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Sample records for human bk channel

  1. BK channel modulators: a comprehensive overview

    DEFF Research Database (Denmark)

    Nardi, Antonio; Olesen, Søren-Peter

    2008-01-01

    and blockers 4) Marketed and/or investigational drugs with BK-modulating side properties and structural analogues 5) Naturally-occurring BK channel openers and structural analogues 6) Synthetic BK channel openers. This review is intended to provide readers with current opinion on the BK channel as a drug...

  2. BK channel activators and their therapeutic perspectives

    DEFF Research Database (Denmark)

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

    2014-01-01

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

  3. 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

  4. Regulation of BK channels by auxiliary γ subunits

    Directory of Open Access Journals (Sweden)

    Jiyuan eZhang

    2014-10-01

    Full Text Available The large-conductance, calcium- and voltage-activated potassium (BK channel has the largest single-channel conductance among potassium channels and can be activated by both membrane depolarization and increases in intracellular calcium concentration. BK channels consist of pore-forming, voltage- and calcium-sensing α subunits, either alone or in association with regulatory subunits. BK channels are widely expressed in various tissues and cells including both excitable and non-excitable cells and display diverse biophysical and pharmacological characteristics. This diversity can be explained in part by posttranslational modifications and alternative splicing of the α subunit, which is encoded by a single gene, KCNMA1, as well as by tissue-specific β subunit modulation. Recently, a leucine-rich repeat-containing membrane protein, LRRC26, was found to interact with BK channels and cause an unprecedented large negative shift (~-140 mV in the voltage dependence of the BK channel activation. LRRC26 allows BK channels to open even at near-physiological calcium concentration and membrane voltage in non-excitable cells. Three LRRC26-related proteins, LRRC52, LRRC55, and LRRC38, were subsequently identified as BK channel modulators. These LRRC proteins are structurally and functionally distinct from the BK channel β subunits and were designated as γ subunits. The discovery of the γ subunits adds a new dimension to BK channel regulation and improves our understanding of the physiological functions of BK channels in various tissues and cell types. Unlike BK channel β subunits, which have been intensively investigated both mechanistically and physiologically, our understanding of the γ subunits is very limited at this stage. This article reviews the structure, modulatory mechanisms, physiological relevance, and potential therapeutic implications of γ subunits as they are currently understood.

  5. Hair cell BK channels interact with RACK1, and PKC increases its expression on the cell surface by indirect phosphorylation

    OpenAIRE

    Surguchev, Alexei; Bai, Jun-Ping; Joshi, Powrnima; Navaratnam, Dhasakumar

    2012-01-01

    Large conductance (BK) calcium activated potassium channels (Slo) are ubiquitous and implicated in a number of human diseases including hypertension and epilepsy. BK channels consist of a pore forming α-subunit (Slo) and a number of accessory subunits. In hair cells of nonmammalian vertebrates these channels play a critical role in electrical resonance, a mechanism of frequency selectivity. Hair cell BK channel clusters on the surface and currents increase along the tonotopic axis and contrib...

  6. Single-channel kinetics of BK (Slo1 channels

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    Yanyan eGeng

    2015-01-01

    Full Text Available Single-channel kinetics has proven a powerful tool to reveal information about the gating mechanisms that control the opening and closing of ion channels. This introductory review focuses on the gating of large conductance Ca2+- and voltage-activated K+ (BK or Slo1 channels at the single-channel level. It starts with single-channel current records and progresses to presentation and analysis of single-channel data and the development of gating mechanisms in terms of discrete state Markov (DSM models. The DSM models are formulated in terms of the tetrameric modular structure of BK channels, consisting of a central transmembrane pore-gate domain (PGD attached to four surrounding transmembrane voltage sensing domains (VSD and a large intracellular cytosolic domain (CTD, also referred to as the gating ring. The modular structure and data analysis shows that the Ca2+ and voltage dependent gating considered separately can each be approximated by 10-state two-tiered models with 5 closed states on the upper tier and 5 open states on the lower tier. The modular structure and joint Ca2+ and voltage dependent gating are consistent with a 50 state two-tiered model with 25 closed states on the upper tier and 25 open states on the lower tier. Adding an additional tier of brief closed (flicker states to the 10-state or 50-state models improved the description of the gating. For fixed experimental conditions a channel would gate in only a subset of the potential number of states. The detected number of states and the correlations between adjacent interval durations are consistent with the tiered models. The examined models can account for the single-channel kinetics and the bursting behavior of gating. Ca2+ and voltage activate BK channels by predominantly increasing the effective opening rate of the channel with a smaller decrease in the effective closing rate. Ca2+ and depolarization thus activate by mainly destabilizing the closed states.

  7. BK channels reveal novel phosphate sensitivity in SNr neurons.

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    Juan Juan Ji

    Full Text Available Whether large conductance Ca(2+-activated potassium (BK channels are present in the substantia nigra pars reticulata (SNr is a matter of debate. Using the patch-clamp technique, we examined the functional expression of BK channels in neurons of the SNr and showed that the channels were activated or inhibited by internal high-energy phosphates (IHEPs at positive and negative membrane potentials, respectively. SNr neurons showed membrane potential hyperpolarization under glucose-deprivation conditions which was attenuated by paxilline, a specific BK channel blocker. In addition, Fluo-3 fluorescence recording detected an increase in the level of internal free calcium ([Ca(2+](i during ischemic hyperpolarization. These results confirm that BK channels are present in SNr neurons and indicate that their unique IHEP sensitivity is requisite in neuronal ischemic responses. Bearing in mind that the K(ATP channel blocker tolbutamide also attenuated the hyperpolarization, we suggest that BK channels may play a protective role in the basal ganglia by modulating the excitability of SNr neurons along with K(ATP channels under ischemic stresses.

  8. Voltage-dependent BK and Hv1 channels expressed in non-excitable tissues: New therapeutics opportunities as targets in human diseases.

    Science.gov (United States)

    Morera, Francisco J; Saravia, Julia; Pontigo, Juan Pablo; Vargas-Chacoff, Luis; Contreras, Gustavo F; Pupo, Amaury; Lorenzo, Yenisleidy; Castillo, Karen; Tilegenova, Cholpon; Cuello, Luis G; Gonzalez, Carlos

    2015-11-01

    Voltage-gated ion channels are the molecular determinants of cellular excitability. This group of ion channels is one of the most important pharmacological targets in excitable tissues such as nervous system, cardiac and skeletal muscle. Moreover, voltage-gated ion channels are expressed in non-excitable cells, where they mediate key cellular functions through intracellular biochemical mechanisms rather than rapid electrical signaling. This review aims at illustrating the pharmacological impact of these ion channels, highlighting in particular the structural details and physiological functions of two of them - the high conductance voltage- and Ca(2+)-gated K(+) (BK) channels and voltage-gated proton (Hv1) channels- in non-excitable cells. BK channels have been implicated in a variety of physiological processes ranging from regulation of smooth muscle tone to modulation of hormone and neurotransmitter release. Interestingly, BK channels are also involved in modulating K(+) transport in the mammalian kidney and colon epithelium with a potential role in the hyperkalemic phenotype observed in patients with familial hyperkalemic hypertension type 2, and in the pathophysiology of hypertension. In addition, BK channels are responsible for resting and stimulated Ca(2+)-activated K(+) secretion in the distal colon. Hv1 channels have been detected in many cell types, including macrophages, blood cells, lung epithelia, skeletal muscle and microglia. These channels have a central role in the phagocytic system. In macrophages, Hv1 channels participate in the generation of reactive oxygen species in the respiratory burst during the process of phagocytosis. Copyright © 2015 Elsevier Ltd. All rights reserved.

  9. 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.

  10. SHAPING OF ACTION POTENTIALS BY TYPE I AND TYPE II BK CHANNELS

    OpenAIRE

    Jaffe, David B.; Wang, Bin; Brenner, Robert

    2011-01-01

    The BK channel is a Ca2+ 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 ...

  11. Western blot analysis of BK channel β1-subunit expression should be interpreted cautiously when using commercially available antibodies.

    Science.gov (United States)

    Bhattarai, Yogesh; Fernandes, Roxanne; Kadrofske, Mark M; Lockwood, Lizbeth R; Galligan, James J; Xu, Hui

    2014-10-01

    Large conductance Ca(2+)-activated K(+) (BK) channels consist of pore-forming α- and accessory β-subunits. There are four β-subunit subtypes (β1-β4), BK β1-subunit is specific for smooth muscle cells (SMC). Reduced BK β1-subunit expression is associated with SMC dysfunction in animal models of human disease, because downregulation of BK β1-subunit reduces channel activity and increases SMC contractility. Several anti-BK β1-subunit antibodies are commercially available; however, the specificity of most antibodies has not been tested or confirmed in the tissues from BK β1-subunit knockout (KO) mice. In this study, we tested the specificity and sensitivity of six commercially available antibodies from five manufacturers. We performed western blot analysis on BK β1-subunit enriched tissues (mesenteric arteries and colons) and non-SM tissue (cortex of kidney) from wild-type (WT) and BK β1-KO mice. We found that antibodies either detected protein bands of the appropriate molecular weight in tissues from both WT and BK β1-KO mice or failed to detect protein bands at the appropriate molecular weight in tissues from WT mice, suggesting that these antibodies may lack specificity for the BK β1-subunit. The absence of BK β1-subunit mRNA expression in arteries, colons, and kidneys from BK β1-KO mice was confirmed by RT-PCR analysis. We conclude that these commercially available antibodies might not be reliable tools for studying BK β1-subunit expression in murine tissues under the denaturing conditions that we have used. Data obtained using commercially available antibodies should be interpreted cautiously. Our studies underscore the importance of proper negative controls in western blot analyses. © 2014 The Authors. Physiological Reports published by Wiley Periodicals, Inc. on behalf of the American Physiological Society and The Physiological Society.

  12. Coronary arterial BK channel dysfunction exacerbates ischemia/reperfusion-induced myocardial injury in diabetic mice.

    Science.gov (United States)

    Lu, Tong; Jiang, Bin; Wang, Xiao-Li; Lee, Hon-Chi

    2016-09-01

    The large conductance Ca(2+)-activated K(+) (BK) channels, abundantly expressed in coronary artery smooth muscle cells (SMCs), play a pivotal role in regulating coronary circulation. A large body of evidence indicates that coronary arterial BK channel function is diminished in both type 1 and type 2 diabetes. However, the consequence of coronary BK channel dysfunction in diabetes is not clear. We hypothesized that impaired coronary BK channel function exacerbates myocardial ischemia/reperfusion (I/R) injury in streptozotocin-induced diabetic mice. Combining patch-clamp techniques and cellular biological approaches, we found that diabetes facilitated the colocalization of angiotensin II (Ang II) type 1 receptors and BK channel α-subunits (BK-α), but not BK channel β1-subunits (BK-β1), in the caveolae of coronary SMCs. This caveolar compartmentation in vascular SMCs not only enhanced Ang II-mediated inhibition of BK-α but also produced a physical disassociation between BK-α and BK-β1, leading to increased infarct size in diabetic hearts. Most importantly, genetic ablation of caveolae integrity or pharmacological activation of coronary BK channels protected the cardiac function of diabetic mice from experimental I/R injury in both in vivo and ex vivo preparations. Our results demonstrate a vascular ionic mechanism underlying the poor outcome of myocardial injury in diabetes. Hence, activation of coronary BK channels may serve as a therapeutic target for cardiovascular complications of diabetes.

  13. Cholesterol Down-Regulates BK Channels Stably Expressed in HEK 293 Cells

    Science.gov (United States)

    Deng, Xiu-Ling; Sun, Hai-Ying; Li, Gui-Rong

    2013-01-01

    Cholesterol is one of the major lipid components of the plasma membrane in mammalian cells and is involved in the regulation of a number of ion channels. The present study investigates how large conductance Ca2+-activated K+ (BK) channels are regulated by membrane cholesterol in BK-HEK 293 cells expressing both the α-subunit hKCa1.1 and the auxiliary β1-subunit or in hKCa1.1-HEK 293 cells expressing only the α-subunit hKCa1.1 using approaches of electrophysiology, molecular biology, and immunocytochemistry. Membrane cholesterol was depleted in these cells with methyl-β-cyclodextrin (MβCD), and enriched with cholesterol-saturated MβCD (MβCD-cholesterol) or low-density lipoprotein (LDL). We found that BK current density was decreased by cholesterol enrichment in BK-HEK 293 cells, with a reduced expression of KCa1.1 protein, but not the β1-subunit protein. This effect was fully countered by the proteasome inhibitor lactacystin or the lysosome function inhibitor bafilomycin A1. Interestingly, in hKCa1.1-HEK 293 cells, the current density was not affected by cholesterol enrichment, but directly decreased by MβCD, suggesting that the down-regulation of BK channels by cholesterol depends on the auxiliary β1-subunit. The reduced KCa1.1 channel protein expression was also observed in cultured human coronary artery smooth muscle cells with cholesterol enrichment using MβCD-cholesterol or LDL. These results demonstrate the novel information that cholesterol down-regulates BK channels by reducing KCa1.1 protein expression via increasing the channel protein degradation, and the effect is dependent on the auxiliary β1-subunit. PMID:24260325

  14. 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.

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

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    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.

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

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    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.

  17. Ethanol modulation of mammalian BK channels in excitable tissues: molecular targets and their possible contribution to alcohol-induced altered behavior

    Directory of Open Access Journals (Sweden)

    Alex M. Dopico

    2014-12-01

    Full Text Available In most tissues, the function of calcium- and voltage-gated potassium (BK channels is modified in response to ethanol concentrations reached in human blood during alcohol intoxication. In general, modification of BK current from ethanol-naïve preparations in response to brief ethanol exposure results from changes in channel open probability without modification of unitary conductance or change in BK protein levels in the membrane. Protracted and/or repeated ethanol exposure, however, may evoke changes in BK expression. The final ethanol effect on BK open probability leading to either BK current potentiation or BK current reduction is determined by an orchestration of molecular factors, including levels of activating ligand (cytosolic calcium, BK subunit composition and posttranslational modifications, and the channel’s lipid microenvironment. These factors seem to allosterically regulate a direct interaction between ethanol and a recognition pocket of discrete dimensions recently mapped to the channel-forming (slo1 subunit. Type of ethanol exposure also plays a role in the final BK response to the drug: in several central nervous system regions (e.g., striatum, primary sensory neurons, and supraoptic nucleus, acute exposure to ethanol reduces neuronal excitability by enhancing BK activity. In contrast, protracted or repetitive ethanol administration may alter BK subunit composition and membrane expression, rendering the BK complex insensitive to further ethanol exposure. In neurohypophysial axon terminals, ethanol potentiation of BK channel activity leads to a reduction in neuropeptide release. In vascular smooth muscle, however, ethanol inhibition of BK current leads to cell contraction and vascular constriction.

  18. A role for BK channels in heart rate regulation in rodents.

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    Wendy L Imlach

    Full Text Available The heart generates and propagates action potentials through synchronized activation of ion channels allowing inward Na(+ and Ca(2+ and outward K(+ currents. There are a number of K(+ channel types expressed in the heart that play key roles in regulating the cardiac cycle. Large conductance calcium-activated potassium (BK ion channels are not thought to be directly involved in heart function. Here we present evidence that heart rate can be significantly reduced by inhibiting the activity of BK channels. Agents that specifically inhibit BK channel activity, including paxilline and lolitrem B, slowed heart rate in conscious wild-type mice by 30% and 42%, respectively. Heart rate of BK channel knock-out mice (Kcnma1(-/- was not affected by these BK channel inhibitors, suggesting that the changes to heart rate were specifically mediated through BK channels. The possibility that these effects were mediated through BK channels peripheral to the heart was ruled out with experiments using isolated, perfused rat hearts, which showed a significant reduction in heart rate when treated with the BK channel inhibitors paxilline (1 microM, lolitrem B (1 microM, and iberiotoxin (0.23 microM, of 34%, 60%, and 42%, respectively. Furthermore, paxilline was shown to decrease heart rate in a dose-dependent manner. These results implicate BK channels located in the heart to be directly involved in the regulation of heart rate.

  19. Downregulation of BK channel expression in the pilocarpine model of temporal lobe epilepsy

    OpenAIRE

    Pacheco Otalora, Luis F.; Hernandez, Eder F.; Arshadmansab, Massoud F.; rancisco, Sebastian F; Willis, Michael; Ermolinsky, Boris; Zarei, Masoud; Knaus, Hans-Guenther; Garrido-Sanabria, Emilio R.

    2008-01-01

    In the hippocampus, BK channels are preferentially localized in presynaptic glutamatergic terminals including mossy fibers where they are thought to play an important role regulating excessive glutamate release during hyperactive states. Large conductance calcium-activated potassium channels (BK, MaxiK, Slo) have recently been implicated in the pathogenesis of genetic epilepsy. However, the role of BK channels in acquired mesial temporal lobe epilepsy (MTLE) remains unknown. Here we used immu...

  20. Conserved BK channel-protein interactions reveal signals relevant to cell death and survival.

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    Bernd Sokolowski

    Full Text Available The large-conductance Ca(2+-activated K(+ (BK channel and its β-subunit underlie tuning in non-mammalian sensory or hair cells, whereas in mammals its function is less clear. To gain insights into species differences and to reveal putative BK functions, we undertook a systems analysis of BK and BK-Associated Proteins (BKAPS in the chicken cochlea and compared these results to other species. We identified 110 putative partners from cytoplasmic and membrane/cytoskeletal fractions, using a combination of coimmunoprecipitation, 2-D gel, and LC-MS/MS. Partners included 14-3-3γ, valosin-containing protein (VCP, stathmin (STMN, cortactin (CTTN, and prohibitin (PHB, of which 16 partners were verified by reciprocal coimmunoprecipitation. Bioinformatics revealed binary partners, the resultant interactome, subcellular localization, and cellular processes. The interactome contained 193 proteins involved in 190 binary interactions in subcellular compartments such as the ER, mitochondria, and nucleus. Comparisons with mice showed shared hub proteins that included N-methyl-D-aspartate receptor (NMDAR and ATP-synthase. Ortholog analyses across six species revealed conserved interactions involving apoptosis, Ca(2+ binding, and trafficking, in chicks, mice, and humans. Functional studies using recombinant BK and RNAi in a heterologous expression system revealed that proteins important to cell death/survival, such as annexinA5, γ-actin, lamin, superoxide dismutase, and VCP, caused a decrease in BK expression. This revelation led to an examination of specific kinases and their effectors relevant to cell viability. Sequence analyses of the BK C-terminus across 10 species showed putative binding sites for 14-3-3, RAC-α serine/threonine-protein kinase 1 (Akt, glycogen synthase kinase-3β (GSK3β and phosphoinositide-dependent kinase-1 (PDK1. Knockdown of 14-3-3 and Akt caused an increase in BK expression, whereas silencing of GSK3β and PDK1 had the opposite

  1. Big Potassium (BK) ion channels in biology, disease and possible targets for cancer immunotherapy.

    Science.gov (United States)

    Ge, Lisheng; Hoa, Neil T; Wilson, Zechariah; Arismendi-Morillo, Gabriel; Kong, Xiao-Tang; Tajhya, Rajeev B; Beeton, Christine; Jadus, Martin R

    2014-10-01

    The Big Potassium (BK) ion channel is commonly known by a variety of names (Maxi-K, KCNMA1, slo, stretch-activated potassium channel, KCa1.1). Each name reflects a different physical property displayed by this single ion channel. This transmembrane channel is found on nearly every cell type of the body and has its own distinctive roles for that tissue type. The BKα channel contains the pore that releases potassium ions from intracellular stores. This ion channel is found on the cell membrane, endoplasmic reticulum, Golgi and mitochondria. Complex splicing pathways produce different isoforms. The BKα channels can be phosphorylated, palmitoylated and myristylated. BK is composed of a homo-tetramer that interacts with β and γ chains. These accessory proteins provide a further modulating effect on the functions of BKα channels. BK channels play important roles in cell division and migration. In this review, we will focus on the biology of the BK channel, especially its role, and its immune response towards cancer. Recent proteomic studies have linked BK channels with various proteins. Some of these interactions offer further insight into the role that BK channels have with cancers, especially with brain tumors. This review shows that BK channels have a complex interplay with intracellular components of cancer cells and still have plenty of secrets to be discovered.

  2. BK channels regulate sinoatrial node firing rate and cardiac pacing in vivo.

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    Lai, Michael H; Wu, Yuejin; Gao, Zhan; Anderson, Mark E; Dalziel, Julie E; Meredith, Andrea L

    2014-11-01

    Large-conductance Ca(2+)- and voltage-activated K(+) (BK) channels play prominent roles in shaping muscle and neuronal excitability. In the cardiovascular system, BK channels promote vascular relaxation and protect against ischemic injury. Recently, inhibition of BK channels has been shown to lower heart rate in intact rodents and isolated hearts, suggesting a novel role in heart function. However, the underlying mechanism is unclear. In the present study, we recorded ECGs from mice injected with paxilline (PAX), a membrane-permeable BK channel antagonist, and examined changes in cardiac conduction. ECGs revealed a 19 ± 4% PAX-induced reduction in heart rate in wild-type but not BK channel knockout (Kcnma1(-/-)) mice. The heart rate decrease was associated with slowed cardiac pacing due to elongation of the sinus interval. Action potential firing recorded from isolated sinoatrial node cells (SANCs) was reduced by 55 ± 15% and 28 ± 9% by application of PAX (3 μM) and iberiotoxin (230 nM), respectively. Furthermore, baseline firing rates from Kcnma1(-/-) SANCs were 33% lower than wild-type SANCs. The slowed firing upon BK current inhibition or genetic deletion was due to lengthening of the diastolic depolarization phase of the SANC action potential. Finally, BK channel immunoreactivity and PAX-sensitive currents were identified in SANCs with HCN4 expression and pacemaker current, respectively, and BK channels cloned from SANCs recapitulated similar activation as the PAX-sensitive current. Together, these data localize BK channels to SANCs and demonstrate that loss of BK current decreases SANC automaticity, consistent with slowed sinus pacing after PAX injection in vivo. Furthermore, these findings suggest BK channels are potential therapeutic targets for disorders of heart rate.

  3. Cochlear function in mice lacking the BK channel alpha, beta1, or beta4 subunits

    NARCIS (Netherlands)

    Pyott, Sonja J; Meredith, Andrea L; Fodor, Anthony A; Vázquez, Ana E; Yamoah, Ebenezer N; Aldrich, Richard W

    2007-01-01

    Large conductance voltage- and calcium-activated potassium (BK) channels are important for regulating many essential cellular functions, from neuronal action potential shape and firing rate to smooth muscle contractility. In amphibians, reptiles, and birds, BK channels mediate the intrinsic frequenc

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

    DEFF Research Database (Denmark)

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

    2009-01-01

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

  5. Down-regulation of BK channel expression in the pilocarpine model of temporal lobe epilepsy.

    Science.gov (United States)

    Pacheco Otalora, Luis F; Hernandez, Eder F; Arshadmansab, Massoud F; Francisco, Sebastian; Willis, Michael; Ermolinsky, Boris; Zarei, Masoud; Knaus, Hans-Guenther; Garrido-Sanabria, Emilio R

    2008-03-20

    In the hippocampus, BK channels are preferentially localized in presynaptic glutamatergic terminals including mossy fibers where they are thought to play an important role regulating excessive glutamate release during hyperactive states. Large conductance calcium-activated potassium channels (BK, MaxiK, Slo) have recently been implicated in the pathogenesis of genetic epilepsy. However, the role of BK channels in acquired mesial temporal lobe epilepsy (MTLE) remains unknown. Here we used immunohistochemistry, laser scanning confocal microscopy (LSCM), Western immunoblotting and RT-PCR to investigate the expression pattern of the alpha-pore-forming subunit of BK channels in the hippocampus and cortex of chronically epileptic rats obtained by the pilocarpine model of MTLE. All epileptic rats experiencing recurrent spontaneous seizures exhibited a significant down-regulation of BK channel immunostaining in the mossy fibers at the hilus and stratum lucidum of the CA3 area. Quantitative analysis of immunofluorescence signals by LSCM revealed a significant 47% reduction in BK channel immunofluorescent signals in epileptic rats when compared to age-matched non-epileptic control rats. These data correlate with a similar reduction in BK channel protein levels and transcripts in the cortex and hippocampus. Our data indicate a seizure-related down-regulation of BK channels in chronically epileptic rats. Further functional assays are necessary to determine whether altered BK channel expression is an acquired channelopathy or a compensatory mechanism affecting the network excitability in MTLE. Moreover, seizure-mediated BK down-regulation may disturb neuronal excitability and presynaptic control at glutamatergic terminals triggering exaggerated glutamate release and seizures.

  6. Interacting influence of diuretics and diet on BK channel-regulated K homeostasis

    Science.gov (United States)

    Wen, Donghai; Cornelius, Ryan J.; Sansom, Steven C.

    2014-01-01

    Large conductance, Ca-activated K channels are abundantly located in cells of vasculature, glomerulus and distal nephron, where they are involved in maintaining blood volume, blood pressure and K homeostasis. In mesangial cells and smooth muscle cells of vessels, the BK-α pore associates with BK-β1 subunits and regulates contraction in a Ca-mediated feedback manner. The BK-β1 also resides in connecting tubule cells of the nephron. BK-β1 knockout mice (β1KO) exhibit fluid retention, hypertension, and compromised K handling. The BK-α/β4resides in acid/base transporting intercalated cells (IC) of the distal nephron, where they mediate K secretion in mammals on a high K, alkaline diet. BK-α expression in IC is increased by a high K diet via aldosterone. The BK-β4 subunit and alkaline urine are necessary for the luminal expression and function of BK-α in mouse IC. In distal nephron cells, membrane BK-α expression is inhibited by WNK4 in in vitro expression systems, indicating a role in the hyperkalemic phenotype in patients with familial hyperkalemic hypertension type 2 (FHHt2). β1KO and BK-β4 knockout mice (β4KO) are hypertensive because of exaggerated ENaC-mediated Na retention in an effort to secrete K via only ROMK. BK hypertension is resistant to thiazides and furosemide, and would be more amenable to ENaC and aldosterone inhibiting drugs. Activators of BK-α/β1 or BK-α/β4 might be effective blood pressure lowering agents for a subset of hypertensive patients. Inhibitors of renal BK would effectively spare K in patients with Bartter Syndrome, a renal K wasting disease. PMID:24721651

  7. Current understanding of iberiotoxin-resistant BK channels in the nervous system.

    Science.gov (United States)

    Wang, Bin; Jaffe, David B; Brenner, Robert

    2014-01-01

    While most large-conductance, calcium-, and voltage-activated potassium channels (BK or Maxi-K type) are blocked by the scorpion venom iberiotoxin, the so-called "type II" subtype has the property of toxin resistance. This property is uniquely mediated by channel assembly with one member of the BK accessory β subunit family, the neuron-enriched β4 subunit. This review will focus on current understanding of iberiotoxin-resistant, β4-containing BK channel properties and their function in the CNS. Studies have shown that β4 dramatically promotes BK channel opening by shifting voltage sensor activation to more negative voltage ranges, but also slows activation to timescales that theoretically preclude BK ability to shape action potentials (APs). In addition, β4 membrane trafficking is regulated through an endoplasmic retention signal and palmitoylation. More recently, the challenge has been to understand the functional role of the iberiotoxin-resistant BK subtype utilizing computational modeling of neurons and neurophysiological approaches. Utilizing iberiotoxin-resistance as a footprint for these channels, they have been identified in dentate gyrus granule neurons and in purkinje neurons of the cerebellum. In these neurons, the role of these channels is largely consistent with slow-gated channels that reduce excitability either through an interspike conductance, such as in purkinje neurons, or by replacing fast-gating BK channels that otherwise facilitate high frequency AP firing, such as in dentate gyrus neurons. They are also observed in presynaptic mossy fiber terminals of the dentate gyrus and posterior pituitary terminals. More recent studies suggest that β4 subunits may also be expressed in some neurons lacking iberiotoxin-resistant BK channels, such as in CA3 hippocampus neurons. Ongoing research using novel, specific blockers and agonists of BK/β4, and β4 knockout mice, will continue to move the field forward in understanding the function of these

  8. Current understanding of iberiotoxin-resistant BK channels in the nervous system

    Directory of Open Access Journals (Sweden)

    Bin eWang

    2014-10-01

    Full Text Available While most large-conductance, calcium- and voltage-activated potassium channels (BK or Maxi-K type are blocked by the scorpion venom iberiotoxin, the so-called type II subtype has the property of toxin resistance. This property is uniquely mediated by channel assembly with one member of the BK accessory β subunit family, the neuron-enriched β4 subunit. This review will focus on current understanding of iberiotoxin-resistant, β4-containing BK channel properties and their function in the CNS. Studies have shown that β4 dramatically promotes BK channel opening by shifting voltage sensor activation to more negative voltage ranges, but also slows activation to timescales that theoretically preclude BK ability to shape action potentials (APs. In addition, β4 membrane trafficking is regulated through an endoplasmic retention signal and palmitoylation. More recently, the challenge has been to understand the functional role of the iberiotoxin-resistant BK subtype utilizing computational modeling of neurons and neurophysiological approaches. Utilizing iberiotoxin-resistance as a footprint for these channels, they have been identified in dentate gyrus granule neurons and in purkinje neurons of the cerebellum. In these neurons, the role of these channels is largely consistent with slow-gated channels that reduce excitability either through an interspike conductance, such as in purkinje neurons, or by replacing fast-gating BK channels that otherwise facilitate high frequency AP firing, such as in dentate gyrus neurons. They are also observed in presynaptic mossy fiber terminals of the dentate gyrus and posterior pituitary terminals. More recent studies suggest that β4 subunits may also be expressed in some neurons lacking iberiotoxin-resistant BK channels, such as in CA3 hippocampus neurons. Ongoing research using novel, specific blockers and agonists of BK/β4, and β4 knockout mice, will continue to move the field forward in understanding the

  9. Current understanding of iberiotoxin-resistant BK channels in the nervous system

    OpenAIRE

    Bin eWang; Jaffe, David B.; Robert eBrenner

    2014-01-01

    While most large-conductance, calcium- and voltage-activated potassium channels (BK or Maxi-K type) are blocked by the scorpion venom iberiotoxin, the so-called type II subtype has the property of toxin resistance. This property is uniquely mediated by channel assembly with one member of the BK accessory β subunit family, the neuron-enriched β4 subunit. This review will focus on current understanding of iberiotoxin-resistant, β4-containing BK channel properties and their function in the CNS. ...

  10. TRPV1 channels are functionally coupled with BK(mSlo1) channels in rat dorsal root ganglion (DRG) neurons.

    Science.gov (United States)

    Wu, Ying; Liu, Yongfeng; Hou, Panpan; Yan, Zonghe; Kong, Wenjuan; Liu, Beiying; Li, Xia; Yao, Jing; Zhang, Yuexuan; Qin, Feng; Ding, Jiuping

    2013-01-01

    The transient receptor potential vanilloid receptor 1 (TRPV1) channel is a nonselective cation channel activated by a variety of exogenous and endogenous physical and chemical stimuli, such as temperature (≥42 °C), capsaicin, a pungent compound in hot chili peppers, and allyl isothiocyanate. Large-conductance calcium- and voltage-activated potassium (BK) channels regulate the electric activities and neurotransmitter releases in excitable cells, responding to changes in membrane potentials and elevation of cytosolic calcium ions (Ca(2+)). However, it is unknown whether the TRPV1 channels are coupled with the BK channels. Using patch-clamp recording combined with an infrared laser device, we found that BK channels could be activated at 0 mV by a Ca(2+) influx through TRPV1 channels not the intracellular calcium stores in submilliseconds. The local calcium concentration around BK is estimated over 10 μM. The crosstalk could be affected by 10 mM BAPTA, whereas 5 mM EGTA was ineffectual. Fluorescence and co-immunoprecipitation experiments also showed that BK and TRPV1 were able to form a TRPV1-BK complex. Furthermore, we demonstrated that the TRPV1-BK coupling also occurs in dosal root ganglion (DRG) cells, which plays a critical physiological role in regulating the "pain" signal transduction pathway in the peripheral nervous system.

  11. TRPV1 channels are functionally coupled with BK(mSlo1 channels in rat dorsal root ganglion (DRG neurons.

    Directory of Open Access Journals (Sweden)

    Ying Wu

    Full Text Available The transient receptor potential vanilloid receptor 1 (TRPV1 channel is a nonselective cation channel activated by a variety of exogenous and endogenous physical and chemical stimuli, such as temperature (≥42 °C, capsaicin, a pungent compound in hot chili peppers, and allyl isothiocyanate. Large-conductance calcium- and voltage-activated potassium (BK channels regulate the electric activities and neurotransmitter releases in excitable cells, responding to changes in membrane potentials and elevation of cytosolic calcium ions (Ca(2+. However, it is unknown whether the TRPV1 channels are coupled with the BK channels. Using patch-clamp recording combined with an infrared laser device, we found that BK channels could be activated at 0 mV by a Ca(2+ influx through TRPV1 channels not the intracellular calcium stores in submilliseconds. The local calcium concentration around BK is estimated over 10 μM. The crosstalk could be affected by 10 mM BAPTA, whereas 5 mM EGTA was ineffectual. Fluorescence and co-immunoprecipitation experiments also showed that BK and TRPV1 were able to form a TRPV1-BK complex. Furthermore, we demonstrated that the TRPV1-BK coupling also occurs in dosal root ganglion (DRG cells, which plays a critical physiological role in regulating the "pain" signal transduction pathway in the peripheral nervous system.

  12. State-dependent FRET reports calcium- and voltage-dependent gating-ring motions in BK channels

    OpenAIRE

    Miranda, Pablo; Contreras, Jorge E.; Plested, Andrew J. R.; Sigworth, Fred J.; Holmgren, Miguel; Giraldez, Teresa

    2013-01-01

    Large-conductance voltage- and calcium-dependent potassium channels (BK, “Big K+”) are important controllers of cell excitability. In the BK channel, a large C-terminal intracellular region containing a “gating-ring” structure has been proposed to transduce Ca2+ binding into channel opening. Using patch-clamp fluorometry, we have investigated the calcium and voltage dependence of conformational changes of the gating-ring region of BK channels, while simultaneously monitoring channel conductan...

  13. KCNMA1 encoded cardiac BK channels afford protection against ischemia-reperfusion injury.

    Directory of Open Access Journals (Sweden)

    Ewa Soltysinska

    Full Text Available Mitochondrial potassium channels have been implicated in myocardial protection mediated through pre-/postconditioning. Compounds that open the Ca2+- and voltage-activated potassium channel of big-conductance (BK have a pre-conditioning-like effect on survival of cardiomyocytes after ischemia/reperfusion injury. Recently, mitochondrial BK channels (mitoBKs in cardiomyocytes were implicated as infarct-limiting factors that derive directly from the KCNMA1 gene encoding for canonical BKs usually present at the plasma membrane of cells. However, some studies challenged these cardio-protective roles of mitoBKs. Herein, we present electrophysiological evidence for paxilline- and NS11021-sensitive BK-mediated currents of 190 pS conductance in mitoplasts from wild-type but not BK-/- cardiomyocytes. Transmission electron microscopy of BK-/- ventricular muscles fibres showed normal ultra-structures and matrix dimension, but oxidative phosphorylation capacities at normoxia and upon re-oxygenation after anoxia were significantly attenuated in BK-/- permeabilized cardiomyocytes. In the absence of BK, post-anoxic reactive oxygen species (ROS production from cardiomyocyte mitochondria was elevated indicating that mitoBK fine-tune the oxidative state at hypoxia and re-oxygenation. Because ROS and the capacity of the myocardium for oxidative metabolism are important determinants of cellular survival, we tested BK-/- hearts for their response in an ex-vivo model of ischemia/reperfusion (I/R injury. Infarct areas, coronary flow and heart rates were not different between wild-type and BK-/- hearts upon I/R injury in the absence of ischemic pre-conditioning (IP, but differed upon IP. While the area of infarction comprised 28±3% of the area at risk in wild-type, it was increased to 58±5% in BK-/- hearts suggesting that BK mediates the beneficial effects of IP. These findings suggest that cardiac BK channels are important for proper oxidative energy supply of

  14. KCNMA1 encoded cardiac BK channels afford protection against ischemia-reperfusion injury.

    Science.gov (United States)

    Soltysinska, Ewa; Bentzen, Bo Hjorth; Barthmes, Maria; Hattel, Helle; Thrush, A Brianne; Harper, Mary-Ellen; Qvortrup, Klaus; Larsen, Filip J; Schiffer, Tomas A; Losa-Reyna, Jose; Straubinger, Julia; Kniess, Angelina; Thomsen, Morten Bækgaard; Brüggemann, Andrea; Fenske, Stefanie; Biel, Martin; Ruth, Peter; Wahl-Schott, Christian; Boushel, Robert Christopher; Olesen, Søren-Peter; Lukowski, Robert

    2014-01-01

    Mitochondrial potassium channels have been implicated in myocardial protection mediated through pre-/postconditioning. Compounds that open the Ca2+- and voltage-activated potassium channel of big-conductance (BK) have a pre-conditioning-like effect on survival of cardiomyocytes after ischemia/reperfusion injury. Recently, mitochondrial BK channels (mitoBKs) in cardiomyocytes were implicated as infarct-limiting factors that derive directly from the KCNMA1 gene encoding for canonical BKs usually present at the plasma membrane of cells. However, some studies challenged these cardio-protective roles of mitoBKs. Herein, we present electrophysiological evidence for paxilline- and NS11021-sensitive BK-mediated currents of 190 pS conductance in mitoplasts from wild-type but not BK-/- cardiomyocytes. Transmission electron microscopy of BK-/- ventricular muscles fibres showed normal ultra-structures and matrix dimension, but oxidative phosphorylation capacities at normoxia and upon re-oxygenation after anoxia were significantly attenuated in BK-/- permeabilized cardiomyocytes. In the absence of BK, post-anoxic reactive oxygen species (ROS) production from cardiomyocyte mitochondria was elevated indicating that mitoBK fine-tune the oxidative state at hypoxia and re-oxygenation. Because ROS and the capacity of the myocardium for oxidative metabolism are important determinants of cellular survival, we tested BK-/- hearts for their response in an ex-vivo model of ischemia/reperfusion (I/R) injury. Infarct areas, coronary flow and heart rates were not different between wild-type and BK-/- hearts upon I/R injury in the absence of ischemic pre-conditioning (IP), but differed upon IP. While the area of infarction comprised 28±3% of the area at risk in wild-type, it was increased to 58±5% in BK-/- hearts suggesting that BK mediates the beneficial effects of IP. These findings suggest that cardiac BK channels are important for proper oxidative energy supply of cardiomyocytes at

  15. Pharmacological consequences of the coexpression of BK channel α and auxiliary β subunits

    Science.gov (United States)

    Torres, Yolima P.; Granados, Sara T.; Latorre, Ramón

    2014-01-01

    Coded by a single gene (Slo1, KCM) and activated by depolarizing potentials and by a rise in intracellular Ca2+ concentration, the large conductance voltage- and Ca2+-activated K+ channel (BK) is unique among the superfamily of K+ channels. BK channels are tetramers characterized by a pore-forming α subunit containing seven transmembrane segments (instead of the six found in voltage-dependent K+ channels) and a large C terminus composed of two regulators of K+ conductance domains (RCK domains), where the Ca2+-binding sites reside. BK channels can be associated with accessory β subunits and, although different BK modulatory mechanisms have been described, greater interest has recently been placed on the role that the β subunits may play in the modulation of BK channel gating due to its physiological importance. Four β subunits have currently been identified (i.e., β1, β2, β3, and β4) and despite the fact that they all share the same topology, it has been shown that every β subunit has a specific tissue distribution and that they modify channel kinetics as well as their pharmacological properties and the apparent Ca2+ sensitivity of the α subunit in different ways. Additionally, different studies have shown that natural, endogenous, and synthetic compounds can modulate BK channels through β subunits. Considering the importance of these channels in different pathological conditions, such as hypertension and neurological disorders, this review focuses on the mechanisms by which these compounds modulate the biophysical properties of BK channels through the regulation of β subunits, as well as their potential therapeutic uses for diseases such as those mentioned above. PMID:25346693

  16. 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...

  17. The brain-specific Beta4 subunit downregulates BK channel cell surface expression.

    Science.gov (United States)

    Shruti, Sonal; Urban-Ciecko, Joanna; Fitzpatrick, James A; Brenner, Robert; Bruchez, Marcel P; Barth, Alison L

    2012-01-01

    The large-conductance K(+) channel (BK channel) can control neural excitability, and enhanced channel currents facilitate high firing rates in cortical neurons. The brain-specific auxiliary subunit β4 alters channel Ca(++)- and voltage-sensitivity, and β4 knock-out animals exhibit spontaneous seizures. Here we investigate β4's effect on BK channel trafficking to the plasma membrane. Using a novel genetic tag to track the cellular location of the pore-forming BKα subunit in living cells, we find that β4 expression profoundly reduces surface localization of BK channels via a C-terminal ER retention sequence. In hippocampal CA3 neurons from C57BL/6 mice with endogenously high β4 expression, whole-cell BK channel currents display none of the characteristic properties of BKα+β4 channels observed in heterologous cells. Finally, β4 knock-out animals exhibit a 2.5-fold increase in whole-cell BK channel current, indicating that β4 also regulates current magnitude in vivo. Thus, we propose that a major function of the brain-specific β4 subunit in CA3 neurons is control of surface trafficking.

  18. The brain-specific Beta4 subunit downregulates BK channel cell surface expression.

    Directory of Open Access Journals (Sweden)

    Sonal Shruti

    Full Text Available The large-conductance K(+ channel (BK channel can control neural excitability, and enhanced channel currents facilitate high firing rates in cortical neurons. The brain-specific auxiliary subunit β4 alters channel Ca(++- and voltage-sensitivity, and β4 knock-out animals exhibit spontaneous seizures. Here we investigate β4's effect on BK channel trafficking to the plasma membrane. Using a novel genetic tag to track the cellular location of the pore-forming BKα subunit in living cells, we find that β4 expression profoundly reduces surface localization of BK channels via a C-terminal ER retention sequence. In hippocampal CA3 neurons from C57BL/6 mice with endogenously high β4 expression, whole-cell BK channel currents display none of the characteristic properties of BKα+β4 channels observed in heterologous cells. Finally, β4 knock-out animals exhibit a 2.5-fold increase in whole-cell BK channel current, indicating that β4 also regulates current magnitude in vivo. Thus, we propose that a major function of the brain-specific β4 subunit in CA3 neurons is control of surface trafficking.

  19. Tuning the mechanosensitivity of a BK channel by changing the linker length

    Institute of Scientific and Technical Information of China (English)

    2008-01-01

    Some large-conductance Ca2+ and voltage-activated K+ (BK) channels are activated by membrane stretch. However, the mechanism of mechano-gating of the BK channels is still not well understood. Previous studies have led to the proposal that the tinker-gating ring complex functions as a passive spring, transducing the force generated by intraceilular Ca2+ to the gate to open the channel. This raises the question as to whether membrane stretch is also transmitted to the gate of mechanosensitive (MS) BK channels via the tinker-gating complex. To study this, we changed the linker length in the stretch-activated BK channel (SAKCaC), and examined the effect of membrane stretch on the gating of the resultant mutant channels. Shortening the tinker increased, whereas extending the tinker reduced, the channel mechanosensitivity both in the presence and in the absence of intracellular Ca2+. However, the voltage and Ca2+ sensitivities were not significantly altered by membrane stretch. Furthermore, the SAKCaC became less sensitive to membrane stretch at relatively high intracellular Ca2+ concentrations or membrane depolarization. These observations suggest that once the channel is in the open-state conformation, tension on the spring is partially released and membrane stretch is less effective. Our results are consistent with the idea that membrane stretch is transferred to the gate via the tinker-gating ring complex of the MS BK channels.

  20. The Brain-Specific Beta4 Subunit Downregulates BK Channel Cell Surface Expression

    OpenAIRE

    Sonal Shruti; Joanna Urban-Ciecko; Fitzpatrick, James A.; Robert Brenner; Bruchez, Marcel P.; Alison L Barth

    2012-01-01

    The large-conductance K(+) channel (BK channel) can control neural excitability, and enhanced channel currents facilitate high firing rates in cortical neurons. The brain-specific auxiliary subunit β4 alters channel Ca(++)- and voltage-sensitivity, and β4 knock-out animals exhibit spontaneous seizures. Here we investigate β4's effect on BK channel trafficking to the plasma membrane. Using a novel genetic tag to track the cellular location of the pore-forming BKα subunit in living cells, we fi...

  1. BK potassium channels control transmitter release at CA3-CA3 synapses in the rat hippocampus.

    Science.gov (United States)

    Raffaelli, Giacomo; Saviane, Chiara; Mohajerani, Majid H; Pedarzani, Paola; Cherubini, Enrico

    2004-05-15

    Large conductance calcium- and voltage-activated potassium channels (BK channels) activate in response to calcium influx during action potentials and contribute to the spike repolarization and fast afterhyperpolarization. BK channels targeted to active zones in presynaptic nerve terminals have been shown to limit calcium entry and transmitter release by reducing the duration of the presynaptic spike at neurosecretory nerve terminals and at the frog neuromuscular junction. However, their functional role in central synapses is still uncertain. In the hippocampus, BK channels have been proposed to act as an 'emergency brake' that would control transmitter release only under conditions of excessive depolarization and accumulation of intracellular calcium. Here we demonstrate that in the CA3 region of hippocampal slice cultures, under basal experimental conditions, the selective BK channel blockers paxilline (10 microM) and iberiotoxin (100 nM) increase the frequency, but not the amplitude, of spontaneously occurring action potential-dependent EPSCs. These drugs did not affect miniature currents recorded in the presence of tetrodotoxin, suggesting that their action was dependent on action potential firing. Moreover, in double patch-clamp recordings from monosynaptically interconnected CA3 pyramidal neurones, blockade of BK channels enhanced the probability of transmitter release, as revealed by the increase in success rate, EPSC amplitude and the concomitant decrease in paired-pulse ratio in response to pairs of presynaptic action potentials delivered at a frequency of 0.05 Hz. BK channel blockers also enhanced the appearance of delayed responses, particularly following the second action potential in the paired-pulse protocol. These results are consistent with the hypothesis that BK channels are powerful modulators of transmitter release and synaptic efficacy in central neurones.

  2. 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.

  3. 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.

  4. Hair cell BK channels interact with RACK1, and PKC increases its expression on the cell surface by indirect phosphorylation.

    Science.gov (United States)

    Surguchev, Alexei; Bai, Jun-Ping; Joshi, Powrnima; Navaratnam, Dhasakumar

    2012-07-15

    Large conductance (BK) calcium activated potassium channels (Slo) are ubiquitous and implicated in a number of human diseases including hypertension and epilepsy. BK channels consist of a pore forming α-subunit (Slo) and a number of accessory subunits. In hair cells of nonmammalian vertebrates these channels play a critical role in electrical resonance, a mechanism of frequency selectivity. Hair cell BK channel clusters on the surface and currents increase along the tonotopic axis and contribute significantly to the responsiveness of these hair cells to sounds of high frequency. In contrast, messenger RNA levels encoding the Slo gene show an opposite decrease in high frequency hair cells. To understand the molecular events underlying this paradox, we used a yeast two-hybrid screen to isolate binding partners of Slo. We identified Rack1 as a Slo binding partner and demonstrate that PKC activation increases Slo surface expression. We also establish that increased Slo recycling of endocytosed Slo is at least partially responsible for the increased surface expression of Slo. Moreover, analysis of several PKC phosphorylation site mutants confirms that the effects of PKC on Slo surface expression are likely indirect. Finally, we show that Slo clusters on the surface of hair cells are also increased by increased PKC activity and may contribute to the increasing amounts of channel clusters on the surface of high-frequency hair cells.

  5. Role of BK channels in the apoptotic volume decrease in native eel intestinal cells

    DEFF Research Database (Denmark)

    Lionetto, Maria Giulia; Giordano, Maria Elena; Calisi, Antonio

    2010-01-01

    of these channels in the Apoptotic Volume Decrease (AVD) of isolated eel enterocytes, and the possible interaction between BK channels and the progression of apoptosis. The detection of apoptosis was performed by confocal microscopy and annexin V and propidium iodide labelling; cell volume changes were monitored...

  6. KCNMA1 encoded cardiac BK channels afford protection against ischemia-reperfusion injury

    DEFF Research Database (Denmark)

    Soltysinska, Ewa; Bentzen, Bo Hjorth; Barthmes, Maria

    2014-01-01

    Mitochondrial potassium channels have been implicated in myocardial protection mediated through pre-/postconditioning. Compounds that open the Ca2+- and voltage-activated potassium channel of big-conductance (BK) have a pre-conditioning-like effect on survival of cardiomyocytes after ischemia/rep...

  7. Differential expression of BK channel isoforms and beta-subunits in rat neuro-vascular tissues

    DEFF Research Database (Denmark)

    Poulsen, Asser Nyander; Johansson, Helle Wulf; Hay-Schmidt, Anders

    2009-01-01

    We investigated the expression of splice variants and beta-subunits of the BK channel (big conductance Ca(2+)-activated K(+) channel, Slo1, MaxiK, K(Ca)1.1) in rat cerebral blood vessels, meninges, trigeminal ganglion among other tissues. An alpha-subunit splice variant X1(+24) was found expresse...

  8. Ca(2+)-BK channel clusters in olfactory receptor neurons and their role in odour coding.

    Science.gov (United States)

    Bao, Guobin; de Jong, Daniëlle; Alevra, Mihai; Schild, Detlev

    2015-12-01

    Olfactory receptor neurons (ORNs) have high-voltage-gated Ca(2+) channels whose physiological impact has remained enigmatic since the voltage-gated conductances in this cell type were first described in the 1980s. Here we show that in ORN somata of Xenopus laevis tadpoles these channels are clustered and co-expressed with large-conductance potassium (BK) channels. We found approximately five clusters per ORN and twelve Ca(2+) channels per cluster. The action potential-triggered activation of BK channels accelerates the repolarization of action potentials and shortens interspike intervals during odour responses. This increases the sensitivity of individual ORNs to odorants. At the level of mitral cells of the olfactory bulb, odour qualities have been shown to be coded by first-spike-latency patterns. The system of Ca(2+) and BK channels in ORNs appears to be important for correct odour coding because the blockage of BK channels not only affects ORN spiking patterns but also changes the latency pattern representation of odours in the olfactory bulb.

  9. Molecular studies of BKCa channels in intracranial arteries

    DEFF Research Database (Denmark)

    Wulf, Helle; Hay-Schmidt, Anders; Poulsen, Asser Nyander

    2008-01-01

    expression of the BK(Ca) channel in rat basilar, middle cerebral, and middle meningeal arteries by reverse transcription polymerase chain reaction (RT-PCR), quantitative real-time PCR, and Western blotting. Distribution patterns were investigated using in situ hybridization and immunofluorescence studies. RT......-PCR and quantitative real-time PCR detected the expression of the BK(Ca) channel mRNA transcript in rat basilar, middle cerebral, and middle meningeal arteries, with the transcript being expressed more abundantly in rat basilar arteries than in middle cerebral and middle meningeal arteries. Western blotting detected...

  10. Overexpression of the Large-Conductance, Ca2+-Activated K+ (BK) Channel Shortens Action Potential Duration in HL-1 Cardiomyocytes.

    Science.gov (United States)

    Stimers, Joseph R; Song, Li; Rusch, Nancy J; Rhee, Sung W

    2015-01-01

    Long QT syndrome is characterized by a prolongation of the interval between the Q wave and the T wave on the electrocardiogram. This abnormality reflects a prolongation of the ventricular action potential caused by a number of genetic mutations or a variety of drugs. Since effective treatments are unavailable, we explored the possibility of using cardiac expression of the large-conductance, Ca2+-activated K+ (BK) channel to shorten action potential duration (APD). We hypothesized that expression of the pore-forming α subunit of human BK channels (hBKα) in HL-1 cells would shorten action potential duration in this mouse atrial cell line. Expression of hBKα had minimal effects on expression levels of other ion channels with the exception of a small but significant reduction in Kv11.1. Patch-clamped hBKα expressing HL-1 cells exhibited an outward voltage- and Ca2+-sensitive K+ current, which was inhibited by the BK channel blocker iberiotoxin (100 nM). This BK current phenotype was not detected in untransfected HL-1 cells or in HL-1 null cells sham-transfected with an empty vector. Importantly, APD in hBKα-expressing HL-1 cells averaged 14.3 ± 2.8 ms (n = 10), which represented a 53% reduction in APD compared to HL-1 null cells lacking BKα expression. APD in the latter cells averaged 31.0 ± 5.1 ms (n = 13). The shortened APD in hBKα-expressing cells was restored to normal duration by 100 nM iberiotoxin, suggesting that a repolarizing K+ current attributed to BK channels accounted for action potential shortening. These findings provide initial proof-of-concept that the introduction of hBKα channels into a cardiac cell line can shorten APD, and raise the possibility that gene-based interventions to increase hBKα channels in cardiac cells may hold promise as a therapeutic strategy for long QT syndrome.

  11. BK channel β1 and β4 auxiliary subunits exert opposite influences on escalated ethanol drinking in dependent mice

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    Max eKreifeldt

    2013-12-01

    Full Text Available Large conductance calcium-activated potassium (BK channels play a key role in the control of neuronal activity. Ethanol is a potent activator of BK channel gating, but how this action may impact ethanol drinking still remains poorly understood. Auxiliary β subunits are known to modulate ethanol-induced potentiation of BK currents. In the present study, we investigated whether BK β1 and β4 subunits influence voluntary ethanol consumption using knockout mice. In a first experiment, mice were first subjected to continuous two-bottle choice (2BC and were then switched to intermittent 2BC, which progressively increased ethanol intake as previously described in wildtype mice. BK β1 or β4 subunit deficiency did not affect ethanol self-administration under either schedule of access. In a second experiment, mice were first trained to drink ethanol in a limited-access 2BC paradigm. BK β1 or β4 deletion did not affect baseline consumption. Weeks of 2BC were then alternated with weeks of chronic intermittent ethanol (CIE or air inhalation. As expected, a gradual escalation of ethanol drinking was observed in dependent wildtype mice, while intake remained stable in non-dependent wildtype mice. However, CIE exposure only produced a mild augmentation of ethanol consumption in BK β4 knockout mice. Conversely, ethanol drinking increased after fewer CIE cycles in BK β1 knockout mice than in wildtype mice. In conclusion, BK β1 or β4 did not influence voluntary ethanol drinking in non-dependent mice, regardless of the pattern of access to ethanol. However, deletion of BK β4 attenuated, while deletion of BK β1 accelerated, the escalation of ethanol drinking during withdrawal from CIE. Our data suggest that BK β1 and β4 subunits have an opposite influence on the negative reinforcing properties of ethanol withdrawal. Modulating the expression, distribution or interactions of BK channel auxiliary subunits may therefore represent a novel avenue for the

  12. Broadening roles for FMRP: big news for big potassium (BK) channels.

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    Contractor, Anis

    2013-02-20

    FMRP is an RNA-binding protein that negatively regulates translation and which is lost in fragile X syndrome. In this issue of Neuron, Deng et al. (2013) demonstrate a novel translation-independent function for FMRP as a regulator of presynaptic BK channels that modulate the dynamics of neurotransmitter release.

  13. Deletion of cytosolic gating ring decreases gate and voltage sensor coupling in BK channels.

    Science.gov (United States)

    Zhang, Guohui; Geng, Yanyan; Jin, Yakang; Shi, Jingyi; McFarland, Kelli; Magleby, Karl L; Salkoff, Lawrence; Cui, Jianmin

    2017-03-06

    Large conductance Ca(2+)-activated K(+) channels (BK channels) gate open in response to both membrane voltage and intracellular Ca(2+) The channel is formed by a central pore-gate domain (PGD), which spans the membrane, plus transmembrane voltage sensors and a cytoplasmic gating ring that acts as a Ca(2+) sensor. How these voltage and Ca(2+) sensors influence the common activation gate, and interact with each other, is unclear. A previous study showed that a BK channel core lacking the entire cytoplasmic gating ring (Core-MT) was devoid of Ca(2+) activation but retained voltage sensitivity (Budelli et al. 2013. Proc. Natl. Acad. Sci. USA http://dx.doi.org/10.1073/pnas.1313433110). In this study, we measure voltage sensor activation and pore opening in this Core-MT channel over a wide range of voltages. We record gating currents and find that voltage sensor activation in this truncated channel is similar to WT but that the coupling between voltage sensor activation and gating of the pore is reduced. These results suggest that the gating ring, in addition to being the Ca(2+) sensor, enhances the effective coupling between voltage sensors and the PGD. We also find that removal of the gating ring alters modulation of the channels by the BK channel's β1 and β2 subunits.

  14. Alcohol modulation of BK channel gating depends on β subunit composition.

    Science.gov (United States)

    Kuntamallappanavar, Guruprasad; Dopico, Alex M

    2016-11-01

    In most mammalian tissues, Ca(2+)i/voltage-gated, large conductance K(+) (BK) channels consist of channel-forming slo1 and auxiliary (β1-β4) subunits. When Ca(2+)i (3-20 µM) reaches the vicinity of BK channels and increases their activity at physiological voltages, β1- and β4-containing BK channels are, respectively, inhibited and potentiated by intoxicating levels of ethanol (50 mM). Previous studies using different slo1s, lipid environments, and Ca(2+)i concentrations-all determinants of the BK response to ethanol-made it impossible to determine the specific contribution of β subunits to ethanol action on BK activity. Furthermore, these studies measured ethanol action on ionic current under a limited range of stimuli, rendering no information on the gating processes targeted by alcohol and their regulation by βs. Here, we used identical experimental conditions to obtain single-channel and macroscopic currents of the same slo1 channel ("cbv1" from rat cerebral artery myocytes) in the presence and absence of 50 mM ethanol. First, we assessed the role five different β subunits (1,2,2-IR, 3-variant d, and 4) in ethanol action on channel function. Thus, two phenotypes were identified: (1) ethanol potentiated cbv1-, cbv1+β3-, and cbv1+β4-mediated currents at low Ca(2+)i while inhibiting current at high Ca(2+)i, the potentiation-inhibition crossover occurring at 20 µM Ca(2+)i; (2) for cbv1+β1, cbv1+wt β2, and cbv1+β2-IR, this crossover was shifted to ∼3 µM Ca(2+)i Second, applying Horrigan-Aldrich gating analysis on both phenotypes, we show that ethanol fails to modify intrinsic gating and the voltage-dependent parameters under examination. For cbv1, however, ethanol (a) drastically increases the channel's apparent Ca(2+) affinity (nine-times decrease in Kd) and (b) very mildly decreases allosteric coupling between Ca(2+) binding and channel opening (C). The decreased Kd leads to increased channel activity. For cbv1+β1, ethanol (a) also decreases Kd

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

    Science.gov (United States)

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

    2016-01-01

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

  16. BK Channels Alleviate Lysosomal Storage Diseases by Providing Positive Feedback Regulation of Lysosomal Ca2+ Release.

    Science.gov (United States)

    Cao, Qi; Zhong, Xi Zoë; Zou, Yuanjie; Zhang, Zhu; Toro, Ligia; Dong, Xian-Ping

    2015-05-26

    Promoting lysosomal trafficking represents a promising therapeutic approach for lysosome storage diseases. Efficient Ca(2+) mobilization from lysosomes is important for lysosomal trafficking. Ca(2+) release from lysosomes could generate a negative potential in the lumen to disturb subsequent Ca(2+) release in the absence of counter ion flux. Here we report that lysosomes express big-conductance Ca(2+)-activated potassium (BK) channels that form physical and functional coupling with the lysosomal Ca(2+) release channel, TRPML1. Ca(2+) release via TRPML1 causes BK activation, which in turn facilitates further lysosomal Ca(2+) release and membrane trafficking. Importantly, BK overexpression rescues the impaired TRPML1-mediated Ca(2+) release and abnormal lysosomal storage in cells from Niemann-Pick C1 patients. Therefore, we have identified a lysosomal K(+) channel that provides a positive feedback mechanism to facilitate TRPML1-mediated Ca(2+) release and membrane trafficking. Our findings suggest that upregulating BK may be a potential therapeutic strategy for certain lysosomal storage diseases and common neurodegenerative disorders.

  17. Renovascular BK(Ca) channels are not activated in vivo under resting conditions and during agonist stimulation

    DEFF Research Database (Denmark)

    Magnusson, Linda; Sørensen, Charlotte Mehlin; Braunstein, Thomas Hartig

    2006-01-01

    We investigated the role of large-conductance Ca(2+)-activated K(+) (BK(Ca)) channels for the basal renal vascular tone in vivo. Furthermore, the possible buffering by BK(Ca) of the vasoconstriction elicited by angiotensin II (ANG II) or norepinephrine (NE) was investigated. The possible activati...

  18. Bimane fluorescence scanning suggests secondary structure near the S3-S4 linker of BK channels.

    Science.gov (United States)

    Semenova, Nina P; Abarca-Heidemann, Karin; Loranc, Eva; Rothberg, Brad S

    2009-04-17

    Gating of large conductance Ca(2+)-activated K(+) channels (BK or maxi-K channels) is controlled by a Ca(2+)-sensor, formed by the channel cytoplasmic C-terminal domain, and a voltage sensor, formed by its S0-S4 transmembrane helices. Here we analyze structural properties of a portion of the BK channel voltage sensing domain, the S3-S4 linker, using fluorescence lifetime spectroscopy. Single residues in the S3-S4 linker region were substituted with cysteine, and the cysteine-substituted mutants were expressed in CHO cells and covalently labeled with the sulfhydryl-reactive fluorophore monobromo-trimethylammonio-bimane (qBBr). qBBr fluorescence is quenched by tryptophan and, to a lesser extent, tyrosine side chains. We found that qBBr fluorescence in several of the labeled cysteine-substituted channels shows position-specific quenching, as indicated by increase of the brief lifetime component of the qBBr fluorescence decay. Quenching was reduced with the mutation W203F (in the S4 segment), suggesting that Trp-203 acts as a quenching group. Our results suggest a working hypothesis for the secondary structure of the BK channel S3-S4 region, and places residues Leu-204, Gly-205, and Leu-206 within the extracellular end of the S4 helix.

  19. Habituation of reflexive and motivated behaviour in mice with deficient BK channel function

    Directory of Open Access Journals (Sweden)

    Marei eTyplt

    2013-11-01

    Full Text Available Habituation is considered the most basic form of learning. It describes the decrease of a behavioural response to a repeated non-threatening sensory stimulus and therefore provides an important sensory filtering mechanism. While some neuronal pathways mediating habituation are well described, underlying cellular/molecular mechanisms are not yet fully understood. In general, there is an agreement that short-term and long-term habituation are based on different mechanisms. Historically, a distinction has also been made between habituation of motivated versus reflexive behaviour. In recent studies in invertebrates the large conductance voltage- and calcium-activated potassium (BK channel has been implicated to be a key player in habituation by regulating synaptic transmission. Here, we tested mice deficient for the pore forming α-subunit of the BK channel for short-term and long-term habituation of the acoustic startle reflex (reflexive behaviour and of the exploratory locomotor behaviour in the open field box (motivated behaviour. Short-term habituation of startle was completely abolished in the BK knock-out mice, whereas neither long-term habituation of startle nor habituation of motivated behaviour was affected by the BK deficiency. Our results support a highly preserved mechanism for short-term habituation of startle across species that is distinct from long-term habituation mechanisms. It also supports the notion that there are different mechanisms underlying habituation of motivated behaviour versus reflexive behaviour.

  20. Science Signaling Podcast for 9 May 2017: Trafficking of BK channel subunits in arterial myocytes.

    Science.gov (United States)

    Jaggar, Jonathan H; VanHook, Annalisa M

    2017-05-09

    This Podcast features a conversation with Jonathan Jaggar, senior author of a Research Article that appears in the 9 May 2017 issue of Science Signaling, about trafficking of big potassium (BK) channel subunits in arterial myocytes. Depolarization of the arterial myocyte membrane causes a rise in intracellular calcium that stimulates the cell to contract, which leads to vasoconstriction. Membrane depolarization also activates BK channels, which allow potassium to flow out of the cell, thus repolarizing the membrane and promoting vasodilation. Leo et al found that a critical aspect of this negative feedback mechanism was the trafficking of the regulatory β1 BK channel subunit to the plasma membrane. Membrane depolarization caused the β1 subunit to translocate to the plasma membrane, where it associated with the pore-forming α subunit to increase the calcium sensitivity of the channel. These findings identify trafficking of regulatory subunits as a mode of regulation for multisubunit ion channels.Listen to Podcast. Copyright © 2017, American Association for the Advancement of Science.

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

    DEFF Research Database (Denmark)

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

    2009-01-01

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

  2. A non-cardiomyocyte autonomous mechanism of cardioprotection involving the SLO1 BK channel

    Directory of Open Access Journals (Sweden)

    Andrew P. Wojtovich

    2013-03-01

    Full Text Available Opening of BK-type Ca2+ activated K+ channels protects the heart against ischemia-reperfusion (IR injury. However, the location of BK channels responsible for cardioprotection is debated. Herein we confirmed that openers of the SLO1 BK channel, NS1619 and NS11021, were protective in a mouse perfused heart model of IR injury. As anticipated, deletion of the Slo1 gene blocked this protection. However, in an isolated cardiomyocyte model of IR injury, protection by NS1619 and NS11021 was insensitive to Slo1 deletion. These data suggest that protection in intact hearts occurs by a non-cardiomyocyte autonomous, SLO1-dependent, mechanism. In this regard, an in-situ assay of intrinsic cardiac neuronal function (tachycardic response to nicotine revealed that NS1619 preserved cardiac neurons following IR injury. Furthermore, blockade of synaptic transmission by hexamethonium suppressed cardioprotection by NS1619 in intact hearts. These results suggest that opening SLO1 protects the heart during IR injury, via a mechanism that involves intrinsic cardiac neurons. Cardiac neuronal ion channels may be useful therapeutic targets for eliciting cardioprotection.

