role. Using H(2)O(2) to cause oxidative stress in microvascular complexes freshly isolated from the adult rat retina, we assessed ionic currents, cell viability, intracellular oxidants, and cell calcium by using perforated-patch recordings, trypan blue dye exclusion, and fura-2 fluorescence, respectively. Supporting a role for the oxidant-sensitive ATP-sensitive K (K(ATP)) channels, we found that these channels are activated during exposure of retinal microvessels to H(2)O(2). Furthermore, their inhibition by glibenclamide significantly lessened H(2)O(2)-induced microvascular cell death. Additional experiments established that by increasing the influx of calcium into microvascular cells, the K(ATP) channel-mediated hyperpolarization boosted the vulnerability of these cells to oxidative stress. In addition to the K(ATP) channel-dependent mechanism for increasing the lethality of oxidative stress, we also found that the vulnerability of cells in the capillaries, but not in the arterioles, was further boosted by a K(ATP) channel-independent mechanism, which our experiments indicated involves the oxidant-induced activation of calcium-permeable nonspecific cation channels. Taken together, our findings support a working model in which both K(ATP) channel-independent and K(ATP) channel-dependent mechanisms render the capillaries of the retina particularly vulnerable to oxidative stress. Identification of these previously unappreciated mechanisms for boosting the lethality of oxidants may provide new targets for pharmacologically limiting damage to the retinal microvasculature during periods of oxidative stress.

  11. Anion Channel Inhibitor NPPB-Inhibited Fluoride Accumulation in Tea Plant (Camellia sinensis) Is Related to the Regulation of Ca2+, CaM and Depolarization of Plasma Membrane Potential

    Science.gov (United States)

    Zhang, Xian-Chen; Gao, Hong-Jian; Yang, Tian-Yuan; Wu, Hong-Hong; Wang, Yu-Mei; Zhang, Zheng-Zhu; Wan, Xiao-Chun

    2016-01-01

    Tea plant is known to be a hyper-accumulator of fluoride (F). Over-intake of F has been shown to have adverse effects on human health, e.g., dental fluorosis. Thus, understanding the mechanisms fluoride accumulation and developing potential approaches to decrease F uptake in tea plants might be beneficial for human health. In the present study, we found that pretreatment with the anion channel inhibitor NPPB reduced F accumulation in tea plants. Simultaneously, we observed that NPPB triggered Ca2+ efflux from mature zone of tea root and significantly increased relative CaM in tea roots. Besides, pretreatment with the Ca2+ chelator (EGTA) and CaM antagonists (CPZ and TFP) suppressed NPPB-elevated cytosolic Ca2+ fluorescence intensity and CaM concentration in tea roots, respectively. Interestingly, NPPB-inhibited F accumulation was found to be significantly alleviated in tea plants pretreated with either Ca2+ chelator (EGTA) or CaM antagonists (CPZ and TFP). In addition, NPPB significantly depolarized membrane potential transiently and we argue that the net Ca2+ and H+ efflux across the plasma membrane contributed to the restoration of membrane potential. Overall, our results suggest that regulation of Ca2+-CaM and plasma membrane potential depolarization are involved in NPPB-inhibited F accumulation in tea plants. PMID:26742036

  12. Anion Channel Inhibitor NPPB-Inhibited Fluoride Accumulation in Tea Plant (Camellia sinensis) Is Related to the Regulation of Ca²⁺, CaM and Depolarization of Plasma Membrane Potential.

    Science.gov (United States)

    Zhang, Xian-Chen; Gao, Hong-Jian; Yang, Tian-Yuan; Wu, Hong-Hong; Wang, Yu-Mei; Zhang, Zheng-Zhu; Wan, Xiao-Chun

    2016-01-05

    Tea plant is known to be a hyper-accumulator of fluoride (F). Over-intake of F has been shown to have adverse effects on human health, e.g., dental fluorosis. Thus, understanding the mechanisms fluoride accumulation and developing potential approaches to decrease F uptake in tea plants might be beneficial for human health. In the present study, we found that pretreatment with the anion channel inhibitor NPPB reduced F accumulation in tea plants. Simultaneously, we observed that NPPB triggered Ca(2+) efflux from mature zone of tea root and significantly increased relative CaM in tea roots. Besides, pretreatment with the Ca(2+) chelator (EGTA) and CaM antagonists (CPZ and TFP) suppressed NPPB-elevated cytosolic Ca(2+) fluorescence intensity and CaM concentration in tea roots, respectively. Interestingly, NPPB-inhibited F accumulation was found to be significantly alleviated in tea plants pretreated with either Ca(2+) chelator (EGTA) or CaM antagonists (CPZ and TFP). In addition, NPPB significantly depolarized membrane potential transiently and we argue that the net Ca(2+) and H⁺ efflux across the plasma membrane contributed to the restoration of membrane potential. Overall, our results suggest that regulation of Ca(2+)-CaM and plasma membrane potential depolarization are involved in NPPB-inhibited F accumulation in tea plants.

  13. Low-dose combination of Rho kinase and L-type Ca(2+) channel antagonists for selective inhibition of depolarization-induced sustained arterial contraction.

    Science.gov (United States)

    Porras-González, Cristina; González-Rodríguez, Patricia; Calderón-Sánchez, Eva; López-Barneo, José; Ureña, Juan

    2014-06-05

    L-type Ca(2+) channels (LTCCs) are involved in the maintenance of tonic arterial contractions and regulate the RhoA/Rho-associated kinase (ROCK) sensitization cascade. We have tested effects of individual and combined low concentrations of LTCCs and ROCK inhibitors to produce arterial relaxation without the adverse side effects of LTCCs antagonists. We have also studied whether this pharmacological strategy alters Ca(2+)-dependent electrical properties of isolated arterial and cardiac myocytes as well as cardiac contractility. Rat basilar, human carotid and coronary arterial rings were mounted on a small-vessel myograph to measure isometric tension and cardiac contractility was measured in Langendorff-perfused rat heart. Simultaneous cytosolic Ca(2+) concentration and arterial diameter were measured in intact pressurized arteries loaded with Fura-2. Patch-clamp techniques were used to measure electrical properties in isolated cardiac and arterial myocytes. Low concentrations of LTCCs and ROCK inhibitors reduced the tonic component of moderate depolarization-evoked contraction, leaving the phasic component practically unaltered. This selective vasorelaxant effect was more marked when the LTCCs and ROCK inhibitors were applied together. In the concentration range used (nM), Ca(2+) currents in arterial myocytes, cardiac action potentials and heart contractility were unaffected by this pharmacological approach. In conclusion, low doses of LTCCs and ROCK inhibitors could be used to selectively relax precontracted arteries in pathologic conditions such as hypertension, and cerebral or coronary spasms with minor side effects on physiological contractile properties of vascular and cardiac myocytes.

  14. Research progress in cation-π interactions

    Institute of Scientific and Technical Information of China (English)

    2008-01-01

    Cation-π interaction is a potent intermolecular interaction between a cation and an aromatic system,which has been viewed as a new kind of binding force,as being compared with the classical interactions(e.g. hydrogen bonding,electrostatic and hydrophobic interactions). Cation-π interactions have been observed in a wide range of biological contexts. In this paper,we present an overview of the typical cation-π interactions in biological systems,the experimental and theoretical investigations on cation-π interactions,as well as the research results on cation-π interactions in our group.

  15. Research progress in cation-π interactions

    Institute of Scientific and Technical Information of China (English)

    CHENG JiaGao; LUO XiaoMin; YAN XiuHua; LI Zhong; TANG Yun; JIANG HuaLiang; ZHU WeiLiang

    2008-01-01

    Cation-π interaction is a potent intermolecular interaction between a cation and an aromatic system, which has been viewed as a new kind of binding force, as being compared with the classical interac-tions (e.g. hydrogen bonding, electrostatic and hydrophobic interactions). Cation-π interactions have been observed in a wide range of biological contexts. In this paper, we present an overview of the typi-cal cation-π interactions in biological systems, the experimental and theoretical investigations on cation-π interactions, as well as the research results on cation-π interactions in our group.

  16. Lack of negatively charged residues at the external mouth of Kir2.2 channels enable the voltage-dependent block by external Mg2+.

    Directory of Open Access Journals (Sweden)

    Junwei Li

    Full Text Available Kir channels display voltage-dependent block by cytosolic cations such as Mg2+ and polyamines that causes inward rectification. In fact, cations can regulate K channel activity from both the extracellular and intracellular sides. Previous studies have provided insight into the up-regulation of Kir channel activity by extracellular K+ concentration. In contrast, extracellular Mg2+ has been found to reduce the amplitude of the single-channel current at milimolar concentrations. However, little is known about the molecular mechanism of Kir channel blockade by external Mg2+ and the relationship between the Mg2+ blockade and activity potentiation by permeant K+ ions. In this study, we applied an interactive approach between theory and experiment. Electrophysiological recordings on Kir2.2 and its mutants were performed by heterologous expression in Xenopus laevis oocytes. Our results confirmed that extracellular Mg2+ could reduce heterologously expressed WT Kir2.2 currents in a voltage dependent manner. The kinetics of inhibition and recovery of Mg2+ exhibit a 3∼4s time constant. Molecular dynamics simulation results revealed a Mg2+ binding site located at the extracellular mouth of Kir2.2 that showed voltage-dependent Mg2+ binding. The mutants, G119D, Q126E and H128D, increased the number of permeant K+ ions and reduced the voltage-dependent blockade of Kir2.2 by extracellular Mg2+.

  17. Increased cation conductance in human erythrocytes artificially aged by glycation.

    Science.gov (United States)

    Kucherenko, Yuliya V; Bhavsar, Shefalee K; Grischenko, Valentin I; Fischer, Uwe R; Huber, Stephan M; Lang, Florian

    2010-06-01

    Excessive glucose concentrations foster glycation and thus premature aging of erythrocytes. The present study explored whether glycation-induced erythrocyte aging is paralleled by features of suicidal erythrocyte death or eryptosis, which is characterized by cell membrane scrambling with subsequent phosphatidylserine exposure at the cell surface and cell shrinkage. Both are triggered by increases of cytosolic Ca(2+) concentration ([Ca(2+)](i)), which may result from activation of Ca(2+) permeable cation channels. Glycation was accomplished by exposure to high glucose concentrations (40 and 100 mM), phosphatidylserine exposure estimated from annexin binding, cell shrinkage from decrease of forward scatter, and [Ca(2+)](i) from Fluo3-fluorescence in analysis via fluorescence-activated cell sorter. Cation channel activity was determined by means of whole-cell patch clamp. Glycation of total membrane proteins, immunoprecipitated TRPC3/6/7, and immunoprecipitated L-type Ca(2+) channel proteins was estimated by Western blot testing with polyclonal antibodies used against advanced glycation end products. A 30-48-h exposure of the cells to 40 or 100 mM glucose in Ringer solution (at 37 degrees C) significantly increased glycation of membrane proteins, hemoglobin (HbA(1c)), TRPC3/6/7, and L-type Ca(2+) channel proteins, enhanced amiloride-sensitive, voltage-independent cation conductance, [Ca(2+)](i), and phosphatidylserine exposure, and led to significant cell shrinkage. Ca(2+) removal and addition of Ca(2+) chelator EGTA prevented the glycation-induced phosphatidylserine exposure and cell shrinkage after glycation. Glycation-induced erythrocyte aging leads to eryptosis, an effect requiring Ca(2+) entry from extracellular space.

