Science.gov

Sample records for channel maintenance activities

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

    SciTech Connect

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

    2001-10-19

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

  2. Functional Apical Large Conductance, Ca2+-activated, and Voltage-dependent K+ Channels Are Required for Maintenance of Airway Surface Liquid Volume*

    PubMed Central

    Manzanares, Dahis; Gonzalez, Carlos; Ivonnet, Pedro; Chen, Ren-Shiang; Valencia-Gattas, Monica; Conner, Gregory E.; Larsson, H. Peter; Salathe, Matthias

    2011-01-01

    Large conductance, Ca2+-activated, and voltage-dependent K+ (BK) channels control a variety of physiological processes in nervous, muscular, and renal epithelial tissues. In bronchial airway epithelia, extracellular ATP-mediated, apical increases in intracellular Ca2+ are important signals for ion movement through the apical membrane and regulation of water secretion. Although other, mainly basolaterally expressed K+ channels are recognized as modulators of ion transport in airway epithelial cells, the role of BK in this process, especially as a regulator of airway surface liquid volume, has not been examined. Using patch clamp and Ussing chamber approaches, this study reveals that BK channels are present and functional at the apical membrane of airway epithelial cells. BK channels open in response to ATP stimulation at the apical membrane and allow K+ flux to the airway surface liquid, whereas no functional BK channels were found basolaterally. Ion transport modeling supports the notion that apically expressed BK channels are part of an apical loop current, favoring apical Cl− efflux. Importantly, apical BK channels were found to be critical for the maintenance of adequate airway surface liquid volume because continuous inhibition of BK channels or knockdown of KCNMA1, the gene coding for the BK α subunit (KCNMA1), lead to airway surface dehydration and thus periciliary fluid height collapse revealed by low ciliary beat frequency that could be fully rescued by addition of apical fluid. Thus, apical BK channels play an important, previously unrecognized role in maintaining adequate airway surface hydration. PMID:21454692

  3. Maintenance of large deltas through channelization

    NASA Astrophysics Data System (ADS)

    Giosan, L.; Constatinescu, S.; Filip, F.

    2013-12-01

    A new paradigm for delta restoration is currently taking shape using primarily Mississippi delta examples. Here we propose an alternative for delta maintenance primarily envisioned for wave-influenced deltas based on Danube delta experiences. Over the last half century, while the total sediment load of the Danube dramatically decreased due to dam construction on tributaries and its mainstem, a grand experiment was inadvertently run in the Danube delta: the construction of a dense network of canals, which almost tripled the water discharge toward the interior of the delta plain. We use core-based and chart-based sedimentation rates and patterns to explore the delta transition from the natural to an anthropogenic regime, to understand the effects of far-field damming and near-field channelization, and to construct a conceptual model for delta development as a function sediment partition between the delta plain and the delta coastal fringe. We show that sediment fluxes increased to the delta plain due to channelization, counteracting sea level rise. In turn, the delta coastal fringe was most impacted by the Danube's sediment load collapse. Furthermore, we show that morphodynamic feedbacks at the river mouth are crucial in trapping sediment near the coast and constructing wave-dominated deltas or lobes or delaying their destruction. As a general conclusion, we suggest that increased channelization that mimics and enhances natural processes may provide a simple solution for keeping delta plains above sea level and that abandonment of wave-dominated lobes may be the most long term efficient solution for protecting the internal fluvial regions of deltas and provide new coastal growth downcoast.

  4. Inlet Spits and Maintenance of Navigation Channels

    DTIC Science & Technology

    2002-06-01

    Fire Island on the south shore of Long Island, NY. This spit has grown from sediments supplied from mainland deposits located updrift – to the east...Figure 1). The U.S Army Engineer District, New York, studied the westward growth of Fire Island in the early 1950s (Gofseyeff 1953; Saville 1960) to...determine maintenance requirements for Fire Island Inlet. Jetty construction from 1939 to 1941 was conducted to arrest growth of the spit, which had

  5. Mechanically Activated Ion Channels.

    PubMed

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

    2015-09-23

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

  6. Mechanically Activated Ion Channels

    PubMed Central

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

    2015-01-01

    Mechanotransduction, the conversion of physical forces into biochemical signals, is an essential component of numerous physiological processes including not only conscious senses of touch and hearing, but also unconscious senses such as blood pressure regulation. Mechanically activated (MA) ion channels have been proposed as sensors of physical force, but the identity of these channels and an understanding of how mechanical force is transduced has remained elusive. A number of recent studies on previously known ion channels along with the identification of novel MA ion channels have greatly transformed our understanding of touch and hearing in both vertebrates and invertebrates. Here, we present an updated review of eukaryotic ion channel families that have been implicated in mechanotransduction processes and evaluate the qualifications of the candidate genes according to specified criteria. We then discuss the proposed gating models for MA ion channels and highlight recent structural studies of mechanosensitive potassium channels. PMID:26402601

  7. Predictive techniques for river channel evolution and maintenance

    USGS Publications Warehouse

    Nelson, J.M.

    1996-01-01

    Predicting changes in alluvial channel morphology associated with anthropogenic and natural changes in flow and/or sediment supply is a critical part of the management of riverine systems. Over the past few years, advances in the understanding of the physics of sediment transport in conjunction with rapidly increasing capabilities in computational fluid dynamics have yielded now approaches to problems in river mechanics. Techniques appropriate for length scales ranging from reaches to bars and bedforms are described here. Examples of the use of these computational approaches are discussed for three cases: (1) the design of diversion scenarios that maintain channel morphology in steep cobble-bedded channels in Colorado, (2) determination of channel maintenance flows for the preservation of channel islands in the Snake River in Idaho, and (3) prediction of the temporal evolution of deposits in lateral separation zones for future assessment of the impacts of various dam release scenarios on lateral separation deposits in the Colorado River in Grand Canyon. With continued development of their scientific and technical components, the methodologies described here can provide powerful tools for the management of river environments in the future.

  8. Maintenance of an obstruction-forced pool in a gravel-bed channel: streamflow, channel morphology, and sediment transport.

    Treesearch

    Richard D. Woodsmith; Marwan A. Hassan

    2005-01-01

    Maintenance of pool morphology in a stream channel with a mobile bed requires hydraulic conditions at moderate to high flows that route bed load through the pool as it is delivered from upstream. Through field measurements of discharge, vertical velocity profiles, bed load transport, and streambed scour, fill, and grain-size distribution, we found that maintenance of a...

  9. Cognitive Aging: Activity Patterns and Maintenance Intentions

    ERIC Educational Resources Information Center

    Gilhooly, K. J.; Gilhooly, M. L.; Phillips, L. H.; Harvey, D.; Murray, A.; Hanlon, P.

    2007-01-01

    This study examined relationships between cognitive functioning in older people and (1) levels of mental, physical and social activities, and (2) intentions regarding maintenance of cognitive functioning. Participants (N = 145) were 70-91 years of age, varied in health status and socio-economic backgrounds. Current cognitive functioning was…

  10. Mechanisms of Activation of Voltage-Gated Potassium Channels

    PubMed Central

    Grizel, A. V.; Glukhov, G. S.; Sokolova, O. S.

    2014-01-01

    Voltage-gated potassium ion channels (Kv) play an important role in a variety of cellular processes, including the functioning of excitable cells, regulation of apoptosis, cell growth and differentiation, the release of neurotransmitters and hormones, maintenance of cardiac activity, etc. Failure in the functioning of Kv channels leads to severe genetic disorders and the development of tumors, including malignant ones. Understanding the mechanisms underlying Kv channels functioning is a key factor in determining the cause of the diseases associated with mutations in the channels, and in the search for new drugs. The mechanism of activation of the channels is a topic of ongoing debate, and a consensus on the issue has not yet been reached. This review discusses the key stages in studying the mechanisms of functioning of Kv channels and describes the basic models of their activation known to date. PMID:25558391

  11. Lubiprostone: a chloride channel activator.

    PubMed

    Lacy, Brian E; Levy, L Campbell

    2007-04-01

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

  12. The Effect of Line Maintenance Activity on Airline Safety Quality

    NASA Technical Reports Server (NTRS)

    Rhoades, Dawna L.; Reynolds, Rosemarie; Waguespack, Blaise, Jr.; Williams, Michael

    2005-01-01

    One of the arguments against deregulation of the airline industry has been the possibility that financially troubled carriers would be tempted to lower line maintenance spending, thus lowering maintenance quality and decreasing the overall safety of the carrier. Given the financial crisis triggered by the events of 9/11: it appears to be a good time to revisit this issue. This paper examines the quality of airline line maintenance activity and examines the impact of maintenance spending on maintenance quality and overall safety. Findings indicate that increased maintenance spending is associated with increased line maintenance activity and increased overall safety quality for the major U.S. carriers.

  13. Impacts of maintenance channel dredging in a northern Adriatic coastal lagoon. II: Effects on macrobenthic assemblages in channels and ponds

    NASA Astrophysics Data System (ADS)

    Ponti, Massimo; Pasteris, Andrea; Guerra, Roberta; Abbiati, Marco

    2009-10-01

    Coastal lagoons are ephemeral habitats whose conservation requires human intervention, such as maintenance dredging of inner channels. Dredging can reduce the abundance of benthic species due to the removal of individuals with the sediment, modify sediment properties, and resuspend fine sediment, nutrients and pollutants, which can lead to eutrophication, hypoxic events and increasing toxicity. Both direct effects in the dredged channel and possible indirect effects in surrounding shallow areas could be expected. This study assesses the effects of the channel maintenance dredging, performed between October 2004 and August 2005, on the invertebrate assemblages both in channels and adjacent ponds in the northern Adriatic coastal lagoon of Pialassa Baiona. The lagoon is affected by eutrophication, chemical and thermal pollution from wastewater treatment and power plants. Three impacted sites were located in the dredged channel and three in the adjacent interconnected shallow water ponds, while three non-impacted sites were located in a channel and in a pond far from the dredged area. Replicate samples were collected from each site one time before and one time after the dredging operations. Despite the extent of the intervention, effects of the dredging on macrobenthic assemblages were detected only within the dredged channel, while in the surrounding ponds no clear and unequivocal effects were found. In particular the dredging could have promoted the increase of the abundance of the polychaete Streblospio shrubsolii in the southern and central parts of the dredged channel and the increase in abundance of the amphipod Corophium insidiosum in the northern side, compared to the controls. Instead, species diversity was reduced in the central and northern parts of the dredged channel. These effects on the macrobenthic invertebrate assemblages could be related to the observed changes of sediment characteristics, contamination and toxicity. Overall, direct effects on benthic

  14. Calcium Activation of Mougeotia Potassium Channels 1

    PubMed Central

    Lew, Roger R.; Serlin, Bruce S.; Schauf, Charles L.; Stockton, Marsha E.

    1990-01-01

    Phytochrome mediates chloroplast movement in the alga Mougeotia, possibly via changes in cytosolic calcium. It is known to regulate a calcium-activated potassium channel in the algal plasma membrane. As part of a characterization of the potassium channel, we examined the properties of calcium activation. The calcium ionophore A23187 activates the channel at external [Ca2+] as low as 20 micromolar. However, external [Ca2+] is not required for activation of the channel by photoactivated phytochrome. Furthermore, when an inhibitor of calcium release from internal stores, 8-(diethylamino)-octyl-3,4,5-trimethoxybenzoate, hydrochloride (TMB-8), is present, red light no longer stimulates channel activity. We conclude that phytochrome activates the plasma membrane potassium channel by releasing calcium from intracellular calcium vesicles; the elevated cytosolic calcium then stimulates channel activity by an unknown mechanism. In the presence of TMB-8, red light does induce chloroplast rotation; thus, potassium channel activation may not be coupled to chloroplast rotation. PMID:16667356

  15. Thermally activated TRPV3 channels.

    PubMed

    Luo, Jialie; Hu, Hongzhen

    2014-01-01

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

  16. 76 FR 26311 - Agency Information Collection Activities: Harbor Maintenance Fee

    Federal Register 2010, 2011, 2012, 2013, 2014

    2011-05-06

    ... SECURITY U.S. Customs and Border Protection Agency Information Collection Activities: Harbor Maintenance... concerning the Harbor Maintenance Fee (CBP Forms 349 and 350). This request for comment is being made... keepers from the collection of information (a total capital/startup costs and operations and maintenance...

  17. 76 FR 50489 - Agency Information Collection Activities: Harbor Maintenance Fee

    Federal Register 2010, 2011, 2012, 2013, 2014

    2011-08-15

    ... SECURITY U.S. Customs and Border Protection Agency Information Collection Activities: Harbor Maintenance... approval in accordance with the Paperwork Reduction Act: Harbor Maintenance Fee (CBP Forms 349 and 350.../startup costs and operations and maintenance costs). The comments that are submitted will be summarized...

  18. Quantifying Channel Maintenance Instream Flows: An Approach for Gravel-Bed Streams in the Western United States

    Treesearch

    Larry J. Schmidt; John P. Potyondy

    2004-01-01

    This paper discusses one approach for quantifying channel maintenance instream flow necessary to achieve the Forest Service Organic Act purpose of securing favorable conditions of water flows. The approach is appropriate for quantifying channel maintenance flows on perennial, unregulated, snowmelt-dominated, gravel-bed streams with alluvial reaches. The approach...

  19. Agonist-activated ion channels

    PubMed Central

    Colquhoun, David

    2006-01-01

    This paper looks at ion channels as an example of the pharmacologist's stock in trade, the action of an agonist on a receptor to produce a response. Looked at in this way, ion channels have been helpful because they are still the only system which is simple enough for quantitative investigation of transduction mechanisms. A short history is given of attempts to elucidate what happens between the time when agonist first binds, and the time when the channel opens. PMID:16402101

  20. [Occupational radiation exposures during maintenance activities at nuclear power plants].

    PubMed

    Imahori, A

    1987-11-01

    Occupational exposures at nuclear power plants occur mostly during maintenance activities rather than during routine reactor operation. In this paper, statistical summaries of occupational exposures during routine maintenance activities for the years 1982-84 at nuclear power plants in Japan are presented, including comparison of the exposure levels by reactor type and by plant age. Average annual collective doses per reactor for BWRs and PWRs are 7.30 man-Sv and 2.84 man-Sv, respectively, and 78% and 89% of annual doses are incurred during maintenance activities. Average annual outage days of BWRs and PWRs for routine maintenance are 102 d and 97 d. Annual collective doses per reactor, most of which occur during maintenance activities, usually increase with plant age. Higher collective doses are observed for routine maintenance performed on older reactors as compared to newer reactors, especially in BWRs. Collective doses accrued during respective routine maintenance activities have a significant correlation with duration of maintenance and number of workers involved in maintenance.

  1. 7 CFR 1470.23 - Conservation activity operation and maintenance.

    Code of Federal Regulations, 2012 CFR

    2012-01-01

    ... 7 Agriculture 10 2012-01-01 2012-01-01 false Conservation activity operation and maintenance. 1470... CREDIT CORPORATION, DEPARTMENT OF AGRICULTURE LOANS, PURCHASES, AND OTHER OPERATIONS CONSERVATION STEWARDSHIP PROGRAM Contracts and Payments § 1470.23 Conservation activity operation and maintenance....

  2. 7 CFR 1470.23 - Conservation activity operation and maintenance.

    Code of Federal Regulations, 2014 CFR

    2014-01-01

    ... 7 Agriculture 10 2014-01-01 2014-01-01 false Conservation activity operation and maintenance. 1470... CREDIT CORPORATION, DEPARTMENT OF AGRICULTURE LOANS, PURCHASES, AND OTHER OPERATIONS CONSERVATION STEWARDSHIP PROGRAM Contracts and Payments § 1470.23 Conservation activity operation and maintenance....

  3. 7 CFR 1470.23 - Conservation activity operation and maintenance.

    Code of Federal Regulations, 2011 CFR

    2011-01-01

    ... 7 Agriculture 10 2011-01-01 2011-01-01 false Conservation activity operation and maintenance. 1470... CREDIT CORPORATION, DEPARTMENT OF AGRICULTURE LOANS, PURCHASES, AND OTHER OPERATIONS CONSERVATION STEWARDSHIP PROGRAM Contracts and Payments § 1470.23 Conservation activity operation and maintenance....

  4. 7 CFR 1470.23 - Conservation activity operation and maintenance.

    Code of Federal Regulations, 2010 CFR

    2010-01-01

    ... 7 Agriculture 10 2010-01-01 2010-01-01 false Conservation activity operation and maintenance. 1470... CREDIT CORPORATION, DEPARTMENT OF AGRICULTURE LOANS, PURCHASES, AND OTHER OPERATIONS CONSERVATION STEWARDSHIP PROGRAM Contracts and Payments § 1470.23 Conservation activity operation and maintenance....

  5. 7 CFR 1470.23 - Conservation activity operation and maintenance.

    Code of Federal Regulations, 2013 CFR

    2013-01-01

    ... 7 Agriculture 10 2013-01-01 2013-01-01 false Conservation activity operation and maintenance. 1470... CREDIT CORPORATION, DEPARTMENT OF AGRICULTURE LOANS, PURCHASES, AND OTHER OPERATIONS CONSERVATION STEWARDSHIP PROGRAM Contracts and Payments § 1470.23 Conservation activity operation and maintenance....

  6. Automobile Maintenance. Reading and Language Activities.

    ERIC Educational Resources Information Center

    Kessman, William A.

    Designed primarily for special needs students in a vocational program in automobile maintenance, this book was written to refine the basic skills of following directions, reading comprehension, vocabulary building, spelling, word usage, and word recognition, while relating these skills to some of the tasks a beginning student in the program must…

  7. BK channel activation: structural and functional insights

    PubMed Central

    Lee, Urvi S.; Cui, Jianmin

    2010-01-01

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

  8. BK channel activators and their therapeutic perspectives

    PubMed Central

    Bentzen, Bo H.; Olesen, Søren-Peter; Rønn, Lars C. B.; Grunnet, Morten

    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 in intracellular calcium to outward hyperpolarizing potassium currents. Consequently, the channel has many important physiological roles including regulation of smooth muscle tone, neurotransmitter release and neuronal excitability. Additionally, cardioprotective roles have been revealed in recent years. After a short introduction to the structure, function and regulation of BK channels, we review the small organic molecules activating BK channels and how these tool compounds have helped delineate the roles of BK channels in health and disease. PMID:25346695

  9. Sediment Transport into the Swinomish Navigation Channel, Puget Sound—Habitat Restoration versus Navigation Maintenance Needs

    DOE PAGES

    Khangaonkar, Tarang; Nugraha, Adi; Hinton, Steve; ...

    2017-04-21

    The 11 mile (1.6 km) Swinomish Federal Navigation Channel provides a safe and short passage to fishing and recreational craft in and out of Northern Puget Sound by connecting Skagit and Padilla Bays, US State abbrev., USA. A network of dikes and jetties were constructed through the Swinomish corridor between 1893 and 1936 to improve navigation functionality. Over the years, these river training dikes and jetties designed to minimize sedimentation in the channel have deteriorated, resulting in reduced protection of the channel. The need to repair or modify dikes/jetties for channel maintenance, however, may conflict with salmon habitat restoration goalsmore » aimed at improving access, connectivity and brackish water habitat. Several restoration projects have been proposed in the Skagit delta involving breaching, lowering, or removal of dikes. To assess relative merits of the available alternatives, a hydrodynamic model of the Skagit River estuary was developed using the Finite Volume Community Ocean Model (FVCOM). Here, in this paper, we present the refinement and calibration of the model using oceanographic data collected from the years 2006 and 2009 with a focus on the sediment and brackish water transport from the river and Skagit Bay tide flats to the Swinomish Channel. The model was applied to assess the feasibility of achieving the desired dual outcome of (a) reducing sedimentation and shoaling in the Swinomish Channel and (b) providing a direct migration pathway and improved conveyance of freshwater into the Swinomish Channel. Finally, the potential reduction in shoaling through site-specific structure repairs is evaluated. Similarly, the potential to significantly improve of brackish water habitat through dike breach restoration actions using the McGlinn Causeway project example, along with its impacts on sediment deposition in the Swinomish Navigation Channel, is examined« less

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

    NASA Astrophysics Data System (ADS)

    Talukdar, Keka; Shantappa, Anil

    2016-05-01

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

  11. The development and applications of dual channel structure based on maintenance-free moisture absorber

    NASA Astrophysics Data System (ADS)

    Su, Wei; Hu, Weimin; Gao, Anliang; Ye, Yu; Huang, Juan; Liu, Xinyu; Yin, Wen

    2017-04-01

    It is necessary to carry out regular maintenance and replacement of the traditional power transformer pressure and humidity absorber in the long running time. When the transformer in use to upgrade or retrofit, usually need to find a simple, practical solution. Conventional moisture absorbers require the use of silica gel particles or similar desiccant to absorb water, the need for regular replacement of desiccant. In order to improve the working efficiency of desiccant and reduce the cost of desiccant replacement, this paper introduces the latest technology of dual-channel maintenance-free moisture absorber, describes the working principle of the device and the advantages of the element, and its application prospects in the power industry Outlook.

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

    PubMed

    Yang, Huanghe; Zhang, Guohui; Cui, Jianmin

    2015-01-01

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

  13. Constitutive Activation of the Shaker Kv Channel

    PubMed Central

    Sukhareva, Manana; Hackos, David H.; Swartz, Kenton J.

    2003-01-01

    In different types of K+ channels the primary activation gate is thought to reside near the intracellular entrance to the ion conduction pore. In the Shaker Kv channel the gate is closed at negative membrane voltages, but can be opened with membrane depolarization. In a previous study of the S6 activation gate in Shaker (Hackos, D.H., T.H. Chang, and K.J. Swartz. 2002. J. Gen. Physiol. 119:521–532.), we found that mutation of Pro 475 to Asp results in a channel that displays a large macroscopic conductance at negative membrane voltages, with only small increases in conductance with membrane depolarization. In the present study we explore the mechanism underlying this constitutively conducting phenotype using both macroscopic and single-channel recordings, and probes that interact with the voltage sensors or the intracellular entrance to the ion conduction pore. Our results suggest that constitutive conduction results from a dramatic perturbation of the closed-open equilibrium, enabling opening of the activation gate without voltage-sensor activation. This mechanism is discussed in the context of allosteric models for activation of Kv channels and what is known about the structure of this critical region in K+ channels. PMID:14557403

  14. The effects of physical activity as maintenance for smoking cessation.

    PubMed

    Russell, P O; Epstein, L H; Johnston, J J; Block, D R; Blair, E

    1988-01-01

    The effects of physical activity as a maintenance strategy for smoking cessation were evaluated. After a standard smoking cessation program, forty-two women were randomly assigned to one of three groups that received equal number of maintenance meetings: a physical activity program, smoking habit change information and contact control. Abstinence decreased from 83% at the end of treatment to 73% at 3 months, 49% at six months and 34% at 18 months. No differences were shown in cessation across groups. Group differences were shown for subjective levels of tension-anxiety, those who exercised showed increased tension anxiety levels compared to subjects in the no activity groups.

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

    PubMed

    Borbiro, Istvan; Badheka, Doreen; Rohacs, Tibor

    2015-02-10

    Capsaicin is an activator of the heat-sensitive TRPV1 (transient receptor potential vanilloid 1) ion channels and has been used as a local analgesic. We found that activation of TRPV1 channels with capsaicin either in dorsal root ganglion neurons or in a heterologous expression system inhibited the mechanosensitive Piezo1 and Piezo2 channels by depleting phosphatidylinositol 4,5-bisphosphate [PI(4,5)P2] and its precursor phosphatidylinositol 4-phosphate [PI(4)P] from the plasma membrane through Ca(2+)-induced phospholipase Cδ (PLCδ) activation. Experiments with chemically inducible phosphoinositide phosphatases and receptor-induced activation of PLCβ indicated that inhibition of Piezo channels required depletion of both PI(4)P and PI(4,5)P2. The mechanically activated current amplitudes decreased substantially in the excised inside-out configuration, where the membrane patch containing Piezo1 channels is removed from the cell. PI(4,5)P2 and PI(4)P applied to these excised patches inhibited this decrease. Thus, we concluded that Piezo channel activity requires the presence of phosphoinositides, and the combined depletion of PI(4,5)P2 and PI(4)P reduces channel activity. In addition to revealing a role for distinct membrane lipids in mechanosensitive ion channel regulation, these data suggest that inhibition of Piezo2 channels may contribute to the analgesic effect of capsaicin. Copyright © 2015, American Association for the Advancement of Science.

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

    PubMed Central

    Borbiro, Istvan; Badheka, Doreen; Rohacs, Tibor

    2015-01-01

    Capsaicin is an activator of the heat-sensitive TRPV1 (transient receptor potential vanilloid 1) ion channels and has been used as a local analgesic. We found that activation of TRPV1 channels with capsaicin either in dorsal root ganglion neurons or in a heterologous expression system inhibited the mechanosensitive Piezo1 and Piezo2 channels by depleting phosphatidylinositol 4,5-bisphosphate [PI(4,5)P2] and its precursor PI(4)P from the plasma membrane through Ca2+-induced phospholipase Cδ (PLCδ) activation. Experiments with chemically inducible phosphoinositide phosphatases and receptor-induced activation of PLCβ indicated that inhibition of Piezo channels required depletion of both PI(4)P and PI(4,5)P2. The mechanically activated current amplitudes decreased substantially in the excised inside-out configuration, where the membrane patch containing Piezo1 channels is removed from the cell. PI(4,5)P2 and PI(4)P applied to these excised patches inhibited this decrease. Thus, we concluded that Piezo channel activity requires the presence of phosphoinositides, and the combined depletion of PI(4,5)P2 or PI(4)P reduces channel activity. In addition to revealing a role for distinct membrane lipids in mechanosensitive ion channel regulation, these data suggest that inhibition of Piezo2 channels may contribute to the analgesic effect of capsaicin. PMID:25670203

  17. Comparing Psychosocial Predictors of Physical Activity Adoption and Maintenance

    PubMed Central

    Williams, David M.; Lewis, Beth A.; Dunsiger, Shira; Whiteley, Jessica A.; Papandonatos, George D.; Napolitano, Melissa A.; Bock, Beth C.; Ciccolo, Joseph T.; Marcus, Bess H.

    2009-01-01

    Background Most health behavior models do not distinguish between determinants of behavior adoption and maintenance. Purpose This study compared psychosocial predictors of physical activity (PA) adoption and predictors of PA maintenance among 205 initially sedentary adults enrolled in a home-based PA promotion trial. Methods Psychosocial variables were measured at 6 months (at which point 107 participants remained inactive and 98 participants adopted regular PA) and used to predict 12-month PA status (an indicator of PA adoption among those inactive at 6 months and an indicator of PA maintenance among those active at 6 months). Results 6-month PA status moderated the relationships between 6-month measures of home access to PA equipment (p = .049), self-efficacy (p = .086), and perceived satisfaction (p = .062) and 12-month PA status. Simple effects analyses revealed that home access to PA equipment was predictive of PA adoption (OR = 1.73; 95% CI: 1.05, 2.85), but not PA maintenance (OR = 0.88; 95% CI: 0.58, 1.35), whereas self-efficacy and perceived satisfaction were predictive of PA maintenance (OR = 2.65; 95% CI: 1.55, 4.52; OR = 1.95; 95% CI: 0.93, 4.06), but not PA adoption (OR = 1.50; 95% CI: 0.87, 2.57; OR = 0.82, CI: 0.44, 1.52). Conclusion Results suggest that these psychosocial variables may operate differently in predicting PA adoption versus maintenance. PMID:18777124

  18. Skylab Experiment M516 - Crew Activities/Maintenance Study

    NASA Technical Reports Server (NTRS)

    Bond, R. L.

    1974-01-01

    Skylab required daily movement about the interior of a 340 cu m vehicle and the handling and transfer of numerous loose items. Planned and unplanned maintenance tasks were also included in the daily routine of activity. Experiment M516, Crew Activities/Maintenance Study, involved an investigation of crew activity during routine daily operations. The overall objective was to secure in-flight data relevant to the performance of tasks in the weightless environment. This paper will present an evaluation of man's ability to handle and transport items of various sizes and masses (logistics management) and to make equipment repairs (maintenance). Results and conclusions are based on subjective crew comments, motion-picture film, and television transmissions.

  19. Modulation of bone remodeling via mechanically activated ion channels

    NASA Technical Reports Server (NTRS)

    Duncan, Randall L. (Principal Investigator)

    1996-01-01

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

  20. Active dendrites, potassium channels and synaptic plasticity.

    PubMed Central

    Johnston, Daniel; Christie, Brian R; Frick, Andreas; Gray, Richard; Hoffman, Dax A; Schexnayder, Lalania K; Watanabe, Shigeo; Yuan, Li-Lian

    2003-01-01

    The dendrites of CA1 pyramidal neurons in the hippocampus express numerous types of voltage-gated ion channel, but the distributions or densities of many of these channels are very non-uniform. Sodium channels in the dendrites are responsible for action potential (AP) propagation from the axon into the dendrites (back-propagation); calcium channels are responsible for local changes in dendritic calcium concentrations following back-propagating APs and synaptic potentials; and potassium channels help regulate overall dendritic excitability. Several lines of evidence are presented here to suggest that back-propagating APs, when coincident with excitatory synaptic input, can lead to the induction of either long-term depression (LTD) or long-term potentiation (LTP). The induction of LTD or LTP is correlated with the magnitude of the rise in intracellular calcium. When brief bursts of synaptic potentials are paired with postsynaptic APs in a theta-burst pairing paradigm, the induction of LTP is dependent on the invasion of the AP into the dendritic tree. The amplitude of the AP in the dendrites is dependent, in part, on the activity of a transient, A-type potassium channel that is expressed at high density in the dendrites and correlates with the induction of the LTP. Furthermore, during the expression phase of the LTP, there are local changes in dendritic excitability that may result from modulation of the functioning of this transient potassium channel. The results support the view that the active properties of dendrites play important roles in synaptic integration and synaptic plasticity of these neurons. PMID:12740112

  1. Regulation of TRPM8 channel activity

    PubMed Central

    Yudin, Yevgen; Rohacs, Tibor

    2011-01-01

    Transient Receptor Potential Melastatin 8 (TRPM8) is a Ca2+ permeable non-selective cation channel directly activated by cold temperatures and chemical agonists such as menthol. It is a well established sensor of environmental cold temperatures, found in peripheral sensory neurons, where its activation evokes depolarization and action potentials. The activity of TRPM8 is regulated by a number of cellular signaling pathways, most notably by phosphoinositides and the activation of phospholipase C. This review will summarize current knowledge on the physiological and pathophysiological roles of TRPM8 and its regulation by various intracellular messenger molecules and signaling pathways. PMID:22061619

  2. Ca2+-activated K channels in parotid acinar cells

    PubMed Central

    Romanenko, Victor G; Thompson, Jill

    2010-01-01

    Fluid secretion relies on a close interplay between Ca2+-activated Cl and K channels. Salivary acinar cells contain both large conductance, BK, and intermediate conductance, IK1, K channels. Physiological fluid secretion occurs with only modest (<500 nM) increases in intracellular Ca2+ levels but BK channels in many cell types and in heterologous expression systems require very high concentrations for significant activation. We report here our efforts to understand this apparent contradiction. We determined the Ca2+ dependence of IK1 and BK channels in mouse parotid acinar cells. IK1 channels activated with an apparent Ca2+ affinity of about 350 nM and a hill coefficient near 3. Native parotid BK channels activated at similar Ca2+ levels unlike the BK channels in other cell types. Since the parotid BK channel is encoded by an uncommon splice variant, we examined this clone in a heterologous expression system. In contrast to the native parotid channel, activation of this expressed “parslo” channel required very high levels of Ca2+. In order to understand the functional basis for the special properties of the native channels, we analyzed the parotid BK channel in the context of the horrigan-Aldrich model of BK channel gating. We found that the shifted activation of parotid BK channels resulted from a hyperpolarizing shift of the voltage dependence of voltage sensor activation and channel opening and included a large change in the coupling of these two processes. PMID:20519930

  3. Operation and Maintenance. 9-Foot Navigation Channel, Upper Mississippi River, Head of Navigation to Guttenberg, Iowa. Volume 1. Narrative.

    DTIC Science & Technology

    1974-08-01

    N ~ V W _ __I’Ip . .71 Miss Marian Mlay Acting Regional Director Region V U.S. Department of Health , Education and Welfare New Post Office Building...VEGETATION AND ANIMAL LIFE 219 Pool Operation 219 Channel Maintenance 221 IMPACTS ON RECREATION 225 IMPACTS ON PUBLIC HEALTH AND SAFETY 227 ECONOMIC...sewer discharges increase bacteria to levels which create a health hazard. However, water quality throughout pool 1 permits a wider usage than in pool 2

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

    PubMed

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

    2016-01-05

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

  5. Metal interactions with voltage- and receptor-activated ion channels.

    PubMed Central

    Vijverberg, H P; Oortgiesen, M; Leinders, T; van Kleef, R G

    1994-01-01

    Effects of Pb and several other metal ions on various distinct types of voltage-, receptor- and Ca-activated ion channels have been investigated in cultured N1E-115 mouse neuroblastoma cells. Experiments were performed using the whole-cell voltage clamp and single-channel patch clamp techniques. External superfusion of nanomolar to submillimolar concentrations of Pb causes multiple effects on ion channels. Barium current through voltage-activated Ca channels is blocked by micromolar concentrations of Pb, whereas voltage-activated Na current appears insensitive. Neuronal type nicotinic acetylcholine receptor-activated ion current is blocked by nanomolar concentrations of Pb and this block is reversed at micromolar concentrations. Serotonin 5-HT3 receptor-activated ion current is much less sensitive to Pb. In addition, external superfusion with micromolar concentrations of Pb as well as of Cd and aluminum induces inward current, associated with the direct activation of nonselective cation channels by these metal ions. In excised inside-out membrane patches of neuroblastoma cells, micromolar concentrations of Ca activate small (SK) and big (BK) Ca-activated K channels. Internally applied Pb activates SK and BK channels more potently than Ca, whereas Cd is approximately equipotent to Pb with respect to SK channel activation, but fails to activate BK channels. The results show that metal ions cause distinct, selective effects on the various types of ion channels and that metal ion interaction sites of ion channels may be highly selective for particular metal ions. PMID:7531139

  6. Human temporal cortical single neuron activity during working memory maintenance.

    PubMed

    Zamora, Leona; Corina, David; Ojemann, George

    2016-06-01

    The Working Memory model of human memory, first introduced by Baddeley and Hitch (1974), has been one of the most influential psychological constructs in cognitive psychology and human neuroscience. However the neuronal correlates of core components of this model have yet to be fully elucidated. Here we present data from two studies where human temporal cortical single neuron activity was recorded during tasks differentially affecting the maintenance component of verbal working memory. In Study One we vary the presence or absence of distracting items for the entire period of memory storage. In Study Two we vary the duration of storage so that distractors filled all, or only one-third of the time the memory was stored. Extracellular single neuron recordings were obtained from 36 subjects undergoing awake temporal lobe resections for epilepsy, 25 in Study one, 11 in Study two. Recordings were obtained from a total of 166 lateral temporal cortex neurons during performance of one of these two tasks, 86 study one, 80 study two. Significant changes in activity with distractor manipulation were present in 74 of these neurons (45%), 38 Study one, 36 Study two. In 48 (65%) of those there was increased activity during the period when distracting items were absent, 26 Study One, 22 Study Two. The magnitude of this increase was greater for Study One, 47.6%, than Study Two, 8.1%, paralleling the reduction in memory errors in the absence of distracters, for Study One of 70.3%, Study Two 26.3% These findings establish that human lateral temporal cortex is part of the neural system for working memory, with activity during maintenance of that memory that parallels performance, suggesting it represents active rehearsal. In 31 of these neurons (65%) this activity was an extension of that during working memory encoding that differed significantly from the neural processes recorded during overt and silent language tasks without a recent memory component, 17 Study one, 14 Study two

  7. Managing Highway Maintenance: Maintenance Activities, Work Units, and Classifying Work, Unit 6, Level 2.

    ERIC Educational Resources Information Center

    Federal Highway Administration (DOT), Washington, DC. Offices of Research and Development.

    Part of the series "Managing Highway Maintenance," the unit explains how maintenance work should be described, measured, and classified. It is designed for supervisors who need to know the mechanics of describing work. The format is a programed, self-instruction approach in which information is presented in progressive segments or…

  8. RPMA (Real Property Maintenance Activities) Consolidation Activities in the National Capital Region. Volume 1. Main Report.

    DTIC Science & Technology

    1984-05-01

    planning the activities of future IPCs. Pre-IPC Activities Participating installations should have been directed through command channels to take the...the task with minimal impact on the existing A workload, and (2) ensure that the request for inforniation is formal and sent through channels . Planners...34... . .•.....¢,.. -...... ,- % * .*. ...... ....... .°. •.-.-.-. •..... 39. The District init inttei the cofl ract Ior <I’MA t i i on;, e i, ( I i HQDA through command channels . 40

  9. Maintenance of Physical Activity among Faculty and Staff in University Settings

    ERIC Educational Resources Information Center

    Whipple, Kerry; Kinney, Judy; Kattenbraker, Mark

    2008-01-01

    Previous studies have placed little emphasis on maintenance of healthy behaviors longer than six months. This study examined factors that contribute to maintenance of physical activity among faculty and staff in university settings. A 55-item survey on physical activity maintenance was used to assess attitudes towards exercise, exercise…

  10. Bisandrographolide from Andrographis paniculata activates TRPV4 channels.

    PubMed

    Smith, Paula L; Maloney, Katherine N; Pothen, Randy G; Clardy, Jon; Clapham, David E

    2006-10-06

    Many transient receptor potential (TRP) channels are activated or blocked by various compounds found in plants; two prominent examples include the activation of TRPV1 channels by capsaicin and the activation of TRPM8 channels by menthol. We sought to identify additional plant compounds that are active on other types of TRP channels. We screened a library of extracts from 50 Chinese herbal plants using a calcium-imaging assay to find compounds active on TRPV3 and TRPV4 channels. An extract from the plant Andrographis paniculata potently activated TRPV4 channels. The extract was fractionated further, and the active compound was identified as bisandrographolide A (BAA). We used purified compound to characterize the activity of BAA on certain TRPV channel subtypes. Although BAA activated TRPV4 channels with an EC(50) of 790-950 nm, it did not activate or block activation of TRPV1, TRPV2, or TRPV3 channels. BAA activated a large TRPV4-like current in immortalized mouse keratinocytes (308 cells) that have been shown to express TRPV4 protein endogenously. This compound also activated TRPV4 currents in cell-free outside-out patches from HEK293T cells overexpressing TRPV4 cDNA, suggesting that BAA can activate the channel in a membrane-delimited manner. Another related compound, andrographolide, found in abundance in the plant Andrographis was unable to activate or block activation of TRPV4 channels. These experiments show that BAA activates TRPV4 channels, and we discuss the possibility that activation of TRPV4 by BAA could play a role in some of the effects of Andrographis extract described in traditional medicine.

  11. Time Use Patterns between Maintenance, Subsistence and Leisure Activities: A Case Study in China

    ERIC Educational Resources Information Center

    Hui-fen, Zhou; Zhen-shan, Li; Dong-qian, Xue; Yang, Lei

    2012-01-01

    The Chinese government conducted its first time use survey of the activities of Chinese individuals in 2008. Activities were classified into three broad types, maintenance activities, subsistence activities and leisure activities. Time use patterns were defined by an individuals' time spent on maintenance, subsistence and leisure activities each…

  12. Time Use Patterns between Maintenance, Subsistence and Leisure Activities: A Case Study in China

    ERIC Educational Resources Information Center

    Hui-fen, Zhou; Zhen-shan, Li; Dong-qian, Xue; Yang, Lei

    2012-01-01

    The Chinese government conducted its first time use survey of the activities of Chinese individuals in 2008. Activities were classified into three broad types, maintenance activities, subsistence activities and leisure activities. Time use patterns were defined by an individuals' time spent on maintenance, subsistence and leisure activities each…

  13. A quantized mechanism for activation of pannexin channels

    PubMed Central

    Chiu, Yu-Hsin; Jin, Xueyao; Medina, Christopher B.; Leonhardt, Susan A.; Kiessling, Volker; Bennett, Brad C.; Shu, Shaofang; Tamm, Lukas K.; Yeager, Mark; Ravichandran, Kodi S.; Bayliss, Douglas A.

    2017-01-01

    Pannexin 1 (PANX1) subunits form oligomeric plasma membrane channels that mediate nucleotide release for purinergic signalling, which is involved in diverse physiological processes such as apoptosis, inflammation, blood pressure regulation, and cancer progression and metastasis. Here we explore the mechanistic basis for PANX1 activation by using wild type and engineered concatemeric channels. We find that PANX1 activation involves sequential stepwise sojourns through multiple discrete open states, each with unique channel gating and conductance properties that reflect contributions of the individual subunits of the hexamer. Progressive PANX1 channel opening is directly linked to permeation of ions and large molecules (ATP and fluorescent dyes) and occurs during both irreversible (caspase cleavage-mediated) and reversible (α1 adrenoceptor-mediated) forms of channel activation. This unique, quantized activation process enables fine tuning of PANX1 channel activity and may be a generalized regulatory mechanism for other related multimeric channels. PMID:28134257

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

    PubMed

    Trattnig, Sarah M; Gasiorek, Agnes; Deeb, Tarek Z; Ortiz, Eydith J Comenencia; Moss, Stephen J; Jensen, Anders A; Davies, Paul A

    2016-03-01

    The zinc-activated channel (ZAC) is a cationic ion channel belonging to the superfamily of Cys-loop receptors, which consists of pentameric ligand-gated ion channels. ZAC is the least understood member of this family so in the present study we sought to characterize the properties of this channel further. We demonstrate that not only zinc (Zn(2+)) but also copper (Cu(2+)) and protons (H(+)) are agonists of ZAC, displaying potencies and efficacies in the rank orders of H(+)>Cu(2+)>Zn(2+) and H(+)>Zn(2+)>Cu(2+), respectively. The responses elicited by Zn(2+), Cu(2+) and H(+) through ZAC are all characterized by low degrees of desensitization. In contrast, currents evoked by high concentrations of the three agonists comprise distinctly different activation and decay components, with transitions to and from an open state being significantly faster for H(+) than for the two metal ions. The permeabilities of ZAC for Na(+) and K(+) relative to Cs(+) are indistinguishable, whereas replacing all of extracellular Na(+) and K(+) with the divalent cations Ca(2+) or Mg(2+) results in complete elimination of Zn(2+)-activated currents at both negative and positive holding potentials. This indicates that ZAC is non-selectively permeable to monovalent cations, whereas Ca(2+) and Mg(2+) inhibit the channel. In conclusion, this is the first report of a Cys-loop receptor being gated by Zn(2+), Cu(2+) and H(+). ZAC could be an important mediator of some of the wide range of physiological functions regulated by or involving Zn(2+), Cu(2+) and H(+). Copyright © 2016 Elsevier Inc. All rights reserved.

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

    PubMed Central

    Trattnig, Sarah M.; Gasiorek, Agnes; Deeb, Tarek Z.; Comenencia Ortiz, Eydith J.; Moss, Stephen J.; Jensen, Anders A.; Davies, Paul A.

    2016-01-01

    The zinc-activated channel (ZAC) is a cationic ion channel belonging to the superfamily of Cys-loop receptors, which consists of pentameric ligand-gated ion channels. ZAC is the least understood member of this family so in the present study we sought to characterize the properties of this channel further. We demonstrate that not only zinc (Zn2+) but also copper (Cu2+) and protons (H+) are agonists of ZAC, displaying potencies and efficacies in the rank orders of H+ > Cu2+ > Zn2+ and H+ > Zn2+ > Cu2+, respectively. The responses elicited by Zn2+, Cu2+ and H+ through ZAC are all characterized by low degrees of desensitization. In contrast, currents evoked by high concentrations of the three agonists comprise distinctly different activation and decay components, with transitions to and from an open state being significantly faster for H+ than for the two metal ions. The permeabilities of ZAC for Na+ and K+ relative to Cs+ are indistinguishable, whereas replacing all of extracellular Na+ and K+ with the divalent cations Ca2+ or Mg2+ results in complete elimination of Zn2+-activated currents at both negative and positive holding potentials. This indicates that ZAC is non-selectively permeable to monovalent cations, whereas Ca2+ and Mg2+ inhibit the channel. In conclusion, this is the first report of a Cys-loop receptor being gated by Zn2+, Cu2+ and H+. ZAC could be an important mediator of some of the wide range of physiological functions regulated by or involving Zn2+, Cu2+ and H+. PMID:26872532

  16. Determinants and benefits of physical activity maintenance in hospital employees.

    PubMed

    Lavoie-Tremblay, Mélanie; Sounan, Charles; Martin, Kara; Trudel, Julie G; Lavigne, Genevieve L; Grover, Steven A; Lowensteyn, Ilka

    2014-01-01

    This study investigated whether the positive behavioral and anthropometric outcomes of a pedometer-based physical activity 8-week challenge were maintained 6 months after the end of the program. It further investigated the motivational profile of those who maintained their physical activity levels in the months following the end of the program and of those who did not. Hospital employees from a university-affiliated multisite health care center in Canada participated using a questionnaire. Of the 235 participants who completed the 8-week challenge, 157 questionnaires were returned 6 months later. Paired-samples t tests were conducted between the baseline and follow-up scores as well as between the postprogram and follow-up scores to detect significant differences between the measurement points. This study shows that the pedometer-based physical activity helped hospital employees maintain a high level of physical activity as well as maintain a healthy body mass index after 6 months. The results demonstrated that during maintenance the high physical activity group obtained higher scores for identified regulation and intrinsic regulation compared with the other groups. The results of the study revealed that identified and intrinsic regulations are important contributors to maintaining physical activity among hospital employees.

  17. Revalidation of the Volatile Organic Analyzer Following a Major On-Orbit Maintenance Activity

    NASA Technical Reports Server (NTRS)

    Limero, Thomas; James, John T.

    2007-01-01

    The Volatile Organic Analyzer (VOA) contributes to the assessment of air quality aboard the International Space Station (ISS) by identifying and quantifying target airborne volatile organic contaminants in the module air. This on-orbit contaminant monitoring capability becomes particularly important during an air quality degradation event such as a system leak. During several ISS air quality degradations, the VOA has generated near real-time data that was used to make decisions or to better understand the contingency. The VOA was operational from January 2002 through June 2003, during which time it was validated by comparing VOA data to simultaneously acquired grab sample containers (GSCs). In January 2003, one of the two analytical channels of the VOA was shutdown because of a component failure, but a redundant channel continued to supply the necessary analytical data. In June 2003, the sole remaining channel was deactivated. Initial assessments of the channel shutdowns pointed to failed fuses or heaters, but neither was considered repairable on orbit. In 2005, it was determined that failed fuses could be replaced on orbit and the crew conducted a diagnostic procedure to identify the failed component. The crew discovered that both channels incurred failed fuses, which lead to a subsequent on orbit maintenance activity and return of the VOA to operational status in December 2005. The VOA has been providing data on the ISS atmosphere since its reactivation in 2005 and this paper will present the VOA data collected during 2006. Special emphasis will be placed upon the revalidation of the repaired VOA using GSCs as well as a summary of the diagnostic and repair procedures.

  18. Single Na+ channels activated by veratridine and batrachotoxin

    PubMed Central

    1987-01-01

    Voltage-sensitive Na+ channels from rat skeletal muscle plasma membrane vesicles were inserted into planar lipid bilayers in the presence of either of the alkaloid toxins veratridine (VT) or batrachotoxin (BTX). Both of these toxins are known to cause persistent activation of Na+ channels. With BTX as the channel activator, single channels remain open nearly all the time. Channels activated with VT open and close on a time scale of 1-10 s. Increasing the VT concentration enhances the probability of channel opening, primarily by increasing the rate constant of opening. The kinetics and voltage dependence of channel block by 21-sulfo-11-alpha-hydroxysaxitoxin are identical for VT and BTX, as is the ionic selectivity sequence determined by bi-ionic reversal potential (Na+ approximately Li+ greater than K+ greater than Rb+ greater than Cs+). However, there are striking quantitative differences in open channel conduction for channels in the presence of the two activators. Under symmetrical solution conditions, the single channel conductance for Na+ is about twice as high with BTX as with VT. Furthermore, the symmetrical solution single channel conductances show a different selectivity for BTX (Na+ greater than Li+ greater than K+) than for VT (Na+ greater than K+ greater than Li+). Open channel current-voltage curves in symmetrical Na+ and Li+ are roughly linear, while those in symmetrical K+ are inwardly rectifying. Na+ currents are blocked asymmetrically by K+ with both BTX and VT, but the voltage dependence of K+ block is stronger with BTX than with VT. The results show that the alkaloid neurotoxins not only alter the gating process of the Na+ channel, but also affect the structure of the open channel. We further conclude that the rate-determining step for conduction by Na+ does not occur at the channel's "selectivity filter," where poorly permeating ions like K+ are excluded. PMID:2435846

  19. Slack sodium-activated potassium channel membrane expression requires p38 mitogen-activated protein kinase phosphorylation.

    PubMed

    Gururaj, Sushmitha; Fleites, John; Bhattacharjee, Arin

    2016-04-01

    p38 MAPK has long been understood as an inducible kinase under conditions of cellular stress, but there is now increasing evidence to support its role in the regulation of neuronal function. Several phosphorylation targets have been identified, an appreciable number of which are ion channels, implicating the possible involvement of p38 MAPK in neuronal excitability. The KNa channel Slack is an important protein to be studied as it is highly and ubiquitously expressed in DRG neurons and is important in the maintenance of their firing accommodation. We sought to examine if the Slack channel could be a substrate of p38 MAPK activity. First, we found that the Slack C-terminus contains two putative p38 MAPK phosphorylation sites that are highly conserved across species. Second, we show via electrophysiology experiments that KNa currents and further, Slack currents, are subject to tonic modulation by p38 MAPK. Third, biochemical approaches revealed that Slack channel regulation by p38 MAPK occurs through direct phosphorylation at the two putative sites of interaction, and mutating both sites prevented surface expression of Slack channels. Based on these results, we conclude that p38 MAPK is an obligate regulator of Slack channel function via the trafficking of channels into the membrane. The present study identifies Slack KNa channels as p38 MAPK substrates.

  20. Structure of Thermally Activated TRP Channels

    PubMed Central

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

    2015-01-01

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

  1. Structure of thermally activated TRP channels.

    PubMed

    Cohen, Matthew R; Moiseenkova-Bell, Vera Y

    2014-01-01

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

  2. Low voltage activated calcium channels: from genes to function.

    PubMed

    Lacinová, L; Klugbauer, N; Hofmann, F

    2000-06-01

    Cloning of three members of low-voltage-activated (LVA) calcium channel family, predominantly neuronal alpha1G and alpha1I, and ubiquitous alpha1H, enabled to investigate directly their electrophysiological and pharmacological profile as well as their putative subunit composition. All the three channels are half-activated at membrane potential about -40 mV and half-inactivated at about -70 mV. Kinetics of alpha1G and alpha1H channels activation and inactivation are similar and faster than that of alpha1I channel. All the three channels are blocked with high affinity by the organic blocker mibefradil. Another high affinity blocker is kurtoxin. Cloned LVA channels are relatively insensitive to antiepileptics, dihydropyridines and omega-conotoxins. Ni2+ is high affinity blocker of alpha1H channel only. Amiloride inhibits the alpha1H channel. The subunit composition of LVA channel remains unclear. Out of known high-voltage-activated calcium channel subunits, alpha2delta-2 and gamma-5 subunits significantly and systematically modified activation and/or inactivation of the current. In contrast, alpha2delta-1, alpha2delta-3, gamma-2 and gamma-4 subunits failed to modulate the current or had only minor effects.

