DOE Office of Scientific and Technical Information (OSTI.GOV)

Yuan, Meichun; Department of Physiology, Hubei University of Medicine, Shiyan; Li, Jianjie

Mast cells play a key role in the pathogenesis of asthma and are a promising target for therapeutic intervention in asthma. This study investigated the effects of polydatin (PD), a resveratrol glucoside, on mast cell degranulation upon cross-linking of the high-affinity IgE receptors (FcεRI), as well as the anti-allergic activity of PD in vivo. Herein, we demonstrated that PD treatment for 30 min suppressed FcεRI-mediated mast cell degranulation in a dose-dependent manner. Concomitantly, PD significantly decreased FcεRI-mediated Ca{sup 2+} increase in mast cells. The suppressive effects of PD on FcεRI-mediated Ca{sup 2+} increase were largely inhibited by using LaCl{sub 3}more » to block the Ca{sup 2+} release-activated Ca{sup 2+} channels (CRACs). Furthermore, PD significantly inhibited Ca{sup 2+} entry through CRACs evoked by thapsigargin (TG). Knocking down protein expression of Orai1, the pore-forming subunit of CRACs, significantly decreased PD suppression of FcεRI-induced intracellular Ca{sup 2+} influx and mast cell degranulation. In a mouse model of mast cell-dependent passive cutaneous anaphylaxis (PCA), in vivo PD administration suppressed mast cell degranulation and inhibited anaphylaxis. Taken together, our data indicate that PD stabilizes mast cells by suppressing FcεRI-induced Ca{sup 2+} mobilization mainly through inhibiting Ca{sup 2+} entry via CRACs, thus exerting a protective effect against PCA. -- Highlights: ► Polydatin can prevent the pathogenesis of passive cutaneous anaphylaxis in mice. ► Polydatin stabilizes mast cells by decreasing FcεRI-mediated degranulation. ► Polydatin suppresses Ca{sup 2+} entry through CRAC channels in mast cells.« less

Complex role of STIM1 in the activation of store-independent Orai1/3 channels

PubMed Central

Zhang, Wei; González-Cobos, José C.; Jardin, Isaac; Romanin, Christoph; Matrougui, Khalid

2014-01-01

Orai proteins contribute to Ca2+ entry into cells through both store-dependent, Ca2+ release–activated Ca2+ (CRAC) channels (Orai1) and store-independent, arachidonic acid (AA)-regulated Ca2+ (ARC) and leukotriene C4 (LTC4)-regulated Ca2+ (LRC) channels (Orai1/3 heteromultimers). Although activated by fundamentally different mechanisms, CRAC channels, like ARC and LRC channels, require stromal interacting molecule 1 (STIM1). The role of endoplasmic reticulum–resident STIM1 (ER-STIM1) in CRAC channel activation is widely accepted. Although ER-STIM1 is necessary and sufficient for LRC channel activation in vascular smooth muscle cells (VSMCs), the minor pool of STIM1 located at the plasma membrane (PM-STIM1) is necessary for ARC channel activation in HEK293 cells. To determine whether ARC and LRC conductances are mediated by the same or different populations of STIM1, Orai1, and Orai3 proteins, we used whole-cell and perforated patch-clamp recording to compare AA- and LTC4-activated currents in VSMCs and HEK293 cells. We found that both cell types show indistinguishable nonadditive LTC4- and AA-activated currents that require both Orai1 and Orai3, suggesting that both conductances are mediated by the same channel. Experiments using a nonmetabolizable form of AA or an inhibitor of 5-lipooxygenase suggested that ARC and LRC currents in both cell types could be activated by either LTC4 or AA, with LTC4 being more potent. Although PM-STIM1 was required for current activation by LTC4 and AA under whole-cell patch-clamp recordings in both cell types, ER-STIM1 was sufficient with perforated patch recordings. These results demonstrate that ARC and LRC currents are mediated by the same cellular populations of STIM1, Orai1, and Orai3, and suggest a complex role for both ER-STIM1 and PM-STIM1 in regulating these store-independent Orai1/3 channels. PMID:24567509

Relevance of CARC and CRAC Cholesterol-Recognition Motifs in the Nicotinic Acetylcholine Receptor and Other Membrane-Bound Receptors.

PubMed

Di Scala, Coralie; Baier, Carlos J; Evans, Luke S; Williamson, Philip T F; Fantini, Jacques; Barrantes, Francisco J

2017-01-01

Cholesterol is a ubiquitous neutral lipid, which finely tunes the activity of a wide range of membrane proteins, including neurotransmitter and hormone receptors and ion channels. Given the scarcity of available X-ray crystallographic structures and the even fewer in which cholesterol sites have been directly visualized, application of in silico computational methods remains a valid alternative for the detection and thermodynamic characterization of cholesterol-specific sites in functionally important membrane proteins. The membrane-embedded segments of the paradigm neurotransmitter receptor for acetylcholine display a series of cholesterol consensus domains (which we have coined "CARC"). The CARC motif exhibits a preference for the outer membrane leaflet and its mirror motif, CRAC, for the inner one. Some membrane proteins possess the double CARC-CRAC sequences within the same transmembrane domain. In addition to in silico molecular modeling, the affinity, concentration dependence, and specificity of the cholesterol-recognition motif-protein interaction have recently found experimental validation in other biophysical approaches like monolayer techniques and nuclear magnetic resonance spectroscopy. From the combined studies, it becomes apparent that the CARC motif is now more firmly established as a high-affinity cholesterol-binding domain for membrane-bound receptors and remarkably conserved along phylogenetic evolution. © 2017 Elsevier Inc. All rights reserved.

Store-operated Ca2+ Entry Modulates the Expression of Enamel Genes.

PubMed

Nurbaeva, M K; Eckstein, M; Snead, M L; Feske, S; Lacruz, R S

2015-10-01

Dental enamel formation is an intricate process tightly regulated by ameloblast cells. The correct spatiotemporal patterning of enamel matrix protein (EMP) expression is fundamental to orchestrate the formation of enamel crystals, which depend on a robust supply of Ca2+. In the extracellular milieu, Ca2+ -EMP interactions occur at different levels. Despite its recognized role in enamel development, the molecular machinery involved in Ca2+ homeostasis in ameloblasts remains poorly understood. A common mechanism for Ca2+ influx is store-operated Ca2+ entry (SOCE). We evaluated the possibility that Ca2+ influx in enamel cells might be mediated by SOCE and the Ca2+ release-activated Ca2+ (CRAC) channel, the prototypical SOCE channel. Using ameloblast-like LS8 cells, we demonstrate that these cells express Ca2+ -handling molecules and mediate Ca2+ influx through SOCE. As a rise in the cytosolic Ca2+ concentration is a versatile signal that can modulate gene expression, we assessed whether SOCE in enamel cells had any effect on the expression of EMPs. Our results demonstrate that stimulating LS8 cells or murine primary enamel organ cells with thapsigargin to activate SOCE leads to increased expression of Amelx, Ambn, Enam, Mmp20. This effect is reversed when cells are treated with a CRAC channel inhibitor. These data indicate that Ca2+ influx in LS8 cells and enamel organ cells is mediated by CRAC channels and that Ca2+ signals enhance the expression of EMPs. Ca2+ plays an important role not only in mineralizing dental enamel but also in regulating the expression of EMPs. © International & American Associations for Dental Research 2015.

Identification of key amino acid residues responsible for internal and external pH sensitivity of Orai1/STIM1 channels.

PubMed

Tsujikawa, Hiroto; Yu, Albert S; Xie, Jia; Yue, Zhichao; Yang, Wenzhong; He, Yanlin; Yue, Lixia

2015-11-18

Changes of intracellular and extracellular pH are involved in a variety of physiological and pathological processes, in which regulation of the Ca(2+) release activated Ca(2+) channel (I CRAC) by pH has been implicated. Ca(2+) entry mediated by I CRAC has been shown to be regulated by acidic or alkaline pH. Whereas several amino acid residues have been shown to contribute to extracellular pH (pHo) sensitivity, the molecular mechanism for intracellular pH (pHi) sensitivity of Orai1/STIM1 is not fully understood. By investigating a series of mutations, we find that the previously identified residue E106 is responsible for pHo sensitivity when Ca(2+) is the charge carrier. Unexpectedly, we identify that the residue E190 is responsible for pHo sensitivity when Na(+) is the charge carrier. Furthermore, the intracellular mutant H155F markedly diminishes the response to acidic and alkaline pHi, suggesting that H155 is responsible for pHi sensitivity of Orai1/STIM1. Our results indicate that, whereas H155 is the intracellular pH sensor of Orai1/STIM1, the molecular mechanism of external pH sensitivity varies depending on the permeant cations. As changes of pH are involved in various physiological/pathological functions, Orai/STIM channels may be an important mediator for various physiological and pathological processes associated with acidosis and alkalinization.

The calcium feedback loop and T cell activation: how cytoskeleton networks control intracellular calcium flux.

PubMed

Joseph, Noah; Reicher, Barak; Barda-Saad, Mira

2014-02-01

During T cell activation, the engagement of a T cell with an antigen-presenting cell (APC) results in rapid cytoskeletal rearrangements and a dramatic increase of intracellular calcium (Ca(2+)) concentration, downstream to T cell antigen receptor (TCR) ligation. These events facilitate the organization of an immunological synapse (IS), which supports the redistribution of receptors, signaling molecules and organelles towards the T cell-APC interface to induce downstream signaling events, ultimately supporting T cell effector functions. Thus, Ca(2+) signaling and cytoskeleton rearrangements are essential for T cell activation and T cell-dependent immune response. Rapid release of Ca(2+) from intracellular stores, e.g. the endoplasmic reticulum (ER), triggers the opening of Ca(2+) release-activated Ca(2+) (CRAC) channels, residing in the plasma membrane. These channels facilitate a sustained influx of extracellular Ca(2+) across the plasma membrane in a process termed store-operated Ca(2+) entry (SOCE). Because CRAC channels are themselves inhibited by Ca(2+) ions, additional factors are suggested to enable the sustained Ca(2+) influx required for T cell function. Among these factors, we focus here on the contribution of the actin and microtubule cytoskeleton. The TCR-mediated increase in intracellular Ca(2+) evokes a rapid cytoskeleton-dependent polarization, which involves actin cytoskeleton rearrangements and microtubule-organizing center (MTOC) reorientation. Here, we review the molecular mechanisms of Ca(2+) flux and cytoskeletal rearrangements, and further describe the way by which the cytoskeletal networks feedback to Ca(2+) signaling by controlling the spatial and temporal distribution of Ca(2+) sources and sinks, modulating TCR-dependent Ca(2+) signals, which are required for an appropriate T cell response. 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. © 2013.

CRAC2 model description

DOE Office of Scientific and Technical Information (OSTI.GOV)

Ritchie, L.T.; Alpert, D.J.; Burke, R.P.

1984-03-01

The CRAC2 computer code is a revised version of CRAC (Calculation of Reactor Accident Consequences) which was developed for the Reactor Safety Study. This document provides an overview of the CRAC2 code and a description of each of the models used. Significant improvements incorporated into CRAC2 include an improved weather sequence sampling technique, a new evacuation model, and new output capabilities. In addition, refinements have been made to the atmospheric transport and deposition model. Details of the modeling differences between CRAC2 and CRAC are emphasized in the model descriptions.

Store-operated Ca2+ entry controls ameloblast cell function and enamel development

PubMed Central

Eckstein, Miriam; Vaeth, Martin; Fornai, Cinzia; Vinu, Manikandan; Bromage, Timothy G.; Nurbaeva, Meerim K.; Sorge, Jessica L.; Coelho, Paulo G.; Idaghdour, Youssef; Feske, Stefan; Lacruz, Rodrigo S.

2017-01-01

Loss-of-function mutations in stromal interaction molecule 1 (STIM1) impair the activation of Ca2+ release–activated Ca2+ (CRAC) channels and store-operated Ca2+ entry (SOCE), resulting in a disease syndrome called CRAC channelopathy that is characterized by severe dental enamel defects. The cause of these enamel defects has remained unclear given a lack of animal models. We generated Stim1/2K14cre mice to delete STIM1 and its homolog STIM2 in enamel cells. These mice showed impaired SOCE in enamel cells. Enamel in Stim1/2K14cre mice was hypomineralized with decreased Ca content, mechanically weak, and thinner. The morphology of SOCE-deficient ameloblasts was altered, showing loss of the typical ruffled border, resulting in mislocalized mitochondria. Global gene expression analysis of SOCE-deficient ameloblasts revealed strong dysregulation of several pathways. ER stress genes associated with the unfolded protein response were increased in Stim1/2-deficient cells, whereas the expression of components of the glutathione system were decreased. Consistent with increased oxidative stress, we found increased ROS production, decreased mitochondrial function, and abnormal mitochondrial morphology in ameloblasts of Stim1/2K14cre mice. Collectively, these data show that loss of SOCE in enamel cells has substantial detrimental effects on gene expression, cell function, and the mineralization of dental enamel. PMID:28352661

Ca(2+) signals mediated by bradykinin type 2 receptors in normal pancreatic stellate cells can be inhibited by specific Ca(2+) channel blockade.

PubMed

Gryshchenko, Oleksiy; Gerasimenko, Julia V; Gerasimenko, Oleg V; Petersen, Ole H

2016-01-15

Bradykinin may play a role in the autodigestive disease acute pancreatitis, but little is known about its pancreatic actions. In this study, we have investigated bradykinin-elicited Ca(2+) signal generation in normal mouse pancreatic lobules. We found complete separation of Ca(2+) signalling between pancreatic acinar (PACs) and stellate cells (PSCs). Pathophysiologically relevant bradykinin concentrations consistently evoked Ca(2+) signals, via B2 receptors, in PSCs but never in neighbouring PACs, whereas cholecystokinin, consistently evoking Ca(2+) signals in PACs, never elicited Ca(2+) signals in PSCs. The bradykinin-elicited Ca(2+) signals were due to initial Ca(2+) release from inositol trisphosphate-sensitive stores followed by Ca(2+) entry through Ca(2+) release-activated channels (CRACs). The Ca(2+) entry phase was effectively inhibited by a CRAC blocker. B2 receptor blockade reduced the extent of PAC necrosis evoked by pancreatitis-promoting agents and we therefore conclude that bradykinin plays a role in acute pancreatitis via specific actions on PSCs. Normal pancreatic stellate cells (PSCs) are regarded as quiescent, only to become activated in chronic pancreatitis and pancreatic cancer. However, we now report that these cells in their normal microenvironment are far from quiescent, but are capable of generating substantial Ca(2+) signals. We have compared Ca(2+) signalling in PSCs and their better studied neighbouring acinar cells (PACs) and found complete separation of Ca(2+) signalling in even closely neighbouring PACs and PSCs. Bradykinin (BK), at concentrations corresponding to the slightly elevated plasma BK levels that have been shown to occur in the auto-digestive disease acute pancreatitis in vivo, consistently elicited substantial Ca(2+) signals in PSCs, but never in neighbouring PACs, whereas the physiological PAC stimulant cholecystokinin failed to evoke Ca(2+) signals in PSCs. The BK-induced Ca(2+) signals were mediated by B2 receptors and B2 receptor blockade protected against PAC necrosis evoked by agents causing acute pancreatitis. The initial Ca(2+) rise in PSCs was due to inositol trisphosphate receptor-mediated release from internal stores, whereas the sustained phase depended on external Ca(2+) entry through Ca(2+) release-activated Ca(2+) (CRAC) channels. CRAC channel inhibitors, which have been shown to protect PACs against damage caused by agents inducing pancreatitis, therefore also inhibit Ca(2+) signal generation in PSCs and this may be helpful in treating acute pancreatitis. © 2015 The Authors. The Journal of Physiology published by John Wiley & Sons Ltd on behalf of The Physiological Society.

Mutations of the central tyrosines of putative cholesterol recognition amino acid consensus (CRAC) sequences modify folding, activity, and sterol-sensing of the human ABCG2 multidrug transporter.

PubMed

Gál, Zita; Hegedüs, Csilla; Szakács, Gergely; Váradi, András; Sarkadi, Balázs; Özvegy-Laczka, Csilla

2015-02-01

Human ABCG2 is a plasma membrane glycoprotein causing multidrug resistance in cancer. Membrane cholesterol and bile acids are efficient regulators of ABCG2 function, while the molecular nature of the sterol-sensing sites has not been elucidated. The cholesterol recognition amino acid consensus (CRAC, L/V-(X)(1-5)-Y-(X)(1-5)-R/K) sequence is one of the conserved motifs involved in cholesterol binding in several proteins. We have identified five potential CRAC motifs in the transmembrane domain of the human ABCG2 protein. In order to define their roles in sterol-sensing, the central tyrosines of these CRACs (Y413, 459, 469, 570 and 645) were mutated to S or F and the mutants were expressed both in insect and mammalian cells. We found that mutation in Y459 prevented protein expression; the Y469S and Y645S mutants lost their activity; while the Y570S, Y469F, and Y645F mutants retained function as well as cholesterol and bile acid sensitivity. We found that in the case of the Y413S mutant, drug transport was efficient, while modulation of the ATPase activity by cholesterol and bile acids was significantly altered. We suggest that the Y413 residue within a putative CRAC motif has a role in sterol-sensing and the ATPase/drug transport coupling in the ABCG2 multidrug transporter. Copyright © 2014. Published by Elsevier B.V.

Roles of Ca(v) channels and AHNAK1 in T cells: the beauty and the beast.

PubMed

Matza, Didi; Flavell, Richard A

2009-09-01

T lymphocytes require Ca2+ entry though the plasma membrane for their activation and function. Recently, several routes for Ca2+ entry through the T-cell plasma membrane after activation have been described. These include calcium release-activated channels (CRAC), transient receptor potential (TRP) channels, and inositol-1,4,5-trisphosphate receptors (IP3Rs). Herein we review the emergence of a fourth new route for Ca2+ entry, composed of Ca(v) channels (also known as L-type voltage-gated calcium channels) and the scaffold protein AHNAK1 (AHNAK/desmoyokin). Both helper (CD4+) and killer (CD8+) T cells express high levels of Ca(v)1 alpha1 subunits (alpha1S, alpha1C, alpha1D, and alpha1F) and AHNAK1 after their differentiation and require these molecules for Ca2+ entry during an immune response. In this article, we describe the observations and open questions that ultimately suggest the involvement of multiple consecutive routes for Ca2+ entry into lymphocytes, one of which may be mediated by Ca(v) channels and AHNAK1.

An aromatic amino acid in the coiled-coil 1 domain plays a crucial role in the auto-inhibitory mechanism of STIM1.

PubMed

Yu, Junwei; Zhang, Haining; Zhang, Mingshu; Deng, Yongqiang; Wang, Huiyu; Lu, Jingze; Xu, Tao; Xu, Pingyong

2013-09-15

STIM1 (stromal interaction molecule 1) is one of the key elements that mediate store-operated Ca²⁺ entry via CRAC (Ca²⁺- release-activated Ca²⁺) channels in immune and non-excitable cells. Under physiological conditions, the intramolecular auto-inhibitions in STIM1 C- and STIM1 N-termini play essential roles in keeping STIM1 in an inactive state. However, the auto-inhibitory mechanism of the STIM1 C-terminus is still unclear. In the present study, we first predicted a short inhibitory domain (residues 310-317) in human STIM1 that might determine the different localizations of human STIM1 from Caenorhabditis elegans STIM1 in resting cells. Next, we confirmed the prediction and further identified an aromatic amino acid residue, Tyr³¹⁶, that played a crucial role in maintaining STIM1 in a closed conformation in quiescent cells. Full-length STIM1-Y316A formed constitutive clusters near the plasma membrane and activated the CRAC channel in the resting state when co-expressed with Orai1. The introduction of a Y316A mutation caused the higher-order oligomerization of the in vitro purified STIM1 fragment containing both the auto-inhibitory domain and CAD(CRAC-activating domain).We propose that the Tyr³¹⁶ residue may be involved in the auto-inhibitory mechanism of the STIM1 C-terminus in the quiescent state. This inhibition could be achieved either by interacting with the CAD using hydrogen and/or hydrophobic bonds, or by an intermolecular interaction using repulsive forces, which maintained a dimeric STIM1.

Calcium-activated K(+) channel (K(Ca)3.1) activity during Ca(2+) store depletion and store-operated Ca(2+) entry in human macrophages.

PubMed

Gao, Ya-dong; Hanley, Peter J; Rinné, Susanne; Zuzarte, Marylou; Daut, Jurgen

2010-07-01

STIM1 'senses' decreases in endoplasmic reticular (ER) luminal Ca(2+) and induces store-operated Ca(2+) (SOC) entry through plasma membrane Orai channels. The Ca(2+)/calmodulin-activated K(+) channel K(Ca)3.1 (previously known as SK4) has been implicated as an 'amplifier' of the Ca(2+)-release activated Ca(2+) (CRAC) current, especially in T lymphocytes. We have previously shown that human macrophages express K(Ca)3.1, and here we used the whole-cell patch-clamp technique to investigate the activity of these channels during Ca(2+) store depletion and store-operated Ca(2+) influx. Using RT-PCR, we found that macrophages express the elementary CRAC channel components Orai1 and STIM1, as well as Orai2, Orai3 and STIM2, but not the putatively STIM1-activated channels TRPC1, TRPC3-7 or TRPV6. In whole-cell configuration, a robust Ca(2+)-induced outwardly rectifying K(+) current inhibited by clotrimazole and augmented by DC-EBIO could be detected, consistent with K(Ca)3.1 channel current (also known as intermediate-conductance IK1). Introduction of extracellular Ca(2+) following Ca(2+) store depletion via P2Y(2) receptors induced a robust charybdotoxin (CTX)- and 2-APB-sensitive outward K(+) current and hyperpolarization. We also found that SOC entry induced by thapsigargin treatment induced CTX-sensitive K(+) current in HEK293 cells transiently expressing K(Ca)3.1. Our data suggest that SOC and K(Ca)3.1 channels are tightly coupled, such that a small Ca(2+) influx current induces a much large K(Ca)3.1 channel current and hyperpolarization, providing the necessary electrochemical driving force for prolonged Ca(2+) signaling and store repletion. Copyright 2010 Elsevier Ltd. All rights reserved.

DOE Office of Scientific and Technical Information (OSTI.GOV)

White, J.E.; Roussin, R.W.; Gilpin, H.

A version of the CRAC2 computer code applicable for use in analyses of consequences and risks of reactor accidents in case work for environmental statements has been implemented for use on the Nuclear Regulatory Commission Data General MV/8000 computer system. Input preparation is facilitated through the use of an interactive computer program which operates on an IBM personal computer. The resulting CRAC2 input deck is transmitted to the MV/8000 by using an error-free file transfer mechanism. To facilitate the use of CRAC2 at NRC, relevant background material on input requirements and model descriptions has been extracted from four reports -more » ''Calculations of Reactor Accident Consequences,'' Version 2, NUREG/CR-2326 (SAND81-1994) and ''CRAC2 Model Descriptions,'' NUREG/CR-2552 (SAND82-0342), ''CRAC Calculations for Accident Sections of Environmental Statements, '' NUREG/CR-2901 (SAND82-1693), and ''Sensitivity and Uncertainty Studies of the CRAC2 Computer Code,'' NUREG/CR-4038 (ORNL-6114). When this background information is combined with instructions on the input processor, this report provides a self-contained guide for preparing CRAC2 input data with a specific orientation toward applications on the MV/8000. 8 refs., 11 figs., 10 tabs.« less

DOE Office of Scientific and Technical Information (OSTI.GOV)

Ritchie, L.T.; Johnson, J.D.; Blond, R.M.

The CRAC2 computer code is a revision of the Calculation of Reactor Accident Consequences computer code, CRAC, developed for the Reactor Safety Study. The CRAC2 computer code incorporates significant modeling improvements in the areas of weather sequence sampling and emergency response, and refinements to the plume rise, atmospheric dispersion, and wet deposition models. New output capabilities have also been added. This guide is to facilitate the informed and intelligent use of CRAC2. It includes descriptions of the input data, the output results, the file structures, control information, and five sample problems.

Characterization of long-chain acyl-CoA synthetases which stimulate secretion of fatty acids in green algae Chlamydomonas reinhardtii.

PubMed

Jia, Bin; Song, Yanzi; Wu, Min; Lin, Baicheng; Xiao, Kang; Hu, Zhangli; Huang, Ying

2016-01-01

Microalgae biofuel has become the most promising renewable energy over the past few years. But limitations still exist because of its high cost. Although, efforts have been made in enhancement of lipid productivity, the major cost problem in harvesting and oil extraction is still intractable. Thus, the idea of fatty acids (FAs) secretion which can massively facilitate algae harvesting and oil extraction was investigated here. The cDNAs of two long-chain acyl-CoA synthetases (LACSs) genes were cloned from Chlamydomonas reinhardtii and named as cracs1 and cracs2. They showed different substrate adaptation in the yeast complementation experiments. Cracs2 could utilize FAs C12:0, C14:0, C16:0, C18:0, C16:1 and C18:1, while crac1 could only utilize substrate C14:0, C16:1 and C18:1. Knockdown of cracs1 and cracs2 in C. reinhardtii resulted in accumulation of intracellular lipids. The total intracellular lipids contents of transgenic algae q-15 (knockdown of cracs1) and p-13 (knockdown of cracs2) were 45 and 55 %, respectively higher than that of cc849. Furthermore, FAs secretion was discovered in both transgenic algae. Secreted FAs can reach 8.19 and 9.66 mg/10(9) cells in q-15 and p-13, respectively. These results demonstrated the possibility of FAs secretion by microalgae and may give a new strategy of low-cost oil extraction. According to our findings, we proposed that FAs secretion may also be achieved in other species besides Chlamydomonas reinhardtii by knocking-down cracs genes, which may promote the future industrial application of microalgae biofuels.

The clinician rating of adult communication (CRAC): a clinician's guide to the assessment of interpersonal communication skill.

PubMed

Basco, M R; Birchler, G R; Kalal, B; Talbott, R; Slater, M A

1991-05-01

This paper reports the results of an initial investigation of the psychometric properties of a new clinical marital communication assessment instrument, the Clinician Rating of Adult Communication (CRAC). The sample consisted of 36 marital communication samples from both maritally satisfied and distressed couples. Reliability results indicated that the CRAC demonstrated high levels of internal consistency, test-retest reliability, and interrater agreement. Support for the validity of the CRAC was found in its correspondence with a marital interaction coding system, its relationship to ratings of marital satisfaction, and its concordance with couples' perceptions of their conflict management behavior. Overall, these findings support the conclusion that the CRAC may provide a useful addition to the measurement armamentarium of the marital clinician and researcher.

Molecular mechanisms of protein-cholesterol interactions in plasma membranes: Functional distinction between topological (tilted) and consensus (CARC/CRAC) domains.

PubMed

Fantini, Jacques; Di Scala, Coralie; Baier, Carlos J; Barrantes, Francisco J

2016-09-01

The molecular mechanisms that control the multiple possible modes of protein association with membrane cholesterol are remarkably convergent. These mechanisms, which include hydrogen bonding, CH-π stacking and dispersion forces, are used by a wide variety of extracellular proteins (e.g. microbial or amyloid) and membrane receptors. Virus fusion peptides penetrate the membrane of host cells with a tilted orientation that is compatible with a transient interaction with cholesterol; this tilted orientation is also characteristic of the process of insertion of amyloid proteins that subsequently form oligomeric pores in the plasma membrane of brain cells. Membrane receptors that are associated with cholesterol generally display linear consensus binding motifs (CARC and CRAC) characterized by a triad of basic (Lys/Arg), aromatic (Tyr/phe) and aliphatic (Leu/Val) amino acid residues. In some cases, the presence of both CARC and CRAC within the same membrane-spanning domain allows the simultaneous binding of two cholesterol molecules, one in each membrane leaflet. In this review the molecular basis and the functional significance of the different modes of protein-cholesterol interactions in plasma membranes are discussed. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

Phosphorylated Protein Kinase C (Zeta/Lambda) Expression in Colorectal Adenocarcinoma and Its Correlation with Clinicopathologic Characteristics and Prognosis.

PubMed

Yeo, Min-Kyung; Kim, Ji Yeon; Seong, In-Ock; Kim, Jin-Man; Kim, Kyung-Hee

2017-01-01

Background: Protein kinase C zeta/lambda (PKCζ/λ) is a family of protein kinase enzymes that contributes to cell proliferation and regulation, which are important for cancer development. PKCζ/λ has been shown to be an important regulator of tumorigenesis in intestinal cancer. The phosphorylated form of PKCζ/λ, p-PKCζ/λ, is suggested as an active form of PKCζ/λ. However, p-PKCζ/λ expression and its clinicopathologic implication in colorectal adenocarcinoma (CRAC) are unclear. Methods: Seven whole-tissue sections of malignant polyps containing both non-neoplastic and neoplastic mucosa, 11 adenomas with low-grade dysplasia, and 173 CRACs were examined by immunohistochemistry and western blot assay for p-PKCζ/λ protein expression. The association of p-PKCζ/λ expression with clinicopathologic factors including patient survival was studied. Results: In non-neoplastic epithelia, p-PKCζ/λ showed a weak cytoplasmic immunostaining. Adenomas and CRACs demonstrated up-regulated p-PKCζ/λ detection. Cytoplasmic p-PKCζ/λ expression was higher in CRAC than in adenoma. In CRACs, p-PKCζ/λ expression was inversely correlated with pathologic TNM stage (I-II versus III-IV) and poor differentiation. Statistical correlations between low expression of p-PKCζ/λ with shortened overall survival and disease-free survival were seen (p=0.004 and p=0.034, respectively). Conclusions: P-PKCζ/λ overexpression is implicated in tumorigenesis but down-regulation was a poor prognostic factor in CRAC.

The role of receptor topology in the vitamin D3 uptake and Ca{sup 2+} response systems

DOE Office of Scientific and Technical Information (OSTI.GOV)

Morrill, Gene A., E-mail: gene.morrill@einstein.yu.edu; Kostellow, Adele B.; Gupta, Raj K.

The steroid hormone, vitamin D{sub 3}, regulates gene transcription via at least two receptors and initiates putative rapid response systems at the plasma membrane. The vitamin D receptor (VDR) binds vitamin D{sub 3} and a second receptor, importin-4, imports the VDR-vitamin D{sub 3} complex into the nucleus via nuclear pores. Here we present evidence that the Homo sapiens VDR homodimer contains two transmembrane (TM) helices ({sup 327}E – D{sup 342}), two TM “half-helix” ({sup 264}K − N{sup 276}), one or more large channels, and 16 cholesterol binding (CRAC/CARC) domains. The importin-4 monomer exhibits 3 pore-lining regions ({sup 226}E – L{supmore » 251}; {sup 768}V – G{sup 783}; {sup 876}S – A{sup 891}) and 16 CRAC/CARC domains. The MEMSAT algorithm indicates that VDR and importin-4 may not be restricted to cytoplasm and nucleus. VDR homodimer TM helix-topology predicts insertion into the plasma membrane, with two 84 residue C-terminal regions being extracellular. Similarly, MEMSAT predicts importin-4 insertion into the plasma membrane with 226 residue extracellular N-terminal regions and 96 residue C-terminal extracellular loops; with the pore-lining regions contributing gated Ca{sup 2+} channels. The PoreWalker algorithm indicates that, of the 427 residues in each VDR monomer, 91 line the largest channel, including two vitamin D{sub 3} binding sites and residues from both the TM helix and “half-helix”. Cholesterol-binding domains also extend into the channel within the ligand binding region. Programmed changes in bound cholesterol may regulate both membrane Ca{sup 2+} response systems and vitamin D{sub 3} uptake as well as receptor internalization by the endomembrane system culminating in uptake of the vitamin D{sub 3}-VDR-importin-4 complex into the nucleus.« less

The anti-proliferative effect of cation channel blockers in T lymphocytes depends on the strength of mitogenic stimulation.

PubMed

Petho, Zoltan; Balajthy, Andras; Bartok, Adam; Bene, Krisztian; Somodi, Sandor; Szilagyi, Orsolya; Rajnavolgyi, Eva; Panyi, Gyorgy; Varga, Zoltan

2016-03-01

Ion channels are crucially important for the activation and proliferation of T lymphocytes, and thus, for the function of the immune system. Previous studies on the effects of channel blockers on T cell proliferation reported variable effectiveness due to differing experimental systems. Therefore our aim was to investigate how the strength of the mitogenic stimulation influences the efficiency of cation channel blockers in inhibiting activation, cytokine secretion and proliferation of T cells under standardized conditions. Human peripheral blood lymphocytes were activated via monoclonal antibodies targeting the TCR-CD3 complex and the co-stimulator CD28. We applied the blockers of Kv1.3 (Anuroctoxin), KCa3.1 (TRAM-34) and CRAC (2-Apb) channels of T cells either alone or in combination with rapamycin, the inhibitor of the mammalian target of rapamycin (mTOR). Five days after the stimulation ELISA and flow cytometric measurements were performed to determine IL-10 and IFN-γ secretion, cellular viability and proliferation. Our results showed that ion channel blockers and rapamycin inhibit IL-10 and IFN-γ secretion and cell division in a dose-dependent manner. Simultaneous application of the blockers for each channel along with rapamycin was the most effective, indicating synergy among the various activation pathways. Upon increasing the extent of mitogenic stimulation the anti-proliferative effect of the ion channel blockers diminished. This phenomenon may be important in understanding the fine-tuning of T cell activation. Copyright © 2016 European Federation of Immunological Societies. Published by Elsevier B.V. All rights reserved.

Synthesis of activated carbon-based amino phosphonic acid chelating resin and its adsorption properties for Ce(III) removal.

PubMed

Chen, Tao; Yan, Chunjie; Wang, Yixia; Tang, Conghai; Zhou, Sen; Zhao, Yuan; Ma, Rui; Duan, Ping

2015-01-01

This work aims to investigate the adsorption of Ce(III) onto chelating resin based on activated carbon (CRAC). The CRAC adsorbent was prepared from activated carbon (AC) followed by oxidation, silane coupling, ammoniation and phosphorylation, and characterized by Fourier transform-infrared spectrometry, nitrogen adsorption measurements and scanning electron microscopy. The effects of solution pH, adsorbent dosage and contact time were studied by batch technique. Langmuir and Freundlich isotherms were used to describe the adsorption behaviour of Ce(III) by CRAC, and the results showed that the adsorption behaviour well fitted the Langmuir model. The maximum uptake capacity (qmax) calculated by using the Langmuir equation for cerium ions was found to be 94.34 mg/g. A comparison of the kinetic models and the overall experimental data was best fitted with the type 1 pseudo second-order kinetic model. The calculated thermodynamic parameters (ΔG°, ΔH° and ΔS°) showed that the adsorption for Ce(III) was feasible, spontaneous and exothermic at 25-45 °C. The CRAC showed an excellent adsorptive selectivity towards Ce(III). Moreover, more than 82% of Ce(III) adsorbed onto CRAC could be desorbed with HCl and could be used several times.

Balloon test project: Cosmic Ray Antimatter Calorimeter (CRAC)

NASA Technical Reports Server (NTRS)

Christy, J. C.; Dhenain, G.; Goret, P.; Jorand, J.; Masse, P.; Mestreau, P.; Petrou, N.; Robin, A.

1984-01-01

Cosmic ray observations from balloon flights are discussed. The cosmic ray antimatter calorimeter (CRAC) experiment attempts to measure the flux of antimatter in the 200-600 Mev/m energy range and the isotopes of light elements between 600 and 1,000 Mev/m.

The WAVE2 complex regulates actin cytoskeletal reorganization and CRAC-mediated calcium entry during T cell activation.

PubMed

Nolz, Jeffrey C; Gomez, Timothy S; Zhu, Peimin; Li, Shuixing; Medeiros, Ricardo B; Shimizu, Yoji; Burkhardt, Janis K; Freedman, Bruce D; Billadeau, Daniel D

2006-01-10

The engagement of the T cell receptor results in actin cytoskeletal reorganization at the immune synapse (IS) and the triggering of biochemical signaling cascades leading to gene regulation and, ultimately, cellular activation. Recent studies have identified the WAVE family of proteins as critical mediators of Rac1-induced actin reorganization in other cell types. However, whether these proteins participate in actin reorganization at the IS or signaling pathways in T cells has not been investigated. By using a combination of biochemical, genetic, and cell biology approaches, we provide evidence that WAVE2 is recruited to the IS, is biochemically modified, and is required for actin reorganization and beta-integrin-mediated adhesion after TCR crosslinking. Moreover, we show that WAVE2 regulates calcium entry at a point distal to PLCgamma1 activation and IP(3)-mediated store release. These data reveal a role for WAVE2 in regulating multiple pathways leading to T cell activation. In particular, this work shows that WAVE2 is a key component of the actin regulatory machinery in T cells and that it also participates in linking intracellular calcium store depletion to calcium release-activated calcium (CRAC) channel activation.

Amphipathic alpha-helices and putative cholesterol binding domains of the influenza virus matrix M1 protein are crucial for virion structure organisation.

PubMed

Tsfasman, Tatyana; Kost, Vladimir; Markushin, Stanislav; Lotte, Vera; Koptiaeva, Irina; Bogacheva, Elena; Baratova, Ludmila; Radyukhin, Victor

2015-12-02

The influenza virus matrix M1 protein is an amphitropic membrane-associated protein, forming the matrix layer immediately beneath the virus raft membrane, thereby ensuring the proper structure of the influenza virion. The objective of this study was to elucidate M1 fine structural characteristics, which determine amphitropic properties and raft membrane activities of the protein, via 3D in silico modelling with subsequent mutational analysis. Computer simulations suggest the amphipathic nature of the M1 α-helices and the existence of putative cholesterol binding (CRAC) motifs on six amphipathic α-helices. Our finding explains for the first time many features of this protein, particularly the amphitropic properties and raft/cholesterol binding potential. To verify these results, we generated mutants of the A/WSN/33 strain via reverse genetics. The M1 mutations included F32Y in the CRAC of α-helix 2, W45Y and W45F in the CRAC of α-helix 3, Y100S in the CRAC of α-helix 6, M128A and M128S in the CRAC of α-helix 8 and a double L103I/L130I mutation in both a putative cholesterol consensus motif and the nuclear localisation signal. All mutations resulted in viruses with unusual filamentous morphology. Previous experimental data regarding the morphology of M1-gene mutant influenza viruses can now be explained in structural terms and are consistent with the pivotal role of the CRAC-domains and amphipathic α-helices in M1-lipid interactions. Copyright © 2015 Elsevier B.V. All rights reserved.

Customized rating assessment of climate suitability (CRACS): climate satisfaction evaluation based on subjective perception.

PubMed

Lin, Tzu-Ping; Yang, Shing-Ru; Matzarakis, Andreas

2015-12-01

Climate not only influences the behavior of people in urban environments but also affects people's schedules and travel plans. Therefore, providing people with appropriate long-term climate evaluation information is crucial. Therefore, we developed an innovative climate assessment system based on field investigations conducted in three cities located in Northern, Central, and Southern Taiwan. The field investigations included the questionnaire surveys and climate data collection. We first analyzed the relationship between the participants and climate parameters comprising physiologically equivalent temperature, air temperature, humidity, wind speed, solar radiation, cloud cover, and precipitation. Second, we established the neutral value, comfort range, and dissatisfied range of each parameter. Third, after verifying that the subjects' perception toward the climate parameters vary based on individual preferences, we developed the customized rating assessment of climate suitability (CRACS) approach, which featured functions such as personalized and default climate suitability information to be used by users exhibiting varying demands. Finally, we performed calculations using the climate conditions of two cities during the past 10 years to demonstrate the performance of the CRACS approach. The results can be used as a reference when planning activities in the city or when organizing future travel plans. The flexibility of the assessment system enables it to be adjusted for varying regions and usage characteristics.

Customized rating assessment of climate suitability (CRACS): climate satisfaction evaluation based on subjective perception

NASA Astrophysics Data System (ADS)

Lin, Tzu-Ping; Yang, Shing-Ru; Matzarakis, Andreas

2015-12-01

Climate not only influences the behavior of people in urban environments but also affects people's schedules and travel plans. Therefore, providing people with appropriate long-term climate evaluation information is crucial. Therefore, we developed an innovative climate assessment system based on field investigations conducted in three cities located in Northern, Central, and Southern Taiwan. The field investigations included the questionnaire surveys and climate data collection. We first analyzed the relationship between the participants and climate parameters comprising physiologically equivalent temperature, air temperature, humidity, wind speed, solar radiation, cloud cover, and precipitation. Second, we established the neutral value, comfort range, and dissatisfied range of each parameter. Third, after verifying that the subjects' perception toward the climate parameters vary based on individual preferences, we developed the customized rating assessment of climate suitability (CRACS) approach, which featured functions such as personalized and default climate suitability information to be used by users exhibiting varying demands. Finally, we performed calculations using the climate conditions of two cities during the past 10 years to demonstrate the performance of the CRACS approach. The results can be used as a reference when planning activities in the city or when organizing future travel plans. The flexibility of the assessment system enables it to be adjusted for varying regions and usage characteristics.

Modified use of a dynamic bite opener--treatment and prevention of trismus in a child with head and neck cancer: a case report.

PubMed

Dijkstra, P U; Kropmans, T J; Tamminga, R Y

1992-10-01

Trismus may be a complication arising during or after treatment of patients with head and neck cancer. Treatment of trismus is difficult, making prevention very important. To prevent and treat trismus in a patient with a nasopharyngeal tumor, the Contract-Relax-Antagonist-Contract (CRAC) technique was applied, with the aid of a custom-made dynamic bite opener (DBO). The CRAC technique in combination with the DBO, as a therapy/prevention program for trismus, is not referred to in the literature. The combination of CRAC and DBO appeared to be a gentle and effective method well tolerated by the patient.

The WAVE2 Complex Regulates Actin Cytoskeletal Reorganization and CRAC-Mediated Calcium Entry during T Cell Activation

PubMed Central

Nolz, Jeffrey C.; Gomez, Timothy S.; Zhu, Peimin; Li, Shuixing; Medeiros, Ricardo B.; Shimizu, Yoji; Burkhardt, Janis K.; Freedman, Bruce D.; Billadeau, Daniel D.

2007-01-01

Summary Background The engagement of the T cell receptor results in actin cytoskeletal reorganization at the immune synapse (IS) and the triggering of biochemical signaling cascades leading to gene regulation and, ultimately, cellular activation. Recent studies have identified the WAVE family of proteins as critical mediators of Rac1-induced actin reorganization in other cell types. However, whether these proteins participate in actin reorganization at the IS or signaling pathways in T cells has not been investigated. Results By using a combination of biochemical, genetic, and cell biology approaches, we provide evidence that WAVE2 is recruited to the IS, is biochemically modified, and is required for actin reorganization and β-integrin-mediated adhesion after TCR crosslinking. Moreover, we show that WAVE2 regulates calcium entry at a point distal to PLCγ1 activation and IP3-mediated store release. Conclusions These data reveal a role for WAVE2 in regulating multiple pathways leading to T cell activation. In particular, this work shows that WAVE2 is a key component of the actin regulatory machinery in T cells and that it also participates in linking intracellular calcium store depletion to calcium release-activated calcium (CRAC) channel activation. PMID:16401421

Environmental impact of highway construction and repair materials on surface and ground waters. Case study: crumb rubber asphalt concrete.

PubMed

Azizian, Mohammad F; Nelson, Peter O; Thayumanavan, Pugazhendhi; Williamson, Kenneth J

2003-01-01

The practice of incorporating certain waste products into highway construction and repair materials (CRMs) has become more popular. These practices have prompted the National Academy of Science, National Cooperative Highway Research Program (NCHRP) to research the possible impacts of these CRMs on the quality of surface and ground waters. State department of transportations (DOTs) are currently experimenting with use of ground tire rubber ( crumb rubber) in bituminous construction and as a crack sealer. Crumb rubber asphalt concrete (CR-AC) leachates contain a mixture of organic and metallic contaminants. Benzothiazole and 2(3H)-benzothiazolone (organic compounds used in tire rubber manufacturing) and the metals mercury and aluminum were leached in potentially harmful concentrations (exceeding toxic concentrations for aquatic toxicity tests). CR-AC leachate exhibited moderate to high toxicity for algae ( Selenastrum capriconutum) and moderate toxicity for water fleas ( Daphnia magna). Benzothiazole was readily removed from CR-AC leachate by the environmental processes of soil sorption, volatilization, and biodegradation. Metals, which do not volatilize or photochemically or biologically degrade, were removed from the leachate by soil sorption. Contaminants from CR-AC leachates are thus degraded or retarded in their transport through nearby soils and ground waters.

