Sample records for water channel proteins

  1. A Rapidly Emerging Field: Water Channel Proteins in the Eye

    Microsoft Academic Search

    Jorge Fischbarg

    After the first water channel was cloned and se- quenced from 1991 to 1992, work in this field has proceeded at a rapid pace. It is clear now that many tissues in the eye have water channels, and that num- ber could continue to grow. In all probability, water channels are the conduits for transcellular and translayer water flow in

  2. Brain water channel proteins in health and disease.

    PubMed

    Benga, Oana; Huber, Vincent J

    2012-01-01

    The aim of this article is to describe the roles of water channel proteins (WCPs) in brain functionality. The fluid compartments of the brain, which include the brain parenchyma (with intracellular and extracellular spaces), the intravascular and the cerebrospinal fluid compartments are presented. Then the localization and functional roles of WCPs found in the brain are described: AQP1, AQP2, AQP3, AQP4, AQP5, AQP7, AQP8, AQP9 and AQP11. In subsequent chapters the involvement of brain WCPs in pathologies are discussed: brain edema, brain trauma, brain tumors, stroke, dementia (Alzheimer's disease, human immunodeficiency virus--HIV-dementia), autism, pain signal transduction and migraine, hydrocephalus and other pathologies with neurological implications: eclampsia, uremia. New WCP ligands for brain imaging are also discussed. PMID:22504060

  3. Urea transport mediated by aquaporin water channel proteins.

    PubMed

    Li, Chunling; Wang, Weidong

    2014-01-01

    Aquaporins (AQPs) are a family of membrane water channels that basically function as regulators of intracellular and intercellular water flow. To date, thirteen aquaporins have been characterized. They are distributed wildly in specific cell types in multiple organs and tissues. Each AQP channel consists of six membrane-spanning alpha-helices that have a central water-transporting pore. Four AQP monomers assemble to form tetramers, which are the functional units in the membrane. Some of AQPs also transport urea, glycerol, ammonia, hydrogen peroxide, and gas molecules. AQP-mediated osmotic water transport across epithelial plasma membranes facilitates transcellular fluid transport and thus water reabsorption. AQP-mediated urea and glycerol transport is involved in energy metabolism and epidermal hydration. AQP-mediated CO2 and NH3 transport across membrane maintains intracellular acid-base homeostasis. AQPs are also involved in the pathophysiology of a wide range of human diseases (including water disbalance in kidney and brain, neuroinflammatory disease, obesity, and cancer). Further work is required to determine whether aquaporins are viable therapeutic targets or reliable diagnostic and prognostic biomarkers. PMID:25298348

  4. Supplementary Information Conformational Changes of an Ion Channel Detected Through Water-Protein

    E-print Network

    Hong, Mei

    S1 Supplementary Information Conformational Changes of an Ion Channel Detected Through Water) 4 ms mixing. (b) 100 ms mixing. Intermolecular water-protein cross peaks are assigned in black membranes after 1 H spin diffusion from water. All spectra were measured using a 1 H T2 filter of 2 ms

  5. On the definition, nomenclature and classification of water channel proteins (aquaporins and relatives).

    PubMed

    Benga, Gheorghe

    2012-01-01

    A water channel protein (WCP) or a water channel can be defined as a transmembrane protein that has a specific three-dimensional structure with a pore that provides a pathway for water permeation across biological membranes. The pore is formed by two highly conserved regions in the amino acid sequence, called NPA boxes (or motifs) with three amino acid residues (asparagine-proline-alanine, NPA) and several surrounding amino acids. The NPA boxes have been called the "signature" sequence of WCPs. WCPs are a family of proteins belonging to the Membrane Intrinsic Proteins (MIPs) superfamily. In addition, in the MIP superfamily (with more than 1000 members) there are also proteins with no channel activity. The WCP family include three subfamilies: aquaporins, aquaglyceroporins and S-aquaporins. (1) The aquaporins (AQPs) are water selective or specific water channels, also named by various authors as "orthodox", "ordinary", "conventional", "classical", "pure", "normal", or "sensu strictu" aquaporins); (2) The aquaglyceroporins are permeable to water, but also to other small uncharged molecules, in particular glycerol; this family includes the glycerol facilitators, abbreviated as GlpFs, from glycerol permease facilitators. The "signature" sequence for aquaglyceroporins is the aspartic acid residue (D) in the second NPA box. (3) The third subfamily of WCPs have little conserved amino acid sequences around the NPA boxes, unclassifiable to the first two subfamilies. I recommend to use always for this subfamily the name S-aquaporins. They are also named "superaquaporins", "aquaporins with unusual (or deviated) NPA boxes", "subcellular aquaporins", or "sip-like aquaporins". I also recommend to use always the spelling aquaporin (not aquaporine), and, for various AQPs, the abbreviation AQP followed immediately by the number, (e.g. AQP1), with no space or--which might create confusions with "minus". PMID:22542572

  6. Water channel proteins (later called aquaporins) and relatives: past, present, and future.

    PubMed

    Benga, Gheorghe

    2009-02-01

    Water channels or water channel proteins (WCPs) are transmembrane proteins that have a specific three-dimensional structure with a pore that can be permeated by water molecules. WCPs are large families (over 450 members) that are present in all kingdoms of life. The first WCP was discovered in the human red blood cell (RBC) membrane in 1980s. In 1990s other WCPs were discovered in plants, microorganisms, various animals, and humans; and it became obvious that the WCPs belong to the superfamily of major intrinsic proteins (MIPs, over 800 members). WCPs include three subfamilies: (a) aquaporins (AQPs), which are water specific (or selective water channels); (b) aquaglyceroporins (and glycerol facilitators), which are permeable to water and/or other small molecules; and (c) "superaquaporins" or subcellular AQPs. WCPs (and MIPs) have several structural characteristics which were better understood after the atomic structure of some MIPs was deciphered. The structure-function relationships of MIPs expressed in microorganisms (bacteria, archaea, yeast, and protozoa), plants, and some multicellular animal species [nematodes, insects, fishes, amphibians, mammals (and humans)] are described. A synthetic overview on the WCPs from RBCs from various species is provided. The physiological roles of WCPs in kidney, gastrointestinal system, respiratory apparatus, central nervous system, eye, adipose tissue, skin are described, and some implications of WCPs in various diseases are briefly presented. References of detailed reviews on each topic are given. This is the first review providing in a condensed form an overview of the whole WCP field that became in the last 20 years a very hot area of research in biochemistry and molecular cell biology, with wide and increasing implications. PMID:19165894

  7. Conformational Changes of an Ion Channel Detected Through Water-Protein Interactions Using Solid-State NMR

    E-print Network

    Hong, Mei

    Conformational Changes of an Ion Channel Detected Through Water-Protein Interactions Using Solid conduction by M2 is known to involve water; however direct experimental evidence of M2-water interaction is scarce. Using 1 H spin diffusion solid-state NMR, we have now determined the water accessibility of the M

  8. Molecular and functional characterization of multiple aquaporin water channel proteins from the western tarnished plant bug, Lygus hesperus

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Aquaporins (AQPs) are integral membrane channel proteins that facilitate the bidirectional transfer of water or other small solutes across biological membranes involved in numerous essential physiological processes. In arthropods, AQPs belong to several subfamilies, which contribute to osmoregulatio...

  9. Immunolocalization of the Mercurial-Insensitive Water Channel and Glycerol Intrinsic Protein in Epithelial Cell Plasma Membranes

    Microsoft Academic Search

    Antonio Frigeri; Michael A. Gropper; C. W. Turck; A. S. Verkman

    1995-01-01

    Two water channel homologs were cloned recently from rat kidney, mercurial-insensitive water channel (MIWC) and glycerol intrinsic protein (GLIP). Polyclonal antibodies were raised against synthetic C-terminal peptides and purified by affinity chromatography. MIWC and GLIP antibodies recognized proteins in rat kidney with an apparent molecular mass of 30 and 27 kDa, respectively, and did not cross-react. By immunofluorescence, MIWC and

  10. The Role of Water Channel Proteins in Facilitating Recovery of Leaf Hydraulic Conductance from Water Stress in Populus trichocarpa

    PubMed Central

    Laur, Joan; Hacke, Uwe G.

    2014-01-01

    Gas exchange is constrained by the whole-plant hydraulic conductance (Kplant). Leaves account for an important fraction of Kplant and may therefore represent a major determinant of plant productivity. Leaf hydraulic conductance (Kleaf) decreases with increasing water stress, which is due to xylem embolism in leaf veins and/or the properties of the extra-xylary pathway. Water flow through living tissues is facilitated and regulated by water channel proteins called aquaporins (AQPs). Here we assessed changes in the hydraulic conductance of Populus trichocarpa leaves during a dehydration-rewatering episode. While leaves were highly sensitive to drought, Kleaf recovered only 2 hours after plants were rewatered. Recovery of Kleaf was absent when excised leaves were bench-dried and subsequently xylem-perfused with a solution containing AQP inhibitors. We examined the expression patterns of 12 highly expressed AQP genes during a dehydration-rehydration episode to identify isoforms that may be involved in leaf hydraulic adjustments. Among the AQPs tested, several genes encoding tonoplast intrinsic proteins (TIPs) showed large increases in expression in rehydrated leaves, suggesting that TIPs contribute to reversing drought-induced reductions in Kleaf. TIPs were localized in xylem parenchyma, consistent with a role in facilitating water exchange between xylem vessels and adjacent living cells. Dye uptake experiments suggested that reversible embolism formation in minor leaf veins contributed to the observed changes in Kleaf. PMID:25406088

  11. Water channel proteins: from their discovery in 1985 in Cluj-Napoca, Romania, to the 2003 Nobel Prize in Chemistry.

    PubMed

    Benga, Gh

    2006-01-01

    Water channel proteins, later called aquaporins, are transmembrane proteins that have as their main(specific) function the water transport across biological membranes. The first water channel protein (WCP), now called aquaporin 1, was identified or "seen" in situ (hence discovered) in the human red blood cell (RBC) membrane in 1985 by Benga's group (Cluj-Napoca, Romania). This was achieved by a very selective radiolabeling of RBC membrane proteins with the water transport inhibitor [203Hg]-p-chloromercuribenzene sulfonate (PCMBS), under conditions of specific inhibition. The presence and location of the WCP was discovered among the polypeptides migrating in the region of 35-60 kDa on the electrophoretogram of RBC membrane proteins. The work was first published in 1986 in Biochemistry and Eur. J. Cell Biol. and reviewed by Benga in several articles in 1988-2004. We have thus a world priority in the discovery of the first water channel in the RBC membrane, that was re-discovered by chance by the group of Agre (Baltimore, USA) in 1988, when they isolated a new protein from the RBC membrane, nick-named CHIP28 (channel-forming integral membrane protein of 28 kDa). However, in addition to the 28 kDa component, this protein had a 35-60 kDa glycosylated component, the one detected by Benga's group. Only in 1992 the Agre's group suggested that "it is likely that CHIP28 is a functional unit of membrane water channels". In 1993 CHIP28 was renamed aquaporin 1. Looking in retrospect, asking the crucial question, when was the first WCP, discovered, a fair and clear cut answer would be: the first WCP, now called aquaporin 1, was identified or "seen" (hence discovered) in situ in the human RBC membrane by Benga and coworkers in 1985. It was again "seen" when it was purified in 1988 and again identified when its water transport property was found byAgre's group in 1992. If we make a comparison with the discovery of New World of America, the first man who has "seen" a part, very small indeed, of The New Land was Columbus; later, others, including Amerigo Vespucci (from whom the name derived), have better "seen" and in the subsequent years many explorers discovered the complexity of the Americas. Consequently, the initial discovery of the first water channel by Benga's group must be properly credited; the omission of Gheorghe Benga from the 2003 Nobel Prize in Chemistry (half of which was awarded to Peter Agre "for the discovery of the water channels") was a new mistake in the award of Nobel Prizes. Benga's claim is presented on the web site of the Ad Astra Association (www.ad-astra.ro/benga). As can be seen on this site his recognition as a discoverer of the first water channel protein from the human RBC membrane is growing. Thousands of science-related professionals from hundreds of academic and research units, as well as participants in several international scientific events, have signed as supporters of Benga; his priority is also mentioned in several comments on the 2003 Nobel Prize as presented on the site. PMID:17543216

  12. A Simple Water Channel

    ERIC Educational Resources Information Center

    White, A. S.

    1976-01-01

    Describes a simple water channel, for use with an overhead projector. It is run from a water tap and may be used for flow visualization experiments, including the effect of streamlining and elementary building aerodynamics. (MLH)

  13. Atomic water channel controlling remarkable properties of a single brain microtubule: correlating single protein to its supramolecular assembly.

    PubMed

    Sahu, Satyajit; Ghosh, Subrata; Ghosh, Batu; Aswani, Krishna; Hirata, Kazuto; Fujita, Daisuke; Bandyopadhyay, Anirban

    2013-09-15

    Microtubule nanotubes are found in every living eukaryotic cells; these are formed by reversible polymerization of the tubulin protein, and their hollow fibers are filled with uniquely arranged water molecules. Here we measure single tubulin molecule and single brain-neuron extracted microtubule nanowire with and without water channel inside to unravel their unique electronic and optical properties for the first time. We demonstrate that the energy levels of a single tubulin protein and single microtubule made of 40,000 tubulin dimers are identical unlike conventional materials. Moreover, the transmitted ac power and the transient fluorescence decay (single photon count) are independent of the microtubule length. Even more remarkable is the fact that the microtubule nanowire is more conducting than a single protein molecule that constitutes the nanowire. Microtubule's vibrational peaks condense to a single mode that controls the emergence of size independent electronic/optical properties, and automated noise alleviation, which disappear when the atomic water core is released from the inner cylinder. We have carried out several tricky state-of-the-art experiments and identified the electromagnetic resonance peaks of single microtubule reliably. The resonant vibrations established that the condensation of energy levels and periodic oscillation of unique energy fringes on the microtubule surface, emerge as the atomic water core resonantly integrates all proteins around it such that the nanotube irrespective of its size functions like a single protein molecule. Thus, a monomolecular water channel residing inside the protein-cylinder displays an unprecedented control in governing the tantalizing electronic and optical properties of microtubule. PMID:23567633

  14. Identification and characterization of functional aquaporin water channel protein from alimentary tract of whitefly, Bemisia tabaci

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Some hemipteran xylem and phloem feeding insects have evolved specialized alimentary structures or filter chambers that rapidly transport water for excretion or osmoregulation. In the whitefly, Bemisia tabaci, mass movement of water through opposing alimentary tract tissues within the filter chamber...

  15. TRPC Channels: Interacting Proteins

    Microsoft Academic Search

    K. Kiselyov; D. M. Shin; J.-Y. Kim; J. P. Yuan; S. Muallem

    TRP channels, in particular the TRPC and TRPV subfamilies, have emerged as important constituents of the receptor-activated\\u000a Ca2+ influx mechanism triggered by hormones, growth factors, and neurotransmitters through activation of phospholipase C (PLC).\\u000a Several TRPC channels are also activated by passive depletion of endoplasmic reticulum (ER) Ca2+. Although in several studies the native TRP channels faithfully reproduce the respective recombinant

  16. Involvement of MAPK ERK Activation in Upregulation of Water Channel Protein Aquaporin 1 in a Mouse Model of Bell's Palsy.

    PubMed

    Fang, Fan; Liu, Cai-Yue; Zhang, Jie; Zhu, Lie; Qian, Yu-Xin; Yi, Jing; Xiang, Zheng-Hua; Wang, Hui; Jiang, Hua

    2015-05-01

    The aim of this study is to immunolocalize the aquaporin 1 water channel protein (AQP1) in Schwann cells of idiopathic facial nerve and explore its possible role during the development of facial palsy induced by herpes simplex virus type 1 (HSV-1). HSV-1 was inoculated into the surface of posterior auricle of mouse to establish a paralyzed animal model. In HSV-1-induced facial palsy mice, protein levels of AQP1 significantly increased on the 9th to 16th day after inoculation of HSV-1. The upregulation of AQP1 was closely related to the intratemporal facial nerve edema in facial nerve canal, which was also consistent with the symptom of facial palsy in mice. In a hypoxia model of Schwann cells in vitro, we found that U0126, an ERK antagonist, inhibited not only morphological changes of cultures Schwann cells but also upregulation of both AQP1 and phosphorylated ERK. Combined with increased phosphorylated ERK in HSV-1-induced facial palsy mice, we inferred that ERK MAPK pathway might also be involved in increased AQP1 in mouse model of Bell's palsy. Although the precise mechanism needs to be further explored, our findings suggest that AQP1 in Schwann cells of intratemporal facial nerve is involved in the evolution of facial palsy induced by HSV-1 and may play an important role in the pathogenesis of this disease. AQP1 might be a potential target, and the ERK antagonist U0126 could be a new drug for the treatment of HSV-1-induced Bell's palsy in an early stage. PMID:25527444

  17. Aquaporin water channels - from atomic structure to clinical medicine

    Microsoft Academic Search

    Peter Agre; Landon S. King; Masato Yasui; Wm B. Guggino; Ole Petter Ottersen; Yoshinori Fujiyoshi; Andreas Engel; Søren Nielsen

    2002-01-01

    The water permeability of biological membranes has been a longstanding problem in physiology, but the proteins responsible for this remained unknown until discovery of the aquaporin 1 (AQP1) water channel protein. AQP1 is selectively permeated by water driven by osmotic gradients. The atomic structure of human AQP1 has recently been defined. Each subunit of the tetramer contains an individual aqueous

  18. Computational optimization of synthetic water channels.

    SciTech Connect

    Rogers, David Michael; Rempe, Susan L. B.

    2012-12-01

    Membranes for liquid and gas separations and ion transport are critical to water purification, osmotic energy generation, fuel cells, batteries, supercapacitors, and catalysis. Often these membranes lack pore uniformity and robustness under operating conditions, which can lead to a decrease in performance. The lack of uniformity means that many pores are non-functional. Traditional membranes overcome these limitations by using thick membrane materials that impede transport and selectivity, which results in decreased performance and increased operating costs. For example, limitations in membrane performance demand high applied pressures to deionize water using reverse osmosis. In contrast, cellular membranes combine high flux and selective transport using membrane-bound protein channels operating at small pressure differences. Pore size and chemistry in the cellular channels is defined uniformly and with sub-nanometer precision through protein folding. The thickness of these cellular membranes is limited to that of the cellular membrane bilayer, about 4 nm thick, which enhances transport. Pores in the cellular membranes are robust under operating conditions in the body. Recent efforts to mimic cellular water channels for efficient water deionization produced a significant advance in membrane function. The novel biomimetic design achieved a 10-fold increase in membrane permeability to water flow compared to commercial membranes and still maintained high salt rejection. Despite this success, there is a lack of understanding about why this membrane performs so well. To address this lack of knowledge, we used highperformance computing to interrogate the structural and chemical environments experienced by water and electrolytes in the newly created biomimetic membranes. We also compared the solvation environments between the biomimetic membrane and cellular water channels. These results will help inform future efforts to optimize and tune the performance of synthetic biomimetic membranes for applications in water purification, energy, and catalysis.

  19. Water channels in platelet volume regulation

    PubMed Central

    Lee, Jin-Sook; Agrawal, Shivani; von Turkovich, Michele; Taatjes, Douglas J; Walz, Daniel A; Jena, Bhanu P

    2012-01-01

    Abstract The regulation of platelet volume significantly affects its function. Because water is the major molecule in cells and its active transport via water channels called aquaporins (AQPs) have been implicated in cellular and organelle volume regulation, the presence of water channels in platelets and their potential role in platelet volume regulation was investigated. G-protein–mediated AQP regulation in secretory vesicle swelling has previously been reported in neurons and in pancreatic acinar cells. Mercuric chloride has been demonstrated to inhibit most AQPs except AQP6, which is stimulated by the compound. Exposure of platelets to HgCl2-induced swelling in a dose-dependent manner, suggesting the presence of AQP6 in platelets. Immunoblot analysis of platelet protein confirmed the presence of AQP6, and also of G?o, G?i–1 and G?i–3 proteins. Results from this study demonstrate for the first time that in platelets AQP6 is involved in cell volume regulation via a G-protein–mediated pathway. PMID:21692982

  20. Implications of water channel proteins in selected neurological disorders: epilepsies, muscular dystrophies, amyotrophic lateral sclerosis, neuromyelitis optica, Parkinson's disease, and spongiform encephalopathies.

    PubMed

    Benga, Ileana; Benga, Oana

    2012-01-01

    The aim of this article is to describe the roles of water channel proteins (WCPs) in some neurological diseases in which the implications of these proteins became obvious in the decades after the discovery of WCPs of their presence in the CNS. The diseases which were selected for this review include: epilepsies, muscular dystrophies, amyotrophic lateral sclerosis, neuromyelitis optica, Parkinson's disease, and spongiform encephalopathies. The priorities of Benga group from Cluj-Napoca, Romania, are mentioned, such as the idea of a generalized membrane defect affecting water permeability in epilepsy and in Duchenne muscular dystrophy. Some of these neurological disorders discussed in this article appeared to be water channelopathies. A typical example is neuromyelitis optica (NMO), in which the identification of the specific marker autoantibody against aquaporin 4 in the sera of patients was a milestone in the diagnosis. This has aided understanding of the pathogenesis of NMO and led to better control of its treatment. However, further studies are needed to characterize the function and regulation of WCPs in other neurological diseases, in particular to determine if modulation of WCP function may provide a novel approach to therapy in such diseases. PMID:22484281

  1. Regulation of Ion Channels by G Proteins

    NSDL National Science Digital Library

    Maria Diverse-Pierluissi (Mount Sinai School of Medicine.; Department of Pharmacology and Biological Chemistry REV)

    2005-08-16

    This Teaching Resource provides lecture notes and slides for a class covering regulation of ion channels by G proteins and is part of the course "Cell Signaling Systems: A Course for Graduate Students." The lecture begins with an overview of calcium channels and then proceeds to describe the interaction of signaling molecules with calcium channels.

  2. The plasma membrane of Arabidopsis thaliana contains a mercury- insensitive aquaporin that is a homolog of the tonoplast water channel protein TIP

    Microsoft Academic Search

    Mark J. Daniels; T. Erik Mirkov; Maarten J. Chrispeels

    1994-01-01

    Plant cells contain proteins that are members of the major intrinsic protein (MIP) family, an ancient family of membrane channel proteins characterized by six membrane-spanning domains and two asparagine-proline-alanine (NPA) amino acid motifs in the two halves of the protein. We recently demonstrated that y- TIP, one of the MIP homologs found in the vacuolar membrane of plant cells, is

  3. Viral proteins function as ion channels.

    PubMed

    Wang, Kai; Xie, Shiqi; Sun, Bing

    2011-02-01

    Viral ion channels are short membrane proteins with 50-120 amino acids and play an important role either in regulating virus replication, such as virus entry, assembly and release or modulating the electrochemical balance in the subcellular compartments of host cells. This review summarizes the recent advances in viral encoded ion channel proteins (or viroporins), including PBCV-1 KcV, influenza M2, HIV-1 Vpu, HCV p7, picornavirus 2B, and coronavirus E and 3a. We focus on their function and mechanisms, and also discuss viral ion channel protein serving as a potential drug target. PMID:20478263

  4. Parameters affecting protein production from brewery waste water in a multi-channel laboratory-scale activated sludge system

    Microsoft Academic Search

    L. Vriens; E. Van den Eynde; H. Verachtert

    1983-01-01

    With the aim of studying the possible utilization of brewery waste water activated sludge for animal feeding, the influence of the solids retention time (SRT) and nitrogen supplementation were investigated, especially with respect to biomass production and biomass composition. It was found that the SRT strongly influenced both parameters. At an SRT of from 4 to 6 days excellent biomass

  5. Rapid stalk elongation in tulip (Tulipa gesneriana L. cv. Apeldoorn) and the combined action of cold-induced invertase and the water-channel protein gammaTIP.

    PubMed

    Balk, P A; de Boer, A D

    1999-09-01

    Many bulbous plants need a low-temperature treatment for flowering. Cold, for example, affects the elongation of the stalk, thereby influencing the quality of the cut flower. How the elongation of the stalk is promoted by cold and which physiological and biochemical mechanisms are involved have remained obscure. As invertase has been shown to be involved in the cold-induced elongation of the flower stalks of tulips (Lambrechts et al., 1994, Plant Physiol 104: 515-520), we further characterized this enzyme by cloning the cDNA and analysing its expression in various tissues of the tulip (Tulipa gesneriana L. cv. Apeldoorn) stalk. In addition, the role of sucrose synthase was investigated. Since turgor pressure is an important force driving cell elongation, the role of a water-channel protein (gammaTIP) was studied in relation to these two enzymes. The mRNA level of the invertase found was substantially up-regulated as a result of cold treatment. Analysis of the amino acid sequence of this invertase revealed the presence of a vacuolar targeting signal. Two different forms of sucrose synthase were found, the expression of one of them appeared to be restricted to the vascular tissue while the other form was present in the surrounding tissue. Both sucrose synthases were present in the stalk during the entire period of bulb storage and after planting, but their activities declined during stalk elongation. The expression of the gammaTIP gene was restricted mainly to the vascular tissue and its expression profile was identical to that of invertase. Simultaneous expression of invertase and gammaTIP possibly leads to an increase in osmotic potential and vacuolar water uptake, thus providing a driving force for stretching the stalk cells. PMID:10502102

  6. A SUBSTRATE CHANNEL IN THE NITROGENASE MoFe PROTEIN

    PubMed Central

    Barney, Brett M.; Yurth, Michael G.; Dos Santos, Patricia C.; Dean, Dennis R.; Seefeldt, Lance C.

    2010-01-01

    Nitrogenase catalyzes the six electron/six proton reduction of N2 to two ammonia molecules at a complex organo-metallocluster called FeMo-cofactor. This cofactor is buried within the ?-subunit of the MoFe protein, with no obvious access for substrates. Examination of high-resolution X-ray crystal structures of MoFe proteins from several organisms has revealed the existence of a water-filled channel that extends from the solvent exposed surface to a specific face of FeMo-cofactor. This channel could provide a pathway for substrate and product access to the active site. In the present work, we examine this possibility by substituting four different amino acids that line the channel with other residues and analyze the impact of these substitutions on substrate reduction kinetic parameters. Each of the MoFe protein variants was purified and kinetic parameters established for the reduction of the substrates N2, acetylene, azide and propyne. For each MoFe protein, Vmax values for the different substrates were found to be nearly unchanged when compared to the wild-type MoFe protein, indicating that electron delivery to the active site is not compromised by the various substitutions. In contrast, the Km values for these substrates were found to increase significantly (up to 22-fold) in some of the MoFe protein variants compared to the wild-type MoFe protein values. Given that each of the amino acids that were substituted is remote from the active site, these results are consistent with the water filled channel functioning as a substrate channel in the MoFe protein. PMID:19458968

  7. Theoretical and computational studies of microscopic water channels

    NASA Astrophysics Data System (ADS)

    Zhu, Fangqiang

    Water channels are ubiquitous in all life forms. A notable example is aquaporins (AQPs), a family of proteins which mainly function as passive water channels in cell membranes. The availability of the crystal structures of several AQPs in recent years allowed us to study them in atomic details. We performed molecular dynamics (MD) simulations on AQPs in lipid bilayers to study water permeation through these channels. We also simulated water conduction in carbon nanotubes, serving as simplified models for biological channels. We developed theories and methodologies aimed to reproduce and predict important experimental quantities of water channels from simulations. We showed that the diffusion permeability (pd), which is measured by tracer diffusion in experiments, can be calculated from equilibrium MD simulations. In order to calculate the osmotic permeability (p f), which is experimentally measured in the presence of a solute concentration difference, we developed a method to induce a hydrostatic pressure difference across the membrane under periodic boundary conditions. We calculated the osmotic permeability for aquaporin-1 using this method, which agrees with experiments. Using a continuous-time random-walk model, we showed that for single-file water channels, the ratio of p f to pd is roughly equal to the number of water molecules in the channel. Proton transfer through single water file was studied theoretically using network thermodynamics. Finally, we proposed a new model for general water channels, which gives a quantitative relationship between water permeations under equilibrium and non-equilibrium conditions, and therefore allows one to calculate pf from equilibrium MD simulations.

  8. Signaling protein complexes associated with neuronal ion channels

    Microsoft Academic Search

    Irwin B Levitan

    2006-01-01

    The pore-forming subunits of many ion channels exist in the membrane as one component of a regulatory protein complex, which may also contain one or more signaling proteins that contribute to the modulation of channel properties. Here I review this field, with emphasis on several different kinds of neuronal potassium channels for which the evidence for ion channel signaling complexes

  9. Water Channel of Horseradish Peroxidase Studied by the Charge-Transfer Absorption Band of Ferric Heme

    E-print Network

    Sharp, Kim

    Water Channel of Horseradish Peroxidase Studied by the Charge-Transfer Absorption Band of Ferric The heme of horseradish peroxidase is buried in the protein, but a channel from the protein surface connects the aqueous solution to the heme site. Ferric horseradish peroxidase has an absorption band at 640

  10. Desformylgramicidin: a model channel with an extremely high water permeability.

    PubMed Central

    Saparov, S M; Antonenko, Y N; Koeppe, R E; Pohl, P

    2000-01-01

    The water conductivity of desformylgramicidin exceeds the permeability of gramicidin A by two orders of magnitude. With respect to its single channel hydraulic permeability coefficient of 1.1.10(-12) cm(3) s(-1), desformylgramicidin may serve as a model for extremely permeable aquaporin water channel proteins (AQP4 and AQPZ). This osmotic permeability exceeds the conductivity that is predicted by the theory of single-file transport. It was derived from the concentration distributions of both pore-impermeable and -permeable cations that were simultaneously measured by double barreled microelectrodes in the immediate vicinity of a planar bilayer. From solvent drag experiments, approximately five water molecules were found to be transported by a single-file process along with one ion through the channel. The single channel proton, potassium, and sodium conductivities were determined to be equal to 17 pS (pH 2.5), 7 and 3 pS, respectively. Under any conditions, the desformyl-channel remains at least 10 times longer in its open state than gramicidin A. PMID:11053127

  11. 1. INTAKE CHANNEL LOOKING NORTHEAST; WATER FROM BEAVER BROOK ENTERS ...

    Library of Congress Historic Buildings Survey, Historic Engineering Record, Historic Landscapes Survey

    1. INTAKE CHANNEL LOOKING NORTHEAST; WATER FROM BEAVER BROOK ENTERS THE INTAKE CHANNEL HERE. - Hondius Water Line, 1.6 miles Northwest of Park headquarters building & 1 mile Northwest of Beaver Meadows entrance station, Estes Park, Larimer County, CO

  12. Water at interface with proteins

    E-print Network

    Giancarlo Franzese; Valentino Bianco; Svilen Iskrov

    2010-12-07

    Water is essential for the activity of proteins. However, the effect of the properties of water on the behavior of proteins is only partially understood. Recently, several experiments have investigated the relation between the dynamics of the hydration water and the dynamics of protein. These works have generated a large amount of data whose interpretation is debated. New experiments measure the dynamics of water at low temperature on the surface of proteins, finding a qualitative change (crossover) that might be related to the slowing down and stop of the protein's activity (protein glass transition), possibly relevant for the safe preservation of organic material at low temperature. To better understand the experimental data several scenarios have been discussed. Here, we review these experiments and discuss their interpretations in relation with the anomalous properties of water. We summarize the results for the thermodynamics and dynamics of supercooled water at an interface. We consider also the effect of water on protein stability, making a step in the direction of understanding, by means of Monte Carlo simulations and theoretical calculations, how the interplay of water cooperativity and hydrogen bonds interfacial strengthening affects the protein cold denaturation.

  13. Aquaporin1 Channels in Human Retinal Pigment Epithelium: Role in Transepithelial Water Movement

    Microsoft Academic Search

    W. Daniel Stamer; Dean Bok; Jane Hu; Glenn J. Jaffe; Brian S. McKay

    2003-01-01

    PURPOSE. Aquaporin (AQP) is a hexahelical integral membrane protein that functions as a constitutive channel for water and regulated channel for cations in fluid transporting tissues, in- cluding many in the eye. Although AQP1 has been cloned from a cDNA library prepared from cultures of retinal pigment epithelial (RPE) cells isolated from human fetal tissue, three separate studies failed with

  14. Biomphalaria species in Alexandria water channels.

    PubMed

    Abou-El-Naga, Iman F; El-Nassery, Suzanne M F; Allam, Sonia R; Shaat, Eman A; Mady, Rasha F M

    2011-09-01

    Of the several species of Biomphalaria snails worldwide that serve as the intermediate host for Schistosoma mansoni, Biomphalaria alexandrina is a species that is indigenous to Egypt. Recently, there has been much debate concerning the presence of Biomphalaria glabrata and the hybrid of the species with Biomphalaria alexandrina. Due to this debate, the absence of a clear explanation for the presence of B. glabrata in Egyptian water channels and the probability that they may be reintroduced, we conducted this field study to identify Biomphalaria species present in Alexandria water channels. Laboratory-adapted susceptible snails to Schistosoma mansoni of the following species were used as a reference; Biomphalaria alexandrina, Biomphalaria glabrata and their hybrid. These snails were used to perpetuate the Schistosoma life cycle at the Theodor Bilharz Research Institute (TBRI), Cairo, Egypt. Morphological and molecular studies were conducted on these reference snails as well as on the first generation of Biomphalaria snails from two areas in the Alexandria governorate. The morphological study included both external shell morphology and internal anatomy of the renal ridge. The molecular study used a species-specific PCR technique. The results demonstrated that there was an absence of Biomphalaria glabrata and the hybrid from Alexandria water channels. Moreover, the susceptibility patterns of these reference snails were studied by measuring the different parasitological parameters. It was found that Biomphalaria glabrata and the hybrid were significantly more susceptible than Biomphalaria alexandrina to the Egyptian strain of Schistosoma mansoni. The results demonstrated that if Biomphalaria glabrata was reintroduced and adapted to the local environment in Egypt, it would have important epidemiologic impacts that would have a serious effect on the health of Egyptian people. PMID:21458594

  15. Ion channel regulation by protein S-acylation

    PubMed Central

    2014-01-01

    Protein S-acylation, the reversible covalent fatty-acid modification of cysteine residues, has emerged as a dynamic posttranslational modification (PTM) that controls the diversity, life cycle, and physiological function of numerous ligand- and voltage-gated ion channels. S-acylation is enzymatically mediated by a diverse family of acyltransferases (zDHHCs) and is reversed by acylthioesterases. However, for most ion channels, the dynamics and subcellular localization at which S-acylation and deacylation cycles occur are not known. S-acylation can control the two fundamental determinants of ion channel function: (1) the number of channels resident in a membrane and (2) the activity of the channel at the membrane. It controls the former by regulating channel trafficking and the latter by controlling channel kinetics and modulation by other PTMs. Ion channel function may be modulated by S-acylation of both pore-forming and regulatory subunits as well as through control of adapter, signaling, and scaffolding proteins in ion channel complexes. Importantly, cross-talk of S-acylation with other PTMs of both cysteine residues by themselves and neighboring sites of phosphorylation is an emerging concept in the control of ion channel physiology. In this review, I discuss the fundamentals of protein S-acylation and the tools available to investigate ion channel S-acylation. The mechanisms and role of S-acylation in controlling diverse stages of the ion channel life cycle and its effect on ion channel function are highlighted. Finally, I discuss future goals and challenges for the field to understand both the mechanistic basis for S-acylation control of ion channels and the functional consequence and implications for understanding the physiological function of ion channel S-acylation in health and disease. PMID:24821965

  16. Leak detection in open water channels Erik Weyer

    E-print Network

    Bastin, Georges

    and minimise potential water losses. Implementation of automatic control systems for regulation of the flows of the channel system. In addition to losses caused by oversupply of water, there are also losses due to faultsLeak detection in open water channels Erik Weyer Georges Bastin Department of Electrical

  17. Water transport by the bacterial channel alpha-hemolysin

    NASA Technical Reports Server (NTRS)

    Paula, S.; Akeson, M.; Deamer, D.

    1999-01-01

    This study is an investigation of the ability of the bacterial channel alpha-hemolysin to facilitate water permeation across biological membranes. alpha-Hemolysin channels were incorporated into rabbit erythrocyte ghosts at varying concentrations, and water permeation was induced by mixing the ghosts with hypertonic sucrose solutions. The resulting volume decrease of the ghosts was followed by time-resolved optical absorption at pH 5, 6, and 7. The average single-channel permeability coefficient of alpha-hemolysin for water ranged between 1.3x10-12 cm/s and 1.5x10-12 cm/s, depending on pH. The slightly increased single-channel permeability coefficient at lower pH-values was attributed to an increase in the effective pore size. The activation energy of water transport through the channel was low (Ea=5.4 kcal/mol), suggesting that the properties of water inside the alpha-hemolysin channel resemble those of bulk water. This conclusion was supported by calculations based on macroscopic hydrodynamic laws of laminar water flow. Using the known three-dimensional structure of the channel, the calculations accurately predicted the rate of water flow through the channel. The latter finding also indicated that water permeation data can provide a good estimate of the pore size for large channels.

  18. BIOCHEMISTRY: TRP Ion Channels--Two Proteins in One

    NSDL National Science Digital Library

    Irwin B. Levitan (University of Pennsylvania School of Medicine; Department of Neuroscience)

    2001-08-17

    Access to the article is free, however registration and sign-in are required. It is well established that some proteins carry out more than one job in the cell, but so far, ion channels do not appear to exhibit this versatility. In their Perspective, Levitan and Cibulsky discuss a cluster of new findings showing that two members of the long TRP ion channel family, LTRPC7 and LTRPC2, are both ion channels and enzymes.

  19. Two configurations of a channel-forming membrane protein

    NASA Astrophysics Data System (ADS)

    Unwin, P. N. T.; Ennis, P. D.

    1984-02-01

    The protein oligomer forming the gap junction channel has been analysed in two Ca2+-sensitive states by electron microscopy of membranes in frozen aqueous solutions. Switching between states occurs by a small cooperative rearrangement involving tilting of the subunits, which may be responsible for the effect of Ca2+ on channel permeability in vivo.

  20. The stream channel incision syndrome and water quality

    Microsoft Academic Search

    F. Douglas Shields Jr.; Richard E. Lizotte Jr.; Scott S. Knight; Charles M. Cooper; Darlene Wilcox

    2010-01-01

    Watershed development often triggers channel incision that leads to radical changes in channel morphology. Although morphologic evolution due to channel incision has been documented and modeled by others, ecological effects, particularly water quality effects, are less well understood. Discharge, basic physical parameters, solids, nutrients (nitrogen and phosphorus), chlorophyll and bacteria were monitored for five years at two sites along a

  1. Small potassium ion channel proteins encoded by chlorella viruses.

    PubMed

    Kang, Ming; Moroni, Anna; Gazzarrini, Sabrina; DiFrancesco, Dario; Thiel, Gerhard; Severino, Maria; Van Etten, James L

    2004-04-13

    Kcv, a 94-aa protein encoded by Paramecium bursaria chlorella virus 1, is the smallest known protein to form a functional potassium ion channel and basically corresponds to the "pore module" of potassium channels. Both viral replication and channel activity are inhibited by the ion channel blockers barium and amantadine but not by cesium. Genes encoding Kcv-like proteins were isolated from 40 additional chlorella viruses. Differences in 16 of the 94 amino acids were detected, producing six Kcv-like proteins with amino acid substitutions occurring in most of the functional domains of the protein (N terminus, transmembrane 1, pore helix, selectivity filter, and transmembrane 2). The six proteins form functional potassium selective channels in Xenopus oocytes with different properties including altered current kinetics and inhibition by cesium. The amino acid changes together with the different properties observed in the six Kcv-like channels will be used to guide site-directed mutations, either singularly or in combination, to identify key amino acids that confer specific properties to Kcv. PMID:14762169

  2. Water hardness influences Flavobacterium columnare pathogenesis in channel catfish

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Studies were conducted to determine aspects of water chemistry responsible for large differences in pathogenesis and mortality rates in challenges of channel catfish Ictalurus punctatus with Flavobacterium columnare; challenges were conducted in water supplying the Stuttgart National Aquaculture Res...

  3. Anoctamin and transmembrane channel-like proteins are evolutionarily related

    PubMed Central

    HAHN, YOONSOO; KIM, DONG SEON; PASTAN, IRA H.; LEE, BYUNGKOOK

    2009-01-01

    Anoctamin (ANO) family of proteins, consisting of 10 members in mammals, are transmembrane proteins that have Ca2+-activated Cl- channel activities. Transmembrane channel-like (TMC) family of proteins, consisting of 8 members in mammals, are also transmembrane proteins of which mutations are implicated in various human conditions, such as hearing loss and epidermodysplasia verruciformis. Here we show that ANO and TMC proteins share high sequence similarity and probably the same membrane topology, indicating that these proteins are evolutionarily related. We found many conserved amino acid residues between the two families of proteins, especially in regions spanning the transmembrane domains TM1, TM4-TM5, and TM6-TM7. These findings imply that these proteins form one large family, which we term ANO/TMC superfamily and that TMC proteins may also function as channels for Cl- or possibly other ions. The ANO/TMC superfamily proteins are present in almost all the diverse groups of eukaryotic organisms, suggesting that the proteins function in important biological processes, such as ion homeostasis, in eukaryotic cells. PMID:19513534

  4. IDENTIFICATION OF RESONANCE WAVES IN OPEN WATER CHANNELS

    Technology Transfer Automated Retrieval System (TEKTRAN)

    This article presents a procedure to determine the characteristics of open water channels required for controller and filter design, with special focus on the resonance waves. Also, a new simplified model structure for open water channels is proposed. The procedure applies System Identification tool...

  5. Regulation of voltage-dependent calcium channels by RGK proteins

    PubMed Central

    Yang, Tingting; Colecraft, Henry M.

    2013-01-01

    RGK proteins belong to the Ras superfamily of monomeric G-proteins, and currently include four members– Rad, Rem, Rem2, and Gem/Kir. RGK proteins are broadly expressed, and are the most potent known intracellular inhibitors of high-voltage-activated Ca2+ (CaV1 and CaV2) channels. Here, we review and discuss the evidence in the literature regarding the functional mechanisms, structural determinants, physiological role, and potential practical applications of RGK-mediated inhibition of CaV1/CaV2 channels. PMID:23063948

  6. Water Stress and Protein Synthesis

    PubMed Central

    Dhindsa, Rajinder S.; Bewley, J. Derek

    1977-01-01

    The effects have been studied of water stress and desiccation on protein synthesis in the drought-tolerant moss Tortula ruralis and the drought-sensitive moss Hygrohypnum luridum. At any particular level of steady state water stress, the inhibition of protein synthesis was greater in H. luridum than in T. ruralis. Water stress-induced changes in the pattern of protein synthesis, as determined by the double label ratio technique, were minor in T. ruralis, but major in H. luridum. Proteins of both mosses were found to be stable during desiccation and subsequent rehydration. Changes in membrane permeability, as indicated by the leakage of amino acid, were observed during rehydration of desiccated moss and were dependent on the rate of desiccation. The leakage was small and reversible in T. ruralis but large and irreversible in H. luridum. Although H. luridum failed to recover from complete desiccation (80% loss in fresh weight), it was able to recover fully from steady state stress under conditions where a maximum loss of 55% in fresh weight was recorded. PMID:16659837

  7. Water Binding in Whey Protein Concentrates

    Microsoft Academic Search

    P. G. Kliman; B. A. Anderson; M. J. Pallansch

    1973-01-01

    Measurements of the heat of fusion of free water in concentrated solutions of purified whey proteins showed that .5 g water\\/g whey protein would not freeze at --40 C. This water was defined as bound. Total bound water in protein solu- tions containing lactose and salts varied between .5 and 1.2 g water\\/g solids, with unfreezable water increasing as the

  8. Interfacial water screens the protein-induced transmembrane voltage.

    PubMed

    Zarzycki, Piotr

    2015-01-29

    Transmembrane proteins are crucial in cellular traffic, signal transduction, and energy storage in a form of transmembrane voltage. These proteins are stabilized by hydrophobic and hydrophilic interactions, in which cytoplasmic and exoplasmic water plays a special role. Water structural ordering generates the dipole potential that typically overcompensates for an intrinsic membrane-protein potential gradient, and thus it modifies and sustains an overall cellular electrostatics. Although the transmembrane voltage has been extensively studied, the dipole potential has attracted very little attention. Here, by using molecular dynamics, we examined water electrostatic response to the transmembrane charge, field, and potential asymmetry introduced by the presence of four integral membrane proteins: typical of inner (?-helix) and outer membrane (?-barrel). In all cases, the protein presence introduces electrostatic directionality in the transmembrane dipole field and voltage. In particular, water generates a deep potential sink if strongly polar residues are densely packed on one side of bilayer, as frequently occurs in a selectivity filter of the K(+) channel. We also found that protein secondary structure is less important than the polar residue distribution along the protein channel. Our findings are relevant for understanding the driving force behind biomembrane conductivity: the ability of biological water to electrostatically screen the transmembrane voltage. PMID:25563965

  9. G protein inhibition of CaV2 calcium channels

    PubMed Central

    2010-01-01

    Voltage-gated Ca2+ channels translate the electrical inputs of excitable cells into biochemical outputs by controlling influx of the ubiquitous second messenger Ca2+. As such the channels play pivotal roles in many cellular functions including the triggering of neurotransmitter and hormone release by CaV2.1 (P/Q-type) and CaV2.2 (N-type) channels. It is well established that G protein coupled receptors (GPCRs) orchestrate precise regulation neurotransmitter and hormone release through inhibition of CaV2 channels. Although the GPCRs recruit a number of different pathways, perhaps the most prominent, and certainly most studied among these is the so-called voltage-dependent inhibition mediated by direct binding of G?? to the ?1 subunit of CaV2 channels. This article will review the basics of Ca2+-channels and G protein signaling, and the functional impact of this now classical inhibitory mechanism on channel function. It will also provide an update on more recent developments in the field, both related to functional effects and crosstalk with other signaling pathways, and advances made toward understanding the molecular interactions that underlie binding of G?? to the channel and the voltage-dependence that is a signature characteristic of this mechanism. PMID:21150298

  10. Single channel recordings of reconstituted ion channel proteins: an improved technique.

    PubMed

    Keller, B U; Hedrich, R; Vaz, W L; Criado, M

    1988-01-01

    Single channel recording of reconstituted ion channels is possible by patch clamp measurements of giant liposomes formed by dehydration-rehydration of lipid films. This "hydration technique" consists of carefully controlled dehydration of a suspension of small vesicles followed by rehydration of the residue resulting in formation of large liposomes. Patch pipettes can be attached to the liposome surface, yielding stable, high resistance seals between membranes and glass pipettes. This method allows the study of the properties of reconstituted ion channels from different tissues. The hydration technique was used to characterize the reconstituted K+-channel of sarcoplasmic reticulum from rabbit skeletal muscle. In a solution of 100 mM KCl, the sarcoplasmic reticulum K+-channel studied displays a conductance gamma K+ of 145 pS. The single channel conductance in 100 mM Rb+ and Na+ is gamma Rb+ = 98 pS and gamma Na+ = 65 pS respectively. A concentration of 0.5 mM decamethonium causes a flickering channel block. These properties are in good agreement with the ones found in sarcoplasmic reticulum K+-channels characterized by other methods. Other ion channels have also been reconstituted and studied by this technique. This improved method is compared with previous approaches and its applicability for the characterization of reconstituted ion channel proteins is discussed. PMID:2451217

  11. CHOLESTEROL REGULATES PROKARYOTIC KIR CHANNEL BY DIRECT BINDING TO CHANNEL PROTEIN

    PubMed Central

    Singh, Dev; Shentu, Tzu-Pin; Enkvetchakul, Decha; Levitan, Irena

    2011-01-01

    Cholesterol is a major regulator of a variety of ion channels but the mechanisms underlying cholesterol sensitivity of ion channels are still poorly understood. The key question is whether cholesterol regulates ion channels by direct binding to the channel protein or by altering the physical environment of lipid bilayer. In this study, we provide the first direct evidence that cholesterol binds to prokaryotic Kir channels, KirBac1.1, and that cholesterol binding is essential for its regulatory effect. Specifically, we show that cholesterol is eluted together with the KirBac1.1 protein when separated on an affinity column and that the amount of bound cholesterol is proportional to the amount of the protein. We also show that cholesterol binding to KirBac1.1 is saturable with a KD of 390 ?M. Moreover, there is clear competition between radioactive and non-radioactive cholesterol for the binding site. There is no competition, however, between cholesterol and 5-Androsten 3?-17 ?-diol, a sterol that we showed previously to have no effect on KirBac1.1 function. Finally, we show that cholesterol-KirBac1.1 binding is significantly inhibited by trifluoperazine, known to inhibit cholesterol binding to other proteins, and that inhibition of cholesterol-KirBac1.1 binding results in full recovery of the channel activity. Collectively, results from this study indicate that cholesterol-induced suppression of KirBac1.1 activity is mediated by direct interaction between cholesterol and the channel protein. PMID:21798234

  12. The Structure of the Aquaporin-1 Water Channel: A Comparison between Cryo-electron Microscopy and

    E-print Network

    de Groot, Bert

    throughout nature, in species ranging from bacteria to humans, and more than 350 proteins have been Switzerland Three different medium-resolution structures of the human water channel aquaporin-1 (AQP1) have and limitations of state of the art cryo-EM methods. We present a detailed comparison between the different models

  13. Conductance and block of hair-cell mechanotransducer channels in transmembrane channel–like protein mutants

    PubMed Central

    Beurg, Maryline; Kim, Kyunghee X.

    2014-01-01

    Transmembrane channel–like (TMC) proteins TMC1 and TMC2 are crucial to the function of the mechanotransducer (MT) channel of inner ear hair cells, but their precise function has been controversial. To provide more insight, we characterized single MT channels in cochlear hair cells from wild-type mice and mice with mutations in Tmc1, Tmc2, or both. Channels were recorded in whole-cell mode after tip link destruction with BAPTA or after attenuating the MT current with GsMTx-4, a peptide toxin we found to block the channels with high affinity. In both cases, the MT channels in outer hair cells (OHCs) of wild-type mice displayed a tonotopic gradient in conductance, with channels from the cochlear base having a conductance (110 pS) nearly twice that of those at the apex (62 pS). This gradient was absent, with channels at both cochlear locations having similar small conductances, with two different Tmc1 mutations. The conductance of MT channels in inner hair cells was invariant with cochlear location but, as in OHCs, was reduced in either Tmc1 mutant. The gradient of OHC conductance also disappeared in Tmc1/Tmc2 double mutants, in which a mechanically sensitive current could be activated by anomalous negative displacements of the hair bundle. This “reversed stimulus–polarity” current was seen with two different Tmc1/Tmc2 double mutants, and with Tmc1/Tmc2/Tmc3 triple mutants, and had a pharmacological sensitivity comparable to that of native MT currents for most antagonists, except dihydrostreptomycin, for which the affinity was less, and for curare, which exhibited incomplete block. The existence in the Tmc1/Tmc2 double mutants of MT channels with most properties resembling those of wild-type channels indicates that proteins other than TMCs must be part of the channel pore. We suggest that an external vestibule of the MT channel may partly account for the channel’s large unitary conductance, high Ca2+ permeability, and pharmacological profile, and that this vestibule is disrupted in Tmc mutants. PMID:24981230

  14. Water Transport in Hydrophilic Channels of Nafion (DMR 0819860)

    E-print Network

    Petta, Jason

    Water Transport in Hydrophilic Channels of Nafion (DMR 0819860) Qiao Zhao, Paul Majsztrik and Jay, consists of sulfonic acid groups dispersed in a teflon-like hydrophobic matrix. It was shown that water of the sulfonic acids; (ii) (10%water forms a hydration shell and forms a hydrophilic percolation pathway

  15. Zn2+ Interaction with Alzheimer Amyloid ? Protein Calcium Channels

    NASA Astrophysics Data System (ADS)

    Arispe, Nelson; Pollard, Harvey B.; Rojas, Eduardo

    1996-02-01

    The Alzheimer disease 40-residue amyloid ? protein (A? P[1-40]) forms cation-selective channels across acidic phospholipid bilayer membranes with spontaneous transitions over a wide range of conductances ranging from 40 to 4000 pS. Zn2+ has been reported to bind to A? P[1-40] with high affinity, and it has been implicated in the formation of amyloid plaques. We now report the functional consequences of such Zn2+ binding for the A? P[1-40] channel. Provided the A? P[1-40] channel is expressed in the low conductance (<400 pS) mode, Zn2+ blocks the open channel in a dosedependent manner. For A? P[1-40] channels in the giant conductance mode (>400 pS), Zn2+ doses in the millimolar range were required to exert substantial blockade. The Zn2+ chelator o-phenanthroline reverses the blockade. We also found that Zn2+ modulates A? P[1-40] channel gating and conductance only from one side of the channel. These data are consistent with predictions of our recent molecular modeling studies on A? P[1-40] channels indicating asymmetric Zn2+-A? P[1-40] interactions at the entrance to the pore.

  16. FAITH Water Channel Flow Visualization - Duration: 0:56.

    NASA Video Gallery

    Water channel flow visualization experiments are performed on a three dimensional model of a small hill. This experiment was part of a series of measurements of the complex fluid flow around the hi...

  17. Conductance and block of hair-cell mechanotransducer channels in transmembrane channel-like protein mutants.

    PubMed

    Beurg, Maryline; Kim, Kyunghee X; Fettiplace, Robert

    2014-07-01

    Transmembrane channel-like (TMC) proteins TMC1 and TMC2 are crucial to the function of the mechanotransducer (MT) channel of inner ear hair cells, but their precise function has been controversial. To provide more insight, we characterized single MT channels in cochlear hair cells from wild-type mice and mice with mutations in Tmc1, Tmc2, or both. Channels were recorded in whole-cell mode after tip link destruction with BAPTA or after attenuating the MT current with GsMTx-4, a peptide toxin we found to block the channels with high affinity. In both cases, the MT channels in outer hair cells (OHCs) of wild-type mice displayed a tonotopic gradient in conductance, with channels from the cochlear base having a conductance (110 pS) nearly twice that of those at the apex (62 pS). This gradient was absent, with channels at both cochlear locations having similar small conductances, with two different Tmc1 mutations. The conductance of MT channels in inner hair cells was invariant with cochlear location but, as in OHCs, was reduced in either Tmc1 mutant. The gradient of OHC conductance also disappeared in Tmc1/Tmc2 double mutants, in which a mechanically sensitive current could be activated by anomalous negative displacements of the hair bundle. This "reversed stimulus-polarity" current was seen with two different Tmc1/Tmc2 double mutants, and with Tmc1/Tmc2/Tmc3 triple mutants, and had a pharmacological sensitivity comparable to that of native MT currents for most antagonists, except dihydrostreptomycin, for which the affinity was less, and for curare, which exhibited incomplete block. The existence in the Tmc1/Tmc2 double mutants of MT channels with most properties resembling those of wild-type channels indicates that proteins other than TMCs must be part of the channel pore. We suggest that an external vestibule of the MT channel may partly account for the channel's large unitary conductance, high Ca(2+) permeability, and pharmacological profile, and that this vestibule is disrupted in Tmc mutants. PMID:24981230

  18. Upregulation of aquaporin 2 water channel expression in pregnant rats.

    PubMed Central

    Ohara, M; Martin, P Y; Xu, D L; St John, J; Pattison, T A; Kim, J K; Schrier, R W

    1998-01-01

    Water retention is characteristic of pregnancy but the mechanism(s) of the altered water metabolism has yet to be elucidated. The collecting duct water channel, aquaporin 2 (AQP2), plays a pivotal role in the renal water regulation, and we hypothesized that AQP2 expression could be modified during pregnancy. Sprague-Dawley female rats were studied on days 7 (P7), 14 (P14), and 20 (P20) of pregnancy, and expression of AQP2 in papillae was examined. Nonpregnant (NP) littermates were used as controls. Plasma osmolalities were significantly lower in pregnant rats by day 7 of gestation (P7 283.8+/-1.82, P14 284.3+/-1.64, P < 0.001, P20 282. 4+/-1.32, P < 0.0001, vs. NP 291.8+/-1.06 mosmol/kgH2O). However, plasma vasopressin concentrations in pregnant rats were not significantly different than in nonpregnant rats (NP 1.03+/-0.14, P7 1.11+/-0.21, P14 1.15+/-0.21, P20 1.36+/-0.24 pg/ml, NS). The mRNA of AQP2 was increased early during pregnancy: AQP2/beta actin: P7 196+/-17.9, P14 200+/-6.8, and P20 208+/-15.5%, P < 0.005 vs. NP (100+/-11.1%). AQP2 protein was also increased during pregnancy: AQP2 protein: P7 269+/-10.0, P14 251+/-12.0, P < 0.0001, and P20 250+/-13.6%, P < 0.001 vs. NP (100+/-12.5%). The effect of V2 vasopressin receptor antagonist, OPC-31260, was then investigated. AQP2 mRNA was suppressed significantly by OPC-31260 administration to P14 rats (AQP2/beta actin: P14 with OPC-31260 39.6+/-1.7%, P < 0.001 vs. P14 with vehicle) and was decreased to the same level of expression as NP rats receiving OPC-31260. Similar findings were found with the analysis of AQP2 protein. The decreased plasma osmolality of P14 rats was not modified by OPC-31260. The results of the study indicate that upregulation of AQP2 contributes to the water retention in pregnancy through a V2 receptor-mediated effect. In addition to vasopressin, other factors may be involved in this upregulation. PMID:9486978

  19. Nanometer-Scale Water- and Proton-Diffusion Heterogeneities across Water Channels in Polymer Electrolyte Membranes.

    PubMed

    Song, Jinsuk; Han, Oc Hee; Han, Songi

    2015-03-16

    Nafion, the most widely used polymer for electrolyte membranes (PEMs) in fuel cells, consists of a fluorocarbon backbone and acidic groups that, upon hydration, swell to form percolated channels through which water and ions diffuse. Although the effects of the channel structures and the acidic groups on water/ion transport have been studied before, the surface chemistry or the spatially heterogeneous diffusivity across water channels has never been shown to directly influence water/ion transport. By the use of molecular spin probes that are selectively partitioned into heterogeneous regions of the PEM and Overhauser dynamic nuclear polarization relaxometry, this study reveals that both water and proton diffusivity are significantly faster near the fluorocarbon and the acidic groups lining the water channels than within the water channels. The concept that surface chemistry at the (sub)nanometer scale dictates water and proton diffusivity invokes a new design principle for PEMs. PMID:25630609

  20. Channel character of uncoupling protein-mediated transport

    Microsoft Academic Search

    Petr Ježek; Martin Jab?rek; Keith D. Garlid

    2010-01-01

    Mitochondrial uncoupling proteins (UCPs) are pure anion uniporters, which mediate fatty acid (FA) uniport leading to FA cycling. Protonated FAs then flip-flop back across the lipid bilayer. An existence of pure proton channel in UCPs is excluded by the equivalent flux-voltage dependencies for uniport of FAs and halide anions, which are best described by the Eyring barrier variant with a

  1. Sansom Group: Simulation of Ion Channels and Membrane Proteins

    NSDL National Science Digital Library

    Dr. Mark Sansom of the Department of Biochemistry at Oxford University (UK) heads up this research group on Simulation of Ion Channels and Membrane Proteins. Dedicated to understanding the physiological properties of ion channels -- conductance, ion selectivity, and gating -- this lab uses molecular modeling (and other approaches) to generate "plausible molecular models of ion channels." Targeting those in the know, the homepage provides a wealth of technical information and resource links. The Introduction section outlines the research project, while technical publications, including FTP access to several papers, are offered in the Bibliography and FTP sections. The heart of the site is the Research section, which gives detailed, color-illustrated descriptions of the (currently) six main projects, including Antimicrobial Peptides and Molecular Dynamics Simulations of Ion Permeation in Channels, among others. A hefty selection of academic links rounds out the site.

  2. Engineering Proteins for Custom Inhibition of CaV Channels

    NSDL National Science Digital Library

    Xianghua Xu (College of Physicians and Surgeons, Columbia University Physiology and Cellular Biophysics)

    2009-08-01

    The influx of Ca2+ ions through voltage-dependent calcium (CaV) channels links electrical signals to physiological responses in all excitable cells. Not surprisingly, blocking CaV channel activity is a powerful method to regulate the function of excitable cells, and this is exploited for both physiological and therapeutic benefit. Nevertheless, the full potential for CaV channel inhibition is not being realized by currently available small-molecule blockers or second-messenger modulators due to limitations in targeting them either to defined groups of cells in an organism or to distinct subcellular regions within a single cell. Here, we review early efforts to engineer protein molecule blockers of CaV channels to fill this crucial niche. This technology would greatly expand the toolbox available to physiologists studying the biology of excitable cells at the cellular and systems level.

  3. Close association of water channel AQP1 with amyloid-? deposition in Alzheimer disease brains

    Microsoft Academic Search

    Tamako Misawa; Kunimasa Arima; Hidehiro Mizusawa; Jun-ichi Satoh

    2008-01-01

    Aquaporin-1 (AQP1), a membrane water channel protein, is expressed exclusively in the choroid plexus epithelium in the central\\u000a nervous system under physiological conditions. However, AQP1 expression is enhanced in reactive astrocytes, accumulating in\\u000a brain lesions of Creutzfeldt-Jakob disease and multiple sclerosis, suggesting a role of AQP1-expressing astrocytes in brain\\u000a water homeostasis under pathological conditions. To clarify a pathological implication of

  4. Regulation of the epithelial sodium channel by accessory proteins.

    PubMed Central

    Gormley, Kelly; Dong, Yanbin; Sagnella, Giuseppe A

    2003-01-01

    The epithelial sodium channel (ENaC) is of fundamental importance in the control of sodium fluxes in epithelial cells. Modulation of sodium reabsorption through the distal nephron ENaC is an important component in the overall control of sodium balance, blood volume and thereby of blood pressure. This is clearly demonstrated by rare genetic disorders of sodium-channel activity (Liddle's syndrome and pseudohypoaldosteronism type 1), associated with contrasting effects on blood pressure. The mineralocorticoid aldosterone is a well-established modulator of sodium-channel activity. Considerable insight has now been gained into the intracellular signalling pathways linking aldosterone-mediated changes in gene transcription with changes in ion transport. Activating pathways include aldosterone-induced proteins and especially the serum- and glucocorticoid-inducible kinase (SGK) and the small G-protein, K-Ras 2A. Targeting of the ENaC for endocytosis and degradation is now emerging as a major mechanism for the down-regulation of channel activity. Several proteins acting in concert are an intrinsic part of this process but Nedd4 (neural precursor cell expressed developmentally down-regulated 4) is of central importance. Other mechanisms known to interact with ENaC and affect sodium transport include channel-activating protease 1 (CAP-1), a membrane-anchored protein, and the cystic fibrosis transmembrane regulator. The implications of research on accessory factors controlling ENaC activity are wide-ranging. Understanding cellular mechanisms controlling ENaC activity may provide a more detailed insight not only of ion-channel abnormalities in cystic fibrosis but also of the link between abnormal renal sodium transport and essential hypertension. PMID:12460120

  5. Water-Protein Interactions: The Secret of Protein Dynamics

    PubMed Central

    Martini, Silvia; Bonechi, Claudia; Foletti, Alberto; Rossi, Claudio

    2013-01-01

    Water-protein interactions help to maintain flexible conformation conditions which are required for multifunctional protein recognition processes. The intimate relationship between the protein surface and hydration water can be analyzed by studying experimental water properties measured in protein systems in solution. In particular, proteins in solution modify the structure and the dynamics of the bulk water at the solute-solvent interface. The ordering effects of proteins on hydration water are extended for several angstroms. In this paper we propose a method for analyzing the dynamical properties of the water molecules present in the hydration shells of proteins. The approach is based on the analysis of the effects of protein-solvent interactions on water protons NMR relaxation parameters. NMR relaxation parameters, especially the nonselective (R1NS ) and selective (R1SE ) spin-lattice relaxation rates of water protons, are useful for investigating the solvent dynamics at the macromolecule-solvent interfaces as well as the perturbation effects caused by the water-macromolecule interactions on the solvent dynamical properties. In this paper we demonstrate that Nuclear Magnetic Resonance Spectroscopy can be used to determine the dynamical contributions of proteins to the water molecules belonging to their hydration shells. PMID:23766672

  6. Heterologous expression of tulip petal plasma membrane aquaporins in Pichia pastoris for water channel analysis.

    PubMed

    Azad, Abul Kalam; Sawa, Yoshihiro; Ishikawa, Takahiro; Shibata, Hitoshi

    2009-05-01

    Water channels formed by aquaporins (AQPs) play an important role in the control of water homeostasis in individual cells and in multicellular organisms. Plasma membrane intrinsic proteins (PIPs) constitute a subclass of plant AQPs. TgPIP2;1 and TgPIP2;2 from tulip petals are members of the PIP family. In this study, we overexpressed TgPIP2;1 and TgPIP2;2 in Pichia pastoris and monitored their water channel activity (WCA) either by an in vivo spheroplast-bursting assay performed after hypo-osmotic shock or by growth assay. Osmolarity, pH, and inhibitors of AQPs, protein kinases (PKs), and protein phosphatases (PPs) affect the WCA of heterologous AQPs in this expression system. The WCA of TgPIP2;2-expressing spheroplasts was affected by inhibitors of PKs and PPs, which indicates that the water channel of this homologue is regulated by phosphorylation in P. pastoris. From the results reported herein, we suggest that P. pastoris can be employed as a heterologous expression system to assay the WCA of PIPs and to monitor the AQP-mediated channel gating mechanism, and it can be developed to screen inhibitors/effectors of PIPs. PMID:19251885

  7. Protein Rearrangements Underlying Slow Inactivation of the Shaker K+ Channel

    PubMed Central

    Loots, E.; Isacoff, E.Y.

    1998-01-01

    Voltage-dependent ion channels transduce changes in the membrane electric field into protein rearrangements that gate their transmembrane ion permeation pathways. While certain molecular elements of the voltage sensor and gates have been identified, little is known about either the nature of their conformational rearrangements or about how the voltage sensor is coupled to the gates. We used voltage clamp fluorometry to examine the voltage sensor (S4) and pore region (P-region) protein motions that underlie the slow inactivation of the Shaker K+ channel. Fluorescent probes in both the P-region and S4 changed emission intensity in parallel with the onset and recovery of slow inactivation, indicative of local protein rearrangements in this gating process. Two sequential rearrangements were observed, with channels first entering the P-type, and then the C-type inactivated state. These forms of inactivation appear to be mediated by a single gate, with P-type inactivation closing the gate and C-type inactivation stabilizing the gate's closed conformation. Such a stabilization was due, at least in part, to a slow rearrangement around S4 that stabilizes S4 in its activated transmembrane position. The fluorescence reports of S4 and P-region fluorophore are consistent with an increased interaction of the voltage sensor and inactivation gate upon gate closure, offering insight into how the voltage-sensing apparatus is coupled to a channel gate. PMID:9758858

  8. 5. GATE 5, INTAKE CHANNEL LOOKING SOUTH; WATER FROM GATE ...

    Library of Congress Historic Buildings Survey, Historic Engineering Record, Historic Landscapes Survey

    5. GATE 5, INTAKE CHANNEL LOOKING SOUTH; WATER FROM GATE 5 ENTERED DITCH AND IRRIGATED HONDIUS' FIELDS. - Hondius Water Line, 1.6 miles Northwest of Park headquarters building & 1 mile Northwest of Beaver Meadows entrance station, Estes Park, Larimer County, CO

  9. Water-protein interactions from high-resolution protein crystallography.

    PubMed Central

    Nakasako, Masayoshi

    2004-01-01

    To understand the role of water in life at molecular and atomic levels, structures and interactions at the protein-water interface have been investigated by cryogenic X-ray crystallography. The method enabled a much clearer visualization of definite hydration sites on the protein surface than at ambient temperature. Using the structural models of proteins, including several hydration water molecules, the characteristics in hydration structures were systematically analysed for the amount, the interaction geometries between water molecules and proteins, and the local and global distribution of water molecules on the surface of proteins. The tetrahedral hydrogen-bond geometry of water molecules in bulk solvent was retained at the interface and enabled the extension of a three-dimensional chain connection of a hydrogen-bond network among hydration water molecules and polar protein atoms over the entire surface of proteins. Networks of hydrogen bonds were quite flexible to accommodate and/or to regulate the conformational changes of proteins such as domain motions. The present experimental results may have profound implications in the understanding of the physico-chemical principles governing the dynamics of proteins in an aqueous environment and a discussion of why water is essential to life at a molecular level. PMID:15306376

  10. Regulation of neurovascular coupling in autoimmunity to water and ion channels.

    PubMed

    Jukkola, Peter; Gu, Chen

    2015-03-01

    Much progress has been made in understanding autoimmune channelopathies, but the underlying pathogenic mechanisms are not always clear due to broad expression of some channel proteins. Recent studies show that autoimmune conditions that interfere with neurovascular coupling in the central nervous system (CNS) can lead to neurodegeneration. Cerebral blood flow that meets neuronal activity and metabolic demand is tightly regulated by local neural activity. This process of reciprocal regulation involves coordinated actions of a number of cell types, including neurons, glia, and vascular cells. In particular, astrocytic endfeet cover more than 90% of brain capillaries to assist blood-brain barrier (BBB) function, and wrap around synapses and nodes of Ranvier to communicate with neuronal activity. In this review, we highlight four types of channel proteins that are expressed in astrocytes, regarding their structures, biophysical properties, expression and distribution patterns, and related diseases including autoimmune disorders. Water channel aquaporin 4 (AQP4) and inwardly rectifying potassium (Kir4.1) channels are concentrated in astrocytic endfeet, whereas some voltage-gated Ca(2+) and two-pore domain K(+) channels are expressed throughout the cell body of reactive astrocytes. More channel proteins are found in astrocytes under normal and abnormal conditions. This research field will contribute to a better understanding of pathogenic mechanisms underlying autoimmune disorders. PMID:25462580

  11. Stimulatory and Inhibitory Regulation of Calcium-Activated Potassium Channels by Guanine Nucleotide-Binding Proteins

    Microsoft Academic Search

    Hiroaki Kume; Michael P. Graziano; Michael I. Kotlikoff

    1992-01-01

    The regulation of membrane ion channels by guanine nucleotide-binding proteins (G proteins) has been described in numerous tissues. This regulation has been shown to involve the membrane-delimited stimulatory action of G proteins on ion channels. We now show that single calcium-activated potassium channels (KCa channels) in airway smooth muscle cells are both stimulated and inhibited by G proteins in membrane

  12. A Stochastic Model of Ionic Permeation in Protein Channels, I

    NASA Astrophysics Data System (ADS)

    Schuss, Zeev

    2003-03-01

    "A Stochastic Model of Ionic Permeation in Protein Channels, I" Z. Schuss, B. Nadler, A. Singer, R.S. Eisenberg Abstract The motion of ions through solution and protein channels is diffusion in an electrostatic field. The phenomenology of permeation through narrow channels is strongly nonlinear, exhibiting blocking, saturation, anomalous flux ratios, selectivity of different ionic species, nonlinear conductivity, and more. The confined geometry of the channels enhances the effect of ion-ion interactions in channel. This interaction is believed to be the cause of many of the nonlinear permeation phenomena. The steady-state diffusion of particles with short- and long-range interactions leads to fundamental problems in non-equilibrium statistical mechanics. In particular, continuum descriptions of such diffusion need to incorporate these interactions in order to reproduce the non-linear phenomena. The pair correlation function, analogous to that of equilibrium statistical mechanics, plays a key role in the description of ionic permeation by continuum equations. It is believed that it accounts for many of the non-linear channel phenomena, including selectivity. We show that the non-equilibrium pair correlation function of interacting ions can be determined from Poisson-Nernst-Planck equations (PNPEs) and we determine the boundary conditions it satisfies. This is a new result in non-equilibrium statistical mechanics. To decouple the PNPEs for the pair correlation function from these for the triplet correlation function, a closure relation is needed. We derive a closure relation for short-range interactions, such as Lennard-Jones repulsion, and propose an approach to the closure problem for long-range interactions, such as coulomb forces.

