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Sample records for active channel width

  1. Channel planform geometry and slopes from freely available high-spatial resolution imagery and DEM fusion: Implications for channel width scalings, erosion proxies, and fluvial signatures in tectonically active landscapes

    NASA Astrophysics Data System (ADS)

    Fisher, G. Burch; Bookhagen, Bodo; Amos, Colin B.

    2013-07-01

    The rapid expansion of high-spatial resolution optical sensors and imagery over the last decade presents exceptional opportunities for quantifying visible attributes of geomorphic systems. In this study, we detail a simple, robust methodology (ChanGeom) to extract continuous channel width and centerline datasets for single-thread channels using freely available high-spatial resolution imagery currently available in Google Earth and Bing Maps. Comparisons with a global dataset of field and lidar-derived channel widths indicate minimal errors associated with the imagery and ChanGeom methodology (< 1% overall), while examples from the Goriganga River (Indian Himalaya) and the Yakima River (WA, USA) emphasize the benefit of empirical width values over established channel width scalings in deciphering fluvial responses to complex landscape forcings in tectonically active regions. Additionally, accurate centerline delineation from the ChanGeom methodology provides improved sinuosity measurements, and when fused with coarse resolution digital elevation models (DEMs), removes along-profile shortening and coincident increases in reach-scale channel slope. Lastly, comparisons of ASTER GDEM V2, SRTM V4.1, and lidar channel profiles extracted in moderate to high-relief regions demonstrate the inferiority of the ASTER GDEM for channel slope calculations, despite the apparent spatial resolution advantages (9 ×). The methodology presented here will facilitate new discoveries in the fluvial environment that have historically been difficult due to access and imagery resolution issues, and provide greater perspective on channel signatures and responses to a host of landscape forcings, especially in tectonically active bedrock and lower order drainage systems.

  2. Preliminary appraisal of ephemeral-streamflow characteristics as related to drainage area, active-channel width, and soils in northwestern New Mexico

    USGS Publications Warehouse

    Hejl, H.R., Jr.

    1980-01-01

    Regression equations are presented to predict ephemeral streamflow characteristics in the San Juan Basin in northwestern New Mexico. The standard error of estimate for predicting runoff for water year 1978 using drainage area as the independent variable was 152 percent. Indications are that reliable equations for predicating annual runoff can be developed and the standard error of estimate might be reduced significantly with additional years of record. The coefficient of regression when relating drainage area to runoff for water year 1978 was significant at the 1-percent level. Preliminary results also indicate it is feasible to predict streamflow characteristics using hyrologic soil-group classifications based on runoff potential. The standard error of estimate for predicting peak discharges with recurrence intervals of 2, 5, 10, 25, 50, and 100 years using active-channel width as the independent variable averaged about 50 percent, and the regression coefficient was significant at the 1-percent level. Using drainage area to predict peak discharges resulted in a standard error of estimate that averaged about 60 percent and a regression coefficient significant at the 5-percent level. The standard error of estimate averaged about 45 percent when active-channel width and drainage area were related to peak discharges in multiple regression analyses. (USGS)

  3. Bank stability and channel width adjustment, East Fork River, Wyoming.

    USGS Publications Warehouse

    Andrews, E.D.

    1982-01-01

    Frequent surveys of eight cross sections located in self-formed reaches of the East Fork River, Wyoming, during the 1974 snowmelt flood showed a close relation between channel morphology and scour and fill. Those cross sections narrower than the mean reach width filled at discharges less than bankfull and scoured at discharges greater than bankfull. Those cross sections wider than the mean reach width scoured at discharges less than bankfull and filled at discharges greater than bankfull. Bank stability, and to some extent the adjustment of stream channel width, in the East Fork River study reach appears to be controlled by the processes of scour and fill. -from Author

  4. Morphodynamics structures induced by variations of the channel width

    NASA Astrophysics Data System (ADS)

    Duro, Gonzalo; Crosato, Alessandra; Tassi, Pablo

    2014-05-01

    In alluvial channels, forcing effects, such as a longitudinally varying width, can induce the formation of steady bars (Olesen, 1984). The type of bars that form, such as alternate, central or multiple, will mainly depend on the local flow width-to-depth ratio and on upstream conditions (Struiksma et al., 1985). The effects on bar formation of varying the channel width received attention only recently and investigations, based on flume experiments and mathematical modelling, are mostly restricted to small longitudinal sinusoidal variations of the channel width (e.g. Repetto et al., 2002; Wu and Yeh, 2005, Zolezzi et al., 2012; Frascati and Lanzoni, 2013). In this work, we analyze the variations in equilibrium bed topography in a longitudinal width-varying channel with characteristic scales of the Waal River (The Netherlands) using two different 2D depth-averaged morphodynamic models, one based on the Delft3D code and one on Telemac-Mascaret system. In particular, we explore the effects of changing the wavelength of sinusoidal width variations in a straight channel, focusing on the effects of the spatial lag between bar formation and forcing that is observed in numerical models and laboratory experiments (e.g. Crosato et al, 2011). We extend the investigations to finite width variations in which longitudinal changes of the width-to-depth ratio are such that they may affect the type of bars that become unstable (alternate, central or multiple bars). Numerical results are qualitatively validated with field observations and the resulting morphodynamic pattern is compared with the physics-based predictor of river bar modes by Crosato and Mosselman (2009). The numerical models are finally used to analyse the experimental conditions of Wu and Yeh (2005). The study should be seen as merely exploratory. The aim is to investigate possible approaches for future research aiming at assessing the effects of artificial river widening and narrowing to control bar formation in

  5. Revised techniques for estimating peak discharges from channel width in Montana

    USGS Publications Warehouse

    Parrett, Charles; Hull, J.A.; Omang, R.J.

    1987-01-01

    This study was conducted to develop new estimating equations based on channel width and the updated flood frequency curves of previous investigations. Simple regression equations for estimating peak discharges with recurrence intervals of 2, 5, 10 , 25, 50, and 100 years were developed for seven regions in Montana. The standard errors of estimates for the equations that use active channel width as the independent variables ranged from 30% to 87%. The standard errors of estimate for the equations that use bankfull width as the independent variable ranged from 34% to 92%. The smallest standard errors generally occurred in the prediction equations for the 2-yr flood, 5-yr flood, and 10-yr flood, and the largest standard errors occurred in the prediction equations for the 100-yr flood. The equations that use active channel width and the equations that use bankfull width were determined to be about equally reliable in five regions. In the West Region, the equations that use bankfull width were slightly more reliable than those based on active channel width, whereas in the East-Central Region the equations that use active channel width were slightly more reliable than those based on bankfull width. Compared with similar equations previously developed, the standard errors of estimate for the new equations are substantially smaller in three regions and substantially larger in two regions. Limitations on the use of the estimating equations include: (1) The equations are based on stable conditions of channel geometry and prevailing water and sediment discharge; (2) The measurement of channel width requires a site visit, preferably by a person with experience in the method, and involves appreciable measurement errors; (3) Reliability of results from the equations for channel widths beyond the range of definition is unknown. In spite of the limitations, the estimating equations derived in this study are considered to be as reliable as estimating equations based on basin and

  6. GIS technology for spatiotemporal measurements of gully channel width evolution

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Field observations of gully evolution in active croplands have often revealed the presence of a less erodible soil layer that is typically associated with tillage practices (i.e. plowpan). This more erosion-resistant layer limits channel incision forcing the gully channel to expand laterally through...

  7. Insights About Channel Width Controls Based on Channel Narrowing Below Dams

    NASA Astrophysics Data System (ADS)

    Schmidt, J. C.

    2007-12-01

    All channels narrow under conditions of flood reduction, such as downstream from dams, but the magnitude of narrowing is highly variable. Channel width changes on 61 dam-impacted reaches covering more than 4000 km of six large rivers of the western United States (Missouri, Rio Grande, Colorado, Trinity, Snake, Deschutes) were reviewed in an extensive literature search, supplemented by field studies on the Rio Grande, Colorado River and its tributaries, and Snake River. These studies demonstrate that narrowing occurs under conditions of post-dam sediment deficit and surplus, indicating that the primary control on width is not sediment mass balance. However, channel width changes are not related to flood reduction in a simple way, such as would be predicted by downstream hydraulic geometry relations. Narrowing is of larger magnitude on rivers with large suspended sediment loads, and is less obvious on gravel-bed rivers in sediment surplus. Narrowing occurs where beds incise and where this is not the case. Narrowing occurs in the presence or absence of invasive, woody riparian vegetation. Large-scale channel organization, such as distinguishing between meandering and debris-fan affected channels, has a strong effect on the magnitude of narrowing, primarily because the mechanism of narrowing differs between these major channel types. We examined channels whose post-dam 2-yr flood is as little as 0.20 times the pre-dam flood and where channel width narrowed by as much as 0.15 times of the pre- dam width. Field studies were based on extensive analysis of historical data as well an dendrogeomorphic interpretation of floodplain sediments.

  8. River channel width change: Dynamics and scaling relationships in the upper Midwestern US

    NASA Astrophysics Data System (ADS)

    Notebaert, Bastiaan; Belmont, Patrick; Donovan, Mitchell

    2016-04-01

    The width of alluvial river channels varies as a function of multiple variables, including flow, sediment supply, bed roughness and riparian vegetation. Changes in channel width are highly variable in space and time, but few have characterized and/or explained the structure and scaling relationships of that variability. Increasing availability of remote sensing data and computational power allows us to measure landscape changes at more detailed spatial and temporal scales than ever. In this study we use historic air photos to study patterns of channel width change and examine the effects data resolution on measurements of channel width change. We digitized 129 km of (vegetated) channel banks for the Root River in Minnesota, USA, for nearly every decade (excluding the 60s and 80s) spanning 1937-2013. Rates of channel widening were calculated at different spatial and temporal scales. Spatial-scaling effects were examined by measuring width changes from a 10-m window to the reach (~10 km) scale. The time interval between measurements varied from 1 year to 76 years. Data show that at small (100 m) spatial scales reaches that widen in one time period have a strong propensity to narrow in the following period. The most active reaches typically exhibit short, punctuated periods of change, but the stretches that are most active varied across decades. When increasing the temporal scale (time period) over which rates are calculated, the rates exhibit an apparent decrease, an effect that is observed for both the recent period and for data from the 1930s-50s. When considering the same time scale, rates are comparable for both periods. In addition to a temporal scaling effect there is also a spatial scaling effect. Changes in width are spatially correlated for distances up to a 3 to 5 times the channel width. Rates measured over shorter stretches are higher than those measured for longer ones. The most extreme changes occurred over shorter time periods along reaches with a

  9. GWD-LR: a satellite-based global database of river channel width

    NASA Astrophysics Data System (ADS)

    Yamazaki, Dai; O'Loughlin, Fiachra; Trigg, Mark; Bates, Paul

    2015-04-01

    River width is a fundamental parameter of river hydrodynamic simulations, but no global-scale river width database based on observed water bodies has yet been developed. Here we present a new algorithm that automatically calculates river width from satellite-based water masks and flow direction maps. The Global Width Database for Large Rivers (GWD-LR) is developed by applying the algorithm to the SRTM Water Body Database and the HydroSHEDS flow direction map. Both bank-to-bank river width and effective river width excluding islands are calculated for river channels between 60S and 60N. The effective river width of GWD-LR is compared with existing river width databases for the Congo and Mississippi Rivers. The effective river width of the GWD-LR is slightly narrower compared to the existing databases, but the relative difference is within +/-20% for most river channels. As the river width of the GWD-LR is calculated along the river channels of the HydroSHEDS flow direction map, it is relatively straightforward to apply the GWD-LR to global- and continental-scale river modeling.

  10. Ephemeral gully channel width and erosion simulation technology

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Concentrated runoff increases erosion and transfers fine sediment and associated agrichemicals from upland areas to stream channels. Ephemeral gully erosion on cropland in the U.S. may contribute up to 40% or more of the sediment delivered to the edge of the field. Typically, conservation practices ...

  11. On how spatial variations of channel width influence river profile curvature

    NASA Astrophysics Data System (ADS)

    Ferrer-Boix, Carles; Chartrand, Shawn M.; Hassan, Marwan A.; Martín-Vide, Juan P.; Parker, Gary

    2016-06-01

    Longitudinal profiles of alluvial rivers usually exhibit upward-concave curvatures at equilibrium. River profile concavity has been primarily attributed to sediment downstream fining and to streamwise increments of water discharge. Conversely, upward-convex profiles have been typically associated with tectonic and geologic controls and with outlet base-level drops. Equations to describe river profiles at equilibrium developed from mass conservation principles do not consider longitudinal changes in channel width. This study addresses how variations in channel width can also act to control the curvature of longitudinal profiles. We develop a new theoretical framework in which the role on river profiles of downstream variations of channel width, flow discharge, bed roughness, and surface texture are explicitly shown. Unlike classical approaches for river profile evolution, this novel framework identifies physical domains for rivers to develop upward-concave/convex longitudinal profiles depending on channel width and flow discharge gradients flow intensity and surface texture.

  12. Effect of Channel Length and Width on NBTI in Ultra Deep Sub-Micron PMOSFETs

    NASA Astrophysics Data System (ADS)

    Cao, Yan-Rong; Ma, Xiao-Hua; Hao, Yue; Tian, Wen-Chao

    2010-03-01

    The effects of channel length and width on the degradation of negative bias temperature instability (NBTI) are studied. With the channel length decreasing, the NBTI degradation increases. As the channel edges have more damage and latent damage for the process reasons, the device can be divided into three parts: the gate and source overlap region, the middle channel region, and the gate and drain overlap region. When the NBTI stress is applied, the non-uniform distribution of the generated defects in the three parts will be generated due to the inhomogeneous degradation. With the decreasing channel length, the channel edge regions will take up a larger ratio to the middle channel region and the degradation of NBTI is enhanced. The channel width also plays an important role in the degradation of NBTI. There is an inflection point during the decreasing channel width. There are two particular factors: the lower vertical electric field effect for the thicker gate oxide thickness of the shallow trench isolation (STI) edge and the STI mechanical stress effecting on the NBTI degradation. The former reduces and the latter intensifies the degradation. Under the mutual compromise of the both factors, when the effect of the STI mechanical stress starts to prevail over the lower vertical electric field effect with the channel width decreasing, the inflection point comes into being.

  13. Width adjustment in experimental gravel-bed channels in response to overbank flows

    NASA Astrophysics Data System (ADS)

    Pitlick, John; Marr, Jeff; Pizzuto, Jim

    2013-06-01

    We conducted a series of flume experiments to investigate the response of self-formed gravel-bed channels to floods of varying magnitude and duration. Floods were generated by increasing the discharge into a channel created in sand- and gravel-sized sediment with a median grain size of 2 mm. Flooding increased the Shields stress along the channel perimeter, causing bank erosion and rapid channel widening. The sediment introduced to the channel by bank erosion was not necessarily deposited on the channel bed, but was rather transported downstream, a process likely facilitated by transient fining of the bed surface. At the end of each experiment, bank sediments were no longer in motion, "partial bed load transport" characterized the flat-bed portion of the channel, and the Shields stress approached a constant value of 0.056, about 1.2 times the critical Shields stress for incipient motion. Furthermore, the discharge was entirely accommodated by flow within the channel: the creation of a stable channel entirely eliminated overbank flows. We speculate that similar processes may occur in nature, but only where bank sediments are non-cohesive and where channel-narrowing processes cannot counteract bank erosion during overbank flows. We also demonstrate that a simple model of lateral bed load transport can reproduce observed channel widening rates, suggesting that simple methods may be appropriate for predicting width increases in channels with non-cohesive, unvegetated banks, even during overbank flows. Last, we present a model for predicting the equilibrium width and depth of a stable gravel-bed channel with a known channel-forming Shields stress.

  14. Latest updates in global flood modelling: channel bifurcation and global river width database

    NASA Astrophysics Data System (ADS)

    Yamazaki, D.; Kanae, S.; Hirabayashi, Y.; O'Loughlin, F.; Trigg, M. A.; Bates, P. D.

    2014-12-01

    Global flood modelling is a relatively new framework in earth system studies, and there still exist many rooms for improving model physics. A typical grid size of global models (generally >5 km) is coarser than the scale of the topography of river channels and floodplains, therefore flood dynamics in global flood models is represented by sub-grid parameterization. Here, we introduce two latest updates in flood dynamics parameterization, i.e. channel bifurcation scheme and global river width database. The upstream-downstream relationship of model grids is prescribed (i.e. parameterized) by a river network map, where each grid has been assumed to have only one downstream grid. We abandoned this "only one downstream" assumption, and succeeded to represent channel bifurcation in a global flood model. The new bifurcation scheme was tested in the Mekong River, and showed the importance of channel bifurcation in mega-delta hydrodynamics. Channel cross-sectional shape has been parameterized using an empirical equation of discharge (or drainage area), and it is a major source of uncertainties in global flood modelling. We recently developed a fully-automated algorithm to calculate river width from satellite water mask. By applying this algorithm to SRTM Water Body Data, the Global Width Database for Large Rivers (GWD-LR) was constructed. The difference between the satellite-based width and empirically-estimated width is very large, suggesting the difficulty of river width parameterization by an empirical equation. Improvement in flood dynamics parameterization reduces uncertainties in global flood simulations. This enables advanced validation/calibration of global flood models, such as direct comparison against satellite altimeters. A future strategy for advanced model validation/calibration will be mentioned in the conference presentation.

  15. Mechanically Activated Ion Channels.

    PubMed

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

    2015-09-23

    Mechanotransduction, the conversion of physical forces into biochemical signals, is essential for various physiological processes such as the conscious sensations of touch and hearing, and the unconscious sensation of blood flow. Mechanically activated (MA) ion channels have been proposed as sensors of physical force, but the identity of these channels and an understanding of how mechanical force is transduced has remained elusive. A number of recent studies on previously known ion channels along with the identification of novel MA ion channels have greatly transformed our understanding of touch and hearing in both vertebrates and invertebrates. Here, we present an updated review of eukaryotic ion channel families that have been implicated in mechanotransduction processes and evaluate the qualifications of the candidate genes according to specified criteria. We then discuss the proposed gating models for MA ion channels and highlight recent structural studies of mechanosensitive potassium channels. PMID:26402601

  16. Do river channels decrease in width downstream on Distributive Fluvial Systems? An evaluation of modern mega-fans

    NASA Astrophysics Data System (ADS)

    Espinoza, T. N.; Scuderi, L. A.; Weissmann, G. S.; Hartley, A. J.

    2014-12-01

    Recent studies on aggradational continental sedimentary basins globally show that fluvial deposits in most modern sedimentary basins are dominated Distributive Fluvial Systems (DFS). DFS's are identified by: (1) pattern of channels and floodplain deposits that radiate outward from an apex located where the river enters the sedimentary basin, (2) deposition where an alluvial system becomes unconfined upon entering the sedimentary basin, (3) broadly fan shaped deposit that is convex upward across the DFS and concave upward down-fan, and (4) if the DFS is incised, an intersection point above which the alluvial system is held in an incised valley and below which it distributes sediment across an active depositional lobe. Several papers about DFS hypothesized that rivers on DFS decrease in size down-fan. We are testing this hypothesis through evaluation of LANDSAT and STRM data from large DFS described by Hartley et al (2010). We use ArcGIS to: (1) open the images and merge them together if there are more than one image corresponding to the DFS being studied, (2) use a Maximum Likelihood Analysis in six classes to segment different features on the DFS (e.g. exposed sands, water, vegetation, and other fan environments), (3) isolate the classes that correspond to the active channel belt (e.g., exposed sand bars and water), (4) divide the active channel belt into 1000 m long sections, (5) determine the area of active channel belt in each section, and (6) calculate the average width of the river in each section (e.g., W = area/1000m). We present our result for each DFS river on a graph that shows the change in width downstream. Our final product will be a dataset that contains width versus distance down-fan from the apex for as many of the large DFS from Hartley et al (2010) as possible. If the hypothesis is supported, the decrease in width could have a substantial predictive significance on sandstone geometry in fluvial successions.

  17. Graphene modulation channel-width field effect transistors enabling high carrier velocity acceleration and bandgap introduction

    NASA Astrophysics Data System (ADS)

    Mohamad, Aizuddin; Awano, Yuji

    2015-11-01

    The electrical properties of an advanced graphene FET structure with local channel width modulation were theoretically investigated with the aim of increasing the local electric field along the channel and creating a bandgap by introducing a graphene nanoribbon (GNR) array. A semi-classical Monte Carlo particle method was used to simulate near ballistic electron transport combination with ab-initio calculation for successfully determining electronic states. Performance improvement was achieved in terms of the carriers’ transit time, even though there was a tradeoff between the bandgap creation and electron mobility in graphene.

  18. Landscape predictors of channel wetted width at baseflow using air photos

    NASA Astrophysics Data System (ADS)

    Rawlins, Barry; Clark, Liam; Boyd, Doreen

    2013-04-01

    Evasion of carbon dioxide from the surface of freshwater channels accounts for a substantial proportion of its flux from the terrestrial biosphere to the atmosphere; accurate estimates of channel wetted width (WW) are required to improve predictions of this flux. We investigated which landscape and climate-related data were statistically significant predictors of WW at baseflow across a large region (2200 km2) of north Wales and western England (UK) where habitat surveys suggest the majority of channels are in a near natural state. We used 25 cm pixel resolution air photos to measure channel WW at baseflow, and quantified the magnitude of the errors in these measurements. We used flow information from local gauging stations to ensure that channels were at or close to baseflow for the days on which the air photos were captured. The root mean squared difference between the field-based and air photo measurements of WW (n=28 sites) was small (0.14 m) in comparison to the median channel WW (3.07 m), and there was very little bias between the two sets of measurements (0.026 m). We created a set of points along those sections of channels which were visible in air photos and used a digital terrain model to create the drainage catchments for the points and computed their catchment area (CA). We removed points with CA

  19. Topographic viscous fingering: fluid-fluid displacement in a channel of non-uniform gap width.

    PubMed

    Woods, Andrew W; Mingotti, Nicola

    2016-10-13

    We consider the displacement of one fluid by a second immiscible fluid through a long, thin permeable channel whose thickness and permeability decrease away from the axis of the channel. We build a model that illustrates how the shape of the fluid-fluid interface evolves in time. We find that if the injected fluid is of the same viscosity as the original fluid, then the cross-channel variations in permeability and thickness tend to focus the flow along the centre of the channel. If the viscosity of the injected fluid is smaller than the original fluid, then this flow focusing intensifies, leading to very poor sweep of the original fluid in the system, with the injected fluid bypassing much of the channel. We also show that if the viscosity ratio of the injected fluid to the original fluid is sufficiently large, then a blunt nose may develop at the leading edge of the injected fluid, whereas the remainder of the fluid-fluid interface becomes stretched out along the edges of the channel. This leads to a much more efficient sweep of the original fluid from the channel. We generalize the model to illustrate how buoyancy forces and capillary pressure affect the evolution of the system and compare our model predictions with some simple laboratory experiments. This partial stabilization of a fluid interface in a channel of non-uniform width represents a generalization of the classical Saffman-Taylor instability, and our nonlinear solutions for the evolution of the interface highlight the importance of cross-channel variations in permeability and thickness in modelling flow in channelled reservoirs.This article is part of the themed issue 'Energy and the subsurface'. PMID:27597790

  20. On the channel width-dependence of the thermal conductivity in ultra-narrow graphene nanoribbons

    NASA Astrophysics Data System (ADS)

    Karamitaheri, Hossein; Neophytou, Neophytos

    2016-08-01

    The thermal conductivity of low-dimensional materials and graphene nanoribbons, in particular, is limited by the strength of line-edge-roughness scattering. One way to characterize the roughness strength is the dependency of the thermal conductivity on the channel's width in the form Wβ. Although in the case of electronic transport, this dependency is very well studied, resulting in W6 for nanowires and quantum wells and W4 for nanoribbons, in the case of phonon transport it is not yet clear what this dependence is. In this work, using lattice dynamics and Non-Equilibrium Green's Function simulations, we examine the width dependence of the thermal conductivity of ultra-narrow graphene nanoribbons under the influence of line edge-roughness. We show that the exponent β is in fact not a single well-defined number, but it is different for different parts of the phonon spectrum depending on whether phonon transport is ballistic, diffusive, or localized. The exponent β takes values β < 1 for semi-ballistic phonon transport, values β ≫ 1 for sub-diffusive or localized phonons, and β = 1 only in the case where the transport is diffusive. The overall Wβ dependence of the thermal conductivity is determined by the width-dependence of the dominant phonon modes (usually the acoustic ones). We show that due to the long phonon mean-free-paths, the width-dependence of thermal conductivity becomes a channel length dependent property, because the channel length determines whether transport is ballistic, diffusive, or localized.

  1. A Novel Joint Problem of Routing, Scheduling, and Variable-Width Channel Allocation in WMNs

    PubMed Central

    Liu, Wan-Yu; Chou, Chun-Hung

    2014-01-01

    This paper investigates a novel joint problem of routing, scheduling, and channel allocation for single-radio multichannel wireless mesh networks in which multiple channel widths can be adjusted dynamically through a new software technology so that more concurrent transmissions and suppressed overlapping channel interference can be achieved. Although the previous works have studied this joint problem, their linear programming models for the problem were not incorporated with some delicate constraints. As a result, this paper first constructs a linear programming model with more practical concerns and then proposes a simulated annealing approach with a novel encoding mechanism, in which the configurations of multiple time slots are devised to characterize the dynamic transmission process. Experimental results show that our approach can find the same or similar solutions as the optimal solutions for smaller-scale problems and can efficiently find good-quality solutions for a variety of larger-scale problems. PMID:24982990

  2. Tracking Hillslope and Channel Changes During Development with the Width Function

    NASA Astrophysics Data System (ADS)

    Jones, D.; Baker, M. E.; Miller, A. J.

    2012-12-01

    Linking watershed geomorphology to observed hydrologic response is a topic of considerable interest in watershed science. For this purpose, the width function remains popular because it is easily obtained from terrain analysis and because it can mimic realistic unit hydrographs while incorporating network topology. Of published studies using this technique, none have analyzed urban or urbanizing landscapes, so its efficacy and utility in modified landscapes remains largely unknown. We applied five sequential LiDAR-derived DEMs spanning the development of two watersheds in Clarksburg, MD to track changes in surface flow pathways and understand differences in the resulting width functions. Both watersheds are within the Clarksburg Special Protection Area and utilize state of the art stormwater management (SWM) techniques to protect local streams; however the purpose of this study is to quantify the effects of development-induced topographic changes prior to consideration of mitigation by SWM. A third, undeveloped forested watershed was also used to assess temporal changes independent of development. Multidirectional flow pathways were determined for each year of LiDAR, explicitly including focused flow along roads and throughput from detention basin drainages. We analyzed patterns of tangential curvature to delineate stream networks, distinguished subsequent hillslope and channel flow distances, and quantified changes in their distributions as development progressed. Velocity-modified width functions generated from distributions of hillslope and channel flow distances were used to assess how idealized watershed response to a unit of rainfall changed during development. As development progressed, modified width functions exhibited a shift towards shorter flowpath lengths, suggesting potential for decreased storm response times. The observed pattern appeared largely driven by artificial increases in drainage density, despite widespread channel burial, as newly

  3. An experimental study of voice communication over a bandlimited channel using variable bit width delta modulation

    NASA Astrophysics Data System (ADS)

    Tumok, N. Nur

    1989-12-01

    A variable bit width delta modulator (VBWDM) demodulator was designed, built and tested to achieve voice and music communication using a bandlimited channel. Only baseband modulation is applied to the input signal. Since there is no clock used during the digitizing process at the modulator, no bit synchronization is required for signal recovery in the receiver. The modulator is a hybrid design using 7 linear and 3 digital integrated circuits (IC), and the demodulator uses 2 linear ICs. A lowpass filter (LPF) is used to simulate the channel. The average number of bits sent over the channel is measured with a frequency counter at the output of the modulator. The minimum bandwidth required for the LPF is determined according to the intelligibility of the recovered message. Measurements indicate an average bit rate required for intelligible voice transmission is in the range of 2 to 4 kilobits per seconds (kbps) and between 2 to 5 kbps for music. The channel 3 dB bandwidth required is determined to be 1.5 kilohertzs. Besides the hardware simplicity, VBWDM provides an option for intelligible digitized voice transmission at very low bit rates without requiring synchronization. Another important feature of the modulator design is that no bits are sent when no signal is present at the input which saves transmitter power (important for mobile stations) and reduces probability of intercept and jamming in military applications.

  4. Influence of Gate Dielectrics, Electrodes and Channel Width on OFET Characteristics

    NASA Astrophysics Data System (ADS)

    Liyana, V. P.; Stephania, A. M.; Shiju, K.; Predeep, P.

    2015-06-01

    Organic Field Effect Transistors (OFET) possess wide applications in large area electronics owing to their attractive features like easy fabrication process, light weight, flexibility, cost effectiveness etc. But instability, high operational voltages and low carrier mobility act as inhibitors to commercialization of OFETs and various approaches were tried on a regular basis so as to make it viable. In this work, Poly 3-hexylthiophene-2,5diyl (P3HT) based OFETs with bottom-contact top-gate configuration using Poly vinyl alcohol (PVA) and Poly (methyl methacrylate) (PMMA) as gate dielectrics, aluminium and copper as source-drain electrodes are investigated. An effort is made to compare the effect of these dielectric materials and electrodes on the performance of OFET. Also, an attempt has been made to optimize the channel width of the device. These devices are characterised with mobility (μ), threshold voltage (VT), on-off ratio (Ion/Ioff) and their comparative analysis is reported.

  5. Investigation of channel width-dependent threshold voltage variation in a-InGaZnO thin-film transistors

    SciTech Connect

    Liu, Kuan-Hsien; Chou, Wu-Ching; Chang, Ting-Chang; Wu, Ming-Siou; Hung, Yi-Syuan; Sze, Simon M.; Hung, Pei-Hua; Chu, Ann-Kuo; Hsieh, Tien-Yu; Yeh, Bo-Liang

    2014-03-31

    This Letter investigates abnormal channel width-dependent threshold voltage variation in amorphous indium-gallium-zinc-oxide (a-IGZO) thin-film transistors. Unlike drain-induced source barrier lowering effect, threshold voltage increases with increasing drain voltage. Furthermore, the wider the channel, the larger the threshold voltage observed. Because of the surrounding oxide and other thermal insulating material and the low thermal conductivity of the IGZO layer, the self-heating effect will be pronounced in wider channel devices and those with a larger operating drain bias. To further clarify the physical mechanism, fast IV measurement is utilized to demonstrate the self-heating induced anomalous channel width-dependent threshold voltage variation.

  6. Investigation of channel width-dependent threshold voltage variation in a-InGaZnO thin-film transistors

    NASA Astrophysics Data System (ADS)

    Liu, Kuan-Hsien; Chang, Ting-Chang; Wu, Ming-Siou; Hung, Yi-Syuan; Hung, Pei-Hua; Hsieh, Tien-Yu; Chou, Wu-Ching; Chu, Ann-Kuo; Sze, Simon M.; Yeh, Bo-Liang

    2014-03-01

    This Letter investigates abnormal channel width-dependent threshold voltage variation in amorphous indium-gallium-zinc-oxide (a-IGZO) thin-film transistors. Unlike drain-induced source barrier lowering effect, threshold voltage increases with increasing drain voltage. Furthermore, the wider the channel, the larger the threshold voltage observed. Because of the surrounding oxide and other thermal insulating material and the low thermal conductivity of the IGZO layer, the self-heating effect will be pronounced in wider channel devices and those with a larger operating drain bias. To further clarify the physical mechanism, fast IV measurement is utilized to demonstrate the self-heating induced anomalous channel width-dependent threshold voltage variation.

  7. Using LandSat and SRTM datasets to develop relationships for estimating bankfull channel widths in the Amazon Basin

    NASA Astrophysics Data System (ADS)

    Gummadi, V.; He, Y.; Beighley, E. R.

    2007-12-01

    Modeling fine scale spatial and temporal processes of the hydrologic cycle over continental to global extents is vital for assessing the potential impacts of climate and land use change on global water resources and related systems. Significant advancement in understanding and predicting the magnitude, trend, timing and partitioning of terrestrial water stores and fluxes requires the development of methodologies and knowledge for extracting representative hydraulic geometries from remote sensing data products and field data, suitable for estimating inundation characteristics and water storage changes which are limited for much of the globe. In this research, relationships between channel and floodplain widths and spatial drainage characteristics are developed for the Amazon Basin. Channel and floodplain widths were measured using SRTM data and LandSat TM/ETM imagery at 510 sites. The study sites were selected based on the Pfafstetter decomposition methodology which provides an irregular model grid based on repeatedly subdividing landscape units into nine subunits consisting of basins and interbasins. The selected sites encompass all possible combinations of Pfafstetter modeling units (ex., basins of interbasins, interbasins of basins, etc.). The 510 study sites are within the Amazon Basin with drainage areas ranging 10 to 5.4 million sq km and mean watershed ground slopes ranging from 0.4 and 30 percent. Preliminary results indicate that channel widths can be predicted using drainage area and mean watershed slope (R2 = 0.85). Floodplain widths can be predicted using channel width and the local slope (R2 = 0.70). Using the Purus watershed, a sub-basin to the Amazon (350,000 sq km), effects of channel and floodplain widths on simulated hydrographs are presented.

  8. Geomorphic Classification and Evaluation of Channel Width and Emergent Sandbar Habitat Relations on the Lower Platte River, Nebraska

    USGS Publications Warehouse

    Elliott, Caroline M.

    2011-01-01

    This report presents a summary of geomorphic characteristics extracted from aerial imagery for three broad segments of the Lower Platte River. This report includes a summary of the longitudinal multivariate classification in Elliott and others (2009) and presents a new analysis of total channel width and habitat variables. Three segments on the lower 102.8 miles of the Lower Platte River are addressed in this report: the Loup River to the Elkhorn River (70 miles long), the Elkhorn River to Salt Creek (6.9 miles long), and Salt Creek to the Missouri River (25.9 miles long). The locations of these segments were determined by the locations of tributaries potentially significant to the hydrology or sediment supply of the Lower Platte River. This report summarizes channel characteristics as mapped from July 2006 aerial imagery including river width, valley width, channel curvature, and in-channel habitat features. In-channel habitat measurements were not made under consistent hydrologic conditions and must be considered general estimates of channel condition in late July 2006. Longitudinal patterns in these features are explored and are summarized in the context of the longitudinal multivariate classification in Elliott and others (2009) for the three Lower Platte River segments. Detailed descriptions of data collection and classification methods are described in Elliott and others (2009). Nesting data for the endangered interior least tern (Sternula antillarum) and threatened piping plover (Charadrius melodus) from 2006 through 2009 are examined within the context of the multivariate classification and Lower Platte River segments. The widest reaches of the Lower Platte River are located in the segment downstream from the Loup River to the Elkhorn River. This segment also has the widest valley and highest degree of braiding of the three segments and many large vegetated islands. The short segment of river between the Elkhorn River and Salt Creek has a fairly low valley

  9. Dynamic Adjustments in Channel Width in Response to a Forced Diversion: Gower Gulch, Death Valley National Park, California

    NASA Astrophysics Data System (ADS)

    Snyder, N. P.; Kammer, L. L.

    2007-12-01

    We study the 1941 diversion of Furnace Creek Wash (drainage area 439 km2) into Gower Gulch (5.8 km2) as an experiment in the transient response of channel geometry to a large change in water and sediment discharge. We measure sequential changes in valley width using a time series of aerial photographs (1948-1995), airborne laser elevation data from 2005, and a field survey. We find that response of the system varies depending on the pre-diversion channel morphology and geology. In two steep knickzone segments, narrowing, knickpoint retreat, and bedrock incision dominates-- a detachment-limited response. In the relatively low-gradient main part of Gower Gulch, fine-grained, soft sedimentary rocks underlie the channel, and widening dominates as the large, coarse post-diversion sediment load covers the channel bed. The response in this section is transport limited, with only modest incision and adjustments in gradient. Two different processes appear to cause the channel to widen. (1) In many reaches, the stream is attacking the valley walls, as evidenced by fresh plucking and scour marks. This probably occurs because the bed in the middle of the channel is alluviated and protected, which minimizes the opportunity for vertical incision. (2) Some reaches have experienced aggradation, which widens the valley by filling it in. This occurs in places where storage space exists (splay deposits in small tributary mouths, fill terraces in the wide valleys at larger tributary mouths) or in reaches upstream of constrictions. Over long periods, the lowering rate of Gower Gulch probably depends on knickpoint retreat, but the present-day response of this non-steady-state system is a hybrid of incision and narrowing in detachment-limited reaches and widening in transport-limited reaches. This system demonstrates the importance of initial conditions and evolving channel geometry in setting the transient response of rivers.

  10. Energy-dependent target widths in a coupled-channel scattering study

    SciTech Connect

    Canton, L.; Fraser, P. R.; Svenne, J. P.; Amos, K.; Knijff, D. van der; Karataglidis, S.

    2011-04-15

    The question of how the scattering cross section changes when the spectra of the colliding nuclei have low-excitation particle-emitting resonances is explored using a multichannel algebraic scattering method. As a test case, the particle-unstable nucleus {sup 8}Be has been considered as a target. Nucleon-nucleus scattering cross sections, as well as the spectra of the compound nuclei formed, have been determined from calculations that consider, as well as those that disregard, particle-emission widths of the target nuclear states. The resonant character of the unstable excited states introduces a problem because the low-energy tails of these resonances can intrude into the subthreshold, bound-state region. This unphysical behavior needs to be corrected by modifying, in an energy-dependent way, the shape of the target resonances from the usual Lorentzian form. The resonance function must smoothly reach zero at the elastic threshold. We explore herein a way of achieving this condition.

  11. Carbon mediated reduction of silicon dioxide and growth of copper silicide particles in uniform width channels

    SciTech Connect

    Pizzocchero, Filippo; Bøggild, Peter; Booth, Timothy J.

    2013-09-21

    We show that surface arc-discharge deposited carbon plays a critical intermediary role in the breakdown of thermally grown oxide diffusion barriers of 90 nm on a silicon wafer at 1035 °C in an Ar/H{sub 2} atmosphere, resulting in the formation of epitaxial copper silicide particles in ≈ 10 μm wide channels, which are aligned with the intersections of the (100) surface of the wafer and the (110) planes on an oxidized silicon wafer, as well as endotaxial copper silicide nanoparticles within the wafer bulk. We apply energy dispersive x-ray spectroscopy, in combination with scanning and transmission electron microscopy of focused ion beam fabricated lammelas and trenches in the structure to elucidate the process of their formation.

  12. Thermally activated TRPV3 channels.

    PubMed

    Luo, Jialie; Hu, Hongzhen

    2014-01-01

    TRPV3 is a temperature-sensitive transient receptor potential (TRP) ion channel. The TRPV3 protein functions as a Ca(2+)-permeable nonselective cation channel with six transmembrane domains forming a tetrameric complex. TRPV3 is known to be activated by warm temperatures, synthetic small-molecule chemicals, and natural compounds from plants. Its function is regulated by a variety of physiological factors including extracellular divalent cations and acidic pH, intracellular adenosine triphosphate, membrane voltage, and arachidonic acid. TRPV3 shows a broad expression pattern in both neuronal and non-neuronal tissues including epidermal keratinocytes, epithelial cells in the gut, endothelial cells in blood vessels, and neurons in dorsal root ganglia and CNS. TRPV3 null mice exhibit abnormal hair morphogenesis and compromised skin barrier function. Recent advances suggest that TRPV3 may play critical roles in inflammatory skin disorders, itch, and pain sensation. Thus, identification of selective TRPV3 activators and inhibitors could potentially lead to beneficial pharmacological interventions in several diseases. The intent of this review is to summarize our current knowledge of the tissue expression, structure, function, and mechanisms of activation of TRPV3. PMID:25366242

  13. Principal curves for lumen center extraction and flow channel width estimation in 3-D arterial networks: theory, algorithm, and validation.

    PubMed

    Wong, Wilbur C K; So, Ronald W K; Chung, Albert C S

    2012-04-01

    We present an energy-minimization-based framework for locating the centerline and estimating the width of tubelike objects from their structural network with a nonparametric model. The nonparametric representation promotes simple modeling of nested branches and n -way furcations, i.e., structures that abound in an arterial network, e.g., a cerebrovascular circulation. Our method is capable of extracting the entire vascular tree from an angiogram in a single execution with a proper initialization. A succinct initial model from the user with arterial network inlets, outlets, and branching points is sufficient for complex vasculature. The novel method is based upon the theory of principal curves. In this paper, theoretical extension to grayscale angiography is discussed, and an algorithm to find an arterial network as principal curves is also described. Quantitative validation on a number of simulated data sets, synthetic volumes of 19 BrainWeb vascular models, and 32 Rotterdam Coronary Artery volumes was conducted. We compared the algorithm to a state-of-the-art method and further tested it on two clinical data sets. Our algorithmic outputs-lumen centers and flow channel widths-are important to various medical and clinical applications, e.g., vasculature segmentation, registration and visualization, virtual angioscopy, and vascular atlas formation and population study. PMID:22167625

  14. Teasing Foggy Memories out of Pines on the California Channel Islands Using Tree-Ring Width and Stable Isotope Approaches

    NASA Astrophysics Data System (ADS)

    Williams, A. P.; Still, C. J.; Fischer, D. T.; Leavitt, S. W.

    2006-12-01

    The coast of California is home to many rare, endemic conifers and other plants that are not well adapted to the Mediterranean climate that prevails across most of the state. It has long been suggested that coastal pines survived the early-Pleistocene transition to a warmer and drier environment because they benefit from frequent fog and low stratus clouds that provide much needed water inputs and shading during the rainless summer. Here, we report evidence for the importance of this summer cloudiness to Torrey pines (Pinus torreyana) growing on Santa Rosa Island in Channel Islands National Park. We developed a tree-ring width chronology and quantified the relative importance of winter/spring precipitation and summer fog by comparing ring widths to nearby rainfall records and airport cloud-ceiling height data. While winter/spring precipitation explains most of the variation in annual tree-ring width (R2 = 0.592), the frequency of summertime fog correlated significantly and positively with annual ring width for 52 years of available fog data when the effect of winter/spring precipitation was removed (R2 = 0.118). The correlation between fog frequency and ring width decreased sharply when the range of possible cloud-ceiling heights deviated from the habitat range of the Torrey pine stand, emphasizing the importance of direct cloud immersion to these pines. In addition, the relationship between fog frequency and ring width was strongest in the 26 years that had enough winter/spring rainfall to maintain above-average soil moisture throughout the dry summer months (R2 = 0.312). This suggests that Torrey pines have an adaptive growing season length and that summer fog-water inputs are supplemental but not substantial enough to sustain tree growth independently. It may also be suggested that when summer growth does occur, the frequency of summer fog and stratus may govern growing season length. This made a "fog signal" difficult to detect in the stable isotope (carbon and

  15. BK channel activation: structural and functional insights

    PubMed Central

    Lee, Urvi S.; Cui, Jianmin

    2010-01-01

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

  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. PMID:25705194

  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. Measurements of Non-thermal Line Widths in Solar Active Regions

    NASA Astrophysics Data System (ADS)

    Brooks, David H.; Warren, Harry P.