Near-infrared photoactivatable control of Ca2+ signaling and optogenetic immunomodulation

PubMed Central

He, Lian; Zhang, Yuanwei; Ma, Guolin; Tan, Peng; Li, Zhanjun; Zang, Shengbing; Wu, Xiang; Jing, Ji; Fang, Shaohai; Zhou, Lijuan; Wang, Youjun; Huang, Yun; Hogan, Patrick G; Han, Gang; Zhou, Yubin

2015-01-01

The application of current channelrhodopsin-based optogenetic tools is limited by the lack of strict ion selectivity and the inability to extend the spectra sensitivity into the near-infrared (NIR) tissue transmissible range. Here we present an NIR-stimulable optogenetic platform (termed 'Opto-CRAC') that selectively and remotely controls Ca2+ oscillations and Ca2+-responsive gene expression to regulate the function of non-excitable cells, including T lymphocytes, macrophages and dendritic cells. When coupled to upconversion nanoparticles, the optogenetic operation window is shifted from the visible range to NIR wavelengths to enable wireless photoactivation of Ca2+-dependent signaling and optogenetic modulation of immunoinflammatory responses. In a mouse model of melanoma by using ovalbumin as surrogate tumor antigen, Opto-CRAC has been shown to act as a genetically-encoded 'photoactivatable adjuvant' to improve antigen-specific immune responses to specifically destruct tumor cells. Our study represents a solid step forward towards the goal of achieving remote and wireless control of Ca2+-modulated activities with tailored function. DOI: http://dx.doi.org/10.7554/eLife.10024.001 PMID:26646180

Career Guidance for the Third Age. Report on a NICEC/CRAC Invitational Policy Consultation (Birmingham, England, October 30-31, 1996). CRAC/NICEC Conference Briefing.

ERIC Educational Resources Information Center

National Inst. for Careers Education and Counselling, Cambridge (England).

This document synthesizes the findings of an invitational policy consultation at which 27 invited participants from England, Wales, and Scotland examined the special career guidance needs of third-age adults (adults age 45 or older) and strategies for meeting those needs. First, the special career- and employment-related problems faced by…

Structure-activity relationship (SAR) analysis of a family of steroids acutely controlling steroidogenesis.

PubMed

Midzak, Andrew; Rammouz, Georges; Papadopoulos, Vassilios

2012-11-01

Steroids metabolically derive from lipid cholesterol, and vertebrate steroids additionally derive from the steroid pregnenolone. Pregnenolone is derived from cholesterol by hydrolytic cleavage of the aliphatic tail by mitochondrial cytochrome P450 enzyme CYP11A1, located in the inner mitochondrial membrane. Delivery of cholesterol to CYP11A1 comprises the principal control step of steroidogenesis, and requires a series of proteins spanning the mitochondrial double membranes. A critical member of this cholesterol translocation machinery is the integral outer mitochondrial membrane translocator protein (18kDa, TSPO), a high-affinity drug- and cholesterol-binding protein. The cholesterol-binding site of TSPO consists of a phylogenetically conserved cholesterol recognition/interaction amino acid consensus (CRAC). Previous studies from our group identified 5-androsten-3β,17,19-triol (19-Atriol) as drug ligand for the TSPO CRAC motif inhibiting cholesterol binding to CRAC domain and steroidogenesis. To further understand 19-Atriol's mechanism of action as well as the molecular recognition by the TSPO CRAC motif, we undertook structure-activity relationship (SAR) analysis of the 19-Atriol molecule with a variety of substituted steroids oxygenated at positions around the steroid backbone. We found that in addition to steroids hydroxylated at carbon C19, hydroxylations at C4, C7, and C11 contributed to inhibition of cAMP-mediated steroidogenesis in a minimal steroidogenic cell model. However, only substituted steroids with C19 hydroxylations exhibited specificity to TSPO, its CRAC motif, and mitochondrial cholesterol transport, as the C4, C7, and C11 hydroxylated steroids inhibited the metabolic transformation of cholesterol by CYP11A1. We thus provide new insights into structure-activity relationships of steroids inhibiting mitochondrial cholesterol transport and steroidogenic cholesterol metabolic enzymes. Copyright © 2012 Elsevier Inc. All rights reserved.

STIM1 as a key regulator for Ca2+ homeostasis in skeletal-muscle development and function

PubMed Central

2011-01-01

Stromal interaction molecules (STIM) were identified as the endoplasmic-reticulum (ER) Ca2+ sensor controlling store-operated Ca2+ entry (SOCE) and Ca2+-release-activated Ca2+ (CRAC) channels in non-excitable cells. STIM proteins target Orai1-3, tetrameric Ca2+-permeable channels in the plasma membrane. Structure-function analysis revealed the molecular determinants and the key steps in the activation process of Orai by STIM. Recently, STIM1 was found to be expressed at high levels in skeletal muscle controlling muscle function and properties. Novel STIM targets besides Orai channels are emerging. Here, we will focus on the role of STIM1 in skeletal-muscle structure, development and function. The molecular mechanism underpinning skeletal-muscle physiology points toward an essential role for STIM1-controlled SOCE to drive Ca2+/calcineurin/nuclear factor of activated T cells (NFAT)-dependent morphogenetic remodeling programs and to support adequate sarcoplasmic-reticulum (SR) Ca2+-store filling. Also in our hands, STIM1 is transiently up-regulated during the initial phase of in vitro myogenesis of C2C12 cells. The molecular targets of STIM1 in these cells likely involve Orai channels and canonical transient receptor potential (TRPC) channels TRPC1 and TRPC3. The fast kinetics of SOCE activation in skeletal muscle seem to depend on the triad-junction formation, favoring a pre-localization and/or pre-formation of STIM1-protein complexes with the plasma-membrane Ca2+-influx channels. Moreover, Orai1-mediated Ca2+ influx seems to be essential for controlling the resting Ca2+ concentration and for proper SR Ca2+ filling. Hence, Ca2+ influx through STIM1-dependent activation of SOCE from the T-tubule system may recycle extracellular Ca2+ losses during muscle stimulation, thereby maintaining proper filling of the SR Ca2+ stores and muscle function. Importantly, mouse models for dystrophic pathologies, like Duchenne muscular dystrophy, point towards an enhanced Ca2+ influx through Orai1 and/or TRPC channels, leading to Ca2+-dependent apoptosis and muscle degeneration. In addition, human myopathies have been associated with dysfunctional SOCE. Immunodeficient patients harboring loss-of-function Orai1 mutations develop myopathies, while patients suffering from Duchenne muscular dystrophy display alterations in their Ca2+-handling proteins, including STIM proteins. In any case, the molecular determinants responsible for SOCE in human skeletal muscle and for dysregulated SOCE in patients of muscular dystrophy require further examination. PMID:21798093

Use of recycled chunk rubber asphalt concrete (CRAC) on low volume roads and use of recycled crumb rubber modifier in asphalt pavements. Final report, June 1993-June 1995

DOE Office of Scientific and Technical Information (OSTI.GOV)

Hossain, M.; Funk, L.P.; Sadeq, M.A.

1995-06-01

The major objective of this project was to formulate a Chunk Rubber Asphalt Concrete (CRAC) mix for use on low volume roads. CRAC is a rubber modified asphalt concrete product produced by the `dry process` where rubber chunks of 1/2 inch size are used as aggregate in a cold mix with a type C fly ash. The second objective of this project was to develop guidelines concerning the use of rubber modified asphalt concrete hot mix to include: (1) Design methods for use of asphalt-rubber mix for new construction and overlay, (2) Mix design method for asphalt-rubber, and (3) Testmore » method for determining the amount of rubber in an asphalt-rubber concrete for quality control purposes.« less

Transcriptome-wide Analysis of Exosome Targets

PubMed Central

Schneider, Claudia; Kudla, Grzegorz; Wlotzka, Wiebke; Tuck, Alex; Tollervey, David

2012-01-01

Summary The exosome plays major roles in RNA processing and surveillance but the in vivo target range and substrate acquisition mechanisms remain unclear. Here we apply in vivo RNA crosslinking (CRAC) to the nucleases (Rrp44, Rrp6), two structural subunits (Rrp41, Csl4) and a cofactor (Trf4) of the yeast exosome. Analysis of wild-type Rrp44 and catalytic mutants showed that both the CUT and SUT classes of non-coding RNA, snoRNAs and, most prominently, pre-tRNAs and other Pol III transcripts are targeted for oligoadenylation and exosome degradation. Unspliced pre-mRNAs were also identified as targets for Rrp44 and Rrp6. CRAC performed using cleavable proteins (split-CRAC) revealed that Rrp44 endonuclease and exonuclease activities cooperate on most substrates. Mapping oligoadenylated reads suggests that the endonuclease activity may release stalled exosome substrates. Rrp6 was preferentially associated with structured targets, which frequently did not associate with the core exosome indicating that substrates follow multiple pathways to the nucleases. PMID:23000172

Haemophilus parasuis encodes two functional cytolethal distending toxins: CdtC contains an atypical cholesterol recognition/interaction region.

PubMed

Zhou, Mingguang; Zhang, Qiang; Zhao, Jianping; Jin, Meilin

2012-01-01

Haemophilus parasuis is the causative agent of Glässer's disease of pigs, a disease associated with fibrinous polyserositis, polyarthritis and meningitis. We report here H. parasuis encodes two copies of cytolethal distending toxins (Cdts), which these two Cdts showed the uniform toxin activity in vitro. We demonstrate that three Cdt peptides can form an active tripartite holotoxin that exhibits maximum cellular toxicity, and CdtA and CdtB form a more active toxin than CdtB and CdtC. Moreover, the cellular toxicity is associated with the binding of Cdt subunits to cells. Further analysis indicates that CdtC subunit contains an atypical cholesterol recognition/interaction amino acid consensus (CRAC) region. The mutation of CRAC site resulted in decreased cell toxicity. Finally, western blot analysis show all the 15 H. parasuis reference strains and 109 clinical isolates expressed CdtB subunit, indicating that Cdt is a conservative putative virulence factor for H. parasuis. This is the first report of the molecular and cellular basis of Cdt host interactions in H. parasuis.

The CRAC cohort model: A computerized low cost registry of interventional cardiology with daily update and long-term follow-up.

PubMed

Rangé, G; Chassaing, S; Marcollet, P; Saint-Étienne, C; Dequenne, P; Goralski, M; Bardiére, P; Beverilli, F; Godillon, L; Sabine, B; Laure, C; Gautier, S; Hakim, R; Albert, F; Angoulvant, D; Grammatico-Guillon, L

2018-05-01

To assess the reliability and low cost of a computerized interventional cardiology (IC) registry to prospectively and systematically collect high-quality data for all consecutive coronary patients referred for coronary angiogram or/and coronary angioplasty. Rigorous clinical practice assessment is a key factor to improve prognosis in IC. A prospective and permanent registry could achieve this goal but, presumably, at high cost and low level of data quality. One multicentric IC registry (CRAC registry), fully integrated to usual coronary activity report software, started in the centre Val-de-Loire (CVL) French region in 2014. Quality assessment of CRAC registry was conducted on five IC CathLab of the CVL region, from January 1st to December 31st 2014. Quality of collected data was evaluated by measuring procedure exhaustivity (comparing with data from hospital information system), data completeness (quality controls) and data consistency (by checking complete medical charts as gold standard). Cost per procedure (global registry operating cost/number of collected procedures) was also estimated. CRAC model provided a high-quality level with 98.2% procedure completeness, 99.6% data completeness and 89% data consistency. The operating cost per procedure was €14.70 ($16.51) for data collection and quality control, including ST-segment elevation myocardial infarction (STEMI) preadmission information and one-year follow-up after angioplasty. This integrated computerized IC registry led to the construction of an exhaustive, reliable and costless database, including all coronary patients entering in participating IC centers in the CVL region. This solution will be developed in other French regions, setting up a national IC database for coronary patients in 2020: France PCI. Copyright © 2018 Elsevier Masson SAS. All rights reserved.

A mirror code for protein-cholesterol interactions in the two leaflets of biological membranes

NASA Astrophysics Data System (ADS)

Fantini, Jacques; di Scala, Coralie; Evans, Luke S.; Williamson, Philip T. F.; Barrantes, Francisco J.

2016-02-01

Cholesterol controls the activity of a wide range of membrane receptors through specific interactions and identifying cholesterol recognition motifs is therefore critical for understanding signaling receptor function. The membrane-spanning domains of the paradigm neurotransmitter receptor for acetylcholine (AChR) display a series of cholesterol consensus domains (referred to as “CARC”). Here we use a combination of molecular modeling, lipid monolayer/mutational approaches and NMR spectroscopy to study the binding of cholesterol to a synthetic CARC peptide. The CARC-cholesterol interaction is of high affinity, lipid-specific, concentration-dependent, and sensitive to single-point mutations. The CARC motif is generally located in the outer membrane leaflet and its reverse sequence CRAC in the inner one. Their simultaneous presence within the same transmembrane domain obeys a “mirror code” controlling protein-cholesterol interactions in the outer and inner membrane leaflets. Deciphering this code enabled us to elaborate guidelines for the detection of cholesterol-binding motifs in any membrane protein. Several representative examples of neurotransmitter receptors and ABC transporters with the dual CARC/CRAC motifs are presented. The biological significance and potential clinical applications of the mirror code are discussed.

Uncontrolled concrete bridge parapet cracking.

DOT National Transportation Integrated Search

2013-06-01

The Ohio Department of Transportation has recently identified the problem of wide-spread premature cracking of concrete bridge : parapets throughout its District 12 region (Northeast Ohio). Many of the bridge decks that contain these prematurely crac...

PAR-CLIP data indicate that Nrd1-Nab3-dependent transcription termination regulates expression of hundreds of protein coding genes in yeast

PubMed Central

2014-01-01

Background Nrd1 and Nab3 are essential sequence-specific yeast RNA binding proteins that function as a heterodimer in the processing and degradation of diverse classes of RNAs. These proteins also regulate several mRNA coding genes; however, it remains unclear exactly what percentage of the mRNA component of the transcriptome these proteins control. To address this question, we used the pyCRAC software package developed in our laboratory to analyze CRAC and PAR-CLIP data for Nrd1-Nab3-RNA interactions. Results We generated high-resolution maps of Nrd1-Nab3-RNA interactions, from which we have uncovered hundreds of new Nrd1-Nab3 mRNA targets, representing between 20 and 30% of protein-coding transcripts. Although Nrd1 and Nab3 showed a preference for binding near 5′ ends of relatively short transcripts, they bound transcripts throughout coding sequences and 3′ UTRs. Moreover, our data for Nrd1-Nab3 binding to 3′ UTRs was consistent with a role for these proteins in the termination of transcription. Our data also support a tight integration of Nrd1-Nab3 with the nutrient response pathway. Finally, we provide experimental evidence for some of our predictions, using northern blot and RT-PCR assays. Conclusions Collectively, our data support the notion that Nrd1 and Nab3 function is tightly integrated with the nutrient response and indicate a role for these proteins in the regulation of many mRNA coding genes. Further, we provide evidence to support the hypothesis that Nrd1-Nab3 represents a failsafe termination mechanism in instances of readthrough transcription. PMID:24393166

Interaction of mammalian seminal plasma protein PDC-109 with cholesterol: implications for a putative CRAC domain.

PubMed

Scolari, Silvia; Müller, Karin; Bittman, Robert; Herrmann, Andreas; Müller, Peter

2010-10-26

Seminal plasma proteins of the fibronectin type II (Fn2) family modulate mammalian spermatogenesis by triggering the release of the lipids phosphatidylcholine and cholesterol from sperm cells. Whereas the specific interaction of these proteins with phosphatidylcholine is well-understood, their selectivity for cholesterol is unknown. To characterize the interaction between the bovine Fn2 protein PDC-109 and cholesterol, we have investigated the effect of PDC-109 on the dynamics of fluorescent cholesterol analogues in lipid vesicles by time-resolved fluorescence anisotropy. The data show that PDC-109 decreases the rotational mobility of cholesterol within the membrane and that the extent of this impact depends on the cholesterol structure, indicating a specific influence of PDC-109 on cholesterol. We propose that the cholesterol recognition/interaction amino acid consensus (CRAC) regions of PDC-109 are involved in the interaction with cholesterol.

Plasma Membrane Cholesterol as a Regulator of Human and Rodent P2X7 Receptor Activation and Sensitization*

PubMed Central

Robinson, Lucy E.; Shridar, Mitesh; Smith, Philip; Murrell-Lagnado, Ruth D.

2014-01-01

P2X7 receptors are nonselective cation channels gated by high extracellular ATP, but with sustained activation, receptor sensitization occurs, whereby the intrinsic pore dilates, making the cell permeable to large organic cations, which eventually leads to cell death. P2X7 receptors associate with cholesterol-rich lipid rafts, but it is unclear how this affects the properties of the receptor channel. Here we show that pore-forming properties of human and rodent P2X7 receptors are sensitive to perturbations of cholesterol levels. Acute depletion of cholesterol with 5 mm methyl-β-cyclodextrin (MCD) caused a substantial increase in the rate of agonist-evoked pore formation, as measured by the uptake of ethidium dye, whereas cholesterol loading inhibited this process. Patch clamp analysis of P2X7 receptor currents carried by Na+ and N-methyl-d-glucamine (NMDG+) showed enhanced activation and current facilitation following cholesterol depletion. This contrasts with the inhibitory effect of methyl-β-cyclodextrin reported for other P2X subtypes. Mutational analysis suggests the involvement of an N-terminal region and a proximal C-terminal region that comprises multiple cholesterol recognition amino acid consensus (CRAC) motifs, in the cholesterol sensitivity of channel gating. These results reveal cholesterol as a negative regulator of P2X7 receptor pore formation, protecting cells from P2X7-mediated cell death. PMID:25281740

Isolation and structure determination of malevamide E, a dolastatin 14 analogue, from the marine cyanobacterium Symploca laete-viridis.

PubMed

Adams, Beatrice; Pörzgen, Peter; Pittman, Emily; Yoshida, Wesley Y; Westenburg, Hans E; Horgen, F David

2008-05-01

A new depsipeptide, malevamide E (1), was isolated from field-collected colonies of the filamentous cyanobacterium Symploca laete-viridis. The gross structure of 1 was determined by spectroscopic analyses, including one- and two-dimensional NMR and accurately measured MS/MS. Chiral HPLC analyses of an acid hydrolysate of 1 allowed the stereochemical assignments of its amino acid residues, which include N-methyl-L-alanine, alpha-N,gamma-N-dimethyl-L-asparagine, N-methyl-L-phenylalanine, L-proline, D-valine, and N-methyl-L-valine. LC-MS/MS analysis of S. laete-viridis fractions established the co-occurrence of malevamide E (1) and its homologue dolastatin 14 (2), which was previously reported in low yield from the sea hare Dolabella auricularia. Malevamide E (1) demonstrated a dose-dependent (2-45 microM) inhibition of store-operated Ca(2+) entry in thapsigargin-treated human embryonic kidney (HEK) cells, indicating an inhibitory effect on Ca(2+) release-activated Ca(2+) (CRAC) channels.

Ca2+ handling remodeling and STIM1L/Orai1/TRPC1/TRPC4 upregulation in monocrotaline-induced right ventricular hypertrophy.

PubMed

Jessica, Sabourin; Angèle, Boet; Catherine, Rucker-Martin; Mélanie, Lambert; Ana-Maria, Gomez; Jean-Pierre, Benitah; Frédéric, Perros; Marc, Humbert; Fabrice, Antigny

2018-05-01

Right ventricular (RV) function is the most important prognostic factor for pulmonary arterial hypertension (PAH) patients. The progressive increase of pulmonary vascular resistance induces RV hypertrophy (RVH) and at term RV failure (RVF). However, the molecular mechanisms of RVH and RVF remain understudied. In this study, we gained insights into cytosolic Ca 2+ signaling remodeling in ventricular cardiomyocytes during the pathogenesis of severe pulmonary hypertension (PH) induced in rats by monocrotaline (MCT) exposure, and we further identified molecular candidates responsible for this Ca 2+ remodeling. After PH induction, hypertrophied RV myocytes presented longer action potential duration, higher and faster [Ca 2+ ] i transients and increased sarcoplasmic reticulum (SR) Ca 2+ content, whereas no changes in these parameters were detected in left ventricular (LV) myocytes. These modifications were associated with increased P-Ser 16 -phospholamban pentamer expression without altering SERCA2a (Sarco/Endoplasmic Reticulum Ca 2+ -ATPase) pump abundance. Moreover, after PH induction, Ca 2+ sparks frequency were higher in hypertrophied RV cells, while total RyR2 (Ryanodine Receptor) expression and phosphorylation were unaffected. Together with cellular hypertrophy, the T-tubules network was disorganized. Hypertrophied RV cardiomyocytes from MCT-exposed rats showed decreased expression of classical STIM1 (Stromal Interaction molecule) associated with increased expression of muscle-specific STIM1 Long isoform, glycosylated-Orai1 channel form, and TRPC1 and TRPC4 channels, which was correlated with an enhanced Ca 2+ -release-activated Ca 2+ (CRAC)-like current. Pharmacological inhibition of TRPCs/Orai1 channels in hypertrophied RV cardiomyocytes normalized [Ca 2+ ] i transients amplitude, the SR Ca 2+ content and cell contractility to control levels. Finally, we showed that most of these changes did not appear in LV cardiomyocytes. These new findings demonstrate RV-specific cellular Ca 2+ cycling remodeling in PH rats with maladaptive RVH and that the STIM1L/Orai1/TRPC1/C4-dependent Ca 2+ current participates in this Ca 2+ remodeling in RVH secondary to PH. Copyright © 2018 Elsevier Ltd. All rights reserved.

Integration of pavement cracking prediction model with asset management and vehicle-infrastructure interaction models.

DOT National Transportation Integrated Search

2015-01-01

Not long after the construction of a pavement or a new pavement surface, various : forms of deterioration begin to accumulate due to the harsh effects of traffic loading : combined with weathering action. In a recent NEXTRANS project, a pavement crac...

Sterol Binding by the Tombusviral Replication Proteins Is Essential for Replication in Yeast and Plants.

PubMed

Xu, Kai; Nagy, Peter D

2017-04-01

Membranous structures derived from various organelles are important for replication of plus-stranded RNA viruses. Although the important roles of co-opted host proteins in RNA virus replication have been appreciated for a decade, the equally important functions of cellular lipids in virus replication have been gaining full attention only recently. Previous work with Tomato bushy stunt tombusvirus (TBSV) in model host yeast has revealed essential roles for phosphatidylethanolamine and sterols in viral replication. To further our understanding of the role of sterols in tombusvirus replication, in this work we showed that the TBSV p33 and p92 replication proteins could bind to sterols in vitro The sterol binding by p33 is supported by cholesterol recognition/interaction amino acid consensus (CRAC) and CARC-like sequences within the two transmembrane domains of p33. Mutagenesis of the critical Y amino acids within the CRAC and CARC sequences blocked TBSV replication in yeast and plant cells. We also showed the enrichment of sterols in the detergent-resistant membrane (DRM) fractions obtained from yeast and plant cells replicating TBSV. The DRMs could support viral RNA synthesis on both the endogenous and exogenous templates. A lipidomic approach showed the lack of enhancement of sterol levels in yeast and plant cells replicating TBSV. The data support the notion that the TBSV replication proteins are associated with sterol-rich detergent-resistant membranes in yeast and plant cells. Together, the results obtained in this study and the previously published results support the local enrichment of sterols around the viral replication proteins that is critical for TBSV replication. IMPORTANCE One intriguing aspect of viral infections is their dependence on efficient subcellular assembly platforms serving replication, virion assembly, or virus egress via budding out of infected cells. These assembly platforms might involve sterol-rich membrane microdomains, which are heterogeneous and highly dynamic nanoscale structures usurped by various viruses. Here, we demonstrate that TBSV p33 and p92 replication proteins can bind to sterol in vitro Mutagenesis analysis of p33 within the CRAC and CARC sequences involved in sterol binding shows the important connection between the abilities of p33 to bind to sterol and to support TBSV replication in yeast and plant cells. Together, the results further strengthen the model that cellular sterols are essential as proviral lipids during viral replication. Copyright © 2017 American Society for Microbiology.

CUP-1 Is a Novel Protein Involved in Dietary Cholesterol Uptake in Caenorhabditis elegans

PubMed Central

Valdes, Victor J.; Athie, Alejandro; Salinas, Laura S.; Navarro, Rosa E.; Vaca, Luis

2012-01-01

Sterols transport and distribution are essential processes in all multicellular organisms. Survival of the nematode Caenorhabditis elegans depends on dietary absorption of sterols present in the environment. However the general mechanisms associated to sterol uptake in nematodes are poorly understood. In the present work we provide evidence showing that a previously uncharacterized transmembrane protein, designated Cholesterol Uptake Protein-1 (CUP-1), is involved in dietary cholesterol uptake in C. elegans. Animals lacking CUP-1 showed hypersensitivity to cholesterol limitation and were unable to uptake cholesterol. A CUP-1-GFP fusion protein colocalized with cholesterol-rich vesicles, endosomes and lysosomes as well as the plasma membrane. Additionally, by FRET imaging, a direct interaction was found between the cholesterol analog DHE and the transmembrane “cholesterol recognition/interaction amino acid consensus” (CRAC) motif present in C. elegans CUP-1. In-silico analysis identified two mammalian homologues of CUP-1. Most interestingly, CRAC motifs are conserved in mammalian CUP-1 homologous. Our results suggest a role of CUP-1 in cholesterol uptake in C. elegans and open up the possibility for the existence of a new class of proteins involved in sterol absorption in mammals. PMID:22479487

STORE-OPERATED CALCIUM ENTRY IS PRESENT IN HL-1 CARDIOMYOCYTES AND CONTRIBUTES TO RESTING CALCIUM

PubMed Central

Touchberry, Chad D.; Elmore, Chris J.; Nguyen, Tien M.; Andresen, Jon J.; Zhao, Xiaoli; Orange, Matthew; Weisleder, Noah; Brotto, Marco; Claycomb, William C.; Wacker, Michael J.

2011-01-01

Store-operated Ca2+ entry (SOCE) has recently been shown to be of physiological and pathological importance in the heart, particularly during cardiac hypertrophy. However, measuring changes in intracellular Ca2+ during SOCE is very difficult to study in adult primary cardiomyocytes. As a result there is a need for a stable and reliable in vitro model of SOCE which can be used to test cardiac drugs and investigate the role of SOCE in cardiac pathology. HL-1 cells are the only immortal cardiomyocyte cell line available that continuously divides and spontaneously contracts while maintaining phenotypic characteristics of the adult cardiomyocyte. To date the role of SOCE has not yet been investigated in the HL-1 cardiac cell line. We report for the first time that these cells express stromal interaction molecule 1 (STIM1) and the Ca2+ release-activated Ca2+ (CRAC) channel Orai1, which are essential components of the SOCE machinery. In addition, SOCE is tightly coupled to sarcoplasmic reticulum (SR)-Ca2+ release in HL-1 cells, and such response was not impaired in the presence of voltage dependent Ca2+ channels (L-type and T-type channels) or reverse mode Na+/ Ca2+ exchanger (NCX) inhibitors. We were able to abolish the SOCE response with known SOCE inhibitors (BTP-2 and SKF-96365) and by targeted knockdown of Orai1 with RNAi. In addition, knockdown of Orai1 resulted in lower baseline Ca2+ and an attenuated response to thapsigargin (TG) and caffeine, indicating that SOCE may play a role in Ca2+ homeostasis during unstressed conditions in cardiomyocytes. Currently, there is little knowledge about SOCE in cardiomyocytes, and the present results suggest that HL-1 cells will be of great utility in investigating the role of SOCE in the heart. PMID:22079292

To die or not to die SGK1-sensitive ORAI/STIM in cell survival.

PubMed

Lang, Florian; Pelzl, Lisann; Hauser, Stefan; Hermann, Andreas; Stournaras, Christos; Schöls, Ludger

2018-05-03

The pore forming Ca 2+ release activated Ca 2+ channel (CRAC) isoforms ORAI1-3 and their regulators STIM1,2 accomplish store operated Ca 2+ entry (SOCE). Activation of SOCE may lead to cytosolic Ca 2+ oscillations, which in turn support cell proliferation and cell survival. ORAI/STIM and thus SOCE are upregulated by the serum and glucocorticoid inducible kinase SGK1, a kinase under powerful genomic regulation and activated by phosphorylation via the phosphoinositol-3-phosphate pathway. SGK1 enhances ORAI1 abundance partially by phosphorylation of Nedd4-2, an ubiquitin ligase priming the channel protein for degradation. The SGK1-phosphorylated Nedd4-2 binds to the protein 14-3-3 and is thus unable to ubiquinate ORAI1. SGK1 further increases the ORAI1 and STIM1 protein abundance by activating nuclear factor kappa B (NF-κB), a transcription factor upregulating the expression of STIM1 and ORAI1. SGK1-sensitive upregulation of ORAI/STIM and thus SOCE is triggered by a wide variety of hormones and growth factors, as well as several cell stressors including ischemia, radiation, and cell shrinkage. SGK1 dependent upregulation of ORAI/STIM confers survival of tumor cells and thus impacts on growth and therapy resistance of cancer. On the other hand, SGK1-dependent upregulation of ORAI1 and STIM1 may support survival of neurons and impairment of SGK1-dependent ORAI/STIM activity may foster neurodegeneration. Clearly, further experimental effort is needed to define the mechanisms linking SGK1-dependent upregulation of ORAI1 and STIM1 to cell survival and to define the impact of SGK1-dependent upregulation of ORAI1 and STIM1 on malignancy and neurodegenerative disease. Copyright © 2018 Elsevier Ltd. All rights reserved.

Beer flavor provokes striatal dopamine release in male drinkers: mediation by family history of alcoholism.

PubMed

Oberlin, Brandon G; Dzemidzic, Mario; Tran, Stella M; Soeurt, Christina M; Albrecht, Daniel S; Yoder, Karmen K; Kareken, David A

2013-08-01

Striatal dopamine (DA) is increased by virtually all drugs of abuse, including alcohol. However, drug-associated cues are also known to provoke striatal DA transmission- a phenomenon linked to the motivated behaviors associated with addiction. To our knowledge, no one has tested if alcohol's classically conditioned flavor cues, in the absence of a significant pharmacologic effect, are capable of eliciting striatal DA release in humans. Employing positron emission tomography (PET), we hypothesized that beer's flavor alone can reduce the binding potential (BP) of [(11)C]raclopride (RAC; a reflection of striatal DA release) in the ventral striatum, relative to an appetitive flavor control. Forty-nine men, ranging from social to heavy drinking, mean age 25, with a varied family history of alcoholism underwent two [(11)C]RAC PET scans: one while tasting beer, and one while tasting Gatorade. Relative to the control flavor of Gatorade, beer flavor significantly increased self-reported desire to drink, and reduced [(11)C]RAC BP, indicating that the alcohol-associated flavor cues induced DA release. BP reductions were strongest in subjects with first-degree alcoholic relatives. These results demonstrate that alcohol-conditioned flavor cues can provoke ventral striatal DA release, absent significant pharmacologic effects, and that the response is strongest in subjects with a greater genetic risk for alcoholism. Striatal DA responses to salient alcohol cues may thus be an inherited risk factor for alcoholism.

Profiling calcium signals of in vitro polarized human effector CD4+ T cells.

PubMed

Kircher, Sarah; Merino-Wong, Maylin; Niemeyer, Barbara A; Alansary, Dalia

2018-06-01

Differentiation of naïve CD4 + T cells into effector subtypes with distinct cytokine profiles and physiological roles is a tightly regulated process, the imbalance of which can lead to an inadequate immune response or autoimmune disease. The crucial role of Ca 2+ signals, mainly mediated by the store operated Ca 2+ entry (SOCE) in shaping the immune response is well described. However, it is unclear if human effector CD4 + T cell subsets show differential Ca 2+ signatures in response to different stimulation methods. Herein, we provide optimized in vitro culture conditions for polarization of human CD4 + effector T cells and characterize their SOCE following both pharmacological store depletion and direct T-cell receptor (TCR) activation. Moreover, we measured whole cell Ca 2+ release activated Ca 2+ currents (I CRAC ) and investigated whether the observed differences correlate to the expression of CRAC genes. Our results show that Ca 2+ profiles of helper CD4 + Th1, Th2 and Th17 are distinct and in part shaped by the intensity of stimulation. Regulatory T cells (Treg) are unique being the subtype with the most prominent SOCE response. Analysis of in vivo differentiated Treg unraveled the role of differential expression of ORAI2 in fine-tuning signals in Treg vs. conventional CD4 + T cells. Copyright © 2018 The Author(s). Published by Elsevier B.V. All rights reserved.

Data Center Energy Efficiency Measurement Assessment Kit Guide and Specification

DOE Office of Scientific and Technical Information (OSTI.GOV)

None

2012-10-26

A portable and temporary wireless mesh assessment kit can be used to speed up and reduce the costs of a data center energy use assessment and overcome the issues with respect to shutdowns. The assessment kit is comprised of temperature, relative humidity, and pressure sensors. Also included are power meters that can be installed on computer room air conditioners (CRACs) without intrusive interruption of data center operations. The assessment kit produces data required for a detailed energy assessment of the data center.

The University for Industry and Local Information, Advice and Guidance Partnerships. Report on a NICEC/CRAC Policy Consultation Held in Association with the National Advisory Council for Careers and Educational Guidance (Cambridge, England, February 24-25, 1999). Conference Briefing.

ERIC Educational Resources Information Center

Watts, Tony

The University for Industry (UFI) and local information, advice, and guidance (IAG) partnerships are two key aspects of the British Government's lifelong learning strategy. UFI's key role is to expand the demand for and supply of learning and to exploit the learning potential of information and communication technologies. The main UFI activities…

"Speeding up the road to recovery": The Complex Recovery Assessment and Consultation (CRAC) service.

PubMed

Davis Le Brun, Stephanie

2015-01-01

The number of bed closures in mental health is on the rise, creating additional pressure on services, including acute mental health services. An efficient way of working is required in order to streamline the acute care pathway and decrease unnecessary delays to length of stay, ensuring all individuals can be offered an inpatient bed when in crisis. The Complex Recovery Assessment and Consultation (CRAC) service was created in order to support acute mental health inpatient clinicians in streamlining hospital stays for service users who present with complex presentations that require lengthier admissions (over 40 days) by offering assessment, advice, and intervention from a rehabilitation perspective. The team was also created to understand why individuals may require a lengthy hospital stay. Preliminary data showed that requiring a placement on discharge proved to be the most significant factor in increased length of stay and so the team took on a new role of discharge coordinator after around a year of operating. This involved assisting in decreasing any delays out of hospital through improved communication and dedicated time to complete tasks, such as completing paperwork for placement referrals and funding panels. Since taking on this role it was found that the time taken for individuals to be discharged to a rehabilitation or specialist placement decreased; a rehabilitation placement by 13.12 days and a specialist placement by 9.22 days. Discharge to a family address also decreased by 2.9 days and a home address by 2.47 days. Those patients with complex presentations benefit from having one dedicated team to coordinate the discharge process. Their lengthier acute inpatient stay is improved through streamlining care pathways, ultimately decreasing delays in discharge.

Air Launch Instrumented Vehicles Evaluation (ALIVE).

DTIC Science & Technology

1977-02-01

propellant .s. The study addressed aging of two 12—inch—diamete r , SRBDM—type motors cast with mode ra te—burning—rate prope l l a n t . The propel lan t...s Ii t ttiis j t .y Factor vs Half Crack Length 86 30 Stress Intensity Factor /Load vs I1~ l 1 Crack Length 87 31 Log Stress I n t c r t s t t y... Factor vs Log Crac k Tip V e l o c i ty for S t r ip Biaxial Specimen 88 32 Log Stress I t i t i n s i t v Factor A d j u s t e d for Stra in

Acid rock drainage passive remediation using alkaline clay and impacts of vegetation and saturated sand barrier

NASA Astrophysics Data System (ADS)

Plaza, F.; Wen, Y.; Liang, X.

2017-12-01

Acid rock drainage (ARD) caused by abundance of coal refuse (CR) deposits in mining regions requires adequate treatment to prevent serious water pollution due to its acidity and high concentrations of sulfate and metals/metalloids. Over the past decades, various approaches have been explored and developed to remediate ARD. This study uses laboratory experiments to investigate the effectiveness and impacts of ARD passive remediation using alkaline clay (AC), a by-product of the aluminum refining process. Twelve column kinetic leaching experiments were set up with CR/AC mixing ratios ranging from 1%AC to 10%AC. Samples were collected from these columns to measure the pH, sulfate, metals/metalloids, acidity and alkalinity. Additional tests of XRD and acid base accounting were also conducted to better characterize the mineral phase in terms of the alkalinity and acidity potential. Based on the leachate measurement results, these columns were further classified into two groups of neutral/near neutral pH and acidic pH for further analysis. In addition, impacts of the vegetation and saturated sand layer on the remediation effectiveness were explored. The results of our long-term (more than three years in some cases) laboratory experiments show that AC is an effective ARD remediation material for the neutralization of leachate pH and immobilization of sulfate and metals such as Fe, Mn, Cu, Zn, Ni, Pb, Cd, Co. The CR/AC mixing ratios higher than 3%AC are found to be effective, with 10% close to optimal. Moreover, the results demonstrate the benefits of using vegetation and a saturated sand barrier. Vegetation acted as a phytoaccumulation/phytoextraction agent, causing an additional immobilization of metals. The saturated sand barrier blocked the oxygen and water diffusion downwards, leading to a reduction of the pyrite oxidation rate. Finally, the proposed remediation approach shows that the acidity consumption will likely occur before all the alkalinity is exhausted, guaranteeing an adequate long-term performance of this remediation approach.

Interaction of the P-Glycoprotein Multidrug Transporter with Sterols.

PubMed

Clay, Adam T; Lu, Peihua; Sharom, Frances J

2015-11-03

The ABC transporter P-glycoprotein (Pgp, ABCB1) actively exports structurally diverse substrates from within the lipid bilayer, leading to multidrug resistance. Many aspects of Pgp function are altered by the phospholipid environment, but its interactions with sterols remain enigmatic. In this work, the functional interaction between purified Pgp and various sterols was investigated in detergent solution and proteoliposomes. Fluorescence studies showed that dehydroergosterol, cholestatrienol, and NBD-cholesterol interact intimately with Pgp, resulting in both quenching of protein Trp fluorescence and enhancement of sterol fluorescence. Kd values indicated binding affinities in the range of 3-9 μM. Collisional quenching experiments showed that Pgp-bound NBD-cholesterol was protected from the external milieu, resonance energy transfer was observed between Pgp Trp residues and the sterol, and the fluorescence emission of bound sterol was enhanced. These observations suggested an intimate interaction of bound sterols with the transporter at a protected nonpolar site. Cholesterol hemisuccinate altered the thermal unfolding of Pgp and greatly stabilized its basal ATPase activity in both a detergent solution and reconstituted proteoliposomes of certain phospholipids. Other sterols, including dehydroergosterol, did not stabilize the basal ATPase activity of detergent-solubilized Pgp, which suggests that this is not a generalized sterol effect. The phospholipid composition and cholesterol hemisuccinate content of Pgp proteoliposomes altered the basal ATPase and drug transport cycles differently. Sterols may interact with Pgp and modulate its structure and function by occupying part of the drug-binding pocket or by binding to putative consensus cholesterol-binding (CRAC/CARC) motifs located within the transmembrane domains.

Silver Nanoparticle-Directed Mast Cell Degranulation Is Mediated through Calcium and PI3K Signaling Independent of the High Affinity IgE Receptor

PubMed Central

Alsaleh, Nasser B.; Persaud, Indushekhar; Brown, Jared M.

2016-01-01

Engineered nanomaterial (ENM)-mediated toxicity often involves triggering immune responses. Mast cells can regulate both innate and adaptive immune responses and are key effectors in allergic diseases and inflammation. Silver nanoparticles (AgNPs) are one of the most prevalent nanomaterials used in consumer products due to their antimicrobial properties. We have previously shown that AgNPs induce mast cell degranulation that was dependent on nanoparticle physicochemical properties. Furthermore, we identified a role for scavenger receptor B1 (SR-B1) in AgNP-mediated mast cell degranulation. However, it is completely unknown how SR-B1 mediates mast cell degranulation and the intracellular signaling pathways involved. In the current study, we hypothesized that SR-B1 interaction with AgNPs directs mast cell degranulation through activation of signal transduction pathways that culminate in an increase in intracellular calcium signal leading to mast cell degranulation. For these studies, we utilized bone marrow-derived mast cells (BMMC) isolated from C57Bl/6 mice and RBL-2H3 cells (rat basophilic leukemia cell line). Our data support our hypothesis and show that AgNP-directed mast cell degranulation involves activation of PI3K, PLCγ and an increase in intracellular calcium levels. Moreover, we found that influx of extracellular calcium is required for the cells to degranulate in response to AgNP exposure and is mediated at least partially via the CRAC channels. Taken together, our results provide new insights into AgNP-induced mast cell activation that are key for designing novel ENMs that are devoid of immune system activation. PMID:27907088

Preclinical assessment of dopaminergic system in rats by MicroPET using three positron-emitting radiopharmaceuticals

DOE Office of Scientific and Technical Information (OSTI.GOV)

Lara-Camacho, V. M., E-mail: victormlc13@hotmail.com; Ávila-García, M. C., E-mail: victormlc13@hotmail.com; Ávila-Rodríguez, M. A., E-mail: victormlc13@hotmail.com

Different diseases associated with dysfunction of dopaminergic system such as Parkinson, Alzheimer, and Schizophrenia are being widely studied with positron emission tomography (PET) which is a noninvasive method useful to assess the stage of these illnesses. In our facility we have recently implemented the production of [{sup 11}C]-DTBZ, [{sup 11}C]-RAC, and [{sup 18}F]-FDOPA, which are among the most common PET radiopharmaceuticals used in neurology applications to get information about the dopamine pathways. In this study two healthy rats were imaged with each of those radiotracers in order to confirm selective striatum uptake as a proof of principle before to releasemore » them for human use.« less

Channel function reconstitution and re-animation: a single-channel strategy in the postcrystal age

PubMed Central

Oiki, Shigetoshi

2015-01-01

The most essential properties of ion channels for their physiologically relevant functions are ion-selective permeation and gating. Among the channel species, the potassium channel is primordial and the most ubiquitous in the biological world, and knowledge of this channel underlies the understanding of features of other ion channels. The strategy applied to studying channels changed dramatically after the crystal structure of the potassium channel was resolved. Given the abundant structural information available, we exploited the bacterial KcsA potassium channel as a simple model channel. In the postcrystal age, there are two effective frameworks with which to decipher the functional codes present in the channel structure, namely reconstitution and re-animation. Complex channel proteins are decomposed into essential functional components, and well-examined parts are rebuilt for integrating channel function in the membrane (reconstitution). Permeation and gating are dynamic operations, and one imagines the active channel by breathing life into the ‘frozen’ crystal (re-animation). Capturing the motion of channels at the single-molecule level is necessary to characterize the behaviour of functioning channels. Advanced techniques, including diffracted X-ray tracking, lipid bilayer methods and high-speed atomic force microscopy, have been used. Here, I present dynamic pictures of the KcsA potassium channel from the submolecular conformational changes to the supramolecular collective behaviour of channels in the membrane. These results form an integrated picture of the active channel and offer insights into the processes underlying the physiological function of the channel in the cell membrane. PMID:25833254

Channel function reconstitution and re-animation: a single-channel strategy in the postcrystal age.

PubMed

Oiki, Shigetoshi

2015-06-15

The most essential properties of ion channels for their physiologically relevant functions are ion-selective permeation and gating. Among the channel species, the potassium channel is primordial and the most ubiquitous in the biological world, and knowledge of this channel underlies the understanding of features of other ion channels. The strategy applied to studying channels changed dramatically after the crystal structure of the potassium channel was resolved. Given the abundant structural information available, we exploited the bacterial KcsA potassium channel as a simple model channel. In the postcrystal age, there are two effective frameworks with which to decipher the functional codes present in the channel structure, namely reconstitution and re-animation. Complex channel proteins are decomposed into essential functional components, and well-examined parts are rebuilt for integrating channel function in the membrane (reconstitution). Permeation and gating are dynamic operations, and one imagines the active channel by breathing life into the 'frozen' crystal (re-animation). Capturing the motion of channels at the single-molecule level is necessary to characterize the behaviour of functioning channels. Advanced techniques, including diffracted X-ray tracking, lipid bilayer methods and high-speed atomic force microscopy, have been used. Here, I present dynamic pictures of the KcsA potassium channel from the submolecular conformational changes to the supramolecular collective behaviour of channels in the membrane. These results form an integrated picture of the active channel and offer insights into the processes underlying the physiological function of the channel in the cell membrane. © 2015 The Authors. The Journal of Physiology © 2015 The Physiological Society.