  13. Secretins: dynamic channels for protein transport across membranes

    PubMed Central

    Korotkov, Konstantin V.; Gonen, Tamir; Hol, Wim G. J.

    2011-01-01

    Secretins form mega-Dalton bacterial membrane channels in at least four sophisticated multi-protein systems that are crucial for translocation of proteins and assembled fibers across the outer membrane of many species of bacteria. Secretin subunits contain multiple domains, which interact with numerous other proteins, including pilotins, secretion system partner proteins and exoproteins. Our understanding of the structure of secretins is rapidly progressing, and we now recognize that features common to all secretins include a cylindrical arrangement of 12–15 subunits, a large periplasmic vestibule with a wide opening on one end and a periplasmic gate at the other end. Secretins might also play a key role in the biogenesis of their cognate secretion systems. PMID:21565514

  14. Zn2+ Interaction with Alzheimer Amyloid beta Protein Calcium Channels

    Microsoft Academic Search

    Nelson Arispe; Harvey B. Pollard; Eduardo Rojas

    1996-01-01

    The Alzheimer disease 40-residue amyloid beta protein (Abeta P[1-40]) forms cation-selective channels across acidic phospholipid bilayer membranes with spontaneous transitions over a wide range of conductances ranging from 40 to 4000 pS. Zn2+ has been reported to bind to Abeta P[1-40] with high affinity, and it has been implicated in the formation of amyloid plaques. We now report the functional

  15. Strain Engineering Water Transport in Graphene Nano-channels

    E-print Network

    Xiong, Wei; Ma, Ming; Xu, Zhiping; Sheridan, John; Zheng, Quanshui

    2011-01-01

    Using equilibrium and non-equilibrium molecular dynamic (MD) simulations, we found that engineering the strain on the graphene planes forming a channel can drastically change the interfacial friction of water transport through it. There is a sixfold change of interfacial friction stress when the strain changes from -10% to 10%. Stretching the graphene walls increases the interfacial shear stress, while compressing the graphene walls reduces it. Detailed analysis of the molecular structure reveals the essential roles of the interfacial potential energy barrier and the structural commensurateness between the solid walls and the first water layer. Our results suggest that the strain engineering is an effective way of controlling the water transport inside nano-channels. The resulting quantitative relations between shear stress and slip velocity and the understanding of the molecular mechanisms will be invaluable in designing graphene nano-channel devices.

  16. Bimodal modulation of the botulinum neurotoxin protein-conducting channel

    PubMed Central

    Fischer, Audrey; Nakai, Yuya; Eubanks, Lisa M.; Clancy, Colin M.; Tepp, William H.; Pellett, Sabine; Dickerson, Tobin J.; Johnson, Eric A.; Janda, Kim D.; Montal, Mauricio

    2009-01-01

    Clostridium botulinum neurotoxin (BoNT) is the causative agent of botulism, a neuroparalytic disease. We describe here a semisynthetic strategy to identify inhibitors based on toosendanin, a traditional Chinese medicine reported to protect from BoNT intoxication. Using a single molecule assay of BoNT serotypes A and E light chain (LC) translocation through the heavy chain (HC) channel in neurons, we discovered that toosendanin and its tetrahydrofuran analog selectively arrest the LC translocation step of intoxication with subnanomolar potency, and increase the unoccluded HC channel propensity to open with micromolar efficacy. The inhibitory profile on LC translocation is accurately recapitulated in 2 different BoNT intoxication assays, namely the mouse protection and the primary rat spinal cord cell assays. Toosendanin has an unprecedented dual mode of action on the protein-conducting channel acting as a cargo-dependent inhibitor of translocation and as cargo-free channel activator. These results imply that the bimodal modulation by toosendanin depends on the dynamic interactions between channel and cargo, highlighting their tight interplay during the progression of LC transit across endosomes. PMID:19164566

  17. Bimodal modulation of the botulinum neurotoxin protein-conducting channel.

    PubMed

    Fischer, Audrey; Nakai, Yuya; Eubanks, Lisa M; Clancy, Colin M; Tepp, William H; Pellett, Sabine; Dickerson, Tobin J; Johnson, Eric A; Janda, Kim D; Montal, Mauricio

    2009-02-01

    Clostridium botulinum neurotoxin (BoNT) is the causative agent of botulism, a neuroparalytic disease. We describe here a semisynthetic strategy to identify inhibitors based on toosendanin, a traditional Chinese medicine reported to protect from BoNT intoxication. Using a single molecule assay of BoNT serotypes A and E light chain (LC) translocation through the heavy chain (HC) channel in neurons, we discovered that toosendanin and its tetrahydrofuran analog selectively arrest the LC translocation step of intoxication with subnanomolar potency, and increase the unoccluded HC channel propensity to open with micromolar efficacy. The inhibitory profile on LC translocation is accurately recapitulated in 2 different BoNT intoxication assays, namely the mouse protection and the primary rat spinal cord cell assays. Toosendanin has an unprecedented dual mode of action on the protein-conducting channel acting as a cargo-dependent inhibitor of translocation and as cargo-free channel activator. These results imply that the bimodal modulation by toosendanin depends on the dynamic interactions between channel and cargo, highlighting their tight interplay during the progression of LC transit across endosomes. PMID:19164566

  18. High-Density Reconstitution of Functional Water Channels into Vesicular and Planar Block Copolymer Membranes

    PubMed Central

    2012-01-01

    The exquisite selectivity and unique transport properties of membrane proteins can be harnessed for a variety of engineering and biomedical applications if suitable membranes can be produced. Amphiphilic block copolymers (BCPs), developed as stable lipid analogs, form membranes that functionally incorporate membrane proteins and are ideal for such applications. While high protein density and planar membrane morphology are most desirable, BCP–membrane protein aggregates have so far been limited to low protein densities in either vesicular or bilayer morphologies. Here, we used dialysis to reproducibly form planar and vesicular BCP membranes with a high density of reconstituted aquaporin-0 (AQP0) water channels. We show that AQP0 retains its biological activity when incorporated at high density in BCP membranes, and that the morphology of the BCP–protein aggregates can be controlled by adjusting the amount of incorporated AQP0. We also show that BCPs can be used to form two-dimensional crystals of AQP0. PMID:23082933

  19. Regulation of Cardiac L-Type Calcium Channels by Protein Kinase A and Protein Kinase C

    Microsoft Academic Search

    Timothy J. Kamp; Johannes W. Hell

    a cAMP\\/PKA pathway. Growing evidence points to an important role of localized signaling complexes involved in the PKA-mediated regulation of ICa, including A-kinase anchor proteins and binding of phosphatase PP2a to the carboxyl terminus of the a1C (Cav1.2) subunit. Both a1C and b2a subunits of the channel are substrates for PKA in vivo. The regulation of L-type Ca21 channels by

  20. Novel Channel Enzyme Fusion Proteins Confer Arsenate Resistance*

    PubMed Central

    Wu, Binghua; Song, Jie; Beitz, Eric

    2010-01-01

    Steady exposure to environmental arsenic has led to the evolution of vital cellular detoxification mechanisms. Under aerobic conditions, a two-step process appears most common among microorganisms involving reduction of predominant, oxidized arsenate (H2AsVO4?/HAsVO42?) to arsenite (AsIII(OH)3) by a cytosolic enzyme (ArsC; Escherichia coli type arsenate reductase) and subsequent extrusion via ArsB (E. coli type arsenite transporter)/ACR3 (yeast type arsenite transporter). Here, we describe novel fusion proteins consisting of an aquaglyceroporin-derived arsenite channel with a C-terminal arsenate reductase domain of phosphotyrosine-phosphatase origin, providing transposable, single gene-encoded arsenate resistance. The fusion occurred in actinobacteria from soil, Frankia alni, and marine environments, Salinispora tropica; Mycobacterium tuberculosis encodes an analogous ACR3-ArsC fusion. Mutations rendered the aquaglyceroporin channel more polar resulting in lower glycerol permeability and enhanced arsenite selectivity. The arsenate reductase domain couples to thioredoxin and can complement arsenate-sensitive yeast strains. A second isoform with a nonfunctional channel may use the mycothiol/mycoredoxin cofactor pool. These channel enzymes constitute prototypes of a novel concept in metabolism in which a substrate is generated and compartmentalized by the same molecule. Immediate diffusion maintains the dynamic equilibrium and prevents toxic accumulation of metabolites in an energy-saving fashion. PMID:20947511

  1. The stream channel incision syndrome and water quality

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Watershed development often triggers channel incision, which accounts for 60-90% of sediments leaving many disturbed watersheds. Impacts of such incision on water quality processes and the implication of such impairment on stream biota are relevant to issues associated with establishing total maxim...

  2. Dual regulation of G proteins and the G-protein-activated K+ channels by lithium.

    PubMed

    Farhy Tselnicker, Isabella; Tsemakhovich, Vladimir; Rishal, Ida; Kahanovitch, Uri; Dessauer, Carmen W; Dascal, Nathan

    2014-04-01

    Lithium (Li(+)) is widely used to treat bipolar disorder (BPD). Cellular targets of Li(+), such as glycogen synthase kinase 3? (GSK3?) and G proteins, have long been implicated in BPD etiology; however, recent genetic studies link BPD to other proteins, particularly ion channels. Li(+) affects neuronal excitability, but the underlying mechanisms and the relevance to putative BPD targets are unknown. We discovered a dual regulation of G protein-gated K(+) (GIRK) channels by Li(+), and identified the underlying molecular mechanisms. In hippocampal neurons, therapeutic doses of Li(+) (1-2 mM) increased GIRK basal current (Ibasal) but attenuated neurotransmitter-evoked GIRK currents (Ievoked) mediated by Gi/o-coupled G-protein-coupled receptors (GPCRs). Molecular mechanisms of these regulations were studied with heterologously expressed GIRK1/2. In excised membrane patches, Li(+) increased Ibasal but reduced GPCR-induced GIRK currents. Both regulations were membrane-delimited and G protein-dependent, requiring both G? and G?? subunits. Li(+) did not impair direct activation of GIRK channels by G??, suggesting that inhibition of Ievoked results from an action of Li(+) on G?, probably through inhibition of GTP-GDP exchange. In direct binding studies, Li(+) promoted GPCR-independent dissociation of G?i(GDP) from G?? by a Mg(2+)-independent mechanism. This previously unknown Li(+) action on G proteins explains the second effect of Li(+), the enhancement of GIRK's Ibasal. The dual effect of Li(+) on GIRK may profoundly regulate the inhibitory effects of neurotransmitters acting via GIRK channels. Our findings link between Li(+), neuronal excitability, and both cellular and genetic targets of BPD: GPCRs, G proteins, and ion channels. PMID:24639496

  3. Human odontoblasts express transient receptor protein and acid-sensing ion channel mechanosensor proteins.

    PubMed

    Solé-Magdalena, Antonio; Revuelta, Enrique G; Menénez-Díaz, Ivan; Calavia, Marta G; Cobo, Teresa; García-Suárez, Olivia; Pérez-Piñera, Pablo; De Carlos, Felix; Cobo, Juan; Vega, Jose A

    2011-05-01

    Diverse proteins of the denegerin/epithelial sodium channel (DEG/ENa(+) C) superfamily, in particular those belonging to the acid-sensing ion channel (ASIC) family, as well as some members of the transient receptor protein (TRP) channel, function as mechanosensors or may be required for mechanosensation in a diverse range of species and cell types. Therefore, we investigated the putative mechanosensitive function of human odontoblasts using immunohistochemistry to detect ENa(+) C subunits (?, ?, and ?) and ASIC (1, 2, 3, and 4) proteins, as well as TRPV4, in these cells. Positive and specific immunoreactivity in the odontoblast soma and/or processes was detected for all proteins studied except ?-ENa(+) C. The intensity of immunostaining was high for ?-ENa(+) C and ASIC2, whereas it was low for ASIC1, ASIC3, ?-ENa(+) C, and TRPV4, being absent for ?-ENa(+) C and ASIC4. These results suggest that human odontoblasts in situ express proteins related to mechanosensitive channels that probably participate in the mechanisms involved in teeth sensory transmission. PMID:20836083

  4. Template-assembled melittin: structural and functional characterization of a designed, synthetic channel-forming protein.

    PubMed Central

    Pawlak, M.; Meseth, U.; Dhanapal, B.; Mutter, M.; Vogel, H.

    1994-01-01

    Template-assembled proteins (TASPs) comprising 4 peptide blocks, each of either the natural melittin sequence (melittin-TASP) or of a truncated melittin sequence (amino acids 6-26, melittin6-26-TASP), C-terminally linked to a (linear or cyclic) 10-amino acid template were synthesized and characterized, structurally by CD, by fluorescence spectroscopy, and by monolayer experiments, and functionally, by electrical conductance measurements on planar bilayers and release experiments on dye-loaded vesicles. Melittin-TASP and the truncated analogue preferentially adopt alpha-helical structures in methanol (56% and 52%, respectively) as in lipid membranes. Unlike in methanol, the melittin-TASP self-aggregates in water. On an air-water interface, the differently sized molecules can be self-assembled and compressed to a compact structure with a molecular area of around 600 A2, compatible with a 4-helix bundle preferentially oriented perpendicular to the interface. The proteins reveal a strong affinity for lipid membranes. A partition coefficient of 1.5 x 10(9) M-1 was evaluated from changes of the Trp fluorescence spectra of the TASP in water and in the lipid bilayer. In planar lipid bilayers, TASP molecules are able to form defined ion channels, exhibiting a small single-channel conductance of 7 pS (in 1 M NaCl). With increasing protein concentration in the lipid bilayer, additional, larger conductance states of up to 1 nS were observed. These states are likely to be formed by aggregated TASP structures as inferred from a strongly voltage-dependent channel activity on membranes of large area. In this respect, melittin-TASP reveals channel features of the native peptide, but with a considerably lower variation in the size of the channel states. Compared to the free peptide, template-assembled melittin has a much higher membrane activity: it is about 100 times more effective in channel formation and 20 times more effective in releasing dye molecules from lipid vesicles. This demonstrates that the lytic properties are not solely related to channel formation. PMID:7531528

  5. Filter gate closure inhibits ion but not water transport through potassium channels

    PubMed Central

    Hoomann, Torben; Jahnke, Nadin; Horner, Andreas; Keller, Sandro; Pohl, Peter

    2013-01-01

    The selectivity filter of K+ channels is conserved throughout all kingdoms of life. Carbonyl groups of highly conserved amino acids point toward the lumen to act as surrogates for the water molecules of K+ hydration. Ion conductivity is abrogated if some of these carbonyl groups flip out of the lumen, which happens (i) in the process of C-type inactivation or (ii) during filter collapse in the absence of K+. Here, we show that K+ channels remain permeable to water, even after entering such an electrically silent conformation. We reconstituted fluorescently labeled and constitutively open mutants of the bacterial K+ channel KcsA into lipid vesicles that were either C-type inactivating or noninactivating. Fluorescence correlation spectroscopy allowed us to count both the number of proteoliposomes and the number of protein-containing micelles after solubilization, providing the number of reconstituted channels per proteoliposome. Quantification of the per-channel increment in proteoliposome water permeability with the aid of stopped-flow experiments yielded a unitary water permeability pf of (6.9 ± 0.6) × 10?13 cm3?s?1 for both mutants. “Collapse” of the selectivity filter upon K+ removal did not alter pf and was fully reversible, as demonstrated by current measurements through planar bilayers in a K+-containing medium to which K+-free proteoliposomes were fused. Water flow through KcsA is halved by 200 mM K+ in the aqueous solution, which indicates an effective K+ dissociation constant in that range for a singly occupied channel. This questions the widely accepted hypothesis that multiple K+ ions in the selectivity filter act to mutually destabilize binding. PMID:23754382

  6. Voltage Gated Ion Channel Function: Gating, Conduction, and the Role of Water and Protons

    SciTech Connect

    Kariev, Alisher M.; Green, Michael E.

    2012-02-26

    Ion channels, which are found in every biological cell, regulate the concentration of electrolytes, and are responsible for multiple biological functions, including in particular the propagation of nerve impulses. The channels with the latter function are gated (opened) by a voltage signal, which allows Na+ into the cell and K+ out. These channels have several positively charged amino acids on a transmembrane domain of their voltage sensor, and it is generally considered, based primarily on two lines of experimental evidence, that these charges move with respect to the membrane to open the channel. At least three forms of motion, with greatly differing extents and mechanisms of motion, have been proposed. There is a “gating current”, a capacitative current preceding the channel opening, that corresponds to several charges (for one class of channel typically 12–13) crossing the membrane field, which may not require protein physically crossing a large fraction of the membrane. The coupling to the opening of the channel would in these models depend on the motion. The conduction itself is usually assumed to require the “gate” of the channel to be pulled apart to allow ions to enter as a section of the protein partially crosses the membrane, and a selectivity filter at the opposite end of the channel determines the ion which is allowed to pass through. We will here primarily consider K+ channels, although Na+ channels are similar. We propose that the mechanism of gating differs from that which is generally accepted, in that the positively charged residues need not move (there may be some motion, but not as gating current). Instead, protons may constitute the gating current, causing the gate to open; opening consists of only increasing the diameter at the gate from approximately 6 Å to approximately 12 Å. We propose in addition that the gate oscillates rather than simply opens, and the ion experiences a barrier to its motion across the channel that is tuned by the water present within the channel. Our own quantum calculations as well as numerous experiments of others are interpreted in terms of this hypothesis. It is also shown that the evidence that supports the motion of the sensor as the gating current can also be consistent with the hypothesis we present.

  7. Channel character of uncoupling protein-mediated transport.

    PubMed

    Jezek, Petr; Jab?rek, Martin; Garlid, Keith D

    2010-05-17

    Mitochondrial uncoupling proteins (UCPs) are pure anion uniporters, which mediate fatty acid (FA) uniport leading to FA cycling. Protonated FAs then flip-flop back across the lipid bilayer. An existence of pure proton channel in UCPs is excluded by the equivalent flux-voltage dependencies for uniport of FAs and halide anions, which are best described by the Eyring barrier variant with a single energy well in the middle of two peaks. Experiments with FAs unable to flip and alkylsulfonates also support this view. Phylogenetically, UCPs took advantage of the common FA-uncoupling function of SLC25 family carriers and dropped their solute transport function. PMID:20206627

  8. Nanosecond Relaxation Dynamics of Hydrated Proteins: Water versus protein contributions

    SciTech Connect

    Khodadadi, S [University of Akron; Curtis, J. E. [National Institute of Standards and Technology (NIST), Gaithersburg, MD; Sokolov, Alexei P [ORNL

    2011-01-01

    We have studied picosecond to nanosecond dynamics of hydrated protein powders using dielectric spectroscopy and molecular dynamics (MD) simulations. Our analysis of hydrogen-atom single particle dynamics from MD simulations focused on main ( main tens of picoseconds) and slow ( slow nanosecond) relaxation processes that were observed in dielectric spectra of similar hydrated protein samples. Traditionally, the interpretation of these processes observed in dielectric spectra has been ascribed to the relaxation behavior of hydration water tightly bounded to a protein and not to protein atoms. Detailed analysis of the MD simulations and comparison to dielectric data indicate that the observed relaxation process in the nanosecond time range of hydrated protein spectra is mainly due to protein atoms. The relaxation processes involve the entire structure of protein including atoms in the protein backbone, side chains, and turns. Both surface and buried protein atoms contribute to the slow processes; however, surface atoms demonstrate slightly faster relaxation dynamics. Analysis of the water molecule residence and dipolar relaxation correlation behavior indicates that the hydration water relaxes at much shorter time scales.

  9. Protein packing defects "heat up" interfacial water.

    PubMed

    Sierra, María Belén; Accordino, Sebastián R; Rodriguez-Fris, J Ariel; Morini, Marcela A; Appignanesi, Gustavo A; Fernández Stigliano, Ariel

    2013-06-01

    Ligands must displace water molecules from their corresponding protein surface binding site during association. Thus, protein binding sites are expected to be surrounded by non-tightly-bound, easily removable water molecules. In turn, the existence of packing defects at protein binding sites has been also established. At such structural motifs, named dehydrons, the protein backbone is exposed to the solvent since the intramolecular interactions are incompletely wrapped by non-polar groups. Hence, dehydrons are sticky since they depend on additional intermolecular wrapping in order to properly protect the structure from water attack. Thus, a picture of protein binding is emerging wherein binding sites should be both dehydrons rich and surrounded by easily removable water. In this work we shall indeed confirm such a link between structure and dynamics by showing the existence of a firm correlation between the degree of underwrapping of the protein chain and the mobility of the corresponding hydration water molecules. In other words, we shall show that protein packing defects promote their local dehydration, thus producing a region of "hot" interfacial water which might be easily removed by a ligand upon association. PMID:23797357

  10. Steric Selectivity in Na Channels Arising from Protein Polarization and Mobile Side Chains

    PubMed Central

    Boda, Dezs?; Nonner, Wolfgang; Valiskó, Mónika; Henderson, Douglas; Eisenberg, Bob; Gillespie, Dirk

    2007-01-01

    Monte Carlo simulations of equilibrium selectivity of Na channels with a DEKA locus are performed over a range of radius R and protein dielectric coefficient ?p. Selectivity arises from the balance of electrostatic forces and steric repulsion by excluded volume of ions and side chains of the channel protein in the highly concentrated and charged (?30 M) selectivity filter resembling an ionic liquid. Ions and structural side chains are described as mobile charged hard spheres that assume positions of minimal free energy. Water is a dielectric continuum. Size selectivity (ratio of Na+ occupancy to K+ occupancy) and charge selectivity (Na+ to Ca2+) are computed in concentrations as low as 10?5 M Ca2+. In general, small R reduces ion occupancy and favors Na+ over K+ because of steric repulsion. Small ?p increases occupancy and favors Na+ over Ca2+ because protein polarization amplifies the pore's net charge. Size selectivity depends on R and is independent of ?p; charge selectivity depends on both R and ?p. Thus, small R and ?p make an efficient Na channel that excludes K+ and Ca2+ while maximizing Na+ occupancy. Selectivity properties depend on interactions that cannot be described by qualitative or verbal models or by quantitative models with a fixed free energy landscape. PMID:17526571

  11. Comparison of Space-Time Water-filling and Spatial Water-filling for MIMO Fading Channels

    E-print Network

    Evans, Brian L.

    -filling algorithm [1] [2]. With only average power constraints, a two-dimensional water-filling in both the temporalComparison of Space-Time Water-filling and Spatial Water-filling for MIMO Fading Channels Zukang the capacities achieved by space-time water-filling and spatial water-filling for MIMO fading channels. Both

  12. Emerging roles for G protein-gated inwardly rectifying potassium (GIRK) channels in health and disease

    Microsoft Academic Search

    Christian Lüscher; Paul A. Slesinger

    2010-01-01

    G protein-gated inwardly rectifying potassium (GIRK) channels hyperpolarize neurons in response to activation of many different G protein-coupled receptors and thus control the excitability of neurons through GIRK-mediated self-inhibition, slow synaptic potentials and volume transmission. GIRK channel function and trafficking are highly dependent on the channel subunit composition. Pharmacological investigations of GIRK channels and studies in animal models suggest that

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

    Microsoft Academic Search

    Nasrin Nejatbakhsh; Zhong-ping Feng

    2011-01-01

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

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

    PubMed Central

    Nejatbakhsh, Nasrin; Feng, Zhong-ping

    2011-01-01

    Calcium ion entry through voltage-gated calcium channels is essential for cellular signalling in a wide variety of cells and multiple physiological processes. Perturbations of voltage-gated calcium channel function can lead to pathophysiological consequences. Calcium binding proteins serve as calcium sensors and regulate the calcium channel properties via feedback mechanisms. This review highlights the current evidences of calcium binding protein-mediated channel regulation in human diseases. PMID:21642945

  15. Sinuous deep-water channels: Genesis, geometry and architecture

    Microsoft Academic Search

    Russell B. Wynn; Bryan T. Cronin; Jeff Peakall

    2007-01-01

    Sinuous deep-water channels display a wide range of geometries and internal architectures. Most modern examples have been documented from large passive-margin fans, supplied by major rivers carrying huge volumes of dominantly fine-grained sediments, e.g. Amazon, Mississippi, Zaire, Bengal, Indus, Rhône and Nile Fans. However, similar examples have also been documented from tectonically active margins, e.g. Magdalena Fan. In most cases,

  16. The role of transmembrane channel–like proteins in the operation of hair cell mechanotransducer channels

    PubMed Central

    Kim, Kyunghee X.; Beurg, Maryline; Hackney, Carole M.; Furness, David N.; Mahendrasingam, Shanthini

    2013-01-01

    Sound stimuli elicit movement of the stereocilia that make up the hair bundle of cochlear hair cells, putting tension on the tip links connecting the stereocilia and thereby opening mechanotransducer (MT) channels. Tmc1 and Tmc2, two members of the transmembrane channel–like family, are necessary for mechanotransduction. To assess their precise role, we recorded MT currents elicited by hair bundle deflections in mice with null mutations of Tmc1, Tmc2, or both. During the first postnatal week, we observed a normal MT current in hair cells lacking Tmc1 or Tmc2; however, in the absence of both isoforms, we recorded a large MT current that was phase-shifted 180°, being evoked by displacements of the hair bundle away from its tallest edge rather than toward it as in wild-type hair cells. The anomalous MT current in hair cells lacking Tmc1 and Tmc2 was blocked by FM1-43, dihydrostreptomycin, and extracellular Ca2+ at concentrations similar to those that blocked wild type. MT channels in the double knockouts carried Ca2+ with a lower permeability than wild-type or single mutants. The MT current in double knockouts persisted during exposure to submicromolar Ca2+, even though this treatment destroyed the tip links. We conclude that the Tmc isoforms do not themselves constitute the MT channel but are essential for targeting and interaction with the tip link. Changes in the MT conductance and Ca2+ permeability observed in the absence of Tmc1 mutants may stem from loss of interaction with protein partners in the transduction complex. PMID:24127526

  17. Dynamics of Protein Hydration Water

    E-print Network

    Wolf, M; Gulich, R; Lunkenheimer, P; Loidl, A

    2014-01-01

    We present the frequency- and temperature-dependent dielectric properties of lysozyme solutions in a broad concentration regime, measured at subzero temperatures and compare the results with measurements above the freezing point of water and on hydrated lysozyme powder. Our experiments allow examining the dynamics of unfreezable hydration water in a broad temperature range including the so-called No Man's Land (160 - 235 K). The obtained results prove the bimodality of the hydration shell dynamics and are discussed in the context of the highly-debated fragile-to-strong transition of water.

  18. Dynamics of Protein Hydration Water

    E-print Network

    M. Wolf; S. Emmert; R. Gulich; P. Lunkenheimer; A. Loidl

    2014-12-08

    We present the frequency- and temperature-dependent dielectric properties of lysozyme solutions in a broad concentration regime, measured at subzero temperatures and compare the results with measurements above the freezing point of water and on hydrated lysozyme powder. Our experiments allow examining the dynamics of unfreezable hydration water in a broad temperature range including the so-called No Man's Land (160 - 235 K). The obtained results prove the bimodality of the hydration shell dynamics and are discussed in the context of the highly-debated fragile-to-strong transition of water.

  19. 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.

  20. Actin directly interacts with different membrane channel proteins and influences channel activities: AQP2 as a model.

    PubMed

    Sasaki, Sei; Yui, Naofumi; Noda, Yumi

    2014-02-01

    The interplay between actin and 10 membrane channel proteins that have been shown to directly bind to actin are reviewed. The 10 membrane channel proteins covered in this review are aquaporin 2 (AQP2), cystic fibrosis transmembrane conductance regulator (CFTR), ClC2, short form of ClC3 (sClC3), chloride intracellular channel 1 (CLIC1), chloride intracellular channel 5 (CLIC5), epithelial sodium channel (ENaC), large-conductance calcium-activated potassium channel (Maxi-K), transient receptor potential vanilloid 4 (TRPV4), and voltage-dependent anion channel (VDAC), with particular attention to AQP2. In regard to AQP2, most reciprocal interactions between actin and AQP2 occur during intracellular trafficking, which are largely mediated through indirect binding. Actin and the actin cytoskeleton work as cables, barriers, stabilizers, and force generators for motility. However, as with ENaC, the effects of actin cytoskeleton on channel gating should be investigated further. This article is part of a Special Issue entitled: Reciprocal influences between cell cytoskeleton and membrane channels, receptors and transporters. Guest Editor: Jean Claude Hervé. PMID:23770358

  1. Molecular dynamics simulations reveal highly permeable oxygen exit channels shared with water uptake channels in photosystem II.

    PubMed

    Vassiliev, Serguei; Zaraiskaya, Tatiana; Bruce, Doug

    2013-10-01

    Photosystem II (PSII) catalyzes the oxidation of water in the conversion of light energy into chemical energy in photosynthesis. Water delivery and oxygen removal from the oxygen evolving complex (OEC), buried deep within PSII, are critical requirements to facilitate the reaction and minimize reactive oxygen damage. It has often been assumed that water and oxygen travel through separate channels within PSII, as demonstrated in cytochrome c oxidase. This study describes all-atom molecular dynamics simulations of PSII designed to investigate channels by fully characterizing the distribution and permeation of both water and oxygen. Interestingly, most channels found in PSII were permeable to both oxygen and water, however individual channels exhibited different energetic barriers for the two solutes. Several routes for oxygen diffusion within PSII with low energy permeation barriers were found, ensuring its fast removal from the OEC. In contrast, all routes for water showed significant energy barriers, corresponding to a much slower permeation rate for water through PSII. Two major factors were responsible for this selectivity: (1) hydrogen bonds between water and channel amino acids, and (2) steric restraints. Our results reveal the presence of a shared network of channels in PSII optimized to both facilitate the quick removal of oxygen and effectively restrict the water supply to the OEC to help stabilize and protect it from small water soluble inhibitors. PMID:23816955

  2. Brain localization and behavioral impact of the G-protein-gated K+ channel subunit GIRK4.

    PubMed

    Wickman, K; Karschin, C; Karschin, A; Picciotto, M R; Clapham, D E

    2000-08-01

    Neuronal G-protein-gated potassium (K(G)) channels are activated by several neurotransmitters and constitute an important mode of synaptic inhibition in the mammalian nervous system. K(G) channels are composed of combinations of four subunits termed G protein-gated inwardly rectifying K(+) channels (GIRK). All four GIRK subunits are expressed in the brain, and there is a general consensus concerning the expression patterns of GIRK1, GIRK2, and GIRK3. The localization pattern of GIRK4, however, remains controversial. In this study, we exploit the negative background of mice lacking a functional GIRK4 gene to identify neuronal populations that contain GIRK4 mRNA. GIRK4 mRNA was detected in only a few regions of the mouse brain, including the deep cortical pyramidal neurons, the endopiriform nucleus and claustrum of the insular cortex, the globus pallidus, the ventromedial hypothalamic nucleus, parafascicular and paraventricular thalamic nuclei, and a few brainstem nuclei (e.g., the inferior olive and vestibular nuclei). Mice lacking GIRK4 were viable and appeared normal and did not display gross deficiencies in locomotor activity, visual tasks, and pain perception. Furthermore, GIRK4-deficient mice performed similarly to wild-type controls in the passive avoidance paradigm, a test of aversive learning. GIRK4 knock-out mice did, however, exhibit impaired performance in the Morris water maze, a test of spatial learning and memory. PMID:10908597

  3. Alcohol modulation of G-protein-gated inwardly rectifying potassium channels: from binding to therapeutics

    PubMed Central

    Bodhinathan, Karthik; Slesinger, Paul A.

    2014-01-01

    Alcohol (ethanol)-induced behaviors may arise from direct interaction of alcohol with discrete protein cavities within brain proteins. Recent structural and biochemical studies have provided new insights into the mechanism of alcohol-dependent activation of G protein-gated inwardly rectifying potassium (GIRK) channels, which regulate neuronal responses in the brain reward circuit. GIRK channels contain an alcohol binding pocket formed at the interface of two adjacent channel subunits. Here, we discuss the physiochemical properties of the alcohol pocket and the roles of G protein ?? subunits and membrane phospholipid PIP2 in regulating the alcohol response of GIRK channels. Some of the features of alcohol modulation of GIRK channels may be common to other alcohol-sensitive brain proteins. We discuss the possibility of alcohol-selective therapeutics that block alcohol access to the pocket. Understanding alcohol recognition and modulation of brain proteins is essential for development of therapeutics for alcohol abuse and addiction. PMID:24611054

  4. Kinetics of gravity-driven water channels under steady rainfall

    NASA Astrophysics Data System (ADS)

    Cejas, Cesare M.; Wei, Yuli; Barrois, Remi; Frétigny, Christian; Durian, Douglas J.; Dreyfus, Rémi

    2014-10-01

    We investigate the formation of fingered flow in dry granular media under simulated rainfall using a quasi-two-dimensional experimental setup composed of a random close packing of monodisperse glass beads. Using controlled experiments, we analyze the finger instabilities that develop from the wetting front as a function of fundamental granular (particle size) and fluid properties (rainfall, viscosity). These finger instabilities act as precursors for water channels, which serve as outlets for water drainage. We look into the characteristics of the homogeneous wetting front and channel size as well as estimate relevant time scales involved in the instability formation and the velocity of the channel fingertip. We compare our experimental results with that of the well-known prediction developed by Parlange and Hill [D. E. Hill and J. Y. Parlange, Soil Sci. Soc. Am. Proc. 36, 697 (1972), 10.2136/sssaj1972.03615995003600050010x]. This model is based on linear stability analysis of the growth of perturbations arising at the interface between two immiscible fluids. Results show that, in terms of morphology, experiments agree with the proposed model. However, in terms of kinetics we nevertheless account for another term that describes the homogenization of the wetting front. This result shows that the manner we introduce the fluid to a porous medium can also influence the formation of finger instabilities. The results also help us to calculate the ideal flow rate needed for homogeneous distribution of water in the soil and minimization of runoff, given the grain size, fluid density, and fluid viscosity. This could have applications in optimizing use of irrigation water.

  5. Kinetics of gravity-driven water channels under steady rainfall.

    PubMed

    Cejas, Cesare M; Wei, Yuli; Barrois, Remi; Frétigny, Christian; Durian, Douglas J; Dreyfus, Rémi

    2014-10-01

    We investigate the formation of fingered flow in dry granular media under simulated rainfall using a quasi-two-dimensional experimental setup composed of a random close packing of monodisperse glass beads. Using controlled experiments, we analyze the finger instabilities that develop from the wetting front as a function of fundamental granular (particle size) and fluid properties (rainfall, viscosity). These finger instabilities act as precursors for water channels, which serve as outlets for water drainage. We look into the characteristics of the homogeneous wetting front and channel size as well as estimate relevant time scales involved in the instability formation and the velocity of the channel fingertip. We compare our experimental results with that of the well-known prediction developed by Parlange and Hill [D. E. Hill and J. Y. Parlange, Soil Sci. Soc. Am. Proc. 36, 697 (1972)]. This model is based on linear stability analysis of the growth of perturbations arising at the interface between two immiscible fluids. Results show that, in terms of morphology, experiments agree with the proposed model. However, in terms of kinetics we nevertheless account for another term that describes the homogenization of the wetting front. This result shows that the manner we introduce the fluid to a porous medium can also influence the formation of finger instabilities. The results also help us to calculate the ideal flow rate needed for homogeneous distribution of water in the soil and minimization of runoff, given the grain size, fluid density, and fluid viscosity. This could have applications in optimizing use of irrigation water. PMID:25375487

  6. Protein expression of G-protein inwardly rectifying potassium channels (GIRK) in breast cancer cells

    Microsoft Academic Search

    Madhu S Dhar; Howard K Plummer III

    2006-01-01

    BACKGROUND: Previous data from our laboratory has indicated that a functional link exists between the G-protein-coupled inwardly rectifying potassium (GIRK) channel and the beta-adrenergic receptor pathway in breast cancer cell lines, and these pathways were involved in growth regulation of these cells. Alcohol is an established risk factor for breast cancer and has been found to open GIRK. In order

  7. Regulation of the ABA-sensitive Arabidopsis potassium channel gene GORK in response to water stress.

    PubMed

    Becker, D; Hoth, S; Ache, P; Wenkel, S; Roelfsema, M R G; Meyerhoff, O; Hartung, W; Hedrich, R

    2003-11-01

    The phytohormone abscisic acid (ABA) regulates many stress-related processes in plants. In this context ABA mediates the responsiveness of plants to environmental stresses such as drought, cold or salt. In response to water stress, ABA induces stomatal closure by activating Ca2+, K+ and anion channels in guard cells. To understand the signalling pathways that regulate these turgor control elements, we studied the transcriptional control of the K+ release channel gene GORK that is expressed in guard cells, roots and vascular tissue. GORK transcription was up-regulated upon onset of drought, salt stress and cold. The wilting hormone ABA that integrates responses to these stimuli induced GORK expression in seedlings in a time- and concentration-dependent manner and this induction was dependent on extracellular Ca2+. ABA-responsive expression of GORK was impaired in the ABA-insensitive mutants abi1-1 and abi2-1, indicating that these protein phosphatases are regulators of GORK expression. Application of ABA to suspension-cultured cells for 2 min followed by a 4 h chase was sufficient to manifest transcriptional activation of the K+ channel gene. As predicted for a process involved in drought adaptation, only 12-24 h after the release of the stress hormone, GORK mRNA slowly decreased. In contrast to other tissues, GORK expression as well as K+(out) channel activity in guard cells is ABA insensitive, allowing the plant to adjust stomatal movement and water status control separately. PMID:14596925

  8. Nitric oxide modulates cardiac Na(+) channel via protein kinase A and protein kinase G.

    PubMed

    Ahmmed, G U; Xu, Y; Hong Dong, P; Zhang, Z; Eiserich, J; Chiamvimonvat, N

    2001-11-23

    We directly tested the effects of nitric oxide (NO) on Na(+) channels in guinea pig and mouse ventricular myocytes using patch-clamp recordings. We have previously shown that NO donors have no observed effects on expressed Na(+) channels. In contrast, NO (half-blocking concentration of 523 nmol/L) significantly reduces peak whole-cell Na(+) current (I(Na)) in isolated ventricular myocytes. The inhibitory effect of NO on I(Na) was not associated with changes in activation, inactivation, or reactivation kinetics. At the single-channel level, the reduction in macroscopic current was mediated by a decrease in open probability and/or a decrease in the number of functional channels with no change in single-channel conductance. Application of cell permeable analogs of cGMP or cAMP mimics the inhibitory effects of NO. Furthermore, the effects of NO on I(Na) can only be blocked by inhibition of both cGMP and cAMP pathways. Sulfhydryl-reducing agent does not reverse the effect of NO. In summary, although NO exerts its action via the known guanylyl cyclase (GC)/cGMP pathway, our findings provide evidence that NO can mediate its function via a GC/cGMP-independent mechanism involving the activation of adynylyl cyclase (AC) and cAMP-dependent protein kinase. PMID:11717157

  9. The effect of water on protein dynamics.

    PubMed Central

    Zaccai, G

    2004-01-01

    Neutron diffraction and spectroscopy were applied to describe the hydration and dynamics of a soluble protein and a natural membrane from extreme halophilic Archaea. The quantitative dependence of protein motions on water activity was clearly illustrated, and it was established that a minimum hydration shell is required for the systems to access their functional resilience, i.e. a dynamics state that allows biological activity. PMID:15306381

  10. Photosynthetic water oxidation: The protein framework

    Microsoft Academic Search

    Wim F. J. Vermaas; Stenbjörn Styring; Wolfgang P. Schröder; Bertil Andersson

    1993-01-01

    Approximately 20 protein subunits are associated with the PS II complex, not counting subunits of peripheral light-harvesting antenna complexes. However, it is not yet established which proteins specifically are involved in the water-oxidation process. Much evidence supports the concept that the D1\\/D2 reaction center heterodimer not only plays a central role in the primary photochemistry of Photosystem II, but also

  11. Selectivity and conductance among the glycerol and water conducting aquaporin family of channels

    E-print Network

    Minireview Selectivity and conductance among the glycerol and water conducting aquaporin family water plus glycerol conducting channel (GlpF), and now of aquaporin Z (AqpZ) from the same species in the glycerol plus water channel paradigm, GlpF. Water molecules form a single hydrogen bonded ¢le throughout

  12. Ordinary High Water Mark in ephemeral and intermittent channels in the arid southwestern United States

    Microsoft Academic Search

    K. E. Curtis; R. Lichvar; L. Dixon

    2010-01-01

    The Ordinary High Water Mark (OHWM) is important for regulating stream channels, as it defines the lateral extent of non-wetland waters, as defined under Waters of the United States in the Clean Water Act (33 CFR Part 328.3). Prior to this study, little was known about the OHWM and geomorphically effective events in arid southwest ephemeral and intermittent channels. The

  13. Thermodynamic stability of water molecules in the bacteriorhodopsin proton channel: a molecular dynamics free energy perturbation study.

    PubMed Central

    Roux, B; Nina, M; Pomès, R; Smith, J C

    1996-01-01

    The proton transfer activity of the light-driven proton pump, bacteriorhodopsin (bR) in the photochemical cycle might imply internal water molecules. The free energy of inserting water molecules in specific sites along the bR transmembrane channel has been calculated using molecular dynamics simulations based on a microscopic model. The existence of internal hydration is related to the free energy change on transfer of a water molecule from bulk solvent into a specific binding site. Thermodynamic integration and perturbation methods were used to calculate free energies of hydration for each hydrated model from molecular dynamics simulations of the creation of water molecules into specific protein-binding sites. A rigorous statistical mechanical formulation allowing the calculation of the free energy of transfer of water molecules from the bulk to a protein cavity is used to estimate the probabilities of occupancy in the putative bR proton channel. The channel contains a region lined primarily by nonpolar side-chains. Nevertheless, the results indicate that the transfer of four water molecules from bulk water to this apparently hydrophobic region is thermodynamically permitted. The column forms a continuous hydrogen-bonded chain over 12 A between a proton donor, Asp 96, and the retinal Schiff base acceptor. The presence of two water molecules in direct hydrogen-bonding association with the Schiff base is found to be strongly favorable thermodynamically. The implications of these results for the mechanism of proton transfer in bR are discussed. PMID:8842206

  14. Highly selective water channel activity measured by voltage clamp: Analysis of planar lipid bilayers reconstituted with purified AqpZ

    PubMed Central

    Pohl, Peter; Saparov, Sapar M.; Borgnia, Mario J.; Agre, Peter

    2001-01-01

    Aquaporins are membrane channels selectively permeated by water or water plus glycerol. Conflicting reports have described ion conductance associated with some water channels, raising the question of whether ion conductance is a general property of the aquaporin family. To clarify this question, a defined system was developed to simultaneously measure water permeability and ion conductance. The Escherichia coli water channel aquaporin-Z (AqpZ) was studied, because it is a highly stable tetramer. Planar lipid bilayers were formed from unilamellar vesicles containing purified AqpZ. The hydraulic conductivity of bilayers made from the total extract of E. coli lipids increased 3-fold if reconstituted with AqpZ, but electric conductance was unchanged. No channel activity was detected under voltage-clamp conditions, indicating that less than one in 109 transport events is electrogenic. Microelectrode measurements were simultaneously undertaken adjacent to the membrane. Changes in sodium concentration profiles accompanying transmembrane water flow permitted calculation of the activation energies: 14 kcal/mol for protein-free lipid bilayers and 4 kcal/mol for lipid bilayers containing AqpZ. Neither the water permeability nor the electric conductivity exhibited voltage dependence. This sensitive system demonstrated that AqpZ is permeated by water but not charged ions and should permit direct analyses of putative electrogenic properties of other aquaporins. PMID:11493683

  15. Highly selective water channel activity measured by voltage clamp: analysis of planar lipid bilayers reconstituted with purified AqpZ.

    PubMed

    Pohl, P; Saparov, S M; Borgnia, M J; Agre, P

    2001-08-14

    Aquaporins are membrane channels selectively permeated by water or water plus glycerol. Conflicting reports have described ion conductance associated with some water channels, raising the question of whether ion conductance is a general property of the aquaporin family. To clarify this question, a defined system was developed to simultaneously measure water permeability and ion conductance. The Escherichia coli water channel aquaporin-Z (AqpZ) was studied, because it is a highly stable tetramer. Planar lipid bilayers were formed from unilamellar vesicles containing purified AqpZ. The hydraulic conductivity of bilayers made from the total extract of E. coli lipids increased 3-fold if reconstituted with AqpZ, but electric conductance was unchanged. No channel activity was detected under voltage-clamp conditions, indicating that less than one in 10(9) transport events is electrogenic. Microelectrode measurements were simultaneously undertaken adjacent to the membrane. Changes in sodium concentration profiles accompanying transmembrane water flow permitted calculation of the activation energies: 14 kcal/mol for protein-free lipid bilayers and 4 kcal/mol for lipid bilayers containing AqpZ. Neither the water permeability nor the electric conductivity exhibited voltage dependence. This sensitive system demonstrated that AqpZ is permeated by water but not charged ions and should permit direct analyses of putative electrogenic properties of other aquaporins. PMID:11493683

  16. Identification of characteristic protein folding channels in a coarse-grained hydrophobic-polar peptide model

    E-print Network

    Bachmann, Michael

    Identification of characteristic protein folding channels in a coarse-grained hydrophobic of protein folding is one of the major challenges of modern interdisciplinary science. Proteins are linear simulations of protein folding are difficult, mainly for two reasons. Firstly, the folding process is so slow

  17. Suppression of aquaporin, a mediator of water channel control in the carcinogenic liver fluke, Opisthorchis viverrini

    PubMed Central

    2014-01-01

    Background Opisthorchiasis and Opisthorchis viverrini-associated bile duct cancer represent major public health threats in Thailand and Laos. The tegument of this food borne fluke plays pivotal roles in parasite metabolism, homeostasis and osmoregulation. Excretory/secretory products also pass from the fluke to the biliary environment, products that likely underlie pathogenesis of liver fluke infection. Aquaporins (AQPs), belong to the major intrinsic protein superfamily of integral plasma membrane channel proteins that selectively transport water across cell membranes. AQPs play key roles as water and ion transport channels through the tegument of helminth parasites. Methods Here, two forms of AQP mRNAs from the adult developmental stage of O. viverrini, termed O. viverrini aquaporin-1 and -2 (Ov-aqp-1 and -2) were investigated. Roles of Ov-aqp-1 and -2 in the movement of water across the tegument of this carcinogenic liver fluke were investigated using RNA interference. Results Ov-AQP-1 and Ov-AQP-2 contain unique characteristic asparagine-proline-alanine (NPA) motifs of AQP transmembrane proteins. Phylogenetic analysis indicated that Ov-AQPs belong to an expanding group of aquaglyceroporin-like water channel proteins characterized from helminth and protozoan parasites, which is pivotal to the specialized requirements of water and solute control during parasitism. Elevated transcription of Ov-aqp-1 was evident in the egg, cercaria, metacercaria and adult stages of O. viverrini, whereas Ov-aqp-2 transcripts were detected at higher level in egg, metacercaria, cercaria and adult stage, respectively. RNA interference using electroporated dsRNA suppressed transcript levels of Ov-aqp-1 and Ov-aqp-2 in adult worms by 58-99% over periods of up to 16 days in vitro. Suppression of Ov-aqp-1 and Ov-aqp-2 in vitro disabled water transport in adult flukes. Conclusion The apparently pivotal roles of Ov-AQP in solute homeostasis at the fluke surface suggest that deeper investigation will be informative for the pathophysiology of O. viverrini, and may uncover intervention targets, particularly in view of the singularly notable predilection of this pathogen for residence within ducts of the biliary tree. PMID:24885060

  18. Morphology of Rain Water Channeling in Systematically Varied Model Sandy Soils

    NASA Astrophysics Data System (ADS)

    Wei, Yuli; Cejas, Cesare M.; Barrois, Rémi; Dreyfus, Rémi; Durian, Douglas J.

    2014-10-01

    We visualize the formation of fingered flow in dry model sandy soils under different rain conditions using a quasi-2D experimental setup and systematically determine the impact of the soil grain diameter and surface wetting properties on the water channeling phenomenon. The model sandy soils we use are random closely packed glass beads with varied diameters and surface treatments. For hydrophilic sandy soils, our experiments show that rain water infiltrates a shallow top layer of soil and creates a horizontal water wetting front that grows downward homogeneously until instabilities occur to form fingered flows. For hydrophobic sandy soils, in contrast, we observe that rain water ponds on the top of the soil surface until the hydraulic pressure is strong enough to overcome the capillary repellency of soil and create narrow water channels that penetrate the soil packing. Varying the raindrop impinging speed has little influence on water channel formation. However, varying the rain rate causes significant changes in the water infiltration depth, water channel width, and water channel separation. At a fixed rain condition, we combine the effects of the grain diameter and surface hydrophobicity into a single parameter and determine its influence on the water infiltration depth, water channel width, and water channel separation. We also demonstrate the efficiency of several soil water improvement methods that relate to the rain water channeling phenomenon, including prewetting sandy soils at different levels before rainfall, modifying soil surface flatness, and applying superabsorbent hydrogel particles as soil modifiers.

  19. Morphology of rain water channelization in systematically varied model sandy soils

    E-print Network

    Y. Wei; C. M. Cejas; R. Barrois; R. Dreyfus; D. J. Durian

    2014-03-13

    We visualize the formation of fingered flow in dry model sandy soils under different raining conditions using a quasi-2d experimental set-up, and systematically determine the impact of soil grain diameter and surface wetting property on water channelization phenomenon. The model sandy soils we use are random closely-packed glass beads with varied diameters and surface treatments. For hydrophilic sandy soils, our experiments show that rain water infiltrates into a shallow top layer of soil and creates a horizontal water wetting front that grows downward homogeneously until instabilities occur to form fingered flows. For hydrophobic sandy soils, in contrast, we observe that rain water ponds on the top of soil surface until the hydraulic pressure is strong enough to overcome the capillary repellency of soil and create narrow water channels that penetrate the soil packing. Varying the raindrop impinging speed has little influence on water channel formation. However, varying the rain rate causes significant changes in water infiltration depth, water channel width, and water channel separation. At a fixed raining condition, we combine the effects of grain diameter and surface hydrophobicity into a single parameter and determine its influence on water infiltration depth, water channel width, and water channel separation. We also demonstrate the efficiency of several soil water improvement methods that relate to rain water channelization phenomenon, including pre-wetting sandy soils at different level before rainfall, modifying soil surface flatness, and applying superabsorbent hydrogel particles as soil modifiers.

  20. Effect of Channel Materials on the Behavior of Water Droplet Emerging From GDL into PEMFC Gas Channels

    E-print Network

    Kandlikar, Satish

    Effect of Channel Materials on the Behavior of Water Droplet Emerging From GDL into PEMFC Gas (PEMFCs) significantly affects the gas diffusion and the overall performance. Hence, understanding (stainless steel, copper, graphite composite, polycarbonate) that are commercially used in PEMFC

  1. Structural Basis for Allosteric Coupling at the Membrane-Protein Interface in Gloeobacter violaceus Ligand-gated Ion Channel (GLIC)*

    PubMed Central

    Velisetty, Phanindra; Chalamalasetti, Sreevatsa V.; Chakrapani, Sudha

    2014-01-01

    Ligand binding at the extracellular domain of pentameric ligand-gated ion channels initiates a relay of conformational changes that culminates at the gate within the transmembrane domain. The interface between the two domains is a key structural entity that governs gating. Molecular events in signal transduction at the interface are poorly defined because of its intrinsically dynamic nature combined with functional modulation by membrane lipid and water vestibules. Here we used electron paramagnetic resonance spectroscopy to delineate protein motions underlying Gloeobacter violaceus ligand-gated ion channel gating in a membrane environment and report the interface conformation in the closed and the desensitized states. Extensive intrasubunit interactions were observed in the closed state that are weakened upon desensitization and replaced by newer intersubunit contacts. Gating involves major rearrangements of the interfacial loops, accompanied by reorganization of the protein-lipid-water interface. These structural changes may serve as targets for modulation of gating by lipids, alcohols, and amphipathic drug molecules. PMID:24338475

  2. Water uptake profile in a model ion-exchange membrane: Conditions for water-rich channels

    NASA Astrophysics Data System (ADS)

    Herbst, Daniel C.; Witten, Thomas A.; Tsai, Tsung-Han; Coughlin, E. Bryan; Maes, Ashley M.; Herring, Andrew M.

    2015-03-01

    Ionic conductivity in a polymeric fuel cell requires water uptake. Previous theoretical studies of water uptake used idealized parameters. We report a parameter-free prediction of the water-swelling behavior of a model fuel cell membrane. The model polymers, poly(methyl-butylene)-block-poly(vinylbenzyl-trimethylamine), form lamellar domains that absorb water in humid air. We use the Scheutjens-Fleer methodology to predict the resulting change in lamellar structure and compare with x-ray scattering. The results suggest locally uniform water distributions. However, under conditions where a PVBTMA and water mixture phase-separate, the two phases arrange into stripes with a dilute stripe sandwiched between two concentrated stripes. A small amount of water enhances conductivity most when it is partitioned into such channels, improving fuel-cell performance.

  3. Water uptake profile in a model ion-exchange membrane: Conditions for water-rich channels.

    PubMed

    Herbst, Daniel C; Witten, Thomas A; Tsai, Tsung-Han; Bryan Coughlin, E; Maes, Ashley M; Herring, Andrew M

    2015-03-21

    Ionic conductivity in a polymeric fuel cell requires water uptake. Previous theoretical studies of water uptake used idealized parameters. We report a parameter-free prediction of the water-swelling behavior of a model fuel cell membrane. The model polymers, poly(methyl-butylene)-block-poly(vinylbenzyl-trimethylamine), form lamellar domains that absorb water in humid air. We use the Scheutjens-Fleer methodology to predict the resulting change in lamellar structure and compare with x-ray scattering. The results suggest locally uniform water distributions. However, under conditions where a PVBTMA and water mixture phase-separate, the two phases arrange into stripes with a dilute stripe sandwiched between two concentrated stripes. A small amount of water enhances conductivity most when it is partitioned into such channels, improving fuel-cell performance. PMID:25796265

  4. Role of aquaporin water channels in eye function

    Microsoft Academic Search

    A. S. Verkman

    2003-01-01

    The aquaporins (AQPs) are a family of more than 10 homologous water transporting proteins expressed in many mammalian epithelia and endothelia. At least five AQPs are expressed in the eye: AQP0 (MIP) in lens fiber, AQP1 in cornea endothelium, ciliary and lens epithelia and trabecular meshwork, AQP3 in conjunctiva, AQP4 in ciliary epithelium and retinal Müller cells, and AQP5 in

  5. Long distance interactions within the potassium channel pore are revealed by molecular diversity of viral proteins.

    PubMed

    Gazzarrini, Sabrina; Kang, Ming; Van Etten, James L; Tayefeh, Sascha; Kast, Stefan M; DiFrancesco, Dario; Thiel, Gerhard; Moroni, Anna

    2004-07-01

    Kcv is a 94-amino acid protein encoded by chlorella virus PBCV-1 that corresponds to the pore module of K(+) channels. Therefore, Kcv can be a model for studying the protein design of K(+) channel pores. We analyzed the molecular diversity generated by approximately 1 billion years of evolution on kcv genes isolated from 40 additional chlorella viruses. Because the channel is apparently required for virus replication, the Kcv variants are all functional and contain multiple and dispersed substitutions that represent a repertoire of allowed sets of amino acid substitutions (from 4 to 12 amino acids). Correlations between amino acid substitutions and the new properties displayed by these channels guided site-directed mutations that revealed synergistic amino acid interactions within the protein as well as previously unknown interactions between distant channel domains. The effects of these multiple changes were not predictable from a priori structural knowledge of the channel pore. PMID:15105432

  6. Amitriptyline inhibits the G protein and K+ channel in the cloned thyroid cell line.

    PubMed

    Yoshida, A; Hisatome, I; Nawada, T; Sasaki, N; Taniguchi, S; Tanaka, Y; Manabe, I; Ahmmed, G U; Sato, R; Mori, A; Hattori, K; Ueta, Y; Mitani, Y; Watanabe, M; Igawa, O; Fujimoto, Y; Shigemasa, C

    1996-09-19

    We have reported that thyroid K+ channel is activated by extracellular application of the thyroid-stimulating hormone (TSH) using single channel recording method performed on cloned normal rat thyroid cell (FRTL-5) membrane. Treatment of dibutyryladenosine cyclic monophosphate (Bt2 cAMP) also activated the TSH-dependent K+ channel. These findings indicate that the thyroid K+ channel is activated through the TSH-adenosine cyclic monophosphate (cAMP)-protein kinase A system. We examined the effects of amitriptyline on TSH-guanosine triphosphate binding protein (G protein)-adenylate cyclase-cAMP-K+ channel system in the cloned normal rat thyroid cell line FRTL-5. Amitriptyline inhibited the cAMP production induced by TSH. Amitriptyline also inhibited the cAMP production induced by cholera toxin, indicating that amitriptyline inhibited the thyroid G protein. Amitriptyline had no effect on TSH-receptor binding and cAMP production by forskolin (adenylate cyclase stimulator). Amitriptyline inhibited the K+ channel activation by cAMP, indicating that the suppressing mechanism is not the inhibition of TSH receptor or G protein but the direct suppression of K+ channel. It was concluded that amitriptyline inhibited the thyroid G protein and K+ channel. PMID:8891586

  7. Protein trafficking and anchoring complexes revealed by proteomic analysis of inward rectifier potassium channel (Kir2.x)-associated proteins.

    PubMed

    Leonoudakis, Dmitri; Conti, Lisa R; Anderson, Scott; Radeke, Carolyn M; McGuire, Leah M M; Adams, Marvin E; Froehner, Stanley C; Yates, John R; Vandenberg, Carol A

    2004-05-21

    Inward rectifier potassium (Kir) channels play important roles in the maintenance and control of cell excitability. Both intracellular trafficking and modulation of Kir channel activity are regulated by protein-protein interactions. We adopted a proteomics approach to identify proteins associated with Kir2 channels via the channel C-terminal PDZ binding motif. Detergent-solubilized rat brain and heart extracts were subjected to affinity chromatography using a Kir2.2 C-terminal matrix to purify channel-interacting proteins. Proteins were identified with multidimensional high pressure liquid chromatography coupled with electrospray ionization tandem mass spectrometry, N-terminal microsequencing, and immunoblotting with specific antibodies. We identified eight members of the MAGUK family of proteins (SAP97, PSD-95, Chapsyn-110, SAP102, CASK, Dlg2, Dlg3, and Pals2), two isoforms of Veli (Veli-1 and Veli-3), Mint1, and actin-binding LIM protein (abLIM) as Kir2.2-associated brain proteins. From heart extract purifications, SAP97, CASK, Veli-3, and Mint1 also were found to associate with Kir2 channels. Furthermore, we demonstrate for the first time that components of the dystrophin-associated protein complex, including alpha1-, beta1-, and beta2-syntrophin, dystrophin, and dystrobrevin, interact with Kir2 channels, as demonstrated by immunoaffinity purification and affinity chromatography from skeletal and cardiac muscle and brain. Affinity pull-down experiments revealed that Kir2.1, Kir2.2, Kir2.3, and Kir4.1 all bind to scaffolding proteins but with different affinities for the dystrophin-associated protein complex and SAP97, CASK, and Veli. Immunofluorescent localization studies demonstrated that Kir2.2 co-localizes with syntrophin, dystrophin, and dystrobrevin at skeletal muscle neuromuscular junctions. These results suggest that Kir2 channels associate with protein complexes that may be important to target and traffic channels to specific subcellular locations, as well as anchor and stabilize channels in the plasma membrane. PMID:15024025

  8. Vasopressin Increases Water Permeability of Kidney Collecting Duct by Inducing Translocation of Aquaporin-CD Water Channels to Plasma Membrane

    Microsoft Academic Search

    Soren Nielsen; Chung-Lin Chou; David Marples; Erik I. Christensen; Bellamkonda K. Kishore; Mark A. Knepper

    1995-01-01

    Water excretion by the kidney is regulated by the peptide hormone vasopressin. Vasopressin increases the water permeability of the renal collecting duct cells, allowing more water to be reabsorbed from collecting duct urine to blood. Despite long-standing interest in this process, the mechanism of the water permeability increase has remained undetermined. Recently, a molecular water channel (AQP-CD) has been cloned

  9. Correlation of Apical Fluid-Regulating Channel Proteins with Lung Function in Human COPD Lungs

    PubMed Central

    Zhao, Meimi; Liu, Shan-Lu; Huang, Yao; Idell, Steven; Li, Xiumin; Ji, Hong-Long

    2014-01-01

    Links between epithelial ion channels and chronic obstructive pulmonary diseases (COPD) are emerging through animal model and in vitro studies. However, clinical correlations between fluid-regulating channel proteins and lung function in COPD remain to be elucidated. To quantitatively measure epithelial sodium channels (ENaC), cystic fibrosis transmembrane conductance regulator (CFTR), and aquaporin 5 (AQP5) proteins in human COPD lungs and to analyze the correlation with declining lung function, quantitative western blots were used. Spearman tests were performed to identify correlations between channel proteins and lung function. The expression of ? and ? ENaC subunits was augmented and inversely associated with lung function. In contrast, both total and alveolar type I (ATI) and II (ATII)-specific CFTR proteins were reduced. The expression level of CFTR proteins was associated with FEV1 positively. Abundance of AQP5 proteins and extracellular superoxide dismutase (SOD3) was decreased and correlated with spirometry test results and gas exchange positively. Furthermore, these channel proteins were significantly associated with severity of disease. Our study demonstrates that expression of ENaC, AQP5, and CFTR proteins in human COPD lungs is quantitatively associated with lung function and severity of COPD. These apically located fluid-regulating channels may thereby serve as biomarkers and potent druggable targets of COPD. PMID:25329998

  10. Cold-acclimation-induced protein hypertrophy in channel catfish and green sunfish

    Microsoft Academic Search

    Jeffrey Kent; Michael Koban; C. Ladd Prosser

    1988-01-01

    Summary 1.Following acclimation of channel catfish to a reduction in temperature from 25° to 15°C, there were approximately two-fold increases in liver mass, cell size, total protein, and total enzyme activity, relative to activity per milligram of protein and per gram wet weight of tissue, indicating tissue hypertrophy. There was no change in either total liver DNA content or protein

  11. Channel inactivation in G-protein regulation Pflugers Arch. Author manuscript

    E-print Network

    Boyer, Edmond

    constructs. G-protein activation wasv produced via -opioid receptor activation using the DAMGO agonist ; Rabbits ; Rats ; Receptors, Opioid, mu ; physiology ; Time Factors Author Keywords N-type calcium channel ; Cav2.2 subunit ; G-protein ; G-protein coupled receptor ; -opioid receptor ; inactivation ; beta

  12. Porin channels in Escherichia coli: studies with liposomes reconstituted from purified proteins.

    PubMed Central

    Nikaido, H; Rosenberg, E Y

    1983-01-01

    Rates of diffusion of uncharged and charged solute molecules through porin channels were determined by using liposomes reconstituted from egg phosphatidylcholine and purified Escherichia coli porins OmpF (protein 1a), OmpC (protein 1b), and PhoE (protein E). All three porin proteins appeared to produce channels of similar size, although the OmpF channel appeared to be 7 to 9% larger than the OmpC and PhoE channels in an equivalent radius. Hydrophobicity of the solute retarded the penetration through all three channels in a similar manner. The presence of one negative charge on the solute resulted in about a threefold reduction in penetration rates through OmpF and OmpC channels, whereas it produced two- to tenfold acceleration of diffusion through the PhoE channel. The addition of the second negatively charged group to the solutes decreased the diffusion rates through OmpF and OmpC channels further, whereas diffusion through the PhoE channel was not affected much. These results suggest that PhoE specializes in the uptake of negatively charged solutes. At the present level of resolution, no sign of true solute specificity was found in OmpF and OmpC channels; peptides, for example, diffused through both of these channels at rates expected from their molecular size, hydrophobicity, and charge. However, the OmpF porin channel allowed influx of more solute molecules per unit time than did the equivalent weight of the OmpC porin when the flux was driven by a concentration gradient of the same size. This apparent difference in "efficiency" became more pronounced with larger solutes, and it is likely to be the consequence of the difference in the sizes of OmpF and OmpC channels. PMID:6294049

  13. Concentrating Toxoplasma gondii and Cyclospora cayetanensis from Surface Water and Drinking Water by Continuous Separation Channel Centrifugation

    EPA Science Inventory

    Aims: To evaluate the effectiveness of continuous separation channel centrifugation for concentrating Toxoplasma gondii and Cyclospora cayetanensis from drinking water and environmental waters. Methods and Results: Ready-to-seed vials with known quantities of Toxoplasma gondii a...

  14. Multiple Scales in the Simulation of Ion Channels and Proteins

    PubMed Central

    Eisenberg, Bob

    2010-01-01

    Computation of living processes creates great promise for the everyday life of mankind and great challenges for physical scientists. Simulations molecular dynamics have great appeal to biologists as a natural extension of structural biology. Once a biologist sees a structure, she/he wants to see it move. Molecular biology has shown that a small number of atoms, sometimes even one messenger ion, like Ca2+, can control biological function on the scale of cells, organs, tissues, and organisms. Enormously concentrated ions—at number densities of ~20 M—in protein channels and enzymes are responsible for many of the characteristics of living systems, just as highly concentrated ions near electrodes are responsible for many of the characteristics of electrochemical systems. Here we confront the reality of the scale differences of ions. We show that the scale differences needed to simulate all the atoms of biological cells are 107 in linear dimension, 1021 in three dimensions, 109 in resolution, 1011 in time, and 1013 in particle number (to deal with concentrations of Ca2+). These scales must be dealt with simultaneously if the simulation is to deal with most biological functions. Biological function extends across all of them, all at once in most cases. We suggest a computational approach using explicit multiscale analysis instead of implicit simulation of all scales. The approach is based on an energy variational principle EnVarA introduced by Chun Liu to deal with complex fluids. Variational methods deal automatically with multiple interacting components and scales. When an additional component is added to the system, the resulting Euler Lagrange field equations change form automatically—by algebra alone—without additional unknown parameters. Multifaceted interactions are solutions of the resulting equations. We suggest that ionic solutions should be viewed as complex fluids with simple components. Highly concentrated solutions—dominated by interactions of components—are easily computed by EnVarA. Successful computation of ions concentrated in special places may be a significant step to understanding the defining characteristics of biological and electrochemical systems. Indeed, computing ions near proteins and nucleic acids may prove as important to molecular biology and chemical technology as computing holes and electrons has been to our semiconductor and digital technology. PMID:21135913

  15. Dramatic nano-fluidic properties of carbon nanotube membranes as a platform for protein channel mimetics

    NASA Astrophysics Data System (ADS)

    Hinds, Bruce

    2013-03-01

    Carbon nanotubes have three key attributes that make them of great interest for novel membrane applications: 1) atomically flat graphite surface allows for ideal fluid slip boundary conditions and extremely fast flow rates 2) the cutting process to open CNTs inherently places functional chemistry at CNT core entrance for chemical selectivity and 3) CNT are electrically conductive allowing for electrochemical reactions and application of electric fields gradients at CNT tips. Pressure driven flux of a variety of solvents (H2O, hexane, decane ethanol, methanol) are 4-5 orders of magnitude higher than conventional Newtonian flow [Nature 2005, 438, 44] due to atomically flat graphite planes inducing nearly ideal slip conditions. However this is eliminated with selective chemical functionalization [ACS Nano 2011 5(5) 3867-3877] needed to give chemical selectivity. These unique properties allow us to explore the hypothesis of producing ``Gatekeeper'' membranes that mimic natural protein channels to actively pump through rapid nm-scale channels. With anionic tip functionality strong electroosmotic flow is induced by unimpeded cation flow with similar 10,000 fold enhancements [Nature Nano 2012 7(2) 133-39]. With enhanced power efficiency, carbon nanotube membranes were employed as the active element of a switchable transdermal drug delivery device that can facilitate more effective treatments of drug abuse and addiction. Recently methods to deposit Pt monolayers on CNT surface have been developed making for highly efficient catalytic platforms. Discussed are other applications of CNT protein channel mimetics, for large area robust engineering platforms, including water purification, flow battery energy storage, and biochemical/biomass separations.

  16. Characterization of two putative mechanosensitive channel proteins of Campylobacter jejuni involved in protection against osmotic downshock.