    2016-03-01

    Spectral line widths are often observed to be larger than can be accounted for by thermal and instrumental broadening alone. This excess broadening is a key observational constraint for both nanoflare and wave dissipation models of coronal heating. Here we present a survey of non-thermal velocities measured in the high temperature loops (1-4 MK) often found in the cores of solar active regions. This survey of Hinode Extreme Ultraviolet Imaging Spectrometer (EIS) observations covers 15 non-flaring active regions that span a wide range of solar conditions. We find relatively small non-thermal velocities, with a mean value of 17.6 ± 5.3 km s-1, and no significant trend with temperature or active region magnetic flux. These measurements appear to be inconsistent with those expected from reconnection jets in the corona, chromospheric evaporation induced by coronal nanoflares, and Alfvén wave turbulence models. Furthermore, because the observed non-thermal widths are generally small, such measurements are difficult and susceptible to systematic effects.

  19. Quantifying the transient response of bedrock channels to Active Normal Faulting: New Field Observations

    NASA Astrophysics Data System (ADS)

    Whittaker, A. C.; Cowie, P. A.; Tucker, G. E.; Attal, M.; Roberts, G.

    2005-12-01

    Understanding the morphological response of the fluvial system to transient tectonic forcing is one of the major challenges facing quantitative geomorphology. In theory, insight gained from studying channel adjustment to changing tectonic rates should provide clear diagnostic tests of the many competing `erosion laws' which aim to quantify stream incision. However, fluvial algorithms in current landscape models tend to be parameterised in terms of hydraulic scaling relationships, which only describe channel width and depth as power-law functions of river discharge or upstream drainage area. Unfortunately, these scaling relationships, which have been derived from channels in tectonically quiescent areas, are not appropriate for bedrock rivers in active settings. This problem is serious for understanding non-equilibrium systems because hydraulic adjustments are an important aspect of the morphodynamic response to tectonic and climatic forcing. Recent theoretical attempts to resolve this issue still rely fundamentally on assumptions of steady-state channel form. To devise an alternative approach we need to collect geometrical data for channels incising in areas where the boundary conditions are well-constrained independently. We address this challenge by providing new and detailed field measurements of valley and bankfull channel width, depth, slope and grain-size data for an out-of-equilibrium channel with a drainage area of 65km2 crossing an active extensional fault near Fiamignano, Italy, where there are excellent constraints on current rates of fault movement, and good evidence for an increase in throw-rate approximately 700 Kyr ago. We show that in this situation channel width becomes strongly decoupled from drainage area immediately upstream of the fault and that channel aspect ratio and median grain-size are correlated with channel slope. The ratio of total stream power to coarse-fraction grain size peaks in precisely the areas where channel width

  20. 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.

  1. Can red cell distribution width be a marker of disease activity in ulcerative colitis?

    PubMed Central

    Ipek, Serkan; Cekic, Cem; Alper, Emrah; Coban, Eyup; Eliacik, Eylem; Arabul, Mahmut; Aslan, Fatih; Vatansever, Sezgin; Yalcin, Hulya; Unsal, Belkis

    2015-01-01

    Aim: The current study aimed to investigate the association between disease activity and red cell distribution width (RDW) levels in ulcerative colitis and to determine whether RDW can be used as a marker of disease activity in non-anemic ulcerative colitis. Methods: The RDW levels of 310 ulcerative colitis patients who underwent colonoscopy were analyzed retrospectively. The patients were divided into two groups (active disease and remission) according to the endoscopic activity index. In addition, the accuracy of RDW in determining disease activity in non-anemic patients was assessed. The efficacy of RDW in determining disease activity was compared to that of white blood cell count, platelet count, C-reactive protein, and erythrocyte sedimentation rate. Results: Two hundred and six (66.5%) patients had active disease, and 104 (33.5%) were in remission. The mean RDW levels in patients with active ulcerative colitis and in those in remission were 16.8±2.9 and 15.5±1.4, respectively (P<0.001). Ninety-six (46.6%) patients in the active disease group and 89 (85.6%) in the remission group were non-anemic, and their respective RDW levels were 15.4±1.2 and 15.3±1.1 (P=0.267). The sensitivity and specificity of RDW in determining inflammation were 41% and 91%, respectively (AUC 0.65, P<0.001). Conclusions: This study demonstrated that RDW can be used as a marker for disease activity in ulcerative colitis, but it did not have the same efficacy in the non-anemic group. PMID:26550336

  2. The 3D pore structure and fluid dynamics simulation of macroporous monoliths: High permeability due to alternating channel width.

    PubMed

    Jungreuthmayer, Christian; Steppert, Petra; Sekot, Gerhard; Zankel, Armin; Reingruber, Herbert; Zanghellini, Jürgen; Jungbauer, Alois

    2015-12-18

    Polymethacrylate-based monoliths have excellent flow properties. Flow in the wide channel interconnected with narrow channels is theoretically assumed to account for favorable permeability. Monoliths were cut into 898 slices in 50nm distances and visualized by serial block face scanning electron microscopy (SBEM). A 3D structure was reconstructed and used for the calculation of flow profiles within the monolith and for calculation of pressure drop and permeability by computational fluid dynamics (CFD). The calculated and measured permeabilities showed good agreement. Small channels clearly flowed into wide and wide into small channels in a repetitive manner which supported the hypothesis describing the favorable flow properties of these materials. This alternating property is also reflected in the streamline velocity which fluctuated. These findings were corroborated by artificial monoliths which were composed of regular (interconnected) cells where narrow cells followed wide cells. In the real monolith and the artificial monoliths with interconnected flow channels similar velocity fluctuations could be observed. A two phase flow simulation showed a lateral velocity component, which may contribute to the transport of molecules to the monolith wall. Our study showed that the interconnection of small and wide pores is responsible for the excellent pressure flow properties. This study is also a guide for further design of continuous porous materials to achieve good flow properties. PMID:26615711

  3. Dynamic adjustments in channel width in response to a forced diversion: Gower Gulch, Death Valley National Park, California

    NASA Astrophysics Data System (ADS)

    Snyder, Noah P.; Kammer, Lisa L.

    2008-02-01

    We studied the 1941 diversion of Furnace Creek Wash (drainagearea 439 km2) into Gower Gulch (5.8 km2) as an experiment inthe transient response of a channel to a large change in waterand sediment discharge. We measured sequential changes in valleywidth using a time series of aerial photographs (1948-1995),airborne laser elevation data from 2005, and a field survey.We found that the response of the system varied depending onthe prediversion channel morphology and bedrock geology. Intwo steep knickzone segments, narrowing, knickpoint retreatand bedrock incision dominated, indicating a detachment-limitedresponse. In the relatively low-gradient main part of GowerGulch, widening dominated as the coarse postdiversion sedimentload covered the channel bed. This transport-limited part ofthe system has undergone only modest incision and adjustmentsin gradient. Over long periods, the lowering rate of Gower Gulchprobably depends on knickpoint retreat, but the present-dayresponse of this non-steady-state system is a hybrid ofincision and narrowing in detachment-limited reaches and wideningin transport-limited reaches. This system demonstrates the importanceof evolving channel geometry in setting the transient responseof rivers to changes in forcing parameters.

  4. 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. PMID:24679405

  5. 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. PMID:27038376

  6. Virilization of the Broad Line Region in Active Galactic Nuclei—connection between shifts and widths of broad emission lines

    NASA Astrophysics Data System (ADS)

    Jonić, S.; Kovačević-Dojčinović, J.; Ilić, D.; Popović, L. Č.

    2016-03-01

    We investigate the virilization of the emission lines {Hβ } and Mg II in the sample of 287 Type 1 Active Galactic Nuclei taken from the Sloan Digital Sky Survey database. We explore the connections between the intrinsic line shifts and full widths at different levels of maximal intensity. We found that: (i) {Hβ} seems to be a good virial estimator of black hole masses, and an intrinsic redshift of {Hβ} is dominantly caused by the gravitational effect, (ii) there is an anti-correlation between the redshift and width of the wings of the Mg II line, (iii) the broad Mg II line can be used as virial estimator only at 50 % of the maximal intensity, while the widths and intrinsic shifts of the line wings cannot be used for this purpose.

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

    PubMed

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

    2016-01-01

    Twelve sodium-activated potassium channel (KCNT1, Slack) genetic mutants have been identified from severe early-onset epilepsy patients. The changes in biophysical properties of these mutants and the underlying mechanisms causing disease remain elusive. Here, we report that seven of the 12 mutations increase, whereas one mutation decreases, the channel's sodium sensitivity. Two of the mutants exhibit channel over-activity only when the intracellular Na(+) ([Na(+)]i) concentration is ∼80 mM. In contrast, single-channel data reveal that all 12 mutants increase the maximal open probability (Po). We conclude that these mutant channels lead to channel over-activity predominantly by increasing the ability of sodium binding to activate the channel, which is indicated by its maximal Po. The sodium sensitivity of these epilepsy causing mutants probably determines the [Na(+)]i concentration at which these mutants exert their pathological effects. PMID:26725113

  8. Dendritic NMDA receptors activate axonal calcium channels

    PubMed Central

    Christie, Jason M.; Jahr, Craig E.

    2008-01-01

    Summary NMDA receptor (NMDAR) activation can alter synaptic strength by regulating transmitter release from a variety of neurons in the CNS. As NMDARs are permeable to Ca2+ and monovalent cations, they could alter release directly by increasing presynaptic Ca2+ or indirectly by axonal depolarization sufficient to activate voltage-sensitive Ca2+ channels (VSCCs). Using two-photon microscopy to measure Ca2+ excursions, we found that somatic depolarization or focal activation of dendritic NMDARs elicited small Ca2+ transients in axon varicosities of cerebellar stellate cell interneurons. These axonal transients resulted from Ca2+ entry through VSCCs that were opened by the electrotonic spread of the NMDAR-mediated depolarization elicited in the dendrites. In contrast, we were unable to detect direct activation of NMDARs on axons indicating an exclusive somatodendritic expression of functional NMDARs. In cerebellar stellate cells, dendritic NMDAR activation masquerades as a presynaptic phenomenon and may influence Ca2+-dependent forms of presynaptic plasticity and release. PMID:18957221

  9. Headwater Stream Temperature Response to Forest Harvesting in Coastal British Columbia, Canada: Influences of Riparian Buffer Width, Channel Morphology and Weather

    NASA Astrophysics Data System (ADS)

    Moore, R.; Gomi, T.; Dhakal, A.

    2003-12-01

    Forest harvesting can influence stream temperature regimes, and the potentially deleterious impacts of higher temperatures on salmonids and other species have generated significant debate. One common approach to protecting streams is to leave a riparian buffer to provide shade. However, little information has been collected on the effectiveness of different buffer widths. We report the results of a 6-year field experiment to evaluate the effects of different riparian buffer widths on stream and riparian ecosystems, including stream temperature response, in headwater streams in coastal British Columbia. The experiment included 13 streams, with at least three being assigned to each of four treatments, including no harvesting (80 yr-old second growth conifer riparian forest), clear-cut harvesting with 10 m and 30 m riparian buffers, and clear-cut harvesting with no buffer. Regression analysis was used to calibrate the pre-harvest data for each treatment stream with one of the control streams, to provide a basis for estimating post-harvest treatment effects. Autoregressive and heteroskedastic errors were included in the regression model, because stream temperature exhibited serial correlation and the error variance increased with stream temperature. Temperature response was substantial in the clearcut treatments with no buffers, with maximum temperatures increasing by up to 8 degrees C. The magnitude of temperature response amongst the no-buffer treatments varied with channel morphology, particularly in relation to bank shading and stream depth. The treatment effect for daily maximum water temperature increased with decreasing flow and increasing maximum air temperature on the current day, and also exhibited significant autocorrelation, indicating that the sequence of daily weather conditions can influence the magnitude of temperature response.

  10. 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

  11. Implications of Lyman-alpha equivalent widths in active galactic nuclei

    NASA Technical Reports Server (NTRS)

    Shields, Joseph C.; Ferland, Gary J.

    1993-01-01

    Results of photoionization calculations that examine the sensitivity of Ly-alpha emission to parameters describing the broadline clouds and incident radiation field are reported. The case of the Seyfert I galaxy NGC 5548 is considered, and good consistency is found with observed properties of the Ly-alpha zone for a cloud density of about 10 exp 11/cu cm and ratio of ionizing photon to hydrogen density near 0.2. Estimation of the cloud covering factor depends on the extrapolation of the EUV continuum. EUV cutoffs at 20 and 60 eV correspond to a range of covering factors between 0.8 and 0.5, which is larger than typically assumed in the past for luminous Seyferts. Equivalent width observations of Fairall 9 show that its continuum cannot be as soft as suggested by previous line ratio analyses and imply that its EUV continuum persists to 20 eV or more before cutting off. A method by which variability measurements can be combined with the present calculations to measure the distances to AGN, and hence the Hubble constant, is outlined.

  12. Structure of Thermally Activated TRP Channels

    PubMed Central

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

    2015-01-01

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

  13. 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

  14. 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

  15. 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.

  16. 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

  17. 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. PMID:26823461

  18. 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

  19. 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

  20. Voltage is a partial activator of rat thermosensitive TRP channels

    PubMed Central

    Matta, José A; Ahern, Gerard P

    2007-01-01

    TRPV1 and TRPM8 are sensory nerve ion channels activated by heating and cooling, respectively. A variety of physical and chemical stimuli activate these receptors in a synergistic manner but the underlying mechanisms are unclear. Both channels are voltage sensitive, and temperature and ligands modulate this voltage dependence. Thus, a voltage-sensing mechanism has become an attractive model to explain the generalized gating of these and other thermo-sensitive TRP channels. We show here using whole-cell and single channel measurements that voltage produces only a partial activation of TRPV1 and TRPM8. At room temperature (20–25°C) membrane depolarization evokes responses that saturate at ∼50–60% of the maximum open probability. Furthermore, high concentrations of capsaicin (10 μm), resiniferatoxin (5 μm) and menthol (6 mm) reveal voltage-independent gating. Similarly, other modes of TRPV1 regulation including heat, protein kinase C-dependent phosphorylation, and protons enhance both the efficacy and sensitivity of voltage activation. In contrast, the TRPV1 antagonist capsazepine produces the opposite effects. These data can be explained by an allosteric model in which voltage, temperature, agonists and inverse agonists are independently coupled, either positively or negatively, to channel gating. Thus, voltage acts separately but in concert with other stimuli to regulate channel activation, and, therefore, a voltage-sensitive mechanism is unlikely to represent a final, gating mechanism for these channels. PMID:17932142

  1. 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

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

    PubMed Central

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

    2001-01-01

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

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

    PubMed Central

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

    2001-01-01

    The genetic disease cystic fibrosis is caused by mutation of the gene coding for the cystic fibrosis transmembrane conductance regulator (CFTR). Controversial studies reported regulation of the epithelial sodium channel (ENaC) by CFTR. We found that uptake of 22Na+ through ENaC is modulated by activation of CFTR in oocytes, coexpressing CFTR and ENaC, depending on extracellular chloride concentration. Furthermore we found that the effect of CFTR activation could be mimicked by other chloride channels. Voltage– and patch–clamp measurements, however, showed neither stimulation nor inhibition of ENaC-mediated conductance by activated CFTR. We conclude that the observed modulation of 22Na+ uptake by activated CFTR is due to the effect of CFTR-mediated chloride conductance on the membrane potential. These findings argue against the notion of a specific influence of CFTR on ENaC and emphasize the chloride channel function of CFTR. PMID:11266369

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

    PubMed

    Nagel, G; Szellas, T; Riordan, J R; Friedrich, T; Hartung, K

    2001-03-01

    The genetic disease cystic fibrosis is caused by mutation of the gene coding for the cystic fibrosis transmembrane conductance regulator (CFTR). Controversial studies reported regulation of the epithelial sodium channel (ENaC) by CFTR. We found that uptake of (22)Na(+) through ENaC is modulated by activation of CFTR in oocytes, coexpressing CFTR and ENaC, depending on extracellular chloride concentration. Furthermore we found that the effect of CFTR activation could be mimicked by other chloride channels. Voltage- and patch-clamp measurements, however, showed neither stimulation nor inhibition of ENaC-mediated conductance by activated CFTR. We conclude that the observed modulation of (22)Na(+) uptake by activated CFTR is due to the effect of CFTR-mediated chloride conductance on the membrane potential. These findings argue against the notion of a specific influence of CFTR on ENaC and emphasize the chloride channel function of CFTR. PMID:11266369

  5. Curcumin stimulates cystic fibrosis transmembrane conductance regulator Cl- channel activity.

    PubMed

    Berger, Allan L; Randak, Christoph O; Ostedgaard, Lynda S; Karp, Philip H; Vermeer, Daniel W; Welsh, Michael J

    2005-02-18

    Compounds that enhance either the function or biosynthetic processing of the cystic fibrosis transmembrane conductance regulator (CFTR) Cl(-) channel may be of value in developing new treatments for cystic fibrosis (CF). Previous studies suggested that the herbal extract curcumin might affect the processing of a common CF mutant, CFTR-DeltaF508. Here, we tested the hypothesis that curcumin influences channel function. Curcumin increased CFTR channel activity in excised, inside-out membrane patches by reducing channel closed time and prolonging the time channels remained open. Stimulation was dose-dependent, reversible, and greater than that observed with genistein, another compound that stimulates CFTR. Curcumin-dependent stimulation required phosphorylated channels and the presence of ATP. We found that curcumin increased the activity of both wild-type and DeltaF508 channels. Adding curcumin also increased Cl(-) transport in differentiated non-CF airway epithelia but not in CF epithelia. These results suggest that curcumin may directly stimulate CFTR Cl(-) channels. PMID:15582996

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

    PubMed Central

    Cho, M H; Spalding, E P

    1996-01-01

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

  7. 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.

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

    PubMed Central

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

    1994-01-01

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

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

    PubMed

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

    1994-09-13

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

  10. 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

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

    PubMed Central

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

    2015-01-01

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

  12. 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

  13. a Linear Model for Meandering Rivers with Arbitrarily Varying Width

    NASA Astrophysics Data System (ADS)

    Frascati, A.; Lanzoni, S.

    2011-12-01

    Alluvial rivers usually exhibit quite complex planforms, characterized by a wide variety of alternating bends, that have attracted the interest of a large number of researchers. Much less attention has been paid to another striking feature observed in alluvial rivers, namely the relatively regular spatial variations attained by the channel width. Actively meandering channels, in fact, generally undergo spatial oscillations systematically correlated with channel curvature, with cross sections wider at bends than at crossings. Some other streams have been observed to exhibit irregular width variations. Conversely, rivers flowing in highly vegetated flood plains, i.e. canaliform rivers, may exhibit an opposite behavior, owing to the combined effects of bank erodibility and floodplain depositional processes which, in turn, are strictly linked to vegetation cover. Similarly to streamline curvatures induced by bends, the presence of along channel width variations may have remarkable effects on the flow field and sediment dynamics and, thereby, on the equilibrium river bed configuration. In particular, spatial distribution of channel curvature typically determines the formation of a rhythmic bar-pool pattern in the channel bed strictly associated with the development of river meanders. Channel width variations are on the contrary characterized by a sequence of narrowing, yielding a central scour, alternated to the downstream development of a widening associated with the formation of a central bar. Here we present a morphodynamic model that predict at a linear level the spatial distribution of the flow field and the equilibrium bed configuration of an alluvial river characterized by arbitrary along channel distributions of both the channel axis curvature and the channel width. The mathematical model is averaged over the depth and describes the steady, non-uniform flow and sediment transport in sinuous channels with a noncohesive bed. The governing two-dimensional equations

  14. Hydrostatic and osmotic pressure activated channel in plant vacuole

    PubMed Central

    Alexandre, Joel; Lassalles, Jean-Paul

    1991-01-01

    The vacuolar membrane of red beet vacuoles contains a channel which was not gated by voltage or Ca2+ ions. Its unit conductance was 20 pS in 200 mM symmetrical KCl solutions. It was stretch activated: the conductance remained constant but the probability of opening was increased by suction or pressure applied to a membrane patch. A 1.5-kNm-2 suction applied to isolated patches or a 0.08-kNm-2 pressure applied to a 45-μm diameter vacuole induced an e-fold change in the mean current. A 75% inhibition of the channel current was obtained with 10 μM Gd3+ on the cytoplasmic side. The channel was more permeable for K+ than for Cl- (PK/PCl ∼ 3). A possible clustering for this channel was suggested by the recordings of the patch current. The channel properties were not significantly affected by a change in sorbitol osmolality in the solutions under isoosmotic conditions, between 0.6 and 1 mol/kg sorbitol. However, the channel was very sensitive to an osmotic gradient. A 0.2-mol/kg sorbitol gradient induced a two-fold increase in unit conductance and a thirty-fold increase in the mean patch current of the channel. A current was measured, when the osmotic gradient was the only driving force applied to the vacuolar membrane. The hydrostatic and osmotic pressure (HOP) activated channel described in this paper could be gated in vivo condition by a change in osmolality, without the need of a change in the turgor pressure in the cell. The HOP channel represents a possible example of an osmoreceptor for plant cells. PMID:19431814

  15. Cyclic nucleotide-activated channels in carp olfactory receptor cells.

    PubMed

    Kolesnikov, S S; Kosolapov, A V

    1993-07-25

    When applied from the cytoplasmic side, cyclic 3',5'-adenosine and guanosine monophosphates reversibly increased the ion permeability of inside-out patches of carp olfactory neuron plasma membrane. The cAMP (cGMP)-induced permeability via cAMP (cGMP) concentration was fitted by Hill's equation with the exponents of 1.07 +/- 0.15 (1.12 +/- 0.05) and EC50 = 1.3 +/- 0.6 microM (0.9 +/- 0.3 microM). Substitution of NaCl in the bathing solution by chlorides of other alkali metals resulted in a slight shift of reversal potential of the cyclic nucleotide-dependent (CN) current, which indicates a weak selectivity of the channels. Permeability coefficients calculated by Goldman-Hodgkin-Katz's equation corresponded to the following relation: PNa/PK/PLi/PRb/PCs = 1:0.98:0.94:0.70:0.61. Ca2+ and Mg2+ in physiological concentrations blocked the channels activated by cyclic nucleotides (CN-channels). In the absence of divalent cations the conductance of single CN-channels was equal to 51 +/- 9 pS in 100 mM NaCl solution. Channel density did not exceed 1 micron-2. The maximal open state probability of the channel (Po) tended towards 1.0 at a high concentration of cAMP or cGMP. Dichlorobenzamil decreased Po without changing the single CN-channel' conductance. CN-channels exhibited burst activity. Mean open and closed times as well as the burst duration depended on agonist concentration. A kinetic model with four states (an inactivated, a closed and two open ones) is suggested to explain the regularities of CN-channel gating and dose-response relations. PMID:8334139

  16. Novel Activation of Voltage-gated K+ Channels by Sevoflurane*

    PubMed Central

    Barber, Annika F.; Liang, Qiansheng; Covarrubias, Manuel

    2012-01-01

    Voltage-gated ion channels are modulated by halogenated inhaled general anesthetics, but the underlying molecular mechanisms are not understood. Alkanols and halogenated inhaled anesthetics such as halothane and isoflurane inhibit the archetypical voltage-gated Kv3 channel homolog K-Shaw2 by stabilizing the resting/closed states. By contrast, sevoflurane, a more heavily fluorinated ether commonly used in general anesthesia, specifically activates K-Shaw2 currents at relevant concentrations (0.05–1 mm) in a rapid and reversible manner. The concentration dependence of this modulation is consistent with the presence of high and low affinity interactions (KD = 0.06 and 4 mm, respectively). Sevoflurane (<1 mm) induces a negative shift in the conductance-voltage relation and increases the maximum conductance. Furthermore, suggesting possible roles in general anesthesia, mammalian Kv1.2 and Kv1.5 channels display similar changes. Quantitative description of the observations by an economical allosteric model indicates that sevoflurane binding favors activation gating and eliminates an unstable inactivated state outside the activation pathway. This study casts light on the mechanism of the novel sevoflurane-dependent activation of Kv channels, which helps explain how closely related inhaled anesthetics achieve specific actions and suggests strategies to develop novel Kv channel activators. PMID:23038249

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

    PubMed

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

    2010-05-01

    Cell-attached patches from isolated epithelial cells from larval bullfrog skin revealed a cation channel that was activated by applying suction (-1 kPa to -4.5 kPa) to the pipette. Activation was characterized by an initial large current spike that rapidly attenuated to a stable value and showed a variable pattern of opening and closing with continuing suction. Current-voltage plots demonstrated linear or inward rectification and single channel conductances of 44-56 pS with NaCl or KCl Ringer's solution as the pipette solution, and a reversal potential (-V(p)) of 20-40 mV. The conductance was markedly reduced with N-methyl-D-glucamide (NMDG)-Cl Ringer's solution in the pipette. Neither amiloride nor ATP, which are known to stimulate an apical cation channel in Ussing chamber preparations of larval frog skin, produced channel activation nor did these compounds affect the response to suction. Stretch activation was not affected by varying the pipette concentrations of Ca(2+) between 0 mmol l(-1) and 4 mmol l(-1) or by varying pH between 6.8 and 8.0. However, conductance was reduced with 4 mmol l(-1) Ca(2+). Western blot analysis of membrane homogenates from larval bullfrog and larval toad skin identified proteins that were immunoreactive with mammalian TRPC1 and TRPC5 (TRPC, canonical transient receptor potential channel) antibodies while homogenates of skin from newly metamorphosed bullfrogs were positive for TRPC1 and TRPC3/6/7 antibodies. The electrophysiological response of larval bullfrog skin resembles that of a stretch-activated cation channel characterized in Xenopus oocytes and proposed to be TRPC1. These results indicate this channel persists in all life stages of anurans and that TRP isoforms may be important for sensory functions of their skin. PMID:20435829

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

    PubMed Central

    Félétou, Michel

    2009-01-01

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

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

    PubMed

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

    2015-04-25

    Cystic fibrosis transmembrane conductance regulator (CFTR), a cAMP-dependent chloride channel, plays key roles in fluid secretion in serous epithelial cells. Previously, we identified two polymethoxylated flavonoids, 3',4',5,5',6,7-hexamethoxyflavone (HMF) and 5-hydroxy-6,7,3',4'-tetramethoxyflavone (HTF) which could potentiate CFTR chloride channel activities. The present study was aimed to investigate the potentiation effects of HMF and HTF on CFTR Cl(-) channel activities by using a cell-based fluorescence assay and the short circuit Ussing chamber assay. The results of cell-based fluorescence assay showed that both HMF and HTF could dose-dependently potentiate CFTR Cl(-) channel activities in rapid and reversible ways, and the activations could be reversed by the CFTR blocker CFTRinh-172. Notably, HMF showed the highest affinity (EC50 = 2 μmol/L) to CFTR protein among the flavonoid CFTR activators identified so far. The activation of CFTR by HMF or HTF was forskolin (FSK) dependent. Both compounds showed additive effect with FSK and 3-Isobutyl-1-methylx (IBMX) in the activation of CFTR, while had no additive effect with genistein (GEN). In ex vivo studies, HMF and HTF could stimulate transepithelial Cl(-) secretion in rat colonic mucosa and enhance fluid secretion in mouse trachea submucosal glands. These results suggest that HMF and HTF may potentiate CFTR Cl(-) channel activities through both elevation of cAMP level and binding to CFTR protein pathways. The results provide new clues in elucidating structure and activity relationship of flavonoid CFTR activators. HMF might be developed as a new drug in the therapy of CFTR-related diseases such as bronchiectasis and habitual constipation. PMID:25896054

  20. 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

  1. 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

  2. Use of Small Fluorescent Molecules to Monitor Channel Activity

    NASA Astrophysics Data System (ADS)

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

    2001-03-01

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

  3. Neuronal modulation of calcium channel activity in cultured rat astrocytes

    SciTech Connect

    Corvalan, V.; Cole, R.; De Vellis, J.; Hagiwara, Susumu )

    1990-06-01

    The patch-clamp technique was used to study whether cocultivation of neurons and astrocytes modulates the expression of calcium channel activity in astrocytes. Whole-cell patch-clamp recordings from rat brain astrocytes cocultured with rat embryonic neurons revealed two types of voltage-dependent inward currents carried by Ca{sup 2+} and blocked by either Cd{sup 2+} or Co{sup 2+} that otherwise were not detected in purified astrocytes. This expression of calcium channel activity in astrocytes was neuron dependent and was not observed when astrocytes were cocultured with purified oligodendrocytes.

  4. 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.

  5. Experimental demonstration of line-width modulation in plasmonic lithography using a solid immersion lens-based active nano-gap control

    SciTech Connect

    Lee, Won-Sup; Kim, Taeseob; Choi, Guk-Jong; Lim, Geon; Joe, Hang-Eun; Gang, Myeong-Gu; Min, Byung-Kwon; Park, No-Cheol; Moon, Hyungbae; Kim, Do-Hyung; Park, Young-Pil

    2015-02-02

    Plasmonic lithography has been used in nanofabrication because of its utility beyond the diffraction limit. The resolution of plasmonic lithography depends on the nano-gap between the nanoaperture and the photoresist surface—changing the gap distance can modulate the line-width of the pattern. In this letter, we demonstrate solid-immersion lens based active non-contact plasmonic lithography, applying a range of gap conditions to modulate the line-width of the pattern. Using a solid-immersion lens-based near-field control system, the nano-gap between the exit surface of the nanoaperture and the media can be actively modulated and maintained to within a few nanometers. The line-widths of the recorded patterns using 15- and 5-nm gaps were 47 and 19.5 nm, respectively, which matched closely the calculated full-width at half-maximum. From these results, we conclude that changing the nano-gap within a solid-immersion lens-based plasmonic head results in varying line-width patterns.

  6. 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

  7. 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.

  8. 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. PMID:25778101

  9. Patterns and Processes of Width Adjustment to Increased Streamflows in Semi-Alluvial Rivers

    NASA Astrophysics Data System (ADS)

    Kelly, S. A.; Belmont, P.

    2015-12-01

    While it is understood that river channel width is determined by fluxes of water and sediment, predictive models of channel width, and especially changes in width under non-stationary conditions, have proven elusive. Classic hydraulic geometry relations commonly used in numerical models and channel design typically scale width as a power law function of discharge, without consideration of bank properties. This study investigates the role of bank material in determining spatial and temporal variability in channel width and widening rates for semi-alluvial rivers that have experienced increases in flow. The 45,000 km2 Minnesota River Basin contains many semi-alluvial rivers that have been rapidly incising into fine-grained glacial deposits over the last 13,400 years in response to a catastrophic base level drop. Large, recent increases in streamflows have caused significant channel widening and migration, exacerbated erosion of channel (alluvial) banks and (consolidated till) bluffs, and dramatically increased sediment supply. Here we leverage multiple decades of aerial photos, repeat lidar surveys, Structure from Motion photogrammetry and sediment gaging to examine past, and predict future, changes in channel width. We use empirical observations and a simple model to examine whether semi-alluvial channels tend toward a single, or multiple, equilibrium channel width(s). Preliminary results suggest that under stationary hydrologic conditions (1930s - 1970s) channel width was relatively consistent among reaches underlain by alluvium versus consolidated till. Since the late 1970s the study area has undergone profound hydrologic changes, with geomorphically-active flows nearly doubling in magnitude. Alluvial reaches widened relatively quickly in response to the increase in flows, whereas reaches underlain by till have not seen the same amount of widening. Aerial lidar-based geomorphic change detection between 2005 - 2012 records channel width changes in response to an

  10. Phosphatidylinositol-3-kinase regulates mast cell ion channel activity.

    PubMed

    Lam, Rebecca S; Shumilina, Ekaterina; Matzner, Nicole; Zemtsova, Irina M; Sobiesiak, Malgorzata; Lang, Camelia; Felder, Edward; Dietl, Paul; Huber, Stephan M; Lang, Florian

    2008-01-01

    Stimulation of the mast cell IgE-receptor (FcepsilonRI) by antigen leads to stimulation of Ca(2+) entry with subsequent mast cell degranulation and release of inflammatory mediators. Ca(2+) further activates Ca(2+)-activated K(+) channels, which in turn provide the electrical driving force for Ca(2+) entry. Since phosphatidylinositol (PI)-3-kinase has previously been shown to be required for mast cell activation and degranulation, we explored, whether mast cell Ca(2+) and Ca(2+)-activated K(+) channels may be sensitive to PI3-kinase activity. Whole-cell patch clamp experiments and Fura-2 fluorescence measurements for determination of cytosolic Ca(2+) concentration were performed in mouse bone marrow-derived mast cells either treated or untreated with the PI3-kinase inhibitors LY-294002 (10 muM) and wortmannin (100 nM). Antigen-stimulated Ca(2+) entry but not Ca(2+) release from the intracellular stores was dramatically reduced upon PI3-kinase inhibition. Ca(2+) entry was further inhibited by TRPV blocker ruthenium red (10 muM). Ca(2+) entry following readdition after Ca(+)-store depletion with thapsigargin was again decreased by LY-294002, pointing to inhibition of store-operated channels (SOCs). Moreover, inhibition of PI3-kinase abrogated IgE-stimulated, but not ionomycin-induced stimulation of Ca(2+)-activated K(+) channels. These observations disclose PI3-kinase-dependent regulation of Ca(2+) entry and Ca(2+)-activated K(+)-channels, which in turn participate in triggering mast cell degranulation. PMID:18769043

  11. TRPV3 channels mediate strontium-induced mouse egg activation

    PubMed Central

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

    2014-01-01

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

  12. 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

  13. Measurement of the w and z cross sections in the electron channel for p anti-p collisions at s**(1/2) = 1.96 TeV and extraction of the w total width from the ratio

    SciTech Connect

    Gardner, John Michael; /Kansas U.

    2005-01-01

    This dissertation presents measurements of the inclusive production cross sections for W and Z gauge bosons decaying through the electron channel with p{bar p} collisions at a center-of-mass energy of 1.96 TeV. The ratio of these cross sections is then used to extract the W total width. The Standard Model (SM) of electroweak and strong interactions is a collection of theories which together encompass what is currently known about the elementary particles that make up matter and the forces through which they interact. Experimentalists are constantly searching for violations of the Standard Model by making precision measurements of predicted interactions. The decay of the W boson is one such interaction. The rate of its decay is reflected in its width which is predicted to high precision using Standard Model-based calculations. Therefore, a high precision experimental width measurement would be very sensitive to any such violation. In principle the W and Z boson production cross sections could also be good Standard Model tests. However, a precise knowledge of integrated luminosity is required which is unfortunately difficult to obtain at the Tevatron. In fact, the W and Z cross section results can be used to obtain a more precise luminosity measurement. The data set consists of a total integrated luminosity of 177 pb{sup -1} collected from September 2002 to September 2003 using the D0 detector at Fermilab.

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

    PubMed Central

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

    2013-01-01

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

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

    PubMed Central

    Chambers, James J.; Kramer, Richard H.

    2009-01-01

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

  16. Statistical Hauser-Feshbach theory with width-fluctuation correction including direct reaction channels for neutron-induced reactions at low energies

    NASA Astrophysics Data System (ADS)

    Kawano, T.; Capote, R.; Hilaire, S.; Chau Huu-Tai, P.

    2016-07-01

    A model to calculate particle-induced reaction cross sections with statistical Hauser-Feshbach theory including direct reactions is given. The energy average of the scattering matrix from the coupled-channels optical model is diagonalized by the transformation proposed by Engelbrecht and Weidenmüller [C. A. Engelbrecht and H. A. Weidenmüller, Phys. Rev. C 8, 859 (1973), 10.1103/PhysRevC.8.859]. The ensemble average of S -matrix elements in the diagonalized channel space is approximated by a model of Moldauer [P. A. Moldauer, Phys. Rev. C 12, 744 (1975), 10.1103/PhysRevC.12.744] using the newly parametrized channel degree-of-freedom νa to better describe the Gaussian orthogonal ensemble (GOE) reference calculations. The Moldauer approximation is confirmed by a Monte Carlo study using a randomly generated S matrix, as well as the GOE threefold integration formula. The method proposed is applied to the 238U(n ,n' ) cross-section calculation in the fast-energy range, showing an enhancement in the inelastic scattering cross sections.

  17. 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.

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

    PubMed

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

    2015-11-01

    Amphetamine (AMPH) and its more potent enantiomer S(+)AMPH are psychostimulants used therapeutically to treat attention deficit hyperactivity disorder and have significant abuse liability. AMPH is a dopamine transporter (DAT) substrate that inhibits dopamine (DA) uptake and is implicated in DA release. Furthermore, AMPH activates ionic currents through DAT that modify cell excitability presumably by modulating voltage-gated channel activity. Indeed, several studies suggest that monoamine transporter-induced depolarization opens voltage-gated Ca(2+) channels (CaV), which would constitute an additional AMPH mechanism of action. In this study we co-express human DAT (hDAT) with Ca(2+) channels that have decreasing sensitivity to membrane depolarization (CaV1.3, CaV1.2 or CaV2.2). Although S(+)AMPH is more potent than DA in transport-competition assays and inward-current generation, at saturating concentrations both substrates indirectly activate voltage-gated L-type Ca(2+) channels (CaV1.3 and CaV1.2) but not the N-type Ca(2+) channel (CaV2.2). Furthermore, the potency to achieve hDAT-CaV electrical coupling is dominated by the substrate affinity on hDAT, with negligible influence of L-type channel voltage sensitivity. In contrast, the maximal coupling-strength (defined as Ca(2+) signal change per unit hDAT current) is influenced by CaV voltage sensitivity, which is greater in CaV1.3- than in CaV1.2-expressing cells. Moreover, relative to DA, S(+)AMPH showed greater coupling-strength at concentrations that induced relatively small hDAT-mediated currents. Therefore S(+)AMPH is not only more potent than DA at inducing hDAT-mediated L-type Ca(2+) channel currents but is a better depolarizing agent since it produces tighter electrical coupling between hDAT-mediated depolarization and L-type Ca(2+) channel activation. PMID:26162812

  19. Activation of peripheral KCNQ channels attenuates inflammatory pain

    PubMed Central

    2014-01-01

    Background Refractory chronic pain dramatically reduces the quality of life of patients. Existing drugs cannot fully achieve effective chronic pain control because of their lower efficacy and/or accompanying side effects. Voltage-gated potassium channels (KCNQ) openers have demonstrated their analgesic effect in preclinical and clinical studies, and are thus considered to be a potential therapeutic target as analgesics. However, these drugs exhibit a narrow therapeutic window due to their imposed central nerve system (CNS) side effects. To clarify the analgesic effect by peripheral KCNQ channel activation, we investigated whether the analgesic effect of the KCNQ channel opener, retigabine, is inhibited by intracerebroventricular (i.c.v.) administration of the KCNQ channel blocker, 10, 10-bis (4-Pyridinylmethyl)-9(10H) -anthracenone dihydrochloride (XE-991) in rats. Results Oral administration (p.o.) of retigabine showed an anticonvulsant effect on maximal electronic seizures and an analgesic effect on complete Freund’s adjuvant-induced thermal hyperalgesia. However, impaired motor coordination and reduced exploratory behavior were also observed at the analgesic doses of retigabine. Administration (i.c.v.) of XE-991 reversed the retigabine-induced anticonvulsant effect, impaired motor coordination and reduced exploratory behavior but not the analgesic effect. Moreover, intraplantar administration of retigabine or an additional KCNQ channel opener, N-(6-Chloro-pyridin-3-yl)-3,4-difluoro-benzamide (ICA-27243), inhibited formalin-induced nociceptive behavior. Conclusions Our findings suggest that the peripheral sensory neuron is the main target for KCNQ channel openers to induce analgesia. Therefore, peripheral KCNQ channel openers that do not penetrate the CNS may be suitable analgesic drugs as they would prevent CNS side effects. PMID:24555569

  20. 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.

  1. 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. PMID:24489780

  2. 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

  3. 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

  4. 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

  5. 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.

  6. 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

  7. Structure and activity of the acid-sensing ion channels

    PubMed Central

    Sherwood, Thomas W.; Frey, Erin N.

    2012-01-01

    The acid-sensing ion channels (ASICs) are a family of proton-sensing channels expressed throughout the nervous system. Their activity is linked to a variety of complex behaviors including fear, anxiety, pain, depression, learning, and memory. ASICs have also been implicated in neuronal degeneration accompanying ischemia and multiple sclerosis. As a whole, ASICs represent novel therapeutic targets for several clinically important disorders. An understanding of the correlation between ASIC structure and function will help to elucidate their mechanism of action and identify potential therapeutics that specifically target these ion channels. Despite the seemingly simple nature of proton binding, multiple studies have shown that proton-dependent gating of ASICs is quite complex, leading to activation and desensitization through distinct structural components. This review will focus on the structural aspects of ASIC gating in response to both protons and the newly discovered activators GMQ and MitTx. ASIC modulatory compounds and their action on proton-dependent gating will also be discussed. This review is dedicated to the memory of Dale Benos, who made a substantial contribution to our understanding of ASIC activity. PMID:22843794

  8. 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).

  9. 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.

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

    PubMed

    Mahaut-Smith, M P; Mason, M J

    1991-08-01

    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

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

    PubMed Central

    Qu, Zhiqiang; Hartzell, H. Criss

    2000-01-01

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

  12. 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

  13. 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

  14. Tissue kallikrein activation of the epithelial Na channel

    PubMed Central

    Patel, Ankit B.; Chao, Julie

    2012-01-01

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

  15. Cognitive recovery by chronic activation of the large-conductance calcium-activated potassium channel in a mouse model of Alzheimer's disease.

