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. Evidence that Ca2+-release-activated Ca2+ channels in rat hepatocytes are required for the maintenance of hormone-induced Ca2+ oscillations.

    PubMed Central

    Gregory, Roland B; Barritt, Gregory J

    2003-01-01

    Store-operated Ca(2+) channels in liver cells have been shown previously to exhibit a high selectivity for Ca(2+) and to have properties indistinguishable from those of Ca(2+)-release-activated Ca(2+) (CRAC) channels in mast cells and lymphocytes [Rychkov, Brereton, Harland and Barritt (2001) Hepatology 33, 938-947]. The role of CRAC channels in the maintenance of hormone-induced oscillations in the cytoplasmic free Ca(2+) concentration ([Ca(2+)](cyt)) in isolated rat hepatocytes was investigated using several inhibitors of CRAC channels. 2-Aminoethyl diphenylborate (2-APB; 75 microM), Gd(3+) (1 microM) and 1-[beta-[3-(4-methoxyphenyl)propoxy]-4-methoxyphenethyl]-1H-imidazole hydrochloride (SK&F 96365; 50 microM) each inhibited vasopressin- and adrenaline (epinephrine)-induced Ca(2+) oscillations (measured using fura-2). The characteristics of this inhibition were similar to those of inhibition caused by decreasing the extracellular Ca(2+) concentration to zero by addition of EGTA. The effect of 2-APB was reversible. In contrast, LOE-908 [( R, S )-(3,4-dihydro-6,7-dimethoxy-isochinolin-1-yl)-2-phenyl- N, N -di[2-(2,3,4-trimethoxyphenyl)ethyl]acetamide mesylate] (30 microM), used commonly to block Ca(2+) inflow through intracellular-messenger-activated, non-selective cation channels, did not inhibit the Ca(2+) oscillations. In the absence of added extracellular Ca(2+), 2-APB, Gd(3+) and SK&F 96365 did not alter the kinetics of the increase in [Ca(2+)](cyt) induced by a concentration of adrenaline or vasopressin that induces continuous Ca(2+) oscillations at the physiological extracellular Ca(2+) concentration. Ca(2+) inflow through non-selective cation channels activated by maitotoxin could not restore Ca(2+) oscillations in cells treated with 2-APB to block Ca(2+) inflow through CRAC channels. Evidence for the specificity of the pharmacological agents for inhibition of CRAC channels under the conditions of the present experiments with hepatocytes is discussed. It

  3. Proposed SOLCOST maintenance activities

    SciTech Connect

    1980-01-01

    This document provides a short description of work that has been accomplished to date and work in progress. A discussion of the program status as it is currently configured follows and finally proposed work by Solar Environmental Engineering Company (SEEC) in its most recently signed contract with the Department of Energy (DOE) is given. Early statements are designed to give the reader a good background so that the suggested SOLCOST maintenance activities will be more easily understood.

  4. 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.

  5. 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

  6. 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.

  7. 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

  8. 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.

  9. 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....

  10. 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....

  11. 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....

  12. 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....

  13. 7 CFR 1470.23 - Conservation activity operation and maintenance.

    Code of Federal Regulations, 2010 CFR

    2010-01-01

    ... STEWARDSHIP PROGRAM Contracts and Payments § 1470.23 Conservation activity operation and maintenance. The... stewardship contract period and additional activities installed and adopted over the term of the conservation stewardship contract....

  14. 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...

  15. 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.

  16. 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.

  17. BK channels: multiple sensors, one activation gate

    PubMed Central

    Yang, Huanghe; Zhang, Guohui; Cui, Jianmin

    2015-01-01

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

  18. TRPC channel activation by extracellular thioredoxin

    PubMed Central

    Xu, Shang-Zhong; Sukumar, Piruthivi; Zeng, Fanning; Li, Jing; Jairaman, Amit; English, Anne; Naylor, Jacqueline; Ciurtin, Coziana; Majeed, Yasser; Milligan, Carol J; Bahnasi, Yahya M; AL-Shawaf, Eman; Porter, Karen E; Jiang, Lin-Hua; Emery, Paul; Sivaprasadarao, Asipu; Beech, David J

    2009-01-01

    Mammalian homologues of Drosophila melanogaster transient receptor potential (TRP) are a large family of multimeric cation channels that act, or putatively act, as sensors of one or more chemical factor1,2. Major research objectives are the identification of endogenous activators and the determination of cellular and tissue functions of these novel channels. Here we show activation of TRPC5 homomultimeric and TRPC5-TRPC1 heteromultimeric channels3-5 by extracellular reduced thioredoxin acting by breaking a disulphide bridge in the predicted extracellular loop adjacent to the ion-selectivity filter of TRPC5. Thioredoxin is an endogenous redox protein with established intracellular functions, but it is also secreted and its extracellular targets are largely unknown6-9. Particularly high extracellular concentrations of thioredoxin are apparent in rheumatoid arthritis8,10-12, an inflammatory joint disease disabling millions of people world-wide13. We show that TRPC5 and TRPC1 are expressed in secretory fibroblast-like synoviocytes from patients with rheumatoid arthritis, endogenous TRPC5-TRPC1 channels of the cells are activated by reduced thioredoxin, and blockade of the channels enhances secretory activity and prevents suppression of secretion by thioredoxin. The data suggest a novel ion channel activation mechanism that couples extracellular thioredoxin to cell function. PMID:18172497

  19. Active channel for Fanno Creek, Oregon

    USGS Publications Warehouse

    Sobieszczyk, Steven

    2011-01-01

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

  20. 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.

  1. Channeling Children's Energy through Vocabulary Activities

    ERIC Educational Resources Information Center

    Schindler, Andrea

    2006-01-01

    In this article, the author shares vocabulary development activities for young learners. These activities channel students' energy and make learning more effective and fun. The author stresses the importance of giving young learners a good language-learning experience, and the challenges of teaching young learners who are not literate in their L1.…

  2. Language Development Activities through the Auditory Channel.

    ERIC Educational Resources Information Center

    Fitzmaurice, Peggy, Comp.; And Others

    Presented primarily for use with educable mentally retarded and learning disabled children are approximately 100 activities for language development through the auditory channel. Activities are grouped under the following three areas: receptive skills (auditory decoding, auditory memory, and auditory discrimination); expressive skills (auditory…

  3. 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.

  4. Determinants of uptake and maintenance of active commuting to school.

    PubMed

    Murtagh, Elaine M; Dempster, Martin; Murphy, Marie H

    2016-07-01

    The objective was to identify determinants of uptake and maintenance of active school travel (AST) over 4 years in children aged 9 at baseline. Data from wave 1 (n=8502) and 2 (n=7479) of the Growing Up in Ireland study were analysed. At 9- and 13-years 25% and 20% engaged in AST. Children were more likely to maintain or take-up AST if they lived in an urban area. Change in distance to school influenced both maintenance and adoption of AST, with a negative impact seen for increased distance between 9 and 13 years and a positive impact seen for decreased distance. Some factors which predict uptake and maintenance of AST are modifiable and can inform intervention development.

  5. Targeting the Channel Activity of Viroporins.

    PubMed

    To, Janet; Surya, Wahyu; Torres, Jaume

    2016-01-01

    Since the discovery that certain small viral membrane proteins, collectively termed as viroporins, can permeabilize host cellular membranes and also behave as ion channels, attempts have been made to link this feature to specific biological roles. In parallel, most viroporins identified so far are virulence factors, and interest has focused toward the discovery of channel inhibitors that would have a therapeutic effect, or be used as research tools to understand the biological roles of viroporin ion channel activity. However, this paradigm is being shifted by the difficulties inherent to small viral membrane proteins, and by the realization that protein-protein interactions and other diverse roles in the virus life cycle may represent an equal, if not, more important target. Therefore, although targeting the channel activity of viroporins can probably be therapeutically useful in some cases, the focus may shift to their other functions in following years. Small-molecule inhibitors have been mostly developed against the influenza A M2 (IAV M2 or AM2). This is not surprising since AM2 is the best characterized viroporin to date, with a well-established biological role in viral pathogenesis combined the most extensive structural investigations conducted, and has emerged as a validated drug target. For other viroporins, these studies are still mostly in their infancy, and together with those for AM2, are the subject of the present review.

  6. Synthetic modulators of TRP channel activity.

    PubMed

    Harteneck, Christian; Klose, Chihab; Krautwurst, Dietmar

    2011-01-01

    In humans, 27 TRP channels from 6 related families contribute to a broad spectrum of cellular functions, such as thermo-, pressure-, volume-, pain- and chemosensation. Pain and inflammation-inducing compounds represent potent plant and animal defense mechanisms explaining the great variety of the naturally occurring, TRPV1-, TRPM8-, and TRPA1-activating ligands. The discovery of the first vanilloid receptor (TRPV1) and its involvement in nociception triggered the euphoria and the hope in novel therapeutic strategies treating pain, and this clear-cut indication inspired the development of TRPV1-selective ligands. On the other hand the nescience in the physiological role and putative clinical indication hampered the development of a selective drug in the case of the other TRP channels. Therefore, currently only a handful of mostly un-selective blocker is available to target TRP channels. Nevertheless, there is an ongoing quest for new, natural or synthetic ligands and modulators. In this chapter, we will give an overview on available broad-range blocker, as well as first TRP channel-selective compounds. PMID:21290290

  7. Epithelial sodium channel modulates platelet collagen activation.

    PubMed

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

    2014-03-01

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

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

    PubMed

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

    2016-01-01

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

  9. 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

  10. 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

  11. Regulated flushing in a gravel-bed river for channel habitat maintenance: A Trinity River fisheries case study

    NASA Astrophysics Data System (ADS)

    Nelson, R. Wayne; Dwyer, John R.; Greenberg, Wendy E.

    1987-08-01

    The operation of Trinity and Lewiston Dams on the Trinity River in northern California in the United States, combined with severe watershed erosion, has jeopardized the existence of prime salmonid fisheries. Extreme streamflow depletion and stream sedimentation below Lewiston have resulted in heavy accumulation of coarse sediment on riffle gravel and filling of streambed pools, causing the destruction of spawning, nursery, and overwintering habitat for prized chinook salmon ( Salmo gairdnerii) and steelhead trout ( Oncorhynchus tschawytscha). Proposals to restore and maintain the degraded habitat include controlled one-time remedial peak flows or annual maintenance peak flows designed to flush the spawning gravel and scour the banks, deltas, and pools. The criteria for effective channel restoration or maintenance by streambed flushing and scouring are examined here, as well as the mechanics involved. The liabilities of releasing mammoth scouring-flushing flows approximating the magnitude that preceded reservoir construction make this option unviable. The resulting damage to fish habitat established under the postproject streamflow regime, as well as damage to human settlements in the floodplain, would be unacceptable, as would the opportunity costs to hydroelectric and irrigation water users. The technical feasibility of annual maintenance flushing flows depends upon associated mechanical and structural measures, particularly instream maintenance dredging of deep pools and construction of a sediment control dam on a tributary where watershed erosion is extreme. The cost effectiveness of a sediment dam with a limited useful economic life, combined with perpetual maintenance dredging, is questionable.

  12. 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 frames. (EA)

  13. 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…

  14. 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…

  15. 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(+).

  16. Ion channels activated by light in Limulus ventral photoreceptors

    PubMed Central

    1986-01-01

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

  17. 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...

  18. 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...

  19. 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.

  20. Active versus passive maintenance of visual nonverbal memory.

    PubMed

    McKeown, Denis; Holt, Jessica; Delvenne, Jean-Francois; Smith, Amy; Griffiths, Benjamin

    2014-08-01

    Forgetting over the short term has challenged researchers for more than a century, largely because of the difficulty of controlling what goes on within the memory retention interval. But the "recent-negative-probe" procedure offers a valuable paradigm, by examining the influences of (presumably) unattended memoranda from prior trials. Here we used a recent-probe task to investigate forgetting for visual nonverbal short-term memory. The target stimuli (two visually presented abstract shapes) on a trial were followed after a retention interval by a probe, and participants indicated whether the probe matched one of the target items. Proactive interference, and hence memory for old trial probes, was observed, whereby participants were slowed in rejecting a nonmatching probe on the current trial that nevertheless matched a target item on the previous trial (a recent-negative probe). The attraction of the paradigm is that, by uncovering proactive influences of past-trial probe stimuli, it can be argued that active maintenance in memory of those probes is unlikely. In two experiments, we recorded such proactive interference of prior-trial items over a range of interstimulus (ISI) and intertrial (ITI) intervals (between 1 and 6 s, respectively). Consistent with a proposed two-process memory conception (the active-passive memory model, or APM), actively maintained memories on current trials decayed, but passively "maintained," or unattended, visual memories of stimuli on past trials did not. PMID:24390797

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

    PubMed Central

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

    2008-01-01

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

  2. 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.

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

    PubMed

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

    2016-07-01

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

  4. Mechanism of activation of the prokaryotic channel ELIC by propylamine: a single-channel study.

    PubMed

    Marabelli, Alessandro; Lape, Remigijus; Sivilotti, Lucia

    2015-01-01

    Prokaryotic channels, such as Erwinia chrysanthemi ligand-gated ion channel (ELIC) and Gloeobacter violaceus ligand-gated ion channel, give key structural information for the pentameric ligand-gated ion channel family, which includes nicotinic acetylcholine receptors. ELIC, a cationic channel from E. chrysanthemi, is particularly suitable for single-channel recording because of its high conductance. Here, we report on the kinetic properties of ELIC channels expressed in human embryonic kidney 293 cells. Single-channel currents elicited by the full agonist propylamine (0.5-50 mM) in outside-out patches at -60 mV were analyzed by direct maximum likelihood fitting of kinetic schemes to the idealized data. Several mechanisms were tested, and their adequacy was judged by comparing the predictions of the best fit obtained with the observable features of the experimental data. These included open-/shut-time distributions and the time course of macroscopic propylamine-activated currents elicited by fast theta-tube applications (50-600 ms, 1-50 mM, -100 mV). Related eukaryotic channels, such as glycine and nicotinic receptors, when fully liganded open with high efficacy to a single open state, reached via a preopening intermediate. The simplest adequate description of their activation, the "Flip" model, assumes a concerted transition to a single intermediate state at high agonist concentration. In contrast, ELIC open-time distributions at saturating propylamine showed multiple components. Thus, more than one open state must be accessible to the fully liganded channel. The "Primed" model allows opening from multiple fully liganded intermediates. The best fits of this type of model showed that ELIC maximum open probability (99%) is reached when at least two and probably three molecules of agonist have bound to the channel. The overall efficacy with which the fully liganded channel opens was ∼ 102 (∼ 20 for α1β glycine channels). The microscopic affinity for the agonist

  5. Serine protease activation of near-silent epithelial Na+ channels.

    PubMed

    Caldwell, Ray A; Boucher, Richard C; Stutts, M Jackson

    2004-01-01

    The regulation of epithelial Na+ channel (ENaC) function is critical for normal salt and water balance. This regulation is achieved through cell surface insertion/retrieval of channels, by changes in channel open probability (Po), or through a combination of these processes. Epithelium-derived serine proteases, including channel activating protease (CAP) and prostasin, regulate epithelial Na+ transport, but the molecular mechanism is unknown. We tested the hypothesis that extracellular serine proteases activate a near-silent ENaC population resident in the plasma membrane. Single-channel events were recorded in outside-out patches from fibroblasts (NIH/3T3) stably expressing rat alpha-, beta-, and gamma-subunits (rENaC), before and during exposure to trypsin, a serine protease homologous to CAP and prostasin. Under baseline conditions, near-silent patches were defined as having rENaC activity (NPo) < 0.03, where N is the number of channels. Within 1-5 min of 3 microg/ml bath trypsin superfusion, NPo increased approximately 66-fold (n = 7). In patches observed to contain a single functional channel, trypsin increased Po from 0.02 +/- 0.01 to 0.57 +/- 0.03 (n = 3, mean +/- SE), resulting from the combination of an increased channel open time and decreased channel closed time. Catalytic activity was required for activation of near-silent ENaC. Channel conductance and the Na+/Li+ current ratio with trypsin were similar to control values. Modulation of ENaC Po by endogenous epithelial serine proteases is a potentially important regulator of epithelial Na+ transport, distinct from the regulation achieved by hormone-induced plasma membrane insertion of channels. PMID:12967915

  6. 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

  7. 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.

  8. 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

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

    PubMed

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

    2016-06-01

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

  10. 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

  11. 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

  12. 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.

  13. Indirect activation of the epithelial Na+ channel by trypsin.

    PubMed

    Bengrine, Abderrahmane; Li, Jinqing; Hamm, L Lee; Awayda, Mouhamed S

    2007-09-14

    We tested the hypothesis that the serine protease trypsin can indirectly activate the epithelial Na(+) channel (ENaC). Experiments were carried out in Xenopus oocytes and examined the effects on the channel formed by all three human ENaC subunits and that formed by Xenopus epsilon and human beta and gamma subunits (epsilonbetagammaENaC). Low levels of trypsin (1-10 ng/ml) were without effects on the oocyte endogenous conductances and were specifically used to test the effects on ENaC. Addition of 1 ng/ml trypsin for 60 min stimulated the amiloride-sensitive human ENaC conductance (g(Na)) by approximately 6-fold. This effect on the g(Na) was [Na(+)]-independent, thereby ruling out an interaction with channel feedback inhibition by Na(+). The indirect nature of this activation was confirmed in cell-attached patch clamp experiments with trypsin added to the outside of the pipette. Trypsin was comparatively ineffective at activating epsilonbetagammaENaC, a channel that exhibited a high spontaneous open probability. These observations, in combination with surface binding experiments, indicated that trypsin indirectly activated membrane-resident channels. Activation by trypsin was also dependent on catalytic activity of this protease but was not accompanied by channel subunit proteolysis. Channel activation was dependent on downstream activation of G-proteins and was blocked by G-protein inhibition by injection of guanyl-5'-yl thiophosphate and by pre-stimulation of phospholipase C. These data indicate a receptor-mediated activation of ENaC by trypsin. This trypsin-activated receptor is distinct from that of protease-activated receptor-2, because the response to trypsin was unaffected by protease-activated receptor-2 overexpression or knockdown. PMID:17627947

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

    PubMed

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

    1999-03-01

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

  15. 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.

  16. 76 FR 50489 - Agency Information Collection Activities: Harbor Maintenance Fee

    Federal Register 2010, 2011, 2012, 2013, 2014

    2011-08-15

    ... previously published in the Federal Register (76 FR 26311) on May 6, 2011, allowing for a 60-day comment... Fee AGENCY: U.S. Customs and Border Protection, Department of Homeland Security. ACTION: 30-Day Notice... approval in accordance with the Paperwork Reduction Act: Harbor Maintenance Fee (CBP Forms 349 and...

  17. 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

  18. 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.

  19. The role of Na(V)1.8 sodium channel in the maintenance of chronic inflammatory hypernociception.

    PubMed

    Villarreal, Cristiane Flora; Sachs, Daniela; Cunha, Fernando de Queiroz; Parada, Carlos Amílcar; Ferreira, Sérgio Henrique

    2005-09-30

    We previously described an animal model of persistent inflammatory sensitization of nociceptors. In this model the hypernociception persists for more than 30 days after the cessation of 2 weeks of daily intraplantar treatment with prostaglandin E(2) (PGE(2)). The tetrodotoxin-resistant (TTX-R) voltage-gated sodium channel Na(V)1.8 is considered a characteristic of primary afferent nociceptive C fibers and plays an important role in acute hypernociception. In the present study, the relevance of the Na(V)1.8 channel was investigated in this model of persistent mechanical hypernociception in rats. In the PGE(2)-induced persistent hypernociception, but not in the single injection-induced acute hypernociception, the mRNA expression (RT-PCR) of Na(V)1.8 in dorsal root ganglia (DRG) was up-regulated. A similar increase of Na(V)1.8 mRNA was observed when DbcAMP was used to induce persistent hypernociception. Four daily intrathecal administrations of oligodeoxynucleotides (ODN) antisense against Na(V)1.8 decreased the mRNA encoding Na(V)1.8 in DRG. The intrathecal administration of ODN antisense prevented the PGE(2)-induced acute hypernociception and significantly reduced ongoing PGE(2)-induced persistent hypernociception. A parallel restoration of the persistent hypernociception and up-regulation of Na(V)1.8 mRNA was observed after the cessation of ODN antisense treatment. These results suggest the participation of Na(V)1.8 channels in the development and maintenance of chronic inflammatory hyperalgesia, and confirm their involvement in the acute inflammatory hypernociception. PMID:16043287

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

    PubMed

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

    2009-11-01

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

  1. 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.

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

    PubMed

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

    2002-08-01

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

  4. 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.

  5. 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

  6. Single Channel Activity from Ion Channels in Engineered Tethered Bilayer Membrane Arrays

    NASA Astrophysics Data System (ADS)

    Keizer, Henk; Fine, Daniel; K"{O}Per, Ingo; Anderson, Peter

    2005-11-01

    The demand for rapid in situ detection of chemical and biological analytes at high sensitivity has increased interest in the development of biosensors like the commercially available compact glucose sensor. Engineered membrane bound ion channels are promising biological receptors since they would allow for the stochastic detection of analytes at high sensitivity, they can be mutated to alter sensitivity, and they produce a well-defined read-out that is inherently suitable for digitization. In order to perform stochastic sensing it is necessary to be able to measure the ion currents associated with single ion channel opening and closing events. Although sensors based on supported bilayers containing various pore forming proteins have been described, none of these systems have recorded single channel activity. Here we describe the measurement of stochastic activity from synthetic single ion channels, based on the nicotinic acetylcholine receptor (nAChR) from Torpedo californica, inserted into individual pixels of a microelectrode array device. The limited size of the gold sense pad surface, 100x100 μm, and the electrical stability of the overlying lipid bilayer membrane make each pixel sensitive enough to measure single ion channel currents in the picoampere range.

  7. Occurrence of Postictal α/β Activity During Maintenance Course of Electroconvulsive Therapy.

    PubMed

    Schultz, Krista; Tham, Joseph; Chan, Peter; Brenner, Colleen; Northcott, Colleen; Vila-Rodriguez, Fidel

    2015-12-01

    We are aware of a report of postictal α activity after seizures in Epilepsy and Behavior. We would like to report another case of a similar phenomenon in a woman receiving maintenance electroconvulsive therapy. PMID:25764162

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

    PubMed

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

    2008-08-01

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

  9. 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

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

    PubMed

    Bansal, V; Fisher, T E

    2016-06-01

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

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

    PubMed

    Kobrinsky, Evgeny

    2015-01-01

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

  12. Reduction of K+ Uptake in Glia Prevents Long-Term Depression Maintenance and Causes Epileptiform Activity

    PubMed Central

    Janigro, Damir; Gasparini, Sonia; D'Ambrosio, Raimondo; II, Guy McKhann; DiFrancesco, Dario

    2014-01-01

    Extracellular cesium causes synchronous, interictal-like bursting and prevents maintenance of long-term depression (LTD) in the CA1 hippocampal region. We have investigated the cellular mechanisms underlying cesium actions. Whole-cell recordings showed that brief (2 min) bath exposures to cesium caused pyramidal cell hyperpolarization associated with decreased membrane conductance attributable to blockade of an inward h-type current. After prolonged (>2 min) exposures, a late depolarizing response was observed; this effect was not associated with changes in cell membrane conductance. Recordings from interneurons revealed that Ih is expressed in a sub-population of cells and that cesium effects on interneurons expressing Ih are comparable to those observed in pyramidal cells. Consistent with this effect, cesium decreased the early component of the IPSP recorded in pyramidal cells. Interneurons lacking Ih were not affected by cesium but developed a depolarizing response when drug applications were paired to orthodromic stimulation. We concluded that cesium actions on LTD and cesium-induced epileptiform activity were not attributable exclusively to its direct effects on neurons. Recordings from hippocampal slice astrocytes revealed that cesium interfered with glial electrical responses during LTD induction. Cesium blocked glial inwardly rectifying potassium channels and increased the amplitude and duration of stimulation-evoked [K+]out increases. Thus, the effects of cesium on CA1 synchronization and synaptic plasticity appear to be mediated predominantly by blockade of glial voltage-dependent potassium uptake. PMID:9092603

  13. 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).

  14. Active Brownian particles escaping a channel in single file

    NASA Astrophysics Data System (ADS)

    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).

  15. 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.

  16. 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.

  17. River Elongation as a Proxy for Lateral Channel Activity

    NASA Astrophysics Data System (ADS)

    Lauer, J. W.

    2009-12-01

    Lateral channel movement is a process that is tightly linked to both channel hydraulics and sediment transport, strongly influences floodplain ecology, and also has great relevance for banktop property owners. The correlation between channel migration rate and channel curvature usually causes meandering river channels to elongate as they migrate laterally. Over the long term, the increase in sinuosity is compensated by a rapid decrease in sinuosity where and when river bends shorten through cutoff processes. However, the elongation for most meander bends in systems free to migrate across wide floodplains often occurs relatively uniformly throughout the system. Consequently, the rate of elongation of individual river bends, integrated across a river reach, offers a simple mechanism for characterizing the reach’s lateral activity. Spatial series of accumulated elongation can also be used to delineate reaches with similar properties. We use aerial imagery pairs to compare rates of lateral channel centerline shifting with channel centerline elongation for reaches many bends long along eight different rivers with widths ranging from 12 to 584 m. Except where bends translate downstream without changing form, elongation rates are closely linked to lateral shifting. In several cases, a change in elongation rate corresponds closely with a change in channel width, discharge, and/or bed material. For reaches free to migrate across a wide, unconfined floodplain and where lateral migration measurements are likely of high quality, the average ratio between the reach average migration rate normalized by channel width and the rate of sinuosity increase (excluding bends that experienced a cutoff between imagery dates) is approximately 5.6. Since elongation rate measurements can be made accurately even from photos that are poorly aligned, the relationship between sinuosity increase and lateral migration potentially provides a means of bypassing time-consuming georeferencing

  18. Detection of single ion channel activity with carbon nanotubes

    PubMed Central

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

    2015-01-01

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

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

    PubMed

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

    2015-01-01

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

  20. 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.

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

    PubMed

    DiFrancesco, D; Tortora, P

    1991-05-01

    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.

  2. Swelling-activated Ca2+ entry via TRPV4 channel is defective in cystic fibrosis airway epithelia.

    PubMed

    Arniges, Maite; Vázquez, Esther; Fernández-Fernández, José M; Valverde, Miguel A

    2004-12-24

    The vertebrate transient receptor potential cationic channel TRPV4 has been proposed as an osmo- and mechanosensor channel. Studies using knock-out animal models have further emphasized the relevance of the TRPV4 channel in the maintenance of the internal osmotic equilibrium and mechanosensation. However, at the cellular level, there is still one important question to answer: does the TRPV4 channel generate the Ca(2+) signal in those cells undergoing a Ca(2+)-dependent regulatory volume decrease (RVD) response? RVD in human airway epithelia requires the generation of a Ca(2+) signal to activate Ca(2+)-dependent K(+) channels. The RVD response is lost in airway epithelia affected with cystic fibrosis (CF), a disease caused by mutations in the cystic fibrosis transmembrane conductance regulator channel. We have previously shown that the defective RVD in CF epithelia is linked to the lack of swelling-dependent activation of Ca(2+)-dependent K(+) channels. In the present study, we show the expression of TRPV4 in normal human airway epithelia, where it functions as the Ca(2+) entry pathway that triggers the RVD response after hypotonic stress, as demonstrated by TRPV4 antisense experiments. However, cell swelling failed to trigger Ca(2+) entry via TRPV4 channels in CF airway epithelia, although the channel's response to a specific synthetic activator, 4 alpha-phorbol 12,13-didecanoate, was maintained. Furthermore, RVD was recovered in CF airway epithelia treated with 4 alpha-phorbol 12,13-didecanoate. Together, these results suggest that defective RVD in CF airway epithelia might be caused by the absence of a TRPV4-mediated Ca(2+) signal and the subsequent activation of Ca(2+)-dependent K(+) channels. PMID:15489228

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

    PubMed Central

    Sandilos, Joanna K; Bayliss, Douglas A

    2012-01-01

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

  4. Stretch-Activated Ion Channels: What Are They?

    PubMed Central

    Sachs, Frederick

    2010-01-01

    Mechanosensitive ion channels (MSCs) exist in all cells, but mechanosensitivity is a phenotype not a genotype. Specialized mechanoreceptors such as the hair cells of the cochlea require elaborate mechanical impedance matching to couple the channels to the external stress. In contrast, MSCs in nonspecialized cells appear activated by stress in the bilayer local to the channel—within about three lipids. Local mechanical stress can be produced by far-field tension, amphipaths, phase separations, the cytoskeleton, the extracellular matrix, and the adhesion energy between the membrane and a patch pipette. Understanding MSC function requires understanding the stimulus. PMID:20134028

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

    NASA Astrophysics Data System (ADS)

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

    2012-07-01

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

  6. Patterned electrical activity modulates sodium channel expression in sensory neurons.

    PubMed

    Klein, Joshua P; Tendi, Elisabetta A; Dib-Hajj, Sulayman D; Fields, R Douglas; Waxman, Stephen G

    2003-10-15

    Peripheral nerve injury induces changes in the level of gene expression for sodium channels Nav1.3, Nav1.8, and Nav1.9 within dorsal root ganglion (DRG) neurons, which may contribute to the development of hyperexcitability, ectopic neuronal discharge, and neuropathic pain. The mechanism of this change in sodium channel expression is unclear. Decreased availability of neurotrophic factors following axotomy contributes to these changes in gene transcription, but the question of whether changes in intrinsic neuronal activity levels alone can trigger changes in the expression of these sodium channels has not been addressed. We examined the effect of electrical stimulation on the expression of Nav1.3, Nav1.8, and Nav1.9 by using cultured embryonic mouse sensory neurons under conditions in which nerve growth factor (NGF) was not limiting. Expression of Nav1.3 was not significantly changed following stimulation. In contrast, we observed activity-dependent down-regulation of Nav1.8 and Nav1.9 mRNA and protein levels after stimulation, as demonstrated by quantitative polymerase chain reaction and immunocytochemistry. These results show that a change in neuronal activity can alter the expression of sodium channel genes in a subtype-specific manner, via a mechanism independent of NGF withdrawal. PMID:14515348

  7. 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.

  8. 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.

  9. 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.

  10. 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

  11. Chemical activation of the mechanotransduction channel Piezo1

    PubMed Central

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

    2015-01-01

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

  12. 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.

  13. 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

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

    PubMed Central

    Stathopulos, Peter B; Ikura, Mitsuhiko

    2013-01-01

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

  15. 25 CFR Appendix A to Subpart G - List of Activities Eligible for Funding Under BIA Transportation Facility Maintenance Program

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ... Transportation Facility Maintenance Program A Appendix A to Subpart G Indians BUREAU OF INDIAN AFFAIRS, DEPARTMENT OF THE INTERIOR LAND AND WATER INDIAN RESERVATION ROADS PROGRAM BIA Road Maintenance Pt. 170... Transportation Facility Maintenance Program The following activities are eligible for BIA Transportation...

  16. 25 CFR Appendix A to Subpart G - List of Activities Eligible for Funding Under BIA Transportation Facility Maintenance Program

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ... Transportation Facility Maintenance Program A Appendix A to Subpart G Indians BUREAU OF INDIAN AFFAIRS, DEPARTMENT OF THE INTERIOR LAND AND WATER INDIAN RESERVATION ROADS PROGRAM BIA Road Maintenance Pt. 170... Transportation Facility Maintenance Program The following activities are eligible for BIA Transportation...

  17. 25 CFR Appendix A to Subpart G - List of Activities Eligible for Funding Under BIA Transportation Facility Maintenance Program

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ... Transportation Facility Maintenance Program A Appendix A to Subpart G Indians BUREAU OF INDIAN AFFAIRS, DEPARTMENT OF THE INTERIOR LAND AND WATER INDIAN RESERVATION ROADS PROGRAM BIA Road Maintenance Pt. 170... Transportation Facility Maintenance Program The following activities are eligible for BIA Transportation...

  18. 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.

  19. 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.

  20. A myelin galactolipid, sulfatide, is essential for maintenance of ion channels on myelinated axon but not essential for initial cluster formation.

    PubMed

    Ishibashi, Tomoko; Dupree, Jeffrey L; Ikenaka, Kazuhiro; Hirahara, Yukie; Honke, Koichi; Peles, Elior; Popko, Brian; Suzuki, Kinuko; Nishino, Hitoo; Baba, Hiroko

    2002-08-01

    Myelinated axons are divided into four distinct regions: the node of Ranvier, paranode, juxtaparanode, and internode, each of which is characterized by a specific set of axonal proteins. Voltage-gated Na+ channels are clustered at high densities at the nodes, whereas shaker-type K+ channels are concentrated at juxtaparanodal regions. These channels are separated by the paranodal regions, where septate-like junctions are formed between the axon and the myelinating glial cells. Although oligodendrocytes and myelin sheaths are believed to play an instructive role in the local differentiation of the axon to distinct domains, the molecular mechanisms involved are poorly understood. In the present study, we have examined the distribution of axonal components in mice incapable of synthesizing sulfatide by disruption of the galactosylceramide sulfotransferase gene. These mice displayed abnormal paranodal junctions in the CNS and PNS, whereas their compact myelin was preserved. Immunohistochemical analysis demonstrated a decrease in Na+ and K+ channel clusters, altered nodal length, abnormal localization of K+ channel clusters appearing primarily in the presumptive paranodal regions, and diffuse distribution of contactin-associated protein along the internode. Similar abnormalities have been reported previously in mice lacking both galactocerebroside and sulfatide. Interestingly, although no demyelination was observed, these channel clusters decreased markedly with age. The initial timing and the number of Na+ channel clusters formed were normal during development. These results indicate a critical role for sulfatide in proper localization and maintenance of ion channels clusters, whereas they do not appear to be essential for initial cluster formation of Na+ channels. PMID:12151530

  1. 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.

  2. 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).

  3. Stimulation of epithelial sodium channel activity by the sulfonylurea glibenclamide.

    PubMed

    Chrabi, A; Horisberger, J D

    1999-07-01

    The amiloride-sensitive epithelial sodium channel (ENaC) contributes to the regulation of the sodium balance and blood pressure because it mediates a rate-limiting step in sodium transport across the epithelium of the distal nephron. The activity of ENaC is regulated by hormones, such as aldosterone and vasopressin, and by other intracellular or extracellular factors, but the mechanisms of these regulations are not yet well understood. It has been proposed that ENaC may be regulated by an associated ATP-binding cassette protein such as the cystic fibrosis conductance regulator or the K channel-associated sulfonylurea receptor. Glibenclamide, a known inhibitor of sulfonylurea receptor and cystic fibrosis conductance regulator, induced a dose-dependent and reversible stimulation (of the order of 40-50%) of the amiloride-sensitive current in oocytes expressing Xenopus ENaC, with a K1/2 of 45 +/- 5 microM. A similar effect was observed in oocytes expressing human ENaC, but not rat ENaC. Measurements performed with various combinations of rat and Xenopus subunits indicated that several subunits are involved in this effect. Glibenclamide also increased the transepithelial Na transport by the A6 Xenopus kidney cell line. Single-channel current recordings showed a doubling of the number of the open channels when glibenclamide was applied locally to the extracellular surface of the cell membrane. These results support the hypothesis of the existence of an associated ATP-binding cassette-type regulatory protein associated with the epithelial sodium channel. PMID:10381797

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

    PubMed Central

    Mahaut-Smith, M P; Mason, M J

    1991-01-01

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

  5. 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.

  6. 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.

  7. Sexual Function, Activity, and Satisfaction among Women Receiving Maintenance Hemodialysis

    PubMed Central

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

    2014-01-01

    Summary Background and objectives 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. Design, setting, participants, & measurements 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. Results 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. Conclusions 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

  8. A common mechanism underlies stretch activation and receptor activation of TRPC6 channels

    PubMed Central

    Spassova, Maria A.; Hewavitharana, Thamara; Xu, Wen; Soboloff, Jonathan; Gill, Donald L.

    2006-01-01

    The TRP family of ion channels transduce an extensive range of chemical and physical signals. TRPC6 is a receptor-activated nonselective cation channel expressed widely in vascular smooth muscle and other cell types. We report here that TRPC6 is also a sensor of mechanically and osmotically induced membrane stretch. Pressure-induced activation of TRPC6 was independent of phospholipase C. The stretch responses were blocked by the tarantula peptide, GsMTx-4, known to specifically inhibit mechanosensitive channels by modifying the external lipid-channel boundary. The GsMTx-4 peptide also blocked the activation of TRPC6 channels by either receptor-induced PLC activation or by direct application of diacylglycerol. The effects of the peptide on both stretch- and diacylglycerol-mediated TRPC6 activation indicate that the mechanical and chemical lipid sensing by the channel has a common molecular mechanism that may involve lateral-lipid tension. The mechanosensing properties of TRPC6 channels highly expressed in smooth muscle cells are likely to play a key role in regulating myogenic tone in vascular tissue. PMID:17056714

  9. 24 CFR 35.935 - Ongoing lead-based paint maintenance activities.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ... 24 Housing and Urban Development 1 2012-04-01 2012-04-01 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...

  10. 24 CFR 35.935 - Ongoing lead-based paint maintenance activities.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ... 24 Housing and Urban Development 1 2014-04-01 2014-04-01 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...

  11. 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...

  12. 24 CFR 35.935 - Ongoing lead-based paint maintenance activities.

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ... 24 Housing and Urban Development 1 2013-04-01 2013-04-01 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...

  13. 24 CFR 35.935 - Ongoing lead-based paint maintenance activities.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... 24 Housing and Urban Development 1 2011-04-01 2011-04-01 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...

  14. 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

  15. Regulation of Hyperpolarization-activated Cyclic Nucleotide-gated (HCN) Channel Activity by cCMP*

    PubMed Central

    Zong, Xiangang; Krause, Stefanie; Chen, Cheng-Chang; Krüger, Jens; Gruner, Christian; Cao-Ehlker, Xiaochun; Fenske, Stefanie; Wahl-Schott, Christian; Biel, Martin

    2012-01-01

    Activation of hyperpolarization-activated cyclic nucleotide-gated (HCN) channels is facilitated in vivo by direct binding of the second messenger cAMP. This process plays a fundamental role in the fine-tuning of HCN channel activity and is critical for the modulation of cardiac and neuronal rhythmicity. Here, we identify the pyrimidine cyclic nucleotide cCMP as another regulator of HCN channels. We demonstrate that cCMP shifts the activation curves of two members of the HCN channel family, HCN2 and HCN4, to more depolarized voltages. Moreover, cCMP speeds up activation and slows down deactivation kinetics of these channels. The two other members of the HCN channel family, HCN1 and HCN3, are not sensitive to cCMP. The modulatory effect of cCMP is reversible and requires the presence of a functional cyclic nucleotide-binding domain. We determined an EC50 value of ∼30 μm for cCMP compared with 1 μm for cAMP. Notably, cCMP is a partial agonist of HCN channels, displaying an efficacy of ∼0.6. cCMP increases the frequency of pacemaker potentials from isolated sinoatrial pacemaker cells in the presence of endogenous cAMP concentrations. Electrophysiological recordings indicated that this increase is caused by a depolarizing shift in the activation curve of the native HCN current, which in turn leads to an enhancement of the slope of the diastolic depolarization of sinoatrial node cells. In conclusion, our findings establish cCMP as a gating regulator of HCN channels and indicate that this cyclic nucleotide has to be considered in HCN channel-regulated processes. PMID:22715094

  16. 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

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

    PubMed

    Braun, Andrew P

    2012-01-01

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

  18. Molecular candidates for cardiac stretch-activated ion channels

    PubMed Central

    Reed, Alistair; Kohl, Peter; Peyronnet, Rémi

    2014-01-01

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

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

    PubMed Central

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

    2016-01-01

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

  20. 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.

  1. 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

    2014-04-01

    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.

  2. 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.

  3. 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.

  4. 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

  5. Photochemical activation of TRPA1 channels in neurons and animals

    PubMed Central

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

    2013-01-01

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

  6. 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

  7. Differential role of GDNF and NGF in the maintenance of two TTX-resistant sodium channels in adult DRG neurons.

    PubMed

    Fjell, J; Cummins, T R; Dib-Hajj, S D; Fried, K; Black, J A; Waxman, S G

    1999-04-20

    Following sciatic nerve transection, the electrophysiological properties of small dorsal root ganglion (DRG) neurons are markedly altered, with attenuation of TTX-R sodium currents and the appearance of rapidly repriming TTX-S currents. The reduction in TTX-R currents has been attributed to a down-regulation of sodium channels SNS/PN3 and NaN. While infusion of exogenous NGF to the transected nerve restores SNS/PN3 transcripts to near-normal levels in small DRG neurons, TTX-R sodium currents are only partially rescued. Binding of the isolectin IB4 distinguishes two subpopulations of small DRG neurons: IB4+ neurons, which express receptors for the GDNF family of neurotrophins, and IB4- neurons that predominantly express TrkA. We show here that SNS/PN3 is expressed in approximately one-half of both IB4+ and IB4- DRG neurons, while NaN is preferentially expressed in IB4+ neurons. Whole-cell patch-clamp studies demonstrate that TTX-R sodium currents in IB4+ neurons have a more hyperpolarized voltage-dependence of activation and inactivation than do IB4- neurons, suggesting different electrophysiological properties for SNS/PN3 and NaN. We confirm that NGF restores SNS/PN3 mRNA levels in DRG neurons in vitro and demonstrate that the trk antagonist K252a blocks this rescue. The down-regulation of NaN mRNA is, nevertheless, not rescued by NGF-treatment in either IB4+ or IB4- neurons and NGF-treatment in vitro does not significantly increase the peak amplitude of the TTX-R current in small DRG neurons. In contrast, GDNF-treatment causes a twofold increase in the peak amplitude of TTX-R sodium currents and restores both SNS/PN3 and NaN mRNA to near-normal levels in IB4+ neurons. These observations provide a mechanism for the partial restoration of TTX-R sodium currents by NGF in axotomized DRG neurons, and demonstrate that the neurotrophins NGF and GDNF differentially regulate sodium channels SNS/PN3 and NaN. PMID:10216225

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

    PubMed Central

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

    2016-01-01

    Large-conductance calcium-activated potassium (BK) channels are currently considered as vital players in a variety of renal physiological processes. In podocytes, BK channels become active in response to stimuli that increase local cytosolic Ca2+, possibly secondary to activation of slit diaphragm TRPC6 channels by chemical or mechanical stimuli. Insulin increases filtration barrier permeability through mobilization of BK channels. In mesangial cells, BK channels co-expressed with β1 subunits act as a major component of the counteractive response to contraction in order to regulate glomerular filtration. This review aims to highlight recent discoveries on the localization, physiological and pathological roles of BK channels in glomerulus. PMID:27445840

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

    PubMed

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

    2016-01-01

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

  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. Activity-Dependent Regulation of Substance P Expression and Topographic Map Maintenance by a Cholinergic Pathway

    PubMed Central

    Tu, Shichun; Butt, Christopher M.; Pauly, James R.; Debski, Elizabeth A.