  3. FMRP regulates neurotransmitter release and synaptic information transmission by modulating action potential duration via BK channels.

    Science.gov (United States)

    Deng, Pan-Yue; Rotman, Ziv; Blundon, Jay A; Cho, Yongcheol; Cui, Jianmin; Cavalli, Valeria; Zakharenko, Stanislav S; Klyachko, Vitaly A

    2013-02-20

    Loss of FMRP causes fragile X syndrome (FXS), but the physiological functions of FMRP remain highly debatable. Here we show that FMRP regulates neurotransmitter release in CA3 pyramidal neurons by modulating action potential (AP) duration. Loss of FMRP leads to excessive AP broadening during repetitive activity, enhanced presynaptic calcium influx, and elevated neurotransmitter release. The AP broadening defects caused by FMRP loss have a cell-autonomous presynaptic origin and can be acutely rescued in postnatal neurons. These presynaptic actions of FMRP are translation independent and are mediated selectively by BK channels via interaction of FMRP with BK channel's regulatory β4 subunits. Information-theoretical analysis demonstrates that loss of these FMRP functions causes marked dysregulation of synaptic information transmission. FMRP-dependent AP broadening is not limited to the hippocampus, but also occurs in cortical pyramidal neurons. Our results thus suggest major translation-independent presynaptic functions of FMRP that may have important implications for understanding FXS neuropathology.

  4. Molecular investigations of BK(Ca) channels and the modulatory beta-subunits in porcine basilar and middle cerebral arteries

    DEFF Research Database (Denmark)

    Johansson, Helle Wulf; Hay-Schmidt, Anders; Poulsen, Asser Nyander

    2009-01-01

    arteries using reverse transcription polymerase chain reaction (RT-PCR) and quantitative real-time PCR. Western blotting was used to detect immunoreactivity for the porcine BK(Ca) channel alpha-subunit and beta-subunit proteins. The BK(Ca) channel alpha-subunit RNA and protein distribution patterns were......-PCR in porcine basilar and middle cerebral arteries. However, at the protein level, only, the beta1-subunit protein was found by western blotting....

  5. BK channels regulate spontaneous action potential rhythmicity in the suprachiasmatic nucleus.

    Directory of Open Access Journals (Sweden)

    Jack Kent

    Full Text Available BACKGROUND: Circadian ( approximately 24 hr rhythms are generated by the central pacemaker localized to the suprachiasmatic nucleus (SCN of the hypothalamus. Although the basis for intrinsic rhythmicity is generally understood to rely on transcription factors encoded by "clock genes", less is known about the daily regulation of SCN neuronal activity patterns that communicate a circadian time signal to downstream behaviors and physiological systems. Action potentials in the SCN are necessary for the circadian timing of behavior, and individual SCN neurons modulate their spontaneous firing rate (SFR over the daily cycle, suggesting that the circadian patterning of neuronal activity is necessary for normal behavioral rhythm expression. The BK K(+ channel plays an important role in suppressing spontaneous firing at night in SCN neurons. Deletion of the Kcnma1 gene, encoding the BK channel, causes degradation of circadian behavioral and physiological rhythms. METHODOLOGY/PRINCIPAL FINDINGS: To test the hypothesis that loss of robust behavioral rhythmicity in Kcnma1(-/- mice is due to the disruption of SFR rhythms in the SCN, we used multi-electrode arrays to record extracellular action potentials from acute wild-type (WT and Kcnma1(-/- slices. Patterns of activity in the SCN were tracked simultaneously for up to 3 days, and the phase, period, and synchronization of SFR rhythms were examined. Loss of BK channels increased arrhythmicity but also altered the amplitude and period of rhythmic activity. Unexpectedly, Kcnma1(-/- SCNs showed increased variability in the timing of the daily SFR peak. CONCLUSIONS/SIGNIFICANCE: These results suggest that BK channels regulate multiple aspects of the circadian patterning of neuronal activity in the SCN. In addition, these data illustrate the characteristics of a disrupted SCN rhythm downstream of clock gene-mediated timekeeping and its relationship to behavioral rhythms.

  6. The coupling of acetylcholine-induced BK channel and calcium channel in guinea pig saccular type II vestibular hair cells.

    Science.gov (United States)

    Kong, Wei-Jia; Guo, Chang-Kai; Zhang, Xiao-Wen; Chen, Xiong; Zhang, Song; Li, Guan-Qiao; Li, Zhi-Wang; Van Cauwenberge, Paul

    2007-01-19

    Molecular biological studies and electrophysiological data have demonstrated that acetylcholine (ACh) is the principal cochlear and vestibular efferent neurotransmitter among mammalians. However, the functional roles of ACh in type II vestibular hair cells (VHCs II) among mammalians are still unclear, with the exception of the well-known alpha9-containing nicotinic ACh receptor (alpha9-containing nAChR)-activated small conductance, calcium-dependent potassium current (SK) in cochlear hair cells and frog saccular hair cells. The activation of SK current was necessary for the calcium influx through the alpha9-containing nAChR. Recently, we have demonstrated that ACh-induced big conductance, calcium-dependent potassium current (BK) was present in VHCs II of the vestibular end-organ of guinea pig. In this study, the nature of calcium influx for the activation of ACh-induced BK current in saccular VHCs II of guinea pig was investigated. Following extracellular perfusion of ACh, saccular VHCs II displayed a sustained outward current, which was sensitive to iberiotoxin (IBTX). High concentration of apamin failed to inhibit the current amplitude of ACh-induced outward current. Intracellular application of Cs(+) completely abolished the current evoked by ACh. ACh-induced current was potently inhibited by nifedipine, nimodipine, Cd(2+) and Ni(2+), respectively. The inhibition potency of these four calcium channel antagonists was nimodipine>nifedipine>cadmium>nickel. The L-type Ca(2+) channels agonist, (-)-Bay-K 8644 mimicked the effect of ACh and activated an IBTX-sensitive current. In addition, partial VHCs II displayed a biphasic waveform. In conclusion, the present data showed that in the guinea pig saccular VHCs II, ACh-induced BK channel was coupled with the calcium channel, but not the receptor. The perfusion of ACh will drive the opening of calcium channels; the influx of calcium ions will then activate the BK current.

  7. Neuronal fast activating and meningeal silent modulatory BK channel splice variants cloned from rat

    DEFF Research Database (Denmark)

    Poulsen, Asser Nyander; Jansen-Olesen, Inger; Olesen, Jes

    2011-01-01

    The big conductance calcium-activated K(+) channel (BK) is involved in regulating neuron and smooth muscle cell excitability. Functional diversity of BK is generated by alpha-subunit splice variation and co-expression with beta subunits. Here, we present six different splice combinations cloned...... from rat brain or cerebral vascular/meningeal tissues, of which at least three variants were previously uncharacterized (X1, X2(92), and X2(188)). An additional variant was identified by polymerase chain reaction but not cloned. Expression in Xenopus oocytes showed that the brain-specific X1 variant...... displays reduced current, faster activation, and less voltage sensitivity than the insert-less Zero variant. Other cloned variants Strex and Slo27,3 showed slower activation than Zero. The X1 variant contains sequence inserts in the S1-S2 extracellular loop (8 aa), between intracellular domains RCK1...

  8. BK channel activity determines the extent of cell degeneration after oxygen and glucose deprivation: a study in organotypical hippocampal slice cultures.

    Science.gov (United States)

    Rundén-Pran, E; Haug, F M; Storm, J F; Ottersen, O P

    2002-01-01

    BK channels are voltage- and calcium-dependent potassium channels whose activation tends to reduce cellular excitability. In hippocampal pyramidal cells, BK channels repolarize somatic action potentials, and recent immunogold and electrophysiological analyses have revealed a presynaptic pool of BK channels that can regulate glutamate release. Agents that modulate BK channel activity would therefore be expected to affect cell excitability and neurotransmitter release also under pathological conditions. We have investigated the role of BK potassium channels in a model of ischemia-induced nerve cell degeneration. Organotypical slice cultures of rat hippocampus were exposed to oxygen and glucose deprivation (OGD), and cell death was assessed by the fluorescent dye propidium iodide. OGD induced cell death in the CA1 region and to a lesser extent in CA3. Treatment with the BK channel blockers, paxilline and iberiotoxin, during and after OGD induced increased cell death in CA1 and CA3. Both BK channel blockers also sensitized the relatively resistant granule cells in fascia dentata to OGD. The effect of paxilline and iberiotoxin was evident from 3 h after OGD, indicating a role of BK channels early in the post-ischemic phase or during OGD itself. The BK channel opener, NS1619, turned out to be gliotoxic, and this effect was not counteracted by paxilline and iberiotoxin. Our data show that blockade of BK channels aggravates OGD-induced cell damage and suggest that BK channels act as a kind of 'emergency brake' during and/or after ischemia. Accordingly, the BK channel is a potential molecular target for neuroprotective therapy in stroke.

  9. Phosphorylation of BK channels modulates the sensitivity to hydrogen sulfide (H2S

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    Guzel F. Sitdikova

    2014-11-01

    Full Text Available Introduction: Gases, such as nitric oxide (NO, carbon monoxide (CO or hydrogen sulfide (H2S, termed gasotransmitters, play an increasingly important role in understanding of how electrical signaling of cells is modulated. H2S is well known to act on various ion channels and receptors. In a previous study we reported that H2S increased calcium-activated potassium (BK channel activity. Aims: The goal of the present study is to investigate the modulatory effect of BK channel phosphorylation on the action of H2S on the channel as well as to recalculate and determine the H2S concentrations in aqueous sodium hydrogen sulfide (NaHS solutions.Methods: Single channel recordings of GH3, GH4 and GH4 STREX cells were used to analyze channel open probability, amplitude and open dwell times. H2S was measured with ananion selective electrode. Results: The concentration of H2S produced from NaHS was recalculated taking pH, temperature salinity of the perfusate and evaporation of H2S into account. The results indicate that from a concentration of 300 µM NaHS, only11-13%, i.e. 34-41 µM is effective as H2S in solution. GH3, GH4 and GH4 STREX cells respond differently to phosphorylation. BK channel open probability (Po of all cells lines used was increased by H2S in ATP containing solutions. PKA prevented the action of H2S on channel Po in GH4 and GH4 STREX, but not in GH3 cells. H2S, high significantly increased Po of all PKG pretreated cells. In the presence of PKC, which lowers channel activity, H2S increased channel Po of GH4 and GH4 STREX, but not those of GH3 cells. H2S increased open dwell times of GH3 cells in the absence of ATP significantly. A significant increase of dwell times with H2S was also observed in the presence of okadaic acid.Conclusions: Our results suggest that phosphorylation by PKG primes the channels for H2S activation and indicate that channel phosphorylation plays an important role in the response to H2S.

  10. Phosphorylation of BK channels modulates the sensitivity to hydrogen sulfide (H2S).

    Science.gov (United States)

    Sitdikova, Guzel F; Fuchs, Roman; Kainz, Verena; Weiger, Thomas M; Hermann, Anton

    2014-01-01

    Gases, such as nitric oxide (NO), carbon monoxide (CO), or hydrogen sulfide (H2S), termed gasotransmitters, play an increasingly important role in understanding of how electrical signaling of cells is modulated. H2S is well-known to act on various ion channels and receptors. In a previous study we reported that H2S increased calcium-activated potassium (BK) channel activity. The goal of the present study is to investigate the modulatory effect of BK channel phosphorylation on the action of H2S on the channel as well as to recalculate and determine the H2S concentrations in aqueous sodium hydrogen sulfide (NaHS) solutions. Single channel recordings of GH3, GH4, and GH4 STREX cells were used to analyze channel open probability, amplitude, and open dwell times. H2S was measured with an anion selective electrode. The concentration of H2S produced from NaHS was recalculated taking pH, temperature salinity of the perfusate, and evaporation of H2S into account. The results indicate that from a concentration of 300 μM NaHS, only 11-13%, i.e., 34-41 μM is effective as H2S in solution. GH3, GH4, and GH4 STREX cells respond differently to phosphorylation. BK channel open probability (Po) of all cells lines used was increased by H2S in ATP-containing solutions. PKA prevented the action of H2S on channel Po in GH4 and GH4 STREX, but not in GH3 cells. H2S, high significantly increased Po of all PKG pretreated cells. In the presence of PKC, which lowers channel activity, H2S increased channel Po of GH4 and GH4 STREX, but not those of GH3 cells. H2S increased open dwell times of GH3 cells in the absence of ATP significantly. A significant increase of dwell times with H2S was also observed in the presence of okadaic acid. Our results suggest that phosphorylation by PKG primes the channels for H2S activation and indicate that channel phosphorylation plays an important role in the response to H2S.

  11. Functional coupling of TRPV4 channels and BK channels in regulating spontaneous contractions of the guinea pig urinary bladder.

    Science.gov (United States)

    Isogai, Ayu; Lee, Ken; Mitsui, Retsu; Hashitani, Hikaru

    2016-09-01

    We investigated the role of TRPV4 channels (TRPV4) in regulating the contractility of detrusor smooth muscle (DSM) and muscularis mucosae (MM) of the urinary bladder. Distribution of TRPV4 in DSM and MM of guinea-pig bladders was examined by fluorescence immunohistochemistry. Changes in the contractility of DSM and MM bundles were measured using isometric tension recording. Intracellular Ca(2+) dynamics were visualized by Cal-520 fluorescent Ca(2+) imaging, while membrane potential changes were recorded using intracellular microelectrode technique. DSM and MM expressed TRPV4 immunoreactivity. GSK1016790A (GSK, 1 nM), a TRPV4 agonist, evoked a sustained contraction in both DSM and MM associated with a cessation of spontaneous phasic contractions in a manner sensitive to HC-067047 (10 μM), a TRPV4 antagonist. Iberiotoxin (100 nM) and paxilline (1 μM), large conductance Ca(2+)-activated K(+) (BK) channel blockers restored the spontaneous contractions in GSK. The sustained contractions in DSM and MM were reduced by nifedipine (10 μM), a blocker of L-type voltage-dependent Ca(2+) channels (LVDCCs) by about 40 % and by nominally Ca(2+)-free solution by some 90 %. GSK (1 nM) abolished spontaneous Ca(2+) transients, increased basal Ca(2+) levels and also prevented spontaneous action potential discharge associated with DSM membrane hyperpolarization. In conclusion, Ca(2+) influx through TRPV4 appears to activate BK channels to suppress spontaneous contractions and thus a functional coupling of TRPV4 with BK channels may act as a self-limiting mechanism for bladder contractility during its storage phase. Despite the membrane hyperpolarization in GSK, Ca(2+) entry mainly through TRPV4 develops the tonic contraction.

  12. Polyomavirus BK Neutralizing Activity in Human Immunoglobulin Preparations

    Science.gov (United States)

    Randhawa, Parmjeet S; Schonder, Kristine; Shapiro, Ron; Farasati, Nousha; Huang, Yuchen

    2011-01-01

    Background Polyomavirus BK (BKV) infection can cause nephropathy in the allograft kidney. No well-established drug treatment is available at this time. Human intravenous immunoglobulins (IVIG) have been used as an empiric therapy without proof of effectiveness. Methods We tested five lots of commercially available IVIG preparations from two different suppliers for polyomavirus neutralizing activity. BKV and mouse polyomavirus were used to infect human and murine host cells, respectively, with or without prior treatment with IVIG. Neutralization activity was measured by quantitation of viral DNA after 7 days in culture. Results Coincubation of BKV but not mouse polyomavirus with clinically relevant concentrations of IVIG derived from healthy and hepatitis B vaccinated subjects caused more than 90% inhibition of viral DNA yield after 7 days in culture. Consistent with a direct neutralizing mechanism, this effect was significantly diminished if viral infection was performed in immunoglobulin pretreated cells or if immunoglobulin treatment was delayed 2 hr after addition of infectious virus. Conclusion Human IVIG preparations contain BKV neutralizing antibodies. Data on neutralizing capacity of these antibodies are presented to aid dose exploration in clinical trials seeking to validate the use of IVIG in patients with BKV infection. PMID:20568674

  13. Recombinant Expression and Functional Characterization of Martentoxin: A Selective Inhibitor for BK Channel (α + β4

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    Jie Tao

    2014-04-01

    Full Text Available Martentoxin (MarTX, a 37-residue peptide purified from the venom of East-Asian scorpion (Buthus martensi Karsch, was capable of blocking large-conductance Ca2+-activated K+ (BK channels. Here, we report an effective expression and purification approach for this toxin. The cDNA encoding martentoxin was expressed by the prokaryotic expression system pGEX-4T-3 which was added an enterokinase cleavage site by PCR. The fusion protein (GST-rMarTX was digested by enterokinase to release hetero-expressed toxin and further purified via reverse-phase HPLC. The molecular weight of the hetero-expressed rMarTX was 4059.06 Da, which is identical to that of the natural peptide isolated from scorpion venom. Functional characterization through whole-cell patch clamp showed that rMarTX selectively and potently inhibited the currents of neuronal BK channels (α + β4 (IC50 = 186 nM, partly inhibited mKv1.3, but hardly having any significant effect on hKv4.2 and hKv3.1a even at 10 μM. Successful expression of martentoxin lays basis for further studies of structure-function relationship underlying martentoxin or other potassium-channel specific blockers.

  14. An alpha-catulin homologue controls neuromuscular function through localization of the dystrophin complex and BK channels in Caenorhabditis elegans.

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    Linu S Abraham

    2010-08-01

    Full Text Available The large conductance, voltage- and calcium-dependent potassium (BK channel serves as a major negative feedback regulator of calcium-mediated physiological processes and has been implicated in muscle dysfunction and neurological disorders. In addition to membrane depolarization, activation of the BK channel requires a rise in cytosolic calcium. Localization of the BK channel near calcium channels is therefore critical for its function. In a genetic screen designed to isolate novel regulators of the Caenorhabditis elegans BK channel, SLO-1, we identified ctn-1, which encodes an α-catulin homologue with homology to the cytoskeletal proteins α-catenin and vinculin. ctn-1 Mutants resemble slo-1 loss-of-function mutants, as well as mutants with a compromised dystrophin complex. We determined that CTN-1 uses two distinct mechanisms to localize SLO-1 in muscles and neurons. In muscles, CTN-1 utilizes the dystrophin complex to localize SLO-1 channels near L-type calcium channels. In neurons, CTN-1 is involved in localizing SLO-1 to a specific domain independent of the dystrophin complex. Our results demonstrate that CTN-1 ensures the localization of SLO-1 within calcium nanodomains, thereby playing a crucial role in muscles and neurons.

  15. Intracellular segment between transmembrane helices S0 and S1 of BK channel α subunit contains two amphipathic helices connected by a flexible loop.

    Science.gov (United States)

    Shi, Pan; Li, Dong; Lai, Chaohua; Zhang, Longhua; Tian, Changlin

    2013-08-02

    The BK channel, a tetrameric potassium channel with very high conductance, has a central role in numerous physiological functions. The BK channel can be activated by intracellular Ca(2+) and Mg(2+), as well as by membrane depolarization. Unlike other tetrameric potassium channels, the BK channel has seven transmembrane helices (S0-S6) including an extra helix S0. The intracellular segment between S0 and S1 (BK-IS1) is essential to BK channel functions and Asp99 in BK-IS1 is reported to be responsible for Mg(2+) coordination. In this study, BK-IS1 (44-113) was over-expressed using a bacterial system and purified in the presence of detergent micelles for multidimensional heteronuclear nuclear magnetic resonance (NMR) structural studies. Backbone resonance assignment and secondary structure analysis showed that BK-IS1 contains two amphipathic helices connected by a 36-residue loop. Amide (1)H-(15)N heteronuclear NOE analysis indicated that the loop is very flexible, while the two amphipathic helices are possibly stabilized through interaction with the membrane. A solution NMR-based titration assay of BK-IS1 was performed with various concentrations of Mg(2+). Two residues (Thr45 and Leu46) with chemical shift changes were observed but no, or very minor, chemical shift difference was observed for Asp99, indicating a possible site for binding divalent ions or other modulation partners.

  16. FMRP Regulates Neurotransmitter Release and Synaptic Information Transmission by Modulating Action Potential Duration via BK channels

    Science.gov (United States)

    Deng, Pan-Yue; Rotman, Ziv; Blundon, Jay A.; Cho, Yongcheol; Cui, Jianmin; Cavalli, Valeria; Zakharenko, Stanislav S.; Klyachko, Vitaly A.

    2013-01-01

    SUMMARY Loss of FMRP causes Fragile X syndrome (FXS), but the physiological functions of FMRP remain highly debatable. Here we show that FMRP regulates neurotransmitter release in CA3 pyramidal neurons by modulating action potential (AP) duration. Loss of FMRP leads to excessive AP broadening during repetitive activity, enhanced presynaptic calcium influx and elevated neurotransmitter release. The AP broadening defects caused by FMRP loss have a cell-autonomous presynaptic origin and can be acutely rescued in postnatal neurons. These presynaptic actions of FMRP are translation-independent and are mediated selectively by BK channels via interaction of FMRP with BK channel’s regulatory β4 subunits. Information-theoretical analysis demonstrates that loss of these FMRP functions causes marked dysregulation of synaptic information transmission. FMRP-dependent AP broadening is not limited to the hippocampus, but also occurs in cortical pyramidal neurons. Our results thus suggest major translation-independent presynaptic functions of FMRP that may have important implications for understanding FXS neuropathology. PMID:23439122

  17. 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.

  18. BK and Kv3.1 potassium channels control different aspects of deep cerebellar nuclear neurons action potentials and spiking activity.

    Science.gov (United States)

    Pedroarena, Christine M

    2011-12-01

    Deep cerebellar nuclear neurons (DCNs) display characteristic electrical properties, including spontaneous spiking and the ability to discharge narrow spikes at high frequency. These properties are thought to be relevant to processing inhibitory Purkinje cell input and transferring well-timed signals to cerebellar targets. Yet, the underlying ionic mechanisms are not completely understood. BK and Kv3.1 potassium channels subserve similar functions in spike repolarization and fast firing in many neurons and are both highly expressed in DCNs. Here, their role in the abovementioned spiking characteristics was addressed using whole-cell recordings of large and small putative-glutamatergic DCNs. Selective BK channel block depolarized DCNs of both groups and increased spontaneous firing rate but scarcely affected evoked activity. After adjusting the membrane potential to control levels, the spike waveforms under BK channel block were indistinguishable from control ones, indicating no significant BK channel involvement in spike repolarization. The increased firing rate suggests that lack of DCN-BK channels may have contributed to the ataxic phenotype previously found in BK channel-deficient mice. On the other hand, block of Kv3.1 channels with low doses of 4-aminopyridine (20 μM) hindered spike repolarization and severely depressed evoked fast firing. Therefore, I propose that despite similar characteristics of BK and Kv3.1 channels, they play different roles in DCNs: BK channels control almost exclusively spontaneous firing rate, whereas DCN-Kv3.1 channels dominate the spike repolarization and enable fast firing. Interestingly, after Kv3.1 channel block, BK channels gained a role in spike repolarization, demonstrating how the different function of each of the two channels is determined in part by their co-expression and interplay.

  19. Modulation of BK channels contributes to activity-dependent increase of excitability through MTORC1 activity in CA1 pyramidal cells of mouse hippocampus

    Directory of Open Access Journals (Sweden)

    Steven eSpringer

    2015-01-01

    Full Text Available Memory acquisition and synaptic plasticity are accompanied by changes in the intrinsic excitability of CA1 pyramidal neurons. These activity-dependent changes in excitability are mediated by modulation of intrinsic currents which alters the responsiveness of the cell to synaptic inputs. The afterhyperpolarization, a major contributor to the regulation of neuronal excitability, is reduced in animals that have acquired several types of hippocampus-dependent memory tasks and also following synaptic potentiation by high frequency stimulation. BK channels underlie the fast afterhyperpolarization and contribute to spike repolarization, and this afterhyperpolarization is reduced in animals that successfully acquired trace-eyeblink conditioning. This suggests that BK channel function is activity-dependent, but the mechanisms are unknown. In this study, we found that blockade of BK channels with paxilline (10µM increased spike half-width and instantaneous frequency in response to a +100pA depolarization. In addition, induction of LTP by theta burst stimulation (TBS in CA1 pyramidal neurons reduced BK channel’s contribution to spike repolarization and instantaneous frequency. This result indicates that BK channel activity is decreased following synaptic potentiation. Interestingly, blockade of mammalian target of rapamycin (MTORC1 with rapamycin (400 nM following synaptic potentiation restored BK channel function, suggesting a role for protein translation in signaling events which decreased postsynaptic BK channel activity following synaptic potentiation.

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

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    Marei Typlt

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

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

    DEFF Research Database (Denmark)

    Gruhn, Nicolai; Boesgaard, Søren; Eiberg, Jonas

    2002-01-01

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

  2. 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.

  3. Role of BKCa channels in cephalic vasodilation induced by CGRP, NO and transcranial electrical stimulation in the rat

    DEFF Research Database (Denmark)

    Gozalov, A.; Jansen-Olesen, I.; Klærke, Dan Arne;

    2007-01-01

    by the NO donor glyceryltrinitrate (GTN) or by CGRP is partially mediated via large conductance calcium-activated potassium (BK(Ca)) channels. The effects of the BK(Ca) channel selective inhibitor iberiotoxin on dural and pial vasodilation induced by CGRP, GTN and endogenously released CGRP by transcranial...... electrical stimulation (TES) were examined. Iberiotoxin significantly attenuated GTN-induced dural and pial artery dilation in vivo and in vitro, but had no effect on vasodilation induced by CGRP and TES. Our results show that GTN- but not CGRP-induced dural and pial vasodilation involves opening of BK...

  4. BK virus infection in human immunodeficiency virus-infected patients.

    Science.gov (United States)

    Ledesma, J; Muñoz, P; Garcia de Viedma, D; Cabrero, I; Loeches, B; Montilla, P; Gijon, P; Rodriguez-Sanchez, B; Bouza, E

    2012-07-01

    The aim of this study is to evaluate the prevalence of BK virus (BKV) infection in HIV-positive patients receiving highly active antiretroviral therapy (HAART) in our hospital. The presence of BKV was analysed in urine and plasma samples from 78 non-selected HIV-infected patients. Clinical data were recorded using a pre-established protocol. We used a nested PCR to amplify a specific region of the BKV T-large antigen. Positive samples were quantified using real-time PCR. Mean CD4 count in HIV-infected patients was 472 cells/mm3 and median HIV viral load was 500 cells/mm3 (74.3% vs 25.7%; p=0.007). Viruria was present in 21.7% of healthy controls (5 out of 23 samples, p=0.02). All viral loads were low (<100 copies/mL), and we could not find any association between BKV infection and renal or neurological manifestations. We provide an update on the prevalence of BKV in HIV-infected patients treated with HAART. BKV viruria was more common in HIV-infected patients; however, no role for BKV has been demonstrated in this population.

  5. Biophysical studies of the membrane location of the voltage-gated sensors in the HsapBK and KvAP K(+) channels.

    Science.gov (United States)

    Biverståhl, Henrik; Lind, Jesper; Bodor, Andrea; Mäler, Lena

    2009-09-01

    The membrane location of two fragments in two different K(+)-channels, the KvAP (from Aeropyrum pernix) and the HsapBK (human) corresponding to the putative "paddle" domains, has been investigated by CD, fluorescence and NMR spectroscopy. Both domains interact with q = 0.5 phospholipid bicelles, DHPC micelles and with POPC vesicles. CD spectra demonstrate that both peptides become largely helical in the presence of phospholipid bicelles. Fluorescence quenching studies using soluble acrylamide or lipid-attached doxyl-groups show that the arginine-rich domains are located within the bilayered region in phospholipid bicelles. Nuclear magnetic relaxation parameters, T(1) and (13)C-(1)H NOE, for DMPC in DMPC/DHPC bicelles and for DHPC in micelles showed that the lipid acyl chains in the bicelles become less flexible in the presence of either of the fragments. An even more pronounced effect is seen on the glycerol carbons. (2)H NMR spectra of magnetically aligned bicelles showed that the peptide derived from KvAP had no or little effect on bilayer order, while the peptide derived from HsapBK had the effect of lowering the order of the bilayer. The present study demonstrates that the fragments derived from the full-length proteins interact with the bilayered interior of model membranes, and that they affect both the local mobility and lipid order of model membrane systems.

  6. Occurrence of BK Virus and Human Papilloma Virus in colorectal cancer

    Directory of Open Access Journals (Sweden)

    Adrian Jarzyński

    2017-09-01

    Developing colorectal cancer can show no symptoms, even for many years. This is why it is so important to become familiar with as many etiological factors as possible. The development of many human neoplasms is often initiated by exposure to infectious agents – such as bacterial or viral infections. Similar to the human papillomavirus, the BK virus was detected in clinical specimens. It seems that HPV and BKV infections can contribute to the neoplastic process, which requires detailed studies on a larger group of patients.

  7. Astaxanthin and Docosahexaenoic Acid Reverse the Toxicity of the Maxi-K (BK) Channel Antagonist Mycotoxin Penitrem A

    Science.gov (United States)

    Goda, Amira A.; Naguib, Khayria M.; Mohamed, Magdy M.; Amra, Hassan A.; Nada, Somaia A.; Abdel-Ghaffar, Abdel-Rahman B.; Gissendanner, Chris R.; El Sayed, Khalid A.

    2016-01-01

    Penitrem A (PA) is a food mycotoxin produced by several terrestrial and few marine Penicillium species. PA is a potent tremorgen through selective antagonism of the calcium-dependent potassium BK (Maxi-K) channels. Discovery of natural products that can prevent the toxic effects of PA is important for food safety. Astaxanthin (AST) is a marine natural xanthophyll carotenoid with documented antioxidant activity. Unlike other common antioxidants, AST can cross blood brain barriers (BBBs), inducing neuroprotective effects. Docosahexaenoic acid (DHA) is polyunsaturated ω-3 fatty acid naturally occurring in fish and algae. DHA is essential for normal neurological and cellular development. This study evaluated the protective activity of AST and DHA against PA-induced toxicity, in vitro on Schwann cells CRL-2765 and in vivo in the worm Caenorhbitidis elegans and Sprague Dawley rat models. PA inhibited the viability of Schwann cells, with an IC50 of 22.6 μM. Dose-dependent treatments with 10–100 μM DHA significantly reversed the PA toxicity at its IC50 dose, and improved the survival of Schwann cells to 70.5%–98.8%. Similarly, dose-dependent treatments with 10–20 μM AST reversed the PA toxicity at its IC50 dose and raised these cells’ survival to 61.7%–70.5%. BK channel inhibition in the nematode C. elegans is associated with abnormal reversal locomotion. DHA and AST counteracted the in vivo PA BK channel antagonistic activity in the C. elegans model. Rats fed a PA-contaminated diet showed high levels of glutamate (GLU), aspartate (ASP), and gamma amino butyric acid (GABA), with observed necrosis or absence of Purkinjie neurons, typical of PA-induced neurotoxicity. Dopamine (DA), serotonin (5-HT), and norepinephrine (NE) levels were abnormal, Nitric Oxide (NO) and Malondialdehyde (MDA) levels were significantly increased, and total antioxidant capacity (TAC) level in serum and brain homogenates was significantly decreased in PA-treated rats. DHA and AST

  8. Astaxanthin and Docosahexaenoic Acid Reverse the Toxicity of the Maxi-K (BK Channel Antagonist Mycotoxin Penitrem A

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    Amira A. Goda

    2016-11-01

    Full Text Available Penitrem A (PA is a food mycotoxin produced by several terrestrial and few marine Penicillium species. PA is a potent tremorgen through selective antagonism of the calcium-dependent potassium BK (Maxi-K channels. Discovery of natural products that can prevent the toxic effects of PA is important for food safety. Astaxanthin (AST is a marine natural xanthophyll carotenoid with documented antioxidant activity. Unlike other common antioxidants, AST can cross blood brain barriers (BBBs, inducing neuroprotective effects. Docosahexaenoic acid (DHA is polyunsaturated ω-3 fatty acid naturally occurring in fish and algae. DHA is essential for normal neurological and cellular development. This study evaluated the protective activity of AST and DHA against PA-induced toxicity, in vitro on Schwann cells CRL-2765 and in vivo in the worm Caenorhbitidis elegans and Sprague Dawley rat models. PA inhibited the viability of Schwann cells, with an IC50 of 22.6 μM. Dose-dependent treatments with 10–100 μM DHA significantly reversed the PA toxicity at its IC50 dose, and improved the survival of Schwann cells to 70.5%–98.8%. Similarly, dose-dependent treatments with 10–20 μM AST reversed the PA toxicity at its IC50 dose and raised these cells’ survival to 61.7%–70.5%. BK channel inhibition in the nematode C. elegans is associated with abnormal reversal locomotion. DHA and AST counteracted the in vivo PA BK channel antagonistic activity in the C. elegans model. Rats fed a PA-contaminated diet showed high levels of glutamate (GLU, aspartate (ASP, and gamma amino butyric acid (GABA, with observed necrosis or absence of Purkinjie neurons, typical of PA-induced neurotoxicity. Dopamine (DA, serotonin (5-HT, and norepinephrine (NE levels were abnormal, Nitric Oxide (NO and Malondialdehyde (MDA levels were significantly increased, and total antioxidant capacity (TAC level in serum and brain homogenates was significantly decreased in PA-treated rats. DHA and AST

  9. Novel mechanism of hydrogen sulfide-induced guinea pig urinary bladder smooth muscle contraction: role of BK channels and cholinergic neurotransmission.

    Science.gov (United States)

    Fernandes, Vítor S; Xin, Wenkuan; Petkov, Georgi V

    2015-07-15

    Hydrogen sulfide (H2S) is a key signaling molecule regulating important physiological processes, including smooth muscle function. However, the mechanisms underlying H2S-induced detrusor smooth muscle (DSM) contractions are not well understood. This study investigates the cellular and tissue mechanisms by which H2S regulates DSM contractility, excitatory neurotransmission, and large-conductance voltage- and Ca(2+)-activated K(+) (BK) channels in freshly isolated guinea pig DSM. We used a multidisciplinary experimental approach including isometric DSM tension recordings, colorimetric ACh measurement, Ca(2+) imaging, and patch-clamp electrophysiology. In isolated DSM strips, the novel slow release H2S donor, P-(4-methoxyphenyl)-p-4-morpholinylphosphinodithioic acid morpholine salt (GYY4137), significantly increased the spontaneous phasic and nerve-evoked DSM contractions. The blockade of neuronal voltage-gated Na(+) channels or muscarinic ACh receptors with tetrodotoxin or atropine, respectively, reduced the stimulatory effect of GYY4137 on DSM contractility. GYY4137 increased ACh release from bladder nerves, which was inhibited upon blockade of L-type voltage-gated Ca(2+) channels with nifedipine. Furthermore, GYY4137 increased the amplitude of the Ca(2+) transients and basal Ca(2+) levels in isolated DSM strips. GYY4137 reduced the DSM relaxation induced by the BK channel opener, NS11021. In freshly isolated DSM cells, GYY4137 decreased the amplitude and frequency of transient BK currents recorded in a perforated whole cell configuration and reduced the single BK channel open probability measured in excised inside-out patches. GYY4137 inhibited spontaneous transient hyperpolarizations and depolarized the DSM cell membrane potential. Our results reveal the novel findings that H2S increases spontaneous phasic and nerve-evoked DSM contractions by activating ACh release from bladder nerves in combination with a direct inhibition of DSM BK channels.

  10. Slo1 tail domains, but not the Ca2+ bowl, are required for the beta 1 subunit to increase the apparent Ca2+ sensitivity of BK channels

    National Research Council Canada - National Science Library

    Qian, Xiang; Nimigean, Crina M; Niu, Xiaowei; Moss, Brenda L; Magleby, Karl L

    2002-01-01

    .... In the first, the tail domain of the alpha subunit, which includes the RCK2 (regulator of K(+) conductance) domain and Ca(2+) bowl, was replaced with the tail domain of Slo3, a BK-related channel that lacks both a Ca...

  11. Slo1 tail domains, but not the Ca2+ bowl, are required for the beta1 subunit to increase the apparent Ca2+ sensitivity of BK channels

    National Research Council Canada - National Science Library

    Xiang Qian; Crina M Nimigean; Xiaowei Niu; Brenda L Moss; Karl L Magleby

    2002-01-01

    .... In the first, the tail domain of the subunit, which includes the RCK2 (regulator of K+ conductance) domain and Ca2+ bowl, was replaced with the tail domain of Slo3, a BK-related channel that lacks both a Ca2...

  12. 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.

  13. The first transmembrane domain (TM1) of β2-subunit binds to the transmembrane domain S1 of α-subunit in BK potassium channels

    Science.gov (United States)

    Morera, Francisco J.; Alioua, Abderrahmane; Kundu, Pallob; Salazar, Marcelo; Gonzalez, Carlos; Martinez, Agustin D.; Stefani, Enrico; Toro, Ligia; Latorre, Ramon

    2012-01-01

    The BK channel is one of the most broadly expressed ion channels in mammals. In many tissues, the BK channel pore-forming α-subunit is associated to an auxiliary β-subunit that modulates the voltage- and Ca2+-dependent activation of the channel. Structural components present in β-subunits that are important for the physical association with the α-subunit are yet unknown. Here, we show through co-immunoprecipitation that the intracellular C-terminus, the second transmembrane domain (TM2) and the extracellular loop of the β2-subunit are dispensable for association with the α-subunit pointing transmembrane domain 1 (TM1) as responsible for the interaction. Indeed, the TOXCAT assay for transmembrane protein–protein interactions demonstrated for the first time that TM1 of the β2-subunit physically binds to the transmembrane S1 domain of the α-subunit. PMID:22710124

  14. Martentoxin: A unique ligand of BK channels%Martentoxin:一种大电导钙激活钾离子通道的独有配体

    Institute of Scientific and Technical Information of China (English)

    陶杰; 施健; 刘志睿; 吉永华

    2012-01-01

    The large-conductance calcium-activated potassium (BK) channels distributed in both excitable and non-excitable cells are key participants in a variety of physiological functions.By employing numerous high-affinity natural toxins originated from scorpion venoms the pharmacological and structural characteristics of these channels tend to be approached A 37-residue short-chain peptide,named as martentoxin,arising from the venom of the East-Asian scorpion (Buthus martensi Karsch) has been investigated with a comparatively higher preference for BK channels over other voltage-gated potassium (Kv) channels.Up to now,since the specific drug tool probing for clarifying structure-function of BK channel subtypes and related pathology remain scarce,it is of importance to illuminate the underlying mechanism of molecular interaction between martentoxin and BK channels.As for it,the current review will address the recent progress on the studies of pharmacological characterizations and molecular determinants of martentoxin targeting on BK channels.%大电导钙激活钾离子(BK)通道广泛分布于可兴奋细胞与非兴奋细胞中,行使着一系列重要的生理功能.以源于蝎粗毒的高亲和性毒素作为研究工具,使BK通道的药理学和结构性质正逐步被揭示.Martentoxin是一种分离提取自东亚短钳蝎(Buthus martensi Karsch)粗毒的短链多肽,由37个氨基酸残基构成.研究表明,其对BK通道的特异性远高于其它各类型的电压门控钾通道(Kv).迄今为止,由于用以探明BK通道亚型结构与功能及相关病理的特异性药物工具仍然稀缺,因此阐明martentoxin与BK通道间的相互作用模式就显得至关重要了.鉴于此原因,本综述将针对martentoxin的药理性质和其与BK通道相互作用的分子机制做进一步阐明.

  15. Gentamicin Blocks the ACh-Induced BK Current in Guinea Pig Type II Vestibular Hair Cells by Competing with Ca2+ at the l-Type Calcium Channel

    Directory of Open Access Journals (Sweden)

    Hong Yu

    2014-04-01

    Full Text Available Type II vestibular hair cells (VHCs II contain big-conductance Ca2+-dependent K+ channels (BK and L-type calcium channels. Our previous studies in guinea pig VHCs II indicated that acetylcholine (ACh evoked the BK current by triggering the influx of Ca2+ ions through l-type Ca2+ channels, which was mediated by M2 muscarinic ACh receptor (mAChRs. Aminoglycoside antibiotics, such as gentamicin (GM, are known to have vestibulotoxicity, including damaging effects on the efferent nerve endings on VHCs II. This study used the whole-cell patch clamp technique to determine whether GM affects the vestibular efferent system at postsynaptic M2-mAChRs or the membrane ion channels. We found that GM could block the ACh-induced BK current and that inhibition was reversible, voltage-independent, and dose-dependent with an IC50 value of 36.3 ± 7.8 µM. Increasing the ACh concentration had little influence on GM blocking effect, but increasing the extracellular Ca2+ concentration ([Ca2+]o could antagonize it. Moreover, 50 µM GM potently blocked Ca2+ currents activated by (--Bay-K8644, but did not block BK currents induced by NS1619. These observations indicate that GM most likely blocks the M2 mAChR-mediated response by competing with Ca2+ at the l-type calcium channel. These results provide insights into the vestibulotoxicity of aminoglycoside antibiotics on mammalian VHCs II.

  16. 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.

  17. Human BK Polyomavirus—The Potential for Head and Neck Malignancy and Disease

    Directory of Open Access Journals (Sweden)

    Raquel Burger-Calderon

    2015-07-01

    Full Text Available Members of the human Polyomaviridae family are ubiquitous and pathogenic among immune-compromised individuals. While only Merkel cell polyomavirus (MCPyV has conclusively been linked to human cancer, all members of the polyomavirus (PyV family encode the oncoprotein T antigen and may be potentially carcinogenic. Studies focusing on PyV pathogenesis in humans have become more abundant as the number of PyV family members and the list of associated diseases has expanded. BK polyomavirus (BKPyV in particular has emerged as a new opportunistic pathogen among HIV positive individuals, carrying harmful implications. Increasing evidence links BKPyV to HIV-associated salivary gland disease (HIVSGD. HIVSGD is associated with elevated risk of lymphoma formation and its prevalence has increased among HIV/AIDS patients. Determining the relationship between BKPyV, disease and tumorigenesis among immunosuppressed individuals is necessary and will allow for expanding effective anti-viral treatment and prevention options in the future.

  18. Molecular mechanisms underlying the effect of the novel BK channel opener GoSlo: involvement of the S4/S5 linker and the S6 segment.

    Science.gov (United States)

    Webb, Timothy I; Kshatri, Aravind Singh; Large, Roddy J; Akande, Adebola Morayo; Roy, Subhrangsu; Sergeant, Gerard P; McHale, Noel G; Thornbury, Keith D; Hollywood, Mark A

    2015-02-17

    GoSlo-SR-5-6 is a novel large-conductance Ca(2+)-activated K(+) (BK) channel agonist that shifts the activation V1/2 of these channels in excess of -100 mV when applied at a concentration of 10 μM. Although the structure-activity relationship of this family of molecules has been established, little is known about how they open BK channels. To help address this, we used a combination of electrophysiology, mutagenesis, and mathematical modeling to investigate the molecular mechanisms underlying the effect of GoSlo-SR-5-6. Our data demonstrate that the effects of this agonist are practically abolished when three point mutations are made: L227A in the S4/S5 linker in combination with S317R and I326A in the S6C region. Our data suggest that GoSlo-SR-5-6 interacts with the transmembrane domain of the channel to enhance pore opening. The Horrigan-Aldrich model suggests that GoSlo-SR-5-6 works by stabilizing the open conformation of the channel and the activated state of the voltage sensors, yet decouples the voltage sensors from the pore gate.

  19. Role of the BK channel (KCa1.1) during activation of electrogenic K+ secretion in guinea pig distal colon.

    Science.gov (United States)

    Zhang, Jin; Halm, Susan T; Halm, Dan R

    2012-12-15

    Secretagogues acting at a variety of receptor types activate electrogenic K(+) secretion in guinea pig distal colon, often accompanied by Cl(-) secretion. Distinct blockers of K(Ca)1.1 (BK, Kcnma1), iberiotoxin (IbTx), and paxilline inhibited the negative short-circuit current (I(sc)) associated with K(+) secretion. Mucosal addition of IbTx inhibited epinephrine-activated I(sc) ((epi)I(sc)) and transepithelial conductance ((epi)G(t)) consistent with K(+) secretion occurring via apical membrane K(Ca)1.1. The concentration dependence of IbTx inhibition of (epi)I(sc) yielded an IC(50) of 193 nM, with a maximal inhibition of 51%. Similarly, IbTx inhibited (epi)G(t) with an IC(50) of 220 nM and maximal inhibition of 48%. Mucosally added paxilline (10 μM) inhibited (epi)I(sc) and (epi)G(t) by ∼50%. IbTx and paxilline also inhibited I(sc) activated by mucosal ATP, supporting apical K(Ca)1.1 as a requirement for this K(+) secretagogue. Responses to IbTx and paxilline indicated that a component of K(+) secretion occurred during activation of Cl(-) secretion by prostaglandin-E(2) and cholinergic stimulation. Analysis of K(Ca)1.1α mRNA expression in distal colonic epithelial cells indicated the presence of the ZERO splice variant and three splice variants for the COOH terminus. The presence of the regulatory β-subunits K(Ca)β1 and K(Ca)β4 also was demonstrated. Immunolocalization supported the presence of K(Ca)1.1α in apical and basolateral membranes of surface and crypt cells. Together these results support a cellular mechanism for electrogenic K(+) secretion involving apical membrane K(Ca)1.1 during activation by several secretagogue types, but the observed K(+) secretion likely required the activity of additional K(+) channel types in the apical membrane.

  20. Resveratrol inhibits BK-induced COX-2 transcription by suppressing acetylation of AP-1 and NF-κB in human rheumatoid arthritis synovial fibroblasts.

    Science.gov (United States)

    Yang, Chuen-Mao; Chen, Yu-Wen; Chi, Pei-Ling; Lin, Chih-Chung; Hsiao, Li-Der

    2017-05-15

    Bradykinin (BK) induces inflammation in rheumatoid arthritis (RA). Resveratrol is a potent activator of Sirt1 which could modulate inflammation through deacetylating histones of transcription factors. Here, we investigated the mechanisms underlying BK-induced COX-2 expression which is modulated by resveratrol/Sirt1 in human rheumatoid arthritis synovial fibroblasts (RASFs). We found that BK-induced COX-2 protein and mRNA expression associated with PGE2 synthesis, and promoter activity was mediated through B2R receptors, which were attenuated by selective B2R antagonist Hoe140 or transfection with B2R siRNA. BK-induced responses were mediated through PKCμ, MAPKs, AP-1 and NF-κB which were inhibited by their respective inhibitors or siRNAs. Up-regulation of Sirt1 by resveratrol suppressed the BK-induced COX-2/PGE2 production through inhibiting the interaction of AP-1 and NF-κB with COX-2 promoter in RASFs. BK-induced COX-2/PGE2 expression is mediated through a B2R-PKCμ-dependent MAPKs, AP-1, and NF-κB cascade. Resveratrol inhibited the phosphorylation and acetylation of p65, c-Jun, and Fos and reduced the binding to the COX-2 promoter, thereby attenuated the COX-2 expression. Therefore, resveratrol may be a promising therapeutic intervention for treatment of inflammatory arthritis. Copyright © 2017 Elsevier Inc. All rights reserved.

  1. The role of potassium BK channels in anticonvulsant effect of cannabidiol in pentylenetetrazole and maximal electroshock models of seizure in mice.

    Science.gov (United States)

    Shirazi-zand, Zahra; Ahmad-Molaei, Leila; Motamedi, Fereshteh; Naderi, Nima

    2013-07-01

    Cannabidiol is a nonpsychoactive member of phytocannabinoids that produces various pharmacological effects that are not mediated through putative CB1/CB2 cannabinoid receptors and their related effectors. In this study, we examined the effect of the i.c.v. administration of potassium BK channel blocker paxilline alone and in combination with cannabidiol in protection against pentylenetetrazol (PTZ)- and maximal electroshock (MES)-induced seizure in mice. In the PTZ-induced seizure model, i.c.v. administration of cannabidiol caused a significant increase in seizure threshold compared with the control group. Moreover, while i.c.v. administration of various doses of paxilline did not produce significant change in the PTZ-induced seizure threshold in mice, coadministration of cannabidiol and paxilline attenuated the antiseizure effect of cannabidiol in PTZ-induced tonic seizures. In the MES model of seizure, both cannabidiol and paxilline per se produced significant increase in percent protection against electroshock-induced seizure. However, coadministration of cannabidiol and paxilline did not produce significant interaction in their antiseizure effect in the MES test. The results of the present study showed a protective effect of cannabidiol in both PTZ and MES models of seizure. These results suggested a BK channel-mediated antiseizure action of cannabidiol in PTZ model of seizure. However, such an interaction might not exist in MES-induced convulsion.

  2. BKCa and KV channels limit conducted vasomotor responses in rat mesenteric terminal arterioles

    DEFF Research Database (Denmark)

    Hald, Bjørn Olav; Jacobsen, Jens Christian Brings; Braunstein, Thomas Hartig

    2012-01-01

    smooth muscle cells (VSMC) simulations of both membrane potential and intracellular [Ca(2+)] were performed. The "characteristic" length constant, ¿, was approximated using a modified cable equation in both experiments and simulations. We hypothesized that K(+) conductance in the arteriolar wall limit......Intracellular Ca(2+) signals underlying conducted vasoconstriction to local application of a brief depolarizing KCl stimulus was investigated in rat mesenteric terminal arterioles (cells (EC) and vascular...... the electrotonic spread of a local depolarization along arterioles by current dissipation across the VSMC plasma membrane. Thus, we anticipated an increased ¿ by inhibition of voltage-activated K(+) channels. Application of the BK(Ca) channel blocker iberiotoxin (100 nM) onto mesenteric arterioles in vitro...

  3. Hydrophobic interaction between contiguous residues in the S6 transmembrane segment acts as a stimuli integration node in the BK channel

    Science.gov (United States)

    Carrasquel-Ursulaez, Willy; Contreras, Gustavo F.; Sepúlveda, Romina V.; Aguayo, Daniel; González-Nilo, Fernando

    2015-01-01

    Large-conductance Ca2+- and voltage-activated K+ channel (BK) open probability is enhanced by depolarization, increasing Ca2+ concentration, or both. These stimuli activate modular voltage and Ca2+ sensors that are allosterically coupled to channel gating. Here, we report a point mutation of a phenylalanine (F380A) in the S6 transmembrane helix that, in the absence of internal Ca2+, profoundly hinders channel opening while showing only minor effects on the voltage sensor active–resting equilibrium. Interpretation of these results using an allosteric model suggests that the F380A mutation greatly increases the free energy difference between open and closed states and uncouples Ca2+ binding from voltage sensor activation and voltage sensor activation from channel opening. However, the presence of a bulky and more hydrophobic amino acid in the F380 position (F380W) increases the intrinsic open–closed equilibrium, weakening the coupling between both sensors with the pore domain. Based on these functional experiments and molecular dynamics simulations, we propose that F380 interacts with another S6 hydrophobic residue (L377) in contiguous subunits. This pair forms a hydrophobic ring important in determining the open–closed equilibrium and, like an integration node, participates in the communication between sensors and between the sensors and pore. Moreover, because of its effects on open probabilities, the F380A mutant can be used for detailed voltage sensor experiments in the presence of permeant cations. PMID:25548136

  4. Orientations and proximities of the extracellular ends of transmembrane helices S0 and S4 in open and closed BK potassium channels.

    Directory of Open Access Journals (Sweden)

    Xiaowei Niu

    Full Text Available The large-conductance potassium channel (BK α subunit contains a transmembrane (TM helix S0 preceding the canonical TM helices S1 through S6. S0 lies between S4 and the TM2 helix of the regulatory β1 subunit. Pairs of Cys were substituted in the first helical turns in the membrane of BK α S0 and S4 and in β1 TM2. One such pair, W22C in S0 and W203C in S4, was 95% crosslinked endogenously. Under voltage-clamp conditions in outside-out patches, this crosslink was reduced by DTT and reoxidized by a membrane-impermeant bis-quaternary ammonium derivative of diamide. The rate constants for this reoxidation were not significantly different in the open and closed states of the channel. Thus, these two residues are approximately equally close in the two states. In addition, 90% crosslinking of a second pair, R20C in S0 and W203C in S4, had no effect on the V50 for opening. Taken together, these findings indicate that separation between residues at the extracellular ends of S0 and S4 is not required for voltage-sensor activation. On the contrary, even though W22C and W203C were equally likely to form a disulfide in the activated and deactivated states, relative immobilization by crosslinking of these two residues favored the activated state. Furthermore, the efficiency of recrosslinking of W22C and W203C on the cell surface was greater in the presence of the β1 subunit than in its absence, consistent with β1 acting through S0 to stabilize its immobilization relative to α S4.

  5. Membrane-perturbing properties of two Arg-rich paddle domains from voltage-gated sensors in the KvAP and HsapBK K(+) channels.

    Science.gov (United States)

    Unnerståle, Sofia; Madani, Fatemeh; Gräslund, Astrid; Mäler, Lena

    2012-05-15

    Voltage-gated K(+) channels are gated by displacement of basic residues located in the S4 helix that together with a part of the S3 helix, S3b, forms a "paddle" domain, whose position is altered by changes in the membrane potential modulating the open probability of the channel. Here, interactions between two paddle domains, KvAPp from the K(v) channel from Aeropyrum pernix and HsapBKp from the BK channel from Homo sapiens, and membrane models have been studied by spectroscopy. We show that both paddle domains induce calcein leakage in large unilamellar vesicles, and we suggest that this leakage represents a general thinning of the bilayer, making movement of the whole paddle domain plausible. The fact that HsapBKp induces more leakage than KvAPp may be explained by the presence of a Trp residue in HsapBKp. Trp residues generally promote localization to the hydrophilic-hydrophobic interface and disturb tight packing. In magnetically aligned bicelles, KvAPp increases the level of order along the whole acyl chain, while HsapBKp affects the morphology, also indicating that KvAPp adapts more to the lipid environment. Nuclear magnetic resonance (NMR) relaxation measurements for HsapBKp show that overall the sequence has anisotropic motions. The S4 helix is well-structured with restricted local motion, while the turn between S4 and S3b is more flexible and undergoes slow local motion. Our results indicate that the calcein leakage is related to the flexibility in this turn region. A possibility by which HsapBKp can undergo structural transitions is also shown by relaxation NMR, which may be important for the gating mechanism.

  6. Regulation of Guinea Pig Detrusor Smooth Muscle Excitability by 17β-Estradiol: The Role of the Large Conductance Voltage- and Ca2+-Activated K+ Channels.

    Science.gov (United States)

    Provence, Aaron; Hristov, Kiril L; Parajuli, Shankar P; Petkov, Georgi V

    2015-01-01

    Estrogen replacement therapies have been suggested to be beneficial in alleviating symptoms of overactive bladder. However, the precise regulatory mechanisms of estrogen in urinary bladder smooth muscle (UBSM) at the cellular level remain unknown. Large conductance voltage- and Ca2+-activated K+ (BK) channels, which are key regulators of UBSM function, are suggested to be non-genomic targets of estrogens. This study provides an electrophysiological investigation into the role of UBSM BK channels as direct targets for 17β-estradiol, the principle estrogen in human circulation. Single BK channel recordings on inside-out excised membrane patches and perforated whole cell patch-clamp were applied in combination with the BK channel selective inhibitor paxilline to elucidate the mechanism of regulation of BK channel activity by 17β-estradiol in freshly-isolated guinea pig UBSM cells. 17β-Estradiol (100 nM) significantly increased the amplitude of depolarization-induced whole cell steady-state BK currents and the frequency of spontaneous transient BK currents in freshly-isolated UBSM cells. The increase in whole cell BK currents by 17β-estradiol was eliminated upon blocking BK channels with paxilline. 17β-Estradiol (100 nM) significantly increased (~3-fold) the single BK channel open probability, indicating direct 17β-estradiol-BK channel interactions. 17β-Estradiol (100 nM) caused a significant hyperpolarization of the membrane potential of UBSM cells, and this hyperpolarization was reversed by blocking the BK channels with paxilline. 17β-Estradiol (100 nM) had no effects on L-type voltage-gated Ca2+ channel currents recorded under perforated patch-clamp conditions. This study reveals a new regulatory mechanism in the urinary bladder whereby BK channels are directly activated by 17β-estradiol to reduce UBSM cell excitability.

  7. TRP Channels in Human Prostate

    Directory of Open Access Journals (Sweden)

    Carl Van Haute

    2010-01-01

    Full Text Available This review gives an overview of morphological and functional characteristics in the human prostate. It will focus on the current knowledge about transient receptor potential (TRP channels expressed in the human prostate, and their putative role in normal physiology and prostate carcinogenesis. Controversial data regarding the expression pattern and the potential impact of TRP channels in prostate function, and their involvement in prostate cancer and other prostate diseases, will be discussed.

  8. Stimulation of large-conductance calcium-activated potassium channels inhibits neurogenic contraction of human bladder from patients with urinary symptoms and reverses acetic acid-induced bladder hyperactivity in rats.

    Science.gov (United States)

    La Fuente, José M; Fernández, Argentina; Cuevas, Pedro; González-Corrochano, Rocío; Chen, Mao Xiang; Angulo, Javier

    2014-07-15

    We have analysed the effects of large-conductance calcium-activated potassium channel (BK) stimulation on neurogenic and myogenic contraction of human bladder from healthy subjects and patients with urinary symptoms and evaluated the efficacy of activating BK to relief bladder hyperactivity in rats. Bladder specimens were obtained from organ donors and from men with benign prostatic hyperplasia (BPH). Contractions elicited by electrical field stimulation (EFS) and carbachol (CCh) were evaluated in isolated bladder strips. in vivo cystometric recordings were obtained in anesthetized rats under control and acetic acid-induced hyperactive conditions. Neurogenic contractions of human bladder were potentiated by blockade of BK and small-conductance calcium-activated potassium channels (SK) but were unaffected by the blockade of intermediate calcium-activated potassium channels (IK). EFS-induced contractions were inhibited by BK stimulation with NS-8 or NS1619 or by SK/IK stimulation with NS309 (3µM). CCh-induced contractions were not modified by blockade or stimulation of BK, IK or SK. The anti-cholinergic agent, oxybutynin (0.3µM) inhibited either neurogenic or CCh-induced contractions. Neurogenic contractions of bladders from BPH patients were less sensitive to BK inhibition and more sensitive to BK activation than healthy bladders. The BK activator, NS-8 (5mg/kg; i.v.), reversed bladder hyperactivity induced by acetic acid in rats, while oxybutynin was ineffective. NS-8 did not significantly impact blood pressure or heart rate. BK stimulation specifically inhibits neurogenic contractions in patients with urinary symptoms and relieves bladder hyperactivity in vivo without compromising bladder contractile capacity or cardiovascular safety, supporting its potential therapeutic use for relieving bladder overactivity.

  9. Archaerhodopsin voltage imaging: synaptic calcium and BK channels stabilize action potential repolarization at the Drosophila neuromuscular junction.

    Science.gov (United States)

    Ford, Kevin J; Davis, Graeme W

    2014-10-29

    The strength and dynamics of synaptic transmission are determined, in part, by the presynaptic action potential (AP) waveform at the nerve terminal. The ion channels that shape the synaptic AP waveform remain essentially unknown for all but a few large synapses amenable to electrophysiological interrogation. The Drosophila neuromuscular junction (NMJ) is a powerful system for studying synaptic biology, but it is not amenable to presynaptic electrophysiology. Here, we demonstrate that Archaerhodopsin can be used to quantitatively image AP waveforms at the Drosophila NMJ without disrupting baseline synaptic transmission or neuromuscular development. It is established that Shaker mutations cause a dramatic increase in neurotransmitter release, suggesting that Shaker is predominantly responsible for AP repolarization. Here we demonstrate that this effect is caused by a concomitant loss of both Shaker and slowpoke (slo) channel activity because of the low extracellular calcium concentrations (0.2-0.5 mM) used typically to assess synaptic transmission in Shaker. In contrast, at physiological extracellular calcium (1.5 mM), the role of Shaker during AP repolarization is limited. We then provide evidence that calcium influx through synaptic CaV2.1 channels and subsequent recruitment of Slo channel activity is important, in concert with Shaker, to ensure proper AP repolarization. Finally, we show that Slo assumes a dominant repolarizing role during repetitive nerve stimulation. During repetitive stimulation, Slo effectively compensates for Shaker channel inactivation, stabilizing AP repolarization and limiting neurotransmitter release. Thus, we have defined an essential role for Slo channels during synaptic AP repolarization and have revised our understanding of Shaker channels at this model synapse.

  10. Temporal and spatial mouse brain expression of cereblon, an ionic channel regulator involved in human intelligence.

    Science.gov (United States)

    Higgins, Joseph J; Tal, Adit L; Sun, Xiaowei; Hauck, Stefanie C R; Hao, Jin; Kosofosky, Barry E; Rajadhyaksha, Anjali M

    2010-03-01

    A mild form of autosomal recessive, nonsyndromal intellectual disability (ARNSID) in humans is caused by a homozygous nonsense mutation in the cereblon gene (mutCRBN). Rodent crbn protein binds to the intracellular C-terminus of the large conductance Ca(2+)-activated K(+)channel (BK(Ca)). An mRNA variant (human SITE 2 INSERT or mouse strex) of the BK(Ca) gene (KCNMA1) that is normally expressed during embryonic development is aberrantly expressed in mutCRBN human lymphoblastoid cell lines (LCLs) as compared to wild-type (wt) LCLs. The present study analyzes the temporal and spatial distribution of crbn and kcnma1 mRNAs in the mouse brain by the quantitative real-time reverse transcriptase-polymerase chain reaction (qPCR). The spatial expression pattern of endogenous and exogenous crbn proteins is characterized by immunostaining. The results show that neocortical (CTX) crbn and kcnma1 mRNA expression increases from embryonic stages to adulthood. The strex mRNA variant is >3.5-fold higher in embryos and decreases rapidly postnatally. Mouse crbn mRNA is abundant in the cerebellum (CRBM), with less expression in the CTX, hippocampus (HC), and striatum (Str) in adult mice. The intracytoplasmic distribution of endogenous crbn protein in the mouse CRBM, CTX, HC, and Str is similar to the immunostaining pattern described previously for the BK(Ca) channel. Exogenous hemagglutinin (HA) epitope-tagged human wt- and mutCRBN proteins using cDNA transfection in HEK293T cell lines showed the same intracellular expression distribution as endogenous mouse crbn protein. The results suggest that mutCRBN may cause ARNSID by disrupting the developmental regulation of BK(Ca) in brain regions that are critical for memory and learning.

  11. Expression of BK Ca channels and the modulatory beta-subunits in the rat and porcine trigeminal ganglion

    DEFF Research Database (Denmark)

    Johansson, Helle Wulf; Hay-Schmidt, Anders; Poulsen, Asser Nyander

    2009-01-01

    (Ca) channel protein was visualized by western blotting and histochemistry. The presence of the modulatory beta1-beta 4 subunit mRNAs was investigated using RT-PCR. beta1-, beta2- and beta 4-subunit mRNAs were expressed in rat TG whereas beta2- and beta 4-subunits were detected in porcine TG. Western blotting...

  12. 1-O-hexadecyloxypropyl cidofovir (CMX001) effectively inhibits polyomavirus BK replication in primary human renal tubular epithelial cells.

    Science.gov (United States)

    Rinaldo, Christine Hanssen; Gosert, Rainer; Bernhoff, Eva; Finstad, Solrun; Hirsch, Hans H

    2010-11-01

    Antiviral drugs for treating polyomavirus BK (BKV) replication in polyomavirus-associated nephropathy or hemorrhagic cystitis are of considerable clinical interest. Unlike cidofovir, the lipid conjugate 1-O-hexadecyloxypropyl cidofovir (CMX001) is orally available and has not caused detectable nephrotoxicity in rodent models or human studies to date. Primary human renal proximal tubular epithelial cells were infected with BKV-Dunlop, and CMX001 was added 2 h postinfection (hpi). The intracellular and extracellular BKV DNA load was determined by quantitative PCR. Viral gene expression was examined by quantitative reverse transcription-PCR, Western blotting, and immunofluorescence microscopy. We also examined host cell viability, proliferation, metabolic activity, and DNA replication. The titration of CMX001 identified 0.31 μM as the 90% effective concentration (EC(90)) for reducing the extracellular BKV load at 72 hpi. BKV large T antigen mRNA and protein expression was unaffected at 24 hpi, but the intracellular BKV genome was reduced by 90% at 48 hpi. Late gene expression was reduced by 70 and 90% at 48 and 72 hpi, respectively. Comparisons of CMX001 and cidofovir EC(90)s from 24 to 96 hpi demonstrated that CMX001 had a more rapid and enduring effect on BKV DNA and infectious progeny at 96 hpi than cidofovir. CMX001 at 0.31 μM had little effect on overall cell metabolism but reduced bromodeoxyuridine incorporation and host cell proliferation by 20 to 30%, while BKV infection increased cell proliferation in both rapidly dividing and near-confluent cultures. We conclude that CMX001 inhibits BKV replication with a longer-lasting effect than cidofovir at 400× lower levels, with fewer side effects on relevant host cells in vitro.