    PubMed

    Kakuda, Tsutomu; Koide, Yuuki; Sakamoto, Aya; Takai, Shinji

    2012-11-01

    Acute hypotonic stress becomes a threat to the survival of bacteria in the environment. Mechanosensitive channels play an essential role in the maintenance of bacterial cell integrity during hypoosmotic shock. A database search suggested that Campylobacter jejuni, a major worldwide cause of bacterial gastroenteritis in humans, possesses two putative mechanosensitive channels, designated Cjj0263 and Cjj1025, in C. jejuni strain 81-176. Osmotic downshock experiments demonstrated that a mutant lacking Cjj0263 showed a severe defect in survival of hypoosmotic shock, while a mutant lacking Cjj1025 exhibited the same survival capacity as the wild type. We further examined the colonization ability of each mutant using the one-day old chick model. Cjj0263 or Cjj1025 mutants were able to colonize chick ceca at the same level as the wild type, but a Cjj0263 Cjj1025 double mutant revealed significantly reduced ability to colonize chick ceca. To examine whether C. jejuni that have grown in the digestive tract of chicks are protected against acute hypotonic stress, bacteria in ceca were directly exposed to water. The wild type was able to survive acute osmotic downshift, but the Cjj0263 mutant suffered a substantial loss of viability when subjected to a rapid osmotic downshock. Immunoblot analysis suggested that both Cjj0263 and Cjj1025 were glycosylated via the N-linked protein glycosylation pathway, but glycan modification of these proteins was unlikely to have a major effect on their function and stability. Our data suggest that Cjj0263, a mechanosensitive channel, has a pivotal role in protection against hypoosmotic stress experienced during environmental transmission. PMID:22608101

  17. Remodeling of channel-forming ORAI proteins determines an oncogenic switch in prostate cancer.

    PubMed

    Dubois, Charlotte; Vanden Abeele, Fabien; Lehen'kyi, V'yacheslav; Gkika, Dimitra; Guarmit, Basma; Lepage, Gilbert; Slomianny, Christian; Borowiec, Anne Sophie; Bidaux, Gabriel; Benahmed, Mohamed; Shuba, Yaroslav; Prevarskaya, Natalia

    2014-07-14

    ORAI family channels have emerged as important players in malignant transformation, yet the way in which they reprogram cancer cells remains elusive. Here we show that the relative expression levels of ORAI proteins in prostate cancer are different from that in noncancerous tissue. By mimicking ORAI protein remodeling observed in primary tumors, we demonstrate in in vitro models that enhanced ORAI3 expression favors heteromerization with ORAI1 to form a novel channel. These channels support store-independent Ca(2+) entry, thereby promoting cell proliferation and a smaller number of functional homomeric ORAI1-based store-operated channels, which are important in supporting susceptibility to apoptosis. Thus, our findings highlight disrupted dynamic equilibrium of channel-forming proteins as an oncogenic mechanism. PMID:24954132

  18. Control of Neuronal Voltage-Gated Calcium Ion Channels From RNA to Protein

    PubMed Central

    Lipscombe, Diane; Allen, Summer E; Toro, Cecilia P.

    2013-01-01

    Voltage-gated calcium (CaV) ion channels convert neuronal activity into rapid intracellular calcium signals to trigger a myriad of cellular responses. Their involvement in major neurological and psychiatric diseases, and importance as therapeutic targets, has propelled interest in subcellular-specific mechanisms that align CaV channel activity to specific tasks. Here we highlight recent studies that delineate mechanisms controlling the expression of CaV channels at the level of RNA and protein. We discuss the roles of RNA editing and alternative pre-mRNA splicing in generating CaV channel isoforms with activities specific to the demands of individual cells; the roles of ubiquitination and accessory proteins in regulating CaV channel expression; and the specific binding partners which contribute to both pre- and post- synaptic CaV channel function. PMID:23907011

  19. Activation and inhibition of G protein-coupled inwardly rectifying potassium (Kir3) channels by G protein ?? subunits

    PubMed Central

    Lei, Qiubo; Jones, Miller B.; Talley, Edmund M.; Schrier, Andrew D.; McIntire, William E.; Garrison, James C.; Bayliss, Douglas A.

    2000-01-01

    G protein-coupled inwardly rectifying potassium (GIRK) channels can be activated or inhibited by different classes of receptors, suggesting a role for G proteins in determining signaling specificity. Because G protein ?? subunits containing either ?1 or ?2 with multiple G? subunits activate GIRK channels, we hypothesized that specificity might be imparted by ?3, ?4, or ?5 subunits. We used a transfection assay in cell lines expressing GIRK channels to examine effects of dimers containing these G? subunits. Inwardly rectifying K+ currents were increased in cells expressing ?3 or ?4, with either ?2 or ?11. Purified, recombinant ?3?2 and ?4?2 bound directly to glutathione-S-transferase fusion proteins containing N- or C-terminal cytoplasmic domains of GIRK1 and GIRK4, indicating that ?3 and ?4, like ?1, form dimers that bind to and activate GIRK channels. By contrast, ?5-containing dimers inhibited GIRK channel currents. This inhibitory effect was obtained with either ?5?2 or ?5?11, was observed with either GIRK1,4 or GIRK1,2 channels, and was evident in the context of either basal or agonist-induced currents, both of which were mediated by endogenous G?? subunits. In cotransfection assays, ?5?2 suppressed ?1?2-activated GIRK currents in a dose-dependent manner consistent with competitive inhibition. Moreover, we found that ?5?2 could bind to the same GIRK channel cytoplasmic domains as other, activating G?? subunits. Thus, ?5-containing dimers inhibit G??-stimulated GIRK channels, perhaps by directly binding to the channels. This suggests that ?5-containing dimers could act as competitive antagonists of other G?? dimers on GIRK channels. PMID:10944236

  20. Bioluminescence methodology for the detection of protein-protein interactions within the voltage-gated sodium channel macromolecular complex.

    PubMed

    Shavkunov, Alexander; Panova, Neli; Prasai, Anesh; Veselenak, Ron; Bourne, Nigel; Stoilova-McPhie, Svetla; Laezza, Fernanda

    2012-04-01

    Protein-protein interactions are critical molecular determinants of ion channel function and emerging targets for pharmacological interventions. Yet, current methodologies for the rapid detection of ion channel macromolecular complexes are still lacking. In this study we have adapted a split-luciferase complementation assay (LCA) for detecting the assembly of the voltage-gated Na+ (Nav) channel C-tail and the intracellular fibroblast growth factor 14 (FGF14), a functionally relevant component of the Nav channelosome that controls gating and targeting of Nav channels through direct interaction with the channel C-tail. In the LCA, two complementary N-terminus and C-terminus fragments of the firefly luciferase were fused, respectively, to a chimera of the CD4 transmembrane segment and the C-tail of Nav1.6 channel (CD4-Nav1.6-NLuc) or FGF14 (CLuc-FGF14). Co-expression of CLuc-FGF14 and CD4-Nav1.6-NLuc in live cells led to a robust assembly of the FGF14:Nav1.6 C-tail complex, which was attenuated by introducing single-point mutations at the predicted FGF14:Nav channel interface. To evaluate the dynamic regulation of the FGF14:Nav1.6 C-tail complex by signaling pathways, we investigated the effect of kinase inhibitors on the complex formation. Through a platform of counter screenings, we show that the p38/MAPK inhibitor, PD169316, and the I?B kinase inhibitor, BAY 11-7082, reduce the FGF14:Nav1.6 C-tail complementation, highlighting a potential role of the p38MAPK and the I?B/NF?B pathways in controlling neuronal excitability through protein-protein interactions. We envision the methodology presented here as a new valuable tool to allow functional evaluations of protein-channel complexes toward probe development and drug discovery targeting ion channels implicated in human disorders. PMID:22364545

  1. A protein kinase-phosphatase pair interacts with an ion channel to regulate ABA signaling in plant guard cells

    PubMed Central

    Lee, Sung Chul; Lan, Wenzhi; Buchanan, Bob B.; Luan, Sheng

    2009-01-01

    The plant hormone abscisic acid (ABA) serves as a physiological monitor to assess the water status of plants and, under drought conditions, induces stomatal pore closure by activating specific ion channels, such as a slow-anion channel (SLAC1) that, in turn, mediate ion efflux from the guard cells. Earlier genetic analyses uncovered a protein kinase (OST1) and several 2C-type phosphatases, as respective positive and negative regulators of ABA-induced stomatal closure. Here we show that the OST1 kinase interacts with the SLAC1 anion channel, leading to its activation via phosphorylation. PP2CA, one of the PP2C phosphatase family members acts in an opposing manner and inhibits the activity of SLAC1 by two mechanisms: (1) direct interaction with SLAC1 itself, and (2) physical interaction with OSTI leading to inhibition of the kinase independently of phosphatase activity. The results suggest that ABA signaling is mediated by a physical interaction chain consisting of several components, including a PP2C member, SnRK2-type kinase (OST1), and an ion channel, SLAC1, to regulate stomatal movements. The findings are in keeping with a paradigm in which a protein kinase-phosphatase pair interacts physically with a target protein to couple a signal with a specific response. PMID:19955427

  2. The jellyfish green fluorescent protein: A new tool for studying ion channel expression and function

    Microsoft Academic Search

    John Marshall; Raymond Molloy; Guy W. J Moss; James R Howe; Thomas E Hughes

    1995-01-01

    Two methods are described for using the jellyfish green fluorescent protein (GFP) as a reporter gene for ion channel expression. GFP fluorescence can be used to identify the transfected cells, and to estimate the relative levels of ion channel expression, in cotransfection experiments. A GFP-NMDAR1 chimera can be constructed that produces a functional, fluorescent receptor subunit. These methods should facilitate

  3. Functional Expression and Characterization of G-protein-gated Inwardly Rectifying K+ Channels Containing GIRK3

    E-print Network

    Clapham, David E.

    Functional Expression and Characterization of G-protein-gated Inwardly Rectifying K+ Channels and ei- ther the GIRK2 or GIRK4 subunits. However, the ho- mologous mouse brain GIRK3 clone failed of GIRK1/GIRK3 were characterized and compared to those of the GIRK1/ GIRK2 and GIRK1/GIRK4 channels. All

  4. Rim1 modulates direct G-protein regulation of Cav2.2 channels Norbert Weiss1,2,

    E-print Network

    Paris-Sud XI, Université de

    1 Rim1 modulates direct G-protein regulation of Cav2.2 channels Norbert Weiss1,2,§ , Alejandro contributed equally to this work Key words: Calcium channel; Cav2.2 channel; N channel; beta subunit; Rim1; G-protein regulation of Cav2.2 channels. Activation of µ-opioid receptors expressed in HEK-293 cells along with Cav2

  5. Protein-directed synthesis of Mn-doped ZnS quantum dots: a dual-channel biosensor for two proteins.

    PubMed

    Wu, Peng; Zhao, Ting; Tian, Yunfei; Wu, Lan; Hou, Xiandeng

    2013-06-01

    Proteins typically have nanoscale dimensions and multiple binding sites with inorganic ions, which facilitates the templated synthesis of nanoparticles to yield nanoparticle-protein hybrids with tailored functionality, water solubility, and tunable frameworks with well-defined structure. In this work, we report a protein-templated synthesis of Mn-doped ZnS quantum dots (QDs) by exploring bovine serum albumin (BSA) as the template. The obtained Mn-doped ZnS QDs give phosphorescence emission centered at 590?nm, with a decay time of about 1.9?ms. A dual-channel sensing system for two different proteins was developed through integration of the optical responses (phosphorescence emission and resonant light scattering (RLS)) of Mn-doped ZnS QDs and recognition of them by surface BSA phosphorescent sensing of trypsin and RLS sensing of lysozyme. Trypsin can digest BSA and remove BSA from the surface of Mn-doped ZnS QDs, thus quenching the phosphorescence of QDs, whereas lysozyme can assemble with BSA to lead to aggregation of QDs and enhanced RLS intensity. The detection limits for trypsin and lysozyme were 40 and 3?nM, respectively. The selectivity of the respective channel for trypsin and lysozyme was evaluated with a series of other proteins. Unlike other protein sensors based on nanobioconjugates, the proposed dual-channel sensor employs only one type of QDs but can detect two different proteins. Further, we found the RLS of QDs can also be useful for studying the BSA-lysozyme binding stoichiometry, which has not been reported in the literature. These successful biosensor applications clearly demonstrate that BSA not only serves as a template for growth of Mn-doped ZnS QDs, but also impacts the QDs for selective recognition of analyte proteins. PMID:23576296

  6. Ancient association between cation leak channels and Mid1 proteins is conserved in fungi and animals

    PubMed Central

    Ghezzi, Alfredo; Liebeskind, Benjamin J.; Thompson, Ammon; Atkinson, Nigel S.; Zakon, Harold H.

    2014-01-01

    Neuronal resting potential can tune the excitability of neural networks, affecting downstream behavior. Sodium leak channels (NALCN) play a key role in rhythmic behaviors by helping set, or subtly changing neuronal resting potential. The full complexity of these newly described channels is just beginning to be appreciated, however. NALCN channels can associate with numerous subunits in different tissues and can be activated by several different peptides and second messengers. We recently showed that NALCN channels are closely related to fungal calcium channels, which they functionally resemble. Here, we use this relationship to predict a family of NALCN-associated proteins in animals on the basis of homology with the yeast protein Mid1, the subunit of the yeast calcium channel. These proteins all share a cysteine-rich region that is necessary for Mid1 function in yeast. We validate this predicted association by showing that the Mid1 homolog in Drosophila, encoded by the CG33988 gene, is coordinately expressed with NALCN, and that knockdown of either protein creates identical phenotypes in several behaviors associated with NALCN function. The relationship between Mid1 and leak channels has therefore persisted over a billion years of evolution, despite drastic changes to both proteins and the organisms in which they exist. PMID:24639627

  7. Translocation of botulinum neurotoxin light chain protease by the heavy chain protein-conducting channel.

    PubMed

    Montal, Mauricio

    2009-10-01

    Clostridial botulinum neurotoxins (BoNTs) inhibit synaptic exocytosis; intoxication requires the di-chain protein to undergo conformational changes in response to pH and redox gradients across the endosomal membrane with consequent formation of a protein-conducting channel by the heavy chain (HC) that translocates the light chain (LC) protease into the cytosol, colocalizing it with the substrate SNARE proteins. We investigate the dynamics of protein translocation across membranes using a sensitive single-molecule assay to track translocation events with millisecond resolution on lipid bilayers and on membrane patches of Neuro 2A cells. Translocation of BoNT/A LC by the HC is observed in real time as changes of channel conductance: the channel is occluded by the light chain during transit, and open after completion of translocation and release of cargo, acting intriguingly similar to the protein-conducting/translocating channels of the endoplasmic reticulum, mitochondria, and chloroplasts. Our findings support the notion of an interdependent, tight interplay between the HC transmembrane chaperone and the LC cargo that prevents LC aggregation and dictates the productive passage of cargo through the channel and completion of translocation. The protein-conducting channel of BoNT, a key element in the process of neurotoxicity, emerges therefore as a target for antidote discovery - a novel paradigm of paramount significance to health science and biodefense. PMID:19111565

  8. Translocation of Botulinum neurotoxin light chain protease by the heavy chain protein-conducting channel

    PubMed Central

    Montal, Mauricio

    2009-01-01

    Clostridial botulinum neurotoxins (BoNTs) inhibit synaptic exocytosis; intoxication requires the di-chain protein to undergo conformational changes in response to pH and redox gradients across the endosomal membrane with consequent formation of a protein-conducting channel by the heavy chain (HC) that translocates the light chain (LC) protease into the cytosol, colocalizing it with the substrate SNARE proteins. We investigate the dynamics of protein-translocation across membranes using a sensitive single molecule assay to track translocation events with millisecond resolution on lipid bilayers and on membrane patches of Neuro 2A cells. Translocation of BoNT/A LC by the HC is observed in real time as changes of channel conductance: the channel is occluded by the light chain during transit, and open after completion of translocation and release of cargo, acting intriguingly similar to the protein-conducting/translocating channels of the endoplasmic reticulum, mitochondria, and chloroplasts. Our findings support the notion of an interdependent, tight interplay between the HC transmembrane chaperone and the LC cargo that prevents LC aggregation and dictates the productive passage of cargo through the channel and completion of translocation. The protein-conducting channel of BoNT, a key element in the process of neurotoxicity, emerges therefore as a target for antidote discovery –a novel paradigm of paramount significance to health science and biodefense. PMID:19111565

  9. EFFECT OF ELEVATED WATER TEMPERATURE ON MACRO-INVERTEBRATE COMMUNITIES IN OUTDOOR EXPERIMENTAL CHANNELS

    EPA Science Inventory

    Macroinvertebrates were sampled monthly from December 1976 to September 1977 in two 520 m long outdoor experimental channels. One channel was maintained at ambient seasonal Mississippi River water temperatures and the other 10C higher. Maximum water temperature reached 31.0C in t...

  10. EFFECT OF ELEVATED WATER TEMPERATURE ON INSECT EMERGENCE IN OUTDOOR EXPERIMENTAL CHANNELS

    EPA Science Inventory

    Emergence of adult aquatic insects was evaluated weekly with plexiglass traps positioned in two outdoor experimental channels from April through August 1977. One channel was seasonal Mississippi River water temperature and the other maintained at 10C above. Maximum water temperat...

  11. Liquid Water Dynamics in a Model Polymer Electrolyte Fuel Cell Flow Channel

    E-print Network

    Victoria, University of

    Liquid Water Dynamics in a Model Polymer Electrolyte Fuel Cell Flow Channel by Chris Miller in a Model Polymer Electrolyte Fuel Cell Flow Channel by Chris Miller Bachelors of Engineering, University in a polymer electrolyte fuel cell is a critical issue in ensuring high cell performance. The water production

  12. Probing membrane protein structure using water polarization transfer solid-state NMR

    NASA Astrophysics Data System (ADS)

    Williams, Jonathan K.; Hong, Mei

    2014-10-01

    Water plays an essential role in the structure and function of proteins, lipid membranes and other biological macromolecules. Solid-state NMR heteronuclear-detected 1H polarization transfer from water to biomolecules is a versatile approach for studying water-protein, water-membrane, and water-carbohydrate interactions in biology. We review radiofrequency pulse sequences for measuring water polarization transfer to biomolecules, the mechanisms of polarization transfer, and the application of this method to various biological systems. Three polarization transfer mechanisms, chemical exchange, spin diffusion and NOE, manifest themselves at different temperatures, magic-angle-spinning frequencies, and pulse irradiations. Chemical exchange is ubiquitous in all systems examined so far, and spin diffusion plays the key role in polarization transfer within the macromolecule. Tightly bound water molecules with long residence times are rare in proteins at ambient temperature. The water polarization-transfer technique has been used to study the hydration of microcrystalline proteins, lipid membranes, and plant cell wall polysaccharides, and to derive atomic-resolution details of the kinetics and mechanism of ion conduction in channels and pumps. Using this approach, we have measured the water polarization transfer to the transmembrane domain of the influenza M2 protein to obtain information on the structure of this tetrameric proton channel. At short mixing times, the polarization transfer rates are site-specific and depend on the pH, labile protons, sidechain conformation, as well as the radial position of the residues in this four-helix bundle. Despite the multiple dependences, the initial transfer rates reflect the periodic nature of the residue positions from the water-filled pore, thus this technique provides a way of gleaning secondary structure information, helix tilt angle, and the oligomeric structure of membrane proteins.

  13. Probing membrane protein structure using water polarization transfer solid-state NMR.

    PubMed

    Williams, Jonathan K; Hong, Mei

    2014-10-01

    Water plays an essential role in the structure and function of proteins, lipid membranes and other biological macromolecules. Solid-state NMR heteronuclear-detected (1)H polarization transfer from water to biomolecules is a versatile approach for studying water-protein, water-membrane, and water-carbohydrate interactions in biology. We review radiofrequency pulse sequences for measuring water polarization transfer to biomolecules, the mechanisms of polarization transfer, and the application of this method to various biological systems. Three polarization transfer mechanisms, chemical exchange, spin diffusion and NOE, manifest themselves at different temperatures, magic-angle-spinning frequencies, and pulse irradiations. Chemical exchange is ubiquitous in all systems examined so far, and spin diffusion plays the key role in polarization transfer within the macromolecule. Tightly bound water molecules with long residence times are rare in proteins at ambient temperature. The water polarization-transfer technique has been used to study the hydration of microcrystalline proteins, lipid membranes, and plant cell wall polysaccharides, and to derive atomic-resolution details of the kinetics and mechanism of ion conduction in channels and pumps. Using this approach, we have measured the water polarization transfer to the transmembrane domain of the influenza M2 protein to obtain information on the structure of this tetrameric proton channel. At short mixing times, the polarization transfer rates are site-specific and depend on the pH, labile protons, sidechain conformation, as well as the radial position of the residues in this four-helix bundle. Despite the multiple dependences, the initial transfer rates reflect the periodic nature of the residue positions from the water-filled pore, thus this technique provides a way of gleaning secondary structure information, helix tilt angle, and the oligomeric structure of membrane proteins. PMID:25228502

  14. The ABC protein turned chloride channel whose failure causes cystic fibrosis

    NASA Astrophysics Data System (ADS)

    Gadsby, David C.; Vergani, Paola; Csanády, László

    2006-03-01

    CFTR chloride channels are encoded by the gene mutated in patients with cystic fibrosis. These channels belong to the superfamily of ABC transporter ATPases. ATP-driven conformational changes, which in other ABC proteins fuel uphill substrate transport across cellular membranes, in CFTR open and close a gate to allow transmembrane flow of anions down their electrochemical gradient. New structural and biochemical information from prokaryotic ABC proteins and functional information from CFTR channels has led to a unifying mechanism explaining those ATP-driven conformational changes.

  15. Structure, dynamics and reactions of protein hydration water.

    PubMed Central

    Smith, Jeremy C; Merzel, Franci; Bondar, Ana-Nicoleta; Tournier, Alexander; Fischer, Stefan

    2004-01-01

    The apparent simplicity of the water molecule belies the wide range of fascinating protein phenomena in which it participates. We review recent computer simulation work on buried, internal water molecules, discussing the thermodynamics of water molecule binding and the participation of water in proton transfer reactions. Surface water molecules are also considered, with emphasis on the modification of average solvent structure on a protein surface, the role of water in the protein dynamical 'glass' transition and a simplified description of the protein motions thereby activated. PMID:15306375

  16. Roles of heterotrimeric G proteins in guard cell ion channel regulation

    PubMed Central

    2011-01-01

    Stomata are formed by pairs of surrounding guard cells and perform important roles in photosynthesis, transpiration and innate immunity of terrestrial plants. Ionic solutes in the cytosol of guard cells are important for cell turgor and volume change. Consequently, trans-membrane flux of ions such as K+, Cl?, and malate2? through K+ channels and anion channels of guard cells are a direct driving force for turgor change, while the opening of calcium permeable channels can serve as a trigger of cytosolic free calcium concentration elevations or oscillations, which play second messenger roles. In plants, heterotrimeric G proteins have fewer members than in animals, but they are well investigated and found to regulate these channels and to play fundamental roles in guard cell function. This mini-review focuses on the recent understanding of G-protein regulation of ion channels on the plasma membrane of guard cells and their participation in stomatal movements. PMID:21617376

  17. Apparent digestibility of alternative plant-protein feedstuffs for channel catfish, Ictalurus punctatus (Rafinesque)

    Technology Transfer Automated Retrieval System (TEKTRAN)

    A study was conducted with channel catfish, Ictalurus puntatus to determine apparent digestibility/availability coefficients of protein, amino acids, lipid and energy for alternative plant-protein feedstuffs: corn gluten feed, corn germ meal, distillers dried grains with solubles, and canola meal, c...

  18. Regulation of CRAC channels by protein interactions and post-translational modification

    PubMed Central

    Srikanth, Sonal; Ribalet, Bernard; Gwack, Yousang

    2013-01-01

    Store-operated Ca2+ entry (SOCE) is a widespread mechanism to elevate the intracellular Ca2+ concentrations and stimulate downstream signaling pathways affecting proliferation, secretion, differentiation and death in different cell types. In immune cells, immune receptor stimulation induces intracellular Ca2+ store depletion that subsequently activates Ca2+-release-activated-Ca2+ (CRAC) channels, a prototype of store-operated Ca2+ (SOC) channels. CRAC channel opening leads to activation of diverse downstream signaling pathways affecting proliferation, differentiation, cytokine production and cell death. Recent identification of STIM1 as the endoplasmic reticulum Ca2+ sensor and Orai1 as the pore subunit of CRAC channels has provided the much-needed molecular tools to dissect the mechanism of activation and regulation of CRAC channels. In this review, we discuss the recent advances in understanding the associating partners and posttranslational modifications of Orai1 and STIM1 proteins that regulate diverse aspects of CRAC channel function. PMID:23454861

  19. Investigation of water droplet dynamics in PEM fuel cell gas channels

    NASA Astrophysics Data System (ADS)

    Gopalan, Preethi

    Water management in Proton Exchange Membrane Fuel Cell (PEMFC) has remained one of the most important issues that need to be addressed before its commercialization in automotive applications. Accumulation of water on the gas diffusion layer (GDL) surface in a PEMFC introduces a barrier for transport of reactant gases through the GDL to the catalyst layer. Despite the fact that the channel geometry is one of the key design parameters of a fluidic system, very limited research is available to study the effect of microchannel geometry on the two-phase flow structure. In this study, the droplet-wall dynamics and two-phase pressure drop across the water droplet present in a typical PEMFC channel, were examined in auto-competitive gas channel designs (0.4 x 0.7 mm channel cross section). The liquid water flow pattern inside the gas channel was analyzed for different air velocities. Experimental data was analyzed using the Concus-Finn condition to determine the wettability characteristics in the corner region. It was confirmed that the channel angle along with the air velocity and the channel material influences the water distribution and holdup within the channel. Dynamic contact angle emerged as an important parameter in controlling the droplet-wall interaction. Experiments were also performed to understand how the inlet location of the liquid droplet on the GDL surface affects the droplet dynamic behavior in the system. It was found that droplets emerging near the channel wall or under the land lead to corner filling of the channel. Improvements in the channel design has been proposed based on the artificial channel roughness created to act as capillary grooves to transport the liquid water away from the land area. For droplets emerging near the center of the channel, beside the filling and no-filling behavior reported in the literature, a new droplet jumping behavior was observed. As droplets grew and touched the sidewalls, they jumped off to the sidewall leaving the whole GDL exposed for gases to diffuse to the catalyst layer. A theoretical model was developed and a criterion was proposed to predict the droplet jumping behavior in the gas channel. A theoretical force balance model was proposed to predict the pressure force and air velocity required to remove the droplet from the channel to avoid complete channel blockage. The overall goal of this work was to identify the gas channel configuration that provides efficient water removal with a lower pressure drop in the system efficiency while meeting the US Department of Energy's specifications for a PEMFC for automotive application.

  20. Natural channel protein inserts and functions in a completely artificial, solid-supported bilayer membrane

    PubMed Central

    Zhang, Xiaoyan; Fu, Wangyang; Palivan, Cornelia G.; Meier, Wolfgang

    2013-01-01

    Reconstitution of membrane proteins in artificial membrane systems creates a platform for exploring their potential for pharmacological or biotechnological applications. Previously, we demonstrated amphiphilic block copolymers as promising building blocks for artificial membranes with long-term stability and tailorable structural parameters. However, the insertion of membrane proteins has not previously been realized in a large-area, stable, and solid-supported artificial membrane. Here, we show the first, preliminary model of a channel membrane protein that is functionally incorporated in a completely artificial polymer, tethered, solid-supported bilayer membrane (TSSBM). Unprecedented ionic transport characteristics that differ from previous results on protein insertion into planar, free-standing membranes, are identified. Our findings mark a change in understanding protein insertion and ion flow within natural channel proteins when inserted in an artificial TSSBM, thus holding great potential for numerous applications such as drug screening, trace analyzing, and biosensing. PMID:23846807

  1. SDS capillary gel electrophoresis of proteins in microfabricated channels

    PubMed Central

    Yao, Shao; Anex, Deon S.; Caldwell, W. Brett; Arnold, Don W.; Smith, Katherine B.; Schultz, Peter G.

    1999-01-01

    Analysis of variations in the concentrations or structures of biomolecules (e.g., mRNAs, proteins, peptides, natural products) that occur either naturally or in response to environmental or genetic perturbations can provide important insight into complex biological processes. Many biological samples are mixtures that require a separation step before quantitation of variations in the individual components. Two-dimensional denaturing gel electrophoresis has been used very effectively to separate complex mixtures of proteins, but it is time consuming and requires considerable amounts of sample. Microchannel-based separations have proven very effective in rapidly separating small amounts of nucleic acids; more recently, isoelectric focusing of proteins also has been adapted to the microchannel format. Here, we describe microchannel-based SDS capillary gel electrophoresis of proteins and demonstrate the speed and high resolution it provides. This development is an important step toward the miniaturization and integration of multidimensional and array separation methods for complex protein mixtures. PMID:10318890

  2. Expression, functional analysis, and in situ hybridization of a cloned rat kidney collecting duct water channel.

    PubMed

    Ma, T; Hasegawa, H; Skach, W R; Frigeri, A; Verkman, A S

    1994-01-01

    The cloning and expression of an apical membrane water channel from rat kidney collecting duct (WCH-CD) homologous to a 28-kDa integral membrane protein (CHIP28) was reported recently (K. Fushimi, S. Uchida, Y. Hara, Y. Hirata, F. Marumo, and S. Sasaki. Nature Lond. 361: 549-552, 1993). We obtained an approximately 1.8-kilobase clone from a rat kidney lambda gt10 cDNA library by a polymerase chain reaction cloning method; whereas the coding sequence (814 base pairs, predicted protein size 29 kDa) was identical to that reported, we identified an in-frame ATG codon at base pair -123 predicting a protein size of 33 kDa. On Northern blots probed by cDNAs corresponding to the WCH-CD coding sequence (base pairs +1 to +814) or 5'-untranslated sequence (-403 to -16), a single band at 1.9 kilobases was observed in kidney medulla greater than in cortex but not in other tissues; mRNA expression was increased strongly by dehydration. Translation and oocyte expression studies were performed to identify the translation start site. The short (base pairs +1 to +814) and long (base pairs -123 to +814) cDNAs were subcloned in vector pSP64 containing the 5'-untranslated Xenopus globin sequence upstream to the ATGs; a 30-base pair c-myc sequence was engineered at the COOH- terminal for antibody recognition.(ABSTRACT TRUNCATED AT 250 WORDS) PMID:7508187

  3. Optimization of 3D Poisson-Nernst-Planck model for fast evaluation of diverse protein channels.

    PubMed

    Dyrka, Witold; Bartuzel, Maciej M; Kotulska, Malgorzata

    2013-10-01

    We show the accuracy and applicability of our fast algorithmic implementation of a three-dimensional Poisson-Nernst-Planck (3D-PNP) flow model for characterizing different protein channels. Due to its high computational efficiency, our model can predict the full current-voltage characteristics of a channel within minutes, based on the experimental 3D structure of the channel or its computational model structure. Compared with other methods, such as Brownian dynamics, which currently needs a few weeks of the computational time, or even much more demanding molecular dynamics modeling, 3D-PNP is the only available method for a function-based evaluation of very numerous tentative structural channel models. Flow model tests of our algorithm and its optimal parametrization are provided for five native channels whose experimental structures are available in the protein data bank (PDB) in an open conductive state, and whose experimental current-voltage characteristics have been published. The channels represent very different geometric and structural properties, which makes it the widest test to date of the accuracy of 3D-PNP on real channels. We test whether the channel conductance, rectification, and charge selectivity obtained from the flow model, could be sufficiently sensitive to single-point mutations, related to unsignificant changes in the channel structure. Our results show that the classical 3D-PNP model, under proper parametrization, is able to achieve a qualitative agreement with experimental data for a majority of the tested characteristics and channels, including channels with narrow and irregular conductivity pores. We propose that although the standard PNP model cannot provide insight into complex physical phenomena due to its intrinsic limitations, its semiquantitative agreement is achievable for rectification and selectivity at a level sufficient for the bioinformatical purpose of selecting the best structural models with a great advantage of a very short computational time. PMID:23720356

  4. Structure of the SecY channel during initiation of protein translocation

    PubMed Central

    Park, Eunyong; Ménétret, Jean-François; Gumbart, James C.; Ludtke, Steven J.; Li, Weikai; Whynot, Andrew

    2013-01-01

    Many secretory proteins are targeted by signal sequences to a protein-conducting channel, formed by prokaryotic SecY- or eukaryotic Sec61-complexes, and are translocated across the membrane during their synthesis1,2. Crystal structures of the inactive channel show that the SecY subunit of the heterotrimeric complex consists of two halves that form an hourglass-shaped pore with a constriction in the middle of the membrane and a lateral gate that faces the lipid phase3-5. The closed channel has an empty cytoplasmic funnel and an extracellular funnel that is filled with a small helical domain, called the plug. During initiation of translocation, a ribosome–nascent chain complex binds to the SecY/Sec61 complex, resulting in insertion of the nascent chain. However, the mechanism of channel opening during translocation is unclear. Here, we have addressed this question by determining structures of inactive and active ribosome–channel complexes with cryo-electron microscopy. Non-translating ribosome–SecY channel complexes derived from Methanococcus jannaschii or Escherichia coli show the channel in its closed state, and indicate that ribosome binding per se causes only minor changes. The structure of an active E. coli ribosome–channel complex demonstrates that the nascent chain opens the channel, causing mostly rigid body movements of the N- and C-terminal halves of SecY. In this early translocation intermediate, the polypeptide inserts as a loop into the SecY channel with the hydrophobic signal sequence intercalated into the open lateral gate. The nascent chain also forms a loop on the cytoplasmic surface of SecY rather than directly entering the channel. PMID:24153188

  5. Aquaporin-4 water channels and synaptic plasticity in the hippocampus

    PubMed Central

    Scharfman, Helen E.; Binder, Devin K.

    2013-01-01

    Aquaporin-4 (AQP4) is the major water channel expressed in the central nervous system (CNS) and is primarily expressed in glial cells. Many studies have shown that AQP4 regulates the response of the CNS to insults or injury, but far less is known about the potential for AQP4 to influence synaptic plasticity or behavior. Recent studies have examined long-term potentiation (LTP), long-term depression (LTD), and behavior in AQP4 knockout (KO) and wild-type mice to gain more insight into its potential role. The results showed a selective effect of AQP4 deletion on LTP of the Schaffer collateral pathway in hippocampus using an LTP induction protocol that simulates pyramidal cell firing during theta oscillations (theta-burst stimulation; TBS). However, a different LTP induction protocol was unaffected by AQP4 deletion. There was also a defect in LTD after low frequency stimulation (LFS) in AQP4 KO mice. Interestingly, some slices from AQP4 KO mice exhibited LTD after TBS instead of LTP, or LTP following LFS instead of LTD. These data suggest that AQP4 and astrocytes influence the polarity of long-term synaptic plasticity (potentiation or depression). These potentially powerful roles expand the influence of AQP4 and astrocytes beyond the original suggestions related to regulation of extracellular potassium and water balance. Remarkably, AQP4 KO mice did not show deficits in basal transmission, suggesting specificity for long-term synaptic plasticity. The mechanism appears to be related to neurotrophins and specifically brain-derived neurotrophic factor (BDNF) because pharmacological blockade of neurotrophin trk receptors or scavenging ligands such as BDNF restored plasticity. The in vitro studies predicted effects in vivo of AQP4 deletion because AQP4 KO mice performed worse using a task that requires memory for the location of objects (object placement). However, performance on other hippocampal-dependent tasks was spared. The results suggest an unanticipated and selective role of AQP4 in synaptic plasticity and spatial memory, and underscore the growing appreciation of the role of glial cells in functions typically attributed to neurons. Implications for epilepsy are discussed because of the previous evidence that AQP4 influences seizures, and the role of synaptic plasticity in epileptogenesis. PMID:23684954

  6. PorH, a new channel-forming protein present in the cell wall of Corynebacterium efficiens and Corynebacterium callunae

    Microsoft Academic Search

    Peter Hunten; Bettina Schiffler; Friedrich Lottspeich; Roland Benz

    2005-01-01

    Corynebacterium callunae and Corynebacterium efficiens are close relatives of the glutamate-producing mycolata species Corynebacterium glutamicum. The properties of the pore-forming proteins, extracted by organic solvents, were studied. The cell extracts contained channel-forming proteins that formed ion-permeable channels with a single-channel conductance of about 2 to 3 nS in 1 M KCl in a lipid bilayer assay. The corresponding proteins from

  7. IUPHAR-DB: the IUPHAR database of G protein-coupled receptors and ion channels

    Microsoft Academic Search

    Anthony J. Harmar; Rebecca A. Hills; Edward M. Rosser; Martin Jones; O. Peter Buneman; Donald R. Dunbar; Stuart D. Greenhill; Valerie A. Hale; Joanna L. Sharman; Tom I. Bonner; William A. Catterall; Anthony P. Davenport; Philippe Delagrange; Colin T. Dollery; Steven M. Foord; George A. Gutman; Vincent Laudet; Richard R. Neubig; Eliot H. Ohlstein; Richard W. Olsen; John Peters; Jean-philippe Pin; Robert R. Ruffolo; David B. Searls; Mathew W. Wright; Michael Spedding

    2009-01-01

    The IUPHAR database (IUPHAR-DB) integrates peer- reviewed pharmacological, chemical, genetic, func- tional and anatomical information on the 354 non- sensory G protein-coupled receptors (GPCRs), 71 ligand-gated ion channel subunits and 141 voltage- gated-like ion channel subunits encoded by the human, rat and mouse genomes. These genes repre- sent the targets of approximately one-third of cur- rently approved drugs and are

  8. Groundwater-surface water ecotones at the upstream part of confluences in former river channels

    Microsoft Academic Search

    Gudrun Bornettel; Andrew R. G. Large

    1995-01-01

    Two slow flowing former channels of the Rhône River, which are supplied by nutrient-poor groundwater and subject to backflows of nutrient-rich water from the Rhone, were studied in order to demonstrate the occurrence of an ecotone located upstream to their confluences with the main channel. In one of the channels, the long-term stability of the boundary zone compared to the

  9. PEDV ORF3 encodes an ion channel protein and regulates virus production.

    PubMed

    Wang, Kai; Lu, Wei; Chen, Jianfei; Xie, Shiqi; Shi, Hongyan; Hsu, Haojen; Yu, Wenjing; Xu, Ke; Bian, Chao; Fischer, Wolfgang B; Schwarz, Wolfgang; Feng, Li; Sun, Bing

    2012-02-17

    Several studies suggest that the open reading frame 3 (ORF3) gene of porcine epidemic diarrhea virus (PEDV) is related to viral infectivity and pathogenicity, but its function remains unknown. Here, we propose a structure model of the ORF3 protein consisting of four TM domains and forming a tetrameric assembly. ORF3 protein can be detected in PEDV-infected cells and it functions as an ion channel in both Xenopus laevis oocytes and yeast. Mutation analysis showed that Tyr170 in TM4 is important for potassium channel activity. Furthermore, viral production is reduced in infected Vero cells when ORF3 gene is silenced by siRNA. Interestingly, the ORF3 gene from an attenuated PEDV encodes a truncated protein with 49 nucleotide deletions, which lacks the ion channel activity. PMID:22245155

  10. Electron Spin-Echo Envelope Modulation (ESEEM) Reveals Water and Phosphate Interactions with the KcsA Potassium Channel

    SciTech Connect

    Cieslak, John A.; Focia, Pamela J.; Gross, Adrian (NWU)

    2010-08-13

    Electron spin-echo envelope modulation (ESEEM) spectroscopy is a well-established technique for the study of naturally occurring paramagnetic metal centers. The technique has been used to study copper complexes, hemes, enzyme mechanisms, micellar water content, and water permeation profiles in membranes, among other applications. In the present study, we combine ESEEM spectroscopy with site-directed spin labeling (SDSL) and X-ray crystallography in order to evaluate the technique's potential as a structural tool to describe the native environment of membrane proteins. Using the KcsA potassium channel as a model system, we demonstrate that deuterium ESEEM can detect water permeation along the lipid-exposed surface of the KcsA outer helix. We further demonstrate that {sup 31}P ESEEM is able to identify channel residues that interact with the phosphate headgroup of the lipid bilayer. In combination with X-ray crystallography, the {sup 31}P data may be used to define the phosphate interaction surface of the protein. The results presented here establish ESEEM as a highly informative technique for SDSL studies of membrane proteins.

  11. Pxmp2 Is a Channel-Forming Protein in Mammalian Peroxisomal Membrane

    PubMed Central

    Rokka, Aare; Soininen, Raija; Immonen, Hanna L.; Pirilä, Päivi L.; Bergmann, Ulrich; Sormunen, Raija T.; Weckström, Matti; Hiltunen, J. Kalervo

    2009-01-01

    Background Peroxisomal metabolic machinery requires a continuous flow of organic and inorganic solutes across peroxisomal membrane. Concerning small solutes, the molecular nature of their traffic has remained an enigma. Methods/Principal Findings In this study, we show that disruption in mice of the Pxmp2 gene encoding Pxmp2, which belongs to a family of integral membrane proteins with unknown function, leads to partial restriction of peroxisomal membrane permeability to solutes in vitro and in vivo. Multiple-channel recording of liver peroxisomal preparations reveals that the channel-forming components with a conductance of 1.3 nS in 1.0 M KCl were lost in Pxmp2?/? mice. The channel-forming properties of Pxmp2 were confirmed with recombinant protein expressed in insect cells and with native Pxmp2 purified from mouse liver. The Pxmp2 channel, with an estimated diameter of 1.4 nm, shows weak cation selectivity and no voltage dependence. The long-lasting open states of the channel indicate its functional role as a protein forming a general diffusion pore in the membrane. Conclusions/Significance Pxmp2 is the first peroxisomal channel identified, and its existence leads to prediction that the mammalian peroxisomal membrane is permeable to small solutes while transfer of “bulky” metabolites, e.g., cofactors (NAD/H, NADP/H, and CoA) and ATP, requires specific transporters. PMID:19352492

  12. Cloning and expression of apical membrane water channel of rat kidney collecting tubule

    Microsoft Academic Search

    Kiyohide Fushimi; Shinichi Uchida; Yukichi Harat; Yukio Hirata; Fumiaki Marumo; Sei Sasaki

    1993-01-01

    CONCENTRATING urine is mandatory for most mammals to prevent water loss from the body. Concentrated urine is produced in response to vasopressin by the transepithelial recovery of water from the lumen of the kidney collecting tubule through highly water-permeable membranes1,2. In this nephron segment, vaso-pressin regulates water permeability by endo- and exocytosis of water channels from or to the apical

  13. Visualizing Water Molecules in Transmembrane Proteins Using Radiolytic Labeling Methods

    SciTech Connect

    Orban, T.; Gupta, S; Palczewski, K; Chance, M

    2010-01-01

    Essential to cells and their organelles, water is both shuttled to where it is needed and trapped within cellular compartments and structures. Moreover, ordered waters within protein structures often colocalize with strategically placed polar or charged groups critical for protein function, yet it is unclear if these ordered water molecules provide structural stabilization, mediate conformational changes in signaling, neutralize charged residues, or carry out a combination of all these functions. Structures of many integral membrane proteins, including G protein-coupled receptors (GPCRs), reveal the presence of ordered water molecules that may act like prosthetic groups in a manner quite unlike bulk water. Identification of 'ordered' waters within a crystalline protein structure requires sufficient occupancy of water to enable its detection in the protein's X-ray diffraction pattern, and thus, the observed waters likely represent a subset of tightly bound functional waters. In this review, we highlight recent studies that suggest the structures of ordered waters within GPCRs are as conserved (and thus as important) as conserved side chains. In addition, methods of radiolysis, coupled to structural mass spectrometry (protein footprinting), reveal dynamic changes in water structure that mediate transmembrane signaling. The idea of water as a prosthetic group mediating chemical reaction dynamics is not new in fields such as catalysis. However, the concept of water as a mediator of conformational dynamics in signaling is just emerging, because of advances in both crystallographic structure determination and new methods of protein footprinting. Although oil and water do not mix, understanding the roles of water is essential to understanding the function of membrane proteins.

  14. Ricean Shadowed Statistical Characterization of Shallow Water Acoustic Channels for Wireless Communications

    E-print Network

    Ruiz-Vega, F; Otero, P; Paris, J F

    2011-01-01

    In this letter, the statistical behaviour of the shallow water acoustic channel for wireless communications is shown to be well characterized by the Ricean shadowed distribution, which has never been proposed for communication purposes on this type of channel. This characterization is clearly motivated from statistical and physical perspectives and has both theoretical and practical advantages compared to previously proposed models.

  15. The Role of Channel Bar Influences on Groundwater \\/ Surface Water Interactions

    Microsoft Academic Search

    C. L. Shope; J. E. Constantz; C. A. Cooper; W. A. McKay

    2010-01-01

    Channel bars are dominant in-stream geomorphic island features present in a large range of river classes throughout the world, particularly in the arid western United States. A quantitative understanding of groundwater and surface water exchange through channel bar features is necessary to understand near-stream hyporheic flow patterns. The Truckee River in northwestern Nevada was used as a research site to

  16. Hydrogen peroxide treatments for channel catfish eggs infected with water molds

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Fungi, or water molds Saprolegnia spp., on channel catfish Ictalurus punctatus eggs can lower fry production. This requires the producer to spawn more catfish or face fingerling shortages. Few treatments have been tested against channel catfish eggs infested with an identified fungus. Hydrogen pe...

  17. Efficient water-filling algorithms for a Gaussian multiaccess channel with ISI

    Microsoft Academic Search

    Chaohuang Zeng; Louise M. C. Hoo; John M. Cioffi

    2000-01-01

    Although the optimum transmit power spectral density (PSD) of each user in a Gaussian multiaccess channel is theoretically available, efficient algorithms are needed to obtain the optimal PSD numerically. This paper presents two water-filling algorithms for the scenario where all users have the same priorities. First we propose for a two-user channel an exact algorithm with a computational complexity of

  18. Drift of macroinvertebrates in a channel carrying heated water from a power plant

    Microsoft Academic Search

    Charles W. Durrett; William D. Pearson

    1975-01-01

    Hourly drift samples were collected once each month over a 24 hour period for 10 months at the mouth of a channel returning heated coolant water from a steam electric generating station to a 330 ha reservoir. Most benthic macroinvertebrates existed at lower densities on the bottom of the channel than in the reservoir. No organisms were found in the

  19. A protein phosphorylation/dephosphorylation network regulates a plant potassium channel

    PubMed Central

    Lee, Sung Chul; Lan, Wen-Zhi; Kim, Beom-Gi; Li, Legong; Cheong, Yong Hwa; Pandey, Girdhar K.; Lu, Guihua; Buchanan, Bob B.; Luan, Sheng

    2007-01-01

    Potassium (K+) is an essential nutrient for plant growth and development. Plants often adapt to low K+ conditions by increasing their K+ uptake capability. Recent studies have led to the identification of a calcium signaling pathway that enables plants to act in this capacity. Calcium is linked to two calcineurin B-like calcium sensors (CBLs) and a target kinase (CBL-interacting protein kinase 23 or CIPK23) that, in turn, appears to phosphorylate and activate the potassium channel, Arabidopsis K+ transporter 1 (AKT1), responsible for K+ uptake in roots. Here, we report evidence that this regulatory mechanism is more elaborate than earlier envisaged. The recently described pathway is part of an extensive network whereby several CBLs interact with multiple CIPKs in the activation of the potassium channel, AKT1. The physical interactions among the CBL, CIPK, and AKT1 components provide a mechanism for specifying the members of the CBL and CIPK families functional in AKT1 regulation. The interaction between the CIPKs and AKT1 was found to involve the kinase domain of the CIPK component and the ankyrin repeat domain of the channel. Furthermore, we identified a 2C-type protein phosphatase that physically interacts and inactivates the AKT1 channel. These findings provide evidence that the calcium-sensitive CBL and CIPK families together with 2C-type protein phosphatases form a protein phoshporylation/dephosphorylation network that regulates the AKT1 channel for K+ transport in plants. PMID:17898163

  20. Building the water edge : a public event for art and artists at Fort Point Channel

    E-print Network

    Godwin, Audrey

    1996-01-01

    The thesis deals with building the water edge at Fort Point Channel, between Congress Street and Summer Street Bridges. It serves as a public event that intends to establish continuity of movement along the waterfront. The ...

  1. Members of the chloride intracellular ion channel protein family demonstrate glutaredoxin-like enzymatic activity.

    PubMed

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

    2015-01-01

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

  2. 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

  3. Ordinary High Water Mark in ephemeral and intermittent channels in the arid southwestern United States

    NASA Astrophysics Data System (ADS)

    Curtis, K. E.; Lichvar, R.; Dixon, L.

    2010-12-01

    The Ordinary High Water Mark (OHWM) is important for regulating stream channels, as it defines the lateral extent of non-wetland waters, as defined under Waters of the United States in the Clean Water Act (33 CFR Part 328.3). Prior to this study, little was known about the OHWM and geomorphically effective events in arid southwest ephemeral and intermittent channels. The hydrogeomorphic floodplain features and flow dynamics are challenging to identify in these channels because of the unstable channel morphology, flashy flood events, and lack of gage data. Through field surveys, LiDAR high-resolution topography data collection, HEC-RAS flow modeling, analysis of aerial photography, and determination of recurrence intervals using gage data, we have developed a methodology to identify the hydrogeomorphic floodplain units and delineate the OHWM. The hydrogeomorphic floodplain units include a migratory low-flow channel, an active floodplain associated with low to moderate discharge events, and a low terrace inundated by moderate to high floods. We identified primary OHWM indicators such as a break in slope, a change in sediment texture, and a change in vegetation characteristics that create a repeatable and reliable signature in the channel morphology that is associated with the OHW boundary between the active floodplain and the low terrace. Secondary indicators such as drift, ripples, and silt deposits are randomly distributed throughout the channel and cannot be linked to a particular inundation extent. In an analysis of 14 gaged ephemeral and intermittent channels, we determined that the recurrence intervals varied from <1 to 15.5 yr. Lower recurrence intervals were typically associated with more stable channels, while channels with a sandy, erodable substrate had higher recurrence intervals. A more complete understanding of the channel morphology and the highly variable OHW recurrence intervals allows more reliable and repeatable delineations in southwest ephemeral and intermittent streams.

  4. Cooperative endocytosis of the endosomal SNARE protein syntaxin-8 and the potassium channel TASK-1

    PubMed Central

    Renigunta, Vijay; Fischer, Thomas; Zuzarte, Marylou; Kling, Stefan; Zou, Xinle; Siebert, Kai; Limberg, Maren M.; Rinné, Susanne; Decher, Niels; Schlichthörl, Günter; Daut, Jürgen

    2014-01-01

    The endosomal SNARE protein syntaxin-8 interacts with the acid-sensitive potassium channel TASK-1. The functional relevance of this interaction was studied by heterologous expression of these proteins (and mutants thereof) in Xenopus oocytes and in mammalian cell lines. Coexpression of syntaxin-8 caused a fourfold reduction in TASK-1 current, a corresponding reduction in the expression of TASK-1 at the cell surface, and a marked increase in the rate of endocytosis of the channel. TASK-1 and syntaxin-8 colocalized in the early endosomal compartment, as indicated by the endosomal markers 2xFYVE and rab5. The stimulatory effect of the SNARE protein on the endocytosis of the channel was abolished when both an endocytosis signal in TASK-1 and an endocytosis signal in syntaxin-8 were mutated. A syntaxin-8 mutant that cannot assemble with other SNARE proteins had virtually the same effect as wild-type syntaxin-8. Total internal reflection fluorescence microscopy showed formation and endocytosis of vesicles containing fluorescence-tagged clathrin, TASK-1, and/or syntaxin-8. Our results suggest that the unassembled form of syntaxin-8 and the potassium channel TASK-1 are internalized via clathrin-mediated endocytosis in a cooperative manner. This implies that syntaxin-8 regulates the endocytosis of TASK-1. Our study supports the idea that endosomal SNARE proteins can have functions unrelated to membrane fusion. PMID:24743596

  5. A conserved cationic motif enhances membrane binding and insertion of the chloride intracellular channel protein 1 transmembrane domain.

    PubMed

    Peter, Bradley; Fanucchi, Sylvia; Dirr, Heini W

    2014-09-01

    The chloride intracellular channel protein 1 (CLIC1) is unique among eukaryotic ion channels in that it can exist as either a soluble monomer or an integral membrane channel. CLIC1 contains no known membrane-targeting signal sequences and the environmental factors which promote membrane binding of the transmembrane domain (TMD) are poorly understood. Here we report a positively charged motif at the C-terminus of the TMD and show that it enhances membrane partitioning and insertion. A 30-mer TMD peptide was synthesized in which the positively charged motif was replaced by three glutamate residues. The peptide was examined in 2,2,2-trifluoroethanol (TFE), sodium dodecyl sulfate micelles and 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine liposomes using size-exclusion chromatography, far-UV CD, and fluorescence spectroscopy. The motif appears to enhance membrane interaction via electrostatic contacts and functions as an electrostatic plug to anchor the TMD in membranes. In addition, the motif is also involved in orientating the TMD with respect to the cis and trans faces of the membrane. These findings shed light on the intrinsic and environmental factors that promote the spontaneous conversion of CLIC1 from a water-soluble to a membrane-bound protein. PMID:24925575

  6. Suspended marine particulate proteins in coastal and oligotrophic waters

    NASA Astrophysics Data System (ADS)

    Bridoux, Maxime C.; Neibauer, Jaqui; Ingalls, Anitra E.; Nunn, Brook L.; Keil, Richard G.

    2015-03-01

    Metaproteomic analyses were performed on suspended sediments collected in one coastal environment (Washington margin, Pacific Ocean, n = 5) and two oligotrophic environments (Atlantic Ocean near BATS, n = 5, and Pacific Ocean near HOTS, n = 5). Using a database of 2.3 million marine proteins developed using the NCBI database, 443 unique peptides were detected from which 363 unique proteins were identified. Samples from the euphotic zone contained on average 2-3x more identifiable proteins than deeper waters (150-1500 m) and these proteins were predominately from photosynthetic organisms. Diatom peptides dominate the spectra of the Washington margin while peptides from cyanobacteria, such as Synechococcus sp. dominated the spectra of both oligotrophic sites. Despite differences in the exact proteins identified at each location, there is good agreement for protein function and cellular location. Proteins in surface waters code for a variety of cellular functions including photosynthesis (24% of detected proteins), energy production (10%), membrane production (9%) and genetic coding and reading (9%), and are split 60-40 between membrane proteins and intracellular cytoplasmic proteins. Sargasso Sea surface waters contain a suite of peptides consistent with proteins involved in circadian rhythms that promote both C and N fixation at night. At depth in the Sargasso Sea, both muscle-derived myosin protein and the muscle-hydrolyzing proteases deseasin MCP-01 and metalloprotease Mcp02 from ?-proteobacteria were observed. Deeper waters contain peptides predominately sourced from ?-proteobacteria (37% of detected proteins) and ?-proteobacteria (26%), although peptides from membrane and photosynthetic proteins attributable to phytoplankton were still observed (13%). Relative to surface values, detection frequencies for bacterial membrane proteins and extracellular enzymes rose from 9 to 16 and 2 to 4% respectively below the thermocline and the overall balance between membrane proteins and intracellular proteins grows to an approximate 75-25 split. Unlike the phytoplankton membrane proteins, which are detrital in nature, the bacterial protein suite at depth is consistent with living biomass.

  7. Identification and characterisation of a functional aquaporin water channel (Anomala cuprea DRIP) in a coleopteran insect.

    PubMed

    Nagae, Tomone; Miyake, Seiji; Kosaki, Shiho; Azuma, Masaaki

    2013-07-15

    Water transport across the plasma membrane depends on the presence of the water channel aquaporin (AQP), which mediates the bulk movement of water through osmotic and pressure gradients. In terrestrial insects, which are solid and/or plant feeders, the entrance and exit of water is primarily executed along the alimentary tract, where the hindgut, particularly the rectum, is the major site of water conservation. A cDNA encoding the homologue of the water-specific Drosophila AQP [Drosophila integral protein (DRIP)] was identified through the RT-PCR of RNA isolated from the rectum of the cupreous chafer larvae, Anomala cuprea, a humus and plant root feeder. This gene (Anocu AQP1) has a predicted molecular mass of 26.471 kDa, similar to the DRIP clade of insect AQPs characterised from caterpillars, flies and several liquid-feeding insects. When expressed in Xenopus laevis oocytes, Anocu AQP1 showed the hallmarks of aquaporin-mediated water transport but no glycerol or urea permeability, and the reversible inhibition of elevated water transport through 1 mmol l(-1) HgCl2. This is the first experimental demonstration of the presence of a water-specific AQP, namely DRIP, in the Coleoptera. The genome of the model beetle Tribolium castaneum contains six putative AQP sequences, one of which (Trica-1a, XP_972862) showed the highest similarity to Anocu AQP1 (~60% amino acid identity). Anocu AQP1 is predominantly expressed in the rectum. Using a specific antibody raised against DRIP in the silkworm Bombyx mori (AQP-Bom1), Anocu AQP1 was localised to the apical plasma membrane of rectal epithelial cells, and lacking in the midgut and gastric caecal epithelia. Based on the BeetleBase prediction, there are three putative AQPs (Trica-3a, 3b, 3c: XP_970728, 970912, 970791) that are homologous to B. mori aquaglyceroporin [AQP-Bom2 (GLP)]. The immunocytochemical studies using the specific anti-peptide antibody against AQP-Bom2 revealed the presence of the GLP homologue at the apical plasma membrane of enterocytes in the midgut and gastric caeca. Thus, DRIP (Anocu AQP1) and the putative GLP share epithelial fluid-transporting roles along the alimentary tract in cupreous chafer larvae. PMID:23531819

  8. Biochemical Identification and Biophysical Characterization of a Channel-Forming Protein from Rhodococcus erythropolis

    Microsoft Academic Search

    THOMAS LICHTINGER; GILA REISS; ROLAND BENZ

    2000-01-01

    Organic solvent extracts of whole cells of the gram-positive bacterium Rhodococcus erythropolis contain a channel-forming protein. It was identified by lipid bilayer experiments and purified to homogeneity by preparative sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE). The pure protein had a rather low molecular mass of about 8.4 kDa, as judged by SDS-PAGE. SDS-resistant oligomers with a molecular mass of

  9. Laser-induced acoustic wave generation/propagation/interaction in water in various internal channels

    NASA Astrophysics Data System (ADS)

    Ko, Seung Hwan; Lee, Daeho; Pan, Heng; Ryu, Sang-Gil; Grigoropoulos, Costas P.; Kladias, Nick; Panides, Elias; Domoto, Gerald A.

    2010-08-01

    Short pulsed laser-induced single acoustic wave generation, propagation, interaction within a water-filled internal channel are experimentally and numerically studied. A large-area, short-duration, single-plane acoustic wave was generated by the thermoelastic interaction of a homogenized nanosecond pulsed laser beam with a liquid-solid interface and propagated at the speed of sound in water. Laser flash Schlieren photography was used to visualize the transient interaction of the plane acoustic wave in various internal channel structures ((a) sudden expansion and contraction channels, (b) bifurcating channels, (c) gradual contraction wall channels and (d) a cylinder). Fairly good agreement between the experimental results and numerical simulation is observed.

  10. Mouse ERG K+ Channel Clones Reveal Differences in Protein Trafficking and Function

    PubMed Central

    Lin, Eric C.; Moungey, Brooke M.; Lim, Evi; Concannon, Sarah P.; Anderson, Corey L.; Kyle, John W.; Makielski, Jonathan C.; Balijepalli, Sadguna Y.; January, Craig T.

    2014-01-01

    Background The mouse ether?a?go?go?related gene 1a (mERG1a, mKCNH2) encodes mERG K+ channels in mouse cardiomyocytes. The mERG channels and their human analogue, hERG channels, conduct IKr. Mutations in hERG channels reduce IKr to cause congenital long?QT syndrome type 2, mostly by decreasing surface membrane expression of trafficking?deficient channels. Three cDNA sequences were originally reported for mERG channels that differ by 1 to 4 amino acid residues (mERG?London, mERG?Waterston, and mERG?Nie). We characterized these mERG channels to test the postulation that they would differ in their protein trafficking and biophysical function, based on previous findings in long?QT syndrome type 2. Methods and Results The 3 mERG and hERG channels were expressed in HEK293 cells and neonatal mouse cardiomyocytes and were studied using Western blot and whole?cell patch clamp. We then compared our findings with the recent sequencing results in the Welcome Trust Sanger Institute Mouse Genomes Project (WTSIMGP). Conclusions First, the mERG?London channel with amino acid substitutions in regions of highly ordered structure is trafficking deficient and undergoes temperature?dependent and pharmacological correction of its trafficking deficiency. Second, the voltage dependence of channel gating would be different for the 3 mERG channels. Third, compared with the WTSIMGP data set, the mERG?Nie clone is likely to represent the wild?type mouse sequence and physiology. Fourth, the WTSIMGP analysis suggests that substrain?specific sequence differences in mERG are a common finding in mice. These findings with mERG channels support previous findings with hERG channel structure–function analyses in long?QT syndrome type 2, in which sequence changes in regions of highly ordered structure are likely to result in abnormal protein trafficking. PMID:25497881

  11. Water masses exchanged through the Channel of Sicily: Evidence for the presence of new water masses on the Tunisian side of the channel

    NASA Astrophysics Data System (ADS)

    Ben Ismail, Sana; Sammari, Chérif; Gasparini, Gian Pietro; Béranger, Karine; Brahim, Mouldi; Aleya, Lotfi

    2012-05-01

    Studies of water masses present in the Channel of Sicily are relevant to understanding not only the overall Mediterranean circulation but also the Tunisian coastal shelf circulation. This study attempts to clarify the dynamics of water masses exchanged through the channel and its variability using data collected during six hydrographic cruises in the western half of the Cap Bon - Mazara del Vallo section during 2003. Hydrographic measurements clearly show the signature of the Atlantic Tunisian Current along the Tunisian coast characterized by important mesoscale variability. Computation of a normalized temperature and salinity standard deviation makes it possible to distinguish areas of high temporal variability located in the transitional layer between the Atlantic Water and the Levantine Intermediate Water. This transitional layer is created by the presence of both Western Intermediate Water and Ionian Water. In addition to highlighting the relevance of the mesoscale activity, a comparison between direct current measurements and adjusted geostrophic currents produced a more confident estimate of the surface and deep transports through the western part of the channel. Our conclusions on seasonal and mesoscale variability are confirmed by a high resolution numerical simulation.

  12. Liquid Water Transport in the Reactant Channels of Proton Exchange Membrane Fuel Cells

    NASA Astrophysics Data System (ADS)

    Banerjee, Rupak

    Water management has been identified as a critical issue in the development of PEM fuel cells for automotive applications. Water is present inside the PEM fuel cell in three phases, i.e. liquid phase, vapor phase and mist phase. Liquid water in the reactant channels causes flooding of the cell and blocks the transport of reactants to the reaction sites at the catalyst layer. Understanding the behavior of liquid water in the reactant channels would allow us to devise improved strategies for removing liquid water from the reactant channels. In situ fuel cell tests have been performed to identify and diagnose operating conditions which result in the flooding of the fuel cell. A relationship has been identified between the liquid water present in the reactant channels and the cell performance. A novel diagnostic technique has been established which utilizes the pressure drop multiplier in the reactant channels to predict the flooding of the cell or the drying-out of the membrane. An ex-situ study has been undertaken to quantify the liquid water present in the reactant channels. A new parameter, the Area Coverage Ratio (ACR), has been defined to identify the interfacial area of the reactant channel which is blocked for reactant transport by the presence of liquid water. A parametric study has been conducted to study the effect of changing temperature and the inlet relative humidity on the ACR. The ACR decreases with increase in current density as the gas flow rates increase, removing water more efficiently. With increase in temperature, the ACR decreases rapidly, such that by 60°C, there is no significant ACR to be reported. Inlet relative humidity of the gases does change the saturation of the gases in the channel, but did not show any significant effect on the ACR. Automotive powertrains, which is the target for this work, are continuously faced with transient changes. Water management under transient operating conditions is significantly more challenging and has not been investigated in detail. This study begins to investigate the effects of changing operating conditions on liquid water transport through the reactant channels. It has been identified that rapidly increasing temperature leads to the dry-out of the membrane and rapidly cooling the cell below 55°C results in the start of cell flooding. In changing the operating load of the PEMFC, overshoot in the pressure drop in the reactant channel has been identified for the first time as part of this investigation. A parametric study has been conducted to identify the factors which influence this overshoot behavior.

  13. Soy protein polymers: Enhancing the water stability property

    Microsoft Academic Search

    Gowrishankar Srinivasan

    2010-01-01

    Soy protein based plastics have been processed in the past by researchers for various short-term applications; however a common issue is the high water sensitivity of these plastics. This work concentrates on resolving this water sensitivity issue of soy protein polymers by employing chemical and mechanical interaction at the molecular level during extrusion. The primary chemical interactions employed were anhydride

  14. Experimental study of two-phase water flow in vertical thin rectangular channels

    NASA Astrophysics Data System (ADS)

    Wright, Christopher T.; O'Brien, James E.; Anderson, Elgin A.

    2001-11-01

    An experimental heat transfer study of two-phase water flow in vertical thin rectangular channels with side vents is conducted. A multiple, heated channel configuration with up- and down-flow conditions is investigated. Parallel heated and unheated flow channels test the effects of cross flow on the onset of nucleate boiling (ONB) and critical heat flux (CHF). The test apparatus provides pressure and substrate temperature data and visual data of the boiling regimes and side-vent flow patterns. The objectives are to determine the two-phase, heat and mass transfer characteristics between adjacent channels as permitted by side-vent cross flow. These data will help develop ONB and CHF correlations for flow geometries typical of plate-type nuclear reactors and heat exchangers. Fundamentally, the data shows how the geometry, flow conditions, and channel configurations affect the heat transfer characteristics of interior channel flows, essential in understanding the ONB and CHF phenomena.

  15. The small envelope protein of porcine reproductive and respiratory syndrome virus possesses ion channel protein-like properties

    SciTech Connect

    Lee, Changhee [Department of Pathobiology, Ontario Veterinary College, University of Guelph, Guelph, Ontario, N1G 2W1 (Canada); Yoo, Dongwan [Department of Pathobiology, Ontario Veterinary College, University of Guelph, Guelph, Ontario, N1G 2W1 (Canada)]. E-mail: dyoo@uoguelph.ca

    2006-11-10

    The small envelope (E) protein of porcine reproductive and respiratory syndrome virus (PRRSV) is a hydrophobic 73 amino acid protein encoded in the internal open reading frame (ORF) of the bicistronic mRNA2. As a first step towards understanding the biological role of E protein during PRRSV replication, E gene expression was blocked in a full-length infectious clone by mutating the ATG translational initiation to GTG, such that the full-length mutant genomic clone was unable to synthesize the E protein. DNA transfection of PRRSV-susceptible cells with the E gene knocked-out genomic clone showed the absence of virus infectivity. P129-{delta}E-transfected cells however produced virion particles in the culture supernatant, and these particles contained viral genomic RNA, demonstrating that the E protein is essential for PRRSV infection but dispensable for virion assembly. Electron microscopy suggests that the P129-{delta}E virions assembled in the absence of E had a similar appearance to the wild-type particles. Strand-specific RT-PCR demonstrated that the E protein-negative, non-infectious P129-{delta}E virus particles were able to enter cells but further steps of replication were interrupted. The entry of PRRSV has been suggested to be via receptor-mediated endocytosis, and lysomotropic basic compounds and known ion-channel blocking agents both inhibited PRRSV replication effectively during the uncoating process. The expression of E protein in Escherichia coli-mediated cell growth arrests and increased the membrane permeability. Cross-linking experiments in cells infected with PRRSV or transfected with E gene showed that the E protein was able to form homo-oligomers. Taken together, our data suggest that the PRRSV E protein is likely an ion-channel protein embedded in the viral envelope and facilitates uncoating of virus and release of the genome in the cytoplasm.

  16. Epithelial sodium channel abundance is decreased by an unfolded protein response induced by hyperosmolality

    PubMed Central

    Crambert, Gilles; Ernandez, Thomas; Lamouroux, Christine; Roth, Isabelle; Dizin, Eva; Martin, Pierre?Yves; Féraille, Eric; Hasler, Udo

    2014-01-01

    Abstract Large shifts of osmolality occur in the kidney medulla as part of the urine concentrating mechanism. Hyperosmotic stress profoundly challenges cellular homeostasis and induces endoplasmic reticulum (ER) stress. Here, we examined the unfolded protein response (UPR) in hyperosmotically?challenged principal cells of the kidney collecting duct (CD) and show its relevance in controlling epithelial sodium channel (ENaC) abundance, responsible for the final adjustment of Na+ excretion. Dehydration increases medullary but not cortical osmolality. Q?PCR analysis of microdissected CD of water?deprived mice revealed increased aquaporin?2 (AQP2) expression in outer medullary and cortical CD while ENaC abundance decreased in outer medullary but not cortical CD. Immunoblotting, Q?PCR and immunofluorescence revealed that hyperosmolality induced a transient ER stress?like response both ex vivo and in cultured CD principal cells and increased activity of the canonical UPR mediators PERK and ATF6. Both hyperosmolality and chemical induction of ER stress decreased ENaC expression in vitro. ENaC depletion by either stimulus was abolished by transcriptional inhibition and by the chemical chaperone 4?phenylbutyric acid and was partly abrogated by either PERK or ATF6 silencing. Our data suggest that induction of the UPR by hyperosmolality may help preserve body fluid homeostasis under conditions of dehydration by uncoupling AQP2 and ENaC abundance in outer medullary CD. PMID:25413317

  17. A virus-encoded potassium ion channel is a structural protein in the chlorovirus Paramecium bursaria chlorella virus 1 virion

    PubMed Central

    Romani, Giulia; Piotrowski, Adrianna; Hillmer, Stefan; Gurnon, James; Van Etten, James L.; Moroni, Anna; Thiel, Gerhard

    2013-01-01

    Most chloroviruses encode small K+ channels, which are functional in electrophysiological assays. The experimental finding that initial steps in viral infection exhibit the same sensitivity to channel inhibitors as the viral K+ channels has led to the hypothesis that the channels are structural proteins located in the internal membrane of the virus particles. This hypothesis was questioned recently because proteomic studies failed to detect the channel protein in virions of the prototype chlorovirus Paramecium bursaria chlorella virus 1 (PBCV-1). Here, we used a mAb raised against the functional K+ channel from chlorovirus MA-1D to search for the viral K+ channel in the virus particle. The results showed that the antibody was specific and bound to the tetrameric channel on the extracellular side. The antibody reacted in a virus-specific manner with protein extracts from chloroviruses that encoded channels similar to that from MA-1D. There was no cross-reactivity with chloroviruses that encoded more diverse channels or with a chlorovirus that lacked a K+ channel gene. Together with electron microscopic imaging, which revealed labelling of individual virus particles with the channel antibody, these results establish that the viral particles contain an active K+ channel, presumably located in the lipid membrane that surrounds the DNA in the mature virions. PMID:23918407

  18. Immobilization of the Plug Domain Inside the SecY Channel Allows Unrestricted Protein Translocation*

    PubMed Central

    Lycklama a Nijeholt, Jelger A.; Bulacu, Monica; Marrink, Siewert Jan; Driessen, Arnold J. M.