    2008-01-01

    We have assessed the role of activity in the adult frog visual system in modulating two aspects of neuronal plasticity: neurotransmitter expression and topographic map maintenance. Chronic treatment of one tectal lobe with the non-NMDA receptor antagonist, 6-cyano-7-nitroquinoxaline-2,3-dione decreased the percentage of substance P-like immunoreactive (SP-IR) tectal cells in the untreated lobe while disrupting topographic map formation in the treated one. Treatment with the NMDA receptor antagonist d-(−)-2-amino-5-phosphonovaleric acid (d-AP-5) disrupted the topographic map but had no affect on SP-IR cells. These results indicate that maintenance of the topographic map is dependent on direct input from the glutamatergic retinal ganglion cells, whereas substance P (SP) expression is being regulated by a pathway that relays activity from one tectal lobe to the other. Such a pathway is provided by the cholinergic nucleus isthmi, which is reciprocally connected to the ipsilateral tectum and sends a projection to the contralateral one. Mecamylamine and atropine, antagonists of nicotinic and muscarinic receptors, respectively, were used together to block all cholinergic activity or alone to block receptor subclass activity. All three treatments decreased SP expression and disrupted the topographic map in the treated tectal lobe. We conclude that both SP expression and topographic map maintenance in the adult optic tectum are activity-dependent processes. Although our results are consistent with the maintenance of the topographic map through an NMDA receptor-based mechanism, they suggest that SP expression is regulated by a cholinergic interaction that depends on retinal ganglion cell input only for its activation. PMID:10884319

  12. NSAIDs attenuate hyperalgesia induced by TRP channel activation

    PubMed Central

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

    2016-01-01

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

  13. The LRRC26 Protein Selectively Alters the Efficacy of BK Channel Activators

    PubMed Central

    Almassy, Janos

    2012-01-01

    Large conductance, Ca2+-activated K channel proteins are involved in a wide range of physiological activities, so there is considerable interest in the pharmacology of large conductance calcium-activated K (BK) channels. One potent activator of BK channels is mallotoxin (MTX), which produces a very large hyperpolarizing shift of the voltage gating of heterologously expressed BK channels and causes a dramatic increase in the activity of BK channels in human smooth muscle cells. However, we found that MTX shifted the steady-state activation of BK channels in native parotid acinar cells by only 6 mV. This was not because the parotid BK isoform (parSlo) is inherently insensitive to MTX as MTX shifted the activation of heterologously expressed parSlo channels by 70 mV. Even though MTX had a minimal effect on steady-state activation of parotid BK channels, it produced an approximate 2-fold speeding of the channel-gating kinetics. The BK channels in parotid acinar cells have a much more hyperpolarized voltage activation range than BK channels in most other cell types. We found that this is probably attributable to an accessory protein, LRRC26, which is expressed in parotid glands: expressed parSlo + LRRC26 channels were resistant to the actions of MTX. Another class of BK activators is the benzimidazalones that includes 1,3-dihydro-1-(2-hydroxy-5-(trifluoromethyl)phenyl)-5-(trifluoromethyl)-2H-benzimidazol-2-one (NS-1619). Although the LRRC26 accessory protein strongly inhibited the ability of MTX to activate BK channels, we found that it had only a small effect on the action of NS-1619 on BK channels. Thus, the LRRC26 BK channel accessory protein selectively alters the pharmacology of BK channels. PMID:21984254

  14. 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.

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

    PubMed

    Kaneko, Toshiyuki; Takahashi, Naoya; Kikuyama, Munehiro

    2009-03-01

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

  16. Activation of endogenously expressed ion channels by active complement in the retinal pigment epithelium.

    PubMed

    Genewsky, Andreas; Jost, Ingmar; Busch, Catharina; Huber, Christian; Stindl, Julia; Skerka, Christine; Zipfel, Peter F; Rohrer, Bärbel; Strauß, Olaf

    2015-10-01

    Defective regulation of the alternative pathway of the complement system is believed to contribute to damage of retinal pigment epithelial (RPE) cells in age-related macular degeneration. Thus we investigated the effect of complement activation on the RPE cell membrane by analyzing changes in membrane conductance via patch-clamp techniques and Ca(2+) imaging. Exposure of human ARPE-19 cells to complement-sufficient normal human serum (NHS) (25 %) resulted in a biphasic increase in intracellular free Ca(2+) ([Ca(2+)]i); an initial peak followed by sustained Ca(2+) increase. C5- or C7-depleted sera did not fully reproduce the signal generated by NHS. The initial peak of the Ca(2+) response was reduced by sarcoplasmic Ca(2+)-ATPase inhibitor thapsigargin, L-type channel blockers (R)-(+)-BayK8644 and isradipine, transient-receptor-potential (TRP) channel blocker ruthenium-red and ryanodine receptor blocker dantrolene. The sustained phase was carried by CaV1.3 L-type channels via tyrosine-phosphorylation. Changes in [Ca(2+)]I were accompanied by an abrupt hyperpolarization, resulting from a transient increase in membrane conductance, which was absent under extracellular Ca(2+)- or K(+)-free conditions and blocked by (R)-(+)-BayK8644 or paxilline, a maxiK channel inhibitor. Single-channel recordings confirmed the contribution of maxiK channels. Primary porcine RPE cells responded to NHS in a comparable manner. Pre-incubation with NHS reduced H2O2-induced cell death. In summary, in a concerted manner, C3a, C5a and sC5b-9 increased [Ca(2+)]i by ryanodine-receptor-dependent activation of L-type channels in addition to maxi-K channels and TRP channels absent from any insertion of a lytic pore.

  17. 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

  18. 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.

  19. Sustained Dorsal Hippocampal Activity is Not Obligatory for Either the Maintenance or Retrieval of Long-Term Spatial Memory

    PubMed Central

    Broadbent, Nicola J.; Squire, Larry R.; Clark, Robert E.

    2011-01-01

    Memories are initially stored in a labile state and are subject to modification by a variety of treatments, including disruption of hippocampal function. We infused a sodium channel blocker (or CNQX) to inactivate the rat dorsal hippocampus reversibly for 1 week following training on a task of spatial memory (the water maze). Previous work with conventional lesions has established that the dorsal hippocampus is essential for both the acquisition and expression of memory in this task. The question in the present study was whether chronic disruption of neuronal activity in the dorsal hippocampus after training would abolish memory or whether memory would survive extended disruption of hippocampal activity. As expected from earlier work, we found that performance was impaired during the infusion period. The critical test occurred 1 week after the lesion was reversed. We found that retention of the water maze recovered to control levels. Accordingly, sustained hippocampal activity following training is not obligatory for either the maintenance of long-term spatial memory or its subsequent retrieval. PMID:19921702

  20. 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.

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

    NASA Astrophysics Data System (ADS)

    Solaro, Christopher R.; Lingle, Christopher J.

    1992-09-01

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

  2. The effect of single cerebroside compounds on activation of BKCa channels.

    PubMed

    Xu, Huina; Qi, Jianhua; Wang, Guangfa; Deng, Hongwen; Qi, Zhi

    2011-02-01

    We have previously shown that a mixture of cerebrosides obtained from dried tubers of herb Typhonium giganteum Engl. plays a neuroprotective role in the ischemic brain through its effect on activation of BK(Ca) channels. It is very curious to know whether a single pure cerebroside compound could activate the BK(Ca) channel as well. This study explored the possible effects of pure cerebroside compounds, termitomycesphins A and B, on the BK(Ca) channel activation. Both termitomycesphins A and B activated the BK(Ca) channels at micromole concentration without significant difference. Termitomycesphin A increased the single channel open probability of the BK(Ca) channels in a dose-dependent manner without modifying the single channel conductance. Termitomycesphin A activated BK(Ca) channel more efficiently when it was applied to the cytoplasmic face of the membrane, suggesting that binding site for termitomycesphin A is located at the cytoplasmic side. Termitomycesphin A shifted the voltage-dependent activation curve to less positive membrane potentials and the Ca(2+)-dependent activation curve of the channel upwards, suggesting that termitomycesphin A could activate the channels even without intracellular free Ca(2+). Furthermore, STREX-deleted BK(Ca) channels were completely insensitive to termitomycesphin A, indicating that STREX domain is required for the activation of the BK(Ca) channel. These data provide evidence that termitomycesphins are potent in stimulating the activity of the BK(Ca) channels. As BK(Ca) channels are associated with pathology of many diseases, termitomycesphins might be used as therapeutic agents for treating these diseases through its regulatory effect on the BK(Ca) channels.

  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. 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. PMID:17281912

  5. 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

    ... Power Plants'' AGENCY: Nuclear Regulatory Commission. ACTION: Notice of withdrawal. SUMMARY: The U.S... (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:...

  6. 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.

  7. 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

    2015-01-01

    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 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.

  8. 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, Dongxian; Miao, Chiyuan; Wu, Jingwen; Jiang, Lin; Duan, Qingyun

    2015-10-01

    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.

  9. Channel

    NASA Technical Reports Server (NTRS)

    2006-01-01

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

    This channel is located south of Iani Chaos.

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

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

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

  10. 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.

  11. Acute exposure of methylglyoxal leads to activation of KATP channels expressed in HEK293 cells

    PubMed Central

    Yang, Yang; Konduru, Anuhya S; Cui, Ningren; Yu, Lei; Trower, Timothy C; Shi, Weiwei; Shi, Yun; Jiang, Chun

    2014-01-01

    Aim: Highly reactive carbonyl methylglyoxal (MGO) is one of the metabolites excessively produced in diabetes. We have showed that prolonged exposure of vascular smooth muscle cells to MGO leads to instability of the mRNA encoding ATP-sensitive potassium (KATP) channel. In the present study we investigated the effects of MGO on the activity of KATP channels. Methods: Kir6.1/ SUR2B, Kir6.2/SUR2B or Kir6.2Δ36 (a truncated Kir6.2 isoform) alone was expressed in HEK293 cells. Whole-cell currents were recorded in the cells with an Axopatch 200B amplifier. Macroscopic currents and single-channel currents were recorded in giant inside-out patches and normal inside-out patches, respectively. Data were analyzed using Clampfit 9 software. Results: The basal activity of Kir6.1/SUR2B channels was low. The specific KATP channel opener pinacidil (10 μmol/L) could fully activate Kir6.1/SUR2B channels, which was inhibited by the specific KATP channel blocker glibenclamide (10 μmol/L). MGO (0.1-10 mmol/L) dose-dependently activated Kir6.1/SUR2B channels with an EC50 of 1.7 mmol/L. The activation of Kir6.1/SUR2B channels by MGO was reversible upon washout, and could be inhibited completely by glibenclamide. Kir6.2Δ36 channels expressed in HEK293 cells could open automatically, and the channel activity was enhanced in the presence of MGO (3 mmol/L). Single channel recordings showed that MGO (3 mmol/L) markedly increased the open probability of Kir6.1/SUR2B channels, leaving the channel conductance unaltered. Conclusion: Acute application of MGO activates KATP channels through direct, non-covalent and reversible interactions with the Kir6 subunits. PMID:24122011

  12. Effect of realistic grounds and atmospheric conditions on single-channel active control of outdoor sound propagation

    NASA Astrophysics Data System (ADS)

    Nakashima, Ann; Hodgson, Murray

    2005-03-01

    Engine run-up tests are a part of routine aircraft maintenance at the Vancouver International Airport. A source of noise complaints is the Dash-8 aircraft, which emits low-frequency, tonal noise. Active noise control is a potentially cost-effective alternative to passive noise-control methods, which are ineffective at controlling low-frequency noise. Since the run-up tests are performed outdoors, the effects of outdoor conditions on the performance of an active control system must be considered. In this paper, the results of a preliminary investigation of the effects of realistic meteorological conditions and ground impedance on the performance of a single-channel active-control system are presented. Computer simulations of single-channel active control of a monopole source were performed using the Green's-function parabolic-equation method. Different realistic atmospheric conditions, and reflective or soft ground, were used in the simulations. The results show that atmospheric refraction causes fluctuations in the noise attenuation achieved by a single-channel control system, and has the overall effect of decreasing its performance, making the system ineffective in some cases. .

  13. Identification and Characterization of Stretch-Activated Ion Channels in Pollen Protoplasts1

    PubMed Central

    Dutta, Rajiv; Robinson, Kenneth R.

    2004-01-01

    Pollen tube growth requires a Ca2+ gradient, with elevated levels of cytosolic Ca2+ at the growing tip. This gradient's magnitude oscillates with growth oscillation but is always maintained. Ca2+ influx into the growing tip is necessary, and its magnitude also oscillates with growth. It has been widely assumed that stretch-activated Ca2+ channels underlie this influx, but such channels have never been reported in either pollen grains or pollen tubes. We have identified and characterized stretch-activated Ca2+ channels from Lilium longiflorum pollen grain and tube tip protoplasts. The channels were localized to a small region of the grain protoplasts associated with the site of tube germination. In addition, we find a stretch-activated K+ channel as well as a spontaneous K+ channel distributed over the entire grain surface, but neither was present at the germination site or at the tip. Neither stretch-activated channel was detected in the grain protoplasts unless the grains were left in germination medium for at least 1 h before protoplast preparation. The stretch-activated channels were inhibited by a spider venom that is known to block stretch-activated channels in animal cells, but the spontaneous channel was unaffected by the venom. The venom also stopped pollen tube germination and elongation and blocked Ca2+ entry into the growing tip, suggesting that channel function is necessary for growth. PMID:15247410

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

    PubMed Central

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

    2014-01-01

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

  15. 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…

  16. 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

  17. 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.

  18. 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. PMID:24588198

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

    PubMed

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

    2015-03-09

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

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

    PubMed Central

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

    2014-01-01

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

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

    PubMed Central

    Gaurav, Rohit; Bewtra, Againdra K.

    2015-01-01

    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. PMID:25514499

  2. 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

  3. Gabapentin activates ROMK1 channels by a protein kinase A (PKA)-dependent mechanism

    PubMed Central

    Lee, C-H; Tsai, T-S; Liou, H-H

    2008-01-01

    Background and purpose: Gabapentin is an effective anticonvulsant. The major physiological function of renal outer medullary potassium (ROMK1) channels is to maintain the resting membrane potential (RMP). We investigated the effect of gabapentin on ROMK1 channels and the mechanism involved. Experimental approach: Xenopus oocytes were injected with mRNA coding for wild-type or mutant ROMK1 channels and giant inside-out patch-clamp recordings were performed. Key results: Gabapentin increased the activity of ROMK1 channels, concentration-dependently and enhanced the activity of wild-type and an intracellular pH (pHi)-gating residue mutant (K80M) channels over a range of pHi. Gabapentin also increased activity of channels mutated at phosphatidylinositol 4,5-bisphosphate (PIP2)-binding sites (R188Q, R217A and K218A). However, gabapentin failed to enhance channel activity in the presence of protein kinase A (PKA) inhibitors and did not activate phosphorylation site mutants (S44A, S219A or S313A), mutants that mimicked the negative charge carried by a phosphate group bound to a serine (S44D, S219D or S313D), or a mutated channel with a positive charge (S219R). These findings show that gabapentin activates ROMK1 channels independently of the pHi and not via a PIP2-dependent pathway. The effects of gabapentin on ROMK1 channels may be due to a PKA-mediated phosphorylation-induced conformational change, but not to charge–charge interactions. Conclusions and implications: ROMK1 channels are the main channels responsible for maintaining the RMP during cellular excitation. Gabapentin increased the activity of ROMK1 channels by a PKA-dependent mechanism, reducing neuronal excitability, and this may play an important role in its antiepileptic effect. PMID:18311184

  4. 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.

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

    PubMed Central

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

    2014-01-01

    Transient Receptor Potential (TRP) proteins are a large family of ion channels, grouped into seven sub-families. Although great advances have been made regarding the activation and modulation of TRP channel activity, detailed molecular mechanisms governing TRP channel gating are still needed. Sensitive to electric, chemical, mechanical, and thermal cues, TRP channels are tightly associated with the detection and integration of sensory input, emerging as a model to study the polymodal activation of ion channel proteins. Among TRP channels, the temperature-activated kind constitute a subgroup by itself, formed by Vanilloid receptors 1–4, Melastatin receptors 2, 4, 5, and 8, TRPC5, and TRPA1. Some of the so-called “thermoTRP” channels participate in the detection of noxious stimuli making them an interesting pharmacological target for the treatment of pain. However, the poor specificity of the compounds available in the market represents an important obstacle to overcome. Understanding the molecular mechanics underlying ligand-dependent modulation of TRP channels may help with the rational design of novel synthetic analgesics. The present review focuses on the structural basis of ligand-dependent activation of TRPV1 and TRPM8 channels. Special attention is drawn to the dissection of ligand-binding sites within TRPV1, PIP2-dependent modulation of TRP channels, and the structure of natural and synthetic ligands. PMID:24847275

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

    PubMed Central

    Lev, Shaya; Katz, Ben; Minke, Baruch

    2012-01-01

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

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

    PubMed Central

    Zhao, Xiaoqi; Gu, Tianxiang

    2016-01-01

    Hyperpolarization-activated cyclic nucleotide-gated (HCN) channels are reverse voltage-dependent, and their activation depends on the hyperpolarization of the membrane and may be directly or indirectly regulated by the cyclic adenosine monophosphate (cAMP) or other signal-transduction cascades. The distribution, quantity and activation states of HCN channels differ in tissues throughout the body. Evidence exhibits that HCN channels play critical roles in the generation and conduction of the electrical impulse and the physiopathological process of some cardiac diseases. They may constitute promising drug targets in the treatment of these cardiac diseases. Pharmacological treatment targeting HCN channels is of benefit to these cardiac conditions. PMID:27556324

  8. Substrate size as a determinant of fibrillatory activity maintenance in a mathematical model of canine atrium.

    PubMed

    Zou, Renqiang; Kneller, James; Leon, L Joshua; Nattel, Stanley

    2005-09-01

    Tissue size has been considered an important determinant of atrial fibrillation (AF), but recent work has questioned the critical size hypothesis. Here, we use a previously developed mathematical model of the two-dimensional canine atrium with realistic action potential, ionic, and conduction properties to address substrate size effects on the maintenance of fibrillatory activity. Cholinergic AF was simulated at different acetylcholine (ACh) concentrations ([ACh]) and distributions, with substrate area varied 11.1-fold. Automated phase singularity detection was used to facilitate the analysis of arrhythmic activity. The duration of activity induced by a single extrastimulus increased with increasing substrate dimensions. Two general mechanisms underlying activity were observed and were differentially affected by substrate size. For large mean [ACh], single primary rotors anchored in low-[ACh] zones maintained activity and substrate dimensions were not critical. At lower mean [ACh], extensive spiral wave meander prevented the emergence of single stable rotors. Prolonged activity was favored when substrate size permitted a sufficiently large number of simultaneous longer-lasting rotors that extinction of all was unlikely. Thus either single dominant rotor or multiple reentrant spiral generator mechanisms could maintain fibrillatory activity in this model and were differentially dependent on substrate size. These results speak to recent debates about the role in AF of single driver rotors versus multiple reentrant circuit mechanisms by suggesting that either may maintain fibrillatory atrial activity depending on atrial size and electrophysiological properties.

  9. Maintenance of Stem Cell Niche Integrity by a Novel Activator of Integrin Signaling

    PubMed Central

    Lee, Joo Yeun; Chang, Karen T.

    2016-01-01

    Stem cells depend critically on the surrounding microenvironment, or niche, for their maintenance and self-renewal. While much is known about how the niche regulates stem cell self-renewal and differentiation, mechanisms for how the niche is maintained over time are not well understood. At the apical tip of the Drosophila testes, germline stem cells (GSCs) and somatic stem cells share a common niche formed by hub cells. Here we demonstrate that a novel protein named Shriveled (Shv) is necessary for the maintenance of hub/niche integrity. Depletion of Shv protein results in age-dependent deterioration of the hub structure and loss of GSCs, whereas upregulation of Shv preserves the niche during aging. We find Shv is a secreted protein that modulates DE-cadherin levels through extracellular activation of integrin signaling. Our work identifies Shv as a novel activator of integrin signaling and suggests a new integration model in which crosstalk between integrin and DE-cadherin in niche cells promote their own preservation by maintaining the niche architecture. PMID:27191715

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

    NASA Astrophysics Data System (ADS)

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

    2016-08-01

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

  11. 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

    2015-07-01

    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.

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

    PubMed

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

    2010-08-01

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

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

    PubMed

    Lees-Green, Rachel; Gibbons, Simon J; Farrugia, Gianrico; Sneyd, James; Cheng, Leo K

    2014-04-15

    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 Ca(2+) oscillations in ICC appear to produce the slow waves by activating pacemaker currents, currently thought to be carried by the Ca(2+)-activated Cl(-) channel anoctamin 1 (Ano1). In this article we present a novel model of small intestinal ICC pacemaker activity that incorporates store-operated Ca(2+) 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 Ca(2+) channel but suggest that small cyclical depolarizations may still occur in ICC in Ano1 knockout mice. The results predict that voltage-dependent Ca(2+) 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.

  14. 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.

  15. 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. PMID:24438736

  16. 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.

  17. [Effects of calcium-activated chloride channels on vascular activity of rat cerebral basilar artery].

    PubMed

    Wang, Rui; Li, Li; Ma, Ke-Tao; Si, Jun-Qiang

    2014-06-25

    This study investigated the role of calcium-activated Cl⁻ channels (CaCCs) in mediating vasomotor activity of cerebral basilar artery (BA) of Wistar rat. Pressure myograph was used to examine the changes in diameter of isolated BA to vasoactive reagents. The results showed that (1) The rate of pressure-induced vasomotor activity was 78.6% (n = 28) in BA from 0 to 100 mmHg working pressure. The contractile phase of the response was faster than the relaxation phase; (2) The amplitude of contraction was (62.6 ± 6.4) µm (n = 22), the frequency of contraction was variable and the highest value was 8.0 ± 2.3 per 5 min at 60 mmHg working pressure (n = 22); (3) The pressure-induced vasomotor activity of BA was markedly attenuated when Ca²⁺ was removed from medium; (4) The pressure-induced vasomotor activity was blocked by voltage dependent Ca²⁺ channel blocker nimodipine; (5) The pressure-induced vasomotor was inhibited by CaCC antagonists NFA and NPPB. These results suggest that the pressure-induced vasomotor activity of isolated BA is associated with Ca²⁺ influx that activates CaCCs.

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

    PubMed Central

    Gincel, Dan; Shoshan-Barmatz, Varda

    2002-01-01

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

  19. 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

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

    PubMed

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

    2014-11-01

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

  1. 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.

  2. Oxidized glutathione mediates cation channel activation in calf vascular endothelial cells during oxidant stress.

    PubMed

    Koliwad, S K; Elliott, S J; Kunze, D L

    1996-08-15

    1. The oxidant, tert-butylhydroperoxide (tBuOOH) depolarizes calf pulmonary artery endothelial cells by activating a non-selective cation channel. To identify the molecular mediator of channel activation during oxidant stress, the patch-clamp technique was used to compare tBuOOH-induced changes in membrane potential and channel activity with those induced by oxidized glutathione (GSSG), a cytosolic product of oxidant metabolism. 2. When recording pipettes contained GSSG (2 mM), whole-cell zero-current potential measured immediately following pipette break-in was not different from control values (-57 mV). However, within 20 min of break-in, zero-current potential was depolarized to -7 mV. The time course of depolarization was dependent on the concentration of GSSG and was accelerated by inhibition of GSSG metabolism. 3. In excised membrane patches, channels were activated by internal GSSG, but not by internal tBuOOH, reduced glutathione (GSH), or external GSSG. Channels were equal in size (28 pS) and in ionic selectivity to those activated by incubation of intact cells with tBuOOH. As little as 20 microM GSSG was sufficient to maximally activate channels. However, the time course of channel activation was concentration dependent between 20 microM and 2 mM GSSG. 4. Channel activation by GSSG was reversed by GSH and by increasing the [GSH]:[GSSG] ratio. Likewise, channel activation by pre-incubation of intact cells with tBuOOH was reversed by GSH applied after patch excision. 5. These results strongly suggest that GSSG is an endogenous intracellular mediator of channel activation and depolarization during oxidant stress. PMID:8866350

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

    PubMed

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

    2015-11-01

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

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

    PubMed

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

    2015-11-01

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

  5. Working memory encoding and maintenance deficits in schizophrenia: neural evidence for activation and deactivation abnormalities.

    PubMed

    Anticevic, Alan; Repovs, Grega; Barch, Deanna M

    2013-01-01

    Substantial evidence implicates working memory (WM) as a core deficit in schizophrenia (SCZ), purportedly due to primary deficits in dorsolateral prefrontal cortex functioning. Recent findings suggest that SCZ is also associated with abnormalities in suppression of certain regions during cognitive engagement--namely the default mode system--that may further contribute to WM pathology. However, no study has systematically examined activation and suppression abnormalities across both encoding and maintenance phases of WM in SCZ. Twenty-eight patients and 24 demographically matched healthy subjects underwent functional magnetic resonance imaging at 3T while performing a delayed match-to-sample WM task. Groups were accuracy matched to rule out performance effects. Encoding load was identical across subjects to facilitate comparisons across WM phases. We examined activation differences using an assumed model approach at the whole-brain level and within meta-analytically defined WM areas. Despite matched performance, we found regions showing less recruitment during encoding and maintenance for SCZ subjects. Furthermore, we identified 2 areas closely matching the default system, which SCZ subjects failed to deactivate across WM phases. Lastly, activation in prefrontal regions predicted the degree of deactivation for healthy but not SCZ subjects. Current results replicate and extend prefrontal recruitment abnormalities across WM phases in SCZ. Results also indicate deactivation abnormalities across WM phases, possibly due to inefficient prefrontal recruitment. Such regional deactivation may be critical for suppressing sources of interference during WM trace formation. Thus, deactivation deficits may constitute an additional source of impairments, which needs to be further characterized for a complete understanding of WM pathology in SCZ.

  6. 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.

  7. 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.

  8. 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.

  9. 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. PMID:23312295

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

    PubMed Central

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

    2007-01-01

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

  11. Mechanosensitive channel activation by diffusio-osmotic force.

    PubMed

    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. PMID:25325663

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

    PubMed Central

    Gage, P W; Hamill, O P

    1980-01-01

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

  13. 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.

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

    PubMed

    Knaus, H G; McManus, O B; Lee, S H; Schmalhofer, W A; Garcia-Calvo, M; Helms, L M; Sanchez, M; Giangiacomo, K; Reuben, J P; Smith, A B

    1994-05-17

    Tremorgenic indole alkaloids produce neurological disorders (e.g., staggers syndromes) in ruminants. The mode of action of these fungal mycotoxins is not understood but may be related to their known effects on neurotransmitter release. To determine whether these effects could be due to inhibition of K+ channels, the interaction of various indole diterpenes with high-conductance Ca(2+)-activated K+ (maxi-K) channels was examined. Paspalitrem A, paspalitrem C, aflatrem, penitrem A, and paspalinine inhibit binding of [125I]charybdotoxin (ChTX) to maxi-K channels in bovine aortic smooth muscle sarcolemmal membranes. In contrast, three structurally related compounds, paxilline, verruculogen, and paspalicine, enhanced toxin binding. As predicted from the binding studies, covalent incorporation of [125I]ChTX into the 31-kDa subunit of the maxi-K channel was blocked by compounds that inhibit [125I]ChTX binding and enhanced by compounds that stimulate [125I]ChTX binding. Modulation of [125I]ChTX binding was due to allosteric mechanisms. Despite their different effects on binding of [125I]ChTX to maxi-K channels, all compounds potently inhibited maxi-K channels in electrophysiological experiments. Other types of voltage-dependent or Ca(2+)-activated K+ channels examined were not affected. Chemical modifications of paxilline indicate a defined structure-activity relationship for channel inhibition. Paspalicine, a deshydroxy analog of paspalinine lacking tremorgenic activity, also potently blocked maxi-K channels. Taken together, these data suggest that indole diterpenes are the most potent nonpeptidyl inhibitors of maxi-K channels identified to date. Some of their pharmacological properties could be explained by inhibition of maxi-K channels, although tremorgenicity may be unrelated to channel block. PMID:7514038

  15. Voltage, calcium, and stretch activated ionic channels and intracellular calcium in bone cells.

    PubMed

    Ypey, D L; Weidema, A F; Höld, K M; Van der Laarse, A; Ravesloot, J H; Van Der Plas, A; Nijweide, P J

    1992-12-01

    Embryonic chick bone cells express various types of ionic channels in their plasma membranes for as yet unresolved functions. Chick osteoclasts (OCL) have the richest spectrum of channel types. Specific for OCL is a K+ channel, which activates (opens) when the inside negative membrane potential (Vm) becomes more negative (hyperpolarization). This is consistent with findings of others on rat OCL. The membrane conductance constituted by these channels is called the inward rectifying K+ conductance (GKi), or inward rectifier, because the hyperpolarization-activated channels cause cell-inward K+ current to pass more easily through the membrane than outward K+ current. Besides GKi channels, OCL may express two other types of voltage-activated K+ channels. One constitutes the transient outward rectifying K+ conductance (GKto), which is activated upon making the membrane potential less negative (depolarization) but has a transient nature. This conductance favors transient K+ conduction in the cell-outward direction. The GKto also occurs in a small percentage of cells in osteoblast (OBL) and periosteal fibroblast (PFB) cultures. The other OCL K+ conductance, the GKCa, is activated by both membrane depolarization and a rise in [Ca2+]i. GKCa channels are also present in the other chick bone cell types, that is, OBL, osteocytes (OCY), and PFB. Furthermore, in excised patches of all bone cell types, channels have been found that conduct anions, including Cl- and phosphate ions. These channels are only active around Vm = 0 mV. While searching for a membrane mechanism for adaptation of bone to mechanical loading, we found stretch-activated channels in chick osteoclasts; other investigators have found stretch-activated cation channels (K+ or aselective) in rat and human osteogenic cell lines. In contrast to other studies on cell lines or OBL from other species, we have not found any of the classic macroscopic voltage-activated calcium conductances (GCa) in any of the chick bone

  16. 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

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

    PubMed

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

    2016-03-01

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

  18. Thermodynamic view of activation energies of proton transfer in various gramicidin A channels.

    PubMed

    Chernyshev, Anatoly; Cukierman, Samuel

    2002-01-01

    The temperature dependencies (range: 5-45 degrees C) of single-channel proton conductances (g(H)) in native gramicidin A (gA) and in two diastereoisomers (SS and RR) of the dioxolane-linked gA channels were measured in glycerylmonooleate/decane (GMO) and diphytanoylphosphatidylcholine/decane (DiPhPC) bilayers. Linear Arrhenius plots (ln (g(H)) versus K(-1)) were obtained for the native gA and RR channels in both types of bilayers, and for the SS channel in GMO bilayers only. The Arrhenius plot for proton transfer in the SS channel in DiPhPC bilayers had a break in linearity around 20 degrees C. This break seems to occur only when protons are the permeating cations in the SS channel. The activation energies (E(a)) for proton transfer in various gA channels (approximately 15 kJ/mol) are consistent with the rate-limiting step being in the channel and/or at the membrane-channel/solution interface, and not in bulk solution. E(a) values for proton transfer in gA channels are considerably smaller than for the permeation of nonproton currents in gA as well as in various other ion channels. The E(a) values for proton transfer in native gA channels are nearly the same in both GMO and DiPhPC bilayers. In contrast, for the dioxolane linked gA dimers, E(a) values were strongly modulated by the lipid environment. The Gibbs activation free energies (Delta G(#)(o)) for protons in various gA channels are within the range of 27-29 kJ/mol in GMO bilayers and of 20-22 kJ/mol in DiPhPC bilayers. The largest difference between Delta G(#)(o) for proton currents occurs between native gA (or SS channels) and the RR channel. In general, the activation entropy (Delta S) is mostly responsible for the differences between g(H) values in various gA channels, and also in distinct bilayers. However, significant differences between the activation enthalpies (Delta H(#)(o)) for proton transfer in the SS and RR channels occur in distinct membranes.

  19. Cyclic GMP-activated channels of salamander retinal rods: spatial distribution and variation of responsiveness.

    PubMed Central

    Karpen, J W; Loney, D A; Baylor, D A

    1992-01-01

    1. Patch-clamp methods were used to investigate the areal density and spatial location of cyclic GMP-activated channels in the surface membrane of salamander rod outer segments. 2. The density of active channels (i.e. channels able to respond to cyclic GMP) in patches excised from outer segments was determined from the number of active channels, N, and the membrane area, A. N was estimated from the current induced by a saturating concentration of cyclic GMP, while A was estimated from the electrical capacitance of the patch. 3. In patches excised from forty-one isolated outer segments prepared in the light the active channel density varied over a remarkable range: 0.34-629 microns-2, with a mean of 166 microns-2. Density was not correlated with patch area in this or any of the conditions studied. 4. The spatial distribution of open channels on the outer segment of a transducing rod was measured by recording the local dark current at various positions with a loose-patch electrode. The apparent density of open channels varied by only about +/- 50% around the circumference of the outer segment and up and down its length. This indicates that the wide range of densities in excised patches did not result from sampling a non-uniform spatial distribution of channels. 5. Patches excised from sixteen dark-adapted whole cells with healthy appearances and saturating light responses of normal size had active channel densities of 1.1-200 microns-2, with a mean of 60 microns-2. Patches from twenty light-adapted whole cells had similar densities. Many densities from the whole cells were much lower than expected. This, and the wide variation in densities, suggests that obtaining a patch often lowered the density of active channels. The number of channels in a patch was quite stable from 1 s to 30 min after excision, ruling out progressive denaturation or adsorption of channels to the glass as a cause for this effect. 6. The mean active channel density in patches excised from whole

  20. 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...

  1. 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.

  2. Molecular bases for the asynchronous activation of sodium and potassium channels required for nerve impulse generation.

    PubMed

    Lacroix, Jérôme J; Campos, Fabiana V; Frezza, Ludivine; Bezanilla, Francisco

    2013-08-21

    Most action potentials are produced by the sequential activation of voltage-gated sodium (Nav) and potassium (Kv) channels. This is mainly achieved by the rapid conformational rearrangement of voltage-sensor (VS) modules in Nav channels, with activation kinetics up to 6-fold faster than Shaker-type Kv channels. Here, using mutagenesis and gating current measurements, we show that a 3-fold acceleration of the VS kinetics in Nav versus Shaker Kv channels is produced by the hydrophilicity of two "speed-control" residues located in the S2 and S4 segments in Nav domains I-III. An additional 2-fold acceleration of the Nav VS kinetics is provided by the coexpression of the β1 subunit, ubiquitously found in mammal tissues. This study uncovers the molecular bases responsible for the differential activation of Nav versus Kv channels, a fundamental prerequisite for the genesis of action potentials.

  3. 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.

  4. 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.

  5. 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

  6. Fe2+ Substrate Transport through Ferritin Protein Cage Ion Channels Influences Enzyme Activity and Biomineralization

    PubMed Central

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

    2015-01-01

    Ferritins, complex protein nanocages, form internal iron-oxy minerals (Fe2O3.H2O), by moving cytoplasmic Fe2+ through intracage ion channels to cage-embedded enzyme (2Fe2+/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 Fe2+ 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 650nm (DFP λmax). The caged biomineral formation, measured at 350 nm in the middle of the broad, nonspecific Fe3+-O absorption band, was slower. Structural differences (protein X-ray crystallography), between ion channels in wild type and ferritin D127E, which correlate with the inhibition of ferritin D127E enzyme activity include: 1. narrower interior ion channel openings/pores, 2. increased numbers of ion channel protein-metal binding sites, and 3. a change in ion channel electrostatics due to carboxylate crowding. The contributions of ion channel size and structure to ferritin activity reflect metal ion transport in ion channels are precisely regulated both in ferritin protein nanocages and membranes of living cells. PMID:26202907

  7. Visual short-term memory: activity supporting encoding and maintenance in retinotopic visual cortex.

    PubMed

    Sneve, Markus H; Alnæs, Dag; Endestad, Tor; Greenlee, Mark W; Magnussen, Svein

    2012-10-15

    Recent studies have demonstrated that retinotopic cortex maintains information about visual stimuli during retention intervals. However, the process by which transient stimulus-evoked sensory responses are transformed into enduring memory representations is unknown. Here, using fMRI and short-term visual memory tasks optimized for univariate and multivariate analysis approaches, we report differential involvement of human retinotopic areas during memory encoding of the low-level visual feature orientation. All visual areas show weaker responses when memory encoding processes are interrupted, possibly due to effects in orientation-sensitive primary visual cortex (V1) propagating across extrastriate areas. Furthermore, intermediate areas in both dorsal (V3a/b) and ventral (LO1/2) streams are significantly more active during memory encoding compared with non-memory (active and passive) processing of the same stimulus material. These effects in intermediate visual cortex are also observed during memory encoding of a different stimulus feature (spatial frequency), suggesting that these areas are involved in encoding processes on a higher level of representation. Using pattern-classification techniques to probe the representational content in visual cortex during delay periods, we further demonstrate that simply initiating memory encoding is not sufficient to produce long-lasting memory traces. Rather, active maintenance appears to underlie the observed memory-specific patterns of information in retinotopic cortex.

  8. 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.

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

    PubMed Central

    Waring, Dennis W.; Turgeon, Judith L.

    2009-01-01

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

  10. 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

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

    PubMed Central

    Shahmoradi, Tahereh; Sepehry, Hamid; Ashrafpour, Manuchehr

    2016-01-01

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

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

    PubMed

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

    2016-01-01

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

  13. Ion Channels in Regulation of Neuronal Regenerative Activities

    PubMed Central

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

    2014-01-01

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

  14. 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.

  15. 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.

  16. 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.

  17. CRL4DCAF1 is required in activated oocytes for follicle maintenance and ovulation.

    PubMed

    Yu, Chao; Xu, Yi-Wen; Sha, Qian-Qian; Fan, Heng-Yu

    2015-02-01

    In mammals, oocytes within the primordial follicles require a number of essential factors to maintain their survival. However, the survival factors for activated oocytes have been poorly characterized. Recently we reported that damaged DNA binding protein-1 (DDB1), the linker subunit of the cullin ring-finger ubiquitin E3 ligase-4 (CRL4) complex, and its substrate adaptor, DDB1-CUL4 associated factor-1 (DCAF1), were essential for primordial follicle maintenance. In this study we specifically deleted these in the oocytes of growing follicles, to investigate if DDB1 and DCAF1 were also survival factors for activated oocytes. In the ovaries of Ddb1(fl/fl);Zp3-Cre mice, the primordial follicle pool was intact, but awakened oocytes and growing follicles beyond the primary stage were rapidly depleted. In the ovaries of Dcaf1(fl/fl);Pten(fl/fl);Gdf9-Cre and Ddb1(fl/fl);Pten(fl/fl);Gdf9-Cre mice, global primordial follicle activation was stimulated by enhanced PI3K signaling, but the awakened oocytes were rapidly lost due to no CRL4(DCAF1) activity. These mouse models provided original evidence that CRL4(DCAF1) was essential for maintaining oocyte survival, not only those in dormancy at the primordial follicle stage, but also naturally awakened oocytes and those awakened by hyper-activation of PI3K signaling. Interestingly, the oocyte-specific Ddb1 or Dcaf1 knockout mice had ovulation defects even before oocyte exhaustion. CRL4(DCAF1) within oocytes was required for cumulus expansion and ovulation-related somatic gene expression in a cell non-autonomous manner. Granulosa cells that surrounded these Ddb1 or Dcaf1-deleted oocytes exhibited increased rates of apoptosis and showed poor responses to ovulation signals. These results suggested that CRL4 in oocytes also regulated granulosa cell functions in a cell non-autonomous manner.

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

    PubMed

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

    2016-08-01

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

  19. 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

  20. Store-operated Ca2+ channels blockers inhibit lipopolysaccharide induced astrocyte activation.

    PubMed

    Li, Jian-Hua; Zhao, Shen-Ting; Wu, Cui-Ying; Cao, Xiong; Peng, Miao-Ru; Li, Shu-Ji; Liu, Xiao-Ai; Gao, Tian-Ming

    2013-10-01

    The destruction of calcium homeostasis is an important factor leading to neurological diseases. Store-operated Ca(2+) (SOC) channels are essential for Ca(2+) homeostasis in many cell types. However, whether SOC channels are involved in astrocyte activation induced by lipopolysaccharide (LPS) still remains unknown. In this study, we used LPS as an exogenous stimulation to investigate the role of SOC channels in astrocyte activation. Using calcium imaging technology, we first found that SOC channels blockers, 1-[h-[3-(4-methoxyphenyl)propoxy]-4-methoxyphenethyl]-1H-imidazole (SKF-96365) and 2-aminoethyldiphenyl borate (2-APB), inhibited LPS induced [Ca(2+)]i increase, which prompted us to speculate that SOC channels may be involved in LPS induced astrocyte activation. Further experiments confirmed our speculation shown as SOC channels blockers inhibited LPS induced astrocyte activation characterized as cell proliferation by MTS and BrdU assay, raise in glial fibrillary acidic protein expression by immunofluorescence and Western Blot and secretion of interleukin 6 (IL-6) and interleukin 1β (IL-1β) by ELISA. So, our studies showed that SOC channels are involved in LPS-induced astrocyte activation.

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

    PubMed

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

    1997-05-01

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

  2. 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

  3. HCN channels contribute to serotonergic modulation of ventral surface chemosensitive neurons and respiratory activity.

    PubMed

    Hawkins, Virginia E; Hawryluk, Joanna M; Takakura, Ana C; Tzingounis, Anastasios V; Moreira, Thiago S; Mulkey, Daniel K

    2015-02-15

    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.

  4. 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.

  5. 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

  6. Maintenance of plastid RNA editing activities independently of their target sites

    PubMed Central

    Tillich, Michael; Poltnigg, Peter; Kushnir, Sergei; Schmitz-Linneweber, Christian

    2006-01-01

    RNA editing in plant organelles is mediated by site-specific, nuclear-encoded factors. Previous data suggested that the maintenance of these factors depends on the presence of their rapidly evolving cognate sites. The surprising ability of allotetraploid Nicotiana tabacum (tobacco) to edit a foreign site in the chloroplast ndhA messenger RNA was thought to be inherited from its diploid male ancestor, Nicotiana tomentosiformis. Here, we show that the same ndhA editing activity is also present in Nicotiana sylvestris, which is the female diploid progenitor of tobacco and which lacks the ndhA site. Hence, heterologous editing is not simply a result of tobacco's allopolyploid genome organization. Analyses of other editing sites after sexual or somatic transfer between land plants showed that heterologous editing occurs at a surprisingly high frequency. This suggests that the corresponding editing activities are conserved despite the absence of their target sites, potentially because they serve other functions in the plant cell. PMID:16415790

  7. Evolutionarily conserved mechanisms for the selection and maintenance of behavioural activity.

    PubMed

    Fiore, Vincenzo G; Dolan, Raymond J; Strausfeld, Nicholas J; Hirth, Frank

    2015-12-19

    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

  8. 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

  9. 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

  10. Memory Maintenance in Synapses with Calcium-Based Plasticity in the Presence of Background Activity

    PubMed Central

    Higgins, David; Graupner, Michael; Brunel, Nicolas

    2014-01-01

    Most models of learning and memory assume that memories are maintained in neuronal circuits by persistent synaptic modifications induced by specific patterns of pre- and postsynaptic activity. For this scenario to be viable, synaptic modifications must survive the ubiquitous ongoing activity present in neural circuits in vivo. In this paper, we investigate the time scales of memory maintenance in a calcium-based synaptic plasticity model that has been shown recently to be able to fit different experimental data-sets from hippocampal and neocortical preparations. We find that in the presence of background activity on the order of 1 Hz parameters that fit pyramidal layer 5 neocortical data lead to a very fast decay of synaptic efficacy, with time scales of minutes. We then identify two ways in which this memory time scale can be extended: (i) the extracellular calcium concentration in the experiments used to fit the model are larger than estimated concentrations in vivo. Lowering extracellular calcium concentration to in vivo levels leads to an increase in memory time scales of several orders of magnitude; (ii) adding a bistability mechanism so that each synapse has two stable states at sufficiently low background activity leads to a further boost in memory time scale, since memory decay is no longer described by an exponential decay from an initial state, but by an escape from a potential well. We argue that both features are expected to be present in synapses in vivo. These results are obtained first in a single synapse connecting two independent Poisson neurons, and then in simulations of a large network of excitatory and inhibitory integrate-and-fire neurons. Our results emphasise the need for studying plasticity at physiological extracellular calcium concentration, and highlight the role of synaptic bi- or multistability in the stability of learned synaptic structures. PMID:25275319

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

    PubMed

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

    2014-07-01

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

  12. Hypoosmotic- and pressure-induced membrane stretch activate TRPC5 channels

    PubMed Central

    Gomis, Ana; Soriano, Sergio; Belmonte, Carlos; Viana, Félix

    2008-01-01

    Transient receptor potential (TRP) channels mediate a wide array of sensory functions. We investigated the role of TRPC5, a poorly characterized channel widely expressed in the central and peripheral nervous system, as a potential osmosensory protein. Here we show that hypoosmotic stimulation activates TRPC5 channels resulting in a large calcium influx. The response to osmotically induced membrane stretch is blocked by GsMTx-4, an inhibitor of stretch activated ion channels. Direct hypoosmotic activation of TRPC5 is independent of phospholipase C function. However, the osmotic response is inhibited in a cell line in which PIP2 levels are reduced by regulated overexpression of a lipid phosphatase. The response was restored by increasing intracellular PIP2 levels through the patch pipette. The mechano-sensitivity of the channel was probed in the whole-cell configuration by application of steps of positive pressure through the patch pipette. Pressure-induced membrane stretch also activated TRPC5 channels, suggesting its role as a transducer of osmo-mechanical stimuli. We also demonstrated the expression of TRPC5 in sensory neurones which together with the osmo-mechanical characteristics of TRPC5 channels suggest its putative role in mechanosensory transduction events. PMID:18832422

  13. 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

  14. 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

  15. 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.