Nuclear BK Channels Regulate Gene Expression via the Control of Nuclear Calcium Signaling

PubMed Central

Li, Boxing; Jie, Wei; Huang, Lianyan; Wei, Peng; Li, Shuji; Luo, Zhengyi; Friedman, Allyson K.; Meredith, Andrea L.; Han, Ming-Hu; Zhu, Xin-Hong; Gao, Tian-Ming

2014-01-01

Ion channels are essential for the regulation of neuronal functions. The significance of plasma membrane, mitochondrial, endoplasmic reticulum, and lysosomal ion channels in the regulation of Ca2+ is well established. In contrast, surprisingly less is known about the function of ion channels on the nuclear envelope (NE). Here we demonstrate the presence of functional large-conductance, calcium-activated potassium channels (BK channels) on the NE of rodent hippocampal neurons. Functionally blockade of nuclear BK channels (nBK channels) induces NE-derived Ca2+ release, nucleoplasmic Ca2+ elevation, and cAMP response element binding protein (CREB)-dependent transcription. More importantly, blockade of nBK channels regulates nuclear Ca2+-sensitive gene expression and promotes dendritic arborization in a nuclear Ca2+-dependent manner. These results suggest that nBK channel functions as a molecular linker between neuronal activity and nuclear Ca2+ to convey the signals from synapse to nucleus and is a new modulator for synaptic activity-dependent neuronal functions at the NE level. PMID:24952642

A selected annotated bibliography of the core biomedical literature pertaining to stroke, cervical spine, manipulation and head/neck movement

PubMed Central

Gotlib, Allan C.; Thiel, Haymo

1985-01-01

This manuscript’s purpose was to establish a knowledge base of information related to stroke and the cervical spine vascular structures, from both historical and current perspectives. The scientific biomedical literatures both indexed (ie. Index Medicus, CRAC) and non-indexed literature systems were scanned and the pertinent manuscripts were annotated. Citation is by occurence in the literature so that historical trends may be viewed more easily. No analysis of the reference material is offered. Suggested however is that: 1. complications to cervical spine manipulation are being recognized and reported with increasing frequency, 2. a cause and effect relationship between stroke and cervical spine manipulation has not been established, 3. a screening mechanism that is valid, reliable and reasonable needs to be established.

The use of dwell time cross-correlation functions to study single-ion channel gating kinetics.

PubMed Central

Ball, F G; Kerry, C J; Ramsey, R L; Sansom, M S; Usherwood, P N

1988-01-01

The derivation of cross-correlation functions from single-channel dwell (open and closed) times is described. Simulation of single-channel data for simple gating models, alongside theoretical treatment, is used to demonstrate the relationship of cross-correlation functions to underlying gating mechanisms. It is shown that time irreversibility of gating kinetics may be revealed in cross-correlation functions. Application of cross-correlation function analysis to data derived from the locust muscle glutamate receptor-channel provides evidence for multiple gateway states and time reversibility of gating. A model for the gating of this channel is used to show the effect of omission of brief channel events on cross-correlation functions. PMID:2462924

Plant Ion Channels: Gene Families, Physiology, and Functional Genomics Analyses

PubMed Central

Ward, John M.; Mäser, Pascal; Schroeder, Julian I.

2016-01-01

Distinct potassium, anion, and calcium channels in the plasma membrane and vacuolar membrane of plant cells have been identified and characterized by patch clamping. Primarily owing to advances in Arabidopsis genetics and genomics, and yeast functional complementation, many of the corresponding genes have been identified. Recent advances in our understanding of ion channel genes that mediate signal transduction and ion transport are discussed here. Some plant ion channels, for example, ALMT and SLAC anion channel subunits, are unique. The majority of plant ion channel families exhibit homology to animal genes; such families include both hyperpolarization-and depolarization-activated Shaker-type potassium channels, CLC chloride transporters/channels, cyclic nucleotide–gated channels, and ionotropic glutamate receptor homologs. These plant ion channels offer unique opportunities to analyze the structural mechanisms and functions of ion channels. Here we review gene families of selected plant ion channel classes and discuss unique structure-function aspects and their physiological roles in plant cell signaling and transport. PMID:18842100

Plant ion channels: gene families, physiology, and functional genomics analyses.

PubMed

Ward, John M; Mäser, Pascal; Schroeder, Julian I

2009-01-01

Distinct potassium, anion, and calcium channels in the plasma membrane and vacuolar membrane of plant cells have been identified and characterized by patch clamping. Primarily owing to advances in Arabidopsis genetics and genomics, and yeast functional complementation, many of the corresponding genes have been identified. Recent advances in our understanding of ion channel genes that mediate signal transduction and ion transport are discussed here. Some plant ion channels, for example, ALMT and SLAC anion channel subunits, are unique. The majority of plant ion channel families exhibit homology to animal genes; such families include both hyperpolarization- and depolarization-activated Shaker-type potassium channels, CLC chloride transporters/channels, cyclic nucleotide-gated channels, and ionotropic glutamate receptor homologs. These plant ion channels offer unique opportunities to analyze the structural mechanisms and functions of ion channels. Here we review gene families of selected plant ion channel classes and discuss unique structure-function aspects and their physiological roles in plant cell signaling and transport.

[K+ channels and lung epithelial physiology].

PubMed

Bardou, Olivier; Trinh, Nguyen Thu Ngan; Brochiero, Emmanuelle

2009-04-01

Transcripts of more than 30 different K(+) channels have been detected in the respiratory epithelium lining airways and alveoli. These channels belong to the 3 main classes of K(+) channels, i.e. i) voltage-dependent or calcium-activated, 6 transmembrane segments (TM), ii) 2-pores 4-TM and iii) inward-rectified 2-TM channels. The physiological and functional significance of this high molecular diversity of lung epithelial K(+) channels is not well understood. Surprisingly, relatively few studies are focused on K(+) channel function in lung epithelial physiology. Nevertheless, several studies have shown that KvLQT1, KCa and K(ATP) K(+) channels play a crucial role in ion and fluid transport, contributing to the control of airway and alveolar surface liquid composition and volume. K(+) channels are involved in other key functions, such as O(2) sensing or the capacity of the respiratory epithelia to repair after injury. This mini-review aims to discuss potential functions of lung K(+) channels.

Loss of Transient Receptor Potential Ankyrin 1 Channel Deregulates Emotion, Learning and Memory, Cognition, and Social Behavior in Mice.

PubMed

Lee, Kuan-I; Lin, Hui-Ching; Lee, Hsueh-Te; Tsai, Feng-Chuan; Lee, Tzong-Shyuan

2017-07-01

The transient receptor potential ankyrin 1 (TRPA1) channel is a non-selective cation channel that helps regulate inflammatory pain sensation and nociception and the development of inflammatory diseases. However, the potential role of the TRPA1 channel and the underlying mechanism in brain functions are not fully resolved. In this study, we demonstrated that genetic deletion of the TRPA1 channel in mice or pharmacological inhibition of its activity increased neurite outgrowth. In vivo study in mice provided evidence of the TRPA1 channel as a negative regulator in hippocampal functions; functional ablation of the TRPA1 channel in mice enhanced hippocampal functions, as evidenced by less anxiety-like behavior, and enhanced fear-related or spatial learning and memory, and novel location recognition as well as social interactions. However, the TRPA1 channel appears to be a prerequisite for motor function; functional loss of the TRPA1 channel in mice led to axonal bundle fragmentation, downregulation of myelin basic protein, and decreased mature oligodendrocyte population in the brain, for impaired motor function. The TRPA1 channel may play a crucial role in neuronal development and oligodendrocyte maturation and be a potential regulator in emotion, cognition, learning and memory, and social behavior.

Basolateral membrane K+ channels in renal epithelial cells

PubMed Central

Devor, Daniel C.

2012-01-01

The major function of epithelial tissues is to maintain proper ion, solute, and water homeostasis. The tubule of the renal nephron has an amazingly simple structure, lined by epithelial cells, yet the segments (i.e., proximal tubule vs. collecting duct) of the nephron have unique transport functions. The functional differences are because epithelial cells are polarized and thus possess different patterns (distributions) of membrane transport proteins in the apical and basolateral membranes of the cell. K+ channels play critical roles in normal physiology. Over 90 different genes for K+ channels have been identified in the human genome. Epithelial K+ channels can be located within either or both the apical and basolateral membranes of the cell. One of the primary functions of basolateral K+ channels is to recycle K+ across the basolateral membrane for proper function of the Na+-K+-ATPase, among other functions. Mutations of these channels can cause significant disease. The focus of this review is to provide an overview of the basolateral K+ channels of the nephron, providing potential physiological functions and pathophysiology of these channels, where appropriate. We have taken a “K+ channel gene family” approach in presenting the representative basolateral K+ channels of the nephron. The basolateral K+ channels of the renal epithelia are represented by members of the KCNK, KCNJ, KCNQ, KCNE, and SLO gene families. PMID:22338089

Fading channel simulator

DOEpatents

Argo, Paul E.; Fitzgerald, T. Joseph

1993-01-01

Fading channel effects on a transmitted communication signal are simulated with both frequency and time variations using a channel scattering function to affect the transmitted signal. A conventional channel scattering function is converted to a series of channel realizations by multiplying the square root of the channel scattering function by a complex number of which the real and imaginary parts are each independent variables. The two-dimensional inverse-FFT of this complex-valued channel realization yields a matrix of channel coefficients that provide a complete frequency-time description of the channel. The transmitted radio signal is segmented to provide a series of transmitted signal and each segment is subject to FFT to generate a series of signal coefficient matrices. The channel coefficient matrices and signal coefficient matrices are then multiplied and subjected to inverse-FFT to output a signal representing the received affected radio signal. A variety of channel scattering functions can be used to characterize the response of a transmitter-receiver system to such atmospheric effects.

K+ channels of Müller glial cells in retinal disorders.

PubMed

Gao, Feng; Xu, Linjie; Zhao, Yuan; Sun, Xinghuai; Wang, Zhongfeng

2018-02-01

Müller cell is the major type glial cell in the vertebrate retina. Müller cells express various types of K+ channels, such as inwardly rectifying K+ (Kir) channels, big conductance Ca2+-activated K+ (BKCa) channels, delayed rectifier K+ channels (KDR), and transient A-type K+ channels. These K+ channels play important roles in maintaining physiological functions of Müller cells. Under some retinal pathological conditions, the changed expression and functions of K+ channels may contribute to retinal pathogenesis. In this article, we reviewed the physiological properties of K+ channels in retinal Müller cells and the functional changes of these channels in retinal disorders. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.

Functional Characterization of Cnidarian HCN Channels Points to an Early Evolution of Ih.

PubMed

Baker, Emma C; Layden, Michael J; van Rossum, Damian B; Kamel, Bishoy; Medina, Monica; Simpson, Eboni; Jegla, Timothy

2015-01-01

HCN channels play a unique role in bilaterian physiology as the only hyperpolarization-gated cation channels. Their voltage-gating is regulated by cyclic nucleotides and phosphatidylinositol 4,5-bisphosphate (PIP2). Activation of HCN channels provides the depolarizing current in response to hyperpolarization that is critical for intrinsic rhythmicity in neurons and the sinoatrial node. Additionally, HCN channels regulate dendritic excitability in a wide variety of neurons. Little is known about the early functional evolution of HCN channels, but the presence of HCN sequences in basal metazoan phyla and choanoflagellates, a protozoan sister group to the metazoans, indicate that the gene family predates metazoan emergence. We functionally characterized two HCN channel orthologs from Nematostella vectensis (Cnidaria, Anthozoa) to determine which properties of HCN channels were established prior to the emergence of bilaterians. We find Nematostella HCN channels share all the major functional features of bilaterian HCNs, including reversed voltage-dependence, activation by cAMP and PIP2, and block by extracellular Cs+. Thus bilaterian-like HCN channels were already present in the common parahoxozoan ancestor of bilaterians and cnidarians, at a time when the functional diversity of voltage-gated K+ channels was rapidly expanding. NvHCN1 and NvHCN2 are expressed broadly in planulae and in both the endoderm and ectoderm of juvenile polyps.

G-protein-coupled inwardly rectifying potassium channels are targets of alcohol action.

PubMed

Lewohl, J M; Wilson, W R; Mayfield, R D; Brozowski, S J; Morrisett, R A; Harris, R A

1999-12-01

G-protein-coupled inwardly rectifying potassium channels (GIRKs) are important for regulation of synaptic transmission and neuronal firing rates. Because of their key role in brain function, we asked if these potassium channels are targets of alcohol action. Ethanol enhanced function of cerebellar granule cell GIRKs coupled to GABAB receptors. Enhancement of GIRK function by ethanol was studied in detail using Xenopus oocytes expressing homomeric or heteromeric channels. Function of all GIRK channels was enhanced by intoxicating concentrations of ethanol, but other, related inwardly rectifying potassium channels were not affected. GIRK2/IRK1 chimeras and GIRK2 truncation mutants were used to identify a region of 43 amino acids in the carboxyl (C) terminus that is critical for the action of ethanol on these channels.

Functional evolution of scorpion venom peptides with an inhibitor cystine knot fold.

PubMed

Gao, Bin; Harvey, Peta J; Craik, David J; Ronjat, Michel; De Waard, Michel; Zhu, Shunyi

2013-06-27

The ICK (inhibitor cystine knot) defines a large superfamily of polypeptides with high structural stability and functional diversity. Here, we describe a new scorpion venom-derived K+ channel toxin (named λ-MeuKTx-1) with an ICK fold through gene cloning, chemical synthesis, nuclear magnetic resonance spectroscopy, Ca2+ release measurements and electrophysiological recordings. λ-MeuKTx-1 was found to adopt an ICK fold that contains a three-strand anti-parallel β-sheet and a 310-helix. Functionally, this peptide selectively inhibits the Drosophila Shaker K+ channel but is not capable of activating skeletal-type Ca2+ release channels/ryanodine receptors, which is remarkably different from the previously known scorpion venom ICK peptides. The removal of two C-terminal residues of λ-MeuKTx-1 led to the loss of the inhibitory activity on the channel, whereas the C-terminal amidation resulted in the emergence of activity on four mammalian K+ channels accompanied by the loss of activity on the Shaker channel. A combination of structural and pharmacological data allows the recognition of three putative functional sites involved in channel blockade of λ-MeuKTx-1. The presence of a functional dyad in λ-MeuKTx-1 supports functional convergence among scorpion venom peptides with different folds. Furthermore, similarities in precursor organization, exon-intron structure, 3D-fold and function suggest that scorpion venom ICK-type K+ channel inhibitors and Ca2+ release channel activators share a common ancestor and their divergence occurs after speciation between buthidae and non-buthids. The structural and functional characterizations of the first scorpion venom ICK toxin with K+ channel-blocking activity sheds light on functionally divergent and convergent evolution of this conserved scaffold of ancient origin.

Pharmacological Correction of Trafficking Defects in ATP-sensitive Potassium Channels Caused by Sulfonylurea Receptor 1 Mutations*

PubMed Central

Martin, Gregory M.; Rex, Emily A.; Devaraneni, Prasanna; Denton, Jerod S.; Boodhansingh, Kara E.; DeLeon, Diva D.; Stanley, Charles A.; Shyng, Show-Ling

2016-01-01

ATP-sensitive potassium (KATP) channels play a key role in mediating glucose-stimulated insulin secretion by coupling metabolic signals to β-cell membrane potential. Loss of KATP channel function due to mutations in ABCC8 or KCNJ11, genes encoding the sulfonylurea receptor 1 (SUR1) or the inwardly rectifying potassium channel Kir6.2, respectively, results in congenital hyperinsulinism. Many SUR1 mutations prevent trafficking of channel proteins from the endoplasmic reticulum to the cell surface. Channel inhibitors, including sulfonylureas and carbamazepine, have been shown to correct channel trafficking defects. In the present study, we identified 13 novel SUR1 mutations that cause channel trafficking defects, the majority of which are amenable to pharmacological rescue by glibenclamide and carbamazepine. By contrast, none of the mutant channels were rescued by KATP channel openers. Cross-linking experiments showed that KATP channel inhibitors promoted interactions between the N terminus of Kir6.2 and SUR1, whereas channel openers did not, suggesting the inhibitors enhance intersubunit interactions to overcome channel biogenesis and trafficking defects. Functional studies of rescued mutant channels indicate that most mutants rescued to the cell surface exhibited WT-like sensitivity to ATP, MgADP, and diazoxide. In intact cells, recovery of channel function upon trafficking rescue by reversible sulfonylureas or carbamazepine was facilitated by the KATP channel opener diazoxide. Our study expands the list of KATP channel trafficking mutations whose function can be recovered by pharmacological ligands and provides further insight into the structural mechanism by which channel inhibitors correct channel biogenesis and trafficking defects. PMID:27573238

Subunit stoichiometry of human muscle chloride channels.

PubMed

Fahlke, C; Knittle, T; Gurnett, C A; Campbell, K P; George, A L

1997-01-01

Voltage-gated Cl- channels belonging to the ClC family appear to function as homomultimers, but the number of subunits needed to form a functional channel is controversial. To determine subunit stoichiometry, we constructed dimeric human skeletal muscle Cl- channels in which one subunit was tagged by a mutation (D136G) that causes profound changes in voltage-dependent gating. Sucrose-density gradient centrifugation experiments indicate that both monomeric and dimeric hClC-1 channels in their native configurations exhibit similar sedimentation properties consistent with a multimeric complex having a molecular mass of a dimer. Expression of the heterodimeric channel in a mammalian cell line results in a homogenous population of Cl- channels exhibiting novel gating properties that are best explained by the formation of heteromultimeric channels with an even number of subunits. Heteromultimeric channels were not evident in cells cotransfected with homodimeric WT-WT and D136G-D136G constructs excluding the possibility that functional hClC-1 channels are assembled from more than two subunits. These results demonstrate that the functional hClC-1 unit consists of two subunits.

Role of potassium ion channels in detrusor smooth muscle function and dysfunction

PubMed Central

Petkov, Georgi V.

2013-01-01

Contraction and relaxation of the detrusor smooth muscle (DSM), which makes up the wall of the urinary bladder, facilitates the storage and voiding of urine. Several families of K+ channels, including voltage-gated K+ (KV) channels, Ca2+-activated K+ (KCa) channels, inward-rectifying ATP-sensitive K+ (Kir, KATP) channels, and two-pore-domain K+ (K2P) channels, are expressed and functional in DSM. They control DSM excitability and contractility by maintaining the resting membrane potential and shaping the action potentials that determine the phasic nature of contractility in this tissue. Defects in DSM K+ channel proteins or in the molecules involved in their regulatory pathways may underlie certain forms of bladder dysfunction, such as overactive bladder. K+ channels represent an opportunity for novel pharmacological manipulation and therapeutic intervention in human DSM. Modulation of DSM K+ channels directly or indirectly by targeting their regulatory mechanisms has the potential to control urinary bladder function. This Review summarizes our current state of knowledge of the functional role of K+ channels in DSM in health and disease, with special emphasis on current advancements in the field. PMID:22158596

Carbamazepine as a novel small molecule corrector of trafficking-impaired ATP-sensitive potassium channels identified in congenital hyperinsulinism.

PubMed

Chen, Pei-Chun; Olson, Erik M; Zhou, Qing; Kryukova, Yelena; Sampson, Heidi M; Thomas, David Y; Shyng, Show-Ling

2013-07-19

ATP-sensitive potassium (KATP) channels consisting of sulfonylurea receptor 1 (SUR1) and the potassium channel Kir6.2 play a key role in insulin secretion by coupling metabolic signals to β-cell membrane potential. Mutations in SUR1 and Kir6.2 that impair channel trafficking to the cell surface lead to loss of channel function and congenital hyperinsulinism. We report that carbamazepine, an anticonvulsant, corrects the trafficking defects of mutant KATP channels previously identified in congenital hyperinsulinism. Strikingly, of the 19 SUR1 mutations examined, only those located in the first transmembrane domain of SUR1 responded to the drug. We show that unlike that reported for several other protein misfolding diseases, carbamazepine did not correct KATP channel trafficking defects by activating autophagy; rather, it directly improved the biogenesis efficiency of mutant channels along the secretory pathway. In addition to its effect on channel trafficking, carbamazepine also inhibited KATP channel activity. Upon subsequent removal of carbamazepine, however, the function of rescued channels was recovered. Importantly, combination of the KATP channel opener diazoxide and carbamazepine led to enhanced mutant channel function without carbamazepine washout. The corrector effect of carbamazepine on mutant KATP channels was also demonstrated in rat and human β-cells with an accompanying increase in channel activity. Our findings identify carbamazepine as a novel small molecule corrector that may be used to restore KATP channel expression and function in a subset of congenital hyperinsulinism patients.

Local calcium signalling is mediated by mechanosensitive ion channels in mesenchymal stem cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Chubinskiy-Nadezhdin, Vladislav I., E-mail: vchubinskiy@gmail.com; Vasileva, Valeria Y.; Pugovkina, Natalia A.

Mechanical forces are implicated in key physiological processes in stem cells, including proliferation, differentiation and lineage switching. To date, there is an evident lack of understanding of how external mechanical cues are coupled with calcium signalling in stem cells. Mechanical reactions are of particular interest in adult mesenchymal stem cells because of their promising potential for use in tissue remodelling and clinical therapy. Here, single channel patch-clamp technique was employed to search for cation channels involved in mechanosensitivity in mesenchymal endometrial-derived stem cells (hMESCs). Functional expression of native mechanosensitive stretch-activated channels (SACs) and calcium-sensitive potassium channels of different conductances inmore » hMESCs was shown. Single current analysis of stretch-induced channel activity revealed functional coupling of SACs and BK channels in plasma membrane. The combination of cell-attached and inside-out experiments have indicated that highly localized Ca{sup 2+} entry via SACs triggers BK channel activity. At the same time, SK channels are not coupled with SACs despite of high calcium sensitivity as compared to BK. Our data demonstrate novel mechanism controlling BK channel activity in native cells. We conclude that SACs and BK channels are clusterized in functional mechanosensitive domains in the plasma membrane of hMESCs. Co-clustering of ion channels may significantly contribute to mechano-dependent calcium signalling in stem cells. - Highlights: • Stretch-induced channel activity in human mesenchymal stem cells was analyzed. • Functional expression of SACs and Ca{sup 2+}-sensitive BK and SK channels was shown. • Local Ca{sup 2+} influx via stretch-activated channels triggers BK channel activity. • SK channels are not coupled with SACs despite higher sensitivity to [Ca{sup 2+}]{sub i}. • Functional clustering of SACs and BK channels in stem cell membrane is proposed.« less

Epigallocatechin-3-gallate (EGCG) up-regulates miR-15b expression thus attenuating store operated calcium entry (SOCE) into murine CD4+ T cells and human leukaemic T cell lymphoblasts.

PubMed

Zhang, Shaqiu; Al-Maghout, Tamer; Bissinger, Rosi; Zeng, Ni; Pelzl, Lisann; Salker, Madhuri S; Cheng, Anchun; Singh, Yogesh; Lang, Florian

2017-10-27

CD4 + T cells are key elements in immune responses and inflammation. Activation of T cell receptors in CD4 + T cells triggers cytosolic Ca 2+ release with subsequent store operated Ca 2+ entry (SOCE), which is accomplished by the pore forming Ca 2+ release activated Ca 2+ (CRAC) channel Orai1 and its regulator stromal cell-interaction molecule 2 (STIM2). Green tea polyphenol epigallocatechin-3-gallate (EGCG) acts as a potent anti-inflammatory and anti-oxidant agent for various types of cells including immune cells. However, how post-transcriptional gene regulators such as miRNAs are involved in the regulation of Ca 2+ influx into murine CD4 + T cells and human Jurkat T cells through EGCG is not defined. EGCG treatment of murine CD4 + T cells significantly down-regulated the expression of STIM2 and Orai1 both at mRNA and protein levels. Furthermore, EGCG significantly decreased SOCE in both murine and human T cells. EGCG treatment increased miRNA-15b (miR-15b) abundance in both murine and human T cells. Bioinformatics analysis reveals that miR-15b, which has a STIM2 binding site, is involved in the down-regulation of SOCE. Overexpression of miR-15b significantly decreased the mRNA and protein expression of STIM2 and Orai1 in murine T cells. Treatment of Jurkat T cells with 10 μM EGCG further decreased mTOR and PTEN protein levels. EGCG decreased mitochondrial membrane potential (MMP) in both human and murine T cells. In conclusion, the observations suggest that EGCG inhibits the Ca 2+ entry into murine and human T cells, an effect accomplished at least in part by up-regulation of miR-15b.

TRP channels in calcium homeostasis: from hormonal control to structure-function relationship of TRPV5 and TRPV6.

PubMed

van Goor, Mark K C; Hoenderop, Joost G J; van der Wijst, Jenny

2017-06-01

Maintaining plasma calcium levels within a narrow range is of vital importance for many physiological functions. Therefore, calcium transport processes in the intestine, bone and kidney are tightly regulated to fine-tune the rate of absorption, storage and excretion. The TRPV5 and TRPV6 calcium channels are viewed as the gatekeepers of epithelial calcium transport. Several calciotropic hormones control the channels at the level of transcription, membrane expression, and function. Recent technological advances have provided the first near-atomic resolution structural models of several TRPV channels, allowing insight into their architecture. While this field is still in its infancy, it has increased our understanding of molecular channel regulation and holds great promise for future structure-function studies of these ion channels. This review will summarize the mechanisms that control the systemic calcium balance, as well as extrapolate structural views to the molecular functioning of TRPV5/6 channels in epithelial calcium transport. Copyright © 2016. Published by Elsevier B.V.

Pharmacological Correction of Trafficking Defects in ATP-sensitive Potassium Channels Caused by Sulfonylurea Receptor 1 Mutations.

PubMed

Martin, Gregory M; Rex, Emily A; Devaraneni, Prasanna; Denton, Jerod S; Boodhansingh, Kara E; DeLeon, Diva D; Stanley, Charles A; Shyng, Show-Ling

2016-10-14

ATP-sensitive potassium (K ATP ) channels play a key role in mediating glucose-stimulated insulin secretion by coupling metabolic signals to β-cell membrane potential. Loss of K ATP channel function due to mutations in ABCC8 or KCNJ11, genes encoding the sulfonylurea receptor 1 (SUR1) or the inwardly rectifying potassium channel Kir6.2, respectively, results in congenital hyperinsulinism. Many SUR1 mutations prevent trafficking of channel proteins from the endoplasmic reticulum to the cell surface. Channel inhibitors, including sulfonylureas and carbamazepine, have been shown to correct channel trafficking defects. In the present study, we identified 13 novel SUR1 mutations that cause channel trafficking defects, the majority of which are amenable to pharmacological rescue by glibenclamide and carbamazepine. By contrast, none of the mutant channels were rescued by K ATP channel openers. Cross-linking experiments showed that K ATP channel inhibitors promoted interactions between the N terminus of Kir6.2 and SUR1, whereas channel openers did not, suggesting the inhibitors enhance intersubunit interactions to overcome channel biogenesis and trafficking defects. Functional studies of rescued mutant channels indicate that most mutants rescued to the cell surface exhibited WT-like sensitivity to ATP, MgADP, and diazoxide. In intact cells, recovery of channel function upon trafficking rescue by reversible sulfonylureas or carbamazepine was facilitated by the K ATP channel opener diazoxide. Our study expands the list of K ATP channel trafficking mutations whose function can be recovered by pharmacological ligands and provides further insight into the structural mechanism by which channel inhibitors correct channel biogenesis and trafficking defects. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

Exclusion of alternative exon 33 of CaV1.2 calcium channels in heart is proarrhythmogenic

PubMed Central

Li, Guang; Wang, Juejin; Liao, Ping; Bartels, Peter; Zhang, Hengyu; Yu, Dejie; Liang, Mui Cheng; Poh, Kian Keong; Yu, Chye Yun; Jiang, Fengli; Yong, Tan Fong; Wong, Yuk Peng; Hu, Zhenyu; Huang, Hua; Zhang, Guangqin; Galupo, Mary Joyce; Bian, Jin-Song; Ponniah, Sathivel; Trasti, Scott Lee; Foo, Roger; Hoppe, Uta C.; Herzig, Stefan; Soong, Tuck Wah

2017-01-01

Alternative splicing changes the CaV1.2 calcium channel electrophysiological property, but the in vivo significance of such altered channel function is lacking. Structure–function studies of heterologously expressed CaV1.2 channels could not recapitulate channel function in the native milieu of the cardiomyocyte. To address this gap in knowledge, we investigated the role of alternative exon 33 of the CaV1.2 calcium channel in heart function. Exclusion of exon 33 in CaV1.2 channels has been reported to shift the activation potential −10.4 mV to the hyperpolarized direction, and increased expression of CaV1.2Δ33 channels was observed in rat myocardial infarcted hearts. However, how a change in CaV1.2 channel electrophysiological property, due to alternative splicing, might affect cardiac function in vivo is unknown. To address these questions, we generated mCacna1c exon 33−/−-null mice. These mice contained CaV1.2Δ33 channels with a gain-of-function that included conduction of larger currents that reflects a shift in voltage dependence and a modest increase in single-channel open probability. This altered channel property underscored the development of ventricular arrhythmia, which is reflected in significantly more deaths of exon 33−/− mice from β-adrenergic stimulation. In vivo telemetric recordings also confirmed increased frequencies in premature ventricular contractions, tachycardia, and lengthened QT interval. Taken together, the significant decrease or absence of exon 33-containing CaV1.2 channels is potentially proarrhythmic in the heart. Of clinical relevance, human ischemic and dilated cardiomyopathy hearts showed increased inclusion of exon 33. However, the possible role that inclusion of exon 33 in CaV1.2 channels may play in the pathogenesis of human heart failure remains unclear. PMID:28490495

Big-conductance Ca2+-activated K+ channels in physiological and pathophysiological urinary bladder smooth muscle cells

PubMed Central

Parajuli, Shankar P.; Zheng, Yun-Min; Levin, Robert; Wang, Yong-Xiao

2016-01-01

ABSTRACT Contraction and relaxation of urinary bladder smooth muscle cells (UBSMCs) represent the important physiological functions of the bladder. Contractile responses in UBSMCs are regulated by a number of ion channels including big-conductance Ca2+- activated K+ (BK) channels. Great progress has been made in studies of BK channels in UBSMCs. The intent of this review is to summarize recent exciting findings with respect to the functional interactions of BK channels with muscarinic receptors, ryanodine receptors (RyRs) and inositol triphosphate receptors (IP3Rs) as well as their functional importance under normal and pathophysiological conditions. BK channels are highly expressed in UBSMCs. Activation of muscarinic M3 receptors inhibits the BK channel activity, facilitates opening of voltage-dependent Ca2+ (CaV) channels, and thereby enhances excitability and contractility of UBSMCs. Signaling molecules and regulatory mechanisms involving RyRs and IP3Rs have a significant effect on functions of BK channels and thereby regulate cellular responses in UBSMCs under normal and pathophysiological conditions including overactive bladders. Moreover, BK channels may represent a novel target for the treatment of bladder dysfunctions. PMID:27101440

Differential subcellular distribution of ion channels and the diversity of neuronal function.

PubMed

Nusser, Zoltan

2012-06-01

Following the astonishing molecular diversity of voltage-gated ion channels that was revealed in the past few decades, the ion channel repertoire expressed by neurons has been implicated as the major factor governing their functional heterogeneity. Although the molecular structure of ion channels is a key determinant of their biophysical properties, their subcellular distribution and densities on the surface of nerve cells are just as important for fulfilling functional requirements. Recent results obtained with high resolution quantitative localization techniques revealed complex, subcellular compartment-specific distribution patterns of distinct ion channels. Here I suggest that within a given neuron type every ion channel has a unique cell surface distribution pattern, with the functional consequence that this dramatically increases the computational power of nerve cells. Copyright © 2011 Elsevier Ltd. All rights reserved.

Functionally heterogenous ryanodine receptors in avian cerebellum.

PubMed

Sierralta, J; Fill, M; Suárez-Isla, B A

1996-07-19

The functional heterogeneity of the ryanodine receptor (RyR) channels in avian cerebellum was defined. Heavy endoplasmic reticulum microsomes had significant levels of ryanodine and inositol 1,4,5-trisphosphate binding. Scatchard analysis and kinetic studies indicated the existence of at least two distinct ryanodine binding sites. Ryanodine binding was calcium-dependent but was not significantly enhanced by caffeine. Incorporation of microsomes into planar lipid bilayers revealed ion channels with pharmacological features (calcium, magnesium, ATP, and caffeine sensitivity) similar to the RyR channels found in mammalian striated muscle. Despite a wide range of unitary conductances (220-500 picosiemens, symmetrical cesium methanesulfonate), ryanodine locked both channels into a characteristic slow gating subconductance state, positively identifying them as RyR channels. Two populations of avian RyR channels were functionally distinguished by single channel calcium sensitivity. One population was defined by a bell-shaped calcium sensitivity analogous to the skeletal muscle RyR isoform (type I). The calcium sensitivity of the second RyR population was sigmoidal and analogous to the cardiac muscle RyR isoform (type II). These data show that there are at least two functionally distinct RyR channel populations in avian cerebellum. This leads to the possibility that these functionally distinct RyR channels are involved in different intracellular calcium signaling pathways.

Physiology and pathophysiology of ClC-K/barttin channels.

PubMed

Fahlke, Christoph; Fischer, Martin

2010-01-01

ClC-K channels form a subgroup of anion channels within the ClC family of anion transport proteins. They are expressed predominantly in the kidney and in the inner ear, and are necessary for NaCl resorption in the loop of Henle and for K+ secretion by the stria vascularis. Subcellular distribution as well as the function of these channels are tightly regulated by an accessory subunit, barttin. Barttin improves the stability of ClC-K channel protein, stimulates the exit from the endoplasmic reticulum and insertion into the plasma membrane and changes its function by modifying voltage-dependent gating processes. The importance of ClC-K/barttin channels is highlighted by several genetic diseases. Dysfunctions of ClC-K channels result in Bartter syndrome, an inherited human condition characterized by impaired urinary concentration. Mutations in the gene encoding barttin, BSND, affect the urinary concentration as well as the sensory function of the inner ear. Surprisingly, there is one BSND mutation that causes deafness without affecting renal function, indicating that kidney function tolerates a reduction of anion channel activity that is not sufficient to support normal signal transduction in inner hair cells. This review summarizes recent work on molecular mechanisms, physiology, and pathophysiology of ClC-K/barttin channels.

Novel regulatory mechanism in human urinary bladder: central role of transient receptor potential melastatin 4 channels in detrusor smooth muscle function

PubMed Central

Hristov, Kiril L.; Smith, Amy C.; Parajuli, Shankar P.; Malysz, John; Rovner, Eric S.

2016-01-01

Transient receptor potential melastatin 4 (TRPM4) channels are Ca2+-activated nonselective cation channels that have been recently identified as regulators of detrusor smooth muscle (DSM) function in rodents. However, their expression and function in human DSM remain unexplored. We provide insights into the functional role of TRPM4 channels in human DSM under physiological conditions. We used a multidisciplinary experimental approach, including RT-PCR, Western blotting, immunohistochemistry and immunocytochemistry, patch-clamp electrophysiology, and functional studies of DSM contractility. DSM samples were obtained from patients without preoperative overactive bladder symptoms. RT-PCR detected mRNA transcripts for TRPM4 channels in human DSM whole tissue and freshly isolated single cells. Western blotting and immunohistochemistry with confocal microscopy revealed TRPM4 protein expression in human DSM. Immunocytochemistry further detected TRPM4 protein expression in DSM single cells. Patch-clamp experiments showed that 9-phenanthrol, a selective TRPM4 channel inhibitor, significantly decreased the transient inward cation currents and voltage step-induced whole cell currents in freshly isolated human DSM cells. In current-clamp mode, 9-phenanthrol hyperpolarized the human DSM cell membrane potential. Furthermore, 9-phenanthrol attenuated the spontaneous phasic, carbachol-induced and nerve-evoked contractions in human DSM isolated strips. Significant species-related differences in TRPM4 channel activity between human, rat, and guinea pig DSM were revealed, suggesting a more prominent physiological role for the TRPM4 channel in the regulation of DSM function in humans than in rodents. In conclusion, TRPM4 channels regulate human DSM excitability and contractility and are critical determinants of human urinary bladder function. Thus, TRPM4 channels could represent promising novel targets for the pharmacological or genetic control of overactive bladder. PMID:26791488

CaV channels and cancer: canonical functions indicate benefits of repurposed drugs as cancer therapeutics.

PubMed

Buchanan, Paul J; McCloskey, Karen D

2016-10-01

The importance of ion channels in the hallmarks of many cancers is increasingly recognised. This article reviews current knowledge of the expression of members of the voltage-gated calcium channel family (Ca V ) in cancer at the gene and protein level and discusses their potential functional roles. The ten members of the Ca V channel family are classified according to expression of their pore-forming α-subunit; moreover, co-expression of accessory α2δ, β and γ confers a spectrum of biophysical characteristics including voltage dependence of activation and inactivation, current amplitude and activation/inactivation kinetics. Ca V channels have traditionally been studied in excitable cells including neurones, smooth muscle, skeletal muscle and cardiac cells, and drugs targeting the channels are used in the treatment of hypertension and epilepsy. There is emerging evidence that several Ca V channels are differentially expressed in cancer cells compared to their normal counterparts. Interestingly, a number of Ca V channels also have non-canonical functions and are involved in transcriptional regulation of the expression of other proteins including potassium channels. Pharmacological studies show that Ca V canonical function contributes to the fundamental biology of proliferation, cell-cycle progression and apoptosis. This raises the intriguing possibility that calcium channel blockers, approved for the treatment of other conditions, could be repurposed to treat particular cancers. Further research will reveal the full extent of both the canonical and non-canonical functions of Ca V channels in cancer and whether calcium channel blockers are beneficial in cancer treatment.

Anandamide Revisited: How Cholesterol and Ceramides Control Receptor-Dependent and Receptor-Independent Signal Transmission Pathways of a Lipid Neurotransmitter.

PubMed

Di Scala, Coralie; Fantini, Jacques; Yahi, Nouara; Barrantes, Francisco J; Chahinian, Henri

2018-05-22

Anandamide is a lipid neurotransmitter derived from arachidonic acid, a polyunsaturated fatty acid. The chemical differences between anandamide and arachidonic acid result in a slightly enhanced solubility in water and absence of an ionisable group for the neurotransmitter compared with the fatty acid. In this review, we first analyze the conformational flexibility of anandamide in aqueous and membrane phases. We next study the interaction of the neurotransmitter with membrane lipids and discuss the molecular basis of the unexpected selectivity of anandamide for cholesterol and ceramide from among other membrane lipids. We show that cholesterol behaves as a binding partner for anandamide, and that following an initial interaction mediated by the establishment of a hydrogen bond, anandamide is attracted towards the membrane interior, where it forms a molecular complex with cholesterol after a functional conformation adaptation to the apolar membrane milieu. The complex is then directed to the anandamide cannabinoid receptor (CB1) which displays a high affinity binding pocket for anandamide. We propose that cholesterol may regulate the entry and exit of anandamide in and out of CB1 by interacting with low affinity cholesterol recognition sites (CARC and CRAC) located in transmembrane helices. The mirror topology of cholesterol binding sites in the seventh transmembrane domain is consistent with the delivery, extraction and flip-flop of anandamide through a coordinated cholesterol-dependent mechanism. The binding of anandamide to ceramide illustrates another key function of membrane lipids which may occur independently of protein receptors. Interestingly, ceramide forms a tight complex with anandamide which blocks the degradation pathway of both lipids and could be exploited for anti-cancer therapies.

The TiPS/TINS lecture: the molecular biology of mammalian glutamate receptor channels.

PubMed

Seeburg, P H

1993-08-01

In native brain membranes the principal excitatory neurotransmitter L-glutamate activates cation-conducting channels with distinct biophysical and pharmacological properties. Molecular cloning has revealed the existence of 16 channel subunits that can assemble in homomeric or heteromeric configurations in vitro to form receptor channels with disparate functional properties. This review describes the different channel types obtained by recombinant means and the genetic mechanisms controlling the expression of functionally important channel structures.

The TINS/TiPS Lecture. The molecular biology of mammalian glutamate receptor channels.

PubMed

Seeburg, P H

1993-09-01

In native brain membranes the principal excitatory neurotransmitter L-glutamate activates cation-conducting channels with distinct biophysical and pharmacological properties. Molecular cloning has revealed the existence of 16 channel subunits that can assemble in homomeric or heteromeric configurations in vitro to form receptor channels with disparate functional properties. This review describes the different channel types obtained by recombinant means and the genetic mechanisms controlling the expression of functionally important channel structures.

Transmembrane S1 mutations in CNGA3 from achromatopsia 2 patients cause loss of function and impaired cellular trafficking of the cone CNG channel.

PubMed

Patel, Kirti A; Bartoli, Kristen M; Fandino, Richard A; Ngatchou, Anita N; Woch, Gustaw; Carey, Jannette; Tanaka, Jacqueline C

2005-07-01

Achromatopsia 2, an inherited retinal disorder resulting in attenuation or loss of cone function, is caused by mutations in the alpha subunit of the cone cyclic nucleotide-gated (CNG) channel gene CNGA3. Examination of mutations that cluster in the first transmembrane segment of the protein may provide insight into its role in CNG channel structure, function, biogenesis, and pathophysiology. The human CNGA3 gene was tagged at the C terminus with green fluorescent protein. Four mutations, Y181C, N182Y, L186F, and C191Y, were expressed in human embryonic kidney cells. Protein expression was evaluated with immunoblot analysis and cellular localization was determined by immunocytochemistry. Channel function was evaluated by patch-clamp electrophysiology. All the mutations result in loss of channel function, as determined by the failure of cGMP to activate wild-type currents in excised patches. Full-length mutant proteins were synthesized but retained in the endoplasmic reticulum. Glycerol treatment did not rescue channel function nor did coexpression with CNGB3, a subunit of native hetero-tetrameric cone channels. A control mutant, C191S, exhibited cGMP current activation with significantly reduced cooperativity, suggesting that mutations in the first transmembrane domain alter in inter- or intrasubunit communication. The results implicate the first transmembrane segment in both maturation and function of CNG channels. The defects are not reversed with glycerol, a chemical chaperone that rescues channel function in some channelopathies. Molecular analysis of achromatopsia 2 mutations may be useful in evaluating potential therapeutic approaches for treatment of this channelopathy.

Lipid microdomains and the regulation of ion channel function

PubMed Central

Dart, Caroline

2010-01-01

Many types of ion channel localize to cholesterol and sphingolipid-enriched regions of the plasma membrane known as lipid microdomains or ‘rafts’. The precise physiological role of these unique lipid microenvironments remains elusive due largely to difficulties associated with studying these potentially extremely small and dynamic domains. Nevertheless, increasing evidence suggests that membrane rafts regulate channel function in a number of different ways. Raft-enriched lipids such as cholesterol and sphingolipids exert effects on channel activity either through direct protein–lipid interactions or by influencing the physical properties of the bilayer. Rafts also appear to selectively recruit interacting signalling molecules to generate subcellular compartments that may be important for efficient and selective signal transduction. Direct interaction with raft-associated scaffold proteins such as caveolin can also influence channel function by altering gating kinetics or by affecting trafficking and surface expression. Selective association of ion channels with specific lipid microenvironments within the membrane is thus likely to be an important and fundamental regulatory aspect of channel physiology. This brief review highlights some of the existing evidence for raft modulation of channel function. PMID:20519314

Functional diversity of potassium channel voltage-sensing domains.

PubMed

Islas, León D

2016-01-01

Voltage-gated potassium channels or Kv's are membrane proteins with fundamental physiological roles. They are composed of 2 main functional protein domains, the pore domain, which regulates ion permeation, and the voltage-sensing domain, which is in charge of sensing voltage and undergoing a conformational change that is later transduced into pore opening. The voltage-sensing domain or VSD is a highly conserved structural motif found in all voltage-gated ion channels and can also exist as an independent feature, giving rise to voltage sensitive enzymes and also sustaining proton fluxes in proton-permeable channels. In spite of the structural conservation of VSDs in potassium channels, there are several differences in the details of VSD function found across variants of Kvs. These differences are mainly reflected in variations in the electrostatic energy needed to open different potassium channels. In turn, the differences in detailed VSD functioning among voltage-gated potassium channels might have physiological consequences that have not been explored and which might reflect evolutionary adaptations to the different roles played by Kv channels in cell physiology.

Functional diversity of potassium channel voltage-sensing domains

PubMed Central

Islas, León D.