    2010-01-01

    The SecYEG complex forms a protein-conducting channel in the inner membrane of Escherichia coli to support the translocation of secretory proteins in their unfolded state. The SecY channel is closed at the periplasmic face of the membrane by a small re-entrance loop that connects transmembrane segment 1 with 2b. This helical domain 2a is termed the plug domain. By the introduction of pairs of cysteines and crosslinkers, the plug domain was immobilized inside the channel and connected to transmembrane segment 10. Translocation was inhibited to various degrees depending on the position and crosslinker spacer length. With one of the crosslinked mutants translocation occurred unrestricted. Biochemical characterization of this mutant as well as molecular dynamics simulations suggest that only a limited movement of the plug domain suffices for translocation. PMID:20489195

  19. Thermodynamics of Water Entry in Hydrophobic Channels of Carbon Nanotubes

    E-print Network

    Hemant Kumar; Biswaroop Mukherjee; Shiang-Tai Lin Chandan Dasgupta; A. K. Sood; Prabal K. Maiti

    2011-08-19

    Experiments and computer simulations demonstrate that water spontaneously fills the hydrophobic cavity of a carbon nanotube. To gain a quantitative thermody- namic understanding of this phenomenon, we use the recently developed Two Phase Thermodynamics (2PT) method to compute translational and rotational entropies of confined water molecules inside single-walled carbon nanotubes and show that the increase in energy of a water molecule inside the nanotube is compensated by the gain in its rotational entropy. The confined water is in equilibrium with the bulk wa- ter and the Helmholtz free energy per water molecule of confined water is the same as that in the bulk within the accuracy of the simulation results. A comparison of translational and rotational spectra of water molecules confined in carbon nanotubes with that of bulk water shows significant shifts in the positions of the spectral peaks that are directly related to the tube radius.

  20. Intra-membrane molecular interactions of K%2B channel proteins : application to problems in biodefense and bioenergy.

    SciTech Connect

    Moczydlowski, Edward G.

    2013-07-01

    Ion channel proteins regulate complex patterns of cellular electrical activity and ionic signaling. Certain K+ channels play an important role in immunological biodefense mechanisms of adaptive and innate immunity. Most ion channel proteins are oligomeric complexes with the conductive pore located at the central subunit interface. The long-term activity of many K+ channel proteins is dependent on the concentration of extracellular K+; however, the mechanism is unclear. Thus, this project focused on mechanisms underlying structural stability of tetrameric K+ channels. Using KcsA of Streptomyces lividans as a model K+ channel of known structure, the molecular basis of tetramer stability was investigated by: 1. Bioinformatic analysis of the tetramer interface. 2. Effect of two local anesthetics (lidocaine, tetracaine) on tetramer stability. 3. Molecular simulation of drug docking to the ion conduction pore. The results provide new insights regarding the structural stability of K+ channels and its possible role in cell physiology.

  1. Translational Hydration Water Dynamics Drives the Protein Glass Transition

    PubMed Central

    Tournier, Alexander L.; Xu, Jiancong; Smith, Jeremy C.

    2003-01-01

    Experimental and computer simulation studies have revealed the presence of a glasslike transition in the internal dynamics of hydrated proteins at ?200 K involving an increase of the amplitude of anharmonic dynamics. This increase in flexibility has been correlated with the onset of protein activity. Here, we determine the driving force behind the protein transition by performing molecular dynamics simulations of myoglobin surrounded by a shell of water. A dual heatbath method is used with which, in any given simulation, the protein and solvent are held at different temperatures, and sets of simulations are performed varying the temperature of the components. The results show that the protein transition is driven by a dynamical transition in the hydration water that induces increased fluctuations primarily in side chains in the external regions of the protein. The water transition involves activation of translational diffusion and occurs even in simulations where the protein atoms are held fixed. PMID:12944299

  2. Effects of Fasting on IGF-Binding Proteins, Glucose, and Cortisol in Channel Catfish (Ictalurus punctatus)

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The effects of fasting on IGF-binding proteins, glucose, and cortisol in channel catfish were examined. Fed fish (controls) were compared to 14-, 30-, and 45-day fasted fish and 45-day fasted fish refed for 15 additional days. Body length and body weight changes, condition factor(CF), hepatosomati...

  3. EVALUATION OF PROTEIN REDUCTION AND LYSINE SUPPLEMENTATION OF PRODUCTION DIETS FOR CHANNEL CATFISH ICTALURUS PUNCTATUS

    Technology Transfer Automated Retrieval System (TEKTRAN)

    A 2-year continuous production trial was conducted in earthen ponds to evaluate lysine supplementation of practical diets as a means to reduce the need for total dietary protein and limit nitrogenous waste production of channel catfish. Experimental diets consisted of three practical diets containi...

  4. The Stoichiometry of G Binding to G-protein-regulated Inwardly Rectifying K Channels (GIRKs)*

    E-print Network

    Clapham, David E.

    channels (6­12), phospho- lipase C (13), adenylyl cyclases (14), G-protein-coupled recep- tor kinases (15), PI1 3-kinase (16), plasma membrane Ca2 pumps (17), Bruton's tyrosine kinase (18), and calmodulin (19-dopa- mine, 2-adrenergic, opiate, somatostatin, and adenosine all employ the G i-G signal transduction

  5. Dramatic transport properties of carbon nanotube membranes for a robust protein channel mimetic platform

    Microsoft Academic Search

    Bruce Hinds

    Carbon nanotube (CNT) membranes offer an exciting opportunity to mimic natural protein channels due to (1) a mechanism for dramatically enhanced fluid flow, (2) ability to place ‘gatekeeper’ chemistry at the entrance to pores, and (3) being electrically conductive to localize electric field or perform electrochemical transformations. The transport mechanisms through CNT membranes are primarily (1) ionic diffusion near bulk

  6. REST: A mammalian silencer protein that restricts sodium channel gene expression to neurons

    Microsoft Academic Search

    Jayhong A Chong; José Tapia-Ramirez; Sandra Kim; Juan J Toledo-Aral; Yingcong Zheng; Michael C Boutros; Yelena M Altshuller; Michael A Frohman; Susan D Kraner; Gail Mandel

    1995-01-01

    Expression of the type II voltage-dependent sodium channel gene is restricted to neurons by a silencer element active in nonneuronal cells. We have cloned cDNA coding for a transcription factor (REST) that binds to this silencer element. Expression of a recombinant REST protein confers the ability to silence type II reporter genes in neuronal cell types lacking the native REST

  7. Water-soluble proteins of the human red cell membrane

    Microsoft Academic Search

    J. Th. Hoogeveen; R. Juliano; J. Coleman; A. Rothstein

    1970-01-01

    Summary Procedures were developed for preparation of red cell membranes almost free of hemoglobin but with minimal loss of membrane proteins. Two water-soluble protein fractions are described, each constituting about 25% of the ghost protein. The first is ionically bonded and can be solubilized in water rapidly at pH 7.0 and more slowly at higher ionic strength solutions, with a

  8. Shear strength and water resistance of modified soy protein adhesives

    Microsoft Academic Search

    Xiuzhi Sun; Ke Bian

    1999-01-01

    Soy protein polymers recently have been considered as alternatives to petroleum polymers to ease environmental pollution.\\u000a The use of soy proteins as adhesives for plywood has been limited because of their low water resistance. The objective of\\u000a this research was to test the water resistance of adhesives containing modified soy proteins in walnut, maple, poplar, and\\u000a pine plywood applications. Gluing

  9. Engineering of an E. coli outer membrane protein FhuA with increased channel diameter

    PubMed Central

    2011-01-01

    Background Channel proteins like FhuA can be an alternative to artificial chemically synthesized nanopores. To reach such goals, channel proteins must be flexible enough to be modified in their geometry, i.e. length and diameter. As continuation of a previous study in which we addressed the lengthening of the channel, here we report the increasing of the channel diameter by genetic engineering. Results The FhuA ?1-159 diameter increase has been obtained by doubling the amino acid sequence of the first two N-terminal ?-strands, resulting in variant FhuA ?1-159 Exp. The total number of ?-strands increased from 22 to 24 and the channel surface area is expected to increase by ~16%. The secondary structure analysis by circular dichroism (CD) spectroscopy shows a high ?-sheet content, suggesting the correct folding of FhuA ?1-159 Exp. To further prove the FhuA ?1-159 Exp channel functionality, kinetic measurement using the HRP-TMB assay (HRP = Horse Radish Peroxidase, TMB = 3,3',5,5'-tetramethylbenzidine) were conducted. The results indicated a 17% faster diffusion kinetic for FhuA ?1-159 Exp as compared to FhuA ?1-159, well correlated to the expected channel surface area increase of ~16%. Conclusion In this study using a simple "semi rational" approach the FhuA ?1-159 diameter was enlarged. By combining the actual results with the previous ones on the FhuA ?1-159 lengthening a new set of synthetic nanochannels with desired lengths and diameters can be produced, broadening the FhuA ?1-159 applications. As large scale protein production is possible our approach can give a contribution to nanochannel industrial applications. PMID:21854627

  10. Mercury-sensitive water channels as possible sensors of water potentials in pollen.

    PubMed

    Shachar-Hill, Bruria; Hill, Adrian E; Powell, Janet; Skepper, Jeremy N; Shachar-Hill, Yair

    2013-11-01

    The growing pollen tube is central to plant reproduction and is a long-standing model for cellular tip growth in biology. Rapid osmotically driven growth is maintained under variable conditions, which requires osmosensing and regulation. This study explores the mechanism of water entry and the potential role of osmosensory regulation in maintaining pollen growth. The osmotic permeability of the plasmalemma of Lilium pollen tubes was measured from plasmolysis rates to be 1.32±0.31×10(-3) cm s(-1). Mercuric ions reduce this permeability by 65%. Simulations using an osmotic model of pollen tube growth predict that an osmosensor at the cell membrane controls pectin deposition at the cell tip; inhibiting the sensor is predicted to cause tip bursting due to cell wall thinning. It was found that adding mercury to growing pollen tubes caused such a bursting of the tips. The model indicates that lowering the osmotic permeability per se does not lead to bursting but rather to thickening of the tip. The time course of induced bursting showed no time lag and was independent of mercury concentration, compatible with a surface site of action. The submaximal bursting response to intermediate mercuric ion concentration was independent of the concentration of calcium ions, showing that bursting is not due to a competitive inhibition of calcium binding or entry. Bursting with the same time course was also shown by cells growing on potassium-free media, indicating that potassium channels (implicated in mechanosensing) are not involved in the bursting response. The possible involvement of mercury-sensitive water channels as osmosensors and current knowledge of these in pollen cells are discussed. PMID:24098048

  11. Mercury-sensitive water channels as possible sensors of water potentials in pollen

    PubMed Central

    Hill, Adrian E.

    2013-01-01

    The growing pollen tube is central to plant reproduction and is a long-standing model for cellular tip growth in biology. Rapid osmotically driven growth is maintained under variable conditions, which requires osmosensing and regulation. This study explores the mechanism of water entry and the potential role of osmosensory regulation in maintaining pollen growth. The osmotic permeability of the plasmalemma of Lilium pollen tubes was measured from plasmolysis rates to be 1.32±0.31×10–3 cm s–1. Mercuric ions reduce this permeability by 65%. Simulations using an osmotic model of pollen tube growth predict that an osmosensor at the cell membrane controls pectin deposition at the cell tip; inhibiting the sensor is predicted to cause tip bursting due to cell wall thinning. It was found that adding mercury to growing pollen tubes caused such a bursting of the tips. The model indicates that lowering the osmotic permeability per se does not lead to bursting but rather to thickening of the tip. The time course of induced bursting showed no time lag and was independent of mercury concentration, compatible with a surface site of action. The submaximal bursting response to intermediate mercuric ion concentration was independent of the concentration of calcium ions, showing that bursting is not due to a competitive inhibition of calcium binding or entry. Bursting with the same time course was also shown by cells growing on potassium-free media, indicating that potassium channels (implicated in mechanosensing) are not involved in the bursting response. The possible involvement of mercury-sensitive water channels as osmosensors and current knowledge of these in pollen cells are discussed. PMID:24098048

  12. Capillary-Channeled Polymer (C-CP) Films as Processing Platforms for Protein Analysis by Matrix-Assisted Laser/Desorption Ionization Mass Spectrometry (MALDI-MS)

    NASA Astrophysics Data System (ADS)

    Pittman, Jennifer J.; Manard, Benjamin T.; Kowalski, Paul J.; Marcus, R. Kenneth

    2012-01-01

    Polypropylene (PP) capillary-channeled polymer (C-CP) films have parallel, ?m-sized channels that induce solution wicking via capillary action. Efficient mass transport from the solution phase to the channel surface leads to adsorption of hydrophobic protein solutes. The basic premise by which C-CP films can be used as media to manipulate analyte solutions (e.g., proteins in buffer), for the purpose of desalting or chromatographic separation prior to MALDI-MS analysis is presented here. Cytochrome c and myoglobin prepared in a Tris-HCl buffer, and ribonuclease A, lysozyme, and transferrin prepared in phosphate buffered saline (PBS), are used as the test solutions to demonstrate the desalting concept. Protein analysis is performed after deposition on a C-CP film with and without a water washing step, followed by spray deposition of a typical sinapinic acid matrix. Extracted MALDI mass spectra exhibit much improved signal-to-noise characteristics after water washing. A mixture of cytochrome c and myoglobin (2 ?L of 2.5 ?M each in Tris-HCl buffer) was applied, washed with water and spatially separated via simple capillary action (wicking) using a reversed-phase solvent composition of 0.1% trifluoroacetic acid (TFA) in 50:50 acetonitrile (ACN):H2O. Subsequent application of sinapinic acid followed by imaging of the film using MALDI-MS reveals that as the protein solution is wicked down the film, separation occurs.

  13. Brain Localization and Behavioral Impact of the G-Protein-Gated K1 Channel Subunit GIRK4

    Microsoft Academic Search

    Kevin Wickman; Christine Karschin; Andreas Karschin; Marina R. Picciotto; David E. Clapham

    Neuronal G-protein-gated potassium (KG) channels are activated by several neurotransmitters and constitute an important mode of synaptic inhibition in the mammalian nervous system. KG channels are composed of combinations of four subunits termed G protein-gated inwardly rectifying K1 channels (GIRK). All four GIRK subunits are expressed in the brain, and there is a general consensus concerning the expression patterns of

  14. Activation of inwardly rectifying potassium channels by muscarinic receptor-linked G protein in isolated human ventricular myocytes

    Microsoft Academic Search

    S.-i. Koumi; R. Sato; K. Nagasawa; H. Hayakawa

    1997-01-01

    Muscarinic receptor-linked G protein, Gi, can directely activate the specific K+ channel (I\\u000a K(ach)) in the atrium and in pacemaker tissues in the heart. Coupling of Gi to the K+ channel in the ventricle has not been well defined. G protein regulation of K+ channels in isolated human ventricular myocytes was examined using the patch-clamp technique. Bath application of 1

  15. Estimating field-scale soil water content dynamics with multi-channel GPR

    NASA Astrophysics Data System (ADS)

    Wollschläger, Ute; Buchner, Jens; Schneider, Stefan; Roth, Kurt

    2010-05-01

    Soil water content is a key quantity, for instance, for the exchange of water and energy between land and the atmosphere, for plant growth, and for groundwater recharge. Due to the textural heterogeneity of natural soils, estimating field-scale soil water content variability is challenging. Non-invasive geophysical measurement techniques provide the opportunity to efficiently capture this variability. Ground-penetrating radar (GPR) is a well-established and still expanding method for measuring soil water content. New multi-channel GPR systems allow to efficiently infer soil architecture and soil water content with high lateral resolution. The evaluation of reflection data gives access to the water content within the whole root zone and even deeper sections of the soil profile. This allows, for instance, to gain information about soil water stores which is of high interest to hydrologists. In this presentation, we use examples of field measurements to demonstrate the multi-channel GPR method for estimating field-scale soil architecture and soil water content. Based on these data we conduct synthetic 2D numerical simulations of vadose zone water content dynamics using natural rainfall conditions and calculate the corresponding water contents which would be measured with GPR. Using these results we discuss the implications for the application of multi-channel GPR as a field-scale method for the measurement of soil water content dynamics and to bridge the gap between traditional small-scale point measurements and large-scale remote sensing data.

  16. KATP channel agonists preserve connexin43 protein in infarcted rats by a protein kinase C-dependent pathway

    PubMed Central

    Lee, Tsung-Ming; Lin, Chih-Chan; Lien, Hsiao-Yin; Chen, Chien-Chang

    2012-01-01

    Abstract Downward remodelling of gap junctional proteins between myocytes may trigger ventricular arrhythmia after myocardial infarction. We have demonstrated that ATP-sensitive potassium (KATP) channel agonists attenuated post-infarction arrhythmias. However, the involved mechanisms remain unclear. The purpose of this study was to determine whether KATP channel agonists can attenuate arrhythmias through preserving protein kinase C (PKC)-–dependent connexin43 level after myocardial infarction. Male Wistar rats after ligating coronary artery were randomized to either vehicle, nicorandil, pinacidil, glibenclamide or a combination of nicorandil and glibenclamide or pinacidil and glibenclamide for 4 weeks. To elucidate the role of PKC in the modulation of connexin43 level, carbachol and myristoylated PKC V1–2 peptide were also assessed. Myocardial connexin43 level was significantly decreased in vehicle-treated infarcted rats compared with sham. Attenuated connexin43 level was blunted after administering KATP channel agonists, assessed by immunofluorescent analysis, Western blotting, and real-time quantitative reverse transcription-PCR of connexin43. Arrhythmic scores during programmed stimulation in the KATP channel agonists-treated rats were significantly lower than those treated with vehicle. The beneficial effects of KATP channel agonists were blocked by either glibenclamide or 5-hydroxydecanoate. Addition of the PKC activator, phorbol 12-myristate 13-acetate and the specific PKC agonist, carbachol, blocked the effects of nicorandil on connexin43 phosphorylation and dye permeability. The specific PKC antagonist, myristoylated PKC V1–2 peptide, did not have additional beneficial effects on connexin43 phosphorylation compared with rats treated with nicorandil alone. Chronic use of KATP channel agonists after infarction, resulting in enhanced connexin43 level through a PKC-dependent pathway, may attenuate the arrhythmogenic response to programmed electrical stimulation. PMID:21692984

  17. Mapping energy transfer channels in fucoxanthin-chlorophyll protein complex.

    PubMed

    Gelzinis, Andrius; Butkus, Vytautas; Songaila, Egidijus; Augulis, Ram?nas; Gall, Andrew; Büchel, Claudia; Robert, Bruno; Abramavicius, Darius; Zigmantas, Donatas; Valkunas, Leonas

    2015-02-01

    Fucoxanthin-chlorophyll protein (FCP) is the key molecular complex performing the light-harvesting function in diatoms, which, being a major group of algae, are responsible for up to one quarter of the total primary production on Earth. These photosynthetic organisms contain an unusually large amount of the carotenoid fucoxanthin, which absorbs the light in the blue-green spectral region and transfers the captured excitation energy to the FCP-bound chlorophylls. Due to the large number of fucoxanthins, the excitation energy transfer cascades in these complexes are particularly tangled. In this work we present the two-color two-dimensional electronic spectroscopy experiments on FCP. Analysis of the data using the modified decay associated spectra permits a detailed mapping of the excitation frequency dependent energy transfer flow with a femtosecond time resolution. PMID:25445318

  18. 33 CFR 207.640 - Sacramento Deep Water Ship Channel Barge Lock and Approach Canals; use, administration, and...

    Code of Federal Regulations, 2011 CFR

    2011-07-01

    33 Navigation and Navigable Waters 3 2011-07-01 2011-07-01 false Sacramento Deep Water Ship Channel Barge Lock and Approach Canals...Section 207.640 Navigation and Navigable Waters CORPS OF ENGINEERS, DEPARTMENT OF...

  19. 33 CFR 207.640 - Sacramento Deep Water Ship Channel Barge Lock and Approach Canals; use, administration, and...

    Code of Federal Regulations, 2013 CFR

    2013-07-01

    33 Navigation and Navigable Waters 3 2013-07-01 2013-07-01 false Sacramento Deep Water Ship Channel Barge Lock and Approach Canals...Section 207.640 Navigation and Navigable Waters CORPS OF ENGINEERS, DEPARTMENT OF...

  20. Computational Analysis of the Soluble Form of the Intracellular Chloride Ion Channel Protein CLIC1

    PubMed Central

    Jones, Peter M.; Curmi, Paul M. G.; Valenzuela, Stella M.; George, Anthony M.

    2013-01-01

    The chloride intracellular channel (CLIC) family of proteins has the remarkable property of maintaining both a soluble form and an integral membrane form acting as an ion channel. The soluble form is structurally related to the glutathione-S-transferase family, and CLIC can covalently bind glutathione via an active site cysteine. We report approximately 0.6??s of molecular dynamics simulations, encompassing the three possible ligand-bound states of CLIC1, using the structure of GSH-bound human CLIC1. Noncovalently bound GSH was rapidly released from the protein, whereas the covalently ligand-bound protein remained close to the starting structure over 0.25??s of simulation. In the unliganded state, conformational changes in the vicinity of the glutathione-binding site resulted in reduced reactivity of the active site thiol. Elastic network analysis indicated that the changes in the unliganded state are intrinsic to the protein architecture and likely represent functional transitions. Overall, our results are consistent with a model of CLIC function in which covalent binding of glutathione does not occur spontaneously but requires interaction with another protein to stabilise the GSH binding site and/or transfer of the ligand. The results do not indicate how CLIC1 undergoes a radical conformational change to form a transmembrane chloride channel but further elucidate the mechanism by which CLICs are redox controlled. PMID:24089665

  1. Small heat shock protein alphaA-crystallin regulates epithelial sodium channel expression.

    PubMed

    Kashlan, Ossama B; Mueller, Gunhild M; Qamar, Mohammad Z; Poland, Paul A; Ahner, Annette; Rubenstein, Ronald C; Hughey, Rebecca P; Brodsky, Jeffrey L; Kleyman, Thomas R

    2007-09-21

    Integral membrane proteins are synthesized on the cytoplasmic face of the endoplasmic reticulum (ER). After being translocated or inserted into the ER, they fold and undergo post-translational modifications. Within the ER, proteins are also subjected to quality control checkpoints, during which misfolded proteins may be degraded by proteasomes via a process known as ER-associated degradation. Molecular chaperones, including the small heat shock protein alphaA-crystallin, have recently been shown to play a role in this process. We have now found that alphaA-crystallin is expressed in cultured mouse collecting duct cells, where apical Na(+) transport is mediated by epithelial Na(+) channels (ENaC). ENaC-mediated Na(+) currents in Xenopus oocytes were reduced by co-expression of alphaA-crystallin. This reduction in ENaC activity reflected a decrease in the number of channels expressed at the cell surface. Furthermore, we observed that the rate of ENaC delivery to the cell surface of Xenopus oocytes was significantly reduced by co-expression of alphaA-crystallin, whereas the rate of channel retrieval remained unchanged. We also observed that alphaA-crystallin and ENaC co-immunoprecipitate. These data are consistent with the hypothesis that small heat shock proteins recognize ENaC subunits at ER quality control checkpoints and can target ENaC subunits for ER-associated degradation. PMID:17664274

  2. Cephalopod consumption by trawl caught sh in Scottish and English Channel waters

    E-print Network

    Pierce, Graham

    Cephalopod consumption by trawl caught ®sh in Scottish and English Channel waters H.I. Dalya , G waters, they are often of limited use for estimating the impact of ®sh predation on cephalopod resources. This is due to either the origin of the ®sh sampled, e.g. from areas with a relatively low cephalopod

  3. Laboratory Modeling of Self-Formed Leveed Channels From Sediment-Laden Flows Entering Still Water

    NASA Astrophysics Data System (ADS)

    Rowland, J. C.; Dietrich, W. E.

    2004-12-01

    Self-formed leveed channels constructed by deposition of suspended sediment from sediment-laden flows entering still water are common features in nature. Such channels drive delta progradation, develop at tidal inlets and occur where mainstem river flows empty into oxbows and blocked valley lakes. Presently there is no theory for the formation of such channels. This lack of theory is partly due to a lack of field or laboratory studies that provide insight about the mechanism controlling these self-formed, propagating channels. The creation of such features in the laboratory, have proved illusive to date. Our ongoing experiments aimed at modeling the formation of floodplain tie channels provide insight into the necessary conditions for levee formation and channel growth. Under conditions of steady water discharge, constant sediment feed rate, unimodal sediment distribution and invariant basin stage we are able to create subaqueous lateral bars (submerged levees) along the margins of a sediment laden jet. Our results highlight the sensitivity of channel formation to issues of scaling and experimental design. In the laboratory, levee formation has only been possible with the use of plastic particles (specific gravity ~1.5); complete bed alluviation and dune formation results from the use of particles with specific gravities of ~ 2.65 across a range grain diameters and shapes. We hypothesize this effect is related to high entrainment thresholds relative to suspension thresholds of small (< 100 mm) natural particles under conditions of reduced turbulence in laboratory scaled flows. Additionally, both the width to depth ratio and the form of the outlet channel introducing the sediment laden flow into the experimental basin exert a strong control on sedimentation pattern and levee growth. Continuing experiments are focused on generating emergent channel levees and a basin ward propagation of the channel by adjusting the form of the feed channel, varying basin stage, and the use of unsteady discharge.

  4. Remote Sensing of Water Vapor and Thin Cirrus Clouds using MODIS Near-IR Channels

    NASA Technical Reports Server (NTRS)

    Gao, Bo-Cai; Kaufman, Yoram J.

    2001-01-01

    The Moderate Resolution Imaging Spectroradiometer (MODIS), a major facility instrument on board the Terra Spacecraft, was successfully launched into space in December of 1999. MODIS has several near-IR channels within and around the 0.94 micrometer water vapor bands for remote sensing of integrated atmospheric water vapor over land and above clouds. MODIS also has a special near-IR channel centered at 1.375-micron with a width of 30 nm for remote sensing of cirrus clouds. In this paper, we describe briefly the physical principles on remote sensing of water vapor and cirrus clouds using these channels. We also present sample water vapor images and cirrus cloud images obtained from MODIS data.

  5. Water slug to drop and film transitions in gas-flow channels.

    PubMed

    Cheah, May Jean; Kevrekidis, Ioannis G; Benziger, Jay B

    2013-12-01

    Water emerging from micrometer-sized pores into millimeter-sized gas-flow channels forms drops. The drops grow until the force from the flowing gas is sufficient to detach the drops as either (1) slugs that completely occlude the cross section of the channel and move at the superficial gas velocity, (2) drops that partially occlude the channel and move at a velocity that is less than the gas velocity, or (3) films that flow continuously, occluding part of the channel. At steady state, small residual water droplets, ?100 ?m in diameter, left in corners and on surface defects from previous drops, are key in determining the shape of water drops at detachment. Slugs are formed at low-gas-phase Reynolds numbers (ReG) in both hydrophilic and hydrophobic channels. Drops are shed in Teflon-coated hydrophobic channels for ReG > 30. Films are formed in acrylic hydrophilic channels for ReG > 30. Slugs form when growing drops encounter residual water droplets that nucleate the drop to slug transition. Drops are shed when the force exerted by the flowing gas on growing drops exceeds the force needed to advance the gas/liquid/solid contact line before they grow to the critical size for the drop to slug transition. Drops grow by "stick-slip" of the solid-liquid-gas contact lines and with pinned contact lines until the force on the drops results in either the downstream contact angle becoming greater than the dynamic advancing contact angle or the upstream contact angle becoming less than the dynamic receding contact angle. The upstream contact line never detaches for hydrophilic channels, which is why films form. The shape of water drops and the detachment energies are shown to be well approximated by the force balance between the force needed to advance the drop's contact lines and the force that the flowing gas exerts on a stationary drop. PMID:24206393

  6. The threshold of vapor channel formation in water induced by pulsed CO2 laser

    NASA Astrophysics Data System (ADS)

    Guo, Wenqing; Zhang, Xianzeng; Zhan, Zhenlin; Xie, Shusen

    2012-12-01

    Water plays an important role in laser ablation. There are two main interpretations of laser-water interaction: hydrokinetic effect and vapor phenomenon. The two explanations are reasonable in some way, but they can't explain the mechanism of laser-water interaction completely. In this study, the dynamic process of vapor channel formation induced by pulsed CO2 laser in static water layer was monitored by high-speed camera. The wavelength of pulsed CO2 laser is 10.64 um, and pulse repetition rate is 60 Hz. The laser power ranged from 1 to 7 W with a step of 0.5 W. The frame rate of high-speed camera used in the experiment was 80025 fps. Based on high-speed camera pictures, the dynamic process of vapor channel formation was examined, and the threshold of vapor channel formation, pulsation period, the volume, the maximum depth and corresponding width of vapor channel were determined. The results showed that the threshold of vapor channel formation was about 2.5 W. Moreover, pulsation period, the maximum depth and corresponding width of vapor channel increased with the increasing of the laser power.

  7. Cytosolic Na+ Controls an Epithelial Na+ Channel Via the Go Guanine Nucleotide-Binding Regulatory Protein

    NASA Astrophysics Data System (ADS)

    Komwatana, P.; Dinudom, A.; Young, J. A.; Cook, D. I.

    1996-07-01

    In tight Na+-absorbing epithelial cells, the rate of Na+ entry through amiloride-sensitive apical membrane Na+ channels is matched to basolateral Na+ extrusion so that cell Na+ concentration and volume remain steady. Control of this process by regulation of apical Na+ channels has been attributed to changes in cytosolic Ca2+ concentration or pH, secondary to changes in cytosolic Na+ concentration, although cytosolic Cl- seems also to be involved. Using mouse mandibular gland duct cells, we now demonstrate that increasing cytosolic Na+ concentration inhibits apical Na+ channels independent of changes in cytosolic Ca2+, pH, or Cl-, and the effect is blocked by GDP-? -S, pertussis toxin, and antibodies against the ? -subunits of guanine nucleotide-binding regulatory proteins (Go). In contrast, the inhibitory effect of cytosolic anions is blocked by antibodies to inhibitory guanine nucleotide-binding regulatory proteins (Gi1/Gi2. It thus appears that apical Na+ channels are regulated by Go and Gi proteins, the activities of which are controlled, respectively, by cytosolic Na+ and Cl-.

  8. KCl Mediates K+ Channel-Activated Mitogen-Activated Protein Kinases Signaling in Wound Healing

    PubMed Central

    Shim, Jung Hee; Lim, Jong Woo; Kim, Byeong Kyu; Park, Soo Jin; Kim, Suk Wha

    2015-01-01

    Background Wound healing is an interaction of a complex signaling cascade of cellular events, including inflammation, proliferation, and maturation. K+ channels modulate the mitogen-activated protein kinase (MAPK) signaling pathway. Here, we investigated whether K+ channel-activated MAPK signaling directs collagen synthesis and angiogenesis in wound healing. Methods The human skin fibroblast HS27 cell line was used to examine cell viability and collagen synthesis after potassium chloride (KCl) treatment by Cell Counting Kit-8 (CCK-8) and western blotting. To investigate whether K+ ion channels function upstream of MAPK signaling, thus affecting collagen synthesis and angiogenesis, we examined alteration of MAPK expression after treatment with KCl (channel inhibitor), NS1619 (channel activator), or kinase inhibitors. To research the effect of KCl on angiogenesis, angiogenesis-related proteins such as thrombospondin 1 (TSP1), anti-angiogenic factor, basic fibroblast growth factor (bFGF) and vascular endothelial growth factor (VEGF), pro-angiogenic factor were assayed by western blot. Results The viability of HS27 cells was not affected by 25 mM KCl. Collagen synthesis increased dependent on time and concentration of KCl exposure. The phosphorylations of MAPK proteins such as extracellular-signal-regulated kinase (ERK) and p38 increased about 2.5-3 fold in the KCl treatment cells and were inhibited by treatment of NS1619. TSP1 expression increased by 100%, bFGF expression decreased by 40%, and there is no significant differences in the VEGF level by KCl treatment, TSP1 was inhibited by NS1619 or kinase inhibitors. Conclusions Our results suggest that KCl may function as a therapeutic agent for wound healing in the skin through MAPK signaling mediated by the K+ ion channel. PMID:25606484

  9. Viral M2 ion channel protein: a promising target for anti-influenza drug discovery.

    PubMed

    Moorthy, N S Hari Narayana; Poongavanam, Vasanthanathan; Pratheepa, V

    2014-01-01

    Influenza virus is an important RNA virus causing pandemics (Spanish Flu (1918), Asian Flu (1957), Hong Kong Flu (1968) and Swine Flu (2009)) over the last decades. Due to the spontaneous mutations of these viral proteins, currently available antiviral and anti-influenza drugs quickly develop resistance. To account this, only limited antiinfluenza drugs have been approved for the therapeutic use. These include amantadine and rimantadine (M2 proton channel blockers), zanamivir, oseltamivir and peramivir (neuraminidase inhibitors), favipravir (polymerase inhibitor) and laninamivir. This review provides an outline on the strategies to develop novel, potent chemotherapeutic agents against M2 proton channel. Primarily, the M2 proton channel blockers elicit pharmacological activity through destabilizing the helices by blocking the proton transport across the transmembrane. The biologically important compounds discovered using the scaffolds such as bisnoradmantane, noradamantane, triazine, spiroadamantane, isoxazole, amino alcohol, azaspiro, spirene, pinanamine, etc are reported to exhibit anti-influenza activity against wild or mutant type (S31N and V27A) of M2 proton channel protein. The reported studies explained that the adamantane based compounds (amantadine and rimantadine) strongly interact with His37 (through hydrogen bonding) and Ala30, Ile33 and Gly34 residues (hydrophobic interactions). The adamantane and the non-adamantane scaffolds fit perfectly in the active site pocket present in the wild type and the charged amino groups (ammonium) create positive electrostatic potential, which blocks the transport of protons across the pore. In the mutated proteins, larger or smaller binding pocket are created by small or large mutant residues, which do not allow the molecules fit in the active site. This causes the channel to be unblocked and the protons are allowed to transfer inside the pore. The structural analysis of the M2 proton channel blockers illustrated that the adamantane derivatives have action against both influenza A and B, but have no effect on the mutants. PMID:25342196

  10. AKAP79/150 signal complexes in G-protein modulation of neuronal ion channels

    PubMed Central

    Zhang, Jie; Bal, Manjot; Bierbower, Sonya; Zaika, Oleg; Shapiro, Mark S.

    2011-01-01

    Voltage-gated M-type (KCNQ) K+ channels play critical roles in regulation of neuronal excitability. Previous work showed A-kinase-anchoring protein (AKAP)79/150-mediated protein kinase C phosphorylation of M channels to be involved in M current (IM) suppression by muscarinic M1, but not bradykinin B2 receptors. In this study, we first explored if purinergic and angiotensin suppression of IM in superior cervical ganglion (SCG) sympathetic neurons involves AKAP79/150. Transfection into rat SCG neurons of ?A-AKAP79, which lacks the A-domain necessary for PKC binding, or the absence of AKAP150 in AKAP150 (?/?) mice, did not affect IM suppression by purinergic agonist or by bradykinin, but reduced IM suppression by muscarinic agonist and angiotensin II. Transfection of AKAP79, but not ?A-AKAP79 or AKAP15, “rescued” suppression of IM by muscarinic receptors in AKAP150 (?/?) neurons. We also tested association of AKAP79 with M1, B2, P2Y6 and AT1 receptors, and KCNQ2 and KCNQ3 channels, via Förster resonance energy transfer on CHO cells under total internal refection fluorescence microscopy, which revealed substantial FRET between AKAP79 and M1 and AT1 receptors, and with the channels, but only weak FRET with P2Y6 or B2 receptors. The involvement of AKAP79/150 in Gq/11-coupled muscarinic regulation of N- and L-type Ca2+ channels and by cAMP/protein kinase A was also studied. We found AKAP79/150 to not play a role in the former, but to be necessary for forskolin-induced up-regulation of L-current. Thus, AKAP79/150 action correlates with the PIP2-depletion mode of IM suppression, but does not generalize to Gq/11-mediated inhibition of N- or L-type Ca2+ channels. PMID:21562284

  11. Homer proteins mediate the interaction between STIM1 and Cav1.2 channels.

    PubMed

    Dionisio, Natalia; Smani, Tarik; Woodard, Geoffrey E; Castellano, Antonio; Salido, Gines M; Rosado, Juan A

    2015-05-01

    STIM1 is a ubiquitous Ca(2+) sensor of the intracellular, agonist-sensitive, Ca(2+) stores that communicates the filling state of the Ca(2+) compartments to plasma membrane store-operated Ca(2+) (SOC) channels. STIM1 has been presented as a point of convergence between store-operated and voltage-operated Ca(2+) influx, both inducing activation of SOC channels while suppressing Cav1.2 channels. Here we report that Homer proteins play a relevant role in the communication between STIM1 and Cav1.2 channels. HEK-293 cells transiently expressing Cav1.2 channel subunits ?1, ?2 and ?2?-1 exhibited a significant Ca(2+) entry upon treatment with a high concentration of KCl. In Cav1.2-expressing cells, treatment with thapsigargin (TG), to induce passive discharge of the intracellular Ca(2+) stores, resulted in Ca(2+) influx that was significantly greater than in cells not expressing Cav1.2 channels, a difference that was abolished by nifedipine and diltiazem. Treatment with TG induces co-immunoprecipitation of Homer1 with STIM1 and the Cav1.2 ?1 subunit. Impairment of Homer function by introduction of the synthetic PPKKFR peptide into cells, which emulates the proline-rich sequences of the PPXXF motif, or using siRNA Homer1, reduced the association of STIM1 and the Cav1.2 ?1 subunit. These findings indicate that Homer is important for the association between both proteins. Finally, treatment with siRNA Homer1 or the PPKKFR peptide enhanced the nifedipine-sensitive component of TG response in Cav1.2-expressing cells. Altogether, these findings provide evidence for a new role of Homer1 supporting the regulation of Cav1.2 channels by STIM1. PMID:25712868

  12. Plant plasma membrane water channels conduct the signalling molecule H2O2.

    PubMed

    Dynowski, Marek; Schaaf, Gabriel; Loque, Dominique; Moran, Oscar; Ludewig, Uwe

    2008-08-15

    H(2)O(2) is a relatively long-lived reactive oxygen species that signals between cells and organisms. H(2)O(2) signalling in plants is essential for response to stress, defence against pathogens and the regulation of programmed cell death. Although H(2)O(2) diffusion across membranes is often considered as a passive property of lipid bilayers, native membranes represent significant barriers for H(2)O(2). In the present study we addressed the question of whether channels might facilitate H(2)O(2) conduction across plasma membranes. The expression of several plant plasma membrane aquaporins in yeast, including PIP2;1 from Arabidopsis (where PIP is plasma membrane intrinsic protein), enhanced the toxicity of H(2)O(2) and increased the fluorescence of dye-loaded yeast when exposed to H(2)O(2). The sensitivity of aquaporin-expressing yeast to H(2)O(2) was altered by mutations that alter gating and the selectivity of the aquaporins. The conduction of water, H(2)O(2) and urea was compared, using molecular dynamics simulations based on the crystal structure of SoPIP2;1 from spinach. The calculations identify differences in the conduction between the substrates and reveal channel residues critically involved in H(2)O(2) conduction. The results of the calculations on tetramers and monomers are in agreement with the biochemical data. Taken together, the results strongly suggest that plasma membrane aquaporin pores determine the efficiency of H(2)O(2) signalling between cells. Aquaporins are present in most species and their capacity to facilitate the diffusion of H(2)O(2) may be of physiological significance in many organisms and particularly in communication between different species. PMID:18462192

  13. Communication: Protein dynamical transition vs. liquid-liquid phase transition in protein hydration water.

    PubMed

    Schirò, Giorgio; Fomina, Margarita; Cupane, Antonio

    2013-09-28

    In this work, we compare experimental data on myoglobin hydrated powders from elastic neutron scattering, broadband dielectric spectroscopy, and differential scanning calorimetry. Our aim is to obtain new insights on the connection between the protein dynamical transition, a fundamental phenomenon observed in proteins whose physical origin is highly debated, and the liquid-liquid phase transition (LLPT) possibly occurring in protein hydration water and related to the existence of a low temperature critical point in supercooled water. Our results provide a consistent thermodynamic/dynamic description which gives experimental support to the LLPT hypothesis and further reveals how fundamental properties of water and proteins are tightly related. PMID:24089711

  14. Communication: Protein dynamical transition vs. liquid-liquid phase transition in protein hydration water

    NASA Astrophysics Data System (ADS)

    Schirò, Giorgio; Fomina, Margarita; Cupane, Antonio

    2013-09-01

    In this work, we compare experimental data on myoglobin hydrated powders from elastic neutron scattering, broadband dielectric spectroscopy, and differential scanning calorimetry. Our aim is to obtain new insights on the connection between the protein dynamical transition, a fundamental phenomenon observed in proteins whose physical origin is highly debated, and the liquid-liquid phase transition (LLPT) possibly occurring in protein hydration water and related to the existence of a low temperature critical point in supercooled water. Our results provide a consistent thermodynamic/dynamic description which gives experimental support to the LLPT hypothesis and further reveals how fundamental properties of water and proteins are tightly related.

  15. Structural Waters Define a Functional Channel Mediating Activation of the GPCR, rhodopsin

    SciTech Connect

    Angel, T.; Gupta, S; Jastrzebska, B; Palczewski, K; Chance, M

    2009-01-01

    Structural water molecules may act as prosthetic groups indispensable for proper protein function. In the case of allosteric activation of G protein-coupled receptors (GPCRs), water likely imparts structural plasticity required for agonist-induced signal transmission. Inspection of structures of GPCR superfamily members reveals the presence of conserved embedded water molecules likely important to GPCR function. Coupling radiolytic hydroxyl radical labeling with rapid H2O18 solvent mixing, we observed no exchange of these structural waters with bulk solvent in either ground state or for the Meta II or opsin states. However, the radiolysis approach permitted labeling of selected side chain residues within the transmembrane helices and revealed activation-induced changes in local structural constraints likely mediated by dynamics of both water and protein. These results suggest both a possible general mechanism for water-dependent communication in family A GPCRs based on structural conservation, and a strategy for probing membrane protein structure.

  16. Establishing homology between mitochondrial calcium uniporters, prokaryotic magnesium channels and chlamydial IncA proteins.

    PubMed

    Lee, Andre; Vastermark, Ake; Saier, Milton H

    2014-08-01

    Mitochondrial calcium uniporters (MCUs) (TC no. 1.A.77) are oligomeric channel proteins found in the mitochondrial inner membrane. MCUs have two well-conserved transmembrane segments (TMSs), connected by a linker, similar to bacterial MCU homologues. These proteins and chlamydial IncA proteins (of unknown function; TC no. 9.B.159) are homologous to prokaryotic Mg(2+) transporters, AtpI and AtpZ, based on comparison scores of up to 14.5 sds. A phylogenetic tree containing all of these proteins showed that the AtpZ proteins cluster coherently as a subset within the large and diverse AtpI cluster, which branches separately from the MCUs and IncAs, both of which cluster coherently. The MCUs and AtpZs share the same two TMS topology, but the AtpIs have four TMSs, and IncAs can have either two (most frequent) or four (less frequent) TMSs. Binary alignments, comparison scores and motif analyses showed that TMSs 1 and 2 align with TMSs 3 and 4 of the AtpIs, suggesting that the four TMS AtpI proteins arose via an intragenic duplication event. These findings establish an evolutionary link interconnecting eukaryotic and prokaryotic Ca(2+) and Mg(2+) transporters with chlamydial IncAs, and lead us to suggest that all members of the MCU superfamily, including IncAs, function as divalent cation channels. PMID:24869855

  17. Leptin Regulates KATP Channel Trafficking in Pancreatic ?-Cells by a Signaling Mechanism Involving AMP-activated Protein Kinase (AMPK) and cAMP-dependent Protein Kinase (PKA)* ?

    PubMed Central

    Chen, Pei-Chun; Kryukova, Yelena N.; Shyng, Show-Ling

    2013-01-01

    Pancreatic ?-cells secrete insulin in response to metabolic and hormonal signals to maintain glucose homeostasis. Insulin secretion is under the control of ATP-sensitive potassium (KATP) channels that play key roles in setting ?-cell membrane potential. Leptin, a hormone secreted by adipocytes, inhibits insulin secretion by increasing KATP channel conductance in ?-cells. We investigated the mechanism by which leptin increases KATP channel conductance. We show that leptin causes a transient increase in surface expression of KATP channels without affecting channel gating properties. This increase results primarily from increased channel trafficking to the plasma membrane rather than reduced endocytosis of surface channels. The effect of leptin on KATP channels is dependent on the protein kinases AMP-activated protein kinase (AMPK) and PKA. Activation of AMPK or PKA mimics and inhibition of AMPK or PKA abrogates the effect of leptin. Leptin activates AMPK directly by increasing AMPK phosphorylation at threonine 172. Activation of PKA leads to increased channel surface expression even in the presence of AMPK inhibitors, suggesting AMPK lies upstream of PKA in the leptin signaling pathway. Leptin signaling also leads to F-actin depolymerization. Stabilization of F-actin pharmacologically occludes, whereas destabilization of F-actin simulates, the effect of leptin on KATP channel trafficking, indicating that leptin-induced actin reorganization underlies enhanced channel trafficking to the plasma membrane. Our study uncovers the signaling and cellular mechanism by which leptin regulates KATP channel trafficking to modulate ?-cell function and insulin secretion. PMID:24100028

  18. Severe Acute Respiratory Syndrome Coronavirus Envelope Protein Ion Channel Activity Promotes Virus Fitness and Pathogenesis

    PubMed Central

    Nieto-Torres, Jose L.; DeDiego, Marta L.; Verdiá-Báguena, Carmina; Jimenez-Guardeño, Jose M.; Regla-Nava, Jose A.; Fernandez-Delgado, Raul; Castaño-Rodriguez, Carlos; Alcaraz, Antonio; Torres, Jaume; Aguilella, Vicente M.; Enjuanes, Luis

    2014-01-01

    Deletion of Severe Acute Respiratory Syndrome Coronavirus (SARS-CoV) envelope (E) gene attenuates the virus. E gene encodes a small multifunctional protein that possesses ion channel (IC) activity, an important function in virus-host interaction. To test the contribution of E protein IC activity in virus pathogenesis, two recombinant mouse-adapted SARS-CoVs, each containing one single amino acid mutation that suppressed ion conductivity, were engineered. After serial infections, mutant viruses, in general, incorporated compensatory mutations within E gene that rendered active ion channels. Furthermore, IC activity conferred better fitness in competition assays, suggesting that ion conductivity represents an advantage for the virus. Interestingly, mice infected with viruses displaying E protein IC activity, either with the wild-type E protein sequence or with the revertants that restored ion transport, rapidly lost weight and died. In contrast, mice infected with mutants lacking IC activity, which did not incorporate mutations within E gene during the experiment, recovered from disease and most survived. Knocking down E protein IC activity did not significantly affect virus growth in infected mice but decreased edema accumulation, the major determinant of acute respiratory distress syndrome (ARDS) leading to death. Reduced edema correlated with lung epithelia integrity and proper localization of Na+/K+ ATPase, which participates in edema resolution. Levels of inflammasome-activated IL-1? were reduced in the lung airways of the animals infected with viruses lacking E protein IC activity, indicating that E protein IC function is required for inflammasome activation. Reduction of IL-1? was accompanied by diminished amounts of TNF and IL-6 in the absence of E protein ion conductivity. All these key cytokines promote the progression of lung damage and ARDS pathology. In conclusion, E protein IC activity represents a new determinant for SARS-CoV virulence. PMID:24788150

  19. A large iris-like expansion of a mechanosensitive channel protein induced by membrane tension

    NASA Technical Reports Server (NTRS)

    Betanzos, Monica; Chiang, Chien-Sung; Guy, H. Robert; Sukharev, Sergei

    2002-01-01

    MscL, a bacterial mechanosensitive channel of large conductance, is the first structurally characterized mechanosensor protein. Molecular models of its gating mechanisms are tested here. Disulfide crosslinking shows that M1 transmembrane alpha-helices in MscL of resting Escherichia coli are arranged similarly to those in the crystal structure of MscL from Mycobacterium tuberculosis. An expanded conformation was trapped in osmotically shocked cells by the specific bridging between Cys 20 and Cys 36 of adjacent M1 helices. These bridges stabilized the open channel. Disulfide bonds engineered between the M1 and M2 helices of adjacent subunits (Cys 32-Cys 81) do not prevent channel gating. These findings support gating models in which interactions between M1 and M2 of adjacent subunits remain unaltered while their tilts simultaneously increase. The MscL barrel, therefore, undergoes a large concerted iris-like expansion and flattening when perturbed by membrane tension.

  20. Dynamic ion-ion and water-ion interactions in ion channels.

    PubMed Central

    Wu, J V

    1992-01-01

    The dynamic interactions among ions and water molecules in ion channels are treated based on an assumption that ions at binding sites can be knocked off by both transient entering ions and local water molecules. The theory, when applied to a single-site model K+ channel, provides solutions for super- and subsaturations, flux-ratio exponent (n') greater than 1, osmotic streaming current, activity-dependent reversal potentials, and anomalous mole-fraction behavior. The analysis predicts that: (a) the saturation may but, in general, does not follow the Michaelis-Menten relation; (b) streaming current results from imbalanced water-ion knock-off interactions; (c) n' greater than 1 even for single-site channels, but it is unlikely to exceed 1.4 unless the pore is occupied by one or more ion(s); (d) in the calculation involving two permeant ion species with similar radii, the heavier ions show higher affinity; the ion-ion knock-off dissociation from the site is more effective when two interacting ions are identical. Therefore, the "multi-ion behaviors" found in most ion channels are the consequences of dynamic ion-ion and water-ion interactions. The presence of these interactions does not require two or more binding sites in channels. PMID:1376158

  1. Hydration-dependent dynamic crossover phenomenon in protein hydration water

    E-print Network

    Wang, Zhe

    The characteristic relaxation time ? of protein hydration water exhibits a strong hydration level h dependence. The dynamic crossover is observed when h is higher than the monolayer hydration level h[subscript c] =0.2–0.25 ...

  2. Regulation of K+ Channels in Maize Roots by Water Stress and Abscisic Acid1

    PubMed Central

    Roberts, Stephen K.

    1998-01-01

    Root cortical and stelar protoplasts were isolated from maize (Zea mays L.) plants that were either well watered or water stressed, and the patch-clamp technique was used to investigate their plasma membrane K+ channel activity. In the root cortex water stress did not significantly affect inward- or outward-rectifying K+ conductances relative to those observed in well-watered plants. In contrast, water stress significantly reduced the magnitude of the outward-rectifying K+ current in the root stele but had little effect on the inward-rectifying K+ current. Pretreating well-watered plants with abscisic acid also significantly affected K+ currents in a way that was consistent with abscisic acid mediating, at least in part, the response of roots to water stress. It is proposed that the K+ channels underlying the K+ currents in the root stelar cells represent pathways that allow K+ exchange between the root symplasm and xylem apoplast. It is suggested that the regulation of K+ channel activity in the root in response to water stress could be part of an important adaptation of the plant to survive drying soils.

  3. An outer membrane channel protein of Mycobacterium tuberculosis with exotoxin activity

    PubMed Central

    Danilchanka, Olga; Sun, Jim; Pavlenok, Mikhail; Maueröder, Christian; Speer, Alexander; Siroy, Axel; Marrero, Joeli; Trujillo, Carolina; Mayhew, David L.; Doornbos, Kathryn S.; Muñoz, Luis E.; Herrmann, Martin; Ehrt, Sabine; Berens, Christian; Niederweis, Michael

    2014-01-01

    The ability to control the timing and mode of host cell death plays a pivotal role in microbial infections. Many bacteria use toxins to kill host cells and evade immune responses. Such toxins are unknown in Mycobacterium tuberculosis. Virulent M. tuberculosis strains induce necrotic cell death in macrophages by an obscure molecular mechanism. Here we show that the M. tuberculosis protein Rv3903c (channel protein with necrosis-inducing toxin, CpnT) consists of an N-terminal channel domain that is used for uptake of nutrients across the outer membrane and a secreted toxic C-terminal domain. Infection experiments revealed that CpnT is required for survival and cytotoxicity of M. tuberculosis in macrophages. Furthermore, we demonstrate that the C-terminal domain of CpnT causes necrotic cell death in eukaryotic cells. Thus, CpnT has a dual function in uptake of nutrients and induction of host cell death by M. tuberculosis. PMID:24753609

  4. An efficient method for modeling kinetic behavior of channel proteins in cardiomyocytes.

    PubMed

    Wang, Chong; Beyerlein, Peter; Pospisil, Heike; Krause, Antje; Nugent, Chris; Dubitzky, Werner

    2012-01-01

    Characterization of the kinetic and conformational properties of channel proteins is a crucial element in the integrative study of congenital cardiac diseases. The proteins of the ion channels of cardiomyocytes represent an important family of biological components determining the physiology of the heart. Some computational studies aiming to understand the mechanisms of the ion channels of cardiomyocytes have concentrated on Markovian stochastic approaches. Mathematically, these approaches employ Chapman-Kolmogorov equations coupled with partial differential equations. As the scale and complexity of such subcellular and cellular models increases, the balance between efficiency and accuracy of algorithms becomes critical. We have developed a novel two-stage splitting algorithm to address efficiency and accuracy issues arising in such modeling and simulation scenarios. Numerical experiments were performed based on the incorporation of our newly developed conformational kinetic model for the rapid delayed rectifier potassium channel into the dynamic models of human ventricular myocytes. Our results show that the new algorithm significantly outperforms commonly adopted adaptive Runge-Kutta methods. Furthermore, our parallel simulations with coupled algorithms for multicellular cardiac tissue demonstrate a high linearity in the speedup of large-scale cardiac simulations. PMID:21576757

  5. Detection of ion channel activity in Xenopus laevis oocytes expressing Influenza C virus CM2 protein.

    PubMed

    Hongo, S; Ishii, K; Mori, K; Takashita, E; Muraki, Y; Matsuzaki, Y; Sugawara, K

    2004-01-01

    To demonstrate the ion channel activity of Influenza C virus CM2, we expressed this protein in oocytes of Xenopus laevis and measured whole cell currents by a two-electrode voltage-clamp method. It was found that the inward currents were induced upon hyperpolarizing the oocyte membranes. The amplitude of the currents increased slowly with time during the hyperpolarizing pulse, and the current-voltage relationship was nonlinear, having a slope that increased with the level of hyperpolarization. These results suggest strongly that CM2 forms a voltage-activated ion channel. The current amplitude was increased to a small extent by lowering the external pH. We also found that the anti-influenza A virus drug amantadine hydrochloride failed to attenuate the inward currents of CM2-expressing oocytes induced by hyperpolarization. The reversal voltage of tail currents was affected by the reduction of [Cl-], but neither by the change of [Na+] nor by that of [K+]. Furthermore, the amplitude of the inward currents was decreased by an anion channel blocker. The data presented here suggest that CM2 protein forms a voltage-activated ion channel permeable to chloride ion. PMID:14689274

  6. TMEM16A Protein: A New Identity for Ca2+-Dependent Cl? Channels

    NSDL National Science Digital Library

    Loretta Ferrera (Istituto Giannina Gaslini)

    2010-12-01

    Ca+-dependent Cl? channels (CaCCs) play a variety of physiological roles in different organs and tissues, including transepithelial Cl? secretion, smooth muscle contraction, regulation of neuronal excitability, and transduction of sensory stimuli. The recent identification of TMEM16A protein as an important component of CaCCs should allow a better understanding of their physiological role, structure-function relationship, and regulatory mechanisms.

  7. Vertical arrival structure of shipping noise in deep water channels

    Microsoft Academic Search

    Zizheng Li; Lisa M. Zurk; Barry Ma

    2010-01-01

    In passive sonar systems, knowledge of low-frequency shipping noise is significant for target detection performance. However, an accurate model for the shipping noise structure is difficult to obtain, because of the varying distributions of ships and complicated underwater environment. This work characterizes low-frequency distant shipping noise observed in deep water environments as a function of receiver depth and vertical arrival

  8. Capacitation and Ca(2+) influx in spermatozoa: role of CNG channels and protein kinase G.

    PubMed

    Cisneros-Mejorado, A; Hernández-Soberanis, L; Islas-Carbajal, M C; Sánchez, D

    2014-01-01

    Cyclic guanosine monophosphate (cGMP) has been recently shown to modulate in vitro capacitation of mammalian spermatozoa, but the mechanisms through which it influences sperm functions have not been clarified. There are at least two targets of cGMP, cyclic nucleotide-gated (CNG) channels and cGMP-dependent protein kinase (PKG), involved in several physiological events in mammalian spermatozoa. It has been suggested that CNG channels allow the influx of Ca(2+) to cytoplasm during capacitation, whereas PKG could trigger a phosphorylation pathway which might also, indirectly, mediate calcium entry. Using the patch-clamp technique in whole-cell configuration, we showed how l-cis-Diltiazem (a CNG-channel inhibitor) and KT5823 (a PKG inhibitor) decreased significantly the amplitude of macroscopic ion currents in a dose-response manner, and decreased in vitro capacitation. The inhibition of CNG channels completely abolishes the Ca(2+) influx induced by cyclic nucleotides in mouse spermatozoa. This work suggests that the downstream cGMP pathway is required in mammalian sperm capacitation and the mechanisms involved include CNG channels and PKG, highlighting these molecules as important therapeutic targets for infertility treatments or to develop new male contraceptives. PMID:24293181

  9. Manipulating L-type calcium channels in cardiomyocytes using split-intein protein transsplicing

    PubMed Central

    Subramanyam, Prakash; Chang, Donald D.; Fang, Kun; Xie, Wenjun; Marks, Andrew R.; Colecraft, Henry M.

    2013-01-01

    Manipulating expression of large genes (>6 kb) in adult cardiomyocytes is challenging because these cells are only efficiently transduced by viral vectors with a 4–7 kb packaging capacity. This limitation impedes understanding structure–function mechanisms of important proteins in heart. L-type calcium channels (LTCCs) regulate diverse facets of cardiac physiology including excitation–contraction coupling, excitability, and gene expression. Many important questions about how LTCCs mediate such multidimensional signaling are best resolved by manipulating expression of the 6.6 kb pore-forming ?1C-subunit in adult cardiomyocytes. Here, we use split-intein–mediated protein transsplicing to reconstitute LTCC ?1C-subunit from two distinct halves, overcoming the difficulty of expressing full-length ?1C in cardiomyocytes. Split-intein–tagged ?1C fragments encoding dihydropyridine-resistant channels were incorporated into adenovirus and reconstituted in cardiomyocytes. Similar to endogenous LTCCs, recombinant channels targeted to dyads, triggered Ca2+ transients, associated with caveolin-3, and supported ?-adrenergic regulation of excitation–contraction coupling. This approach lowers a longstanding technical hurdle to manipulating large proteins in cardiomyocytes. PMID:24003157

  10. Coupled Radon and Water Temperature Measurements to Characterize the Effects of Altered Stream Channel Planform

    NASA Astrophysics Data System (ADS)

    Amerson, B. E.; Poole, G. C.; O'Daniel, S. J.

    2013-12-01

    In summer 2011, a 2.6 km reach of Meacham Creek, Oregon, USA, was altered from a straight, steep wall-based channel to more a sinuous, low-gradient channel. Key objectives of this restoration project were to increase the rate and magnitude of hyporheic exchange. The overarching goal was to initiate increased buffering and lagging of water temperature in the subsurface to mitigate warm surface water temperature in Meacham Creek, an important spawning and rearing stream for depressed populations of Chinook salmon and summer steelhead. To evaluate progress toward project goals and objectives, stream temperature and groundwater temperature in 22 wells have been measured hourly at the restoration site since March 2011. In addition, the radioactive isotope 222Rn was measured in each well and in the surface water on two occasions. The relative residence time of down welling stream water measured in the wells can be determined by ranked amplitude depression and lagged phase of annual temperature signals in the wells relative to that of the open channel flow. Residence times predicted by annual temperature signal dynamics are corroborated by 222Rn concentrations in each well. The data collected to date provide a foundation for developing a groundwater thermal model to predict the effects of channel reconfiguration on ground-surface water exchange and associated temperature effects at the reach scale.

  11. 2D IR spectroscopy reveals the role of water in the binding of channel-blocking drugs to the influenza M2 channel

    NASA Astrophysics Data System (ADS)

    Ghosh, Ayanjeet; Wang, Jun; Moroz, Yurii S.; Korendovych, Ivan V.; Zanni, Martin; DeGrado, William F.; Gai, Feng; Hochstrasser, Robin M.

    2014-06-01

    Water is an integral part of the homotetrameric M2 proton channel of the influenza A virus, which not only assists proton conduction but could also play an important role in stabilizing channel-blocking drugs. Herein, we employ two dimensional infrared (2D IR) spectroscopy and site-specific IR probes, i.e., the amide I bands arising from isotopically labeled Ala30 and Gly34 residues, to probe how binding of either rimantadine or 7,7-spiran amine affects the water dynamics inside the M2 channel. Our results show, at neutral pH where the channel is non-conducting, that drug binding leads to a significant increase in the mobility of the channel water. A similar trend is also observed at pH 5.0 although the difference becomes smaller. Taken together, these results indicate that the channel water facilitates drug binding by increasing its entropy. Furthermore, the 2D IR spectral signatures obtained for both probes under different conditions collectively support a binding mechanism whereby amantadine-like drugs dock in the channel with their ammonium moiety pointing toward the histidine residues and interacting with a nearby water cluster, as predicted by molecular dynamics simulations. We believe these findings have important implications for designing new anti-influenza drugs.

  12. Functional reconstitution of a rice aquaporin water channel, PIP1;1, by a micro-batchwise methodology.

    PubMed

    Scalera, Vito; Gena, Patrizia; Mastrodonato, Maria; Kitagawa, Yoshichika; Carulli, Salvatore; Svelto, Maria; Calamita, Giuseppe

    2014-10-30

    Assessing the selectivity, regulation and physiological relevance of aquaporin membrane channels (AQPs) requires structural and functional studies of wild type and modified proteins. In particular, when characterizing their transport properties, reconstitution in isolation from native cellular or membrane processes is of pivotal importance. Here, we describe rapid and efficient incorporation of OsPIP1;1, a rice AQP, in liposomes at analytical scale. PIP1;1 was produced as a histidine-tagged form, 10His-OsPIP1;1, in an Escherichia coli-based expression system. The recombinant protein was purified by affinity chromatography and incorporated into liposomes by a micro-batchwise technology using egg-yolk phospholipids and the non-polar Amberlite resin. PIP1;1 proteoliposomes and control empty liposomes had good size homogeneity as seen by quasi-elastic light scattering and electron microscopy analyses. By stopped-flow light scattering, indicating correct protein folding of the incorporated protein, the osmotic water permeability exhibited by the PIP1;1 proteoliposomes was markedly higher than empty liposomes. Functional reconstitution of OsPIP1;1 was further confirmed by the low Arrhenius activation energy (3.37 kcal/mol) and sensitivity to HgCl2, a known AQP blocker, of the PIP1;1-mediated osmotic water conductance. These results provide a valuable contribution in fully elucidating the regulation and water-conducting property of PIP1;1, an AQP that needs to hetero-multimerize with AQPs of the PIP2 subgroup to reach the native plasma membrane and play its role. The micro-batchwise methodology is suitable for the functional reconstitution of whichever AQPs and other membrane transport proteins. PMID:25394803

  13. More than one dynamic crossover in protein hydration water

    PubMed Central

    Mazza, Marco G.; Stokely, Kevin; Pagnotta, Sara E.; Bruni, Fabio; Stanley, H. Eugene; Franzese, Giancarlo

    2011-01-01

    Studies of liquid water in its supercooled region have helped us better understand the structure and behavior of water. Bulk water freezes at its homogeneous nucleation temperature (approximately 235 K), but protein hydration water avoids this crystallization because each water molecule binds to a protein. Here, we study the dynamics of the hydrogen bond (HB) network of a percolating layer of water molecules and compare the measurements of a hydrated globular protein with the results of a coarse-grained model that successfully reproduces the properties of hydration water. Using dielectric spectroscopy, we measure the temperature dependence of the relaxation time of proton charge fluctuations. These fluctuations are associated with the dynamics of the HB network of water molecules adsorbed on the protein surface. Using Monte Carlo simulations and mean-field calculations, we study the dynamics and thermodynamics of the model. Both experimental and model analyses are consistent with the interesting possibility of two dynamic crossovers, (i) at approximately 252 K and (ii) at approximately 181 K. Because the experiments agree with the model, we can relate the two crossovers to the presence at ambient pressure of two specific heat maxima. The first is caused by fluctuations in the HB formation, and the second, at a lower temperature, is due to the cooperative reordering of the HB network. PMID:22135473

  14. Adenosine regulates a chloride channel via protein kinase C and a G protein in a rabbit cortical collecting duct cell line.

    PubMed Central

    Schwiebert, E M; Karlson, K H; Friedman, P A; Dietl, P; Spielman, W S; Stanton, B A

    1992-01-01

    We examined the regulation by adenosine of a 305-pS chloride (Cl-) channel in the apical membrane of a continuous cell line derived from rabbit cortical collecting duct (RCCT-28A) using the patch clamp technique. Stimulation of A1 adenosine receptors by N6-cyclohexyladenosine (CHA) activated the channel in cell-attached patches. Phorbol 12,13-didecanoate and 1-oleoyl 2-acetylglycerol, activators of protein kinase C (PKC), mimicked the effect of CHA, whereas the PKC inhibitor H7 blocked the action of CHA. Stimulation of A1 adenosine receptors also increased the production of diacylglycerol, an activator of PKC. Exogenous PKC added to the cytoplasmic face of inside-out patches also stimulated the Cl- channel. Alkaline phosphatase reversed PKC activation. These results show that stimulation of A1 adenosine receptors activates a 305-pS Cl-channel in the apical membrane by a phosphorylation-dependent pathway involving PKC. In previous studies, we showed that the protein G alpha i-3 activated the 305-pS Cl- channel (Schwiebert et al. 1990. J. Biol. Chem. 265:7725-7728). We, therefore, tested the hypothesis that PKC activates the channel by a G protein-dependent pathway. In inside-out patches, pertussis toxin blocked PKC activation of the channel. In contrast, H7 did not prevent G protein activation of the channel. We conclude that adenosine activates a 305-pS Cl- channel in the apical membrane of RCCT-28A cells by a membrane-delimited pathway involving an A1 adenosine receptor, phospholipase C, diacylglycerol, PKC, and a G protein. Because we have shown, in previous studies, that this Cl- channel participates in the regulatory volume decrease subsequent to cell swelling, adenosine release during ischemic cell swelling may activate the Cl-channel and restore cell volume. Images PMID:1311718

  15. Slip effects on mixed convective peristaltic transport of copper-water nanofluid in an inclined channel.

    PubMed

    Abbasi, Fahad Munir; Hayat, Tasawar; Ahmad, Bashir; Chen, Guo-Qian

    2014-01-01

    Peristaltic transport of copper-water nanofluid in an inclined channel is reported in the presence of mixed convection. Both velocity and thermal slip conditions are considered. Mathematical modelling has been carried out using the long wavelength and low Reynolds number approximations. Resulting coupled system of equations is solved numerically. Quantities of interest are analyzed through graphs. Numerical values of heat transfer rate at the wall for different parameters are obtained and examined. Results showed that addition of copper nanoparticles reduces the pressure gradient, axial velocity at the center of channel, trapping and temperature. Velocity slip parameter has a decreasing effect on the velocity near the center of channel. Temperature of nanofluid increases with increase in the Grashoff number and channel inclination angle. It is further concluded that the heat transfer rate at the wall increases considerably in the presence of copper nanoparticles. PMID:25170908

  16. Slip Effects on Mixed Convective Peristaltic Transport of Copper-Water Nanofluid in an Inclined Channel

    PubMed Central

    Abbasi, Fahad Munir; Hayat, Tasawar; Ahmad, Bashir; Chen, Guo-Qian

    2014-01-01

    Peristaltic transport of copper-water nanofluid in an inclined channel is reported in the presence of mixed convection. Both velocity and thermal slip conditions are considered. Mathematical modelling has been carried out using the long wavelength and low Reynolds number approximations. Resulting coupled system of equations is solved numerically. Quantities of interest are analyzed through graphs. Numerical values of heat transfer rate at the wall for different parameters are obtained and examined. Results showed that addition of copper nanoparticles reduces the pressure gradient, axial velocity at the center of channel, trapping and temperature. Velocity slip parameter has a decreasing effect on the velocity near the center of channel. Temperature of nanofluid increases with increase in the Grashoff number and channel inclination angle. It is further concluded that the heat transfer rate at the wall increases considerably in the presence of copper nanoparticles. PMID:25170908

  17. The Dynamic Transition of Protein Hydration Water

    E-print Network

    W. Doster; S. Busch; A. M. Gaspar; M. -S. Appavou; J. Wuttke; H. Scheer

    2010-02-12

    Thin layers of water on biomolecular and other nanostructured surfaces can be supercooled to temperatures not accessible with bulk water. Chen et al. [PNAS 103, 9012 (2006)] suggested that anomalies near 220 K observed by quasi-elastic neutron scattering can be explained by a hidden critical point of bulk water. Based on more sensitive measurements of water on perdeuterated phycocyanin, using the new neutron backscattering spectrometer SPHERES, and an improved data analysis, we present results that show no sign of such a fragile-to-strong transition. The inflection of the elastic intensity at 220 K has a dynamic origin that is compatible with a calorimetric glass transition at 170 K. The temperature dependence of the relaxation times is highly sensitive to data evaluation; it can be brought into perfect agreement with the results of other techniques, without any anomaly.

  18. Requirement for the synaptic protein interaction site for reconstitution of synaptic transmission by P\\/Q-type calcium channels

    Microsoft Academic Search

    Sumiko Mochida; Ruth E. Westenbroek; Charles T. Yokoyama; Huijun Zhong; Scott J. Myers; Todd Scheuer; Kanako Itoh; William A. Catterall

    2003-01-01

    Cav2.1 channels, which conduct P\\/Q-type Ca2+ currents, were expressed in superior cervical ganglion neurons in cell culture, and neurotransmission initiated by these exogenously expressed Ca2+ channels was measured. Deletions in the synaptic protein interaction (synprint) site in the intracellular loop between domains II and III of Cav2.1 channels reduced their effectiveness in synaptic transmission. Surprisingly, this effect was correlated with

  19. Heat shock proteins and p53 play a critical role in K + channel-mediated tumor cell proliferation and apoptosis

    Microsoft Academic Search

    Xiaobing Han; Fang Wang; Weixing Yao; Hui Xing; Danhui Weng; Xiaohong Song; Gang Chen; Ling Xi; Tao Zhu; Jianfeng Zhou; Gang Xu; Shixuan Wang; Li Meng; Costantino Iadecola; Gang Wang; Ding Ma

    2007-01-01

    Plasma membrane potassium (K+) channels are required for tumor cell proliferation and apoptosis. However, the signal transduction mechanisms underlying\\u000a K+ channel-dependent tumor cell proliferation or apoptosis remains elusive. Using HeLa and A2780 cells as study models, we tested\\u000a the hypothesis that apoptotic proteins are linked with K+ channel-dependent tumor cell cycle and apoptosis. The patch-clamping study using the whole-cell mode

  20. Pore waters regulate ion permeation in a calcium release-activated calcium channel

    PubMed Central

    Dong, Hao; Fiorin, Giacomo; Carnevale, Vincenzo; Treptow, Werner; Klein, Michael L.

