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Sample records for channel mediating activation

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

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

    2014-05-01

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

  2. TRPV3 Channels Mediate Strontium-Induced Mouse-Egg Activation

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    Ingrid Carvacho

    2013-12-01

    Full Text Available In mammals, calcium influx is required for oocyte maturation and egg activation. The molecular identities of the calcium-permeant channels that underlie the initiation of embryonic development are not established. Here, we describe a transient receptor potential (TRP ion channel current activated by TRP agonists that is absent in TrpV3−/− eggs. TRPV3 current is differentially expressed during oocyte maturation, reaching a peak of maximum density and activity at metaphase of meiosis II (MII, the stage of fertilization. Selective activation of TRPV3 channels provokes egg activation by mediating massive calcium entry. Widely used to activate eggs, strontium application is known to yield normal offspring in combination with somatic cell nuclear transfer. We show that TRPV3 is required for strontium influx, because TrpV3−/− eggs failed to conduct Sr2+ or undergo strontium-induced activation. We propose that TRPV3 is a major mediator of calcium influx in mouse eggs and is a putative target for artificial egg activation.

  3. Antispasmodic activity of Symplocos paniculata is mediated through opening of ATP-dependent K+ channel

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    Khalid Hussain Janbaz

    2016-06-01

    Full Text Available Symplocos paniculata is a medicinal plant used by native healers to manage gastrointestinal ailments. The crude methanolic extract of S. paniculata was screened pharmacologically both in vitro and in vivo for the validation of its therapeutic potential. It suppressed the spontaneous activity of isolated rabbit jejunum preparations and also caused inhibition of the low K+ (20 mM- induced spastic contractions in isolated rabbit jejunum preparations in a manner comparable to cromakalim. The relaxant effect was found to be blocked following glibenclamide exposure of the isolated tissue preparations similar to cromakalim, suggesting that observed response was likely to be mediated through opening of ATP dependent K+ channels. Following oral administration to mice provided protection against castor oil-induced diarrhea in a manner similar to loperamide. The plant material was found safe in toxicity study up to oral dose of 8 g/kg in mice. Hence, present study provides a scientific basis for the vernacular use of S. paniculata in gastro-intestinal system.

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

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

    1999-02-26

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

  5. Ethanol affects network activity in cultured rat hippocampus: mediation by potassium channels.

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    Eduard Korkotian

    Full Text Available The effects of ethanol on neuronal network activity were studied in dissociated cultures of rat hippocampus. Exposure to low (0.25-0.5% ethanol concentrations caused an increase in synchronized network spikes, and a decrease in the duration of individual spikes. Ethanol also caused an increase in rate of miniature spontaneous excitatory postsynaptic currents. Higher concentrations of ethanol eliminated network spikes. These effects were reversible upon wash. The effects of the high, but not the low ethanol were blocked by the GABA antagonist bicuculline. The enhancing action of low ethanol was blocked by apamin, an SK potassium channel antagonist, and mimicked by 1-EBIO, an SK channel opener. It is proposed that in cultured hippocampal networks low concentration of ethanol is associated with SK channel activity, rather than the GABAergic receptor.

  6. PLC-mediated PI(4,5)P2 hydrolysis regulates activation and inactivation of TRPC6/7 channels.

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    Itsuki, Kyohei; Imai, Yuko; Hase, Hideharu; Okamura, Yasushi; Inoue, Ryuji; Mori, Masayuki X

    2014-02-01

    Transient receptor potential classical (or canonical) (TRPC)3, TRPC6, and TRPC7 are a subfamily of TRPC channels activated by diacylglycerol (DAG) produced through the hydrolysis of phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2) by phospholipase C (PLC). PI(4,5)P2 depletion by a heterologously expressed phosphatase inhibits TRPC3, TRPC6, and TRPC7 activity independently of DAG; however, the physiological role of PI(4,5)P2 reduction on channel activity remains unclear. We used Förster resonance energy transfer (FRET) to measure PI(4,5)P2 or DAG dynamics concurrently with TRPC6 or TRPC7 currents after agonist stimulation of receptors that couple to Gq and thereby activate PLC. Measurements made at different levels of receptor activation revealed a correlation between the kinetics of PI(4,5)P2 reduction and those of receptor-operated TRPC6 and TRPC7 current activation and inactivation. In contrast, DAG production correlated with channel activation but not inactivation; moreover, the time course of channel inactivation was unchanged in protein kinase C-insensitive mutants. These results suggest that inactivation of receptor-operated TRPC currents is primarily mediated by the dissociation of PI(4,5)P2. We determined the functional dissociation constant of PI(4,5)P2 to TRPC channels using FRET of the PLCδ Pleckstrin homology domain (PHd), which binds PI(4,5)P2, and used this constant to fit our experimental data to a model in which channel gating is controlled by PI(4,5)P2 and DAG. This model predicted similar FRET dynamics of the PHd to measured FRET in either human embryonic kidney cells or smooth muscle cells, whereas a model lacking PI(4,5)P2 regulation failed to reproduce the experimental data, confirming the inhibitory role of PI(4,5)P2 depletion on TRPC currents. Our model also explains various PLC-dependent characteristics of channel activity, including limitation of maximum open probability, shortening of the peak time, and the bell-shaped response of total

  7. Light-activated control of protein channel assembly mediated by membrane mechanics

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    Miller, David M.; Findlay, Heather E.; Ces, Oscar; Templer, Richard H.; Booth, Paula J.

    2016-12-01

    Photochemical processes provide versatile triggers of chemical reactions. Here, we use a photoactivated lipid switch to modulate the folding and assembly of a protein channel within a model biological membrane. In contrast to the information rich field of water-soluble protein folding, there is only a limited understanding of the assembly of proteins that are integral to biological membranes. It is however possible to exploit the foreboding hydrophobic lipid environment and control membrane protein folding via lipid bilayer mechanics. Mechanical properties such as lipid chain lateral pressure influence the insertion and folding of proteins in membranes, with different stages of folding having contrasting sensitivities to the bilayer properties. Studies to date have relied on altering bilayer properties through lipid compositional changes made at equilibrium, and thus can only be made before or after folding. We show that light-activation of photoisomerisable di-(5-[[4-(4-butylphenyl)azo]phenoxy]pentyl)phosphate (4-Azo-5P) lipids influences the folding and assembly of the pentameric bacterial mechanosensitive channel MscL. The use of a photochemical reaction enables the bilayer properties to be altered during folding, which is unprecedented. This mechanical manipulation during folding, allows for optimisation of different stages of the component insertion, folding and assembly steps within the same lipid system. The photochemical approach offers the potential to control channel assembly when generating synthetic devices that exploit the mechanosensitive protein as a nanovalve.

  8. Mechanoprotection by Polycystins against Apoptosis Is Mediated through the Opening of Stretch-Activated K2P Channels

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    Rémi Peyronnet

    2012-03-01

    Full Text Available How renal epithelial cells respond to increased pressure and the link with kidney disease states remain poorly understood. Pkd1 knockout or expression of a PC2 pathogenic mutant, mimicking the autosomal dominant polycystic kidney disease, dramatically enhances mechanical stress-induced tubular apoptotic cell death. We show the presence of a stretch-activated K+ channel dependent on the TREK-2 K2P subunit in proximal convoluted tubule epithelial cells. Our findings further demonstrate that polycystins protect renal epithelial cells against apoptosis in response to mechanical stress, and this function is mediated through the opening of stretch-activated K2P channels. Thus, to our knowledge, we establish for the first time, both in vitro and in vivo, a functional relationship between mechanotransduction and mechanoprotection. We propose that this mechanism is at play in other important pathologies associated with apoptosis and in which pressure or flow stimulation is altered, including heart failure or atherosclerosis.

  9. Activation of L-type calcium channels is required for gap junction-mediated intercellular calcium signaling in osteoblastic cells

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    Jorgensen, Niklas Rye; Teilmann, Stefan Cuoni; Henriksen, Zanne; Civitelli, Roberto; Sorensen, Ole Helmer; Steinberg, Thomas H.

    2003-01-01

    The propagation of mechanically induced intercellular calcium waves (ICW) among osteoblastic cells occurs both by activation of P2Y (purinergic) receptors by extracellular nucleotides, resulting in "fast" ICW, and by gap junctional communication in cells that express connexin43 (Cx43), resulting in "slow" ICW. Human osteoblastic cells transmit intercellular calcium signals by both of these mechanisms. In the current studies we have examined the mechanism of slow gap junction-dependent ICW in osteoblastic cells. In ROS rat osteoblastic cells, gap junction-dependent ICW were inhibited by removal of extracellular calcium, plasma membrane depolarization by high extracellular potassium, and the L-type voltage-operated calcium channel inhibitor, nifedipine. In contrast, all these treatments enhanced the spread of P2 receptor-mediated ICW in UMR rat osteoblastic cells. Using UMR cells transfected to express Cx43 (UMR/Cx43) we confirmed that nifedipine sensitivity of ICW required Cx43 expression. In human osteoblastic cells, gap junction-dependent ICW also required activation of L-type calcium channels and influx of extracellular calcium.

  10. Activation of L-type calcium channels is required for gap junction-mediated intercellular calcium signaling in osteoblastic cells

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    Jørgensen, Niklas Rye; Teilmann, Stefan Cuoni; Henriksen, Zanne

    2003-01-01

    The propagation of mechanically induced intercellular calcium waves (ICW) among osteoblastic cells occurs both by activation of P2Y (purinergic) receptors by extracellular nucleotides, resulting in "fast" ICW, and by gap junctional communication in cells that express connexin43 (Cx43), resulting...... in "slow" ICW. Human osteoblastic cells transmit intercellular calcium signals by both of these mechanisms. In the current studies we have examined the mechanism of slow gap junction-dependent ICW in osteoblastic cells. In ROS rat osteoblastic cells, gap junction-dependent ICW were inhibited by removal...... of extracellular calcium, plasma membrane depolarization by high extracellular potassium, and the L-type voltage-operated calcium channel inhibitor, nifedipine. In contrast, all these treatments enhanced the spread of P2 receptor-mediated ICW in UMR rat osteoblastic cells. Using UMR cells transfected to express Cx...

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

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    Gruhn, Nicolai; Boesgaard, Søren; Eiberg, Jonas;

    2002-01-01

    /kg/min). Iberiotoxin reduced the vasorelaxant effect of nitroglycerin (E(max)) by 60% in endothelium-intact arteries and 13% in endothelium-denuded arteries (P0.05) and (compared to arterial segments) veins were less sensitive to BK(Ca) channel blockade (30% reduction in E(max)) or endothelial removal. The results...

  12. Transduction for pheromones in the main olfactory epithelium is mediated by the Ca2+ -activated channel TRPM5.

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    López, Fabián; Delgado, Ricardo; López, Roberto; Bacigalupo, Juan; Restrepo, Diego

    2014-02-26

    Growing evidence suggests that the main olfactory epithelium contains a subset of olfactory sensory neurons (OSNs) responding to pheromones. One candidate subpopulation expresses the calcium activated cation channel TRPM5 (transient receptor potential channel M5). Using GFP driven by the TRPM5 promoter in mice, we show that this subpopulation responds to putative pheromones, urine, and major histocompatibility complex peptides, but not to regular odors or a pheromone detected by other species. In addition, this subpopulation of TRPM5-GFP+ OSNs uses novel transduction. In regular OSNs, odorants elicit activation of the cyclic nucleotide-gated (CNG) channel, leading to Ca2+ gating of Cl- channels; in TRPM5-GFP+ OSNs, the Ca2+ -activated Cl- ANO2 (anoctamin 2) channel is not expressed, and pheromones elicit activation of the CNG channel leading to Ca2+ gating of TRPM5. In conclusion, we show that OSNs expressing TRPM5 respond to pheromones, but not to regular odors through the opening of CNG channels leading to Ca2+ gating of TRPM5.

  13. Activation of ATP-sensitive potassium channels enhances DMT1-mediated iron uptake in SK-N-SH cells in vitro

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    Du, Xixun; Xu, Huamin; Shi, Limin; Jiang, Zhifeng; Song, Ning; Jiang, Hong; Xie, Junxia

    2016-01-01

    Iron importer divalent metal transporter 1 (DMT1) plays a crucial role in the nigal iron accumulation in Parkinson’s disease (PD). Membrane hyperpolarization is one of the factors that could affect its iron transport function. Besides iron, selective activation of the ATP-sensitive potassium (KATP) channels also contributes to the vulnerability of dopaminergic neurons in PD. Interestingly, activation of KATP channels could induce membrane hyperpolarization. Therefore, it is of vital importance to study the effects of activation of KATP channels on DMT1-mediated iron uptake function. In the present study, activation of KATP channels by diazoxide resulted in the hyperpolarization of the membrane potential and increased DMT1-mediated iron uptake in SK-N-SH cells. This led to an increase in intracellular iron levels and a subsequent decrease in the mitochondrial membrane potential and an increase in ROS production. Delayed inactivation of the Fe2+-evoked currents by diazoxide was recorded by patch clamp in HEK293 cells, which demonstrated that diazoxide could prolonged DMT1-facilitated iron transport. While inhibition of KATP channels by glibenclamide could block ferrous iron influx and the subsequent cell damage. Overexpression of Kir6.2/SUR1 resulted in an increase in iron influx and intracellular iron levels, which was markedly increased after diazoxide treatment. PMID:27646472

  14. Calcium-activated potassium channels mediated blood-brain tumor barrier opening in a rat metastatic brain tumor model

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    Ong John M

    2007-03-01

    Full Text Available Abstract Background The blood-brain tumor barrier (BTB impedes the delivery of therapeutic agents to brain tumors. While adequate delivery of drugs occurs in systemic tumors, the BTB limits delivery of anti-tumor agents into brain metastases. Results In this study, we examined the function and regulation of calcium-activated potassium (KCa channels in a rat metastatic brain tumor model. We showed that intravenous infusion of NS1619, a KCa channel agonist, and bradykinin selectively enhanced BTB permeability in brain tumors, but not in normal brain. Iberiotoxin, a KCa channel antagonist, significantly attenuated NS1619-induced BTB permeability increase. We found KCa channels and bradykinin type 2 receptors (B2R expressed in cultured human metastatic brain tumor cells (CRL-5904, non-small cell lung cancer, metastasized to brain, human brain microvessel endothelial cells (HBMEC and human lung cancer brain metastasis tissues. Potentiometric assays demonstrated the activity of KCa channels in metastatic brain tumor cells and HBMEC. Furthermore, we detected higher expression of KCa channels in the metastatic brain tumor tissue and tumor capillary endothelia as compared to normal brain tissue. Co-culture of metastatic brain tumor cells and brain microvessel endothelial cells showed an upregulation of KCa channels, which may contribute to the overexpression of KCa channels in tumor microvessels and selectivity of BTB opening. Conclusion These findings suggest that KCa channels in metastatic brain tumors may serve as an effective target for biochemical modulation of BTB permeability to enhance selective delivery of chemotherapeutic drugs to metastatic brain tumors.

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

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

    2008-09-12

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

  16. Calmodulin and calcium interplay in the modulation of TRPC5 channel activity. Identification of a novel C-terminal domain for calcium/calmodulin-mediated facilitation.

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    Ordaz, Benito; Tang, Jisen; Xiao, Rui; Salgado, Alfonso; Sampieri, Alicia; Zhu, Michael X; Vaca, Luis

    2005-09-02

    TRPC5 forms Ca2+-permeable nonselective cation channels important for neurite outgrowth and growth cone morphology of hippocampal neurons. Here we studied the activation of mouse TRPC5 expressed in Chinese hamster ovary and human embryonic kidney 293 cells by agonist stimulation of several receptors that couple to the phosphoinositide signaling cascade and the role of calmodulin (CaM) on the activation. We showed that exogenous application of 10 microM CaM through patch pipette accelerated the agonist-induced channel activation by 2.8-fold, with the time constant for half-activation reduced from 4.25 +/- 0.4 to 1.56 +/- 0.85 min. We identified a novel CaM-binding site located at the C terminus of TRPC5, 95 amino acids downstream from the previously determined common CaM/IP3R-binding (CIRB) domain for all TRPC proteins. Deletion of the novel CaM-binding site attenuated the acceleration in channel activation induced by CaM. However, disruption of the CIRB domain from TRPC5 rendered the channel irresponsive to agonist stimulation without affecting the cell surface expression of the channel protein. Furthermore, we showed that high (>5 microM) intracellular free Ca2+ inhibited the current density without affecting the time course of TRPC5 activation by receptor agonists. These results demonstrated that intracellular Ca2+ has dual and opposite effects on the activation of TRPC5. The novel CaM-binding site is important for the Ca2+/CaM-mediated facilitation, whereas the CIRB domain is critical for the overall response of receptor-induced TRPC5 channel activation.

  17. Reduced KCNQ4-encoded voltage-dependent potassium channel activity underlies impaired β-adrenoceptor-mediated relaxation of renal arteries in hypertension.

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    Chadha, Preet S; Zunke, Friederike; Zhu, Hai-Lei; Davis, Alison J; Jepps, Thomas A; Olesen, Søren P; Cole, William C; Moffatt, James D; Greenwood, Iain A

    2012-04-01

    KCNQ4-encoded voltage-dependent potassium (Kv7.4) channels are important regulators of vascular tone that are severely compromised in models of hypertension. However, there is no information as to the role of these channels in responses to endogenous vasodilators. We used a molecular knockdown strategy, as well as pharmacological tools, to examine the hypothesis that Kv7.4 channels contribute to β-adrenoceptor-mediated vasodilation in the renal vasculature and underlie the vascular deficit in spontaneously hypertensive rats. Quantitative PCR and immunohistochemistry confirmed gene and protein expression of KCNQ1, KCNQ3, KCNQ4, KCNQ5, and Kv7.1, Kv7.4, and Kv7.5 in rat renal artery. Isoproterenol produced concentration-dependent relaxation of precontracted renal arteries and increased Kv7 channel currents in isolated smooth muscle cells. Application of the Kv7 blocker linopirdine attenuated isoproterenol-induced relaxation and current. Isoproterenol-induced relaxations were also reduced in arteries incubated with small interference RNAs targeted to KCNQ4 that produced a ≈60% decrease in Kv7.4 protein level. Relaxation to isoproterenol and the Kv7 activator S-1 were abolished in arteries from spontaneously hypertensive rats, which was associated with ≈60% decrease in Kv7.4 abundance. This study provides the first evidence that Kv7 channels contribute to β-adrenoceptor-mediated vasodilation in the renal vasculature and that abrogation of Kv7.4 channels is strongly implicated in the impaired β-adrenoceptor pathway in spontaneously hypertensive rats. These findings may provide a novel pathogenic link between arterial dysfunction and hypertension.

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

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    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.

  19. Activation of PPARβ/δ prevents hyperglycaemia-induced impairment of Kv7 channels and cAMP-mediated relaxation in rat coronary arteries.

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    Morales-Cano, Daniel; Moreno, Laura; Barreira, Bianca; Briones, Ana M; Pandolfi, Rachele; Moral-Sanz, Javier; Callejo, Maria; Mondejar-Parreño, Gema; Cortijo, Julio; Salaices, Mercedes; Duarte, Juan; Perez-Vizcaino, Francisco; Cogolludo, Angel

    2016-10-01

    PPARβ/δ activation protects against endothelial dysfunction in diabetic models. Elevated glucose is known to impair cAMP-induced relaxation and Kv channel function in coronary arteries (CA). Herein, we aimed to analyse the possible protective effects of the PPARβ/δ agonist GW0742 on the hyperglycaemic-induced impairment of cAMP-induced relaxation and Kv channel function in rat CA. As compared with low glucose (LG), incubation under high glucose (HG) conditions attenuated the relaxation induced by the adenylate cyclase activator forskolin in CA and this was prevented by GW0742. The protective effect of GW0742 was supressed by a PPARβ/δ antagonist. In myocytes isolated from CA under LG, forskolin enhanced Kv currents and induced hyperpolarization. In contrast, when CA were incubated with HG, Kv currents were diminished and the electrophysiological effects of forskolin were abolished. These deleterious effects were prevented by GW0742. The protective effects of GW0742 on forskolin-induced relaxation and Kv channel function were confirmed in CA from type-1 diabetic rats. In addition, the differences in the relaxation induced by forskolin in CA incubated under LG, HG or HG + GW0742 were abolished by the Kv7 channel inhibitor XE991. Accordingly, GW0742 prevented the down-regulation of Kv7 channels induced by HG. Finally, the preventive effect of GW0742 on oxidative stress and cAMP-induced relaxation were overcome by the pyruvate dehydrogenase kinase 4 (PDK4) inhibitor dichloroacetate (DCA). Our results reveal that the PPARβ/δ agonist GW0742 prevents the impairment of the cAMP-mediated relaxation in CA under HG. This protective effect was associated with induction of PDK4, attenuation of oxidative stress and preservation of Kv7 channel function.

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

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    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.

  1. Augmented activity of the pelvic nerve afferent mediated by TRP channels in dextran sulfate sodium (DSS)-induced colitis of rats.

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    Makimura, Yukitoshi; Ito, Koichi; Kuwahara, Masayoshi; Tsubone, Hirokazu

    2012-08-01

    Enteritis has been recognized as a major symptom in domestic animals and human patients suffering from feed and food poisonings. The aim of the present study was to clarify the excitatory mechanism of the pelvic nerve afferent which may influence the occurrence of enteritis in response to nociceptive chemical stimuli of the colon in normal and abnormal rats with colitis induced by dextran sulfate sodium (DSS). The pelvic nerve afferent activity was markedly increased by colonic instillation of solution (0.5 ml) of acetic acid (5-25%) and capsaicin (100 μg/ml). The nerve activity was augmented by colonic instillation of capsaicin to a greater extent in rats with DSS-induced colitis than in normal control rats. This augmented activity by capsaicin was more prominent at one day (DSS-1) than at 8 day (DSS-8) after the administration of DSS. The increased nerve activity caused by capsaicin in DSS-1 and DSS-8 was significantly inhibited by pretreatment with ruthenium red, which is a nonselective inhibitor of TRP channels of unmyelinated C-fibers (nociceptors). In conclusion, it was elucidated that the nociceptive function of the pelvic nerve was largely elevated at one day after DSS-induced colitis and such increased function was mostly mediated by TRP channels.

  2. Effects of K+ channel agonists cromakalim and pinacidil on rat basilar artery smooth muscle cells are mediated by Ca(++)-activated K+ channels.

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    Stockbridge, N; Zhang, H; Weir, B

    1991-11-27

    Whole-cell and cell-free inside-out patch-clamp recording techniques were used to examine the actions of potassium channel openers pinacidil and cromakalim in enzymatically isolated smooth muscle cells of rat basilar artery. Delayed rectifier and calcium-dependent potassium currents were identified from the whole-cell recordings. Only the calcium-dependent potassium current was increased by cromakalim and pinacidil. Recordings from inside-out membrane patches revealed a large conductance voltage- and calcium-dependent potassium channel, which was blocked by charybdotoxin but unaffected by ATP less than 10 mM. Cromakalim and pinacidil increased the open probability of this channel. On the basis of these results, we suggest that such drugs, acting on cerebral arterial smooth muscle cell potassium channels, may be of some benefit in the treatment of cerebral vasospasm following subarachnoid hemorrhage.

  3. Reduced KCNQ4-encoded voltage-dependent potassium channel activity underlies impaired ß-adrenoceptor-mediated relaxation of renal arteries in hypertension

    DEFF Research Database (Denmark)

    Chadha, Preet S; Zunke, Friederike; Zhu, Hai-Lei;

    2012-01-01

    KCNQ4-encoded voltage-dependent potassium (Kv7.4) channels are important regulators of vascular tone that are severely compromised in models of hypertension. However, there is no information as to the role of these channels in responses to endogenous vasodilators. We used a molecular knockdown...... strategy, as well as pharmacological tools, to examine the hypothesis that Kv7.4 channels contribute to ß-adrenoceptor-mediated vasodilation in the renal vasculature and underlie the vascular deficit in spontaneously hypertensive rats. Quantitative PCR and immunohistochemistry confirmed gene and protein...... spontaneously hypertensive rats, which was associated with ˜60% decrease in Kv7.4 abundance. This study provides the first evidence that Kv7 channels contribute to ß-adrenoceptor-mediated vasodilation in the renal vasculature and that abrogation of Kv7.4 channels is strongly implicated in the impaired ß...

  4. Activation of a TRP-like channel and intracellular Ca2+ dynamics during phospholipase-C-mediated cell death.

    Science.gov (United States)

    Gonçalves, A Pedro; Cordeiro, J Miguel; Monteiro, João; Muñoz, Alberto; Correia-de-Sá, Paulo; Read, Nick D; Videira, Arnaldo

    2014-09-01

    The model organism Neurospora crassa undergoes programmed cell death when exposed to staurosporine. Here, we show that staurosporine causes defined changes in cytosolic free Ca(2+) ([Ca(2+)]c) dynamics and a distinct Ca(2+) signature that involves Ca(2+) influx from the external medium and internal Ca(2+) stores. We investigated the molecular basis of this Ca(2+) response by using [Ca(2+)]c measurements combined with pharmacological and genetic approaches. Phospholipase C was identified as a pivotal player during cell death, because modulation of the phospholipase C signaling pathway and deletion of PLC-2, which we show to be involved in hyphal development, results in an inability to trigger the characteristic staurosporine-induced Ca(2+) signature. Using Δcch-1, Δfig-1 and Δyvc-1 mutants and a range of inhibitors, we show that extracellular Ca(2+) entry does not occur through the hitherto described high- and low-affinity Ca(2+) uptake systems, but through the opening of plasma membrane channels with properties resembling the transient receptor potential (TRP) family. Partial blockage of the response to staurosporine after inhibition of a putative inositol-1,4,5-trisphosphate (IP3) receptor suggests that Ca(2+) release from internal stores following IP3 formation combines with the extracellular Ca(2+) influx.

  5. Pannexin Channels Mediate the Acquisition of Myogenic Commitment in C2C12 Reserve Cells Promoted by P2 Receptor Activation

    Directory of Open Access Journals (Sweden)

    Manuel Antonio Riquelme

    2015-05-01

    Full Text Available The acquisition of myoblast commitment to the myogenic linage requires rises in intracellular free Ca2+ concentration ([Ca2+]i. Putative cell membrane pathways involved in these [Ca2+]i increments are P2 receptors (P2Rs as well as connexin (Cx and/or pannexin (Panx hemichannels and channels (Cx HChs and Panx Chs, respectively, which are known to permeate Ca2+. Reserve cells (RCs are uncommitted myoblasts obtained from differentiated C2C12 cell cultures, which acquire commitment upon replating. Regarding these cells, we found that extracellular ATP increases the [Ca2+]i via P2Rs. Moreover, ATP increases the plasma membrane permeability to small molecules and a non-selective membrane current, both of which were inhibited by Cx HCh/Panx1Ch blockers. However, RCs exposed to divalent cation-free saline solution, which is known to activate Cx HChs (but not Panx Chs, did not enhance membrane permeability, thus ruling out the possible involvement of Cx HChs. Moreover, ATP-induced membrane permeability was inhibited with blockers of P2Rs that activate Panx Chs. In addition, exogenous ATP induced the expression of myogenic commitment and increased MyoD levels, which was prevented by the inhibition of P2Rs or knockdown of Panx1 Chs. Similarly, increases in MyoD levels induced by ATP released by RCs were inhibited by Panx Ch/Cx HCh blockers. Myogenic commitment acquisition thus requires a feed-forward mechanism mediated by extracellular ATP, P2Rs and Panx Chs.

  6. Lubiprostone: a chloride channel activator.

    Science.gov (United States)

    Lacy, Brian E; Levy, L Campbell

    2007-04-01

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

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

    DEFF Research Database (Denmark)

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

    2009-01-01

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

  8. Protein kinase A and mitogen-activated protein kinase pathways mediate cAMP induction of alpha-epithelial Na+ channels (alpha-ENaC).

    Science.gov (United States)

    Mustafa, Shamimunisa B; Castro, Robert; Falck, Alison J; Petershack, Jean A; Henson, Barbara M; Mendoza, Yvonne M; Choudary, Ahsan; Seidner, Steven R

    2008-04-01

    A major mechanism for Na+ transport across epithelia occurs through epithelial Na+ channels (ENaC). ENaC is a multimeric channel consisting of three subunits (alpha, beta, and gamma). The alpha-subunit is critical for ENaC function. In specific culture conditions, the rat submandibular gland epithelial cell line (SMG-C6) demonstrates minimal Na+ transport properties and exposure to dibutyryl cAMP (DbcAMP) for up to 48 h caused an elevation of alpha-ENaC mRNA and protein expression and amiloride-sensitive short-circuit current (I(SC)). Here we examined the early signaling pathways evoked by DbcAMP which contribute to the eventual increase in Na+ transport is present. Treatment with either of the protein kinase A (PKA) inhibitors KT5720 or H-89 followed by exposure to 1 mM DbcAMP for 24 h markedly attenuated DbcAMP-induced alpha-ENaC protein formation and I(SC). Exposure of SMG-C6 cells to 1 mM DbcAMP induced a rapid, transient phosphorylation of the cAMP response element binding protein (CREB). This response was attenuated in the presence of either KT5720 or H-89. Dominant-negative CREB decreased DbcAMP-induced alpha-ENaC expression. Suppression of the extracellular signal-regulated protein kinase (ERK 1,2) with PD98059 or the p38 mitogen-activated protein kinase (MAPK) pathway with SB203580 reduced DbcAMP-induced alpha-ENaC protein levels in SMG-C6 cells. DbcAMP-induced phosphorylation of CREB was markedly attenuated by PD98059 or SB203580. DbcAMP-induced activation of the either the p38 or the ERK 1,2 MAPK pathways was abolished by either of the PKA inhibitors, H-89 or KT5720. Cross talk between these signaling pathways induced by DbcAMP via the activation of CREB appears to contribute to increased levels of alpha-ENaC observed after 24 h of treatment in SMG-C6 epithelial cells.

  9. Network excitability in a model of chronic temporal lobe epilepsy critically depends on SK channel-mediated AHP currents.

    Science.gov (United States)

    Schulz, Robert; Kirschstein, Timo; Brehme, Hannes; Porath, Katrin; Mikkat, Ulrike; Köhling, Rüdiger

    2012-01-01

    Hippocampal CA1 pyramidal neurons generate an after-hyperpolarization (AHP) whose medium component is thought to be generated by small-conductance Ca(2+)-activated K(+) channels (SK channels). Neuronal excitability is increased in epilepsy, and the AHP in turn is fundamentally involved in regulation of cellular excitability. We therefore investigated the involvement of the SK channel-mediated AHP in controlling cell and network excitability in the pilocarpine model epilepsy. Both acutely isolated CA1 pyramidal cells and isolated hippocampal slices were investigated in terms of the impact of SK channel-mediated AHP on hyperexcitability. Our findings show that pilocarpine-treated chronically epileptic rats exhibit significantly reduced SK channel-mediated hyperpolarizing outward current which was accompanied by a significant decrease in the somatic AHP. Paradoxically, inhibiting SK channels strongly exacerbated 0-Mg(2+)-induced epileptiform activity in slices from pilocarpine-treated animals, while having a significantly smaller effect in control tissue. This suggests that in chronically epileptic tissue, network excitability very critically depends on the remaining SK-channel mediated AHP. Additional real-time RT-PCR and semiquantitative Western blot experiments revealed that both the SK2 channel transcript and protein were significantly downregulated in the epileptic CA1 region. We conclude that SK2 channels are down-regulated in chronic epilepsy underlying the impaired SK channel function in CA1 pyramidal cells, and a further reduction of the remaining critical mass of SK channels results in an acute network decompensation.

  10. BK channel activators and their therapeutic perspectives

    DEFF Research Database (Denmark)

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

    2014-01-01

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

  11. CRMP-2 peptide mediated decrease of high and low voltage-activated calcium channels, attenuation of nociceptor excitability, and anti-nociception in a model of AIDS therapy-induced painful peripheral neuropathy

    Directory of Open Access Journals (Sweden)

    Piekarz Andrew D

    2012-07-01

    Full Text Available Abstract Background The ubiquity of protein-protein interactions in biological signaling offers ample opportunities for therapeutic intervention. We previously identified a peptide, designated CBD3, that suppressed inflammatory and neuropathic behavioral hypersensitivity in rodents by inhibiting the ability of collapsin response mediator protein 2 (CRMP-2 to bind to N-type voltage-activated calcium channels (CaV2.2 [Brittain et al. Nature Medicine 17:822–829 (2011]. Results and discussion Here, we utilized SPOTScan analysis to identify an optimized variation of the CBD3 peptide (CBD3A6K that bound with greater affinity to Ca2+ channels. Molecular dynamics simulations demonstrated that the CBD3A6K peptide was more stable and less prone to the unfolding observed with the parent CBD3 peptide. This mutant peptide, conjugated to the cell penetrating motif of the HIV transduction domain protein TAT, exhibited greater anti-nociception in a rodent model of AIDS therapy-induced peripheral neuropathy when compared to the parent TAT-CBD3 peptide. Remarkably, intraperitoneal administration of TAT-CBD3A6K produced none of the minor side effects (i.e. tail kinking, body contortion observed with the parent peptide. Interestingly, excitability of dissociated small diameter sensory neurons isolated from rats was also reduced by TAT-CBD3A6K peptide suggesting that suppression of excitability may be due to inhibition of T- and R-type Ca2+ channels. TAT-CBD3A6K had no effect on depolarization-evoked calcitonin gene related peptide (CGRP release compared to vehicle control. Conclusions Collectively, these results establish TAT-CBD3A6K as a peptide therapeutic with greater efficacy in an AIDS therapy-induced model of peripheral neuropathy than its parent peptide, TAT-CBD3. Structural modifications of the CBD3 scaffold peptide may result in peptides with selectivity against a particular subset of voltage-gated calcium channels resulting in a multipharmacology of

  12. 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.

  13. TRPA1 channels mediate acute neurogenic inflammation and pain produced by bacterial endotoxins

    Science.gov (United States)

    Meseguer, Victor; Alpizar, Yeranddy A.; Luis, Enoch; Tajada, Sendoa; Denlinger, Bristol; Fajardo, Otto; Manenschijn, Jan-Albert; Fernández-Peña, Carlos; Talavera, Arturo; Kichko, Tatiana; Navia, Belén; Sánchez, Alicia; Señarís, Rosa; Reeh, Peter; Pérez-García, María Teresa; López-López, José Ramón; Voets, Thomas; Belmonte, Carlos; Talavera, Karel; Viana, Félix

    2014-01-01

    Gram-negative bacterial infections are accompanied by inflammation and somatic or visceral pain. These symptoms are generally attributed to sensitization of nociceptors by inflammatory mediators released by immune cells. Nociceptor sensitization during inflammation occurs through activation of the Toll-like receptor 4 (TLR4) signalling pathway by lipopolysaccharide (LPS), a toxic by-product of bacterial lysis. Here we show that LPS exerts fast, membrane delimited, excitatory actions via TRPA1, a transient receptor potential cation channel that is critical for transducing environmental irritant stimuli into nociceptor activity. Moreover, we find that pain and acute vascular reactions, including neurogenic inflammation (CGRP release) caused by LPS are primarily dependent on TRPA1 channel activation in nociceptive sensory neurons, and develop independently of TLR4 activation. The identification of TRPA1 as a molecular determinant of direct LPS effects on nociceptors offers new insights into the pathogenesis of pain and neurovascular responses during bacterial infections and opens novel avenues for their treatment.

  14. Is ion channel selectivity mediated by confined water?

    CERN Document Server

    Prada-Gracia, Diego

    2012-01-01

    Ion channels form pores across the lipid bilayer, selectively allowing inorganic ions to cross the membrane down their electrochemical gradient. While the study of ion desolvation free-energies have attracted much attention, the role of water inside the pore is less clear. Here, molecular dynamics simulations of a reduced model of the KcsA selectivity filter indicate that the equilibrium position of Na+, but not of K+, is strongly influenced by confined water. The latter forms a stable complex with Na+, moving the equilibrium position of the ion to the plane of the backbone carbonyls. Almost at the centre of the binding site, the water molecule is trapped by favorable electrostatic interactions and backbone hydrogen-bonds. In the absence of confined water the equilibrium position of both Na+ and K+ is identical. Our observations strongly suggest a previously unnoticed active role of confined water in the selectivity mechanism of ion channels.

  15. Hydralazine-induced vasodilation involves opening of high conductance Ca2+-activated K+ channels

    DEFF Research Database (Denmark)

    Bang, Lone; Nielsen-Kudsk, J E; Gruhn, N;

    1998-01-01

    The purpose of this study was to investigate whether high conductance Ca2+-activated K+ channels (BK(Ca)) are mediating the vasodilator action of hydralazine. In isolated porcine coronary arteries, hydralazine (1-300 microM), like the K+ channel opener levcromakalim, preferentially relaxed...

  16. Involvement of anion channels in mediating elicitor-induced ATP efflux in Salvia miltiorrhiza hairy roots.

    Science.gov (United States)

    Wu, Shu-Jing; Siu, Ka-Chai; Wu, Jian-Yong

    2011-01-15

    This study examines the roles of anion channels and ATP binding cassette (ABC) protein transporters in mediating elicitor-induced ATP release in Salvia miltiorrhiza hairy root cultures. The elicitor-induced ATP release was effectively blocked by two putative membrane anion channel blockers, niflumic acid and Zn(2+), but not by a specific Cl(-) channel blocker, phenylanthranilic acid. The elicitor-induced ATP release was also significantly suppressed by two ABC inhibitors, glibenclamide and ethacrynic acid. Notable ATP release from the hairy roots was also induced by verapamil (2mM), an ABC activator in animal cells. The verapamil-induced ATP release was effectively blocked by niflumic acid, but only slightly inhibited by the ABC inhibitors. Another notable effect of verapamil was the induction of exocytosis, the secretion of vesicle-like particles to the root surface. The verapamil-induced exocytosis was not inhibited by nifulumic acid and YE did not induce the exocytosis. Overall, the results suggest a significant role of anion channels, a possible involvement of ABC proteins and no significant involvement of exocytosis in mediating the ATP efflux in hairy root cells.

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

    Science.gov (United States)

    Zhang, Xiaoli; Li, Xinran; Xu, Haoxing

    2012-07-10

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

  18. Channel-Mediated Lactate Release by K+-Stimulated Astrocytes

    KAUST Repository

    Sotelo-Hitschfeld, T.

    2015-03-11

    Excitatory synaptic transmission is accompanied by a local surge in interstitial lactate that occurs despite adequate oxygen availability, a puzzling phenomenon termed aerobic glycolysis. In addition to its role as an energy substrate, recent studies have shown that lactate modulates neuronal excitability acting through various targets, including NMDA receptors and G-protein-coupled receptors specific for lactate, but little is known about the cellular and molecular mechanisms responsible for the increase in interstitial lactate. Using a panel of genetically encoded fluorescence nanosensors for energy metabolites, we show here that mouse astrocytes in culture, in cortical slices, and in vivo maintain a steady-state reservoir of lactate. The reservoir was released to the extracellular space immediately after exposure of astrocytes to a physiological rise in extracellular K+ or cell depolarization. Cell-attached patch-clamp analysis of cultured astrocytes revealed a 37 pS lactate-permeable ion channel activated by cell depolarization. The channel was modulated by lactate itself, resulting in a positive feedback loop for lactate release. A rapid fall in intracellular lactate levels was also observed in cortical astrocytes of anesthetized mice in response to local field stimulation. The existence of an astrocytic lactate reservoir and its quick mobilization via an ion channel in response to a neuronal cue provides fresh support to lactate roles in neuronal fueling and in gliotransmission.

  19. 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.

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

    Directory of Open Access Journals (Sweden)

    Ravshan Z. Sabirov

    2011-12-01

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

  1. Calcium binding protein-mediated regulation of voltage-gated calcium channels linked to human diseases

    Institute of Scientific and Technical Information of China (English)

    Nasrin NFJATBAKHSH; Zhong-ping FENG

    2011-01-01

    Calcium ion entry through voltage-gated calcium channels is essential for cellular signalling in a wide variety of cells and multiple physiological processes. Perturbations of voltage-gated calcium channel function can lead to pathophysiological consequences. Calcium binding proteins serve as calcium sensors and regulate the calcium channel properties via feedback mechanisms. This review highlights the current evidences of calcium binding protein-mediated channel regulation in human diseases.

  2. Mediatized Extreme Right Activism and Discourse

    DEFF Research Database (Denmark)

    Peters, Rikke Alberg

    2015-01-01

    and mediatized activism. In order to analyse of the protest form, the visual aesthetics and the discourse of ‘The Immortals’, the paper mobilises two concepts from media and communication studies: mediation and mediatization. It will be argued that that the current transformation of the extreme right: that is...

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

    OpenAIRE

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

    2014-01-01

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

  4. Epithelial sodium channel modulates platelet collagen activation.

    Science.gov (United States)

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

    2014-03-01

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

  5. 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.

  6. 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.

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

    Science.gov (United States)

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

    2007-10-01

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

  8. 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.

  9. Impact of Volcanic Activity on AMC Channel Operations

    Science.gov (United States)

    2014-06-13

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

  10. Acrolein-mediated conduction loss is partially restored by K⁺ channel blockers.

    Science.gov (United States)

    Yan, Rui; Page, Jessica C; Shi, Riyi

    2016-02-01

    Acrolein-mediated myelin damage is thought to be a critical mechanism leading to conduction failure following neurotrauma and neurodegenerative diseases. The exposure and activation of juxtaparanodal voltage-gated K(+) channels due to myelin damage leads to conduction block, and K(+) channel blockers have long been studied as a means for restoring axonal conduction in spinal cord injury (SCI) and multiple sclerosis (MS). In this study, we have found that 100 μM K(+) channel blockers 4-aminopyridine-3-methanol (4-AP-3-MeOH), and to a lesser degree 4-aminopyridine (4-AP), can significantly restore compound action potential (CAP) conduction in spinal cord tissue following acrolein-mediated myelin damage using a well-established ex vivo SCI model. In addition, 4-AP-3-MeOH can effectively restore CAP conduction in acrolein-damaged axons with a range of concentrations from 0.1 to 100 μM. We have also shown that while both compounds at 100 μM showed no preference of small- and large-caliber axons when restoring CAP conduction, 4-AP-3-MeOH, unlike 4-AP, is able to augment CAP amplitude while causing little change in axonal responsiveness measured in refractory periods and response to repetitive stimuli. In a prior study, we show that 4-AP-3-MeOH was able to functionally rescue mechanically injured axons. In this investigation, we conclude that 4-AP-3-MeOH is an effective K(+) channel blocker in restoring axonal conduction following both primary (physical) and secondary (chemical) insults. These findings also suggest that 4-AP-3-MeOH is a viable alternative of 4-AP for treating myelin damage and improving function following central nervous system trauma and neurodegenerative diseases.

  11. Fps1p channel is the mediator of the major part of glycerol passive diffusion in Saccharomyces cerevisiae : artefacts and re-definitions

    OpenAIRE

    Oliveira, Rui Pedro Soares de; Lages, Fernanda; Graça, Magda Maria Couto da Silva; Lucas, Cândida

    2003-01-01

    Glycerol has been shown to cross Saccharomyces cerevisiae plasma membrane (1) through a H+/symport detected in cells grown on non-fermentable carbon sources, (2) by passive diffusion and (3) through the constitutive Fps1p channel. This has been named a facilitator, for mediating glycerol low affinity transport of the facilitated diffusion type. We present experimental evidence that this kinetics is an artefact created by glycerol kinase activity. Instead, the channel is shown to mediate the m...

  12. 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

  13. The two-pore channel TPCN2 mediates NAADP-dependent Ca(2+)-release from lysosomal stores.

    Science.gov (United States)

    Zong, Xiangang; Schieder, Michael; Cuny, Hartmut; Fenske, Stefanie; Gruner, Christian; Rötzer, Katrin; Griesbeck, Oliver; Harz, Hartmann; Biel, Martin; Wahl-Schott, Christian

    2009-09-01

    Second messenger-induced Ca(2+)-release from intracellular stores plays a key role in a multitude of physiological processes. In addition to 1,4,5-inositol trisphosphate (IP(3)), Ca(2+), and cyclic ADP ribose (cADPR) that trigger Ca(2+)-release from the endoplasmatic reticulum (ER), nicotinic acid adenine dinucleotide phosphate (NAADP) has been identified as a cellular metabolite that mediates Ca(2+)-release from lysosomal stores. While NAADP-induced Ca(2+)-release has been found in many tissues and cell types, the molecular identity of the channel(s) conferring this release remained elusive so far. Here, we show that TPCN2, a novel member of the two-pore cation channel family, displays the basic properties of native NAADP-dependent Ca(2+)-release channels. TPCN2 transcripts are widely expressed in the body and encode a lysosomal protein forming homomers. TPCN2 mediates intracellular Ca(2+)-release after activation with low-nanomolar concentrations of NAADP while it is desensitized by micromolar concentrations of this second messenger and is insensitive to the NAADP analog nicotinamide adenine dinucleotide phosphate (NADP). Furthermore, TPCN2-mediated Ca(2+)-release is almost completely abolished when the capacity of lysosomes for storing Ca(2+) is pharmacologically blocked. By contrast, TPCN2-specific Ca(2+)-release is unaffected by emptying ER-based Ca(2+) stores. In conclusion, these findings indicate that TPCN2 is a major component of the long-sought lysosomal NAADP-dependent Ca(2+)-release channel.

  14. Nanobody mediated crystallization of an archeal mechanosensitive channel.

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    Christian Löw

    Full Text Available Mechanosensitive channels (MS are integral membrane proteins and allow bacteria to survive sudden changes in external osmolarity due to transient opening of their pores. The efflux of cytoplasmic osmolytes reduces the membrane tension and prevents membrane rupture. Therefore these channels serve as emergency valves when experiencing significant environmental stress. The preparation of high quality crystals of integral membrane proteins is a major bottleneck for structure determination by X-ray crystallography. Crystallization chaperones based on various protein scaffolds have emerged as promising tool to increase the crystallization probability of a selected target protein. So far archeal mechanosensitive channels of small conductance have resisted crystallization in our hands. To structurally analyse these channels, we selected nanobodies against an archeal MS channel after immunization of a llama with recombinant expressed, detergent solubilized and purified protein. Here we present the characterization of 23 different binders regarding their interaction with the channel protein using analytical gel filtration, western blotting and surface plasmon resonance. Selected nanobodies bound the target with affinities in the pico- to nanomolar range and some binders had a profound effect on the crystallization of the MS channel. Together with previous data we show that nanobodies are a versatile and valuable tool in structural biology by widening the crystallization space for highly challenging proteins, protein complexes and integral membrane proteins.

  15. 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...

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

    Science.gov (United States)

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

    2001-09-25

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

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

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    Takayuki Teramoto

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

  18. SK channels mediate NADPH oxidase-independent reactive oxygen species production and apoptosis in granulocytes.

    Science.gov (United States)

    Fay, Alex J; Qian, Xiang; Jan, Yuh Nung; Jan, Lily Yeh

    2006-11-14

    Neutrophils are immune cells that bind to, engulf, and destroy bacterial and fungal pathogens in infected tissue, and their clearance by apoptosis is essential for the resolution of inflammation. Killing involves both oxidative and nonoxidative processes, the oxidative pathway requiring electrogenic production of superoxide by the membrane-bound NADPH oxidase complex. A variety of stimuli, from bacterial chemotactic peptides to complement- or IgG-opsonized microbes, can induce the production of reactive oxygen species (ROS) by neutrophils, presumably by means of NADPH oxidase. We report here that 1-ethyl-2-benzimidazolinone (1-EBIO), an activator of Ca2+-activated potassium channels of small conductance (SK) and intermediate conductance (IK), causes production of superoxide and hydrogen peroxide by neutrophils and granulocyte-differentiated PLB-985 cells. This response can be partially inhibited by the SK blocker apamin, which inhibits a Ca2+-activated K+ current in these cells. Analysis of RNA transcripts indicates that channels encoded by the SK3 gene carry this current. The effects of 1-EBIO and apamin are independent of the NADPH oxidase pathway, as demonstrated by using a PLB-985 cell line lacking the gp91phox subunit. Rather, 1-EBIO and apamin modulate mitochondrial ROS production. Consistent with the enhanced ROS production and K+ efflux mediated by 1-EBIO, we found that this SK opener increased apoptosis of PLB-985 cells. Together, these findings suggest a previously uncharacterized mechanism for the regulation of neutrophil ROS production and programmed cell death.