  16. 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

  17. 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

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

    PubMed Central

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

    2012-01-01

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

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

    PubMed Central

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

    2016-01-01

    The key trigger for Hebbian synaptic plasticity is influx of Ca2+ into postsynaptic dendritic spines. The magnitude of [Ca2+] increase caused by NMDA-receptor (NMDAR) and voltage-gated Ca2+ -channel (VGCC) activation is thought to determine both the amplitude and direction of synaptic plasticity by differential activation of Ca2+ -sensitive enzymes such as calmodulin. Ca2+ influx is negatively regulated by Ca2+ -activated K+ channels (SK-channels) which are in turn inhibited by neuromodulators such as acetylcholine. However, the precise mechanisms by which SK-channels control the induction of synaptic plasticity remain unclear. Using a 3-dimensional model of Ca2+ and calmodulin dynamics within an idealised, but biophysically-plausible, dendritic spine, we show that SK-channels regulate calmodulin activation specifically during neuron-firing patterns associated with induction of spike timing-dependent plasticity. SK-channel activation and the subsequent reduction in Ca2+ influx through NMDARs and L-type VGCCs results in an order of magnitude decrease in calmodulin (CaM) activation, providing a mechanism for the effective gating of synaptic plasticity induction. This provides a common mechanism for the regulation of synaptic plasticity by neuromodulators. PMID:27232631

  20. 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

  1. Reduced Frontoparietal Activity in Schizophrenia Is Linked to a Specific Deficit in Goal Maintenance: A Multisite Functional Imaging Study.

    PubMed

    Poppe, Andrew B; Barch, Deanna M; Carter, Cameron S; Gold, James M; Ragland, John Daniel; Silverstein, Steven M; MacDonald, Angus W

    2016-09-01

    Patients with schizophrenia (SZ) previously demonstrated specific deficits in an executive function known as goal maintenance, associated with reduced middle frontal gyrus (MFG) activity. This study aimed to validate a new tool-the Dot Pattern Expectancy (DPX) task-developed to facilitate multisite imaging studies of goal maintenance deficits in SZ or other disorders. Additionally, it sought to arrive at recommendations for scan length for future studies using the DPX. Forty-seven SZ and 56 healthy controls (HC) performed the DPX in 3-Tesla functional magnetic resonance imaging (fMRI) scanners at 5 sites. Group differences in DPX-related activity were examined with whole brain voxelwise analyses. SZs showed the hypothesized specific performance deficits with as little as 1 block of data. Reduced activity in SZ compared with HC was observed in bilateral frontal pole/MFG, as well as left posterior parietal lobe. Efficiency analyses found significant group differences in activity using 18 minutes of scan data but not 12 minutes. Several behavioral and imaging findings from the goal maintenance literature were robustly replicated despite the use of different scanners at different sites. We did not replicate a previous correlation with disorganization symptoms among patients. Results were consistent with an executive/attention network dysfunction in the higher levels of a cascading executive system responsible for goal maintenance. Finally, efficiency analyses found that 18 minutes of scanning during the DPX task is sufficient to detect group differences with a similar sample size.

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

    PubMed

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

    2014-12-15

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

  3. Properties of cyclic nucleotide-gated channels mediating olfactory transduction. Activation, selectivity, and blockage

    PubMed Central

    1992-01-01

    Cyclic nucleotide-gated channels (cng channels) in the sensory membrane of olfactory receptor cells, activated after the odorant-induced increase of cytosolic cAMP concentration, conduct the receptor current that elicits electrical excitation of the receptor neurons. We investigated properties of cng channels from frog and rat using inside- out and outside-out membrane patches excised from isolated olfactory receptor cells. Channels were activated by cAMP and cGMP with activation constants of 2.5-4.0 microM for cAMP and 1.0-1.8 for cGMP. Hill coefficients of dose-response curves were 1.4-1.8, indicating cooperativity of ligand binding. Selectivity for monovalent alkali cations and the Na/Li mole-fraction behavior identified the channel as a nonselective cation channel, having a cation-binding site of high field strength in the pore. Cytosolic pH effects suggest the presence of an additional titratable group which, when protonated, inhibits the cAMP-induced current with an apparent pK of 5.0-5.2. The pH effects were not voltage dependent. Several blockers of Ca2+ channels also blocked olfactory cng channels. Amiloride, D 600, and diltiazem inhibited the cAMP-induced current from the cytosolic side. Inhibition constants were voltage dependent with values of, respectively, 0.1, 0.3, and 1 mM at -60 mV, and 0.03, 0.02, and 0.2 mM at +60 mV. Our results suggest functional similarity between frog and rat cng channels, as well as marked differences to cng channels from photoreceptors and other tissues. PMID:1324972

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

    PubMed Central

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

    2014-01-01

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

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

    PubMed Central

    2015-01-01

    The basal fungus Allomyces macrogynus (A. macrogynus) produces motile male gametes displaying well-studied chemotaxis toward their female counterparts. This chemotaxis is driven by sirenin, a sexual pheromone released by the female gametes. The pheromone evokes a large calcium influx in the motile gametes, which could proceed through the cation channel of sperm (CatSper) complex. Herein, we report the total synthesis of sirenin in 10 steps and 8% overall yield and show that the synthetic pheromone activates the CatSper channel complex, indicated by a concentration-dependent increase in intracellular calcium in human sperm. Sirenin activation of the CatSper channel was confirmed using whole-cell patch clamp electrophysiology with human sperm. Based on this proficient synthetic route and confirmed activation of CatSper, analogues of sirenin can be designed as blockers of the CatSper channel that could provide male contraceptive agents. PMID:26674547

  6. Calmodulin regulation of TMEM16A and 16B Ca(2+)-activated chloride channels.

    PubMed

    Yang, Tingting; Colecraft, Henry M

    2016-01-01

    Ca(2+)-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 Ca(2+) 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 Ca(2+)-free CaM (apoCaM) is pre-associated with functioning TMEM16A and 16B channel complexes in live cells. Further, the pre-associated apoCaM mediates Ca(2+)-dependent sensitization of activation (CDSA) and Ca(2+)-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 Ca(2+)-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.

  7. 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. PMID:27245091

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

    PubMed

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

    2016-01-01

    W1282X is a common nonsense mutation among cystic fibrosis patients that results in the production of a truncated Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) channel. Here we show that the channel activity of the W1282X-CFTR polypeptide is exceptionally low in excised membrane patches at normally saturating doses of ATP and PKA (single channel open probability (PO) < 0.01). However, W1282X-CFTR channels were stimulated by two CFTR modulators, the FDA-approved VX-770 and the dietary compound curcumin. Each of these compounds is an allosteric modulator of CFTR gating that promotes channel activity in the absence of the native ligand, ATP. Although W1282X-CFTR channels were stimulated by VX-770 in the absence of ATP their activities remained dependent on PKA phosphorylation. Thus, activated W1282X-CFTR channels should remain under physiologic control by cyclic nucleotide signaling pathways in vivo. VX-770 and curcumin exerted additive effects on W1282X-CFTR channel gating (opening/closing) in excised patches such that the Po of the truncated channel approached unity (> 0.9) when treated with both modulators. VX-770 and curcumin also additively stimulated W1282X-CFTR mediated currents in polarized FRT epithelial monolayers. In this setting, however, the stimulated W1282X-CFTR currents were smaller than those mediated by wild type CFTR (3-5%) due presumably to lower expression levels or cell surface targeting of the truncated protein. Combining allosteric modulators of different mechanistic classes is worth considering as a treatment option for W1282X CF patients perhaps when coupled with maneuvers to increase expression of the truncated protein. PMID:27007499

  9. 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

  10. Modulation of BK channel activities by calcium-sensing receptor in rat bronchopulmonary sensory neurons.

    PubMed

    Vysotskaya, Zhanna V; Moss, Charles R; Gilbert, Carolyn A; Gabriel, Sabry A; Gu, Qihai

    2014-11-01

    This study was carried out to investigate the expression of large-conductance Ca(2+)-activated potassium (BK) channels and to explore the possible modulation of BK channel activities by calcium-sensing receptors (CaSR) in rat bronchopulmonary sensory neurons. The expression of BK channels was demonstrated by immunohistochemistry and RT-PCR. Results from whole-cell patch-clamp recordings demonstrated that activation of CaSR with its agonist spermine or NPS R-568 showed a dual regulating effect on BK channel activities: it potentiated BK currents in cells exhibiting low baseline BK activity while slightly inhibited BK currents in cells with high baseline BK activity. Blocking CaSR with its antagonist NPS 2143 significantly inhibited BK currents. Our results further showed that the modulation of BK currents by CaSR activation or blockade was completely abolished when the intracellular Ca(2+) was chelated by BAPTA-AM. In summary, our data suggest that CaSR plays an integrative role in bronchopulmonary afferent signaling, at least partially through the regulation of BK channel activities.

  11. Blockade by local anaesthetics of the single Ca(2+)-activated K+ channel in rat hippocampal neurones.

    PubMed

    Oda, M; Yoshida, A; Ikemoto, Y

    1992-01-01

    1. Effects of local anaesthetics on single Ca(2+)-activated K+ channels were investigated using the inside-out configuration of the patch-clamp technique in single pyramidal neurones, which were freshly dissociated from rat hippocampus by use of proteolytic enzymes. 2. No significant effect was observed when 2 mM benzocaine was applied on either side of the membrane patch, or when 2 mM lignocaine or QX-314 was applied to the external surface of the membrane. 3. Lignocaine 1 mM, applied to the internal surface, slightly reduced the amplitude of the single K+ channel current. When applied to the internal surface QX-314 reduced the amplitude of the K+ channel current, accompanied by an increase in noise in the open channel current, suggesting a fast flickering block. The blocking effect of QX-314 on the outward current increased with depolarization, suggesting a binding site for the drug at an electrical distance of about 0.5 across the membrane field. 4. The open time histogram showed one exponential component and the closed time histogram showed at least two components. The mean open time of the outward current was increased when the amplitude was reduced by the drugs. 5. The ionized form of the local anaesthetics had a similar action on the Ca(2+)-activated K+ channels to that on Na+ channels, that is, they enter into the channel from the cytoplasmic side to induce open channel block. The blocking kinetics, however, might be so fast that they were beyond the frequency response of our recording apparatus, thus the recorded current amplitude was decreased. In contrast the K+ channel was not accessible via hydrophobic pathways for the neutral form, which is also known to block the sodium channel.

  12. 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.

  13. Alternative mechanism of activation of the epithelial na+ channel by cleavage.

    PubMed

    Hu, John Cong; Bengrine, Abderrahmane; Lis, Agnieszka; Awayda, Mouhamed S

    2009-12-25

    We examined activation of the human epithelial sodium channel (ENaC) by cleavage. We focused on cleavage of alphaENaC using the serine protease subtilisin. Trimeric channels formed with alphaFM, a construct with point mutations in both furin cleavage sites (R178A/R204A), exhibited marked reduction in spontaneous cleavage and an approximately 10-fold decrease in amiloride-sensitive whole cell conductance as compared with alphaWT (2.2 versus 21.2 microsiemens (microS)). Both alphaWT and alphaFM were activated to similar levels by subtilisin cleavage. Channels formed with alphaFD, a construct that deleted the segment between the two furin sites (Delta175-204), exhibited an intermediate conductance of 13.2 microS. More importantly, alphaFD retained the ability to be activated by subtilisin to 108.8 +/- 20.9 microS, a level not significantly different from that of subtilisin activated alphaWT (125.6 +/- 23.9). Therefore, removal of the tract between the two furin sites is not the main mechanism of channel activation. In these experiments the levels of the cleaved 22-kDa N-terminal fragment of alpha was low and did not match those of the C-terminal 65-kDa fragment. This indicated that cleavage may activate ENaC by the loss of the smaller fragment and the first transmembrane domain. This was confirmed in channels formed with alphaLD, a construct that extended the deleted sequence of alphaFD by 17 amino acids (Delta175-221). Channels with alphaLD were uncleaved, exhibited low baseline activity (4.1 microS), and were insensitive to subtilisin. Collectively, these data support an alternative hypothesis of ENaC activation by cleavage that may involve the loss of the first transmembrane domain from the channel complex. PMID:19858199

  14. Chronic hypoxia suppresses pregnancy-induced upregulation of large-conductance Ca2+-activated K+ channel activity in uterine arteries.

    PubMed

    Hu, Xiang-Qun; Xiao, Daliao; Zhu, Ronghui; Huang, Xiaohui; Yang, Shumei; Wilson, Sean M; Zhang, Lubo

    2012-07-01

    Our previous study demonstrated that increased Ca(2+)-activated K(+) (BK(Ca)) channel activity played a key role in the normal adaptation of reduced myogenic tone of uterine arteries in pregnancy. The present study tested the hypothesis that chronic hypoxia during gestation inhibits pregnancy-induced upregulation of BK(Ca) channel function in uterine arteries. Resistance-sized uterine arteries were isolated from nonpregnant and near-term pregnant sheep maintained at sea level (≈ 300 m) or exposed to high-altitude (3801 m) hypoxia for 110 days. Hypoxia during gestation significantly inhibited pregnancy-induced upregulation of BK(Ca) channel activity and suppressed BK(Ca) channel current density in pregnant uterine arteries. This was mediated by a selective downregulation of BK(Ca) channel β1 subunit in the uterine arteries. In accordance, hypoxia abrogated the role of the BK(Ca) channel in regulating pressure-induced myogenic tone of uterine arteries that was significantly elevated in pregnant animals acclimatized to chronic hypoxia. In addition, hypoxia abolished the steroid hormone-mediated increase in the β1 subunit and BK(Ca) channel current density observed in nonpregnant uterine arteries. Although the activation of protein kinase C inhibited BK(Ca) channel current density in pregnant uterine arteries of normoxic sheep, this effect was ablated in the hypoxic animals. The results demonstrate that selectively targeting BK(Ca) channel β1 subunit plays a critical role in the maladaption of uteroplacental circulation caused by chronic hypoxia, which contributes to the increased incidence of preeclampsia and fetal intrauterine growth restriction associated with gestational hypoxia. PMID:22665123

  15. Effects of antiarrhythmic drugs on the hyperpolarization-activated cyclic nucleotide-gated channel current.

    PubMed

    Tamura, Atsushi; Ogura, Takehiko; Uemura, Hiroko; Reien, Yoshie; Kishimoto, Takashi; Nagai, Toshio; Komuro, Issei; Miyazaki, Masaru; Nakaya, Haruaki

    2009-06-01

    After the report of the Cardiac Arrhythmia Suppression Trial, a tabular framework of the Sicilian Gambit has been proposed to display actions of antiarrhythmic drugs on ion channels and receptors and to provide more rational pharmacotherapy of arrhythmias. However, because effects of antiarrhythmic drugs on If have not been thoroughly examined, we used patch clamp techniques to determine the effects of various antiarrhythmic drugs on the HCN (hyperpolarization-activated cyclic nucleotide-gated) channel currents. HCN4 channels, a dominant isoform of HCN channels in the heart, were expressed in HEK293 cells. Amiodarone and bepridil potently inhibited the HCN4 channel current with IC50 values of 4.5 and 4.9 microM, respectively, which were close to their therapeutic concentrations. The inhibitory effects of quinidine, disopyramide, cibenzoline, lidocaine, mexiletine, aprindine, propafenone, flecainide, propranolol, and verapamil on the HCN4 channel current were weak in their therapeutic concentrations, with IC50 values of 78.3, 249, 46.8, 276, 309, 43.7, 14.3, 1700, 50.5, and 44.9 microM, respectively, suggesting that the inhibitory effects on If would be clinically small. D,L-Sotalol hardly affected the HCN4 channel current. Information about the HCN4-channel effects of many antiarrhythmic drugs may be useful for determining the appropriate drug for treatment of various arrhythmias while minimizing adverse effects. PMID:19498275

  16. Trimethyloxonium modification of batrachotoxin-activated Na channels alters functionally important protein residues.

    PubMed Central

    Cherbavaz, D B

    1995-01-01

    The extracellular side of single batrachotoxin-activated voltage-dependent Na channels isolated from rat skeletal muscle membranes incorporated into neutral planar lipid bilayers were treated in situ with the carboxyl methylating reagent, trimethyloxonium (TMO). These experiments were designed to determine whether TMO alters Na channel function by a general through-space electrostatic mechanism or by methylating specific carboxyl groups essential to channel function. TMO modification reduced single-channel conductance by decreasing the maximal turnover rate. Modification increased channel selectivity for sodium ions relative to potassium ions as measured under biionic conditions. TMO modification increased the mu-conotoxin (muCTX) off-rate by three orders of magnitude. Modification did not alter the muCTX on-rate at low ionic strength or Na channel voltage-dependent gating characteristics. These data demonstrate that TMO does not act via a general electrostatic mechanism. Instead, TMO targets protein residues specifically involved in ion conduction, ion selectivity, and muCTX binding. These data support the hypothesis that muCTX blocks open-channel current by physically obstructing the ion channel pore. PMID:7787022

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

    PubMed

    Zhang, Xiao-Xue; Min, Xiao-Chun; Xu, Xu-Lin; Zheng, Min; Guo, Lian-Jun

    2016-05-01

    The selective hyperpolarization-activated cyclic nucleotide-gated (HCN) channel blocker 4-(N-ethyl-N-phenylamino)-1,2-dimethyl-6-(methylamino) pyrimidinium chloride (ZD7288) blocks the induction of long-term potentiation in the perforant path-CA3 region in rat hippocampus in vivo. To explore the mechanisms underlying the action of ZD7288, we recorded excitatory postsynaptic potentials in perforant path-CA3 synapses in male Sprague-Dawley rats. We measured glutamate content in the hippocampus and in cultured hippocampal neurons using high performance liquid chromatography, and determined intracellular Ca(2+) concentration [Ca(2+)]i) using Fura-2. ZD7288 inhibited the induction and maintenance of long-term potentiation, and these effects were mirrored by the nonspecific HCN channel blocker cesium. ZD7288 also decreased glutamate release in hippocampal tissue and in cultured hippocampal neurons. Furthermore, ZD7288 attenuated glutamate-induced rises in [Ca(2+)]i in a concentration-dependent manner and reversed 8-Br-cAMP-mediated facilitation of these glutamate-induced [Ca(2+)]i rises. Our results suggest that ZD7288 inhibits hippocampal synaptic plasticity both glutamate release and resultant [Ca(2+)]i increases in rat hippocampal neurons. PMID:27335562

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

    PubMed

    Chen, Qijing; Tao, Jie; Hei, Hongya; Li, Fangping; Wang, Yunman; Peng, Wen; Zhang, Xuemei

    2015-01-01

    Large conductance Ca2+-activated potassium channels (BK) are targets for research that explores therapeutic means to various diseases, owing to the roles of the channels in mediating multiple physiological processes in various cells and tissues. We investigated the pharmacological effects of curcumin, a compound isolated from the herb Curcuma longa, on BK channels. As recorded by whole-cell patch-clamp, curcumin increased BK (α) and BK (α+β1) currents in transfected HEK293 cells as well as the current density of BK in A7r5 smooth muscle cells in a dose-dependent manner. By incubating with curcumin for 24 hours, the current density of exogenous BK (α) in HEK293 cells and the endogenous BK in A7r5 cells were both enhanced notably, though the steady-state activation of the channels did not shift significantly, except for BK (α+β1). Curcumin up-regulated the BK protein expression without changing its mRNA level in A7r5 cells. The surface expression and the half-life of BK channels were also increased by curcumin in HEK293 cells. These effects of curcumin were abolished by MG-132, a proteasome inhibitor. Curcumin also increased ERK 1/2 phosphorylation, while inhibiting ERK by U0126 attenuated the curcumin-induced up-regulation of BK protein expression. We also observed that the curcumin-induced relaxation in the isolated rat aortic rings was significantly attenuated by paxilline, a BK channel specific blocker. These results show that curcumin enhances the activity of the BK channels by interacting with BK directly as well as enhancing BK protein expression through inhibiting proteasomal degradation and activating ERK signaling pathway. The findings suggest that curcumin is a potential BK channel activator and provide novel insight into its complicated pharmacological effects and the underlying mechanisms. PMID:26672753

  19. [The activation effect of nobiletin on cystic fibrosis transmembrane conductance regulator chloride channel].

    PubMed

    Yang, Shuang; Yu, Bo; Zhang, Yao-Fang; Wang, Xue; Yang, Hong

    2013-06-01

    Aim of the present study is to investigate activation effect of nobiletin on cystic fibrosis transmembrane conductance regulator (CFTR) chloride channel activity. CFTR-mediated iodide influx assay and patch-clamp tests were done on FRT cells stably co-transfected with human CFTR and EYFP/H148Q. Nobiletin potently activated CFTR chloride channel activity in a dose- and time-dependent manner. The CFTR blocker CFTR(inh)-172 could completely reverse the effect. Preliminary mechanism study indicated that nobiletin activated CFTR chloride channel through a direct binding way. In addition, ex vivo tests done on mice trachea showed that nobiletin time-dependently stimulated submucosal gland fluid secretion. Nobiletin may be a therapeutic lead compound in treating CFTR-related diseases including disseminated bronchiectasis.

  20. Flow activates an endothelial potassium channel to release an endogenous nitrovasodilator.

    PubMed Central

    Cooke, J P; Rossitch, E; Andon, N A; Loscalzo, J; Dzau, V J

    1991-01-01

    Flow-mediated vasodilation is endothelium dependent. We hypothesized that flow activates a potassium channel on the endothelium, and that activation of this channel leads to the release of the endogenous nitrovasodilator, nitric oxide. To test this hypothesis, rabbit iliac arteries were perfused at varying flow rates, at a constant pressure of 60 mm Hg. Increments in flow induced proportional increases in vessel diameter, which were abolished by L,N-mono-methylarginine (the antagonist of nitric-oxide synthesis). Barium chloride, depolarizing solutions of potassium, verapamil, calcium-free medium, and antagonists of the KCa channel (charybdotoxin, iberiotoxin) also blocked flow-mediated vasodilation. Conversely, responses to other agonists of endothelium-dependent and independent vasodilation were unaffected by charybdotoxin or iberiotoxin. To confirm that flow activated a specific potassium channel to induce the release of nitric oxide, endothelial cells cultured on micro-carrier beads were added to a flow chamber containing a vascular ring without endothelium. Flow-stimulated endothelial cells released a diffusible vasodilator; the degree of vasorelaxation was dependent upon the flow rate. Relaxation was abrogated by barium, tetraethylammonium ion, or charybdotoxin, but was not affected by apamin, glybenclamide, tetrodotoxin, or ouabain. The data suggest that transmission of a hyperpolarizing current from endothelium to the vascular smooth muscle is not necessary for flow-mediated vasodilation. Flow activates a potassium channel (possibly the KCa channel) on the endothelial cell membrane that leads to the release of nitric oxide. Images PMID:1719029

  1. RIM Promotes Calcium Channel Accumulation at Active Zones of the Drosophila Neuromuscular Junction

    PubMed Central

    Graf, Ethan R.; Valakh, Vera; Wright, Christina M.; Wu, Chunlai; Liu, Zhihua; Zhang, Yong Q.; DiAntonio, Aaron

    2012-01-01

    Summary Synaptic communication requires the controlled release of synaptic vesicles from presynaptic axon terminals. Release efficacy is regulated by the many proteins that comprise the presynaptic release apparatus, including Ca2+ channels and proteins that influence Ca2+ channel accumulation at release sites. Here we identify Drosophila RIM and demonstrate that it localizes to active zones at the larval neuromuscular junction. In Drosophila RIM mutants, there is a large decrease in evoked synaptic transmission, due to a significant reduction in both the clustering of Ca2+ channels and the size of the readily releasable pool of synaptic vesicles at active zones. Hence, RIM plays an evolutionarily conserved role in regulating synaptic calcium channel localization and readily releasable pool size. Since RIM has traditionally been studied as an effector of Rab3 function, we investigate whether RIM is involved in the newly identified function of Rab3 in the distribution of presynaptic release machinery components across release sites. Bruchpilot (Brp), an essential component of the active zone cytomatrix T bar, is unaffected by RIM disruption, indicating that Brp localization and distribution across active zones does not require wild type RIM. In addition, larvae containing mutations in both RIM and rab3 have reduced Ca2+ channel levels and a Brp distribution that is very similar to that of the rab3 single mutant, indicating that RIM functions to regulate Ca2+ channel accumulation but is not a Rab3 effector for release machinery distribution across release sites. PMID:23175814

  2. Na+ activation of the muscarinic K+ channel by a G-protein-independent mechanism

    PubMed Central

    1996-01-01

    Muscarinic potassium channels (KACh) are composed of two subunits, GIRK1 and GIRK4 (or CIR), and are directly gated by G proteins. We have identified a novel gating mechanism of KACh, independent of G-protein activation. This mechanism involved functional modification of KACh which required hydrolysis of physiological levels of intracellular ATP and was manifested by an increase in the channel mean open time. The ATP-modified channels could in turn be gated by intracellular Na+, starting at approximately 3 mM with an EC50 of approximately 40 mM. The Na(+)-gating of KACh was operative both in native atrial cells and in a heterologous system expressing recombinant channel subunits. Block of the Na+/K+ pump (e.g., by cardiac glycosides) caused significant activation of KACh in atrial cells, with a time course similar to that of Na+ accumulation and in a manner indistinguishable from that of Na(+)-mediated activation of the channel, suggesting that cardiac glycosides activated KACh by increasing intracellular Na+ levels. These results demonstrate for the first time a direct effect of cardiac glycosides on atrial myocytes involving ion channels which are critical in the regulation of cardiac rhythm. PMID:8923264

  3. Long-term maintenance of channel distribution in a central pattern generator neuron by neuromodulatory inputs revealed by decentralization in organ culture.

    PubMed

    Mizrahi, A; Dickinson, P S; Kloppenburg, P; Fénelon, V; Baro, D J; Harris-Warrick, R M; Meyrand, P; Simmers, J

    2001-09-15

    Organotypic cultures of the lobster (Homarus gammarus) stomatogastric nervous system (STNS) were used to assess changes in membrane properties of neurons of the pyloric motor pattern-generating network in the long-term absence of neuromodulatory inputs to the stomatogastric ganglion (STG). Specifically, we investigated decentralization-induced changes in the distribution and density of the transient outward current, I(A), which is encoded within the STG by the shal gene and plays an important role in shaping rhythmic bursting of pyloric neurons. Using an antibody against lobster shal K(+) channels, we found shal immunoreactivity in the membranes of neuritic processes, but not somata, of STG neurons in 5 d cultured STNS with intact modulatory inputs. However, in 5 d decentralized STG, shal immunoreactivity was still seen in primary neurites but was likewise present in a subset of STG somata. Among the neurons displaying this altered shal localization was the pyloric dilator (PD) neuron, which remained rhythmically active in 5 d decentralized STG. Two-electrode voltage clamp was used to compare I(A) in synaptically isolated PD neurons in long-term decentralized STG and nondecentralized controls. Although the voltage dependence and kinetics of I(A) changed little with decentralization, the maximal conductance of I(A) in PD neurons increased by 43.4%. This increase was consistent with the decentralization-induced increase in shal protein expression, indicating an alteration in the density and distribution of functional A-channels. Our results suggest that, in addition to the short-term regulation of network function, modulatory inputs may also play a role, either directly or indirectly, in controlling channel number and distribution, thereby maintaining the biophysical character of neuronal targets on a long-term basis. PMID:11549743

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

    PubMed

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

    2016-02-01

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

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

    PubMed

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

    2012-02-19

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

  6. Glial potassium channels activated by neuronal firing or intracellular cyclic AMP in Helix.

    PubMed Central

    Gommerat, I; Gola, M

    1996-01-01

    1. Cell-attached and whole cell patch clamp experiments were performed on satellite glial cells adhering to the cell body of neurones in situ within the nervous system of the snail Helix pomatia. The underlying neurone was under current or voltage-clamp control. 2. Neuronal firing induced a delayed (20-30 s) persistent (3-4 min) increase in the opening probability of glial K+ channels. The channels were also activated by perfusing the ganglion with a depolarizing high-K+ saline, except when the underlying neurone was prevented from depolarizing under voltage-clamp conditions. 3. Two K(+)-selective channels were detected in the glial membrane. The channel responding to neuronal firing was present in 95% of the patches (n = 393). It had a unitary conductance of 56 pS, a Na+ :K+ permeability ratio < 0.02 and displayed slight inward rectification in symmetrical [K+] conditions. It was sensitive to TEA, Ba2+ and Cs+. The following results refer to this channel as studied in the cell-attached configuration. 4. The glial K+ channel was activated by bath application of the membrane-permeant cyclic AMP derivatives 8-bromo-cAMP and dibutyryl-cAMP, the adenylyl cyclase activator forskolin and the diesterase inhibitors IBMX, theophylline and caffeine. It was insensitive to cyclic GMP activators and to conditions that might alter the intracellular [Ca2+] (ionomycin, low-Ca2+ saline and Ca2+ channel blockers). 5. The forskolin-induced changes in channel behaviour (open and closed time distributions, burst duration, short and long gaps within bursts) could be accounted for by a four-state model (3 closed states, 1 open state) by simply changing one of the six rate parameters. 6. The present results suggest that the signal sent by an active neurone to satellite glial cells is confined to the glial cells round that neurone. The effect of this signal on the class of glial K+ channels studied can be mimicked by an increase in glial cAMP concentration. The subsequent delayed opening

  7. Nitric oxide directly activates calcium-dependent potassium channels in vascular smooth muscle.

    PubMed

    Bolotina, V M; Najibi, S; Palacino, J J; Pagano, P J; Cohen, R A

    1994-04-28

    Nitric oxide is the major endothelium-derived relaxing factor (EDRF), and it is thought to relax smooth muscle cells by stimulation of guanylate cyclase, accumulation of its product cyclic GMP, and cGMP-dependent modification of several intracellular processes, including activation of potassium channels through cGMP-dependent protein kinase. Here we present evidence that both exogenous nitric oxide and native EDRF can directly activate single Ca(2+)-dependent K+ channels (K+Ca) in cell-free membrane patches without requiring cGMP. Under conditions when guanylate cyclase was inhibited by methylene blue, considerable relaxation of rabbit aorta to nitric oxide persisted which was blocked by charybdotoxin, a specific inhibitor of K+Ca channels. These studies demonstrate a novel direct action of nitric oxide on K+Ca channels. PMID:7512692

  8. Faster voltage-dependent activation of Na+ channels in growth cones versus somata of neuroblastoma N1E-115 cells.

    PubMed Central

    Zhang, J; Loew, L M; Davidson, R M

    1996-01-01

    Kinetics of voltage-gated ionic channels fundamentally reflect the response of the channels to local electric fields. In this report cell-attached patch-clamp studies reveal that the voltage-dependent activation rate of sodium channels residing in the growth cone membrane differs from that of soma sodium channels in differentiating N1E-115 neuroblastoma cells. Because other electrophysiological properties of these channels do not differ, this finding may be a reflection of the difference in intramembrane electric field in these two regions of the cell. This represents a new mechanism for channels to attain a range of activities both within and between cells. PMID:8913589

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

    PubMed Central

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

    2015-01-01

    Functional expression of voltage-gated Na+ channels (VGSCs) has been demonstrated in multiple cancer cell types where channel activity induces invasive activity. The signaling mechanisms by which VGSCs promote oncogenesis remain poorly understood. We explored the signal transduction process critical to VGSC-mediated invasion on the basis of reports linking channel activity to gene expression changes in excitable cells. Coincidentally, many genes transcriptionally regulated by the SCN5A isoform in colon cancer have an over-representation of cis-acting sites for transcription factors phosphorylated by ERK1/2 MAPK. We hypothesized that VGSC activity promotes MAPK activation to induce transcriptional changes in invasion-related genes. Using pharmacological inhibitors/activators and siRNA-mediated gene knockdowns, we correlated channel activity with Rap1-dependent persistent MAPK activation in the SW620 human colon cancer cell line. We further demonstrated that VGSC activity induces downstream changes in invasion-related gene expression via a PKA/ERK/c-JUN/ELK-1/ETS-1 transcriptional pathway. This is the first study illustrating a molecular mechanism linking functional activity of VGSCs to transcriptional activation of invasion-related genes. PMID:26096612

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

    NASA Astrophysics Data System (ADS)

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

    2015-06-01

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

  11. STIM1 dimers undergo unimolecular coupling to activate Orai1 channels

    PubMed Central

    Zhou, Yandong; Wang, Xizhuo; Wang, Xianming; Loktionova, Natalia A.; Cai, Xiangyu; Nwokonko, Robert M.; Vrana, Erin; Wang, Youjun; Rothberg, Brad S.; Gill, Donald L.

    2015-01-01

    The endoplasmic reticulum (ER) Ca2+ sensor, STIM1, becomes activated when ER-stored Ca2+ is depleted and translocates into ER–plasma membrane junctions where it tethers and activates Orai1 Ca2+ entry channels. The dimeric STIM1 protein contains a small STIM-Orai-activating region (SOAR)—the minimal sequence sufficient to activate Orai1 channels. Since SOAR itself is a dimer, we constructed SOAR concatemer–dimers and introduced mutations at F394, which is critical for Orai1 coupling and activation. The F394H mutation in both SOAR monomers completely blocks dimer function, but F394H introduced in only one of the dimeric SOAR monomers has no effect on Orai1 binding or activation. This reveals an unexpected unimolecular coupling between STIM1 and Orai1 and argues against recent evidence suggesting dimeric interaction between STIM1 and two adjacent Orai1 channel subunits. The model predicts that STIM1 dimers may be involved in crosslinking between Orai1 channels with implications for the kinetics and localization of Orai1 channel opening. PMID:26399906

  12. STIM1 dimers undergo unimolecular coupling to activate Orai1 channels

    NASA Astrophysics Data System (ADS)

    Zhou, Yandong; Wang, Xizhuo; Wang, Xianming; Loktionova, Natalia A.; Cai, Xiangyu; Nwokonko, Robert M.; Vrana, Erin; Wang, Youjun; Rothberg, Brad S.; Gill, Donald L.

    2015-09-01

    The endoplasmic reticulum (ER) Ca2+ sensor, STIM1, becomes activated when ER-stored Ca2+ is depleted and translocates into ER-plasma membrane junctions where it tethers and activates Orai1 Ca2+ entry channels. The dimeric STIM1 protein contains a small STIM-Orai-activating region (SOAR)--the minimal sequence sufficient to activate Orai1 channels. Since SOAR itself is a dimer, we constructed SOAR concatemer-dimers and introduced mutations at F394, which is critical for Orai1 coupling and activation. The F394H mutation in both SOAR monomers completely blocks dimer function, but F394H introduced in only one of the dimeric SOAR monomers has no effect on Orai1 binding or activation. This reveals an unexpected unimolecular coupling between STIM1 and Orai1 and argues against recent evidence suggesting dimeric interaction between STIM1 and two adjacent Orai1 channel subunits. The model predicts that STIM1 dimers may be involved in crosslinking between Orai1 channels with implications for the kinetics and localization of Orai1 channel opening.

  13. GIRK channel activation via adenosine or muscarinic receptors has similar effects on rat atrial electrophysiology.

    PubMed

    Wang, Xiaodong; Liang, Bo; Skibsbye, Lasse; Olesen, Søren-Peter; Grunnet, Morten; Jespersen, Thomas

    2013-08-01

    G protein-coupled inwardly rectifying K⁺ channels (GIRK) are important in the regulation of heart rate and atrial electrophysiology. GIRK channels are activated by G protein-coupled receptors, including muscarinic M₂ receptors and adenosine A₁ receptors. The aim of this study was to characterize and compare the electrophysiological effects of acetylcholine (ACh) and adenosine on GIRK channels in rat atria. Action potential duration at 90% repolarization (APD₉₀), effective refractory period (ERP), and resting membrane potential (RMP) were investigated in isolated rat atria by intracellular recordings. Both the adenosine analog N6-cyclopentyladenosine (CPA) and ACh profoundly shortened APD₉₀ and ERP and hyperpolarized the RMP. No additive or synergistic effect of CPA and ACh coapplication was observed. To antagonize GIRK channel activation, the specific inhibitor rTertiapin Q (TTQ) was applied. The coapplication of TTQ reversed the CPA and ACh-induced effects. When TTQ was applied without exogenous receptor activator, both APD₉₀ and ERP were prolonged and RMP was depolarized, confirming a basal activity of the GIRK current. The results reveal that activation of A₁ and M₂ receptors has a profound and equal effect on the electrophysiology in rat atrium. This effect is to a major extent mediated through GIRK channels. Furthermore, these results support the notion that atrial GIRK currents from healthy hearts have a basal component and additional activation can be mediated via at least 2 different receptor mechanisms. PMID:23609329

  14. Kinetics of xylem loading, membrane potential maintenance, and sensitivity of K(+) -permeable channels to reactive oxygen species: physiological traits that differentiate salinity tolerance between pea and barley.

    PubMed

    Bose, Jayakumar; Shabala, Lana; Pottosin, Igor; Zeng, Fanrong; Velarde-Buendía, Ana-Maria; Massart, Amandine; Poschenrieder, Charlotte; Hariadi, Yuda; Shabala, Sergey

    2014-03-01

    Salt sensitive (pea) and salt tolerant (barley) species were used to understand the physiological basis of differential salinity tolerance in crops. Pea plants were much more efficient in restoring otherwise depolarized membrane potential thereby effectively decreasing K(+) efflux through depolarization-activated outward rectifying potassium channels. At the same time, pea root apex was 10-fold more sensitive to physiologically relevant H2 O2 concentration and accumulated larger amounts of H2 O2 under saline conditions. This resulted in a rapid loss of cell viability in the pea root apex. Barley plants rapidly loaded Na(+) into the xylem; this increase was only transient, and xylem and leaf Na(+) concentration remained at a steady level for weeks. On the contrary, pea plants restricted xylem Na(+) loading during the first few days of treatment but failed to prevent shoot Na(+) elevation in the long term. It is concluded that superior salinity tolerance of barley plants compared with pea is conferred by at least three different mechanisms: (1) efficient control of xylem Na(+) loading; (2) efficient control of H2 O2 accumulation and reduced sensitivity of non-selective cation channels to H2 O2 in the root apex; and (3) higher energy saving efficiency, with less ATP spent to maintain membrane potential under saline conditions.

  15. Proton Channel Activity of Influenza A Virus Matrix Protein 2 Contributes to Autophagy Arrest

    PubMed Central

    Ren, Yizhong; Feng, Liqiang; Pan, Weiqi; Li, Liang; Wang, Qian; Li, Jiashun; Li, Na; Han, Ling; Zheng, Xuehua; Niu, Xuefeng; Sun, Caijun

    2015-01-01

    Influenza A virus infection can arrest autophagy, as evidenced by autophagosome accumulation in infected cells. Here, we report that this autophagosome accumulation can be inhibited by amantadine, an antiviral proton channel inhibitor, in amantadine-sensitive virus infected cells or cells expressing influenza A virus matrix protein 2 (M2). Thus, M2 proton channel activity plays a role in blocking the fusion of autophagosomes with lysosomes, which might be a key mechanism for arresting autophagy. PMID:26468520

  16. Development of a Theory-Based Internet Program Promoting Maintenance of Diet and Physical Activity Change to 8-Year-Old African American Girls

    ERIC Educational Resources Information Center

    Thompson, Debbe; Baranowski, Janice; Cullen, Karen; Baranowski, Tom

    2007-01-01

    Obesity and chronic disease risk factors are rising among youth. The Internet offers promise as a channel for delivering behavior change programs in a manner that is both available and accessible. This manuscript describes how theory informed the development of an Internet-based program promoting the maintenance of healthy eating and physical…

  17. Variomics screen identifies the re-entrant loop of the calcium-activated chloride channel ANO1 that facilitates channel activation.

    PubMed

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

    2015-01-01

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

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

    PubMed Central

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

    2015-01-01

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

  19. Role of calcium-activated potassium channels in transmitter release at the squid giant synapse.

    PubMed Central

    Augustine, G J; Charlton, M P; Horn, R

    1988-01-01

    1. Several compounds known to block Ca2+-activated K+ channels were microinjected into squid 'giant' presynaptic terminals to test the hypothesis that these channels mediate Ca2+-dependent neurotransmitter release. 2. Injection of tetrapentylammonium, nonyl-triethylammonium and decamethonium all reversibly blocked transmission evoked by presynaptic action potentials. 3. All three of these compounds blocked presynaptic Ca2+ channels. The actions of tetrapentylammonium on presynaptic Ca2+ influx were examined in detail and found to be quantitatively sufficient to account for the ability of this compound to inhibit transmitter release. 4. Injection of Ba2+, another agent known to block Ca2+-activated K+ channels, also reversibly blocked evoked transmitter release. Ba2+ simultaneously enhanced basal (asynchronous) transmitter release and thus may be decreasing evoked release by depleting transmitter quanta available for release. 5. None of these results provide any support for the hypothesis that Ca2+-activated K+ channels mediate Ca2+-dependent release of transmitter at the squid synapse. However, our results have identified a new class of compounds that block Ca2+ channels from their cytoplasmic surface. PMID:2455797

  20. Enhanced activation of the transient receptor potential channel TRPA1 by ajoene, an allicin derivative.

    PubMed

    Yassaka, Ricardo Tsuneo; Inagaki, Hidetoshi; Fujino, Tsuchiyoshi; Nakatani, Kei; Kubo, Tai

    2010-01-01

    TRPA1 is a calcium-permeable, nonselective cation channel expressed in the dorsal root ganglion and trigeminal ganglia nociceptive neurons. It is activated by the pungent compounds in mustard oil (AITC, allyl isothiocyanate), cinnamon (cinnamaldehyde), garlic (allicin), and is believed to mediate the inflammatory actions of environmental irritants and proalgesic agents. Thiosulfinate (allicin) and isothiocyanate (AITC) compounds contain reactive electrophilic chemical groups that react with cysteine residues within the TRPA1 channel N terminus, leading to channel activation. Ajoene also contains reactive electrophilic chemical groups likely to target TRPA1 channel. Here, we have used voltage-clamp recordings to show that TRPA1-responses are enhanced by ajoene application in a Xenopus oocyte expression system. Though ajoene alone did not activate TRPA1, subsequent application of ajoene enhanced the AITC-, allicin- and depolarization-induced responses of TRPA1. Moreover, when increasing concentrations of ajoene were applied along with constant concentrations of allicin or AITC, stronger responses were elicited. These findings suggest that ajoene is a novel TRPA1 channel enhancer, operating in a channel-opening-dependent manner.

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

    PubMed

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

    2009-05-01

    The sensation of cold or heat depends on the activation of specific nerve endings in the skin. This involves heat- and cold-sensitive excitatory transient receptor potential (TRP) channels. However, we show here that the mechano-gated and highly temperature-sensitive potassium channels of the TREK/TRAAK family, which normally work as silencers of the excitatory channels, are also implicated. They are important for the definition of temperature thresholds and temperature ranges in which excitation of nociceptor takes place and for the intensity of excitation when it occurs. They are expressed with thermo-TRP channels in sensory neurons. TRAAK and TREK-1 channels control pain produced by mechanical stimulation and both heat and cold pain perception in mice. Expression of TRAAK alone or in association with TREK-1 controls heat responses of both capsaicin-sensitive and capsaicin-insensitive sensory neurons. Together TREK-1 and TRAAK channels are important regulators of nociceptor activation by cold, particularly in the nociceptor population that is not activated by menthol.

  2. Apical Ca2+-activated potassium channels in mouse parotid acinar cells.

    PubMed

    Almassy, Janos; Won, Jong Hak; Begenisich, Ted B; Yule, David I

    2012-02-01

    Ca(2+) activation of Cl and K channels is a key event underlying stimulated fluid secretion from parotid salivary glands. Cl channels are exclusively present on the apical plasma membrane (PM), whereas the localization of K channels has not been established. Mathematical models have suggested that localization of some K channels to the apical PM is optimum for fluid secretion. A combination of whole cell electrophysiology and temporally resolved digital imaging with local manipulation of intracellular [Ca(2+)] was used to investigate if Ca(2+)-activated K channels are present in the apical PM of parotid acinar cells. Initial experiments established Ca(2+)-buffering conditions that produced brief, localized increases in [Ca(2+)] after focal laser photolysis of caged Ca(2+). Conditions were used to isolate K(+) and Cl(-) conductances. Photolysis at the apical PM resulted in a robust increase in K(+) and Cl(-) currents. A localized reduction in [Ca(2+)] at the apical PM after photolysis of Diazo-2, a caged Ca(2+) chelator, resulted in a decrease in both K(+) and Cl(-) currents. The K(+) currents evoked by apical photolysis were partially blocked by both paxilline and TRAM-34, specific blockers of large-conductance "maxi-K" (BK) and intermediate K (IK), respectively, and almost abolished by incubation with both antagonists. Apical TRAM-34-sensitive K(+) currents were also observed in BK-null parotid acini. In contrast, when the [Ca(2+)] was increased at the basal or lateral PM, no increase in either K(+) or Cl(-) currents was evoked. These data provide strong evidence that K and Cl channels are similarly distributed in the apical PM. Furthermore, both IK and BK channels are present in this domain, and the density of these channels appears higher in the apical versus basolateral PM. Collectively, this study provides support for a model in which fluid secretion is optimized after expression of K channels specifically in the apical PM.