  3. Phenotypes influencing low physical activity in maintenance dialysis.

    PubMed

    Panaye, Marine; Kolko-Labadens, Anne; Lasseur, Catherine; Paillasseur, Jean-Louis; Guillodo, Marie Paule; Levannier, Martial; Teta, Daniel; Fouque, Denis

    2015-01-01

    The "Pas à Pas" initiative aimed at evaluating the weekly physical activity (PA) and its determinants in a large cohort of dialysis patients. Physical inactivity is a risk factor for mortality in maintenance dialysis patients and is still poorly documented in this population. A prospective national epidemiological study was performed. A total of 1,163 patients on maintenance dialysis (hemodialysis and peritoneal dialysis) were included. PA was recorded during seven consecutive days using a pedometer to measure daily step numbers. Median age was 63 years (Q1 51-Q3 75). Sixty-three percent were sedentary (<5000 steps/day) with a median of 3,688 steps/day (1,866-6,271)]. PA level was similar between hemodialysis patients and those on peritoneal dialysis (3,693 steps [1,896-6,307] vs. 3,320 [1,478-5,926], P = .33). In hemodialysis patients, PA was lower on dialysis days compared with nondialysis days (2,912 [1,439-5,232] vs. 4,054 [2,136-7,108], respectively, P < .01). PA gradually decreased with age, 57% being sedentary between 50 and 65 years and 83% of patients after 80 years. Beyond this age effect, we identified, for the first time, specific phenotypes of patients with lower PA, such as inflammation, cardiovascular disease, protein energy wasting, obesity, and diabetes. By contrast, previous kidney transplantation and a higher muscle mass were associated with higher PA. Dialysis patients present a very low level of PA with high sedentary. Acting on patient's modifiable phenotypes may help to increase PA to improve morbidity, mortality, and quality of life. Copyright © 2015 National Kidney Foundation, Inc. Published by Elsevier Inc. All rights reserved.

  4. Resveratrol attenuates cortical neuron activity: roles of large conductance calcium-activated potassium channels and voltage-gated sodium channels.

    PubMed

    Wang, Ya-Jean; Chan, Ming-Huan; Chen, Linyi; Wu, Sheng-Nan; Chen, Hwei-Hisen

    2016-05-21

    Resveratrol, a phytoalexin found in grapes and red wine, exhibits diverse pharmacological activities. However, relatively little is known about whether resveratrol modulates the ion channels in cortical neurons. The large-conductance calcium-activated potassium channels (BKCa) and voltage-gated sodium channels were expressed in cortical neurons and play important roles in regulation of neuronal excitability. The present study aimed to determine the effects of resveratrol on BKCa currents and voltage-gated sodium currents in cortical neurons. Resveratrol concentration-dependently increased the current amplitude and the opening activity of BKCa channels, but suppressed the amplitude of voltage-gated sodium currents. Similar to the BKCa channel opener NS1619, resveratrol decreased the firing rate of action potentials. In addition, the enhancing effects of BKCa channel blockers tetraethylammonium (TEA) and paxilline on action potential firing were sensitive to resveratrol. Our results indicated that the attenuation of action potential firing rate by resveratrol might be mediated through opening the BKCa channels and closing the voltage-gated sodium channels. As BKCa channels and sodium channels are critical molecular determinants for seizure generation, our findings suggest that regulation of these two channels in cortical neurons probably makes a considerable contribution to the antiseizure activity of resveratrol.

  5. Regulation of Sodium Channel Activity by Capping of Actin Filaments

    PubMed Central

    Shumilina, Ekaterina V.; Negulyaev, Yuri A.; Morachevskaya, Elena A.; Hinssen, Horst; Khaitlina, Sofia Yu

    2003-01-01

    Ion transport in various tissues can be regulated by the cortical actin cytoskeleton. Specifically, involvement of actin dynamics in the regulation of nonvoltage-gated sodium channels has been shown. Herein, inside-out patch clamp experiments were performed to study the effect of the heterodimeric actin capping protein CapZ on sodium channel regulation in leukemia K562 cells. The channels were activated by cytochalasin-induced disruption of actin filaments and inactivated by G-actin under ionic conditions promoting rapid actin polymerization. CapZ had no direct effect on channel activity. However, being added together with G-actin, CapZ prevented actin-induced channel inactivation, and this effect occurred at CapZ/actin molar ratios from 1:5 to 1:100. When actin was allowed to polymerize at the plasma membrane to induce partial channel inactivation, subsequent addition of CapZ restored the channel activity. These results can be explained by CapZ-induced inhibition of further assembly of actin filaments at the plasma membrane due to the modification of actin dynamics by CapZ. No effect on the channel activity was observed in response to F-actin, confirming that the mechanism of channel inactivation does not involve interaction of the channel with preformed filaments. Our data show that actin-capping protein can participate in the cytoskeleton-associated regulation of sodium transport in nonexcitable cells. PMID:12686620

  6. Fast activation of dihydropyridine-sensitive calcium channels of skeletal muscle. Multiple pathways of channel gating

    PubMed Central

    1996-01-01

    Dihydropyridine (DHP) receptors of the transverse tubule membrane play two roles in excitation-contraction coupling in skeletal muscle: (a) they function as the voltage sensor which undergoes fast transition to control release of calcium from sarcoplasmic reticulum, and (b) they provide the conducting unit of a slowly activating L-type calcium channel. To understand this dual function of the DHP receptor, we studied the effect of depolarizing conditioning pulse on the activation kinetics of the skeletal muscle DHP-sensitive calcium channels reconstituted into lipid bilayer membranes. Activation of the incorporated calcium channel was imposed by depolarizing test pulses from a holding potential of -80 mV. The gating kinetics of the channel was studied with ensemble averages of repeated episodes. Based on a first latency analysis, two distinct classes of channel openings occurred after depolarization: most had delayed latencies, distributed with a mode of 70 ms (slow gating); a small number of openings had short first latencies, < 12 ms (fast gating). A depolarizing conditioning pulse to +20 mV placed 200 ms before the test pulse (-10 mV), led to a significant increase in the activation rate of the ensemble averaged-current; the time constant of activation went from tau m = 110 ms (reference) to tau m = 45 ms after conditioning. This enhanced activation by the conditioning pulse was due to the increase in frequency of fast open events, which was a steep function of the intermediate voltage and the interval between the conditioning pulse and the test pulse. Additional analysis demonstrated that fast gating is the property of the same individual channels that normally gate slowly and that the channels adopt this property after a sojourn in the open state. The rapid secondary activation seen after depolarizing prepulses is not compatible with a linear activation model for the calcium channel, but is highly consistent with a cyclical model. A six- state cyclical model is

  7. Upregulation of apical sodium-chloride cotransporter and basolateral chloride channels is responsible for the maintenance of salt-sensitive hypertension.

    PubMed

    Capasso, Giovambattista; Rizzo, Maria; Garavaglia, Maria Lisa; Trepiccione, Francesco; Zacchia, Miriam; Mugione, Alessandra; Ferrari, Patrizia; Paulmichl, Markus; Lang, Florian; Loffing, Johannes; Carrel, Monique; Damiano, Sara; Wagner, Carsten A; Bianchi, Giuseppe; Meyer, Giuliano

    2008-08-01

    We investigated which of the NaCl transporters are involved in the maintenance of salt-sensitive hypertension. Milan hypertensive (MHS) rats were studied 3 mo after birth. In MHS, compared with normotensive strain (MNS), mRNA abundance, quantified by competitive PCR on isolated tubules, was unchanged, both for Na+/H+ isoform 3 (NHE3) and Na+-K+-2Cl- (NKCC2), but higher (119%, n = 5, P < 0.005) for Na+-Cl- (NCC) in distal convoluted tubules (DCT). These results were confirmed by Western blots, which revealed: 1) unchanged NHE3 in the cortex and NKCC2 in the outer medulla; 2) a significant increase (52%, n = 6, P < 0.001) of NCC in the cortex; 3) alpha- and beta-sodium channels [epithelial Na+ channel (ENaC)] unaffected in renal cortex and slightly reduced in the outer medulla, while gamma-ENaC remained unchanged. Pendrin protein expression was unaffected. The role of NCC was reinforced by immunocytochemical studies showing increased NCC on the apical membrane of DCT cells of MHS animals, and by clearance experiments demonstrating a larger sensitivity (P < 0.001) to bendroflumethiazide in MHS rats. Kidney-specific chloride channels (ClC-K) were studied by Western blot experiments on renal cortex and by patch-clamp studies on primary culture of DCT dissected from MNS and MHS animals. Electrophysiological characteristics of ClC-K channels were unchanged in MHS rats, but the number of active channels in a patch was 0.60 +/- 0.21 (n = 35) in MNS rats and 2.17 +/- 0.59 (n = 23) in MHS rats (P < 0.05). The data indicate that, in salt-sensitive hypertension, there is a strong upregulation, both of NCC and ClC-K along the DCT, which explains the persistence of hypertension.

  8. Distinct contributions of small and large conductance Ca2+-activated K+ channels to rat Purkinje neuron function

    PubMed Central

    Edgerton, Jeremy R; Reinhart, Peter H

    2003-01-01

    The cerebellum is important for many aspects of behaviour, from posture maintenance and goal-oriented reaching movements to timing tasks and certain forms of learning. In every case, information flowing through the cerebellum passes through Purkinje neurons, which receive input from the two primary cerebellar afferents and generate continuous streams of action potentials that constitute the sole output from the cerebellar cortex to the deep nuclei. The tonic firing behaviour observed in Purkinje neurons in vivo is maintained in brain slices even when synaptic inputs are blocked, suggesting that Purkinje neuron activity relies to a significant extent on intrinsic conductances. Previous research has suggested that the interplay between Ca2+ currents and Ca2+-activated K+ channels (KCa channels) is important for Purkinje cell activity, but how many different KCa channel types are present and what each channel type contributes to cell behaviour remains unclear. In order to better understand the ionic mechanisms that control the behaviour of these neurons, we investigated the effects of different Ca2+ channel and KCa channel antagonists on Purkinje neurons in acute slices of rat cerebellum. Our data show that Ca2+ entering through P-type voltage-gated Ca2+ channels activates both small-conductance (SK) and large-conductance (BK) KCa channels. SK channels play a role in setting the intrinsic firing frequency, while BK channels regulate action potential shape and may contribute to the unique climbing fibre response. PMID:12576503

  9. Molecular mechanism of pharmacological activation of BK channels

    PubMed Central

    Gessner, Guido; Cui, Yong-Mei; Otani, Yuko; Ohwada, Tomohiko; Soom, Malle; Hoshi, Toshinori; Heinemann, Stefan H.

    2012-01-01

    Large-conductance voltage- and Ca2+-activated K+ (Slo1 BK) channels serve numerous cellular functions, and their dysregulation is implicated in various diseases. Drugs activating BK channels therefore bear substantial therapeutic potential, but their deployment has been hindered in part because the mode of action remains obscure. Here we provide mechanistic insight into how the dehydroabietic acid derivative Cym04 activates BK channels. As a representative of NS1619-like BK openers, Cym04 reversibly left-shifts the half-activation voltage of Slo1 BK channels. Using an established allosteric BK gating model, the Cym04 effect can be simulated by a shift of the voltage sensor and the ion conduction gate equilibria toward the activated and open state, respectively. BK activation by Cym04 occurs in a splice variant-specific manner; it does not occur in such Slo1 BK channels using an alternative neuronal exon 9, which codes for the linker connecting the transmembrane segment S6 and the cytosolic RCK1 domain—the S6/RCK linker. In addition, Cym04 does not affect Slo1 BK channels with a two-residue deletion within this linker. Mutagenesis and model-based gating analysis revealed that BK openers, such as Cym04 and NS1619 but not mallotoxin, activate BK channels by functionally interacting with the S6/RCK linker, mimicking site-specific shortening of this purported passive spring, which transmits force from the cytosolic gating ring structure to open the channel's gate. PMID:22331907

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

    PubMed

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

    2016-07-05

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

  11. Hypoglycemia-activated K+ channels in hippocampal neurons.

    PubMed

    Tromba, C; Salvaggio, A; Racagni, G; Volterra, A

    1992-08-31

    Channels linking the electrical and metabolic activities of cells (KATP channels) have been described in various tissues, including some brain areas (hypothalamus, cerebral cortex and substantia nigra). Here we report the existence in hippocampal neurons of K+ permeant channels whose activity is regulated by extracellular glucose. They are open at the cell resting potential and respond to transient hypoglycemia with a reversible increase in activity. The one type so far characterized has a conductance of approximately 100 pS in isotonic K+, is inhibited by the sulphonylurea glibenclamide (1 microM), and is activated by the potassium channel opener lemakalim (0.1-1 microM). These data provide a direct demonstration of the presence, in hippocampal neurons, of glucose-sensitive channels that could belong to the KATP family.

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

    PubMed

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

    2006-05-01

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

  13. Measures of the risk impacts of testing and maintenance activities

    SciTech Connect

    Vesely, W.E.; Davis, T.C.; Saltos, N.

    1983-11-01

    This report is a companion to Measures of Risk Importance and Their Applications (NUREG/CR-3385). This report focuses on quantifying the importance of online tests, maintenances, repairs in controlling the risk. The importance measures which are defined are applied to evaluate the corrective and preventative benefits of online testing, maintenance, and repair. The corrective and preventative benefits are measured in terms of the impacts in risk which result. The corrective value of a repair or unscheduled maintenance action is termed the restoration worth of the action and is defined to be the reduction in risk which results from restoring a failed component or system. The preventative value of a test or maintenance action is termed the expected worth of the action and is defined to be the expected risk reduction from performing the action. There is a great deal of information obtainable from these kinds of measures which can be simply calculated in any PRA and enhance the usefulness of the PRA. If there is a general message from this work regarding risk assurance programs and inspection programs it is on the usefulness of risk analysis techniques in these programs. To be most effective with regard to risk control inspection programs and risk assurance programs directed at testing and maintenance should be guided by the risk impacts of the test or maintenance.

  14. Role of KATP channels in the maintenance of ventricular fibrillation in cardiomyopathic human hearts.

    PubMed

    Farid, Talha A; Nair, Krishnakumar; Massé, Stephéne; Azam, Mohammed Ali; Maguy, Ange; Lai, Patrick F H; Umapathy, Karthikeyan; Dorian, Paul; Chauhan, Vijay; Varró, András; Al-Hesayen, Abdul; Waxman, Menashe; Nattel, Stanley; Nanthakumar, Kumaraswamy

    2011-11-11

    Ventricular fibrillation (VF) leads to global ischemia. The modulation of ischemia-dependent pathways may alter the electrophysiological evolution of VF. We addressed the hypotheses that there is regional disease-related expression of K(ATP) channels in human cardiomyopathic hearts and that K(ATP) channel blockade promotes spontaneous VF termination by attenuating spatiotemporal dispersion of refractoriness. In a human Langendorff model, electric mapping of 6 control and 9 treatment (10 μmol/L glibenclamide) isolated cardiomyopathic hearts was performed. Spontaneous defibrillation was studied and mean VF cycle length was compared regionally at VF onset and after 180 seconds between control and treatment groups. K(ATP) subunit gene expression was compared between LV endocardium versus epicardium in myopathic hearts. Spontaneous VF termination occurred in 1 of 6 control hearts and 7 of 8 glibenclamide-treated hearts (P=0.026). After 180 seconds of ischemia, a transmural dispersion in VF cycle length was observed between epicardium and endocardium (P=0.001), which was attenuated by glibenclamide. There was greater gene expression of all K(ATP) subunit on the endocardium compared with the epicardium (P<0.02). In an ischemic rat heart model, transmural dispersion of refractoriness (ΔERP(Transmural)=ERP(Epicardium)-ERP(Endocardium)) was verified with pacing protocols. ΔERP(Transmural) in control was 5 ± 2 ms and increased to 36 ± 5 ms with ischemia. This effect was greatly attenuated by glibenclamide (ΔERP(Transmural) for glibenclamide+ischemia=4.9 ± 4 ms, P=0.019 versus control ischemia). K(ATP) channel subunit gene expression is heterogeneously altered in the cardiomyopathic human heart. Blockade of K(ATP) channels promotes spontaneous defibrillation in cardiomyopathic human hearts by attenuating the ischemia-dependent spatiotemporal heterogeneity of refractoriness during early VF.

  15. Repair, Evaluation, Maintenance, and Rehabilitation Research Program. Geotechnical Aspects of Rock Erosion in Emergency Spillway Channels

    DTIC Science & Technology

    1986-08-01

    Formations arp exposed at the damsite. The Grafford Formation is covered ini the valley but is well exposed in the lower end of the spillway discharge...channel. The formation consista prodominantly of soft-to-moderately hard multicolored shales and a 20+ ft-thick sandstone bed occurring approximately...20 ft below the base of the overlying Winchell Formation . 39 7/ "LAN Mai 10 1 ’* C- ’Sao isvefl - - w~ -cIsm ,ISO Figure 19. Spillway plan and

  16. Flow-activated ion channels in vascular endothelium.

    PubMed

    Gautam, Mamta; Gojova, Andrea; Barakat, Abdul I

    2006-01-01

    The ability of vascular endothelial cells (ECs) to respond to fluid mechanical forces associated with blood flow is essential for flow-mediated vasoregulation and arterial wall remodeling. Abnormalities in endothelial responses to flow also play a role in the development of atherosclerosis. Although our understanding of the endothelial signaling pathways stimulated by flow has greatly increased over the past two decades, the mechanisms by which ECs sense flow remain largely unknown. Activation of flow-sensitive ion channels is among the fastest known endothelial responses to flow; therefore, these ion channels have been proposed as candidate flow sensors. This review focuses on: 1) describing the various types of flow-sensitive ion channels that have been reported in ECs, 2) discussing the implications of activation of these ion channels for endothelial function, and 3) proposing candidate mechanisms for activation of flow-sensitive ion channels.

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

    PubMed Central

    Kaczmarek, Leonard K.

    2013-01-01

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

  18. Impacts of maintenance channel dredging in a northern Adriatic coastal lagoon. I: Effects on sediment properties, contamination and toxicity

    NASA Astrophysics Data System (ADS)

    Guerra, Roberta; Pasteris, Andrea; Ponti, Massimo

    2009-10-01

    Conservation and management of coastal lagoons envisage direct human intervention. To prevent siltation and to preserve the hydrodynamics features of the lagoon system, the inner channels undergo regular maintenance dredging. Sediment properties (RDP, organic matter, grain size), trace metals (Cd, Cu, Cr, Hg, Ni, and Pb), and toxicity vs. the amphipod Corophium insidiosum and the luminescent bacterium Vibrio fischeri, were analysed before and after dredging operations in a coastal lagoon (Pialassa Baiona, Italy). To detect the actual impacts, disturbed sites were contrasted with multiple controls in two distinct times, i.e. before and after disturbance, according to a sampling design based on Beyond BACI principles. The integrated methodology here adopted suggests that dredging operations carried out are not likely to pose dramatic effects on environmental quality of the lagoon.

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

    PubMed

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

    2016-04-01

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

  20. Open-channel block by internally applied amines inhibits activation gate closure in batrachotoxin-activated sodium channels.

    PubMed Central

    Zamponi, G W; French, R J

    1994-01-01

    We have studied the action of several pore-blocking amines on voltage-dependent activation gating of batrachotoxin(BTX)-activated sodium channels, from bovine heart and rat skeletal muscle, incorporated into planar lipid bilayers. Although structurally simpler, the compounds studied show general structural features and channel-inhibiting actions that resemble those of lidocaine. When applied to the cytoplasmic end of the channel, these compounds cause a rapid, voltage-dependent, open-channel block seen as a reduction in apparent single-channel amplitude (companion paper). Internal application of phenylpropanolamine, phenylethylamine, phenylmethylamine, and diethylamine, as well as causing open-channel block, reduces the probability of channel closure, producing a shift of the steady-state activation curve toward more hyperpolarizing potentials. These gating effects were observed for both cardiac and skeletal muscle channels and were not evoked by addition of equimolar N-Methyl-D-Glucamine, suggesting a specific interaction of the blockers with the channel rather than a surface charge effect. Kinetic analysis of phenylpropanolamine action on skeletal muscle channels indicated that phenylpropanolamine reduced the closed probability via two separate mechanisms. First, mean closed durations were slightly abbreviated in its presence. Second, and more important, the frequency of the gating closures was reduced. This action was correlated with the degree, and the voltage dependence, of open-channel block, suggesting that the activation gate cannot close while the pore is occluded by the blocker. Such a mechanism might underlie the previously reported immobilization of gating charge associated with local anesthetic block of unmodified sodium channels. PMID:7811914

  1. Small Conductance Ca2+-Activated K+ Channels and Cardiac Arrhythmias

    PubMed Central

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

    2015-01-01

    Small conductance Ca2+-activated K+ (SK, KCa2) channels are unique in that they are gated solely by changes in intracellular Ca2+ and hence, function to integrate intracellular Ca2+ and membrane potentials on a beat-to-beat basis. Recent studies have provided evidence for the existence and functional significance of SK channels in the heart. Indeed, our knowledge of cardiac SK channels has been greatly expanded over the past decade. Interests in cardiac SK channels are further driven by recent studies suggesting the critical roles of SK channels in human atrial fibrillation, SK channel as a possible novel therapeutic target in atrial arrhythmias and up-regulation of SK channels in heart failure (HF) in animal models and human HF. However, there remain critical gaps in our knowledge. Specifically, blockade of SK channels in cardiac arrhythmias has been shown to be both anti-arrhythmic and proarrhythmic. This contemporary review will provide an overview of the literature on the role of cardiac SK channels in cardiac arrhythmias and to serve as a discussion platform for the current clinical perspectives. At the translational level, development of SK channel blockers as a new therapeutic target in the treatment of atrial fibrillation and the possible pro-arrhythmic effects merit further considerations and investigations. PMID:25956967

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

    PubMed

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

    2016-06-05

    Small-conductance calcium-activated potassium (SK) channels mediate a potassium conductance in the brain and are involved in synaptic plasticity, learning, and memory. SK channels show a distinct subcellular localization that is crucial for their neuronal functions. However, the mechanisms that control this spatial distribution are unknown. We imaged SK channels labeled with fluorophore-tagged apamin and monitored SK channel nanoclustering at the single molecule level by combining atomic force microscopy and toxin (i.e., apamin) pharmacology. Using these two complementary approaches, we found that native SK channel distribution in pyramidal neurons, across the somatodendritic domain, depends on ongoing cyclic adenosine monophosphate (cAMP)-protein kinase A (PKA) levels, strongly limiting SK channel expression at the pyramidal neuron soma. Furthermore, tonic cAMP-PKA levels also controlled whether SK channels were expressed in nanodomains as single entities or as a group of multiple channels. Our study reveals a new level of regulation of SK channels by cAMP-PKA and suggests that ion channel topography and nanoclustering might be under the control of second messenger cascades. Copyright © 2016 Elsevier Ltd. All rights reserved.

  3. Coexpression and activation of TRPV1 suppress the activity of the KCNQ2/3 channel

    PubMed Central

    Zhang, Xu-Feng; Han, Ping; Neelands, Torben R.; McGaraughty, Steve; Honore, Prisca; Surowy, Carol S.

    2011-01-01

    Transient receptor potential vanilloid 1 (TRPV1) is a ligand-gated nonselective cation channel expressed predominantly in peripheral nociceptors. By detecting and integrating diverse noxious thermal and chemical stimuli, and as a result of its sensitization by inflammatory mediators, the TRPV1 receptor plays a key role in inflammation-induced pain. Activation of TRPV1 leads to a cascade of pro-nociceptive mechanisms, many of which still remain to be identified. Here, we report a novel effect of TRPV1 on the activity of the potassium channel KCNQ2/3, a negative regulator of neuronal excitability. Using ion influx assays, we revealed that TRPV1 activation can abolish KCNQ2/3 activity, but not vice versa, in human embryonic kidney (HEK)293 cells. Electrophysiological studies showed that coexpression of TRPV1 caused a 7.5-mV depolarizing shift in the voltage dependence of KCNQ2/3 activation compared with control expressing KCNQ2/3 alone. Furthermore, activation of TRPV1 by capsaicin led to a 54% reduction of KCNQ2/3-mediated current amplitude and attenuation of KCNQ2/3 activation. The inhibitory effect of TRPV1 appears to depend on Ca2+ influx through the activated channel followed by Ca2+-sensitive depletion of phosphatidylinositol 4,5-bisphosphate and activation of protein phosphatase calcineurin. We also identified physical interactions between TRPV1 and KCNQ2/3 coexpressed in HEK293 cells and in rat dorsal root ganglia neurons. Mutation studies established that this interaction is mediated predominantly by the membrane-spanning regions of the respective proteins and correlates with the shift of KCNQ2/3 activation. Collectively, these data reveal that TRPV1 activation may deprive neurons from inhibitory control mediated by KCNQ2/3. Such neurons may thus have a lower threshold for activation, which may indirectly facilitate TRPV1 in integrating multiple noxious signals and/or in the establishment or maintenance of chronic pain. PMID:21844219

  4. Finding of No Significant Impact, Skiffes Creek Federal Navigation Channel Maintenance Dredging, Joint Base Langley-Eustis, Fort Eustis, Virginia

    DTIC Science & Technology

    2014-06-06

    sediment controls and best management practices will be applied to maintenance activities involving upland soil disturbance at the Fort Eustis Dredged...Virginia’s erosion and sediment control program was created “for the effective control of soil erosion, sediment deposition and nonagricultural runoff...However, this level was within the mean soil background range of 0.1 to 40 mg/kg. Kepone was not detected in sediment samples in April 2002 (IMS

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

    PubMed Central

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

    2015-01-01

    All cells contain ion channels in their outer (plasma) and inner (organelle) membranes. Ion channels, similar to other proteins, are targets of oxidative impact, which modulates ion fluxes across membranes. Subsequently, these ion currents affect electrical excitability, such as action potential discharge (in neurons, muscle, and receptor cells), alteration of the membrane resting potential, synaptic transmission, hormone secretion, muscle contraction or coordination of the cell cycle. In this chapter we summarize effects of oxidative stress and redox mechanisms on some ion channels, in particular on maxi calcium-activated potassium (BK) channels which play an outstanding role in a plethora of physiological and pathophysiological functions in almost all cells and tissues. We first elaborate on some general features of ion channel structure and function and then summarize effects of oxidative alterations of ion channels and their functional consequences. PMID:26287261

  6. Channel sialic acids limit hERG channel activity during the ventricular action potential.

    PubMed

    Norring, Sarah A; Ednie, Andrew R; Schwetz, Tara A; Du, Dongping; Yang, Hui; Bennett, Eric S

    2013-02-01

    Activity of human ether-a-go-go-related gene (hERG) 1 voltage-gated K(+) channels is responsible for portions of phase 2 and phase 3 repolarization of the human ventricular action potential. Here, we questioned whether and how physiologically and pathophysiologically relevant changes in surface N-glycosylation modified hERG channel function. Voltage-dependent hERG channel gating and activity were evaluated as expressed in a set of Chinese hamster ovary (CHO) cell lines under conditions of full glycosylation, no sialylation, no complex N-glycans, and following enzymatic deglycosylation of surface N-glycans. For each condition of reduced glycosylation, hERG channel steady-state activation and inactivation relationships were shifted linearly by significant depolarizing ∼9 and ∼18 mV, respectively. The hERG window current increased significantly by 50-150%, and the peak shifted by a depolarizing ∼10 mV. There was no significant change in maximum hERG current density. Deglycosylated channels were significantly more active (20-80%) than glycosylated controls during phases 2 and 3 of action potential clamp protocols. Simulations of hERG current and ventricular action potentials corroborated experimental data and predicted reduced sialylation leads to a 50-70-ms decrease in action potential duration. The data describe a novel mechanism by which hERG channel gating is modulated through physiologically and pathophysiologically relevant changes in N-glycosylation; reduced channel sialylation increases hERG channel activity during the action potential, thereby increasing the rate of action potential repolarization.

  7. Chlorpromazine confers neuroprotection against brain ischemia by activating BKCa channel.

    PubMed

    Li, Hua-Juan; Zhang, Yu-Jiao; Zhou, Li; Han, Feng; Wang, Ming-Yan; Xue, Mao-Qiang; Qi, Zhi

    2014-07-15

    Chlorpromazine (CPZ) is a well-known antipsychotic drug, still widely being used to treat symptoms of schizophrenia, psychotic depression and organic psychoses. We have previously reported that CPZ activates the BKCa (KCa1.1) channel at whole cell level. In the present study, we demonstrated that CPZ increased the single channel open probability of the BKCa channels without changing its single channel amplitude. As BKCa channel is one of the molecular targets of brain ischemia, we explored a possible new use of this old drug on ischemic brain injury. In middle cerebral artery occlusion (MCAO) focal cerebral ischemia, a single intraperitoneal injection of CPZ at several dosages (5mg/kg, 10mg/kg and 20mg/kg) could exert a significant neuroprotective effect on the brain damage in a dose- and time-dependent manner. Furthermore, blockade of BKCa channels abolished the neuroprotective effect of CPZ on MCAO, suggesting that the effect of CPZ is mediated by activation of the BKCa channel. These results demonstrate that CPZ could reduce focal cerebral ischemic damage through activating BKCa channels and merits exploration as a potential therapeutic agent for treating ischemic stroke. Copyright © 2014 Elsevier B.V. All rights reserved.

  8. Permeability properties of chick myotube acetylcholine-activated channels.

    PubMed Central

    Dwyer, T M; Farley, J M

    1984-01-01

    The acetylcholine-(ACh-)activated channels of chick myotubes were studied by the patch-clamp method. Single-channel amplitudes were measured over a wide range of potentials in solutions of cesium, arginine, and three small amines. Symmetrical, isotonic cesium solutions gave a linear I-V relationship with the single-channel conductance, gamma, of 42 pS at 11 degrees C. Dilutions of cesium by mannitol shifted the reversal potential 23.9 mV per e-fold change in internal cesium concentration. Selectivity, as defined by reversal potential criteria, depended on the molecular size of the permeant cation. The Q10 of gamma for the symmetrical isotonic cesium solutions as well as internal isotonic methylamine was 1.3-1.4. These properties are qualitatively similar to those seen at the ACh-activated channel of the frog neuromuscular junction. Partially substituting arginine for internal cesium depressed outward currents. 80 mM arginine acted equally well from the inside or the outside, as if arginine transiently blocks the ACh-activated channel in a current dependent way. Diluting internal cesium almost 10-fold, from 320 to 40 mM, increased the permeability of the channel calculated from Goldman-Hodgkin-Katz equations by almost threefold. Thus, cesium itself appears to block with a dissociation constant of 135 mM. Methylamine blocked the channel approximately as well as did cesium. Ammonia and ethylamine blocked the channel somewhat more than cesium. We conclude that (a) the channel is qualitatively similar to that of frog neuromuscular junction, (b) cations bind within the channel, and (c) arginine decreases channel conductance equally whether applied from the inside or the outside. PMID:6324917

  9. Memory reconsolidation and its maintenance depend on L-voltage-dependent calcium channels and CaMKII functions regulating protein turnover in the hippocampus.

    PubMed

    Da Silva, Weber Cláudio; Cardoso, Gabriela; Bonini, Juliana Sartori; Benetti, Fernando; Izquierdo, Ivan

    2013-04-16

    Immediate postretrieval bilateral blockade of long-acting voltage-dependent calcium channels (L-VDCCs), but not of glutamatergic NMDA receptors, in the dorsal CA1 region of the hippocampus hinders retention of long-term spatial memory in the Morris water maze. Immediate postretrieval bilateral inhibition of calcium/calmodulin-dependent protein kinase (CaMK) II in dorsal CA1 does not affect retention of this task 24 h later but does hinder it 5 d later. These two distinct amnesic effects are abolished if protein degradation by proteasomes is inhibited concomitantly. These results indicate that spatial memory reconsolidation depends on the functionality of L-VDCC in dorsal CA1, that maintenance of subsequent reconsolidated memory trace depends on CaMKII, and these results also suggest that the role played by both L-VDCC and CaMKII is to promote the retrieval-dependent, synaptically localized enhancement of protein synthesis necessary to counteract a retrieval-dependent, synaptic-localized enhancement of protein degradation, which has been described as underlying the characteristic labilization of the memory trace triggered by retrieval. Thus, conceivably, L-VDCC and CaMKII would enhance activity-dependent localized protein renewal, which may account for the improvement of the long-term efficiency of the synapses responsible for the maintenance of reactivated long-term spatial memory.

  10. Memory reconsolidation and its maintenance depend on L-voltage-dependent calcium channels and CaMKII functions regulating protein turnover in the hippocampus

    PubMed Central

    Da Silva, Weber Cláudio; Cardoso, Gabriela; Bonini, Juliana Sartori; Benetti, Fernando; Izquierdo, Ivan

    2013-01-01

    Immediate postretrieval bilateral blockade of long-acting voltage–dependent calcium channels (L-VDCCs), but not of glutamatergic NMDA receptors, in the dorsal CA1 region of the hippocampus hinders retention of long-term spatial memory in the Morris water maze. Immediate postretrieval bilateral inhibition of calcium/calmodulin-dependent protein kinase (CaMK) II in dorsal CA1 does not affect retention of this task 24 h later but does hinder it 5 d later. These two distinct amnesic effects are abolished if protein degradation by proteasomes is inhibited concomitantly. These results indicate that spatial memory reconsolidation depends on the functionality of L-VDCC in dorsal CA1, that maintenance of subsequent reconsolidated memory trace depends on CaMKII, and these results also suggest that the role played by both L-VDCC and CaMKII is to promote the retrieval-dependent, synaptically localized enhancement of protein synthesis necessary to counteract a retrieval-dependent, synaptic-localized enhancement of protein degradation, which has been described as underlying the characteristic labilization of the memory trace triggered by retrieval. Thus, conceivably, L-VDCC and CaMKII would enhance activity-dependent localized protein renewal, which may account for the improvement of the long-term efficiency of the synapses responsible for the maintenance of reactivated long-term spatial memory. PMID:23576750

  11. Flow-induced activation of TRPV5 and TRPV6 channels stimulates Ca(2+)-activated K(+) channel causing membrane hyperpolarization.

    PubMed

    Cha, Seung-Kuy; Kim, Ji-Hee; Huang, Chou-Long

    2013-12-01

    TRPV5 and TRPV6 channels are expressed in distal renal tubules and play important roles in the transcellular Ca(2+) reabsorption in kidney. They are regulated by multiple intracellular factors including protein kinases A and C, membrane phospholipid PIP2, protons, and divalent ions Ca(2+) and Mg(2+). Here, we report that fluid flow that generates shear force within the physiological range of distal tubular fluid flow activated TRPV5 and TRPV6 channels expressed in HEK cells. Flow-induced activation of channel activity was reversible and did not desensitize over 2min. Fluid flow stimulated TRPV5 and 6-mediated Ca(2+) entry and increased intracellular Ca(2+) concentration. N-glycosylation-deficient TRPV5 channel was relatively insensitive to fluid flow. In cells coexpressing TRPV5 (or TRPV6) and Slo1-encoded maxi-K channels, fluid flow induced membrane hyperpolarization, which could be prevented by the maxi-K blocker iberiotoxin or TRPV5 and 6 blocker La(3+). In contrast, fluid flow did not cause membrane hyperpolarization in cells coexpressing ROMK1 and TRPV5 or 6 channel. These results reveal a new mechanism for the regulation of TRPV5 and TRPV6 channels. Activation of TRPV5 and TRPV6 by fluid flow may play a role in the regulation of flow-stimulated K(+) secretion via maxi-K channels in distal renal tubules and in the mechanism of pathogenesis of thiazide-induced hypocalciuria.

  12. Flow-Induced Activation of TRPV5 and TRPV6 Channel Stimulates Ca2+-Activated K+ Channel Causing Membrane Hyperpolarization

    PubMed Central

    Cha, Seung-Kuy; Kim, Ji-Hee; Huang, Chou-Long

    2014-01-01

    TRPV5 and TRPV6 channels are expressed in distal renal tubules and play important roles in the transcellular Ca2+ reabsorption in kidney. They are regulated by multiple intracellular factors including protein kinase A and C, membrane phospholipid PIP2, protons, and divalent ions Ca2+ and Mg2+. Here, we report that fluid flow that generates shear force within the physiological range of distal tubular fluid flow activated TRPV5 and TRPV6 channels expressed in HEK cells. Flow-induced activation of channel activity was reversible and did not desensitize over 2 minutes. Fluid flow stimulated TRPV5 and 6-mediated Ca2+ entry and increased intracellular Ca2+ concentration. N-glycosylation-deficient TRPV5 channel was relatively insensitive to fluid flow. In cells coexpressing TRPV5 (or TRPV6) and Slo1-encoded maxi-K channels, fluid flow induced membrane hyperpolarization, which could be prevented by the maxi-K blocker iberiotoxin or TRPV5 and 6 blocker La3+. In contrast, fluid flow did not cause membrane hyperpolarization in cells coexpressing ROMK1 and TRPV5 or 6 channels. These results reveal a new mechanism for regulation of TRPV5 and TRPV6 channels. Activation of TRPV5 and TRPV6 by fluid flow may play a role in the regulation of flow-stimulated K+ secretion via maxi-K channels in distal renal tubules and in the mechanism of pathogenesis of thiazide-induced hypocalciuria. PMID:24001793

  13. Chloride dependence of hyperpolarization-activated chloride channel gates

    PubMed Central

    Pusch, Michael; Jordt, Sven-Eric; Stein, Valentin; Jentsch, Thomas J

    1999-01-01

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

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

    PubMed Central

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

    2001-01-01

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

  15. The NCA sodium leak channel is required for persistent motor circuit activity that sustains locomotion.

    PubMed

    Gao, Shangbang; Xie, Lin; Kawano, Taizo; Po, Michelle D; Guan, Sihui; Zhen, Mei; Pirri, Jennifer K; Alkema, Mark J

    2015-02-26

    Persistent neural activity, a sustained circuit output that outlasts the stimuli, underlies short-term or working memory, as well as various mental representations. Molecular mechanisms that underlie persistent activity are not well understood. Combining in situ whole-cell patch clamping and quantitative locomotion analyses, we show here that the Caenorhabditis elegans neuromuscular system exhibits persistent rhythmic activity, and such an activity contributes to the sustainability of basal locomotion, and the maintenance of acceleration after stimulation. The NALCN family sodium leak channel regulates the resting membrane potential and excitability of invertebrate and vertebrate neurons. Our molecular genetics and electrophysiology analyses show that the C. elegans NALCN, NCA, activates a premotor interneuron network to potentiate persistent motor circuit activity and to sustain C. elegans locomotion. Collectively, these results reveal a mechanism for, and physiological function of, persistent neural activity using a simple animal model, providing potential mechanistic clues for working memory in other systems.

  16. Imaging Large Cohorts of Single Ion Channels and Their Activity

    PubMed Central

    Hiersemenzel, Katia; Brown, Euan R.; Duncan, Rory R.

    2013-01-01

    As calcium is the most important signaling molecule in neurons and secretory cells, amongst many other cell types, it follows that an understanding of calcium channels and their regulation of exocytosis is of vital importance. Calcium imaging using calcium dyes such as Fluo3, or FRET-based dyes that have been used widely has provided invaluable information, which combined with modeling has estimated the subtypes of channels responsible for triggering the exocytotic machinery as well as inferences about the relative distances away from vesicle fusion sites these molecules adopt. Importantly, new super-resolution microscopy techniques, combined with novel Ca2+ indicators and imaginative imaging approaches can now define directly the nano-scale locations of very large cohorts of single channel molecules in relation to single vesicles. With combinations of these techniques the activity of individual channels can be visualized and quantified using novel Ca2+ indicators. Fluorescently labeled specific channel toxins can also be used to localize endogenous assembled channel tetramers. Fluorescence lifetime imaging microscopy and other single-photon-resolution spectroscopic approaches offer the possibility to quantify protein–protein interactions between populations of channels and the SNARE protein machinery for the first time. Together with simultaneous electrophysiology, this battery of quantitative imaging techniques has the potential to provide unprecedented detail describing the locations, dynamic behaviors, interactions, and conductance activities of many thousands of channel molecules and vesicles in living cells. PMID:24027557

  17. Toward a unifying model of malaria-induced channel activity

    PubMed Central

    Bouyer, Guillaume; Egée, Stéphane; Thomas, Serge L. Y.

    2007-01-01

    Infection of RBC by the malaria parasite Plasmodium falciparum activates, at the trophozoite stage, a membrane current 100- to 150-fold larger than in uninfected RBC. This current is carried by small anion channels initially described in supraphysiological ion concentrations (1.115 M Cl−) and named plasmodial surface anion channels (PSAC), suggesting their plasmodial origin. Our results obtained with physiological ion concentrations (0.145 M Cl−) support the notion that the parasite-induced channels represent enhanced activity versions of anion channels already present in uninfected RBCs. Among them, an 18-pS inwardly rectifying anion channel (IRC) and a 4- to 5-pS small conductance anion channel (SCC) were present in most single-channel recordings of infected membranes. The aim of this study was to clarify disparities in the reported electrophysiological data and to investigate possible technical reasons why these discrepancies have arisen. We demonstrate that PSAC is the supraphysiological correlate of the SCC and is inhibited by Zn2+, suggesting that it is a ClC-2 channel. We show that in physiological solutions 80% of the membrane conductance in infected cells can be accounted for by IRC and 20% can be accounted for by SCC whereas in supraphysiological conditions the membrane conductance is almost exclusively carried by SCC (PSAC) because the IRC is functionally turned off. PMID:17576926

  18. Effective contractile response to voltage-gated Na+ channels revealed by a channel activator.

    PubMed

    Ho, W-S Vanessa; Davis, Alison J; Chadha, Preet S; Greenwood, Iain A

    2013-04-15

    This study investigated the molecular identity and impact of enhancing voltage-gated Na(+) (Na(V)) channels in the control of vascular tone. In rat isolated mesenteric and femoral arteries mounted for isometric tension recording, the vascular actions of the Na(V) channel activator veratridine were examined. Na(V) channel expression was probed by molecular techniques and immunocytochemistry. In mesenteric arteries, veratridine induced potent contractions (pEC(50) = 5.19 ± 0.20, E(max) = 12.0 ± 2.7 mN), which were inhibited by 1 μM TTX (a blocker of all Na(V) channel isoforms, except Na(V)1.5, Na(V)1.8, and Na(V)1.9), but not by selective blockers of Na(V)1.7 (ProTx-II, 10 nM) or Na(V)1.8 (A-80347, 1 μM) channels. The responses were insensitive to endothelium removal but were partly (~60%) reduced by chemical destruction of sympathetic nerves by 6-hydroxydopamine (2 mM) or antagonism at the α1-adrenoceptor by prazosin (1 μM). KB-R7943, a blocker of the reverse mode of the Na(+)/Ca(2+) exchanger (3 μM), inhibited veratridine contractions in the absence or presence of prazosin. T16A(inh)-A01, a Ca(2+)-activated Cl(-) channel blocker (10 μM), also inhibited the prazosin-resistant contraction to veratridine. Na(V) channel immunoreactivity was detected in freshly isolated mesenteric myocytes, with apparent colocalization with the Na(+)/Ca(2+) exchanger. Veratridine induced similar contractile effects in the femoral artery, and mRNA transcripts for Na(V)1.2 and Na(V)1.3 channels were evident in both vessel types. We conclude that, in addition to sympathetic nerves, NaV channels are expressed in vascular myocytes, where they are functionally coupled to the reverse mode of Na(+)/Ca(2+) exchanger and subsequent activation of Ca(2+)-activated Cl(-) channels, causing contraction. The TTX-sensitive Na(V)1.2 and Na(V)1.3 channels are likely involved in vascular control.

  19. Does proximity to physical activity infrastructures predict maintenance of organized and unorganized physical activities in youth?

    PubMed Central

    MacKenzie, Jason; Brunet, Jennifer; Boudreau, Jonathan; Iancu, Horia-Daniel; Bélanger, Mathieu

    2015-01-01

    Physical activity (PA) infrastructures can provide youth chances to engage in PA. As determinants of organized and unorganized PA (OPA and UPA) may differ, we investigated if proximity to PA infrastructures (proximity) was associated with maintenance of OPA and UPA over 3 years. Youth from New Brunswick, Canada (n = 187; 10–12 years at baseline) reported participation in OPA and UPA every 4 months from 2011 to 2014 as part of the MATCH study. Proximity data were drawn from parent's questionnaires. Proximity scores were divided into tertiles. Kaplan–Meier and Cox proportional hazard models were used to assess associations between proximity and maintenance of OPA and UPA. There were no crude or adjusted differences in average maintenance of participation in OPA [mean number of survey cycle participation (95%CI) was 6.6 (5.7–7.5), 6.3 (5.5–7.1), and 5.8 (5.1–6.6)] or UPA [6.8 (6.2–7.4), 5.9 (5.3–6.5), and 6.6 (5.9–7.3)] across low, moderate, and high tertiles of proximity, respectively. Findings suggest that proximity does not affect maintenance of participation in OPA or UPA during adolescence. Other environmental aspects may have a greater effect. Further research is needed before conclusions can be made. PMID:26844149

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

    PubMed

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

    2013-06-01

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

  1. 77 FR 66958 - Agency Information Collection Activities; Comment Request; Form for Maintenance of Effort Waiver...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2012-11-08

    ... Agency Information Collection Activities; Comment Request; Form for Maintenance of Effort Waiver Requests... notice will be considered public records. Title of Collection: Form for Maintenance of Effort Waiver... (SEA) finds that either the combined fiscal effort per student or the aggregate expenditures of the...

  2. School Bus and School Pupil Activity Bus Inspection and Maintenance Guide.

    ERIC Educational Resources Information Center

    Sowash, Fred W.

    This publication deals with suggested maintenance standards that apply to vehicles used for pupil transportation in California and covers all the safety-related systems that are common to most buses. The guide has been prepared to help all school bus and school pupil activity bus operators set up an inspection and preventive maintenance program…

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

    PubMed

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

    2009-11-01

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

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

    NASA Technical Reports Server (NTRS)

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

    1996-01-01

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

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

    NASA Technical Reports Server (NTRS)

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

    1996-01-01

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

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

    PubMed Central

    Cho, M H; Spalding, E P

    1996-01-01

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

  7. Collateral response to activation of potassium channels in vivo.

    PubMed

    Lamping, K G

    1998-04-01

    Activation of ATP-sensitive K+ channels is involved in the coronary vascular response to decreases in perfusion pressure and ischemia. Since activation of ATP-sensitive K+ channels in collateral vessels may be important in determining flow to collateral-dependent myocardium, the ability of collaterals to respond to activation of the channel was tested. In the beating heart of dogs, we compared responses of non-collaterals less than 100 microns in diameter to collaterals of similar size using computer-controlled stroboscopic epi-illumination of the left ventricle coupled to a microscope-video system. Aprikalim, a selective activator of ATP-sensitive K+ channels (0.1-10 microM) produced similar dose-dependent dilation of non-collaterals and collaterals. Relaxation was decreased by inhibition of ATP-sensitive K+ channels with glibenclamide, but not by inhibition of nitric oxide synthase with nitro-L-arginine. Bradykinin (10-100 microM) produced similar dilation of non-collaterals and collaterals which was decreased by nitro-L-arginine but not glibenclamide. Thus, in microvascular collaterals, relaxation to both nitric oxide and activation of ATP-sensitive K+ channels is similar to non-collaterals.

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

    PubMed Central

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

    1994-01-01

    The cystic fibrosis transmembrane conductance regulator (CFTR) chloride channel is regulated by phosphorylation and dephosphorylation at multiple sites. Although activation by protein kinases has been studied in some detail, the dephosphorylation step has received little attention. This report examines the mechanisms responsible for the dephosphorylation and spontaneous deactivation ("rundown") of CFTR chloride channels excised from transfected Chinese hamster ovary (CHO) and human airway epithelial cells. We report that the alkaline phosphatase inhibitors bromotetramisole, 3-isobutyl-1-methylxanthine, theophylline, and vanadate slow the rundown of CFTR channel activity in excised membrane patches and reduce dephosphorylation of CFTR protein in isolated membranes. It was also found that in unstimulated cells, CFTR channels can be activated by exposure to phosphatase inhibitors alone. Most importantly, exposure of mammalian cells to phosphatase inhibitors alone activates CFTR channels that have disease-causing mutations, provided the mutant channels are present in the plasma membrane (R117H, G551D, and delta F508 after cooling). These results suggest that CFTR dephosphorylation is dynamic and that membrane-associated phosphatase activity may be a potential therapeutic target for the treatment of cystic fibrosis. Images PMID:7522329

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

    PubMed

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

    2002-08-09

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

  10. Pharmacological Targeting of Native CatSper Channels Reveals a Required Role in Maintenance of Sperm Hyperactivation

    PubMed Central

    Carlson, Anne E.; Burnett, Lindsey A.; del Camino, Donato; Quill, Timothy A.; Hille, Bertil; Chong, Jayhong A.; Moran, Magdalene M.; Babcock, Donner F.

    2009-01-01

    The four sperm-specific CatSper ion channel proteins are required for hyperactivated motility and male fertility, and for Ca2+ entry evoked by alkaline depolarization. In the absence of external Ca2+, Na+ carries current through CatSper channels in voltage-clamped sperm. Here we show that CatSper channel activity can be monitored optically with the [Na+]i-reporting probe SBFI in populations of intact sperm. Removal of external Ca2+ increases SBFI signals in wild-type but not CatSper2-null sperm. The rate of the indicated rise of [Na+]i is greater for sperm alkalinized with NH4Cl than for sperm acidified with propionic acid, reflecting the alkaline-promoted signature property of CatSper currents. In contrast, the [Na+]i rise is slowed by candidate CatSper blocker HC-056456 (IC50 ∼3 µM). HC-056456 similarly slows the rise of [Ca2+]i that is evoked by alkaline depolarization and reported by fura-2. HC-056456 also selectively and reversibly decreased CatSper currents recorded from patch-clamped sperm. HC-056456 does not prevent activation of motility by HCO3− but does prevent the development of hyperactivated motility by capacitating incubations, thus producing a phenocopy of the CatSper-null sperm. When applied to hyperactivated sperm, HC-056456 causes a rapid, reversible loss of flagellar waveform asymmetry, similar to the loss that occurs when Ca2+ entry through the CatSper channel is terminated by removal of external Ca2+. Thus, open CatSper channels and entry of external Ca2+ through them sustains hyperactivated motility. These results indicate that pharmacological targeting of the CatSper channel may impose a selective late-stage block to fertility, and that high-throughput screening with an optical reporter of CatSper channel activity may identify additional selective blockers with potential for male-directed contraception. PMID:19718436

  11. KC-46 Workforce Requirements for Depot Maintenance Activation

    DTIC Science & Technology

    2014-03-27

    Community College of the Air force CFR: Code of Federal Regulation CLS: Contract Logistic Service CoF: Complex of the Future CSR : Case Study...subject. Therefore, the method of choice for this research study is to seek expert opinion from those in the field of aircraft maintenance, gather...training to ensure airmen become experts in their field . However, personnel at the depot do not move around, in fact they may stay their whole

  12. A Naval Aviation Maintenance Organizational Activity Strategic Information System (OASIS)

    DTIC Science & Technology

    1990-03-01

    goals are to: " Increase efficient and economical management of human , monetary, and material resources , " Ensure the maintenance, manufacture, and...Rcf. 65 51]), but are organized to be more consistent with an OMA’s primary goal: opti- mum management of human , monetary and material resources . They...dealing with human resources are addressed first. In view of cur- rent events in the world, and the much discussed "peace dividend", the second priority

  13. Maintenance of Mouse Gustatory Terminal Field Organization Is Disrupted following Selective Removal of Peripheral Sodium Salt Taste Activity at Adulthood.

    PubMed

    Skyberg, Rolf; Sun, Chengsan; Hill, David L

    2017-08-09

    Neural activity plays a critical role in the development of central circuits in sensory systems. However, the maintenance of these circuits at adulthood is usually not dependent on sensory-elicited neural activity. Recent work in the mouse gustatory system showed that selectively deleting the primary transduction channel for sodium taste, the epithelial sodium channel (ENaC), throughout development dramatically impacted the organization of the central terminal fields of three nerves that carry taste information to the nucleus of the solitary tract. More specifically, deleting ENaCs during development prevented the normal maturation of the fields. The present study was designed to extend these findings by testing the hypothesis that the loss of sodium taste activity impacts the maintenance of the normal adult terminal field organization in male and female mice. To do this, we used an inducible Cre-dependent genetic recombination strategy to delete ENaC function after terminal field maturation occurred. We found that removal of sodium taste neural activity at adulthood resulted in significant reorganization of mature gustatory afferent terminal fields in the nucleus of the solitary tract. Specifically, the chorda tympani and greater superficial petrosal nerve terminal fields were 1.4× and 1.6× larger than age-matched controls, respectively. By contrast, the glossopharyngeal nerve, which is not highly sensitive to sodium taste stimulation, did not undergo terminal field reorganization. These surprising results suggest that gustatory nerve terminal fields remain plastic well into adulthood, which likely impacts central coding of taste information and taste-related behaviors with altered taste experience.SIGNIFICANCE STATEMENT Neural activity plays a major role in the development of sensory circuits in the mammalian brain. However, the importance of sensory-driven activity in maintaining these circuits at adulthood, especially in subcortical structures, appears to be

  14. The Ca2+-activated Cl- channel ANO1/TMEM16A regulates primary ciliogenesis.

    PubMed

    Ruppersburg, Chelsey Chandler; Hartzell, H Criss

    2014-06-01

    Many cells possess a single, nonmotile, primary cilium highly enriched in receptors and sensory transduction machinery that plays crucial roles in cellular morphogenesis. Although sensory transduction requires ion channels, relatively little is known about ion channels in the primary cilium (with the exception of TRPP2). Here we show that the Ca(2+)-activated Cl ((-)) channel anoctamin-1 (ANO1/TMEM16A) is located in the primary cilium and that blocking its channel function pharmacologically or knocking it down with short hairpin RNA interferes with ciliogenesis. Before ciliogenesis, the channel becomes organized into a torus-shaped structure ("the nimbus") enriched in proteins required for ciliogenesis, including the small GTPases Cdc42 and Arl13b and the exocyst complex component Sec6. The nimbus excludes F-actin and coincides with a ring of acetylated microtubules. The nimbus appears to form before, or independent of, apical docking of the mother centriole. Our data support a model in which the nimbus provides a scaffold for staging of ciliary components for assembly very early in ciliogenesis and chloride transport by ANO1/TMEM16A is required for the genesis or maintenance of primary cilia.