  19. Ca2+ channels as integrators of G protein-mediated signaling in neurons.

    Science.gov (United States)

    Strock, Jesse; Diversé-Pierluissi, María A

    2004-11-01

    The observations from Dunlap and Fischbach that transmitter-mediated shortening of the duration of action potentials could be caused by a decrease in calcium conductance led to numerous studies of the mechanisms of modulation of voltage-dependent calcium channels. Calcium channels are well known targets for inhibition by receptor-G protein pathways, and multiple forms of inhibition have been described. Inhibition of Ca(2+) channels can be mediated by G protein betagamma-subunits or by kinases, such as protein kinase C and tyrosine kinases. In the last few years, it has been shown that integration of G protein signaling can take place at the level of the calcium channel by regulation of the interaction of the channel pore-forming subunit with different cellular proteins.

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

    DEFF Research Database (Denmark)

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

    2002-01-01

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

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

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    Qiong-Yao Tang

    2016-01-01

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

  2. Follicle-stimulating hormone receptor-mediated uptake of sup 45 Ca sup 2+ by proteoliposomes and cultured rat sertoli cells: Evidence for involvement of voltage-activated and voltage-independent calcium channels

    Energy Technology Data Exchange (ETDEWEB)

    Grasso, P.; Reichert, L.E. Jr. (Albany Medical College, NY (USA))

    1989-12-01

    We have previously reported incorporation into liposomes of Triton X-100-solubilized FSH receptor-G-protein complexes derived from purified bovine calf testis membranes. In the present study we have used this model system to show that FSH induces flux of 45Ca2+ into such proteoliposomes in a hormone-specific concentration-dependent manner. FSH, inactivated by boiling, had no stimulatory effect on 45Ca2+ flux, nor did isolated alpha- or beta-subunits of FSH. Addition of GTP (or its analogs 5'-guanylylimidodiphosphate and guanosine-5'-O-(3-thiotriphosphate)) or sodium fluoride (in the presence or absence of GTP or its analogs) failed to induce 45Ca2+ flux into proteoliposomes, suggesting that the uptake of 45Ca2+ was receptor, and not G-protein, related. Voltage-independent (ruthenium red and gadolinium chloride) and voltage-activated (methyoxyverapamil and nifedipine) calcium channel-blocking agents reduced FSH-stimulated 45Ca2+ flux into proteoliposomes to control levels. FSH also induced uptake of 45Ca2+ by cultured rat Sertoli cells. Ruthenium red and gadolinium chloride had no effect on basal levels of 45Ca2+ uptake or estradiol secretion by cultured rat Sertoli cells, nor did methoxyverapamil or nifedipine. All four calcium channel blockers, however, were able to reduce FSH-induced 45Ca2+ uptake to basal levels and FSH-stimulated conversion of androstenedione to estradiol by up to 50%, indicating an involvement of Ca2+ in FSH-stimulated steroidogenesis. Our results suggest that the well documented changes in intracellular calcium levels consequent to FSH binding may be due, at least in part, to an influx of calcium through FSH receptor-regulated calcium channels.

  3. 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.

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

    Science.gov (United States)

    Xu, Jian Wei; Slaughter, Malcolm M

    2005-08-17

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

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

    Science.gov (United States)

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

    2015-06-01

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

  6. Biphasic somatic A-type K channel downregulation mediates intrinsic plasticity in hippocampal CA1 pyramidal neurons.

    Directory of Open Access Journals (Sweden)

    Sung-Cherl Jung

    Full Text Available Since its original description, the induction of synaptic long-term potentiation (LTP has been known to be accompanied by a lasting increase in the intrinsic excitability (intrinsic plasticity of hippocampal neurons. Recent evidence shows that dendritic excitability can be enhanced by an activity-dependent decrease in the activity of A-type K(+ channels. In the present manuscript, we examined the role of A-type K(+ channels in regulating intrinsic excitability of CA1 pyramidal neurons of the hippocampus after synapse-specific LTP induction. In electrophysiological recordings we found that LTP induced a potentiation of excitability which was accompanied by a two-phased change in A-type K(+ channel activity recorded in nucleated patches from organotypic slices of rat hippocampus. Induction of LTP resulted in an immediate but short lasting hyperpolarization of the voltage-dependence of steady-state A-type K(+ channel inactivation along with a progressive, long-lasting decrease in peak A-current density. Blocking clathrin-mediated endocytosis prevented the A-current decrease and most measures of intrinsic plasticity. These results suggest that two temporally distinct but overlapping mechanisms of A-channel downregulation together contribute to the plasticity of intrinsic excitability. Finally we show that intrinsic plasticity resulted in a global enhancement of EPSP-spike coupling.

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

    Directory of Open Access Journals (Sweden)

    Jean-Yves eTano

    2014-10-01

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

  8. Ca2+ paradox injury mediated through TRPC channels in mouse ventricular myocytes.

    Science.gov (United States)

    Kojima, Akiko; Kitagawa, Hirotoshi; Omatsu-Kanbe, Mariko; Matsuura, Hiroshi; Nosaka, Shuichi

    2010-12-01

    BACKGROUND AND PURPOSE The Ca(2+) paradox is an important phenomenon associated with Ca(2+) overload-mediated cellular injury in myocardium. The present study was undertaken to elucidate molecular and cellular mechanisms for the development of the Ca(2+) paradox. EXPERIMENTAL APPROACH Fluorescence imaging was performed on fluo-3 loaded quiescent mouse ventricular myocytes using confocal laser scanning microscope. KEY RESULTS The Ca(2+) paradox was readily evoked by restoration of the extracellular Ca(2+) following 10-20 min of nominally Ca(2+)-free superfusion. The Ca(2+) paradox was significantly reduced by blockers of transient receptor potential canonical (TRPC) channels (2-aminoethoxydiphenyl borate, Gd(3+), La(3+)) and anti-TRPC1 antibody. The sarcoplasmic reticulum (SR) Ca(2+) content, assessed by caffeine application, gradually declined during Ca(2+)-free superfusion, which was further accelerated by metabolic inhibition. Block of SR Ca(2+) leak by tetracaine prevented Ca(2+) paradox. The Na(+) /Ca(2+) exchange (NCX) blocker KB-R7943 significantly inhibited Ca(2+) paradox when applied throughout superfusion period, but had little effect when added for a period of 3 min before and during Ca(2+) restoration. The SR Ca(2+) content was better preserved during Ca(2+) depletion by KB-R7943. Immunocytochemistry confirmed the expression of TRPC1, in addition to TRPC3 and TRPC4, in mouse ventricular myocytes. CONCLUSIONS AND IMPLICATIONS These results provide evidence that (i) the Ca(2+) paradox is primarily mediated by Ca(2+) entry through TRPC (probably TRPC1) channels that are presumably activated by SR Ca(2+) depletion; and (ii) reverse mode NCX contributes little to the Ca(2+) paradox, whereas inhibition of NCX during Ca(2+) depletion improves SR Ca(2+) loading, and is associated with reduced incidence of Ca(2+) paradox in mouse ventricular myocytes.

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

    Science.gov (United States)

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

    2016-08-01

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

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

    Science.gov (United States)

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

    2017-02-01

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

  11. Activation of CFTR-mediated Cl- Transport by Magnolin

    Institute of Scientific and Technical Information of China (English)

    JIN Ling-ling; LIU Xin; SUN Yan; LIN Sen; ZHOU Na; XU Li-na; YU BO; HOU Shu-guang; YANG Hong

    2008-01-01

    Magnolin is a herbal compound from Magnolia biondii Pamp.It possesses numerous biological activities.Cystic fibrosis transmembrane conductance regulator(CFTR)is all epithelial chloride channel that plays a key role in the fluid secretion of various exocrine organs.In the present study,the activation of CFTR-mediated chloride transport by magnolin is indentified and characterized.In CFTR stably trailsfected FRT cells.magnolin increases CFTR Cl- currents in a concentration-dependent manner.The activation of magnolin on CFTR is rapid,reversible,and cAMP-dependent.Magnolin does not elevate cellular cAMP level.indicating that it activates CFTR by direct binding and interaction with CFTR protein.Magnolin selectively activates wildtype CFTR rather than mutant CFTIL Magnolin may present a novel class of therapeutic lead compound for the treatment of diseases associated with reduced CFTR function such as keratoconjunctivitis sicca,idiopathic chronic pancreatiti,and chromc constipation.

  12. Slow synaptic transmission mediated by TRPV1 channels in CA3 interneurons of the hippocampus.

    Science.gov (United States)

    Eguchi, Noriomi; Hishimoto, Akitoyo; Sora, Ichiro; Mori, Masahiro

    2016-03-11

    Metabotropic glutamate receptors (mGluRs) modulate various neuronal functions in the central nervous system. Many studies reported that mGluRs have linkages to neuronal disorders such as schizophrenia and autism related disorders, indicating that mGluRs are involved in critical functions of the neuronal circuits. To study this possibility further, we recorded mGluR-induced synaptic responses in the interneurons of the CA3 stratum radiatum using rat hippocampal organotypic slice cultures. Electrical stimulation in the CA3 pyramidal cell layer evoked a slow inward current in the interneurons at a holding potential of -70mV in the presence of antagonists for AMPA/kainate receptors, NMDA receptors, GABAA receptors and GABAB receptors. The slow inward current was blocked in the absence of extracellular calcium, suggesting that this was a synaptic response. The slow excitatory postsynaptic current (EPSC) reversed near 0mV, reflecting an increase in a non-selective cationic conductance. The slow EPSC is mediated by group I mGluRs, as it was blocked by AP3, a group I mGluR antagonist. Neither a calcium chelator BAPTA nor a phospholipase C (PLC) inhibitor U73122 affected the slow EPSC. La(3+), a general TRP channel blocker or capsazepine, a selective TRPV1 channel antagonist significantly suppressed the slow EPSC. DHPG, a selective group I mGluRs agonist induced an inward current, which was suppressed by capsazepine. These results indicate that in the interneurons of the hippocampal CA3 stratum radiatum group I mGluRs activate TRPV1 channels independently of PLC and intracellular Ca(2+), resulting in the slow EPSC in the interneurons.

  13. TRESK channel as a potential target to treat T-cell mediated immune dysfunction

    Energy Technology Data Exchange (ETDEWEB)

    Han, Jaehee [Medical Research Center for Neural Dysfunction, Department of Physiology, Institute of Health Sciences, Gyeongsang National University, School of Medicine, Jinju 660-751 (Korea, Republic of); Kang, Dawon, E-mail: dawon@gnu.ac.kr [Medical Research Center for Neural Dysfunction, Department of Physiology, Institute of Health Sciences, Gyeongsang National University, School of Medicine, Jinju 660-751 (Korea, Republic of)

    2009-12-25

    In this review, we propose that TRESK background K{sup +} channel could serve as a potential therapeutic target for T-cell mediated immune dysfunction. TRESK has many immune function-related properties. TRESK is abundantly expressed in the thymus, the spleen, and human leukemic T-lymphocytes. TRESK is highly activated by Ca{sup 2+}, calcineurin, acetylcholine, and histamine which induce hypertrophy, whereas TRESK is inhibited by immunosuppressants, such as cyclosporin A and FK506. Cyclosporine A and FK506 target the binding site of nuclear factor of activated T-cells (NFAT) to inhibit calcineurin. Interestingly, TRESK possesses an NFAT-like docking site that is present at its intracellular loop. Calcineurin has been found to interact with TRESK via specific NFAT-like docking site. When the T-cell is activated, calcineurin can bind to the NFAT-docking site of TRESK. The activation of both TRESK and NFAT via Ca{sup 2+}-calcineurin-NFAT/TRESK pathway could modulate the transcription of new genes in addition to regulating several aspects of T-cell function.

  14. Activation of Slo2.1 channels by niflumic acid

    OpenAIRE

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

    2010-01-01

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

  15. Decrease of a Current Mediated by Kv1.3 Channels Causes Striatal Cholinergic Interneuron Hyperexcitability in Experimental Parkinsonism

    Directory of Open Access Journals (Sweden)

    Cecilia Tubert

    2016-09-01

    Full Text Available The mechanism underlying a hypercholinergic state in Parkinson’s disease (PD remains uncertain. Here, we show that disruption of the Kv1 channel-mediated function causes hyperexcitability of striatal cholinergic interneurons in a mouse model of PD. Specifically, our data reveal that Kv1 channels containing Kv1.3 subunits contribute significantly to the orphan potassium current known as IsAHP in striatal cholinergic interneurons. Typically, this Kv1 current provides negative feedback to depolarization that limits burst firing and slows the tonic activity of cholinergic interneurons. However, such inhibitory control of cholinergic interneuron excitability by Kv1.3-mediated current is markedly diminished in the parkinsonian striatum, suggesting that targeting Kv1.3 subunits and their regulatory pathways may have therapeutic potential in PD therapy. These studies reveal unexpected roles of Kv1.3 subunit-containing channels in the regulation of firing patterns of striatal cholinergic interneurons, which were thought to be largely dependent on KCa channels.

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

    Institute of Scientific and Technical Information of China (English)

    凌琰; 谢益宽

    1996-01-01

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

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

    Directory of Open Access Journals (Sweden)

    Wei Wang

    Full Text Available W1282X is a common nonsense mutation among cystic fibrosis patients that results in the production of a truncated Cystic Fibrosis Transmembrane Conductance Regulator (CFTR channel. Here we show that the channel activity of the W1282X-CFTR polypeptide is exceptionally low in excised membrane patches at normally saturating doses of ATP and PKA (single channel open probability (PO 0.9 when treated with both modulators. VX-770 and curcumin also additively stimulated W1282X-CFTR mediated currents in polarized FRT epithelial monolayers. In this setting, however, the stimulated W1282X-CFTR currents were smaller than those mediated by wild type CFTR (3-5% due presumably to lower expression levels or cell surface targeting of the truncated protein. Combining allosteric modulators of different mechanistic classes is worth considering as a treatment option for W1282X CF patients perhaps when coupled with maneuvers to increase expression of the truncated protein.

  18. Phosphatase inhibitors activate normal and defective CFTR chloride channels

    OpenAIRE

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

    1994-01-01

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

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

    Institute of Scientific and Technical Information of China (English)

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

    2012-01-01

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

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

    Directory of Open Access Journals (Sweden)

    Anton Hermann

    2015-08-01

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

  1. Characterization of Two-pore Channel 2 (TPCN2)-mediated Ca2+ Currents in Isolated Lysosomes*

    OpenAIRE

    Schieder, Michael; Rötzer, Katrin; Brüggemann, Andrea; Biel, Martin; Wahl-Schott, Christian A.

    2010-01-01

    Two-pore channels (TPCNs) have been proposed to form lysosomal Ca2+ release channels that are activated by nicotinic acid adenine dinucleotide phosphate. Here, we employ a glass chip-based method to record for the first time nicotinic acid adenine dinucleotide phosphate -dependent currents through a two-pore channel (TPCN2) from intact lysosomes. We show that TPCN2 is a highly selective Ca2+ channel that is regulated by intralysosomal pH. Using site-directed mutagenesis, we identify an amino ...

  2. Characterization of two-pore channel 2 (TPCN2)-mediated Ca2+ currents in isolated lysosomes.

    Science.gov (United States)

    Schieder, Michael; Rötzer, Katrin; Brüggemann, Andrea; Biel, Martin; Wahl-Schott, Christian A

    2010-07-01

    Two-pore channels (TPCNs) have been proposed to form lysosomal Ca(2+) release channels that are activated by nicotinic acid adenine dinucleotide phosphate. Here, we employ a glass chip-based method to record for the first time nicotinic acid adenine dinucleotide phosphate -dependent currents through a two-pore channel (TPCN2) from intact lysosomes. We show that TPCN2 is a highly selective Ca(2+) channel that is regulated by intralysosomal pH. Using site-directed mutagenesis, we identify an amino acid residue in the putative pore region that is crucial for conferring high Ca(2+) selectivity. Our glass chip-based method will provide electrophysiological access not only to lysosomal TPCN channels but also to a broad range of other intracellular ion channels.

  3. Characterization of Two-pore Channel 2 (TPCN2)-mediated Ca2+ Currents in Isolated Lysosomes*

    Science.gov (United States)

    Schieder, Michael; Rötzer, Katrin; Brüggemann, Andrea; Biel, Martin; Wahl-Schott, Christian A.

    2010-01-01

    Two-pore channels (TPCNs) have been proposed to form lysosomal Ca2+ release channels that are activated by nicotinic acid adenine dinucleotide phosphate. Here, we employ a glass chip-based method to record for the first time nicotinic acid adenine dinucleotide phosphate -dependent currents through a two-pore channel (TPCN2) from intact lysosomes. We show that TPCN2 is a highly selective Ca2+ channel that is regulated by intralysosomal pH. Using site-directed mutagenesis, we identify an amino acid residue in the putative pore region that is crucial for conferring high Ca2+ selectivity. Our glass chip-based method will provide electrophysiological access not only to lysosomal TPCN channels but also to a broad range of other intracellular ion channels. PMID:20495006

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

    Directory of Open Access Journals (Sweden)

    Veronika Matschke

    2016-12-01

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

  5. 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.

  6. The vacuolar channel VvALMT9 mediates malate and tartrate accumulation in berries of Vitis vinifera.

    Science.gov (United States)

    De Angeli, Alexis; Baetz, Ulrike; Francisco, Rita; Zhang, Jingbo; Chaves, Maria Manuela; Regalado, Ana

    2013-08-01

    Vitis vinifera L. represents an economically important fruit species. Grape and wine flavour is made from a complex set of compounds. The acidity of berries is a major parameter in determining grape berry quality for wine making and fruit consumption. Despite the importance of malic and tartaric acid (TA) storage and transport for grape berry acidity, no vacuolar transporter for malate or tartrate has been identified so far. Some members of the aluminium-activated malate transporter (ALMT) anion channel family from Arabidopsis thaliana have been shown to be involved in mediating malate fluxes across the tonoplast. Therefore, we hypothesised that a homologue of these channels could have a similar role in V. vinifera grape berries. We identified homologues of the Arabidopsis vacuolar anion channel AtALMT9 through a TBLASTX search on the V. vinifera genome database. We cloned the closest homologue of AtALMT9 from grape berry cDNA and designated it VvALMT9. The expression profile revealed that VvALMT9 is constitutively expressed in berry mesocarp tissue and that its transcription level increases during fruit maturation. Moreover, we found that VvALMT9 is targeted to the vacuolar membrane. Using patch-clamp analysis, we could show that, besides malate, VvALMT9 mediates tartrate currents which are higher than in its Arabidopsis homologue. In summary, in the present study we provide evidence that VvALMT9 is a vacuolar malate channel expressed in grape berries. Interestingly, in V. vinifera, a tartrate-producing plant, the permeability of the channel is apparently adjusted to TA.

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

    Directory of Open Access Journals (Sweden)

    Qijing Chen

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

  8. Activation of CFTR by ASBT-mediated bile salt absorption

    NARCIS (Netherlands)

    Bijvelds, MJC; Jorna, H; Verkade, HJ; Bot, AGM; Hofmann, F; Agellon, LB; Sinaasappel, M; de Jonge, HR

    2005-01-01

    In cholangiocytes, bile salt (BS) uptake via the apical sodium-dependent bile acid transporter (ASBT) may evoke ductular flow by enhancing cAMP-mediated signaling to the cystic fibrosis transmembrane conductance regulator (CFTR) anion channel. We considered that ASBT-mediated BS uptake in the distal

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

    Science.gov (United States)

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

    2013-06-01

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

  10. Phospholipase C not protein kinase C is required for the activation of TRPC5 channels by cholecystokinin.

    Science.gov (United States)

    Grisanti, Laurel A; Kurada, Lalitha; Cilz, Nicholas I; Porter, James E; Lei, Saobo

    2012-08-15

    Cholecystokinin (CCK) is one of the most abundant neuropeptides in the brain where it interacts with two G protein-coupled receptors (CCK1 and CCK2). Both types of CCK receptors are coupled to G(q/11) proteins resulting in increased function of phospholipase C (PLC) pathway. Whereas CCK has been suggested to increase neuronal excitability in the brain via activation of cationic channels, the types of cationic channels have not yet been identified. Here, we co-expressed CCK2 receptors and TRPC5 channels in human embryonic kidney (HEK) 293 cells and studied the effects of CCK on TRPC5 channels using patch-clamp techniques. Our results demonstrate that activation of CCK2 receptors robustly potentiates the function of TRPC5 channels. CCK-induced activation of TRPC5 channels requires the functions of G-proteins and PLC and depends on extracellular Ca(2+). The activation of TRPC5 channels mediated by CCK2 receptors is independent of IP(3) receptors and protein kinase C. CCK-induced opening of TRPC5 channels is not store-operated because application of thapsigargin to deplete intracellular Ca(2+) stores failed to alter CCK-induced TRPC5 channel currents significantly. Bath application of CCK also significantly increased the open probability of TRPC5 single channel currents in cell-attached patches. Because CCK exerts extensive effects in the brain, our results may provide a novel mechanism to explain its roles in modulating neuronal excitability.

  11. Opening of small and intermediate calcium-activated potassium channels induces relaxation mainly mediated by nitric-oxide release in large arteries and endothelium-derived hyperpolarizing factor in small arteries from rat

    DEFF Research Database (Denmark)

    Stankevicius, Edgaras; Dalsgaard, Thomas; Kroigaard, Christel

    2011-01-01

    mesenteric arteries, NS309 relaxations and NO release were inhibited by both N(G),N(G)-asymmetric dimethyl-l-arginine (ADMA) (300 μM), an inhibitor of NO synthase, and apamin (0.5 μM) plus 1-[(2-chlorophenyl)diphenylmethyl]-1H-pyrazole (TRAM-34) (1 μM), blockers of SK(Ca) and IK(Ca) channels, respectively...

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

    Science.gov (United States)

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

    2016-03-01

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

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

    Science.gov (United States)

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

    2006-05-01

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

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

    Directory of Open Access Journals (Sweden)

    Robert C Cannon

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

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

    Science.gov (United States)

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

    2008-08-01

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

  16. Intracellular ion channel CLIC1: involvement in microglia-mediated β-amyloid peptide(1-42) neurotoxicity.

    Science.gov (United States)

    Skaper, Stephen D; Facci, Laura; Giusti, Pietro

    2013-09-01

    Microglia can exacerbate central nervous system disorders, including stroke and chronic progressive neurodegenerative diseases such as Alzheimer disease. Mounting evidence points to ion channels expressed by microglia as contributing to these neuropathologies. The Chloride Intracellular Channel (CLIC) family represents a class of chloride intracellular channel proteins, most of which are localized to intracellular membranes. CLICs are unusual in that they possess both soluble and integral membrane forms. Amyloid β-peptide (Aβ) accumulation in plaques is a hallmark of familial Alzheimer disease. The truncated Aβ25-35 species was shown previously to increase the expression of CLIC1 chloride conductance in cortical microglia and to provoke microglial neurotoxicity. However, the highly pathogenic and fibrillogenic full-length Aβ1-42 species was not examined, nor was the potential role of CLIC1 in mediating microglial activation and neurotoxicity by other stimuli (e.g. ligands for the Toll-like receptors). In the present study, we utilized a two chamber Transwell™ cell culture system to allow separate treatment of microglia and neurons while examining the effect of pharmacological blockade of CLIC1 in protecting cortical neurons from toxicity caused by Aβ1-42- and lipopolysaccaride-stimulated microglia. Presentation of Aβ1-42 to the upper, microglia-containing chamber resulted in a progressive loss of neurons over 3 days. Neuronal cell injury was prevented by the CLIC1 ion channel blockers IAA-94 [(R(+)-[(6,7-dichloro-2-cyclopentyl-2,3-dihydro-2-methyl-1-oxo-1H-inden-5yl)-oxy] acetic acid)] and niflumic acid (2-{[3-(trifluoromethyl)phenyl]amino}nicotinic acid) when presented to the upper chamber only. Incubation of microglia with lipopolysaccharide plus interferon-γ led to neuronal cell injury which, however, was insensitive to inhibition by the CLIC1 channel blockers, suggesting a degree of selectivity in agents leading to CLIC1 activation.

  17. ROMK1 channel activity is regulated by monoubiquitination.

    Science.gov (United States)

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

    2005-03-22

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

  18. Tetrodotoxin-resistant sodium channels in sensory neurons generate slow resurgent currents that are enhanced by inflammatory mediators.

    Science.gov (United States)

    Tan, Zhi-Yong; Piekarz, Andrew D; Priest, Birgit T; Knopp, Kelly L; Krajewski, Jeffrey L; McDermott, Jeff S; Nisenbaum, Eric S; Cummins, Theodore R

    2014-05-21

    Resurgent sodium currents contribute to the regeneration of action potentials and enhanced neuronal excitability. Tetrodotoxin-sensitive (TTX-S) resurgent currents have been described in many different neuron populations, including cerebellar and dorsal root ganglia (DRG) neurons. In most cases, sodium channel Nav1.6 is the major contributor to these TTX-S resurgent currents. Here we report a novel TTX-resistant (TTX-R) resurgent current recorded from rat DRG neurons. The TTX-R resurgent currents are similar to classic TTX-S resurgent currents in many respects, but not all. As with TTX-S resurgent currents, they are activated by membrane repolarization, inhibited by lidocaine, and enhanced by a peptide-mimetic of the β4 sodium channel subunit intracellular domain. However, the TTX-R resurgent currents exhibit much slower kinetics, occur at more depolarized voltages, and are sensitive to the Nav1.8 blocker A803467. Moreover, coimmunoprecipitation experiments from rat DRG lysates indicate the endogenous sodium channel β4 subunits associate with Nav1.8 in DRG neurons. These results suggest that slow TTX-R resurgent currents in DRG neurons are mediated by Nav1.8 and are generated by the same mechanism underlying TTX-S resurgent currents. We also show that both TTX-S and TTX-R resurgent currents in DRG neurons are enhanced by inflammatory mediators. Furthermore, the β4 peptide increased excitability of small DRG neurons in the presence of TTX. We propose that these slow TTX-R resurgent currents contribute to the membrane excitability of nociceptive DRG neurons under normal conditions and that enhancement of both types of resurgent currents by inflammatory mediators could contribute to sensory neuronal hyperexcitability associated with inflammatory pain.

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

    Directory of Open Access Journals (Sweden)

    Kwok Wai-Meng

    2009-03-01

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

  20. Molecular Basis of Regulating High Voltage-Activated Calcium Channels by S-Nitrosylation.

    Science.gov (United States)

    Zhou, Meng-Hua; Bavencoffe, Alexis; Pan, Hui-Lin

    2015-12-18

    Nitric oxide (NO) is involved in a variety of physiological processes, such as vasoregulation and neurotransmission, and has a complex role in the regulation of pain transduction and synaptic transmission. We have shown previously that NO inhibits high voltage-activated Ca(2+) channels in primary sensory neurons and excitatory synaptic transmission in the spinal dorsal horn. However, the molecular mechanism involved in this inhibitory action remains unclear. In this study, we investigated the role of S-nitrosylation in the NO regulation of high voltage-activated Ca(2+) channels. The NO donor S-nitroso-N-acetyl-DL-penicillamine (SNAP) rapidly reduced N-type currents when Cav2.2 was coexpressed with the Cavβ1 or Cavβ3 subunits in HEK293 cells. In contrast, SNAP only slightly inhibited P/Q-type and L-type currents reconstituted with various Cavβ subunits. SNAP caused a depolarizing shift in voltage-dependent N-type channel activation, but it had no effect on Cav2.2 protein levels on the membrane surface. The inhibitory effect of SNAP on N-type currents was blocked by the sulfhydryl-specific modifying reagent methanethiosulfonate ethylammonium. Furthermore, the consensus motifs of S-nitrosylation were much more abundant in Cav2.2 than in Cav1.2 and Cav2.1. Site-directed mutagenesis studies showed that Cys-805, Cys-930, and Cys-1045 in the II-III intracellular loop, Cys-1835 and Cys-2145 in the C terminus of Cav2.2, and Cys-346 in the Cavβ3 subunit were nitrosylation sites mediating NO sensitivity of N-type channels. Our findings demonstrate that the consensus motifs of S-nitrosylation in cytoplasmically accessible sites are critically involved in post-translational regulation of N-type Ca(2+) channels by NO. S-Nitrosylation mediates the feedback regulation of N-type channels by NO.

  1. Activism and the Online Mediation Opportunity Structure

    DEFF Research Database (Denmark)

    Uldam, Julie

    2013-01-01

    The annual United Nations (UN) Framework Convention on Climate Change conferences provides a transnational mediation opportunity structure for activist networks to contest policies that favor market-based models for solving the climate crisis. Online technologies, including commercial social media...... to climate change activism. This impedes possibilities for using online media to protest at the radical end of the climate justice movement spectrum. This article explores this interrelationship between activist demands and (online) modes of action through a focus on the mobilization efforts of London...

  2. Putative calcium-binding domains of the Caenorhabditis elegans BK channel are dispensable for intoxication and ethanol activation.

    Science.gov (United States)

    Davis, S J; Scott, L L; Ordemann, G; Philpo, A; Cohn, J; Pierce-Shimomura, J T

    2015-07-01

    Alcohol modulates the highly conserved, voltage- and calcium-activated potassium (BK) channel, which contributes to alcohol-mediated behaviors in species from worms to humans. Previous studies have shown that the calcium-sensitive domains, RCK1 and the Ca(2+) bowl, are required for ethanol activation of the mammalian BK channel in vitro. In the nematode Caenorhabditis elegans, ethanol activates the BK channel in vivo, and deletion of the worm BK channel, SLO-1, confers strong resistance to intoxication. To determine if the conserved RCK1 and calcium bowl domains were also critical for intoxication and basal BK channel-dependent behaviors in C. elegans, we generated transgenic worms that express mutated SLO-1 channels predicted to have the RCK1, Ca(2+) bowl or both domains rendered insensitive to calcium. As expected, mutating these domains inhibited basal function of SLO-1 in vivo as neck and body curvature of these mutants mimicked that of the BK null mutant. Unexpectedly, however, mutating these domains singly or together in SLO-1 had no effect on intoxication in C. elegans. Consistent with these behavioral results, we found that ethanol activated the SLO-1 channel in vitro with or without these domains. By contrast, in agreement with previous in vitro findings, C. elegans harboring a human BK channel with mutated calcium-sensing domains displayed resistance to intoxication. Thus, for the worm SLO-1 channel, the putative calcium-sensitive domains are critical for basal in vivo function but unnecessary for in vivo ethanol action.

  3. Statins attenuate polymethylmethacrylate-mediated monocyte activation.

    LENUS (Irish Health Repository)

    Laing, Alan J

    2012-02-03

    BACKGROUND: Periprosthetic osteolysis precipitates aseptic loosening of components, increases the risk of periprosthetic fracture and, through massive bone loss, complicates revision surgery and ultimately is the primary cause for failure of joint arthroplasty. The anti-inflammatory properties of HMG-CoA reductase inhibitors belonging to the statin family are well recognized. We investigated a possible role for status in initiating the first stage of the osteolytic cycle, namely monocytic activation. METHODS: We used an in vitro model of the human monocyte\\/macrophage inflammatory response to poly-methylmethacrylate (PMMA) particles after pretreat-ing cells with cerivastatin, a potent member of the statin family. Cell activation based upon production of TNF-alpha and MCP-1 cytokines was analyzed and the intracellular Raf-MEK-ERK signal transduction pathway was evaluated using western blot analysis, to identify its role in cell activation and in any cerivastatin effects observed. RESULTS: We found that pretreatment with cerivastatin significantly abrogates the production of inflammatory cytokines TNF-alpha and MCP-1 by human monocytes in response to polymethylmethacrylate particle activation. This inflammatory activation and attenuation appear to be mediated through the intracellular Raf-MEK-ERK pathway. INTERPRETATION: We propose that by intervening at the upstream activation stage, subsequent osteoclast activation and osteolysis can be suppressed. We believe that the anti-inflammatory properties of statins may potentially play a prophylactic role in the setting of aseptic loosening, and in so doing increase implant longevity.

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

    Science.gov (United States)

    Premkumar, L S

    2005-04-01

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

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

    OpenAIRE

    Chambers, James J.; Richard H Kramer

    2008-01-01

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

  6. Small-conductance Ca2+-activated potassium type 2 channels regulate the formation of contextual fear memory.

    Directory of Open Access Journals (Sweden)

    Saravana R K Murthy

    Full Text Available Small-conductance, Ca2+ activated K+ channels (SK channels are expressed at high levels in brain regions responsible for learning and memory. In the current study we characterized the contribution of SK2 channels to synaptic plasticity and to different phases of hippocampal memory formation. Selective SK2 antisense-treatment facilitated basal synaptic transmission and theta-burst induced LTP in hippocampal brain slices. Using the selective SK2 antagonist Lei-Dab7 or SK2 antisense probes, we found that hippocampal SK2 channels are critical during two different time windows: 1 blockade of SK2 channels before the training impaired fear memory, whereas, 2 blockade of SK2 channels immediately after the training enhanced contextual fear memory. We provided the evidence that the post-training cleavage of the SK2 channels was responsible for the observed bidirectional effect of SK2 channel blockade on memory consolidation. Thus, Lei-Dab7-injection before training impaired the C-terminal cleavage of SK2 channels, while Lei-Dab7 given immediately after training facilitated the C-terminal cleavage. Application of the synthetic peptide comprising a leucine-zipper domain of the C-terminal fragment to Jurkat cells impaired SK2 channel-mediated currents, indicating that the endogenously cleaved fragment might exert its effects on memory formation by blocking SK2 channel-mediated currents. Our present findings suggest that SK2 channel proteins contribute to synaptic plasticity and memory not only as ion channels but also by additionally generating a SK2 C-terminal fragment, involved in both processes. The modulation of fear memory by down-regulating SK2 C-terminal cleavage might have applicability in the treatment of anxiety disorders in which fear conditioning is enhanced.

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

    Science.gov (United States)

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

    2008-08-01

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

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

    Institute of Scientific and Technical Information of China (English)

    2002-01-01

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

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

    Science.gov (United States)

    Kobrinsky, Evgeny

    2015-01-01

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

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

    Science.gov (United States)

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

    2010-03-01

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

  11. Activating mutations of the TRPML1 channel revealed by proline-scanning mutagenesis.

    Science.gov (United States)

    Dong, Xian-ping; Wang, Xiang; Shen, Dongbiao; Chen, Su; Liu, Meiling; Wang, Yanbin; Mills, Eric; Cheng, Xiping; Delling, Markus; Xu, Haoxing

    2009-11-13

    The mucolipin TRP (TRPML) proteins are a family of endolysosomal cation channels with genetically established importance in humans and rodent. Mutations of human TRPML1 cause type IV mucolipidosis, a devastating pediatric neurodegenerative disease. Our recent electrophysiological studies revealed that, although a TRPML1-mediated current can only be recorded in late endosome and lysosome (LEL) using the lysosome patch clamp technique, a proline substitution in TRPML1 (TRPML1(V432P)) results in a large whole cell current. Thus, it remains unknown whether the large TRPML1(V432P)-mediated current results from an increased surface expression (trafficking), elevated channel activity (gating), or both. Here we performed systemic Pro substitutions in a region previously implicated in the gating of various 6 transmembrane cation channels. We found that several Pro substitutions displayed gain-of-function (GOF) constitutive activities at both the plasma membrane (PM) and endolysosomal membranes. Although wild-type TRPML1 and non-GOF Pro substitutions localized exclusively in LEL and were barely detectable in the PM, the GOF mutations with high constitutive activities were not restricted to LEL compartments, and most significantly, exhibited significant surface expression. Because lysosomal exocytosis is Ca(2+)-dependent, constitutive Ca(2+) permeability due to Pro substitutions may have resulted in stimulus-independent intralysosomal Ca(2+) release, hence the surface expression and whole cell current of TRPML1. Indeed, surface staining of lysosome-associated membrane protein-1 (Lamp-1) was dramatically increased in cells expressing GOF TRPML1 channels. We conclude that TRPML1 is an inwardly rectifying, proton-impermeable, Ca(2+) and Fe(2+)/Mn(2+) dually permeable cation channel that may be gated by unidentified cellular mechanisms through a conformational change in the cytoplasmic face of the transmembrane 5 (TM5). Furthermore, activation of TRPML1 in LEL may lead to the

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

    OpenAIRE

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

    2001-01-01

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

  13. Self-consistent Dark Matter simplified models with an s-channel scalar mediator

    Science.gov (United States)

    Bell, Nicole F.; Busoni, Giorgio; Sanderson, Isaac W.

    2017-03-01

    We examine Simplified Models in which fermionic DM interacts with Standard Model (SM) fermions via the exchange of an s-channel scalar mediator. The single-mediator version of this model is not gauge invariant, and instead we must consider models with two scalar mediators which mix and interfere. The minimal gauge invariant scenario involves the mixing of a new singlet scalar with the Standard Model Higgs boson, and is tightly constrained. We construct two Higgs doublet model (2HDM) extensions of this scenario, where the singlet mixes with the 2nd Higgs doublet. Compared with the one doublet model, this provides greater freedom for the masses and mixing angle of the scalar mediators, and their coupling to SM fermions. We outline constraints on these models, and discuss Yukawa structures that allow enhanced couplings, yet keep potentially dangerous flavour violating processes under control. We examine the direct detection phenomenology of these models, accounting for interference of the scalar mediators, and interference of different quarks in the nucleus. Regions of parameter space consistent with direct detection measurements are determined.

  14. Role of gp91phox -containing NADPH oxidase in mediating the effect of K restriction on ROMK channels and renal K excretion.

    Science.gov (United States)

    Babilonia, Elisa; Lin, Daohong; Zhang, Yan; Wei, Yuan; Yue, Peng; Wang, Wen-Hui

    2007-07-01

    Previous study has demonstrated that superoxide and the related products are involved in mediating the effect of low K intake on renal K secretion and ROMK channel activity in the cortical collecting duct (CCD). This study investigated the role of gp91(phox)-containing NADPH oxidase (NOXII) in mediating the effect of low K intake on renal K excretion and ROMK channel activity in gp91(-/-) mice. K depletion increased superoxide levels, phosphorylation of c-Jun, expression of c-Src, and tyrosine phosphorylation of ROMK in renal cortex and outer medulla in wild-type (WT) mice. In contrast, tempol treatment in WT mice abolished whereas deletion of gp91 significantly attenuated the effect of low K intake on superoxide production, c-Jun phosphorylation, c-Src expression, and tyrosine phosphorylation of ROMK. Patch-clamp experiments demonstrated that low K intake decreased mean product of channel number (N) and open probability (P) (NP(o)) of ROMK channels from 1.1 to 0.4 in the CCD. However, the effect of low K intake on ROMK channel activity was significantly attenuated in the CCD from gp91(-/-) mice and completely abolished by tempol treatment. Immunocytochemical staining also was used to examine the ROMK distribution in WT, gp91(-/-), and WT mice with tempol treatment in response to K restriction. K restriction decreased apical staining of ROMK in WT mice. In contrast, a sharp apical ROMK staining was observed in the tempol-treated WT or gp91(-/-) mice. Metabolic cage study further showed that urinary K loss is significantly higher in gp91(-/-) mice than in WT mice. It is concluded that superoxide anions play a key role in suppressing K secretion during K restriction and that NOXII is involved in mediating the effect of low K intake on renal K secretion and ROMK channel activity.

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

    Energy Technology Data Exchange (ETDEWEB)

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

    1990-05-01

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

  16. Ionic channel mechanisms mediating the intrinsic excitability of Kenyon cells in the mushroom body of the cricket brain.

    Science.gov (United States)

    Inoue, Shigeki; Murata, Kaoru; Tanaka, Aiko; Kakuta, Eri; Tanemura, Saori; Hatakeyama, Shiori; Nakamura, Atsunao; Yamamoto, Chihiro; Hasebe, Masaharu; Kosakai, Kumiko; Yoshino, Masami

    2014-09-01

    Intrinsic neurons within the mushroom body of the insect brain, called Kenyon cells, play an important role in olfactory associative learning. In this study, we examined the ionic mechanisms mediating the intrinsic excitability of Kenyon cells in the cricket Gryllus bimaculatus. A perforated whole-cell clamp study using β-escin indicated the existence of several inward and outward currents. Three types of inward currents (INaf, INaP, and ICa) were identified. The transient sodium current (INaf) activated at -40 mV, peaked at -26 mV, and half-inactivated at -46.7 mV. The persistent sodium current (INaP) activated at -51 mV, peaked at -23 mV, and half-inactivated at -30.7 mV. Tetrodotoxin (TTX; 1 μM) completely blocked both INaf and INaP, but 10nM TTX blocked INaf more potently than INaP. Cd(2+) (50 μM) potently blocked INaP with little effect on INaf. Riluzole (>20 μM) nonselectively blocked both INaP and INaf. The voltage-dependent calcium current (ICa) activated at -30 mV, peaked at -11.3 mV, and half-inactivated at -34 mV. The Ca(2+) channel blocker verapamil (100 μM) blocked ICa in a use-dependent manner. Cell-attached patch-clamp recordings showed the presence of a large-conductance Ca(2+)-activated K(+) (BK) channel, and the activity of this channel was decreased by removing the extracellular Ca(2+) or adding verapamil or nifedipine, and increased by adding the Ca(2+) agonist Bay K8644, indicating that Ca(2+) entry via the L-type Ca(2+) channel regulates BK channel activity. Under the current-clamp condition, membrane depolarization generated membrane oscillations in the presence of 10nM TTX or 100 μM riluzole in the bath solution. These membrane oscillations disappeared with 1 μM TTX, 50 μM Cd(2+), replacement of external Na(+) with choline, and blockage of Na(+)-activated K(+) current (IKNa) with 50 μM quinidine, indicating that membrane oscillations are primarily mediated by INaP in cooperation with IKNa. The plateau potentials observed either in

  17. Radar Waveform Design in Active Communications Channel

    OpenAIRE

    Ric A. Romero; Shepherd, Kevin D.

    2013-01-01

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

  18. 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

  19. Evaluating Active U: an internet-mediated physical activity program

    Directory of Open Access Journals (Sweden)

    Goodrich David E

    2009-09-01

    Full Text Available Abstract Background Engaging in regular physical activity can be challenging, particularly during the winter months. To promote physical activity at the University of Michigan during the winter months, an eight-week Internet-mediated program (Active U was developed providing participants with an online physical activity log, goal setting, motivational emails, and optional team participation and competition. Methods This study is a program evaluation of Active U. Approximately 47,000 faculty, staff, and graduate students were invited to participate in the online Active U intervention in the winter of 2007. Participants were assigned a physical activity goal and were asked to record each physical activity episode into the activity log for eight weeks. Statistics for program reach, effectiveness, adoption, and implementation were calculated using the Re-Aim framework. Multilevel regression analyses were used to assess the decline in rates of data entry and goal attainment during the program, to assess the likelihood of joining a team by demographic characteristics, to test the association between various predictors and the number of weeks an individual met his or her goal, and to analyze server load. Results Overall, 7,483 individuals registered with the Active U website (≈16% of eligible, and 79% participated in the program by logging valid data at least once. Staff members, older participants, and those with a BMI P Conclusion Internet-mediated physical activity interventions that focus on physical activity logging and goal setting while incorporating team competition may help a significant percentage of the target population maintain their physical activity during the winter months.

  20. Ligand-Gated Ion Channels: Permeation and Activation1

    Science.gov (United States)

    Lynch, Joseph W.; Barry, Peter H.

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

  1. T Cell Receptor Mediated Calcium Entry Requires Alternatively Spliced Cav1.1 Channels.

    Directory of Open Access Journals (Sweden)

    Didi Matza

    Full Text Available The process of calcium entry in T cells is a multichannel and multi-step process. We have studied the requirement for L-type calcium channels (Cav1.1 α1S subunits during calcium entry after TCR stimulation. High expression levels of Cav1.1 channels were detected in activated T cells. Sequencing and cloning of Cav1.1 channel cDNA from T cells revealed that a single splice variant is expressed. This variant lacks exon 29, which encodes the linker region adjacent to the voltage sensor, but contains five new N-terminal exons that substitute for exons 1 and 2, which are found in the Cav1.1 muscle counterpart. Overexpression studies using cloned T cell Cav1.1 in 293HEK cells (that lack TCR suggest that the gating of these channels was altered. Knockdown of Cav1.1 channels in T cells abrogated calcium entry after TCR stimulation, suggesting that Cav1.1 channels are controlled by TCR signaling.

  2. Adrenaline-induced colonic K+ secretion is mediated by KCa1.1 (BK) channels

    DEFF Research Database (Denmark)

    Sørensen, Mads Vaarby; Sausbier, Matthias; Ruth, Peter

    2010-01-01

    secretory K(+) channel in the apical membrane of the murine distal colon. The BK channel is responsible for both resting and Ca(2+)-activated colonic K(+) secretion and is up-regulated by aldosterone. Agonists (e.g. adrenaline) that elevate cAMP are potent activators of distal colonic K(+) secretion....... However, the secretory K(+) channel responsible for cAMP-induced K(+) secretion remains to be defined. In this study we used the Ussing chamber to identify adrenaline-induced electrogenic K(+) secretion. We found that the adrenaline-induced electrogenic ion secretion is a compound effect dominated...... by anion secretion and a smaller electrically opposing K(+) secretion. Using tissue from (i) BK wildtype (BK(+/+)) and knockout (BK(/)) and (ii) cystic fibrosis transmembrane regulator (CFTR) wildtype (CFTR(+/+)) and knockout (CFTR(/)) mice we were able to isolate the adrenaline-induced K(+) secretion. We...

  3. The intact CFTR protein mediates ATPase rather than adenylate kinase activity.

    Science.gov (United States)

    Ramjeesingh, Mohabir; Ugwu, Francisca; Stratford, Fiona L L; Huan, Ling-Jun; Li, Canhui; Bear, Christine E

    2008-06-01

    The two NBDs (nucleotide-binding domains) of ABC (ATP-binding-cassette) proteins function in a complex to mediate ATPase activity and this activity has been linked to their regulated transport activity. A similar model has been proposed for CFTR (cystic fibrosis transmembrane conductance regulator), the chloride channel defective in cystic fibrosis, wherein ATP binding and hydrolysis regulate the channel gate. Recently, it was shown that the individual NBDs isolated from CFTR primarily mediate adenylate kinase activity, raising the possibility that this activity may also contribute to gating of the CFTR channel. However, this present study shows that whereas the isolated NBDs exhibit adenylate kinase activity, the full-length purified and reconstituted CFTR protein functions as an ATPase, arguing that the enzymatic activity of the NBDs is dependent on their molecular context and appropriate domain-domain assembly. As expected, the disease-causing mutant bearing a mutation in the ABC signature motif, CFTR-G551D, exhibited a markedly reduced ATPase activity. Furthermore, mutation of the putative catalytic base in CFTR caused a reduction in ATPase activity, with the CFTR-E1371Q mutant supporting a low level of residual activity. Neither of these mutants exhibited detectable adenylate kinase activity. Together, these findings support the concept that the molecular mechanism of action of CFTR is dependent on ATP binding and hydrolysis, and that the structure of prokaryotic ABC ATPases provide a useful template for understanding their mechanism of action.

  4. 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.

  5. Small conductance Ca2+-activated K+ channels regulate firing properties and excitability in parasympathetic cardiac motoneurons in the nucleus ambiguus.

    Science.gov (United States)

    Lin, Min; Hatcher, Jeff T; Chen, Qin-Hui; Wurster, Robert D; Cheng, Zixi Jack

    2010-12-01

    Small conductance Ca(2+)-activated K(+) channels (SK) regulate action potential (AP) firing properties and excitability in many central neurons. However, the functional roles of SK channels of parasympathetic cardiac motoneurons (PCMNs) in the nucleus ambiguus have not yet been well characterized. In this study, the tracer X-rhodamine-5 (and 6)-isothiocyanate (XRITC) was injected into the pericardial sac to retrogradely label PCMNs in FVB mice at postnatal days 7-9. Two days later, XRITC-labeled PCMNs in brain stem slices were identified. With the use of whole cell current clamp, single APs and spike trains of different frequencies were evoked by current injections. We found that 1) PCMNs have two different firing patterns: the majority of PCMNs (90%) exhibited spike frequency adaptation (SFA) and the rest (10%) showed less or no adaptation; 2) application of the specific SK channel blocker apamin significantly increased spike half-width in single APs and trains and reduced the spike frequency-dependent AP broadening in trains; 3) SK channel blockade suppressed afterhyperpolarization (AHP) amplitude following single APs and trains and abolished spike-frequency dependence of AHP in trains; and 4) SK channel blockade increased the spike frequency but did not alter the pattern of SFA. Using whole cell voltage clamp, we measured outward currents and afterhyperpolarization current (I(AHP)). SK channel blockade revealed that SK-mediated outward currents had both transient and persistent components. After bath application of apamin and Ca(2+)-free solution, we found that apamin-sensitive and Ca(2+)-sensitive I(AHP) were comparable, confirming that SK channels may contribute to a major portion of Ca(2+)-activated K(+) channel-mediated I(AHP). These results suggest that PCMNs have SK channels that significantly regulate AP repolarization, AHP, and spike frequency but do not affect SFA. We conclude that activation of SK channels underlies one of the mechanisms for negative

  6. 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.