    2013-01-01

    The recent crystal structure of Orai, the pore unit of a calcium release-activated calcium (CRAC) channel, is used as the starting point for molecular dynamics and free-energy calculations designed to probe this channel’s conduction properties. In free molecular dynamics simulations, cations localize preferentially at the extracellular channel entrance near the ring of Glu residues identified in the crystal structure, whereas anions localize in the basic intracellular half of the pore. To begin to understand ion permeation, the potential of mean force (PMF) was calculated for displacing a single Na+ ion along the pore of the CRAC channel. The computed PMF indicates that the central hydrophobic region provides the major hindrance for ion diffusion along the permeation pathway, thereby illustrating the nonconducting nature of the crystal structure conformation. Strikingly, further PMF calculations demonstrate that the mutation V174A decreases the free energy barrier for conduction, rendering the channel effectively open. This seemingly dramatic effect of mutating a nonpolar residue for a smaller nonpolar residue in the pore hydrophobic region suggests an important role for the latter in conduction. Indeed, our computations show that even without significant channel-gating motions, a subtle change in the number of pore waters is sufficient to reshape the local electrostatic field and modulate the energetics of conduction, a result that rationalizes recent experimental findings. The present work suggests the activation mechanism for the wild-type CRAC channel is likely regulated by the number of pore waters and hence pore hydration governs the conductance. PMID:24101457

  1. 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.

  2. Biogas production from water hyacinth and channel grass used for phytoremediation of industrial effluents

    Microsoft Academic Search

    V Singhal; J. P. N Rai

    2003-01-01

    The paper reports on the biogas production from water hyacinth (Eichhornia crassipes) and channel grass (Vallisneria spiralis) employed separately for phytoremediation of lignin and metal-rich pulp and paper mill and highly acidic distillery effluents. These plants eventually grow well in diluted effluent up to 40% (i.e., 2.5-times dilution with deionized water) and often take up metals and toxic materials from

  3. Integration of thermal and osmotic regulation of water homeostasis: the role of TRPV channels

    PubMed Central

    Johnson, Alan Kim

    2013-01-01

    Maintenance of body water homeostasis is critical for preventing hyperthermia, because evaporative cooling is the most efficient means of dissipating excess body heat. Water homeostasis is achieved by regulation of water intake and water loss by the kidneys. The former is achieved by sensations of thirst that motivate water acquisition, whereas the latter is regulated by the antidiuretic action of vasopressin. Vasopressin secretion and thirst are stimulated by increases in the osmolality of the extracellular fluid as well as decreases in blood pressure and/or blood volume, signals that are precipitated by water depletion associated with the excess evaporative water loss required to prevent hyperthermia. In addition, they are stimulated by increases in body temperature. The sites and molecular mechanisms involved in integrating thermal and osmotic regulation of thirst and vasopressin secretion are reviewed here with a focus on the role of the thermal and mechanosensitive transient receptor potential-vanilloid (TRPV) family of ion channels. PMID:23883678

  4. Integration of thermal and osmotic regulation of water homeostasis: the role of TRPV channels.

    PubMed

    Sladek, Celia D; Johnson, Alan Kim

    2013-10-01

    Maintenance of body water homeostasis is critical for preventing hyperthermia, because evaporative cooling is the most efficient means of dissipating excess body heat. Water homeostasis is achieved by regulation of water intake and water loss by the kidneys. The former is achieved by sensations of thirst that motivate water acquisition, whereas the latter is regulated by the antidiuretic action of vasopressin. Vasopressin secretion and thirst are stimulated by increases in the osmolality of the extracellular fluid as well as decreases in blood pressure and/or blood volume, signals that are precipitated by water depletion associated with the excess evaporative water loss required to prevent hyperthermia. In addition, they are stimulated by increases in body temperature. The sites and molecular mechanisms involved in integrating thermal and osmotic regulation of thirst and vasopressin secretion are reviewed here with a focus on the role of the thermal and mechanosensitive transient receptor potential-vanilloid (TRPV) family of ion channels. PMID:23883678

  5. Inhibition of Protein Kinases in Rat Pheochromocytoma (PC12) Cells Promotes Morphological Differentiation and Down-Regulates Ion Channel Expression

    Microsoft Academic Search

    H. Reuter; A. Bouron; R. Neuhaus; C. Becker; B. F. X. Reber

    1992-01-01

    We have studied morphological differentiation and ion channel expression in PC12 cells under different culture conditions. Differentiation mediated by nerve growth factor (NGF) was compared with that induced by depletion and inhibition of protein kinases (phorbol ester beta -PMA plus staurosporine). Morphological differentiation was similar under both conditions. However, ion channel densities, studied by means of the patch-clamp technique, were

  6. Regulation of the ABA-sensitive Arabidopsis potassium channel gene GORK in response to water stress

    Microsoft Academic Search

    D. Becker; S. Hoth; P. Ache; S. Wenkel; M. R. G. Roelfsema; O. Meyerhoff; W. Hartung; R. Hedrich

    2003-01-01

    The phytohormone abscisic acid (ABA) regulates many stress-related processes in plants. In this context ABA mediates the responsiveness of plants to environmental stresses such as drought, cold or salt. In response to water stress, ABA induces stomatal closure by activating Ca2+, K+ and anion channels in guard cells. To understand the signalling pathways that regulate these turgor control elements, we

  7. Low-Level Water Vapor Fields from the VISSR Atmospheric Sounder (VAS) `Split Window' Channels

    Microsoft Academic Search

    Dennis Chesters; Louis W. Uccellini; Wayne D. Robinson

    1983-01-01

    A simple physical algorithm is developed which calculates the water vapor content of the lower troposphere from the 11 and 12 m (split window) channels on the VISSR Atmospheric Sounder (VAS) on the Geostationary Operational Environmental Satellites (GOES). The algorithm is applied to a time series of VAS split window radiances observed at 15 km horizontal resolution over eastern North

  8. Distyrylbenzene-aldehydes: identification of proteins in water.

    PubMed

    Kumpf, Jan; Freudenberg, Jan; Bunz, Uwe H F

    2015-05-01

    Three different, water soluble, aldehyde-appended distyrylbenzene (DSB) derivatives were prepared. Their interaction with different albumin variants (human, porcine, bovine, lactalbumin, ovalbumin) was investigated (pH 11). All three fluorophores exhibit graded, protein-dependent fluorescence turn-on at slightly differing wavelengths. Linear discriminant analysis (LDA) differentiated all of the investigated albumins and was used to discern commercially available protein shakes. The three DSB derivatives barely react with the constituting amino acids but cysteine. In the proteins significant fluorescence signals are generated, probably due to a combination of imine/N,S-aminal formation and hydrophobic interactions between the DSBs and the proteins. PMID:25803009

  9. Interannual variability of water mass properties in the Tunisia-Sicily Channel

    NASA Astrophysics Data System (ADS)

    Ben Ismail, Sana; Schroeder, Katrin; Sammari, Chérif; Gasparini, Gian Pietro; Borghini, Mireno; Aleya, Lotfi

    2014-07-01

    We analysed in situ CTD data collected on 36 joint Tunisian-Italian oceanographic cruises in the Tunisia-Sicily Channel (Cap Bon-Mazara del Vallo section) from 1995 to 2009 in order to identify the water masses in the region and to estimate the interannual variability of their hydrological characteristics. Besides the well-known AW (Atlantic Water) and LIW (Levantine Intermediate Water), other water masses have been identified though their presence is neither as steady nor as stable as the aforementioned ones. The WIW (Western Intermediate Water) flows beneath the AW towards the eastern basin while the IW (Ionian Water), with a highly intermittent character, flows within the subsurface layer towards the western basin. The flow of subsurface water (WIW and IW) is affected by intense mixing which modifies the water masses, tending to make them disappear. Moreover, the same hydrological time series produced interesting results concerning the increase in both temperature and salinity in the Tunisia-Sicily Channel. This trend especially concerns the deeper layers (LIW and tEMDW, i.e. transitional Eastern Mediterranean Deep Water) and we hypothesise that this is a direct response to climatic change occurring in the eastern basin.

  10. On the turbulent flow around water turbines placed in an open channel: an experimental study

    NASA Astrophysics Data System (ADS)

    Sotiropoulos, F.; Chamorro, L. P.; Arndt, R.

    2010-12-01

    A growing interest in water turbines (using tidal, river, marine currents) has been observed during the last few years. Fundamental understanding of the turbulent flow around the water turbines is crucial to predict the potential effects of these structures on the local morphology, water flow and power available in the current, among others. In this study, a series of model water turbines (single and an aligned array) of 50 cm rotor diameter were placed in the main channel of the Saint Anthony Falls Laboratory at the University of Minnesota. The main channel is approx 2.5 m wide, 1.8 m height and 85 m long. Flow around the water turbines were analyzed under subcritical conditions. Turbine hub heights coincided with the channel mid height. A series of acoustic Doppler anemometers (ADV) were used to obtain 3 velocity components of the flow at a rate of 200 Hz. Selected streamwise and spanwise vertical planes were measured to describe the kinematics around the water turbines. Potential interactions with the lateral walls were also addressed. High order statistics (mean velocity, turbulence intensities and Reynolds stresses) as well as two point correlations and spectra were computed to infer fundamental differences and similitude with their counterparts, the wind turbines.

  11. MM-PBSA Captures Key Role of Intercalating Water Molecules at a Protein?Protein Interface

    PubMed Central

    2009-01-01

    The calculation of protein interaction energetics is of fundamental interest, yet accurate quantities are difficult to obtain due to the complex and dynamic nature of protein interfaces. This is further complicated by the presence of water molecules, which can exhibit transient interactions of variable duration and strength with the protein surface. The T-cell receptor (TCR) and its staphylococcal enterotoxin 3 (SEC3) binding partner are well-characterized examples of a protein?protein interaction system exhibiting interfacial plasticity, cooperativity, and additivity among mutants. Specifically engineered mutants induce intercalating interfacial water molecules, which subsequently enhance protein?protein binding affinity. In this work, we perform a set of molecular mechanics (MM) Poisson?Boltzmann (PB) surface area (SA) calculations on the wild type and two mutant TCR-SEC3 systems and show that the method is able to discriminate between weak and strong binders only when key explicit water molecules are included in the analysis. The results presented here point to the promise of MM-PBSA toward rationalizing molecular recognition at protein?protein interfaces, while establishing a general approach to handle explicit interfacial water molecules in such calculations. PMID:19461869

  12. Inhibition of G Protein-Activated Inwardly Rectifying K+ Channels by Different Classes of Antidepressants

    PubMed Central

    Kobayashi, Toru; Washiyama, Kazuo; Ikeda, Kazutaka

    2011-01-01

    Various antidepressants are commonly used for the treatment of depression and several other neuropsychiatric disorders. In addition to their primary effects on serotonergic or noradrenergic neurotransmitter systems, antidepressants have been shown to interact with several receptors and ion channels. However, the molecular mechanisms that underlie the effects of antidepressants have not yet been sufficiently clarified. G protein-activated inwardly rectifying K+ (GIRK, Kir3) channels play an important role in regulating neuronal excitability and heart rate, and GIRK channel modulation has been suggested to have therapeutic potential for several neuropsychiatric disorders and cardiac arrhythmias. In the present study, we investigated the effects of various classes of antidepressants on GIRK channels using the Xenopus oocyte expression assay. In oocytes injected with mRNA for GIRK1/GIRK2 or GIRK1/GIRK4 subunits, extracellular application of sertraline, duloxetine, and amoxapine effectively reduced GIRK currents, whereas nefazodone, venlafaxine, mianserin, and mirtazapine weakly inhibited GIRK currents even at toxic levels. The inhibitory effects were concentration-dependent, with various degrees of potency and effectiveness. Furthermore, the effects of sertraline were voltage-independent and time-independent during each voltage pulse, whereas the effects of duloxetine were voltage-dependent with weaker inhibition with negative membrane potentials and time-dependent with a gradual decrease in each voltage pulse. However, Kir2.1 channels were insensitive to all of the drugs. Moreover, the GIRK currents induced by ethanol were inhibited by sertraline but not by intracellularly applied sertraline. The present results suggest that GIRK channel inhibition may reveal a novel characteristic of the commonly used antidepressants, particularly sertraline, and contributes to some of the therapeutic effects and adverse effects. PMID:22164246

  13. Ancient Origins of RGK Protein Function: Modulation of Voltage-Gated Calcium Channels Preceded the Protostome and Deuterostome Split

    PubMed Central

    Puhl, Henry L.; Lu, Van B.; Won, Yu-Jin; Sasson, Yehezkel; Hirsch, Joel A.; Ono, Fumihito; Ikeda, Stephen R.

    2014-01-01

    RGK proteins, Gem, Rad, Rem1, and Rem2, are members of the Ras superfamily of small GTP-binding proteins that interact with Ca2+ channel ? subunits to modify voltage-gated Ca2+ channel function. In addition, RGK proteins affect several cellular processes such as cytoskeletal rearrangement, neuronal dendritic complexity, and synapse formation. To probe the phylogenetic origins of RGK protein–Ca2+ channel interactions, we identified potential RGK-like protein homologs in genomes for genetically diverse organisms from both the deuterostome and protostome animal superphyla. RGK-like protein homologs cloned from Danio rerio (zebrafish) and Drosophila melanogaster (fruit flies) expressed in mammalian sympathetic neurons decreased Ca2+ current density as reported for expression of mammalian RGK proteins. Sequence alignments from evolutionarily diverse organisms spanning the protostome/deuterostome divide revealed conservation of residues within the RGK G-domain involved in RGK protein – Cav? subunit interaction. In addition, the C-terminal eleven residues were highly conserved and constituted a signature sequence unique to RGK proteins but of unknown function. Taken together, these data suggest that RGK proteins, and the ability to modify Ca2+ channel function, arose from an ancestor predating the protostomes split from deuterostomes approximately 550 million years ago. PMID:24992013

  14. The Missing Link: the Role of Floodplain Tie Channels in Connecting Off River Water Bodies to Lowland Rivers

    Microsoft Academic Search

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

    2005-01-01

    Along lowland river systems across the globe the exchange of water, sediment, carbon, nutrients and biota between main stem rivers and off-river water bodies (ORWB) is facilitated by the presence of stable secondary channels referred to here as tie channels. Sixty five percent of the ORWB along the middle Fly River in Papua New Guinea connect to the river through

  15. Modulation of rat cardiac sodium channel by the stimulatory G protein ? subunit

    PubMed Central

    Lu, Tong; Lee, Hon-Chi; Kabat, Julia A; Shibata, Erwin F

    1999-01-01

    Modulation of cardiac sodium currents (INa) by the G protein stimulatory ? subunit (Gs?) was studied using patch-clamp techniques on freshly dissociated rat ventricular myocytes.Whole-cell recordings showed that stimulation of ?-adrenergic receptors with 10 ?M isoprenaline (isoproterenol, ISO) enhanced INa by 68·4 ± 9·6 % (mean ±s.e.m.; n= 7, P < 0·05vs. baseline). With the addition of 22 ?g ml?1 protein kinase A inhibitor (PKI) to the pipette solution, 10 ?M ISO enhanced INa by 30·5 ± 7·0 % (n= 7, P < 0·05vs. baseline). With the pipette solution containing both PKI and 20 ?g ml?1 anti-Gs? IgG or 20 ?g ml?1 anti-Gs? IgG alone, 10 ?M ISO produced no change in INa.The effect of Gs? on INa was not due to changes in the steady-state activation or inactivation curves, the time course of current decay, the development of inactivation, or the recovery from inactivation.Whole-cell INa was increased by 45·2 ± 5·3 % (n= 13, P < 0·05vs. control) with pipette solution containing 1 ?M Gs?27-42 peptide (amino acids 27-42 of rat brain Gs?) without altering the properties of Na+ channel kinetics. Furthermore, application of 1 nM Gs?27-42 to Na+ channels in inside-out macropatches increased the ensemble-averaged INa by 32·5 ± 6·8 % (n= 8, P < 0·05vs. baseline). The increase in INa was reversible upon Gs?27-42 peptide washout. Single channel experiments showed that the Gs?27-42 peptide did not alter the Na+ single channel current amplitude, the mean open time or the mean closed time, but increased the number of functional channels (N) in the patch.Application of selected short amino acid segments (Gs?27-36, Gs?33-42 and Gs?30-39) of the 16 amino acid Gs? peptide (Gs?27-42 peptide) showed that only the C-terminal segment of this peptide (Gs?33-42) significantly increased INa in a dose-dependent fashion. These results show that cardiac INa is regulated by Gs? via a mechanism independent of PKA that results in an increase in the number of functional Na+ channels. In addition, a 10 residue domain (amino acids 33-42) near the N-terminus of Gs? is important in modulating cardiac Na+ channels. PMID:10381586

  16. Role of protein sulfation in vasodilation induced by minoxidil sulfate, a K+ channel opener

    SciTech Connect

    Meisheri, K.D.; Oleynek, J.J.; Puddington, L. (Cardiovascular Diseases Research, Upjohn Laboratories, Upjohn Company, Kalamazoo, MI (United States))

    1991-09-01

    Evidence from contractile, radioisotope ion flux and electrophysiological studies suggest that minoxidil sulfate (MNXS) acts as a K+ channel opener in vascular smooth muscle. This study was designed to examine possible biochemical mechanisms by which MNXS exerts such an effect. Experiments performed in the isolated rabbit mesenteric artery (RMA) showed that MNXS, 5 microM, but not the parent compound minoxidil, was a potent vasodilator. Whereas the relaxant effects of an another K+ channel opener vasodilator, BRL-34915 (cromakalim), were removed by washing with physiological saline solution, the effects of MNXS persisted after repeated washout attempts. Furthermore, after an initial exposure of segments of intact RMA to (35S) MNXS, greater than 30% of the radiolabel was retained 2 hr after removal of the drug. In contrast, retention of radiolabel was not detected with either (3H)MNXS (label on the piperidine ring of MNXS) or (3H)minoxidil (each less than 3% after a 2-hr washout). These data suggested that the sulfate moiety from MNXS was closely associated with the vascular tissue. To determine if proteins were the acceptors of sulfate from MNXS, intact RMAs were incubated with (35S)MNXS, and then 35S-labeled proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and analyzed by fluorography. Preferential labeling of a 116 kD protein was detected by 2 and 5 min of treatment. A 43 kD protein (resembling actin) also showed significant labeling. A similar profile of 35S-labeled proteins was observed in (35S) MNXS-treated A7r5 rat aortic smooth muscle cells, suggesting that the majority of proteins labeled by (35S)MNXS in intact RMA were components of smooth muscle cells.

  17. Physiological roles and diseases of tmem16/anoctamin proteins: are they all chloride channels?

    PubMed Central

    Duran, Charity; Hartzell, H Criss

    2011-01-01

    The Tmem16 gene family was first identified by bioinformatic analysis in 2004. In 2008, it was shown independently by 3 laboratories that the first two members (Tmem16A and Tmem16B) of this 10-gene family are Ca2+-activated Cl? channels. Because these proteins are thought to have 8 transmembrane domains and be anion-selective channels, the alternative name, Anoctamin (anion and octa=8), has been proposed. However, it remains unclear whether all members of this family are, in fact, anion channels or have the same 8-transmembrane domain topology. Since 2008, there have been nearly 100 papers published on this gene family. The excitement about Tmem16 proteins has been enhanced by the finding that Ano1 has been linked to cancer, mutations in Ano5 are linked to several forms of muscular dystrophy (LGMDL2 and MMD-3), mutations in Ano10 are linked to autosomal recessive spinocerebellar ataxia, and mutations in Ano6 are linked to Scott syndrome, a rare bleeding disorder. Here we review some of the recent developments in understanding the physiology and structure-function of the Tmem16 gene family. PMID:21642943

  18. Generation, Comparison, and Merging of Pathways between Protein Conformations: Gating in K-Channels

    PubMed Central

    Enosh, Angela; Raveh, Barak; Furman-Schueler, Ora; Halperin, Dan; Ben-Tal, Nir

    2008-01-01

    We present a general framework for the generation, alignment, comparison, and hybridization of motion pathways between two known protein conformations. The framework, which is rooted in probabilistic motion-planning techniques in robotics, allows for the efficient generation of collision-free motion pathways, while considering a wide range of degrees of freedom involved in the motion. Within the framework, we provide the means to hybridize pathways, thus producing, the motion pathway of the lowest energy barrier out of the many pathways proposed by our algorithm. This method for comparing and hybridizing pathways is modular, and may be used within the context of molecular dynamics and Monte Carlo simulations. The framework was implemented within the Rosetta software suite, where the protein is represented in atomic detail. The K-channels switch between open and closed conformations, and we used the overall framework to investigate this transition. Our analysis suggests that channel-opening may follow a three-phase pathway. First, the channel unlocks itself from the closed state; second, it opens; and third, it locks itself in the open conformation. A movie that depicts the proposed pathway is available in the Supplementary Material (Movie S1) and at http://www.cs.tau.ac.il/?angela/SuppKcsA.html. PMID:18621834

  19. Membrane topology and multimeric structure of a mechanosensitive channel protein of Escherichia coli.

    PubMed Central

    Blount, P; Sukharev, S I; Moe, P C; Schroeder, M J; Guy, H R; Kung, C

    1996-01-01

    We have studied the membrane topology and multimeric structure of a mechanosensitive channel, MscL, which we previously isolated and cloned from Escherichia coli. We have localized this 15-kDa protein to the inner membrane and, by PhoA fusion, have shown that it contains two transmembrane domains with both the amino and carboxyl termini on the cytoplasmic side. Mutation of the glutamate at position 56 to histidine led to changes in channel kinetics which were dependent upon the pH on the periplasmic, but not cytoplasmic side of the membrane, providing additional evidence for the periplasmic positioning of this part of the molecule. Tandems of two MscL subunits expressed as a single polypeptide formed functional channels, suggesting an even number of transmembrane domains per subunit (amino and carboxyl termini on the same side of the membrane), and an even number of subunits per functional complex. Finally, cross-linking studies suggest that the functional MscL complex is a homohexamer. In summary, these data are all consistent with a protein domain assignment and topological model which we propose and discuss. Images PMID:8890153

  20. RING finger protein 121 facilitates the degradation and membrane localization of voltage-gated sodium channels.

    PubMed

    Ogino, Kazutoyo; Low, Sean E; Yamada, Kenta; Saint-Amant, Louis; Zhou, Weibin; Muto, Akira; Asakawa, Kazuhide; Nakai, Junichi; Kawakami, Koichi; Kuwada, John Y; Hirata, Hiromi

    2015-03-01

    Following their synthesis in the endoplasmic reticulum (ER), voltage-gated sodium channels (NaV) are transported to the membranes of excitable cells, where they often cluster, such as at the axon initial segment of neurons. Although the mechanisms by which NaV channels form and maintain clusters have been extensively examined, the processes that govern their transport and degradation have received less attention. Our entry into the study of these processes began with the isolation of a new allele of the zebrafish mutant alligator, which we found to be caused by mutations in the gene encoding really interesting new gene (RING) finger protein 121 (RNF121), an E3-ubiquitin ligase present in the ER and cis-Golgi compartments. Here we demonstrate that RNF121 facilitates two opposing fates of NaV channels: (i) ubiquitin-mediated proteasome degradation and (ii) membrane localization when coexpressed with auxiliary NaV? subunits. Collectively, these results indicate that RNF121 participates in the quality control of NaV channels during their synthesis and subsequent transport to the membrane. PMID:25691753

  1. Structure-function of proteins interacting with the ?1 pore-forming subunit of high-voltage-activated calcium channels

    PubMed Central

    Neely, Alan; Hidalgo, Patricia

    2014-01-01

    Openings of high-voltage-activated (HVA) calcium channels lead to a transient increase in calcium concentration that in turn activate a plethora of cellular functions, including muscle contraction, secretion and gene transcription. To coordinate all these responses calcium channels form supramolecular assemblies containing effectors and regulatory proteins that couple calcium influx to the downstream signal cascades and to feedback elements. According to the original biochemical characterization of skeletal muscle Dihydropyridine receptors, HVA calcium channels are multi-subunit protein complexes consisting of a pore-forming subunit (?1) associated with four additional polypeptide chains ?, ?2, ?, and ?, often referred to as accessory subunits. Twenty-five years after the first purification of a high-voltage calcium channel, the concept of a flexible stoichiometry to expand the repertoire of mechanisms that regulate calcium channel influx has emerged. Several other proteins have been identified that associate directly with the ?1-subunit, including calmodulin and multiple members of the small and large GTPase family. Some of these proteins only interact with a subset of ?1-subunits and during specific stages of biogenesis. More strikingly, most of the ?1-subunit interacting proteins, such as the ?-subunit and small GTPases, regulate both gating and trafficking through a variety of mechanisms. Modulation of channel activity covers almost all biophysical properties of the channel. Likewise, regulation of the number of channels in the plasma membrane is performed by altering the release of the ?1-subunit from the endoplasmic reticulum, by reducing its degradation or enhancing its recycling back to the cell surface. In this review, we discuss the structural basis, interplay and functional role of selected proteins that interact with the central pore-forming subunit of HVA calcium channels. PMID:24917826

  2. Structure-function of proteins interacting with the ?1 pore-forming subunit of high-voltage-activated calcium channels.

    PubMed

    Neely, Alan; Hidalgo, Patricia

    2014-01-01

    Openings of high-voltage-activated (HVA) calcium channels lead to a transient increase in calcium concentration that in turn activate a plethora of cellular functions, including muscle contraction, secretion and gene transcription. To coordinate all these responses calcium channels form supramolecular assemblies containing effectors and regulatory proteins that couple calcium influx to the downstream signal cascades and to feedback elements. According to the original biochemical characterization of skeletal muscle Dihydropyridine receptors, HVA calcium channels are multi-subunit protein complexes consisting of a pore-forming subunit (?1) associated with four additional polypeptide chains ?, ?2, ?, and ?, often referred to as accessory subunits. Twenty-five years after the first purification of a high-voltage calcium channel, the concept of a flexible stoichiometry to expand the repertoire of mechanisms that regulate calcium channel influx has emerged. Several other proteins have been identified that associate directly with the ?1-subunit, including calmodulin and multiple members of the small and large GTPase family. Some of these proteins only interact with a subset of ?1-subunits and during specific stages of biogenesis. More strikingly, most of the ?1-subunit interacting proteins, such as the ?-subunit and small GTPases, regulate both gating and trafficking through a variety of mechanisms. Modulation of channel activity covers almost all biophysical properties of the channel. Likewise, regulation of the number of channels in the plasma membrane is performed by altering the release of the ?1-subunit from the endoplasmic reticulum, by reducing its degradation or enhancing its recycling back to the cell surface. In this review, we discuss the structural basis, interplay and functional role of selected proteins that interact with the central pore-forming subunit of HVA calcium channels. PMID:24917826

  3. Channel Surface Patterning of Alternating Biomimetic Protein Combinations for Enhanced Microfluidic Tumor Cell Isolation

    PubMed Central

    Launiere, Cari; Gaskill, Marissa; Czaplewski, Gregory; Myung, Ja Hye; Hong, Seungpyo; Eddington, David T.

    2012-01-01

    Here we report a new method for multi-component protein patterning in a microchannel and also a technique for improving immunoaffinity-based circulating tumor cell (CTC) capture by patterning regions of alternating adhesive proteins using the new method. The first of two proteins, anti-epithelial cell adhesion molecule (anti-EpCAM), provides the specificity for CTC capture. The second, E-selectin, increases CTC capture under shear. Patterning regions with and without E-selectin allows captured leukocytes, which also bind E-selectin and are unwanted impurities in CTC isolation, to roll a short distance and detach from the capture surface. This reduces leukocyte capture by up to 82%. The patterning is combined with a leukocyte elution step in which a calcium chelating buffer effectively deactivates E-selectin so that leukocytes may be rinsed away 60% more efficiently than with a buffer containing calcium. The alternating patterning of this biomimetic protein combination, used in conjunction with the elution step, reduce capture of leukocytes while maintaining a high tumor capture efficiency that is up to 1.9 times higher than the tumor cell capture efficiency of a surface with only anti-EpCAM. The new patterning technique described here does not require mask alignment and can be used to spatially control the immobilization of any 2 proteins or protein mixtures inside a sealed microfluidic channel. PMID:22482510

  4. Epithelial Na+ channel proteins are mechanotransducers of myogenic constriction in rat posterior cerebral arteries.

    PubMed

    Kim, Eok-Cheon; Ahn, Duck-Sun; Yeon, Soo-In; Lim, Mihwa; Lee, Young-Ho

    2012-04-01

    It has been suggested that mechanosensitive ion channels initiate myogenic responses in vessels; however, the molecular identity of the mechanosensitive ion channel complex is unknown. Although previous reports have suggested that epithelial Na(+) channel (ENaC) proteins are mechanotransducers in arteries, experimental evidence demonstrating that ENaC proteins are mechanotransducers are not fully elucidated. The goal of the present study was to determine whether the ENaC is a mechanotransducer for the myogenic response by providing supporting evidence in the rat posterior cerebral artery (PCA). We measured the effect of ENaC inhibition on the pressure-induced myogenic response, Ca(2+) concentration and 20 kDa myosin light chain (MLC(20)) phosphorylation. We detected expression of ?ENaC and ?ENaC subunits in rat PCA by Western blots and immunofluorescence. Inhibition of ENaCs with amiloride, ethyl isopropyl amiloride or benzamil blocked the myogenic response. Moreover, the myogenic response was inhibited in rat PCA transfected with ?ENaC and ?ENaC small interfering RNA. The myogenic response was inhibited by elimination of external Na(+), which was replaced with N-methyl-d-glucamine. Amiloride and nifedipine inhibited the pressure-induced increase in Ca(2+) concentration. Finally, MLC(20) increased when the intraluminal pressure was raised, and the pressure-induced increase in MLC(20) phosphorylation was inhibited by pretreatment with amiloride, and in arteries transfected with ?ENaC or ?ENaC small interfering RNA. Our results suggest that ENaCs may play an important role as mechanosensitive ion channels initiating pressure-induced myogenic responses in rat PCA. PMID:22090066

  5. Ethanol inhibition of a T-type Ca2+channel through activity of protein kinase C

    PubMed Central

    Shan, Hong Qu; Hammarback, James A.; Godwin, Dwayne W.

    2015-01-01

    Background T-type calcium channels are widely distributed in the central and peripheral nervous system, where they mediate calcium entry and regulate the intrinsic excitability of neurons. T-channels are dysregulated in response to alcohol administration and withdrawal. We therefore investigated acute ethanol effects and the underlying mechanism of action in Human Embryonic Kidney (HEK) 293 cell lines, as well as effects on native currents recorded from dorsal root ganglion (DRG) neurons cultured from Long-Evans Rats. MATERIALS and Methods Whole cell voltage-clamp recordings were performed at 32-34°C in both HEK cell lines and DRG neurons. The recordings were taken after a 10 min application of ethanol or protein kinase C (PKC) activator (phorbol 12-myristate 13-acetate, PMA). Results We recorded T-currents from three channel isoforms (CaV3.1, CaV3.2 and CaV3.3) before and during administration of ethanol. We found that only one isoform, CaV3.2, was significantly affected by ethanol. Ethanol reduced current density as well as producing a hyperpolarizing shift in steady state inactivation of both CaV3.2 currents from HEK 293 cell lines and in native T currents from DRG neurons that are known to be enriched in CaV3.2. A protein kinase C peptide inhibitor (MPI) blocked the major ethanol effects, in both the cell lines and the DRG neurons. However, PMA effects were more complex. Lower concentration PMA (100 nM) replicated the major effects of ethanol, while higher concentration PMA (1 ?M) significantly increased current density, suggesting that the ethanol effect may operate at lower PKC activation levels. Conclusions Ethanol primarily affects the CaV3.2 isoform of T-type Ca2+channels acting through PKC, highlighting a novel target and mechanism for ethanol effects on excitable membranes. PMID:23488970

  6. Substrate channels revealed in the trimeric Lactobacillus reuteri bacterial microcompartment shell protein PduB.

    PubMed

    Pang, Allan; Liang, Mingzhi; Prentice, Michael B; Pickersgill, Richard W

    2012-12-01

    Lactobacillus reuteri metabolizes two similar three-carbon molecules, 1,2-propanediol and glycerol, within closed polyhedral subcellular bacterial organelles called bacterial microcompartments (metabolosomes). The outer shell of the propanediol-utilization (Pdu) metabolosome is composed of hundreds of mainly hexagonal protein complexes made from six types of protein subunits that share similar domain structures. The structure of the bacterial microcompartment protein PduB has a tandem structural repeat within the subunit and assembles into a trimer with pseudo-hexagonal symmetry. This trimeric structure forms sheets in the crystal lattice and is able to fit within a polymeric sheet of the major shell component PduA to assemble a facet of the polyhedron. There are three pores within the trimer and these are formed between the tandem repeats within the subunits. The structure shows that each of these pores contains three glycerol molecules that interact with conserved residues, strongly suggesting that these subunit pores channel glycerol substrate into the metabolosome. In addition to the observation of glycerol occupying the subunit channels, the presence of glycerol on the molecular threefold symmetry axis suggests a role in locking closed the central region. PMID:23151629

  7. 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.

  8. Inhibition of protein kinases in rat pheochromocytoma (PC12) cells promotes morphological differentiation and down-regulates ion channel expression.

    PubMed

    Reuter, H; Bouron, A; Neuhaus, R; Becker, C; Reber, B F

    1992-08-22

    We have studied morphological differentiation and ion channel expression in PC12 cells under different culture conditions. Differentiation mediated by nerve growth factor (NGF) was compared with that induced by depletion and inhibition of protein kinases (phorbol ester beta-PMA plus staurosporine). Morphological differentiation was similar under both conditions. However, ion channel densities, studied by means of the patch-clamp technique, were enhanced by NGF and reduced by beta-PMA+staurosporine. Similar changes were also observed for omega-conotoxin-sensitive Ca2+ channels by measuring radioligand binding. The decrease in Ca2+ channel density, after treatment of the cells with beta-PMA+staurosporine, resulted in a reduced increase in the intracellular Ca2+ concentration during K+ depolarization. We conclude that morphological differentiation, but not ion channel expression, can occur during depression of protein kinase activities in PC12 cells. PMID:1280837

  9. Current inhibition of human EAG1 potassium channels by the Ca2+ binding protein S100B.

    PubMed

    Sahoo, Nirakar; Tröger, Jessica; Heinemann, Stefan H; Schönherr, Roland

    2010-09-24

    Voltage-dependent human ether à go-go (hEAG1) potassium channels are implicated in neuronal signaling as well as in cancer cell proliferation. Unique sensitivity of the channel to intracellular Ca(2+) is mediated by calmodulin (CaM) binding to the intracellular N- and C-termini of the channel. Here we show that application of the acidic calcium-binding protein S100B to inside-out patches of Xenopus oocytes causes Ca(2+)-dependent inhibition of expressed hEAG1 channels. Protein pull-down assays and fluorescence correlation spectroscopy (FCS) revealed that S100B binds to hEAG1 and shares the same binding sites with CaM. Thus, S100B is a potential alternative calcium sensor for hEAG1 potassium channels. PMID:20708613

  10. Effect of channelization of Rio Puerto Nuevo on ground-water levels in the San Juan metropolitan area, Puerto Rico

    USGS Publications Warehouse

    Padilla, Ingrid

    1991-01-01

    Channelization and concrete lining of the Rio Puerto Nuevo and its tributaries in the San Juan Metropolitan area has been proposed to control flooding in low lying areas adjacent to the stream. Concern about the effect of these channel modifications on the ground-water system prompted the U.S. Geological Survey in cooperation with the U.S. Army Corps of Engineers to conduct an investigation of surface-water and ground-water interactions in the Rio Puerto Nuevo basin in 1988. A principal objective of this investigation was to determine the potential effect of channelization of the Rio Puerto Nuevo on ground-water levels.

  11. Adaptive resolution simulation of an atomistic protein in MARTINI water

    SciTech Connect

    Zavadlav, Julija [Laboratory for Molecular Modeling, National Institute of Chemistry, Hajdrihova 19, SI-1001 Ljubljana (Slovenia)] [Laboratory for Molecular Modeling, National Institute of Chemistry, Hajdrihova 19, SI-1001 Ljubljana (Slovenia); Melo, Manuel Nuno; Marrink, Siewert J., E-mail: s.j.marrink@rug.nl [Groningen Biomolecular Sciences and Biotechnology Institute and Zernike Institute for Advanced Materials, University of Groningen, Nijenborgh 7, 9747 AG Groningen (Netherlands); Praprotnik, Matej, E-mail: praprot@cmm.ki.si [Laboratory for Molecular Modeling, National Institute of Chemistry, Hajdrihova 19, SI-1001 Ljubljana (Slovenia) [Laboratory for Molecular Modeling, National Institute of Chemistry, Hajdrihova 19, SI-1001 Ljubljana (Slovenia); Department of Physics, Faculty of Mathematics and Physics, University of Ljubljana, Jadranska 19, SI-1000 Ljubljana (Slovenia)

    2014-02-07

    We present an adaptive resolution simulation of protein G in multiscale water. We couple atomistic water around the protein with mesoscopic water, where four water molecules are represented with one coarse-grained bead, farther away. We circumvent the difficulties that arise from coupling to the coarse-grained model via a 4-to-1 molecule coarse-grain mapping by using bundled water models, i.e., we restrict the relative movement of water molecules that are mapped to the same coarse-grained bead employing harmonic springs. The water molecules change their resolution from four molecules to one coarse-grained particle and vice versa adaptively on-the-fly. Having performed 15?ns long molecular dynamics simulations, we observe within our error bars no differences between structural (e.g., root-mean-squared deviation and fluctuations of backbone atoms, radius of gyration, the stability of native contacts and secondary structure, and the solvent accessible surface area) and dynamical properties of the protein in the adaptive resolution approach compared to the fully atomistically solvated model. Our multiscale model is compatible with the widely used MARTINI force field and will therefore significantly enhance the scope of biomolecular simulations.

  12. Exploration of cone cyclic nucleotide-gated channel-interacting proteins using affinity purification and mass spectrometry.

    PubMed

    Ding, Xi-Qin; Matveev, Alexander; Singh, Anil; Komori, Naoka; Matsumoto, Hiroyuki

    2014-01-01

    Photopic (cone) vision essential for color sensation, central vision, and visual acuity is mediated by the activation of photoreceptor cyclic nucleotide-gated (CNG) channels. Naturally occurring mutations in the cone channel subunits CNGA3 and CNGB3 are associated with achromatopsia and cone dystrophies. This work investigated the functional modulation of cone CNG channel by exploring the channel-interacting proteins. Retinal protein extracts prepared from cone-dominant Nrl (- / -) mice were used in CNGA3 antibody affinity purification, followed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) separation and matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry analysis. The peptide mass fingerprinting of the tryptic digests and database search identified a number of proteins including spectrin alpha-2, ATPase (Na(+)/K(+) transporting) alpha-3, alpha and beta subunits of ATP synthase (H(+) transporting, mitochondrial F1 complex), and alpha-2 subunit of the guanine nucleotide-binding protein. In addition, the affinity-binding assays demonstrated an interaction between cone CNG channel and calmodulin but not cone Na(+)/Ca(2+)-K(+) exchanger in the mouse retina. Results of this study provide insight into our understanding of cone CNG channel-interacting proteins and the functional modulations. PMID:24664681

  13. Liquid Crystalline Properties of Amyloid Protein Fibers in Water

    Microsoft Academic Search

    Raffaele Mezzenga; Jin-Mi Jung

    2010-01-01

    We have studied the liquid crystalline features of two colloidal systems consisting of food protein amyloid fibrils in water, obtained by heat-denaturation and aggregation of beta-lactoglobulin, a globular dairy protein. The resulting fibrils, have a monodisperse cross section of about 4 nm and two groups of polydisperse contour lengths: (i) fibrils 1-10 mum long, showing semiflexible polyeletrolyte-like behaviour and (ii)

  14. Water and Protein Structure in Photoaged and Chronically Aged Skin

    Microsoft Academic Search

    Monika Gniadecka; Ole Faurskov Nielsen; Sonja Wessel; Michael Heidenheim; Daniel Højgaard Christensen; Hans Christian Wulf

    1998-01-01

    Changes in the structural proteins and hydration during aging is responsible for altered skin morphologic and mechanical properties manifested as wrinkling, sagging, loss of elasticity, or apparent dryness. To gain insight into the age-related alterations in protein conformation and water structure, we obtained Raman spectra from the sun-protected buttock skin representing chronologic aging and the sun-exposed forearm skin representing combined

  15. Channel Incision and Water-Table Decline Along a Recently Formed Proglacial Stream, Mendenhall Valley, Southeastern Alaska

    USGS Publications Warehouse

    Neal, Edward G.

    2009-01-01

    Retreat of the Mendenhall Glacier, in southeastern Alaska, resulted in the formation of Mendenhall Lake, which has reduced the supply of coarse sediment to the proglacial Mendenhall River. Channel geometry surveys conducted in 1969 and 1998 over a 5.3 km reach of the Mendenhall River revealed reductions in mean bed elevations ranging from 0.4 to 1.5 meters based on cross sections replicated at 7 locations. Channel incision in the Mendenhall River is believed to be the result of a combination of factors resulting from localized and region-wide glacial retreat. In addition to a reduction of river stage due to channel incision, a decline in water-table elevations of about 0.6 m during a 17-year period from 1984 to 2001 was identified in an observation well located 250 m from the incising stream channel. Water-table elevations 600 m from the incising channel in the adjacent alluvial outwash aquifer respond in phase to changes in river stage, indicating water-levels in the adjacent aquifer are declining in response to river-channel incision. This study suggests channel incision can rapidly lower water-table elevations for large distances in the adjacent aquifer, potentially modifying the hydrology to a degree capable of influencing adjacent surface-water features, such as off-channel wetlands and flood-plain side channels.

  16. Hydration-dependent dynamic crossover phenomenon in protein hydration water.

    PubMed

    Wang, Zhe; Fratini, Emiliano; Li, Mingda; Le, Peisi; Mamontov, Eugene; Baglioni, Piero; Chen, Sow-Hsin

    2014-10-01

    The characteristic relaxation time ? of protein hydration water exhibits a strong hydration level h dependence. The dynamic crossover is observed when h is higher than the monolayer hydration level hc=0.2-0.25 and becomes more visible as h increases. When h is lower than hc, ? only exhibits Arrhenius behavior in the measured temperature range. The activation energy of the Arrhenius behavior is insensitive to h, indicating a local-like motion. Moreover, the h dependence of the crossover temperature shows that the protein dynamic transition is not directly or solely induced by the dynamic crossover in the hydration water. PMID:25375521

  17. Why do proteins aggregate? “Intrinsically insoluble proteins” and “dark mediators” revealed by studies on “insoluble proteins” solubilized in pure water

    PubMed Central

    Song, Jianxing

    2013-01-01

    In 2008, I reviewed and proposed a model for our discovery in 2005 that unrefoldable and insoluble proteins could in fact be solubilized in unsalted water. Since then, this discovery has offered us and other groups a powerful tool to characterize insoluble proteins, and we have further addressed several fundamental and disease-relevant issues associated with this discovery. Here I review these results, which are conceptualized into several novel scenarios. 1) Unlike 'misfolded proteins', which still retain the capacity to fold into well-defined structures but are misled to 'off-pathway' aggregation, unrefoldable and insoluble proteins completely lack this ability and will unavoidably aggregate in vivo with ~150 mM ions, thus designated as 'intrinsically insoluble proteins (IIPs)' here. IIPs may largely account for the 'wastefully synthesized' DRiPs identified in human cells. 2) The fact that IIPs including membrane proteins are all soluble in unsalted water, but get aggregated upon being exposed to ions, logically suggests that ions existing in the background play a central role in mediating protein aggregation, thus acting as 'dark mediators'. Our study with 14 salts confirms that IIPs lack the capacity to fold into any well-defined structures. We uncover that salts modulate protein dynamics and anions bind proteins with high selectivity and affinity, which is surprisingly masked by pre-existing ions. Accordingly, I modified my previous model. 3) Insoluble proteins interact with lipids to different degrees. Remarkably, an ALS-causing P56S mutation transforms the ?-sandwich MSP domain into a helical integral membrane protein. Consequently, the number of membrane-interacting proteins might be much larger than currently recognized. To attack biological membranes may represent a common mechanism by which aggregated proteins initiate human diseases. 4) Our discovery also implies a solution to the 'chicken-and-egg paradox' for the origin of primitive membranes embedded with integral membrane proteins, if proteins originally emerged in unsalted prebiotic media. PMID:24555050

  18. Channel Catfish, Ictalurus punctatus Rafinesque 1818, Tetraspanin Membrane Protein Family: Characterization and Expression Analysis of CD81 cDNA

    Technology Transfer Automated Retrieval System (TEKTRAN)

    CD81, also known as the target of an antiproliferative antibody 1 (TAPA-1), is a member of tetraspanin integral membrane protein family. This protein plays many important roles in immune functions. In this report, we characterized and analyzed expression of the channel catfish CD81 transcript. T...

  19. Protein tyrosine kinase PYK2 involved in Ca2+-induced regulation of ion channel and MAP kinase functions

    Microsoft Academic Search

    S. Lev; H. Moreno; R. Martinez; P. Canoll; E. Peles; J. M. Musacchio; G. D. Plowman; B. Rudy; J. Schlessinger

    1995-01-01

    The protein tyrosine kinase PYK2, which is highly expressed in the central nervous system, is rapidly phosphorylated on tyrosine residues in response to various stimuli that elevate the intracellular calcium concentration, as well as by protein kinase C activation. Activation of PYK2 leads to modulation of ion channel function and activation of the MAP kinase signalling pathway. PYK2 activation may

  20. Grazing Land Management Strongly Controls Water Quality, Sediment and Channel Dynamics in Tallgrass Prairie Headwater Networks

    NASA Astrophysics Data System (ADS)

    Grudzinski, B. G.; Daniels, M. D.

    2013-12-01

    In the prairie remnants of North America, watershed sediment regimes are heavily influenced by livestock grazing practices. Despite dramatic declines in stream water quality and ecosystem function concomitant with increasing gazing pressures, there have been no studies to quantitatively assess the relationship between various grazing treatments and sediment production in natural grassland ecosystems. In this study, we evaluate suspended sediment transport and channel morphology in the Flint Hills physiographic province using a paired whole-watershed approach, including 2 replicates of high density cattle grazing, 2 replicates of low density cattle grazing, 3 replicates of bison grazing and 3 replicates of no grazing. As expected, results demonstrate that cattle grazing operations increase e-coli, sediment concentrations and increase channel width. However, no significant differences in e-coli, suspended sediment dynamics or channel geomorphology were found between bison grazed and ungrazed watersheds.

  1. Stability analysis of a square rod bundle sub-channel in supercritical water reactor

    NASA Astrophysics Data System (ADS)

    Hai-jun, Wang; Ting, You; Lei, Zhang; Hong-fang, Gu; Yu-shan, Luo; Ji-lian, Bian

    2013-07-01

    Extensive investigations on the flow and heat transfer behavior in SCWR fuel assembly have been undertaken worldwide. However, stability analysis of supercritical water in the sub-channels of tight lattices is still lacking. In this paper, the flow stability of a fuel bundle channel with square pitches has been analyzed using commercial CFD code-ANSYS Fluent. Typical dynamic instability of Density Wave Oscillation (DWO) has occurred in heated channel containing fluids at supercritical pressure. A further discussion about the impacts of various operational parameters (e.g. power input, system pressure, mass velocity, inlet temperature, etc) shows that the system becomes more stable as system pressure and/or mass flow rate increases. An increase in inlet temperature also has a stabilizing effect on the system.

  2. A Novel Plant Major Intrinsic Protein in Physcomitrella patens Most Similar to Bacterial Glycerol Channels1

    PubMed Central

    Gustavsson, Sofia; Lebrun, Anne-Sophie; Nordén, Kristina; Chaumont, François; Johanson, Urban

    2005-01-01

    A gene encoding a novel fifth type of major intrinsic protein (MIP) in plants has been identified in the moss Physcomitrella patens. Phylogenetic analyses show that this protein, GlpF-like intrinsic protein (GIP1;1), is closely related to a subclass of glycerol transporters in bacteria that in addition to glycerol are highly permeable to water. A likely explanation of the occurrence of this bacterial-like MIP in P. patens is horizontal gene transfer. The expressed P. patens GIP1;1 gene contains five introns and encodes a unique C-loop extension of approximately 110 amino acid residues that has no obvious similarity with any other known protein. Based on alignments and structural comparisons with other MIPs, GIP1;1 is suggested to have retained the permeability for glycerol but not for water. Studies on heterologously expressed GIP1;1 in Xenopus laevis oocytes confirm the predicted substrate specificity. Interestingly, proteins of one of the plant-specific subgroups of MIPs, the NOD26-like intrinsic proteins, are also facilitating the transport of glycerol and have previously been suggested to have evolved from a horizontally transferred bacterial gene. Further studies on localization and searches for GIP1;1 homologs in other plants will clarify the function and significance of this new plant MIP. PMID:16113222

  3. Microfiltration: Effect of retentate protein concentration on limiting flux and serum protein removal with 4-mm-channel ceramic microfiltration membranes.

    PubMed

    Hurt, E E; Adams, M C; Barbano, D M

    2015-04-01

    The objective of our study was to determine if the limiting flux and serum protein (SP) removal were different at 8, 9, or 10% true protein (TP) in the microfiltration (MF) retentate recirculation loop using 0.1-µm ceramic graded permeability membranes with 4-mm-channel diameters operated at 50°C using a diluted milk protein concentrate with 85% protein on a total solids basis (MPC85) as the MF feed. The limiting flux for the MF of diluted MPC85 was determined at 3 TP concentrations in the recirculation loop (8, 9, and 10%). The experiment was replicated 3 times for a total of 9 runs. On the morning of each run, MPC85 was diluted with reverse osmosis water to an MF feed TP concentration of 5.4%. In all runs, the starting flux was 55kg/m(2) per hour, the flux was increased in steps until the limiting flux was reached. The minimum flux increase was 10kg/m(2) per hour. The limiting flux decreased as TP concentration in the recirculation loop increased. The limiting flux was 154±0.3, 133±0.7, and 117±3.3kg/m(2) per hour at recirculation loop TP concentrations of 8.2±0.07, 9.2±0.04, and 10.2±0.09%, respectively. No effect of recirculation loop TP concentration on the SP removal factor was detected. However, the SP removal factor decreased from 0.80±0.02 to 0.75±0.02 as flux was increased from the starting flux of 55kg/m(2) per hour to the limiting flux, with a similar decrease seen at all recirculation loop TP concentrations. PMID:25682139

  4. Interaction of the Faroe Bank Channel overflow with Iceland Basin intermediate waters

    NASA Astrophysics Data System (ADS)

    Ullgren, Jenny E.; Fer, Ilker; Darelius, Elin; Beaird, Nicholas

    2014-01-01

    The narrow and deep Faroe Bank Channel (FBC) is an important pathway for cold, dense waters from the Nordic Seas to flow across the Iceland-Scotland ridge into the North Atlantic. The swift, turbulent FBC overflow is associated with strong vertical mixing. Hydrographic profiles from a shipboard survey and two Slocum electric gliders deployed during a cruise in May-June 2012 show an intermediate water mass characterized by low salinity and low oxygen concentration between the upper waters of Atlantic origin and the dense overflow water. A weak low-salinity signal originating north-east of Iceland is discernible at the exit of the FBC, but smeared out by intense mixing. Further west (downstream) marked salinity and oxygen minima are found, which we hypothesize are indicators of a mixture of Labrador Sea Water and Intermediate Water from the Iceland Basin. Water mass characteristics vary strongly on short time scales. Low-salinity, low-oxygen water in the stratified interface above the overflow plume is shown to move along isopycnals toward the Iceland-Faroe Front as a result of eddy stirring and a secondary, transverse circulation in the plume interface. The interaction of low-salinity, low-oxygen intermediate waters with the overflow plume already at a short distance downstream of the sill, here reported for the first time, affects the final properties of the overflow waters through entrainment and mixing.

  5. An algorithm to detect tropical deep convective clouds through AMSU-B water vapor channels

    NASA Astrophysics Data System (ADS)

    Xu, Xu; Georg, Heygster; Zhang, Suping

    2009-03-01

    An algorithm to detect tropical deep convective clouds and deep convective overshootings based on the measurements from the three water vapor channels (183.3 GHz±1 GHz, 183.3 GHz±3 GHz and 183.3 GHz±7 GHz) of the Advanced Microwave Sounding Unit-B (AMSU-B) is presented. This algorithm is an improved version of the method of Hong et al. (2005). The proposed procedure is based on the statistical analysis of seven years’ (2001-2007) measurements from AMSU-B on NOAA-16. From the 1-d histograms of the brightness temperature of the three water vapor channels and the 2-d histograms of the brightness temperature difference between these channels, new thresholds for brightness temperature differences and the brightness temperature of channel 18 (183.3 GHz±1 GHz) are suggested. The new algorithm is employed to investigate the mean distribution of tropical deep convective clouds and convective overshootings from 30°S to 30°N for the years 2001 to 2007. The major concentration of deep convective clouds and convective overshootings is found over the Intertropical Convergence Zone (ITCZ), the South Pacific Convergence Zone (SPCZ), tropical Africa, South America, the Indian Ocean and Indonesia with an average fraction of 0.4%. In terms of these clouds we identify the secondary Intertropical Convergence Zone located in the eastern South Pacific and parallel to the main ITCZ in the North Pacific. The convective overshooting is more frequently observed over land than over the ocean.

  6. Modulation of proton transfer in the water wire of dioxolane-linked gramicidin channels by lipid membranes.

    PubMed

    de Godoy, C M; Cukierman, S

    2001-09-01

    Proton conductance (g(H)) in single SS stereoisomers of dioxolane-linked gramicidin A (gA) channels were measured in different phospholipid bilayers at different HCl concentrations. In particular, measurements were obtained in bilayers made of 1,2-diphytanoyl 3-phosphocholine (DiPhPC) or its ethylated derivative 1,2-diphytanoyl 3-ethyl-phosphocholine (et-DiPhPC,). The difference between these phospholipids is that in et-DiPhPC one of the phosphate oxygens is covalently linked to an ethyl group and cannot be protonated. In relatively dilute acid solutions, g(H) in DiPhPC is significantly higher than in et-DiPhPC. At high acid concentrations, g(H) is the same in both diphytanoyl bilayers. Such differences in g(H) can be accounted for by surface charge effects at the membrane/solution interfaces. In the linear portion of the log g(H)-log [H] relationship, g(H) values in diphytanoyl bilayers were significantly larger (approximately 10-fold) than in neutral glyceryl monooleate (GMO) membranes. The slopes of the linear log-log relationships between g(H) and [H] in diphytanoyl and GMO bilayers are essentially the same (approximately 0.76). This slope is significantly lower than the slope of the log-log plot of proton conductivity versus proton concentration in aqueous solutions (approximately 1.00). Because the chemical composition of the membrane-channel/solution interface is strikingly different in GMO and diphytanoyl bilayers, the reduced slope in g(H)-[HCl] relationships may be a characteristic of proton transfer in the water wire inside the SS channel. Values of g(H) in diphytanoyl bilayers were also significantly larger than in membranes made of the more common biological phospholipids 1-palmitoyl 2-oleoyl phosphocholine (POPC) or 1-palmitoyl 2-oleoyl phosphoethanolamine (POPE). These differences, however, cannot be accounted for by different surface charge effects or by different internal dipole potentials. On the other hand, maximum g(H) measured in the SS channel does not depend on the composition of the bilayer and is determined essentially by the reduced mobility of protons in concentrated acid solutions. Finally, no experimental evidence was found in support of a lateral proton movement at the phospholipid/solution interface contributing to g(H) in single SS channels. Protein-lipid interactions are likely to modulate g(H) in the SS channel. PMID:11509357

  7. Bidirectional Regulation of Dendritic Voltage-gated Potassium Channels by the Fragile X Mental Retardation Protein

    PubMed Central

    Lee, Hye Young; Ge, Woo-Ping; Huang, Wendy; He, Ye; Wang, Gordon X.; Rowson-Baldwin, Ashley; Smith, Stephen J; Jan, Yuh Nung; Jan, Lily Yeh

    2012-01-01

    Summary How transmitter receptors modulate neuronal signaling by regulating voltage-gated ion channel expression remains an open question. Here we report dendritic localization of mRNA of Kv4.2 voltage-gated potassium channel, which regulates synaptic plasticity, and its local translational regulation by fragile X mental retardation protein (FMRP) linked to fragile X syndrome (FXS), the most common heritable mental retardation. FMRP suppression of Kv4.2 is revealed by elevation of Kv4.2 in neurons from fmr1 knockout (KO) mice, and in neurons expressing Kv4.2-3?UTR that binds FMRP. Moreover, treating hippocampal slices from fmr1 KO mice with Kv4 channel blocker restores long-term potentiation (LTP) induced by moderate stimuli. Surprisingly, recovery of Kv4.2 after N-methyl-D-aspartate receptor (NMDAR)-induced degradation also requires FMRP, likely due to NMDAR-induced FMRP dephosphorylation, which turns off FMRP suppression of Kv4.2. Our study of FMRP regulation of Kv4.2 reveals a novel aspect of NMDAR signaling and a new FMRP target of potential relevance to FXS. PMID:22099464

  8. Moving Fe2+ from ferritin ion channels to catalytic OH centers depends on conserved protein cage carboxylates

    PubMed Central

    Behera, Rabindra K.; Theil, Elizabeth C.

    2014-01-01

    Ferritin biominerals are protein-caged metabolic iron concentrates used for iron–protein cofactors and oxidant protection (Fe2+ and O2 sequestration). Fe2+ passage through ion channels in the protein cages, like membrane ion channels, required for ferritin biomineral synthesis, is followed by Fe2+ substrate movement to ferritin enzyme (Fox) sites. Fe2+ and O2 substrates are coupled via a diferric peroxo (DFP) intermediate, ?max 650 nm, which decays to [Fe3+–O–Fe3+] precursors of caged ferritin biominerals. Structural studies show multiple conformations for conserved, carboxylate residues E136 and E57, which are between ferritin ion channel exits and enzymatic sites, suggesting functional connections. Here we show that E136 and E57 are required for ferritin enzyme activity and thus are functional links between ferritin ion channels and enzymatic sites. DFP formation (Kcat and kcat/Km), DFP decay, and protein-caged hydrated ferric oxide accumulation decreased in ferritin E57A and E136A; saturation required higher Fe2+ concentrations. Divalent cations (both ion channel and intracage binding) selectively inhibit ferritin enzyme activity (block Fe2+ access), Mn2+ << Co2+ < Cu2+ < Zn2+, reflecting metal ion–protein binding stabilities. Fe2+–Cys126 binding in ferritin ion channels, observed as Cu2+–S–Cys126 charge-transfer bands in ferritin E130D UV-vis spectra and resistance to Cu2+ inhibition in ferritin C126S, was unpredicted. Identifying E57 and E136 links in Fe2+ movement from ferritin ion channels to ferritin enzyme sites completes a bucket brigade that moves external Fe2+ into ferritin enzymatic sites. The results clarify Fe2+ transport within ferritin and model molecular links between membrane ion channels and cytoplasmic destinations. PMID:24843174

  9. Downregulation of the renal outer medullary K(+) channel ROMK by the AMP-activated protein kinase.

    PubMed

    Siraskar, Balasaheb; Huang, Dan Yang; Pakladok, Tatsiana; Siraskar, Gulab; Sopjani, Mentor; Alesutan, Ioana; Kucherenko, Yulia; Almilaji, Ahmad; Devanathan, Vasudharani; Shumilina, Ekaterina; Föller, Michael; Munoz, Carlos; Lang, Florian

    2013-02-01

    The 5'-adenosine monophosphate-activated serine/threonine protein kinase (AMPK) is stimulated by energy depletion, increase in cytosolic Ca(2+) activity, oxidative stress, and nitric oxide. AMPK participates in the regulation of the epithelial Na(+) channel ENaC and the voltage-gated K(+) channel KCNE1/KCNQ1. It is partially effective by decreasing PIP(2) formation through the PI3K pathway. The present study explored whether AMPK regulates the renal outer medullary K(+) channel ROMK. To this end, cRNA encoding ROMK was injected into Xenopus oocytes with and without additional injection of constitutively active AMPK(?R70Q) (AMPK(?1)-HA+AMPK(?1)-Flag+AMPK?1(R70Q)), or of inactive AMPK(?K45R) (AMPK(?1K45R)+AMPK(?1)-Flag+AMPK(?1)-HA), and the current determined utilizing two-electrode voltage-clamp and single channel patch clamp. ROMK protein abundance was measured utilizing chemiluminescence in Xenopus oocytes and western blot in whole kidney tissue. Moreover, renal Na(+) and K(+) excretion were determined in AMPK(?1)-deficient mice (ampk ( -/- )) and wild-type mice (ampk ( +/+ )) prior to and following an acute K(+) load (111 mM KCl, 30 mM NaHCO(3), 4.7 mM NaCl, and 2.25 g/dl BSA) at a rate of 500 ?l/h. As a result, coexpression of AMPK(?R70Q) but not of AMPK(?K45R) significantly decreased the current in ROMK1-expressing Xenopus oocytes. Injection of phosphatidylinositol PI((4,5))P(2) significantly increased the current in ROMK1-expressing Xenopus oocytes, an effect reversed in the presence of AMPK(?R70Q). Under control conditions, no significant differences between ampk ( -/- ) and ampk ( +/+ ) mice were observed in glomerular filtration rate (GFR), urinary flow rate, serum aldosterone, plasma Na(+), and K(+) concentrations as well as absolute and fractional Na(+) and K(+) excretion. Following an acute K(+) load, GFR, urinary flow rate, serum aldosterone, plasma Na(+), and K(+) concentration were again similar in both genotypes, but renal absolute and fractional Na(+) and K(+) excretion were higher in ampk ( -/- ) than in ampk ( +/+ ) mice. According to micropuncture following a K(+) load, delivery of Na(+) to the early distal tubule but not delivery of K(+) to late proximal and early distal tubules was increased in ampk (-/-) mice. The upregulation of renal ROMK1 protein expression by acute K(+) load was more pronounced in ampk (-/-) than in ampk ( +/+ ) mice. In conclusion, AMPK downregulates ROMK, an effect compromising the ability of the kidney to excrete K(+) following an acute K(+) load. PMID:23179379

  10. Subunit-specific inhibition of acid sensing ion channels by stomatin-like protein 1

    PubMed Central

    Kozlenkov, Alexey; Lapatsina, Liudmila; Lewin, Gary R; Smith, Ewan St John

    2014-01-01

    There are five mammalian stomatin-domain genes, all of which encode peripheral membrane proteins that can modulate ion channel function. Here we examined the ability of stomatin-like protein 1 (STOML1) to modulate the proton-sensitive members of the acid-sensing ion channel (ASIC) family. STOML1 profoundly inhibits ASIC1a, but has no effect on the splice variant ASIC1b. The inactivation time constant of ASIC3 is also accelerated by STOML1. We examined STOML1 null mutant mice with a ?-galactosidase-neomycin cassette gene-trap reporter driven from the STOML1 gene locus, which indicated that STOML1 is expressed in at least 50% of dorsal root ganglion (DRG) neurones. Patch clamp recordings from mouse DRG neurones identified a trend for larger proton-gated currents in neurones lacking STOML1, which was due to a contribution of effects upon both transient and sustained currents, at different pH, a finding consistent with an endogenous inhibitory function for STOML1. PMID:24247984

  11. A Dual-Channel Microwave Radiometer for Measurement of Precipitable Water Vapor and Liquid

    Microsoft Academic Search

    Fred Guiraud; Joe Howard; David Hogg

    1979-01-01

    The design and performance of a two-channel ground-based microwave radiometer (20.6 and 31.6 GHz) for measurement of total integrated water vapor and cloud liquid in a vertical column are discussed. Unique features of this instrument are a single antenna producing equal beamwidth for the two frequencies, and incorporation of two stable reference loads in a three-way Dicke switching sequence. The

  12. Role of Aquaporin Water Channels in Airway Fluid Transport, Humidification, and Surface Liquid Hydration

    Microsoft Academic Search

    Yuanlin Song; Sujatha Jayaraman; Baoxue Yang; Michael A. Matthay; A. S. Verkman

    2001-01-01

    Several aquaporin-type water channels are expressed in mammalian airways and lung: AQP1 in mi- crovascular endothelia, AQP3 in upper airway epithelia, AQP4 in upper and lower airway epithelia, and AQP5 in al- veolar epithelia. Novel quantitative methods were developed to compare airway fluid transport-related functions in wild-type mice and knockout mice deficient in these aquaporins. Lower airway humidification, measured from

  13. Optical Detection of Disordered Water Within a Protein Cavity

    PubMed Central

    Goldbeck, Robert A.; Pillsbury, Marlisa L.; Jensen, Russell A.; Mendoza, Juan L.; Nguyen, Rosa L.; Olson, John S.; Soman, Jayashree; Kliger, David S.; Esquerra, Raymond M.

    2009-01-01

    Internal water molecules are important to protein structure and function, but positional disorder and low occupancies can obscure their detection by x-ray crystallography. Here we show that water can be detected within the distal cavities of myoglobin mutants by subtle changes in the absorbance spectrum of pentacoordinate heme, even when the presence of solvent is not readily observed in the corresponding crystal structures. A well defined, non-coordinated water molecule hydrogen bonded to the distal histidine (His64) is seen within the distal heme pocket in the crystal structure of wild type (wt) deoxymyoglobin. Displacement of this water decreases the rate of ligand entry into wt Mb, and we have shown previously that the entry of this water is readily detected optically after laser photolysis of MbCO complexes. However, for L29F and V68L Mb no discrete positions for solvent molecules are seen in the electron density maps of the crystal structures even though His64 is still present and slow rates of ligand binding indicative of internal water are observed. In contrast, time-resolved perturbations of the visible absorption bands of L29F and V68L deoxyMb generated after laser photolysis detect the entry and significant occupancy of water within the distal pockets of these variants. Thus, the spectral perturbation of pentacoordinate heme offers a potentially robust system for measuring non-specific hydration of the active sites of heme proteins. PMID:19655795

  14. Fluctuations, exchange processes, and water diffusion in aqueous protein systems

    PubMed Central

    Kimmich, R.; Gneiting, T.; Kotitschke, K.; Schnur, G.

    1990-01-01

    Experimental frequency, concentration, and temperature dependences of the deuteron relaxation times T1 and T2 of D2O solutions of bovine serum albumin are reported and theoretically described in a closed form without formal parameters. Crucial processes of the theoretical concept are material exchange, translational diffusion of water molecules on the rugged surfaces of proteins, and tumbling of the macromolecules. It is also concluded that, apart from averaging of the relaxation rates in the diverse deuteron phases, material exchange contributes to transverse relaxation by exchange modulation of the Larmor frequency. The rate limiting factor of macromolecular tumbling is determined by the free water content. In a certain analogy to the classical free-volume theory, a “free-water-volume theory” is presented. There are two characteristic water mass fractions indicating the saturation of the hydration shells (Cs ? 0.3) and the onset of protein tumbling (C0 ? 0.6). The existence of the translational degrees of freedom of water molecules in the hydration shells has been verified by direct measurement of the diffusion coefficient using an NMR field-gradient technique. The concentration and temperature dependences show phenomena indicating a percolation transition of clusters of free water. The threshold water content was found to be Ccw ? 0.43. PMID:19431772

  15. Performance analysis of quantum key distribution based on air-water channel

    NASA Astrophysics Data System (ADS)

    Zhou, Yuan-yuan; Zhou, Xue-jun

    2015-03-01

    Considering the air-water interface and ocean water's optical attenuation, the performance of quantum key distribution (QKD) based on air-water channel is studied. The effects of photons' various incident angles to air-water interface on quantum bit error rate (QBER) and the maximum secure transmission distance are analyzed. Taking the optical attenuation of ocean water into account, the performance bounds of QKD in different types of ocean water are discussed. The simulation results show that the maximum secure transmission distance of QKD gradually reduces as the incident angle from air to ocean water increases. In the clearest ocean water with the lowest attenuation, the maximum secure transmission distance of photons far exceeds the the working depth of underwater vehicles. In intermediate and murky ocean waters with higher attenuation, the secure transmission distance shortens, but the underwater vehicle can deploy other accessorial methods for QKD with perfect security. So the implementation of OKD between the satellite and the underwater vehicle is feasible.

  16. Laser-induced acoustic wave generation\\/propagation\\/interaction in water in various internal channels

    Microsoft Academic Search

    Seung Hwan Ko; Daeho Lee; Heng Pan; Sang-Gil Ryu; Costas P. Grigoropoulos; Nick Kladias; Elias Panides; Gerald A. Domoto

    2010-01-01

    Short pulsed laser-induced single acoustic wave generation, propagation, interaction within a water-filled internal channel\\u000a are experimentally and numerically studied. A large-area, short-duration, single-plane acoustic wave was generated by the\\u000a thermoelastic interaction of a homogenized nanosecond pulsed laser beam with a liquid–solid interface and propagated at the\\u000a speed of sound in water. Laser flash Schlieren photography was used to visualize the

  17. Comparisons of the hydraulics of water flows in Martian outflow channels with flows of similar scale on earth

    NASA Technical Reports Server (NTRS)

    Komar, P. D.

    1979-01-01

    The hydraulics of channelized water flows on Mars and the resulting sediment transport rates are calculated, and similar computations are performed for such terrestrial analogs as the Mississippi River and the catastrophic Lake Missoula floods that formed the Channeled Scabland in eastern Washington State. The morphologies of deep-sea channels formed by catastrophic turbidity currents are compared with the Martian channels, many similarities are pointed out, and the hydraulics of the various flows are compared. The results indicate that the velocities, discharges, bottom shear stresses, and sediment-transport capacity of water flows along the Martian channels would be comparable to those of the oceanic turbidity currents and the Lake Missoula floods. It is suggested that the submarine canyons from which turbidity currents originate are the terrestrial counterparts to the chaotic-terrain areas or craters that serve as sources for many of the Martian channels.

  18. Soy protein polymers: Enhancing the water stability property

    NASA Astrophysics Data System (ADS)

    Srinivasan, Gowrishankar

    Soy protein based plastics have been processed in the past by researchers for various short-term applications; however a common issue is the high water sensitivity of these plastics. This work concentrates on resolving this water sensitivity issue of soy protein polymers by employing chemical and mechanical interaction at the molecular level during extrusion. The primary chemical interactions employed were anhydride chemistries such as maleic anhydride (MA), phthalic anhydride (PTA), and butylated hydroxyanisole (BHA). These were respectively used in conjunction with glycerol as a plasticizer to produce relatively water stable soy protein based plastics. Formulations with varying additive levels of the chemistries were extruded and injection molded to form the samples for characterization. The additive levels of anhydrides were varied between 3-10% tw/tw (total mass). Results indicated that phthalic anhydride formulations resulted in highest water stability. Plastic formulations with concentration up to 10% phthalic anhydride were observed to have water absorption as low as 21.5% after 24 hrs of exposure to water with respect to 250% for the control formulation. Fourier transform infrared spectroscopy (FTIR) was utilized to characterize and confirm the fundamental mechanisms of water stability achieved by phthalic and maleic anhydride chemistries. In addition, the anhydride formulations were modified by inclusion of cotton fibers and pretreated cotton powder in order to improve mechanical properties. The incorporation of cotton fibers improved the dry strength by 18%, but did not significantly improve the wet state strength of the plastics. It was also observed that the butylated-hydroxy anisole (BHA) formulation exhibited high extension values in the dry state and had inferior water absorption properties in comparison with anhydride formulations.