  18. Post-Translational Modifications of TRP Channels

    Directory of Open Access Journals (Sweden)

    Olaf Voolstra

    2014-04-01

    Full Text Available Transient receptor potential (TRP channels constitute an ancient family of cation channels that have been found in many eukaryotic organisms from yeast to human. TRP channels exert a multitude of physiological functions ranging from Ca2+ homeostasis in the kidney to pain reception and vision. These channels are activated by a wide range of stimuli and undergo covalent post-translational modifications that affect and modulate their subcellular targeting, their biophysical properties, or channel gating. These modifications include N-linked glycosylation, protein phosphorylation, and covalent attachment of chemicals that reversibly bind to specific cysteine residues. The latter modification represents an unusual activation mechanism of ligand-gated ion channels that is in contrast to the lock-and-key paradigm of receptor activation by its agonists. In this review, we summarize the post-translational modifications identified on TRP channels and, when available, explain their physiological role.

  19. Tumor Necrosis Factor Alpha Inhibits L-Type Ca2+ Channels in Sensitized Guinea Pig Airway Smooth Muscle through ERK 1/2 Pathway

    Directory of Open Access Journals (Sweden)

    Jorge Reyes-García

    2016-01-01

    Full Text Available Tumor necrosis factor alpha (TNF-α is a potent proinflammatory cytokine that plays a significant role in the pathogenesis of asthma by inducing hyperresponsiveness and airway remodeling. TNF-α diminishes the L-type voltage dependent Ca2+ channel (L-VDCC current in cardiac myocytes, an observation that seems paradoxical. In guinea pig sensitized tracheas KCl responses were lower than in control tissues. Serum from sensitized animals (Ser-S induced the same phenomenon. In tracheal myocytes from nonsensitized (NS and sensitized (S guinea pigs, an L-VDCC current (ICa was observed and diminished by Ser-S. The same decrease was detected in NS myocytes incubated with TNF-α, pointing out that this cytokine might be present in Ser-S. We observed that a small-molecule inhibitor of TNF-α (SMI-TNF and a TNF-α receptor 1 (TNFR1 antagonist (WP9QY reversed ICa decrease induced by Ser-S in NS myocytes, confirming the former hypothesis. U0126 (a blocker of ERK 1/2 kinase also reverted the decrease in ICa. Neither cycloheximide (a protein synthesis inhibitor nor actinomycin D (a transcription inhibitor showed any effect on the TNF-α-induced ICa reduction. We found that CaV1.2 and CaV1.3 mRNA and proteins were expressed in tracheal myocytes and that sensitization did not modify them. In cardiac myocytes, ERK 1/2 phosphorylates two sites of the L-VDCC, augmenting or decreasing ICa; we postulate that, in guinea pig tracheal smooth muscle, TNF-α diminishes ICa probably by phosphorylating the L-VDCC site that reduces its activity through the ERK1/2 MAP kinase pathway.

  20. Tumor Necrosis Factor Alpha Inhibits L-Type Ca(2+) Channels in Sensitized Guinea Pig Airway Smooth Muscle through ERK 1/2 Pathway.

    Science.gov (United States)

    Reyes-García, Jorge; Flores-Soto, Edgar; Solís-Chagoyán, Héctor; Sommer, Bettina; Díaz-Hernández, Verónica; García-Hernández, Luz María; Montaño, Luis M

    2016-01-01

    Tumor necrosis factor alpha (TNF-α) is a potent proinflammatory cytokine that plays a significant role in the pathogenesis of asthma by inducing hyperresponsiveness and airway remodeling. TNF-α diminishes the L-type voltage dependent Ca(2+) channel (L-VDCC) current in cardiac myocytes, an observation that seems paradoxical. In guinea pig sensitized tracheas KCl responses were lower than in control tissues. Serum from sensitized animals (Ser-S) induced the same phenomenon. In tracheal myocytes from nonsensitized (NS) and sensitized (S) guinea pigs, an L-VDCC current (ICa) was observed and diminished by Ser-S. The same decrease was detected in NS myocytes incubated with TNF-α, pointing out that this cytokine might be present in Ser-S. We observed that a small-molecule inhibitor of TNF-α (SMI-TNF) and a TNF-α receptor 1 (TNFR1) antagonist (WP9QY) reversed ICa decrease induced by Ser-S in NS myocytes, confirming the former hypothesis. U0126 (a blocker of ERK 1/2 kinase) also reverted the decrease in ICa. Neither cycloheximide (a protein synthesis inhibitor) nor actinomycin D (a transcription inhibitor) showed any effect on the TNF-α-induced ICa reduction. We found that CaV1.2 and CaV1.3 mRNA and proteins were expressed in tracheal myocytes and that sensitization did not modify them. In cardiac myocytes, ERK 1/2 phosphorylates two sites of the L-VDCC, augmenting or decreasing ICa; we postulate that, in guinea pig tracheal smooth muscle, TNF-α diminishes ICa probably by phosphorylating the L-VDCC site that reduces its activity through the ERK1/2 MAP kinase pathway.

  1. 公丁香提取物抑制CFTR氯离子通道的发现与研究%The extract of clove inhibits CFTR chloride channel

    Institute of Scientific and Technical Information of China (English)

    栾剑; 张耀方; 杨红

    2015-01-01

    Cystic fibrosis transmembrane conductance regulator (CFTR) is an epithelial chloride chan‐nel .In recent years ,the blockers of CFTR become the new hot spot in the treatment of secretory di‐arrhea .The aim of this research is using high‐throughput screening techniques screened blockers of CFTR chloride channel from traditional Chinese medicine .In this study ,after 40000 fractions of Chi‐nese herbal medicine have been screened ,clove extract was found .In cell‐based fluorescence assays and voltage clamp experiments ,the best active fraction‐E06 significantly blocks CFTR chloride chan‐nel .Therefore ,clove extract screened from traditional Chinese medicine blocks CFTR chloride chan‐nel and provides a theoretical basis for the in‐depth study of anti‐diarrheal drugs .%囊性纤维化跨膜电导调节因子(CFTR)是一种上皮细胞顶膜中表达的氯离子通道,是近年来治疗分泌型腹泻的新热点。利用高通量筛选技术,自中国传统中药中筛选能够抑制CFTR氯离子通道的中药组分。结果显示,自500种中草药的40000种中药组分中筛选到公丁香。经细胞荧光实验和电压膜片钳实验验证公丁香最佳活性孔———E06对CFTR具有明显的抑制作用,IC50=103 mg/L 。本研究结果为深入探讨公丁香的抗泻药物研发提供理论依据。

  2. Measurement of antioxidant activity with trifluoperazine dihydrochloride radical cation

    Directory of Open Access Journals (Sweden)

    M.N. Asghar

    2008-06-01

    Full Text Available A novel, rapid and cost-effective trifluoperazine dihydrochloride (TFPH decolorization assay is described for the screening of antioxidant activity. A chromogenic reaction between TFPH and potassium persulfate at low pH produces an orange-red radical cation with maximum absorption at 502 nm in its first-order derivative spectrum. TFPH was dissolved in distilled water to give a 100 mM solution. The TFPH radical cation solution was made by reacting 0.5 mL of the solution with K2S2O8 (final concentration: 0.1 mM and diluting to 100 mL with 4 M H2SO4 solution. A linear inhibition of color production was observed with linearly increasing amounts of antioxidants, with correlation coefficients (R² ranging from 0.999 to 0.983. The antioxidant capacity of standard solutions of an antioxidant was evaluated by comparing with the inhibition curve using Trolox as the standard. Comparison of antioxidant capacity determined with this newly developed TFPH assay and with the well-known 2,2'-azinobis-[3-ethylbenzthiazoline-6-sulfonic acid] (ABTS-persulfate decolorization assay indicated the efficacy and sensitivity of the procedure. The proposed assay is less expensive (costs about US$4 per 100 assays and requires only 20 min for preparation of radical cation solution in comparison with ABTS assay, in which almost 12-16 h are required for preparation of a stable ABTS radical cation solution. The present assay has the advantage over ABTS assay that it can be used to measure the antioxidant activity of the samples, which are naturally found at a pH as low as 1, because the radical cation itself has been stabilized at low pH.

  3. Differential effects of bitter compounds on the taste transduction channels TRPM5 and IP3 receptor type 3.

    Science.gov (United States)

    Gees, Maarten; Alpizar, Yeranddy A; Luyten, Tomas; Parys, Jan B; Nilius, Bernd; Bultynck, Geert; Voets, Thomas; Talavera, Karel

    2014-05-01

    Transient receptor potential cation channel subfamily M member 5 (TRPM5) is a Ca(2+)-activated nonselective cation channel involved in the transduction of sweet, bitter, and umami tastes. We previously showed that TRPM5 is a locus for the modulation of taste perception by temperature changes, and by quinine and quinidine, 2 bitter compounds that suppress gustatory responses. Here, we determined whether other bitter compounds known to modulate taste perception also affect TRPM5. We found that nicotine inhibits TRPM5 currents with an effective inhibitory concentration of ~1.3mM at -50 mV. This effect may contribute to the inhibitory effect of nicotine on gustatory responses in therapeutic and experimental settings, where nicotine is often employed at millimolar concentrations. In addition, it implies the existence of a TRPM5-independent pathway for the detection of nicotine bitterness. Nicotine seems to act from the extracellular side of the channel, reducing the maximal whole-cell conductance and inducing an acceleration of channel closure that leads to a negative shift of the activation curve. TRPM5 currents were unaffected by nicotine's metabolite cotinine, the intensive sweetener saccharin or by the bitter xanthines caffeine, theobromine, and theophylline. We also tested the effects of bitter compounds on another essential element of the sweet taste transduction pathway, the type 3 IP3 receptor (IP3R3). We found that IP3R3-mediated Ca(2+) flux is slightly enhanced by nicotine, not affected by saccharin, modestly inhibited by caffeine, theobromine, and theophylline, and strongly inhibited by quinine. Our results demonstrate that bitter compounds have differential effects on key elements of the sweet taste transduction pathway, suggesting for heterogeneous mechanisms of bitter-sweet taste interactions.

  4. The adjuvant mechanism of cationic dimethyldioctadecylammonium liposomes

    DEFF Research Database (Denmark)

    Korsholm, Karen Smith; Agger, Else Marie; Foged, Camilla;

    2007-01-01

    Cationic liposomes are being used increasingly as efficient adjuvants for subunit vaccines but their precise mechanism of action is still unknown. Here, we investigated the adjuvant mechanism of cationic liposomes based on the synthetic amphiphile dimethyldioctadecylammonium (DDA). The liposomes ...

  5. Tripodal Receptors for Cation and Anion Sensors

    NARCIS (Netherlands)

    Kuswandi, Bambang; Nuriman,; Verboom, Willem; Reinhoudt, David N.