  7. KCNQ4 channel activation by BMS-204352 and retigabine

    DEFF Research Database (Denmark)

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

    2001-01-01

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

  8. 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.

  9. Contribution of Kv7 channels to natriuretic peptide mediated vasodilation in normal and hypertensive rats

    DEFF Research Database (Denmark)

    Stott, Jennifer B; Barrese, Vincenzo; Jepps, Thomas Andrew;

    2015-01-01

    -cAMP-linked vasodilator pathways has not been investigated. Natriuretic peptides are potent vasodilators, which operate primarily through the activation of a cGMP-dependent signaling pathway. This study investigated the putative role of Kv7 channels in natriuretic peptide-dependent relaxations in the vasculature...

  10. Shaping of action potentials by type I and type II large-conductance Ca²+-activated K+ channels.

    Science.gov (United States)

    Jaffe, D B; Wang, B; Brenner, R

    2011-09-29

    The BK channel is a Ca(2+) and voltage-gated conductance responsible for shaping action potential waveforms in many types of neurons. Type II BK channels are differentiated from type I channels by their pharmacology and slow gating kinetics. The β4 accessory subunit confers type II properties on BK α subunits. Empirically derived properties of BK channels, with and without the β4 accessory subunit, were obtained using a heterologous expression system under physiological ionic conditions. These data were then used to study how BK channels alone (type I) and with the accessory β4 subunit (type II) modulate action potential properties in biophysical neuron models. Overall, the models support the hypothesis that it is the slower kinetics provided by the β4 subunit that endows the BK channel with type II properties, which leads to broadening of action potentials and, secondarily, to greater recruitment of SK channels reducing neuronal excitability. Two regions of parameter space distinguished type II and type I effects; one where the range of BK-activating Ca(2+) was high (>20 μM) and the other where BK-activating Ca(2+) was low (∼0.4-1.2 μM). The latter required an elevated BK channel density, possibly beyond a likely physiological range. BK-mediated sharpening of the spike waveform associated with the lack of the β4 subunit was sensitive to the properties of voltage-gated Ca(2+) channels due to electrogenic effects on spike duration. We also found that depending on Ca(2+) dynamics, type II BK channels may have the ability to contribute to the medium AHP, a property not generally ascribed to BK channels, influencing the frequency-current relationship. Finally, we show how the broadening of action potentials conferred by type II BK channels can also indirectly increase the recruitment of SK-type channels decreasing the excitability of the neuron.

  11. 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.

  12. Role of the S4-S5 linker in CNG channel activation.

    Science.gov (United States)

    Kusch, Jana; Zimmer, Thomas; Holschuh, Jascha; Biskup, Christoph; Schulz, Eckhard; Nache, Vasilica; Benndorf, Klaus

    2010-10-20

    Cyclic nucleotide-gated (CNG) channels mediate sensory signal transduction in retinal and olfactory cells. The channels are activated by the binding of cyclic nucleotides to a cyclic nucleotide-binding domain (CNBD) in the C-terminus that is located at the intracellular side. The molecular events translating the ligand binding to the pore opening are still unknown. We investigated the role of the S4-S5 linker in the activation process by quantifying its interaction with other intracellular regions. To this end, we constructed chimeric channels in which the N-terminus, the S4-S5 linker, the C-linker, and the CNBD of the retinal CNGA1 subunit were systematically replaced by the respective regions of the olfactory CNGA2 subunit. Macroscopic concentration-response relations were analyzed, yielding the apparent affinity to cGMP and the Hill coefficient. The degree of functional coupling of intracellular regions in the activation gating was determined by thermodynamic double-mutant cycle analysis. We observed that all four intracellular regions, including the relatively short S4-S5 linker, are involved in controlling the apparent affinity of the channel to cGMP and, moreover, in determining the degree of cooperativity between the subunits, as derived from the Hill coefficient. The interaction energies reveal an interaction of the S4-S5 linker with both the N-terminus and the C-linker, but no interaction with the CNBD.

  13. TRPC1 regulates calcium-activated chloride channels in salivary gland cells.

    Science.gov (United States)

    Sun, Yuyang; Birnbaumer, Lutz; Singh, Brij B

    2015-11-01

    Calcium-activated chloride channel (CaCC) plays an important role in modulating epithelial secretion. It has been suggested that in salivary tissues, sustained fluid secretion is dependent on Ca(2+) influx that activates ion channels such as CaCC to initiate Cl(-) efflux. However direct evidence as well as the molecular identity of the Ca(2+) channel responsible for activating CaCC in salivary tissues is not yet identified. Here we provide evidence that in human salivary cells, an outward rectifying Cl(-) current was activated by increasing [Ca(2+)]i, which was inhibited by the addition of pharmacological agents niflumic acid (NFA), an antagonist of CaCC, or T16Ainh-A01, a specific TMEM16a inhibitor. Addition of thapsigargin (Tg), that induces store-depletion and activates TRPC1-mediated Ca(2+) entry, potentiated the Cl(-) current, which was inhibited by the addition of a non-specific TRPC channel blocker SKF96365 or removal of external Ca(2+). Stimulation with Tg also increased plasma membrane expression of TMEM16a protein, which was also dependent on Ca(2+) entry. Importantly, in salivary cells, TRPC1 silencing, but not that of TRPC3, inhibited CaCC especially upon store depletion. Moreover, primary acinar cells isolated from submandibular gland also showed outward rectifying Cl(-) currents upon increasing [Ca(2+)]i. These Cl(-) currents were again potentiated with the addition of Tg, but inhibited in the presence of T16Ainh-A01. Finally, acinar cells isolated from the submandibular glands of TRPC1 knockout mice showed significant inhibition of the outward Cl(-) currents without decreasing TMEM16a expression. Together the data suggests that Ca(2+) entry via the TRPC1 channels is essential for the activation of CaCC.

  14. SUMOylation of the Hyperpolarization-Activated Cyclic Nucleotide-Gated Channel 2 Increases Surface Expression and the Maximal Conductance of the Hyperpolarization-Activated Current

    Science.gov (United States)

    Parker, Anna R.; Welch, Meghyn A.; Forster, Lori A.; Tasneem, Sarah M.; Dubhashi, Janhavi A.; Baro, Deborah J.

    2017-01-01

    Small Ubiquitin-like Modifier (SUMO) is a ∼10 kDa peptide that can be post-translationally added to a lysine (K) on a target protein to facilitate protein–protein interactions. Recent studies have found that SUMOylation can be regulated in an activity-dependent manner and that ion channel SUMOylation can alter the biophysical properties and surface expression of the channel. Hyperpolarization-activated cyclic nucleotide-gated (HCN) channel surface expression can be regulated in an activity-dependent manner through unknown processes. We hypothesized that SUMOylation might influence the surface expression of HCN2 channels. In this manuscript, we show that HCN2 channels are SUMOylated in the mouse brain. Baseline levels of SUMOylation were also observed for a GFP-tagged HCN2 channel stably expressed in Human embryonic kidney (Hek) cells. Elevating GFP-HCN2 channel SUMOylation above baseline in Hek cells led to an increase in surface expression that augmented the hyperpolarization-activated current (Ih) mediated by these channels. Increased SUMOylation did not alter Ih voltage-dependence or kinetics of activation. There are five predicted intracellular SUMOylation sites on HCN2. Site-directed mutagenesis indicated that more than one K on the GFP-HCN2 channel was SUMOylated. Enhancing SUMOylation at one of the five predicted sites, K669, led to the increase in surface expression and Ih Gmax. The role of SUMOylation at additional sites is currently unknown. The SUMOylation site at K669 is also conserved in HCN1 channels. Aberrant SUMOylation has been linked to neurological diseases that also display alterations in HCN1 and HCN2 channel expression, such as seizures and Parkinson’s disease. This work is the first report that HCN channels can be SUMOylated and that this can regulate surface expression and Ih. PMID:28127275

  15. A monomer is the minimum functional unit required for channel and ATPase activity of the cystic fibrosis transmembrane conductance regulator.

    Science.gov (United States)

    Ramjeesingh, M; Li, C; Kogan, I; Wang, Y; Huan, L J; Bear, C E

    2001-09-04

    The cystic fibrosis transmembrane conductance regulator (CFTR) normally functions as a phosphorylation-regulated chloride channel on the apical surface of epithelial cells, and lack of this function is the primary cause for the fatal disease cystic fibrosis (CF). Previous studies showed that purified, reconstituted CFTR can function as a chloride channel and, further, that its intrinsic ATPase activity is required to regulate opening and closing of the channel gate. However, these previous studies did not identify the quaternary structure required to mediate conduction and catalysis. Our present studies show that CFTR molecules may self-associate in CHO and Sf9 membranes, as complexes close to the predicted size of CFTR dimers can be captured by chemical cross-linking reagents and detected using nondissociative PAGE. However, CFTR function does not require a multimeric complex for function as we determined that purified, reconstituted CFTR monomers are sufficient to mediate regulated chloride conduction and ATPase activity.

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

    Science.gov (United States)

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

    2016-09-01

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

  17. The role of DPO-1 and XE991-sensitive potassium channels in perivascular adipose tissue-mediated regulation of vascular tone

    Directory of Open Access Journals (Sweden)

    Dmitry Tsvetkov

    2016-08-01

    Full Text Available The anti-contractile effect of perivascular adipose tissue (PVAT is an important mechanism in the modulation of vascular tone in peripheral arteries. Recent evidence has implicated the XE991-sensitive voltage-gated Kv (KCNQ channels in the regulation of arterial tone by PVAT. However, until now the in vivo pharmacology of the involved vascular Kv channels with regard to XE991 remains undetermined, since XE991 effects may involve Ca2+ activated BKCa channels and/or voltage-dependent Kv1.5 channels sensitive to diphenyl phosphine oxide-1 (DPO-1. In this study, we tested whether Kv1.5 channels are involved in the control of mesenteric arterial tone and its regulation by PVAT. Our study was also aimed at extending our current knowledge on the in situ vascular pharmacology of DPO-1 and XE991 regarding Kv1.5 and BKCa channels, in helping to identify the nature of K+ channels that could contribute to PVAT-mediated relaxation. XE991 at 30 µM reduced the anti-contractile response of PVAT, but had no effects on vasocontraction induced by phenylephrine (PE in the absence of PVAT. Similar effects were observed for XE991 at 0.3 µM, which is known to almost completely inhibit mesenteric artery VSMC Kv currents. 30 µM XE991 did not affect BKCa currents in VSMCs. Kcna5-/- arteries and wild-type arteries incubated with 1 µM DPO-1 showed normal vasocontractions in response to PE in the presence and absence of PVAT. Kv current density and inhibition by 30 µM XE991 were normal in mesenteric artery VSMCs isolated from Kcna5-/- mice. We conclude that Kv channels are involved in the control of arterial vascular tone by PVAT. These channels are present in VSMCs and very potently inhibited by the KCNQ channel blocker XE991. BKCa channels and/or DPO-1 sensitive Kv1.5 channels in VSMCs are not the downstream mediators of the XE991 effects on PVAT-dependent arterial vasorelaxation. Further studies will need to be undertaken to examine the role of other Kv channels in

  18. Increased anion channel activity is an unavoidable event in ozone-induced programmed cell death.

    Directory of Open Access Journals (Sweden)

    Takashi Kadono

    Full Text Available BACKGROUND: Ozone is a major secondary air pollutant often reaching high concentrations in urban areas under strong daylight, high temperature and stagnant high-pressure systems. Ozone in the troposphere is a pollutant that is harmful to the plant. PRINCIPAL FINDINGS: By exposing cells to a strong pulse of ozonized air, an acute cell death was observed in suspension cells of Arabidopsis thaliana used as a model. We demonstrated that O(3 treatment induced the activation of a plasma membrane anion channel that is an early prerequisite of O(3-induced cell death in A. thaliana. Our data further suggest interplay of anion channel activation with well known plant responses to O(3, Ca(2+ influx and NADPH-oxidase generated reactive oxygen species (ROS in mediating the oxidative cell death. This interplay might be fuelled by several mechanisms in addition to the direct ROS generation by O(3; namely, H(2O(2 generation by salicylic and abscisic acids. Anion channel activation was also shown to promote the accumulation of transcripts encoding vacuolar processing enzymes, a family of proteases previously reported to contribute to the disruption of vacuole integrity observed during programmed cell death. SIGNIFICANCE: Collectively, our data indicate that anion efflux is an early key component of morphological and biochemical events leading to O(3-induced programmed cell death. Because ion channels and more specifically anion channels assume a crucial position in cells, an understanding about the underlying role(s for ion channels in the signalling pathway leading to programmed cell death is a subject that warrants future investigation.

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

    Science.gov (United States)

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

    2015-01-01

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

  20. DYRK1A-mediated phosphorylation of GluN2A at Ser1048 regulates the surface expression and channel activity of GluN1/GluN2A receptors

    Directory of Open Access Journals (Sweden)

    Cristina eGrau

    2014-10-01

    Full Text Available N-methyl-D-aspartate glutamate receptors (NMDARs play a pivotal role in neural development and synaptic plasticity, as well as in neurological disease. Since NMDARs exert their function at the cell surface, their density in the plasma membrane is finely tuned by a plethora of molecules that regulate their production, trafficking, docking and internalization in response to external stimuli. In addition to transcriptional regulation, the density of NMDARs is also influenced by post-translational mechanisms like phosphorylation, a modification that also affects their biophysical properties. We previously described the increased surface expression of GluN1/GluN2A receptors in transgenic mice overexpressing the Dual specificity tyrosine-phosphorylation-regulated kinase 1A (DYRK1A, suggesting that DYRK1A regulates NMDARs. Here we have further investigated whether the density and activity of NMDARs was modulated by DYRK1A phosphorylation. Accordingly, we show that endogenous DYRK1A is recruited to GluN2A-containing NMDARs in the adult mouse brain, and we identify a DYRK1A phosphorylation site at Ser1048 of GluN2A, within its intracellular C-terminal domain. Mechanistically, the DYRK1A-dependent phosphorylation of GluN2A at Ser1048 hinders the internalization of GluN1/GluN2A, causing an increase of surface GluN1/GluN2A in heterologous systems, as well as in primary cortical neurons. Furthermore, GluN2A phosphorylation at Ser1048 increases the current density and potentiates the gating of GluN1/GluN2A receptors. We conclude that DYRK1A is a direct regulator of NMDA receptors and we propose a novel mechanism for the control of NMDAR activity in neurons.

  1. Plasmin in nephrotic urine activates the epithelial sodium channel

    DEFF Research Database (Denmark)

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

    2008-01-01

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

  2. Self-cleavage of Human CLCA1 Protein by a Novel Internal Metalloprotease Domain Controls Calcium-activated Chloride Channel Activation*♦

    Science.gov (United States)

    Yurtsever, Zeynep; Sala-Rabanal, Monica; Randolph, David T.; Scheaffer, Suzanne M.; Roswit, William T.; Alevy, Yael G.; Patel, Anand C.; Heier, Richard F.; Romero, Arthur G.; Nichols, Colin G.; Holtzman, Michael J.; Brett, Tom J.

    2012-01-01

    The chloride channel calcium-activated (CLCA) family are secreted proteins that regulate both chloride transport and mucin expression, thus controlling the production of mucus in respiratory and other systems. Accordingly, human CLCA1 is a critical mediator of hypersecretory lung diseases, such as asthma, chronic obstructive pulmonary disease, and cystic fibrosis, that manifest mucus obstruction. Despite relevance to homeostasis and disease, the mechanism of CLCA1 function remains largely undefined. We address this void by showing that CLCA proteins contain a consensus proteolytic cleavage site recognized by a novel zincin metalloprotease domain located within the N terminus of CLCA itself. CLCA1 mutations that inhibit self-cleavage prevent activation of calcium-activated chloride channel (CaCC)-mediated chloride transport. CaCC activation requires cleavage to unmask the N-terminal fragment of CLCA1, which can independently gate CaCCs. Gating of CaCCs mediated by CLCA1 does not appear to involve proteolytic cleavage of the channel because a mutant N-terminal fragment deficient in proteolytic activity is able to induce currents comparable with that of the native fragment. These data provide both a mechanistic basis for CLCA1 self-cleavage and a novel mechanism for regulation of chloride channel activity specific to the mucosal interface. PMID:23112050

  3. Self-cleavage of human CLCA1 protein by a novel internal metalloprotease domain controls calcium-activated chloride channel activation.

    Science.gov (United States)

    Yurtsever, Zeynep; Sala-Rabanal, Monica; Randolph, David T; Scheaffer, Suzanne M; Roswit, William T; Alevy, Yael G; Patel, Anand C; Heier, Richard F; Romero, Arthur G; Nichols, Colin G; Holtzman, Michael J; Brett, Tom J

    2012-12-07

    The chloride channel calcium-activated (CLCA) family are secreted proteins that regulate both chloride transport and mucin expression, thus controlling the production of mucus in respiratory and other systems. Accordingly, human CLCA1 is a critical mediator of hypersecretory lung diseases, such as asthma, chronic obstructive pulmonary disease, and cystic fibrosis, that manifest mucus obstruction. Despite relevance to homeostasis and disease, the mechanism of CLCA1 function remains largely undefined. We address this void by showing that CLCA proteins contain a consensus proteolytic cleavage site recognized by a novel zincin metalloprotease domain located within the N terminus of CLCA itself. CLCA1 mutations that inhibit self-cleavage prevent activation of calcium-activated chloride channel (CaCC)-mediated chloride transport. CaCC activation requires cleavage to unmask the N-terminal fragment of CLCA1, which can independently gate CaCCs. Gating of CaCCs mediated by CLCA1 does not appear to involve proteolytic cleavage of the channel because a mutant N-terminal fragment deficient in proteolytic activity is able to induce currents comparable with that of the native fragment. These data provide both a mechanistic basis for CLCA1 self-cleavage and a novel mechanism for regulation of chloride channel activity specific to the mucosal interface.

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

    Science.gov (United States)

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

    2007-10-05

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

  5. Pregnenolone sulfate activates basic region leucine zipper transcription factors in insulinoma cells: role of voltage-gated Ca2+ channels and transient receptor potential melastatin 3 channels.

    Science.gov (United States)

    Müller, Isabelle; Rössler, Oliver G; Thiel, Gerald

    2011-12-01

    The neurosteroid pregnenolone sulfate activates a signaling cascade in insulinoma cells involving activation of extracellular signal-regulated protein kinase and enhanced expression of the transcription factor Egr-1. Here, we show that pregnenolone sulfate stimulation leads to a significant elevation of activator protein-1 (AP-1) activity in insulinoma cells. Expression of the basic region leucine zipper (bZIP) transcription factors c-Jun and c-Fos is up-regulated in insulinoma cells and pancreatic β-cells in primary culture after pregnenolone sulfate stimulation. Up-regulation of a chromatin-embedded c-Jun promoter/luciferase reporter gene transcription in pregnenolone sulfate-stimulated insulinoma cells was impaired when the AP-1 binding sites were mutated, indicating that these motifs function as pregnenolone sulfate response elements. In addition, phosphorylation of cAMP response element (CRE)-binding protein is induced and transcription of a CRE-controlled reporter gene is stimulated after pregnenolone sulfate treatment, indicating that the CRE functions as a pregnenolone sulfate response element as well. Pharmacological and genetic experiments revealed that both L-type Ca(2+) channels and transient receptor potential melastatin 3 (TRPM3) channels are essential for connecting pregnenolone sulfate stimulation with enhanced AP-1 activity and bZIP-mediated transcription in insulinoma cells. In contrast, pregnenolone sulfate stimulation did not enhance AP-1 activity or c-Jun and c-Fos expression in pituitary corticotrophs that express functional L-type Ca(2+) channels but only trace amounts of TRPM3. We conclude that expression of L-type Ca(2+) channels is not sufficient to activate bZIP-mediated gene transcription by pregnenolone sulfate. Rather, additional expression of TRPM3 or depolarization of the cells is required to connect pregnenolone sulfate stimulation with enhanced gene transcription.

  6. 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...

  7. The complete structure of an activated open sodium channel

    Science.gov (United States)

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

    2017-01-01

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

  8. Information Systems as Dialectic, Tool - Mediated Activity

    Directory of Open Access Journals (Sweden)

    Helen Hasan

    2002-11-01

    Full Text Available Information Systems (IS draws its significance from the uniqueness of computer-based information and communications tools and their place in shaping recent human history. Advances in the field come from a better understanding of how to develop and use these tools and what impact they have on the way we work, and live. As IS is still an evolving field of study, two views, the objective and the subjective, are in constant tension and, though these may be considered complementary, it is rare that they come together as a unified whole. A more balanced, and integrated, foundation for IS may be found in the subject-object dialectic arising out of the German philosophical tradition. An extension of this approach from the Cultural-Historical Activity Theory is presented in this paper. This theory views all human endeavour as a purposeful, dynamic, dialectic relationship between subject and object, mediated by tools, such as technology and information, and by the social environment or community. An adaptation of this holistic theory, to incorporate the best of other theoretical approaches used by IS researchers, could span and integrate the breadth of the field IS providing it with unity and identity.

  9. 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.

  10. Atomic basis for therapeutic activation of neuronal potassium channels

    DEFF Research Database (Denmark)

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

    2015-01-01

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

  11. NS19504: a novel BK channel activator with relaxing effect on bladder smooth muscle spontaneous phasic contractions.

    Science.gov (United States)

    Nausch, Bernhard; Rode, Frederik; Jørgensen, Susanne; Nardi, Antonio; Korsgaard, Mads P G; Hougaard, Charlotte; Bonev, Adrian D; Brown, William D; Dyhring, Tino; Strøbæk, Dorte; Olesen, Søren-Peter; Christophersen, Palle; Grunnet, Morten; Nelson, Mark T; Rønn, Lars C B

    2014-09-01

    Large-conductance Ca(2+)-activated K(+) channels (BK, KCa1.1, MaxiK) are important regulators of urinary bladder function and may be an attractive therapeutic target in bladder disorders. In this study, we established a high-throughput fluorometric imaging plate reader-based screening assay for BK channel activators and identified a small-molecule positive modulator, NS19504 (5-[(4-bromophenyl)methyl]-1,3-thiazol-2-amine), which activated the BK channel with an EC50 value of 11.0 ± 1.4 µM. Hit validation was performed using high-throughput electrophysiology (QPatch), and further characterization was achieved in manual whole-cell and inside-out patch-clamp studies in human embryonic kidney 293 cells expressing hBK channels: NS19504 caused distinct activation from a concentration of 0.3 and 10 µM NS19504 left-shifted the voltage activation curve by 60 mV. Furthermore, whole-cell recording showed that NS19504 activated BK channels in native smooth muscle cells from guinea pig urinary bladder. In guinea pig urinary bladder strips, NS19504 (1 µM) reduced spontaneous phasic contractions, an effect that was significantly inhibited by the specific BK channel blocker iberiotoxin. In contrast, NS19504 (1 µM) only modestly inhibited nerve-evoked contractions and had no effect on contractions induced by a high K(+) concentration consistent with a K(+) channel-mediated action. Collectively, these results show that NS19504 is a positive modulator of BK channels and provide support for the role of BK channels in urinary bladder function. The pharmacologic profile of NS19504 indicates that this compound may have the potential to reduce nonvoiding contractions associated with spontaneous bladder overactivity while having a minimal effect on normal voiding.

  12. Distance-dependent homeostatic synaptic scaling mediated by A-type potassium channels

    Directory of Open Access Journals (Sweden)

    Hiroshi T Ito

    2009-11-01

    Full Text Available Many lines of evidence suggest that the efficacy of synapses on CA1 pyramidal neuron dendrites increases as a function of distance from the cell body. The strength of an individual synapse is also dynamically modulated by activity-dependent synaptic plasticity, which raises the question as to how a neuron can reconcile individual synaptic changes with the maintenance of the proximal-to-distal gradient of synaptic strength along the dendrites. As the density of A-type potassium channels exhibits a similar gradient from proximal (low-to-distal (high dendrites, the A-current may play a role in coordinating local synaptic changes with the global synaptic strength gradient. Here we describe a form of homeostatic plasticity elicited by conventional activity blockade (with TTX coupled with a block of the A-type potassium channel. Following A-type potassium channel inhibition for 12 hrs, recordings from CA1 somata revealed a significantly higher miniature excitatory postsynaptic current (mEPSC frequency, whereas in dendritic recordings, there was no change in mEPSC frequency. Consistent with mEPSC recordings, we observed a significant increase in AMPA receptor density in stratum pyramidale but not stratum radiatum. Based on these data, we propose that the differential distribution of A-type potassium channels along the apical dendrites may create a proximal-to-distal membrane potential gradient. This gradient may regulate AMPA receptor distribution along the same axis. Taken together, our results indicate that A-type potassium channels play an important role in controlling synaptic strength along the dendrites, which may help to maintain the computational capacity of the neuron.

  13. Protection of protease-activated receptor 2 mediated vasodilatation against angiotensin II-induced vascular dysfunction in mice

    OpenAIRE

    Chia, Elizabeth; Kagota, Satomi; Wijekoon, Enoka P; McGuire, John J

    2011-01-01

    Background Under conditions of cardiovascular dysfunction, protease-activated receptor 2 (PAR2) agonists maintain vasodilatation activity, which has been attributed to increased cyclooxygenase-2, nitric oxide synthase and calcium-activated potassium channel (SK3.1) activities. Protease-activated receptor 2 agonist mediated vasodilatation is unknown under conditions of dysfunction caused by angiotensin II. The main purpose of our study was to determine whether PAR2-induced vasodilatation of re...

  14. 980-nm infrared laser modulation of sodium channel kinetics in a neuron cell linearly mediated by photothermal effect

    Science.gov (United States)

    Li, Xinyu; Liu, Jia; Liang, Shanshan; Sun, Changsen

    2014-10-01

    Photothermal effect (PE) plays a major role in the near-infrared laser interaction with biological tissue. But, quite few interactions can be quantitatively depicted. Here, a two-step model is proposed to describe a 980-nm infrared laser interaction with neuron cell in vitro. First, the laser-induced temperature rises in the cell surrounding area were measured by using an open pipette method and also calculated by solving the heat conduction equation. Second, we recorded the modifications on sodium (Na) channel current in neuron cells directly by using a patch clamp to synchronize the 980-nm laser irradiation and obtained how the electrophysiological function of neuron cells respond to the temperature rise. Then, the activation time constants, τm, were extracted by fitting the sodium currents with the Hodgkin-Huxley model. The infrared laser modulation effect on sodium currents kinetics was examined by taking a ratio between the time constants with and without the laser irradiations. The analysis revealed that the averaged ratio at a specific laser exposure could be well related to the temperature properties of the Na channel protein. These results proved that the modulation of sodium current kinetics of a neuron cell in vitro by 980-nm laser with different-irradiation levels was linearly mediated corresponding to the laser-induced PE.

  15. AtHKT1;1 mediates nernstian sodium channel transport properties in Arabidopsis root stelar cells.

    Directory of Open Access Journals (Sweden)

    Shaowu Xue

    Full Text Available The Arabidopsis AtHKT1;1 protein was identified as a sodium (Na⁺ transporter by heterologous expression in Xenopus laevis oocytes and Saccharomyces cerevisiae. However, direct comparative in vivo electrophysiological analyses of a plant HKT transporter in wild-type and hkt loss-of-function mutants has not yet been reported and it has been recently argued that heterologous expression systems may alter properties of plant transporters, including HKT transporters. In this report, we analyze several key functions of AtHKT1;1-mediated ion currents in their native root stelar cells, including Na⁺ and K⁺ conductances, AtHKT1;1-mediated outward currents, and shifts in reversal potentials in the presence of defined intracellular and extracellular salt concentrations. Enhancer trap Arabidopsis plants with GFP-labeled root stelar cells were used to investigate AtHKT1;1-dependent ion transport properties using patch clamp electrophysiology in wild-type and athkt1;1 mutant plants. AtHKT1;1-dependent currents were carried by sodium ions and these currents were not observed in athkt1;1 mutant stelar cells. However, K⁺ currents in wild-type and athkt1;1 root stelar cell protoplasts were indistinguishable correlating with the Na⁺ over K⁺ selectivity of AtHKT1;1-mediated transport. Moreover, AtHKT1;1-mediated currents did not show a strong voltage dependence in vivo. Unexpectedly, removal of extracellular Na⁺ caused a reduction in AtHKT1;1-mediated outward currents in Columbia root stelar cells and Xenopus oocytes, indicating a role for external Na⁺ in regulation of AtHKT1;1 activity. Shifting the NaCl gradient in root stelar cells showed a Nernstian shift in the reversal potential providing biophysical evidence for the model that AtHKT1;1 mediates passive Na⁺ channel transport properties.

  16. Experience with ActiveX control for simple channel access

    Energy Technology Data Exchange (ETDEWEB)

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

    2003-05-15

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

  17. [Properties of cholinergic receptor-mediated ion channels on type I vestibular hair cells of guinea pigs].

    Science.gov (United States)

    Zhu, Yun; Kong, Wei-Jia; Xia, Jiao; Zhang, Yu; Cheng, Hua-Mao; Guo, Chang-Kai

    2008-06-25

    To confirm the existence of cholinergic receptors on type I vestibular hair cells (VHCs I) of guinea pigs and to study the properties of the cholinergic receptor-mediated ion channels on VHCs I, electrophysiological responses of isolated VHCs I to external ACh were examined by means of whole-cell patch-clamp recordings. The results showed that 7.5% (21/279) VHCs I were found to be sensitive to ACh (10-1000 μmol/L). ACh generated an outward current in a steady, slow, dose-dependent [EC(50) was (63.78±2.31) μmol/L] and voltage-independent manner. In standard extracellular solution, ACh at the concentration of 100 μmol/L triggered a calcium-dependent current of (170±15) pA at holding potential of -50 mV, and the current amplitude could be depressed by extracellularly added calcium-dependent potassium channel antagonist TEA. The time interval for the next complete activation of ACh-sensitive current was no less than 1 min. The ion channels did not shut off even when they were exposed to ACh for an extended period of time (8 min). The results suggest that dose-dependent, calcium-dependent and voltage-independent cholinergic receptors were located on a few of the VHCs I investibular epithelium of guinea pigs. The cholinergic receptors did not show desensitization to ACh. This work reveals the existence of efferent neurotransmitter receptors on VHCs I and helps in understanding the function of vestibular efferent nervous system, and may provide some useful information on guiding the clinical rehabilitative treatment of vertigo.

  18. MicroRNA-Mediated Downregulation of the Potassium Channel Kv4.2 Contributes to Seizure Onset

    Directory of Open Access Journals (Sweden)

    Christina Gross

    2016-09-01

    Full Text Available Seizures are bursts of excessive synchronized neuronal activity, suggesting that mechanisms controlling brain excitability are compromised. The voltage-gated potassium channel Kv4.2, a major mediator of hyperpolarizing A-type currents in the brain, is a crucial regulator of neuronal excitability. Kv4.2 expression levels are reduced following seizures and in epilepsy, but the underlying mechanisms remain unclear. Here, we report that Kv4.2 mRNA is recruited to the RNA-induced silencing complex shortly after status epilepticus in mice and after kainic acid treatment of hippocampal neurons, coincident with reduction of Kv4.2 protein. We show that the microRNA miR-324-5p inhibits Kv4.2 protein expression and that antagonizing miR-324-5p is neuroprotective and seizure suppressive. MiR-324-5p inhibition also blocks kainic-acid-induced reduction of Kv4.2 protein in vitro and in vivo and delays kainic-acid-induced seizure onset in wild-type but not in Kcnd2 knockout mice. These results reveal an important role for miR-324-5p-mediated silencing of Kv4.2 in seizure onset.

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

    LENUS (Irish Health Repository)

    Irnaten, Mustapha

    2009-01-01

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

  20. T-type Ca(2+) channels facilitate NO-formation, vasodilatation and NO-mediated modulation of blood pressure

    DEFF Research Database (Denmark)

    Svenningsen, Per; Andersen, Kenneth; Thuesen, Anne D

    2014-01-01

    Voltage-gated calcium channels are involved in the vascular excitation-contraction mechanism and regulation of arterial blood pressure. It was hypothesized that T-type channels promote formation of nitric oxide from the endothelium. The present experiments determine the involvement of T-type chan......Voltage-gated calcium channels are involved in the vascular excitation-contraction mechanism and regulation of arterial blood pressure. It was hypothesized that T-type channels promote formation of nitric oxide from the endothelium. The present experiments determine the involvement of T......-type channels in depolarization-dependent dilatation of mesenteric arteries and blood pressure regulation in Cav3.1 knock-out mice. Nitric oxide-dependent vasodilatation following depolarization-mediated vasoconstriction was reduced significantly in mesenteric arteries from Cav3.1(-/-) compared to wild type...

  1. Drosophila mushroom body Kenyon cells generate spontaneous calcium transients mediated by PLTX-sensitive calcium channels.

    Science.gov (United States)

    Jiang, Shaojuan Amy; Campusano, Jorge M; Su, Hailing; O'Dowd, Diane K

    2005-07-01

    Spontaneous calcium oscillations in mushroom bodies of late stage pupal and adult Drosophila brains have been implicated in memory consolidation during olfactory associative learning. This study explores the cellular mechanisms regulating calcium dynamics in Kenyon cells, principal neurons in mushroom bodies. Fura-2 imaging shows that Kenyon cells cultured from late stage Drosophila pupae generate spontaneous calcium transients in a cell autonomous fashion, at a frequency similar to calcium oscillations in vivo (10-20/h). The expression of calcium transients is up regulated during pupal development. Although the ability to generate transients is a property intrinsic to Kenyon cells, transients can be modulated by bath application of nicotine and GABA. Calcium transients are blocked, and baseline calcium levels reduced, by removal of external calcium, addition of cobalt, or addition of Plectreurys toxin (PLTX), an insect-specific calcium channel antagonist. Transients do not require calcium release from intracellular stores. Whole cell recordings reveal that the majority of voltage-gated calcium channels in Kenyon cells are PLTX-sensitive. Together these data show that influx of calcium through PLTX-sensitive voltage-gated calcium channels mediates spontaneous calcium transients and regulates basal calcium levels in cultured Kenyon cells. The data also suggest that these calcium transients represent cellular events underlying calcium oscillations in the intact mushroom bodies. However, spontaneous calcium transients are not unique to Kenyon cells as they are present in approximately 60% of all cultured central brain neurons. This suggests the calcium transients play a more general role in maturation or function of adult brain neurons.

  2. Activation of steroid-sensitive TRPM3 channels potentiates glutamatergic transmission at cerebellar Purkinje neurons from developing rats.

    Science.gov (United States)

    Zamudio-Bulcock, Paula A; Everett, Julie; Harteneck, Christian; Valenzuela, C Fernando

    2011-11-01

    The functional implications of transient receptor potential melastatin 3 (TRPM3) activation, the most recently described member of the melastatin subfamily of cation permeable TRP channels, have begun to be elucidated in recent years. The discovery of TRPM3 activation by the steroid pregnenolone sulfate (PregS) has shed new light on the physiological role of this channel. For example, TRPM3 activation enhances insulin secretion from β pancreatic cells, induces contraction of vascular smooth muscle, and is also involved in the detection of noxious heat. Although TRPM3 expression has been detected in several regions of the developing and mature brain, little is known about the roles of TRPM3 in brain physiology. In this study, we demonstrate the abundant expression of TRPM3 steroid-sensitive channels in the developing cerebellar cortex. We also show that TRPM3-like channels are expressed at glutamatergic synapses in neonatal Purkinje cells. We recently showed that PregS potentiates spontaneous glutamate release onto neonatal Purkinje cells during a period of active glutamatergic synapse formation; we now show that this effect of PregS is mediated by TRPM3-like channels. Mefenamic acid, a recently discovered TRPM3 antagonist, blocked the effect of PregS on glutamate release. The PregS effect on glutamate release was mimicked by other TRPM3 agonists (nifedipine and epipregnanolone sulfate) but not by a TRMP3-inactive steroid (progesterone). Our findings identify TRPM3 channels as novel modulators of glutamatergic transmission in the developing brain.

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

    DEFF Research Database (Denmark)

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

    2004-01-01

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

  4. Noxious cold ion channel TRPA1 is activated by pungent compounds and bradykinin.

    Science.gov (United States)

    Bandell, Michael; Story, Gina M; Hwang, Sun Wook; Viswanath, Veena; Eid, Samer R; Petrus, Matt J; Earley, Taryn J; Patapoutian, Ardem

    2004-03-25

    Six members of the mammalian transient receptor potential (TRP) ion channels respond to varied temperature thresholds. The natural compounds capsaicin and menthol activate noxious heat-sensitive TRPV1 and cold-sensitive TRPM8, respectively. The burning and cooling perception of capsaicin and menthol demonstrate that these ion channels mediate thermosensation. We show that, in addition to noxious cold, pungent natural compounds present in cinnamon oil, wintergreen oil, clove oil, mustard oil, and ginger all activate TRPA1 (ANKTM1). Bradykinin, an inflammatory peptide acting through its G protein-coupled receptor, also activates TRPA1. We further show that phospholipase C is an important signaling component for TRPA1 activation. Cinnamaldehyde, the most specific TRPA1 activator, excites a subset of sensory neurons highly enriched in cold-sensitive neurons and elicits nociceptive behavior in mice. Collectively, these data demonstrate that TRPA1 activation elicits a painful sensation and provide a potential molecular model for why noxious cold can paradoxically be perceived as burning pain.

  5. Expression of calcium-activated chloride channels Ano1 and Ano2 in mouse taste cells.

    Science.gov (United States)

    Cherkashin, Alexander P; Kolesnikova, Alisa S; Tarasov, Michail V; Romanov, Roman A; Rogachevskaja, Olga A; Bystrova, Marina F; Kolesnikov, Stanislav S

    2016-02-01

    Specialized Ca(2+)-dependent ion channels ubiquitously couple intracellular Ca(2+) signals to a change in cell polarization. The existing physiological evidence suggests that Ca(2+)-activated Cl(-) channels (CaCCs) are functional in taste cells. Because Ano1 and Ano2 encode channel proteins that form CaCCs in a variety of cells, we analyzed their expression in mouse taste cells. Transcripts for Ano1 and Ano2 were detected in circumvallate (CV) papillae, and their expression in taste cells was confirmed using immunohistochemistry. When dialyzed with CsCl, taste cells of the type III exhibited no ion currents dependent on cytosolic Ca(2+). Large Ca(2+)-gated currents mediated by TRPM5 were elicited in type II cells by Ca(2+) uncaging. When TRPM5 was inhibited by triphenylphosphine oxide (TPPO), ionomycin stimulated a small but resolvable inward current that was eliminated by anion channel blockers, including T16Ainh-A01 (T16), a specific Ano1 antagonist. This suggests that CaCCs, including Ano1-like channels, are functional in type II cells. In type I cells, CaCCs were prominently active, blockable with the CaCC antagonist CaCCinh-A01 but insensitive to T16. By profiling Ano1 and Ano2 expressions in individual taste cells, we revealed Ano1 transcripts in type II cells only, while Ano2 transcripts were detected in both type I and type II cells. P2Y agonists stimulated Ca(2+)-gated Cl(-) currents in type I cells. Thus, CaCCs, possibly formed by Ano2, serve as effectors downstream of P2Y receptors in type I cells. While the role for TRPM5 in taste transduction is well established, the physiological significance of expression of CaCCs in type II cells remains to be elucidated.

  6. Fungal mediator tail subunits contain classical transcriptional activation domains.

    Science.gov (United States)

    Liu, Zhongle; Myers, Lawrence C

    2015-04-01

    Classical activation domains within DNA-bound eukaryotic transcription factors make weak interactions with coactivator complexes, such as Mediator, to stimulate transcription. How these interactions stimulate transcription, however, is unknown. The activation of reporter genes by artificial fusion of Mediator subunits to DNA binding domains that bind to their promoters has been cited as evidence that the primary role of activators is simply to recruit Mediator. We have identified potent classical transcriptional activation domains in the C termini of several tail module subunits of Saccharomyces cerevisiae, Candida albicans, and Candida dubliniensis Mediator, while their N-terminal domains are necessary and sufficient for their incorporation into Mediator but do not possess the ability to activate transcription when fused to a DNA binding domain. This suggests that Mediator fusion proteins actually are functioning in a manner similar to that of a classical DNA-bound activator rather than just recruiting Mediator. Our finding that deletion of the activation domains of S. cerevisiae Med2 and Med3, as well as C. dubliniensis Tlo1 (a Med2 ortholog), impairs the induction of certain genes shows these domains function at native promoters. Activation domains within coactivators are likely an important feature of these complexes and one that may have been uniquely leveraged by a common fungal pathogen.

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

    DEFF Research Database (Denmark)

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

    2013-01-01

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

  8. Acid extrusion from human spermatozoa is mediated by flagellar voltage-gated proton channel.

    Science.gov (United States)

    Lishko, Polina V; Botchkina, Inna L; Fedorenko, Andriy; Kirichok, Yuriy

    2010-02-05

    Human spermatozoa are quiescent in the male reproductive system and must undergo activation once introduced into the female reproductive tract. This process is known to require alkalinization of sperm cytoplasm, but the mechanism responsible for transmembrane proton extrusion has remained unknown because of the inability to measure membrane conductance in human sperm. Here, by successfully patch clamping human spermatozoa, we show that proton channel Hv1 is their dominant proton conductance. Hv1 is confined to the principal piece of the sperm flagellum, where it is expressed at unusually high density. Robust flagellar Hv1-dependent proton conductance is activated by membrane depolarization, an alkaline extracellular environment, endocannabinoid anandamide, and removal of extracellular zinc, a potent Hv1 blocker. Hv1 allows only outward transport of protons and is therefore dedicated to inducing intracellular alkalinization and activating spermatozoa. The importance of Hv1 for sperm activation makes it an attractive target for controlling male fertility.

  9. Mediated Electrochemical Measurements of Intracellular Catabolic Activities of Yeast Cells

    Institute of Scientific and Technical Information of China (English)

    Jin Sheng ZHAO; Zhen Yu YANG; Yao LU; Zheng Yu YANG

    2005-01-01

    Coupling with the dual mediator system menadione/ferricyanide, microelectrode voltammetric measurements were undertaken to detect the ferrocyanide accumulations arising from the mediated reduction of ferricyanide by yeast cells. The results indicate that the dual mediator system menadione/ferricyanide could be used as a probe to detect cellular catabolic activities in yeast cells and the electrochemical response has a positive relationship with the specific growth rate of yeast cells.

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

    Science.gov (United States)

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

    2016-01-01

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

  11. Calcium channel blocker prevents stress-induced activation of renin and aldosterone in conscious pig

    Energy Technology Data Exchange (ETDEWEB)

    Ceremuzynski, L.K.; Klos, J.; Barcikowski, B.; Herbaczynska-Cedro, K. (Department of Cardiology, Postgraduate Medical School, Warsaw (Poland))

    1991-06-01

    A considerable amount of data suggest the involvement of calcium-mediated processes in the activation of the renin-angiotensin-aldosterone (RAA) cascade. To investigate the effect of calcium-channel inhibition on the RAA system, the authors studied 21 conscious pigs. Blood renin and aldosterone levels increased by subjecting animals to 24 hours of immobilization stress. Renin and aldosterone levels were repeatedly measured by radioimmunoassay in blood samples taken periodically over 24 hours from a chronically implanted arterial cannula. Pretreatment of the animals (N = 11) with nisoldipine, 2 {times} 20 mg p.o. daily for 2 days before and on the day of immobilization, transiently attenuated the stress-induced increase of plasma renin activity and completely prevented the rise of aldosterone, as compared to nontreated controls (N = 10). The finding that nisoldipine suppresses RAA activation induced by a nonpharmacologic stimulus in the conscious intact animal may have clinical implications.

  12. Phentolamine relaxes human corpus cavernosum by a nonadrenergic mechanism activating ATP-sensitive K+ channel.

    Science.gov (United States)

    Silva, L F G; Nascimento, N R F; Fonteles, M C; de Nucci, G; Moraes, M E; Vasconcelos, P R L; Moraes, M O

    2005-01-01

    To investigate the pharmacodynamics of phentolamine in human corpus cavernosum (HCC) with special attention to the role of the K+ channels. Strips of HCC precontracted with nonadrenergic stimuli and kept in isometric organ bath immersed in a modified Krebs-Henseleit solution enriched with guanethidine and indomethacine were used in order to study the mechanism of the phentolamine-induced relaxation. Phentolamine caused relaxation (approximately 50%) in HCC strips precontracted with K+ 40 mM. This effect was not blocked by tetrodotoxin (1 microM) (54.6+/-4.6 vs 48.9+/-6.4%) or (atropine (10 microM) (52.7+/-6.5 vs 58.6+/-5.6%). However, this relaxation was significantly attenuated by L-NAME (100 microM) (59.7+/-5.8 vs 27.8+/-7.1%; Pphentolamine relaxations (54.6+/-4.6 vs 59.3+/-5.2%). Glibenclamide (100 microM), an inhibitor of K(ATP)-channel, caused a significant inhibition (56.7+/-6.3 vs 11.3+/-2.3%; Pphentolamine-induced relaxation. In addition, the association of glibenclamide and L-NAME almost abolished the phentolamine-mediated relaxation (54.6+/-5.6 vs 5.7+/-1.4%; Pphentolamine relaxes HCC by a nonadrenergic-noncholinergic mechanism dependent on nitric oxide synthase activity and activation of K(ATP)-channel.

  13. Channel Response to Gravel Mining Activities in Mountain Rivers

    Institute of Scientific and Technical Information of China (English)

    José Luis López S.

    2004-01-01

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

  14. Hyperforin modulates dendritic spine morphology in hippocampal pyramidal neurons by activating Ca(2+) -permeable TRPC6 channels.

    Science.gov (United States)

    Leuner, Kristina; Li, Wei; Amaral, Michelle D; Rudolph, Stephanie; Calfa, Gaston; Schuwald, Anita M; Harteneck, Christian; Inoue, Takafumi; Pozzo-Miller, Lucas

    2013-01-01

    The standardized extract of the St. John's wort plant (Hypericum perforatum) is commonly used to treat mild to moderate depression. Its active constituent is hyperforin, a phloroglucinol derivative that reduces the reuptake of serotonin and norepinephrine by increasing intracellular Na(+) concentration through the activation of nonselective cationic TRPC6 channels. TRPC6 channels are also Ca(2+) -permeable, resulting in intracellular Ca(2+) elevations. Indeed, hyperforin activates TRPC6-mediated currents and Ca(2+) transients in rat PC12 cells, which induce their differentiation, mimicking the neurotrophic effect of nerve growth factor. Here, we show that hyperforin modulates dendritic spine morphology in CA1 and CA3 pyramidal neurons of hippocampal slice cultures through the activation of TRPC6 channels. Hyperforin also evoked intracellular Ca(2+) transients and depolarizing inward currents sensitive to the TRPC channel blocker La(3+) , thus resembling the actions of the neurotrophin brain-derived neurotrophic factor (BDNF) in hippocampal pyramidal neurons. These results suggest that the antidepressant actions of St. John's wort are mediated by a mechanism similar to that engaged by BDNF.

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

    DEFF Research Database (Denmark)

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

    2016-01-01

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

  16. Analysis of TRPV channel activation by stimulation of FCεRI and MRGPR receptors in mouse peritoneal mast cells

    Science.gov (United States)

    Solís-López, A.; Kriebs, U.; Marx, A.; Mannebach, S.; Liedtke, W. B.; Caterina, M. J.; Freichel, M.; Tsvilovskyy, V. V.

    2017-01-01

    The activation of mast cells (MC) is part of the innate and adaptive immune responses and depends on Ca2+ entry across the plasma membrane, leading to the release of preformed inflammatory mediators by degranulation or by de novo synthesis. The calcium conducting channels of the TRPV family, known by their thermo and osmotic sensitivity, have been proposed to be involved in the MC activation in murine, rat, and human mast cell models. So far, immortalized mast cell lines and nonspecific TRPV blockers have been employed to characterize the role of TRPV channels in MC. The aim of this work was to elucidate the physiological role of TRPV channels by using primary peritoneal mast cells (PMCs), a model of connective tissue type mast cells. Our RT-PCR and NanoString analysis identified the expression of TRPV1, TRPV2, and TRPV4 channels in PMCs. For determination of the functional role of the expressed TRPV channels we performed measurements of intracellular free Ca2+ concentrations and beta-hexosaminidase release in PMCs obtained from wild type and mice deficient for corresponding TRPV1, TRPV2 and TRPV4 in response to various receptor-mediated and physical stimuli. Furthermore, substances known as activators of corresponding TRPV-channels were also tested using these assays. Our results demonstrate that TRPV1, TRPV2, and TRPV4 do not participate in activation pathways triggered by activation of the high-affinity receptors for IgE (FcεRI), Mrgprb2 receptor, or Endothelin-1 receptor nor by heat or osmotic stimulation in mouse PMCs. PMID:28158279

  17. 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.