  15. Active integrated filters for RF-photonic channelizers.

    PubMed

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

    2011-01-01

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

  16. Active Integrated Filters for RF-Photonic Channelizers

    PubMed Central

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

    2011-01-01

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

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

    PubMed

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

    2015-10-01

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

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

    PubMed Central

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

    2015-01-01

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

  19. 15 CFR 908.11 - Maintenance of records of related activities.

    Code of Federal Regulations, 2010 CFR

    2010-01-01

    ... REGULATIONS MAINTAINING RECORDS AND SUBMITTING REPORTS ON WEATHER MODIFICATION ACTIVITIES § 908.11 Maintenance of records of related activities. (a) Persons whose activities relate to weather modification activities, other than persons engaged in weather modification activities, shall maintain records concerning...

  20. 15 CFR 908.11 - Maintenance of records of related activities.

    Code of Federal Regulations, 2011 CFR

    2011-01-01

    ... REGULATIONS MAINTAINING RECORDS AND SUBMITTING REPORTS ON WEATHER MODIFICATION ACTIVITIES § 908.11 Maintenance of records of related activities. (a) Persons whose activities relate to weather modification activities, other than persons engaged in weather modification activities, shall maintain records concerning...

  1. Effect of Cytoskeletal Reagents on Stretch Activated Ion Channels

    DTIC Science & Technology

    1992-11-12

    transduction. Biophys J59: 1143-1145, 1991. 23. SACHS, F., W. SIGURDSON, A. RUKNUDIN, AND C. BOWMAN. Single- channel mechanosensitive currents. Science 253: 800... mechanosensitive ion channels . In: Advances in Comparative and Environmental Physiology, v0C, edited by F. Ito. Berlin: Springer-Verlag, 1992, p. 55-77. Report of Inventions: None 4 ...EFFECT OF CYTOSKELETAL REAGENTS ON STRETCH ACTIVATED ION CHANNELS b lfli..3-f-I’- o0*’t 6. AUTHOR(S) Dr.-Frederick Sachs DI 7. PERFORMING ORGANIZATION NAME

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

    PubMed

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

    1995-01-01

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

  3. ROMK1 channel activity is regulated by monoubiquitination.

    PubMed

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

    2005-03-22

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

  4. ROMK1 channel activity is regulated by monoubiquitination

    PubMed Central

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

    2005-01-01

    The ubiquitination of proteins can signal their degradation, modify their activity or target them to specific membranes or cellular organelles. Here, we show that monoubiquitination regulates the plasma membrane abundance and function of the potassium channel, ROMK. Immunoprecipitation of proteins obtained from renal cortex and outer medulla with ROMK antibody revealed that this channel was monoubiquitinated. To determine the ubiquitin binding site on ROMK1, all intracellular lysine (Lys) residues of ROMK1 were individually mutated to arginine (Arg), and a two-electrode voltage clamp was used to measure the ROMK1 channel activity in Xenopus oocytes. ROMK1 channel activity increased from 8.1 to 27.2 μA only when Lys-22 was mutated to Arg. Furthermore, Western blotting failed to detect the ubiquitinated ROMK1 in oocytes injected with R1K22R. Patch-clamp experiments showed that biophysical properties of R1K22R were identical to those of wild-type ROMK1. Although total protein expression levels of GFP-ROMK1 and GFP-R1K22R in oocytes were similar, confocal microscopy showed that the surface fluorescence intensity in oocytes injected with GFP-R1K22R was higher than that of GFP-ROMK1. In addition, biotin labeling of ROMK1 and R1K22R proteins expressed in HEK293 cells showed increased surface expression of the Lys-22 mutant channel. Finally, expression of R1K22R in COS7 cells significantly stimulated the surface expression of ROMK1. We conclude that ROMK1 can be monoubiquitinated and that Lys-22 is an ubiquitin-binding site. Thus, monoubiquitination of ROMK1 regulates channel activity by reducing the surface expression of channel protein. This finding implicates the linking of a single ubiquitin molecule to channels as an important posttranslational regulatory signal. PMID:15767585

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

    PubMed

    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.

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

    PubMed Central

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

    2009-01-01

    A number of potassium channels including members of the KCNQ family and the Ca2+ 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 (∼50 μm2 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. PMID:19289549

  7. Activation of purified calcium channels by stoichiometric protein phosphorylation

    SciTech Connect

    Nunoki, K.; Florio, V.; Catterall, W.A. )

    1989-09-01

    Purified dihydropyridine-sensitive calcium channels from rabbit skeletal muscle were reconstituted into phosphatidylcholine vesicles to evaluate the effect of phosphorylation by cyclic AMP-dependent protein kinase (PK-A) on their function. Both the rate and extent of {sup 45}Ca{sup 2+} uptake into vesicles containing reconstituted calcium channels were increased severalfold after incubation with ATP and PK-A. The degree of stimulation of {sup 45}Ca{sup 2+} uptake was linearly proportional to the extent of phosphorylation of the alpha 1 and beta subunits of the calcium channel up to a stoichiometry of approximately 1 mol of phosphate incorporated into each subunit. The calcium channels activated by phosphorylation were determined to be incorporated into the reconstituted vesicles in the inside-out orientation and were completely inhibited by low concentrations of dihydropyridines, phenylalkylamines, Cd{sup 2+}, Ni{sup 2+}, and Mg{sup 2+}. The results demonstrate a direct relationship between PK-A-catalyzed phosphorylation of the alpha 1 and beta subunits of the purified calcium channel and activation of the ion conductance activity of the dihydropyridine-sensitive calcium channels.

  8. The Value Assessment Method for Evaluating Preventive Maintenance Activities.

    DTIC Science & Technology

    1991-08-01

    Managemnt and Budget, Paperwork Reduction Prolec (0704-0188). Washington. DC 205M. 1. AGENCY USE ONLY (Leave Blank) 12. REPORT DATE j3. REPORT TYPE...Office. COL Everett R . Thomas is Commander and Director of USACERL, and Dr. L.R. Shaffer is Technical Director. Accesion For / ---- NTIS CR J&I T % ~O...automated system also provides a data base for easy input or recall o17 the maintenance history for each unit serviced. Data base r -quirements for a

  9. Big Bay Harbor Operation and Maintenance Activities, Marquette County, Michigan.

    DTIC Science & Technology

    1975-04-01

    water disposal of polluted sediments. Resulting recommendations, however, noted that confinement of polluted dredged material for a period of years...combined with elimination of the sources of channel and harbor pollution , would result in improved water quality in the Great Lakes. 1.733 Present...Wisconsin boatyard and is packaged to be trans- ported to any Lake Superior site to clean up accidental oil spills. Adverse effects on air quality may result

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

    PubMed

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

    2010-05-01

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

  11. Stretch-activated cation channel from larval bullfrog skin

    PubMed Central

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

    2010-01-01

    Cell-attached patches from isolated epithelial cells from larval bullfrog skin revealed a cation channel that was activated by applying suction (−1 kPa to −4.5 kPa) to the pipette. Activation was characterized by an initial large current spike that rapidly attenuated to a stable value and showed a variable pattern of opening and closing with continuing suction. Current–voltage plots demonstrated linear or inward rectification and single channel conductances of 44–56 pS with NaCl or KCl Ringer's solution as the pipette solution, and a reversal potential (−Vp) of 20–40 mV. The conductance was markedly reduced with N-methyl-D-glucamide (NMDG)-Cl Ringer's solution in the pipette. Neither amiloride nor ATP, which are known to stimulate an apical cation channel in Ussing chamber preparations of larval frog skin, produced channel activation nor did these compounds affect the response to suction. Stretch activation was not affected by varying the pipette concentrations of Ca2+ between 0 mmol l−1 and 4 mmol l−1 or by varying pH between 6.8 and 8.0. However, conductance was reduced with 4 mmol l−1 Ca2+. Western blot analysis of membrane homogenates from larval bullfrog and larval toad skin identified proteins that were immunoreactive with mammalian TRPC1 and TRPC5 (TRPC, canonical transient receptor potential channel) antibodies while homogenates of skin from newly metamorphosed bullfrogs were positive for TRPC1 and TRPC3/6/7 antibodies. The electrophysiological response of larval bullfrog skin resembles that of a stretch-activated cation channel characterized in Xenopus oocytes and proposed to be TRPC1. These results indicate this channel persists in all life stages of anurans and that TRP isoforms may be important for sensory functions of their skin. PMID:20435829

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

    PubMed

    Premkumar, L S

    2005-04-01

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

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

    PubMed Central

    Félétou, Michel

    2009-01-01

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

  14. Atomic determinants of BK channel activation by polyunsaturated fatty acids

    PubMed Central

    Tian, Yutao; Aursnes, Marius; Hansen, Trond Vidar; Tungen, Jørn Eivind; Galpin, Jason D.; Leisle, Lilia; Ahern, Christopher A.; Xu, Rong; Heinemann, Stefan H.; Hoshi, Toshinori

    2016-01-01

    Docosahexaenoic acid (DHA), a polyunsaturated ω-3 fatty acid enriched in oily fish, contributes to better health by affecting multiple targets. Large-conductance Ca2+- and voltage-gated Slo1 BK channels are directly activated by nanomolar levels of DHA. We investigated DHA–channel interaction by manipulating both the fatty acid structure and the channel composition through the site-directed incorporation of unnatural amino acids. Electrophysiological measurements show that the para-group of a Tyr residue near the ion conduction pathway has a critical role. To robustly activate the channel, ionization must occur readily by a fatty acid for a good efficacy, and a long nonpolar acyl tail with a Z double bond present at the halfway position for a high affinity. The results suggest that DHA and the channel form an ion–dipole bond to promote opening and demonstrate the channel druggability. DHA, a marine-derived nutraceutical, represents a promising lead compound for rational drug design and discovery. PMID:27849612

  15. Caffeine activates mouse TRPA1 channels but suppresses human TRPA1 channels

    PubMed Central

    Nagatomo, Katsuhiro; Kubo, Yoshihiro

    2008-01-01

    Caffeine has various well-characterized pharmacological effects, but in mammals there are no known plasma membrane receptors or ion channels activated by caffeine. We observed that caffeine activates mouse transient receptor potential A1 (TRPA1) in heterologous expression systems by Cai2+ imaging and electrophysiological analyses. These responses to caffeine were confirmed in acutely dissociated dorsal root ganglion sensory neurons from WT mice, which are known to express TRPA1, but were not seen in neurons from TRPA1 KO mice. Expression of TRPA1 was detected immunohistochemically in nerve fibers and bundles in the mouse tongue. Moreover, WT mice, but not KO mice, showed a remarkable aversion to caffeine-containing water. These results demonstrate that mouse TRPA1 channels expressed in sensory neurons cause an aversion to drinking caffeine-containing water, suggesting they mediate the perception of caffeine. Finally, we observed that caffeine does not activate human TRPA1; instead, it suppresses its activity. PMID:18988737

  16. Use of Small Fluorescent Molecules to Monitor Channel Activity

    NASA Astrophysics Data System (ADS)

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

    2001-03-01

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

  17. Intracellular calcium strongly potentiates agonist-activated TRPC5 channels

    PubMed Central

    Blair, Nathaniel T.; Kaczmarek, J. Stefan

    2009-01-01

    TRPC5 is a calcium (Ca2+)-permeable nonselective cation channel expressed in several brain regions, including the hippocampus, cerebellum, and amygdala. Although TRPC5 is activated by receptors coupled to phospholipase C, the precise signaling pathway and modulatory signals remain poorly defined. We find that during continuous agonist activation, heterologously expressed TRPC5 currents are potentiated in a voltage-dependent manner (∼5-fold at positive potentials and ∼25-fold at negative potentials). The reversal potential, doubly rectifying current–voltage relation, and permeability to large cations such as N-methyl-d-glucamine remain unchanged during this potentiation. The TRPC5 current potentiation depends on extracellular Ca2+: replacement by Ba2+ or Mg2+ abolishes it, whereas the addition of 10 mM Ca2+ accelerates it. The site of action for Ca2+ is intracellular, as simultaneous fura-2 imaging and patch clamp recordings indicate that potentiation is triggered at ∼1 µM [Ca2+]. This potentiation is prevented when intracellular Ca2+ is tightly buffered, but it is promoted when recording with internal solutions containing elevated [Ca2+]. In cell-attached and excised inside-out single-channel recordings, increases in internal [Ca2+] led to an ∼10–20-fold increase in channel open probability, whereas single-channel conductance was unchanged. Ca2+-dependent potentiation should result in TRPC5 channel activation preferentially during periods of repetitive firing or coincident neurotransmitter receptor activation. PMID:19398778

  18. Activation of peripheral KCNQ channels relieves gout pain

    PubMed Central

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

    2015-01-01

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

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

    PubMed

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

    2015-04-25

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

  20. Somatostatin stimulates Ca(2+)-activated K+ channels through protein dephosphorylation.

    PubMed

    White, R E; Schonbrunn, A; Armstrong, D L

    1991-06-13

    The neuropeptide somatostatin inhibits secretion from electrically excitable cells in the pituitary, pancreas, gut and brain. In mammalian pituitary tumour cells somatostatin inhibits secretion through two distinct pertussis toxin-sensitive mechanisms. One involves inhibition of adenylyl cyclase, the other an unidentified cyclic AMP-independent mechanism that reduces Ca2+ influx by increasing membrane conductance to potassium. Here we demonstrate that the predominant electrophysiological effect of somatostatin on metabolically intact pituitary tumour cells is a large, sustained increase in the activity of the large-conductance Ca(2+)- and voltage-activated K+ channels (BK). This action of somatostatin does not involve direct effects of Ca2+, cAMP or G proteins on the channels. Our results indicate instead that somatostatin stimulates BK channel activity through protein dephosphorylation.

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

    PubMed Central

    Ponissery Saidu, Samsudeen; Stephan, Aaron B.; Talaga, Anna K.

    2013-01-01

    Anoctamin (ANO)2 (or TMEM16B) forms a cell membrane Ca2+-activated Cl− channel that is present in cilia of olfactory receptor neurons, vomeronasal microvilli, and photoreceptor synaptic terminals. Alternative splicing of Ano2 transcripts generates multiple variants with the olfactory variants skipping exon 14 and having alternative splicing of exon 4. In the present study, 5′ rapid amplification of cDNA ends analysis was conducted to characterize the 5′ end of olfactory Ano2 transcripts, which showed that the most abundant Ano2 transcripts in the olfactory epithelium contain a novel starting exon that encodes a translation initiation site, whereas transcripts of the publically available sequence variant, which has an alternative and longer 5′ end, were present in lower abundance. With two alternative starting exons and alternative splicing of exon 4, four olfactory ANO2 isoforms are thus possible. Patch-clamp experiments in transfected HEK293T cells expressing these isoforms showed that N-terminal sequences affect Ca2+ sensitivity and that the exon 4–encoded sequence is required to form functional channels. Coexpression of the two predominant isoforms, one with and one without the exon 4 sequence, as well as coexpression of the two rarer isoforms showed alterations in channel properties, indicating that different isoforms interact with each other. Furthermore, channel properties observed from the coexpression of the predominant isoforms better recapitulated the native channel properties, suggesting that the native channel may be composed of two or more splicing isoforms acting as subunits that together shape the channel properties. PMID:23669718

  2. Characterization of three novel mechanosensitive channel activities in Escherichia coli.

    PubMed

    Edwards, Michelle D; Black, Susan; Rasmussen, Tim; Rasmussen, Akiko; Stokes, Neil R; Stephen, Terri-Leigh; Miller, Samantha; Booth, Ian R

    2012-01-01

    Mechanosensitive channels sense elevated membrane tension that arises from rapid water influx occurring when cells move from high to low osmolarity environments (hypoosmotic shock). These non-specific channels in the cytoplasmic membrane release osmotically-active solutes and ions. The two major mechanosensitive channels in Escherichia coli are MscL and MscS. Deletion of both proteins severely compromises survival of hypoosmotic shock. However, like many bacteria, E. coli cells possess other MscS-type genes (kefA, ybdG, ybiO, yjeP and ynaI). Two homologs, MscK (kefA) and YbdG, have been characterized as mechanosensitive channels that play minor roles in maintaining cell integrity. Additional channel openings are occasionally observed in patches derived from mutants lacking MscS, MscK and MscL. Due to their rare occurrence, little is known about these extra pressure-induced currents or their genetic origins. Here we complete the identification of the remaining E. coli mechanosensitive channels YnaI, YbiO and YjeP. The latter is the major component of the previously described MscM activity (~300 pS), while YnaI (~100 pS) and YbiO (~1000 pS) were previously unknown. Expression of native YbiO is NaCl-specific and RpoS-dependent. A Δ7 strain was created with all seven E. coli mechanosensitive channel genes deleted. High level expression of YnaI, YbiO or YjeP proteins from a multicopy plasmid in the Δ7 strain (MJFGH) leads to substantial protection against hypoosmotic shock. Purified homologs exhibit high molecular masses that are consistent with heptameric assemblies. This work reveals novel mechanosensitive channels and discusses the regulation of their expression in the context of possible additional functions.

  3. Activation of Slo2.1 channels by niflumic acid

    PubMed Central

    Dai, Li; Garg, Vivek

    2010-01-01

    Slo2.1 channels conduct an outwardly rectifying K+ current when activated by high [Na+]i. Here, we show that gating of these channels can also be activated by fenamates such as niflumic acid (NFA), even in the absence of intracellular Na+. In Xenopus oocytes injected with <10 ng cRNA, heterologously expressed human Slo2.1 current was negligible, but rapidly activated by extracellular application of NFA (EC50 = 2.1 mM) or flufenamic acid (EC50 = 1.4 mM). Slo2.1 channels activated by 1 mM NFA exhibited weak voltage dependence. In high [K+]e, the conductance–voltage (G-V) relationship had a V1/2 of +95 mV and an effective valence, z, of 0.48 e. Higher concentrations of NFA shifted V1/2 to more negative potentials (EC50 = 2.1 mM) and increased the minimum value of G/Gmax (EC50 = 2.4 mM); at 6 mM NFA, Slo2.1 channel activation was voltage independent. In contrast, V1/2 of the G-V relationship was shifted to more positive potentials when [K+]e was elevated from 1 to 300 mM (EC50 = 21.2 mM). The slope conductance measured at the reversal potential exhibited the same [K+]e dependency (EC50 = 23.5 mM). Conductance was also [Na+]e dependent. Outward currents were reduced when Na+ was replaced with choline or mannitol, but unaffected by substitution with Rb+ or Li+. Neutralization of charged residues in the S1–S4 domains did not appreciably alter the voltage dependence of Slo2.1 activation. Thus, the weak voltage dependence of Slo2.1 channel activation is independent of charged residues in the S1–S4 segments. In contrast, mutation of R190 located in the adjacent S4–S5 linker to a neutral (Ala or Gln) or acidic (Glu) residue induced constitutive channel activity that was reduced by high [K+]e. Collectively, these findings indicate that Slo2.1 channel gating is modulated by [K+]e and [Na+]e, and that NFA uncouples channel activation from its modulation by transmembrane voltage and intracellular Na+. PMID:20176855

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

    PubMed

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

    2010-03-01

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

  5. Influence of malfunctions of the maintenance activities on the urban buses fuel consumption

    NASA Astrophysics Data System (ADS)

    George, Crişan; Nicolae, Filip

    2014-06-01

    Optimization of activities with the aim to provide quality service in conditions of high profitability, is one of the main objectives chased by managers in transportation companies. As a consequence, directing the attention towards monitoring of maintenance activities of vehicles fleet, can achieve desired results. Two of the most important issues related to the maintenance activity, is the increase of reliability and reduction of fuel consumption of the vehicles fleet. Aforementioned actions represents a way forward for raising the quality and profitability of services offered. In this paper, the main ways of monitoring the fuel consumption, in order to reduce it and increase the reliability of transportation vehicles fleet, are presented. For the evaluation of the maintenance system and the degree of influence of malfunctions recorded on the fuel consumption, using the Pareto -ABC method, following case study on a fleet of buses for urban public transport has been conducted. Results obtained highlights the deficiencies of the maintenance process carried out and constitutes a solid base for the reorganization of the maintenance activity, involving preventive maintenance activities, in order to contribute decisively to the results targeted by the management of transport companies.

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

    SciTech Connect

    Schmid-Antomarchi, H.; Amoroso, S.; Fosset, M.; Lazdunski, M. )

    1990-05-01

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

  7. 77 FR 70846 - Regulatory Guide 1.182, “Assessing and Managing Risk Before Maintenance Activities at Nuclear...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2012-11-27

    ... COMMISSION Regulatory Guide 1.182, ``Assessing and Managing Risk Before Maintenance Activities at Nuclear... (Rev.) 0, ``Assessing and Managing Risk Before Maintenance Activities at Nuclear Power Plants... 1.160, ``Monitoring the Effectiveness of Maintenance at Nuclear Power Plants.'' ADDRESSES: Please...

  8. Bacteria-Based Analysis of HIV-1 Vpu Channel Activity

    PubMed Central

    Taube, Robert; Alhadeff, Raphael; Assa, Dror; Krugliak, Miriam; Arkin, Isaiah T.

    2014-01-01

    HIV-1 Vpu is a small, single-span membrane protein with two attributed functions that increase the virus' pathogenicity: degradation of CD4 and inactivation of BST-2. Vpu has also been shown to posses ion channel activity, yet no correlation has been found between this attribute and Vpu's role in viral release. In order to gain further insight into the channel activity of Vpu we devised two bacteria-based assays that can examine this function in detail. In the first assay Vpu was over-expressed, such that it was deleterious to bacterial growth due to membrane permeabilization. In the second and more sensitive assay, the channel was expressed at low levels in K+ transport deficient bacteria. Consequently, Vpu expression enabled the bacteria to grow at otherwise non permissive low K+ concentrations. Hence, Vpu had the opposite impact on bacterial growth in the two assays: detrimental in the former and beneficial in the latter. Furthermore, we show that channel blockers also behave reciprocally in the two assays, promoting growth in the first assay and hindering it in the second assay. Taken together, we investigated Vpu's channel activity in a rapid and quantitative approach that is amenable to high-throughput screening, in search of novel blockers. PMID:25272035

  9. Bacteria-based analysis of HIV-1 Vpu channel activity.

    PubMed

    Taube, Robert; Alhadeff, Raphael; Assa, Dror; Krugliak, Miriam; Arkin, Isaiah T

    2014-01-01

    HIV-1 Vpu is a small, single-span membrane protein with two attributed functions that increase the virus' pathogenicity: degradation of CD4 and inactivation of BST-2. Vpu has also been shown to possess ion channel activity, yet no correlation has been found between this attribute and Vpu's role in viral release. In order to gain further insight into the channel activity of Vpu we devised two bacteria-based assays that can examine this function in detail. In the first assay Vpu was over-expressed, such that it was deleterious to bacterial growth due to membrane permeabilization. In the second and more sensitive assay, the channel was expressed at low levels in K(+) transport deficient bacteria. Consequently, Vpu expression enabled the bacteria to grow at otherwise non permissive low K(+) concentrations. Hence, Vpu had the opposite impact on bacterial growth in the two assays: detrimental in the former and beneficial in the latter. Furthermore, we show that channel blockers also behave reciprocally in the two assays, promoting growth in the first assay and hindering it in the second assay. Taken together, we investigated Vpu's channel activity in a rapid and quantitative approach that is amenable to high-throughput screening, in search of novel blockers.

  10. Active Brownian particles escaping a channel in single file.

    PubMed

    Locatelli, Emanuele; Baldovin, Fulvio; Orlandini, Enzo; Pierno, Matteo

    2015-02-01

    Active particles may happen to be confined in channels so narrow that they cannot overtake each other (single-file conditions). This interesting situation reveals nontrivial physical features as a consequence of the strong interparticle correlations developed in collective rearrangements. We consider a minimal two-dimensional model for active Brownian particles with the aim of studying the modifications introduced by activity with respect to the classical (passive) single-file picture. Depending on whether their motion is dominated by translational or rotational diffusion, we find that active Brownian particles in single file may arrange into clusters that are continuously merging and splitting (active clusters) or merely reproduce passive-motion paradigms, respectively. We show that activity conveys to self-propelled particles a strategic advantage for trespassing narrow channels against external biases (e.g., the gravitational field).

  11. Heme Regulates Allosteric Activation of the Slo1 BK Channel

    PubMed Central

    Horrigan, Frank T.; Heinemann, Stefan H.; Hoshi, Toshinori

    2005-01-01

    Large conductance calcium-dependent (Slo1 BK) channels are allosterically activated by membrane depolarization and divalent cations, and possess a rich modulatory repertoire. Recently, intracellular heme has been identified as a potent regulator of Slo1 BK channels (Tang, X.D., R. Xu, M.F. Reynolds, M.L. Garcia, S.H. Heinemann, and T. Hoshi. 2003. Nature. 425:531–535). Here we investigated the mechanism of the regulatory action of heme on heterologously expressed Slo1 BK channels by separating the influences of voltage and divalent cations. In the absence of divalent cations, heme generally decreased ionic currents by shifting the channel's G–V curve toward more depolarized voltages and by rendering the curve less steep. In contrast, gating currents remained largely unaffected by heme. Simulations suggest that a decrease in the strength of allosteric coupling between the voltage sensor and the activation gate and a concomitant stabilization of the open state account for the essential features of the heme action in the absence of divalent ions. At saturating levels of divalent cations, heme remained similarly effective with its influence on the G–V simulated by weakening the coupling of both Ca2+ binding and voltage sensor activation to channel opening. The results thus show that heme dampens the influence of allosteric activators on the activation gate of the Slo1 BK channel. To account for these effects, we consider the possibility that heme binding alters the structure of the RCK gating ring and thereby disrupts both Ca2+- and voltage-dependent gating as well as intrinsic stability of the open state. PMID:15955873

  12. Heme regulates allosteric activation of the Slo1 BK channel.

    PubMed

    Horrigan, Frank T; Heinemann, Stefan H; Hoshi, Toshinori

    2005-07-01

    Large conductance calcium-dependent (Slo1 BK) channels are allosterically activated by membrane depolarization and divalent cations, and possess a rich modulatory repertoire. Recently, intracellular heme has been identified as a potent regulator of Slo1 BK channels (Tang, X.D., R. Xu, M.F. Reynolds, M.L. Garcia, S.H. Heinemann, and T. Hoshi. 2003. Nature. 425:531-535). Here we investigated the mechanism of the regulatory action of heme on heterologously expressed Slo1 BK channels by separating the influences of voltage and divalent cations. In the absence of divalent cations, heme generally decreased ionic currents by shifting the channel's G-V curve toward more depolarized voltages and by rendering the curve less steep. In contrast, gating currents remained largely unaffected by heme. Simulations suggest that a decrease in the strength of allosteric coupling between the voltage sensor and the activation gate and a concomitant stabilization of the open state account for the essential features of the heme action in the absence of divalent ions. At saturating levels of divalent cations, heme remained similarly effective with its influence on the G-V simulated by weakening the coupling of both Ca(2+) binding and voltage sensor activation to channel opening. The results thus show that heme dampens the influence of allosteric activators on the activation gate of the Slo1 BK channel. To account for these effects, we consider the possibility that heme binding alters the structure of the RCK gating ring and thereby disrupts both Ca(2+)- and voltage-dependent gating as well as intrinsic stability of the open state.

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

    SciTech Connect

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

    1990-04-01

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

  14. Detection of single ion channel activity with carbon nanotubes

    PubMed Central

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

    2015-01-01

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

  15. Detection of single ion channel activity with carbon nanotubes

    NASA Astrophysics Data System (ADS)

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

    2015-03-01

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

  16. Ligand-Gated Ion Channels: Permeation and Activation1

    NASA Astrophysics Data System (ADS)

    Lynch, Joseph W.; Barry, Peter H.

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

  17. Ion channel activity in lobster skeletal muscle membrane.

    PubMed

    Worden, M K; Rahamimoff, R; Kravitz, E A

    1993-09-01

    Ion channel activity in the sarcolemmal membrane of muscle fibers is critical for regulating the excitability, and therefore the contractility, of muscle. To begin the characterization of the biophysical properties of the sarcolemmal membrane of lobster exoskeletal muscle fibers, recordings were made from excised patches of membrane from enzymatically induced muscle fiber blebs. Blebs formed as evaginations of the muscle sarcolemmal membrane and were sufficiently free of extracellular debris to allow the formation of gigaohm seals. Under simple experimental conditions using bi-ionic symmetrical recording solutions and maintained holding potentials, a variety of single channel types with conductances in the range 32-380 pS were detected. Two of these ion channel species are described in detail, both are cation channels selective for potassium. They can be distinguished from each other on the basis of their single-channel conductance and gating properties. The results suggest that current flows through a large number of ion channels that open spontaneously in bleb membranes in the absence of exogenous metabolites or hormones.

  18. Understand spiciness: mechanism of TRPV1 channel activation by capsaicin.

    PubMed

    Yang, Fan; Zheng, Jie

    2017-03-01

    Capsaicin in chili peppers bestows the sensation of spiciness. Since the discovery of its receptor, transient receptor potential vanilloid 1 (TRPV1) ion channel, how capsaicin activates this channel has been under extensive investigation using a variety of experimental techniques including mutagenesis, patch-clamp recording, crystallography, cryo-electron microscopy, computational docking and molecular dynamic simulation. A framework of how capsaicin binds and activates TRPV1 has started to merge: capsaicin binds to a pocket formed by the channel's transmembrane segments, where it takes a "tail-up, head-down" configuration. Binding is mediated by both hydrogen bonds and van der Waals interactions. Upon binding, capsaicin stabilizes the open state of TRPV1 by "pull-and-contact" with the S4-S5 linker. Understanding the ligand-host interaction will greatly facilitate pharmaceutical efforts to develop novel analgesics targeting TRPV1.

  19. Animal peptides targeting voltage-activated sodium channels.

    PubMed

    Billen, Bert; Bosmans, Frank; Tytgat, Jan

    2008-01-01

    Throughout millions of years of evolution, nature has supplied various organisms with a massive arsenal of venoms to defend themselves against predators or to hunt prey. These venoms are rich cocktails of diverse bioactive compounds with divergent functions, extremely effective in immobilizing or killing the recipient. In fact, venom peptides from various animals have been shown to specifically act on ion channels and other cellular receptors, and impair their normal functioning. Because of their key role in the initiation and propagation of electrical signals in excitable tissue, it is not very surprising that several isoforms of voltage-activated sodium channels are specifically targeted by many of these venom peptides. Therefore, these peptide toxins provide tremendous opportunities to design drugs with a higher efficacy and fewer undesirable side effects. This review puts venom peptides from spiders, scorpions and cone snails that target voltage-activated sodium channels in the spotlight, and addresses their potential therapeutical applications.

  20. Direct activation of cardiac pacemaker channels by intracellular cyclic AMP.

    PubMed

    DiFrancesco, D; Tortora, P

    1991-05-09

    Cyclic AMP acts as a second messenger in the modulation of several ion channels that are typically controlled by a phosphorylation process. In cardiac pacemaker cells, adrenaline and acetylcholine regulate the hyperpolarization-activated current (if), but in opposite ways; this current is involved in the generation and modulation of pacemaker activity. These actions are mediated by cAMP and underlie control of spontaneous rate by neurotransmitters. Whether the cAMP modulation of if is mediated by channel phosphorylation is, however, still unknown. Here we investigate the action of cAMP on if in excised patches of cardiac pacemaker cells and find that cAMP activates if by a mechanism independent of phosphorylation, involving a direct interaction with the channels at their cytoplasmic side. Cyclic AMP activates if by shifting its activation curve to more positive voltages, in agreement with whole-cell results. This is the first evidence of an ion channel whose gating is dually regulated by voltage and direct cAMP binding.

  1. Single Channel Properties and Regulated Expression of Ca2+ Release-Activated Ca2+ (Crac) Channels in Human T Cells

    PubMed Central

    Fomina, Alla F.; Fanger, Christopher M.; Kozak, J. Ashot; Cahalan, Michael D.

    2000-01-01

    Although the crucial role of Ca2+ influx in lymphocyte activation has been well documented, little is known about the properties or expression levels of Ca2+ channels in normal human T lymphocytes. The use of Na+ as the permeant ion in divalent-free solution permitted Ca2+ release-activated Ca2+ (CRAC) channel activation, kinetic properties, and functional expression levels to be investigated with single channel resolution in resting and phytohemagglutinin (PHA)-activated human T cells. Passive Ca2+ store depletion resulted in the opening of 41-pS CRAC channels characterized by high open probabilities, voltage-dependent block by extracellular Ca2+ in the micromolar range, selective Ca2+ permeation in the millimolar range, and inactivation that depended upon intracellular Mg2+ ions. The number of CRAC channels per cell increased greatly from ∼15 in resting T cells to ∼140 in activated T cells. Treatment with the phorbol ester PMA also increased CRAC channel expression to ∼60 channels per cell, whereas the immunosuppressive drug cyclosporin A (1 μM) suppressed the PHA-induced increase in functional channel expression. Capacitative Ca2+ influx induced by thapsigargin was also significantly enhanced in activated T cells. We conclude that a surprisingly low number of CRAC channels are sufficient to mediate Ca2+ influx in human resting T cells, and that the expression of CRAC channels increases ∼10-fold during activation, resulting in enhanced Ca2+ signaling. PMID:10995447

  2. The Antibacterial Activity of Human Neutrophils and Eosinophils Requires Proton Channels but Not BK Channels

    PubMed Central

    Femling, Jon K.; Cherny, Vladimir V.; Morgan, Deri; Rada, Balázs; Davis, A. Paige; Czirják, Gabor; Enyedi, Peter; England, Sarah K.; Moreland, Jessica G.; Ligeti, Erzsébet; Nauseef, William M.; DeCoursey, Thomas E.

    2006-01-01

    Electrophysiological events are of central importance during the phagocyte respiratory burst, because NADPH oxidase is electrogenic and voltage sensitive. We investigated the recent suggestion that large-conductance, calcium-activated K+ (BK) channels, rather than proton channels, play an essential role in innate immunity (Ahluwalia, J., A. Tinker, L.H. Clapp, M.R. Duchen, A.Y. Abramov, S. Page, M. Nobles, and A.W. Segal. 2004. Nature. 427:853–858). In PMA-stimulated human neutrophils or eosinophils, we did not detect BK currents, and neither of the BK channel inhibitors iberiotoxin or paxilline nor DPI inhibited any component of outward current. BK inhibitors did not inhibit the killing of bacteria, nor did they affect NADPH oxidase-dependent degradation of bacterial phospholipids by extracellular gIIA-PLA2 or the production of superoxide anion (\\documentclass[10pt]{article} \\usepackage{amsmath} \\usepackage{wasysym} \\usepackage{amsfonts} \\usepackage{amssymb} \\usepackage{amsbsy} \\usepackage{mathrsfs} \\usepackage{pmc} \\usepackage[Euler]{upgreek} \\pagestyle{empty} \\oddsidemargin -1.0in \\begin{document} \\begin{equation*}{\\mathrm{O}}_{2^{.}}^{-}\\end{equation*}\\end{document}). Moreover, an antibody against the BK channel did not detect immunoreactive protein in human neutrophils. A required role for voltage-gated proton channels is demonstrated by Zn2+ inhibition of NADPH oxidase activity assessed by H2O2 production, thus validating previous studies showing that Zn2+ inhibited \\documentclass[10pt]{article} \\usepackage{amsmath} \\usepackage{wasysym} \\usepackage{amsfonts} \\usepackage{amssymb} \\usepackage{amsbsy} \\usepackage{mathrsfs} \\usepackage{pmc} \\usepackage[Euler]{upgreek} \\pagestyle{empty} \\oddsidemargin -1.0in \\begin{document} \\begin{equation*}{\\mathrm{O}}_{2^{.}}^{-}\\end{equation*}\\end{document} production when assessed by cytochrome c reduction. In conclusion, BK channels were not detected in human neutrophils or eosinophils, and

  3. Channels

    NASA Image and Video Library

    2014-04-29

    Two channels are visible in this image from NASA 2001 Mars Odyssey spacecraft . The smaller one near the bottom did not carve as deeply as the larger channel at the top. The channel near the top of the image is near the origin of Mamers Valles.

  4. TRPV3 channels mediate strontium-induced mouse egg activation

    PubMed Central

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

    2014-01-01

    SUMMARY In mammals, calcium influx is required for oocyte maturation and egg activation. The molecular identities of the calcium-permeant channels that underlie the initiation of embryonic development are not established. Here, we describe a Transient Receptor Potential (TRP) ion channel current activated by TRP agonists that is absent in TrpV3−/− eggs. TRPV3 current is differentially expressed during oocyte maturation, reaching a peak of maximum density and activity at metaphase of meiosis II (MII), the stage of fertilization. Selective activation of TRPV3 channels provokes egg activation by mediating massive calcium entry. Widely used to activate eggs, strontium application is known to yield normal offspring in combination with somatic cell nuclear transfer. We show that TRPV3 is required for strontium influx, as TrpV3−/− eggs failed to permeate Sr2+ or undergo strontium-induced activation. We propose that TRPV3 is the major mediator of calcium influx in mouse eggs and is a putative target for artificial egg activation. PMID:24316078

  5. Nav channel mechanosensitivity: activation and inactivation accelerate reversibly with stretch.

    PubMed

    Morris, Catherine E; Juranka, Peter F

    2007-08-01

    Voltage-gated sodium channels (Nav) are modulated by many bilayer mechanical amphiphiles, but whether, like other voltage-gated channels (Kv, HCN, Cav), they respond to physical bilayer deformations is unknown. We expressed human heart Nav1.5 pore alpha-subunit in oocytes (where, unlike alphaNav1.4, alphaNav1.5 exhibits normal kinetics) and measured small macroscopic currents in cell-attached patches. Pipette pressure was used to reversibly stretch the membrane for comparison of I(Na)(t) before, during, and after stretch. At all voltages, and in a dose-dependent fashion, stretch accelerated the I(Na)(t) time course. The sign of membrane curvature was not relevant. Typical stretch stimuli reversibly accelerated both activation and inactivation by approximately 1.4-fold; normalization of peak I(Na)(t) followed by temporal scaling ( approximately 1.30- to 1.85-fold) resulted in full overlap of the stretch/no-stretch traces. Evidently the rate-limiting outward voltage sensor motion in the Nav1.5 activation path (as in Kv1) accelerated with stretch. Stretch-accelerated inactivation occurred even with activation saturated, so an independently stretch-modulated inactivation transition is also a possibility. Since Nav1.5 channel-stretch modulation was both reliable and reversible, and required stretch stimuli no more intense than what typically activates putative mechanotransducer channels (e.g., stretch-activated TRPC1-based currents), Nav channels join the ranks of putative mechanotransducers. It is noteworthy that at voltages near the activation threshold, moderate stretch increased the peak I(Na) amplitude approximately 1.5-fold. It will be important to determine whether stretch-modulated Nav current contributes to cardiac arrhythmias, to mechanosensory responses in interstitial cells of Cajal, to touch receptor responses, and to neuropathic (i.e., hypermechanosensitive) and/or normal pain reception.

  6. Proton and non-proton activation of ASIC channels

    PubMed Central

    Gautschi, Ivan; van Bemmelen, Miguel Xavier; Schild, Laurent

    2017-01-01

    The Acid-Sensing Ion Channels (ASIC) exhibit a fast desensitizing current when activated by pH values below 7.0. By contrast, non-proton ligands are able to trigger sustained ASIC currents at physiological pHs. To analyze the functional basis of the ASIC desensitizing and sustained currents, we have used ASIC1a and ASIC2a mutants with a cysteine in the pore vestibule for covalent binding of different sulfhydryl reagents. We found that ASIC1a and ASIC2a exhibit two distinct currents, a proton-induced desensitizing current and a sustained current triggered by sulfhydryl reagents. These currents differ in their pH dependency, their sensitivity to the sulfhydryl reagents, their ionic selectivity and their relative magnitude. We propose a model for ASIC1 and ASIC2 activity where the channels can function in two distinct modes, a desensitizing mode and a sustained mode depending on the activating ligands. The pore vestibule of the channel represents a functional site for binding non-proton ligands to activate ASIC1 and ASIC2 at neutral pH and to prevent channel desensitization. PMID:28384246

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

    PubMed Central

    Chambers, James J.; Kramer, Richard H.

    2009-01-01

    Light-activated ion channels provide a new opportunity to precisely and remotely control neuronal activity for experimental applications in neurobiology. In the past few years, several strategies have arisen that allow light to control ion channels and therefore neuronal function. Light-based triggers for ion channel control include caged compounds, which release active neurotransmitters when photolyzed with light, and natural photoreceptive proteins, which can be expressed exogenously in neurons. More recently, a third type of light trigger has been introduced: a photoisomerizable tethered ligand that directly controls ion channel activity in a light-dependent manner. Beyond the experimental applications for light-gated ion channels, there may be clinical applications in which these light-sensitive ion channels could prove advantageous over traditional methods. Electrodes for neural stimulation to control disease symptoms are invasive and often difficult to reposition between cells in tissue. Stimulation by chemical agents is difficult to constrain to individual cells and has limited temporal accuracy in tissue due to diffusional limitations. In contrast, ion channels that can be directly activated with light allow control with unparalleled spatial and temporal precision. The goal of this chapter is to describe light-regulated ion channels and how they have been tailored to control different aspects of neural activity, and how to use these channels to manipulate and better understand development, function, and plasticity of neurons and neural circuits. PMID:19195553

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

    PubMed Central

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

    2013-01-01

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

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

    PubMed

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

    2015-11-01

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

  10. The preventive effect of physical activity on weight maintenance in overweight and obese women.

    PubMed

    Dashti, Sareh; Esfehani, Ali Jafarzadeh; Leonard Joseph, H J

    2014-01-01

    Prevalence of obesity has increased recently especially in women. Obesity is related to mortality due to non-communicable diseases and has become a public health issue. Among the two important factors to reduce weight calorie limitation alone is modestly effective in initial weight but can result in weight gain after primary weight reduction is common. Therefore adding physical activity to weight maintenance program can reduce weight gain rebound. The aim of this review article was to identify the preventive effect of physical activity on weight maintenance in overweight women. Articles were selected from PubMed database and screened for the relativity to the study objectives, using scoring systems. Eleven studies were found appropriate. No statistical test was done on the data except simple mean and some descriptive analyses. Physical activity is proved to have a significant effect in weight loss/maintenance both in induction and maintenance period. This effect was more significant in higher intensities. Sever intensity physical activity can be effective in weight maintenance in long term but the effect of moderate and light physical activity could not be evaluated due to lack of data.

  11. Office of Inspector General audit report on maintenance activities at the Y-12 Plant

    SciTech Connect

    1999-05-01

    The Office of Inspector General has not reviewed maintenance activities at the Y-12 Plant in recent years. However, an audit was performed of maintenance activities at the East Tennessee Technology Park (formerly the K-25 Site) in February 1994. The audit concluded that Lockheed Martin (formerly Martin Marietta Energy Systems) had not effectively used engineered performance standards to estimate maintenance hours, and had not adequately analyzed variances between actual and estimated hours to identify and correct maintenance inefficiencies. The objective of this audit was to determine whether Lockheed Martin used performance measures to identify and correct inefficiencies in its maintenance program. Lockheed Martin did not adequately use performance measures to identify and correct inefficiencies in its maintenance program. Specifically, Lockheed Martin did not adequately apply engineered time standards in estimating jobs, nor did it use variance analysis to resolve deviations from job plans. This condition occurred because Lockheed Martin did not fully implement Departmental guidelines. As a result, Lockheed martin missed opportunities to improve its performance and cost-effectiveness.

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

    PubMed

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

    2015-01-01

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

  13. GABA-Activated Chloride Channels in Secretory Nerve Endings

    NASA Astrophysics Data System (ADS)

    Zhang, Shuanglin J.; Jackson, Meyer B.

    1993-01-01

    Neurotransmitters acting on presynaptic terminals regulate synaptic transmission and plasticity. Because of the difficulty of direct electrophysiological recording from small presynaptic terminals, little is known about the ion channels that mediate these actions or about the mechanisms by which transmitter secretion is altered. The patch-clamp technique is used to show that the predominant inhibitory presynaptic neurotransmitter, γ-aminobutyric acid (GABA), activates a GABA_A receptor and gates a chloride channel in the membranes of peptidergic nerve terminals of the posterior pituitary. The opening of a chloride channel by GABA weakly depolarizes the nerve terminal membrane and blocks action potentials. In this way, GABA limits secretion by retarding the spread of excitation into the terminal arborization.

  14. A cyclic AMP-activated K+ channel in Drosophila larval muscle is persistently activated in dunce.

    PubMed

    Delgado, R; Hidalgo, P; Diaz, F; Latorre, R; Labarca, P

    1991-01-15

    Single-channel recording from longitudinal ventrolateral Drosophila larval muscle reveals the presence of a potassium-selective channel that is directly and reversibly activated by cAMP in a dose-dependent fashion. Activation is specific and it cannot be mimicked by a series of agents that include AMP, cGMP, ATP, inositol trisphosphate, and Ca2+. Channel current records obtained from larval muscle in different dunce mutants possessing abnormally high levels of cAMP show that, in the mutants, the channel displays an increased probability of opening.

  15. A cyclic AMP-activated K+ channel in Drosophila larval muscle is persistently activated in dunce.

    PubMed Central

    Delgado, R; Hidalgo, P; Diaz, F; Latorre, R; Labarca, P

    1991-01-01

    Single-channel recording from longitudinal ventrolateral Drosophila larval muscle reveals the presence of a potassium-selective channel that is directly and reversibly activated by cAMP in a dose-dependent fashion. Activation is specific and it cannot be mimicked by a series of agents that include AMP, cGMP, ATP, inositol trisphosphate, and Ca2+. Channel current records obtained from larval muscle in different dunce mutants possessing abnormally high levels of cAMP show that, in the mutants, the channel displays an increased probability of opening. PMID:1846445

  16. Activation and Inhibition of TMEM16A Calcium-Activated Chloride Channels

    PubMed Central

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

    2014-01-01

    Calcium-activated chloride channels (CaCC) encoded by family members of transmembrane proteins of unknown function 16 (TMEM16) have recently been intensely studied for functional properties as well as their physiological roles as chloride channels in various tissues. One technical hurdle in studying these channels is the well-known channel rundown that frequently impairs the precision of electrophysiological measurements for the channels. Using experimental protocols that employ fast-solution exchange, we circumvented the problem of channel rundown by normalizing the Ca2+-induced current to the maximally-activated current obtained within a time period in which the channel rundown was negligible. We characterized the activation of the TMEM16A-encoded CaCC (also called ANO1) by Ca2+, Sr2+, and Ba2+, and discovered that Mg2+ competes with Ca2+ in binding to the divalent-cation binding site without activating the channel. We also studied the permeability of the ANO1 pore for various anions and found that the anion occupancy in the pore–as revealed by the permeability ratios of these anions–appeared to be inversely correlated with the apparent affinity of the ANO1 inhibition by niflumic acid (NFA). On the other hand, the NFA inhibition was neither affected by the degree of the channel activation nor influenced by the types of divalent cations used for the channel activation. These results suggest that the NFA inhibition of ANO1 is likely mediated by altering the pore function but not through changing the channel gating. Our study provides a precise characterization of ANO1 and documents factors that can affect divalent cation activation and NFA inhibition of ANO1. PMID:24489780

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

    PubMed

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

    2014-01-01

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

  18. Remote maintenance design activities and research and development accomplishments for the Compact Ignition Tokamak

    SciTech Connect

    Spampinato, P.T.

    1988-01-01

    The use of deuterium-tritium (D-T) fuel for the Compact Ignition Tokamak (CIT) requires the use of remote handling technology to carry out maintenance operations. The remote operations consist of removing and replacing such components as first wall armor protection tiles, radio-frequency (rf) heating modules, and diagnostic modules. The major pieces of equipment being developed for maintenance activities internal to the vacuum vessel include an articulated boom manipulator (ABM), an inspection manipulator, and special tooling. For activities external to the vessel, the equipment includes a bridge-mounted manipulator system, decontamination equipment, hot cell equipment, and solid radiation-waste (rad-waste) handling and packaging equipment. The CIT Project is completing the conceptual design phase; research and development (R and D) activities, which include demonstrations of remote maintenance operations on full-size partial mock-ups are under way. 5 figs.

  19. Zinc activates damage-sensing TRPA1 ion channels

    PubMed Central

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

    2009-01-01

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

  20. TRP channels activated by extracellular hypo-osmoticity in epithelia.

    PubMed

    Harteneck, C; Reiter, B

    2007-02-01

    TRP (transient receptor potential) channels comprise a superfamily of non-selective cation channels with at least seven subfamilies. The variety of subfamilies corresponds to the differences in the activation mechanisms and functions. TRPM3 (TRP melastatin 3) and TRPV4 (TRP vanilloid 3) have been characterized as cation channels activated by extracellular hypo-osmoticity. In addition, TRPV4 is activated by metabolites of arachidonic acid as well as alpha-isomers of phorbol esters known to be ineffective in stimulating proteins of the protein kinase C family. TRPM3 is responsive to sphingosine derivatives. The detection of splice variants with probably different activation mechanisms supports the idea that TRPM3 may have diverse cellular functions depending on the expression of a particular variant. The expression of TRPV4 in many epithelial cell types raised the question of the role of TRPV4 in epithelial physiology. Single-cell experiments as well as approaches using epithelial layers show that multiple cellular responses are triggered by TRPV4 activation and subsequent elevation of intracellular calcium. The TRPV4-induced responses increasing transcellular ion flux as well as paracellular permeability may allow the cells to adjust to changes in extracellular osmolarity. In summary, TRPV4 plays a central role in epithelial homoeostasis by modulating epithelial barrier function.

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

    PubMed

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

    2007-10-05

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

  2. Atomic basis for therapeutic activation of neuronal potassium channels

    NASA Astrophysics Data System (ADS)

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

    2015-09-01

    Retigabine is a recently approved anticonvulsant that acts by potentiating neuronal M-current generated by KCNQ2-5 channels, interacting with a conserved Trp residue in the channel pore domain. Using unnatural amino-acid mutagenesis, we subtly altered the properties of this Trp to reveal specific chemical interactions required for retigabine action. Introduction of a non-natural isosteric H-bond-deficient Trp analogue abolishes channel potentiation, indicating that retigabine effects rely strongly on formation of a H-bond with the conserved pore Trp. Supporting this model, substitution with fluorinated Trp analogues, with increased H-bonding propensity, strengthens retigabine potency. In addition, potency of numerous retigabine analogues correlates with the negative electrostatic surface potential of a carbonyl/carbamate oxygen atom present in most KCNQ activators. These findings functionally pinpoint an atomic-scale interaction essential for effects of retigabine and provide stringent constraints that may guide rational improvement of the emerging drug class of KCNQ channel activators.