    2006-01-01

    This review discusses different types of artificial tripodal receptors for the selectiverecognition and sensing of cations and anions. Examples on the relationship between structure andselectivity towards cations and anions are described. Furthermore, their applications as potentiometricion sensing

  6. A WATER-SOLUBLE DUAL-CHANNEL FLUORESCENCE-ENHANCED BIOSENSOR FOR HEPARIN BASED ON A CATIONIC CONJUGATED POLYELECTROLYTE CONTAINING TETRAPHENYLETHENE AND 2,1,3-BENZOTHIADIAZOLE%荧光增强型共轭聚电解质的合成及对肝素的双通道检测

    Institute of Scientific and Technical Information of China (English)

    吴燕梅; 石建兵; 佟斌; 支俊格; 董宇平

    2012-01-01

    A conjugated polymer ( P0) containing tetraphenylethene ( TPE) group and benzothiadiazole (BT) group has been designed and synthesized by Suzuki coupling reaction of 1,2-di[4-(6-bromohexyloxy) phenyl]-l ,2-di(4-bromophenyl)ethene (Ml) , 1,2-di[4-(6-bromohexyloxy) phenyl]-1 ,2-di(4-(4,4,5,5-tetramethyl-1 ,3, 2-dioxaborolan-2-yl) phenyl) ethylene ( M2 ) and 4, 7-dibromo-2 , 1 , 3-benzothiadiazole (M3). The ratio of TPE unit to BT unit at 0. 803: 0. 197 was determined by 1H-NMR spectrum. A water-soluble cationic conjugated polyelectrolyte (P1) is obtained by quaternization P0 with trimethylamine,which is completely soluble in water and insoluble in common organic solvents such as THF and acetone. When P1 is aggregated by adding a poor solvent such as THF into P1 aqueous solution, the fluorescence intensity and color of P1 change drastically. The intensity increases up to seven times and the maximum emission peak is red-shifted up to 30 nm. The color is changing from khaki ( no aggregation) to orange ( aggregation) under ultraviolet irradiation. Therefore, dual-channel fluorescence response as the unique properties of P1 was applied to detect heparin, a highly sulfated glycosaminoglycan with the highest negative charge density among the known biological molecules. The fluorescence intensity gradually increased upon addition of heparin into P1 aqueous solution with a linear response to concentration of heparin, and the correlation coefficient was up to 0. 9999. In addition, a linear red shift of the maximum emission peak was observed as the concentration of heparin increased with a correlation coefficient up to 0. 9998. These results indicate that P1 is a potential material for developing dual-channel fluorescence probe for heparin quantification. The UV spectra of P1/ heparin complexing titration don' t show any change. This indicates that there is no inter-chains or intra-chains π-π stacking of P1. DLS result indicates some aggregates can be formed through

  7. Acid-Sensing Ion Channels and Pain

    Directory of Open Access Journals (Sweden)

    Qihai Gu

    2010-05-01

    Full Text Available Pathophysiological conditions such as inflammation, ischemia, infection and tissue injury can all evoke pain, and each is accompanied by local acidosis. Acid sensing ion channels (ASICs are proton-gated cation channels expressed in both central and peripheral nervous systems. Increasing evidence suggests that ASICs represent essential sensors for tissue acidosis-related pain. This review provides an update on the role of ASICs in pain sensation and discusses their therapeutic potential for pain management.

  8. Schisandrin B inhibits the proliferation of airway smooth muscle cells via microRNA-135a suppressing the expression of transient receptor potential channel 1.

    Science.gov (United States)

    Zhang, Xiao-Yu; Zhang, Luo-Xian; Guo, Ya-Li; Zhao, Li-Min; Tang, Xue-Yi; Tian, Cui-Jie; Cheng, Dong-Jun; Chen, Xian-Liang; Ma, Li-Jun; Chen, Zhuo-Chang

    2016-07-01

    Airway smooth muscle cell (ASMC) was known to involve in the pathophysiology of asthma. Schisandrin B was reported to have anti-asthmatic effects in a murine asthma model. However, the molecular mechanism involving in the effect of Schisandrin B on ASMCs remains poorly understood. Sprague-Dawley rats were divided into three groups: rats as the control (Group 1), sensitized rats (Group 2), sensitized rats and intragastric-administrated Schisandrin B (Group 3). The expression of miR-135a and TRPC1 was detected in the rats from three groups. Platelet-derived growth factor (PDGF)-BB was used to induce the proliferation of isolated ASMCs, and the expression of miR-135a and TRPC1 was detected in PDGF-BB-treated ASMCs. Cell viability was examined in ASMCs transfected with miR-135a inhibitor or si-TRPC1. The expression of TRPC1 was examined in A10 cells pretreated with miR-135a inhibitor or miR-135a mimic. In this study, we found that Schisandrin B attenuated the inspiratory and expiratory resistances in sensitized rats. Schisandrin B upregulated the mRNA level of miR-135a and decreased the expression of TRPC1 in sensitized rats. In addition, Schisandrin B reversed the expression of miR-135a and TRPC1 in PDGF-BB-induced ASMCs. Si-TRPC1 abrogated the increasing proliferation of ASMCs induced by miR-135a inhibitor. We also found that miR-135a regulated the expression of TRPC1 in the A10 cells. These results demonstrate that Schisandrin B inhibits the proliferation of ASMCs via miR-135a suppressing the expression of TRPC1.

  9. Uptake Rate of Cationic Mitochondrial Inhibitor MKT-077 Determines Cellular Oxygen Consumption Change in Carcinoma Cells

    OpenAIRE

    John L Chunta; Vistisen, Kerry S.; Zeinab Yazdi; Braun, Rod D.

    2012-01-01

    OBJECTIVE: Since tumor radiation response is oxygen-dependent, radiosensitivity can be enhanced by increasing tumor oxygenation. Theoretically, inhibiting cellular oxygen consumption is the most efficient way to increase oxygen levels. The cationic, rhodacyanine dye-analog MKT-077 inhibits mitochondrial respiration and could be an effective metabolic inhibitor. However, the relationship between cellular MKT-077 uptake and metabolic inhibition is unknown. We hypothesized that rat and human mam...

  10. Relevance of lysine snorkeling in the outer transmembrane domain of small viral potassium ion channels.

    Science.gov (United States)

    Gebhardt, Manuela; Henkes, Leonhard M; Tayefeh, Sascha; Hertel, Brigitte; Greiner, Timo; Van Etten, James L; Baumeister, Dirk; Cosentino, Cristian; Moroni, Anna; Kast, Stefan M; Thiel, Gerhard

    2012-07-17

    Transmembrane domains (TMDs) are often flanked by Lys or Arg because they keep their aliphatic parts in the bilayer and their charged groups in the polar interface. Here we examine the relevance of this so-called "snorkeling" of a cationic amino acid, which is conserved in the outer TMD of small viral K(+) channels. Experimentally, snorkeling activity is not mandatory for Kcv(PBCV-1) because K29 can be replaced by most of the natural amino acids without any corruption of function. Two similar channels, Kcv(ATCV-1) and Kcv(MT325), lack a cytosolic N-terminus, and neutralization of their equivalent cationic amino acids inhibits their function. To understand the variable importance of the cationic amino acids, we reanalyzed molecular dynamics simulations of Kcv(PBCV-1) and N-terminally truncated mutants; the truncated mutants mimic Kcv(ATCV-1) and Kcv(MT325). Structures were analyzed with respect to membrane positioning in relation to the orientation of K29. The results indicate that the architecture of the protein (including the selectivity filter) is only weakly dependent on TMD length and protonation of K29. The penetration depth of Lys in a given protonation state is independent of the TMD architecture, which leads to a distortion of shorter proteins. The data imply that snorkeling can be important for K(+) channels; however, its significance depends on the architecture of the entire TMD. The observation that the most severe N-terminal truncation causes the outer TMD to move toward the cytosolic side suggests that snorkeling becomes more relevant if TMDs are not stabilized in the membrane by other domains.

  11. Antiviral effect of cationic compounds on bacteriophages

    Directory of Open Access Journals (Sweden)

    Mai Huong eChatain-Ly

    2013-03-01

    Full Text Available The antiviral activity of several cationic compounds - cetytrimethylammonium (CTAB, chitosan, nisin and lysozyme - was investigated on the bacteriophage c2 (DNA head and non-contractile tail infecting Lactococcus strains and the bacteriophage MS2 (F-specific RNA infecting E.coli. Firstly, these activities were evaluated in a phosphate buffer pH 7- 10 mM. The CTAB had a virucidal effect on the Lactococcus bacteriophages, but not on the MS2. After 1 min of contact with 0.125 mM CTAB, the c2 population was reduced from 6 log(pfu/mL to 1,5 log(pfu/mL and completely deactivated at 1 mM. On the contrary, chitosan inhibited the MS2 more than it did the bacteriophages c2. No antiviral effect was observed for the nisin or the lysozyme on bacteriophages after 1 min of treatment. A 1 and 2.5 log reduction was respectively observed for nisin and lysozyme when the treatment time increased (5 or 10 min. These results showed that the antiviral effect depended both on the virus and structure of the antimicrobial compounds. The antiviral activity of these compounds was also evaluated in different physico-chemical conditions and in complex matrices. The antiviral activity of CTAB was impaired in acid pH and with an increase of the ionic strength. These results might be explained by the electrostatic interactions between cationic compounds and negatively charged particles such as bacteriophages or other compounds in a matrix. Milk proved to be protective suggesting the components of food could interfere with antimicrobial compounds.

  12. Marine Toxins Targeting Ion Channels

    Directory of Open Access Journals (Sweden)

    Hugo R. Arias

    2006-04-01

    Full Text Available Abstract: This introductory minireview points out the importance of ion channels for cell communication. The basic concepts on the structure and function of ion channels triggered by membrane voltage changes, the so-called voltage-gated ion channels (VGICs, as well as those activated by neurotransmitters, the so-called ligand-gated ion channel (LGICs, are introduced. Among the most important VGIC superfamiles, we can name the voltage-gated Na+ (NaV, Ca2+ (CaV, and K+ (KV channels. Among the most important LGIC super families, we can include the Cys-loop or nicotinicoid, the glutamate-activated (GluR, and the ATP-activated (P2XnR receptor superfamilies. Ion channels are transmembrane proteins that allow the passage of different ions in a specific or unspecific manner. For instance, the activation of NaV, CaV, or KV channels opens a pore that is specific for Na+, Ca2+, or K+, respectively. On the other hand, the activation of certain LGICs such as nicotinic acetylcholine receptors, GluRs, and P2XnRs allows the passage of cations (e.g., Na+, K+, and/or Ca2+, whereas the activation of other LGICs such as type A γ-butyric acid and glycine receptors allows the passage of anions (e.g., Cl− and/or HCO3−. In this regard, the activation of NaV and CaV as well as ligand-gated cation channels produce membrane depolarization, which finally leads to stimulatory effects in the cell, whereas the activation of KV as well as ligand-gated anion channels induce membrane hyperpolarization that finally leads to inhibitory effects in the cell. The importance of these ion channel superfamilies is emphasized by considering their physiological functions throughout the body as well as their pathophysiological implicance in several neuronal diseases. In this regard, natural molecules, and especially marine toxins, can be potentially used as modulators (e.g., inhibitors or prolongers of ion channel functions to treat or to alleviate a specific

  13. Tissue transglutaminase inhibits the TRPV5-dependent calcium transport in an N-glycosylation-dependent manner

    DEFF Research Database (Denmark)

    Boros, Sandor; Xi, Qi; Dimke, Henrik Anthony;

    2011-01-01

    Tissue transglutaminase (tTG) is a multifunctional Ca(2+)-dependent enzyme, catalyzing protein crosslinking. The transient receptor potential vanilloid (TRPV) family of cation channels was recently shown to contribute to the regulation of TG activities in keratinocytes and hence skin barrier...... of polarized cultures of rabbit connecting tubule and cortical collecting duct (CNT/CCD) cells. Extracellular application of tTG significantly reduced TRPV5 activity in human embryonic kidney cells transiently expressing the channel. Similarly, a strong inhibition of transepithelial Ca(2+) transport...... was observed after apical application of purified tTG to polarized rabbit CNT/CCD cells. Furthermore, tTG promoted the aggregation of the plasma membrane-associated fraction of TRPV5. Using patch clamp analysis, we observed a reduction in the pore diameter after tTG treatment, suggesting distinct structural...

  14. The role of transient receptor potential channels in kidney disease.

    NARCIS (Netherlands)

    Woudenberg-Vrenken, T.E.; Bindels, R.J.M.; Hoenderop, J.G.J.