2016-01-01

Abstract Voltage-gated potassium channels or Kv's are membrane proteins with fundamental physiological roles. They are composed of 2 main functional protein domains, the pore domain, which regulates ion permeation, and the voltage-sensing domain, which is in charge of sensing voltage and undergoing a conformational change that is later transduced into pore opening. The voltage-sensing domain or VSD is a highly conserved structural motif found in all voltage-gated ion channels and can also exist as an independent feature, giving rise to voltage sensitive enzymes and also sustaining proton fluxes in proton-permeable channels. In spite of the structural conservation of VSDs in potassium channels, there are several differences in the details of VSD function found across variants of Kvs. These differences are mainly reflected in variations in the electrostatic energy needed to open different potassium channels. In turn, the differences in detailed VSD functioning among voltage-gated potassium channels might have physiological consequences that have not been explored and which might reflect evolutionary adaptations to the different roles played by Kv channels in cell physiology. PMID:26794852

IA channels: diverse regulatory mechanisms.

PubMed

Carrasquillo, Yarimar; Nerbonne, Jeanne M

2014-04-01

In many peripheral and central neurons, A-type K(+) currents, IA, have been identified and shown to be key determinants in shaping action potential waveforms and repetitive firing properties, as well as in the regulation of synaptic transmission and synaptic plasticity. The functional properties and physiological roles of native neuronal IA, however, have been shown to be quite diverse in different types of neurons. Accumulating evidence suggests that this functional diversity is generated by multiple mechanisms, including the expression and subcellular distributions of IA channels encoded by different voltage-gated K(+) (Kv) channel pore-forming (α) subunits, interactions of Kv α subunits with cytosolic and/or transmembrane accessory subunits and regulatory proteins and post-translational modifications of channel subunits. Several recent reports further suggest that local protein translation in the dendrites of neurons and interactions between IA channels with other types of voltage-gated ion channels further expands the functional diversity of native neuronal IA channels. Here, we review the diverse molecular mechanisms that have been shown or proposed to underlie the functional diversity of native neuronal IA channels.

Oxidative Modulation of Voltage-Gated Potassium Channels

PubMed Central

Sahoo, Nirakar; Hoshi, Toshinori

2014-01-01

Abstract Significance: Voltage-gated K+ channels are a large family of K+-selective ion channel protein complexes that open on membrane depolarization. These K+ channels are expressed in diverse tissues and their function is vital for numerous physiological processes, in particular of neurons and muscle cells. Potentially reversible oxidative regulation of voltage-gated K+ channels by reactive species such as reactive oxygen species (ROS) represents a contributing mechanism of normal cellular plasticity and may play important roles in diverse pathologies including neurodegenerative diseases. Recent Advances: Studies using various protocols of oxidative modification, site-directed mutagenesis, and structural and kinetic modeling provide a broader phenomenology and emerging mechanistic insights. Critical Issues: Physicochemical mechanisms of the functional consequences of oxidative modifications of voltage-gated K+ channels are only beginning to be revealed. In vivo documentation of oxidative modifications of specific amino-acid residues of various voltage-gated K+ channel proteins, including the target specificity issue, is largely absent. Future Directions: High-resolution chemical and proteomic analysis of ion channel proteins with respect to oxidative modification combined with ongoing studies on channel structure and function will provide a better understanding of how the function of voltage-gated K+ channels is tuned by ROS and the corresponding reducing enzymes to meet cellular needs. Antioxid. Redox Signal. 21, 933–952. PMID:24040918

Functional characterization of Kv11.1 (hERG) potassium channels split in the voltage-sensing domain.

PubMed

de la Peña, Pilar; Domínguez, Pedro; Barros, Francisco

2018-03-23

Voltage-dependent KCNH family potassium channel functionality can be reconstructed using non-covalently linked voltage-sensing domain (VSD) and pore modules (split channels). However, the necessity of a covalent continuity for channel function has not been evaluated at other points within the two functionally independent channel modules. We find here that by cutting Kv11.1 (hERG, KCNH2) channels at the different loops linking the transmembrane spans of the channel core, not only channels split at the S4-S5 linker level, but also those split at the intracellular S2-S3 and the extracellular S3-S4 loops, yield fully functional channel proteins. Our data indicate that albeit less markedly, channels split after residue 482 in the S2-S3 linker resemble the uncoupled gating phenotype of those split at the C-terminal end of the VSD S4 transmembrane segment. Channels split after residues 514 and 518 in the S3-S4 linker show gating characteristics similar to those of the continuous wild-type channel. However, breaking the covalent link at this level strongly accelerates the voltage-dependent accessibility of a membrane impermeable methanethiosulfonate reagent to an engineered cysteine at the N-terminal region of the S4 transmembrane helix. Thus, besides that of the S4-S5 linker, structural integrity of the intracellular S2-S3 linker seems to constitute an important factor for proper transduction of VSD rearrangements to opening and closing the cytoplasmic gate. Furthermore, our data suggest that the short and probably rigid characteristics of the extracellular S3-S4 linker are not an essential component of the Kv11.1 voltage sensing machinery.

Enhanced store-operated Ca2+ influx and ORAI1 expression in ventricular fibroblasts from human failing heart

PubMed Central

Ross, Gracious R.; Bajwa, Tanvir; Edwards, Stacie; Emelyanova, Larisa; Rizvi, Farhan; Holmuhamedov, Ekhson L.; Werner, Paul; Downey, Francis X.; Tajik, A. Jamil

2017-01-01

ABSTRACT Excessive cardiac fibrosis, characterized by increased collagen-rich extracellular matrix (ECM) deposition, is a major predisposing factor for mechanical and electrical dysfunction in heart failure (HF). The human ventricular fibroblast (hVF) remodeling mechanisms that cause excessive collagen deposition in HF are unclear, although reports suggest a role for intracellular free Ca2+ in fibrosis. Therefore, we determined the association of differences in cellular Ca2+ dynamics and collagen secretion/deposition between hVFs from failing and normal (control) hearts. Histology of left ventricle sections (Masson trichrome) confirmed excessive fibrosis in HF versus normal. In vitro, hVFs from HF showed increased secretion/deposition of soluble collagen in 48 h of culture compared with control [85.9±7.4 µg/106 cells vs 58.5±8.8 µg/106 cells, P<0.05; (Sircol™ assay)]. However, collagen gene expressions (COL1A1 and COL1A2; RT-PCR) were not different. Ca2+ imaging (fluo-3) of isolated hVFs showed no difference in the thapsigargin-induced intracellular Ca2+ release capacity (control 16±1.4% vs HF 17±1.1%); however, Ca2+ influx via store-operated Ca2+ entry/Ca2+ release-activated channels (SOCE/CRAC) was significantly (P≤0.05) greater in HF-hVFs (47±3%) compared with non-failing (35±5%). Immunoblotting for ICRAC channel components showed increased ORAI1 expression in HF-hVFs compared with normal without any difference in STIM1 expression. The Pearson's correlation coefficient for co-localization of STIM1/ORAI1 was significantly (P<0.01) greater in HF (0.5±0.01) than control (0.4±0.01) hVFs. The increase in collagen secretion of HF versus control hVFs was eliminated by incubation of hVFs with YM58483 (10 µM), a selective ICRAC inhibitor, for 48 h (66.78±5.87 µg/106 cells vs 55.81±7.09 µg/106 cells, P=0.27). In conclusion, hVFs from HF have increased collagen secretion capacity versus non-failing hearts and this is related to increase in Ca2+ entry via SOCE and enhanced expression of ORAI, the pore-forming subunit. Therapeutic inhibition of SOCE may reduce the progression of cardiac fibrosis/HF. PMID:28126709

Transient Receptor Potential Channels in the Vasculature

PubMed Central

Earley, Scott; Brayden, Joseph E.

2015-01-01

The mammalian genome encodes 28 distinct members of the transient receptor potential (TRP) superfamily of cation channels, which exhibit varying degrees of selectivity for different ionic species. Multiple TRP channels are present in all cells and are involved in diverse aspects of cellular function, including sensory perception and signal transduction. Notably, TRP channels are involved in regulating vascular function and pathophysiology, the focus of this review. TRP channels in vascular smooth muscle cells participate in regulating contractility and proliferation, whereas endothelial TRP channel activity is an important contributor to endothelium-dependent vasodilation, vascular wall permeability, and angiogenesis. TRP channels are also present in perivascular sensory neurons and astrocytic endfeet proximal to cerebral arterioles, where they participate in the regulation of vascular tone. Almost all of these functions are mediated by changes in global intracellular Ca2+ levels or subcellular Ca2+ signaling events. In addition to directly mediating Ca2+ entry, TRP channels influence intracellular Ca2+ dynamics through membrane depolarization associated with the influx of cations or through receptor- or store-operated mechanisms. Dysregulation of TRP channels is associated with vascular-related pathologies, including hypertension, neointimal injury, ischemia-reperfusion injury, pulmonary edema, and neurogenic inflammation. In this review, we briefly consider general aspects of TRP channel biology and provide an in-depth discussion of the functions of TRP channels in vascular smooth muscle cells, endothelial cells, and perivascular cells under normal and pathophysiological conditions. PMID:25834234

Molecular Aspects of Structure, Gating, and Physiology of pH-Sensitive Background K2P and Kir K+-Transport Channels

PubMed Central

Sepúlveda, Francisco V.; Pablo Cid, L.; Teulon, Jacques; Niemeyer, María Isabel

2015-01-01

K+ channels fulfill roles spanning from the control of excitability to the regulation of transepithelial transport. Here we review two groups of K+ channels, pH-regulated K2P channels and the transport group of Kir channels. After considering advances in the molecular aspects of their gating based on structural and functional studies, we examine their participation in certain chosen physiological and pathophysiological scenarios. Crystal structures of K2P and Kir channels reveal rather unique features with important consequences for the gating mechanisms. Important tasks of these channels are discussed in kidney physiology and disease, K+ homeostasis in the brain by Kir channel-equipped glia, and central functions in the hearing mechanism in the inner ear and in acid secretion by parietal cells in the stomach. K2P channels fulfill a crucial part in central chemoreception probably by virtue of their pH sensitivity and are central to adrenal secretion of aldosterone. Finally, some unorthodox behaviors of the selectivity filters of K2P channels might explain their normal and pathological functions. Although a great deal has been learned about structure, molecular details of gating, and physiological functions of K2P and Kir K+-transport channels, this has been only scratching at the surface. More molecular and animal studies are clearly needed to deepen our knowledge. PMID:25540142

Conopeptide Vt3.1 preferentially inhibits BK potassium channels containing β4 subunits via electrostatic interactions.

PubMed

Li, Min; Chang, Shan; Yang, Longjin; Shi, Jingyi; McFarland, Kelli; Yang, Xiao; Moller, Alyssa; Wang, Chunguang; Zou, Xiaoqin; Chi, Chengwu; Cui, Jianmin

2014-02-21

BK channel β subunits (β1-β4) modulate the function of channels formed by slo1 subunits to produce tissue-specific phenotypes. The molecular mechanism of how the homologous β subunits differentially alter BK channel functions and the role of different BK channel functions in various physiologic processes remain unclear. By studying channels expressed in Xenopus laevis oocytes, we show a novel disulfide-cross-linked dimer conopeptide, Vt3.1 that preferentially inhibits BK channels containing the β4 subunit, which is most abundantly expressed in brain and important for neuronal functions. Vt3.1 inhibits the currents by a maximum of 71%, shifts the G-V relation by 45 mV approximately half-saturation concentrations, and alters both open and closed time of single channel activities, indicating that the toxin alters voltage dependence of the channel. Vt3.1 contains basic residues and inhibits voltage-dependent activation by electrostatic interactions with acidic residues in the extracellular loops of the slo1 and β4 subunits. These results suggest a large interaction surface between the slo1 subunit of BK channels and the β4 subunit, providing structural insight into the molecular interactions between slo1 and β4 subunits. The results also suggest that Vt3.1 is an excellent tool for studying β subunit modulation of BK channels and for understanding the physiological roles of BK channels in neurophysiology.

The Role of Auxiliary Subunits for the Functional Diversity of Voltage-Gated Calcium Channels

PubMed Central

Campiglio, Marta; Flucher, Bernhard E

2015-01-01

Voltage-gated calcium channels (VGCCs) represent the sole mechanism to convert membrane depolarization into cellular functions like secretion, contraction, or gene regulation. VGCCs consist of a pore-forming α1 subunit and several auxiliary channel subunits. These subunits come in multiple isoforms and splice-variants giving rise to a stunning molecular diversity of possible subunit combinations. It is generally believed that specific auxiliary subunits differentially regulate the channels and thereby contribute to the great functional diversity of VGCCs. If auxiliary subunits can associate and dissociate from pre-existing channel complexes, this would allow dynamic regulation of channel properties. However, most auxiliary subunits modulate current properties very similarly, and proof that any cellular calcium channel function is indeed modulated by the physiological exchange of auxiliary subunits is still lacking. In this review we summarize available information supporting a differential modulation of calcium channel functions by exchange of auxiliary subunits, as well as experimental evidence in support of alternative functions of the auxiliary subunits. At the heart of the discussion is the concept that, in their native environment, VGCCs function in the context of macromolecular signaling complexes and that the auxiliary subunits help to orchestrate the diverse protein–protein interactions found in these calcium channel signalosomes. Thus, in addition to a putative differential modulation of current properties, differential subcellular targeting properties and differential protein–protein interactions of the auxiliary subunits may explain the need for their vast molecular diversity. J. Cell. Physiol. 999: 00–00, 2015. © 2015 The Authors. Journal of Cellular Physiology Published by Wiley Periodicals, Inc. J. Cell. Physiol. 230: 2019–2031, 2015. © 2015 Wiley Periodicals, Inc. PMID:25820299

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

PubMed Central

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

2015-01-01

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

pH and external Ca(2+) regulation of a small conductance Cl(-) channel in kidney distal tubule.

PubMed

Sauvé, R; Cai, S; Garneau, L; Klein, H; Parent, L

2000-12-20

A single channel characterization of the Cl(-) channels in distal nephron was undertaken using vesicles prepared from plasma membranes of isolated rabbit distal tubules. The presence in this vesicle preparation of ClC-K type Cl(-) channels was first established by immunodetection using an antibody raised against ClC-K isoforms. A ClC-K1 based functional characterization was next performed by investigating the pH and external Ca(2+) regulation of a small conductance Cl(-) channel which we identified previously by channel incorporation experiments. Acidification of the cis (external) solution from pH 7.4 to 6.5 led to a dose-dependent inhibition of the channel open probability P(O). Similarly, changing the trans pH from 7.4 to 6.8 resulted in a 4-fold decrease of the channel P(O) with no effect on the channel conductance. Channel activity also appeared to be regulated by cis (external) Ca(2+) concentration, with a dose-dependent increase in channel activity as a function of the cis Ca(2+) concentration. It is concluded on the basis of these results that the small conductance Cl(-) channel present in rabbit distal tubules is functionally equivalent to the ClC-K1 channel in the rat. In addition, the present work constitutes the first single channel evidence for a chloride channel regulated by external Ca(2+).

Regulation of T-type Ca2+ channel expression by herpes simplex virus-1 infection in sensory-like ND7 cells

PubMed Central

Zhang, Qiaojuan; Hsia, Shao-Chung

2017-01-01

Infection of sensory neurons by herpes simplex virus (HSV)-1 disrupts electrical excitability, altering pain sensory transmission. Because of their low threshold for activation, functional expression of T-type Ca2+ channels regulates various cell functions, including neuronal excitability and neuronal communication. In this study, we have tested the effect of HSV-1 infection on the functional expression of T-type Ca2+ channels in differentiated ND7-23 sensory-like neurons. Voltage-gated Ca2+ currents were measured using whole cell patch clamp recordings in differentiated ND7-23 neurons under various culture conditions. Differentiation of ND7-23 cells evokes a significant increase in T-type Ca2+ current densities. Increased T-type Ca2+ channel expression promotes the morphological differentiation of ND7-23 cells and triggers a rebound depolarization. HSV-1 infection of differentiated ND7-23 cells causes a significant loss of T-type Ca2+ channels from the membrane. HSV-1 evoked reduction in the functional expression of T-type Ca2+ channels is mediated by several factors, including decreased expression of Cav3.2 T-type Ca2+ channel subunits and disruption of endocytic transport. Decreased functional expression of T-type Ca2+ channels by HSV-1 infection requires protein synthesis and viral replication, but occurs independently of Egr-1 expression. These findings suggest that infection of neuron-like cells by HSV-1 causes a significant disruption in the expression of T-type Ca2+ channels, which can results in morphological and functional changes in electrical excitability. PMID:28639215

Protons stabilize the closed conformation of gain-of-function mutants of the TRPV1 channel.

PubMed

Boukalova, Stepana; Teisinger, Jan; Vlachova, Viktorie

2013-03-01

The vanilloid transient receptor potential channel TRPV1 is a molecular integrator of noxious stimuli, including capsaicin, heat and protons. Despite clear similarities between the overall architecture of TRPV1 and voltage-dependent potassium (Kv) channels, the extent of conservation in the molecular logic for gating is unknown. In Kv channels, a small contact surface between S1 and the pore-helix is required for channel functioning. To explore the function of S1 in TRPV1, we used tryptophan-scanning mutagenesis and characterized the responses to capsaicin and protons. Wild-type-like currents were generated in 9 out of 17 mutants; three mutants (M445W, A452W, R455W) were non-functional. The conservative mutation R455K in the extracellular extent of S1 slowed down capsaicin-induced activation and prevented normal channel closure. This mutant was neither activated nor potentiated by protons, on the contrary, protons promoted a rapid deactivation of its currents. Similar phenotypes were found in two other gain-of-function mutants and also in the pore-helix mutant T633A, known to uncouple proton activation. We propose that the S1 domain contains a functionally important region that may be specifically involved in TRPV1 channel gating, and thus be important for the energetic coupling between S1-S4 sensor activation and gate opening. Analogous to Kv channels, the S1-pore interface might serve to stabilize conformations associated with TRPV1 channel gating. Copyright © 2012 Elsevier B.V. All rights reserved.

20 CFR 200.2 - The general course and method by which the Board's functions are channeled and determined.

Code of Federal Regulations, 2010 CFR

2010-04-01

... 20 Employees' Benefits 1 2010-04-01 2010-04-01 false The general course and method by which the Board's functions are channeled and determined. 200.2 Section 200.2 Employees' Benefits RAILROAD... the Board's functions are channeled and determined. (a) Retirement and death benefits. (1) Retirement...

Differential Effects of TRPA and TRPV Channels on Behaviors of Caenorhabditis elegans

PubMed Central

Thies, Jennifer; Neutzler, Vanessa; O’Leary, Fidelma; Liu, He

2016-01-01

TRPA and TRPV ion channels are members of the transient receptor potential (TRP) cation channel superfamily, which mediates various sensory transductions. In Caenorhabditis elegans, the TRPV channels are known to affect chemosensation, while the TRPA-1 channel is associated with thermosensation and mechanosensation. We examined thermosensation, chemosensation, and osmosensation in strains lacking TRPA-1 or TRPV channels. We found that TRPV channel knockout worms exhibited similar behavioral deficits associated with thermotaxis as the TRPA-1 channel knockout, suggesting a dual role for TRPV channels. In contrast, chemosensation responses, assessed by both avoidance reversal behavior and NaCl osmosensation, were dependent on TRPV channels but seemed independent of TRPA-1 channel. Our findings suggest that, in addition to TRPA-1 channel, TRPV channels are necessary for thermotaxis and may activate, or modulate, the function of TRPA-1 channels. In contrast, TRPA-1 channels do not have a dual responsibility, as they have no functional role in odorant avoidance or osmosensation. PMID:27168724

Combined effects of VX-770 and VX-809 on several functional abnormalities of F508del-CFTR channels.

PubMed

Kopeikin, Z; Yuksek, Z; Yang, H-Y; Bompadre, S G

2014-09-01

The most common cystic fibrosis-associated mutation, the deletion of phenylalanine 508 (F508del), results in channels with poor membrane expression and impaired function. VX-770, a clinically approved drug for treatment of CF patients carrying the G551D mutation, and VX-809, a corrector shown in vitro to increase membrane expression of mutant channels, are currently undergoing clinical trials, but functional data at the molecular level is still lacking. The effect of VX-770 and VX-809 on the multiple functional defects of F508del-CFTR was assessed via excised inside-out patch-clamp experiments. VX-770 completely restores the low opening-rate of F508del-CFTR, with smaller open-time increase, in temperature-corrected and VX-809-treated channels. The shorter locked-open time of hydrolysis-deficient F508del-CFTR is also prolonged by VX-770. VX-809 does not improve channel function by itself as previously reported. The results from these studies can be interpreted as an equilibrium shift toward the open-channel conformation of F508del-CFTR channels. Copyright © 2014 European Cystic Fibrosis Society. Published by Elsevier B.V. All rights reserved.

Ether-à-go-go family voltage-gated K+ channels evolved in an ancestral metazoan and functionally diversified in a cnidarian-bilaterian ancestor.

PubMed

Li, Xiaofan; Martinson, Alexandra S; Layden, Michael J; Diatta, Fortunay H; Sberna, Anna P; Simmons, David K; Martindale, Mark Q; Jegla, Timothy J

2015-02-15

We examined the evolutionary origins of the ether-à-go-go (EAG) family of voltage-gated K(+) channels, which have a strong influence on the excitability of neurons. The bilaterian EAG family comprises three gene subfamilies (Eag, Erg and Elk) distinguished by sequence conservation and functional properties. Searches of genome sequence indicate that EAG channels are metazoan specific, appearing first in ctenophores. However, phylogenetic analysis including two EAG family channels from the ctenophore Mnemiopsis leidyi indicates that the diversification of the Eag, Erg and Elk gene subfamilies occurred in a cnidarian/bilaterian ancestor after divergence from ctenophores. Erg channel function is highly conserved between cnidarians and mammals. Here we show that Eag and Elk channels from the sea anemone Nematostella vectensis (NvEag and NvElk) also share high functional conservation with mammalian channels. NvEag, like bilaterian Eag channels, has rapid kinetics, whereas NvElk activates at extremely hyperpolarized voltages, which is characteristic of Elk channels. Potent inhibition of voltage activation by extracellular protons is conserved between mammalian and Nematostella EAG channels. However, characteristic inhibition of voltage activation by Mg(2+) in Eag channels and Ca(2+) in Erg channels is reduced in Nematostella because of mutation of a highly conserved aspartate residue in the voltage sensor. This mutation may preserve sub-threshold activation of Nematostella Eag and Erg channels in a high divalent cation environment. mRNA in situ hybridization of EAG channels in Nematostella suggests that they are differentially expressed in distinct cell types. Most notable is the expression of NvEag in cnidocytes, a cnidarian-specific stinging cell thought to be a neuronal subtype. © 2015. Published by The Company of Biologists Ltd.

[Architecture of receptor-operated ionic channels of biological membranes].

PubMed

Bregestovski, P D

2011-01-01

Ion channels of biological membranes are the key proteins, which provide bioelectric functioning of living systems. These proteins are homo- or heterooligomers assembled from several identical or different subunits. Understanding the architectural organization and functioning of ion channels has been significantly extended due to resolving the crystal structure of several types of voltage-gated and receptor-operated channels. This review summarizes the information obtained from crystal structures of potassium, nicotinic acetylcholine receptor, P2X, and other ligand-gated ion channels. Despite the differences in the function, topology, ionic selectivity, and the subunit stoichiometry, a high similarity in the principles of organization of these macromolecular complexes has been revealed.

TMEM16 proteins: unknown structure and confusing functions.

PubMed

Picollo, Alessandra; Malvezzi, Mattia; Accardi, Alessio

2015-01-16

The TMEM16 family of membrane proteins, also known as anoctamins, plays key roles in a variety of physiological functions that range from ion transport to phospholipid scrambling and to regulating other ion channels. The first two family members to be functionally characterized, TMEM16A (ANO1) and TMEM16B (ANO2), form Ca(2+)-activated Cl(-) channels and are important for transepithelial ion transport, olfaction, phototransduction, smooth muscle contraction, nociception, cell proliferation and control of neuronal excitability. The roles of other family members, such as TMEM16C (ANO3), TMEM16D (ANO4), TMEM16F (ANO6), TMEM16G (ANO7) and TMEM16J (ANO9), remain poorly understood and controversial. These homologues were reported to be phospholipid scramblases, ion channels, to have both functions or to be regulatory subunits of other channels. Mutations in TMEM16F cause Scott syndrome, a bleeding disorder caused by impaired Ca(2+)-dependent externalization of phosphatidylserine in activated platelets, suggesting that this homologue might be a scramblase. However, overexpression of TMEM16F has also been associated with a remarkable number of different ion channel types, raising the possibility that this protein might be involved in both ion and lipid transports. The recent identification of an ancestral TMEM16 homologue with intrinsic channel and scramblase activities supports this hypothesis. Thus, the TMEM16 family might have diverged in two or three different subclasses, channels, scramblases and dual-function channel/scramblases. The structural bases and functional implication of such a functional diversity within a single protein family remain to be elucidated and the links between TMEM16 functions and human physiology and pathologies need to be investigated. Copyright © 2014 Elsevier Ltd. All rights reserved.

Connexin channels and phospholipids: association and modulation

PubMed Central

Locke, Darren; Harris, Andrew L

2009-01-01

Background For membrane proteins, lipids provide a structural framework and means to modulate function. Paired connexin hemichannels form the intercellular channels that compose gap junction plaques while unpaired hemichannels have regulated functions in non-junctional plasma membrane. The importance of interactions between connexin channels and phospholipids is poorly understood. Results Endogenous phospholipids most tightly associated with purified connexin26 or connexin32 hemichannels or with junctional plaques in cell membranes, those likely to have structural and/or modulatory effects, were identified by tandem electrospray ionization-mass spectrometry using class-specific interpretative methods. Phospholipids were characterized by headgroup class, charge, glycerol-alkyl chain linkage and by acyl chain length and saturation. The results indicate that specific endogenous phospholipids are uniquely associated with either connexin26 or connexin32 channels, and some phospholipids are associated with both. Functional effects of the major phospholipid classes on connexin channel activity were assessed by molecular permeability of hemichannels reconstituted into liposomes. Changes to phospholipid composition(s) of the liposome membrane altered the activity of connexin channels in a manner reflecting changes to the surface charge/potential of the membrane and, secondarily, to cholesterol content. Together, the data show that connexin26 and connexin32 channels have a preference for tight association with unique anionic phospholipids, and that these, independent of headgroup, have a positive effect on the activity of both connexin26 and connexin32 channels. Additionally, the data suggest that the likely in vivo phospholipid modulators of connexin channel structure-function that are connexin isoform-specific are found in the cytoplasmic leaflet. A modulatory role for phospholipids that promote negative curvature is also inferred. Conclusion This study is the first to identify (endogenous) phospholipids that tightly associate with connexin channels. The finding that specific phospholipids are associated with different connexin isoforms suggests connexin-specific regulatory and/or structural interactions with lipid membranes. The results are interpreted in light of connexin channel function and cell biology, as informed by current knowledge of lipid-protein interactions and membrane biophysics. The intimate involvement of distinct phospholipids with different connexins contributes to channel structure and/or function, as well as plaque integrity, and to modulation of connexin channels by lipophilic agents. PMID:19686581

A computer program for analyzing channel geometry

USGS Publications Warehouse

Regan, R.S.; Schaffranek, R.W.

1985-01-01

The Channel Geometry Analysis Program (CGAP) provides the capability to process, analyze, and format cross-sectional data for input to flow/transport simulation models or other computational programs. CGAP allows for a variety of cross-sectional data input formats through use of variable format specification. The program accepts data from various computer media and provides for modification of machine-stored parameter values. CGAP has been devised to provide a rapid and efficient means of computing and analyzing the physical properties of an open-channel reach defined by a sequence of cross sections. CGAP 's 16 options provide a wide range of methods by which to analyze and depict a channel reach and its individual cross-sectional properties. The primary function of the program is to compute the area, width, wetted perimeter, and hydraulic radius of cross sections at successive increments of water surface elevation (stage) from data that consist of coordinate pairs of cross-channel distances and land surface or channel bottom elevations. Longitudinal rates-of-change of cross-sectional properties are also computed, as are the mean properties of a channel reach. Output products include tabular lists of cross-sectional area, channel width, wetted perimeter, hydraulic radius, average depth, and cross-sectional symmetry computed as functions of stage; plots of cross sections; plots of cross-sectional area and (or) channel width as functions of stage; tabular lists of cross-sectional area and channel width computed as functions of stage for subdivisions of a cross section; plots of cross sections in isometric projection; and plots of cross-sectional area at a fixed stage as a function of longitudinal distance along an open-channel reach. A Command Procedure Language program and Job Control Language procedure exist to facilitate program execution on the U.S. Geological Survey Prime and Amdahl computer systems respectively. (Lantz-PTT)

Calcium channels in chicken sperm regulate motility and the acrosome reaction.

PubMed

Nguyen, Thi Mong Diep; Duittoz, Anne; Praud, Christophe; Combarnous, Yves; Blesbois, Elisabeth

2016-05-01

Intracellular cytoplasmic calcium ([Ca(2+) ]i ) has an important regulatory role in gamete functions. However, the biochemical components involved in Ca(2+) transport are still unknown in birds, an animal class that has lost functional sperm-specific CatSper channels. Here, we provide evidence for the presence and expression of various Ca(2+) channels in chicken sperm, including high voltage-activated channels (L and R types), the store-operated Ca(2+) channel (SOC) component Orai1, the transient receptor potential channel (TRPC1) and inositol-1,4,5-trisphosphate receptors (IP3 R1). L- and R-type channels were mainly localized in the acrosome and the midpiece, and T-type channels were not detected in chicken sperm. Orai1 was found in all compartments, but with a weak, diffuse signal in the flagellum. TRCP1 was mainly localized in the acrosome and the midpiece, but a weak diffuse signal was also observed in the nucleus and the flagellum. IP3 R1 was mainly detected in the nucleus. The L-type channel inhibitor nifedipine, the R-type channel inhibitor SNX-482 and the SOC inhibitors MRS-1845, 2-APB and YM-58483 decreased [Ca(2+) ]i sperm motility and acrosome reaction capability, with the SOC inhibitors inhibiting these functions most efficiently. Furthermore, we showed that Ca(2+) -mediated induction of AMP-activated protein kinase (AMPK) phosphorylation was blocked by SOC inhibition. Our identification of important regulators of Ca(2+) signaling in avian sperm suggests that SOCs play a predominant role in gamete function, whereas T-type channels may not be involved. In addition, Ca(2+) entry via SOCs appears to be the most likely pathway for AMPK activation and energy-requiring sperm functions such as motility and the acrosome reaction. © 2016 Federation of European Biochemical Societies.

Subconductance states of mitochondrial chloride channels: implication for functionally-coupled tetramers.

PubMed

Tomasek, Milan; Misak, Anton; Grman, Marian; Tomaskova, Zuzana

2017-08-01

Recently, it has been discovered that isoforms of intracellular chloride channels (CLIC) are present in cardiac mitochondria. By reconstituting rat cardiac mitochondrial chloride channels into bilayer lipid membranes, we detected three equally separated subconductance states with conductance increment of 45 pS and < 2% occupancy. The observed rare events of channel decomposition into substates, accompanied by disrupted gating, provide an insight into channel quaternary structure. Our findings suggest that the observed channels work as four functionally coupled subunits with synchronized gating. We discuss the putative connection of channel activity from native mitochondria with the recombinant CLIC channels. However, conclusive evidence is needed to prove this connection. © 2017 Federation of European Biochemical Societies.

Pharmacological rescue of trafficking-impaired ATP-sensitive potassium channels

PubMed Central

Martin, Gregory M.; Chen, Pei-Chun; Devaraneni, Prasanna; Shyng, Show-Ling

2013-01-01

ATP-sensitive potassium (KATP) channels link cell metabolism to membrane excitability and are involved in a wide range of physiological processes including hormone secretion, control of vascular tone, and protection of cardiac and neuronal cells against ischemic injuries. In pancreatic β-cells, KATP channels play a key role in glucose-stimulated insulin secretion, and gain or loss of channel function results in neonatal diabetes or congenital hyperinsulinism, respectively. The β-cell KATP channel is formed by co-assembly of four Kir6.2 inwardly rectifying potassium channel subunits encoded by KCNJ11 and four sulfonylurea receptor 1 subunits encoded by ABCC8. Many mutations in ABCC8 or KCNJ11 cause loss of channel function, thus, congenital hyperinsulinism by hampering channel biogenesis and hence trafficking to the cell surface. The trafficking defects caused by a subset of these mutations can be corrected by sulfonylureas, KATP channel antagonists that have long been used to treat type 2 diabetes. More recently, carbamazepine, an anticonvulsant that is thought to target primarily voltage-gated sodium channels has been shown to correct KATP channel trafficking defects. This article reviews studies to date aimed at understanding the mechanisms by which mutations impair channel biogenesis and trafficking and the mechanisms by which pharmacological ligands overcome channel trafficking defects. Insight into channel structure-function relationships and therapeutic implications from these studies are discussed. PMID:24399968

G protein modulation of CaV2 voltage-gated calcium channels.

PubMed

Currie, Kevin P M

2010-01-01

Voltage-gated Ca(2+) channels translate the electrical inputs of excitable cells into biochemical outputs by controlling influx of the ubiquitous second messenger Ca(2+) . 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 Gβγ to the α1 subunit of CaV2 channels. This article will review the basics of Ca(2+) -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 Gβγ to the channel and the voltage-dependence that is a signature characteristic of this mechanism.

Two-Dimensional Covalent Organic Frameworks for Carbon Dioxide Capture through Channel-Wall Functionalization

PubMed Central

Huang, Ning; Chen, Xiong; Krishna, Rajamani; Jiang, Donglin

2015-01-01

Ordered open channels found in two-dimensional covalent organic frameworks (2D COFs) could enable them to adsorb carbon dioxide. However, the frameworks’ dense layer architecture results in low porosity that has thus far restricted their potential for carbon dioxide adsorption. Here we report a strategy for converting a conventional 2D COF into an outstanding platform for carbon dioxide capture through channel-wall functionalization. The dense layer structure enables the dense integration of functional groups on the channel walls, creating a new version of COFs with high capacity, reusability, selectivity, and separation productivity for flue gas. These results suggest that channel-wall functional engineering could be a facile and powerful strategy to develop 2D COFs for high-performance gas storage and separation. PMID:25613010

The CSM and the NCO Support Channel.

DTIC Science & Technology

1988-03-30

the NCO support channel functions . And it would be wrong to blame the CSM for conflicts between the chain of command and the NCO support channel. It is...model for all noncommissioned officers and soldiers of the unit. However, Command Sergeant Majors are conditioned to function in a dual channel of ... the premier role model for all noncommissioned officers and soldiers of the unit.

Channels Active in the Excitability of Nerves and Skeletal Muscles across the Neuromuscular Junction: Basic Function and Pathophysiology

ERIC Educational Resources Information Center

Goodman, Barbara E.

2008-01-01

Ion channels are essential for the basic physiological function of excitable cells such as nerve, skeletal, cardiac, and smooth muscle cells. Mutations in genes that encode ion channels have been identified to cause various diseases and disorders known as channelopathies. An understanding of how individual ion channels are involved in the…

Cellular Mechanism of Inner Ear Genetic Disease, roles of Kv7.1 (KCNQ1) Channel

NASA Astrophysics Data System (ADS)

Mousavi Nik, Atefeh

Potassium channels are the most diverse and widely distributed membrane protein in all living organisms. They have various roles in the body such as controlling membrane potential, cell volume, and cell migration. Many studies have shown that mutation in these channels is associated with different diseases for example: Hearing Defect, Cardiac Arrhythmia, Episodic Ataxia, Seizure and Neuromyotonia. One of the most important diseases associated with K+ channel mutations is called Jervell and Lange-Nielsen syndrome (JLNS). This disease causes bilateral congenital deafness and the patients also suffer from Long QT and they usually experience syncopal episodes in their life and eventually die as a result of cardiac arrest. The gene KCNQ1 encodes the Kv7.1 voltage gated potassium channel. This channel expresses in apical membrane of marginal cell in stria vasularis of cochlea and secret K+ ion to endolymp to keep the endocochlear potential stable, which is necessary for the inner ear to function properly. Kv7.1 channel also expresses in cardiac myocytes and mutation in this gene is associated with another syndrome called Romano-Ward syndrome (RWS). Although Romano-Ward patients have mutation in KCNQ1, similar to Jervell and Lange-Nielsen patients, they only suffer from cardiac defect, and their hearing is completely normal. Several studies identified that mutations in Kv7.1 gene is associated with JLNS and RWS, but the biophysical and cellular mechanisms of these mutations are still unknown. To determine the cellular mechanisms of JLNS and RWS, and to provide mechanistic insight on the functional outputs of JLNS versus RWS mutations, we generated several mutant forms of the human Kv7.1 ( KCNQ1) clone, using site-directed mutagenesis to define their sub-cellular localization and examined their electrophysiological properties. We identified JLNS and RWS mutations at the S4-S5-linker, the pore loop (P-loop) and the C-terminus of hKv7.1 which have been found to control channel gating, permeation and modulation, respectively. The result showed that for JLNS, all P-loop and C-terminal mutations (seven mutations) yielded non-functional channels when expressed alone. Moreover, the W248F at the end of the S4 domain yielded a functional current, but it became inactivated at positive step potentials, therefore the channel essentially is non-functional. All the JLNS mutant channels are non-functional, and have impaired membrane trafficking. In contrast, the RWS mutants showed wide-ranging functional phenotypes consisting of channels with large current, channel with no measurable current when expressed alone, but significant current upon addition of the WT subunit and finally channel with no measurable current even in presence of WT subunit. The RWS mutants, however, produced dominant negative effect. These findings provide integrated cellular and molecular mechanisms of hKv7.1 functions and the consequent diseased phenotype in JLNS and RWS may come from the tissue-specific function of the channel.

D1 receptors physically interact with N-type calcium channels to regulate channel distribution and dendritic calcium entry.

PubMed

Kisilevsky, Alexandra E; Mulligan, Sean J; Altier, Christophe; Iftinca, Mircea C; Varela, Diego; Tai, Chao; Chen, Lina; Hameed, Shahid; Hamid, Jawed; Macvicar, Brian A; Zamponi, Gerald W

2008-05-22

Dopamine signaling through D1 receptors in the prefrontal cortex (PFC) plays a critical role in the maintenance of higher cognitive functions, such as working memory. At the cellular level, these functions are predicated to involve alterations in neuronal calcium levels. The dendrites of PFC neurons express D1 receptors and N-type calcium channels, yet little information exists regarding their coupling. Here, we show that D1 receptors potently inhibit N-type channels in dendrites of rat PFC neurons. Using coimmunoprecipitation, we demonstrate the existence of a D1 receptor-N-type channel signaling complex in this region, and we provide evidence for a direct receptor-channel interaction. Finally, we demonstrate the importance of this complex to receptor-channel colocalization in heterologous systems and in PFC neurons. Our data indicate that the N-type calcium channel is an important physiological target of D1 receptors and reveal a mechanism for D1 receptor-mediated regulation of cognitive function in the PFC.

Channel Access in Erlang

DOE Office of Scientific and Technical Information (OSTI.GOV)

Nicklaus, Dennis J.

2013-10-13

We have developed an Erlang language implementation of the Channel Access protocol. Included are low-level functions for encoding and decoding Channel Access protocol network packets as well as higher level functions for monitoring or setting EPICS process variables. This provides access to EPICS process variables for the Fermilab Acnet control system via our Erlang-based front-end architecture without having to interface to C/C++ programs and libraries. Erlang is a functional programming language originally developed for real-time telecommunications applications. Its network programming features and list management functions make it particularly well-suited for the task of managing multiple Channel Access circuits and PVmore » monitors.« less

SLO-2 potassium channel is an important regulator of neurotransmitter release in Caenorhabditis elegans

PubMed Central

Liu, Ping; Chen, Bojun; Wang, Zhao-Wen

2014-01-01

Slo2 channels are prominent K+ channels in mammalian neurons but their physiological functions are not well understood. Here we investigate physiological functions and regulation of the C. elegans homologue SLO-2 in motor neurons through electrophysiological analyses of wild-type and mutant worms. We find that SLO-2 is the primary K+ channel conducting delayed outward current in cholinergic motor neurons, and one of two K+ channels with this function in GABAergic motor neurons. Loss-of-function mutation of slo-2 increases the duration and charge transfer rate of spontaneous postsynaptic current bursts at the neuromuscular junction, which are physiological signals used by motor neurons to control muscle cells, without altering postsynaptic receptor sensitivity. SLO-2 activity in motor neurons depends on Ca2+ entry through EGL-19, an L-type voltage-gated Ca2+ channel (CaV1), but not on other proteins implicated in either Ca2+ entry or intracellular Ca2+ release. Thus, SLO-2 is functionally coupled with CaV1 and regulates neurotransmitter release. PMID:25300429

Expression and function of CLC and cystic fibrosis transmembrane conductance regulator chloride channels in renal epithelial tubule cells: pathophysiological implications.

PubMed

Vandewalle, Alain

2007-01-01

Cl(-) channels play important roles in the regulation of a variety of functions, including electrical excitability, cell volume regulation, transepithelial transport and acidification of cellular organelles. They are expressed in plasma membranes or reside in intracellular organelles. A large number of Cl(-) channels with different functions have been identified. Some of them are highly expressed in the kidney. They include members of the CLC Cl(-) channel family: ClC-K1 (or ClC-Ka), ClC-K2 (or ClC-Kb) and ClC-5. The identification of mutations responsible for human inherited diseases (Bartter syndrome for ClC-Kb and Dent's disease for ClC-5) and studies on knockout mice models have evidenced the physiological importance of these CLC Cl(-) channels, permitting better understanding on their functions in renal tubule epithelial cells. The cystic fibrosis transmembrane conductance regulator (CFTR) Cl(-) channel, also expressed in renal tubule epithelial cells, is involved in the transepithelial transport of Cl(-) in the distal nephron. This short review focuses on intrarenal distribution, subcellular localization and function of the ClK(-1), ClC-K2 and ClC-5 Cl(-) channels in renal tubule epithelial cells, and the role of the CFTR Cl(-) channel in chloride fluxes elicited by vasopressin in the distal nephron.

Inhibition of cardiac inward rectifier currents by cationic amphiphilic drugs.

PubMed

van der Heyden, M A G; Stary-Weinzinger, A; Sanchez-Chapula, J A

2013-09-01

Cardiac inward rectifier channels belong to three different classes of the KIR channel protein family. The KIR2.x proteins generate the classical inward rectifier current, IK1, while KIR3 and KIR6 members are responsible for the acetylcholine responsive and ATP sensitive inward rectifier currents IKAch and IKATP, respectively. Aberrant function of these channels has been correlated with severe cardiac arrhythmias, indicating their significant contribution to normal cardiac electrophysiology. A common feature of inward rectifier channels is their dependence on the lipid phosphatidyl-4,5-bisphospate (PIP2) interaction for functional activity. Cationic amphiphilic drugs (CADs) are one of the largest classes of pharmaceutical compounds. Several widely used CADs have been associated with inward rectifier current disturbances, and recent evidence points to interference of the channel-PIP2 interaction as the underlying mechanism of action. Here, we will review how six of these well known drugs, used for treatment in various different conditions, interfere in cardiac inward rectifier functioning. In contrast, KIR channel inhibition by the anionic anesthetic thiopental is achieved by a different mechanism of channel-PIP2 interference. We will discuss the latest basic science insights of functional inward rectifier current characteristics, recently derived KIR channel structures and specific PIP2-receptor interactions at the molecular level and provide insight in how these drugs interfere in the structure-function relationships.

International Union of Basic and Clinical Pharmacology. LXXVI. Current Progress in the Mammalian TRP Ion Channel Family

PubMed Central

Wu, Long-Jun; Sweet, Tara-Beth

2010-01-01

Transient receptor potential (TRP) channels are a large family of ion channel proteins, surpassed in number in mammals only by voltage-gated potassium channels. TRP channels are activated and regulated through strikingly diverse mechanisms, making them suitable candidates for cellular sensors. They respond to environmental stimuli such as temperature, pH, osmolarity, pheromones, taste, and plant compounds, and intracellular stimuli such as Ca2+ and phosphatidylinositol signal transduction pathways. However, it is still largely unknown how TRP channels are activated in vivo. Despite the uncertainties, emerging evidence using TRP channel knockout mice indicates that these channels have broad function in physiology. Here we review the recent progress on the physiology, pharmacology and pathophysiological function of mammalian TRP channels. PMID:20716668

Micro- and nanofabrication methods for ion channel reconstitution in bilayer lipid membranes

NASA Astrophysics Data System (ADS)

Tadaki, Daisuke; Yamaura, Daichi; Arata, Kohei; Ohori, Takeshi; Ma, Teng; Yamamoto, Hideaki; Niwano, Michio; Hirano-Iwata, Ayumi

2018-03-01

The self-assembled bilayer lipid membrane (BLM) forms the basic structure of the cell membrane and serves as a major barrier against ion movement. Ion channel proteins function as gated pores that permit ion permeation across the BLM. The reconstitution of ion channel proteins in artificially formed BLMs represents a well-defined system for investigating channel functions and screening drug effects on ion channels. In this review, we will discuss our recent microfabrication approaches to the formation of stable BLMs containing ion channel proteins as a potential platform for next-generation drug screening systems. BLMs formed in a microaperture having a tapered edge exhibited highly stable properties, such as a lifetime of ∼65 h and tolerance to solution changes even after the incorporation of the human ether-a-go-go-related gene (hERG) channel. We also explore a new method of efficiently incorporating human ion channels into BLMs by centrifugation. Our approaches to the formation of stable BLMs and efficient channel incorporation markedly improve the experimental efficiency of BLM reconstitution systems, leading to the realization of a BLM-based high-throughput platform for functional assays of various ion channels.