  3. Atomic basis for therapeutic activation of neuronal potassium channels

    PubMed Central

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

    2015-01-01

    Retigabine is a recently approved anticonvulsant that acts by potentiating neuronal M-current generated by KCNQ2–5 channels, interacting with a conserved Trp residue in the channel pore domain. Using unnatural amino-acid mutagenesis, we subtly altered the properties of this Trp to reveal specific chemical interactions required for retigabine action. Introduction of a non-natural isosteric H-bond-deficient Trp analogue abolishes channel potentiation, indicating that retigabine effects rely strongly on formation of a H-bond with the conserved pore Trp. Supporting this model, substitution with fluorinated Trp analogues, with increased H-bonding propensity, strengthens retigabine potency. In addition, potency of numerous retigabine analogues correlates with the negative electrostatic surface potential of a carbonyl/carbamate oxygen atom present in most KCNQ activators. These findings functionally pinpoint an atomic-scale interaction essential for effects of retigabine and provide stringent constraints that may guide rational improvement of the emerging drug class of KCNQ channel activators. PMID:26333338

  4. Atomic basis for therapeutic activation of neuronal potassium channels.

    PubMed

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

    2015-09-03

    Retigabine is a recently approved anticonvulsant that acts by potentiating neuronal M-current generated by KCNQ2-5 channels, interacting with a conserved Trp residue in the channel pore domain. Using unnatural amino-acid mutagenesis, we subtly altered the properties of this Trp to reveal specific chemical interactions required for retigabine action. Introduction of a non-natural isosteric H-bond-deficient Trp analogue abolishes channel potentiation, indicating that retigabine effects rely strongly on formation of a H-bond with the conserved pore Trp. Supporting this model, substitution with fluorinated Trp analogues, with increased H-bonding propensity, strengthens retigabine potency. In addition, potency of numerous retigabine analogues correlates with the negative electrostatic surface potential of a carbonyl/carbamate oxygen atom present in most KCNQ activators. These findings functionally pinpoint an atomic-scale interaction essential for effects of retigabine and provide stringent constraints that may guide rational improvement of the emerging drug class of KCNQ channel activators.

  5. Surveillance and maintenance activities of waste area groupings at Oak Ridge National Laboratory

    SciTech Connect

    Ford, M.K.; Holder, L. Jr.; Jones, R.G.

    1991-12-01

    Surveillance and maintenance (S M) of 75 sites were conductd by the Remedial Action Section for the Environmental Restoration Program for surplus facilities and sites contaminated with radioactive materials and/or hazardous chemicals. S M activities on these sites were conducted from the end of their operating life until final facility disposal or site stabilization. The objectives of the Waste Area Grouping S M Program are met by maintaining a program of routine S M as well as by implementing interim corrective maintenance when deemed necessary as a result of site surveillance. This report briefly presents this program's activities and includes tables indicating tank levels and dry well data for FY 1991.

  6. Cilostazol induces vasodilation through the activation of Ca(2+)-activated K(+) channels in aortic smooth muscle.

    PubMed

    Li, Hongliang; Hong, Da Hye; Son, Youn Kyoung; Na, Sung Hun; Jung, Won-Kyo; Bae, Young Min; Seo, Eun Young; Kim, Sung Joon; Choi, Il-Whan; Park, Won Sun

    2015-07-01

    We investigated the vasorelaxant effect of cilostazol and related signaling pathways in phenylephrine (Phe)-induced pre-contracted aortic rings. Cilostazol induced vasorelaxation in a concentration-dependent manner when aortic rings were pre-contracted with Phe. Application of the voltage-dependent K(+) (Kv) channel inhibitor 4-AP, the ATP-sensitive K(+) (K(ATP)) channel inhibitor glibenclamide, and the inwardly rectifying K(+) (Kir) channel inhibitor Ba(2+) did not alter the vasorelaxant effect of cilostazol; however, pre- and post-treatment with the big-conductance Ca(2+)-activated K(+) (BK(Ca)) channel inhibitor paxilline inhibited the vasorelaxant effect of cilostazol. This vasorelaxant effect of cilostazol was reduced in the presence of an adenylyl cyclase or a protein kinase A (PKA) inhibitor, but not a protein kinase G inhibitor. Inside-out single channel recordings revealed that cilostazol induced the activation of BK(Ca) channel activity. The vasorelaxant effect of cilostazol was not affected by removal of the endothelium. In addition, application of a nitric oxide synthase inhibitor and a small-conductance Ca(2+)-activated K(+) (SK(Ca)) channel inhibitor did not affect cilostazol-induced vasorelaxation. We conclude that cilostazol induced vasorelaxation of the aorta through activation of BK(Ca) channel via a PKA-dependent signaling mechanism independent of endothelium.

  7. Differential effects of quercetin glycosides on GABAC receptor channel activity.

    PubMed

    Kim, Hyeon-Joong; Lee, Byung-Hwan; Choi, Sun-Hye; Jung, Seok-Won; Kim, Hyun-Sook; Lee, Joon-Hee; Hwang, Sung-Hee; Pyo, Mi-Kyung; Kim, Hyoung-Chun; Nah, Seung-Yeol

    2015-01-01

    Quercetin, a representative flavonoid, is a compound of low molecular weight found in various colored plants and vegetables. Quercetin shows a wide range of neuropharmacological activities. In fact, quercetin naturally exists as monomer-(quercetin-3-O-rhamnoside) (Rham1), dimer-(Rutin), or trimer-glycosides [quercetin-3-(2(G)-rhamnosylrutinoside)] (Rham2) at carbon-3 in fruits and vegetables. The carbohydrate components are removed after ingestion into gastrointestinal systems. The role of the glycosides attached to quercetin in the regulation of γ-aminobutyric acid class C (GABAC) receptor channel activity has not been determined. In the present study, we examined the effects of quercetin glycosides on GABAC receptor channel activity by expressing human GABAC alone in Xenopus oocytes using a two-electrode voltage clamp technique and also compared the effects of quercetin glycosides with quercetin. We found that GABA-induced inward current (I GABA ) was inhibited by quercetin or quercetin glycosides. The inhibitory effects of quercetin and its glycosides on I GABA were concentration-dependent and reversible in the order of Rutin ≈ quercetin ≈ Rham 1 > Rham 2. The inhibitory effects of quercetin and its glycosides on I GABA were noncompetitive and membrane voltage-insensitive. These results indicate that quercetin and its glycosides regulate GABAC receptor channel activity through interaction with a different site from that of GABA, and that the number of carbohydrate attached to quercetin might play an important role in the regulation of GABAC receptor channel activity.

  8. The complete structure of an activated open sodium channel

    PubMed Central

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

    2017-01-01

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

  9. Swell activated chloride channel function in human neutrophils

    SciTech Connect

    Salmon, Michael D.; Ahluwalia, Jatinder

    2009-04-17

    Non-excitable cells such as neutrophil granulocytes are the archetypal inflammatory immune cell involved in critical functions of the innate immune system. The electron current generated (I{sub e}) by the neutrophil NADPH oxidase is electrogenic and rapidly depolarises the membrane potential. For continuous function of the NADPH oxidase, I{sub e} has to be balanced to preserve electroneutrality, if not; sufficient depolarisation would prevent electrons from leaving the cell and neutrophil function would be abrogated. Subsequently, the depolarisation generated by the neutrophil NADPH oxidase I{sub e} must be counteracted by ion transport. The finding that depolarisation required counter-ions to compensate electron transport was followed by the observation that chloride channels activated by swell can counteract the NADPH oxidase membrane depolarisation. In this mini review, we discuss the research findings that revealed the essential role of swell activated chloride channels in human neutrophil function.

  10. Fluctuation driven active molecular transport in passive channel proteins

    NASA Astrophysics Data System (ADS)

    Kosztin, Ioan

    2006-03-01

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

  11. Increasing SK2 channel activity impairs associative learning

    PubMed Central

    McKay, Bridget M.; Oh, M. Matthew; Galvez, Roberto; Burgdorf, Jeffrey; Kroes, Roger A.; Weiss, Craig; Adelman, John P.; Moskal, Joseph R.

    2012-01-01

    Enhanced intrinsic neuronal excitability of hippocampal pyramidal neurons via reductions in the postburst afterhyperpolarization (AHP) has been hypothesized to be a biomarker of successful learning. This is supported by considerable evidence that pharmacologic enhancement of neuronal excitability facilitates learning. However, it has yet to be demonstrated that pharmacologic reduction of neuronal excitability restricted to the hippocampus can retard acquisition of a hippocampus-dependent task. Thus, the present study was designed to address this latter point using a small conductance potassium (SK) channel activator NS309 focally applied to the dorsal hippocampus. SK channels are important contributors to intrinsic excitability, as measured by the medium postburst AHP. NS309 increased the medium AHP and reduced excitatory postsynaptic potential width of CA1 neurons in vitro. In vivo, NS309 reduced the spontaneous firing rate of CA1 pyramidal neurons and impaired trace eyeblink conditioning in rats. Conversely, trace eyeblink conditioning reduced levels of SK2 channel mRNA and protein in the hippocampus. Therefore, the present findings indicate that modulation of SK channels is an important cellular mechanism for associative learning and further support postburst AHP reductions in hippocampal pyramidal neurons as a biomarker of successful learning. PMID:22552186

  12. Peptides of arachnid venoms with insecticidal activity targeting sodium channels.

    PubMed

    De Lima, M E; Figueiredo, S G; Pimenta, A M C; Santos, D M; Borges, M H; Cordeiro, M N; Richardson, M; Oliveira, L C; Stankiewicz, M; Pelhate, M

    2007-01-01

    Arachnids have a venom apparatus and secrete a complex chemical mixture of low molecular mass organic molecules, enzymes and polypeptide neurotoxins designed to paralyze or kill their prey. Most of these toxins are specific for membrane voltage-gated sodium channels, although some may also target calcium or potassium channels and other membrane receptors. Scorpions and spiders have provided the greatest number of the neurotoxins studied so far, for which, a good number of primary and 3D structures have been obtained. Structural features, comprising a folding that determines a similar spatial distribution of charged and hydrophobic side chains of specific amino acids, are strikingly common among the toxins from spider and scorpion venoms. Such similarities are, in turn, the key feature to target and bind these proteins to ionic channels. The search for new insecticidal compounds, as well as the study of their modes of action, constitutes a current approach to rationally design novel insecticides. This goal tends to be more relevant if the resistance to the conventional chemical products is considered. A promising alternative seems to be the biotechnological approach using toxin-expressing recombinant baculovirus. Spider and scorpion toxins having insecticidal activity are reviewed here considering their structures, toxicities and action mechanisms in sodium channels of excitable membranes.

  13. Formation and maintenance of single-thread tie channels entering floodplain lakes: Observations from three diverse river systems

    NASA Astrophysics Data System (ADS)

    Rowland, J. C.; Dietrich, W. E.; Day, G.; Parker, G.

    2009-06-01

    Tie channels connect rivers to floodplain lakes on many lowland rivers and thereby play a central role in floodplain sedimentology and ecology; yet they are generally unrecognized and little studied. Here we report the results of field studies focused on tie channel origin and morphodynamics in the following three contrasting systems: the Middle Fly River (Papua New Guinea), the Lower Mississippi River, and Birch Creek in Alaska. Across these river systems, tie channels vary by an order of magnitude in size but exhibit the same characteristic morphology and appear to develop and evolve by a similar set of processes. In all three systems, the channels are characterized by a narrow, leveed, single-thread morphology with maximum width approximately one tenth the width of the mainstem river. The channels typically have a V-shaped cross section, unlike most fluvial channels. These channels develop as lakes become isolated from the river by sedimentation. Narrowing of the connection between river and lake causes a sediment-laden jet to develop. Levees develop along the margins of the jet leading to channel emergence and eventual levee aggradation to the height of the mainstem levees. Bidirectional flow in these channels is common. Outflows from the lake scour sediment and prevent channel blockage. We propose that channel geometry and size are then controlled by a dynamic balance between channel narrowing by suspended sediment deposition and incision and widening by mass failure of banks during outflows. Tie channels are laterally stable and may convey flow for hundreds to a few thousand of years.

  14. The formation and maintenance of single-thread tie channels entering floodplain lakes: observations from three diverse river systems

    SciTech Connect

    Rowland, Joel C; Dietrich, William E; Day, Geoff; Parker, Gary

    2009-01-01

    Tie channels connect rivers to floodplain lakes on many lowland rivers and thereby play a central role in floodplain sedimentology and ecology, yet they are generally unrecognized and little studied. here we report the results of field studies focused on tie channel origin and morphodynamics in three contrasting systems: the Middle Fly River, Papua New Guinea, the Lower Mississippi River, and Birch Creek in Alaska. Across these river systems, tie channels vary by an order of magnitude in size but exhibit the same characteristic morphology and appear to develop and evolve by a similar set of processes. In all three systems, the channels are characterized by a narrow, leveed single-thread morphology with maximum width approximately one tenth the width of the mainstem river. The channels typically have a V shaped cross-section, unlike most fluvial channels. These channels develop as lakes become isolated from the river by sedimentation. Narrowing of the connection between river and lake causes a sediment-laden jet to develop. Levees develop along the margins of the jet leading to channel emergence and eventual levee aggradation to the height of the mainstem levees. Bi-directional flow in these channels is common. Outflows from the lake scour sediment and prevent channel blockage. We propose that channel geometry and size are then controlled by a dynamic balance between channel narrowing by suspended sediment deposition and incision and widening by mass failure of banks during outflows. Tie channels are laterally stable and may convey flow for hundreds to a few thousand of years.

  15. Activation of protein kinase C inhibits calcium-activated potassium channels in rat pituitary tumour cells.

    PubMed Central

    Shipston, M J; Armstrong, D L

    1996-01-01

    1. The regulation of large-conductance, calcium- and voltage-dependent potassium (BK) channels by protein kinase C (PKC) was investigated in clonal rat anterior pituitary cells (GH4C1), which were voltage clamped at -40 mV in a physiological potassium gradient through amphotericin-perforated patches. 2. Maximal activation of PKC by 100 nM phorbol 12, 13-dibutyrate (PdBu) almost completely inhibited the voltage-activated outward current through BK channels. In contrast PdBu had no significant effect on the residual outward current after block of BK channels with 2 mM TEA or 30 nM charybdotoxin. In single-channel recordings from cell-attached patches, PdBu reduced the open probability of BK channels more than eightfold with no significant effect on mean open lifetime or unitary conductance. 3. The effects of PdBu on BK channels were not mimicked by the 4 alpha-isomer, which does not activate PKC, and were blocked almost completely by 25 microM chelerythrine, a specific, noncompetitive PKC inhibitor. 4. PdBu had no significant effect on the amplitude of the pharmacologically isolated, high voltage-activated calcium current. 5. Inhibition of BK channel activity by PKC provides the first molecular mechanism linking hormonal activation of phospholipase C to sustained excitability in pituitary cells. PMID:8799890

  16. Participation of Ca2(+)-activated K+ channels in electrical activity of canine gastric smooth muscle.

    PubMed Central

    Carl, A; McHale, N G; Publicover, N G; Sanders, K M

    1990-01-01

    1. The hypothesis that Ca2(+)-activated K+ channels participate in the repolarization of electrical slow waves was tested in isolated cells and intact muscles of the canine gastric antrum. 2. Freshly dispersed cells from the gastric antrum liberally express large conductance channels that were characterized as Ca2(+)-activated K+ channels by several criteria. 3. Mean slope conductance of these channels in symmetrical 140 mM-KCl solutions was 265 +/- 25 pS and reversal potential was 1.3 +/- 3.3 mV. The reversal potential was shifted when K+ was partially replaced with Na+ in a manner consistent with the Nernst equation for the K+ gradient. 4. Open probability was studied in excised patches in solutions containing 10(-7)-10(-6) M-Ca2+ with holding potentials ranging from -100 to +100 mV. Resulting activation curves were fitted by Boltzmann functions. 5. Increasing [Ca2+] from 10(-7) to 10(-6) M shifted the half-maximal activation from +99 to 0 mV. These data suggest that Ca2(+)-activated K+ channels may be activated in the voltage range and [Ca2+]i occurring during the plateau phase of the slow wave. 6. In intact muscles loaded with the photolabile Ca2+ chelator, nitr-5, photo-activated release of Ca2+ during the slow wave cycle produced changes consistent with activation of Ca2(+)-dependent outward currents. 7. The data are consistent with the idea that Ca2+ build-up during electrical slow waves shifts the activation voltage of Ca2(+)-activated K+ channels into the range of the plateau potential. Activation of these channels yields outward current and repolarization. 8. Since the force of contractions depends on slow wave amplitude and duration, regulation of these channels may be important in controlling gastric motility. PMID:2126041

  17. Participation of Ca2(+)-activated K+ channels in electrical activity of canine gastric smooth muscle.

    PubMed

    Carl, A; McHale, N G; Publicover, N G; Sanders, K M

    1990-10-01

    1. The hypothesis that Ca2(+)-activated K+ channels participate in the repolarization of electrical slow waves was tested in isolated cells and intact muscles of the canine gastric antrum. 2. Freshly dispersed cells from the gastric antrum liberally express large conductance channels that were characterized as Ca2(+)-activated K+ channels by several criteria. 3. Mean slope conductance of these channels in symmetrical 140 mM-KCl solutions was 265 +/- 25 pS and reversal potential was 1.3 +/- 3.3 mV. The reversal potential was shifted when K+ was partially replaced with Na+ in a manner consistent with the Nernst equation for the K+ gradient. 4. Open probability was studied in excised patches in solutions containing 10(-7)-10(-6) M-Ca2+ with holding potentials ranging from -100 to +100 mV. Resulting activation curves were fitted by Boltzmann functions. 5. Increasing [Ca2+] from 10(-7) to 10(-6) M shifted the half-maximal activation from +99 to 0 mV. These data suggest that Ca2(+)-activated K+ channels may be activated in the voltage range and [Ca2+]i occurring during the plateau phase of the slow wave. 6. In intact muscles loaded with the photolabile Ca2+ chelator, nitr-5, photo-activated release of Ca2+ during the slow wave cycle produced changes consistent with activation of Ca2(+)-dependent outward currents. 7. The data are consistent with the idea that Ca2+ build-up during electrical slow waves shifts the activation voltage of Ca2(+)-activated K+ channels into the range of the plateau potential. Activation of these channels yields outward current and repolarization. 8. Since the force of contractions depends on slow wave amplitude and duration, regulation of these channels may be important in controlling gastric motility.

  18. Sexual function, activity, and satisfaction among women receiving maintenance hemodialysis.

    PubMed

    Mor, Maria K; Sevick, Mary Ann; Shields, Anne Marie; Green, Jamie A; Palevsky, Paul M; Arnold, Robert M; Fine, Michael J; Weisbord, Steven D

    2014-01-01

    Past studies that demonstrated that sexual dysfunction is common among women receiving chronic hemodialysis did not distinguish sexual dysfunction/difficulty from sexual inactivity. This study sought to differentiate these in order to elucidate the prevalence of true "sexual dysfunction" in this population. As part of a clinical trial of symptom management strategies in patients receiving chronic hemodialysis, female sexual function was prospectively assessed monthly for 6 months and quarterly thereafter using the Female Sexual Function Index, to which questions were added differentiating sexual dysfunction/difficulty from sexual inactivity. Beginning in month 7, patients were asked three questions about sexual activity, difficulty, and satisfaction monthly. Of the women enrolled in the clinical trial,125 participants completed 1721 assessments between 2009 and 2011. Scores on 574 of 643 (89%) quarterly Female Sexual Function Index assessments were consistent with sexual dysfunction, due largely to sexual inactivity, which was reported on 525 (82%) quarterly assessments. When reported (n=1663), the most frequently described reasons for sexual inactivity were lack of interest in sex (n=715; 43%) and lack of a partner (n=647; 39%), but rarely sexual difficulty (n=36; 2%). When reported (n=1582), women were moderately to very satisfied with their sexual life on 1020 (64%) assessments and on 513 of 671 (76%) assessments in which lack of interest was cited as a reason for sexual inactivity. Women indicated an interest in learning about the causes of and treatment for sexual dysfunction on just 5% of all assessments. Although many women receiving chronic hemodialysis are sexually inactive, few describe sexual difficulty. Most, including those with a lack of interest in sex, are satisfied with their sexual life and few wish to learn about treatment options. These findings suggest that true sexual dysfunction is uncommon in this population and that treatment opportunities

  19. 24 CFR 35.935 - Ongoing lead-based paint maintenance activities.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... 24 Housing and Urban Development 1 2010-04-01 2010-04-01 false Ongoing lead-based paint..., Department of Housing and Urban Development LEAD-BASED PAINT POISONING PREVENTION IN CERTAIN RESIDENTIAL STRUCTURES Rehabilitation § 35.935 Ongoing lead-based paint maintenance activities. In the case of a rental...

  20. 24 CFR 35.1220 - Ongoing lead-based paint maintenance activities.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... 24 Housing and Urban Development 1 2010-04-01 2010-04-01 false Ongoing lead-based paint..., Department of Housing and Urban Development LEAD-BASED PAINT POISONING PREVENTION IN CERTAIN RESIDENTIAL STRUCTURES Tenant-Based Rental Assistance § 35.1220 Ongoing lead-based paint maintenance activities...

  1. 24 CFR 35.935 - Ongoing lead-based paint maintenance activities.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ..., Department of Housing and Urban Development LEAD-BASED PAINT POISONING PREVENTION IN CERTAIN RESIDENTIAL STRUCTURES Rehabilitation § 35.935 Ongoing lead-based paint maintenance activities. In the case of a rental... 24 Housing and Urban Development 1 2011-04-01 2011-04-01 false Ongoing lead-based...

  2. 24 CFR 35.935 - Ongoing lead-based paint maintenance activities.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ..., Department of Housing and Urban Development LEAD-BASED PAINT POISONING PREVENTION IN CERTAIN RESIDENTIAL STRUCTURES Rehabilitation § 35.935 Ongoing lead-based paint maintenance activities. In the case of a rental... 24 Housing and Urban Development 1 2014-04-01 2014-04-01 false Ongoing lead-based...

  3. 24 CFR 35.935 - Ongoing lead-based paint maintenance activities.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ..., Department of Housing and Urban Development LEAD-BASED PAINT POISONING PREVENTION IN CERTAIN RESIDENTIAL STRUCTURES Rehabilitation § 35.935 Ongoing lead-based paint maintenance activities. In the case of a rental... 24 Housing and Urban Development 1 2012-04-01 2012-04-01 false Ongoing lead-based...

  4. 24 CFR 35.935 - Ongoing lead-based paint maintenance activities.

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ..., Department of Housing and Urban Development LEAD-BASED PAINT POISONING PREVENTION IN CERTAIN RESIDENTIAL STRUCTURES Rehabilitation § 35.935 Ongoing lead-based paint maintenance activities. In the case of a rental... 24 Housing and Urban Development 1 2013-04-01 2013-04-01 false Ongoing lead-based...

  5. Emerging roles of calcium-activated K channels and TRPV4 channels in lung oedema and pulmonary circulatory collapse.

    PubMed

    Simonsen, U; Wandall-Frostholm, C; Oliván-Viguera, A; Köhler, R

    2017-01-01

    It has been suggested that the transient receptor potential cation (TRP) channel subfamily V (vanilloid) type 4 (TRPV4) and intermediate conductance calcium-activated potassium (KCa3.1) channels contribute to endothelium-dependent vasodilation. Here, we summarize very recent evidence for a synergistic interplay of TRPV4 and KCa3.1 channels in lung disease. Among the endothelial Ca(2+) -permeable TRPs, TRPV4 is best characterized and produces arterial dilation by stimulating Ca(2+) -dependent nitric oxide synthesis and endothelium-dependent hyperpolarization. Besides these roles, some TRP channels control endothelial/epithelial barrier functions and vascular integrity, while KCa3.1 channels provide the driving force required for Cl(-) and water transport in some cells and most secretory epithelia. The three conditions, increased pulmonary venous pressure caused by left heart disease, high inflation pressure and chemically induced lung injury, may lead to activation of TRPV4 channels followed by Ca(2+) influx leading to activation of KCa3.1 channels in endothelial cells ultimately leading to acute lung injury. We find that a deficiency in KCa3.1 channels protects against TRPV4-induced pulmonary arterial relaxation, fluid extravasation, haemorrhage, pulmonary circulatory collapse and cardiac arrest in vivo. These data identify KCa3.1 channels as crucial molecular components in downstream TRPV4 signal transduction and as a potential target for the prevention of undesired fluid extravasation, vasodilatation and pulmonary circulatory collapse. © 2016 Scandinavian Physiological Society. Published by John Wiley & Sons Ltd.

  6. Leptin excites POMC neurons via activation of TRPC channels

    PubMed Central

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

    2010-01-01

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

  7. Maintenance: organizational modes, activities and health and safety. Use of a French national survey and in-situ analyses.

    PubMed

    Grusenmeyer, Corinne

    2014-12-01

    Maintenance activities are identified as critical both to operator safety and to systems safety and reliability. However, it is still difficult to identify maintenance workers in French occupational accident and disease statistics. Moreover, few analyses of these activities and of organizational changes in this field have been conducted. This paper presents two different approaches to this same issue. Analyses were aimed firstly at identifying the occupational exposures of these operators and at comparing them with occupational exposures of production staff and, secondly at developing understanding of normal real maintenance activities, i.e. maintenance activities that are normally actually carried out, while taking into account the socio-technical system and maintenance organization within which they lie. The use of the French SUMER 2003 survey shows that occupational exposures of maintenance staff to various constraints are more frequent than occupational exposures of their production colleagues. However, maintenance staff appear to have greater independence. Analyses were also conducted in a subcontracting urban public transport company, who outsources some maintenance work. Those analyses highlight a complex network of companies involved in maintenance activities, a substantial number of work interruptions and a significant fragmentation of the internal technicians' activities that can be cognitively costly, reduce anticipation possibilities and lead to incidents or accidents. Above all they underline internal technicians' contributions to the completion of outsourced interventions and interdependent relationships between the activities of the internal and the external technicians. Outsourcing maintenance interventions thus raises the question of risks associated with the interdependence of actual work activities undertaken by the different types of staff, since they contribute to the same maintenance intervention. This study therefore pinpoints the need to

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

    PubMed Central

    Qu, Zhiqiang; Hartzell, H. Criss

    2000-01-01

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

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

    PubMed Central

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

    2011-01-01

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

  10. Electrical resonance of Amphotericin B channel activity in lipidic membranes

    NASA Astrophysics Data System (ADS)

    Récamier, Karla S.; Ortega-Blake, Iván; Parmananda, P.

    2017-05-01

    In our previous work [J. Membrane Biol. 237, 31 (2010)], we showed the dependence of the time average conductance of Nystatin channels as a function of the applied potential. Specifically, it was observed that greater potential induced enhanced channel activity. This indicates that the supramolecular structure could be stabilized by a large field, possibly by giving a preferential orientation to the monomers. In the present work, we entertain the notion that the process of pore formation in the lipidic membranes has an underlying deterministic component. To verify this hypothesis, experiments were performed under potentio-dynamic conditions, i.e., a square train of pulses of different frequencies (0.05-2 Hz) were applied to a 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine membrane having 30 mol. % cholesterol and the presence of 35 μM Amphotericin B. An emergence of a resonant frequency, in the present experiments, is tantamount to observing fingerprints of determinism in the activity of these channels in lipidic membranes.

  11. A highly polarized excitable cell separates sodium channels from sodium-activated potassium channels by more than a millimeter

    PubMed Central

    Smith, Benjamin E.; Markham, Michael R.

    2015-01-01

    The bioelectrical properties and resulting metabolic demands of electrogenic cells are determined by their morphology and the subcellular localization of ion channels. The electric organ cells (electrocytes) of the electric fish Eigenmannia virescens generate action potentials (APs) with Na+ currents >10 μA and repolarize the AP with Na+-activated K+ (KNa) channels. To better understand the role of morphology and ion channel localization in determining the metabolic cost of electrocyte APs, we used two-photon three-dimensional imaging to determine the fine cellular morphology and immunohistochemistry to localize the electrocytes' ion channels, ionotropic receptors, and Na+-K+-ATPases. We found that electrocytes are highly polarized cells ∼1.5 mm in anterior-posterior length and ∼0.6 mm in diameter, containing ∼30,000 nuclei along the cell periphery. The cell's innervated posterior region is deeply invaginated and vascularized with complex ultrastructural features, whereas the anterior region is relatively smooth. Cholinergic receptors and Na+ channels are restricted to the innervated posterior region, whereas inward rectifier K+ channels and the KNa channels that terminate the electrocyte AP are localized to the anterior region, separated by >1 mm from the only sources of Na+ influx. In other systems, submicrometer spatial coupling of Na+ and KNa channels is necessary for KNa channel activation. However, our computational simulations showed that KNa channels at a great distance from Na+ influx can still terminate the AP, suggesting that KNa channels can be activated by distant sources of Na+ influx and overturning a long-standing assumption that AP-generating ion channels are restricted to the electrocyte's posterior face. PMID:25925327

  12. Relationship between volition, physical activity and weight loss maintenance: Study rationale, design, methods and baseline characteristics.

    PubMed

    Dandanell, Sune; Elbe, Anne-Marie; Pfister, Gertrud; Elsborg, Peter; W Helge, Jørn

    2017-05-01

    To investigate the relationship between volition, physical activity and weight loss maintenance. We recruited 84 sedentary (maximal oxygen uptake: 25 ± 5 ml/min), overweight and obese (Body mass index (BMI) 38 ± 7 m/h(2), fat 44 ± 7 %) women ( n = 55) and men ( n = 29) for an interdisciplinary prospective study with follow-up. The change in lifestyle and weight loss is promoted via a 3-month intensive lifestyle intervention at a private health school. The intervention consists of supervised training (1-3 hours/day), a healthy hypo-caloric diet (-500 to -700 kCal/day) and education in healthy lifestyle in classes/groups. The participants' body weight and composition (Dual Energy X-ray absorptiometry), volitional skills (questionnaire), physical activity level (heart rate accelerometer/questionnaire) and maximal oxygen uptake (indirect calorimetry) are to be monitored before, after, and 3 and 12 months after the intervention. At the 12-month follow-up, three different groups will be established: Clinical weight loss maintenance (> 10% weight loss from baseline), moderate weight loss maintenance (1-10% weight loss) and no weight loss (or weight regain). A linear mixed model analysis will be used to compare levels of volitional skills, physical activity and maximal oxygen uptake over time, between the three groups. Correlational analyses will be used to investigate possible associations between volition, maximal oxygen uptake, physical activity level and weight loss maintenance. If specific volitional skills are identified as predictors of adherence to physical activity and success in clinical weight loss maintenance, these can be trained in future intensive lifestyle interventions in order to optimize the success rate.

  13. Long-term maintenance of Na+ channels at nodes of Ranvier depends on glial contact mediated by gliomedin and NrCAM.

    PubMed

    Amor, Veronique; Feinberg, Konstantin; Eshed-Eisenbach, Yael; Vainshtein, Anya; Frechter, Shahar; Grumet, Martin; Rosenbluth, Jack; Peles, Elior

    2014-04-09

    Clustering of Na(+) channels at the nodes of Ranvier is coordinated by myelinating glia. In the peripheral nervous system, axoglial contact at the nodes is mediated by the binding of gliomedin and glial NrCAM to axonal neurofascin 186 (NF186). This interaction is crucial for the initial clustering of Na(+) channels at heminodes. As a result, it is not clear whether continued axon-glial contact at nodes of Ranvier is required to maintain these channels at the nodal axolemma. Here, we report that, in contrast to mice that lack either gliomedin or NrCAM, absence of both molecules (and hence the glial clustering signal) resulted in a gradual loss of Na(+) channels and other axonal components from the nodes, the formation of binary nodes, and dysregulation of nodal gap length. Therefore, these mice exhibit neurological abnormalities and slower nerve conduction. Disintegration of the nodes occurred in an orderly manner, starting with the disappearance of neurofascin 186, followed by the loss of Na(+) channels and ankyrin G, and then βIV spectrin, a sequence that reflects the assembly of nodes during development. Finally, the absence of gliomedin and NrCAM led to the invasion of the outermost layer of the Schwann cell membrane beyond the nodal area and the formation of paranodal-like junctions at the nodal gap. Our results reveal that axon-glial contact mediated by gliomedin, NrCAM, and NF186 not only plays a role in Na(+) channel clustering during development, but also contributes to the long-term maintenance of Na(+) channels at nodes of Ranvier.

  14. Long-Term Maintenance of Na+ Channels at Nodes of Ranvier Depends on Glial Contact Mediated by Gliomedin and NrCAM

    PubMed Central

    Amor, Veronique; Feinberg, Konstantin; Eshed-Eisenbach, Yael; Vainshtein, Anya; Frechter, Shahar; Grumet, Martin; Rosenbluth, Jack

    2014-01-01

    Clustering of Na+ channels at the nodes of Ranvier is coordinated by myelinating glia. In the peripheral nervous system, axoglial contact at the nodes is mediated by the binding of gliomedin and glial NrCAM to axonal neurofascin 186 (NF186). This interaction is crucial for the initial clustering of Na+ channels at heminodes. As a result, it is not clear whether continued axon-glial contact at nodes of Ranvier is required to maintain these channels at the nodal axolemma. Here, we report that, in contrast to mice that lack either gliomedin or NrCAM, absence of both molecules (and hence the glial clustering signal) resulted in a gradual loss of Na+ channels and other axonal components from the nodes, the formation of binary nodes, and dysregulation of nodal gap length. Therefore, these mice exhibit neurological abnormalities and slower nerve conduction. Disintegration of the nodes occurred in an orderly manner, starting with the disappearance of neurofascin 186, followed by the loss of Na+ channels and ankyrin G, and then βIV spectrin, a sequence that reflects the assembly of nodes during development. Finally, the absence of gliomedin and NrCAM led to the invasion of the outermost layer of the Schwann cell membrane beyond the nodal area and the formation of paranodal-like junctions at the nodal gap. Our results reveal that axon-glial contact mediated by gliomedin, NrCAM, and NF186 not only plays a role in Na+ channel clustering during development, but also contributes to the long-term maintenance of Na+ channels at nodes of Ranvier. PMID:24719088

  15. Development and maintenance of a telescoping debris flow fan in response to human-induced fan surface channelization, Chalk Creek Valley Natural Debris Flow Laboratory, Colorado, USA

    NASA Astrophysics Data System (ADS)

    Wasklewicz, T.; Scheinert, C.

    2016-01-01

    Channel change has been a constant theme throughout William L. Graf's research career. Graf's work has examined channel changes in the context of natural environmental fluctuations, but more often has focused on quantifying channel change in the context of anthropogenic modifications. Here, we consider how channelization of a debris flows along a bajada has perpetuated and sustained the development of 'telescoping' alluvial fan. Two-dimensional debris-flow modeling shows the importance of the deeply entrenched channelized flow in the development of a telescoping alluvial fan. GIS analyses of repeat (five different debris flows), high-resolution (5 cm) digital elevation models (DEMs) generated from repeat terrestrial laser scanning (TLS) data elucidate sediment and topographic dynamics of the new telescoping portion of the alluvial fan (the embryonic fan). Flow constriction from channelization helps to perpetuate debris-flow runout and to maintain the embryonic fan and telescoping nature of the alluvial fan complex. Embryonic fan development, in response to five debris flows, proceeds with a major portion of the flows depositing on the southern portion of the embryonic fan. The third through the fifth debris flows also begin to shift some deposition to the northern portion of the embryonic. The transfer of sediment from a higher portion of the embryonic fan to a lower portion continues currently on the embryonic fan. While channelized flow has been shown to be critical to the maintenance of the telescoping fan, the flow constriction has led to higher than background levels of sediment deposition in Chalk Creek, a tributary of the Arkansas River. A majority of the sediment from each debris flow is incorporated into Chalk Creek as opposed to being stored on the embryonic fan.

  16. Low intravascular pressure activates endothelial cell TRPV4 channels, local Ca2+ events, and IKCa channels, reducing arteriolar tone

    PubMed Central

    Bagher, Pooneh; Beleznai, Timea; Kansui, Yasuo; Mitchell, Ray; Garland, Christopher J.; Dora, Kim A.

    2012-01-01

    Endothelial cell (EC) Ca2+-activated K channels (SKCa and IKCa channels) generate hyperpolarization that passes to the adjacent smooth muscle cells causing vasodilation. IKCa channels focused within EC projections toward the smooth muscle cells are activated by spontaneous Ca2+ events (Ca2+ puffs/pulsars). We now show that transient receptor potential, vanilloid 4 channels (TRPV4 channels) also cluster within this microdomain and are selectively activated at low intravascular pressure. In arterioles pressurized to 80 mmHg, ECs generated low-frequency (∼2 min−1) inositol 1,4,5-trisphosphate receptor-based Ca2+ events. Decreasing intraluminal pressure below 50 mmHg increased the frequency of EC Ca2+ events twofold to threefold, an effect blocked with the TRPV4 antagonist RN1734. These discrete events represent both TRPV4-sparklet- and nonsparklet-evoked Ca2+ increases, which on occasion led to intracellular Ca2+ waves. The concurrent vasodilation associated with increases in Ca2+ event frequency was inhibited, and basal myogenic tone was increased, by either RN1734 or TRAM-34 (IKCa channel blocker), but not by apamin (SKCa channel blocker). These data show that intraluminal pressure influences an endothelial microdomain inversely to alter Ca2+ event frequency; at low pressures the consequence is activation of EC IKCa channels and vasodilation, reducing the myogenic tone that underpins tissue blood-flow autoregulation. PMID:23071308

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

    PubMed Central

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

    2016-01-01

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

  18. Ca(2+)-activated K(+) channels as therapeutic targets for myocardial and vascular protection.

    PubMed

    Clements, Richard T; Terentyev, Dmitry; Sellke, Frank W

    2015-01-01

    Small- and large-conductance Ca(2+)-activated K(+)channels (SKCa and BKCa, respectively) may be important targets for therapeutic interventions in a variety of cardiac conditions. In cardiomyocytes, BKCa channels are localized to mitochondria where they beneficially modulate reactive oxygen species, mitochondrial Ca(2+), and respiration. In vascular smooth muscle cells, BKCa channels regulate vascular tone and promote vasodilation. Activation of BKCa channels has demonstrated significant cardioprotection following ischemic injury, including improved function and reduced infarct size. SKCa channels are expressed in both the membrane and mitochondria of cardiomyocytes. Modulation of cardiomyocyte SKCa channels may be beneficial for arrhythmia, heart failure, and ischemia. Mitochondrial SKCa channels may provide similar benefit to BKCa channels. In addition, activation of SKCa channels on the endothelium promotes vasodilation. This mini-review focuses on the modulation of cardiomyocyte BKCa and SKCa channels for cardioprotection and briefly address associated potential therapeutic benefits in the coronary circulation.

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

    PubMed Central

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

    2016-01-01

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

  20. Natural resources management activities and biodiversity maintenance. Progress report, July 1, 1993--June 30, 1994

    SciTech Connect

    Caudell, M.B.

    1994-05-01

    This progress report briefly outlines activities at the Crackerneck Wildlife Management Area. Thirty-nine days were spent administering hunts with 1524 people participating. Biological data was collected on 89 deer, 6 turkeys, 8 feral hogs, 40 ducks of two species, 24 bobwhite, 3 rabbits, 2 gray squirrels, and 313 fish of 8 species. Public relations, maintenance and hunt preparation activities are summarized as well.

  1. Tissue kallikrein activation of the epithelial Na channel

    PubMed Central

    Patel, Ankit B.; Chao, Julie

    2012-01-01

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

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

    PubMed Central

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

    2016-01-01

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

  3. Brain activation during associative short-term memory maintenance is not predictive for subsequent retrieval

    PubMed Central

    Bergmann, Heiko C.; Daselaar, Sander M.; Beul, Sarah F.; Rijpkema, Mark; Fernández, Guillén; Kessels, Roy P. C.

    2015-01-01

    Performance on working memory (WM) tasks may partially be supported by long-term memory (LTM) processing. Hence, brain activation recently being implicated in WM may actually have been driven by (incidental) LTM formation. We examined which brain regions actually support successful WM processing, rather than being confounded by LTM processes, during the maintenance and probe phase of a WM task. We administered a four-pair (faces and houses) associative delayed-match-to-sample (WM) task using event-related functional MRI (fMRI) and a subsequent associative recognition LTM task, using the same stimuli. This enabled us to analyze subsequent memory effects for both the WM and the LTM test by contrasting correctly recognized pairs with incorrect pairs for either task. Critically, with respect to the subsequent WM effect, we computed this analysis exclusively for trials that were forgotten in the subsequent LTM recognition task. Hence, brain activity associated with successful WM processing was less likely to be confounded by incidental LTM formation. The subsequent LTM effect, in contrast, was analyzed exclusively for pairs that previously had been correctly recognized in the WM task, disclosing brain regions involved in successful LTM formation after successful WM processing. Results for the subsequent WM effect showed no significantly activated brain areas for WM maintenance, possibly due to an insensitivity of fMRI to mechanisms underlying active WM maintenance. In contrast, a correct decision at WM probe was linked to activation in the “retrieval success network” (anterior and posterior midline brain structures). The subsequent LTM analyses revealed greater activation in left dorsolateral prefrontal cortex and posterior parietal cortex in the early phase of the maintenance stage. No supra-threshold activation was found during the WM probe. Together, we obtained clearer insights in which brain regions support successful WM and LTM without the potential confound

  4. Brain activation during associative short-term memory maintenance is not predictive for subsequent retrieval.

    PubMed

    Bergmann, Heiko C; Daselaar, Sander M; Beul, Sarah F; Rijpkema, Mark; Fernández, Guillén; Kessels, Roy P C

    2015-01-01

    Performance on working memory (WM) tasks may partially be supported by long-term memory (LTM) processing. Hence, brain activation recently being implicated in WM may actually have been driven by (incidental) LTM formation. We examined which brain regions actually support successful WM processing, rather than being confounded by LTM processes, during the maintenance and probe phase of a WM task. We administered a four-pair (faces and houses) associative delayed-match-to-sample (WM) task using event-related functional MRI (fMRI) and a subsequent associative recognition LTM task, using the same stimuli. This enabled us to analyze subsequent memory effects for both the WM and the LTM test by contrasting correctly recognized pairs with incorrect pairs for either task. Critically, with respect to the subsequent WM effect, we computed this analysis exclusively for trials that were forgotten in the subsequent LTM recognition task. Hence, brain activity associated with successful WM processing was less likely to be confounded by incidental LTM formation. The subsequent LTM effect, in contrast, was analyzed exclusively for pairs that previously had been correctly recognized in the WM task, disclosing brain regions involved in successful LTM formation after successful WM processing. Results for the subsequent WM effect showed no significantly activated brain areas for WM maintenance, possibly due to an insensitivity of fMRI to mechanisms underlying active WM maintenance. In contrast, a correct decision at WM probe was linked to activation in the "retrieval success network" (anterior and posterior midline brain structures). The subsequent LTM analyses revealed greater activation in left dorsolateral prefrontal cortex and posterior parietal cortex in the early phase of the maintenance stage. No supra-threshold activation was found during the WM probe. Together, we obtained clearer insights in which brain regions support successful WM and LTM without the potential confound of

  5. Financial Motivation Undermines Maintenance in an Intensive Diet and Activity Intervention

    PubMed Central

    Moller, Arlen C.; McFadden, H. Gene; Hedeker, Donald; Spring, Bonnie

    2012-01-01

    Financial incentives are widely used in health behavior interventions. However, self-determination theory posits that emphasizing financial incentives can have negative consequences if experienced as controlling. Feeling controlled into performing a behavior tends to reduce enjoyment and undermine maintenance after financial contingencies are removed (the undermining effect). We assessed participants' context-specific financial motivation to participate in the Make Better Choices trial—a trial testing four different strategies for improving four health risk behaviors: low fruit and vegetable intake, high saturated fat intake, low physical activity, and high sedentary screen time. The primary outcome was overall healthy lifestyle change; weight loss was a secondary outcome. Financial incentives were contingent upon meeting behavior goals for 3 weeks and became contingent upon merely providing data during the 4.5-month maintenance period. Financial motivation for participation was assessed at baseline using a 7-item scale (α = .97). Across conditions, a main effect of financial motivation predicted a steeper rate of weight regained during the maintenance period, t(165) = 2.15, P = .04. Furthermore, financial motivation and gender interacted significantly in predicting maintenance of healthy diet and activity changes, t(160) = 2.42, P = .016, such that financial motivation had a more deleterious influence among men. Implications for practice and future research on incentivized lifestyle and weight interventions are discussed. PMID:22548152

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

    PubMed

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

    2016-04-01

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

  7. Curcumin inhibits activation of TRPM2 channels in rat hepatocytes

    PubMed Central

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

    2015-01-01

    Oxidative stress is a hallmark of many liver diseases including viral and drug-induced hepatitis, ischemia-reperfusion injury, and non-alcoholic steatohepatitis. One of the consequences of oxidative stress in the liver is deregulation of Ca2+ homeostasis, resulting in a sustained elevation of the free cytosolic Ca2+ concentration ([Ca2+]c) in hepatocytes, which leads to irreversible cellular damage. Recently it has been shown that liver damage induced by paracetamol and subsequent oxidative stress is, in large part, mediated by Ca2+ entry through Transient Receptor Potential Melastatin 2 (TRPM2) channels. Involvement of TRPM2 channels in hepatocellular damage induced by oxidative stress makes TRPM2 a potential therapeutic target for treatment of a range of oxidative stress-related liver diseases. We report here the identification of curcumin ((1E,6E)-1,7-bis(4-hydroxy-3-methoxyphenyl)-1,6-heptadiene-3,5-dione), a natural plant-derived polyphenol in turmeric spice, as a novel inhibitor of TRPM2 channel. Presence of 5 µM curcumin in the incubation medium prevented the H2O2- and paracetamol-induced [Ca2+]c rise in rat hepatocytes. Furthermore, in patch clamping experiments incubation of hepatocytes with curcumin inhibited activation of TRPM2 current by intracellular ADPR with IC50 of approximately 50 nM. These findings enhance understanding of the actions of curcumin and suggest that the known hepatoprotective properties of curcumin are, at least in part, mediated through inhibition of TRPM2 channels. PMID:26609559

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

    PubMed

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

    2014-05-01

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

  9. Physical activity maintenance in patients with rheumatoid arthritis: a qualitative study.

    PubMed

    Loeppenthin, K; Esbensen, Ba; Ostergaard, M; Jennum, P; Thomsen, T; Midtgaard, J

    2014-03-01

    To describe the experience of physical activity maintenance in patients with rheumatoid arthritis. A qualitative salutogenic-oriented interview study. A rheumatology outpatient clinic. A purposive sample of 16 physically active patients (mean age 50, range 37-67) diagnosed with rheumatoid arthritis on average 21 years previously (range 4-46 years). In-depth interviews were conducted using a semi-structured interview guide to illuminate how the phenomenon 'physical activity maintenance' was experienced by patients with rheumatoid arthritis. The interviews were analysed using systematic text condensation, inspired by Giorgi's descriptive phenomenological methodology. The analysis revealed three categories: (1) knowing and enjoying the body; (2) responsibility and challenges; (3) autonomy and social belonging. On the basis of these categories, the essential meaning of the phenomenon of physical activity maintenance for patients with rheumatoid arthritis was summarized into 'striving for a transparent body and participation', pointing to experiences of sensations of wellbeing, liberation from restrictions and social participation on equal terms with non-arthritis populations. This study demonstrates that physical activity in patients with rheumatoid arthritis may be understood as a resource to resist disability and to feel and stay healthy while creating and sustaining meaningfulness in life.

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

    PubMed

    Banerjee, Kinshuk; Das, Biswajit; Gangopadhyay, Gautam

    2013-04-28

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

  11. Activation of rat transient receptor potential cation channel subfamily V member 1 channels by 2-aminoethoxydiphenyl borate.

    PubMed

    Mamatova, Knara Nazaralievna; Kang, Tong Mook

    2013-09-01

    The transient receptor potential cation channel subfamily V member 1 (TRPV1) channel has been proved to be a molecular integrator of inflammatory pain sensation. 2-Aminoethoxydiphenyl borate (2-APB) and its analogs have been noticed as attractive candidates for the development of a selective TRPV1 agonist and/or antagonist. However, selectivity and effectiveness, species dependence, and the binding site(s) of 2-APB on TRPV1 channel protein remain controversial. The present study aimed to characterize acting sites of 2-APB on heterologously expressed rat TRPV1 (rTRPV1) channels in HEK 293 cells. Rat TRPV1 currents were recorded by cell-free, excised patch clamp techniques. In inside-out and outside-out patch modes, 2-APB applied either side of the membrane dose-dependently activated rTRPV1 channels. 2-APB dose-dependently potentiated rTRPV1 currents, that activated by capsaicin, protons, or noxious heat. 2-APB potentiated the capsaicin-activated rTRPV1 current from both side of the patch membrane. A structural analogue of 2-APB, diphenylboronic anhydride, showed the same potentiation effect on the capsaicin-activated rTRPV1 current. It is suggested that 2-APB directly opens rTRPV1 channels from both sides of the membrane and potentiates the opening of channels by inflammatory stimuli.

  12. Channels

    NASA Image and Video Library

    2015-11-20

    Today's VIS image shows a number of unnamed channels located on the northeastern margin of Terra Sabaea. Orbit Number: 61049 Latitude: 33.5036 Longitude: 58.6967 Instrument: VIS Captured: 2015-09-18 12:54 http://photojournal.jpl.nasa.gov/catalog/PIA20097

  13. Zn2+ activates large conductance Ca2+-activated K+ channel via an intracellular domain.

    PubMed

    Hou, Shangwei; Vigeland, Leif E; Zhang, Guangping; Xu, Rong; Li, Min; Heinemann, Stefan H; Hoshi, Toshinori

    2010-02-26

    Zinc is an essential trace element and plays crucial roles in normal development, often as an integral structural component of transcription factors and enzymes. Recent evidence suggests that intracellular Zn(2+) functions as a signaling molecule, mediating a variety of important physiological phenomena. However, the immediate effectors of intracellular Zn(2+) signaling are not well known. We show here that intracellular Zn(2+) potently and reversibly activates large-conductance voltage- and Ca(2+)-activated Slo1 K(+) (BK) channels. The full effect of Zn(2+) requires His(365) in the RCK1 (regulator of conductance for K(+)) domain of the channel. Furthermore, mutation of two nearby acidic residues, Asp(367) and Glu(399), also reduced activation of the channel by Zn(2+), suggesting a possible structural arrangement for Zn(2+) binding by the aforementioned residues. Extracellular Zn(2+) activated Slo1 BK channels when coexpressed with Zn(2+)-permeable TRPM7 (transient receptor potential melastatin 7) channels. The results thus demonstrate that Slo1 BK channels represent a positive and direct effector of Zn(2+) signaling and may participate in sculpting cellular response to an increase in intracellular Zn(2+) concentration.

  14. Single-channel properties of BK-type calcium-activated potassium channels at a cholinergic presynaptic nerve terminal

    PubMed Central

    Sun, Xiao-Ping; Schlichter, Lyanne C; Stanley, Elis F

    1999-01-01

    A high-conductance calcium-activated potassium channel (BK KCa) was characterized at a cholinergic presynaptic nerve terminal using the calyx synapse isolated from the chick ciliary ganglion.The channel had a conductance of 210 pS in a 150 mM:150 mM K+ gradient, was highly selective for K+ over Na+, and was sensitive to block by external charybdotoxin or tetraethylammonium (TEA) and by internal Ba2+. At +60 mV it was activated by cytoplasmic calcium [Ca2+]i with a Kd of ≈0.5 μM and a Hill coefficient of ≈2.0. At 10 μM [Ca2+]i the channel was 50 % activated (V½) at -8.0 mV with a voltage dependence (Boltzmann slope-factor) of 32.7 mV. The V½ values hyperpolarized with an increase in [Ca2+]i while the slope factors decreased. There were no overt differences in conductance or [Ca2+]i sensitivity between BK channels from the transmitter release face and the non-release face.Open and closed times were fitted by two and three exponentials, respectively. The slow time constants were strongly affected by both [Ca2+]i and membrane potential changes.In cell-attached patch recordings BK channel opening was enhanced by a prepulse permissive for calcium influx through the patch, suggesting that the channel can be activated by calcium ion influx through neighbouring calcium channels.The properties of the presynaptic BK channel are well suited for rapid activation during the presynaptic depolarization and Ca2+ influx that are associated with transmitter release. This channel may play an important role in terminating release by rapid repolarization of the action potential. PMID:10420003

  15. Considerations in repetitive activation of light sensitive ion channels for long-term studies: Channel rhodopsin in the Drosophila model.