    PubMed

    Wang, Li; Kang, Huicong; Li, Yongzhi; Shui, Yuan; Yamamoto, Ryo; Sugai, Tokio; Kato, Nobuo

    2015-05-01

    We previously showed that activity of the large conductance calcium-activated potassium (Big-K; BK) channels is suppressed in 3xTg Alzheimer disease (AD) model mice. However, its behavioral significance is not known. In the present report, ventricular injection of the BK channel activator isopimaric acid (ISO) was conducted to examine whether BK channel activation ameliorates cognition in 3xTg mice. The novel object recognition (NOR) test revealed that chronic injection of ISO improved non-spatial memory in 3xTg mice. In the Morris water maze, the probe test demonstrated an improved spatial memory after ISO injection. Electrophysiological underpinnings of the ISO effect were then examined in slices obtained from the mice after behavior. At hippocampal CA1 synapses, the basic synaptic transmission was abnormally elevated and long-term potentiation (LTP) was partially suppressed in 3xTg mice. These were both recovered by ISO treatment. We then confirmed suppressed BK channel activity in 3xTg mice by measuring the half-width of evoked action potentials. This was also recovered by ISO treatment. We previously showed that the recovery of BK channel activity accompanies reduction of neuronal excitability in pyramidal cells. Here again, pyramidal cell excitability, as assessed by calculating the frequency of evoked spikes, was elevated in the 3xTg mouse and was normalized by ISO. ELISA experiments demonstrated an ISO-induced reduction of Aβ1-42 content in hippocampal tissue in 3xTg mice. The present study thus suggests a potential therapeutic utility of BK channel activators for AD. PMID:25577958

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

    PubMed Central

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

    2016-01-01

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

  17. Relaxin stimulates myometrial calcium-activated potassium channel activity via protein kinase A.

    PubMed

    Meera, P; Anwer, K; Monga, M; Oberti, C; Stefani, E; Toro, L; Sanborn, B M

    1995-08-01

    Relaxin, a hormone that is elevated during pregnancy, can suppress myometrial contractile activity. Ca(2+)-activated K+ channels (KCa) play a role in the modulation of uterine contractions and myometrial Ca2+ homeostasis and have been implicated in the control of smooth muscle excitability. We now show that relaxin stimulates KCa channels in cell-attached patches in a cell line derived from term pregnant human myometrium. This effect was prevented by the protein kinase A (PKA) antagonist, the Rp diastereomer of adenosine 3',5'-cyclic monophosphothioate (Rp-cAMPS). After patch excision, the channel was activated by PKA and inhibited by alkaline phosphatase. These data suggest that relaxin may promote myometrial quiescence in part by stimulation of KCa channels via a PKA-mediated mechanism. PMID:7653512

  18. 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

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

    PubMed

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

    2016-08-01

    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 Ca(2+) and ATP together to control life and death'. PMID:27377720

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

    PubMed Central

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

    2016-01-01

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

  1. Using fluvial channel morphology to obtain the neotectonic characteristics of the Liuchia fault, an important active structure in southwestern Taiwan

    NASA Astrophysics Data System (ADS)

    Shyu, J. H.; Du, K.

    2013-12-01

    The Liuchia fault in southwestern Taiwan has been considered as one of the major active faults in the active Taiwan orogen. It is identified by its clear geomorphic features, and forms a major geologic boundary of Taiwan's Western Foothills. No unanimous historical evidence for seismic activity of the Liuchia fault exists, thus the fault poses large earthquake hazard potentials for the populous southwestern Taiwan. Here we attempted to analyze the characteristics of the fault from fluvial channel morphology of the Kueichung River that flows across the fault. We also calculated actual river incision rates from the age of river terraces along the river to obtain the rock uplift rates of the hanging-wall block of the fault. We have obtained a detailed river long profile of the Kueichung River from surveys using RTK-GPS, and a channel width profile from actual field measurements using a Laser Rangefinder. The fluvial channel morphology of the Kueichung River appears to have been affected by active folding in the hanging-wall block of the Liuchia fault. Such active deformation pattern is also evident from river incision rate patterns. Combining these different datasets, we constructed a realistic model of the subsurface geometry of the Liuchia fault in southwestern Taiwan, and calculated the long-term slip rates of this important active structure in southwestern Taiwan.

  2. 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

  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. PMID:26609559

  4. 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

  5. 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

  6. 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.

  7. NSAIDs attenuate hyperalgesia induced by TRP channel activation.

    PubMed

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

    2016-03-01

    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

  8. 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

  9. Membrane stretch and cytoplasmic Ca2+ independently modulate stretch-activated BK channel activity.

    PubMed

    Zhao, Hu-Cheng; Agula, Hasi; Zhang, Wei; Wang, Fa; Sokabe, Masahiro; Li, Lu-Ming

    2010-11-16

    Large conductance Ca(2+)-activated K(+) (BK) channels are responsible for changes in chemical and physical signals such as Ca(2+), Mg(2+) and membrane potentials. Previously, we reported that a BK channel cloned from chick heart (SAKCaC) is activated by membrane stretch. Molecular cloning and subsequent functional characterization of SAKCaC have shown that both the membrane stretch and intracellular Ca(2+) signal allosterically regulate the channel activity via the linker of the gating ring complex. Here we investigate how these two gating principles interact with each other. We found that stretch force activated SAKCaC in the absence of cytoplasmic Ca(2+). Lack of Ca(2+) bowl (a calcium binding motif) in SAKCaC diminished the Ca(2+)-dependent activation, but the mechanosensitivity of channel was intact. We also found that the abrogation of STREX (a proposed mechanosensing apparatus) in SAKCaC abolished the mechanosensitivity without altering the Ca(2+) sensitivity of channels. These observations indicate that membrane stretch and intracellular Ca(2+) could independently modulate SAKCaC activity. PMID:20673577

  10. 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

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

    PubMed Central

    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. PMID:8700850

  12. 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.

  13. 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

  14. 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.

  15. 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.

  16. 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.

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

    PubMed

    Bock, Tobias; Stuart, Greg J

    2016-03-01

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

  18. Zero Additional Process, Local Charge Trap, Embedded Flash Memory with Drain-Side Assisted Erase Scheme Using Minimum Channel Length/Width Standard Complemental Metal-Oxide-Semiconductor Single Transistor Cell

    NASA Astrophysics Data System (ADS)

    Miyaji, Kousuke; Shinozuka, Yasuhiro; Takeuchi, Ken

    2012-04-01

    This paper proposes for the first time the completely complemental metal-oxide-semiconductor (CMOS) compatible embedded flash memory with the small cell size as well as the lowest process cost. The single transistor cell with the minimum channel length and width realizes the ideal smallest cell. The non-volatile memory operation is realized with locally injected electrons at the drain-edge by the hot electron injection. This paper also proposes the novel forward-bias assisted erase. The proposed memory is experimentally demonstrated with the 65 nm standard CMOS process without additional process or mask. The cell size is 10F2 with the 65 nm CMOS logic design rule. The excellent reliability such as 100-times program/erase endurance, 10-year data retention and high immunity to the read/program/erase disturb is also experimentally demonstrated. The proposed cell is the ideal candidate for the code-storage embedded non-volatile memories in system-on-chip and microcontroller unit.

  19. 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.

  20. Measurement of the W and Z cross sections in the electron channel for proton-antiproton collisions at center of mass energy = 1.96 TeV and extraction of the W total width from the ratio

    NASA Astrophysics Data System (ADS)

    Gardner, John Michael

    This dissertation presents measurements of the inclusive production cross sections for W and Z gauge bosons decaying through the electron channel with pp¯ collisions at a center-of-mass energy of 1.96 TeV. The ratio of these cross sections is then used to extract the W total width. The Standard Model (SM) of electroweak and strong interactions is a collection of theories which together encompass what is currently known about the elementary particles that make up matter and the forces through which they interact. Experimentalists are constantly searching for violations of the Standard Model by making precision measurements of predicted interactions. The decay of the W boson is one such interaction. The rate of its decay is reflected in its width which is predicted to high precision using Standard Model-based calculations. Therefore, a high precision experimental width measurement would be very sensitive to any such violation. In principle the W and Z boson production cross sections could also be good Standard Model tests. However, a precise knowledge of integrated luminosity is required which is unfortunately difficult to obtain at the Tevatron. In fact, the W and Z cross section results can be used to obtain a more precise luminosity measurement. The data set consists of a total integrated luminosity of 177 pb -1 collected from September 2002 to September 2003 using the DO detector at Fermilab. From this, 97757 W → enu and 7928 Z → ee candidates are found where at least one electron having a matched track is present within the central region of the detector (|etadet| < 1.05). The inclusive cross sections and their ratio are found to be s WxBW→e+/-n =2929+/-9 stat+/-57 sys+56-28 pdf+/-190 lumipb , s ZxBZ→e+e- =267.7+/-3.0 stat+/-4.8 sys+4.0-3.3 pdf+/-17.4 lumipb , and R≡ sWxBW→e +/-ns ZxBZ→e+ e-=10.94+/- 0.13stat +/-0.16sys +0.12-0.08 pdf. Sources of uncertainty arise from limited statistics (stat), systematic effects (sys), parton distribution

  1. Mechanisms of caffeine activation of single calcium-release channels of sheep cardiac sarcoplasmic reticulum.

    PubMed Central

    Sitsapesan, R; Williams, A J

    1990-01-01

    1. Calcium-release channels of sheep cardiac junctional sarcoplasmic reticulum were incorporated into planar phospholipid bilayers. Single-channel current fluctuations were recorded under voltage clamp conditions. 2. Channels incorporate into the bilayer with a fixed orientation and channel open probability is regulated by the calcium concentration at the cytosolic face of the membrane. 3. Addition of caffeine (0.5-2.0 mM) to the cytosolic side of the membrane increased the open probability of the calcium-activated calcium-release channel by increasing the frequency of opening without significant alteration to the durations of open events. This effect was observed at both 0.1 and 10 microM-activating cytosolic calcium. 4. Caffeine (0.5-2.0 mM) did not activate the channel at a subactivating cytosolic calcium concentration (80 pM). 5. At subactivating calcium concentrations, channels could be activated by higher concentrations of caffeine (greater than 5.0 mM) revealing a second, calcium-independent, mechanism for channel activation. Channel openings induced by these high concentrations of caffeine at subactivating calcium concentrations displayed different kinetics from those observed with calcium as the sole activating ligand or with combinations of calcium and low concentrations of caffeine. 6. Activation of channel opening by caffeine in the presence of calcium did not affect single-channel conductance. Channel openings produced by caffeine at subactivating cytosolic calcium concentrations had identical conductance and relative permeability to those seen on calcium activation. 7. Channels activated by caffeine at both activating and subactivating calcium concentrations were characteristically modified by ryanodine, Ruthenium Red, ATP and magnesium, implying that the same channel is involved under both conditions. PMID:2167363

  2. Adult feeding moths (Sphingidae) differ from non-adult feeding ones (Saturniidae) in activity-timing overlap and temporal niche width.

    PubMed

    de Camargo, Nícholas F; de Camargo, Willian R F; Corrêa, Danilo do C V; de Camargo, Amabílio J A; Vieira, Emerson M

    2016-02-01

    According to classic ecology, resource partitioning by segregation along at least one of the three main niche axes (time, food, and space) must take place for the coexistence of species with similar ecological requirements. We used nocturnal light traps to investigate the assemblage structuration of two moth families: Sphingidae (23 species) and Saturniidae (13 species). Because competition for food among adults potentially occurs only among sphingids, only for this family did we expect less overlap of diel activity patterns than expected by chance and also a greater temporal niche width compared to saturniids. Moreover, we expected a greater number of sphingid species pairs to differ in activity timing compared to saturniid pairs. We also hypothesized that in the case of a lack of temporal structuration, sphingids would be morphologically structured in relation to proboscis length. Contrary to what we expected, both families overlapped their activity patterns more than expected by chance alone and sphingid moths were not morphologically structured. Nevertheless, there were 173 significant pairwise differences in temporal activity between sphingids, contrasting with no interspecific differences between saturniids. Sphingid species also showed a wider temporal niche width than saturniids, as expected. Predation risk and abiotic factors may have caused the overall similarities in activity patterns for both families. The temporal niche seemed not to be determinant for the assemblage structuration of moths as a whole for either of the studied families, but segregation along the temporal niche axis of some potentially competing species pairs can be a relevant factor for the coexistence of nectar-feeding species. PMID:26104275

  3. 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

  4. 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.

  5. 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…

  6. Dual actions of procainamide on batrachotoxin-activated sodium channels: open channel block and prevention of inactivation.

    PubMed Central

    Zamponi, G W; Sui, X; Codding, P W; French, R J

    1993-01-01

    We have investigated the action of procainamide on batrachotoxin (BTX)-activated sodium channels from bovine heart and rat skeletal muscle. When applied to the intracellular side, procainamide induced rapid, open-channel block. We estimated rate constants using amplitude distribution analysis (Yellen, G. 1984. J. Gen. Physiol. 84:157). Membrane depolarization increased the blocking rate and slowed unblock. The rate constants were similar in both magnitude and voltage dependence for cardiac and skeletal muscle channels. Qualitatively, this block resembled the fast open-channel block by lidocaine (Zamponi, G. W., D. D. Doyle, and R. J. French. 1993. Biophys. J. 65:80), but procainamide was about sevenfold less potent. Molecular modeling suggests that the difference in potency between procainamide and lidocaine might arise from the relative orientation of their aromatic rings, or from differences in the structure of the aryl-amine link. For the cardiac channels, procainamide reduced the frequency of transitions to a long-lived closed state which shows features characteristic of inactivation (Zamponi, G. W., D. D. Doyle, and R. J. French. 1993. Biophys J. 65:91). Mean durations of kinetically identified closed states were not affected. The degree of fast block and of inhibition of the slow closures were correlated. Internally applied QX-314, a lidocaine derivative and also a fast blocker, produced a similar effect. Thus, drug binding to the fast blocking site appears to inhibit inactivation in BTX-activated cardiac channels. Images FIGURE 6 PMID:8312472

  7. Arachidonic acid activation of a new family of K+ channels in cultured rat neuronal cells.

    PubMed Central

    Kim, D; Sladek, C D; Aguado-Velasco, C; Mathiasen, J R

    1995-01-01

    1. The presence and properties of K+ channels activated by arachidonic acid were studied in neuronal cells cultured from the mesencephalic and hypothalamic areas of rat brain. 2. Arachidonic acid produced a concentration-dependent (5-50 microM) and reversible activation of whole-cell currents. 3. In excised membrane patches, arachidonic acid applied to the cytoplasmic or extracellular side of the membrane caused opening of three types of channels whose current-voltage relationships were slightly outwardly rectifying, inwardly rectifying and linear, and whose single channel slope conductances at +60 mV were 143, 45 and 52 pS, respectively. 4. All three currents were K+ selective and blocked by 2 mM Ba2+ but not by other K+ channel blockers such as tetraethylammonium chloride, 4-aminopyridine and quinidine. The outwardly and inwardly rectifying currents were slightly voltage dependent with higher channel activity at more depolarized potentials. 5. Arachidonic acid activated the K+ channels in cells treated with cyclo-oxygenase and lipoxygenase inhibitors (indomethacin and nordihydroguaiaretic acid), indicating that arachidonic acid itself can directly activate the channels. Alcohol and methyl ester derivatives of arachidonic acid failed to activate the K+ channels, indicating that the charged carboxyl group is important for activation. 6. Certain unsaturated fatty acids (linoleic, linolenic and docosahexaenoic acids), but not saturated fatty acids (myristic, palmitic, stearic acids), also reversibly activated all three types of K+ channel. 7. All three K+ channels were activated by pressure applied to the membrane (i.e. channels were stretch sensitive) with a half-maximal pressure of approximately 18 mmHg. The K+ channels were not blocked by 100 microM GdCl3. 8. A decrease in intracellular pH (over the range 5.6-7.2) caused a reversible, pH-dependent increase in channel activity whether the channel was initially activated by arachidonic acid or stretch. 9. Glutamate

  8. 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.

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

    PubMed

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

    2014-09-01

    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

  10. 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

  11. Ca(2+) influx through L-type Ca(2+) channels and transient receptor potential channels activates pathological hypertrophy signaling.

    PubMed

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

    2012-11-01

    Common cardiovascular diseases such as hypertension and myocardial infarction require that myocytes develop greater than normal force to maintain cardiac pump function. This requires increases in [Ca(2+)]. These diseases induce cardiac hypertrophy and increases in [Ca(2+)] are known to be an essential proximal signal for activation of hypertrophic genes. However, the source of "hypertrophic" [Ca(2+)] is not known and is the topic of this study. The role of Ca(2+) influx through L-type Ca(2+) channels (LTCC), T-type Ca(2+) channels (TTCC) and transient receptor potential (TRP) channels on the activation of calcineurin (Cn)-nuclear factor of activated T cells (NFAT) signaling and myocyte hypertrophy was studied. Neonatal rat ventricular myocytes (NRVMs) and adult feline ventricular myocytes (AFVMs) were infected with an adenovirus containing NFAT-GFP, to determine factors that could induce NFAT nuclear translocation. Four millimolar Ca(2+) or pacing induced NFAT nuclear translocation. This effect was blocked by Cn inhibitors. In NRVMs Nifedipine (Nif, LTCC antagonist) blocked high Ca(2+)-induced NFAT nuclear translocation while SKF-96365 (TRP channel antagonist) and Nickel (Ni, TTCC antagonist) were less effective. The relative potency of these antagonists against Ca(2+) induced NFAT nuclear translocation (Nif>SKF-96365>Ni) was similar to their effects on Ca(2+) transients and the LTCC current. Infection of NRVM with viruses containing TRP channels also activated NFAT-GFP nuclear translocation and caused myocyte hypertrophy. TRP effects were reduced by SKF-96365, but were more effectively antagonized by Nif. These experiments suggest that Ca(2+) influx through LTCCs is the primary source of Ca(2+) to activate Cn-NFAT signaling in NRVMs and AFVMs. While TRP channels cause hypertrophy, they appear to do so through a mechanism involving Ca(2+) entry via LTCCs. PMID:22921230

  12. Lithologic controls on valley width and strath terrace formation

    NASA Astrophysics Data System (ADS)

    Schanz, Sarah A.; Montgomery, David R.

    2016-04-01

    Valley width and the degree of bedrock river terrace development vary with lithology in the Willapa and Nehalem river basins, Pacific Northwest, USA. Here, we present field-based evidence for the mechanisms by which lithology controls floodplain width and bedrock terrace formation in erosion-resistant and easily friable lithologies. We mapped valley surfaces in both basins, dated straths using radiocarbon, compared valley width versus drainage area for basalt and sedimentary bedrock valleys, and constructed slope-area plots. In the friable sedimentary bedrock, valleys are 2 to 3 times wider, host flights of strath terraces, and have concavity values near 1; whereas the erosion-resistant basalt bedrock forms narrow valleys with poorly developed, localized, or no bedrock terraces and a channel steepness index half that of the friable bedrock and an average channel concavity of about 0.5. The oldest dated strath terrace on the Willapa River, T2, was active for nearly 10,000 years, from 11,265 to 2862 calibrated years before present (cal YBP), whereas the youngest terrace, T1, is Anthropocene in age and recently abandoned. Incision rates derived from terrace ages average 0.32 mm y- 1 for T2 and 11.47 mm y- 1 for T1. Our results indicate bedrock weathering properties influence valley width through the creation of a dense fracture network in the friable bedrock that results in high rates of lateral erosion of exposed bedrock banks. Conversely, the erosion-resistant bedrock has concavity values more typical of detachment-limited streams, exhibits a sparse fracture network, and displays evidence for infrequent episodic block erosion and plucking. Lithology thereby plays a direct role on the rates of lateral erosion, influencing valley width and the potential for strath terrace planation and preservation.

  13. 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

  14. 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.

  15. 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

  16. Structural elements in the Girk1 subunit that potentiate G protein-gated potassium channel activity.

    PubMed

    Wydeven, Nicole; Young, Daniele; Mirkovic, Kelsey; Wickman, Kevin

    2012-12-26

    G protein-gated inwardly rectifying K(+) (Girk/K(IR)3) channels mediate the inhibitory effect of many neurotransmitters on excitable cells. Girk channels are tetramers consisting of various combinations of four mammalian Girk subunits (Girk1 to -4). Although Girk1 is unable to form functional homomeric channels, its presence in cardiac and neuronal channel complexes correlates with robust channel activity. This study sought to better understand the potentiating influence of Girk1, using the GABA(B) receptor and Girk1/Girk2 heteromer as a model system. Girk1 did not increase the protein levels or alter the trafficking of Girk2-containing channels to the cell surface in transfected cells or hippocampal neurons, indicating that its potentiating influence involves enhancement of channel activity. Structural elements in both the distal carboxyl-terminal domain and channel core were identified as key determinants of robust channel activity. In the distal carboxyl-terminal domain, residue Q404 was identified as a key determinant of receptor-induced channel activity. In the Girk1 core, three unique residues in the pore (P) loop (F137, A142, Y150) were identified as a collective potentiating influence on both receptor-dependent and receptor-independent channel activity, exerting their influence, at least in part, by enhancing mean open time and single-channel conductance. Interestingly, the potentiating influence of the Girk1 P-loop is tempered by residue F162 in the second membrane-spanning domain. Thus, discontinuous and sometime opposing elements in Girk1 underlie the Girk1-dependent potentiation of receptor-dependent and receptor-independent heteromeric channel activity. PMID:23236146

  17. 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.

  18. A Ca2+-activated channel from Xenopus laevis oocyte membranes reconstituted into planar bilayers.

    PubMed Central

    Young, G P; Young, J D; Deshpande, A K; Goldstein, M; Koide, S S; Cohn, Z A

    1984-01-01

    Plasma membrane fractions from Xenopus laevis oocytes were incorporated into planar lipid bilayers. We show the existence of numerous Ca2+-activated nonspecific channels that are more permeable to anions. These channels are activated by Ca2+ at micromolar concentration but not by Mg2+, Zn2+, or Mn2+, even at millimolar concentrations. Decreasing Ca2+ concentration to less than 1 microM decreases the time of channel opening until channels close completely in the absence of Ca2+ and in the presence of EGTA. I- and Br- are more permeable through this channel than Cl-. The time during which the channels remain open is also voltage-dependent, with the channels switching off at higher voltages in both polarities. Single-channel activity shows a conductance of 380 pS in 1 M NaCl and 1 mM CaCl2, with an average open lifetime of 1.5 s at 40 mV. Similar channels are found in different stages of oocyte maturation. These observations support the hypothesis that an increase in oocyte-free Ca2+ activates directly these channels, and the resultant Cl- efflux forms the ionic basis for the fertilization potential in X. laevis. PMID:6089180

  19. Extremely Elevated Room-Temperature Kinetic Isotope Effects Quantify the Critical Role of Barrier Width in Enzymatic C–H Activation

    PubMed Central

    2015-01-01

    The enzyme soybean lipoxygenase (SLO) has served as a prototype for hydrogen-tunneling reactions, as a result of its unusual kinetic isotope effects (KIEs) and their temperature dependencies. Using a synergy of kinetic, structural, and theoretical studies, we show how the interplay between donor–acceptor distance and active-site flexibility leads to catalytic behavior previously predicted by quantum tunneling theory. Modification of the size of two hydrophobic residues by site-specific mutagenesis in SLO reduces the reaction rate 104-fold and is accompanied by an enormous and unprecedented room-temperature KIE. Fitting of the kinetic data to a non-adiabatic model implicates an expansion of the active site that cannot be compensated by donor–acceptor distance sampling. A 1.7 Å resolution X-ray structure of the double mutant further indicates an unaltered backbone conformation, almost identical side-chain conformations, and a significantly enlarged active-site cavity. These findings show the compelling property of room-temperature hydrogen tunneling within a biological context and demonstrate the very high sensitivity of such tunneling to barrier width. PMID:24884374

  20. 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

  1. Voltage-induced membrane displacement in patch pipettes activates mechanosensitive channels

    PubMed Central

    Gil, Ziv; Silberberg, Shai D.; Magleby, Karl L.

    1999-01-01

    The patch-clamp technique allows currents to be recorded through single ion channels in patches of cell membrane in the tips of glass pipettes. When recording, voltage is typically applied across the membrane patch to drive ions through open channels and to probe the voltage-sensitivity of channel activity. In this study, we used video microscopy and single-channel recording to show that prolonged depolarization of a membrane patch in borosilicate pipettes results in delayed slow displacement of the membrane into the pipette and that this displacement is associated with the activation of mechanosensitive (MS) channels in the same patch. The membrane displacement, ≈1 μm with each prolonged depolarization, occurs after variable delays ranging from tens of milliseconds to many seconds and is correlated in time with activation of MS channels. Increasing the voltage step shortens both the delay to membrane displacement and the delay to activation. Preventing depolarization-induced membrane displacement by applying positive pressure to the shank of the pipette or by coating the tips of the borosilicate pipettes with soft glass prevents the depolarization-induced activation of MS channels. The correlation between depolarization-induced membrane displacement and activation of MS channels indicates that the membrane displacement is associated with sufficient membrane tension to activate MS channels. Because membrane tension can modulate the activity of various ligand and voltage-activated ion channels as well as some transporters, an apparent voltage dependence of a channel or transporter in a membrane patch in a borosilicate pipette may result from voltage-induced tension rather than from direct modulation by voltage. PMID:10588750

  2. The Clinically Tested Gardos Channel Inhibitor Senicapoc Exhibits Antimalarial Activity

    PubMed Central

    Tubman, Venée N.; Mejia, Pedro; Shmukler, Boris E.; Bei, Amy K.; Alper, Seth L.; Mitchell, James R.

    2015-01-01

    Senicapoc, a Gardos channel inhibitor, prevented erythrocyte dehydration in clinical trials of patients with sickle cell disease. We tested the hypothesis that senicapoc-induced blockade of the Gardos channel inhibits Plasmodium growth. Senicapoc inhibited in vitro growth of human and primate plasmodia during the clinical blood stage. Senicapoc treatment suppressed P. yoelii parasitemia in vivo in C57BL/6 mice. The reassuring safety and biochemical profile of senicapoc encourage its use in antimalarial development. PMID:26459896

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

    PubMed Central

    Zamponi, G W; French, R J

    1994-01-01

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

  4. Oscillating activity of a calcium-activated K+ channel in normal and cancerous mammary cells in culture.

    PubMed

    Enomoto, K; Furuya, K; Maeno, T; Edwards, C; Oka, T

    1991-01-01

    Calcium-activated potassium channels were the channels most frequently observed in primary cultured normal mammary cell and in the established mammary tumor cell, MMT060562. In both cells, single-channel and whole-cell clamp recordings sometimes showed slow oscillations of the Ca2(+)-gated K+ current. The characteristics of the Ca2(+)-activated K+ channels in normal and cancerous mammary cells were quite similar. The slope conductances changed from 8 to 70 pS depending on the mode of recording and the ionic composition in the patch electrode. The open probability of this channel increased between 0.1 to 1 microM of the intracellular Ca2+, but it was independent of the membrane potential. Charybdotoxin reduced the activity of the Ca2(+)-activated K+ channel and the oscillation of the membrane current, but apamin had no apparent effect. The application of tetraethylammonium (TEA) from outside and BaCl2 from inside of the cell diminished the activity of the channel. The properties of this channel were different from those of both the large conductance (BK or MAXI K) and small conductance (SK) type Ca2(+)-activated K+ channels. PMID:1710671

  5. 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.

  6. [Osmoregulatory reactions of frog erythrocytes under conditions of activation and blockade of Ca2+-channels].

    PubMed

    Skorkina, M Iu

    2012-01-01

    The kinetics of cell osmoregulatory reactions under conditions of activation and blockade of Ca2+-channels was studied on a model of frog polyfunctional nuclear erythrocyte. Both activation and blockade of Ca2+-channels has been established to promote swelling of nuclei and an increase of the nuclear-cytoplasmic ratios under conditions of hypotonic exposure. The osmoregulatory cell reactions after activation of Ca2+-channels are expressed as a decrease of the cell volume. The blockator of Ca2+-channels verapamil produces an alternated increase and decrease of the erythrocyte volume with time intervals of 30 and 60 s. The clearly expressed functional activity of the nuclear membrane in response to the hypotonic action under conditions of activation and blockade of Ca2+-channels indicates participation of Ca2+ ions in mechanisms of the nuclear-cytoplasmic transfer. PMID:22645976

  7. 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

  8. Low-dose photon irradiation alters cell differentiation via activation of hIK channels.

    PubMed

    Roth, Bastian; Gibhardt, Christine S; Becker, Patrick; Gebhardt, Manuela; Knoop, Jan; Fournier, Claudia; Moroni, Anna; Thiel, Gerhard

    2015-08-01

    To understand the impact of ionizing irradiation from diagnostics and radiotherapy on cells, we examined K(+) channel activity before and immediately after exposing cells to X-rays. Already, low dose in the cGy range caused in adenocarcinoma A549 cells within minutes a hyperpolarization following activation of the human intermediate-conductance Ca(2+)-activated K(+) channel (hIK). The response was specific for cells, which functionally expressed hIK channels and in which hIK activity was low before irradiation. HEK293 cells, which do not respond to X-ray irradiation, accordingly develop a sensitivity to this stress after heterologous expression of hIK channels. The data suggest that hIK activation involves a Ca(2+)-mediated signaling cascade because channel activation is suppressed by a strong cytosolic Ca(2+) buffer. The finding that an elevation of H2O2 causes an increase in the concentration of cytosolic Ca(2+) suggests that radicals, which emerge early in response to irradiation, trigger this Ca(2+) signaling cascade. Inhibition of hIK channels by specific blockers clotrimazole and TRAM-34 slowed cell proliferation and migration in "wound" scratch assays; ionizing irradiation, in turn, stimulated the latter process presumably via its activation of the hIK channels. These data stress an indirect radiosensitivity of hIK channels with an impact on cell differentiation. PMID:25277267

  9. 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

  10. Allosteric interactions and the modular nature of the voltage- and Ca2+-activated (BK) channel

    PubMed Central

    Latorre, Ramon; Morera, Francisco J; Zaelzer, Cristian

    2010-01-01

    The high conductance voltage- and Ca2+-activated K+ channel is one of the most broadly expressed channels in mammals. This channel is named BK for ‘big K’ because of its single-channel conductance that can be as large as 250 pS in 100 mm symmetrical K+. BK channels increase their activity by membrane depolarization or an increase in cytosolic Ca2+. One of the key features that defines the behaviour of BK channels is that neither Ca2+ nor voltage is strictly necessary for channel activation. This and several other observations led to the idea that both Ca2+ and voltage increase the open probability by an allosteric mechanism. In this type of mechanism, the processes of voltage sensor displacement, Ca2+ binding and pore opening are independent equilibria that interact allosterically with each other. These allosteric interactions in BK channels reside in the structural characteristics of the BK channel in the sense that voltage and Ca2+ sensors and the pore need to be contained in different structures or ‘modules’. Through electrophysiological, mutagenesis, biochemical and fluorescence studies these modules have been identified and, more important, some of the interactions between them have been unveiled. In this review, we have covered the main advances achieved during the last few years in the elucidation of the structure of the BK channel and how this is related with its function as an allosteric protein. PMID:20603335

  11. Calcium-activated chloride channels in cultured embryonic Xenopus spinal neurons.

    PubMed

    Hussy, N

    1992-12-01

    1. Single-channel currents were recorded from Xenopus spinal neurons developing in vitro using the patch-clamp technique, to identify the channels underlying the large and small macroscopic Ca(2+)-activated Cl- currents (ICl(Ca)) present in these cells. 2. Channels of large (maxi-channels; 310 pS) and smaller conductance (mini-channels; 50-60 pS) are activated by elevation of cytoplasmic Ca2+ concentration. Channel activity is not altered by subsequent removal of Ca2+ from the bath, arguing against a direct ligand-type Ca2+ dependence. The much higher incidence of channel activation in cell-attached patches from cells permeabilized with the Ca2+ ionophore A23187 than in excised patches also suggests the involvement of some unidentified intracellular factor. 3. The reversal potential of maxi-Cl- channels is not altered by changes in Na+ concentration, but is shifted in the negative direction by the substitution of Cl- by methanesulfonate on the intracellular side of the patch, indicating their anionic selectivity. 4. Maxi-Cl- channels exhibited the presence of multiple probable subconductance states and showed marked voltage-dependent inactivation above and below +/- 20 mV. 5. Examination of maxi-Cl- channels at early times in culture (6-9 h) and 24 h later did not reveal any developmental change in the characteristics described above. However, the mean open duration of the channel was found to increase twofold during this period of time. 6. The simultaneous presence of maxi- and mini-Cl- channels prevented detailed characterization of the latter. The anionic selectivity of mini-Cl- channels is suggested by their reversal potential that lies close to the Cl- equilibrium potential.(ABSTRACT TRUNCATED AT 250 WORDS) PMID:1283407

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

    PubMed Central

    Cui, Yuanyuan; Yang, Fan; Cao, Xu; Yarov-Yarovoy, Vladimir

    2012-01-01

    The capsaicin receptor transient receptor potential vanilloid (TRPV)1 is a highly heat-sensitive ion channel. Although chemical activation and heat activation of TRPV1 elicit similar pungent, painful sensation, the molecular mechanism underlying synergistic activation remains mysterious. In particular, where the temperature sensor is located and whether heat and capsaicin share a common activation pathway are debated. To address these fundamental issues, we searched for channel mutations that selectively affected one form of activation. We found that deletion of the first 10 amino acids of the pore turret significantly reduced the heat response amplitude and shifted the heat activation threshold, whereas capsaicin activation remained unchanged. Removing larger portions of the turret disrupted channel function. Introducing an artificial sequence to replace the deleted region restored sensitive capsaicin activation in these nonfunctional channels. The heat activation, however, remained significantly impaired, with the current exhibiting diminishing heat sensitivity to a level indistinguishable from that of a voltage-gated potassium channel, Kv7.4. Our results demonstrate that heat and capsaicin activation of TRPV1 are structurally and mechanistically distinct processes, and the pore turret is an indispensible channel structure involved in the heat activation process but is not part of the capsaicin activation pathway. Synergistic effect of heat and capsaicin on TRPV1 activation may originate from convergence of the two pathways on a common activation gate. PMID:22412190

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

    PubMed Central

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

    2015-01-01

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

  14. 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

  15. Tubular Unimolecular Transmembrane Channels: Construction Strategy and Transport Activities.

    PubMed

    Si, Wen; Xin, Pengyang; Li, Zhan-Ting; Hou, Jun-Li

    2015-06-16

    Lipid bilayer membranes separate living cells from their environment. Membrane proteins are responsible for the processing of ion and molecular inputs and exports, sensing stimuli and signals across the bilayers, which may operate in a channel or carrier mechanism. Inspired by these wide-ranging functions of membrane proteins, chemists have made great efforts in constructing synthetic mimics in order to understand the transport mechanisms, create materials for separation, and develop therapeutic agents. Since the report of an alkylated cyclodextrin for transporting Cu(2+) and Co(2+) by Tabushi and co-workers in 1982, chemists have constructed a variety of artificial transmembrane channels by making use of either the multimolecular self-assembly or unimolecular strategy. In the context of the design of unimolecular channels, important advances have been made, including, among others, the tethering of natural gramicidin A or alamethicin and the modification of various macrocycles such as crown ethers, cyclodextrins, calixarenes, and cucurbiturils. Many of these unimolecular channels exhibit high transport ability for metal ions, particularly K(+) and Na(+). Concerning the development of artificial channels based on macrocyclic frameworks, one straightforward and efficient approach is to introduce discrete chains to reinforce their capability to insert into bilayers. Currently, this approach has found the widest applications in the systems of crown ethers and calixarenes. We envisioned that for macrocycle-based unimolecular channels, control of the arrangement of the appended chains in the upward and/or downward direction would favor the insertion of the molecular systems into bilayers, while the introduction of additional interactions among the chains would further stabilize a tubular conformation. Both factors should be helpful for the formation of new efficient channels. In this Account, we discuss our efforts in designing new unimolecular artificial channels from

  16. 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

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

    PubMed

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

    2016-08-01

    Seizure activity is linked to astrocyte activation as well as dysfunctional cortical neuron excitability produced from changes in calcium-activated potassium (KCa) channel function. Ciliary neurotrophic factor-treated astrocyte conditioned medium (CNTF-ACM) can be used to investigate the peripheral effects of activated astrocytes upon cortical neurons. However, CNTF-ACM's effect upon KCa channel activity in cultured cortical neurons has not yet been investigated. Whole-cell patch clamp recordings were performed in rat cortical neurons to evaluate CNTF-ACM's effects upon charybdotoxin-sensitive large-conductance KCa (BK) channel currents and apamin-sensitive small-conductance KCa (SK) channel current. Biotinylation and RT-PCR were applied to assess CNTF-ACM's effects upon the protein and mRNA expression, respectively, of the SK channel subunits SK2 and SK3 and the BK channel subunits BKα1 and BKβ3. An anti-fibroblast growth factor-2 (FGF-2) monoclonal neutralizing antibody was used to assess the effects of the FGF-2 component of CNTF-ACM. CNTF-ACM significantly increased KCa channel current density, which was predominantly attributable to gains in BK channel activity (p < 0.05). CNTF-ACM produced a significant increase in BKα1 and BKβ3 expression (p < 0.05) but had no significant effect upon SK2 or SK3 expression (p > 0.05). Blocking FGF-2 produced significant reductions in KCa channel current density (p > 0.05) as well as BKα1 and BKβ3 expression in CNTF-ACM-treated neurons (p > 0.05). CNTF-ACM significantly enhances BK channel activity in rat cortical neurons and that FGF-2 is partially responsible for these effects. CNTF-induced astrocyte activation results in secretion of neuroactive factors which may affect neuronal excitability and resultant seizure activity in mammalian cortical neurons. PMID:27097551

  18. 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

  19. Regulation of Human Kv1.4 Channel Activity by the Antidepressant Metergoline.

    PubMed

    Yeom, Hye Duck; Lee, Jun-Ho

    2016-01-01

    Metergoline is an ergot-derived psychoactive drug that is a ligand for various serotonin and dopamine receptors. Little is known about the effect of metergoline on different types of receptors and ion channels. Potassium channels are the most diverse group of ion channels. Kv1.4, a shaker family K channel alpha subunit, is one of a family of voltage gated K channels that mediates transient and rapid inactivating A-type currents and N-type inactivation. We demonstrated previously that metergoline inhibited the activity of neuronal voltage-dependent Na(+) channels in Xenopus laevis oocytes (Acta Pharmacol. Sin., 35, 2014, Lee et al.). In this study, we sought to elucidate the regulatory effects underlying metergoline-induced human Kv1.4 channel inhibition. We used the two electrode voltage-clamp (TEVC) technique to investigate the effect of metergoline on human Kv1.4 channel currents in Xenopus laevis oocytes expressing human Kv1.4 alpha subunits. Interestingly, metergoline treatment also induced inhibition of peak currents in human Kv1.4 channels in a concentration-dependent manner. The IC50 of peak currents of hKv1.4 currents was 3.6±0.6 µM. These results indicate that metergoline might regulate the human Kv1.4 channel activity that is expressed in X. laevis oocytes. Further, this regulation of potassium currents by metergoline might be one of the pharmacological actions of metergoline-mediated psychoactivity. PMID:27251511

  20. Resonances and resonance widths

    SciTech Connect

    Collins, T.

    1986-05-01

    Two-dimensional betatron resonances are much more important than their simple one-dimensional counterparts and exhibit a strong dependence on the betatron phase advance per cell. A practical definition of ''width'' is expanded upon in order to display these relations in tables. A primarily pedagogical introduction is given to explain the tables, and also to encourage a wider capability for deriving resonance behavior and wider use of ''designer'' resonances.

  1. Narrow Width Pentaquarks

    NASA Astrophysics Data System (ADS)

    Buccella, F.; Sorba, P.

    A general study of pentaquarks built with four quarks in a L=1 state and an antiquark in S-wave shows that several of such states are forbidden by a selection rule, which holds in the limit of flavor symmetry, to decay into a baryon and a meson final state. We identify the most promising /line{10} multiplet for the classification of the Θ+ and Ξ-- particles recently discovered with the prediction of a narrow width for both of them.

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

    PubMed

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

    1994-05-17

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

  3. Mean annual runoff and peak flow estimates based on channel geometry of streams in southeastern Montana

    USGS Publications Warehouse

    Omang, R.J.; Parrett, Charles; Hull, J.A.

    1983-01-01

    Equations using channel-geometry measurements were developed for estimating mean runoff and peak flows of ungaged streams in southeastern Montana. Two separate sets of esitmating equations were developed for determining mean annual runoff: one for perennial streams and one for ephemeral and intermittent streams. Data from 29 gaged sites on perennial streams and 21 gaged sites on ephemeral and intermittent streams were used in these analyses. Data from 78 gaged sites were used in the peak-flow analyses. Southeastern Montana was divided into three regions and separate multiple-regression equations for each region were developed that relate channel dimensions to peak discharge having recurrence intervals of 2, 5, 10, 25, 50, and 100 years. Channel-geometery relations were developed using measurements of the active-channel width and bankfull width. Active-channel width and bankfull width were the most significant channel features for estimating mean annual runoff for al types of streams. Use of this method requires that onsite measurements be made of channel width. The standard error of estimate for predicting mean annual runoff ranged from about 38 to 79 percent. The standard error of estimate relating active-channel width or bankfull width to peak flow ranged from about 37 to 115 percent. (USGS)

  4. 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

  5. Calcium-mediated agonists activate an inwardly rectified K+ channel in colonic secretory cells.

    PubMed

    Devor, D C; Frizzell, R A

    1993-11-01

    Single-channel recording techniques were used to identify and characterize the K+ channel activated by Ca(2+)-mediated secretory agonists in T84 cells. Carbachol (CCh; 100 microM) and taurodeoxycholate (TDC; 0.75 mM) stimulated oscillatory outward K+ currents. With K gluconate in bath and pipette, cell-attached single-channel K+ currents stimulated by CCh and ionomycin (2 microM) were inwardly rectified and reversed at 0 mV. The single-channel chord conductance was 32 pS at -90 mV and 14 pS at +90 mV. Similar properties were observed in excised inside-out patches in symmetric K+, permitting further characterization of channel properties. Partial substitution of bath or pipette K+ with Na+ gave a K(+)-to-Na+ selectivity ratio of 5.5:1. Channel activity increased with increasing bath Ca2+ concentration in the physiological range of 50-800 nM. Maximal channel activity occurred at intracellular pH 7.2 and decreased at more acidic or alkaline pH values. Extracellular charybdotoxin (CTX; 50 nM) blocked inward but not outward currents. Extracellular tetraethylammonium (TEA; 10 mM) reduced single-channel amplitude at all voltages. No apparent block of the channel was observed with extracellular Ba2+ (1 mM), apamin (1 microM), 4-aminopyridine (4-AP; 4 mM), quinine (500 microM), or glyburide (10 microM). Cytosolic quinine and 4-AP blocked both inward and outward currents, whereas Ba2+ blocked only outward currents. Apamin, CTX, TEA, and glyburide did not affect channel activity. The agonist activation and pharmacological profile of this inwardly rectified K+ channel indicate that it is responsible for the increase in basolateral K+ conductance stimulated by Ca(2+)-mediated agonists in T84 cells. PMID:7694492

  6. Control of Inward Rectifier K Channel Activity by Lipid Tethering of Cytoplasmic Domains

    PubMed Central

    Enkvetchakul, Decha; Jeliazkova, Iana; Bhattacharyya, Jaya; Nichols, Colin G.