  18. Functional BK channels facilitate the β3-adrenoceptor agonist-mediated relaxation of nerve-evoked contractions in rat urinary bladder smooth muscle isolated strips.

    Science.gov (United States)

    Afeli, Serge A Y; Petkov, Georgi V

    2013-07-05

    The large-conductance voltage- and Ca(2+)-activated K(+) (BK) channel is a major regulator of detrusor smooth muscle (DSM) contractility thus facilitating urinary bladder function. Recent findings suggest that activation of β3-adrenoceptors causes DSM relaxation. However, it is unknown whether the β3-adrenoceptor-mediated DSM relaxation is BK channel-dependent during nerve-evoked contractions. To test this hypothesis, we induced nerve-evoked contractions in rat DSM isolated strips by using a tissue bath system equipped with platinum electrodes for electrical field stimulation (EFS). (±)-(R(*),R(*))-[4-[2-[[2-(3-chlorophenyl)-2-hydroxyethyl]amino]propyl]phenoxy] acetic acid sodium hydrate (BRL37344), a β3-adrenoceptor agonist, significantly decreased the amplitude and muscle force of the 20 Hz EFS-induced DSM contractions in a concentration-dependent manner. The BRL37344 inhibitory effect was significantly antagonized by 1-(2-ethylphenoxy)-3-[[(1S)-1,2,3,4-tetrahydro-1-naphthalenyl]amino]-(2S)-2-propanol hydrochloride (SR59230A), a β3-adrenoceptor antagonist. We further isolated the cholinergic from the purinergic component of the 0.5-50 Hz EFS-induced DSM contractions by using selective inhibitors, atropine as well as suramin and α,β-methylene-ATP. We found that BRL37344 inhibited both the purinergic and cholinergic components of the nerve-evoked contractions in rat DSM isolated strips. The pharmacological blockade of the BK channels with iberiotoxin, a selective BK channel inhibitor, increased the amplitude and muscle force of the 20 Hz EFS-induced contractions in rat DSM isolated strips. In the presence of iberiotoxin, there was a significant reduction in the BRL37344-induced inhibition of the 20 Hz EFS-induced contractions in rat DSM isolated strips. These latter findings suggest that BK channels play a critical role in the β3-adrenoceptor-mediated inhibition of rat DSM nerve-evoked contractions.

  19. PIC Activation through Functional Interplay between Mediator and TFIIH.

    Science.gov (United States)

    Malik, Sohail; Molina, Henrik; Xue, Zhu

    2017-01-06

    The multiprotein Mediator coactivator complex functions in large part by controlling the formation and function of the promoter-bound preinitiation complex (PIC), which consists of RNA polymerase II and general transcription factors. However, precisely how Mediator impacts the PIC, especially post-recruitment, has remained unclear. Here, we have studied Mediator effects on basal transcription in an in vitro transcription system reconstituted from purified components. Our results reveal a close functional interplay between Mediator and TFIIH in the early stages of PIC development. We find that under conditions when TFIIH is not normally required for transcription, Mediator actually represses transcription. TFIIH, whose recruitment to the PIC is known to be facilitated by the Mediator, then acts to relieve Mediator-induced repression to generate an active form of the PIC. Gel mobility shift analyses of PICs and characterization of TFIIH preparations carrying mutant XPB translocase subunit further indicate that this relief of repression is achieved through expending energy via ATP hydrolysis, suggesting that it is coupled to TFIIH's established promoter melting activity. Our interpretation of these results is that Mediator functions as an assembly factor that facilitates PIC maturation through its various stages. Whereas the overall effect of the Mediator is to stimulate basal transcription, its initial engagement with the PIC generates a transcriptionally inert PIC intermediate, which necessitates energy expenditure to complete the process.

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

    Directory of Open Access Journals (Sweden)

    Ivaniš Predrag

    2004-01-01

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

  1. Effect of mitochondrial potassium channel on the renal protection mediated by sodium thiosulfate against ethylene glycol induced nephrolithiasis in rat model

    Directory of Open Access Journals (Sweden)

    N. Baldev

    2015-12-01

    Full Text Available Purpose: Sodium thiosulfate (STS is clinically reported to be a promising drug in preventing nephrolithiasis. However, its mechanism of action remains unclear. In the present study, we investigated the role of mitochondrial KATP channel in the renal protection mediated by STS. Materials and Methods: Nephrolithiasis was induced in Wistar rats by administrating 0.4% ethylene glycol (EG along with 1% ammonium chloride for one week in drinking water followed by only 0.75% EG for two weeks. Treatment groups received STS, mitochondrial KATP channel opener and closer exclusively or in combination with STS for two weeks. Results: Animals treated with STS showed normal renal tissue architecture, supported by near normal serum creatinine, urea and ALP activity. Diazoxide (mitochondria KATP channel opening treatment to the animal also showed normal renal tissue histology and improved serum chemistry. However, an opposite result was shown by glibenclamide (mitochondria KATP channel closer treated rats. STS administered along with diazoxide negated the renal protection rendered by diazoxide alone, while it imparted protection to the glibenclamide treated rats, formulating a mitochondria modulated STS action. Conclusion: The present study confirmed that STS render renal protection not only through chelation and antioxidant effect but also by modulating the mitochondrial KATP channel for preventing urolithiasis.

  2. Modulation of voltage-gated sodium channels hyperpolarizes the voltage threshold for activation in spinal motoneurones.

    Science.gov (United States)

    Power, Kevin E; Carlin, Kevin P; Fedirchuk, Brent

    2012-03-01

    Previous work has shown that motoneurone excitability is enhanced by a hyperpolarization of the membrane potential at which an action potential is initiated (V(th)) at the onset, and throughout brainstem-evoked fictive locomotion in the adult decerebrate cat and neonatal rat. Modeling work has suggested the modulation of Na(+) conductance as a putative mechanism underlying this state-dependent change in excitability. This study sought to determine whether modulation of voltage-gated sodium channels could induce V(th) hyperpolarization. Whole-cell patch-clamp recordings were made from antidromically identified lumbar spinal motoneurones in an isolated neonatal rat spinal cord preparation. Recordings were made with and without the bath application of veratridine, a plant alkaloid neurotoxin that acts as a sodium channel modulator. As seen in HEK 293 cells expressing Nav1.2 channels, veratridine-modified channels demonstrated a hyperpolarizing shift in their voltage-dependence of activation and a slowing of inactivation that resulted in an enhanced inward current in response to voltage ramp stimulations. In the native rat motoneurones, veratridine-modified sodium channels induced a hyperpolarization of V(th) in all 29 neonatal rat motoneurones examined (mean hyperpolarization: -6.6 ± 4.3 mV). V(th) hyperpolarization was not due to the effects on Ca(2+) and/or K(+) channels as blockade of these currents did not alter V(th). Veratridine also significantly increased the amplitude of persistent inward currents (PICs; mean increase: 72.5 ± 98.5 pA) evoked in response to slow depolarizing current ramps. However, the enhancement of the PIC amplitude had a slower time course than the hyperpolarization of V(th), and the PIC onset voltage could be either depolarized or hyperpolarized, suggesting that PIC facilitation did not mediate the V(th) hyperpolarization. We therefore suggest that central neuronal circuitry in mammals could affect V(th) in a mechanism similar to that of

  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. The prostaglandin E2/EP4 receptor/cyclic AMP/T-type Ca(2+) channel pathway mediates neuritogenesis in sensory neuron-like ND7/23 cells.

    Science.gov (United States)

    Mitani, Kenji; Sekiguchi, Fumiko; Maeda, Takashi; Tanaka, Yukari; Yoshida, Shigeru; Kawabata, Atsufumi

    2016-03-01

    We investigated mechanisms for the neuritogenesis caused by prostaglandin E2 (PGE2) or intracellular cyclic AMP (cAMP) in sensory neuron-like ND7/23 cells. PGE2 caused neuritogenesis, an effect abolished by an EP4 receptor antagonist or inhibitors of adenylyl cyclase (AC) or protein kinase A (PKA) and mimicked by the AC activator forskolin, dibutyryl cAMP (db-cAMP), and selective activators of PKA or Epac. ND7/23 cells expressed both Cav3.1 and Cav3.2 T-type Ca(2+) channels (T-channels). The neuritogenesis induced by db-cAMP or PGE2 was abolished by T-channel blockers. T-channels were functionally upregulated by db-cAMP. The PGE2/EP4/cAMP/T-channel pathway thus appears to mediate neuritogenesis in sensory neurons.

  5. 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...

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

    Science.gov (United States)

    Roth, Alexander; Hammerer, Klemens

    2016-10-01

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

  7. Cooperative gating between ion channels.

    Science.gov (United States)

    Choi, Kee-Hyun

    2014-01-01

    Cooperative gating between ion channels, i.e. the gating of one channel directly coupled to the gating of neighboring channels, has been observed in diverse channel types at the single-channel level. Positively coupled gating could enhance channel-mediated signaling while negative coupling may effectively reduce channel gating noise. Indeed, the physiological significance of cooperative channel gating in signal transduction has been recognized in several in vivo studies. Moreover, coupled gating of ion channels was reported to be associated with some human disease states. In this review, physiological roles for channel cooperativity and channel clustering observed in vitro and in vivo are introduced, and stimulation-induced channel clustering and direct channel cross linking are suggested as the physical mechanisms of channel assembly. Along with physical clustering, several molecular mechanisms proposed as the molecular basis for functional coupling of neighboring channels are covered: permeant ions as a channel coupling mediator, concerted channel activation through the membrane, and allosteric mechanisms. Also, single-channel analysis methods for cooperative gating such as the binomial analysis, the variance analysis, the conditional dwell time density analysis, and the maximum likelihood fitting analysis are reviewed and discussed.

  8. Regulation of high-voltage-activated Ca(2+) channel function, trafficking, and membrane stability by auxiliary subunits.

    Science.gov (United States)

    Felix, Ricardo; Calderón-Rivera, Aida; Andrade, Arturo

    2013-09-01

    Voltage-gated Ca(2+) (CaV) channels mediate Ca(2+) ions influx into cells in response to depolarization of the plasma membrane. They are responsible for initiation of excitation-contraction and excitation-secretion coupling, and the Ca(2+) that enters cells through this pathway is also important in the regulation of protein phosphorylation, gene transcription, and many other intracellular events. Initial electrophysiological studies divided CaV channels into low-voltage-activated (LVA) and high-voltage-activated (HVA) channels. The HVA CaV channels were further subdivided into L, N, P/Q, and R-types which are oligomeric protein complexes composed of an ion-conducting CaVα1 subunit and auxiliary CaVα2δ, CaVβ, and CaVγ subunits. The functional consequences of the auxiliary subunits include altered functional and pharmacological properties of the channels as well as increased current densities. The latter observation suggests an important role of the auxiliary subunits in membrane trafficking of the CaVα1 subunit. This includes the mechanisms by which CaV channels are targeted to the plasma membrane and to appropriate regions within a given cell. Likewise, the auxiliary subunits seem to participate in the mechanisms that remove CaV channels from the plasma membrane for recycling and/or degradation. Diverse studies have provided important clues to the molecular mechanisms involved in the regulation of CaV channels by the auxiliary subunits, and the roles that these proteins could possibly play in channel targeting and membrane Stabilization.

  9. 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.

  10. Control of sensory neuron excitability by serotonin involves 5HT2C receptors and Ca(2+)-activated chloride channels.

    Science.gov (United States)

    Salzer, Isabella; Gantumur, Enkhbileg; Yousuf, Arsalan; Boehm, Stefan

    2016-11-01

    Serotonin (5HT) is a constituent of the so-called "inflammatory soup" that sensitizes nociceptors during inflammation. Nevertheless, receptors and signaling mechanisms that mediate an excitation of dorsal root ganglion (DRG) neurons by 5HT remained controversial. Therefore, capsaicin-sensitive nociceptive neurons dissociated from rat DRGs were used to investigate effects of 5HT on membrane excitability and currents through ligand- as well as voltage-gated ion channels. In 58% of the neurons tested, 5HT increased action potential firing, an effect that was abolished by the 5HT2 receptor antagonist ritanserin, but not by the 5HT3 antagonist tropisetron. Unlike other algogenic mediators, such as PGE2 and bradykinin, 5HT did not affect currents through TTX-resistant Na(+) channels or Kv7 K(+) channels. In all neurons investigated, 5HT potentiated capsaicin-evoked currents through TRPV1 channels, an effect that was attenuated by antagonists at 5HT2A (4 F 4 PP), 5HT2B (SB 204741), as well as 5HT2C (RS 102221) receptors. 5HT triggered slowly arising inward Cl(-) currents in 53% of the neurons. This effect was antagonized by the 5HT2C receptor blocker only, and the current was prevented by an inhibitor of Ca(2+)-activated chloride channels (CaCC). The 5HT-induced increase in action potential firing was also abolished by this CaCC blocker and by the TRPV1 inhibitor capsazepine. Amongst the subtype selective 5HT2 antagonists, only RS 102221 (5HT2C-selectively) counteracted the rise in action potential firing elicited by 5HT. These results show that 5HT excites DRG neurons mainly via 5HT2C receptors which concomitantly mediate a sensitization of TRPV1 channels and an opening of CaCCs.

  11. Gingerol activates noxious cold ion channel TRPA1 in gastrointestinal tract

    Institute of Scientific and Technical Information of China (English)

    YANG Meng-Qi; YE Lin-Lan; LIU Xiao-Ling; QI Xiao-Ming; LV Jia-Di; WANG Gang; FARHAN Ulah-Khan

    2016-01-01

    TRPA1 channels are non-selective cation channels that could be activated by plant-derived pungent products,including gingerol,a main active constituent of ginger.Ginger could improve the digestive function;however whether ginger improves the digestive function through activating TRPA1 receptor in gastrointestinal tract has not been investigated.In the present study,gingerol was used to stimulate cell lines (RIN14B or STC-1) while depletion of extracellular calcium.TRPA1 inhibitor (rethenium red) and TRPA1 gene silencing via TRPAl-specific siRNA were also used for mechanistic studies.The intracellular calcium and secretion of serotonin or cholecystokinin were measured by fura-2/AM and ELISA.Stimulation of those cells with gingerol increased intracellular calcium levels and the serotonin or cholecystokinin secretion.The gingerol-induced intracellular calcium increase and secretion (serotonin or cholecystokinin) release were completely blocked by ruthenium red,EGTA,and TRPAl-specific siRNA.In summary,our results suggested that gingerol derived from ginger might improve the digestive function through secretion releasing from endocrine cells of the gut by inducing TRPA1-mediated calcium influx.

  12. Biophysical characterization of KV3.1 potassium channel activating compounds.

    Science.gov (United States)

    Taskin, Bahar; von Schoubye, Nadia Lybøl; Sheykhzade, Majid; Bastlund, Jesper Frank; Grunnet, Morten; Jespersen, Thomas

    2015-07-05

    The effect of two positive modulators, RE1 and EX15, on the voltage-gated K(+) channel Kv3.1 was investigated using the whole-cell patch-clamp technique on HEK293 cells expressing Kv3.1a. RE1 and EX15 increased the Kv3.1 currents in a concentration-dependent manner with an EC50 value of 4.5 and 1.3µM, respectively. However, high compound concentrations caused an inhibition of the Kv3.1 current. The compound-induced activation of Kv3.1 channels showed a profound hyperpolarized shift in activation kinetics. 30µM RE1 shifted V1/2 from 5.63±0.31mV to -9.71±1.00mV and 10µM EX15 induced a shift from 10.77±0.32mV to -15.11±1.57mV. The activation time constant (Tauact) was reduced for both RE1 and EX15, with RE1 being the fastest activator. The deactivation time constant (Taudeact) was also markedly reduced for both RE1 and EX15, with EX15 inducing the most prominent effect. Furthermore, subjected to depolarizing pulses at 30Hz, both compounds were showing a use-dependent effect resulting in a reduction of the compound-mediated effect. However, during these conditions, RE1- and EX15-modified current amplitudes still exceeded the control condition amplitudes by up to 200%. In summary, the present study introduces the first detailed biophysical characterization of two new Kv3.1 channel modifying compounds with different modulating properties.

  13. Angiotensin-2-mediated Ca2+ signaling in the retinal pigment epithelium: role of angiotensin-receptor-associated-protein and TRPV2 channel.

    Directory of Open Access Journals (Sweden)

    Rene Barro-Soria

    Full Text Available Angiotensin II (AngII receptor (ATR is involved in pathologic local events such as neovascularisation and inflammation including in the brain and retina. The retinal pigment epithelium (RPE expresses ATR in its AT1R form, angiotensin-receptor-associated protein (Atrap, and transient-receptor-potential channel-V2 (TRPV2. AT1R and Atrap co-localize to the basolateral membrane of the RPE, as shown by immunostaining. Stimulation of porcine RPE (pRPE cells by AngII results in biphasic increases in intracellular free Ca(2+inhibited by losartan. Xestospongin C (xest C and U-73122, blockers of IP3R and PLC respectively, reduced AngII-evoked Ca(2+response. RPE cells from Atrap(-/- mice showed smaller AngII-evoked Ca(2+peak (by 22% and loss of sustained Ca(2+elevation compared to wild-type. The TRPV channel activator cannabidiol (CBD at 15 µM stimulates intracellular Ca(2+-rise suggesting that porcine RPE cells express TRPV2 channels. Further evidence supporting the functional expression of TRPV2 channels comes from experiments in which 100 µM SKF96365 (a TRPV channel inhibitor reduced the cannabidiol-induced Ca(2+-rise. Application of SKF96365 or reduction of TRPV2 expression by siRNA reduced the sustained phase of AngII-mediated Ca(2+transients by 53%. Thus systemic AngII, an effector of the local renin-angiotensin system stimulates biphasic Ca(2+transients in the RPE by releasing Ca(2+from cytosolic IP3-dependent stores and activating ATR/Atrap and TRPV2 channels to generate a sustained Ca(2+elevation.

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

    Energy Technology Data Exchange (ETDEWEB)

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

    2001-10-19

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

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

    Institute of Scientific and Technical Information of China (English)

    魏珂; 闵苏; 龙村

    2004-01-01

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

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

    Science.gov (United States)

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

    2013-09-01

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

  17. Characteristics of single large-conductance Ca2+-activated K+ channels and their regulation of action potentials and excitability in parasympathetic cardiac motoneurons in the nucleus ambiguus.

    Science.gov (United States)

    Lin, Min; Hatcher, Jeff T; Wurster, Robert D; Chen, Qin-Hui; Cheng, Zixi Jack

    2014-01-15

    Large-conductance Ca2(+)-activated K+ channels (BK) regulate action potential (AP) properties and excitability in many central neurons. However, the properties and functional roles of BK channels in parasympathetic cardiac motoneurons (PCMNs) in the nucleus ambiguus (NA) have not yet been well characterized. In this study, the tracer X-rhodamine-5 (and 6)-isothiocyanate (XRITC) was injected into the pericardial sac to retrogradely label PCMNs in FVB mice at postnatal 7-9 days. Two days later, XRITC-labeled PCMNs in brain stem slices were identified. Using excised patch single-channel recordings, we identified voltage-gated and Ca(2+)-dependent BK channels in PCMNs. The majority of BK channels exhibited persistent channel opening during voltage holding. These BK channels had a conductance of 237 pS and a 50% opening probability at +27.9 mV, the channel open time constant was 3.37 ms at +20 mV, and dwell time increased exponentially as the membrane potential depolarized. At the +20-mV holding potential, the [Ca2+]50 was 15.2 μM with a P0.5 of 0.4. Occasionally, some BK channels showed a transient channel opening and fast inactivation. Using whole cell voltage clamp, we found that BK channel mediated outward currents and afterhyperpolarization currents (IAHP). Using whole cell current clamp, we found that application of BK channel blocker iberiotoxin (IBTX) increased spike half-width and suppressed fast afterhyperpolarization (fAHP) amplitude following single APs. In addition, IBTX application increased spike half-width and reduced the spike frequency-dependent AP broadening in trains and spike frequency adaption (SFA). Furthermore, BK channel blockade decreased spike frequency. Collectively, these results demonstrate that PCMNs have BK channels that significantly regulate AP repolarization, fAHP, SFA, and spike frequency. We conclude that activation of BK channels underlies one of the mechanisms for facilitation of PCMN excitability.

  18. Kv3.4 potassium channel-mediated electrosignaling controls cell cycle and survival of irradiated leukemia cells.

    Science.gov (United States)

    Palme, Daniela; Misovic, Milan; Schmid, Evi; Klumpp, Dominik; Salih, Helmut R; Rudner, Justine; Huber, Stephan M

    2013-08-01

    Aberrant ion channel expression in the plasma membrane is characteristic for many tumor entities and has been attributed to neoplastic transformation, tumor progression, metastasis, and therapy resistance. The present study aimed to define the function of these "oncogenic" channels for radioresistance of leukemia cells. Chronic myeloid leukemia cells were irradiated (0-6 Gy X ray), ion channel expression and activity, Ca(2+)- and protein signaling, cell cycle progression, and cell survival were assessed by quantitative reverse transcriptase-polymerase chain reaction, patch-clamp recording, fura-2 Ca(2+)-imaging, immunoblotting, flow cytometry, and clonogenic survival assays, respectively. Ionizing radiation-induced G2/M arrest was preceded by activation of Kv3.4-like voltage-gated potassium channels. Channel activation in turn resulted in enhanced Ca(2+) entry and subsequent activation of Ca(2+)/calmodulin-dependent kinase-II, and inactivation of the phosphatase cdc25B and the cyclin-dependent kinase cdc2. Accordingly, channel inhibition by tetraethylammonium and blood-depressing substance-1 and substance-2 or downregulation by RNA interference led to release from radiation-induced G2/M arrest, increased apoptosis, and decreased clonogenic survival. Together, these findings indicate the functional significance of voltage-gated K(+) channels for the radioresistance of myeloid leukemia cells.

  19. How to save the WIMP. Global analysis of a dark matter model with two s-channel mediators

    Energy Technology Data Exchange (ETDEWEB)

    Duerr, Michael; Kahlhoefer, Felix; Schmidt-Hoberg, Kai [Deutsches Elektronen-Synchrotron (DESY), Hamburg (Germany); Schwetz, Thomas; Vogl, Stefan [Karlsruher Institut fuer Technologie (KIT), Karlsruhe (Germany). Inst. fuer Kernphysik

    2016-06-15

    A reliable comparison of different dark matter (DM) searches requires models that satisfy certain consistency requirements like gauge invariance and perturbative unitarity. As a well-motivated example, we study two-mediator DM (2MDM). The model is based on a spontaneously broken U(1){sup '} gauge symmetry and contains a Majorana DM particle as well as two s-channel mediators, one vector (the Z{sup '}) and one scalar (the dark Higgs). We perform a global scan over the parameters of the model assuming that the DM relic density is obtained by thermal freeze-out in the early Universe and imposing a large set of constraints: direct and indirect DM searches, monojet, dijet and dilepton searches at colliders, Higgs observables, electroweak precision tests and perturbative unitarity. We conclude that thermal DM is only allowed either close to an s-channel resonance or if at least one mediator is lighter than the DM particle. In these cases a thermal DM abundance can be obtained although DM couplings to the Standard Model are tiny. Interestingly, we find that vector-mediated DM-nucleon scattering leads to relevant constraints despite the velocity-suppressed cross section, and that indirect detection can be important if DM annihilations into both mediators are kinematically allowed.

  20. Acid-sensing ion channel 3 mediates peripheral anti-hyperalgesia effects of acupuncture in mice inflammatory pain

    Directory of Open Access Journals (Sweden)

    Chen Wei-Hsin

    2011-11-01

    Full Text Available Abstract Background Peripheral tissue inflammation initiates hyperalgesia accompanied by tissue acidosis, nociceptor activation, and inflammation mediators. Recent studies have suggested a significantly increased expression of acid-sensing ion channel 3 (ASIC3 in both carrageenan- and complete Freund's adjuvant (CFA-induced inflammation. This study tested the hypothesis that acupuncture is curative for mechanical hyperalgesia induced by peripheral inflammation. Methods Here we used mechanical stimuli to assess behavioral responses in paw and muscle inflammation induced by carrageenan or CFA. We also used immunohistochemistry staining and western blot methodology to evaluate the expression of ASIC3 in dorsal root ganglion (DRG neurons. Results In comparison with the control, the inflammation group showed significant mechanical hyperalgesia with both intraplantar carrageenan and CFA-induced inflammation. Interestingly, both carrageenan- and CFA-induced hyperalgesia were accompanied by ASIC3 up-regulation in DRG neurons. Furthermore, electroacupuncture (EA at the ST36 rescued mechanical hyperalgesia through down-regulation of ASIC3 overexpression in both carrageenan- and CFA-induced inflammation. Conclusions In addition, electrical stimulation at the ST36 acupoint can relieve mechanical hyperalgesia by attenuating ASIC3 overexpression.

  1. Persistent CaMKII activation mediates learning-induced long-lasting enhancement of synaptic inhibition.

    Science.gov (United States)

    Ghosh, Sourav; Reuveni, Iris; Lamprecht, Raphael; Barkai, Edi

    2015-01-07

    Training rats in a particularly difficult olfactory-discrimination task results in acquisition of high skill to perform the task superbly, termed "rule learning" or "learning set." Such complex learning results in enhanced intrinsic neuronal excitability of piriform cortex pyramidal neurons, and in their excitatory synaptic interconnections. These changes, while subserving memory maintenance, must be counterbalanced by modifications that prevent overspreading of activity and uncontrolled synaptic strengthening. Indeed, we have previously shown that the average amplitude of GABAA-mediated miniature IPSCs (mIPSCs) in these neurons is enhanced for several days after learning, an enhancement mediated via a postsynaptic mechanism. To unravel the molecular mechanism of this long-term inhibition enhancement, we tested the role of key second-messenger systems in maintaining such long-lasting modulation. The calcium/calmodulin-dependent kinase II (CaMKII) blocker, KN93, significantly reduced the average mIPSC amplitude in neurons from trained rats only to the average pretraining level. A similar effect was obtained by the CaMKII peptide inhibitor, tatCN21. Such reduction resulted from decreased single-channel conductance and not in the number of activated channels. The PKC inhibitor, GF109203X, reduced the average mIPSC amplitude in neurons from naive, pseudo-trained, and trained animals, and the difference between the trained and control groups remained. Such reduction resulted from a decrease in the number of activated channels. The PKA inhibitor H89 dihydrochloride did not affect the average mIPSC amplitude in neurons from any of the three groups. We conclude that learning-induced enhancement of GABAA-mediated synaptic inhibition is maintained by persistent CaMKII activation.

  2. Plant-derived cannabinoids modulate the activity of transient receptor potential channels of ankyrin type-1 and melastatin type-8.

    Science.gov (United States)

    De Petrocellis, Luciano; Vellani, Vittorio; Schiano-Moriello, Aniello; Marini, Pietro; Magherini, Pier Cosimo; Orlando, Pierangelo; Di Marzo, Vincenzo

    2008-06-01

    The plant cannabinoids (phytocannabinoids), cannabidiol (CBD), and Delta(9)-tetrahydrocannabinol (THC) were previously shown to activate transient receptor potential channels of both vanilloid type 1 (TRPV1) and ankyrin type 1 (TRPA1), respectively. Furthermore, the endocannabinoid anandamide is known to activate TRPV1 and was recently found to antagonize the menthol- and icilin-sensitive transient receptor potential channels of melastatin type 8 (TRPM8). In this study, we investigated the effects of six phytocannabinoids [i.e., CBD, THC, CBD acid, THC acid, cannabichromene (CBC), and cannabigerol (CBG)] on TRPA1- and TRPM8-mediated increase in intracellular Ca2+ in either HEK-293 cells overexpressing the two channels or rat dorsal root ganglia (DRG) sensory neurons. All of the compounds tested induced TRPA1-mediated Ca2+ elevation in HEK-293 cells with efficacy comparable with that of mustard oil isothiocyanates (MO), the most potent being CBC (EC(50) = 60 nM) and the least potent being CBG and CBD acid (EC(50) = 3.4-12.0 microM). CBC also activated MO-sensitive DRG neurons, although with lower potency (EC(50) = 34.3 microM). Furthermore, although none of the compounds tested activated TRPM8-mediated Ca2+ elevation in HEK-293 cells, they all, with the exception of CBC, antagonized this response when it was induced by either menthol or icilin. CBD, CBG, THC, and THC acid were equipotent (IC(50) = 70-160 nM), whereas CBD acid was the least potent compound (IC(50) = 0.9-1.6 microM). CBG inhibited Ca2+ elevation also in icilin-sensitive DRG neurons with potency (IC(50) = 4.5 microM) similar to that of anandamide (IC(50) = 10 microM). Our findings suggest that phytocannabinoids and cannabis extracts exert some of their pharmacological actions also by interacting with TRPA1 and TRPM8 channels, with potential implications for the treatment of pain and cancer.

  3. A key role for STIM1 in store operated calcium channel activation in airway smooth muscle

    Directory of Open Access Journals (Sweden)

    Peel Samantha E

    2006-09-01

    Full Text Available Abstract Background Control of cytosolic calcium plays a key role in airway myocyte function. Changes in intracellular Ca2+ stores can modulate contractile responses, modulate proliferation and regulate synthetic activity. Influx of Ca2+ in non excitable smooth muscle is believed to be predominantly through store operated channels (SOC or receptor operated channels (ROC. Whereas agonists can activate both SOC and ROC in a range of smooth muscle types, the specific trigger for SOC activation is depletion of the sarcoplasmic reticulum Ca2+ stores. The mechanism underlying SOC activation following depletion of intracellular Ca2+ stores in smooth muscle has not been identified. Methods To investigate the roles of the STIM homologues in SOC activation in airway myocytes, specific siRNA sequences were utilised to target and selectively suppress both STIM1 and STIM2. Quantitative real time PCR was employed to assess the efficiency and the specificity of the siRNA mediated knockdown of mRNA. Activation of SOC was investigated by both whole cell patch clamp electrophysiology and a fluorescence based calcium assay. Results Transfection of 20 nM siRNA specific for STIM1 or 2 resulted in robust decreases (>70% of the relevant mRNA. siRNA targeted at STIM1 resulted in a reduction of SOC associated Ca2+ influx in response to store depletion by cyclopiazonic acid (60% or histamine but not bradykinin. siRNA to STIM2 had no effect on these responses. In addition STIM1 suppression resulted in a more or less complete abrogation of SOC associated inward currents assessed by whole cell patch clamp. Conclusion Here we show that STIM1 acts as a key signal for SOC activation following intracellular Ca2+ store depletion or following agonist stimulation with histamine in human airway myocytes. These are the first data demonstrating a role for STIM1 in a physiologically relevant, non-transformed endogenous expression cell model.

  4. L-type calcium channels and calcium/calmodulin-dependent kinase II differentially mediate behaviors associated with nicotine withdrawal in mice.

    Science.gov (United States)

    Jackson, K J; Damaj, M I

    2009-07-01

    Smoking is a widespread health problem. Because the nicotine withdrawal syndrome is a major contributor to continued smoking and relapse, it is important to understand the molecular and behavioral mechanisms of nicotine withdrawal to generate more effective smoking cessation therapies. Studies suggest a role for calcium-dependent mechanisms, such as L-type calcium channels and calcium/calmodulin-dependent protein kinase II (CaMKII), in the effects of nicotine dependence; however, the role of these mechanisms in nicotine-mediated behaviors is unclear. Thus, the goal of this study was to elucidate the role of L-type calcium channels and CaMKII in nicotine withdrawal behaviors. Using both pharmacological and genetic methods, our results show that L-type calcium channels are involved in physical, but not affective, nicotine withdrawal behaviors. Although our data do provide evidence of a role for CaMKII in nicotine withdrawal behaviors, our pharmacological and genetic assessments yielded different results concerning the specific role of the kinase. Pharmacological data suggest that CaMKII is involved in somatic signs and affective nicotine withdrawal, and activity level is decreased after nicotine withdrawal, whereas the genetic assessments yielded results suggesting that CaMKII is involved only in the anxiety-related response, yet the kinase activity may be increased after nicotine withdrawal; thus, future studies are necessary to clarify the precise behavioral specifics of the relevance of CaMKII in nicotine withdrawal behaviors. Overall, our data show that L-type calcium channels and CaMKII are relevant in nicotine withdrawal and differentially mediate nicotine withdrawal behaviors.

  5. Neuroprotective Activity of (--Epigallocatechin Gallate against Lipopolysaccharide-Mediated Cytotoxicity

    Directory of Open Access Journals (Sweden)

    Jin-Biao Liu

    2016-01-01

    Full Text Available Lipopolysaccharide- (LPS- mediated systemic inflammation plays a critical role in neurodegenerative diseases. The present study was conducted to evaluate the protective effects of epigallocatechin gallate (EGCG, the major component in green tea, on LPS-mediated inflammation and neurotoxicity. LPS treatment of macrophages induced expression of proinflammatory cytokines (TNF-α, IL-1β, and IL-6. However, EGCG pretreatment of macrophages significantly inhibited LPS-mediated induction of these cytokines. In addition, EGCG significantly diminished LPS-induced inflammatory cytokines in the peripheral mononuclear blood cells (PBMCs. Supernatant from EGCG-pretreated and LPS-activated macrophage cultures was found to be less cytotoxic to neurons than that from non-EGCG-pretreated and LPS-activated macrophage cultures. Furthermore, EGCG treatment of neurons could inhibit LPS-induced production of reactive oxygen species (ROS. Thus EGCG represents a potent and useful neuroprotective agent for inflammation-mediated neurological disorders.

  6. Non-IgE mediated mast cell activation.

    Science.gov (United States)

    Yu, Yingxin; Blokhuis, Bart R; Garssen, Johan; Redegeld, Frank A

    2016-05-05

    Mast cells are crucial effector cells in allergic reactions, where IgE is the best known mechanism to trigger their degranulation and release of a vast array of allergic mediators. However, IgE is not the only component to stimulate these cells to degranulate, while mast cell activation can also result in differential release of mediators. There is a plethora of stimuli, such as IgG, complement components, TLR ligands, neuropeptides, cytokines, chemokines and other inflammatory products, that can directly trigger mast cell degranulation, cause selective release of mediators, and stimulate proliferation, differentiation and/or migration. Moreover, some of these stimuli have a synergic effect on the IgE-mediated mast cell activation. Because of the ability to respond to a large repertoire of stimuli, mast cells may act as a versatile cell in various physiological and pathological conditions. In this review, we discuss current knowledge on non-IgE stimuli for (human) mast cells.

  7. 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.

  8. 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.

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

    Science.gov (United States)

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

    2015-01-01

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

  10. Immunolocalization and expression of small-conductance calcium-activated potassium channels in human myometrium

    DEFF Research Database (Denmark)

    Rosenbaum, Sofia T; Svalø, Julie; Nielsen, Karsten;

    2012-01-01

    Small-conductance calcium-activated potassium (SK3) channels have been detected in human myometrium and we have previously shown a functional role of SK channels in human myometrium in vitro. The aims of this study were to identify the precise localization of SK3 channels and to quantify SK3 mRNA...

  11. Baker's Yeast Mediated Reduction of Optically Active Diketone

    Institute of Scientific and Technical Information of China (English)

    ZHENG, Guo-Jun(郑国君); GAO, Xiao-Lei(高晓蕾); CHEN, Jin-Chun(陈锦春); LI, Yu-Lin(李裕林)

    2004-01-01

    Baker's yeast mediated reduction of optically active diketone is described. The two keto groups are efficiently differentiated and the ee value of the recovered material is considerably raised. It affords highly optically active key intermediates efficiently for the synthesis of natural polyhydroxylated agarofuran products.

  12. Nitric oxide and Kir6.1 potassium channel mediate isoquercitrin-induced endothelium-dependent and independent vasodilation in the mesenteric arterial bed of rats.

    Science.gov (United States)

    Gasparotto Junior, Arquimedes; Dos Reis Piornedo, Renê; Assreuy, Jamil; Da Silva-Santos, José Eduardo

    2016-10-05

    The vascular effect of flavonoid isoquercitrin was investigated in the perfused mesenteric vascular bed of rats. In preparations with functional endothelium isoquercitrin (100, 300 and 1000nmol) dose-dependently reduced the perfusion pressure by 13±2.2, 33±3.9, and 58±3.7mm Hg, respectively. Endothelium removal or inhibition of the nitric oxide synthase enzymes by l-NAME did not change the effects of 100 and 300 nmol isoquercitrin, but reduced by 30-40% the vasodilation induced by 1000 nmol isoquercitrin. Perfusion with nutritive solution containing 40mM KCl abolished the vasodilatory effect of all isoquercitrin doses. Treatment with glibenclamide, a Kir6.1 (ATP-sensitive) potassium channel blocker, inhibited vasodilation induced by 100 and 300 nmol isoquercitrin, but only partially reduced the effect of 1000 nmol isoquercitrin. The non-selective KCa (calcium-activated) potassium channel blocker tetraethylammonium, but not the selective KCa1.1 channel blocker iberiotoxin, reduced by around 60% vasodilation induced by all isoquercitrin doses. In addition, association of tetraethylammonium and glibenclamide, or l-NAME and glibenclamide, fully inhibited isoquercitrin-induced vasodilation. Our study shows that isoquercitrin induces vasodilation in resistance arteries, an effect mediated by K(+) channel opening and endothelial nitric oxide production.

  13. Differential calcium signaling mediated by voltage-gated calcium channels in rat retinal ganglion cells and their unmyelinated axons.

    Directory of Open Access Journals (Sweden)

    Allison Sargoy

    Full Text Available Aberrant calcium regulation has been implicated as a causative factor in the degeneration of retinal ganglion cells (RGCs in numerous injury models of optic neuropathy. Since calcium has dual roles in maintaining homeostasis and triggering apoptotic pathways in healthy and injured cells, respectively, investigation of voltage-gated Ca channel (VGCC regulation as a potential strategy to reduce the loss of RGCs is warranted. The accessibility and structure of the retina provide advantages for the investigation of the mechanisms of calcium signalling in both the somata of ganglion cells as well as their unmyelinated axons. The goal of the present study was to determine the distribution of VGCC subtypes in the cell bodies and axons of ganglion cells in the normal retina and to define their contribution to calcium signals in these cellular compartments. We report L-type Ca channel α1C and α1D subunit immunoreactivity in rat RGC somata and axons. The N-type Ca channel α1B subunit was in RGC somata and axons, while the P/Q-type Ca channel α1A subunit was only in the RGC somata. We patch clamped isolated ganglion cells and biophysically identified T-type Ca channels. Calcium imaging studies of RGCs in wholemounted retinas showed that selective Ca channel antagonists reduced depolarization-evoked calcium signals mediated by L-, N-, P/Q- and T-type Ca channels in the cell bodies but only by L-type Ca channels in the axons. This differential contribution of VGCC subtypes to calcium signals in RGC somata and their axons may provide insight into the development of target-specific strategies to spare the loss of RGCs and their axons following injury.

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

    Science.gov (United States)

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

    2017-02-22

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

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

    Science.gov (United States)

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

    2017-01-01

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

  16. Development and Testing of an In-Vitro Assay for Screening of Potential Therapeutic Agents Active against Na Channel Neurotoxins

    Science.gov (United States)

    1988-02-08

    effect to inhibit the binding of [3H]BTX-B is not mediated through an interaction at sodium ...compounds acting at the sodium channel 29 Figure 2. Sodium channel /ot-scorpion toxin sequence homology 30 Figure 3. Effects of nesacaine, benzimidazole...the channel , and alters the single channel conductance. In considering this broad spectrum of effects , one is led to invoke a model for sodium channel

  17. Interaction between Cl- channels and CRAC-related Ca2+ signaling during T lymphocyte activation and proliferation

    Institute of Scientific and Technical Information of China (English)

    Guan-lei WANG; Yan QIAN; Qin-ying QIU; Xiu-jian LAN; Hua HE; Yong-yuan GUAN

    2006-01-01

    Aim:To test the hypothesis that Cl- channel blockers affect T cell proliferation through Ca2+-release-activated Ca2+ (CRAC) signaling and examine the effects of the combination of a CRAC channel blocker and a Cl- channel blocker on concanavalin A (ConA;5 mg/mL) -induced Ca2+ signaling,gene expression and cellular proliferation in human peripheral T lymphocytes.Methods:[3H]Thymidine incorporation,Fura-2 fluorescent probe,RNase protection assay,and reverse transcription.polymerase chain reaction were used.Results:The Cl- channel blocker 4,4'-diisothiocvanostilbene-2,2'-disulfonic acid (DIDS) inhibited ConA-induced Ca2+influx.interleukin-2 mRNA expression and T lymphocyte proliferation in a concentration.dependent manner,and also enhanced the inhibitory effects of 1-{beta-[3-(4-methoxyphenyl)propoxyl]-4-methoxyphenethyl}-1H-imidazole (SK&F96365) on the above key events during T cell activation.A combination ofDIDS (1μmol/L) and SK&F96365 (1μmol/L) significantly diminished ConA-induced ClC-3 mRNA expression by 64%,whereas DIDS (1μmol/L) or SK&F96365 (1μmol/L) alone decreased ConA-induced ClC-3 mRNA expression by only 16% and 9%.respectively.Conclusion:These results suggest that there is an interaction between CRAC-mediated Ca2+ signaling and DIDS-sensitive C1-channels during ConA-induced T cell activation and proliferation.Moreover,the DIDS-sensitive Cl-channels may be related to the ClC-3 Cl- channels.

  18. Flavonoid Myricetin Modulates GABAA Receptor Activity through Activation of Ca2+ Channels and CaMK-II Pathway

    Directory of Open Access Journals (Sweden)

    Xiao Hu Zhang

    2012-01-01

    Full Text Available The flavonoid myricetin is found in several sedative herbs, for example, the St. John's Wort, but its influence on sedation and its possible mechanism of action are unknown. Using patch-clamp technique on a brain slice preparation, the present study found that myricetin promoted GABAergic activity in the neurons of hypothalamic paraventricular nucleus (PVN by increasing the decay time and frequency of the inhibitory currents mediated by GABAA receptor. This effect of myricetin was not blocked by the GABAA receptor benzodiazepine- (BZ- binding site antagonist flumazenil, but by KN-62, a specific inhibitor of the Ca2+/calmodulin-stimulated protein kinase II (CaMK-II. Patch clamp and live Ca2+ imaging studies found that myricetin could increase Ca2+ current and intracellular Ca2+ concentration, respectively, via T- and L-type Ca2+ channels in rat PVN neurons and hypothalamic primary culture neurons. Immunofluorescence staining showed increased phosphorylation of CaMK-II after myricetin incubation in primary culture of rat hypothalamic neurons, and the myricetin-induced CaMK-II phosphorylation was further confirmed by Western blotting in PC-12 cells. The present results suggest that myricetin enhances GABAA receptor activity via calcium channel/CaMK-II dependent mechanism, which is distinctively different from that of most existing BZ-binding site agonists of GABAA receptor.

  19. 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...

  20. Flavaglines Stimulate Transient Receptor Potential Melastatin Type 6 (TRPM6) Channel Activity.

    Science.gov (United States)

    Blanchard, Maxime G; de Baaij, Jeroen H F; Verkaart, Sjoerd A J; Lameris, Anke L; Basmadjian, Christine; Zhao, Qian; Désaubry, Laurent; Bindels, René J M; Hoenderop, Joost G J

    2015-01-01

    Magnesium (Mg2+) is essential for enzymatic activity, brain function and muscle contraction. Blood Mg2+ concentrations are tightly regulated between 0.7 and 1.1 mM by Mg2+ (re)absorption in kidney and intestine. The apical entry of Mg2+ in (re)absorbing epithelial cells is mediated by the transient receptor potential melastatin type 6 (TRPM6) ion channel. Here, flavaglines are described as a novel class of stimulatory compounds for TRPM6 activity. Flavaglines are a group of natural and synthetic compounds that target the ubiquitously expressed prohibitins and thereby affect cellular signaling. By whole-cell patch clamp analyses, it was demonstrated that nanomolar concentrations of flavaglines increases TRPM6 activity by ∼2 fold. The stimulatory effects were dependent on the presence of the alpha-kinase domain of TRPM6, but did not require its phosphotransferase activity. Interestingly, it was observed that two natural occurring TRPM6 mutants with impaired insulin-sensitivity, TRPM6-p.Val1393Ile and TRPM6-p.Lys1584Glu, are not sensitive to flavagline stimulation. In conclusion, we have identified flavaglines as potent activators of TRPM6 activity. Our results suggest that flavaglines stimulate TRPM6 via the insulin receptor signaling pathway.

  1. Flavaglines Stimulate Transient Receptor Potential Melastatin Type 6 (TRPM6 Channel Activity.

    Directory of Open Access Journals (Sweden)

    Maxime G Blanchard

    Full Text Available Magnesium (Mg2+ is essential for enzymatic activity, brain function and muscle contraction. Blood Mg2+ concentrations are tightly regulated between 0.7 and 1.1 mM by Mg2+ (reabsorption in kidney and intestine. The apical entry of Mg2+ in (reabsorbing epithelial cells is mediated by the transient receptor potential melastatin type 6 (TRPM6 ion channel. Here, flavaglines are described as a novel class of stimulatory compounds for TRPM6 activity. Flavaglines are a group of natural and synthetic compounds that target the ubiquitously expressed prohibitins and thereby affect cellular signaling. By whole-cell patch clamp analyses, it was demonstrated that nanomolar concentrations of flavaglines increases TRPM6 activity by ∼2 fold. The stimulatory effects were dependent on the presence of the alpha-kinase domain of TRPM6, but did not require its phosphotransferase activity. Interestingly, it was observed that two natural occurring TRPM6 mutants with impaired insulin-sensitivity, TRPM6-p.Val1393Ile and TRPM6-p.Lys1584Glu, are not sensitive to flavagline stimulation. In conclusion, we have identified flavaglines as potent activators of TRPM6 activity. Our results suggest that flavaglines stimulate TRPM6 via the insulin receptor signaling pathway.

  2. Flavaglines Stimulate Transient Receptor Potential Melastatin Type 6 (TRPM6) Channel Activity

    Science.gov (United States)

    Verkaart, Sjoerd A. J.; Lameris, Anke L.; Basmadjian, Christine; Zhao, Qian; Désaubry, Laurent; Bindels, René J. M.; Hoenderop, Joost G. J.

    2015-01-01

    Magnesium (Mg2+) is essential for enzymatic activity, brain function and muscle contraction. Blood Mg2+ concentrations are tightly regulated between 0.7 and 1.1 mM by Mg2+ (re)absorption in kidney and intestine. The apical entry of Mg2+ in (re)absorbing epithelial cells is mediated by the transient receptor potential melastatin type 6 (TRPM6) ion channel. Here, flavaglines are described as a novel class of stimulatory compounds for TRPM6 activity. Flavaglines are a group of natural and synthetic compounds that target the ubiquitously expressed prohibitins and thereby affect cellular signaling. By whole-cell patch clamp analyses, it was demonstrated that nanomolar concentrations of flavaglines increases TRPM6 activity by ∼2 fold. The stimulatory effects were dependent on the presence of the alpha-kinase domain of TRPM6, but did not require its phosphotransferase activity. Interestingly, it was observed that two natural occurring TRPM6 mutants with impaired insulin-sensitivity, TRPM6-p.Val1393Ile and TRPM6-p.Lys1584Glu, are not sensitive to flavagline stimulation. In conclusion, we have identified flavaglines as potent activators of TRPM6 activity. Our results suggest that flavaglines stimulate TRPM6 via the insulin receptor signaling pathway. PMID:25774985

  3. Aldose reductase mediates retinal microglia activation.

    Science.gov (United States)

    Chang, Kun-Che; Shieh, Biehuoy; Petrash, J Mark

    2016-04-29

    Retinal microglia (RMG) are one of the major immune cells in charge of surveillance of inflammatory responses in the eye. In the absence of an inflammatory stimulus, RMG reside predominately in the ganglion layer and inner or outer plexiform layers. However, under stress RMG become activated and migrate into the inner nuclear layer (INL) or outer nuclear layer (ONL). Activated RMG in cell culture secrete pro-inflammatory cytokines in a manner sensitive to downregulation by aldose reductase inhibitors. In this study, we utilized CX3CR1(GFP) mice carrying AR mutant alleles to evaluate the role of AR on RMG activation and migration in vivo. When tested on an AR(WT) background, IP injection of LPS induced RMG activation and migration into the INL and ONL. However, this phenomenon was largely prevented by AR inhibitors or in AR null mice, or was exacerbated in transgenic mice that over-express AR. LPS-induced increases in ocular levels of TNF-α and CX3CL-1 in WT mice were substantially lower in AR null mice or were reduced by AR inhibitor treatment. These studies demonstrate that AR expression in RMG may contribute to the proinflammatory phenotypes common to various eye diseases such as uveitis and diabetic retinopathy.