    PubMed

    Higgins, Jake; Hermanns, Christina; Malloy, Cole; Cooper, Robin L

    2017-07-17

    Optogenetics is a technique used in various animal models and holds a potential for therapeutic possibilities in mammals. There are technical issues with the use of light sensitive ion channels: reproducible effects over time, controlling where the non-native proteins are targeted within the cell and changes in the biophysical properties of the cells they are expressed in. We used a variant of channel rhodopsin (ChR2-XXL) and targeted expression in neurons of larval Drosophila to investigate the acute and chronic activation, with light pulses, of the channels on synaptic function. The rhodopsin channel modifier all trans retinal (ATR) also plays a role in the sensitivity of the channel to light. Periods of acute, repetitive, and pulsatile blue light exposure over larval development produced attenuated responses. These blue light sensitive ion channels, with ATR, show accommodation and produce an electrical refractory period in inducing synaptic responses. The biological significance and aim of this study is to demonstrate that in controlling particular neurons or neuronal circuits with optogenetics, over time and throughout development, one will have to understand the dynamic nature of activating and silencing the light sensitive channels as well as the biophysical effects on neuronal activity. Copyright © 2017 Elsevier Ireland Ltd and Japan Neuroscience Society. All rights reserved.

  16. Nicotine activates the chemosensory cation channel TRPA1.

    PubMed

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

    2009-10-01

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

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

    PubMed

    Kaneko, Toshiyuki; Takahashi, Naoya; Kikuyama, Munehiro

    2009-03-01

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

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

    NASA Astrophysics Data System (ADS)

    Roth, Alexander; Hammerer, Klemens

    2016-10-01

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

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

    NASA Technical Reports Server (NTRS)

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

    1996-01-01

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

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

    NASA Technical Reports Server (NTRS)

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

    1996-01-01

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

  1. Src Tyrosine Kinase Alters Gating of Hyperpolarization-Activated HCN4 Pacemaker Channel through Tyr531

    PubMed Central

    Li, Chen-Hong; Zhang, Qi; Teng, Bunyen; Mustafa, S. Jamal; Huang, Jian-Ying; Yu, Han-Gang

    2009-01-01

    We recently discovered that the constitutively active Src tyrosine kinase can enhance the HCN4 channel activity by binding to the channel protein. To investigate the mechanism of modulation by Src of HCN channels, we studied the effects of a selective inhibitor of Src tyrosine kinase, PP2, on HCN4 and its mutant channels ex pressed in HEK293 cells using whole-cell patch clamp technique. We found that PP2 can inhibit HCN4 currents by negatively shifting the voltage dependence of channel activation, decreasing the whole-cell channel conductance, and slowing activation and deactivation kinetics. Screening putative tyrosine residues subject to phosphorylation yielded two candidates: Tyr531 and Tyr554. Substituting HCN4-Tyr531 with phenylalanine largely abolished the effects of PP2 on HCN4 channels. Replacing HCN4-Tyr554 by phenylalanine did not abolish the effects of PP2 on voltage-dependent activation, but did eliminate PP2-induced slowing of channel kinetics. The inhibitory effects of HCN channels associated with reduced Src tyrosine activity is confirmed in HL-1 cardiomyocytes. Finally, we found that PP2 can decrease the heart rate in a mouse model. These results demonstrate that Src tyrosine kinase enhances HCN4 currents by shifting their activation to more positive potentials and increasing the whole-cell channel conductance as well as speeding the channel kinetics. The tyrosine residue that mediates most of Src actions on HCN4 channels is Tyr531. PMID:17977941

  2. Determinants of Physical Activity Maintenance in Breast Cancer Survivors After a Community-Based Intervention.

    PubMed

    Lee, C Ellen; Von Ah, Diane; Szuck, Beth; Lau, Yiu-Keung James

    2016-01-01

    To determine whether empirically selected and social cognitive theory-based factors, including baseline characteristics and modifiable behavioral and psychosocial factors, were determinants of physical activity (PA) maintenance in breast cancer survivors (BCSs) six months after a PA intervention.
. Single-group longitudinal study.
. The Breast Health Centre in Winnipeg, Manitoba, Canada.
. 42 survivors with stage 0-III breast cancer who completed chemotherapy and/or radiation therapy.
. The community-based PA intervention included six weekly education and practice sessions on home-based aerobic, resistance, balance, and flexibility exercises.
. The dependent variable, PA maintenance, was determined based on PA level measurement at six months postintervention. The independent variables of baseline characteristics (age, stage of cancer, and chronic musculoskeletal symptoms) and modifiable behavioral and psychosocial factors (PA level, fatigue, PA self-efficacy in overcoming barriers and performing tasks) were assessed at baseline and postintervention.
. Multivariate regression analyses revealed that baseline fatigue and chronic musculoskeletal symptoms were the only factors significantly associated with PA maintenance.
. Baseline fatigue level and chronic musculoskeletal symptoms were significant determinants of PA maintenance in breast cancer survivors who had completed a community-based PA intervention. However, other key factors were considered.
. Prior to participation in community-based PA interventions, clinicians should take into account the effects of high baseline fatigue levels and chronic musculoskeletal symptoms on potential PA maintenance, and consider additional assessments and support for BCSs to sustain their PA behavioral change.
.

  3. Optimal Medical Equipment Maintenance Service Proposal Decision Support System combining Activity Based Costing (ABC) and the Analytic Hierarchy Process (AHP).

    PubMed

    da Rocha, Leticia; Sloane, Elliot; M Bassani, Jose

    2005-01-01

    This study describes a framework to support the choice of the maintenance service (in-house or third party contract) for each category of medical equipment based on: a) the real medical equipment maintenance management system currently used by the biomedical engineering group of the public health system of the Universidade Estadual de Campinas located in Brazil to control the medical equipment maintenance service, b) the Activity Based Costing (ABC) method, and c) the Analytic Hierarchy Process (AHP) method. Results show the cost and performance related to each type of maintenance service. Decision-makers can use these results to evaluate possible strategies for the categories of equipment.

  4. Characterization of Underwater Sounds Produced by a Hydraulic Cutterhead Dredge during Maintenance Dredging in the Stockton Deepwater Shipping Channel, California

    DTIC Science & Technology

    2014-03-01

    recorder running MDR_SLM Software. The ST1400ENV is a self-contained system designed specifically to record underwater sounds while simultaneously...Approved for public release; distribution is unlimited. ERDC TN-DOER-E38 March 2014 Characterization of Underwater Sounds Produced by a...PURPOSE: The purpose of this study was to record and analyze underwater sounds generated by a small hydraulic cutterhead dredge during maintenance

  5. Operation and Maintenance, 9-Foot Navigation Channel, Upper Mississippi River, Head of Navigation to Guttenberg, Iowa. Volume 2. Exhibits.

    DTIC Science & Technology

    1974-08-01

    Flying Squirrel (llaucorrs volans Occasional p Plains Pocket Gopher Geomys bursarius Occasional S Beaver Castor canadensis Common Western Harvest Mouse... TYPE OF REPORT & PERIOD COVERED STATEMENT, OPERATION AND MAINTENANCE 9-FOOT Final NAVIGATION CHANNGEL, UPPER MISSISSIPPI RIVER HEAD OF NAVIGATION TO...Population 151 of Major Types of Livestock and Poultry in the Area of Pool 3 127 Commercial Lockages in Pool 3, 1960 - 1972 152 128 Pool 3 Waterborne

  6. Activation and Regulation of Purinergic P2X Receptor Channels

    PubMed Central

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

    2011-01-01

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

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

    NASA Technical Reports Server (NTRS)

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

    1989-01-01

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

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

    NASA Technical Reports Server (NTRS)

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

    1989-01-01

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

  9. Activation of the cell integrity pathway is channelled through diverse signalling elements in fission yeast.

    PubMed

    Barba, Gregorio; Soto, Teresa; Madrid, Marisa; Núñez, Andrés; Vicente, Jeronima; Gacto, Mariano; Cansado, José

    2008-04-01

    MAPK Pmk1p is the central element of a cascade involved in the maintenance of cell integrity and other functions in Schizosaccharomyces pombe. Pmk1p becomes activated by multiple stressing situations and also during cell separation. GTPase Rho2p acts upstream of the protein kinase C homolog Pck2p to activate the Pmk1 signalling pathway through direct interaction with MAPKKK Mkh1p. In this work we analyzed the functional significance of both Rho2p and Pck2p in the transduction of various stress signals by the cell integrity pathway. The results indicate that basal Pmk1p activity can be positively regulated by alternative mechanisms which are independent on the control by Rho2p and/or Pck2p. Unexpectedly, Pck1p, another protein kinase C homolog, negatively modulates Pmk1p basal activity by an unknown mechanism. Moreover, different elements appear to regulate the stress-induced activation of Pmk1p depending on the nature of the triggering stimuli. Whereas Pmk1p activation induced by hyper- or hypotonic stresses is channeled through Rho2p-Pck2p, other stressors, like glucose deprivation or cell wall disturbance, are transduced via other pathways in addition to that of Rho2p-Pck2p. On the contrary, Pmk1p activation observed during cell separation or after treatment with hydrogen peroxide does not involve Rho2p-Pck2p. Finally, Pck2p function is critical to maintain a Pmk1p basal activity that allows Pmk1p activation induced by heat stress. These data demonstrate the existence of a complex signalling network modulating Pmk1p activation in response to a variety of stresses in fission yeast.

  10. Bi-objective analysis of water-sediment regulation for channel scouring and delta maintenance: A study of the lower Yellow River

    NASA Astrophysics Data System (ADS)

    Kong, D.; Miao, C.; Duan, Q.

    2016-12-01

    Long-term hydrological data and remotely-sensed satellite images were used to analyze the effects of the water-sediment regulation scheme (WSRS) implemented in the lower Yellow River (LYR), China, between 1983 and 2013. The WSRS aimed to control channel scouring in the LYR and maintain the Yellow River Delta (YRD). Channel erosion in the LYR has primarily depended on the incoming sediment concentration at Xiaolangdi, where the concentration must be lower than approximately 9.17 × 10-3 t m-3 to avoid rising of the riverbed. In 1996, an artificial diversion altered the evolution of the YRD. To maintain delta equilibrium, an average sediment load of about 441 × 106 t year-1 was required before 1996, after which this value decreased to 167 × 106 t year-1. We provide a preliminary estimate of the incoming water and sediment conditions required at the Xiaolangdi station to guarantee both LYR channel scouring and maintenance of the YRD. Our results show that it is feasible to transport sediment originally deposited in the LYR to the river mouth to maintain the delta, which is of great significance for the future management and environmental protection of the LYR.

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

    PubMed Central

    Bock, Tobias

    2016-01-01

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

  12. Two salt bridges differentially contribute to the maintenance of cystic fibrosis transmembrane conductance regulator (CFTR) channel function.

    PubMed

    Cui, Guiying; Freeman, Cody S; Knotts, Taylor; Prince, Chengyu Z; Kuang, Christopher; McCarty, Nael A

    2013-07-12

    Previous studies have identified two salt bridges in human CFTR chloride ion channels, Arg(352)-Asp(993) and Arg(347)-Asp(924), that are required for normal channel function. In the present study, we determined how the two salt bridges cooperate to maintain the open pore architecture of CFTR. Our data suggest that Arg(347) not only interacts with Asp(924) but also interacts with Asp(993). The tripartite interaction Arg(347)-Asp(924)-Asp(993) mainly contributes to maintaining a stable s2 open subconductance state. The Arg(352)-Asp(993) salt bridge, in contrast, is involved in stabilizing both the s2 and full (f) open conductance states, with the main contribution being to the f state. The s1 subconductance state does not require either salt bridge. In confirmation of the role of Arg(352) and Asp(993), channels bearing cysteines at these sites could be latched into a full open state using the bifunctional cross-linker 1,2-ethanediyl bismethanethiosulfonate, but only when applied in the open state. Channels remained latched open even after washout of ATP. The results suggest that these interacting residues contribute differently to stabilizing the open pore in different phases of the gating cycle.

  13. Two Salt Bridges Differentially Contribute to the Maintenance of Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) Channel Function*

    PubMed Central

    Cui, Guiying; Freeman, Cody S.; Knotts, Taylor; Prince, Chengyu Z.; Kuang, Christopher; McCarty, Nael A.

    2013-01-01

    Previous studies have identified two salt bridges in human CFTR chloride ion channels, Arg352-Asp993 and Arg347-Asp924, that are required for normal channel function. In the present study, we determined how the two salt bridges cooperate to maintain the open pore architecture of CFTR. Our data suggest that Arg347 not only interacts with Asp924 but also interacts with Asp993. The tripartite interaction Arg347-Asp924-Asp993 mainly contributes to maintaining a stable s2 open subconductance state. The Arg352-Asp993 salt bridge, in contrast, is involved in stabilizing both the s2 and full (f) open conductance states, with the main contribution being to the f state. The s1 subconductance state does not require either salt bridge. In confirmation of the role of Arg352 and Asp993, channels bearing cysteines at these sites could be latched into a full open state using the bifunctional cross-linker 1,2-ethanediyl bismethanethiosulfonate, but only when applied in the open state. Channels remained latched open even after washout of ATP. The results suggest that these interacting residues contribute differently to stabilizing the open pore in different phases of the gating cycle. PMID:23709221

  14. Hippocampal hypoglycaemia-activated K+ channels: single-channel analysis of glucose and voltage dependence.

    PubMed

    Tromba, C; Salvaggio, A; Racagni, G; Volterra, A

    1994-11-01

    The effect of glucose on kinetics and the voltage-dependent characteristics of glucose-sensitive channels in hippocampal neurons were examined with the cell-attached mode of the patch-clamp technique. Recordings of a 100-pS K+ channel in the presence or absence of glucose demonstrate that the increase in channel open state probability (Po) induced by glucose deprivation (40- to 400-times the control in high-glucose medium) was largely due to a decrease in the global amount of time spent by the channel in a long-lived closed state. The Po value of the same 100-pS channel was also found to increase (by approx. 80-times) following a depolarization of 40 mV from rest, the main factor responsible for this being a dramatic shortening of the long closed-times on depolarization. Another glucose-sensitive channel of smaller conductance (approx. 10 pS) showed a similar dependence of Po on glucose, but different dependence on voltage, with long openings at the same hyperpolarized potentials where the 100-pS channel was almost always closed. Our results indicate that the action of glucose on the kinetics of hippocampal channels closely resembles that of ATP-sensitive channels in pancreatic beta-cells. Furthermore, they indicate that the two types of glucose-sensitive channels found in hippocampal neurons, differing not only in their single-channel conductance but also in the dependence on voltage, could play different roles in the responses of these cells to modified energetic supply.

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

    PubMed

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

    2015-09-23

    Ion channel opening and closing are fundamental to cellular signalling and homeostasis. Gates that control K(+) channel activity were found both at an intracellular pore constriction and within the selectivity filter near the extracellular side but the specific location of the gate that opens Ca(2+)-activated K(+) channels has remained elusive. Using the Methanobacterium thermoautotrophicum homologue (MthK) and a stopped-flow fluorometric assay for fast channel activation, we show that intracellular quaternary ammonium blockers bind to closed MthK channels. Since the blockers are known to bind inside a central channel cavity, past the intracellular entryway, the gate must be within the selectivity filter. Furthermore, the blockers access the closed channel slower than the open channel, suggesting that the intracellular entryway narrows upon pore closure, without preventing access of either the blockers or the smaller K(+). Thus, Ca(2+)-dependent gating in MthK occurs at the selectivity filter with coupled movement of the intracellular helices.

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

    NASA Astrophysics Data System (ADS)

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

    2015-09-01

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

  17. The role of 'know-how' in maintenance activities and reliability in a high-risk process control plant.

    PubMed

    Garrigou, A; Carballeda, G; Daniellou, F

    1998-04-01

    Using observation of a maintenance operator's activity and 'the history' that it produced as a basis, we will discuss the role of 'know-how' in maintenance activities, and particularly the problems raised by putting this knowledge into words. Second, we will underline the contribution of this know-how to the operational reliability of the facilities and we will investigate its being taken into account in work instructions. With such issues as a basis, we will conclude with the need for ergonomists to develop modes of interviewing to help people to put know-how into words, so that it is recognized and considered in maintenance activity organization.

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

    PubMed

    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.

  19. Natural resources management activities and biodiversity maintenance. Progress report, July 1, 1994--June 30, 1995

    SciTech Connect

    Caudell, M.B.

    1995-05-01

    This progress report for the Natural Resources Management Activities and Biodiversity Maintenance for the time period July 1, 1994 - June 30, 1995 was submitted to the DOE by the South Carolina Department of Natural Resources. The activities devoted to revitilization of wildlife areas and reintroduction of various animal species to wildlife areas (such as Crackerneck Wildlife Management Area) are described. Other information regarding site characterization, land use and resource management in South Carolina is provided. Also, a description of attendence to various meetings and certification seminars is covered.

  20. Activation by divalent cations of a Ca2+-activated K+ channel from skeletal muscle membrane

    PubMed Central

    1988-01-01

    Several divalent cations were studied as agonists of a Ca2+-activated K+ channel obtained from rat muscle membranes and incorporated into planar lipid bilayers. The effect of these agonists on single-channel currents was tested in the absence and in the presence of Ca2+. Among the divalent cations that activate the channel, Ca2+ is the most effective, followed by Cd2+, Sr2+, Mn2+, Fe2+, and Co2+. Mg2+, Ni2+, Ba2+, Cu2+, Zn2+, Hg2+, and Sn2+ are ineffective. The voltage dependence of channel activation is the same for all the divalent cations. The time-averaged probability of the open state is a sigmoidal function of the divalent cation concentration. The sigmoidal curves are described by a dissociation constant K and a Hill coefficient N. The values of these parameters, measured at 80 mV are: N = 2.1, K = 4 X 10(- 7) mMN for Ca2+; N = 3.0, K = 0.02 mMN for Cd2+; N = 1.45, K = 0.63 mMN for Sr2+; N = 1.7, K = 0.94 mMN for Mn2+; N = 1.1, K = 3.0 mMN for Fe2+; and N = 1.1 K = 4.35 mMN for Co2+. In the presence of Ca2+, the divalent cations Cd2+, Co2+, Mn2+, Ni2+, and Mg2+ are able to increase the apparent affinity of the channel for Ca2+ and they increase the Hill coefficient in a concentration-dependent fashion. These divalent cations are only effective when added to the cytoplasmic side of the channel. We suggest that these divalent cations can bind to the channel, unmasking new Ca2+ sites. PMID:3171535

  1. A new focus on operation and maintenance activities and the role of EPA

    SciTech Connect

    Griffith, J.A.; Hopkins, N.; Taylor, J.

    1996-12-31

    The Environmental Protection Agency`s (EPA) Superfund program, which was established pursuant to the Comprehensive Environmental Response. Compensation, and Liability Act (CERCLA) of 1980, as amended by the Superfund Amendments and Reauthorization Act (SARA) of 1986, governs the cleanup of domestic hazardous waste sites. After Superfund sites undergo the cleanup process, additional site activities may be necessary to ensure that the remedy continues to operate effectively and the cleanup continues to protect human health and the environment. Examples of these operation and maintenance (O and M) activities include the maintenance of contaminated soil covers to prevent erosion, the continuing operation of treatment systems for contaminated groundwater or surface water, and the ongoing monitoring of land or water use restrictions. According to a May 1995 General Accounting Office (GAO) report, Information on Operations and Maintenance Activities and Costs, roughly 62 percent of the 229 National Priorities List (NPL) sites reviewed where the remedy had been constructed required some long-term actions resulting from the remedy choice. EPA monitors the O and M activities of states and PRPs to ensure that they are being performed. Monitoring O and M activities, which is vital to preserving the integrity of cleanup remedies, includes the requirement that EPA review certain sites every five years. This paper will highlight EPA`s policies for managing O and M issues throughout the Superfund pipeline, transferring O and M to the state, performing O and M review, and termination of O and M. This paper also generally covers O and M issues of concern to PRPs.

  2. Physical activity in relation to long-term maintenance after intentional weight loss in premenopausal women

    PubMed Central

    Mekary, Rania A.; Feskanich, Diane; Hu, Frank B.; Willett, Walter C.; Field, Alison E.

    2009-01-01

    The type and amount of physical activity needed for prevention of weight regain are not well understood. We prospectively examined the associations between patterns of discretionary physical activity and 6-year maintenance of intentional weight loss among 4,558 healthy premenopausal women who were 26–45 years old in 1991 and had lost >5% of their body weight in the previous two years. Participants reported their physical activity and weight in 1991 and 1997. The outcome was weight regain, defined as regaining in 1997 >30% of the lost weight between 1989 and 1991. Between 1991 and 1997, 80% of women regained >30% of their previous intentional weight loss. An increase of 30 minutes/day in total discretionary activity between 1991 and 1997 was associated with less weight regain (−1.36kg, 95% confidence interval (CI) = −1.61, −1.12), particularly among overweight women (body mass index ≥25) (−2.45kg, −3.12 to −1.78). Increased jogging or running was associated with less weight regain (−3.26kg; −4.41 to −2.10) than increased brisk walking (−1.69kg; −2.15 to −1.22) or other activities (−1.26kg; −1.65 to −0.87). Compared to women who remained sedentary, women were less likely to regain >30% of the lost weight if they maintained 30+ minutes/day of discretionary physical activity (OR=0.69, 0.53 to 0.89) or increased to this activity level (OR=0.48, 0.39 to 0.60). Conversely, risk was elevated in women who decreased their activity. Increased physical activity, particularly high intensity activities, is associated with better maintenance of weight loss. The benefits of activity were greater among overweight/obese than normal weight women. PMID:19498346

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

    PubMed Central

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

    2008-01-01

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

  4. Teaching children with autism to use photographic activity schedules: maintenance and generalization of complex response chains.

    PubMed

    MacDuff, G S; Krantz, P J; McClannahan, L E

    1993-01-01

    We used a graduated guidance procedure to teach 4 boys with autism to follow photographic activity schedules to increase on-task and on-schedule behavior. The multiple baseline across participants design included baseline, teaching, maintenance, resequencing of photographs, and generalization to novel photographs phases. The results indicated that photographic activity schedules (albums depicting after-school activities) produced sustained engagement, and skills generalized to a new sequence of photographs and to new photographs. The acquisition of schedule-following skills enabled these children with severe developmental disabilities to display lengthy response chains, independently change activities, and change activities in different group home settings in the absence of immediate supervision and prompts from others.

  5. Repair, Evaluation, Maintenance, and Rehabilitation Research Program: Geotechnical Aspects of Rock Erosion in Emergency Spillway Channels. Report 3. Remediation

    DTIC Science & Technology

    1988-09-01

    flood- control and water-storage projects buiit and managed by the US Army Corps of Engineers (CE), other Federal agencies, state, and local interests...this problem area to CE personnel and other interested parties in Federal, state, and local agencies. Scope 3. Geotechnical factors control the...earth mate- rials often control the location and geometry of channel gradient changes (knickpoints) which can occur as abrupt waterfalls, a series of

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

    ERIC Educational Resources Information Center

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

    2015-01-01

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

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

    ERIC Educational Resources Information Center

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

    2015-01-01

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

  8. Telomere maintenance and telomerase activity are differentially regulated in asexual and sexual worms

    PubMed Central

    Tan, Thomas C. J.; Rahman, Ruman; Jaber-Hijazi, Farah; Felix, Daniel A.; Chen, Chen; Louis, Edward J.; Aboobaker, Aziz

    2012-01-01

    In most sexually reproducing animals, replication and maintenance of telomeres occurs in the germ line and during early development in embryogenesis through the use of telomerase. Somatic cells generally do not maintain telomere sequences, and these cells become senescent in adults as telomeres shorten to a critical length. Some animals reproduce clonally and must therefore require adult somatic mechanisms for maintaining their chromosome ends. Here we study the telomere biology of planarian flatworms with apparently limitless regenerative capacity fueled by a population of highly proliferative adult stem cells. We show that somatic telomere maintenance is different in asexual and sexual animals. Asexual animals maintain telomere length somatically during reproduction by fission or when regeneration is induced by amputation, whereas sexual animals only achieve telomere elongation through sexual reproduction. We demonstrate that this difference is reflected in the expression and alternate splicing of the protein subunit of the telomerase enzyme. Asexual adult planarian stem cells appear to maintain telomere length over evolutionary timescales without passage through a germ-line stage. The adaptations we observe demonstrate indefinite somatic telomerase activity in proliferating stem cells during regeneration or reproduction by fission, and establish planarians as a pertinent model for studying telomere structure, function, and maintenance. PMID:22371573

  9. 2-Aminoethoxydiphenyl Borate as a Common Activator of TRPV1, TRPV2, and TRPV3 Channels

    PubMed Central

    Colton, C. K.; Zhu, M. X.

    2015-01-01

    2-Aminoethoxydiphenyl borate (2APB) had been depicted as a universal blocker of transient receptor potential (TRP) channels. While evidence has accumulated showing that some TRP channels are indeed inhibited by 2APB, especially in heterologous expression systems, there are other TRP channels that are unaffected or affected very little by this compound. More interestingly, the thermosensitive TRPV1, TRPV2, and TRPV3 channels are activated by 2APB. This has been demonstrated both in heterologous systems and in native tissues that express these channels. A number of 2APB analogs have been examined for their effects on native store-operated channels and heterologously expressed TRPV3. These studies revealed a complex mechanism of action for 2APB and its analogs on ion channels. In this review, we have summarized the current results on 2APB-induced activation of TRPV1–3 and discussed the potential mechanisms by which 2APB may regulate TRP channels. PMID:17217057

  10. Purinergic regulation of CFTR and Ca(2+)-activated Cl(-) channels and K(+) channels in human pancreatic duct epithelium.

    PubMed

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

    2013-04-01

    Purinergic agonists have been considered for the treatment of respiratory epithelia in cystic fibrosis (CF) patients. The pancreas, one of the most seriously affected organs in CF, expresses various purinergic receptors. Studies on the rodent pancreas show that purinergic signaling regulates pancreatic secretion. In the present study we aim to identify Cl(-) and K(+) channels in human pancreatic ducts and their regulation by purinergic receptors. Human pancreatic duct epithelia formed by Capan-1 or CFPAC-1 cells were studied in open-circuit Ussing chambers. In Capan-1 cells, ATP/UTP effects were 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, KCa3.1). The apical effects of ATP/UTP were greatly potentiated by the IK channel opener DC-EBIO. Determination of RNA and protein levels revealed that Capan-1 cells have high expression of TMEM16A (ANO1), a likely CaCC candidate. We conclude that in human pancreatic duct cells ATP/UTP regulates via purinergic 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 function should consider sidedness of purinergic signaling and the essential role of K(+) channels.

  11. Activation of Drosophila Sodium Channels Promotes Modification by Deltamethrin

    PubMed Central

    Vais, Horia; Williamson, Martin S.; Goodson, Susannah J.; Devonshire, Alan L.; Warmke, Jeffrey W.; Usherwood, Peter N.R.; Cohen, Charles J.

    2000-01-01

    kdr and super-kdr are mutations in houseflies and other insects that confer 30- and 500-fold resistance to the pyrethroid deltamethrin. They correspond to single (L1014F) and double (L1014F+M918T) mutations in segment IIS6 and linker II(S4–S5) of Na channels. We expressed Drosophila para Na channels with and without these mutations and characterized their modification by deltamethrin. All wild-type channels can be modified by <10 nM deltamethrin, but high affinity binding requires channel opening: (a) modification is promoted more by trains of brief depolarizations than by a single long depolarization, (b) the voltage dependence of modification parallels that of channel opening, and (c) modification is promoted by toxin II from Anemonia sulcata, which slows inactivation. The mutations reduce channel opening by enhancing closed-state inactivation. In addition, these mutations reduce the affinity for open channels by 20- and 100-fold, respectively. Deltamethrin inhibits channel closing and the mutations reduce the time that channels remain open once drug has bound. The super-kdr mutations effectively reduce the number of deltamethrin binding sites per channel from two to one. Thus, the mutations reduce both the potency and efficacy of insecticide action. PMID:10694259

  12. Potassium channel activation inhibits proliferation of breast cancer cells by activating a senescence program.

    PubMed

    Lansu, K; Gentile, S

    2013-06-06

    Traditionally the hERG1 potassium channel has been known to have a fundamental role in membrane excitability of several mammalian cells including cardiac myocytes. hERG1 has recently been found to be expressed in non-excitable cancer cells of different histogenesis, but the role of this channel in cancer biology is unknown. Results form recent studies on the effect hERG1 inhibition in some breast cancer cells are controversial as it can lead to apoptosis or protect against cell death. Nevertheless, these data suggest that the hERG1 channel could have an important role in cancer biology. Here we report the effects of hyperstimulation of hERG1 channel in human mammary gland adenocarcinoma-derived cells. Application of the hERG1 activator, the diphenylurea derivative NS1643, inhibits cell proliferation irreversibly. This event is accompanied by a preferential arrest of the cell cycle in G0/G1 phase without the occurrence of apoptotic events. Consequently, cells responded to NS1643 by developing a senescence-like phenotype associated with increased protein levels of the tumor suppressors p21 and p16(INK4a) and by a positive β-galactosidase assay. These data suggest that prolonged stimulation of the hERG1 potassium channel may activate a senescence program and offers a compelling opportunity to develop a potential antiproliferative cancer therapy.

  13. Requirement of calcium-activated chloride channels in the activation of mouse vomeronasal neurons

    PubMed Central

    Kim, SangSeong; Ma, Limei; Yu, C. Ron

    2011-01-01

    In terrestrial vertebrates, the vomeronasal organ (VNO) detects and transduces pheromone signals. VNO activation is thought to be mediated by the transient receptor potential C2 (TRPC2) channel. The aberrant behavioural phenotypes observed in TRPC2−/− mice are generally attributed to the lost VNO function. Recently, calcium-activated chloride channels have been shown to contribute to VNO activation. Here we show that CACCs can be activated in VNO slice preparations from the TRPC2−/− mice and this activation is blocked by pharmacological agents that inhibit intracellular Ca2+ release. Urine-evoked Cl− current is sufficient to drive spiking changes in VNO neurons from both wild-type (WT) and TRPC2−/− mice. Moreover, blocking Cl− conductance essentially abolishes VNO activation in WT neurons. These results suggest a TRPC2-independent signalling pathway in the VNO and the requirement of calcium-activated chloride channels currents to mediate pheromone activation. Our data further suggest that TRPC2−/− mice retain partial VNO function. PMID:21694713

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

    PubMed Central

    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

  15. Forcing Open TRP channels: mechanical gating as a unifying activation mechanism

    PubMed Central

    Liu, Chao; Montell, Craig

    2015-01-01

    Transient receptor potential (TRP) proteins are cation channels that comprise a superfamily of molecular sensors that enable animals to detect a wide variety of environmental stimuli. This versatility enables vertebrate and invertebrate TRP channels to function in a diversity of senses, ranging from vision to taste, smell, touch, hearing, proprioception and thermosensation. Moreover, many individual TRP channels are activated through a surprising range of sensory stimuli. The multitasking nature of TRP channels raises the question as to whether seemingly disparate activators gate TRPs through common strategies. In this regard, a recent major advance is the discovery that a phospholipase C (PLC)-dependent signaling cascade activates the TRP channels in Drosophila photoreceptor cells through generation of force in the lipid-bilayer. The premise of this review is that mechanical force is a unifying, common strategy for gating TRP channels. In addition to several TRP channels that function in mechanosensation and are gated by force applied to the cells, changes in temperature and in the concentration of lipophilic second messengers through stimulation of signaling cascades, cause architectural modifications of the cell membrane, which in turn activate TRP channels through mechanical force. Consequently, TRPs are capable of functioning as stretch-activated channels, even in cases in which the stimuli that initiate the signaling cascades are not mechanical. We propose that most TRPs are actually mechanosensitive channels (MSCs), which undergo conformational changes in response to tension imposed on the lipid bilayer, resulting in channel gating. PMID:25998730

  16. Functional Reconstitution and Channel Activity Measurements of Purified Wildtype and Mutant CFTR Protein

    PubMed Central

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

    2015-01-01

    The Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) is a unique channel-forming member of the ATP Binding Cassette (ABC) superfamily of transporters. The phosphorylation and nucleotide dependent chloride channel activity of CFTR has been frequently studied in whole cell systems and as single channels in excised membrane patches. Many Cystic Fibrosis-causing mutations have been shown to alter this activity. While a small number of purification protocols have been published, a fast reconstitution method that retains channel activity and a suitable method for studying population channel activity in a purified system have been lacking. Here rapid methods are described for purification and functional reconstitution of the full-length CFTR protein into proteoliposomes of defined lipid composition that retains activity as a regulated halide channel. This reconstitution method together with a novel flux-based assay of channel activity is a suitable system for studying the population channel properties of wild type CFTR and the disease-causing mutants F508del- and G551D-CFTR. Specifically, the method has utility in studying the direct effects of phosphorylation, nucleotides and small molecules such as potentiators and inhibitors on CFTR channel activity. The methods are also amenable to the study of other membrane channels/transporters for anionic substrates. PMID:25867140

  17. Activation of Ca(2+) -activated Cl(-) channel ANO1 by localized Ca(2+) signals.

    PubMed

    Jin, Xin; Shah, Sihab; Du, Xiaona; Zhang, Hailin; Gamper, Nikita

    2016-01-01

    Ca(2+)-activated chloride channels (CaCCs) regulate numerous physiological processes including epithelial transport, smooth muscle contraction and sensory processing. Anoctamin-1 (ANO1, TMEM16A) is a principal CaCC subunit in many cell types, yet our understanding of the mechanisms of ANO1 activation and regulation are only beginning to emerge. Ca(2+) sensitivity of ANO1 is rather low and at negative membrane potentials the channel requires several micromoles of intracellular Ca(2+) for activation. However, global Ca(2+) levels in cells rarely reach such levels and, therefore, there must be mechanisms that focus intracellular Ca(2+) transients towards the ANO1 channels. Recent findings indeed indicate that ANO1 channels often co-localize with sources of intracellular Ca(2+) signals. Interestingly, it appears that in many cell types ANO1 is particularly tightly coupled to the Ca(2+) release sites of the intracellular Ca(2+) stores. Such preferential coupling may represent a general mechanism of ANO1 activation in native tissues.

  18. Voltage-dependent potassium channels in activated rat microglia.

    PubMed Central

    Nörenberg, W; Gebicke-Haerter, P J; Illes, P

    1994-01-01

    equimolar concentration of Cs+, and the extracellular application of tetraethylammonium and quinine inhibited both currents. 7. An increase of extracellular Ca2+ from 2 to 20 mM resulted in outwardly rectifying K+ channels activating at more positive potentials. Omission of Ca2+ from the extracellular medium had the opposite effect. When the intracellular free Ca2+ was increased from 0.01 to 1 microM, the outward current amplitudes were depressed. The Ca2+ ionophore A23187 had a similar effect. 8. LPS-treated microglial cells possess inwardly and outwardly rectifying K+ channels. The physiological and pharmacological characteristics of these two channel populations are markedly different.(ABSTRACT TRUNCATED AT 400 WORDS) PMID:7514664

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

    PubMed

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

    2012-11-23

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

  20. Glycosylation-dependent activation of epithelial sodium channel by solnatide.

    PubMed

    Shabbir, Waheed; Tzotzos, Susan; Bedak, Minela; Aufy, Mohammad; Willam, Anita; Kraihammer, Martin; Holzner, Alexander; Czikora, Istvan; Scherbaum-Hazemi, Parastoo; Fischer, Hendrik; Pietschmann, Helmut; Fischer, Bernhard; Lucas, Rudolf; Lemmens-Gruber, Rosa

    2015-12-15

    Dysfunction of the epithelial sodium channel (ENaC), which regulates salt and water homeostasis in epithelia, causes several human pathological conditions, including pulmonary oedema. This is a potentially lethal complication of acute lung injury at least partially caused by dysfunctional alveolar liquid clearance, which in turn impairs alveolar gas exchange. Solnatide (named TIP-peptide, AP301), a 17 residue peptide mimicking the lectin-like domain of TNF has been shown to activate ENaC in several experimental animal models of acute lung injury and is being evaluated as a potential therapy for pulmonary oedema. The peptide has recently completed phase 1 and 2a clinical trials. In this study, we identify a glycosylation-dependent mechanism that preserves ENaC function and expression. Since our previous data suggested that the pore-forming subunits of ENaC are essential for maximal current activation by solnatide, we performed single- and multi-N-glycosylation site mutations in αN232,293,312,397,511Q- and δN166,211,384Q-subunits, in order to identify crucial residues for interaction with solnatide within the extracellular loop of the channel. Additionally, we generated αL576X and αN232,293,312,397,511Q,L576X deletion mutants of ENaC-α, since we have previously demonstrated that the carboxy terminal domain of this subunit is also involved in its interaction with solnatide. In cells expressing αN232,293,312,397,511Q,L576Xβγ-hENaC or δN166,311,384Q,D552Xβγ-hENaC activation by solnatide, as measured in whole cell patch clamp mode, was completely abolished, whereas it was attenuated in αL576Xβγ-hENaC- and δD552Xβγ-hENaC-expressing cells. Taken together, our findings delineate an N-glycan dependent interaction between the TIP-peptide and ENaC leading to normalization of both sodium and fluid absorption in oedematous alveoli to non-oedematous levels.

  1. Effects of N-glycosylation on hyperpolarization-activated cyclic nucleotide-gated (HCN) channels.

    PubMed

    Li, Mo; Tonggu, Lige; Tang, Lan; Wang, Liguo

    2015-02-15

    Hyperpolarization-activated cyclic nucleotide-gated (HCN) channels are activated by membrane hyperpolarization and conduct an inward cation current, which contributes to rhythmic electrical activity of neural and cardiac pacemaker cells. HCN channels have been shown to undergo N-linked glycosylation, and the N-glycosylation has been shown to be required for membrane trafficking and possibly function. In this study, recombinant wild-type (WT) and glycosylation-defective N380Q HCN2 channels were individually or co-expressed in HEK-293 cells. We demonstrate that glycosylation is required for trafficking to the plasma membrane and for the stability of HCN channels in the cell. Interestingly, the heteromeric HCN2 channels of WT and glycosylation-defective N380Q have been observed on cell membranes, indicating that not all four subunits of a tetrameric HCN2 channel need to be glycosylated for HCN2 channels to traffic to plasma membranes. Subsequently, we investigate the effect of N-glycosylation on the function of HCN2 channels. We developed a fluorescence-based flux assay, which makes it possible to establish a negative potential inside liposomes to open HCN2 channels. Using this flux assay, we demonstrate that glycosylation-defective N380Q HCN2 channels reconstituted into liposomes function similarly to WT HCN2 channels. This suggests that N-glycosylation is not required for HCN2 channels to function.

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

    PubMed Central

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

    2012-01-01

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

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

    PubMed

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

    2014-09-02

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

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

    NASA Astrophysics Data System (ADS)

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

    2014-02-01

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

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

    PubMed

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

    2014-02-28

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

  6. Ca2+ influx through L-type Ca2+ channels and transient receptor potential channels activate pathological hypertrophy signaling

    PubMed Central

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

    2012-01-01

    Common cardiovascular diseases such as hypertension and myocardial infarction require that myocytes develop greater than normal force to maintain cardiac pump function. This requires increases in [Ca2+]. These diseases induce cardiac hypertrophy and increases in [Ca2+] are known to be an essential proximal signal for activation of hypertrophic genes. However, the source of “hypertrophic” [Ca2+] is not known and is the topic of this study. The role of Ca2+ influx through L-type Ca2+ channels (LTCC), T-type Ca2+ channels (TTCC) and transient receptor potential (TRP) channels on the activation of Calcineurin (Cn) – Nuclear Factor of Activated T cells (NFAT) signaling and myocyte hypertrophy was studied. Neonatal rat (NRVMs) and adult feline (AFVM) ventricular myocytes were infected with an adenovirus containing NFAT-GFP, to determine factors that could induce NFAT nuclear translocation. Four millimolar Ca2+ or pacing induced NFAT nuclear translocation. This effect was blocked by Cn inhibitors. In NRVMs Nifedipine (Nif, LTCC antagonist) blocked high Ca2+-induced NFAT nuclear translocation while SKF-96365 (TRP channel antagonist) and Nickel (Ni, TTCC antagonist) were less effective. The relative potency of these antagonists against Ca2+ induced NFAT nuclear translocation (Nif>SKF-96365>Ni) was similar to their effects on Ca2+ transients and the LTCC current. Infection of NRVM with viruses containing TRP channels also activated NFAT-GFP nuclear translocation and caused myocyte hypertrophy. TRP effects were reduced by SKF-96365, but were more effectively antagonized by Nif. These experiments suggest that Ca2+ influx through LTCCs is the primary source of Ca2+ to activate Cn-NFAT signaling in NRVMs and AFVMs. While TRP channels cause hypertrophy, they appear to do so through a mechanism involving Ca2+ entry via LTCCs. PMID:22921230

  7. Mathematical Modelling of Liner Piston Maintenance Activity using Field Data to Minimize Overhauling Time and Human Energy Consumption

    NASA Astrophysics Data System (ADS)

    Belkhode, Pramod Namdeorao

    2017-06-01

    Field data based model is proposed to reduce the overhauling time and human energy consumed in liner piston maintenance activity so as to increase the productivity of liner piston maintenance activity. The independent variables affecting the phenomenon such as anthropometric parameters of workers (Eastman Kodak Co. Ltd in Section VIA Appendix-A: Anthropometric Data. Ergonomic Design for People at Work, Van Nostrans Reinhold, New York, 1), workers parameters, specification of liner piston data, specification of tools used in liner piston maintenance activity, specification of solvents, axial clearance of big end bearing and bolt elongation, workstation data (Eastman Kodak Co. Ltd in Work Place Ergonomic Design for People at Work, Van Nostrans Reinhold, New York, 2) and extraneous variables, namely, temperature, humidity at workplace, illumination at workplace and noise at workplace (Eastman Kodak Co. Ltd in Chapter V Environment Ergonomic Design for People at Work, Van Nostrans Reinhold, New York, 3) are taken into account. The model is formulated for dependent variables of liner piston maintenance activity to minimize the overhauling time and human energy consumption so as to improve the productivity of liner piston maintenance activity. The developed model can predict the performance of liner piston maintenance activity which involves man and machine system (Schenck in Theories of Engineering Experimentation, Mc-Graw Hill, New York 4). The model is then optimized by optimization technique and the sensitivity analysis of the model has also been estimated.

  8. Coupling of activation and inactivation gate in a K+-channel: potassium and ligand sensitivity.

    PubMed

    Ader, Christian; Schneider, Robert; Hornig, Sönke; Velisetty, Phanindra; Vardanyan, Vitya; Giller, Karin; Ohmert, Iris; Becker, Stefan; Pongs, Olaf; Baldus, Marc

    2009-09-16

    Potassium (K(+))-channel gating is choreographed by a complex interplay between external stimuli, K(+) concentration and lipidic environment. We combined solid-state NMR and electrophysiological experiments on a chimeric KcsA-Kv1.3 channel to delineate K(+), pH and blocker effects on channel structure and function in a membrane setting. Our data show that pH-induced activation is correlated with protonation of glutamate residues at or near the activation gate. Moreover, K(+) and channel blockers distinctly affect the open probability of both the inactivation gate comprising the selectivity filter of the channel and the activation gate. The results indicate that the two gates are coupled and that effects of the permeant K(+) ion on the inactivation gate modulate activation-gate opening. Our data suggest a mechanism for controlling coordinated and sequential opening and closing of activation and inactivation gates in the K(+)-channel pore.

  9. Chloride Channel 3 Channels in the Activation and Migration of Human Blood Eosinophils in Allergic Asthma.

    PubMed

    Gaurav, Rohit; Bewtra, Againdra K; Agrawal, Devendra K

    2015-08-01

    Nicotinamide adenine dinucleotide phosphate (NADPH) oxidase is responsible for respiratory burst in immune cells. Chloride channel 3 (CLC3) has been linked to the respiratory burst in eosinophils and neutrophils. The effect of cytokines and the involvement of CLC3 in the regulation of NADPH-dependent oxidative stress and on cytokine-mediated migration of eosinophils are not known. Human peripheral blood eosinophils were isolated from healthy individuals and from individuals with asthma by negative selection. Real-time PCR was used to detect the expression of NADPH oxidases in eosinophils. Intracellular reactive oxygen species (ROS) measurement was done with flow cytometry. Superoxide generation was measured with transforming growth factor (TGF)-β, eotaxin, and CLC3 blockers. CLC3 dependence of eosinophils in TGF-β- and eotaxin-induced migration was also examined. The messenger RNA (mRNA) transcripts of NADPH oxidase (NOX) 2, dual oxidase (DUOX) 1, and DUOX2 were detected in blood eosinophils, with very low expression of NOX1, NOX3, and NOX5 and no NOX4 mRNA. The level of NOX2 mRNA transcripts increased with disease severity in the eosinophils of subjects with asthma compared with healthy nonatopic volunteers. Change in granularity and size in eosinophils, but no change in intracellular ROS, was observed with phorbol myristate acetate (PMA). PMA, TGF-β, and eotaxin used the CLC3-dependent pathway to increase superoxide radicals. TGF-β and eotaxin induced CLC3-dependent chemotaxis of eosinophils. These findings support the requirement of CLC3 in the activation and migration of human blood eosinophils and may provide a potential novel therapeutic target to regulate eosinophil hyperactivity in allergic airway inflammation in asthma.

  10. L-type Calcium Channel Cav1.2 Is Required for Maintenance of Auditory Brainstem Nuclei*

    PubMed Central

    Ebbers, Lena; Satheesh, Somisetty V.; Janz, Katrin; Rüttiger, Lukas; Blosa, Maren; Hofmann, Franz; Morawski, Markus; Griesemer, Désirée; Knipper, Marlies; Friauf, Eckhard; Nothwang, Hans Gerd

    2015-01-01

    Cav1.2 and Cav1.3 are the major L-type voltage-gated Ca2+ channels in the CNS. Yet, their individual in vivo functions are largely unknown. Both channel subunits are expressed in the auditory brainstem, where Cav1.3 is essential for proper maturation. Here, we investigated the role of Cav1.2 by targeted deletion in the mouse embryonic auditory brainstem. Similar to Cav1.3, loss of Cav1.2 resulted in a significant decrease in the volume and cell number of auditory nuclei. Contrary to the deletion of Cav1.3, the action potentials of lateral superior olive (LSO) neurons were narrower compared with controls, whereas the firing behavior and neurotransmission appeared unchanged. Furthermore, auditory brainstem responses were nearly normal in mice lacking Cav1.2. Perineuronal nets were also unaffected. The medial nucleus of the trapezoid body underwent a rapid cell loss between postnatal days P0 and P4, shortly after circuit formation. Phosphorylated cAMP response element-binding protein (CREB), nuclear NFATc4, and the expression levels of p75NTR, Fas, and FasL did not correlate with cell death. These data demonstrate for the first time that both Cav1.2 and Cav1.3 are necessary for neuronal survival but are differentially required for the biophysical properties of neurons. Thus, they perform common as well as distinct functions in the same tissue. PMID:26242732

  11. Nitric Oxide Regulates Neuronal Activity via Calcium-Activated Potassium Channels

    PubMed Central

    Zhong, Lei Ray; Estes, Stephen; Artinian, Liana; Rehder, Vincent

    2013-01-01

    Nitric oxide (NO) is an unconventional membrane-permeable messenger molecule that has been shown to play various roles in the nervous system. How NO modulates ion channels to affect neuronal functions is not well understood. In gastropods, NO has been implicated in regulating the feeding motor program. The buccal motoneuron, B19, of the freshwater pond snail Helisoma trivolvis is active during the hyper-retraction phase of the feeding motor program and is located in the vicinity of NO-producing neurons in the buccal ganglion. Here, we asked whether B19 neurons might serve as direct targets of NO signaling. Previous work established NO as a key regulator of growth cone motility and neuronal excitability in another buccal neuron involved in feeding, the B5 neuron. This raised the question whether NO might modulate the electrical activity and neuronal excitability of B19 neurons as well, and if so whether NO acted on the same or a different set of ion channels in both neurons. To study specific responses of NO on B19 neurons and to eliminate indirect effects contributed by other cells, the majority of experiments were performed on single cultured B19 neurons. Addition of NO donors caused a prolonged depolarization of the membrane potential and an increase in neuronal excitability. The effects of NO could mainly be attributed to the inhibition of two types of calcium-activated potassium channels, apamin-sensitive and iberiotoxin-sensitive potassium channels. NO was found to also cause a depolarization in B19 neurons in situ, but only after NO synthase activity in buccal ganglia had been blocked. The results suggest that NO acts as a critical modulator of neuronal excitability in B19 neurons, and that calcium-activated potassium channels may serve as a common target of NO in neurons. PMID:24236040

  12. Selectivity filter gating in large-conductance Ca(2+)-activated K+ channels.

    PubMed

    Thompson, Jill; Begenisich, Ted

    2012-03-01

    Membrane voltage controls the passage of ions through voltage-gated K (K(v)) channels, and many studies have demonstrated that this is accomplished by a physical gate located at the cytoplasmic end of the pore. Critical to this determination were the findings that quaternary ammonium ions and certain peptides have access to their internal pore-blocking sites only when the channel gates are open, and that large blocking ions interfere with channel closing. Although an intracellular location for the physical gate of K(v) channels is well established, it is not clear if such a cytoplasmic gate exists in all K(+) channels. Some studies on large-conductance, voltage- and Ca(2+)-activated K(+) (BK) channels suggest a cytoplasmic location for the gate, but other findings question this conclusion and, instead, support the concept that BK channels are gated by the pore selectivity filter. If the BK channel is gated by the selectivity filter, the interactions between the blocking ions and channel gating should be influenced by the permeant ion. Thus, we tested tetrabutyl ammonium (TBA) and the Shaker "ball" peptide (BP) on BK channels with either K(+) or Rb(+) as the permeant ion. When tested in K(+) solutions, both TBA and the BP acted as open-channel blockers of BK channels, and the BP interfered with channel closing. In contrast, when Rb(+) replaced K(+) as the permeant ion, TBA and the BP blocked both closed and open BK channels, and the BP no longer interfered with channel closing. We also tested the cytoplasmically gated Shaker K channels and found the opposite behavior: the interactions of TBA and the BP with these K(v) channels were independent of the permeant ion. Our results add significantly to the evidence against a cytoplasmic gate in BK channels and represent a positive test for selectivity filter gating.

  13. Selectivity filter gating in large-conductance Ca2+-activated K+ channels

    PubMed Central

    Thompson, Jill

    2012-01-01

    Membrane voltage controls the passage of ions through voltage-gated K (Kv) channels, and many studies have demonstrated that this is accomplished by a physical gate located at the cytoplasmic end of the pore. Critical to this determination were the findings that quaternary ammonium ions and certain peptides have access to their internal pore-blocking sites only when the channel gates are open, and that large blocking ions interfere with channel closing. Although an intracellular location for the physical gate of Kv channels is well established, it is not clear if such a cytoplasmic gate exists in all K+ channels. Some studies on large-conductance, voltage- and Ca2+-activated K+ (BK) channels suggest a cytoplasmic location for the gate, but other findings question this conclusion and, instead, support the concept that BK channels are gated by the pore selectivity filter. If the BK channel is gated by the selectivity filter, the interactions between the blocking ions and channel gating should be influenced by the permeant ion. Thus, we tested tetrabutyl ammonium (TBA) and the Shaker “ball” peptide (BP) on BK channels with either K+ or Rb+ as the permeant ion. When tested in K+ solutions, both TBA and the BP acted as open-channel blockers of BK channels, and the BP interfered with channel closing. In contrast, when Rb+ replaced K+ as the permeant ion, TBA and the BP blocked both closed and open BK channels, and the BP no longer interfered with channel closing. We also tested the cytoplasmically gated Shaker K channels and found the opposite behavior: the interactions of TBA and the BP with these Kv channels were independent of the permeant ion. Our results add significantly to the evidence against a cytoplasmic gate in BK channels and represent a positive test for selectivity filter gating. PMID:22371364

  14. Food activities and identity maintenance in old age: a systematic review and meta-synthesis

    PubMed Central

    Plastow, Nicola Ann; Atwal, Anita; Gilhooly, Mary

    2015-01-01

    Objectives: Services provided to older people should be developed based on active ageing policies. Nutrition is one aspect of active ageing, but little is known about how food activities contribute to psychological well-being in later life. This is a systematic review of qualitative and quantitative research that answers the question ‘What is known about the relationship between food activities and the maintenance of identities in old age?’. Methods: We followed the preferred reporting items for systematic reviews and meta-analyses guidelines and used quality assessment parameters to complete a systematic review and narrative synthesis. Academic Search Premier, MEDLINE, CINAHL Plus, and PsycINFO databases were searched. Results: We initially identified 8016 articles, of which 167 full-text articles were screened for inclusion. Twenty-two articles were included in the review. There was moderate evidence from nine qualitative and two quantitative studies, of variable quality, that food activities contribute to the maintenance of women's gendered identities, the ethnic identities of men and women, and community identities. There was moderate evidence from 10 qualitative studies, of variable quality, that a change in food choice and deteriorating health changed food activity participation. These changes threatened identities. Most studies included both younger adults and older adults. Conclusion: In later life, there are many life experiences leading to change. Further research is needed to develop understanding of how identity and mental well-being are maintained, despite changes in everyday activities like cooking and eating. This may enable health care professionals to meet psychological needs alongside biological needs during nutritional interventions. PMID:25373998

  15. Intrinsic disorder in the C-terminal domain of the Shaker voltage-activated K+ channel modulates its interaction with scaffold proteins

    PubMed Central

    Magidovich, Elhanan; Orr, Irit; Fass, Deborah; Abdu, Uri; Yifrach, Ofer

    2007-01-01

    The interaction of membrane-embedded voltage-activated potassium channels (Kv) with intracellular scaffold proteins, such as the postsynaptic density 95 (PSD-95) protein, is mediated by the channel C-terminal segment. This interaction underlies Kv channel clustering at unique membrane sites and is important for the proper assembly and functioning of the synapse. In the current study, we address the molecular mechanism underlying Kv/PSD-95 interaction. We provide experimental evidence, based on hydrodynamic and spectroscopic analyses, indicating that the isolated C-terminal segment of the archetypical Shaker Kv channel (ShB-C) is a random coil, suggesting that ShB-C belongs to the recently defined class of intrinsically disordered proteins. We show that isolated ShB-C is still able to bind its scaffold protein partner and support protein clustering in vivo, indicating that unfoldedness is compatible with ShB-C activity. Pulldown experiments involving C-terminal chains differing in flexibility or length further demonstrate that intrinsic disorder in the C-terminal segment of the Shaker channel modulates its interaction with the PSD-95 protein. Our results thus suggest that the C-terminal domain of the Shaker Kv channel behaves as an entropic chain and support a “fishing rod” molecular mechanism for Kv channel binding to scaffold proteins. The importance of intrinsically disordered protein segments to the complex processes of synapse assembly, maintenance, and function is discussed. PMID:17666528

  16. Mutating three residues in the bovine rod cyclic nucleotide-activated channel can switch a nucleotide from inactive to active.