    2007-01-01

    Interactions between nontransmembrane domains and the lipid membrane are proposed to modulate activity of many ion channels. In Kir channels, the so-called “slide-helix” is proposed to interact with the lipid headgroups and control channel gating. We examined this possibility directly in a cell-free system consisting of KirBac1.1 reconstituted into pure lipid vesicles. Cysteine substitution of positively charged slide-helix residues (R49C and K57C) leads to loss of channel activity that is rescued by in situ restoration of charge following modification by MTSET+ or MTSEA+, but not MTSES− or neutral MMTS. Strikingly, activity is also rescued by modification with long-chain alkyl-MTS reagents. Such reagents are expected to partition into, and hence tether the side chain to, the membrane. Systematic scanning reveals additional slide-helix residues that are activated or inhibited following alkyl-MTS modification. A pattern emerges whereby lipid tethering of the N terminus, or C terminus, of the slide-helix, respectively inhibits, or activates, channel activity. This study establishes a critical role of the slide-helix in Kir channel gating, and directly demonstrates that physical interaction of soluble domains with the membrane can control ion channel activity. PMID:17698595

  7. Natural Bile Acids and Synthetic Analogues Modulate Large Conductance Ca2+-activated K+ (BKCa) Channel Activity in Smooth Muscle Cells

    PubMed Central

    Dopico, Alejandro M.; Walsh, John V.; Singer, Joshua J.

    2002-01-01

    Bile acids have been reported to produce relaxation of smooth muscle both in vitro and in vivo. The cellular mechanisms underlying bile acid–induced relaxation are largely unknown. Here we demonstrate, using patch-clamp techniques, that natural bile acids and synthetic analogues reversibly increase BKCa channel activity in rabbit mesenteric artery smooth muscle cells. In excised inside-out patches bile acid–induced increases in channel activity are characterized by a parallel leftward shift in the activity-voltage relationship. This increase in BKCa channel activity is not due to Ca2+-dependent mechanism(s) or changes in freely diffusible messengers, but to a direct action of the bile acid on the channel protein itself or some closely associated component in the cell membrane. For naturally occurring bile acids, the magnitude of bile acid–induced increase in BKCa channel activity is inversely related to the number of hydroxyl groups in the bile acid molecule. By using synthetic analogues, we demonstrate that such increase in activity is not affected by several chemical modifications in the lateral chain of the molecule, but is markedly favored by polar groups in the side of the steroid rings opposite to the side where the methyl groups are located, which stresses the importance of the planar polarity of the molecule. Bile acid–induced increases in BKCa channel activity are also observed in smooth muscle cells freshly dissociated from rabbit main pulmonary artery and gallbladder, raising the possibility that a direct activation of BKCa channels by these planar steroids is a widespread phenomenon in many smooth muscle cell types. Bile acid concentrations that increase BKCa channel activity in mesenteric artery smooth muscle cells are found in the systemic circulation under a variety of human pathophysiological conditions, and their ability to enhance BKCa channel activity may explain their relaxing effect on smooth muscle. PMID:11865021

  8. A synthetic prostone activates apical chloride channels in A6 epithelial cells

    PubMed Central

    Bao, Hui Fang; Liu, Lian; Self, Julie; Duke, Billie Jeanne; Ueno, Ryuji; Eaton, Douglas C.

    2008-01-01

    The bicyclic fatty acid lubiprostone (formerly known as SPI-0211) activates two types of anion channels in A6 cells. Both channel types are rarely, if ever, observed in untreated cells. The first channel type was activated at low concentrations of lubiprostone (<100 nM) in >80% of cell-attached patches and had a unit conductance of ∼3–4 pS. The second channel type required higher concentrations (>100 nM) of lubiprostone to activate, was observed in ∼30% of patches, and had a unit conductance of 8–9 pS. The properties of the first type of channel were consistent with ClC-2 and the second with CFTR. ClC-2's unit current strongly inwardly rectified that could be best fit by models of the channel with multiple energy barrier and multiple anion binding sites in the conductance pore. The open probability and mean open time of ClC-2 was voltage dependent, decreasing dramatically as the patches were depolarized. The order of anion selectivity for ClC-2 was Cl > Br > NO3 > I > SCN, where SCN is thiocyanate. ClC-2 was a “double-barreled” channel favoring even numbers of levels over odd numbers as if the channel protein had two conductance pathways that opened independently of one another. The channel could be, at least, partially blocked by glibenclamide. The properties of the channel in A6 cells were indistinguishable from ClC-2 channels stably transfected in HEK293 cells. CFTR in the patches had a selectivity of Cl > Br ≫ NO3 ≅ SCN ≅ I. It outwardly rectified as expected for a single-site anion channel. Because of its properties, ClC-2 is uniquely suitable to promote anion secretion with little anion reabsorption. CFTR, on the other hand, could promote either reabsorption or secretion depending on the anion driving forces. PMID:18511742

  9. Let It Go and Open Up, an Ensemble of Ion Channel Active States.

    PubMed

    Minor, Daniel L

    2016-02-11

    Ligand binding usually moves the target protein from an ensemble of inactive states to a well-defined active conformation. Matthies et al. flip this scheme around, finding that, for the magnesium channel CorA, loss of ligand binding induces an ensemble of conformations that turn the channel on. PMID:26871624

  10. Endogenous KCNE Subunits Govern Kv2.1 K+ Channel Activation Kinetics in Xenopus Oocyte Studies

    PubMed Central

    Gordon, Earl; Roepke, Torsten K.; Abbott, Geoffrey W.

    2006-01-01

    Kv2.1 is a voltage-gated potassium (Kv) channel that generates delayed rectifier currents in mammalian heart and brain. The biophysical properties of Kv2.1 and other ion channels have been characterized by functional expression in heterologous systems, and most commonly in Xenopus laevis oocytes. A number of previous oocyte-based studies of mammalian potassium channels have revealed expression-level-dependent changes in channel properties, leading to the suggestion that endogenous oocyte factors regulate channel gating. Here, we show that endogenous oocyte potassium channel KCNE ancillary subunits xMinK and xMiRP2 slow the activation of oocyte-expressed mammalian Kv2.1 channels two-to-fourfold. This produces a sigmoidal relationship between Kv2.1 current density and activation rate in oocyte-based two-electrode voltage clamp studies. The effect of endogenous xMiRP2 and xMinK on Kv2.1 activation is diluted at high Kv2.1 expression levels, or by RNAi knockdown of either endogenous subunit. RNAi knockdown of both xMiRP2 and xMinK eliminates the correlation between Kv2.1 expression level and activation kinetics. The data demonstrate a molecular basis for expression-level-dependent changes in Kv channel gating observed in heterologous expression studies. PMID:16326911

  11. Ion channel modifying agents influence the electrical activity generated by canine intrinsic cardiac neurons in situ.

    PubMed

    Thompson, G W; Horackova, M; Armour, J A

    2000-04-01

    This study was designed to establish whether agents known to modify neuronal ion channels influence the behavior of mammalian intrinsic cardiac neurons in situ and, if so, in a manner consistent with that found previously in vitro. The activity generated by right atrial neurons was recorded extracellularly in varying numbers of anesthetized dogs before and during continuous local arterial infusion of several neuronal ion channel modifying agents. Veratridine (7.5 microM), the specific modifier of Na+-selective channels, increased neuronal activity (95% above control) in 80% of dogs tested (n = 25). The membrane depolarizing agent potassium chloride (40 mM) reduced neuronal activity (43% below control) in 84% of dogs tested (n = 19). The inhibitor of voltage-sensitive K+ channels, tetraethylammonium (10 mM), decreased neuronal activity (42% below control) in 73% of dogs tested (n = 11). The nonspecific potassium channel inhibitor barium chloride (5 mM) excited neurons (47% above control) in 13 of 19 animals tested. Cadmium chloride (200 microM), which inhibits Ca2+-selective channels and Ca2+-dependent K+ channels, increased neuronal activity (65% above control) in 79% of dogs tested (n = 14). The specific L-type Ca2+ channel blocking agent nifedipine (5 microM) reduced neuronal activity (52% blow control in 72% of 11 dogs tested), as did the nonspecific inhibitor of L-type Ca2+ channels, nickel chloride (5 mM) (36% below control in 69% of 13 dogs tested). Each agent induced either excitatory or inhibitory responses, depending on the agent tested. It is concluded that specific ion channels (I(Na), I(CaL), I(Kv), and I(KCa)) that have been associated with intrinsic cardiac neurons in vitro are involved in their capacity to generate action potentials in situ. PMID:10772056

  12. Calmodulin-dependent activation and inactivation of anoctamin calcium-gated chloride channels

    PubMed Central

    Vocke, Kerstin; Dauner, Kristin; Hahn, Anne; Ulbrich, Anne; Broecker, Jana; Keller, Sandro; Frings, Stephan

    2013-01-01

    Calcium-dependent chloride channels serve critical functions in diverse biological systems. Driven by cellular calcium signals, the channels codetermine excitatory processes and promote solute transport. The anoctamin (ANO) family of membrane proteins encodes three calcium-activated chloride channels, named ANO 1 (also TMEM16A), ANO 2 (also TMEM16B), and ANO 6 (also TMEM16F). Here we examined how ANO 1 and ANO 2 interact with Ca2+/calmodulin using nonstationary current analysis during channel activation. We identified a putative calmodulin-binding domain in the N-terminal region of the channel proteins that is involved in channel activation. Binding studies with peptides indicated that this domain, a regulatory calmodulin-binding motif (RCBM), provides two distinct modes of interaction with Ca2+/calmodulin, one at submicromolar Ca2+ concentrations and one in the micromolar Ca2+ range. Functional, structural, and pharmacological data support the concept that calmodulin serves as a calcium sensor that is stably associated with the RCBM domain and regulates the activation of ANO 1 and ANO 2 channels. Moreover, the predominant splice variant of ANO 2 in the brain exhibits Ca2+/calmodulin-dependent inactivation, a loss of channel activity within 30 s. This property may curtail ANO 2 activity during persistent Ca2+ signals in neurons. Mutagenesis data indicated that the RCBM domain is also involved in ANO 2 inactivation, and that inactivation is suppressed in the retinal ANO 2 splice variant. These results advance the understanding of Ca2+ regulation in anoctamin Cl− channels and its significance for the physiological function that anoctamin channels subserve in neurons and other cell types. PMID:24081981

  13. Neutron field for activation experiments in horizontal channel of training reactor VR-1

    NASA Astrophysics Data System (ADS)

    Stefanik, Milan; Katovsky, Karel; Vins, Miroslav; Soltes, Jaroslav; Zavorka, Lukas

    2014-11-01

    The experimental channels of nuclear reactors often serve for nuclear data measurement and validation. The dosimetry-foils activation technique was employed to measure neutron field parameters in the horizontal radial channel of the training reactor VR-1, and to test the possibility of using the reactor for scientific purposes. The reaction rates, energy spectral indexes, and neutron spectrum at several irradiation positions of the experimental channel were determined. The experimental results show the feasibility of the radial channel for irradiating experiments and open new possibilities for data validation by using this nuclear facility.

  14. 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. PMID:26202907

  15. 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

  16. Tamoxifen inhibits BK channels in chick cochlea without alterations in voltage-dependent activation.

    PubMed

    Tong, Mingjie; Duncan, R Keith

    2009-07-01

    Large-conductance, Ca(2+)-activated, and voltage-gated potassium channels (BK, BK(Ca), or Maxi-K) play an important role in electrical tuning in nonmammalian vertebrate hair cells. Systematic changes in tuning frequency along the tonotopic axis largely result from variations in BK channel kinetics, but the molecular changes underpinning these functional variations remain unknown. Auxiliary beta(1) have been implicated in low-frequency tuning at the cochlear apex because these subunits dramatically slow channel kinetics. Tamoxifen (Tx), a (xeno)estrogen compound known to activate BK channels through the beta-subunit, was used to test for the functional presence of beta(1). The hypotheses were that Tx would activate the majority of BK channels in hair cells from the cochlear apex due to the presence of beta(1) and that the level of activation would exhibit a tonotopic gradient following the expression profile of beta(1). Outside-out patches of BK channels were excised from tall hair cells along the apical half of the chicken basilar papilla. In low-density patches, single-channel conductance was reduced and the averaged open probability was unaffected by Tx. In high-density patches, the amplitude of ensemble-averaged BK current was inhibited, whereas half-activation potential and activation kinetics were unaffected by Tx. In both cases, no tonotopic Tx-dependent activation of channel activity was observed. Therefore, contrary to the hypotheses, electrophysiological assessment suggests that molecular mechanisms other than auxiliary beta-subunits are involved in generating a tonotopic distribution of BK channel kinetics and electric tuning in chick basilar papilla. PMID:19439526

  17. Deletion of phenylalanine 508 causes attenuated phosphorylation-dependent activation of CFTR chloride channels.

    PubMed

    Wang, F; Zeltwanger, S; Hu, S; Hwang, T C

    2000-05-01

    In cell-attached patches stimulated with cAMP agonists, the single-channel open probability (Po) of the phenylalanine 508-deleted cystic fibrosis transmembrane conductance regulator (DeltaF508-CFTR) channel, the most common disease-associated mutation in cystic fibrosis, was abnormally low (a functional defect). To investigate the mechanism for the poor response of DeltaF508-CFTR to cAMP stimulation, we examined, in excised inside-out patches, protein kinase A (PKA)-dependent phosphorylation activation and ATP-dependent gating of wild-type (WT) and DeltaF508-CFTR channels expressed in NIH3T3 mouse fibroblasts. For WT-CFTR, the activation time course of CFTR channel current upon addition of PKA and ATP followed a sigmoidal function with time constants that decreased as [PKA] was increased. The curvilinear relationship between [PKA] and the apparent activation rate suggests an incremental phosphorylation-dependent activation of CFTR at multiple phosphorylation sites. The time course of PKA-dependent activation of DeltaF508-CFTR channel current also followed a sigmoidal function, but the rate of activation was at least 7-fold slower than that with WT channels. This result suggests that deletion of phenylalanine 508 causes attenuated PKA-dependent phosphorylation of the CFTR chloride channel. Once DeltaF508-CFTR channels were maximally activated with PKA, the mutant channel and WT channel had indistinguishable steady-state Po values, ATP dose-response relationships and single-channel kinetics, indicating that DeltaF508-CFTR is not defective in ATP-dependent gating. By measuring whole-cell current density, we compared the number of functional channels in WT- and DeltaF508-CFTR cell membrane. Our data showed that the estimated channel density for DeltaF508-CFTR was approximately 10-fold lower than that for WT-CFTR, but the cAMP-dependent whole-cell current density differed by approximately 200-fold. We thus conclude that the functional defect (a decrease in Po) of Delta

  18. Deletion of phenylalanine 508 causes attenuated phosphorylation-dependent activation of CFTR chloride channels

    PubMed Central

    Wang, Fei; Zeltwanger, Shawn; Hu, Shenghui; Hwang, Tzyh-Chang

    2000-01-01

    In cell-attached patches stimulated with cAMP agonists, the single-channel open probability (Po) of the phenylalanine 508-deleted cystic fibrosis transmembrane conductance regulator (ΔF508-CFTR) channel, the most common disease-associated mutation in cystic fibrosis, was abnormally low (a functional defect). To investigate the mechanism for the poor response of ΔF508-CFTR to cAMP stimulation, we examined, in excised inside-out patches, protein kinase A (PKA)-dependent phosphorylation activation and ATP-dependent gating of wild-type (WT) and ΔF508-CFTR channels expressed in NIH3T3 mouse fibroblasts.For WT-CFTR, the activation time course of CFTR channel current upon addition of PKA and ATP followed a sigmoidal function with time constants that decreased as [PKA] was increased. The curvilinear relationship between [PKA] and the apparent activation rate suggests an incremental phosphorylation-dependent activation of CFTR at multiple phosphorylation sites.The time course of PKA-dependent activation of ΔF508-CFTR channel current also followed a sigmoidal function, but the rate of activation was at least 7-fold slower than that with WT channels. This result suggests that deletion of phenylalanine 508 causes attenuated PKA-dependent phosphorylation of the CFTR chloride channel.Once ΔF508-CFTR channels were maximally activated with PKA, the mutant channel and WT channel had indistinguishable steady-state Po values, ATP dose-response relationships and single-channel kinetics, indicating that ΔF508-CFTR is not defective in ATP-dependent gating.By measuring whole-cell current density, we compared the number of functional channels in WT- and ΔF508-CFTR cell membrane. Our data showed that the estimated channel density for ΔF508-CFTR was ∼10-fold lower than that for WT-CFTR, but the cAMP-dependent whole-cell current density differed by ∼200-fold. We thus conclude that the functional defect (a decrease in Po) of ΔF508-CFTR is as important as the trafficking defect (a

  19. 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. PMID:27094086

  20. 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

  1. Stretch-activated channels in single early distal tubule cells of the frog.

    PubMed Central

    Hurst, A M; Hunter, M

    1990-01-01

    1. Single stretch-activated channels have been studied in cell-attached and excised patches from single early distal tubule (diluting segment) cells of Rana temporaria. 2. The channels can be reversibly activated, in both cell-attached and excised patches, by the application of negative pressure to the pipette causing mechanical stretching of the cell membrane. In cell-attached patches, application of 14.8 cmH2O negative pressure to the patch pipette increased reversibly the open probably from 0.11 to 0.87. 3. The channel conductance in the cell-attached configuration with standard Ringer solution in the pipette is 21.3 pS. 4. The channel is non-specific. In excised inside-out patches ion substitution experiments show that the channel does not discriminate between sodium and potassium ions, nor does it appear to select for cations over anions. 5. The channel is voltage sensitive such that depolarizing the cell opens the channel. The open probability at the resting membrane potential, 0.89, was reduced to 0.26 at a hyperpolarizing potential of 100 mV (holding pressure of -20.1 cmH2O or -206 Pa). 6. The sensitivity of the channel to mechanical stretching suggests that the channel may be involved in cell volume regulation. PMID:1707964

  2. The regulation of BK channel activity by pre- and post-translational modifications

    PubMed Central

    Kyle, Barry D.; Braun, Andrew P.

    2014-01-01

    Large conductance, Ca2+-activated K+ (BK) channels represent an important pathway for the outward flux of K+ ions from the intracellular compartment in response to membrane depolarization, and/or an elevation in cytosolic free [Ca2+]. They are functionally expressed in a range of mammalian tissues (e.g., nerve and smooth muscles), where they can either enhance or dampen membrane excitability. The diversity of BK channel activity results from the considerable alternative mRNA splicing and post-translational modification (e.g., phosphorylation) of key domains within the pore-forming α subunit of the channel complex. Most of these modifications are regulated by distinct upstream cell signaling pathways that influence the structure and/or gating properties of the holo-channel and ultimately, cellular function. The channel complex may also contain auxiliary subunits that further affect channel gating and behavior, often in a tissue-specific manner. Recent studies in human and animal models have provided strong evidence that abnormal BK channel expression/function contributes to a range of pathologies in nerve and smooth muscle. By targeting the upstream regulatory events modulating BK channel behavior, it may be possible to therapeutically intervene and alter BK channel expression/function in a beneficial manner. PMID:25202279

  3. 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

  4. [Cooperative phenomena in the activity of single ion channels].

    PubMed

    Geletiuk, V I; Kazachenko, V N

    1989-01-01

    Using the patch-voltage-clamp method kinetics of the fast potential-dependent K+-channels in molluscan neurones was investigated. It was found that under given experimental conditions the amplitudes of single current impulses have a wide spectrum. The amplitudes are proportional to a number of the current substates involved. Averaged fronts of the current impulses are S-shaped, and have duration greater than 1 ms. Averaged duration of the current impulses increases (from 0.25 to 30-40 ms) with the impulse amplitude (or with the number of the substates involved). There is a sharp bend of the dependence at the impulse amplitude 0.6-0.7 of maximal value. The phenomena investigated reflect, probably, cooperativity of the channel transitions between the substates. The degree of the cooperativity depends on the membrane potential value. PMID:2804147

  5. Diatomic predissociation line widths

    NASA Technical Reports Server (NTRS)

    Child, M. S.

    1973-01-01

    Predissociation by rotation and curve crossing in diatomic molecules is discussed. The pattern of predissociation line widths is seen as providing a highly sensitive yardstick for the determination of unknown potential curves. In addition, the computation of such a pattern for given potential curves is considered a matter of routine, unless the predissociation happens to occur from an adiabatic potential curve. Analytic formulas are used to provide physical insight into the details of the predissociation pattern, to the extent that a direct inversion procedure is developed for determination of the repulsive potential curves for Type 1 predissociations.

  6. 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

  7. 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

  8. Regulation of large conductance calcium- and voltage-activated potassium (BK) channels by S-palmitoylation.

    PubMed

    Shipston, Michael J

    2013-02-01

    BK (large conductance calcium- and voltage-activated potassium) channels are important determinants of physiological control in the nervous, endocrine and vascular systems with channel dysfunction associated with major disorders ranging from epilepsy to hypertension and obesity. Thus the mechanisms that control channel surface expression and/or activity are important determinants of their (patho)physiological function. BK channels are S-acylated (palmitoylated) at two distinct sites within the N- and C-terminus of the pore-forming α-subunit. Palmitoylation of the N-terminus controls channel trafficking and surface expression whereas palmitoylation of the C-terminal domain determines regulation of channel activity by AGC-family protein kinases. Recent studies are beginning to reveal mechanistic insights into how palmitoylation controls channel trafficking and cross-talk with phosphorylation-dependent signalling pathways. Intriguingly, each site of palmitoylation is regulated by distinct zDHHCs (palmitoyl acyltransferases) and APTs (acyl thioesterases). This supports that different mechanisms may control substrate specificity by zDHHCs and APTs even within the same target protein. As palmitoylation is dynamically regulated, this fundamental post-translational modification represents an important determinant of BK channel physiology in health and disease. PMID:23356260

  9. Human monocytes kill M-CSF-expressing glioma cells by BK channel activation.

    PubMed

    Hoa, Neil T; Zhang, Jian Gang; Delgado, Christina L; Myers, Michael P; Callahan, Linda L; Vandeusen, Gerald; Schiltz, Patric M; Wepsic, H Terry; Jadus, Martin R

    2007-02-01

    In this study, human monocytes/macrophages were observed to kill human U251 glioma cells expressing membrane macrophage colony-stimulating factor (mM-CSF) via a swelling and vacuolization process called paraptosis. Human monocytes responded to the mM-CSF-transduced U251 glioma cells, but not to viral vector control U251 glioma cells (U251-VV), by producing a respiratory burst within 20 min. Using patch clamp techniques, functional big potassium (BK) channels were observed on the membrane of the U251 glioma cell. It has been previously reported that oxygen indirectly regulates BK channel function. In this study, it was demonstrated that prolonged BK channel activation in response to the respiratory burst induced by monocytes initiates paraptosis in selected glioma cells. Forced BK channel opening within the glioma cells by BK channel activators (phloretin or pimaric acid) induced U251 glioma cell swelling and vacuolization occurred within 30 min. U251 glioma cell cytotoxicity, induced by using BK channel activators, required between 8 and 12 h. Swelling and vacuolization induced by phloretin and pimaric acid was prevented by iberiotoxin, a specific BK channel inhibitor. Confocal fluorescence microscopy demonstrated BK channels co-localized with the endoplasmic reticulum and mitochondria, the two targeted organelles affected in paraptosis. Iberiotoxin prevented monocytes from producing death in mM-CSF-expressing U251glioma cells in a 24 h assay. This study demonstrates a novel mechanism whereby monocytes can induce paraptosis via the disruption of internal potassium ion homeostasis. PMID:17318194

  10. Potent KCNQ2/3-Specific Channel Activator Suppresses In Vivo Epileptic Activity and Prevents the Development of Tinnitus

    PubMed Central

    Kalappa, Bopanna I.; Soh, Heun; Duignan, Kevin M.; Furuya, Takeru; Edwards, Scott

    2015-01-01

    Voltage-gated Kv7 (KCNQ) channels are voltage-dependent potassium channels that are activated at resting membrane potentials and therefore provide a powerful brake on neuronal excitability. Genetic or experience-dependent reduction of KCNQ2/3 channel activity is linked with disorders that are characterized by neuronal hyperexcitability, such as epilepsy and tinnitus. Retigabine, a small molecule that activates KCNQ2–5 channels by shifting their voltage-dependent opening to more negative voltages, is an US Food and Drug Administration (FDA) approved anti-epileptic drug. However, recently identified side effects have limited its clinical use. As a result, the development of improved KCNQ2/3 channel activators is crucial for the treatment of hyperexcitability-related disorders. By incorporating a fluorine substituent in the 3-position of the tri-aminophenyl ring of retigabine, we synthesized a small-molecule activator (SF0034) with novel properties. Heterologous expression of KCNQ2/3 channels in HEK293T cells showed that SF0034 was five times more potent than retigabine at shifting the voltage dependence of KCNQ2/3 channels to more negative voltages. Moreover, unlike retigabine, SF0034 did not shift the voltage dependence of either KCNQ4 or KCNQ5 homomeric channels. Conditional deletion of Kcnq2 from cerebral cortical pyramidal neurons showed that SF0034 requires the expression of KCNQ2/3 channels for reducing the excitability of CA1 hippocampal neurons. Behavioral studies demonstrated that SF0034 was a more potent and less toxic anticonvulsant than retigabine in rodents. Furthermore, SF0034 prevented the development of tinnitus in mice. We propose that SF0034 provides, not only a powerful tool for investigating ion channel properties, but, most importantly, it provides a clinical candidate for treating epilepsy and preventing tinnitus. PMID:26063916

  11. Screening of Transient Receptor Potential Canonical Channel Activators Identifies Novel Neurotrophic Piperazine Compounds.

    PubMed

    Sawamura, Seishiro; Hatano, Masahiko; Takada, Yoshinori; Hino, Kyosuke; Kawamura, Tetsuya; Tanikawa, Jun; Nakagawa, Hiroshi; Hase, Hideharu; Nakao, Akito; Hirano, Mitsuru; Rotrattanadumrong, Rachapun; Kiyonaka, Shigeki; Mori, Masayuki X; Nishida, Motohiro; Hu, Yaopeng; Inoue, Ryuji; Nagata, Ryu; Mori, Yasuo

    2016-03-01

    Transient receptor potential canonical (TRPC) proteins form Ca(2+)-permeable cation channels activated upon stimulation of metabotropic receptors coupled to phospholipase C. Among the TRPC subfamily, TRPC3 and TRPC6 channels activated directly by diacylglycerol (DAG) play important roles in brain-derived neurotrophic factor (BDNF) signaling, promoting neuronal development and survival. In various disease models, BDNF restores neurologic deficits, but its therapeutic potential is limited by its poor pharmacokinetic profile. Elucidation of a framework for designing small molecules, which elicit BDNF-like activity via TRPC3 and TRPC6, establishes a solid basis to overcome this limitation. We discovered, through library screening, a group of piperazine-derived compounds that activate DAG-activated TRPC3/TRPC6/TRPC7 channels. The compounds [4-(5-chloro-2-methylphenyl)piperazin-1-yl](3-fluorophenyl)methanone (PPZ1) and 2-[4-(2,3-dimethylphenyl)piperazin-1-yl]-N-(2-ethoxyphenyl)acetamide (PPZ2) activated, in a dose-dependent manner, recombinant TRPC3/TRPC6/TRPC7 channels, but not other TRPCs, in human embryonic kidney cells. PPZ2 activated native TRPC6-like channels in smooth muscle cells isolated from rabbit portal vein. Also, PPZ2 evoked cation currents and Ca(2+) influx in rat cultured central neurons. Strikingly, both compounds induced BDNF-like neurite growth and neuroprotection, which were abolished by a knockdown or inhibition of TRPC3/TRPC6/TRPC7 in cultured neurons. Inhibitors of Ca(2+) signaling pathways, except calcineurin, impaired neurite outgrowth promotion induced by PPZ compounds. PPZ2 increased activation of the Ca(2+)-dependent transcription factor, cAMP response element-binding protein. These findings suggest that Ca(2+) signaling mediated by activation of DAG-activated TRPC channels underlies neurotrophic effects of PPZ compounds. Thus, piperazine-derived activators of DAG-activated TRPC channels provide important insights for future development of a

  12. 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. PMID:24158430

  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. Toward the rational design of constitutively active KCa3.1 mutant channels.

    PubMed

    Garneau, Line; Klein, Hélène; Parent, Lucie; Sauvé, Rémy

    2010-01-01

    The Ca²+ activated potassium channel of intermediate conductance KCa3.1 is now emerging as a therapeutic target for a large variety of health disorders. KCa3.1 is a tetrameric membrane protein with each subunit formed of six transmembrane helices (S1-S6). Ca²+ sensitivity is conferred by the Ca²+ binding protein calmodulin (CaM), with the CaM C-lobe constitutively bound to an intracellular domain of the channel C-terminus, located proximal to the membrane and connected to the S6 transmembrane segment. Patch clamp single channel recordings have demonstrated that binding of Ca²+ to CaM allows the channel to transit dose dependently from a nonconducting to an ion-conducting configuration. Here we present a general strategy to generate KCa3.1 mutant channels that remain in an ion-conducting state in the absence of Ca²+. Our strategy is first based on the production of a 3D model of the channel pore region, followed by SCAM experiments to confirm that residues along each of the channel S6 transmembrane helix form the channel pore lumen as predicted. In a simple model, constitutive activity can be obtained by removing the steric hindrances inside the channel pore susceptible to prevent ion flow when the channel is in the closed configuration. Using charged MTS reagents and Ag+ ions as probes acting on Cys residues engineered in the pore lumen, we found that the S6 transmembrane helices of KCa3.1 cannot form a pore constriction tight enough to prevent ion flow for channels in the closed state. These observations ruled out experimental strategies where constitutive activity would be generated by producing a "leaky" closed channel. A more successful approach consisted however in perturbing the channel open/closed state equilibrium free energy. In particular, we found that substituting the hydrophobic residue V282 in S6 by hydrophilic amino acids could lock the channel in an open-like state, resulting in channels that were ion conducting in the absence of Ca²+. PMID

  15. Differential distribution of the sodium-activated potassium channels slick and slack in mouse brain.

    PubMed

    Rizzi, Sandra; Knaus, Hans-Günther; Schwarzer, Christoph

    2016-07-01

    The sodium-activated potassium channels Slick (Slo2.1, KCNT2) and Slack (Slo2.2, KCNT1) are high-conductance potassium channels of the Slo family. In neurons, Slick and Slack channels are involved in the generation of slow afterhyperpolarization, in the regulation of firing patterns, and in setting and stabilizing the resting membrane potential. The distribution and subcellular localization of Slick and Slack channels in the mouse brain have not yet been established in detail. The present study addresses this issue through in situ hybridization and immunohistochemistry. Both channels were widely distributed and exhibited distinct distribution patterns. However, in some brain regions, their expression overlapped. Intense Slick channel immunoreactivity was observed in processes, varicosities, and neuronal cell bodies of the olfactory bulb, granular zones of cortical regions, hippocampus, amygdala, lateral septal nuclei, certain hypothalamic and midbrain nuclei, and several regions of the brainstem. The Slack channel showed primarily a diffuse immunostaining pattern, and labeling of cell somata and processes was observed only occasionally. The highest Slack channel expression was detected in the olfactory bulb, lateral septal nuclei, basal ganglia, and distinct areas of the midbrain, brainstem, and cerebellar cortex. In addition, comparing our data obtained from mouse brain with a previously published study on rat brain revealed some differences in the expression and distribution of Slick and Slack channels in these species. J. Comp. Neurol. 524:2093-2116, 2016. © 2015 The Authors The Journal of Comparative Neurology Published by Wiley Periodicals, Inc. PMID:26587966

  16. 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

  17. 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

  18. Paradoxical contribution of SK3 and GIRK channels to the activation of mouse vomeronasal organ.

    PubMed

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

    2012-09-01

    The vomeronasal organ (VNO) is essential for intraspecies communication in many terrestrial vertebrates. The ionic mechanisms of VNO activation remain unclear. We found that the calcium-activated potassium channel SK3 and the G protein-activated potassium channel GIRK are part of an independent pathway for VNO activation. In slice preparations, the potassium channels attenuated inward currents carried by TRPC2 and calcium-activated chloride channels (CACCs). In intact tissue preparations, paradoxically, the potassium channels enhanced urine-evoked inward currents. This discrepancy resulted from the loss of a high concentration of lumenal potassium, which enabled the influx of potassium ions to depolarize the VNO neurons in vivo. Both Sk3 (also known as Kcnn3) and Girk1 (also known as Kcnj3) homozygous null mice showed deficits in mating and aggressive behaviors, and the deficiencies in Sk3(-/-) mice were exacerbated by Trpc2 knockout. Our results suggest that VNO activation is mediated by TRPC2, CACCs and two potassium channels, all of which contributed to the in vivo depolarization of VNO neurons. PMID:22842147

  19. 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

  20. Dendrotoxins: structure-activity relationships and effects on potassium ion channels.

    PubMed

    Harvey, A L; Robertson, B

    2004-12-01

    Dendrotoxins are small proteins isolated from mamba (Dendroaspis) snakes. The original dendrotoxin was found in venom of the Eastern green mamba, Dendroaspis angusticeps, and related proteins were subsequently found in other mamba venoms. The dendrotoxins contain 57-60 amino acid residues cross-linked by three disulphide bridges, and they are homologous to Kunitz-type serine protease inhibitors, such as aprotinin (BPTI). The dendrotoxins have little or no anti-protease activity, but they block particular subtypes of voltage-dependent potassium channels of the Kv1 subfamily in neurones. Alpha-dendrotoxin from green mamba Dendroaspis angusticeps and toxin I from the black mamba Dendroaspis polylepis block cloned Kv1.1, Kv1.2 and Kv1.6 channels in the low nanomolar range; toxin K, also from the black mamba Dendroaspis polylepis, preferentially blocks Kv1.1 channels and is active at picomolar concentrations. Structural modifications and mutations to dendrotoxins have helped to define the molecular recognition properties of different types of K+ channels, although more work is needed to characterise the chemical features of the toxins that underlie their selectivity and potency at particular subtypes of channels. Dendrotoxins have been useful markers of subtypes of K+ channels in vivo, and dendrotoxins have become widely used as probes for studying the function of K+ channels in physiology and pathophysiology. With some pathological conditions being associated with voltage-gated K+ channels, analogues of dendrotoxins might have therapeutic potential. PMID:15579000

  1. 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

  2. Mechanisms of Rose Bengal inhibition on SecA ATPase and ion channel activities.

    PubMed

    Hsieh, Ying-Hsin; Huang, Ying-Ju; Jin, Jin-Shan; Yu, Liyan; Yang, Hsiuchin; Jiang, Chun; Wang, Binghe; Tai, Phang C

    2014-11-14

    SecA is an essential protein possessing ATPase activity in bacterial protein translocation for which Rose Bengal (RB) is the first reported sub-micromolar inhibitor in ATPase activity and protein translocation. Here, we examined the mechanisms of inhibition on various forms of SecA ATPase by conventional enzymatic assays, and by monitoring the SecA-dependent channel activity in the semi-physiological system in cells. We build on the previous observation that SecA with liposomes form active protein-conducting channels in the oocytes. Such ion channel activity is enhanced by purified Escherichia coli SecYEG-SecDF·YajC liposome complexes. Inhibition by RB could be monitored, providing correlation of in vitro activity and intact cell functionality. In this work, we found the intrinsic SecA ATPase is inhibited by RB competitively at low ATP concentration, and non-competitively at high ATP concentrations while the translocation ATPase with precursors and SecYEG is inhibited non-competitively by RB. The Inhibition by RB on SecA channel activity in the oocytes with exogenous ATP-Mg(2+), mimicking translocation ATPase activity, is also non-competitive. The non-competitive inhibition on channel activity has also been observed with SecA from other bacteria which otherwise would be difficult to examine without the cognate precursors and membranes. PMID:25450394

  3. Active Urbanization and Channel Adjustment in Apple Creek, Appleton, WI

    NASA Astrophysics Data System (ADS)

    Clark, J. J.

    2002-12-01

    Headwaters of the Apple Creek watershed have been and continue to be rapidly developed as part of the City of Appleton's long-term growth plan. Concurrent with early development, and prior to development over the past 4 years, two regional stormwater management facilities were constructed. Cross-sectional surveys and core transects were used to determine channel response to urbanization mitigated by stormwater management. The reach immediately downstream of the first pond complex has a narrow, but well established, wooded riparian zone and has not changed in size or shape over the past two years. An engineered reach approximately one mile downstream, however has exhibited widespread bed aggradation. Cross-sectional area decreased an average of 51% over the past four years. Despite the use of sediment and erosion control BMPs, sediment concentrations exceeding 1000 mg/L during base flow are not uncommon downstream of construction sites adjacent to the stream. The artificially widened channel, a reduction in stream gradient, and the backwater effect from downstream ponds caused much of this sediment to remain within the engineered reach. It is estimated that approximately 21,000 Mg of sediment is stored in this mile-long reach. As this sediment migrates downstream, the forebay of the second set of stormwater ponds will begin to fill, reducing storage capacity and thereby limiting its effectiveness in mitigating peak discharges and sequestering nutrients.

  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. Exploring the biophysical evidence that mammalian two-pore channels are NAADP-activated calcium-permeable channels.

    PubMed

    Pitt, Samantha J; Reilly-O'Donnell, Benedict; Sitsapesan, Rebecca

    2016-08-01

    Nicotinic acid adenine dinucleotide phosphate (NAADP) potently releases Ca(2+) from acidic intracellular endolysosomal Ca(2+) stores. It is widely accepted that two types of two-pore channels, termed TPC1 and TPC2, are responsible for the NAADP-mediated Ca(2+) release but the underlying mechanisms regulating their gating appear to be different. For example, although both TPC1 and TPC2 are activated by NAADP, TPC1 appears to be additionally regulated by cytosolic Ca(2+) . Ion conduction and permeability also differ markedly. TPC1 and TPC2 are permeable to a range of cations although biophysical experiments suggest that TPC2 is slightly more selective for Ca(2+) over K(+) than TPC1 and hence capable of releasing greater quantities of Ca(2+) from acidic stores. TPC1 is also permeable to H(+) and therefore may play a role in regulating lysosomal and cytosolic pH, possibly creating localised acidic domains. The significantly different gating and ion conducting properties of TPC1 and TPC2 suggest that these two ion channels may play complementary physiological roles as Ca(2+) -release channels of the endolysosomal system. PMID:26872338

  6. Preferential Phosphorylation of R-domain Serine 768 Dampens Activation of CFTR Channels by PKA

    PubMed Central

    Csanády, László; Seto-Young, Donna; Chan, Kim W.; Cenciarelli, Cristina; Angel, Benjamin B.; Qin, Jun; McLachlin, Derek T.; Krutchinsky, Andrew N.; Chait, Brian T.; Nairn, Angus C.; Gadsby, David C.

    2005-01-01

    CFTR (cystic fibrosis transmembrane conductance regulator), the protein whose dysfunction causes cystic fibrosis, is a chloride ion channel whose gating is controlled by interactions of MgATP with CFTR's two cytoplasmic nucleotide binding domains, but only after several serines in CFTR's regulatory (R) domain have been phosphorylated by cAMP-dependent protein kinase (PKA). Whereas eight R-domain serines have previously been shown to be phosphorylated in purified CFTR, it is not known how individual phosphoserines regulate channel gating, although two of them, at positions 737 and 768, have been suggested to be inhibitory. Here we show, using mass spectrometric analysis, that Ser 768 is the first site phosphorylated in purified R-domain protein, and that it and five other R-domain sites are already phosphorylated in resting Xenopus oocytes expressing wild-type (WT) human epithelial CFTR. The WT channels have lower activity than S768A channels (with Ser 768 mutated to Ala) in resting oocytes, confirming the inhibitory influence of phosphoserine 768. In excised patches exposed to a range of PKA concentrations, the open probability (Po) of mutant S768A channels exceeded that of WT CFTR channels at all [PKA], and the half-maximally activating [PKA] for WT channels was twice that for S768A channels. As the open burst duration of S768A CFTR channels was almost double that of WT channels, at both low (55 nM) and high (550 nM) [PKA], we conclude that the principal mechanism by which phosphoserine 768 inhibits WT CFTR is by hastening the termination of open channel bursts. The right-shifted Po-[PKA] curve of WT channels might explain their slower activation, compared with S768A channels, at low [PKA]. The finding that phosphorylation kinetics of WT or S768A R-domain peptides were similar provides no support for an alternative explanation, that early phosphorylation of Ser 768 in WT CFTR might also impair subsequent phosphorylation of stimulatory R-domain serines. The

  7. 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. PMID:25456079

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

    PubMed

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

    2016-02-19

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

  9. 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

  10. On Application Of Langevin Dynamics In Logarithmic Potential To Model Ion Channel Gate Activity.

    PubMed

    Wawrzkiewicz-Jałowiecka, Agata; Borys, Przemysław; Grzywna, Zbigniew J

    2015-12-01

    We model the activity of an ion channel gate by Langevin dynamics in a logarithmic potential. This approach enables one to describe the power-law dwell-time distributions of the considered system, and the long-term correlations between the durations of the subsequent channel states, or fractal scaling of statistical characteristics of the gate's movement with time. Activity of an ion channel gate is described as an overdamped motion of the reaction coordinate in a confining logarithmic potential, which ensures great flexibility of the model. Depending on the chosen parameters, it allows one to reproduce many types of gate dynamics within the family of non-Markovian, anomalous conformational diffusion processes. In this study we apply the constructed model to largeconductance voltage and Ca2+-activated potassium channels (BKCa). The interpretation of model assumptions and parameters is provided in terms of this biological system. Our results show good agreement with the experimental data. PMID:26317442

  11. Compensation effects and relation between the activation energy of spin transition and the hysteresis loop width for an iron(ii) complex.