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

    Science.gov (United States)

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

    2017-01-01

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

  5. 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.

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

    Science.gov (United States)

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

    2015-03-09

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

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

    CERN Document Server

    Roth, Alexander

    2016-01-01

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

  8. 5-HT1A receptors modulate small-conductance Ca2+-activated K+ channels

    DEFF Research Database (Denmark)

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

    2004-01-01

    Small-conductance calcium-activated potassium channels (SK) are responsible for the medium afterhyperpolarisation (mAHP) following action potentials in neurons. Here we tested the ability of serotonin (5-HT) to modulate the activity of SK channels by coexpressing 5-HT1A receptors with different...... subtypes of SK channels (SK1, SK2, and SK3) in Xenopus laevis oocytes. SK channels were activated by intracellular injection of Cd2+. Subsequent activation of 5-HT1A receptors by 8-OH-DPAT always produced an inhibition of the SK current, showing the existence of a specific pathway between the receptor...

  9. Sodium-calcium exchanger and R-type Ca(2+) channels mediate spontaneous [Ca(2+)]i oscillations in magnocellular neurones of the rat supraoptic nucleus.

    Science.gov (United States)

    Kortus, Stepan; Srinivasan, Chinnapaiyan; Forostyak, Oksana; Zapotocky, Martin; Ueta, Yoichi; Sykova, Eva; Chvatal, Alexandr; Verkhratsky, Alexei; Dayanithi, Govindan

    2016-06-01

    Isolated supraoptic neurones generate spontaneous [Ca(2+)]i oscillations in isolated conditions. Here we report in depth analysis of the contribution of plasmalemmal ion channels (Ca(2+), Na(+)), Na(+)/Ca(2+) exchanger (NCX), intracellular Ca(2+) release channels (InsP3Rs and RyRs), Ca(2+) storage organelles, plasma membrane Ca(2+) pump and intracellular signal transduction cascades into spontaneous Ca(2+) activity. While removal of extracellular Ca(2+) or incubation with non-specific voltage-gated Ca(2+) channel (VGCC) blocker Cd(2+) suppressed the oscillations, neither Ni(2+) nor TTA-P2, the T-type VGCC blockers, had an effect. Inhibitors of VGCC nicardipine, ω-conotoxin GVIA, ω-conotoxin MVIIC, ω-agatoxin IVA (for L-, N-, P and P/Q-type channels, respectively) did not affect [Ca(2+)]i oscillations. In contrast, a specific R-type VGCC blocker SNX-482 attenuated [Ca(2+)]i oscillations. Incubation with TTX had no effect, whereas removal of the extracellular Na(+) or application of an inhibitor of the reverse operation mode of Na(+)/Ca(2+) exchanger KB-R7943 blocked the oscillations. The mitochondrial uncoupler CCCP irreversibly blocked spontaneous [Ca(2+)]i activity. Exposure of neurones to Ca(2+) mobilisers (thapsigargin, cyclopiazonic acid, caffeine and ryanodine); 4-aminopyridine (A-type K(+) current blocker); phospholipase C and adenylyl cyclase pathways blockers U-73122, Rp-cAMP, SQ-22536 and H-89 had no effect. Oscillations were blocked by GABA, but not by glutamate, apamin or dynorphin. In conclusion, spontaneous oscillations in magnocellular neurones are mediated by a concerted action of R-type Ca(2+) channels and the NCX fluctuating between forward and reverse modes.

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

    Directory of Open Access Journals (Sweden)

    Lei Ray Zhong

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

  11. 酸敏感离子通道参与伤害性感受的研究%Advance in nociception mediated by acid sensing ion channels

    Institute of Scientific and Technical Information of China (English)

    刘鹤; 曹君利

    2013-01-01

    背景 组织酸化是炎症、缺血/缺氧、骨质破坏等多种疼痛条件下的共同病理特征.酸敏感离子通道(acid-sensingion channels,ASICs)是一类兴奋性阳离子通道,表达在神经系统,可直接被细胞外质子激活,介导组织酸化所致的伤害性感受. 目的 以ASICs为疼痛治疗靶标,将为疼痛治疗提供一条新途径. 内容 综述ASICs参与组织酸化所致伤害性感受的相关研究. 趋向 近年来,研究发现ASICs在介导组织酸化所致伤害性感受过程中发挥重要作用,以ASICs为靶点,将为开发新型镇痛药物和疼痛治疗提供新思路.%Background Tissue acidosis is a common pathological feature of many painful conditions including inflammation,ischemia and bone destruction.Acid sensing ion channels (ASICs) are excitatory cation channels directly activated by extracellular protons that are expressed in the nervous system,and mediate nociception indcued by tissue acidosis.Objective It will provide a new approach to take ASICs for pain treatment targets.Content The studies of ASICs in mediating nociception associated with tissue acidosis is reviewed.Trend Recent studies show that ASICs play a key role in mediating nociception associated with tissue acidosis,and it will provide a novel approach for development new analgesic drugs and pain treatment targeted ASICs.

  12. N- and L-type voltage-gated calcium channels mediate fast calcium transients in axonal shafts of mouse peripheral nerve.

    Directory of Open Access Journals (Sweden)

    Ruxandra eBarzan

    2016-06-01

    Full Text Available In the peripheral nervous system a vast number of axons are accommodated within fiber bundles that constitute peripheral nerves. A major function of peripheral axons is to propagate action potentials along their length, and hence they are equipped with Na+ and K+ channels, which ensure successful generation, conduction and termination of each action potential. However little is known about Ca2+ ion channels expressed along peripheral axons and their possible functional significance. The goal of the present study was to test whether voltage-gated Ca2+ channels (VGCCs are present along peripheral nerve axons in situ and mediate rapid activity-dependent Ca2+ elevations under physiological circumstances. To address this question we used mouse sciatic nerve slices, Ca2+ indicator Oregon Green BAPTA-1, and 2-photon Ca2+ imaging in fast line scan mode (500 Hz. We report that transient increases in intra-axonal Ca2+ concentration take place along peripheral nerve axons in situ when axons are stimulated electrically with single pulses. Furthermore, we show for the first time that Ca2+ transients in peripheral nerves are fast, i.e. occur in a millisecond time-domain. Combining Ca2+ imaging and pharmacology with specific blockers of different VGCCs subtypes we demonstrate that Ca2+ transients in peripheral nerves are mediated mainly by N-type and L-type VGCCs. Discovery of fast Ca2+ entry into the axonal shafts through VGCCs in peripheral nerves suggests that Ca2+ may be involved in regulation of action potential propagation and/or properties in this system, or mediate neurotransmitter release along peripheral axons as it occurs in the optic nerve and white matter of the central nervous system.

  13. N- and L-Type Voltage-Gated Calcium Channels Mediate Fast Calcium Transients in Axonal Shafts of Mouse Peripheral Nerve.

    Science.gov (United States)

    Barzan, Ruxandra; Pfeiffer, Friederike; Kukley, Maria

    2016-01-01

    In the peripheral nervous system (PNS) a vast number of axons are accommodated within fiber bundles that constitute peripheral nerves. A major function of peripheral axons is to propagate action potentials along their length, and hence they are equipped with Na(+) and K(+) channels, which ensure successful generation, conduction and termination of each action potential. However little is known about Ca(2+) ion channels expressed along peripheral axons and their possible functional significance. The goal of the present study was to test whether voltage-gated Ca(2+) channels (VGCCs) are present along peripheral nerve axons in situ and mediate rapid activity-dependent Ca(2+) elevations under physiological circumstances. To address this question we used mouse sciatic nerve slices, Ca(2+) indicator Oregon Green BAPTA-1, and 2-photon Ca(2+) imaging in fast line scan mode (500 Hz). We report that transient increases in intra-axonal Ca(2+) concentration take place along peripheral nerve axons in situ when axons are stimulated electrically with single pulses. Furthermore, we show for the first time that Ca(2+) transients in peripheral nerves are fast, i.e., occur in a millisecond time-domain. Combining Ca(2+) imaging and pharmacology with specific blockers of different VGCCs subtypes we demonstrate that Ca(2+) transients in peripheral nerves are mediated mainly by N-type and L-type VGCCs. Discovery of fast Ca(2+) entry into the axonal shafts through VGCCs in peripheral nerves suggests that Ca(2+) may be involved in regulation of action potential propagation and/or properties in this system, or mediate neurotransmitter release along peripheral axons as it occurs in the optic nerve and white matter of the central nervous system (CNS).

  14. The Voltage-dependent Anion Channel 1 Mediates Amyloid β Toxicity and Represents a Potential Target for Alzheimer Disease Therapy.

    Science.gov (United States)

    Smilansky, Angela; Dangoor, Liron; Nakdimon, Itay; Ben-Hail, Danya; Mizrachi, Dario; Shoshan-Barmatz, Varda

    2015-12-25

    The voltage-dependent anion channel 1 (VDAC1), found in the mitochondrial outer membrane, forms the main interface between mitochondrial and cellular metabolisms, mediates the passage of a variety of molecules across the mitochondrial outer membrane, and is central to mitochondria-mediated apoptosis. VDAC1 is overexpressed in post-mortem brains of Alzheimer disease (AD) patients. The development and progress of AD are associated with mitochondrial dysfunction resulting from the cytotoxic effects of accumulated amyloid β (Aβ). In this study we demonstrate the involvement of VDAC1 and a VDAC1 N-terminal peptide (VDAC1-N-Ter) in Aβ cell penetration and cell death induction. Aβ directly interacted with VDAC1 and VDAC1-N-Ter, as monitored by VDAC1 channel conductance, surface plasmon resonance, and microscale thermophoresis. Preincubated Aβ interacted with bilayer-reconstituted VDAC1 and increased its conductance ∼ 2-fold. Incubation of cells with Aβ resulted in mitochondria-mediated apoptotic cell death. However, the presence of non-cell-penetrating VDAC1-N-Ter peptide prevented Aβ cellular entry and Aβ-induced mitochondria-mediated apoptosis. Likewise, silencing VDAC1 expression by specific siRNA prevented Aβ entry into the cytosol as well as Aβ-induced toxicity. Finally, the mode of Aβ-mediated action involves detachment of mitochondria-bound hexokinase, induction of VDAC1 oligomerization, and cytochrome c release, a sequence of events leading to apoptosis. As such, we suggest that Aβ-mediated toxicity involves mitochondrial and plasma membrane VDAC1, leading to mitochondrial dysfunction and apoptosis induction. The VDAC1-N-Ter peptide targeting Aβ cytotoxicity is thus a potential new therapeutic strategy for AD treatment.

  15. SUMOylation of p53 mediates interferon activities

    Science.gov (United States)

    Marcos-Villar, Laura; Pérez-Girón, José V; Vilas, Jéssica M; Soto, Atenea; de la Cruz-Hererra, Carlos F; Lang, Valerie; Collado, Manuel; Vidal, Anxo; Rodríguez, Manuel S; Muñoz-Fontela, César; Rivas, Carmen

    2013-01-01

    There is growing evidence that many host proteins involved in innate and intrinsic immunity are regulated by SUMOylation, and that SUMO contributes to the regulatory process that governs the initiation of the type I interferon (IFN) response. The tumor suppressor p53 is a modulator of the IFN response that plays a role in virus-induced apoptosis and in IFN-induced senescence. Here we demonstrate that IFN treatment increases the levels of SUMOylated p53 and induces cellular senescence through a process that is partially dependent upon SUMOylation of p53. Similarly, we show that vesicular stomatitis virus (VSV) infection induces p53 SUMOylation, and that this modification favors the control of VSV replication. Thus, our study provides evidence that IFN signaling induces p53 SUMOylation, which results in the activation of a cellular senescence program and contributes to the antiviral functions of interferon. PMID:23966171

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

    Science.gov (United States)

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

    2015-12-01

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

  17. LIME mediates immunological synapse formation through activation of VAV.

    Science.gov (United States)

    Son, Myoungsun; Park, Inyoung; Lee, Ok-Hee; Rhee, Inmoo; Park, Changwon; Yun, Yungdae

    2012-04-01

    Lck Interacting Membrane protein (LIME) was previously characterized as a transmembrane adaptor protein mediating TCR-dependent T cell activation. Here, we show that LIME associates with Vav in response to TCR stimulation and is required for Vav guanine nucleotide exchange factor (GEF) activity for Rac1. Consistent with this finding, actin polymerization at the immunological synapse (IS) was markedly enhanced by overexpression of LIME, but was reduced by expression of a LIME shRNA. Moreover, TCR-mediated cell adhesion to ICAM-1, laminin, or fibronectin was downregulated by expression of LIME shRNA. In addition, in the IS, LIME but not LAT was found to localize at the peripheral-supramolecular activation cluster (p-SMAC) where the integrins were previously shown to be localized. Together, these results establish LIME as a transmembrane adaptor protein linking TCR stimulation to IS formation and integrin activation through activation of Vav.

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

    OpenAIRE

    Cerda, Oscar; Trimmer, James S.

    2011-01-01

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

  19. KCNN Genes that Encode Small-Conductance Ca2+-Activated K+ Channels Influence Alcohol and Drug Addiction.

    Science.gov (United States)

    Padula, Audrey E; Griffin, William C; Lopez, Marcelo F; Nimitvilai, Sudarat; Cannady, Reginald; McGuier, Natalie S; Chesler, Elissa J; Miles, Michael F; Williams, Robert W; Randall, Patrick K; Woodward, John J; Becker, Howard C; Mulholland, Patrick J

    2015-07-01

    Small-conductance Ca(2+)-activated K(+) (KCa2) channels control neuronal excitability and synaptic plasticity, and have been implicated in substance abuse. However, it is unknown if genes that encode KCa2 channels (KCNN1-3) influence alcohol and drug addiction. In the present study, an integrative functional genomics approach shows that genetic datasets for alcohol, nicotine, and illicit drugs contain the family of KCNN genes. Alcohol preference and dependence QTLs contain KCNN2 and KCNN3, and Kcnn3 transcript levels in the nucleus accumbens (NAc) of genetically diverse BXD strains of mice predicted voluntary alcohol consumption. Transcript levels of Kcnn3 in the NAc negatively correlated with alcohol intake levels in BXD strains, and alcohol dependence enhanced the strength of this association. Microinjections of the KCa2 channel inhibitor apamin into the NAc increased alcohol intake in control C57BL/6J mice, while spontaneous seizures developed in alcohol-dependent mice following apamin injection. Consistent with this finding, alcohol dependence enhanced the intrinsic excitability of medium spiny neurons in the NAc core and reduced the function and protein expression of KCa2 channels in the NAc. Altogether, these data implicate the family of KCNN genes in alcohol, nicotine, and drug addiction, and identify KCNN3 as a mediator of voluntary and excessive alcohol consumption. KCa2.3 channels represent a promising novel target in the pharmacogenetic treatment of alcohol and drug addiction.

  20. Hepatic ATGL mediates PPAR-α signaling and fatty acid channeling through an L-FABP independent mechanism.

    Science.gov (United States)

    Ong, Kuok Teong; Mashek, Mara T; Davidson, Nicholas O; Mashek, Douglas G

    2014-05-01

    Adipose TG lipase (ATGL) catalyzes the rate-limiting step in TG hydrolysis in most tissues. We have shown that hepatic ATGL preferentially channels hydrolyzed FAs to β-oxidation and induces PPAR-α signaling. Previous studies have suggested that liver FA binding protein (L-FABP) transports FAs from lipid droplets to the nucleus for ligand delivery and to the mitochondria for β-oxidation. To determine if L-FABP is involved in ATGL-mediated FA channeling, we used adenovirus-mediated suppression or overexpression of hepatic ATGL in either WT or L-FABP KO mice. Hepatic ATGL knockdown increased liver weight and TG content of overnight fasted mice regardless of genotype. L-FABP deletion did not impair the effects of ATGL overexpression on the oxidation of hydrolyzed FAs in primary hepatocyte cultures or on serum β-hydroxybutyrate concentrations in vivo. Moreover, L-FABP deletion did not influence the effects of ATGL knockdown or overexpression on PPAR-α target gene expression. Taken together, we conclude that L-FABP is not required to channel ATGL-hydrolyzed FAs to mitochondria for β-oxidation or the nucleus for PPAR-α regulation.

  1. The Pyrexia transient receptor potential channel mediates circadian clock synchronization to low temperature cycles in Drosophila melanogaster.

    Science.gov (United States)

    Wolfgang, Werner; Simoni, Alekos; Gentile, Carla; Stanewsky, Ralf

    2013-10-07

    Circadian clocks are endogenous approximately 24 h oscillators that temporally regulate many physiological and behavioural processes. In order to be beneficial for the organism, these clocks must be synchronized with the environmental cycles on a daily basis. Both light : dark and the concomitant daily temperature cycles (TCs) function as Zeitgeber ('time giver') and efficiently entrain circadian clocks. The temperature receptors mediating this synchronization have not been identified. Transient receptor potential (TRP) channels function as thermo-receptors in animals, and here we show that the Pyrexia (Pyx) TRP channel mediates temperature synchronization in Drosophila melanogaster. Pyx is expressed in peripheral sensory organs (chordotonal organs), which previously have been implicated in temperature synchronization. Flies deficient for Pyx function fail to synchronize their behaviour to TCs in the lower range (16-20°C), and this deficit can be partially rescued by introducing a wild-type copy of the pyx gene. Synchronization to higher TCs is not affected, demonstrating a specific role for Pyx at lower temperatures. In addition, pyx mutants speed up their clock after being exposed to TCs. Our results identify the first TRP channel involved in temperature synchronization of circadian clocks.

  2. Development and Testing of an In Vitro Assay for Screening of Potential Therapeutic Agents Active against Na Channel Neurotoxins

    Science.gov (United States)

    1991-04-12

    to produce approximately half-maximal effects mediated through these different sodium channel sites in the assay. Thus, the binding of [3HJBTX-B should...experiments with I3H]STX, yielding the unexpected result that effects of HM-197 are not mediated through the TTX/STX sodium channel binding site. Additional...Scorpion toxin; Screening; nA Pyrethroids; Radioligand binding; Synaptoneurosomes; RA 1 ; nA I ~ I ITherapeutic agents; Sodium channel 19. ABSTRACT

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

    Science.gov (United States)

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

    2017-03-21

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

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

    DEFF Research Database (Denmark)

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

    2003-01-01

    Big-conductance Ca(2+)-activated K(+) channels (BK channels) may play an important role in the regulation of epithelial salt and water transport, but little is known about the expression level and the precise localization of BK channels in epithelia. The aim of the present study was to quantify...... cells. In conclusion, the significant and distinct expression of BK channels in epithelia, combined with their strict regulation, indicate that these channels may play an important role in the overall regulation of salt and water transport....

  5. Non-IgE mediated mast cell activation

    NARCIS (Netherlands)

    Yu, Yingxin; Blokhuis, Bart R; Garssen, Johan; Redegeld, Frank A

    2016-01-01

    Mast cells are crucial effector cells in allergic reactions, where IgE is the best known mechanism to trigger their degranulation and release of a vast array of allergic mediators. However, IgE is not the only component to stimulate these cells to degranulate, while mast cell activation can also res

  6. Non-IgE mediated mast cell activation

    NARCIS (Netherlands)

    Yu, Yingxin; Blokhuis, Bart R; Garssen, Johan; Redegeld, Frank A

    2015-01-01

    Mast cells are crucial effector cells in allergic reactions, where IgE is the best known mechanism to trigger their degranulation and release of a vast array of allergic mediators. However, IgE is not the only component to stimulate these cells to degranulate, while mast cell activation can also res

  7. 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.

  8. How to save the WIMP: global analysis of a dark matter model with two s-channel mediators

    CERN Document Server

    Duerr, Michael; Schmidt-Hoberg, Kai; Schwetz, Thomas; Vogl, Stefan

    2016-01-01

    A reliable comparison of different dark matter (DM) searches requires models that satisfy certain consistency requirements like gauge invariance and perturbative unitarity. As a well-motivated example, we study two-mediator DM (2MDM). The model is based on a spontaneously broken $U(1)'$ gauge symmetry and contains a Majorana DM particle as well as two $s$-channel mediators, one vector (the $Z'$) and one scalar (the dark Higgs). We perform a global scan over the parameters of the model assuming that the DM relic density is obtained by thermal freeze-out in the early Universe and imposing a large set of constraints: direct and indirect DM searches, monojet, dijet and dilepton searches at colliders, Higgs observables, electroweak precision tests and perturbative unitarity. We conclude that thermal DM is only allowed either close to an $s$-channel resonance or if at least one mediator is lighter than the DM particle. In these cases a thermal DM abundance can be obtained although DM couplings to the Standard Model...

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

    Science.gov (United States)

    Xu, Yan; Shinomiya, Misato; Harada, Atsushi

    2016-03-16

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

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

    Science.gov (United States)

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

    2010-01-22

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

  11. CFTR mediates bicarbonate-dependent activation of miR-125b in preimplantation embryo development

    Institute of Scientific and Technical Information of China (English)

    Yong Chao Lu; Alvin Chun Hang Ma; Anskar Yu Hung Leung; He Feng Huang; Hsiao Chang Chan; Hui Chen; Kin Lam Fok; Lai Ling Tsang; Mei Kuen Yu; Xiao Hu Zhang; Jing Chen; Xiaohua Jiang; Yiu Wa Chung

    2012-01-01

    Although HCO3-is known to be required for early embryo development,its exact role remains elusive.Here we report that HCO3-acts as an environmental cue in regulating miR-125b expression through CFTR-mediated influx during preimplantation embryo development.The results show that the effect of HCO3-on preimplantation embryo development can be suppressed by interfering the function of a HCO3--conducting channel,CFTR,by a specific inhibitor or gene knockout.Removal of extracellular HCO3-or inhibition of CFTR reduces miR-125b expression in 2 cell-stage mouse embryos.Knockdown of miR-125b mimics the effect of HCO3-removal and CFTR inhibition,while injection of miR-125b precursor reverses it.Downregulation of miR-125b upregulates p53 cascade in both human and mouse embryos.The activation of miR-125b is shown to be mediated by sAC/PKA-dependent nuclear shuttling of NF-KB.These results have revealed a critical role of CFTR in signal transduction linking the environmental HCO3-to activation of miR-125b during preimplantation embryo development and indicated the importance of ion channels in regulation of miRNAs.

  12. Dehydrocostuslactone, a sesquiterpene lactone activates wild-type and ΔF508 mutant CFTR chloride channel.

    Science.gov (United States)

    Wang, Xue; Zhang, Yao-Fang; Yu, Bo; Yang, Shuang; Luan, Jian; Liu, Xin; Yang, Hong

    2013-01-01

    Cystic fibrosis transmembrane conductance regulator (CFTR) represents the main cAMP-activated Cl⁻ channel expressed in the apical membrane of serous epithelial cells. Both deficiency and overactivation of CFTR may cause fluid and salt secretion related diseases. The aim of this study was to identify natural compounds that are able to stimulate wild-type (wt) and ΔF508 mutant CFTR channel activities in CFTR-expressing Fischer rat thyroid (FRT) cells. We found that dehydrocostuslactone [DHC, (3aS, 6aR, 9aR, 9bS)-decahydro-3,6,9-tris (methylene) azuleno [4,5-b] furan-2(3H)-one)] dose dependently potentiates both wt and ΔF508 mutant CFTR-mediated iodide influx in cell-based fluorescent assays and CFTR-mediated Cl⁻ currents in short-circuit current studies, and the activations could be reversed by the CFTR inhibitor CFTRinh-172. Maximal CFTR-mediated apical Cl⁻ current secretion in CFTR-expressing FRT cells was stimulated by 100 μM DHC. Determination of intracellular cAMP content showed that DHC modestly but significantly increased cAMP level in FRT cells, but cAMP elevation effects contributed little to DHC-stimulated iodide influx. DHC also stimulated CFTR-mediated apical Cl⁻ current secretion in FRT cells expressing ΔF508-CFTR. Subsequent studies demonstrated that activation of CFTR by DHC is forskolin dependent. DHC represents a new class of CFTR potentiators that may have therapeutic potential in CFTR-related diseases.

  13. Opposing effects of the anesthetic propofol at pentameric ligand-gated ion channels mediated by a common site

    DEFF Research Database (Denmark)

    Lynagh, Timothy Peter; Laube, Bodo

    2014-01-01

    Propofol is an intravenous general anesthetic that alters neuronal excitability by modulating agonist responses of pentameric ligand-gated ion channels (pLGICs). Evidence suggests that propofol enhancement of anion-selective pLGICs is mediated by a binding site between adjacent subunits, whereas...... in Xenopus laevis oocytes with electrophysiology. The Haemonchus contortus AVR-14B GluCl was inhibited by propofol with an IC50 value of 252 ± 48 μM, providing the first example of propofol inhibition of an anion-selective pLGIC. Remarkably, inhibition was converted to enhancement by a single I18'S...... substitution in the channel-forming M2 helix (EC50 = 979 ± 88 μM). When a previously identified site between adjacent subunits was disrupted by the M3 G329I substitution, both propofol inhibition and enhancement of GluCls were severely impaired (IC50 and EC50 values could not be calculated). Similarly, when...

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

    Science.gov (United States)

    Thompson, Jill L; Shuttleworth, Trevor J

    2012-01-01

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

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

    DEFF Research Database (Denmark)

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

    2009-01-01

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

  16. Prolactin potentiates the activity of acid-sensing ion channels in female rat primary sensory neurons.

    Science.gov (United States)

    Liu, Ting-Ting; Qu, Zu-Wei; Ren, Cuixia; Gan, Xiong; Qiu, Chun-Yu; Hu, Wang-Ping

    2016-04-01

    Prolactin (PRL) is a polypeptide hormone produced and released from the pituitary and extrapituitary tissues. It regulates activity of nociceptors and causes hyperalgesia in pain conditions, but little is known the molecular mechanism. We report here that PRL can exert a potentiating effect on the functional activity of acid-sensing ion channels (ASICs), key sensors for extracellular protons. First, PRL dose-dependently increased the amplitude of ASIC currents with an EC50 of (5.89 ± 0.28) × 10(-8) M. PRL potentiation of ASIC currents was also pH dependent. Second, PRL potentiation of ASIC currents was blocked by Δ1-9-G129R-hPRL, a PRL receptor antagonist, and removed by intracellular dialysis of either protein kinase C inhibitor GF109203X, protein interacting with C-kinase 1(PICK1) inhibitor FSC-231, or PI3K inhibitor AS605240. Third, PRL altered acidosis-evoked membrane excitability of DRG neurons and caused a significant increase in the amplitude of the depolarization and the number of spikes induced by acid stimuli. Four, PRL exacerbated nociceptive responses to injection of acetic acid in female rats. Finally, PRL displayed a stronger effect on ASIC mediated-currents and nociceptive behavior in intact female rats than OVX female and male rats and thus modulation of PRL may be gender-dependent. These results suggest that PRL up-regulates the activity of ASICs and enhances ASIC mediated nociceptive responses in female rats, which reveal a novel peripheral mechanism underlying PRL involvement in hyperalgesia.

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

    Science.gov (United States)

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

    2012-04-01

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

  18. Activation of mitochondrial ATP-sensitive potassium channels delays ischemia-induced cellular uncoupling in rat heart

    Institute of Scientific and Technical Information of China (English)

    Yue-liangSHEN; Ying-yingCHEN; Xun-dongWU; IainCBRUCE; QiangXIA

    2004-01-01

    AIM: To test the hypothesis that cellular uncoupling induced by myocardial ischemia is mediated by activation of mitochondrial ATP-sensitive potassium channels (mitoKATP). METHODS: Rat hearts were perfused on a Langendorff apparatus and subjected to 40-min ischemia followed by 30-min reperfusion (I/R). Changes in cellular coupling were monitored by measuring whole-tissue resistance. RESULTS: (1) In hearts subjected to I/R, the onset of uncoupling started at (13.3± 1.0) min of ischemia; (2) Ischemic preconditioning (IPC) delayed the onset of uncoupling until (22.7±1.3) min. Blocking mitoKATP channels with 5-hydroxydecanoate (5-HD) before the IPC abolishedthe uncoupling delay [(12.6±1.6)min]; (3) Calcium preconditioning (CPC) had the same effect as IPC. And this effect was reversed by blocking the mitoKATP channel again. In the CPC group the onset of uncoupling occurred after (20.6±1.3) min, and this was canceled by 5-HD [(13.6±0.8) min]; (4) In hearts pretreated with the specific mitoKATP channel opener diazoxide before sustained ischemia, the onset was delayed to (18.4±1.4) min; (5) 5-HD canceled the protective effects of diazoxide (12.6±1.0) min; and both the L-type Ca2+ channel inhibitor verapamiland the free radical scavenger N-(2-mercaptopropionyl)glycine, reduced the extended onset time induced by diazoxide[to (13.3±1.8) min and (13.4±2.1) min, respectively]. CONCLUSION: IPC and CPC delay the onset of cellular uncoupling induced by acute ischemia in rat heart, and the underlying mechanism involves activation of the mitoKATP channels.

  19. Phorbol Ester Modulation of Ca2+ Channels Mediates Nociceptive Transmission in Dorsal Horn Neurones

    Directory of Open Access Journals (Sweden)

    Gary J. Stephens

    2013-05-01

    Full Text Available Phorbol esters are analogues of diacylglycerol which activate C1 domain proteins, such as protein kinase C (PKC. Phorbol ester/PKC pathways have been proposed as potential therapeutic targets for chronic pain states, potentially by phosphorylating proteins involved in nociception, such as voltage-dependent Ca2+ channels (VDCCs. In this brief report, we investigate the potential involvement of CaV2 VDCC subtypes in functional effects of the phorbol ester, phorbol 12-myristate 13-acetate (PMA on nociceptive transmission in the spinal cord. Effects of PMA and of selective pharmacological blockers of CaV2 VDCC subtypes on nociceptive transmission at laminae II dorsal horn neurones were examined in mouse spinal cord slices. Experiments were extended to CaV2.3(−/− mice to complement pharmacological studies. PMA increased the mean frequency of spontaneous postsynaptic currents (sPSCs in dorsal horn neurones, without an effect on event amplitude or half-width. sPSC frequency was reduced by selective VDCC blockers, w-agatoxin-IVA (AgTX; CaV2.1, w-conotoxin-GVIA (CTX; CaV2.2 or SNX-482 (CaV2.3. PMA effects were attenuated in the presence of each VDCC blocker and, also, in CaV2.3(−/− mice. These initial data demonstrate that PMA increases nociceptive transmission at dorsal horn neurones via actions on different CaV2 subtypes suggesting potential anti-nociceptive targets in this system.

  20. Interactions between permeation and gating in the TMEM16B/anoctamin2 calcium-activated chloride channel

    OpenAIRE

    Betto, Giulia; Cherian, O. Lijo; Pifferi, Simone; Cenedese, Valentina; Boccaccio, Anna; Menini, Anna

    2014-01-01

    At least two members of the TMEM16/anoctamin family, TMEM16A (also known as anoctamin1) and TMEM16B (also known as anoctamin2), encode Ca2+-activated Cl− channels (CaCCs), which are found in various cell types and mediate numerous physiological functions. Here, we used whole-cell and excised inside-out patch-clamp to investigate the relationship between anion permeation and gating, two processes typically viewed as independent, in TMEM16B expressed in HEK 293T cells. The permeability ratio se...

  1. Low-Dose Ethanol Preconditioning Protects Against Oxygen-Glucose Deprivation/Reoxygenation-Induced Neuronal Injury By Activating Large Conductance, Ca(2+)-Activated K(+) Channels In Vitro.

    Science.gov (United States)

    Su, Fang; Guo, An-Chen; Li, Wei-Wei; Zhao, Yi-Long; Qu, Zheng-Yi; Wang, Yong-Jun; Wang, Qun; Zhu, Yu-Lan

    2017-02-01

    Increasing evidence suggests that low to moderate ethanol ingestion protects against the deleterious effects of subsequent ischemia/reperfusion; however, the underlying mechanism has not been elucidated. In the present study, we showed that expression of the neuronal large-conductance, Ca(2+)-activated K(+) channel (BKCa) α-subunit was upregulated in cultured neurons exposed to oxygen-glucose deprivation/reoxygenation (OGD/R) compared with controls. Preconditioning with low-dose ethanol (10 mmol/L) increased cell survival rate in neurons subjected to OGD/R, attenuated the OGD/R-induced elevation of cytosolic Ca(2+) levels, and reduced the number of apoptotic neurons. Western blots revealed that ethanol preconditioning upregulated expression of the anti-apoptotic protein Bcl-2 and downregulated the pro-apoptotic protein Bax. The protective effect of ethanol preconditioning was antagonized by a BKCa channel inhibitor, paxilline. Inside-out patches in primary neurons also demonstrated the direct activation of the BKCa channel by 10 mmol/L ethanol. The above results indicated that low-dose ethanol preconditioning exerts its neuroprotective effects by attenuating the elevation of cytosolic Ca(2+) and preventing neuronal apoptosis, and this is mediated by BKCa channel activation.

  2. Dark-matter production through loop-induced processes at the LHC: the s-channel mediator case.

    Science.gov (United States)

    Mattelaer, Olivier; Vryonidou, Eleni

    We show how studies relevant for mono-X searches at the LHC in simplified models featuring a dark-matter candidate and an s-channel mediator can be performed within the MadGraph5_aMC@NLO framework. We focus on gluon-initiated loop-induced processes, mostly relevant to the case where the mediator couples preferentially to third generation quarks and in particular to the top quark. Our implementation allows us to study signatures at hadron colliders involving missing transverse energy plus jets or plus neutral bosons ([Formula: see text]), possibly including the effects of extra radiation by multi-parton merging and matching to the parton shower.

  3. Dark-matter production through loop-induced processes at the LHC: the s-channel mediator case

    Energy Technology Data Exchange (ETDEWEB)

    Mattelaer, Olivier [Durham University, Institute for Particle Physics Phenomenology (IPPP), Durham (United Kingdom); Vryonidou, Eleni [Universite catholique de Louvain, Centre for Cosmology, Particle Physics and Phenomenology (CP3), Louvain-la-Neuve (Belgium)

    2015-09-15

    We show how studies relevant for mono-X searches at the LHC in simplified models featuring a dark-matter candidate and an s-channel mediator can be performed within the MadGraph5{sub a}MC rate at NLO framework. We focus on gluon-initiated loop-induced processes, mostly relevant to the case where the mediator couples preferentially to third generation quarks and in particular to the top quark. Our implementation allows us to study signatures at hadron colliders involving missing transverse energy plus jets or plus neutral bosons (γ,Z,H), possibly including the effects of extra radiation by multi-parton merging and matching to the parton shower. (orig.)

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

    Science.gov (United States)

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

    2012-12-01

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

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

    Directory of Open Access Journals (Sweden)

    Ximena eSteinberg

    2014-05-01

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

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

    Science.gov (United States)

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

    2016-08-01

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

  7. The mechano-activated K+ channels TRAAK and TREK-1 control both warm and cold perception

    OpenAIRE

    Noël, Jacques; Zimmermann, Katharina; Busserolles, Jérome; Deval, Emanuel; Alloui, Abdelkrim; Diochot, Sylvie; Guy, Nicolas; Borsotto, Marc; Reeh, Peter; Eschalier, Alain; Lazdunski, Michel

    2009-01-01

    The sensation of cold or heat depends on the activation of specific nerve endings in the skin. This involves heat- and cold-sensitive excitatory transient receptor potential (TRP) channels. However, we show here that the mechano-gated and highly temperature-sensitive potassium channels of the TREK/TRAAK family, which normally work as silencers of the excitatory channels, are also implicated. They are important for the definition of temperature thresholds and temperature ranges in which excita...

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

    OpenAIRE

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

    2004-01-01

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

  9. Caspase-9 mediates Puma activation in UCN-01-induced apoptosis.

    Science.gov (United States)

    Nie, C; Luo, Y; Zhao, X; Luo, N; Tong, A; Liu, X; Yuan, Z; Wang, C; Wei, Y

    2014-10-30

    The protein kinase inhibitor 7-hydroxystaurosporine (UCN-01) is one of the most potent and frequently used proapoptotic stimuli. The BH3-only molecule of Bcl-2 family proteins has been reported to contribute to UCN-01-induced apoptosis. Here we have found that UCN-01 triggers Puma-induced mitochondrial apoptosis pathway. Our data confirmed that Akt-FoxO3a pathway mediated Puma activation. Importantly, we elucidate the detailed mechanisms of Puma-induced apoptosis. Our data have also demonstrated that caspase-9 is a decisive molecule of Puma induction after UCN-01 treatment. Caspase-9 mediates apoptosis through two kinds of feedback loops. On the one hand, caspase-9 enhances Puma activation by cleaving Bcl-2 and Bcl-xL independent of caspase-3. On the other hand, caspase-9 directly activated caspase-3 in the presence of caspase-3. Caspase-3 could cleave XIAP in an another positive feedback loop to further sensitize cancer cells to UCN-01-induced apoptosis. Therefore, caspase-9 mediates Puma activation to determine the threshold for overcoming chemoresistance in cancer cells.

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

    DEFF Research Database (Denmark)

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

    2003-01-01

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

  11. Cellular Mechanisms of Calcium-Mediated Triggered Activity

    Science.gov (United States)

    Song, Zhen

    Life-threatening cardiac arrhythmias continue to pose a major health problem. Ventricular fibrillation, which is a complex form of electrical wave turbulence in the lower chambers of the heart, stops the heart from pumping and is the largest cause of natural death in the United States. Atrial fibrillation, a related form of wave turbulence in the upper heart chambers, is in turn the most common arrhythmia diagnosed in clinical practice. Despite extensive research to date, mechanisms of cardiac arrhythmias remain poorly understood. It is well established that both spatial disorder of the refractory period of heart cells and triggered activity (TA) jointly contribute to the initiation and maintenance of arrhythmias. TA broadly refers to the abnormal generation of a single or a sequence of abnormal excitation waves from a small submillimeter region of the heart in the interval of time between two normal waves generated by the heart's natural pacemaker (the sinoatrial node). TA has been widely investigated experimentally and occurs in several pathological conditions where the intracellular concentration of free Ca2+ ions in heart cells becomes elevated. Under such conditions, Ca2+ can be spontaneously released from intracellular stores, thereby driving an electrogenic current that exchanges 3Na+ ions for one Ca2+ ion across the cell membrane. This current in turn depolarizes the membrane of heart cells after a normal excitation. If this calcium-mediated "delayed after depolarization'' (DAD) is sufficiently large, it can generate an action potential. While the arrhythmogenic importance of spontaneous Ca2+ release and DADs is well appreciated, the conditions under which they occur in heart pathologies remain poorly understood. Calcium overload is only one factor among several other factors that can promote DADs, including sympathetic nerve stimulation, different expression levels of membrane ion channels and calcium handling proteins, and different mutations of those

  12. Ferroelectric active models of ion channels in biomembranes.

    Science.gov (United States)

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

    1994-06-21

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

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

    Science.gov (United States)

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

    2014-05-21

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

  14. 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.

  15. PLCγ-activated signalling is essential for TrkB mediated sensory neuron structural plasticity

    Directory of Open Access Journals (Sweden)

    Rocha-Sanchez Sonia M

    2010-10-01

    Full Text Available Abstract Background The vestibular system provides the primary input of our sense of balance and spatial orientation. Dysfunction of the vestibular system can severely affect a person's quality of life. Therefore, understanding the molecular basis of vestibular neuron survival, maintenance, and innervation of the target sensory epithelia is fundamental. Results Here we report that a point mutation at the phospholipase Cγ (PLCγ docking site in the mouse neurotrophin tyrosine kinase receptor TrkB (Ntrk2 specifically impairs fiber guidance inside the vestibular sensory epithelia, but has limited effects on the survival of vestibular sensory neurons and growth of afferent processes toward the sensory epithelia. We also show that expression of the TRPC3 cation calcium channel, whose activity is known to be required for nerve-growth cone guidance induced by brain-derived neurotrophic factor (BDNF, is altered in these animals. In addition, we find that absence of the PLCγ mediated TrkB signalling interferes with the transformation of bouton type afferent terminals of vestibular dendrites into calyces (the largest synaptic contact of dendrites known in the mammalian nervous system on type I vestibular hair cells; the latter are normally distributed in these mutants as revealed by an unaltered expression pattern of the potassium channel KCNQ4 in these cells. Conclusions These results demonstrate a crucial involvement of the TrkB/PLCγ-mediated intracellular signalling in structural aspects of sensory neuron plasticity.

  16. Higher-order QCD predictions for dark matter production at the LHC in simplified models with s-channel mediators.

    Science.gov (United States)

    Backović, Mihailo; Krämer, Michael; Maltoni, Fabio; Martini, Antony; Mawatari, Kentarou; Pellen, Mathieu

    Weakly interacting dark matter particles can be pair-produced at colliders and detected through signatures featuring missing energy in association with either QCD/EW radiation or heavy quarks. In order to constrain the mass and the couplings to standard model particles, accurate and precise predictions for production cross sections and distributions are of prime importance. In this work, we consider various simplified models with s-channel mediators. We implement such models in the FeynRules/MadGraph5_aMC@NLO framework, which allows to include higher-order QCD corrections in realistic simulations and to study their effect systematically. As a first phenomenological application, we present predictions for dark matter production in association with jets and with a top-quark pair at the LHC, at next-to-leading order accuracy in QCD, including matching/merging to parton showers. Our study shows that higher-order QCD corrections to dark matter production via s-channel mediators have a significant impact not only on total production rates, but also on shapes of distributions. We also show that the inclusion of next-to-leading order effects results in a sizeable reduction of the theoretical uncertainties.

  17. Simplified DM models with the full SM gauge symmetry : the case of $t$-channel colored scalar mediators

    CERN Document Server

    Ko, P; Park, Myeonghun; Yokoya, Hiroshi

    2016-01-01

    The general strategy for dark matter (DM) searches at colliders currently relies on simplified models. In this paper, we propose a new $t$-channel UV-complete simplified model that improves the existing simplified DM models in two important respects: (i) we impose the full SM gauge symmetry including the fact that the left-handed and the right-handed fermions have two independent mediators with two independent couplings, and (ii) we include the renormalization group evolution when we derive the effective Lagrangian for DM-nucleon scattering from the underlying UV complete models by integrating out the $t$-channel mediators. The first improvement will introduce a few more new parameters compared with the existing simplified DM models. In this study we look at the effect this broader set of free parameters has on direct detection and the mono-$X$ + MET ($X$=jet,$W,Z$) signatures at 13 TeV LHC while maintaining gauge invariance of the simplified model under the full SM gauge group. We find that the direct detect...

  18. Higher-order QCD predictions for dark matter production at the LHC in simplified models with s-channel mediators

    Energy Technology Data Exchange (ETDEWEB)

    Backović, Mihailo [Centre for Cosmology, Particle Physics and Phenomenology (CP3), Université catholique de Louvain, 1348, Louvain-la-Neuve (Belgium); Krämer, Michael [Institute for Theoretical Particle Physics and Cosmology, RWTH Aachen University, 52056, Aachen (Germany); Maltoni, Fabio; Martini, Antony [Centre for Cosmology, Particle Physics and Phenomenology (CP3), Université catholique de Louvain, 1348, Louvain-la-Neuve (Belgium); Mawatari, Kentarou, E-mail: kentarou.mawatari@vub.ac.be [Theoretische Natuurkunde and IIHE/ELEM, Vrije Universiteit Brussel, and International Solvay Institutes, Pleinlaan 2, 1050, Brussels (Belgium); Pellen, Mathieu [Institute for Theoretical Particle Physics and Cosmology, RWTH Aachen University, 52056, Aachen (Germany)

    2015-10-07

    Weakly interacting dark matter particles can be pair-produced at colliders and detected through signatures featuring missing energy in association with either QCD/EW radiation or heavy quarks. In order to constrain the mass and the couplings to standard model particles, accurate and precise predictions for production cross sections and distributions are of prime importance. In this work, we consider various simplified models with s-channel mediators. We implement such models in the FeynRules/MadGraph5{sub a}MC@NLO framework, which allows to include higher-order QCD corrections in realistic simulations and to study their effect systematically. As a first phenomenological application, we present predictions for dark matter production in association with jets and with a top-quark pair at the LHC, at next-to-leading order accuracy in QCD, including matching/merging to parton showers. Our study shows that higher-order QCD corrections to dark matter production via s-channel mediators have a significant impact not only on total production rates, but also on shapes of distributions. We also show that the inclusion of next-to-leading order effects results in a sizeable reduction of the theoretical uncertainties.

  19. Higher-order QCD predictions for dark matter production at the LHC in simplified models with s-channel mediators

    Energy Technology Data Exchange (ETDEWEB)

    Backovic, Mihailo; Maltoni, Fabio; Martini, Antony [Universite catholique de Louvain, Centre for Cosmology, Particle Physics and Phenomenology (CP3), Louvain-la-Neuve (Belgium); Kraemer, Michael; Pellen, Mathieu [RWTH Aachen University, Institute for Theoretical Particle Physics and Cosmology, Aachen (Germany); Mawatari, Kentarou [Theoretische Natuurkunde and IIHE/ELEM, Vrije Universiteit Brussel, and International Solvay Institutes, Brussels (Belgium)

    2015-10-15

    Weakly interacting dark matter particles can be pair-produced at colliders and detected through signatures featuring missing energy in association with either QCD/EW radiation or heavy quarks. In order to constrain the mass and the couplings to standard model particles, accurate and precise predictions for production cross sections and distributions are of prime importance. In this work, we consider various simplified models with s-channel mediators. We implement such models in the FeynRules/MadGraph5{sub a}MC rate at NLO framework, which allows to include higher-order QCD corrections in realistic simulations and to study their effect systematically. As a first phenomenological application, we present predictions for dark matter production in association with jets and with a top-quark pair at the LHC, at next-to-leading order accuracy in QCD, including matching/merging to parton showers. Our study shows that higher-order QCD corrections to dark matter production via s-channel mediators have a significant impact not only on total production rates, but also on shapes of distributions. We also show that the inclusion of next-to-leading order effects results in a sizeable reduction of the theoretical uncertainties. (orig.)

  20. Calcium-sensing receptor activation contributed to apoptosis stimulates TRPC6 channel in rat neonatal ventricular myocytes

    Energy Technology Data Exchange (ETDEWEB)

    Sun, Yi-hua [Department of Clinical Laboratory, The Second Affiliated Hospital of Harbin Medical University, Harbin 150086 (China); Li, Yong-quan [Harbin Medical University, Harbin 150086 (China); Feng, Shan-li [Department of Clinical Laboratory, The Second Affiliated Hospital of Harbin Medical University, Harbin 150086 (China); Li, Bao-xin; Pan, Zhen-wei [Department of Pharmacology, Harbin Medical University, Harbin 150086 (China); Xu, Chang-qing [Department of Pathophysiology, Harbin Medical University, Harbin 150086 (China); Li, Ting-ting [Department of Clinical Laboratory, The Second Affiliated Hospital of Harbin Medical University, Harbin 150086 (China); Yang, Bao-feng, E-mail: syh200415@yahoo.com.cn [Department of Pharmacology, Harbin Medical University, Harbin 150086 (China)

    2010-04-16

    Capacitative calcium entry (CCE) refers to the influx of calcium through plasma membrane channels activated on depletion of endoplasmic sarcoplasmic/reticulum (ER/SR) Ca{sup 2+} stores, which is performed mainly by the transient receptor potential (TRP) channels. TRP channels are expressed in cardiomyocytes. Calcium-sensing receptor (CaR) is also expressed in rat cardiac tissue and plays an important role in mediating cardiomyocyte apoptosis. However, there are no data regarding the link between CaR and TRP channels in rat heart. In this study, in rat neonatal myocytes, by Ca{sup 2+} imaging, we found that the depletion of ER/SR Ca{sup 2+} stores by thapsigargin (TG) elicited a transient rise in cytoplasmic Ca{sup 2+} ([Ca{sup 2+}]{sub i}), followed by sustained increase depending on extracellular Ca{sup 2+}. But, TRP channels inhibitor (SKF96365), not L-type channels or the Na{sup +}/Ca{sup 2+} exchanger inhibitors, inhibited [Ca{sup 2+}]{sub i} relatively high. Then, we found that the stimulation of CaR with its activator gadolinium chloride (GdCl{sub 3}) or by an increased extracellular Ca{sup 2+}([Ca{sup 2+}]{sub o}) increased the concentration of intracelluar Ca{sup 2+}, whereas, the sustained elevation of [Ca{sup 2+}]{sub i} was reduced in the presence of SKF96365. Similarly, the duration of [Ca{sup 2+}]{sub i} increase was also shortened in the absence of extracellular Ca{sup 2+}. Western blot analysis showed that GdCl{sub 3} increased the expression of TRPC6, which was reversed by SKF96365. Additionally, SKF96365 reduced cardiomyocyte apoptosis induced by GdCl{sub 3}. Our results suggested that CCE exhibited in rat neonatal myocytes and CaR activation induced Ca{sup 2+}-permeable cationic channels TRPCs to gate the CCE, for which TRPC6 was one of the most likely candidates. TRPC6 channel was functionally coupled with CaR to enhance the cardiomyocyte apoptosis.