  19. The Outer Membrane Protein OmpW Forms an Eight-Stranded beta-Barrel with a Hydrophobic Channel

    SciTech Connect

    Hong,H.; Patel, D.; Tamm, L.; van den Berg, B.

    2006-01-01

    Escherichia coli OmpW belongs to a family of small outer membrane (OM) proteins that are widespread in Gram-negative bacteria. Their functions are unknown, but recent data suggest that they may be involved in the protection of bacteria against various forms of environmental stress. In order to gain insight into the function of these proteins we have determined the crystal structure of Escherichia coli OmpW to 2.7 Angstroms resolution. The structure shows that OmpW forms an eight-stranded beta-barrel with a long and narrow hydrophobic channel that contains a bound LDAO detergent molecule. Single channel conductance experiments show that OmpW functions as an ion channel in planar lipid bilayers. The channel activity can be blocked by the addition of LDAO. Taken together, the data suggest that members of the OmpW family could be involved in the transport of small hydrophobic molecules across the bacterial OM.

  20. Coupling of Retinal, Protein, and Water Dynamics in Squid Rhodopsin

    PubMed Central

    Jardón-Valadez, Eduardo; Bondar, Ana-Nicoleta; Tobias, Douglas J.

    2010-01-01

    The light-induced isomerization of the retinal from 11-cis to all-trans triggers changes in the conformation of visual rhodopsins that lead to the formation of the activated state, which is ready to interact with the G protein. To begin to understand how changes in the structure and dynamics of the retinal are transmitted to the protein, we performed molecular dynamics simulations of squid rhodopsin with 11-cis and all-trans retinal, and with two different force fields for describing the retinal molecule. The results indicate that structural rearrangements in the binding pocket, albeit small, propagate toward the cytoplasmic side of the protein, and affect the dynamics of internal water molecules. The sensitivity of the active-site interactions on the retinal force-field parameters highlights the coupling between the retinal molecule and its immediate protein environment. PMID:20923654

  1. Water stress inhibits hydraulic conductance and leaf growth in rice seedlings but not the transport of water via mercury-sensitive water channels in the root

    PubMed

    Lu; Neumann

    1999-05-01

    The mechanisms by which moderate water stress (adding polyethylene glycol 6000 to the root medium) induces a sustained inhibition of growth in emerging first leaves of intact rice (Oryza sativa) seedlings was investigated under growth-chamber conditions. Early (24 h) inhibition of leaf growth was not related to changes in root size or in osmotic potential gradients and cell wall-yielding characteristics in the leaf-expansion zone of stressed seedlings. However, reductions in root-to-leaf hydraulic conductance (L) were measured in two rice cultivars after 4 or 24 h at various levels of water stress, and these reductions correlated well with the inhibition of leaf growth. We assayed L by a psychrometric method and, in intact seedlings, by a novel osmotic-jump method. The addition of 0.5 mM HgCl2 to the root medium to inhibit water transport through Hg-sensitive water channels in the roots did not inhibit leaf growth in unstressed seedlings. However, both leaf growth and L were additionally reduced (by 49% and 43%, respectively) within minutes of adding HgCl2 to roots of water-stressed seedlings. Water stress therefore appeared to increase the transport of water via Hg-sensitive water channels. Other mechanisms were apparently involved in inhibiting overall L and leaf growth. PMID:10318692

  2. Channel Slope From SRTM Water Surface Elevations in the Amazon Basin

    NASA Astrophysics Data System (ADS)

    Hendricks, G. A.; Alsdorf, D. E.; Pavelsky, T. M.; Sheng, Y.

    2003-12-01

    A lack of hydrologic observations exists globally, particularly for large rivers in remote, non-industrialized regions. Considering the combined global decline in gauging stations as well as a prohibitive cost in increasing gauges to a reliable number for discharge prediction, remote sensing observations are both a necessary and efficient means for measuring certain hydraulic variables. Remote sensing observations allow for an unparalleled spatial scale and consistency in temporal acquisition. The Shuttle Radar Topography Mission (SRTM) digital elevation model (DEM) provides an initial means of measuring surface water elevations. Typically, open water flowing in channels yields specular radar pulse travel paths, however a roughened water surface can allow for partial radar return to the antennae. These water surface elevations combined with associated distances can be used to determine channel slope, which is an important component of the Manning equation and ultimately can be used to predict river discharge. Distance along each channel was extracted from the SRTM DEM by first determining flow vectors for each pixel from evaluation of the surrounding eight pixels, and then by linking the vectors. The resulting water slopes were calculated for the area from 0S-8S, 72W-54W. The Amazon mainstem was found to have a slope of 3.26 cm/km upstream of Manaus, while the slope is only 0.64 cm/km for the reach downstream of Manaus. The Tapajos slopes ranged from nearly flat near the mouth (0.29 cm/km) to a greater slope (17.57 cm/km to 11.47 cm/km) in the middle and upper reaches respectively, which are drained from a higher relief region. The Japura, Branco, Ica, and Madiera tributaries drain relatively higher relief regions and have slopes that range from 4.79 to 3.65 cm/km. The Purus and the Negro are more moderate with slopes of 3.09 cm/km and 2.47 cm/km respectively. These values are consistent with topographic relief and show that channel surface slopes can be measured on a vast spatial scale from SRTM provided that water surfaces are roughened. These slopes will be used in conjunction with depth values attained from nautical charts (for the mainstem Amazon) and width measurements obtained from classifications of JERS-1 low water data to estimate discharge of the Amazon and its major tributaries.

  3. Estimating suspended sediment concentrations in turbid coastal waters of the Santa Barbara Channel with SeaWiFS

    E-print Network

    Siegel, David A.

    Estimating suspended sediment concentrations in turbid coastal waters of the Santa Barbara Channel- tions of turbid coastal waters with remotely sensed multi-spectral data. The method improves upon many calibration in excessively turbid waters and influences of dissolved organic materials, chlorophyll pigments

  4. Potent Neutralization of Influenza A Virus by a Single-Domain Antibody Blocking M2 Ion Channel Protein

    Microsoft Academic Search

    Guowei Wei; Weixu Meng; Haijiang Guo; Weiqi Pan; Jinsong Liu; Tao Peng; Ling Chen; Chang-You Chen

    2011-01-01

    Influenza A virus poses serious health threat to humans. Neutralizing antibodies against the highly conserved M2 ion channel is thought to offer broad protection against influenza A viruses. Here, we screened synthetic Camel single-domain antibody (VHH) libraries against native M2 ion channel protein. One of the isolated VHHs, M2-7A, specifically bound to M2-expressed cell membrane as well as influenza A

  5. Maxi K + channels are stimulated by cyclic guanosine monophosphate-dependent protein kinase in canine coronary artery smooth muscle cells

    Microsoft Academic Search

    Junichi Taniguchi; Ken-Ichi Furukawa; Munekazu Shigekawa

    1993-01-01

    By using a patch clamp technique, we examined the effect of cyclic guanosine monophosphate (cGMP)-dependent protein kinase (G kinase) on Ca2+-activated maxi K+ channels in canine coronary artery smooth muscle cells. Maxi K+ channels (274±4 pS in symmetrical 140 mM KCl at 24–26°C) were activated by cytoplasmic Ca2+ and were completely blocked by 100 nM charybdotoxin (CTX). G kinase (300

  6. Evidence for Functional Diversity between the Voltage-Gated Proton Channel Hv1 and Its Closest Related Protein HVRP1

    PubMed Central

    Kim, Iris H.; Hevezi, Peter; Varga, Csaba; Pathak, Medha M.; Hong, Liang; Ta, Dennis; Tran, Chau T.; Zlotnik, Albert; Soltesz, Ivan; Tombola, Francesco

    2014-01-01

    The Hv1 channel and voltage-sensitive phosphatases share with voltage-gated sodium, potassium, and calcium channels the ability to detect changes in membrane potential through voltage-sensing domains (VSDs). However, they lack the pore domain typical of these other channels. NaV, KV, and CaV proteins can be found in neurons and muscles, where they play important roles in electrical excitability. In contrast, VSD-containing proteins lacking a pore domain are found in non-excitable cells and are not involved in neuronal signaling. Here, we report the identification of HVRP1, a protein related to the Hv1 channel (from which the name Hv1 Related Protein 1 is derived), which we find to be expressed primarily in the central nervous system, and particularly in the cerebellum. Within the cerebellar tissue, HVRP1 is specifically expressed in granule neurons, as determined by in situ hybridization and immunohistochemistry. Analysis of subcellular distribution via electron microscopy and immunogold labeling reveals that the protein localizes on the post-synaptic side of contacts between glutamatergic mossy fibers and the granule cells. We also find that, despite the similarities in amino acid sequence and structural organization between Hv1 and HVRP1, the two proteins have distinct functional properties. The high conservation of HVRP1 in vertebrates and its cellular and subcellular localizations suggest an important function in the nervous system. PMID:25165868

  7. Real-time visualization of oxygen partial pressures in straight channels of running polymer electrolyte fuel cell with water plugging

    NASA Astrophysics Data System (ADS)

    Nagase, Katsuya; Suga, Takeo; Nagumo, Yuzo; Uchida, Makoto; Inukai, Junji; Nishide, Hiroyuki; Watanabe, Masahiro

    2015-01-01

    Visualization inside polymer electrolyte fuel cells (PEFCs) for elucidating the reaction distributions is expected to improve the performance, durability, and stability. An oxygen-sensitive film of a luminescent porphyrin was used to visualize the oxygen partial pressures in five straight gas-flow channels of a running PEFC with liquid-water blockages formed at the end of the channels. The blockage greatly lowered and unstabilized the cell voltage. The oxygen partial pressure decreased nearly to 0 kPa in the blocked channel. With a water blockage in a channel, the oxygen partial pressures in the adjacent channels were lowered due to an extra demand of oxygen consumption. When the number of the blocked channels increased, the oxygen partial pressure in the unblocked channels became much lowered. When the water blockages disappeared, the oxygen partial pressures quickly returned to the values before plugging. The influence of the cross flows of air through the gas diffusion layers in straight channels was much smaller than that in serpentine flow channels.

  8. Inspection Head Design for the In-Service Inspection of Fuel Channels of Pressurized Heavy Water Reactors

    Microsoft Academic Search

    Amit Kumar Haruray; R. D. Veerapur; R. K. Puri; Manjit Singh

    2006-01-01

    This paper discusses the challenges associated with the mechanical design of Inspection Head for the in-service inspection (ISI) of fuel channels of Indian Pressurized Heavy Water Reactors (PHWRs). ISI is carried out during shut down period in the reactor. Non Destructive Examination (NDE) of fuel channels is a mandatory requirement to acquire knowledge about the structural condition. A typical 220

  9. Numerical investigation of interfacial transport resistance due to water droplets in proton exchange membrane fuel cell air channels

    E-print Network

    Kandlikar, Satish

    Nusselt number a b s t r a c t Oxygen transport resistance at the air flow channel and gas diffusion layerNumerical investigation of interfacial transport resistance due to water droplets in proton exchange membrane fuel cell air channels Mustafa Koz a , Satish G. Kandlikar a,b,* a Microsystems

  10. ABA signaling in guard cells entails a dynamic protein-protein interaction relay from the PYL-RCAR family receptors to ion channels.

    PubMed

    Lee, Sung Chul; Lim, Chae Woo; Lan, Wenzhi; He, Kai; Luan, Sheng

    2013-03-01

    Plant hormone abscisic acid (ABA) serves as an integrator of environmental stresses such as drought to trigger stomatal closure by regulating specific ion channels in guard cells. We previously reported that SLAC1, an outward anion channel required for stomatal closure, was regulated via reversible protein phosphorylation events involving ABA signaling components, including protein phosphatase 2C members and a SnRK2-type kinase (OST1). In this study, we reconstituted the ABA signaling pathway as a protein-protein interaction relay from the PYL/RCAR-type receptors, to the PP2C-SnRK2 phosphatase-kinase pairs, to the ion channel SLAC1. The ABA receptors interacted with and inhibited PP2C phosphatase activity against the SnRK2-type kinase, releasing active SnRK2 kinase to phosphorylate, and activate the SLAC1 channel, leading to reduced guard cell turgor and stomatal closure. Both yeast two-hybrid and bimolecular fluorescence complementation assays were used to verify the interactions among the components in the pathway. These biochemical assays demonstrated activity modifications of phosphatases and kinases by their interaction partners. The SLAC1 channel activity was used as an endpoint readout for the strength of the signaling pathway, depending on the presence of different combinations of signaling components. Further study using transgenic plants overexpressing one of the ABA receptors demonstrated that changing the relative level of interacting partners would change ABA sensitivity. PMID:22935148

  11. A trace component of ginseng that inhibits Ca2+ channels through a pertussis toxin-sensitive G protein.

    PubMed Central

    Nah, S Y; Park, H J; McCleskey, E W

    1995-01-01

    A crude extract from ginseng root inhibits high-threshold, voltage-dependent Ca2+ channels through an unknown receptor linked to a pertussis toxin-sensitive G protein. We now have found the particular compound that seems responsible for the effect: it is a saponin, called ginsenoside Rf (Rf), that is present in only trace amounts within ginseng. At saturating concentrations, Rf rapidly and reversibly inhibits N-type, and other high-threshold, Ca2+ channels in rat sensory neurons to the same degree as a maximal dose of opioids. The effect is dose-dependent (half-maximal inhibition: 40 microM) and it is virtually eliminated by pretreatment of the neurons with pertussis toxin, an inhibitor of G(o) and Gi GTP-binding proteins. Other ginseng saponins--ginsenosides Rb1, Rc, Re, and Rg1--caused relatively little inhibition of Ca2+ channels, and lipophilic components of ginseng root had no effect. Antagonists of a variety of neurotransmitter receptors that inhibit Ca2+ channels fail to alter the effect of Rf, raising the possibility that Rf acts through another G protein-linked receptor. Rf also inhibits Ca2+ channels in the hybrid F-11 cell line, which might, therefore, be useful for molecular characterization of the putative receptor for Rf. Because it is not a peptide and it shares important cellular and molecular targets with opioids, Rf might be useful in itself or as a template for designing additional modulators of neuronal Ca2+ channels. PMID:7568008

  12. Crystal Structure of the Mammalian GIRK2 KplusChannel and Gating Regulation by G Proteins PIP2 and Sodium

    SciTech Connect

    M Whorton; R MacKinnon

    2011-12-31

    G protein-gated K{sup +} channels (Kir3.1--Kir3.4) control electrical excitability in many different cells. Among their functions relevant to human physiology and disease, they regulate the heart rate and govern a wide range of neuronal activities. Here, we present the first crystal structures of a G protein-gated K{sup +} channel. By comparing the wild-type structure to that of a constitutively active mutant, we identify a global conformational change through which G proteins could open a G loop gate in the cytoplasmic domain. The structures of both channels in the absence and presence of PIP{sub 2} suggest that G proteins open only the G loop gate in the absence of PIP{sub 2}, but in the presence of PIP{sub 2} the G loop gate and a second inner helix gate become coupled, so that both gates open. We also identify a strategically located Na{sup +} ion-binding site, which would allow intracellular Na{sup +} to modulate GIRK channel activity. These data provide a structural basis for understanding multiligand regulation of GIRK channel gating.

  13. Crystal Structure of the Mammalian GIRK2 K+ Channel and Gating Regulation by G-Proteins, PIP2 and Sodium

    PubMed Central

    Whorton, Matthew R.; MacKinnon, Roderick

    2011-01-01

    Summary G-protein-gated K+ channels (Kir3.1–Kir3.4) control electrical excitability in many different cells. Among their functions relevant to human physiology and disease, they regulate the heart rate and govern a wide range of neuronal activities. Here we present the first crystal structures of a G-protein-gated K+ channel. By comparing the wild-type structure to that of a constitutively active mutant, we identify a global conformational change through which G-proteins could open a G-loop gate in the cytoplasmic domain. The structures of both channels in the absence and presence of PIP2 show that G-proteins open only the G-loop gate in the absence of PIP2, but in the presence of PIP2 the G-loop gate and a second inner helix gate become coupled, so that both gates open. We also identify a strategically located Na+ ion-binding site, which would allow intracellular Na+ to modulate GIRK channel activity. These data provide a mechanistic description of multi-ligand regulation of GIRK channel gating. PMID:21962516

  14. Generation of intense x-rays during ejection of a fast water jet from a metal channel to atmosphere

    Microsoft Academic Search

    A. A. Kornilova; V. I. Vysotskii; N. N. Sysoev; N. K. Litvin; V. I. Tomak; A. A. Barzov

    2010-01-01

    The radiation processes associated with a supersonic water jet exhausting from a narrow channel are considered. It has been\\u000a found for the first time that the output of the channel and the initial portion of the jet are sources of intense X-radiation,\\u000a generation of which is related to cavitation processes in the water jet bulk and subsequent excitation of shock

  15. Bio-optical properties of coastal waters in the Eastern English Channel

    NASA Astrophysics Data System (ADS)

    Vantrepotte, Vincent; Brunet, Christophe; Mériaux, Xavier; Lécuyer, Eric; Vellucci, Vincenzo; Santer, Richard

    2007-03-01

    Strong tidal currents, shallow water and numerous freshwater inputs characterize the coastal waters of the eastern English Channel. These case 2 waters were investigated through an intensive sampling effort in 2000 aiming to study the distribution and variability of the Chromophoric Dissolved Organic Matter (CDOM), Non-Algal Particles (NAP) and phytoplankton absorption at the mesoscale. Four cruises were carried out in February, March, May and July and more than 80 stations each cruise were sampled for hydrographical, chemical and bio-optical analyses. Results showed two distinct situations, the winter period characterized by the strong dominance of CDOM absorption over the particulate matter, and the spring-summer period when phytoplankton and CDOM represented the same contribution. Meteorology was the main factor driving the bio-optical properties of the water column in winter whereas in spring-summer the biological activity seemed to be the more active driving force. The algal community composition in term of dominant cell size and, therefore pigment packaging, is the main factor driving the phytoplankton specific absorption in the water column. Photoprotective pigments did not significantly influence algal absorption, due to turbid and highly mixed water masses. This feature also explained the bio-optical homogeneity found along the water column. On the mesoscale, distinct bio-optical provinces were defined in relation with the observed bio-hydrographical variability.

  16. Recovery from Slow Inactivation in K+ Channels is Controlled by Water Molecules

    PubMed Central

    Ostmeyer, Jared; Chakrapani, Sudha; Pan, Albert C.; Perozo, Eduardo; Roux, Benoît

    2013-01-01

    The bacterial K+ channel KcsA can be used to help elucidate questions about channel inactivation and recovery at the atomic level. Although KcsA contains only a pore domain, without voltage-sensing machinery, it has the structural elements necessary for ion conduction, activation and inactivation1–7. Available X-ray structures of KcsA provide an atomic view of the four most important functional states in which the intracellular gate is either closed or open, and the selectivity filter is either conductive or inactivated8–10. Application of a specific stimulus opens the intracellular gate of a K+ channel (activation), yielding a transient period of ion conduction until the selectivity filter spontaneously undergoes a conformational change toward a non-conductive state (inactivation). Removal of the stimulus closes the gate and allows the selectivity filter to interconvert back to its conductive conformation (recovery). In this manuscript, a series of long molecular dynamics (MD) simulations reveal how the selectivity filter is sterically locked in the inactive conformation by buried water molecules bound behind the selectivity filter. Potential of mean force calculations show how the recovery process is affected by the buried waters and the rebinding of an external K+ ion. A kinetic model deduced from the simulations shows how releasing the buried waters can stretch the timescale of recovery to seconds. This leads to the prediction that reducing the occupancy of the buried waters by imposing a high osmotic stress should accelerate the rate of recovery, which was verified experimentally by measuring the recovery rate in the presence of 2M sucrose. PMID:23892782

  17. Bordetella pertussis major outer membrane porin protein forms small, anion-selective channels in lipid bilayer membranes.

    PubMed Central

    Armstrong, S K; Parr, T R; Parker, C D; Hancock, R E

    1986-01-01

    The major outer membrane protein of molecular weight 40,000 (the 40K protein) of a virulent isolate of Bordetella pertussis was purified to apparent homogeneity. The purified protein formed an oligomer band (of apparent molecular weight 90,000) on sodium dodecyl sulfate-polyacrylamide gels after solubilization at low temperatures. The porin function of this protein was characterized by the black lipid bilayer method. The 40K protein formed channels smaller than all other constitutive major outer membrane porins studied to date. The average single-channel conductance in 1 M KCl was 0.56 nS. This was less than a third of the conductance previously observed for Escherichia coli porins. Zero-current potential measurements made of the porin to determine its ion selectivity revealed the porin to be more than 100-fold selective for anions over cations. The single-channel conductance was measured as a function of salt concentration. The data could be fitted to a Lineweaver-Burk plot suggesting an anion binding site with a Kd of 1.17 M Cl- and a maximum possible conductance through the channel of 1.28 nS. Images PMID:2420780

  18. Sensitivity of polarization fluctuations to the nature of protein-water interactions: study of biological water in four different protein-water systems.

    PubMed

    Ghosh, Rikhia; Banerjee, Saikat; Hazra, Milan; Roy, Susmita; Bagchi, Biman

    2014-12-14

    Since the time of Kirkwood, observed deviations in magnitude of the dielectric constant of aqueous protein solution from that of neat water (?80) and slower decay of polarization have been subjects of enormous interest, controversy, and debate. Most of the common proteins have large permanent dipole moments (often more than 100 D) that can influence structure and dynamics of even distant water molecules, thereby affecting collective polarization fluctuation of the solution, which in turn can significantly alter solution's dielectric constant. Therefore, distance dependence of polarization fluctuation can provide important insight into the nature of biological water. We explore these aspects by studying aqueous solutions of four different proteins of different characteristics and varying sizes, chicken villin headpiece subdomain (HP-36), immunoglobulin binding domain protein G (GB1), hen-egg white lysozyme (LYS), and Myoglobin (MYO). We simulate fairly large systems consisting of single protein molecule and 20000-30000 water molecules (varied according to the protein size), providing a concentration in the range of ?2-3 mM. We find that the calculated dielectric constant of the system shows a noticeable increment in all the cases compared to that of neat water. Total dipole moment auto time correlation function of water ??MW(0)?MW(t)? is found to be sensitive to the nature of the protein. Surprisingly, dipole moment of the protein and total dipole moment of the water molecules are found to be only weakly coupled. Shellwise decomposition of water molecules around protein reveals higher density of first layer compared to the succeeding ones. We also calculate heuristic effective dielectric constant of successive layers and find that the layer adjacent to protein has much lower value (?50). However, progressive layers exhibit successive increment of dielectric constant, finally reaching a value close to that of bulk 4-5 layers away. We also calculate shellwise orientational correlation function and tetrahedral order parameter to understand the local dynamics and structural re-arrangement of water. Theoretical analysis providing simple method for calculation of shellwise local dielectric constant and implication of these findings are elaborately discussed in the present work. PMID:25494802

  19. Sensitivity of polarization fluctuations to the nature of protein-water interactions: Study of biological water in four different protein-water systems

    NASA Astrophysics Data System (ADS)

    Ghosh, Rikhia; Banerjee, Saikat; Hazra, Milan; Roy, Susmita; Bagchi, Biman

    2014-12-01

    Since the time of Kirkwood, observed deviations in magnitude of the dielectric constant of aqueous protein solution from that of neat water (˜80) and slower decay of polarization have been subjects of enormous interest, controversy, and debate. Most of the common proteins have large permanent dipole moments (often more than 100 D) that can influence structure and dynamics of even distant water molecules, thereby affecting collective polarization fluctuation of the solution, which in turn can significantly alter solution's dielectric constant. Therefore, distance dependence of polarization fluctuation can provide important insight into the nature of biological water. We explore these aspects by studying aqueous solutions of four different proteins of different characteristics and varying sizes, chicken villin headpiece subdomain (HP-36), immunoglobulin binding domain protein G (GB1), hen-egg white lysozyme (LYS), and Myoglobin (MYO). We simulate fairly large systems consisting of single protein molecule and 20000-30000 water molecules (varied according to the protein size), providing a concentration in the range of ˜2-3 mM. We find that the calculated dielectric constant of the system shows a noticeable increment in all the cases compared to that of neat water. Total dipole moment auto time correlation function of water ??MW(0)?MW(t)? is found to be sensitive to the nature of the protein. Surprisingly, dipole moment of the protein and total dipole moment of the water molecules are found to be only weakly coupled. Shellwise decomposition of water molecules around protein reveals higher density of first layer compared to the succeeding ones. We also calculate heuristic effective dielectric constant of successive layers and find that the layer adjacent to protein has much lower value (˜50). However, progressive layers exhibit successive increment of dielectric constant, finally reaching a value close to that of bulk 4-5 layers away. We also calculate shellwise orientational correlation function and tetrahedral order parameter to understand the local dynamics and structural re-arrangement of water. Theoretical analysis providing simple method for calculation of shellwise local dielectric constant and implication of these findings are elaborately discussed in the present work.

  20. Protein kinase CK2 triggers cytosolic zinc signaling pathways by phosphorylation of zinc channel ZIP7

    PubMed Central

    Taylor, Kathryn M.; Hiscox, Stephen; Nicholson, Robert I.; Hogstrand, Christer; Kille, Peter

    2012-01-01

    The transition element zinc, which has recently been identified as an intracellular second messenger, has been implicated in various signaling pathways, including those leading to cell proliferation. Zinc channels of the ZIP protein family (Solute Carrier Family 39A, SLC39A) transiently increase the cytosolic free zinc (Zn2+) concentration in response to extracellular signals. Here, we show that phosphorylation of evolutionarily conserved residues in zinc transporter ZIP7 is associated with the gated release of Zn2+ from intracellular stores, leading to activation of tyrosine kinases and the phosphorylation of AKT and extracellular signal-regulated kinases 1 and 2 (ERK1/2). Through pharmacological manipulation, proximity assay, and mutagenesis, we identified CK2 as the kinase responsible for ZIP7 activation. Together, the present results show that eukaryotic transition element channels can be activated post-translationally by phosphorylation eliciting a cell signaling cascade. Our study links the regulated release of zinc from intracellular stores to phosphorylation of kinases involved in proliferative responses and cell migration, suggesting a functional role for ZIP7 and zinc signals for these events which are characteristic of cancerous cells. Furthermore, the interaction of ZIP7 with CK2, a kinase that is antiapoptotoc and promotes cell division, highlights the potential for ZIP7 as a target for anti-cancer drug development. PMID:22317921

  1. ANOs 3–7 in the anoctamin/Tmem16 Cl? channel family are intracellular proteins

    PubMed Central

    Duran, Charity; Qu, Zhiqiang; Osunkoya, Adeboye O.; Cui, Yuanyuan

    2012-01-01

    Ca2+-activated Cl? channels (CaCCs) participate in numerous physiological functions such as neuronal excitability, sensory transduction, and transepithelial fluid transport. Recently, it was shown that heterologously expressed anoctamins ANO1 and ANO2 generate currents that resemble native CaCCs. The anoctamin family (also called Tmem16) consists of 10 members, but it is not known whether all members of the family are CaCCs. Expression of ANOs 3–7 in HEK293 cells did not generate Cl? currents activated by intracellular Ca2+, as determined by whole cell patch clamp electrophysiology. With the use of confocal imaging, only ANO1 and ANO2 traffic to the plasma membrane when expressed heterologously. Furthermore, endogenously expressed ANO7 in the human prostate is predominantly intracellular. We took a chimeric approach to identify regions critical for channel trafficking and function. However, none of the chimeras of ANO1 and ANO5/7 that we made trafficked to the plasma membrane. Our results suggest that intracellular anoctamins may be endoplasmic reticulum proteins, although it remains unknown whether these family members are CaCCs. Determining the role of anoctamin family members in ion transport will be critical to understanding their functions in physiology and disease. PMID:22075693

  2. ANOs 3-7 in the anoctamin/Tmem16 Cl- channel family are intracellular proteins.

    PubMed

    Duran, Charity; Qu, Zhiqiang; Osunkoya, Adeboye O; Cui, Yuanyuan; Hartzell, H Criss

    2012-02-01

    Ca(2+)-activated Cl(-) channels (CaCCs) participate in numerous physiological functions such as neuronal excitability, sensory transduction, and transepithelial fluid transport. Recently, it was shown that heterologously expressed anoctamins ANO1 and ANO2 generate currents that resemble native CaCCs. The anoctamin family (also called Tmem16) consists of 10 members, but it is not known whether all members of the family are CaCCs. Expression of ANOs 3-7 in HEK293 cells did not generate Cl(-) currents activated by intracellular Ca(2+), as determined by whole cell patch clamp electrophysiology. With the use of confocal imaging, only ANO1 and ANO2 traffic to the plasma membrane when expressed heterologously. Furthermore, endogenously expressed ANO7 in the human prostate is predominantly intracellular. We took a chimeric approach to identify regions critical for channel trafficking and function. However, none of the chimeras of ANO1 and ANO5/7 that we made trafficked to the plasma membrane. Our results suggest that intracellular anoctamins may be endoplasmic reticulum proteins, although it remains unknown whether these family members are CaCCs. Determining the role of anoctamin family members in ion transport will be critical to understanding their functions in physiology and disease. PMID:22075693

  3. Characterization of vegetative storage protein (VSP) and low molecular proteins induced by water deficit in stolon of white clover.

    PubMed

    Lee, Bok-Rye; Lee, Dong-Gi; Avice, Jean-Christophe; Kim, Tae-Hwan

    2014-01-01

    In stolon of white clover (Trifolium repens L.), the 17.3 kDa protein has been newly identified as a vegetative storage protein (VSP) which has preponderant roles in N accumulation and mobilization to sustain growth when capacity of N uptake is strongly reduced. To characterize the water deficit effect on this protein, the kinetic pattern of soluble protein, SDS-PAGE, Western blotting, and proteomic analysis was studied in the stolon of white clover during 28 days of water-deficit. Water deficit led to decrease protein concentration. SDS-PAGE revealed that two major proteins of 17.3 and 16 kDa were accumulated to high level in response to water stress. These proteins cross-reacted positively with antibodies raised against the 17.3 kDa VSP, a protein which shared biochemical features with stress proteins implied in dehydration tolerance. Using two-dimensional electrophoresis (2-DE) gel and matrix-assisted laser desorption/ionization time-of-flight mass spectrometer (MALDI-TOF-MS) analysis, it was demonstrated that 19.5 and 17.3 kDa protein spots were up-regulated by water stress, and both spots were identical to nucleoside diphosphate kinase (NDPK) and lipid transfer proteins (LTPs), respectively. These results suggest that low molecular proteins induced by water-deficit in the stolon of white clover act as an alternative N reserves or play significant roles in plant protection against water-deficit stress. PMID:24299955

  4. The Mycobacterium tuberculosis Outer Membrane Channel Protein CpnT Confers Susceptibility to Toxic Molecules.

    PubMed

    Danilchanka, Olga; Pires, David; Anes, Elsa; Niederweis, Michael

    2015-04-01

    Mycobacterium tuberculosis, the causative agent of tuberculosis, is protected from toxic solutes by an effective outer membrane permeability barrier. Recently, we showed that the outer membrane channel protein CpnT is required for efficient nutrient uptake by M. tuberculosis and Mycobacterium bovis BCG. In this study, we found that the cpnT mutant of M. bovis BCG is more resistant than the wild type to a large number of drugs and antibiotics, including rifampin, ethambutol, clarithromycin, tetracycline, and ampicillin, by 8- to 32-fold. Furthermore, the cpnT mutant of M. bovis BCG was 100-fold more resistant to nitric oxide, a major bactericidal agent required to control M. tuberculosis infections in mice. Thus, CpnT constitutes the first outer membrane susceptibility factor in slow-growing mycobacteria. The dual functions of CpnT in uptake of nutrients and mediating susceptibility to toxic molecules are reflected in macrophage infection experiments: while loss of CpnT was detrimental for M. bovis BCG in macrophages that enable bacterial replication, presumably due to inadequate nutrient uptake, it conferred a survival advantage in macrophages that mount a strong bactericidal response. Importantly, the cpnT gene showed a significantly higher density of nonsynonymous mutations in drug-resistant clinical M. tuberculosis strains, indicating that CpnT is under selective pressure in human tuberculosis and/or during chemotherapy. Our results indicate that the CpnT channel constitutes an outer membrane gateway controlling the influx of nutrients and toxic molecules into slow-growing mycobacteria. This study revealed that reducing protein-mediated outer membrane permeability might constitute a new drug resistance mechanism in slow-growing mycobacteria. PMID:25645841

  5. The elusive character of discontinuous deep-water channels: New insights from Lucia Chica channel system, offshore California

    USGS Publications Warehouse

    Maier, K.L.; Fildani, A.; Paull, C.K.; Graham, S.A.; McHargue, T.R.; Caress, D.W.; McGann, M.

    2011-01-01

    New high-resolution autonomous underwater vehicle (AUV) seafloor images, with 1 m lateral resolution and 0.3 m vertical resolution, reveal unexpected seafloor rugosity and low-relief (<10 m), discontinuous conduits over ~70 km2. Continuous channel thalwegs were interpreted originally from lower-resolution images, but newly acquired AUV data indicate that a single sinuous channel fed a series of discontinuous lower-relief channels. These discontinuous channels were created by at least four avulsion events. Channel relief, defined as the height from the thalweg to the levee crest, controls avulsions and overall stratigraphic architecture of the depositional area. Flowstripped turbidity currents separated into and reactivated multiple channels to create a distributary pattern and developed discontinuous trains of cyclic scours and megaflutes, which may be erosional precursors to continuous channels. The diverse features now imaged in the Lucia Chica channel system (offshore California) are likely common in modern and ancient systems with similar overall morphologies, but have not been previously mapped with lower-resolution detection methods in any of these systems. ?? 2011 Geological Society of America.

  6. Solvent effects on squid sodium channels are attributable to movements of a flexible protein structure in gating currents and to hydration in a pore

    PubMed Central

    Kukita, Fumio

    2000-01-01

    Solvent effects on the time course of gating and sodium currents were analysed in squid sodium channels using four non-electrolytes of different size, glycerol, erythritol, glucose and sucrose, to separate effects of viscosity from those of osmolarity and to obtain viscosity and osmolarity parameters that were independent of molecular size. The gating and sodium currents were reversibly slowed in a voltage-independent manner as the non-electrolyte concentration increased. Solvent effects were analysed using a model in which the percentage change in time constant was expressed by an equation involving the viscosity parameter ? and the osmolarity parameter ?: t/t0 =?((?/?0) – 1 + 100??1)exp(???), where ?/?0 is solution viscosity and ?? is increase in osmolarity. Since the solution viscosity was found experimentally to be a function of the solution osmolarity, solvent effects are described by an equation with one independent variable ?/?0 or ??. Voltage sensor movement, reflected in gating currents, was primarily sensitive to viscosity, as its decay time constant was a function of ?/?0, with only a minor sensitivity to osmolarity (? was 2–3 water molecules). For sodium currents, ? was equal to that of gating currents but ? was 2–3 times greater, suggesting that the final channel opening was primarily sensitive to osmolarity (?? was 5 water molecules). The relative ineffectiveness of the largest non-electrolyte, sucrose, suggested that this osmolarity-sensitive step in channel opening occurred in the narrow pore region. Sodium channel inactivation was primarily sensitive to osmolarity (?? was 8–12 water molecules). The observed viscosity dependence of the sodium current activation and inactivation processes was attributable to the viscosity-dependent process accompanying the gating current. This model explains why non-electrolytes slow sodium currents while electrolytes do not. Viscosity effects on gating currents can be explained by a process in which non-electrolytes interact with the flexible hydrophilic parts of sodium channel proteins, but osmolarity effects on the final step need to be explained by a local interaction of several water molecules with fluctuating protein segments in the pore. PMID:10713962

  7. Regulation of Cardiac ATP-sensitive Potassium Channel Surface Expression by Calcium/Calmodulin-dependent Protein Kinase II*

    PubMed Central

    Sierra, Ana; Zhu, Zhiyong; Sapay, Nicolas; Sharotri, Vikas; Kline, Crystal F.; Luczak, Elizabeth D.; Subbotina, Ekaterina; Sivaprasadarao, Asipu; Snyder, Peter M.; Mohler, Peter J.; Anderson, Mark E.; Vivaudou, Michel; Zingman, Leonid V.; Hodgson-Zingman, Denice M.

    2013-01-01

    Cardiac ATP-sensitive potassium (KATP) channels are key sensors and effectors of the metabolic status of cardiomyocytes. Alteration in their expression impacts their effectiveness in maintaining cellular energy homeostasis and resistance to injury. We sought to determine how activation of calcium/calmodulin-dependent protein kinase II (CaMKII), a central regulator of calcium signaling, translates into reduced membrane expression and current capacity of cardiac KATP channels. We used real-time monitoring of KATP channel current density, immunohistochemistry, and biotinylation studies in isolated hearts and cardiomyocytes from wild-type and transgenic mice as well as HEK cells expressing wild-type and mutant KATP channel subunits to track the dynamics of KATP channel surface expression. Results showed that activation of CaMKII triggered dynamin-dependent internalization of KATP channels. This process required phosphorylation of threonine at 180 and 224 and an intact 330YSKF333 endocytosis motif of the KATP channel Kir6.2 pore-forming subunit. A molecular model of the ?2 subunit of the endocytosis adaptor protein, AP2, complexed with Kir6.2 predicted that ?2 docks by interaction with 330YSKF333 and Thr-180 on one and Thr-224 on the adjacent Kir6.2 subunit. Phosphorylation of Thr-180 and Thr-224 would favor interactions with the corresponding arginine- and lysine-rich loops on ?2. We concluded that calcium-dependent activation of CaMKII results in phosphorylation of Kir6.2, which promotes endocytosis of cardiac KATP channel subunits. This mechanism couples the surface expression of cardiac KATP channels with calcium signaling and reveals new targets to improve cardiac energy efficiency and stress resistance. PMID:23223335

  8. Vpr Protein of Human Immunodeficiency Virus Type 1 Forms Cation-Selective Channels in Planar Lipid Bilayers

    NASA Astrophysics Data System (ADS)

    Piller, S. C.; Ewart, G. D.; Premkumar, A.; Cox, G. B.; Gage, P. W.

    1996-01-01

    A small (96-aa) protein, virus protein R (Vpr), of human immunodeficiency virus type 1 contains one hydrophobic segment that could form a membrane-spanning helix. Recombinant Vpr, expressed in Escherichia coli and purified by affinity chromatography, formed ion channels in planar lipid bilayers when it was added to the cis chamber and when the trans chamber was held at a negative potential. The channels were more permeable to Na+ than to Cl- ions and were inhibited when the trans potential was made positive. Similar channel activity was caused by Vpr that had a truncated C terminus, but the potential dependence of channel activity was no longer seen. Antibody raised to a peptide mimicking part of the C terminus of Vpr (AbC) inhibited channel activity when added to the trans chamber but had no effect when added to the cis chamber. Antibody to the N terminus of Vpr (AbN) increased channel activity when added to the cis chamber but had no effect when added to the trans chamber. The effects of potential and antibodies on channel activity are consistent with a model in which the positive C-terminal end of dipolar Vpr is induced to traverse the bilayer membrane when the opposite (trans) side of the membrane is at a negative potential. The C terminus of Vpr would then be available for interaction with AbC in the trans chamber, and the N terminus would be available for interaction with AbN in the cis chamber. The ability of Vpr to form ion channels in vitro suggests that channel formation by Vpr in vivo is possible and may be important in the life cycle of human immunodeficiency virus type 1 and/or may cause changes in cells that contribute to AIDS-related pathologies.

  9. Evidence for Recent Liquid Water on Mars: Channeled Aprons in a Small Crater within Newton Crater

    NASA Technical Reports Server (NTRS)

    2000-01-01

    [figure removed for brevity, see original site]

    Newton Crater is a large basin formed by an asteroid impact that probably occurred more than 3 billion years ago. It is approximately 287 kilometers (178 miles) across. The picture shown here (top) highlights the north wall of a specific, smaller crater located in the southwestern quarter of Newton Crater (above). The crater of interest was also formed by an impact; it is about 7 km (4.4 mi) across, which is about 7 times bigger than the famous Meteor Crater in northern Arizona in North America.

    The north wall of the small crater has many narrow gullies eroded into it. These are hypothesized to have been formed by flowing water and debris flows. Debris transported with the water created lobed and finger-like deposits at the base of the crater wall where it intersects the floor (bottom center top image). Many of the finger-like deposits have small channels indicating that a liquid--most likely water--flowed in these areas. Hundreds of individual water and debris flow events might have occurred to create the scene shown here. Each outburst of water from higher upon the crater slopes would have constituted a competition between evaporation, freezing, and gravity.

    The individual deposits at the ends of channels in this MOC image mosaic were used to get a rough estimate of the minimum amount of water that might be involved in each flow event. This is done first by assuming that the deposits are like debris flows on Earth. In a debris flow, no less than about 10% (and no more than 30%) of their volume is water. Second, the volume of an apron deposit is estimated by measuring the area covered in the MOC image and multiplying it by a conservative estimate of thickness, 2 meters (6.5 feet). For a flow containing only 10% water, these estimates conservatively suggest that about 2.5 million liters (660,000 gallons) of water are involved in each event; this is enough to fill about 7 community-sized swimming pools or enough to supply 20 people with their water needs for a year.

    The MOC high resolution view is located near 41.1oS, 159.8oW and is a mosaic of three different pictures acquired between January and May 2000. The MOC scene is illuminated from the left; north is up. The context picture was acquired in 1977 by the Viking 1 orbiter and is illuminated from the upper right.

  10. Early Fluid and Protein Shifts in Men During Water Immersion

    NASA Technical Reports Server (NTRS)

    Hinghofer-Szalkay, H.; Harrison, M. H.; Greenleaf, J. E.

    1987-01-01

    High precision blood and plasma densitometry was used to measure transvascular fluid shifts during water immersion to the neck. Six men (28-49 years) undertook 30 min of standing immersion in water at 35.0 +/- 0.2 C; immersion was preceded by 30 min control standing in air at 28 +/- 1 C. Blood was sampled from an antecubital catheter for determination of Blood Density (BD), Plasma Density (PD), Haematocrit (Ht), total Plasma Protein Concentration (PPC), and Plasma Albumin Concentration (PAC). Compared to control, significant decreases (p less than 0.01) in all these measures were observed after 20 min immersion. At 30 min, plasma volume had increased by 11.0 +/- 2.8%; the average density of the fluid shifted from extravascular fluid into the vascular compartment was 1006.3 g/l; albumin moved with the fluid and its albumin concentration was about one-third of the plasma protein concentration during early immersion. These calculations are based on the assumption that the F-cell ratio remained unchanged. No changes in erythrocyte water content during immersion were found. Thus, immersion-induced haemodilution is probably accompanied by protein (mainly albumin) augmentation which accompanies the intra-vascular fluid shift.

  11. Protein structure, stability and solubility in water and other solvents.

    PubMed Central

    Pace, C Nick; Treviño, Saul; Prabhakaran, Erode; Scholtz, J Martin

    2004-01-01

    Proteins carry out the most difficult tasks in living cells. They do so by interacting specifically with other molecules. This requires that they fold to a unique, globular conformation that is only marginally more stable than the large ensemble of unfolded states. The folded state is stabilized mainly by the burial and tight packing of over 80% of the peptide groups and non-polar side chains. If life as we know it is to exist in a solvent other than water, the folded state must be stable and soluble in the new solvent. Our analysis suggests that proteins will be unstable in most polar solvents such as ethanol, extremely stable in non-polar solvents such as cyclohexane, and even more stable in a vacuum. Our solubility studies suggest that protein solubility will be markedly lower in polar solvents such as ethanol and that proteins will be essentially insoluble in non-polar solvents such as cyclohexane. For these and other reasons it seems unlikely that the life we know could exist in any solvent system other than water. PMID:15306378

  12. Estimating seepage flux from ephemeral stream channels using surface water and groundwater level data

    NASA Astrophysics Data System (ADS)

    Noorduijn, Saskia L.; Shanafield, Margaret; Trigg, Mark A.; Harrington, Glenn A.; Cook, Peter G.; Peeters, L.

    2014-02-01

    Seepage flux from ephemeral streams can be an important component of the water balance in arid and semiarid regions. An emerging technique for quantifying this flux involves the measurement and simulation of a flood wave as it moves along an initially dry channel. This study investigates the usefulness of including surface water and groundwater data to improve model calibration when using this technique. We trialed this approach using a controlled flow event along a 1387 m reach of artificial stream channel. Observations were then simulated using a numerical model that combines the diffusion-wave approximation of the Saint-Vénant equations for streamflow routing, with Philip's infiltration equation and the groundwater flow equation. Model estimates of seepage flux for the upstream segments of the study reach, where streambed hydraulic conductivities were approximately 101 m d-1, were on the order of 10-4 m3 d-1 m-2. In the downstream segments, streambed hydraulic conductivities were generally much lower but highly variable (˜10-3 to 10-7 m d-1). A Latin Hypercube Monte Carlo sensitivity analysis showed that the flood front timing, surface water stage, groundwater heads, and the predicted streamflow seepage were most influenced by specific yield. Furthermore, inclusion of groundwater data resulted in a higher estimate of total seepage estimates than if the flood front timing were used alone.

  13. Role of water in protein aggregation and amyloid polymorphism.

    PubMed

    Thirumalai, D; Reddy, Govardhan; Straub, John E

    2012-01-17

    A variety of neurodegenerative diseases are associated with amyloid plaques, which begin as soluble protein oligomers but develop into amyloid fibrils. Our incomplete understanding of this process underscores the need to decipher the principles governing protein aggregation. Mechanisms of in vivo amyloid formation involve a number of coconspirators and complex interactions with membranes. Nevertheless, understanding the biophysical basis of simpler in vitro amyloid formation is considered important for discovering ligands that preferentially bind regions harboring amyloidogenic tendencies. The determination of the fibril structure of many peptides has set the stage for probing the dynamics of oligomer formation and amyloid growth through computer simulations. Most experimental and simulation studies, however, have been interpreted largely from the perspective of proteins: the role of solvent has been relatively overlooked in oligomer formation and assembly to protofilaments and amyloid fibrils. In this Account, we provide a perspective on how interactions with water affect folding landscapes of amyloid beta (A?) monomers, oligomer formation in the A?16-22 fragment, and protofilament formation in a peptide from yeast prion Sup35. Explicit molecular dynamics simulations illustrate how water controls the self-assembly of higher order structures, providing a structural basis for understanding the kinetics of oligomer and fibril growth. Simulations show that monomers of A? peptides sample a number of compact conformations. The formation of aggregation-prone structures (N*) with a salt bridge, strikingly similar to the structure in the fibril, requires overcoming a high desolvation barrier. In general, sequences for which N* structures are not significantly populated are unlikely to aggregate. Oligomers and fibrils generally form in two steps. First, water is expelled from the region between peptides rich in hydrophobic residues (for example, A?16-22), resulting in disordered oligomers. Then the peptides align along a preferred axis to form ordered structures with anti-parallel ?-strand arrangement. The rate-limiting step in the ordered assembly is the rearrangement of the peptides within a confining volume. The mechanism of protofilament formation in a polar peptide fragment from the yeast prion, in which the two sheets are packed against each other and create a dry interface, illustrates that water dramatically slows self-assembly. As the sheets approach each other, two perfectly ordered one-dimensional water wires form. They are stabilized by hydrogen bonds to the amide groups of the polar side chains, resulting in the formation of long-lived metastable structures. Release of trapped water from the pore creates a helically twisted protofilament with a dry interface. Similarly, the driving force for addition of a solvated monomer to a preformed fibril is water release; the entropy gain and favorable interpeptide hydrogen bond formation compensate for entropy loss in the peptides. We conclude by offering evidence that a two-step model, similar to that postulated for protein crystallization, must also hold for higher order amyloid structure formation starting from N*. Distinct water-laden polymorphic structures result from multiple N* structures. Water plays multifarious roles in all of these protein aggregations. In predominantly hydrophobic sequences, water accelerates fibril formation. In contrast, water-stabilized metastable intermediates dramatically slow fibril growth rates in hydrophilic sequences. PMID:21761818

  14. Role of mixed boundaries on flow in open capillary channels with curved air-water interfaces.

    PubMed

    Zheng, Wenjuan; Wang, Lian-Ping; Or, Dani; Lazouskaya, Volha; Jin, Yan

    2012-09-01

    Flow in unsaturated porous media or in engineered microfluidic systems is dominated by capillary and viscous forces. Consequently, flow regimes may differ markedly from conventional flows, reflecting strong interfacial influences on small bodies of flowing liquids. In this work, we visualized liquid transport patterns in open capillary channels with a range of opening sizes from 0.6 to 5.0 mm using laser scanning confocal microscopy combined with fluorescent latex particles (1.0 ?m) as tracers at a mean velocity of ?0.50 mm s(-1). The observed velocity profiles indicate limited mobility at the air-water interface. The application of the Stokes equation with mixed boundary conditions (i.e., no slip on the channel walls and partial slip or shear stress at the air-water interface) clearly illustrates the increasing importance of interfacial shear stress with decreasing channel size. Interfacial shear stress emerges from the velocity gradient from the adjoining no-slip walls to the center where flow is trapped in a region in which capillary forces dominate. In addition, the increased contribution of capillary forces (relative to viscous forces) to flow on the microscale leads to increased interfacial curvature, which, together with interfacial shear stress, affects the velocity distribution and flow pattern (e.g., reverse flow in the contact line region). We found that partial slip, rather than the commonly used stress-free condition, provided a more accurate description of the boundary condition at the confined air-water interface, reflecting the key role that surface/interface effects play in controlling flow behavior on the nanoscale and microscale. PMID:22867425

  15. TRP channel-associated factors are a novel protein family that regulates TRPM8 trafficking and activity.

    PubMed

    Gkika, Dimitra; Lemonnier, Loic; Shapovalov, George; Gordienko, Dmitri; Poux, Céline; Bernardini, Michela; Bokhobza, Alexandre; Bidaux, Gabriel; Degerny, Cindy; Verreman, Kathye; Guarmit, Basma; Benahmed, Mohamed; de Launoit, Yvan; Bindels, Rene J M; Fiorio Pla, Alessandra; Prevarskaya, Natalia

    2015-01-01

    TRPM8 is a cold sensor that is highly expressed in the prostate as well as in other non-temperature-sensing organs, and is regulated by downstream receptor-activated signaling pathways. However, little is known about the intracellular proteins necessary for channel function. Here, we identify two previously unknown proteins, which we have named "TRP channel-associated factors" (TCAFs), as new TRPM8 partner proteins, and we demonstrate that they are necessary for channel function. TCAF1 and TCAF2 both bind to the TRPM8 channel and promote its trafficking to the cell surface. However, they exert opposing effects on TRPM8 gating properties. Functional interaction of TCAF1/TRPM8 also leads to a reduction in both the speed and directionality of migration of prostate cancer cells, which is consistent with an observed loss of expression of TCAF1 in metastatic human specimens, whereas TCAF2 promotes migration. The identification of TCAFs introduces a novel mechanism for modulation of TRPM8 channel activity. PMID:25559186

  16. Guard cell anion channel SLAC1 is regulated by CDPK protein kinases with distinct Ca2+ affinities

    PubMed Central

    Geiger, D.; Scherzer, S.; Mumm, P.; Marten, I.; Ache, P.; Matschi, S.; Liese, A.; Wellmann, C.; Al-Rasheid, K. A. S.; Grill, E.; Romeis, T.; Hedrich, R.

    2010-01-01

    In response to drought stress, the phytohormone abscisic acid (ABA) induces stomatal closure. Thereby the stress hormone activates guard cell anion channels in a calcium-dependent, as well as –independent, manner. Open stomata 1 protein kinase (OST1) and ABI1 protein phosphatase (ABA insensitive 1) represent key components of calcium-independent ABA signaling. Recently, the guard cell anion channel SLAC1 was identified. When expressed heterologously SLAC1 remained electrically silent. Upon coexpression with Ca2+-independent OST1, however, SLAC1 anion channels appear activated in an ABI1-dependent manner. Mutants lacking distinct calcium-dependent protein kinases (CPKs) appeared impaired in ABA stimulation of guard cell ion channels, too. To study SLAC1 activation via the calcium-dependent ABA pathway, we studied the SLAC1 response to CPKs in the Xenopus laevis oocyte system. Split YFP-based protein–protein interaction assays, using SLAC1 as the bait, identified guard cell expressed CPK21 and 23 as major interacting partners. Upon coexpression of SLAC1 with CPK21 and 23, anion currents document SLAC1 stimulation by these guard cell protein kinases. Ca2+-sensitive activation of SLAC1, however, could be assigned to the CPK21 pathway only because CPK23 turned out to be rather Ca2+-insensitive. In line with activation by OST1, CPK activation of the guard cell anion channel was suppressed by ABI1. Thus the CPK and OST1 branch of ABA signal transduction in guard cells seem to converge on the level of SLAC1 under the control of the ABI1/ABA-receptor complex. PMID:20385816

  17. C. elegans TRP family protein TRP-4 is a pore-forming subunit of a native mechanotransduction channel

    PubMed Central

    Kang, Lijun; Gao, Jingwei; Schafer, William R.; Xie, Zhixiong; Xu, X. Z. Shawn

    2010-01-01

    Summary Mechanotransduction channels mediate several common sensory modalities such as hearing, touch, and proprioception; however, very little is known about the molecular identities of these channels. Many TRP family channels have been implicated in mechanosensation, but none of them has been demonstrated to form a mechanotransduction channel, raising the question of whether TRP proteins simply play indirect roles in mechanosensation. Using C. elegans as a model, here we have recorded a mechanosensitive conductance in a ciliated mechanosensory neuron in vivo. This conductance develops very rapidly upon mechanical stimulation with its latency and activation time constant reaching the range of micro-seconds, consistent with mechanical gating of the conductance. TRP-4, a TRPN (NOMPC) subfamily channel, is required for this conductance. Importantly, point mutations in the predicted pore region of TRP-4 alter the ion selectivity of the conductance. These results identify TRP-4 as the first TRP protein that functions as an essential pore-forming subunit of a native mechanotransduction channel. PMID:20696377

  18. Water-surface elevation controls on sediment-transport dynamics in channel-flat environments of intertidal flats

    NASA Astrophysics Data System (ADS)

    Nowacki, D. J.; Ogston, A. S.

    2010-12-01

    Tidal flats are thought to have a balanced sediment budget between export through channels and import via more diffuse processes over flat boundaries. However, little has been done to understand the mechanisms of sediment transport between channels and flats that span multiple morphological and temporal scales. The muddy flats of southeastern Willapa Bay, Washington, are tidally dominated and receive relatively little direct freshwater influence. We use data from instrumented tripods in representative channel and flat pairs of different orders to a) better understand sediment dynamics in each morphological setting, b) investigate whether sediment fluxes are balanced between channels and flats, and c) determine the importance of channel order on these sediment dynamics. Data from intensive field efforts as well as longer-term deployments help to inform how the hydrodynamic regimes of each environment serve to export or retain sediment and to further characterize the total sediment budget of intertidal flats. Results from two month-long deployments in winter 2009-2010 show channels of all orders in southeastern Willapa Bay were flood dominated. This was driven by longer durations of and sustained higher velocities during flooding tides, and suggests that larger circulation patterns were active within the tidal flat complex. The deployment periods were characterized by a range of meteorological conditions, including rain and several wind events. The wind events were correlated with increased flood dominance of water and sediment transport. Near-bed observations of velocity and suspended-sediment concentration (SSC) give insight to processes active when water levels are shallow over the flat. These processes are important in determining the net flux of water and sediment of the system. High-resolution water-column velocity and backscatter profiles reveal complex sediment-flux dynamics between channel and flat environments. Pulses of velocity and SSC were observed in the channel during flooding and ebbing tides when water levels were near the flat elevation, a phenomenon often observed in tidal flats and salt marshes. Instrumentation deployed near the bed on the flat measured elevated SSC when flat water depth dropped below 10 cm. This “skimming” of sediment on the flat contributed to the SSC pulse in the channel during ebbing tides. Water convergence from the flat led to increased channel bottom stresses and resuspension of freshly deposited sediment temporarily stored within the channel. These fine-scale observations allow us to address the mechanisms that govern the total sediment balance of channels and flats within tidal flat systems.

  19. Spacial Distribution of Salinity and the Mechanism of Saltwater Intrusion in the Modaomen Water Channel of Pear River Estuary

    NASA Astrophysics Data System (ADS)

    Liu, J. B.; Bao, Y.

    2011-09-01

    Modaomen channel is an important fresh water resource in Pearl River Delta. It has been impacted by saltwater intrusion frequently in the last decade. This has drawn more and more attention from scientists and engineers. The hydrodynamic mechanism of saltwater intrusion is still impercipient. In the present paper, hydrographs of velocity and salinity in the channel are analyzed based on field observations of velocity and salinity of upper, middle, and lower water layers at several stations along the Modaomen channel. It is found that the transport of salinity in Modaomen channel is obviously different from other estuaries. As the tidal range increases from neap to spring tide, the salinity in each water layer decreases unexpectedly. This peculiar phenomenon is attributed to the extraordinary flow process in the channel. When salinity value in each layer and vertical salinity gradient are lower during spring tide, no matter on rising or ebbing tide, the flow velocity monotonously decreases from water surface to the bottom, which is suggested by common sense. However, when salinity values and vertical salinity gradient are higher during neap tide, the flow velocity unexpectedly increases from water surface to the bottom during flood period, and flood duration of the bottom current is surprisingly as long as 15-18 hours. In addition, an inflexional velocity profile may remain amazingly for about 9 hours. This could be driven by the baroclinic pressure under the condition of tides, topography and upstream runoff discharge of this channel.

  20. Automatic Measurement of Water Levels by Using Image Identification Method in Open Channel

    NASA Astrophysics Data System (ADS)

    Chung Yang, Han; Xue Yang, Jia

    2014-05-01

    Water level data is indispensable to hydrology research, and it is important information for hydraulic engineering and overall utilization of water resources. The information of water level can be transmitted to management office by the network so that the management office may well understand whether the river level is exceeding the warning line. The existing water level measurement method can only present water levels in a form of data without any of images, the methods which make data just be a data and lack the sense of reality. Those images such as the rising or overflow of river level that the existing measurement method cannot obtain simultaneously. Therefore, this research employs a newly, improved method for water level measurement. Through the Video Surveillance System to record the images on site, an image of water surface will be snapped, and then the snapped image will be pre-processed and be compared with its altitude reference value to obtain a water level altitude value. With the ever-growing technology, the application scope of image identification is widely in increase. This research attempts to use image identification technology to analyze water level automatically. The image observation method used in this research is one of non-contact water level gage but it is quite different from other ones; the image observation method is cheap and the facilities can be set up beside an embankment of river or near the houses, thus the impact coming from external factors will be significantly reduced, and a real scene picture will be transmitted through wireless transmission. According to the dynamic water flow test held in an indoor experimental channel, the results of the research indicated that all of error levels of water level identification were less than 2% which meant the image identification could achieve identification result at different water levels. This new measurement method can offer instant river level figures and on-site video so that a disaster prevention measures can be made accordingly. Keywords: Image identification; Water Level; Video surveillance system.

  1. Water Dynamics and Dewetting Transitions in the Small Mechanosensitive Channel MscS

    PubMed Central

    Anishkin, Andriy; Sukharev, Sergei

    2004-01-01

    The dynamics of confined water in capillaries and nanotubes suggests that gating of ion channels may involve not only changes of the pore geometry, but also transitions between water-filled and empty states in certain locations. The recently solved heptameric structure of the small mechanosensitive channel of Escherichia coli, MscS, has revealed a relatively wide (7–15 Å) yet highly hydrophobic transmembrane pore. Continuum estimations based on the properties of pore surface suggest low conductance and a thermodynamic possibility of dewetting. To test the predictions we performed molecular dynamics simulations of MscS filled with flexible TIP3P water. Irrespective to the initial conditions, several independent 6-ns simulations converged to the same stable state with the pore water-filled in the wider part, but predominantly empty in the narrow hydrophobic part, displaying intermittent vapor-liquid transitions. The polar gain-of-function substitution L109S in the constriction resulted in a stable hydration of the entire pore. Steered passages of Cl? ions through the narrow part of the pore consistently produced partial ion dehydration and required a force of 200–400 pN to overcome an estimated barrier of 10–20 kcal/mole, implying negligibly low conductance. We conclude that the crystal structure of MscS does not represent an open state. We infer that MscS gate, which is similar to that of the nicotinic ACh receptor, involves a vapor-lock mechanism where limited changes of geometry or surface polarity can locally switch the regime between water-filled (conducting) and empty (nonconducting) states. PMID:15111405

  2. A multi-channel gel electrophoresis and continuous fraction collection apparatus for high throughput protein separation and characterization

    SciTech Connect

    Choi, Megan; Nordmeyer, Robert A.; Cornell, Earl; Dong, Ming; Biggin, Mark D.; Jin, Jian

    2009-10-02

    To facilitate a direct interface between protein separation by PAGE and protein identification by mass spectrometry, we developed a multichannel system that continuously collects fractions as protein bands migrate off the bottom of gel electrophoresis columns. The device was constructed using several short linear gel columns, each of a different percent acrylamide, to achieve a separation power similar to that of a long gradient gel. A Counter Free-Flow elution technique then allows continuous and simultaneous fraction collection from multiple channels at low cost. We demonstrate that rapid, high-resolution separation of a complex protein mixture can be achieved on this system using SDS-PAGE. In a 2.5 h electrophoresis run, for example, each sample was separated and eluted into 48-96 fractions over a mass range of 10-150 kDa; sample recovery rates were 50percent or higher; each channel was loaded with up to 0.3 mg of protein in 0.4 mL; and a purified band was eluted in two to three fractions (200 L/fraction). Similar results were obtained when running native gel electrophoresis, but protein aggregation limited the loading capacity to about 50 g per channel and reduced resolution.

  3. tassel-less1 encodes a boron channel protein required for inflorescence development in maize.

    PubMed

    Leonard, April; Holloway, Beth; Guo, Mei; Rupe, Mary; Yu, GongXin; Beatty, Mary; Zastrow-Hayes, Gina; Meeley, Robert; Llaca, Victor; Butler, Karlene; Stefani, Tony; Jaqueth, Jennifer; Li, Bailin

    2014-06-01

    tassel-less1 (tls1) is a classical maize (Zea mays) inflorescence mutant. Homozygous mutant plants have no tassels or very small tassels, and ear development is also impaired. Using a positional cloning approach, ZmNIP3;1 (a NOD26-like intrinsic protein) was identified as the candidate gene for tls1. The ZmNIP3;1 gene is completely deleted in the tls1 mutant genome. Two Mutator-insertional TUSC alleles of ZmNIP3;1 exhibited tls1-like phenotypes, and allelism tests confirmed that the tls1 gene encodes ZmNIP3;1. Transgenic plants with an RNA interference (RNAi) construct to down-regulate ZmNIP3;1 also showed tls1-like phenotypes, further demonstrating that TLS1 is ZmNIP3;1. Sequence analysis suggests that ZmNIP3;1 is a boron channel protein. Foliar application of boron could rescue the tls1 phenotypes and restore the normal tassel and ear development. Gene expression analysis indicated that in comparison with that of the wild type or tls1 plants treated with boron, the transition from the vegetative to reproductive phase or the development of the floral meristem is impaired in the shoot apical meristem of the tls1 mutant plants. It is concluded that the tls1 mutant phenotypes are caused by impaired boron transport, and boron is essential for inflorescence development in maize. PMID:24685595

  4. Activation and Inhibition of Neuronal G Protein-Gated Inwardly Rectifying K Channels by P2Y Nucleotide Receptors

    E-print Network

    Brown, David

    Activation and Inhibition of Neuronal G Protein-Gated Inwardly Rectifying K Channels by P2Y Nucleotide Receptors Alexander K. Filippov, Jose M. Fern ´andez-Fern ´andez, Stephen J. Marsh, Joseph SimonSADP) induced activation of GIRK current (IGIRK) followed by inhibition. In contrast, stimulation of endogenous

  5. Fine-tuning synaptic plasticity by modulation of CaV2.1 channels with Ca2+ sensor proteins

    PubMed Central

    Leal, Karina; Mochida, Sumiko; Scheuer, Todd; Catterall, William A.

    2012-01-01

    Modulation of P/Q-type Ca2+ currents through presynaptic voltage-gated calcium channels (CaV2.1) by binding of Ca2+/calmodulin contributes to short-term synaptic plasticity. Ca2+-binding protein-1 (CaBP1) and Visinin-like protein-2 (VILIP-2) are neurospecific calmodulin-like Ca2+ sensor proteins that differentially modulate CaV2.1 channels, but how they contribute to short-term synaptic plasticity is unknown. Here, we show that activity-dependent modulation of presynaptic CaV2.1 channels by CaBP1 and VILIP-2 has opposing effects on short-term synaptic plasticity in superior cervical ganglion neurons. Expression of CaBP1, which blocks Ca2+-dependent facilitation of P/Q-type Ca2+ current, markedly reduced facilitation of synaptic transmission. VILIP-2, which blocks Ca2+-dependent inactivation of P/Q-type Ca2+ current, reduced synaptic depression and increased facilitation under conditions of high release probability. These results demonstrate that activity-dependent regulation of presynaptic CaV2.1 channels by differentially expressed Ca2+ sensor proteins can fine-tune synaptic responses to trains of action potentials and thereby contribute to the diversity of short-term synaptic plasticity. PMID:23027954

  6. Students' Understanding of External Representations of the Potassium Ion Channel Protein Part II: Structure-Function Relationships and Fragmented Knowledge

    ERIC Educational Resources Information Center

    Harle, Marissa; Towns, Marcy H.

    2012-01-01

    Research that has focused on external representations in biochemistry has uncovered student difficulties in comprehending and interpreting external representations. This study focuses on students' understanding of three external representations (ribbon diagram, wireframe, and hydrophobic/hydrophilic) of the potassium ion channel protein. Analysis…

  7. Replacement of fish meal in juvenile channel catfish, Ictalurus punctatus, diets using a yeast-derived protein source

    Technology Transfer Automated Retrieval System (TEKTRAN)

    We examined the effects of a yeast-derived protein source (NuPro) as a replacement for menhaden fish meal on weight gain, specific growth rate (SGR), food conversion ratio (FCR), whole-body composition, and disease resistance in juvenile channel catfish. NuPro replaced 0, 20, 40, 60, 80, and 100% o...

  8. Experimental study of Cu-water nanofluid forced convective flow inside a louvered channel

    NASA Astrophysics Data System (ADS)

    Khoshvaght-Aliabadi, M.; Hormozi, F.; Zamzamian, A.

    2015-03-01

    Heat transfer enhancement plays a very important role for energy saving in plate-fin heat exchangers. In the present study, the influences of simultaneous utilization of a louvered plate-fin channel and copper-base deionized water nanofluid on performance of these exchangers are experimentally explored. The effects of flow rate (2-5 l/min) and nanoparticles weight fraction (0-0.4 %) on heat transfer and pressure drop characteristics are determined. Experimental results indicate that the use of louvered channel instead of the plain one can improve the heat transfer performance. Likewise, addition of small amounts of copper nanoparticles to the base fluid augments the convective heat transfer coefficient remarkably. The maximum rise of 21.7 % in the convective heat transfer coefficient is observed for the 0.4 % wt nanofluid compared to the base fluid. Also, pumping power for the base fluid and nanofluids are calculated based on the measured pressure drop in the louvered channel. The average increase in pumping power is 11.8 % for the nanofluid with 0.4 % wt compared to the base fluid. Applied performance criterion shows a maximum performance index of 1.167 for the nanofluid with 0.1 % wt Finally, two correlations are proposed for Nusselt number and friction factor which fit the experimental data with in ±10 %.

  9. Identification of two distinct uncoupling protein 2 (UCP2) messenger RNAs and the relationship between expression, food intake, and growth in channel catfish

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Uncoupling protein 2 is a member of the mitochondrial channel proteins that regulate the flow of hydrogen ions and ATP generation. The relationship between UCP2 and nutrient metabolism has been well-defined in humans but unclear in fish. We hypothesized that increased muscle growth in channel catf...

  10. Quantitative analysis of the water occupancy around the selectivity filter of a K+ channel in different gating modes.

    PubMed

    Weingarth, Markus; van der Cruijsen, Elwin A W; Ostmeyer, Jared; Lievestro, Sylke; Roux, Benoît; Baldus, Marc

    2014-02-01

    Recovery in K(+) channels, that is, the transition from the inactivated nonconductive selectivity filter conformation toward the conductive conformation, occurs on a time scale of the order of seconds, which is astonishingly long, given that the structural differences among the filter conformations are faint (<1 Å). Computational studies and electrophysiological measurements suggested that buried water molecules bound behind the selectivity filter are at the origin of the slowness of recovery in K(+) channels. Using a combination of solid-state NMR spectroscopy (ssNMR) and long molecular dynamics simulations, we sketch a high-resolution map of the spatial and temporal distribution of water behind the selectivity filter of a membrane-embedded K(+) channel in two different gating modes. Our study demonstrates that buried water molecules with long residence times are spread all along the rear of the inactivated filter, which explains the recovery kinetics. In contrast, the same region of the structure appears to be dewetted when the selectivity filter is in the conductive state. Using proton-detected ssNMR on fully protonated channels, we demonstrate the presence of a pathway that allows for the interchange of buried and bulk water, as required for a functional influence of buried water on recovery and slow inactivation. Furthermore, we provide direct experimental evidence for the presence of additional ordered water molecules that surround the filter and that are modulated by the channel's gating mode. PMID:24410583

  11. Channel water balance and exchange with subsurface flow along a mountain headwater stream in Montana, United States

    USGS Publications Warehouse

    Payn, R.A.; Gooseff, M.N.; McGlynn, B.L.; Bencala, K.E.; Wondzell, S.M.