A Cyclic Nucleotide-Gated Channel Mutation Associated with Canine Daylight Blindness Provides Insight into a Role for the S2 Segment Tri-Asp motif in Channel Biogenesis

PubMed Central

Tanaka, Naoto; Delemotte, Lucie; Klein, Michael L.; Komáromy, András M.; Tanaka, Jacqueline C.

2014-01-01

Cone cyclic nucleotide-gated channels are tetramers formed by CNGA3 and CNGB3 subunits; CNGA3 subunits function as homotetrameric channels but CNGB3 exhibits channel function only when co-expressed with CNGA3. An aspartatic acid (Asp) to asparagine (Asn) missense mutation at position 262 in the canine CNGB3 (D262N) subunit results in loss of cone function (daylight blindness), suggesting an important role for this aspartic acid residue in channel biogenesis and/or function. Asp 262 is located in a conserved region of the second transmembrane segment containing three Asp residues designated the Tri-Asp motif. This motif is conserved in all CNG channels. Here we examine mutations in canine CNGA3 homomeric channels using a combination of experimental and computational approaches. Mutations of these conserved Asp residues result in the absence of nucleotide-activated currents in heterologous expression. A fluorescent tag on CNGA3 shows mislocalization of mutant channels. Co-expressing CNGB3 Tri-Asp mutants with wild type CNGA3 results in some functional channels, however, their electrophysiological characterization matches the properties of homomeric CNGA3 channels. This failure to record heteromeric currents suggests that Asp/Asn mutations affect heteromeric subunit assembly. A homology model of S1–S6 of the CNGA3 channel was generated and relaxed in a membrane using molecular dynamics simulations. The model predicts that the Tri-Asp motif is involved in non-specific salt bridge pairings with positive residues of S3/S4. We propose that the D262N mutation in dogs with CNGB3-day blindness results in the loss of these inter-helical interactions altering the electrostatic equilibrium within in the S1–S4 bundle. Because residues analogous to Tri-Asp in the voltage-gated Shaker potassium channel family were implicated in monomer folding, we hypothesize that destabilizing these electrostatic interactions impairs the monomer folding state in D262N mutant CNG channels during biogenesis. PMID:24586388

Man-induced channel adjustment in Tennessee streams

USGS Publications Warehouse

Robbins, C.H.; Simon, Andrew

1983-01-01

Channel modifications in Tennessee, particularly in the western part, have led to large-scale instabilities in the channelized rivers and may have contributed to several bridge failures. These modifications, together with land-use practices, led to downcutting, headward erosion, downstream aggradation, accelerated scour, and bank instabilities. Changes in gradient by channel straightening caused more severe channel response than did dredging or clearing. Large-scale changes continue to occur in all the channelized rivers: the Obion River, its forks, and the South Fork Forked Deer River. However, the non-channelized Hatchie River in west Tennessee not only withstood the natural stresses imposed by the wet years of 1973 to 1975 but continues to exhibit characteristics of stability. Water-surface slope, the primary dependent variable, proved to be a sensitive and descriptive parameter useful in determining channel adjustment. Adjustments to man-induced increases in channel-slope are described by inverse exponential functions of the basic form S=ae(-b(t)); where ' S ' is some function describing channel-slope, ' t ' is the number of years since completion of channel work, and ' a ' and ' b ' are coefficients. Response times for the attainment of ' equilibrium ' channel slopes are a function of the magnitude and extent of the imposed modifications. The adjusted profile gradients attained by the streams following channelization are similar to the predisturbed profile gradients, where no alteration to channel length was made. Where the channels were straightened by constructing cut-offs, thus shortening channel length, then slope adjustments (reduction) proceed past the predisturbed profile gradients, to new profiles with lower gradients. (USGS)

Insights in KIR2.1 channel structure and function by an evolutionary approach; cloning and functional characterization of the first reptilian inward rectifier channel KIR2.1, derived from the California kingsnake (Lampropeltis getula californiae).

PubMed

Houtman, Marien J C; Korte, Sanne M; Ji, Yuan; Kok, Bart; Vos, Marc A; Stary-Weinzinger, Anna; van der Heyden, Marcel A G

2014-10-03

Potassium inward rectifier KIR2.1 channels contribute to the stable resting membrane potential in a variety of muscle and neuronal cell-types. Mutations in the KIR2.1 gene KCNJ2 have been associated with human disease, such as cardiac arrhythmias and periodic paralysis. Crystal structure and homology modelling of KIR2.1 channels combined with functional current measurements provided valuable insights in mechanisms underlying channel function. KIR2.1 channels have been cloned and analyzed from all main vertebrate phyla, except reptilians. To address this lacuna, we set out to clone reptilian KIR2.1 channels. Using a degenerated primer set we cloned the KCNJ2 coding regions from muscle tissue of turtle, snake, bear, quail and bream, and compared their deduced amino acid sequences with those of KIR2.1 sequences from 26 different animal species obtained from Genbank. Furthermore, expression constructs were prepared for functional electrophysiological studies of ectopically expressed KIR2.1 ion channels. In general, KCNJ2 gene evolution followed normal phylogenetic patterns, however turtle KIR2.1 ion channel sequence is more homologues to avians than to snake. Alignment of all 31 KIR2.1 sequences showed that all disease causing KIR2.1 mutations, except V93I, V123G and N318S, are fully conserved. Homology models were built to provide structural insights into species specific amino acid substitutions. Snake KIR2.1 channels became expressed at the plasmamembrane and produced typical barium sensitive (IC50 ∼6μM) inward rectifier currents. Copyright © 2014 Elsevier Inc. All rights reserved.

Structure, function and translational relevance of aquaporin dual water and ion channels.

PubMed

Yool, Andrea J; Campbell, Ewan M

2012-01-01

Aquaporins have been assumed to be selective for water alone, and aquaglyceroporins are accepted as carrying water and small uncharged solutes including glycerol. This review presents an expanded view of aquaporins as channels with more complex mechanisms of regulation and diverse repertoires of substrate permeabilities than were originally appreciated in the early establishment of the field. The role of aquaporins as dual water and gated ion channels is likely to have physiological and potentially translational relevance, and can be evaluated with newly developed molecular and pharmacological tools. Ion channel activity has been shown for Aquaporins -0, -1, and -6, Drosphila Big Brain, and plant Nodulin-26. Although the concept of ion channel function in aquaporins remains controversial, research advances are beginning to define not only the ion channel function but also the detailed molecular mechanisms that govern and mediate the multifunctional capabilities. With regard to physiological relevance, the adaptive benefit of expression of ion channel activity in aquaporins, implied by amino acid sequence conservation of the ion channel gating domains, suggests they provide more than water or glycerol and solute transport. Dual ion and water channels are of interest for understanding the modulation of transmembrane fluid gradients, volume regulation, and possible signal transduction in tissues expressing classes of aquaporins that have the dual function capability. Other aquaporin classes might be found in future work to have ion channel activities, pending identification of the possible signaling pathways that could govern activation. Copyright © 2012 Elsevier Ltd. All rights reserved.

Directed translocation of a flexible polymer through a cone-shaped nano-channel

NASA Astrophysics Data System (ADS)

Nikoofard, Narges; Khalilian, Hamidreza; Fazli, Hossein

2013-08-01

Translocation of a flexible polymer through a cone-shaped channel is studied, theoretically and using computer simulations. Our simulations show that the shape of the channel causes the polymer translocation to be a driven process. The effective driving force of entropic origin acting on the polymer is calculated as a function of the length and the apex-angle of the channel, theoretically. It is found that the translocation time is a non-monotonic function of the apex-angle of the channel. By increasing the apex-angle from zero, the translocation time shows a minimum and then a maximum. Also, it is found that regardless of the value of the apex-angle, the translocation time is a uniformly decreasing function of the channel length. The results of the theory and the simulation are in good qualitative agreement.

Control channels in the brain and their influence on brain executive functions

NASA Astrophysics Data System (ADS)

Meng, Qinglei; Choa, Fow-Sen; Hong, Elliot; Wang, Zhiguang; Islam, Mohammad

2014-05-01

In a computer network there are distinct data channels and control channels where massive amount of visual information are transported through data channels but the information streams are routed and controlled by intelligent algorithm through "control channels". Recent studies on cognition and consciousness have shown that the brain control channels are closely related to the brainwave beta (14-40 Hz) and alpha (7-13 Hz) oscillations. The high-beta wave is used by brain to synchronize local neural activities and the alpha oscillation is for desynchronization. When two sensory inputs are simultaneously presented to a person, the high-beta is used to select one of the inputs and the alpha is used to deselect the other so that only one input will get the attention. In this work we demonstrated that we can scan a person's brain using binaural beats technique and identify the individual's preferred control channels. The identified control channels can then be used to influence the subject's brain executive functions. In the experiment, an EEG measurement system was used to record and identify a subject's control channels. After these channels were identified, the subject was asked to do Stroop tests. Binaural beats was again used to produce these control-channel frequencies on the subject's brain when we recorded the completion time of each test. We found that the high-beta signal indeed speeded up the subject's executive function performance and reduced the time to complete incongruent tests, while the alpha signal didn't seem to be able to slow down the executive function performance.

Further characterization of the effect of ethanol on voltage-gated Ca(2+) channel function in developing CA3 hippocampal pyramidal neurons.

PubMed

Morton, Russell A; Valenzuela, C Fernando

2016-02-15

Developmental ethanol exposure damages the hippocampus, a brain region involved in learning and memory. Alterations in synaptic transmission and plasticity may play a role in this effect of ethanol. We previously reported that acute and repeated exposure to ethanol during the third trimester-equivalent inhibits long-term potentiation of GABAA receptor-dependent synaptic currents in CA3 pyramidal neurons through a mechanism that depends on retrograde release of brain-derived neurotrophic factor driven by activation of voltage-gated Ca(2+) channels (Zucca and Valenzuela, 2010). We found evidence indicating that voltage-gated Ca(2+) channels are inhibited in the presence of ethanol, an effect that may play a role in its mechanism of action. Here, we further investigated the acute effect of ethanol on the function of voltage-gated Ca(2+) channels in CA3 pyramidal neurons using Ca(2+) imaging techniques. These experiments revealed that acute ethanol exposure inhibits voltage-gated Ca(2+) channels both in somatic and proximal dendritic compartments. To investigate the long-term consequences of ethanol on voltage-gated Ca(2+) channels, we used patch-clamp electrophysiological techniques to assess the function of L-type voltage-gated Ca(2+) channels during and following ten days of vapor ethanol exposure. During ethanol withdrawal periods, the function of these channels was not significantly affected by vapor chamber exposure. Taken together with our previous findings, our results suggest that 3(rd) trimester-equivalent ethanol exposure transiently inhibits L-type voltage-gated Ca(2+) channel function in CA3 pyramidal neurons and that compensatory mechanisms restore their function during ethanol withdrawal. Transient inhibition of these channels by ethanol may be, in part, responsible for the hippocampal abnormalities associated with developmental exposure to this agent. Copyright © 2015 Elsevier B.V. All rights reserved.

Inherited macular degeneration-associated mutations in CNGB3 increase the ligand sensitivity and spontaneous open probability of cone cyclic nucleotide-gated channels

PubMed Central

Meighan, Peter C.; Peng, Changhong; Varnum, Michael D.

2015-01-01

Cyclic nucleotide gated (CNG) channels are a critical component of the visual transduction cascade in the vertebrate retina. Mutations in the genes encoding these channels have been associated with a spectrum of inherited retinal disorders. To gain insight into their pathophysiological mechanisms, we have investigated the functional consequences of several CNGB3 mutations, previously associated with macular degeneration (Y469D and L595F) or complete achromatopsia (S156F, P309L, and G558C), by expressing these subunits in combination with wild-type CNGA3 in Xenopus oocytes and characterizing them using patch-clamp recordings in the inside-out configuration. These mutations did not prevent the formation of functional heteromeric channels, as indicated by sensitivity to block by L-cis-diltiazem. With the exception of S156F, each of the mutant channels displayed electrophysiological properties reflecting enhanced channel activity at physiological concentrations of cGMP (i.e., a gain-of-function phenotype). The increased channel activity produced by these mutations resulted from either increased functional expression levels, or increased sensitivity to cyclic nucleotides. Furthermore, L595F increased the spontaneous open probability in the absence of activating ligand, signifying a ligand independent gain-of-function change. In addition to the CNGB3 disease-associate mutations, we characterized the effects of several common CNGB3 and CNGA3 single-nucleotide polymorphisms (SNPs) on heteromeric CNGA3+CNGB3 channel function. Two of the SNPs examined (A3-T153M, and B3-W234C) produced decreased ligand sensitivity for heteromeric CNG channels. These changes may contribute to background disease susceptibility when combined with other genetic or non-genetic factors. Together, these studies help to define the underlying molecular phenotype for mutations relating to CNG channel disease pathogenesis. PMID:26106334

Computer Aided Synthesis or Measurement Schemes for Telemetry applications

DTIC Science & Technology

1997-09-02

5.2.5. Frame structure generation The algorithm generating the frame structure should take as inputs the sampling frequency requirements of the channels...these channels into the frame structure. Generally there can be a lot of ways to divide channels among groups. The algorithm implemented in...groups) first. The algorithm uses the function "try_permutation" recursively to distribute channels among the groups, and the function "try_subtable

Acid stress mediated adaptive divergence in ion channel function during embryogenesis in Rana arvalis

PubMed Central

Shu, Longfei; Laurila, Anssi; Räsänen, Katja

2015-01-01

Ion channels and pumps are responsible for ion flux in cells, and are key mechanisms mediating cellular function. Many environmental stressors, such as salinity and acidification, are known to severely disrupt ionic balance of organisms thereby challenging fitness of natural populations. Although ion channels can have several vital functions during early life-stages (e.g. embryogenesis), it is currently not known i) how developing embryos maintain proper intracellular conditions when exposed to environmental stress and ii) to what extent environmental stress can drive intra-specific divergence in ion channels. Here we studied the moor frog, Rana arvalis, from three divergent populations to investigate the role of different ion channels and pumps for embryonic survival under acid stress (pH 4 vs 7.5) and whether populations adapted to contrasting acidities differ in the relative role of different ion channel/pumps. We found that ion channels that mediate Ca2+ influx are essential for embryonic survival under acidic pH, and, intriguingly, that populations differ in calcium channel function. Our results suggest that adaptive divergence in embryonic acid stress tolerance of amphibians may in part be mediated by Ca2+ balance. We suggest that ion flux may mediate adaptive divergence of natural populations at early life-stages in the face of environmental stress. PMID:26381453

Technical Note: Statistical dependences between channels in radiochromic film readings. Implications in multichannel dosimetry.

PubMed

González-López, Antonio; Vera-Sánchez, Juan Antonio; Ruiz-Morales, Carmen

2016-05-01

This note studies the statistical relationships between color channels in radiochromic film readings with flatbed scanners. The same relationships are studied for noise. Finally, their implications for multichannel film dosimetry are discussed. Radiochromic films exposed to wedged fields of 6 MV energy were read in a flatbed scanner. The joint histograms of pairs of color channels were used to obtain the joint and conditional probability density functions between channels. Then, the conditional expectations and variances of one channel given another channel were obtained. Noise was extracted from film readings by means of a multiresolution analysis. Two different dose ranges were analyzed, the first one ranging from 112 to 473 cGy and the second one from 52 to 1290 cGy. For the smallest dose range, the conditional expectations of one channel given another channel can be approximated by linear functions, while the conditional variances are fairly constant. The slopes of the linear relationships between channels can be used to simplify the expression that estimates the dose by means of the multichannel method. The slopes of the linear relationships between each channel and the red one can also be interpreted as weights in the final contribution to dose estimation. However, for the largest dose range, the conditional expectations of one channel given another channel are no longer linear functions. Finally, noises in different channels were found to correlate weakly. Signals present in different channels of radiochromic film readings show a strong statistical dependence. By contrast, noise correlates weakly between channels. For the smallest dose range analyzed, the linear behavior between the conditional expectation of one channel given another channel can be used to simplify calculations in multichannel film dosimetry.

Technical Note: Statistical dependences between channels in radiochromic film readings. Implications in multichannel dosimetry

DOE Office of Scientific and Technical Information (OSTI.GOV)

González-López, Antonio, E-mail: antonio.gonzalez7@carm.es; Vera-Sánchez, Juan Antonio; Ruiz-Morales, Carmen

Purpose: This note studies the statistical relationships between color channels in radiochromic film readings with flatbed scanners. The same relationships are studied for noise. Finally, their implications for multichannel film dosimetry are discussed. Methods: Radiochromic films exposed to wedged fields of 6 MV energy were read in a flatbed scanner. The joint histograms of pairs of color channels were used to obtain the joint and conditional probability density functions between channels. Then, the conditional expectations and variances of one channel given another channel were obtained. Noise was extracted from film readings by means of a multiresolution analysis. Two different dosemore » ranges were analyzed, the first one ranging from 112 to 473 cGy and the second one from 52 to 1290 cGy. Results: For the smallest dose range, the conditional expectations of one channel given another channel can be approximated by linear functions, while the conditional variances are fairly constant. The slopes of the linear relationships between channels can be used to simplify the expression that estimates the dose by means of the multichannel method. The slopes of the linear relationships between each channel and the red one can also be interpreted as weights in the final contribution to dose estimation. However, for the largest dose range, the conditional expectations of one channel given another channel are no longer linear functions. Finally, noises in different channels were found to correlate weakly. Conclusions: Signals present in different channels of radiochromic film readings show a strong statistical dependence. By contrast, noise correlates weakly between channels. For the smallest dose range analyzed, the linear behavior between the conditional expectation of one channel given another channel can be used to simplify calculations in multichannel film dosimetry.« less

Drosophila TRP and TRPL are assembled as homomultimeric channels in vivo.

PubMed

Katz, Ben; Oberacker, Tina; Richter, David; Tzadok, Hanan; Peters, Maximilian; Minke, Baruch; Huber, Armin

2013-07-15

Family members of the cationic transient receptor potential (TRP) channels serve as sensors and transducers of environmental stimuli. The ability of different TRP channel isoforms of specific subfamilies to form heteromultimers and the structural requirements for channel assembly are still unresolved. Although heteromultimerization of different mammalian TRP channels within single subfamilies has been described, even within a subfamily (such as TRPC) not all members co-assemble with each other. In Drosophila photoreceptors two TRPC channels, TRP and TRP-like protein (TRPL) are expressed together in photoreceptors where they generate the light-induced current. The formation of functional TRP-TRPL heteromultimers in cell culture and in vitro has been reported. However, functional in vivo assays have shown that each channel functions independently of the other. Therefore, the issue of whether TRP and TRPL form heteromultimers in vivo is still unclear. In the present study we investigated the ability of TRP and TRPL to form heteromultimers, and the structural requirements for channel assembly, by studying assembly of GFP-tagged TRP and TRPL channels and chimeric TRP and TRPL channels, in vivo. Interaction studies of tagged and native channels as well as native and chimeric TRP-TRPL channels using co-immunoprecipitation, immunocytochemistry and electrophysiology, critically tested the ability of TRP and TRPL to interact. We found that TRP and TRPL assemble exclusively as homomultimeric channels in their native environment. The above analyses revealed that the transmembrane regions of TRP and TRPL do not determine assemble specificity of these channels. However, the C-terminal regions of both TRP and TRPL predominantly specify the assembly of homomeric TRP and TRPL channels.

Alternative Splicing Generates a Novel Truncated Cav1.2 Channel in Neonatal Rat Heart*

PubMed Central

Liao, Ping; Yu, Dejie; Hu, Zhenyu; Liang, Mui Cheng; Wang, Jue Jin; Yu, Chye Yun; Ng, Gandi; Yong, Tan Fong; Soon, Jia Lin; Chua, Yeow Leng; Soong, Tuck Wah

2015-01-01

L-type Cav1.2 Ca2+ channel undergoes extensive alternative splicing, generating functionally different channels. Alternatively spliced Cav1.2 Ca2+ channels have been found to be expressed in a tissue-specific manner or under pathological conditions. To provide a more comprehensive understanding of alternative splicing in Cav1.2 channel, we systematically investigated the splicing patterns in the neonatal and adult rat hearts. The neonatal heart expresses a novel 104-bp exon 33L at the IVS3-4 linker that is generated by the use of an alternative acceptor site. Inclusion of exon 33L causes frameshift and C-terminal truncation. Whole-cell electrophysiological recordings of Cav1.233L channels expressed in HEK 293 cells did not detect any current. However, when co-expressed with wild type Cav1.2 channels, Cav1.233L channels reduced the current density and altered the electrophysiological properties of the wild type Cav1.2 channels. Interestingly, the truncated 3.5-domain Cav1.233L channels also yielded a dominant negative effect on Cav1.3 channels, but not on Cav3.2 channels, suggesting that Cavβ subunits is required for Cav1.233L regulation. A biochemical study provided evidence that Cav1.233L channels enhanced protein degradation of wild type channels via the ubiquitin-proteasome system. Although the physiological significance of the Cav1.233L channels in neonatal heart is still unknown, our report demonstrates the ability of this novel truncated channel to modulate the activity of the functional Cav1.2 channels. Moreover, the human Cav1.2 channel also contains exon 33L that is developmentally regulated in heart. Unexpectedly, human exon 33L has a one-nucleotide insertion that allowed in-frame translation of a full Cav1.2 channel. An electrophysiological study showed that human Cav1.233L channel is a functional channel but conducts Ca2+ ions at a much lower level. PMID:25694430

Downregulation of BK Channel Function and Protein Expression in Coronary Arteriolar Smooth Muscle Cells of Type 2 Diabetic Patients.

PubMed

Lu, Tong; Chai, Qiang; Jiao, Guoqing; Wang, Xiao-Li; Sun, Xiaojing; Furuseth, Jonathan D; Stulak, John M; Daly, Richard C; Greason, Kevin L; Cha, Yong-Mei; Lee, Hon-Chi

2018-05-30

Type 2 diabetes (T2D) is strongly associated with cardiovascular morbidity and mortality in patients. Vascular large conductance Ca2+-activated potassium (BK) channels, composed of four pore-forming α subunits (BK-α) and four regulatory β1 subunits (BK-β1), are densely expressed in coronary arterial smooth muscle cells (SMCs) and play an important role in regulating vascular tone and myocardial perfusion. However, the role of BK channels in coronary microvascular dysfunction of human subjects with diabetes is unclear. In this study, we examined BK channel function and protein expression, and BK channel-mediated vasodilation in freshly isolated coronary arterioles from T2D patients. Atrial tissues were obtained from 25 patients with T2D and 16 matched non-diabetic subjects during cardiopulmonary bypass procedure. Microvessel videomicroscopy and immunoblot analysis were performed in freshly dissected coronary arterioles and inside-out single BK channel currents was recorded in enzymatically isolated coronary arteriolar SMCs. We found that BK channel sensitivity to physiological Ca2+ concentration and voltage was downregulated in the coronary arteriolar SMCs of diabetic patients, compared with non-diabetic controls. BK channel kinetics analysis revealed that there was significant shortening of the mean open time and prolongation of the mean closed time in diabetic patients, resulting in a remarkable reduction of the channel open probability. Functional studies showed that BK channel activation by dehydrosoyasaponin-1 was diminished and that BK channel-mediated vasodilation in response to shear stress was impaired in diabetic coronary arterioles. Immunoblot experiments confirmed that the protein expressions of BK-α and BK-β1 subunits were significantly downregulated, but the ratio of BK-α/BK-β1 was unchanged in the coronary arterioles of T2D patients. Our results demonstrated for the first time that BK channel function and BK channel-mediated vasodilation were abnormal in the coronary microvasculature of diabetic patients, due to decreased protein expression and altered intrinsic properties of BK channels.

The Ketogenic Diet and Potassium Channel Function

DTIC Science & Technology

2015-11-01

1 AWARD NUMBER: W81XWH-13-1-0463 TITLE: The Ketogenic Diet and Potassium Channel Function PRINCIPAL INVESTIGATOR: Dr. Geoffrey Murphy...NUMBER The Ketogenic Diet and Potassium Channel Function 5b. GRANT NUMBER W81XWH-13-1-0463 5c. PROGRAM ELEMENT NUMBER 6. AUTHOR(S) Geoffrey Murphy...The overall objective of this Discovery Award was to explore the hypothesis the ketogenic diet (KD) regulates neuronal excitability by influencing

TRP channels in the skin.

PubMed

Tóth, Balázs I; Oláh, Attila; Szöllősi, Attila Gábor; Bíró, Tamás

2014-05-01

Emerging evidence suggests that transient receptor potential (TRP) ion channels not only act as 'polymodal cellular sensors' on sensory neurons but are also functionally expressed by a multitude of non-neuronal cell types. This is especially true in the skin, one of the largest organs of the body, where they appear to be critically involved in regulating various cutaneous functions both under physiological and pathophysiological conditions. In this review, we focus on introducing the roles of several cutaneous TRP channels in the regulation of the skin barrier, skin cell proliferation and differentiation, and immune functions. Moreover, we also describe the putative involvement of several TRP channels in the development of certain skin diseases and identify future TRP channel-targeted therapeutic opportunities. © 2013 The British Pharmacological Society.

Lattice model for calcium dynamics

NASA Astrophysics Data System (ADS)

Guisoni, Nara; de Oliveira, Mario José

2005-06-01

We present a simplified lattice model to study calcium dynamics in the endoplasmic reticulum membrane. Calcium channels and calcium ions are placed in two interpenetrating square lattices which are connected in two ways: (i) via calcium release and (ii) because transitions between channel states are calcium dependent. The opening or closing of a channel is a stochastic process controlled by two functions which depend on the calcium density on the channel neighborhood. The model is studied through mean field calculations and simulations. We show that the critical behavior of the model changes drastically depending on the opening/closing functions. For certain choices of these functions, all channels are closed at very low and high calcium densities and the model presents one absorbing state.

[Comparisons and analysis of the spectral response functions' difference between FY-2E's and FY2C's split window channels].

PubMed

Zhang, Yong; Li, Yuan; Rong, Zhi-Guo

2010-06-01

Remote sensors' channel spectral response function (SRF) was one of the key factors to influence the quantitative products' inversion algorithm, accuracy and the geophysical characteristics. Aiming at the adjustments of FY-2E's split window channels' SRF, detailed comparisons between the FY-2E and FY-2C corresponding channels' SRF differences were carried out based on three data collections: the NOAA AVHRR corresponding channels' calibration look up tables, field measured water surface radiance and atmospheric profiles at Lake Qinghai and radiance calculated from the PLANK function within all dynamic range of FY-2E/C. The results showed that the adjustments of FY-2E's split window channels' SRF would result in the spectral range's movements and influence the inversion algorithms of some ground quantitative products. On the other hand, these adjustments of FY-2E SRFs would increase the brightness temperature differences between FY-2E's two split window channels within all dynamic range relative to FY-2C's. This would improve the inversion ability of FY-2E's split window channels.

TRPC Channel Structure and Properties.

PubMed

Feng, Shengjie

2017-01-01

TRPC channels are the first identified members in the TRP family. They function as either homo- or heterotetramers regulating intracellular Ca 2+ concentration in response to numerous physiological or pathological stimuli. TRPC channels are nonselective cation channels permeable to Ca 2+ . The properties and the functional domains of TRPC channels have been identified by electrophysiological and biochemical methods. However, due to the large size, instability, and flexibility of their complexes, the structures of the members in TRPC family remain unrevealed. More efforts should be made on structure analysis and generating good tools, including specific antibodies, agonist, and antagonist.

Small Ion Channel Linking Molecular Simulations and Electrophysiology

NASA Technical Reports Server (NTRS)

Pohorille, Andrzej

2017-01-01

Ion channels are pore-forming protein assemblies that mediate the transport of small ions across cell membranes. Otherwise, membrane bilayers would be almost impermeable to ions incapable to traverse the low dielectric constant, hydrophobic membrane core. Ion channels are ubiquitous to all life forms. In humans and other higher organisms they play the central role in conducting nerve impulses, cardiac functions, muscle contraction and apoptosis. On the other extreme of biological complexity, viral ion channels (viroporins) influence many stages of the virus infection cycle either through regulating virus replication, such as entry, assembly and release or modulating the electrochemical balance in the subcellular compartments of host cells. Ion channels were crucial components of protocells. Their emergence facilitated adaptation of nascent life to different environmental conditions. The earliest ion channels must have been much simpler than most of their modern ancestors. Viral channels are among only a few naturally occurring models to study the structure, function and evolution of primordial channels. Experimental studies of these properties are difficult and often unreliable. In principle, computational methods, and molecular dynamics (MD) simulations in particular, can aid in providing information about both the structure and the function of ion channels. However, MD suffers from its own problems, such as inability to access sufficiently long time scales or limited accuracy of force fields. It is, therefore, essential to determine the reliability of MD simulations. We propose to do so on the basis of two criteria. One is channel stability on time scales that extend for several microseconds or longer. The other is the ability to reproduce the measured ionic conductance as a function of applied voltage. If both the stability and the calculated ionic conductance are satisfactory it will greatly increase our confidence that the structure and the function of a channel are described sufficiently accurately. To our knowledge, long time scale stability (approx.10 micro-sec) and the correct electrophysiology have been shown so far for only one channel - the synthetic LS3 hexamer). In this presentation, this approach will be discussed in application to two viral channels - Vpu, encoded by the HIV-1 genome and p7 of hepatitis C.

Functional description of signal processing in the Rogue GPS receiver

NASA Technical Reports Server (NTRS)

Thomas, J. B.

1988-01-01

Over the past year, two Rogue GPS prototype receivers have been assembled and successfully subjected to a variety of laboratory and field tests. A functional description is presented of signal processing in the Rogue receiver, tracing the signal from RF input to the output values of group delay, phase, and data bits. The receiver can track up to eight satellites, without time multiplexing among satellites or channels, simultaneously measuring both group delay and phase for each of three channels (L1-C/A, L1-P, L2-P). The Rogue signal processing described requires generation of the code for all three channels. Receiver functional design, which emphasized accuracy, reliability, flexibility, and dynamic capability, is summarized. A detailed functional description of signal processing is presented, including C/A-channel and P-channel processing, carrier-aided averaging of group delays, checks for cycle slips, acquistion, and distinctive features.

Design of two-dimensional channels with prescribed velocity distributions along the channel walls

NASA Technical Reports Server (NTRS)

Stanitz, John D

1953-01-01

A general method of design is developed for two-dimensional unbranched channels with prescribed velocities as a function of arc length along the channel walls. The method is developed for both compressible and incompressible, irrotational, nonviscous flow and applies to the design of elbows, diffusers, nozzles, and so forth. In part I solutions are obtained by relaxation methods; in part II solutions are obtained by a Green's function. Five numerical examples are given in part I including three elbow designs with the same prescribed velocity as a function of arc length along the channel walls but with incompressible, linearized compressible, and compressible flow. One numerical example is presented in part II for an accelerating elbow with linearized compressible flow, and the time required for the solution by a Green's function in part II was considerably less than the time required for the same solution by relaxation methods in part I.

The Segregated Expression of Voltage-Gated Potassium and Sodium Channels in Neuronal Membranes: Functional Implications and Regulatory Mechanisms

PubMed Central

Duménieu, Maël; Oulé, Marie; Kreutz, Michael R.; Lopez-Rojas, Jeffrey

2017-01-01

Neurons are highly polarized cells with apparent functional and morphological differences between dendrites and axon. A critical determinant for the molecular and functional identity of axonal and dendritic segments is the restricted expression of voltage-gated ion channels (VGCs). Several studies show an uneven distribution of ion channels and their differential regulation within dendrites and axons, which is a prerequisite for an appropriate integration of synaptic inputs and the generation of adequate action potential (AP) firing patterns. This review article will focus on the signaling pathways leading to segmented expression of voltage-gated potassium and sodium ion channels at the neuronal plasma membrane and the regulatory mechanisms ensuring segregated functions. We will also discuss the relevance of proper ion channel targeting for neuronal physiology and how alterations in polarized distribution contribute to neuronal pathology. PMID:28484374

Structural determinants of PIP(2) regulation of inward rectifier K(ATP) channels.

PubMed

Shyng, S L; Cukras, C A; Harwood, J; Nichols, C G

2000-11-01

Phosphatidylinositol 4,5-bisphosphate (PIP(2)) activates K(ATP) and other inward rectifier (Kir) channels. To determine residues important for PIP(2) regulation, we have systematically mutated each positive charge in the COOH terminus of Kir6.2 to alanine. The effects of these mutations on channel function were examined using (86)Rb efflux assays on intact cells and inside-out patch-clamp methods. Both methods identify essentially the same basic residues in two narrow regions (176-222 and 301-314) in the COOH terminus that are important for the maintenance of channel function and interaction with PIP(2). Only one residue (R201A) simultaneously affected ATP and PIP(2) sensitivity, which is consistent with the notion that these ligands, while functionally competitive, are unlikely to bind to identical sites. Strikingly, none of 13 basic residues in the terminal portion (residues 315-390) of the COOH terminus affected channel function when neutralized. The data help to define the structural requirements for PIP(2) sensitivity of K(ATP) channels. Moreover, the regions and residues defined in this study parallel those uncovered in recent studies of PIP(2) sensitivity in other inward rectifier channels, indicating a common structural basis for PIP(2) regulation.

Disturbed Processing of Contextual Information in HCN3 Channel Deficient Mice

PubMed Central

Stieglitz, Marc S.; Fenske, Stefanie; Hammelmann, Verena; Becirovic, Elvir; Schöttle, Verena; Delorme, James E.; Schöll-Weidinger, Martha; Mader, Robert; Deussing, Jan; Wolfer, David P.; Seeliger, Mathias W.; Albrecht, Urs; Wotjak, Carsten T.; Biel, Martin; Michalakis, Stylianos; Wahl-Schott, Christian

2018-01-01

Hyperpolarization-activated cyclic nucleotide-gated channels (HCNs) in the nervous system are implicated in a variety of neuronal functions including learning and memory, regulation of vigilance states and pain. Dysfunctions or genetic loss of these channels have been shown to cause human diseases such as epilepsy, depression, schizophrenia, and Parkinson's disease. The physiological functions of HCN1 and HCN2 channels in the nervous system have been analyzed using genetic knockout mouse models. By contrast, there are no such genetic studies for HCN3 channels so far. Here, we use a HCN3-deficient (HCN3−/−) mouse line, which has been previously generated in our group to examine the expression and function of this channel in the CNS. Specifically, we investigate the role of HCN3 channels for the regulation of circadian rhythm and for the determination of behavior. Contrary to previous suggestions we find that HCN3−/− mice show normal visual, photic, and non-photic circadian function. In addition, HCN3−/− mice are impaired in processing contextual information, which is characterized by attenuated long-term extinction of contextual fear and increased fear to a neutral context upon repeated exposure. PMID:29375299

ACIRF user's guide: Theory and examples

NASA Astrophysics Data System (ADS)

Dana, Roger A.

1989-12-01

Design and evaluation of radio frequency systems that must operate through ionospheric disturbances resulting from high altitude nuclear detonations requires an accurate channel model. This model must include the effects of high gain antennas that may be used to receive the signals. Such a model can then be used to construct realizations of the received signal for use in digital simulations of trans-ionospheric links or for use in hardware channel simulators. The FORTRAN channel model ACIRF (Antenna Channel Impulse Response Function) generates random realizations of the impulse response function at the outputs of multiple antennas. This user's guide describes the FORTRAN program ACIRF (version 2.0) that generates realizations of channel impulse response functions at the outputs of multiple antennas with arbitrary beamwidths, pointing angles, and relatives positions. This channel model is valid under strong scattering conditions when Rayleigh fading statistics apply. Both frozen-in and turbulent models for the temporal fluctuations are included in this version of ACIRF. The theory of the channel model is described and several examples are given.

Single-channel analysis of functional epithelial sodium channel (ENaC) stability at the apical membrane of A6 distal kidney cells

PubMed Central

Yu, Ling; Helms, My N.; Yue, Qiang; Eaton, Douglas C.

2008-01-01

Epithelial sodium channels (ENaC) play an essential role in maintaining total body fluid and electrolyte homeostasis. As such, abnormal expression of ENaC at the cell surface is linked to several important human diseases. Although the stability of ENaC subunits has been extensively studied by protein biochemical analysis, the half-life of the functional channel in the apical membrane remains controversial. Because the functional stability of the multisubunit channel may be more physiologically relevant than the stability of individual subunit proteins, we performed studies of functional ENaC channels using A6 epithelial cells, a Xenopus laevis distal nephron cell line. We recorded single-channel activity in over 400 cells with the translation blockers cycloheximide (CHX) or puromycin, as well as the intracellular protein trafficking inhibitors brefeldin A (BFA) or nocodazole. Our cell-attached, single-channel recordings allow us to quantify the channel density in the apical membrane, as well as to determine channel open probability (Po) from control (untreated) cells and from cells at different times of drug treatment. The data suggest that the half-life of ENaC channels is ∼3.5 h following puromycin, BFA, and nocodazole treatment. Furthermore, these three drugs had no significant effect on the Po of ENaC for at least 6 h after exposure. A decrease in apical channel number and Po was observed following 2 h of CHX inhibition of protein synthesis, and the apparent channel half-life was closer to 1.5 h following CHX treatment. Treatment of cells with the translation inhibitors does not alter the expression of the protease furin, and therefore changes in protease activity cannot explain changes in ENaC Po. Confocal images show that BFA and nocodazole both disrupt most of the Golgi apparatus after 1-h exposure. In cells with the Golgi totally disrupted by overnight exposure to BFA, 20% of apical ENaC channels remained functional. This result suggests that ENaC is delivered to the apical membrane via a pathway that might bypass the Golgi vesicular trafficking pathway, or that there might be two pools of channels with markedly different half-lives in the apical membrane. PMID:18784262

Single-channel analysis of functional epithelial sodium channel (ENaC) stability at the apical membrane of A6 distal kidney cells.

PubMed

Yu, Ling; Helms, My N; Yue, Qiang; Eaton, Douglas C

2008-11-01

Epithelial sodium channels (ENaC) play an essential role in maintaining total body fluid and electrolyte homeostasis. As such, abnormal expression of ENaC at the cell surface is linked to several important human diseases. Although the stability of ENaC subunits has been extensively studied by protein biochemical analysis, the half-life of the functional channel in the apical membrane remains controversial. Because the functional stability of the multisubunit channel may be more physiologically relevant than the stability of individual subunit proteins, we performed studies of functional ENaC channels using A6 epithelial cells, a Xenopus laevis distal nephron cell line. We recorded single-channel activity in over 400 cells with the translation blockers cycloheximide (CHX) or puromycin, as well as the intracellular protein trafficking inhibitors brefeldin A (BFA) or nocodazole. Our cell-attached, single-channel recordings allow us to quantify the channel density in the apical membrane, as well as to determine channel open probability (Po) from control (untreated) cells and from cells at different times of drug treatment. The data suggest that the half-life of ENaC channels is approximately 3.5 h following puromycin, BFA, and nocodazole treatment. Furthermore, these three drugs had no significant effect on the Po of ENaC for at least 6 h after exposure. A decrease in apical channel number and Po was observed following 2 h of CHX inhibition of protein synthesis, and the apparent channel half-life was closer to 1.5 h following CHX treatment. Treatment of cells with the translation inhibitors does not alter the expression of the protease furin, and therefore changes in protease activity cannot explain changes in ENaC Po. Confocal images show that BFA and nocodazole both disrupt most of the Golgi apparatus after 1-h exposure. In cells with the Golgi totally disrupted by overnight exposure to BFA, 20% of apical ENaC channels remained functional. This result suggests that ENaC is delivered to the apical membrane via a pathway that might bypass the Golgi vesicular trafficking pathway, or that there might be two pools of channels with markedly different half-lives in the apical membrane.

Phosphatidylinositol-4,5-bisphosphate is required for KCNQ1/KCNE1 channel function but not anterograde trafficking.

PubMed

Royal, Alice A; Tinker, Andrew; Harmer, Stephen C

2017-01-01

The slow delayed-rectifier potassium current (IKs) is crucial for human cardiac action potential repolarization. The formation of IKs requires co-assembly of the KCNQ1 α-subunit and KCNE1 β-subunit, and mutations in either of these subunits can lead to hereditary long QT syndrome types 1 and 5, respectively. It is widely recognised that the KCNQ1/KCNE1 (Q1/E1) channel requires phosphatidylinositol-4,5-bisphosphate (PIP2) binding for function. We previously identified a cluster of basic residues in the proximal C-terminus of KCNQ1 that form a PIP2/phosphoinositide binding site. Upon charge neutralisation of these residues we found that the channel became more retained in the endoplasmic reticulum, which raised the possibility that channel-phosphoinositide interactions could play a role in channel trafficking. To explore this further we used a chemically induced dimerization (CID) system to selectively deplete PIP2 and/or phosphatidylinositol-4-phosphate (PI(4)P) at the plasma membrane (PM) or Golgi, and we subsequently monitored the effects on both channel trafficking and function. The depletion of PIP2 and/or PI(4)P at either the PM or Golgi did not alter channel cell-surface expression levels. However, channel function was extremely sensitive to the depletion of PIP2 at the PM, which is in contrast to the response of other cardiac potassium channels tested (Kir2.1 and Kv11.1). Surprisingly, when using the CID system IKs was dramatically reduced even before dimerization was induced, highlighting limitations regarding the utility of this system when studying processes highly sensitive to PIP2 depletion. In conclusion, we identify that the Q1/E1 channel does not require PIP2 or PI(4)P for anterograde trafficking, but is heavily reliant on PIP2 for channel function once at the PM.

Colloid-Colloid Hydrodynamic Interaction Around a Bend in a Quasi-One-Dimensional Channel

NASA Astrophysics Data System (ADS)

Liepold, Christopher; Zarcone, Ryan; Heumann, Tibor; Lin, Binhua; Rice, Stuart

We report a study of the correlation between a pair of particles in a colloid suspension in a bent quasi-one-dimensional (q1d) channel as a function of bend angle. As the bend angle becomes more acute, we observe an increasing depletion of particles in the vicinity of the bend and an increase in the nearest-neighbor separation in the pair correlation function for particles on opposite sides of the bend. Further, we observe that the peak value of D12, the coupling term in the pair diffusion tensor that characterizes the effect of the motion of particle 1 on particle 2, coincides with the first peak in the pair correlation function, and that the pair separation dependence of D12 mimics that of the pair correlation function. We show that the observed behavior is a consequence of the geometric constraints imposed by the single-file requirement that the particle centers lie on the centerline of the channel and the requirement that the hydrodynamic flow must follow the channel around the bend. We find that the correlation between a pair of particles in a colloidal suspension in a bent q1D channel has the same functional dependence on the pair correlation function as in a straight q1D channel when measured in a coordinate system that follows the centerline of the bent channel. NSF MRSEC (DMR-1420709), Dreyfus Foundation (SI-14-014).

TRP channels in the skin

PubMed Central

Tóth, Balázs I; Oláh, Attila; Szöllősi, Attila Gábor; Bíró, Tamás

2014-01-01

Emerging evidence suggests that transient receptor potential (TRP) ion channels not only act as ‘polymodal cellular sensors’ on sensory neurons but are also functionally expressed by a multitude of non-neuronal cell types. This is especially true in the skin, one of the largest organs of the body, where they appear to be critically involved in regulating various cutaneous functions both under physiological and pathophysiological conditions. In this review, we focus on introducing the roles of several cutaneous TRP channels in the regulation of the skin barrier, skin cell proliferation and differentiation, and immune functions. Moreover, we also describe the putative involvement of several TRP channels in the development of certain skin diseases and identify future TRP channel-targeted therapeutic opportunities. Linked Articles This article is part of a themed section on the pharmacology of TRP channels. To view the other articles in this section visit http://dx.doi.org/10.1111/bph.2014.171.issue-10 PMID:24372189

Functional conversion between A-type and delayed rectifier K+ channels by membrane lipids.