  13. Single Channel Recordings Reveal Differential β2 Subunit Modulations Between Mammalian and Drosophila BKCa(β2) Channels

    Science.gov (United States)

    Zhong, Ling; Guo, Xiying; Weng, Anxi; Xiao, Feng; Zeng, Wenping; Zhang, Yan; Ding, Jiuping; Hou, Panpan

    2016-01-01

    Large-conductance Ca2+- and voltage-activated potassium (BK) channels are widely expressed in tissues. As a voltage and calcium sensor, BK channels play significant roles in regulating the action potential frequency, neurotransmitter release, and smooth muscle contraction. After associating with the auxiliary β2 subunit, mammalian BK(β2) channels (mouse or human Slo1/β2) exhibit enhanced activation and complete inactivation. However, how the β2 subunit modulates the Drosophila Slo1 channel remains elusive. In this study, by comparing the different functional effects on heterogeneous BK(β2) channel, we found that Drosophila Slo1/β2 channel exhibits “paralyzed”-like and incomplete inactivation as well as slow activation. Further, we determined three different modulations between mammalian and Drosophila BK(β2) channels: 1) dSlo1/β2 doesn’t have complete inactivation. 2) β2(K33,R34,K35) delays the dSlo1/Δ3-β2 channel activation. 3) dSlo1/β2 channel has enhanced pre-inactivation than mSlo1/β2 channel. The results in our study provide insights into the different modulations of β2 subunit between mammalian and Drosophila Slo1/β2 channels and structural basis underlie the activation and pre-inactivation of other BK(β) complexes. PMID:27755549

  14. Computed Tomography Findings of Human Polyomavirus BK (BKV)-Associated Cystitis in Allogeneic Hematopoietic Stem Cell Transplant Recipients

    Energy Technology Data Exchange (ETDEWEB)

    Schulze, M.; Beck, R.; Igney, A.; Vogel, M.; Maksimovic, O.; Claussen, C.D.; Faul, C.; Horger, M. [Dept. of Diagnostic Radiology, Dept. of Internal Medicine-Oncology, and Inst. of Medical Virology, Eberhard-Karls Univ., Tbingen (Germany)

    2008-12-15

    Background: Over 70% of the general population worldwide is positive for antibodies against polyomavirus hominis type 1 (BKV). Polyomavirus can be reactivated in immunocompromised patients and thereby induce urogenital tract infection, including cystitis. Purpose: To describe the computed tomography (CT) findings of human polyomavirus-induced cystitis in adult patients after allogeneic hematopoietic stem cell transplantation (allogeneic HCT). Material and Methods: The study population was a retrospective cohort of 11 consecutive adult patients (eight men, three women; age range 22-59 years, mean 42.9 years) who received allogeneic HCT between December 2003 and December 2007 and were tested positive for urinary BKV infection. All CT scans were evaluated with regard to bladder wall thickness, mucosal enhancement, distinct layering of thickened bladder wall, and presence of intravesical clots, perivesical stranding as well as attenuation values of intravesical urine. Clinical data concerning transplant and conditioning regimen variables and laboratory parameters were correlated with degree and extent of imaging findings. Results: All patients had clinical signs of cystitis with different degrees of thickening of the urinary bladder wall. Well-delineated urinary bladder layers were present in six patients. Thickening of the urinary bladder wall was continuous in nine of 11 patients. Increased attenuation of intravesical urine was found in seven patients with hemorrhagic cystitis. Four patients had intraluminal clots. Perivesical stranding was not a major CT finding, occurring in a mild fashion in three of 11 patients. The clinical classification of hemorrhagic cystitis did not correlate with the analyzed imaging parameters. Patient outcome was not influenced by this infectious complication. Conclusion: CT findings in patients with polyomavirus BK cystitis consist of different degrees of bladder wall thickening usually with good delineation of all mural layers and

  15. Computed Tomography Findings of Human Polyomavirus BK (BKV)-Associated Cystitis in Allogeneic Hematopoietic Stem Cell Transplant Recipients

    Energy Technology Data Exchange (ETDEWEB)

    Schulze, M.; Beck, R.; Igney, A.; Vogel, M.; Maksimovic, O.; Claussen, C.D.; Faul, C.; Horger, M. (Dept. of Diagnostic Radiology, Dept. of Internal Medicine-Oncology, and Inst. of Medical Virology, Eberhard-Karls Univ., Tbingen (Germany))

    2008-12-15

    Background: Over 70% of the general population worldwide is positive for antibodies against polyomavirus hominis type 1 (BKV). Polyomavirus can be reactivated in immunocompromised patients and thereby induce urogenital tract infection, including cystitis. Purpose: To describe the computed tomography (CT) findings of human polyomavirus-induced cystitis in adult patients after allogeneic hematopoietic stem cell transplantation (allogeneic HCT). Material and Methods: The study population was a retrospective cohort of 11 consecutive adult patients (eight men, three women; age range 22-59 years, mean 42.9 years) who received allogeneic HCT between December 2003 and December 2007 and were tested positive for urinary BKV infection. All CT scans were evaluated with regard to bladder wall thickness, mucosal enhancement, distinct layering of thickened bladder wall, and presence of intravesical clots, perivesical stranding as well as attenuation values of intravesical urine. Clinical data concerning transplant and conditioning regimen variables and laboratory parameters were correlated with degree and extent of imaging findings. Results: All patients had clinical signs of cystitis with different degrees of thickening of the urinary bladder wall. Well-delineated urinary bladder layers were present in six patients. Thickening of the urinary bladder wall was continuous in nine of 11 patients. Increased attenuation of intravesical urine was found in seven patients with hemorrhagic cystitis. Four patients had intraluminal clots. Perivesical stranding was not a major CT finding, occurring in a mild fashion in three of 11 patients. The clinical classification of hemorrhagic cystitis did not correlate with the analyzed imaging parameters. Patient outcome was not influenced by this infectious complication. Conclusion: CT findings in patients with polyomavirus BK cystitis consist of different degrees of bladder wall thickening usually with good delineation of all mural layers and

  16. WNK4 kinase-mediated inhibitory effect on expression of BK channel via lysosomal pathway%WNK4激酶通过溶酶体途径抑制BK通道表达

    Institute of Scientific and Technical Information of China (English)

    庄捷秋; 王德选; 张益前; 牛伟辉; 陈方旋; 施珍; 潘殊方; 谷定英

    2012-01-01

    Objective To investigate the mechanism underlying the WNK4 kinasemediated inhibitory effect on BK channel. Methods Cos-7 cells were cotransfected with BK in combination with either CD4 (control group) or wild type WNK4 (WNK4-WT).Immunostaining and confocal microscopy,chemiluminescence,Western blotting analysis were then employed to determine the BK localization in cells,BK surface expression and total protein level,respectively.To further investigate whether the reduction of BK protein expression is due to an increase in degradation through a lysosomal pathway,BK protein level was determined after treated with bafilomycin A1(Baf A1),a proton pump inhibitor affecting lysosomal degradation. Results Immunostaining and confocal microscopic study showed that BK was localized both in plasma membrane and cytosol in the control group.After cells transfected with WNK4-WT,BK expression was markedly reduced.Chemiluminescent assay found that BK surface expression level was 299.9±18.6 in the control group,whereas it was significantly reduced (148.4±13.7,P<0.01) in the WNK4-WT group.Western blotting analysis showed that total BK protein level was markedly reduced in the presence of WNK4-WT compared to the control group.WNK4-WT was shown to significantly reduce the BK total protein level (42.3%±15.2%) compared to the control group (100%) (P<0.01).When the cells was treated with Bafilomycin A1 (Baf A1,0.5 μmol/L),WNK4-mediated reduction in BK protein was reversed (82.2%±12.1%,P<0.05). Conclusions WNK4 inhibits total and surface protein expression of BK in Cos-7 cells whick is likely due to an increase in BK degradation through a lysosomal pathway.%目的 研究WNK4激酶对BK通道的调节作用及机制.方法 将BK和WNK4野生型(WNK4-WT)或CD4(对照)质粒DNA共同转染进Cos-7细胞中,采用免疫染色-共聚焦激光显微镜、化学发光法、Western印迹法检测BK在细胞上的分布、细胞膜表面蛋白和总蛋

  17. Contribution of SK and BK channels in the control of catecholamine release by electrical stimulation of the cat adrenal gland.

    Science.gov (United States)

    Montiel, C; López, M G; Sánchez-García, P; Maroto, R; Zapater, P; García, A G

    1995-07-15

    on catecholamine release induced by electrical stimulation was observed at low but not at high [Ca2+]o. 6. Simultaneous release of acetylcholine and catecholamines upon electrical stimulation was achieved in glands in which the endogenous acetylcholine stores in the splanchnic nerve terminals had been prelabelled by perfusion with [3H]choline. While apamin enhanced more than 2-fold the postsynaptic release of catecholamines, the presynaptic release of acetylcholine remained unaffected. 7. The results are compatible with the hypothesis that, under physiological conditions, Ca(2+)-activated SK channels present in chromaffin cells control the firing patterns of action potentials induced by the acetylcholine released from splanchnic nerves during stress.(ABSTRACT TRUNCATED AT 400 WORDS)

  18. Contribution of SK and BK channels in the control of catecholamine release by electrical stimulation of the cat adrenal gland.

    Science.gov (United States)

    Montiel, C; López, M G; Sánchez-García, P; Maroto, R; Zapater, P; García, A G

    1995-01-01

    on catecholamine release induced by electrical stimulation was observed at low but not at high [Ca2+]o. 6. Simultaneous release of acetylcholine and catecholamines upon electrical stimulation was achieved in glands in which the endogenous acetylcholine stores in the splanchnic nerve terminals had been prelabelled by perfusion with [3H]choline. While apamin enhanced more than 2-fold the postsynaptic release of catecholamines, the presynaptic release of acetylcholine remained unaffected. 7. The results are compatible with the hypothesis that, under physiological conditions, Ca(2+)-activated SK channels present in chromaffin cells control the firing patterns of action potentials induced by the acetylcholine released from splanchnic nerves during stress.(ABSTRACT TRUNCATED AT 400 WORDS) Images Figure 3 Figure 4 Figure 6 PMID:7473208

  19. APE1/Ref-1 promotes the effect of angiotensin II on Ca2+ -activated K+ channel in human endothelial cells via suppression of NADPH oxidase.

    Science.gov (United States)

    Park, Won Sun; Ko, Eun A; Jung, In Duk; Son, Youn Kyoung; Kim, Hyoung Kyu; Kim, Nari; Park, So Youn; Hong, Ki Whan; Park, Yeong-Min; Choi, Tae-Hoon; Han, Jin

    2008-10-01

    The effects of angiotensin II (Ang II) on whole-cell large conductance Ca(2+)-activated K(+) (BK(Ca)) currents was investigated in control and Apurinic/apyrimidinic endonuclease1/redox factor 1 (APE1/Ref-1)-overexpressing human umbilical vein endothelial cells (HUVECs). Ang II blocked the BK(Ca) current in a dose-dependent fashion, and this inhibition was greater in APE1/Ref-1-overexpressing HUVECs than in control HUVECs (half-inhibition values of 102.81+/-9.54 nM and 11.34+/-0.39 nM in control and APE1/Ref-1-overexpressing HUVECs, respectively). Pretreatment with the NADPH oxidase inhibitor diphenyleneiodonium (DPI) or knock down of NADPH oxidase (p22 phox) using siRNA increased the inhibitory effect of Ang II on the BK(Ca) currents, similar to the effect of APE1/Ref-1 overexpression. In addition, application of Ang II increased the superoxide and hydrogen peroxide levels in the control HUVECs but not in APE1/Ref-1-overexpressing HUVECs. Furthermore, direct application of hydrogen peroxide increased BK(Ca) channel activity. Finally, the inhibitory effect of Ang II on the BK(Ca) current was blocked by an antagonist of the Ang II type 1 (AT(1)) receptor in both control and APE1/Ref-1-overexpressing HUVECs. From these results, we conclude that the inhibitory effect of Ang II on BK(Ca) channel function is NADPH oxidase-dependent and may be promoted by APE1/Ref-1.

  20. Human endothelial cells allow passage of an archetypal BK virus (BKV) strain--a tool for cultivation and functional studies of natural BKV strains.

    Science.gov (United States)

    Hanssen Rinaldo, C; Hansen, H; Traavik, T

    2005-07-01

    The ubiquitous human polyomavirus BK (BKV) causes the serious condition BKV-nephropathy in an increasing number of renal-transplant patients. The lack of authentic cell cultures for multiplication of naturally occurring strains has hampered cultivation and functional studies of BKV. Here we demonstrate that the most common urine shed BKV strain, the archetype, multiplies in the human endothelial cell line HUV-EC-C. Additional variants with deletions in the non-coding control region (NCCR) appear upon prolonged propagation. Although the titer produced was low, at the present HUV-EC-C is the only cell line shown to allow propagation of archetypal BKV. HUV-EC-C may therefore be a useful tool for BKV cultivation as well as functional studies.

  1. Genetic activation of BK currents in vivo generates bidirectional effects on neuronal excitability.

    Science.gov (United States)

    Montgomery, Jenna R; Meredith, Andrea L

    2012-11-13

    Large-conductance calcium-activated potassium channels (BK) are potent negative regulators of excitability in neurons and muscle, and increasing BK current is a novel therapeutic strategy for neuro- and cardioprotection, disorders of smooth muscle hyperactivity, and several psychiatric diseases. However, in some neurons, enhanced BK current is linked with seizures and paradoxical increases in excitability, potentially complicating the clinical use of agonists. The mechanisms that switch BK influence from inhibitory to excitatory are not well defined. Here we investigate this dichotomy using a gain-of-function subunit (BK(R207Q)) to enhance BK currents. Heterologous expression of BK(R207Q) generated currents that activated at physiologically relevant voltages in lower intracellular Ca(2+), activated faster, and deactivated slower than wild-type currents. We then used BK(R207Q) expression to broadly augment endogenous BK currents in vivo, generating a transgenic mouse from a circadian clock-controlled Period1 gene fragment (Tg-BK(R207Q)). The specific impact on excitability was assessed in neurons of the suprachiasmatic nucleus (SCN) in the hypothalamus, a cell type where BK currents regulate spontaneous firing under distinct day and night conditions that are defined by different complements of ionic currents. In the SCN, Tg-BK(R207Q) expression converted the endogenous BK current to fast-activating, while maintaining similar current-voltage properties between day and night. Alteration of BK currents in Tg-BK(R207Q) SCN neurons increased firing at night but decreased firing during the day, demonstrating that BK currents generate bidirectional effects on neuronal firing under distinct conditions.

  2. Persistence of DNA sequences of BK virus and JC virus in normal human tissues and in diseased tissues.

    Science.gov (United States)

    Chesters, P M; Heritage, J; McCance, D J

    1983-04-01

    Available evidence suggests that BK virus (BKV) and JC virus (JCV) persist in the kidneys of healthy individuals after primary infection and may reactivate when the host's immune response is impaired. Data supporting this hypothesis are presented. A previous study had shown BKV to be present in the kidneys of eight (57%) of 14 subjects. In the present study, which extended the investigation to a total of 30 subjects, BKV DNA was found in the renal tissues of 10 (33%) subjects, and JCV DNA was found in the renal tissues of three (10%) subjects. The viral DNA detected appeared not to be integrated with host DNA and to be isolated in foci. Investigation of normal and diseased brain tissue, including tissue from six subjects with multiple sclerosis, failed to reveal the presence of either JCV DNA or BKV DNA.

  3. Prevalence of Polyoma BK Virus (BKPyV), Epstein-Barr Virus (EBV) and Human Papilloma Virus (HPV) in Oropharyngeal Cancer.

    Science.gov (United States)

    Polz-Gruszka, Dorota; Morshed, Kamal; Jarzyński, Adrian; Polz-Dacewicz, Małgorzata

    2015-01-01

    The aim of this study was to analyze the prevalence of BK virus, Human Papillomavirus and Epstein-Barr virus in oropharyngeal cancer, and to test our hypothesis that BKV/HPV/EBV co-infection plays a role in oropharyngeal squamous cell carcinoma. The correlation between viral infection, OSCC, anatomic location, pre-treatment staging, evidence of metastases to lymph nodes, and grading was also investigated. The examination samples were collected from 62 patients from paraffin tissue blocks. Males (90.3%) with, smoking (83.9%) and alcohol abuse (67.7%) problems prevailed in the studied group. G2 histological type was recognized in 80.6% cases. T4 (77.4%) and N2 (56.5%) traits occurred in the majority of patients. No cases of metastasis were observed (M0 100%). HPV - 24.2%, EBV - 27.4% and BKV 17.7% were detected in the studied samples. We observed co-infection EBV/BKV in 8% of cases, HPV/BKV in 4.8%, and HPV/EBV in 9% cases. Only in two cases co-infection of all three viruses was found.

  4. Effects of sodium metabisulfite on the expression of BK(Ca), K(ATP), and L-Ca(2+) channels in rat aortas in vivo and in vitro.

    Science.gov (United States)

    Zhang, Quanxi; Bai, Yunlong; Tian, Jingjing; Lei, Xiaodong; Li, Mei; Yang, Zhenhua; Meng, Ziqiang

    2015-03-01

    Sodium metabisulfite (SMB) is most commonly used as the preservative in many food preparations and drugs. So far, few studies about its negative effects were reported. The purpose of this study was to investigate the effect of SMB on the expression of big-conductance Ca(2+)-activated K(+) (BKCa), ATP-sensitive K(+) (KATP), and L-type calcium (L-Ca(2+)) channels in rat aorta in vivo and in vitro. The results showed that the mRNA and protein levels of the BKCa channel subunits α and β1 of aorta in rats were increased by SMB in vivo and in vitro. Similarly, the expression of the KATP channel subunits Kir6.1, Kir6.2, and SUR2B were increased by SMB. However, SMB at the highest concentration significantly decreased the expression of the L-Ca(2+) channel subunits Cav1.2 and Cav1.3. These results suggest that SMB can activate BKCa and KATP channels by increasing the expression of α, β1, and Kir6.1, Kir6.2, SUR2B respectively, while also inhibit L-Ca(2+) channels by decreasing the expression of Cav1.2 and Cav1.3 of aorta in rats. The molecular mechanism of SMB-induced vasorelaxant effect might be related to the expression changes of BKCa, KATP, and L-Ca(2+) channels subunits. Further work is needed to determine the relative contribution of each channel in SMB-mediated vasorelaxant effect.

  5. Expression of BKCa channels and the modulatory ß-subunits in the rat and porcine trigeminal ganglion

    DEFF Research Database (Denmark)

    Wulf-Johansson, Helle; Hay-Schmidt, Anders; Poulsen, Asser Nyander

    2009-01-01

    (Ca) channel protein was visualized by western blotting and histochemistry. The presence of the modulatory beta1-beta 4 subunit mRNAs was investigated using RT-PCR. beta1-, beta2- and beta 4-subunit mRNAs were expressed in rat TG whereas beta2- and beta 4-subunits were detected in porcine TG. Western blotting...

  6. Overexpression of Large-Conductance Calcium-Activated Potassium Channels in Human Glioblastoma Stem-Like Cells and Their Role in Cell Migration.

    Science.gov (United States)

    Rosa, Paolo; Sforna, Luigi; Carlomagno, Silvia; Mangino, Giorgio; Miscusi, Massimo; Pessia, Mauro; Franciolini, Fabio; Calogero, Antonella; Catacuzzeno, Luigi

    2017-09-01

    Glioblastomas (GBMs) are brain tumors characterized by diffuse invasion of cancer cells into the healthy brain parenchyma, and establishment of secondary foci. GBM cells abundantly express large-conductance, calcium-activated potassium (BK) channels that are thought to promote cell invasion. Recent evidence suggests that the GBM high invasive potential mainly originates from a pool of stem-like cells, but the expression and function of BK channels in this cell subpopulation have not been studied. We investigated the expression of BK channels in GBM stem-like cells using electrophysiological and immunochemical techniques, and assessed their involvement in the migratory process of this important cell subpopulation. In U87-MG cells, BK channel expression and function were markedly upregulated by growth conditions that enriched the culture in GBM stem-like cells (U87-NS). Cytofluorimetric analysis further confirmed the appearance of a cell subpopulation that co-expressed high levels of BK channels and CD133, as well as other stem cell markers. A similar association was also found in cells derived from freshly resected GBM biopsies. Finally, transwell migration tests showed that U87-NS cells migration was much more sensitive to BK channel block than U87-MG cells. Our data show that BK channels are highly expressed in GBM stem-like cells, and participate to their high migratory activity. J. Cell. Physiol. 232: 2478-2488, 2017. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

  7. 豚鼠Ⅱ型前庭毛细胞乙酰胆碱敏感性大电导钙依赖性钾通道与L型钙通道共存%Co-location of Ach-sensitive BK channels and L-type calcium channels in type Ⅱ vestibular hair cells of guinea pig

    Institute of Scientific and Technical Information of China (English)

    郭长凯; 李冠乔; 孔维佳; 张松; 吴婷婷; 李家荔; 李擎天

    2008-01-01

    Objective To explore the mechanisms of the influx of calcium ions during the activation of Ach-sensitive BK channel(big conductance,calcium-dependent potassium channel)in type Ⅱ vestibular hair cells of guinea pigs. Methods Type Ⅱ vestibular hair cells were isolated by collagenase type IA.Under the whole-cell patch mode,the sensitivity of Ach-sensitive BK current to the calcium channels blockers was investigated,the pharmacological property of L-type calcium channel activator-sensitive current and Ach-sensitive BK current was compared. Results Following application of Ach, type Ⅱ vestibular hair cells displayed a sustained outward potassium current,with a reversal potential of(-70.5±10.6)mV(-x±s,n=10). At the holding potential of -50 mV, the current amplitude of Ach-sensitive potassium current activated by 100 μmol/L Ach was(267±106) pA(n=11). Ach-sensitive potassium current was potently sensitive to the BK current blocker, IBTX(iberiotoxin, 200 nmol/L). Apamin,the well-known small conductance, calcium-dependent potassium current blocker, failed to inhibit the amplitude of Ach-sensitive potassium current at a dose of 1 μmol/L. Ach-sensitive BK current was sensitive to NiCl2 and potently inhibited by CdCl2. NiCl2 and CdCl2 showed a dose-dependent blocking effect with a half inhibitionmaximal response of(135.5±18.5)μmol/L(n=7) and (23.4±2.6) μmol/L(n=7). The L-type calcium channel activator,(-)-Bay-K 8644(10 μmol/L),mimicked the role of Ach and activated the IBTX-sensitive outward current. Conclusion Ach-sensitive BK and L-type calcium channels are co-located in type Ⅱ vestibular hair cells of guinea pigs.%目的 研究豚鼠Ⅱ型前庭毛细胞乙酰胆碱(acetylcholine,ACh)敏感性大电导钙依赖性钾通道(big conductance,calcium-dependent potassium channel,BK)激活过程中的钙离子内流机制.方法 健康杂色豚鼠52只,断头后取出前庭终器,经胶原酶IA消化后获取Ⅱ型前庭毛细胞.采用全细胞膜片钳技术

  8. Role of BK(Ca) Potassium Channels in the Mechanisms of Modulatory Effects of IL-10 on Hypoxia-Induced Changes in Activity of Hippocampal Neurons.

    Science.gov (United States)

    Levin, S G; Konakov, M V; Godukhin, O V

    2016-03-01

    We studied the contribution of large conductance Ca(2+)-activated potassium channels (BKCa) in the mechanisms of neuromodulatory effects of anti-inflammatory cytokine IL-10 on hypoxiainduced changes in activity of CA1 pyramidal neurons in rat hippocampus. We used the method of registration of population spikes from CA1 pyramidal neurons in hippocampal slices before, during, and after exposure to short-term episodes of hypoxia. Selective blocker (iberiotoxin) and selective activator of BKCa (BMS-191011) were used to evaluate the contribution of these channels in the mechanisms of suppressive effects of IL-10 on changes in neuronal activity during hypoxia and development of post-hypoxic hyperexcitability. It was shown that BKCa are involved in the modulatory effects of IL-10 on hypoxia-induced suppression of activity of CA1 pyramidal neurons in the hippocampus and development of post-hypoxic hyperexcitability in these neurons.

  9. Niflumic acid hyperpolarizes the smooth muscle cells by opening BK(Ca) channels through ryanodine-sensitive Ca(2+) release in spiral modiolar artery.

    Science.gov (United States)

    Li, Li; Ma, Ke-Tao; Zhao, Lei; Si, Jun-Qiang

    2008-12-25

    The mechanism by which niflumic acid (NFA), a Cl(-) channel antagonist, hyperpolarizes the smooth muscle cells (SMCs) of cochlear spiral modiolar artery (SMA) was explored. Guinea pigs were used as subjects and perforated patch clamp and intracellular recording technique were used to observe NFA-induced response of SMC in the acutely isolated SMA preparation. The results showed that bath application of NFA, indanyloxyacetic acid 94 (IAA-94) and disodium 4,4'-diisothiocyanatostilbene-2,2'-disulfonate (DIDS) caused hyperpolarization and evoked outward currents in all cells at low resting potential (RP), but had no effects in cells at high RP. In the low RP SMCs, the average RP was about (-42.47+/-1.38) mV (n=24). Application of NFA (100 mumol/L), IAA-94 (10 mumol/L) and DIDS (200 mumol/L) shifted the RP to (13.7+/-4.3) mV (n=9, P<0.01), (11.4+/-4.2) mV (n=7, P<0.01) and (12.3+/-3.7) mV (n=8, P<0.01), respectively. These drug-induced responses were in a concentration-dependent manner. NFA-induced hyperpolarization and outward current were almost blocked by charybdotoxin (100 nmol/L), iberiotoxin (100 nmol/L), tetraethylammonium (10 mmol/L), BAPTA-AM (50 mumol/L), ryanodine (10 mumol/L) and caffeine (0.1-10 mmol/L), respectively, but not by nifedipine (100 mumol/L), CdCl2 (100 mumol/L) and Ca(2+)-free medium. It is concluded that NFA induces a release of intracellular calcium from the Ca(2+) stores and the released intracellular calcium in turn causes concentration-dependent and reversible hyperpolarization and evokes outward currents in the SMCs of the cochlear SMA via activation of the Ca(2+)-activated potassium channels.

  10. Alchemy: A web 2.0 real-time quality assurance platform for human immunodeficiency Virus, hepatitis C Virus, and BK Virus quantitation assays

    Directory of Open Access Journals (Sweden)

    Emmanuel Agosto-Arroyo

    2017-01-01

    Full Text Available Background: The molecular diagnostics laboratory faces the challenge of improving test turnaround time (TAT. Low and consistent TATs are of great clinical and regulatory importance, especially for molecular virology tests. Laboratory information systems (LISs contain all the data elements necessary to do accurate quality assurance (QA reporting of TAT and other measures, but these reports are in most cases still performed manually: a time-consuming and error-prone task. The aim of this study was to develop a web-based real-time QA platform that would automate QA reporting in the molecular diagnostics laboratory at our institution, and minimize the time expended in preparing these reports. Methods: Using a standard Linux, Nginx, MariaDB, PHP stack virtual machine running atop a Dell Precision 5810, we designed and built a web-based QA platform, code-named Alchemy. Data files pulled periodically from the LIS in comma-separated value format were used to autogenerate QA reports for the human immunodeficiency virus (HIV quantitation, hepatitis C virus (HCV quantitation, and BK virus (BKV quantitation. Alchemy allowed the user to select a specific timeframe to be analyzed and calculated key QA statistics in real-time, including the average TAT in days, tests falling outside the expected TAT ranges, and test result ranges. Results: Before implementing Alchemy, reporting QA for the HIV, HCV, and BKV quantitation assays took 45–60 min of personnel time per test every month. With Alchemy, that time has decreased to 15 min total per month. Alchemy allowed the user to select specific periods of time and analyzed the TAT data in-depth without the need of extensive manual calculations. Conclusions: Alchemy has significantly decreased the time and the human error associated with QA report generation in our molecular diagnostics laboratory. Other tests will be added to this web-based platform in future updates. This effort shows the utility of informatician

  11. Reactivation of BK polyomavirus in patients with multiple sclerosis receiving natalizumab therapy.

    LENUS (Irish Health Repository)

    Lonergan, Roisin M

    2012-02-01

    Natalizumab therapy in multiple sclerosis has been associated with JC polyomavirus-induced progressive multifocal leucoencephalopathy. We hypothesized that natalizumab may also lead to reactivation of BK, a related human polyomavirus capable of causing morbidity in immunosuppressed groups. Patients with relapsing remitting multiple sclerosis treated with natalizumab were prospectively monitored for reactivation of BK virus in blood and urine samples, and for evidence of associated renal dysfunction. In this cohort, JC and BK DNA in blood and urine; cytomegalovirus (CMV) DNA in blood and urine; CD4 and CD8 T-lymphocyte counts and ratios in peripheral blood; and renal function were monitored at regular intervals. BK subtyping and noncoding control region sequencing was performed on samples demonstrating reactivation. Prior to commencement of natalizumab therapy, 3 of 36 patients with multiple sclerosis (8.3%) had BK viruria and BK reactivation occurred in 12 of 54 patients (22.2%). BK viruria was transient in 7, continuous in 2 patients, and persistent viruria was associated with transient viremia. Concomitant JC and CMV viral loads were undetectable. CD4:CD8 ratios fluctuated, but absolute CD4 counts did not fall below normal limits. In four of seven patients with BK virus reactivation, transient reductions in CD4 counts were observed at onset of BK viruria: these resolved in three of four patients on resuppression of BK replication. No renal dysfunction was observed in the cohort. BK virus reactivation can occur during natalizumab therapy; however, the significance in the absence of renal dysfunction is unclear. We propose regular monitoring for BK reactivation or at least for evidence of renal dysfunction in patients receiving natalizumab.

  12. BK Virus in Recipients of Kidney Transplants.

    Science.gov (United States)

    Hendrix, Kelly M

    2014-01-01

    Since its discovery in 1971, the BK virus, a human polyomavirus, has emerged as a significant cause of renal dysfunction and transplant graft loss in kidney transplant recipients. Improved screening methods have been effective in assisting in the early identification of the virus, and thus, prompt intervention to prevent the progression of the disease. Treatment options for the virus are limited; therefore, lowering immunosuppressive medications should be considered the first line of treatment. Current adjunctive therapies are not guaranteed to control the viral activity and may have limited therapeutic value.

  13. BK virus in solid organ transplant recipients: an emerging syndrome.

    Science.gov (United States)

    Mylonakis, E; Goes, N; Rubin, R H; Cosimi, A B; Colvin, R B; Fishman, J A

    2001-11-27

    BK virus is a human polyomavirus associated with a range of clinical presentations from asymptomatic viruria with pyuria to ureteral ulceration with ureteral stenosis in renal transplant patients or hemorrhagic cystitis in bone marrow transplant recipients. Infection of renal allografts has been associated with diminished graft function in some individuals. Fortunately, however, the majority of patients with BK virus infections are asymptomatic. The type, duration, and intensity of immunosuppression are major contributors to susceptibility to the activation of BK virus infection. Histopathology is required for the demonstration of renal parenchymal involvement; urine cytology and viral polymerase chain reaction methods are useful adjunctive diagnostic tools. Current, treatment of immunosuppressed patients with polyomavirus viruria is largely supportive and directed toward minimizing immunosuppression. Improved diagnostic tools and antiviral therapies are needed for polyomavirus infections.

  14. 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...

  15. A Case of BK Nephropathy without Detectable Viremia or Viruria

    OpenAIRE

    Kamel, Mahmoud; Kadian, Manish; Salazar, Maria Nieva; Mohan, Prince; Self, Sally; Srinivas, Titte; Salas, Maria Aurora Posadas

    2015-01-01

    Patient: Male, 49 Final Diagnosis: BK nephropathy without detectable viremia or viruria Symptoms: — Medication: — Clinical Procedure: Kidney biopsy Specialty: Nephrology Objective: Unusual clinical course Background: BK nephropathy is an evolving challenge among kidney transplant recipients. Diagnosis of BK nephropathy depends on the presence of BK viral inclusions on renal biopsy. Most cases of BK nephropathy are preceded by BK viremia or viruria. Case Report: We report a case of BK nephropa...

  16. Potassium channels and human epileptic phenotypes: an updated overview

    Directory of Open Access Journals (Sweden)

    Chiara eVilla

    2016-03-01

    Full Text Available Potassium (K+ channels are expressed in almost every cells and are ubiquitous in neuronal and glial cell membranes. These channels have been implicated in different disorders, in particular in epilepsy. K+ channel diversity depends on the presence in the human genome of a large number of genes either encoding pore-forming or accessory subunits. More than 80 genes encoding the K+ channels were cloned and they represent the largest group of ion channels regulating the electrical activity of cells in different tissues, including the brain. It is therefore not surprising that mutations in these genes lead to K+ channels dysfunctions linked to inherited epilepsy in humans and non-human model animals.This article reviews genetic and molecular progresses in exploring the pathogenesis of different human epilepsies, with special emphasis on the role of K+ channels in monogenic forms.

  17. Calcium activated K⁺ channels in the electroreceptor of the skate confirmed by cloning. Details of subunits and splicing.

    Science.gov (United States)

    King, Benjamin L; Shi, Ling Fang; Kao, Peter; Clusin, William T

    2016-03-01

    Elasmobranchs detect small potentials using excitable cells of the ampulla of Lorenzini which have calcium-activated K(+) channels, first described in 1974. A distinctive feature of the outward current in voltage clamped ampullae is its apparent insensitivity to voltage. The sequence of a BK channel α isoform expressed in the ampulla of the skate was characterized. A signal peptide is present at the beginning of the gene. When compared to human isoform 1 (the canonical sequence), the largest difference was absence of a 59 amino acid region from the S8-S9 intra-cellular linker that contains the strex regulatory domain. The ampulla isoform was also compared with the isoform predicted in late skate embryos where strex was also absent. The BK voltage sensors were conserved in both skate isoforms. Differences between the skate and human BK channel included alternative splicing. Alternative splicing occurs at seven previously defined sites that are characteristic for BK channels in general and hair cells in particular. Skate BK sequences were highly similar to the Australian ghost shark and several other vertebrate species. Based on alignment of known BK sequences with the skate genome and transcriptome, there are at least two isoforms of Kcnma1α expressed in the skate. One of the β subunits (β4), which is known to decrease voltage sensitivity, was also identified in the skate genome and transcriptome and in the ampulla. These studies advance our knowledge of BK channels and suggest further studies in the ampulla and other excitable tissues. Copyright © 2015 Elsevier B.V. All rights reserved.

  18. Box model for channels of human migration

    CERN Document Server

    Vitanov, Nikolay K

    2016-01-01

    We discuss a mathematical model of migration channel based on the truncated Waring distribution. The truncated Waring distribution is obtained for a more general model of motion of substance through a channel containing finite number of boxes. The model is applied then for case of migrants moving through a channel consisting of finite number of countries or cities. The number of migrants in the channel strongly depends on the number of migrants that enter the channel through the country of entrance. It is shown that if the final destination country is very popular then large percentage of migrants may concentrate there.

  19. DDESC: Dragon database for exploration of sodium channels in human

    Directory of Open Access Journals (Sweden)

    Radovanovic Aleksandar

    2008-12-01

    Full Text Available Abstract Background Sodium channels are heteromultimeric, integral membrane proteins that belong to a superfamily of ion channels. The mutations in genes encoding for sodium channel proteins have been linked with several inherited genetic disorders such as febrile epilepsy, Brugada syndrome, ventricular fibrillation, long QT syndrome, or channelopathy associated insensitivity to pain. In spite of these significant effects that sodium channel proteins/genes could have on human health, there is no publicly available resource focused on sodium channels that would support exploration of the sodium channel related information. Results We report here Dragon Database for Exploration of Sodium Channels in Human (DDESC, which provides comprehensive information related to sodium channels regarding different entities, such as "genes and proteins", "metabolites and enzymes", "toxins", "chemicals with pharmacological effects", "disease concepts", "human anatomy", "pathways and pathway reactions" and their potential links. DDESC is compiled based on text- and data-mining. It allows users to explore potential associations between different entities related to sodium channels in human, as well as to automatically generate novel hypotheses. Conclusion DDESC is first publicly available resource where the information related to sodium channels in human can be explored at different levels. This database is freely accessible for academic and non-profit users via the worldwide web http://apps.sanbi.ac.za/ddesc.

  20. Unsupervised Idealization of Ion Channel Recordings by Minimum Description Length: Application to Human PIEZO1-Channels

    DEFF Research Database (Denmark)

    Gnanasambandam, Radhakrishnan; Nielsen, Morten S; Nicolai, Christopher

    2017-01-01

    on inputs and supervision by the user, thus requiring some prior knowledge of underlying processes. Channels with unknown gating and/or functional sub-states and the presence in the recording of currents from uncorrelated background channels present substantial challenges to such analyses. Here we describe...... channel currents and their substates from recordings with multiple channels, even under conditions of high noise. We then tested the MDL algorithm on real experimental data from human PIEZO1 channels and found that our method revealed the presence of substates with alternate conductances....

  1. [BK virus infection in a pediatric renal transplant recipient].

    Science.gov (United States)

    Bonaventura, R; Vázquez, A; Exeni, A; Rivero, K; Freire, M C

    2005-01-01

    BK Human Polyomavirus causes an asymptomatic primary infection in children, then establishing latency mainly in the urinary tratt. Viral reactivation can lead to renal pathology in individuals with impaired cellular immune response. This is particularly important in pediatric transplant recipients, who can suffer a primary infection when immunosupressed. We followed up the case of a 5 years old patient who received a renal transplant in October 2003, and presented damaged graft 45 days after the intervention. The patient suffered 3 episodes of renal function failure between October 2003 and June 2004. Blood, urine, renal biopsy and lymphocele liquid samples were analyzed. A differential diagnosis between acute rejection and infectious causes was established by testing for BK, CMV and ADV viruses, and the cytological study of renal tissue. Laboratory findings together with clinical signs suggest the patient was infected by BK virus. As a final consideration, the great importance of differentiating between acute rejection and BK infection is emphasized, since immunosuppressant management is opposite in each case.

  2. Human sperm cells swimming in micro-channels

    CERN Document Server

    Denissenko, Petr; Smith, David; Kirkman-Brown, Jackson

    2012-01-01

    The migratory abilities of motile human spermatozoa in vivo are essential for natural fertility, but it remains a mystery what properties distinguish the tens of cells which find an egg from the millions of cells ejaculated. To reach the site of fertilization, sperm must traverse narrow and convoluted channels, filled with viscous fluids. To elucidate individual and group behaviors that may occur in the complex three-dimensional female tract environment, we examine the behavior of migrating sperm in assorted micro-channel geometries. Cells rarely swim in the central part of the channel cross-section, instead traveling along the intersection of the channel walls (`channel corners'). When the channel turns sharply, cells leave the corner, continuing ahead until hitting the opposite wall of the channel, with a distribution of departure angles, the latter being modulated by fluid viscosity. If the channel bend is smooth, cells depart from the inner wall when the curvature radius is less than a threshold value clo...

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

    DEFF Research Database (Denmark)

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

    2004-01-01

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

  4. Recent genetic discoveries implicating ion channels in human cardiovascular diseases.

    Science.gov (United States)

    George, Alfred L

    2014-04-01

    The term 'channelopathy' refers to human genetic disorders caused by mutations in genes encoding ion channels or their interacting proteins. Recent advances in this field have been enabled by next-generation DNA sequencing strategies such as whole exome sequencing with several intriguing and unexpected discoveries. This review highlights important discoveries implicating ion channels or ion channel modulators in cardiovascular disorders including cardiac arrhythmia susceptibility, cardiac conduction phenotypes, pulmonary and systemic hypertension. These recent discoveries further emphasize the importance of ion channels in the pathophysiology of human disease and as important druggable targets.

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

    Science.gov (United States)

    Bernheim, Laurent; Bader, Charles R

    2002-02-01

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

  6. 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.

  7. Cholesterol tuning of BK ethanol response is enantioselective, and is a function of accompanying lipids.

    Directory of Open Access Journals (Sweden)

    Chunbo Yuan

    Full Text Available In the search to uncover ethanol's molecular mechanisms, the calcium and voltage activated, large conductance potassium channel (BK has emerged as an important molecule. We examine how cholesterol content in bilayers of 1,2-dioleoyl-3-phosphatidylethanolamine (DOPE/sphingomyelin (SPM and 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphatidylethanolamine (POPE/1-palmitoyl-2-oleoyl-sn-glycero-3-phosphatidylserine (POPS affect the function and ethanol sensitivity of BK. In addition, we examine how manipulation of cholesterol in biological membranes modulates ethanol's actions on BK. We report that cholesterol levels regulate the change in BK channel open probability elicited by 50 mM ethanol. Low levels of cholesterol (<20%, molar ratio supports ethanol activation, while high levels of cholesterol leads to ethanol inhibition of BK. To determine if cholesterol affects BK and its sensitivity to ethanol through a direct cholesterol-protein interaction or via an indirect action on the lipid bilayer, we used the synthetic enantiomer of cholesterol (ent-CHS. We found that 20% and 40% ent-CHS had little effect on the ethanol sensitivity of BK, when compared with the same concentration of nat-CHS. We accessed the effects of ent-CHS and nat-CHS on the molecular organization of DOPE/SPM monolayers at the air/water interface. The isotherm data showed that ent-CHS condensed DOPE/SPM monolayer equivalently to nat-CHS at a 20% concentration, but slightly less at a 40% concentration. Atomic force microscopy (AFM images of DOPE/SPM membranes in the presence of ent-CHS or nat-CHS prepared with LB technique or vesicle deposition showed no significant difference in topographies, supporting the interpretation that the differences in actions of nat-CHS and ent-CHS on BK channel are not likely from a generalized action on bilayers. We conclude that membrane cholesterol influences ethanol's modulation of BK in a complex manner, including an interaction with the channel protein

  8. Large-conductance K+ channel openers NS1619 and NS004 as inhibitors of mitochondrial function in glioma cells.

    Science.gov (United States)

    Debska, Grazyna; Kicinska, Anna; Dobrucki, Jerzy; Dworakowska, Beata; Nurowska, Ewa; Skalska, Jolanta; Dolowy, Krzysztof; Szewczyk, Adam

    2003-06-01

    Recently, it has been reported that large-conductance Ca(2+)-activated potassium channels, also known as BK(Ca)-type potassium channels, are present in the inner mitochondrial membrane of the human glioma LN229 cell line. Hence, in the present study, we have investigated whether BK(Ca)-channel openers (BK(Ca)COs), such as the benzimidazolone derivatives NS004 (5-trifluoromethyl-1-(5-chloro-2-hydroxyphenyl)-1,3-dihydro-2H-benzimidazole-2-one) and NS1619 (1,3-dihydro-1-[2-hydroxy-5-(trifluoromethyl)phenyl]-5-(trifluoromethyl)-2H-benzimidazol-2-one), affect the functioning of LN229 glioma cell mitochondria in situ. We examined the effect of BK(Ca)COs on mitochondrial membrane potential, mitochondrial respiration and plasma membrane potassium current in human glioma cell line LN229. We found that BK(Ca)COs decrease the mitochondrial membrane potential with an EC(50) value of 3.6+/-0.4 microM for NS1619 and 5.4+/-0.8 microM for NS004. This mitochondrial depolarization was accompanied by an inhibition of the mitochondrial respiratory chain. Both BK(Ca)COs induced whole-cell potassium current blocked by charybdotoxin, as measured by the patch-clamp technique. The BK(Ca)COs had no effect on membrane bilayer conductance. Moreover, the inhibition of mitochondrial function by NS004 and NS1619 was without effect on cell survival, as measured by lactate dehydrogenase release from the cells.

  9. TRESK potassium channel in human T lymphoblasts

    Energy Technology Data Exchange (ETDEWEB)

    Sánchez-Miguel, Dénison Selene, E-mail: amurusk@hotmail.com [Center for Biomedical Research, University of Colima, Av. 25 de Julio 965, Villa San Sebastian, C.P. 28045 Colima (Mexico); García-Dolores, Fernando, E-mail: garciaddf@yahoo.com [Department of Pathology, Institute of Forensic Sciences, Av. Niños Héroes 130, Col. Doctores, C.P. 06720 Mexico, DF (Mexico); Rosa Flores-Márquez, María, E-mail: mariafo31@yahoo.com.mx [National Medical Center of Occident (CMNO) IMSS, Belisario Dominguez 735, Col. Independencia Oriente, C.P. 44340 Guadalajara, Jalisco (Mexico); Delgado-Enciso, Iván [University of Colima, School of Medicine, Av. Universidad 333, Col. Las Viboras, C.P. 28040 Colima (Mexico); Pottosin, Igor, E-mail: pottosin@ucol.mx [Center for Biomedical Research, University of Colima, Av. 25 de Julio 965, Villa San Sebastian, C.P. 28045 Colima (Mexico); Dobrovinskaya, Oxana, E-mail: oxana@ucol.mx [Center for Biomedical Research, University of Colima, Av. 25 de Julio 965, Villa San Sebastian, C.P. 28045 Colima (Mexico)

    2013-05-03

    Highlights: • TRESK (KCNK18) mRNA is present in different T lymphoblastic cell lines. • KCNK18 mRNA was not found in resting peripheral blood lymphocytes. • Clinical samples of T lymphoblastic leukemias and lymphomas were positive for TRESK. • TRESK in T lymphoblasts has dual localization, in plasma membrane and intracellular. -- Abstract: TRESK (TWIK-related spinal cord K{sup +}) channel, encoded by KCNK18 gene, belongs to the double-pore domain K{sup +} channel family and in normal conditions is expressed predominantly in the central nervous system. In our previous patch-clamp study on Jurkat T lymphoblasts we have characterized highly selective K{sup +} channel with pharmacological profile identical to TRESK. In the present work, the presence of KCNK18 mRNA was confirmed in T lymphoblastic cell lines (Jurkat, JCaM, H9) but not in resting peripheral blood lymphocytes of healthy donors. Positive immunostaining for TRESK was demonstrated in lymphoblastic cell lines, in germinal centers of non-tumoral lymph nodes, and in clinical samples of T acute lymphoblastic leukemias/lymphomas. Besides detection in the plasma membrane, intracellular TRESK localization was also revealed. Possible involvement of TRESK channel in lymphocyte proliferation and tumorigenesis is discussed.

  10. 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.

  11. Early monitoring of the human polyomavirus BK replication and sequencing analysis in a cohort of adult kidney transplant patients treated with basiliximab

    Directory of Open Access Journals (Sweden)

    Mischitelli Monica

    2011-08-01

    Full Text Available Abstract Background Nowadays, better immunosuppressors have decreased the rates of acute rejection in kidney transplantation, but have also led to the emergence of BKV-associated nephropathy (BKVAN. Therefore, we prospectively investigated BKV load in plasma and urine samples in a cohort of kidney transplants, receiving basiliximab combined with a mycophenolate mofetil-based triple immunotherapy, to evaluate the difference between BKV replication during the first 3 months post-transplantation, characterized by the non-depleting action of basiliximab, versus the second 3 months, in which the maintenance therapy acts alone. We also performed sequencing analysis to assess whether a particular BKV subtype/subgroup or transcriptional control region (TCR variants were present. Methods We monitored BK viruria and viremia by quantitative polymerase chain reaction (Q-PCR at 12 hours (Tx, 1 (T1, 3 (T2 and 6 (T3 months post-transplantation among 60 kidney transplant patients. Sequencing analysis was performed by nested-PCR with specific primers for TCR and VP1 regions. Data were statistically analyzed using χ2 test and Student's t-test. Results BKV was detected at Tx in 4/60 urine and in 16/60 plasma, with median viral loads of 3,70 log GEq/mL and 3,79 log GEq/mL, respectively, followed by a significant increase of both BKV-positive transplants (32/60 and median values of viruria (5,78 log GEq/mL and viremia (4,52 log GEq/mL at T2. Conversely, a significantly decrease of patients with viruria and viremia (17/60 was observed at T3, together with a reduction of the median urinary and plasma viral loads (4,09 log GEq/mL and 4,00 log GEq/mL, respectively. BKV TCR sequence analysis always showed the presence of archetypal sequences, with a few single-nucleotide substitutions and one nucleotide insertion that, interestingly, were all representative of the particular subtypes/subgroups we identified by VP1 sequencing analysis: I/b-2 and IV/c-2. Conclusions Our

  12. Characterization of highly frequent epitope-specific CD45RA+/CCR7+/- T lymphocyte responses against p53-binding domains of the human polyomavirus BK large tumor antigen in HLA-A*0201+ BKV-seropositive donors

    Directory of Open Access Journals (Sweden)

    Zajac Paul

    2006-11-01

    Full Text Available Abstract Human polyomavirus BK (BKV has been implicated in oncogenic transformation. Its ability to replicate is determined by the binding of its large tumor antigen (LTag to products of tumor-suppressor genes regulating cell cycle, as specifically p53. We investigated CD8+ T immune responses to BKV LTag portions involved in p53 binding in HLA-A*0201+ BKV LTag experienced individuals. Peptides selected from either p53-binding region (LTag351–450 and LTag533–626 by current algorithms and capacity to bind HLA-A*0201 molecule were used to stimulate CD8+ T responses, as assessed by IFN-γ gene expression ex vivo and detected by cytotoxicity assays following in vitro culture. We observed epitope-specific immune responses in all HLA-A*0201+ BKV LTag experienced individuals tested. At least one epitope, LTag579–587; LLLIWFRPV, was naturally processed in non professional antigen presenting cells and induced cytotoxic responses with CTL precursor frequencies in the order of 1/20'000. Antigen specific CD8+ T cells were only detectable in the CD45RA+ subset, in both CCR7+ and CCR7- subpopulations. These data indicate that widespread cellular immune responses against epitopes within BKV LTag-p53 binding regions exist and question their roles in immunosurveillance against tumors possibly associated with BKV infection.

  13. K ATP channels in pig and human intracranial arteries

    DEFF Research Database (Denmark)

    Ploug, Kenneth Beri; Sørensen, Mette Aaskov; Strøbech, Lotte Bjørg

    2008-01-01

    Clinical trials suggest that synthetic ATP-sensitive K(+) (K(ATP)) channel openers may cause headache and migraine by dilating cerebral and meningeal arteries. We studied the mRNA expression profile of K(ATP) channel subunits in the pig and human middle meningeal artery (MMA) and in the pig middle...... cerebral artery (MCA). We determined the order of potency of four K(ATP) channel openers when applied to isolated pig MMA and MCA, and we examined the potential inhibitory effects of the Kir6.1 subunit specific K(ATP) channel blocker PNU-37883A on K(ATP) channel opener-induced relaxation of the isolated...... pig MMA and MCA. Using conventional RT-PCR, we detected the mRNA transcripts of the K(ATP) channel subunits Kir6.1 and SUR2B in all the examined pig and human intracranial arteries. Application of K(ATP) channel openers to isolated pig MMA and MCA in myographs caused a concentration...

  14. Molecular dynamics insights into human aquaporin 2 water channel.

    Science.gov (United States)

    Binesh, A R; Kamali, R

    2015-12-01

    In this study, the first molecular dynamics simulation of the human aquaporin 2 is performed and for a better understanding of the aquaporin 2 permeability performance, the characteristics of water transport in this protein channel and key biophysical parameters of AQP2 tetramer including osmotic and diffusive permeability constants and the pore radius are investigated. For this purpose, recently recovered high resolution X-ray crystal structure of` the human aquaporin 2 is used to perform twenty nanosecond molecular dynamics simulation of fully hydrated tetramer of this protein embedded in a lipid bilayer. The resulting water permeability characteristics of this protein channel showed that the water permeability of the human AQP2 is in a mean range in comparison with other human aquaporins family. Finally, the results reported in this research demonstrate that molecular dynamics simulation of human AQP2 provided useful insights into the mechanisms of water permeation and urine concentration in the human kidney.

  15. Bilirubin oxidation end products directly alter K+ channels important in the regulation of vascular tone.

    Science.gov (United States)

    Hou, Shangwei; Xu, Rong; Clark, Joseph F; Wurster, William L; Heinemann, Stefan H; Hoshi, Toshinori

    2011-01-01

    The exact etiology of delayed cerebral vasospasm following cerebral hemorrhage is not clear, but a family of compounds termed 'bilirubin oxidation end products (BOXes)' derived from heme has been implicated. As proper regulation of vascular smooth muscle tone involves large-conductance Ca(2+)- and voltage-dependent Slo1 K(+) (BK, maxiK, K(Ca)1.1) channels, we examined whether BOXes altered functional properties of the channel. Electrophysiological measurements of Slo1 channels heterologously expressed in a human cell line and of native mouse BK channels in isolated cerebral myocytes showed that BOXes markedly diminished open probability. Biophysically, BOXes specifically stabilized the conformations of the channel with its ion conduction gate closed. The results of chemical amino-acid modifications and molecular mutagenesis together suggest that two specific lysine residues in the structural element linking the transmembrane ion-permeation domain to the carboxyl cytosolic domain of the Slo1 channel are critical in determining the sensitivity of the channel to BOXes. Inhibition of Slo1 BK channels by BOXes may contribute to the development of delayed cerebral vasospasm following brain hemorrhage.

  16. Efficacy of intravenous immunoglobulin in the treatment of persistent BK viremia and BK virus nephropathy in renal transplant recipients.

    Science.gov (United States)

    Vu, D; Shah, T; Ansari, J; Naraghi, R; Min, D

    2015-03-01

    BK virus-associated nephropathy (BKVN) can cause clinically significant viral infection in renal transplant recipients, leading to allograft dysfunction and loss. The usual management of BKVN involves the reduction of immunosuppression and the addition of leflunomide, quinolones, and cidofovir, but the rate of graft loss remains high. The aim of this study was to assess the impact of treatment with intravenous human immunoglobulin (IVIG) on the outcome of BKVN in renal transplant recipients. Upon diagnosis of BKVN, patients remained on anti-polyomavirus treatment, consisting of the reduction of immunosuppression and the use of leflunomide therapy. Treatment with IVIG was given only to patients who did not respond to 8 weeks of the adjustment of immunosuppression and leflunomide. All 30 patients had persistent BKV viremia and BKVN with their mean BK viral loads higher than the baseline (range, 15,000-2 million copies/mL). Mean peak BK load was 205,314 copies/mL compared with 697 copies/mL after 1 year of follow-up. Twenty-seven patients (90%) had a positive response in clearing viremia. The actuarial patient and graft survival rates after 12 months were 100% and 96.7%, respectively. IVIG administration appeared to be safe and effective in treating BKV viremia and BKVN and preventing graft loss in patients who had inadequate response to immunosuppression reduction and leflunomide therapy. Copyright © 2015. Published by Elsevier Inc.

  17. Molecular Structure of the Human CFTR Ion Channel.

    Science.gov (United States)

    Liu, Fangyu; Zhang, Zhe; Csanády, László; Gadsby, David C; Chen, Jue

    2017-03-23

    The cystic fibrosis transmembrane conductance regulator (CFTR) is an ATP-binding cassette (ABC) transporter that uniquely functions as an ion channel. Here, we present a 3.9 Å structure of dephosphorylated human CFTR without nucleotides, determined by electron cryomicroscopy (cryo-EM). Close resemblance of this human CFTR structure to zebrafish CFTR under identical conditions reinforces its relevance for understanding CFTR function. The human CFTR structure reveals a previously unresolved helix belonging to the R domain docked inside the intracellular vestibule, precluding channel opening. By analyzing the sigmoid time course of CFTR current activation, we propose that PKA phosphorylation of the R domain is enabled by its infrequent spontaneous disengagement, which also explains residual ATPase and gating activity of dephosphorylated CFTR. From comparison with MRP1, a feature distinguishing CFTR from all other ABC transporters is the helix-loop transition in transmembrane helix 8, which likely forms the structural basis for CFTR's channel function.

  18. An Apical-Membrane Chloride Channel in Human Tracheal Epithelium

    Science.gov (United States)

    Welsh, Michael J.

    1986-06-01

    The mechanism of chloride transport by airway epithelia has been of substantial interest because airway and sweat gland-duct epithelia are chloride-impermeable in cystic fibrosis. The decreased chloride permeability prevents normal secretion by the airway epithelium, thereby interfering with mucociliary clearance and contributing to the morbidity and mortality of the disease. Because chloride secretion depends on and is regulated by chloride conductance in the apical cell membrane, the patch-clamp technique was used to directly examine single-channel currents in primary cultures of human tracheal epithelium. The cells contained an anion-selective channel that was not strongly voltage-gated or regulated by calcium in cell-free patches. The channel was also blocked by analogs of carboxylic acid that decrease apical chloride conductance in intact epithelia. When attached to the cell, the channel was activated by isoproterenol, although the channel was also observed to open spontaneously. However, in some cases, the channel was only observed after the patch was excised from the cell. These results suggest that this channel is responsible for the apical chloride conductance in airway epithelia.

  19. Dysregulation of large-conductance Ca2+-activated K+ channel expression in nonsyndromal mental retardation due to a cereblon p.R419X mutation.

    Science.gov (United States)

    Higgins, Joseph J; Hao, Jin; Kosofsky, Barry E; Rajadhyaksha, Anjali M

    2008-07-01

    A nonsense mutation (R419X) in the human cereblon gene [mutation (mut) CRBN] causes a mild type of autosomal recessive nonsyndromal mental retardation (ARNSMR). CRBN, a cytosolic protein, regulates the assembly and neuronal surface expression of large-conductance Ca(2+)-activated K(+) channels (BK(Ca)) in brain regions involved in memory and learning. Using the real-time quantitative polymerase chain reaction, we show that mut CRBN disturbs the development of adult brain BK(Ca) isoforms. These changes are predicted to result in BK(Ca) channels with a higher intracellular Ca(2+) sensitivity, faster activation, and slower deactivation kinetics. Such alterations may contribute to cognitive impairments in patients with mild ARNSMR.

  20. 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.

  1. 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.

  2. Unique action of sodium tanshinone II-A sulfonate (DS-201) on the Ca(2+) dependent BK(Ca) activation in mouse cerebral arterial smooth muscle cells.

    Science.gov (United States)

    Tan, Xiaoqiu; Yang, Yan; Cheng, Jun; Li, Pengyun; Inoue, Isao; Zeng, Xiaorong

    2011-04-10

    Sodium tanshinone II-A sulfonate (DS-201) is a water-soluble derivative of tanshinone IIA, a main active constituent of Salvia miltiorrhiza which has been used for treatments of cardio- and cerebro-vascular diseases. DS-201 activates large conductance Ca(2+)-sensitive K(+) channels (BK(Ca)) in arterial smooth muscle cells, and reduces the vascular tone. Here we investigated the effect of DS-201 on the BK(Ca) channel kinetics by analyzing single channel currents. Smooth muscle cells were freshly isolated from mouse cerebral arteries. Single channel currents of BK(Ca) were recorded by patch clamp. DS-201 increased the total open probability (NPo) of BK(Ca) in a concentration-dependent manner. But this action required intracellular Ca(2+), and the effect depended on the Ca(2+) concentration ([Ca(2+)](free)). DS-201 activated BK(Ca) with the half maximal effective concentration (EC(50)) of 111.5μM at 0.01μM [Ca(2+)](free), and 68.5μM at 0.1μM [Ca(2+)](free.) The effect of DS-201 on NPo was particularly strong in the range of [Ca(2+)](free) between 0.1 and 1μM. Analysis of the channel kinetics revealed that DS-201 had only the effect on the channel closing without affecting the channel opening, which was a striking contrast to the effect of [Ca(2+)](free), that is characterized by changing the channel opening without changing the channel closing. DS-201 may be bound to the open state of BK(Ca), and have an inhibitory effect on the transition from the open to closed state. By this way DS-201 may enhance the activity of BK(Ca), and exhibit a strong vasodilating effect against vasoconstriction in the range of [Ca(2+)](free) between 0.1 and 1μM. Copyright © 2011 Elsevier B.V. All rights reserved.

  3. Changes in channel morphology over human time scales [Chapter 32

    Science.gov (United States)

    John M. Buffington

    2012-01-01

    Rivers are exposed to changing environmental conditions over multiple spatial and temporal scales, with the imposed environmental conditions and response potential of the river modulated to varying degrees by human activity and our exploitation of natural resources. Watershed features that control river morphology include topography (valley slope and channel...

  4. Off-the-Shelf Virus-Specific T Cells to Treat BK Virus, Human Herpesvirus 6, Cytomegalovirus, Epstein-Barr Virus, and Adenovirus Infections After Allogeneic Hematopoietic Stem-Cell Transplantation.

    Science.gov (United States)

    Tzannou, Ifigeneia; Papadopoulou, Anastasia; Naik, Swati; Leung, Kathryn; Martinez, Caridad A; Ramos, Carlos A; Carrum, George; Sasa, Ghadir; Lulla, Premal; Watanabe, Ayumi; Kuvalekar, Manik; Gee, Adrian P; Wu, Meng-Fen; Liu, Hao; Grilley, Bambi J; Krance, Robert A; Gottschalk, Stephen; Brenner, Malcolm K; Rooney, Cliona M; Heslop, Helen E; Leen, Ann M; Omer, Bilal

    2017-08-07

    Purpose Improvement of cure rates for patients treated with allogeneic hematopoietic stem-cell transplantation (HSCT) will require efforts to decrease treatment-related mortality from severe viral infections. Adoptively transferred virus-specific T cells (VSTs) generated from eligible, third-party donors could provide broad antiviral protection to recipients of HSCT as an immediately available off-the-shelf product. Patient and Methods We generated a bank of VSTs that recognized five common viral pathogens: Epstein-Barr virus (EBV), adenovirus (AdV), cytomegalovirus (CMV), BK virus (BKV), and human herpesvirus 6 (HHV-6). The VSTs were administered to 38 patients with 45 infections in a phase II clinical trial. Results A single infusion produced a cumulative complete or partial response rate of 92% (95% CI, 78.1% to 98.3%) overall and the following rates by virus: 100% for BKV (n = 16), 94% for CMV (n = 17), 71% for AdV (n = 7), 100% for EBV (n = 2), and 67% for HHV-6 (n = 3). Clinical benefit was achieved in 31 patients treated for one infection and in seven patients treated for multiple coincident infections. Thirteen of 14 patients treated for BKV-associated hemorrhagic cystitis experienced complete resolution of gross hematuria by week 6. Infusions were safe, and only two occurrences of de novo graft-versus host disease (grade 1) were observed. VST tracking by epitope profiling revealed persistence of functional VSTs of third-party origin for up to 12 weeks. Conclusion The use of banked VSTs is a feasible, safe, and effective approach to treat severe and drug-refractory infections after HSCT, including infections from two viruses (BKV and HHV-6) that had never been targeted previously with an off-the-shelf product. Furthermore, the multispecificity of the VSTs ensures extensive antiviral coverage, which facilitates the treatment of patients with multiple infections.

  5. Ionic Selectivity and Permeation Properties of Human PIEZO1 Channels.

    Science.gov (United States)

    Gnanasambandam, Radhakrishnan; Bae, Chilman; Gottlieb, Philip A; Sachs, Frederick

    2015-01-01

    Members of the eukaryotic PIEZO family (the human orthologs are noted hPIEZO1 and hPIEZO2) form cation-selective mechanically-gated channels. We characterized the selectivity of human PIEZO1 (hPIEZO1) for alkali ions: K+, Na+, Cs+ and Li+; organic cations: TMA and TEA, and divalents: Ba2+, Ca2+, Mg2+ and Mn2+. All monovalent ions permeated the channel. At a membrane potential of -100 mV, Cs+, Na+ and K+ had chord conductances in the range of 35-55 pS with the exception of Li+, which had a significantly lower conductance of ~ 23 pS. The divalents decreased the single-channel permeability of K+, presumably because the divalents permeated slowly and occupied the open channel for a significant fraction of the time. In cell-attached mode, 90 mM extracellular divalents had a conductance for inward currents carried by the divalents of: 25 pS for Ba2+ and 15 pS for Ca2+ at -80 mV and 10 pS for Mg2+ at -50 mV. The organic cations, TMA and TEA, permeated slowly and attenuated K+ currents much like the divalents. As expected, the channel K+ conductance increased with K+ concentration saturating at ~ 45 pS and the KD of K+ for the channel was 32 mM. Pure divalent ion currents were of lower amplitude than those with alkali ions and the channel opening rate was lower in the presence of divalents than in the presence of monovalents. Exposing cells to the actin disrupting reagent cytochalasin D increased the frequency of openings in cell-attached patches probably by reducing mechanoprotection.