    PubMed

    Bushuev, Mark B; Pishchur, Denis P; Nikolaenkova, Elena B; Krivopalov, Viktor P

    2016-06-22

    The enthalpy-entropy compensation was observed for the cooperative → spin transition (the phase is a mononuclear complex [FeL2](BF4)2, L is 4-(3,5-dimethyl-1H-pyrazol-1-yl)-2-(pyridin-2-yl)-6-methylpyrimidine). The physical origin of this effect is the fact that the → spin transition is the first order phase transition accompanied by noticeable variations in the Tonset↑, ΔH and ΔS values. Higher ΔH and ΔS values are correlated with higher Tonset↑ values. The higher the enthalpy and entropy of the spin transition, the wider the hysteresis loop. The kinetic compensation effect, i.e. a linear relationship between ln A and Ea, was observed for the → spin transition. Moreover, an isokinetic relationship was detected in this system: the Arrhenius lines (ln k vs. 1/T) obtained from magnetochemical data for different samples of the phase undergoing the → transition show a common point of intersection (Tiso = 490 ± 2 K, ln kiso = -6.0 ± 0.2). The validity of this conclusion was confirmed by the Exner-Linert statistical method. This means that the isokinetic relationship and the kinetic compensation effect (ln A vs. Ea) in this system are true ones. The existence of a true kinetic compensation effect is supported independently by the fact that the hysteresis loop width for the cooperative spin transition ↔ increases with increasing activation barrier height. Estimating the energy of excitations for the phase with Tiso ∼ 490 K yields wavenumbers of ca. 340 cm(-1) corresponding to the frequencies of the stretching vibrations of the Fe(LS)-N bonds, i.e. the bonds directly involved in the mechanism of the spin transition. This is the first observation of the kinetic compensation effect (ln A vs. Ea) and the isokinetic relationship for a cooperative spin crossover system showing thermal hysteresis. Our results provide the first experimental evidence that the higher the activation barrier for the spin transition, the wider the hysteresis loop for a

  12. Block by ruthenium red of the ryanodine-activated calcium release channel of skeletal muscle

    PubMed Central

    1993-01-01

    The effects of ruthenium red and the related compounds tetraamine palladium (4APd) and tetraamine platinum (4APt) were studied on the ryanodine activated Ca2+ release channel reconstituted in planar bilayers with the immunoaffinity purified ryanodine receptor. Ruthenium red, applied at submicromolar concentrations to the myoplasmic side (cis), induced an all-or-none flickery block of the ryanodine activated channel. The blocking effect was strongly voltage dependent, as large positive potentials that favored the movement of ruthenium red into the channel conduction pore produced stronger block. The half dissociation constants (Kd) for ruthenium red block of the 500 pS channel were 0.22, 0.38, and 0.62 microM, at +100, +80, and +60 mV, respectively. Multiple ruthenium red molecules seemed to be involved in the inhibition, because a Hill coefficient of close to 2 was obtained from the dose response curve. The half dissociation constant of ruthenium red block of the lower conductance state of the ryanodine activated channel (250 pS) was higher (Kd = 0.82 microM at +100 mV), while the Hill coefficient remained approximately the same (nH = 2.7). Ruthenium red block of the channel was highly asymmetric, as trans ruthenium red produced a different blocking effect. The blocking and unblocking events (induced by cis ruthenium red) can be resolved at the single channel level at a cutoff frequency of 2 kHz. The closing rate of the channel in the presence of ruthenium red increased linearly with ruthenium red concentration, and the unblocking rate of the channel was independent of ruthenium red concentrations. This suggests that ruthenium red block of the channel occurred via a simple blocking mechanism. The on-rate of ruthenium red binding to the channel was 1.32 x 10(9) M-1 s-1, and the off-rate of ruthenium red binding was 0.75 x 10(3) s-1 at +60 mV, in the presence of 200 nM ryanodine. The two related compounds, 4APd and 4APt, blocked the channel in a similar way to that

  13. Single-Channel Kinetic Analysis for Activation and Desensitization of Homomeric 5-HT3A Receptors

    PubMed Central

    Corradi, Jeremías; Gumilar, Fernanda; Bouzat, Cecilia

    2009-01-01

    Abstract The 5-HT3A receptor is a member of the Cys-loop family of ligand-gated ion channels. To perform kinetic analysis, we mutated the 5-HT3A subunit to obtain a high-conductance form so that single-channel currents can be detected. At all 5-HT concentrations (>0.1 μM), channel activity appears as openings in quick succession that form bursts, which coalesce into clusters. By combining single-channel and macroscopic data, we generated a kinetic model that perfectly describes activation, deactivation, and desensitization. The model shows that full activation arises from receptors with three molecules of agonist bound. It reveals an earlier conformational change of the fully liganded receptor that occurs while the channel is still closed. From this pre-open closed state, the receptor enters into an open-closed cycle involving three open states, which form the cluster whose duration parallels the time constant of desensitization. A similar model lacking the pre-open closed state can describe the data only if the opening rates are fixed to account for the slow activation rate. The application of the model to M4 mutant receptors shows that position 10′ contributes to channel opening and closing rates. Thus, our kinetic model provides a foundation for understanding structural bases of activation and drug action. PMID:19720021

  14. Calcium-channel number critically influences synaptic strength and plasticity at the active zone

    PubMed Central

    Sheng, Jiansong; He, Liming; Zheng, Hongwei; Xue, Lei; Luo, Fujun; Shin, Wonchul; Sun, Tao; Kuner, Thomas; Yue, David T; Wu, Ling-Gang

    2016-01-01

    How synaptic-vesicle release is controlled at the basic release structure, the active zone, is poorly understood. By performing cell-attached current and capacitance recordings predominantly at single active zones in rat calyces, we found that single active zones contained 5-218 (mean, 42) calcium channels and 1–10 (mean, 5) readily releasable vesicles (RRVs) and released 0–5 vesicles during a 2-ms depolarization. Large variation in the number of calcium channels caused wide variation in release strength (measured during a 2-ms depolarization) by regulating the RRV release probability (PRRV) and the RRV number. Consequently, an action potential opened ~1–35 (mean, ~7) channels, resulting in different release probabilities at different active zones. As the number of calcium-channels determined PRRV, it critically influenced whether subsequent release would be facilitated or depressed. Regulating calcium channel density at active zones may thus be a major mechanism to yield synapses with different release properties and plasticity. These findings may explain large differences reported at synapses regarding release strength (release of 0, 1 or multiple vesicles), PRRV, short-term plasticity, calcium transients and the requisite calcium-channel number for triggering release. PMID:22683682

  15. Activation of TRPC cationic channels by mercurial compounds confers the cytotoxicity of mercury exposure.

    PubMed

    Xu, Shang-Zhong; Zeng, Bo; Daskoulidou, Nikoleta; Chen, Gui-Lan; Atkin, Stephen L; Lukhele, Bhekithemba

    2012-01-01

    Mercury is an established worldwide environmental pollutant with well-known toxicity affecting neurodevelopment in humans, but the molecular basis of cytotoxicity and the detoxification procedure are still unclear. Here we examined the involvement of the canonical transient receptor potential (TRPC) channel in the mercury-induced cytotoxicity and the potential detoxification strategy. Whole-cell and excised patches, Ca(2+) imaging, and site-directed mutagenesis were used to determine the mechanism of action of mercurial compounds on TRPC channels overexpressed in HEK293 cells, and cytotoxicity and preventive effect were investigated in cell culture models using small interfering RNA and pharmacological blockers. Mercury potently activates TRPC4 and TRPC5 channels. The extracellular cysteine residues (C(553) and C(558)) near the channel pore region of TRPC5 are the molecular targets for channel activation by mercury. The sensitivity of mercury to TRPC5 is presumed to be specific because other divalent heavy metal pollutants, such as Cd(2+), Ni(2+), and Zn(2+), had no stimulating effect, and TRPC3, TRPC6, TRPV1, and TRPM2 were resistant to mercurial compounds. The channel activity of TRPC5, as well as TRPC4, induced by mercury, was prevented by 2-aminoethoxydiphenyl borate and modified by a reducing environment. The inhibition of TRPC5 channels by specific TRPC5 pore-blocking antibody or by SKF-96365 alleviated the cytotoxicity, whereas the mercury chelator, meso-2,3-dimercaptosuccinic acid, showed nonselective prevention of cell survival. Silencing of the TRPC5 gene reduced the mercury-induced neuronal damage. These results indicate that mercurial compounds are activators for TRPC5 and TRPC4 channels. Blockade of TRPC channels could be a novel strategy for preventing mercury-induced cytotoxicity and neurodevelopment impairment. PMID:21984481

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

    PubMed Central

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

    2016-01-01

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

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

    PubMed

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

    2016-01-01

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

  18. Modulation of Ca2+ oscillation and melatonin secretion by BKCa channel activity in rat pinealocytes.

    PubMed

    Mizutani, Hiroya; Yamamura, Hisao; Muramatsu, Makoto; Hagihara, Yumiko; Suzuki, Yoshiaki; Imaizumi, Yuji

    2016-05-01

    The pineal glands regulate circadian rhythm through the synthesis and secretion of melatonin. The stimulation of nicotinic acetylcholine receptor due to parasympathetic nerve activity causes an increase in intracellular Ca(2+) concentration and eventually downregulates melatonin production. Our previous report shows that rat pinealocytes have spontaneous and nicotine-induced Ca(2+) oscillations that are evoked by membrane depolarization followed by Ca(2+) influx through voltage-dependent Ca(2+) channels (VDCCs). These Ca(2+) oscillations are supposed to contribute to the inhibitory mechanism of melatonin secretion. Here we examined the involvement of large-conductance Ca(2+)-activated K(+) (BKCa) channel conductance on the regulation of Ca(2+) oscillation and melatonin production in rat pinealocytes. Spontaneous Ca(2+) oscillations were markedly enhanced by BKCa channel blockers (1 μM paxilline or 100 nM iberiotoxin). Nicotine (100 μM)-induced Ca(2+) oscillations were also augmented by paxilline. In contrast, spontaneous Ca(2+) oscillations were abolished by BKCa channel opener [3 μM 12,14-dichlorodehydroabietic acid (diCl-DHAA)]. Under whole cell voltage-clamp configurations, depolarization-elicited outward currents were significantly activated by diCl-DHAA and blocked by paxilline. Expression analyses revealed that the α and β3 subunits of BKCa channel were highly expressed in rat pinealocytes. Importantly, the activity of BKCa channels modulated melatonin secretion from whole pineal gland of the rat. Taken together, BKCa channel activation attenuates these Ca(2+) oscillations due to depolarization-synchronized Ca(2+) influx through VDCCs and results in a recovery of reduced melatonin secretion during parasympathetic nerve activity. BKCa channels may play a physiological role for melatonin production via a negative-feedback mechanism. PMID:26791489

  19. Alterations in Membrane Caveolae and BKCa Channel Activity in Skin Fibroblasts in Smith-Lemli-Opitz Syndrome

    PubMed Central

    Ren, Gongyi; Jacob, Robert F.; Kaulin, Yuri; DiMuzio, Paul; Xie, Yi; Mason, R. Preston; Tint, G. Stephen; Steiner, Robert D.; Roulett, Jean-Baptiste; Merkens, Louise; Whitaker-Mendez, Diana; Frank, Phillipe G.; Lisanti, Michael; Cox, Robert H.; Tulenko, Thomas N.

    2011-01-01

    The Smith-Lemli-Opitz syndrome (SLOS) is an inherited disorder of cholesterol synthesis caused by mutations in DHCR7 which encodes the final enzyme in the cholesterol synthesis pathway. The immediate precursor to cholesterol synthesis, 7-dehydrocholesterol (7-DHC) accumulates in the plasma and cells of SLOS patients which has led to the idea that the accumulation of abnormal sterols and/or reduction in cholesterol underlies the phenotypic abnormalities of SLOS. We tested the hypothesis that 7-DHC accumulates in membrane caveolae where it disturbs caveolar bilayer structure-function. Membrane caveolae from skin fibroblasts obtained from SLOS patients were isolated and found to accumulate 7-DHC. In caveolar-like model membranes containing 7-DHC, subtle, but complex alterations in intermolecular packing, lipid order and membrane width were observed. In addition, the BKCa K+ channel, which co-migrates with caveolin-1 in a membrane fraction enriched with cholesterol, was impaired in SLOS cells as reflected by reduced single channel conductance and a 50 mV rightward shift in the channel activation voltage. In addition, a marked decrease in BKCa protein but not mRNA expression levels were seen suggesting post-translational alterations. Accompanying these changes was a reduction in caveolin-1 protein and mRNA levels, but membrane caveolar structure was not altered. These results are consistent with the hypothesis that 7-DHC accumulation in the caveolar membrane results in defective caveolar signaling. However, additional cellular alterations beyond mere changes associated with abnormal sterols in the membrane likely contribute to the pathogenesis of SLOS. PMID:21724437

  20. Cortical Axons, Isolated in Channels, Display Activity-Dependent Signal Modulation as a Result of Targeted Stimulation

    PubMed Central

    Lewandowska, Marta K.; Radivojević, Miloš; Jäckel, David; Müller, Jan; Hierlemann, Andreas R.

    2016-01-01

    Mammalian cortical axons are extremely thin processes that are difficult to study as a result of their small diameter: they are too narrow to patch while intact, and super-resolution microscopy is needed to resolve single axons. We present a method for studying axonal physiology by pairing a high-density microelectrode array with a microfluidic axonal isolation device, and use it to study activity-dependent modulation of axonal signal propagation evoked by stimulation near the soma. Up to three axonal branches from a single neuron, isolated in different channels, were recorded from simultaneously using 10–20 electrodes per channel. The axonal channels amplified spikes such that propagations of individual signals along tens of electrodes could easily be discerned with high signal to noise. Stimulation from 10 up to 160 Hz demonstrated similar qualitative results from all of the cells studied: extracellular action potential characteristics changed drastically in response to stimulation. Spike height decreased, spike width increased, and latency increased, as a result of reduced propagation velocity, as the number of stimulations and the stimulation frequencies increased. Quantitatively, the strength of these changes manifested itself differently in cells at different frequencies of stimulation. Some cells' signal fidelity fell to 80% already at 10 Hz, while others maintained 80% signal fidelity at 80 Hz. Differences in modulation by axonal branches of the same cell were also seen for different stimulation frequencies, starting at 10 Hz. Potassium ion concentration changes altered the behavior of the cells causing propagation failures at lower concentrations and improving signal fidelity at higher concentrations. PMID:27013945

  1. Cortical Axons, Isolated in Channels, Display Activity-Dependent Signal Modulation as a Result of Targeted Stimulation.

    PubMed

    Lewandowska, Marta K; Radivojević, Miloš; Jäckel, David; Müller, Jan; Hierlemann, Andreas R

    2016-01-01

    Mammalian cortical axons are extremely thin processes that are difficult to study as a result of their small diameter: they are too narrow to patch while intact, and super-resolution microscopy is needed to resolve single axons. We present a method for studying axonal physiology by pairing a high-density microelectrode array with a microfluidic axonal isolation device, and use it to study activity-dependent modulation of axonal signal propagation evoked by stimulation near the soma. Up to three axonal branches from a single neuron, isolated in different channels, were recorded from simultaneously using 10-20 electrodes per channel. The axonal channels amplified spikes such that propagations of individual signals along tens of electrodes could easily be discerned with high signal to noise. Stimulation from 10 up to 160 Hz demonstrated similar qualitative results from all of the cells studied: extracellular action potential characteristics changed drastically in response to stimulation. Spike height decreased, spike width increased, and latency increased, as a result of reduced propagation velocity, as the number of stimulations and the stimulation frequencies increased. Quantitatively, the strength of these changes manifested itself differently in cells at different frequencies of stimulation. Some cells' signal fidelity fell to 80% already at 10 Hz, while others maintained 80% signal fidelity at 80 Hz. Differences in modulation by axonal branches of the same cell were also seen for different stimulation frequencies, starting at 10 Hz. Potassium ion concentration changes altered the behavior of the cells causing propagation failures at lower concentrations and improving signal fidelity at higher concentrations. PMID:27013945

  2. Vanilloid receptor-related osmotically activated channel (VR-OAC), a candidate vertebrate osmoreceptor

    PubMed Central

    Liedtke, Wolfgang; Choe, Yong; Martí-Renom, Marc A.; Bell, Andrea M.; Denis, Charlotte S.; Šali, Andrej; Hudspeth, A. J.; Friedman, Jeffrey M.; Heller, Stefan

    2008-01-01

    SUMMARY The detection of osmotic stimuli is essential for all organisms, yet few osmoreceptive proteins are known, none of them in vertebrates. By employing a candidate-gene approach based on genes encoding members of the TRP superfamily of ion channels, we cloned cDNAs encoding the vanilloid receptor-related osmotically activated channel (VR-OAC) from the rat, mouse, human, and chicken. This novel cation-selective channel is gated by exposure to hypotonicity within the physiological range. In the central nevous system, the channel is expressed neurons of the circumventricular organs, neurosensory cells responsive to systemic osmotic pressure. The channel also occurs in other neurosensory cells, including inner-ear hair cells, sensory neurons, and Merkel cells. PMID:11081638

  3. Mechanisms of STIM1 Activation of Store-Independent Leukotriene C4-Regulated Ca2+ Channels

    PubMed Central

    Zhang, Xuexin; González-Cobos, José C.; Schindl, Rainer; Muik, Martin; Ruhle, Brian; Motiani, Rajender K.; Bisaillon, Jonathan M.; Zhang, Wei; Fahrner, Marc; Barroso, Margarida; Matrougui, Khalid; Romanin, Christoph

    2013-01-01

    We recently showed, in primary vascular smooth muscle cells (VSMCs), that the platelet-derived growth factor activates canonical store-operated Ca2+ entry and Ca2+ release-activated Ca2+ currents encoded by Orai1 and STIM1 genes. However, thrombin activates store-independent Ca2+ selective channels contributed by both Orai3 and Orai1. These store-independent Orai3/Orai1 channels are gated by cytosolic leukotriene C4 (LTC4) and require STIM1 downstream LTC4 action. However, the source of LTC4 and the signaling mechanisms of STIM1 in the activation of this LTC4-regulated Ca2+ (LRC) channel are unknown. Here, we show that upon thrombin stimulation, LTC4 is produced through the sequential activities of phospholipase C, diacylglycerol lipase, 5-lipo-oxygenease, and leukotriene C4 synthase. We show that the endoplasmic reticulum-resident STIM1 is necessary and sufficient for LRC channel activation by thrombin. STIM1 does not form sustained puncta and does not colocalize with Orai1 either under basal conditions or in response to thrombin. However, STIM1 is precoupled to Orai3 and Orai3/Orai1 channels under basal conditions as shown using Forster resonance energy transfer (FRET) imaging. The second coiled-coil domain of STIM1 is required for coupling to either Orai3 or Orai3/Orai1 channels and for LRC channel activation. We conclude that STIM1 employs distinct mechanisms in the activation of store-dependent and store-independent Ca2+ entry pathways. PMID:23878392

  4. Gadolinium inhibits mechanoelectrical transduction in rabbit carotid baroreceptors. Implication of stretch-activated channels.

    PubMed Central

    Hajduczok, G; Chapleau, M W; Ferlic, R J; Mao, H Z; Abboud, F M

    1994-01-01

    Gadolinium (Gd3+) has been shown to prevent mechanoelectrical transduction believed to be mediated through stretch-activated channels. We investigated the possible role of Gd(3+)-sensitive channels in mediating baroreceptor activity in the carotid sinus of rabbits. Baroreceptor activity induced by a ramp increase of carotid sinus pressure was reduced significantly during exposure to Gd3+. The inhibition was dose-related and reversible, and was not associated with alteration of carotid sinus wall mechanics as the pressure-strain relationship was unaffected. Veratrine triggered action potentials from single- and multiple-baroreceptor fibers when their response to pressure was inhibited by Gd3+. This suggests that the effect of Gd3+ on baroreceptors in the isolated carotid sinus was specific to their mechanical activation. The results suggest that stretch-activated ion channels sensitive to Gd3+ may be the mechanoelectrical transducers of rabbit carotid sinus baroreceptors. PMID:7527431

  5. 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

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

    PubMed Central

    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

  7. Chaotic model and memory in single calcium-activated potassium channel kinetics

    NASA Astrophysics Data System (ADS)

    Bandeira, Heliovânio T.; Barbosa, Catão T. F.; Campos De Oliveira, Regina A.; Aguiar, José F.; Nogueira, Romildo A.

    2008-09-01

    Ion channels are pores formed by proteins and responsible for carrying ion fluxes through cellular membranes. The ion channels can assume conformational states thereby controlling ion flow. Physically, the conformational transitions from one state to another are associated with energy barriers between them and are dependent on stimulus, such as, electrical field, ligands, second messengers, etc. Several models have been proposed to describe the kinetics of ion channels. The classical Markovian model assumes that a future transition is independent of the time that the ion channel stayed in a previous state. Others models as the fractal and the chaotic assume that the rate of transitions between the states depend on the time that the ionic channel stayed in a previous state. For the calcium activated potassium channels of Leydig cells the R/S Hurst analysis has indicated that the channels are long-term correlated with a Hurst coefficient H around 0.7, showing a persistent memory in this kinetic. Here, we applied the R /S analysis to the opening and closing dwell time series obtained from simulated data from a chaotic model proposed by L. Liebovitch and T. Tóth [J. Theor. Biol. 148, 243 (1991)] and we show that this chaotic model or any model that treats the set of channel openings and closings as independent events is inadequate to describe the long-term correlation (memory) already described for the experimental data.

  8. The vacuolar Ca2+-activated channel TPC1 regulates germination and stomatal movement.

    PubMed

    Peiter, Edgar; Maathuis, Frans J M; Mills, Lewis N; Knight, Heather; Pelloux, Jérôme; Hetherington, Alistair M; Sanders, Dale

    2005-03-17

    Cytosolic free calcium ([Ca2+]cyt) is a ubiquitous signalling component in plant cells. Numerous stimuli trigger sustained or transient elevations of [Ca2+]cyt that evoke downstream stimulus-specific responses. Generation of [Ca2+]cyt signals is effected through stimulus-induced opening of Ca2+-permeable ion channels that catalyse a flux of Ca2+ into the cytosol from extracellular or intracellular stores. Many classes of Ca2+ current have been characterized electrophysiologically in plant membranes. However, the identity of the ion channels that underlie these currents has until now remained obscure. Here we show that the TPC1 ('two-pore channel 1') gene of Arabidopsis thaliana encodes a class of Ca2+-dependent Ca2+-release channel that is known from numerous electrophysiological studies as the slow vacuolar channel. Slow vacuolar channels are ubiquitous in plant vacuoles, where they form the dominant conductance at micromolar [Ca2+]cyt. We show that a tpc1 knockout mutant lacks functional slow vacuolar channel activity and is defective in both abscisic acid-induced repression of germination and in the response of stomata to extracellular calcium. These studies unequivocally demonstrate a critical role of intracellular Ca2+-release channels in the physiological processes of plants. PMID:15772667

  9. 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

  10. Thermosensitive TRP channel pore turret is part of the temperature activation pathway

    PubMed Central

    Yang, Fan; Cui, Yuanyuan; Wang, KeWei; Zheng, Jie

    2010-01-01

    Temperature sensing is crucial for homeotherms, including human beings, to maintain a stable body core temperature and respond to the ambient environment. A group of exquisitely temperature-sensitive transient receptor potential channels, termed thermoTRPs, serve as cellular temperature sensors. How thermoTRPs convert thermal energy (heat) into protein conformational changes leading to channel opening remains unknown. Here we demonstrate that the pathway for temperature-dependent activation is distinct from those for ligand- and voltage-dependent activation and involves the pore turret. We found that mutant channels with an artificial pore turret sequence lose temperature sensitivity but maintain normal ligand responses. Using site-directed fluorescence recordings we observed that temperature change induces a significant rearrangement of TRPV1 pore turret that is coupled to channel opening. This movement is specifically associated to temperature-dependent activation and is not observed during ligand- and voltage-dependent channel activation. These observations suggest that the turret is part of the temperature-sensing apparatus in thermoTRP channels, and its conformational change may give rise to the large entropy that defines high temperature sensitivity. PMID:20351268

  11. Long-term correlation in single calcium-activated potassium channel kinetics

    NASA Astrophysics Data System (ADS)

    Campos de Oliveira, R. A.; Barbosa, C. T. F.; Consoni, L. H. A.; Rodrigues, A. R. A.; Varanda, W. A.; Nogueira, R. A.

    2006-05-01

    Ion channels are protein molecules found in biological membranes, which can assume distinct open and closed conformational states, a phenomenon called ion channel kinetics. The transitions from one state to another are dependent on the potential energy barrier that separates them and can be controlled by the electrical field, ions and/or drugs. Both Markovian and fractal models have been used for modeling the ion channel kinetics. Ion single channel records are characterized by successive openings and closings, which are correlated in time. Here the rescaled range analysis ( R/S Hurst analysis) is used to test for the occurrence of long-term correlation in the kinetics of a calcium-activated potassium channel of Leydig cells. A Hurst coefficient H=0.640±0.064 ( n=5) was found for the single calcium-activated potassium channel clamped at -80 mV and exposed to a free Ca 2+ concentration equal to 10 nM. This numerical value indicates the presence of long-term correlation (memory) in this kinetic process. However, when the R/ S analysis was applied to ion channel data simulated using Markovian and fractal models, it could not account for the long-term correlation previously found in the experimental data. In summary, in this work we show that: (i) opening and closing dwell times for the single calcium-activated potassium channel of Leydig cells present long-term correlation and (ii) Markovian and fractal models, which describe well the dwell time distributions, are not adequate to describe the memory found in the kinetics of this channel.

  12. 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.

  13. Divalent cations potentiate TRPV1 channel by lowering the heat activation threshold

    PubMed Central

    Cao, Xu; Ma, Linlin; Yang, Fan

    2014-01-01

    Transient receptor potential vanilloid type 1 (TRPV1) channel responds to a wide spectrum of physical and chemical stimuli. In doing so, it serves as a polymodal cellular sensor for temperature change and pain. Many chemicals are known to strongly potentiate TRPV1 activation, though how this is achieved remains unclear. In this study we investigated the molecular mechanism underlying the gating effects of divalent cations Mg2+ and Ba2+. Using a combination of fluorescence imaging and patch-clamp analysis, we found that these cations potentiate TRPV1 gating by most likely promoting the heat activation process. Mg2+ substantially lowers the activation threshold temperature; as a result, a significant fraction of channels are heat-activated at room temperature. Although Mg2+ also potentiates capsaicin- and voltage-dependent activation, these processes were found either to be not required (in the case of capsaicin) or insufficient (in the case of voltage) to mediate the activating effect. In support of a selective effect on heat activation, Mg2+ and Ba2+ cause a Ca2+-independent desensitization that specifically prevents heat-induced channel activation but does not prevent capsaicin-induced activation. These results can be satisfactorily explained within an allosteric gating framework in which divalent cations strongly promote the heat-dependent conformational change or its coupling to channel activation, which is further coupled to the voltage- and capsaicin-dependent processes. PMID:24344247

  14. 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. PMID:26748289

  15. Modulation of channel activity and gadolinium block of MscL by static magnetic fields.

    PubMed

    Petrov, Evgeny; Martinac, Boris

    2007-02-01

    The magnetic field of the Earth has for long been known to influence the behaviour and orientation of a variety of living organisms. Experimental studies of the magnetic sense have, however, been impaired by the lack of a plausible cellular and/or molecular mechanism providing meaningful explanation for detection of magnetic fields by these organisms. Recently, mechanosensitive (MS) ion channels have been implied to play a role in magnetoreception. In this study we have investigated the effect of static magnetic fields (SMFs) of moderate intensity on the activity and gadolinium block of MscL, the bacterial MS channel of large conductance, which has served as a model channel to study the basic physical principles of mechanosensory transduction in living cells. In addition to showing that direct application of the magnetic field decreased the activity of the MscL channel, our study demonstrates for the first time that SMFs can reverse the effect of gadolinium, a well-known blocker of MS channels. The results of our study are consistent with a notion that (1) the effects of SMFs on the MscL channels may result from changes in physical properties of the lipid bilayer due to diamagnetic anisotropy of phospholipid molecules and consequently (2) cooperative superdiamagnetism of phospholipid molecules under influence of SMFs could cause displacement of Gd(3+) ions from the membrane bilayer and thus remove the MscL channel block. PMID:17089151

  16. 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

  17. Urinary Bladder-Relaxant Effect of Kurarinone Depending on Potentiation of Large-Conductance Ca2+-Activated K+ Channels.

    PubMed

    Lee, Sojung; Chae, Mee Ree; Lee, Byoung-Cheol; Kim, Yong-Chul; Choi, Jae Sue; Lee, Sung Won; Cheong, Jae Hoon; Park, Chul-Seung

    2016-08-01

    The large-conductance calcium-activated potassium channel (BKCa channel) plays critical roles in smooth muscle relaxation. In urinary bladder smooth muscle, BKCa channel activity underlies the maintenance of the resting membrane potential and repolarization of the spontaneous action potential triggering the phasic contraction. To identify novel BKCa channel activators, we screened a library of natural compounds using a cell-based fluorescence assay and a hyperactive mutant BKCa channel (Lee et al., 2013). From 794 natural compounds, kurarinone, a flavanone from Sophora flavescens, strongly potentiated BKCa channels. When treated from the extracellular side, this compound progressively shifted the conductance-voltage relationship of BKCa channels to more negative voltages and increased the maximum conductance in a dose-dependent manner. Whereas kurarinone strongly potentiated the homomeric BKCa channel composed of only the α subunit, its effects were much smaller on heteromeric channels coassembled with auxiliary β subunits. Although the activation kinetics was not altered significantly, the deactivation of BKCa channels was dramatically slowed by kurarinone treatment. At the single-channel level, kurarinone increased the open probability of the BKCa channel without affecting its single-channel conductance. Kurarinone potently relaxed acetylcholine-induced contraction of rat bladder smooth muscle and thus decreased the micturition frequency of rats with overactive bladder symptoms. These results indicate that kurarinone can directly potentiate BKCa channels and demonstrate the therapeutic potentials of kurarinone and its derivatives for developing antioveractive bladder medications and supplements. PMID:27251362

  18. Width of nonlinear resonance

    SciTech Connect

    Ohnuma, S.

    1984-03-01

    Two approximations are made, one essential and the other not so essential but convenient to keep the analytical treatment manageable: (1) Only one nonlinear resonance is considered at a time so that the treatment is best suited when the tune is close to one resonance only. To improve this approximation, one must go to the next order which involves a canonical transformation of dynamical variables. Analytical treatment of more than one resonance is not possible for general cases. (2) In the formalism using the action-angle variables, the Hamiltonian can have terms which are independent of the angle variables. These terms are called phase-independent terms or shear terms. The tune is then a function of the oscillation amplitudes. In the lowest-order treatment, the (4N)-pole components but not the (4N + 2)-pole components contribute to this dependence. In deriving the resonance width analytically, one ignores these terms in the Hamiltonian for the sake of simplicity. If these are retained, one needs at least three extra parameters and the analytical treatment becomes rather unwieldy.

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

    PubMed

    Gururaj, Sushmitha; Fleites, John; Bhattacharjee, Arin

    2016-04-01

    p38 MAPK has long been understood as an inducible kinase under conditions of cellular stress, but there is now increasing evidence to support its role in the regulation of neuronal function. Several phosphorylation targets have been identified, an appreciable number of which are ion channels, implicating the possible involvement of p38 MAPK in neuronal excitability. The KNa channel Slack is an important protein to be studied as it is highly and ubiquitously expressed in DRG neurons and is important in the maintenance of their firing accommodation. We sought to examine if the Slack channel could be a substrate of p38 MAPK activity. First, we found that the Slack C-terminus contains two putative p38 MAPK phosphorylation sites that are highly conserved across species. Second, we show via electrophysiology experiments that KNa currents and further, Slack currents, are subject to tonic modulation by p38 MAPK. Third, biochemical approaches revealed that Slack channel regulation by p38 MAPK occurs through direct phosphorylation at the two putative sites of interaction, and mutating both sites prevented surface expression of Slack channels. Based on these results, we conclude that p38 MAPK is an obligate regulator of Slack channel function via the trafficking of channels into the membrane. The present study identifies Slack KNa channels as p38 MAPK substrates. PMID:26721627

  20. Calcium-Activated Potassium Channels at Nodes of Ranvier Secure Axonal Spike Propagation

    PubMed Central

    Gründemann, Jan; Clark, Beverley A.

    2015-01-01

    Summary Functional connectivity between brain regions relies on long-range signaling by myelinated axons. This is secured by saltatory action potential propagation that depends fundamentally on sodium channel availability at nodes of Ranvier. Although various potassium channel types have been anatomically localized to myelinated axons in the brain, direct evidence for their functional recruitment in maintaining node excitability is scarce. Cerebellar Purkinje cells provide continuous input to their targets in the cerebellar nuclei, reliably transmitting axonal spikes over a wide range of rates, requiring a constantly available pool of nodal sodium channels. We show that the recruitment of calcium-activated potassium channels (IK, KCa3.1) by local, activity-dependent calcium (Ca2+) influx at nodes of Ranvier via a T-type voltage-gated Ca2+ current provides a powerful mechanism that likely opposes depolarizing block at the nodes and is thus pivotal to securing continuous axonal spike propagation in spontaneously firing Purkinje cells. PMID:26344775

  1. Calcium-Activated Potassium Channels at Nodes of Ranvier Secure Axonal Spike Propagation.

    PubMed

    Gründemann, Jan; Clark, Beverley A

    2015-09-22

    Functional connectivity between brain regions relies on long-range signaling by myelinated axons. This is secured by saltatory action potential propagation that depends fundamentally on sodium channel availability at nodes of Ranvier. Although various potassium channel types have been anatomically localized to myelinated axons in the brain, direct evidence for their functional recruitment in maintaining node excitability is scarce. Cerebellar Purkinje cells provide continuous input to their targets in the cerebellar nuclei, reliably transmitting axonal spikes over a wide range of rates, requiring a constantly available pool of nodal sodium channels. We show that the recruitment of calcium-activated potassium channels (IK, K(Ca)3.1) by local, activity-dependent calcium (Ca(2+)) influx at nodes of Ranvier via a T-type voltage-gated Ca(2+) current provides a powerful mechanism that likely opposes depolarizing block at the nodes and is thus pivotal to securing continuous axonal spike propagation in spontaneously firing Purkinje cells. PMID:26344775

  2. Pinostrobin from Cajanus cajan (L.) Millsp. inhibits sodium channel-activated depolarization of mouse brain synaptoneurosomes.

    PubMed

    Nicholson, Russell A; David, Laurence S; Pan, Rui Le; Liu, Xin Min

    2010-10-01

    This investigation focuses on the in vitro neuroactive properties of pinostrobin, a substituted flavanone from Cajanus cajan (L.) Millsp. of the Fabaceae family. We demonstrate that pinostrobin inhibits voltage-gated sodium channels of mammalian brain (IC(50)=23 µM) based on the ability of this substance to suppress the depolarizing effects of the sodium channel-selective activator veratridine in a synaptoneurosomal preparation from mouse brain. The resting membrane potential of synaptoneurosomes was unaffected by pinostrobin. The pharmacological profile of pinostrobin resembles that of depressant drugs that block sodium channels. PMID:20472040

  3. 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.

  4. Afferent Arteriolar Dilation to 11,12-EET Analogs Involves PP2A Activity and Ca2+-Activated K+ Channels

    PubMed Central

    Imig, John D.; Dimitropoulou, Christiana; Reddy, D. Sudarshan; White, Richard E.; Falck, John R.

    2008-01-01

    The epoxygenase metabolite, 11,12-epoxyeicosatrienoic acid (11,12-EET), has renal vascular actions. 11,12-EET analogs have been developed to determine the structure activity relationship for 11,12-EET and as a tool to investigate signaling mechanisms responsible for afferent arteriolar dilation. We hypothesized that 11,12-EET mediated afferent arteriolar dilation involves increased phosphoprotein phosphatase 2A (PP2A) and large-conductance calcium activated K+ (KCa) channels. We evaluated the chemically and/or metabolically stable 11,12-EET analogs: 11,12-EET-N-methylsulfonimide (11,12-EET-SI), 11-nonyloxy-undec-8(Z)-enoic acid (11,12-ether-EET-8-ZE), and 11,12-trans-oxidoeicosa-8(Z)-eonoic acid (11,12-tetra-EET-8-ZE). Afferent arteriolar responses were assessed. Activation of KCa channels by 11,12-EET analogs were established by single cell channel recordings in renal myocytes. Assessment of renal vascular responses revealed that 11,12-EET analogs increased afferent arteriolar diameter. Vasodilator responses to 11,12-EET analogs were abolished by K+ channel or PP2A inhibition. 11,12-EET analogs activated renal myocyte large-conductance KCa channels. 11,12-EET analogs increased cAMP by 2-fold and PP2A activity increased 3-8 fold in renal myocytes. PP2A inhibition did not significantly affect the 11,12-EET analog mediated increase in cAMP and PP2A increased renal myocyte KCa channel activity to a much greater extent than PKA. These data support the concept that 11,12-EET utilizes PP2A dependent pathways to activate large-conductance KCa channels and dilate the afferent arteriole. PMID:18260004

  5. 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

  6. 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

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

    PubMed

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

    2016-03-01

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

  8. 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

  9. Endoxifen, the active metabolite of tamoxifen, inhibits cloned hERG potassium channels.

    PubMed

    Chae, Yun Ju; Lee, Keon Jin; Lee, Hong Joon; Sung, Ki-Wug; Choi, Jin-Sung; Lee, Eun Hui; Hahn, Sang June

    2015-04-01

    The effects of tamoxifen, and its active metabolite endoxifen (4-hydroxy-N-desmethyl-tamoxifen), on hERG currents stably expressed in HEK cells were investigated using the whole-cell patch-clamp technique and an immunoblot assay. Tamoxifen and endoxifen inhibited hERG tail currents at -50mV in a concentration-dependent manner with IC50 values of 1.2 and 1.6μM, respectively. The steady-state activation curve of the hERG currents was shifted to the hyperpolarizing direction in the presence of endoxifen. The voltage-dependent inhibition of hERG currents by endoxifen increased steeply in the voltage range of channel activation. The inhibition by endoxifen displayed a shallow voltage dependence (δ=0.18) in the full activation voltage range. A fast application of endoxifen induced a reversible block of hERG tail currents during repolarization in a concentration-dependent manner, which suggested an interaction with the open state of the channel. Endoxifen also decreased the hERG current elicited by a 5s depolarizing pulse to +60mV to inactivate the hERG currents, suggesting an interaction with the activated (open and/or inactivated) states of the channels. Tamoxifen and endoxifen inhibited the hERG channel protein trafficking to the plasma membrane in a concentration-dependent manner with endoxifen being more potent than tamoxifen. These results indicated that tamoxifen and endoxifen inhibited the hERG current by direct channel blockage and by the disruption of channel trafficking to the plasma membrane in a concentration-dependent manner. A therapeutic concentration of endoxifen inhibited the hERG current by preferentially interacting with the activated (open and/or inactivated) states of the channel. PMID:25680947

  10. Hormone-regulated K+ channels in follicle-enclosed oocytes are activated by vasorelaxing K+ channel openers and blocked by antidiabetic sulfonylureas.

    PubMed Central

    Honoré, E; Lazdunski, M

    1991-01-01

    Follicular oocytes from Xenopus laevis contain K+ channels activated by members of the recently recognized class of vasorelaxants that include cromakalim and pinacidil and blocked by antidiabetic sulfonylureas, such as glibenclamide. These channels are situated on the adherent follicular cells and are not present in denuded oocytes. Cromakalim-activated K+ channels are also activated by increases in intracellular cAMP, and cAMP-activated K+ channels are blocked by glibenclamide. Although cromakalim and cAMP effects are synergistic, cromakalim activation of K+ channels is drastically reduced or abolished by treatments that stimulate protein kinase C (e.g., muscarinic effectors, phorbol esters). Gonadotropins, known to play an essential role in ovarian physiology, also activate cromakalim and sulfonylurea-sensitive K+ channels. Follicular oocytes constitute an excellent system for studying regulation of cromakalim-sensitive K+ channels that are important in relation to a variety of disease processes, such as cardiovascular dysfunction and asthma, as well as brain function. PMID:1647032

  11. 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

  12. Role of CFTR's intrinsic adenylate kinase activity in gating of the Cl(-) channel.

    PubMed

    Randak, Christoph O; Welsh, Michael J

    2007-12-01

    The cystic fibrosis transmembrane conductance regulator (CFTR) is a Cl(-)channel in the ATP-binding cassette (ABC) transporter protein family. CFTR features the modular design characteristic of ABC transporters, which includes two membrane-spanning domains forming the channel pore, and two ABC nucleotide-binding domains that interact with ATP and contain the enzymatic activity coupled to normal gating. Like other ABC transporters CFTR is an ATPase (ATP + H(2)O --> ADP + Pi). Recent work has shown that CFTR also possesses intrinsic adenylate kinase activity (ATP + AMP left arrow over right arrow ADP + ADP). This finding raises important questions: How does AMP influence CFTR gating? Why does ADP inhibit CFTR current? Which enzymatic activity gates CFTR in vivo? Are there implications for other ABC transporters? This minireview attempts to shed light on these questions by summarizing recent advances in our understanding of the role of the CFTR adenylate kinase activity for channel gating. PMID:17965924

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

    PubMed Central

    Almassy, Janos; Won, Jong Hak; Begenisich, Ted B.

    2012-01-01

    Ca2+ 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 [Ca2+] was used to investigate if Ca2+-activated K channels are present in the apical PM of parotid acinar cells. Initial experiments established Ca2+-buffering conditions that produced brief, localized increases in [Ca2+] after focal laser photolysis of caged Ca2+. 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 [Ca2+] at the apical PM after photolysis of Diazo-2, a caged Ca2+ 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 [Ca2+] 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. PMID:22291145

  14. Mouse cortical collecting duct cells show nonselective cation channel activity and express a gene related to the cGMP-gated rod photoreceptor channel.

    PubMed Central

    Ahmad, I; Korbmacher, C; Segal, A S; Cheung, P; Boulpaep, E L; Barnstable, C J

    1992-01-01

    Apical nonselective cation channels with an average single-channel conductance of 34 +/- 2.3 pS were found in M-1 mouse cortical collecting duct cells. Channel activity is increased by depolarization and abolished by cytoplasmic calcium removal. Cytoplasmic application of 0.1 mM cGMP decreases channel open probability by 27%. cDNAs corresponding to approximately 40% of the coding region of the photoreceptor channel were isolated by the polymerase chain reaction from M-1 cells and a rat kidney cDNA library. The rat kidney-derived sequence differs by a single base, and the M-1-cell-derived sequence differs by only two bases, from the photoreceptor sequence. A second clone from M-1 cells differs by 20 out of 426 bases from the photoreceptor sequence. In all three clones, the deduced amino acid sequence is identical to that of the rat photoreceptor channel. Northern blot analysis of poly(A)+ RNA from M-1 cells reveals the presence of a 3.2-kilobase band hybridizing with a retinal cGMP-gated cation channel probe. The results suggest the expression in M-1 cells of more than one gene coding for nonselective cation channels or channel subunits, one of which is identical to the cGMP-gated cation channel gene of rod photoreceptors. Images PMID:1279673

  15. G-protein–gated TRP-like Cationic Channel Activated by Muscarinic Receptors

    PubMed Central

    Zholos, Alexander V.; Zholos, Andrey A.; Bolton, Thomas B.