  3. Pyrethroid modifications of the activation and inactivation kinetics of the sodium channels in squid giant axons.

    PubMed

    de Weille, J R; Brown, L D; Narahashi, T

    1990-03-26

    The kinetics of sodium channel activation and inactivation were analyzed in the squid giant axons internally treated with various pyrethroids. Pyrethroids increased the steady-state sodium current in squid giant axons by removing the inactivation. The steady-state sodium conductances in control and pyrethroid-treated axons showed the same voltage dependence, indicating that the removal of inactivation by pyrethroids did not lead to an alteration of gating charge transfer. The pyrethroid-modified sodium channels were activated with a biphasic time course involving the movement of at least two gating particles, and both components were voltage-dependent. The slower component was abolished by treatment with either pronase or N-bromoacetamide. The net elementary charges transported in the electric membrane field were reduced in the course of slow activation of the pyrethroid-induced sodium current. It appears that the 'immobilization' of gating charge is related to the slow activation rather than the inactivation of the sodium channel.

  4. KCNQ channel openers reverse depressive symptoms via an active resilience mechanism

    PubMed Central

    Friedman, Allyson K.; Juarez, Barbara; Ku, Stacy M.; Zhang, Hongxing; Calizo, Rhodora C.; Walsh, Jessica J.; Chaudhury, Dipesh; Zhang, Song; Hawkins, Angel; Dietz, David M.; Murrough, James W.; Ribadeneira, Maria; Wong, Erik H.; Neve, Rachael L.; Han, Ming-Hu

    2016-01-01

    Less than half of patients suffering from major depressive disorder, a leading cause of disability worldwide, achieve remission with current antidepressants, making it imperative to develop more effective treatment. A new therapeutic direction is emerging from the increased understanding of natural resilience as an active stress-coping process. It is known that potassium (K+) channels in the ventral tegmental area (VTA) are an active mediator of resilience. However, no druggable targets have been identified to potentiate active resilience mechanisms. In the chronic social defeat stress model of depression, we report that KCNQ-type K+ channel openers, including FDA-approved drug retigabine (ezogabine), show antidepressant efficacy. We demonstrate that overexpression of KCNQ channels in the VTA dopaminergic neurons and either local infusion or systemic administration of retigabine normalized neuronal hyperactivity and depressive behaviours. These findings identify KCNQ as a target for conceptually novel antidepressants that function through the potentiation of active resilience mechanisms. PMID:27216573

  5. Space exploration by dendritic cells requires maintenance of myosin II activity by IP3 receptor 1.

    PubMed

    Solanes, Paola; Heuzé, Mélina L; Maurin, Mathieu; Bretou, Marine; Lautenschlaeger, Franziska; Maiuri, Paolo; Terriac, Emmanuel; Thoulouze, Maria-Isabel; Launay, Pierre; Piel, Matthieu; Vargas, Pablo; Lennon-Duménil, Ana-Maria

    2015-03-12

    Dendritic cells (DCs) patrol the interstitial space of peripheral tissues. The mechanisms that regulate their migration in such constrained environment remain unknown. We here investigated the role of calcium in immature DCs migrating in confinement. We found that they displayed calcium oscillations that were independent of extracellular calcium and more frequently observed in DCs undergoing strong speed fluctuations. In these cells, calcium spikes were associated with fast motility phases. IP₃ receptors (IP₃Rs) channels, which allow calcium release from the endoplasmic reticulum, were identified as required for immature DCs to migrate at fast speed. The IP₃R1 isoform was further shown to specifically regulate the locomotion persistence of immature DCs, that is, their capacity to maintain directional migration. This function of IP₃R1 results from its ability to control the phosphorylation levels of myosin II regulatory light chain (MLC) and the back/front polarization of the motor protein. We propose that by upholding myosin II activity, constitutive calcium release from the ER through IP₃R1 maintains DC polarity during migration in confinement, facilitating the exploration of their environment.

  6. Analysis of G-protein-activated inward rectifying K(+) (GIRK) channel currents upon GABAB receptor activation in rat supraoptic neurons.

    PubMed

    Harayama, Nobuya; Kayano, Tomohiko; Moriya, Taiki; Kitamura, Naoki; Shibuya, Izumi; Tanaka-Yamamoto, Keiko; Uezono, Yasuhito; Ueta, Yoichi; Sata, Takeyoshi

    2014-12-01

    While magnocellular neurons in the supraoptic nucleus (SON) possess rich Gi/o-mediated mechanisms, molecular and cellular properties of G-protein-activated inwardly rectifying K(+) (GIRK) channels have been controversial. Here, properties of GIRK channels are examined by RT-PCR and whole-cell patch-clamp techniques in rat SON neurons. Patch clamp experiments showed that the selective GABAB agonist, baclofen, enhanced currents in a high K(+) condition. The baclofen-enhanced currents exhibited evident inward rectification and were blocked by the selective GABAB antagonist, CGP55845A, the IRK channel blocker, Ba(2+), and the selective GIRK channel blocker, tertiapin, indicating that baclofen activates GIRK channels via GABAB receptors. The GIRK currents were abolished by N-ethylmaleimide pretreatment, and prolonged by GTPγS inclusion in the patch pipette, suggesting that Gi/o proteins are involved. RT-PCR analysis revealed mRNAs for all four GIRK 1-4 channels and for both GABABR1 and GABABR2 receptors in rat SON. However, the concentration-dependency of the baclofen-induced activation of GIRK currents had an EC50 of 110 µM, which is about 100 times higher than that of baclofen-induced inhibition of voltage-dependent Ca(2+) channels. Moreover, baclofen caused no significant changes in the membrane potential and the firing rate. These results suggest that although GIRK channels can be activated by GABAB receptors via the Gi/o pathway, this occurs at high agonist concentrations, and thus may not be a physiological mechanism regulating the function of SON neurons. This property that the membrane potential receives little influence from GIRK currents seems to be uncommon for CNS neurons possessing rich Gi/o-coupled receptors, and could be a special feature of rat SON neurons.

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

    PubMed

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

    2016-01-01

    Secretory diarrhea remains a global health burden and causes major mortality in children. There have been some focuses on antidiarrheal therapies that may reduce fluid losses and intestinal motility in diarrheal diseases. In the present study, we identified shikonin as an inhibitor of TMEM16A chloride channel activity using cell-based fluorescent-quenching assay. The IC50 value of shikonin was 6.5 μM. Short-circuit current measurements demonstrated that shikonin inhibited Eact-induced Cl(-) current in a dose-dependent manner, with IC50 value of 1.5 μM. Short-circuit current measurement showed that shikonin exhibited inhibitory effect against CCh-induced Cl(-) currents in mouse colonic epithelia but did not affect cytoplasmic Ca(2+) concentration as well as the other major enterocyte chloride channel conductance regulator. Characterization study found that shikonin inhibited basolateral K(+) channel activity without affecting Na(+)/K(+)-ATPase activities. In vivo studies revealed that shikonin significantly delayed intestinal motility in mice and reduced stool water content in a neonatal mice model of rotaviral diarrhea without affecting the viral infection process in vivo. Taken together, the results suggested that shikonin inhibited enterocyte calcium-activated chloride channels, the inhibitory effect was partially through inhbition of basolateral K(+) channel activity, and shikonin could be a lead compound in the treatment of rotaviral secretory diarrhea.

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

    PubMed Central

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

    2014-01-01

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

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

    PubMed Central

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

    2016-01-01

    Secretory diarrhea remains a global health burden and causes major mortality in children. There have been some focuses on antidiarrheal therapies that may reduce fluid losses and intestinal motility in diarrheal diseases. In the present study, we identified shikonin as an inhibitor of TMEM16A chloride channel activity using cell-based fluorescent-quenching assay. The IC50 value of shikonin was 6.5 μM. Short-circuit current measurements demonstrated that shikonin inhibited Eact-induced Cl- current in a dose-dependent manner, with IC50 value of 1.5 μM. Short-circuit current measurement showed that shikonin exhibited inhibitory effect against CCh-induced Cl- currents in mouse colonic epithelia but did not affect cytoplasmic Ca2+ concentration as well as the other major enterocyte chloride channel conductance regulator. Characterization study found that shikonin inhibited basolateral K+ channel activity without affecting Na+/K+-ATPase activities. In vivo studies revealed that shikonin significantly delayed intestinal motility in mice and reduced stool water content in a neonatal mice model of rotaviral diarrhea without affecting the viral infection process in vivo. Taken together, the results suggested that shikonin inhibited enterocyte calcium-activated chloride channels, the inhibitory effect was partially through inhbition of basolateral K+ channel activity, and shikonin could be a lead compound in the treatment of rotaviral secretory diarrhea. PMID:27601995

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

    PubMed

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

    2016-01-01

    Secretory diarrhea remains a global health burden and causes major mortality in children. There have been some focuses on antidiarrheal therapies that may reduce fluid losses and intestinal motility in diarrheal diseases. In the present study, we identified shikonin as an inhibitor of TMEM16A chloride channel activity using cell-based fluorescent-quenching assay. The IC50 value of shikonin was 6.5 μM. Short-circuit current measurements demonstrated that shikonin inhibited Eact-induced Cl(-) current in a dose-dependent manner, with IC50 value of 1.5 μM. Short-circuit current measurement showed that shikonin exhibited inhibitory effect against CCh-induced Cl(-) currents in mouse colonic epithelia but did not affect cytoplasmic Ca(2+) concentration as well as the other major enterocyte chloride channel conductance regulator. Characterization study found that shikonin inhibited basolateral K(+) channel activity without affecting Na(+)/K(+)-ATPase activities. In vivo studies revealed that shikonin significantly delayed intestinal motility in mice and reduced stool water content in a neonatal mice model of rotaviral diarrhea without affecting the viral infection process in vivo. Taken together, the results suggested that shikonin inhibited enterocyte calcium-activated chloride channels, the inhibitory effect was partially through inhbition of basolateral K(+) channel activity, and shikonin could be a lead compound in the treatment of rotaviral secretory diarrhea. PMID:27601995

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

    PubMed Central

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

    2016-01-01

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

  12. SecA Alone Can Promote Protein Translocation and Ion Channel Activity

    PubMed Central

    Hsieh, Ying-hsin; Zhang, Hao; Lin, Bor-ruei; Cui, Ningren; Na, Bing; Yang, Hsiuchin; Jiang, Chun; Sui, Sen-fang; Tai, Phang C.

    2011-01-01

    SecA is an essential component of the Sec-dependent protein translocation pathway across cytoplasmic membranes in bacteria. Escherichia coli SecA binds to cytoplasmic membranes at SecYEG high affinity sites and at phospholipid low affinity sites. It has been widely viewed that SecYEG functions as the essential protein-conducting channel through which precursors cross the membranes in bacterial Sec-dependent pathways, and that SecA functions as a motor to hydrolyze ATP in translocating precursors through SecYEG channels. We have now found that SecA alone can promote precursor translocation into phospholiposomes. Moreover, SecA-liposomes elicit ionic currents in Xenopus oocytes. Patch-clamp recordings further show that SecA alone promotes signal peptide- or precursor-dependent single channel activity. These activities were observed with the functional SecA at about 1–2 μm. The results show that SecA alone is sufficient to promote protein translocation into liposomes and to elicit ionic channel activity at the phospholipids low affinity binding sites, thus indicating that SecA is able to form the protein-conducting channels. Even so, such SecA-liposomes are less efficient than those with a full complement of Sec proteins, and lose the signal-peptide proofreading function, resembling the effects of PrlA mutations. Addition of purified SecYEG restores the signal peptide specificity and increases protein translocation and ion channel activities. These data show that SecA can promote protein translocation and ion channel activities both when it is bound to lipids at low affinity sites and when it is bound to SecYEG with high affinity. The latter of the two interactions confers high efficiency and specificity. PMID:22033925

  13. The bile acid-sensitive ion channel (BASIC) is activated by alterations of its membrane environment.

    PubMed

    Schmidt, Axel; Lenzig, Pia; Oslender-Bujotzek, Adrienne; Kusch, Jana; Lucas, Susana Dias; Gründer, Stefan; Wiemuth, Dominik

    2014-01-01

    The bile acid-sensitive ion channel (BASIC) is a member of the DEG/ENaC family of ion channels. Channels of this family are characterized by a common structure, their physiological functions and modes of activation, however, are diverse. Rat BASIC is expressed in brain, liver and intestinal tract and activated by bile acids. The physiological function of BASIC and its mechanism of bile acid activation remain a puzzle. Here we addressed the question whether amphiphilic bile acids activate BASIC by directly binding to the channel or indirectly by altering the properties of the surrounding membrane. We show that membrane-active substances other than bile acids also affect the activity of BASIC and that activation by bile acids and other membrane-active substances is non-additive, suggesting that BASIC is sensitive for changes in its membrane environment. Furthermore based on results from chimeras between BASIC and ASIC1a, we show that the extracellular and the transmembrane domains are important for membrane sensitivity.

  14. Discovery, structure-activity relationship study, and oral analgesic efficacy of cyproheptadine derivatives possessing N-type calcium channel inhibitory activity.

    PubMed

    Yamamoto, Takashi; Niwa, Seiji; Iwayama, Satoshi; Koganei, Hajime; Fujita, Shin-ichi; Takeda, Tomoko; Kito, Morikazu; Ono, Yukitsugu; Saitou, Yuki; Takahara, Akira; Iwata, Seinosuke; Yamamoto, Hiroshi; Shoji, Masataka

    2006-08-01

    Antiallergic drug cyproheptadine (Cyp) is known to have inhibitory activities for L-type calcium channels in addition to histamine and serotonin receptors. Since we found that Cyp had an inhibitory activity against N-type calcium channel, Cyp was optimized to obtain more selective N-type calcium channel blocker with analgesic action. As a consequence of the optimization, we found 13 with potent N-type calcium channel inhibitory activity which had lower inhibitory activities against L-type calcium channel, histamine (H1), and serotonin (5-HT2A) receptors than those of Cyp. 13 showed an oral analgesic activity in rat formalin-induced pain model.

  15. Influence of proline position upon the ion channel activity of alamethicin.

    PubMed Central

    Kaduk, C; Duclohier, H; Dathe, M; Wenschuh, H; Beyermann, M; Molle, G; Bienert, M

    1997-01-01

    Alamethicin, a 20-residue peptaibol, induces voltage-dependent ion channels in lipid bilayers according to the barrel-stave model. To study relationships between the proline-14-induced kink region and the channel-forming behavior of the peptide, a set of alamethicin analogs with proline incorporated at positions 11, 12, 13, 14, 15, 16, and 17, respectively, as well as an analog with alanine instead of proline at position 14 were synthesized. Macroscopic conductance experiments show that the voltage dependence of the peptides is conserved although slightly influenced, but the apparent mean number of monomers forming the channels is significantly reduced when proline is not located at position 14. This is confirmed in single-channel experiments. The analogs with proline next to position 14 (i.e., 13, 15, 16) show stable conductance levels, but of reduced number, which follows the order Alam-P14 > Alam-P15 > Alam-P16 > Alam-P13. This reduction in the number of levels is connected with changes in the lifetime of the channels. Analogs with proline at position 11, 12, or 17 produce erratic, extremely short-lived current events that could not be resolved. The changes in functional properties are related to structural properties as probed by circular dichroism. The results indicate that proline at position 14 results in optimal channel activity, whereas channels formed by the analogs bearing proline at different positions are considerably less stable. PMID:9129817

  16. TRPM8 Channel Activation Induced by Monoterpenoid Rotundifolone Underlies Mesenteric Artery Relaxation

    PubMed Central

    Silva, Darizy Flavia; de Almeida, Monica Moura; Chaves, Cinthia Guedes; Braz, Ana Letícia; Gomes, Maria Aparecida; Pinho-da-Silva, Leidiane; Pesquero, Jorge Luiz; Andrade, Viviane Aguiar; Leite, Maria de Fátima; de Albuquerque, José George Ferreira; Araujo, Islania Giselia Albuquerque; Nunes, Xirley Pereira; Barbosa-Filho, José Maria; Cruz, Jader dos Santos; Correia, Nadja de Azevedo; de Medeiros, Isac Almeida

    2015-01-01

    In this study, our aims were to investigate transient receptor potential melastatin-8 channels (TRPM8) involvement in rotundifolone induced relaxation in the mesenteric artery and to increase the understanding of the role of these thermosensitive TRP channels in vascular tissue. Thus, message and protein levels of TRPM8 were measured by semi-quantitative PCR and western blotting in superior mesenteric arteries from 12 week-old Spague-Dawley (SD) rats. Isometric tension recordings evaluated the relaxant response in mesenteric rings were also performed. Additionally, the intracellular Ca2+ changes in mesenteric artery myocytes were measured using confocal microscopy. Using PCR and western blotting, both TRPM8 channel mRNA and protein expression was measured in SD rat mesenteric artery. Rotundifolone and menthol induced relaxation in the isolated superior mesenteric artery from SD rats and improved the relaxant response induced by cool temperatures. Also, this monoterpene induced an increase in transient intracellular Ca2+. These responses were significantly attenuated by pretreatment with capsazepine or BCTC, both TRPM8 channels blockers. The response induced by rotundifolone was not significantly attenuated by ruthenium red, a non-selective TRP channels blocker, or following capsaicin-mediated desensitization of TRPV1. Our findings suggest that rotundifolone induces relaxation by activating TRPM8 channels in rat superior mesenteric artery, more selectively than menthol, the classic TRPM8 agonist, and TRPM8 channels participates in vasodilatory pathways in isolated rat mesenteric arteries. PMID:26599698

  17. Molecular mechanism of ATP binding and ion channel activation in P2X receptors

    SciTech Connect

    Hattori, Motoyuki; Gouaux, Eric

    2012-10-24

    P2X receptors are trimeric ATP-activated ion channels permeable to Na{sup +}, K{sup +} and Ca{sup 2+}. The seven P2X receptor subtypes are implicated in physiological processes that include modulation of synaptic transmission, contraction of smooth muscle, secretion of chemical transmitters and regulation of immune responses. Despite the importance of P2X receptors in cellular physiology, the three-dimensional composition of the ATP-binding site, the structural mechanism of ATP-dependent ion channel gating and the architecture of the open ion channel pore are unknown. Here we report the crystal structure of the zebrafish P2X4 receptor in complex with ATP and a new structure of the apo receptor. The agonist-bound structure reveals a previously unseen ATP-binding motif and an open ion channel pore. ATP binding induces cleft closure of the nucleotide-binding pocket, flexing of the lower body {beta}-sheet and a radial expansion of the extracellular vestibule. The structural widening of the extracellular vestibule is directly coupled to the opening of the ion channel pore by way of an iris-like expansion of the transmembrane helices. The structural delineation of the ATP-binding site and the ion channel pore, together with the conformational changes associated with ion channel gating, will stimulate development of new pharmacological agents.

  18. The activation effect of hainantoxin-I, a peptide toxin from the Chinese spider, Ornithoctonus hainana, on intermediate-conductance Ca2+-activated K+ channels.

    PubMed

    Huang, Pengfei; Zhang, Yiya; Chen, Xinyi; Zhu, Li; Yin, Dazhong; Zeng, Xiongzhi; Liang, Songping

    2014-08-21

    Intermediate-conductance Ca2+-activated K+ (IK) channels are calcium/calmodulin-regulated voltage-independent K+ channels. Activation of IK currents is important in vessel and respiratory tissues, rendering the channels potential drug targets. A variety of small organic molecules have been synthesized and found to be potent activators of IK channels. However, the poor selectivity of these molecules limits their therapeutic value. Venom-derived peptides usually block their targets with high specificity. Therefore, we searched for novel peptide activators of IK channels by testing a series of toxins from spiders. Using electrophysiological experiments, we identified hainantoxin-I (HNTX-I) as an IK-channel activator. HNTX-I has little effect on voltage-gated Na+ and Ca2+ channels from rat dorsal root ganglion neurons and on the heterologous expression of voltage-gated rapidly activating delayed rectifier K+ channels (human ether-à-go-go-related gene; human ERG) in HEK293T cells. Only 35.2% ± 0.4% of the currents were activated in SK channels, and there was no effect on BK channels. We demonstrated that HNTX-I was not a phrenic nerve conduction blocker or acutely toxic. This is believed to be the first report of a peptide activator effect on IK channels. Our study suggests that the activity and selectivity of HNTX-I on IK channels make HNTX-I a promising template for designing new drugs for cardiovascular diseases.

  19. Factors affecting daily physical activity and physical performance in maintenance dialysis patients.

    PubMed

    Kopple, Joel D; Kim, Jun C; Shapiro, Bryan B; Zhang, Min; Li, Yinan; Porszasz, Janos; Bross, Rachelle; Feroze, Usama; Upreti, Rajeev; Kalantar-Zadeh, Kamyar

    2015-03-01

    Maintenance hemodialysis (MHD) patients display reduced daily physical activity (DPA) and physical performance (PP). Previous studies did not differentiate the effects of kidney failure and MHD treatments from comorbidities as causes for reduced DPA and PP. In relatively healthy MHD patients and normal adults, we evaluated DPA and PP and examined relationships between DPA and PP and possible associations between anxiety or depression and DPA and PP. DPA, 6-minute walk distance (6-MWD), sit-to-stand (STS), and stair-climbing tests were measured in 72 MHD patients (40% diabetics) with limited comorbidities and 39 normal adults of similar age and gender mix. Anxiety and depression were measured by the Beck anxiety and depression inventories. DPA, time-averaged over 7 days, and all 3 PP tests were impaired in MHD patients, to about 60% to 70% of normal values (P < .0001 for each measurement). MHD patients spent more time sleeping or physically inactive (P < .0001) and less time in ≥ moderate activity (P < .0001). Adjusted DPA correlated with 6-MWD but not STS or stair-climbing. Anxiety and depression were identified in 43% and 33% of MHD patients and 2.5% and 5.1% of normals (P < .0001 for each comparison). Most of the impairment in DPA and PP tests were also observed in MHD patients without anxiety or depression. However, MHD patients with both anxiety and depression generally had the most impaired DPA and PP. In MHD patients, higher adjusted anxiety scores were correlated with impaired 6-MWD and STS, whereas adjusted average DPA was negatively correlated with depression (r = -0.33, P = .006) but not anxiety. DPA on the hemodialysis day (P = .01), day after dialysis (P = .03), and day 2 after dialysis (P = .03) each correlated negatively with degree of depression but not with anxiety. MHD patients displayed negative-adjusted correlations between anxiety and 6-MWD (P = .03) and STS (P = .04). In relatively healthy MHD patients, DPA and PP are substantially impaired

  20. Multi-ion occupancy alters gating in high-conductance, Ca(2+)-activated K+ channels

    PubMed Central

    1991-01-01

    In this study, single-channel recordings of high-conductance Ca(2+)- activated K+ channels from rat skeletal muscle inserted into planar lipid bilayer were used to analyze the effects of two ionic blockers, Ba2+ and Na+, on the channel's gating reactions. The gating equilibrium of the Ba(2+)-blocked channel was investigated through the kinetics of the discrete blockade induced by Ba2+ ions. Gating properties of Na(+)- blocked channels could be directly characterized due to the very high rates of Na+ blocking/unblocking reactions. While in the presence of K+ (5 mM) in the external solution Ba2+ is known to stabilize the open state of the blocked channel (Miller, C., R. Latorre, and I. Reisin. 1987. J. Gen. Physiol. 90:427-449), we show that the divalent blocker stabilizes the closed-blocked state if permeant ions are removed from the external solution (K+ less than 10 microM). Ionic substitutions in the outer solution induce changes in the gating equilibrium of the Ba(2+)-blocked channel that are tightly correlated to the inhibition of Ba2+ dissociation by external monovalent cations. In permeant ion-free external solutions, blockade of the channel by internal Na+ induces a shift (around 15 mV) in the open probability--voltage curve toward more depolarized potentials, indicating that Na+ induces a stabilization of the closed-blocked state, as does Ba2+ under the same conditions. A kinetic analysis of the Na(+)-blocked channel indicates that the closed- blocked state is favored mainly by a decrease in opening rate. Addition of 1 mM external K+ completely inhibits the shift in the activation curve without affecting the Na(+)-induced reduction in the apparent single-channel amplitude. The results suggest that in the absence of external permeant ions internal blockers regulate the permeant ion occupancy of a site near the outer end of the channel. Occupancy of this site appears to modulate gating primarily by speeding the rate of channel opening. PMID:2056305

  1. Maintenance of Activity and Eating Change Following a Clinical Trial of Tailored Newsletters with Older Rural Women

    PubMed Central

    Walker, Susan Noble; Pullen, Carol H.; Hageman, Patricia A.; Boeckner, Linda S.; Hertzog, Melody; Oberdorfer, Maureen K.; Rutledge, Matthew J.

    2010-01-01

    Background In the Wellness for Women Project, a randomized-by-site 1-year controlled clinical trial, the efficacy of generic newsletters and tailored newsletters on Health Promotion Model behavior-specific cognitions, eating change, and activity change were compared among 225 women aged 50 to 69 years. Objectives To compare the maintenance of change in healthy eating and physical activity over 12 months following the tailored versus generic mailed newsletter intervention. Method Outcomes at 18 and 24 months included behavioral markers and biomarkers of physical activity and eating. Data were analyzed using the multivariate approach to repeated measures analysis of variance and generalized estimating equations (α < .05). Results At 18 months, the tailored group maintained levels of all eating and activity behaviors, while the generic group maintained levels of fruit and vegetable servings and moderate or greater activity, stretching exercise, lower body strength, and flexibility, but increased saturated fat intake and declined in weekly strength exercise and cardiorespiratory fitness. At 24 months, both groups maintained or returned to 12-month levels of all eating behaviors and moderate or greater activity, stretching exercise, and flexibility, but declined in cardiorespiratory fitness; the tailored group maintained levels of strength exercise and lower body strength, while the generic group decreased in both. A greater proportion of women who received tailored newsletters continued to achieve most Healthy People 2010 criteria for eating and activity. Discussion Mailed tailored print newsletters were more effective than generic newsletters in facilitating maintenance of change in eating and activity for 6 months postintervention. Both tailored and generic newsletters facilitated the maintenance of change in eating behaviors and in moderate or greater physical activity and stretching exercise, while tailored newsletters were more effective in maintaining change in

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

    PubMed

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

    2008-08-01

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

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

    PubMed

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

    2004-10-01

    Intraerythrocytic growth of the human malaria parasite Plasmodium falciparum depends on delivery of nutrients. Moreover, infection challenges cell volume constancy of the host erythrocyte requiring enhanced activity of cell volume regulatory mechanisms. Patch clamp recording demonstrated inwardly and outwardly rectifying anion channels in infected but not in control erythrocytes. The molecular identity of those channels remained elusive. We show here for one channel type that voltage dependence, cell volume sensitivity, and activation by oxidation are identical to ClC-2. Moreover, Western blots and FACS analysis showed protein and functional ClC-2 expression in human erythrocytes and erythrocytes from wild type (Clcn2(+/+)) but not from Clcn2(-/-) mice. Finally, patch clamp recording revealed activation of volume-sensitive inwardly rectifying channels in Plasmodium berghei-infected Clcn2(+/+) but not Clcn2(-/-) erythrocytes. Erythrocytes from infected mice of both genotypes differed in cell volume and inhibition of ClC-2 by ZnCl(2) (1 mm) induced an increase of cell volume only in parasitized Clcn2(+/+) erythrocytes. Lack of ClC-2 did not inhibit P. berghei development in vivo nor substantially affect the mortality of infected mice. In conclusion, activation of host ClC-2 channels participates in the altered permeability of Plasmodium-infected erythrocytes but is not required for intraerythrocytic parasite survival. PMID:15272009

  4. Activation of Plant Plasma Membrane Ca2+-Permeable Channels by Race-Specific Fungal Elicitors.

    PubMed Central

    Gelli, A.; Higgins, V. J.; Blumwald, E.

    1997-01-01

    The response of plant cells to invading pathogens is regulated by fluctuations in cytosolic Ca2+ levels that are mediated by Ca2+-permeable channels located at the plasma membrane of the host cell. The mechanisms by which fungal elicitors can induce Ca2+ uptake by the host cell were examined by the application of conventional patch-clamp techniques. Whole-cell and single-channel experiments on tomato (Lycopersicon esculentum L.) protoplasts revealed a race-specific fungal elicitor-induced activation of a plasma membrane Ca2+-permeable channel. The presence of the fungal elicitor resulted in a greater probability of channel opening. Guanosine 5[prime]-[[beta]-thio]diphosphate, a GDP analog that locks heterotrimeric G-proteins into their inactivated state, abolished the channel activation induced by the fungal elicitor, whereas guanosine 5[prime][[gamma]-thio]triphosphate, a nonhydrolyzable GTP analog that locks heterotrimeric G-proteins into their activated state, produced an effect similar to that observed with the fungal elicitor. Mastoparan, which stimulates GTPase activity, mimicked the effect of GTP[[gamma

  5. Energetics of Ion Permeation in an Open-Activated TRPV1 Channel.

    PubMed

    Jorgensen, Christian; Furini, Simone; Domene, Carmen

    2016-09-20

    Ion channels enable diffusion of ions down physiological electrochemical gradients. Modulation of ion permeation is crucial for the physiological functioning of cells, and misregulation of ion channels is linked to a myriad of channelopathies. The ion permeation mechanism in the transient receptor potential (TRP) ion channel family is currently not understood at an atomistic level. In this work, we employed a simulation strategy for ion permeation (molecular-dynamics simulations with bias-exchange metadynamics) to study and compare monovalent (Na(+), K(+)) ion permeation in the open-activated TRP vanniloid-1 (TRPV1) ion channel. Using ∼3.6 μs of simulation trajectories, we obtained atomistic evidence for the nonselective nature of TRPV1. Our analysis shows that solvated monovalent ions permeate through the selectivity filter with comparable energetic barriers via a two-site mechanism. Finally, we confirmed that an intracellular binding site is located between the intracellular gate residues I679 and E684. PMID:27653480

  6. Energetics of Ion Permeation in an Open-Activated TRPV1 Channel.

    PubMed

    Jorgensen, Christian; Furini, Simone; Domene, Carmen

    2016-09-20

    Ion channels enable diffusion of ions down physiological electrochemical gradients. Modulation of ion permeation is crucial for the physiological functioning of cells, and misregulation of ion channels is linked to a myriad of channelopathies. The ion permeation mechanism in the transient receptor potential (TRP) ion channel family is currently not understood at an atomistic level. In this work, we employed a simulation strategy for ion permeation (molecular-dynamics simulations with bias-exchange metadynamics) to study and compare monovalent (Na(+), K(+)) ion permeation in the open-activated TRP vanniloid-1 (TRPV1) ion channel. Using ∼3.6 μs of simulation trajectories, we obtained atomistic evidence for the nonselective nature of TRPV1. Our analysis shows that solvated monovalent ions permeate through the selectivity filter with comparable energetic barriers via a two-site mechanism. Finally, we confirmed that an intracellular binding site is located between the intracellular gate residues I679 and E684.

  7. Impedance spectroscopy of micro-Droplets reveals activation of Bacterial Mechanosensitive Channels in Hypotonic Solutions

    NASA Astrophysics Data System (ADS)

    Ebrahimi, Aida; Alam, Muhammad A.

    Rapid detection of bacterial pathogens is of great importance in healthcare, food safety, environmental monitoring, and homeland security. Most bacterial detection platforms rely on binary fission (i.e. cell growth) to reach a threshold cell population that can be resolved by the sensing method. Since cell division depends on the bacteria type, the detection time of such methods can vary from hours to days. In contrast, in this work, we show that bacteria cells can be detected within minutes by relying on activation of specific protein channels, i.e. mechanosensitive channels (MS channels). When cells are exposed to hypotonic solutions, MS channels allow efflux of solutes to the external solution which leads to release the excessive membrane tension. Release of the cytoplasmic solutes, in turn, results in increase of the electrical conductance measured by droplet-based impedance sensing. The approach can be an effective technique for fast, pre-screening of bacterial contamination at ultra-low concentration.

  8. Structural and functional characterization of a calcium-activated cation channel from Tsukamurella paurometabola

    PubMed Central

    Dhakshnamoorthy, Balasundaresan; Rohaim, Ahmed; Rui, Huan; Blachowicz, Lydia; Roux, Benoît

    2016-01-01

    The selectivity filter is an essential functional element of K+ channels that is highly conserved both in terms of its primary sequence and its three-dimensional structure. Here, we investigate the properties of an ion channel from the Gram-positive bacterium Tsukamurella paurometabola with a selectivity filter formed by an uncommon proline-rich sequence. Electrophysiological recordings show that it is a non-selective cation channel and that its activity depends on Ca2+ concentration. In the crystal structure, the selectivity filter adopts a novel conformation with Ca2+ ions bound within the filter near the pore helix where they are coordinated by backbone oxygen atoms, a recurrent motif found in multiple proteins. The binding of Ca2+ ion in the selectivity filter controls the widening of the pore as shown in crystal structures and in molecular dynamics simulations. The structural, functional and computational data provide a characterization of this calcium-gated cationic channel. PMID:27678077

  9. Backward reflection analysis of transmitting channel of active laser ranging optics

    NASA Astrophysics Data System (ADS)

    Hong, Jinsuk; Koh, Hae Seog

    2013-09-01

    The designed Active LDR(Laser Detection and Ranging) System contains high-power Laser and its diameter is approximately 24mm. Although the laser transmitting channel and receiving optic channel are completely separated from each other and doesn't share any of the optical components in design, each channel shares 4 wedge scanners, which are to overcome the narrow FOV(Field of View) of the optical system. Any backward reflection back to the fiber laser end must be carefully studied since it can damage the LD(Laser Diodes), the inner components of the laser unit because of the high amplification factor of the laser unit. In this study, the stray light caused by the transmitting channel's laser and inner reflection by optical components were analyzed by ASAP(Advanced System Analysis Program) software. We also can confirm the operability and stability of the system by more than 6 months of operation of the system.

  10. Structural and functional characterization of a calcium-activated cation channel from Tsukamurella paurometabola

    NASA Astrophysics Data System (ADS)

    Dhakshnamoorthy, Balasundaresan; Rohaim, Ahmed; Rui, Huan; Blachowicz, Lydia; Roux, Benoît

    2016-09-01

    The selectivity filter is an essential functional element of K+ channels that is highly conserved both in terms of its primary sequence and its three-dimensional structure. Here, we investigate the properties of an ion channel from the Gram-positive bacterium Tsukamurella paurometabola with a selectivity filter formed by an uncommon proline-rich sequence. Electrophysiological recordings show that it is a non-selective cation channel and that its activity depends on Ca2+ concentration. In the crystal structure, the selectivity filter adopts a novel conformation with Ca2+ ions bound within the filter near the pore helix where they are coordinated by backbone oxygen atoms, a recurrent motif found in multiple proteins. The binding of Ca2+ ion in the selectivity filter controls the widening of the pore as shown in crystal structures and in molecular dynamics simulations. The structural, functional and computational data provide a characterization of this calcium-gated cationic channel.

  11. Hyaluronan modulates TRPV1 channel opening, reducing peripheral nociceptor activity and pain.

    PubMed

    Caires, Rebeca; Luis, Enoch; Taberner, Francisco J; Fernandez-Ballester, Gregorio; Ferrer-Montiel, Antonio; Balazs, Endre A; Gomis, Ana; Belmonte, Carlos; de la Peña, Elvira

    2015-01-01

    Hyaluronan (HA) is present in the extracellular matrix of all body tissues, including synovial fluid in joints, in which it behaves as a filter that buffers transmission of mechanical forces to nociceptor nerve endings thereby reducing pain. Using recombinant systems, mouse-cultured dorsal root ganglia (DRG) neurons and in vivo experiments, we found that HA also modulates polymodal transient receptor potential vanilloid subtype 1 (TRPV1) channels. HA diminishes heat, pH and capsaicin (CAP) responses, thus reducing the opening probability of the channel by stabilizing its closed state. Accordingly, in DRG neurons, HA decreases TRPV1-mediated impulse firing and channel sensitization by bradykinin. Moreover, subcutaneous HA injection in mice reduces heat and capsaicin nocifensive responses, whereas the intra-articular injection of HA in rats decreases capsaicin joint nociceptor fibres discharge. Collectively, these results indicate that extracellular HA reduces the excitability of the ubiquitous TRPV1 channel, thereby lowering impulse activity in the peripheral nociceptor endings underlying pain.

  12. ANO2 is the cilial calcium-activated chloride channel that may mediate olfactory amplification

    PubMed Central

    Stephan, Aaron B.; Shum, Eleen Y.; Hirsh, Sarah; Cygnar, Katherine D.; Reisert, Johannes; Zhao, Haiqing

    2009-01-01

    For vertebrate olfactory signal transduction, a calcium-activated chloride conductance serves as a major amplification step. However, the molecular identity of the olfactory calcium-activated chloride channel (CaCC) is unknown. Here we report a proteomic screen for cilial membrane proteins of mouse olfactory sensory neurons (OSNs) that identified all the known olfactory transduction components as well as Anoctamin 2 (ANO2). Ano2 transcripts were expressed specifically in OSNs in the olfactory epithelium, and ANO2::EGFP fusion protein localized to the OSN cilia when expressed in vivo using an adenoviral vector. Patch-clamp analysis revealed that ANO2, when expressed in HEK-293 cells, forms a CaCC and exhibits channel properties closely resembling the native olfactory CaCC. Considering these findings together, we propose that ANO2 constitutes the olfactory calcium-activated chloride channel. PMID:19561302

  13. EMERGENCE OF HELICAL FLUX AND THE FORMATION OF AN ACTIVE REGION FILAMENT CHANNEL

    SciTech Connect

    Lites, B. W.; Kubo, M.; Berger, T.; Frank, Z.; Shine, R.; Tarbell, T.; Title, A.; Okamoto, T. J.; Otsuji, K.

    2010-07-20

    We present comprehensive observations of the formation and evolution of a filament channel within NOAA Active Region (AR) 10978 from Hinode/Solar Optical Telescope and TRACE. We employ sequences of Hinode spectro-polarimeter maps of the AR, accompanying Hinode Narrowband Filter Instrument magnetograms in the Na I D1 line, Hinode Broadband Filter Instrument filtergrams in the Ca II H line and G-band, Hinode X-ray telescope X-ray images, and TRACE Fe IX 171 A image sequences. The development of the channel resembles qualitatively that presented by Okamoto et al. in that many indicators point to the emergence of a pre-existing sub-surface magnetic flux rope. The consolidation of the filament channel into a coherent structure takes place rapidly during the course of a few hours, and the filament form then gradually shrinks in width over the following two days. Particular to this filament channel is the observation of a segment along its length of horizontal, weak (500 G) flux that, unlike the rest of the filament channel, is not immediately flanked by strong vertical plage fields of opposite polarity on each side of the filament. Because this isolated horizontal field is observed in photospheric lines, we infer that it is unlikely that the channel formed as a result of reconnection in the corona, but the low values of inferred magnetic fill fraction along the entire length of the filament channel suggest that the bulk of the field resides somewhat above the low photosphere. Correlation tracking of granulation in the G band presents no evidence for either systematic flows toward the channel or systematic shear flows along it. The absence of these flows, along with other indications of these data from multiple sources, reinforces (but does not conclusively demonstrate) the picture of an emerging flux rope as the origin of this AR filament channel.

  14. Active membrane having uniform physico-chemically functionalized ion channels

    DOEpatents

    Gerald, II, Rex E; Ruscic, Katarina J; Sears, Devin N; Smith, Luis J; Klingler, Robert J; Rathke, Jerome W

    2012-09-24

    The present invention relates to a physicochemically-active porous membrane for electrochemical cells that purports dual functions: an electronic insulator (separator) and a unidirectional ion-transporter (electrolyte). The electrochemical cell membrane is activated for the transport of ions by contiguous ion coordination sites on the interior two-dimensional surfaces of the trans-membrane unidirectional pores. One dimension of the pore surface has a macroscopic length (1 nm-1000 .mu.m) and is directed parallel to the direction of an electric field, which is produced between the cathode and the anode electrodes of an electrochemical cell. The membrane material is designed to have physicochemical interaction with ions. Control of the extent of the interactions between the ions and the interior pore walls of the membrane and other materials, chemicals, or structures contained within the pores provides adjustability of the ionic conductivity of the membrane.

  15. Identification of a stretch-activated monovalent cation channel from teleost urinary bladder cells.

    PubMed

    Chang, W H; Loretz, C A

    1991-09-01

    The urinary bladder of euryhaline teleost is an important osmoregulatory organ which absorbs Na+, Cl-, and water from urine. Using patch clamp technique, single stretch-activated channels, which were permeable to K+ and Na+ (PNa/PK approximately 0.75) and had conductances of 55 and 116 pS, were studied. In excised, inside-out patches which were voltage-clamped in the physiological range of membrane potential, the single-channel open probability (Po) was low (approximately 0.02), and increased to a maximum of 0.9 with applied pipette suction. Single-channel conductance also increased with suction. The channels showed adaptation to applied suction and relaxed to a steady-state activity about 20 seconds after application of suction. The Po increased up to 0.9 with strong membrane depolarization (Vm = 0 to +80 mV); however, there was little dependence of Po on membrane potential in the physiological range. The kinetic data suggest that there is one conducting state and at least two non-conducting states of the channel. The open-time constant increased with suction but remained unchanged with membrane potential (Vm = -70 to +60 mV). The mean closed-time of the channel decreased with suction and membrane depolarization. These results demonstrate the presence of a non-selective monovalent cation channel which may be involved in cell volume regulation in the goby urinary bladder. Additionally, this channel may function as an enhancer of Na+ influx and K+ efflux across the bladder cell as part of transepithelial ion transport if it is located in apical membrane.

  16. Thalamic Kv7 channels: pharmacological properties and activity control during noxious signal processing

    PubMed Central

    Cerina, Manuela; Szkudlarek, Hanna J; Coulon, Philippe; Meuth, Patrick; Kanyshkova, Tatyana; Nguyen, Xuan Vinh; Göbel, Kerstin; Seidenbecher, Thomas; Meuth, Sven G; Pape, Hans-Christian; Budde, Thomas

    2015-01-01

    Background and Purpose The existence of functional Kv7 channels in thalamocortical (TC) relay neurons and the effects of the K+-current termed M-current (IM) on thalamic signal processing have long been debated. Immunocytochemical evidence suggests their presence in this brain region. Therefore, we aimed to verify their existence, pharmacological properties and function in regulating activity in neurons of the ventrobasal thalamus (VB). Experimental Approach Characterization of Kv7 channels was performed by combining in vitro, in vivo and in silico techniques with a pharmacological approach. Retigabine (30 μM) and XE991 (20 μM), a specific Kv7 channel enhancer and blocker, respectively, were applied in acute brain slices during electrophysiological recordings. The effects of intrathalamic injection of retigabine (3 mM, 300 nL) and/or XE991 (2 mM, 300 nL) were investigated in freely moving animals during hot-plate tests by recording behaviour and neuronal activity. Key Results Kv7.2 and Kv7.3 subunits were found to be abundantly expressed in TC neurons of mouse VB. A slow K+-current with properties of IM was activated by retigabine and inhibited by XE991. Kv7 channel activation evoked membrane hyperpolarization, a reduction in tonic action potential firing, and increased burst firing in vitro and in computational models. Single-unit recordings and pharmacological intervention demonstrated a specific burst-firing increase upon IM activation in vivo. A Kv7 channel-mediated increase in pain threshold was associated with fewer VB units responding to noxious stimuli, and increased burst firing in responsive neurons. Conclusions and Implications Kv7 channel enhancement alters somatosensory activity and may reflect an anti-nociceptive mechanism during acute pain processing. PMID:25684311

  17. Institutional Maintenance Management and Services. Student Activity Book [and] Reference Book.

    ERIC Educational Resources Information Center

    Koukel, Sonja D.