    2009-01-01

    The transient receptor potential (TRP) superfamily consists, in mammals, of six protein subfamilies, TRPC, TRPM, TRPV, TRPA, TRPML and TRPP. TRPs are cation channels involved in many physiological processes and in the pathogenesis of various disorders. In the kidney, TRP channels are expressed along

  15. SNF8, a member of the ESCRT-II complex, interacts with TRPC6 and enhances its channel activity

    Directory of Open Access Journals (Sweden)

    Carrasquillo Robert

    2012-11-01

    Full Text Available Abstract Background Transient receptor potential canonical (TRPC channels are non-selective cation channels involved in receptor-mediated calcium signaling in diverse cells and tissues. The canonical transient receptor potential 6 (TRPC6 has been implicated in several pathological processes, including focal segmental glomerulosclerosis (FSGS, cardiac hypertrophy, and pulmonary hypertension. The two large cytoplasmic segments of the cation channel play a critical role in the proper regulation of channel activity, and are involved in several protein-protein interactions. Results Here we report that SNF8, a component of the endosomal sorting complex for transport-II (ESCRT-II complex, interacts with TRPC6. The interaction was initially observed in a yeast two-hybrid screen using the amino-terminal cytoplasmic domain of TRPC6 as bait, and confirmed by co-immunoprecipitation from eukaryotic cell extracts. The amino-terminal 107 amino acids are necessary and sufficient for the interaction. Overexpression of SNF8 enhances both wild-type and gain-of-function mutant TRPC6-mediated whole-cell currents in HEK293T cells. Furthermore, activation of NFAT-mediated transcription by gain-of-function mutants is enhanced by overexpression of SNF8, and partially inhibited by RNAi mediated knockdown of SNF8. Although the ESCRT-II complex functions in the endocytosis and lysosomal degradation of transmembrane proteins, SNF8 overexpression does not alter the amount of TRPC6 present on the cell surface. Conclusion SNF8 is novel binding partner of TRPC6, binding to the amino-terminal cytoplasmic domain of the channel. Modulating SNF8 expression levels alters the TRPC6 channel current and can modulate activation of NFAT-mediated transcription downstream of gain-of-function mutant TRPC6. Taken together, these results identify SNF8 as a novel regulator of TRPC6.

  16. TRP Channels in Skin Biology and Pathophysiology

    Science.gov (United States)

    Caterina, Michael J.; Pang, Zixuan

    2016-01-01

    Ion channels of the Transient Receptor Potential (TRP) family mediate the influx of monovalent and/or divalent cations into cells in response to a host of chemical or physical stimuli. In the skin, TRP channels are expressed in many cell types, including keratinocytes, sensory neurons, melanocytes, and immune/inflammatory cells. Within these diverse cell types, TRP channels participate in physiological processes ranging from sensation to skin homeostasis. In addition, there is a growing body of evidence implicating abnormal TRP channel function, as a product of excessive or deficient channel activity, in pathological skin conditions such as chronic pain and itch, dermatitis, vitiligo, alopecia, wound healing, skin carcinogenesis, and skin barrier compromise. These diverse functions, coupled with the fact that many TRP channels possess pharmacologically accessible sites, make this family of proteins appealing therapeutic targets for skin disorders. PMID:27983625

  17. TRP Channels in Skin Biology and Pathophysiology

    Directory of Open Access Journals (Sweden)

    Michael J. Caterina

    2016-12-01

    Full Text Available Ion channels of the Transient Receptor Potential (TRP family mediate the influx of monovalent and/or divalent cations into cells in response to a host of chemical or physical stimuli. In the skin, TRP channels are expressed in many cell types, including keratinocytes, sensory neurons, melanocytes, and immune/inflammatory cells. Within these diverse cell types, TRP channels participate in physiological processes ranging from sensation to skin homeostasis. In addition, there is a growing body of evidence implicating abnormal TRP channel function, as a product of excessive or deficient channel activity, in pathological skin conditions such as chronic pain and itch, dermatitis, vitiligo, alopecia, wound healing, skin carcinogenesis, and skin barrier compromise. These diverse functions, coupled with the fact that many TRP channels possess pharmacologically accessible sites, make this family of proteins appealing therapeutic targets for skin disorders.

  18. Pore size matters for potassium channel conductance

    Science.gov (United States)

    Moldenhauer, Hans; Pincuntureo, Matías

    2016-01-01

    Ion channels are membrane proteins that mediate efficient ion transport across the hydrophobic core of cell membranes, an unlikely process in their absence. K+ channels discriminate K+ over cations with similar radii with extraordinary selectivity and display a wide diversity of ion transport rates, covering differences of two orders of magnitude in unitary conductance. The pore domains of large- and small-conductance K+ channels share a general architectural design comprising a conserved narrow selectivity filter, which forms intimate interactions with permeant ions, flanked by two wider vestibules toward the internal and external openings. In large-conductance K+ channels, the inner vestibule is wide, whereas in small-conductance channels it is narrow. Here we raise the idea that the physical dimensions of the hydrophobic internal vestibule limit ion transport in K+ channels, accounting for their diversity in unitary conductance. PMID:27619418

  19. L-type Ca2+ channels in the enteric nervous system mediate oscillatory Cl- secretion in guinea pig colon.

    Science.gov (United States)

    Nishikitani, Mariko; Yasuoka, Yukiko; Kawada, Hideaki; Kawahara, Katsumasa

    2007-02-01

    The enteric nervous system regulates epithelial ion and fluid secretion. Our previous study has shown that the low (0.2-1 mM) concentrations of Ba2+, a K+ channel inhibitor, evoke Ca2+-dependent oscillatory Cl- secretion via activation of submucosal cholinergic neurons in guinea pig distal colon. However, it is still unclear which types of Ca2+ channels are involved in the oscillation at the neuroepithelial junction. We investigated the inhibitory effects of organic and inorganic Ca2+ channel antagonists on the short circuit current (I(sc)) of colonic epithelia (mucosa-submucosa sheets) mounted in Ussing chambers. The amplitude (412 +/- 37 microA cm(-2)) and frequency (2.6 +/- 0.1 cycles min(-1)) of the Ba2+-induced I(sc) in normal (1.8 mM) Ca2+ solution (n = 26) significantly decreased by 37.6% and 38.5%, respectively, in the low (0.1 mM) Ca2+ solution (n = 14). The I(sc) amplitude was reversibly inhibited by either verapamil (an L-type Ca2+ channel antagonist) or divalent cations (Cd2+, Mn2+, Ni2+) in a concentration-dependent manner. The concentration of verapamil for half-maximum inhibition (IC50) was 4 and 2 microM in normal and low Ca2+ solution, respectively. The relative blocking potencies of metal ions were Cd2+ > Mn2+, Ni2+ in normal Ca2+ solution. In contrast, the frequency of I(sc) was unchanged over the range of concentrations of the Ca2+ channel antagonists used. Our results show that the oscillatory I(sc) evoked by Ba2+ involves L-type voltage-gated Ca2+ channels. We conclude that L-type Ca2+ channels play a key role in the oscillation at the neuroepithelial junctions of guinea pig colon.

  20. Differential Effects of TRPA and TRPV Channels on Behaviors of Caenorhabditis elegans

    Science.gov (United States)

    Thies, Jennifer; Neutzler, Vanessa; O’Leary, Fidelma; Liu, He

    2016-01-01

    TRPA and TRPV ion channels are members of the transient receptor potential (TRP) cation channel superfamily, which mediates various sensory transductions. In Caenorhabditis elegans, the TRPV channels are known to affect chemosensation, while the TRPA-1 channel is associated with thermosensation and mechanosensation. We examined thermosensation, chemosensation, and osmosensation in strains lacking TRPA-1 or TRPV channels. We found that TRPV channel knockout worms exhibited similar behavioral deficits associated with thermotaxis as the TRPA-1 channel knockout, suggesting a dual role for TRPV channels. In contrast, chemosensation responses, assessed by both avoidance reversal behavior and NaCl osmosensation, were dependent on TRPV channels but seemed independent of TRPA-1 channel. Our findings suggest that, in addition to TRPA-1 channel, TRPV channels are necessary for thermotaxis and may activate, or modulate, the function of TRPA-1 channels. In contrast, TRPA-1 channels do not have a dual responsibility, as they have no functional role in odorant avoidance or osmosensation. PMID:27168724

  1. The Free Tricoordinated Silyl Cation Problem

    Directory of Open Access Journals (Sweden)

    Čičak, H.

    2010-03-01

    Full Text Available As the importance and abundance of silicon in our environment is large, it has been thought that silicon might take the place of carbon in forming a host of similar compounds and silicon-based life. However, until today there is no experimental evidence for such a hypothesis and carbon is still unique among the elements in the vast number and variety of compounds it can form. Also, the corresponding derivatives of the two elements show considerable differences in their chemical properties.The essential debate concerning organosilicon chemistry relates to the existence of the free planar tricoordinated silyl cations in condensed phase (R3Si+, in analogy to carbocations (R3C+ which have been known and characterized as free species. Although silyl cations are thermodynamically more stable than their carbon analogs, they are very reactive due to their high inherent electrophilicity and the ability of hypervalent coordination. On the other hand, stabilization by inductive and hyperconjugative effects and larger steric effects of carbocations make them less sensitive to solvation or other environmental effects than silyl cations. Hence, observation of free silyl cations in the condensed phase proved extremely difficult and the actual problem is the question of the degree of the (remaining silyl cation character.The first free silyl cation, trimesitylsilyl cation, and in analogy with it tridurylsilyl cation, were synthesized by Lambert et al. Free silyl cations based on analogy to aromatic ions (homocyclopropenylium and tropylium have also been prepared. However, in these silyl cations the cationic character is reduced by internal π -conjugation. Čičak et al. prepared some silyl-cationic intermediates (Me3Si--CH≡CR+in solid state. With the help of quantum-mechanical calculations it was concluded that these adducts have much more silyl cation than carbocation character.

  2. Cholecystokinin facilitates neuronal excitability in the entorhinal cortex via activation of TRPC-like channels.

    Science.gov (United States)

    Wang, Shouping; Zhang, An-Ping; Kurada, Lalitha; Matsui, Toshimitsu; Lei, Saobo

    2011-09-01

    Cholecystokinin (CCK) is one of the most abundant neuropeptides in the brain, where it interacts with two G protein-coupled receptors (CCK-1 and CCK-2). Activation of both CCK receptors increases the activity of PLC, resulting in increases in intracellular calcium ion (Ca(2+)) release and activation of PKC. Whereas high density of CCK receptors has been detected in the superficial layers of the entorhinal cortex (EC), the functions of CCK in this brain region have not been determined. Here, we studied the effects of CCK on neuronal excitability of layer III pyramidal neurons in the EC. Our results showed that CCK remarkably increased the firing frequency of action potentials (APs). The effects of CCK on neuronal excitability were mediated via activation of CCK-2 receptors and required the functions of G proteins and PLC. However, CCK-mediated facilitation of neuronal excitability was independent of inositol trisphosphate receptors and PKC. CCK facilitated neuronal excitability by activating a cationic channel to generate membrane depolarization. The effects of CCK were suppressed by the generic, nonselective cationic channel blockers, 2-aminoethyldiphenyl borate and flufenamic acid, but potentiated by gadolinium ion and lanthanum ion at 100 μM. Depletion of extracellular Ca(2+) also counteracted CCK-induced increases in AC firing frequency. Moreover, CCK-induced enhancement of neuronal excitability was inhibited significantly by intracellular application of the antibody to transient receptor potential channel 5 (TRPC5), suggesting the involvement of TRPC5 channels. Our results provide a cellular and molecular mechanism to help explain the functions of CCK in vivo.

  3. Flufenamic acid decreases neuronal excitability through modulation of voltage-gated sodium channel gating.

    Science.gov (United States)

    Yau, Hau-Jie; Baranauskas, Gytis; Martina, Marco

    2010-10-15

    The electrophysiological phenotype of individual neurons critically depends on the biophysical properties of the voltage-gated channels they express. Differences in sodium channel gating are instrumental in determining the different firing phenotypes of pyramidal cells and interneurons; moreover, sodium channel modulation represents an important mechanism of action for many widely used CNS drugs. Flufenamic acid (FFA) is a non-steroidal anti-inflammatory drug that has been long used as a blocker of calcium-dependent cationic conductances. Here we show that FFA inhibits voltage-gated sodium currents in hippocampal pyramidal neurons; this effect is dose-dependent with IC(50) = 189 μm. We used whole-cell and nucleated patch recordings to investigate the mechanisms of FFA modulation of TTX-sensitive voltage-gated sodium current. Our data show that flufenamic acid slows down the inactivation process of the sodium current, while shifting the inactivation curve ~10 mV toward more hyperpolarized potentials. The recovery from inactivation is also affected in a voltage-dependent way, resulting in slower recovery at hyperpolarized potentials. Recordings from acute slices demonstrate that FFA reduces repetitive- and abolishes burst-firing in CA1 pyramidal neurons. A computational model based on our data was employed to better understand the mechanisms of FFA action. Simulation data support the idea that FFA acts via a novel mechanism by reducing the voltage dependence of the sodium channel fast inactivation rates. These effects of FFA suggest that it may be an effective anti-epileptic drug.