    PubMed Central

    Scott, S P; Cummings, J; Joe, J C; Tanaka, J C

    2000-01-01

    Cyclic nucleotide-gated (CNG) channels, which were initially studied in retina and olfactory neurons, are activated by cytoplasmic cGMP or cAMP. Detailed comparisons of nucleotide-activated currents using nucleotide analogs and mutagenesis revealed channel-specific residues in the nucleotide-binding domain that regulate the binding and channel-activation properties. Of particular interest are N(1)-oxide cAMP, which does not activate bovine rod channels, and Rp-cGMPS, which activates bovine rod, but not catfish, olfactory channels. Previously, we showed that four residues coordinate the purine interactions in the binding domain and that three of these residues vary in the alpha subunits of the bovine rod, catfish, and rat olfactory channels. Here we show that both N(1)-oxide cAMP and Rp-cGMPS activate rat olfactory channels. A mutant of the bovine rod alpha subunit, substituted with residues from the rat olfactory channel at the three variable positions, was weakly activated by N(1)-oxide cAMP, and a catfish olfactory-like bovine rod mutant lost activation by Rp-cGMPS. These experiments underscore the functional importance of purine contacts with three residues in the cyclic nucleotide-binding domain. Molecular models of nucleotide analogs in the binding domains, constructed with AMMP, showed differences in the purine contacts among the channels that might account for activation differences. PMID:10777730

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

    NASA Astrophysics Data System (ADS)

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

    2015-12-01

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

  18. Function and regulation of large conductance Ca(2+)-activated K+ channel in vascular smooth muscle cells.

    PubMed

    Hu, Xiang-Qun; Zhang, Lubo

    2012-09-01

    Large conductance Ca(2+)-activated K(+) (BK(Ca)) channels are abundantly expressed in vascular smooth muscle cells. Activation of BK(Ca) channels leads to hyperpolarization of cell membrane, which in turn counteracts vasoconstriction. Therefore, BK(Ca) channels have an important role in regulation of vascular tone and blood pressure. The activity of BK(Ca) channels is subject to modulation by various factors. Furthermore, the function of BK(Ca) channels are altered in both physiological and pathophysiological conditions, such as pregnancy, hypertension and diabetes, which has dramatic impacts on vascular tone and hemodynamics. Consequently, compounds and genetic manipulation that alter activity and expression of the channel might be of therapeutic interest.

  19. Voltage-induced slow activation and deactivation of mechanosensitive channels in Xenopus oocytes.

    PubMed Central

    Silberberg, S D; Magleby, K L

    1997-01-01

    1. The relationship between stretch and voltage activation of mechanosensitive (MS) channels from Xenopus oocytes was studied in excised patches of membrane using the patch clamp technique. 2. As is characteristic of MS channels to oocytes, stretching the membrane by applying negative pressure to the patch pipette at -50 mV activated the MS channels rapidly. The channels then deactivated rapidly when the stretch was removed. The stretch-activated MS channels entered a main conductance level (45 pS) and one or more subconductance levels in the range of about 75-90% of the main conductance level. 3. In the absence of stretch, a depolarizing step from -50 to +50 mV activated apparent MS channels after long delays of typically 1-20 s (range, 100 ms to 6 min). Upon repolarization, the channels deactivated slowly with a single exponential (mean time constant of 4 s) or double exponential (mean time constants of 0.8 and 3 s) time course. 4. Delayed activation with depolarization and slow deactivation upon repolarization were also observed for apparent MS channels in on-cell patches. 5. The voltage-activated channels were cation selective and had the same selectivity and conductance levels as the stretch activated MS channels. Applying stretch during voltage-induced channel activity did not activate any additional channels, and the same maximal number of channels were typically activated by either stretch or by voltage. These observations suggest that voltage activates the same MS channels that are activated by stretch. 6. The opening of MS channels following steps to +50 mV occurred in an apparently co-operative manner in 70% of the excised patches containing multiple MS channels. 7. In the absence of stretch, the opening frequency and open probability of MS channels increased with depolarization in the examined voltage range of -60 to -20 mV. 8. Applying a brief stretch during the delay to activation at +50 mV activated the MS channels rapidly, which then remained active

  20. The pseudokinase CaMKv is required for the activity-dependent maintenance of dendritic spines

    PubMed Central

    Liang, Zhuoyi; Zhan, Yi; Shen, Yang; Wong, Catherine C. L.; Yates, John R.; Plattner, Florian; Lai, Kwok-On; Ip, Nancy Y.

    2016-01-01

    Dendritic spine stabilization depends on afferent synaptic input and requires changes in actin cytoskeleton dynamics and protein synthesis. However, the underlying molecular mechanism remains unclear. Here we report the identification of ‘calmodulin kinase-like vesicle-associated' (CaMKv), a pseudokinase of the CaMK family with unknown function, as a synaptic protein crucial for dendritic spine maintenance. CaMKv mRNA localizes at dendrites, and its protein synthesis is regulated by neuronal activity. CaMKv function is inhibited upon phosphorylation by cyclin-dependent kinase 5 (Cdk5) at Thr345. Furthermore, CaMKv knockdown in mouse hippocampal CA1 pyramidal neurons impairs synaptic transmission and plasticity in vivo, resulting in hyperactivity and spatial memory impairment. These findings collectively indicate that the precise regulation of CaMKv through activity-dependent synthesis and post-translational phosphorylation is critical for dendritic spine maintenance, revealing an unusual signalling pathway in the regulation of synaptic transmission and brain function that involves a pseudokinase. PMID:27796283

  1. Src tyrosine kinase alters gating of hyperpolarization-activated HCN4 pacemaker channel through Tyr531.

    PubMed

    Li, Chen-Hong; Zhang, Qi; Teng, Bunyen; Mustafa, S Jamal; Huang, Jian-Ying; Yu, Han-Gang

    2008-01-01

    We recently discovered that the constitutively active Src tyrosine kinase can enhance hyperpolarization-activated, cyclic nucleotide-gated (HCN) 4 channel activity by binding to the channel protein. To investigate the mechanism of modulation by Src of HCN channels, we studied the effects of a selective inhibitor of Src tyrosine kinase, 4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine (PP2), on HCN4 and its mutant channels expressed in HEK 293 cells by using a whole cell patch-clamp technique. We found that PP2 can inhibit HCN4 currents by negatively shifting the voltage dependence of channel activation, decreasing the whole cell channel conductance, and slowing activation and deactivation kinetics. Screening putative tyrosine residues subject to phosphorylation yielded two candidates: Tyr(531) and Tyr(554). Substituting HCN4-Tyr(531) with phenylalanine largely abolished the effects of PP2 on HCN4 channels. Replacing HCN4-Tyr(554) with phenylalanine did not abolish the effects of PP2 on voltage-dependent activation but did eliminate PP2-induced slowing of channel kinetics. The inhibitory effects of HCN channels associated with reduced Src tyrosine activity is confirmed in HL-1 cardiomyocytes. Finally, we found that PP2 can decrease the heart rate in a mouse model. These results demonstrate that Src tyrosine kinase enhances HCN4 currents by shifting their activation to more positive potentials and increasing the whole cell channel conductance as well as speeding the channel kinetics. The tyrosine residue that mediates most of Src's actions on HCN4 channels is Tyr(531).

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

    PubMed

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

    2017-03-21

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

  3. A large-conductance calcium-activated potassium channel in potato (Solanum tuberosum) tuber mitochondria.

    PubMed

    Koszela-Piotrowska, Izabela; Matkovic, Karolina; Szewczyk, Adam; Jarmuszkiewicz, Wieslawa

    2009-11-11

    In the present study, we describe the existence of a novel potassium channel in the plant [potato (Solanum tuberosum) tuber] mitochondrial inner membrane. We found that substances known to modulate large-conductance calcium-activated potassium channel activity influenced the bioenergetics of potato tuber mitochondria. In isolated mitochondria, Ca2+ and NS1619 {1,3-dihydro-1-[2-hydroxy-5-(trifluoromethyl)phenyl]-5-(trifluoromethyl)-2H-ben-zimidazole-2-one; a potassium channel opener} were found to depolarize the mitochondrial membrane potential and to stimulate resting respiration. These effects were blocked by iberiotoxin (a potassium channel inhibitor) in a potassium-dependent manner. Additionally, the electrophysiological properties of the large-conductance potassium channel present in the potato tuber inner mitochondrial membrane are described in a reconstituted system, using planar lipid bilayers. After incorporation in 50/450 mM KCl gradient solutions, we recorded large-conductance potassium channel activity with conductance from 502+/-15 to 615+/-12 pS. The probability of channel opening was increased by Ca2+ and reduced by iberiotoxin. Immunological analysis with antibodies raised against the mammalian plasma-membrane large-conductance Ca2+-dependent K+ channel identified a pore-forming alpha subunit and an auxiliary beta2 subunit of the channel in potato tuber mitochondrial inner membrane. These results suggest that a large-conductance calcium-activated potassium channel similar to that of mammalian mitochondria is present in potato tuber mitochondria.

  4. Activation of olfactory-type cyclic nucleotide-gated channels is highly cooperative

    PubMed Central

    Nache, Vasilica; Schulz, Eckhard; Zimmer, Thomas; Kusch, Jana; Biskup, Christoph; Koopmann, Rolf; Hagen, Volker; Benndorf, Klaus

    2005-01-01

    Cyclic nucleotide-gated (CNG) ion channels play a key role in the sensory transduction of vision and olfaction. The channels are opened by the binding of cyclic nucleotides. Native olfactory CNG channels are heterotetramers of CNGA2, CNGA4, and CNGB1b subunits. Upon heterologous expression, only CNGA2 subunits can form functional homotetrameric channels. It is presently not known how the binding of the ligands to the four subunits is translated to channel opening. We studied activation of olfactory CNG channels by photolysis-induced jumps of cGMP or cAMP, two cyclic nucleotides with markedly different apparent affinity. It is shown that at equal degree of activation, the activation time course of homotetrameric channels is similar with cGMP and cAMP and it is also similar in homo- and heterotetrameric channels with the same cyclic nucleotide. Kinetic models were globally fitted to activation time courses of homotetrameric channels. While all models containing equivalent binding sites failed, a model containing three binding sites with a ligand affinity high–low–high described the data adequately. Only the second binding step switches from a very low to a very high open probability. We propose a unique gating mechanism for homotetrameric and heterotetrameric channels that involves only three highly cooperative binding steps. PMID:16081488

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

    PubMed

    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.

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

    PubMed Central

    Zamponi, G W; French, R J

    1994-01-01

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

  7. Persistent neural activity during the maintenance of spatial position in working memory.

    PubMed

    Srimal, Riju; Curtis, Clayton E

    2008-01-01

    The mechanism for the short-term maintenance of information involves persistent neural activity during the retention interval, which forms a bridge between the cued memoranda and its later contingent response. Here, we used event-related functional magnetic resonance imaging to identify cortical areas with activity that persists throughout working memory delays with the goal of testing if such activity represents visuospatial attention or prospective saccade goals. We did so by comparing two spatial working memory tasks. During a memory-guided saccade (MGS) task, a location was maintained during a delay after which a saccade was generated to the remembered location. During a spatial item recognition (SIR) task identical to MGS until after the delay, a button press indicated whether a newly cued location matched the remembered location. Activity in frontal and parietal areas persisted above baseline and was greater in the hemisphere contralateral to the cued visual field. However, delay-period activity did not differ between the tasks. Notably, in the putative frontal eye field (FEF), delay period activity did not differ despite that the precise metrics of the memory-guided saccade were known during the MGS delay and saccades were never made in SIR. Persistent FEF activity may therefore represent a prioritized attentional map of space, rather than the metrics for saccades.

  8. The role of exercise and physical activity in weight loss and maintenance.

    PubMed

    Swift, Damon L; Johannsen, Neil M; Lavie, Carl J; Earnest, Conrad P; Church, Timothy S

    2014-01-01

    This review explores the role of physical activity (PA) and exercise training (ET) in the prevention of weight gain, initial weight loss, weight maintenance, and the obesity paradox. In particular, we will focus the discussion on the expected initial weight loss from different ET programs, and explore intensity/volume relationships. Based on the present literature, unless the overall volume of aerobic ET is very high, clinically significant weight loss is unlikely to occur. Also, ET also has an important role in weight regain after initial weight loss. Overall, aerobic ET programs consistent with public health recommendations may promote up to modest weight loss (~2 kg), however the weight loss on an individual level is highly heterogeneous. Clinicians should educate their patients on reasonable expectations of weight loss based on their physical activity program and emphasize that numerous health benefits occur from PA programs in the absence of weight loss. © 2014.

  9. Dissociation among in vitro telomerase activity, telomere maintenance, and cellular immortalization

    PubMed Central

    Counter, Christopher M.; Hahn, William C.; Wei, Wenyi; Caddle, Stephanie Dickinson; Beijersbergen, Roderick L.; Lansdorp, Peter M.; Sedivy, John M.; Weinberg, Robert A.

    1998-01-01

    The immortalization of human cells is a critical step during tumorigenesis. In vitro, normal human somatic cells must overcome two proliferative blockades, senescence and crisis, to become immortal. Transformation with viral oncogenes extends the life span of human cells beyond senescence. Such transformed cells eventually succumb to crisis, a period of widespread cellular death that has been proposed to be the result of telomeric shortening. We now show that ectopic expression of the telomerase catalytic subunit (human telomerase reverse transcriptase or hTERT) and subsequent activation of telomerase can allow postsenescent cells to proliferate beyond crisis, the last known proliferative blockade to cellular immortality. Moreover, we demonstrate that alteration of the carboxyl terminus of human telomerase reverse transcriptase does not affect telomerase enzymatic activity but impedes the ability of this enzyme to maintain telomeres. Telomerase-positive cells expressing this mutant enzyme fail to undergo immortalization, further tightening the connection between telomere maintenance and immortalization. PMID:9843956

  10. The Role of Exercise and Physical Activity in Weight Loss and Maintenance

    PubMed Central

    Swift, Damon L.; Johannsen, Neil M.; Lavie, Carl J.; Earnest, Conrad P.; Church, Timothy S.

    2013-01-01

    This review explores the role of physical activity (PA) and exercise training (ET) in the prevention of weight gain, initial weight loss, weight maintenance, and the obesity paradox. In particular, we will focus the discussion on the expected initial weight loss from different ET programs, and explore intensity/volume relationships. Based on the present literature, unless the overall volume of aerobic ET is very high, clinically significant weight loss is unlikely to occur. Also, ET also has an important role in weight regain after initial weight loss. Overall, aerobic ET programs consistent with public health recommendations may promote up to modest weight loss (~2 kg), however the weight loss on an individual level is highly heterogeneous. Clinicians should educate their patients on reasonable expectations of weight loss based on their physical activity program and emphasize that numerous health benefits occur from PA programs in the absence of weight loss. PMID:24438736

  11. Computational modeling of anoctamin 1 calcium-activated chloride channels as pacemaker channels in interstitial cells of Cajal

    PubMed Central

    Gibbons, Simon J.; Farrugia, Gianrico; Sneyd, James; Cheng, Leo K.

    2014-01-01

    Interstitial cells of Cajal (ICC) act as pacemaker cells in the gastrointestinal tract by generating electrical slow waves to regulate rhythmic smooth muscle contractions. Intrinsic Ca2+ oscillations in ICC appear to produce the slow waves by activating pacemaker currents, currently thought to be carried by the Ca2+-activated Cl− channel anoctamin 1 (Ano1). In this article we present a novel model of small intestinal ICC pacemaker activity that incorporates store-operated Ca2+ entry and a new model of Ano1 current. A series of simulations were carried out with the ICC model to investigate current controversies about the reversal potential of the Ano1 Cl− current in ICC and to predict the characteristics of the other ion channels that are necessary to generate slow waves. The model results show that Ano1 is a plausible pacemaker channel when coupled to a store-operated Ca2+ channel but suggest that small cyclical depolarizations may still occur in ICC in Ano1 knockout mice. The results predict that voltage-dependent Ca2+ current is likely to be negligible during the slow wave plateau phase. The model shows that the Cl− equilibrium potential is an important modulator of slow wave morphology, highlighting the need for a better understanding of Cl− dynamics in ICC. PMID:24481603

  12. Cell membrane stretch activates intermediate-conductance Ca2+-activated K+ channels in arterial smooth muscle cells.

    PubMed

    Hayabuchi, Yasunobu; Nakaya, Yutaka; Mawatari, Kazuaki; Inoue, Miki; Sakata, Miho; Kagami, Shoji

    2011-01-01

    The aim of this study is to determine the signal transduction of membrane stretch on intermediate-conductance Ca(2+)-activated K(+) (IKca) channels in rat aorta smooth muscle cells using the patch-clamp technique. To stretch the cell membrane, both suction to the rear end of patch pipette and hypotonic shock were used. In cell-attached and inside-out patch configurations, the open probability of IKca channels increased when 20- to 45-mmHg suction was applied. Hyposmotic swelling efficiently increased IKca channel current. When the Ca(2+)-free solution was superfused, the activation of IKca current by the hyposmotic swelling was reduced. Furthermore, gadolinium (Gd(3+)) attenuated the activation of IKca channels induced by hyposmotic swelling, whereas nicardipine did not. In the experiments with Ca(2+)-free bath solution, pretreatment with GF109203X, a protein kinase C (PKC) inhibitor, completely abolished the stretch-induced activation of IKca currents. The stretch-induced activation of IKca channels was strongly inhibited by cytochalasin D, indicating a role for the F-actin in modulation of IKca channels by changes in cell stretching. These data suggest that cell membrane stretch activates IKca channels. In addition, the activation is associated with extracellular Ca(2+) influx through stretch-activated nonselective cation channels, and is also modulated by the F-actin cytoskeleton and the activation of PKC.

  13. Folding of active calcium channel beta(1b) -subunit by size-exclusion chromatography and its role on channel function.

    PubMed

    Neely, Alan; Garcia-Olivares, Jennie; Voswinkel, Stephan; Horstkott, Hannelore; Hidalgo, Patricia

    2004-05-21

    Voltage-gated calcium channels mediate the influx of Ca(2+) ions into eukaryotic cells in response to membrane depolarization. They are hetero-multimer membrane proteins formed by at least three subunits, the poreforming alpha(1)-subunit and the auxiliary beta- and alpha(2)delta-subunits. The beta-subunit is essential for channel performance because it regulates two distinct features of voltage-gated calcium channels, the surface expression and the channel activity. Four beta-subunit genes have been cloned, beta(1-4), with molecular masses ranging from 52 to 78 kDa, and several splice variants have been identified. The beta(1b)-subunit, expressed at high levels in mammalian brain, has been used extensively to study the interaction between the pore forming alpha(1)- and the regulatory beta-subunit. However, structural characterization has been impaired for its tendency to form aggregates when expressed in bacteria. We applied an on-column refolding procedure based on size exclusion chromatography to fold the beta(1b)-subunit of the voltage gated-calcium channels from Escherichia coli inclusion bodies. The beta(1b)-subunit refolds into monomers, as shown by sucrose gradient analysis, and binds to a glutathione S-transferase protein fused to the known target in the alpha(1)-subunit (the alpha-interaction domain). Using the cut-open oocyte voltage clamp technique, we measured gating and ionic currents in Xenopus oocytes expressing cardiac alpha(1)-subunit (alpha(1C)) co-injected with folded-beta(1b)-protein or beta(1b)-cRNA. We demonstrate that the co-expression of the alpha(1C)-subunit with either folded-beta(1b)-protein or beta(1b)-cRNA increases ionic currents to a similar extent and with no changes in charge movement, indicating that the beta(1b)-subunit primarily modulates channel activity, rather than expression.

  14. 2011 Operations and Maintenance Activities in the East Region of UNAVCO's Plate Boundary Observatory

    NASA Astrophysics Data System (ADS)

    Dittmann, T.; Feaux, K.; Kasmer, D.; Jenkins, F.; Mencin, D.

    2011-12-01

    2011 marked Year 3 of Operations and Maintenance of the Plate Boundary Observatory (PBO). In the East Region of PBO, it was a year characterized by several major projects as well as scheduled ongoing maintenance activities. The most significant major project was a USGS/ARRA funded communications upgrade in Yellowstone National Park. This upgrade consisted of bringing 8 existing PBO stations within the Yellowstone volcanic region to near real-time communications. This work will be completed on time and in collaboration with the National Park Service. The upgrade promises to provide much faster latency for invaluable data being recorded for one of the most geodetically critical regions of the current PBO network. Another significant ongoing project in the East Region has been supporting the community that continues to use PBO data. In particular, support of Kristine Larson (Univ of CO) both in installing webcams at PBO sites for monitoring snow depth as well as supporting vegetative surveys at current PBO sites. Similarly, the East Region responded promptly to the community with requests for data quality issues that are station hardware related, including replacing GPS antennae and receivers. With regards to ongoing operations and maintenance projects, reasons for site visits in 2011 were dominated by two significant situations: battery replacement and CDMA modem swaps. 83 site visits were required as part of the Operations and Maintenance strategic battery plan of 5 year battery replacements. This proved to be a considerable challenge due to the scale and geography of the scheduled replacements- the sites were spread throughout the entire network, east to west and north to south. 20 station visits were required due to a Verizon upgrade of the older Alltel network purchased by Verizon. These stations are predominantly in the Rocky Mountain region, but often times had limited access to due weather. Overall, despite record snowfalls throughout the west, state of health

  15. Activation of the SK potassium channel-calmodulin complex by nanomolar concentrations of terbium

    PubMed Central

    Li, Weiyan; Aldrich, Richard W.

    2009-01-01

    Small conductance Ca2+-activated K+ (SK) channels sense intracellular Ca2+ concentrations via the associated Ca2+-binding protein calmodulin. Structural and functional studies have revealed essential properties of the interaction between calmodulin and SK channels. However, it is not fully understood how the binding of Ca2+ to calmodulin leads to channel opening. Drawing on previous biochemical studies of free calmodulin using lanthanide ions as Ca2+ substitutes, we have used the lanthanide ion, Tb3+, as an alternative ligand to study the activation properties of SK channels. We found that SK channels can be fully activated by nanomolar concentrations of Tb3+, indicating an apparent affinity >100-fold higher than Ca2+. Competition experiments show that Tb3+ binds to the same sites as Ca2+ to activate the channels. Additionally, SK channels activated by Tb3+ demonstrate a remarkably slow deactivation process. Comparison of our results with previous biochemical studies suggests that in the intact SK channel complex, the N-lobe of calmodulin provides ligand-binding sites for channel gating, and that its ligand-binding properties are comparable to those of the N-lobe in isolated calmodulin. PMID:19144926

  16. Escherichia coli membranes depleted of SecYEG elicit SecA-dependent ion-channel activity but lose signal peptide specificity.

    PubMed

    Lin, Bor-Ruei; Hsieh, Ying-Hsin; Jiang, Chun; Tai, Phang C

    2012-11-01

    We have developed a sensitive method to detect the opening of SecA-dependent, protein-conducting channels in Xenopus oocytes. In this study, we determined the ionic current activities of the SecA-dependent channel from membrane vesicles depleted of SecYEG. We found that these SecYEG-depleted membranes produced SecA-dependent ionic currents in the oocytes, as did membranes containing SecYEG. However, reconstituted membranes depleted of SecYEG required higher concentrations of SecA to elicit ionic currents like those in membranes containing SecYEG. In contrast to membranes containing SecYEG, the proofreading capacity of signal peptides was lost for those membranes lacking SecYEG. These findings are consistent with loss of signal peptide specificity in channel activity from membranes of SecY suppressor or SecY plug domain mutants. The signal peptide specificity of the reconstituted membranes, like SecA-liposomes, can be restored by the addition of SecYEG proteoliposomes. On the other hand, the channel activity efficiency of reconstituted membranes was fully restored, while SecA-liposomes could only be partially enhanced by the addition of SecYEG, indicating that, in addition to SecYEG, other membrane proteins contribute to the efficiency of channel activity. The SecA-dependent channels in membranes that lacked SecYEG also lost ion selectivity to monovalent cations but retained selective permeability to large anions. Thus, the electrophysiological evidence presented here indicates that SecYEG is not obligatory for the channel activity of Escherichia coli membranes, as previously shown for protein translocation, and that SecYEG is important for maintenance of the efficiency and specificity of SecA-dependent channels.

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

    NASA Astrophysics Data System (ADS)

    Brayden, Joseph E.; Nelson, Mark T.

    1992-04-01

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

  18. A cyclic model for bimodal activation of calcium activated potassium channels in radish vacuoles.

    PubMed

    Carpaneto, A

    2001-01-01

    This paper presents the mathematical framework of a cyclic model proposed for describing the transition between a fast and a slow mode (fast-slow effect) induced by the application of step membrane potentials to ion channels from radish vacuoles. A voltage stimulation pulse with frequency in the range of 2 Hz or higher increased the activation time (slow mode) of the recorded currents. When the frequency of the stimulation pattern was restored to 0.1 Hz the activation time decreased twofold (fast mode). This experimental result cannot be explained by classical kinetic theory. The model, based on a simple extension of the Hodgkin and Huxley chain, describes the whole current experimental data and provides hints on the structural conformation of ion channels.

  19. Selective activation of heteromeric SK channels contributes to action potential repolarization in mouse atrial myocytes.

    PubMed

    Hancock, Jane M; Weatherall, Kate L; Choisy, Stéphanie C; James, Andrew F; Hancox, Jules C; Marrion, Neil V

    2015-05-01

    Activation of small conductance calcium-activated potassium (SK) channels is proposed to contribute to repolarization of the action potential in atrial myocytes. This role is controversial, as these cardiac SK channels appear to exhibit an uncharacteristic pharmacology. The objectives of this study were to resolve whether activation of SK channels contributes to atrial action potential repolarization and to determine the likely subunit composition of the channel. The effect of 2 SK channel inhibitors was assessed on outward current evoked in voltage clamp and on action potential duration in perforated patch and whole-cell current clamp recording from acutely isolated mouse atrial myocytes. The presence of SK channel subunits was assessed using immunocytochemistry. A significant component of outward current was reduced by the SK channel blockers apamin and UCL1684. Block by apamin displayed a sensitivity indicating that this current was carried by homomeric SK2 channels. Action potential duration was significantly prolonged by UCL1684, but not by apamin. This effect was accompanied by an increase in beat-to-beat variability and action potential triangulation. This pharmacology was matched by that of expressed heteromeric SK2-SK3 channels in HEK293 cells. Immunocytochemistry showed that atrial myocytes express both SK2 and SK3 channels with an overlapping expression pattern. Only proposed heteromeric SK2-SK3 channels are physiologically activated to contribute to action potential repolarization, which is indicated by the difference in pharmacology of evoked outward current and prolongation of atrial action potential duration. The effect of blocking this channel on the action potential suggests that SK channel inhibition during cardiac function has the potential to be proarrhythmic. Copyright © 2015 Heart Rhythm Society. Published by Elsevier Inc. All rights reserved.

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

    PubMed Central

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

    2014-01-01

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

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

    PubMed

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

    2014-11-04

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

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

    PubMed

    Thompson, Jill L; Shuttleworth, Trevor J

    2012-01-01

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

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

    PubMed Central

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

    2015-01-01

    Abstract: Small-conductance Ca2+-activated potassium (SK) channels are relative newcomers within the field of cardiac electrophysiology. In recent years, an increased focus has been given to these channels because they might constitute a relatively atrial-selective target. This review will give a general introduction to SK channels followed by their proposed function in the heart under normal and pathophysiological conditions. It is revealed how antiarrhythmic effects can be obtained by SK channel inhibition in a number of species in situations of atrial fibrillation. On the contrary, the beneficial effects of SK channel inhibition in situations of heart failure are questionable and still needs investigation. The understanding of cardiac SK channels is rapidly increasing these years, and it is hoped that this will clarify whether SK channel inhibition has potential as a new anti–atrial fibrillation principle. PMID:25830485

  4. Dual effects of microwaves on single Ca(2+)-activated K+ channels in cultured kidney cells Vero.

    PubMed

    Geletyuk, V I; Kazachenko, V N; Chemeris, N K; Fesenko, E E

    1995-02-06

    Using the patch voltage-clamp method, possible effects of millimetre microwaves (42.25 GHz) on single Ca(2+)-activated K+ channels in cultured kidney cells (Vero) were investigated. It was found that exposure to the field of non-thermal power (about 100 microW/cm2) for 20-30 min greatly modifies both the Hill coefficient and an apparent affinity of the channels for Ca2+(i). The data suggest that the field alters both cooperativity and binding characteristics of the channel activation by internal Ca2+. The effects depend on initial sensitivity of the channels to Ca2+ and the Ca2+ concentration applied.

  5. Guideline to good practices for types of maintenance activities at DOE nuclear facilities

    SciTech Connect

    Not Available

    1993-03-01

    The purpose of the Guideline to Good Practices for Types of Maintenance at DOE Nuclear Facilities is to provide contractor maintenance organizations with information that may be used for the development and implementation of a properly balanced corrective, preventive and predictive maintenance program at DOE nuclear facilities. This document is intended to be an example guideline for the implementation of DOE Order 4330.4A, Maintenance Management Program, Chapter II, Element 4. DOE contractors should not feel obligated to adopt all parts of this guide. Rather, they should use the information contained herein as a guide for developing maintenance programs that are applicable to their facility.

  6. Tamoxifen does not inhibit the swell activated chloride channel in human neutrophils during the respiratory burst

    SciTech Connect

    Ahluwalia, Jatinder

    2008-10-31

    Effective functioning of neutrophils relies upon electron translocation through the NADPH oxidase (NOX). The electron current generated (I{sub e}) by the neutrophil NADPH oxidase is electrogenic and rapidly depolarises the membrane potential in activated human neutrophils. Swelling activated chloride channels have been demonstrated in part to counteract the depolarisation generated by the NADPH oxidase I{sub e}. In the present study, the effects of inhibitors of swell activated chloride channels on ROS production and on the swelling activated chloride conductance was investigated in activated human neutrophils. Tamoxifen (10 {mu}M), a specific inhibitor for swell activated chloride channels in neutrophils, completely inhibited both the PMA and FMLP stimulated respiratory burst. This inhibition of the neutrophil respiratory burst was not due to the blocking effect of tamoxifen on the swelling activated chloride conductance in these cells. These results demonstrate that a tamoxifen insensitive swell activated chloride channel has important significance during the neutrophil respiratory burst.

  7. A natural product telomerase activator as part of a health maintenance program: metabolic and cardiovascular response.

    PubMed

    Harley, Calvin B; Liu, Weimin; Flom, Peter L; Raffaele, Joseph M

    2013-10-01

    A short average telomere length is associated with low telomerase activity and certain degenerative diseases. Studies in animals and with human cells confirm a causal mechanism for cell or tissue dysfunction triggered by critically short telomeres, suggesting that telomerase activation may be an approach to health maintenance. Previously, we reported on positive immune remodeling in humans taking a commercial health maintenance program, PattonProtocol-1, composed of TA-65® (a natural product-derived telomerase activator) and other dietary supplements. In over a 5-year period and an estimated 7000 person-years of use, no adverse events or effects have been attributed to TA-65 by physicians licensed to sell the product. Here we report on changes in metabolic markers measured at baseline (n=97-107 subjects) and every 3-6 months (n=27-59 subjects) during the first 12 months of study. Rates of change per year from baseline determined by a multi-level model were -3.72 mg/dL for fasting glucose (p=0.02), -1.32 mIU/mL for insulin (p=0.01), -13.2 and -11.8 mg/dL for total cholesterol and low-density lipoprotein cholesterol (LDL-C) (p=0.002, p=0.002, respectively), -17.3 and -4.2 mmHg for systolic and diastolic blood pressure (p=0.007 and 0.001, respectively), and -3.6 μmole/L homocysteine (p=0.001). In a subset of individuals with bone mineral density (BMD) measured at baseline and 12 months, density increased 2.0% in the spine (p=0.003). We conclude that in addition to apparent positive immune remodeling, PattonProtocol-1 may improve markers of metabolic, bone, and cardiovascular health.

  8. Activation of the human, intermediate-conductance, Ca2+-activated K+ channel by methylxanthines.

    PubMed

    Schrøder, R L; Jensen, B S; Strøbaek, D; Olesen, S P; Christophersen, P

    2000-10-01

    This study demonstrated that the methylxanthines, theophylline, IBMX and caffeine, activate the human, intermediate-conductance, Ca2+-activated K+ channel (hIK) stably expressed in HEK-293 cells. Whole-cell voltage-clamp experiments showed that the hIK current increased reversibly and voltage independently after the addition of methylxanthines. In current-clamp experiments, theophylline dose-dependently hyperpolarised the cell membrane from a resting potential of -18 mV to -56 mV. The methylxanthines did not affect large-conductance (BK) or small-conductance (SK2), Ca2+-activated K+ channels, demonstrating that the effects were not secondary to a rise in intracellular Ca2+. However, the activation of hIK by theophylline required an intracellular [Ca2+] above 30 nM. The hIK current was insensitive to 8-bromoadenosine cyclic 3',5'-monophosphate (8-bromo-cAMP), forskolin, 8-bromoguanosine cyclic 3',5'-monophosphate (8-bromo-cGMP) and sodium nitroprusside. Moreover, in the presence of inhibitors of protein kinase A (PKA) or protein kinase G (PKG) theophylline still activated the current. Finally, mutation of the putative PKA/PKG consensus phosphorylation site (Ser334) had no effect on the theophylline-induced activation of hIK. Since the observed activation is independent of changes in PKA/PKG-phosphorylation and of fluctuations in intracellular Ca2+, we suggest that the methylxanthines interact directly with the hIK protein.

  9. Burrowing activity in channel levees: impact of the invasive red swamp crayfish Procambarus clarkii

    NASA Astrophysics Data System (ADS)

    Solari, L.; Bendoni, M.; Consumi, L.; Haubrock, P.; Inghilesi, A.; Mazza, G.; Torrini, M.; Tricarico, E.

    2016-12-01

    The effect of animal burrowing, as an example of bioturbation on the stability of river levees has been recently raised to the scientific community as a consequence of the levee collapses of Secchia and Foenna rivers in Italy (Camici et al., 2010, 2014; Orlandini et al., 2015). Indeed, these authors showed that the presence of animal burrows is crucial in promoting the collapse of the bank. The American red swamp Crayfish Procambarus clarkii is an invasive species in Europe, mostly introduced for commercial purposes related to livestock. It is rapidly spreading throughout the Italian peninsula due to its plasticity, dispersal capability and high reproduction rate (Souty-Grosset et al., 2016). As well as the negative effects on local biodiversity, it damages the levees of the irrigation channel leading to disastrous collapses, relevant repairing and maintenance costs. In this work, we present an experimental activity where specimens of P. clarkii were monitored while burrowing into a small-scale physical model of an earthen levee, coupled with the mathematical modelling of the variations induced by the burrows on the seepage flow patterns through the levee.Preliminary results show the burrowing structure was quite irregular. Generally, crayfish start burrowing under the water level, developing tunnels (diameter ranging 4-7cm) both horizontally and heading upward, also above the water level. Some tunnels showed one or more circular chambers. The highest burrowing activity was observed during the experiments carried out in summer, when the species has a peak of maximum activity due to the higher temperature. Mathematical modelling shows that, for given boundary conditions and experimental duration, the presence of burrows in the levee raises the phreatic line. Critical conditions for levee integrity may be associated either to the internal erosion and stability of the system of tunnels and to the emergence of the phreatic line of the landside of the levee slope. These

  10. Activation of single cardiac and skeletal ryanodine receptor channels by flash photolysis of caged Ca2+.

    PubMed Central

    Györke, S; Vélez, P; Suárez-Isla, B; Fill, M

    1994-01-01

    Single ryanodine-sensitive sarcoplasmic reticulum (SR) Ca2+ release channels isolated from rabbit skeletal and canine cardiac muscle were reconstituted in planar lipid bilayers. Single channel activity was measured in simple solutions (no ATP or Mg2+) with 250 mM symmetrical Cs+ as charge carrier. A laser flash was used to photolyze caged-Ca2+ (DM-nitrophen) in a small volume directly in front of the bilayer. The free [Ca2+] in this small volume and in the bulk solution was monitored with Ca2+ electrodes. This setup allowed fast, calibrated free [Ca2+] stimuli to be applied repetitively to single SR Ca2+ release channels. A standard photolytically induced free [Ca2+] step (pCa 7-->6) was applied to both the cardiac and skeletal release channels. The rate of channel activation was determined by fitting a single exponential to ensemble currents generated from at least 50 single channel sweeps. The time constants of activation were 1.43 +/- 0.65 ms (mean +/- SD; n = 5) and 1.28 +/- 0.61 ms (n = 5) for cardiac and skeletal channels, respectively. This study presents a method for defining the fast Ca2+ regulation kinetics of single SR Ca2+ release channels and shows that the activation rate of skeletal SR Ca2+ release channels is consistent with a role for CICR in skeletal muscle excitation-contraction coupling. PMID:8075325

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

    PubMed

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

    2012-12-01

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

  12. Repair, Evaluation, Maintenance, and Rehabilitation Research Program. Compliance requirements for environmental laws applicable to REMR activities. Final report

    SciTech Connect

    Henderson, J.E.; Peyman-Dove, L.D.

    1988-08-01

    Operation and maintenance (OandM) activities of the Corps of Engineers must comply with the environmental laws and regulations just as other Corps activities and private actions. The OandM procedures developed under the Repair, Evaluation, Maintenance, and Rehabilitation (REMR) Research Program might result in water pollution, use or disposal of hazardous or toxic materials, or other actions that are regulated by environmental laws. A previous review of environmental laws for applicability to REMR activities identified 14 laws, regulations, or executive orders that may require compliance actions (Technical Report REMR-EI-1).

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

    PubMed Central

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

    2012-01-01

    The capsaicin receptor transient receptor potential vanilloid (TRPV)1 is a highly heat-sensitive ion channel. Although chemical activation and heat activation of TRPV1 elicit similar pungent, painful sensation, the molecular mechanism underlying synergistic activation remains mysterious. In particular, where the temperature sensor is located and whether heat and capsaicin share a common activation pathway are debated. To address these fundamental issues, we searched for channel mutations that selectively affected one form of activation. We found that deletion of the first 10 amino acids of the pore turret significantly reduced the heat response amplitude and shifted the heat activation threshold, whereas capsaicin activation remained unchanged. Removing larger portions of the turret disrupted channel function. Introducing an artificial sequence to replace the deleted region restored sensitive capsaicin activation in these nonfunctional channels. The heat activation, however, remained significantly impaired, with the current exhibiting diminishing heat sensitivity to a level indistinguishable from that of a voltage-gated potassium channel, Kv7.4. Our results demonstrate that heat and capsaicin activation of TRPV1 are structurally and mechanistically distinct processes, and the pore turret is an indispensible channel structure involved in the heat activation process but is not part of the capsaicin activation pathway. Synergistic effect of heat and capsaicin on TRPV1 activation may originate from convergence of the two pathways on a common activation gate. PMID:22412190

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

    PubMed

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

    2012-04-01

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

  15. Effects of cytoplasmic Mg2+ on slowly activating channels in isolated vacuoles of Beta vulgaris.

    PubMed

    Carpaneto, A; Cantù, A M; Gambale, F

    2001-07-01

    The slow vacuolar (SV) channel can mediate a large part of the ionic current in plant tonoplasts, but its actual physiological role is still unclear. We demonstrate that in vacuoles from the taproots of sugar beet (Beta vulgaris L.), besides Ca2+, cytoplasmic Mg2+ also plays an important role in promoting the activation of the SV channel. An increase in Mg2+ concentration decreases the time constants of channel activation and deactivation, and determines a consistent shift, towards negative voltages, of the conductance characteristic; as an example, when the free concentration of Mg2+ was increased from the micromolar range up to 10 mM the activation shifted by about -60 mV. The experimental results obtained, which are based on a fast perfusion procedure allowing us to change the solution bathing the vacuole in a few milliseconds, suggest that magnesium-binding is a faster process than the voltage-activation gating of the channel, which constitutes the rate-limiting step controlling channel opening. Interestingly, the activation of the channel mediated by Mg2+ depends on the cooperative binding of at least three magnesium ions. We verified that cytoplasmic magnesium favours the activation of SV channels in the presence of nanomolar cytoplasmic calcium concentrations. A critical discussion on the Calcium Induced Calcium Release (CICR) mechanism proposed for the SV channel is presented.

  16. Cryogenics maintenance strategy

    NASA Astrophysics Data System (ADS)

    Cruzat, Fabiola

    2012-09-01

    ALMA is an interferometer composed of 66 independent systems, with specific maintenance requirements for each subsystem. To optimize the observation time and reduce downtime maintenance, requirements are very demanding. One subsystem with high maintenance efforts is cryogenics and vacuum. To organize the maintenance, the Cryogenic and Vacuum department is using and implementing different tools. These are monitoring and problem reporting systems and CMMS. This leads to different maintenance approaches: Preventive Maintenance, Corrective Maintenance and Condition Based Maintenance. In order to coordinate activities with other departments the preventive maintenance schedule is kept as flexible as systems allow. To cope with unavoidable failures, the team has to be prepared to work under any condition with the spares on time. Computerized maintenance management system (CMMS) will help to manage inventory control for reliable spare part handling, the correct record of work orders and traceability of maintenance activities. For an optimized approach the department is currently evaluating where preventive or condition based maintenance applies to comply with the individual system demand. Considering the change from maintenance contracts to in-house maintenance will help to minimize costs and increase availability of parts. Due to increased number of system and tasks the cryo team needs to grow. Training of all staff members is mandatory, in depth knowledge must be built up by doing complex maintenance activities in the Cryo group, use of advanced computerized metrology systems.

  17. Ion Channel Activity of Vpu Proteins Is Conserved throughout Evolution of HIV-1 and SIV

    PubMed Central

    Greiner, Timo; Bolduan, Sebastian; Hertel, Brigitte; Groß, Christine; Hamacher, Kay; Schubert, Ulrich; Moroni, Anna; Thiel, Gerhard

    2016-01-01

    The human immunodeficiency virus type 1 (HIV-1) protein Vpu is encoded exclusively by HIV-1 and related simian immunodeficiency viruses (SIVs). The transmembrane domain of the protein has dual functions: it counteracts the human restriction factor tetherin and forms a cation channel. Since these two functions are causally unrelated it remains unclear whether the channel activity has any relevance for viral release and replication. Here we examine structure and function correlates of different Vpu homologs from HIV-1 and SIV to understand if ion channel activity is an evolutionary conserved property of Vpu proteins. An electrophysiological testing of Vpus from different HIV-1 groups (N and P) and SIVs from chimpanzees (SIVcpz), and greater spot-nosed monkeys (SIVgsn) showed that they all generate channel activity in HEK293T cells. This implies a robust and evolutionary conserved channel activity and suggests that cation conductance may also have a conserved functional significance. PMID:27916968

  18. Ion Channel Activity of Vpu Proteins Is Conserved throughout Evolution of HIV-1 and SIV.

    PubMed

    Greiner, Timo; Bolduan, Sebastian; Hertel, Brigitte; Groß, Christine; Hamacher, Kay; Schubert, Ulrich; Moroni, Anna; Thiel, Gerhard

    2016-12-01

    The human immunodeficiency virus type 1 (HIV-1) protein Vpu is encoded exclusively by HIV-1 and related simian immunodeficiency viruses (SIVs). The transmembrane domain of the protein has dual functions: it counteracts the human restriction factor tetherin and forms a cation channel. Since these two functions are causally unrelated it remains unclear whether the channel activity has any relevance for viral release and replication. Here we examine structure and function correlates of different Vpu homologs from HIV-1 and SIV to understand if ion channel activity is an evolutionary conserved property of Vpu proteins. An electrophysiological testing of Vpus from different HIV-1 groups (N and P) and SIVs from chimpanzees (SIVcpz), and greater spot-nosed monkeys (SIVgsn) showed that they all generate channel activity in HEK293T cells. This implies a robust and evolutionary conserved channel activity and suggests that cation conductance may also have a conserved functional significance.

  19. Hybrid Particle Swarm Optimization for Hybrid Flowshop Scheduling Problem with Maintenance Activities

    PubMed Central

    Li, Jun-qing; Pan, Quan-ke; Mao, Kun

    2014-01-01

    A hybrid algorithm which combines particle swarm optimization (PSO) and iterated local search (ILS) is proposed for solving the hybrid flowshop scheduling (HFS) problem with preventive maintenance (PM) activities. In the proposed algorithm, different crossover operators and mutation operators are investigated. In addition, an efficient multiple insert mutation operator is developed for enhancing the searching ability of the algorithm. Furthermore, an ILS-based local search procedure is embedded in the algorithm to improve the exploitation ability of the proposed algorithm. The detailed experimental parameter for the canonical PSO is tuning. The proposed algorithm is tested on the variation of 77 Carlier and Néron's benchmark problems. Detailed comparisons with the present efficient algorithms, including hGA, ILS, PSO, and IG, verify the efficiency and effectiveness of the proposed algorithm. PMID:24883414

  20. Cross talk between activation and slow inactivation gates of Shaker potassium channels.

    PubMed

    Panyi, Gyorgy; Deutsch, Carol

    2006-11-01

    This study addresses the energetic coupling between the activation and slow inactivation gates of Shaker potassium channels. To track the status of the activation gate in inactivated channels that are nonconducting, we used two functional assays: the accessibility of a cysteine residue engineered into the protein lining the pore cavity (V474C) and the liberation by depolarization of a Cs(+) ion trapped behind the closed activation gate. We determined that the rate of activation gate movement depends on the state of the inactivation gate. A closed inactivation gate favors faster opening and slower closing of the activation gate. We also show that hyperpolarization closes the activation gate long before a channel recovers from inactivation. Because activation and slow inactivation are ubiquitous gating processes in potassium channels, the cross talk between them is likely to be a fundamental factor in controlling ion flux across membranes.

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

    PubMed

    Teramoto, Takayuki; Sternick, Laura A; Kage-Nakadai, Eriko; Sajjadi, Shirine; Siembida, Jakub; Mitani, Shohei; Iwasaki, Kouichi; Lambie, Eric J

    2010-03-08

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

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

    PubMed

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

    1994-05-17

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

  3. Mechanosensitive channel activation by diffusio-osmotic force.

    PubMed

    Bonthuis, Douwe Jan; Golestanian, Ramin

    2014-10-03

    For ion channel gating, the appearance of two distinct conformational states and the discrete transitions between them are essential, and therefore of crucial importance to all living organisms. We show that the physical interplay between two structural elements that are commonly present in bacterial mechanosensitive channels--namely, a charged vestibule and a hydrophobic constriction--creates two distinct conformational states, open and closed, as well as the gating between them. We solve the nonequilibrium Stokes-Poisson-Nernst-Planck equations, extended to include a molecular potential of mean force, and show that a first order transition between the closed and open states arises naturally from the diffusio-osmotic stress caused by the ions and the water inside the channel and the elastic restoring force from the membrane.

  4. Three-dimensional flow dynamics of an active submarine channel

    NASA Astrophysics Data System (ADS)

    Sumner, E. J.; Dorrell, R. M.; Peakall, J.; Darby, S. E.; Parsons, D. R.; Wynn, R.

    2012-12-01

    Field scale submarine channel gravity currents are notoriously difficult to measure and thus directly investigate due to their inaccessible location and infrequent nature, which is compounded by present sea-level high-stand. An exception to this is the almost continuous density-driven current that results from the inflow of saline Mediterranean water, via the Bosporus strait, into the Black Sea. This flow has carved a sinuous channel system in water depths of 70 to 120 m. The relatively shallow depths of the channel and the continuous nature of this current provide a rare opportunity to study three-dimensional flow dynamics and the interaction of the flow with a seafloor channel network. Thus, it provides a rare analogue for channelized dilute sediment-laden turbidity currents. Sediment erosion, transport and deposition within submarine channel bends is primarily controlled by the magnitude and direction of near bed flow. Flow around channel bends is characterized by a helical or spiralling structure. In rivers this helical flow is characterized by near-surface fluid moving toward the outer bank and near-bed fluid moving toward the inner bank. Following fierce debate over the last decade, it is now accepted that helical flow in submarine channel bends can display a variety of complex structures. Most importantly for understanding sediment transport, near bed flow can be directed towards the outer bank, which is in the opposite sense to in a river. The next challenge is to understand what the exact controls on the orientation of helical flow cells within submarine flows are, and their spatial evolution around bends. We present data from the Black Sea showing how the three-dimensional velocity and density of a submarine gravity current evolves at multiple cross sections as the flow travels around a bend. We use this data to calculate the magnitude, relative importance and interaction of centrifugal, coriolis and pressure gradients in controlling the structure of

  5. Ferroelectric active models of ion channels in biomembranes.

    PubMed

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

    1994-06-21

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

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

    PubMed

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

    2016-08-01

    Seizure activity is linked to astrocyte activation as well as dysfunctional cortical neuron excitability produced from changes in calcium-activated potassium (KCa) channel function. Ciliary neurotrophic factor-treated astrocyte conditioned medium (CNTF-ACM) can be used to investigate the peripheral effects of activated astrocytes upon cortical neurons. However, CNTF-ACM's effect upon KCa channel activity in cultured cortical neurons has not yet been investigated. Whole-cell patch clamp recordings were performed in rat cortical neurons to evaluate CNTF-ACM's effects upon charybdotoxin-sensitive large-conductance KCa (BK) channel currents and apamin-sensitive small-conductance KCa (SK) channel current. Biotinylation and RT-PCR were applied to assess CNTF-ACM's effects upon the protein and mRNA expression, respectively, of the SK channel subunits SK2 and SK3 and the BK channel subunits BKα1 and BKβ3. An anti-fibroblast growth factor-2 (FGF-2) monoclonal neutralizing antibody was used to assess the effects of the FGF-2 component of CNTF-ACM. CNTF-ACM significantly increased KCa channel current density, which was predominantly attributable to gains in BK channel activity (p < 0.05). CNTF-ACM produced a significant increase in BKα1 and BKβ3 expression (p < 0.05) but had no significant effect upon SK2 or SK3 expression (p > 0.05). Blocking FGF-2 produced significant reductions in KCa channel current density (p > 0.05) as well as BKα1 and BKβ3 expression in CNTF-ACM-treated neurons (p > 0.05). CNTF-ACM significantly enhances BK channel activity in rat cortical neurons and that FGF-2 is partially responsible for these effects. CNTF-induced astrocyte activation results in secretion of neuroactive factors which may affect neuronal excitability and resultant seizure activity in mammalian cortical neurons.