  6. Ionic Selectivity and Permeation Properties of Human PIEZO1 Channels.

    Directory of Open Access Journals (Sweden)

    Radhakrishnan Gnanasambandam

    Full Text Available Members of the eukaryotic PIEZO family (the human orthologs are noted hPIEZO1 and hPIEZO2 form cation-selective mechanically-gated channels. We characterized the selectivity of human PIEZO1 (hPIEZO1 for alkali ions: K+, Na+, Cs+ and Li+; organic cations: TMA and TEA, and divalents: Ba2+, Ca2+, Mg2+ and Mn2+. All monovalent ions permeated the channel. At a membrane potential of -100 mV, Cs+, Na+ and K+ had chord conductances in the range of 35-55 pS with the exception of Li+, which had a significantly lower conductance of ~ 23 pS. The divalents decreased the single-channel permeability of K+, presumably because the divalents permeated slowly and occupied the open channel for a significant fraction of the time. In cell-attached mode, 90 mM extracellular divalents had a conductance for inward currents carried by the divalents of: 25 pS for Ba2+ and 15 pS for Ca2+ at -80 mV and 10 pS for Mg2+ at -50 mV. The organic cations, TMA and TEA, permeated slowly and attenuated K+ currents much like the divalents. As expected, the channel K+ conductance increased with K+ concentration saturating at ~ 45 pS and the KD of K+ for the channel was 32 mM. Pure divalent ion currents were of lower amplitude than those with alkali ions and the channel opening rate was lower in the presence of divalents than in the presence of monovalents. Exposing cells to the actin disrupting reagent cytochalasin D increased the frequency of openings in cell-attached patches probably by reducing mechanoprotection.

  7. Human and automatic speaker recognition over telecommunication channels

    CERN Document Server

    Fernández Gallardo, Laura

    2016-01-01

    This work addresses the evaluation of the human and the automatic speaker recognition performances under different channel distortions caused by bandwidth limitation, codecs, and electro-acoustic user interfaces, among other impairments. Its main contribution is the demonstration of the benefits of communication channels of extended bandwidth, together with an insight into how speaker-specific characteristics of speech are preserved through different transmissions. It provides sufficient motivation for considering speaker recognition as a criterion for the migration from narrowband to enhanced bandwidths, such as wideband and super-wideband.

  8. Infección por virus BK en paciente pediátrico trasplantado renal BK virus infection in a pediatric renal transplant recipient

    Directory of Open Access Journals (Sweden)

    R. Bonaventura

    2005-09-01

    Full Text Available El poliomavirus humano BK causa infección primaria asintomática en la niñez, estableciendo latencia principalmente en el tracto urinario. En individuos con alteración en la inmunidad celular se puede producir su reactivación desencadenando patología a nivel renal. Por estas razones es particularmente importante en la población pediátrica trasplantada renal, en la que puede producir la infección primaria cuando el paciente está inmunosuprimido. En nuestro trabajo se realizó el seguimiento de un paciente de 5 años trasplantado renal en octubre de 2003 que 45 días post-trasplante sufrió un deterioro del órgano injertado. Desde la fecha del trasplante hasta junio de 2004 se produjeron 3 episodios de alteración en la función renal, durante los cuales se analizaron muestras de sangre, orina, biopsia renal y líquido de linfocele. Para el diagnóstico difererencial entre rechazo agudo versus causa infecciosa se emplearon técnicas de detección para los virus BK, CMV y ADV, además del estudio citológico del tejido renal. Los resultados obtenidos junto con la clínica del paciente indican un probable caso de infección por BK. La importancia de realizar el diagnóstico diferencial entre rechazo agudo y la infección por BK radica en que la conducta en cuanto a la terapia inmunosupresora es opuesta en cada caso.BK Human Polyomavirus causes an asymptomatic primary infection in children, then establishing latency mainly in the urinary tract. Viral reactivation can lead to renal pathology in individuals with impaired cellular immune response. This is particularly important in pediatric transplant recipients, who can suffer a primary infection when immunosupressed. We followed up the case of a 5 years old patient who received a renal transplant in October 2003, and presented damaged graft 45 days after the intervention. The patient suffered 3 episodes of renal function failure between October 2003 and June 2004. Blood, urine, renal biopsy

  9. Impact of two different commercial DNA extraction methods on BK virus viral load

    Directory of Open Access Journals (Sweden)

    Massimiliano Bergallo

    2016-03-01

    Full Text Available Background and aim: BK virus, a member of human polyomavirus family, is a worldwide distributed virus characterized by a seroprevalence rate of 70-90% in adult population. Monitoring of viral replication is made by evaluation of BK DNA by quantitative polymerase chain reaction. Many different methods can be applied for extraction of nucleic acid from several specimens. The aim of this study was to assess the impact of two different DNA extraction procedure on BK viral load. Materials and methods: DNA extraction procedure including the Nuclisens easyMAG platform (bioMerieux, Marcy l’Etoile, France and manual QIAGEN extraction (QIAGEN Hilden, Germany. BK DNA quantification was performed by Real Time TaqMan PCR using a commercial kit. Result and discussion: The samples capacity, cost and time spent were compared for both systems. In conclusion our results demonstrate that automated nucleic acid extraction method using Nuclisense easyMAG was superior to manual protocol (QIAGEN Blood Mini kit, for the extraction of BK virus from serum and urine specimens.

  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. Noise analysis and single-channel observations of 4 pS chloride channels in human airway epithelia.

    OpenAIRE

    Duszyk, M; French, A S; Man, S F

    1992-01-01

    Apical membranes of human airway epithelial cells have significant chloride permeability, which is reduced in cystic fibrosis (CF), causing abnormal electrochemistry and impaired mucociliary clearance. At least four types of chloride channels have been identified in these cells, but their relative roles in total permeability and CF are unclear. Noise analysis was used to measure the conductance of chloride channels in human nasal epithelial cells. The data indicate that channels with a mean c...

  12. TRPM8, a versatile channel in human sperm.

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    Gerardo A De Blas

    Full Text Available BACKGROUND: The transient receptor potential channel (TRP family includes more than 30 proteins; they participate in various Ca(2+ dependent processes. TRPs are functionally diverse involving thermal, chemical and mechanical transducers which modulate the concentration of intracellular Ca(2+ ([Ca(2+]i. Ca(2+ triggers and/or regulates principal sperm functions during fertilization such as motility, capacitation and the acrosome reaction. Nevertheless, the presence of the TRPM subfamily in sperm has not been explored. PRINCIPAL FINDINGS: Here we document with RT-PCR, western blot and immunocitochemistry analysis the presence of TRPM8 in human sperm. We also examined the participation of this channel in sperm function using specific agonists (menthol and temperature and antagonists (BCTC and capsazepine. Computer-aided sperm analysis revealed that menthol did not significantly alter human sperm motility. In contrast, menthol induced the acrosome reaction in human sperm. This induction was inhibited about 70% by capsazepine (20 microM and 80% by BCTC (1.6 microM. Activation of TRPM8 either by temperature or menthol induced [Ca(2+]i increases in human sperm measured by fluorescence in populations or individual sperm cells, effect that was also inhibited by capsazepine (20 microM and BCTC (1.6 microM. However, the progesterone and ZP3-induced acrosome reaction was not inhibited by capsazepine or BCTC, suggesting that TRPM8 activation triggers this process by a different signaling pathway. CONCLUSIONS: This is the first report dealing with the presence of a thermo sensitive channel (TRPM8 in human sperm. This channel could be involved in cell signaling events such as thermotaxis or chemotaxis.

  13. TRPM8, a Versatile Channel in Human Sperm

    Science.gov (United States)

    Ocampo, Ana Y.; Serrano, Carmen J.; Castellano, Laura E.; Hernández-González, Enrique O.; Chirinos, Mayel; Larrea, Fernando; Beltrán, Carmen; Treviño, Claudia L.

    2009-01-01

    Background The transient receptor potential channel (TRP) family includes more than 30 proteins; they participate in various Ca2+ dependent processes. TRPs are functionally diverse involving thermal, chemical and mechanical transducers which modulate the concentration of intracellular Ca2+ ([Ca2+]i). Ca2+ triggers and/or regulates principal sperm functions during fertilization such as motility, capacitation and the acrosome reaction. Nevertheless, the presence of the TRPM subfamily in sperm has not been explored. Principal Findings Here we document with RT-PCR, western blot and immunocitochemistry analysis the presence of TRPM8 in human sperm. We also examined the participation of this channel in sperm function using specific agonists (menthol and temperature) and antagonists (BCTC and capsazepine). Computer-aided sperm analysis revealed that menthol did not significantly alter human sperm motility. In contrast, menthol induced the acrosome reaction in human sperm. This induction was inhibited about 70% by capsazepine (20 µM) and 80% by BCTC (1.6 µM). Activation of TRPM8 either by temperature or menthol induced [Ca2+]i increases in human sperm measured by fluorescence in populations or individual sperm cells, effect that was also inhibited by capsazepine (20 µM) and BCTC (1.6 µM). However, the progesterone and ZP3-induced acrosome reaction was not inhibited by capsazepine or BCTC, suggesting that TRPM8 activation triggers this process by a different signaling pathway. Conclusions This is the first report dealing with the presence of a thermo sensitive channel (TRPM8) in human sperm. This channel could be involved in cell signaling events such as thermotaxis or chemotaxis. PMID:19582168

  14. Evaluating the BK 21 Program. Research Brief

    Science.gov (United States)

    Seong, Somi; Popper, Steven W.; Goldman, Charles A.; Evans, David K.; Grammich, Clifford A.

    2008-01-01

    The Brain Korea 21 program (BK21), an effort to improve Korean universities and research, has attracted a great deal of attention in Korea, producing the need to understand how well the program is meeting its goals. RAND developed a logic model for identifying program goals and dynamics, suggested quantitative and qualitative evaluation methods,…

  15. Polyoma BK Virus: An Oncogenic Virus?

    Directory of Open Access Journals (Sweden)

    Syed Hassan

    2013-01-01

    Full Text Available We report a case of a 65-year-old gentleman with a history of end stage renal disease who underwent a successful cadaveric donor kidney transplant four years ago. He subsequently developed BK virus nephropathy related to chronic immunosuppressant therapy. Three years later, misfortune struck again, and he developed adenocarcinoma of the bladder.

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

    DEFF Research Database (Denmark)

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

    2009-01-01

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

  17. Functional properties of human neuronal Kv11 channels

    DEFF Research Database (Denmark)

    Einarsen, Karoline; Calloe, Kirstine; Grunnet, Morten

    2009-01-01

    Kv11 potassium channels are important for regulation of the membrane potential. Kv11.2 and Kv11.3 are primarily found in the nervous system, where they most likely are involved in the regulation of neuronal excitability. Two isoforms of human Kv11.2 have been published so far. Here, we present...... a new splice variant that is present in human brain as demonstrated by reverse transcription PCR. Heterologous expression in Xenopus laevis oocytes revealed a 30-mV shift in the voltage dependence of activation to more depolarized potentials and slower activation together with faster deactivation...

  18. Dynamic Propagation Channel Characterization and Modeling for Human Body Communication

    Directory of Open Access Journals (Sweden)

    Lei Wang

    2012-12-01

    Full Text Available This paper presents the first characterization and modeling of dynamic propagation channels for human body communication (HBC. In-situ experiments were performed using customized transceivers in an anechoic chamber. Three HBC propagation channels, i.e., from right leg to left leg, from right hand to left hand and from right hand to left leg, were investigated under thirty-three motion scenarios. Snapshots of data (2,800,000 were acquired from five volunteers. Various path gains caused by different locations and movements were quantified and the statistical distributions were estimated. In general, for a given reference threshold è = −10 dB, the maximum average level crossing rate of the HBC was approximately 1.99 Hz, the maximum average fade time was 59.4 ms, and the percentage of bad channel duration time was less than 4.16%. The HBC exhibited a fade depth of −4 dB at 90% complementary cumulative probability. The statistical parameters were observed to be centered for each propagation channel. Subsequently a Fritchman model was implemented to estimate the burst characteristics of the on-body fading. It was concluded that the HBC is motion-insensitive, which is sufficient for reliable communication link during motions, and therefore it has great potential for body sensor/area networks.

  19. Dynamic propagation channel characterization and modeling for human body communication.

    Science.gov (United States)

    Nie, Zedong; Ma, Jingjing; Li, Zhicheng; Chen, Hong; Wang, Lei

    2012-12-18

    This paper presents the first characterization and modeling of dynamic propagation channels for human body communication (HBC). In-situ experiments were performed using customized transceivers in an anechoic chamber. Three HBC propagation channels, i.e., from right leg to left leg, from right hand to left hand and from right hand to left leg, were investigated under thirty-three motion scenarios. Snapshots of data (2,800,000) were acquired from five volunteers. Various path gains caused by different locations and movements were quantified and the statistical distributions were estimated. In general, for a given reference threshold è = -10 dB, the maximum average level crossing rate of the HBC was approximately 1.99 Hz, the maximum average fade time was 59.4 ms, and the percentage of bad channel duration time was less than 4.16%. The HBC exhibited a fade depth of -4 dB at 90% complementary cumulative probability. The statistical parameters were observed to be centered for each propagation channel. Subsequently a Fritchman model was implemented to estimate the burst characteristics of the on-body fading. It was concluded that the HBC is motion-insensitive, which is sufficient for reliable communication link during motions, and therefore it has great potential for body sensor/area networks.

  20. 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...

  1. BK Virus Nephropathy in Heart Transplant Recipients.

    Science.gov (United States)

    Joseph, Alin; Pilichowska, Monika; Boucher, Helen; Kiernan, Michael; DeNofrio, David; Inker, Lesley A

    2015-06-01

    Polyomavirus-associated nephropathy (PVAN) has become an important cause of kidney failure in kidney transplant recipients. PVAN is reported to affect 1% to 7% of kidney transplant recipients, leading to premature transplant loss in approximately 30% to 50% of diagnosed cases. PVAN occurring in the native kidneys of solid-organ transplant recipients other than kidney only recently has been noted. We report 2 cases of PVAN in heart transplant recipients, which brings the total of reported cases to 7. We briefly review the literature on the hypothesized causes of PVAN in kidney transplant recipients and comment on whether these same mechanisms also may cause PVAN in other solid-organ transplant recipients. PVAN should be considered in the differential diagnosis when evaluating worsening kidney function. BK viremia surveillance studies of nonkidney solid-organ recipients should be conducted to provide data to assist the transplantation community in deciding whether regular monitoring of nonkidney transplant recipients for BK viremia is indicated.

  2. Exposing the Molecular Machinery of BK Polyomavirus.

    Science.gov (United States)

    Buck, Christopher B

    2016-04-05

    BK polyomavirus (BKV) is an opportunistic pathogen that poses a serious threat to organ transplant recipients. In this issue of Structure, Hurdiss and colleagues' (Hurdiss et al., 2016) beautiful new high-resolution cryo-EM reconstruction of BKV provides a structural roadmap for the ongoing development of therapeutic antibodies and vaccines targeting this potentially deadly virus. The study also serves as a platform for exploring the basic biology of virion assembly and infectious entry.

  3. Aquaporin water channels: molecular mechanisms for human diseases.

    Science.gov (United States)

    Agre, Peter; Kozono, David

    2003-11-27

    Although water is the major component of all biological fluids, the molecular pathways for water transport across cell membranes eluded identification until the discovery of the aquaporin family of water channels. The atomic structure of mammalian AQP1 illustrates how this family of proteins is freely permeated by water but not protons (hydronium ions, H3O+). Definition of the subcellular sites of expression predicted their physiological functions and potential clinical disorders. Analysis of several human disease states has confirmed that aquaporins are involved in multiple different illnesses including abnormalities of kidney function, loss of vision, onset of brain edema, starvation, and arsenic toxicity.

  4. Progression from Sustained BK Viruria to Sustained BK Viremia with Immunosuppression Reduction Is Not Associated with Changes in the Noncoding Control Region of the BK Virus Genome

    Science.gov (United States)

    Memon, Imran A.; Parikh, Bijal A.; Gaudreault-Keener, Monique; Skelton, Rebecca; Storch, Gregory A.; Brennan, Daniel C.

    2012-01-01

    Changes in the BK virus archetypal noncoding control region (NCCR) have been associated with BK-virus-associated nephropathy (BKVAN). Whether sustained viremia, a surrogate for BKVAN, is associated with significant changes in the BK-NCCR is unknown. We performed PCR amplification and sequencing of (1) stored urine and (2) plasma samples from the time of peak viremia from 11 patients with sustained viremia who participated in a 200-patient clinical trial. The antimetabolite was withdrawn for BK viremia and reduction of the calcineurin inhibitor for sustained BK viremia. DNA sequencing from the 11 patients with sustained viremia revealed 8 insertions, 16 transversions, 3 deletions, and 17 transitions. None were deemed significant. No patient developed clinically evident BKVAN. Our data support, at a genomic level, the effectiveness of reduction of immunosuppression for prevention of progression from viremia to BKVAN. PMID:22701777

  5. 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...

  6. Fusion and quasifission dynamics in the reactions 48Ca+249Bk and 50Ti+249Bk using a time-dependent Hartree-Fock approach

    Science.gov (United States)

    Umar, A. S.; Oberacker, V. E.; Simenel, C.

    2016-08-01

    Background: Synthesis of superheavy elements (SHEs) with fusion-evaporation reactions is strongly hindered by the quasifission (QF) mechanism which prevents the formation of an equilibrated compound nucleus and which depends on the structure of the reactants. New SHEs have been recently produced with doubly-magic 48Ca beams. However, SHE synthesis experiments with single-magic 50Ti beams have so far been unsuccessful. Purpose: In connection with experimental searches for Z =117 ,119 superheavy elements, we perform a theoretical study of fusion and quasifission mechanisms in 48Ca,50Ti+249Bk reactions in order to investigate possible differences in reaction mechanisms induced by these two projectiles. Methods: The collision dynamics and the outcome of the reactions are studied using unrestricted time-dependent Hartree-Fock (TDHF) calculations as well as the density-constrained TDHF method to extract the nucleus-nucleus potentials and the excitation energy in each fragment. Results: Nucleus-nucleus potentials, nuclear contact times, masses and charges of the fragments, as well as their kinetic and excitation energies strongly depend on the orientation of the prolate 249Bk nucleus. Long contact times associated with fusion are observed in collisions of both projectiles with the side of the 249Bk nucleus, but not on collisions with its tip. The energy and impact parameter dependencies of the fragment properties, as well as their mass-angle and mass-total kinetic energy correlations are investigated. Conclusions: Entrance channel reaction dynamics are similar with both 48Ca and 50Ti projectiles. Both are expected to lead to the formation of a compound nucleus by fusion if they have enough energy to get in contact with the side of the 249Bk target.

  7. Twist decomposition of proton structure from BFKL and BK amplitudes

    CERN Document Server

    Motyka, Leszek

    2014-01-01

    An analysis of twist composition of Balitsky-Kovchegov (BK) amplitude is performed in the double logarithmic limit. In this limit the BK evolution of color dipole -- proton scattering is equivalent to BFKL evolution which follows from vanishing of the Bartels vertex in the collinear limit. We perform twist decomposition of the BFKL/BK amplitude for proton structure functions and find compact analytic expressions that provide accurate approximations for higher twist amplitudes. The BFKL/BK higher twist amplitudes are much smaller than those following from eikonal saturation models.

  8. 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.

  9. 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

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

    DEFF Research Database (Denmark)

    Grunnet, Morten; Kaufmann, Walter A

    2004-01-01

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

  11. Systematic study of spatiotemporal dynamics of intense femtosecond laser pulses in BK-7 glass

    Indian Academy of Sciences (India)

    Ram Gopal; V Deepak; S Sivaramakrishnan

    2007-04-01

    In this paper we present a systematic study of the spatial and temporal effects of intense femtosecond laser pulses in BK-7 over a broad range of input powers, 1–1000 times the critical power for self-focusing (cr) by numerically solving the nonlinear Schrödinger equation (NLS). Most numerical studies have not been extended to such high powers. A clear-cut classification of spatio-temporal dynamics up to very high powers into three regimes – the group-velocity dispersion (GVD) regime, the ionization regime and the dominant plasma regime – as done here, is a significant step towards a better understanding. Further, we examine in detail the role of GVD in channel formation by comparing BK-7 to an `artificial' medium. Our investigations bring forth the important observation that diffraction plays a minimal role in the formation of multiple cones and that plasma plays a diffraction-like role at very high powers. A detailed study of the spatio-temporal dynamics in any condensed medium over this range of powers has not been reported hitherto, to the best of our knowledge. We also suggest appropriate operational powers for various applications employing BK-7 on the basis of our results.

  12. 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.

  13. Localization and function of ATP-sensitive potassium channels in human skeletal muscle

    DEFF Research Database (Denmark)

    Nielsen, Jens Jung; Kristensen, Michael; Hellsten, Ylva

    2003-01-01

    The present study investigated the localization of ATP-sensitive K+ (KATP) channels in human skeletal muscle and the functional importance of these channels for human muscle K+ distribution at rest and during muscle activity. Membrane fractionation based on the giant vesicle technique...

  14. High glucose-induced oxidative stress increases transient receptor potential channel expression in human monocytes

    DEFF Research Database (Denmark)

    Wuensch, Tilo; Thilo, Florian; Krueger, Katharina;

    2010-01-01

    Transient receptor potential (TRP) channel-induced cation influx activates human monocytes, which play an important role in the pathogenesis of atherosclerosis. In the present study, we investigated the effects of high glucose-induced oxidative stress on TRP channel expression in human monocytes....

  15. Molecular Networks Involved in the Immune Control of BK Polyomavirus

    Directory of Open Access Journals (Sweden)

    Eva Girmanova

    2012-01-01

    Full Text Available BK polyomavirus infection is the important cause of virus-related nephropathy following kidney transplantation. BK virus reactivates in 30%–80% of kidney transplant recipients resulting in BK virus-related nephropathy in 1%–10% of cases. Currently, the molecular processes associated with asymptomatic infections in transplant patients infected with BK virus remain unclear. In this study we evaluate intrarenal molecular processes during different stages of BKV infection. The gene expression profiles of 90 target genes known to be associated with immune response were evaluated in kidney graft biopsy material using TaqMan low density array. Three patient groups were examined: control patients with no evidence of BK virus reactivation (n=11, infected asymptomatic patients (n=9, and patients with BK virus nephropathy (n=10. Analysis of biopsies from asymptomatic viruria patients resulted in the identification of 5 differentially expressed genes (CD3E, CD68, CCR2, ICAM-1, and SKI (P<0.05, and functional analysis showed a significantly heightened presence of costimulatory signals (e.g., CD40/CD40L; P<0.05. Gene ontology analysis revealed several biological networks associated with BKV immune control in comparison to the control group. This study demonstrated that asymptomatic BK viruria is associated with a different intrarenal regulation of several genes implicating in antiviral immune response.

  16. Molecular networks involved in the immune control of BK polyomavirus.

    Science.gov (United States)

    Girmanova, Eva; Brabcova, Irena; Klema, Jiri; Hribova, Petra; Wohlfartova, Mariana; Skibova, Jelena; Viklicky, Ondrej

    2012-01-01

    BK polyomavirus infection is the important cause of virus-related nephropathy following kidney transplantation. BK virus reactivates in 30%-80% of kidney transplant recipients resulting in BK virus-related nephropathy in 1%-10% of cases. Currently, the molecular processes associated with asymptomatic infections in transplant patients infected with BK virus remain unclear. In this study we evaluate intrarenal molecular processes during different stages of BKV infection. The gene expression profiles of 90 target genes known to be associated with immune response were evaluated in kidney graft biopsy material using TaqMan low density array. Three patient groups were examined: control patients with no evidence of BK virus reactivation (n = 11), infected asymptomatic patients (n = 9), and patients with BK virus nephropathy (n = 10). Analysis of biopsies from asymptomatic viruria patients resulted in the identification of 5 differentially expressed genes (CD3E, CD68, CCR2, ICAM-1, and SKI) (P < 0.05), and functional analysis showed a significantly heightened presence of costimulatory signals (e.g., CD40/CD40L; P < 0.05). Gene ontology analysis revealed several biological networks associated with BKV immune control in comparison to the control group. This study demonstrated that asymptomatic BK viruria is associated with a different intrarenal regulation of several genes implicating in antiviral immune response.

  17. Identification of TRPM7 channels in human intestinal interstitial cells of Cajal

    Institute of Scientific and Technical Information of China (English)

    Byung Joo Kim; Kyu Joo Park; Hyung Woo Kim; Seok Choi; Jae Yeoul Jun; In Youb Chang; Ju-Hong Jeon; Insuk So; Seon Jeong Kim

    2009-01-01

    AIM: To investigate the characteristics of slow electrical waves and the presence of transient receptor potential melastatin-type 7 (TRPM7) in the human gastrointestinal (GI) tract. METHODS: Conventional microelectrode techniques were used to record intracellular electrical responses from human GI smooth muscle tissue. Immunohistochemistry was used to identify TRPM7 channels in interstitial cells of Cajal (ICCs). RESULTS: The human GI tract generated slow electrical waves and had ICCs which functioned as pacemaker cells. Flufenamic acid, a nonselective cation channel blocker, and 2-APB (2-aminoethoxydiphenyl borate) and La3~+, TRPM7 channel blockers, inhibited the slow waves. Also, TRPM7 channels were expressed in ICCs in human tissue. CONCLUSION: These results suggest that the human GI tract generates slow waves and that TRPM7 channels expressed in the ICCs may be involved in the generation of the slow waves.

  18. Increased BK viremia and progression to BK-virus nephropathy following high-dose intravenous immunoglobulin for acute cellular rejection.

    Science.gov (United States)

    Boonyapredee, Maytee; Knight, Kendral; Little, Dustin

    2014-06-01

    BK virus nephropathy and cellular rejection are common causes of allograft dysfunction in renal transplant recipients. The two can be difficult to distinguish on allograft biopsy and can be present simultaneously. Management of the patient with coexistent BK infection and rejection is complicated by the conflicting ideals of decreasing immunosuppression to treat the former and increasing immunosuppression to treat the latter. The authors present the case of a 57-year-old renal transplant recipient who underwent allograft biopsy 8 weeks post-transplant for evaluation of increased serum creatinine in the setting of BK viremia (BKV). Biopsy revealed Banff classification 1b acute cellular rejection, with insufficient evidence to diagnose BK virus-associated nephropathy. The patient was administered intravenous immune globulin (IVIG), with no other changes in immunosuppressive therapy. Plasma and urine BK increased exponentially following IVIG administration, and allograft function further deteriorated. Repeat biopsy showed overt BK viral nephropathy, and BKV and creatinine decreased only after reduction in immunosuppression and initiation of leflunomide. Although case series have suggested a potential role for IVIG in the setting of BK infection, further study is needed to define the safety and efficacy of this approach. Reprint & Copyright © 2014 Association of Military Surgeons of the U.S.

  19. BK/TD models for analyzing in vitro impedance data on cytotoxicity.

    Science.gov (United States)

    Teng, S; Barcellini-Couget, S; Beaudouin, R; Brochot, C; Desousa, G; Rahmani, R; Pery, A R R

    2015-06-01

    The ban of animal testing has enhanced the development of new in vitro technologies for cosmetics safety assessment. Impedance metrics is one such technology which enables monitoring of cell viability in real time. However, analyzing real time data requires moving from static to dynamic toxicity assessment. In the present study, we built mechanistic biokinetic/toxicodynamic (BK/TD) models to analyze the time course of cell viability in cytotoxicity assay using impedance. These models account for the fate of the tested compounds during the assay. BK/TD models were applied to analyze HepaRG cell viability, after single (48 h) and repeated (4 weeks) exposures to three hepatotoxic compounds (coumarin, isoeugenol and benzophenone-2). The BK/TD models properly fit the data used for their calibration that was obtained for single or repeated exposure. Only for one out of the three compounds, the models calibrated with a single exposure were able to predict repeated exposure data. We therefore recommend the use of long-term exposure in vitro data in order to adequately account for chronic hepatotoxic effects. The models we propose here are capable of being coupled with human biokinetic models in order to relate dose exposure and human hepatotoxicity.

  20. Changes in tumor-antigen expression proifle as human small-cell lung cancers progress

    Institute of Scientific and Technical Information of China (English)

    Li-Sheng Ge; Neil T Hoa; Nils Lambrecht; Maria Dacosta-Iyer; Yi Ouyang; Amir Abolhoda; Martin R Jadus

    2015-01-01

    AbstrAct Objective:Our group has previously observed that in patients with small-cell lung cancers (SCLCs), the expression of a tumor antigen, glioma big potassium (gBK) ion channel, is higher at the time of death than when the cancer is ifrst treated by surgical resection. This study aimed to determine whether this dichotomy was common in other potential lung tumor antigens by examining the same patient samples using our more extensive proifle analysis of tumor-antigen precursor protein (TAPP). We then tested the hypothesis that therapeutic intervention may inadvertently cause this increased gBK production. Methods:SCLC samples (eight surgical resections and three autopsy samples) and three control lungs were examined by quantitative real-time polymerase chain reaction for 42 potential TAPPs that represent potential T-cell-mediated immunological targets. Results:Twenty-two TAPP mRNAs displayed the same profile as gBK, i.e., more mRNAs were expressed at autopsy than in their surgical counterparts. B-cyclin and mouse double minute 2, human homolog of P53-binding protein were elevated in both autopsy and surgical specimens above the normal-lung controls. When HTB119 cells were incubated with doxorubicin, gBK was strongly induced, as conifrmed by intracellular lfow cytometry with a gBK-speciifc antibody. Conclusion:Our findings suggested that more immunological targets became available as the tumor responded to chemotherapy and proceeded toward its terminal stages.

  1. Cloning and characterization of a human delayed rectifier potassium channel gene.

    Science.gov (United States)

    Albrecht, B; Lorra, C; Stocker, M; Pongs, O

    1993-01-01

    A human genomic DNA library was screened for sequences homologues to the rat delayed rectifier Kv 2.1 (DRK1) K+ channel cDNA. Three phages were isolated which hybridized to Kv 2.1 cDNA probes. Alignment of the human genomic DNA sequence with the rat cDNA sequence indicated that the open reading frame (ORF) is interrupted by a large intervening sequence, that separates exons encoding the membrane spanning core region of the K+ channel polypeptide. The Kv 2.1 gene occurs once in the human genome and has been mapped to chromosome 20. The human, mouse and rat Kv 2.1 proteins have been highly conserved, showing only a few substitutions outside of the membrane spanning domains in the amino- and carboxy-terminal cytoplasmic domains. Nevertheless, expression of human DRK1 channels in Xenopus oocytes showed that mouse, rat and human Kv 2.1 channels have distinct pharmacological and electrophysiological properties. The observed differences in activation, voltage-dependence, 4-aminopyridine sensitivity and single-channel conductance have to be attributed to amino acid substitutions in the amino-and/or carboxy-terminal cytoplasmic domains. Obviously, these domains of Kv 2.1 channels influence biophysical K+ channel properties, which are thought to be determined solely by the membrane spanning core domain of potassium channels.

  2. Ion-exchanged tapered-waveguide laser in neodymium-doped BK7 glass.

    Science.gov (United States)

    Hettrick, S J; Mackenzie, J I; Harris, R D; Wilkinson, J S; Shepherd, D P; Tropper, A C

    2000-10-01

    We report what is to our knowledge the first operation of a planar dielectric tapered-waveguide laser. The waveguide laser is fabricated by potassium-ion exchange in Nd(3+) -doped BK7 glass and consists of a single-mode channel waveguide of a few micrometers' width followed by a linear taper up to a broad region with a width of ~180microm . A slope efficiency of 42% is found both in the tapers and in standard channel waveguides fabricated upon the same substrate, indicating that the tapers and the channels have similar internal losses; hence the low-loss nature of the tapered beam expansion. The output from either end of the tapered structure is found to be nearly diffraction limited.

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

    Directory of Open Access Journals (Sweden)

    David L Prole

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

  4. New disguises for an old channel: MaxiK channel beta-subunits.

    Science.gov (United States)

    Orio, Patricio; Rojas, Patricio; Ferreira, Gonzalo; Latorre, Ramón

    2002-08-01

    Ca(2+)-activated K(+) channels of large conductance (MaxiK or BK channels) control a large variety of physiological processes, including smooth muscle tone, neurosecretion, and hearing. Despite being coded by a single gene (Slowpoke), the diversity of MaxiK channels is great. Regulatory b-subunits, splicing, and metabolic regulation create this diversity fundamental to the adequate function of many tissues.

  5. Polyomavirus specific cellular immunity: from BK-virus-specific cellular immunity to BK-virus-associated nephropathy ?

    Directory of Open Access Journals (Sweden)

    manon edekeyser

    2015-06-01

    Full Text Available In renal transplantation, BK-virus-associated nephropathy has emerged as a major complication, with a prevalence of 5–10% and graft loss in >50% of cases. BK-virus is a member of the Polyomavirus family and rarely induces apparent clinical disease in the general population. However, replication of polyomaviruses, associated with significant organ disease, is observed in patients with acquired immunosuppression, which suggests a critical role for virus-specific cellular immunity to control virus replication and prevent chronic disease. Monitoring of specific immunity combined with viral load could be used to individually assess the risk of viral reactivation and virus control. We review the current knowledge on BK-virus specific cellular immunity and, more specifically, in immunocompromised patients. In the future, immune-based therapies could allow us to treat and prevent BK-virus-associated nephropathy.

  6. Human beta-defensin 1, a new animal toxin-like blocker of potassium channel.

    Science.gov (United States)

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

    2016-04-01

    The discovery of human β-defensin 2 (hBD2), as a Kv1.3 channel inhibitor with the unique molecular mechanism and novel immune modulatory function, suggests that human β-defensins are a novel class of channel ligands. Here, the function and mechanism of the human β-defensin 1 (hBD1) binding to potassium channels was investigated. Based on the structural similarity between hBD1 and Kv1.3 channel-sensitive hBD2, hBD1 was found to selectively inhibit human and mouse Kv1.3 channels with IC50 values of 11.8 ± 3.1 μM and 13.2 ± 4.0 μM, respectively. Different from hBD2 modifying Kv1.3 channel activation and increasing activation time constant, hBD1 did not affect the activation feature of both human and mouse Kv1.3 channels. In comparison with hBD2 simultaneously interacting with the extracellular S1-S2 linker and pore region of Kv1.3 channel, the chimeric channel and mutagenesis experiments showed that hBD1 only bound to the extracellular pore region of Kv1.3 channel instead of extracellular S1-S2 linker or S3-S4 linker. Together, these findings enhance knowledge of hBD1 as a new immune-related Kv1.3 channel blocker and highlight the major functional differences between hBD1 and hBD2 to explore in future research.

  7. BK virus-associated hemorrhagıc cystitis in patients wıth allogeneıc hematopoıetıc cell transplantation: report of three cases

    Directory of Open Access Journals (Sweden)

    Duygu Mert

    2017-06-01

    Full Text Available BK virus is a human polyoma virus. It is acquired in early childhood and remains life-long latent in the genitourinary system. BK virus replication is more common in receiving immunosuppressive therapy receiving patients and transplant patients. BK virus could cause hemorrhagic cystitis in patients with allogeneic stem cell transplantation. Hemorrhagic cystitis is a serious complication of hematopoietic stem cell transplantation. Hemorrhagic cystitis could cause morbidity and long stay in the hospital. Diagnosis is more frequently determined by the presence of BK virus DNA detected with quantitative or real-time PCR testing in serum or plasma and less often in urine. The reduction of immunosuppression is effective in the treatment of BK virus infection. There are also several agents with anti-BK virus activity. Cidofovir is an active agent against a variety of DNA viruses including poliomyoma viruses and it is a cytosine nucleotide analogue. Intravenous immunoglobulin IgG (IVIG also includes antibodies against BK and JC (John Cunningham viruses. Hereby, we report three cases of hemorrhagic cystitis. Hemorrhagic cystitis developed in all these three cases of allogeneic stem cell transplantation due to acute myeloid leukemia (AML. BK virus were detected as the cause of hemorrhagic cystitis in these patients. Irrigation of the bladder was performed. Then levofloxacin 1×750 mg intravenous and IVIG 0.5 gr/kg were started. But the hematuria did not decreased. In the first case, treatment with leflunomide was started, but patient died due to refractory AML and severe graft-versus-host disease after 4th day of leflunamide and levofloxacin treatments. Cidofovir treatment and the reduction of immunosuppressive treatment decreased the BK virus load and resulted symptomatic improvement in the second case. Initiation of cidofovir was planned in the third case. Administration of cidofovir together with the reduction of immunosuppression in the treatment of

  8. Presynaptic Ca2+-activated K+ channels in glutamatergic hippocampal terminals and their role in spike repolarization and regulation of transmitter release.

    Science.gov (United States)

    Hu, H; Shao, L R; Chavoshy, S; Gu, N; Trieb, M; Behrens, R; Laake, P; Pongs, O; Knaus, H G; Ottersen, O P; Storm, J F

    2001-12-15

    Large-conductance Ca(2+)-activated K(+) channels (BK, also called Maxi-K or Slo channels) are widespread in the vertebrate nervous system, but their functional roles in synaptic transmission in the mammalian brain are largely unknown. By combining electrophysiology and immunogold cytochemistry, we demonstrate the existence of functional BK channels in presynaptic terminals in the hippocampus and compare their functional roles in somata and terminals of CA3 pyramidal cells. Double-labeling immunogold analysis with BK channel and glutamate receptor antibodies indicated that BK channels are targeted to the presynaptic membrane facing the synaptic cleft in terminals of Schaffer collaterals in stratum radiatum. Whole-cell, intracellular, and field-potential recordings from CA1 pyramidal cells showed that the presynaptic BK channels are activated by calcium influx and can contribute to repolarization of the presynaptic action potential (AP) and negative feedback control of Ca(2+) influx and transmitter release. This was observed in the presence of 4-aminopyridine (4-AP, 40-100 microm), which broadened the presynaptic compound action potential. In contrast, the presynaptic BK channels did not contribute significantly to regulation of action potentials or transmitter release under basal experimental conditions, i.e., without 4-AP, even at high stimulation frequencies. This is unlike the situation in the parent cell bodies (CA3 pyramidal cells), where BK channels contribute strongly to action potential repolarization. These results indicate that the functional role of BK channels depends on their subcellular localization.

  9. 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

  10. The sedimentary dynamics in natural and human-influenced delta channel belts

    NARCIS (Netherlands)

    Hobo, N.

    2015-01-01

    This study investigates the increased anthropogenic influence on the within-channel belt sedimentary dynamics in the Rhine delta. To make this investigation, the sedimentary dynamics within the life-cycle of a single channel belt were reconstructed for three key periods of increasing human impact, w

  11. Progression from Sustained BK Viruria to Sustained BK Viremia with Immunosuppression Reduction Is Not Associated with Changes in the Noncoding Control Region of the BK Virus Genome

    Directory of Open Access Journals (Sweden)

    Imran A. Memon

    2012-01-01

    We performed PCR amplification and sequencing of (1 stored urine and (2 plasma samples from the time of peak viremia from 11 patients with sustained viremia who participated in a 200-patient clinical trial. The antimetabolite was withdrawn for BK viremia and reduction of the calcineurin inhibitor for sustained BK viremia. DNA sequencing from the 11 patients with sustained viremia revealed 8 insertions, 16 transversions, 3 deletions, and 17 transitions. None were deemed significant. No patient developed clinically evident BKVAN. Our data support, at a genomic level, the effectiveness of reduction of immunosuppression for prevention of progression from viremia to BKVAN.

  12. Tetrachromacy of human vision: spectral channels and primary colors

    Science.gov (United States)

    Gavrik, Vitali V.

    2002-06-01

    Full-color imaging requires four channels as, in contrast to a colorimeter, can add no primary to matched scene colors themselves. An ideal imaging channel should have the same spectral sensitivity of scene recording as a retinal receptor and evoke the same primary color sensation. The alternating matching functions of a triad of real primaries are inconsistent with the three cones but explicable of two pairs of independent opponent receptors with their alternating blue-yellow and green-red chromatic axes in the color space. Much other controversy of trichromatic approach can also be explained with the recently proposed intra- receptor processes in the photopic rod and cone, respectively. Each of their four primary sensations, unmixed around 465, 495, 575, and 650 nm, is evoked within a different spectral region. The current trichromatic photographic systems have been found separately to approximate the blue and red receptors, as well as their spectral opponency against the respective yellow and blue- green receptors simulated with a single middle-wave imaging channel. The channel sensitivities are delimited by the neutral points of rod and cone and cannot simulate the necessary overlap of non-opponent channels for properly to render some mixed colors. The yellow and cyan positive dyes closely control the brightness of blue and red sensations, respectively. Those red and blue respectively to control the yellow and blue-green sensations on brightness scales are replaced by magenta dye, controlling them together. Accurate rendering of natural saturation metameric colors, problematic blue-green, purple-red, and low-illumination colors requires to replace the hybrid 'green' channel with the blue-green and yellow channels.

  13. 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.

  14. Large-conductance voltage- and Ca2+-activated K+ channel regulation by protein kinase C in guinea pig urinary bladder smooth muscle.

    Science.gov (United States)

    Hristov, Kiril L; Smith, Amy C; Parajuli, Shankar P; Malysz, John; Petkov, Georgi V

    2014-03-01

    Large-conductance voltage- and Ca(2+)-activated K(+) (BK) channels are critical regulators of detrusor smooth muscle (DSM) excitability and contractility. PKC modulates the contraction of DSM and BK channel activity in non-DSM cells; however, the cellular mechanism regulating the PKC-BK channel interaction in DSM remains unknown. We provide a novel mechanistic insight into BK channel regulation by PKC in DSM. We used patch-clamp electrophysiology, live-cell Ca(2+) imaging, and functional studies of DSM contractility to elucidate BK channel regulation by PKC at cellular and tissue levels. Voltage-clamp experiments showed that pharmacological activation of PKC with PMA inhibited the spontaneous transient BK currents in native freshly isolated guinea pig DSM cells. Current-clamp recordings revealed that PMA significantly depolarized DSM membrane potential and inhibited the spontaneous transient hyperpolarizations in DSM cells. The PMA inhibitory effects on DSM membrane potential were completely abolished by the selective BK channel inhibitor paxilline. Activation of PKC with PMA did not affect the amplitude of the voltage-step-induced whole cell steady-state BK current or the single BK channel open probability (recorded in cell-attached mode) upon inhibition of all major Ca(2+) sources for BK channel activation with thapsigargin, ryanodine, and nifedipine. PKC activation with PMA elevated intracellular Ca(2+) levels in DSM cells and increased spontaneous phasic and nerve-evoked contractions of DSM isolated strips. Our results support the concept that PKC activation leads to a reduction of BK channel activity in DSM via a Ca(2+)-dependent mechanism, thus increasing DSM contractility.

  15. Localization of large conductance calcium-activated potassium channels and their effect on calcitonin gene-related peptide release in the rat trigemino-neuronal pathway

    DEFF Research Database (Denmark)

    Wulf-Johansson, H.; Amrutkar, D.V.; Hay-Schmidt, Anders

    2010-01-01

    Large conductance calcium-activated potassium (BK(Ca)) channels are membrane proteins contributing to electrical propagation through neurons. Calcitonin gene-related peptide (CGRP) is a neuropeptide found in the trigeminovascular system (TGVS). Both BK(Ca) channels and CGRP are involved in migraine...

  16. BK viruria and viremia in children with systemic lupus erythematosus.

    Science.gov (United States)

    Gupta, Nirupama; Nguyen, Cuong Q; Modica, Renee F; Elder, Melissa E; Garin, Eduardo H

    2017-04-11

    BK virus (BKV) is a ubiquitous polyoma virus that lies dormant in the genitourinary tract once acquired in early childhood. In states of cellular immunodeficiency, the virus can reactivate to cause hemorrhagic cystitis and nephritis. Children with systemic lupus erythematosus (SLE) have an increased risk of developing infectious complications secondary to their immunocompromised state from the administration of several immuno-modulatory drugs. Currently, there are no data regarding the prevalence of BK viruria or viremia in children with SLE. We conducted a prospective cohort study involving children with SLE of 18 years and younger. We obtained urine and blood samples at baseline and every 3 months up to 1 year for BK virus detection by real-time, quantitative polymerase chain reaction analysis. A comprehensive review of demographic information, clinical characteristics and medication history was also obtained. Thirty-two pediatric patients (26 females and 6 males) with SLE were enrolled. Median age at the time of SLE diagnosis and enrollment into study was 13.6 years and 16.0 years old, respectively. The prevalence at enrollment was 3.1% (1/32) for BK viruria and 6.2% (2/32) for BK viremia. During the study period, 3 patients had viruria, 5 had viremia and 4 had both viruria and viremia. Of the 12 patients with BKV reactivation, only one was positive for microscopic hematuria, all others were asymptomatic. A total of nine of 97(9.2%) urine samples and 10 of 96(10.4%) blood samples were positive for BK virus. The most commonly utilized biologics in this cohort group were Rituximab (90.6%), Abatacept (12.5%), and Belimumab (9.3%). The type of medication exposure and clinical characteristics did not statistically differ between the groups that did or did not have BK viruria and/or viremia. Our study suggests that pediatric patients with SLE have BK viremia and/or viruria at a higher rate than the general healthy population, although the significance of the

  17. Localization of large conductance calcium-activated potassium channels and their effect on calcitonin gene-related peptide release in the rat trigemino-neuronal pathway.

    Science.gov (United States)

    Wulf-Johansson, H; Amrutkar, D V; Hay-Schmidt, A; Poulsen, A N; Klaerke, D A; Olesen, J; Jansen-Olesen, I

    2010-06-02

    Large conductance calcium-activated potassium (BK(Ca)) channels are membrane proteins contributing to electrical propagation through neurons. Calcitonin gene-related peptide (CGRP) is a neuropeptide found in the trigeminovascular system (TGVS). Both BK(Ca) channels and CGRP are involved in migraine pathophysiology. Here we study the expression and localization of BK(Ca) channels and CGRP in the rat trigeminal ganglion (TG) and the trigeminal nucleus caudalis (TNC) as these structures are involved in migraine pain. Also the effect of the BK(Ca) channel blocker iberiotoxin and the BK(Ca) channel opener NS11021 on CGRP release from isolated TG and TNC was investigated. By RT-PCR, BK(Ca) channel mRNA was detected in the TG and the TNC. A significant difference in BK(Ca) channel mRNA transcript levels were found using qPCR between the TNC as compared to the TG. The BK(Ca) channel protein was more expressed in the TNC as compared to the TG shown by western blotting. Immunohistochemistry identified BK(Ca) channels in the nerve cell bodies of the TG and the TNC. The beta2- and beta4-subunit proteins were found in the TG and the TNC. They were both more expressed in the TNC as compared to TG shown by western blotting. In isolated TNC, the BK(Ca) channel blocker iberiotoxin induced a concentration-dependent release of CGRP that was attenuated by the BK(Ca) channel opener NS11021. No effect on basal CGRP release was found by NS11021 in isolated TG or TNC or by iberiotoxin in TG. In conclusion, we found both BK(Ca) channel mRNA and protein expression in the TG and the TNC. The BK(Ca) channel protein and the modulatory beta2- and beta4-subunt proteins were more expressed in the TNC than in the TG. Iberiotoxin induced an increase in CGRP release from the TNC that was attenuated by NS11021. Thus, BK(Ca) channels might have a role in trigeminovascular pain transmission.

  18. Functional KCa1.1 channels are crucial for regulating the proliferation, migration and differentiation of human primary skeletal myoblasts

    Science.gov (United States)

    Tajhya, Rajeev B; Hu, Xueyou; Tanner, Mark R; Huq, Redwan; Kongchan, Natee; Neilson, Joel R; Rodney, George G; Horrigan, Frank T; Timchenko, Lubov T; Beeton, Christine

    2016-01-01

    Myoblasts are mononucleated precursors of myofibers; they persist in mature skeletal muscles for growth and regeneration post injury. During myotonic dystrophy type 1 (DM1), a complex autosomal-dominant neuromuscular disease, the differentiation of skeletal myoblasts into functional myotubes is impaired, resulting in muscle wasting and weakness. The mechanisms leading to this altered differentiation are not fully understood. Here, we demonstrate that the calcium- and voltage-dependent potassium channel, KCa1.1 (BK, Slo1, KCNMA1), regulates myoblast proliferation, migration, and fusion. We also show a loss of plasma membrane expression of the pore-forming α subunit of KCa1.1 in DM1 myoblasts. Inhibiting the function of KCa1.1 in healthy myoblasts induced an increase in cytosolic calcium levels and altered nuclear factor kappa B (NFκB) levels without affecting cell survival. In these normal cells, KCa1.1 block resulted in enhanced proliferation and decreased matrix metalloproteinase secretion, migration, and myotube fusion, phenotypes all observed in DM1 myoblasts and associated with disease pathogenesis. In contrast, introducing functional KCa1.1 α-subunits into DM1 myoblasts normalized their proliferation and rescued expression of the late myogenic marker Mef2. Our results identify KCa1.1 channels as crucial regulators of skeletal myogenesis and suggest these channels as novel therapeutic targets in DM1. PMID:27763639

  19. Reconstitution of Human Ion Channels into Solvent-free Lipid Bilayers Enhanced by Centrifugal Forces.

    Science.gov (United States)

    Hirano-Iwata, Ayumi; Ishinari, Yutaka; Yoshida, Miyu; Araki, Shun; Tadaki, Daisuke; Miyata, Ryusuke; Ishibashi, Kenichi; Yamamoto, Hideaki; Kimura, Yasuo; Niwano, Michio

    2016-05-24

    Artificially formed bilayer lipid membranes (BLMs) provide well-defined systems for functional analyses of various membrane proteins, including ion channels. However, difficulties associated with the integration of membrane proteins into BLMs limit the experimental efficiency and usefulness of such BLM reconstitution systems. Here, we report on the use of centrifugation to more efficiently reconstitute human ion channels in solvent-free BLMs. The method improves the probability of membrane fusion. Membrane vesicles containing the human ether-a-go-go-related gene (hERG) channel, the human cardiac sodium channel (Nav1.5), and the human GABAA receptor (GABAAR) channel were formed, and the functional reconstitution of the channels into BLMs via vesicle fusion was investigated. Ion channel currents were recorded in 67% of the BLMs that were centrifuged with membrane vesicles under appropriate centrifugal conditions (14-55 × g). The characteristic channel properties were retained for hERG, Nav1.5, and GABAAR channels after centrifugal incorporation into the BLMs. A comparison of the centrifugal force with reported values for the fusion force revealed that a centrifugal enhancement in vesicle fusion was attained, not by accelerating the fusion process but by accelerating the delivery of membrane vesicles to the surface of the BLMs, which led to an increase in the number of membrane vesicles that were available for fusion. Our method for enhancing the probability of vesicle fusion promises to dramatically increase the experimental efficiency of BLM reconstitution systems, leading to the realization of a BLM-based, high-throughput platform for functional assays of various membrane proteins.

  20. Data of evolutionary structure change: 1BK9A-2QOGD [Confc[Archive

    Lifescience Database Archive (English)

    Full Text Available 1BK9A-2QOGD 1BK9 2QOG A D SLIQFETLIMKVAKKSGMFWYSNYGCYCGWGGQGRPQDA...TDRCCFVHDCCYGKVTGCDPKMDVYSFSEENGDIVCGGDDPCKKEICECDRAAAICFRDNLTLYNDKKYWAFGAKNCPQEESEPC SLLQFNKMI.../pdbChain> 2QOGD LSTYK-NEYMF

  1. BK Virus-Associated Nephropathy without Viremia in an Adolescent Kidney Transplant Recipient

    Directory of Open Access Journals (Sweden)

    Kraisoon Lomjansook, M.D.

    2017-09-01

    Full Text Available BK virus can reactivate in kidney transplant recipients leading to BK virus-associated nephropathy (BKVAN and allograft dysfunction. Pathogenesis begins with viral replication, follows by viruria, viremia and nephropathy. Screening tools recommended for viral detection are urine and blood BK viral load. Viremia has higher positive predictive value than viruria, thus several guidelines recommend using viremia to determine whether renal biopsy, a gold standard for diagnosis of BKVAN is needed. We present a 16-year-old boy who developed BKVAN five months after deceased donor kidney transplantation. He had increased serum creatinine with negative blood BK viral load. BK nephropathy was diagnosed in kidney graft biopsy. The urine showed BK viruria. Immunosuppressant was reduced and ciprofloxacin given. Viruria disappeared and repeated graft biopsy was normal 4 months later. BK viremia was negative through 1 year follow up. We conclude that BKVAN may occur even without viremia and BK viruria may be considered for screening tool.

  2. Urinary BK virus excretion in children newly diagnosed with acute lymphoblastic leukemia

    Directory of Open Access Journals (Sweden)

    Nahid Raeesi

    2012-01-01

    Conclusion: To demonstrate the role of BK virus in inducing ALL or increasing the number of relapses, prospective studies on larger scale of population and evaluating both serum and urine for BK virus are recommended.

  3. Internalisasi Mind Skills Mahasiswa Bimbingan Konseling (BK Melalui Experiential Learning

    Directory of Open Access Journals (Sweden)

    Ribut Purwaningrum

    2014-06-01

    Full Text Available Tujuan penelitian ini adalah mengetahui bagaimana menerapkan model pembelajaran experi-ential learning untuk internalisasi mind skills mahasiswa jurusan BK. Rancangan penelitian ini menggunakan penelitian tindakan kelas. Subjek penelitian adalah mahasiswa BK offering C angkatan 2011 peserta matakuliah Konseling Individual. Penelitian dilakukan selama tiga siklus dengan jabaran siklus I sebanyak 6 pertemuan, siklus II sebanyak 6 pertemuan, dan siklus III sebanyak 3 pertemuan. Pengumpulan data dilakukan menggunakan metode kuantitatif dan kualitatif. Data kuantitatif diolah menggunakan statistik dan diinterpretasi secara kualitatif. Analisis data dilakukan secara kualitatif merujuk pada Miles dan Huberman, meliputi reduksi data, penyajian data, dan pengambilan simpulan. Hasil penelitian menunjukkan bahwa model pembelajaran experiential learning mampu digunakan sebagai strategi internalisasi mind skills mahasiswa BK adalah experiential learning yang dilakukan secara berkesinambungan dalam tahapnya, sehingga mampu menyentuh aspek ‘feeling’,‘watching or describing’,‘thinking’ dan ‘doing’. Kata kunci: internalisasi, mind skills, experiential learning

  4. Transient receptor potential canonical type 3 channels and blood pressure in humans

    DEFF Research Database (Denmark)

    Thilo, Florian; Baumunk, Daniel; Krause, Hans;

    2009-01-01

    There is evidence that transient receptor potential canonical type 3 (TRPC3) cation channels are involved in the regulation of blood pressure, but this has not been studied using human renal tissue. We tested the hypothesis that the expression of TRPC3 in human renal tissue is associated with blood...

  5. Statistical characterization of the dynamic human body communication channel at 45 MHz.

    Science.gov (United States)

    Nie, Zedong; Ma, Jingjing; Chen, Hong; Wang, Lei

    2013-01-01

    The dynamic human body communication (HBC) propagation channel at 45 MHz was statistical characterized in this paper. A large amount of measurement data has been gathered in practical environment with real activities -treadmill running at different speeds in a lab room. The received power between two lower legs was acquired from three volunteers, with more than 60,000 snap shot of data in total. The statistical analyses confirmed that the HBC propagation channel at 45 MHz followed the Gamma and Lognormal distributions at the slower (2 km/h and 4 km/h) and faster (6 km/h and 8 km/h) running activities, respectively. The channel is insensitive to body motion with the maximum average fade duration is 0.0413 s and the most averaging bad channel duration time being less than 60 ms with the percentage of the bad channel duration time being less than 4.35%.

  6. 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.

  7. 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.

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

    Science.gov (United States)

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

    1999-01-01

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

  9. Molecular diversity of L-type Ca2+ channel transcripts in human fibroblasts.

    OpenAIRE

    Soldatov, N M

    1992-01-01

    The nucleotide sequence of cDNA encoding the human fibroblast Ca2+ channel of L type (HFCC) has been determined. It is highly homologous to L-type channels previously cloned from rabbit lung and heart as well as from rat brain. At least four sites of molecular diversity were identified in the nucleotide sequence of HFCC. Three of these include regions encoding the transmembrane segments IIS6, IIIS2, and IVS3, which are known to be important for channel gating properties. The positions of thes...

  10. Functional coupling between large-conductance potassium channels and Cav3.2 voltage-dependent calcium channels participates in prostate cancer cell growth

    Directory of Open Access Journals (Sweden)

    Florian Gackière

    2013-07-01

    It is strongly suspected that potassium (K+ channels are involved in various aspects of prostate cancer development, such as cell growth. However, the molecular nature of those K+ channels implicated in prostate cancer cell proliferation and the mechanisms through which they control proliferation are still unknown. This study uses pharmacological, biophysical and molecular approaches to show that the main voltage-dependent K+ current in prostate cancer LNCaP cells is carried by large-conductance BK channels. Indeed, most of the voltage-dependent current was inhibited by inhibitors of BK channels (paxillin and iberiotoxin and by siRNA targeting BK channels. In addition, we reveal that BK channels constitute the main K+ channel family involved in setting the resting membrane potential in LNCaP cells at around −40 mV. This consequently promotes a constitutive calcium entry through T-type Cav3.2 calcium channels. We demonstrate, using single-channel recording, confocal imaging and co-immunoprecipitation approaches, that both channels form macromolecular complexes. Finally, using flow cytometry cell cycle measurements, cell survival assays and Ki67 immunofluorescent staining, we show that both BK and Cav3.2 channels participate in the proliferation of prostate cancer cells.

  11. Study of the properties of the superheavy nuclei Z = 117 produced in the 249Bk + 48Ca reaction

    Science.gov (United States)

    Oganessian, Yu. Ts.; Abdullin, F. Sh.; Alexander, C.; Binder, J.; Boll, R. A.; Dmitriev, S. N.; Ezold, J.; Felker, K.; Gostic, J. M.; Grzywacz, R. K.; Hamilton, J. H.; Henderson, R. A.; Itkis, M. G.; Miernik, K.; Miller, D.; Moody, K. J.; Polyakov, A. N.; Ramayya, A. V.; Roberto, J. B.; Ryabinin, M. A.; Rykaczewski, K. P.; Sagaidak, R. N.; Shaughnessy, D. A.; Shirokovsky, I. V.; Shumeiko, M. V.; Stoyer, M. A.; Stoyer, N. J.; Subbotin, V. G.; Sukhov, A. M.; Tsyganov, Yu. S.; Utyonkov, V. K.; Voinov, A. A.; Vostokin, G. K.

    2014-03-01

    The reaction of 249Bk with 48Ca have been reinvestigated to provide new evidence for the discovery of element 117 on a larger number of events. The experiments were performed at five projectile energies and with a total beam dose of 48Ca of about 4.6×1019. Two isotopes 293,294117 were synthesized in the 249Bk+48Ca reaction, providing excitation functions and α-decay spectra of the produced isotopes that establishes these nuclei to be the products of the 4n- and 3n-evaporation channels, respectively. Decay properties of 293,294117 and of all the daughter products agree with the data of the experiment in which these nuclei were synthesized for the first time in 2010. The new 289115 events, populated by α decay of 293117, demonstrate the same decay properties as those observed for 289115 produced in the 243Am(48Ca,2n) reaction thus providing crossbombardment evidence. In addition, a single decay of 294118 was observed from the reaction with 249Cf - a result of the in-growth of 249Cf in the 249Bk target.

  12. Study of the properties of the superheavy nuclei Z = 117 produced in the 249Bk + 48Ca reaction

    Directory of Open Access Journals (Sweden)

    Oganessian Yu. Ts.

    2014-03-01

    Full Text Available The reaction of 249Bk with 48Ca have been reinvestigated to provide new evidence for the discovery of element 117 on a larger number of events. The experiments were performed at five projectile energies and with a total beam dose of 48Ca of about 4.6×1019. Two isotopes 293,294117 were synthesized in the 249Bk+48Ca reaction, providing excitation functions and α-decay spectra of the produced isotopes that establishes these nuclei to be the products of the 4n- and 3n-evaporation channels, respectively. Decay properties of 293,294117 and of all the daughter products agree with the data of the experiment in which these nuclei were synthesized for the first time in 2010. The new 289115 events, populated by α decay of 293117, demonstrate the same decay properties as those observed for 289115 produced in the 243Am(48Ca,2n reaction thus providing crossbombardment evidence. In addition, a single decay of 294118 was observed from the reaction with 249Cf – a result of the in-growth of 249Cf in the 249Bk target.

  13. 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...

  14. Communication channel modeling of human forearm with muscle fiber tissue characteristics.

    Science.gov (United States)

    Zhang, Shuang; Pun, Sio Hang; Mak, Peng Un; Qin, Yu-Ping; Liu, Yi-He; Vai, Mang I

    2016-09-14

    Human-Body Communication (HBC) is a wireless communication method using the human body tissue as a transmission medium for signals. This paper on the basis of human muscle fiber tissues' characteristics, it is first proposed to establish the analytical model of galvanic coupling human-body communication channel. In this model, the parallel and the transverse electrical characteristics of muscular tissue are fully considered, and the model accurately presents the transmission mechanism of galvanic coupling human-body communication signals in the channel. At last, through compare with the experimental results and calculation results, the maximum error of the model is 22.4% and the average error is 14.2% within the frequency range.

  15. Ion channels in human red blood cell membrane: actors or relics?

    Science.gov (United States)

    Thomas, Serge L Y; Bouyer, Guillaume; Cueff, Anne; Egée, Stéphane; Glogowska, Edyta; Ollivaux, Céline

    2011-04-15

    During the past three decades, electrophysiological studies revealed that human red blood cell membrane is endowed with a large variety of ion channels. The physiological role of these channels, if any, remains unclear; they do not participate in red cell homeostasis which is rather based on the almost total absence of cationic permeability and minute anionic conductance. They seem to be inactive in the "resting cell." However, when activated experimentally, ion channels can lead to a very high single cell conductance and potentially induce disorders, with the major risks of fast dehydration and dissipation of gradients. Could there be physiological conditions under which the red cell needs to activate these high conductances, or are ion channels relics of a function lost in anucleated cells? It has been demonstrated that they play a key role in diseases such as sickle cell anemia or malaria. This short overview of ion channels identified to-date in the human red cell membrane is an attempt to propose a dynamic role for these channels in circulating cells in health and disease.

  16. Correlation of apical fluid-regulating channel proteins with lung function in human COPD lungs.

    Directory of Open Access Journals (Sweden)

    Runzhen Zhao

    Full Text Available Links between epithelial ion channels and chronic obstructive pulmonary diseases (COPD are emerging through animal model and in vitro studies. However, clinical correlations between fluid-regulating channel proteins and lung function in COPD remain to be elucidated. To quantitatively measure epithelial sodium channels (ENaC, cystic fibrosis transmembrane conductance regulator (CFTR, and aquaporin 5 (AQP5 proteins in human COPD lungs and to analyze the correlation with declining lung function, quantitative western blots were used. Spearman tests were performed to identify correlations between channel proteins and lung function. The expression of α and β ENaC subunits was augmented and inversely associated with lung function. In contrast, both total and alveolar type I (ATI and II (ATII-specific CFTR proteins were reduced. The expression level of CFTR proteins was associated with FEV1 positively. Abundance of AQP5 proteins and extracellular superoxide dismutase (SOD3 was decreased and correlated with spirometry test results and gas exchange positively. Furthermore, these channel proteins were significantly associated with severity of disease. Our study demonstrates that expression of ENaC, AQP5, and CFTR proteins in human COPD lungs is quantitatively associated with lung function and severity of COPD. These apically located fluid-regulating channels may thereby serve as biomarkers and potent druggable targets of COPD.

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

    Science.gov (United States)

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

    2015-08-01

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

  18. 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...

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

    DEFF Research Database (Denmark)

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

    2006-01-01

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

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

    Science.gov (United States)

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

    2009-05-15

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

  1. Anion conductance of the human red cell is carried by a maxi-anion channel

    DEFF Research Database (Denmark)

    Glogowska, Edyta; Dyrda, Agnieszka; Cueff, Anne

    2010-01-01

    played by serum in the recruitment of multiple new conductance levels showing very complex kinetics and gating properties upon serum addition. These channels, which seem to be dormant under normal physiological conditions, are potentially activable and could confer a far higher anion conductance...... channels proper has never been clarified, and the informations obtained by different groups of electrophysiologists are rather badly matched. This study, using the cell-attached configuration of the patch-clamp technique, rationalizes and explains earlier confusing results by demonstrating...... that the diversity of anionic channel activities recorded in human erythrocytes corresponds to different kinetic modalities of a unique type of maxi-anion channel with multiple conductance levels and probably multiple gating properties and pharmacology, depending on conditions. It demonstrates the role of activator...

  2. L-type calcium channel gating is modulated by bradykinin with a PKC-dependent mechanism in NG108-15 cells.

    Science.gov (United States)

    Toselli, Mauro; Taglietti, Vanni

    2005-05-01

    Bradykinin (BK) excites dorsal root ganglion cells, leading to the sensation of pain. The actions of BK are thought to be mediated by heterotrimeric G protein-regulated pathways. Indeed there is strong evidence that in different cell types BK is involved in phosphoinositide breakdown following activation of G(q/11). In the present study we show that the Ca(2+) current flowing through L-type voltage-gated Ca(2+) channels in NG108-15 cells (differentiated in vitro to acquire a neuronal phenotype), measured using the whole-cell patch clamp configuration, is reversibly inhibited by BK in a voltage-independent fashion, suggesting a cascade process where a second messenger system is involved. This inhibitory action of BK is mimicked by the application of 1,2-oleoyl-acetyl glycerol (OAG), an analog of diacylglycerol that activates PKC. Interestingly, OAG occluded the effects of BK and both effects were blocked by selective PKC inhibitors. The down modulation of single L-type Ca(2+) channels by BK and OAG was also investigated in cell-attached patches. Our results indicate that the inhibitory action of BK involves activation of PKC and mainly shows up in a significant reduction of the probability of channel opening, caused by an increase and clustering of null sweeps in response to BK.