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

    DEFF Research Database (Denmark)

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

    2011-01-01

    Accessory ß-subunits of the KCNE gene family modulate the function of various cation channel a-subunits by the formation of heteromultimers. Among the most dramatic changes of biophysical properties of a voltage-gated channel by KCNEs are the effects of KCNE1 on KCNQ1 channels. KCNQ1 and KCNE1 ar...

  5. Function and regulation of the channel-kinase TRPM7 in health and disease

    NARCIS (Netherlands)

    Visser, D.; Middelbeek, J.; Leeuwen, F.N. van; Jalink, K.

    2014-01-01

    Transient receptor potential (TRP) cation channels represent a large and diverse family of ion channels that act as important transducers of sensory information. The Melastatin subfamily member TRPM7 has garnered much interest due to its functional kinase domain; a unique feature among ion channels.

  6. Interplay between TRP channels and the cytoskeleton in health and disease.

    NARCIS (Netherlands)

    Clark, K.; Middelbeek, J.; Leeuwen, F.N. van

    2008-01-01

    Transient receptor potential (TRP) channels are a family of cation channels that play a key role in ion homeostasis and cell volume regulation. In addition, TRP channels are considered universal integrators of sensory information required for taste, vision, hearing, touch, temperature, and the detec

  7. Reactions of atomic cations with methane: gas phase room-temperature kinetics and periodicities in reactivity.

    Science.gov (United States)

    Shayesteh, Alireza; Lavrov, Vitali V; Koyanagi, Gregory K; Bohme, Diethard K

    2009-05-14

    Reactions of methane have been measured with 59 atomic metal cations at room temperature in helium bath gas at 0.35 Torr using an inductively-coupled plasma/selected-ion flow tube (ICP/SIFT) tandem mass spectrometer. The atomic cations were produced at approximately 5500 K in an ICP source and allowed to decay radiatively and to thermalize by collisions with argon and helium atoms prior to reaction. Rate coefficients and product distributions are reported for the reactions of fourth-row atomic cations from K(+) to Se(+), of fifth-row atomic cations from Rb(+) to Te(+) (excluding Tc(+)), of sixth-row atomic cations from Cs(+) to Bi(+), and of the lanthanide cations from La(+) to Lu(+) (excluding Pm(+)). Two primary reaction channels were observed: C-H bond insertion with elimination of H(2), and CH(4) addition. The bimolecular H(2) elimination was observed in the reactions of CH(4) with As(+), Nb(+), and some sixth-row metal cations, i.e., Ta(+), W(+), Os(+), Ir(+), Pt(+); secondary and higher-order H(2) elimination was observed exclusively for Ta(+), W(+), and Ir(+) ions. All other transition-metal cations except Mn(+) and Re(+) were observed to react with CH(4) exclusively by addition, and up to two methane molecules were observed to add sequentially to most transition-metal ions. CH(4) addition was also observed for Ge(+), Se(+), La(+), Ce(+), and Gd(+) ions, while the other main-group and lanthanide cations did not react measurably with methane.

  8. TRP channels and psychiatric disorders.

    Science.gov (United States)

    Chahl, Loris A

    2011-01-01

    Depression and schizophrenia are major psychiatric disorders that cause much human suffering. Current treatments have major limitations and new drug targets are eagerly sought. Study of transient receptor potential (TRP) channels in these disorders is at an early stage and the potential of agents that activate or inhibit these channels remains speculative. The findings that TRPC6 channels promote dendritic growth and are selectively activated by hyperforin, the key constitutent of St John's wort, suggest that TRPC6 channels might prove to be a new target for antidepressant drug development. There is now considerable evidence that TRPV1 antagonists have anxiolytic activity but there is no direct evidence that they have antidepressant activity. There is also no direct evidence that TRP channels play a role in schizophrenia. However, the findings that TRPC channels are involved in neuronal development and fundamental synaptic mechanisms, and that TRPV1 channels play a role in central dopaminergic and cannabinoid mechanisms is suggestive of potential roles of these channels in schizophrenia. Investigation of TRP channels in psychiatric disorders holds the promise of yielding further understanding of the aetiology of psychiatric disorders and the development of new drug treatments.

  9. Mechanosensitive Channels

    Science.gov (United States)

    Martinac, Boris

    Living cells are exposed to a variety of mechanical stimuli acting throughout the biosphere. The range of the stimuli extends from thermal molecular agitation to potentially destructive cell swelling caused by osmotic pressure gradients. Cellular membranes present a major target for these stimuli. To detect mechanical forces acting upon them cell membranes are equipped with mechanosensitive (MS) ion channels. Functioning as molecular mechanoelectrical transducers of mechanical forces into electrical and/or chemical intracellular signals these channels play a critical role in the physiology of mechanotransduction. Studies of prokaryotic MS channels and recent work on MS channels of eukaryotes have significantly increased our understanding of their gating mechanism, physiological functions, and evolutionary origins as well as their role in the pathology of disease.

  10. Afrikaans Syllabification Patterns

    Directory of Open Access Journals (Sweden)

    Tilla Fick

    2010-01-01

    Full Text Available In contrast to English, automatic hyphenation by computer of Afrikaans words is a problem that still needs to be addressed, since errors are still often encountered in printed text. An initial step in this task is the ability to automatically syllabify words. Since new words are created continuously by joining words, it is necessary to develop an “intelligent” technique for syllabification. As a first phase of the research, we consider only the orthographic information of words, and disregard both syntactic and morphological information. This approach allows us to use machine-learning techniques such as artificial neural networks and decision trees that are known for their pattern recognition abilities. Both these techniques are trained with isolated patterns consisting of input patterns and corresponding outputs (or targets that indicate whether the input pattern should be split at a certain position, or not. In the process of compiling a list of syllabified words from which to generate training data for the  syllabification problem, irregular patterns were identified. The same letter patterns are split differently in different words and complete words that are spelled identically are split differently due to meaning. We also identified irregularities in and between  the different dictionaries that we used. We examined the influence range of letters that are involved in irregularities. For example, for their in agter-ente and vaste-rente we have to consider three letters to the left of r to be certain where the hyphen should be inserted. The influence range of the k in verstek-waarde and kleinste-kwadrate is four to the left and three to the right. In an analysis of letter patterns in Afrikaans words we found that the letter e has the highest frequency overall (16,2% of all letters in the word list. The frequency of words starting with s is the highest, while the frequency of words ending with e is the highest. It is important to

  11. A Bioluminescence Assay System for Imaging Metal Cationic Activities in Urban Aerosols.

    Science.gov (United States)

    Kim, Sung-Bae; Naganawa, Ryuichi; Murata, Shingo; Nakayama, Takayoshi; Miller, Simon; Senda, Toshiya

    2016-01-01

    A bioluminescence-based assay system was fabricated for an efficient determination of the activities of air pollutants. The following four components were integrated into this assay system: (1) an 8-channel assay platform uniquely designed for simultaneously sensing multiple optical samples, (2) single-chain probes illuminating toxic chemicals or heavy metal cations from air pollutants, (3) a microfluidic system for circulating medium mimicking the human body, and (4) the software manimulating the above system. In the protocol, we briefly introduce how to integrate the components into the system and the application to the illumination of the metal cationic activities in air pollutants.

  12. Regulation of Intestinal Glucose Absorption by Ion Channels and Transporters.

    Science.gov (United States)

    Chen, Lihong; Tuo, Biguang; Dong, Hui

    2016-01-14

    The absorption of glucose is electrogenic in the small intestinal epithelium. The major route for the transport of dietary glucose from intestinal lumen into enterocytes is the Na⁺/glucose cotransporter (SGLT1), although glucose transporter type 2 (GLUT2) may also play a role. The membrane potential of small intestinal epithelial cells (IEC) is important to regulate the activity of SGLT1. The maintenance of membrane potential mainly depends on the activities of cation channels and transporters. While the importance of SGLT1 in glucose absorption has been systemically studied in detail, little is currently known about the regulation of SGLT1 activity by cation channels and transporters. A growing line of evidence suggests that cytosolic calcium ([Ca(2+)]cyt) can regulate the absorption of glucose by adjusting GLUT2 and SGLT1. Moreover, the absorption of glucose and homeostasis of Ca(2+) in IEC are regulated by cation channels and transporters, such as Ca(2+) channels, K⁺ channels, Na⁺/Ca(2+) exchangers, and Na⁺/H⁺ exchangers. In this review, we consider the involvement of these cation channels and transporters in the regulation of glucose uptake in the small intestine. Modulation of them may be a potential strategy for the management of obesity and diabetes.

  13. Regulation of Intestinal Glucose Absorption by Ion Channels and Transporters

    Directory of Open Access Journals (Sweden)

    Lihong Chen

    2016-01-01

    Full Text Available The absorption of glucose is electrogenic in the small intestinal epithelium. The major route for the transport of dietary glucose from intestinal lumen into enterocytes is the Na+/glucose cotransporter (SGLT1, although glucose transporter type 2 (GLUT2 may also play a role. The membrane potential of small intestinal epithelial cells (IEC is important to regulate the activity of SGLT1. The maintenance of membrane potential mainly depends on the activities of cation channels and transporters. While the importance of SGLT1 in glucose absorption has been systemically studied in detail, little is currently known about the regulation of SGLT1 activity by cation channels and transporters. A growing line of evidence suggests that cytosolic calcium ([Ca2+]cyt can regulate the absorption of glucose by adjusting GLUT2 and SGLT1. Moreover, the absorption of glucose and homeostasis of Ca2+ in IEC are regulated by cation channels and transporters, such as Ca2+ channels, K+ channels, Na+/Ca2+ exchangers, and Na+/H+ exchangers. In this review, we consider the involvement of these cation channels and transporters in the regulation of glucose uptake in the small intestine. Modulation of them may be a potential strategy for the management of obesity and diabetes.

  14. Ion Permeation and Mechanotransduction Mechanisms of Mechanosensitive Piezo Channels.

    Science.gov (United States)

    Zhao, Qiancheng; Wu, Kun; Geng, Jie; Chi, Shaopeng; Wang, Yanfeng; Zhi, Peng; Zhang, Mingmin; Xiao, Bailong

    2016-03-16

    Piezo proteins have been proposed as the long-sought-after mechanosensitive cation channels in mammals that play critical roles in various mechanotransduction processes. However, the molecular bases that underlie their ion permeation and mechanotransduction have remained functionally undefined. Here we report our finding of the miniature pore-forming module of Piezo1 that resembles the pore architecture of other trimeric channels and encodes the essential pore properties. We further identified specific residues within the pore module that determine unitary conductance, pore blockage and ion selectivity for divalent and monovalent cations and anions. The non-pore-containing region of Piezo1 confers mechanosensitivity to mechano-insensitive trimeric acid-sensing ion channels, demonstrating that Piezo1 channels possess intrinsic mechanotransduction modules separate from their pore modules. In conclusion, this is the first report on the bona fide pore module and mechanotransduction components of Piezo channels, which define their ion-conducting properties and gating by mechanical stimuli, respectively.