PubMed

Oliver, Dominik; Lien, Cheng-Chang; Soom, Malle; Baukrowitz, Thomas; Jonas, Peter; Fakler, Bernd

2004-04-09

Voltage-gated potassium (Kv) channels control action potential repolarization, interspike membrane potential, and action potential frequency in excitable cells. It is thought that the combinatorial association between distinct alpha and beta subunits determines whether Kv channels function as non-inactivating delayed rectifiers or as rapidly inactivating A-type channels. We show that membrane lipids can convert A-type channels into delayed rectifiers and vice versa. Phosphoinositides remove N-type inactivation from A-type channels by immobilizing the inactivation domains. Conversely, arachidonic acid and its amide anandamide endow delayed rectifiers with rapid voltage-dependent inactivation. The bidirectional control of Kv channel gating by lipids may provide a mechanism for the dynamic regulation of electrical signaling in the nervous system.

A novel PKD2L1 C-terminal domain critical for trimerization and channel function.

PubMed

Zheng, Wang; Hussein, Shaimaa; Yang, JungWoo; Huang, Jun; Zhang, Fan; Hernandez-Anzaldo, Samuel; Fernandez-Patron, Carlos; Cao, Ying; Zeng, Hongbo; Tang, Jingfeng; Chen, Xing-Zhen

2015-03-30

As a transient receptor potential (TRP) superfamily member, polycystic kidney disease 2-like-1 (PKD2L1) is also called TRPP3 and has similar membrane topology as voltage-gated cation channels. PKD2L1 is involved in hedgehog signaling, intestinal development, and sour tasting. PKD2L1 and PKD1L3 form heterotetramers with 3:1 stoichiometry. C-terminal coiled-coil-2 (CC2) domain (G699-W743) of PKD2L1 was reported to be important for its trimerization but independent studies showed that CC2 does not affect PKD2L1 channel function. It thus remains unclear how PKD2L1 proteins oligomerize into a functional channel. By SDS-PAGE, blue native PAGE and mutagenesis we here identified a novel C-terminal domain called C1 (K575-T622) involved in stronger homotrimerization than the non-overlapping CC2, and found that the PKD2L1 N-terminus is critical for dimerization. By electrophysiology and Xenopus oocyte expression, we found that C1, but not CC2, is critical for PKD2L1 channel function. Our co-immunoprecipitation and dynamic light scattering experiments further supported involvement of C1 in trimerization. Further, C1 acted as a blocking peptide that inhibits PKD2L1 trimerization as well as PKD2L1 and PKD2L1/PKD1L3 channel function. Thus, our study identified C1 as the first PKD2L1 domain essential for both PKD2L1 trimerization and channel function, and suggest that PKD2L1 and PKD2L1/PKD1L3 channels share the PKD2L1 trimerization process.

Role of TRP channels in the cardiovascular system

PubMed Central

Yue, Zhichao; Xie, Jia; Yu, Albert S.; Stock, Jonathan; Du, Jianyang

2014-01-01

The transient receptor potential (TRP) superfamily consists of a large number of nonselective cation channels with variable degree of Ca2+-permeability. The 28 mammalian TRP channel proteins can be grouped into six subfamilies: canonical, vanilloid, melastatin, ankyrin, polycystic, and mucolipin TRPs. The majority of these TRP channels are expressed in different cell types including both excitable and nonexcitable cells of the cardiovascular system. Unlike voltage-gated ion channels, TRP channels do not have a typical voltage sensor, but instead can sense a variety of other stimuli including pressure, shear stress, mechanical stretch, oxidative stress, lipid environment alterations, hypertrophic signals, and inflammation products. By integrating multiple stimuli and transducing their activity to downstream cellular signal pathways via Ca2+ entry and/or membrane depolarization, TRP channels play an essential role in regulating fundamental cell functions such as contraction, relaxation, proliferation, differentiation, and cell death. With the use of targeted deletion and transgenic mouse models, recent studies have revealed that TRP channels are involved in numerous cellular functions and play an important role in the pathophysiology of many diseases in the cardiovascular system. Moreover, several TRP channels are involved in inherited diseases of the cardiovascular system. This review presents an overview of current knowledge concerning the physiological functions of TRP channels in the cardiovascular system and their contributions to cardiovascular diseases. Ultimately, TRP channels may become potential therapeutic targets for cardiovascular diseases. PMID:25416190

Role of TRP channels in the cardiovascular system.

PubMed

Yue, Zhichao; Xie, Jia; Yu, Albert S; Stock, Jonathan; Du, Jianyang; Yue, Lixia

2015-02-01

The transient receptor potential (TRP) superfamily consists of a large number of nonselective cation channels with variable degree of Ca(2+)-permeability. The 28 mammalian TRP channel proteins can be grouped into six subfamilies: canonical, vanilloid, melastatin, ankyrin, polycystic, and mucolipin TRPs. The majority of these TRP channels are expressed in different cell types including both excitable and nonexcitable cells of the cardiovascular system. Unlike voltage-gated ion channels, TRP channels do not have a typical voltage sensor, but instead can sense a variety of other stimuli including pressure, shear stress, mechanical stretch, oxidative stress, lipid environment alterations, hypertrophic signals, and inflammation products. By integrating multiple stimuli and transducing their activity to downstream cellular signal pathways via Ca(2+) entry and/or membrane depolarization, TRP channels play an essential role in regulating fundamental cell functions such as contraction, relaxation, proliferation, differentiation, and cell death. With the use of targeted deletion and transgenic mouse models, recent studies have revealed that TRP channels are involved in numerous cellular functions and play an important role in the pathophysiology of many diseases in the cardiovascular system. Moreover, several TRP channels are involved in inherited diseases of the cardiovascular system. This review presents an overview of current knowledge concerning the physiological functions of TRP channels in the cardiovascular system and their contributions to cardiovascular diseases. Ultimately, TRP channels may become potential therapeutic targets for cardiovascular diseases. Copyright © 2015 the American Physiological Society.

Redox regulation of neuronal voltage-gated calcium channels.

PubMed

Todorovic, Slobodan M; Jevtovic-Todorovic, Vesna

2014-08-20

Voltage-gated calcium channels are ubiquitously expressed in neurons and are key regulators of cellular excitability and synaptic transmitter release. There is accumulating evidence that multiple subtypes of voltage-gated calcium channels may be regulated by oxidation and reduction. However, the redox mechanisms involved in the regulation of channel function are not well understood. Several studies have established that both T-type and high-voltage-activated subtypes of voltage-gated calcium channel can be redox-regulated. This article reviews different mechanisms that can be involved in redox regulation of calcium channel function and their implication in neuronal function, particularly in pain pathways and thalamic oscillation. A current critical issue in the field is to decipher precise mechanisms of calcium channel modulation via redox reactions. In this review we discuss covalent post-translational modification via oxidation of cysteine molecules and chelation of trace metals, and reactions involving nitric oxide-related molecules and free radicals. Improved understanding of the roles of redox-based reactions in regulation of voltage-gated calcium channels may lead to improved understanding of novel redox mechanisms in physiological and pathological processes. Identification of redox mechanisms and sites on voltage-gated calcium channel may allow development of novel and specific ion channel therapies for unmet medical needs. Thus, it may be possible to regulate the redox state of these channels in treatment of pathological process such as epilepsy and neuropathic pain.

Functional and Molecular Evidence for Kv7 Channel Subtypes in Human Detrusor from Patients with and without Bladder Outflow Obstruction

PubMed Central

Svalø, Julie; Sheykhzade, Majid; Nordling, Jørgen; Matras, Christina; Bouchelouche, Pierre

2015-01-01

The aim of the study was to investigate whether Kv7 channels and their ancillary β-subunits, KCNE, are functionally expressed in the human urinary bladder. Kv7 channels were examined at the molecular level and by functional studies using RT-qPCR and myography, respectively. We found mRNA expression of KCNQ1, KCNQ3-KCNQ5 and KCNE1-5 in the human urinary bladder from patients with normal bladder function (n = 7) and in patients with bladder outflow obstruction (n = 3). Interestingly, a 3.4-fold up-regulation of KCNQ1 was observed in the latter. The Kv7 channel subtype selective modulators, ML277 (activator of Kv7.1 channels, 10 μM) and ML213 (activator of Kv7.2, Kv7.4, Kv7.4/7.5 and Kv7.5 channels, 10 μM), reduced the tone of 1 μM carbachol pre-constricted bladder strips. XE991 (blocker of Kv7.1–7.5 channels, 10 μM) had opposing effects as it increased contractions achieved with 20 mM KPSS. Furthermore, we investigated if there is interplay between Kv7 channels and β-adrenoceptors. Using cumulative additions of isoprenaline (β-adrenoceptor agonist) and forskolin (adenylyl cyclase activator) in combination with the Kv7 channel activator and blocker, retigabine and XE991, we did not find interplay between Kv7 channels and β-adrenoceptors in the human urinary bladder. The performed gene expression analysis combined with the organ bath studies imply that compounds that activate Kv7 channels could be useful for treatment of overactive bladder syndrome. PMID:25692982

Functional and molecular evidence for Kv7 channel subtypes in human detrusor from patients with and without bladder outflow obstruction.

PubMed

Svalø, Julie; Sheykhzade, Majid; Nordling, Jørgen; Matras, Christina; Bouchelouche, Pierre

2015-01-01

The aim of the study was to investigate whether Kv7 channels and their ancillary β-subunits, KCNE, are functionally expressed in the human urinary bladder. Kv7 channels were examined at the molecular level and by functional studies using RT-qPCR and myography, respectively. We found mRNA expression of KCNQ1, KCNQ3-KCNQ5 and KCNE1-5 in the human urinary bladder from patients with normal bladder function (n = 7) and in patients with bladder outflow obstruction (n = 3). Interestingly, a 3.4-fold up-regulation of KCNQ1 was observed in the latter. The Kv7 channel subtype selective modulators, ML277 (activator of Kv7.1 channels, 10 μM) and ML213 (activator of Kv7.2, Kv7.4, Kv7.4/7.5 and Kv7.5 channels, 10 μM), reduced the tone of 1 μM carbachol pre-constricted bladder strips. XE991 (blocker of Kv7.1-7.5 channels, 10 μM) had opposing effects as it increased contractions achieved with 20 mM KPSS. Furthermore, we investigated if there is interplay between Kv7 channels and β-adrenoceptors. Using cumulative additions of isoprenaline (β-adrenoceptor agonist) and forskolin (adenylyl cyclase activator) in combination with the Kv7 channel activator and blocker, retigabine and XE991, we did not find interplay between Kv7 channels and β-adrenoceptors in the human urinary bladder. The performed gene expression analysis combined with the organ bath studies imply that compounds that activate Kv7 channels could be useful for treatment of overactive bladder syndrome.

Reconfigurable virtual electrowetting channels.

PubMed

Banerjee, Ananda; Kreit, Eric; Liu, Yuguang; Heikenfeld, Jason; Papautsky, Ian

2012-02-21

Lab-on-a-chip systems rely on several microfluidic paradigms. The first uses a fixed layout of continuous microfluidic channels. Such lab-on-a-chip systems are almost always application specific and far from a true "laboratory." The second involves electrowetting droplet movement (digital microfluidics), and allows two-dimensional computer control of fluidic transport and mixing. The merging of the two paradigms in the form of programmable electrowetting channels takes advantage of both the "continuous" functionality of rigid channels based on which a large number of applications have been developed to date and the "programmable" functionality of digital microfluidics that permits electrical control of on-chip functions. In this work, we demonstrate for the first time programmable formation of virtual microfluidic channels and their continuous operation with pressure driven flows using an electrowetting platform. Experimental, theoretical, and numerical analyses of virtual channel formation with biologically relevant electrolyte solutions and electrically-programmable reconfiguration are presented. We demonstrate that the "wall-less" virtual channels can be formed reliably and rapidly, with propagation rates of 3.5-3.8 mm s(-1). Pressure driven transport in these virtual channels at flow rates up to 100 μL min(-1) is achievable without distortion of the channel shape. We further demonstrate that these virtual channels can be switched on-demand between multiple inputs and outputs. Ultimately, we envision a platform that would provide rapid prototyping of microfluidic concepts and would be capable of a vast library of functions and benefitting applications from clinical diagnostics in resource-limited environments to rapid system prototyping to high throughput pharmaceutical applications.

New Aspects of the Contribution of ER to SOCE Regulation: TRPC Proteins as a Link Between Plasma Membrane Ion Transport and Intracellular Ca2+ Stores.

PubMed

Bavencoffe, Alexis; Zhu, Michael Xi; Tian, Jin-Bin

2017-01-01

Transient receptor potential canonical (TRPC) proteins were identified as molecular candidates of receptor- and/or store-operated channels because of their close homology to the Drosophila TRP and TRPL. Functional studies have revealed that TRPC channels play an integrated part of phospholipase C-transduced cell signaling, mediating the influx of both Ca 2+ and Na + into cells. As a consequence, the TRPC channels have diverse functional roles in different cell types, including metabotropic receptor-evoked membrane depolarization and intracellular Ca 2+ concentration elevation. Depending on the cellular environment and the protein partners present in the channel complex, the TRPC channels display different biophysical properties and mechanisms of regulation, including but not limited to the Ca 2+ filling state of the endoplasmic reticulum. Despite the overwhelming focus on STIM-regulated Orai channels for store-operated Ca 2+ entry, evidence is growing for STIM-operated TRPC channel activities in various cell types, demonstrating both store-dependent and store-independent mechanisms of TRPC channel gating. The existence of physical and functional interactions between plasma membrane-localized TRPC channels and other proteins involved in sensing and regulating the intracellular Ca 2+ store contents, such as inositol trisphosphate receptors, Junctate, and Homer, further argues for the role of TRPC proteins in linking plasma membrane ion transport with intracellular Ca 2+ stores. The interplay among these proteins will likely define the functional significance of TRPC channel activation in different cellular contexts and under different modes of stimulations.

What You Don't Know Won't Hurt Me: Impression Management Functions of Communication Channels in Relationships.

ERIC Educational Resources Information Center

O'Sullivan, Patrick B.

2000-01-01

Addresses the implications of interpersonal communication technology use for personal relationships. Tests elements of an impression management model, which specifies the processes and outcomes of strategic uses of channel and message for self-presentational goals. Supports a functional perspective that views mediated communication channels as a…

Polyester modification of the mammalian TRPM8 channel protein: Implications for structure and function

PubMed Central

Bikard, Yann; Chen, Wei; Liu, Tong; Li, Hong; Jendrossek, Dieter; Cohen, Alejandro; Pavlov, Evgeny; Rohacs, Tibor; Zakharian, Eleonora

2013-01-01

SUMMARY The TRPM8 ion channel is expressed in sensory neurons and is responsible for sensing environmental cues such as cold temperatures and chemical compounds, including menthol and icilin. The channel functional activity is regulated by various physical and chemical factors, and is likely to be pre-conditioned by its molecular composition. Our studies indicate that TRPM8 channel forms a structural-functional complex with the polyester, poly-(R)-3hydroxybutyrate (PHB). We identified by mass spectrometry a number of PHB-modified peptides in the N-terminus of the TRPM8 protein and in its extracellular S3–S4 linker. Removal of PHB by enzymatic hydrolysis, and site-directed mutagenesis of both the serine residues that serve as covalent anchors for PHB and adjacent hydrophobic residues that interact with the methyl groups of the polymer, resulted in significant inhibition of TRPM8 channel activity. We conclude that the TRPM8 channel undergoes post-translational modification by PHB and that this modification is required for its normal function. PMID:23850286

A novel crystallization method for visualizing the membrane localization of potassium channels.

PubMed Central

Lopatin, A N; Makhina, E N; Nichols, C G

1998-01-01

The high permeability of K+ channels to monovalent thallium (Tl+) ions and the low solubility of thallium bromide salt were used to develop a simple yet very sensitive approach to the study of membrane localization of potassium channels. K+ channels (Kir1.1, Kir2.1, Kir2.3, Kv2.1), were expressed in Xenopus oocytes and loaded with Br ions by microinjection. Oocytes were then exposed to extracellular thallium. Under conditions favoring influx of Tl+ ions (negative membrane potential under voltage clamp, or high concentration of extracellular Tl+), crystals of TlBr, visible under low-power microscopy, formed under the membrane in places of high density of K+ channels. Crystals were not formed in uninjected oocytes, but were formed in oocytes expressing as little as 5 microS K+ conductance. The number of observed crystals was much lower than the estimated number of functional channels. Based on the pattern of crystal formation, K+ channels appear to be expressed mostly around the point of cRNA injection when injected either into the animal or vegetal hemisphere. In addition to this pseudopolarized distribution of K+ channels due to localized microinjection of cRNA, a naturally polarized (animal/vegetal side) distribution of K+ channels was also frequently observed when K+ channel cRNA was injected at the equator. A second novel "agarose-hemiclamp" technique was developed to permit direct measurements of K+ currents from different hemispheres of oocytes under two-microelectrode voltage clamp. This technique, together with direct patch-clamping of patches of membrane in regions of high crystal density, confirmed that the localization of TlBr crystals corresponded to the localization of functional K+ channels and suggested a clustered organization of functional channels. With appropriate permeant ion/counterion pairs, this approach may be applicable to the visualization of the membrane distribution of any functional ion channel. PMID:9591643

A novel crystallization method for visualizing the membrane localization of potassium channels.

PubMed

Lopatin, A N; Makhina, E N; Nichols, C G

1998-05-01

The high permeability of K+ channels to monovalent thallium (Tl+) ions and the low solubility of thallium bromide salt were used to develop a simple yet very sensitive approach to the study of membrane localization of potassium channels. K+ channels (Kir1.1, Kir2.1, Kir2.3, Kv2.1), were expressed in Xenopus oocytes and loaded with Br ions by microinjection. Oocytes were then exposed to extracellular thallium. Under conditions favoring influx of Tl+ ions (negative membrane potential under voltage clamp, or high concentration of extracellular Tl+), crystals of TlBr, visible under low-power microscopy, formed under the membrane in places of high density of K+ channels. Crystals were not formed in uninjected oocytes, but were formed in oocytes expressing as little as 5 microS K+ conductance. The number of observed crystals was much lower than the estimated number of functional channels. Based on the pattern of crystal formation, K+ channels appear to be expressed mostly around the point of cRNA injection when injected either into the animal or vegetal hemisphere. In addition to this pseudopolarized distribution of K+ channels due to localized microinjection of cRNA, a naturally polarized (animal/vegetal side) distribution of K+ channels was also frequently observed when K+ channel cRNA was injected at the equator. A second novel "agarose-hemiclamp" technique was developed to permit direct measurements of K+ currents from different hemispheres of oocytes under two-microelectrode voltage clamp. This technique, together with direct patch-clamping of patches of membrane in regions of high crystal density, confirmed that the localization of TlBr crystals corresponded to the localization of functional K+ channels and suggested a clustered organization of functional channels. With appropriate permeant ion/counterion pairs, this approach may be applicable to the visualization of the membrane distribution of any functional ion channel.

Conformational heterogeneity in closed and open states of the KcsA potassium channel in lipid bicelles

PubMed Central

Kim, Dorothy M.; Dikiy, Igor; Upadhyay, Vikrant; Posson, David J.

2016-01-01

The process of ion channel gating—opening and closing—involves local and global structural changes in the channel in response to external stimuli. Conformational changes depend on the energetic landscape that underlies the transition between closed and open states, which plays a key role in ion channel gating. For the prokaryotic, pH-gated potassium channel KcsA, closed and open states have been extensively studied using structural and functional methods, but the dynamics within each of these functional states as well as the transition between them is not as well understood. In this study, we used solution nuclear magnetic resonance (NMR) spectroscopy to investigate the conformational transitions within specific functional states of KcsA. We incorporated KcsA channels into lipid bicelles and stabilized them into a closed state by using either phosphatidylcholine lipids, known to favor the closed channel, or mutations designed to trap the channel shut by disulfide cross-linking. A distinct state, consistent with an open channel, was uncovered by the addition of cardiolipin lipids. Using selective amino acid labeling at locations within the channel that are known to move during gating, we observed at least two different slowly interconverting conformational states for both closed and open channels. The pH dependence of these conformations and the predictable disruptions to this dependence observed in mutant channels with altered pH sensing highlight the importance of conformational heterogeneity for KcsA gating. PMID:27432996

Functional and pharmacological consequences of the distribution of voltage-gated calcium channels in the renal blood vessels.

PubMed

Hansen, P B L

2013-04-01

Calcium channel blockers are widely used to treat hypertension because they inhibit voltage-gated calcium channels that mediate transmembrane calcium influx in, for example, vascular smooth muscle and cardiomyocytes. The calcium channel family consists of several subfamilies, of which the L-type is usually associated with vascular contractility. However, the L-, T- and P-/Q-types of calcium channels are present in the renal vasculature and are differentially involved in controlling vascular contractility, thereby contributing to regulation of kidney function and blood pressure. In the preglomerular vascular bed, all the three channel families are present. However, the T-type channel is the only channel in cortical efferent arterioles which is in contrast to the juxtamedullary efferent arteriole, and that leads to diverse functional effects of L- and T-type channel inhibition. Furthermore, by different mechanisms, T-type channels may contribute to both constriction and dilation of the arterioles. Finally, P-/Q-type channels are involved in the regulation of human intrarenal arterial contractility. The calcium blockers used in the clinic affect not only L-type but also P-/Q- and T-type channels. Therefore, the distinct effect obtained by inhibiting a given subtype or set of channels under experimental settings should be considered when choosing a calcium blocker for treatment. T-type channels seem to be crucial for regulating the GFR and the filtration fraction. Use of blockers is expected to lead to preferential efferent vasodilation, reduction of glomerular pressure and proteinuria. Therefore, renovascular T-type channels might provide novel therapeutic targets, and may have superior renoprotective effects compared to conventional calcium blockers. Acta Physiologica © 2013 Scandinavian Physiological Society.

Drosophila TRP and TRPL are assembled as homomultimeric channels in vivo

PubMed Central

Katz, Ben; Oberacker, Tina; Richter, David; Tzadok, Hanan; Peters, Maximilian; Minke, Baruch; Huber, Armin

2013-01-01

Summary Family members of the cationic transient receptor potential (TRP) channels serve as sensors and transducers of environmental stimuli. The ability of different TRP channel isoforms of specific subfamilies to form heteromultimers and the structural requirements for channel assembly are still unresolved. Although heteromultimerization of different mammalian TRP channels within single subfamilies has been described, even within a subfamily (such as TRPC) not all members co-assemble with each other. In Drosophila photoreceptors two TRPC channels, TRP and TRP-like protein (TRPL) are expressed together in photoreceptors where they generate the light-induced current. The formation of functional TRP–TRPL heteromultimers in cell culture and in vitro has been reported. However, functional in vivo assays have shown that each channel functions independently of the other. Therefore, the issue of whether TRP and TRPL form heteromultimers in vivo is still unclear. In the present study we investigated the ability of TRP and TRPL to form heteromultimers, and the structural requirements for channel assembly, by studying assembly of GFP-tagged TRP and TRPL channels and chimeric TRP and TRPL channels, in vivo. Interaction studies of tagged and native channels as well as native and chimeric TRP–TRPL channels using co-immunoprecipitation, immunocytochemistry and electrophysiology, critically tested the ability of TRP and TRPL to interact. We found that TRP and TRPL assemble exclusively as homomultimeric channels in their native environment. The above analyses revealed that the transmembrane regions of TRP and TRPL do not determine assemble specificity of these channels. However, the C-terminal regions of both TRP and TRPL predominantly specify the assembly of homomeric TRP and TRPL channels. PMID:23687378

Proton transfer in the K-channel analog of B-type Cytochrome c oxidase from Thermus thermophilus.

PubMed

Woelke, Anna Lena; Wagner, Anke; Galstyan, Gegham; Meyer, Tim; Knapp, Ernst-Walter

2014-11-04

A key enzyme in aerobic metabolism is cytochrome c oxidase (CcO), which catalyzes the reduction of molecular oxygen to water in the mitochondrial and bacterial membranes. Substrate electrons and protons are taken up from different sides of the membrane and protons are pumped across the membrane, thereby generating an electrochemical gradient. The well-studied A-type CcO uses two different entry channels for protons: the D-channel for all pumped and two consumed protons, and the K-channel for the other two consumed protons. In contrast, the B-type CcO uses only a single proton input channel for all consumed and pumped protons. It has the same location as the A-type K-channel (and thus is named the K-channel analog) without sharing any significant sequence homology. In this study, we performed molecular-dynamics simulations and electrostatic calculations to characterize the K-channel analog in terms of its energetic requirements and functionalities. The function of Glu-15B as a proton sink at the channel entrance is demonstrated by its rotational movement out of the channel when it is deprotonated and by its high pKA value when it points inside the channel. Tyr-244 in the middle of the channel is identified as the valve that ensures unidirectional proton transfer, as it moves inside the hydrogen-bond gap of the K-channel analog only while being deprotonated. The electrostatic energy landscape was calculated for all proton-transfer steps in the K-channel analog, which functions via proton-hole transfer. Overall, the K-channel analog has a very stable geometry without large energy barriers.

Proton Transfer in the K-Channel Analog of B-Type Cytochrome c Oxidase from Thermus thermophilus

PubMed Central

Woelke, Anna Lena; Wagner, Anke; Galstyan, Gegham; Meyer, Tim; Knapp, Ernst-Walter

2014-01-01

A key enzyme in aerobic metabolism is cytochrome c oxidase (CcO), which catalyzes the reduction of molecular oxygen to water in the mitochondrial and bacterial membranes. Substrate electrons and protons are taken up from different sides of the membrane and protons are pumped across the membrane, thereby generating an electrochemical gradient. The well-studied A-type CcO uses two different entry channels for protons: the D-channel for all pumped and two consumed protons, and the K-channel for the other two consumed protons. In contrast, the B-type CcO uses only a single proton input channel for all consumed and pumped protons. It has the same location as the A-type K-channel (and thus is named the K-channel analog) without sharing any significant sequence homology. In this study, we performed molecular-dynamics simulations and electrostatic calculations to characterize the K-channel analog in terms of its energetic requirements and functionalities. The function of Glu-15B as a proton sink at the channel entrance is demonstrated by its rotational movement out of the channel when it is deprotonated and by its high pKA value when it points inside the channel. Tyr-244 in the middle of the channel is identified as the valve that ensures unidirectional proton transfer, as it moves inside the hydrogen-bond gap of the K-channel analog only while being deprotonated. The electrostatic energy landscape was calculated for all proton-transfer steps in the K-channel analog, which functions via proton-hole transfer. Overall, the K-channel analog has a very stable geometry without large energy barriers. PMID:25418102

Regulation of the cellular localization and function of human transient receptor potential channel 1 by other members of the TRPC family.

PubMed

Alfonso, Salgado; Benito, Ordaz; Alicia, Sampieri; Angélica, Zepeda; Patricia, Glazebrook; Diana, Kunze; Vaca, Luis; Luis, Vaca

2008-04-01

Members of the Canonical Transient Receptor Potential (TRPC) family of ionic channels are able to form homo- and heterotetrameric channels. Depending on the study, TRPC1 has been detected on both the surface and inside the cell, probably in the endoplasmic reticulum (ER). Likewise, TRPC1 has been described both as a store-operated channel and as one unable to function when forming a homotetramer. It is possible that the apparent differences in the expression and function of TRPC1 are due to its association with other proteins, possibly from the same TRPC family. In the present study we used confocal microscopy and a fluorescently tagged TRPC1 to examine the localization of this protein when co-expressed with other members of the TRPC family. Whole-cell and single channel electrophysiological recordings were conducted to study the function of TRPC1 expressed alone or co-expressed with other members of the TRPC family. A FRET-based calcium sensor fused to TRPC1 was used to assess the functionality of the intracellular TRPC1. Our results showed that TRPC4 and TRPC5 were able to increase the amount of membrane-expressed TRPC1 as evaluated by confocal microscopy and patch clamp recordings. The FRET-based calcium sensor fused to TRPC1 strongly suggests that this protein forms ER-expressed functional homotetrameric channels activated by agonists coupled to the IP(3) cascade. These results indicate that TRPC1 is a multifunctional protein able to form intracellular calcium release channels when expressed alone, and plasma membrane channels when co-expressed with TRPC4 or TRPC5, but not TRPC3 or TRPC6. Both (ER and plasma membrane) forms of the channel are activated upon addition of agonists coupled to the IP(3) cascade.

Beamspace Multiple Input Multiple Output. Part II: Steerable Antennas in Mobile Ad Hoc Networks

DTIC Science & Technology

2016-09-01

to the transmitter with half the channel transfer function power , since the actual receiver dwells on each channel only half the time. Fourth diagram...steering in a wireless network to maximize signal power and minimize interference [8–10]. The ability to switch beams adds another diversity dimension to...channel transfer function power , since the actual receiver dwells on each channel only half the time. Fourth diagram: The transmit array sends four

Impact of the F508del mutation on ovine CFTR, a Cl− channel with enhanced conductance and ATP-dependent gating

PubMed Central

Cai, Zhiwei; Palmai-Pallag, Timea; Khuituan, Pissared; Mutolo, Michael J; Boinot, Clément; Liu, Beihui; Scott-Ward, Toby S; Callebaut, Isabelle; Harris, Ann; Sheppard, David N

2015-01-01

Cross-species comparative studies are a powerful approach to understanding the epithelial Cl− channel cystic fibrosis transmembrane conductance regulator (CFTR), which is defective in the genetic disease cystic fibrosis (CF). Here, we investigate the single-channel behaviour of ovine CFTR and the impact of the most common CF mutation, F508del-CFTR, using excised inside-out membrane patches from transiently transfected CHO cells. Like human CFTR, ovine CFTR formed a weakly inwardly rectifying Cl− channel regulated by PKA-dependent phosphorylation, inhibited by the open-channel blocker glibenclamide. However, for three reasons, ovine CFTR was noticeably more active than human CFTR. First, single-channel conductance was increased. Second, open probability was augmented because the frequency and duration of channel openings were increased. Third, with enhanced affinity and efficacy, ATP more strongly stimulated ovine CFTR channel gating. Consistent with these data, the CFTR modulator phloxine B failed to potentiate ovine CFTR Cl− currents. Similar to its impact on human CFTR, the F508del mutation caused a temperature-sensitive folding defect, which disrupted ovine CFTR protein processing and reduced membrane stability. However, the F508del mutation had reduced impact on ovine CFTR channel gating in contrast to its marked effects on human CFTR. We conclude that ovine CFTR forms a regulated Cl− channel with enhanced conductance and ATP-dependent channel gating. This phylogenetic analysis of CFTR structure and function demonstrates that subtle changes in structure have pronounced effects on channel function and the consequences of the CF mutation F508del. Key points Malfunction of the cystic fibrosis transmembrane conductance regulator (CFTR), a gated pathway for chloride movement, causes the common life-shortening genetic disease cystic fibrosis (CF). Towards the development of a sheep model of CF, we have investigated the function of sheep CFTR. We found that sheep CFTR was noticeably more active than human CFTR, while the most common CF mutation, F508del, had reduced impact on sheep CFTR function. Our results demonstrate that subtle changes in protein structure have marked effects on CFTR function and the consequences of the CF mutation F508del. PMID:25763566

Proximal clustering between BK and CaV1.3 channels promotes functional coupling and BK channel activation at low voltage

PubMed Central

Vivas, Oscar; Moreno, Claudia M; Santana, Luis F; Hille, Bertil

2017-01-01

CaV-channel dependent activation of BK channels is critical for feedback control of both calcium influx and cell excitability. Here we addressed the functional and spatial interaction between BK and CaV1.3 channels, unique CaV1 channels that activate at low voltages. We found that when BK and CaV1.3 channels were co-expressed in the same cell, BK channels started activating near −50 mV, ~30 mV more negative than for activation of co-expressed BK and high-voltage activated CaV2.2 channels. In addition, single-molecule localization microscopy revealed striking clusters of CaV1.3 channels surrounding clusters of BK channels and forming a multi-channel complex both in a heterologous system and in rat hippocampal and sympathetic neurons. We propose that this spatial arrangement allows tight tracking between local BK channel activation and the gating of CaV1.3 channels at quite negative membrane potentials, facilitating the regulation of neuronal excitability at voltages close to the threshold to fire action potentials. DOI: http://dx.doi.org/10.7554/eLife.28029.001 PMID:28665272

Tyrosine Phosphatases ε and α Perform Specific and Overlapping Functions in Regulation of Voltage-gated Potassium Channels in Schwann Cells

PubMed Central

Tiran, Zohar; Peretz, Asher; Sines, Tal; Shinder, Vera; Sap, Jan; Attali, Bernard

2006-01-01

Tyrosine phosphatases (PTPs) ε and α are closely related and share several molecular functions, such as regulation of Src family kinases and voltage-gated potassium (Kv) channels. Functional interrelationships between PTPε and PTPα and the mechanisms by which they regulate K+ channels and Src were analyzed in vivo in mice lacking either or both PTPs. Lack of either PTP increases Kv channel activity and phosphorylation in Schwann cells, indicating these PTPs inhibit Kv current amplitude in vivo. Open probability and unitary conductance of Kv channels are unchanged, suggesting an effect on channel number or organization. PTPα inhibits Kv channels more strongly than PTPε; this correlates with constitutive association of PTPα with Kv2.1, driven by membranal localization of PTPα. PTPα, but not PTPε, activates Src in sciatic nerve extracts, suggesting Src deregulation is not responsible exclusively for the observed phenotypes and highlighting an unexpected difference between both PTPs. Developmentally, sciatic nerve myelination is reduced transiently in mice lacking either PTP and more so in mice lacking both PTPs, suggesting both PTPs support myelination but are not fully redundant. We conclude that PTPε and PTPα differ significantly in their regulation of Kv channels and Src in the system examined and that similarity between PTPs does not necessarily result in full functional redundancy in vivo. PMID:16870705

Use of a purified and functional recombinant calcium-channel beta4 subunit in surface-plasmon resonance studies.

PubMed Central

Geib, Sandrine; Sandoz, Guillaume; Mabrouk, Kamel; Matavel, Alessandra; Marchot, Pascale; Hoshi, Toshinori; Villaz, Michel; Ronjat, Michel; Miquelis, Raymond; Lévêque, Christian; de Waard, Michel

2002-01-01

Native high-voltage-gated calcium channels are multi-subunit complexes comprising a pore-forming subunit Ca(v) and at least two auxiliary subunits alpha(2)delta and beta. The beta subunit facilitates cell-surface expression of the channel and contributes significantly to its biophysical properties. In spite of its importance, detailed structural and functional studies are hampered by the limited availability of native beta subunit. Here, we report the purification of a recombinant calcium-channel beta(4) subunit from bacterial extracts by using a polyhistidine tag. The purified protein is fully functional since it binds on the alpha1 interaction domain, its main Ca(v)-binding site, and regulates the activity of P/Q calcium channel expressed in Xenopus oocytes in a similar way to the beta(4) subunit produced by cRNA injection. We took advantage of the functionality of the purified material to (i) develop an efficient surface-plasmon resonance assay of the interaction between two calcium channel subunits and (ii) measure, for the first time, the affinity of the recombinant His-beta(4) subunit for the full-length Ca(v)2.1 channel. The availability of this purified material and the development of a surface-plasmon resonance assay opens two immediate research perspectives: (i) drug screening programmes applied to the Ca(v)/beta interaction and (ii) crystallographic studies of the calcium-channel beta(4) subunit. PMID:11988102

β1-Adrenergic blocker bisoprolol reverses down-regulated ion channels in sinoatrial node of heart failure rats.

PubMed

Du, Yuan; Zhang, Junbo; Xi, Yutao; Wu, Geru; Han, Ke; Huang, Xin; Ma, Aiqun; Wang, Tingzhong

2016-06-01

Bisoprolol, an antagonist of β1-adrenergic receptors, is effective in reducing the morbidity and mortality in patients with heart failure (HF). It has been found that HF is accompanied with dysfunction of the sinoatrial node (SAN). However, whether bisoprolol reverses the decreased SAN function in HF and how the relevant ion channels in SAN change were relatively less studied. SAN function and messenger RNA (mRNA) expression of sodium channels and hyperpolarization-activated cyclic nucleotide-gated (HCN) channel subunits were assessed in sham-operated rats, abdominal arterio-venous shunt (volume overload)-induced HF rats, and bisoprolol- treated HF rats. SAN cells of rats were isolated by laser capture microdissection. Quantitative real-time PCR analysis was used to quantify mRNA expression of sodium channels and HCN channel subunits in SAN. Intrinsic heart rate declined and sinus node recovery time prolonged in HF rats, indicating the suppressed SAN function, which could be improved by bisoprolol treatment. Nav1.1, Nav1.6, and HCN4 mRNA expressions were reduced in SAN in HF rats compared with that in control rats. Treatment with bisoprolol could reverse both the SAN function and the Nav1.1, Nav1.6, and HCN4 mRNA expression partially. These data indicated that bisoprolol is effective in HF treatment partially due to improved SAN function by reversing the down-regulation of sodium channels (Nav1.1 and Nav1.6) and HCN channel (HCN4) subunits in SAN in failing hearts.

Brownian motion and thermophoresis effects on Peristaltic slip flow of a MHD nanofluid in a symmetric/asymmetric channel

NASA Astrophysics Data System (ADS)

Sucharitha, G.; Sreenadh, S.; Lakshminarayana, P.; Sushma, K.

2017-11-01

The slip and heat transfer effects on MHD peristaltic transport of a nanofluid in a non-uniform symmetric/asymmetric channel have studied under the assumptions of elongated wave length and negligible Reynolds number. From the simplified governing equations, the closed form solutions for velocity, stream function, temperature and concentrations are obtained. Also dual solutions are discussed for symmetric and asymmetric channel cases. The effects of important physical parameters are explained graphically. The slip parameter decreases the fluid velocity in middle of the channel whereas it increases the velocity at the channel walls. Temperature and concentration are decreasing and increasing functions of radiation parameter respectively. Moreover, velocity, temperature and concentrations are high in symmetric channel when compared with asymmetric channel.

Phosphatidylinositol-4,5-bisphosphate is required for KCNQ1/KCNE1 channel function but not anterograde trafficking

PubMed Central

Royal, Alice A.

2017-01-01

The slow delayed-rectifier potassium current (IKs) is crucial for human cardiac action potential repolarization. The formation of IKs requires co-assembly of the KCNQ1 α-subunit and KCNE1 β-subunit, and mutations in either of these subunits can lead to hereditary long QT syndrome types 1 and 5, respectively. It is widely recognised that the KCNQ1/KCNE1 (Q1/E1) channel requires phosphatidylinositol-4,5-bisphosphate (PIP2) binding for function. We previously identified a cluster of basic residues in the proximal C-terminus of KCNQ1 that form a PIP2/phosphoinositide binding site. Upon charge neutralisation of these residues we found that the channel became more retained in the endoplasmic reticulum, which raised the possibility that channel–phosphoinositide interactions could play a role in channel trafficking. To explore this further we used a chemically induced dimerization (CID) system to selectively deplete PIP2 and/or phosphatidylinositol-4-phosphate (PI(4)P) at the plasma membrane (PM) or Golgi, and we subsequently monitored the effects on both channel trafficking and function. The depletion of PIP2 and/or PI(4)P at either the PM or Golgi did not alter channel cell-surface expression levels. However, channel function was extremely sensitive to the depletion of PIP2 at the PM, which is in contrast to the response of other cardiac potassium channels tested (Kir2.1 and Kv11.1). Surprisingly, when using the CID system IKs was dramatically reduced even before dimerization was induced, highlighting limitations regarding the utility of this system when studying processes highly sensitive to PIP2 depletion. In conclusion, we identify that the Q1/E1 channel does not require PIP2 or PI(4)P for anterograde trafficking, but is heavily reliant on PIP2 for channel function once at the PM. PMID:29020060

A Co-operative Regulation of Neuronal Excitability by UNC-7 Innexin and NCA/NALCN Leak Channel

PubMed Central

2011-01-01

Gap junctions mediate the electrical coupling and intercellular communication between neighboring cells. Some gap junction proteins, namely connexins and pannexins in vertebrates, and innexins in invertebrates, may also function as hemichannels. A conserved NCA/Dmα1U/NALCN family cation leak channel regulates the excitability and activity of vertebrate and invertebrate neurons. In the present study, we describe a genetic and functional interaction between the innexin UNC-7 and the cation leak channel NCA in Caenorhabditis elegans neurons. While the loss of the neuronal NCA channel function leads to a reduced evoked postsynaptic current at neuromuscular junctions, a simultaneous loss of the UNC-7 function restores the evoked response. The expression of UNC-7 in neurons reverts the effect of the unc-7 mutation; moreover, the expression of UNC-7 mutant proteins that are predicted to be unable to form gap junctions also reverts this effect, suggesting that UNC-7 innexin regulates neuronal activity, in part, through gap junction-independent functions. We propose that, in addition to gap junction-mediated functions, UNC-7 innexin may also form hemichannels to regulate C. elegans' neuronal activity cooperatively with the NCA family leak channels. PMID:21489288

47 CFR 22.575 - Use of mobile channel for remote control of station functions.

Code of Federal Regulations, 2010 CFR

2010-10-01

... 47 Telecommunication 2 2010-10-01 2010-10-01 false Use of mobile channel for remote control of...) COMMON CARRIER SERVICES PUBLIC MOBILE SERVICES Paging and Radiotelephone Service One-Way Or Two-Way Mobile Operation § 22.575 Use of mobile channel for remote control of station functions. Carriers may...

Application of amphipols for structure-functional analysis of TRP channels.

PubMed

Huynh, Kevin W; Cohen, Matthew R; Moiseenkova-Bell, Vera Y

2014-10-01

Amphipathic polymers (amphipols), such as A8-35 and SApol, are a new tool for stabilizing integral membrane proteins in detergent-free conditions for structural and functional studies. Transient receptor potential (TRP) ion channels function as tetrameric protein complexes in a diverse range of cellular processes including sensory transduction. Mammalian TRP channels share ~20 % sequence similarity and are categorized into six subfamilies: TRPC (canonical), TRPV (vanilloid), TRPA (ankyrin), TRPM (melastatin), TRPP (polycystin), and TRPML (mucolipin). Due to the inherent difficulties in purifying eukaryotic membrane proteins, structural studies of TRP channels have been limited. Recently, A8-35 was essential in resolving the molecular architecture of the nociceptor TRPA1 and led to the determination of a high-resolution structure of the thermosensitive TRPV1 channel by cryo-EM. Newly developed maltose-neopentyl glycol (MNG) detergents have also proven to be useful in stabilizing TRP channels for structural analysis. In this review, we will discuss the impacts of amphipols and MNG detergents on structural studies of TRP channels by cryo-EM. We will compare how A8-35 and MNG detergents interact with the hydrophobic transmembrane domains of TRP channels. In addition, we will discuss what these cryo-EM studies reveal on the importance of screening different types of surfactants toward determining high-resolution structures of TRP channels.

Application of amphipols for structure-functional analysis of TRP channels

PubMed Central

Huynh, Kevin W.; Cohen, Matthew R.; Moiseenkova-Bell, Vera Y.

2014-01-01

Amphipathic polymers (amphipols), such as A8-35 and SApol, are a new tool for stabilizing integral membrane proteins in detergent-free conditions for structural and functional studies. Transient receptor potential (TRP) ion channels function as tetrameric protein complexes in a diverse range of cellular processes including sensory transduction. Mammalian TRP channels share ~20% sequence similarity and are categorized into six subfamilies: TRPC (canonical), TRPV (vanilloid), TRPA (ankyrin), TRPM (melastatin), TRPP (polycystin), and TRPML (mucolipin). Due to the inherent difficulties in purifying eukaryotic membrane proteins, structural studies of TRP channels have been limited. Recently, A8-35 was essential in resolving the molecular architecture of the nociceptor TRPA1 and led to the determination of a high resolution structure of the thermosensitive TRPV1 channel by cryo-EM. Newly developed maltose-neopentyl glycol (MNG) detergents have also proven useful in stabilizing TRP channels for structural analysis. In this review, we will discuss the impact of amphipols and MNG detergents on structural studies of TRP channels by cryo-EM. We will compare how A8-35 and MNG detergents interact with the hydrophobic transmembrane (TM) domains of TRP channels. In addition, we will discuss what these cryo-EM studies reveal on the importance of screening different types of surfactants towards determining high resolution structures of TRP channels. PMID:24894720

Diverse Roles for Auxiliary Subunits in Phosphorylation-Dependent Regulation of Mammalian Brain Voltage-Gated Potassium Channels

PubMed Central

Vacher, Helene; Trimmer, James S.