  3. Electrogenic transport and K(+) ion channel expression by the human endolymphatic sac epithelium.

    Science.gov (United States)

    Kim, Sung Huhn; Kim, Bo Gyung; Kim, Jin Young; Roh, Kyung Jin; Suh, Michelle J; Jung, JinSei; Moon, In Seok; Moon, Sung K; Choi, Jae Young

    2015-12-14

    The endolymphatic sac (ES) is a cystic organ that is a part of the inner ear and is connected to the cochlea and vestibule. The ES is thought to be involved in inner ear ion homeostasis and fluid volume regulation for the maintenance of hearing and balance function. Many ion channels, transporters, and exchangers have been identified in the ES luminal epithelium, mainly in animal studies, but there has been no functional study investigating ion transport using human ES tissue. We designed the first functional experiments on electrogenic transport in human ES and investigated the contribution of K(+) channels in the electrogenic transport, which has been rarely identified, even in animal studies, using electrophysiological/pharmacological and molecular biological methods. As a result, we identified functional and molecular evidence for the essential participation of K(+) channels in the electrogenic transport of human ES epithelium. The identified K(+) channels involved in the electrogenic transport were KCNN2, KCNJ14, KCNK2, and KCNK6, and the K(+) transports via those channels are thought to play an important role in the maintenance of the unique ionic milieu of the inner ear fluid.

  4. Inhibitory effects of cortisone and hydrocortisone on human Kv1.5 channel currents.

    Science.gov (United States)

    Yu, Jing; Park, Mi-Hyeong; Jo, Su-Hyun

    2015-01-05

    Glucocorticoids are the primary hormones that respond to stress and protect organisms from dangerous situations. The glucocorticoids hydrocortisone and its dormant form, cortisone, affect the cardiovascular system with changes such as increased blood pressure and cardioprotection. Kv1.5 channels play a critical role in the maintenance of cellular membrane potential and are widely expressed in pancreatic β-cells, neurons, myocytes, and smooth muscle cells of the pulmonary vasculature. We examined the electrophysiological effects of both cortisone and hydrocortisone on human Kv1.5 channels expressed in Xenopus oocytes using a two-microelectrode voltage clamp technique. Both cortisone and hydrocortisone rapidly and irreversibly suppressed the amplitude of Kv1.5 channel current with IC50 values of 50.2±4.2μM and 33.4±3.2μM, respectively, while sustained the current trace shape of Kv1.5 current. The inhibitory effect of cortisone on Kv1.5 decreased progressively from -10mV to +30mV, while hydrocortisone׳s inhibition of the channel did not change across the same voltage range. Both cortisone and hydrocortisone blocked Kv1.5 channel currents in a non-use-dependent manner and neither altered the channel׳s steady-state activation or inactivation curves. These results show that cortisone and hydrocortisone inhibited Kv1.5 channel currents differently, and that Kv1.5 channels were more sensitive to hydrocortisone than to cortisone. Copyright © 2014 Elsevier B.V. All rights reserved.

  5. Na+-permeable channels of human sperm membrane re- assembled into giant liposome

    Institute of Scientific and Technical Information of China (English)

    2001-01-01

    Previous data showed that a Na+-transmembrane flux was accompanied with acrosome reaction of sperm. However, the electrophysiological recording and characterization of Na+ current in human sperm membrane have not been yet reported. In the present investigation, membrane proteins extracted from human sperms were reassembled into liposome bilayer, and then the liposomes were fused by dehydration-rehydration into giant liposomes with the diameter of more than 10 mm. By patch clamping the giant liposomes two kinds of single channel currents were recorded in a NaCl solution system. Both of them were Na+-carried, TTX-sensitive and strongly rectifying, but with different unit conductance and open probability. Moreover, bursting activity and channel-substates as well as two open time constants were observed in the larger channel.

  6. Expression and function of K(V)2-containing channels in human urinary bladder smooth muscle.

    Science.gov (United States)

    Hristov, Kiril L; Chen, Muyan; Afeli, Serge A Y; Cheng, Qiuping; Rovner, Eric S; Petkov, Georgi V

    2012-06-01

    The functional role of the voltage-gated K(+) (K(V)) channels in human detrusor smooth muscle (DSM) is largely unexplored. Here, we provide molecular, electrophysiological, and functional evidence for the expression of K(V)2.1, K(V)2.2, and the electrically silent K(V)9.3 subunits in human DSM. Stromatoxin-1 (ScTx1), a selective inhibitor of K(V)2.1, K(V)2.2, and K(V)4.2 homotetrameric channels and of K(V)2.1/9.3 heterotetrameric channels, was used to examine the role of these channels in human DSM function. Human DSM tissues were obtained during open bladder surgeries from patients without a history of overactive bladder. Freshly isolated human DSM cells were studied using RT-PCR, immunocytochemistry, live-cell Ca(2+) imaging, and the perforated whole cell patch-clamp technique. Isometric DSM tension recordings of human DSM isolated strips were conducted using tissue baths. RT-PCR experiments showed mRNA expression of K(V)2.1, K(V)2.2, and K(V)9.3 (but not K(V)4.2) channel subunits in human isolated DSM cells. K(V)2.1 and K(V)2.2 protein expression was confirmed by Western blot analysis and immunocytochemistry. Perforated whole cell patch-clamp experiments revealed that ScTx1 (100 nM) inhibited the amplitude of the voltage step-induced K(V) current in freshly isolated human DSM cells. ScTx1 (100 nM) significantly increased the intracellular Ca(2+) level in DSM cells. In human DSM isolated strips, ScTx1 (100 nM) increased the spontaneous phasic contraction amplitude and muscle force, and enhanced the amplitude of the electrical field stimulation-induced contractions within the range of 3.5-30 Hz stimulation frequencies. These findings reveal that ScTx1-sensitive K(V)2-containing channels are key regulators of human DSM excitability and contractility and may represent new targets for pharmacological or genetic intervention for bladder dysfunction.

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

    Directory of Open Access Journals (Sweden)

    Antonio Cejudo-Roman

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

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

    NARCIS (Netherlands)

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

    2013-01-01

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

  9. Cholesterol binding to ion channels

    Directory of Open Access Journals (Sweden)

    Irena eLevitan

    2014-02-01

    Full Text Available Numerous studies demonstrated that membrane cholesterol is a major regulator of ion channel function. The goal of this review is to discuss significant advances that have been recently achieved in elucidating the mechanisms responsible for cholesterol regulation of ion channels. The first major insight that comes from growing number of studies that based on the sterol specificity of cholesterol effects, show that several types of ion channels (nAChR, Kir, BK, TRPV are regulated by specific sterol-protein interactions. This conclusion is supported by demonstrating direct saturable binding of cholesterol to a bacterial Kir channel. The second major advance in the field is the identification of putative cholesterol binding sites in several types of ion channels. These include sites at locations associated with the well-known cholesterol binding motif CRAC and its reversed form CARC in nAChR, BK, and TRPV, as well as novel cholesterol binding regions in Kir channels. Notably, in the majority of these channels, cholesterol is suggested to interact mainly with hydrophobic residues in non-annular regions of the channels being embedded in between transmembrane protein helices. We also discuss how identification of putative cholesterol binding sites is an essential step to understand the mechanistic basis of cholesterol-induced channel regulation. Clearly, however, these are only the first few steps in obtaining a general understanding of cholesterol-ion channels interactions and their roles in cellular and organ functions.

  10. Functional expression of voltage-gated calcium channels in human melanoma.

    Science.gov (United States)

    Das, A; Pushparaj, C; Bahí, N; Sorolla, A; Herreros, J; Pamplona, R; Vilella, R; Matias-Guiu, X; Martí, R M; Cantí, C

    2012-03-01

    The expression of voltage-gated calcium channels (VGCCs) has not been reported previously in melanoma cells in spite of increasing evidence of a role of VGCCs in tumorigenesis and tumour progression. To address this issue we have performed an extensive RT-PCR analysis of VGCC expression in human melanocytes and a range of melanoma cell lines and biopsies. In addition, we have tested the functional expression of these channels using Ca(2+) imaging techniques and examined their relevance for the viability and proliferation of the melanoma cells. Our results show that control melanocytes and melanoma cells express channel isoforms belonging to the Ca(v) 1 and Ca(v) 2 gene families. Importantly, the expression of low voltage-activated Ca(v) 3 (T-type) channels is restricted to melanoma. We have confirmed the function of T-type channels as mediators of constitutive Ca(2+) influx in melanoma cells. Finally, pharmacological and gene silencing approaches demonstrate a role for T-type channels in melanoma viability and proliferation. These results encourage the analysis of T-type VGCCs as targets for therapeutic intervention in melanoma tumorigenesis and/or tumour progression. © 2012 John Wiley & Sons A/S.

  11. T-type calcium channel expression in cultured human neuroblastoma cells

    Institute of Scientific and Technical Information of China (English)

    Xianjie Wen; Shiyuan Xu; Lingling Wang; Hua Liang; Chengxiang Yang; Hanbing Wang; Hongzhen Liu

    2011-01-01

    Human neuroblastoma cells (SH-SY5Y) have similar structures and functions as neural cells and have been frequently used for cell culture studies of neural cell functions. Previous studies have revealed Land N-type calcium channels in SH-SY5Y cells. However, the distribution of the low -voltage activated calcium channel (namely called T-type calcium channel, including Cav3.1, Cav3.2, and Cav3.3) in SH-SY5Y cells remains poorly understood. The present study detected mRNA and protein expression of the T-type calcium channel (Cav3.1, Cav3.2, and Cav3.3) in cultured SH-SY5Y cells using real-time polymerase chain reaction (PCR) and western blot analysis. Results revealed mRNA and protein expression from all three T-type calcium channel subtypes in SH-SY5Y cells. Moreover,Cav3.1 was the predominant T-type calcium channel subtype in SH-SY5Y cells.

  12. Comparison of the channelized Hotelling and human observers for lesion detection in hepatic SPECT imaging

    Science.gov (United States)

    King, Michael A.; de Vries, Daniel J.; Soares, Edward J.

    1997-04-01

    The relative rankings of the channelized Hotelling model observer were compared to those of the human observers for the task of detecting 'hot' tumors in simulated hepatic SPECT slices. The signal-to-noise ratios (SNRs) were determined using eighty images for each of three slice locations. The acquisition and processing strategies investigated were: (1) imaging solely primary photons, (2) imaging primary plus scatter within a 20% symmetric energy window for Tc-99m, (3) imaging with primary plus an elevated amount of scatter, (4) energy-spectrum-based scatter compensation of the primary plus scatter acquisitions, and (5) energy-spectrum-based scatter compensation of the acquisitions with an elevated amount of scatter. Both square non-overlapping channels (SQR), and overlapping difference- of-Gaussian channels (DOG) were incorporated into the Hotelling model observer. When the scatter compensation results were excluded, both channelized Hotelling model observers exhibited a strong correlation with the rankings of the human-observers. With the inclusion of the scatter compensation results, only with the DOG model observer was the null-hypothesis of no correlation rejected at the p equals 0.05 level. It is concluded that further investigation of the channel model used with the Hotelling observer is indicated to determine if better correlation can be obtained.

  13. Human Digital Meissner Corpuscles Display Immunoreactivity for the Multifunctional Ion Channels Trpc6 and Trpv4.

    Science.gov (United States)

    Alonso-González, Paula; Cabo, Roberto; San José, Isabel; Gago, Angel; Suazo, Iván C; García-Suárez, Olivia; Cobo, Juan; Vega, José A

    2017-06-01

    Ion channels are at the basis of the sensory processes including mechanosensing. Some members of the transient receptor potential (TRP) ion channel superfamily have been proposed as mechanosensors, but their putative role in mechanotransduction is controversial. Among them there are TRP canonical 6 (TRPC6) and TRP vanilloid 4 (TRPV4) ion channels, which are known to cooperate in mechanical hyperalgesia. Here, we investigated the occurrence, distribution, and possible colocalization of TRPC6 and TRPV4 in human digital Meissner sensory corpuscles using immunohistochemistry and double immunofluorescence (associate with markers for specific corpuscular constituents). TRPC6 immunoreactivity was restricted to the axon of Meissner corpuscles, whereas TRPV4 was detected in the axon but also in the lamellar cells. Moreover, axonal colocalization of TRPV4 and TRPC6 was found in the digital Meissner corpuscles. Present results demonstrate for the first time the occurrence and colocalization of two ion channels candidates to mechanosensors in human cutaneous mechanoreceptors. The functional significance of these ion channels in that place remains to be clarified, but should be related to different properties of mechanosensitivity. Anat Rec, 300:1022-1031, 2017. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

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

    Science.gov (United States)

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

    2011-08-15

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

  15. 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.

  16. Can robots patch-clamp as well as humans? Characterization of a novel sodium channel mutation.

    Science.gov (United States)

    Estacion, M; Choi, J S; Eastman, E M; Lin, Z; Li, Y; Tyrrell, L; Yang, Y; Dib-Hajj, S D; Waxman, S G

    2010-06-01

    Ion channel missense mutations cause disorders of excitability by changing channel biophysical properties. As an increasing number of new naturally occurring mutations have been identified, and the number of other mutations produced by molecular approaches such as in situ mutagenesis has increased, the need for functional analysis by patch-clamp has become rate limiting. Here we compare a patch-clamp robot using planar-chip technology with human patch-clamp in a functional assessment of a previously undescribed Nav1.7 sodium channel mutation, S211P, which causes erythromelalgia. This robotic patch-clamp device can increase throughput (the number of cells analysed per day) by 3- to 10-fold. Both modes of analysis show that the mutation hyperpolarizes activation voltage dependence (8 mV by manual profiling, 11 mV by robotic profiling), alters steady-state fast inactivation so that it requires an additional Boltzmann function for a second fraction of total current (approximately 20% manual, approximately 40% robotic), and enhances slow inactivation (hyperpolarizing shift--15 mV by human,--13 mV robotic). Manual patch-clamping demonstrated slower deactivation and enhanced (approximately 2-fold) ramp response for the mutant channel while robotic recording did not, possibly due to increased temperature and reduced signal-to-noise ratio on the robotic platform. If robotic profiling is used to screen ion channel mutations, we recommend that each measurement or protocol be validated by initial comparison to manual recording. With this caveat, we suggest that, if results are interpreted cautiously, robotic patch-clamp can be used with supervision and subsequent confirmation from human physiologists to facilitate the initial profiling of a variety of electrophysiological parameters of ion channel mutations.

  17. Effects of Shensong Yangxin capsule on pacemaker channels encoded by human HCN4 gene

    Institute of Scientific and Technical Information of China (English)

    SUN Li-ping; LI Ning; WU Yi-ling; PU Jie-lin

    2010-01-01

    @@ Shensong Yangxin (SSYX) is one of the compound recipes of Chinese materia medica including 12ingredients such as Panax ginseng, dwarf lilyturf tuber,nardostachys root, etc. Small-scale randomized multi-centre clinical trials suggested that SSYX reduced the number of ventricular extrasystoles in patients with or without structural heart disease.1 Besides excellent antiarrhythmic efficacy,2 SSYX also improved bradycardia in some patients, which was evidenced by animal studies3 as well. However, the antiarrhythmic mechanisms of SSYX have not been fully understood.Our previous studies have explored effect of SSYX on many channels except hyperpolarization-activated cation channel encoded by human hHCN4 gene.4

  18. Channel Modeling of Human Somatosensory Nanonetwork: Body Discriminative Touch and Proprioception Perspective

    Directory of Open Access Journals (Sweden)

    Partha Pratim Ray

    2013-10-01

    Full Text Available Nanonetwork design and analysis has become a very interesting topic in recent years. Though this area of research is in its formative stage, it definitely posses a strong integrity in finding out numerous applications in medical and allied sciences. Nanonetworking is indeed a nature built foundation which comprises human intra body communications. Somatosensory system is the one of the critical and must have systems of human body. This literature concentrates on the body discriminative touch and proprioception mechanism of somatosensory system. This particular system is well architecture by medial lemniscal pathway, in human body for transduction of touch and proprioceptive information. This paper seeks out the novel communication channel model of somatosensory system. The working principle of the channel model is established by an equivalent Moore machine. A novel algorithm MLP is proposed after its name, medial lemniscal pathway. A novel naomachine and appropriate processing unit are also devised, based on the automaton.

  19. 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.

  20. Functional Importance of L- and P/Q-Type Voltage-Gated Calcium Channels in Human Renal Vasculature

    DEFF Research Database (Denmark)

    Hansen, Pernille B; Poulsen, Christian B; Walter, Steen

    2011-01-01

    in kidney function. It was hypothesized that human renal vascular excitation-contraction coupling involves different subtypes of channels. In human renal artery and dissected intrarenal blood vessels from nephrectomies, PCR analysis showed expression of L-type (Ca(v) 1.2), P/Q-type (Ca(v) 2.1), and T-type......, and L- and P/Q-type channels are of functional importance for the depolarization-induced vasoconstriction. The contribution of P/Q-type channels to contraction in the human vasculature is a novel mechanism for the regulation of renal blood flow and suggests that clinical treatment with calcium blockers......Calcium channel blockers are widely used for treatment of hypertension, because they decrease peripheral vascular resistance through inhibition of voltage-gated calcium channels. Animal studies of renal vasculature have shown expression of several types of calcium channels that are involved...

  1. Molecular diversity of L-type Ca2+ channel transcripts in human fibroblasts.

    Science.gov (United States)

    Soldatov, N M

    1992-05-15

    The nucleotide sequence of cDNA encoding the human fibroblast Ca2+ channel of L type (HFCC) has been determined. It is highly homologous to L-type channels previously cloned from rabbit lung and heart as well as from rat brain. At least four sites of molecular diversity were identified in the nucleotide sequence of HFCC. Three of these include regions encoding the transmembrane segments IIS6, IIIS2, and IVS3, which are known to be important for channel gating properties. The positions of these sites correlate with RNA splice sites, indicating that the molecular diversity of the transcripts is a result of alternative splicing. The fourth diversity region is located at the C-terminal region and comprises insertions and deletions. It is suggested that these variations may give rise to multiple subforms of HFCC with altered electrophysiological properties.

  2. Supernova 2008bk and Its Red Supergiant Progenitor

    CERN Document Server

    Van Dyk, Schuyler D; Elias-Rosa, Nancy; Taubenberger, Stefan; Li, Weidong; Howerton, Stanley; Pignata, Giuliano; Morrell, Nidia; Hamuy, Mario; Filippenko, Alexei V

    2010-01-01

    We have observed Supernova (SN) 2008bk in NGC 7793, both photometrically and spectroscopically, primarily at late times. We find that it is a Type II-Plateau (II-P) SN, which most closely resembles the low-luminosity SN 1999br in NGC 4900. Given the overall similarity between the observed light curves and colors of SNe 2008bk and 1999br, we infer that the total visual extinction to SN 2008bk must be almost entirely due to the Galactic foreground, similar to that for SN 1999br: A_V=0.065 mag, which is substantially less than the 1.0 +/- 0.5 mag assumed by Mattila et al. (2008). Furthermore, we confirm the identification of the putative red supergiant progenitor star of the SN in high-quality g'r'i' Gemini-South images from 2007. Little ambiguity exists in this progenitor identification; besides the connection between the star Sk -69 202 and SN 1987A, it qualifies as one of the best SN progenitor identifications to date. From a combination of the Gemini images with archival, pre-SN, Very Large Telescope JHK_s i...

  3. 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.

  4. BIOPSY-PROVEN BK VIRUS NEPHROPATHY WITHOUT DETECTABLE BK VIREMIA IN A ONE-YEAR POST-KIDNEY TRANSPLANT RECIPIENT.

    Science.gov (United States)

    Ruangkanchanasetr, Prajej; Pumchandh, Norawee; Satirapoj, Bancha; Termmathurapoj, Sumeth; Pongthanapisith, Viroj

    2015-07-01

    BK virus nephropathy (BKVN) is an important clinical problem in kidney transplant (KT) recipients. The sequence of disease is usually viruria, viremia and then nephropathy. Diagnosis of BK virus (BKV) infection includes checking BKV DNA in the urine, in the plasma and histology on renal biopsy. This last method is used to diagnose BKVN. We describe a KT patient with BKVN without detectable BK viremia. A 62-year-old female with hypertensive nephropathy underwent renal transplant from a living relative donor in December 2011. Fourteen months after transplantation, her serum creatinine(SCr) rose up from 1.2 to 1.6 mg/dl with biopsy-proven acute antibody-mediated and cellular rejection. After pulse methylprednisolone, plasmapheresis and intravenous immunoglobulin, her SCr decreased to baseline but she subsequently developed cytomegalovirus infection with pancytopenia and transaminitis. The SCr rose to 1.9 mg/dl despite ganciclovir treatment. Renal ultrasound and antegrade pyelogram showed partial obstruction of the proximal ureter with moderate hydronephrosis. A quantitative polymerase chain reaction (PCR) assay for BKV DNA was negative (less than 10 copies/ml). A renal biopsy was performed and the pathology revealed viral cytopathic changes in the tubular epithelium with interstitial inflammation. The renal biopsy also showed BKV nucleic acid sequences by in-situ hybridization confirming BKVN. Immunosuppression regimen was changed to cyclosporine, low-dose prednisolone and leflunomide. A temporary percutaneous nephrostomy was performed. Her renal function improved within one week. The diagnosis of BKVN should be considered in a KT recipient with a rising SCr with or without BK viremia and should be made by renal biopsy.

  5. Otilonium bromide inhibits calcium entry through L-type calcium channels in human intestinal smooth muscle.

    Science.gov (United States)

    Strege, P R; Evangelista, S; Lyford, G L; Sarr, M G; Farrugia, G

    2004-04-01

    Otilonium bromide (OB) is used as an intestinal antispasmodic. The mechanism of action of OB is not completely understood. As Ca(2+) entry into intestinal smooth muscle is required to trigger contractile activity, our hypothesis was that OB blocked Ca(2+) entry through L-type Ca(2+) channels. Our aim was to determine the effects of OB on Ca(2+), Na(+) and K(+) ion channels in human jejunal circular smooth muscle cells and on L-type Ca(2+) channels expressed heterologously in HEK293 cells. Whole cell currents were recorded using standard patch clamp techniques. Otilonium bromide (0.09-9 micromol L(-1)) was used as this reproduced clinical intracellular concentrations. In human circular smooth muscle cells, OB inhibited L-type Ca(2+) current by 25% at 0.9 micromol L(-1) and 90% at 9 micromol L(-1). Otilonium bromide had no effect on Na(+) or K(+) currents. In HEK293 cells, 1 micromol L(-1) OB significantly inhibited the expressed L-type Ca(2+) channels. Truncation of the alpha(1C) subunit C and N termini did not block the inhibitory effects of OB. Otilonium bromide inhibited Ca(2+) entry through L-type Ca(2+) at concentrations similar to intestinal tissue levels. This effect may underlie the observed muscle relaxant effects of the drug.

  6. Urban river restoration: implications on channel sedimentation patterns and associated ecosystem and human health

    Science.gov (United States)

    Gibbs, H.; Gurnell, A.; Heppell, K.; Spencer, K.

    2012-04-01

    Urban river restoration, which alters the physical and hydraulic conditions of rivers, creates rivers favourable to increased sedimentation through greater sediment availability and heterogeneous flow patterns. Sediments, particularly finer-grained, store contaminants including metals which can have detrimental impacts upon aquatic ecosystems and potentially human health. This research therefore looks at the effect of urban river restoration practices upon sedimentation patterns, the associated changes in sediment metal storage and the potential impact upon river function and use in terms of the aquatic ecosystem and human health. Research was undertaken at four sites on urban rivers in London. The spatial extent of different bed sediment types (unvegetated gravel, sand, finer and sediment around in-channel vegetation) in adjacent restored and unrestored stretches was mapped in July 2010. Additionally, sediments were sampled through the year and analysed for a range of metals and sediment characteristics. Two sites (Chinbrook Meadows and Sutcliffe Park) showed a clear difference in bed sediment type channel cover between the restored and unrestored stretches. The majority of the concrete-lined unrestored stretch at Chinbrook Meadows had no sediment deposition, whereas the restored stretch had over half of the channel occupied by finer sediment either on the open channel bed or accumulated around in-channel vegetation. At Sutcliffe Park, the dominant bed sediment type in the restored stretch was finer sediment on the open bed and accumulated around in-channel vegetation, whereas in the unrestored stretch the dominant bed sediment type was gravel. At both sites there were significant differences in metal concentrations and sediment characteristics between bed sediment types. Metal concentrations, organic matter and % <63µm were generally higher in the finer sediment whether on the open bed or around in-channel vegetation. Total loadings of all metals were greater in

  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. Geometry of human vocal folds and glottal channel for mathematical and biomechanical modeling of voice production.

    Science.gov (United States)

    Sidlof, Petr; Svec, Jan G; Horácek, Jaromír; Veselý, Jan; Klepácek, Ivo; Havlík, Radan

    2008-01-01

    Current models of the vocal folds derive their shape from approximate information rather than from exactly measured data. The objective of this study was to obtain detailed measurements on the geometry of human vocal folds and the glottal channel in phonatory position. A non-destructive casting methodology was developed to capture the vocal fold shape from excised human larynges on both medial and superior surfaces. Two female larynges, each in two different phonatory configurations corresponding to low and high fundamental frequency of the vocal fold vibrations, were measured. A coordinate measuring machine was used to digitize the casts yielding 3D computer models of the vocal fold shape. The coronal sections were located in the models, extracted and fitted by piecewise-defined cubic functions allowing a mathematical expression of the 2D shape of the glottal channel. Left-right differences between the cross-sectional shapes of the vocal folds were found in both the larynges.

  9. 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....

  10. Pharmacological Investigation of Voltage-dependent Ca2+ Channels in Human Ejaculatory Sperm in vitro

    Institute of Scientific and Technical Information of China (English)

    LI Lu; LIU Jihong; LI Jiagui; YE Zhangqun

    2006-01-01

    The types of the voltage-dependent calcium channels (VDCCs) in human ejaculatory sperm and the effects of calcium channel blocker (CCB) on human sperm motility parameters in vitro were investigated. The human sperm motility parameters in vitro in response to the pharmacological agents nifedipine (NIF, inhibitor of L-type VDCC) and ω-conotoxin (GVIA, inhibitor of N-type VDCC) were compared and analyzed statistically. The results showed that NIF (1, 5, 10 μmol/L)could not only significantly affect human sperm's shape but also spermatozoa motility after incubated at least 10 min in vitro (P<0.001). GVIA (0.1, 0.5 and 1 μmol/L) could just only significantly affect human sperm's progressive motility (a %+b %) after incubated for 20 min in vitro (P<0.01), but they both could not significantly affect spermic abnormality rate. It is suggested that L-type VDCC, non L-type VDCCs and isoform of L-type VDCC exist in the cell membrane of human sperm solely or together, and they participate in the spermic physiological processes especially the spermic motility.

  11. Reconstitution of synaptic Ion channels from rodent and human brain in Xenopus oocytes: a biochemical and electrophysiological characterization.

    Science.gov (United States)

    Mazzo, Francesca; Zwart, Ruud; Serratto, Giulia Maia; Gardinier, Kevin M; Porter, Warren; Reel, Jon; Maraula, Giovanna; Sher, Emanuele

    2016-08-01

    Disruption in the expression and function of synaptic proteins, and ion channels in particular, is critical in the pathophysiology of human neuropsychiatric and neurodegenerative diseases. However, very little is known regarding the functional and pharmacological properties of native synaptic human ion channels, and their potential changes in pathological conditions. Recently, an electrophysiological technique has been enabled for studying the functional and pharmacological properties of ion channels present in crude membrane preparation obtained from post-mortem frozen brains. We here extend these studies by showing that human synaptic ion channels also can be studied in this way. Synaptosomes purified from different regions of rodent and human brain (control and Alzheimer's) were characterized biochemically for enrichment of synaptic proteins, and expression of ion channel subunits. The same synaptosomes were also reconstituted in Xenopus oocytes, in which the functional and pharmacological properties of the native synaptic ion channels were characterized using the voltage clamp technique. We show that we can detect GABA, (RS)-α-Amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid, and NMDA receptors, and modulate them pharmacologically with selective agonists, antagonists, and allosteric modulators. Furthermore, changes in ion channel expression and function were detected in synaptic membranes from Alzheimer's brains. Our present results demonstrate the possibility to investigate synaptic ion channels from healthy and pathological brains. This method of synaptosomes preparation and injection into oocytes is a significant improvement over the earlier method. It opens the way to directly testing, on native ion channels, the effects of novel drugs aimed at modulating important classes of synaptic targets. Disruption in the expression and function of synaptic ion channels is critical in the pathophysiology of human neurodegenerative diseases. We here show that

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

    DEFF Research Database (Denmark)

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

    2016-01-01

    , by shifting the conductance-versus-voltage curve towards more negative voltages (by -7.4 to -11.3 mV by 70 μM). Uncharged docosahexaenoic acid methyl ester and monounsaturated oleic acid did not facilitate opening of the human KV 7.2/3 channel. CONCLUSIONS: These findings suggest that circulating...... acids reduce neuronal excitability. This article is protected by copyright. All rights reserved....

  13. Measurement and analysis of channel attenuation characteristics for an implantable galvanic coupling human-body communication.

    Science.gov (United States)

    Zhang, Shuang; Pun, Sio Hang; Mak, Peng Un; Qin, Yu-Ping; Liu, Yi-He; Vai, Mang I

    2016-11-14

    In this study, an experiment was designed to verify the low power consumption of galvanic coupling human-body communication. A silver electrode (silver content: 99%) is placed in a pig leg and a sine wave signal with the power of 0 dBm is input. Compared with radio frequency communication and antenna transmission communication, attenuation is reduced by approximately 10 to 15 dB, so channel characteristics are highly improved.

  14. Immature human dendritic cells enhance their migration through KCa3.1 channel activation.

    Science.gov (United States)

    Crottès, David; Félix, Romain; Meley, Daniel; Chadet, Stéphanie; Herr, Florence; Audiger, Cindy; Soriani, Olivier; Vandier, Christophe; Roger, Sébastien; Angoulvant, Denis; Velge-Roussel, Florence

    2016-04-01

    Migration capacity is essential for dendritic cells (DCs) to present antigen to T cells for the induction of immune response. The DC migration is supposed to be a calcium-dependent process, while not fully understood. Here, we report a role of the KCa3.1/IK1/SK4 channels in the migration capacity of both immature (iDC) and mature (mDC) human CD14(+)-derived DCs. KCa3.1 channels were shown to control the membrane potential of human DC and the Ca(2+) entry, which is directly related to migration capacities. The expression of migration marker such as CCR5 and CCR7 was modified in both types of DCs by TRAM-34 (100nM). But, only the migration of iDC was decreased by use of both TRAM-34 and KCa3.1 siRNA. Confocal analyses showed a close localization of CCR5 with KCa3.1 in the steady state of iDC. Finally, the implication of KCa3.1 seems to be limited to the migration capacities as T cell activation of DCs appeared unchanged. Altogether, these results demonstrated that KCa3.1 channels have a pro-migratory effect on iDC migration. Our findings suggest that KCa3.1 in human iDC play a major role in their migration and constitute an attractive target for the cell therapy optimization.

  15. Effects of environmental changes and human impact on the functioning of mountain river channels, Carpathians, southern Poland

    Directory of Open Access Journals (Sweden)

    Krzemień Kazimierz

    2015-09-01

    Full Text Available In the northern slope of the Carpathian Mountains and in their foreland, river and stream channels have been significantly transformed by human impact. These transformations result from changing land use in river basins and direct interference with river channels (alluvia extraction, engineering infrastructure, channel straightening. Anthropogenic impacts cause significant changes in the channel system patterns leading to increased impact of erosion. This mainly leads to the channelling of the fluvial system. This article reviews studies of structure and dynamics of Carpathian river channels conducted based on the methodology of collection of data on channel systems, developed in the Department of Geomorphology of the Institute of Geography and Spatial Management, Jagiellonian University.

  16. A novel potassium channel in skeletal muscle mitochondria.

    Science.gov (United States)

    Skalska, Jolanta; Piwońska, Marta; Wyroba, Elzbieta; Surmacz, Liliana; Wieczorek, Rafal; Koszela-Piotrowska, Izabela; Zielińska, Joanna; Bednarczyk, Piotr; Dołowy, Krzysztof; Wilczynski, Grzegorz M; Szewczyk, Adam; Kunz, Wolfram S

    2008-01-01

    In this work we provide evidence for the potential presence of a potassium channel in skeletal muscle mitochondria. In isolated rat skeletal muscle mitochondria, Ca(2+) was able to depolarize the mitochondrial inner membrane and stimulate respiration in a strictly potassium-dependent manner. These potassium-specific effects of Ca(2+) were completely abolished by 200 nM charybdotoxin or 50 nM iberiotoxin, which are well-known inhibitors of large conductance, calcium-activated potassium channels (BK(Ca) channel). Furthermore, NS1619, a BK(Ca)-channel opener, mimicked the potassium-specific effects of calcium on respiration and mitochondrial membrane potential. In agreement with these functional data, light and electron microscopy, planar lipid bilayer reconstruction and immunological studies identified the BK(Ca) channel to be preferentially located in the inner mitochondrial membrane of rat skeletal muscle fibers. We propose that activation of mitochondrial K(+) transport by opening of the BK(Ca) channel may be important for myoprotection since the channel opener NS1619 protected the myoblast cell line C2C12 against oxidative injury.

  17. Electrical coupling between the human serotonin transporter and voltage-gated Ca(2+) channels.

    Science.gov (United States)

    Ruchala, Iwona; Cabra, Vanessa; Solis, Ernesto; Glennon, Richard A; De Felice, Louis J; Eltit, Jose M

    2014-07-01

    Monoamine transporters have been implicated in dopamine or serotonin release in response to abused drugs such as methamphetamine or ecstasy (MDMA). In addition, monoamine transporters show substrate-induced inward currents that may modulate excitability and Ca(2+) mobilization, which could also contribute to neurotransmitter release. How monoamine transporters modulate Ca(2+) permeability is currently unknown. We investigate the functional interaction between the human serotonin transporter (hSERT) and voltage-gated Ca(2+) channels (CaV). We introduce an excitable expression system consisting of cultured muscle cells genetically engineered to express hSERT. Both 5HT and S(+)MDMA depolarize these cells and activate the excitation-contraction (EC)-coupling mechanism. However, hSERT substrates fail to activate EC-coupling in CaV1.1-null muscle cells, thus implicating Ca(2+) channels. CaV1.3 and CaV2.2 channels are natively expressed in neurons. When these channels are co-expressed with hSERT in HEK293T cells, only cells expressing the lower-threshold L-type CaV1.3 channel show Ca(2+) transients evoked by 5HT or S(+)MDMA. In addition, the electrical coupling between hSERT and CaV1.3 takes place at physiological 5HT concentrations. The electrical coupling between monoamine neurotransmitter transporters and Ca(2+) channels such as CaV1.3 is a novel mechanism by which endogenous substrates (neurotransmitters) or exogenous substrates (like ecstasy) could modulate Ca(2+)-driven signals in excitable cells.

  18. Effect of propionyl-L-carnitine on L-type calcium channels in human heart sarcolemma

    Energy Technology Data Exchange (ETDEWEB)

    Bevilacqua, M.; Vago, T.; Norbiato, G. (Servizio di Endocrinologia, Milano, (Italy))

    1991-02-01

    Propionyl-L-carnitine (PC) protects perfused rat hearts against damage by ischemia-reperfusion. Activation of L-type calcium channel play a role on ischemia-reperfusion damage. Therefore, we studied the effect of PC on some properties of L-type calcium channels in an in vitro preparation from human myocardium sarcolemma (from patients with idiopathic dilated cardiomyopathy). Binding of the L-type calcium channel blockers isradipine ({sup 3}H)-PN 200-110 (PN) to plasma membrane preparations revealed a single population of binding sites (total number: Bmax = 213 +/- 34 fM/mg protein and affinity: Kd = 152 +/- 19 nM; n = 6). The characteristics of these binding sites were evaluated in the presence and in the absence of Ca{sup 2}{sup +} and of calcium blockers (D-888, a verapamillike drug, and diltiazem). Incubation in a Ca{sup 2}{sup +}-containing buffer increased the affinity of PN binding sites. Binding sites for PN were modulated by organic calcium channel blockers; in competition isotherms at 37{degree}C, D-888 (desmethoxyverapamil) decreased the PN binding, whereas diltiazem increased it. These results strongly suggest that the site labelled by PN is the voltage-operated calcium channel of the human myocardium. The addition of PC (1 mM) to plasma membranes labelled with PN at 37{degree}C decreased the affinity of the binding; this effect was counteracted by the addition of Ca{sup 2}{sup +} to the medium. This result was consistent with a competition between Ca{sup 2}{sup +} and PC. The effect of PC incubation at 4{degree}C was the opposite; at this temperature PC increased the affinity of the binding sites and the effect was obscured by Ca{sup 2}{sup +}.

  19. Evaluation of six channelized Hotelling observers in combination with a contrast sensitivity function to predict human observer performance

    Science.gov (United States)

    Goffi, Marco; Veldkamp, Wouter J. H.; van Engen, Ruben E.; Bouwman, Ramona W.

    2015-03-01

    Standard methods to quantify image quality (IQ) may not be adequate for clinical images since they depend on uniform backgrounds and linearity. Statistical model observers are not restricted to these limitations and might be suitable for IQ evaluation of clinical images. One of these statistical model observers is the channelized Hotelling observer (CHO), where the images are filtered by a set of channels. The aim of this study was to evaluate six different channel sets, with an additional filter to simulate the human contrast sensitivity function (CSF), in their ability to predict human observer performance. For this evaluation a two alternative forced choice experiment was performed with two types of background structures (white noise (WN) and clustered lumpy background (CLB)), 5 disk-shaped objects with different diameters and 3 different signal energies. The results show that the correlation between human and model observers have a diameter dependency for some channel sets in combination with CLBs. The addition of the CSF reduces this diameter dependency and in some cases improves the correlation coefficient between human- and model observer. For the CLB the Partial Least Squares channel set shows the highest correlation with the human observer (r2=0.71) and for WN backgrounds it was the Gabor-channel set with CSF (r2=0.72). This study showed that for some channels there is a high correlation between human and model observer, which suggests that the CHO has potential as a tool for IQ analysis of digital mammography systems.

  20. Detection of BK virus DNA in nasopharyngeal aspirates from children with respiratory infections but not in saliva from immunodeficient and immunocompetent adult patients.

    OpenAIRE

    SUNDSFJORD, A.; Spein, A R; Lucht, E.; Flaegstad, T; Seternes, O M; Traavik, T.

    1994-01-01

    Our understanding of important stages in the pathogenesis of the human polyomavirus BK virus (BKV) and JC virus (JCV) infections is limited. In this context, nasopharyngeal aspirates from 201 children with respiratory diseases and saliva from 60 human immunodeficiency virus type 1-infected adults and 10 healthy adult controls were collected and analyzed for the presence of BKV and JCV DNA by PCR. Neither BKV nor JCV DNA was detected in the saliva specimens. We demonstrated BKV DNA, but no inf...

  1. Inhalation of the BK(Ca-opener NS1619 attenuates right ventricular pressure and improves oxygenation in the rat monocrotaline model of pulmonary hypertension.

    Directory of Open Access Journals (Sweden)

    Marc Revermann

    Full Text Available BACKGROUND: Right heart failure is a fatal consequence of chronic pulmonary hypertension (PH. The development of PH is characterized by increased proliferation of vascular cells, in particular pulmonary artery smooth muscle cells (PASMCs and pulmonary artery endothelial cells. In the course of PH, an escalated right ventricular (RV afterload occurs, which leads to increased perioperative morbidity and mortality. BK(Ca channels are ubiquitously expressed in vascular smooth muscle cells and their opening induces cell membrane hyperpolarization followed by vasodilation. Moreover, BK activation induces anti-proliferative effects in a multitude of cell types. On this basis, we hypothesized that treatment with the nebulized BK channel opener NS1619 might be a therapy option for pulmonary hypertension and tested this in rats. METHODS: (1 Rats received monocrotaline injection for PH induction. Twenty-four days later, rats were anesthetized and NS1619 or the solvent was administered by inhalation. Systemic hemodynamic parameters, RV hemodynamic parameters, and blood gas analyses were measured before as well as 30 and 120 minutes after inhalation. (2 Rat PASMCs were stimulated with PDGF-BB in the presence and absence of NS1619. AKT, ERK1 and ERK2 activation were investigated by western blot analyses, and relative cell number was determined 48 hours after stimulation. RESULTS: Inhalation of a 12 µM and 100 µM NS1619 solution significantly reduced RV pressure without affecting systemic arterial pressure. Blood gas analyses demonstrated significantly reduced carbon dioxide and improved oxygenation in NS1619-treated animals pointing towards a considerable pulmonary shunt-reducing effect. In PASMC's, NS1619 (100 µM significantly attenuated PASMC proliferation by a pathway independent of AKT and ERK1/2 activation. CONCLUSION: NS1619 inhalation reduces RV pressure and improves oxygen supply and its application inhibits PASMC proliferation in vitro. Hence, BK

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

    Directory of Open Access Journals (Sweden)

    Andrias O O'Reilly

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

  3. Expression and pharmacology of endogenous Cav channels in SH-SY5Y human neuroblastoma cells.

    Directory of Open Access Journals (Sweden)

    Silmara R Sousa

    Full Text Available SH-SY5Y human neuroblastoma cells provide a useful in vitro model to study the mechanisms underlying neurotransmission and nociception. These cells are derived from human sympathetic neuronal tissue and thus, express a number of the Cav channel subtypes essential for regulation of important physiological functions, such as heart contraction and nociception, including the clinically validated pain target Cav2.2. We have detected mRNA transcripts for a range of endogenous expressed subtypes Cav1.3, Cav2.2 (including two Cav1.3, and three Cav2.2 splice variant isoforms and Cav3.1 in SH-SY5Y cells; as well as Cav auxiliary subunits α2δ1-3, β1, β3, β4, γ1, γ4-5, and γ7. Both high- and low-voltage activated Cav channels generated calcium signals in SH-SY5Y cells. Pharmacological characterisation using ω-conotoxins CVID and MVIIA revealed significantly (∼ 10-fold higher affinity at human versus rat Cav2.2, while GVIA, which interacts with Cav2.2 through a distinct pharmacophore had similar affinity for both species. CVID, GVIA and MVIIA affinity was higher for SH-SY5Y membranes vs whole cells in the binding assays and functional assays, suggesting auxiliary subunits expressed endogenously in native systems can strongly influence Cav2.2 channels pharmacology. These results may have implications for strategies used to identify therapeutic leads at Cav2.2 channels.

  4. High glucose modifies transient receptor potential canonical type 6 channels via increased oxidative stress and syndecan-4 in human podocytes

    DEFF Research Database (Denmark)

    Thilo, Florian; Lee, Marlene; Xia, Shengqiang

    2014-01-01

    Transient receptor potential canonical (TRPC) channels type 6 play an important role in the function of human podocytes. Diabetic nephropathy is characterized by altered TRPC6 expression and functions of podocytes. Thus, we hypothesized that high glucose modifies TRPC6 channels via increased oxid...

  5. A novel LQT3 mutation implicates the human cardiac sodium channel domain IVS6 in inactivation kinetics

    NARCIS (Netherlands)

    Groenewegen, WA; Bezzina, CR; van Tintelen, JP; Hoorntje, TM; Mannens, MMAM; Wilde, AAM; Jongsma, HJ; Rook, MB

    2003-01-01

    The Long QT3 syndrome is associated with mutations in the cardiac sodium channel gene SCN5A. Objective: The aim of the present study was the identification and functional characterization of a mutation in a family with the long QT3 syndrome. Methods: The human cardiac sodium channel gene SCN5A was s

  6. Human aquaporin-11 is a water and glycerol channel and localizes in the vicinity of lipid droplets in human adipocytes.

    Science.gov (United States)

    Madeira, Ana; Fernández-Veledo, Sonia; Camps, Marta; Zorzano, Antonio; Moura, Teresa F; Ceperuelo-Mallafré, Victoria; Vendrell, Joan; Soveral, Graça

    2014-09-01

    For a long time Aquaporin-7 has been the only aquaporin associated with the adipose tissue, and its dysregulation has been linked to the underlying mechanisms of obesity. However, the presence of alternative glycerol channels within the adipose tissue has been postulated, which has prompted us to the search of alternate glycerol transport routes in adipocytes. In view of this, it is hypothesized that Aquaporin-11 (AQP11) would have a role in adipocyte cell biology. The expression, the localization and the function of human AQP11 (hAQP11) in cultured differentiated adipocytes were investigated. Gene expression analysis revealed the presence of AQP11 in both subcutaneous and visceral human mature adipocytes. It is found that hAQP11 is primarily located intracellularly in human adipocytes and partially colocalizes with perilipin, pointing towards AQP11 preferential location in the vicinity of lipid droplets. Overexpression of hAQP11 in 3T3-L1 adipocytes enabled to validate its function as a water channel and reveal its glycerol permeation activity. hAQP11 permeates both water and glycerol, localizing in the vicinity of lipid droplets in human adipocytes. © 2014 The Obesity Society.

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

    DEFF Research Database (Denmark)

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

    2005-01-01

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

  8. Calcium and Vitamin D increase mRNA levels for the growth control hIK1 channel in human epidermal keratinocytes but functional channels are not observed

    Directory of Open Access Journals (Sweden)

    Rossie Sandra

    2004-06-01

    Full Text Available Abstract Background Intermediate-conductance, calcium-activated potassium channels (IKs modulate proliferation and differentiation in mesodermal cells by enhancing calcium influx, and they contribute to the physiology of fluid movement in certain epithelia. Previous reports suggest that IK channels stimulate proliferative growth in a keratinocyte cell line; however, because these channels indirectly promote calcium influx, a critically unique component of the keratinocyte differentiation program, an alternative hypothesis is that they would be anti-proliferative and pro-differentiating. This study addresses these hypotheses. Methods Real-time PCR, patch clamp electrophysiology, and proliferation assays were used to determine if human IK1 (hIK1 expression and function are correlated with either proliferation or differentiation in cultured human skin epidermal keratinocytes, and skin biopsies grown in explant culture. Results hIK1 mRNA expression in human keratinocytes and skin was increased in response to anti-proliferative/pro-differentiating stimuli (elevated calcium and Vitamin D. Correspondingly, the hIK1 agonist 1-EBIO inhibited keratinocyte proliferation suggesting that the channel could be anti-proliferative and pro-differentiating. However, this proliferative inhibition by 1-EBIO was not reversed by a panel of hIK1 blockers, calling into question the mechanism of 1-EBIO action. Subsequent patch clamp electrophysiological analysis failed to detect hIK1 channel currents in keratinocytes, even those expressing substantial hIK1 mRNA in response to calcium and Vitamin D induced differentiation. Identical electrophysiological recording conditions were then used to observe robust IK1 currents in fibroblasts which express IK1 mRNA levels comparable to those of keratinocytes. Thus, the absence of observable hIK1 currents in keratinocytes was not a function of the electrophysiological techniques. Conclusion Human keratinocyte differentiation is

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

    Directory of Open Access Journals (Sweden)

    Steven B. Symington

    2009-01-01

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

  10. Environment-Sensitive Fluorescent Probe for the Human Ether-a-go-go-Related Gene Potassium Channel

    OpenAIRE

    Liu, Zhenzhen; Jiang, Tianyu; Wang, Beilei; Ke, Bowen; Zhou, Yubin; Du, Lupei; Li, Minyong

    2016-01-01

    A novel environment-sensitive probe S2 with turn-on switch for Human Ether-a-go-go-Related Gene (hERG) potassium channel was developed herein. After careful evaluation, this fluorescent probe showed high binding affinity with hERG potassium channel with an IC50 value of 41.65 nM and can be well applied to hERG channel imaging or cellular distribution study for hERG channel blockers. Compared with other imaging techniques, such as immunofluorescence and fluorescent protein-based approaches, th...

  11. Acacetin Blocks Kv1.3 Channels and Inhibits Human T Cell Activation

    Directory of Open Access Journals (Sweden)

    Ning Zhao

    2014-10-01

    Full Text Available Backgrounds/Aims: Acacetin, a natural flavonoid compound, has been proven to exert anti-inflammatory and immunomodulatory effects. Kv1.3 channels, highly expressed in human T cells, are attractive therapeutic targets to treat inflammatory and immunological disorders. The present study was designed to characterize the inhibition of Kv1.3 channels by Acacetin in human T cells and examine its role in T cell activation. Methods: Whole-cell patch-clamp was applied to record the Kv1.3 and KCa currents in human T cells; Western blot was used to detect Kv1.3 expression as well as NFAT1 and NF-κB activity; Fluo-4, CCK-8 and an ELISA kit were used to measure Ca2+ influx, proliferation, and IL-2 secretion, respectively. Results: Acacetin decreased the Kv1.3 current, accelerated the decay rate and negatively shifted the steady-state inactivation curves in a concentration-dependent manner. The IC50 values at +40 mV for peak and the current at end of pulse were 21.09 ± 2.75 and 3.63 ± 0.25 µmol/L, respectively. Treatment with Acacetin for 24 h significantly inhibited Kv1.3 protein expression. Additionally, paralleling Kv1.3 inhibition, Acacetin also inhibited Ca2+ influx, the Ca2+-activated transcription factors NFAT1, NF-κB p65/p50 activity, and proliferation as well as IL-2 production. Small interfering RNA against Kv1.3 reduced the inhibitory effect of Acacetin on IL-2 secretion. Conclusions: Acacetin blocks the Kv1.3 channel and inhibits human T cell activation. This action most likely contributes to its immunomodulatory and anti-inflammatory actions.

  12. Transmural expression of ion channels and transporters in human nondiseased and end-stage failing hearts

    DEFF Research Database (Denmark)

    Soltysinska, Ewa; Olesen, Søren-Peter; Christ, Torsten

    2009-01-01

    The cardiac action potential is primarily shaped by the orchestrated function of several different types of ion channels and transporters. One of the regional differences believed to play a major role in the progression and stability of the action potential is the transmural gradient of electrical...... activity across the ventricular wall. An altered balance in the ionic currents across the free wall is assumed to be a substrate for arrhythmia. A large fraction of patients with heart failure experience ventricular arrhythmia. However, the underlying substrate of these functional changes is not well......-established as expression analyses of human heart failure (HF) are sparse. We have investigated steady-state RNA levels by quantitative polymerase chain reaction of ion channels, transporters, connexin 43, and miR-1 in 11 end-stage HF and seven nonfailing (NF) hearts. The quantifications were performed on endo-, mid...

  13. 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.

  14. Inhibitory Interactions between BK and JC Virus among Kidney Transplant Recipients

    Science.gov (United States)

    Cheng, Xingxing S.; Bohl, Daniel L.; Storch, Gregory A.; Ryschkewitsch, Caroline; Gaudreault-Keener, Monique; Major, Eugene O.; Randhawa, Parmjeet; Hardinger, Karen L.

    2011-01-01

    BK and JC polyomaviruses can reactivate after transplantation, causing renal dysfunction and graft loss. The incidence of JC reactivation after renal transplant is not well understood. Here, we characterized JC reactivation using samples collected during the first year after transplantation from 200 kidney recipients. We detected BK and JC viruses in the urine of 35 and 16% of transplant recipients, respectively. The median viral load in the urine was 400 times higher for BK virus than JC virus. The presence of BK viruria made concurrent JC viruria less likely: JC viruria was detected in 22% of non-BK viruric recipients compared with 4% of BK viruric recipients (P = 0.001). The co-detection rate was 1.5%, which is less than the expected 5.6% if reactivation of each virus was independent (P = 0.001). We did not observe JC viremia, JC nephropathy, or progressive multifocal leukoencephalopathy. The onset of JC viruria was associated with donor, but not recipient, JC-specific antibody in a titer-dependent fashion and inversely associated with donor and recipient BK-specific antibody. Donor and recipient JC seropositivity did not predict BK viruria or viremia. In conclusion, among renal transplant recipients, infection with one polyomavirus inversely associates with infection with the other. PMID:21511831

  15. 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.

  16. Collinearly-improved BK evolution meets the HERA data

    Directory of Open Access Journals (Sweden)

    E. Iancu

    2015-11-01

    Full Text Available In a previous publication, we have established a collinearly-improved version of the Balitsky–Kovchegov (BK equation, which resums to all orders the radiative corrections enhanced by large double transverse logarithms. Here, we study the relevance of this equation as a tool for phenomenology, by confronting it to the HERA data. To that aim, we first improve the perturbative accuracy of our resummation, by including two classes of single-logarithmic corrections: those generated by the first non-singular terms in the DGLAP splitting functions and those expressing the one-loop running of the QCD coupling. The equation thus obtained includes all the next-to-leading order corrections to the BK equation which are enhanced by (single or double collinear logarithms. We then use numerical solutions to this equation to fit the HERA data for the electron–proton reduced cross-section at small Bjorken x. We obtain good quality fits for physically acceptable initial conditions. Our best fit, which shows a good stability up to virtualities as large as Q2=400 GeV2 for the exchanged photon, uses as an initial condition the running-coupling version of the McLerran–Venugopalan model, with the QCD coupling running according to the smallest dipole prescription.

  17. TRPV channel expression in human skin and possible role in thermally induced cell death.

    Science.gov (United States)

    Radtke, Christine; Sinis, Nektarios; Sauter, Michael; Jahn, Sabrina; Kraushaar, Udo; Guenther, Elke; Rodemann, H Peter; Rennekampff, Hans-Oliver

    2011-01-01

    Cell death via necrosis and apoptosis is a hallmark of deep dermal to full-thickness cutaneous burn injuries. Keratinocytes might act as thermosensory cells that transmit information regarding ambient temperature via heat-gated transient receptor potential vanilloid (TRPV) ion channels. The aim of this study was to investigate the distribution of TRPV1, 2, 3, and 4 in uninjured and thermally burned skin. The authors investigated warmth-evoked currents in keratinocytes and cell kinetics of thermally injured keratinocytes in culture with agonists and antagonists of TRPV channels. Specimens of uninjured normal skin and discarded tissue of thermally injured skin were stained for TRPV1, 2, 3, and 4. Cultured primary human keratinocytes were heated for 5 minutes at the following temperatures: 37°C (control), 42°C, and 60°C and thereafter cultured for 24 or 48 hours at 37°C. Thermally stressed cells were treated with TRPV antagonists capsazepine or ruthenium red, and cell viability capacity was determined. TRPV1, TRPV2, TRPV3, and TRPV4 immunoreactivity was differentially identified on basal and suprabasal keratinocytes of healthy human skin. Patch clamp analysis showed a functional response of human keratinocytes at temperatures >40°C. Cell death of keratinocytes after heating at 42°C was reduced by 15 and 5% with ruthenium red and by 20 and 30% by capsazepine at 24 and 48 hours, respectively. Cell death after treatment at 60°C was significantly reduced at 24 hours with capsazepine (22%) or ruthenium red (18%) but only minimally affected after 48 hours postinjury. Interaction with TRPV channels on keratinocytes may offer a new strategy to counteract cell death after thermal injury.

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

    DEFF Research Database (Denmark)

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

    2015-01-01

    The epithelial sodium channel (ENaC) of the kidney is necessary for extracellular volume homeostasis and normal arterial BP. Activity of ENaC is enhanced by proteolytic cleavage of the gamma-subunit and putative release of a 43-amino acid inhibitory tract from the gamma-subunit ectodomain. We......ENaC was detected consistently only in tissue from patients with proteinuria and observed in collecting ducts. In conclusion, human kidney gammaENaC is subject to proteolytic cleavage, yielding fragments compatible with furin cleavage, and proteinuria is associated with cleavage at the putative prostasin...

  19. Development of membrane ion channels during neural differentiation from human embryonic stem cells.

    Science.gov (United States)

    Mirsadeghi, Sara; Shahbazi, Ebrahim; Hemmesi, Katayoun; Nemati, Shiva; Baharvand, Hossein; Mirnajafi-Zadeh, Javad; Kiani, Sahar

    2017-09-09

    For human embryonic stem cells (hESCs) to differentiate into neurons, enormous changes has to occur leading to trigger action potential and neurotransmitter release. We attempt to determine the changes in expression of voltage gated channels (VGCs) and their electrophysiological properties during neural differentiation. The relative expressions of α-subunit of voltage gated potassium, sodium and calcium channels were characterized by qRT-PCR technique. Patch clamp recording was performed to characterize the electrophysiological properties of hESCs during their differentiation into neuron-like cells. Relative expression of α-subunit of channels changed significantly. 4-AP and TEA sensitive outward currents were observed in all stages, although TEA sensitive currents were recorded once in rosette structure. Nifedipine and QX314 sensitive inward currents were recorded only in neuron-like cells. K(+) currents were recorded in hESCs and rosette structure cells. Inward currents, sensitive to Nifedipine and QX314, were recorded in neuron-like cells. Copyright © 2017 Elsevier Inc. All rights reserved.

  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. 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.

  2. Mechanisms of sustained high firing rates in two classes of vestibular nucleus neurons: differential contributions of resurgent Na, Kv3, and BK currents.

    Science.gov (United States)

    Gittis, Aryn H; Moghadam, Setareh H; du Lac, Sascha

    2010-09-01

    To fire at high rates, neurons express ionic currents that work together to minimize refractory periods by ensuring that sodium channels are available for activation shortly after each action potential. Vestibular nucleus neurons operate around high baseline firing rates and encode information with bidirectional modulation of firing rates up to several hundred Hz. To determine the mechanisms that enable these neurons to sustain firing at high rates, ionic currents were measured during firing by using the action potential clamp technique in vestibular nucleus neurons acutely dissociated from transgenic mice. Although neurons from the YFP-16 line fire at rates higher than those from the GIN line, both classes of neurons express Kv3 and BK currents as well as both transient and resurgent Na currents. In the fastest firing neurons, Kv3 currents dominated repolarization at all firing rates and minimized Na channel inactivation by rapidly transitioning Na channels from the open to the closed state. In slower firing neurons, BK currents dominated repolarization at the highest firing rates and sodium channel availability was protected by a resurgent blocking mechanism. Quantitative differences in Kv3 current density across neurons and qualitative differences in immunohistochemically detected expression of Kv3 subunits could account for the difference in firing range within and across cell classes. These results demonstrate how divergent firing properties of two neuronal populations arise through the interplay of at least three ionic currents.

  3. Identification of BACE1 cleavage sites in human voltage-gated sodium channel beta 2 subunit

    Directory of Open Access Journals (Sweden)

    Kovacs Dora M

    2010-12-01

    Full Text Available Abstract Background The voltage-gated sodium channel β2 subunit (Navβ2 is a physiological substrate of BACE1 (β-site APP cleaving enzyme and γ-secretase, two proteolytic enzymes central to Alzheimer's disease pathogenesis. Previously, we have found that the processing of Navβ2 by BACE1 and γ-secretase regulates sodium channel metabolism in neuronal cells. In the current study we identified the BACE1 cleavage sites in human Navβ2. Results We found a major (147-148 L↓M, where ↓ indicates the cleavage site and a minor (144145 L↓Q BACE1 cleavage site in the extracellular domain of human Navβ2 using a cell-free BACE1 cleavage assay followed by mass spectrometry. Next, we introduced two different double mutations into the identified major BACE1 cleavage site in human Navβ2: 147LM/VI and 147LM/AA. Both mutations dramatically decreased the cleavage of human Navβ2 by endogenous BACE1 in cell-free BACE1 cleavage assays. Neither of the two mutations affected subcellular localization of Navβ2 as confirmed by confocal fluorescence microscopy and subcellular fractionation of cholesterol-rich domains. Finally, wildtype and mutated Navβ2 were expressed along BACE1 in B104 rat neuroblastoma cells. In spite of α-secretase still actively cleaving the mutant proteins, Navβ2 cleavage products decreased by ~50% in cells expressing Navβ2 (147LM/VI and ~75% in cells expressing Navβ2 (147LM/AA as compared to cells expressing wildtype Navβ2. Conclusion We identified a major (147-148 L↓M and a minor (144-145 L↓Q BACE1 cleavage site in human Navβ2. Our in vitro and cell-based results clearly show that the 147-148 L↓M is the major BACE1 cleavage site in human Navβ2. These findings expand our understanding of the role of BACE1 in voltage-gated sodium channel metabolism.

  4. Effect of blockers of Kv and BKCa channels on muscle tension of rabbit oddi sphincter in vivo and th e regulatory effect of paeoniflorin%Kv 和 BK Ca通道阻断剂对家兔离体 Oddi括约肌肌环张力的作用及芍药甙的调控作用

    Institute of Scientific and Technical Information of China (English)

    雒建瑞; 王芳; 冯骅; 王长淼

    2014-01-01

    Objective It is to investigate the effect of blockers of voltage-gated potassium channels ( Kv) , large-conduct-ance calcium-activated potassium channel ( BKCa) on sphincter of Oddi ( sphincter of Oddi , SO) from rabbits muscle ten-sion rings, and to explore the regulatory effect of the active ingredients of traditional Chinese medicine paeoniflorin on them . Methods Isolated rabbit sphincter of Oddi muscle rings specimens were prepared and placed on the smooth muscle perfusion bath temperature to observe the contraction effect of Kv channel blocker 4-aminopyridine (4-AP), BKCa channel blocker tetraethylammonium chloride( TEA) on isolated rabbit SO muscle rings and the effects of paeoniflorin on the contraction effects induced by 4-AP and TEA.Resul ts 4-AP and TEA both could cause contraction of SO muscle rings , with the the concen-trations increased , the contraction degrees increased .The contraction effects induced by 4-AP and TEA could be inhibited by paeoniflorin .Conclusion In vitro condition , Kv channel and BKCa channel play a major role in the maintenance of sphincter of Oddi resting membrane potential of cells .Paeoniflorin may influence the relaxant responses of SO cells possibly through the regulation of Kv and BKCa channels .%目的:探讨电压依赖性钾通道( Kv)、大电导钙激活钾通道( BKCa )阻断剂对家兔离体Oddi 括约肌( SO)肌环张力的作用及中药芍药的有效成分芍药甙对其调控作用。方法制备离体兔Oddi 括约肌肌环标本,放置于平滑肌恒温灌流浴槽中,观察Kv通道阻断剂4-aminopyridine (4-AP)、BKCa通道阻断剂tetraethylammonium chloride ( TEA)对家兔离体SO肌环的收缩作用;观察芍药甙对4-AP和TEA引起的收缩作用的影响。结果4-AP、TEA均可以引起SO肌环收缩,且随4-AP、TEA浓度增加,收缩程度也在增强。4-AP和TEA对SO肌环的收缩作用可以被芍药甙抑制。结论在离体条件下,Kv通道和BKCa通道

  5. 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.

  6. An optofluidic channel model for in vivo nanosensor networks in human blood

    Science.gov (United States)

    Johari, Pedram; Jornet, Josep M.

    2017-05-01

    In vivo Wireless Nanosensor Networks (iWNSNs) consist of nano-sized communicating devices with unprece- dented sensing and actuation capabilities, which are able to operate inside the human body. iWNSNs are a disruptive technology that enables the monitoring and control of biological processes at the cellular and sub- cellular levels. Compared to ex vivo measurements, which are conducted on samples extracted from the human body, iWNSNs can track (sub) cellular processes when and where they occur. Major progress in the field of na- noelectronics, nanophotonics and wireless communication is enabling the interconnection of nanosensors. Among others, plasmonic nanolasers with sub-micrometric footprint, plasmonic nano-antennas able to confine light in nanometric structures, and single-photon detectors with unrivaled sensitivity, enable the communication among implanted nanosensors in the near infrared and optical transmission windows. Motivated by these results, in this paper, an optofluidic channel model is developed to investigate the communication properties and temporal dynamics between a pair of in vivo nanosensors in the human blood. The developed model builds upon the authors' recent work on light propagation modeling through multi-layered single cells and cell assemblies and takes into account the geometric, electromagnetic and microfluidic properties of red blood cells in the human circulatory system. The proposed model guides the development of practical communication strategies among nanosensors, and paves the way through new nano-biosensing strategies able to identify diseases by detecting the slight changes in the channel impulse response, caused by either the change in shape of the blood cells or the presence of pathogens.

  7. 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

  8. The effect of pH and ion channel modulators on human placental arteries.

    Directory of Open Access Journals (Sweden)

    Tayyba Y Ali

    Full Text Available Chorionic plate arteries (CPA are located at the maternofetal interface where they are able to respond to local metabolic changes. Unlike many other types of vasculature, the placenta lacks nervous control and requires autoregulation for controlling blood flow. The placental circulation, which is of low-resistance, may become hypoxic easily leading to fetal acidosis and fetal distress however the role of the ion channels in these circumstances is not well-understood. Active potassium channel conductances that are subject to local physicochemical modulation may serve as pathways through which such signals are transduced. The aim of this study was to investigate the modulation of CPA by pH and the channels implicated in these responses using wire myography. CPA were isolated from healthy placentae and pre-contracted with U46619 before testing the effects of extracellular pH using 1 M lactic acid over the pH range 7.4-6.4 in the presence of a variety of ion channel modulators. A change from pH 7.4 to 7.2 produced a 29±3% (n = 9 relaxation of CPA which increased to 61±4% at the lowest pH of 6.4. In vessels isolated from placentae of women with pre-eclampsia (n = 6, pH responses were attenuated. L-methionine increased the relaxation to 67±7% (n = 6; p<0.001 at pH 6.4. Similarly the TASK 1/3 blocker zinc chloride (1 mM gave a maximum relaxation of 72±5% (n = 8; p<0.01 which compared with the relaxation produced by the TREK-1 opener riluzole (75±5%; n = 6. Several other modulators induced no significant changes in vascular responses. Our study confirmed expression of several ion channel subtypes in CPA with our results indicating that extracellular pH within the physiological range has an important role in controlling vasodilatation in the human term placenta.