    2004-01-01

    There is little information about the mechanisms by which G-protein–coupled receptors gate ion channels although many ionotropic receptors are well studied. We have investigated gating of the muscarinic cationic channel, which mediates the excitatory effect of acetylcholine in smooth muscles, and proposed a scheme consisting of four pairs of closed and open states. Channel kinetics appeared to be the same in cell-attached or outside-out patches whether the channel was activated by carbachol application or by intracellular dialysis with GTPγS. Since in the latter case G-proteins are permanently active, it is concluded that the cationic channel is the major determinant of its own gating, similarly to the KACh channel (Ivanova-Nikolova, T.T., and G.E. Breitwieser. 1997. J. Gen. Physiol. 109:245–253). Analysis of adjacent-state dwell times revealed connections between the states that showed features conserved among many other ligand-gated ion channels (e.g., nAChR, BKCa channel). Open probability (PO) of the cationic channel was increased by membrane depolarization consistent with the prominent U-shaped I-V relationship of the muscarinic whole-cell current at negative potentials. Membrane potential affected transitions within each closed-open state pair but had little effect on transitions between pairs; thus, the latter are likely to be caused by interactions of the channel with its ligands, e.g., Ca2+ and Gαo-GTP. Channel activity was highly heterogeneous, as was evident from the prominent cycling behavior when PO was measured over 5-s intervals. This was related to the variable frequency of openings (as in the KACh channel) and, especially, to the number of long openings between consecutive long shuttings. Analysis of the underlying Markov chain in terms of probabilities allowed us to evaluate the contribution of each open state to the integral current (from shortest to longest open state: 0.1, 3, 24, and 73%) as PO increased 525-fold in three stages. PMID

  16. 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

  17. Transient Receptor Potential Canonical 7 (TRPC7): A Diacylglycerol-Activated Non-Selective Cation Channel

    PubMed Central

    Zhang, Xuexin

    2016-01-01

    Transient receptor potential canonical 7 (TRPC7) channel is the seventh member of the mammalian TRPC channel family. TRPC7 mRNA, protein and channel activity have been detected in many tissues and organs from mouse, rat and human. TRPC7 has high sequence homology with TRPC3 and TRPC6 and all three channels are activated by membrane receptors that couple to isoforms of phospholipase C (PLC) and mediate non-selective cation currents. TRPC7, along with TRPC3 and TRPC6 can be activated by direct exogenous application of diacylglycerol (DAG) analogs and by pharmacological maneuvers that increase endogenous DAG in cells. TRPC7 shows distinct properties of activation, such as constitutive activity, susceptibility to negative regulation by extracellular Ca2+ and by protein kinase C. TRPC7 can form heteromultimers with TRPC3 and TRPC6. Although TRPC7 remains one of the least studied TRPC channel, its role in various cell types and physiological and pathophysiological conditions is begining to emerge. PMID:24756707

  18. 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

  19. 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

  20. 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

  1. Vasodilation of retinal arterioles induced by activation of BKCa channels is attenuated in diabetic rats.

    PubMed

    Mori, Asami; Suzuki, Sachi; Sakamoto, Kenji; Nakahara, Tsutomu; Ishii, Kunio

    2011-11-01

    The large-conductance Ca(2+)-activated K(+) (BK(Ca)) channels modulate the retinal vascular tone, but question of whether the impairment of the channel function contributes to abnormalities of retinal circulation has not yet been completely elucidated. The purpose of this study was to examine effects of diabetes on the vasodilation induced by activation of BK(Ca) channels. Male Wistar rats were treated with streptozotocin and experiments were performed 2 weeks later. The streptozotocin-treated animals were given drinking water containing 5% d-glucose to shorten the term in the development of retinal vascular dysfunction. The retinal vascular responses were assessed by measuring diameter of retinal arterioles in the fundus images that were captured with an original fundus camera system. In non-diabetic rats, vasodilator effects of acetylcholine on retinal arterioles were significantly reduced by iberiotoxin, an inhibitor of BK(Ca) channels. However, the inhibitory effect of iberiotoxin was not observed in diabetic rats, and the responses to the BK(Ca) channel opener BMS-191011 were almost completely abolished. The retinal vasodilator response to acetylcholine, possibly an endothelium-derived hyperpolarizing factor-mediated response, observed after treatment with N(G)-nitro-l-arginine methyl ester and indomethacin was markedly reduced in diabetic rats. The responses to pinacidil, an opener of ATP-sensitive K(+) channels, were unchanged. These results suggest that the retinal vasodilator response mediated through mechanisms involving activation of BK(Ca) channels is diminished at the early stage of diabetes in rats. The impairment of BK(Ca) channel function may contribute to abnormal retinal hemodynamics in diabetes and consequently play an important role in the pathogenesis of diabetic retinopathy. PMID:21871885

  2. Odorant receptors activated by amino acids in sensory neurons of the channel catfish Ictalurus punctatus.

    PubMed

    Ivanova, T T; Caprio, J

    1993-12-01

    Odorant receptors activated by amino acids were investigated with patch-clamp techniques in olfactory receptor neurons of the channel catfish, Ictalurus punctatus. The L-isomers of alanine, norvaline, arginine, and glutamate, known to act predominantly on different olfactory receptor sites, activated nondesensitizing inward currents with amplitudes of -2.5 to -280 pA in olfactory neurons voltage-clamped at membrane potentials of -72 or -82 mV. Different amino acids were shown to induce responses in the same sensory neurons; however, the amplitude and the kinetics of the observed whole cell currents differed among the stimuli and may therefore reflect activation of different amino acid receptor types or combinations of receptor types in these cells. Amino acid-induced currents appeared to have diverse voltage dependence and could also be classified according to the amplitude of the spontaneous channel fluctuations underlying the macroscopic currents. A mean single-channel conductance (gamma) of 360 fS was estimated from small noise whole-cell currents evoked by arginine within the same olfactory neuron in which a mean gamma value of 23.6 pS was estimated from 'large noise' response to norvaline. Quiescent olfactory neurons fired bursts of action potentials in response to either amino acid stimulation or application of 8-Br-cyclic GMP (100 microM), and voltage-gated channels underlying generation of action potentials were similar in these neurons. However, in whole-cell voltage-clamp, 8-Br-cyclic GMP evoked large rectangular current pulses, and single-channel conductances of 275, 220, and 110 pS were obtained from the discrete current levels. These results suggest that in addition to the cyclic nucleotide-gated transduction channels, olfactory neurons of the channel catfish possess a variety of odor receptors coupled to different types of transduction channels. PMID:8133240

  3. The Activation Effect of Hainantoxin-I, a Peptide Toxin from the Chinese Spider, Ornithoctonus hainana, on Intermediate-Conductance Ca2+-Activated K+ Channels

    PubMed Central

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

    2014-01-01

    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. PMID:25153257

  4. 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

  5. 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.

  6. 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

  7. Swelling-Activated Ca2+ Channels Trigger Ca2+ Signals in Merkel Cells

    PubMed Central

    Haeberle, Henry; Bryan, Leigh A.; Vadakkan, Tegy J.; Dickinson, Mary E.; Lumpkin, Ellen A.

    2008-01-01

    Merkel cell-neurite complexes are highly sensitive touch receptors comprising epidermal Merkel cells and sensory afferents. Based on morphological and molecular studies, Merkel cells are proposed to be mechanosensory cells that signal afferents via neurotransmission; however, functional studies testing this hypothesis in intact skin have produced conflicting results. To test this model in a simplified system, we asked whether purified Merkel cells are directly activated by mechanical stimulation. Cell shape was manipulated with anisotonic solution changes and responses were monitored by Ca2+ imaging with fura-2. We found that hypotonic-induced cell swelling, but not hypertonic solutions, triggered cytoplasmic Ca2+ transients. Several lines of evidence indicate that these signals arise from swelling-activated Ca2+-permeable ion channels. First, transients were reversibly abolished by chelating extracellular Ca2+, demonstrating a requirement for Ca2+ influx across the plasma membrane. Second, Ca2+ transients were initially observed near the plasma membrane in cytoplasmic processes. Third, voltage-activated Ca2+ channel (VACC) antagonists reduced transients by half, suggesting that swelling-activated channels depolarize plasma membranes to activate VACCs. Finally, emptying internal Ca2+ stores attenuated transients by 80%, suggesting Ca2+ release from stores augments swelling-activated Ca2+ signals. To identify candidate mechanotransduction channels, we used RT-PCR to amplify ion-channel transcripts whose pharmacological profiles matched those of hypotonic-evoked Ca2+ signals in Merkel cells. We found 11 amplicons, including PKD1, PKD2, and TRPC1, channels previously implicated in mechanotransduction in other cells. Collectively, these results directly demonstrate that Merkel cells are activated by hypotonic-evoked swelling, identify cellular signaling mechanisms that mediate these responses, and support the hypothesis that Merkel cells contribute to touch reception

  8. Bounding the Higgs boson width through interferometry.

    PubMed

    Dixon, Lance J; Li, Ye

    2013-09-13

    We study the change in the diphoton-invariant-mass distribution for Higgs boson decays to two photons, due to interference between the Higgs resonance in gluon fusion and the continuum background amplitude for gg→γγ. Previously, the apparent Higgs mass was found to shift by around 100 MeV in the standard model in the leading-order approximation, which may potentially be experimentally observable. We compute the next-to-leading-order QCD corrections to the apparent mass shift, which reduce it by about 40%. The apparent mass shift may provide a way to measure, or at least bound, the Higgs boson width at the Large Hadron Collider through "interferometry." We investigate how the shift depends on the Higgs width, in a model that maintains constant Higgs boson signal yields. At Higgs widths above 30 MeV, the mass shift is over 200 MeV and increases with the square root of the width. The apparent mass shift could be measured by comparing with the ZZ* channel, where the shift is much smaller. It might be possible to measure the shift more accurately by exploiting its strong dependence on the Higgs transverse momentum. PMID:24074073

  9. Up-Regulation of Pressure-activated Ca2+-permeable Cation Channel in Intact Vascular Endothelium of Hypertensive Rats

    NASA Astrophysics Data System (ADS)

    Hoyer, J.; Kohler, R.; Haase, W.; Distler, A.

    1996-10-01

    In endothelial cells, stretch-activated cation channels have been proposed to act as mechanosensors for changes in hemodynamic forces. We have identified a novel mechanosensitive pressure-activated channel in intact endothelium from rat aorta and mesenteric artery. The 18-pS cation channel responded with a multifold increase in channel activity when positive pressure was applied to the luminal cell surface with the patch pipette and inactivated at negative pipette pressure. Channel permeability ratio for K+, Na+, and Ca2+ ions was 1:0.98:0.23. Ca2+ influx through the channel was sufficient to activate a neighboring Ca2+-dependent K+ channel. Hemodynamic forces are chronically disturbed in arterial hypertension. Endothelial cell dysfunction has been implicated in the pathogenesis of arterial hypertension. In two comparative studies, density of the pressure-activated channel was found to be significantly higher in spontaneously hypertensive rats and renovascular hypertensive rats compared with their respective normotensive controls. Channel activity presumably leads to mechanosensitive Ca2+ influx and induces cell hyperpolarization by K+ channel activity. Both Ca2+ influx and hyperpolarization are known to induce a vasodilatory endothelial response by stimulating endothelial nitric oxide (NO) production. Up-regulation of channel density in hypertension could, therefore, represent a counterregulatory mechanism of vascular endothelium.

  10. Members of the Chloride Intracellular Ion Channel Protein Family Demonstrate Glutaredoxin-Like Enzymatic Activity

    PubMed Central

    Al Khamici, Heba; Brown, Louise J.; Hossain, Khondker R.; Hudson, Amanda L.; Sinclair-Burton, Alxcia A.; Ng, Jane Phui Mun; Daniel, Elizabeth L.; Hare, Joanna E.; Cornell, Bruce A.; Curmi, Paul M. G.; Davey, Mary W.; Valenzuela, Stella M.

    2015-01-01

    The Chloride Intracellular Ion Channel (CLIC) family consists of six evolutionarily conserved proteins in humans. Members of this family are unusual, existing as both monomeric soluble proteins and as integral membrane proteins where they function as chloride selective ion channels, however no function has previously been assigned to their soluble form. Structural studies have shown that in the soluble form, CLIC proteins adopt a glutathione S-transferase (GST) fold, however, they have an active site with a conserved glutaredoxin monothiol motif, similar to the omega class GSTs. We demonstrate that CLIC proteins have glutaredoxin-like glutathione-dependent oxidoreductase enzymatic activity. CLICs 1, 2 and 4 demonstrate typical glutaredoxin-like activity using 2-hydroxyethyl disulfide as a substrate. Mutagenesis experiments identify cysteine 24 as the catalytic cysteine residue in CLIC1, which is consistent with its structure. CLIC1 was shown to reduce sodium selenite and dehydroascorbate in a glutathione-dependent manner. Previous electrophysiological studies have shown that the drugs IAA-94 and A9C specifically block CLIC channel activity. These same compounds inhibit CLIC1 oxidoreductase activity. This work for the first time assigns a functional activity to the soluble form of the CLIC proteins. Our results demonstrate that the soluble form of the CLIC proteins has an enzymatic activity that is distinct from the channel activity of their integral membrane form. This CLIC enzymatic activity may be important for protecting the intracellular environment against oxidation. It is also likely that this enzymatic activity regulates the CLIC ion channel function. PMID:25581026

  11. 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

  12. 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

  13. Small-Conductance Ca2+-Activated Potassium Channels Negatively Regulate Aldosterone Secretion in Human Adrenocortical Cells.

    PubMed

    Yang, Tingting; Zhang, Hai-Liang; Liang, Qingnan; Shi, Yingtang; Mei, Yan-Ai; Barrett, Paula Q; Hu, Changlong

    2016-09-01

    Aldosterone, which plays a key role in maintaining water and electrolyte balance, is produced by zona glomerulosa cells of the adrenal cortex. Autonomous overproduction of aldosterone from zona glomerulosa cells causes primary hyperaldosteronism. Recent clinical studies have highlighted the pathological role of the KCNJ5 potassium channel in primary hyperaldosteronism. Our objective was to determine whether small-conductance Ca(2+)-activated potassium (SK) channels may also regulate aldosterone secretion in human adrenocortical cells. We found that apamin, the prototypic inhibitor of SK channels, decreased membrane voltage, raised intracellular Ca(2+) and dose dependently increased aldosterone secretion from human adrenocortical H295R cells. By contrast, 1-Ethyl-2-benzimidazolinone, an agonist of SK channels, antagonized apamin's action and decreased aldosterone secretion. Commensurate with an increase in aldosterone production, apamin increased mRNA expression of steroidogenic acute regulatory protein and aldosterone synthase that control the early and late rate-limiting steps in aldosterone biosynthesis, respectively. In addition, apamin increased angiotensin II-stimulated aldosterone secretion, whereas 1-Ethyl-2-benzimidazolinone suppressed both angiotensin II- and high K(+)-stimulated production of aldosterone in H295R cells. These findings were supported by apamin-modulation of basal and angiotensin II-stimulated aldosterone secretion from acutely prepared slices of human adrenals. We conclude that SK channel activity negatively regulates aldosterone secretion in human adrenocortical cells. Genetic association studies are necessary to determine whether mutations in SK channel subtype 2 genes may also drive aldosterone excess in primary hyperaldosteronism. PMID:27432863

  14. The sodium-activated potassium channel Slack is required for optimal cognitive flexibility in mice.

    PubMed

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

    2015-07-01

    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 development. In particular, recent findings have shown that human Slack mutations produce very severe intellectual disability and that Slack channels interact directly with the Fragile X mental retardation protein (FMRP), a protein that when missing or mutated results in Fragile X syndrome (FXS), the most common form of inherited intellectual disability and autism in humans. We have now analyzed a recently developed Kcnt1 null mouse model in several behavioral tasks to assess which aspects of memory and learning are dependent on Slack. We demonstrate that Slack deficiency results in mildly altered general locomotor activity, but normal working memory, reference memory, as well as cerebellar control of motor functions. In contrast, we find that Slack channels are required for cognitive flexibility, including reversal learning processes and the ability to adapt quickly to unfamiliar situations and environments. Our data reveal that hippocampal-dependent spatial learning capabilities require the proper function of Slack channels. PMID:26077685

  15. The secret life of CFTR as a calcium-activated chloride channel.

    PubMed

    Billet, Arnaud; Hanrahan, John W

    2013-11-01

    cAMP-stimulated anion conductance is defective in cystic fibrosis (CF). The regulatory domain of CFTR, the anion channel protein encoded by the CF gene, possesses an unusually high density of consensus sequences for phosphorylation by protein kinase A (14 in a stretch of <200 amino acids). Thus it is not surprising that CFTR is viewed primarily as a cAMP-stimulated anion channel, and most studies have focused on this mode of activation. However, there is growing evidence that CFTR also responds to Ca(2+)-mobilizing secretagogues and contributes substantially to cholinergic and purinergic responses in native tissues. G protein-coupled receptors that signal through Gαq can stimulate CFTR channels by activating Ca(2+)-dependent adenylyl cyclase and tyrosine kinases, and also by inhibiting protein phosphatase type 2A. Here we review evidence for these novel mechanisms of CFTR activation and discuss how they may help explain previous observations. PMID:23959675

  16. Ion conductance of the Ca(2+)-activated maxi-K+ channel from the embryonic rat brain.

    PubMed

    Mienville, J M; Clay, J R

    1997-01-01

    By using single-channel recording techniques, we measured the conductance (gK) of the Ca(2+)-activated Maxi-K+ channel from the embryonic rat brain, and examined its dependence on K+ ions present in equimolar concentrations on both sides of the membrane patch. With ionic strength maintained constant by substitution of N-methyl-D-glucamine for K+, gK has a sigmoidal dependence upon [K+]. This result has been obscured in previous work by variations in ionic strength, which has a marked effect on single-channel conductance, especially in the limit for which this variable approaches zero. The gK versus [K+] relationship is described, theoretically, by a three-barrier, two-binding-site model in which the barrier that an ion must cross to leave the channel is decreased as [K+] is increased. PMID:8994603

  17. 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.

  18. 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.

  19. Characterization of apamin-sensitive Ca(2+)-activated potassium channels in human leukaemic T lymphocytes.

    PubMed

    Hanselmann, C; Grissmer, S

    1996-11-01

    1. The whole-cell recording mode of the patch-clamp technique was used to study the effect of extracellularly applied ions, toxins and drugs on voltage-independent, apamin-sensitive Ca(2+)-activated K+ channels, K(Ca), expressed in the Jurkat human leukaemic T cell line. 2. Extracellular Ba2+ and Sr+ produced a voltage-dependent block. The equilibrium dissociation constant of the Ba2+/K(Ca) channel complex increased e-fold for a 20 mV change of potential. Ba2+ block of Jurkat K(Ca) channels is therefore as steep as expected from the movement of a single divalent cation about half-way into the electric field of the membrane from the outside. 3. We determined the ion selectivity as well as the conductance of these channels. Calculated permeability ratios, PX/PK, for these K(Ca) channels were 1.0, 0.96, 0.26 and 0.53 for K+, Rb+, Cs+ and NH4+, respectively. Conductance ratios, gX/gK, for the same ions were 1.0, 1.0, 0.67 and 0.11, respectively. Most strikingly this channel can also carry significant current with Cs+ as current carrier. 4. Scyllatoxin (ScTX), a thirty-one amino acid peptide toxin, reduced current through these K(Ca) channels with a half-blocking concentration of approximately 0.3 nM independent of the pH. Other drugs that were able to reduce current through these channels include the classical calcium antagonists diltiazem and verapamil. In contrast, nifedipine, clotrimazole and kaliotoxin (100 nM) were unable to block current through these channels in Jurkat T cells. PMID:8930831

  20. Burst activity and ultrafast activation kinetics of CaV1.3 Ca2+ channels support presynaptic activity in adult gerbil hair cell ribbon synapses

    PubMed Central

    Zampini, Valeria; Johnson, Stuart L; Franz, Christoph; Knipper, Marlies; Holley, Matthew C; Magistretti, Jacopo; Masetto, Sergio; Marcotti, Walter

    2013-01-01

    Auditory information transfer to afferent neurons relies on precise triggering of neurotransmitter release at the inner hair cell (IHC) ribbon synapses by Ca2+ entry through CaV1.3 Ca2+ channels. Despite the crucial role of CaV1.3 Ca2+ channels in governing synaptic vesicle fusion, their elementary properties in adult mammals remain unknown. Using near-physiological recording conditions we investigated Ca2+ channel activity in adult gerbil IHCs. We found that Ca2+ channels are partially active at the IHC resting membrane potential (−60 mV). At −20 mV, the large majority (>70%) of Ca2+ channel first openings occurred with an estimated delay of about 50 μs in physiological conditions, with a mean open time of 0.5 ms. Similar to other ribbon synapses, Ca2+ channels in IHCs showed a low mean open probability (0.21 at −20 mV), but this increased significantly (up to 0.91) when Ca2+ channel activity switched to a bursting modality. We propose that IHC Ca2+ channels are sufficiently rapid to transmit fast signals of sound onset and support phase-locking. Short-latency Ca2+ channel opening coupled to multivesicular release would ensure precise and reliable signal transmission at the IHC ribbon synapse. PMID:23713031

  1. 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

  2. Excessive activation of cyclic nucleotide-gated channels contributes to neuronal degeneration of photoreceptors.

    PubMed

    Vallazza-Deschamps, Géraldine; Cia, David; Gong, Jie; Jellali, Abdeljelil; Duboc, Agnès; Forster, Valérie; Sahel, Jose A; Tessier, Luc-Henri; Picaud, Serge

    2005-09-01

    In different animal models, photoreceptor degeneration was correlated to an abnormal increase in cGMP concentration. The cGMP-induced photoreceptor toxicity was demonstrated by applying the phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine on retinal explants. To assess the role of cGMP-gated channels in this cGMP toxicity, the Ca(2+) channel blockers verapamil and L- and D-diltiazem, which block cGMP-gated channels with different efficacies, were applied to in vitro animal models of photoreceptor degeneration. These models included: (i) adult rat retinal explants incubated with zaprinast, a more specific inhibitor of the rod phosphodiesterase than 3-isobutyl-1-methylxanthine and (ii) rd mouse retinal explants. Photoreceptor apoptosis was assessed by terminal dUTP nick end labelling and caspase 3 activation. Effects of the blockers on the synaptic rod Ca(2+) channels were measured by patch-clamp recording. In the zaprinast-induced photoreceptor degeneration model, both diltiazem isomers rescued photoreceptors whereas verapamil had no influence. Their neuroprotective efficacy was correlated to their inhibition of cGMP-gated channels (l-diltiazem>d-diltiazem>verapamil=0). In contrast, all three Ca(2+) channel blockers suppressed rod Ca(2+) channel currents similarly. This suppression of the currents by the diltiazem isomers was very weak (16.5%) at the neuroprotective concentration (10 microm). In rd retinal explants, both diltiazem isomers also slowed down rod degeneration in contrast to verapamil. L-diltiazem exhibited this effect at concentrations ranging from 1 to 20 microm. This study further supports the photoreceptor neuroprotection by diltiazem particularly in the rd mouse retina, whereas the absence of neuroprotection by verapamil further suggests the role of cGMP-gated channel activation in the induction of photoreceptor degeneration. PMID:16176343

  3. Reporting Sodium Channel Activity Using Calcium Flux: Pharmacological Promiscuity of Cardiac Nav1.5

    PubMed Central

    Zhang, Hongkang; Zou, Beiyan; Du, Fang; Xu, Kaiping

    2015-01-01

    Voltage-gated sodium (Nav) channels are essential for membrane excitability and represent therapeutic targets for treating human diseases. Recent reports suggest that these channels, e.g., Nav1.3 and Nav1.5, are inhibited by multiple structurally distinctive small molecule drugs. These studies give reason to wonder whether these drugs collectively target a single site or multiple sites in manifesting such pharmacological promiscuity. We thus investigate the pharmacological profile of Nav1.5 through systemic analysis of its sensitivity to diverse compound collections. Here, we report a dual-color fluorescent method that exploits a customized Nav1.5 [calcium permeable Nav channel, subtype 5 (SoCal5)] with engineered-enhanced calcium permeability. SoCal5 retains wild-type (WT) Nav1.5 pharmacological profiles. WT SoCal5 and SoCal5 with the local anesthetics binding site mutated (F1760A) could be expressed in separate cells, each with a different-colored genetically encoded calcium sensor, which allows a simultaneous report of compound activity and site dependence. The pharmacological profile of SoCal5 reveals a hit rate (>50% inhibition) of around 13% at 10 μM, comparable to that of hERG. The channel activity is susceptible to blockage by known drugs and structurally diverse compounds. The broad inhibition profile is highly dependent on the F1760 residue in the inner cavity, which is a residue conserved among all nine subtypes of Nav channels. Both promiscuity and dependence on F1760 seen in Nav1.5 were replicated in Nav1.4. Our evidence of a broad inhibition profile of Nav channels suggests a need to consider off-target effects on Nav channels. The site-dependent promiscuity forms a foundation to better understand Nav channels and compound interactions. PMID:25422141

  4. Stretch-activated single ion channel currents in tissue-cultured embryonic chick skeletal muscle.

    PubMed Central

    Guharay, F; Sachs, F

    1984-01-01

    The membrane of tissue-cultured chick pectoral muscle contains an ionic channel which is activated by membrane stretch. Nicotinic channels and Ca2+-activated K+ channels are not affected by stretch. In 150 mM-external K+ and 150 mM-internal Na+ the channel has a conductance of 70 pS, linear current-voltage relationship between -50 and -140 mV and a reversal potential of +30 mV. Kinetic analysis of single-channel records indicates that there are one open (O) and three closed (C) states. The data can be fitted by the reaction scheme: C1-C2-C3-O. Only the rate constant that governs the C1-C2 transition (k1,2) is stretch-sensitive. None of the rates are voltage-sensitive. The rate constant k1,2 varies with the square of the tension as k1, 2 = k0 X e alpha T2, where alpha is a constant describing the sensitivity to stretch and T is the tension. A typical value of alpha is 0.08 (dyn cm-1)-2. Following exposure to cytochalasin B the channel becomes more sensitive to stretch. The stretch-sensitivity constant, alpha, increases from 0.08 to 2.4 (dyn cm-1)-2. The probability of the channel being open is strongly dependent upon the extracellular K+ concentration. With a suction of 2 cmHg the probability increases from 0.004 in normal saline (5 mM-K+) to 0.26 in 150 mM-K+. The channel appears to gather force from a large area of membrane (greater than 3 X 10(5) A2), probably by a cytochalasin-resistant cytoskeletal network. PMID:6086918

  5. Effects of the small molecule HERG activator NS1643 on Kv11.3 channels.

    PubMed

    Bilet, Arne; Bauer, Christiane K

    2012-01-01

    NS1643 is one of the small molecule HERG (Kv11.1) channel activators and has also been found to increase erg2 (Kv11.2) currents. We now investigated whether NS1643 is also able to act as an activator of Kv11.3 (erg3) channels expressed in CHO cells. Activation of rat Kv11.3 current occurred in a dose-dependent manner and maximal current increasing effects were obtained with 10 µM NS1643. At this concentration, steady-state outward current increased by about 80% and the current increase was associated with a significant shift in the voltage dependence of activation to more negative potentials by about 15 mV. In addition, activation kinetics were accelerated, whereas deactivation was slowed. There was no significant effect on the kinetics of inactivation and recovery from inactivation. The strong current-activating agonistic effect of NS1643 did not result from a shift in the voltage dependence of Kv11.3 channel inactivation and was independent from external Na(+) or Ca(2+). At the higher concentration of 20 µM, NS1643 induced clearly less current increase. The left shift in the voltage dependence of activation reversed and the voltage sensitivity of activation dramatically decreased along with a slowing of Kv11.3 channel activation. These data show that, in comparison to other Kv11 family members, NS1643 exerts distinct effects on Kv11.3 channels with especially pronounced partial antagonistic effects at higher concentration. PMID:23226420

  6. 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

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

    PubMed

    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. Formation of cellular projections in neural progenitor cells depends on SK3 channel activity.

    PubMed

    Liebau, Stefan; Vaida, Bianca; Proepper, Christian; Grissmer, Stephan; Storch, Alexander; Boeckers, Tobias M; Dietl, Paul; Wittekindt, Oliver H

    2007-06-01

    Ion channels are potent modulators for developmental processes in progenitor cells. In a screening approach for different ion channels in neural progenitor cells (NPCs) we observed a 1-ethyl-2-benzimidazolinone (1-EBIO) activated inward current, which could be blocked by scyllatoxin (ScTX, IC50=2+/- 0.3 nmol/L). This initial evidence for the expression of the small conductance Ca2+ activated K+-channel SK3 was confirmed by the detection of SK3 transcripts and protein in NPCs. Interestingly, SK3 proteins were highly expressed in non-differentiated NPCs with a focused localization in lamellipodia as well as filopodial structures. The activation of SK3 channels using 1-EBIO lead to an immediate filopodial sprouting and the translocation of the protein into these novel filopodial protrusions. Both effects could be prevented by the pre-incubation of NPCs with ScTX. Our study gives first evidence that the formation and prolongation of filopodia in NPCs is, at least in part, effectively induced and regulated by SK3 channels. PMID:17459146

  9. 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

  10. Transmembrane helix straightening and buckling underlies activation of mechanosensitive and thermosensitive K(2P) channels.

    PubMed

    Lolicato, Marco; Riegelhaupt, Paul M; Arrigoni, Cristina; Clark, Kimberly A; Minor, Daniel L

    2014-12-17

    Mechanical and thermal activation of ion channels is central to touch, thermosensation, and pain. The TRAAK/TREK K(2P) potassium channel subfamily produces background currents that alter neuronal excitability in response to pressure, temperature, signaling lipids, and anesthetics. How such diverse stimuli control channel function is unclear. Here we report structures of K(2P)4.1 (TRAAK) bearing C-type gate-activating mutations that reveal a tilting and straightening of the M4 inner transmembrane helix and a buckling of the M2 transmembrane helix. These conformational changes move M4 in a direction opposite to that in classical potassium channel activation mechanisms and open a passage lateral to the pore that faces the lipid bilayer inner leaflet. Together, our findings uncover a unique aspect of K(2P) modulation, indicate a means for how the K(2P) C-terminal cytoplasmic domain affects the C-type gate which lies ∼40Å away, and suggest how lipids and bilayer inner leaflet deformations may gate the channel. PMID:25500157

  11. 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

  12. 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

  13. A Semi-Synthetic Ion Channel Platform for Detection of Phosphatase and Protease Activity

    PubMed Central

    Macrae, Michael X.; Blake, Steven; Jiang, Xiayun; Capone, Ricardo; Estes, Daniel J.; Mayer, Michael; Yang, Jerry

    2009-01-01

    Sensitive methods to probe the activity of enzymes are important for clinical assays and for elucidating the role of these proteins in complex biochemical networks. This paper describes a semi-synthetic ion channel platform for detecting the activity of two different classes of enzymes with high sensitivity. In the first case, this method uses single ion channel conductance measurements to follow the enzyme-catalyzed hydrolysis of a phosphate group attached to the C-terminus of gramicidin A (gA, an ion channel-forming peptide) in the presence of alkaline phosphatase (AP). Enzymatic hydrolysis of this phosphate group removes negative charges from the entrance of the gA pore, resulting in a product with measurably reduced single ion channel conductance compared to the original gA-phosphate substrate. This technique employs a standard, commercial bilayer setup and takes advantage of the catalytic turnover of enzymes and the amplification characteristics of ion flux through individual gA pores to detect picomolar concentrations of active AP in solution. Furthermore, this technique makes it possible to study the kinetics of an enzyme and provides an estimate for the observed rate constant (kcat) and the Michaelis constant (KM) by following the conversion of the gA-phosphate substrate to product over time in the presence of different concentrations of AP. In the second case, modification of gA with a substrate for proteolytic cleavage by anthrax lethal factor (LF) afforded a sensitive method for detection of LF activity, illustrating the utility of ion channel-based sensing for detection of a potential biowarfare agent. This ion channel-based platform represents a powerful, novel approach to monitor the activity of femtomoles to picomoles of two different classes of enzymes in solution. Furthermore, this platform has the potential for realizing miniaturized, cost-effective bioanalytical assays that complement currently established assays. PMID:19860382

  14. 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

    2014-01-01

    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. PMID:24980701

  15. Increased TMEM16A-encoded calcium-activated chloride channel activity is associated with pulmonary hypertension.

    PubMed

    Forrest, Abigail S; Joyce, Talia C; Huebner, Marissa L; Ayon, Ramon J; Wiwchar, Michael; Joyce, John; Freitas, Natalie; Davis, Alison J; Ye, Linda; Duan, Dayue D; Singer, Cherie A; Valencik, Maria L; Greenwood, Iain A; Leblanc, Normand

    2012-12-15

    Pulmonary artery smooth muscle cells (PASMCs) are more depolarized and display higher Ca(2+) levels in pulmonary hypertension (PH). Whether the functional properties and expression of Ca(2+)-activated Cl- channels (Cl(Ca)), an important excitatory mechanism in PASMCs, are altered in PH is unknown. The potential role of Cl(Ca) channels in PH was investigated using the monocrotaline (MCT)-induced PH model in the rat. Three weeks postinjection with a single dose of MCT (50 mg/kg ip), the animals developed right ventricular hypertrophy (heart weight measurements) and changes in pulmonary arterial flow (pulse-waved Doppler imaging) that were consistent with increased pulmonary arterial pressure and PH. Whole cell patch experiments revealed an increase in niflumic acid (NFA)-sensitive Ca(2+)-activated Cl(-) current [I(Cl(Ca))] density in PASMCs from large conduit and small intralobar pulmonary arteries of MCT-treated rats vs. aged-matched saline-injected controls. Quantitative RT-PCR and Western blot analysis revealed that the alterations in I(Cl(Ca)) were accompanied by parallel changes in the expression of TMEM16A, a gene recently shown to encode for Cl(Ca) channels. The contraction to serotonin of conduit and intralobar pulmonary arteries from MCT-treated rats exhibited greater sensitivity to nifedipine (1 μM), an l-type Ca(2+) channel blocker, and NFA (30 or 100 μM, with or without 10 μM indomethacin to inhibit cyclooxygenases) or T16A(Inh)-A01 (10 μM), TMEM16A/Cl(Ca) channel inhibitors, than that of control animals. In conclusion, augmented Cl(Ca)/TMEM16A channel activity is a major contributor to the changes in electromechanical coupling of PA in this model of PH. TMEM16A-encoded channels may therefore represent a novel therapeutic target in this disease. PMID:23034390

  16. Calibration activities on the BepiColombo High-Resolution Channel (HRIC) of SIMBIO-SYS instrument

    NASA Astrophysics Data System (ADS)

    Della Corte, V.; Zusi, M.; Palumbo, P.; Baroni, M.; Ficai Veltroni, I.; Flamini, E.; Mugnuolo, R.

    2015-10-01

    HRIC (High Resolution Imaging Channel) is the high resolution channel of the SIMBIO-SYS instrument on- board the ESA BepiColombo Mission. Calibration activities were performed at SelexES premises in spring- summer 2014 in order to check for Channel performances (radiometric performances, quality image and geometrical performances) and to obtain data necessary to setup a calibration pipeline necessary to process the raw images acquired by the channel when in operative scenario.

  17. 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

  18. Apamin-sensitive, small-conductance, calcium-activated potassium channels mediate cholinergic inhibition of chick auditory hair cells.

    PubMed

    Yuhas, W A; Fuchs, P A

    1999-11-01

    Acetylcholine released from efferent neurons in the cochlea causes inhibition of mechanosensory hair cells due to the activation of calcium-dependent potassium channels. Hair cells are known to have large-conductance, "BK"-type potassium channels associated with the afferent synapse, but these channels have different properties than those activated by acetylcholine. Whole-cell (tight-seal) and cell-attached patch-clamp recordings were made from short (outer) hair cells isolated from the chicken basilar papilla (cochlea equivalent). The peptides apamin and charybdotoxin were used to distinguish the calcium-activated potassium channels involved in the acetylcholine response from the BK-type channels associated with the afferent synapse. Differential toxin blockade of these potassium currents provides definitive evidence that ACh activates apamin-sensitive, "SK"-type potassium channels, but does not activate carybdotoxin-sensitive BK channels. This conclusion is supported by tentative identification of small-conductance, calcium-sensitive but voltage-insensitive potassium channels in cell-attached patches. The distinction between these channel types is important for understanding the segregation of opposing afferent and efferent synaptic activity in the hair cell, both of which depend on calcium influx. These different calcium-activated potassium channels serve as sensitive indicators for functionally significant calcium influx in the hair cell. PMID:10573868

  19. Synthesis and evaluation of 1,4-dihydropyridine derivatives with calcium channel blocking activity.

    PubMed

    Bladen, Chris; Gündüz, Miyase Gözde; Şimşek, Rahime; Şafak, Cihat; Zamponi, Gerald W

    2014-07-01

    1,4-Dihydropyridines (DHPs) are an important class of L-type calcium channel blockers that are used to treat conditions such as hypertension and angina. Their primary target in the cardiovascular system is the Cav1.2 L-type calcium channel isoform, however, a number of DHPs also block low-voltage-activated T-type calcium channels. Here, we describe the synthesis of a series of novel DHP derivatives that have a condensed 1,4-DHP ring system (hexahydroquinoline) and report on their abilities to block both L- and T-type calcium channels. Within this series of compounds, modification of a key ester moiety not only regulates the blocking affinity for both L- and T-type channels, but also allows for the development of DHPs with 30-fold selectivity for T-type channels over the L-type. Our data suggest that a condensed dihydropyridine-based scaffold may serve as a pharmacophore for a new class of T-type selective inhibitors. PMID:24149495

  20. Inhibition of parathyroid hormone release by maitotoxin, a calcium channel activator

    SciTech Connect

    Fitzpatrick, L.A.; Yasumoto, T.; Aurbach, G.D.

    1989-01-01

    Maitotoxin, a toxin derived from a marine dinoflagellate, is a potent activator of voltage-sensitive calcium channels. To further test the hypothesis that inhibition of PTH secretion by calcium is mediated via a calcium channel we studied the effect of maitotoxin on dispersed bovine parathyroid cells. Maitotoxin inhibited PTH release in a dose-dependent fashion, and inhibition was maximal at 1 ng/ml. Chelation of extracellular calcium by EGTA blocked the inhibition of PTH by maitotoxin. Maitotoxin enhanced the effects of the dihydropyridine calcium channel agonist (+)202-791 and increased the rate of radiocalcium uptake in parathyroid cells. Pertussis toxin, which ADP-ribosylates and inactivates a guanine nucleotide regulatory protein that interacts with calcium channels in the parathyroid cell, did not affect the inhibition of PTH secretion by maitotoxin. Maitotoxin, by its action on calcium channels allows entry of extracellular calcium and inhibits PTH release. Our results suggest that calcium channels are involved in the release of PTH. Inhibition of PTH release by maitotoxin is not sensitive to pertussis toxin, suggesting that maitotoxin may act distal to the site interacting with a guanine nucleotide regulatory protein, or maitotoxin could interact with other ions or second messengers to inhibit PTH release.

  1. 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

  2. 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

  3. 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

  4. 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

  5. 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

  6. Large Conductance, Ca2+-Activated K+ Channels (BKCa) and Arteriolar Myogenic Signaling

    PubMed Central

    Hill, Michael A.; Yang, Yan; Ella, Srikanth R.; Davis, Michael J.; Braun, Andrew P.