    2009-01-01

    Channel water balances of contiguous reaches along streams represent a poorly understood scale of stream-subsurface interaction. We measured reach water balances along a headwater stream in Montana, United States, during summer base flow recessions. Reach water balances were estimated from series of tracer tests in 13 consecutive reaches delineated evenly along a 2.6 km valley segment. For each reach, we estimated net change in discharge, gross hydrologic loss, and gross hydrologic gain from tracer dilution and mass recovery. Four series of tracer tests were performed during relatively high, intermediate, and low base flow conditions. The relative distribution of channel water along the stream was strongly related to a transition in valley structure, with a general increase in gross losses through the recession. During tracer tests at intermediate and low flows, there were frequent substantial losses of tracer mass (>10%) that could not be explained by net loss in flow over the reach, indicating that many of the study reaches were concurrently losing and gaining water. For example, one reach with little net change in discharge exchanged nearly 20% of upstream flow with gains and losses along the reach. These substantial bidirectional exchanges suggest that some channel interactions with subsurface flow paths were not measurable by net change in flow or transient storage of recovered tracer. Understanding bidirectional channel water balances in stream reaches along valleys is critical to an accurate assessment of stream solute fate and transport and to a full assessment of exchanges between the stream channel and surrounding subsurface.

  12. Sensitivity to Abscisic Acid of Guard-Cell K^+ Channels is Suppressed by abi1-1, a Mutant Arabidopsis Gene Encoding a Putative Protein Phosphatase

    Microsoft Academic Search

    Fiona Armstrong; Jeffrey Leung; Alexander Grabov; Jane Brearley; Jerome Giraudat; Michael R. Blatt

    1995-01-01

    Abscisic acid (ABA) modulates the activities of three major classes of ion channels-inward- and outward-rectifying K^+ channels (IK,in and IK,out, respectively) and anion channels-at the guard-cell plasma membrane to achieve a net efflux of osmotica and stomatal closure. Disruption of ABA sensitivity in wilty abi1-1 mutants of Arabidopsis and evidence that this gene encodes a protein phosphatase suggest that protein

  13. The TRPC Ion Channels: Association with Orai1 and STIM1 Proteins and Participation in Capacitative and Non-capacitative Calcium Entry

    Microsoft Academic Search

    Gines M. Salido; Isaac Jardín; Juan A. Rosado

    \\u000a Transient receptor potential (TRP) proteins are involved in a large number of non-selective cation channels that are permeable\\u000a to both monovalent and divalent cations. Two general classes of receptor-mediated Ca2+ entry has been proposed: one of then is conduced by receptor-operated Ca2+ channels (ROC), the second is mediated by channels activated by the emptying of intracellular Ca2+ stores (store-operated channels

  14. Chip-type asymmetrical flow field-flow fractionation channel coupled with mass spectrometry for top-down protein identification.

    PubMed

    Kim, Ki Hun; Moon, Myeong Hee

    2011-11-15

    A chip-type design asymmetrical flow field-flow fractionation (AF4) channel has been developed for high-speed separation of proteins and top-down proteomic analysis using online coupled electrospray ionization mass spectrometry (ESI-MS). The new miniaturized AF4 channel was assembled by stacking multilayer thin stainless steel (SS, 1.5 mm each) plates embedded with an SS frit in such a way that the total thickness of the channel assembly was about 6 mm. The efficiency of the miniaturized AF4 channel at different channel lengths was examined with the separation of protein standards by adjusting flow rates in which an identical effective channel flow rate or an identical void time can be maintained at different channels. Detection limit, overloading effect, reproducibility, and influence of channel membrane materials on separation efficiency were investigated. Desalting and purification of proteins achieved during the AF4 operation by the action of an exiting crossflow and the use of aqueous mass-spectrometry-compatible (MS-compatible) buffer were advantageous for online coupling of the chip-type AF4 with ESI-MS. The direct coupling of AF4 and ESI-MS capabilities was demonstrated for the high-speed separation and identification of carbonic anhydrase (29 kDa) and transferrin (78 kDa) by full scan MS and for the first top-down identification of proteins with AF4-ESI-MS-MS using collision-induced fragmentation (CID). The presence of intact dimers (156 kDa) of transferrin was confirmed by AF4-ESI-MS via size separation of the dimers from monomers, followed by multiply charged ion spectral analysis of the dimers and molecular mass determinations. It was also found from these experiments that AF4-ESI-MS analysis of transferrin exhibited an increased signal-to-noise ratio compared to that of direct ESI-MS analysis due to online purification of the protein sample and size separation of dimers with AF4. PMID:21981549

  15. 3-D imaging beneath water-bottom channels in the Gippsland Basin: A case study

    SciTech Connect

    Young, K.T.J.; Schneider, W.A. Jr. (Exxon Production Research Co., Houston, TX (United States)); Moore, J.F. (Esso Australia Limited, Melbourne (Australia)) (and others)

    1996-01-01

    Water-bottom channels in the Gippsland Basin cause nonhyperbolic moveout and time delays resulting in disrupted and distorted structural images on time sections. In 3-D processing, prestack depth migration is the ultimate processing technique for resolving nonhyperbolic moveout and producing correct depth images. But, the high cost and slow turnaround time of 3-D prestack depth migration limits its application. An alternative 3-D processing approach, consisting of prestack 3-D replacement dynamics, stacking, poststack inverse 3-D replacement dynamics, and 3-D poststack depth migration, was applied to a 3-D survey located near the shelf-slope edge of the Gippsland Basin. Application of 3-D replacement dynamics reduced nonhyperbolic moveout caused by variable water depth and improved the quality of stacked traces. We found that the interval velocity distribution below the sea floor is largely controlled by compaction or depth below the sea floor. Poststack depth migration, with a compaction-based velocity field, removed the structural distortion beneath the channels, resulting in an accurate 3-D depth image.

  16. 3-D imaging beneath water-bottom channels in the Gippsland Basin: A case study

    SciTech Connect

    Young, K.T.J.; Schneider, W.A. Jr. [Exxon Production Research Co., Houston, TX (United States); Moore, J.F. [Esso Australia Limited, Melbourne (Australia)] [and others

    1996-12-31

    Water-bottom channels in the Gippsland Basin cause nonhyperbolic moveout and time delays resulting in disrupted and distorted structural images on time sections. In 3-D processing, prestack depth migration is the ultimate processing technique for resolving nonhyperbolic moveout and producing correct depth images. But, the high cost and slow turnaround time of 3-D prestack depth migration limits its application. An alternative 3-D processing approach, consisting of prestack 3-D replacement dynamics, stacking, poststack inverse 3-D replacement dynamics, and 3-D poststack depth migration, was applied to a 3-D survey located near the shelf-slope edge of the Gippsland Basin. Application of 3-D replacement dynamics reduced nonhyperbolic moveout caused by variable water depth and improved the quality of stacked traces. We found that the interval velocity distribution below the sea floor is largely controlled by compaction or depth below the sea floor. Poststack depth migration, with a compaction-based velocity field, removed the structural distortion beneath the channels, resulting in an accurate 3-D depth image.

  17. On the behavior of water at subfreezing temperatures in a protein crystal: evidence of higher mobility than in bulk water.

    PubMed

    Wang, Dongqi; Böckmann, Anja; Dolenc, Jožica; Meier, Beat H; van Gunsteren, Wilfred F

    2013-10-01

    NMR experiments have shown that water molecules in the crystal of the protein Crh are still mobile at temperatures well below 273 K. In order to investigate this water anomaly, a molecular dynamics (MD) simulation study of crystalline Crh was carried out to determine the mobility of water in this crystal. The simulations were carried out at three temperatures, 150, 200, and 291 K. Simulations of bulk water at these temperatures were also done to obtain the properties of the simple point charge (SPC) water model used at these temperatures and to allow a comparison of the properties of water in the Crh crystal with those of bulk water at the same temperatures. According to the simulations, water is immobilized at 150 K both in crystal and in bulk water. As expected, at 291 K it diffuses and rotates more slowly in the protein crystal than in bulk water. However, at 200 K, the translational and rotational mobility of the water molecules is larger in the crystal than in bulk water. The enhancement of water mobility in the crystal at 200 K was further investigated by MD simulations in which the backbone or all protein atoms were positionally restrained, and in which additionally the electrostatic protein-water interactions were removed. Of these changes in the environment of the water molecules, rigidifying the protein backbones slightly enhanced water diffusion, while it slowed down rotation. In contrast, removal of electrostatic protein-water interactions did not change water diffusion but enhanced rotational motion significantly. Further investigations are required to delineate particular features of the protein crystal that induce the anomalous behavior of water at 200 K. PMID:23998392

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

    PubMed

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

    2013-04-16

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

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

    PubMed Central

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

    2013-01-01

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

  20. Effects of dietary protein source and protein–lipid source interaction on channel catfish ( Ictalurus punctatus) egg biochemical composition, egg production and quality, and fry hatching percentage and performance

    Microsoft Academic Search

    Todd D. Sink; Rebecca T. Lochmann; Camilo Pohlenz; Alejandro Buentello; Delbert Gatlin III

    2010-01-01

    The relative importance of proteins (amino acids) and protein–lipid interactions for reproduction of channel catfish can be assessed qualitatively. We conducted a feeding\\/spawning trial to determine the effects of protein source and protein–lipid interactions of channel catfish broodstock diets on egg biochemical composition and egg and fry production. A general linear mixed model was used to examine statistical significance (?=0.05)

  1. Interlayer Water Regulates the Bio-nano Interface of a ?-sheet Protein stacking on Graphene

    NASA Astrophysics Data System (ADS)

    Lv, Wenping; Xu, Guiju; Zhang, Hongyan; Li, Xin; Liu, Shengju; Niu, Huan; Xu, Dongsheng; Wu, Ren'an

    2015-01-01

    Using molecular dynamics simulations, we investigated an integrated bio-nano interface consisting of a ?-sheet protein stacked onto graphene. We found that the stacking assembly of the model protein on graphene could be controlled by water molecules. The interlayer water filled within interstices of the bio-nano interface could suppress the molecular vibration of surface groups on protein, and could impair the CH...? interaction driving the attraction of the protein and graphene. The intermolecular coupling of interlayer water would be relaxed by the relative motion of protein upon graphene due to the interaction between water and protein surface. This effect reduced the hindrance of the interlayer water against the assembly of protein on graphene, resulting an appropriate adsorption status of protein on graphene with a deep free energy trap. Thereby, the confinement and the relative sliding between protein and graphene, the coupling of protein and water, and the interaction between graphene and water all have involved in the modulation of behaviors of water molecules within the bio-nano interface, governing the hindrance of interlayer water against the protein assembly on hydrophobic graphene. These results provide a deep insight into the fundamental mechanism of protein adsorption onto graphene surface in water.

  2. Translational diffusion of hydration water correlates with functional motions in folded and intrinsically disordered proteins

    NASA Astrophysics Data System (ADS)

    Schirò, Giorgio; Fichou, Yann; Gallat, Francois-Xavier; Wood, Kathleen; Gabel, Frank; Moulin, Martine; Härtlein, Michael; Heyden, Matthias; Colletier, Jacques-Philippe; Orecchini, Andrea; Paciaroni, Alessandro; Wuttke, Joachim; Tobias, Douglas J.; Weik, Martin

    2015-03-01

    Hydration water is the natural matrix of biological macromolecules and is essential for their activity in cells. The coupling between water and protein dynamics has been intensively studied, yet it remains controversial. Here we combine protein perdeuteration, neutron scattering and molecular dynamics simulations to explore the nature of hydration water motions at temperatures between 200 and 300?K, across the so-called protein dynamical transition, in the intrinsically disordered human protein tau and the globular maltose binding protein. Quasi-elastic broadening is fitted with a model of translating, rotating and immobile water molecules. In both experiment and simulation, the translational component markedly increases at the protein dynamical transition (around 240?K), regardless of whether the protein is intrinsically disordered or folded. Thus, we generalize the notion that the translational diffusion of water molecules on a protein surface promotes the large-amplitude motions of proteins that are required for their biological activity.

  3. Translational diffusion of hydration water correlates with functional motions in folded and intrinsically disordered proteins.

    PubMed

    Schirò, Giorgio; Fichou, Yann; Gallat, Francois-Xavier; Wood, Kathleen; Gabel, Frank; Moulin, Martine; Härtlein, Michael; Heyden, Matthias; Colletier, Jacques-Philippe; Orecchini, Andrea; Paciaroni, Alessandro; Wuttke, Joachim; Tobias, Douglas J; Weik, Martin

    2015-01-01

    Hydration water is the natural matrix of biological macromolecules and is essential for their activity in cells. The coupling between water and protein dynamics has been intensively studied, yet it remains controversial. Here we combine protein perdeuteration, neutron scattering and molecular dynamics simulations to explore the nature of hydration water motions at temperatures between 200 and 300?K, across the so-called protein dynamical transition, in the intrinsically disordered human protein tau and the globular maltose binding protein. Quasi-elastic broadening is fitted with a model of translating, rotating and immobile water molecules. In both experiment and simulation, the translational component markedly increases at the protein dynamical transition (around 240?K), regardless of whether the protein is intrinsically disordered or folded. Thus, we generalize the notion that the translational diffusion of water molecules on a protein surface promotes the large-amplitude motions of proteins that are required for their biological activity. PMID:25774711

  4. Translational diffusion of hydration water correlates with functional motions in folded and intrinsically disordered proteins

    PubMed Central

    Schirò, Giorgio; Fichou, Yann; Gallat, Francois-Xavier; Wood, Kathleen; Gabel, Frank; Moulin, Martine; Härtlein, Michael; Heyden, Matthias; Colletier, Jacques-Philippe; Orecchini, Andrea; Paciaroni, Alessandro; Wuttke, Joachim; Tobias, Douglas J.; Weik, Martin

    2015-01-01

    Hydration water is the natural matrix of biological macromolecules and is essential for their activity in cells. The coupling between water and protein dynamics has been intensively studied, yet it remains controversial. Here we combine protein perdeuteration, neutron scattering and molecular dynamics simulations to explore the nature of hydration water motions at temperatures between 200 and 300?K, across the so-called protein dynamical transition, in the intrinsically disordered human protein tau and the globular maltose binding protein. Quasi-elastic broadening is fitted with a model of translating, rotating and immobile water molecules. In both experiment and simulation, the translational component markedly increases at the protein dynamical transition (around 240?K), regardless of whether the protein is intrinsically disordered or folded. Thus, we generalize the notion that the translational diffusion of water molecules on a protein surface promotes the large-amplitude motions of proteins that are required for their biological activity. PMID:25774711

  5. The channels of Mars

    NASA Technical Reports Server (NTRS)

    Baker, Victor R.

    1988-01-01

    The geomorphology of Mars is discussed, focusing on the Martian channels. The great flood channels of Mars, the processes of channel erosion, and dendritic channel networks, are examined. The topography of the Channeled Scabland region of the northwestern U.S. is described and compared to the Martian channels. The importance of water in the evolution of the channel systems is considered.

  6. Tentative Study on Performance of Darriues-Type Hydroturbine Operated in Small Open Water Channel

    NASA Astrophysics Data System (ADS)

    Matsushita, D.; Moriyama, R.; Nakashima, K.; Watanabe, S.; Okuma, K.; Furukawa, A.

    2014-12-01

    The development of small hydropower is one of the realistic and preferable utilizations of renewable energy, but the extra-low head hydropower less than 2 m is almost undeveloped yet for some reasons. The authors have developed several types of Darrieus-type hydro-turbine system, and among them, the Darrieus-turbine with a wear and a nozzle installed upstream of turbine is so far in success to obtain more output power, i.e. more shaft torque, by gathering all water into the turbine. However, there can several cases exist, in which installing the wear covering all the flow channel width is unrealistic. Then, in the present study, the hydraulic performances of Darrieus-type hydro-turbine with the inlet nozzle is investigated, putting alone in a small open channel without upstream wear. In the experiment, the five-bladed Darrieus-type runner with the pitch-circle diameter of 300 mm and the blade span of 300 mm is vertically installed in the open channel with the width of 1,200 mm. The effectiveness of the shape of the inlet nozzle is also examined using two types of two-dimensional symmetric nozzle, the straight line nozzle (SL nozzle) with the converging angle of 45 degrees and the half diameter curved nozzle (HD nozzle) whose radius is a half diameter of runner pitch circle. Inlet and outlet nozzle widths are in common for the both nozzles, which are 540 mm and 240 mm respectively. All the experiments are carried out under the conditions with constant flow rate and downstream water level, and performances are evaluated by measured output torque and the measured head difference between the water levels upstream and downstream of the turbine. As a result, it is found that the output power is remarkably increased by installing the inlet nozzle, and the turbine with SL nozzle produces larger power than that with HD nozzle. However, the peak efficiency is deteriorated in both cases. The speed ratio defined by the rotor speed divided by the downstream water velocity at the peak efficiency is larger in both cases with the inlet nozzle, partly due to the increase of inflow velocity into the turbine. In order to understand the cause of the differences of power, i.e. torque characteristics of the turbine with SL and HD nozzles, twodimensional CFD simulation is carried out. It is found that the instantaneous torque variation is important for the overall turbine performances, indicating the possibility of further performance improvement through the optimization of nozzle geometry.

  7. Electron transfer activation of a second water channel for proton transport in [FeFe]-hydrogenase

    NASA Astrophysics Data System (ADS)

    Sode, Olaseni; Voth, Gregory A.

    2014-12-01

    Hydrogenase enzymes are important because they can reversibly catalyze the production of molecular hydrogen. Proton transport mechanisms have been previously studied in residue pathways that lead to the active site of the enzyme via residues Cys299 and Ser319. The importance of this pathway and these residues has been previously exhibited through site-specific mutations, which were shown to interrupt the enzyme activity. It has been shown recently that a separate water channel (WC2) is coupled with electron transport to the active site of the [FeFe]-hydrogenase. The water-mediated proton transport mechanisms of the enzyme in different electronic states have been studied using the multistate empirical valence bond reactive molecular dynamics method, in order to understand any role WC2 may have in facilitating the residue pathway in bringing an additional proton to the enzyme active site. In a single electronic state A2-, a water wire was formed through which protons can be transported with a low free energy barrier. The remaining electronic states were shown, however, to be highly unfavorable to proton transport in WC2. A double amino acid substitution is predicted to obstruct proton transport in electronic state A2- by closing a cavity that could otherwise fill with water near the proximal Fe of the active site.

  8. Electron transfer activation of a second water channel for proton transport in [FeFe]-hydrogenase.

    PubMed

    Sode, Olaseni; Voth, Gregory A

    2014-12-14

    Hydrogenase enzymes are important because they can reversibly catalyze the production of molecular hydrogen. Proton transport mechanisms have been previously studied in residue pathways that lead to the active site of the enzyme via residues Cys299 and Ser319. The importance of this pathway and these residues has been previously exhibited through site-specific mutations, which were shown to interrupt the enzyme activity. It has been shown recently that a separate water channel (WC2) is coupled with electron transport to the active site of the [FeFe]-hydrogenase. The water-mediated proton transport mechanisms of the enzyme in different electronic states have been studied using the multistate empirical valence bond reactive molecular dynamics method, in order to understand any role WC2 may have in facilitating the residue pathway in bringing an additional proton to the enzyme active site. In a single electronic state A(2-), a water wire was formed through which protons can be transported with a low free energy barrier. The remaining electronic states were shown, however, to be highly unfavorable to proton transport in WC2. A double amino acid substitution is predicted to obstruct proton transport in electronic state A(2-) by closing a cavity that could otherwise fill with water near the proximal Fe of the active site. PMID:25494798

  9. Intuitive, but not simple: including explicit water molecules in protein-protein docking simulations improves model quality.

    PubMed

    Parikh, Hardik I; Kellogg, Glen E

    2014-06-01

    Characterizing the nature of interaction between proteins that have not been experimentally cocrystallized requires a computational docking approach that can successfully predict the spatial conformation adopted in the complex. In this work, the Hydropathic INTeractions (HINT) force field model was used for scoring docked models in a data set of 30 high-resolution crystallographically characterized "dry" protein-protein complexes and was shown to reliably identify native-like models. However, most current protein-protein docking algorithms fail to explicitly account for water molecules involved in bridging interactions that mediate and stabilize the association of the protein partners, so we used HINT to illuminate the physical and chemical properties of bridging waters and account for their energetic stabilizing contributions. The HINT water Relevance metric identified the "truly" bridging waters at the 30 protein-protein interfaces and we utilized them in "solvated" docking by manually inserting them into the input files for the rigid body ZDOCK program. By accounting for these interfacial waters, a statistically significant improvement of ?24% in the average hit-count within the top-10 predictions the protein-protein dataset was seen, compared to standard "dry" docking. The results also show scoring improvement, with medium and high accuracy models ranking much better than incorrect ones. These improvements can be attributed to the physical presence of water molecules that alter surface properties and better represent native shape and hydropathic complementarity between interacting partners, with concomitantly more accurate native-like structure predictions. PMID:24214407

  10. Identification of a family of calcium sensors as protein ligands of inositol trisphosphate receptor Ca2+ release channels

    PubMed Central

    Yang, Jun; McBride, Sean; Mak, Don-On Daniel; Vardi, Noga; Palczewski, Krzysztof; Haeseleer, Françoise; Foskett, J. Kevin

    2002-01-01

    The inositol trisphosphate (InsP3) receptor (InsP3R) is a ubiquitously expressed intracellular Ca2+ channel that mediates complex cytoplasmic Ca2+ signals, regulating diverse cellular processes, including synaptic plasticity. Activation of the InsP3R channel is normally thought to require binding of InsP3 derived from receptor-mediated activation of phosphatidylinositol lipid hydrolysis. Here we identify a family of neuronal Ca2+-binding proteins as high-affinity protein agonists of the InsP3R, which bind to the channel and activate gating in the absence of InsP3. CaBP/caldendrin, a subfamily of the EF-hand-containing neuronal calcium sensor family of calmodulin-related proteins, bind specifically to the InsP3-binding region of all three InsP3R channel isoforms with high affinity (Ka ? 25 nM) in a Ca2+-dependent manner (Ka ? 1 ?M). Binding activates single-channel gating as efficaciously as InsP3, dependent on functional EF-hands in CaBP. In contrast, calmodulin neither bound with high affinity nor activated channel gating. CaBP1 and the type 1 InsP3R associate in rat whole brain and cerebellum lysates, and colocalize extensively in subcellular regions in cerebellar Purkinje neurons. Thus, InsP3R-mediated Ca2+ signaling in cells is possible even in the absence of InsP3 generation, a process that may be particularly important in responding to and shaping changes in intracellular Ca2+ concentration by InsP3-independent pathways and for localizing InsP3-mediated Ca2+ signals to individual synapses. PMID:12032348

  11. ATP-Sensitive Potassium Channel Traffic Regulation by Adenosine and Protein Kinase C

    Microsoft Academic Search

    Keli Hu; Cindy Shen Huang; Yuh Nung Jan; Lily Yeh Jan

    2003-01-01

    ATP-sensitive potassium (KATP) channels activate under metabolic stress to protect neurons and cardiac myocytes. However, excessive channel activation may cause arrhythmia in the heart and silence neurons in the brain. Here, we report that PKC-mediated downregulation of KATP channel number, via dynamin-dependent channel internalization, can act as a brake mechanism to control KATP activation. A dileucine motif in the pore-lining

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

    PubMed Central

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

    2007-01-01

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

  13. Selection of ion channel elements in the serine and aspartate methyl-accepting chemotaxis proteins of bacteria.

    PubMed

    Kosower, E M

    1983-09-15

    Two plausible, transmembrane ion channel elements (These 'elements' are alpha-helical sequences of 24 amino acids in which polar, hydrophilic side chains occupy one side and hydrophobic side chains the other) have been identified in the serine chemoreceptor-methyl-accepting chemotaxis protein (MCP) (SerR) of E. coli and the aspartate chemoreceptor-MCP (AspR) of S. typhimurium. That the chemoreceptor might serve as, or activate, an ion channel is supported strongly by the occurrence of membrane depolarization, specific peptide methylation and neurotoxin inhibition of response in the chemotaxis of S. aurantia (E.P. Greenberg, refs. 13-18). PMID:6312987

  14. Evidence for Recent Liquid Water on Mars: Channels and Aprons in East Gorgonum Crater

    NASA Technical Reports Server (NTRS)

    2000-01-01

    [figure removed for brevity, see original site] Warning!This link leads to a very large image that may be too long for some web browsers (in these cases, you must save the link to your desktop and view with other software) [figure removed for brevity, see original site]

    This suite of Mars Global Surveyor (MGS) Mars Orbiter Camera (MOC) pictures provides a vista of martian gullies on the northern wall of a 12 kilometer-(7.4 mile)-wide meteor impact crater east of the Gorgonum Chaos region on the red planet.

    The first picture (above left) is a composite of three different high resolution MOC views obtained in 1999 and 2000. The second picture (above right)shows the location of the high resolution views relative to the whole crater as it appeared in the highest resolution image previously acquired of the area, taken by the Viking 1 orbiter in 1978. The release image (top) shows a close-up of one of the channels and debris aprons found in the northwestern quarter of the impact crater.

    Some of the channels in this crater are deeply-entrenched and cut into lighter-toned deposits. The numerous channels and apron deposits indicate that many tens to hundreds of individual events involving the flow of water and debris have occurred here. The channels and aprons have very crisp, sharp relief and there are no small meteor impact craters on them, suggesting that these features are extremely young relative to the 4.5 billion year history of Mars. It is possible that these landforms are still being created by water seeping from the layered rock in the crater wall today.

    The crater has no name and it is located near 37.4oS, 168.0oW. The composite view in (above left) includes a picture taken by MOC on September 10, 1999, a picture obtained April 26, 2000, and another on May 22, 2000. The scene from left to right (including the dark gap between photos) covers an area approximately 7.6 kilometers (4.7 miles) wide by 18 km (11.1 mi) long. Sunlight illuminates the scene from the upper left. MOC high resolution images are taken black-and-white (grayscale); the color seen here has been synthesized from the colors of Mars observed by the MOC wide angle cameras and by the Viking Orbiters in the late 1970s.

  15. Inhibition of Ca(2+)-induced calcitonin secretion by somatostatin: roles of voltage dependent Ca2+ channels and G-proteins.

    PubMed

    Scherübl, H; Hescheler, J; Schultz, G; Kliemann, D; Zink, A; Ziegler, R; Raue, F

    1992-01-01

    Somatostatin has recently been applied therapeutically for hypercalcitonemia in patients with calcitonin-producing tumours. Using calcitonin-secreting cells (C-cells) of the medullary thyroid carcinoma cell line rMTC 44-2, we investigated the inhibitory action of somatostatin on calcitonin release, cytosolic Ca2+ and Ca2+ channel currents. The Ca(2+)-induced rises of the cytosolic Ca2+ and calcitonin secretion were greatly inhibited by somatostatin or its stable analogue octreotide. The effects of somatostatin were pertussis toxin-sensitive. Under voltage clamp conditions, C-cells exhibited slowly inactivating Ca2+ channel currents. Bath application of 100 nM somatostatin reversibly reduced the Ca2+ channel current by about 30%. The Ca2+ channel current and its inhibition by somatostatin were not affected by intracellularly applied cyclic AMP. Moreover, pretreating the cells with pertussis toxin had no effect on the control Ca2+ channel currents but greatly abolished its inhibition by somatostatin. The data show that somatostatin suppresses the Ca(2+)-stimulated calcitonin secretion by inhibiting voltage-dependent Ca2+ channel currents and by lowering cytosolic Ca2+. These actions of somatostatin involve pertussis toxin-sensitive G-proteins and occur independently of changes in the cyclic AMP concentration. PMID:1349229

  16. Surimi wash water treatment for protein recovery: effect of chitosan–alginate complex concentration and treatment time on protein adsorption

    Microsoft Academic Search

    Singgih Wibowo; Gonzalo Velazquez; Vivek Savant; J. Antonio Torres

    2005-01-01

    Chitosan (Chi), a protein recovery agent for the treatment of aqueous food processing streams, appears to work by mechanical entrapment and electrostatic interaction of chitosan amino groups with anionic groups on proteins. Chitosan effectiveness for recovering soluble proteins from surimi wash water (SWW) is increased by complexation with alginate (Alg) and by adjusting complex concentration and treatment time. Flocculation at

  17. Shape dependence of the radial distribution function of hydration water around proteins

    NASA Astrophysics Data System (ADS)

    Rani, Pooja; Biswas, Parbati

    2014-08-01

    The characterization of hydration water in proteins is important to understand their structure, function and folding properties. A calculation of the shape parameters reveals considerable asymmetry in the shapes of globular proteins. The present study suggests a generalized approach for the calculation of radial distribution of hydration water by accounting for the shape asymmetry in proteins. The surface and radial distribution function is analyzed for three groups of high resolution globular proteins and nonglobular proteins. This generalized approach depicts a considerable difference in the hydration water distribution pattern around aspherical proteins as compared to the earlier method and this difference is more pronounced for nonglobular proteins. The peaks for normalized RDF are found to be sharper compared to those of bulk water. The normalized RDF of hydration water exactly coincides with that of the bulk beyond 8.0 Å. The radial distribution of hydration water as a function of the water-protein distance matches with the experimentally observed distribution of hydration water around myoglobin. The results reveal that the distribution of hydration water is dependent on the shapes of proteins and hence a generalized approach should be used for the calculation of hydration water distribution around proteins, especially for nonglobular proteins.

  18. Shape dependence of the radial distribution function of hydration water around proteins.

    PubMed

    Rani, Pooja; Biswas, Parbati

    2014-08-20

    The characterization of hydration water in proteins is important to understand their structure, function and folding properties. A calculation of the shape parameters reveals considerable asymmetry in the shapes of globular proteins. The present study suggests a generalized approach for the calculation of radial distribution of hydration water by accounting for the shape asymmetry in proteins. The surface and radial distribution function is analyzed for three groups of high resolution globular proteins and nonglobular proteins. This generalized approach depicts a considerable difference in the hydration water distribution pattern around aspherical proteins as compared to the earlier method and this difference is more pronounced for nonglobular proteins. The peaks for normalized RDF are found to be sharper compared to those of bulk water. The normalized RDF of hydration water exactly coincides with that of the bulk beyond 8.0 ?. The radial distribution of hydration water as a function of the water-protein distance matches with the experimentally observed distribution of hydration water around myoglobin. The results reveal that the distribution of hydration water is dependent on the shapes of proteins and hence a generalized approach should be used for the calculation of hydration water distribution around proteins, especially for nonglobular proteins. PMID:25053697

  19. Inhibition of the KCa3.1 channels by AMP-activated protein kinase in human airway epithelial cells

    PubMed Central

    Klein, Hélène; Garneau, Line; Trinh, Nguyen Thu Ngan; Privé, Anik; Dionne, François; Goupil, Eugénie; Thuringer, Dominique; Parent, Lucie; Brochiero, Emmanuelle; Sauvé, Rémy

    2009-01-01

    The vectorial transport of ions and water across epithelial cells depends to a large extent on the coordination of the apical and basolateral ion fluxes with energy supply. In this work we provide the first evidence for a regulation by the 5?-AMP-activated protein kinase (AMPK) of the calcium-activated potassium channel KCa3.1 expressed at the basolateral membrane of a large variety of epithelial cells. Inside-out patch-clamp experiments performed on human embryonic kidney (HEK) cells stably transfected with KCa3.1 first revealed a decrease in KCa3.1 activity following the internal addition of AMP at a fixed ATP concentration. This effect was dose dependent with half inhibition at 140 ?M AMP in 1 mM ATP. Evidence for an interaction between the COOH-terminal region of KCa3.1 and the ?1-subunit of AMPK was next obtained by two-hybrid screening and pull-down experiments. Our two-hybrid analysis confirmed in addition that the amino acids extending from Asp380 to Ala400 in COOH-terminal were essential for the interaction AMPK-?1/KCa3.1. Inside-out experiments on cells coexpressing KCa3.1 with the dominant negative AMPK-?1-R299G mutant showed a reduced sensitivity of KCa3.1 to AMP, arguing for a functional link between KCa3.1 and the ?1-subunit of AMPK. More importantly, coimmunoprecipitation experiments carried out on bronchial epithelial NuLi cells provided direct evidence for the formation of a KCa3.1/AMPK-?1 complex at endogenous AMPK and KCa3.1 expression levels. Finally, treating NuLi monolayers with the membrane permeant AMPK activator 5-aminoimidazole-4-carboxamide-1-?-d-ribofuranoside (AICAR) caused a significant decrease of the KCa3.1-mediated short-circuit currents, an effect reversible by coincubation with the AMPK inhibitor Compound C. These observations argue for a regulation of KCa3.1 by AMPK in a functional epithelium through protein/protein interactions involving the ?1-subunit of AMPK. PMID:19052260

  20. Cotranslational Partitioning of Nascent Prion Protein into Multiple Populations at the Translocation Channel

    PubMed Central

    Kim, Soo Jung; Hegde, Ramanujan S.

    2002-01-01

    The decisive events that direct a single polypeptide such as the prion protein (PrP) to be synthesized at the endoplasmic reticulum in both fully translocated and transmembrane forms are poorly understood. In this study, we demonstrate that the topological heterogeneity of PrP is determined cotranslationally, while at the translocation channel. By evaluating sequential intermediates during PrP topogenesis, we find that signal sequence-mediated initiation of translocation results in an interaction between nascent PrP and endoplasmic reticulum chaperones, committing the N terminus to the lumen. Synthesis of the transmembrane domain before completion of this step allows it to direct the generation of CtmPrP, a transmembrane form with its N terminus in the cytosol. Thus, segregation of nascent PrP into different topological configurations is critically dependent on the precise timing of signal-mediated initiation of N-terminus translocation. Consequently, this step could be experimentally tuned to modify PrP topogenesis, including complete reversal of the elevated CtmPrP caused by disease-associated mutations in the transmembrane domain. These results delineate the sequence of events involved in PrP biogenesis, explain the mechanism of action of CtmPrP-favoring mutations associated with neurodegenerative disease, and more generally, reveal that translocation substrates can be cotranslationally partitioned into multiple populations at the translocon. PMID:12429823

  1. A Geophysical Investigation along a Fractured Bedrock River Channel: Implications to Groundwater-Surface Water Interaction

    NASA Astrophysics Data System (ADS)

    Steelman, C. M.; Kennedy, C.; Parker, B. L.; Cherry, J.

    2012-12-01

    Fractured sedimentary rock aquifers represent an important source of water for many communities around the world. The Eramosa River - a major tributary of the Speed River within the Grand River Watershed, Ontario, Canada - resides upon a bedrock aquifer of densely fractured dolostone with dissolution-enhanced channels and karst features. While this aquifer represents a major component of the total water supply for the surrounding region, the potential effects of increased groundwater extraction on the overlying river and surrounding environment are not yet fully understood. A comprehensive assessment of groundwater-surface water interaction over a 12 km reach of the river has been initiated using a discrete fracture network framework, i.e., based on the idea that groundwater flow occurs in networks of discrete fractures or channels that are interconnected. Preliminary measurements using airborne infrared thermography and surface-water temperature have been collected in an attempt to delineate hydraulically-active fracture zones; however, these measurements do not provide any information about the physical geometry of features potentially controlling hydraulic interactions. A nest of small-diameter vertical coreholes have been drilled through the upper 15 m to characterize the geology and hydrogeology at a single location along the river. While these coreholes provide the typical information used to understand flow systems, well installation can be improved using prior knowledge of the shallow subsurface and river flow system. Geophysical techniques such as ground-penetrating radar (GPR) and frequency-domain electromagnetics (FDEM) can provide detailed information about and shallow rock environment, thereby increasing the likelihood of identifying hydraulically-active zones along a river channel. To examine the suitability of GPR and FDEM for the characterization of bedrock river environments, a geophysical investigation has been initiated along a 0.5 km reach of the river in close proximity to the vertical coreholes. The study area contains outcrops along the riverbank which enable direct imaging into the upper 15-20 m rock. GPR reflection profiles and common-midpoint (CMP) soundings were collected using 50 MHz and 100 MHz antennas along two main transects situated on either side of the river to examine the nature of the formation. FDEM measurements using an EM-34, -31 and -38 were concurrently collected along the GPR transects to identify variations in depth to rock and riverbed sedimentation. In addition, a 3D GPR reflection grid and azimuthal CMP and EM-31 measurements were collected over a 10 × 25 m plot immediately adjacent to the river to better understand fracture orientation and karst development within the formations. These geophysical data provide valuable insight into the nature of the structurally-controlled features expected to strongly influence groundwater discharge and surface water recharge along the bedrock river channel. This information will guide the site-selection process and placement of future coreholes, ultimately minimizing the invasiveness of small-diameter drilling activities.

  2. GTP-Binding Proteins Inhibit cAMP Activation of Chloride Channels in Cystic Fibrosis Airway Epithelial Cells

    NASA Astrophysics Data System (ADS)

    Schwiebert, Erik M.; Kizer, Neil; Gruenert, Dieter C.; Stanton, Bruce A.

    1992-11-01

    Cystic fibrosis (CF) is a genetic disease characterized, in part, by defective regulation of Cl^- secretion by airway epithelial cells. In CF, cAMP does not activate Cl^- channels in the apical membrane of airway epithelial cells. We report here whole-cell patch-clamp studies demonstrating that pertussis toxin, which uncouples heterotrimeric GTP-binding proteins (G proteins) from their receptors, and guanosine 5'-[?-thio]diphosphate, which prevents G proteins from interacting with their effectors, increase Cl^- currents and restore cAMP-activated Cl^- currents in airway epithelial cells isolated from CF patients. In contrast, the G protein activators guanosine 5'-[?-thio]triphosphate and AlF^-_4 reduce Cl^- currents and inhibit cAMP from activating Cl^- currents in normal airway epithelial cells. In CF cells treated with pertussis toxin or guanosine 5'-[?-thio]diphosphate and in normal cells, cAMP activates a Cl^- conductance that has properties similar to CF transmembrane-conductance regulator Cl^- channels. We conclude that heterotrimeric G proteins inhibit cAMP-activated Cl^- currents in airway epithelial cells and that modulation of the inhibitory G protein signaling pathway may have the therapeutic potential for improving cAMP-activated Cl^- secretion in CF.

  3. Stream channel surface water - groundwater interactions in a fire impacted watershed

    NASA Astrophysics Data System (ADS)

    Russo, T. A.; Fisher, A. T.

    2010-12-01

    We are conducting a study of surface water - groundwater interactions within the Scott Creek watershed, a 4th order catchment of 76.6 km2 in central coastal California, to assess the impacts of fire on channel and riparian conditions. Scott Creek and its tributaries are valuable spawning habitat for Coho salmon and Steelhead trout. The Scott Creek watershed is located on the western (windward) side of the Santa Cruz Mountains, where the most intense precipitation falls from November to April, and includes a mixture of protected land and areas used for agriculture, grazing, and selective timber harvesting. 37% of the watershed was burned in a fire in August 2009, and we hypothesize that this could result in enhanced delivery of fine grained hill slope sediments to stream channels for several years post fire, reducing the extent of hyporheic exchange downstream of burned areas. This could reduce the survival rates of Coho and Steelhead redds (egg nests), which are dependent on surface water - groundwater exchange for regulation of water nutrient content and temperature. We are monitoring streambed seepage rates and hydraulic conductivity, and performing repeated tracer discharge experiments at three sites on Scott Creek, two within and one upstream of the area burned in the 2009 fire. Streambed seepage rates are calculated using a time series method applied to heat as a tracer, using naturally occurring diurnal changes in stream temperature, and extended to calculations of streambed hydraulic conductivity based on measured head gradients. Hyporheic exchange parameters are assessed using tracer breakthrough data, as fit by an optimized model of one-dimensional advection, dispersion and transient storage. Variations in hydrologic characteristics (e.g., transient storage area, exchange coefficient) over time at each site are being used to assess the magnitude and timing of channel modifications independent to, and associated with, the burning of catchment hill slopes. Preliminary data suggest that there is more variability in both streambed seepage rates and streambed hydraulic conductivity in burned areas relative to the unburned reach. Fires in undeveloped and urbanized areas seem likely to become more common throughout the western U.S., and other parts of the world, as a result of land use practices and climate change. Studies such as ours are important for understanding, anticipating, and mitigating the hydrologic response of critical habitat to fires and other events.

  4. Liquid Crystalline Properties of Amyloid Protein Fibers in Water

    NASA Astrophysics Data System (ADS)

    Mezzenga, Raffaele; Jung, Jin-Mi

    2010-03-01

    We have studied the liquid crystalline features of two colloidal systems consisting of food protein amyloid fibrils in water, obtained by heat-denaturation and aggregation of ?-lactoglobulin, a globular dairy protein. The resulting fibrils, have a monodisperse cross section of about 4 nm and two groups of polydisperse contour lengths: (i) fibrils 1-10 ?m long, showing semiflexible polyeletrolyte-like behaviour and (ii) rigid rods 100-200 nm long. In both systems, the fibers are highly charged (+5 e/nm) and stable in water at low ionic strength (0.01 M) and low pH (pH 2). The physical properties of these systems are studied using a polymer physics approach and phase diagrams of these two systems are obtained by changing concentration and pH. Both systems exhibit rich phase behaviours. Interestingly, the experimentally measured isotropic-nematic phase transition was found to occur at concentrations more than one order of magnitude lower than what expected based on Onsager theory. Experimental results are revisited in terms of the Flory theory developed for rigid polymers in solvent of varying conditions.

  5. Protein film photoelectrochemistry of the water oxidation enzyme photosystem II.

    PubMed

    Kato, Masaru; Zhang, Jenny Z; Paul, Nicholas; Reisner, Erwin

    2014-09-21

    Photosynthesis is responsible for the sunlight-powered conversion of carbon dioxide and water into chemical energy in the form of carbohydrates and the release of O2 as a by-product. Although many proteins are involved in photosynthesis, the fascinating machinery of Photosystem II (PSII) is at the heart of this process. This tutorial review describes an emerging technique named protein film photoelectrochemistry (PF-PEC), which allows for the light-dependent activity of PSII adsorbed onto an electrode surface to be studied. The technique is straightforward to use, does not require highly specialised and/or expensive equipment, is highly selective for the active fractions of the adsorbed enzyme, and requires a small amount of enzyme sample. The use of PF-PEC to study PSII can yield insights into its activity, stability, quantum yields, redox behaviour, and interfacial electron transfer pathways. It can also be used in PSII inhibition studies and chemical screening, which may prove useful in the development of biosensors. PSII PF-PEC cells also serve as proof-of-principle solar water oxidation systems; here, a comparison is made against PSII-inspired synthetic photocatalysts and materials for artificial photosynthesis. PMID:24668258

  6. Hydrolysis of whey protein isolate using subcritical water.

    PubMed

    Espinoza, Ashley D; Morawicki, Rubén O; Hager, Tiffany

    2012-01-01

    Hydrolyzed whey protein isolate (WPI) is used in the food industry for protein enrichment and modification of functional properties. The purpose of the study was to determine the feasibility of subcritical water hydrolysis (SWH) on WPI and to determine the temperature and reaction time effects on the degree of hydrolysis (DH) and the production of peptides and free amino acids (AAs). Effects of temperature (150 to 320 °C) and time (0 to 20 min) were initially studied with a central composite rotatable design followed by a completely randomized factorial design with temperature (250 and 300 °C) and time (0 to 50 min) as factors. SWH was conducted in an electrically heated, 100-mL batch, high pressure vessel. The DH was determined by a spectrophotometric method after derivatization. The peptide molecular weights (MWs) were analyzed by gel electrophoresis and mass spectrometry, and AAs were quantified by high-performance liquid chromotography. An interaction of temperature and time significantly affected the DH and AA concentration. As the DH increased, the accumulation of lower MW peptides also increased following SWH (and above 10% DH, the majority of peptides were <1000 Da). Hydrolysis at 300 °C for 40 min generated the highest total AA concentration, especially of lysine (8.894 mg/g WPI). Therefore, WPI was successfully hydrolyzed by subcritical water, and with adjustment of treatment parameters there is reasonable control of the end-products. PMID:22122092

  7. CRMP2 Protein SUMOylation Modulates NaV1.7 Channel Trafficking*

    PubMed Central

    Dustrude, Erik T.; Wilson, Sarah M.; Ju, Weina; Xiao, Yucheng; Khanna, Rajesh

    2013-01-01

    Voltage-gated sodium channel (NaV) trafficking is incompletely understood. Post-translational modifications of NaVs and/or auxiliary subunits and protein-protein interactions have been posited as NaV-trafficking mechanisms. Here, we tested if modification of the axonal collapsin response mediator protein 2 (CRMP2) by a small ubiquitin-like modifier (SUMO) could affect NaV trafficking; CRMP2 alters the extent of NaV slow inactivation conferred by the anti-epileptic (R)-lacosamide, implying NaV-CRMP2 functional coupling. Expression of a CRMP2 SUMOylation-incompetent mutant (CRMP2-K374A) in neuronal model catecholamine A differentiated (CAD) cells did not alter lacosamide-induced NaV slow inactivation compared with CAD cells expressing wild type CRMP2. Like wild type CRMP2, CRMP2-K374A expressed robustly in CAD cells. Neurite outgrowth, a canonical CRMP2 function, was moderately reduced by the mutation but was still significantly higher than enhanced GFP-transfected cortical neurons. Notably, huwentoxin-IV-sensitive NaV1.7 currents, which predominate in CAD cells, were significantly reduced in CAD cells expressing CRMP2-K374A. Increasing deSUMOylation with sentrin/SUMO-specific protease SENP1 or SENP2 in wild type CRMP2-expressing CAD cells decreased NaV1.7 currents. Consistent with a reduction in current density, biotinylation revealed a significant reduction in surface NaV1.7 levels in CAD cells expressing CRMP2-K374A; surface NaV1.7 expression was also decreased by SENP1 + SENP2 overexpression. Currents in HEK293 cells stably expressing NaV1.7 were reduced by CRMP2-K374A in a manner dependent on the E2-conjugating enzyme Ubc9. No decrement in current density was observed in HEK293 cells co-expressing CRMP2-K374A and NaV1.1 or NaV1.3. Diminution of sodium currents, largely NaV1.7, was recapitulated in sensory neurons expressing CRMP2-K374A. Our study elucidates a novel regulatory mechanism that utilizes CRMP2 SUMOylation to choreograph NaV1.7 trafficking. PMID:23836888

  8. A conceptual model for river water and sediment dispersal in the Santa Barbara Channel, California

    USGS Publications Warehouse

    Warrick, J.A.; Mertes, L.A.K.; Washburn, L.; Siegel, D.A.

    2004-01-01

    The ephemeral Santa Clara River delivers large amounts of freshwater and sediment to the eastern Santa Barbara Channel during brief, episodic discharge events. This discharge into the channel was characterized here with shipboard measurements during floods of 1997 and 1998. Within approximately 1-km of the river mouth, the river discharge quickly stratifies into a freshened, turbid surface plume and a bottom nephloid layer. Observations immediately off the Santa Clara River mouth on a peak day of river discharge revealed that sediment rapidly settled from the freshened surface waters, as suspended sediment in the freshened surface plume contained only ???6% of the sediment mass expected if the sediment mixed conservatively. On the two subsequent days the reduction of sediment mass in the surface plume continued at ???50% per day. These observations suggest that river sediment undergoes rapid initial settling within ???1-km of the river mouth, followed by somewhat slower rates of settling. Although we did not measure sedimentation or bottom boundary layer processes, our mass balance results suggest that almost all of the river sediment either escapes along or deposits upon the inner shelf seabed.

  9. A conceptual model for river water and sediment dispersal in the Santa Barbara Channel, California

    NASA Astrophysics Data System (ADS)

    Warrick, Jonathan A.; Mertes, Leal A. K.; Washburn, Libe; Siegel, David A.

    2004-11-01

    The ephemeral Santa Clara River delivers large amounts of freshwater and sediment to the eastern Santa Barbara Channel during brief, episodic discharge events. This discharge into the channel was characterized here with shipboard measurements during floods of 1997 and 1998. Within approximately 1-km of the river mouth, the river discharge quickly stratifies into a freshened, turbid surface plume and a bottom nephloid layer. Observations immediately off the Santa Clara River mouth on a peak day of river discharge revealed that sediment rapidly settled from the freshened surface waters, as suspended sediment in the freshened surface plume contained only ˜6% of the sediment mass expected if the sediment mixed conservatively. On the two subsequent days the reduction of sediment mass in the surface plume continued at ˜50% per day. These observations suggest that river sediment undergoes rapid initial settling within ˜1-km of the river mouth, followed by somewhat slower rates of settling. Although we did not measure sedimentation or bottom boundary layer processes, our mass balance results suggest that almost all of the river sediment either escapes along or deposits upon the inner shelf seabed.

  10. A device to achieve low Reynolds numbers in an open surface water channel

    NASA Astrophysics Data System (ADS)

    Radi, Alexander; Lo Jacono, David; Sheridan, John

    2014-05-01

    When investigating flow structures, and especially flow transitions, research projects often seek to increase insight using complementary numerical and physical experiments. Obtaining exact Reynolds number correspondence can frequently be difficult in experiments, particularly when relatively low values are required. Often, available test facilities were designed and optimised for a specific velocity range, meaning they have restrictions on the minimum flow velocity. This study describes a device to reduce the flow velocity locally in an open surface water channel. The underlying idea is to divert a controlled fraction of the incoming flow from the working section by increasing the pressure there, resulting in reduced velocity. This idea is realised using a `sub-channel' that can be inserted into the main test chamber, with a variable porosity perforated screen at its downstream end. This study assesses and optimises the flow quality inside this structure, such as usable test section length, uniformity of the velocity profiles and turbulence intensity. The results demonstrate that the device creates high quality low Reynolds number flows, which is exemplified with the canonical circular cylinder in cross-flow.

  11. Molecular mechanisms of somatostatin's inhibition of hormone release: participation of voltage-gated calcium channels and G-proteins.

    PubMed

    Scherübl, H; Hescheler, J; Riecken, E O

    1993-01-01

    Somatostatin is known to inhibit hormone release from various neuroendocrine cells. In order to understand the mechanisms underlying somatostatin's action we performed patch-clamp experiments in GH3 pituitary, rMTC 44-2 thyroid and BON carcinoid cells. Calcium-mediated hormone release depended on the intracellular calcium concentration and thus on the calcium influx through voltage-gated calcium channels. In addition to inhibiting the cAMP-dependent secretory pathway, somatostatin reduced the calcium inward currents and thereby hormone release. The inhibition of voltage-gated calcium channels by somatostatin was mediated by "signal transducing" Go proteins. Thus, somatostatin's actions on hormone release involve both cAMP and intracellular calcium as second messengers. Patch-clamp experiments of voltage-gated calcium channels allow functional studies on the coupling of somatostatin receptors to cellular effector systems. PMID:8101178

  12. The role of water exchange between a stream channel and its hyporheic zone in nitrogen cycling at the terrestrial-aquatic interface

    USGS Publications Warehouse

    Triska, F.J.; Duff, J.H.; Avanzino, R.J.

    1993-01-01

    The subsurface riparian zone was examined as an ecotone with two interfaces. Inland is a terrestrial boundary, where transport of water and dissolved solutes is toward the channel and controlled by watershed hydrology. Streamside is an aquatic boundary, where exchange of surface water and dissolved solutes is bi-directional and flux is strongly influenced by channel hydraulics. Streamside, bi-directional exchange of water was qualitatively defined using biologically conservative tracers in a third order stream. In several experiments, penetration of surface water extended 18 m inland. Travel time of water from the channel to bankside sediments was highly variable. Subsurface chemical gradients were indirectly related to the travel time. Sites with long travel times tended to be low in nitrate and DO (dissolved oxygen) but high in ammonium and DOC (dissolved organic carbon). Sites with short travel times tended to be high in nitrate and DO but low in ammonium and DOC. Ammonium concentration of interstitial water also was influenced by sorption-desorption processes that involved clay minerals in hyporheic sediments. Denitrification potential in subsurface sediments increased with distance from the channel, and was limited by nitrate at inland sites and by DO in the channel sediments. Conversely, nitrification potential decreased with distance from the channel, and was limited by DO at inland sites and by ammonium at channel locations. Advection of water and dissolved oxygen away from the channel resulted in an oxidized subsurface habitat equivalent to that previously defined as the hyporheic zone. The hyporheic zone is viewed as stream habitat because of its high proportion of surface water and the occurrence of channel organisms. Beyond the channel's hydrologic exchange zone, interstitial water is often chemically reduced. Interstitial water that has not previously entered the channel, groundwater, is viewed as a terrestrial component of the riparian ecotone. Thus, surface water habitats may extend under riparian vegetation, and terrestrial groundwater habitats may be found beneath the stream channel. ?? 1993 Kluwer Academic Publishers.

  13. A computational study of water and CO migration sites and channels inside myoglobin.

    PubMed

    Lapelosa, Mauro; Abrams, Cameron F

    2013-02-12

    Pathways are computed for transport of H2O and CO in myoglobin (Mb), using the single sweep and zero-temperature string methods in a fully atomistic, explicitly solvated model system. Our predictions of sites and barriers in the pathways for CO transport agree with previous studies. For H2O, we predict a binding site in the distal pocket (DP), in agreement with crystallographic observations, and another one close to Leu 29 which explains the importance of this residue in controlling the pocket's hydrophobicity, as well as disordered minima in the largely apolar xenon cavities. In particular, H2O can occupy and transition among the xenon cavities, Xe4, Xe2, and Xe3. Our results support the hypothesis that the thermodynamically most favorable entry/exit portal for H2O is the so-called histidine gate (HG), the same as for CO. This result, along with the observation of water occupation of both DP and apolar Xe cavities, suggest that water and small gas molecules like CO compete for access to the protein interior, and therefore models of gas molecule transport within proteins should also explicitly consider water transport. PMID:23505344

  14. DEFECTIVE TRAFFICKING OF CONE PHOTORECEPTOR CNG CHANNELS INDUCES THE UNFOLDED PROTEIN RESPONSE AND ER STRESS-ASSOCIATED CELL DEATH

    PubMed Central

    Duricka, Deborah L.; Brown, R. Lane; Varnum, Michael D.

    2011-01-01

    SYNOPSIS Mutations that perturb the function of photoreceptor cyclic nucleotide-gated (CNG) channels are associated with several human retinal disorders, but the molecular and cellular mechanisms leading to photoreceptor dysfunction and degeneration remain unclear. Many loss-of-function mutations result in intracellular accumulation of CNG channel subunits. Accumulation of proteins in the endoplasmic reticulum (ER) is known to cause ER stress and trigger the unfolded protein response (UPR), an evolutionarily conserved cellular program that results in either adaptation via increased protein processing capacity or apoptotic cell death. We hypothesize that defective trafficking of cone photoreceptor CNG channels can induce UPR-mediated cell death. To test this idea, CNGA3 subunits bearing the R563H and Q655X mutations were expressed in photoreceptor-derived 661W cells with CNGB3 subunits. Compared to wild type, R563H and Q655X subunits displayed altered degradation rates and/or were retained in the ER. ER retention was associated with increased expression of UPR-related markers of ER stress and with decreased cell viability. Chemical and pharmacological chaperones (TUDCA, 4PBA, and the cGMP analog CPT-cGMP) differentially reduced degradation and/or promoted plasma-membrane localization of defective subunits. Improved subunit maturation was concordant with reduced expression of ER stress markers and improved viability of cells expressing localization-defective channels. These results indicate that ER stress can arise from expression of localization defective CNG channels, and may represent a contributing factor for photoreceptor degeneration. PMID:21992067

  15. A lanthanum chelate possessing an open-channel framework with water nanotubes: properties and desalination.

    PubMed

    Chen, Mao-Long; Guo, Yi-Chao; Yang, Fang; Liang, Jin-Xia; Cao, Ze-Xing; Zhou, Zhao-Hui

    2014-04-28

    A new type of thermally stable chelate {La(H2O)4[La(1,3-pdta)(H2O)]3}n · 12nH2O (1) [1,3-H4pdtaCH2[CH2N(CH2CO2H)2]2] with an open-channel shows significant and unusual solvent transport properties and demonstrates a use for low-pressure desalination, which is constructed by cheap and available lanthanum salt and 1,3-propanediaminetetraacetate. The chelate could be converted reversibly to its trihydrate {La(H2O)4[La(1,3-pdta)(H2O)]3}n · 3nH2O (1a), dehydrated product {La(H2O)4[La(1,3-pdta)(H2O)]3}n (1b) and ethanol adduct {La(H2O)4[La(1,3-pdta)(H2O)]3}n · 3nH2O · 3nEtOH (1c). The latter nano-confined ethanol shows a remarkable downfield shift (?? = 6.0 ppm) for the methylene group in the solid 13C NMR spectrum compared with that of the free EtOH. Crystal 1 with a regular hexagonal appearance can be used directly for saline water desalination on a small-scale at an ambient temperature, demonstrating a low energy consumption and environmentally friendly method. This is attributed to the 10.0 Å hydrophobic open-channel containing water nanotubes (WNTs, ? = 4.2 Å). The nano-confined WNTs can be removed at a low temperature (45 °C). PMID:24382489

  16. Water in the Polar and Nonpolar Cavities of the Protein Interleukin-1 Guogang Feng,

    E-print Network

    Clore, G. Marius

    Water in the Polar and Nonpolar Cavities of the Protein Interleukin-1 Hao Yin, Guogang Feng, G ReceiVed: September 13, 2010; ReVised Manuscript ReceiVed: October 19, 2010 Water in the protein by water on the basis of some experiments and simulations and to be empty on the basis of others. Here we

  17. Structural ordering and ice-like glass transition on cooling the nano-channel water formed within a crystalline framework

    NASA Astrophysics Data System (ADS)

    Watanabe, Keisuke; Oguni, Masaharu; Tadokoro, Makoto; Nakamura, Ryouhei

    2006-10-01

    Heat capacities and enthalpy relaxation rates were measured for crystalline [Ni(cyclam)(H2O)2]3(TMA)2·24H2O, where cyclam is 1,4,8,11-tetraazacyclotetradecane, TMA is 1,3,5-benzene tricarboxylic acid, and 24H2O represents the water forming a nano-channel. A phase transition was found to occur at 196.9 K, and a glass transition at 87 K. A potential picture is given for the progress of the ordering of water molecules and hydrogen atoms in the channel. At room temperature, interfacial water molecules form hydrogen bonds with the oxygen atoms of the channel wall, and the aggregation of water molecules is recognized, as the average structure, to be in a crystalline state with a long-range order. The bond formation plays important role in the stabilization of the crystalline framework with a channel structure. The aggregate transforms to a more stable crystalline state at 196.9 K. All the water molecules should be fixed completely there. The positions of the hydrogen atoms on the network are, however, essentially in the disordered state while keeping an ice rule, and freeze at 87 K only with a short-range order in the arrangement. The progress of such ordering of channel water reveals a striking contrast to the behaviour of the water within meso-porous silicas, in which the molecules in the interface are always in the non-crystalline state and those in the pore centre tend to crystallize only when the pore diameter is greater than 2 or 3 nm.

  18. Transport of Small Molecules Across Cell Membranes: Water Channels and Urea Transporters

    NSDL National Science Digital Library

    PhD Barbara E. Goodman (University of South Dakota School of Medicine Division of Basic Biomedical Sciences)

    2002-09-01

    How do small hydrophilic nonelectrolytes cross cell membranes? Which pathways are most important for small lipid insoluble molecules to cross cell membranes? These are questions that have been basic to membrane transport physiology for decades. More importantly, these are questions whose answers have changed significantly within the last 10 years. This review discusses the evidence that pathways other than the lipid bilayer itself exist for the transport across cell membranes of specific small hydrophilic nonelectrolytes. The description begins with briefly analyzing the relevance of well accepted basic mathematical models for transport for understanding the permeability of representative physiologically important molecules across actual cell membranes. Particular emphasis is placed on describing recently discovered proteins that facilitate the transport of some of the smallest physiologically important lipid-insoluble molecules, water, and urea. Evidence also exists for transport proteins that selectively enhance the transmembrane transport of other small lipid-insoluble molecules. Do nonselective pores for small molecules exist in cell membranes?

  19. Activation of mitochondrial voltage-dependent anion channel by apro-apoptotic BH3-only protein Bim

    Microsoft Academic Search

    Tomoyasu Sugiyama; Shigeomi Shimizu; Yosuke Matsuoka; Yoshihiro Yoneda; Yoshihide Tsujimoto

    2002-01-01

    Bcl-2 family of proteins regulates apoptosis by controlling mitochondrial membrane permeability. We have previously shown that the voltage-dependent anion channel (VDAC) plays a crucial role in apoptotic changes of the mitochondria and its activity is directly regulated by some Bcl-2 family members, including Bcl-2\\/Bcl-xL and Bax\\/Bak but not Bid. Here, we showed that in isolated mitochondria, Bim induced loss of

  20. Using protein backbone mutagenesis to dissect the link between ion occupancy and C-type inactivation in K+ channels

    PubMed Central

    Matulef, Kimberly; Komarov, Alexander G.; Costantino, Corey A.; Valiyaveetil, Francis I.

    2013-01-01

    K+ channels distinguish K+ from Na+ in the selectivity filter, which consists of four ion-binding sites (S1–S4, extracellular to intracellular) that are built mainly using the carbonyl oxygens from the protein backbone. In addition to ionic discrimination, the selectivity filter regulates the flow of ions across the membrane in a gating process referred to as C-type inactivation. A characteristic of C-type inactivation is a dependence on the permeant ion, but the mechanism by which permeant ions modulate C-type inactivation is not known. To investigate, we used amide-to-ester substitutions in the protein backbone of the selectivity filter to alter ion binding at specific sites and determined the effects on inactivation. The amide-to-ester substitutions in the protein backbone were introduced using protein semisynthesis or in vivo nonsense suppression approaches. We show that an ester substitution at the S1 site in the KcsA channel does not affect inactivation whereas ester substitutions at the S2 and S3 sites dramatically reduce inactivation. We determined the structure of the KcsA S2 ester mutant and found that the ester substitution eliminates K+ binding at the S2 site. We also show that an ester substitution at the S2 site in the KvAP channel has a similar effect of slowing inactivation. Our results link C-type inactivation to ion occupancy at the S2 site. Furthermore, they suggest that the differences in inactivation of K+ channels in K+ compared with Rb+ are due to different ion occupancies at the S2 site. PMID:24128761

  1. The transient receptor potential, TRP4, cation channel is a novel member of the family of calmodulin binding proteins.

    PubMed Central

    Trost, C; Bergs, C; Himmerkus, N; Flockerzi, V

    2001-01-01

    The mammalian gene products, transient receptor potential (trp)1 to trp7, are related to the Drosophila TRP and TRP-like ion channels, and are candidate proteins underlying agonist-activated Ca(2+)-permeable ion channels. Recently, the TRP4 protein has been shown to be part of native store-operated Ca(2+)-permeable channels. These channels, most likely, are composed of other proteins in addition to TRP4. In the present paper we report the direct interaction of TRP4 and calmodulin (CaM) by: (1) retention of in vitro translated TRP4 and of TRP4 protein solubilized from bovine adrenal cortex by CaM-Sepharose in the presence of Ca(2+), and (2) TRP4-glutathione S-transferase pull-down experiments. Two domains of TRP4, amino acid residues 688-759 and 786-848, were identified as being able to interact with CaM. The binding of CaM to both domains occurred only in the presence of Ca(2+) concentrations above 10 microM, with half maximal binding occurring at 16.6 microM (domain 1) and 27.9 microM Ca(2+) (domain 2). Synthetic peptides, encompassing the two putative CaM binding sites within these domains and covering amino acid residues 694-728 and 829-853, interacted directly with dansyl-CaM with apparent K(d) values of 94-189 nM. These results indicate that TRP4/Ca(2+)-CaM are parts of a signalling complex involved in agonist-induced Ca(2+) entry. PMID:11311128

  2. Suprachiasmatic nucleus function and circadian entrainment are modulated by G protein-coupled inwardly rectifying (GIRK) channels.

    PubMed

    Hablitz, L M; Molzof, H E; Paul, J R; Johnson, R L; Gamble, K L

    2014-11-15

    G protein signalling within the central circadian oscillator, the suprachiasmatic nucleus (SCN), is essential for conveying time-of-day information. We sought to determine whether G protein-coupled inwardly rectifying potassium channels (GIRKs) modulate SCN physiology and circadian behaviour. We show that GIRK current and GIRK2 protein expression are greater during the day. Pharmacological inhibition of GIRKs and genetic loss of GIRK2 depolarized the day-time resting membrane potential of SCN neurons compared to controls. Behaviourally, GIRK2 knockout (KO) mice failed to shorten free running period in response to wheel access in constant darkness and entrained more rapidly to a 6 h advance of a 12 h:12 h light-dark (LD) cycle than wild-type (WT) littermate controls. We next examined whether these effects were due to disrupted signalling of neuropeptide Y (NPY), which is known to mediate non-photic phase shifts, attenuate photic phase shifts and activate GIRKs. Indeed, GIRK2 KO SCN slices had significantly fewer silent cells in response to NPY, likely contributing to the absence of NPY-induced phase advances of PER2::LUC rhythms in organotypic SCN cultures from GIRK2 KO mice. Finally, GIRK channel activation is sufficient to cause a non-photic-like phase advance of PER2::LUC rhythms on a Per2(Luc+/-) background. These results suggest that rhythmic regulation of GIRK2 protein and channel function in the SCN contributes to day-time resting membrane potential, providing a mechanism for the fine tuning responses to non-photic and photic stimuli. Further investigation could provide insight into disorders with circadian disruption comorbidities such as epilepsy and addiction, in which GIRK channels have been implicated. PMID:25217379

  3. Identification of the Hydrophobic Thickness of a Membrane Protein Using Fluorescence Spectroscopy:  Studies with the Mechanosensitive Channel MscL † , 1

    Microsoft Academic Search

    Andrew M. Powl; J. Neville Wright; J. Malcolm East; Anthony G. Lee

    2005-01-01

    The hydrophobic thickness of a membrane protein is an important parameter, defining how the protein sits within the hydrocarbon core of the lipid bilayer that surrounds it in a membrane. Here we show that Trp scanning mutagenesis combined with fluorescence spectroscopy can be used to define the hydrophobic thickness of a membrane protein. The mechanosensitive channel of large conductance (MscL)

  4. Probing Single-Molecule Protein Conformational Folding-Unfolding Dynamics: The multiple-State and Multiple-Channel Energy Landscape

    NASA Astrophysics Data System (ADS)

    Lu, H. Peter; Wang, Zhijiang; He, Yufan

    2013-03-01

    The folding-unfolding dynamics of protein provides an important understanding of the protein conformational dynamics and functions. We have used single-molecule fluorescence resonance energy transfer combined with statistical data analysis to characterize enzyme and signaling protein fundamental conformational dynamics of Calmodulin (CaM) and kinase (6-Hydroxymethyl-7,8-dihydropterin pyrophosphokinase, HPPK). The concentration dependence of FRET efficiency of GdmCl indicates the unfolding conformational transition of the proteins. At 2M of denaturant solvent, the majority of the HPPK and CaM protein molecules are under fluctuating folding-unfolding conformational changes, spending about half time in their native state and half time in their unfolded state. We obtained the fluctuation rates from the autocorrelation function analyses of the protein conformational fluctuation trajectories, and we have identified multiple intermediate states involving in bunched time dynamics and the related energy landscape. We had also analyzed the protein folding-unfolding pathways using detailed balance theoretical model analysis in order to understand the complex multiple-state and multiple-channel protein dynamics.

  5. Functional assay for T4 lysozyme-engineered G Protein-Coupled Receptors with an ion channel reporter

    PubMed Central

    Niescierowicz, Katarzyna; Caro, Lydia; Cherezov, Vadim; Vivaudou, Michel; Moreau, Christophe J.

    2013-01-01

    Summary: Structural studies of G protein-coupled receptors (GPCRs) extensively use the insertion of globular soluble protein domains in order to facilitate their crystallization. However, when inserted in the third intracellular loop (i3 loop), the soluble protein domain disrupts their coupling to G proteins and impedes the GPCRs functional characterization by standard G protein-based assays. Therefore, activity tests of crystallization-optimized GPCRs are essentially limited to their ligand binding properties using radioligand binding assays. Functional characterization of additional thermostabilizing mutations requires the insertion of similar mutations in the wild-type receptor to allow G protein-activation tests. We demonstrate that Ion Channel-Coupled Receptor technology is a complementary approach for a comprehensive functional characterization of crystallization-optimized GPCRs and potentially of any engineered GPCR. Ligand-induced conformational changes of the GPCRs are translated into electrical signal and detected by simple current recordings, even though binding of G proteins is sterically blocked by the added soluble protein domain. PMID:24268646

  6. Fragile X mental retardation protein controls synaptic vesicle exocytosis by modulating N-type calcium channel density

    NASA Astrophysics Data System (ADS)

    Ferron, Laurent; Nieto-Rostro, Manuela; Cassidy, John S.; Dolphin, Annette C.

    2014-04-01

    Fragile X syndrome (FXS), the most common heritable form of mental retardation, is characterized by synaptic dysfunction. Synaptic transmission depends critically on presynaptic calcium entry via voltage-gated calcium (CaV) channels. Here we show that the functional expression of neuronal N-type CaV channels (CaV2.2) is regulated by fragile X mental retardation protein (FMRP). We find that FMRP knockdown in dorsal root ganglion neurons increases CaV channel density in somata and in presynaptic terminals. We then show that FMRP controls CaV2.2 surface expression by targeting the channels to the proteasome for degradation. The interaction between FMRP and CaV2.2 occurs between the carboxy-terminal domain of FMRP and domains of CaV2.2 known to interact with the neurotransmitter release machinery. Finally, we show that FMRP controls synaptic exocytosis via CaV2.2 channels. Our data indicate that FMRP is a potent regulator of presynaptic activity, and its loss is likely to contribute to synaptic dysfunction in FXS.

  7. Stimulation of arterial smooth muscle L-type calcium channels by hydrogen peroxide requires protein kinase C

    PubMed Central

    Chaplin, Nathan L.; Amberg, Gregory C.

    2012-01-01

    Changes in intracellular calcium regulate countless biological processes. In arterial smooth muscle, voltage-dependent L-type calcium channels are major conduits for calcium entry with the primary function being determination of arterial diameter. Similarly, changes in intracellular redox status, either discrete controlled changes or global pathological perturbations, are also critical determinants of cell function. We recently reported that in arterial smooth muscle cells, local generation of hydrogen peroxide leads to colocalized calcium entry through L-type calcium channels. Here we extend our investigation into mechanisms linking hydrogen peroxide to calcium influx through L-type calcium channels by focusing on the role of protein kinase C (PKC). Our data indicate that stimulation of L-type calcium channels by hydrogen peroxide requires oxidant-dependent increases in PKC catalytic activity. This effect is independent of classical cofactor-dependent activation of PKC by diacylglycerol. These data provide additional experimental evidence supporting the concept of oxidative stimulation of L-type calcium channels. PMID:22907102

  8. Fragile X mental retardation protein controls synaptic vesicle exocytosis by modulating N-type calcium channel density

    PubMed Central

    Ferron, Laurent; Nieto-Rostro, Manuela; Cassidy, John S.; Dolphin, Annette C.