  1. Activation of TRPC6 channels is essential for lung ischaemia–reperfusion induced oedema in mice

    Science.gov (United States)

    Weissmann, Norbert; Sydykov, Akylbek; Kalwa, Hermann; Storch, Ursula; Fuchs, Beate; Schnitzler, Michael Mederos y; Brandes, Ralf P.; Grimminger, Friedrich; Meissner, Marcel; Freichel, Marc; Offermanns, Stefan; Veit, Florian; Pak, Oleg; Krause, Karl-Heinz; Schermuly, Ralph T.; Brewer, Alison C; Schmidt, Harald H.H.W.; Seeger, Werner; Shah, Ajay M.; Gudermann, Thomas; Ghofrani, Hossein A.; Dietrich, Alexander

    2012-01-01

    Lung ischaemia–reperfusion-induced oedema (LIRE) is a life-threatening condition that causes pulmonary oedema induced by endothelial dysfunction. Here we show that lungs from mice lacking nicotinamide adenine dinucleotide phosphate (NADPH) oxidase (Nox2y/−) or the classical transient receptor potential channel 6 (TRPC6−/−) are protected from LIR-induced oedema (LIRE). Generation of chimeric mice by bone marrow cell transplantation and endothelial-specific Nox2 deletion showed that endothelial Nox2, but not leukocytic Nox2 or TRPC6, are responsible for LIRE. Lung endothelial cells from Nox2- or TRPC6-deficient mice showed attenuated ischaemia-induced Ca2+ influx, cellular shape changes and impaired barrier function. Production of reactive oxygen species was completely abolished in Nox2y/− cells. A novel mechanistic model comprising endothelial Nox2-derived production of superoxide, activation of phospholipase C-γ, inhibition of diacylglycerol (DAG) kinase, DAG-mediated activation of TRPC6 and ensuing LIRE is supported by pharmacological and molecular evidence. This mechanism highlights novel pharmacological targets for the treatment of LIRE. PMID:22337127

  2. 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.

  3. Ca2+-activated K+ (BK) channel inactivation contributes to spike broadening during repetitive firing in the rat lateral amygdala.

    Science.gov (United States)

    Faber, E S Louise; Sah, Pankaj

    2003-10-15

    In many neurons, trains of action potentials show frequency-dependent broadening. This broadening results from the voltage-dependent inactivation of K+ currents that contribute to action potential repolarisation. In different neuronal cell types these K+ currents have been shown to be either slowly inactivating delayed rectifier type currents or rapidly inactivating A-type voltage-gated K+ currents. Recent findings show that inactivation of a Ca2+-dependent K+ current, mediated by large conductance BK-type channels, also contributes to spike broadening. Here, using whole-cell recordings in acute slices, we examine spike broadening in lateral amygdala projection neurons. Spike broadening is frequency dependent and is reversed by brief hyperpolarisations. This broadening is reduced by blockade of voltage-gated Ca2+ channels and BK channels. In contrast, broadening is not blocked by high concentrations of 4-aminopyridine (4-AP) or alpha-dendrotoxin. We conclude that while inactivation of BK-type Ca2+-activated K+ channels contributes to spike broadening in lateral amygdala neurons, inactivation of another as yet unidentified outward current also plays a role.

  4. Stress-mediated p38 activation promotes somatic cell reprogramming

    Institute of Scientific and Technical Information of China (English)

    Xinxiu Xu; Quan Wang; Yuan Long; Ru Zhang; Xiaoyuan Wei; Mingzhe Xing; Haifeng Gu

    2013-01-01

    Environmental stress-mediated adaptation plays essential roles in the evolution of life.Cellular adaptation mechanisms usually involve the regulation of chromatin structure,transcription,mRNA stability and translation,which eventually lead to efficient changes in gene expression.Global epigenetic change is also involved in the reprogramming of somatic cells into induced pluripotent stem (iPS) cells by defined factors.Here we report that environmental stress such as hyperosmosis not only facilitates four factor-mediated reprogramming,but also enhances two or one factor-induced iPS cell generation.Hyperosmosis-induced p38 activation plays a critical role in this process.Constitutive active p38 mimics the positive effect of hyperosmosis,while dominant negative p38 and p38 inhibitor block the effect of hyperosmosis.Further study indicates stress-mediated p38 activation may promote reprogramming by reducing the global DNA methylation level and enhancing the expression of pluripotency genes.Our results demonstrate how simple environmental stress like hyperosmosis helps to alter the fate of cells via intracellular signaling and epigenetic modulation.

  5. Short-term plasticity in turtle dorsal horn neurons mediated by L-type Ca2+ channels

    DEFF Research Database (Denmark)

    Russo, R E; Hounsgaard, J

    1994-01-01

    Windup--the gradual increase of the response--of dorsal horn neurons to repeated activation of primary afferents is an elementary form of short-term plasticity that may mediate central sensitization to pain. In deep dorsal horn neurons of the turtle spinal cord in vitro we report windup of the re......Windup--the gradual increase of the response--of dorsal horn neurons to repeated activation of primary afferents is an elementary form of short-term plasticity that may mediate central sensitization to pain. In deep dorsal horn neurons of the turtle spinal cord in vitro we report windup...

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

    DEFF Research Database (Denmark)

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

    2015-01-01

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

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

    Institute of Scientific and Technical Information of China (English)

    Xiao-liangWANG; Ya-pingPAN

    2004-01-01

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

  8. 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...

  9. Sensitization of voltage activated calcium channel currents for capsaicin in nociceptive neurons by tumor-necrosis-factor-alpha.

    Science.gov (United States)

    Hagenacker, T; Czeschik, J C; Schäfers, M; Büsselberg, D

    2010-01-15

    It is known that application of tumor-necrosis-factor-alpha (TNF-alpha) sensitizes neuronal calcium channels for heat stimuli in rat models of neuropathic pain. This study examines whether TNF-alpha modulates the capsaicin-induced effects after transient receptor potential vanilloid (TRPV)-1 receptor activation on voltage activated calcium channel currents (I(Ca(V))). TRPV-1 receptors are activated by heat and play an important role in the pathogenesis of thermal hyperalgesia in neuropathic pain syndromes, while voltage activated channels are essential for transmission of neuronal signals. Eliciting I(Ca(V)) in DRG neurons of rats by a depolarization from the resting potential to 0 mV, TNF-alpha (100 ng/ml) reduces I(Ca(V)) by 16.9+/-2.2%, while capsaicin (0.1 microM) decreases currents by 27+/-4.3%. Pre-application of TNF-alpha (100 ng/ml) for 24h results in a sensitization of I(Ca(V)) to capsaicin (0.1 microM) with a reduction of 42.8+/-4.4% mediated by TRPV-1. While L-type (36.6+/-5.2%) and P/Q-type currents (35.6+/-4.1%) are also sensitized by TRPV-1 activation, N-type channel currents are most sensitive (74.5+/-7.3%). The capsaicin-induced shift towards the hyperpolarizing voltage range does not occur when TNF-alpha is applied. Summarizing, TNF-alpha sensitizes nociceptive neurons for capsaicin.

  10. Interneuron-mediated inhibition synchronizes neuronal activity during slow oscillation

    Science.gov (United States)

    Chen, Jen-Yung; Chauvette, Sylvain; Skorheim, Steven; Timofeev, Igor; Bazhenov, Maxim

    2012-01-01

    The signature of slow-wave sleep in the electroencephalogram (EEG) is large-amplitude fluctuation of the field potential, which reflects synchronous alternation of activity and silence across cortical neurons. While initiation of the active cortical states during sleep slow oscillation has been intensively studied, the biological mechanisms which drive the network transition from an active state to silence remain poorly understood. In the current study, using a combination of in vivo electrophysiology and thalamocortical network simulation, we explored the impact of intrinsic and synaptic inhibition on state transition during sleep slow oscillation. We found that in normal physiological conditions, synaptic inhibition controls the duration and the synchrony of active state termination. The decline of interneuron-mediated inhibition led to asynchronous downward transition across the cortical network and broke the regular slow oscillation pattern. Furthermore, in both in vivo experiment and computational modelling, we revealed that when the level of synaptic inhibition was reduced significantly, it led to a recovery of synchronized oscillations in the form of seizure-like bursting activity. In this condition, the fast active state termination was mediated by intrinsic hyperpolarizing conductances. Our study highlights the significance of both intrinsic and synaptic inhibition in manipulating sleep slow rhythms. PMID:22641778

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

    Science.gov (United States)

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

    2015-08-01

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

  12. Definition of two agonist types at the mammalian cold-activated channel TRPM8

    Science.gov (United States)

    Janssens, Annelies; Gees, Maarten; Toth, Balazs Istvan; Ghosh, Debapriya; Mulier, Marie; Vennekens, Rudi; Vriens, Joris; Talavera, Karel; Voets, Thomas

    2016-01-01

    Various TRP channels act as polymodal sensors of thermal and chemical stimuli, but the mechanisms whereby chemical ligands impact on TRP channel gating are poorly understood. Here we show that AITC (allyl isothiocyanate; mustard oil) and menthol represent two distinct types of ligands at the mammalian cold sensor TRPM8. Kinetic analysis of channel gating revealed that AITC acts by destabilizing the closed channel, whereas menthol stabilizes the open channel, relative to the transition state. Based on these differences, we classify agonists as either type I (menthol-like) or type II (AITC-like), and provide a kinetic model that faithfully reproduces their differential effects. We further demonstrate that type I and type II agonists have a distinct impact on TRPM8 currents and TRPM8-mediated calcium signals in excitable cells. These findings provide a theoretical framework for understanding the differential actions of TRP channel ligands, with important ramifications for TRP channel structure-function analysis and pharmacology. DOI: http://dx.doi.org/10.7554/eLife.17240.001 PMID:27449282

  13. Endothelin potentiates TRPV1 via ETA receptor-mediated activation of protein kinase C

    Directory of Open Access Journals (Sweden)

    Furkert Jens

    2007-11-01

    Full Text Available Abstract Background Endothelin-1 (ET-1 both stimulates nociceptors and sensitizes them to noxious stimuli, an effect probably mediated by the ETA receptor (ETAR expressed in sensory neurons. The cellular mechanisms of this ET-1-mediated effect are only poorly understood. TRPV1, the heat-, pH- and capsaicin-sensitive cation channel already known to be modulated by a number of cellular mediators released in response to noxious stimuli and during inflammation, is a potential target for the action of ET-1. Results We studied the effects of ET-1 on TRPV1 in sensory neurons from the dorsal root ganglion (DRG and in HEK293 cells coexpressing TRPV1 and the ETAR. Specific 125I-ET-1 binding sites (817 ± 92 fmol/mg were detected in membrane preparations of DRG with an ETAR/ETBR ratio of 60:40. In an immunofluorescence analysis, coexpression of TRPV1 and the ETAR was found in a subpopulation of primary sensory neurons. ET-1 strongly potentiated capsaicin-induced TRPV1 currents in some neurons, and in HEK293 cells co-expressing TRPV1 and the ETAR. Weaker potentiation was observed in HEK293 cells coexpressing TRPV1 and the ETBR. ETAR activation also increased responses to low pH and heat. In HEK293 cells, strong potentiation of TRPV1 like that induced by ET-1 via the ETAR could be induced by PKC activation, but not with activators of the adenylyl cyclase or the PKA pathway. Furthermore, inhibition of PKC with bisindolylmaleimide X (BIM X or mutation of the PKC phosphorylation site S800 completely prevented ETAR-mediated potentiation. Conclusion We conclude that ET-1 potentiates TRPV1 by a PKC-dependent mechanism and that this could play a major role in the algogenic and hyperalgesic effects of ET-1 described in previous studies.

  14. Calcium Transients in Dendrites of Neocortical Neurons Evoked by Single Subthreshold Excitatory Postsynaptic Potentials via Low-Voltage-Activated Calcium Channels

    Science.gov (United States)

    Markram, Henry; Sakmann, Bert

    1994-05-01

    Simultaneous recordings of membrane voltage and concentration of intracellular Ca2+ ([Ca2+]_i) were made in apical dendrites of layer 5 pyramidal cells of rat neocortex after filling dendrites with the fluorescent Ca2+ indicator Calcium Green-1. Subthreshold excitatory postsynaptic potentials (EPSPs), mediated by the activation of glutamate receptor channels, caused a brief increase in dendritic [Ca2+]_i. This rise in dendritic [Ca2+]_i was mediated by the opening of low-voltage-activated Ca2+ channels in the dendritic membrane. The results provide direct evidence that dendrites do not function as passive cables even at low-frequency synaptic activity; rather, a single subthreshold EPSP changes the dendritic membrane conductance by opening Ca2+ channels and generating a [Ca2+]_i transient that may propagate towards the soma. The activation of these Ca2+ channels at a low-voltage threshold is likely to influence the way in which dendritic EPSPs contribute to the electrical activity of the neuron.

  15. Pharmacological activation of small conductance calcium-activated potassium channels with naphtho[1,2-d]thiazol-2-ylamine decreases guinea pig detrusor smooth muscle excitability and contractility.

    Science.gov (United States)

    Parajuli, Shankar P; Soder, Rupal P; Hristov, Kiril L; Petkov, Georgi V

    2012-01-01

    Small conductance Ca²⁺-activated K⁺ (SK) and intermediate conductance Ca(2+)-activated K⁺ (IK) channels are thought to be involved in detrusor smooth muscle (DSM) excitability and contractility. Using naphtho[1,2-d]thiazol-2-ylamine (SKA-31), a novel and highly specific SK/IK channel activator, we investigated whether pharmacological activation of SK/IK channels reduced guinea pig DSM excitability and contractility. We detected the expression of all known isoforms of SK (SK1-SK3) and IK channels at mRNA and protein levels in DSM by single-cell reverse transcription-polymerase chain reaction and Western blot. Using the perforated patch-clamp technique on freshly isolated DSM cells, we observed that SKA-31 (10 μM) increased SK currents, which were blocked by apamin (1 μM), a selective SK channel inhibitor. In current-clamp mode, SKA-31 (10 μM) hyperpolarized the cell resting membrane potential, which was blocked by apamin (1 μM) but not by 1-[(2-chlorophenyl)diphenylmethyl]-1H-pyrazole (TRAM-34) (1 μM), a selective IK channel inhibitor. SKA-31 (10 nM-10 μM) significantly inhibited the spontaneous phasic contraction amplitude, frequency, duration, and muscle force in DSM isolated strips. The SKA-31 inhibitory effects on DSM contractility were blocked by apamin (1 μM) but not by TRAM-34 (1 μM), which did not per se significantly affect DSM spontaneous contractility. SK channel activation with SKA-31 reduced contractions evoked by electrical field stimulation. SKA-31 effects were reversible upon washout. In conclusion, SK channels, but not IK channels, mediate SKA-31 effects in guinea pig DSM. Pharmacological activation of SK channels reduces DSM excitability and contractility and therefore may provide a novel therapeutic approach for controlling bladder dysfunction.

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

    Science.gov (United States)

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

    2015-06-19

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

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

    Directory of Open Access Journals (Sweden)

    Bernat Elvira

    2016-04-01

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

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

    Science.gov (United States)

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

    2012-01-01

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

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

    Directory of Open Access Journals (Sweden)

    Xiaohui eSun

    2012-04-01

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

  1. Cardiac mechanosensitivity and stretch-activated ion channels.

    Science.gov (United States)

    Bett, G C; Sachs, F

    1997-01-01

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

  2. SUMOylation of NaV1.2 channels mediates the early response to acute hypoxia in central neurons

    Science.gov (United States)

    Plant, Leigh D; Marks, Jeremy D; Goldstein, Steve AN

    2016-01-01

    The mechanism for the earliest response of central neurons to hypoxia—an increase in voltage-gated sodium current (INa)—has been unknown. Here, we show that hypoxia activates the Small Ubiquitin-like Modifier (SUMO) pathway in rat cerebellar granule neurons (CGN) and that SUMOylation of NaV1.2 channels increases INa. The time-course for SUMOylation of single NaV1.2 channels at the cell surface and changes in INa coincide, and both are prevented by mutation of NaV1.2-Lys38 or application of a deSUMOylating enzyme. Within 40 s, hypoxia-induced linkage of SUMO1 to the channels is complete, shifting the voltage-dependence of channel activation so that depolarizing steps evoke larger sodium currents. Given the recognized role of INa in hypoxic brain damage, the SUMO pathway and NaV1.2 are identified as potential targets for neuroprotective interventions. DOI: http://dx.doi.org/10.7554/eLife.20054.001 PMID:28029095

  3. Phosphorylation mediated structural and functional changes in pentameric ligand-gated ion channels: implications for drug discovery.

    Science.gov (United States)

    Talwar, Sahil; Lynch, Joseph W

    2014-08-01

    Pentameric ligand-gated ion channels (pLGICs) mediate numerous physiological processes, including fast neurotransmission in the brain. They are targeted by a large number of clinically-important drugs and disruptions to their function are associated with many neurological disorders. The phosphorylation of pLGICs can result in a wide range of functional consequences. Indeed, many neurological disorders result from pLGIC phosphorylation. For example, chronic pain is caused by the protein kinase A-mediated phosphorylation of α3 glycine receptors and nicotine addiction is mediated by the phosphorylation of α4- or α7-containing nicotinic receptors. A recent study demonstrated that phosphorylation can induce a global conformational change in a pLGIC that propagates to the neurotransmitter-binding site. Here we present evidence that phosphorylation-induced global conformational changes may be a universal phenomenon in pLGICs. This raises the possibility of designing drugs to specifically treat disease-modified pLGICs. This review summarizes some of the opportunities available in this area.

  4. Epithelial Sodium Channel-Mediated Sodium Transport Is Not Dependent on the Membrane-Bound Serine Protease CAP2/Tmprss4.

    Directory of Open Access Journals (Sweden)

    Anna Keppner

    Full Text Available The membrane-bound serine protease CAP2/Tmprss4 has been previously identified in vitro as a positive regulator of the epithelial sodium channel (ENaC. To study its in vivo implication in ENaC-mediated sodium absorption, we generated a knockout mouse model for CAP2/Tmprss4. Mice deficient in CAP2/Tmprss4 were viable, fertile, and did not show any obvious histological abnormalities. Unexpectedly, when challenged with sodium-deficient diet, these mice did not develop any impairment in renal sodium handling as evidenced by normal plasma and urinary sodium and potassium electrolytes, as well as normal aldosterone levels. Despite minor alterations in ENaC mRNA expression, we found no evidence for altered proteolytic cleavage of ENaC subunits. In consequence, ENaC activity, as monitored by the amiloride-sensitive rectal potential difference (ΔPD, was not altered even under dietary sodium restriction. In summary, ENaC-mediated sodium balance is not affected by lack of CAP2/Tmprss4 expression and thus, does not seem to directly control ENaC expression and activity in vivo.

  5. Characteristics of Gintonin-Mediated Membrane Depolarization of Pacemaker Activity in Cultured Interstitial Cells of Cajal

    Directory of Open Access Journals (Sweden)

    Byung Joo Kim

    2014-08-01

    Full Text Available Background/Aims: Ginseng regulates gastrointestinal (GI motor activity but the underlying components and molecular mechanisms are unknown. We investigated the effect of gintonin, a novel ginseng-derived G protein-coupled lysophosphatidic acid (LPA receptor ligand, on the pacemaker activity of the interstitial cells of Cajal (ICC in murine small intestine and GI motility. Materials and Methods: Enzymatic digestion was used to dissociate ICC from mouse small intestines. The whole-cell patch-clamp configuration was used to record pacemaker potentials and currents from cultured ICC in the absence or presence of gintonin. In vivo effects of gintonin on gastrointestinal (GI motility were investigated by measuring the intestinal transit rate (ITR of Evans blue in normal and streptozotocin (STZ-induced diabetic mice. Results: We investigated the effects of gintonin on pacemaker potentials and currents in cultured ICC from mouse small intestine. Gintonin caused membrane depolarization in current clamp mode but this action was blocked by Ki16425, an LPA1/3 receptor antagonist, and by the addition of GDPβS, a GTP-binding protein inhibitor, into the ICC. To study the gintonin signaling pathway, we examined the effects of U-73122, an active PLC inhibitor, and chelerythrine and calphostin, which inhibit PKC. All inhibitors blocked gintonin actions on pacemaker potentials, but not completely. Gintonin-mediated depolarization was lower in Ca2+-free than in Ca2+-containing external solutions and was blocked by thapsigargin. We found that, in ICC, gintonin also activated Ca2+-activated Cl- channels (TMEM16A, ANO1, but not TRPM7 channels. In vivo, gintonin (10-100 mg/kg, p.o. not only significantly increased the ITR in normal mice but also ameliorated STZ-induced diabetic GI motility retardation in a dose-dependent manner. Conclusions: Gintonin-mediated membrane depolarization of pacemaker activity and ANO1 activation are coupled to the stimulation of GI

  6. 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.

  7. Computer-mediated communication as a channel for social resistance : The strategic side of SIDE

    NARCIS (Netherlands)

    Spears, R; Lea, M; Corneliussen, RA; Postmes, T; Ter Haar, W

    2002-01-01

    In two studies, the authors tested predictions derived from the social identity model of deindividuation effects (SIDE) concerning the potential of computer-mediated communication (CMC) to serve as a means to resist powerful out-groups. Earlier research using the SIDE model indicates that the anonym

  8. Search for Gauge Mediated Supersymmetry in the gamma gamma missing ET Channel

    Energy Technology Data Exchange (ETDEWEB)

    Kesisoglou, Stilianos Isaak [Brown Univ., Providence, RI (United States)

    2005-05-01

    We present results on a search for Gauge Mediated Supersymmetry in the di-photon final state using Run II data collected by the D0 Experiment at the Fermilab Tevatron Collider. We discuss event selection, Standard Model backgrounds, and the lower limits on the lightest neutralino and chargino masses resulted from this analysis.

  9. Blockade of IP[subscript 3]-Mediated SK Channel Signaling in the Rat Medial Prefrontal Cortex Improves Spatial Working Memory

    Science.gov (United States)

    Brennan, Avis R.; Dolinsky, Beth; Vu, Mai-Anh T.; Stanley, Marion; Yeckel, Mark F.; Arnsten, Amy F. T.

    2008-01-01

    Planning and directing thought and behavior require the working memory (WM) functions of prefrontal cortex. WM is compromised by stress, which activates phosphatidylinositol (PI)-mediated IP[subscript 3]-PKC intracellular signaling. PKC overactivation impairs WM operations and in vitro studies indicate that IP[subscript 3] receptor (IP[subscript…

  10. Ion channels regulating mast cell biology.

    Science.gov (United States)

    Ashmole, I; Bradding, P

    2013-05-01

    Mast cells play a central role in the pathophysiology of asthma and related allergic conditions. Mast cell activation leads to the degranulation of preformed mediators such as histamine and the secretion of newly synthesised proinflammatory mediators such as leukotrienes and cytokines. Excess release of these mediators contributes to allergic disease states. An influx of extracellular Ca2+ is essential for mast cell mediator release. From the Ca2+ channels that mediate this influx, to the K+ , Cl- and transient receptor potential channels that set the cell membrane potential and regulate Ca2+ influx, ion channels play a critical role in mast cell biology. In this review we provide an overview of our current knowledge of ion channel expression and function in mast cells with an emphasis on how channels interact to regulate Ca2+ signalling.

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

    Directory of Open Access Journals (Sweden)

    Guoxing Liu

    2015-11-01

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

  12. K(ATP channel opener diazoxide prevents neurodegeneration: a new mechanism of action via antioxidative pathway activation.

    Directory of Open Access Journals (Sweden)

    Noemí Virgili

    Full Text Available Pharmacological modulation of ATP-sensitive potassium channels has become a promising new therapeutic approach for the treatment of neurodegenerative diseases due to their role in mitochondrial and cellular protection. For instance, diazoxide, a well-known ATP-sensitive potassium channel activator with high affinity for mitochondrial component of the channel has been proved to be effective in animal models for different diseases such as Alzheimer's disease, stroke or multiple sclerosis. Here, we analyzed the ability of diazoxide for protecting neurons front different neurotoxic insults in vitro and ex vivo. Results showed that diazoxide effectively protects NSC-34 motoneurons from glutamatergic, oxidative and inflammatory damage. Moreover, diazoxide decreased neuronal death in organotypic hippocampal slice cultures after exicitotoxicity and preserved myelin sheath in organotypic cerebellar cultures exposed to pro-inflammatory demyelinating damage. In addition, we demonstrated that one of the mechanisms of actions implied in the neuroprotective role of diazoxide is mediated by the activation of Nrf2 expression and nuclear translocation. Nrf2 expression was increased in NSC-34 neurons in vitro as well as in the spinal cord of experimental autoimmune encephalomyelitis animals orally administered with diazoxide. Thus, diazoxide is a neuroprotective agent against oxidative stress-induced damage and cellular dysfunction that can be beneficial for diseases such as multiple sclerosis.

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

    Science.gov (United States)

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

    1999-09-01

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

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

    Institute of Scientific and Technical Information of China (English)

    2008-01-01

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

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

    Institute of Scientific and Technical Information of China (English)

    GONG YuBing; XU Bo; MA XiaoGuang; HAN JiQu

    2008-01-01

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

  16. Decrease of a Current Mediated by K(v)1.3 Channels Causes Striatal Cholinergic Interneuron Hyperexcitability in Experimental Parkinsonism

    OpenAIRE

    Cecilia Tubert; Irene R.E. Taravini; Eden Flores-Barrera; Gonzalo M. Sánchez; María Alejandra Prost; María Elena Avale; Kuei Y. Tseng; Lorena Rela; Mario Gustavo Murer

    2016-01-01

    The mechanism underlying a hypercholinergic state in Parkinsons disease (PD) remains uncertain. Here, we show that disruption of the K(v)1 channel-mediated function causes hyperexcitability of striatal cholinergic interneurons in a mouse model of PD. Specifically, our data reveal that Kv1 channels containing K(v)1.3 subunits contribute significantly to the orphan potassium current known as I-sAHP in striatal cholinergic interneurons. Typically, this Kv1 current provides negative feedback to d...

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

    Directory of Open Access Journals (Sweden)

    Thom Griffith

    2016-05-01

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

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

    OpenAIRE

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

    2005-01-01

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

  19. 15 CFR 930.44 - Availability of mediation for disputes concerning proposed activities.

    Science.gov (United States)

    2010-01-01

    ... 15 Commerce and Foreign Trade 3 2010-01-01 2010-01-01 false Availability of mediation for disputes... PROGRAMS Consistency for Federal Agency Activities § 930.44 Availability of mediation for disputes..., either party may request the Secretarial mediation or OCRM mediation services provided for in subpart G....

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

    Science.gov (United States)

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

    2016-02-19

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

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

    Directory of Open Access Journals (Sweden)

    Xiaoqi Zhao

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

  2. The sodium channel β1 subunit mediates outgrowth of neurite-like processes on breast cancer cells and promotes tumour growth and metastasis.

    Science.gov (United States)

    Nelson, Michaela; Millican-Slater, Rebecca; Forrest, Lorna C; Brackenbury, William J

    2014-11-15

    Voltage-gated Na(+) channels (VGSCs) are heteromeric proteins composed of pore-forming α subunits and smaller β subunits. The β subunits are multifunctional channel modulators and are members of the immunoglobulin superfamily of cell adhesion molecules (CAMs). β1, encoded by SCN1B, is best characterized in the central nervous system (CNS), where it plays a critical role in regulating electrical excitability, neurite outgrowth and migration during development. β1 is also expressed in breast cancer (BCa) cell lines, where it regulates adhesion and migration in vitro. In the present study, we found that SCN1B mRNA/β1 protein were up-regulated in BCa specimens, compared with normal breast tissue. β1 upregulation substantially increased tumour growth and metastasis in a xenograft model of BCa. β1 over-expression also increased vascularization and reduced apoptosis in the primary tumours, and β1 over-expressing tumour cells had an elongate morphology. In vitro, β1 potentiated outgrowth of processes from BCa cells co-cultured with fibroblasts, via trans-homophilic adhesion. β1-mediated process outgrowth in BCa cells required the presence and activity of fyn kinase, and Na(+) current, thus replicating the mechanism by which β1 regulates neurite outgrowth in CNS neurons. We conclude that when present in breast tumours, β1 enhances pathological growth and cellular dissemination. This study is the first demonstration of a functional role for β1 in tumour growth and metastasis in vivo. We propose that β1 warrants further study as a potential biomarker and targeting β1-mediated adhesion interactions may have value as a novel anti-cancer therapy.

  3. AhR-mediated and antiestrogenic activity of humic substances.

    Science.gov (United States)

    Janosek, J; Bittner, M; Hilscherová, K; Bláha, L; Giesy, J P; Holoubek, I

    2007-04-01

    Humic substances (HS) were for decades regarded as inert in the ecosystems with respect to their possible toxicity. However, HS have been recently shown to elicit various adverse effects generally attributed to xenobiotics. In our study, we used MVLN and H4IIE-luc cell lines stably transfected with luciferase gene under control of estrogen receptor (ER) and Ah receptor (AhR; receptor connected with so-called dioxin-like toxicity) for assessment of anti/estrogenic and AhR-mediated effects of 12 commercially available humic substances. Out of those, five humic acids were shown to induce AhR-mediated activity with relative potencies related to TCDD 2.6 x 10(-8)-7.4 x 10(-8). Organic extracts of HS solutions also elicited high activities what means that lipophilic molecules are responsible for a great part of effect. However, relatively high activity remaining in extracted solution suggests also presence of polar AhR-agonists. Contribution of persistent organic compounds to the observed effects was ruled out by H(2)SO(4) treatment. Eight out of twelve HS elicited significant antiestrogenic effects with IC(50) ranging from 40 to 164 mg l(-1). The possible explanations of the antiestrogenic effect include sorption of 17-beta-estradiol (E2) on HS, changes in membrane permeability for E2 or another specific mechanism.

  4. LIF Mediates Proinvasive Activation of Stromal Fibroblasts in Cancer

    Directory of Open Access Journals (Sweden)

    Jean Albrengues

    2014-06-01

    Full Text Available Signaling crosstalk between tumor cells and fibroblasts confers proinvasive properties to the tumor microenvironment. Here, we identify leukemia inhibitory factor (LIF as a tumor promoter that mediates proinvasive activation of stromal fibroblasts independent of alpha-smooth muscle actin (α-SMA expression. We demonstrate that a pulse of transforming growth factor β (TGF-β establishes stable proinvasive fibroblast activation by inducing LIF production in both fibroblasts and tumor cells. In fibroblasts, LIF mediates TGF-β-dependent actomyosin contractility and extracellular matrix remodeling, which results in collective carcinoma cell invasion in vitro and in vivo. Accordingly, carcinomas from multiple origins and melanomas display strong LIF upregulation, which correlates with dense collagen fiber organization, cancer cell collective invasion, and poor clinical outcome. Blockade of JAK activity by Ruxolitinib (JAK inhibitor counteracts fibroblast-dependent carcinoma cell invasion in vitro and in vivo. These findings establish LIF as a proinvasive fibroblast producer independent of α-SMA and may open novel therapeutic perspectives for patients with aggressive primary tumors.

  5. Activating Mutations of the TRPML1 Channel Revealed by Proline-scanning Mutagenesis*

    OpenAIRE

    2009-01-01

    The mucolipin TRP (TRPML) proteins are a family of endolysosomal cation channels with genetically established importance in humans and rodent. Mutations of human TRPML1 cause type IV mucolipidosis, a devastating pediatric neurodegenerative disease. Our recent electrophysiological studies revealed that, although a TRPML1-mediated current can only be recorded in late endosome and lysosome (LEL) using the lysosome patch clamp technique, a proline substitution in TRPML1 (TRPML1V432P) results in a...

  6. Human mediator subunit MED15 promotes transcriptional activation.

    Science.gov (United States)

    Nakatsubo, Takuya; Nishitani, Saori; Kikuchi, Yuko; Iida, Satoshi; Yamada, Kana; Tanaka, Aki; Ohkuma, Yoshiaki

    2014-10-01

    In eukaryotes, the Mediator complex is an essential transcriptional cofactor of RNA polymerase II (Pol II). In humans, it contains up to 30 subunits and consists of four modules: head, middle, tail, and CDK/Cyclin. One of the subunits, MED15, is located in the tail module, and was initially identified as Gal11 in budding yeast, where it plays an essential role in the transcriptional regulation of galactose metabolism with the potent transcriptional activator Gal4. For this reason, we investigated the function of the human MED15 subunit (hMED15) in transcriptional activation. First, we measured the effect of hMED15 knockdown on cell growth in HeLa cells. The growth rate was greatly reduced. By immunostaining, we observed the colocalization of hMED15 with the general transcription factors TFIIE and TFIIH in the nucleus. We measured the effects of siRNA-mediated knockdown of hMED15 on transcriptional activation using two different transcriptional activators, VP16 and SREBP1a. Treatment with siRNAs reduced transcriptional activation, and this reduction could be rescued by overexpression of HA/Flag-tagged, wild-type hMED15. To investigate hMED15 localization, we treated human MCF-7 cells with the MDM2 inhibitor Nutlin-3, thus inducing p21 transcription. We found that hMED15 localized to both the p53 binding site and the p21 promoter region, along with TFIIE and TFIIH. These results indicate that hMED15 promotes transcriptional activation.

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

    Science.gov (United States)

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

    2017-01-21

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

  8. Gustatory-mediated avoidance of bacterial lipopolysaccharides via TRPA1 activation in Drosophila.

    Science.gov (United States)

    Soldano, Alessia; Alpizar, Yeranddy A; Boonen, Brett; Franco, Luis; López-Requena, Alejandro; Liu, Guangda; Mora, Natalia; Yaksi, Emre; Voets, Thomas; Vennekens, Rudi; Hassan, Bassem A; Talavera, Karel

    2016-06-14

    Detecting pathogens and mounting immune responses upon infection is crucial for animal health. However, these responses come at a high metabolic price (McKean and Lazzaro, 2011, Kominsky et al., 2010), and avoiding pathogens before infection may be advantageous. The bacterial endotoxins lipopolysaccharides (LPS) are important immune system infection cues (Abbas et al., 2014), but it remains unknown whether animals possess sensory mechanisms to detect them prior to infection. Here we show that Drosophila melanogaster display strong aversive responses to LPS and that gustatory neurons expressing Gr66a bitter receptors mediate avoidance of LPS in feeding and egg laying assays. We found the expression of the chemosensory cation channel dTRPA1 in these cells to be necessary and sufficient for LPS avoidance. Furthermore, LPS stimulates Drosophila neurons in a TRPA1-dependent manner and activates exogenous dTRPA1 channels in human cells. Our findings demonstrate that flies detect bacterial endotoxins via a gustatory pathway through TRPA1 activation as conserved molecular mechanism.

  9. Expression of postsynaptic Ca2+-activated K+ (SK) channels at C-bouton synapses in mammalian lumbar α-motoneurons

    Science.gov (United States)

    Deardorff, Adam S; Romer, Shannon H; Deng, Zhihui; Bullinger, Katie L; Nardelli, Paul; Cope, Timothy C; Fyffe, Robert E W

    2013-01-01

    Small-conductance calcium-activated potassium (SK) channels mediate medium after-hyperpolarization (AHP) conductances in neurons throughout the central nervous system. However, the expression profile and subcellular localization of different SK channel isoforms in lumbar spinal α-motoneurons (α-MNs) is unknown. Using immunohistochemical labelling of rat, mouse and cat spinal cord, we reveal a differential and overlapping expression of SK2 and SK3 isoforms across specific types of α-MNs. In rodents, SK2 is expressed in all α-MNs, whereas SK3 is expressed preferentially in small-diameter α-MNs; in cats, SK3 is expressed in all α-MNs. Function-specific expression of SK3 was explored using post hoc immunostaining of electrophysiologically characterized rat α-MNs in vivo. These studies revealed strong relationships between SK3 expression and medium AHP properties. Motoneurons with SK3-immunoreactivity exhibit significantly longer AHP half-decay times (24.67 vs. 11.02 ms) and greater AHP amplitudes (3.27 vs. 1.56 mV) than MNs lacking SK3-immunoreactivity. We conclude that the differential expression of SK isoforms in rat and mouse spinal cord may contribute to the range of medium AHP durations across specific MN functional types and may be a molecular factor distinguishing between slow- and fast-type α-MNs in rodents. Furthermore, our results show that SK2- and SK3-immunoreactivity is enriched in distinct postsynaptic domains that contain Kv2.1 channel clusters associated with cholinergic C-boutons on the soma and proximal dendrites of α-MNs. We suggest that this remarkably specific subcellular membrane localization of SK channels is likely to represent the basis for a cholinergic mechanism for effective regulation of channel function and cell excitability. PMID:23129791

  10. Serum inflammatory mediators as markers of human Lyme disease activity.

    Science.gov (United States)

    Soloski, Mark J; Crowder, Lauren A; Lahey, Lauren J; Wagner, Catriona A; Robinson, William H; Aucott, John N

    2014-01-01

    Chemokines and cytokines are key signaling molecules that orchestrate the trafficking of immune cells, direct them to sites of tissue injury and inflammation and modulate their states of activation and effector cell function. We have measured, using a multiplex-based approach, the levels of 58 immune mediators and 7 acute phase markers in sera derived from of a cohort of patients diagnosed with acute Lyme disease and matched controls. This analysis identified a cytokine signature associated with the early stages of infection and allowed us to identify two subsets (mediator-high and mediator-low) of acute Lyme patients with distinct cytokine signatures that also differed significantly (p<0.0005) in symptom presentation. In particular, the T cell chemokines CXCL9 (MIG), CXCL10 (IP-10) and CCL19 (MIP3B) were coordinately increased in the mediator-high group and levels of these chemokines could be associated with seroconversion status and elevated liver function tests (p = 0.027 and p = 0.021 respectively). There was also upregulation of acute phase proteins including CRP and serum amyloid A. Consistent with the role of CXCL9/CXCL10 in attracting immune cells to the site of infection, CXCR3+ CD4 T cells are reduced in the blood of early acute Lyme disease (p = 0.01) and the decrease correlates with chemokine levels (p = 0.0375). The levels of CXCL9/10 did not relate to the size or number of skin lesions but elevated levels of serum CXCL9/CXCL10 were associated with elevated liver enzymes levels. Collectively these results indicate that the levels of serum chemokines and the levels of expression of their respective chemokine receptors on T cell subsets may prove to be informative biomarkers for Lyme disease and related to specific disease manifestations.

  11. Serum inflammatory mediators as markers of human Lyme disease activity.

    Directory of Open Access Journals (Sweden)

    Mark J Soloski

    Full Text Available Chemokines and cytokines are key signaling molecules that orchestrate the trafficking of immune cells, direct them to sites of tissue injury and inflammation and modulate their states of activation and effector cell function. We have measured, using a multiplex-based approach, the levels of 58 immune mediators and 7 acute phase markers in sera derived from of a cohort of patients diagnosed with acute Lyme disease and matched controls. This analysis identified a cytokine signature associated with the early stages of infection and allowed us to identify two subsets (mediator-high and mediator-low of acute Lyme patients with distinct cytokine signatures that also differed significantly (p<0.0005 in symptom presentation. In particular, the T cell chemokines CXCL9 (MIG, CXCL10 (IP-10 and CCL19 (MIP3B were coordinately increased in the mediator-high group and levels of these chemokines could be associated with seroconversion status and elevated liver function tests (p = 0.027 and p = 0.021 respectively. There was also upregulation of acute phase proteins including CRP and serum amyloid A. Consistent with the role of CXCL9/CXCL10 in attracting immune cells to the site of infection, CXCR3+ CD4 T cells are reduced in the blood of early acute Lyme disease (p = 0.01 and the decrease correlates with chemokine levels (p = 0.0375. The levels of CXCL9/10 did not relate to the size or number of skin lesions but elevated levels of serum CXCL9/CXCL10 were associated with elevated liver enzymes levels. Collectively these results indicate that the levels of serum chemokines and the levels of expression of their respective chemokine receptors on T cell subsets may prove to be informative biomarkers for Lyme disease and related to specific disease manifestations.

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

    Science.gov (United States)

    Gu, Yuanzheng; Barry, Joshua; Gu, Chen

    2013-05-15

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

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

    Directory of Open Access Journals (Sweden)

    Berg Ulrike

    2009-04-01

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

  14. Acute p38-mediated modulation of tetrodotoxin-resistant sodium channels in mouse sensory neurons by tumor necrosis factor-alpha.

    Science.gov (United States)

    Jin, Xiaochun; Gereau, Robert W

    2006-01-04

    Tumor necrosis factor-alpha (TNFalpha) is a proinflammatory cytokine involved in the development and maintenance of inflammatory and neuropathic pain conditions. TNFalpha can have long-lasting effects by regulating the expression of a variety of inflammatory mediators, including other cytokines and TNFalpha itself. However, the speed with which TNFalpha induces tactile and thermal hypersensitivity suggests that transcriptional regulation cannot fully account for its sensitizing effects, and some recent findings suggest that TNFalpha may act directly on primary afferent neurons to induce pain hypersensitivity. In the present study, we show that peripheral administration of TNFalpha induces thermal hypersensitivity in wild-type mice but not in transient receptor potential vanilloid receptor TRPV1(-/-) mice. In contrast, TNFalpha produced equivalent mechanical hypersensitivity in TRPV1(-/-) mice and wild-type littermates, suggesting a role for TRPV1 in TNFalpha-induced thermal, but not mechanical, hypersensitivity. Because tetrodotoxin (TTX)-resistant Na+ channels are a critical site of modulation underlying mechanical hypersensitivity in inflammatory and neuropathic pain conditions, we tested the effects of TNFalpha on these channels in isolated mouse dorsal root ganglion (DRG) neurons. We report that acute application of TNFalpha rapidly enhances TTX-resistant Na+ currents in isolated DRG neurons. This potentiation of TTX-resistant currents by TNFalpha is dramatically reduced in DRG neurons from TNF receptor 1 (TNFR1) knock-out mice and is blocked by the p38 mitogen-activated protein kinase inhibitor SB202190 [4-(4-fluorophenyl)-2-(4-hydroxyphenyl)-5-(4-pyridyl)1H-imidazole]. Mechanical hypersensitivity induced by peripherally applied TNFalpha is also significantly reduced by SB202190. These results suggest that TNFalpha may induce acute peripheral mechanical sensitization by acting directly on TNFR1 in primary afferent neurons, resulting in p38-dependent modulation

  15. The activity of the epithelial sodium channels is regulated by caveolin-1 via a Nedd4-2-dependent mechanism.

    Science.gov (United States)

    Lee, Il-Ha; Campbell, Craig R; Song, Sung-Hee; Day, Margot L; Kumar, Sharad; Cook, David I; Dinudom, Anuwat

    2009-05-08

    It has recently been shown that the epithelial Na(+) channel (ENaC) is compartmentalized in caveolin-rich lipid rafts and that pharmacological depletion of membrane cholesterol, which disrupts lipid raft formation, decreases the activity of ENaC. Here we show, for the first time, that a signature protein of caveolae, caveolin-1 (Cav-1), down-regulates the activity and membrane surface expression of ENaC. Physical interaction between ENaC and Cav-1 was also confirmed in a coimmunoprecipitation assay. We found that the effect of Cav-1 on ENaC requires the activity of Nedd4-2, a ubiquitin protein ligase of the Nedd4 family, which is known to induce ubiquitination and internalization of ENaC. The effect of Cav-1 on ENaC requires the proline-rich motifs at the C termini of the beta- and gamma-subunits of ENaC, the binding motifs that mediate interaction with Nedd4-2. Taken together, our data suggest that Cav-1 inhibits the activity of ENaC by decreasing expression of ENaC at the cell membrane via a mechanism that involves the promotion of Nedd4-2-dependent internalization of the channel.

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

    Science.gov (United States)

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

    2005-07-01

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

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

    Science.gov (United States)

    McGarry, S J; Williams, A J

    1993-04-01

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

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

    Science.gov (United States)

    Lishko, Polina V; Kirichok, Yuriy

    2010-12-01

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

  19. Modeling CaMKII-mediated regulation of L-type Ca2+ channels and ryanodine receptors in the heart

    Directory of Open Access Journals (Sweden)

    Joseph L Greenstein

    2014-04-01

    Full Text Available Excitation-contraction coupling (ECC in the cardiac myocyte is mediated by a number of highly integrated mechanisms of intracellular Ca2+ transport. Voltage- and Ca2+-dependent L-type Ca2+ channels (LCCs allow for Ca2+ entry into the myocyte, which then binds to nearby ryanodine receptors (RyRs and triggers Ca2+ release from the sarcoplasmic reticulum in a process known as Ca2+-induced Ca2+ release. The highly coordinated Ca2+-mediated interaction between LCCs and RyRs is further regulated by the cardiac isoform of the Ca2+/calmodulin-dependent protein kinase (CaMKII. Because CaMKII targets and modulates the function of many ECC proteins, elucidation of its role in ECC and integrative cellular function is challenging and much insight has been gained through the use of detailed computational models. Multiscale models that can both reconstruct the detailed nature of local signaling events within the cardiac dyad and predict their functional consequences at the level of the whole cell have played an important role in advancing our understanding of CaMKII function in ECC. Here, we review experimentally based models of CaMKII function with a focus on LCC and RyR regulation, and the mechanistic insights that have been gained through their application.

  20. Fluorescence-tracking of activation gating in human ERG channels reveals rapid S4 movement and slow pore opening.

    Directory of Open Access Journals (Sweden)

    Zeineb Es-Salah-Lamoureux

    Full Text Available BACKGROUND: hERG channels are physiologically important ion channels which mediate cardiac repolarization as a result of their unusual gating properties. These are very slow activation compared with other mammalian voltage-gated potassium channels, and extremely rapid inactivation. The mechanism of slow activation is not well understood and is investigated here using fluorescence as a direct measure of S4 movement and pore opening. METHODS AND FINDINGS: Tetramethylrhodamine-5-maleimide (TMRM fluorescence at E519 has been used to track S4 voltage sensor movement, and channel opening and closing in hERG channels. Endogenous cysteines (C445 and C449 in the S1-S2 linker bound TMRM, which caused a 10 mV hyperpolarization of the V((1/2 of activation to -27.5+/-2.0 mV, and showed voltage-dependent fluorescence signals. Substitution of S1-S2 linker cysteines with valines allowed unobstructed recording of S3-S4 linker E519C and L520C emission signals. Depolarization of E519C channels caused rapid initial fluorescence quenching, fit with a double Boltzmann relationship, F-V(ON, with V((1/2 (,1 = -37.8+/-1.7 mV, and V((1/2 (,2 = 43.5+/-7.9 mV. The first phase, V((1/2 (,1, was approximately 20 mV negative to the conductance-voltage relationship measured from ionic tail currents (G-V((1/2 = -18.3+/-1.2 mV, and relatively unchanged in a non-inactivating E519C:S620T mutant (V((1/2 = -34.4+/-1.5 mV, suggesting the fast initial fluorescence quenching tracked S4 voltage sensor movement. The second phase of rapid quenching was absent in the S620T mutant. The E519C fluorescence upon repolarization (V((1/2 = -20.6+/-1.2, k = 11.4 mV and L520C quenching during depolarization (V((1/2 = -26.8+/-1.0, k = 13.3 mV matched the respective voltage dependencies of hERG ionic tails, and deactivation time constants from -40 to -110 mV, suggesting they detected pore-S4 rearrangements related to ionic current flow during pore opening and closing. CONCLUSION: THE DATA INDICATE: 1

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

    Science.gov (United States)

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

    2012-02-19

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

  2. Presynaptic Localization and Possible Function of Calcium-Activated Chloride Channel Anoctamin 1 in the Mammalian Retina.

    Directory of Open Access Journals (Sweden)

    Ji Hyun Jeon

    Full Text Available Calcium (Ca(2+-activated chloride (Cl(- channels (CaCCs play a role in the modulation of action potentials and synaptic responses in the somatodendritic regions of central neurons. In the vertebrate retina, large Ca(2+-activated Cl(- currents (ICl(Ca regulate synaptic transmission at photoreceptor terminals; however, the molecular identity of CaCCs that mediate ICl(Ca remains unclear. The transmembrane protein, TMEM16A, also called anoctamin 1 (ANO1, has been recently validated as a CaCC and is widely expressed in various secretory epithelia and nervous tissues. Despite the fact that tmem16a was first cloned in the retina, there is little information on its cellular localization and function in the mammalian retina. In this study, we found that ANO1 was abundantly expressed as puncta in 2 synaptic layers. More specifically, ANO1 immunoreactivity was observed in the presynaptic terminals of various retinal neurons, including photoreceptors. ICl(Ca was first detected in dissociated rod bipolar cells expressing ANO1. ICl(Ca was abolished by treatment with the Ca(2+ channel blocker Co(2+, the L-type Ca(2+ channel blocker nifedipine, and the Cl(- channel blockers 5-nitro-2-(3-phenylpropylamino benzoic acid (NPPB and niflumic acid (NFA. More specifically, a recently discovered ANO1-selective inhibitor, T16Ainh-A01, and a neutralizing antibody against ANO1 inhibited ICl(Ca in rod bipolar cells. Under a current-clamping mode, the suppression of ICl(Ca by using NPPB and T16Ainh-A01 caused a prolonged Ca(2+ spike-like depolarization evoked by current injection in dissociated rod bipolar cells. These results suggest that ANO1 confers ICl(Ca in retinal neurons and acts as an intrinsic regulator of the presynaptic membrane potential during synaptic transmission.