  15. Localization versus delocalization in diamine radical cations

    DEFF Research Database (Denmark)

    Brouwer, A.M.; Wiering, P.G.; Zwier, J.M.;

    1997-01-01

    The optical absorption spectrum of the radical cation of 1,4-diphenylpiperazine 2a shows a strong transition in the near-IR, and only a weak band at 445 nm, in the region where aniline radical cations normally absorb strongly. This indicates that the charge and spin are delocalized over the two...

  16. Advancements in Anion Exchange Membrane Cations

    Energy Technology Data Exchange (ETDEWEB)

    Sturgeon, Matthew R. [National Renewable Energy Lab. (NREL), Golden, CO (United States); Long, Hai [National Renewable Energy Lab. (NREL), Golden, CO (United States); Park, Andrew M. [National Renewable Energy Lab. (NREL), Golden, CO (United States); Pivovar, Bryan S. [National Renewable Energy Lab. (NREL), Golden, CO (United States)

    2015-10-15

    Anion-exchange membrane fuel cells (AME-FCs) are of increasingly popular interest as they enable the use of non-Pt fuel cell catalysts, the primary cost limitation of proton exchange membrane fuel cells. Benzyltrimethyl ammonium (BTMA) is the standard cation that has historically been utilized as the hydroxide conductor in AEMs. Herein we approach AEMs from two directions. First and foremost we study the stability of several different cations in a hydroxide solution at elevated temperatures. We specifically targeted BTMA and methoxy and nitro substituted BTMA. We've also studied the effects of adding an akyl spacer units between the ammonium cation and the phenyl group. In the second approach we use computational studies to predict stable ammonium cations, which are then synthesized and tested for stability. Our unique method to study cation stability in caustic conditions at elevated temperatures utilizes Teflon Parr reactors suitable for use under various temperatures and cation concentrations. NMR analysis was used to determine remaining cation concentrations at specific time points with GCMS analysis verifying product distribution. We then compare the experimental results with calculated modeling stabilities. Our studies show that the electron donating methoxy groups slightly increase stability (compared to that of BTMA), while the electron withdrawing nitro groups greatly decrease stability in base. These results give insight into possible linking strategies to be employed when tethering a BTMA like ammonium cation to a polymeric backbone; thus synthesizing an anion exchange membrane.

  17. Cation diffusion in the natural zeolite clinoptilolite

    Energy Technology Data Exchange (ETDEWEB)

    Dyer, A.; White, K.J. [Science Research Institute, Chemistry Division, Cockcroft Building, University of Salford, Salford (United Kingdom)

    1999-12-14

    The natural zeolite clinoptilolite is mined commercially in many parts of the world. It is a selective exchanger for the ammonium cation and this has prompted its use in waste water treatment, swimming pools and in fish farming. It is also used to scavenge radioisotopes in nuclear waste clean-up. Further potential uses for clinoptilolite are in soil amendment and remediation. The work described herein provides thermodynamic data on cation exchange processes in clinoptilolite involving the NH{sub 4}, Na, K, Ca, and Mg cations. The data includes estimates of interdiffusion coefficients together with free energies, entropies and energies of activation for the cation exchanges studied. Suggestions are made as to the mechanisms of cation-exchanges involved.

  18. MITOCHONDRIAL BKCa CHANNEL

    Directory of Open Access Journals (Sweden)

    Enrique eBalderas

    2015-03-01

    Full Text Available Since its discovery in a glioma cell line 15 years ago, mitochondrial BKCa channel (mitoBKCa has been studied in brain cells and cardiomyocytes sharing general biophysical properties such as high K+ conductance (~300 pS, voltage-dependency and Ca2+-sensitivity. Main advances in deciphering the molecular composition of mitoBKCa have included establishing that it is encoded by the Kcnma1 gene, that a C-terminal splice insert confers mitoBKCa ability to be targeted to cardiac mitochondria, and evidence for its potential coassembly with β subunits. Notoriously, β1 subunit directly interacts with cytochrome c oxidase and mitoBKCa can be modulated by substrates of the respiratory chain. mitoBKCa channel has a central role in protecting the heart from ischemia, where pharmacological activation of the channel impacts the generation of reactive oxygen species and mitochondrial Ca2+ preventing cell death likely by impeding uncontrolled opening of the mitochondrial transition pore. Supporting this view, inhibition of mitoBKCa with Iberiotoxin, enhances cytochrome c release from glioma mitochondria. Many tantalizing questions remain. Some of them are: how is mitoBKCa coupled to the respiratory chain? Does mitoBKCa play non-conduction roles in mitochondria physiology? Which are the functional partners of mitoBKCa? What are the roles of mitoBKCa in other cell types? Answers to these questions are essential to define the impact of mitoBKCa channel in mitochondria biology and disease.

  19. Interactions between alpha-latrotoxin and trivalent cations in rat striatal synaptosomal preparations

    Energy Technology Data Exchange (ETDEWEB)

    Scheer, H.W.

    1989-05-01

    The interactions between alpha-latrotoxin (alpha-LTx), a neurosecretagogue purified from the venom of the black widow spider, and the trivalent cations Al3+, Y3+, La3+, Gd3+, and Yb3+ were investigated in rat striatal synaptosomal preparations. All trivalent cations tested were inhibitors of alpha-LTx-induced (/sup 3/H)dopamine ((/sup 3/H)DA) release (order of potency: Yb3+ greater than Gd3+ approximately Y3+ greater than La3+ greater than Al3+). Only with Al3+ could inhibition of (/sup 3/H)DA release be attributed to a block of /sup 125/I-alpha-LTx specific binding to synaptosomal preparations. The inhibitory effect of trivalent ions was reversible provided synaptosomes were washed with buffer containing EDTA. Trivalent ions also inhibited alpha-LTx-induced (/sup 3/H)DA release at times when alpha-LTx-stimulated release was already evident. alpha-LTx-induced synaptosomal membrane depolarization was blocked by La3+, but not affected by Gd3+, Y3+, and Yb3+. alpha-LTx-stimulated uptake of /sup 45/Ca/sup 2 +/ was inhibited by all trivalent cations tested. These results demonstrate that there exist at least three means by which trivalent cations can inhibit alpha-LTx action in rat striatal synaptosomal preparations: (1) inhibition of alpha-LTx binding (Al3+); (2) inhibition of alpha-LTx-induced depolarization (La3+); and (3) inhibition of alpha-LTx-induced /sup 45/Ca/sup 2 +/ uptake (Gd3+, Y3+, Yb3+, La3+).

  20. Leptin excites POMC neurons via activation of TRPC channels

    Science.gov (United States)

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

    2010-01-01

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

  1. Cationic Bolaamphiphiles for Gene Delivery

    Science.gov (United States)

    Tan, Amelia Li Min; Lim, Alisa Xue Ling; Zhu, Yiting; Yang, Yi Yan; Khan, Majad

    2014-05-01

    Advances in medical research have shed light on the genetic cause of many human diseases. Gene therapy is a promising approach which can be used to deliver therapeutic genes to treat genetic diseases at its most fundamental level. In general, nonviral vectors are preferred due to reduced risk of immune response, but they are also commonly associated with low transfection efficiency and high cytotoxicity. In contrast to viral vectors, nonviral vectors do not have a natural mechanism to overcome extra- and intracellular barriers when delivering the therapeutic gene into cell. Hence, its design has been increasingly complex to meet challenges faced in targeting of, penetration of and expression in a specific host cell in achieving more satisfactory transfection efficiency. Flexibility in design of the vector is desirable, to enable a careful and controlled manipulation of its properties and functions. This can be met by the use of bolaamphiphile, a special class of lipid. Unlike conventional lipids, bolaamphiphiles can form asymmetric complexes with the therapeutic gene. The advantage of having an asymmetric complex lies in the different purposes served by the interior and exterior of the complex. More effective gene encapsulation within the interior of the complex can be achieved without triggering greater aggregation of serum proteins with the exterior, potentially overcoming one of the great hurdles faced by conventional single-head cationic lipids. In this review, we will look into the physiochemical considerations as well as the biological aspects of a bolaamphiphile-based gene delivery system.

  2. Brands & Channels

    Institute of Scientific and Technical Information of China (English)

    Alice Yang

    2009-01-01

    @@ "Brands" and "Channels" are the two most important things in Ku-Hai Chen's eyes when doing business with Main-land China. Ku-Hai Chen, Executive Director of the International Trade Institute of Taiwan External Trade Development Council (TAITRA), flies frequently between Chinese Taipei and Mainland China, and was in Beijing earlier this month for his seminar.

  3. Positron Channeling

    CERN Document Server

    Badikyan, Karen

    2016-01-01

    The possibility of channeling the low-energy relativistic positrons around separate crystallographic axes with coaxial symmetry of negative ions in some types of crystals is shown. The process of annihilation of positrons with electrons of medium was studied in detail.

  4. Functional role of anion channels in cardiac diseases

    Institute of Scientific and Technical Information of China (English)

    Da-yue DUAN; Luis LH LIU; Nathan BOZEAT; Z Maggie HUANG; Sunny Y XIANG; Guan-lei WANG; Linda YE; Joseph R HUME

    2005-01-01

    In comparison to cation (K+, Na+, and Ca2+) channels, much less is currently known about the functional role of anion (Cl-) channels in cardiovascular physiology and pathophysiology. Over the past 15 years, various types of Cl- currents have been recorded in cardiac cells from different species including humans. All cardiac Cl- channels described to date may be encoded by five different Cl- channel genes: the PKA- and PKC-activated cystic fibrosis tansmembrane conductance regulator (CFTR), the volume-regulated ClC-2 and ClC-3, and the Ca2+-activated CLCA or Bestrophin. Recent studies using multiple approaches to examine the functional role of Cl- channels in the context of health and disease have demonstrated that Cl- channels might contribute to: 1) arrhythmogenesis in myocardial injury; 2) cardiac ischemic preconditioning; and 3) the adaptive remodeling of the heart during myocardial hypertrophy and heart failure. Therefore,anion channels represent very attractive novel targets for therapeutic approaches to the treatment of heart diseases. Recent evidence suggests that Cl- channels,like cation channels, might function as a multiprotein complex or functional module.In the post-genome era, the emergence of functional proteomics has necessitated a new paradigm shift to the structural and functional assessment of integrated Cl- channel multiprotein complexes in the heart, which could provide new insight into our understanding of the underlying mechanisms responsible for heart disease and protection.

  5. Cation distributions on rapidly solidified cobalt ferrite

    Science.gov (United States)

    De Guire, Mark R.; Kalonji, Gretchen; O'Handley, Robert C.

    1990-01-01

    The cation distributions in two rapidly solidified cobalt ferrites have been determined using Moessbauer spectroscopy at 4.2 K in an 8-T magnetic field. The samples were obtained by gas atomization of a Co0-Fe2O3-P2O5 melt. The degree of cation disorder in both cases was greater than is obtainable by cooling unmelted cobalt ferrite. The more rapidly cooled sample exhibited a smaller departure from the equilibrium cation distribution than did the more slowly cooled sample. This result is explained on the basis of two competing effects of rapid solidification: high cooling rate of the solid, and large undercooling.

  6. Hydroxyproline-induced Helical Disruption in Conantokin Rl-B Affects Subunit-selective Antagonistic Activities toward Ion Channels of N-Methyl-d-aspartate Receptors.