    These publications are two of three that comprise a course that provides occupationally specific training designed to develop knowledge and skills for employment in the area of institutional maintenance management and services. The reference book is a student text that provides information needed by employees in the institutional maintenance…

  18. 24 CFR 35.1220 - Ongoing lead-based paint maintenance activities.

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ... 24 Housing and Urban Development 1 2013-04-01 2013-04-01 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...

  19. 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...

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

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ... 24 Housing and Urban Development 1 2012-04-01 2012-04-01 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...

  1. 24 CFR 35.1220 - Ongoing lead-based paint maintenance activities.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... 24 Housing and Urban Development 1 2011-04-01 2011-04-01 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...

  2. 24 CFR 35.1220 - Ongoing lead-based paint maintenance activities.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ... 24 Housing and Urban Development 1 2014-04-01 2014-04-01 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...

  3. Criterion Referenced Measures of Technical Proficiency in Maintenance Activities. Final Report.

    ERIC Educational Resources Information Center

    Foley, John P., Jr.

    The paper concerns instruments used in the determination of task performance efficiency of mechanical and electronic maintenance men. Composite results of unvalidated knowledge tests, theory tests, school marks, and supervisors' ratings are presented which indicate that these measurement instruments have low empirical validity. The limitations of…

  4. Dissection of the components for PIP2 activation and thermosensation in TRP channels

    PubMed Central

    Brauchi, Sebastian; Orta, Gerardo; Mascayano, Carolina; Salazar, Marcelo; Raddatz, Natalia; Urbina, Hector; Rosenmann, Eduardo; Gonzalez-Nilo, Fernando; Latorre, Ramon

    2007-01-01

    Phosphatidylinositol 4,5-bisphosphate (PIP2) plays a central role in the activation of several transient receptor potential (TRP) channels. The role of PIP2 on temperature gating of thermoTRP channels has not been explored in detail, and the process of temperature activation is largely unexplained. In this work, we have exchanged different segments of the C-terminal region between cold-sensitive (TRPM8) and heat-sensitive (TRPV1) channels, trying to understand the role of the segment in PIP2 and temperature activation. A chimera in which the proximal part of the C-terminal of TRPV1 replaces an equivalent section of TRPM8 C-terminal is activated by PIP2 and confers the phenotype of heat activation. PIP2, but not temperature sensitivity, disappears when positively charged residues contained in the exchanged region are neutralized. Shortening the exchanged segment to a length of 11 aa produces voltage-dependent and temperature-insensitive channels. Our findings suggest the existence of different activation domains for temperature, PIP2, and voltage. We provide an interpretation for channel–PIP2 interaction using a full-atom molecular model of TRPV1 and PIP2 docking analysis. PMID:17548815

  5. Channel-Forming Activities in the Glycosomal Fraction from the Bloodstream Form of Trypanosoma brucei

    PubMed Central

    Miinalainen, Ilkka J.; Hiltunen, J. Kalervo; Michels, Paul A. M.; Antonenkov, Vasily D.

    2012-01-01

    Background Glycosomes are a specialized form of peroxisomes (microbodies) present in unicellular eukaryotes that belong to the Kinetoplastea order, such as Trypanosoma and Leishmania species, parasitic protists causing severe diseases of livestock and humans in subtropical and tropical countries. The organelles harbour most enzymes of the glycolytic pathway that is responsible for substrate-level ATP production in the cell. Glycolysis is essential for bloodstream-form Trypanosoma brucei and enzymes comprising this pathway have been validated as drug targets. Glycosomes are surrounded by a single membrane. How glycolytic metabolites are transported across the glycosomal membrane is unclear. Methods/Principal Findings We hypothesized that glycosomal membrane, similarly to membranes of yeast and mammalian peroxisomes, contains channel-forming proteins involved in the selective transfer of metabolites. To verify this prediction, we isolated a glycosomal fraction from bloodstream-form T.brucei and reconstituted solubilized membrane proteins into planar lipid bilayers. The electrophysiological characteristics of the channels were studied using multiple channel recording and single channel analysis. Three main channel-forming activities were detected with current amplitudes 70–80 pA, 20–25 pA, and 8–11 pA, respectively (holding potential +10 mV and 3.0 M KCl as an electrolyte). All channels were in fully open state in a range of voltages ±150 mV and showed no sub-conductance transitions. The channel with current amplitude 20–25 pA is anion-selective (PK+/PCl−∼0.31), while the other two types of channels are slightly selective for cations (PK+/PCl− ratios ∼1.15 and ∼1.27 for the high- and low-conductance channels, respectively). The anion-selective channel showed an intrinsic current rectification that may suggest a functional asymmetry of the channel's pore. Conclusions/Significance These results indicate that the membrane of glycosomes apparently

  6. A chimeric prokaryotic pentameric ligand–gated channel reveals distinct pathways of activation

    PubMed Central

    Schmandt, Nicolaus; Velisetty, Phanindra; Chalamalasetti, Sreevatsa V.; Stein, Richard A.; Bonner, Ross; Talley, Lauren; Parker, Mark D.; Mchaourab, Hassane S.; Yee, Vivien C.; Lodowski, David T.

    2015-01-01

    Recent high resolution structures of several pentameric ligand–gated ion channels have provided unprecedented details of their molecular architecture. However, the conformational dynamics and structural rearrangements that underlie gating and allosteric modulation remain poorly understood. We used a combination of electrophysiology, double electron–electron resonance (DEER) spectroscopy, and x-ray crystallography to investigate activation mechanisms in a novel functional chimera with the extracellular domain (ECD) of amine-gated Erwinia chrysanthemi ligand–gated ion channel, which is activated by primary amines, and the transmembrane domain of Gloeobacter violaceus ligand–gated ion channel, which is activated by protons. We found that the chimera was independently gated by primary amines and by protons. The crystal structure of the chimera in its resting state, at pH 7.0 and in the absence of primary amines, revealed a closed-pore conformation and an ECD that is twisted with respect to the transmembrane region. Amine- and pH-induced conformational changes measured by DEER spectroscopy showed that the chimera exhibits a dual mode of gating that preserves the distinct conformational changes of the parent channels. Collectively, our findings shed light on both conserved and divergent features of gating mechanisms in this class of channels, and will facilitate the design of better allosteric modulators. PMID:26415570

  7. Fly DPP10 acts as a channel ancillary subunit and possesses peptidase activity

    PubMed Central

    Shiina, Yohei; Muto, Tomohiro; Zhang, Zhili; Baihaqie, Ahmad; Yoshizawa, Takamasa; Lee, Hye-in J.; Park, Eulsoon; Tsukiji, Shinya; Takimoto, Koichi

    2016-01-01

    Mammalian DPP6 (DPPX) and DPP10 (DPPY) belong to a family of dipeptidyl peptidases, but lack enzyme activity. Instead, these proteins form complexes with voltage-gated K+ channels in Kv4 family to control their gating and other properties. Here, we find that the fly DPP10 ortholog acts as an ancillary subunit of Kv4 channels and digests peptides. Similarly to mammalian DPP10, the fly ortholog tightly binds to rat Kv4.3 protein. The association causes negative shifts in voltage dependence of channel activation and steady state inactivation. It also results in faster inactivation and recovery from inactivation. In addition to its channel regulatory role, fly DPP10 exhibits significant dipeptidyl peptidase activity with Gly-Pro-MCA (glycyl-L-proline 4-methylcoumaryl-7-amide) as a substrate. Heterologously expressed Flag-tagged fly DPP10 and human DPP4 show similar Km values towards this substrate. However, fly DPP10 exhibits approximately a 6-times-lower relative kcat value normalized with anti-Flag immunoreactivity than human DPP4. These results demonstrate that fly DPP10 is a dual functional protein, controlling Kv4 channel gating and removing bioactive peptides. PMID:27198182

  8. Chlorotoxin does not inhibit volume-regulated, calcium-activated and cyclic AMP-activated chloride channels.

    PubMed

    Maertens, C; Wei, L; Tytgat, J; Droogmans, G; Nilius, B

    2000-02-01

    It was the aim of this study to look for a high-affinity and selective polypeptide toxin, which could serve as a probe for the volume-regulated anion channel (VRAC) or the calcium-activated chloride channel (CaCC). We have partially purified chlorotoxin, including new and homologous short chain insectotoxins, from the crude venom of Leiurus quinquestriatus quinquestriatus (Lqq) by means of gel filtration chromatography. Material eluting between 280 and 420 min, corresponding to fractions 15-21, was lyophilized and tested on VRAC and CaCC, using the whole-cell patch-clamp technique. We have also tested the commercially available chlorotoxin on VRAC, CaCC, the cystic fibrosis transmembrane conductance regulator (CFTR) and on the glioma specific chloride channel (GCC). VRAC and the correspondent current, I(Cl,swell), was activated in Cultured Pulmonary Artery Endothelial (CPAE) cells by a 25% hypotonic solution. Neither of the fractions 16-21 significantly inhibited I(Cl,swell) (n=4-5). Ca(2+)-activated Cl(-) currents, I(Cl,Ca), activated by loading T84 cells via the patch pipette with 1 microM free Ca(2+), were not inhibited by any of the tested fractions (15-21), (n=2-5). Chlorotoxin (625 nM) did neither effect I(Cl,swell) nor I(Cl,Ca) (n=4-5). The CFTR channel, transiently transfected in COS cells and activated by a cocktail containing IBMX and forskolin, was not affected by 1.2 microM chlorotoxin (n=5). In addition, it did not affect currents through GCC. We conclude that submicromolar concentrations of chlorotoxin do not block volume-regulated, Ca(2+)-activated and CFTR chloride channels and that it can not be classified as a general chloride channel toxin.

  9. Human activities impact on mountain river channels (case study of Kamchatka peninsula rivers)

    NASA Astrophysics Data System (ADS)

    Ermakova, Aleksandra S.

    2010-05-01

    Human-induced driving factors along with natural environmental changes greatly impact on fluvial regime of rivers. On mountain and semi-mountain territories these processes are developed in the most complicated manner due to man-made activities diversity throughout river basins. Besides these processes are significantly enhanced because of the disastrous natural processes (like volcanic and mud-flow activity) frequent occurrences in mountainous regions. On of the most striking example on the matter is Kamchatka peninsula which is located at the North-West part of Russian Federation. This paper contributes to the study of human activities impact on fluvial systems in this volcanic mountain region. Human effects on rivers directly alter channel morphology and deformations, dynamics of water and sediment movement, aquatic communities or indirectly affect streams by altering the movement of water and sediment into the channel. In case study of Kamchatka peninsula human activities affect fluvial systems through engineering works including construction of bridges, dams and channel diversions and placer mining. These processes are characterized by spatial heterogeneity because of irregular population distribution. Due to specific natural conditions of the peninsula the most populated areas are the valleys of big rivers (rivers Kamchatka, Avacha, Bistraya (Bolshaya), etc) within piedmont and plain regions. These rivers are characterized by very unstable channels. Both with man-made activities this determines wide range of fluvial system changes. Firstly bridges construction leads to island and logjam formation directly near their piers and intensification of channels patterns shifts. Furthermore rivers of the peninsula are distinguished for high water flow velocities and water rate. Incorrect bridge constructions both with significant channel deformations lead to the destructions of the bridges themselves due to intensive bank erosion. Secondly, intensive water flow

  10. Facilities maintenance handbook

    NASA Technical Reports Server (NTRS)

    1991-01-01

    This handbook is a guide for facilities maintenance managers. Its objective is to set minimum facilities maintenance standards. It also provides recommendations on how to meet the standards to ensure that NASA maintains its facilities in a manner that protects and preserves its investment in the facilities in a cost-effective manner while safely and efficiently performing its mission. This handbook implements NMI 8831.1, which states NASA facilities maintenance policy and assigns organizational responsibilities for the management of facilities maintenance activities on all properties under NASA jurisdiction. It is a reference for facilities maintenance managers, not a step-by-step procedural manual. Because of the differences in NASA Field Installation organizations, this handbook does not assume or recommend a typical facilities maintenance organization. Instead, it uses a systems approach to describe the functions that should be included in any facilities maintenance management system, regardless of its organizational structure. For documents referenced in the handbook, the most recent version of the documents is applicable. This handbook is divided into three parts: Part 1 specifies common definitions and facilities maintenance requirements and amplifies the policy requirements contained in NMI 8831. 1; Part 2 provides guidance on how to meet the requirements of Part 1, containing recommendations only; Part 3 contains general facilities maintenance information. One objective of this handbook is to fix commonality of facilities maintenance definitions among the Centers. This will permit the application of uniform measures of facilities conditions, of the relationship between current replacement value and maintenance resources required, and of the backlog of deferred facilities maintenance. The utilization of facilities maintenance system functions will allow the Centers to quantitatively define maintenance objectives in common terms, prepare work plans, and

  11. Blocking mechanisms of batrachotoxin-activated Na channels in artificial bilayers.

    PubMed

    Uehara, A; Moczydlowski, E

    1986-01-01

    The effects of various pharmacological agents that block single batrachotoxin-activated Na channels from rat muscle can be described in terms of three modes of action that correspond to at least three different binding sites. Guanidinium toxins such as tetrodotoxin, saxitoxin, and a novel polypeptide, mu-conotoxin GIIIA, act only from the extra-cellular side and induce discrete blocked states that correspond to residence times of individual toxin molecules. Such toxins apparently do not deeply penetrate the channel pore since the voltage dependence of block is insensitive to toxin charge and block is not relieved by internal Na+. Many nonspecific organic cations, including charged anesthetics, exhibit a voltage-dependent block that is enhanced by depolarization when present on the inside of the channel. This site is probably within the pore, but binding to this site is weak, as indicated by fast blockade that often appears as lowered channel conductance. A separate class of neutral and tertiary amine anesthetics such as benzocaine and procaine induce discrete closed states when added to either side of the membrane. This blocking effect can be explained by preferential binding to closed states of the channel and appears to be due to a modulation of channel gating.

  12. Hyperpolarization-activated, cyclic nucleotide-gated cation channels in Aplysia: Contribution to classical conditioning

    PubMed Central

    Yang, Qizong; Kuzyk, Pavlo; Antonov, Igor; Bostwick, Caleb J.; Kohn, Andrea B.; Moroz, Leonid L.; Hawkins, Robert D.

    2015-01-01

    Hyperpolarization-activated, cyclic nucleotide-gated cation (HCN) channels are critical regulators of neuronal excitability, but less is known about their possible roles in synaptic plasticity and memory circuits. Here, we characterized the HCN gene organization, channel properties, distribution, and involvement in associative and nonassociative forms of learning in Aplysia californica. Aplysia has only one HCN gene, which codes for a channel that has many similarities to the mammalian HCN channel. The cloned acHCN gene was expressed in Xenopus oocytes, which displayed a hyperpolarization-induced inward current that was enhanced by cGMP as well as cAMP. Similarly to its homologs in other animals, acHCN is permeable to K+ and Na+ ions, and is selectively blocked by Cs+ and ZD7288. We found that acHCN is predominantly expressed in inter- and motor neurons, including LFS siphon motor neurons, and therefore tested whether HCN channels are involved in simple forms of learning of the siphon-withdrawal reflex in a semiintact preparation. ZD7288 (100 μM) significantly reduced an associative form of learning (classical conditioning) but had no effect on two nonassociative forms of learning (intermediate-term sensitization and unpaired training) or baseline responses. The HCN current is enhanced by nitric oxide (NO), which may explain the postsynaptic role of NO during conditioning. HCN current in turn enhances the NMDA-like current in the motor neurons, suggesting that HCN channels contribute to conditioning through this pathway. PMID:26668355

  13. Molecular Interactions between Tarantula Toxins and Low-Voltage-Activated Calcium Channels

    PubMed Central

    Salari, Autoosa; Vega, Benjamin S.; Milescu, Lorin S.; Milescu, Mirela

    2016-01-01

    Few gating-modifier toxins have been reported to target low-voltage-activated (LVA) calcium channels, and the structural basis of toxin sensitivity remains incompletely understood. Studies of voltage-gated potassium (Kv) channels have identified the S3b–S4 “paddle motif,” which moves at the protein-lipid interface to drive channel opening, as the target for these amphipathic neurotoxins. Voltage-gated calcium (Cav) channels contain four homologous voltage sensor domains, suggesting multiple toxin binding sites. We show here that the S3–S4 segments within Cav3.1 can be transplanted into Kv2.1 to examine their individual contributions to voltage sensing and pharmacology. With these results, we now have a more complete picture of the conserved nature of the paddle motif in all three major voltage-gated ion channel types (Kv, Nav, and Cav). When screened with tarantula toxins, the four paddle sequences display distinct toxin binding properties, demonstrating that gating-modifier toxins can bind to Cav channels in a domain specific fashion. Domain III was the most commonly and strongly targeted, and mutagenesis revealed an acidic residue that is important for toxin binding. We also measured the lipid partitioning strength of all toxins tested and observed a positive correlation with their inhibition of Cav3.1, suggesting a key role for membrane partitioning. PMID:27045173

  14. Anoctamin Calcium-Activated Chloride Channels May Modulate Inhibitory Transmission in the Cerebellar Cortex.

    PubMed

    Zhang, Weiping; Schmelzeisen, Steffen; Parthier, Daniel; Frings, Stephan; Möhrlen, Frank

    2015-01-01

    Calcium-activated chloride channels of the anoctamin (alias TMEM16) protein family fulfill critical functions in epithelial fluid transport, smooth muscle contraction and sensory signal processing. Little is known, however, about their contribution to information processing in the central nervous system. Here we examined the recent finding that a calcium-dependent chloride conductance impacts on GABAergic synaptic inhibition in Purkinje cells of the cerebellum. We asked whether anoctamin channels may underlie this chloride conductance. We identified two anoctamin channel proteins, ANO1 and ANO2, in the cerebellar cortex. ANO1 was expressed in inhibitory interneurons of the molecular layer and the granule cell layer. Both channels were expressed in Purkinje cells but, while ANO1 appeared to be retained in the cell body, ANO2 was targeted to the dendritic tree. Functional studies confirmed that ANO2 was involved in a calcium-dependent mode of ionic plasticity that reduces the efficacy of GABAergic synapses. ANO2 channels attenuated GABAergic transmission by increasing the postsynaptic chloride concentration, hence reducing the driving force for chloride influx. Our data suggest that ANO2 channels are involved in a Ca2+-dependent regulation of synaptic weight in GABAergic inhibition. Thus, in balance with the chloride extrusion mechanism via the co-transporter KCC2, ANO2 appears to regulate ionic plasticity in the cerebellum.

  15. Anoctamin Calcium-Activated Chloride Channels May Modulate Inhibitory Transmission in the Cerebellar Cortex.

    PubMed

    Zhang, Weiping; Schmelzeisen, Steffen; Parthier, Daniel; Frings, Stephan; Möhrlen, Frank

    2015-01-01

    Calcium-activated chloride channels of the anoctamin (alias TMEM16) protein family fulfill critical functions in epithelial fluid transport, smooth muscle contraction and sensory signal processing. Little is known, however, about their contribution to information processing in the central nervous system. Here we examined the recent finding that a calcium-dependent chloride conductance impacts on GABAergic synaptic inhibition in Purkinje cells of the cerebellum. We asked whether anoctamin channels may underlie this chloride conductance. We identified two anoctamin channel proteins, ANO1 and ANO2, in the cerebellar cortex. ANO1 was expressed in inhibitory interneurons of the molecular layer and the granule cell layer. Both channels were expressed in Purkinje cells but, while ANO1 appeared to be retained in the cell body, ANO2 was targeted to the dendritic tree. Functional studies confirmed that ANO2 was involved in a calcium-dependent mode of ionic plasticity that reduces the efficacy of GABAergic synapses. ANO2 channels attenuated GABAergic transmission by increasing the postsynaptic chloride concentration, hence reducing the driving force for chloride influx. Our data suggest that ANO2 channels are involved in a Ca2+-dependent regulation of synaptic weight in GABAergic inhibition. Thus, in balance with the chloride extrusion mechanism via the co-transporter KCC2, ANO2 appears to regulate ionic plasticity in the cerebellum. PMID:26558388

  16. Anoctamin Calcium-Activated Chloride Channels May Modulate Inhibitory Transmission in the Cerebellar Cortex

    PubMed Central

    Parthier, Daniel; Frings, Stephan; Möhrlen, Frank

    2015-01-01

    Calcium-activated chloride channels of the anoctamin (alias TMEM16) protein family fulfill critical functions in epithelial fluid transport, smooth muscle contraction and sensory signal processing. Little is known, however, about their contribution to information processing in the central nervous system. Here we examined the recent finding that a calcium-dependent chloride conductance impacts on GABAergic synaptic inhibition in Purkinje cells of the cerebellum. We asked whether anoctamin channels may underlie this chloride conductance. We identified two anoctamin channel proteins, ANO1 and ANO2, in the cerebellar cortex. ANO1 was expressed in inhibitory interneurons of the molecular layer and the granule cell layer. Both channels were expressed in Purkinje cells but, while ANO1 appeared to be retained in the cell body, ANO2 was targeted to the dendritic tree. Functional studies confirmed that ANO2 was involved in a calcium-dependent mode of ionic plasticity that reduces the efficacy of GABAergic synapses. ANO2 channels attenuated GABAergic transmission by increasing the postsynaptic chloride concentration, hence reducing the driving force for chloride influx. Our data suggest that ANO2 channels are involved in a Ca2+-dependent regulation of synaptic weight in GABAergic inhibition. Thus, in balance with the chloride extrusion mechanism via the co-transporter KCC2, ANO2 appears to regulate ionic plasticity in the cerebellum. PMID:26558388

  17. Hyperpolarization-activated, cyclic nucleotide-gated cation channels in Aplysia: Contribution to classical conditioning.

    PubMed

    Yang, Qizong; Kuzyk, Pavlo; Antonov, Igor; Bostwick, Caleb J; Kohn, Andrea B; Moroz, Leonid L; Hawkins, Robert D

    2015-12-29

    Hyperpolarization-activated, cyclic nucleotide-gated cation (HCN) channels are critical regulators of neuronal excitability, but less is known about their possible roles in synaptic plasticity and memory circuits. Here, we characterized the HCN gene organization, channel properties, distribution, and involvement in associative and nonassociative forms of learning in Aplysia californica. Aplysia has only one HCN gene, which codes for a channel that has many similarities to the mammalian HCN channel. The cloned acHCN gene was expressed in Xenopus oocytes, which displayed a hyperpolarization-induced inward current that was enhanced by cGMP as well as cAMP. Similarly to its homologs in other animals, acHCN is permeable to K(+) and Na(+) ions, and is selectively blocked by Cs(+) and ZD7288. We found that acHCN is predominantly expressed in inter- and motor neurons, including LFS siphon motor neurons, and therefore tested whether HCN channels are involved in simple forms of learning of the siphon-withdrawal reflex in a semiintact preparation. ZD7288 (100 μM) significantly reduced an associative form of learning (classical conditioning) but had no effect on two nonassociative forms of learning (intermediate-term sensitization and unpaired training) or baseline responses. The HCN current is enhanced by nitric oxide (NO), which may explain the postsynaptic role of NO during conditioning. HCN current in turn enhances the NMDA-like current in the motor neurons, suggesting that HCN channels contribute to conditioning through this pathway. PMID:26668355

  18. Molecular Interactions between Tarantula Toxins and Low-Voltage-Activated Calcium Channels.

    PubMed

    Salari, Autoosa; Vega, Benjamin S; Milescu, Lorin S; Milescu, Mirela

    2016-01-01

    Few gating-modifier toxins have been reported to target low-voltage-activated (LVA) calcium channels, and the structural basis of toxin sensitivity remains incompletely understood. Studies of voltage-gated potassium (Kv) channels have identified the S3b-S4 "paddle motif," which moves at the protein-lipid interface to drive channel opening, as the target for these amphipathic neurotoxins. Voltage-gated calcium (Cav) channels contain four homologous voltage sensor domains, suggesting multiple toxin binding sites. We show here that the S3-S4 segments within Cav3.1 can be transplanted into Kv2.1 to examine their individual contributions to voltage sensing and pharmacology. With these results, we now have a more complete picture of the conserved nature of the paddle motif in all three major voltage-gated ion channel types (Kv, Nav, and Cav). When screened with tarantula toxins, the four paddle sequences display distinct toxin binding properties, demonstrating that gating-modifier toxins can bind to Cav channels in a domain specific fashion. Domain III was the most commonly and strongly targeted, and mutagenesis revealed an acidic residue that is important for toxin binding. We also measured the lipid partitioning strength of all toxins tested and observed a positive correlation with their inhibition of Cav3.1, suggesting a key role for membrane partitioning. PMID:27045173

  19. Hyperpolarization-activated, cyclic nucleotide-gated cation channels in Aplysia: Contribution to classical conditioning.

    PubMed

    Yang, Qizong; Kuzyk, Pavlo; Antonov, Igor; Bostwick, Caleb J; Kohn, Andrea B; Moroz, Leonid L; Hawkins, Robert D

    2015-12-29

    Hyperpolarization-activated, cyclic nucleotide-gated cation (HCN) channels are critical regulators of neuronal excitability, but less is known about their possible roles in synaptic plasticity and memory circuits. Here, we characterized the HCN gene organization, channel properties, distribution, and involvement in associative and nonassociative forms of learning in Aplysia californica. Aplysia has only one HCN gene, which codes for a channel that has many similarities to the mammalian HCN channel. The cloned acHCN gene was expressed in Xenopus oocytes, which displayed a hyperpolarization-induced inward current that was enhanced by cGMP as well as cAMP. Similarly to its homologs in other animals, acHCN is permeable to K(+) and Na(+) ions, and is selectively blocked by Cs(+) and ZD7288. We found that acHCN is predominantly expressed in inter- and motor neurons, including LFS siphon motor neurons, and therefore tested whether HCN channels are involved in simple forms of learning of the siphon-withdrawal reflex in a semiintact preparation. ZD7288 (100 μM) significantly reduced an associative form of learning (classical conditioning) but had no effect on two nonassociative forms of learning (intermediate-term sensitization and unpaired training) or baseline responses. The HCN current is enhanced by nitric oxide (NO), which may explain the postsynaptic role of NO during conditioning. HCN current in turn enhances the NMDA-like current in the motor neurons, suggesting that HCN channels contribute to conditioning through this pathway.

  20. Role of phospholipase D and diacylglycerol in activating constitutive TRPC-like cation channels in rabbit ear artery myocytes.

    PubMed

    Albert, A P; Piper, A S; Large, W A

    2005-08-01

    Previously we have described a constitutively active Ca2+-permeable non-selective cation channel in freshly dispersed rabbit ear artery myocytes that has similar properties to canonical transient receptor potential (TRPC) channel proteins. In the present study we have investigated the transduction pathways responsible for stimulating constitutive channel activity in these myocytes. Application of the pharmacological inhibitors of phosphatidylcholine-phospholipase D (PC-PLD), butan-1-ol and C2 ceramide, produced marked inhibition of constitutive channel activity in cell-attached patches and also butan-1-ol produced pronounced suppression of resting membrane conductance measured with whole-cell recording whereas the inactive isomer butan-2-ol had no effect on constitutive whole-cell or channel activity. In addition butan-1-ol had no effect on channel activity evoked by the diacylglycerol (DAG) analogue 1-oleoyl-2-acetyl-sn-glycerol (OAG). Inhibitors of PC-phospholipase C (PC-PLC) and phospholipase A2 (PLA2) had no effect on constitutive channel activity. Application of a purified PC-PLD enzyme and its metabolite phosphatidic acid to inside-out patches markedly increased channel activity. The phosphatidic acid phosphohydrolase (PAP) inhibitor dl-propranolol also inhibited constitutive and phosphatidic acid-induced increases in channel activity but had no effect on OAG-evoked responses. The DAG lipase and DAG kinase inhibitors, RHC80267 and R59949 respectively, which inhibit DAG metabolism, produced transient increases in channel activity which were mimicked by relatively high concentrations (40 microm) of OAG. The protein kinase C (PKC) inhibitor chelerythrine did not prevent channel activation by OAG but blocked the secondary inhibitory response of OAG. It is proposed that endogenous DAG is involved in the activation of channel activity and that its effects on channel activity are concentration-dependent with higher concentrations of DAG also inhibiting channel

  1. Role of phospholipase D and diacylglycerol in activating constitutive TRPC-like cation channels in rabbit ear artery myocytes.

    PubMed

    Albert, A P; Piper, A S; Large, W A

    2005-08-01

    Previously we have described a constitutively active Ca2+-permeable non-selective cation channel in freshly dispersed rabbit ear artery myocytes that has similar properties to canonical transient receptor potential (TRPC) channel proteins. In the present study we have investigated the transduction pathways responsible for stimulating constitutive channel activity in these myocytes. Application of the pharmacological inhibitors of phosphatidylcholine-phospholipase D (PC-PLD), butan-1-ol and C2 ceramide, produced marked inhibition of constitutive channel activity in cell-attached patches and also butan-1-ol produced pronounced suppression of resting membrane conductance measured with whole-cell recording whereas the inactive isomer butan-2-ol had no effect on constitutive whole-cell or channel activity. In addition butan-1-ol had no effect on channel activity evoked by the diacylglycerol (DAG) analogue 1-oleoyl-2-acetyl-sn-glycerol (OAG). Inhibitors of PC-phospholipase C (PC-PLC) and phospholipase A2 (PLA2) had no effect on constitutive channel activity. Application of a purified PC-PLD enzyme and its metabolite phosphatidic acid to inside-out patches markedly increased channel activity. The phosphatidic acid phosphohydrolase (PAP) inhibitor dl-propranolol also inhibited constitutive and phosphatidic acid-induced increases in channel activity but had no effect on OAG-evoked responses. The DAG lipase and DAG kinase inhibitors, RHC80267 and R59949 respectively, which inhibit DAG metabolism, produced transient increases in channel activity which were mimicked by relatively high concentrations (40 microm) of OAG. The protein kinase C (PKC) inhibitor chelerythrine did not prevent channel activation by OAG but blocked the secondary inhibitory response of OAG. It is proposed that endogenous DAG is involved in the activation of channel activity and that its effects on channel activity are concentration-dependent with higher concentrations of DAG also inhibiting channel

  2. Intramembrane Proton Binding Site Linked to Activation of Bacterial Pentameric Ion Channel*

    PubMed Central

    Wang, Hai-Long; Cheng, Xiaolin; Sine, Steven M.

    2012-01-01

    Prokaryotic orthologs of eukaryotic Cys-loop receptor channels recently emerged as structural and mechanistic surrogates to investigate this superfamily of intercellular signaling proteins. Here, we examine proton activation of the prokaryotic ortholog GLIC using patch clamp electrophysiology, mutagenesis, and molecular dynamics (MD) simulations. Whole-cell current recordings from human embryonic kidney (HEK) 293 cells expressing GLIC show half-maximal activation at pH 6, close to the pKa of histidine, implicating the three native His residues in proton sensing linked to activation. The mutation H235F abolishes proton activation, H277Y is without effect, and all nine mutations of His-127 prevent expression on the cell surface. In the GLIC crystal structure, His-235 on transmembrane (TM) α-helix 2, hydrogen bonds to the main chain carbonyl oxygen of Ile-259 on TM α-helix 3. MD simulations show that when His-235 is protonated, the hydrogen bond persists, and the channel remains in the open conformation, whereas when His-235 is deprotonated, the hydrogen bond dissociates, and the channel closes. Mutations of the proximal Tyr-263, which also links TM α-helices 2 and 3 via a hydrogen bond, alter proton sensitivity over a 1.5 pH unit range. MD simulations show that mutations of Tyr-263 alter the hydrogen bonding capacity of His-235. The overall findings show that His-235 in the TM region of GLIC is a novel proton binding site linked to channel activation. PMID:22084238

  3. Single-channel basis for the slow activation of the repolarizing cardiac potassium current, I(Ks).

    PubMed

    Werry, Daniel; Eldstrom, Jodene; Wang, Zhuren; Fedida, David

    2013-03-12

    Coassembly of potassium voltage-gated channel, KQT-like subfamily, member 1 (KCNQ1) with potassium voltage-gated channel, Isk-related family, member 1 (KCNE1) the delayed rectifier potassium channel I(Ks). Its slow activation is critically important for membrane repolarization and for abbreviating the cardiac action potential, especially during sympathetic activation and at high heart rates. Mutations in either gene can cause long QT syndrome, which can lead to fatal arrhythmias. To understand better the elementary behavior of this slowly activating channel complex, we quantitatively analyzed direct measurements of single-channel I(Ks). Single-channel recordings from transiently transfected mouse ltk(-) cells confirm a channel that has long latency periods to opening (1.67 ± 0.073 s at +60 mV) but that flickers rapidly between multiple open and closed states in non-deactivating bursts at positive membrane potentials. Channel activity is cyclic with periods of high activity followed by quiescence, leading to an overall open probability of only ∼0.15 after 4 s under our recording conditions. The mean single-channel conductance was determined to be 3.2 pS, but unlike any other known wild-type human potassium channel, long-lived subconductance levels coupled to activation are a key feature of both the activation and deactivation time courses of the conducting channel complex. Up to five conducting levels ranging from 0.13 to 0.66 pA could be identified in single-channel recordings at 60 mV. Fast closings and overt subconductance behavior of the wild-type I(Ks) channel required modification of existing Markov models to include these features of channel behavior. PMID:23431135

  4. Shaking stack model of ion conduction through the Ca(2+)-activated K+ channel.

    PubMed Central

    Schumaker, M F

    1992-01-01

    Motivated by the results of Neyton and Miller (1988. J. Gen. Physiol. 92:549-586), suggesting that the Ca(2+)-activated K+ channel has four high affinity ion binding sites, we propose a physically attractive variant of the single-vacancy conduction mechanism for this channel. Simple analytical expressions for conductance, current, flux ratio exponent, and reversal potential under bi-ionic conditions are found. A set of conductance data are analyzed to determine a realistic range of parameter values. Using these, we find qualitative agreement with a variety of experimental results previously reported in the literature. The exquisite selectivity of the Ca(2+)-activated K+ channel may be explained as a consequence of the concerted motion of the "stack" in the proposed mechanism. PMID:1420923

  5. Activation of Na+ channels in cell membrane by capacitive stimulation with silicon chip

    NASA Astrophysics Data System (ADS)

    Schoen, Ingmar; Fromherz, Peter

    2005-11-01

    Sodium channels are the crucial electrical elements of neuronal excitation. As a step toward hybrid neuron-semiconductor devices, we studied the activation of recombinant NaV1.4 sodium channels in human embryonic kidney (HEK293) cells by stimulation from an electrolyte/oxide/silicon (EOS) capacitor. HfO2 was used as an insulator to attain a high capacitance. An effective activation was achieved by decaying voltage ramps at constant intracellular voltage at a depleted NaCl concentration in the bath to enhance the resistance of the cell-chip contact. We were also able to open sodium channels at a NaCl concentration close to physiological conditions. This experiment provides a basis for noninvasive capacitive stimulation of nerve cells with semiconductor chips.

  6. A comprehensive search for calcium binding sites critical for TMEM16A calcium-activated chloride channel activity.

    PubMed

    Tien, Jason; Peters, Christian J; Wong, Xiu Ming; Cheng, Tong; Jan, Yuh Nung; Jan, Lily Yeh; Yang, Huanghe

    2014-06-30

    TMEM16A forms calcium-activated chloride channels (CaCCs) that regulate physiological processes such as the secretions of airway epithelia and exocrine glands, the contraction of smooth muscles, and the excitability of neurons. Notwithstanding intense interest in the mechanism behind TMEM16A-CaCC calcium-dependent gating, comprehensive surveys to identify and characterize potential calcium sensors of this channel are still lacking. By aligning distantly related calcium-activated ion channels in the TMEM16 family and conducting systematic mutagenesis of all conserved acidic residues thought to be exposed to the cytoplasm, we identify four acidic amino acids as putative calcium-binding residues. Alterations of the charge, polarity, and size of amino acid side chains at these sites alter the ability of different divalent cations to activate the channel. Furthermore, TMEM16A mutant channels containing double cysteine substitutions at these residues are sensitive to the redox potential of the internal solution, providing evidence for their physical proximity and solvent accessibility.

  7. Mechanosensitive channels are activated by stress in the actin stress fibres, and could be involved in gravity sensing in plants.

    PubMed

    Tatsumi, H; Furuichi, T; Nakano, M; Toyota, M; Hayakawa, K; Sokabe, M; Iida, H

    2014-01-01

    Mechanosensitive (MS) channels are expressed in a variety of cells. The molecular and biophysical mechanism involved in the regulation of MS channel activities is a central interest in basic biology. MS channels are thought to play crucial roles in gravity sensing in plant cells. To date, two mechanisms have been proposed for MS channel activation. One is that tension development in the lipid bilayer directly activates MS channels. The second mechanism proposes that the cytoskeleton is involved in the channel activation, because MS channel activities are modulated by pharmacological treatments that affect the cytoskeleton. We tested whether tension in the cytoskeleton activates MS channels. Mammalian endothelial cells were microinjected with phalloidin-conjugated beads, which bound to stress fibres, and a traction force to the actin cytoskeleton was applied by dragging the beads with optical tweezers. MS channels were activated when the force was applied, demonstrating that a sub-pN force to the actin filaments activates a single MS channel. Plants may use a similar molecular mechanism in gravity sensing, since the cytoplasmic Ca(2+) concentration increase induced by changes in the gravity vector was attenuated by potential MS channel inhibitors, and by actin-disrupting drugs. These results support the idea that the tension increase in actin filaments by gravity-dependent sedimentation of amyloplasts activates MS Ca(2+) -permeable channels, which can be the molecular mechanism of a Ca(2+) concentration increase through gravistimulation. We review recent progress in the study of tension sensing by actin filaments and MS channels using advanced biophysical methods, and discuss their possible roles in gravisensing.

  8. Nuclear pore ion channel activity in live syncytial nuclei.

    PubMed

    Bustamante, Jose Omar

    2002-05-01

    Nuclear pore complexes (NPCs) are important nanochannels for the control of gene activity and expression. Most of our knowledge of NPC function has been derived from isolated nuclei and permeabilized cells in cell lysates/extracts. Since recent patch-clamp work has challenged the dogma that NPCs are freely permeable to small particles, a preparation of isolated living nuclei in their native liquid environment was sought and found: the syncytial nuclei in the water of the coconut Cocos nucifera. These nuclei have all properties of NPC-mediated macromolecular transport (MMT) and express foreign green fluorescent protein (GFP) plasmids. They display chromatin movement, are created by particle aggregation or by division, can grow by throwing filaments to catch material, etc. This study shows, for the first time, that living NPCs engaged in MMT do not transport physiological ions - a phenomenon that explains observations of nucleocytoplasmic ion gradients. Since coconuts are inexpensive (less than US$1/nut per litre), this robust preparation may contribute to our understanding of NPCs and cell nucleus and to the development of biotechnologies for the production of DNA, RNA and proteins.

  9. Multi-channel Kalman filters for active noise control.

    PubMed

    van Ophem, S; Berkhoff, A P

    2013-04-01

    By formulating the feed-forward broadband active noise control problem as a state estimation problem it is possible to achieve a faster rate of convergence than the filtered reference least mean squares algorithm and possibly also a better tracking performance. A multiple input/multiple output Kalman algorithm is derived to perform this state estimation. To make the algorithm more suitable for real-time applications, the Kalman filter is written in a fast array form and the secondary path state matrices are implemented in output normal form. The resulting filter implementation is tested in simulations and in real-time experiments. It was found that for a constant primary path the filter has a fast rate of convergence and is able to track changes in the frequency spectrum. For a forgetting factor equal to unity the system is robust but the filter is unable to track rapid changes in the primary path. A forgetting factor lower than 1 gives a significantly improved tracking performance but leads to a numerical instability for the fast array form of the algorithm. PMID:23556580

  10. Apparent intermediate K conductance channel hyposmotic activation in human lens epithelial cells.

    PubMed

    Lauf, Peter K; Misri, Sandeep; Chimote, Ameet A; Adragna, Norma C

    2008-03-01

    This study explores the nature of K fluxes in human lens epithelial cells (LECs) in hyposmotic solutions. Total ion fluxes, Na-K pump, Cl-dependent Na-K-2Cl (NKCC), K-Cl (KCC) cotransport, and K channels were determined by 85Rb uptake and cell K (Kc) by atomic absorption spectrophotometry, and cell water gravimetrically after exposure to ouabain +/- bumetanide (Na-K pump and NKCC inhibitors), and ion channel inhibitors in varying osmolalities with Na, K, or methyl-d-glucamine and Cl, sulfamate, or nitrate. Reverse transcriptase polymerase chain reaction (RT-PCR), Western blot analyses, and immunochemistry were also performed. In isosmotic (300 mosM) media approximately 90% of the total Rb influx occurred through the Na-K pump and NKCC and approximately 10% through KCC and a residual leak. Hyposmotic media (150 mosM) decreased K(c) by a 16-fold higher K permeability and cell water, but failed to inactivate NKCC and activate KCC. Sucrose replacement or extracellular K to >57 mM, but not Rb or Cs, in hyposmotic media prevented Kc and water loss. Rb influx equaled Kc loss, both blocked by clotrimazole (IC50 approximately 25 microM) and partially by 1-[(2-chlorophenyl) diphenylmethyl]-1H-pyrazole (TRAM-34) inhibitors of the IK channel KCa3.1 but not by other K channel or connexin hemichannel blockers. Of several anion channel blockers (dihydro-indenyl)oxy]alkanoic acid (DIOA), 4-2(butyl-6,7-dichloro-2-cyclopentylindan-1-on-5-yl)oxybutyric acid (DCPIB), and phloretin totally or partially inhibited Kc loss and Rb influx, respectively. RT-PCR and immunochemistry confirmed the presence of KCa3.1 channels, aside of the KCC1, KCC2, KCC3 and KCC4 isoforms. Apparently, IK channels, possibly in parallel with volume-sensitive outwardly rectifying Cl channels, effect regulatory volume decrease in LECs. PMID:18184876

  11. Ca(2+)-activated chloride channel activity during Ca(2+) alternans in ventricular myocytes.

    PubMed

    Kanaporis, Giedrius; Blatter, Lothar A

    2016-11-01

    Cardiac alternans, defined beat-to-beat alternations in contraction, action potential (AP) morphology or cytosolic Ca transient (CaT) amplitude, is a high risk indicator for cardiac arrhythmias. We investigated mechanisms of cardiac alternans in single rabbit ventricular myocytes. CaTs were monitored simultaneously with membrane currents or APs recorded with the patch clamp technique. A strong correlation between beat-to-beat alternations of AP morphology and CaT alternans was observed. During CaT alternans application of voltage clamp protocols in form of pre-recorded APs revealed a prominent Ca(2+)-dependent membrane current consisting of a large outward component coinciding with AP phases 1 and 2, followed by an inward current during AP repolarization. Approximately 85% of the initial outward current was blocked by Cl(-) channel blocker DIDS or lowering external Cl(-) concentration identifying it as a Ca(2+)-activated Cl(-) current (ICaCC). The data suggest that ICaCC plays a critical role in shaping beat-to-beat alternations in AP morphology during alternans.

  12. Annual report on surveillance and maintenance activities at Oak Ridge National Laboratory, Oak Ridge, Tennessee, fiscal year 1996

    SciTech Connect

    1996-11-01

    In fiscal year (FY) 1995, the sites and facilities from both the Remedial Action (RA) and Decontamination and Decommissioning (D and D) programs were combined to form the Oak Ridge National Laboratory (ORNL) Environmental Restoration (ER) Surveillance and Maintenance (S and M) Program. Surveillance and Maintenance activities were conducted throughout FY 1996 at the RA facilities. Overall, the RA S and M Program consists of approximately 650 acres that include 14 waste area groupings with approximately 200 sites. These sites include 46 major facilities, several leak and contaminated soil sites, 38 inactive tanks, approximately 50 environmental study areas and approximately 2,973 wells and boreholes. Site inspections were conducted at established frequencies on appropriate sites in the RA S and M Program in accordance with the established S and M FY 1996 Incentive Task Order (ITO).