  3. Functional analysis of Ficolin-3 mediated complement activation

    DEFF Research Database (Denmark)

    Hein, Estrid; Honoré, Christian Le Fèvre; Skjoedt, Mikkel-Ole;

    2010-01-01

    The recognition molecules of the lectin complement pathway are mannose-binding lectin and Ficolin -1, -2 and -3. Recently deficiency of Ficolin-3 was found to be associated with life threatening infections. Thus, we aimed to develop a functional method based on the ELISA platform for evaluating...... Ficolin-3 mediated complement activation that could be applicable for research and clinical use. Bovine serum albumin (BSA) was acetylated (acBSA) and chosen as a solid phase ligand for Ficolins in microtiter wells. Binding of Ficolins on acBSA was evaluated, as was functional complement activation...... assessed by C4, C3 and terminal complement complex (TCC) deposition. Serum Ficolin-3 bound to acBSA in a calcium dependent manner, while only minimal binding of Ficolin-2 and no binding of Ficolin-1 were observed. No binding to normal BSA was seen for any of the Ficolins. Serum C4, C3 and TCC deposition...

  4. The antileishmanial activity of xanthohumol is mediated by mitochondrial inhibition.

    Science.gov (United States)

    Monzote, Lianet; Lackova, Alexandra; Staniek, Katrin; Steinbauer, Silvia; Pichler, Gerald; Jäger, Walter; Gille, Lars

    2016-12-12

    Xanthohumol (Xan) is a natural constituent of human nutrition. Little is known about its actions on leishmanial parasites and their mitochondria as putative target. Therefore, we determined the antileishmanial activity of Xan and resveratrol (Res, as alternative compound with antileishmanial activity) with respect to mitochondria in Leishmania amazonensis promastigotes/amastigotes (LaP/LaA) in comparison with their activity in peritoneal macrophages from mouse (PMM) and macrophage cell line J774A.1 (J774). Mechanistic studies were conducted in Leishmania tarentolae promastigotes (LtP) and mitochondrial fractions isolated from LtP. Xan and Res demonstrated antileishmanial activity in LaA [half inhibitory concentration (IC50): Xan 7 µ m, Res 14 µ m]; while they had less influence on the viability of PMM (IC50: Xan 70 µ m, Res >438 µ m). In contrast to Res, Xan strongly inhibited oxygen consumption in Leishmania (LtP) but not in J774 cells. This was based on the inhibition of the mitochondrial electron transfer complex II/III by Xan, which was less pronounced with Res. Neither Xan nor Res increased mitochondrial superoxide release in LtP, while both decreased the mitochondrial membrane potential in LtP. Bioenergetic studies showed that LtP mitochondria have no spare respiratory capacity in contrast to mitochondria in J774 cells and can therefore much less adapt to stress by mitochondrial inhibitors, such as Xan. These data show that Xan may have antileishmanial activity, which is mediated by mitochondrial inhibition.

  5. CaV3.2 T-type Ca²⁺ channels in H₂S-mediated hypoxic response of the carotid body.

    Science.gov (United States)

    Makarenko, Vladislav V; Peng, Ying-Jie; Yuan, Guoxiang; Fox, Aaron P; Kumar, Ganesh K; Nanduri, Jayasri; Prabhakar, Nanduri R

    2015-01-15

    Arterial blood O2 levels are detected by specialized sensory organs called carotid bodies. Voltage-gated Ca(2+) channels (VGCCs) are important for carotid body O2 sensing. Given that T-type VGCCs contribute to nociceptive sensation, we hypothesized that they participate in carotid body O2 sensing. The rat carotid body expresses high levels of mRNA encoding the α1H-subunit, and α1H protein is localized to glomus cells, the primary O2-sensing cells in the chemoreceptor tissue, suggesting that CaV3.2 is the major T-type VGCC isoform expressed in the carotid body. Mibefradil and TTA-A2, selective blockers of the T-type VGCC, markedly attenuated elevation of hypoxia-evoked intracellular Ca(2+) concentration, secretion of catecholamines from glomus cells, and sensory excitation of the rat carotid body. Similar results were obtained in the carotid body and glomus cells from CaV3.2 knockout (Cacna1h(-/-)) mice. Since cystathionine-γ-lyase (CSE)-derived H2S is a critical mediator of the carotid body response to hypoxia, the role of T-type VGCCs in H2S-mediated O2 sensing was examined. Like hypoxia, NaHS, a H2S donor, increased intracellular Ca(2+) concentration and augmented carotid body sensory nerve activity in wild-type mice, and these effects were markedly attenuated in Cacna1h(-/-) mice. In wild-type mice, TTA-A2 markedly attenuated glomus cell and carotid body sensory nerve responses to hypoxia, and these effects were absent in CSE knockout mice. These results demonstrate that CaV3.2 T-type VGCCs contribute to the H2S-mediated carotid body response to hypoxia.

  6. CaV3.2 T-type Ca2+ channels in H2S-mediated hypoxic response of the carotid body

    Science.gov (United States)

    Makarenko, Vladislav V.; Peng, Ying-Jie; Yuan, Guoxiang; Fox, Aaron P.; Kumar, Ganesh K.; Nanduri, Jayasri

    2014-01-01

    Arterial blood O2 levels are detected by specialized sensory organs called carotid bodies. Voltage-gated Ca2+ channels (VGCCs) are important for carotid body O2 sensing. Given that T-type VGCCs contribute to nociceptive sensation, we hypothesized that they participate in carotid body O2 sensing. The rat carotid body expresses high levels of mRNA encoding the α1H-subunit, and α1H protein is localized to glomus cells, the primary O2-sensing cells in the chemoreceptor tissue, suggesting that CaV3.2 is the major T-type VGCC isoform expressed in the carotid body. Mibefradil and TTA-A2, selective blockers of the T-type VGCC, markedly attenuated elevation of hypoxia-evoked intracellular Ca2+ concentration, secretion of catecholamines from glomus cells, and sensory excitation of the rat carotid body. Similar results were obtained in the carotid body and glomus cells from CaV3.2 knockout (Cacna1h−/−) mice. Since cystathionine-γ-lyase (CSE)-derived H2S is a critical mediator of the carotid body response to hypoxia, the role of T-type VGCCs in H2S-mediated O2 sensing was examined. Like hypoxia, NaHS, a H2S donor, increased intracellular Ca2+ concentration and augmented carotid body sensory nerve activity in wild-type mice, and these effects were markedly attenuated in Cacna1h−/− mice. In wild-type mice, TTA-A2 markedly attenuated glomus cell and carotid body sensory nerve responses to hypoxia, and these effects were absent in CSE knockout mice. These results demonstrate that CaV3.2 T-type VGCCs contribute to the H2S-mediated carotid body response to hypoxia. PMID:25377087

  7. Polyoxometalate active charge-transfer material for mediated redox flow battery

    Energy Technology Data Exchange (ETDEWEB)

    Anderson, Travis Mark; Hudak, Nicholas; Staiger, Chad; Pratt, Harry

    2017-01-17

    Redox flow batteries including a half-cell electrode chamber coupled to a current collecting electrode are disclosed herein. In a general embodiment, a separator is coupled to the half-cell electrode chamber. The half-cell electrode chamber comprises a first redox-active mediator and a second redox-active mediator. The first redox-active mediator and the second redox-active mediator are circulated through the half-cell electrode chamber into an external container. The container includes an active charge-transfer material. The active charge-transfer material has a redox potential between a redox potential of the first redox-active mediator and a redox potential of the second redox-active mediator. The active charge-transfer material is a polyoxometalate or derivative thereof. The redox flow battery may be particularly useful in energy storage solutions for renewable energy sources and for providing sustained power to an electrical grid.

  8. Hydrogen sulfide-induced itch requires activation of Cav3.2 T-type calcium channel in mice

    Science.gov (United States)

    Wang, Xue-Long; Tian, Bin; Huang, Ya; Peng, Xiao-Yan; Chen, Li-Hua; Li, Jun-Cheng; Liu, Tong

    2015-01-01

    The contributions of gasotransmitters to itch sensation are largely unknown. In this study, we aimed to investigate the roles of hydrogen sulfide (H2S), a ubiquitous gasotransmitter, in itch signaling. We found that intradermal injection of H2S donors NaHS or Na2S, but not GYY4137 (a slow-releasing H2S donor), dose-dependently induced scratching behavior in a μ-opioid receptor-dependent and histamine-independent manner in mice. Interestingly, NaHS induced itch via unique mechanisms that involved capsaicin-insensitive A-fibers, but not TRPV1-expressing C-fibers that are traditionally considered for mediating itch, revealed by depletion of TRPV1-expressing C-fibers by systemic resiniferatoxin treatment. Moreover, local application of capsaizapine (TRPV1 blocker) or HC-030031 (TRPA1 blocker) had no effects on NaHS-evoked scratching. Strikingly, pharmacological blockade and silencing of Cav3.2 T-type calcium channel by mibefradil, ascorbic acid, zinc chloride or Cav3.2 siRNA dramatically decreased NaHS-evoked scratching. NaHS induced robust alloknesis (touch-evoked itch), which was inhibited by T-type calcium channels blocker mibefradil. Compound 48/80-induced itch was enhanced by an endogenous precursor of H2S (L-cysteine) but attenuated by inhibitors of H2S-producing enzymes cystathionine γ-lyase and cystathionine β-synthase. These results indicated that H2S, as a novel nonhistaminergic itch mediator, may activates Cav3.2 T-type calcium channel, probably located at A-fibers, to induce scratching and alloknesis in mice. PMID:26602811

  9. Evaluation of a loop-mediated isothermal amplification method for rapid detection of channel catfish Ictalurus punctatus important bacterial pathogen Edwardsiella ictaluri.

    Science.gov (United States)

    Channel catfish Ictalurus punctatus infected with Edwardsiella ictaluri results in $40 - 50 million annual losses in profits to catfish producers. Early detection of this pathogen is necessary for disease control and reduction of economic loss. In this communication, the loop-mediated isothermal a...

  10. The mechano-activated K+ channels TRAAK and TREK-1 control both warm and cold perception.

    Science.gov (United States)

    Noël, Jacques; Zimmermann, Katharina; Busserolles, Jérome; Deval, Emanuel; Alloui, Abdelkrim; Diochot, Sylvie; Guy, Nicolas; Borsotto, Marc; Reeh, Peter; Eschalier, Alain; Lazdunski, Michel

    2009-05-01

    The sensation of cold or heat depends on the activation of specific nerve endings in the skin. This involves heat- and cold-sensitive excitatory transient receptor potential (TRP) channels. However, we show here that the mechano-gated and highly temperature-sensitive potassium channels of the TREK/TRAAK family, which normally work as silencers of the excitatory channels, are also implicated. They are important for the definition of temperature thresholds and temperature ranges in which excitation of nociceptor takes place and for the intensity of excitation when it occurs. They are expressed with thermo-TRP channels in sensory neurons. TRAAK and TREK-1 channels control pain produced by mechanical stimulation and both heat and cold pain perception in mice. Expression of TRAAK alone or in association with TREK-1 controls heat responses of both capsaicin-sensitive and capsaicin-insensitive sensory neurons. Together TREK-1 and TRAAK channels are important regulators of nociceptor activation by cold, particularly in the nociceptor population that is not activated by menthol.

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

    Science.gov (United States)

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

    2016-10-24

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

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

    Science.gov (United States)

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

    2007-10-01

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

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

    Science.gov (United States)

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

    2012-02-01

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

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

    Directory of Open Access Journals (Sweden)

    Yiliu Liao

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

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

    Science.gov (United States)

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

    2010-12-30

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

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

    Directory of Open Access Journals (Sweden)

    Mahmut ÖZER

    2003-03-01

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

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

    Institute of Scientific and Technical Information of China (English)

    2006-01-01

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

  18. Central functions of bicarbonate in S-type anion channel activation and OST1 protein kinase in CO 2 signal transduction in guard cell

    KAUST Repository

    Xue, Shaowu

    2011-03-18

    Plants respond to elevated CO(2) via carbonic anhydrases that mediate stomatal closing, but little is known about the early signalling mechanisms following the initial CO(2) response. It remains unclear whether CO(2), HCO(3)(-) or a combination activates downstream signalling. Here, we demonstrate that bicarbonate functions as a small-molecule activator of SLAC1 anion channels in guard cells. Elevated intracellular [HCO(3)(-)](i) with low [CO(2)] and [H(+)] activated S-type anion currents, whereas low [HCO(3)(-)](i) at high [CO(2)] and [H(+)] did not. Bicarbonate enhanced the intracellular Ca(2+) sensitivity of S-type anion channel activation in wild-type and ht1-2 kinase mutant guard cells. ht1-2 mutant guard cells exhibited enhanced bicarbonate sensitivity of S-type anion channel activation. The OST1 protein kinase has been reported not to affect CO(2) signalling. Unexpectedly, OST1 loss-of-function alleles showed strongly impaired CO(2)-induced stomatal closing and HCO(3)(-) activation of anion channels. Moreover, PYR/RCAR abscisic acid (ABA) receptor mutants slowed but did not abolish CO(2)/HCO(3)(-) signalling, redefining the convergence point of CO(2) and ABA signalling. A new working model of the sequence of CO(2) signalling events in gas exchange regulation is presented.

  19. Functional analysis of Ficolin-3 mediated complement activation.

    Directory of Open Access Journals (Sweden)

    Estrid Hein

    Full Text Available The recognition molecules of the lectin complement pathway are mannose-binding lectin and Ficolin -1, -2 and -3. Recently deficiency of Ficolin-3 was found to be associated with life threatening infections. Thus, we aimed to develop a functional method based on the ELISA platform for evaluating Ficolin-3 mediated complement activation that could be applicable for research and clinical use. Bovine serum albumin (BSA was acetylated (acBSA and chosen as a solid phase ligand for Ficolins in microtiter wells. Binding of Ficolins on acBSA was evaluated, as was functional complement activation assessed by C4, C3 and terminal complement complex (TCC deposition. Serum Ficolin-3 bound to acBSA in a calcium dependent manner, while only minimal binding of Ficolin-2 and no binding of Ficolin-1 were observed. No binding to normal BSA was seen for any of the Ficolins. Serum C4, C3 and TCC deposition on acBSA were dependent only on Ficolin-3 in appropriate serum dilutions. Deposition of down stream complement components correlated highly significantly with the serum concentration of Ficolin-3 but not with Ficolin-2 in healthy donors. To make the assay robust for clinical use a chemical compound was applied to the samples that inhibited interference from the classical pathway due to the presence of anti-BSA antibodies in some sera. We describe a novel functional method for measuring complement activation mediated by Ficolin-3 in human serum up to the formation of TCC. The assay provides the possibility to diagnose functional and genetic defects of Ficolin-3 and down stream components in the lectin complement pathway.

  20. Biodegradable microsphere-mediated cell perforation in microfluidic channel using femtosecond laser

    Science.gov (United States)

    Ishii, Atsuhiro; Ariyasu, Kazumasa; Mitsuhashi, Tatsuki; Heinemann, Dag; Heisterkamp, Alexander; Terakawa, Mitsuhiro

    2016-05-01

    The use of small particles has expanded the capability of ultrashort pulsed laser optoinjection technology toward simultaneous treatment of multiple cells. The microfluidic platform is one of the attractive systems that has obtained synergy with laser-based technology for cell manipulation, including optoinjection. We have demonstrated the delivery of molecules into suspended-flowing cells in a microfluidic channel by using biodegradable polymer microspheres and a near-infrared femtosecond laser pulse. The use of polylactic-co-glycolic acid microspheres realized not only a higher optoinjection ratio compared to that with polylactic acid microspheres but also avoids optical damage to the microfluidic chip, which is attributable to its higher optical intensity enhancement at the localized spot under a microsphere. Interestingly, optoinjection ratios to nucleus showed a difference for adhered cells and suspended cells. The use of biodegradable polymer microspheres provides high throughput optoinjection; i.e., multiple cells can be treated in a short time, which is promising for various applications in cell analysis, drug delivery, and ex vivo gene transfection to bone marrow cells and stem cells without concerns about residual microspheres.

  1. Role of TRPM7 channels in hyperglycemia-mediated injury of vascular endothelial cells.

    Directory of Open Access Journals (Sweden)

    Huawei Sun

    Full Text Available This study investigated the change of transient receptor potential melastatin 7 (TRPM7 expression by high glucose and its role in hyperglycemia induced injury of vascular endothelial cells. Human umbilical vein endothelial cells (HUVECs were incubated in the presence or absence of high concentrations of D-glucose (HG for 72 h. RT-PCR, Real-time PCR, Western blotting, Immunofluorescence staining and whole-cell patch-clamp recordings showed that TRPM7 mRNA, TRPM7 protein expression and TRPM7-like currents were increased in HUVECs following exposure to HG. In contrast to D-glucose, exposure of HUVECs to high concentrations of L-glucose had no effect. HG increased reactive oxygen species (ROS generation, cytotoxicity and decreased endothelial nitric oxide synthase protein expression, which could be attenuated by knockdown of TRPM7 with TRPM7 siRNA. The protective effect of silencing TRPM7 against HG induced endothelial injury was abolished by U0126, an inhibitor of the extracellular signal-regulated kinase signaling pathway. These observations suggest that TRPM7 channels play an important role in hyperglycemia-induced injury of vascular endothelial cells.

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

    Science.gov (United States)

    Morachevskaya, Elena; Sudarikova, Anastasiya; Negulyaev, Yuri

    2007-04-01

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

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

    Directory of Open Access Journals (Sweden)

    Pengfei Huang

    2014-08-01

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

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

    Directory of Open Access Journals (Sweden)

    Krishna Samanta

    2015-07-01

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

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

    Science.gov (United States)

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

    2016-11-22

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

  6. H2S-mediated thermal and photochemical methane activation.

    Science.gov (United States)

    Baltrusaitis, Jonas; de Graaf, Coen; Broer, Ria; Patterson, Eric V

    2013-12-02

    Sustainable, low-temperature methods for natural gas activation are critical in addressing current and foreseeable energy and hydrocarbon feedstock needs. Large portions of natural gas resources are still too expensive to process due to their high content of hydrogen sulfide gas (H2S) mixed with methane, deemed altogether as sub-quality or "sour" gas. We propose a unique method of activation to form a mixture of sulfur-containing hydrocarbon intermediates, CH3SH and CH3SCH3 , and an energy carrier such as H2. For this purpose, we investigated the H2S-mediated methane activation to form a reactive CH3SH species by means of direct photolysis of sub-quality natural gas. Photoexcitation of hydrogen sulfide in the CH4 + H2S complex resulted in a barrierless relaxation by a conical intersection to form a ground-state CH3SH + H2 complex. The resulting CH3SH could further be coupled over acidic catalysts to form higher hydrocarbons, and the resulting H2 used as a fuel. This process is very different from conventional thermal or radical-based processes and can be driven photolytically at low temperatures, with enhanced control over the conditions currently used in industrial oxidative natural gas activation. Finally, the proposed process is CO2 neutral, as opposed to the current industrial steam methane reforming (SMR).

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

    Energy Technology Data Exchange (ETDEWEB)

    Hattori, Motoyuki; Gouaux, Eric (Oregon HSU)

    2012-10-24

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

  8. Channel-mediated lactic acid transport: a novel function for aquaglyceroporins in bacteria.

    Science.gov (United States)

    Bienert, Gerd P; Desguin, Benoît; Chaumont, François; Hols, Pascal

    2013-09-15

    MIPs (major intrinsic proteins), also known as aquaporins, are membrane proteins that channel water and/or uncharged solutes across membranes in all kingdoms of life. Considering the enormous number of different bacteria on earth, functional information on bacterial MIPs is scarce. In the present study, six MIPs [glpF1 (glycerol facilitator 1)-glpF6] were identified in the genome of the Gram-positive lactic acid bacterium Lactobacillus plantarum. Heterologous expression in Xenopus laevis oocytes revealed that GlpF2, GlpF3 and GlpF4 each facilitated the transmembrane diffusion of water, dihydroxyacetone and glycerol. As several lactic acid bacteria have GlpFs in their lactate racemization operon (GlpF1/F4 phylogenetic group), their ability to transport this organic acid was tested. Both GlpF1 and GlpF4 facilitated the diffusion of D/L-lactic acid. Deletion of glpF1 and/or glpF4 in Lb. plantarum showed that both genes were involved in the racemization of lactic acid and, in addition, the double glpF1 glpF4 mutant showed a growth delay under conditions of mild lactic acid stress. This provides further evidence that GlpFs contribute to lactic acid metabolism in this species. This lactic acid transport capacity was shown to be conserved in the GlpF1/F4 group of Lactobacillales. In conclusion, we have functionally analysed the largest set of bacterial MIPs and demonstrated that the lactic acid membrane permeability of bacteria can be regulated by aquaglyceroporins.

  9. Intermolecular Interactions in the TMEM16A Dimer Controlling Channel Activity

    Science.gov (United States)

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

    2016-12-01

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

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

    Science.gov (United States)

    Billet, Arnaud; Hanrahan, John W

    2013-11-01

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

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

    Science.gov (United States)

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

    1992-06-01

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

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

    Science.gov (United States)

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

    2017-01-01

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

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

    Science.gov (United States)

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

    2012-09-24

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

  14. A Critical Role of TRPM7 As an Ion Channel Protein in Mediating the Mineralization of the Craniofacial Hard Tissues

    Science.gov (United States)

    Nakano, Yukiko; Le, Michael H.; Abduweli, Dawud; Ho, Sunita P.; Ryazanova, Lillia V.; Hu, Zhixian; Ryazanov, Alexey G.; Den Besten, Pamela K.; Zhang, Yan

    2016-01-01

    Magnesium ion (Mg2+) is the fourth most common cation in the human body, and has a crucial role in many physiological functions. Mg2+ homeostasis is an important contributor to bone development, however, its roles in the development of dental mineralized tissues have not yet been well known. We identified that transient receptor potential cation channel, subfamily M, member 7 (TRPM7), was significantly upregulated in the mature ameloblasts as compared to other ameloblasts through our whole transcript microarray analyses of the ameloblasts. TRPM7, an ion channel for divalent metal cations with an intrinsic serine/threonine protein kinase activity, has been characterized as a key regulator of whole body Mg2+ homeostasis. Semi-quantitative PCR and immunostaining for TRMP7 confirmed its upregulation during the maturation stage of enamel formation, at which ameloblasts direct rapid mineralization of the enamel matrix. The significantly hypomineralized craniofacial structures, including incisors, molars, and cranial bones were demonstrated by microCT analysis, von Kossa and trichrome staining in Trpm7Δkinase∕+ mice. A previously generated heterozygous mouse model with the deletion of the TRPM7 kinase domain. Interestingly, the skeletal phenotype of Trpm7Δkinase∕+ mice resembled those found in the tissue-nonspecific alkaline phosphatase (Alpl) KO mice, thus we further examined whether ALPL protein content and alkaline phosphatase (ALPase) activity in ameloblasts, odontoblasts and osteoblasts were affected in those mice. While ALPL protein in Trpm7Δkinase∕+ mice remained at the similar level as that in wt mice, ALPase activities in the Trpm7Δkinase∕+ mice were almost nonexistent. Supplemented magnesium successfully rescued the activities of ALPase in ameloblasts, odontoblasts and osteoblasts of Trpm7Δkinase∕+ mice. These results suggested that TRPM7 is essential for mineralization of enamel as well as dentin and bone by providing sufficient Mg2+ for the ALPL

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

    Directory of Open Access Journals (Sweden)

    Jonathan V Rocheleau

    2006-02-01

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

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

    Science.gov (United States)

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

    2006-02-01

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

  17. H2O2 generated by NADPH oxidase 4 contributes to transient receptor potential vanilloid 1 channel-mediated mechanosensation in the rat kidney.

    Science.gov (United States)

    Lin, Chian-Shiung; Lee, Shang-Hsing; Huang, Ho-Shiang; Chen, Yih-Sharng; Ma, Ming-Chieh

    2015-08-15

    The presence of NADPH oxidase (Nox) in the kidney, especially Nox4, results in H2O2 production, which regulates Na(+) excretion and urine formation. Redox-sensitive transient receptor potential vanilloid 1 channels (TRPV1s) are distributed in mechanosensory fibers of the renal pelvis and monitor changes in intrapelvic pressure (IPP) during urine formation. The present study tested whether H2O2 derived from Nox4 affects TRPV1 function in renal sensory responses. Perfusion of H2O2 into the renal pelvis dose dependently increased afferent renal nerve activity and substance P (SP) release. These responses were attenuated by cotreatment with catalase or TRPV1 blockers. In single unit recordings, H2O2 activated afferent renal nerve activity in response to rising IPP but not high salt. Western blots revealed that Nox2 (gp91(phox)) and Nox4 are both present in the rat kidney, but Nox4 is abundant in the renal pelvis and originates from dorsal root ganglia. This distribution was associated with expression of the Nox4 regulators p22(phox) and polymerase δ-interacting protein 2. Coimmunoprecipitation experiments showed that IPP increases polymerase δ-interacting protein 2 association with Nox4 or p22(phox) in the renal pelvis. Interestingly, immunofluorescence labeling demonstrated that Nox4 colocalizes with TRPV1 in sensory fibers of the renal pelvis, indicating that H2O2 generated from Nox4 may affect TRPV1 activity. Stepwise increases in IPP and saline loading resulted in H2O2 and SP release, sensory activation, diuresis, and natriuresis. These effects, however, were remarkably attenuated by Nox inhibition. Overall, these results suggest that Nox4-positive fibers liberate H2O2 after mechanostimulation, thereby contributing to a renal sensory nerve-mediated diuretic/natriuretic response.

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

    OpenAIRE

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

    2010-01-01

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

  19. Characterization of neuronal membrane K + and Ca 2+ channels operated under steady magnetic fields exposure

    Science.gov (United States)

    Azanza, María J.

    1990-01-01

    Two populations of ionic channels seem to be involved in the responses elicited on single neurons under static magnetic fields exposure: Ca 2+-dependent-K +-channels and Ca 2+-specific-channels. The application of specific activating and blocking drugs shows that those channels could effectively be the mediators of the electrophysiological responses described.

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

    Science.gov (United States)

    Sanguinetti, M C; Xu, Q P

    1999-02-01

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

  1. Intracellular calcium-dependent regulation of the sperm-specific calcium-activated potassium channel, hSlo3, by the BKCa activator LDD175

    Science.gov (United States)

    Wijerathne, Tharaka Darshana; Kim, Jihyun; Yang, Dongki

    2017-01-01

    Plasma membrane hyperpolarization associated with activation of Ca2+-activated K+ channels plays an important role in sperm capacitation during fertilization. Although Slo3 (slowpoke homologue 3), together with the auxiliary γ2-subunit, LRRC52 (leucine-rich-repeat–containing 52), is known to mediate the pH-sensitive, sperm-specific K+ current KSper in mice, the molecular identity of this channel in human sperm remains controversial. In this study, we tested the classical BKCa activators, NS1619 and LDD175, on human Slo3, heterologously expressed in HEK293 cells together with its functional interacting γ2 subunit, hLRRC52. As previously reported, Slo3 K+ current was unaffected by iberiotoxin or 4-aminopyridine, but was inhibited by ~50% by 20 mM TEA. Extracellular alkalinization potentiated hSlo3 K+ current, and internal alkalinization and Ca2+ elevation induced a leftward shift its activation voltage. NS1619, which acts intracellularly to modulate hSlo1 gating, attenuated hSlo3 K+ currents, whereas LDD175 increased this current and induced membrane potential hyperpolarization. LDD175-induced potentiation was not associated with a change in the half-activation voltage at different intracellular pHs (pH 7.3 and pH 8.0) in the absence of intracellular Ca2+. In contrast, elevation of intracellular Ca2+ dramatically enhanced the LDD175-induced leftward shift in the half-activation potential of hSlo3. Therefore, the mechanism of action does not involve pH-dependent modulation of hSlo3 gating; instead, LDD175 may modulate Ca2+-dependent activation of hSlo3. Thus, LDD175 potentially activates native KSper and may induce membrane hyperpolarization-associated hyperactivation in human sperm.

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

    Science.gov (United States)

    Schoen, Ingmar; Fromherz, Peter

    2005-11-01

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

  3. Age-dependent impact of CaV3.2 T-type calcium channel deletion on myogenic tone and flow-mediated vasodilatation in small arteries

    DEFF Research Database (Denmark)

    Mikkelsen, Miriam F.; Björling, Karl; Jensen, Lars Jørn

    2016-01-01

    .2-dependent and -independent effects. No changes in mRNA expression of several important K(+) and Ca(2+) channel genes were induced by CaV3.2 knock-out. However, the expression of the other T-type channel isoform (CaV3.1) was reduced at the mRNA and protein level in mature adult compared to young WT arteries......The myogenic response and flow-mediated vasodilatation are important regulators of local blood perfusion and total peripheral resistance, and are known to entail a calcium influx into vascular smooth muscle cells (VSMCs) and endothelial cells (ECs), respectively. CaV3.2 T-type calcium channels...... are expressed in both VSMCs and ECs of small arteries. The T-type channels are important drug targets but due to the lack of specific antagonists our understanding of the role of CaV3.2 channels in vasomotor tone at various ages is scarce. We evaluated the myogenic response, flow-mediated vasodilatation...

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

    DEFF Research Database (Denmark)

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

    2005-01-01

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

  5. Role of low voltage activated calcium channels in neuritogenesis and active migration of embryonic neural progenitor cells.

    Science.gov (United States)

    Louhivuori, Lauri M; Louhivuori, Verna; Wigren, Henna-Kaisa; Hakala, Elina; Jansson, Linda C; Nordström, Tommy; Castrén, Maija L; Akerman, Karl E

    2013-04-15

    The central role of calcium influx and electrical activity in embryonic development raises important questions about the role and regulation of voltage-dependent calcium influx. Using cultured neural progenitor cell (NPC) preparations, we recorded barium currents through voltage-activated channels using the whole-cell configuration of the patch-clamp technique and monitored intracellular free calcium concentrations with Fura-2 digital imaging. We found that NPCs as well as expressing high-voltage-activated (HVA) calcium channels express functional low-threshold voltage-dependent calcium channels in the very early stages of differentiation (5 h to 1 day). The size of the currents recorded at -50 versus -20 mV after 1 day in differentiation was dependent on the nature of the charge carrier. Peak currents measured at -20 mV in the presence 10 mM Ca2+ instead of 10 mM Ba2+ had a tendency to be smaller, whereas the nature of the divalent species did not influence the amplitude measured at -50 mV. The T-type channel blockers mibefradil and NNC 55-0396 significantly reduced the calcium responses elicited by depolarizing with extracellular potassium, while the overall effect of the HVA calcium channel blockers was small at differentiation day 1. At differentiation day 20, the calcium responses were effectively blocked by nifedipine. Time-lapse imaging of differentiating neurospheres cultured in the presence of low-voltage-activated (LVA) blockers showed a significant decrease in the number of active migrating neuron-like cells and neurite extensions. Together, these data provide evidence that LVA calcium channels are involved in the physiology of differentiating and migrating NPCs.

  6. [6]-Shogaol Induces Ca2+ Signals by Activating the TRPV1 Channels in the Rat Insulinoma INS-1E Cells

    Directory of Open Access Journals (Sweden)

    Paola Rebellato

    2014-01-01

    Full Text Available Context [6]-shogaol is a vanilloid compound present in steamed ginger (Zingiber officinale, a commonly used spice. Pancreatic beta-cells respond to nutrients like glucose, amino acids and fatty acids, by an increase in the cytoplasmic free Ca2+ concentration ([Ca2+]i, which mediates diverse cellular processes in these cells. Some vanilloid compounds activate the transient receptor potential vanilloid receptor type 1 (TRPV1 channel. Objective We investigated whether [6]-shogaol could trigger Ca2+ signals in the beta-cell. Methods [Ca2+]i was measured from single INS-1E cells by microscope-based fluorometry using fura-2 as the Ca2+ indicator. Results In fura-2 loaded single rat insulinoma INS-1E cells, a widely used model of beta-cell, [6]-shogaol increased [Ca2+]i in a concentration-dependent manner. [Ca2+]i increase by [6]-shogaol was completely blocked when Ca2+ was omitted from the extracellular medium. Capsazepine, an inhibitor of the TRPV1 ion channel completely inhibited the [6]-shogaol-induced [Ca2+]i increase. [Ca2+]i increase obtained by 1 μM [6]-shogaol was greater than that obtained by 10 mM glucose. Moreover, a sub-stimulatory concentration of [6]-shogaol (300 nM, significantly enhanced the glucose-induced [Ca2+]i increase in these cells. Conclusion We conclude that [6]-shogaol induces Ca2+ signals in the beta-cell by activating the TRPV1 channels, and it sensitizes the beta-cells to stimulation by glucose.

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

    Science.gov (United States)

    Kanaporis, Giedrius; Blatter, Lothar A

    2016-11-01

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

  8. Stochastically gating ion channels enable patterned spike firing through activity-dependent modulation of spike probability.

    Directory of Open Access Journals (Sweden)

    Joshua T Dudman

    2009-02-01

    Full Text Available The transformation of synaptic input into patterns of spike output is a fundamental operation that is determined by the particular complement of ion channels that a neuron expresses. Although it is well established that individual ion channel proteins make stochastic transitions between conducting and non-conducting states, most models of synaptic integration are deterministic, and relatively little is known about the functional consequences of interactions between stochastically gating ion channels. Here, we show that a model of stellate neurons from layer II of the medial entorhinal cortex implemented with either stochastic or deterministically gating ion channels can reproduce the resting membrane properties of stellate neurons, but only the stochastic version of the model can fully account for perithreshold membrane potential fluctuations and clustered patterns of spike output that are recorded from stellate neurons during depolarized states. We demonstrate that the stochastic model implements an example of a general mechanism for patterning of neuronal output through activity-dependent changes in the probability of spike firing. Unlike deterministic mechanisms that generate spike patterns through slow changes in the state of model parameters, this general stochastic mechanism does not require retention of information beyond the duration of a single spike and its associated afterhyperpolarization. Instead, clustered patterns of spikes emerge in the stochastic model of stellate neurons as a result of a transient increase in firing probability driven by activation of HCN channels during recovery from the spike afterhyperpolarization. Using this model, we infer conditions in which stochastic ion channel gating may influence firing patterns in vivo and predict consequences of modifications of HCN channel function for in vivo firing patterns.

  9. Levamisole-activated single-channel currents from muscle of the nematode parasite Ascaris suum.

    Science.gov (United States)

    Robertson, S. J.; Martin, R. J.

    1993-01-01

    1. The patch-clamp technique was used to examine levamisole-activated channels in muscle vesicles from Ascaris suum. Cell-attached and isolated inside-out patches were used. 2. Levamisole (1-90 microM), applied to the extracellular surface, activated channels which had apparent mean open-times in the range 0.80-2.85 ms and linear I/V relationships with conductances in the range 19-46 pS. Ion-replacement experiments showed the channels to be cation selective. 3. The kinetics of the channels were analysed. Generally open- and closed-time distributions were best fitted by two, and three exponentials respectively, indicating the presence of at least two open states and at least three closed states. The distributions of burst-times were best-fitted by two exponentials. 4. Channel open- and burst-times were voltage-sensitive: at low levamisole concentrations (1-10 microM), they increased with hyperpolarization. At higher concentrations of levamisole (30 microM and 90 microM) flickering channel-block was observed at hyperpolarized potentials. Using a simple channel-block model, values for the blocking dissociation constant, KB were determined as 123 microM at -50 mV, 46 microM at -75 mV and 9.4 microM at -100 mV. 5. At the higher concentration of levamisole (30 microM and 90 microM) long closed-times separating 'clusters' of bursts were observed, at both hyperpolarized and depolarized membrane potentials and this was interpreted as desensitization. PMID:7679027

  10. Voltage-dependent gating of hyperpolarization-activated, cyclic nucleotide-gated pacemaker channels: molecular coupling between the S4-S5 and C-linkers.

    Science.gov (United States)

    Decher, Niels; Chen, Jun; Sanguinetti, Michael C

    2004-04-02

    Hyperpolarization-activated, cyclic nucleotide-gated (HCN) channels have a transmembrane topology that is highly similar to voltage-gated K(+) channels, yet HCN channels open in response to membrane hyperpolarization instead of depolarization. The structural basis for the "inverted" voltage dependence of HCN gating and how voltage sensing by the S1-S4 domains is coupled to the opening of the intracellular gate formed by the S6 domain are unknown. Coupling could arise from interaction between specific residues or entire transmembrane domains. We previously reported that the mutation of specific residues in the S4-S5 linker of HCN2 (i.e. Tyr-331 and Arg-339) prevented normal channel closure presumably by disruption of a crucial interaction with the activation gate. Here we hypothesized that the C-linker, a carboxyl terminus segment that connects S6 to the cyclic nucleotide binding domain, interacts with specific residues of the S4-S5 linker to mediate coupling. The recently solved structure of the C-linker of HCN2 indicates that an alpha-helix (the A'-helix) is located near the end of each S6 domain, the presumed location of the activation gate. Ala-scanning mutagenesis of the end of S6 and the A'-helix identified five residues that were important for normal gating as mutations disrupted channel closure. However, partial deletion of the C-linker indicated that the presence of only two of these residues was required for normal coupling. Further mutation analyses suggested that a specific electrostatic interaction between Arg-339 of the S4-S5 linker and Asp-443 of the C-linker stabilizes the closed state and thus participates in the coupling of voltage sensing and activation gating in HCN channels.

  11. Activation of KCNQ5 channels stably expressed in HEK293 cells by BMS-204352

    DEFF Research Database (Denmark)

    Dupuis, Delphine S; Schrøder, Rikke L; Jespersen, Thomas;

    2002-01-01

    The novel anti-ischemic compound, BMS-204352 ((3S)-(+)-(5-chloro-2-methoxyphenyl)-1,3-dihydro-3-fluoro-6-(trifluoromethyl)-2H-indol-2-one)), strongly activates the voltage-gated K+ channel KCNQ5 in a concentration-dependent manner with an EC50 of 2.4 microM. At 10 microM, BMS-204352 increased the...

  12. Activation of KCNQ5 channels stably expressed in HEK293 cells by BMS-204352

    DEFF Research Database (Denmark)

    Dupuis, Delphine S.; Schrøder, Rikke Louise K.; Jespersen, Thomas;

    2002-01-01

    time constant increased up to 10-fold. The Mcurrent blockers, linopirdine (DuP 996; 3,3 -bis (4-pyridinylmethyl)-l-phenylindolin-2 -one) and XE991 (10,10-bis(4-pyridinylmethyl)9(10H)-anthracenone), inhibited the activation of the KCNQ5 channel induced by the BMS-204352. Thus, BMS-204352 appears...

  13. Functional insights into modulation of BKCa channel activity to alter myometrial contractility

    Directory of Open Access Journals (Sweden)

    Ramón A Lorca

    2014-07-01

    Full Text Available The large-conductance voltage- and Ca2+-activated K+ channel (BKCa is an important regulator of membrane excitability in a wide variety of cells and tissues. In myometrial smooth muscle, activation of BKCa plays essential roles in buffering contractility to maintain uterine quiescence during pregnancy and in the transition to a more contractile state at the onset of labor. Multiple mechanisms of modulation have been described to alter BKCa channel activity, expression, and cellular localization. In the myometrium, BKCa is regulated by alternative splicing, protein targeting to the plasma membrane, compartmentation in membrane microdomains, and posttranslational modifications. In addition, interaction with auxiliary proteins (i.e., β1- and β2-subunits, association with G-protein coupled receptor signaling pathways, such as those activated by adrenergic and oxytocin receptors, and hormonal regulation provide further mechanisms of variable modulation of BKCa channel function in myometrial smooth muscle. Here, we provide an overview of these mechanisms of BKCa channel modulation and provide a context for them in relation to myometrial function.

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

    DEFF Research Database (Denmark)

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

    2011-01-01

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

  15. 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

  16. Microparticle-mediated transfer of the viral receptors CAR and CD46, and the CFTR channel in a CHO cell model confers new functions to target cells.

    Directory of Open Access Journals (Sweden)

    Gaëlle Gonzalez

    Full Text Available Cell microparticles (MPs released in the extracellular milieu can embark plasma membrane and intracellular components which are specific of their cellular origin, and transfer them to target cells. The MP-mediated, cell-to-cell transfer of three human membrane glycoproteins of different degrees of complexity was investigated in the present study, using a CHO cell model system. We first tested the delivery of CAR and CD46, two monospanins which act as adenovirus receptors, to target CHO cells. CHO cells lack CAR and CD46, high affinity receptors for human adenovirus serotype 5 (HAdV5, and serotype 35 (HAdV35, respectively. We found that MPs derived from CHO cells (MP-donor cells constitutively expressing CAR (MP-CAR or CD46 (MP-CD46 were able to transfer CAR and CD46 to target CHO cells, and conferred selective permissiveness to HAdV5 and HAdV35. In addition, target CHO cells incubated with MP-CD46 acquired the CD46-associated function in complement regulation. We also explored the MP-mediated delivery of a dodecaspanin membrane glycoprotein, the CFTR to target CHO cells. CFTR functions as a chloride channel in human cells and is implicated in the genetic disease cystic fibrosis. Target CHO cells incubated with MPs produced by CHO cells constitutively expressing GFP-tagged CFTR (MP-GFP-CFTR were found to gain a new cellular function, the chloride channel activity associated to CFTR. Time-course analysis of the appearance of GFP-CFTR in target cells suggested that MPs could achieve the delivery of CFTR to target cells via two mechanisms: the transfer of mature, membrane-inserted CFTR glycoprotein, and the transfer of CFTR-encoding mRNA. These results confirmed that cell-derived MPs represent a new class of promising therapeutic vehicles for the delivery of bioactive macromolecules, proteins or mRNAs, the latter exerting the desired therapeutic effect in target cells via de novo synthesis of their encoded proteins.

  17. CaV3.2 T-type Ca2+ channels mediate the augmented calcium influx in carotid body glomus cells by chronic intermittent hypoxia.

    Science.gov (United States)

    Makarenko, Vladislav V; Ahmmed, Gias U; Peng, Ying-Jie; Khan, Shakil A; Nanduri, Jayasri; Kumar, Ganesh K; Fox, Aaron P; Prabhakar, Nanduri R

    2016-01-01

    Chronic intermittent hypoxia (CIH) is a hallmark manifestation of sleep apnea. A heightened carotid body activity and the resulting chemosensory reflex mediate increased sympathetic nerve activity by CIH. However, the mechanisms underlying heightened carotid body activity by CIH are not known. An elevation of intracellular calcium ion concentration ([Ca(2+)]i) in glomus cells, the primary oxygen-sensing cells, is an essential step for carotid body activation by hypoxia. In the present study, we examined the effects of CIH on the glomus cell [Ca(2+)]i response to hypoxia and assessed the underlying mechanisms. Glomus cells were harvested from adult rats or wild-type mice treated with 10 days of either room air (control) or CIH (alternating cycles of 15 s of hypoxia and 5 min of room air; 9 episodes/h; 8 h/day). CIH-treated glomus cells exhibited an enhanced [Ca(2+)]i response to hypoxia, and this effect was absent in the presence of 2-(4-cyclopropylphenyl)-N-((1R)-1-[5-[(2,2,2-trifluoroethyl)oxo]-pyridin-2-yl]ethyl)acetamide (TTA-A2), a specific inhibitor of T-type Ca(2+) channels, and in voltage-gated calcium channel, type 3.2 (CaV3.2), null glomus cells. CaV3.2 knockout mice exhibited an absence of CIH-induced hypersensitivity of the carotid body. CIH increased reactive oxygen species (ROS) levels in glomus cells. A ROS scavenger prevented the exaggerated TTA-A2-sensitive [Ca(2+)]i response to hypoxia. CIH had no effect on CaV3.2 mRNA levels. CIH augmented Ca(2+) currents and increased CaV3.2 protein in plasma membrane fractions of human embryonic kidney-293 cells stably expressing CaV3.2, and either a ROS scavenger or brefeldin-A, an inhibitor of protein trafficking, prevented these effects. These findings suggest that CIH leads to an augmented Ca(2+) influx via ROS-dependent facilitation of CaV3.2 protein trafficking to the plasma membrane.

  18. Mitochondrial ATP-sensitive potassium channel activity and hypoxic preconditioning are independent of an inwardly rectifying potassium channel subunit in Caenorhabditis elegans.

    Science.gov (United States)

    Wojtovich, Andrew P; DiStefano, Peter; Sherman, Teresa; Brookes, Paul S; Nehrke, Keith

    2012-02-17

    Hypoxic preconditioning (HP) is an evolutionarily-conserved mechanism that protects an organism against stress. The mitochondrial ATP-sensitive K(+) channel (mK(ATP)) plays an essential role in the protective signaling, but remains molecularly undefined. Several lines of evidence suggest that mK(ATP) may arise from an inward rectifying K(+) channel (Kir). The genetic model organism Caenorhabditis elegans exhibits HP and displays mK(ATP) activity. Here, we investigate the tissue expression profile of the three C. elegans Kir genes and demonstrate that mutant strains where the irk genes have been deleted either individually or in combination can be protected by HP and exhibit robust mK(ATP) channel activity in purified mitochondria. These data suggest that the mK(ATP) in C. elegans does not arise from a Kir derived channel.

  19. Mitochondrial ATP-sensitive potassium channel activity and hypoxic preconditioning are independent of an inwardly rectifying potassium channel subunit in C. elegans

    Science.gov (United States)

    Wojtovich, Andrew P.; DiStefano, Peter; Sherman, Teresa; Brookes, Paul S.; Nehrke, Keith

    2012-01-01

    Hypoxic preconditioning (HP) is an evolutionarily-conserved mechanism that protects an organism against stress. The mitochondrial ATP-sensitive K+ channel (mKATP) plays an essential role in the protective signaling, but remains molecularly undefined. Several lines of evidence suggest that mKATP may arise from an inward rectifying K+ channel (Kir). The genetic model organism C. elegans exhibits HP and displays mKATP activity. Here, we investigate the tissue expression profile of the three C. elegans Kir genes and demonstrate that mutant strains where the irk genes have been deleted either individually or in combination can be protected by HP and exhibit robust mKATP channel activity in purified mitochondria. These data suggest that the mKATP in C. elegans does not arise from a Kir derived channel. PMID:22281198

  20. 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.

  1. Possible Selves and Physical Activity in Retirees: The Mediating Role of Identity.

    Science.gov (United States)

    Perras, Mélanie G M; Strachan, Shaelyn M; Fortier, Michelle S

    2016-11-01

    Many retirees remain insufficiently physically active for health benefits. Self-perceptions can influence physical activity. Possible selves and identity are two self-perceptions that, when examined relative to physical activity, may help explain physical activity levels among retirees. Scholars claim that a focus on possible selves may impact identity, which, in the physical activity domain, is a known physical activity correlate. The aim of this study was to examine the relationship between these variables, and more specifically, to determine whether exercise identity mediates the relationship between physical activity possible selves and physical activity. We examined the proposed mediation relationship in 531 new retirees. Data were collected across three time points (1 month apart). We conducted mediation analyses with bootstrapping. Identity mediated the relationship between possible selves (content and processes) and behavior-all related to physical activity. These findings represent an important first step toward designing relevant physical activity interventions for retirees.

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

    Directory of Open Access Journals (Sweden)

    Smith Paula L

    2011-09-01

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

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

    Science.gov (United States)

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

    2016-06-01

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

  4. 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.

  5. Relationship between Customer Perception about CSR activities and Purchase Intention: The Role of CSR Communication Channels

    OpenAIRE

    SONG, JINWEN; Fang, Qi; Wang, Jieru

    2015-01-01

    With the incorporation of businesses as major players in a country’s economy and society, Corporate Social Responsibility is becoming a very important aspect of corporate activity. This field is greatly understudied and only limited research has been done on the consequences of conducting CSR activities through different channels of communication. This thesis therefore contributes to a better understanding of the relationship between customer perception and purchase intention in the setting o...

  6. 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.

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

    Science.gov (United States)

    Yeung, Ella W; Allen, David G

    2004-08-01

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

  8. A multichannel integrated circuit for electrical recording of neural activity, with independent channel programmability.