  9. Neurogenic detrusor overactivity is associated with decreased expression and function of the large conductance voltage- and Ca(2+-activated K(+ channels.

    Directory of Open Access Journals (Sweden)

    Kiril L Hristov

    Full Text Available Patients suffering from a variety of neurological diseases such as spinal cord injury, Parkinson's disease, and multiple sclerosis often develop neurogenic detrusor overactivity (NDO, which currently lacks a universally effective therapy. Here, we tested the hypothesis that NDO is associated with changes in detrusor smooth muscle (DSM large conductance Ca(2+-activated K(+ (BK channel expression and function. DSM tissue samples from 33 patients were obtained during open bladder surgeries. NDO patients were clinically characterized preoperatively with pressure-flow urodynamics demonstrating detrusor overactivity, in the setting of a clinically relevant neurological condition. Control patients did not have overactive bladder and did not have a clinically relevant neurological disease. We conducted quantitative polymerase chain reactions (qPCR, perforated patch-clamp electrophysiology on freshly-isolated DSM cells, and functional studies on DSM contractility. qPCR experiments revealed that DSM samples from NDO patients showed decreased BK channel mRNA expression in comparison to controls. Patch-clamp experiments demonstrated reduced whole cell and transient BK currents (TBKCs in freshly-isolated DSM cells from NDO patients. Functional studies on DSM contractility showed that spontaneous phasic contractions had a decreased sensitivity to iberiotoxin, a selective BK channel inhibitor, in DSM strips isolated from NDO patients. These results reveal the novel finding that NDO is associated with decreased DSM BK channel expression and function leading to increased DSM excitability and contractility. BK channel openers or BK channel gene transfer could be an alternative strategy to control NDO. Future clinical trials are needed to evaluate the value of BK channel opening drugs or gene therapies for NDO treatment and to identify any possible adverse effects.

  10. 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)).

  11. Search for the Decay B+-->K+ tau-/+ mu+/-.

    Science.gov (United States)

    Aubert, B; Bona, M; Boutigny, D; Karyotakis, Y; Lees, J P; Poireau, V; Prudent, X; Tisserand, V; Zghiche, A; Garra Tico, J; Grauges, E; Lopez, L; Palano, A; Pappagallo, M; Eigen, G; Stugu, B; Sun, L; Abrams, G S; Battaglia, M; Brown, D N; Button-Shafer, J; Cahn, R N; Groysman, Y; Jacobsen, R G; Kadyk, J A; Kerth, L T; Kolomensky, Yu G; Kukartsev, G; Lopes Pegna, D; Lynch, G; Mir, L M; Orimoto, T J; Osipenkov, I L; Ronan, M T; Tackmann, K; Tanabe, T; Wenzel, W A; del Amo Sanchez, P; Hawkes, C M; Watson, A T; Held, T; Koch, H; Pelizaeus, M; Schroeder, T; Steinke, M; Walker, D; Asgeirsson, D J; Cuhadar-Donszelmann, T; Fulsom, B G; Hearty, C; Mattison, T S; McKenna, J A; Khan, A; Saleem, M; Teodorescu, L; Blinov, V E; Bukin, A D; Druzhinin, V P; Golubev, V B; Onuchin, A P; Serednyakov, S I; Skovpen, Yu I; Solodov, E P; Todyshev, K Yu; Bondioli, M; Curry, S; Eschrich, I; Kirkby, D; Lankford, A J; Lund, P; Mandelkern, M; Martin, E C; Stoker, D P; Abachi, S; Buchanan, C; Foulkes, S D; Gary, J W; Liu, F; Long, O; Shen, B C; Zhang, L; Paar, H P; Rahatlou, S; Sharma, V; Berryhill, J W; Campagnari, C; Cunha, A; Dahmes, B; Hong, T M; Kovalskyi, D; Richman, J D; Beck, T W; Eisner, A M; Flacco, C J; Heusch, C A; Kroseberg, J; Lockman, W S; Schalk, T; Schumm, B A; Seiden, A; Wilson, M G; Winstrom, L O; Chen, E; Cheng, C H; Fang, F; Hitlin, D G; Narsky, I; Piatenko, T; Porter, F C; Andreassen, R; Mancinelli, G; Meadows, B T; Mishra, K; Sokoloff, M D; Blanc, F; Bloom, P C; Chen, S; Ford, W T; Hirschauer, J F; Kreisel, A; Nagel, M; Nauenberg, U; Olivas, A; Smith, J G; Ulmer, K A; Wagner, S R; Zhang, J; Gabareen, A M; Soffer, A; Toki, W H; Wilson, R J; Winklmeier, F; Altenburg, D D; Feltresi, E; Hauke, A; Jasper, H; Merkel, J; Petzold, A; Spaan, B; Wacker, K; Klose, V; Kobel, M J; Lacker, H M; Mader, W F; Nogowski, R; Schubert, J; Schubert, K R; Schwierz, R; Sundermann, J E; Volk, A; Bernard, D; Bonneaud, G R; Latour, E; Lombardo, V; Thiebaux, Ch; Verderi, M; Clark, P J; Gradl, W; Muheim, F; Playfer, S; Robertson, A I; Watson, J E; Xie, Y; Andreotti, M; Bettoni, D; Bozzi, C; Calabrese, R; Cecchi, A; Cibinetto, G; Franchini, P; Luppi, E; Negrini, M; Petrella, A; Piemontese, L; Prencipe, E; Santoro, V; Anulli, F; Baldini-Ferroli, R; Calcaterra, A; de Sangro, R; Finocchiaro, G; Pacetti, S; Patteri, P; Peruzzi, I M; Piccolo, M; Rama, M; Zallo, A; Buzzo, A; Contri, R; Lo Vetere, M; Macri, M M; Monge, M R; Passaggio, S; Patrignani, C; Robutti, E; Santroni, A; Tosi, S; Chaisanguanthum, K S; Morii, M; Wu, J; Dubitzky, R S; Marks, J; Schenk, S; Uwer, U; Bard, D J; Dauncey, P D; Flack, R L; Nash, J A; Panduro Vazquez, W; Tibbetts, M; Behera, P K; Chai, X; Charles, M J; Mallik, U; Ziegler, V; Cochran, J; Crawley, H B; Dong, L; Eyges, V; Meyer, W T; Prell, S; Rosenberg, E I; Rubin, A E; Gao, Y Y; Gritsan, A V; Guo, Z J; Lae, C K; Denig, A G; Fritsch, M; Schott, G; Arnaud, N; Béquilleux, J; D'Orazio, A; Davier, M; Grosdidier, G; Höcker, A; Lepeltier, V; Le Diberder, F; Lutz, A M; Pruvot, S; Rodier, S; Roudeau, P; Schune, M H; Serrano, J; Sordini, V; Stocchi, A; Wang, W F; Wormser, G; Lange, D J; Wright, D M; Bingham, I; Burke, J P; Chavez, C A; Forster, I J; Fry, J R; Gabathuler, E; Gamet, R; Hutchcroft, D E; Payne, D J; Schofield, K C; Touramanis, C; Bevan, A J; George, K A; Di Lodovico, F; Menges, W; Sacco, R; Cowan, G; Flaecher, H U; Hopkins, D A; Paramesvaran, S; Salvatore, F; Wren, A C; Brown, D N; Davis, C L; Allison, J; Barlow, N R; Barlow, R J; Chia, Y M; Edgar, C L; Lafferty, G D; West, T J; Yi, J I; Anderson, J; Chen, C; Jawahery, A; Roberts, D A; Simi, G; Tuggle, J M; Blaylock, G; Dallapiccola, C; Hertzbach, S S; Li, X; Moore, T B; Salvati, E; Saremi, S; Cowan, R; Dujmic, D; Fisher, P H; Koeneke, K; Sciolla, G; Sekula, S J; Spitznagel, M; Taylor, F; Yamamoto, R K; Zhao, M; Zheng, Y; Mclachlin, S E; Patel, P M; Robertson, S H; Lazzaro, A; Palombo, F; Bauer, J M; Cremaldi, L; Eschenburg, V; Godang, R; Kroeger, R; Sanders, D A; Summers, D J; Zhao, H W; Brunet, S; Côté, D; Simard, M; Taras, P; Viaud, F B; Nicholson, H; De Nardo, G; Fabozzi, F; Lista, L; Monorchio, D; Sciacca, C; Baak, M A; Raven, G; Snoek, H L; Jessop, C P; Knoepfel, K J; LoSecco, J M; Benelli, G; Corwin, L A; Honscheid, K; Kagan, H; Kass, R; Morris, J P; Rahimi, A M; Regensburger, J J; Wong, Q K; Blount, N L; Brau, J; Frey, R; Igonkina, O; Kolb, J A; Lu, M; Rahmat, R; Sinev, N B; Strom, D; Strube, J; Torrence, E; Gagliardi, N; Gaz, A; Margoni, M; Morandin, M; Pompili, A; Posocco, M; Rotondo, M; Simonetto, F; Stroili, R; Voci, C; Ben-Haim, E; Briand, H; Calderini, G; Chauveau, J; David, P; Del Buono, L; de la Vaissière, Ch; Hamon, O; Leruste, Ph; Malclès, J; Ocariz, J; Perez, A; Prendki, J; Gladney, L; Biasini, M; Covarelli, R; Manoni, E; Angelini, C; Batignani, G; Bettarini, S; Carpinelli, M; Cenci, R; Cervelli, A; Forti, F; Giorgi, M A; Lusiani, A; Marchiori, G; Mazur, M A; Morganti, M; Neri, N; Paoloni, E; Rizzo, G; Walsh, J J; Haire, M; Biesiada, J; Elmer, P; Lau, Y P; Lu, C; Olsen, J; Smith, A J S; Telnov, A V; Baracchini, E; Bellini, F; Cavoto, G; del Re, D; Di Marco, E; Faccini, R; Ferrarotto, F; Ferroni, F; Gaspero, M; Jackson, P D; Gioi, L Li; Mazzoni, M A; Morganti, S; Piredda, G; Polci, F; Renga, F; Voena, C; Ebert, M; Hartmann, T; Schröder, H; Waldi, R; Adye, T; Castelli, G; Franek, B; Olaiya, E O; Ricciardi, S; Roethel, W; Wilson, F F; Emery, S; Escalier, M; Gaidot, A; Ganzhur, S F; Hamel de Monchenault, G; Kozanecki, W; Vasseur, G; Yèche, Ch; Zito, M; Chen, X R; Liu, H; Park, W; Purohit, M V; Wilson, J R; Allen, M T; Aston, D; Bartoldus, R; Bechtle, P; Berger, N; Claus, R; Coleman, J P; Convery, M R; Dingfelder, J C; Dorfan, J; Dubois-Felsmann, G P; Dunwoodie, W; Field, R C; Glanzman, T; Gowdy, S J; Graham, M T; Grenier, P; Hast, C; Hryn'ova, T; Innes, W R; Kaminski, J; Kelsey, M H; Kim, H; Kim, P; Kocian, M L; Leith, D W G S; Li, S; Luitz, S; Luth, V; Lynch, H L; MacFarlane, D B; Marsiske, H; Messner, R; Muller, D R; O'Grady, C P; Ofte, I; Perazzo, A; Perl, M; Pulliam, T; Ratcliff, B N; Roodman, A; Salnikov, A A; Schindler, R H; Schwiening, J; Snyder, A; Stelzer, J; Su, D; Sullivan, M K; Suzuki, K; Swain, S K; Thompson, J M; Va'vra, J; van Bakel, N; Wagner, A P; Weaver, M; Wisniewski, W J; Wittgen, M; Wright, D H; Yarritu, A K; Yi, K; Young, C C; Burchat, P R; Edwards, A J; Majewski, S A; Petersen, B A; Wilden, L; Ahmed, S; Alam, M S; Bula, R; Ernst, J A; Jain, V; Pan, B; Saeed, M A; Wappler, F R; Zain, S B; Krishnamurthy, M; Spanier, S M; Eckmann, R; Ritchie, J L; Ruland, A M; Schilling, C J; Schwitters, R F; Izen, J M; Lou, X C; Ye, S; Bianchi, F; Gallo, F; Gamba, D; Pelliccioni, M; Bomben, M; Bosisio, L; Cartaro, C; Cossutti, F; Della Ricca, G; Lanceri, L; Vitale, L; Azzolini, V; Lopez-March, N; Martinez-Vidal, F; Milanes, D A; Oyanguren, A; Albert, J; Banerjee, Sw; Bhuyan, B; Hamano, K; Kowalewski, R; Nugent, I M; Roney, J M; Sobie, R J; Harrison, P F; Ilic, J; Latham, T E; Mohanty, G B; Band, H R; Chen, X; Dasu, S; Flood, K T; Hollar, J J; Kutter, P E; Pan, Y; Pierini, M; Prepost, R; Wu, S L; Neal, H

    2007-11-16

    We present a search for the lepton flavor violating decay B+-->K+ tau-/+ mu+/- using 383 x 10;{6} BB[over ] events collected by the BABAR experiment. The branching fraction for this decay can be substantially enhanced in new physics models. The kinematics of the tau from the signal B decay are inferred from the K+, mu, and other B in the event, which is fully reconstructed in one of a variety of hadronic decay modes, allowing the signal B candidate to be fully reconstructed. We observe no excess of events over the expected background and set a limit of B(B+-->K+ tau mu)<7.7 x 10(-5) at 90% confidence level, where the branching fraction is for the sum of the K+ tau- mu+ and K+ tau+mu- final states. We use this result to improve a model-independent bound on the energy scale of flavor-changing new physics.

  12. Covariance fitting of highly correlated $B_K$ data

    CERN Document Server

    Yoon, Boram; Jung, Chulwoo; Lee, Weonjong

    2011-01-01

    We present the reason why we use the diagonal approximation (uncorrelated fitting) when we perform the data analysis of highly correlated $B_K$ data on the basis of the SU(2) staggered chiral perturbation theory. Basically, the essence of the problem is that we do not have enough statistics to determine the small eigenvalues of the covariance matrix with a high precision. As a result, we have the smallest eigenvalue, which is smaller than the statistical error of the covariance matrix, corresponding to an unphysical eigenmode. We have applied a number of prescriptions available in the market such as the cutoff method and modified covariance matrix method. It turns out that the cutoff method is not a good prescription and the modified covariance matrix method is an even worse one. The diagonal approximation turns out to be a good prescription if the data points are somehow correlated and the statistics are relatively poor.

  13. Block of Human Cardiac Sodium Channels by Lacosamide: Evidence for Slow Drug Binding along the Activation Pathway

    OpenAIRE

    Wang, Ging Kuo; Wang, Sho-Ya

    2014-01-01

    Lacosamide is an anticonvulsant hypothesized to enhance slow inactivation of neuronal Na+ channels for its therapeutic action. Cardiac Na+ channels display less and incomplete slow inactivation, but their sensitivity toward lacosamide remains unknown. We therefore investigated the action of lacosamide in human cardiac Nav1.5 and Nav1.5-CW inactivation-deficient Na+ channels. Lacosamide showed little effect on hNav1.5 Na+ currents at 300 µM when cells were held at −140 mV. With 30-second condi...

  14. Emerging role of calcium-activated potassium channel in the regulation of cell viability following potassium ions challenge in HEK293 cells and pharmacological modulation.

    Directory of Open Access Journals (Sweden)

    Domenico Tricarico

    Full Text Available Emerging evidences suggest that Ca(2+activated-K(+-(BK channel is involved in the regulation of cell viability. The changes of the cell viability observed under hyperkalemia (15 mEq/L or hypokalemia (0.55 mEq/L conditions were investigated in HEK293 cells expressing the hslo subunit (hslo-HEK293 in the presence or absence of BK channel modulators. The BK channel openers(10(-11-10(-3M were: acetazolamide(ACTZ, Dichlorphenamide(DCP, methazolamide(MTZ, bendroflumethiazide(BFT, ethoxzolamide(ETX, hydrochlorthiazide(HCT, quercetin(QUERC, resveratrol(RESV and NS1619; and the BK channel blockers(2 x 10(-7M-5 x 10(-3M were: tetraethylammonium(TEA, iberiotoxin(IbTx and charybdotoxin(ChTX. Experiments on cell viability and channel currents were performed using cell counting kit-8 and patch-clamp techniques, respectively. Hslo whole-cell current was potentiated by BK channel openers with different potency and efficacy in hslo-HEK293. The efficacy ranking of the openers at -60 mV(Vm was BFT> ACTZ >DCP ≥RESV≥ ETX> NS1619> MTZ≥ QUERC; HCT was not effective. Cell viability after 24 h of incubation under hyperkalemia was enhanced by 82+6% and 33+7% in hslo-HEK293 cells and HEK293 cells, respectively. IbTx, ChTX and TEA enhanced cell viability in hslo-HEK293. BK openers prevented the enhancement of the cell viability induced by hyperkalemia or IbTx in hslo-HEK293 showing an efficacy which was comparable with that observed as BK openers. BK channel modulators failed to affect cell currents and viability under hyperkalemia conditions in the absence of hslo subunit. In contrast, under hypokalemia cell viability was reduced by -22+4% and -23+6% in hslo-HEK293 and HEK293 cells, respectively; the BK channel modulators failed to affect this parameter in these cells. In conclusion, BK channel regulates cell viability under hyperkalemia but not hypokalemia conditions. BFT and ACTZ were the most potent drugs either in activating the BK current and in preventing the

  15. Emerging role of calcium-activated potassium channel in the regulation of cell viability following potassium ions challenge in HEK293 cells and pharmacological modulation.

    Science.gov (United States)

    Tricarico, Domenico; Mele, Antonietta; Calzolaro, Sara; Cannone, Gianluigi; Camerino, Giulia Maria; Dinardo, Maria Maddalena; Latorre, Ramon; Conte Camerino, Diana

    2013-01-01

    Emerging evidences suggest that Ca(2+)activated-K(+)-(BK) channel is involved in the regulation of cell viability. The changes of the cell viability observed under hyperkalemia (15 mEq/L) or hypokalemia (0.55 mEq/L) conditions were investigated in HEK293 cells expressing the hslo subunit (hslo-HEK293) in the presence or absence of BK channel modulators. The BK channel openers(10(-11)-10(-3)M) were: acetazolamide(ACTZ), Dichlorphenamide(DCP), methazolamide(MTZ), bendroflumethiazide(BFT), ethoxzolamide(ETX), hydrochlorthiazide(HCT), quercetin(QUERC), resveratrol(RESV) and NS1619; and the BK channel blockers(2 x 10(-7)M-5 x 10(-3)M) were: tetraethylammonium(TEA), iberiotoxin(IbTx) and charybdotoxin(ChTX). Experiments on cell viability and channel currents were performed using cell counting kit-8 and patch-clamp techniques, respectively. Hslo whole-cell current was potentiated by BK channel openers with different potency and efficacy in hslo-HEK293. The efficacy ranking of the openers at -60 mV(Vm) was BFT> ACTZ >DCP ≥RESV≥ ETX> NS1619> MTZ≥ QUERC; HCT was not effective. Cell viability after 24 h of incubation under hyperkalemia was enhanced by 82+6% and 33+7% in hslo-HEK293 cells and HEK293 cells, respectively. IbTx, ChTX and TEA enhanced cell viability in hslo-HEK293. BK openers prevented the enhancement of the cell viability induced by hyperkalemia or IbTx in hslo-HEK293 showing an efficacy which was comparable with that observed as BK openers. BK channel modulators failed to affect cell currents and viability under hyperkalemia conditions in the absence of hslo subunit. In contrast, under hypokalemia cell viability was reduced by -22+4% and -23+6% in hslo-HEK293 and HEK293 cells, respectively; the BK channel modulators failed to affect this parameter in these cells. In conclusion, BK channel regulates cell viability under hyperkalemia but not hypokalemia conditions. BFT and ACTZ were the most potent drugs either in activating the BK current and in preventing

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

    Science.gov (United States)

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

    2015-01-01

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

  17. Novel pharmacological activity of loperamide and CP-339,818 on human HCN channels characterized with an automated electrophysiology assay.

    Science.gov (United States)

    Lee, Yan T; Vasilyev, Dmitry V; Shan, Qin J; Dunlop, John; Mayer, Scott; Bowlby, Mark R

    2008-02-26

    Hyperpolarization-activated cyclic nucleotide-gated (HCN) channels underlie the pacemaker currents in neurons (I(h)) and cardiac (I(f)) cells. As such, the identification and characterization of novel blockers of HCN channels is important to enable the dissection of their function in vivo. Using a new IonWorks HT electrophysiology assay with human HCN1 and HCN4 expressed stably in cell lines, four HCN channel blockers are characterized. Two blockers known for their activity at opioid/Ca(2+) channels and K(+) channels, loperamide and CP-339,818 (respectively), are described to block HCN1 more potently than HCN4. The known HCN blocker ZD7288 was also found to be more selective for HCN1 over HCN4, while the HCN blocker DK-AH269 was equipotent on HCN4 and HCN1. Partial replacement of the intracellular Cl(-) with gluconate reduced the potency on both channels, but to varying degrees. For both HCN1 and HCN4, ZD7288 was most sensitive in lower Cl(-) solutions, while the potency of loperamide was not affected by the differing solutions. The block of HCN1 for all compounds was voltage-dependent, being relieved at more negative potentials. The voltage-dependent, Cl(-) dependent, HCN1 preferring compounds described here elaborate on the current known pharmacology of HCN channels and may help provide novel tools and chemical starting points for the investigation of HCN channel function in natively expressing systems.

  18. Production and decay of the heaviest odd-Z nuclei in the 249Bk + 48Ca reaction

    Science.gov (United States)

    Oganessian, Yu Ts; Abdullin, F. Sh; Alexander, C.; Binder, J.; Boll, R. A.; Dmitriev, S. N.; Ezold, J.; Felker, K.; Gostic, J. M.; Grzywacz, R. K.; Hamilton, J. H.; Henderson, R. A.; Itkis, M. G.; Miernik, K.; Miller, D.; Moody, K. J.; Polyakov, A. N.; Ramayya, A. V.; Roberto, J. B.; Ryabinin, M. A.; Rykaczewski, K. P.; Sagaidak, R. N.; Shaughnessy, D. A.; Shirokovsky, I. V.; Shumeiko, M. V.; Stoyer, M. A.; Stoyer, N. J.; Subbotin, V. G.; Sukhov, A. M.; Tsyganov, Yu S.; Utyonkov, V. K.; Voinov, A. A.; Vostokin, G. K.

    2015-02-01

    The reaction of 249Bk with 48Ca has been investigated with an aim of synthesizing and studying the decay properties of isotopes of the new element 117. The experiments were performed at five projectile energies (in two runs, in 2009-2010 and 2012) and with a total beam dose of 48Ca ions of about 9x1019 The experiments yielded data on a-decay characteristics and excitation functions of the produced nuclei that establish these to be 293117 and 294117 - the products of the 4n- and 3n-evaporation channels, respectively. In total, we have observed 20 decay chains of Z=117 nuclides. The cross sections were measured to be 1.1 pb for the 3n and 2.4 pb for the 4n-reaction channel. The new 289115 events, populated by α decay of 117, demonstrate the same decay properties as those observed for 115 produced in the 243Am(48Ca,2n) reaction thus providing cross-bombardment evidence. In addition, a single decay of 294118 was observed from the reaction with 249Cf - a result of the in-growth of 249Cf in the 249Bk target. The observed decay chain of 294118 is in good agreement with decay properties obtained in 2002-2005 in the experiments with the reaction 249Cf(48Ca,3n)294118. The energies and half-lives of the odd-Z isotopes observed in the 117 decay chains together with the results obtained for lower-Z superheavy nuclei demonstrate enhancement of nuclear stability with increasing neutron number towards the predicted new magic number N=184.

  19. Sustained calcium entry through P2X nucleotide receptor channels in human airway epithelial cells.

    Science.gov (United States)

    Zsembery, Akos; Boyce, Amanda T; Liang, Lihua; Peti-Peterdi, János; Bell, P Darwin; Schwiebert, Erik M

    2003-04-11

    Purinergic receptor stimulation has potential therapeutic effects for cystic fibrosis (CF). Thus, we explored roles for P2Y and P2X receptors in stably increasing [Ca(2+)](i) in human CF (IB3-1) and non-CF (16HBE14o(-)) airway epithelial cells. Cytosolic Ca(2+) was measured by fluorospectrometry using the fluorescent dye Fura-2/AM. Expression of P2X receptor (P2XR) subtypes was assessed by immunoblotting and biotinylation. In IB3-1 cells, ATP and other P2Y agonists caused only a transient increase in [Ca(2+)](i) derived from intracellular stores in a Na(+)-rich environment. In contrast, ATP induced an increase in [Ca(2+)](i) that had transient and sustained components in a Na(+)-free medium; the sustained plateau was potentiated by zinc or increasing extracellular pH. Benzoyl-benzoyl-ATP, a P2XR-selective agonist, increased [Ca(2+)](i) only in Na(+)-free medium, suggesting competition between Na(+) and Ca(2+) through P2XRs. Biochemical evidence showed that the P2X(4) receptor is the major subtype shared by these airway epithelial cells. A role for store-operated Ca(2+) channels, voltage-dependent Ca(2+) channels, or Na(+)/Ca(2+) exchanger in the ATP-induced sustained Ca(2+) signal was ruled out. In conclusion, these data show that epithelial P2X(4) receptors serve as ATP-gated calcium entry channels that induce a sustained increase in [Ca(2+)](i). In airway epithelia, a P2XR-mediated Ca(2+) signal may have therapeutic benefit for CF.

  20. Changes to channel sediments resulting from complex human impacts in a gravel-bed river, Polish Carpathians

    Science.gov (United States)

    Zawiejska, Joanna; Wyżga, Bartłomiej; Hajdukiewicz, Hanna; Radecki-Pawlik, Artur; Mikuś, Paweł

    2016-04-01

    deficit downstream as large volumes of finer bed material are flushed out from the incising channel section. Grain-size analyses of bulk gravels and measurements of 100 coarsest particles within the channel sediment ranging in age from 5200 years BP to the present, performed in this deeply incised section, indicated that grain size of channel sediments changed relatively little since mid-Holocene to the 1960s, but has increased rapidly over the last half-century as a result of human interventions and rapidly progressing channel incision. This study was performed within the scope of the Research Project DEC-2013/09/B/ST10/00056 financed by the National Science Centre of Poland.

  1. Fusion and quasifission dynamics in the reactions $^{48}$Ca+$^{249}$Bk and $^{50}$Ti+$^{249}$Bk using TDHF

    CERN Document Server

    Umar, A S; Simenel, C

    2016-01-01

    Background: Synthesis of superheavy elements (SHE) with fusion-evaporation reactions is strongly hindered by the quasifission (QF) mechanism which prevents the formation of an equilibrated compound nucleus and which depends on the structure of the reactants. New SHE have been recently produced with doubly-magic $^{48}$Ca beams. However, SHE synthesis experiments with single-magic $^{50}$Ti beams have so far been unsuccessful. Purpose: In connection with experimental searches for $Z=117,119$ superheavy elements, we perform a theoretical study of fusion and quasifission mechanisms in $^{48}$Ca,$^{50}$Ti+$^{249}$Bk reactions in order to investigate possible differences in reaction mechanisms induced by these two projectiles. Methods: The collision dynamics and the outcome of the reactions are studied using unrestricted time-dependent Hartree-Fock (TDHF) calculations as well as the density-constrained TDHF method to extract the nucleus-nucleus potentials and the excitation energy in each fragment. Results: Nucleu...

  2. Cadmium regulates the expression of the CFTR chloride channel in human airway epithelial cells.

    Science.gov (United States)

    Rennolds, Jessica; Butler, Susie; Maloney, Kevin; Boyaka, Prosper N; Davis, Ian C; Knoell, Daren L; Parinandi, Narasimham L; Cormet-Boyaka, Estelle

    2010-07-01

    Cadmium is a toxic heavy metal ranked seventh on the Priority List of Hazardous Substances. As a byproduct of smelters, cadmium is a prevalent environmental contaminant. It is also a major component of cigarette smoke, and its inhalation is associated with decreased pulmonary function, lung cancer, and chronic obstructive pulmonary disease. Ion channels, including the cystic fibrosis transmembrane conductance regulator (CFTR), play a central role in maintaining fluid homeostasis and lung functions. CFTR is mostly expressed in epithelial cells, and little is known about the effect of cadmium exposure on lung epithelial cell function. We show that exposure to cadmium decreases the expression of the CFTR protein and subsequent chloride transport in human airway epithelial cells in vitro. Impairment of CFTR protein expression was also observed in vivo in the lung of mice after intranasal instillation of cadmium. We established that the inhibitory effect of cadmium was not a nonspecific effect of heavy metals, as nickel had no effect on CFTR protein levels. Finally, we show that selected antioxidants, including alpha-tocopherol (vitamin E), but not N-acetylcysteine, can prevent the cadmium-induced suppression of CFTR. In summary, we have identified cadmium as a regulator of the CFTR chloride channel present in lung epithelial cells. Future strategies to prevent the deleterious effect of cadmium on epithelial cells and lung functions may benefit from the finding that alpha-tocopherol protects CFTR expression and function.

  3. Molecular modeling and docking simulations of scorpion toxins and related analogs on human SKCa2 and SKCa3 channels.

    Science.gov (United States)

    Andreotti, Nicolas; di Luccio, Eric; Sampieri, François; De Waard, Michel; Sabatier, Jean-Marc

    2005-07-01

    The small-conductance Ca2+-activated K+ (SKCa) channels modulate cytosolic Ca2+ concentration in excitable and non-excitable tissues by regulating the membrane potential and are responsible of slow action potential after hyperpolarization that inhibits cell firing. Among these, human SKCa2 and SKCa3 channels differ in the pore region by only two residues: Ala331 and Asn367 (human small-conductance calcium-activated potassium channel, hSKCa2) instead of Val485 and His521 (hSKCa3). To design highly selective blockers of hSKCa channels, a number of known hSKCa2 and/or hSKCa3-active peptides (i.e. scorpion toxins and analogs thereof) were analyzed for their interactions and selectivities toward these channels. Molecular models of hSKCa2 and hSKCa3 channels (S5-H5-S6 portion) were generated, and scorpion toxins/peptides of unsolved three-dimensional (3D) structures were modeled. Models of toxin-channel complexes were generated by the bimolecular complex generation with global evaluation, and ranking (BiGGER) docking software and selected by using a screening method of the docking solutions. A high degree of correlation was found to exist between docking energies and experimental Kd values of peptides that blocked hSKCa2 and/or hSKCa3 channels, suggesting it could be appropriate to predict Kd values of other bioactive peptides. The best scoring complexes were also used to identify key residues of both interacting partners, indicating that such an approach should help the design of more active and/or selective peptide blockers of targeted ion channels.

  4. Pulsatile atheroprone shear stress affects the expression of transient receptor potential channels in human endothelial cells

    DEFF Research Database (Denmark)

    Thilo, Florian; Vorderwülbecke, Bernd J; Marki, Alex

    2012-01-01

    as measured by quantitative real-time RT-PCR and normalized to GAPDH expression. Thereby, TRPC6 and TRPV1 mRNA expressions were significantly increased after 24 hours of exposure to an atheroprone flow profile compared with an atheroprotective flow profile. Furthermore, the expression of transcription factors......The goal of the study was to assess whether pulsatile atheroprone shear stress modulates the expression of transient receptor potential (TRP) channels, TRPC3, TRPC6, TRPM7, and TRPV1 mRNA, in human umbilical vascular endothelial cells. Exposure of cultured vascular endothelial cells to defined...... shear stress, producing a constant laminar flow (generating a shear stress of 6 dyne/cm(2)), laminar pulsatile atheroprotective flow (with a mean shear stress of 20 dyne/cm(2)), or laminar atheroprone bidirectional flow (with a mean shear stress of 0 dyne/cm(2)) differentially induced TRPC6 and TRPV1 mRNA...

  5. Origin and fate of the nucleolar channel system of normal human endometium

    Institute of Scientific and Technical Information of China (English)

    WANGTZUNENG; JSCHNEIDER

    1992-01-01

    Human normal endometrium was examined in ultrathin sections.Nucleolar channel system(NCS) appeared in the endometrial epithelial cells during the early and mid secretory phase of menstrual cycle.The NCS was a hollow ball like structure of different sizes and was composed of 2 to 5 rows of tubules embedded in an amporphous matrix.On its surface there were numerous electron dense particles resembling ribosomes,It was usually located within or associated with the nucleolus,SOmetimes,it was close to the nuclear envelope or protruding out from the nucleus .On occasion,NCS with simplified structure was found in the perinuclear cytoplasm.Concepts concerning the genesis,involution and function(s) of the NCS were disussed.

  6. Transient receptor potential vanilloid type-1 (TRPV-1) channels contribute to cutaneous thermal hyperaemia in humans.

    Science.gov (United States)

    Wong, Brett J; Fieger, Sarah M

    2010-11-01

    The initial, rapid increase in skin blood flow in response to direct application of heat is thought to be mediated by an axon reflex, which is dependent on intact cutaneous sensory nerves. We tested the hypothesis that inhibition of transient receptor potential vanilloid type 1 (TRPV-1) channels, which are putative channels located on sensory nerves, would attenuate the skin blood flow response to local heating in humans. Ten subjects were equipped with four microdialysis fibres which were randomly assigned one of four treatments: (1) vehicle control (90% propylene glycol + 10% lactated Ringer solution); (2) 20 mm capsazepine to inhibit TRPV-1 channels; (3) 10 mm l-NAME to inhibit NO synthase; and (4) combined 20 mm capsazepine + 10 mm l-NAME. Following baseline measurements, the temperature of skin heaters was increased from 33°C to 42°C at a rate of 1.0°C every 10 s and local temperature was held at 42°C for 20-30 min until a stable plateau in skin blood flow was achieved. An index of skin blood flow was measured directly over each microdialysis site via laser-Doppler flowmetry (LDF). Beat-by-beat blood pressure was measured via photoplethysmography and verified via automated brachial auscultation. At the end of the local heating protocol, temperature of the heaters was increased to 43°C and 28 mm nitroprusside was infused to achieve maximal vasodilatation. Cutaneous vascular conductance (CVC) was calculated as LDF/mean arterial pressure and normalized to maximal values (%CVCmax). Initial peak in capsazepine (44 ± 4%CVCmax), l-NAME (56 ± 4%CVCmax) and capsazepine + l-NAME (32 ± 6%CVCmax) sites was significantly attenuated compared to control (87 ± 5%CVCmax; P thermal hyperaemia was significantly attenuated in capsazepine (73 ± 6%CVCmax), l-NAME (47 ± 5%CVCmax) and capsazepine + l-NAME (31 ± 7%CVCmax) sites compared to control (92 ± 5%CVCmax; P thermal hyperaemia. These data further suggest a portion of the NO component of thermal hyperaemia may be

  7. Fire history on the California Channel Islands spanning human arrival in the Americas.

    Science.gov (United States)

    Hardiman, Mark; Scott, Andrew C; Pinter, Nicholas; Anderson, R Scott; Ejarque, Ana; Carter-Champion, Alice; Staff, Richard A

    2016-06-05

    Recent studies have suggested that the first arrival of humans in the Americas during the end of the last Ice Age is associated with marked anthropogenic influences on landscape; in particular, with the use of fire which, would have given even small populations the ability to have broad impacts on the landscape. Understanding the impact of these early people is complicated by the dramatic changes in climate occurring with the shift from glacial to interglacial conditions. Despite these difficulties, we here attempt to test the extent of anthropogenic influence using the California Channel Islands as a smaller, landscape-scale test bed. These islands are famous for the discovery of the 'Arlington Springs Man', which are some of the earliest human remains in the Americas. A unifying sedimentary charcoal record is presented from Arlington Canyon, Santa Rosa Island, based on over 20 detailed sedimentary sections from eight key localities. Radiocarbon dating was based on thin, fragile, long fragments of charcoal in order to avoid the 'inbuilt' age problem. Radiocarbon dating of 49 such fragments has allowed inferences regarding the fire and landscape history of the Canyon ca 19-11 ka BP. A significant period of charcoal deposition is identified approximately 14-12.5 ka BP and bears remarkable closeness to an estimated age range of the first human arrival on the islands.This article is part of the themed issue 'The interaction of fire and mankind'.

  8. TRPM7 channel inhibition mediates midazolam-induced proliferation loss in human malignant glioma.

    Science.gov (United States)

    Chen, Jingkao; Dou, Yunling; Zheng, Xiaoke; Leng, Tiandong; Lu, Xiaofang; Ouyang, Ying; Sun, Huawei; Xing, Fan; Mai, Jialuo; Gu, Jiayu; Lu, Bingzheng; Yan, Guangmei; Lin, Jun; Zhu, Wenbo

    2016-11-01

    The melastatin-like transient receptor potential 7 (TRPM7) has been implicated in proliferation or apoptosis of some cancers, indicating the potential of TRPM7 as an anti-anaplastic target. Here, we identified the characteristic TRPM7 channel currents in human malignant glioma MGR2 cells, which could be blocked by a pharmacologic inhibitor Gd(3+). We mined the clinical sample data from Oncomine Database and found that human malignant glioma tissues expressed higher TRPM7 mRNA than normal brain ones. Importantly, we identified a widely used clinical anesthetic midazolam as a TRPM7 inhibitor. Midazolam treatment for seconds suppressed the TRPM7 currents and calcium influx, and treatment for 48 h inhibited the TRPM7 expression. The inhibitory effect on TRPM7 accounts for the proliferation loss and G0/G1 phase cell cycle arrest induced by midazolam. Our data demonstrates that midazolam represses proliferation of human malignant glioma cells through inhibiting TRPM7 currents, which may be further potentiated by suppressing the expression of TRPM7. Our result indicates midazolam as a pharmacologic lead compound with brain-blood barrier permeability for targeting TRPM7 in the glioma.

  9. Characterization and variation of a human inwardly-rectifying-K-channel gene (KCNJ6): a putative ATP-sensitive K-channel subunit.

    Science.gov (United States)

    Sakura, H; Bond, C; Warren-Perry, M; Horsley, S; Kearney, L; Tucker, S; Adelman, J; Turner, R; Ashcroft, F M

    1995-06-26

    The ATP-sensitive K-channel plays a central role in insulin release from pancreatic beta-cells. We report here the cloning of the gene (KCNJ6) encoding a putative subunit of a human ATP-sensitive K-channel expressed in brain and beta-cells, and characterisation of its exon-intron structure. Screening of a somatic cell mapping panel and fluorescent in situ hybridization place the gene on chromosome 21 (21q22.1-22.2). Analysis of single-stranded conformational polymorphisms revealed the presence of two silent polymorphisms (Pro-149: CCG-CCA and Asp-328: GAC-GAT) with similar frequencies in normal and non-insulin-dependent diabetic patients.

  10. Transcriptional regulation of the sodium channel gene (SCN5A) by GATA4 in human heart.

    Science.gov (United States)

    Tarradas, Anna; Pinsach-Abuin, Mel Lina; Mackintosh, Carlos; Llorà-Batlle, Oriol; Pérez-Serra, Alexandra; Batlle, Montserrat; Pérez-Villa, Félix; Zimmer, Thomas; Garcia-Bassets, Ivan; Brugada, Ramon; Beltran-Alvarez, Pedro; Pagans, Sara

    2017-01-01

    Aberrant expression of the sodium channel gene (SCN5A) has been proposed to disrupt cardiac action potential and cause human cardiac arrhythmias, but the mechanisms of SCN5A gene regulation and dysregulation still remain largely unexplored. To gain insight into the transcriptional regulatory networks of SCN5A, we surveyed the promoter and first intronic regions of the SCN5A gene, predicting the presence of several binding sites for GATA transcription factors (TFs). Consistent with this prediction, chromatin immunoprecipitation (ChIP) and sequential ChIP (Re-ChIP) assays show co-occupancy of cardiac GATA TFs GATA4 and GATA5 on promoter and intron 1 SCN5A regions in fresh-frozen human left ventricle samples. Gene reporter experiments show GATA4 and GATA5 synergism in the activation of the SCN5A promoter, and its dependence on predicted GATA binding sites. GATA4 and GATA6 mRNAs are robustly expressed in fresh-frozen human left ventricle samples as measured by highly sensitive droplet digital PCR (ddPCR). GATA5 mRNA is marginally but still clearly detected in the same samples. Importantly, GATA4 mRNA levels are strongly and positively correlated with SCN5A transcript levels in the human heart. Together, our findings uncover a novel mechanism of GATA TFs in the regulation of the SCN5A gene in human heart tissue. Our studies suggest that GATA5 but especially GATA4 are main contributors to SCN5A gene expression, thus providing a new paradigm of SCN5A expression regulation that may shed new light into the understanding of cardiac disease. Copyright © 2017 The Authors. Published by Elsevier Ltd.. All rights reserved.

  11. A Novel Case of Symptomatic BK Viraemia in a Patient Undergoing Treatment for Hodgkin Lymphoma

    Directory of Open Access Journals (Sweden)

    Jacinta Perram

    2014-01-01

    Full Text Available Symptomatic BK viral infection in the immunocompromised host is well described, most commonly seen in renal transplant recipients, bone marrow transplant recipients, and HIV positive patients. The present case describes a novel clinical scenario of symptomatic urological BK virus infection in a patient receiving treatment for Hodgkin lymphoma. This case highlights the importance of casting a wide diagnostic net for adverse events encountered with novel therapeutic agents or regimens.

  12. Environment-Sensitive Fluorescent Probe for the Human Ether-a-go-go-Related Gene Potassium Channel.

    Science.gov (United States)

    Liu, Zhenzhen; Jiang, Tianyu; Wang, Beilei; Ke, Bowen; Zhou, Yubin; Du, Lupei; Li, Minyong

    2016-02-02

    A novel environment-sensitive probe S2 with turn-on switch for Human Ether-a-go-go-Related Gene (hERG) potassium channel was developed herein. After careful evaluation, this fluorescent probe showed high binding affinity with hERG potassium channel with an IC50 value of 41.65 nM and can be well applied to hERG channel imaging or cellular distribution study for hERG channel blockers. Compared with other imaging techniques, such as immunofluorescence and fluorescent protein-based approaches, this method is convenient and affordable, especially since a washing procedure is not needed. Meanwhile, this environment-sensitive turn-on design strategy may provide a good example for the probe development for these targets that have no reactive or catalytic activity.

  13. Definitive Endoderm Formation from Plucked Human Hair-Derived Induced Pluripotent Stem Cells and SK Channel Regulation

    Directory of Open Access Journals (Sweden)

    Anett Illing

    2013-01-01

    Full Text Available Pluripotent stem cells present an extraordinary powerful tool to investigate embryonic development in humans. Essentially, they provide a unique platform for dissecting the distinct mechanisms underlying pluripotency and subsequent lineage commitment. Modest information currently exists about the expression and the role of ion channels during human embryogenesis, organ development, and cell fate determination. Of note, small and intermediate conductance, calcium-activated potassium channels have been reported to modify stem cell behaviour and differentiation. These channels are broadly expressed throughout human tissues and are involved in various cellular processes, such as the after-hyperpolarization in excitable cells, and also in differentiation processes. To this end, human induced pluripotent stem cells (hiPSCs generated from plucked human hair keratinocytes have been exploited in vitro to recapitulate endoderm formation and, concomitantly, used to map the expression of the SK channel (SKCa subtypes over time. Thus, we report the successful generation of definitive endoderm from hiPSCs of ectodermal origin using a highly reproducible and robust differentiation system. Furthermore, we provide the first evidence that SKCas subtypes are dynamically regulated in the transition from a pluripotent stem cell to a more lineage restricted, endodermal progeny.

  14. Patch clamp studies of human sperm under physiological ionic conditions reveal three functionally and pharmacologically distinct cation channels.

    Science.gov (United States)

    Mansell, S A; Publicover, S J; Barratt, C L R; Wilson, S M

    2014-05-01

    Whilst fertilizing capacity depends upon a K(+) conductance (GK) that allows the spermatozoon membrane potential (Vm) to be held at a negative value, the characteristics of this conductance in human sperm are virtually unknown. We therefore studied the biophysical/pharmacological properties of the K(+) conductance in spermatozoa from normal donors held under voltage/current clamp in the whole cell recording configuration. Our standard recording conditions were designed to maintain quasi-physiological, Na(+), K(+) and Cl(-) gradients. Experiments that explored the effects of ionic substitution/ion channel blockers upon membrane current/potential showed that resting Vm was dependent upon a hyperpolarizing K(+) current that flowed via channels that displayed only weak voltage dependence and limited (∼7-fold) K(+) versus Na(+) selectivity. This conductance was blocked by quinidine (0.3 mM), bupivacaine (3 mM) and clofilium (50 µM), NNC55-0396 (2 µM) and mibefradil (30 µM), but not by 4-aminopyridine (2 mM, 4-AP). Progesterone had no effect upon the hyperpolarizing K(+) current. Repolarization after a test depolarization consistently evoked a transient inward 'tail current' (ITail) that flowed via a second population of ion channels with poor (∼3-fold) K(+) versus Na(+) selectivity. The activity of these channels was increased by quinidine, 4-AP and progesterone. Vm in human sperm is therefore dependent upon a hyperpolarizing K(+) current that flows via channels that most closely resemble those encoded by Slo3. Although 0.5 µM progesterone had no effect upon these channels, this hormone did activate the pharmacologically distinct channels that mediate ITail. In conclusion, this study reveals three functionally and pharmacologically distinct cation channels: Ik, ITail, ICatSper.

  15. A critical role for the transient receptor potential channel type 6 in human platelet activation.

    Directory of Open Access Journals (Sweden)

    Hari Priya Vemana

    Full Text Available While calcium signaling is known to play vital roles in platelet function, the mechanisms underlying its receptor-operated calcium entry component (ROCE remain poorly understood. It has been proposed, but never proven in platelets, that the canonical transient receptor potential channel-6 (TRPC6 mediates ROCE. Nonetheless, we have previously shown that the mouse TRPC6 regulates hemostasis, thrombogenesis by regulating platelet aggregation. In the present studies, we used a pharmacological approach to characterize the role of TRPC6 in human platelet biology. Thus, interestingly, we observed that a TRPC6 inhibitor exerted significant inhibitory effects on human platelet aggregation in a thromboxane receptor (TPR-selective manner; no additional inhibition was observed in the presence of the calcium chelator BAPTA. This inhibitor also significantly inhibited human platelet secretion (dense and alpha granules, integrin IIb-IIIa, Akt and ERK phosphorylation, again, in a TPR-selective manner; no effects were observed in response to ADP receptor stimulation. Furthermore, there was a causal relationship between these inhibitory effects, and the capacity of the TRPC6 inhibitor to abrogate elevation in intracellular calcium, that was again found to be TPR-specific. This effect was not found to be due to antagonism of TPR, as the TRPC6 inhibitor did not displace the radiolabeled antagonist [3H]SQ29,548 from its binding sites. Finally, our studies also revealed that TRPC6 regulates human clot retraction, as well as physiological hemostasis and thrombus formation, in mice. Taken together, our findings demonstrate, for the first time, that TRPC6 directly regulates TPR-dependent ROCE and platelet function. Moreover, these data highlight TRPC6 as a novel promising therapeutic strategy for managing thrombotic disorders.

  16. Identification and characterization of a selective allosteric antagonist of human P2X4 receptor channels.

    Science.gov (United States)

    Ase, Ariel R; Honson, Nicolette S; Zaghdane, Helmi; Pfeifer, Tom A; Séguéla, Philippe

    2015-04-01

    P2X4 is an ATP-gated nonselective cation channel highly permeable to calcium. There is increasing evidence that this homomeric purinoceptor, which is expressed in several neuronal and immune cell types, is involved in chronic pain and inflammation. The current paucity of unambiguous pharmacological tools available to interrogate or modulate P2X4 function led us to pursue the search for selective antagonists. In the high-throughput screen of a compound library, we identified the phenylurea BX430 (1-(2,6-dibromo-4-isopropyl-phenyl)-3-(3-pyridyl)urea, molecular weight = 413), with antagonist properties on human P2X4-mediated calcium uptake. Patch-clamp electrophysiology confirmed direct inhibition of P2X4 currents by extracellular BX430, with submicromolar potency (IC50 = 0.54 µM). BX430 is highly selective, having virtually no functional impact on all other P2X subtypes, namely, P2X1-P2X3, P2X5, and P2X7, at 10-100 times its IC50. Unexpected species differences were noticed, as BX430 is a potent antagonist of zebrafish P2X4 but has no effect on rat and mouse P2X4 orthologs. The concentration-response curve for ATP on human P2X4 in the presence of BX430 shows an insurmountable blockade, indicating a noncompetitive allosteric mechanism of action. Using a fluorescent dye uptake assay, we observed that BX430 also effectively suppresses ATP-evoked and ivermectin-potentiated membrane permeabilization induced by P2X4 pore dilation. Finally, in single-cell calcium imaging, we validated its selective inhibitory effects on native P2X4 channels at the surface of human THP-1 cells that were differentiated into macrophages. In summary, this ligand provides a novel molecular probe to assess the specific role of P2X4 in inflammatory and neuropathic conditions, where ATP signaling has been shown to be dysfunctional.

  17. Multi-channel microstrip transceiver arrays using harmonics for high field MR imaging in humans.

    Science.gov (United States)

    Wu, Bing; Wang, Chunsheng; Lu, Jonathan; Pang, Yong; Nelson, Sarah J; Vigneron, Daniel B; Zhang, Xiaoliang

    2012-02-01

    Radio-frequency (RF) transceiver array design using primary and higher order harmonics for in vivo parallel magnetic resonance imaging imaging (MRI) and spectroscopic imaging is proposed. The improved electromagnetic decoupling performance, unique magnetic field distributions and high-frequency operation capabilities of higher-order harmonics of resonators would benefit transceiver arrays for parallel MRI, especially for ultrahigh field parallel MRI. To demonstrate this technique, microstrip transceiver arrays using first and second harmonic resonators were developed for human head parallel imaging at 7T. Phantom and human head images were acquired and evaluated using the GRAPPA reconstruction algorithm. The higher-order harmonic transceiver array design technique was also assessed numerically using FDTD simulation. Compared with regular primary-resonance transceiver designs, the proposed higher-order harmonic technique provided an improved g-factor and increased decoupling among resonant elements without using dedicated decoupling circuits, which would potentially lead to a better parallel imaging performance and ultimately faster and higher quality imaging. The proposed technique is particularly suitable for densely spaced transceiver array design where the increased mutual inductance among the elements becomes problematic. In addition, it also provides a simple approach to readily upgrade the channels of a conventional primary resonator microstrip array to a larger number for faster imaging.

  18. Ion channels generating complex spikes in cartwheel cells of the dorsal cochlear nucleus.

    Science.gov (United States)

    Kim, Yuil; Trussell, Laurence O

    2007-02-01

    Cartwheel cells are glycinergic interneurons that modify somatosensory input to the dorsal cochlear nucleus. They are characterized by firing of mixtures of both simple and complex action potentials. To understand what ion channels determine the generation of these two types of spike waveforms, we recorded from cartwheel cells using the gramicidin perforated-patch technique in brain slices of mouse dorsal cochlear nucleus and applied channel-selective blockers. Complex spikes were distinguished by whether they arose directly from a negative membrane potential or later during a long depolarization. Ca(2+) channels and Ca(2+)-dependent K(+) channels were major determinants of complex spikes. Onset complex spikes required T-type and possibly R-type Ca(2+) channels and were shaped by BK and SK K(+) channels. Complex spikes arising later in a depolarization were dependent on P/Q- and L-type Ca(2+) channels as well as BK and SK channels. BK channels also contributed to fast repolarization of simple spikes. Simple spikes featured an afterdepolarization that is probably the trigger for complex spiking and is shaped by T/R-type Ca(2+) and SK channels. Fast spikes were dependent on Na(+) channels; a large persistent Na(+) current may provide a depolarizing drive for spontaneous activity in cartwheel cells. Thus the diverse electrical behavior of cartwheel cells is determined by the interaction of a wide variety of ion channels with a prominent role played by Ca(2+).

  19. Mutations in sodium-channel gene SCN9A cause a spectrum of human genetic pain disorders.

    NARCIS (Netherlands)

    Drenth, J.P.H.; Waxman, S.G.

    2007-01-01

    The voltage-gated sodium-channel type IX alpha subunit, known as Na(v)1.7 and encoded by the gene SCN9A, is located in peripheral neurons and plays an important role in action potential production in these cells. Recent genetic studies have identified Na(v)1.7 dysfunction in three different human pa

  20. Mutations in sodium-channel gene SCN9A cause a spectrum of human genetic pain disorders.

    NARCIS (Netherlands)

    Drenth, J.P.H.; Waxman, S.G.

    2007-01-01

    The voltage-gated sodium-channel type IX alpha subunit, known as Na(v)1.7 and encoded by the gene SCN9A, is located in peripheral neurons and plays an important role in action potential production in these cells. Recent genetic studies have identified Na(v)1.7 dysfunction in three different human pa

  1. Relaxant effect of a novel calcium-activated potassium channel modulator on human myometrial spontaneous contractility in vitro

    DEFF Research Database (Denmark)

    Rosenbaum, S.T.; Larsen, T.; Joergensen, J.C.

    2012-01-01

    Aim: To investigate the effect of 4,5-dichloro-1,3-diethyl-1,3-dihydro-benzoimidazol-2-one (NS4591), a novel SK/IK channels positive modulator, on human myometrial activity. Methods: Organ bath studies were performed on myometrial preparations obtained from women undergoing elective caesarean...

  2. A BK inequality for randomly drawn subsets of fixed size

    CERN Document Server

    Berg, J van den

    2011-01-01

    The BK inequality, proved by van den Berg and Kesten \\cite{BeKe85}, says that, for product measures on $\\Om := \\{0,1\\}^n$, the probability that two increasing events $A$ and $B$ 'occur disjointly' is smaller than or equal to the product of the two individual probabilities. Their conjecture that this holds for {\\em all} events was proved by Reimer \\cite{Re00}. In spite of Reimer's work, several fundamental problems in this area remained open. For instance, although it is easy to see that non-product measures can not satisfy the above inequality for all events, there are several such measures on $\\Om$ which, intuitively, should satisfy the inequality for all increasing events. One of the most 'natural' candidates is what we call here the $k$-out-of-$n$ measure, the measure assigning equal probabilities to all elements of $\\Om$ with exactly $k$ 1's (and probability 0 to all other elements). The main contribution of this paper is a proof for these measures. Remarkably, although the $k$-out-of-$n$ measures are not...

  3. Mitochondrial changes in cidofovir therapy for BK virus nephropathy.

    Science.gov (United States)

    Talmon, G; Cornell, L D; Lager, D J

    2010-06-01

    Polyoma (BK) virus nephropathy (BKVN) is often treated with the nucleotide analog cidofovir. An adverse effect of this drug class is proximal tubular toxicity, and ultrastructural abnormalities in proximal tubular mitochondria have been observed in patients treated with similar drugs for other viral infections. We report similar changes in biopsies from BKVN treated with cidofovir. Renal allograft biopsies showing BKVN, on which electron microscopy was performed, were categorized into 3 groups: initial diagnosis (BD), postcidofovir treatment (CT), and posttreatment with immunosuppression reduction (IR). Nineteen cases from each group were randomly selected. Mitochondrial changes were present in 6 biopsies from patients receiving CT therapy (31.5%), ranging from diffuse mitochondrial swelling to profound morphologic changes. No similar abnormalities were seen in other groups. In those with atypical mitochondria, the mean number of cidofovir doses was 2.67, with an average interval between last dose and biopsy of 2.17 weeks. CT patients without mitochondrial changes had a mean of 4.6 doses and an average interval between last dose and biopsy of 27.2 weeks. Some renal transplant patients treated with cidofovir display alterations in proximal tubular mitochondria akin to those seen with similar drugs. The findings support the mitochondrial toxicity of nucleotide analogs.

  4. The voltage-dependent K+ channels Kv1.3 and Kv1.5 in human cancer

    Science.gov (United States)

    Comes, Núria; Bielanska, Joanna; Vallejo-Gracia, Albert; Serrano-Albarrás, Antonio; Marruecos, Laura; Gómez, Diana; Soler, Concepció; Condom, Enric; Ramón y Cajal, Santiago; Hernández-Losa, Javier; Ferreres, Joan C.; Felipe, Antonio

    2013-01-01

    Voltage-dependent K+ channels (Kv) are involved in a number of physiological processes, including immunomodulation, cell volume regulation, apoptosis as well as differentiation. Some Kv channels participate in the proliferation and migration of normal and tumor cells, contributing to metastasis. Altered expression of Kv1.3 and Kv1.5 channels has been found in several types of tumors and cancer cells. In general, while the expression of Kv1.3 apparently exhibits no clear pattern, Kv1.5 is induced in many of the analyzed metastatic tissues. Interestingly, evidence indicates that Kv1.5 channel shows inversed correlation with malignancy in some gliomas and non-Hodgkin's lymphomas. However, Kv1.3 and Kv1.5 are similarly remodeled in some cancers. For instance, expression of Kv1.3 and Kv1.5 correlates with a certain grade of tumorigenicity in muscle sarcomas. Differential remodeling of Kv1.3 and Kv1.5 expression in human cancers may indicate their role in tumor growth and their importance as potential tumor markers. However, despite of this increasing body of information, which considers Kv1.3 and Kv1.5 as emerging tumoral markers, further research must be performed to reach any conclusion. In this review, we summarize what it has been lately documented about Kv1.3 and Kv1.5 channels in human cancer. PMID:24133455

  5. The voltage-dependent K(+) channels Kv1.3 and Kv1.5 in human cancer.

    Science.gov (United States)

    Comes, Núria; Bielanska, Joanna; Vallejo-Gracia, Albert; Serrano-Albarrás, Antonio; Marruecos, Laura; Gómez, Diana; Soler, Concepció; Condom, Enric; Ramón Y Cajal, Santiago; Hernández-Losa, Javier; Ferreres, Joan C; Felipe, Antonio

    2013-10-10

    Voltage-dependent K(+) channels (Kv) are involved in a number of physiological processes, including immunomodulation, cell volume regulation, apoptosis as well as differentiation. Some Kv channels participate in the proliferation and migration of normal and tumor cells, contributing to metastasis. Altered expression of Kv1.3 and Kv1.5 channels has been found in several types of tumors and cancer cells. In general, while the expression of Kv1.3 apparently exhibits no clear pattern, Kv1.5 is induced in many of the analyzed metastatic tissues. Interestingly, evidence indicates that Kv1.5 channel shows inversed correlation with malignancy in some gliomas and non-Hodgkin's lymphomas. However, Kv1.3 and Kv1.5 are similarly remodeled in some cancers. For instance, expression of Kv1.3 and Kv1.5 correlates with a certain grade of tumorigenicity in muscle sarcomas. Differential remodeling of Kv1.3 and Kv1.5 expression in human cancers may indicate their role in tumor growth and their importance as potential tumor markers. However, despite of this increasing body of information, which considers Kv1.3 and Kv1.5 as emerging tumoral markers, further research must be performed to reach any conclusion. In this review, we summarize what it has been lately documented about Kv1.3 and Kv1.5 channels in human cancer.

  6. OCT Study of Mechanical Properties Associated with Trabecular Meshwork and Collector Channel Motion in Human Eyes

    Science.gov (United States)

    Xin, Chen; Johnstone, Murray; Wang, Ningli; Wang, Ruikang K.

    2016-01-01

    We report the use of a high-resolution optical coherence tomography (OCT) imaging platform to identify and quantify pressure-dependent aqueous outflow system (AOS) tissue relationships and to infer mechanical stiffness through examination of tissue properties in ex vivo human eyes. Five enucleated human eyes are included in this study, with each eye prepared with four equal-sized quadrants, each encompassing 90 degrees of the limbal circumference. In radial limbal segments perfusion pressure within Schlemm’s canal (SC) is controlled by means of a perfusion cannula inserted into the canal lumen, while the other end of the cannula leads to a reservoir at a height that can control the pressure in the cannula. The OCT system images the sample with a spatial resolution of about 5 μm from the trabecular meshwork (TM) surface. Geometric parameters are quantified from the 2D OCT images acquired from the sample subjected to controlled changes in perfusion pressures; parameters include area and height of the lumen of SC, collector channel entrances (CCE) and intrascleral collector channels (ISCC). We show that 3D OCT imaging permits the identification of 3-D relationships of the SC, CCE and ISCC lumen dimensions. Collagen flaps or leaflets are found at CCE that are attached or hinged at only one end, whilst the flaps are connected to the TM by cylindrical structures spanning SC. Increasing static SC pressures resulted in SC lumen enlargement with corresponding enlargement of the CCE and ISCC lumen. Pressure-dependent SC lumen area and height changes are significant at the 0.01 levels for ANOVA, and at the 0.05 for both polynomial curves and Tukey paired comparisons. Dynamic measurements demonstrate a synchronous increase in SC, CCE and ISCC lumen height in response to pressure changes from 0 to 10, 30 or 50 mm Hg, respectively, and the response time is within the 50-millisecond range. From the measured SC volume and corresponding IOP values, we demonstrate that an

  7. Molecular studies of BKCa channels in intracranial arteries: presence and localization

    DEFF Research Database (Denmark)

    Johansson, Helle Wulf; Hay-Schmidt, Anders; Poulsen, Asser Nyander

    2008-01-01

    of the BK(Ca) channel in rat basilar, middle cerebral, and middle meningeal arteries by reverse transcription polymerase chain reaction (RT-PCR), quantitative real-time PCR, and Western blotting. Distribution patterns were investigated using in situ hybridization and immunofluorescence studies. RT......-PCR and quantitative real-time PCR detected the expression of the BK(Ca) channel mRNA transcript in rat basilar, middle cerebral, and middle meningeal arteries, with the transcript being expressed more abundantly in rat basilar arteries than in middle cerebral and middle meningeal arteries. Western blotting detected...

  8. NMR investigation of the isolated second voltage-sensing domain of human Nav1.4 channel.

    Science.gov (United States)

    Paramonov, A S; Lyukmanova, E N; Myshkin, M Yu; Shulepko, M A; Kulbatskii, D S; Petrosian, N S; Chugunov, A O; Dolgikh, D A; Kirpichnikov, M P; Arseniev, A S; Shenkarev, Z O

    2017-03-01

    Voltage-gated Na(+) channels are essential for the functioning of cardiovascular, muscular, and nervous systems. The α-subunit of eukaryotic Na(+) channel consists of ~2000 amino acid residues and encloses 24 transmembrane (TM) helices, which form five membrane domains: four voltage-sensing (VSD) and one pore domain. The structural complexity significantly impedes recombinant production and structural studies of full-sized Na(+) channels. Modular organization of voltage-gated channels gives an idea for studying of the isolated second VSD of human skeletal muscle Nav1.4 channel (VSD-II). Several variants of VSD-II (~150a.a., four TM helices) with different N- and C-termini were produced by cell-free expression. Screening of membrane mimetics revealed low stability of VSD-II samples in media containing phospholipids (bicelles, nanodiscs) associated with the aggregation of electrically neutral domain molecules. The almost complete resonance assignment of (13)C,(15)N-labeled VSD-II was obtained in LPPG micelles. The secondary structure of VSD-II showed similarity with the structures of bacterial Na(+) channels. The fragment of S4 TM helix between the first and second conserved Arg residues probably adopts 310-helical conformation. Water accessibility of S3 helix, observed by the Mn(2+) titration, pointed to the formation of water-filled crevices in the micelle embedded VSD-II. (15)N relaxation data revealed characteristic pattern of μs-ms time scale motions in the VSD-II regions sharing expected interhelical contacts. VSD-II demonstrated enhanced mobility at ps-ns time scale as compared to isolated VSDs of K(+) channels. These results validate structural studies of isolated VSDs of Na(+) channels and show possible pitfalls in application of this 'divide and conquer' approach.

  9. Activator-induced dynamic disorder and molecular memory in human two-pore domain hTREK1 K channel.

    Science.gov (United States)

    Nayak, Tapan Kumar; Dana, Saswati; Raha, Soumyendu; Sikdar, Sujit K

    2011-04-01

    Ion channels are fundamental molecules in the nervous system that catalyze the flux of ions across the cell membrane. Ion channel flux activity is comparable to the catalytic activity of enzyme molecules. Saturating concentrations of substrate induce "dynamic disorder" in the kinetic rate processes of single-enzyme molecules and consequently, develop correlative "memory" of the previous history of activities. Similarly, binding of ions as substrate alone or in presence of agonists affects the catalytic turnover of single-ion channels. Here, we investigated the possible existence of dynamic disorder and molecular memory in the single human-TREK1-channel due to binding of substrate/agonist using the excised inside-out patch-clamp technique. Our results suggest that the single-hTREK1-channel behaves as a typical Michaelis-Menten enzyme molecule with a high-affinity binding site for K(+) ion as substrate. But, in contrast to enzyme, dynamic disorder in single-hTREK1-channel was not induced by substrate K(+) binding, but required allosteric modification of the channel molecule by the agonist, trichloroethanol. In addition, interaction of trichloroethanol with hTREK1 induced strong correlation in the waiting time and flux intensity, exemplified by distinct mode-switching between high and low flux activities. This suggested the induction of molecular memory in the channel molecule by the agonist, which persisted for several decades in time. Our mathematical modeling studies identified the kinetic rate processes associated with dynamic disorder. It further revealed the presence of multiple populations of distinct conformations that contributed to the "heterogeneity" and consequently, to the molecular memory phenomenon that we observed. The online version of this article (doi:10.1007/s12154-010-0053-3) contains supplementary material, which is available to authorized users.