    Science.gov (United States)

    Kunda, Shailaja; Yuan, Yue; Balsara, Rashna D; Zajicek, Jaroslav; Castellino, Francis J

    2015-07-17

    Conantokins are ~20-amino acid peptides present in predatory marine snail venoms that function as allosteric antagonists of ion channels of the N-methyl-d-aspartate receptor (NMDAR). These peptides possess a high percentage of post-/co-translationally modified amino acids, particularly γ-carboxyglutamate (Gla). Appropriately spaced Gla residues allow binding of functional divalent cations, which induces end-to-end α-helices in many conantokins. A smaller number of these peptides additionally contain 4-hydroxyproline (Hyp). Hyp should prevent adoption of the metal ion-induced full α-helix, with unknown functional consequences. To address this disparity, as well as the role of Hyp in conantokins, we have solved the high resolution three-dimensional solution structure of a Gla/Hyp-containing 18-residue conantokin, conRl-B, by high field NMR spectroscopy. We show that Hyp(10) disrupts only a small region of the α-helix of the Mn(2+)·peptide complex, which displays cation-induced α-helices on each terminus of the peptide. The function of conRl-B was examined by measuring its inhibition of NMDA/Gly-mediated current through NMDAR ion channels in mouse cortical neurons. The conRl-B displays high inhibitory selectivity for subclasses of NMDARs that contain the functionally important GluN2B subunit. Replacement of Hyp(10) with N(8)Q results in a Mg(2+)-complexed end-to-end α-helix, accompanied by attenuation of NMDAR inhibitory activity. However, replacement of Hyp(10) with Pro(10) allowed the resulting peptide to retain its inhibitory property but diminished its GluN2B specificity. Thus, these modified amino acids, in specific peptide backbones, play critical roles in their subunit-selective inhibition of NMDAR ion channels, a finding that can be employed to design NMDAR antagonists that function at ion channels of distinct NMDAR subclasses.

  7. The mechanism of kaolin clay flocculation by a cation-independent bioflocculant produced by Chryseobacterium daeguense W6

    Directory of Open Access Journals (Sweden)

    Weijie Liu

    2015-03-01

    Full Text Available In recent years, several novel cation-independent bioflocculants have been reported, which can avoid the secondary contamination caused by addition of cations. However, compared with cation-dependent bioflocculants, the flocculating mechanism of cation-independent bioflocculants is largely unknown. In this study, a cation-independent bioflocculant MBF-W6 produced by Chryseobacterium daeguense W6 was used as a model to investigate the flocculating mechanism. The results showed that the major flocculating component of MBF-W6 is a complex of proteins and polysaccharides. The zeta potential results indicated that kaolin clay particles were not precipitated due to charge neutralization and the bridging mediated by cations did not play a major role in the flocculating process. These results are consistent with the fact that MBF-W6 is a cation-independent bioflocculant. Further scanning electron microscopic observation showed that MBF-W6 induced flocs formed tight packed structure, suggesting that the kaolin clay particles maybe directly attached and bridged by bioflocculant MBF-W6. In addition, we also found out that Fe3+ ions inhibit the flocculating activity of MBF-W6 by affecting –COO− and –NH groups. Therefore this study can improve our understanding on flocculating mechanism of cation-independent bioflocculants.

  8. Interaction of polyamines with proteins of photosystem II: Cation binding and photosynthetic oxygen evolution

    Science.gov (United States)

    Beauchemin, R.; Harnois, J.; Rouillon, R.; Tajmir-Riahi, H. A.; Carpentier, R.

    2007-05-01

    Polyamines are organic cations that function in diverse physiological processes that share as a common thread a close relationship to cell proliferation and growth. Polyamines also affect photosynthetic oxygen evolution and therefore, this study was designed to investigate the interaction of 1,3-diaminopropane, 1,4-diaminobutane (putrescine), and 1,5-diaminopentane (cadaverine) cations with proteins of photosystem II (PSII) using PSII-enriched submembrane fractions with diamine concentrations between 0.01 and 20 mM. Fourier transformed infrared (FTIR) difference spectroscopy with its self-deconvolution and second derivative resolution enhancement, as well as curve-fitting procedures were applied in order to determine the diamine binding mode, the protein conformational changes, and the structural properties of diamine-protein complexes. Spectroscopic evidence showed that diamines interact with proteins (H-bonding) through polypeptide C dbnd O groups with no major perturbations of protein secondary structure. At very low diamine concentration (0.01 mM), no inhibition of oxygen-evolution occurred, while at higher diamine content (5-10 mM), 100% inhibition was observed. Chorophyll fluorescence measurements demonstrated that the inhibition mainly affects the oxygen evolving complex of PSII. Comparisons of the effects of these dipositive organic cations with divalent metal cations on one hand and with polyvalent spermine and spermidine on the other hand, show major alterations of the protein secondary structure as positive charge increases.

  9. Channel Power in Multi-Channel Environments

    NARCIS (Netherlands)

    M.G. Dekimpe (Marnik); B. Skiera (Bernd)

    2004-01-01

    textabstractIn the literature, little attention has been paid to instances where companies add an Internet channel to their direct channel portfolio. However, actively managing multiple sales channels requires knowing the customers’ channel preferences and the resulting channel power. Two key compon

  10. Uptake of photosensitizers by bacteria is influenced by the presence of cations

    Science.gov (United States)

    Kishen, A.; George, S.

    2007-05-01

    This investigation studies the influence of cations on photosensitizer uptake by Enterococcus faecalis (gram positive) and Actinobacillus actinomycetemcomitans (gram negative). Methods- The uptake of Methylene blue (MB) and Indocyanine Green (ICG), by bacteria were studied under the influence of divalent cations (CaCl II & MgCl II) and EDTA. Further, E. faecalis cells subjected to trypsinisation and calcium channel blocker (verapamil) were also analysed for MB and ICG uptake inorder to understand the mechanism of photosensitizer uptake. Results- Uptake of ICG was enhanced in the presence of divalent cations in E. faecalis and A. actinomycetemcomitans. Treating cells with EDTA had no significant effect on the photosensitizer uptake, although the highest concentration tested showed an enhancement of uptake. In contrast to ICG, MB showed a decreased uptake by bacterial cells on subjecting them to divalent cations and EDTA. Calcium channel blocker had no significant inhibitory effect on photosensitizers uptake. However, trypsin treatment resulted in significant reduction of ICG uptake. The result suggested that ICG uptake by bacteria is mediated through specific transporter protein while MB is associated with the outer surface structures of bacterial cells.

  11. Cationic ruthenium alkylidene catalysts bearing phosphine ligands.

    Science.gov (United States)

    Endo, Koji; Grubbs, Robert H

    2016-02-28

    The discovery of highly active catalysts and the success of ionic liquid immobilized systems have accelerated attention to a new class of cationic metathesis catalysts. We herein report the facile syntheses of cationic ruthenium catalysts bearing bulky phosphine ligands. Simple ligand exchange using silver(i) salts of non-coordinating or weakly coordinating anions provided either PPh3 or chelating Ph2P(CH2)nPPh2 (n = 2 or 3) ligated cationic catalysts. The structures of these newly reported catalysts feature unique geometries caused by ligation of the bulky phosphine ligands. Their activities and selectivities in standard metathesis reactions were also investigated. These cationic ruthenium alkylidene catalysts reported here showed moderate activity and very similar stereoselectivity when compared to the second generation ruthenium dichloride catalyst in ring-closing metathesis, cross metathesis, and ring-opening metathesis polymerization assays.

  12. Cation locations and dislocations in zeolites

    Science.gov (United States)

    Smith, Luis James

    The focus of this dissertation is the extra-framework cation sites in a particular structural family of zeolites, chabazite. Cation sites play a particularly important role in the application of these sieves for ion exchange, gas separation, catalysis, and, when the cation is a proton, acid catalysis. Structural characterization is commonly performed through the use of powder diffraction and Rietveld analysis of powder diffraction data. Use of high-resolution nuclear magnetic resonance, in the study of the local order of the various constituent nuclei of zeolites, complements well the long-range order information produced by diffraction. Recent developments in solid state NMR techniques allow for increased study of disorder in zeolites particularly when such phenomena test the detection limits of diffraction. These two powerful characterization techniques, powder diffraction and NMR, offer many insights into the complex interaction of cations with the zeolite framework. The acids site locations in SSZ-13, a high silica chabazite, and SAPO-34, a silicoaluminophosphate with the chabazite structure, were determined. The structure of SAPO-34 upon selective hydration was also determined. The insensitivity of X-rays to hydrogen was avoided through deuteration of the acid zeolites and neutron powder diffraction methods. Protons at inequivalent positions were found to have different acid strengths in both SSZ-13 and SAPO-34. Other light elements are incorporated into zeolites in the form of extra-framework cations, among these are lithium, sodium, and calcium. Not amenable by X-ray powder diffraction methods, the positions of such light cations in fully ion-exchanged versions of synthetic chabazite were determined through neutron powder diffraction methods. The study of more complex binary cation systems were conducted. Powder diffraction and solid state NMR methods (MAS, MQMAS) were used to examine cation site preferences and dislocations in these mixed-akali chabazites

  13. The hyperpolarization-activated non-specific cation current (In ) adjusts the membrane properties, excitability, and activity pattern of the giant cells in the rat dorsal cochlear nucleus.

    Science.gov (United States)

    Rusznák, Zoltán; Pál, Balázs; Kőszeghy, Aron; Fu, Yuhong; Szücs, Géza; Paxinos, George

    2013-03-01

    Giant cells of the cochlear nucleus are thought to integrate multimodal sensory inputs and participate in monaural sound source localization. Our aim was to explore the significance of a hyperpolarization-activated current in determining the activity of giant neurones in slices prepared from 10 to 14-day-old rats. When subjected to hyperpolarizing stimuli, giant cells produced a 4-(N-ethyl-N-phenylamino)-1,2-dimethyl-6-(methylamino) pyridinium chloride (ZD7288)-sensitive inward current with a reversal potential and half-activation voltage of -36 and -88 mV, respectively. Consequently, the current was identified as the hyperpolarization-activated non-specific cationic current (Ih ). At the resting membrane potential, 3.5% of the maximum Ih conductance was available. Immunohistochemistry experiments suggested that hyperpolarization-activated, cyclic nucleotide-gated, cation non-selective (HCN)1, HCN2, and HCN4 subunits contribute to the assembly of the functional channels. Inhibition of Ih hyperpolarized the membrane by 6 mV and impeded spontaneous firing. The frequencies of spontaneous inhibitory and excitatory postsynaptic currents reaching the giant cell bodies were reduced but no significant change was observed when evoked postsynaptic currents were recorded. Giant cells are affected by biphasic postsynaptic currents consisting of an excitatory and a subsequent inhibitory component. Inhibition of Ih reduced the frequency of these biphasic events by 65% and increased the decay time constants of the inhibitory component. We conclude that Ih adjusts the resting membrane potential, contributes to spontaneous action potential firing, and may participate in the dendritic integration of the synaptic inputs of the giant neurones. Because its amplitude was higher in young than in adult rats, Ih of the giant cells may be especially important during the postnatal maturation of the auditory system.

  14. Nonlinear channelizer

    Science.gov (United States)

    In, Visarath; Longhini, Patrick; Kho, Andy; Neff, Joseph D.; Leung, Daniel; Liu, Norman; Meadows, Brian K.; Gordon, Frank; Bulsara, Adi R.; Palacios, Antonio

    2012-12-01

    The nonlinear channelizer is an integrated circuit made up of large parallel arrays of analog nonlinear oscillators, which, collectively, serve as a broad-spectrum analyzer with the ability to receive complex signals containing multiple frequencies and instantaneously lock-on or respond to a received signal in a few oscillation cycles. The concept is based on the generation of internal oscillations in coupled nonlinear systems that do not normally oscillate in the absence of coupling. In particular, the system consists of unidirectionally coupled bistable nonlinear elements, where the frequency and other dynamical characteristics of the emergent oscillations depend on the system's internal parameters and the received signal. These properties and characteristics are being employed to develop a system capable of locking onto any arbitrary input radio frequency signal. The system is efficient by eliminating the need for high-speed, high-accuracy analog-to-digital converters, and compact by making use of nonlinear coupled systems to act as a channelizer (frequency binning and channeling), a low noise amplifier, and a frequency down-converter in a single step which, in turn, will reduce the size, weight, power, and cost of the entire communication system. This paper covers the theory, numerical simulations, and some engineering details that validate the concept at the frequency band of 1-4 GHz.