  13. Active Sites of Spinoxin, a Potassium Channel Scorpion Toxin, Elucidated by Systematic Alanine Scanning.

    PubMed

    Peigneur, Steve; Yamaguchi, Yoko; Kawano, Chihiro; Nose, Takeru; Nirthanan, Selvanayagam; Gopalakrishnakone, Ponnampalam; Tytgat, Jan; Sato, Kazuki

    2016-05-31

    Peptide toxins from scorpion venoms constitute the largest group of toxins that target the voltage-gated potassium channel (Kv). Spinoxin (SPX) isolated from the venom of scorpion Heterometrus spinifer is a 34-residue peptide neurotoxin cross-linked by four disulfide bridges. SPX is a potent inhibitor of Kv1.3 potassium channels (IC50 = 63 nM), which are considered to be valid molecular targets in the diagnostics and therapy of various autoimmune disorders and cancers. Here we synthesized 25 analogues of SPX and analyzed the role of each amino acid in SPX using alanine scanning to study its structure-function relationships. All synthetic analogues showed similar disulfide bond pairings and secondary structures as native SPX. Alanine replacements at Lys(23), Asn(26), and Lys(30) resulted in loss of activity against Kv1.3 potassium channels, whereas replacements at Arg(7), Met(14), Lys(27), and Tyr(32) also largely reduced inhibitory activity. These results suggest that the side chains of these amino acids in SPX play an important role in its interaction with Kv1.3 channels. In particular, Lys(23) appears to be a key residue that underpins Kv1.3 channel inhibition. Of these seven amino acid residues, four are basic amino acids, suggesting that the positive electrostatic potential on the surface of SPX is likely required for high affinity interaction with Kv1.3 channels. This study provides insight into the structure-function relationships of SPX with implications for the rational design of new lead compounds targeting potassium channels with high potency. PMID:27159046

  14. Activation of TRPC6 channels promotes endocannabinoid biosynthesis in neuronal CAD cells

    PubMed Central

    Bardell, Tamera K.; Barker, Eric L.

    2010-01-01

    Calcium influx activates biosynthesis of the endogenous cannabinoids 2-arachidonyl glycerol (2-AG) and anandamide (AEA). The calcium channel involved with endocannabinoid synthesis and release in neurons is still unknown. The canonical TRP (TRPC) channels are calcium-permeable channels that are a homology-based subdivision of the broader class of TRP channels. TRPC3, 6, and 7 are G-protein-gated nonselective cation channels that have been localized to lipid rafts and shown to colocalize with caveolin 1. Because endocannabinoid synthesis has been found to occur “on demand” in a calcium-dependent manner and has been linked to lipid rafts, we explored the potential role of transient receptor potential (TRP) channels in this process. Previously, we observed that after metabolism AEA and arachidonic acid (ArA) can be recycled into new endocannabinoid molecules. Consistent with these previous findings, we found that Cath.a differentiated (CAD) cells pretreated with radiolabeled ArA exhibited a robust increase in 2-AG release in response to TRPC stimulation with the diacylglycerol (DAG) analogue, 1-oleoyl-2-acetyl-sn-glycerol (OAG). Furthermore, cells pretreated with [3H]AEA produced a significant amount of AEA and 2-AG upon stimulation of TRPC channels. This process was not mediated through protein kinase C activation. Reverse transcriptase-polymerase chain reaction (RT-PCR) analysis revealed that only TRPC6 was present in the CAD cells. siRNA-induced knockdown of TRPC6 in the CAD cells abolished OAG-stimulated production of the endocannabionids. This evidence suggests that TRPC6 may be capable of promoting endocannabinoid synthesis in neuronal cells. PMID:20466028

  15. Evaluating, Migrating, and Consolidating Databases and Applications for Long-Term Surveillance and Maintenance Activities at the Rocky Flats Site

    SciTech Connect

    Surovchak, S.; Marutzky, S.; Thompson, B.; Miller, K.; Labonte, E.

    2006-07-01

    The U.S. Department of Energy (DOE) Office of Legacy Management (LM) is assuming responsibilities for long-term surveillance and maintenance (LTS and M) activities at the Rocky Flats Environmental Technology Site (RFETS) during fiscal year 2006. During the transition, LM is consolidating databases and applications that support these various functions into a few applications which will streamline future management and retrieval of data. This paper discussed the process of evaluating, migrating, and consolidating these databases and applications for LTS and M activities and provides lessons learned that will benefit future transitions. (authors)

  16. Quantifying the transition from fluvial- to wave-dominance for river deltas with multiple active channels

    NASA Astrophysics Data System (ADS)

    Nienhuis, J.; Ashton, A. D.; Giosan, L.

    2012-12-01

    The plan-view morphologies of fluvial- and wave-dominated deltas are clearly distinctive, but transitional forms are numerous. A quantitative, process-based description of this transition remains unexplored, particularly for river deltas with multiple active channels. Previous studies focused on general attributes of the fluvial and marine environment, such as the balance between wave energy and river discharge. Here, we propose that the transition between fluvial and wave dominance is directly related to the magnitude of the fluvial bedload flux to the nearshore region versus the alongshore sediment transport capacity of waves removing sediment away from the mouth. In the case of a single-channel delta, this balance can be computed for a given distribution of waves approaching shore. Fluvial dominance occurs when fluvial sediment input exceeds the wave-sustained maximum alongshore sediment transport for all potential shoreline orientations both up- and downdrift of the river mouth. However, deltaic channels have the tendency to bifurcate with increasing fluvial strength. Initial bifurcation splits the fluvial sediment flux among individual channels, while the potential sediment transport by waves remains constant for both river mouths. At higher bifurcation orders, multiple channels interact with each other alongshore, a situation more complicated than the single channel case and one that cannot be simple addressed analytically. We apply a model of plan-view shoreline evolution to simulate the evolution of a deltaic environment with multiple active channels. A highly simplified fluvial domain is represented by deposition of sediment where channels meet the coast. We investigate two scenarios of fluvial delivery. The first scenario deposits fluvial sediment alongshore on a self-similar predefined network of channels. We analyze the effects of different network geometrical parameters, such as bifurcation length, bifurcation angle, and sediment partitioning. In the

  17. Effects of urine composition on epithelial Na+ channel-targeted protease activity

    PubMed Central

    Berman, Jonathan M; Awayda, Ryan G; Awayda, Mouhamed S

    2015-01-01

    We examined human urinary proteolytic activity toward the Epithelial Sodium Channel (ENaC). We focused on two sites in each of alpha and gamma ENaC that are targets of endogenous and exogenous proteases. We examined the effects of ionic strength, pH and urinary H+-buffers, metabolic intermediates, redox molecules, and large urinary proteins. Monoatomic cations caused the largest effect, with sodium inhibiting activity in the 15–515 mEq range. Multivalent cations zinc and copper inhibited urinary proteolytic activity at concentrations below 100 μmol/L. Similar to sodium, urea caused a 30% inhibition in the 0–500 mmol/L range. This was not observed with acetone and ethanol. Modulating urinary redox status modified activity with H2O2 stimulated and ascorbate inhibited activity. Minimal effects (<10%) were observed with caffeine, glucose, several TCA cycle intermediates, salicylic acid, inorganic phosphate, albumin, creatinine, and Tamm–Horsfall protein. The cumulative activity of ENaC-cleaving proteases was highest at neutral pH, however, alpha and gamma proteases exhibited an inverse dependence with alpha stimulated at acidic and gamma stimulated at alkaline pH. These data indicate that ENaC-targeting urinary proteolytic activity is sensitive to sodium, urea and pH and changes in these components can modify channel cleavage and activation status, and likely downstream sodium absorption unrelated to changes in protein or channel density. PMID:26564065

  18. TRPV3 channels mediate strontium-induced mouse-egg activation.

    PubMed

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

    2013-12-12

    In mammals, calcium influx is required for oocyte maturation and egg activation. The molecular identities of the calcium-permeant channels that underlie the initiation of embryonic development are not established. Here, we describe a transient receptor potential (TRP) ion channel current activated by TRP agonists that is absent in TrpV3(-/-) eggs. TRPV3 current is differentially expressed during oocyte maturation, reaching a peak of maximum density and activity at metaphase of meiosis II (MII), the stage of fertilization. Selective activation of TRPV3 channels provokes egg activation by mediating massive calcium entry. Widely used to activate eggs, strontium application is known to yield normal offspring in combination with somatic cell nuclear transfer. We show that TRPV3 is required for strontium influx, because TrpV3(-/-) eggs failed to conduct Sr(2+) or undergo strontium-induced activation. We propose that TRPV3 is a major mediator of calcium influx in mouse eggs and is a putative target for artificial egg activation. PMID:24316078

  19. Effects of urine composition on epithelial Na+ channel-targeted protease activity.

    PubMed

    Berman, Jonathan M; Awayda, Ryan G; Awayda, Mouhamed S

    2015-11-01

    We examined human urinary proteolytic activity toward the Epithelial Sodium Channel (ENaC). We focused on two sites in each of alpha and gamma ENaC that are targets of endogenous and exogenous proteases. We examined the effects of ionic strength, pH and urinary H(+)-buffers, metabolic intermediates, redox molecules, and large urinary proteins. Monoatomic cations caused the largest effect, with sodium inhibiting activity in the 15-515 mEq range. Multivalent cations zinc and copper inhibited urinary proteolytic activity at concentrations below 100 μmol/L. Similar to sodium, urea caused a 30% inhibition in the 0-500 mmol/L range. This was not observed with acetone and ethanol. Modulating urinary redox status modified activity with H2O2 stimulated and ascorbate inhibited activity. Minimal effects (<10%) were observed with caffeine, glucose, several TCA cycle intermediates, salicylic acid, inorganic phosphate, albumin, creatinine, and Tamm-Horsfall protein. The cumulative activity of ENaC-cleaving proteases was highest at neutral pH, however, alpha and gamma proteases exhibited an inverse dependence with alpha stimulated at acidic and gamma stimulated at alkaline pH. These data indicate that ENaC-targeting urinary proteolytic activity is sensitive to sodium, urea and pH and changes in these components can modify channel cleavage and activation status, and likely downstream sodium absorption unrelated to changes in protein or channel density. PMID:26564065

  20. Channel-forming activities of peroxisomal membrane proteins from the yeast Saccharomyces cerevisiae.

    PubMed

    Grunau, Silke; Mindthoff, Sabrina; Rottensteiner, Hanspeter; Sormunen, Raija T; Hiltunen, J Kalervo; Erdmann, Ralf; Antonenkov, Vasily D

    2009-03-01

    Highly-purified peroxisomes from the yeast Saccharomyces cerevisiae grown on oleic acid were investigated for the presence of channel (pore)-forming proteins in the membrane of these organelles. Solubilized membrane proteins were reconstituted in planar lipid bilayers and their pore-forming activity was studied by means of multiple-channel monitoring or single-channel analysis. Two abundant pore-forming activities were detected with an average conductance of 0.2 and 0.6 nS in 1.0 m KCl, respectively. The high-conductance pore (0.6 nS in 1.0 m KCl) is slightly selective to cations (P(K+)/P(Cl-) approximately 1.3) and showed an unusual flickering at elevated (> +/-40 mV) holding potentials directed upward relative to the open state of the channel. The data obtained for the properties of the low-conductance pore (0.2 nS in 1.0 m KCl) support the notion that the high-conductance channel represents a cluster of two low-conductance pores. The results lead to conclusion that the yeast peroxisomes contain membrane pore-forming proteins that may aid the transfer of small solutes between the peroxisomal lumen and cytoplasm.

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

    PubMed

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

    2015-11-01

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

  2. Probing the energy landscape of activation gating of the bacterial potassium channel KcsA.

    PubMed

    Linder, Tobias; de Groot, Bert L; Stary-Weinzinger, Anna

    2013-01-01

    The bacterial potassium channel KcsA, which has been crystallized in several conformations, offers an ideal model to investigate activation gating of ion channels. In this study, essential dynamics simulations are applied to obtain insights into the transition pathways and the energy profile of KcsA pore gating. In agreement with previous hypotheses, our simulations reveal a two phasic activation gating process. In the first phase, local structural rearrangements in TM2 are observed leading to an intermediate channel conformation, followed by large structural rearrangements leading to full opening of KcsA. Conformational changes of a highly conserved phenylalanine, F114, at the bundle crossing region are crucial for the transition from a closed to an intermediate state. 3.9 µs umbrella sampling calculations reveal that there are two well-defined energy barriers dividing closed, intermediate, and open channel states. In agreement with mutational studies, the closed state was found to be energetically more favorable compared to the open state. Further, the simulations provide new insights into the dynamical coupling effects of F103 between the activation gate and the selectivity filter. Investigations on individual subunits support cooperativity of subunits during activation gating.

  3. Functional insights into modulation of BKCa channel activity to alter myometrial contractility

    PubMed Central

    Lorca, Ramón A.; Prabagaran, Monali; England, Sarah K.

    2014-01-01

    The large-conductance voltage- and Ca2+-activated K+ channel (BKCa) is an important regulator of membrane excitability in a wide variety of cells and tissues. In myometrial smooth muscle, activation of BKCa plays essential roles in buffering contractility to maintain uterine quiescence during pregnancy and in the transition to a more contractile state at the onset of labor. Multiple mechanisms of modulation have been described to alter BKCa channel activity, expression, and cellular localization. In the myometrium, BKCa is regulated by alternative splicing, protein targeting to the plasma membrane, compartmentation in membrane microdomains, and posttranslational modifications. In addition, interaction with auxiliary proteins (i.e., β1- and β2-subunits), association with G-protein coupled receptor signaling pathways, such as those activated by adrenergic and oxytocin receptors, and hormonal regulation provide further mechanisms of variable modulation of BKCa channel function in myometrial smooth muscle. Here, we provide an overview of these mechanisms of BKCa channel modulation and provide a context for them in relation to myometrial function. PMID:25132821

  4. Essential role of lncRNA binding for WDR5 maintenance of active chromatin and embryonic stem cell pluripotency

    PubMed Central

    Yang, Yul W; Flynn, Ryan A; Chen, Yong; Qu, Kun; Wan, Bingbing; Wang, Kevin C; Lei, Ming; Chang, Howard Y

    2014-01-01

    The WDR5 subunit of the MLL complex enforces active chromatin and can bind RNA; the relationship between these two activities is unclear. Here we identify a RNA binding pocket on WDR5, and discover a WDR5 mutant (F266A) that selectively abrogates RNA binding without affecting MLL complex assembly or catalytic activity. Complementation in ESCs shows that WDR5 F266A mutant is unable to accumulate on chromatin, and is defective in gene activation, maintenance of histone H3 lysine 4 trimethylation, and ESC self renewal. We identify a family of ESC messenger and lncRNAs that interact with wild type WDR5 but not F266A mutant, including several lncRNAs known to be important for ESC gene expression. These results suggest that specific RNAs are integral inputs into the WDR5-MLL complex for maintenance of the active chromatin state and embryonic stem cell fates. DOI: http://dx.doi.org/10.7554/eLife.02046.001 PMID:24521543

  5. External protons destabilize the activated voltage sensor in hERG channels.

    PubMed

    Shi, Yu Patrick; Cheng, Yen May; Van Slyke, Aaron C; Claydon, Tom W

    2014-03-01

    Extracellular acidosis shifts hERG channel activation to more depolarized potentials and accelerates channel deactivation; however, the mechanisms underlying these effects are unclear. External divalent cations, e.g., Ca(2+) and Cd(2+), mimic these effects and coordinate within a metal ion binding pocket composed of three acidic residues in hERG: D456 and D460 in S2 and D509 in S3. A common mechanism may underlie divalent cation and proton effects on hERG gating. Using two-electrode voltage clamp, we show proton sensitivity of hERG channel activation (pKa = 5.6), but not deactivation, was greatly reduced in the presence of Cd(2+) (0.1 mM), suggesting a common binding site for the Cd(2+) and proton effect on activation and separable effects of protons on activation and deactivation. Mutational analysis confirmed that D509 plays a critical role in the pH dependence of activation, as shown previously, and that cooperative actions involving D456 and D460 are also required. Importantly, neutralization of all three acidic residues abolished the proton-induced shift of activation, suggesting that the metal ion binding pocket alone accounts for the effects of protons on hERG channel activation. Voltage-clamp fluorimetry measurements demonstrated that protons shifted the voltage dependence of S4 movement to more depolarized potentials. The data indicate a site and mechanism of action for protons on hERG activation gating; protonation of D456, D460 and D509 disrupts interactions between these residues and S4 gating charges to destabilize the activated configuration of S4.

  6. GPR119 Agonist AS1269574 Activates TRPA1 Cation Channels to Stimulate GLP-1 Secretion.

    PubMed

    Chepurny, Oleg G; Holz, George G; Roe, Michael W; Leech, Colin A

    2016-06-01

    GPR119 is a G protein-coupled receptor expressed on intestinal L cells that synthesize and secrete the blood glucose-lowering hormone glucagon-like peptide-1 (GLP-1). GPR119 agonists stimulate the release of GLP-1 from L cells, and for this reason there is interest in their potential use as a new treatment for type 2 diabetes mellitus. AS1269574 is one such GPR119 agonist, and it is the prototype of a series of 2,4,6 trisubstituted pyrimidines that exert positive glucoregulatory actions in mice. Here we report the unexpected finding that AS1269574 stimulates GLP-1 release from the STC-1 intestinal cell line by directly promoting Ca(2+) influx through transient receptor potential ankyrin 1 (TRPA1) cation channels. These GPR119-independent actions of AS1269574 are inhibited by TRPA1 channel blockers (AP-18, A967079, HC030031) and are not secondary to intracellular Ca(2+) release or cAMP production. Patch clamp studies reveal that AS1269574 activates an outwardly rectifying membrane current with properties expected of TRPA1 channels. However, the TRPA1 channel-mediated action of AS1269574 to increase intracellular free calcium concentration is not replicated by GPR119 agonists (AR231453, oleoylethanolamide) unrelated in structure to AS1269574. Using human embryonic kidney-293 cells expressing recombinant rat TRPA1 channels but not GPR119, direct TRPA1 channel activating properties of AS1269574 are validated. Because we find that AS1269574 also acts in a conventional GPR119-mediated manner to stimulate proglucagon gene promoter activity in the GLUTag intestinal L cell line, new findings reported here reveal the surprising capacity of AS1269574 to act as a dual agonist at two molecular targets (GPR119/TRPA1) important to the control of L-cell function and type 2 diabetes mellitus drug discovery research. PMID:27082897

  7. Increased Anion Channel Activity Is an Unavoidable Event in Ozone-Induced Programmed Cell Death

    PubMed Central

    Errakhi, Rafik; Hiramatsu, Takuya; Meimoun, Patrice; Briand, Joël; Iwaya-Inoue, Mari; Kawano, Tomonori; Bouteau, François

    2010-01-01

    Background Ozone is a major secondary air pollutant often reaching high concentrations in urban areas under strong daylight, high temperature and stagnant high-pressure systems. Ozone in the troposphere is a pollutant that is harmful to the plant. Principal Findings By exposing cells to a strong pulse of ozonized air, an acute cell death was observed in suspension cells of Arabidopsis thaliana used as a model. We demonstrated that O3 treatment induced the activation of a plasma membrane anion channel that is an early prerequisite of O3-induced cell death in A. thaliana. Our data further suggest interplay of anion channel activation with well known plant responses to O3, Ca2+ influx and NADPH-oxidase generated reactive oxygen species (ROS) in mediating the oxidative cell death. This interplay might be fuelled by several mechanisms in addition to the direct ROS generation by O3; namely, H2O2 generation by salicylic and abscisic acids. Anion channel activation was also shown to promote the accumulation of transcripts encoding vacuolar processing enzymes, a family of proteases previously reported to contribute to the disruption of vacuole integrity observed during programmed cell death. Significance Collectively, our data indicate that anion efflux is an early key component of morphological and biochemical events leading to O3-induced programmed cell death. Because ion channels and more specifically anion channels assume a crucial position in cells, an understanding about the underlying role(s) for ion channels in the signalling pathway leading to programmed cell death is a subject that warrants future investigation. PMID:20967217

  8. ENaC activity requires CFTR channel function independently of phosphorylation in sweat duct.

    PubMed

    Reddy, M M; Quinton, P M

    2005-09-01

    We previously showed that activation of the cystic fibrosis (CF) transmembrane conductance regulator (CFTR) Cl- conductance (gCFTR) supports parallel activation of amiloride-sensitive epithelial Na+ channel (ENaC) in the native human sweat duct. However, it is not clear whether phosphorylated CFTR, phosphorylated ENaC, or only Cl(-) -channel function is required for activation. We used basilaterally alpha-toxin-permeabilized human sweat ducts to test the hypothesis that ENaC activation depends only on Cl(-) -channel function and not on phosphorylation of either CFTR or ENaC. CFTR is classically activated by PKA plus millimolar ATP, but cytosolic glutamate activation of gCFTR is independent of ATP and phosphorylation. We show here that both phosphorylation-dependent (PKA) and phosphorylation-independent (glutamate) activation of CFTR Cl- channel function support gENaC activation. We tested whether cytosolic application of 5 mM ATP alone, phosphorylation by cAMP, cGMP, G-protein dependent kinases (all in the presence of 100 microM ATP), or glutamate could support ENaC activation in the absence of gCFTR. We found that none of these agonists activated gENaC by themselves when Cl- current (I(Cl-)) through CFTR was blocked by: 1) Cl- removal, 2) DIDS inhibition, 3) lowering the ATP concentration to 100 microM (instead of 5 mM required to support CFTR channel function), or 4) mutant CFTR (homozygous DeltaF508 CF ducts). However, Cl- gradients in the direction of absorption supported, while Cl- gradients in the direction of secretion prevented ENaC activation. We conclude that the interaction between CFTR and ENaC is dependent on activated I(Cl-) through CFTR in the direction of absorption (Cl- gradient from lumen to cell). But such activation of ENaC is independent of phosphorylation and ATP. However, reversing I(Cl-) through CFTR in the direction of secretion (Cl- gradient from cell to lumen) prevents ENaC activation even in the presence of I(Cl-) through CFTR. PMID

  9. Myrsinane, Premyrsinane, and Cyclomyrsinane Diterpenes from Euphorbia falcata as Potassium Ion Channel Inhibitors with Selective G Protein-Activated Inwardly Rectifying Ion Channel (GIRK) Blocking Effects.

    PubMed

    Vasas, Andrea; Forgo, Peter; Orvos, Péter; Tálosi, László; Csorba, Attila; Pinke, Gyula; Hohmann, Judit

    2016-08-26

    GIRK channels are activated by a large number of G protein-coupled receptors and regulate the electrical activity of neurons, cardiac atrial myocytes, and β-pancreatic cells. Abnormalities in GIRK channel function have been implicated in the pathophysiology of neuropathic pain, drug addiction, and cardiac arrhythmias. In the heart, GIRK channels are selectively expressed in the atrium, and their activation inhibits pacemaker activity, thereby slowing the heart rate. In the present study, 19 new diterpenes, falcatins A-S (1-19), and the known euphorprolitherin D (20) were isolated from Euphorbia falcata. The compounds were assayed on stable transfected HEK-hERG (Kv11.1) and HEK-GIRK1/4 (Kir3.1 and Kir3.4) cells. Blocking activity on GIRK channels was exerted by 13 compounds (61-83% at 10 μM), and, among them, five possessed low potency on the hERG channel (4-20% at 10 μM). These selective activities suggest that myrsinane-related diterpenes are potential lead compounds for the treatment of atrial fibrillation. PMID:27441737

  10. Potency of irritation by benzylidenemalononitriles in humans correlates with TRPA1 ion channel activation

    PubMed Central

    Lindsay, Christopher D.; Green, Christopher; Bird, Mike; Jones, James T. A.; Riches, James R.; McKee, Katherine K.; Sandford, Mark S.; Wakefield, Debra A.; Timperley, Christopher M.

    2015-01-01

    We show that the physiological activity of solid aerosolized benzylidenemalononitriles (BMNs) including ‘tear gas’ (CS) in historic human volunteer trials correlates with activation of the human transient receptor potential ankyrin 1 ion channel (hTRPA1). This suggests that the irritation caused by the most potent of these compounds results from activation of this channel. We prepared 50 BMNs and measured their hTRPA1 agonist potencies. A mechanism of action consistent with their physiological activity, involving their dissolution in water on contaminated body surfaces, cell membrane penetration and reversible thiolation by a cysteine residue of hTRPA1, supported by data from nuclear magnetic resonance experiments with a model thiol, explains the structure–activity relationships. The correlation provides evidence that hTRPA1 is a receptor for irritants on nociceptive neurons involved in pain perception; thus, its activation in the eye, nose, mouth and skin would explain the symptoms of lachrymation, sneezing, coughing and stinging, respectively. The structure–activity results and the use of the BMNs as pharmacological tools in future by other researchers may contribute to a better understanding of the TRPA1 channel in humans (and other animals) and help facilitate the discovery of treatments for human diseases involving this receptor. PMID:26064575

  11. Single-channel biophysical and pharmacological characterizations of native human large-conductance calcium-activated potassium channels in freshly isolated detrusor smooth muscle cells.

    PubMed

    Malysz, John; Rovner, Eric S; Petkov, Georgi V

    2013-07-01

    Recent studies have demonstrated the importance of large-conductance Ca(2+)-activated K(+) (BK) channels in detrusor smooth muscle (DSM) function in vitro and in vivo. However, in-depth characterization of human native DSM single BK channels has not yet been provided. Here, we conducted single-channel recordings from excised patches from native human DSM cells. Inside-out and outside-out recordings in high K(+) symmetrical solution (containing 140 mM KCl and ~300 nM free Ca(2+)) showed single-channel conductance of 215-220 pS, half-maximum constant for activation of ~+75 to +80 mV, and low probability of opening (P o) at +20 mV that increased ~10-fold at +40 mV and ~60-fold at +60 mV. Using the inside-out configuration at +30 mV, reduction of intracellular [Ca(2+)] from ~300 nM to Ca(2+)-free decreased the P o by ~85 %, whereas elevation to ~800 nM increased P o by ~50-fold. The BK channel activator NS1619 (10 μM) enhanced the P o by ~10-fold at +30 mV; subsequent application of the selective BK channel inhibitor paxilline (500 nM) blocked the activity. Changes in intracellular [Ca(2+)] or the addition of NS1619 did not significantly alter the current amplitude or single-channel conductance. This is the first report to provide biophysical and pharmacological profiles of native human DSM single BK channels highlighting their importance in regulating human DSM excitability.

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

    PubMed

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

    2016-01-01

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

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

    PubMed

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

    2015-02-01

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

  14. TRPC channels underlie cholinergic plateau potentials and persistent activity in entorhinal cortex.

    PubMed

    Zhang, Zizhen; Reboreda, Antonio; Alonso, Angel; Barker, Philip A; Séguéla, Philippe

    2011-04-01

    Persistent neuronal activity lasting seconds to minutes has been proposed to allow for the transient storage of memory traces in entorhinal cortex and thus could play a major role in working memory. Nonsynaptic plateau potentials induced by acetylcholine account for persistent firing in many cortical and subcortical structures. The expression of these intrinsic properties in cortical neurons involves the recruitment of a non-selective cation conductance. Despite its functional importance, the identity of the cation channels remains unknown. Here we show that, in layer V of rat medial entorhinal cortex, muscarinic receptor-evoked plateau potentials and persistent firing induced by carbachol require phospholipase C activation, decrease of PIP(2) levels, and permissive intracellular Ca(2+) concentrations. Plateau potentials and persistent activity were suppressed by the generic nonselective cation channel blockers FFA (100 μM) and 2-APB (100 μM), as well as by the TRPC channel blocker SKF-96365 (50 μM). However, plateau potentials were not affected by the TRPV channel blocker ruthenium red (40 μM). The TRPC3/6/7 activator OAG did not induce or enhance persistent firing evoked by carbachol. Voltage clamp recordings revealed a carbachol-activated, nonselective cationic current with a heteromeric TRPC-like phenotype. Moreover, plateau potentials and persistent firing were inhibited by intracellular application of the peptide EQVTTRL that disrupts interactions between the C-terminal domain of TRPC4/5 subunits and associated PDZ proteins. Altogether, our data suggest that TRPC cation channels mediating persistent muscarinic currents significantly contribute to the firing and mnemonic properties of projection neurons in the entorhinal cortex.

  15. Proteolytic fragmentation of inositol 1,4,5-trisphosphate receptors: a novel mechanism regulating channel activity?

    PubMed

    Wang, Liwei; Alzayady, Kamil J; Yule, David I

    2016-06-01

    Inositol 1,4,5-trisphosphate receptors (IP3 Rs) are a family of ubiquitously expressed intracellular Ca(2+) release channels. Regulation of channel activity by Ca(2+) , nucleotides, phosphorylation, protein binding partners and other cellular factors is thought to play a major role in defining the specific spatiotemporal characteristics of intracellular Ca(2+) signals. These properties are, in turn, believed pivotal for the selective and specific physiological activation of Ca(2+) -dependent effectors. IP3 Rs are also substrates for the intracellular cysteine proteases, calpain and caspase. Cleavage of the IP3 R has been proposed to play a role in apoptotic cell death by uncoupling regions important for IP3 binding from the channel domain, leaving an unregulated leaky Ca(2+) pore. Contrary to this hypothesis, we demonstrate following proteolysis that N- and C-termini of IP3 R1 remain associated, presumably through non-covalent interactions. Further, we show that complementary fragments of IP3 R1 assemble into tetrameric structures and retain their ability to be regulated robustly by IP3 . While peptide continuity is clearly not necessary for IP3 -gating of the channel, we propose that cleavage of the IP3 R peptide chain may alter other important regulatory events to modulate channel activity. In this scenario, stimulation of the cleaved IP3 R may support distinct spatiotemporal Ca(2+) signals and activation of specific effectors. Notably, in many adaptive physiological events, the non-apoptotic activities of caspase and calpain are demonstrated to be important, but the substrates of the proteases are poorly defined. We speculate that proteolytic fragmentation may represent a novel form of IP3 R regulation, which plays a role in varied adaptive physiological processes.

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

    PubMed

    Zeraatpisheh, Zahra; Vatanparast, Jafar

    2015-10-01

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

  17. Activation and maintenance of peripheral semantic features of unambiguous words after right hemisphere brain damage in adults

    PubMed Central

    Tompkins, Connie A.; Fassbinder, Wiltrud; Scharp, Victoria L.; Meigh, Kimberly M.

    2009-01-01

    Background The right cerebral hemisphere (RH) sustains activation of subordinate, secondary, less common, and/or distantly related meanings of words. Much of the pertinent data come from studies of homonyms, but some evidence also suggests that the RH has a unique maintenance function in relation to unambiguous nouns. In a divided visual field priming study, Atchley, Burgess, and Keeney (1999) reported that only left visual field/RH presentation yielded evidence of continuing activation of peripheral semantic features that were incompatible with the most common image or representation of their corresponding nouns (e.g., rotten for “apple”). Activation for weakly related features that were compatible with the dominant representation (e.g., crunchy) was sustained over time regardless of the visual field/hemisphere of initial stimulus input. Several studies report that unilateral right hemisphere brain damage (RHD) in adults affects the RH’s meaning maintenance function, but this work also has centred on homonyms, and/or more recently metonymic and metaphoric polysemous words. Aims The current investigation examined whether RHD deficits in processing secondary and/or distantly related meanings of words, typically observed in studies of homonyms, would extend to peripheral, weakly related semantic features of unambiguous nouns. Methods & Procedures Participants were 28 adults with unilateral RHD from cerebrovascular accident, and 38 adults without brain damage. Participants listened to spoken sentences that ended with an unambiguous noun. Each sentence was followed by a spoken target phoneme string. Targets included peripheral semantic features of the sentence-final noun that were either compatible or incompatible with the dominant mental images of the noun, and were presented at two intervals after that noun. A lexical decision task was used to gauge both the early activation and maintenance of activation for these weakly related semantic features. Outcomes

  18. Twelve years of repeated wild hog activity promotes population maintenance of an invasive clonal plant in a coastal dune ecosystem.

    PubMed

    Oldfield, Callie A; Evans, Jonathan P

    2016-04-01

    Invasive animals can facilitate the success of invasive plant populations through disturbance. We examined the relationship between the repeated foraging disturbance of an invasive animal and the population maintenance of an invasive plant in a coastal dune ecosystem. We hypothesized that feral wild hog (Sus scrofa) populations repeatedly utilized tubers of the clonal perennial, yellow nutsedge (Cyperus esculentus) as a food source and evaluated whether hog activity promoted the long-term maintenance of yellow nutsedge populations on St. Catherine's Island, Georgia, United States. Using generalized linear mixed models, we tested the effect of wild hog disturbance on permanent sites for yellow nutsedge culm density, tuber density, and percent cover of native plant species over a 12-year period. We found that disturbance plots had a higher number of culms and tubers and a lower percentage of native live plant cover than undisturbed control plots. Wild hogs redisturbed the disturbed plots approximately every 5 years. Our research provides demographic evidence that repeated foraging disturbances by an invasive animal promote the long-term population maintenance of an invasive clonal plant. Opportunistic facultative interactions such as we demonstrate in this study are likely to become more commonplace as greater numbers of introduced species are integrated into ecological communities around the world.

  19. Twelve years of repeated wild hog activity promotes population maintenance of an invasive clonal plant in a coastal dune ecosystem.

    PubMed

    Oldfield, Callie A; Evans, Jonathan P

    2016-04-01

    Invasive animals can facilitate the success of invasive plant populations through disturbance. We examined the relationship between the repeated foraging disturbance of an invasive animal and the population maintenance of an invasive plant in a coastal dune ecosystem. We hypothesized that feral wild hog (Sus scrofa) populations repeatedly utilized tubers of the clonal perennial, yellow nutsedge (Cyperus esculentus) as a food source and evaluated whether hog activity promoted the long-term maintenance of yellow nutsedge populations on St. Catherine's Island, Georgia, United States. Using generalized linear mixed models, we tested the effect of wild hog disturbance on permanent sites for yellow nutsedge culm density, tuber density, and percent cover of native plant species over a 12-year period. We found that disturbance plots had a higher number of culms and tubers and a lower percentage of native live plant cover than undisturbed control plots. Wild hogs redisturbed the disturbed plots approximately every 5 years. Our research provides demographic evidence that repeated foraging disturbances by an invasive animal promote the long-term population maintenance of an invasive clonal plant. Opportunistic facultative interactions such as we demonstrate in this study are likely to become more commonplace as greater numbers of introduced species are integrated into ecological communities around the world. PMID:27110354

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

    PubMed Central

    Farley, J.; Rudy, B.

    1988-01-01

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

  1. Measurement of orexin (hypocretin) and substance P effects on constitutively active inward rectifier K(+) channels in brain neurons.

    PubMed

    Nakajima, Yasuko; Nakajima, Shigehiro

    2010-01-01

    Electrophysiological experiments in our laboratory have led to the discovery that the cholinergic neurons in the nucleus basalis in the rat forebrain possess constitutively active inward rectifier K(+) channels. Unlike cloned inward rectifier K(+) channels, these constitutively active inward rectifier K(+) channels were found to have unique properties, and thus were named "KirNB" (inward rectifier K(+) channels in the nucleus basalis). We found that slow excitatory transmitters, such as orexin (hypocretin) and substance P, suppress the KirNB channel, resulting in neuronal excitation. Furthermore, it was discovered that suppression of KirNB channels by these transmitters is through protein kinase C (PKC). This chapter describes detailed electrophysiological techniques for investigating the effects of orexin and substance P on constitutively active KirNB channels. For this purpose, we also present a method for culturing nucleus basalis cholinergic neurons in which KirNB channels exist. Then, we describe the procedures through which PKC has been determined to mediate inhibition of KirNB channels by orexin and substance P. There are probably many other transmitters which may produce effects on KirNB channels. This chapter will enable researchers to investigate the effects of such transmitters on KirNB channels and their roles in neuronal functions.

  2. Prolactin stimulates cell proliferation through a long form of prolactin receptor and K+ channel activation.

    PubMed Central

    Van Coppenolle, Fabien; Skryma, Roman; Ouadid-Ahidouch, Halima; Slomianny, Christian; Roudbaraki, Morad; Delcourt, Philippe; Dewailly, Etienne; Humez, Sandrine; Crépin, Alexandre; Gourdou, Isabelle; Djiane, Jean; Bonnal, Jean-Louis; Mauroy, Brigitte; Prevarskaya, Natalia

    2004-01-01

    PRL (prolactin) has been implicated in the proliferation and differentiation of numerous tissues, including the prostate gland. However, the PRL-R (PRL receptor) signal transduction pathway, leading to the stimulation of cell proliferation, remains unclear and has yet to be mapped. The present study was undertaken to develop a clear understanding of the mechanisms involved in this pathway and, in particular, to determine the role of K(+) channels. We used androgen-sensitive prostate cancer (LNCaP) cells whose proliferation is known to be stimulated by PRL. Reverse transcriptase PCR analysis showed that LNCaP cells express a long form of PRL-R, but do not produce its intermediate isoform. Patch-clamp techniques showed that the application of 5 nM PRL increased both the macroscopic K(+) current amplitude and the single K(+)-channel open probability. This single-channel activity increase was reduced by the tyrosine kinase inhibitors genistein, herbimycin A and lavandustine A, thereby indicating that tyrosine kinase phosphorylation is required in PRL-induced K(+) channel stimulation. PRL enhances p59( fyn ) phosphorylation by a factor of 2 after a 10 min application in culture. In addition, where an antip59( fyn ) antibody is present in the patch pipette, PRL no longer increases K(+) current amplitude. Furthermore, the PRL-stimulated proliferation is inhibited by the K(+) channel inhibitors alpha-dendrotoxin and tetraethylammonium. Thus, as K(+) channels are known to be involved in LNCaP cell proliferation, we suggest that K(+) channel modulation by PRL, via p59( fyn ) pathway, is the primary ionic event in PRL signal transduction, triggering cell proliferation. PMID:14565846

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

    PubMed

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

    2005-01-10

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

  4. Molecular mechanism underlying β1 regulation in voltage- and calcium-activated potassium (BK) channels.

    PubMed

    Castillo, Karen; Contreras, Gustavo F; Pupo, Amaury; Torres, Yolima P; Neely, Alan; González, Carlos; Latorre, Ramon

    2015-04-14

    Being activated by depolarizing voltages and increases in cytoplasmic Ca(2+), voltage- and calcium-activated potassium (BK) channels and their modulatory β-subunits are able to dampen or stop excitatory stimuli in a wide range of cellular types, including both neuronal and nonneuronal tissues. Minimal alterations in BK channel function may contribute to the pathophysiology of several diseases, including hypertension, asthma, cancer, epilepsy, and diabetes. Several gating processes, allosterically coupled to each other, control BK channel activity and are potential targets for regulation by auxiliary β-subunits that are expressed together with the α (BK)-subunit in almost every tissue type where they are found. By measuring gating currents in BK channels coexpressed with chimeras between β1 and β3 or β2 auxiliary subunits, we were able to identify that the cytoplasmic regions of β1 are responsible for the modulation of the voltage sensors. In addition, we narrowed down the structural determinants to the N terminus of β1, which contains two lysine residues (i.e., K3 and K4), which upon substitution virtually abolished the effects of β1 on charge movement. The mechanism by which K3 and K4 stabilize the voltage sensor is not electrostatic but specific, and the α (BK)-residues involved remain to be identified. This is the first report, to our knowledge, where the regulatory effects of the β1-subunit have been clearly assigned to a particular segment, with two pivotal amino acids being responsible for this modulation.

  5. TMCO1 Is an ER Ca(2+) Load-Activated Ca(2+) Channel.

    PubMed

    Wang, Qiao-Chu; Zheng, Qiaoxia; Tan, Haiyan; Zhang, Bing; Li, Xiaoling; Yang, Yuxiu; Yu, Jie; Liu, Yang; Chai, Hao; Wang, Xi; Sun, Zhongshuai; Wang, Jiu-Qiang; Zhu, Shu; Wang, Fengli; Yang, Maojun; Guo, Caixia; Wang, Heng; Zheng, Qingyin; Li, Yang; Chen, Quan; Zhou, Aimin; Tang, Tie-Shan

    2016-06-01

    Maintaining homeostasis of Ca(2+) stores in the endoplasmic reticulum (ER) is crucial for proper Ca(2+) signaling and key cellular functions. The Ca(2+)-release-activated Ca(2+) (CRAC) channel is responsible for Ca(2+) influx and refilling after store depletion, but how cells cope with excess Ca(2+) when ER stores are overloaded is unclear. We show that TMCO1 is an ER transmembrane protein that actively prevents Ca(2+) stores from overfilling, acting as what we term a "Ca(2+) load-activated Ca(2+) channel" or "CLAC" channel. TMCO1 undergoes reversible homotetramerization in response to ER Ca(2+) overloading and disassembly upon Ca(2+) depletion and forms a Ca(2+)-selective ion channel on giant liposomes. TMCO1 knockout mice reproduce the main clinical features of human cerebrofaciothoracic (CFT) dysplasia spectrum, a developmental disorder linked to TMCO1 dysfunction, and exhibit severe mishandling of ER Ca(2+) in cells. Our findings indicate that TMCO1 provides a protective mechanism to prevent overfilling of ER stores with Ca(2+) ions. PMID:27212239

  6. Molecular dioxygen enters the active site of 12/15-lipoxygenase via dynamic oxygen access channels.

    PubMed

    Saam, Jan; Ivanov, Igor; Walther, Matthias; Holzhütter, Hermann-Georg; Kuhn, Hartmut

    2007-08-14

    Cells contain numerous enzymes that use molecular oxygen for their reactions. Often, their active sites are buried deeply inside the protein, which raises the question whether there are specific access channels guiding oxygen to the site of catalysis. Choosing 12/15-lipoxygenase as a typical example for such oxygen-dependent enzymes, we determined the oxygen distribution within the protein and defined potential routes for oxygen access. For this purpose, we have applied an integrated strategy of structural modeling, molecular dynamics simulations, site-directed mutagenesis, and kinetic measurements. First, we computed the 3D free-energy distribution for oxygen, which led to identification of four oxygen channels in the protein. All channels connect the protein surface with a region of high oxygen affinity at the active site. This region is localized opposite to the nonheme iron providing a structural explanation for the reaction specificity of this lipoxygenase isoform. The catalytically most relevant path can be obstructed by L367F exchange, which leads to a strongly increased Michaelis constant for oxygen. The blocking mechanism is explained in detail by reordering the hydrogen-bonding network of water molecules. Our results provide strong evidence that the main route for oxygen access to the active site of the enzyme follows a channel formed by transiently interconnected cavities whereby the opening and closure are governed by side chain dynamics. PMID:17675410

  7. Chloride channel activity of ClC-2 is modified by the actin cytoskeleton.

    PubMed Central

    Ahmed, N; Ramjeesingh, M; Wong, S; Varga, A; Garami, E; Bear, C E

    2000-01-01

    The chloride channel ClC-2 has been implicated in essential physiological functions, including cell-volume regulation and fluid secretion by specific epithelial tissues. Although ClC-2 is known to be activated by hyperpolarization and hypo-osmotic shock, the molecular basis for the regulation of this channel remains unclear. Here we show in the Xenopus oocyte expression system that the chloride-channel activity of ClC-2 is enhanced after treatment with the actin-disrupting agents cytochalasin and latrunkulin. These findings suggest that the actin cytoskeleton normally exerts an inhibitory effect on ClC-2 activity. An inhibitory domain was previously defined in the N-terminus of ClC-2, so we sought to determine whether this domain might interact directly with actin in binding assays in vitro. We found that a glutathione S-transferase fusion protein containing the inhibitory domain was capable of binding actin in overlay and co-sedimentation assays. Further, the binding of actin to this relatively basic peptide (pI 8.4) might be mediated through electrostatic interactions because binding was inhibited at high concentrations of NaCl with a half-maximal decrease in signal at 180 mM NaCl. This work suggests that electrostatic interactions between the N-terminus of ClC-2 and the actin cytoskeleton might have a role in the regulation of this channel. PMID:11104687

  8. A multichannel integrated circuit for electrical recording of neural activity, with independent channel programmability.