  10. High affinity complexes of pannexin channels and L-type calcium channel splice-variants in human lung: Possible role in clevidipine-induced dyspnea relief in acute heart failure

    Directory of Open Access Journals (Sweden)

    Gerhard P. Dahl

    2016-08-01

    Research in Context: Clevidipine lowers blood pressure by inhibiting calcium channels in vascular smooth muscle. In patients with acute heart failure, clevidipine was shown to relieve breathing problems. This was only partially related to the blood pressure lowering actions of clevidipine and not conferred by another calcium channel inhibitor. We here found calcium channel variants in human lung that are more selectively inhibited by clevidipine, especially when associated with pannexin channels. This study gives a possible mechanism for clevidipine's relief of breathing problems and supports future clinical trials testing the role of clevidipine in the treatment of acute heart failure.

  11. Voltage-dependent anion channels (VDACs, porin) expressed in the plasma membrane regulate the differentiation and function of human osteoclasts.

    Science.gov (United States)

    Kotake, Shigeru; Yago, Toru; Kawamoto, Manabu; Nanke, Yuki

    2013-01-01

    Fewer molecules have been identified on human than murine osteoclasts, the former differing from murine osteoclasts in many ways. We show that voltage-dependent anion channels (VDACs, porin) are expressed in the plasma membrane of human osteoclasts. A search for novel proteins expressed in the plasma membrane of human osteoclasts identified VDAC. Anti-VDAC antibodies inhibited human osteoclastogenesis in vitro. VDAC expression was detected in membranes by immunoelectron microscopy and immunocytochemical double staining. The VDAC protein functions as a Cl(-) channel. VDACs regulate bone resorption, which show using Osteologic™ plates. The epitope of the antibody lay within a 10-amino acid sequence in the VDAC. The findings suggest that the VDAC is, at least partly, a novel Cl(-) channel regulating the differentiation and function of human osteoclasts. VDACs may play a crucial role in acidifying the resorption lacunae between osteoclasts and bone. Inhibitors of VDACs could be used to treat diseases involving increased resorption, such as osteoporosis, rheumatoid arthritis, and Paget's disease. © 2012 International Federation for Cell Biology.

  12. Characterisation of K+ Channels in Human Fetoplacental Vascular Smooth Muscle Cells

    OpenAIRE

    Brereton, Melissa F.; Mark Wareing; Rebecca L Jones; Greenwood, Susan L.

    2013-01-01

    Adequate blood flow through placental chorionic plate resistance arteries (CPAs) is necessary for oxygen and nutrient transfer to the fetus and a successful pregnancy. In non-placental vascular smooth muscle cells (SMCs), K(+) channels regulate contraction, vascular tone and blood flow. Previous studies showed that K(+) channel modulators alter CPA tone, but did not distinguish between effects on K(+) channels in endothelial cells and SMCs. In this study, we developed a preparation of freshly...

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

    Science.gov (United States)

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

    2015-07-05

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

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

    Science.gov (United States)

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

    2016-12-15

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

  15. Expression, Distribution and Role of Aquaporin Water Channels in Human and Animal Stomach and Intestines.

    Science.gov (United States)

    Zhu, Cui; Chen, Zhuang; Jiang, Zongyong

    2016-08-29

    Stomach and intestines are involved in the secretion of gastrointestinal fluids and the absorption of nutrients and fluids, which ensure normal gut functions. Aquaporin water channels (AQPs) represent a major transcellular route for water transport in the gastrointestinal tract. Until now, at least 11 AQPs (AQP1-11) have been found to be present in the stomach, small and large intestines. These AQPs are distributed in different cell types in the stomach and intestines, including gastric epithelial cells, gastric glands cells, absorptive epithelial cells (enterocytes), goblet cells and Paneth cells. AQP1 is abundantly distributed in the endothelial cells of the gastrointestinal tract. AQP3 and AQP4 are mainly distributed in the basolateral membrane of epithelial cells in the stomach and intestines. AQP7, AQP8, AQP10 and AQP11 are distributed in the apical of enterocytes in the small and large intestines. Although AQP-null mice displayed almost no phenotypes in gastrointestinal tracts, the alterations of the expression and localization of these AQPs have been shown to be associated with the pathology of gastrointestinal disorders, which suggests that AQPs play important roles serving as potential therapeutic targets. Therefore, this review provides an overview of the expression, localization and distribution of AQPs in the stomach, small and large intestine of human and animals. Furthermore, this review emphasizes the potential roles of AQPs in the physiology and pathophysiology of stomach and intestines.

  16. CFTR anion channel modulates expression of human transmembrane mucin MUC3 through the PDZ protein GOPC.

    Science.gov (United States)

    Pelaseyed, Thaher; Hansson, Gunnar C

    2011-09-15

    The transmembrane mucins in the enterocyte are type 1 transmembrane proteins with long and rigid mucin domains, rich in proline, threonine and serine residues that carry numerous O-glycans. Three of these mucins, MUC3, MUC12 and MUC17 are unique in harboring C-terminal class I PDZ motifs, making them suitable ligands for PDZ proteins. A screening of 123 different human PDZ domains for binding to MUC3 identified a strong interaction with the PDZ protein GOPC (Golgi-associated PDZ and coiled-coil motif-containing protein). This interaction was mediated by the C-terminal PDZ motif of MUC3, binding to the single GOPC PDZ domain. GOPC is also a binding partner for cystic fibrosis transmembrane conductance regulator (CFTR) that directs CFTR for degradation. Overexpression of GOPC downregulated the total levels of MUC3, an effect that was reversed by introducing CFTR. The results suggest that CFTR and MUC3 compete for binding to GOPC, which in turn can regulate levels of these two proteins. For the first time a direct coupling between mucins and the CFTR channel is demonstrated, a finding that will shed further light on the still poorly understood relationship between cystic fibrosis and the mucus phenotype of this disease.

  17. Expression, Distribution and Role of Aquaporin Water Channels in Human and Animal Stomach and Intestines

    Directory of Open Access Journals (Sweden)

    Cui Zhu

    2016-08-01

    Full Text Available Stomach and intestines are involved in the secretion of gastrointestinal fluids and the absorption of nutrients and fluids, which ensure normal gut functions. Aquaporin water channels (AQPs represent a major transcellular route for water transport in the gastrointestinal tract. Until now, at least 11 AQPs (AQP1–11 have been found to be present in the stomach, small and large intestines. These AQPs are distributed in different cell types in the stomach and intestines, including gastric epithelial cells, gastric glands cells, absorptive epithelial cells (enterocytes, goblet cells and Paneth cells. AQP1 is abundantly distributed in the endothelial cells of the gastrointestinal tract. AQP3 and AQP4 are mainly distributed in the basolateral membrane of epithelial cells in the stomach and intestines. AQP7, AQP8, AQP10 and AQP11 are distributed in the apical of enterocytes in the small and large intestines. Although AQP-null mice displayed almost no phenotypes in gastrointestinal tracts, the alterations of the expression and localization of these AQPs have been shown to be associated with the pathology of gastrointestinal disorders, which suggests that AQPs play important roles serving as potential therapeutic targets. Therefore, this review provides an overview of the expression, localization and distribution of AQPs in the stomach, small and large intestine of human and animals. Furthermore, this review emphasizes the potential roles of AQPs in the physiology and pathophysiology of stomach and intestines.

  18. The Involvement of Ser1898 of the Human L-Type Calcium Channel in Evoked Secretion

    Directory of Open Access Journals (Sweden)

    Niv Bachnoff

    2011-01-01

    Full Text Available A PKA consensus phosphorylation site S1928 at the α11.2 subunit of the rabbit cardiac L-type channel, CaV1.2, is involved in the regulation of CaV1.2 kinetics and affects catecholamine secretion. This mutation does not alter basal CaV1.2 current properties or regulation of CaV1.2 current by PKA and the beta-adrenergic receptor, but abolishes CaV1.2 phosphorylation by PKA. Here, we test the contribution of the corresponding PKA phosphorylation site of the human α11.2 subunit S1898, to the regulation of catecholamine secretion in bovine chromaffin cells. Chromaffin cells were infected with a Semliki-Forest viral vector containing either the human wt or a mutated S1898A α11.2 subunit. Both subunits harbor a T1036Y mutation conferring nifedipine insensitivity. Secretion evoked by depolarization in the presence of nifedipine was monitored by amperometry. Depolarization-triggered secretion in cells infected with either the wt α11.2 or α11.2/S1898A mutated subunit was elevated to a similar extent by forskolin. Forskolin, known to directly activate adenylyl-cyclase, increased the rate of secretion in a manner that is largely independent of the presence of S1898. Our results are consistent with the involvement of additional PKA regulatory site(s at the C-tail of α11.2, the pore forming subunit of CaV1.2.

  19. Effect of dexamethasone on voltage-gated Na+ channel in cultured human bronchial smooth muscle cells.

    Science.gov (United States)

    Nakajima, Toshiaki; Jo, Taisuke; Meguro, Kentaro; Oonuma, Hitoshi; Ma, Ji; Kubota, Nami; Imuta, Hiroyuki; Takano, Haruhito; Iida, Haruko; Nagase, Takahide; Nagata, Taiji

    2008-06-06

    Voltage-gated Na(+) channel (I(Na)) encoded by SCN9A mRNA is expressed in cultured human bronchial smooth muscle cells. We investigated the effects of dexamethasone on I(Na), by using whole-cell voltage clamp techniques, reverse transcriptase/polymerase chain reaction (RT-PCR), and quantitative real-time RT-PCR. Acute application of dexamethasone (10(-6) M) did not affect I(Na). However, the percentage of the cells with I(Na) was significantly less in cells pretreated with dexamethasone for 48 h, and the current-density of I(Na) adjusted by cell capacitance in cells with I(Na) was also decreased in cells treated with dexamethasone. RT-PCR analysis showed that alpha and beta subunits mRNA of I(Na) mainly consisted of SCN9A and SCN1beta, respectively. Treatment with dexamethasone for 24-48 h inhibited the expression of SCN9A mRNA. The inhibitory effect of dexamethasone was concentration-dependent, and was observed at a concentration higher than 0.1 nM. The effect of dexamethasone on SCN9A mRNA was not blocked by spironolactone, but inhibited by mifepristone. The inhibitory effects of dexamethasone on SCN9A mRNA could not be explained by the changes of the stabilization of mRNA measured by using actinomycin D. These results suggest that dexamethasone inhibited I(Na) encoded by SCN9A mRNA in cultured human bronchial smooth muscle cells by inhibiting the transcription via the glucocorticoid receptor.

  20. Activation of endothelial and epithelial KCa2.3 calcium-activated potassium channels by NS309 relaxes human small pulmonary arteries and bronchioles

    DEFF Research Database (Denmark)

    Kroigaard, Christel; Dalsgaard, Thomas; Nielsen, Gorm

    2012-01-01

    BACKGROUND AND PURPOSE: Small (K(Ca) 2) and intermediate (K(Ca) 3.1) conductance calcium-activated potassium channels (K(Ca) ) may contribute to both epithelium- and endothelium-dependent relaxations, but this has not been established in human pulmonary arteries and bronchioles. Therefore, we...... investigated the expression of K(Ca) 2.3 and K(Ca) 3.1 channels, and hypothesized that activation of these channels would produce relaxation of human bronchioles and pulmonary arteries. EXPERIMENTAL APPROACH: Channel expression and functional studies were conducted in human isolated small pulmonary arteries.......1 activator, NS309, induced concentration-dependent relaxations. NS309 was equally potent in relaxing pulmonary arteries, but less potent in bronchioles, than salbutamol. NS309 relaxations were blocked by the K(Ca) 2 channel blocker apamin, while the K(Ca) 3.1 channel blocker, charybdotoxin failed to reduce...

  1. Molecular cloning of a K+ channel from the malaria parasite Plasmodium falciparum

    DEFF Research Database (Denmark)

    Ellekvist, Peter; Ricke, Christina Høier; Litman, Thomas

    2004-01-01

    concentrations of K(+) when inside the erythrocyte and low concentrations when in plasma. In the recently published genome of P. falciparum, we have identified a gene, pfkch1, encoding a potential K(+) channel, which to some extent resembles the big-conductance (BK) K(+) channel. We have cloned the approximately...

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

    Science.gov (United States)

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

    2012-01-01

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

  3. Activity of the anticonvulsant lacosamide in experimental and human epilepsy via selective effects on slow Na(+) channel inactivation.

    Science.gov (United States)

    Holtkamp, Dominik; Opitz, Thoralf; Niespodziany, Isabelle; Wolff, Christian; Beck, Heinz

    2017-01-01

    In human epilepsy, pharmacoresistance to antiepileptic drug therapy is a major problem affecting ~30% of patients with epilepsy. Many classical antiepileptic drugs target voltage-gated sodium channels, and their potent activity in inhibiting high-frequency firing has been attributed to their strong use-dependent blocking action. In chronic epilepsy, a loss of use-dependent block has emerged as a potential cellular mechanism of pharmacoresistance for anticonvulsants acting on voltage-gated sodium channels. The anticonvulsant drug lacosamide (LCM) also targets sodium channels, but has been shown to preferentially affect sodium channel slow inactivation processes, in contrast to most other anticonvulsants. We used whole-cell voltage clamp recordings in acutely isolated cells to investigate the effects of LCM on transient Na(+) currents. Furthermore, we used whole-cell current clamp recordings to assess effects on repetitive action potential firing in hippocampal slices. We show here that LCM exerts its effects primarily via shifting the slow inactivation voltage dependence to more hyperpolarized potentials in hippocampal dentate granule cells from control and epileptic rats, and from patients with epilepsy. It is important to note that this activity of LCM was maintained in chronic experimental and human epilepsy. Furthermore, we demonstrate that the efficacy of LCM in inhibiting high-frequency firing is undiminished in chronic experimental and human epilepsy. Taken together, these results show that LCM exhibits maintained efficacy in chronic epilepsy, in contrast to conventional use-dependent sodium channel blockers such as carbamazepine. They also establish that targeting slow inactivation may be a promising strategy for overcoming target mechanisms of pharmacoresistance. Wiley Periodicals, Inc. © 2016 International League Against Epilepsy.

  4. First evidence of TRPV5 and TRPV6 channels in human parathyroid glands: possible involvement in neoplastic transformation.

    Science.gov (United States)

    Giusti, Laura; Cetani, Filomena; Da Valle, Ylenia; Pardi, Elena; Ciregia, Federica; Donadio, Elena; Gargini, Claudia; Piano, Ilaria; Borsari, Simona; Jaber, Ali; Caputo, Antonella; Basolo, Fulvio; Giannaccini, Gino; Marcocci, Claudio; Lucacchini, Antonio

    2014-10-01

    The parathyroid glands play an overall regulatory role in the systemic calcium (Ca(2+)) homeostasis. The purpose of the present study was to demonstrate the presence of the Ca(2+) channels transient receptor potential vanilloid (TRPV) 5 and TRPV6 in human parathyroid glands. Semi-quantitative and quantitative PCR was carried out to evaluate the presence of TRPV5 and TRPV6 mRNAs in sporadic parathyroid adenomas and normal parathyroid glands. Western blot and immunocytochemical assays were used to assess protein expression, cellular localization and time expression in primary cultures from human parathyroid adenoma. TRPV5 and TRPV6 transcripts were then identified both in normal and pathological tissues. Predominant immunoreactive bands were detected at 75-80 kD for both vanilloid channels. These channels co-localized with the calcium-sensing receptor (CASR) on the membrane surface, but immunoreactivity was also detected in the cytosol and around the nuclei. Our data showed that western blotting recorded an increase of protein expression of both channels in adenoma samples compared with normal glands suggesting a potential relation with the cell calcium signalling pathway and the pathological processes of these glands.

  5. The opening of maitotoxin-sensitive calcium channels induces the acrosome reaction in human spermatozoa: differences from the zona pellucida

    Institute of Scientific and Technical Information of China (English)

    Julio C Chávez; Claudia L Trevi(n)o; Gerardo A de Blas; José L de la Vega-Beltrán; Takuya Nishigaki; Mayel Chirinos; María Elena González-González; Fernando Larrea; Alejandra Solís; Alberto Darszon

    2011-01-01

    The acrosome reaction(AR),an absolute requirement for spermatozoa and egg fusion,requires the influx of Ca2+into the spermatozoa through voltage-dependent Ca2+channels and store-operated channels.Maitotoxin(MTx),a Ca2+-mobilizing agent,has been shown to be a potent inducer of the mouse sperm AR,with a pharmacology similar to that of the zona pellucida(ZP),possibly suggesting a common pathway for both inducers.Using recombinant human ZP3(rhZP3),mouse ZP and two MTx channel blockers(U73122 and U73343),we investigated and compared the MTx-and ZP-induced ARs in human and mouse spermatozoa.Herein,we report that MTx induced AR and elevated intracellular Ca2+([Ca2+]1)in human spermatozoa,both of which were blocked by U73122 and U73343.These two compounds also inhibited the MTx-induced AR in mouse spermatozoa.In disagreement with our previous proposal,the AR triggered by rhZP3 or mouse ZP was not blocked by U73343,indicating that in human and mouse spermatozoa,the AR induction by the physiologicalligands or by MTx occurred through distinct pathways.U73122,but not U73343(inactive analogue),can block phospholipase C(PLC).Another PLC inhibitor,edelfosine,also blocked the rhZP3-and ZP-induced ARs.These findings confirmed the participation of a PLC-dependent signalling pathway in human and mouse zona protein-induced AR.Notably,edelfosine also inhibited the MTx-induced mouse sperm AR but not that of the human,suggesting that toxin-induced AR is PLC-dependent in mice and PLC-independent in humans.

  6. FMRP Regulates Neurotransmitter Release and Synaptic Information Transmission by Modulating Action Potential Duration via BK channels

    OpenAIRE

    Deng, Pan-Yue; Rotman, Ziv; Blundon, Jay A.; Cho, Yongcheol; Cui, Jianmin; Cavalli, Valeria; Zakharenko, Stanislav S; Klyachko, Vitaly A.

    2013-01-01

    Loss of FMRP causes Fragile X syndrome (FXS), but the physiological functions of FMRP remain highly debatable. Here we show that FMRP regulates neurotransmitter release in CA3 pyramidal neurons by modulating action potential (AP) duration. Loss of FMRP leads to excessive AP broadening during repetitive activity, enhanced presynaptic calcium influx and elevated neurotransmitter release. The AP broadening defects caused by FMRP loss have a cell-autonomous presynaptic origin and can be acutely r...

  7. Kidney retransplantation for BK virus nephropathy with active viremia without allograft nephrectomy.

    Science.gov (United States)

    Huang, Jingbo; Danovitch, Gabriel; Pham, Phuong-Thu; Bunnapradist, Suphamai; Huang, Edmund

    2015-12-01

    BK virus nephropathy is an important cause of kidney allograft failure. Retransplantation has been successfully performed for patients with previous allograft loss due to BK virus nephropathy; however, whether allograft nephrectomy and viral clearance are required prior to retransplantation is controversial. Some recent studies have suggested that retransplantion can be successfully achieved without allograft nephrectomy if viremia is cleared prior to retransplant. The only published experience of successful retransplantation in the presence of active viremia occurred in the presence of concomitant allograft nephrectomy of the failing kidney. In this report, we describe a case of successful repeat kidney transplant in a patient with high-grade BK viremia and fulminant hepatic failure without concomitant allograft nephrectomy performed under the setting of a simultaneous liver-kidney transplant.

  8. 75 FR 48617 - Airworthiness Directives; Eurocopter Deutschland GmbH (ECD) Model MBB-BK 117 C-2 Helicopters

    Science.gov (United States)

    2010-08-11

    ... vibration, and subsequent loss of control of the helicopter. Actions and Compliance (e) Before further... Deutschland GmbH (ECD) Model MBB-BK 117 C-2 Helicopters AGENCY: Federal Aviation Administration (FAA), DOT... directive (AD) for ECD Model MBB-BK 117 C-2 helicopters. This proposed AD results from a...

  9. 75 FR 66657 - Airworthiness Directives; Eurocopter Deutschland GmbH Model MBB-BK 117 C-2 Helicopters

    Science.gov (United States)

    2010-10-29

    ... separation of dynamic weights, severe vibration, and subsequent loss of control of the helicopter. Actions... Deutschland GmbH Model MBB- BK 117 C-2 Helicopters AGENCY: Federal Aviation Administration, DOT. ACTION: Final... Deutschland GmbH (ECD) Model MBB BK 117 C-2 helicopters. This amendment results from a mandatory...

  10. Conserved archetypal configuration of the transcriptional control region during the course of BK polyomavirus evolution.

    Science.gov (United States)

    Yogo, Yoshiaki; Zhong, Shan; Xu, Yawei; Zhu, Mengyun; Chao, Yuegen; Sugimoto, Chie; Ikegaya, Hiroshi; Shibuya, Ayako; Kitamura, Tadaichi

    2008-08-01

    BK polyomavirus (BKV) is widespread among humans, asymptomatically infecting children and then persisting in renal tissue. The transcriptional control region (TCR) of the BKV genome is variable among clinical isolates. Thus, archetypal TCRs with a common basic configuration generally occur in BKV isolates from the urine of immunocompromised patients, but rearranged TCRs that possibly arise from the archetypal configuration have also been detected in clinical specimens. To examine the hypothesis that archetypal strains represent wild-type strains circulating in the human population (the archetype hypothesis), we analysed 145 complete viral genomes amplified directly from the urine of non-immunocompromised individuals worldwide. These genomes included 82, three, two and 58 sequences classified as belonging to subtypes I, II, III and IV, respectively. Rearranged TCRs with long duplications or deletions were detected from two subtype I and two subtype IV genomes, but not from the other 141 genomes (thus, the TCRs of these genomes were judged to be archetypal). The variations in the archetypal TCRs were nucleotide substitutions and single-nucleotide deletions, most of which were unique to particular subtypes or subgroups. We confirmed that the four complete BKV genomes with rearranged TCRs did not form a unique lineage on a phylogenetic tree. Collectively, the findings demonstrate that the archetypal TCR configuration has been conserved during the evolution of BKV, providing support for the archetype hypothesis. Additionally, we suggest that 'archetype' should be used as a conceptual term that denotes a prototypical structure that can generate various rearranged TCRs during viral growth in vivo and in vitro.

  11. A user-friendly wearable single-channel EOG-based human-computer interface for cursor control

    OpenAIRE

    2015-01-01

    This paper presents a novel wearable single-channel electrooculography (EOG) based human-computer interface (HCI) with a simple system design and robust performance. In the proposed system, EOG signals for control are generated from double eye blinks, collected by a commercial wearable device (the NeuroSky MindWave headset), and then converted into a sequence of commands that can control cursor navigations and actions. The EOG-based cursor control system was tested on 8 subjects in indoor or ...

  12. How Human Resource Professionals Use Electronic Channels to Communicate CSR : A case study focused on Solvay's French industrial sites

    OpenAIRE

    Fournet, Clara; Pauly, Marissa

    2015-01-01

    Corporate Social Responsibility (CSR) has become a large concern for many companies with the rise of globalization. Oftentimes, companies are encouraged to communicate CSR externally, but not internally. This research focuses upon the internal communication of CSR, specifically how Human Resource (HR) professionals use electronic channels to communicate to employees. The scope of this research is focused solely upon HR professionals within Solvay’s French industrial sites, which produce chemi...

  13. Structural and Biochemical Consequences of Disease-Causing Mutations in the Ankyrin Repeat Domain of the Human TRPV4 Channel

    Energy Technology Data Exchange (ETDEWEB)

    Inada, Hitoshi; Procko, Erik; Sotomayor, Marcos; Gaudet, Rachelle (Harvard-Med); (Harvard)

    2012-10-23

    The TRPV4 calcium-permeable cation channel plays important physiological roles in osmosensation, mechanosensation, cell barrier formation, and bone homeostasis. Recent studies reported that mutations in TRPV4, including some in its ankyrin repeat domain (ARD), are associated with human inherited diseases, including neuropathies and skeletal dysplasias, probably because of the increased constitutive activity of the channel. TRPV4 activity is regulated by the binding of calmodulin and small molecules such as ATP to the ARD at its cytoplasmic N-terminus. We determined structures of ATP-free and -bound forms of human TRPV4-ARD and compared them with available TRPV-ARD structures. The third inter-repeat loop region (Finger 3 loop) is flexible and may act as a switch to regulate channel activity. Comparisons of TRPV-ARD structures also suggest an evolutionary link between ARD structure and ATP binding ability. Thermal stability analyses and molecular dynamics simulations suggest that ATP increases stability in TRPV-ARDs that can bind ATP. Biochemical analyses of a large panel of TRPV4-ARD mutations associated with human inherited diseases showed that some impaired thermal stability while others weakened ATP binding ability, suggesting molecular mechanisms for the diseases.

  14. Hydronephrosis Resulting from Bilateral Ureteral Stenosis: A Late Complication of Polyoma BK Virus Cystitis?

    Directory of Open Access Journals (Sweden)

    N. Basara

    2010-01-01

    Full Text Available We report here a case of acute lymphoblastic leukemia in remission presenting a late-onset bilateral hydronephrosis probably due to polyoma BK virus-induced proliferation of bladder endothelium on both ostii. The diagnosis was made virologically by BK virus Polymerase Chain Reaction (PCR detection in the absence of any other bladder disease. Awareness of this late complication is necessary not only in patients after renal transplantation but also in patients after hematopoietic stem cell transplantation from matched unrelated donor.

  15. Pengaruh pH dan Perubahan Temperatur Terhadap Pembentukan Spora Bacillus sp. BK17

    OpenAIRE

    2014-01-01

    Bacterial spores are the surviving structure under unfavourable physical and chemical conditions. Bacillus sp. BK17 is a spore forming bacteria that has been reported to have an ability to inhibit the growth of various pathogenic fungi.This study aims to determine the best pH and temperature for the formation of spore. The result showed that Bacillus sp. BK17 has the highest spore formation at the initial pH of media of 5,0 and at a heat shock of 70° C for 60 minutes. 090805025

  16. Karakterisasi Enzim Kitinase dari Bacillus sp. BK17, Isolat Potensial Pengendali Hayati Jamur Patogen Tanaman

    OpenAIRE

    Maimunah, Siti

    2016-01-01

    Characterization of chitinase of including pH and temperature, Km and Vmax of Bacillus sp. BK 17 has been conducted. Crude extract of Bacillus sp. BK17 growing in minimum salt medium with colloidal chitin for 5 days was precipited with ammonium sulphate. Optimum chitinase activity was found in 50% ammonium sulphate precipitation with specific activity of 0.545 Units. Chitinase activity in homogenated mycelia of Sclerotium rolfsii was 0.0012 U/ml. The Km and Vmax of the enzyme was 0.46 μg and ...

  17. Comparison of Topas cyclic olefin copolymers to BK7 glass in night vision goggle objectives

    Science.gov (United States)

    Stevens, James S.

    2004-09-01

    The objective of this study was to determine the suitability of Topas cyclic olefin copolymers (COC) as an optical plastic for use in military-grade night vision goggle (NVG) lens objectives. Test objective lenses that could include either a Topas COC window element or BK7 glass window element were manufactured. The test objectives were evaluated for low light resolution, MTF, off-axis veiling glare, and on-axis stray light. Additionally, the spectral transmittance of the individual windows elements was measured. This paper compares the evaluation results of test objectives containing Topas COC with test objectives containing BK7 glass.

  18. Human Nav1.6 Channels Generate Larger Resurgent Currents than Human Nav1.1 Channels, but the Navβ4 Peptide Does Not Protect Either Isoform from Use-Dependent Reduction.

    Directory of Open Access Journals (Sweden)

    Reesha R Patel

    Full Text Available Voltage-gated sodium channels are responsible for the initiation and propagation of action potentials (APs. Two brain isoforms, Nav1.1 and Nav1.6, have very distinct cellular and subcellular expression. Specifically, Nav1.1 is predominantly expressed in the soma and proximal axon initial segment of fast-spiking GABAergic neurons, while Nav1.6 is found at the distal axon initial segment and nodes of Ranvier of both fast-spiking GABAergic and excitatory neurons. Interestingly, an auxiliary voltage-gated sodium channel subunit, Navβ4, is also enriched in the axon initial segment of fast-spiking GABAergic neurons. The C-terminal tail of Navβ4 is thought to mediate resurgent sodium current, an atypical current that occurs immediately following the action potential and is predicted to enhance excitability. To better understand the contribution of Nav1.1, Nav1.6 and Navβ4 to high frequency firing, we compared the properties of these two channel isoforms in the presence and absence of a peptide corresponding to part of the C-terminal tail of Navβ4. We used whole-cell patch clamp recordings to examine the biophysical properties of these two channel isoforms in HEK293T cells and found several differences between human Nav1.1 and Nav1.6 currents. Nav1.1 channels exhibited slower closed-state inactivation but faster open-state inactivation than Nav1.6 channels. We also observed a greater propensity of Nav1.6 to generate resurgent currents, most likely due to its slower kinetics of open-state inactivation, compared to Nav1.1. These two isoforms also showed differential responses to slow and fast AP waveforms, which were altered by the Navβ4 peptide. Although the Navβ4 peptide substantially increased the rate of recovery from apparent inactivation, Navβ4 peptide did not protect either channel isoform from undergoing use-dependent reduction with 10 Hz step-pulse stimulation or trains of slow or fast AP waveforms. Overall, these two channels have

  19. Polyoma (BK) virus associated urothelial carcinoma originating within a renal allograft five years following resolution of polyoma virus nephropathy.

    Science.gov (United States)

    Salvatore, Steven P; Myers-Gurevitch, Patricia M; Chu, Stacy; Robinson, Brian D; Dadhania, Darshana; Seshan, Surya V

    2016-03-01

    A direct role for BK polyomavirus infection in malignant tumors of renal allografts and urinary tract is emerging. Case reports suggest a link between BK virus (BKV) reactivation and development of malignancy in renal allograft recipients. Herein we describe the first case of BKV positive invasive urothelial carcinoma within the renal allograft, presenting with chronic diarrhea and weight loss 5 years following resolution of BK viremia/nephropathy (BKVN). Unique to our case was the remote history of BK viremia/BKVN, rising titer of anti-HLA antibody and presence of renal limited urothelial carcinoma with microinvasion of malignant cells staining positive for SV40 large T antigen (T-Ag). These findings suggest that persistence of subclinical BKV infection within the renal allograft may play a role in the malignant transformation of epithelial cells. Patients with history of BKVN may be at risk for kidney and urinary tract malignancy despite resolution of BK viremia/BKVN.

  20. Functional characterization of three ethylene response factor genes from Bupleurum kaoi indicates that BkERFs mediate resistance to Botrytis cinerea.

    Science.gov (United States)

    Liu, Wen-Yu; Chiou, Shu-Jiau; Ko, Chia-Yun; Lin, Tsai-Yun

    2011-03-01

    Three novel ethylene response factor (ERF) genes, BkERF1, BkERF2.1 and BkERF2.2, were isolated from a medicinal plant, Bupleurum kaoi. The deduced BkERFs contain a canonical nuclear localization signal and an ERF/AP2 DNA binding domain. RNA gel blot analysis revealed that BkERF1 and BkERF2.1 were ubiquitously expressed at low levels in all parts of mature plants, and that BkERF2.2 was expressed at moderate levels in vegetative tissues. Exogenous application of methyl jasmonate induced BkERF1/2.1/2.2 transcripts. BkERF2.2 transcript levels were slightly increased by addition of ethephon and salicylic acid. BkERFs were localized in the plant nucleus and functioned as transcriptional activators. In B. kaoi cells overexpressing BKERFs, inoculation with Botrytis cinerea increased expression of some defense genes which are associated with enhanced disease resistance. Similarly, overexpression of BkERFs in transgenic Arabidopsis thaliana resulted in elevated mRNA levels of the defense gene PDF1.2, and in enhanced resistance to B. cinerea. Collectively, these results provide evidence that BkERFs mediate the expression of defense-related genes in plants.

  1. The Realization of Command Channel-Mechanism in a System for Human to Human Interaction and Co-operative%人人交互与协作系统中命令通道机制的实现

    Institute of Scientific and Technical Information of China (English)

    田友胜; 侯义斌

    2000-01-01

    The concept of command channel-mechanism has been introduced into the command channelmechanism system for human to human interaction and co-operative over the Internet/Intranet.A new type of interaction and co-operative environment has been built up.This paper discusses the running mechanism and software realization of the command channel.

  2. Electrophysiological characterisation of KCNQ channel modulators

    DEFF Research Database (Denmark)

    Schrøder, R.L

    -cell configuration by the patch-clamp technique. Voltage-activated KCNQ currents were enhanced by extracellular application of retigabine, and also by the novel BK channel opener Compound 1 (( )-(5-chloro-2-metoxyphenyl)-1.3-didydroxy-3-fluoro-6-(trifluoromethyl)-2H-indol-2-one) (Gribkoff et al. 2001). The effects......, was sensitive to linopirdine and XE991, and had a nearly linear I-V relationship. Moreover, development of the voltage-independent current did not require a preceding voltage-dependent activation of the channel. This effect of Compound 1 may have profound hyperpolarising actions on cells expressing the KCNQ4......Potassium (K+) ion channels are ubiquitously expressed in mammalian cells, and each channel serves a precise physiological role due to its specific biophysical characteristics and expression pattern. A few K+ channels are targets for certain drugs, and in this thesis it is suggested that the KCNQ K...

  3. 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...

  4. Modulation of the transient outward current (Ito) in rat cardiac myocytes and human Kv4.3 channels by mefloquine.

    Science.gov (United States)

    Perez-Cortes, E J; Islas, A A; Arevalo, J P; Mancilla, C; Monjaraz, E; Salinas-Stefanon, E M

    2015-10-15

    The antimalarial drug mefloquine, is known to be a potassium channel blocker, although its mechanism of action has not being elucidated and its effects on the transient outward current (Ito) and the molecular correlate, the Kv4.3 channel has not being studied. Here, we describe the mefloquine-induced inhibition of the rat ventricular Ito and of CHO cells co-transfected with human Kv4.3 and its accessory subunit hKChIP2C by whole-cell voltage-clamp. Mefloquine inhibited rat Ito and hKv4.3+KChIP2C currents in a concentration-dependent manner with a limited voltage dependence and similar potencies (IC50=8.9μM and 10.5μM for cardiac myocytes and Kv4.3 channels, respectively). In addition, mefloquine did not affect the activation of either current but significantly modified the hKv4.3 steady-state inactivation and recovery from inactivation. The effects of this drug was compared with that of 4-aminopyridine (4-AP), a well-known potassium channel blocker and its binding site does not seem to overlap with that of 4-AP.

  5. Cloning, chromosomal localization, and functional expression of the alpha 1 subunit of the L-type voltage-dependent calcium channel from normal human heart

    NARCIS (Netherlands)

    Schultz, D; Mikala, G; Yatani, A; Engle, D B; Iles, D E; Segers, B; Sinke, R J; Weghuis, D O; Klöckner, U; Wakamori, M

    1993-01-01

    A unique structural variant of the cardiac L-type voltage-dependent calcium channel alpha 1 subunit cDNA was isolated from libraries derived from normal human heart mRNA. The deduced amino acid sequence shows significant homology to other calcium channel alpha 1 subunits. However, differences from t

  6. High glucose enhances transient receptor potential channel canonical type 6-dependent calcium influx in human platelets via phosphatidylinositol 3-kinase-dependent pathway

    DEFF Research Database (Denmark)

    Liu, Daoyan; Maier, Alexandra; Scholze, Alexandra;

    2008-01-01

    Transient receptor potential canonical type 6 (TRPC6) channels mediating 1-oleoyl-2-acetyl-sn-glycerol (OAG)-induced calcium entry have been identified on human platelets. In the present study we tested the hypothesis that hyperglycemia increases the expression of TRPC6 channels....

  7. Block of human cardiac sodium channels by lacosamide: evidence for slow drug binding along the activation pathway.

    Science.gov (United States)

    Wang, Ging Kuo; Wang, Sho-Ya

    2014-05-01

    Lacosamide is an anticonvulsant hypothesized to enhance slow inactivation of neuronal Na(+) channels for its therapeutic action. Cardiac Na(+) channels display less and incomplete slow inactivation, but their sensitivity toward lacosamide remains unknown. We therefore investigated the action of lacosamide in human cardiac Nav1.5 and Nav1.5-CW inactivation-deficient Na(+) channels. Lacosamide showed little effect on hNav1.5 Na(+) currents at 300 µM when cells were held at -140 mV. With 30-second conditioning pulses from -90 to -50 mV; however, hNav1.5 Na(+) channels became sensitive to lacosamide with IC50 (50% inhibitory concentration) around 70-80 µM. Higher IC50 values were found at -110 and -30 mV. The development of lacosamide block at -70 mV was slow in wild-type Na(+) channels (τ; 8.04 ± 0.39 seconds, n = 8). This time constant was significantly accelerated in hNav1.5-CW inactivation-deficient counterparts. The recovery from lacosamide block at -70 mV for 10 seconds was relatively rapid in wild-type Na(+) channels (τ; 639 ± 90 milliseconds, n = 8). This recovery was accelerated further in hNav1.5-CW counterparts. Unexpectedly, lacosamide elicited a time-dependent block of persistent hNav1.5-CW Na(+) currents with an IC50 of 242 ± 19 µM (n = 5). Furthermore, both hNav1.5-CW/F1760K mutant and batrachotoxin-activated hNav1.5 Na(+) channels became completely lacosamide resistant, indicating that the lacosamide receptor overlaps receptors for local anesthetics and batrachotoxin. Our results together suggest that lacosamide targets the intermediate preopen and open states of hNav1.5 Na(+) channels. Lacosamide may thus track closely the conformational changes at the hNav1.5-F1760 region along the activation pathway.

  8. Nonlocal atlas-guided multi-channel forest learning for human brain labeling

    Energy Technology Data Exchange (ETDEWEB)

    Ma, Guangkai [Space Control and Inertial Technology Research Center, Harbin Institute of Technology, Harbin 150001, China and Department of Radiology and BRIC, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina 27599 (United States); Gao, Yaozong; Wu, Guorong [Department of Computer Science, Department of Radiology, and BRIC, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina 27599 (United States); Wu, Ligang [Space Control and Inertial Technology Research Center, Harbin Institute of Technology, Harbin 150001 (China); Shen, Dinggang, E-mail: dgshen@med.unc.edu [Department of Radiology and BRIC, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina 27599 and Department of Brain and Cognitive Engineering, Korea University, Seoul 02841 (Korea, Republic of)

    2016-02-15

    Purpose: It is important for many quantitative brain studies to label meaningful anatomical regions in MR brain images. However, due to high complexity of brain structures and ambiguous boundaries between different anatomical regions, the anatomical labeling of MR brain images is still quite a challenging task. In many existing label fusion methods, appearance information is widely used. However, since local anatomy in the human brain is often complex, the appearance information alone is limited in characterizing each image point, especially for identifying the same anatomical structure across different subjects. Recent progress in computer vision suggests that the context features can be very useful in identifying an object from a complex scene. In light of this, the authors propose a novel learning-based label fusion method by using both low-level appearance features (computed from the target image) and high-level context features (computed from warped atlases or tentative labeling maps of the target image). Methods: In particular, the authors employ a multi-channel random forest to learn the nonlinear relationship between these hybrid features and target labels (i.e., corresponding to certain anatomical structures). Specifically, at each of the iterations, the random forest will output tentative labeling maps of the target image, from which the authors compute spatial label context features and then use in combination with original appearance features of the target image to refine the labeling. Moreover, to accommodate the high inter-subject variations, the authors further extend their learning-based label fusion to a multi-atlas scenario, i.e., they train a random forest for each atlas and then obtain the final labeling result according to the consensus of results from all atlases. Results: The authors have comprehensively evaluated their method on both public LONI-LBPA40 and IXI datasets. To quantitatively evaluate the labeling accuracy, the authors use the

  9. Stimulation of BK virus DNA replication by NFI family transcription factors.

    Science.gov (United States)

    Liang, Bo; Tikhanovich, Irina; Nasheuer, Heinz Peter; Folk, William R

    2012-03-01

    BK polyomavirus (BKV) establishes persistent, low-level, and asymptomatic infections in most humans and causes polyomavirus-associated nephropathy (PVAN) and other pathologies in some individuals. The activation of BKV replication following kidney transplantation, leading to viruria, viremia, and, ultimately, PVAN, is associated with immune suppression as well as inflammation and stress from ischemia-reperfusion injury of the allograft, but the stimuli and molecular mechanisms leading to these pathologies are not well defined. The replication of BKV DNA in cell cultures is regulated by the viral noncoding control region (NCCR) comprising the core origin and flanking sequences, to which BKV T antigen (Tag), cellular proteins, and small regulatory RNAs bind. Six nuclear factor I (NFI) binding sites occur in sequences flanking the late side of the core origin (the enhancer) of the archetype virus, and their mutation, either individually or in toto, reduces BKV DNA replication when placed in competition with templates containing intact BKV NCCRs. NFI family members interacted with the helicase domain of BKV Tag in pulldown assays, suggesting that NFI helps recruit Tag to the viral core origin and may modulate its function. However, Tag may not be the sole target of the replication-modulatory activities of NFI: the NFIC/CTF1 isotype stimulates BKV template replication in vitro at low concentrations of DNA polymerase-α primase (Pol-primase), and the p58 subunit of Pol-primase associates with NFIC/CTF1, suggesting that NFI also recruits Pol-primase to the NCCR. These results suggest that NFI proteins (and the signaling pathways that target them) activate BKV replication and contribute to the consequent pathologies caused by acute infection.

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

    Science.gov (United States)

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

    2013-03-28

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

  11. The Piezo1 protein ion channel functions in human nucleus pulposus cell apoptosis by regulating mitochondrial dysfunction and the endoplasmic reticulum stress signal pathway.

    Science.gov (United States)

    Li, Xiao-Fei; Leng, Ping; Zhang, Zhao; Zhang, Hai-Ning

    2017-09-15

    The Piezo1 protein ion channel is a novel mechanical stretch-activated ion channel (SAC) closely related to mechanical signals. Mechanotransduction plays a crucial role in organ development and homeostasis. Previous studies identified Piezo1 and demonstrated that it is distinct from other ion channels with well-established roles in lower organisms. Mechanical stretch-activated ion channels from other organisms are not conserved in mammals or do not act as mechanically activated channels in mammals. In the current study, we explored the role of the Piezo1 ion channel in human nucleus pulposus cell (NP cell) apoptosis through mechanical force-induced mitochondrial dysfunction and endoplasmic reticulum stress. Reverse Transcription Polymerase chain reaction (RT-PCR), immunofluorescence, immunohistochemistry and Annexin V binding and propidium iodide analyses revealed that the Piezo1 protein ion channel was highly expressed in human NP cells, which are the primary cells that comprise the intervertebral disc. In patients with intervertebral disc degeneration (IVDD), the Piezo1 protein may play a crucial role in human NP cell apoptosis through mitochondrial dysfunction and endoplasmic reticulum stress under abnormal loading conditions. This study also verified that human NP cells have an intimate connection with the cytoskeleton upon treatment of the cells with the Piezo1 blocking peptide GsMTx4 from tarantula venom. In summary, Piezo1 functions in human NP cell apoptosis, which may be one underlying mechanism of apoptosis induced by abnormal loading in IVDD patients. Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.

  12. Natural and human-induced driving factors in the evolution of tidal channels: case studies in the Venice Lagoon (Italy).

    Science.gov (United States)

    Rizzetto, Federica

    2013-04-01

    Coastal wetlands are largely affected by a complex variety of both natural and anthropogenic factors, which induce evident, often irreversible, geomorphological transformations. In particular, this research focuses on the main processes that influence the evolution of tidal channels in salt marshes and shows the results derived from the analysis of some case studies in the Venice Lagoon (northwestern Adriatic Sea, Italy). Here tidal network has been recognized as significantly sensitive to sea-level rise and tide oscillations (Rizzetto and Tosi, 2011; Rizzetto and Tosi, 2012), but it is also vulnerable to human impact. The sites were selected in areas characterized by low anthropogenic pressure to prevent strong human interferences from completely masking the effects of natural forces. The interpretation of a large number of high-resolution aerial photographs, taken since the mid 1930s, allowed identifying in detail tidal channel evolution, both in the long- and in the short-term. The observation of historical and recent topographic maps completed the study and provided other important data to define the modifications occurred in the past two centuries. The channel planform changes were determined through the morphometric analysis of the tidal network, carried out using a Geographic Information System software. These modifications were interpreted in the light of sea-level oscillations (i.e. relative sea-level rise and strength/frequency of high tides, which are increasing owing to climate changes), variations of sediment supply, and human activities occurred in the past century. The joint analysis of all the data allowed distinguishing the changes induced by both relative sea-level rise and high tides on planform pattern and evolution of tidal channels, and identifying the effects of human interferences, which magnified the impact of natural factors (e.g. groundwater exploitation responsible for high subsidence rates between 1950 and 1970 and, consequently, for an

  13. Estradiol rapidly induces the translocation and activation of the intermediate conductance calcium activated potassium channel in human eccrine sweat gland cells.

    LENUS (Irish Health Repository)

    Muchekehu, Ruth W

    2009-02-01

    Steroid hormones target K+ channels as a means of regulating electrolyte and fluid transport. In this study, ion transporter targets of Estradiol (E2) were investigated in the human eccrine sweat gland cell line NCL-SG3.

  14. Effects on Humans Elicited by Inhaling the Fragrance of Essential Oils: Sensory Test, Multi-Channel Thermometric Study and Forehead Surface Potential Wave Measurement on Basil and Peppermint

    National Research Council Canada - National Science Library

    SATOH, Tomoko; SUGAWARA, Yoshiaki

    2003-01-01

    The effects on humans inhaling the fragrance of essential oils were examined in terms of a sensory test, a multi-channel skin thermometer study and a portable forehead surface electroencephalographic (IBVA-EEG) measurement...

  15. Gap-junction coupling and ATP-sensitive potassium channels in human β -cell clusters: Effects on emergent dynamics

    Science.gov (United States)

    Loppini, A.; Pedersen, M. G.; Braun, M.; Filippi, S.

    2017-09-01

    The importance of gap-junction coupling between β cells in pancreatic islets is well established in mouse. Such ultrastructural connections synchronize cellular activity, confine biological heterogeneity, and enhance insulin pulsatility. Dysfunction of coupling has been associated with diabetes and altered β -cell function. However, the role of gap junctions between human β cells is still largely unexplored. By using patch-clamp recordings of β cells from human donors, we previously estimated electrical properties of these channels by mathematical modeling of pairs of human β cells. In this work we revise our estimate by modeling triplet configurations and larger heterogeneous clusters. We find that a coupling conductance in the range 0.005 -0.020 nS/pF can reproduce experiments in almost all the simulated arrangements. We finally explore the consequence of gap-junction coupling of this magnitude between β cells with mutant variants of the ATP-sensitive potassium channels involved in some metabolic disorders and diabetic conditions, translating studies performed on rodents to the human case. Our results are finally discussed from the perspective of therapeutic strategies. In summary, modeling of more realistic clusters with more than two β cells slightly lowers our previous estimate of gap-junction conductance and gives rise to patterns that more closely resemble experimental traces.

  16. Combination of Leflunomide and Everolimus for treatment of BK virus nephropathy.

    Science.gov (United States)

    Jaw, Juli; Hill, Prue; Goodman, David

    2017-04-01

    BK nephropathy (BKN) is a common cause of graft dysfunction following kidney transplantation. Minimization of immunosuppressive therapy remains the first line of therapy, but this may lead to rejection and graft loss. In some cases, despite lowering immunosuppression, BK infection can persist, leading to chronic damage and kidney failure. Currently, there is no specific anti-BK viral therapy. Recent in vitro experiments have demonstrated a reduction in BK viral replication when infected cells are treated with the combination of Leflunomide and Everolimus. This study aims to explore the effect of this drugs combination on viral clearance and graft function in patients with persistent disease despite reduction in immunosuppression. We treated three patients with combination Leflunomide and Everolimus. Data on medical history, biochemical parameters and viral loads were collected. Significant improvement in viral loads was observed in two cases with resolution of viremia in another (Table 1). Two recipients had preserved allograft function. The remaining graft was lost because of combination of obstruction and BKN. No adverse reactions such as bone marrow toxicity were observed. Combination of Leflunomide and Everolimus is safe and should be considered as a rescue therapy in treatment of BKN, especially in those who fail to clear this infection despite reduction of immunosuppressive therapy.

  17. Influence of BK7 Substrate Solarization on the Performance of Hafnia and Silica Multilayer Mirrors

    Energy Technology Data Exchange (ETDEWEB)

    Stolz, C J; Menapace, J A; Genin, F; Ehrmann, P; Miller, P; Rogowski, G

    2002-11-26

    Transport mirrors within the National Ignition Facility, a 192-beam 4-MJ fusion laser at 1053 nm, will be exposed to backscattered light from plasmas created from fusion targets and backlighters. This backscattered light covers the UV and visible spectrum from 351-600 nm. The transport mirror BK7 substrates will be intentionally solarized to absorb >95% of the backscattered light to prevent damage to the metallic mechanical support hardware. Solarization has minimal impact on the 351- and 1053-nm laser-induced damage threshold or the reflected wavefront of the multilayer hafnia silica coating. Radiation sources of various energies were examined for BK7 darkening efficiency within the UV and visible region with 1.1 MeV gamma rays from a Cobalt 60 source ultimately being selected. Finally, bleaching rates were measured at elevated temperatures to generate a model for predicting the lifetime at ambient conditions (20 C), before solarized BK7 substrates exceed 5% transmission in the UV and visible region. Over a 30-mm thickness, BK7 glass will bleach in 10 years to 5% transmission at 600 nm, the most transmissive wavelengths over the 351-600 nm regions.

  18. New properties of BK-spaces defined by using regular matrix of Fibonacci numbers

    Science.gov (United States)

    Ercan, Sinan; Bektaş, ćiǧdem A.

    2016-06-01

    In the present paper, we studied the new properties of BK-spaces which were defined using regular matrix of Fibonacci numbers in [1]. We computed alpha-, beta-, gamma- duals of these spaces and obtained Schauder basis. We also derived some topological properties of these spaces.

  19. Application of fibrin glue to damaged bladder mucosa in a case of BK viral hemorrhagic cystitis.

    Science.gov (United States)

    Purves, J Todd; Graham, Michael L; Ramakumar, Sanjay

    2005-09-01

    BK virus is a common cause of severe hemorrhagic cystitis refractory to standard treatment. We describe a technique to achieve hemostasis after failed conservative therapy using fibrin glue applied suprapubically while visualizing and insufflating the bladder through a cystoscope. Long-term hemostasis was achieved using this novel procedure.

  20. Test du Module BECKHOFF (BK7420) Entrées/Sorties deportees sur FIPIO de SCHNEIDER

    CERN Document Server

    Palluel, J; CERN. Geneva. AB Department

    2004-01-01

    Cette note présente le test du nouveau coupleur I/O déporté sur FIPIO de Beckhoff référencé BK7420 (voir photo ci-dessous), et notamment son évaluation sur différentes longueurs par rapport à un module semblable de Schneider (Momentum 170 FNT 110 01).

  1. Effect of beta-adrenoceptor blockers on human ether-a-go-go-related gene (HERG) potassium channels

    DEFF Research Database (Denmark)

    Dupuis, Delphine S; Klaerke, Dan A; Olesen, Søren-Peter

    2005-01-01

    Patients with congenital long QT syndrome may develop arrhythmias under conditions of increased sympathetic tone. We have addressed whether some of the beta-adrenoceptor blockers commonly used to prevent the development of these arrhythmias could per se block the cardiac HERG (Human Ether....... These data showed that HERG blockade by beta-adrenoceptor blockers occurred only at high micromolar concentrations, which are significantly above the recently established safe margin of 100 (Redfern et al., 2003).......-1H-inden-4-yl)oxy]-3-[(1-methylethyl)amino]-2-butanol hydrochloride) blocked the HERG channel with similar affinity, whereas the beta1-receptor antagonists metoprolol and atenolol showed weak effects. Further, the four compounds blocked HERG channels expressed in a mammalian HEK293 cell line...

  2. Computational analysis of the effects of the hERG channel opener NS1643 in a human ventricular cell model

    DEFF Research Database (Denmark)

    Peitersen, Torben; Grunnet, Morten; Benson, Alan P

    2008-01-01

    BACKGROUND: Dysfunction or pharmacologic inhibition of repolarizing cardiac ionic currents can lead to fatal arrhythmias. The hERG potassium channel underlies the repolarizing current I(Kr), and mutations therein can produce both long and short QT syndromes (LQT2 and SQT1). We previously reported...... on the diphenylurea compound NS1643, which acts on hERG channels in two distinct ways: by increasing overall conductance and by shifting the inactivation curve in the depolarized direction. OBJECTIVE: The purpose of this study was to determine which of the two components contributes more to the antiarrhythmic effects...... of NS1643 under normokalemic and hypokalemic conditions. METHODS: The study consisted of mathematical simulation of action potentials in a human ventricular ionic cell model in single cell and string of 100 cells. RESULTS: Regardless of external potassium concentration or diastolic interval used, NS1643...

  3. Voltage-gated sodium channel expressed in cultured human smooth muscle cells: involvement of SCN9A.

    Science.gov (United States)

    Jo, Taisuke; Nagata, Taiji; Iida, Haruko; Imuta, Hiroyuki; Iwasawa, Kuniaki; Ma, Ji; Hara, Kei; Omata, Masao; Nagai, Ryozo; Takizawa, Hajime; Nagase, Takahide; Nakajima, Toshiaki

    2004-06-04

    Voltage-gated Na(+) channel (I(Na)) is expressed under culture conditions in human smooth muscle cells (hSMCs) such as coronary myocytes. The aim of this study is to clarify the physiological, pharmacological and molecular characteristics of I(Na) expressed in cultured hSMCs obtained from bronchus, main pulmonary and coronary artery. I(Na), was recorded in these hSMCs and inhibited by tetrodotoxin (TTX) with an IC(50) value of approximately 10 nM. Reverse transcriptase/polymerase chain reaction (RT-PCR) analysis of mRNA showed the prominent expression of transcripts for SCN9A, which was consistent with the results of real-time quantitative RT-PCR. These results provide novel evidence that TTX-sensitive Na(+) channel expressed in cultured hSMCs is mainly composed of Na(v)1.7.

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

    Science.gov (United States)

    Shipston, Michael J

    2017-06-05

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

  5. Rapid effects of 17beta-estradiol on epithelial TRPV6 Ca2+ channel in human T84 colonic cells.

    LENUS (Irish Health Repository)

    Irnaten, Mustapha

    2008-11-01

    The control of calcium homeostasis is essential for cell survival and is of crucial importance for several physiological functions. The discovery of the epithelial calcium channel Transient Receptor Potential Vaniloid (TRPV6) in intestine has uncovered important Ca(2+) absorptive pathways involved in the regulation of whole body Ca(2+) homeostasis. The role of steroid hormone 17beta-estradiol (E(2)), in [Ca(2+)](i) regulation involving TRPV6 has been only limited at the protein expression levels in over-expressing heterologous systems. In the present study, using a combination of calcium-imaging, whole-cell patch-clamp techniques and siRNA technology to specifically knockdown TRPV6 protein expression, we were able to (i) show that TRPV6 is natively, rather than exogenously, expressed at mRNA and protein levels in human T84 colonic cells, (ii) characterize functional TRPV6 channels and (iii) demonstrate, for the first time, the rapid effects of E(2) in [Ca(2+)](i) regulation involving directly TRPV6 channels in T84 cells. Treatment with E(2) rapidly (<5 min) enhanced [Ca(2+)](i) and this increase was partially but significantly prevented when cells were pre-treated with ruthenium red and completely abolished in cells treated with siRNA specifically targeting TRPV6 protein expression. These results indicate that when cells are stimulated by E(2), Ca(2+) enters the cell through TRPV6 channels. TRPV6 channels in T84 cells contribute to the Ca(2+) entry\\/signalling pathway that is sensitive to 17beta-estradiol.

  6. Basolateral potassium channels of rabbit colon epithelium: role in sodium absorption and chloride secretion.

    Science.gov (United States)

    Turnheim, Klaus; Plass, Herbert; Wyskovsky, Wolfgang

    2002-02-18

    In order to assess the role of different classes of K(+) channels in recirculation of K(+) across the basolateral membrane of rabbit distal colon epithelium, the effects of various K(+) channel inhibitors were tested on the activity of single K(+) channels from the basolateral membrane, on macroscopic basolateral K(+) conductance, and on the rate of Na(+) absorption and Cl(-) secretion. In single-channel measurements using the lipid bilayer reconstitution system, high-conductance (236 pS), Ca(2+)-activated K(+) (BK(Ca)) channels were most frequently detected; the second most abundant channel was a low-conductance K(+) channel (31 pS) that exhibited channel rundown. In addition to Ba(2+) and charybdotoxin (ChTX), the BK(Ca) channels were inhibited by quinidine, verapamil and tetraethylammonium (TEA), the latter only when present on the side of the channel from which K(+) flow originates. Macroscopic basolateral K(+) conductance, determined in amphotericin-permeabilised epithelia, was also markedly reduced by quinidine and verapamil, TEA inhibited only from the lumen side, and serosal ChTX was without effect. The chromanol 293B and the sulphonylurea tolbutamide did not affect BK(Ca) channels and had no or only a small inhibitory effect on macroscopic basolateral K(+) conductance. Transepithelial Na(+) absorption was partly inhibited by Ba(2+), quinidine and verapamil, suggesting that BK(Ca) channels are involved in basolateral recirculation of K(+) during Na(+) absorption in rabbit colon. The BK(Ca) channel inhibitors TEA and ChTX did not reduce Na(+) absorption, probably because TEA does not enter intact cells and ChTX is 'knocked off' its extracellular binding site by K(+) outflow from the cell interior. Transepithelial Cl(-) secretion was inhibited completely by Ba(2+) and 293B, partly by quinidine but not by the other K(+) channel blockers, indicating that the small (<3 pS) K(V)LQT1 channels are responsible for basolateral K(+) exit during Cl(-) secretion. Hence

  7. Single Particle Image Reconstruction of the Human Recombinant Kv2.1 Channel

    OpenAIRE

    Adair, Brian; Nunn, Rashmi; Lewis, Shannon; Dukes, Iain; Philipson, Louis; Yeager, Mark

    2008-01-01

    Kv2.1 channels are widely expressed in neuronal and endocrine cells and generate slowly activating K+ currents, which contribute to repolarization in these cells. Kv2.1 is expressed at high levels in the mammalian brain and is a major component of the delayed rectifier current in the hippocampus. In addition, Kv2.1 channels have been implicated in the regulation of membrane repolarization, cytoplasmic calcium levels, and insulin secretion in pancreatic β-cells. They are therefore an important...

  8. Sodium leak channel, non-selective contributes to the leak current in human myometrial smooth muscle cells from pregnant women.

    Science.gov (United States)

    Reinl, Erin L; Cabeza, Rafael; Gregory, Ismail A; Cahill, Alison G; England, Sarah K

    2015-10-01

    Uterine contractions are tightly regulated by the electrical activity of myometrial smooth muscle cells (MSMCs). These cells require a depolarizing current to initiate Ca(2+) influx and induce contraction. Cationic leak channels, which permit a steady flow of cations into a cell, are known to cause membrane depolarization in many tissue types. Previously, a Gd(3+)-sensitive, Na(+)-dependent leak current was identified in the rat myometrium, but the presence of such a current in human MSMCs and the specific ion channel conducting this current was unknown. Here, we report the presence of a Na(+)-dependent leak current in human myometrium and demonstrate that the Na(+)-leak channel, NALCN, contributes to this current. We performed whole-cell voltage-clamp on fresh and cultured MSMCs from uterine biopsies of term, non-laboring women and isolated the leak currents by using Ca(2+) and K(+) channel blockers in the bath solution. Ohmic leak currents were identified in freshly isolated and cultured MSMCs with normalized conductances of 14.6 pS/pF and 10.0 pS/pF, respectively. The myometrial leak current was significantly reduced (P < 0.01) by treating cells with 10 μM Gd(3+) or by superfusing the cells with a Na(+)-free extracellular solution. Reverse transcriptase PCR and immunoblot analysis of uterine biopsies from term, non-laboring women revealed NALCN messenger RNA and protein expression in the myometrium. Notably, ∼90% knockdown of NALCN protein expression with lentivirus-delivered shRNA reduced the Gd(3+)-sensitive leak current density by 42% (P < 0.05). Our results reveal that NALCN, in part, generates the leak current in MSMCs and provide the basis for future research assessing NALCN as a potential molecular target for modulating uterine excitability.

  9. Reach-scale channel sensitivity to multiple human activities and natural events: Lower Santa Clara River, California, USA

    Science.gov (United States)

    Downs, Peter W.; Dusterhoff, Scott R.; Sears, William A.

    2013-05-01

    Understanding the cumulative impact of natural and human influences on the sensitivity of channel morphodynamics, a relative measure between the drivers for change and the magnitude of channel response, requires an approach that accommodates spatial and temporal variability in the suite of primary stressors. Multiple historical data sources were assembled to provide a reach-scale analysis of the lower Santa Clara River (LSCR) in Ventura County, California, USA. Sediment supply is naturally high due to tectonic activity, earthquake-generated landslides, wildfires, and high magnitude flow events during El Niño years. Somewhat typically for the region, the catchment has been subject to four reasonably distinct land use and resource management combinations since European-American settlement. When combined with analysis of channel morphological response (quantifiable since ca. 1930), reach-scale and temporal differences in channel sensitivity become apparent. Downstream reaches have incised on average 2.4 m and become narrower by almost 50% with changes focused in a period of highly sensitive response after about 1950 followed by forced insensitivity caused by structural flood embankments and a significant grade control structure. In contrast, the middle reaches have been responsive but are morphologically resilient, and the upstream reaches show a mildly sensitive aggradational trend. Superimposing the natural and human drivers for change reveals that large scale stressors (related to ranching and irrigation) have been replaced over time by a suite of stressors operating at multiple spatial scales. Lower reaches have been sensitive primarily to 'local' scale impacts (urban growth, flood control, and aggregate mining) whereas, upstream, catchment-scale influences still prevail (including flow regulation and climate-driven sediment supply factors). These factors illustrate the complexity inherent to cumulative impact assessment in fluvial systems, provide evidence for a

  10. Fluoroquinolone prophylaxis in preventing BK polyomavirus infection after renal transplant: A systematic review and meta-analysis.

    Science.gov (United States)

    Song, Tu-Run; Rao, Zheng-Sheng; Qiu, Yang; Liu, Jin-Peng; Huang, Zhong-Li; Wang, Xian-Ding; Lin, Tao

    2016-03-01

    Previous studies regarding the prevention of BK viremia following renal transplantation with fluoroquinolone have yielded conflicting results. The purpose of this systematic review was to examine the evidence regarding the efficacy of fluoroquinolone in preventing BK polyomavirus infection following renal transplantation. We searched PubMed, Embase, and the Cochrane Central Register of Controlled Trials for research articles published prior to January 2015 using keywords such as "fluoroquinolone," "BK viremia," and "renal transplantation." We extracted all types of study published in English. The primary outcome was BK viremia and viruria at 1 year post-transplantation. Secondary outcomes were BK virus-associated nephropathy (BKVN), graft failure, and fluoroquinolone-resistant infection. We identified eight trials, including a total of 1477 participants with a mean duration of fluoroquinolone prophylaxis of >1 month. At 1 year, fluoroquinolone prophylaxis was not associated with a decreased incidence of BK viremia [risk ratio (RR), 0.84; 95% confidence interval (95% CI), 0.58-1.20). No significant differences in BKVN (RR, 0.88; 95% CI, 0.37-2.11), risk of graft failure due to BKVN (RR, 0.68; 95% CI, 0.29-1.59), or fluoroquinolone-resistant infection (RR, 1.08; 95% CI, 0.64-1.83) were observed between the fluoroquinolone prophylaxis and control groups. The results of this study suggest that fluoroquinolone is ineffective in preventing BK polyomavirus infection following renal transplantation. Copyright © 2016. Published by Elsevier Taiwan.

  11. Surveillance of polyomavirus BK in relation to immunosuppressive therapy in kidney transplantation

    Directory of Open Access Journals (Sweden)

    Cristina Costa

    2012-03-01

    Full Text Available Introduction. Reactivation of polyomavirus BK in kidney transplant recipients has been associated to the development of nephropathy (polyomavirus-associated nephropathy, PVAN, possibly leading to the loss of the transplanted organ. Immunosuppression is the condicio sine qua non for the onset of PVAN; however, a lower incidence of BK viremia has been reported with low-level tacrolimus based immunosuppressive protocols in comparison to cyclosporine A.Aim of this study was to compare the two immunosuppressive protocols. Methods. Virological monitoring of BK was performed in 468 consecutive renal transplant patients over a period of 3 years (2370 urine e 2370 serum specimens: in particular, 1780 specimens from 362 patients treated with tacrolimus and 590 from 106 treated with cyclosporine A. Results. BK viremia was evidenced in 124 (7.0% and 12 (2.0% specimens from 40 (11.0% and 11 (10.4% patients treated with tacrolimus and cyclosporine A, respectively; similarly, BK viruria in 289 (16.2% and 58 (9.8% specimens from 67 (18.5% and 27 (25.5% patients, being the difference of incidence highly significant (p <0.0001 for both viremia and viruria at comparison between specimens and not significant for patients. No case of PVAN was diagnosed at histophatology evaluation. Conclusions. The incidence of viremia and viruria was similar to that previously reported. Our results evidenced that with low-level tacrolimus-based protocols the overall incidence of reactivation in renal transplant patients is not significantly different and there is no increased risk of PVAN, nevertheless the higher incidence of episodes of reactivation.