    2014-01-01

    Fragile X syndrome (FXS), the most common heritable form of mental retardation, is characterized by synaptic dysfunction. Synaptic transmission depends critically on presynaptic calcium entry via voltage-gated calcium (CaV) channels. Here we show that the functional expression of neuronal N-type CaV channels (CaV2.2) is regulated by fragile X mental retardation protein (FMRP). We find that FMRP knockdown in dorsal root ganglion neurons increases CaV channel density in somata and in presynaptic terminals. We then show that FMRP controls CaV2.2 surface expression by targeting the channels to the proteasome for degradation. The interaction between FMRP and CaV2.2 occurs between the carboxy-terminal domain of FMRP and domains of CaV2.2 known to interact with the neurotransmitter release machinery. Finally, we show that FMRP controls synaptic exocytosis via CaV2.2 channels. Our data indicate that FMRP is a potent regulator of presynaptic activity, and its loss is likely to contribute to synaptic dysfunction in FXS. PMID:24709664

  9. The membrane protein Pannexin1 forms two open channel conformations depending on the mode of activation

    PubMed Central

    Wang, Junjie; Ambrosi, Cinzia; Qiu, Feng; Jackson, David G.; Sosinsky, Gina; Dahl, Gerhard

    2014-01-01

    Pannexin1 (Panx1) participates in several signaling events that involve ATP release, including the innate immune response, ciliary beat in airway epithelia and oxygen supply in the vasculature. The view that Panx1 forms a large ATP-release channel has been challenged by the association of a low conductance, small anion-selective channel with the presence of Panx1. We showed that Panx1 membrane channels can function in two distinct modes with different conductances and permeabilities when heterologously expressed in Xenopus oocytes. When stimulated by potassium ions (K+), Panx1 formed a high conductance channel of ~500 pS that was permeable to ATP. Various physiological stimuli can induce this ATP-permeable conformation of the channel in several cell types. In contrast, the channel had a low conductance (~50 pS) with no detectable ATP permeability when activated by voltage in the absence of K+. The two channel states were associated with different reactivities of the terminal cysteine of Panx1 to thiol reagents, suggesting different conformations. Single particle electron microscopic analysis revealed that K+ stimulated the formation of channels with a larger pore diameter than those formed in the absence of K+. These data suggest that different stimuli lead to distinct channel structures with distinct biophysical properties. PMID:25056878

  10. Viruses infecting marine picoplancton encode functional potassium ion channels.

    PubMed

    Siotto, Fenja; Martin, Corinna; Rauh, Oliver; Van Etten, James L; Schroeder, Indra; Moroni, Anna; Thiel, Gerhard

    2014-10-01

    Phycodnaviruses are dsDNA viruses, which infect algae. Their large genomes encode many gene products, like small K(+) channels, with homologs in prokaryotes and eukaryotes. Screening for K(+) channels revealed their abundance in viruses from fresh-water habitats. Recent sequencing of viruses from marine algae or from salt water in Antarctica revealed sequences with the predicted characteristics of K(+) channels but with some unexpected features. Two genes encode either 78 or 79 amino acid proteins, which are the smallest known K(+) channels. Also of interest is an unusual sequence in the canonical ?-helixes in K(+) channels. Structural prediction algorithms indicate that the new channels have the conserved ?-helix folds but the algorithms failed to identify the expected transmembrane domains flanking the K(+) channel pores. In spite of these unexpected properties electophysiological studies confirmed that the new proteins are functional K(+) channels. PMID:25441713

  11. Negative inotropic effect of carbachol and interaction between acetylcholine receptor-operated potassium channel (K.ACh channel) and GTP binding protein in mouse isolated atrium--a novel methodological trial.

    PubMed

    Okada, Muneyoshi; Noma, Chihiro; Yamawaki, Hideyuki; Hara, Yukio

    2013-01-01

    Interaction between acetylcholine receptor-operated potassium channel (K.ACh channel) and GTP binding protein was examined by an immunoprecipitation-Western blotting system in mouse isolated atrium. The carbachol-induced negative inotropic action in indomethacin-pretreated mouse atrium was significantly inhibited by a K.ACh channel blocker, tertiapin or atropine. Kir3.1 K.ACh channel (Kir3.1) was immunoprecipitated with a mouse anti-Kir3.1 antibody. Coprecipitating G? with Kir3.1, detected by Western blotting, was significantly augmented by carbachol. Atropine, but not tertiapin, significantly inhibited the carbachol-induced coprecipitating G? with Kir3.1. The data indicate that immunoprecipitation with Kir3.1 and Western blotting of G? system is a useful method for assessing interaction between K.ACh channel and GTP binding protein in mouse atrium. PMID:23090691

  12. Development of heart failure is independent of K+ channel-interacting protein 2 expression.

    PubMed

    Speerschneider, Tobias; Grubb, Søren; Metoska, Artina; Olesen, Søren-Peter; Calloe, Kirstine; Thomsen, Morten B

    2013-12-01

    Abnormal ventricular repolarization in ion channelopathies and heart disease is a major cause of ventricular arrhythmias and sudden cardiac death. K(+) channel-interacting protein 2 (KChIP2) expression is significantly reduced in human heart failure (HF), contributing to a loss of the transient outward K(+) current (Ito). We aim to investigate the possible significance of a changed KChIP2 expression on the development of HF and proarrhythmia. Transverse aortic constrictions (TAC) and sham operations were performed in wild-type (WT) and KChIP2(-/-) mice. Echocardiography was performed before and every 2 weeks after the operation. Ten weeks post-surgery, surface ECG was recorded and we paced the heart in vivo to induce arrhythmias. Afterwards, tissue from the left ventricle was used for immunoblotting. Time courses of HF development were comparable in TAC-operated WT and KChIP2(-/-) mice. Ventricular protein expression of KChIP2 was reduced by 70% after 10 weeks TAC in WT mice. The amplitudes of the J and T waves were enlarged in KChIP2(-/-) control mice. Ventricular effective refractory period, RR, QRS and QT intervals were longer in mice with HF compared to sham-operated mice of either genotype. Pacing-induced ventricular tachycardia (VT) was observed in 5/10 sham-operated WT mice compared with 2/10 HF WT mice with HF. Interestingly, and contrary to previously published data, sham-operated KChIP2(-/-) mice were resistant to pacing-induced VT resulting in only 1/10 inducible mice. KChIP2(-/-) with HF mice had similar low vulnerability to inducible VT (1/9). Our results suggest that although KChIP2 is downregulated in HF, it is not orchestrating the development of HF. Moreover, KChIP2 affects ventricular repolarization and lowers arrhythmia susceptibility. Hence, downregulation of KChIP2 expression in HF may be antiarrhythmic in mice via reduction of the fast transient outward K(+) current. PMID:24099801

  13. Stomatin-domain protein interactions with acid-sensing ion channels modulate nociceptor mechanosensitivity

    PubMed Central

    Moshourab, Rabih A; Wetzel, Christiane; Martinez-Salgado, Carlos; Lewin, Gary R

    2013-01-01

    Acid-sensing ion channels (ASICs) and their interaction partners of the stomatin family have all been implicated in sensory transduction. Single gene deletion of asic3, asic2, stomatin, or stoml3 all result in deficits in the mechanosensitivity of distinct cutaneous afferents in the mouse. Here, we generated asic3?/?:stomatin?/?, asic3?/?:stoml3?/? and asic2?/?:stomatin?/? double mutant mice to characterize the functional consequences of stomatin–ASIC protein interactions on sensory afferent mechanosensitivity. The absence of ASIC3 led to a clear increase in mechanosensitivity in rapidly adapting mechanoreceptors (RAMs) and a decrease in the mechanosensitivity in both A?- and C-fibre nociceptors. The increased mechanosensitivity of RAMs could be accounted for by a loss of adaptation which could be mimicked by local application of APETx2 a toxin that specifically blocks ASIC3. There is a substantial loss of mechanosensitivity in stoml3?/? mice in which ?35% of the myelinated fibres lack a mechanosensitive receptive field and this phenotype was found to be identical in asic3?/?:stoml3?/? mutant mice. However, A?-nociceptors showed much reduced mechanosensitivity in asic3?/?:stoml3?/? mutant mice compared to asic3?/? controls. Interestingly, in asic2?/?:stomatin?/? mutant mice many A?-nociceptors completely lost their mechanosensitivity which was not observed in asic2?/? or stomatin?/? mice. Examination of stomatin?/?:stoml3?/? mutant mice indicated that a stomatin/STOML3 interaction is unlikely to account for the greater A?-nociceptor deficits in double mutant mice. A key finding from these studies is that the loss of stomatin or STOML3 in asic3?/? or asic2?/? mutant mice markedly exacerbates deficits in the mechanosensitivity of nociceptors without affecting mechanoreceptor function. PMID:23959680

  14. Magnetic field effect on nanoparticles migration and heat transfer of water/alumina nanofluid in a channel

    NASA Astrophysics Data System (ADS)

    Malvandi, A.; Ganji, D. D.

    2014-08-01

    The present study is a theoretical investigation of the laminar flow and convective heat transfer of water/alumina nanofluid inside a parallel-plate channel in the presence of a uniform magnetic field. A modified two-component, four-equation, nonhomogeneous equilibrium model was employed for the alumina/water nanofluid, which fully accounted for the effect of the nanoparticle volume fraction distribution. The no-slip condition of the fluid-solid interface is abandoned in favor of a slip condition which appropriately represents the non-equilibrium region near the interface at micro/nano channels. The results obtained indicated that nanoparticles move from the heated walls (nanoparticles depletion) toward the core region of the channel (nanoparticles accumulation) and construct a non-uniform nanoparticles distribution. Moreover, in the presence of the magnetic field, the near wall velocity gradients increase, enhancing the slip velocity and thus the heat transfer rate and pressure drop increase.

  15. Development of an Imaging System for the Observation of Water Behavior in a Channel in PEMFC

    NASA Astrophysics Data System (ADS)

    Nojima, T.; Yasuda, R.; Takenaka, N.; Katagiri, M.; Iikura, H.; Sakai, T.; Matsubayashi, M.

    A new imaging system was developed for observing water distribution in a polymer electrolyte membrane fuel cell (PEMFC) under operation. This imaging system realizes both low-noise and high-sensitivity imaging, enables image capture with a relatively short exposure time (approximately sub-seconds) and effectively visualizes the internal events of a PEMFC. It consists of an electron multiplying charge coupled device (EMCCD) camera, new 6LiF/ZnS scintillator screen, and slit system. The EMCCD camera has a wide dynamic range and high sensitivity, and the brightness of the new 6LiF/ZnS scintillator screen is approximately twice as high as that of conventional 6LiF/ZnS scintillator screen. The slit system is used to reduce the white dot noise caused by the primary and/or secondary prompt gamma rays. By a characteristic test, it was demonstrated that water behavior in the channel of a PEMFC can be clearly and qualitatively observed using the new imaging system.

  16. Low-level water vapor fields from the VISSR atmospheric sounder (VAS) split window channels at 11 and 12 microns

    Microsoft Academic Search

    D. Chesters; L. Uccellini; W. Robinson

    1982-01-01

    A series of high-resolution water vapor fields were derived from the 11 and 12 micron channels of the VISSR Atmospheric Sounder (VAS) on GOES-5. The low-level tropospheric moisture content was separated from the surface and atmospheric radiances by using the differential adsorption across the 'split window' along with the average air temperature from imbedded radiosondes. Fields of precipitable water are

  17. Mechanism of Interaction between the General Anesthetic Halothane and a Model Ion Channel Protein, II: Fluorescence and Vibrational Spectroscopy Using a Cyanophenylalanine Probe

    SciTech Connect

    Liu, J.; Strzalka, J; Tronin, A; Johansson, J; Blasie, J

    2009-01-01

    We demonstrate that cyano-phenylalanine (PheCN) can be utilized to probe the binding of the inhalational anesthetic halothane to an anesthetic-binding, model ion channel protein hbAP-PheCN. The Trp to PheCN mutation alters neither the a-helical conformation nor the 4-helix bundle structure. The halothane binding properties of this PheCN mutant hbAP-PheCN, based on fluorescence quenching, are consistent with those of the prototype, hbAP1. The dependence of fluorescence lifetime as a function of halothane concentration implies that the diffusion of halothane in the nonpolar core of the protein bundle is one-dimensional. As a consequence, at low halothane concentrations, the quenching of the fluorescence is dynamic, whereas at high concentrations the quenching becomes static. The 4-helix bundle structure present in aqueous detergent solution and at the air-water interface, is preserved in multilayer films of hbAP-PheCN, enabling vibrational spectroscopy of both the protein and its nitrile label (-CN). The nitrile groups' stretching vibration band shifts to higher frequency in the presence of halothane, and this blue-shift is largely reversible. Due to the complexity of this amphiphilic 4-helix bundle model membrane protein, where four PheCN probes are present adjacent to the designed cavity forming the binding site within each bundle, all contributing to the infrared absorption, molecular dynamics (MD) simulation is required to interpret the infrared results. The MD simulations indicate that the blue-shift of -CN stretching vibration induced by halothane arises from an indirect effect, namely an induced change in the electrostatic protein environment averaged over the four probe oscillators, rather than a direct interaction with the oscillators. hbAP-PheCN therefore provides a successful template for extending these investigations of the interactions of halothane with the model membrane protein via vibrational spectroscopy, using cyano-alanine residues to form the anesthetic binding cavity.

  18. Nighttime Cirrus Detection using Atmospheric Infrared Sounder Window Channels and Total Column Water Vapor

    NASA Technical Reports Server (NTRS)

    Kahn, Brian H.; Liou, Kuo Nan; Lee, Sung-Yung; Fishbein, Evan F.; DeSouza-Machado, Sergio; Eldering, Annmarie; Fetzer, Eric J.; Hannon, Scott E.; Strow, L. Larrabee

    2005-01-01

    A method of cirrus detection at nighttime is presented that utilizes 3.8 and 10.4 (micro)m infrared (IR) window brightness temperature differences (dBT) and total column precipitable water (PW) measurements. This technique is applied to the Atmospheric Infrared Sounder (AIRS) and Advanced Microwave Sounding Unit A (AMSU-A) instrument suite on board EOS-Aqua, where dBT is determined from sets of carefully selected AIRS window channels, while PW is derived from the synergistic AIRS and AMSU-A water vapor retrievals. Simulated and observed dBT for a particular value of PW are not constant; several physical factors impact dBT, including the variability in temperature and relative humidity profiles, surface emissivity, instrument noise, and skin/ near-surface air temperature differences. We simulate clear-sky dBT over a realistic range of PWs using 8350 radiosondes that have varying temperature and relative humidity profiles. Thresholds between cloudy and uncertain sky conditions are derived once the scatter in the clear-sky dBT is determined. Simulations of optically thin cirrus indicate that this technique is most sensitive to cirrus optical depth in the 10 (micro)m window of 0.1-0.15 or greater over the tropical and subtropical oceans, where surface emissivity and skin/near-surface air temperature impacts on the IR radiances are minimal. The method at present is generally valid over oceanic regions only, specifically, the tropics and subtropics. The detection of thin cirrus, and other cloud types, is validated using observations at the Atmospheric Radiation Measurement (ARM) program site located at Manus Island in the tropical western Pacific for 89 coincident EOS-Aqua overpasses. Even though the emphasis of this work is on the detection of thin cirrus at nighttime, this technique is sensitive to a broad cloud morphology. The cloud detection technique agrees with ARM-detected clouds 82-84% of the time, which include thin cirrus, as well as other cloud types. Most of the disagreements are well explained by AIRS footprint-scale heterogeneity compared to ARM point measurements, cirrus overlying lower-layer water clouds, possible mixed phase microphysics in midlevel clouds, and significant IR channel noise for cold BT scenes over deep convective towers.

  19. Steady-State Modulation of Voltage-Gated K+ Channels in Rat Arterial Smooth Muscle by Cyclic AMP-Dependent Protein Kinase and Protein Phosphatase 2B

    PubMed Central

    Brignell, Jennifer L.; Perry, Matthew D.; Nelson, Carl P.; Willets, Jonathon M.; Challiss, R. A. John; Davies, Noel W.

    2015-01-01

    Voltage-gated potassium channels (Kv) are important regulators of membrane potential in vascular smooth muscle cells, which is integral to controlling intracellular Ca2+ concentration and regulating vascular tone. Previous work indicates that Kv channels can be modulated by receptor-driven alterations of cyclic AMP-dependent protein kinase (PKA) activity. Here, we demonstrate that Kv channel activity is maintained by tonic activity of PKA. Whole-cell recording was used to assess the effect of manipulating PKA signalling on Kv and ATP-dependent K+ channels of rat mesenteric artery smooth muscle cells. Application of PKA inhibitors, KT5720 or H89, caused a significant inhibition of Kv currents. Tonic PKA-mediated activation of Kv appears maximal as application of isoprenaline (a ?-adrenoceptor agonist) or dibutyryl-cAMP failed to enhance Kv currents. We also show that this modulation of Kv by PKA can be reversed by protein phosphatase 2B/calcineurin (PP2B). PKA-dependent inhibition of Kv by KT5720 can be abrogated by pre-treatment with the PP2B inhibitor cyclosporin A, or inclusion of a PP2B auto-inhibitory peptide in the pipette solution. Finally, we demonstrate that tonic PKA-mediated modulation of Kv requires intact caveolae. Pre-treatment of the cells with methyl-?-cyclodextrin to deplete cellular cholesterol, or adding caveolin-scaffolding domain peptide to the pipette solution to disrupt caveolae-dependent signalling each attenuated PKA-mediated modulation of the Kv current. These findings highlight a novel, caveolae-dependent, tonic modulatory role of PKA on Kv channels providing new insight into mechanisms and the potential for pharmacological manipulation of vascular tone. PMID:25793374

  20. The 2 subunits of voltage-gated calcium channels form GPI-anchored proteins, a posttranslational

    E-print Network

    Dolphin, Annette C.

    the traffick- ing and properties of CaV1 and CaV2 channels. The 2 subunits have been described as type I | electrophysiology | immunocytochemistry Voltage-gated (CaV) calcium channels of the CaV1 and CaV2 classes

  1. Management implications of the relationships between water chemistry and fishes within channelized headwater streams in the midwestern United States

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Many headwater streams in the midwestern United States were channelized for agricultural drainage. Conservation practices are implemented to reduce nutrient and pesticide loadings within these altered streams. The impact of these practices is uncertain because the influence of water chemistry on str...

  2. Optimizing copper sulfate treatments for fungus control on channel catfish eggs in high alkalinity/moderate hardness water

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Interest in the use of copper sulfate (CuSO4) for fungus (Saprolegnia sp.) control in catfish hatcheries has developed over the past few years. A range-finding study was designed to determine the optimum concentration needed for fungus control on channel catfish eggs in 23.5°C well water at the Stu...

  3. The AHNAKs are a class of giant propeller-like proteins that associate with calcium channel proteins of cardiomyocytes and other cells

    PubMed Central

    Komuro, Akihiko; Masuda, Yutaka; Kobayashi, Koichi; Babbitt, Roger; Gunel, Murat; Flavell, Richard A.; Marchesi, Vincent T.

    2004-01-01

    To explore the function of the giant AHNAK molecule, first described in 1992 [Shtivelman, E., Cohen, F. E. & Bishop, J. M. (1992) Proc. Natl. Acad. Sci. USA 89, 5472–5476], we created AHNAK null mice by homologous recombination. Homozygous knockouts showed no obvious phenotype, but revealed instead a second AHNAK-like molecule, provisionally designated AHNAK2. Like the original AHNAK, AHNAK2 is a 600-kDa protein composed of a large number of highly conserved repeat segments. Structural predictions suggest that the repeat segments of both AHNAKs may have as their basic framework a series of linked, antiparallel ?-strands similar to those found in ?-propeller proteins. Both AHNAKs appear to localize to Z-band regions of mouse cardiomyocytes and cosediment with membrane vesicles containing the dihydropyridine receptor, which is consistent with earlier reports that the AHNAKs are linked to L-type calcium channels and can be phosphorylated by protein kinase A. The localization of the AHNAKs in close proximity to transverse tubule membranes and Z-band regions of cardiac sarcomeres raise the possibility that they might be involved in regulating excitation/contraction coupling of cardiomyocytes, but other studies indicate that the association of AHNAKs with calcium channel proteins is more widespread. AHNAK2 is predicted to have a PDZ domain within its N-terminal, nonrepeating domain, which may mediate these interactions. PMID:15007166

  4. The ? 1-subunit is essential for modulation by protein kinase C of an human and a non-human L-type Ca 2+ channel

    Microsoft Academic Search

    Alexandre Bouron; Nikolai M. Soldatov; Harald Reuter

    1995-01-01

    We have investigated in Xenopus oocytes the effects of phorbol ester-induced protein kinase C (PKC) stimulation on dihydropyridine (DHP)-insensitive and -sensitive Ca2+ channels. DHP-insensitive Ba2+ currents (IBa) were recorded from endogenous channels in non-injected oocytes and in oocytes injected with cRNAs encoding the auxiliary rabbit ?2\\/? and ?1 Ca2+ channel subunits. A human ?1C cRNA, injected alone or in combination

  5. Myristoylated alanine-rich C kinase substrate coordinates native TRPC1 channel activation by phosphatidylinositol 4,5-bisphosphate and protein kinase C in vascular smooth muscle

    PubMed Central

    Shi, Jian; Birnbaumer, Lutz; Large, William A.; Albert, Anthony P.

    2014-01-01

    Canonical transient receptor potential 1 (TRPC1) Ca2+-permeable cation channels contribute to vascular tone and blood vessel remodeling and represent potential therapeutic targets for cardiovascular disease. Protein kinase C (PKC) and phosphatidylinositol 4,5-bisphosphate [PI(4,5)P2] are obligatory for native TRPC1 channel activation in vascular smooth muscle cells (VSMCs) but how PKC and PI(4,5)P2 act together to induce channel gating remains unresolved. The present study reveals that myristoylated alanine-rich C kinase substrate (MARCKS) protein coordinates activation of TRPC1 channels by PKC and PI(4,5)P2. TRPC1 channels and MARCKS form signaling complexes with PI(4,5)P2 bound to MARCKS; in this configuration TRPC1 channels are closed. Activators of TRPC1 channels induce PKC phosphorylation of TRPC1 proteins, which causes dissociation of TRPC1 subunits from MARCKS and release of PI(4,5)P2 from MARCKS; PI(4,5)P2 subsequently binds to TRPC1 subunits to induce channel opening. Calmodulin acting at, or upstream of, MARCKS is also required for TRPC1 channel opening through a similar gating mechanism involving PKC and PI(4,5)P2. These novel findings show that MARCKS coordinates native TRPC1 channel activation in VSMCs by acting as a reversible PI(4,5)P2 buffer, which is regulated by PKC-mediated TRPC1 phosphorylation. Moreover, our data provide evidence that PI(4,5)P2 is a gating ligand of TRPC1 channels.—Shi, J., Birnbaumer, L., Large, W. A., and Albert, A. P. Myristoylated alanine-rich C kinase substrate coordinates native TRPC1 channel activation by phosphatidylinositol 4,5-bisphosphate and protein kinase C in vascular smooth muscle. PMID:24022404

  6. The effect of the half-width of the 22GHz water vapor line on retrievals of temperature and water vapor profiles with a 12-channel microwave radiometer

    Microsoft Academic Search

    James C. Liljegren; Sid-Ahmed Boukabara; Karen Cady-Pereira; Shepard A. Clough

    2005-01-01

    We show that observed biases in retrievals of temperature and water vapor profiles from a 12-channel microwave radiometer arise from systematic differences between the observed and model-calculated brightness temperatures at five measurement frequencies between 22 and 30 GHz. Replacing the value for the air-broadened half-width of the 22-GHz water vapor line used in the Rosenkranz absorption model with the 5%

  7. Unzipping Ion Channels

    NSDL National Science Digital Library

    Stacey Nee MacFarlane (University of Pennsylvania School of Medicine; Department of Neuroscience REV)

    2001-09-04

    The functions of ion channels can be regulated by their phosphorylation state. Protein kinases and protein phosphatases tightly control the activity of channels, thereby regulating the flow of ions across cell membranes. Channel proteins and kinases or phosphatases can associate directly or through intermediate adaptor proteins. An interaction domain termed the leucine zipper (LZ), once thought to be unique to some families of transcription factors, has been identified in channel proteins and their cognate binding proteins. MacFarlane and Levitan discuss what roles LZ-containing proteins might have in controlling channel function.

  8. The water channel aquaporin-1 contributes to renin cell recruitment during chronic stimulation of renin production.

    PubMed

    Tinning, Anne R; Jensen, Boye L; Schweda, Frank; Machura, Katharina; Hansen, Pernille B L; Stubbe, Jane; Gramsbergen, Jan Bert; Madsen, Kirsten

    2014-12-01

    Both the processing and release of secretory granules involve water movement across granule membranes. It was hypothesized that the water channel aquaporin (AQP)1 directly contributes to the recruitment of renin-positive cells in the afferent arteriole. AQP1(-/-) and AQP1(+/+) mice were fed a low-salt (LS) diet [0.004% (wt/wt) NaCl] for 7 days and given enalapril [angiotensin-converting enzyme inhibitor (ACEI), 0.1 mg/ml] in drinking water for 3 days. There were no differences in plasma renin concentration at baseline. After LS-ACEI, plasma renin concentrations increased markedly in both genotypes but was significantly lower in AQP1(-/-) mice compared with AQP1(+/+) mice. Tissue renin concentrations were higher in AQP1(-/-) mice, and renin mRNA levels were not different between genotypes. Mean arterial blood pressure was not different at baseline and during LS diet but decreased significantly in both genotypes after the addition of ACEI; the response was faster in AQP1(-/-) mice but then stabilized at a similar level. Renin release after 200 ?l blood withdrawal was not different. Isoprenaline-stimulated renin release from isolated perfused kidneys did not differ between genotypes. Cortical tissue norepinephrine concentrations were lower after LS-ACEI compared with baseline with no difference between genotypes. Plasma nitrite/nitrate concentrations were unaffected by genotype and LS-ACEI. In AQP1(-/-) mice, the number of afferent arterioles with recruitment was significantly lower compared with AQP1(+/+) mice after LS-ACEI. We conclude that AQP1 is not necessary for acutely stimulated renin secretion in vivo and from isolated perfused kidneys, whereas recruitment of renin-positive cells in response to chronic stimulation is attenuated or delayed in AQP1(-/-) mice. PMID:25339696

  9. Anchored Clathrate Waters Bind Antifreeze Proteins to Ice

    SciTech Connect

    C Garnham; R Campbell; P Davies

    2011-12-31

    The mechanism by which antifreeze proteins (AFPs) irreversibly bind to ice has not yet been resolved. The ice-binding site of an AFP is relatively hydrophobic, but also contains many potential hydrogen bond donors/acceptors. The extent to which hydrogen bonding and the hydrophobic effect contribute to ice binding has been debated for over 30 years. Here we have elucidated the ice-binding mechanism through solving the first crystal structure of an Antarctic bacterial AFP. This 34-kDa domain, the largest AFP structure determined to date, folds as a Ca{sup 2+}-bound parallel beta-helix with an extensive array of ice-like surface waters that are anchored via hydrogen bonds directly to the polypeptide backbone and adjacent side chains. These bound waters make an excellent three-dimensional match to both the primary prism and basal planes of ice and in effect provide an extensive X-ray crystallographic picture of the AFP{vert_ellipsis}ice interaction. This unobstructed view, free from crystal-packing artefacts, shows the contributions of both the hydrophobic effect and hydrogen bonding during AFP adsorption to ice. We term this mode of binding the 'anchored clathrate' mechanism of AFP action.

  10. The role of water exchange between a stream channel and its hyporheic zone in nitrogen cycling at the terrestrial-aquatic interface

    Microsoft Academic Search

    Frank J. Triska; John H. Duff; Ronald J. Avanzino

    1993-01-01

    The subsurface riparian zone was examined as an ecotone with two interfaces. Inland is a terrestrial boundary, where transport of water and dissolved solutes is toward the channel and controlled by watershed hydrology. Streamside is an aquatic boundary, where exchange of surface water and dissolved solutes is bi-directional and flux is strongly influenced by channel hydraulics. Streamside, bi-directional exchange of

  11. A two-channel, tunable diode laser-based hygrometer for measurement of water vapor and cirrus cloud ice water content in the upper troposphere and lower stratosphere

    NASA Astrophysics Data System (ADS)

    Thornberry, T. D.; Rollins, A. W.; Gao, R. S.; Watts, L. A.; Ciciora, S. J.; McLaughlin, R. J.; Fahey, D. W.

    2015-01-01

    The recently developed NOAA Water instrument is a two-channel, closed-path, tunable diode laser absorption spectrometer designed for the measurement of upper troposphere/lower stratosphere water vapor and enhanced total water (vapor + inertially enhanced condensed phase) from the NASA Global Hawk unmanned aircraft system (UAS) or other high-altitude research aircraft. The instrument utilizes wavelength-modulated spectroscopy with second harmonic detection near 2694 nm to achieve high precision with a 79 cm double-pass optical path. The detection cells are operated under constant temperature, pressure, and flow conditions to maintain a constant sensitivity to H2O independent of the ambient sampling environment. An onboard calibration system is used to perform periodic in situ calibrations to verify the stability of the instrument sensitivity during flight. For the water vapor channel, ambient air is sampled perpendicular to the flow past the aircraft in order to reject cloud particles, while the total water channel uses a heated, forward-facing inlet to sample both water vapor and cloud particles. The total water inlet operates subisokinetically, thereby inertially enhancing cloud particle number in the sample flow and affording increased cloud water content sensitivity. The NOAA Water instrument was flown for the first time during the second deployment of the Airborne Tropical TRopopause EXperiment (ATTREX) in February-March 2013 on the NASA Global Hawk UAS. The instrument demonstrated a typical in-flight precision (1 s, 1?) of better than 0.17 parts per million (ppm, 10-6 mol mol-1), with an overall H2O vapor measurement uncertainty of 5% ± 0.23 ppm. The inertial enhancement for cirrus cloud particle sampling under ATTREX flight conditions ranged from 33 to 48 for ice particles larger than 8 ?m in diameter, depending primarily on aircraft altitude. The resulting ice water content detection limit (2?) was 0.023-0.013 ppm, corresponding to approximately 2 ?g m-3, with an estimated overall uncertainty of 20%.

  12. Yellow Fluorescent Protein-Based Assay to Measure GABAA Channel Activation and Allosteric Modulation in CHO-K1 Cells

    PubMed Central

    Johansson, Teres; Norris, Tyrrell; Peilot-Sjögren, Helena

    2013-01-01

    The ?-aminobutyric acid A (GABAA) ion channels are important drug targets for treatment of neurological and psychiatric disorders. Finding GABAA channel subtype selective allosteric modulators could lead to new improved treatments. However, the progress in this area has been obstructed by the challenging task of developing functional assays to support screening efforts and the generation of cells expressing functional GABAA ion channels with the desired subtype composition. To address these challenges, we developed a yellow fluorescent protein (YFP)-based assay to be able to study allosteric modulation of the GABAA ion channel using cryopreserved, transiently transfected, assay-ready cells. We show for the first time how the MaxCyte STX electroporation instrument can be used to generate CHO-K1 cells expressing functional GABAA ?2?3?2 along with a halide sensing YFP-H148Q/I152L (YFP-GABAA2 cells). As a basis for a cell-based assay capable of detecting allosteric modulators, experiments with antagonist, ion channel blocker and modulators were used to verify GABAA subunit composition and functionality. We found that the I? concentration used in the YFP assay affected both basal quench of YFP and potency of GABA. For the first time the assay was used to study modulation of GABA with 7 known modulators where statistical analysis showed that the assay can distinguish modulatory pEC50 differences of 0.15. In conclusion, the YFP assay proved to be a robust, reproducible and inexpensive assay. These data provide evidence that the assay is suitable for high throughput screening (HTS) and could be used to discover novel modulators acting on GABAA ion channels. PMID:23516634

  13. Rampant exchange of the structure and function of extramembrane domains between membrane and water soluble proteins.

    PubMed

    Nam, Hyun-Jun; Han, Seong Kyu; Bowie, James U; Kim, Sanguk

    2013-01-01

    Of the membrane proteins of known structure, we found that a remarkable 67% of the water soluble domains are structurally similar to water soluble proteins of known structure. Moreover, 41% of known water soluble protein structures share a domain with an already known membrane protein structure. We also found that functional residues are frequently conserved between extramembrane domains of membrane and soluble proteins that share structural similarity. These results suggest membrane and soluble proteins readily exchange domains and their attendant functionalities. The exchanges between membrane and soluble proteins are particularly frequent in eukaryotes, indicating that this is an important mechanism for increasing functional complexity. The high level of structural overlap between the two classes of proteins provides an opportunity to employ the extensive information on soluble proteins to illuminate membrane protein structure and function, for which much less is known. To this end, we employed structure guided sequence alignment to elucidate the functions of membrane proteins in the human genome. Our results bridge the gap of fold space between membrane and water soluble proteins and provide a resource for the prediction of membrane protein function. A database of predicted structural and functional relationships for proteins in the human genome is provided at sbi.postech.ac.kr/emdmp. PMID:23555228

  14. ATP-sensitive potassium channel traffic regulation by adenosine and protein kinase C.

    PubMed

    Hu, Keli; Huang, Cindy Shen; Jan, Yuh Nung; Jan, Lily Yeh

    2003-05-01

    ATP-sensitive potassium (K(ATP)) channels activate under metabolic stress to protect neurons and cardiac myocytes. However, excessive channel activation may cause arrhythmia in the heart and silence neurons in the brain. Here, we report that PKC-mediated downregulation of K(ATP) channel number, via dynamin-dependent channel internalization, can act as a brake mechanism to control K(ATP) activation. A dileucine motif in the pore-lining Kir6.2 subunit of K(ATP), but not the site of PKC phosphorylation for channel activation, is essential for PKC downregulation. Whereas K(ATP) activation results in a rapid shortening of the action potential duration (APD) in metabolically inhibited ventricular myocytes, adenosine receptor stimulation and consequent PKC-mediated K(ATP) channel internalization can act as a brake to lessen this APD shortening. Likewise, in hippocampal CA1 neurons under metabolic stress, PKC-mediated, dynamin-dependent K(ATP) channel internalization can also act as a brake to dampen the rapid decline of excitability due to K(ATP) activation. PMID:12741989

  15. Decentralised water retention along the river channels in a mesoscale catchment in south-eastern Germany

    NASA Astrophysics Data System (ADS)

    Reinhardt, Christian; Bölscher, Jens; Schulte, Achim; Wenzel, Robert

    2011-01-01

    Throughout the Ore Mountains, a low mountain area located in the German-Czech border region, storm runoff frequently causes severe damage in headwater areas as well as in lower reaches. Settlements along smaller tributaries and towns at the receiving water are affected simultaneously, so measures distributed throughout the entire drainage area (decentralised measures) have to be considered for flood protection planning in such areas. The concept of decentralised flood protection, which is well established in the German literature, offers a large number of potential flood control schemes including measures along the river channels, in agriculture and forestry as well as in settlements. The investigations presented here focus on the group of measures along the river channels, including small, distributed retarding basins, river renaturation and afforestation of floodplains. Based on rainfall-runoff models, its aim is to show how such measures influence flood hydrographs in low mountain areas with a 100-year recurrence interval, using the example of the Upper Flöha watershed in the Central Ore Mountains. The results indicate that along the tributaries of the Flöha very high local peak reductions can be achieved with small retarding basins. The efficiency of the basins is related to the available storage capacity in the valleys upstream of the settlements. On a supralocal level, i.e. at the Flöha River, an additional reduction of the peak discharge occurs in the model. Other significant supralocal effects can be observed for the scenarios with an increased floodplain roughness (afforestation). In a combination of both scenarios the supralocal effects increase further, whereas the local effects are as high as in the retarding basin scenario. By contrast, the river renaturation scenario does not show a significant impact on the flood hydrographs. However, the limited effect is a result of the local characteristics of the study area, where the number of suitable river sections is limited and the slope gradients are high. On the whole, it can be concluded that decentralised measures along the rivers can be efficient elements in the framework of flood protection strategies. The reduction of flood peaks includes not only the receiving water, but also the tributaries, so that an improvement of flood protection extending across the entire watershed can be achieved.

  16. Cocaine sensitization increases Ih current channel subunit 2 (HCN2) protein expression in structures of the Mesocorticolimbic System

    PubMed Central

    Santos-Vera, Bermary; Vázquez-Torres, Rafael; García Marrero, Hermes G.; Ramos Acevedo, Juan M.; Arencibia-Albite, Francisco; Vélez-Hernández, María E.; Miranda, Jorge D.; Jiménez-Rivera, Carlos A.

    2013-01-01

    Alteration of the biological activity among neuronal components of the Mesocorticolimbic (MCL) system has been implicated in the pathophysiology of drug abuse. Changes in the electrophysiological properties of neurons involved in the reward circuit seem to be of utmost importance in addiction. The Hyperpolarization-Activated Cyclic-Nucleotide Current, Ih, is a prominent mixed cation current present in neurons. The biophysical properties of the Ih and its potential modulatory role in cell excitability depend on the expression profile of the Hyperpolarization-activated cyclic nucleotide gated channel (HCN) subunits. We investigated whether cocaine-induced behavioral sensitization, an animal model of drug addiction, elicits region-specific changes in the expression of the HCN2 channel’s subunit in the MCL system. Tissue samples from the ventral tegmental area, prefrontal cortex, nucleus accumbens and hippocampus were analyzed using Western Blot. Our findings demonstrate that cocaine treatment induced a significant increase in the expression profile of the HCN2 subunit in both, its glycosylated and non-glycosylated protein isoforms, in all areas tested. The increase in the glycosylated isoform was only observed in the ventral tegmental area. Together, these data suggest that the observed changes in MCL excitability during cocaine addiction might be associated to alterations in the subunit composition of their HCN channels. PMID:23203153

  17. Concentrations of polychlorinated biphenyls (PCBs) in water, sediment, and aquatic biota in the Houston Ship Channel, Texas.

    PubMed

    Howell, Nathan L; Suarez, Monica P; Rifai, Hanadi S; Koenig, Larry

    2008-01-01

    Polychlorinated biphenyls (PCBs) were quantified in water, sediment, and catfish and crab tissue collected from the Houston Ship Channel (HSC) in Texas. The total concentrations of the 209 PCB congeners ranged from 0.49 to 12.49 ng l(-1), 4.18 to 4601 ng g(-1) dry wt, 4.13 to 1596 ng g(-1) wet wt, and 3.44 to 169 ng g(-1) wet wt, in water, sediment, catfish and crab tissue, respectively. All media showed maximum concentrations greater than studies in other regions with the highest concentrations occurring in the most industrialized segments of the channel. Inter-media correlations suggested that sediment is a source to water. Galveston Bay sediment concentrations compared to a previous study showed a declining trend though the rate of the decline may be slowing. Detailed homolog profiles revealed that the industrialized part of the channel may be receiving PCB-laden sediment from its tributaries. An unusually high fraction of the deca-chlorinated congener (PCB-209) was found in all media. Seen in only a few other studies and in previous air concentrations in the channel, this may point to unusual Aroclor mixtures used in the history of the HSC or to contemporary sources from local industry. A comparison of PCB concentrations obtained using Aroclor, representative congener, and all congener methods, indicated that Aroclors are not an appropriate surrogate for total PCBs and that the NOAA NST method is more representative than the NOAA EPA method. PMID:17850847

  18. Unimolecular and hydrolysis channels for the detachment of water from microsolvated alkaline earth dication (Mg2+, Ca2+, Sr2+, Ba2+) clusters

    SciTech Connect

    Miliordos, Evangelos; Xantheas, Sotiris S.

    2014-02-07

    We examine theoretically the three channels that are associated with the detachment of a single water molecule from the aqueous clusters of the alkaline earth dications, [M(H2O)n]2+, M = Mg, Ca, Sr, Ba, n ? 6. These are the unimolecular water loss (M2+(H2O)n-1 + H2O) and the two hydrolysis channels resulting to the loss of hydronium ([MOH(H2O)n-2]+ + H3O+) and Zundel ([MOH(H2O)n-3]+ + H3O+(H2O)) cations. The Potential Energy Curves (PECs) corresponding to those three channels were constructed at the Møller-Plesset second order perturbation (MP2) level of theory with basis sets of double- and triple-? quality. We furthermore investigated the water and hydronium loss channels from the mono-hydroxide water clusters with up to four water molecules, [MOH(H2O)n]+, 1 ? n ? 4. Our results indicate the preference of the hydronium loss and possibly the Zundel cation loss channels for the smallest size clusters, whereas the unimolecular water loss channel is preferred for the larger ones as well as the mono-hydroxide clusters. Although the charge separation (hydronium and Zundel cation loss) channels produce more stable products when compared to the ones for the unimolecular water loss, they also require the surmounting of high energy barriers, a fact that makes the experimental observation of fragments related to these hydrolysis channels difficult.

  19. Ca2+-binding proteins tune Ca2+-feedback to Cav1.3 channels in mouse auditory hair cells

    PubMed Central

    Cui, Guiying; Meyer, Alexander C; Calin-Jageman, Irina; Neef, Jakob; Haeseleer, Françoise; Moser, Tobias; Lee, Amy

    2007-01-01

    Sound coding at the auditory inner hair cell synapse requires graded changes in neurotransmitter release, triggered by sustained activation of presynaptic Cav1.3 voltage-gated Ca2+ channels. Central to their role in this regard, Cav1.3 channels in inner hair cells show little Ca2+-dependent inactivation, a fast negative feedback regulation by incoming Ca2+ ions, which depends on calmodulin association with the Ca2+ channel ?1 subunit. Ca2+-dependent inactivation characterizes nearly all voltage-gated Ca2+ channels including Cav1.3 in other excitable cells. The mechanism underlying the limited autoregulation of Cav1.3 in inner hair cells remains a mystery. Previously, we established calmodulin-like Ca2+-binding proteins in the brain and retina (CaBPs) as essential modulators of voltage-gated Ca2+ channels. Here, we demonstrate that CaBPs differentially modify Ca2+ feedback to Cav1.3 channels in transfected cells and explore their significance for Cav1.3 regulation in inner hair cells. Of multiple CaBPs detected in inner hair cells (CaBP1, CaBP2, CaBP4 and CaBP5), CaBP1 most efficiently blunts Ca2+-dependent inactivation of Cav1.3. CaBP1 and CaBP4 both interact with calmodulin-binding sequences in Cav1.3, but CaBP4 more weakly inhibits Ca2+-dependent inactivation than CaBP1. Ca2+-dependent inactivation is marginally greater in inner hair cells from CaBP4?/? than from wild-type mice, yet CaBP4?/? mice are not hearing-impaired. In contrast to CaBP4, CaBP1 is strongly localized at the presynaptic ribbon synapse of adult inner hair cells both in wild-type and CaBP4?/? mice and therefore is positioned to modulate native Cav1.3 channels. Our results reveal unexpected diversity in the strengths of CaBPs as Ca2+ channel modulators, and implicate CaBP1 rather than CaBP4 in conferring the anomalous slow inactivation of Cav1.3 Ca2+ currents required for auditory transmission. PMID:17947313

  20. Shut-off of a geopressured water channel behind casing via coiled tubing utilizing a dual slurry cement system: A case history

    SciTech Connect

    Nowak, T.W.; Lange, K.J.; Grant, W.H.; Patout, T.S.

    1995-12-31

    This paper presents a case history involving a unique dual cement system to shut off a geopressured water channel behind casing utilizing coiled tubing. The channeling problem was identified and documented using water flow logging techniques. Logging indicated the lower gravel packed selective could produce salt water if perforated without eliminating the suspected water channel. Reserves did not warrant a major rig workover, making a non-rig workover via coiled tubing the only viable option to repair the well. A unique dual cement system tested on a hesitation squeeze schedule pumped through coiled tubing with extremely limited thickening time was necessary to repair the primary cement job.

  1. Aquaporin water channel genes are differentially expressed and regulated by ovarian steroids during the periimplantation period in the mouse.

    PubMed

    Richard, Charissa; Gao, Ju; Brown, Naoko; Reese, Jeff

    2003-04-01

    The periimplantation period is marked by edematous changes in the uterus. In the mouse, increased uterine vascular permeability occurs in response to estrogen and certain vasoactive mediators, but the mechanisms that regulate fluid transport during implantation are not fully understood. Aquaporins (AQPs) are a family of membrane channel proteins that facilitate bulk water transport. To assess their role in implantation, we examined the expression of AQPs 0-9 in the mouse uterus on d 1-8 of pregnancy. Our results show distinct uterine expression patterns for AQP1, AQP4, and AQP5. AQP1 is localized to the inner circular myometrium throughout the periimplantation period. AQP4 is highly expressed in the luminal epithelium on d 1 of pregnancy but barely detectable at the time of implantation. AQP5 is expressed at low levels in the glandular epithelium during early pregnancy but is markedly increased on d 5. By immunohistochemistry, AQP5 is localized in the basolateral region of the uterine glands. Treatment of adult ovariectomized mice with replacement steroids demonstrates an estrogen-induced shift in AQP1 signals from the myometrium to the uterine stromal vasculature, suggesting a role in uterine fluid imbibition. In contrast, AQP5 is induced only in estrogen-treated, progesterone-primed uteri. We also observed expression of AQP8 in the inner-cell mass and AQP9 in the mural trophectoderm of the implanting blastocyst. Collectively, these results suggest that members of the AQP family are involved in embryo and uterine fluid homeostasis during implantation. PMID:12639938

  2. Empirical nanotube model: Applications to water channels and nano-oscillators

    NASA Astrophysics Data System (ADS)

    Lu, Deyu

    Carbon nanotubes are building blocks of the fast-developing nanotechnology that leads to revolutionary breakthroughs in material science and engineering. The extraordinary mechanical, electrical, and optical properties of nanotubes combined with novel design concepts enable a variety of applications including high-strength materials, logic circuit units, field emission devices, and hydrogen storage media. In particular, nanobiotechnology, a synergy of nanotechnology and biology, aims at characterizing, building, and utilizing nano-devices to benefit human health as well as employ life forms for technical purposes. Examples are nanotube-based biosensors and drug delivery devices. However, development of the principles in this multi-disciplinary field is still in its infancy. Joint efforts from physics, chemistry, and biology are required to achieve new insights. To this end, an empirical carbon nanotube model is developed in this thesis to describe the interaction between nanotubes and the biological environment. Special emphasis is placed on an accurate and efficient description of the electrostatics of nanotubes, which plays a key role in determining molecular transport dynamics through nanotubes. In the proposed model, atomic partial charges are calculated from a quantum chemistry approach, and the polarizability of the nanotube is modeled through a self-consistent tight-binding method. The suitability of the model is demonstrated through studies of a nanotube water channel and a K+-nanotube complex. It is found in the former case that atomic partial charges on the tube edges greatly contribute to the total interaction energy, while the polarization of the nanotube lowers the electrostatic energy once a water molecule moves inside the nanotube. In the latter case, quantum mechanics/molecular mechanics simulations reveal that a K+ ion induces a strong dielectric response in the nanotube wall, which helps to trap the ion inside the tube and force the ion to oscillate at a terahertz frequency. Such a nano-oscillator may hold potential applications as a room temperature terahertz wave detector.

  3. Role of Aquaporin Water Channels in Airway Fluid Transport, Humidification, and Surface Liquid Hydration

    PubMed Central

    Song, Yuanlin; Jayaraman, Sujatha; Yang, Baoxue; Matthay, Michael A.; Verkman, A.S.

    2001-01-01

    Several aquaporin-type water channels are expressed in mammalian airways and lung: AQP1 in microvascular endothelia, AQP3 in upper airway epithelia, AQP4 in upper and lower airway epithelia, and AQP5 in alveolar epithelia. Novel quantitative methods were developed to compare airway fluid transport–related functions in wild-type mice and knockout mice deficient in these aquaporins. Lower airway humidification, measured from the moisture content of expired air during mechanical ventilation with dry air through a tracheotomy, was 54–56% efficient in wild-type mice, and reduced by only 3–4% in AQP1/AQP5 or AQP3/AQP4 double knockout mice. Upper airway humidification, measured from the moisture gained by dry air passed through the upper airways in mice breathing through a tracheotomy, decreased from 91 to 50% with increasing ventilation from 20 to 220 ml/min, and reduced by 3–5% in AQP3/AQP4 knockout mice. The depth and salt concentration of the airway surface liquid in trachea was measured in vivo using fluorescent probes and confocal and ratio imaging microscopy. Airway surface liquid depth was 45 ± 5 ?m and [Na+] was 115 ± 4 mM in wild-type mice, and not significantly different in AQP3/AQP4 knockout mice. Osmotic water permeability in upper airways, measured by an in vivo instillation/sample method, was reduced by ?40% by AQP3/AQP4 deletion. In doing these measurements, we discovered a novel amiloride-sensitive isosmolar fluid absorption process in upper airways (13% in 5 min) that was not affected by aquaporin deletion. These results establish the fluid transporting properties of mouse airways, and indicate that aquaporins play at most a minor role in airway humidification, ASL hydration, and isosmolar fluid absorption. PMID:11382807

  4. Interactions of lipids and detergents with a viral ion channel protein: molecular dynamics simulation studies.

    PubMed

    Rouse, Sarah L; Sansom, Mark S P

    2015-01-22

    Structural studies of membrane proteins have highlighted the likely influence of membrane mimetic environments (i.e., lipid bilayers versus detergent micelles) on the conformation and dynamics of small ?-helical membrane proteins. We have used molecular dynamics simulations to compare the conformational dynamics of BM2 (a small ?-helical protein from the membrane of influenza B) in a model phospholipid bilayer environment with its behavior in protein-detergent complexes with either the zwitterionic detergent dihexanoylphosphatidylcholine (DHPC) or the nonionic detergent dodecylmaltoside (DDM). We find that DDM more closely resembles the lipid bilayer in terms of its interaction with the protein, while the short-tailed DHPC molecule forms "nonphysiological" interactions with the protein termini. We find that the intrinsic micelle properties of each detergent are conserved upon formation of the protein-detergent complex. This implies that simulations of detergent micelles may be used to help select optimal conditions for experimental studies of membrane proteins. PMID:25286030

  5. Pressure drop and heat transfer characteristics of boiling water in sub-hundred micron channel

    SciTech Connect

    Bhide, R.R.; Singh, S.G.; Sridharan, Arunkumar; Duttagupta, S.P.; Agrawal, Amit [Department of Mechanical Engineering, Indian Institute of Technology Bombay, Powai, Mumbai 400 076 (India)

    2009-09-15

    The current work focuses on the pressure drop, heat transfer and stability in two phase flow in microchannels with hydraulic diameter of less than one hundred microns. Experiments were conducted in smooth microchannels of hydraulic diameter of 45, 65 {mu}m, and a rough microchannel of hydraulic diameter of 70 {mu}m, with deionised water as the working fluid. The local saturation pressure and temperature vary substantially over the length of the channel. In order to correctly predict the local saturation temperature and subsequently the heat transfer characteristics, numerical techniques have been used in conjunction with the conventional two phase pressure drop models. The Lockhart-Martinelli (liquid-laminar, vapour-laminar) model is found to predict the two phase pressure drop data within 20%. The instability in two phase flow is quantified; it is found that microchannels of smaller hydraulic diameter have lesser instabilities as compared to their larger counterparts. The experiments also suggest that surface characteristics strongly affect flow stability in the two phase flow regime. The effect of hydraulic diameter and surface characteristics on the flow characteristics and stability in two phase flow is seldom reported, and is of considerable practical relevance. (author)

  6. Calibration of the 1064 nm lidar channel using water phase and cirrus clouds.

    PubMed

    Wu, Yonghua; Gan, Chuen Meei; Cordero, Lina; Gross, Barry; Moshary, Fred; Ahmed, Sam

    2011-07-20

    Calibration is essential to derive aerosol backscatter coefficients from elastic scattering lidar. Unlike the visible UV wavelengths where calibration is based on a molecular reference, calibration of the 1064 nm lidar channel requires other approaches, which depend on various assumptions. In this paper, we analyze two independent calibration methods which use (i) low-altitude water phase clouds and (ii) high cirrus clouds. In particular, we show that to achieve optimal performance, aerosol attenuation below the cloud base and cloud multiple scattering must be accounted for. When all important processes are considered, we find that these two independent methods can provide a consistent calibration constant with relative differences less than 15%. We apply these calibration techniques to demonstrate the stability of our lidar on a monthly scale, along with a natural reduction of the lidar efficiency on an annual scale. Furthermore, our calibration procedure allows us to derive consistent aerosol backscatter coefficients and angstrom coefficient profiles (532-1064 nm) along with column extinction-to-backscatter ratios which are in good agreement with sky radiometer inversions. PMID:21772382

  7. Initiation of a discharge channel in water by means of electrical explosion of aluminum foil

    NASA Astrophysics Data System (ADS)

    Sil'nikov, M. V.; Krivosheev, S. I.; Kulakov, K. S.; Kulakov, S. L.

    2013-12-01

    This paper reports the results of an experimental investigation into initiation of the electric discharge in service water by means of explosion of aluminum foil having various mass and dimensions. The electric discharge was formed in a chamber with a movable wall (the piston). As an electric energy storage, the capacitor bank having the capacity C = 200-600 ?F with charging voltage U 0 = 2-5 kV (stored energy Q 0 = 0.4-7.5 kJ) and the rate of rise of the discharging current dI/ dt = (3-4) × 109 A/s. The results of experiments showed that destruction (loss of conductivity) of foil occurs at the value of the integral of the current density h j = (0.3-0.65) × 109 (A2/cm4)/s. The stage of the repeated breakdown in the electric discharge occurs when the value of the intensity of the electric field along the discharge channel is of E rb ? 50 V/mm. Geometric dimensions and mass of the initiating conductor that provide the maximum efficiency of conversion of the value of Q 0 into kinetic energy of the piston have been determined.

  8. Protein interactions central to stabilizing the K[superscript +] channel selectivity filter in a four-sited configuration for selective K[superscript +] permeation

    SciTech Connect

    Sauer, David B.; Zeng, Weizhong; Raghunathan, Srinivasan; Jiang, Youxing (UTSMC)

    2011-11-18

    The structural and functional conversion of the nonselective NaK channel to a K{sup +} selective channel (NaK2K) allows us to identify two key residues, Tyr and Asp in the filter sequence of TVGYGD, that participate in interactions central to stabilizing the K{sup +} channel selectivity filter. By using protein crystallography and channel electrophysiology, we demonstrate that the K{sup +} channel filter exists as an energetically strained structure and requires these key protein interactions working in concert to hold the filter in the precisely defined four-sited configuration that is essential for selective K{sup +} permeation. Disruption of either interaction, as tested on both the NaK2K and eukaryotic K{sub v}1.6 channels, can reduce or completely abolish K{sup +} selectivity and in some cases may also lead to channel inactivation due to conformational changes at the filter. Additionally, on the scaffold of NaK we recapitulate the protein interactions found in the filter of the Kir channel family, which uses a distinct interaction network to achieve similar stabilization of the filter.

  9. Nifedipine represses ion channels, transporters and Ca{sup 2+}-binding proteins in hearts of spontaneously hypertensive rats

    SciTech Connect

    Zwadlo, Carolin [Fraunhofer Institute of Toxicology and Experimental Medicine, Drug Research and Clinical Inhalation, Nicolai-Fuchs-Str. 1, D-30625 Hannover (Germany); Borlak, Juergen [Fraunhofer Institute of Toxicology and Experimental Medicine, Drug Research and Clinical Inhalation, Nicolai-Fuchs-Str. 1, D-30625 Hannover (Germany)]. E-mail: borlak@item.fraunhofer.de

    2006-06-15

    The Ca{sup 2+} antagonists nifedipine has been used for more than three decades to treat hypertension, but its effects on the transcriptional regulation of cardiac genes are basically unknown. We therefore studied expression of genes coding for ion channels, ion transporters and associated partners as well as Ca{sup 2+}-binding proteins in ventricular tissue of normotensive and spontaneously hypertensive (SH) rats after repeated intraperitoneally (i.p.) dosing of nifedipine. Notably, we observed significant (P < 0.05) repression in transcript levels of most of the genes investigated, including cardiac Na{sup +}, K{sup +}, Ca{sup 2+}-channels (L-type Ca{sup 2+}-channel, K{sub ir}3.4, K{sub ir}6.1, Na{sub v}1.5), ATP-driven ion exchangers (Na{sup +}-K{sup +}-ATPase, NCX-1, PMCA 2 and 4, SERCA 2a and 2b) and their associated partners (phospholamban, RyR-2) as well as cytoskeletal proteins ({alpha} and {beta}-MHC, {alpha} cardiac and {alpha} skeletal actin, troponin T and I). Repression in transcript levels was, however, only seen in ventricular tissue of hypertensive animals. This points to fundamental differences in the mode of action of nifedipine in diseased and healthy animals. Indeed, this preponderance of repressed genes will promote disturbed ion homeostasis to result in contractile dysfunction. It is of considerable importance that repressed gene expression was also seen in end-stage human heart failure [Borlak, J., Thum, T., 2003. Hallmarks of ion channel gene expression in end-stage heart failure. FASEB J. 17, 1592-1608]. We propose repression of cardiac-specific gene expression as a hallmark of nifedipine treatment in hypertrophic hearts.

  10. Interaction with 14-3-3 proteins promotes functional expression of the potassium channels TASK-1 and TASK-3

    PubMed Central

    Rajan, Sindhu; Preisig-Müller, Regina; Wischmeyer, Erhard; Nehring, Ralf; Hanley, Peter J; Renigunta, Vijay; Musset, Boris; Schlichthörl, Günter; Derst, Christian; Karschin, Andreas; Daut, Jürgen

    2002-01-01

    The two-pore-domain potassium channels TASK-1, TASK-3 and TASK-5 possess a conserved C-terminal motif of five amino acids. Truncation of the C-terminus of TASK-1 strongly reduced the currents measured after heterologous expression in Xenopus oocytes or HEK293 cells and decreased surface membrane expression of GFP-tagged channel proteins. Two-hybrid analysis showed that the C-terminal domain of TASK-1, TASK-3 and TASK-5, but not TASK-4, interacts with isoforms of the adapter protein 14-3-3. A pentapeptide motif at the extreme C-terminus of TASK-1, RRx(S/T)x, was found to be sufficient for weak but significant interaction with 14-3-3, whereas the last 40 amino acids of TASK-1 were required for strong binding. Deletion of a single amino acid at the C-terminal end of TASK-1 or TASK-3 abolished binding of 14-3-3 and strongly reduced the macroscopic currents observed in Xenopus oocytes. TASK-1 mutants that failed to interact with 14-3-3 isoforms (V411*, S410A, S410D) also produced only very weak macroscopic currents. In contrast, the mutant TASK-1 S409A, which interacts with 14-3-3-like wild-type channels, displayed normal macroscopic currents. Co-injection of 14-3-3? cRNA increased TASK-1 current in Xenopus oocytes by about 70 %. After co-transfection in HEK293 cells, TASK-1 and 14-3-3? (but not TASK-1?C5 and 14-3-3?) could be co-immunoprecipitated. Furthermore, TASK-1 and 14-3-3 could be co-immunoprecipitated in synaptic membrane extracts and postsynaptic density membranes. Our findings suggest that interaction of 14-3-3 with TASK-1 or TASK-3 may promote the trafficking of the channels to the surface membrane. PMID:12433946

  11. Channel catfish (Ictalurus punctatus) muscle protein isolate performance processed under different acid and alkali pH values.

    PubMed

    Davenport, M P; Kristinsson, H G

    2011-04-01

    Channel catfish (Ictalurus punctatus) muscle was subjected to 6 protein extraction and precipitation techniques using acid solubilization (pH 2.0, 2.5, and 3.0) or alkaline solubilization (pH 10.5, 11.0, 11.5) followed by precipitation at pH 5.5. The catfish protein isolate was compared with ground defatted white muscle. Alkali-processed catfish showed increased gel rigidity, gel strength, and gel flexibility compared to acid-processed catfish, which exhibited inconsistent functional performance, increasing and decreasing gel rigidity, gel strength, and gel flexibility. The gel rigidity (G') at pH 3.0 in the absence of salt had the highest?G' of the acid treatments and was not significantly different from the alkaline-treated catfish muscle (P>0.05). However in the presence of added salt pH treatment it had the lowest?G' and was different from alkaline treatments (P<0.05) during break force testing. These results show that pH-shift processing of channel catfish muscle provides highly functional isolates with a potentially broad range of applications. This range of applications is possible due to the modification of the textural properties of catfish muscle protein produced using different acidic or alkaline pH solubility treatments. PMID:21535823

  12. Small Heat Shock Protein  A-crystallin Regulates Epithelial Sodium Channel Expression

    Microsoft Academic Search

    Ossama B. Kashlan; Gunhild M. Mueller; Mohammad Z. Qamar; Paul A. Poland; Annette Ahner; Ronald C. Rubenstein; Rebecca P. Hughey; Jeffrey L. Brodsky; Thomas R. Kleyman

    2007-01-01

    Integral membrane proteins are synthesized on the cytoplas- mic face of the endoplasmic reticulum (ER). After being trans- located or inserted into the ER, they fold and undergo post- translational modifications. Within the ER, proteins are also subjected to quality control checkpoints, during which mis- folded proteins may be degraded by proteasomes via a process known as ER-associated degradation. Molecular

  13. Hydration and protein folding in water and in reverse micelles: compressibility and volume changes.

    PubMed Central

    Valdez, D; Le Huérou, J Y; Gindre, M; Urbach, W; Waks, M

    2001-01-01

    The partial specific volume and adiabatic compressibility of proteins reflect the hydration properties of the solvent-exposed protein surface, as well as changes in conformational states. Reverse micelles, or water-in-oil microemulsions, are protein-sized, optically-clear microassemblies in which hydration can be experimentally controlled. We explore, by densimetry and ultrasound velocimetry, three basic proteins: cytochrome c, lysozyme, and myelin basic protein in reverse micelles made of sodium bis (2-ethylhexyl) sulfosuccinate, water, and isooctane and in aqueous solvents. For comparison, we use beta-lactoglobulin (pI = 5.1) as a reference protein. We examine the partial specific volume and adiabatic compressibility of the proteins at increasing levels of micellar hydration. For the lowest water content compatible with complete solubilization, all proteins display their highest compressibility values, independent of their amino acid sequence and charge. These values lie within the range of empirical intrinsic protein compressibility estimates. In addition, we obtain volumetric data for the transition of myelin basic protein from its initially unfolded state in water free of denaturants, to a folded, compact conformation within the water-controlled microenvironment of reverse micelles. These results disclose yet another aspect of the protein structural properties observed in membrane-mimetic molecular assemblies. PMID:11371450

  14. Dual Activation of a Sex Pheromone-Dependent Ion Channel from Insect Olfactory Dendrites by Protein Kinase C Activators and Cyclic GMP

    NASA Astrophysics Data System (ADS)

    Zufall, Frank; Hatt, Hanns

    1991-10-01

    Olfactory transduction is thought to take place in the outer dendritic membrane of insect olfactory receptor neurons. Here we show that the outer dendritic plasma membrane of silkmoth olfactory receptor neurons seems to be exclusively equipped with a specific ion channel activated by low concentrations of the species-specific sex pheromone component. This so-called AC_1 channel has a conductance of 56 pS and is nonselectively permeable to cations. The AC_1 channel can be activated from the intracellular side by protein kinase C activators such as diacylglycerol and phorbolester and by cGMP but not by Ca2+, inositol 1,4,5-trisphosphate, or cAMP. Our results imply that phosphorylation of this ion channel by protein kinase C could be the crucial step in channel opening by sex pheromones.

  15. Influence of protein type on oxidation and digestibility of fish oil-in-water emulsions: gliadin, caseinate, and whey protein.

    PubMed

    Qiu, Chaoying; Zhao, Mouming; Decker, Eric Andrew; McClements, David Julian

    2015-05-15

    The influence of three surface-active proteins on the oxidative stability and lipase digestibility of emulsified ?-3 oils was examined: deamidated wheat gliadin (gliadin); sodium caseinate (CN); whey protein isolate (WPI). Gliadin and WPI were more effective at inhibiting lipid oxidation (hydroperoxides and TBARS) of fish oil-in-water emulsions than CN. Protein oxidation during storage was determined by measuring the loss of tryptophan fluorescence. The CN-emulsions exhibited the highest loss of tryptophan fluorescence during aging, as well as the highest amount of lipid oxidation. Potential reasons for the differences in oxidative stability of the emulsions with different proteins include differences in interfacial film thickness, protein chelating ability, and antioxidant amino acids profiles. During in vitro digestion, gliadin-stabilized emulsions had the lowest digestion rate of the three proteins. These results have important implications for using proteins to fabricate emulsion-based delivery systems for ?-3 oils. PMID:25577077

  16. Modes of sediment transport in channelized water flows with ramifications to the erosion of the Martian outflow channels

    NASA Technical Reports Server (NTRS)

    Komar, P. D.

    1980-01-01

    The paper discusses application to Martian water flows of the criteria that determine which grain-size ranges are transported as bed load, suspension, and wash load. The results show nearly all sand-sized material and finer would have been transported as wash load and that basalt pebbles and even cobbles could have been transported at rapid rates of suspension. An analysis of the threshold of sediment motion on Mars further indicates that the flows would have been highly competent, the larger flows having been able to transport boulder-sized material. Comparisons with terrestrial rivers which transport hyperconcentration levels of sediments suggest that the Martian water flows could have achieved sediment concentrations up to 70% in weight. Although it is possible that flows could have picked up enough sediment to convert to pseudolaminar mud flows, they probably remained at hyperconcentration levels and fully turbulent in flow character.

  17. Hierarchic Theory of Condensed Matter: Role of water in protein dynamics, function and cancer emergency

    E-print Network

    Alex Kaivarainen

    2000-03-29

    1. Role of inter-domain water clusters in large-scale dynamics of proteins; 2. Description of large-scale dynamics of proteins based on generalized Stokes-Einstein and Eyring-Polany equation; 3. Dynamic model of protein-ligand complexes formation; 4. The life-time of quasiparticles and frequencies of their excitation; 5. Mesoscopic mechanism of enzyme catalysis; 6. The mechanism of ATP hydrolysis energy utilization in muscle contraction and protein polymerization; 7. Water activity as a regulative factor in the intra- and inter-cell processes; 8. Water and cancer.

  18. Assembly of water-soluble chlorophyll-binding proteins with native hydrophobic chlorophylls in water-in-oil emulsions.

    PubMed

    Bednarczyk, Dominika; Takahashi, Shigekazu; Satoh, Hiroyuki; Noy, Dror

    2015-03-01

    The challenges involved in studying cofactor binding and assembly, as well as energy- and electron transfer mechanisms in the large and elaborate transmembrane protein complexes of photosynthesis and respiration have prompted considerable interest in constructing simplified model systems based on their water-soluble protein analogs. Such analogs are also promising templates and building blocks for artificial bioinspired energy conversion systems. Yet, development is limited by the challenge of introducing the essential cofactors of natural proteins that are highly water-insoluble into the water-soluble protein analogs. Here we introduce a new efficient method based on water-in-oil emulsions for overcoming this challenge. We demonstrate the effectiveness of the method in the assembly of native chlorophylls with four recombinant variants of the water-soluble chlorophyll-binding protein of Brassicaceae plants. We use the method to gain new insights into the protein-chlorophyll assembly process, and demonstrate its potential as a fast screening system for developing novel chlorophyll-protein complexes. PMID:25511505

  19. Differential Detergent Extraction of Mycobacterium marinum Cell Envelope Proteins Identifies an Extensively Modified Threonine-Rich Outer Membrane Protein with Channel Activity

    PubMed Central

    van der Woude, Aniek D.; Mahendran, Kozhinjampara R.; Ummels, Roy; Piersma, Sander R.; Pham, Thang V.; Jiménez, Connie R.; de Punder, Karin; van der Wel, Nicole N.; Winterhalter, Mathias; Luirink, Joen; Bitter, Wilbert

    2013-01-01

    A striking characteristic of mycobacteria is the presence of an unusual outer membrane which forms a thick permeability barrier and provides resistance to many antibiotics. Although specialized proteins must reside in this layer, only few mycolate outer membrane (MOM) proteins have been identified to date. Their discovery is complicated by difficulties in obtaining good separation of mycobacterial inner and outer membranes. During our efforts to identify novel mycobacterial outer membrane proteins (MOMPs), we discovered that we can enrich for MOMPs using differential solubilization of mycobacterial cell envelopes. Subsequently, these different fractions were analyzed by nano liquid chromatography-tandem mass spectrometry (nanoLC-MS/MS). This proteomic analysis confirmed that our marker proteins for inner membrane and MOM were found in their expected fractions and revealed a few interesting candidate MOMPs. A number of these putative MOMPs were further analyzed for their expression and localization in the cell envelope. One identified MOMP, MMAR_0617 of Mycobacterium marinum, was purified and demonstrated to form a large oligomeric complex. Importantly, this protein showed a clear single-channel conductance of 0.8 ± 0.1 ns upon reconstitution into artificial planar lipid bilayers. The most surprising feature of MMAR_0617 is a long C-terminal threonine-rich domain with extensive modifications. In summary, we have identified a novel mycobacterial outer membrane porin with unusual properties. PMID:23457249

  20. Src family protein tyrosine kinase (PTK) modulates the effect of SGK1 and WNK4 on ROMK channels.

    PubMed

    Yue, Peng; Lin, Dao-Hong; Pan, Chun-Yang; Leng, Qiang; Giebisch, Gerhard; Lifton, Richard P; Wang, Wen-Hui

    2009-09-01

    WNK4 (with no lysine kinase 4) inhibits ROMK channel activity in the distal nephron by stimulating clathrin-dependent endocytosis, an effect attenuated by SGK1 (serum-glucocorticoids-induced kinase)-mediated phosphorylation. It has been suggested that increased ROMK activity because of SGK1-mediated inhibition of WNK4 plays a role in promoting renal K secretion in response to elevated serum K or high K (HK) intake. In contrast, intravascular volume depletion also increases SGK1 activity but fails to stimulate ROMK channels and K secretion. Because HK intake decreases Src family protein tyrosine kinase (PTK) activity an inhibitor of ROMK channels, it is possible that Src family PTK may modulate the effects of SGK1 on WNK4. Here, we show that c-Src prevents SGK1 from attenuating WNK4's inhibition of ROMK activity. This effect of c-Src was WNK4-dependent because c-Src had no effect on ROMK harboring mutation at the site of c-Src phosphorylation (R1Y337A) in the absence of WNK4. Moreover, expression c-Src diminished the SGK1-mediated increase in serine phosphorylation of WNK4, suggesting that c-Src enhances WNK4-mediated inhibition of ROMK channels by suppressing the SGK1-induced phosphorylation. This notion is also supported by the observation that c-Src was not able to modulate the interaction between SGK1 and WNK4 mutants (WNK4(S1169A) or WNK4(S1169D)) in which an SGK1-phosphorylation site (serine 1169) was mutated by alanine or aspartate. We conclude that c-Src inhibits SGK1-mediated phosphorylation hereby restoring the WNK4-mediated inhibition of ROMK channels thus suppressing K secretion. PMID:19706464