    PubMed

    Mora Lopez, Carolina; Prodanov, Dimiter; Braeken, Dries; Gligorijevic, Ivan; Eberle, Wolfgang; Bartic, Carmen; Puers, Robert; Gielen, Georges

    2012-04-01

    Since a few decades, micro-fabricated neural probes are being used, together with microelectronic interfaces, to get more insight in the activity of neuronal networks. The need for higher temporal and spatial recording resolutions imposes new challenges on the design of integrated neural interfaces with respect to power consumption, data handling and versatility. In this paper, we present an integrated acquisition system for in vitro and in vivo recording of neural activity. The ASIC consists of 16 low-noise, fully-differential input channels with independent programmability of its amplification (from 100 to 6000 V/V) and filtering (1-6000 Hz range) capabilities. Each channel is AC-coupled and implements a fourth-order band-pass filter in order to steeply attenuate out-of-band noise and DC input offsets. The system achieves an input-referred noise density of 37 nV/√Hz, a NEF of 5.1, a CMRR > 60 dB, a THD < 1% and a sampling rate of 30 kS/s per channel, while consuming a maximum of 70 μA per channel from a single 3.3 V. The ASIC was implemented in a 0.35 μm CMOS technology and has a total area of 5.6 × 4.5 mm². The recording system was successfully validated in in vitro and in vivo experiments, achieving simultaneous multichannel recordings of cell activity with satisfactory signal-to-noise ratios.

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

    PubMed

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

    2010-10-20

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

  10. Voltage-dependent drug blockade of L-glutamate activated channels of the crayfish.

    PubMed Central

    Dekin, M S; Edwards, C

    1983-01-01

    The actions of d-tubocurarine (d-TC) and local anaesthetics on the L-glutamate activated channel at the voltage-clamped crayfish neuromuscular junction were studied. The effect of d-TC and local anaesthetics on the dose-response relationship between ionophoretically applied L-glutamate and synaptic current suggested that both acted as non-competitive inhibitors. The amount of inhibition was voltage dependent, and increased as the membrane potential was hyperpolarized. This voltage-dependent block was also manifest in a flattening of the I-V relationship between L-glutamate induced current and membrane potential in the presence of d-TC. However, the reversal potential for the L-glutamate activated channel was not affected; it was about +7 mV in both the presence and absence of d-TC. The neurally evoked excitatory post-synaptic current (e.p.s.c.) was depressed in the presence of these drugs and this effect was also voltage dependent. The time course of the e.p.s.c. was affected, but less so than expected if the L-glutamate activated channel were identical to the channel opened by acetylcholine at the vertebrate neuromuscular junction. Possible reasons for this discrepancy are discussed. PMID:6312026

  11. Polymodal activation of the TREK-2 K2P channel produces structurally distinct open states.

    PubMed

    McClenaghan, Conor; Schewe, Marcus; Aryal, Prafulla; Carpenter, Elisabeth P; Baukrowitz, Thomas; Tucker, Stephen J

    2016-06-01

    The TREK subfamily of two-pore domain (K2P) K(+) channels exhibit polymodal gating by a wide range of physical and chemical stimuli. Crystal structures now exist for these channels in two main states referred to as the "up" and "down" conformations. However, recent studies have resulted in contradictory and mutually exclusive conclusions about the functional (i.e., conductive) status of these two conformations. To address this problem, we have used the state-dependent TREK-2 inhibitor norfluoxetine that can only bind to the down state, thereby allowing us to distinguish between these two conformations when activated by different stimuli. Our results reconcile these previously contradictory gating models by demonstrating that activation by pressure, temperature, voltage, and pH produce more than one structurally distinct open state and reveal that channel activation does not simply involve switching between the up and down conformations. These results also highlight the diversity of structural mechanisms that K2P channels use to integrate polymodal gating signals. PMID:27241700

  12. Allosteric coupling of the inner activation gate to the outer pore of a potassium channel.

    PubMed

    Peters, Christian J; Fedida, David; Accili, Eric A

    2013-10-23

    In potassium channels, functional coupling of the inner and outer pore gates may result from energetic interactions between residues and conformational rearrangements that occur along a structural path between them. Here, we show that conservative mutations of a residue near the inner activation gate of the Shaker potassium channel (I470) modify the rate of C-type inactivation at the outer pore, pointing to this residue as part of a pathway that couples inner gate opening to changes in outer pore structure and reduction of ion flow. Because they remain equally sensitive to rises in extracellular potassium, altered inactivation rates of the mutant channels are not secondary to modified binding of potassium to the outer pore. Conservative mutations of I470 also influence the interaction of the Shaker N-terminus with the inner gate, which separately affects the outer pore.

  13. Maintenance of Physical Activity and Exercise Capacity After Rehabilitation in Coronary Heart Disease: A Randomized Controlled Trial

    PubMed Central

    Aliabad, Hassan Okati; Vafaeinasab, Mohammadreza; Morowatisharifabad, Mohammad Ali; Afshani, Seyed Alireza; Firoozabadi, Mahdieh Ghanbari; Forouzannia, Seyed Khalil

    2014-01-01

    Background and Objectives: Physical activity is one of the core components in cardiac rehabilitation and secondary prevention programs. This study investigated the effect of an intervention based on the health action process approach (HAPA) together with family support in the maintenance of physical activity and exercise capacity in coronary heart disease after discharge from rehabilitation. Method and Materials: In this randomized controlled trial, 96 patients with coronary heart disease were randomly assigned to control and intervention groups at the end of a rehabilitation program at Afshar Hospital, Yazd, Iran. HAPA Constructs and family support using a self-reported questionnaire and maximal oxygen uptake through a treadmill exercise test were measured prior to and 4 months after the intervention. Results: HAPA-based intervention together with family support increased scores of HAPA constructs and family support in the intervention group compared with the control group. The results showed that physical activity and exercise capacity in the intervention group was significantly higher than the control group after the intervention. Conclusion: HAPA-based intervention together with family support can be a useful tool for maintenance of physical activity and exercise capacity in coronary heart disease. PMID:25363124

  14. Activation and proton transport mechanism in influenza A M2 channel.

    PubMed

    Wei, Chenyu; Pohorille, Andrew

    2013-11-01

    Molecular dynamics trajectories 2 μs in length have been generated for the pH-activated, tetrameric M2 proton channel of the influenza A virus in all protonation states of the pH sensor located at the His(37) tetrad. All simulated structures are in very good agreement with high-resolution structures. Changes in the channel caused by progressive protonation of His(37) provide insight into the mechanism of proton transport. The channel is closed at both His(37) and Trp(41) sites in the singly and doubly protonated states, but it opens at Trp(41) upon further protonation. Anions access the charged His(37) and by doing so stabilize the protonated states of the channel. The narrow opening at the His(37) site, further blocked by anions, is inconsistent with the water-wire mechanism of proton transport. Instead, conformational interconversions of His(37) correlated with hydrogen bonding to water molecules indicate that these residues shuttle protons in high-protonation states. Hydrogen bonds between charged and uncharged histidines are rare. The valve at Val(27) remains on average quite narrow in all protonation states but fluctuates sufficiently to support water and proton transport. A proton transport mechanism in which the channel, depending on pH, opens at either the histidine or valine gate is only partially supported by the simulations. PMID:24209848

  15. Single channel activity of OmpF-like porin from Yersinia pseudotuberculosis.

    PubMed

    Rokitskaya, Tatyana I; Kotova, Elena A; Naberezhnykh, Gennadiy A; Khomenko, Valentina A; Gorbach, Vladimir I; Firsov, Alexander M; Zelepuga, Elena A; Antonenko, Yuri N; Novikova, Olga D

    2016-04-01

    To gain a mechanistic insight in the functioning of the OmpF-like porin from Yersinia pseudotuberculosis (YOmpF), we compared the effect of pH variation on the ion channel activity of the protein in planar lipid bilayers and its binding to lipid membranes. The behavior of YOmpF channels upon acidification was similar to that previously described for Escherichia coli OmpF. In particular, a decrease in pH of the bathing solution resulted in a substantial reduction of YOmpF single channel conductance, accompanied by the emergence of subconductance states. Similar subconductance substates were elicited by the addition of lysophosphatidylcholine. This observation, made with porin channels for the first time, pointed to the relevance of lipid-protein interactions, in particular, the lipid curvature stress, to the appearance of subconductance states at acidic pH. Binding of YOmpF to membranes displayed rather modest dependence on pH, whereas the channel-forming potency of the protein tremendously decreased upon acidification.

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

    PubMed Central

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

    2016-01-01

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

  17. Sodium channel activation in the squid giant axon. Steady state properties

    PubMed Central

    1985-01-01

    Treatment of giant axons from the squid, Loligo pealei, with pronase removes Na channel inactivation. It was found that the peak Na current is increased, but the activation kinetics are not significantly altered, by pronase. Measurements of the fraction of open channels as a function of voltage (F-V) showed an e-folding at 7 mV and a center point near -15 mV. The rate of e-folding implies that a minimum of 4 e- /channel must cross the membrane field to open the channel. The charge vs. voltage (Q-V) curve measured in a pronase-treated axon is not significantly different from that measured when inactivation is intact: approximately 1,850 e-/micron2 were measured over the voltage range - 150 to 50 mV, and the center point was near -30 mV. Normalizing these two curves (F-V and Q-V) and plotting them together reveals that they cross when inactivation is intact but saturate together when inactivation is removed. This illustrates the error one makes when measuring peak conductance with intact inactivation and interpreting that to be the fraction of open channels. A model is described that was used to interpret these results. In the model, we propose that inactivation must be slightly voltage dependent and that an interaction occurs between the inactivating particle and the gating charge. A linear sequence of seven states (a single open state with six closed states) is sufficient to describe the data presented here for Na channel activation in pronase-treated axons. PMID:2578549

  18. Oxygen causes fetal pulmonary vasodilation through activation of a calcium-dependent potassium channel.

    PubMed

    Cornfield, D N; Reeve, H L; Tolarova, S; Weir, E K; Archer, S

    1996-07-23

    At birth, pulmonary vasodilation occurs as air-breathing life begins. The mechanism of O2-induced pulmonary vasodilation is unknown. We proposed that O2 causes fetal pulmonary vasodilation through activation of a calcium-dependent potassium channel (KCa) via a cyclic nucleotide-dependent kinase. We tested this hypothesis in hemodynamic studies in acutely prepared fetal lambs and in patch-clamp studies on resistance fetal pulmonary artery smooth muscle cells. Fetal O2 tension (PaO2) was increased by ventilating the ewe with 100% O2, causing fetal total pulmonary resistance to decrease from 1.18 +/- 0.14 to 0.41 +/- 0.03 mmHg per ml per min. Tetraethylammonium and iberiotoxin, preferential KCa-channel inhibitors, attenuated O2-induced fetal pulmonary vasodilation, while glibenclamide, an ATP-sensitive K+-channel antagonist, had no effect. Treatment with either a guanylate cyclase antagonist (LY83583) or cyclic nucleotide-dependent kinase inhibitors (H-89 and KT 5823) significantly attenuated O2-induced fetal pulmonary vasodilation. Under hypoxic conditions (PaO2 = 25 mmHg), whole-cell K+-channel currents (Ik) were small and were inhibited by 1 mM tetraethylammonium or 100 nM charybdotoxin (CTX; a specific KCa-channel blocker). Normoxia (PaO2 = 120 mmHg) increased Ik by more than 300%, and this was reversed by 100 nM CTX. Nitric oxide also increased Ik. Resting membrane potential was -37.2 +/- 1.9 mV and cells depolarized on exposure to CTX, while hyperpolarizing in normoxia. We conclude that O2 causes fetal pulmonary vasodilation by stimulating a cyclic nucleotide-dependent kinase, resulting in KCa-channel activation, membrane hyperpolarization, and vasodilation. PMID:8755608

  19. Bestrophin-1 encodes for the Ca2+-activated anion channel in hippocampal astrocytes.

    PubMed

    Park, Hyungju; Oh, Soo-Jin; Han, Kyung-Seok; Woo, Dong Ho; Park, Hyekyung; Mannaioni, Guido; Traynelis, Stephen F; Lee, C Justin

    2009-10-14

    In mammalian brain, neurons and astrocytes are reported to express various chloride and anion channels, but the evidence for functional expression of Ca(2+)-activated anion channel (CAAC) and its molecular identity have been lacking. Here we report electrophysiological evidence for the CAAC expression and its molecular identity by mouse Bestrophin 1 (mBest1) in astrocytes of the mouse brain. Using Ca(2+) imaging and perforated-patch-clamp analysis, we demonstrate that astrocytes displayed an inward current at holding potential of -70 mV that was dependent on an increase in intracellular Ca(2+) after G(alphaq)-coupled receptor activation. This current was mediated mostly by anions and was sensitive to well known anion channel blockers such as niflumic acid, 5-nitro-2(3-phenylpropylamino)-benzoic acid, and flufenamic acid. To find the molecular identity of the anion channel responsible for the CAAC current, we analyzed the expression of candidate genes and found that the mRNA for mouse mBest1 is predominantly expressed in acutely dissociated or cultured astrocytes. Whole-cell patch-clamp analysis using HEK293T cells heterologously expressing full-length mBest1 showed a Ca(2+)-dependent current mediated by mBest1, with a complete impairment of the current by a putative pore mutation, W93C. Furthermore, mBest1-mediated CAAC from cultured astrocytes was significantly reduced by expression of mBest1-specific short hairpin RNA (shRNA), suggesting that the CAAC is mediated by a channel encoded by mBest1. Finally, hippocampal CA1 astrocytes in hippocampal slice also showed mBest1-mediated CAAC because it was inhibited by mBest1-specific shRNA. Collectively, these data provide molecular evidence that the mBest1 channel is responsible for CAAC function in astrocytes.

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

    PubMed Central

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

    2009-01-01

    The mucolipin TRP (TRPML) proteins are a family of endolysosomal cation channels with genetically established importance in humans and rodent. Mutations of human TRPML1 cause type IV mucolipidosis, a devastating pediatric neurodegenerative disease. Our recent electrophysiological studies revealed that, although a TRPML1-mediated current can only be recorded in late endosome and lysosome (LEL) using the lysosome patch clamp technique, a proline substitution in TRPML1 (TRPML1V432P) results in a large whole cell current. Thus, it remains unknown whether the large TRPML1V432P-mediated current results from an increased surface expression (trafficking), elevated channel activity (gating), or both. Here we performed systemic Pro substitutions in a region previously implicated in the gating of various 6 transmembrane cation channels. We found that several Pro substitutions displayed gain-of-function (GOF) constitutive activities at both the plasma membrane (PM) and endolysosomal membranes. Although wild-type TRPML1 and non-GOF Pro substitutions localized exclusively in LEL and were barely detectable in the PM, the GOF mutations with high constitutive activities were not restricted to LEL compartments, and most significantly, exhibited significant surface expression. Because lysosomal exocytosis is Ca2+-dependent, constitutive Ca2+ permeability due to Pro substitutions may have resulted in stimulus-independent intralysosomal Ca2+ release, hence the surface expression and whole cell current of TRPML1. Indeed, surface staining of lysosome-associated membrane protein-1 (Lamp-1) was dramatically increased in cells expressing GOF TRPML1 channels. We conclude that TRPML1 is an inwardly rectifying, proton-impermeable, Ca2+ and Fe2+/Mn2+ dually permeable cation channel that may be gated by unidentified cellular mechanisms through a conformational change in the cytoplasmic face of the transmembrane 5 (TM5). Furthermore, activation of TRPML1 in LEL may lead to the appearance of TRPML

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

    PubMed

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

    2015-07-01

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

  2. Annual summary report on the surveillance and maintenance activities for the Oak Rige National Laboratory Environmental Restoration Program for fiscal year 1995

    SciTech Connect

    1995-11-01

    This Annual Summary Report on the Surveillance and Maintenance Activities for the Oak Ridge National Laboratory Environmental Restoration Program for Fiscal Year 1995 was prepared to communicate the accomplishments of the Program during fiscal year 1995. This work was performed under work breakdown structure element 1.4.12.6.1.14.20 (activity data sheet 3314, ``Remedial Action Surveillance and Maintenance``). Publication of this document meets the Life Cycle Baseline milestone date of November 30, 1995. This document provides the accomplishments for both the Remedial Action and Decontamination and Decommissioning Surveillance and Maintenance programs.

  3. A framework for the design of a voice-activated, intelligent, and hypermedia-based aircraft maintenance manual

    NASA Astrophysics Data System (ADS)

    Patankar, Manoj Shashikant

    Federal Aviation Regulations require Aviation Maintenance Technicians (AMTs) to refer to approved maintenance manuals when performing maintenance on airworthy aircraft. Because these manuals are paper-based, larger the size of the aircraft, more cumbersome are the manuals. Federal Aviation Administration (FAA) recognized the difficulties associated with the use of large manuals and conducted studies on the use of electronic media as an alternative to the traditional paper format. However, these techniques do not employ any artificial intelligence technologies and the user interface is limited to either a keyboard or a stylus pen. The primary emphasis of this research was to design a generic framework that would allow future development of voice-activated, intelligent, and hypermedia-based aircraft maintenance manuals. A prototype (VIHAMS-Voice-activated, Intelligent, and Hypermedia-based Aircraft Maintenance System) was developed, as a secondary emphasis, using the design and development techniques that evolved from this research. An evolutionary software design approach was used to design the proposed framework and the structured rapid prototyping technique was used to produce the VIHAMS prototype. VoiceAssist by Creative Labs was used to provide the voice interface so that the users (AMTs) could keep their hands free to work on the aircraft while maintaining complete control over the computer through discrete voice commands. KnowledgePro for Windows sp{TM}, an expert system shell, provided "intelligence" to the prototype. As a result of this intelligence, the system provided expert guidance to the user. The core information contained in conventional manuals was available in a hypermedia format. The prototype's operating hardware included a notebook computer with a fully functional audio system. An external microphone and the built-in speaker served as the input and output devices (along with the color monitor), respectively. Federal Aviation Administration

  4. Mechanism of memantine block of NMDA-activated channels in rat retinal ganglion cells: uncompetitive antagonism.

    PubMed

    Chen, H S; Lipton, S A

    1997-02-15

    1. N-methyl-D-aspartic acid (NMDA)-activated currents were recorded from dissociated rat retinal ganglion cells using whole-cell recording. The NMDA open-channel blocking drug memantine was evaluated for non-competitive and/or uncompetitive components of antagonism. A rapid superfusion system was used to apply various drugs for kinetic analysis. 2. Dose-response data revealed that memantine blocked 200 microM NMDA-evoked responses with a 50% inhibition constant (IC50) of approximately 1 microM at -60 mV and an empirical Hill coefficient of approximately 1. The antagonism followed a bimolecular reaction process. This 1:1 stoichiometry is supported by the fact that the macroscopic blocking rate of memantine (kon) increased linearly with memantine concentration and the macroscopic unblocking rate (koff) was independent of it. The estimated pseudo-first order rate constant for macroscopic blockade was 4 x 10(5) M-1 S-1 and the rate constant for unblocking was 0.44 s-1. Both the blocking and unblocking actions of memantine were well fitted by a single exponential process. 3. The kon for 2 microM memantine decreased with decreasing concentrations of NMDA. By analysing kon behaviour, we estimate that memantine has minimal interaction with the closed-unliganded state of the channel. As channel open probability (Po) approached zero, a small residual action of memantine may be explained by the presence of endogenous glutamate and glycine. 4. Memantine could be trapped within the NMDA-gated channel if it was suddenly closed by fast washout of agonist. The measured gating process of channel activation and deactivation appeared at least 10-20-fold faster than the kinetics of memantine action. By combining the agonist and voltage dependence of antagonism, a trapping scheme was established for further kinetic analysis. 5. With low agonist concentrations, NMDA-gated channels recovered slowly from memantine blockade. By analysing the probability of a channel remaining blocked, we

  5. Mechanism of memantine block of NMDA-activated channels in rat retinal ganglion cells: uncompetitive antagonism.

    PubMed Central

    Chen, H S; Lipton, S A

    1997-01-01

    1. N-methyl-D-aspartic acid (NMDA)-activated currents were recorded from dissociated rat retinal ganglion cells using whole-cell recording. The NMDA open-channel blocking drug memantine was evaluated for non-competitive and/or uncompetitive components of antagonism. A rapid superfusion system was used to apply various drugs for kinetic analysis. 2. Dose-response data revealed that memantine blocked 200 microM NMDA-evoked responses with a 50% inhibition constant (IC50) of approximately 1 microM at -60 mV and an empirical Hill coefficient of approximately 1. The antagonism followed a bimolecular reaction process. This 1:1 stoichiometry is supported by the fact that the macroscopic blocking rate of memantine (kon) increased linearly with memantine concentration and the macroscopic unblocking rate (koff) was independent of it. The estimated pseudo-first order rate constant for macroscopic blockade was 4 x 10(5) M-1 S-1 and the rate constant for unblocking was 0.44 s-1. Both the blocking and unblocking actions of memantine were well fitted by a single exponential process. 3. The kon for 2 microM memantine decreased with decreasing concentrations of NMDA. By analysing kon behaviour, we estimate that memantine has minimal interaction with the closed-unliganded state of the channel. As channel open probability (Po) approached zero, a small residual action of memantine may be explained by the presence of endogenous glutamate and glycine. 4. Memantine could be trapped within the NMDA-gated channel if it was suddenly closed by fast washout of agonist. The measured gating process of channel activation and deactivation appeared at least 10-20-fold faster than the kinetics of memantine action. By combining the agonist and voltage dependence of antagonism, a trapping scheme was established for further kinetic analysis. 5. With low agonist concentrations, NMDA-gated channels recovered slowly from memantine blockade. By analysing the probability of a channel remaining blocked, we

  6. Maintenance and induction of naphthalene degradation activity in Pseudomonas putida and an Alcaligenes sp. under different culture conditions

    SciTech Connect

    Guerin, W.F.; Boyd, S.A.

    1995-11-01

    The expression of xenobiotic-degradative genes in indigenous bacteria or in bacteria introduced into an ecosystem is essential for the successful bioremediation of contaminated environments. The maintenance of naphthalene utilization activity is studied in Pseudomonas putida (ATCC 17484) and an Alcaligenes sp. (strain NP-Alk) under different batch culture conditions. Levels of activity decreased exponentially in stationary phase with half-lives of 43 and 13 h for strains ATCC 17484 nad NP-Alk, respectively. Activity half-lives were 2.7 and 5.3 times longer, respectively, in starved cultures than in stationary-phase cultures following growth on naphthalene. The treatment of starved cultures with chloramphenicol caused a loss of activity more rapid than that measured in untreated starved cultures, suggesting a continued enzyme synthesis in starved cultures in the absence of a substrate. Following growth in nutrient medium, activity decreased to undetectable levels in the Alcaligenes sp. but remained at measureable levels int he pseudomonad even after 9 months. The induction of naphthalene degradation activities in these cultures, when followed by radiorespirometry with {sup 14}C-labeled naphthalene as the substrate, was consistent with activity maintenance data. In the pseudomonad, naphthalene degradation activity was present constitutively at low levels under all growth conditions and was rapidly (in approximately 15 min) induced to high levels upon exposure to naphthalene. Adaptation in the uninduced Alcaligenes sp. occurred after many hours of exposure to naphthalene. In vivo labeling with {sup 35}S, to monitor the extent of de novo enzyme synthesis by naphthalene-challenged cells, provided an independent confirmation of the results. 43 refs., 9 figs., 1 tab.

  7. Influenza matrix protein 2 alters CFTR expression and function through its ion channel activity.

    PubMed

    Londino, James D; Lazrak, Ahmed; Jurkuvenaite, Asta; Collawn, James F; Noah, James W; Matalon, Sadis

    2013-05-01

    The human cystic fibrosis transmembrane conductance regulator (CFTR) is a cyclic AMP-activated chloride (Cl(-)) channel in the lung epithelium that helps regulate the thickness and composition of the lung epithelial lining fluid. We investigated whether influenza M2 protein, a pH-activated proton (H(+)) channel that traffics to the plasma membrane of infected cells, altered CFTR expression and function. M2 decreased CFTR activity in 1) Xenopus oocytes injected with human CFTR, 2) epithelial cells (HEK-293) stably transfected with CFTR, and 3) human bronchial epithelial cells (16HBE14o-) expressing native CFTR. This inhibition was partially reversed by an inhibitor of the ubiquitin-activating enzyme E1. Next we investigated whether the M2 inhibition of CFTR activity was due to an increase of secretory organelle pH by M2. Incubation of Xenopus oocytes expressing CFTR with ammonium chloride or concanamycin A, two agents that alkalinize the secretory pathway, inhibited CFTR activity in a dose-dependent manner. Treatment of M2- and CFTR-expressing oocytes with the M2 ion channel inhibitor amantadine prevented the loss in CFTR expression and activity; in addition, M2 mutants, lacking the ability to transport H(+), did not alter CFTR activity in Xenopus oocytes and HEK cells. Expression of an M2 mutant retained in the endoplasmic reticulum also failed to alter CFTR activity. In summary, our data show that M2 decreases CFTR activity by increasing secretory organelle pH, which targets CFTR for destruction by the ubiquitin system. Alteration of CFTR activity has important consequences for fluid regulation and may potentially modify the immune response to viral infection.

  8. Degenerin sites mediate proton activation of deltabetagamma-epithelial sodium channel.

    PubMed

    Ji, Hong-Long; Benos, Dale J

    2004-06-25

    The delta-subunit of epithelial Na(+) channels (ENaC) is predominately expressed in brain, heart, and pancreas. The amiloride sensitivity, Na(+) conductance, and critical domains for gating are characterized as a cross between proton-activated Na(+) channels and alpha-ENaC. The hypothesis that external protons may activate human delta-ENaC was addressed by expressing deltabetagamma-hENaC in Xenopus oocytes and evaluating proton-activated current with the two-electrode voltage clamp technique. Our results showed that protons transiently evoked a Na(+) current with an EC(50) of pH 6 overlapped on the basal current of deltabetagamma-hENaC. Proton-activated current was not observed in uninjected oocytes. Studies on gating kinetics revealed that activation, desensitization, and recovery times of proton-activated Na(+) current were 3.8 +/- 0.5 s, 253 +/- 9.5 s, and 10 +/- 3.6 s, respectively (n = 4-12). Alkali metal cation selectivity of the proton-activated current was identical to that of the basal current of deltabetagamma-hENaC. The metabolic acids, lactate, pyruvate, and formate, modified the proton-activated current, as did hypo-osmotic stress. EDTA, hypo-osmolarity, and lactate enhanced proton activation synergistically. Our results suggest that delta-hENaC subunit is essential for proton-activated current and gamma-subunit may potentially regulate the response of delta-hENaC to protons. We have concluded that deltabetagamma-hENaC is a proton-activated cation channel whose closing gate can be regulated by a proton-induced conformational change. Proton-sensitivity of deltabetagamma-hENaC may be an important mechanism for integrating external ischemic signals in inflamed and hypoxic tissues. PMID:15084585

  9. Influenza matrix protein 2 alters CFTR expression and function through its ion channel activity

    PubMed Central

    Londino, James D.; Lazrak, Ahmed; Jurkuvenaite, Asta; Collawn, James F.; Noah, James W.

    2013-01-01

    The human cystic fibrosis transmembrane conductance regulator (CFTR) is a cyclic AMP-activated chloride (Cl−) channel in the lung epithelium that helps regulate the thickness and composition of the lung epithelial lining fluid. We investigated whether influenza M2 protein, a pH-activated proton (H+) channel that traffics to the plasma membrane of infected cells, altered CFTR expression and function. M2 decreased CFTR activity in 1) Xenopus oocytes injected with human CFTR, 2) epithelial cells (HEK-293) stably transfected with CFTR, and 3) human bronchial epithelial cells (16HBE14o−) expressing native CFTR. This inhibition was partially reversed by an inhibitor of the ubiquitin-activating enzyme E1. Next we investigated whether the M2 inhibition of CFTR activity was due to an increase of secretory organelle pH by M2. Incubation of Xenopus oocytes expressing CFTR with ammonium chloride or concanamycin A, two agents that alkalinize the secretory pathway, inhibited CFTR activity in a dose-dependent manner. Treatment of M2- and CFTR-expressing oocytes with the M2 ion channel inhibitor amantadine prevented the loss in CFTR expression and activity; in addition, M2 mutants, lacking the ability to transport H+, did not alter CFTR activity in Xenopus oocytes and HEK cells. Expression of an M2 mutant retained in the endoplasmic reticulum also failed to alter CFTR activity. In summary, our data show that M2 decreases CFTR activity by increasing secretory organelle pH, which targets CFTR for destruction by the ubiquitin system. Alteration of CFTR activity has important consequences for fluid regulation and may potentially modify the immune response to viral infection. PMID:23457187

  10. Chloride channels activated by swell can regulate the NADPH oxidase generated membrane depolarisation in activated human neutrophils

    SciTech Connect

    Ahluwalia, Jatinder

    2008-01-11

    Chloride channels activated by swell have important functions in many physiological processes. The phagocyte NADPH oxidase is essential for host defence and it generates superoxide by transferring electrons from the donor NADPH to the acceptor O{sub 2}. This electron current, induces a depolarisation of the plasma membrane. In this study, I report that chloride channels activated by swell can counteract the depolarisation induced by the NADPH oxidase. When a chloride conductance was activated by swelling, its inhibition by either 50 {mu}M NPPB or removing external chloride, depolarised the plasma membrane potential to +26 mV {+-} 3.1 (n = 4) and +40 {+-} 1 mV (n = 4), respectively. These channels were partially inhibited by the NADPH oxidase inhibitor AEBSF (1 mM) and potently inhibited by ZnCl{sub 2} (3 mM). These currents were not activated by a phosphorylation step and elevations in intracellular calcium did not appear to activate chloride currents similar to those activated by swell.

  11. Piezo1 and Piezo2 are essential components of distinct mechanically-activated cation channels

    PubMed Central

    Coste, Bertrand; Mathur, Jayanti; Schmidt, Manuela; Earley, Taryn J.; Ranade, Sanjeev; Petrus, Matt J.; Dubin, Adrienne E.; Patapoutian, Ardem

    2011-01-01

    Mechanical stimuli drive many physiological processes, including touch and pain sensation, hearing, and blood pressure regulation. Mechanically-activated (MA) cation channel activities have been recorded in many cells, but the responsible molecules have not been identified. We characterized a rapidly-adapting MA current in a mouse neuroblastoma cell line. Expression profiling and RNAi knockdown of candidate genes identified Piezo1 (Fam38A) to be required for MA currents in these cells. Piezo1 and related Piezo2 (Fam38B) are vertebrate multipass transmembrane proteins with homologs in invertebrates, plants, and protozoa. Overexpression of mouse Piezo1 or Piezo2 induced two kinetically-distinct MA currents. Piezos are expressed in several tissues, and knockdown of Piezo2 in dorsal root ganglia neurons specifically reduced rapidly-adapting MA currents. We propose that Piezos are components of mechanically-activated cation channels. PMID:20813920

  12. Detection and localization of a putative cyclic-GMP-activated channel protein in the protozoan ciliate Stentor coeruleus.

    PubMed

    Walerczyk, M; Fabczak, H; Fabczak, S

    2006-05-01

    Immunoblotting and immunocytochemical assays were employed to identify and localize a channel protein activated by cyclic GMP (cGMP) in the protozoan ciliate Stentor coeruleus. Analysis of whole-cell homogenate with antibodies raised against the alpha-subunit of the cGMP-activated channel protein from bovine rod outer segments and against cGMP revealed four major protein bands with molecular masses of 40 kDa, 63 kDa, and over 120 kDa, which bound cGMP. However, only a cGMP-binding protein of 63 kDa, corresponding to the alpha-subunit of the cGMP-activated ion channel protein from bovine rod outer segments, was found in the ciliate cortex fraction. The functional cGMP-activated channel protein was also shown to be present in the cortex fraction of S. coeruleus by patch-clamp measurements of artificial liposomes. Incorporation of the cortex fraction into liposomes resulted in the appearance of ion channel activity related to cGMP. The reconstituted protein channels were strongly inhibited by l-cis-diltiazem, a known potent blocker of many types of cyclic-nucleotide-activated channels. The results presented here are the first demonstration of the existence and localization of a putative cGMP-activated channel protein in the ciliate S. coeruleus. Cyclic-nucleotide-activated channel proteins are nonspecific cation channels which mediate the receptor potentials in photoreceptor cells and in cells of the olfactory epithelium. On the basis of these data, we suggest that the 63 kDa protein identified in Stentor coeruleus is also a cGMP-activated ion channel and that it may be involved as an effector in the photosensory transduction pathway leading to the motile photophobic response in this ciliate protist. PMID:16736256

  13. Activation of hERG3 channel stimulates autophagy and promotes cellular senescence in melanoma

    PubMed Central

    Perez-Neut, Mathew; Haar, Lauren; Rao, Vidhya; Santha, Sreevidya; Lansu, Katherine; Rana, Basabi; Jones, Walter K.; Gentile, Saverio

    2016-01-01

    Ion channels play a major factor in maintaining cellular homeostasis but very little is known about the role of these proteins in cancer biology. In this work we have discovered that, the Kv11.3 (hERG3) a plasma-membrane potassium channel plays a critical role in the regulation of autophagy in a cancer cell model. We have found that pharmacologic stimulation of the Kv11.3 channel with a small molecule activator, NS1643 induced autophagy via activation of an AMPK-dependent signaling pathway in melanoma cell line. In addition, we have found that NS1643 produced a strong inhibition of cell proliferation by activating a cellular senescence program. Furthermore, inhibition of autophagy via siRNA targeting AMPK or treatment with hydroxychloroquine an autophagy inhibitor activates apoptosis in NS1643-treated cells. Thus, we propose that, Kv11.3 is a novel mediator of autophagy, autophagy can be a survival mechanism contributing to cellular senescence, and that use of a combinatorial pharmacologic approach of Kv11.3 activator with inhibitors of autophagy represents a novel therapeutic approach against melanoma. PMID:26942884

  14. Pungent products from garlic activate the sensory ion channel TRPA1.

    PubMed

    Bautista, Diana M; Movahed, Pouya; Hinman, Andrew; Axelsson, Helena E; Sterner, Olov; Högestätt, Edward D; Julius, David; Jordt, Sven-Eric; Zygmunt, Peter M

    2005-08-23

    Garlic belongs to the Allium family of plants that produce organosulfur compounds, such as allicin and diallyl disulfide (DADS), which account for their pungency and spicy aroma. Many health benefits have been ascribed to Allium extracts, including hypotensive and vasorelaxant activities. However, the molecular mechanisms underlying these effects remain unknown. Intriguingly, allicin and DADS share structural similarities with allyl isothiocyanate, the pungent ingredient in wasabi and other mustard plants that induces pain and inflammation by activating TRPA1, an excitatory ion channel on primary sensory neurons of the pain pathway. Here we show that allicin and DADS excite an allyl isothiocyanate-sensitive subpopulation of sensory neurons and induce vasodilation by activating capsaicin-sensitive perivascular sensory nerve endings. Moreover, allicin and DADS activate the cloned TRPA1 channel when expressed in heterologous systems. These and other results suggest that garlic excites sensory neurons primarily through activation of TRPA1. Thus different plant genera, including Allium and Brassica, have developed evolutionary convergent strategies that target TRPA1 channels on sensory nerve endings to achieve chemical deterrence. PMID:16103371

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

    NASA Astrophysics Data System (ADS)

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

    2013-12-01

    Models of how mountain belts grow and erode through time largely rely on the paradigm of fluvial bedrock incision as the main motor of response to differences in rock uplift, thus setting base levels of erosion in tectonically active landscapes. Dynamic feedbacks between rock uplift, bedrock river geometry, and mass wasting have been encapsulated within the concept of threshold hillslopes that attain a mechanically critical inclination capable of adjusting to fluvial incision rates via decreased stability and commensurately more frequent landsliding. Here we provide data that challenge the widely held view that channel steepness records tectonic forcing more faithfully than hillslope inclination despite much robust empirical evidence of such links between bedrock-river geometry and hillslope mass wasting. We show that the volume mobilized by mass wasting depends more on local topographic relief and the sinuosity of bedrock rivers than their mean normalized channel steepness. We derive this counterintuitive observation from an unprecedented inventory of ~300,000 landslides covering the tectonically active Japanese archipelago with substantial differences in seismicity, lithology, vertical surface deformation, topography, and precipitation variability. Both total landslide number and volumes increase nonlinearly with mean local relief even in areas where the fraction of steepest channel segments attains a constant threshold well below the maximum topographic relief. Our data document for the first time that mass wasting increases systematically with preferential steepening of flatter channel segments. Yet concomitant changes in mean channel steepness are negligible such that it remains a largely insensitive predictor of landslide denudation. Further, minute increases in bedrock-river sinuosity lead to substantial reduction in landslide abundance and volumes. Our results underline that sinuosity (together with mean local relief) is a key morphometric variable for

  16. Mechanism for phosphoinositide selectivity and activation of TRPV1 ion channels

    PubMed Central

    Ufret-Vincenty, Carmen A.; Klein, Rebecca M.; Collins, Marcus D.; Rosasco, Mario G.; Martinez, Gilbert Q.

    2015-01-01

    Although PI(4,5)P2 is believed to play an essential role in regulating the activity of numerous ion channels and transporters, the mechanisms by which it does so are unknown. Here, we used the ability of the TRPV1 ion channel to discriminate between PI(4,5)P2 and PI(4)P to localize the region of TRPV1 sequence that interacts directly with the phosphoinositide. We identified a point mutation in the proximal C-terminal region after the TRP box, R721A, that inverted the selectivity of TRPV1. Although the R721A mutation produced only a 30% increase in the EC50 for activation by PI(4,5)P2, it decreased the EC50 for activation by PI(4)P by more than two orders of magnitude. We used chemically induced and voltage-activated phosphatases to determine that PI(4)P continued to support TRPV1 activity even after depletion of PI(4,5)P2 from the plasma membrane. Our data cannot be explained by a purely electrostatic mechanism for interaction between the phosphoinositide and the protein, similar to that of the MARCKS (myristoylated alanine-rich C kinase substrate) effector domain or the EGF receptor. Rather, conversion of a PI(4,5)P2-selective channel to a PI(4)P-selective channel indicates that a structured phosphoinositide-binding site mediates the regulation of TRPV1 activity and that the amino acid at position 721 likely interacts directly with the moiety at the 5′ position of the phosphoinositide. PMID:25918361

  17. The MuSK activator agrin has a separate role essential for postnatal maintenance of neuromuscular synapses.

    PubMed

    Tezuka, Tohru; Inoue, Akane; Hoshi, Taisuke; Weatherbee, Scott D; Burgess, Robert W; Ueta, Ryo; Yamanashi, Yuji

    2014-11-18

    The motoneural control of skeletal muscle contraction requires the neuromuscular junction (NMJ), a midmuscle synapse between the motor nerve and myotube. The formation and maintenance of NMJs are orchestrated by the muscle-specific receptor tyrosine kinase (MuSK). Motor neuron-derived agrin activates MuSK via binding to MuSK's coreceptor Lrp4, and genetic defects in agrin underlie a congenital myasthenic syndrome (an NMJ disorder). However, MuSK-dependent postsynaptic differentiation of NMJs occurs in the absence of a motor neuron, indicating a need for nerve/agrin-independent MuSK activation. We previously identified the muscle protein Dok-7 as an essential activator of MuSK. Although NMJ formation requires agrin under physiological conditions, it is dispensable for NMJ formation experimentally in the absence of the neurotransmitter acetylcholine, which inhibits postsynaptic specialization. Thus, it was hypothesized that MuSK needs agrin together with Lrp4 and Dok-7 to achieve sufficient activation to surmount inhibition by acetylcholine. Here, we show that forced expression of Dok-7 in muscle enhanced MuSK activation in mice lacking agrin or Lrp4 and restored midmuscle NMJ formation in agrin-deficient mice, but not in Lrp4-deficient mice, probably due to the loss of Lrp4-dependent presynaptic differentiation. However, these NMJs in agrin-deficient mice rapidly disappeared after birth, and postsynaptic specializations emerged ectopically throughout myotubes whereas exogenous Dok-7-mediated MuSK activation was maintained. These findings demonstrate that the MuSK activator agrin plays another role essential for the postnatal maintenance, but not for embryonic formation, of NMJs and also for the postnatal, but not prenatal, midmuscle localization of postsynaptic specializations, providing physiological and pathophysiological insight into NMJ homeostasis. PMID:25368159

  18. The MuSK activator agrin has a separate role essential for postnatal maintenance of neuromuscular synapses

    PubMed Central

    Tezuka, Tohru; Inoue, Akane; Hoshi, Taisuke; Weatherbee, Scott D.; Burgess, Robert W.; Ueta, Ryo; Yamanashi, Yuji

    2014-01-01

    The motoneural control of skeletal muscle contraction requires the neuromuscular junction (NMJ), a midmuscle synapse between the motor nerve and myotube. The formation and maintenance of NMJs are orchestrated by the muscle-specific receptor tyrosine kinase (MuSK). Motor neuron-derived agrin activates MuSK via binding to MuSK’s coreceptor Lrp4, and genetic defects in agrin underlie a congenital myasthenic syndrome (an NMJ disorder). However, MuSK-dependent postsynaptic differentiation of NMJs occurs in the absence of a motor neuron, indicating a need for nerve/agrin-independent MuSK activation. We previously identified the muscle protein Dok-7 as an essential activator of MuSK. Although NMJ formation requires agrin under physiological conditions, it is dispensable for NMJ formation experimentally in the absence of the neurotransmitter acetylcholine, which inhibits postsynaptic specialization. Thus, it was hypothesized that MuSK needs agrin together with Lrp4 and Dok-7 to achieve sufficient activation to surmount inhibition by acetylcholine. Here, we show that forced expression of Dok-7 in muscle enhanced MuSK activation in mice lacking agrin or Lrp4 and restored midmuscle NMJ formation in agrin-deficient mice, but not in Lrp4-deficient mice, probably due to the loss of Lrp4-dependent presynaptic differentiation. However, these NMJs in agrin-deficient mice rapidly disappeared after birth, and postsynaptic specializations emerged ectopically throughout myotubes whereas exogenous Dok-7–mediated MuSK activation was maintained. These findings demonstrate that the MuSK activator agrin plays another role essential for the postnatal maintenance, but not for embryonic formation, of NMJs and also for the postnatal, but not prenatal, midmuscle localization of postsynaptic specializations, providing physiological and pathophysiological insight into NMJ homeostasis. PMID:25368159

  19. The distribution of intermediate-conductance, calcium-activated, potassium (IK) channels in epithelial cells.

    PubMed

    Thompson-Vest, Nichola; Shimizu, Yasutake; Hunne, Billie; Furness, John B

    2006-02-01

    Intermediate-conductance, calcium-activated, potassium (IK) channels were first identified by their roles in cell volume regulation, and were later shown to be involved in control of proliferation of lymphocytes and to provide a K+ current for epithelial secretory activity. Until now, there has been no systematic investigation of IK channel localization within different epithelia. IK channel immunoreactivity was present in most epithelia, where it occurred in surface membranes of epithelial cells. It was found in all stratified epithelia, including skin, cornea, oral mucosa, vaginal mucosa, urothelium and the oesophageal lining. It occurred in the ducts of fluid-secreting glands, the salivary glands, lacrimal glands and pancreas, and in the respiratory epithelium. A low level of expression was seen in serous acinar cells. It was also found in other epithelia with fluid-exchange properties, the choroid plexus epithelium, the ependyma, visceral pleura and peritoneum, bile ducts and intestinal lining epithelium. However, there was little or no expression in vascular endothelial cells, kidney tubules or collecting ducts, lung alveoli, or in sebaceous glands. It is concluded that the channel is present in surface epithelia (e.g. skin) where it has a cell-protective role against osmotic challenge, and in epithelia where there is anion secretion that is facilitated by a K+ current-dependent hyperpolarization. It was also in some epithelial cells where its roles are as yet unknown. PMID:16441566

  20. Calcium-activated chloride channels anoctamin 1 and 2 promote murine uterine smooth muscle contractility

    PubMed Central

    Bernstein, Kyra; Vink, Joy Y; Fu, Xiao Wen; Wakita, Hiromi; Danielsson, Jennifer; Wapner, Ronald; Gallos, George

    2014-01-01

    Objective To determine the presence of calcium activated chloride channels anoctamin 1 and 2 in human and murine uterine smooth muscle and evaluate the physiologic role for these ion channels in murine myometrial contractility. Study Design We performed reverse transcription polymerase chain reaction to determine if anoctamin 1 and 2 are expressed in human and murine uterine tissue to validate the study of this protein in mouse models. Immunohistochemical staining of anoctamin 1 and 2 was then performed to determine protein expression in murine myometrial tissue. The function of anoctamin 1 and 2 in murine uterine tissue was evaluated using electrophysiological studies, organ bath, and calcium flux experiments. Results Anoctamin 1 and 2 are expressed in human and murine USM cells. Functional studies show that selective antagonism of these channels promotes relaxation of spontaneous murine uterine smooth muscle contractions. Blockade of anoctamin 1 and 2 inhibits both agonist-induced and spontaneous transient inward currents and abolishes G-protein coupled receptor (oxytocin) mediated elevations in intracellular calcium. Conclusion The calcium activated chloride channels ANO 1 and 2 are present in human and murine myometrial tissue and may provide novel potential therapeutic targets to achieve effective tocolysis. PMID:24928056

  1. Multi-channel NIRS of the primary motor cortex to discriminate hand from foot activity

    NASA Astrophysics Data System (ADS)

    Koenraadt, K. L. M.; Duysens, J.; Smeenk, M.; Keijsers, N. L. W.