2012-01-01

Voltage-gated ion channels are a diverse family of signaling proteins that mediate rapid electrical signaling events. Among these, voltage-gated potassium or Kv channels are the most diverse, in part due to the large number of principal (or α) subunits and auxiliary subunits that can assemble in different combinations to generate Kv channel complexes with distinct structures and functions. The diversity of Kv channels underlies much of the variability in the active properties between different mammalian central neurons, and the dynamic changes that lead to experience-dependent plasticity in intrinsic excitability. Recent studies have revealed that Kv channel α subunits and auxiliary subunits are extensively phosphorylated, contributing to additional structural and functional diversity. Here we highlight recent studies that show that auxiliary subunits exert some of their profound effects on dendritic Kv4 and axonal Kv1 channels through phosphorylation-dependent mechanisms, either due to phosphorylation on the auxiliary subunit itself, or by influencing the extent and/or impact of α subunit phosphorylation. The complex effects of auxiliary subunits and phosphorylation provide a potent mechanism to generate additional diversity in the structure and function of Kv4 and Kv1 channels, as well as allowing for dynamic reversible regulation of these important ion channels. PMID:21822597

The TRP channel superfamily: insights into how structure, protein-lipid interactions and localization influence function.

PubMed

Reaves, B J; Wolstenholme, A J

2007-02-01

TRP (transient receptor potential) cationic channels are key molecules that are involved in a variety of diverse biological processes ranging from fertility to osmosensation and nociception. Increasing our knowledge of these channels will help us to understand a range of physiological and pathogenic processes, as well as highlighting potential therapeutic drug targets. The founding members of the TRP family, Drosophila TRP and TRPL (TRP-like) proteins, were identified within the last two decades and there has been a subsequent explosion in the number and type of TRP channel described. Although information is accumulating as to the function of some of the TRP channels, the activation and inactivation mechanisms, structure, and interacting proteins of many, if not most, are awaiting elucidation. The Cell and Molecular Biology of TRP Channels Meeting held at the University of Bath included speakers working on a number of the different subfamilies of TRP channels and provided a basis for highlighting both similarities and differences between these groups. As the TRP channels mediate diverse functions, this meeting also brought together an audience with wide-ranging research interests, including biochemistry, cell biology, physiology and neuroscience, and inspired lively discussion on the issues reviewed herein.

Potassium Channels in Peripheral Pain Pathways: Expression, Function and Therapeutic Potential

PubMed Central

Du, Xiaona; Gamper, Nikita

2013-01-01

Electrical excitation of peripheral somatosensory nerves is a first step in generation of most pain signals in mammalian nervous system. Such excitation is controlled by an intricate set of ion channels that are coordinated to produce a degree of excitation that is proportional to the strength of the external stimulation. However, in many disease states this coordination is disrupted resulting in deregulated peripheral excitability which, in turn, may underpin pathological pain states (i.e. migraine, neuralgia, neuropathic and inflammatory pains). One of the major groups of ion channels that are essential for controlling neuronal excitability is potassium channel family and, hereby, the focus of this review is on the K+ channels in peripheral pain pathways. The aim of the review is threefold. First, we will discuss current evidence for the expression and functional role of various K+ channels in peripheral nociceptive fibres. Second, we will consider a hypothesis suggesting that reduced functional activity of K+ channels within peripheral nociceptive pathways is a general feature of many types of pain. Third, we will evaluate the perspectives of pharmacological enhancement of K+ channels in nociceptive pathways as a strategy for new analgesic drug design. PMID:24396338

Novel treatment strategies for smooth muscle disorders: Targeting Kv7 potassium channels.

PubMed

Haick, Jennifer M; Byron, Kenneth L

2016-09-01

Smooth muscle cells provide crucial contractile functions in visceral, vascular, and lung tissues. The contractile state of smooth muscle is largely determined by their electrical excitability, which is in turn influenced by the activity of potassium channels. The activity of potassium channels sustains smooth muscle cell membrane hyperpolarization, reducing cellular excitability and thereby promoting smooth muscle relaxation. Research over the past decade has indicated an important role for Kv7 (KCNQ) voltage-gated potassium channels in the regulation of the excitability of smooth muscle cells. Expression of multiple Kv7 channel subtypes has been demonstrated in smooth muscle cells from viscera (gastrointestinal, bladder, myometrial), from the systemic and pulmonary vasculature, and from the airways of the lung, from multiple species, including humans. A number of clinically used drugs, some of which were developed to target Kv7 channels in other tissues, have been found to exert robust effects on smooth muscle Kv7 channels. Functional studies have indicated that Kv7 channel activators and inhibitors have the ability to relax and contact smooth muscle preparations, respectively, suggesting a wide range of novel applications for the pharmacological tool set. This review summarizes recent findings regarding the physiological functions of Kv7 channels in smooth muscle, and highlights potential therapeutic applications based on pharmacological targeting of smooth muscle Kv7 channels throughout the body. Published by Elsevier Inc.

Touch, Tension, and Transduction – the Function and Regulation of Piezo Ion Channels

PubMed Central

Wu, Jason; Lewis, Amanda; Grandl, Jörg

2016-01-01

In 2010, two proteins, Piezo1 and Piezo2, were identified as the long-sought molecular carriers of an excitatory mechanically activated current found in many cells. This discovery has opened the floodgates for studying a vast number of mechanotransduction processes. Over the past six years, groundbreaking research has identified Piezos as ion channels that sense light touch, proprioception, and vascular blood flow, ruled out roles for Piezos in several other mechanotransduction processes, and revealed the basic structural and functional properties of the channel. Here, we review these findings and discuss the many aspects of Piezo function that remain mysterious, including how Piezos convert a variety of mechanical stimuli into channel activation and subsequent inactivation, and what molecules and mechanisms modulate Piezo function. PMID:27743844

Touch, Tension, and Transduction - The Function and Regulation of Piezo Ion Channels.

PubMed

Wu, Jason; Lewis, Amanda H; Grandl, Jörg

2017-01-01

In 2010, two proteins, Piezo1 and Piezo2, were identified as the long-sought molecular carriers of an excitatory mechanically activated current found in many cells. This discovery has opened the floodgates for studying a vast number of mechanotransduction processes. Over the past 6 years, groundbreaking research has identified Piezos as ion channels that sense light touch, proprioception, and vascular blood flow, ruled out roles for Piezos in several other mechanotransduction processes, and revealed the basic structural and functional properties of the channel. Here, we review these findings and discuss the many aspects of Piezo function that remain mysterious, including how Piezos convert a variety of mechanical stimuli into channel activation and subsequent inactivation, and what molecules and mechanisms modulate Piezo function. Copyright © 2016 Elsevier Ltd. All rights reserved.

Threading the biophysics of mammalian Slo1 channels onto structures of an invertebrate Slo1 channel

PubMed Central

2017-01-01

For those interested in the machinery of ion channel gating, the Ca2+ and voltage-activated BK K+ channel provides a compelling topic for investigation, by virtue of its dual allosteric regulation by both voltage and intracellular Ca2+ and because its large-single channel conductance facilitates detailed kinetic analysis. Over the years, biophysical analyses have illuminated details of the allosteric regulation of BK channels and revealed insights into the mechanism of BK gating, e.g., inner cavity size and accessibility and voltage sensor-pore coupling. Now the publication of two structures of an Aplysia californica BK channel—one liganded and one metal free—promises to reinvigorate functional studies and interpretation of biophysical results. The new structures confirm some of the previous functional inferences but also suggest new perspectives regarding cooperativity between Ca2+-binding sites and the relationship between voltage- and Ca2+-dependent gating. Here we consider the extent to which the two structures explain previous functional data on pore-domain properties, voltage-sensor motions, and divalent cation binding and activation of the channel. PMID:29025867

T-type calcium channels in synaptic plasticity

PubMed Central

Lambert, Régis C.

2017-01-01

ABSTRACT The role of T-type calcium currents is rarely considered in the extensive literature covering the mechanisms of long-term synaptic plasticity. This situation reflects the lack of suitable T-type channel antagonists that till recently has hampered investigations of the functional roles of these channels. However, with the development of new pharmacological and genetic tools, a clear involvement of T-type channels in synaptic plasticity is starting to emerge. Here, we review a number of studies showing that T-type channels participate to numerous homo- and hetero-synaptic plasticity mechanisms that involve different molecular partners and both pre- and post-synaptic modifications. The existence of T-channel dependent and independent plasticity at the same synapse strongly suggests a subcellular localization of these channels and their partners that allows specific interactions. Moreover, we illustrate the functional importance of T-channel dependent synaptic plasticity in neocortex and thalamus. PMID:27653665

A cost-effective protocol for the over-expression and purification of fully-functional and more stable Erwinia chrisanthemi ligand-gated ion channel

PubMed Central

Elberson, Benjamin W.; Whisenant, Ty E.; Cortes, D. Marien; Cuello, Luis G.

2017-01-01

The Erwinia chrisanthemi ligand-gated ion channel, ELIC, is considered an excellent structural and functional surrogate for the whole pentameric ligand-gated ion channel family. Despite its simplicity, ELIC is structurally capable of undergoing ligand-dependent activation and a concomitant desensitization process. To determine at the molecular level the structural changes underlying ELIC’s function, it is desirable to produce large quantities of protein. This protein should be properly folded, fully-functional and amenable to structural determinations. In the current paper, we report a completely new protocol for the expression and purification of milligram quantities of fully-functional, more stable and crystallizable ELIC. The use of an autoinduction media and inexpensive detergents during ELIC extraction, in addition to the high-quality and large quantity of the purified channel, are the highlights of this improved biochemical protocol. PMID:28279818

TRPM4 channel: a new player in urinary bladder smooth muscle function in rats

PubMed Central

Smith, Amy C.; Parajuli, Shankar P.; Hristov, Kiril L.; Cheng, Qiuping; Soder, Rupal P.; Afeli, Serge A. Y.; Earley, Scott; Xin, Wenkuan; Malysz, John

2013-01-01

The TRPM4 channel is a Ca2+-activated, monovalent cation-selective channel of the melastatin transient receptor potential (TRPM) family. The TRPM4 channel is implicated in the regulation of many cellular processes including the immune response, insulin secretion, and pressure-induced vasoconstriction of cerebral arteries. However, the expression and function of the TRPM4 channels in detrusor smooth muscle (DSM) have not yet been explored. Here, we provide the first molecular, electrophysiological, and functional evidence for the presence of TRPM4 channels in rat DSM. We detected the expression of TRPM4 channels at mRNA and protein levels in freshly isolated DSM single cells and DSM tissue using RT-PCR, Western blotting, immunohistochemistry, and immunocytochemistry. 9-Hydroxyphenanthrene (9-phenanthrol), a novel selective inhibitor of TRPM4 channels, was used to examine their role in DSM function. In perforated patch-clamp recordings using freshly isolated rat DSM cells, 9-phenanthrol (30 μM) decreased the spontaneous inward current activity at −70 mV. Real-time DSM live-cell Ca2+ imaging showed that selective inhibition of TRPM4 channels with 9-phenanthrol (30 μM) significantly reduced the intracellular Ca2+ levels. Isometric DSM tension recordings revealed that 9-phenanthrol (0.1–30 μM) significantly inhibited the amplitude, muscle force integral, and frequency of the spontaneous phasic and pharmacologically induced contractions of rat DSM isolated strips. 9-Phenanthrol also decreased the amplitude and muscle force integral of electrical field stimulation-induced contractions. In conclusion, this is the first study to examine the expression and provide evidence for TRPM4 channels as critical regulators of rat DSM excitability and contractility. PMID:23283997

A Kv7.2 mutation associated with early onset epileptic encephalopathy with suppression-burst enhances Kv7/M channel activity.

PubMed

Devaux, Jérôme; Abidi, Affef; Roubertie, Agathe; Molinari, Florence; Becq, Hélène; Lacoste, Caroline; Villard, Laurent; Milh, Mathieu; Aniksztejn, Laurent

2016-05-01

Mutations in the KCNQ2 gene encoding the voltage-gated potassium channel subunit Kv7.2 cause early onset epileptic encephalopathy (EOEE). Most mutations have been shown to induce a loss of function or to affect the subcellular distribution of Kv7 channels in neurons. Herein, we investigated functional consequences and subcellular distribution of the p.V175L mutation of Kv7.2 (Kv7.2(V175L) ) found in a patient presenting EOEE. We observed that the mutation produced a 25-40 mV hyperpolarizing shift of the conductance-voltage relationship of both the homomeric Kv7.2(V175L) and heteromeric Kv7.2(V175L) /Kv7.3 channels compared to wild-type channels and a 10 mV hyperpolarizing shift of Kv7.2(V175L) /Kv7.2/Kv7.3 channels in a 1:1:2 ratio mimicking the patient situation. Mutant channels also displayed faster activation kinetics and an increased current density that was prevented by 1 μm linopirdine. The p.V175L mutation did not affect the protein expression of Kv7 channels and its localization at the axon initial segment. We conclude that p.V175L is a gain of function mutation. This confirms previous observations showing that mutations having opposite consequences on M channels can produce EOEE. These findings alert us that drugs aiming to increase Kv7 channel activity might have adverse effects in EOEE in the case of gain-of-function variants. Wiley Periodicals, Inc. © 2016 International League Against Epilepsy.

Complex expression and localization of inactivating Kv channels in cultured hippocampal astrocytes.

PubMed

Bekar, Lane K; Loewen, Matthew E; Cao, Kun; Sun, Xianfeng; Leis, Jerome; Wang, Rui; Forsyth, George W; Walz, Wolfgang

2005-03-01

Voltage-gated potassium channels are well established as critical for setting action potential frequency, membrane potential, and neurotransmitter release in neurons. However, their role in the "nonexcitable" glial cell type is yet to be fully understood. We used whole cell current kinetics, pharmacology, immunocytochemistry, and RT-PCR to characterize A-type current in hippocampal astrocyte cultures to better understand its function. Pharmacological analysis suggests that approximately 70, 10, and <5% of total A current is associated with Kv4, Kv3, and Kv1 channels, respectively. In addition, pharmacology and kinetics provide evidence for a significant contribution of KChIP accessory proteins to astrocytic A-channel composition. Localization of the Shaw Kv3.4 channel to astrocytic processes and the Shal Kv4.3 channel to soma suggest that these channels serve a specific function. Given this complex A-type channel expression pattern, we assessed the role of A currents in membrane voltage oscillations in response to current injections. Although TEA-sensitive delayed-rectifying currents are involved in the extent of repolarization, 4-AP-sensitive A currents serve to increase the rate. As in neurons, this effect may enable astrocytes to respond rapidly to high-frequency synaptic events. Our results indicate that hippocampal astrocytes in vitro express multiple A-type Kv channel alpha-subunits with accessory, possibly Ca(2+)-sensitive, cytoplasmic subunits that appear to be specifically localized to subcellular membrane compartments. Function of these channels remains to be determined in a physiological setting. However, this study suggests that they enable astrocytes to respond rapidly with membrane voltage oscillations to high-frequency incoming signals, possibly synchronizing astrocyte function to neuronal activity.

Smooth Muscle Ion Channels and Regulation of Vascular Tone in Resistance Arteries and Arterioles

PubMed Central

Tykocki, Nathan R.; Boerman, Erika M.; Jackson, William F.

2017-01-01

Vascular tone of resistance arteries and arterioles determines peripheral vascular resistance, contributing to the regulation of blood pressure and blood flow to, and within the body’s tissues and organs. Ion channels in the plasma membrane and endoplasmic reticulum of vascular smooth muscle cells (SMCs) in these blood vessels importantly contribute to the regulation of intracellular Ca2+ concentration, the primary determinant of SMC contractile activity and vascular tone. Ion channels provide the main source of activator Ca2+ that determines vascular tone, and strongly contribute to setting and regulating membrane potential, which, in turn, regulates the open-state-probability of voltage gated Ca2+ channels (VGCCs), the primary source of Ca2+ in resistance artery and arteriolar SMCs. Ion channel function is also modulated by vasoconstrictors and vasodilators, contributing to all aspects of the regulation of vascular tone. This review will focus on the physiology of VGCCs, voltage-gated K+ (KV) channels, large-conductance Ca2+-activated K+ (BKCa) channels, strong-inward-rectifier K+ (KIR) channels, ATP-sensitive K+ (KATP) channels, ryanodine receptors (RyRs), inositol 1,4,5-trisphosphate receptors (IP3Rs), and a variety of transient receptor potential (TRP) channels that contribute to pressure-induced myogenic tone in resistance arteries and arterioles, the modulation of the function of these ion channels by vasoconstrictors and vasodilators, their role in the functional regulation of tissue blood flow and their dysfunction in diseases such as hypertension, obesity, and diabetes. PMID:28333380

Redox Regulation of Neuronal Voltage-Gated Calcium Channels

PubMed Central

Jevtovic-Todorovic, Vesna

2014-01-01

Abstract Significance: Voltage-gated calcium channels are ubiquitously expressed in neurons and are key regulators of cellular excitability and synaptic transmitter release. There is accumulating evidence that multiple subtypes of voltage-gated calcium channels may be regulated by oxidation and reduction. However, the redox mechanisms involved in the regulation of channel function are not well understood. Recent Advances: Several studies have established that both T-type and high-voltage-activated subtypes of voltage-gated calcium channel can be redox-regulated. This article reviews different mechanisms that can be involved in redox regulation of calcium channel function and their implication in neuronal function, particularly in pain pathways and thalamic oscillation. Critical Issues: A current critical issue in the field is to decipher precise mechanisms of calcium channel modulation via redox reactions. In this review we discuss covalent post-translational modification via oxidation of cysteine molecules and chelation of trace metals, and reactions involving nitric oxide-related molecules and free radicals. Improved understanding of the roles of redox-based reactions in regulation of voltage-gated calcium channels may lead to improved understanding of novel redox mechanisms in physiological and pathological processes. Future Directions: Identification of redox mechanisms and sites on voltage-gated calcium channel may allow development of novel and specific ion channel therapies for unmet medical needs. Thus, it may be possible to regulate the redox state of these channels in treatment of pathological process such as epilepsy and neuropathic pain. Antioxid. Redox Signal. 21, 880–891. PMID:24161125

TMEM16A is associated with voltage-gated calcium channels in mouse retina and its function is disrupted upon mutation of the auxiliary α2δ4 subunit

PubMed Central

Caputo, Antonella; Piano, Ilaria; Demontis, Gian Carlo; Bacchi, Niccolò; Casarosa, Simona; Santina, Luca Della; Gargini, Claudia

2015-01-01

Photoreceptors rely upon highly specialized synapses to efficiently transmit signals to multiple postsynaptic targets. Calcium influx in the presynaptic terminal is mediated by voltage-gated calcium channels (VGCC). This event triggers neurotransmitter release, but also gates calcium-activated chloride channels (TMEM), which in turn regulate VGCC activity. In order to investigate the relationship between VGCC and TMEM channels, we analyzed the retina of wild type (WT) and Cacna2d4 mutant mice, in which the VGCC auxiliary α2δ4 subunit carries a nonsense mutation, disrupting the normal channel function. Synaptic terminals of mutant photoreceptors are disarranged and synaptic proteins as well as TMEM16A channels lose their characteristic localization. In parallel, calcium-activated chloride currents are impaired in rods, despite unaltered TMEM16A protein levels. Co-immunoprecipitation revealed the interaction between VGCC and TMEM16A channels in the retina. Heterologous expression of these channels in tsA-201 cells showed that TMEM16A associates with the CaV1.4 subunit, and the association persists upon expression of the mutant α2δ4 subunit. Collectively, our experiments show association between TMEM16A and the α1 subunit of VGCC. Close proximity of these channels allows optimal function of the photoreceptor synaptic terminal under physiological conditions, but also makes TMEM16A channels susceptible to changes occurring to calcium channels. PMID:26557056

Different Ligands of the TRPV3 Cation Channel Cause Distinct Conformational Changes as Revealed by Intrinsic Tryptophan Fluorescence Quenching*

PubMed Central

Billen, Bert; Brams, Marijke; Debaveye, Sarah; Remeeva, Alina; Alpizar, Yeranddy A.; Waelkens, Etienne; Kreir, Mohamed; Brüggemann, Andrea; Talavera, Karel; Nilius, Bernd; Voets, Thomas; Ulens, Chris

2015-01-01

TRPV3 is a thermosensitive ion channel primarily expressed in epithelial tissues of the skin, nose, and tongue. The channel has been implicated in environmental thermosensation, hyperalgesia in inflamed tissues, skin sensitization, and hair growth. Although transient receptor potential (TRP) channel research has vastly increased our understanding of the physiological mechanisms of nociception and thermosensation, the molecular mechanics of these ion channels are still largely elusive. In order to better comprehend the functional properties and the mechanism of action in TRP channels, high-resolution three-dimensional structures are indispensable, because they will yield the necessary insights into architectural intimacies at the atomic level. However, structural studies of membrane proteins are currently hampered by difficulties in protein purification and in establishing suitable crystallization conditions. In this report, we present a novel protocol for the purification of membrane proteins, which takes advantage of a C-terminal GFP fusion. Using this protocol, we purified human TRPV3. We show that the purified protein is a fully functional ion channel with properties akin to the native channel using planar patch clamp on reconstituted channels and intrinsic tryptophan fluorescence spectroscopy. Using intrinsic tryptophan fluorescence spectroscopy, we reveal clear distinctions in the molecular interaction of different ligands with the channel. Altogether, this study provides powerful tools to broaden our understanding of ligand interaction with TRPV channels, and the availability of purified human TRPV3 opens up perspectives for further structural and functional studies. PMID:25829496

From The Cover: Microtransplantation of functional receptors and channels from the Alzheimer's brain to frog oocytes

NASA Astrophysics Data System (ADS)

Miledi, R.; Dueñas, Z.; Martinez-Torres, A.; Kawas, C. H.; Eusebi, F.

2004-02-01

About a decade ago, cell membranes from the electric organ of Torpedo and from the rat brain were transplanted to frog oocytes, which thus acquired functional Torpedo and rat neurotransmitter receptors. Nevertheless, the great potential that this method has for studying human diseases has remained virtually untapped. Here, we show that cell membranes from the postmortem brains of humans that suffered Alzheimer's disease can be microtransplanted to the plasma membrane of Xenopus oocytes. We show also that these postmortem membranes carry neurotransmitter receptors and voltage-operated channels that are still functional, even after they have been kept frozen for many years. This method provides a new and powerful approach to study directly the functional characteristics and structure of receptors, channels, and other membrane proteins of the Alzheimer's brain. This knowledge may help in understanding the basis of Alzheimer's disease and also help in developing new treatments. -aminobutyric acid receptors | sodium channels | calcium channels | postmortem brain

Strong potential wave functions with elastic channel distortion

DOE Office of Scientific and Technical Information (OSTI.GOV)

Macek, J.; Taulbjerg, K.

1989-06-01

The strong-potential Born approximation is analyzed in a channel-distorted-wave approach. Channel-distorted SPB wave functions are reduced to a conventional form in which the standard off-energy-shell factor /ital g/ has been replaced by a modified factor ..gamma.., which represents a suitable average of /ital g/ over the momentum distribution of the distorted-channel function. The modified factor is evaluated in a physically realistic model for the distortion potential, and it is found that ..gamma.. is well represented by a slowly varying phase factor. The channel-distorted SPB approximation is accordingly identical to the impulse approximation if the phase variation of ..gamma.. can bemore » ignored. This is generally the case in applications to radiative electron capture and to a good approximation for ordinary capture at not too small velocities.« less

Functional role of hCngb3 in regulation of human cone cng channel: effect of rod monochromacy-associated mutations in hCNGB3 on channel function.

PubMed

Okada, Akira; Ueyama, Hisao; Toyoda, Futoshi; Oda, Sanae; Ding, Wei-Guang; Tanabe, Shoko; Yamade, Shinichi; Matsuura, Hiroshi; Ohkubo, Iwao; Kani, Kazutaka

2004-07-01

The human cone photoreceptor cyclic nucleotide-gated (CNG) channel comprises alpha- and beta-subunits, which are respectively encoded by hCNGA3 and hCNGB3. The purpose was to examine the functional role of hCNGB3 in modulation of human cone CNG channels and to characterize functional consequences of rod monochromacy-associated mutations in hCNGB3 (S435F and D633G). Macroscopic patch currents were recorded from human embryonic kidney (HEK) 293 cells expressing homomeric (hCNGA3 and hCNGB3) and heteromeric (hCNGA3/hCNGB3, hCNGA3/hCNGB3-S435F, and hCNGA3/hCNGB3-D633G) channels using inside-out patch-clamp technique. Both hCNGA3 homomeric and hCNGA3/hCNGB3 heteromeric channels were activated by cGMP, with half-maximally activating concentration (K(1/2)) of 11.1 +/- 1.0 and 26.2 +/- 1.9 micro M, respectively. The hCNGA3 channels appeared to be more sensitive to inhibition by extracellular Ca(2+) compared with hCNGA3/hCNGB3 channels, when assessed by the degree of outward rectification. Coexpression of either of rod monochromacy-associated mutants of hCNGB3 with hCNGA3 significantly reduced K(1/2) value for cGMP but little affected the sensitivity to extracellular Ca(2+), compared with wild-type heteromeric channels. The selectivity of hCNGA3, hCNGA3/hCNGB3, hCNGA3/hCNGB3-S435F, and hCNGA3/hCNGB3-D633G channels for monovalent cations were largely similar. Immunoprecipitation experiments showed association of hCNGA3 subunit with both of wild-type and mutant hCNGB3 subunits. The hCNGB3 plays an important modulatory role in the function of human cone CNG channels with respect to cGMP and extracellular Ca(2+) sensitivities. The rod monochromacy-associated S435F and D633G mutations in hCNGB3 evokes a significant increase in the apparent affinity for cGMP, which should alter cone function and thereby contribute at least partly to pathogenesis of the disease.

KATP Channel Mutations and Neonatal Diabetes.

PubMed

Shimomura, Kenju; Maejima, Yuko

2017-09-15

Since the discovery of the K ATP channel in 1983, numerous studies have revealed its physiological functions. The K ATP channel is expressed in various organs, including the pancreas, brain and skeletal muscles. It functions as a "metabolic sensor" that converts the metabolic status to electrical activity. In pancreatic beta-cells, the K ATP channel regulates the secretion of insulin by sensing a change in the blood glucose level and thus maintains glucose homeostasis. In 2004, heterozygous gain-of-function mutations in the KCNJ11 gene, which encodes the Kir6.2 subunit of the K ATP channel, were found to cause neonatal diabetes. In some mutations, diabetes is accompanied by severe neurological symptoms [developmental delay, epilepsy, neonatal diabetes (DEND) syndrome]. This review focuses on mutations of Kir6.2, the pore-forming subunit and sulfonylurea receptor (SUR) 1, the regulatory subunit of the K ATP channel, which cause neonatal diabetes/DEND syndrome and also discusses the findings of the pathological mechanisms that are associated with neonatal diabetes, and its neurological features.

Targeting mechanisms of high voltage-activated Ca2+ channels.

PubMed

Herlitze, Stefan; Xie, Mian; Han, Jing; Hümmer, Alexander; Melnik-Martinez, Katya V; Moreno, Rosa L; Mark, Melanie D

2003-12-01

Functional voltage-dependent Ca2+ channel complexes are assembled by three to four subunits: alpha1, beta, alpha2delta subunits (C. Leveque et al., 1994, J. Biol Chem. 269, 6306-6312; M. W. McEnery et al., 1991, Proc. Natl. Acad. Sci. U.S.A. 88, 11095-11099) and at least in muscle cells also y subunits (B. M. Curtis and W. A. Catterall, 1984, Biochemistry 23, 2113-2118). Ca2+ channels mediate the voltage-dependent Ca2+ influx in subcellular compartments, triggering such diverse processes as neurotransmitter release, dendritic action potentials, excitation-contraction, and excitation-transcription coupling. The targeting of biophysically defined Ca2+ channel complexes to the correct subcellular structures is, thus, critical to proper cell and physiological functioning. Despite their importance, surprisingly little is known about the targeting mechanisms by which Ca2+ channel complexes are transported to their site of function. Here we summarize what we know about the targeting of Ca2+ channel complexes through the cell to the plasma membrane and subcellular structures.

Towards an understanding of the structural and functional properties of MscL, a mechanosensitive channel in bacteria

NASA Technical Reports Server (NTRS)

Blount, P.; Sukharev, S. I.; Moe, P. C.; Nagle, S. K.; Kung, C.

1996-01-01

Whether it be to sense a touch, arterial pressure, or an osmotic gradient across a cell membrane, essentially all living organisms require the capability of detecting mechanical force. Electrophysiological evidence has suggested that mechanosensitive ion channels play a major role in many systems where mechanical force is detected. But, despite their biological importance, determination of the most basic structural and functional features of mechanosensitive channels has only recently become possible. A gene called mscL, which was isolated from Escherichia coli, was the first gene shown to encode a mechanosensitive channel activity. This channel directly responds to tension in the membrane; no other proteins are required. MscL appears to be a homohexamer of a 136 amino acid polypeptide that is highly alpha helical, contains two transmembrane domains, and has both the amino and carboxyl termini in the cytoplasm. The study of the MscL protein remains, to date, one of the most viable options for understanding the structural and functional characteristics of a mechanosensitive channel.

Positrons vs electrons channeling in silicon crystal: energy levels, wave functions and quantum chaos manifestations

NASA Astrophysics Data System (ADS)

Shul'ga, N. F.; Syshchenko, V. V.; Tarnovsky, A. I.; Solovyev, I. I.; Isupov, A. Yu.

2018-01-01

The motion of fast electrons through the crystal during axial channeling could be regular and chaotic. The dynamical chaos in quantum systems manifests itself in both statistical properties of energy spectra and morphology of wave functions of the individual stationary states. In this report, we investigate the axial channeling of high and low energy electrons and positrons near [100] direction of a silicon crystal. This case is particularly interesting because of the fact that the chaotic motion domain occupies only a small part of the phase space for the channeling electrons whereas the motion of the channeling positrons is substantially chaotic for the almost all initial conditions. The energy levels of transverse motion, as well as the wave functions of the stationary states, have been computed numerically. The group theory methods had been used for classification of the computed eigenfunctions and identification of the non-degenerate and doubly degenerate energy levels. The channeling radiation spectrum for the low energy electrons has been also computed.

Functional Annotation of Ion Channel Structures by Molecular Simulation.

PubMed

Trick, Jemma L; Chelvaniththilan, Sivapalan; Klesse, Gianni; Aryal, Prafulla; Wallace, E Jayne; Tucker, Stephen J; Sansom, Mark S P

2016-12-06

Ion channels play key roles in cell membranes, and recent advances are yielding an increasing number of structures. However, their functional relevance is often unclear and better tools are required for their functional annotation. In sub-nanometer pores such as ion channels, hydrophobic gating has been shown to promote dewetting to produce a functionally closed (i.e., non-conductive) state. Using the serotonin receptor (5-HT 3 R) structure as an example, we demonstrate the use of molecular dynamics to aid the functional annotation of channel structures via simulation of the behavior of water within the pore. Three increasingly complex simulation analyses are described: water equilibrium densities; single-ion free-energy profiles; and computational electrophysiology. All three approaches correctly predict the 5-HT 3 R crystal structure to represent a functionally closed (i.e., non-conductive) state. We also illustrate the application of water equilibrium density simulations to annotate different conformational states of a glycine receptor. Copyright © 2016 The Authors. Published by Elsevier Ltd.. All rights reserved.

Computing rates of Markov models of voltage-gated ion channels by inverting partial differential equations governing the probability density functions of the conducting and non-conducting states.

PubMed

Tveito, Aslak; Lines, Glenn T; Edwards, Andrew G; McCulloch, Andrew

2016-07-01

Markov models are ubiquitously used to represent the function of single ion channels. However, solving the inverse problem to construct a Markov model of single channel dynamics from bilayer or patch-clamp recordings remains challenging, particularly for channels involving complex gating processes. Methods for solving the inverse problem are generally based on data from voltage clamp measurements. Here, we describe an alternative approach to this problem based on measurements of voltage traces. The voltage traces define probability density functions of the functional states of an ion channel. These probability density functions can also be computed by solving a deterministic system of partial differential equations. The inversion is based on tuning the rates of the Markov models used in the deterministic system of partial differential equations such that the solution mimics the properties of the probability density function gathered from (pseudo) experimental data as well as possible. The optimization is done by defining a cost function to measure the difference between the deterministic solution and the solution based on experimental data. By evoking the properties of this function, it is possible to infer whether the rates of the Markov model are identifiable by our method. We present applications to Markov model well-known from the literature. Copyright © 2016 The Authors. Published by Elsevier Inc. All rights reserved.

Functional coupling between sodium-activated potassium channels and voltage-dependent persistent sodium currents in cricket Kenyon cells.

PubMed

Takahashi, Izumi; Yoshino, Masami

2015-10-01

In this study, we examined the functional coupling between Na(+)-activated potassium (KNa) channels and Na(+) influx through voltage-dependent Na(+) channels in Kenyon cells isolated from the mushroom body of the cricket Gryllus bimaculatus. Single-channel activity of KNa channels was recorded with the cell-attached patch configuration. The open probability (Po) of KNa channels increased with increasing Na(+) concentration in a bath solution, whereas it decreased by the substitution of Na(+) with an equimolar concentration of Li(+). The Po of KNa channels was also found to be reduced by bath application of a high concentration of TTX (1 μM) and riluzole (100 μM), which inhibits both fast (INaf) and persistent (INaP) Na(+) currents, whereas it was unaffected by a low concentration of TTX (10 nM), which selectively blocks INaf. Bath application of Cd(2+) at a low concentration (50 μM), as an inhibitor of INaP, also decreased the Po of KNa channels. Conversely, bath application of the inorganic Ca(2+)-channel blockers Co(2+) and Ni(2+) at high concentrations (500 μM) had little effect on the Po of KNa channels, although Cd(2+) (500 μM) reduced the Po of KNa channels. Perforated whole cell clamp analysis further indicated the presence of sustained outward currents for which amplitude was dependent on the amount of Na(+) influx. Taken together, these results indicate that KNa channels could be activated by Na(+) influx passing through voltage-dependent persistent Na(+) channels. The functional significance of this coupling mechanism was discussed in relation to the membrane excitability of Kenyon cells and its possible role in the formation of long-term memory. Copyright © 2015 the American Physiological Society.

Electrogenic transport and K+ ion channel expression by the human endolymphatic sac epithelium

PubMed Central

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

2015-01-01

The endolymphatic sac (ES) is a cystic organ that is a part of the inner ear and is connected to the cochlea and vestibule. The ES is thought to be involved in inner ear ion homeostasis and fluid volume regulation for the maintenance of hearing and balance function. Many ion channels, transporters, and exchangers have been identified in the ES luminal epithelium, mainly in animal studies, but there has been no functional study investigating ion transport using human ES tissue. We designed the first functional experiments on electrogenic transport in human ES and investigated the contribution of K+ channels in the electrogenic transport, which has been rarely identified, even in animal studies, using electrophysiological/pharmacological and molecular biological methods. As a result, we identified functional and molecular evidence for the essential participation of K+ channels in the electrogenic transport of human ES epithelium. The identified K+ channels involved in the electrogenic transport were KCNN2, KCNJ14, KCNK2, and KCNK6, and the K+ transports via those channels are thought to play an important role in the maintenance of the unique ionic milieu of the inner ear fluid. PMID:26655723

Identification of a tetrameric assembly domain in the C terminus of heat-activated TRPV1 channels.

PubMed

Zhang, Feng; Liu, Shuang; Yang, Fan; Zheng, Jie; Wang, KeWei

2011-04-29

Transient receptor potential (TRP) channels as cellular sensors are thought to function as tetramers. Yet, the molecular determinants governing channel multimerization remain largely elusive. Here we report the identification of a segment comprising 21 amino acids (residues 752-772 of mouse TRPV1) after the known TRP-like domain in the channel C terminus that functions as a tetrameric assembly domain (TAD). Purified recombinant C-terminal proteins of TRPV1-4, but not the N terminus, mediated the protein-protein interaction in an in vitro pulldown assay. Western blot analysis combined with electrophysiology and calcium imaging demonstrated that TAD exerted a robust dominant-negative effect on wild-type TRPV1. When fused with the membrane-tethered peptide Gap43, the TAD blocked the formation of stable homomultimers. Calcium imaging and current recordings showed that deletion of the TAD in a poreless TRPV1 mutant subunit suppressed its dominant-negative phenotype, confirming the involvement of the TAD in assembly of functional channels. Our findings suggest that the C-terminal TAD in TRPV1 channels functions as a domain that is conserved among TRPV1-4 and mediates a direct subunit-subunit interaction for tetrameric assembly.

Basal activity of GIRK5 isoforms.

PubMed

Salvador, Carolina; Mora, Silvia I; Ordaz, Benito; Antaramian, Anaid; Vaca, Luis; Escobar, Laura I

2003-02-14

G protein-coupled inwardly rectifying K(+) channels (GIRK or Kir3) form functional heterotetramers gated by Gbetagamma subunits. GIRK channels are critical for functions as diverse as heart rate modulation and neuronal post-synaptic inhibition. GIRK5 (Kir3.5) is the oocyte homologue of the mammalian GIRK subunits that conform the K(ACh) channel. It has been claimed that even when the oocytes express GIRK5 proteins they do not form functional channels. However, the GIRK5 gene shows three initiation sites that suggest the existence of three isoforms. In a previous work we demonstrated the functionality of homomultimers of the shortest isoform overexpressed in the own oocytes. Remarkably, the basal GIRK5-Delta25 inward currents were not coupled to the activation of a G-protein receptor in the oocytes. These results encouraged us to study this channel in another expression system. In this work we show that Sf21 insect cells can be successfully transfected with this channel. GIRK5-Delta25 homomultimers produce time-dependent inward currents only with GTPgammaS in the recording pipette. Therefore, alternative modes of stimulus input to heterotrimeric G-proteins should be present in the oocytes to account for these results.

Voltage-Gated Potassium Channels: A Structural Examination of Selectivity and Gating

PubMed Central

Kim, Dorothy M.; Nimigean, Crina M.

2016-01-01

Voltage-gated potassium channels play a fundamental role in the generation and propagation of the action potential. The discovery of these channels began with predictions made by early pioneers, and has culminated in their extensive functional and structural characterization by electrophysiological, spectroscopic, and crystallographic studies. With the aid of a variety of crystal structures of these channels, a highly detailed picture emerges of how the voltage-sensing domain reports changes in the membrane electric field and couples this to conformational changes in the activation gate. In addition, high-resolution structural and functional studies of K+ channel pores, such as KcsA and MthK, offer a comprehensive picture on how selectivity is achieved in K+ channels. Here, we illustrate the remarkable features of voltage-gated potassium channels and explain the mechanisms used by these machines with experimental data. PMID:27141052

A theoretical study on the impact of particle scattering on the channel characteristics of underwater optical communication system

NASA Astrophysics Data System (ADS)

Sahu, Sanjay Kumar; Shanmugam, Palanisamy

2018-02-01

Scattering by water molecules and particulate matters determines the path and distance of photon propagation in underwater medium. Consequently, photon angle of scattering (given by scattering phase function) requires to be considered in addition to the extinction coefficient of the aquatic medium governed by the absorption and scattering coefficients in channel characterization for an underwater wireless optical communication (UWOC) system. This study focuses on analyzing the received signal power and impulse response of UWOC channel based on Monte-Carlo simulations for different water types, link distances, link geometries and transceiver parameters. A newly developed scattering phase function (referred to as SS phase function), which represents the real water types more accurately like the Petzold phase function, is considered for quantification of the channel characteristics along with the effects of absorption and scattering coefficients. A comparison between the results simulated using various phase function models and the experimental measurements of Petzold revealed that the SS phase function model predicts values closely matching with the actual values of the Petzold's phase function, which further establishes the importance of using a correct scattering phase function model while estimating the channel capacity of UWOC system in terms of the received power and channel impulse response. Results further demonstrate a great advantage of considering the nonzero probability of receiving scattered photons in estimating channel capacity rather than considering the reception of only ballistic photons as in Beer's Law, which severely underestimates the received power and affects the range of communication especially in the scattering water column. The received power computed based on the Monte-Carlo method by considering the receiver aperture sizes and field of views in different water types are further analyzed and discussed. These results are essential for evaluating the underwater link budget and constructing different system and design parameters for an UWOC system.

The Phe932Ile mutation in KCNT1 channels associated with severe epilepsy, delayed myelination and leukoencephalopathy produces a loss-of-function channel phenotype.

PubMed

Evely, Katherine M; Pryce, Kerri D; Bhattacharjee, Arin

2017-05-20

Sodium-activated potassium (K Na ) channels contribute to firing frequency adaptation and slow after hyperpolarization. The KCNT1 gene (also known as SLACK) encodes a K Na subunit that is expressed throughout the central and peripheral nervous systems. Missense mutations of the SLACK C-terminus have been reported in several patients with rare forms of early onset epilepsy and in some cases severely delayed myelination. To date, such mutations identified in patients with autosomal dominant nocturnal frontal lobe epilepsy (ADNFLE), epilepsy of infancy with migrating focal seizures (EIMFS) and Ohtahara syndrome (OS) have been reported to be gain-of-function mutations (Villa and Combi, 2016). An exome sequencing study identified a p.Phe932Ile KCNT1 mutation as the disease-causing change in a child with severe early infantile epileptic encephalopathy and abnormal myelination (Vanderver et al., 2014). We characterized an analogous mutation in the rat Slack channel and unexpectedly found this mutation to produce a loss-of-function phenotype. In an effort to restore current, we tested the known Slack channel opener loxapine. Loxapine exhibited no effect, indicating that this mutation either caused the channel to be insensitive to this established opener or proper translation and trafficking to the membrane was disrupted. Protein analysis confirmed that while total mutant protein did not differ from wild type, membrane expression of the mutant channel was substantially reduced. Although gain-of-function mutations to the Slack channel are linked to epileptic phenotypes, this is the first reported loss-of-function mutation linked to severe epilepsy and delayed myelination. Copyright © 2017 IBRO. Published by Elsevier Ltd. All rights reserved.

Different structural requirements for functional ion pore transplantation suggest different gating mechanisms of NMDA and kainate receptors.

PubMed

Villmann, Carmen; Hoffmann, Jutta; Werner, Markus; Kott, Sabine; Strutz-Seebohm, Nathalie; Nilsson, Tanja; Hollmann, Michael

2008-10-01

Although considerable progress has been made in characterizing the physiological function of the high-affinity kainate (KA) receptor subunits KA1 and KA2, no homomeric ion channel function has been shown. An ion channel transplantation approach was employed in this study to directly test if homomerically expressed KA1 and KA2 pore domains are capable of conducting currents. Transplantation of the ion pore of KA1 or KA2 into GluR6 generated perfectly functional ion channels that allowed characterization of those electrophysiological and pharmacological properties that are determined exclusively by the ion pore of KA1 or KA2. This demonstrates for the first time that KA1 and KA2 ion pore domains are intrinsically capable of conducting ions even in homomeric pore assemblies. NMDA receptors, similar to KA1- or KA2-containing receptors, function only as heteromeric complexes. They are composed of NR1 and NR2 subunits, which both are non-functional when expressed homomerically. In contrast to NR1, the homomeric NR2B ion pore failed to translate ligand binding into pore opening when transplanted into GluR6. Similarly, heteromeric coexpression of the ion channel domains of both NR1 and NR2 inserted into GluR6 failed to produce functional channels. Therefore, we conclude that the mechanism underlying the ion channel opening in the obligatorily heterotetrameric NMDA receptors differs significantly from that in the facultatively heterotetrameric alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionate and KA receptors.

Phosphorylation-Dependent Regulation of Ryanodine Receptors

PubMed Central

Marx, Steven O.; Reiken, Steven; Hisamatsu, Yuji; Gaburjakova, Marta; Gaburjakova, Jana; Yang, Yi-Ming; Rosemblit, Nora; Marks, Andrew R.