  7. Transcriptional Activation by Oct4 Is Sufficient for the Maintenance and Induction of Pluripotency

    PubMed Central

    Hammachi, Fella; Morrison, Gillian M.; Sharov, Alexei A.; Livigni, Alessandra; Narayan, Santosh; Papapetrou, Eirini P.; O'Malley, James; Kaji, Keisuke; Ko, Minoru S.H.; Ptashne, Mark; Brickman, Joshua M.

    2012-01-01

    Summary Oct4 is an essential regulator of pluripotency in vivo and in vitro in embryonic stem cells, as well as a key mediator of the reprogramming of somatic cells into induced pluripotent stem cells. It is not known whether activation and/or repression of specific genes by Oct4 is relevant to these functions. Here, we show that fusion proteins containing the coding sequence of Oct4 or Xlpou91 (the Xenopus homolog of Oct4) fused to activating regions, but not those fused to repressing regions, behave as Oct4, suppressing differentiation and promoting maintenance of undifferentiated phenotypes in vivo and in vitro. An Oct4 activation domain fusion supported embryonic stem cell self-renewal in vitro at lower concentrations than that required for Oct4 while alleviating the ordinary requirement for the cytokine LIF. At still lower levels of the fusion, LIF dependence was restored. We conclude that the necessary and sufficient function of Oct4 in promoting pluripotency is to activate specific target genes. PMID:22832160

  8. Characterization of a Ca2+-activated nonselective cation channel during dedifferentiation of cultured rat ventricular cardiomyocytes.

    PubMed

    Guinamard, R; Rahmati, M; Lenfant, J; Bois, P

    2002-07-15

    Cardiac hypertrophy is associated with electrical activity modifications, including sustained depolarization, that lead to a propensity for arrhythmias. The ionic currents underlying the sustained depolarization are not well defined. Similar modifications were reported on adult rat cardiomyocytes in primary culture undergoing dedifferentiation. Using the single-channel measurements on these cells, we identified the appearance of a Ca2+-activated nonselective cation channel (NSCCa) during the dedifferentiation process. In excised inside-out patches the channel presented a linear I/V relationship with a conductance of 26.5 pS. It was equally selective for Na+ and K+ and impermeable to Cl- and Ca2+ ions. The open probability increased with depolarization and with rise in intracellular calcium concentration. The channel activity was reduced by intracellular ATP and suppressed by flufenamic acid. Channel detection increased after incubation with a purinergic receptor agonist (ATPgS) or a PKC activator (PMA). Furthermore, occurrence of the channel developed during the culture. Absent at one day in vitro (d.i.v.), channel activity was present in 5, 46, 27 and 19% of patches after 4, 7, 14 and 21 d.i.v., respectively. We suggest that the channel may be associated with pro-arrhythmic signaling, in particular during the release of transmitters from autonomic nerve endings in the hypertrophied hearts.

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

    PubMed

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

    2016-03-01

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

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

    PubMed

    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.

  11. Activation of KV7 channels stimulates vasodilatation of human placental chorionic plate arteries.

    PubMed

    Mills, T A; Greenwood, S L; Devlin, G; Shweikh, Y; Robinson, M; Cowley, E; Hayward, C E; Cottrell, E C; Tropea, T; Brereton, M F; Dalby-Brown, W; Wareing, M

    2015-06-01

    Potassium (K(+)) channels are key regulators of vascular smooth muscle cell (VSMC) excitability. In systemic small arteries, Kv7 channel expression/activity has been noted and a role in vascular tone regulation demonstrated. We aimed to demonstrate functional Kv7 channels in human fetoplacental small arteries. Human placental chorionic plate arteries (CPAs) were obtained at term. CPA responses to Kv7 channel modulators was determined by wire myography. Presence of Kv7 channel mRNA (encoded by KCNQ1-5) and protein expression were assessed by RT-PCR and immunohistochemistry/immunofluorescence, respectively. Kv7 channel blockade with linopirdine increased CPA basal tone and AVP-induced contraction. Pre-contracted CPAs (AVP; 80 mM K(+) depolarization solution) exhibited significant relaxation to flupirtine, retigabine, the acrylamide (S)-1, and (S) BMS-204352, differential activators of Kv7.1 - Kv7.5 channels. All CPAs assessed expressed KCNQ1 and KCNQ3-5 mRNA; KCNQ2 was expressed only in a subset of CPAs. Kv7 protein expression was confirmed in intact CPAs and isolated VSMCs. Kv7 channels are present and active in fetoplacental vessels, contributing to vascular tone regulation in normal pregnancy. Targeting these channels may represent a therapeutic intervention in pregnancies complicated by increased vascular resistance. Copyright © 2015 Elsevier Ltd. All rights reserved.

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

    PubMed Central

    Larsen, EH; Gabriei, SE; Stutts, MJ; Fullton, J; Price, EM; Boucher, RC

    1996-01-01

    The endogenous Cl- conductance of Spodoptera frugiperda (Sf9) cells was studied 20-35 h after plating out of either uninfected cells or cells infected by a baculovirus vector carrying the cloned beta-galactosidase gene (beta-Gal cells). With the cation Tris+ in the pipette and Na+ in the bath, the reversal potential of whole-cell currents was governed by the prevailing Cl- equilibrium potential and could be fitted by the Goldman-Hodgkin-Katz equation with similar permeabilities for uninfected and beta-Gal cells. In the frequency range 0.12 < f < 300 Hz, the power density spectrum of whole-cell Cl- currents could be fitted by three Lorentzians. Independent of membrane potential, >50% of the total variance of whole-cell current fluctuations was accounted for by the low frequency Lorentzian (fc = 0.40 +/- 0.03 Hz, n = 6). Single-Cl- channels showed complex gating kinetics with long lasting (seconds) openings interrupted by similar long closures. In the open state, channels exhibited fast burst-like closures. Since the patches normally contained more than a single channel, it was not possible to measure open and closed dwell-time distributions for comparing single-Cl- channel activity with the kinetic features of whole-cell currents. However, the power density spectrum of Cl- currents of cell-attached and excised outside-out patches contained both high and low frequency Lorentzian components, with the corner frequency of the slow component (fc = 0.40 +/- 0.02 Hz, n = 4) similar to that of whole-cell current fluctuations. Chloride channels exhibited multiple conductance states with similar Goldman-Hodgkin-Katz-type rectification. Single-channel permeabilities covered the range from approximately 0.6.10(-14) cm5/s to approximately 6.10(-14) cm3/s, corresponding to a limiting conductance (gamma 150/150) of approximately 3.5 pS and approximately 35 pS, respectively. All states reversed near the same membrane potential, and they exhibited similar halide ion selectivity, P1

  13. Regulation of Proteome Maintenance Gene Expression by Activators of Peroxisome Proliferator-Activated Receptor a (PPARa)

    EPA Science Inventory

    The nuclear receptor peroxisome proliferator-activated receptor alpha (PPARa) is activated by a large number of xenobiotic and hypolipidemic compounds called peroxisome proliferator chemicals (PPC). One agonist of PPARa (WY-14,643) regulates responses in the mouse liver to chemic...

  14. Regulation of Proteome Maintenance Gene Expression by Activators of Peroxisome Proliferator-Activated Receptor a (PPARa)

    EPA Science Inventory

    The nuclear receptor peroxisome proliferator-activated receptor alpha (PPARa) is activated by a large number of xenobiotic and hypolipidemic compounds called peroxisome proliferator chemicals (PPC). One agonist of PPARa (WY-14,643) regulates responses in the mouse liver to chemic...

  15. Membrane-pipette interactions underlie delayed voltage activation of mechanosensitive channels in Xenopus oocytes.

    PubMed Central

    Gil, Z; Magleby, K L; Silberberg, S D

    1999-01-01

    To investigate the mechanism for the delayed activation by voltage of the predominant mechanosensitive (MS) channel in Xenopus oocytes, currents were recorded from on-cell and excised patches of membrane with the patch clamp technique and from intact oocytes with the two-electrode voltage clamp technique. MS channels could be activated by stretch in inside-out, on-cell, and outside-out patch configurations, using pipettes formed of either borosilicate or soft glass. In inside-out patches formed with borosilicate glass pipettes, depolarizing voltage steps activated MS channels in a cooperative manner after delays of seconds. This voltage-dependent activation was not observed for outside-out patches. Voltage-dependent activation was also not observed when the borosilicate pipettes were either replaced with soft glass pipettes or coated with soft glass. When depolarizing voltage steps were applied to the whole oocyte with a two-electrode voltage clamp, currents that could be attributed to MS channels were not observed. Yet the same depolarizing steps activated MS channels in on-cell patches formed with borosilicate pipettes on the same oocyte. These observations suggest that the delayed cooperative activation of MS channels by depolarization is not an intrinsic property of the channels, but requires interaction between the membrane and patch pipette. PMID:10354436

  16. Thermodynamics of Activation Gating in Olfactory-Type Cyclic Nucleotide-Gated (CNGA2) Channels

    PubMed Central

    Nache, Vasilica; Kusch, Jana; Biskup, Christoph; Schulz, Eckhard; Zimmer, Thomas; Hagen, Volker; Benndorf, Klaus

    2008-01-01

    Olfactory-type cyclic nucleotide-gated (CNG) ion channels open by the binding of cyclic nucleotides to a binding domain in the C-terminus. Employing the Eyring rate theory, we performed a thermodynamic analysis of the activation gating in homotetrameric CNGA2 channels. Lowering the temperature shifted the concentration-response relationship to lower concentrations, resulting in a decrease of both the enthalpy ΔH and entropy ΔS upon channel opening, suggesting that the order of an open CNGA2 channel plus its environment is higher than that of the closed channel. Activation time courses induced by cGMP concentration jumps were used to study thermodynamics of the transition state. The activation enthalpies ΔH‡ were positive at all cGMP concentrations. In contrast, the activation entropy ΔS‡ was positive at low cGMP concentrations and became then negative at increasing cGMP concentrations. The enthalpic and entropic parts of the activation energies approximately balance each other at all cGMP concentrations, leaving the free enthalpy of activation in the range between 19 and 21 kcal/mol. We conclude that channel activation proceeds through different pathways at different cGMP concentrations. Compared to the unliganded channel, low cGMP concentrations generate a transitional state of lower order whereas high cGMP concentrations generate a transitional state of higher order. PMID:18567637

  17. A pervasive mechanism for analgesia: activation of GIRK2 channels.

    PubMed

    Blednov, Y A; Stoffel, M; Alva, H; Harris, R A

    2003-01-07

    G protein-coupled inwardly rectifying potassium channels (GIRKs) provide a common link between numerous neurotransmitter receptors and the regulation of synaptic transmission. We asked whether GIRKs specify a single behavioral action that is produced by drugs acting on the diverse receptors coupled with GIRKs. By using GIRK2-null mutant mice, we found marked reduction or complete elimination of the antinociceptive (hot plate test) effects of ethanol, oxotremorine, nicotine, baclofen, clonidine, and the cannabinoid receptor agonist WIN 55,212. However, ketamine analgesia remained intact. For most drugs, there was a sex difference in antinociceptive action, and the impact of deletion of the GIRK2 channel was less in female mice. The deletion of the GIRK2 channel blocks the opioid-dependent component of stress-induced analgesia (SIA), whereas nonopioid SIA was not changed. We propose that opioid, alpha adrenergic, muscarinic cholinergic, gamma-aminobutyric acid-B, and cannabinoid receptors are coupled with postsynaptic GIRK2 channels in vivo. Furthermore, this pathway accounts for essentially all of the antinociceptive effects in males, although females appear to recruit additional signal transduction mechanisms for some analgesic drugs.

  18. Hypotonic medium increases calcium permeant channels activity in human normal and dystrophic myotubes.

    PubMed

    Vandebrouck, Clarisse; Duport, Gérard; Raymond, Guy; Cognard, Christian

    2002-05-03

    Duchenne muscular dystrophy (DMD) is characterized by the absence of dystrophin and an elevated intracellular calcium level. Single-channel recordings were performed with the cell-attached configuration of the patch-clamp technique. The present study shows, on human co-cultured normal and dystrophic muscle cells, the evidence for an increased activity of calcium permeant cationic mechano-sensitive channels under hypotonic medium stimulation. This activity was particularly enhanced in DMD cells. The hypotonic medium induced drastic changes in the single-channel activity characteristics, which are: a large increase of the calcium over potassium permeability ratio; and a great enhancement of the quantity of current crossing through these channels. These channels could contribute to a significant calcium entry, which could participate in the abnormal calcium homeostasis observed in DMD muscle.

  19. Fragile X mental retardation protein controls gating of the sodium-activated potassium channel Slack

    PubMed Central

    Brown, Maile R.; Kronengold, Jack; Gazula, Valeswara-Rao; Chen, Yi; Strumbos, John G.; Sigworth, Fred J.; Navaratnam, Dhasakumar; Kaczmarek, Leonard K.

    2010-01-01

    In humans, absence of Fragile X mental retardation protein (FMRP), an RNA-binding protein, results in Fragile X syndrome (FXS), the most common inherited form of intellectual disability. Here we report through biochemical and electrophysiological studies that FMRP binds the C-terminus of the Slack sodium-activated potassium channel to activate the channel. The findings suggest that Slack activity may provide a link between patterns of neuronal firing and changes in protein translation. PMID:20512134

  20. Zinc pyrithione-mediated activation of voltage-gated KCNQ potassium channels rescues epileptogenic mutants.

    PubMed

    Xiong, Qiaojie; Sun, Haiyan; Li, Min

    2007-05-01

    KCNQ potassium channels are activated by changes in transmembrane voltage and play an important role in controlling electrical excitability. Human mutations of KCNQ2 and KCNQ3 potassium channel genes result in reduction or loss of channel activity and cause benign familial neonatal convulsions (BFNCs). Thus, small molecules capable of augmenting KCNQ currents are essential both for understanding the mechanism of channel activity and for developing therapeutics. We performed a high-throughput screen in search for agonistic compounds potentiating KCNQ potassium channels. Here we report identification of a new opener, zinc pyrithione (1), which activates both recombinant and native KCNQ M currents. Interactions with the channel protein cause an increase of single-channel open probability that could fully account for the overall conductance increase. Separate point mutations have been identified that either shift the concentration dependence or affect potentiation efficacy, thereby providing evidence for residues influencing ligand binding and downstream events. Furthermore, zinc pyrithione is capable of rescuing the mutant channels causal to BFNCs.

  1. CAMKII activation is not required for maintenance of learning-induced enhancement of neuronal excitability.

    PubMed

    Liraz, Ori; Rosenblum, Kobi; Barkai, Edi

    2009-01-01

    Pyramidal neurons in the piriform cortex from olfactory-discrimination trained rats show enhanced intrinsic neuronal excitability that lasts for several days after learning. Such enhanced intrinsic excitability is mediated by long-term reduction in the post-burst after-hyperpolarization (AHP) which is generated by repetitive spike firing. AHP reduction is due to decreased conductance of a calcium-dependent potassium current, the sI(AHP). We have previously shown that learning-induced AHP reduction is maintained by persistent protein kinase C (PKC) and extracellular regulated kinase (ERK) activation. However, the molecular machinery underlying this long-lasting modulation of intrinsic excitability is yet to be fully described. Here we examine whether the CaMKII, which is known to be crucial in learning, memory and synaptic plasticity processes, is instrumental for the maintenance of learning-induced AHP reduction. KN93, that selectively blocks CaMKII autophosphorylation at Thr286, reduced the AHP in neurons from trained and control rat to the same extent. Consequently, the differences in AHP amplitude and neuronal adaptation between neurons from trained rats and controls remained. Accordingly, the level of activated CaMKII was similar in pirifrom cortex samples taken form trained and control rats. Our data show that although CaMKII modulates the amplitude of AHP of pyramidal neurons in the piriform cortex, its activation is not required for maintaining learning-induced enhancement of neuronal excitability.

  2. CAMKII Activation Is Not Required for Maintenance of Learning-Induced Enhancement of Neuronal Excitability

    PubMed Central

    Liraz, Ori; Rosenblum, Kobi; Barkai, Edi

    2009-01-01

    Pyramidal neurons in the piriform cortex from olfactory-discrimination trained rats show enhanced intrinsic neuronal excitability that lasts for several days after learning. Such enhanced intrinsic excitability is mediated by long-term reduction in the post-burst after-hyperpolarization (AHP) which is generated by repetitive spike firing. AHP reduction is due to decreased conductance of a calcium-dependent potassium current, the sIAHP. We have previously shown that learning-induced AHP reduction is maintained by persistent protein kinase C (PKC) and extracellular regulated kinase (ERK) activation. However, the molecular machinery underlying this long-lasting modulation of intrinsic excitability is yet to be fully described. Here we examine whether the CaMKII, which is known to be crucial in learning, memory and synaptic plasticity processes, is instrumental for the maintenance of learning-induced AHP reduction. KN93, that selectively blocks CaMKII autophosphorylation at Thr286, reduced the AHP in neurons from trained and control rat to the same extent. Consequently, the differences in AHP amplitude and neuronal adaptation between neurons from trained rats and controls remained. Accordingly, the level of activated CaMKII was similar in pirifrom cortex samples taken form trained and control rats. Our data show that although CaMKII modulates the amplitude of AHP of pyramidal neurons in the piriform cortex, its activation is not required for maintaining learning-induced enhancement of neuronal excitability. PMID:19172997

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

    PubMed

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

    2017-12-01

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

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

    PubMed

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

    2013-10-01

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

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

    PubMed Central

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

    2013-01-01

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

  6. Fragile X mental retardation protein controls ion channel expression and activity.

    PubMed

    Ferron, Laurent

    2016-10-15

    Fragile X-associated disorders are a family of genetic conditions resulting from the partial or complete loss of fragile X mental retardation protein (FMRP). Among these disorders is fragile X syndrome, the most common cause of inherited intellectual disability and autism. FMRP is an RNA-binding protein involved in the control of local translation, which has pleiotropic effects, in particular on synaptic function. Analysis of the brain FMRP transcriptome has revealed hundreds of potential mRNA targets encoding postsynaptic and presynaptic proteins, including a number of ion channels. FMRP has been confirmed to bind voltage-gated potassium channels (Kv 3.1 and Kv 4.2) mRNAs and regulates their expression in somatodendritic compartments of neurons. Recent studies have uncovered a number of additional roles for FMRP besides RNA regulation. FMRP was shown to directly interact with, and modulate, a number of ion channel complexes. The sodium-activated potassium (Slack) channel was the first ion channel shown to directly interact with FMRP; this interaction alters the single-channel properties of the Slack channel. FMRP was also shown to interact with the auxiliary β4 subunit of the calcium-activated potassium (BK) channel; this interaction increases calcium-dependent activation of the BK channel. More recently, FMRP was shown to directly interact with the voltage-gated calcium channel, Cav 2.2, and reduce its trafficking to the plasma membrane. Studies performed on animal models of fragile X syndrome have revealed links between modifications of ion channel activity and changes in neuronal excitability, suggesting that these modifications could contribute to the phenotypes observed in patients with fragile X-associated disorders.

  7. Calcium-activated potassium channels in the endothelium of intact rat aorta.

    PubMed Central

    Marchenko, S M; Sage, S O

    1996-01-01

    1. Single K+ channel currents and membrane potential were recorded in the endothelium of excised intact rat aorta. 2. Two types of K+ channel were found in excised patches, KCh and KAp. With Na+ and K+ as the main external and internal cations, outward conductances were 6.7 pS (KCh) and 2.8 pS (KAp). In symmetric 150 mM K+, the inward conductances were 18 and 9.1 pS. 3. Activation by Ca2+ was concentration dependent. KCh channels were activated by [Ca2+] > 0.1 microM and KAp by [Ca2+] > 0.5 microM. 4. Apamin at concentrations > 1 nM inhibited KAp Channels. Block was complete at 10 nM. KAp channels were insensitive to charybdotoxin. KCh channels were inhibited by charybdotoxin at concentrations > 50 nM, but were insensitive to apamin. 5. d-Tubocurarine (dTC) evoked flickering activity of KAp channels at concentrations > 5 microM and complete block at 100 microM. At these doses, dTC did not affect KCh channels, but at concentrations > 1 mM it decreased the single channel amplitude. 6. Hyperpolarization evoked by acetylcholine was unaffected by apamin or dTC at low concentrations ( < or = 100 microM), but inhibited by high concentrations of charybdotoxin ( > 50 nM) or dTC ( > 1 mM). 7. These data suggest that KCh channels are novel Ca(2+)-activated K+ channels responsible for the ACh-evoked hyperpolarization in the endothelium of rat aorta. PMID:8730582

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

    PubMed

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

    2015-09-01

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

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

    PubMed

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

    2015-08-01

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

  10. Arachidonic Acid and Other Fatty Acids Directly Activate Potassium Channels in Smooth Muscle Cells

    NASA Astrophysics Data System (ADS)

    Ordway, Richard W.; Walsh, John V.; Singer, Joshua J.

    1989-06-01

    Arachidonic acid, as well as fatty acids that are not substrates for cyclooxygenase and lipoxygenase enzymes, activated a specific type of potassium channel in freshly dissociated smooth muscle cells. Activation occurred in excised membrane patches in the absence of calcium and all nucleotides. Therefore signal transduction pathways that require such soluble factors, including the NADPH-dependent cytochrome P450 pathway, do not mediate the response. Thus, fatty acids directly activate potassium channels and so may constitute a class of signal molecules that regulate ion channels.

  11. Lysophosphatidylcholine- and MCP-1-induced chemotaxis of monocytes requires potassium channel activity.

    PubMed

    Schilling, Tom; Eder, Claudia

    2009-11-01

    One of the earliest cellular responses in atherogenesis is the focal recruitment of circulating monocytes, while the most important atherogenic chemoattractants are monocyte chemoattractant protein-1 (MCP-1) and lysophosphatidylcholine (LPC). Invading monocytes transform into activated macrophages and foam cells, which stimulate inflammatory processes and promote atherosclerosis. In this study, we have searched for common mechanisms involved in MCP-1- and LPC-stimulated monocyte migration. We have found that migration of THP-1 monocytes stimulated with MCP-1 was reduced upon inhibition of G(i/o) proteins with pertussis toxin and upon inhibition of platelet activating factor receptors with BN52021, whereas LPC-stimulated monocyte chemotaxis remained unaffected by both inhibitors. Furthermore, Cl(-) channels were only required for MCP-1-induced chemotaxis. However, activity of voltage-gated K+ channels and of Ca2+-activated K+ channels was found to be involved in migration of monocytes stimulated with either MCP-1 or LPC. Inhibition of voltage-gated K+ channels with 4-aminopyridine or margatoxin partially inhibited MCP-1- and LPC-stimulated migration of monocytes. Blockade of Ca2+-activated K+ channels with TRAM-34 also partially reduced migration of MCP-1- and LPC-stimulated monocytes. Simultaneous inhibition of voltage-gated and Ca2+-activated K+ channels abolished MCP-1- and LPC-induced chemotaxis of monocytes. Thus, K+ channel inhibition may represent a novel powerful strategy to reduce monocyte infiltration and subsequent inflammation in atherosclerosis.

  12. Activation of TRPM7 channels by small molecules under physiological conditions.

    PubMed

    Hofmann, T; Schäfer, S; Linseisen, M; Sytik, L; Gudermann, T; Chubanov, V

    2014-12-01

    Transient receptor potential cation channel, subfamily M, member 7 (TRPM7) is a cation channel covalently linked to a protein kinase domain. TRPM7 is ubiquitously expressed and regulates key cellular processes such as Mg(2+) homeostasis, motility, and proliferation. TRPM7 is involved in anoxic neuronal death, cardiac fibrosis, and tumor growth. The goal of this work was to identify small molecule activators of the TRPM7 channel and investigate their mechanism of action. We used an aequorin bioluminescence-based assay to screen for activators of the TRPM7 channel. Valid candidates were further characterized using patch clamp electrophysiology. We identified 20 drug-like compounds with various structural backbones that can activate the TRPM7 channel. Among them, the δ opioid antagonist naltriben was studied in greater detail. Naltriben's action was selective among the TRP channels tested. Naltriben activates TRPM7 currents without prior depletion of intracellular Mg(2+) even under conditions of low PIP2. Moreover, naltriben interfered with the effect of the TRPM7 inhibitor NS8593. Finally, our experiments with TRPM7 variants carrying mutations in the pore, TRP, and kinase domains indicate that the site of TRPM7 activation by this small-molecule ligand is most likely located in or near the TRP domain. In conclusion, we identified the first organic small-molecule activators of TRPM7 channels, thus providing new experimental tools to study TRPM7 function in native cellular environments.

  13. ZD7288 inhibits low-threshold Ca(2+) channel activity and regulates sperm function.

    PubMed

    Felix, Ricardo; Sandoval, Alejandro; Sánchez, Daniel; Gómora, Juan Carlos; De la Vega-Beltrán, José L; Treviño, Claudia L; Darszon, Alberto

    2003-11-07

    In this study, ZD7288, a blocker of hyperpolarization-activated and cyclic nucleotide-gated (HCN) channels, has been found to inhibit the mouse sperm acrosome reaction (AR). HCN channels have not yet been either recorded or implicated in mouse sperm AR, but low-threshold (T-type) Ca(2+) channels have. Interestingly, ZD7288 blocked native T-type Ca(2+) currents in mouse spermatogenic cells with an IC(50) of about 100 microM. This blockade was more effective at voltages producing low levels of inactivation, suggesting a differential affinity of ZD7288 for different channel conformations. Furthermore, ZD7288 inhibited all cloned T-type but not high-threshold N-type channels heterologously expressed in HEK-293 cells. Our results further support the role of T-type Ca(2+) channels in the mouse sperm AR.

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

    PubMed

    Kyle, Barry D; Braun, Andrew P

    2014-01-01

    Large conductance, Ca(2+)-activated K(+) (BK) channels represent an important pathway for the outward flux of K(+) ions from the intracellular compartment in response to membrane depolarization, and/or an elevation in cytosolic free [Ca(2+)]. They are functionally expressed in a range of mammalian tissues (e.g., nerve and smooth muscles), where they can either enhance or dampen membrane excitability. The diversity of BK channel activity results from the considerable alternative mRNA splicing and post-translational modification (e.g., phosphorylation) of key domains within the pore-forming α subunit of the channel complex. Most of these modifications are regulated by distinct upstream cell signaling pathways that influence the structure and/or gating properties of the holo-channel and ultimately, cellular function. The channel complex may also contain auxiliary subunits that further affect channel gating and behavior, often in a tissue-specific manner. Recent studies in human and animal models have provided strong evidence that abnormal BK channel expression/function contributes to a range of pathologies in nerve and smooth muscle. By targeting the upstream regulatory events modulating BK channel behavior, it may be possible to therapeutically intervene and alter BK channel expression/function in a beneficial manner.

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

    PubMed Central

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

    2016-01-01

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

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

    PubMed Central

    Kyle, Barry D.; Braun, Andrew P.

    2014-01-01

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

  17. Maintenance of Efficacy and Continuing Safety of Golimumab for Active Ulcerative Colitis: PURSUIT-SC Maintenance Study Extension Through 1 Year

    PubMed Central

    Gibson, Peter R; Feagan, Brian G; Sandborn, William J; Marano, Colleen; Strauss, Richard; Johanns, Jewel; Padgett, Lakshmi; Collins, Judith; Tarabar, Dino; Hebzda, Zbigniew; Rutgeerts, Paul; Reinisch, Walter

    2016-01-01

    Objectives: The safety and efficacy of subcutaneous golimumab through 2 years of maintenance therapy was evaluated in patients with moderate-to-severe ulcerative colitis (UC). Methods: Patients completing treatment through week 52 (placebo, golimumab 50, 100, every-4-weeks (q4w)) and evaluations at week 54 were eligible for this long-term extension (LTE) trial. Patients receiving placebo or golimumab 50 mg with worsening disease during the LTE could receive golimumab 100 mg. Efficacy assessments included the Mayo physician's global assessment (PGA) subscore, inflammatory bowel disease questionnaire (IBDQ), and corticosteroid use. Patients who were randomized to golimumab at PURSUIT-Maintenance baseline and continued receiving golimumab during the LTE were analyzed for efficacy (using intention-to-treat and “as observed” analyses; N=195) and safety (N=200). Patients treated with golimumab at any time from induction baseline through week 104 (N=1240) constituted the overall safety population. Results: Baseline demographics and disease characteristics of patients entering the LTE receiving golimumab were similar to those of all patients randomized to golimumab maintenance at baseline. At week 104, 80.5% (157/195) of patients had a PGA=0/1 (range weeks 56–104: 80.5–91.8%) and 56.4% (110/195) had a PGA=0 (weeks 56–104: range: 53.8–58.5%). Through week 104, 86% of patients maintained inactive or mild disease activity. Among 174 corticosteroid-free patients at week 54, 88.5% remained corticosteroid-free at week 104. At week 104, 62.2% (120/193) had an IBDQ score ≥170. Tuberculosis, opportunistic infection, and malignancy rates were low, and the overall safety profile was similar to that reported through week 54. Two non-melanoma skin cancers, one metastatic colon cancer, and two deaths (biventricular heart dysfunction, sepsis) occurred between weeks 54 and 104. Conclusion: Subcutaneous golimumab q4w through 2 years maintained clinical benefit and reduced

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

    PubMed

    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. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

  19. Brain Activation Patterns Associated with the Effects of Emotional Distracters during Working Memory Maintenance in Patients with Generalized Anxiety Disorder.

    PubMed

    Park, Jong-Il; Kim, Gwang-Won; Jeong, Gwang-Woo; Chung, Gyung Ho; Yang, Jong-Chul

    2016-01-01

    Few studies have assessed the neural mechanisms of the effects of emotion on cognition in generalized anxiety disorder (GAD) patients. In this functional MRI (fMRI), we investigated the effects of emotional interference on working memory (WM) maintenance in GAD patients. Fifteen patients with GAD participated in this study. Event-related fMRI data were obtained while the participants performed a WM task (face recognition) with neutral and anxiety-provoking distracters. The GAD patients showed impaired performance in WM task during emotional distracters and showed greater activation on brain regions such as DLPFC, VLPFC, amygdala, hippocampus which are responsible for the active maintenance of goal relevant information in WM and emotional processing. Although our results are not conclusive, our finding cautiously suggests the cognitive-affective interaction in GAD patients which shown interfering effect of emotional distracters on WM maintenance.

  20. Activation of inositol trisphosphate-sensitive Ca2+ channels of sarcoplasmic reticulum from frog skeletal muscle.

    PubMed Central

    Suárez-Isla, B A; Alcayaga, C; Marengo, J J; Bull, R

    1991-01-01

    1. The modulation by Ca2+ of the activation by inositol 1,4,5-trisphosphate (IP3) of Ca2+ channels present in native sarcoplasmic reticulum membranes from frog skeletal muscle was studied after channel incorporation into planar phospholipid bilayers in the presence of Ca2+ or Ba2+ as current carrier species. 2. Channel activity expressed as fractional open time (Po) was low (less than or equal to 0.15) in the presence of varying free Ca2+ concentrations bathing the myoplasmic face of the channel (cis side), and did not increase significantly between 0.01 and 30 microM-Ca2+. 3. Channel activation mediated by IP3 could be elicited from free Ca2+ levels similar to those of resting skeletal muscle (about 0.1 microM) and was found to be strongly regulated by the free Ca2+ concentration present at the myoplasmic moiety of the channel. 4. Channel activation by 10 microM-IP3 depended on the Ca2+ concentration on the cis side. Po reached a maximum between pCa 7.0 and 6.0, but decreased at higher concentrations of free Ca2+. Thus, Ca2+ exerted a modulatory influence on IP3-mediated activation in a concentration range where the channel was insensitive to Ca2+. 5. The results indicate that Ca2+ ions act as modulators of IP3 efficacy to open the channel. This could arise from an interaction of Ca2+ with the channel gating mechanism or with the agonist binding site. PMID:1667801

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

    PubMed

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

    2014-08-01

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

  2. Inverse coupling in leak and voltage-activated K+ channel gates underlies distinct roles in electrical signaling.

    PubMed

    Ben-Abu, Yuval; Zhou, Yufeng; Zilberberg, Noam; Yifrach, Ofer

    2009-01-01

    Voltage-activated (Kv) and leak (K(2P)) K(+) channels have key, yet distinct, roles in electrical signaling in the nervous system. Here we examine how differences in the operation of the activation and slow inactivation pore gates of Kv and K(2P) channels underlie their unique roles in electrical signaling. We report that (i) leak K(+) channels possess a lower activation gate, (ii) the activation gate is an important determinant controlling the conformational stability of the K(+) channel pore, (iii) the lower activation and upper slow inactivation gates of leak channels cross-talk and (iv) unlike Kv channels, where the two gates are negatively coupled, these two gates are positively coupled in K(2P) channels. Our results demonstrate how basic thermodynamic properties of the K(+) channel pore, particularly conformational stability and coupling between gates, underlie the specialized roles of Kv and K(2P) channel families in electrical signaling.

  3. Mechanosensitive Channel Activation by Diffusio-Osmotic Force

    NASA Astrophysics Data System (ADS)

    Bonthuis, Douwe Jan; Golestanian, Ramin

    2014-10-01

    For ion channel gating, the appearance of two distinct conformational states and the discrete transitions between them are essential, and therefore of crucial importance to all living organisms. We show that the physical interplay between two structural elements that are commonly present in bacterial mechanosensitive channels—namely, a charged vestibule and a hydrophobic constriction—creates two distinct conformational states, open and closed, as well as the gating between them. We solve the nonequilibrium Stokes-Poisson-Nernst-Planck equations, extended to include a molecular potential of mean force, and show that a first order transition between the closed and open states arises naturally from the diffusio-osmotic stress caused by the ions and the water inside the channel and the elastic restoring force from the membrane.

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

    PubMed

    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. Putative chanzyme activity of TRPM2 cation channel is unrelated to pore gating

    PubMed Central

    Tóth, Balázs; Iordanov, Iordan; Csanády, László

    2014-01-01

    Transient receptor potential melastatin 2 (TRPM2) is a Ca2+-permeable cation channel expressed in immune cells of phagocytic lineage, pancreatic β cells, and brain neurons and is activated under oxidative stress. TRPM2 activity is required for immune cell activation and insulin secretion and is responsible for postischemic neuronal cell death. TRPM2 is opened by binding of ADP ribose (ADPR) to its C-terminal cytosolic nudix-type motif 9 (NUDT9)-homology (NUDT9-H) domain, which, when expressed in isolation, cleaves ADPR into AMP and ribose-5-phosphate. A suggested coupling of this enzymatic activity to channel gating implied a potentially irreversible gating cycle, which is a unique feature of a small group of channel enzymes known to date. The significance of such a coupling lies in the conceptually distinct pharmacologic strategies for modulating the open probability of channels obeying equilibrium versus nonequilibrium gating mechanisms. Here we examine the potential coupling of TRPM2 enzymatic activity to pore gating. Mutation of several residues proposed to enhance or eliminate NUDT9-H catalytic activity all failed to affect channel gating kinetics. An ADPR analog, α-β-methylene-ADPR (AMPCPR), was shown to be entirely resistant to hydrolysis by NUDT9, but nevertheless supported TRPM2 channel gating, albeit with reduced apparent affinity. The rate of channel deactivation was not slowed but, rather, accelerated in AMPCPR. These findings, as well as detailed analyses of steady-state gating kinetics of single channels recorded in the presence of a range of concentrations of ADPR or AMPCPR, identify TRPM2 as a simple ligand-gated channel that obeys an equilibrium gating mechanism uncoupled from its enzymatic activity. PMID:25385633

  6. Modulation of high-voltage activated Ca(2+) channels by membrane phosphatidylinositol 4,5-bisphosphate.

    PubMed

    Suh, Byung-Chang; Leal, Karina; Hille, Bertil

    2010-07-29

    Modulation of voltage-gated Ca(2+) channels controls activities of excitable cells. We show that high-voltage activated Ca(2+) channels are regulated by membrane phosphatidylinositol 4,5-bisphosphate (PIP(2)) with different sensitivities. Plasma membrane PIP(2) depletion by rapamycin-induced translocation of an inositol lipid 5-phosphatase or by a voltage-sensitive 5-phosphatase (VSP) suppresses Ca(V)1.2 and Ca(V)1.3 channel currents by approximately 35% and Ca(V)2.1 and Ca(V)2.2 currents by 29% and 55%, respectively. Other Ca(V) channels are less sensitive. Inhibition is not relieved by strong depolarizing prepulses. It changes the voltage dependence of channel gating little. Recovery of currents from inhibition needs intracellular hydrolysable ATP, presumably for PIP(2) resynthesis. When PIP(2) is increased by overexpressing PIP 5-kinase, activation and inactivation of Ca(V)2.2 current slow and voltage-dependent gating shifts to slightly higher voltages. Thus, endogenous membrane PIP(2) supports high-voltage activated L-, N-, and P/Q-type Ca(2+) channels, and stimuli that activate phospholipase C deplete PIP(2) and reduce those Ca(2+) channel currents.

  7. Modulation of High-Voltage Activated Ca2+ Channels by Membrane Phosphatidylinositol 4,5-Bisphosphate

    PubMed Central

    Suh, Byung-Chang; Leal, Karina; Hille, Bertil

    2010-01-01

    SUMMARY Modulation of voltage-gated Ca2+ channels controls activities of excitable cells. We show that high-voltage activated Ca2+ channels are regulated by membrane phosphatidylinositol 4,5-bisphosphate (PIP2) with different sensitivities. Plasma membrane PIP2 depletion by rapamycin-induced translocation of an inositol lipid 5-phosphatase or by a voltage-sensitive 5-phosphatase (VSP) suppresses CaV1.2 and CaV1.3 channel currents by ~35%, and CaV2.1 and CaV2.2 currents by 29 and 55%, respectively. Other CaV channels are less sensitive. Inhibition is not relieved by strong depolarizing prepulses. It changes the voltage dependence of channel gating little. Recovery of currents from inhibition needs intracellular hydrolysable ATP, presumably for PIP2 resynthesis. When PIP2 is increased by overexpressing PIP 5-kinase, activation and inactivation of CaV2.2 current slow and voltage-dependent gating shifts to slightly higher voltages. Thus, endogenous membrane PIP2 supports high-voltage activated L-, N-, and P/Q- type Ca2+ channels, and stimuli that activate phospholipase C deplete PIP2 and reduce those Ca2+ channel currents. PMID:20670831

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

    PubMed

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

    2012-01-01

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

  9. Channel-forming activity in the venom of the cockroach-hunting wasp, Ampulex compressa.

    PubMed

    Gincel, Dan; Haspel, Gal; Libersat, Frederic

    2004-05-01

    The parasitoid solitary wasp Ampulex compressa uses the cockroach Periplaneta americana as a food supply for its larvae. To subdue its prey, the wasp injects a venom cocktail into the brain of the cockroach. We investigated channel activity of A. compressa venom by collecting venom and incorporating it into a planar lipid bilayer. The venom, reconstituted into the bilayer, showed ion channel activity, forming a fast-fluctuating channel with a small conductance of 20+/-0.1pS, with no voltage sensitivity. These channels were not observed when the venom was digested with proteases before application to the bilayer, but were not affected by exposure to protease after their incorporation into the bilayer, indicating that the active venom component is a peptide. The channels were found to be cation selective with similar selectivity for the monovalent cations K(+), Li(+) and Na(+), but showed high selectivity against anions (Cl(-)) and divalent cations (Ca(2+) and Mg(2+)). This study is the first demonstration and biophysical characterization of channel activity in the venom of A. compressa. The possible functional significance of this channel activity is discussed in light of the unusual nature of the effects of this wasp venom on the behavior of its prey.

  10. Dihydroxyeicosatrienoic acids are potent activators of Ca2+-activated K+ channels in isolated rat coronary arterial myocytes

    PubMed Central

    Lu, Tong; Katakam, Prasad V G; VanRollins, Mike; Weintraub, Neal L; Spector, Arthur A; Lee, Hon-Chi

    2001-01-01

    Dihydroxyeicosatrienoic acids (DHETs), which are metabolites of arachidonic acid (AA) and epoxyeicosatrienoic acids (EETs), have been identified as highly potent endogenous vasodilators, but the mechanisms by which DHETs induce relaxation of vascular smooth muscle are unknown. Using inside-out patch clamp techniques, we examined the effects of DHETs on the large conductance Ca2+-activated K+ (BK) channels in smooth muscle cells from rat small coronary arteries (150–300 μm diameter). 11,12-DHET potently activated BK channels with an EC50 of 1.87 ± 0.57 nm (n = 5). Moreover, the three other regioisomers 5,6-, 8,9- and 14,15-DHET were equipotent with 11,12-DHET in activating BK channels. The efficacy of 11,12-DHET in opening BK channels was much greater than that of its immediate precursor 11,12-EET. In contrast, AA did not significantly affect BK channel activity. The voltage dependence of BK channels was dramatically modulated by 11,12-DHET. With physiological concentrations of cytoplasmic Ca2+ (200 nm), the voltage at which the channel open probability was half-maximal (V1/2) was shifted from a baseline of 115.6 ± 6.5 mV to 95.0 ± 10.1 mV with 5 nm 11,12-DHET, and to 60.0 ± 8.4 mV with 50 nm 11,12-DHET. 11,12-DHET also enhanced the sensitivity of BK channels to Ca2+ but did not activate the channels in the absence of Ca2+. 11,12-DHET (50 nm) reduced the Ca2+ EC50 of BK channels from a baseline of 1.02 ± 0.07 μm to 0.42 ± 0.11 μm. Single channel kinetic analysis indicated that 11,12-DHET did not alter BK channel conductance but did reduce the first latency of BK channel openings in response to a voltage step. 11,12-DHET dose-dependently increased the open dwell times, abbreviated the closed dwell times, and decreased the transition rates from open to closed states. We conclude that DHETs hyperpolarize vascular smooth muscle cells through modulation of the BK channel gating behaviour, and by enhancing the channel sensitivities to Ca2+ and voltage. Hence

  11. HCN channels contribute to serotonergic modulation of ventral surface chemosensitive neurons and respiratory activity

    PubMed Central

    Hawkins, Virginia E.; Hawryluk, Joanna M.; Takakura, Ana C.; Tzingounis, Anastasios V.; Moreira, Thiago S.

    2014-01-01

    Chemosensitive neurons in the retrotrapezoid nucleus (RTN) provide a CO2/H+-dependent drive to breathe and function as an integration center for the respiratory network, including serotonergic raphe neurons. We recently showed that serotonergic modulation of RTN chemoreceptors involved inhibition of KCNQ channels and activation of an unknown inward current. Hyperpolarization-activated cyclic-nucleotide-gated (HCN) channels are the molecular correlate of the hyperpolarization-activated inward current (Ih) and have a high propensity for modulation by serotonin. To investigate whether HCN channels contribute to basal activity and serotonergic modulation of RTN chemoreceptors, we characterize resting activity and the effects of serotonin on RTN chemoreceptors in vitro and on respiratory activity of anesthetized rats in the presence or absence of blockers of KCNQ (XE991) and/or HCN (ZD7288, Cs+) channels. We found in vivo that bilateral RTN injections of ZD7288 increased respiratory activity and in vitro HCN channel blockade increased activity of RTN chemoreceptors under control conditions, but this was blunted by KCNQ channel inhibition. Furthermore, in vivo unilateral RTN injection of XE991 plus ZD7288 eliminated the serotonin response, and in vitro serotonin sensitivity was eliminated by application of XE991 and ZD7288 or SQ22536 (adenylate cyclase blocker). Serotonin-mediated activation of RTN chemoreceptors was blocked by a 5-HT7-receptor blocker and mimicked by a 5-HT7-receptor agonist. In addition, serotonin caused a depolarizing shift in the voltage-dependent activation of Ih. These results suggest that HCN channels contribute to resting chemoreceptor activity and that serotonin activates RTN chemoreceptors and breathing in part by a 5-HT7 receptor-dependent mechanism and downstream activation of Ih. PMID:25429115

  12. Maintenance of genomic imprinting at the Arabidopsis medea locus requires zygotic DDM1 activity.

    PubMed

    Vielle-Calzada, J P; Thomas, J; Spillane, C; Coluccio, A; Hoeppner, M A; Grossniklaus, U

    1999-11-15

    In higher plants, seed development requires maternal gene activity in the haploid (gametophytic) as well as diploid (sporophytic) tissues of the developing ovule. The Arabidopsis thaliana gene MEDEA (MEA) encodes a SET-domain protein of the Polycomb group that regulates cell proliferation by exerting a gametophytic maternal control during seed development. Seeds derived from female gametocytes (embryo sacs) carrying a mutant mea allele abort and exhibit cell proliferation defects in both the embryo and the endosperm. In this study we show that the mea mutation affects an imprinted gene expressed maternally in cells of the female gametophyte and after fertilization only from maternally inherited MEA alleles. Paternally inherited MEA alleles are transcriptionally silent in both the young embryo and endosperm. Mutations at the decrease in DNA methylation1 (ddm1) locus are able to rescue mea seeds by functionally reactivating paternally inherited MEA alleles during seed development. Rescued seeds are larger than the wild type and exhibit some of the abnormalities found in aborting mea seeds. Our results indicate that the maintenance of the genomic imprint at the mea locus requires zygotic DDM1 activity. Because DDM1 encodes a putative chromatin remodeling factor, chromatin structure is likely to be interrelated with genomic imprinting in Arabidopsis.

  13. Role of SUMO activating enzyme in cancer stem cell maintenance and self-renewal

    PubMed Central

    Du, Li; Li, Yi-Jia; Fakih, Marwan; Wiatrek, Rebecca L.; Duldulao, Marjun; Chen, Zhenbin; Chu, Peiguo; Garcia-Aguilar, Julio; Chen, Yuan

    2016-01-01

    Cancer stem cells (CSCs) have key roles in treatment resistance, tumour metastasis and relapse. Using colorectal cancer (CC) cell lines, patient-derived xenograft (PDX) tissues and patient tissues, here we report that CC CSCs, which resist chemoradiation, have higher SUMO activating enzyme (E1) and global SUMOylation levels than non-CSCs. Knockdown of SUMO E1 or SUMO conjugating enzyme (E2) inhibits CC CSC maintenance and self-renewal, while overexpression of SUMO E1 or E2 increases CC cell stemness. We found that SUMOylation regulates CSCs through Oct-1, a transcription factor for aldehyde dehydrogenases (ALDHs). ALDH activity is not only a marker for CSCs but also important in CSC biology. SUMO does not modify Oct-1 directly, but regulates the expression of TRIM21 that enhances Oct-1 ubiquitination and, consequently, reducing Oct-1 stability. In summary, our findings suggest that SUMOylation could be a target to inhibit CSCs and ultimately to reduce treatment resistance, tumour metastasis and relapse. PMID:27465491

  14. Evolutionarily conserved mechanisms for the selection and maintenance of behavioural activity

    PubMed Central

    Fiore, Vincenzo G.; Dolan, Raymond J.; Strausfeld, Nicholas J.; Hirth, Frank

    2015-01-01

    Survival and reproduction entail the selection of adaptive behavioural repertoires. This selection manifests as phylogenetically acquired activities that depend on evolved nervous system circuitries. Lorenz and Tinbergen already postulated that heritable behaviours and their reliable performance are specified by genetically determined programs. Here we compare the functional anatomy of the insect central complex and vertebrate basal ganglia to illustrate their role in mediating selection and maintenance of adaptive behaviours. Comparative analyses reveal that central complex and basal ganglia circuitries share comparable lineage relationships within clusters of functionally integrated neurons. These clusters are specified by genetic mechanisms that link birth time and order to their neuronal identities and functions. Their subsequent connections and associated functions are characterized by similar mechanisms that implement dimensionality reduction and transition through attractor states, whereby spatially organized parallel-projecting loops integrate and convey sensorimotor representations that select and maintain behavioural activity. In both taxa, these neural systems are modulated by dopamine signalling that also mediates memory-like processes. The multiplicity of similarities between central complex and basal ganglia suggests evolutionarily conserved computational mechanisms for action selection. We speculate that these may have originated from ancestral ground pattern circuitries present in the brain of the last common ancestor of insects and vertebrates. PMID:26554043

  15. Using Visual Methods to Understand Physical Activity Maintenance following Cardiac Rehabilitation

    PubMed Central

    Hardcastle, Sarah J.

    2015-01-01

    Few studies have explored the factors associated with long-term maintenance of exercise following cardiac rehabilitation. The present study used auto-photography and interviews to explore the factors that influence motivation and continued participation in physical activity among post cardiac rehabilitation patients. Twenty-three semi-structured interviews were conducted alongside participant-selected photographs or drawings with participants that had continued participation in physical activity for at least two years following the cardiac rehabilitation programme. Participants were recruited from circuit training classes in East Sussex in the UK. Thematic content analysis revealed seven main themes: fear of death and ill health avoidance, critical incidents, overcoming aging, social influences, being able to enjoy life, provision of routine and structure, enjoyment and psychological well-being. Fear of death, illness avoidance, overcoming aging, and being able to enjoy life were powerful motives for continued participation in exercise. The social nature of the exercise class was also identified as a key facilitator of continued participation. Group-based exercise suited those that continued exercise participation post cardiac rehabilitation and fostered adherence. PMID:26381147

  16. Using Visual Methods to Understand Physical Activity Maintenance following Cardiac Rehabilitation.

    PubMed

    Hardcastle, Sarah J; McNamara, Keira; Tritton, Larette

    2015-01-01

    Few studies have explored the factors associated with long-term maintenance of exercise following cardiac rehabilitation. The present study used auto-photography and interviews to explore the factors that influence motivation and continued participation in physical activity among post cardiac rehabilitation patients. Twenty-three semi-structured interviews were conducted alongside participant-selected photographs or drawings with participants that had continued participation in physical activity for at least two years following the cardiac rehabilitation programme. Participants were recruited from circuit training classes in East Sussex in the UK. Thematic content analysis revealed seven main themes: fear of death and ill health avoidance, critical incidents, overcoming aging, social influences, being able to enjoy life, provision of routine and structure, enjoyment and psychological well-being. Fear of death, illness avoidance, overcoming aging, and being able to enjoy life were powerful motives for continued participation in exercise. The social nature of the exercise class was also identified as a key facilitator of continued participation. Group-based exercise suited those that continued exercise participation post cardiac rehabilitation and fostered adherence.

  17. Structure-activity studies on 1,4-dihydropyridine calcium channel antagonists and activators

    SciTech Connect

    Joslyn, A.F.

    1986-01-01

    Four series of 1,4-dihydropyridine Ca{sup 2+} channel antagonists related to mifedipine were synthesized by a modified Hantzsch procedure to determine the effects of ester (C{sub 3} = CO{sub 2}Me, C{sub 5} = CO{sub 2}R) and phenyl (C{sub 4}) substituents on pharmacological and radioligand binding ((H)nitrendipine) activities in guinea pig ileal longitudinal smooth muscle. Two series of Ca{sup 2+} channel activator 1,4-dihydropyridines, BAY K 8644 (C{sub 3} = NO{sub 2}, C{sub 5} = CO{sub 2}Me) and CGP 28392 (C{sub 2,3} = lactone, C{sub 5} = CO{sub 2}Me) were biochemically evaluated by inhibition of ({sup 3}H)nitrendipine binding in guinea pig ileal longitudinal smooth muscle membranes to establish fundamental structure-activity requirements. A homologous series of bis-1,4-dihydropyridines were synthesized, pharmacologically and biochemically evaluated in an attempt to explore the distribution of the 1,4-dihydropyridine receptor in guinea pig ileal longitudinal smooth muscle membranes. Several potential affinity labels including ester substituted 3- and 4-fluorosulfonyl benzoyl and isothiocyanate derivatives were synthesized and evaluated by inhibition of ({sup 3}H)nitrendipine binding.