    2012-08-01

    The poor spatial resolution of near-infrared spectroscopy (NIRS) makes it difficult to distinguish two closely located cortical areas from each other. Here, a combination of multi-channel NIRS and a centre of gravity (CoG) approach (widely accepted in the field of transcranial magnetic stimulation; TMS) was used to discriminate between closely located cortical areas activated during hand and foot movements. Similarly, the possibility of separating the more anteriorly represented discrete movements from rhythmic movements was studied. Thirteen healthy right-handed subjects performed rhythmic or discrete (‘task’) hand or foot (‘extremity’) tapping. Hemodynamic responses were measured using an 8-channel NIRS setup. For oxyhemoglobin (OHb) and deoxyhemoglobin (HHb), a CoG was determined for each condition using the mean hemodynamic responses and the coordinates of the channels. Significant hemodynamic responses were found for hand and foot movements. Based on the HHb responses, the NIRS-CoG of hand movements was located 0.6 cm more laterally compared to the NIRS-CoG of foot movements. For OHb responses no difference in NIRS-CoG was found for ‘extremity’ nor for ‘task’. This is the first NIRS study showing hemodynamic responses for isolated foot movements. Furthermore, HHb responses have the potential to be used in multi-channel NIRS experiments requiring differential activation of motor cortex areas linked to either hand or foot movements.

  2. Integrin-mediated neurite outgrowth in neuroblastoma cells depends on the activation of potassium channels

    PubMed Central

    1993-01-01

    Ba2+ and Cs+. By moving patched cells in contact with FN-coated beads, it was shown that KIR channel activation was responsible for the FN-mediated hyperpolarization of Vrest. Treatment with Pertuxis toxin (PTX) abolished this hyperpolarization and neurite outgrowth, indicating that a G protein is interposed between integrins and KIR channels and that the activation of these channels is required for neuritogenesis. In fact, the block of KIR channels by Cs+ abolished both hyperpolarization and neurite outgrowth, provided that the cation was supplied during the first two hours after N1 cell contact with FN.(ABSTRACT TRUNCATED AT 400 WORDS) PMID:8354696

  3. K(+) channel activity and redox status are differentially required for JNK activation by UV and reactive oxygen species.

    PubMed

    Gao, Jie; Wu, Dan; Guo, Taylor B; Ruan, Qin; Li, Tie; Lu, Zhenyu; Xu, Ming; Dai, Wei; Lu, Luo

    2004-07-15

    Upon exposure to ultraviolet (UV) radiation, osmotic changes or the presence of reactive oxygen species (ROS) c-Jun N-terminal kinases (JNKs) are rapidly activated. Extensive studies have elucidated molecular components that mediate the activation of JNKs. However, it remains unclear whether activation of JNKs by various stress signals involves different pathways. Here we show that K(+) channel activity is involved in mediating apoptosis induced by UV but not by H(2)O(2) in myelocytic leukemic ML-1 cells. Specifically, JNKs were rapidly phosphorylated upon treatment of ML-1 cells with UV and H(2)O(2). UV-induced, but not H(2)O(2)-induced, JNK-1 phosphorylation was inhibited by pretreatment with 4-aminopyridine (4-AP), a K(+) channel blocker. 4-AP also blocked UV-induced increase in JNK activity as well as p38 phosphorylation. Immunofluorescent microscopy revealed that phosphorylated JNKs were concentrated at centrosomes in ML-1 cells and that these proteins underwent rapid subcellular translocation upon UV treatment. Consistently, the subcellular translocation of JNKs induced by UV was largely blocked by 4-AP. Furthermore, UV-induced JNK activation was blocked by NEM, a sulfhydryl alkylating agent also affecting K(+) current. Both UV- and H(2)O(2)-induced JNK activities were inhibited by glutathione, suggesting that the redox status does play an important role in the activation of JNKs. Taken together, our findings suggest that JNK activation by UV and H(2)O(2) is mediated by distinct yet overlapping pathways and that K(+) channel activity and redox status are differentially required for UV- and H(2)O(2)-induced activation of JNKs.

  4. Partial Agonist and Antagonist Activities of a Mutant Scorpion β-Toxin on Sodium Channels*

    PubMed Central

    Karbat, Izhar; Ilan, Nitza; Zhang, Joel Z.; Cohen, Lior; Kahn, Roy; Benveniste, Morris; Scheuer, Todd; Catterall, William A.; Gordon, Dalia; Gurevitz, Michael

    2010-01-01

    Scorpion β-toxin 4 from Centruroides suffusus suffusus (Css4) enhances the activation of voltage-gated sodium channels through a voltage sensor trapping mechanism by binding the activated state of the voltage sensor in domain II and stabilizing it in its activated conformation. Here we describe the antagonist and partial agonist properties of a mutant derivative of this toxin. Substitution of seven different amino acid residues for Glu15 in Css4 yielded toxin derivatives with both increased and decreased affinities for binding to neurotoxin receptor site 4 on sodium channels. Css4E15R is unique among this set of mutants in that it retained nearly normal binding affinity but lost its functional activity for modification of sodium channel gating in our standard electrophysiological assay for voltage sensor trapping. More detailed analysis of the functional effects of Css4E15R revealed weak voltage sensor trapping activity, which was very rapidly reversed upon repolarization and therefore was not observed in our standard assay of toxin effects. This partial agonist activity of Css4E15R is observed clearly in voltage sensor trapping assays with brief (5 ms) repolarization between the conditioning prepulse and the test pulse. The effects of Css4E15R are fit well by a three-step model of toxin action involving concentration-dependent toxin binding to its receptor site followed by depolarization-dependent activation of the voltage sensor and subsequent voltage sensor trapping. Because it is a partial agonist with much reduced efficacy for voltage sensor trapping, Css4E15R can antagonize the effects of wild-type Css4 on sodium channel activation and can prevent paralysis by Css4 when injected into mice. Our results define the first partial agonist and antagonist activities for scorpion toxins and open new avenues of research toward better understanding of the structure-function relationships for toxin action on sodium channel voltage sensors and toward potential toxin

  5. Differential Activation of TRP Channels in the Adult Rat Spinal Substantia Gelatinosa by Stereoisomers of Plant-Derived Chemicals

    PubMed Central

    Kumamoto, Eiichi; Fujita, Tsugumi

    2016-01-01

    Activation of TRPV1, TRPA1 or TRPM8 channel expressed in the central terminal of dorsal root ganglion (DRG) neuron increases the spontaneous release of l-glutamate onto spinal dorsal horn lamina II (substantia gelatinosa; SG) neurons which play a pivotal role in regulating nociceptive transmission. The TRP channels are activated by various plant-derived chemicals. Although stereoisomers activate or modulate ion channels in a distinct manner, this phenomenon is not fully addressed for TRP channels. By applying the whole-cell patch-clamp technique to SG neurons of adult rat spinal cord slices, we found out that all of plant-derived chemicals, carvacrol, thymol, carvone and cineole, increase the frequency of spontaneous excitatory postsynaptic current, a measure of the spontaneous release of l-glutamate from nerve terminals, by activating TRP channels. The presynaptic activities were different between stereoisomers (carvacrol and thymol; (−)-carvone and (+)-carvone; 1,8-cineole and 1,4-cineole) in the extent or the types of TRP channels activated, indicating that TRP channels in the SG are activated by stereoisomers in a distinct manner. This result could serve to know the properties of the central terminal TRP channels that are targets of drugs for alleviating pain. PMID:27483289

  6. Electric Pole Maintenance in Nagoya City Science Museum as a Cooperative Activity of Industry, School and Local Government

    NASA Astrophysics Data System (ADS)

    Mabuchi, Koichi

    The object of this paper is to suggest an education model as a cooperative activity of industry, school and local government in science museums. Nagoya City Science Museum has opened the new exhibit on electric pole maintenance since 2002, which makes a visitor a temporary electrician working at a height of 3 meters. The exhibit, named “Be an Electrician”, is focusing on stimulating young people's interest in industrial technology. The electric pole and equipments on the pole like transformer, electric wires and so forth were donated from an electric power company to the museum. The museum manages volunteers including an active electrician and students to study engineering, who instruct the visitors in the bucket how to change the insulator on the pole. The active electrician also instructs some technical high school students in practical works at height. This new exhibit indicates the science museum positioned between companies and schools will make it possible to extend internship.

  7. Distribution of High-Conductance Calcium-Activated Potassium Channels in Rat Vestibular Epithelia

    PubMed Central

    Schweizer, Felix E.; Savin, David; Luu, Cindy; Sultemeier, David R.; Hoffman, Larry F.

    2011-01-01

    Voltage- and calcium-activated potassium channels (BK) are important regulators of neuronal excitability. BK channels seem to be crucial for frequency tuning in nonmammalian vestibular and auditory hair cells. However, there are a paucity of data concerning BK expression in mammalian vestibular hair cells. We therefore investigated the localization of BK channels in mammalian vestibular hair cells, specifically in rat vestibular neuroepithelia. We find that only a subset of hair cells in the utricle and the crista ampullaris express BK channels. BK-positive hair cells are located mainly in the medial striolar region of the utricle, where they constitute at most 12% of hair cells, and in the central zone of the horizontal crista. A majority of BK-positive hair cells are encapsulated by a calretinin-positive calyx defining them as type I cells. The remainder are either type I cells encapsulated by a calretinin-negative calyx or type II hair cells. Surprisingly, the number of BK-positive hair cells in the utricle peaks in juvenile rats and declines in early adulthood. BK channels were not found in vestibular afferent dendrites or somata. Our data indicate that BK channel expression in the mammalian vestibular system differs from the expression pattern in the mammalian auditory and the nonmammalian vestibular system. The molecular diversity of vestibular hair cells indicates a functional diversity that has not yet been fully characterized. The predominance of BK-positive hair cells within the medial striola of juvenile animals suggests that they contribute to a scheme of highly lateralized coding of linear head movements during late development. PMID:19731297

  8. Eugenol dilates mesenteric arteries and reduces systemic BP by activating endothelial cell TRPV4 channels

    PubMed Central

    Peixoto-Neves, Dieniffer; Wang, Qian; Leal-Cardoso, Jose H; Rossoni, Luciana V; Jaggar, Jonathan H

    2015-01-01

    Background and Purpose Eugenol, a vanilloid molecule found in some dietary plants, relaxes vasculature in part via an endothelium-dependent process; however, the mechanisms involved are unclear. Here, we investigated the endothelial cell-mediated mechanism by which eugenol modulates rat mesenteric artery contractility and systemic BP. Experimental Approach The isometric tension of rat mesenteric arteries (size 200–300 μm) was measured using wire myography; non-selective cation currents (ICat) were recorded in endothelial cells using patch clamp electrophysiology. Mean arterial pressure (MAP) and heart rate (HR) were determined in anaesthetized rats. Key Results Eugenol relaxed endothelium-intact arteries in a concentration-dependent manner and this effect was attenuated by endothelium denudation. L-NAME, a NOS inhibitor, a combination of TRAM-34 and apamin, selective blockers of intermediate and small conductance Ca2+-activated K+ channels, respectively, and HC-067047, a TRPV4 channel inhibitor, but not indomethacin, a COX inhibitor, reduced eugenol-induced relaxation in endothelium-intact arteries. Eugenol activated HC-067047-sensitive ICat in mesenteric artery endothelial cells. Short interfering RNA (siRNA)-mediated TRPV4 knockdown abolished eugenol-induced ICat activation. An i.v. injection of eugenol caused an immediate, transient reduction in both MAP and HR, which was followed by prolonged, sustained hypotension in anaesthetized rats. This sustained hypotension was blocked by HC-067047. Conclusions and Implications Eugenol activates TRPV4 channels in mesenteric artery endothelial cells, leading to vasorelaxation, and reduces systemic BP in vivo. Eugenol may be therapeutically useful as an antihypertensive agent and is a viable molecular candidate from which to develop second-generation TRPV4 channel activators that reduce BP. PMID:25832173

  9. Reactive oxygen species inhibit polycystin-2 (TRPP2) cation channel activity in term human syncytiotrophoblast.

    PubMed

    Montalbetti, N; Cantero, M R; Dalghi, M G; Cantiello, H F

    2008-06-01

    Pregnancy is often associated with oxidative stress (OS) and lower antioxidant defences, which are both implicated in the pathophysiology of preeclampsia, free radical-induced birth defects, and abortions, as well as gestational diabetes mellitus (GDM), where products of lipid peroxidation are increased. The molecular target(s) of increased oxygen free radicals and consequent lipid peroxidation in the human placenta remains ill defined. The human syncytiotrophoblast (hST) expresses abundant polycystin-2 (PC2, TRPP2), a TRP-type Ca(2+)-permeable non-selective cation channel. Here, we explored the effect of reactive oxygen species (ROS) on PC2 channel activity of term hST. Apical membranes of the hST were reconstituted in a lipid bilayer chamber. Addition of either hydrogen-peroxide (H(2)O(2)) or tert-butyl hydroperoxide (tBHP) to the cis chamber (intracellular side) rapidly and completely inhibited PC2-mediated cation channel activity in reconstituted hST vesicles. A dose-response titration with increasing concentrations of H(2)O(2) gave an IC(50)=131 nM. The effect of H(2)O(2) on the isolated protein from in vitro transcribed/translated material was significantly different. H(2)O(2) inhibited PC2 cation channel activity, with a much lower affinity (IC(50)=193 microM). To correlate these findings with H(2)O(2)-induced lipid peroxidation, TBARS where measured in hST apical membranes incubated with H(2)O(2). Increased TBARS by exposure of hST apical membranes to H(2)O(2) (625 microM) returned to control value in the presence of catalase (167 microg/ml). Taken together these data indicate that ROS affect PC2 channel function by targetting both membrane lipids and the channel protein. Thus, OS in human pregnancy may be linked to dysregulation of channels such as PC2, which allow the transport of Ca(2+) into the placenta. Oxidative complications in pregnancy may implicate dysfunctional cation transfer between mother and fetus. PMID:18417208

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

    PubMed Central

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

    2013-01-01

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

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

    PubMed

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

    2015-07-01

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

  12. Increased Asynchronous Release and Aberrant Calcium Channel Activation in Amyloid Precursor Protein Deficient Neuromuscular Synapses

    PubMed Central

    Yang, Li; Wang, Baiping; Long, Cheng; Wu, Gangyi; Zheng, Hui

    2007-01-01

    Despite the critical roles of the amyloid precursor protein (APP) in Alzheimer's disease pathogenesis, its physiological function remains poorly established. Our previous studies implicated a structural and functional activity of the APP family of proteins in the developing neuromuscular junction (NMJ). Here we performed comprehensive analyses of neurotransmission in mature neuromuscular synapse of APP deficient mice. We found that APP deletion led to reduced paired-pulse facilitation and increased depression of synaptic transmission with repetitive stimulation. Readily releasable pool size and total releasable vesicles were not affected, but probability of release was significantly increased. Strikingly, the amount of asynchronous release, a measure sensitive to presynaptic calcium concentration, was dramatically increased, and pharmacological studies revealed that it was attributed to aberrant activation of N- and L-type Ca2+ channels. We propose that APP modulates synaptic transmission at the NMJ by ensuring proper Ca2+ channel function. PMID:17919826

  13. The calcium-activated chloride channel anoctamin 1 acts as a heat sensor in nociceptive neurons.

    PubMed

    Cho, Hawon; Yang, Young Duk; Lee, Jesun; Lee, Byeongjoon; Kim, Tahnbee; Jang, Yongwoo; Back, Seung Keun; Na, Heung Sik; Harfe, Brian D; Wang, Fan; Raouf, Ramin; Wood, John N; Oh, Uhtaek

    2012-05-27

    Nociceptors are a subset of small primary afferent neurons that respond to noxious chemical, thermal and mechanical stimuli. Ion channels in nociceptors respond differently to noxious stimuli and generate electrical signals in different ways. Anoctamin 1 (ANO1 also known as TMEM16A) is a Ca(2+)-activated chloride channel that is essential for numerous physiological functions. We found that ANO1 was activated by temperatures over 44 °C with steep heat sensitivity. ANO1 was expressed in small sensory neurons and was highly colocalized with nociceptor markers, which suggests that it may be involved in nociception. Application of heat ramps to dorsal root ganglion (DRG) neurons elicited robust ANO1-dependent depolarization. Furthermore, knockdown or deletion of ANO1 in DRG neurons substantially reduced nociceptive behavior in thermal pain models. These results indicate that ANO1 is a heat sensor that detects nociceptive thermal stimuli in sensory neurons and possibly mediates nociception.

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

    PubMed

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

    2016-02-01

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

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

    NASA Technical Reports Server (NTRS)

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

    1997-01-01

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

  16. The UHRF1 Protein Stimulates the Activity and Specificity of the Maintenance DNA Methyltransferase DNMT1 by an Allosteric Mechanism*

    PubMed Central

    Bashtrykov, Pavel; Jankevicius, Gytis; Jurkowska, Renata Z.; Ragozin, Sergey; Jeltsch, Albert

    2014-01-01

    The ubiquitin-like, containing PHD and RING finger domains protein 1 (UHRF1) is essential for maintenance DNA methylation by DNA methyltransferase 1 (DNMT1). UHRF1 has been shown to recruit DNMT1 to replicated DNA by the ability of its SET and RING-associated (SRA) domain to bind to hemimethylated DNA. Here, we demonstrate that UHRF1 also increases the activity of DNMT1 by almost 5-fold. This stimulation is mediated by a direct interaction of both proteins through the SRA domain of UHRF1 and the replication focus targeting sequence domain of DNMT1, and it does not require DNA binding by the SRA domain. Disruption of the interaction between DNMT1 and UHRF1 by replacement of key residues in the replication focus targeting sequence domain led to a strong reduction of DNMT1 stimulation. Additionally, the interaction with UHRF1 increased the specificity of DNMT1 for methylation of hemimethylated CpG sites. These findings show that apart from the targeting of DNMT1 to the replicated DNA UHRF1 increases the activity and specificity of DNMT1, thus exerting a multifaceted influence on the maintenance of DNA methylation. PMID:24368767

  17. FY 1994 annual summary report of the surveillance and maintenance activities for the Oak Ridge National Laboratory Environmental Restoration Program

    SciTech Connect

    Not Available

    1994-11-01

    The Oak Ridge National Laboratory (ORNL) Environmental Restoration (ER) Surveillance and Maintenance (S and M) Program was initiated to manage former waste management and environmental research sites contaminated with radioactive materials and/or hazardous chemicals. The S and M Program is responsible for managing designated sites/facilities from the end of their operating lives until final disposition or site stabilization. To effectively manage and perform the various S and M Program responsibilities, five summary-level work breakdown structure (WBS) elements have been established: S and M Preliminary Investigations, Special Projects, Routine S and M, Inactive Groundwater Wells, and Project Management. Routine S and M activities were conducted as scheduled throughout fiscal years (FY) 1994 at applicable inactive waste management (WM) and other contaminated areas. Overall, the ER S and M Program maintains 47 facilities, performs vegetation maintenance on approximately 230 acres, maintains 54 inactive tanks, and provides overall site management on over 700 acres. In addition to the routine S and M activities, detailed site inspections were conducted at established frequencies on appropriate sites in the ER S and M Program. This document provides a summary of the FY 1994 ORNL ER S and M Program accomplishments.

  18. Calcium-activated chloride channels in bovine pulmonary artery endothelial cells.

    PubMed Central

    Nilius, B; Prenen, J; Szücs, G; Wei, L; Tanzi, F; Voets, T; Droogmans, G

    1997-01-01

    1. We characterized Ca(2+)-activated Cl- currents in calf pulmonary artery endothelial (CPAE) cells by using a combined patch clamp and fura-2 microfluorescence technique to simultaneously measure ionic currents and the intracellular Ca2+ concentration, [Ca2+]i. 2. Various procedures that increased [Ca2+]i, such as stimulation with ATP or ionomycin, or loading the cells with Ca2+ via the patch pipette, activated a strongly outwardly rectifying current with a reversal potential close to the Cl- equilibrium potential. Changing the extracellular Cl- concentration shifted this reversal potential as predicted for a Cl- current. Buffering Ca2+ rises with BAPTA prevented ATP from activating the current. 3. Ca(2+)-activated Cl- currents could be distinguished from volume-activated Cl- currents, which were sometimes coactivated in the same cell. The latter showed much less outward rectification, their activation was voltage independent, and they could be inhibited by exposing the cells to hypertonic solutions. 4. The permeability ratio for the Ca(2+)-activated conductance of the anions iodide:chloride: gluconate was 1.71 +/- 0.06:1:0.39 +/- 0.03 (n = 12). 5. This Ca(2+)-activated Cl- current, ICl, Ca, inactivated rapidly at negative potentials and activated slowly at positive potentials. Outward tail currents were slowly decaying, while inward tail currents decayed much faster. 6. 4,4'-Diisothiocyanatostilbene-2,2'-disulphonic-acid (DIDS) and niflumic acid inhibited Icl,Ca in a voltage-dependent manner, i.e. they exerted a more potent block at positive potentials. The block by N-phenylanthracilic acid (NPA), 5-nitro-2-(3-phenylpropylamino)-benzoate (NPPB) and tamoxifen was voltage independent. Niflumic acid and tamoxifen were the most potent blockers. 7. The single-channel conductance was 7.9 +/- 0.7 pS (n = 15) at 300 mM extracellular Cl-. The channel open probability was high at positive potentials, but very small at negative potentials. 8. It is concluded that [Ca2+]i

  19. Activity of Palythoa caribaeorum Venom on Voltage-Gated Ion Channels in Mammalian Superior Cervical Ganglion Neurons

    PubMed Central

    Lazcano-Pérez, Fernando; Castro, Héctor; Arenas, Isabel; García, David E.; González-Muñoz, Ricardo; Arreguín-Espinosa, Roberto

    2016-01-01

    The Zoanthids are an order of cnidarians whose venoms and toxins have been poorly studied. Palythoa caribaeorum is a zoanthid commonly found around the Mexican coastline. In this study, we tested the activity of P. caribaeorum venom on voltage-gated sodium channel (NaV1.7), voltage-gated calcium channel (CaV2.2), the A-type transient outward (IA) and delayed rectifier (IDR) currents of KV channels of the superior cervical ganglion (SCG) neurons of the rat. These results showed that the venom reversibly delays the inactivation process of voltage-gated sodium channels and inhibits voltage-gated calcium and potassium channels in this mammalian model. The compounds responsible for these effects seem to be low molecular weight peptides. Together, these results provide evidence for the potential use of zoanthids as a novel source of cnidarian toxins active on voltage-gated ion channels. PMID:27164140

  20. Activity of Palythoa caribaeorum Venom on Voltage-Gated Ion Channels in Mammalian Superior Cervical Ganglion Neurons.

    PubMed

    Lazcano-Pérez, Fernando; Castro, Héctor; Arenas, Isabel; García, David E; González-Muñoz, Ricardo; Arreguín-Espinosa, Roberto

    2016-01-01

    The Zoanthids are an order of cnidarians whose venoms and toxins have been poorly studied. Palythoa caribaeorum is a zoanthid commonly found around the Mexican coastline. In this study, we tested the activity of P. caribaeorum venom on voltage-gated sodium channel (NaV1.7), voltage-gated calcium channel (CaV2.2), the A-type transient outward (IA) and delayed rectifier (IDR) currents of KV channels of the superior cervical ganglion (SCG) neurons of the rat. These results showed that the venom reversibly delays the inactivation process of voltage-gated sodium channels and inhibits voltage-gated calcium and potassium channels in this mammalian model. The compounds responsible for these effects seem to be low molecular weight peptides. Together, these results provide evidence for the potential use of zoanthids as a novel source of cnidarian toxins active on voltage-gated ion channels. PMID:27164140

  1. KCa2 channels activation prevents [Ca2+]i deregulation and reduces neuronal death following glutamate toxicity and cerebral ischemia

    PubMed Central

    Dolga, A M; Terpolilli, N; Kepura, F; Nijholt, I M; Knaus, H-G; D'Orsi, B; Prehn, J H M; Eisel, U L M; Plant, T; Plesnila, N; Culmsee, C

    2011-01-01

    Exacerbated activation of glutamate receptor-coupled calcium channels and subsequent increase in intracellular calcium ([Ca2+]i) are established hallmarks of neuronal cell death in acute and chronic neurological diseases. Here we show that pathological [Ca2+]i deregulation occurring after glutamate receptor stimulation is effectively modulated by small conductance calcium-activated potassium (KCa2) channels. We found that neuronal excitotoxicity was associated with a rapid downregulation of KCa2.2 channels within 3 h after the onset of glutamate exposure. Activation of KCa2 channels preserved KCa2 expression and significantly reduced pathological increases in [Ca2+]i providing robust neuroprotection in vitro and in vivo. These data suggest a critical role for KCa2 channels in excitotoxic neuronal cell death and propose their activation as potential therapeutic strategy for the treatment of acute and chronic neurodegenerative disorders. PMID:21509037

  2. Early Life Manipulations of the Nonapeptide System Alter Pair Maintenance Behaviors and Neural Activity in Adult Male Zebra Finches

    PubMed Central

    Baran, Nicole M.; Tomaszycki, Michelle L.; Adkins-Regan, Elizabeth

    2016-01-01

    Adult zebra finches (T. guttata) form socially monogamous pair bonds characterized by proximity, vocal communication, and contact behaviors. In this experiment, we tested whether manipulations of the nonapeptide hormone arginine vasotocin (AVT, avian homolog of vasopressin) and the V1a receptor (V1aR) early in life altered species-typical pairing behavior in adult zebra finches of both sexes. Although there was no effect of treatment on the tendency to pair in either sex, males in different treatments exhibited profoundly different profiles of pair maintenance behavior. Following a brief separation, AVT-treated males were highly affiliative with their female partner but sang very little compared to Controls. In contrast, males treated with a V1aR antagonist sang significantly less than Controls, but did not differ in affiliation. These effects on behavior in males were also reflected in changes in the expression of V1aR and immediate early gene activity in three brain regions known to be involved in pairing behavior in birds: the medial amygdala, medial bed nucleus of the stria terminalis, and the lateral septum. AVT males had higher V1aR expression in the medial amygdala than both Control and antagonist-treated males and immediate early gene activity of V1aR neurons in the medial amygdala was positively correlated with affiliation. Antagonist treated males showed decreased activity in the medial amygdala. In addition, there was a negative correlation between the activity of V1aR cells in the medial bed nucleus of the stria terminalis and singing. Treatment also affected the expression of V1aR and activity in the lateral septum, but this was not correlated with any behaviors measured. These results provide evidence that AVT and V1aR play developmental roles in specific pair maintenance behaviors and the neural substrate underlying these behaviors in a bird. PMID:27065824

  3. Early Life Manipulations of the Nonapeptide System Alter Pair Maintenance Behaviors and Neural Activity in Adult Male Zebra Finches.

    PubMed

    Baran, Nicole M; Tomaszycki, Michelle L; Adkins-Regan, Elizabeth

    2016-01-01

    Adult zebra finches (T. guttata) form socially monogamous pair bonds characterized by proximity, vocal communication, and contact behaviors. In this experiment, we tested whether manipulations of the nonapeptide hormone arginine vasotocin (AVT, avian homolog of vasopressin) and the V1a receptor (V1aR) early in life altered species-typical pairing behavior in adult zebra finches of both sexes. Although there was no effect of treatment on the tendency to pair in either sex, males in different treatments exhibited profoundly different profiles of pair maintenance behavior. Following a brief separation, AVT-treated males were highly affiliative with their female partner but sang very little compared to Controls. In contrast, males treated with a V1aR antagonist sang significantly less than Controls, but did not differ in affiliation. These effects on behavior in males were also reflected in changes in the expression of V1aR and immediate early gene activity in three brain regions known to be involved in pairing behavior in birds: the medial amygdala, medial bed nucleus of the stria terminalis, and the lateral septum. AVT males had higher V1aR expression in the medial amygdala than both Control and antagonist-treated males and immediate early gene activity of V1aR neurons in the medial amygdala was positively correlated with affiliation. Antagonist treated males showed decreased activity in the medial amygdala. In addition, there was a negative correlation between the activity of V1aR cells in the medial bed nucleus of the stria terminalis and singing. Treatment also affected the expression of V1aR and activity in the lateral septum, but this was not correlated with any behaviors measured. These results provide evidence that AVT and V1aR play developmental roles in specific pair maintenance behaviors and the neural substrate underlying these behaviors in a bird. PMID:27065824

  4. MicroRNA-194 (miR-194) regulates ROMK channel activity by targeting intersectin 1.

    PubMed

    Lin, Dao-Hong; Yue, Peng; Zhang, Chengbiao; Wang, Wen-Hui

    2014-01-01

    The aim of the study is to explore the role of miR-194 in mediating the effect of high-K (HK) intake on ROMK channel. Northern blot analysis showed that miR-194 was expressed in kidney and that HK intake increased while low-K intake decreased the expression of miR-194. Real-time PCR analysis further demonstrated that HK intake increased the miR-194 expression in the cortical collecting duct. HK intake decreased the expression of intersectin 1 (ITSN1) which enhanced With-No-Lysine Kinase (WNK)-induced endocytosis of ROMK. Expression of miR-194 mimic decreased luciferase reporter gene activity in HEK293 T cells transfected with ITSN-1-3'UTR containing the complementary seed sequence for miR-194. In contrast, transfection of miR-194 inhibitor increased the luciferase activity. This effect was absent in the cells transfected with mutated 3'UTR of ITSN1 in which the complimentary seed sequence was deleted. Moreover, the inhibition of miR-194 expression increased the protein level of endogenous ITSN1 in HEK293T cells. Expression of miR-194 mimic also decreased the translation of exogenous ITSN1 in the cells transfected with the ITSN1 containing 3'UTR but not with 3'UTR-free ITSN1. Expression of pre-miR-194 increased K currents and ROMK expression in the plasma membrane in ROMK-transfected cells. Coexpression of ITSN1 reversed the stimulatory effect of miR-194 on ROMK channels. This effect was reversed by coexpression of ITSN1. We conclude that miR-194 regulates ROMK channel activity by modulating ITSN1 expression thereby enhancing ITSN1/WNK-dependent endocytosis. It is possible that miR-194 is involved in mediating the effect of a HK intake on ROMK channel activity. PMID:24197061

  5. Targeting solid tumours with potassium channel activators. A return to fundamentals?

    PubMed

    Trechot, Philippe

    2014-01-01

    From a pharmacological point of view nicotinamide and minoxidil are potassium channel activators. Nicotinamide is used as a radiosensitizer in ARCON (accelerated radiotherapy combined with carbogen breathing and nicotinamide) therapeutic strategy with promising results but not confirmed so far. Minoxidil has never been considered by radiotherapists. Based from recent pathophysiological considerations we suggest a new perspective for the use of these two "old" molecules in order to target solid tumours. PMID:25371295

  6. Direct tests of micro channel plates as the active element of a new shower maximum detector

    DOE PAGESBeta

    Ronzhin, A.; Los, S.; Ramberg, E.; Apresyan, A.; Xie, S.; Spiropulu, M.; Kim, H.

    2015-05-22

    We continue the study of micro channel plates (MCP) as the active element of a shower maximum (SM) detector. We present below test beam results obtained with MCPs detecting directly secondary particles of an electromagnetic shower. The MCP efficiency to shower particles is close to 100%. Furthermore, the time resolution obtained for this new type of the SM detector is at the level of 40 ps.

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

    PubMed

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

    2015-02-27

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

  8. Trapping channel block of NMDA-activated responses by amantadine and memantine.

    PubMed

    Blanpied, T A; Boeckman, F A; Aizenman, E; Johnson, J W

    1997-01-01

    We investigated the mechanisms by which the antiparkinsonian and neuroprotective agents amantadine and memantine inhibit responses to N-methyl-D-aspartic acid (NMDA). Whole cell recordings were performed using cultured rat cortical neurons or Chinese hamster ovary (CHO) cells expressing NMDA receptors. Both amantadine and memantine blocked NMDA-activated channels by binding to a site at which they could be trapped after channel closure and agonist unbinding. For neuronal receptors, the IC50s of amantadine and memantine at -67 mV were 39 and 1.4 microM, respectively. When memantine and agonists were washed off after steady-state block, one-sixth of the blocked channels released rather than trapped the blocker; memantine exhibited "partial trapping." Thus memantine appears to have a lesser tendency to be trapped than do phencyclidine or (5R,10S)-(+)-5-methyl-10,11-dihydro-5H-dibenzo[1,d]cyclihepten-5,1 0-imine (MK-801). We next investigated mechanisms that might underlie partial trapping. Memantine blocked and could be trapped by recombinant NMDA receptors composed of NR1 and either NR2A or NR2B subunits. In these receptors, as in the native receptors, the drug was released from one-sixth of blocked channels rather than being trapped in all of them. The partial trapping we observed therefore was not due to variability in the action of memantine on a heterogeneous population of NMDA receptors in cultured cortical neurons. Amantadine and memantine each noncompetitively inhibited NMDA-activated responses by binding at a second site with roughly 100-fold lower affinity, but this form of inhibition had little effect on the extent to which memantine was trapped. A simple kinetic model of blocker action was used to demonstrate that partial trapping can result if the presence of memantine in the channel affects the gating transitions or agonist affinity of the NMDA receptor. Partial trapping guarantees that during synaptic communication in the presence of blocker, some

  9. Down-regulation of the small conductance calcium-activated potassium channels in diabetic mouse atria.

    PubMed

    Yi, Fu; Ling, Tian-You; Lu, Tong; Wang, Xiao-Li; Li, Jingchao; Claycomb, William C; Shen, Win-Kuang; Lee, Hon-Chi

    2015-03-13

    The small conductance Ca(2+)-activated K(+) (SK) channels have recently been found to be expressed in the heart, and genome-wide association studies have shown that they are implicated in atrial fibrillation. Diabetes mellitus is an independent risk factor of atrial fibrillation, but the ionic mechanism underlying this relationship remains unclear. We hypothesized that SK channel function is abnormal in diabetes mellitus, leading to altered cardiac electrophysiology. We found that in streptozotocin-induced diabetic mice, the expression of SK2 and SK3 isoforms was down-regulated by 85 and 92%, respectively, whereas that of SK1 was not changed. SK currents from isolated diabetic mouse atrial myocytes were significantly reduced compared with controls. The resting potentials of isolated atrial preparations were similar between control and diabetic mice, but action potential durations were significantly prolonged in the diabetic atria. Exposure to apamin significantly prolonged action potential durations in control but not in diabetic atria. Production of reactive oxygen species was significantly increased in diabetic atria and in high glucose-cultured HL-1 cells, whereas exposure of HL-1 cells in normal glucose culture to H2O2 reduced the expression of SK2 and SK3. Tyrosine nitration in SK2 and SK3 was significantly increased by high glucose culture, leading to accelerated channel turnover. Treatment with Tiron prevented these changes. Our results suggest that increased oxidative stress in diabetes results in SK channel-associated electrical remodeling in diabetic atria and may promote arrhythmogenesis. PMID:25605734

  10. Hyaluronan modulates TRPV1 channel opening, reducing peripheral nociceptor activity and pain

    PubMed Central

    Caires, Rebeca; Luis, Enoch; Taberner, Francisco J.; Fernandez-Ballester, Gregorio; Ferrer-Montiel, Antonio; Balazs, Endre A.; Gomis, Ana; Belmonte, Carlos; de la Peña, Elvira

    2015-01-01

    Hyaluronan (HA) is present in the extracellular matrix of all body tissues, including synovial fluid in joints, in which it behaves as a filter that buffers transmission of mechanical forces to nociceptor nerve endings thereby reducing pain. Using recombinant systems, mouse-cultured dorsal root ganglia (DRG) neurons and in vivo experiments, we found that HA also modulates polymodal transient receptor potential vanilloid subtype 1 (TRPV1) channels. HA diminishes heat, pH and capsaicin (CAP) responses, thus reducing the opening probability of the channel by stabilizing its closed state. Accordingly, in DRG neurons, HA decreases TRPV1-mediated impulse firing and channel sensitization by bradykinin. Moreover, subcutaneous HA injection in mice reduces heat and capsaicin nocifensive responses, whereas the intra-articular injection of HA in rats decreases capsaicin joint nociceptor fibres discharge. Collectively, these results indicate that extracellular HA reduces the excitability of the ubiquitous TRPV1 channel, thereby lowering impulse activity in the peripheral nociceptor endings underlying pain. PMID:26311398

  11. High-speed microfluidic thermal stimulator for temperature-activated ion channel studies

    NASA Astrophysics Data System (ADS)

    Pennell, Thomas; Wang, Jianbin; Hua, Susan Z.

    2007-04-01

    In this paper we have designed and built a microfluidic thermal chip that provides rapid temperature changes in the solution combined with accurate temperature control. The thermal chip was designed to facilitate the patch-clamp to study temperature dependent activities of ion channels. The device consists of a fluid channel for perfusing solution connected to an accessible reservoir for making patch-clamp measurements on individual cells. A thin film platinum heater was used to generate rapid temperature change and the temperature was monitored using a thin film resistor. The thermal chip was constructed using SU-8 materials on glass wafer to minimize the heat loss to the substrate and channel walls. The chip was characterized for various flow rates ranging from 0.0093 mL/min to 0.0507 mL/min with heater power ranging from 2.7 to 19.4 mW. The heating element is capable of alternating the temperature ranging from bath temperature (20°C) to 90°C at maximum heating rate of 1°C/10 ms. Using the chip, patch clamp recordings were made on cultured HEK cells as the temperature was rapidly varied. The results demonstrated that the thermal chip could be used as a thermal clamp for many thermosensitive ion channel studies.

  12. Interfacial gating triad is crucial for electromechanical transduction in voltage-activated potassium channels

    PubMed Central

    Chowdhury, Sandipan; Haehnel, Benjamin M.

    2014-01-01

    Voltage-dependent potassium channels play a crucial role in electrical excitability and cellular signaling by regulating potassium ion flux across membranes. Movement of charged residues in the voltage-sensing domain leads to a series of conformational changes that culminate in channel opening in response to changes in membrane potential. However, the molecular machinery that relays these conformational changes from voltage sensor to the pore is not well understood. Here we use generalized interaction-energy analysis (GIA) to estimate the strength of site-specific interactions between amino acid residues putatively involved in the electromechanical coupling of the voltage sensor and pore in the outwardly rectifying KV channel. We identified candidate interactors at the interface between the S4–S5 linker and the pore domain using a structure-guided graph theoretical approach that revealed clusters of conserved and closely packed residues. One such cluster, located at the intracellular intersubunit interface, comprises three residues (arginine 394, glutamate 395, and tyrosine 485) that interact with each other. The calculated interaction energies were 3–5 kcal, which is especially notable given that the net free-energy change during activation of the Shaker KV channel is ∼14 kcal. We find that this triad is delicately maintained by balance of interactions that are responsible for structural integrity of the intersubunit interface while maintaining sufficient flexibility at a critical gating hinge for optimal transmission of force to the pore gate. PMID:25311635

  13. HCN channels in behavior and neurological disease: too hyper, or not active enough?

    PubMed Central

    Lewis, Alan S.; Chetkovich, Dane M.

    2010-01-01

    The roles of cells within the nervous system are based on their properties of excitability, which are in part governed by voltage-gated ion channels. HCN channels underlie the hyperpolarization-activated current, Ih, an important regulator of excitability and rhythmicity through control of basic membrane properties. Ih is present in multiple neuronal types and regions of the central nervous system, and changes in Ih alter cellular input-output properties and neuronal circuitry important for behavior such as learning and memory. Furthermore, the pathophysiology of neurological diseases of both the central and peripheral nervous system involves defects in excitability, rhythmicity, and signaling, and animal models of many of these disorders have implicated changes in HCN channels and Ih as critical for pathogenesis. In this review, we focus on recent research elucidating the role of HCN channels and Ih in behavior and disease. These studies have utilized knockout mice as well as animal models of disease to examine how Ih may be important in regulating learning and memory, sleep, and consciousness, as well as how misregulation of Ih may contribute to epilepsy, chronic pain, and other neurological disorders. This review will help guide future studies aimed at further understanding the function of this unique conductance in both health and disease of the mammalian brain. PMID:21130878

  14. Interfacial gating triad is crucial for electromechanical transduction in voltage-activated potassium channels.

    PubMed

    Chowdhury, Sandipan; Haehnel, Benjamin M; Chanda, Baron

    2014-11-01

    Voltage-dependent potassium channels play a crucial role in electrical excitability and cellular signaling by regulating potassium ion flux across membranes. Movement of charged residues in the voltage-sensing domain leads to a series of conformational changes that culminate in channel opening in response to changes in membrane potential. However, the molecular machinery that relays these conformational changes from voltage sensor to the pore is not well understood. Here we use generalized interaction-energy analysis (GIA) to estimate the strength of site-specific interactions between amino acid residues putatively involved in the electromechanical coupling of the voltage sensor and pore in the outwardly rectifying KV channel. We identified candidate interactors at the interface between the S4-S5 linker and the pore domain using a structure-guided graph theoretical approach that revealed clusters of conserved and closely packed residues. One such cluster, located at the intracellular intersubunit interface, comprises three residues (arginine 394, glutamate 395, and tyrosine 485) that interact with each other. The calculated interaction energies were 3-5 kcal, which is especially notable given that the net free-energy change during activation of the Shaker KV channel is ∼14 kcal. We find that this triad is delicately maintained by balance of interactions that are responsible for structural integrity of the intersubunit interface while maintaining sufficient flexibility at a critical gating hinge for optimal transmission of force to the pore gate.

  15. A proton-activated, outwardly rectifying chloride channel in human umbilical vein endothelial cells

    SciTech Connect

    Ma Zhiyong; Zhang Wei; Chen Liang; Wang Rong; Kan Xiaohong; Sun Guizhen; Liu Chunxi; Li Li Zhang Yun

    2008-07-04

    Extracellular acidic pH-activated chloride channel I{sub Cl,acid}, has been characterized in HEK 293 cells and mammalian cardiac myocytes. This study was designed to characterize I{sub Cl,acid} in human umbilical vein endothelial cells(HUVECs). The activation and deactivation of the current rapidly and repeatedly follows the change of the extracellular solution at pH 4.3, with the threshold pH 5.3. In addition, at very positive potentials, the current displays a time-dependent facilitation. pH-response relationship for I{sub Cl,acid} revealed that EC{sub 50} is pH 4.764 with a threshold pH value of pH 5.3 and nH of 14.545. The current can be blocked by the Cl{sup -} channel inhibitor DIDS (100 {mu}M). In summary, for the first time we report the presence of proton-activated, outwardly rectifying chloride channel in HUVECs. Because an acidic environment can develop in local myocardium under pathological conditions such as myocardial ischemia, I{sub Cl,acid} would play a role in regulation of EC function under these pathological conditions.

  16. Photocontrol of Voltage-Gated Ion Channel Activity by Azobenzene Trimethylammonium Bromide in Neonatal Rat Cardiomyocytes

    PubMed Central

    Frolova, Sheyda R.; Gaiko, Olga; Tsvelaya, Valeriya A.; Pimenov, Oleg Y.; Agladze, Konstantin I.

    2016-01-01

    The ability of azobenzene trimethylammonium bromide (azoTAB) to sensitize cardiac tissue excitability to light was recently reported. The dark, thermally relaxed trans- isomer of azoTAB suppressed spontaneous activity and excitation propagation speed, whereas the cis- isomer had no detectable effect on the electrical properties of cardiomyocyte monolayers. As the membrane potential of cardiac cells is mainly controlled by activity of voltage-gated ion channels, this study examined whether the sensitization effect of azoTAB was exerted primarily via the modulation of voltage-gated ion channel activity. The effects of trans- and cis- isomers of azoTAB on voltage-dependent sodium (INav), calcium (ICav), and potassium (IKv) currents in isolated neonatal rat cardiomyocytes were investigated using the whole-cell patch-clamp technique. The experiments showed that azoTAB modulated ion currents, causing suppression of sodium (Na+) and calcium (Ca2+) currents and potentiation of net potassium (K+)