    Science.gov (United States)

    Mora Lopez, Carolina; Prodanov, Dimiter; Braeken, Dries; Gligorijevic, Ivan; Eberle, Wolfgang; Bartic, Carmen; Puers, Robert; Gielen, Georges

    2012-04-01

    Since a few decades, micro-fabricated neural probes are being used, together with microelectronic interfaces, to get more insight in the activity of neuronal networks. The need for higher temporal and spatial recording resolutions imposes new challenges on the design of integrated neural interfaces with respect to power consumption, data handling and versatility. In this paper, we present an integrated acquisition system for in vitro and in vivo recording of neural activity. The ASIC consists of 16 low-noise, fully-differential input channels with independent programmability of its amplification (from 100 to 6000 V/V) and filtering (1-6000 Hz range) capabilities. Each channel is AC-coupled and implements a fourth-order band-pass filter in order to steeply attenuate out-of-band noise and DC input offsets. The system achieves an input-referred noise density of 37 nV/√Hz, a NEF of 5.1, a CMRR > 60 dB, a THD < 1% and a sampling rate of 30 kS/s per channel, while consuming a maximum of 70 μA per channel from a single 3.3 V. The ASIC was implemented in a 0.35 μm CMOS technology and has a total area of 5.6 × 4.5 mm². The recording system was successfully validated in in vitro and in vivo experiments, achieving simultaneous multichannel recordings of cell activity with satisfactory signal-to-noise ratios.

  9. Molecular mechanism underlying β1 regulation in voltage- and calcium-activated potassium (BK) channels.

    Science.gov (United States)

    Castillo, Karen; Contreras, Gustavo F; Pupo, Amaury; Torres, Yolima P; Neely, Alan; González, Carlos; Latorre, Ramon

    2015-04-14

    Being activated by depolarizing voltages and increases in cytoplasmic Ca(2+), voltage- and calcium-activated potassium (BK) channels and their modulatory β-subunits are able to dampen or stop excitatory stimuli in a wide range of cellular types, including both neuronal and nonneuronal tissues. Minimal alterations in BK channel function may contribute to the pathophysiology of several diseases, including hypertension, asthma, cancer, epilepsy, and diabetes. Several gating processes, allosterically coupled to each other, control BK channel activity and are potential targets for regulation by auxiliary β-subunits that are expressed together with the α (BK)-subunit in almost every tissue type where they are found. By measuring gating currents in BK channels coexpressed with chimeras between β1 and β3 or β2 auxiliary subunits, we were able to identify that the cytoplasmic regions of β1 are responsible for the modulation of the voltage sensors. In addition, we narrowed down the structural determinants to the N terminus of β1, which contains two lysine residues (i.e., K3 and K4), which upon substitution virtually abolished the effects of β1 on charge movement. The mechanism by which K3 and K4 stabilize the voltage sensor is not electrostatic but specific, and the α (BK)-residues involved remain to be identified. This is the first report, to our knowledge, where the regulatory effects of the β1-subunit have been clearly assigned to a particular segment, with two pivotal amino acids being responsible for this modulation.

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

    Science.gov (United States)

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

    2015-07-03

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

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

    Directory of Open Access Journals (Sweden)

    Jose L. Nieto-Torres

    2015-07-01

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

  12. Activation of the cold-sensing TRPM8 channel triggers UCP1-dependent thermogenesis and prevents obesity

    Institute of Scientific and Technical Information of China (English)

    Shuangtao Ma; Zhenyu Zhu; Li Li; Jian Zhong; Daoyan Liu; Bernd Nilius; Zhiming Zhu; Hao Yu; Zhigang Zhao; Zhidan Luo; Jing Chen; Yinxing Ni; Rongbing Jin; Liqun Ma; Peijian Wang

    2012-01-01

    Brown adipose tissue (BAT) is an energy-expending organ that produces heat.Expansion or activation of BAT prevents obesity and diabetes.Chronic cold exposure enhances thermogenesis in BAT through uncoupling protein 1 (UCP1) activation triggered via a β-adrenergic pathway.Here,we report that the cold-sensing transient receptor potential melastatin 8 (TRPM8) is functionally present In mouse BAT.Challenging brown adipocytes with menthol,a TRPM8 agonist,up-regulates UCP1 expression and requires protein kinase A activation.Upon mimicking long-term cold exposure with chronic dietary menthol application,menthol significantly increased the core temperatures and locomotor activity in wild-type mice; these effects were absent in both TRPM8-/- and UCP1-/- mice.Dietary obesity and glucose abnormalities were also prevented by menthol treatment.Our results reveal a previously unrecognized role for TRPM8,suggesting that stimulation of this channel mediates BAT thermogenesis,which could constitute a promising way to treat obesity.

  13. Antischistosomal activity of a calcium channel antagonist on schistosomula and adult Schistosoma mansoni worms

    Directory of Open Access Journals (Sweden)

    Vanessa Silva-Moraes

    2013-08-01

    Full Text Available Current schistosomiasis control strategies are largely based on chemotherapeutic agents and a limited number of drugs are available today. Praziquantel (PZQ is the only drug currently used in schistosomiasis control programs. Unfortunately, this drug shows poor efficacy in patients during the earliest infection phases. The effects of PZQ appear to operate on the voltage-operated Ca2+channels, which are located on the external Schistosoma mansoni membrane. Because some Ca2+channels have dihydropyridine drug class (a class that includes nifedipine sensitivity, an in vitro analysis using a calcium channel antagonist (clinically used for cardiovascular hypertension was performed to determine the antischistosomal effects of nifedipine on schistosomula and adult worm cultures. Nifedipine demonstrated antischistosomal activity against schistosomula and significantly reduced viability at all of the concentrations used alone or in combination with PZQ. In contrast, PZQ did not show significant efficacy when used alone. Adult worms were also affected by nifedipine after a 24 h incubation and exhibited impaired motility, several lesions on the tegument and intense contractility. These data support the idea of Ca2+channels subunits as drug targets and favour alternative therapeutic schemes when drug resistance has been reported. In this paper, strong arguments encouraging drug research are presented, with a focus on exploring schistosomal Ca2+channels.

  14. Treatment of Na(v)1.7-mediated pain in inherited erythromelalgia using a novel sodium channel blocker.

    Science.gov (United States)

    Goldberg, Yigal Paul; Price, Nicola; Namdari, Rostam; Cohen, Charles Jay; Lamers, Mieke H; Winters, Conrad; Price, James; Young, Clint E; Verschoof, Henry; Sherrington, Robin; Pimstone, Simon Neil; Hayden, Michael Reuben

    2012-01-01

    Mutations in the SCN9A gene leading to deficiency of its protein product, Na(v)1.7, cause congenital indifference to pain (CIP). CIP is characterized by the absence of the ability to sense pain associated with noxious stimuli. In contrast, the opposite phenotype to CIP, inherited erythromelalgia (IEM), is a disorder of spontaneous pain caused by missense mutations resulting in gain-of-function in Na(v)1.7 that promote neuronal hyperexcitability. The primary aim of this study was to demonstrate that Na(v)1.7 antagonism could alleviate the pain of IEM, thereby demonstrating the utility of this opposite phenotype model as a tool for rapid proof-of-concept for novel analgesics. An exploratory, randomized, double-blind, 2-period crossover study was conducted in 4 SCN9A mutation-proven IEM patients. In each treatment period (2days), separated by a 2-day washout period, patients were orally administered XEN402 (400mg twice daily) or matching placebo. In 3 patients, pain was induced by heat or exercise during each treatment arm. A fourth patient, in constant severe pain, required no induction. Patient-reported outcomes of pain intensity and/or relief were recorded, and the time taken to induce pain was measured. The ability to induce pain in IEM patients was significantly attenuated by XEN402 compared with placebo. XEN402 increased the time to maximal pain induction and significantly reduced the amount of pain (42% less) after induction (P=.014). This pilot study showed that XEN402 blocks Na(v)1.7-mediated pain associated with IEM, thereby demonstrating target engagement in humans and underscoring the use of rare genetic disorders with mutant target channels as a novel approach to rapid proof-of-concept.

  15. Influenza matrix protein 2 alters CFTR expression and function through its ion channel activity.

    Science.gov (United States)

    Londino, James D; Lazrak, Ahmed; Jurkuvenaite, Asta; Collawn, James F; Noah, James W; Matalon, Sadis

    2013-05-01

    The human cystic fibrosis transmembrane conductance regulator (CFTR) is a cyclic AMP-activated chloride (Cl(-)) channel in the lung epithelium that helps regulate the thickness and composition of the lung epithelial lining fluid. We investigated whether influenza M2 protein, a pH-activated proton (H(+)) channel that traffics to the plasma membrane of infected cells, altered CFTR expression and function. M2 decreased CFTR activity in 1) Xenopus oocytes injected with human CFTR, 2) epithelial cells (HEK-293) stably transfected with CFTR, and 3) human bronchial epithelial cells (16HBE14o-) expressing native CFTR. This inhibition was partially reversed by an inhibitor of the ubiquitin-activating enzyme E1. Next we investigated whether the M2 inhibition of CFTR activity was due to an increase of secretory organelle pH by M2. Incubation of Xenopus oocytes expressing CFTR with ammonium chloride or concanamycin A, two agents that alkalinize the secretory pathway, inhibited CFTR activity in a dose-dependent manner. Treatment of M2- and CFTR-expressing oocytes with the M2 ion channel inhibitor amantadine prevented the loss in CFTR expression and activity; in addition, M2 mutants, lacking the ability to transport H(+), did not alter CFTR activity in Xenopus oocytes and HEK cells. Expression of an M2 mutant retained in the endoplasmic reticulum also failed to alter CFTR activity. In summary, our data show that M2 decreases CFTR activity by increasing secretory organelle pH, which targets CFTR for destruction by the ubiquitin system. Alteration of CFTR activity has important consequences for fluid regulation and may potentially modify the immune response to viral infection.

  16. Francisella tularensis Catalase Restricts Immune Function by Impairing TRPM2 Channel Activity.

    Science.gov (United States)

    Shakerley, Nicole L; Chandrasekaran, Akshaya; Trebak, Mohamed; Miller, Barbara A; Melendez, J Andrés

    2016-02-19

    As an innate defense mechanism, macrophages produce reactive oxygen species that weaken pathogens and serve as secondary messengers involved in immune function. The Gram-negative bacterium Francisella tularensis utilizes its antioxidant armature to limit the host immune response, but the mechanism behind this suppression is not defined. Here we establish that F. tularensis limits Ca(2+) entry in macrophages, thereby limiting actin reorganization and IL-6 production in a redox-dependent fashion. Wild type (live vaccine strain) or catalase-deficient F. tularensis (ΔkatG) show distinct profiles in their H2O2 scavenging rates, 1 and 0.015 pm/s, respectively. Murine alveolar macrophages infected with ΔkatG display abnormally high basal intracellular Ca(2+) concentration that did not increase further in response to H2O2. Additionally, ΔkatG-infected macrophages displayed limited Ca(2+) influx in response to ionomycin, as a result of ionophore H2O2 sensitivity. Exogenously added H2O2 or H2O2 generated by ΔkatG likely oxidizes ionomycin and alters its ability to transport Ca(2+). Basal increases in cytosolic Ca(2+) and insensitivity to H2O2-mediated Ca(2+) entry in ΔkatG-infected cells are reversed by the Ca(2+) channel inhibitors 2-aminoethyl diphenylborinate and SKF-96365. 2-Aminoethyl diphenylborinate but not SKF-96365 abrogated ΔkatG-dependent increases in macrophage actin remodeling and IL-6 secretion, suggesting a role for H2O2-mediated Ca(2+) entry through the transient receptor potential melastatin 2 (TRPM2) channel in macrophages. Indeed, increases in basal Ca(2+), actin polymerization, and IL-6 production are reversed in TRPM2-null macrophages infected with ΔkatG. Together, our findings provide compelling evidence that F. tularensis catalase restricts reactive oxygen species to temper macrophage TRPM2-mediated Ca(2+) signaling and limit host immune function.

  17. 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.

  18. Molecular and functional expression of high conductance Ca 2+ activated K+ channels in the eel intestinal epithelium

    DEFF Research Database (Denmark)

    Lionetto, Maria G; Rizzello, Antonia; Giordano, Maria E;

    2008-01-01

    Several types of K(+) channels have been identified in epithelial cells. Among them high conductance Ca(2+)-activated K(+) channels (BK channels) are of relevant importance for their involvement in regulatory volume decrease (RVD) response following hypotonic stress. The aim of the present work...... and morphometric analysis on the intact tissue. BK(Ca) channels appeared to be localized along all the plasma membrane of the enterocytes; the apical part of the villi showed the most intense immunostaining. These channels were silent in basal condition, but were activated on both membranes (apical and basolateral......) by increasing intracellular Ca(2+) concentration with the Ca(2+) ionophore ionomycin (1 microM). BK(Ca) channels were also activated on both membranes by hypotonic swelling of the epithelium and their inhibition by 100 nM iberiotoxin (specific BK(Ca) inhibitor) abolished the Regulatory Volume Decrease (RVD...

  19. Insulin Excites Anorexigenic Proopiomelanocortin Neurons via Activation of Canonical Transient Receptor Potential Channels

    Science.gov (United States)

    Qiu, Jian; Zhang, Chunguang; Borgquist, Amanda; Nestor, Casey C; Smith, Arik W.; Bosch, Martha A.; Ku, Stephen; Wagner, Edward J.; Rønnekleiv, Oline K.; Kelly, Martin J.

    2014-01-01

    SUMMARY Proopiomelanocortin (POMC) neurons within the hypothalamic arcuate nucleus are vital anorexigenic neurons. Although both the leptin receptor and insulin receptor are coupled to activation of phosphatidylinositide3-kinase (PI3K) in POMC neurons, they are thought to have disparate actions on POMC excitability. Using whole-cell recording and selective pharmacological tools, we have found that similar to leptin, purified insulin depolarized POMC, and adjacent kisspeptin neurons via activation of TRPC5 channels, which are highly expressed in these neurons. In contrast, insulin hyperpolarized and inhibited NPY/AgRP neurons via activation of KATP channels. Moreover, Zn2+, which is found in insulin formulations at nanomolar concentrations, inhibited POMC neurons via activation of KATP channels. Finally as predicted, insulin given intracerebroventrically robustly inhibited food intake and activated c-fos expression in arcuate POMC neurons. Our results show that purified insulin excites POMC neurons in the arcuate nucleus, which we propose is a major mechanism by which insulin regulates energy homeostasis. PMID:24703699

  20. Mapping ECoG channel contributions to trajectory and muscle activity prediction in human sensorimotor cortex

    Science.gov (United States)

    Nakanishi, Yasuhiko; Yanagisawa, Takufumi; Shin, Duk; Kambara, Hiroyuki; Yoshimura, Natsue; Tanaka, Masataka; Fukuma, Ryohei; Kishima, Haruhiko; Hirata, Masayuki; Koike, Yasuharu

    2017-01-01

    Studies on brain-machine interface techniques have shown that electrocorticography (ECoG) is an effective modality for predicting limb trajectories and muscle activity in humans. Motor control studies have also identified distributions of “extrinsic-like” and “intrinsic-like” neurons in the premotor (PM) and primary motor (M1) cortices. Here, we investigated whether trajectories and muscle activity predicted from ECoG were obtained based on signals derived from extrinsic-like or intrinsic-like neurons. Three participants carried objects of three different masses along the same counterclockwise path on a table. Trajectories of the object and upper arm muscle activity were predicted using a sparse linear regression. Weight matrices for the predictors were then compared to determine if the ECoG channels contributed more information about trajectory or muscle activity. We found that channels over both PM and M1 contributed highly to trajectory prediction, while a channel over M1 was the highest contributor for muscle activity prediction. PMID:28361947

  1. The amiodarone derivative 2-methyl-3-(3,5-diiodo-4-carboxymethoxybenzyl)benzofuran (KB130015) opens large-conductance Ca2+-activated K+ channels and relaxes vascular smooth muscle.

    Science.gov (United States)

    Gessner, Guido; Heller, Regine; Hoshi, Toshinori; Heinemann, Stefan H

    2007-01-26

    2-methyl-3-(3,5-diiodo-4-carboxymethoxybenzyl)benzofuran (KB130015) has been developed to retain the antiarrhythmic properties of the parent molecule amiodarone but to eliminate its undesired side effects. In patch-clamp experiments, KB130015 activated large-conductance, Ca2+-activated BK(Ca) channels formed by hSlo1 (alpha) subunits in HEK 293 cells. Channels were reversibly activated by shifting the open-probability/voltage (P(o)/V) relationship by about -60 mV in 3 muM intracellular free Ca2+ ([Ca2+]in). No effect on the single-channel conductance was observed. KB130015-mediated activation of BK(Ca) channels was half-maximal at 20 microM with a Hill coefficient of 2.8. BK(Ca) activation by KB130015 did not require the presence of Ca2+ and still occurred with saturating (100 microM) [Ca2+]in. Effects of the prototypic BK(Ca) activator NS1619 (1,3-dihydro-1-[2-hydroxy-5-(trifluoromethyl)phenyl]-5-(trifluoromethyl)-2H-benzimidazol-2-one) and those of KB130015 were not additive suggesting that both activators may at least partially share a common mechanism of action. KB130015-mediated activation was observed also for BK(Ca) channels from insects and for human BK(Ca) channels with already profoundly left-shifted voltage-dependence. In contrast, human intermediate conductance Ca2+-activated channels were inhibited by KB130015. Using segments of porcine pulmonary arteries, KB130015 induced endothelium-independent vasorelaxation, half-maximal at 43 microM KB130015. Relaxation was inhibited by 1 mM tetraethylammonium, suggesting that KB130015 can activate vascular smooth muscle type BK(Ca) channels under physiological conditions. Interestingly, the shift in the P(o)/V relationship was considerably stronger (-90 mV in 3 microM [Ca2+]in) for BK(Ca) channels containing Slo-beta1 subunits. Thus, KB130015 belongs to a novel class of BK(Ca) channel openers that exert an effect depending on the subunit composition of the channel complex.

  2. Schisandra polysaccharide evokes immunomodulatory activity through TLR 4-mediated activation of macrophages.

    Science.gov (United States)

    Zhao, Ting; Feng, Yun; Li, Jing; Mao, Riwen; Zou, Ye; Feng, Weiwei; Zheng, Daheng; Wang, Wei; Chen, Yao; Yang, Liuqing; Wu, Xiangyang

    2014-04-01

    Schisandra chinensis (Turcz.) Baill has been used in traditional Chinese medicine for centuries. Previous studies have shown that Schisandra polysaccharide (SCPP11) has robust antitumor activity in vivo. In this study, the immunomodulatory activity and mechanisms of action of SCPP11 were investigated further to reveal its mechanism of action against tumors. Results showed that SCPP11 increased the thymus and spleen indices, pinocytic activity of peritoneal macrophages, and hemolysin formation in CTX-induced immunosuppressed mice. Moreover, SCPP11 significantly increased immunoglobulin levels, cytokines levels in vivo and induced RAW264.7 cells to secrete cytokines in vitro. RAW264.7 cells pretreated with SCPP11 significantly inhibited the proliferation of HepG-2 cells. In addition, SCPP11 promoted both the expression of iNOS protein and of iNOS and TNF-α mRNA. TLR-4 is a possible receptor for SCPP11-mediated macrophage activation. Therefore, the data suggest that SCPP11 exerted its antitumor activity by improving immune system functions through TLR-4-mediated up-regulation of NO and TNF-α.

  3. Prenatal nicotine exposure enhances Cx43 and Panx1 unopposed channel activity in brain cells of adult offspring mice fed a high-fat/cholesterol diet

    Directory of Open Access Journals (Sweden)

    Juan Andrés Orellana

    2014-12-01

    Full Text Available Nicotine, the most important neuroteratogen of tobacco smoke, can reproduce brain and cognitive disturbances per se when administered prenatally. However, it is still unknown if paracrine signaling among brain cells participates in prenatal nicotine-induced brain impairment of adult offspring. Paracrine signaling is partly mediated by unopposed channels formed by connexins (hemichannels and pannexins serving as aqueous pores permeable to ions and small signaling molecules, allowing exchange between the intra- and extracellular milieus. Our aim was to address whether prenatal nicotine exposure changes the activity of those channels in adult mice offspring under control conditions or subjected to a second challenge during young ages: high-fat/cholesterol (HFC diet. To induce prenatal exposure to nicotine, osmotic minipumps were implanted in CF1 pregnant mice at gestational day 5 to deliver nicotine bitartrate or saline (control solutions. After weaning, offspring of nicotine-treated or untreated pregnant mice were fed ad libitum with chow or HFC diets for 8 weeks. The functional state of Cx43 and Panx1 unopposed channels was evaluated by dye uptake experiments in hippocampal slices from 11-week-old mice. We found that prenatal nicotine increased the opening of Cx43 hemichannels in astrocytes, and Panx1 channels in microglia and neurons only if offspring mice were fed with HFC diet. Blockade of iNOS, COX2 and EP1, P2X7 and NMDA receptors, showed differential inhibition of prenatal nicotine-induced channel opening in glial cells and neurons. Importantly, inhibition of the above mentioned enzymes and receptors, or blockade of Cx43 and Panx1 unopposed channels greatly reduced ATP and glutamate release from hippocampal slices of prenatally nicotine-exposed offspring. We propose that unregulated gliotransmitter release through Cx43 and Panx1 unopposed channels may participate in brain alterations observed in offspring of mothers exposed to tobacco smoke

  4. The facilitating effect of systemic administration of Kv7/M channel blocker XE991 on LTP induction in the hippocampal CA1 area independent of muscarinic activation.

    Science.gov (United States)

    Song, Ming-Ke; Cui, Yong-Yao; Zhang, Wei-Wei; Zhu, Liang; Lu, Yang; Chen, Hong-Zhuan

    2009-09-11

    A large amount of in vitro studies demonstrate suppression of M-current in hippocampal neurons by Kv7/M channel blocker results in depolarization of membrane potential and release of neurotransmitters, such as acetylcholine and glutamate, suggesting that Kv7/M channel may play important roles in regulating synaptic plasticity. In the present study, we examined the in vivo effect of Kv7/M channel inhibition on the long-term potentiation (LTP) induction at basal dendrites in hippocampal CA1 area of urethane-anaesthetized rats. The Kv7/M channel was inhibited by intraperitoneal injection of XE991 (10mg/kg) and the LTP of field excitatory postsynaptic potential (fEPSP) was induced by supra-threshold high frequency stimulation (S1 HFS). A weak protocol which was just below the threshold for evoking LTP was used as sub-threshold high frequency stimulation (S2 HFS). XE991 did not significantly alter the slope of fEPSP and the magnitude of LTP induced by S1 HFS, suggesting that Kv7/M channel inhibition had little or no effect on glutamatergic transmission under basal conditions. However, XE991 could make S2 HFS evoke LTP even after the application of the muscarinic cholinergic (mACh) receptor antagonist scopolamine, suggesting that Kv7/M channel inhibition lowered the threshold for LTP induction and the effect was independent of muscarinic activation. Based on the above findings, we concluded that the facilitating effect of XE991 on LTP induction is not mediated by its ability to enhance the release of acetylcholine; therefore, Kv7/M channel blockers may provide a therapeutic benefit to cholinergic deficiency-related cognitive impairment, e.g., Alzheimer's disease.

  5. 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....

  6. Rate-dependent activation failure in isolated cardiac cells and tissue due to Na+ channel block.

    Science.gov (United States)

    Varghese, Anthony; Spindler, Anthony J; Paterson, David; Noble, Denis

    2015-11-15

    While it is well established that class-I antiarrhythmics block cardiac sodium channels, the mechanism of action of therapeutic levels of these drugs is not well understood. Using a combination of mathematical modeling and in vitro experiments, we studied the failure of activation of action potentials in single ventricular cells and in tissue caused by Na(+) channel block. Our computations of block and unblock of sodium channels by a theoretical class-Ib antiarrhythmic agent predict differences in the concentrations required to cause activation failure in single cells as opposed to multicellular preparations. We tested and confirmed these in silico predictions with in vitro experiments on isolated guinea-pig ventricular cells and papillary muscles stimulated at various rates (2-6.67 Hz) and exposed to various concentrations (5 × 10(-6) to 500 × 10(-6) mol/l) of lidocaine. The most salient result was that whereas large doses (5 × 10(-4) mol/l or higher) of lidocaine were required to inhibit action potentials temporarily in single cells, much lower doses (5 × 10(-6) mol/l), i.e., therapeutic levels, were sufficient to have the same effect in papillary muscles: a hundredfold difference. Our experimental results and mathematical analysis indicate that the syncytial nature of cardiac tissue explains the effects of clinically relevant doses of Na(+) channel blockers.

  7. 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.

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

    Science.gov (United States)

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

    2010-01-01

    Scorpion β-toxin 4 from Centruroides suffusus suffusus (Css4) enhances the activation of voltage-gated sodium channels through a voltage sensor trapping mechanism by binding the activated state of the voltage sensor in domain II and stabilizing it in its activated conformation. Here we describe the antagonist and partial agonist properties of a mutant derivative of this toxin. Substitution of seven different amino acid residues for Glu15 in Css4 yielded toxin derivatives with both increased and decreased affinities for binding to neurotoxin receptor site 4 on sodium channels. Css4E15R is unique among this set of mutants in that it retained nearly normal binding affinity but lost its functional activity for modification of sodium channel gating in our standard electrophysiological assay for voltage sensor trapping. More detailed analysis of the functional effects of Css4E15R revealed weak voltage sensor trapping activity, which was very rapidly reversed upon repolarization and therefore was not observed in our standard assay of toxin effects. This partial agonist activity of Css4E15R is observed clearly in voltage sensor trapping assays with brief (5 ms) repolarization between the conditioning prepulse and the test pulse. The effects of Css4E15R are fit well by a three-step model of toxin action involving concentration-dependent toxin binding to its receptor site followed by depolarization-dependent activation of the voltage sensor and subsequent voltage sensor trapping. Because it is a partial agonist with much reduced efficacy for voltage sensor trapping, Css4E15R can antagonize the effects of wild-type Css4 on sodium channel activation and can prevent paralysis by Css4 when injected into mice. Our results define the first partial agonist and antagonist activities for scorpion toxins and open new avenues of research toward better understanding of the structure-function relationships for toxin action on sodium channel voltage sensors and toward potential toxin

  10. Partial agonist and antagonist activities of a mutant scorpion beta-toxin on sodium channels.

    Science.gov (United States)

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

    2010-10-01

    Scorpion β-toxin 4 from Centruroides suffusus suffusus (Css4) enhances the activation of voltage-gated sodium channels through a voltage sensor trapping mechanism by binding the activated state of the voltage sensor in domain II and stabilizing it in its activated conformation. Here we describe the antagonist and partial agonist properties of a mutant derivative of this toxin. Substitution of seven different amino acid residues for Glu(15) in Css4 yielded toxin derivatives with both increased and decreased affinities for binding to neurotoxin receptor site 4 on sodium channels. Css4(E15R) is unique among this set of mutants in that it retained nearly normal binding affinity but lost its functional activity for modification of sodium channel gating in our standard electrophysiological assay for voltage sensor trapping. More detailed analysis of the functional effects of Css4(E15R) revealed weak voltage sensor trapping activity, which was very rapidly reversed upon repolarization and therefore was not observed in our standard assay of toxin effects. This partial agonist activity of Css4(E15R) is observed clearly in voltage sensor trapping assays with brief (5 ms) repolarization between the conditioning prepulse and the test pulse. The effects of Css4(E15R) are fit well by a three-step model of toxin action involving concentration-dependent toxin binding to its receptor site followed by depolarization-dependent activation of the voltage sensor and subsequent voltage sensor trapping. Because it is a partial agonist with much reduced efficacy for voltage sensor trapping, Css4(E15R) can antagonize the effects of wild-type Css4 on sodium channel activation and can prevent paralysis by Css4 when injected into mice. Our results define the first partial agonist and antagonist activities for scorpion toxins and open new avenues of research toward better understanding of the structure-function relationships for toxin action on sodium channel voltage sensors and toward

  11. Induction of aryl hydrocarbon receptor-mediated and estrogen receptor-mediated activities, and modulation of cell proliferation by dinaphthofurans.

    Science.gov (United States)

    Vondrácek, Jan; Chramostová, Katerina; Plísková, Martina; Bláha, Ludek; Brack, Werner; Kozubík, Alois; Machala, Miroslav

    2004-09-01

    A group of heterocyclic aromatic compounds, dinaphthofurans (DNFs), recently have been identified as potentially significant contaminants in freshwater sediments. In the present study, a battery of in vitro assays was used for detection of toxic effects of DNFs that are potentially associated with endocrine disruption and tumor promotion. Dinaphthofurans were found to act as relatively potent inducers of aryl hydrocarbon receptor (AhR)-mediated activity in the chemical-activated luciferase reporter gene expression DR-CALUX assay. The relative AhR-inducing potencies of DNFs were similar or even higher than relative potencies of unsubstituted polycyclic aromatic hydrocarbons (PAHs), with dinaphtho[1,2-b;2'3'-d]furan being the most potent AhR agonist. Two compounds, dinaphtho[2,1-b;2'3'-d]furan and dinaphtho[1,2-b;1'2'-d]furan, induced estrogen receptor (ER)-mediated activity in the estrogen receptor-mediated CALUX (the ER-CALUX) assay. Two types of potential tumor-promoting effects of DNFs were investigated, using in vitro bioassays for detection of inhibition of gap-junctional intercellular communication and detection of a release from contact inhibition. Although the acute inhibition of gap-junctional intercellular communication was not observed, all six tested DNFs were able to release rat liver epithelial WB-F344 cells from contact inhibition at concentrations as low as 100 nM. In summary, the present study indicated that DNFs can exert multiple biological effects in vitro, including induction of the AhR-mediated activity, release of cells from contact inhibition, and induction of ER-mediated activity.

  12. 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

  13. Flavaglines Stimulate Transient Receptor Potential Melastatin Type 6 (TRPM6) Channel Activity.

    NARCIS (Netherlands)

    Blanchard, M.G.; Baaij, J.H. dr.; Verkaart, S.A.J.; Lameris, A.L.L.; Basmadjian, C.; Zhao, Q.; Desaubry, L.; Bindels, R.J.M.; Hoenderop, J.G.J.

    2015-01-01

    Magnesium (Mg2+) is essential for enzymatic activity, brain function and muscle contraction. Blood Mg2+ concentrations are tightly regulated between 0.7 and 1.1 mM by Mg2+ (re)absorption in kidney and intestine. The apical entry of Mg2+ in (re)absorbing epithelial cells is mediated by the transient

  14. 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.

  15. Activation of volume-regulated Cl− channels by ACh and ATP in Xenopus follicles

    Science.gov (United States)

    Pérez-Samartín, Alberto L; Miledi, Ricardo; Arellano, Rogelio O

    2000-01-01

    Osmolarity-dependent ionic currents from follicle-enclosed Xenopus oocytes (follicles) were studied using electrophysiological techniques. Whole follicle currents were monitored using a two-electrode voltage clamp and single-channel activity was measured using the patch-clamp technique.In follicles held at -60 mV two chloride currents were activated in external hyposmotic solutions. One was the habitual volume-regulated current elicited by external hyposmolarity (ICl,swell), and the second was a slow and smooth current (Sin) generated by ACh or ATP application.In follicles, the permeability ratios for different anions with respect to Cl− were similar for both ICl,swell and Sin, with a sequence of: SCN− > I− > Br−≥ NO3−≥ Cl− > gluconate ≥ cyclamate > acetate > SO42−.Extracellular ATP blocked the outward component of Sin. Also, extracellular pH modulated the inactivation kinetics of Sin elicited by ACh; e.g. inactivation at +80 mV was ∼100% slower at pH 8.0 compared with that at pH 6.0.Lanthanides inhibited ICl,swell and Sin. La3+ completely inhibited ICl,swell with a half-maximal inhibitory concentration (IC50) of 17 ± 1.9 μm, while Sin was blocked up to 55% with an apparent IC50 of 36 ± 2.6 μm.Patch-clamp recordings in follicular cells showed that hyposmotic challenge opened inward single-channel currents. The single channel conductance (4.7 ± 0.4 pS) had a linear current-voltage relationship with a reversal membrane potential close to −20 mV. This single-channel activity was increased by application of ACh or ATP.The ICl,swell generation was not affected by pirenzepine or metoctramine, and did not affect the purinergic activation of the chloride current named Fin. Thus, ICl,swell was not generated via neurotransmitters released during cellular swelling.All together, equal discrimination for different anions, similar modulatory effects by extracellular pH, the blocking effects by ATP and La3+, and the same single-channel activity

  16. Increased store-operated and 1-oleoyl-2-acetyl-sn-glycerol-induced calcium influx in monocytes is mediated by transient receptor potential canonical channels in human essential hypertension

    DEFF Research Database (Denmark)

    Liu, Dao Yan; Thilo, Florian; Scholze, Alexandra;

    2007-01-01

    Activation of nonselective cation channels of the transient receptor potential canonical (TRPC) family has been associated with hypertension. Whether store-operated channels, which are activated after depletion of intracellular stores, or second-messenger-operated channels, which are activated by 1......-oleoyl-2-acetyl-sn-glycerol, are affected in essential hypertension is presently unknown....

  17. Activity of Palythoa caribaeorum Venom on Voltage-Gated Ion Channels in Mammalian Superior Cervical Ganglion Neurons

    Directory of Open Access Journals (Sweden)

    Fernando Lazcano-Pérez

    2016-05-01

    Full Text Available The Zoanthids are an order of cnidarians whose venoms and toxins have been poorly studied. Palythoa caribaeorum is a zoanthid commonly found around the Mexican coastline. In this study, we tested the activity of P. caribaeorum venom on voltage-gated sodium channel (NaV1.7, voltage-gated calcium channel (CaV2.2, the A-type transient outward (IA and delayed rectifier (IDR currents of KV channels of the superior cervical ganglion (SCG neurons of the rat. These results showed that the venom reversibly delays the inactivation process of voltage-gated sodium channels and inhibits voltage-gated calcium and potassium channels in this mammalian model. The compounds responsible for these effects seem to be low molecular weight peptides. Together, these results provide evidence for the potential use of zoanthids as a novel source of cnidarian toxins active on voltage-gated ion channels.

  18. Activation of transient receptor potential vanilloid type-1 channel prevents adipogenesis and obesity

    DEFF Research Database (Denmark)

    Zhang, Li Li; Yan Liu, Dao; Ma, Li Qun

    2007-01-01

    in visceral adipose tissue from obese humans was accompanied by reduced capsaicin-induced calcium influx. The oral administration of capsaicin for 120 days prevented obesity in male wild type mice but not in TRPV1 knockout mice assigned to high fat diet. We conclude that the activation of TRPV1 channels......We tested the hypothesis that activation of transient receptor potential vanilloid type-1 (TRPV1) by capsaicin prevents adipogenesis. TRPV1 channels in 3T3-L1-preadipocytes and visceral adipose tissue from mice and humans were detected by immunoblotting and quantitative real-time RT-PCR. The effect...... of TRPV1 on cytosolic calcium was determined fluorometrically in 3T3-L1-preadipocytes and in human visceral fat tissue. Adipogenesis in stimulated 3T3-L1-preadipocytes was determined by oil red O-staining of intracellular lipid droplets, triglyceride levels, expression of peroxisome proliferator...

  19. 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.

  20. Direct tests of micro channel plates as the active element of a new shower maximum detector

    Energy Technology Data Exchange (ETDEWEB)

    Ronzhin, A., E-mail: ronzhin@fnal.gov [Fermilab, Batavia, IL 60510 (United States); Los, S.; Ramberg, E. [Fermilab, Batavia, IL 60510 (United States); Apresyan, A.; Xie, S.; Spiropulu, M. [California Institute of Technology, Pasadena, CA (United States); Kim, H. [University of Chicago, Chicago, IL 60637 (United States)

    2015-09-21

    We continue the study of micro channel plates (MCP) as the active element of a shower maximum (SM) detector. We present below test beam results obtained with MCPs detecting directly secondary particles of an electromagnetic shower. The MCP efficiency to shower particles is close to 100%. The time resolution obtained for this new type of the SM detector is at the level of 40 ps.

  1. Anti-Convulsant Activity of Boerhaavia diffusa: Plausible Role of Calcium Channel Antagonism

    OpenAIRE

    Mandeep Kaur; Rajesh Kumar Goel

    2011-01-01

    “Ethnopharmacological” use of roots of Boerhaavia diffusa (B. diffusa) in the treatment of epilepsy in Nigerian folk medicine and reports showing the presence of a calcium channel antagonistic compound “liriodendrin” in its roots, led us to undertake the present study. The study was designed to investigate the methanolic root extract of B. diffusa and its different fractions including liriodendrin-rich fraction for exploring the possible role of liriodendrin in its anti-convulsant activity. A...

  2. 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.

  3. Effect of a chloride channel activator, lubiprostone, on colonic sensory and motor functions in healthy subjects

    OpenAIRE

    Sweetser, Seth; Busciglio, Irene A.; Camilleri, Michael; Bharucha, Adil E.; Szarka, Lawrence A.; Papathanasopoulos, Athanasios; Burton, Duane D.; Eckert, Deborah J.; Zinsmeister, Alan R.

    2008-01-01

    Lubiprostone, a bicyclic fatty acid chloride channel activator, is efficacious in treatment of chronic constipation and constipation-predominant irritable bowel syndrome. The study aim was to compare effects of lubiprostone and placebo on colonic sensory and motor functions in humans. In double-blind, randomized fashion, 60 healthy adults received three oral doses of placebo or 24 μg lubiprostone per day in a parallel-group, placebo-controlled trial. A barostat-manometry tube was placed in th...

  4. Graphene incorporated, N doped activated carbon as catalytic electrode in redox active electrolyte mediated supercapacitor

    Science.gov (United States)

    Gao, Zhiyong; Liu, Xiao; Chang, Jiuli; Wu, Dapeng; Xu, Fang; Zhang, Lingcui; Du, Weimin; Jiang, Kai

    2017-01-01

    Graphene incorporated, N doped activated carbons (GNACs) are synthesized by alkali activation of graphene-polypyrrole composite (G-PPy) at different temperatures for application as electrode materials of supercapacitors. Under optimal activation temperature of 700 °C, the resultant samples, labeled as GNAC700, owns hierarchically porous texture with high specific surface area and efficient ions diffusion channels, N, O functionalized surface with apparent pseudocapacitance contribution and high wettability, thus can deliver a moderate capacitance, a high rate capability and a good cycleability when used as supercapacitor electrode. Additionally, the GNAC700 electrode demonstrates high catalytic activity for the redox reaction of pyrocatechol/o-quinone pair in H2SO4 electrolyte, thus enables a high pseudocapacitance from electrolyte. Under optimal pyrocatechol concentration in H2SO4 electrolyte, the electrode capacitance of GNAC700 increases by over 4 folds to 512 F g-1 at 1 A g-1, an excellent cycleability is also achieved simultaneously. Pyridinic- N is deemed to be responsible for the high catalytic activity. This work provides a promising strategy to ameliorate the capacitive performances of supercapacitors via the synergistic interaction between redox-active electrolyte and catalytic electrodes.

  5. Mediated Electron Transfer at Redox Active Monolayers. Part 4: Kinetics of Redox Enzymes Coupled With Electron Mediators

    Directory of Open Access Journals (Sweden)

    Michael E.G. Lyons

    2003-01-01

    Full Text Available A detailed kinetic analysis of the pertinent physical processes underlying the operation of enzyme electrodes immobilized within alkane thiol self assembled monolayers is developed. These electrodes utilize a soluble mediator, which partitions into the monolayer, regenerates the active catalytic form of the enzyme and is re-oxidized at the underlying support electrode surface giving rise to a current which reflects kinetic events at the enzyme surface. Both the enzyme/substrate and enzyme mediator kinetics have been quantified fully in terms of a ping-pong mechanism for the former and Michaelis-Menten kinetics for the latter. The effect of substrate and mediator diffusion in solution have also been specifically considered and the latter processes have been shown to result in a complex expression for the reaction flux. Four limiting kinetic cases have been enumerated and simple expressions for the reaction flux in each of these rate limiting situations have been developed. Kinetic case diagrams have been presented as an aid to mechanistic diagnosis. The complicating effects of diffusive loss of reduced mediator from the enzyme layer have also been examined and the relation between the observed flux corresponding to reduced mediator oxidation at the support electrode and the substrate reaction flux in the enzyme layer have been quantified in terms of an efficiency factor. Results extracted from recently published practical realizations of immobilized monolayer enzyme systems have been discussed in the context of the proposed model analysis.

  6. Hydraphiles: A Rigorously Studied Class of Synthetic Channel Compounds with In Vivo Activity

    Directory of Open Access Journals (Sweden)

    Saeedeh Negin

    2013-01-01

    Full Text Available Hydraphiles are a class of synthetic ion channels that now have a twenty-year history of analysis and success. In early studies, these compounds were rigorously validated in a wide range of in vitro assays including liposomal ion flow detected by NMR or ion-selective electrodes, as well as biophysical experiments in planar bilayers. During the past decade, biological activity was observed for these compounds including toxicity to bacteria, yeast, and mammalian cells due to stress caused by the disruption of ion homeostasis. The channel mechanism was verified in cells using membrane polarity sensitive dyes, as well as patch clamping studies. This body of work has provided a solid foundation with which hydraphiles have recently demonstrated acute biological toxicity in the muscle tissue of living mice, as measured by whole animal fluorescence imaging and histological studies. Here we review the critical structure-activity relationships in the hydraphile family of compounds and the in vitro and in cellulo experiments that have validated their channel behavior. This report culminates with a description of recently reported efforts in which these molecules have demonstrated activity in living mice.

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

    Directory of Open Access Journals (Sweden)

    Sheyda R Frolova

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

  8. Tamoxifen mediated estrogen receptor activation protects against early impairment of hippocampal neuron excitability in an oxygen/glucose deprivation brain slice ischemia model.

    Science.gov (United States)

    Zhang, Huaqiu; Xie, Minjie; Schools, Gary P; Feustel, Paul F; Wang, Wei; Lei, Ting; Kimelberg, Harold K; Zhou, Min

    2009-01-09

    Pretreatment of ovarectomized rats with estrogen shows long-term protection via activation of the estrogen receptor (ER). However, it remains unknown whether activation of the ER can provide protection against early neuronal damage when given acutely. We simulated ischemic conditions by applying oxygen and glucose deprived (OGD) solution to acute male rat hippocampal slices and examined the neuronal electrophysiological changes. Pyramidal neurons and interneurons showed a time-dependent membrane potential depolarization and reduction in evoked action potential frequency and amplitude over a 10 to 15 min OGD exposure. These changes were largely suppressed by 10 microM TAM. The TAM effect was neuron-specific as the OGD-induced astrocytic membrane potential depolarization was not altered. The TAM effect was mediated through ER activation because it could be simulated by 17beta-estradiol and was completely inhibited by the ER inhibitor ICI 182, 780, and is therefore an example of TAM's selective estrogen receptor modulator (SERM) action. We further show that TAM's effects on OGD-induced impairment of neuronal excitability was largely due to activation of neuroprotective BK channels, as the TAM effect was markedly attenuated by the BK channel inhibitor paxilline at 10 microM. TAM also significantly reduced the frequency and amplitude of AMPA receptor mediated spontaneous excitatory postsynaptic currents (sEPSCs) in pyramidal neurons which is an early consequence of OGD. Altogether, this study demonstrates that both 17beta-estradiol and TAM attenuate neuronal excitability impairment early on in a simulated ischemia model via ER activation mediated potentiation of BK K(+) channels and reduction in enhanced neuronal AMPA/NMDA receptor-mediated excitotoxicity.

  9. Glutamate transporter activity promotes enhanced Na+/K+-ATPase-mediated extracellular K+ management during neuronal activity

    DEFF Research Database (Denmark)

    Larsen, Brian Roland; Holm, Rikke; Vilsen, Bente;

    2016-01-01

    , in addition, Na+/K+-ATPase-mediated K+ clearance could be governed by astrocytic [Na+]i. During most neuronal activity, glutamate is released in the synaptic cleft and is re-absorbed by astrocytic Na+-coupled glutamate transporters, thereby elevating [Na+]i. It thus remains unresolved whether the different Na...... constellations in Xenopus oocytes and determined their apparent Na+ affinity in intact oocytes and isolated membranes. The Na+/K+-ATPase was not fully saturated at basal astrocytic [Na+]i, irrespective of isoform constellation, although the β1 subunit conferred lower apparent Na+ affinity to the α1 and α2...

  10. Active video games: the mediating effect of aerobic fitness on body composition

    Directory of Open Access Journals (Sweden)

    Maddison Ralph

    2012-05-01

    Full Text Available Abstract Background Increased understanding of why and how physical activity impacts on health outcomes is needed to increase the effectiveness of physical activity interventions. A recent randomized controlled trial of an active video game (PlayStation EyeToy™ intervention showed a statistically significant treatment effect on the primary outcome, change from baseline in body mass index (BMI, which favored the intervention group at 24 weeks. In this short paper we evaluate the mediating effects of the secondary outcomes. Objective To identify mediators of the effect of an active video games intervention on body composition. Methods Data from a two-arm parallel randomized controlled trial of an active video game intervention (n = 322 were analyzed. The primary outcome was change from baseline in BMI. A priori secondary outcomes were considered as potential mediators of the intervention on BMI, including aerobic fitness (VO2Max, time spent in moderate-to-vigorous physical activity (MVPA, and food snacking at 24 weeks. Results Only aerobic fitness at 24 weeks met the conditions for mediation, and was a significant mediator of BMI. Conclusion Playing active video games can have a positive effect on body composition in overweight or obese children and this effect is most likely mediated through improved aerobic fitness. Future trials should examine other potential mediators related to this type of intervention. Trial registration Australian New Zealand Clinical Trials Registry Website: http://www.anzctr.org.au Study ID number: ACTRN12607000632493

  11. External bioenergy-induced increases in intracellular free calcium concentrations are mediated by Na+/Ca2+ exchanger and L-type calcium channel.

    Science.gov (United States)

    Kiang, Juliann G; Ives, John A; Jonas, Wayne B

    2005-03-01

    External bioenergy (EBE, energy emitted from a human body) has been shown to increase intracellular calcium concentration ([Ca2+]i, an important factor in signal transduction) and regulate the cellular response to heat stress in cultured human lymphoid Jurkat T cells. In this study, we wanted to elucidate the underlying mechanisms. A bioenergy specialist emitted bioenergy sequentially toward tubes of cultured Jurkat T cells for one 15-minute period in buffers containing different ion compositions or different concentrations of inhibitors. [Ca2+], was measured spectrofluorometrically using the fluorescent probe fura-2. The resting [Ca2+]i in Jurkat T cells was 70 +/- 3 nM (n = 130) in the normal buffer. Removal of external calcium decreased the resting [Ca2+]i to 52 +/- 2 nM (n = 23), indicating that Ca2+ entry from the external source is important for maintaining the basal level of [Ca2+]i. Treatment of Jurkat T cells with EBE for 15 min increased [Ca2+]i by 30 +/- 5% (P EBE did not attenuate [Ca2+]i responsiveness to EBE. Removal of external Ca2+ or Na+, but not Mg2+, inhibited the EBE-induced increase in [Ca2+]i. Dichlorobenzamil, an inhibitor of Na+/Ca2+ exchangers, also inhibited the EBE-induced increase in [Ca2+]i in a concentration-dependent manner with an IC50 of 0.11 +/- 0.02 nM. When external [K+] was increased from 4.5 mM to 25 mM, EBE decreased [Ca2+]i. The EBE-induced increase was also blocked by verapamil, an L-type voltage-gated Ca2+ channel blocker. These results suggest that the EBE-induced [Ca2+]i increase may serve as an objective means for assessing and validating bioenergy effects and those specialists claiming bioenergy capability. The increase in [Ca2+]i is mediated by activation of Na+/Ca2+ exchangers and opening of L-type voltage-gated Ca2+ channels.

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

    Science.gov (United States)

    Takahashi, Izumi; Yoshino, Masami

    2015-10-01

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

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

    Directory of Open Access Journals (Sweden)

    Michael J Shipston

    2014-08-01

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

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

    Directory of Open Access Journals (Sweden)

    Jose L Nieto-Torres

    2014-05-01

    Full Text Available Deletion of Severe Acute Respiratory Syndrome Coronavirus (SARS-CoV envelope (E gene attenuates the virus. E gene encodes a small multifunctional protein that possesses ion channel (IC activity, an important function in virus-host interaction. To test the contribution of E protein IC activity in virus pathogenesis, two recombinant mouse-adapted SARS-CoVs, each containing one single amino acid mutation that suppressed ion conductivity, were engineered. After serial infections, mutant viruses, in gene