  15. Cationized Carbohydrate Gas-Phase Fragmentation Chemistry

    Science.gov (United States)

    Bythell, Benjamin J.; Abutokaikah, Maha T.; Wagoner, Ashley R.; Guan, Shanshan; Rabus, Jordan M.

    2016-11-01

    We investigate the fragmentation chemistry of cationized carbohydrates using a combination of tandem mass spectrometry, regioselective labeling, and computational methods. Our model system is D-lactose. Barriers to the fundamental glyosidic bond cleavage reactions, neutral loss pathways, and structurally informative cross-ring cleavages are investigated. The most energetically favorable conformations of cationized D-lactose were found to be similar. In agreement with the literature, larger group I cations result in structures with increased cation coordination number which require greater collision energy to dissociate. In contrast with earlier proposals, the B n -Y m fragmentation pathways of both protonated and sodium-cationized analytes proceed via protonation of the glycosidic oxygen with concerted glycosidic bond cleavage. Additionally, for the sodiated congeners our calculations support sodiated 1,6-anhydrogalactose B n ion structures, unlike the preceding literature. This affects the subsequent propensity of formation and prediction of B n /Y m branching ratio. The nature of the anomeric center (α/β) affects the relative energies of these processes, but not the overall ranking. Low-energy cross-ring cleavages are observed for the metal-cationized analytes with a retro-aldol mechanism producing the 0,2 A 2 ion from the sodiated forms. Theory and experiment support the importance of consecutive fragmentation processes, particularly for the protonated congeners at higher collision energies.

  16. Graphene Oxide Nanofiltration Membranes Stabilized by Cationic Porphyrin for High Salt Rejection.

    Science.gov (United States)

    Xu, Xiao-Ling; Lin, Fu-Wen; Du, Yong; Zhang, Xi; Wu, Jian; Xu, Zhi-Kang

    2016-05-25

    Swelling has great influences on the structure stability and separation performance of graphene oxide laminate membranes (GOLMs) for water desalination and purification. Herein, we report cross-linked GOLMs from GO assembled with cationic tetrakis(1-methyl-pyridinium-4-yl)porphyrin (TMPyP) by a vacuum-assisted strategy. The concave nonoxide regions (G regions) of GO are used as cross-linking sites for the first time to precisely control the channel size for water permeation and salt ion retention. Channels around 1 nm are constructed by modulating the assembly ratio of TMPyP/GO, and these cross-linked GOLMs show high salt rejection.

  17. Differential effect of HOE642 on two separate monovalent cation transporters in the human red cell membrane

    DEFF Research Database (Denmark)

    Bernhardt, Ingolf; Weiss, Erwin; Robinson, Hannah C

    2007-01-01

    Residual K(+) fluxes in red blood cells can be stimulated in conditions of low ionic strength. Previous studies have identified both the non-selective, voltage-dependent cation (NSVDC) channel and the K(+)(Na(+))/H(+) exchanger as candidate pathways mediating this effect, although it is possible ...

  18. Involvement of peripheral TRPV1 channels in the analgesic effects of thalidomide.

    Science.gov (United States)

    Song, Tieying; Wang, Liwen; Gu, Kunfeng; Yang, Yunliang; Yang, Lijun; Ma, Pengyu; Ma, Xiaojing; Zhao, Jianhui; Yan, Ruyv; Guan, Jiao; Wang, Chunping; Qi, Yan; Ya, Jian

    2015-01-01

    Thalidomide was introduced to the market in 1957 as a sedative and antiemetic agent, and returned to the market for the treatment of myelodysplastic syndrome and multiple myeloma. There are reports and studies of thalidomide as an analgesic or analgesic adjuvant in clinic. However, the underlying mechanism is quite elusive. Many studies suggest that the analgesic effect of thalidomide may be due to its immunomodulatory and anti-inflammatory properties as it suppresses the production of tumor necrosis factor α (TNF-α) selectively. However, it is not clear whether any other mechanisms are implicated in the pain relief. In this study, we demonstrated that the peripheral vanilloid receptor 1 (TRPV1) channel was also involved in the analgesic effect of thalidomide in different cell and animal models. During the activation by its agonist capsaicin, the cation inward influx through TRPV1 channels and the whole-cell current significantly decreased after TRPV1-overexpressed HEK293 cells or dorsal root ganglion (DRG) neurons were pre-treated with thalidomide for 20 minutes. And such attenuation in the TRPV1 activity was in a dose-dependent manner of thalidomide. In an acetic acid writhing test, pre-treatment of thalidomide decreased the writhing number in the wild type mice, while it did not happen in TRPV1 knockout mice, suggesting that the TRPV1 channel was involved in the pain relief by thalidomide. Taken together, the study showed that TRPV1 channels were involved in the analgesic effects of thalidomide. Such alteration in the action of TRPV1 channels by thalidomide may help understand how thalidomide takes analgesic effect in the body in addition to its selective inhibition of TNF-α production.

  19. TRP channels in microglia: role in physiology and disease

    OpenAIRE

    2015-01-01

    Microglia are responsible for modulating the cellular environment in the nervous system, where they have neuroprotective effects. In these cells has been reported the expression of different ion channels, including those belonging to the transient receptor potential (TRP) family. TRP are nonselective cation channels that have six transmembrane domains and both N-terminus and C-terminus domain located in the cytoplasm. These proteins can be activated by mechanical, thermal and chemical stimuli...

  20. Current scenario of peptide-based drugs: the key roles of cationic antitumor and antiviral peptides

    Directory of Open Access Journals (Sweden)

    Kelly eMulder

    2013-10-01

    Full Text Available Cationic antimicrobial peptides (AMPs and host defense peptides (HDPs show vast potential as peptide-based drugs. Great effort has been made in order to exploit their mechanisms of action, aiming to identify their targets as well as to enhance their activity and bioavailability. In this review, we will focus on both naturally occurring and designed antiviral and antitumor cationic peptides, including those here called promiscuous, in which multiple targets are associated with a single peptide structure. Emphasis will be given to their bio-chemical features, selectivity against extra targets and molecular mechanisms. Peptides which possess antitumor activity against different cancer cell lines will be discussed, as well as peptides which inhibit virus replication, focusing on their applications for human health, animal health and agriculture, and their potential as new therapeutic drugs. Moreover, the development of production and nano-delivery systems for both classes of cationic peptides and perspectives on improving them will be considered.

  1. Structural Insights into Mitochondrial Calcium Uniporter Regulation by Divalent Cations.

    Science.gov (United States)

    Lee, Samuel K; Shanmughapriya, Santhanam; Mok, Mac C Y; Dong, Zhiwei; Tomar, Dhanendra; Carvalho, Edmund; Rajan, Sudarsan; Junop, Murray S; Madesh, Muniswamy; Stathopulos, Peter B

    2016-09-22

    Calcium (Ca(2+)) flux into the matrix is tightly controlled by the mitochondrial Ca(2+) uniporter (MCU) due to vital roles in cell death and bioenergetics. However, the precise atomic mechanisms of MCU regulation remain unclear. Here, we solved the crystal structure of the N-terminal matrix domain of human MCU, revealing a β-grasp-like fold with a cluster of negatively charged residues that interacts with divalent cations. Binding of Ca(2+) or Mg(2+) destabilizes and shifts the self-association equilibrium of the domain toward monomer. Mutational disruption of the acidic face weakens oligomerization of the isolated matrix domain and full-length human protein similar to cation binding and markedly decreases MCU activity. Moreover, mitochondrial Mg(2+) loading or blockade of mitochondrial Ca(2+) extrusion suppresses MCU Ca(2+)-uptake rates. Collectively, our data reveal that the β-grasp-like matrix region harbors an MCU-regulating acidic patch that inhibits human MCU activity in response to Mg(2+) and Ca(2+) binding.

  2. Association of transient receptor potential canonical type 3 (TRPC3) channel transcripts with proinflammatory cytokines

    DEFF Research Database (Denmark)

    Thilo, Florian; Scholze, Alexandra; Liu, Dao Yan;

    2008-01-01

    We investigated whether expression of non-selective cation channels of the transient receptor potential canonical (TRPC) channel family are associated with proinflammatory cytokines in monocytes. Using quantitative RT-PCR we studied the expression of TRPC3, interleukin-1beta (IL-1beta), and tumor...

  3. Mechanotransduction in mouse inner ear hair cells requires transmembrane channel-like genes

    NARCIS (Netherlands)

    Kawashima, Yoshiyuki; Geleoc, Gwenaelle S. G.; Kurima, Kiyoto; Labay, Valentina; Lelli, Andrea; Asai, Yukako; Makishima, Tomoko; Wu, Doris K.; Della Santina, Charles C.; Holt, Jeffrey R.; Griffith, Andrew J.

    2011-01-01

    Inner ear hair cells convert the mechanical stimuli of sound, gravity, and head movement into electrical signals. This mechanotransduction process is initiated by opening of cation channels near the tips of hair cell stereocilia. Since the identity of these ion channels is unknown, and mutations in

  4. High glucose-induced oxidative stress increases transient receptor potential channel expression in human monocytes

    DEFF Research Database (Denmark)

    Wuensch, Tilo; Thilo, Florian; Krueger, Katharina;

    2010-01-01

    Transient receptor potential (TRP) channel-induced cation influx activates human monocytes, which play an important role in the pathogenesis of atherosclerosis. In the present study, we investigated the effects of high glucose-induced oxidative stress on TRP channel expression in human monocytes....

  5. A highly calcium-selective cation current activated by intracellular calcium release in MDCK cells.

    Science.gov (United States)

    Delles, C; Haller, T; Dietl, P

    1995-08-01

    1. The whole-cell patch clamp technique and fluorescence microscopy with the Ca2+ indicators fura-2 and fluo-3 were used to measure the whole-cell current and the free intracellular Ca2+ concentration ([Ca2+]i) in Madin-Darby canine kidney (MDCK) cells. 2. In a Ca(2+)-free bath solution, thapsigargin (TG) caused a transient increase of [Ca2+]i. Subsequent addition of Ca2+ caused a long lasting elevation of [Ca2+]i. 3. In a Ca(2+)-free bath solution, extracellular application of TG, ATP or ionomycin, or intracellular application of inositol 1,4,5-trisphosphate (IP3), caused a small but significant inward current (Iin) and a transient outward Ca(2+)-dependent K+ current (IK(Ca)), consistent with intracellular Ca2+ release. Subsequent addition of Ca2+ induced a prominent Iin with a current density of -4.2 +/- 0.7 pA pF-1. This Iin was unaffected by inositol 1,3,4,5-tetrakisphosphate (IP4). 4. Na+ replacement by mannitol, N-methyl-D-glucamine+ (NMG+), aminomethylidin-trimethanol+ (Tris+) or choline+ reduced Iin by 54, 65, 52 and 56%, respectively. This indicates an apparent Ca2+ selectivity over Na+ of 26:1. Iin was, however, unaffected by replacing Cl- with gluconate- or by the K+ channel blocker charybdotoxin (CTX). 5. Iin was completely blocked by La3+ (IC50 = 0.77 microM). Consistently, La3+ completely reversed the TG-induced elevation of [Ca2+]i. SK&F 96365 (1-[3-(4-methoxyphenyl)-propoxyl]-1-(4-methoxy-phenyl)-ethyl-1H-im idazole) HCl did not inhibit the TG-induced Iin. It did, however, exhibit a biphasic effect on [Ca2+]i, consisting of an initial Ca2+ decay and a subsequent Ca2+ elevation. La3+ completely reversed the SK&F 96365-induced elevation of [Ca2+]i. 6. In the absence of Na+, Iin was dependent on the bath Ca2+ concentration (EC50 = 1.02 mM). Ca2+ replacement by Ba2+ or Mn2+ resulted in a reduction of Iin by 95 and 94%, respectively. 7. From these experiments we conclude that Ca2+ release from intracellular Ca2+ stores, induced by different independent

  6. Interaction of hydrogen sulfide w