    2010-01-01

    Summary Myogenic, or pressure-induced, vasoconstriction is critical for local blood flow autoregulation. Underlying this VSM response are events including membrane depolarization, Ca2+ entry and mobilization, and activation of contractile proteins. BKCa has been implicated in several of these steps including, 1) channel closure causing membrane depolarization, and 2) channel opening causing hyperpolarization to oppose excessive pressure-induced vasoconstriction. As multiple mechanisms regulate BKCa activity, (subunit composition, Em and Ca2+ levels, post-translational modification) tissue level diversity is predicted. Importantly, heterogeneity may contribute to tissue-specific differences in regulation of myogenic vasoconstriction, allowing local hemodynamics to be matched to metabolic requirements. Knowledge of such variability will be important to exploiting the BKCa channel as a therapeutic target and understanding systemic effects of its pharmacological manipulation. PMID:20178789

  7. 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

  8. The Width of a Proof

    ERIC Educational Resources Information Center

    Hanna, Gila

    2014-01-01

    This paper's aim is to discuss the concept of width of a proof put forward by Timothy Gowers. It explains what this concept means and attempts to show how it relates to other concepts discussed in the existing literature on proof and proving. It also explores how the concept of width of a proof might be used productively in the mathematics…

  9. Functional Roles of a Ca2+-Activated K+ Channel (SK2) in Atrioventricular Nodes

    PubMed Central

    Zhang, Qian; Timofeyev, Valeriy; Lu, Ling; Li, Ning; Singapuri, Anil; Long, Melissa K.; Bond, Chris T.; Adelman, John P.; Chiamvimonvat, Nipavan

    2013-01-01

    Since the first description of the anatomical atrioventricular nodes (AVN), a large number of studies have provided insights into the heterogeneity of the structure as well as a repertoire of ion channel proteins which govern this complex conduction pathway between the atria and ventricles. These studies have revealed the intricate organization of multiple nodal and nodal-like myocytes contributing to the unique electrophysiology of the AVN in health and diseases. On the other hand, information regarding contribution of specific ion channels to the function of the AVN remains incomplete. We reason that the identification of AVN-specific ion channels may provide a more direct and rationale design of therapeutic target in the control of AVN conduction in atrial flutter/fibrillation, one of the most common arrhythmias seen clinically. In this study, we took advantage of two genetically altered mouse models with over-expression or null mutation of one of the small conductance Ca2+-activated K+ channel isoform, SK2 channel and demonstrated robust phenotypes of AVN dysfunction in these experimental models. Over-expression of SK2 channels results in the shortening of the spontaneous action potentials (APs) of the AVN cells and an increase in the firing frequency. On the other hand, ablation of the SK2 channel results in the opposite effects on the spontaneous APs of the AVN. Furthermore, we directly documented the expression of SK2 channel in mouse AVN using multiple techniques. The new insights may have important implications in providing novel drug targets for the modification of AVN conduction in the treatment of atrial arrhythmias. PMID:18096820

  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. 17Beta-Estradiol Inhibits Calcium-Activated Potassium Channel Expressions in Rat Whole Bladder

    PubMed Central

    2016-01-01

    Purpose: To investigate the effect of estrogen on the expression of calcium-activated potassium (KCa) channels in an overactive bladder rat model. To this end, mRNA and protein levels of KCa channel subtypes in the bladder of ovariectomized rats were measured by reverse transcription polymerase chain reaction and western blotting, respectively. Methods: Ten-week-old female Sprague-Dawley rats were divided randomly into 3 groups: sham-operated control group (n=11), ovariectomy group (n=11), and the group treated with estrogen after ovariectomy (n=12). Rats in the last group were subcutaneously injected with 17β-estradiol (50 μg/kg) every other day for 2 weeks, whereas rats in the other 2 groups received vehicle (soybean oil) alone. Two weeks after treatment, the whole bladder was excised for mRNA and protein measurements. Results: Protein levels of the large-conductance KCa (BK) channels in the ovariectomy group were 1.5 folds higher than those in the sham-operated control group. However, the protein levels of the other KCa channel subtypes did not change significantly upon bilateral ovariectomy. Treatment with 17β-estradiol after ovariectomy restored BK channel protein levels to the control value. In contrast, BK channel mRNA levels were not significantly affected by either ovariectomy alone or 17β-estradiol treatment. The small-conductance KCa type 3 channel (SK3) mRNA and protein levels decreased to 75% of control levels upon 17β-estradiol treatment. Conclusions: These results suggest that 17β-estradiol may influence urinary bladder function by modulating BK and SK3 channel expression. PMID:27032553

  12. Stoichiometry of altered hERG1 channel gating by small molecule activators.

    PubMed

    Wu, Wei; Sachse, Frank B; Gardner, Alison; Sanguinetti, Michael C

    2014-04-01

    Voltage-gated K(+) channels are tetramers formed by coassembly of four identical or highly related subunits. All four subunits contribute to formation of the selectivity filter, the narrowest region of the channel pore which determines K(+) selective conductance. In some K(+) channels, the selectivity filter can undergo a conformational change to reduce K(+) flux by a mechanism called C-type inactivation. In human ether-a-go-go-related gene 1 (hERG1) K(+) channels, C-type inactivation is allosterically inhibited by ICA-105574, a substituted benzamide. PD-118057, a 2-(phenylamino) benzoic acid, alters selectivity filter gating to enhance open probability of channels. Both compounds bind to a hydrophobic pocket located between adjacent hERG1 subunits. Accordingly, a homotetrameric channel contains four identical activator binding sites. Here we determine the number of binding sites required for maximal drug effect and determine the role of subunit interactions in the modulation of hERG1 gating by these compounds. Concatenated tetramers were constructed to contain a variable number (zero to four) of wild-type and mutant hERG1 subunits, either L646E to inhibit PD-118057 binding or F557L to inhibit ICA-105574 binding. Enhancement of hERG1 channel current magnitude by PD-118057 and attenuated inactivation by ICA-105574 were mediated by cooperative subunit interactions. Maximal effects of the both compounds required the presence of all four binding sites. Understanding how hERG1 agonists allosterically modify channel gating may facilitate mechanism-based drug design of novel agents for treatment of long QT syndrome. PMID:24638994

  13. Stoichiometry of altered hERG1 channel gating by small molecule activators

    PubMed Central

    Wu, Wei; Sachse, Frank B.; Gardner, Alison

    2014-01-01

    Voltage-gated K+ channels are tetramers formed by coassembly of four identical or highly related subunits. All four subunits contribute to formation of the selectivity filter, the narrowest region of the channel pore which determines K+ selective conductance. In some K+ channels, the selectivity filter can undergo a conformational change to reduce K+ flux by a mechanism called C-type inactivation. In human ether-a-go-go–related gene 1 (hERG1) K+ channels, C-type inactivation is allosterically inhibited by ICA-105574, a substituted benzamide. PD-118057, a 2-(phenylamino) benzoic acid, alters selectivity filter gating to enhance open probability of channels. Both compounds bind to a hydrophobic pocket located between adjacent hERG1 subunits. Accordingly, a homotetrameric channel contains four identical activator binding sites. Here we determine the number of binding sites required for maximal drug effect and determine the role of subunit interactions in the modulation of hERG1 gating by these compounds. Concatenated tetramers were constructed to contain a variable number (zero to four) of wild-type and mutant hERG1 subunits, either L646E to inhibit PD-118057 binding or F557L to inhibit ICA-105574 binding. Enhancement of hERG1 channel current magnitude by PD-118057 and attenuated inactivation by ICA-105574 were mediated by cooperative subunit interactions. Maximal effects of the both compounds required the presence of all four binding sites. Understanding how hERG1 agonists allosterically modify channel gating may facilitate mechanism-based drug design of novel agents for treatment of long QT syndrome. PMID:24638994

  14. Role of thromboxane A₂-activated nonselective cation channels in hypoxic pulmonary vasoconstriction of rat.

    PubMed

    Yoo, Hae Young; Park, Su Jung; Seo, Eun-Young; Park, Kyung Sun; Han, Jung-A; Kim, Kyung Soo; Shin, Dong Hoon; Earm, Yung E; Zhang, Yin-Hua; Kim, Sung Joon

    2012-01-01

    Hypoxia-induced pulmonary vasoconstriction (HPV) is critical for matching of ventilation/perfusion in lungs. Although hypoxic inhibition of K(+) channels has been a leading hypothesis for depolarization of pulmonary arterial smooth muscle cells (PASMCs) under hypoxia, pharmacological inhibition of K(+) channels does not induce significant contraction in rat pulmonary arteries. Because a partial contraction by thromboxane A(2) (TXA(2)) is required for induction of HPV, we hypothesize that TXA(2) receptor (TP) stimulation might activate depolarizing nonselective cation channels (NSCs). Consistently, we found that 5-10 nM U46619, a stable agonist for TP, was indispensible for contraction of rat pulmonary arteries by 4-aminopyridine, a blocker of voltage-gated K(+) channel (K(v)). Whole cell voltage clamp with rat PASMC revealed that U46619 induced a NSC current (I(NSC,TXA2)) with weakly outward rectifying current-voltage relation. I(NSC,TXA2) was blocked by ruthenium red (RR), an antagonist of the transient receptor potential vanilloid-related channel (TRPV) subfamily. 2-Aminoethoxydiphenyl borate, an agonist for TRPV1-3, consistently activated NSC channels in PASMCs. In contrast, agonists for TRPV1 (capsaicin), TRPV3 (camphor), or TRPV4 (α-PDD) rarely induced an increase in the membrane conductance of PASMCs. RT-PCR analysis showed the expression of transcripts for TRPV2 and -4 in rat PASMCs. Finally, it was confirmed that pretreatment with RR largely inhibited HPV in the presence of U46619. The pretreatment with agonists for TRPV1 (capsaicin) and TRPV4 (α-PDD) was ineffective as pretone agents for HPV. Taken together, it is suggested that the concerted effects of I(NSC,TXA2) activation and K(v) inhibition under hypoxia induce membrane depolarization sufficient for HPV. TRPV2 is carefully suggested as the TXA(2)-activated NSC in rat PASMC. PMID:21998141

  15. Molecular and functional significance of Ca2+-activated Cl− channels in pulmonary arterial smooth muscle

    PubMed Central

    Forrest, Abigail S.; Ayon, Ramon J.; Wiwchar, Michael; Angermann, Jeff E.; Pritchard, Harry A. T.; Singer, Cherie A.; Valencik, Maria L.; Britton, Fiona; Greenwood, Iain A.

    2015-01-01

    Abstract Increased peripheral resistance of small distal pulmonary arteries is a hallmark signature of pulmonary hypertension (PH) and is believed to be the consequence of enhanced vasoconstriction to agonists, thickening of the arterial wall due to remodeling, and increased thrombosis. The elevation in arterial tone in PH is attributable, at least in part, to smooth muscle cells of PH patients being more depolarized and displaying higher intracellular Ca2+ levels than cells from normal subjects. It is now clear that downregulation of voltage-dependent K+ channels (e.g., Kv1.5) and increased expression and activity of voltage-dependent (Cav1.2) and voltage-independent (e.g., canonical and vanilloid transient receptor potential [TRPC and TRPV]) Ca2+ channels play an important role in the functional remodeling of pulmonary arteries in PH. This review focuses on an anion-permeable channel that is now considered a novel excitatory mechanism in the systemic and pulmonary circulations. It is permeable to Cl− and is activated by a rise in intracellular Ca2+ concentration (Ca2+-activated Cl− channel, or CaCC). The first section outlines the biophysical and pharmacological properties of the channel and ends with a description of the molecular candidate genes postulated to encode for CaCCs, with particular emphasis on the bestrophin and the newly discovered TMEM16 and anoctamin families of genes. The second section provides a review of the various sources of Ca2+ activating CaCCs, which include stimulation by mobilization from intracellular Ca2+ stores and Ca2+ entry through voltage-dependent and voltage-independent Ca2+ channels. The third and final section summarizes recent findings that suggest a potentially important role for CaCCs and the gene TMEM16A in PH. PMID:26064450

  16. Phase width reduction project summary

    SciTech Connect

    Clark, D.J.; Xie, Z.Q.; McMahan, M. A.

    1999-11-01

    The purpose of the phase width reduction project, 1993--96, was to reduce the phase width of the 88-Inch Cyclotron beam on target from 5--10 ns to 1--2 ns for certain experiments, such as Gammasphere, which use time-of-flight identification. Since reducing the phase width also reduces beam intensity, tuning should be done to also optimize the transmission. The Multi-turn Collimator slits in the cyclotron center region were used to collimate the early turns radially, thus reducing the phase width from about 5 ns to 1--2 ns FWHM for a Gammasphere beam. The effect of the slits on phase width was verified with a Fast Faraday Cup and with particle and gamma-ray detectors in the external beamline.

  17. Hexachlorophene Is a Potent KCNQ1/KCNE1 Potassium Channel Activator Which Rescues LQTs Mutants

    PubMed Central

    Zheng, Yueming; Zhu, Xuejing; Zhou, Pingzheng; Lan, Xi; Xu, Haiyan; Li, Min; Gao, Zhaobing

    2012-01-01

    The voltage-gated KCNQ1 potassium channel is expressed in cardiac tissues, and coassembly of KCNQ1 with an auxiliary KCNE1 subunit mediates a slowly activating current that accelerates the repolarization of action potential in cardiomyocytes. Mutations of KCNQ1 genes that result in reduction or loss of channel activity cause prolongation of repolarization during action potential, thereby causing long QT syndrome (LQTs). Small molecule activators of KCNQ1/KCNE1 are useful both for understanding the mechanism of the complex activity and for developing therapeutics for LQTs. In this study we report that hexachlorophene (HCP), the active component of the topical anti-infective prescription drug pHisoHex, is a KCNQ1/KCNE1 activator. HCP potently increases the current amplitude of KCNQ1/KCNE1 expressed by stabilizing the channel in an open state with an EC50 of 4.61±1.29 μM. Further studies in cardiomyocytes showed that HCP significantly shortens the action potential duration at 1 μM. In addition, HCP is capable of rescuing the loss of function of the LQTs mutants caused by either impaired activation gating or phosphatidylinositol-4,5-bisphosphate (PIP2) binding affinity. Our results indicate HCP is a novel KCNQ1/KCNE1 activator and may be a useful tool compound for the development of LQTs therapeutics. PMID:23251633

  18. 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

  19. 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

  20. Stochastically Gating Ion Channels Enable Patterned Spike Firing through Activity-Dependent Modulation of Spike Probability

    PubMed Central

    Dudman, Joshua T.; Nolan, Matthew F.

    2009-01-01

    The transformation of synaptic input into patterns of spike output is a fundamental operation that is determined by the particular complement of ion channels that a neuron expresses. Although it is well established that individual ion channel proteins make stochastic transitions between conducting and non-conducting states, most models of synaptic integration are deterministic, and relatively little is known about the functional consequences of interactions between stochastically gating ion channels. Here, we show that a model of stellate neurons from layer II of the medial entorhinal cortex implemented with either stochastic or deterministically gating ion channels can reproduce the resting membrane properties of stellate neurons, but only the stochastic version of the model can fully account for perithreshold membrane potential fluctuations and clustered patterns of spike output that are recorded from stellate neurons during depolarized states. We demonstrate that the stochastic model implements an example of a general mechanism for patterning of neuronal output through activity-dependent changes in the probability of spike firing. Unlike deterministic mechanisms that generate spike patterns through slow changes in the state of model parameters, this general stochastic mechanism does not require retention of information beyond the duration of a single spike and its associated afterhyperpolarization. Instead, clustered patterns of spikes emerge in the stochastic model of stellate neurons as a result of a transient increase in firing probability driven by activation of HCN channels during recovery from the spike afterhyperpolarization. Using this model, we infer conditions in which stochastic ion channel gating may influence firing patterns in vivo and predict consequences of modifications of HCN channel function for in vivo firing patterns. PMID:19214199

  1. Natural and synthetic modulators of SK (Kca2) potassium channels inhibit magnesium-dependent activity of the kinase-coupled cation channel TRPM7

    PubMed Central

    Chubanov, V; Mederos y Schnitzler, M; Meißner, M; Schäfer, S; Abstiens, K; Hofmann, T; Gudermann, T

    2012-01-01

    BACKGROUND AND PURPOSE Transient receptor potential cation channel subfamily M member 7 (TRPM7) is a bifunctional protein comprising a TRP ion channel segment linked to an α-type protein kinase domain. TRPM7 is essential for proliferation and cell growth. Up-regulation of TRPM7 function is involved in anoxic neuronal death, cardiac fibrosis and tumour cell proliferation. The goal of this work was to identify non-toxic inhibitors of the TRPM7 channel and to assess the effect of blocking endogenous TRPM7 currents on the phenotype of living cells. EXPERIMENTAL APPROACH We developed an aequorin bioluminescence-based assay of TRPM7 channel activity and performed a hypothesis-driven screen for inhibitors of the channel. The candidates identified were further assessed electrophysiologically and in cell biological experiments. KEY RESULTS TRPM7 currents were inhibited by modulators of small conductance Ca2+-activated K+ channels (KCa2.1–2.3; SK) channels, including the antimalarial plant alkaloid quinine, CyPPA, dequalinium, NS8593, SKA31 and UCL 1684. The most potent compound NS8593 (IC50 1.6 µM) specifically targeted TRPM7 as compared with other TRP channels, interfered with Mg2+-dependent regulation of TRPM7 channel and inhibited the motility of cultured cells. NS8593 exhibited full and reversible block of native TRPM7-like currents in HEK 293 cells, freshly isolated smooth muscle cells, primary podocytes and ventricular myocytes. CONCLUSIONS AND IMPLICATIONS This study reveals a tight overlap in the pharmacological profiles of TRPM7 and KCa2.1–2.3 channels. NS8593 acts as a negative gating modulator of TRPM7 and is well-suited to study functional features and cellular roles of endogenous TRPM7. PMID:22242975

  2. 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

  3. Calcium-activated chloride channels in the apical region of mouse vomeronasal sensory neurons.

    PubMed

    Dibattista, Michele; Amjad, Asma; Maurya, Devendra Kumar; Sagheddu, Claudia; Montani, Giorgia; Tirindelli, Roberto; Menini, Anna

    2012-07-01

    The rodent vomeronasal organ plays a crucial role in several social behaviors. Detection of pheromones or other emitted signaling molecules occurs in the dendritic microvilli of vomeronasal sensory neurons, where the binding of molecules to vomeronasal receptors leads to the influx of sodium and calcium ions mainly through the transient receptor potential canonical 2 (TRPC2) channel. To investigate the physiological role played by the increase in intracellular calcium concentration in the apical region of these neurons, we produced localized, rapid, and reproducible increases in calcium concentration with flash photolysis of caged calcium and measured calcium-activated currents with the whole cell voltage-clamp technique. On average, a large inward calcium-activated current of -261 pA was measured at -50 mV, rising with a time constant of 13 ms. Ion substitution experiments showed that this current is anion selective. Moreover, the chloride channel blockers niflumic acid and 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid partially inhibited the calcium-activated current. These results directly demonstrate that a large chloride current can be activated by calcium in the apical region of mouse vomeronasal sensory neurons. Furthermore, we showed by immunohistochemistry that the calcium-activated chloride channels TMEM16A/anoctamin1 and TMEM16B/anoctamin2 are present in the apical layer of the vomeronasal epithelium, where they largely colocalize with the TRPC2 transduction channel. Immunocytochemistry on isolated vomeronasal sensory neurons showed that TMEM16A and TMEM16B coexpress in the neuronal microvilli. Therefore, we conclude that microvilli of mouse vomeronasal sensory neurons have a high density of calcium-activated chloride channels that may play an important role in vomeronasal transduction. PMID:22732308

  4. Hyperpolarization-activated ion channels as targets for nitric oxide signalling in deep cerebellar nuclei

    PubMed Central

    Wilson, Gary W; Garthwaite, John

    2010-01-01

    Most biological effects of nitric oxide (NO) in the brain are mediated by guanylyl cyclase-coupled NO receptors, whose activation results in increased intracellular cGMP levels. Apart from protein kinase activation little is known about subsequent cGMP signal transduction. In optic nerve axons, hyperpolarization-activated cyclic nucleotide-modulated cation (HCN) channels, which bind cGMP or cAMP directly, were recently suggested to be a target. The aim here was to test this possibility more directly. Neurones of the rat deep cerebellar nuclei were selected for this purpose, their suitability being attested by immunocytochemistry showing that the principal neurones expressed guanylyl cyclase protein and that NO synthase-containing fibres were abundant in the neuropil. Using whole-cell voltage-clamp recording, HCN channels in the neurones were activated in response to isoprenaline and exogenous cAMP but only occasionally did they respond to NO, although exogenous cGMP was routinely effective. With the less invasive sharp microelectrode recording technique, however, exogenous NO modulated the channels reproducibly, as measured by the size of the HCN channel-mediated voltage sag following hyperpolarization. Moreover, NO also blunted the subsequent rebound depolarizing potentials, consistent with it increasing the hyperpolarization-activated current. Optimizing the whole-cell solution to improve the functioning of NO-activated guanylyl cyclase failed to restore NO sensitivity. Minimizing cellular dialysis by using the perforated-patch technique, however, was successful. The results provide evidence that HCN channels are potential downstream mediators of NO signalling in deep cerebellar nuclei neurones and suggest that the more general importance of this transduction pathway may have been overlooked previously because of unsuitable recording methods. PMID:20529121

  5. Basolateral K channel activated by carbachol in the epithelial cell line T84.

    PubMed

    Tabcharani, J A; Harris, R A; Boucher, A; Eng, J W; Hanrahan, J W

    1994-11-01

    Cholinergic stimulation of chloride secretion involves the activation of a basolateral membrane potassium conductance, which maintains the electrical gradient favoring apical Cl efflux and allows K to recycle at the basolateral membrane. We have used transepithelial short-circuit current (Isc), fluorescence imaging, and patch clamp studies to identify and characterize the K channel that mediates this response in T84 cells. Carbachol had little effect on Isc when added alone but produced large, transient currents if added to monolayers prestimulated with cAMP. cAMP also enhanced the subsequent Isc response to calcium ionophores. Carbachol (100 microM) transiently elevated intracellular free calcium ([Ca2+]i) by approximately 3-fold in confluent cells cultured on glass coverslips with a time course resembling the Isc response of confluent monolayers that had been grown on porous supports. In parallel patch clamp experiments, carbachol activated an inwardly rectifying potassium channel on the basolateral aspect of polarized monolayers which had been dissected from porous culture supports. The same channel was transiently activated on the surface of subconfluent monolayers during stimulation by carbachol. Activation was more prolonged when cells were exposed to calcium ionophores. The conductance of the inward rectifier in cell-attached patches was 55 pS near the resting membrane potential (-54 mV) with pipette solution containing 150 mM KCl (37 degrees C). This rectification persisted when patches were bathed in symmetrical 150 mM KCl solutions. The selectivity sequence was 1 K > 0.88 Rb > 0.18 Na > Cs based on permeability ratios under bi-ionic conditions. The channel exhibited fast block by external sodium ions, was weakly inhibited by external TEA, was relatively insensitive to charybdotoxin, kaliotoxin, 4-aminopyridine and quinidine, and was unaffected by external 10 mM barium. It is referred to as the KBIC channel based on its most distinctive properties (Ba

  6. Activation of the melastatin-related cation channel TRPM3 by D-erythro-sphingosine [corrected].

    PubMed

    Grimm, Christian; Kraft, Robert; Schultz, Günter; Harteneck, Christian

    2005-03-01

    TRPM3, a member of the melastatin-like transient receptor potential channel subfamily (TRPM), is predominantly expressed in human kidney and brain. TRPM3 mediates spontaneous Ca2+ entry and nonselective cation currents in transiently transfected human embryonic kidney 293 cells. Using measurements with the Ca2+-sensitive fluorescent dye fura-2 and the whole-cell patch-clamp technique, we found that D-erythro-sphingosine, a metabolite arising during the de novo synthesis of cellular sphingolipids, activated TRPM3. Other transient receptor potential (TRP) channels tested [classic or canonical TRP (TRPC3, TRPC4, TRPC5), vanilloid-like TRP (TRPV4, TRPV5, TRPV6), and melastatin-like TRP (TRPM2)] did not significantly respond to application of sphingosine. Sphingosine-induced TRPM3 activation was not mediated by inhibition of protein kinase C, depletion of intracellular Ca2+ stores, and intracellular conversion of sphingosine to sphingosine-1-phosphate. Although sphingosine-1-phosphate and ceramides had no effect, two structural analogs of sphingosine, dihydro-D-erythro-sphingosine and N,N-dimethyl-D-erythro-sphingosine, also activated TRPM3. Sphingolipids, including sphingosine, are known to have inhibitory effects on a variety of ion channels. Thus, TRPM3 is the first ion channel activated by sphingolipids. PMID:15550678

  7. Emodin augments calcium activated chloride channel in colonic smooth muscle cells by Gi/Go protein.

    PubMed

    Xu, Long; Ting-Lou; Lv, Nonghua; Zhu, Xuan; Chen, Youxiang; Yang, Jing

    2009-08-01

    Emodin is a natural anthraquinone in rhubarb. It has been identified as a prokinetic drug for gastrointestinal motility in Chinese traditional medicine. Emodin contracts smooth muscle by increasing the concentration of intracellular Ca(2+). In many smooth muscles, increasing intracellular Ca(2+) activates Ca(2+)-activated Cl(-) channels (ClCA). The study was aimed to investigate the effects of emodin on ClCA channels in colonic smooth muscle. 4 channel physiology signal acquire system was used to measure isometric contraction of smooth muscle strips. ClCA currents were recorded by EPC10 with perforated whole cell model. Emodin contracted strips and cells in colonic smooth muscle and augmented ClCA currents. Niflumic acid (NFA) and 4', 4'-diisothiostilbene-2, 2-disulfonic acid (DIDS) blocked the effects. Gi/Go protein inhibits protein kinase A (PKA) and protein kinase C (PKC), and PKA and PKC reduced ClCA currents. Pertussis toxin (PTX, a special inhibitor of Gi/Go protein), 8-bromoadenosine 38, 58-cyclic monophosphate (8-BrcAMP, a membrane-permeant protein kinase A activator) and Phorbol-12-myristate-13-acetate (PMA, a membrane-permeant protein kinase C activator) inhibited the effects on ClCA currents significantly. Our findings suggest that emodin augments ClCA channels to contract smooth muscle in colon, and the effect is induced mostly by enhancement of membrane Gi/Go protein signal transducer pathway. PMID:19409890

  8. 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

  9. Synthesis and characterization of a Redox-active artificial ion channel.

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The synthesis and characterization of an artificial ion channel containing both fluorescent and redox-active centers is described. fluorescence spectroscopy was used to study qualitative and quantitative aspects of the coordination of alkali metal cations and black lipid membrane studies were used ...

  10. Ethylene activates a plasma membrane Ca(2+)-permeable channel in tobacco suspension cells.

    PubMed

    Zhao, Min-Gui; Tian, Qiu-Ying; Zhang, Wen-Hao

    2007-01-01

    Here, the effects of the ethylene-releasing compound, ethephon, and the ethylene precursor, 1-aminocyclopropane-1-carboxylic acid (ACC), on ionic currents across plasma membranes and on the cytosolic Ca(2+) activity ([Ca(2+)](c)) of tobacco (Nicotiana tabacum) suspension cells were characterized using a patch-clamp technique and confocal laser scanning microscopy. Exposure of tobacco protoplasts to ethephon and ACC led to activation of a plasma membrane cation channel that was permeable to Ba(2+), Mg(2+) and Ca(2+), and inhibited by La(3+), Gd(3+) and Al(3+). The ethephon- and ACC-induced Ca(2+)-permeable channel was abolished by the antagonist of ethylene perception (1-metycyclopropene) and by the inhibitor of ACC synthase (aminovinylglycin), indicating that activation of the Ca(2+)-permeable channels results from ethylene. Ethephon elicited an increase in the [Ca(2+)](c) of tobacco suspension cells, as visualized by the Ca(2+)-sensitive probe Fluo-3 and confocal microscopy. The ethephon-induced elevation of [Ca(2+)](c) was markedly inhibited by Gd(3+) and BAPTA, suggesting that an influx of Ca(2+) underlies the elevation of [Ca(2+)](c). These results indicate that an elevation of [Ca(2+)](c), resulting from activation of the plasma membrane Ca(2+)-permeable channels by ethylene, is an essential component in ethylene signaling in plants. PMID:17447907

  11. Evaluation of partial widths and branching ratios from resonance wave functions

    SciTech Connect

    Goldzak, Tamar; Gilary, Ido; Moiseyev, Nimrod

    2010-11-15

    A quantum system in a given resonance state has different open channels for decay. Partial widths are the decay rates of the resonance (metastable) state into the different open channels. Here we present a rigorous derivation of the partial widths from the solution of a time-dependent Schroedinger equation with outgoing boundary conditions. We show that the sum of the partial widths obtained from the resonance wave function is equal to the total width. The difference with respect to previous studies on partial widths and branching ratios is discussed.

  12. Tuning voltage-gated channel activity and cellular excitability with a sphingomyelinase

    PubMed Central

    Combs, David J.; Shin, Hyeon-Gyu; Xu, Yanping; Ramu, Yajamana

    2013-01-01

    Voltage-gated ion channels generate action potentials in excitable cells and help set the resting membrane potential in nonexcitable cells like lymphocytes. It has been difficult to investigate what kinds of phospholipids interact with these membrane proteins in their native environments and what functional impacts such interactions create. This problem might be circumvented if we could modify specific lipid types in situ. Using certain voltage-gated K+ (KV) channels heterologously expressed in Xenopus laevis oocytes as a model, our group has shown previously that sphingomyelinase (SMase) D may serve this purpose. SMase D is known to remove the choline group from sphingomyelin, a phospholipid primarily present in the outer leaflet of plasma membranes. This SMase D action lowers the energy required for voltage sensors of a KV channel to enter the activated state, causing a hyperpolarizing shift of the Q-V and G-V curves and thus activating them at more hyperpolarized potentials. Here, we find that this SMase D effect vanishes after removing most of the voltage-sensor paddle sequence, a finding supporting the notion that SMase D modification of sphingomyelin molecules alters these lipids’ interactions with voltage sensors. Then, using SMase D to probe lipid–channel interactions, we find that SMase D not only similarly stimulates voltage-gated Na+ (NaV) and Ca2+ channels but also markedly slows NaV channel inactivation. However, the latter effect is not observed in tested mammalian cells, an observation highlighting the profound impact of the membrane environment on channel function. Finally, we directly demonstrate that SMase D stimulates both native KV1.3 in nonexcitable human T lymphocytes at their typical resting membrane potential and native NaV channels in excitable cells, such that it shifts the action potential threshold in the hyperpolarized direction. These proof-of-concept studies illustrate that the voltage-gated channel activity in both excitable and

  13. Downstream variation in bankfull width of wadeable streams across the conterminous United States

    EPA Science Inventory

    Bankfull channel width is a fundamental measure of stream size and a key parameter of interest for many applications in hydrology, fluvial geomorphology, and stream ecology. We developed downstream hydraulic geometry relationships for bankfull channel width w as a function of dra...

  14. Pharmacological and electrophysiological characterization of AZSMO-23, an activator of the hERG K+ channel

    PubMed Central

    Mannikko, R; Bridgland-Taylor, M H; Pye, H; Swallow, S; Abi-Gerges, N; Morton, M J; Pollard, C E

    2015-01-01

    Background and Purpose We aimed to characterize the pharmacology and electrophysiology of N-[3-(1H-benzimidazol-2-yl)-4-chloro-phenyl]pyridine-3-carboxamide (AZSMO-23), an activator of the human ether-a-go-go-related gene (hERG)-encoded K+ channel (Kv11.1). Experimental Approach Automated electrophysiology was used to study the pharmacology of AZSMO-23 on wild-type (WT), Y652A, F656T or G628C/S631C hERG, and on other cardiac ion channels. Its mechanism of action was characterized with conventional electrophysiology. Key Results AZSMO-23 activated WT hERG pre-pulse and tail current with EC50 values of 28.6 and 11.2 μM respectively. At 100 μM, pre-pulse current at +40 mV was increased by 952 ± 41% and tail current at −30 mV by 238 ± 13% compared with vehicle values. The primary mechanism for this effect was a 74.5 mV depolarizing shift in the voltage dependence of inactivation, without any shift in the voltage dependence of activation. Structure–activity relationships for this effect were remarkably subtle, with close analogues of AZSMO-23 acting as hERG inhibitors. AZSMO-23 blocked the mutant channel, hERG Y652A, but against another mutant channel, hERG F656T, its activator activity was enhanced. It inhibited activity of the G628C/S631C non-inactivating hERG mutant channel. AZSMO-23 was not hERG selective, as it blocked hKv4.3-hKChIP2.2, hCav3.2 and hKv1.5 and activated hCav1.2/β2/α2δ channels. Conclusion and Implications The activity of AZSMO-23 and those of its close analogues suggest these compounds may be of value to elucidate the mechanism of type 2 hERG activators to better understand the pharmacology of this area from both a safety perspective and in relation to treatment of congenital long QT syndrome. PMID:25684549

  15. 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

  16. Activation of Ca2+-dependent K+ channels in human B lymphocytes by anti-immunoglobulin.

    PubMed Central

    MacDougall, S L; Grinstein, S; Gelfand, E W

    1988-01-01

    Many mammalian cell types exhibit Ca2+-dependent K+ channels, and activation of these channels by increasing intracellular calcium generally leads to a hyperpolarization of the plasma membrane. Their presence in B lymphocytes is as yet uncertain. Crosslinking Ig on the surface of B lymphocytes is known to increase the level of free cytoplasmic calcium ([Ca2+]i). However, rather than hyperpolarization, a depolarization has been reported to occur after treatment of B lymphocytes with anti-Ig. To determine if Ca2+-dependent K+ channels are present in B lymphocytes, and to examine the relationship between intracellular free calcium and membrane potential, we monitored [Ca2+]i by means of indo-1 and transmembrane potential using bis(1,3-diethylthiobarbituric)trimethine oxonol in human tonsillar B cells activated by anti-IgM. Treatment with anti-IgM induced a biphasic increase in [Ca2+]i and a simultaneous hyperpolarization. A similar hyperpolarization was induced by ionomycin, a Ca2+ ionophore. Delaying the development of the [Ca2+]i response by increasing the cytoplasmic Ca2+-buffering power delayed the hyperpolarization. Conversely, eliminating the sustained phase of the [Ca2+]i response by omission of external Ca2+ abolished the prolonged hyperpolarization. In fact, a sizable Na+-dependent depolarization was unmasked. This study demonstrates that in human B lymphocytes, Ca2+-dependent K+ channels can be activated by crosslinking of surface IgM. Moreover, it is likely that, by analogy with voltage-sensitive Ca2+ channels, Na+ can permeate through these ligand-gated Ca2+ "channels" in the absence of extracellular Ca2+. PMID:2448342

  17. NMR Structure and Ion Channel Activity of the p7 Protein from Hepatitis C Virus*

    PubMed Central

    Montserret, Roland; Saint, Nathalie; Vanbelle, Christophe; Salvay, Andrés Gerardo; Simorre, Jean-Pierre; Ebel, Christine; Sapay, Nicolas; Renisio, Jean-Guillaume; Böckmann, Anja; Steinmann, Eike; Pietschmann, Thomas; Dubuisson, Jean; Chipot, Christophe; Penin, François

    2010-01-01

    The small membrane protein p7 of hepatitis C virus forms oligomers and exhibits ion channel activity essential for virus infectivity. These viroporin features render p7 an attractive target for antiviral drug development. In this study, p7 from strain HCV-J (genotype 1b) was chemically synthesized and purified for ion channel activity measurements and structure analyses. p7 forms cation-selective ion channels in planar lipid bilayers and at the single-channel level by the patch clamp technique. Ion channel activity was shown to be inhibited by hexamethylene amiloride but not by amantadine. Circular dichroism analyses revealed that the structure of p7 is mainly α-helical, irrespective of the membrane mimetic medium (e.g. lysolipids, detergents, or organic solvent/water mixtures). The secondary structure elements of the monomeric form of p7 were determined by 1H and 13C NMR in trifluoroethanol/water mixtures. Molecular dynamics simulations in a model membrane were combined synergistically with structural data obtained from NMR experiments. This approach allowed us to determine the secondary structure elements of p7, which significantly differ from predictions, and to propose a three-dimensional model of the monomeric form of p7 associated with the phospholipid bilayer. These studies revealed the presence of a turn connecting an unexpected N-terminal α-helix to the first transmembrane helix, TM1, and a long cytosolic loop bearing the dibasic motif and connecting TM1 to TM2. These results provide the first detailed experimental structural framework for a better understanding of p7 processing, oligomerization, and ion channel gating mechanism. PMID:20667830

  18. Structure-activity studies on a novel series of cholinergic channel activators based on a heteroaryl ether framework.

    PubMed

    Lin, N H; Abreo, M A; Gunn, D E; Lebold, S A; Lee, E L; Wasicak, J T; Hettinger, A M; Daanen, J F; Garvey, D S; Campbell, J E; Sullivan, J P; Williams, M; Arneric, S P

    1999-09-20

    Analogs of compound 1 with a variety of azacycles and heteroaryl groups were synthesized. These analogs exhibited Ki values ranging from 0.15 to > 10,000 nM when tested in vitro for cholinergic channel receptor binding activity (displacement of [3H](-) cytisine from whole rat brain synaptic membranes). PMID:10509928

  19. Ginseng Gintonin Activates the Human Cardiac Delayed Rectifier K+ Channel: Involvement of Ca2+/Calmodulin Binding Sites

    PubMed Central

    Choi, Sun-Hye; Lee, Byung-Hwan; Kim, Hyeon-Joong; Jung, Seok-Won; Kim, Hyun-Sook; Shin, Ho-Chul; Lee, Jun-Hee; Kim, Hyoung-Chun; Rhim, Hyewhon; Hwang, Sung-Hee; Ha, Tal soo; Kim, Hyun-Ji; Cho, Hana; Nah, Seung-Yeol

    2014-01-01

    Gintonin, a novel, ginseng-derived G protein-coupled lysophosphatidic acid (LPA) receptor ligand, elicits [Ca2+]i transients in neuronal and non-neuronal cells via pertussis toxin-sensitive and pertussis toxin-insensitive G proteins. The slowly activating delayed rectifier K+ (IKs) channel is a cardiac K+ channel composed of KCNQ1 and KCNE1 subunits. The C terminus of the KCNQ1 channel protein has two calmodulin-binding sites that are involved in regulating IKs channels. In this study, we investigated the molecular mechanisms of gintonin-mediated activation of human IKs channel activity by expressing human IKs channels in Xenopus oocytes. We found that gintonin enhances IKs channel currents in concentration- and voltage-dependent manners. The EC50 for the IKs channel was 0.05 ± 0.01 μg/ml. Gintonin-mediated activation of the IKs channels was blocked by an LPA1/3 receptor antagonist, an active phospholipase C inhibitor, an IP3 receptor antagonist, and the calcium chelator BAPTA. Gintonin-mediated activation of both the IKs channel was also blocked by the calmodulin (CaM) blocker calmidazolium. Mutations in the KCNQ1 [Ca2+]i/CaM-binding IQ motif sites (S373P, W392R, or R539W)blocked the action of gintonin on IKs channel. However, gintonin had no effect on hERG K+ channel activity. These results show that gintonin-mediated enhancement of IKs channel currents is achieved through binding of the [Ca2+]i/CaM complex to the C terminus of KCNQ1 subunit. PMID:25234465

  20. 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. PMID:25619656

  1. 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

  2. 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

  3. 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. PMID:26486785

  4. 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. PMID:26134504

  5. Pharmacological evidence for Orai channel activation as a source of cardiac abnormal automaticity.

    PubMed

    Wolkowicz, Paul E; Huang, Jian; Umeda, Patrick K; Sharifov, Oleg F; Tabengwa, Edlue; Halloran, Brian A; Urthaler, Ferdinand; Grenett, Hernan E

    2011-10-01

    Calcium transport through plasma membrane voltage-independent calcium channels is vital for signaling events in non-excitable and excitable cells. Following up on our earlier work, we tested the hypothesis that this type of calcium transport can disrupt myocardial electromechanical stability. Our Western and immunofluorescence analyses show that left atrial and ventricular myocytes express the Orai1 and the Orai3 calcium channels. Adding the Orai activator 2-aminoethoxydiphenyl borate (2-APB) to the superfusate of rat left atria causes these non-automatic muscles to contract spontaneously and persistently at rates of up to 10 Hz, and to produce normal action potentials from normal resting potentials, all in the absence of external stimulation. 2-APB likewise induces such automatic activity in superfused rat left ventricular papillary muscles, and the EC(50)s at which 2-APB induces this activity in both muscles are similar to the concentrations which activate Orais. Importantly, the voltage-independent calcium channel inhibitor 1-[2-(4-methoxyphenyl)-2-[3-(4-methoxyphenyl) propoxy]ethyl-1H-imidazole (SKF-96365) suppresses this automaticity with an IC(50) of 11 ± 0.6 μM in left atria and 6 ± 1.6 μM in papillary muscles. 1-(5-Iodonaphthalene-1-sulfonyl)-hexahydro-1,4-diazepine (ML-7), a second voltage-independent calcium channel inhibitor, and two calmodulin inhibitors also prevent 2-APB automaticity while two calmodulin-dependent protein kinase II inhibitors do not. Thus an activator of the Orai calcium channels provokes a novel type of high frequency automaticity in non-automatic heart muscle. PMID:21745466

  6. Properties of the Ca-activated K+ channel in pancreatic beta-cells.

    PubMed

    Atwater, I; Rosario, L; Rojas, E

    1983-12-01

    The existence of [Ca2+]i-activated K+-channels in the pancreatic beta-cell membrane is based in two observations: quinine inhibits K+-permeability and, increasing intracellular Ca2+ stimulates it. The changes in K+-permeability of the beta-cell have been monitored electrically by combining measurements of the dependence of the membrane potential on external K+ concentration and input resistance. The changes in the passive 42K and 86Rb efflux from the whole islet have been measured directly. Intracellular Ca2+ has been increased by various means, including increasing extracellular Ca2+, addition of the Ca2+-ionophore A23187 or noradrenaline and application of mitochondrial uncouplers and blockers. In addition to quinine, many other substances have been found to inhibit or modulate the [Ca2+]i-activated K+-channel. The most important of these is the natural stimulus for insulin secretion, glucose. Glucose may inhibit K+-permeability by lowering intracellular Ca2+. Glibenclamide, a hypoglycaemic sulphonylurea, is about 25 times more active than quinine in blocking the K+-channel in beta-cells. The methylxanthines, c-AMP, various calmodulin inhibitors and Ba2+ also inhibit K+-permeability. Genetically diabetic mice have been studied and show an alteration in the [Ca2+]i-activated K+-channel. It is concluded that the [Ca2+]i-activated K+-channel plays a major role in the normal function of the pancreatic beta-cell. The study of its properties should prove valuable for the understanding and treatment of diabetes. PMID:6323007

  7. International Union of Basic and Clinical Pharmacology. LXXXV: Calcium-Activated Chloride Channels

    PubMed Central

    Huang, Fen; Wong, Xiuming

    2012-01-01

    Calcium-activated chloride channels (CaCCs) are widely expressed in various tissues and implicated in physiological processes such as sensory transduction, epithelial secretion, and smooth muscle contraction. Transmembrane proteins with unknown function 16 (TMEM16A) has recently been identified as a major component of CaCCs. Detailed molecular analysis of TMEM16A will be needed to understand its structure-function relationships. The role this channel plays in physiological systems remains to be established and is currently a subject of intense investigation. PMID:22090471

  8. Cloning and Distribution of Ca2+-activated K+ channels in Lobster Panulirus interruptus

    PubMed Central

    Ouyang, Qing; Patel, Vinay; Vanderburgh, Jacqueline; Harris-Warrick, Ronald M.

    2010-01-01

    Large conductance Ca2+-activated potassium (BK) channels play important roles in controlling neuronal excitability. We cloned the PISlo gene encoding BK channels from the spiny lobster, Panulirus interruptus. This gene shows 81–98% sequence identity to Slo genes previously found in other organisms. We isolated a number of splice variants of the PISlo cDNA within Panulirus interruptus nervous tissue. Sequence analysis indicated that there are at least 7 alternative splice sites in PISlo, each with multiple alternative segments. Using immunohistochemistry, we found that the PISlo proteins are distributed in the synaptic neuropil, axon and soma of STG neurons. PMID:20682332

  9. Blockade of TRPM7 Channel Activity and Cell Death by Inhibitors of 5-Lipoxygenase

    PubMed Central

    Chen, Hsiang-Chin; Xie, Jia; Zhang, Zheng; Su, Li-Ting; Yue, Lixia; Runnels, Loren W.