  18. STIM1 carboxyl-terminus activates native SOC, I(crac) and TRPC1 channels.

    PubMed

    Huang, Guo N; Zeng, Weizhong; Kim, Joo Young; Yuan, Joseph P; Han, Linhuang; Muallem, Shmuel; Worley, Paul F

    2006-09-01

    Receptor-evoked Ca2+ signalling involves Ca2+ release from the endoplasmic reticulum, followed by Ca2+ influx across the plasma membrane. Ca2+ influx is essential for many cellular functions, from secretion to transcription, and is mediated by Ca2+-release activated Ca2+ (I(crac)) channels and store-operated calcium entry (SOC) channels. Although the molecular identity and regulation of I(crac) and SOC channels have not been precisely determined, notable recent findings are the identification of STIM1, which has been indicated to regulate SOC and I(crac) channels by functioning as an endoplasmic reticulum Ca2+ sensor, and ORAI1 (ref. 7) or CRACM1 (ref. 8)--both of which may function as I(crac) channels or as an I(crac) subunit. How STIM1 activates the Ca2+ influx channels and whether STIM1 contributes to the channel pore remains unknown. Here, we identify the structural features that are essential for STIM1-dependent activation of SOC and I(crac) channels, and demonstrate that they are identical to those involved in the binding and activation of TRPC1. Notably, the cytosolic carboxyl terminus of STIM1 is sufficient to activate SOC, I(crac) and TRPC1 channels even when native STIM1 is depleted by small interfering RNA. Activity of STIM1 requires an ERM domain, which mediates the selective binding of STIM1 to TRPC1, 2 and 4, but not to TRPC3, 6 or 7, and a cationic lysine-rich region, which is essential for gating of TRPC1. Deletion of either region in the constitutively active STIM1(D76A) yields dominant-negative mutants that block native SOC channels, expressed TRPC1 in HEK293 cells and I(crac) in Jurkat cells. These observations implicate STIM1 as a key regulator of activity rather than a channel component, and reveal similar regulation of SOC, I(crac) and TRPC channel activation by STIM1.

  19. Importance of the Maintenance Pathway in the Regulation of the Activity of Escherichia coli Ribonucleotide Reductase†

    PubMed Central

    2008-01-01

    Ribonucleotide reductases (RNRs) catalyze the conversion of nucleotides to deoxynucleotides in all organisms. The Escherichia coli class Ia RNR is composed of α and β subunits that form an α2β2 active complex. β contains the diferric tyrosyl radical (Y•) cofactor that is essential for the reduction process that occurs on α. [Y•] in vitro is proportional to RNR activity, and its regulation in vivo potentially represents a mechanism for controlling RNR activity. To examine this thesis, N- and C-terminal StrepII-tagged β under the control of an l-arabinose promoter were constructed. Using these constructs and with [l-arabinose] varying from 0 to 0.5 mM in the growth medium, [β] could be varied from 4 to 3300 µM. [Y•] in vivo and on affinity-purified Strep-β in vitro was determined by EPR spectroscopy and Western analysis. In both cases, there was 0.1–0.3 Y• radical per β. To determine if the substoichiometric Y• level was associated with apo β or diferric β, titrations of crude cell extracts from these growths were carried out with reduced YfaE, a 2Fe2S ferredoxin involved in cofactor maintenance and assembly. Each titration, followed by addition of O2 to assemble the cofactor and EPR analysis to quantitate Y•, revealed that β is completely loaded with a diferric cluster even when its concentration in vivo is 244 µM. These titrations, furthermore, resulted in 1 Y• radical per β, the highest levels reported. Whole cell Mössbauer analysis on cells induced with 0.5 mM arabinose supports high iron loading in β. These results suggest that modulation of the level of Y• in vivo in E. coli is a mechanism of regulating RNR activity. PMID:18314964

  20. Fast and slow activation of voltage-dependent ion channels in radish vacuoles.

    PubMed Central

    Gambale, F; Cantu, A M; Carpaneto, A; Keller, B U

    1993-01-01

    The molecular processes associated with voltage-dependent opening and closing (gating) of ion channels were investigated using a new preparation from plant cells, i.e., voltage and calcium-activated ion channels in radish root vacuoles. These channels display a main single channel conductance of approximately 90 pS and are characterized by long activation times lasting several hundreds of milliseconds. Here, we demonstrate that these channels have a second kinetically distinct activation mode which is characterized by even longer activation times. Different membrane potential protocols allowed to switch between the fast and the slow mode in a controlled and reversible manner. At transmembrane potentials of -100 mV, the ratio between the fast and slow activation time constant was around 1:5. Correspondingly, activation times lasting several seconds were observed in the slow mode. The molecular process controlling fast and slow activation may represent an effective modulator of voltage-dependent gating of ion channels in other plant and animal systems. PMID:7507716

  1. Upregulation of the large conductance voltage- and Ca2+-activated K+ channels by Janus kinase 2.

    PubMed

    Hosseinzadeh, Zohreh; Almilaji, Ahmad; Honisch, Sabina; Pakladok, Tatsiana; Liu, GuoXing; Bhavsar, Shefalee K; Ruth, Peter; Shumilina, Ekaterina; Lang, Florian

    2014-06-01

    The iberiotoxin-sensitive large conductance voltage- and Ca(2+)-activated potassium (BK) channels (maxi-K(+)-channels) hyperpolarize the cell membrane thus supporting Ca(2+) entry through Ca(2+)-release activated Ca(2+) channels. Janus kinase-2 (JAK2) has been identified as novel regulator of ion transport. To explore whether JAK2 participates in the regulation of BK channels, cRNA encoding Ca(2+)-insensitive BK channels (BK(M513I+Δ899-903)) was injected into Xenopus oocytes with or without cRNA encoding wild-type JAK2, gain-of-function (V617F)JAK2, or inactive (K882E)JAK2. K(+) conductance was determined by dual electrode voltage clamp and BK-channel protein abundance by confocal microscopy. In A204 alveolar rhabdomyosarcoma cells, iberiotoxin-sensitive K(+) current was determined utilizing whole cell patch clamp. A204 cells were further transfected with JAK2 and BK-channel transcript, and protein abundance was quantified by RT-PCR and Western blotting, respectively. As a result, the K(+) current in BK(M513I+Δ899-903)-expressing oocytes was significantly increased following coexpression of JAK2 or (V617F)JAK2 but not (K882E)JAK2. Coexpression of the BK channel with (V617F)JAK2 but not (K882E)JAK2 enhanced BK-channel protein abundance in the oocyte cell membrane. Exposure of BK-channel and (V617F)JAK2-expressing oocytes to the JAK2 inhibitor AG490 (40 μM) significantly decreased K(+) current. Inhibition of channel insertion by brefeldin A (5 μM) decreased the K(+) current to a similar extent in oocytes expressing the BK channel alone and in oocytes expressing the BK channel and (V617F)JAK2. The iberiotoxin (50 nM)-sensitive K(+) current in rhabdomyosarcoma cells was significantly decreased by AG490 pretreatment (40 μM, 12 h). Moreover, overexpression of JAK2 in A204 cells significantly enhanced BK channel mRNA and protein abundance. In conclusion, JAK2 upregulates BK channels by increasing channel protein abundance in the cell membrane.

  2. Crizotinib Inhibits Hyperpolarization-activated Cyclic Nucleotide-Gated Channel 4 Activity

    PubMed Central

    Zhang, Zhushan; Huang, Tai-Qin; Nepliouev, Igor; Zhang, Hengtao; Barnett, Adam S.; Rosenberg, Paul B.; Ou, Sai-Hong I.; Stiber, Jonathan A.

    2017-01-01

    Background Sinus bradycardia is frequently observed in patients treated with crizotinib, a receptor tyrosine kinase inhibitor used for the treatment of anaplastic lymphoma kinase (ALK)-rearranged non-small cell lung cancer (NSCLC). We investigated whether crizotinib could influence heart rate (HR) through direct cardiac effects. Methods The direct effect of crizotinib on HR was studied using ECG analysis of Langendorff-perfused mouse hearts. The whole-cell patch clamp technique was used to measure the effects of crizotinib on the hyperpolarization-activated funny current, If, in mouse sinoatrial node cells (SANCs) and hyperpolarization-activated cyclic nucleotide-gated channel 4 (HCN4) activity in HEK-293 cells stably expressing human HCN4. Results Crizotinib resulted in a dose-dependent reduction in HR in isolated intact mouse hearts with a half maximal inhibitory concentration (IC50) of 1.7 ± 0.4 μmol/L. Because ECG analysis revealed that crizotinib (0–5 μmol/L) resulted in significant reductions in HR in isolated mouse hearts without changes in PR, QRS, or QT intervals, we performed whole-cell patch clamp recordings of SANCs which showed that crizotinib inhibited If which regulates cardiac pacemaker activity. Crizotinib resulted in diminished current density of HCN4, the major molecular determinant of If, with an IC50 of 1.4 ± 0.3 μmol/L. Crizotinib also slowed HCN4 activation and shifted the activation curve to the left towards more hyperpolarized potentials. Conclusions Our results suggest that crizotinib’s effects on HCN4 channels play a significant role in mediating its observed effects on HR. PMID:28217366

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

    PubMed

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

    2016-07-01

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

  4. Differential distribution of the sodium‐activated potassium channels slick and slack in mouse brain

    PubMed Central

    Knaus, Hans‐Günther; Schwarzer, Christoph

    2015-01-01

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

  5. Fear conditioning suppresses large-conductance calcium-activated potassium channels in lateral amygdala neurons.

    PubMed

    Sun, P; Zhang, Q; Zhang, Y; Wang, F; Wang, L; Yamamoto, R; Sugai, T; Kato, N

    2015-01-01

    It was previously shown that depression-like behavior is accompanied with suppression of the large-conductance calcium activated potassium (BK) channel in cingulate cortex pyramidal cells. To test whether BK channels are also involved in fear conditioning, we studied neuronal properties of amygdala principal cells in fear conditioned mice. After behavior, we made brain slices containing the amygdala, the structure critically relevant to fear memory. The resting membrane potential in lateral amygdala (LA) neurons obtained from fear conditioned mice (FC group) was more depolarized than in neurons from naïve controls. The frequencies of spikes evoked by current injections were higher in neurons from FC mice, demonstrating that excitability of LA neurons was elevated by fear conditioning. The depolarization in neurons from FC mice was shown to depend on BK channels by using the BK channel blocker charybdotoxin. Suppression of BK channels in LA neurons from the FC group was further confirmed on the basis of the spike width, since BK channels affect the descending phase of spikes. Spikes were broader in the FC group than those in the naïve control in a manner dependent on BK channels. Consistently, quantitative real-time PCR revealed a decreased expression of BK channel mRNA. The present findings suggest that emotional disorder manifested in the forms of fear conditioning is accompanied with BK channel suppression in the amygdala, the brain structure critical to this emotional disorder.

  6. The membrane protein Pannexin1 forms two open channel conformations depending on the mode of activation

    PubMed Central

    Wang, Junjie; Ambrosi, Cinzia; Qiu, Feng; Jackson, David G.; Sosinsky, Gina; Dahl, Gerhard

    2014-01-01

    Pannexin1 (Panx1) participates in several signaling events that involve ATP release, including the innate immune response, ciliary beat in airway epithelia and oxygen supply in the vasculature. The view that Panx1 forms a large ATP-release channel has been challenged by the association of a low conductance, small anion-selective channel with the presence of Panx1. We showed that Panx1 membrane channels can function in two distinct modes with different conductances and permeabilities when heterologously expressed in Xenopus oocytes. When stimulated by potassium ions (K+), Panx1 formed a high conductance channel of ~500 pS that was permeable to ATP. Various physiological stimuli can induce this ATP-permeable conformation of the channel in several cell types. In contrast, the channel had a low conductance (~50 pS) with no detectable ATP permeability when activated by voltage in the absence of K+. The two channel states were associated with different reactivities of the terminal cysteine of Panx1 to thiol reagents, suggesting different conformations. Single particle electron microscopic analysis revealed that K+ stimulated the formation of channels with a larger pore diameter than those formed in the absence of K+. These data suggest that different stimuli lead to distinct channel structures with distinct biophysical properties. PMID:25056878

  7. Adenosine triphosphate regulates the activity of guinea pig Cav1.2 channel by direct binding to the channel in a dose-dependent manner.

    PubMed

    Feng, Rui; Xu, Jianjun; Minobe, Etsuko; Kameyama, Asako; Yang, Lei; Yu, Lifeng; Hao, Liying; Kameyama, Masaki

    2014-05-01

    The present study is to investigate the mechanism by which ATP regulates Cav1.2 channel activity. Ventricular tissue was obtained from adult guinea pig hearts using collagenase. Ca(2+) channel activity was monitored using the patch-clamp technique. Proteins were purified using wheat germ agglutinin-Sepharose, and the concentration was determined using the Coomassie brilliant blue technique. ATP binding to the Cav1.2 channel was examined using the photoaffinity method. EDA-ATP-biotin maintains Ca(2+) channel activity in inside-out membrane patches. ATP directly bound to the Cav1.2 channel in a dose-dependent manner, and at least two molecules of ATP bound to one molecule of the Cav1.2 channel. Low levels of calmodulin (CaM) increased ATP binding to the Cav1.2 channel, but higher levels of CaM decreased ATP binding to the Cav1.2 channel. In addition, Ca(2+) was another regulator for ATP binding to the Cav1.2 channel. Furthermore, ATP bound to GST-fusion peptides of NH2-terminal region (amino acids 6-140) and proximal COOH-terminal region (amino acids 1,509-1,789) of the main subunit (α1C) of the Cav1.2 channel. Our data suggest that ATP might regulate Cav1.2 channel activity by directly binding to the Cav1.2 channel in a dose-dependent manner. In addition, the ATP-binding effect to the Cav1.2 channel was both CaM- and Ca(2+) dependent.

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

    PubMed Central

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

    2007-01-01

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

  9. Members of the chloride intracellular ion channel protein family demonstrate glutaredoxin-like enzymatic activity.

    PubMed

    Al Khamici, Heba; Brown, Louise J; Hossain, Khondker R; Hudson, Amanda L; Sinclair-Burton, Alxcia A; Ng, Jane Phui Mun; Daniel, Elizabeth L; Hare, Joanna E; Cornell, Bruce A; Curmi, Paul M G; Davey, Mary W; Valenzuela, Stella M

    2015-01-01

    The Chloride Intracellular Ion Channel (CLIC) family consists of six evolutionarily conserved proteins in humans. Members of this family are unusual, existing as both monomeric soluble proteins and as integral membrane proteins where they function as chloride selective ion channels, however no function has previously been assigned to their soluble form. Structural studies have shown that in the soluble form, CLIC proteins adopt a glutathione S-transferase (GST) fold, however, they have an active site with a conserved glutaredoxin monothiol motif, similar to the omega class GSTs. We demonstrate that CLIC proteins have glutaredoxin-like glutathione-dependent oxidoreductase enzymatic activity. CLICs 1, 2 and 4 demonstrate typical glutaredoxin-like activity using 2-hydroxyethyl disulfide as a substrate. Mutagenesis experiments identify cysteine 24 as the catalytic cysteine residue in CLIC1, which is consistent with its structure. CLIC1 was shown to reduce sodium selenite and dehydroascorbate in a glutathione-dependent manner. Previous electrophysiological studies have shown that the drugs IAA-94 and A9C specifically block CLIC channel activity. These same compounds inhibit CLIC1 oxidoreductase activity. This work for the first time assigns a functional activity to the soluble form of the CLIC proteins. Our results demonstrate that the soluble form of the CLIC proteins has an enzymatic activity that is distinct from the channel activity of their integral membrane form. This CLIC enzymatic activity may be important for protecting the intracellular environment against oxidation. It is also likely that this enzymatic activity regulates the CLIC ion channel function.

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

    PubMed

    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

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

    PubMed Central

    Honnuraiah, Suraj; Narayanan, Rishikesh

    2013-01-01

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

  12. Antidiarrhoeal, antisecretory and antispasmodic activities of Matricaria chamomilla are mediated predominantly through K(+)-channels activation.

    PubMed

    Mehmood, Malik Hassan; Munir, Siraj; Khalid, Uzair Ali; Asrar, Mudassir; Gilani, Anwarul Hassan

    2015-03-24

    insensitive to GB or 4-AP. When assessed for Ca(++) antagonist like activity, Mc.Cr at high concentrations caused rightward shift in the Ca(++) concentration-response curves with suppression of the maximum response, similar to the effect of verapamil, while cromakalim did not show similar effect. This study indicates that Matricaria chamomilla possesses antidiarrhoeal, antisecretory and antispasmodic activities mediated predominantly through K(+)-channels activation along with weak Ca(++) antagonist effect.

  13. Aminopyridines potentiate synaptic and neuromuscular transmission by targeting the voltage-activated calcium channel beta subunit.

    PubMed

    Wu, Zi-Zhen; Li, De-Pei; Chen, Shao-Rui; Pan, Hui-Lin

    2009-12-25

    Aminopyridines such as 4-aminopyridine (4-AP) are widely used as voltage-activated K(+) (Kv) channel blockers and can improve neuromuscular function in patients with spinal cord injury, myasthenia gravis, or multiple sclerosis. Here, we present novel evidence that 4-AP and several of its analogs directly stimulate high voltage-activated Ca(2+) channels (HVACCs) in acutely dissociated neurons. 4-AP, 4-(aminomethyl)pyridine, 4-(methylamino)pyridine, and 4-di(methylamino)pyridine profoundly increased HVACC, but not T-type, currents in dissociated neurons from the rat dorsal root ganglion, superior cervical ganglion, and hippocampus. The widely used Kv channel blockers, including tetraethylammonium, alpha-dendrotoxin, phrixotoxin-2, and BDS-I, did not mimic or alter the effect of 4-AP on HVACCs. In HEK293 cells expressing various combinations of N-type (Cav2.2) channel subunits, 4-AP potentiated Ca(2+) currents primarily through the intracellular beta(3) subunit. In contrast, 4-AP had no effect on Cav3.2 channels expressed in HEK293 cells. Furthermore, blocking Kv channels did not mimic or change the potentiating effects of 4-AP on neurotransmitter release from sensory and motor nerve terminals. Thus, our findings challenge the conventional view that 4-AP facilitates synaptic and neuromuscular transmission by blocking Kv channels. Aminopyridines can directly target presynaptic HVACCs to potentiate neurotransmitter release independent of Kv channels.

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

    PubMed Central

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

    2015-01-01

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

  15. Physical basis of apparent pore-dilation of ATP-activated P2X receptor channels

    PubMed Central

    Li, Mufeng; Toombes, Gilman E S; Silberberg, Shai D; Swartz, Kenton J

    2016-01-01

    The selectivity of ion channels is fundamental for their roles in electrical and chemical signaling, and ion homeostasis. Although most ion channels exhibit stable ion selectivity, the prevailing view for purinergic P2X receptor channels, transient receptor potential V1 (TRPV1) channels and acid sensing ion channels (ASICs) is that their ion conduction pores dilate upon prolonged activation. We investigated this mechanism in P2X receptors and found that the hallmark shift in equilibrium potential observed with prolonged channel activation does not result from pore dilation, but from time-dependent alterations in the concentration of intracellular ions. We derived a physical model to calculate ion concentration changes during patch-clamp recordings, which validates our experimental findings and provides a quantitative guideline for effectively controlling ion concentration. Our results have fundamental implications for understanding ion permeation and gating in P2X receptor channels, and more broadly for using patch-clamp techniques to study ion channels and neuronal excitability. PMID:26389841

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

    PubMed

    Harvey, A L; Robertson, B

    2004-12-01

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

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

    PubMed Central

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

    2014-01-01

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

  18. A calcium-permeable stretch-activated cation channel in renal proximal tubule.

    PubMed

    Filipovic, D; Sackin, H

    1991-01-01

    Isolated Necturus proximal tubules were split to expose the apical membrane surface for patch clamping. When both pipette and bath solutions contained only Ca, N-methyl-D-glucamine, and methanesulfonate, inwardly directed Ca currents were observed through a stretch-activated (SA) cation channel with conductance of 18 +/- 1 pS (n = 19). The SA cation channel exhibited little discrimination among Na, K, and Ca but was at least nine times more selective for cations than anions. The channel was not significantly gated by either membrane potential or cytosolic Ca. However, application of 15 cmH2O suction to patch pipette significantly increased the mean number of open channels by a factor of 6.5, from 0.04 +/- 0.02 to 0.26 +/- 0.08 (n = 11). Ca currents through the SA cation channel were reversibly blocked by 10 microM gadolinium, which was applied to outside surface of excised patches. This is similar to gadolinium block of stretch-activated channels in Xenopus oocytes (X.-C. Yang and F. Sachs. Science Wash. DC 243: 1068-1071, 1989). A Ca-dependent, maxi-K channel (92 +/- 9 pS, n = 5) was also found at the apical membrane of the same proximal tubules. In some cases this maxi-K channel appeared to be indirectly activated by pipette suction, raising the possibility that Ca influx through the SA cation channel may regulate K efflux via the maxi-K channel. Such a process could mediate cell volume regulation and maintain electrolyte homeostasis during normal variations in Na-substrate cotransport.

  19. Notch pathway activation is essential for maintenance of stem-like cells in early tongue cancer

    PubMed Central

    Kaur, Ekjot; Aich, Jyotirmoi; Dani, Prachi; Sethunath, Vidyalakshmi; Gardi, Nilesh; Chandrani, Pratik; Godbole, Mukul; Sonawane, Kavita; Prasad, Ratnam; Kannan, Sadhana; Agarwal, Beamon; Kane, Shubhada; Gupta, Sudeep; Dutt, Shilpee; Dutt, Amit

    2016-01-01

    Background Notch pathway plays a complex role depending on cellular contexts: promotes stem cell maintenance or induces terminal differentiation in potential cancer-initiating cells; acts as an oncogene in lymphocytes and mammary tissue or plays a growth-suppressive role in leukemia, liver, skin, and head and neck cancer. Here, we present a novel clinical and functional significance of NOTCH1 alterations in early stage tongue squamous cell carcinoma (TSCC). Patients and Methods We analyzed the Notch signaling pathway in 68 early stage TSCC primary tumor samples by whole exome and transcriptome sequencing, real-time PCR based copy number, expression, immuno-histochemical, followed by cell based biochemical and functional assays. Results We show, unlike TCGA HNSCC data set, NOTCH1 harbors significantly lower frequency of inactivating mutations (4%); is somatically amplified; and, overexpressed in 31% and 37% of early stage TSCC patients, respectively. HNSCC cell lines over expressing NOTCH1, when plated in the absence of attachment, are enriched in stem cell markers and form spheroids. Furthermore, we show that inhibition of NOTCH activation by gamma secretase inhibitor or shRNA mediated knockdown of NOTCH1 inhibits spheroid forming capacity, transformation, survival and migration of the HNSCC cells suggesting an oncogenic role of NOTCH1 in TSCC. Clinically, Notch pathway activation is higher in tumors of non-smokers compared to smokers (50% Vs 18%, respectively, P=0.026) and is also associated with greater nodal positivity compared to its non-activation (93% Vs 64%, respectively, P=0.029). Conclusion We anticipate that these results could form the basis for therapeutic targeting of NOTCH1 in tongue cancer. PMID:27391340

  20. Supporting activity engagement by family carers at home: maintenance of agency and personhood in dementia

    PubMed Central

    Chung, Pat Yin Fan; Ellis-Hill, Caroline; Coleman, Peter

    2017-01-01

    ABSTRACT An explorative paper to describe how family carers, through the caregiving journey, reaffirm and promote the agency of people with dementia. Agency is an important concept in dementia care and is crucial to the promotion of wellbeing and the delivery of person-centred care. This article is based on one of the key findings of a study that explored family carers’ experiences of engaging their relatives in daily activities in domestic settings. Following research governance and ethical approval, 30 in-depth interviews (initial and follow-up) were carried out with 15 resident-carers of people with dementia who were recruited via local community mental health teams. Then five focus groups were conducted with 21 participants accessed through carers support groups. Interviews and focus groups were transcribed, coded and analysed using a grounded theory method. Findings showed the process in which family carers encouraged and sustained a sense of autonomy and control (agency) in their relative’s daily activities. Key strategies used by carers included: being non-judgemental; facilitating a sense of worth; taking calculated risks; maintaining the continuity of their relative’s identity; enhancing a sense of connection with their relative’s role and identity using enjoyable activities; preventing inactivity and attending to the bodily source of the agency. Lack of support for carers could ultimately pose a risk to the maintenance of the agency of people with dementia. This study provides a deeper insight into the process used by home carers to support the agency of people with dementia. This is essential if practitioners are to identify and develop more realistic intervention strategies and to work in effective partnership with family carers. The implications for the creation of dementia-friendly communities are discussed. PMID:28452609

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

    NASA Astrophysics Data System (ADS)

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

    2017-09-01

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

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

    PubMed

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

    2014-11-14

    SecA is an essential protein possessing ATPase activity in bacterial protein translocation for which Rose Bengal (RB) is the first reported sub-micromolar inhibitor in ATPase activity and protein translocation. Here, we examined the mechanisms of inhibition on various forms of SecA ATPase by conventional enzymatic assays, and by monitoring the SecA-dependent channel activity in the semi-physiological system in cells. We build on the previous observation that SecA with liposomes form active protein-conducting channels in the oocytes. Such ion channel activity is enhanced by purified Escherichia coli SecYEG-SecDF·YajC liposome complexes. Inhibition by RB could be monitored, providing correlation of in vitro activity and intact cell functionality. In this work, we found the intrinsic SecA ATPase is inhibited by RB competitively at low ATP concentration, and non-competitively at high ATP concentrations while the translocation ATPase with precursors and SecYEG is inhibited non-competitively by RB. The Inhibition by RB on SecA channel activity in the oocytes with exogenous ATP-Mg(2+), mimicking translocation ATPase activity, is also non-competitive. The non-competitive inhibition on channel activity has also been observed with SecA from other bacteria which otherwise would be difficult to examine without the cognate precursors and membranes. Copyright © 2014 Elsevier Inc. All rights reserved.

  3. Impact of a lead-safe training program on workers conducting renovation, painting, and maintenance activities.

    PubMed Central

    Buzzetti, Alan J.; Greene, Frank; Needham, Dottie

    2005-01-01

    OBJECTIVE: An important source of lead exposure is lead-based paint that is disturbed when unsafe work practices are used during renovation, remodeling, and maintenance activities. This study explores the success of a pilot lead-safe skills training program for home improvement contractors and their employees (including renovators, remodelers, and painters) and small property owners. METHODS: The study evaluates whether attendees at eight-hour lead-safe work practices training courses learned and retained information about lead exposure; developed and retained positive attitudes toward lead-safe work practices; and developed lasting, positive behavioral intentions to use lead-safe work practice skills and techniques. A questionnaire was administered immediately before, immediately following, and several months following the training program. Coded data from the questionnaires were analyzed using SPSS software. RESULTS: Respondents showed statistically significant changes from before to after the training program, and the changes were maintained over time. Knowledge improved, and attitudes and behavioral intentions changed in a favorable direction. CONCLUSION: These results suggest that lead-safe training can be successful and can create lasting changes in lead-safe knowledge, attitudes, and behaviors. PMID:15736328

  4. Impact of a lead-safe training program on workers conducting renovation, painting, and maintenance activities.

    PubMed

    Buzzetti, Alan J; Greene, Frank; Needham, Dottie

    2005-01-01

    An important source of lead exposure is lead-based paint that is disturbed when unsafe work practices are used during renovation, remodeling, and maintenance activities. This study explores the success of a pilot lead-safe skills training program for home improvement contractors and their employees (including renovators, remodelers, and painters) and small property owners. The study evaluates whether attendees at eight-hour lead-safe work practices training courses learned and retained information about lead exposure; developed and retained positive attitudes toward lead-safe work practices; and developed lasting, positive behavioral intentions to use lead-safe work practice skills and techniques. A questionnaire was administered immediately before, immediately following, and several months following the training program. Coded data from the questionnaires were analyzed using SPSS software. Respondents showed statistically significant changes from before to after the training program, and the changes were maintained over time. Knowledge improved, and attitudes and behavioral intentions changed in a favorable direction. These results suggest that lead-safe training can be successful and can create lasting changes in lead-safe knowledge, attitudes, and behaviors.

  5. NODULES WITH ACTIVATED DEFENSE 1 is required for maintenance of rhizobial endosymbiosis in Medicago truncatula.

    PubMed

    Wang, Chao; Yu, Haixiang; Luo, Li; Duan, Liujian; Cai, Liuyang; He, Xinxing; Wen, Jiangqi; Mysore, Kirankumar S; Li, Guoliang; Xiao, Aifang; Duanmu, Deqiang; Cao, Yangrong; Hong, Zonglie; Zhang, Zhongming

    2016-10-01

    The symbiotic interaction between legume plants and rhizobia results in the formation of root nodules, in which symbiotic plant cells host and harbor thousands of nitrogen-fixing rhizobia. Here, a Medicago truncatula nodules with activated defense 1 (nad1) mutant was identified using reverse genetics methods. The mutant phenotype was characterized using cell and molecular biology approaches. An RNA-sequencing technique was used to analyze the transcriptomic reprogramming of nad1 mutant nodules. In the nad1 mutant plants, rhizobial infection and propagation in infection threads are normal, whereas rhizobia and their symbiotic plant cells become necrotic immediately after rhizobia are released from infection threads into symbiotic cells of nodules. Defense-associated responses were detected in nad1 nodules. NAD1 is specifically present in root nodule symbiosis plants with the exception of Morus notabilis, and the transcript is highly induced in nodules. NAD1 encodes a small uncharacterized protein with two predicted transmembrane helices and is localized at the endoplasmic reticulum. Our data demonstrate a positive role for NAD1 in the maintenance of rhizobial endosymbiosis during nodulation.

  6. Effects of a DVD-Delivered Exercise Intervention on Maintenance of Physical Activity in Older Adults.

    PubMed

    Fanning, Jason; Awick, Elizabeth A; Wójcicki, Thomas R; Gothe, Neha; Roberts, Sarah; Ehlers, Diane K; Motl, Robert W; McAuley, Edward

    2016-06-01

    Previous research supports the efficacy of a 6-month DVD-delivered program for enhancing physical activity (PA) in older adults. In the current study, we examined the degree to which intervention-related increases in PA were maintained after a 6-month, no-contact follow-up. Follow-up assessments of PA via accelerometry and the Godin Leisure-Time Exercise Questionnaire (GLTEQ) were collected in a sample of older adults (N = 238). Repeated measures analyses of variance were conducted to examine changes in PA over the course of the follow-up period. For accelerometer measured PA, there was a significant time × treatment × age group interaction, F1,203 =11.319, P = .001, η2 = .053, such that younger (≤70 years) intervention participants maintained high levels of PA across the follow-up period, while PA in older intervention and young control participants declined significantly. Rates of PA in older control participants remained low over the course of the follow-up period. Analyses of GLTEQ scores revealed similar, though less significant patterns. DVD-based exercise programs may be effective for maintaining PA in younger members of the older adult population; however, there remains a need to develop better strategies for promoting PA maintenance in older individuals when using home-based designs.

  7. Method for including operation and maintenance costs in the economic analysis of active solar energy systems

    SciTech Connect

    Short, W.D.

    1986-08-01

    For a developing technology such as solar energy, the costs for operation and maintenance (O and M) can be substantial. In the past, most economic analyses included these costs by simply assuming that an annual cost will be incurred that is proportional to the initial cost of the system. However, in assessing the economics of new systems proposed for further research and development, such a simplification can obscure the issues. For example, when the typical method for including O and M costs in an economic analysis is used, the O and M costs associated with a newly developed, more reliable, and slightly more expensive controller will be assumed to increase - an obvious inconsistency. The method presented in this report replaces this simplistic approach with a representation of the O and M costs that explicitly accounts for the uncertainties and risks inherent in the operation of any equipment. A detailed description of the data inputs required by the method is included as well as a summary of data sources and an example of the method as applied to an active solar heating system.

  8. Mg2+ mediates interaction between the voltage sensor and cytosolic domain to activate BK channels.

    PubMed

    Yang, Huanghe; Hu, Lei; Shi, Jingyi; Delaloye, Kelli; Horrigan, Frank T; Cui, Jianmin

    2007-11-13

    The voltage-sensor domain (VSD) of voltage-dependent ion channels and enzymes is critical for cellular responses to membrane potential. The VSD can also be regulated by interaction with intracellular proteins and ligands, but how this occurs is poorly understood. Here, we show that the VSD of the BK-type K(+) channel is regulated by a state-dependent interaction with its own tethered cytosolic domain that depends on both intracellular Mg(2+) and the open state of the channel pore. Mg(2+) bound to the cytosolic RCK1 domain enhances VSD activation by electrostatic interaction with Arg-213 in transmembrane segment S4. Our results demonstrate that a cytosolic domain can come close enough to the VSD to regulate its activity electrostatically, thereby elucidating a mechanism of Mg(2+)-dependent activation in BK channels and suggesting a general pathway by which intracellular factors can modulate the function of voltage-dependent proteins.

  9. Transmission probability and active pumping ability of the channel with moving partition plane

    NASA Astrophysics Data System (ADS)

    Luo, X.; Day, Chr.

    2016-11-01

    In this paper we present Test Particle Monte Carlo (TPMC) simulations of the transmission probability and the active pumping ability of a rectangular channel of different physical parameters. The novel feature of this problem is that the channel has a partition plane in the middle which is moving up and down, which makes it to a dynamical system. The system was simulated by our TPMC code ProVac3D developed with a new subroutine considering the moving partition plane. In order to obtain precise simulation results, at least 1011 test particles were simulated in parallel on the supercomputer Helios in Japan. It is found that the amplitude and the frequency of the movement will affect both transmission probability and the active pumping ability of the channel. Moreover, it is also found that transmission probability and active pumping ability of the channel with the moving partition plane are no longer independent of the mass of the gas species.

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

    PubMed

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

    2016-02-19

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

  11. Permeation Mechanisms in the TMEM16B Calcium-Activated Chloride Channels.

    PubMed

    Pifferi, Simone

    2017-01-01

    TMEM16A and TMEM16B encode for Ca2+-activated Cl- channels (CaCC) and are expressed in many cell types and play a relevant role in many physiological processes. Here, I performed a site-directed mutagenesis study to understand the molecular mechanisms of ion permeation of TMEM16B. I mutated two positive charged residues R573 and K540, respectively located at the entrance and inside the putative channel pore and I measured the properties of wild-type and mutant TMEM16B channels expressed in HEK-293 cells using whole-cell and excised inside-out patch clamp experiments. I found evidence that R573 and K540 control the ion permeability of TMEM16B depending both on which side of the membrane the ion substitution occurs and on the level of channel activation. Moreover, these residues contribute to control blockage or activation by permeant anions. Finally, R573 mutation abolishes the anomalous mole fraction effect observed in the presence of a permeable anion and it alters the apparent Ca2+-sensitivity of the channel. These findings indicate that residues facing the putative channel pore are responsible both for controlling the ion selectivity and the gating of the channel, providing an initial understanding of molecular mechanism of ion permeation in TMEM16B.

  12. Permeation Mechanisms in the TMEM16B Calcium-Activated Chloride Channels

    PubMed Central

    2017-01-01

    TMEM16A and TMEM16B encode for Ca2+-activated Cl− channels (CaCC) and are expressed in many cell types and play a relevant role in many physiological processes. Here, I performed a site-directed mutagenesis study to understand the molecular mechanisms of ion permeation of TMEM16B. I mutated two positive charged residues R573 and K540, respectively located at the entrance and inside the putative channel pore and I measured the properties of wild-type and mutant TMEM16B channels expressed in HEK-293 cells using whole-cell and excised inside-out patch clamp experiments. I found evidence that R573 and K540 control the ion permeability of TMEM16B depending both on which side of the membrane the ion substitution occurs and on the level of channel activation. Moreover, these residues contribute to control blockage or activation by permeant anions. Finally, R573 mutation abolishes the anomalous mole fraction effect observed in the presence of a permeable anion and it alters the apparent Ca2+-sensitivity of the channel. These findings indicate that residues facing the putative channel pore are responsible both for controlling the ion selectivity and the gating of the channel, providing an initial understanding of molecular mechanism of ion permeation in TMEM16B. PMID:28046119

  13. Distance constraints on activation of TRPV4 channels by AKAP150-bound PKCα in arterial myocytes.

    PubMed

    Tajada, Sendoa; Moreno, Claudia M; O'Dwyer, Samantha; Woods, Sean; Sato, Daisuke; Navedo, Manuel F; Santana, L Fernando

    2017-06-05

    TRPV4 (transient receptor potential vanilloid 4) channels are Ca(2+)-permeable channels that play a key role in regulating vascular tone. In arterial myocytes, opening of TRPV4 channels creates local increases in Ca(2+) influx, detectable optically as "TRPV4 sparklets." TRPV4 sparklet activity can be enhanced by the action of the vasoconstrictor angiotensin II (AngII). This modulation depends on the activation of subcellular signaling domains that comprise protein kinase C α (PKCα) bound to the anchoring protein AKAP150. Here, we used super-resolution nanoscopy, patch-clamp electrophysiology, Ca(2+) imaging, and mathematical modeling approaches to test the hypothesis that AKAP150-dependent modulation of TRPV4 channels is critically dependent on the distance between these two proteins in the sarcolemma of arterial myocytes. Our data show that the distance between AKAP150 and TRPV4 channel clusters varies with sex and arterial bed. Consistent with our hypothesis, we further find that basal and AngII-induced TRPV4 channel activity decays exponentially as the distance between TRPV4 and AKAP150 increases. Our data suggest a maximum radius of action of ∼200 nm for local modulation of TRPV4 channels by AKAP150-associated PKCα. © 2017 Tajada et al.

  14. Calmodulin regulation of TMEM16A and 16B Ca2+-activated chloride channels

    PubMed Central

    Yang, Tingting; Colecraft, Henry M

    2016-01-01

    Ca2+-activated chloride channels encoded by TMEM16A and 16B are important for regulating epithelial mucus secretion, cardiac and neuronal excitability, smooth muscle contraction, olfactory transduction, and cell proliferation. Whether and how the ubiquitous Ca2+ sensor calmodulin (CaM) regulates the activity of TMEM16A and 16B channels has been controversial and the subject of an ongoing debate. Recently, using a bioengineering approach termed ChIMP (Channel Inactivation induced by Membrane-tethering of an associated Protein) we argued that Ca2+-free CaM (apoCaM) is pre-associated with functioning TMEM16A and 16B channel complexes in live cells. Further, the pre-associated apoCaM mediates Ca2+-dependent sensitization of activation (CDSA) and Ca2+-dependent inactivation (CDI) of some TMEM16A splice variants. In this review, we discuss these findings in the context of previous and recent results relating to Ca2+-dependent regulation of TMEM16A/16B channels and the putative role of CaM. We further discuss potential future directions for these nascent ideas on apoCaM regulation of TMEM16A/16B channels, noting that such future efforts will benefit greatly from the pioneering work of Dr. David T. Yue and colleagues on CaM regulation of voltage-dependent calcium channels. PMID:26083059

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

    PubMed Central

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

    2017-01-01

    Voltage-gated Ca2+ channels are involved in numerous physiological functions and various mechanisms finely tune their activity, including the Ca2+ ion itself. This is well exemplified by the Ca2+-dependent inactivation of L-type Ca2+ channels, whose alteration contributes to the dramatic disease Timothy Syndrome. For T-type Ca2+ channels, a long-held view is that they are not regulated by intracellular Ca2+. Here we challenge this notion by using dedicated electrophysiological protocols on both native and expressed T-type Ca2+ channels. We demonstrate that a rise in submembrane Ca2+ induces a large decrease in T-type current amplitude due to a hyperpolarizing shift in the steady-state inactivation. Activation of most representative Ca2+-permeable ionotropic receptors similarly regulate T-type current properties. Altogether, our data clearly establish that Ca2+ entry exerts a feedback control on T-type channel activity, by modulating the channel availability, a mechanism that critically links cellular properties of T-type Ca2+ channels to their physiological roles. DOI: http://dx.doi.org/10.7554/eLife.22331.001 PMID:28109159

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

    PubMed Central

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

    2016-01-01

    W1282X is a common nonsense mutation among cystic fibrosis patients that results in the production of a truncated Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) channel. Here we show that the channel activity of the W1282X-CFTR polypeptide is exceptionally low in excised membrane patches at normally saturating doses of ATP and PKA (single channel open probability (PO) < 0.01). However, W1282X-CFTR channels were stimulated by two CFTR modulators, the FDA-approved VX-770 and the dietary compound curcumin. Each of these compounds is an allosteric modulator of CFTR gating that promotes channel activity in the absence of the native ligand, ATP. Although W1282X-CFTR channels were stimulated by VX-770 in the absence of ATP their activities remained dependent on PKA phosphorylation. Thus, activated W1282X-CFTR channels should remain under physiologic control by cyclic nucleotide signaling pathways in vivo. VX-770 and curcumin exerted additive effects on W1282X-CFTR channel gating (opening/closing) in excised patches such that the Po of the truncated channel approached unity (> 0.9) when treated with both modulators. VX-770 and curcumin also additively stimulated W1282X-CFTR mediated currents in polarized FRT epithelial monolayers. In this setting, however, the stimulated W1282X-CFTR currents were smaller than those mediated by wild type CFTR (3–5%) due presumably to lower expression levels or cell surface targeting of the truncated protein. Combining allosteric modulators of different mechanistic classes is worth considering as a treatment option for W1282X CF patients perhaps when coupled with maneuvers to increase expression of the truncated protein. PMID:27007499

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

    PubMed

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

    2016-01-01

    W1282X is a common nonsense mutation among cystic fibrosis patients that results in the production of a truncated Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) channel. Here we show that the channel activity of the W1282X-CFTR polypeptide is exceptionally low in excised membrane patches at normally saturating doses of ATP and PKA (single channel open probability (PO) < 0.01). However, W1282X-CFTR channels were stimulated by two CFTR modulators, the FDA-approved VX-770 and the dietary compound curcumin. Each of these compounds is an allosteric modulator of CFTR gating that promotes channel activity in the absence of the native ligand, ATP. Although W1282X-CFTR channels were stimulated by VX-770 in the absence of ATP their activities remained dependent on PKA phosphorylation. Thus, activated W1282X-CFTR channels should remain under physiologic control by cyclic nucleotide signaling pathways in vivo. VX-770 and curcumin exerted additive effects on W1282X-CFTR channel gating (opening/closing) in excised patches such that the Po of the truncated channel approached unity (> 0.9) when treated with both modulators. VX-770 and curcumin also additively stimulated W1282X-CFTR mediated currents in polarized FRT epithelial monolayers. In this setting, however, the stimulated W1282X-CFTR currents were smaller than those mediated by wild type CFTR (3-5%) due presumably to lower expression levels or cell surface targeting of the truncated protein. Combining allosteric modulators of different mechanistic classes is worth considering as a treatment option for W1282X CF patients perhaps when coupled with maneuvers to increase expression of the truncated protein.

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

    PubMed Central

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

    2008-01-01

    SUMMARY The detection of osmotic stimuli is essential for all organisms, yet few osmoreceptive proteins are known, none of them in vertebrates. By employing a candidate-gene approach based on genes encoding members of the TRP superfamily of ion channels, we cloned cDNAs encoding the vanilloid receptor-related osmotically activated channel (VR-OAC) from the rat, mouse, human, and chicken. This novel cation-selective channel is gated by exposure to hypotonicity within the physiological range. In the central nevous system, the channel is expressed neurons of the circumventricular organs, neurosensory cells responsive to systemic osmotic pressure. The channel also occurs in other neurosensory cells, including inner-ear hair cells, sensory neurons, and Merkel cells. PMID:11081638

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

    PubMed

    Liedtke, W; Choe, Y; Martí-Renom, M A; Bell, A M; Denis, C S; Sali, A; Hudspeth, A J; Friedman, J M; Heller, S

    2000-10-27

    The detection of osmotic stimuli is essential for all organisms, yet few osmoreceptive proteins are known, none of them in vertebrates. By employing a candidate-gene approach based on genes encoding members of the TRP superfamily of ion channels, we cloned cDNAs encoding the vanilloid receptor-related osmotically activated channel (VR-OAC) from the rat, mouse, human, and chicken. This novel cation-selective channel is gated by exposure to hypotonicity within the physiological range. In the central nervous system, the channel is expressed in neurons of the circumventricular organs, neurosensory cells responsive to systemic osmotic pressure. The channel also occurs in other neurosensory cells, including inner-ear hair cells, sensory neurons, and Merkel cells.

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

    PubMed

    Gu, Yuanzheng; Barry, Joshua; Gu, Chen

    2013-05-15

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

  1. Gating of Recombinant Small-Conductance Ca-activated K+ Channels by Calcium

    PubMed Central

    Hirschberg, Birgit; Maylie, James; Adelman, John P.; Marrion, Neil V.

    1998-01-01

    Small-conductance Ca-activated K+ channels play an important role in modulating excitability in many cell types. These channels are activated by submicromolar concentrations of intracellular Ca2+, but little is known about the gating kinetics upon activation by Ca2+. In this study, single channel currents were recorded from Xenopus oocytes expressing the apamin-sensitive clone rSK2. Channel activity was detectable in 0.2 μM Ca2+ and was maximal above 2 μM Ca2+. Analysis of stationary currents revealed two open times and three closed times, with only the longest closed time being Ca dependent, decreasing with increasing Ca2+ concentrations. In addition, elevated Ca2+ concentrations resulted in a larger percentage of long openings and short closures. Membrane voltage did not have significant effects on either open or closed times. The open probability was ∼0.6 in 1 μM free Ca2+. A lower open probability of ∼0.05 in 1 μM Ca2+ was also observed, and channels switched spontaneously between behaviors. The occurrence of these switches and the amount of time channels spent displaying high open probability behavior was Ca2+ dependent. The two behaviors shared many features including the open times and the short and intermediate closed times, but the low open probability behavior was characterized by a different, long Ca2+-dependent closed time in the range of hundreds of milliseconds to seconds. Small-conductance Ca- activated K+ channel gating was modeled by a gating scheme consisting of four closed and two open states. This model yielded a close representation of the single channel data and predicted a macroscopic activation time course similar to that observed upon fast application of Ca2+ to excised inside-out patches. PMID:9524139

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

    PubMed

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

    2016-05-01

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

  3. PKA and phosphatases attached to the Ca(V)1.2 channel regulate channel activity in cell-free patches.

    PubMed

    Xu, Jianjun; Yu, Lifeng; Minobe, Etsuko; Lu, Liting; Lei, Ming; Kameyama, Masaki

    2016-01-15

    Calmodulin (CaM) + ATP can reprime voltage-gated L-type Ca(2+) channels (Ca(V)1.2) in inside-out patches for activation, but this effect decreases time dependently. This suggests that the Ca(V)1.2 channel activity is regulated by additional cytoplasmic factors. To test this hypothesis, we examined the role of cAMP-dependent protein kinase A (PKA) and protein phosphatases in the regulation of Ca(V)1.2 channel activity in the inside-out mode in guinea pig ventricular myocytes. Ca(V)1.2 channel activity quickly disappeared after the patch was excised from the cell and recovered to only 9% of that in the cell-attached mode on application of CaM + ATP at 10 min after the inside out. However, immediate exposure of the excised patch to the catalytic subunit of PKA + ATP or the nonspecific phosphatase inhibitor okadaic acid significantly increased the Ca(V)1.2 channel activity recovery by CaM + ATP (114 and 96%, respectively) at 10 min. Interestingly, incubation of the excised patches with cAMP + ATP also increased CaM/ATP-induced Ca(V)1.2 channel activity recovery (108%), and this effect was blocked by the nonspecific protein kinase inhibitor K252a. The channel activity in the inside-out mode was not maintained by either catalytic subunit of PKA or cAMP + ATP in the absence of CaM, but was stably maintained in the presence of CaM for more than 40 min. These results suggest that PKA and phosphatase(s) attached on or near the Ca(V)1.2 channel regulate the basal channel activity, presumably through modulation of the dynamic CaM interaction with the channel.

  4. Voltage dependence and pH regulation of human polycystin-2-mediated cation channel activity.

    PubMed

    Gonzalez-Perrett, Silvia; Batelli, Marisa; Kim, Keetae; Essafi, Makram; Timpanaro, Gustavo; Moltabetti, Nicolas; Reisin, Ignacio L; Arnaout, M Amin; Cantiello, Horacio F

    2002-07-12

    Polycystin-2, the product of the human PKD2 gene, whose mutations cause autosomal dominant polycystic kidney disease, is a large conductance, Ca(2+)-permeable non-selective cation channel. Polycystin-2 is functionally expressed in the apical membrane of the human syncytiotrophoblast, where it may play a role in the control of fetal electrolyte homeostasis. Little is known, however, about the mechanisms that regulate polycystin-2 channel function. In this study, the role of pH in the regulation of polycystin-2 was assessed by ion channel reconstitution of both apical membranes of human syncytiotrophoblast and the purified FLAG-tagged protein from in vitro transcribed/translated material. A kinetic analysis of single channel currents, including dwell time histograms, confirmed two open and two close states for spontaneous channel behavior and a strong voltage dependence of the open probability of the channel (P(o)). A reduction of cis pH (pH(cis)) decreased P(o) and shifted the voltage dependence of channel function but had no effect on the single channel conductance. An increase in pH(cis), in contrast, increased NP(o) (channel number times P(o)). Elimination of the H(+) chemical gradient did not reverse the low pH(cis) inhibition of polycystin-2. Similar findings confirmed the pH effect on the in vitro translated, FLAG-tagged purified polycystin-2. The data indicate the presence of an H(+) ion regulatory site in the channel protein, which is accessible from the cytoplasmic side of the protein. This protonation site controls polycystin-2 cation-selective channel activity.

  5. Calcium activated potassium channel expression during human iPS cell-derived neurogenesis.

    PubMed

    Linta, Leonhard; Boeckers, Tobias M; Kleger, Alexander; Liebau, Stefan

    2013-07-01

    The family of calcium activated potassium channels of low and intermediate conductance, known as SK channels, consists of four members (SK1-4). These channels are widely expressed throughout the organism and involved in various cellular processes, such as the afterhyperpolarization in excitable cells but also in differentiation processes of various tissues. To date, the role of SK channels in developmental processes has been merely a marginal focus of investigation, although it is well accepted that cell differentiation and maturation affect the expression patterns of certain ion channels. Recently, several studies from our laboratory delineated the influence of SK channel expression and their respective activity on cytoskeletal reorganization in neural and pluripotent stem cells and regulation of cell fate determination toward the cardiac lineage in human and mouse pluripotent stem cells. Herein, we have now analyzed SK channel expression patterns and distribution at various stages of human induced pluripotent stem cell-derived neurogenesis particularly focusing on undifferentiated iPS cells, neural progenitors and mature neurons. All family members could be detected starting at the iPS cell level and were differentially expressed during the subsequent maturation process. Intriguingly, we found obvious discrepancies between mRNA and protein expression pointing toward a complex regulatory mechanism. Inhibition of SK channels with either apamin or clotrimazol did not have any significant effects on the speed or amount of neurogenesis in vitro. The abundance and specific regulation of SK channel expression during iPS cell differentiation indicates distinct roles of these ion channels not only for the cardiac but also for neuronal cell differentiation and in vitro neurogenesis.

  6. Activation of the ATP-sensitive K+ channel by decavanadate in guinea-pig ventricular myocytes.

    PubMed

    Nakashima, H; Kakei, M; Tanaka, H

    1993-03-23

    To evaluate the effects of decavanadate on the ATP-sensitive K+ (KATP) channel, we applied the inside-out membrane patch-clamp technique to ventricular myocytes isolated from guinea-pig hearts. Decavanadate increased the probability of the KATP channel being open in a dose-dependent manner over the range of 0.1 to 5 mM in the presence of 0.3 mM ATP. Half-maximal activation occurred at 540 microM decavanadate and a Hill coefficient of 1.3 was obtained when the Hill equation was used to fit the dose-dependent activation for the channel by decavanadate. The half-maximum inhibition for the channel by ATP (K1/2) in the presence of 2 mM Mg2+ was 19 and 74 microM in its absence. In the presence of decavanadate, both curves shifted toward the higher concentration of ATP without a change in steepness of the slope (Hill coefficient = 2). The effect of decavanadate could be expressed by a model in which its binding prevents ATP binding from closing the channel. The estimated dissociation constant of decavanadate was 1.5 microM in the presence and 22.8 microM in the absence of Mg2+. Decavanadate reactivated the rundown channel in the absence of Mg2+ and ATP. Neither the single channel slope conductance nor the mean open and closed lifetime within the bursts of channel openings were affected by decavanadate. We conclude that internal Mg2+ is not required for the modulation produced by decavanadate, but this ion influences the channel and changes the dissociation constant of both ATP and decavanadate to the channel.

  7. Measuring reflection on participation in quality improvement activities for maintenance of certification.

    PubMed

    Wittich, Christopher M; Reed, Darcy A; Ting, Henry H; Berger, Richard A; Nowicki, Kelly M; Blachman, Morris J; Mandrekar, Jayawant N; Beckman, Thomas J

    2014-10-01

    To validate a measure of reflection on participation in quality improvement (QI) activities and to identify associations with characteristics of QI projects, participants, and teams. This was a prospective validation study of all Mayo Clinic team participants who submitted QI projects for maintenance of certification (MOC) credit from 2010 to 2012. The authors developed a measure of reflect