  12. Pre-transplant immune factors may be associated with BK polyomavirus reactivation in kidney transplant recipients.

    Science.gov (United States)

    DeWolfe, David; Gandhi, Jinal; Mackenzie, Matthew R; Broge, Thomas A; Bord, Evelyn; Babwah, Amaara; Mandelbrot, Didier A; Pavlakis, Martha; Cardarelli, Francesca; Viscidi, Raphael; Chandraker, Anil; Tan, Chen S

    2017-01-01

    BK polyomavirus (BKPyV) reactivation in kidney transplant recipients can lead to allograft damage and loss. The elements of the adaptive immune system that are permissive of reactivation and responsible for viral control remain incompletely described. We performed a prospective study evaluating BKPyV-specific T-cell response, humoral response and overall T-cell phenotype beginning pre-transplant through one year post-transplant in 28 patients at two centers. We performed an exploratory analysis of risk factors for the development of viremia and viruria as well as compared the immune response to BKPyV in these groups and those who remained BK negative. 6 patients developed viruria and 3 developed viremia. BKPyV-specific CD8+ T-cells increased post-transplant in viremic and viruric but not BK negative patients. BKPyV-specific CD4+ T-cells increased in viremic, but not viruric or BK negative patients. Anti-BKPyV IgG antibodies increased in viruric and viremic patients but remained unchanged in BK negative patients. Viremic patients had a greater proportion of CD8+ effector cells pre-transplant and at 12 months post-transplant. Viremic patients had fewer CD4+ effector memory cells at 3 months post-transplant. Exploratory analysis demonstrated lower CD4 and higher total CD8 proportions, higher anti-BKPyV antibody titers and the cause of renal failure were associated BKPyV reactivation. In conclusion, low CD4, high CD8 and increased effector CD8 cells were found pre-transplant in patients who became viremic, a phenotype associated with immune senescence. This pre-transplant T-cell senescence phenotype could potentially be used to identify patients at increased risk of BKPyV reactivation.

  13. Heterologous expression and purification of an active human TRPV3 ion channel

    DEFF Research Database (Denmark)

    Kol, Stefan; Braun, Christian; Thiel, Gerhard

    2013-01-01

    selected a suitable detergent and buffer system using analytical size‐exclusion chromatography and a thermal stability assay. We demonstrate that the recombinant purified protein contains high α‐helical content and migrates as dimers and tetramers on native PAGE. Furthermore, the purified channel also...

  14. Cx36 makes channels coupling human pancreatic beta-cells, and correlates with insulin expression

    NARCIS (Netherlands)

    Serre-Beinier, Veronique; Bosco, Domenico; Zulianello, Laurence; Charollais, Anne; Caille, Dorothee; Charpantier, Eric; Gauthier, Benoit R.; Diaferia, Giuseppe R.; Giepmans, Ben N.; Lupi, Roberto; Marchetti, Piero; Deng, Shaoping; Buhler, Leo; Berney, Thierry; Cirulli, Vincenzo; Meda, Paolo

    2009-01-01

    Previous studies have documented that the insulin-producing beta-cells of laboratory rodents are coupled by gap junction channels made solely of the connexin36 (Cx36) protein, and have shown that loss of this protein desynchronizes beta-cells, leading to secretory defects reminiscent of those observ

  15. Cx36 makes channels coupling human pancreatic beta-cells, and correlates with insulin expression

    NARCIS (Netherlands)

    Serre-Beinier, Veronique; Bosco, Domenico; Zulianello, Laurence; Charollais, Anne; Caille, Dorothee; Charpantier, Eric; Gauthier, Benoit R.; Diaferia, Giuseppe R.; Giepmans, Ben N.; Lupi, Roberto; Marchetti, Piero; Deng, Shaoping; Buhler, Leo; Berney, Thierry; Cirulli, Vincenzo; Meda, Paolo

    2009-01-01

    Previous studies have documented that the insulin-producing beta-cells of laboratory rodents are coupled by gap junction channels made solely of the connexin36 (Cx36) protein, and have shown that loss of this protein desynchronizes beta-cells, leading to secretory defects reminiscent of those observ

  16. Efficacy of Levofloxacin in the Treatment of BK Viremia: A Multicenter, Double-Blinded, Randomized, Placebo-Controlled Trial

    Science.gov (United States)

    Lee, Belinda T.; Gabardi, Steven; Grafals, Monica; Hofmann, R. Michael; Akalin, Enver; Aljanabi, Aws; Mandelbrot, Didier A.; Adey, Deborah B.; Heher, Eliot; Fan, Pang-Yen; Conte, Sarah; Dyer-Ward, Christine

    2014-01-01

    Background and objectives BK virus reactivation in kidney transplant recipients can lead to progressive allograft injury. Reduction of immunosuppression remains the cornerstone of treatment for active BK infection. Fluoroquinolone antibiotics are known to have in vitro antiviral properties, but the evidence for their use in patients with BK viremia is inconclusive. The objective of the study was to determine the efficacy of levofloxacin in the treatment of BK viremia. Design, setting, participants, & measurements Enrollment in this prospective, multicenter, double-blinded, placebo-controlled trial occurred from July 2009 to March 2012. Thirty-nine kidney transplant recipients with BK viremia were randomly assigned to receive levofloxacin, 500 mg daily, or placebo for 30 days. Immunosuppression in all patients was adjusted on the basis of standard clinical practices at each institution. Plasma BK viral load and serum creatinine were measured monthly for 3 months and at 6 months. Results At the 3-month follow-up, the percentage reductions in BK viral load were 70.3% and 69.1% in the levofloxacin group and the placebo group, respectively (P=0.93). The percentage reductions in BK viral load were also equivalent at 1 month (58% versus and 67.1%; P=0.47) and 6 months (82.1% versus 90.5%; P=0.38). Linear regression analysis of serum creatinine versus time showed no difference in allograft function between the two study groups during the follow-up period. Conclusions A 30-day course of levofloxacin does not significantly improve BK viral load reduction or allograft function when used in addition to overall reduction of immunosuppression. PMID:24482066

  17. Human endogenous retrovirus W family envelope gene activates the small conductance Ca2+-activated K+ channel in human neuroblastoma cells through CREB.

    Science.gov (United States)

    Li, S; Liu, Z C; Yin, S J; Chen, Y T; Yu, H L; Zeng, J; Zhang, Q; Zhu, F

    2013-09-05

    Numerous studies have shown that human endogenous retrovirus W family (HERV-W) envelope gene (env) is related to various diseases but the underlying mechanism has remained poorly understood. Our previous study showed that there was abnormal expression of HERV-W env in sera of patients with schizophrenia. In this paper, we reported that overexpression of the HERV-W env elevated the levels of small conductance Ca(2+)-activated K(+) channel protein 3 (SK3) in human neuroblastoma cells. Using a luciferase reporter system and RNA interference method, we found that functional cAMP response element site was required for the expression of SK3 triggered by HERV-W env. In addition, it was also found that the SK3 channel was activated by HERV-W env. Further study indicated that cAMP response element-binding protein (CREB) was required for the activation of the SK3 channel. Thus, a novel signaling mechanism of how HERV-W env influences neuronal activity and contributes to mental illnesses such as schizophrenia was proposed.

  18. Silencing of Kv4.1 potassium channels inhibits cell proliferation of tumorigenic human mammary epithelial cells

    Energy Technology Data Exchange (ETDEWEB)

    Jang, Soo Hwa [Laboratories of Veterinary Pharmacology, College of Veterinary Medicine, Seoul National University, San 56-1 Sillim-Dong Kwanak-Gu, Seoul 151-742 (Korea, Republic of); Choi, Changsun [Department of Food and Nutrition, College of Human Ecology, Chung-Ang University, Anseong, Gyeonggi (Korea, Republic of); Hong, Seong-Geun; Yarishkin, Oleg V. [Department of Physiology, College of Medicine, Gyeongsang National University, Jinju (Korea, Republic of); Bae, Young Min; Kim, Jae Gon [Department of Physiology, College of Medicine, Konkuk University, Seoul (Korea, Republic of); O' Grady, Scott M. [Department of Physiology, 495 Animal Science/Veterinary Medicine Bldg., St. Paul, University of Minnesota, MN (United States); Yoon, Kyong-Ah [Research Institute and Hospital, National Cancer Center, Goyang, Gyeonggi (Korea, Republic of); Kang, Kyung-Sun [Veterinary Public Health, College of Veterinary Medicine, Seoul National University, San 56-1 Sillim-Dong Kwanak-Gu, Seoul (Korea, Republic of); Ryu, Pan Dong [Laboratories of Veterinary Pharmacology, College of Veterinary Medicine, Seoul National University, San 56-1 Sillim-Dong Kwanak-Gu, Seoul 151-742 (Korea, Republic of); Lee, So Yeong, E-mail: leeso@snu.ac.kr [Laboratories of Veterinary Pharmacology, College of Veterinary Medicine, Seoul National University, San 56-1 Sillim-Dong Kwanak-Gu, Seoul 151-742 (Korea, Republic of)

    2009-06-26

    Potassium channel activity has been shown to facilitate cell proliferation in cancer cells. In the present study, the role of Kv4.1 channels in immortal and tumorigenic human mammary epithelial cells was investigated. Kv4.1 protein expression was positively correlated with tumorigenicity. Moreover, transfection with siRNAs targeting Kv4.1 mRNA suppressed proliferation of tumorigenic mammary epithelial cells. Experiments using mRNA isolated from human breast cancer tissues revealed that the level of Kv4.1 mRNA expression varied depending on the stage of the tumor. Kv4.1 protein expression increased during stages T2 and T3 compared to normal tissue. These results demonstrated that Kv4.1 plays a role in proliferation of tumorigenic human mammary epithelial cells. In addition, elevated Kv4.1 expression may be useful as a diagnostic marker for staging mammary tumors and selective blockers of Kv4.1 may serve to suppress tumor cell proliferation.

  19. Motor disturbances in mice with deficiency of the sodium channel gene Scn8a show features of human dystonia.

    Science.gov (United States)

    Hamann, Melanie; Meisler, Miriam H; Richter, Angelika

    2003-12-01

    The med(J) mouse with twisting movements related to deficiency of the sodium channel Scn8a has been proposed as a model of kinesiogenic dystonia. This prompted us to examine the phenotype of these mice in more detail. By cortical electroencephalographic (EEG) recordings, we could not detect any changes, demonstrating that the motor disturbances are not epileptic in nature, an important similarity to human dystonia. The significantly decreased body weight of med(J) mice was related to reduced food intake. Observations in the open field and by video recordings revealed that the mice exhibit sustained abnormal postures and movements of limbs, trunk and tail not only during locomotor activity but also at rest. With the exception of the head tremor, the other motor impairments were persistent rather than paroxysmal. When several neurological reflexes were tested, alterations were restricted to the posture and righting reflexes. Results of the wire hang test confirmed the greatly reduced muscle strength in the med(J) mouse. In agreement with different types of human dystonia, biperiden, haloperidol and diazepam moderately reduced the severity of motor disturbances in med(J) mice. In view of the sodium channel deficiency in med(J) mice, the beneficial effects of the sodium channel blocker phenytoin was an unexpected finding. By immunohistochemical examinations, the density of nigral dopaminergic neurons was found to be unaltered, substantiating the absence of pathomorphological abnormalities within the brain of med(J) mice shown by previous studies. With the exception of muscle weakness, many of the features of the med(J) mouse are similar to human idiopathic dystonia.

  20. Positions of the cytoplasmic end of BK α S0 helix relative to S1-S6 and of β1 TM1 and TM2 relative to S0-S6.

    Science.gov (United States)

    Liu, Guoxia; Zakharov, Sergey I; Yao, Yongneng; Marx, Steven O; Karlin, Arthur

    2015-03-01

    The large-conductance, voltage- and Ca(2+)-gated K(+) (BK) channel consists of four α subunits, which form a voltage- and Ca(2+)-gated channel, and up to four modulatory β subunits. The β1 subunit is expressed in smooth muscle, where it slows BK channel kinetics and shifts the conductance-voltage (G-V) curve to the left at [Ca(2+)] > 2 µM. In addition to the six transmembrane (TM) helices, S1-S6, conserved in all voltage-dependent K(+) channels, BK α has a unique seventh TM helix, S0, which may contribute to the unusual rightward shift in the G-V curve of BK α in the absence of β1 and to a leftward shift in its presence. Such a role is supported by the close proximity of S0 to S3 and S4 in the voltage-sensing domain. Furthermore, on the extracellular side of the membrane, one of the two TM helices of β1, TM2, is adjacent to S0. We have now analyzed induced disulfide bond formation between substituted Cys residues on the cytoplasmic side of the membrane. There, in contrast, S0 is closest to the S2-S3 loop, from which position it is displaced on the addition of β1. The cytoplasmic ends of β1 TM1 and TM2 are adjacent and are located between the S2-S3 loop of one α subunit and S1 of a neighboring α subunit and are not adjacent to S0; i.e., S0 and TM2 have different trajectories through the membrane. In the absence of β1, 70% of disulfide bonding of W43C (S0) and L175C (S2-S3) has no effect on V50 for activation, implying that the cytoplasmic end of S0 and the S2-S3 loop move in concert, if at all, during activation. Otherwise, linking them together in one state would obstruct the transition to the other state, which would certainly change V50.

  1. The mystery is solved-CatSper is the principal calcium channel activated by progesterone in human spermatozoa

    Institute of Scientific and Technical Information of China (English)

    Christopher LR Barratt

    2011-01-01

    @@ Aremarkable advance in sperm physiology has recently been published in Nature.Two groups using patch clamping techniques on human sperm have solved a mystery about the sperm cell that has puzzled both andrologists and those involved in non-genomic cellular signalling for over 20 years.In these papers, Lishko1 and Strunker2 independently demonstrate that the universal characteristic effect of progesterone on sperm-a rapid influx of calcium-is via a sperm-specific channel CatSper.

  2. Mutations in sodium-channel gene SCN9A cause a spectrum of human genetic pain disorders

    OpenAIRE

    Drenth, J.P.H.; Waxman, S G

    2007-01-01

    The voltage-gated sodium-channel type IX alpha subunit, known as Na(v)1.7 and encoded by the gene SCN9A, is located in peripheral neurons and plays an important role in action potential production in these cells. Recent genetic studies have identified Na(v)1.7 dysfunction in three different human pain disorders. Gain-of-function missense mutations in Na(v)1.7 have been shown to cause primary erythermalgia and paroxysmal extreme pain disorder, while nonsense mutations in Na(v)1.7 result in los...

  3. FKBP52 is involved in the regulation of SOCE channels in the human platelets and MEG 01 cells.

    Science.gov (United States)

    López, Esther; Berna-Erro, Alejandro; Salido, Ginés M; Rosado, Juan A; Redondo, Pedro C

    2013-03-01

    Immunophilins are FK506-binding proteins that have been involved in the regulation of calcium homeostasis, either by modulating Ca(2+) channels located in the plasma membrane or in the rough endoplasmic reticulum (RE). We have investigated whether immunophilins would participate in the regulation of stored-operated Ca(2+) entry (SOCE) in human platelets and MEG 01. Both cell types were loaded with fura-2 for determining cytosolic calcium concentration changes ([Ca(2+)](c)), or stimulated and fixed to evaluate the protein interaction profile by performing immunoprecipitation and western blotting. We have found that incubation of platelets with FK506 increases Ca(2+) mobilization. Thapsigargin (TG)-evoked, Thr-evoked SOCE and TG-evoked Mn(2+) entry resulted in significant reduction by treatment of platelets with immunophilin antagonists. We confirmed by immunoprecipitation that immunophilins interact with transient receptor potential channel 1 (TRPC1) and Orai1 in human platelets. FK506 and rapamycin reduced the association between TRPC1 and Orai1 with FK506 binding protein (52) (FKBP52) in human platelets, and between TRPC1 and the type II IP(3)R, which association is known to be crucial for the maintenance of SOCE in human platelets. FKBP52 role in SOCE activation was confirmed by silencing FKBP52 using SiRNA FKBP52 in MEG 01 as demonstrated by single cell configuration imaging technique. TRPC1 silencing and depletion of cell of TRPC1 and FKBP52 simultaneously, impair activation of SOCE evoked by TG in MEG 01. Finally, in MEG 01 incubated with FK506 we observed a reduction in TRPC1/FKBP52 coupling, and similarly, FKBP52 silencing reduced the association between IP3R type II and TRPC1 during SOCE. All together, these results demonstrate that immunophilins participate in the regulation of SOCE in human platelets.

  4. Structural analysis of the S4-S5 linker of the human KCNQ1 potassium channel.

    Science.gov (United States)

    Gayen, Shovanlal; Li, Qingxin; Kang, CongBao

    2015-01-02

    KCNQ1 plays important roles in the cardiac action potential and consists of an N-terminal domain, a voltage-sensor domain, a pore domain and a C-terminal domain. KCNQ1 is a voltage-gated potassium channel and its channel activity is regulated by membrane potentials. The linker between transmembrane helices 4 and 5 (S4-S5 linker) is important for transferring the conformational changes from the voltage-sensor domain to the pore domain. In this study, the structure of the S4-S5 linker of KCNQ1 was investigated by solution NMR, circular dichroism and fluorescence spectroscopic studies. The S4-S5 linker adopted a helical structure in detergent micelles. The W248 may interact with the cell membrane.

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

    DEFF Research Database (Denmark)

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

    2012-01-01

    cycle, including its direction, is reproduced by a model with 2×2 discrete states: the normal open/closed states and two different states of "gate tension". Rates of transitions between the two branches of the hysteresis curve are modeled with single-barrier kinetics by introducing a real......-valued "reaction coordinate" parametrizing the protein's conformational change between the two states of gate tension. The resulting scenario suggests a reanalysis of former experiments with NSVDC channels....

  6. Microarray-Based Comparisons of Ion Channel Expression Patterns: Human Keratinocytes to Reprogrammed hiPSCs to Differentiated Neuronal and Cardiac Progeny

    Directory of Open Access Journals (Sweden)

    Leonhard Linta

    2013-01-01

    Full Text Available Ion channels are involved in a large variety of cellular processes including stem cell differentiation. Numerous families of ion channels are present in the organism which can be distinguished by means of, for example, ion selectivity, gating mechanism, composition, or cell biological function. To characterize the distinct expression of this group of ion channels we have compared the mRNA expression levels of ion channel genes between human keratinocyte-derived induced pluripotent stem cells (hiPSCs and their somatic cell source, keratinocytes from plucked human hair. This comparison revealed that 26% of the analyzed probes showed an upregulation of ion channels in hiPSCs while just 6% were downregulated. Additionally, iPSCs express a much higher number of ion channels compared to keratinocytes. Further, to narrow down specificity of ion channel expression in iPS cells we compared their expression patterns with differentiated progeny, namely, neurons and cardiomyocytes derived from iPS cells. To conclude, hiPSCs exhibit a very considerable and diverse ion channel expression pattern. Their detailed analysis could give an insight into their contribution to many cellular processes and even disease mechanisms.

  7. Neuropatía por virus BK post trasplante renal diagnostico y seguimiento por PCR en tiempo real BK virus nephropathy after renal transplantation: Diagnosis and prognosis by real time PCR

    Directory of Open Access Journals (Sweden)

    Marcela Echavarria

    2007-12-01

    Full Text Available La nefropatía producida por el virus BK puede llevar a la pérdida del trasplante renal. El diagnóstico etiológico es importante debido a que la clínica no permite diferenciar entre nefropatía por virus BK y rechazo agudo, en donde los tratamientos de estas dos entidades son diametralmente opuestos. El desarrollo reciente de métodos moleculares muy sensibles y específicos como PCR y PCR en tiempo real para virus BK permiten un diagnóstico de certeza en forma rápida y cuantificar la carga viral presente. El diagnóstico de nefropatía por virus BK se realiza por inmunohistoquímica en una biopsia renal, pero dada la naturaleza multifocal de las lesiones, la sensibilidad no siempre es del 100%. Los nuevos métodos de PCR para detectar virus BK en sangre y orina contribuyen al diagnóstico de nefropatía de una manera más normatizada y menos invasiva. Más aún, la cuantificación del virus BK en sangre por PCR en tiempo real, ha demostrado ser útil en el diagnóstico y monitoreo de esta enfermedad. En este trabajo se presenta el caso de una paciente transplantada renal con nefropatía por virus BK y el desarrollo de un método de PCR en tiempo real para la detección de virus BK en sangre y orina. Esta nueva metodología confirmó el diagnóstico de nefropatía por virus BK lo que permitió un cambio en el esquema de inmunosupresión y la instauración de un tratamiento que pudo ser monitorizado utilizando la carga viral.BK virus nephropathy may lead to kidney transplant failure. BK infection and acute rejection are clinically undistinguishable, therefore diagnosis of these entities is critical to establish the correct treatment. The new molecular methods using PCR and real time PCR have significantly contributed to the rapid and sensitive diagnosis of BK virus. Furthermore, viral load determination in plasma has significantly been associated with BK virus nephropathy. Definite diagnosis of nephropathy requires renal biopsy, although

  8. Latest Holocene evolution and human disturbance of a channel segment in the Hudson River Estuary

    Science.gov (United States)

    Klingbeil, A.D.; Sommerfield, C.K.

    2005-01-01

    The latest Holocene sedimentary record of a cohesive channel and subtidal shoal in the lower Hudson River Estuary was examined to elucidate natural (sea-level rise, sediment transport) and anthropogenic (bulkheading, dredging) influences on the recent morphodynamic evolution of the system. To characterize the seafloor and shallow subbottom, ??? 100 km of high-resolution seismic reflection profiles (chirp) were collected within a 20-km reach of the estuary and correlated with sediment lithologies provided by eight vibracores recovered along seismic lines. Sediment geochronology with 137Cs and 14C was used to estimate intermediate and long-term sedimentation rates, respectively, and historical bathymetric data were analyzed to identify regional patterns of accretion and erosion, and to quantify changes in channel geometry and sediment volume. The shoal lithosome originated around 4 ka presumably with decelerating eustatic sea level rise during the latest Holocene. Long-term sedimentation rates on the shoal (2.3-2.6 mm/yr) are higher than in the channel (2 mm/yr) owing to hydrodynamic conditions that preferentially sequester suspended sediment on the western side of the estuary. As a result, the shoal accretes oblique to the principal axis of tidal transport, and more rapidly than the channel to produce an asymmetric cross-section. Shoal deposits consist of tidally bedded muds and are stratified by minor erosion surfaces that seismic profiles reveal to extend for 10s of meters to kilometers. The frequency and continuity of these surfaces suggest that the surficial shoal is catastrophically stripped on decadal-centennial time scales by elevated tidal flows; tidal erosion maintains the shoal at a uniform depth below sea level and prevents it from transitioning to an intertidal environment. Consequently, the long-term sedimentation rate approximates the rate of sea-level rise in the lower estuary (1-3 mm/yr). After the mid 1800s, the natural geometry of the lower Hudson

  9. The large conductance calcium-activated K(+) channel interacts with the small GTPase Rab11b.

    Science.gov (United States)

    Sokolowski, Sophia; Harvey, Margaret; Sakai, Yoshihisa; Jordan, Amy; Sokolowski, Bernd

    2012-09-21

    The transduction of sound by the receptor or hair cells of the cochlea leads to the activation of ion channels found in the basal and lateral regions of these cells. Thus, the processing of these transduced signals to the central nervous system is tied to the regulation of baso-lateral ion channels. The large conductance calcium-activated potassium or BK channel was revealed to interact with the small GTPase, Rab11b, which is one of many Rabs found in various endosomal pathways. Immunoelectron microscopy showed the colocalization of these two proteins in receptor cells and auditory neurons. Using Chinese hamster ovary cells as a heterologous expression system, Rab11b increased or decreased BK expression, depending on the overexpression or RNAi knockdown of Rab, respectively. Additional mutation analyses, using a yeast two-hybrid assay, suggested that this GTPase moderately interacts within a region of BK exclusive of the N- or C-terminal tails. These data suggest that this small GTPase regulates BK in a slow recycling process through the endocytic compartment and to the plasmalemma.

  10. Mutations in sodium-channel gene SCN9A cause a spectrum of human genetic pain disorders.

    Science.gov (United States)

    Drenth, Joost P H; Waxman, Stephen G

    2007-12-01

    The voltage-gated sodium-channel type IX alpha subunit, known as Na(v)1.7 and encoded by the gene SCN9A, is located in peripheral neurons and plays an important role in action potential production in these cells. Recent genetic studies have identified Na(v)1.7 dysfunction in three different human pain disorders. Gain-of-function missense mutations in Na(v)1.7 have been shown to cause primary erythermalgia and paroxysmal extreme pain disorder, while nonsense mutations in Na(v)1.7 result in loss of Na(v)1.7 function and a condition known as channelopathy-associated insensitivity to pain, a rare disorder in which affected individuals are unable to feel physical pain. This review highlights these recent developments and discusses the critical role of Na(v)1.7 in pain sensation in humans.

  11. Isospin dependence of reactions $^{48}$Ca+$^{243-251}$Bk

    CERN Document Server

    Shen, Caiwan; Boilley, Davoid; Kosenko, Grigory; Zhao, Enguang

    2008-01-01

    The fusion process of $^{48}$Ca induced reactions is studied with the two-step model. In this model, the fusion process is devided into two stages: first, the sticking stage where projectile and target come to the touching point over the Coulomb barrier from infinite distance, and second, the formation stage where the di-nucleus formed with projectile and target evolve to form the spherical compound nucleus from the touching point. By the use of the statistical evaporation model, the residue cross sections for different neutron evaporation channels are analyzed. From the results, optimum reactions are given to synthesize $Z$ = 117 element with $^{48}$Ca induced reactions.

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

    Directory of Open Access Journals (Sweden)

    Yiangou Yiangos

    2010-06-01

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

  13. Assignment of human G-protein-coupled inward rectifier K{sup +} channel homolog GIRK3 gene to chromosome 1q21-q23

    Energy Technology Data Exchange (ETDEWEB)

    Lesage, F.; Fink, M.; Barhanin, J. [CNRS, Valbonne (France)] [and others

    1995-10-10

    More than 20 genes that encode voltage-gated and Ca{sup 2+}-dependent K{sup +} channels have been identified. These channels are involved in a wide variety of biological functions such as neuronal and muscle excitability, hormone secretion, and osmotic regulation. Two voltage-gated K{sup +} channel genes, KCNA1 and HERG, have been related to neurological and cardiac inherited disorders in humans. Missense mutations in the KCNA1 gene lead to episodic ataxia/myokimia syndrome. Missense, splice donor, and deletion mutations in the HERG gene have been shown to cause long QT syndrome. These two channels belong to the superfamily of cationic channels, which share the characteristic structural features of six transmembrane domains and one segment (called 115) involved in pore formation. 17 refs., 1 fig.

  14. Difference of Sodium Currents between Pediatric and Adult Human Atrial Myocytes: Evidence for Developmental Changes of Sodium Channels

    Directory of Open Access Journals (Sweden)

    Benzhi Cai, Xiaoqin Mu, Dongmei Gong, Shulin Jiang, Jianping Li, Qingxin Meng, Yunlong Bai, Yanju Liu, Xinyue Wang, Xueying Tan, Baofeng Yang, Yanjie Lu

    2011-01-01

    Full Text Available Voltage-gated calcium currents and potassium currents were shown to undergo developmental changes in postnatal human and animal cardiomocytes. However, so far, there is no evidence whether sodium currents also presented the developmental changes in postnatal human atrial cells. The aim of this study was to observe age-related changes of sodium currents between pediatric and adult atrial myocytes. Human atrial myocytes were acutely isolated and the whole-cell patch clamp technique was used to record sodium currents isolated from pediatric and adult atrial cardiomocytes. The peak amplitude of sodium currents recorded in adult atrial cells was significantly larger than that in pediatric atrial myocytes. However, there was no significant difference of the activation voltage for peak sodium currents between two kinds of atrial myocytes. The time constants for the activation and inactivation of sodium currents were smaller in adult atria than pediatric atria. The further study revealed that the voltage-dependent inactivation of sodium currents were more slow in adult atrial cardiomyocytes than pediatric atrial cells. A significant difference was also observed in the recovery process of sodium channel from inactivation. In summary, a few significant differences were demonstrated in sodium currents characteristics between pediatric and adult atrial myocytes, which indicates that sodium currents in human atria also undergo developmental changes.

  15. types sat 1 and sat 2 in bhk, bk, vero and lk cell

    African Journals Online (AJOL)

    BSN

    highest virus titres (6.85 log10 TCID 50/ml) in BK cells follo\\\\ed ti: BHK eel' (5 6lo; TUD,. ml) while the lowest titres v.ere obtained ... controlled by slaughter and immunization with chemically inactivated whole virus vaccine (Eric ... Viruses: 1\\ total or 14 F~~[) ,·1rus isolates \\\\l'l"L' used. ..... R. W. ( 1984) Inactivation, Purification.

  16. 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.

  17. Identification and functional characterization of cereblon as a binding protein for large-conductance calcium-activated potassium channel in rat brain.

    Science.gov (United States)

    Jo, Sooyeon; Lee, Kwang-Hee; Song, Sungmin; Jung, Yong-Keun; Park, Chul-Seung

    2005-09-01

    Large-conductance Ca2+-activated K+ (BK(Ca)) channels are activated by membrane depolarization and modulated by intracellular Ca2+. Here, we report the direct interaction of cereblon (CRBN) with the cytosolic carboxy-terminus of the BK(Ca) channel alpha subunit (Slo). Rat CRBN contained the N-terminal domain of the Lon protease, a 'regulators of G protein-signaling' (RGS)-like domain, a leucine zipper (LZ) motif, and four putative protein kinase C (PKC) phosphorylation sites. RNA messages of rat cereblon (rCRBN) were widely distributed in different tissues with especially high-levels of expression in the brain. Direct association of rCRBN with the BK(Ca) channel was confirmed by immunoprecipitation in brain lysate, and the two proteins were co-localized in cultured rat hippocampal neurons. Ionic currents evoked by the rSlo channel were dramatically suppressed upon coexpression of rCRBN. rCRBN decreased the formation of the tetrameric rSlo complex thus reducing the surface expression of functional channels. Therefore, we suggest that CRBN may play an important role in assembly and surface expression of functional BK(Ca) channels by direct interaction with the cytosolic C-terminus of its alpha-subunit.

  18. Differential regulation of human Eag1 channel expression by serum and epidermal growth factor in lung and breast cancer cells

    Directory of Open Access Journals (Sweden)

    Acuña-Macías I

    2015-10-01

    Full Text Available Isabel Acuña-Macías,1 Eunice Vera,1 Alma Yolanda Vázquez-Sánchez,1 María Eugenia Mendoza-Garrido,2 Javier Camacho1 1Department of Pharmacology, 2Department of Physiology, Biophysics and Neurosciences, Centro de Investigación y de Estudios Avanzados del Instituto Politécnico Nacional, Mexico City, Mexico Abstract: Oncogenic ether à-go-go-1 (Eag1 potassium channels are overexpressed in most primary human solid tumors. Low oxygen and nutrient/growth factor concentrations play critical roles in tumorigenesis. However, the mechanisms by which tumor cells survive and proliferate under growth factor-depleted conditions remain elusive. Here, we investigated whether serum-deprived conditions and epidermal growth factor (EGF regulate Eag1 expression in human lung and breast cancer cells. The human cancer cell lines A549 and MCF-7 (from the lungs and breast, respectively were obtained from the American Type Culture Collection and cultured following the manufacturer’s recommendations. Eag1 gene and protein expression were studied by real-time PCR and immunocytochemistry, respectively. Cell proliferation was evaluated using the 3-(4,5-dimethylthiazol-2-yl-2,5-diphenyltetrazolium bromide assay, and ERK1/2 phosphorylation was investigated by Western blot. Serum-deprived conditions increased Eag1 mRNA and protein expression in both cell lines. This Eag1 upregulation was prevented by EGF and the ERK1/2 inhibitor U0126 in only lung cancer cells; vascular endothelial growth factor did not prevent Eag1 upregulation. Our results suggest that Eag1 may act as a survival and mitogenic factor under low-serum and nutrient conditions and may be a clinical target during the early stages of tumor development. Keywords: lung cancer, serum deprivation, ether à-go-go, potassium channels, EGF, epidermal growth factor, ERK 1/2

  19. Comparison of Channelized Hotelling and Human Observers inDetermining Optimum OS-EM Reconstruction Parameters for MyocardialSPECT

    Energy Technology Data Exchange (ETDEWEB)

    Gilland, Karen L.; Tsui, Benjamin M.W.; Qi, Yujin; Gullberg,Grant T.

    2005-07-01

    The performance of the Channelized Hotelling Observer (CHO)was compared to that of human observers for determining optimumparameters for the iterative OS-EM image reconstruction method for thetask of defect detection in myocardial SPECT images. The optimumparameters were those that maximized defect detectability in the SPECTimages. Low noise, parallel SPECT projection data, with and without ananterior, inferior or lateral LV wall defect, were simulated using theMonte Carlo method. Poisson noise was added to generate noisyrealizations. Data were reconstructed using OS-EM at 1&4subsets/iteration and at 1, 3, 5, 7&9 iterations. Images wereconverted to 2D short-axis slices with integer pixel values. The CHO used3 radially-symmetric, 2D channels, with varying levels of internalobserver noise. For each parameter setting, 600 defect-present and 600defect-absent image vectors were used to calculate the detectabilityindex (dA). The human observers rated the likelihood that a defect waspresent in a specified location. For each parameter setting, the AUC wasestimated from 48 defect-present and 48 defect-absent images. Thecombined human observer results showed the optimum parameter settingcould be in the range 5-36 updates ([number of subsets]/iteration enumber of iterations). The CHO results showed the optimum parametersetting to be 4-5 updates. The performance of the CHO was much moresensitive to the reconstruction parameter setting than was that of thehuman observers. The rankings of the CHO detectability values did notchange with varying levels of internal noise.

  20. 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.

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

    Science.gov (United States)

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

    2013-01-01

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

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

    Directory of Open Access Journals (Sweden)

    Leopoldo Raul Beltran

    2013-11-01

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

  3. A whole-genome RNAi screen uncovers a novel role for human potassium channels in cell killing by the parasite Entamoeba histolytica.

    Science.gov (United States)

    Marie, Chelsea; Verkerke, Hans P; Theodorescu, Dan; Petri, William A

    2015-09-08

    The parasite Entamoeba histolytica kills human cells resulting in ulceration, inflammation and invasion of the colonic epithelium. We used the cytotoxic properties of ameba to select a genome-wide RNAi library to reveal novel host factors that control susceptibility to amebic killing. We identified 281 candidate susceptibility genes and bioinformatics analyses revealed that ion transporters were significantly enriched among susceptibility genes. Potassium (K(+)) channels were the most common transporter identified. Their importance was further supported by colon biopsy of humans with amebiasis that demonstrated suppressed K(+) channel expression. Inhibition of human K(+) channels by genetic silencing, pharmacologic inhibitors and with excess K(+) protected diverse cell types from E. histolytica-induced death. Contact with E. histolytica parasites triggered K(+) channel activation and K(+) efflux by intestinal epithelial cells, which preceded cell killing. Specific inhibition of Ca(2+)-dependent K(+) channels was highly effective in preventing amebic cytotoxicity in intestinal epithelial cells and macrophages. Blockade of K(+) efflux also inhibited caspase-1 activation, IL-1β secretion and pyroptotic death in THP-1 macrophages. We concluded that K(+) channels are host mediators of amebic cytotoxicity in multiple cells types and of inflammasome activation in macrophages.

  4. BK virus as a potential oncovirus for bladder cancer in a renal transplant patient.

    Science.gov (United States)

    Yin, Wen-Yao; Lee, Ming-Che; Lai, Ning-Sheng; Lu, Ming-Chi

    2015-04-01

    Renal transplant patients have high risk for bladder cancer. The reactivation of BK virus is common in renal transplant patients especially in the urinary tract. There was some evidence suggesting that the reactivation of BK virus (BKV) in renal transplant patients may associate with the development of bladder cancer. Here we demonstrated that a patient that had persistent elevated BKV viruria (urine BKV DNA concentration more than 10(11) copies/ml) after renal transplantation. Then, bladder cancer was found in 13 months after kidney transplantation. The urine BKV DNA concentration was detected by real-time PCR and the BKV DNA in the bladder tumor was detected by PCR. BKV DNA was found in the marginal and central part of the bladder tumor. After removal of the bladder cancer, the urine BKV viral load in this patients dropped dramatically to <10(2) copies/ml. However, the urine viral load had increased modestly to 10(6) copies/ml in 3 months after surgery. Since there is a close correlation between the urine BK viral load and the presence of bladder cancer, we suggested that there might be a causal relationship between the reactivation of BKV and the development of bladder cancer in renal transplant patient.

  5. Caracterização da virulência da cepa de Escherichia coli - BK99

    Directory of Open Access Journals (Sweden)

    Brito Benito Guimarães de

    2001-01-01

    Full Text Available Com o objetivo de identificar a patogenicidade e resistência a antimicrobianos da cepa de E. coli BK99, foram utilizados alguns testes: aglutinação em lâmina para detecção da fímbria F5, produção de STa, ensaios para hemolisinas e colicinas, patogenicidade em leitões e antibiograma. A cepa BK99 apresentou o seguinte perfil: F1+, F5+, STa+, Col V+, Hly-, ST R, KA R, NO R, TT R SF R e foi capaz de provocar a doença clínica e morte em leitões inoculados; também foi possível o resgate dessa cepa de fezes diarréicas e do conteúdo intestinal dos leitões revelando, assim, alto índice de recuperação de colônias portadoras da fímbria F5+. Os resultados permitem concluir que a cepa de E. coli BK99 é produtora de fatores de virulência e reproduz experimentalmente a colibacilose suína neonatal.

  6. A study of the low-luminosity Type II-Plateau supernova 2008bk

    CERN Document Server

    Lisakov, Sergey; Hillier, D John; Waldman, Roni; Livne, Eli

    2016-01-01

    Supernova (SN) 2008bk is a well observed low-luminosity Type II event visually associated with a low-mass red-supergiant progenitor. To model SN 2008bk, we evolve a 12Msun star from the main sequence until core collapse, when it has a total mass of 9.88Msun, a He-core mass of 3.22Msun, and a radius of 502Rsun. We then artificially trigger an explosion that produces 8.29Msun of ejecta with a total energy of 2.5x10^50erg and ~0.009Msun of 56Ni. We model the subsequent evolution of the ejecta with non-Local-Thermodynamic-Equilibrium time-dependent radiative transfer. Although somewhat too luminous and energetic, this model reproduces satisfactorily the multi-band light curves and multi-epoch spectra of SN 2008bk, confirming the suitability of a low-mass massive star progenitor. As in other low-luminosity SNe II, the structured Halpha profile at the end of the plateau phase is probably caused by BaII 6496.9A rather than asphericity. We discuss the sensitivity of our results to changes in progenitor radius and mas...

  7. Enhanced large conductance K+ channel activity contributes to the impaired myogenic response in the cerebral vasculature of Fawn Hooded Hypertensive rats

    Science.gov (United States)

    Pabbidi, Mallikarjuna R.; Mazur, Olga; Fan, Fan; Farley, Jerry M.; Gebremedhin, Debebe; Harder, David R.

    2014-01-01

    Recent studies have indicated that the myogenic response (MR) in cerebral arteries is impaired in Fawn Hooded Hypertensive (FHH) rats and that transfer of a 2.4 megabase pair region of chromosome 1 (RNO1) containing 15 genes from the Brown Norway rat into the FHH genetic background restores MR in a FHH.1BN congenic strain. However, the mechanisms involved remain to be determined. The present study examined the role of the large conductance calcium-activated potassium (BK) channel in impairing the MR in FHH rats. Whole-cell patch-clamp studies of cerebral vascular smooth muscle cells (VSMCs) revealed that iberiotoxin (IBTX; BK inhibitor)-sensitive outward potassium (K+) channel current densities are four- to fivefold greater in FHH than in FHH.1BN congenic strain. Inside-out patches indicated that the BK channel open probability (NPo) is 10-fold higher and IBTX reduced NPo to a greater extent in VSMCs isolated from FHH than in FHH.1BN rats. Voltage sensitivity of the BK channel is enhanced in FHH as compared with FHH.1BN rats. The frequency and amplitude of spontaneous transient outward currents are significantly greater in VSMCs isolated from FHH than in FHH.1BN rats. However, the expression of the BK-α and -β-subunit proteins in cerebral vessels as determined by Western blot is similar between the two groups. Middle cerebral arteries (MCAs) isolated from FHH rats exhibited an impaired MR, and administration of IBTX restored this response. These results indicate that there is a gene on RNO1 that impairs MR in the MCAs of FHH rats by enhancing BK channel activity. PMID:24464756

  8. 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.

  9. Biophysical assessment of human aquaporin-7 as a water and glycerol channel in 3T3-L1 adipocytes.

    Directory of Open Access Journals (Sweden)

    Ana Madeira

    Full Text Available The plasma membrane aquaporin-7 (AQP7 has been shown to be expressed in adipose tissue and its role in glycerol release/uptake in adipocytes has been postulated and correlated with obesity onset. However, some studies have contradicted this view. Based on this situation, we have re-assessed the precise localization of AQP7 in adipose tissue and analyzed its function as a water and/or glycerol channel in adipose cells. Fractionation of mice adipose tissue revealed that AQP7 is located in both adipose and stromal vascular fractions. Moreover, AQP7 was the only aquaglyceroporin expressed in adipose tissue and in 3T3-L1 adipocytes. By overexpressing the human AQP7 in 3T3-L1 adipocytes it was possible to ascertain its role as a water and glycerol channel in a gain-of-function scenario. AQP7 expression had no effect in equilibrium cell volume but AQP7 loss of function correlated with higher triglyceride content. Furthermore it is also reported for the first time a negative correlation between water permeability and the cell non-osmotic volume supporting the observation that AQP7 depleted cells are more prone to lipid accumulation. Additionally, the strong positive correlation between the rates of water and glycerol transport highlights the role of AQP7 as both a water and a glycerol channel and reflects its expression levels in cells. In all, our results clearly document a direct involvement of AQP7 in water and glycerol transport, as well as in triglyceride content in adipocytes.

  10. Implications of Human Transient Receptor Potential Melastatin 8 (TRPM8) Channel Gating from Menthol Binding Studies of the Sensing Domain.

    Science.gov (United States)

    Rath, Parthasarathi; Hilton, Jacob K; Sisco, Nicholas J; Van Horn, Wade D

    2016-01-12

    The transient receptor potential melastatin 8 (TRPM8) ion channel is the primary cold sensor in humans. TRPM8 is gated by physiologically relevant cold temperatures and chemical ligands that induce cold sensations, such as the analgesic compound menthol. Characterization of TRPM8 ligand-gated channel activation will lead to a better understanding of the fundamental mechanisms that underlie TRPM8 function. Here, the direct binding of menthol to the isolated hTRPM8 sensing domain (transmembrane helices S1-S4) is investigated. These data are compared with two mutant sensing domain proteins, Y745H (S2 helix) and R842H (S4 helix), which have been previously identified in full length TRPM8 to be menthol insensitive. The data presented herein show that menthol specifically binds to the wild type, Y745H, and R842H TRPM8 sensing domain proteins. These results are the first to show that menthol directly binds to the TRPM8 sensing domain and indicates that Y745 and R842 residues, previously identified in functional studies as crucial to menthol sensitivity, do not affect menthol binding but instead alter coupling between the sensing domain and the pore domain.

  11. Inhibition of the Human Ether-a-go-go-related Gene (HERG) K+ Channels by Lindera erythrocarpa

    Science.gov (United States)

    Hong, Hee-Kyung; Yoon, Weon-Jong; Kim, Young Ho; Yoo, Eun-Sook

    2009-01-01

    Lindera erythrocarpa Makino (Lauraceae) is used as a traditional medicine for analgesic, antidote, and antibacterial purposes and shows anti-tumor activity. We studied the effects of Lindera erythrocarpa on the human ether-a-go-go-related gene (HERG) channel, which appears of importance in favoring cancer progression in vivo and determining cardiac action potential duration. Application of MeOH extract of Lindera erythrocarpa showed a dose-dependent decrease in the amplitudes of the outward currents measured at the end of the pulse (IHERG) and the tail currents of HERG (Itail). When the BuOH fraction and H2O fraction of Lindera erythrocarpa were added to the perfusate, both IHERG and Itail were suppressed, while the hexane fraction, CHCl3 fraction, and EtOAc fraction did not inhibit either IHERG or Itail. The potential required for half-maximal activation caused by EtOAc fraction, BuOH fraction, and H2O fraction shifted significantly. The BuOH fraction and H2O fraction (100 µg/mL) decreased gmax by 59.6% and 52.9%, respectively. The H2O fraction- and BuOH fraction-induced blockades of Itail progressively decreased with increasing depolarization, showing the voltage-dependent block. Our findings suggest that Lindera erythrocarpa, a traditional medicine, blocks HERG channel, which could contribute to its anticancer and cardiac arrhythmogenic effect. PMID:19949665

  12. N-terminal tetrapeptide T/SPLH motifs contribute to multimodal activation of human TRPA1 channel

    Science.gov (United States)

    Hynkova, Anna; Marsakova, Lenka; Vaskova, Jana; Vlachova, Viktorie

    2016-06-01

    Human transient receptor potential ankyrin channel 1 (TRPA1) is a polymodal sensor implicated in pain, inflammation and itching. An important locus for TRPA1 regulation is the cytoplasmic N-terminal domain, through which various exogenous electrophilic compounds such as allyl-isothiocyanate from mustard oil or cinnamaldehyde from cinnamon activate primary afferent nociceptors. This major region is comprised of a tandem set of 17 ankyrin repeats (AR1-AR17), five of them contain a strictly conserved T/SPLH tetrapeptide motif, a hallmark of an important and evolutionarily conserved contribution to conformational stability. Here, we characterize the functional consequences of putatively stabilizing and destabilizing mutations in these important structural units and identify AR2, AR6, and AR11-13 to be distinctly involved in the allosteric activation of TRPA1 by chemical irritants, cytoplasmic calcium, and membrane voltage. Considering the potential involvement of the T/SP motifs as putative phosphorylation sites, we also show that proline-directed Ser/Thr kinase CDK5 modulates the activity of TRPA1, and that T673 outside the AR-domain is its only possible target. Our data suggest that the most strictly conserved N-terminal ARs define the energetics of the TRPA1 channel gate and contribute to chemical-, calcium- and voltage-dependence.

  13. Potent suppression of Kv1.3 potassium channel and IL-2 secretion by diphenyl phosphine oxide-1 in human T cells.

    Directory of Open Access Journals (Sweden)

    Ning Zhao

    Full Text Available Diphenyl phosphine oxide-1 (DPO-1 is a potent Kv1.5 channel inhibitor that has therapeutic potential for the treatment of atrial fibrillation. Many other Kv1.5 channel blockers also potently inhibit the Kv1.3 channel, but whether DPO-1 blocks Kv1.3 channels has not been investigated. The Kv1.3 channel is highly expressed in activated T cells, which is considered a favorable target for immunomodulation. Accordingly, we hypothesized that DPO-1 may exert immunosuppressive and anti-inflammatory effects by inhibiting Kv1.3 channel activity. In this study, DPO-1 blocked Kv1.3 current in a voltage-dependent and concentration-dependent manner, with IC₅₀ values of 2.58 µM in Jurkat cells and 3.11 µM in human peripheral blood T cells. DPO-1 also accelerated the inactivation rate and negatively shifted steady-state inactivation. Moreover, DPO-1 at 3 µM had no apparent effect on the Ca²⁺ activated potassium channel (K(Ca current in both Jurkat cells and human peripheral blood T cells. In Jurkat cells, pre-treatment with DPO-1 for 24 h decreased Kv1.3 current density, and protein expression by 48±6% and 60±9%, at 3 and 10 µM, respectively (both p<0.05. In addition, Ca²⁺ influx to Ca²⁺-depleted cells was blunted and IL-2 production was also reduced in activated Jurkat cells. IL-2 secretion was also inhibited by the Kv1.3 inhibitors margatoxin and charybdotoxin. Our results demonstrate for the first time that that DPO-1, at clinically relevant concentrations, blocks Kv1.3 channels, decreases Kv1.3 channel expression and suppresses IL-2 secretion. Therefore, DPO-1 may be a useful treatment strategy for immunologic disorders.

  14. Reconstruction of Cell Surface Densities of Ion Pumps, Exchangers, and Channels from mRNA Expression, Conductance Kinetics, Whole-Cell Calcium, and Current-Clamp Voltage Recordings, with an Application to Human Uterine Smooth Muscle Cells.

    Directory of Open Access Journals (Sweden)

    Jolene Atia

    2016-04-01

    Full Text Available Uterine smooth muscle cells remain quiescent throughout most of gestation, only generating spontaneous action potentials immediately prior to, and during, labor. This study presents a method that combines transcriptomics with biophysical recordings to characterise the conductance repertoire of these cells, the 'conductance repertoire' being the total complement of ion channels and transporters expressed by an electrically active cell. Transcriptomic analysis provides a set of potential electrogenic entities, of which the conductance repertoire is a subset. Each entity within the conductance repertoire was modeled independently and its gating parameter values were fixed using the available biophysical data. The only remaining free parameters were the surface densities for each entity. We characterise the space of combinations of surface densities (density vectors consistent with experimentally observed membrane potential and calcium waveforms. This yields insights on the functional redundancy of the system as well as its behavioral versatility. Our approach couples high-throughput transcriptomic data with physiological behaviors in health and disease, and provides a formal method to link genotype to phenotype in excitable systems. We accurately predict current densities and chart functional redundancy. For example, we find that to evoke the observed voltage waveform, the BK channel is functionally redundant whereas hERG is essential. Furthermore, our analysis suggests that activation of calcium-activated chloride conductances by intracellular calcium release is the key factor underlying spontaneous depolarisations.

  15. Reconstruction of Cell Surface Densities of Ion Pumps, Exchangers, and Channels from mRNA Expression, Conductance Kinetics, Whole-Cell Calcium, and Current-Clamp Voltage Recordings, with an Application to Human Uterine Smooth Muscle Cells.

    Science.gov (United States)

    Atia, Jolene; McCloskey, Conor; Shmygol, Anatoly S; Rand, David A; van den Berg, Hugo A; Blanks, Andrew M

    2016-04-01

    Uterine smooth muscle cells remain quiescent throughout most of gestation, only generating spontaneous action potentials immediately prior to, and during, labor. This study presents a method that combines transcriptomics with biophysical recordings to characterise the conductance repertoire of these cells, the 'conductance repertoire' being the total complement of ion channels and transporters expressed by an electrically active cell. Transcriptomic analysis provides a set of potential electrogenic entities, of which the conductance repertoire is a subset. Each entity within the conductance repertoire was modeled independently and its gating parameter values were fixed using the available biophysical data. The only remaining free parameters were the surface densities for each entity. We characterise the space of combinations of surface densities (density vectors) consistent with experimentally observed membrane potential and calcium waveforms. This yields insights on the functional redundancy of the system as well as its behavioral versatility. Our approach couples high-throughput transcriptomic data with physiological behaviors in health and disease, and provides a formal method to link genotype to phenotype in excitable systems. We accurately predict current densities and chart functional redundancy. For example, we find that to evoke the observed voltage waveform, the BK channel is functionally redundant whereas hERG is essential. Furthermore, our analysis suggests that activation of calcium-activated chloride conductances by intracellular calcium release is the key factor underlying spontaneous depolarisations.

  16. Non-perturbative renormalization of four-quark operators and B_K with Schroedinger functional scheme in quenched domain-wall QCD

    OpenAIRE

    Nakamura, Yousuke; Taniguchi, Yusuke; Collaboration, for CP-PACS

    2007-01-01

    We present non-perturbative renormalization factors for $\\Delta S=2$ four-quark operators in quenched domain-wall QCD using the Schroedinger functional method. Non-perturbative renormalization factor for $B_K$ is evaluated at hadronic scale. Combined with the non-perturbative RG running obtained by the Alpha collaboration, our result yields renormalization factor which converts lattice bare $B_K$ to the renormalization group invariant one. We apply the renormalization factor to bare $B_K$ pre...

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

    DEFF Research Database (Denmark)

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

    2015-01-01

    hypothesized that proteolytic processing of gammaENaC occurs in the human kidney under physiologic conditions and that proteinuria contributes to aberrant proteolytic activation. Here, we used monoclonal antibodies (mAbs) with specificity to the human 43-mer inhibitory tract (N and C termini, mAbinhibit, and m...... higher abundance of full-length and furin-cleaved gammaENaC, with no significant change in the furin-cleaved-to-full-length gammaENaC ratio. In patients with proteinuria (n=6), the inhibitory tract was detected only in full-length gammaENaC by mAbinhibit. Prostasin/kallikrein-cleaved gamma......ENaC was detected consistently only in tissue from patients with proteinuria and observed in collecting ducts. In conclusion, human kidney gammaENaC is subject to proteolytic cleavage, yielding fragments compatible with furin cleavage, and proteinuria is associated with cleavage at the putative prostasin...

  18. The bradykinin B2 receptor induces multiple cellular responses leading to the proliferation of human renal carcinoma cell lines

    Directory of Open Access Journals (Sweden)

    Kramarenko II

    2012-07-01

    Full Text Available Inga I Kramarenko1, Thomas A Morinelli1,2, Marlene A Bunni1,2, John R Raymond Sr3, Maria N Garnovskaya11Department of Medicine (Nephrology Division, Medical University of South Carolina, Charleston, SC, USA; 2Medical and Research Services of the Ralph H Johnson Veterans Affairs Medical Center, Charleston, SC, USA; 3Medical College of Wisconsin, Milwaukee, WI, USABackground: The vasoactive peptide bradykinin (BK acts as a potent growth factor for normal kidney cells, but there have been few studies on the role of BK in renal cell carcinomas.Purpose: In this study, we tested the hypothesis that BK also acts as a mitogen in kidney carcinomas, and explored the effects of BK in human renal carcinoma A498 cells.Methods: The presence of mRNAs for BK B1 and BK B2 receptors in A498 cells was demonstrated by reverse transcription–polymerase chain reaction. To study BK signaling pathways, we employed fluorescent measurements of intracellular Ca2+, measured changes in extracellular pH as a reflection of Na+/H+ exchange (NHE with a Cytosensor microphysiometer, and assessed extracellular signal-regulated kinase (ERK activation by Western blotting.Results: Exposure to 100 nM of BK resulted in the rapid elevation of intracellular Ca2+, caused a ≥30% increase in NHE activity, and a ≥300% increase in ERK phosphorylation. All BK signals were blocked by HOE140, a BK B2 receptor antagonist, but not by a B1 receptor antagonist. Inhibitor studies suggest that BK-induced ERK activation requires phospholipase C and protein kinase C activities, and is Ca2+/calmodulin-dependent. The amiloride analog 5-(N-methyl-N-isobutyl-amiloride (MIA blocked short-term NHE activation and inhibited ERK phosphorylation, suggesting that NHE is critical for ERK activation by BK. BK induced an approximately 40% increase in the proliferation of A498 cells as assessed by bromodeoxyuridine uptake. This effect was blocked by the ERK inhibitor PD98059, and was dependent on NHE activity

  19. Multi-channel photon migration study in visible Chinese human muscle for optical detection of deep vein thrombosis

    Science.gov (United States)

    Sun, Yunlong; Li, Ting

    2016-03-01

    Deep vein thrombosis (DVT) always induced venous thrombosis. Most cases of venous thrombosis were induced by deep vein thrombosis (DVT), with high incidence rate of >60% in >60 years old people. Near-infrared spectroscopy (NIRS) were reported recently to be an intriguing and potential technique in detecting DVT in clinics. However, the photon transport is still unclear, which is crucial for the image reconstruction of the updated development called as NIRS-based DVT imager. Here we employed the Monte Carlo simulation software for 3D voxelized media (MCVM) and the Visible Chinese Human (VCH) model, which segmentation is finest in the world, to simulate multi-channel photon migration in calf muscle. And the image reconstruction of DVT hemodynamic distribution was achieved. This study, for the first time, provides the most realistic 3-D multichannel photon migration for NIRS study on DVT, and explored the image reconstruction for furtherly developing a NIRS-based DVT imager.

  20. Regulation of the human ether-a-go-go-related gene (hERG) potassium channel by Nedd4 family interacting proteins (Ndfips).

    Science.gov (United States)

    Kang, Yudi; Guo, Jun; Yang, Tonghua; Li, Wentao; Zhang, Shetuan

    2015-11-15

    The cardiac electrical disorder long QT syndrome (LQTS) pre-disposes affected individuals to ventricular arrhythmias and sudden death. Dysfunction of the human ether-a-go-go-related gene (hERG)-encoded rapidly activating delayed rectifier K(+) channel (IKr) is a major cause of LQTS. The expression of hERG channels is controlled by anterograde trafficking of newly synthesized channels to and retrograde degradation of existing channels from the plasma membrane. We have previously shown that the E3 ubiquitin (Ub) ligase Nedd4-2 (neural precursor cell expressed developmentally down-regulated protein 4-2) targets the PY motif of hERG channels to initiate channel degradation. Although both immature and mature hERG channels contain the PY motif, Nedd4-2 selectively mediates the degradation of mature hERG channels. In the present study, we demonstrate that Nedd4-2 is directed to specific cellular compartments by the Nedd4 family interacting proteins, Nedd4 family-interacting protein 1 (Ndfip1) and Ndfip2. Ndfip1 is primarily localized in the Golgi apparatus where it recruits Nedd4-2 to mediate the degradation of mature hERG proteins during channel trafficking to the plasma membrane. Although Ndfip2 directs Nedd4-2 to the Golgi apparatus, it also recruits Nedd4-2 to the multivesicular bodies (MVBs), which may impair MVB function and impede the degradation of mature hERG proteins mediated by Nedd4-2. These findings extend our understanding of hERG channel regulation and provide information which may be useful for the rescue of impaired hERG function in LQTS.

  1. Channel-opening kinetic mechanism for human wild-type GluK2 and the M867I mutant kainate receptor.

    Science.gov (United States)

    Han, Yan; Wang, Congzhou; Park, Jae Seon; Niu, Li

    2010-11-02

    GluK2 is a kainate receptor subunit that is alternatively spliced at the C-terminus. Previous studies implicated GluK2 in autism. In particular, the methionine-to-isoleucine replacement at amino acid residue 867 (M867I) that can only occur in the longest isoform of the human GluK2 (hGluK2), as the disease (autism) mutation, is thought to cause gain-of-function. However, the kinetic properties of the wild-type hGluK2 and the functional consequence of this gain-of-function mutation at the molecular level are not well understood. To investigate whether the M867I mutation affects the channel properties of the human GluK2 kainate receptor, we have systematically characterized the rate and the equilibrium constants pertinent to channel opening and channel desensitization for this mutant and the wild-type hGluK2 receptor, along with the wild-type rat GluK2 kainate receptor (rGluK2) as the control. Our results show that the M867I mutation does not affect either the rate or the equilibrium constants of the channel opening but does slow down the channel desensitization rate by ~1.6-fold at saturating glutamate concentrations. It is possible that a consequence of this mutation on the desensitization rate is linked to facilitating the receptor trafficking and membrane expression, given the close proximity of M867 to the forward trafficking motif in the C-terminal sequence. By comparing the kinetic data of the wild-type human and rat GluK2 receptors, we also find that the human GluK2 has a ~3-fold smaller channel-opening rate constant but an identical channel-closing rate constant and thus a channel-opening probability of 0.85 vs 0.96 for rGluK2. Furthermore, the intrinsic equilibrium dissociation constant K(1) for hGluK2, like the EC(50) value, is ~2-fold lower than rGluK2. Our results therefore suggest that the human GluK2 is relatively a slowly activating channel but more sensitive to glutamate, as compared to the rat ortholog, despite the fact that the human and rat forms

  2. Identification and characterization of human neuronal voltage-gated calcium channel gamma 3 subunit gene

    Institute of Scientific and Technical Information of China (English)

    2000-01-01

    By homologous expressed sequence tag (EST) searching,one EST (GenBank: W29095) was obtained,which shows 75% identity in 435 bp overlap with the coding sequence of mouse Cacng2 gene. A 1 545 bp cDNA fragment was obtained from the nested polymerase chain reaction (PCR) and rapid applification of cDNA end (RACE) reaction in the human brain prefrontal cortex cDNA library and the human brain Ready cDNA with the primers designed on W29095. The fragment contained a 948-bp open reading frame (ORF) encoding 315 amino acids,and was named CACNG3. As it was identical to a BAC clone (GenBank: AC004125) from chromosome 16p12-p13.1,the CACNG3 gene was mapped to human chromosome 16p12-p13.1,and the coding region was composed of 4 exons. Reverse transcription PCR (RT-PCR) analysis showed that the CACNG3 gene expressed in human adult brain and fetal brain. Single strand comformation polymorphism (SSCP) analysis was performed in 3 pedigrees with autosomal recessive retinitis pigmentosa,8 pedigrees with autosomal recessive retinitis pigmentosa accompanied by deafness and 2 pedigrees with epilepsy,but no mutation was detected.

  3. Ocular Hypotensive Effects of the ATP-Sensitive Potassium Channel Opener Cromakalim in Human and Murine Experimental Model Systems.

    Directory of Open Access Journals (Sweden)

    Uttio Roy Chowdhury

    Full Text Available Elevated intraocular pressure (IOP is the most prevalent and only treatable risk factor for glaucoma, a leading cause of irreversible blindness worldwide. Unfortunately, all current therapeutics used to treat elevated IOP and glaucoma have significant and sometimes irreversible side effects necessitating the development of novel compounds. We evaluated the IOP lowering ability of the broad spectrum KATP channel opener cromakalim. Cultured human anterior segments when treated with 2 μM cromakalim showed a decrease in pressure (19.33 ± 2.78 mmHg at 0 hours to 13.22 ± 2.64 mmHg at 24 hours; p<0.001 when compared to vehicle treated controls (15.89 ± 5.33 mmHg at 0 h to 15.56 ± 4.88 mmHg at 24 hours; p = 0.89. In wild-type C57BL/6 mice, cromakalim reduced IOP by 18.75 ± 2.22% compared to vehicle treated contralateral eyes (17.01 ± 0.32 mmHg at 0 hours to 13.82 ± 0.37 mmHg at 24 hours; n = 10, p = 0.002. Cromakalim demonstrated an additive effect when used in conjunction with latanoprost free acid, a common ocular hypotensive drug prescribed to patients with elevated IOP. To examine KATP channel subunit specificity, Kir6.2(-/- mice were treated with cromakalim, but unlike wild-type animals, no change in IOP was noted. Histologic analysis of treated and control eyes in cultured human anterior segments and in mice showed similar cell numbers and extracellular matrix integrity within the trabecular meshwork, with no disruptions in the inner and outer walls of Schlemm's canal. Together, these studies suggest that cromakalim is a potent ocular hypotensive agent that lowers IOP via activation of Kir6.2 containing KATP channels, its effect is additive when used in combination with the commonly used glaucoma drug latanoprost, and is not toxic to cells and tissues of the aqueous humor outflow pathway, making it a candidate for future therapeutic development.

  4. Receptor channel TRPC6 orchestrate the activation of human hepatic stellate cell under hypoxia condition

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    Iyer, Soumya C, E-mail: chidambaram.soumya@gmail.com [Unit of Biochemistry, Department of Zoology, School of Life Sciences, University of Madras, Guindy Campus, Chennai 600025, Tamilnadu (India); Kannan, Anbarasu [Department of Biochemistry, University of Madras, Guindy Campus, Chennai 600025, Tamilnadu (India); Gopal, Ashidha [Unit of Biochemistry, Department of Zoology, School of Life Sciences, University of Madras, Guindy Campus, Chennai 600025, Tamilnadu (India); Devaraj, Niranjali [Department of Biochemistry, University of Madras, Guindy Campus, Chennai 600025, Tamilnadu (India); Halagowder, Devaraj [Unit of Biochemistry, Department of Zoology, School of Life Sciences, University of Madras, Guindy Campus, Chennai 600025, Tamilnadu (India)

    2015-08-01

    Hepatic stellate cells (HSCs), a specialized stromal cytotype have a great impact on the biological behaviors of liver diseases. Despite this fact, the underlying mechanism that regulates HSC still remains poorly understood. The aim of the present study was to understand the role of TRPC6 signaling in regulating the molecular mechanism of HSCs in response to hypoxia. In the present study we showed that under hypoxia condition, the upregulated Hypoxia Inducible Factor 1α (HIF1α) increases NICD activation, which in turn induces the expression of transient receptor potential channel 6 (TRPC6) in HSC line lx-2. TRPC6 causes a sustained elevation of intracellular calcium which is coupled with the activation of the calcineurin-nuclear factor of activated T-cell (NFAT) pathway which activates the synthesis of extracellular matrix proteins. TRPC6 also activates SMAD2/3 dependent TGF-β signaling