    2010-01-01

    TRPM7 is a ubiquitous divalent-selective ion channel with its own kinase domain. Recent studies have shown that suppression of TRPM7 protein expression by RNA interference increases resistance to ischemia-induced neuronal cell death in vivo and in vitro, making the channel a potentially attractive pharmacological target for molecular intervention. Here, we report the identification of the 5-lipoxygenase inhibitors, NDGA, AA861, and MK886, as potent blockers of the TRPM7 channel. Using a cell-based assay, application of these compounds prevented cell rounding caused by overexpression of TRPM7 in HEK-293 cells, whereas inhibitors of 12-lipoxygenase and 15-lipoxygenase did not prevent the change in cell morphology. Application of the 5-lipoxygenase inhibitors blocked heterologously expressed TRPM7 whole-cell currents without affecting the protein's expression level or its cell surface concentration. All three inhibitors were also effective in blocking the native TRPM7 current in HEK-293 cells. However, two other 5-lipoxygenase specific inhibitors, 5,6-dehydro-arachidonic acid and zileuton, were ineffective in suppressing TRPM7 channel activity. Targeted knockdown of 5-lipoxygenase did not reduce TRPM7 whole-cell currents. In addition, application of 5-hydroperoxyeicosatetraenoic acid (5-HPETE), the product of 5-lipoxygenase, or 5-HPETE's downstream metabolites, leukotriene B4 and leukotriene D4, did not stimulate TRPM7 channel activity. These data suggested that NDGA, AA861, and MK886 reduced the TRPM7 channel activity independent of their effect on 5-lipoxygenase activity. Application of AA861 and NDGA reduced cell death for cells overexpressing TRPM7 cultured in low extracellular divalent cations. Moreover, treatment of HEK-293 cells with AA861 increased cell resistance to apoptotic stimuli to a level similar to that obtained for cells in which TRPM7 was knocked down by RNA interference. In conclusion, NDGA, AA861, and MK886 are potent blockers of the TRPM7 channel

  10. Cloning and distribution of Ca2+-activated K+ channels in lobster Panulirus interruptus.

    PubMed

    Ouyang, Q; Patel, V; Vanderburgh, J; Harris-Warrick, R M

    2010-10-27

    Large conductance Ca(2+)-activated potassium (BK) channels play important roles in controlling neuronal excitability. We cloned the PISlo gene encoding BK channels from the spiny lobster, Panulirus interruptus. This gene shows 81-98% sequence identity to Slo genes previously found in other organisms. We isolated a number of splice variants of the PISlo cDNA within Panulirus interruptus nervous tissue. Sequence analysis indicated that there are at least seven alternative splice sites in PISlo, each with multiple alternative segments. Using immunohistochemistry, we found that the PISlo proteins are distributed in the synaptic neuropil, axon and soma of stomatogastric ganglion (STG) neurons. PMID:20682332

  11. 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

  12. The TRPC6 channel activator hyperforin induces the release of zinc and calcium from mitochondria.

    PubMed

    Tu, Peng; Gibon, Julien; Bouron, Alexandre

    2010-01-01

    Hyperforin, an extract of the medicinal plant hypericum perforatum (also named St John's wort), possesses antidepressant properties. Recent data showed that it elevates the intracellular concentration of Ca(2+) by activating diacylglycerol-sensitive C-class of transient receptor potential (TRPC6) channels without activating the other isoforms (TRPC1, TRPC3, TRPC4, TRPC5, and TRPC7). This study was undertaken to further characterize the cellular neuronal responses induced by hyperforin. Experiments conducted on cortical neurons in primary culture and loaded with fluorescent probes for Ca(2+) (Fluo-4) and Zn(2+) (FluoZin-3) showed that it not only controls the activity of plasma membrane channels but it also mobilizes these two cations from internal pools. Experiments conducted on isolated brain mitochondria indicated that hyperforin, like the inhibitor of oxidative phosphorylation, carbonyl cyanide 4-(trifluoromethoxy)phenylhydrazone (FCCP), collapses the mitochondrial membrane potential. Furthermore, it promotes the release of Ca(2+) and Zn(2+) from these organelles via a ruthenium red-sensitive transporter. In fact, hyperforin exerts complex actions on CNS neurons. This antidepressant not only triggers the entry of cations via plasma membrane TRPC6 channels but it displays protonophore-like properties. As hyperforin is now use to probe the functions of native TRPC6 channels, our data indicate that caution is required when interpreting results obtained with this antidepressant. PMID:19845832

  13. 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. PMID:15659289

  14. 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

  15. 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

  16. Synthesis and Evaluation of Potent KCNQ2/3-Specific Channel Activators.

    PubMed

    Kumar, Manoj; Reed, Nicholas; Liu, Ruiting; Aizenman, Elias; Wipf, Peter; Tzounopoulos, Thanos

    2016-06-01

    KQT-like subfamily (KCNQ) channels are voltage-gated, noninactivating potassium ion channels, and their down-regulation has been implicated in several hyperexcitability-related disorders, including epilepsy, neuropathic pain, and tinnitus. Activators of these channels reduce the excitability of central and peripheral neurons, and, as such, have therapeutic utility. Here, we synthetically modified several moieties of the KCNQ2-5 channel activator retigabine, an anticonvulsant approved by the U.S. Food and Drug Administration. By introducing a CF3-group at the 4-position of the benzylamine moiety, combined with a fluorine atom at the 3-position of the aniline ring, we generated Ethyl (2-amino-3-fluoro-4-((4-(trifluoromethyl)benzyl)amino)phenyl)carbamate (RL648_81), a new KCNQ2/3-specific activator that is >15 times more potent and also more selective than retigabine. We suggest that RL648_81 is a promising clinical candidate for treating or preventing neurologic disorders associated with neuronal hyperexcitability. PMID:27005699

  17. Low-frequency summation of synaptically activated TRP channel-mediated depolarizations

    PubMed Central

    Petersson, Marcus E.; Yoshida, Motoharu; Fransén, Erik A.

    2011-01-01

    Neurons sum their input by spatial and temporal integration. Temporally, presynaptic firing rates are converted to dendritic membrane depolarizations by postsynaptic receptors and ion channels. In several regions of the brain, including higher association areas, a majority of firing rates are low. For rates below 20 Hz the ionotropic receptors AMPA and NMDA will not produce effective temporal summation. We hypothesized that depolarization mediated by transient receptor potential (TRP) channels activated by metabotropic glutamate receptors would be more effective due to their slow kinetics. Based on voltage-clamp and current-clamp recordings from a rat slice preparation, we constructed a computational model of the TRP channel and its intracellular activation pathway, including the metabotropic glutamate receptor. We show that synaptic input frequencies down to 3-4 Hz and inputs consisting of as few as 3-5 pulses can be effectively summed. We further show that the time constant of integration increases with increasing stimulation frequency and duration. We suggest that the temporal summation characteristics of TRP channels may be important at distal dendritic arbors where spatial summation is limited by the number of concurrently active synapses. It may be particularly important in regions characterized by low and irregular rates. PMID:21777305

  18. 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

  19. 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

  20. 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

  1. Relevance of Viroporin Ion Channel Activity on Viral Replication and Pathogenesis.

    PubMed

    Nieto-Torres, Jose L; Verdiá-Báguena, Carmina; Castaño-Rodriguez, Carlos; Aguilella, Vicente M; Enjuanes, Luis

    2015-07-01

    Modification of host-cell ionic content is a significant issue for viruses, as several viral proteins displaying ion channel activity, named viroporins, have been identified. Viroporins interact with different cellular membranes and self-assemble forming ion conductive pores. In general, these channels display mild ion selectivity, and, eventually, membrane lipids play key structural and functional roles in the pore. Viroporins stimulate virus production through different mechanisms, and ion channel conductivity has been proved particularly relevant in several cases. Key stages of the viral cycle such as virus uncoating, transport and maturation are ion-influenced processes in many viral species. Besides boosting virus propagation, viroporins have also been associated with pathogenesis. Linking pathogenesis either to the ion conductivity or to other functions of viroporins has been elusive for a long time. This article summarizes novel pathways leading to disease stimulated by viroporin ion conduction, such as inflammasome driven immunopathology. PMID:26151305

  2. Relevance of Viroporin Ion Channel Activity on Viral Replication and Pathogenesis

    PubMed Central

    Nieto-Torres, Jose L.; Verdiá-Báguena, Carmina; Castaño-Rodriguez, Carlos; Aguilella, Vicente M.; Enjuanes, Luis

    2015-01-01

    Modification of host-cell ionic content is a significant issue for viruses, as several viral proteins displaying ion channel activity, named viroporins, have been identified. Viroporins interact with different cellular membranes and self-assemble forming ion conductive pores. In general, these channels display mild ion selectivity, and, eventually, membrane lipids play key structural and functional roles in the pore. Viroporins stimulate virus production through different mechanisms, and ion channel conductivity has been proved particularly relevant in several cases. Key stages of the viral cycle such as virus uncoating, transport and maturation are ion-influenced processes in many viral species. Besides boosting virus propagation, viroporins have also been associated with pathogenesis. Linking pathogenesis either to the ion conductivity or to other functions of viroporins has been elusive for a long time. This article summarizes novel pathways leading to disease stimulated by viroporin ion conduction, such as inflammasome driven immunopathology. PMID:26151305

  3. 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. PMID:26034200

  4. 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

  5. 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

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

    PubMed

    Ruppersburg, Chelsey Chandler; Hartzell, H Criss

    2014-06-01

    Many cells possess a single, nonmotile, primary cilium highly enriched in receptors and sensory transduction machinery that plays crucial roles in cellular morphogenesis. Although sensory transduction requires ion channels, relatively little is known about ion channels in the primary cilium (with the exception of TRPP2). Here we show that the Ca(2+)-activated Cl ((-)) channel anoctamin-1 (ANO1/TMEM16A) is located in the primary cilium and that blocking its channel function pharmacologically or knocking it down with short hairpin RNA interferes with ciliogenesis. Before ciliogenesis, the channel becomes organized into a torus-shaped structure ("the nimbus") enriched in proteins required for ciliogenesis, including the small GTPases Cdc42 and Arl13b and the exocyst complex component Sec6. The nimbus excludes F-actin and coincides with a ring of acetylated microtubules. The nimbus appears to form before, or independent of, apical docking of the mother centriole. Our data support a model in which the nimbus provides a scaffold for staging of ciliary components for assembly very early in ciliogenesis and chloride transport by ANO1/TMEM16A is required for the genesis or maintenance of primary cilia. PMID:24694595

  7. Sensing Nature's Electric Fields: Ion Channels as Active Elements of Linear Amplification

    NASA Astrophysics Data System (ADS)

    Bezrukov, Sergey M.

    2003-05-01

    Given the parameters of familiar cellular elements — voltage-sensitive ion channels, carriers, pumps, phospholipid insulators, and electrolytic conductors — is it possible to construct an amplifier whose sensitivity matches the 5 nV/cm threshold found in behavioral experiments on elasmobranch fish? Or, in addition to clever circuitry that uses commonly known elements and principles, do we need something else to understand this sensitivity? The resolution of this question is important not only for studies in sensory biophysics seeking to reveal underlying mechanisms and molecular structures. More generally, it deepens our appreciation of the stochastic nature of inter- and intra-cellular control circuits. Here I analyze a simplified circuit involving negative differential resistance of voltage-sensitive ion channels. The analysis establishes an off-equilibrium criterion for amplification, shows that ion channels are the dominant noise sources, and, by minimizing channel noise within the given constraints, demonstrates that generic voltage-sensitive ion channels are likely candidates for the active elements of the linear cellular amplifiers. Finally, I highlight a number of unsolved issues.

  8. 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. PMID:26854962

  9. Short channel amorphous In-Ga-Zn-O thin-film transistor arrays for ultra-high definition active matrix liquid crystal displays: Electrical properties and stability

    NASA Astrophysics Data System (ADS)

    Kim, Soo Chang; Kim, Young Sun; Yu, Eric Kai-Hsiang; Kanicki, Jerzy

    2015-09-01

    The electrical properties and stability of ultra-high definition (UHD) amorphous In-Ga-Zn-O (a-IGZO) thin-film transistor (TFT) arrays with short channel (width/length = 12/3 μm) were examined. A-IGZO TFT arrays have a mobility of ∼6 cm2/V s, subthreshold swing (S.S.) of 0.34 V/decade, threshold voltage of 3.32 V, and drain current (Id) on/off ratio of <109 with Ioff below 10-13 A. Overall these devices showed slightly different electrical characteristics as compared to the long channel devices; non-saturation of output curve at high drain-to-source voltage (Vds), negative shift of threshold voltage with increasing Vds, and the mobility reduction at high gate voltage (Vgs) were observed. The second derivative method adopting Tikhonov's regularization theory is suggested for the robust threshold voltage extraction. The temperature dependency of γ-value was established after taking into consideration the impact of source/drain contact resistances. The AC bias-temperature stress was used to simulate the actual operation of active matrix liquid crystal displays (AM-LCDs). The threshold voltage shift had a dependency on the magnitude of drain bias stress, frequency, and duty cycle due to the impact ionization accelerated at high temperature. This study demonstrates that the short channel effects, source/drain contact resistances and impact ionization have to be taken into account during optimization of UHD AM-LCDs.

  10. 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

  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. 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

  13. Insulin Excites Anorexigenic Proopiomelanocortin Neurons via Activation of Canonical Transient Receptor Potential Channels

    PubMed Central

    Qiu, Jian; Zhang, Chunguang; Borgquist, Amanda; Nestor, Casey C; Smith, Arik W.; Bosch, Martha A.; Ku, Stephen; Wagner, Edward J.; Rønnekleiv, Oline K.; Kelly, Martin J.

    2014-01-01

    SUMMARY Proopiomelanocortin (POMC) neurons within the hypothalamic arcuate nucleus are vital anorexigenic neurons. Although both the leptin receptor and insulin receptor are coupled to activation of phosphatidylinositide3-kinase (PI3K) in POMC neurons, they are thought to have disparate actions on POMC excitability. Using whole-cell recording and selective pharmacological tools, we have found that similar to leptin, purified insulin depolarized POMC, and adjacent kisspeptin neurons via activation of TRPC5 channels, which are highly expressed in these neurons. In contrast, insulin hyperpolarized and inhibited NPY/AgRP neurons via activation of KATP channels. Moreover, Zn2+, which is found in insulin formulations at nanomolar concentrations, inhibited POMC neurons via activation of KATP channels. Finally as predicted, insulin given intracerebroventrically robustly inhibited food intake and activated c-fos expression in arcuate POMC neurons. Our results show that purified insulin excites POMC neurons in the arcuate nucleus, which we propose is a major mechanism by which insulin regulates energy homeostasis. PMID:24703699

  14. Canonical transient receptor potential channels promote cardiomyocyte hypertrophy through activation of calcineurin signaling.

    PubMed

    Bush, Erik W; Hood, David B; Papst, Philip J; Chapo, Joseph A; Minobe, Wayne; Bristow, Michael R; Olson, Eric N; McKinsey, Timothy A

    2006-11-01

    The calcium/calmodulin-dependent phosphatase calcineurin plays a central role in the control of cardiomyocyte hypertrophy in response to pathological stimuli. Although calcineurin is present at high levels in normal heart, its activity appears to be unaffected by calcium during the course of a cardiac cycle. The mechanism(s) whereby calcineurin is selectively activated by calcium under pathological conditions has remained unclear. Here, we demonstrate that diverse signals for cardiac hypertrophy stimulate expression of canonical transient receptor potential (TRPC) channels. TRPC consists of a family of seven membrane-spanning nonselective cation channels that have been implicated in the nonvoltage-gated influx of calcium in response to G protein-coupled receptor signaling, receptor tyrosine kinase signaling, and depletion of internal calcium stores. TRPC3 expression is up-regulated in multiple rodent models of pathological cardiac hypertrophy, whereas TRPC5 expression is induced in failing human heart. We demonstrate that TRPC promotes cardiomyocyte hypertrophy through activation of calcineurin and its downstream effector, the nuclear factor of activated T cells transcription factor. These results define a novel role for TRPC channels in the control of cardiac growth, and suggest that a TRPC-derived pool of calcium contributes to selective activation of calcineurin in diseased heart. PMID:16950785

  15. 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

  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. Nordihydroguaiaretic acid depletes ATP and inhibits a swelling-activated, ATP-sensitive taurine channel.

    PubMed

    Ballatori, N; Wang, W

    1997-05-01

    The mechanism by which nordihydroguaiaretic acid (NDGA), a lipoxygenase inhibitor, prevents swelling-activated organic osmolyte efflux was examined in the human hepatoma cell line Hep G2. When swollen in hypotonic medium, Hep G2 cell exhibited a regulatory volume decrease that was associated with the release of intracellular taurine, an amino acid found at a concentrations of 22.0 +/- 2.5 nmol/mg protein (approximately 5 mM) in these cells. Rate coefficients for swelling-activated [3H]taurine uptake and efflux were unaffected when extracellular taurine was increased from 0.1 to 25 mM, indicating that taurine is released via a channel. Taurine efflux was rapidly activated after cell swelling and immediately inactivated when cells were returned to normal size by restoration of isotonicity. Swelling-activated taurine efflux was not altered by replacement of extracellular Na+ with choline+ or K+ but was inhibited when cellular ATP levels were decreased with a variety of chemical agents, consistent with an ATP-regulated channel previously described in other cell types. NDGA inhibited swelling-activated [3H]taurine efflux in Hep G2 cells at concentrations of 50-150 microM; however, these same concentrations of NDGA also lowered cell ATP levels. Likewise, ketoconazole, an inhibitor of cytochrome P-450 monoxygenases, inhibited [3H]taurine efflux only at concentrations at which cell ATP levels were also lowered. In contrast, other inhibitors of cyclooxygenase (indomethacin, 100 microM) or of lipoxygenases (caffeic acid, 100 microM), as well as arachidonic acid itself (100 microM), had no effect on either taurine efflux or cell ATP. The present findings characterize a swelling-activated, ATP-sensitive osmolyte channel in Hep G2 cells and demonstrate that inactivation of the channel by NDGA is related to the ability of this drug to deplete cellular ATP. PMID:9176131

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

    PubMed

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

    2015-05-01

    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

  19. Functional characterization of neurotransmitter activation and modulation in a nematode model ligand-gated ion channel.

    PubMed

    Heusser, Stephanie A; Yoluk, Özge; Klement, Göran; Riederer, Erika A; Lindahl, Erik; Howard, Rebecca J

    2016-07-01

    The superfamily of pentameric ligand-gated ion channels includes neurotransmitter receptors that mediate fast synaptic transmission in vertebrates, and are targets for drugs including alcohols, anesthetics, benzodiazepines, and anticonvulsants. However, the mechanisms of ion channel opening, gating, and modulation in these receptors leave many open questions, despite their pharmacological importance. Subtle conformational changes in both the extracellular and transmembrane domains are likely to influence channel opening, but have been difficult to characterize given the limited structural data available for human membrane proteins. Recent crystal structures of a modified Caenorhabditis elegans glutamate-gated chloride channel (GluCl) in multiple states offer an appealing model system for structure-function studies. However, the pharmacology of the crystallographic GluCl construct is not well established. To establish the functional relevance of this system, we used two-electrode voltage-clamp electrophysiology in Xenopus oocytes to characterize activation of crystallographic and native-like GluCl constructs by L-glutamate and ivermectin. We also tested modulation by ethanol and other anesthetic agents, and used site-directed mutagenesis to explore the role of a region of Loop F which was implicated in ligand gating by molecular dynamics simulations. Our findings indicate that the crystallographic construct functionally models concentration-dependent agonism and allosteric modulation of pharmacologically relevant receptors. Specific substitutions at residue Leu174 in loop F altered direct L-glutamate activation, consistent with computational evidence for this region's role in ligand binding. These insights demonstrate conservation of activation and modulation properties in this receptor family, and establish a framework for GluCl as a model system, including new possibilities for drug discovery. In this study, we elucidate the validity of a modified glutamate

  20. 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

  1. THERMAL INSTABILITY OF ΔF508 CFTR CHANNEL FUNCTION: PROTECTION BY SINGLE SUPPRESSOR MUTATIONS AND INHIBITING CHANNEL ACTIVITY

    PubMed Central

    Liu, Xuehong; O’Donnell, Nicolette; Landstrom, Allison; Skach, William R.; Dawson, David C.

    2012-01-01

    Deletion of Phe508 from CFTR results in a temperature-sensitive folding defect that impairs protein maturation and chloride channel function. Both of these adverse effects, however, can be mitigated to varying extents by second-site, suppressor mutations. To better understand the impact of second-site mutations on channel function, we compared the thermal sensitivity of CFTR channels in Xenopus oocytes. CFTR-mediated conductance of oocytes expressing wt or ΔF508 CFTR was stable at 22°C and increased at 28°C; a temperature permissive for ΔF508 CFTR expression in mammalian cells. At 37°C, however, CFTR-mediated conductance was further enhanced, whereas that due to ΔF508 CFTR channels decreased rapidly towards background, a phenomenon referred to here as “thermal inactivation.” Thermal inactivation of ΔF508 was mitigated by each of five suppressor mutations, I539T, R553M, G550E, R555K and R1070W; but each exerted unique effects on the severity of, and recovery from, thermal inactivation. Another mutation, K1250A, known to increase open probability (Po) of ΔF508 CFTR channels, exacerbated thermal inactivation. Application of potentiators known to increase Po of ΔF508 CFTR channels at room temperature failed to protect channels from inactivation at 37°C and one, PG-01, actually exacerbated thermal inactivation. Unstimulated ΔF508CFTR channels or those inhibited by CFTRinh-172, were partially protected from thermal inactivation, suggesting a possible inverse relationship between thermal stability and gating transitions. Thermal stability of channel function and temperature-sensitive maturation of the mutant protein appear to reflect related, but distinct facets of the ΔF508 CFTR conformational defect, both of which must be addressed by effective therapeutic modalities. PMID:22680785

  2. Modulation and pharmacology of low voltage-activated ("T-Type") calcium channels.

    PubMed

    Yunker, Anne Marie R

    2003-12-01

    Although T-type calcium channel currents were observed almost 30 years ago, the genes that encode the pore-forming subunits have only been recently reported. When expressed in heterologous systems, three distinct alpha1 subunits (alpha1G (Cav3.1), alpha1H (Car3.2), and alpha1I (Cav3.3)) conduct T-type currents with insert similar but not identical electrophysiological characteristics that. Alpha 1G, alpha 1H, and alpha 1I transcripts are found throughout neural and nonneural tissues, suggesting multiple types of T-type channels (also called low voltage-activated calcium channels (LVAs)) are coexpressed by many tissues. The study of endogenous LVAs has been hampered by a lack of highly selective antagonists that differentiate between LVA subtypes. Furthermore, many pharmacological agents attenuate currents conducted by LVA and high voltage-activated calcium channels (HVAs). At least 15 classes of pharmacological agents affect T-type currents, and the therapeutic use of many of these drugs has implicated LVAs in the etiology of a variety of diseases. Comparison of the responses of recombinant and native LVAs to pharmacological agents and endogenous modulatory molecules will lead to a better understanding of LVAs in normal and diseased cells. PMID:15000521

  3. 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.

  4. Activation of chloride channels in normal and cystic fibrosis airway epithelial cells by multifunctional calcium/calmodulin-dependent protein kinase

    NASA Astrophysics Data System (ADS)

    Wagner, John A.; Cozens, Alison L.; Schulman, Howard; Gruenert, Dieter C.; Stryer, Lubert; Gardner, Phyllis

    1991-02-01

    CYSTIC fibrosis is associated with defective regulation of apical membrane chloride channels in airway epithelial cells. These channels in normal cells are activated by cyclic AMP-dependent protein kinase1,2 and protein kinase C3,4. In cystic fibrosis these kinases fail to activate otherwise normal Cl- channels1-4. But Cl- flux in cystic fibrosis cells, as in normal cells, can be activated by raising intracellular Ca2+ (refs 5-10). We report here whole-cell patch clamp studies of normal and cystic fibrosis-derived airway epithelial cells showing that Cl- channel activation by Ca2+ is mediated by multifunctional Ca2+/calmodulin-dependent protein kinase. We find that intracellular application of activated kinase and ATP activates a Cl- current similar to that activated by a Ca2+ ionophore, that peptide inhibitors of either the kinase or calmodulin block Ca2+-dependent activation of Cl- channels, and that a peptide inhibitor of protein kinase C does not block Ca2+-dependent activation. Ca2+/calmodulin activation of Cl- channels presents a pathway with therapeutic potential for circumventing defective regulation of Cl- channels in cystic fibrosis.

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

    PubMed

    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. Activity and Ca2+ regulate the mobility of TRPV1 channels in the plasma membrane of sensory neurons

    PubMed Central

    Senning, Eric N; Gordon, Sharona E

    2015-01-01

    TRPV1 channels are gated by a variety of thermal, chemical, and mechanical stimuli. We used optical recording of Ca2+ influx through TRPV1 to measure activity and mobility of single TRPV1 molecules in isolated dorsal root ganglion neurons and cell lines. The opening of single TRPV1 channels produced sparklets, representing localized regions of elevated Ca2+. Unlike sparklets reported for L-type Ca2+ channels, TRPV4 channels, and AchR channels, TRPV1 channels diffused laterally in the plasma membrane as they gated. Mobility was highly variable from channel-to-channel and, to a smaller extent, from cell to cell. Most surprisingly, we found that mobility decreased upon channel activation by capsaicin, but only in the presence of extracellular Ca2+. We propose that decreased mobility of open TRPV1 could act as a diffusion trap to concentrate channels in cell regions with high activity. DOI: http://dx.doi.org/10.7554/eLife.03819.001 PMID:25569155

  7. 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

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

    PubMed Central

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

    2006-01-01

    Transient receptor potential (TRP) channel, melastatin subfamily (TRPM)4 is a Ca2+-activated monovalent cation channel that depolarizes the plasma membrane and thereby modulates Ca2+ influx through Ca2+-permeable pathways. A typical feature of TRPM4 is its rapid desensitization to intracellular Ca2+ ([Ca2+]i). Here we show that phosphatidylinositol 4,5-biphosphate (PIP2) counteracts desensitization to [Ca2+]i in inside-out patches and rundown of TRPM4 currents in whole-cell patch-clamp experiments. PIP2 shifted the voltage dependence of TRPM4 activation towards negative potentials and increased the channel's Ca2+ sensitivity 100-fold. Conversely, activation of the phospholipase C (PLC)-coupled M1 muscarinic receptor or pharmacological depletion of cellular PIP2 potently inhibited currents through TRPM4. Neutralization of basic residues in a C-terminal pleckstrin homology (PH) domain accelerated TRPM4 current desensitization and strongly attenuated the effect of PIP2, whereas mutations to the C-terminal TRP box and TRP domain had no effect on the PIP2 sensitivity. Our data demonstrate that PIP2 is a strong positive modulator of TRPM4, and implicate the C-terminal PH domain in PIP2 action. PLC-mediated PIP2 breakdown may constitute a physiologically important brake on TRPM4 activity. PMID:16424899

  10. TMEM16F (Anoctamin 6), an anion channel of delayed Ca2+ activation

    PubMed Central

    Grubb, Søren; Poulsen, Kristian A.; Juul, Christian Ammitzbøll; Kyed, Tania; Klausen, Thomas K.

    2013-01-01

    Members of the TMEM16 (Anoctamin) family of membrane proteins have been shown to be essential constituents of the Ca2+-activated Cl− channel (CaCC) in many cell types. In this study, we have investigated the electrophysiological properties of mouse TMEM16F. Heterologous expression of TMEM16F in HEK293 cells resulted in plasma membrane localization and an outwardly rectifying ICl,Ca that was activated with a delay of several minutes. Furthermore, a significant Na+ current was activated, and the two permeabilities were correlated according to PNa = 0.3 PCl. The current showed an EC50 of 100 µM intracellular free Ca2+ concentration and an Eisenman type 1 anion selectivity sequence of PSCN > PI > PBr > PCl > PAsp. The mTMEM16F-associated ICl,Ca was abolished in one mutant of the putative pore region (R592E) but retained in two other mutants (K616E and R636E). The mutant K616E had a lower relative permeability to iodide, and the mutant R636E had an altered anion selectivity sequence (PSCN = PI = PBr = PCl > PAsp). Our data provide evidence that TMEM16F constitutes a Ca2+-activated anion channel or a pore-forming subunit of an anion channel with properties distinct from TMEM16A. PMID:23630341

  11. 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

  12. AKAP-Anchored PKA Maintains Neuronal L-type Calcium Channel Activity and NFAT Transcriptional Signaling

    PubMed Central

    Murphy, Jonathan G.; Sanderson, Jennifer L.; Gorski, Jessica A.; Scott, John D.; Catterall, William A.; Sather, William A.; Dell’Acqua, Mark L.

    2014-01-01

    Summary In neurons, Ca2+ influx through L-type voltage-gated Ca2+ channels (LTCC) couples electrical activity to changes in transcription. LTCC activity is elevated by the cAMP-dependent protein kinase (PKA) and depressed by the Ca2+-dependent phosphatase calcineurin (CaN), with both enzymes localized to the channel by A-kinase anchoring protein (AKAP) 79/150. AKAP79/150 anchoring of CaN also promotes LTCC activation of transcription through dephosphorylation of the nuclear factor of activated T-cells (NFAT). We report here that genetic disruption of PKA anchoring to AKAP79/150 also interferes with LTCC activation of CaN-NFAT signaling in neurons. Disruption of AKAP-PKA anchoring promoted redistribution of the kinase out of dendritic spines, profound decreases in LTCC phosphorylation and Ca2+ influx, and impaired NFAT movement to the nucleus and activation of transcription. Our findings support a model wherein basal activity of AKAP79/150-anchored PKA opposes CaN to preserve LTCC phosphorylation, thereby sustaining LTCC activation of CaN-NFAT signaling to the neuronal nucleus. PMID:24835999

  13. Estrogen ameliorates microglial activation by inhibiting the Kir2.1 inward-rectifier K(+) channel.

    PubMed

    Wu, Shih-Ying; Chen, Yun-Wen; Tsai, Sheng-Feng; Wu, Sheng-Nan; Shih, Yao-Hsiang; Jiang-Shieh, Ya-Fen; Yang, Ting-Ting; Kuo, Yu-Min

    2016-01-01

    Microglial activation is implicated in the pathogenesis of Parkinson's disease (PD). Although the etiology of PD remains unclear, age and male gender are known PD risk factors. By comparing microglia and dopaminergic (DA) neurons in the substantia nigra (SN) of male and female mice of different ages, we found that the degrees of microglial activation and DA neuron loss increased with age in both genders, but were more pronounced in males, as were peripheral lipopolysaccharide (LPS)-induced microglial activation and DA neuron loss. A bilateral ovariectomy (OVX) eliminated the female-associated protection against age- and LPS-induced microglial activation, which suggests that ovary hormones are involved in gender-specific responses. Treating female mice with 17β-estradiol supplements reduced the age-associated microglial activation in OVX mice. Moreover, pretreating mouse BV2 microglial cells with 17β-estradiol inhibited LPS-induced elevation of Toll-like receptor 4, phosphorylated p38, and TNF-α levels. We then examined the effect of 17β-estradiol on inward-rectifier K(+) channel Kir2.1, a known regulator of microglial activation. We found that 17β-estradiol inhibited the Kir2.1 activity of BV2 cells by reducing the probability that the channel would be open. We conclude that age- and inflammation-associated microglial activation is attenuated by ovarian estrogen, because it inhibits Kir2.1. PMID:26960267

  14. Estrogen ameliorates microglial activation by inhibiting the Kir2.1 inward-rectifier K+ channel

    PubMed Central

    Wu, Shih-Ying; Chen, Yun-Wen; Tsai, Sheng-Feng; Wu, Sheng-Nan; Shih, Yao-Hsiang; Jiang-Shieh, Ya-Fen; Yang, Ting-Ting; Kuo, Yu-Min

    2016-01-01

    Microglial activation is implicated in the pathogenesis of Parkinson’s disease (PD). Although the etiology of PD remains unclear, age and male gender are known PD risk factors. By comparing microglia and dopaminergic (DA) neurons in the substantia nigra (SN) of male and female mice of different ages, we found that the degrees of microglial activation and DA neuron loss increased with age in both genders, but were more pronounced in males, as were peripheral lipopolysaccharide (LPS)-induced microglial activation and DA neuron loss. A bilateral ovariectomy (OVX) eliminated the female-associated protection against age- and LPS-induced microglial activation, which suggests that ovary hormones are involved in gender-specific responses. Treating female mice with 17β-estradiol supplements reduced the age-associated microglial activation in OVX mice. Moreover, pretreating mouse BV2 microglial cells with 17β-estradiol inhibited LPS-induced elevation of Toll-like receptor 4, phosphorylated p38, and TNF-α levels. We then examined the effect of 17β-estradiol on inward-rectifier K+ channel Kir2.1, a known regulator of microglial activation. We found that 17β-estradiol inhibited the Kir2.1 activity of BV2 cells by reducing the probability that the channel would be open. We conclude that age- and inflammation-associated microglial activation is attenuated by ovarian estrogen, because it inhibits Kir2.1. PMID:26960267

  15. 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

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

    PubMed Central

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

    2016-01-01

    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 Ca2+ 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, Ca2+ 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. PMID:26113050

  17. Calcium influx through stretch-activated channels mediates microfilament reorganization in osteoblasts under simulated weightlessness

    NASA Astrophysics Data System (ADS)

    Luo, Mingzhi; Yang, Zhouqi; Li, Jingbao; Xu, Huiyun; Li, Shengsheng; Zhang, Wei; Qian, Airong; Shang, Peng

    2013-06-01

    We have explored the role of Ca2+ signaling in microfilament reorganization of osteoblasts induced by simulated weightlessness using a random positioning machine (RPM). The RPM-induced alterations of cell morphology, microfilament distribution, cell proliferation, cell migration, cytosol free calcium concentration ([Ca2+]i), and protein expression in MG63 osteoblasts were investigated. Simulated weightlessness reduced cell size, disrupted microfilament, inhibited cellular proliferation and migration, and induced an increase in [Ca2+]i in MG63 human osteosarcoma cells. Gadolinium chloride (Gd), an inhibitor for stretch-activated channels, attenuated the increase in [Ca2+]i and microfilament disruption. Further, the expression of calmodulin was significantly increased by simulated weightlessness, and an inhibitor of calmodulin, W-7, aggravated microfilament disruption. Our findings demonstrate that simulated weightlessness induces Ca2+ influx through stretch-activated channels, then results in microfilament disruption.

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

    PubMed

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

    2010-10-01

    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 RNA interference 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 MA cation channels. PMID:20813920

  19. Modeling Fluvial Incision and Transient Landscape Evolution: Influence of Dynamic Channel Adjustment

    NASA Astrophysics Data System (ADS)

    Attal, M.; Tucker, G. E.; Cowie, P. A.; Whittaker, A. C.; Roberts, G. P.

    2007-12-01

    Channel geometry exerts a fundamental control on fluvial processes. Recent work has shown that bedrock channel width (W) depends on a number of parameters, including channel slope, and is not only a function of drainage area (A) as is commonly assumed. The present work represents the first attempt to investigate the consequences, for landscape evolution, of using a static expression of channel width (W ~ A0.5) versus a relationship that allows channels to dynamically adjust to changes in slope. We consider different models for the evolution of the channel geometry, including constant width-to-depth ratio (after Finnegan et al., Geology, v. 33, no. 3, 2005), and width-to-depth ratio varying as a function of slope (after Whittaker et al., Geology, v. 35, no. 2, 2007). We use the Channel-Hillslope Integrated Landscape Development (CHILD) model to analyze the response of a catchment to a given tectonic disturbance. The topography of a catchment in the footwall of an active normal fault in the Apennines (Italy) is used as a template for the study. We show that, for this catchment, the transient response can be fairly well reproduced using a simple detachment-limited fluvial incision law. We also show that, depending on the relationship used to express channel width, initial steady-state topographies differ, as do transient channel width, slope, and the response time of the fluvial system. These differences lead to contrasting landscape morphologies when integrated at the scale of a whole catchment. Our results emphasize the importance of channel width in controlling fluvial processes and landscape evolution. They stress the need for using a dynamic hydraulic scaling law when modeling landscape evolution, particularly when the uplift field is non-uniform.

  20. 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

  1. Dependence of spontaneous electrical activity and basal prolactin release on nonselective cation channels in pituitary lactotrophs.

    PubMed

    Kučka, M; Kretschmannová, K; Stojilkovic, S S; Zemková, H; Tomić, M

    2012-01-01

    All secretory anterior pituitary cells fire action potentials spontaneously and exhibit a high resting cation conductance, but the channels involved in the background permeability have not been identified. In cultured lactotrophs and immortalized GH(3) cells, replacement of extracellular Na(+) with large organic cations, but not blockade of voltage-gated Na(+) influx, led to an instantaneous hyperpolarization of cell membranes that was associated with a cessation of spontaneous firing. When cells were clamped at -50 mV, which was close to the resting membrane potential in these cells, replacement of bath Na(+) with organic cations resulted in an outward-like current, reflecting an inhibition of the inward holding membrane current and indicating loss of a background-depolarizing conductance. Quantitative RT-PCR analysis revealed the high expression of mRNA transcripts for TRPC1 and much lower expression of TRPC6 in both lactotrophs and GH(3) cells. Very low expression of TRPC3, TRPC4, and TRPC5 mRNA transcripts were also present in pituitary but not GH(3) cells. 2-APB and SKF-96365, relatively selective blockers of TRPC channels, inhibited electrical activity, Ca(2+) influx and prolactin release in a concentration-dependent manner. Gd(3+), a common Ca(2+) channel blocker, and flufenamic acid, an inhibitor of non-selective cation channels, also inhibited electrical activity, Ca(2+) influx and prolactin release. These results indicate that nonselective cation channels, presumably belonging to the TRPC family, contribute to the background depolarizing conductance and firing of action potentials with consequent contribution to Ca(2+) influx and hormone release in lactotrophs and GH(3) cells. PMID:22480423

  2. 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.

  3. 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. PMID:26530828

  4. Cl- Channels in CF: Lack of Activation by Protein Kinase C and cAMP-Dependent Protein Kinase

    NASA Astrophysics Data System (ADS)

    Hwang, Tzyh-Chang; Lu, Luo; Zeitlin, Pamela L.; Gruenert, Dieter C.; Huganir, Richard; Guggino, William B.

    1989-06-01

    Secretory chloride channels can be activated by adenosine 3',5'-monophosphate (cAMP)-dependent protein kinase in normal airway epithelial cells but not in cells from individuals with cystic fibrosis (CF). In excised, inside-out patches of apical membrane of normal human airway cells and airway cells from three patients with CF, the chloride channels exhibited a characteristic outwardly rectifying current-voltage relation and depolarization-induced activation. Channels from normal tissues were activated by both cAMP-dependent protein kinase and protein kinase C. However, chloride channels from CF patients could not be activated by either kinase. Thus, gating of normal epithelial chloride channels is regulated by both cAMP-dependent protein kinase and protein kinase C, and regulation by both kinases is defective in CF.

  5. 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. PMID:25555911

  6. 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

  7. 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

  8. 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

  9. Direct tests of micro channel plates as the active element of a new shower maximum detector

    SciTech Connect

    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.

  10. Direct tests of micro channel plates as the active element of a new shower maximum detector

    NASA Astrophysics Data System (ADS)

    Ronzhin, A.; Los, S.; Ramberg, E.; Apresyan, A.; Xie, S.; Spiropulu, M.; Kim, H.

    2015-09-01

    We continue the study of micro channel plates (MCP) as the active element of a shower maximum (SM) detector. We present below test beam results obtained with MCPs detecting directly secondary particles of an electromagnetic shower. The MCP efficiency to shower particles is close to 100%. The time resolution obtained for this new type of the SM detector is at the level of 40 ps.

  11. TRP channel mediated neuronal activation and ablation in freely behaving zebrafish.

    PubMed

    Chen, Shijia; Chiu, Cindy N; McArthur, Kimberly L; Fetcho, Joseph R; Prober, David A

    2016-02-01

    The zebrafish (Danio rerio) is a useful vertebrate model system in which to study neural circuits and behavior, but tools to modulate neurons in freely behaving animals are limited. As poikilotherms that live in water, zebrafish are amenable to thermal and pharmacological perturbations. We exploit these properties by using transient receptor potential (TRP) channels to activate or ablate specific neuronal populations using the chemical and thermal agonists of heterologously expressed TRPV1, TRPM8 and TRPA1. PMID:26657556

  12. Evolution of thermal response properties in a cold-activated TRP channel.

    PubMed

    Myers, Benjamin R; Sigal, Yaron M; Julius, David

    2009-01-01

    Animals sense changes in ambient temperature irrespective of whether core body temperature is internally maintained (homeotherms) or subject to environmental variation (poikilotherms). Here we show that a cold-sensitive ion channel, TRPM8, displays dramatically different thermal activation ranges in frogs versus mammals or birds, consistent with variations in these species' cutaneous and core body temperatures. Thus, somatosensory receptors are not static through evolution, but show functional diversity reflecting the characteristics of an organism's ecological niche. PMID:19492038

  13. Non-acidic activation of pain-related Acid-Sensing Ion Channel 3 by lipids.

    PubMed

    Marra, Sébastien; Ferru-Clément, Romain; Breuil, Véronique; Delaunay, Anne; Christin, Marine; Friend, Valérie; Sebille, Stéphane; Cognard, Christian; Ferreira, Thierry; Roux, Christian; Euller-Ziegler, Liana; Noel, Jacques; Lingueglia, Eric; Deval, Emmanuel

    2016-02-15

    Extracellular pH variations are seen as the principal endogenous signal that triggers activation of Acid-Sensing Ion Channels (ASICs), which are basically considered as proton sensors, and are involved in various processes associated with tissue acidification. Here, we show that human painful inflammatory exudates, displaying non-acidic pH, induce a slow constitutive activation of human ASIC3 channels. This effect is largely driven by lipids, and we identify lysophosphatidylcholine (LPC) and arachidonic acid (AA) as endogenous activators of ASIC3 in the absence of any extracellular acidification. The combination of LPC and AA evokes robust depolarizing current in DRG neurons at physiological pH 7.4, increases nociceptive C-fiber firing, and induces pain behavior in rats, effects that are all prevented by ASIC3 blockers. Lipid-induced pain is also significantly reduced in ASIC3 knockout mice. These findings open new perspectives on the roles of ASIC3 in the absence of tissue pH variation, as well as on the contribution of those channels to lipid-mediated signaling. PMID:26772186

  14. 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.

  15. Photocontrol of Voltage-Gated Ion Channel Activity by Azobenzene Trimethylammonium Bromide in Neonatal Rat Cardiomyocytes.

    PubMed

    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+) currents. This finding confirms that azoTAB-effect on cardiac tissue excitability do indeed result from modulation of voltage-gated ion channels responsible for action potential. PMID:27015602

  16. Hydraphiles: A Rigorously Studied Class of Synthetic Channel Compounds with In Vivo Activity

    PubMed Central

    Negin, Saeedeh; Smith, Bryan A.; Unger, Alexandra; Leevy, W. Matthew; Gokel, George W.

    2013-01-01

    Hydraphiles are a class of synthetic ion channels that now have a twenty-year history of analysis and success. In early studies, these compounds were rigorously validated in a wide range of in vitro assays including liposomal ion flow detected by NMR or ion-selective electrodes, as well as biophysical experiments in planar bilayers. During the past decade, biological activity was observed for these compounds including toxicity to bacteria, yeast, and mammalian cells due to stress caused by the disruption of ion homeostasis. The channel mechanism was verified in cells using membrane polarity sensitive dyes, as well as patch clamping studies. This body of work has provided a solid foundation with which hydraphiles have recently demonstrated acute biological toxicity in the muscle tissue of living mice, as measured by whole animal fluorescence imaging and histological studies. Here we review the critical structure-activity relationships in the hydraphile family of compounds and the in vitro and in cellulo experiments that have validated their channel behavior. This report culminates with a description of recently reported efforts in which these molecules have demonstrated activity in living mice. PMID:23401675

  17. 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+) currents. This finding confirms that azoTAB-effect on cardiac tissue excitability do indeed result from modulation of voltage-gated ion channels responsible for action potential. PMID:27015602