2001-01-01

Ryanodine receptors (RyRs), intracellular calcium release channels required for cardiac and skeletal muscle contraction, are macromolecular complexes that include kinases and phosphatases. Phosphorylation/dephosphorylation plays a key role in regulating the function of many ion channels, including RyRs. However, the mechanism by which kinases and phosphatases are targeted to ion channels is not well understood. We have identified a novel mechanism involved in the formation of ion channel macromolecular complexes: kinase and phosphatase targeting proteins binding to ion channels via leucine/isoleucine zipper (LZ) motifs. Activation of kinases and phosphatases bound to RyR2 via LZs regulates phosphorylation of the channel, and disruption of kinase binding via LZ motifs prevents phosphorylation of RyR2. Elucidation of this new role for LZs in ion channel macromolecular complexes now permits: (a) rapid mapping of kinase and phosphatase targeting protein binding sites on ion channels; (b) predicting which kinases and phosphatases are likely to regulate a given ion channel; (c) rapid identification of novel kinase and phosphatase targeting proteins; and (d) tools for dissecting the role of kinases and phosphatases as modulators of ion channel function. PMID:11352932

High temperature sensitivity is intrinsic to voltage-gated potassium channels

PubMed Central

Yang, Fan; Zheng, Jie

2014-01-01

Temperature-sensitive transient receptor potential (TRP) ion channels are members of the large tetrameric cation channels superfamily but are considered to be uniquely sensitive to heat, which has been presumed to be due to the existence of an unidentified temperature-sensing domain. Here we report that the homologous voltage-gated potassium (Kv) channels also exhibit high temperature sensitivity comparable to that of TRPV1, which is detectable under specific conditions when the voltage sensor is functionally decoupled from the activation gate through either intrinsic mechanisms or mutations. Interestingly, mutations could tune Shaker channel to be either heat-activated or heat-deactivated. Therefore, high temperature sensitivity is intrinsic to both TRP and Kv channels. Our findings suggest important physiological roles of heat-induced variation in Kv channel activities. Mechanistically our findings indicate that temperature-sensing TRP channels may not contain a specialized heat-sensor domain; instead, non-obligatory allosteric gating permits the intrinsic heat sensitivity to drive channel activation, allowing temperature-sensitive TRP channels to function as polymodal nociceptors. DOI: http://dx.doi.org/10.7554/eLife.03255.001 PMID:25030910

Small wash functions and effects of their disturbance on vegetation of a Mojave Desert bajada

NASA Astrophysics Data System (ADS)

Sandquist, D. R.; Bedford, D.; Macias, M.; Miller, D. M.; Newlander, A.; Schwinning, S.

2011-12-01

The extensive network of small washes and channels that pervades most desert bajadas usually represents only a small proportion of the bajada's spatial area and are usually devoid of vegetation. However, these channels may be the most important geomorphic feature influencing vegetation properties and processes in arid lands. To evaluate the functional influence of small channels on the vegetation of a Mojave Desert bajada, we conducted a series of observations and experiments across a ~100 year old linear disturbance (railroad and parallel road) that disrupts the natural flow path of the channel network. In areas below the railroad where flow has been either increased due to channel diversion and coalescence through culverts, or cut off due to diversion, plant community structure and cover has changed relative to undisturbed areas. Plant physiological responses to simulated runoff experiments, conducted in active (undisturbed) and inactive (cut-off) channels, revealed subtle differences that, when compounded through time, are likely to contribute to these shifts in vegetation. Measurements of water potential on the two dominant plant species, Larrea tridentata and Ambrosia dumosa, indicate that Larrea within 3 m of a channel, and Ambrosia within 2 m, access water from the channel. However, the water potential responses were less pronounced, shorter in duration and more variable for plants adjacent to inactive channels than for those near active channels. Stomatal conductance and sap-flow measurements on Larrea corroborated these findings, suggesting that root patterns and functions associated with channels are altered when water flow is reduced or eliminated over extended periods of time. These findings indicate that disturbance of small desert washes and channels can lead to vegetation shifts through time with consequences that are not yet fully understood. Small desert washes and channels may represent a minor spatial component of the vast bajada landscape, but runoff and higher infiltration rates, coupled with the breadth of their spatial influence on adjacent plants, suggests that these modest geomorphic features may have a disproportionate impact on plant function and community properties in arid ecosystems.

Investigating ion channel conformational changes using voltage clamp fluorometry.

PubMed

Talwar, Sahil; Lynch, Joseph W

2015-11-01

Ion channels are membrane proteins whose functions are governed by conformational changes. The widespread distribution of ion channels, coupled with their involvement in most physiological and pathological processes and their importance as therapeutic targets, renders the elucidation of these conformational mechanisms highly compelling from a drug discovery perspective. Thanks to recent advances in structural biology techniques, we now have high-resolution static molecular structures for members of the major ion channel families. However, major questions remain to be resolved about the conformational states that ion channels adopt during activation, drug modulation and desensitization. Patch-clamp electrophysiology has long been used to define ion channel conformational states based on functional criteria. It achieves this by monitoring conformational changes at the channel gate and cannot detect conformational changes occurring in regions distant from the gate. Voltage clamp fluorometry involves labelling cysteines introduced into domains of interest with environmentally sensitive fluorophores and inferring structural rearrangements from voltage or ligand-induced fluorescence changes. Ion channel currents are monitored simultaneously to verify the conformational status. By defining real time conformational changes in domains distant from the gate, this technique provides unexpected new insights into ion channel structure and function. This review aims to summarise the methodology and highlight recent innovative applications of this powerful technique. This article is part of the Special Issue entitled 'Fluorescent Tools in Neuropharmacology'. Copyright © 2015 Elsevier Ltd. All rights reserved.

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

PubMed

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

2016-09-01

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

Ion channel expression patterns in glioblastoma stem cells with functional and therapeutic implications for malignancy.

PubMed

Pollak, Julia; Rai, Karan G; Funk, Cory C; Arora, Sonali; Lee, Eunjee; Zhu, Jun; Price, Nathan D; Paddison, Patrick J; Ramirez, Jan-Marino; Rostomily, Robert C

2017-01-01

Ion channels and transporters have increasingly recognized roles in cancer progression through the regulation of cell proliferation, migration, and death. Glioblastoma stem-like cells (GSCs) are a source of tumor formation and recurrence in glioblastoma multiforme, a highly aggressive brain cancer, suggesting that ion channel expression may be perturbed in this population. However, little is known about the expression and functional relevance of ion channels that may contribute to GSC malignancy. Using RNA sequencing, we assessed the enrichment of ion channels in GSC isolates and non-tumor neural cell types. We identified a unique set of GSC-enriched ion channels using differential expression analysis that is also associated with distinct gene mutation signatures. In support of potential clinical relevance, expression of selected GSC-enriched ion channels evaluated in human glioblastoma databases of The Cancer Genome Atlas and Ivy Glioblastoma Atlas Project correlated with patient survival times. Finally, genetic knockdown as well as pharmacological inhibition of individual or classes of GSC-enriched ion channels constrained growth of GSCs compared to normal neural stem cells. This first-in-kind global examination characterizes ion channels enriched in GSCs and explores their potential clinical relevance to glioblastoma molecular subtypes, gene mutations, survival outcomes, regional tumor expression, and experimental responses to loss-of-function. Together, the data support the potential biological and therapeutic impact of ion channels on GSC malignancy and provide strong rationale for further examination of their mechanistic and therapeutic importance.

Ion channel expression patterns in glioblastoma stem cells with functional and therapeutic implications for malignancy

PubMed Central

Pollak, Julia; Rai, Karan G.; Funk, Cory C.; Arora, Sonali; Lee, Eunjee; Zhu, Jun; Price, Nathan D.; Paddison, Patrick J.; Ramirez, Jan-Marino; Rostomily, Robert C.

2017-01-01

Ion channels and transporters have increasingly recognized roles in cancer progression through the regulation of cell proliferation, migration, and death. Glioblastoma stem-like cells (GSCs) are a source of tumor formation and recurrence in glioblastoma multiforme, a highly aggressive brain cancer, suggesting that ion channel expression may be perturbed in this population. However, little is known about the expression and functional relevance of ion channels that may contribute to GSC malignancy. Using RNA sequencing, we assessed the enrichment of ion channels in GSC isolates and non-tumor neural cell types. We identified a unique set of GSC-enriched ion channels using differential expression analysis that is also associated with distinct gene mutation signatures. In support of potential clinical relevance, expression of selected GSC-enriched ion channels evaluated in human glioblastoma databases of The Cancer Genome Atlas and Ivy Glioblastoma Atlas Project correlated with patient survival times. Finally, genetic knockdown as well as pharmacological inhibition of individual or classes of GSC-enriched ion channels constrained growth of GSCs compared to normal neural stem cells. This first-in-kind global examination characterizes ion channels enriched in GSCs and explores their potential clinical relevance to glioblastoma molecular subtypes, gene mutations, survival outcomes, regional tumor expression, and experimental responses to loss-of-function. Together, the data support the potential biological and therapeutic impact of ion channels on GSC malignancy and provide strong rationale for further examination of their mechanistic and therapeutic importance. PMID:28264064

Effects of channel tap spacing on delay-lock tracking

NASA Astrophysics Data System (ADS)

Dana, Roger A.; Milner, Brian R.; Bogusch, Robert L.

1995-12-01

High fidelity simulations of communication links operating through frequency selective fading channels require both accurate channel models and faithful reproduction of the received signal. In modern radio receivers, processing beyond the analog-to-digital converter (A/D) is done digitally, so a high fidelity simulation is actually an emulation of this digital signal processing. The 'simulation' occurs in constructing the output of the A/D. One approach to constructing the A/D output is to convolve the channel impulse response function with the combined impulse response of the transmitted modulation and the A/D. For both link simulations and hardware channel simulators, the channel impulse response function is then generated with a finite number of samples per chip, and the convolution is implemented in a tapped delay line. In this paper we discuss the effects of the channel model tap spacing on the performance of delay locked loops (DLLs) in both direct sequence and frequency hopped spread spectrum systems. A frequency selective fading channel is considered, and the channel impulse response function is constructed with an integer number of taps per modulation symbol or chip. The tracking loop time delay is computed theoretically for this tapped delay line channel model and is compared to the results of high fidelity simulations of actual DLLs. A surprising result is obtained. The performance of the DLL depends strongly on the number of taps per chip. As this number increases the DLL delay approaches the theoretical limit.

Ultrasonic alignment of bio-functionalized magnetic beads and live cells in PDMS micro-fluidic channel.

PubMed

Islam, Afroja T; Siddique, Ariful H; Ramulu, T S; Reddy, Venu; Eu, Young-Jae; Cho, Seung Hyun; Kim, CheolGi

2012-12-01

In this work, we demonstrated the alignment of polystyrene latex microspheres (diameter of 1 ~45 μm), bio-functionalized superparamagnetic beads (diameter 2.8 μm), and live cells (average diameter 1 ~2 μm) using an ultrasonic standing wave (USW) in a PDMS microfluidic channel (330 μm width) attached on a Si substrate for bio-medical applications. To generate a standing wave inside the channel, ultrasound of 2.25 MHz resonance frequency (for the channel width) was applied by two ultrasound transducers installed at both sides of the channel which caused the radiation force to concentrate the micro-particles at the single pressure nodal plane of USW. By increasing the frequency to the next resonance condition of the channel, the particles were concentrated in dual nodal planes. Migration time of the micro-particles towards the single nodal plane was recorded as 108 s, 17 s, and 115 s for polystyrene particles of 2 μm diameter, bio-functionalized magnetic beads, and live cells, respectively. These successful alignments of the bio-functionalized magnetic beads along the desired part of the channel can enhance the performance of a sensor which is applicable for the bio-hybrid system and the alignment of live cells without any damage can be used for sample pre-treatment for the application of lab-on-a-chip type bioassays.

A Functional Nuclear Localization Sequence in the C. elegans TRPV Channel OCR-2

PubMed Central

Ezak, Meredith J.; Ferkey, Denise M.

2011-01-01

The ability to modulate gene expression in response to sensory experience is critical to the normal development and function of the nervous system. Calcium is a key activator of the signal transduction cascades that mediate the process of translating a cellular stimulus into transcriptional changes. With the recent discovery that the mammalian Cav1.2 calcium channel can be cleaved, enter the nucleus and act as a transcription factor to control neuronal gene expression, a more direct role for the calcium channels themselves in regulating transcription has begun to be appreciated. Here we report the identification of a nuclear localization sequence (NLS) in the C. elegans transient receptor potential vanilloid (TRPV) cation channel OCR-2. TRPV channels have previously been implicated in transcriptional regulation of neuronal genes in the nematode, although the precise mechanism remains unclear. We show that the NLS in OCR-2 is functional, being able to direct nuclear accumulation of a synthetic cargo protein as well as the carboxy-terminal cytosolic tail of OCR-2 where it is endogenously found. Furthermore, we discovered that a carboxy-terminal portion of the full-length channel can localize to the nucleus of neuronal cells. These results suggest that the OCR-2 TRPV cation channel may have a direct nuclear function in neuronal cells that was not previously appreciated. PMID:21957475

Engineering biosynthetic excitable tissues from unexcitable cells for electrophysiological and cell therapy studies.

PubMed

Kirkton, Robert D; Bursac, Nenad

2011-01-01

Patch-clamp recordings in single-cell expression systems have been traditionally used to study the function of ion channels. However, this experimental setting does not enable assessment of tissue-level function such as action potential (AP) conduction. Here we introduce a biosynthetic system that permits studies of both channel activity in single cells and electrical conduction in multicellular networks. We convert unexcitable somatic cells into an autonomous source of electrically excitable and conducting cells by stably expressing only three membrane channels. The specific roles that these expressed channels have on AP shape and conduction are revealed by different pharmacological and pacing protocols. Furthermore, we demonstrate that biosynthetic excitable cells and tissues can repair large conduction defects within primary 2- and 3-dimensional cardiac cell cultures. This approach enables novel studies of ion channel function in a reproducible tissue-level setting and may stimulate the development of new cell-based therapies for excitable tissue repair.

Role of partial linear momentum transfer on incomplete fusion reaction

NASA Astrophysics Data System (ADS)

Ali, Sabir; Ahmad, Tauseef; Kumar, Kamal; Gull, Muntazir; Rizvi, I. A.; Agarwal, Avinash; Ghugre, S. S.; Sinha, A. K.; Chaubey, A. K.

2018-04-01

Measurements of forward recoil range distributions (FRRDs) of the evaporation residues, populated in the 20Ne+51V reaction at E_{lab}≈ 145 MeV, have been carried out using the offline characteristic γ-ray detection method. The observation does corroborate the presence of complete fusion (CF) process in the population of p xn channel residues and both complete as well as incomplete fusion (ICF) processes in the population of α emitting channel residues. The FRRDs of p xn channel residues comprise single peak only, whereas α emitting channel residues have multiple peaks in their FRRDs. CF cross section data were used to extract the fusion functions. Extracted fusion functions were found to be suppressed with respect to the universal fusion function which is used as a uniform standard reference. The observed contribution arising from the ICF process in the population of α emitting channel residues is explained in terms of breakup fusion model.

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

NASA Astrophysics Data System (ADS)

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

2016-09-01

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

Blind channel estimation and deconvolution in colored noise using higher-order cumulants

NASA Astrophysics Data System (ADS)

Tugnait, Jitendra K.; Gummadavelli, Uma

1994-10-01

Existing approaches to blind channel estimation and deconvolution (equalization) focus exclusively on channel or inverse-channel impulse response estimation. It is well-known that the quality of the deconvolved output depends crucially upon the noise statistics also. Typically it is assumed that the noise is white and the signal-to-noise ratio is known. In this paper we remove these restrictions. Both the channel impulse response and the noise model are estimated from the higher-order (fourth, e.g.) cumulant function and the (second-order) correlation function of the received data via a least-squares cumulant/correlation matching criterion. It is assumed that the noise higher-order cumulant function vanishes (e.g., Gaussian noise, as is the case for digital communications). Consistency of the proposed approach is established under certain mild sufficient conditions. The approach is illustrated via simulation examples involving blind equalization of digital communications signals.

Allosteric mechanism of water channel gating by Ca2+–calmodulin

PubMed Central

Reichow, Steve L.; Clemens, Daniel M.; Freites, J. Alfredo; Németh-Cahalan, Karin L.; Heyden, Matthias; Tobias, Douglas J.; Hall, James E.; Gonen, Tamir

2013-01-01

Calmodulin (CaM) is a universal regulatory protein that communicates the presence of calcium to its molecular targets and correspondingly modulates their function. This key signaling protein is important for controlling the activity of hundreds of membrane channels and transporters. However, our understanding of the structural mechanisms driving CaM regulation of full-length membrane proteins has remained elusive. In this study, we determined the pseudo-atomic structure of full-length mammalian aquaporin-0 (AQP0, Bos Taurus) in complex with CaM using electron microscopy to understand how this signaling protein modulates water channel function. Molecular dynamics and functional mutation studies reveal how CaM binding inhibits AQP0 water permeability by allosterically closing the cytoplasmic gate of AQP0. Our mechanistic model provides new insight, only possible in the context of the fully assembled channel, into how CaM regulates multimeric channels by facilitating cooperativity between adjacent subunits. PMID:23893133

An assembly of steadily translating bubbles in a Hele-Shaw channel

NASA Astrophysics Data System (ADS)

Crowdy, Darren

2009-01-01

This paper presents new solutions for any finite number of bubbles steadily translating along a Hele-Shaw channel. This constitutes a nonlinear free boundary problem. The solutions can be written down explicitly in terms of a special transcendental function called the Schottky-Klein prime function. This work generalizes the exact solutions for a single bubble in a channel found by Tanveer (1987 Phys. Fluids 30 651-8) as well as the solutions for streams of bubbles aligned along the channel centreline due to Vasconcelos (1994 Phys. Rev. E 50 R3306-9).

STIM1 converts TRPC1 from a receptor-operated to a store-operated channel: moving TRPC1 in and out of lipid rafts.

PubMed

Alicia, Sampieri; Angélica, Zepeda; Carlos, Saldaña; Alfonso, Salgado; Vaca, Luis

2008-11-01

While the role of members from the TRPC family of channels as receptor-operated channels (ROC) is well established and supported by numerous studies, the role of this family of channels as store-operated channels (SOC) has been the focus of a heated controversy over the last few years. In the present study, we have explored the modulation of STIM1 on human TRPC1 channel. We show that the association of STIM1 to TRPC1 favors the insertion of TRPC1 into lipid rafts, where TRPC1 functions as a SOC. In the absence of STIM1, TRPC1 associates to other members from the TRPC family of channels to form ROCs. A novel TIRFM-FRET method illustrates the relevance of the dynamic association between STIM1 and TRPC1 for the activation of SOC and the lipid raft localization of the STIM1-TRPC1 complex. This study provides new evidence about the dual activity of TRPC1 (forming ROC or SOC) and the partners needed to determine TRPC1 functional fate. It highlights also the role of plasma membrane microdomains and ER-PM junctions in modulating TRPC1 channel function and its association to STIM1.

New light on ion channel imaging by total internal reflection fluorescence (TIRF) microscopy.

PubMed

Yamamura, Hisao; Suzuki, Yoshiaki; Imaizumi, Yuji

2015-05-01

Ion channels play pivotal roles in a wide variety of cellular functions; therefore, their physiological characteristics, pharmacological responses, and molecular structures have been extensively investigated. However, the mobility of an ion channel itself in the cell membrane has not been examined in as much detail. A total internal reflection fluorescence (TIRF) microscope allows fluorophores to be imaged in a restricted region within an evanescent field of less than 200 nm from the interface of the coverslip and plasma membrane in living cells. Thus the TIRF microscope is useful for selectively visualizing the plasmalemmal surface and subplasmalemmal zone. In this review, we focused on a single-molecule analysis of the dynamic movement of ion channels in the plasma membrane using TIRF microscopy. We also described two single-molecule imaging techniques under TIRF microscopy: fluorescence resonance energy transfer (FRET) for the identification of molecules that interact with ion channels, and subunit counting for the determination of subunit stoichiometry in a functional channel. TIRF imaging can also be used to analyze spatiotemporal Ca(2+) events in the subplasmalemma. Single-molecule analyses of ion channels and localized Ca(2+) signals based on TIRF imaging provide beneficial pharmacological and physiological information concerning the functions of ion channels. Copyright © 2015 The Authors. Production and hosting by Elsevier B.V. All rights reserved.

Tip-localized Ca2+ -permeable channels control pollen tube growth via kinase-dependent R- and S-type anion channel regulation.

PubMed

Gutermuth, Timo; Herbell, Sarah; Lassig, Roman; Brosché, Mikael; Romeis, Tina; Feijó, José Alberto; Hedrich, Rainer; Konrad, Kai Robert

2018-05-01

Pollen tubes (PTs) are characterized by having tip-focused cytosolic calcium ion (Ca 2+ ) concentration ([Ca 2+ ] cyt ) gradients, which are believed to control PT growth. However, the mechanisms by which the apical [Ca 2+ ] cyt orchestrates PT growth are not well understood. Here, we aimed to identify these mechanisms by combining reverse genetics, cell biology, electrophysiology, and live-cell Ca 2+ and anion imaging. We triggered Ca 2+ -channel activation by applying hyperpolarizing voltage pulses and observed that the evoked [Ca 2+ ] cyt increases were paralleled by high anion channel activity and a decrease in the cytosolic anion concentration at the PT tip. We confirmed a functional correlation between these patterns by showing that inhibition of Ca 2+ -permeable channels eliminated the [Ca 2+ ] cyt increase, resulting in the abrogation of anion channel activity via Ca 2+ -dependent protein kinases (CPKs). Functional characterization of CPK and anion-channel mutants revealed a CPK2/20/6-dependent activation of SLAH3 and ALMT12/13/14 anion channels. The impaired growth phenotypes of anion channel and CPK mutants support the physiological significance of a kinase- and Ca 2+ -dependent pathway to control PT growth via anion channel activation. Other than unveiling this functional link, our membrane hyperpolarization method allows for unprecedented manipulation of the [Ca 2+ ] cyt gradient or oscillations in the PT tips and opens an array of opportunities for channel screenings. © 2018 The Authors. New Phytologist © 2018 New Phytologist Trust.

Mitochondrial Ion Channels in Cancer Transformation

PubMed Central

Madamba, Stephen M.; Damri, Kevin N.; Dejean, Laurent M.; Peixoto, Pablo M.

2015-01-01

Cancer transformation involves reprograming of mitochondrial function to avert cell death mechanisms, monopolize energy metabolism, accelerate mitotic proliferation, and promote metastasis. Mitochondrial ion channels have emerged as promising therapeutic targets because of their connection to metabolic and apoptotic functions. This mini review discusses how mitochondrial channels may be associated with cancer transformation and expands on the possible involvement of mitochondrial protein import complexes in pathophysiological process. PMID:26090338

Rescue of protein expression defects may not be enough to abolish the pro-arrhythmic phenotype of long QT type 2 mutations.

PubMed

Perry, Matthew D; Ng, Chai Ann; Phan, Kevin; David, Erikka; Steer, Kieran; Hunter, Mark J; Mann, Stefan A; Imtiaz, Mohammad; Hill, Adam P; Ke, Ying; Vandenberg, Jamie I

2016-07-15

Most missense long QT syndrome type 2 (LQTS2) mutations result in Kv11.1 channels that show reduced levels of membrane expression. Pharmacological chaperones that rescue mutant channel expression could have therapeutic potential to reduce the risk of LQTS2-associated arrhythmias and sudden cardiac death, but only if the mutant Kv11.1 channels function normally (i.e. like WT channels) after membrane expression is restored. Fewer than half of mutant channels exhibit relatively normal function after rescue by low temperature. The remaining rescued missense mutant Kv11.1 channels have perturbed gating and/or ion selectivity characteristics. Co-expression of WT subunits with gating defective missense mutations ameliorates but does not eliminate the functional abnormalities observed for most mutant channels. For patients with mutations that affect gating in addition to expression, it may be necessary to use a combination therapy to restore both normal function and normal expression of the channel protein. In the heart, Kv11.1 channels pass the rapid delayed rectifier current (IKr ) which plays critical roles in repolarization of the cardiac action potential and in the suppression of arrhythmias caused by premature stimuli. Over 500 inherited mutations in Kv11.1 are known to cause long QT syndrome type 2 (LQTS2), a cardiac electrical disorder associated with an increased risk of life threatening arrhythmias. Most missense mutations in Kv11.1 reduce the amount of channel protein expressed at the membrane and, as a consequence, there has been considerable interest in developing pharmacological agents to rescue the expression of these channels. However, pharmacological chaperones will only have clinical utility if the mutant Kv11.1 channels function normally after membrane expression is restored. The aim of this study was to characterize the gating phenotype for a subset of LQTS2 mutations to assess what proportion of mutations may be suitable for rescue. As an initial screen we used reduced temperature to rescue expression defects of mutant channels expressed in Xenopus laevis oocytes. Over half (∼56%) of Kv11.1 mutants exhibited functional gating defects that either dramatically reduced the amount of current contributing to cardiac action potential repolarization and/or reduced the amount of protective current elicited in response to premature depolarizations. Our data demonstrate that if pharmacological rescue of protein expression defects is going to have clinical utility in the treatment of LQTS2 then it will be important to assess the gating phenotype of LQTS2 mutations before attempting rescue. © 2016 The Authors. The Journal of Physiology © 2016 The Physiological Society.

Multi-channel distributed coordinated function over single radio in wireless sensor networks.

PubMed

Campbell, Carlene E-A; Loo, Kok-Keong Jonathan; Gemikonakli, Orhan; Khan, Shafiullah; Singh, Dhananjay

2011-01-01

Multi-channel assignments are becoming the solution of choice to improve performance in single radio for wireless networks. Multi-channel allows wireless networks to assign different channels to different nodes in real-time transmission. In this paper, we propose a new approach, Multi-channel Distributed Coordinated Function (MC-DCF) which takes advantage of multi-channel assignment. The backoff algorithm of the IEEE 802.11 distributed coordination function (DCF) was modified to invoke channel switching, based on threshold criteria in order to improve the overall throughput for wireless sensor networks (WSNs) over 802.11 networks. We presented simulation experiments in order to investigate the characteristics of multi-channel communication in wireless sensor networks using an NS2 platform. Nodes only use a single radio and perform channel switching only after specified threshold is reached. Single radio can only work on one channel at any given time. All nodes initiate constant bit rate streams towards the receiving nodes. In this work, we studied the impact of non-overlapping channels in the 2.4 frequency band on: constant bit rate (CBR) streams, node density, source nodes sending data directly to sink and signal strength by varying distances between the sensor nodes and operating frequencies of the radios with different data rates. We showed that multi-channel enhancement using our proposed algorithm provides significant improvement in terms of throughput, packet delivery ratio and delay. This technique can be considered for WSNs future use in 802.11 networks especially when the IEEE 802.11n becomes popular thereby may prevent the 802.15.4 network from operating effectively in the 2.4 GHz frequency band.

Multi-Channel Distributed Coordinated Function over Single Radio in Wireless Sensor Networks

PubMed Central

Campbell, Carlene E.-A.; Loo, Kok-Keong (Jonathan); Gemikonakli, Orhan; Khan, Shafiullah; Singh, Dhananjay

2011-01-01

Multi-channel assignments are becoming the solution of choice to improve performance in single radio for wireless networks. Multi-channel allows wireless networks to assign different channels to different nodes in real-time transmission. In this paper, we propose a new approach, Multi-channel Distributed Coordinated Function (MC-DCF) which takes advantage of multi-channel assignment. The backoff algorithm of the IEEE 802.11 distributed coordination function (DCF) was modified to invoke channel switching, based on threshold criteria in order to improve the overall throughput for wireless sensor networks (WSNs) over 802.11 networks. We presented simulation experiments in order to investigate the characteristics of multi-channel communication in wireless sensor networks using an NS2 platform. Nodes only use a single radio and perform channel switching only after specified threshold is reached. Single radio can only work on one channel at any given time. All nodes initiate constant bit rate streams towards the receiving nodes. In this work, we studied the impact of non-overlapping channels in the 2.4 frequency band on: constant bit rate (CBR) streams, node density, source nodes sending data directly to sink and signal strength by varying distances between the sensor nodes and operating frequencies of the radios with different data rates. We showed that multi-channel enhancement using our proposed algorithm provides significant improvement in terms of throughput, packet delivery ratio and delay. This technique can be considered for WSNs future use in 802.11 networks especially when the IEEE 802.11n becomes popular thereby may prevent the 802.15.4 network from operating effectively in the 2.4 GHz frequency band. PMID:22346614

Endoplasmic reticulum membrane potassium channel dysfunction in high fat diet induced stress in rat hepatocytes

PubMed Central

Khodaee, Naser; Ghasemi, Maedeh; Saghiri, Reza; Eliassi, Afsaneh

2014-01-01

In a previous study we reported the presence of a large conductance K+ channel in the membrane of endoplasmic reticulum (ER) from rat hepatocytes. The channel open probability (Po) appeared voltage dependent and reached to a minimum 0.2 at +50 mV. Channel activity in this case was found to be totally inhibited at ATP concentration 2.5 mM, glibenclamide 100 µM and tolbutamide 400 µM. Existing evidence indicates an impairment of endoplasmic reticulum functions in ER stress condition. Because ER potassium channels have been involved in several ER functions including cytoprotection, apoptosis and calcium homeostasis, a study was carried out to consider whether the ER potassium channel function is altered in a high fat diet model of ER stress. Male Wistar rats were made ER stress for 2 weeks with a high fat diet. Ion channel incorporation of ER stress model into the bilayer lipid membrane allowed the characterization of K+ channel. Our results indicate that the channel Po was significantly increased at voltages above +30 mV. Interestingly, addition of ATP 7.5 mM, glibenclamide 400 µM and tolbutamide 2400 µM totally inhibited the channel activities, 3-fold, 4-fold and 6-fold higher than that in the control groups, respectively. Our results thus demonstrate a modification in the ER K+ channel gating properties and decreased sensitivity to drugs in membrane preparations coming from ER high fat model of ER stress, an effect potentially linked to a change in ER K+ channel subunits in ER stress condition. Our results may provide new insights into the cellular mechanisms underlying ER dysfunctions in ER stress. PMID:26417322

Function and dysfunction of CNG channels: insights from channelopathies and mouse models.

PubMed

Biel, Martin; Michalakis, Stylianos

2007-06-01

Channels directly gated by cyclic nucleotides (CNG channels) are important cellular switches that mediate influx of Na+ and Ca2+ in response to increases in the intracellular concentration of cAMP and cGMP. In photoreceptors and olfactory receptor neurons, these channels serve as final targets for cGMP and cAMP signaling pathways that are initiated by the absorption of photons and the binding of odorants, respectively. CNG channels have been also found in other types of neurons and in non-excitable cells. However, in most of these cells, the physiological role of CNG channels has yet to be determined. CNG channels have a complex heteromeric structure. The properties of individual subunits that assemble in specific stoichiometries to the native channels have been extensively investigated in heterologous expression systems. Recently, mutations in human CNG channel genes leading to inherited diseases (so-called channelopathies) have been functionally characterized. Moreover, mouse knockout models were generated to define the role of CNG channel proteins in vivo. In this review, we will summarize recent insights into the physiological and pathophysiological role of CNG channel proteins that have emerged from genetic studies in mice and humans.

VOLTAGE-GATED POTASSIUM CHANNELS AT THE CROSSROADS OF NEURONAL FUNCTION, ISCHEMIC TOLERANCE, AND NEURODEGENERATION

PubMed Central

Shah, Niyathi Hegde; Aizenman, Elias

2013-01-01

Voltage-gated potassium (Kv) channels are widely expressed in the central and peripheral nervous system, and are crucial mediators of neuronal excitability. Importantly, these channels also actively participate in cellular and molecular signaling pathways that regulate the life and death of neurons. Injury-mediated increased K+ efflux through Kv2.1 channels promotes neuronal apoptosis, contributing to widespread neuronal loss in neurodegenerative disorders such as Alzheimer’s disease and stroke. In contrast, some forms of neuronal activity can dramatically alter Kv2.1 channel phosphorylation levels and influence their localization. These changes are normally accompanied by modifications in channel voltage-dependence, which may be neuroprotective within the context of ischemic injury. Kv1 and Kv7 channel dysfunction leads to neuronal hyperexcitability that critically contributes to the pathophysiology of human clinical disorders such as episodic ataxia and epilepsy. This review summarizes the neurotoxic, neuroprotective, and neuroregulatory roles of Kv channels, and highlights the consequences of Kv channel dysfunction on neuronal physiology. The studies described in this review thus underscore the importance of normal Kv channel function in neurons, and emphasize the therapeutic potential of targeting Kv channels in the treatment of a wide range of neurological diseases. PMID:24323720

Sperm-specific ion channels: targets holding the most potential for male contraceptives in development.

PubMed

Zheng, Li-Ping; Wang, Hua-Feng; Li, Bao-Ming; Zeng, Xu-Hui

2013-10-01

There is a global need for an ideal method of male contraception. However, the development of male contraceptives has not been well successful. Research on sperm-specific ion channels, especially the recent advance obtained from electrophysiological studies, has emphasized the conception that those channels are targets with the most potential to develop non-hormonal male contraceptives. While summarizing the general options for male contraception, this review focuses on the properties and functions of sperm ion channels together with the attempts of utilizing these channels to develop male contraceptives. We believe that a deeper insight into the signaling and molecular mechanisms by which ion channels regulate sperm functions will pave the way for developing novel male-based contraceptives. Copyright © 2013 Elsevier Inc. All rights reserved.

Cholesterol up-regulates neuronal G protein-gated inwardly rectifying potassium (GIRK) channel activity in the hippocampus

PubMed Central

Bukiya, Anna N.; Noskov, Sergei; Rosenhouse-Dantsker, Avia

2017-01-01

Hypercholesterolemia is a well known risk factor for the development of neurodegenerative disease. However, the underlying mechanisms are mostly unknown. In recent years, it has become increasingly evident that cholesterol-driven effects on physiology and pathophysiology derive from its ability to alter the function of a variety of membrane proteins including ion channels. Yet, the effect of cholesterol on G protein-gated inwardly rectifying potassium (GIRK) channels expressed in the brain is unknown. GIRK channels mediate the actions of inhibitory brain neurotransmitters. As a result, loss of GIRK function can enhance neuron excitability, whereas gain of GIRK function can reduce neuronal activity. Here we show that in rats on a high-cholesterol diet, cholesterol levels in hippocampal neurons are increased. We also demonstrate that cholesterol plays a critical role in modulating neuronal GIRK currents. Specifically, cholesterol enrichment of rat hippocampal neurons resulted in enhanced channel activity. In accordance, elevated currents upon cholesterol enrichment were also observed in Xenopus oocytes expressing GIRK2 channels, the primary GIRK subunit expressed in the brain. Furthermore, using planar lipid bilayers, we show that although cholesterol did not affect the unitary conductance of GIRK2, it significantly enhanced the frequency of channel openings. Last, combining computational and functional approaches, we identified two putative cholesterol-binding sites in the transmembrane domain of GIRK2. These findings establish that cholesterol plays a critical role in modulating GIRK activity in the brain. Because up-regulation of GIRK function can reduce neuronal activity, our findings may lead to novel approaches for prevention and therapy of cholesterol-driven neurodegenerative disease. PMID:28213520

Creation of a genetic calcium channel blocker by targeted gem gene transfer in the heart.

PubMed

Murata, Mitsushige; Cingolani, Eugenio; McDonald, Amy D; Donahue, J Kevin; Marbán, Eduardo

2004-08-20

Calcium channel blockers are among the most commonly used therapeutic drugs. Nevertheless, the utility of calcium channel blockers for heart disease is limited because of the potent vasodilatory effect that causes hypotension, and other side effects attributable to blockade of noncardiac channels. Therefore, focal calcium channel blockade by gene transfer is highly desirable. With a view to creating a focally applicable genetic calcium channel blocker, we overexpressed the ras-related small G-protein Gem in the heart by somatic gene transfer. Adenovirus-mediated delivery of Gem markedly decreased L-type calcium current density in ventricular myocytes, resulting in the abbreviation of action potential duration. Furthermore, transduction of Gem resulted in a significant shortening of the electrocardiographic QTc interval and reduction of left ventricular systolic function. Focal delivery of Gem to the atrioventricular (AV) node significantly slowed AV nodal conduction (prolongation of PR and AH intervals), which was effective in the reduction of heart rate during atrial fibrillation. Thus, these results indicate that gene transfer of Gem functions as a genetic calcium channel blocker, the local application of which can effectively modulate cardiac electrical and contractile function.

Ion Trapping with Fast-Response Ion-Selective Microelectrodes Enhances Detection of Extracellular Ion Channel Gradients

PubMed Central

Messerli, Mark A.; Collis, Leon P.; Smith, Peter J.S.

2009-01-01

Previously, functional mapping of channels has been achieved by measuring the passage of net charge and of specific ions with electrophysiological and intracellular fluorescence imaging techniques. However, functional mapping of ion channels using extracellular ion-selective microelectrodes has distinct advantages over the former methods. We have developed this method through measurement of extracellular K+ gradients caused by efflux through Ca2+-activated K+ channels expressed in Chinese hamster ovary cells. We report that electrodes constructed with short columns of a mechanically stable K+-selective liquid membrane respond quickly and measure changes in local [K+] consistent with a diffusion model. When used in close proximity to the plasma membrane (<4 μm), the ISMs pose a barrier to simple diffusion, creating an ion trap. The ion trap amplifies the local change in [K+] without dramatically changing the rise or fall time of the [K+] profile. Measurement of extracellular K+ gradients from activated rSlo channels shows that rapid events, 10–55 ms, can be characterized. This method provides a noninvasive means for functional mapping of channel location and density as well as for characterizing the properties of ion channels in the plasma membrane. PMID:19217875

TRPA1 Channels in Drosophila and Honey Bee Ectoparasitic Mites Share Heat Sensitivity and Temperature-Related Physiological Functions

PubMed Central

Peng, Guangda; Kashio, Makiko; Li, Tianbang; Dong, Xiaofeng; Tominaga, Makoto; Kadowaki, Tatsuhiko

2016-01-01

The transient receptor potential cation channel, subfamily A, member 1 (TRPA1) is conserved between many arthropods, and in some has been shown to function as a chemosensor for noxious compounds. Activation of arthropod TRPA1 channels by temperature fluctuations has been tested in only a few insect species, and all of them were shown to be activated by heat. The recent identification of chemosensitive TRPA1 channels from two honey bee ectoparasitic mite species (VdTRPA1 and TmTRPA1) have provided an opportunity to study the temperature-dependent activation and the temperature-associated physiological functions of TRPA1 channels in non-insect arthropods. We found that both mite TRPA1 channels are heat sensitive and capable of rescuing the temperature-related behavioral defects of a Drosophila melanogaster trpA1 mutant. These results suggest that heat-sensitivity of TRPA1 could be conserved between many arthropods despite its amino acid sequence diversity. Nevertheless, the ankyrin repeats (ARs) 6 and 7 are well-conserved between six heat-sensitive arthropod TRPA1 channels and have critical roles for the heat activation of VdTRPA1. PMID:27761115

A Crash Course in Calcium Channels.

PubMed

Zamponi, Gerald W

2017-12-20

Much progress has been made in understanding the molecular physiology and pharmacology of calcium channels. Recently, there have been tremendous advances in learning about calcium channel structure and function through crystallography and cryo-electron microscopy studies. Here, I will give an overview of our knowledge about calcium channels, and highlight two recent studies that give important insights into calcium channel structure.

Potassium channels in articular chondrocytes

PubMed Central

Mobasheri, Ali; Lewis, Rebecca; Ferreira-Mendes, Alexandrina; Rufino, Ana; Dart, Caroline; Barrett-Jolley, Richard

2012-01-01

Chondrocytes are the resident cells of cartilage, which synthesize and maintain the extracellular matrix. The range of known potassium channels expressed by these unique cells is continually increasing. Since chondrocytes are non-excitable, and do not need to be repolarized following action potentials, the function of potassium channels in these cells has, until recently, remained completely unknown. However, recent advances in both traditional physiology and “omic” technologies have enhanced our knowledge and understanding of the chondrocyte channelome. A large number of potassium channels have been identified and a number of putative, but credible, functions have been proposed. Members of each of the potassium channel sub-families (calcium activated, inward rectifier, voltage-gated and tandem pore) have all been identified. Mechanotransduction, cell volume regulation, apoptosis and chondrogenesis all appear to involve potassium channels. Since evidence suggests that potassium channel gene transcription is altered in osteoarthritis, future studies are needed that investigate potassium channels as potential cellular biomarkers and therapeutic targets for treatment of degenerative joint conditions. PMID:23064164

Biophysics of BK Channel Gating.

PubMed

Pantazis, A; Olcese, R

2016-01-01

BK channels are universal regulators of cell excitability, given their exceptional unitary conductance selective for K(+), joint activation mechanism by membrane depolarization and intracellular [Ca(2+)] elevation, and broad expression pattern. In this chapter, we discuss the structural basis and operational principles of their activation, or gating, by membrane potential and calcium. We also discuss how the two activation mechanisms interact to culminate in channel opening. As members of the voltage-gated potassium channel superfamily, BK channels are discussed in the context of archetypal family members, in terms of similarities that help us understand their function, but also seminal structural and biophysical differences that confer unique functional properties. © 2016 Elsevier Inc. All rights reserved.

ENaC/DEG in Tumor Development and Progression

PubMed Central

Liu, Cui; Zhu, Li-Li; Xu, Si-Guang; Ji, Hong-Long; Li, Xiu-Min

2016-01-01

The epithelial Na+ channel/degenerin (ENaC/DEG) superfamily, including the acid-sensing ion channels (ASICs), is characterized by a high degree of similarity in structure but highly diverse in physiological functions. These ion channels have been shown to be important in several physiological functions of normal epithelial cells, including salt homeostasis, fluid transportation and cell mobility. There is increasing evidence suggesting that ENaC/DEG channels are critically engaged in cancer cell biology, such as proliferation, migration, invasion and apoptosis, playing a role in tumor development and progression. In this review, we will discuss recent studies showing the role of ENaC and ASIC channels in epithelial cells and its relationship to the oncogenesis. PMID:27698929

TRP channel functions in the gastrointestinal tract.

PubMed

Yu, Xiaoyun; Yu, Mingran; Liu, Yingzhe; Yu, Shaoyong

2016-05-01

Transient receptor potential (TRP) channels are predominantly distributed in both somatic and visceral sensory nervous systems and play a crucial role in sensory transduction. As the largest visceral organ system, the gastrointestinal (GI) tract frequently accommodates external inputs, which stimulate sensory nerves to initiate and coordinate sensory and motor functions in order to digest and absorb nutrients. Meanwhile, the sensory nerves in the GI tract are also able to detect potential tissue damage by responding to noxious irritants. This nocifensive function is mediated through specific ion channels and receptors expressed in a subpopulation of spinal and vagal afferent nerve called nociceptor. In the last 18 years, our understanding of TRP channel expression and function in GI sensory nervous system has been continuously improved. In this review, we focus on the expressions and functions of TRPV1, TRPA1, and TRPM8 in primary extrinsic afferent nerves innervated in the esophagus, stomach, intestine, and colon and briefly discuss their potential roles in relevant GI disorders.

Pharmacological enhancement of calcium-activated potassium channel function reduces the effects of repeated stress on fear memory

PubMed Central

Atchley, Derek; Hankosky, Emily R.; Gasparotto, Kaylyn; Rosenkranz, J. Amiel

2012-01-01

Repeated stress impacts emotion, and can induce mood and anxiety disorders. These disorders are characterized by imbalance of emotional responses. The amygdala is fundamental in expression of emotion, and is hyperactive in many patients with mood or anxiety disorders. Stress also leads to hyperactivity of the amygdala in humans. In rodent studies, repeated stress causes hyperactivity of the amygdala, and increases fear conditioning behavior that is mediated by the basolateral amygdala (BLA). Calcium-activated potassium (KCa) channels regulate BLA neuronal activity, and evidence suggests reduced small conductance KCa (SK) channel function in male rats exposed to repeated stress. Pharmacological enhancement of SK channels reverses the BLA neuronal hyperexcitability caused by repeated stress. However, it is not known if pharmacological targeting of SK channels can repair the effects of repeated stress on amygdala-dependent behaviors. The purpose of this study was to test whether enhancement of SK channel function reverses the effects of repeated restraint on BLA-dependent auditory fear conditioning. We found that repeated restraint stress increased the expression of cued conditioned fear in male rats. However, 1-EBIO (1 or 10 mg/kg) or CyPPA (5 mg/kg) administered 30 minutes prior to testing of fear expression brought conditioned freezing to control levels, with little impact on fear expression in control handled rats. These results demonstrate that enhancement of SK channel function can reduce the abnormalities of BLA-dependent fear memory caused by repeated stress. Furthermore, this indicates that pharmacological targeting of SK channels may provide a novel target for alleviation of psychiatric symptoms associated with amygdala hyperactivity. PMID:22487247

Functional characterization of a prokaryotic Kir channel.

PubMed

Enkvetchakul, Decha; Bhattacharyya, Jaya; Jeliazkova, Iana; Groesbeck, Darcy K; Cukras, Catherine A; Nichols, Colin G

2004-11-05

The Kir gene family encodes inward rectifying K+ (Kir) channels that are widespread and critical regulators of excitability in eukaryotic cells. A related gene family (KirBac) has recently been identified in prokaryotes. While a crystal structure of one member, Kir-Bac1.1, has been solved, there has been no functional characterization of any KirBac gene products. Here we present functional characterization of KirBac1.1 reconstituted in liposomes. Utilizing a 86Rb+ uptake assay, we demonstrate that KirBac1.1 generates a K+ -selective permeation path that is inhibited by extraliposomal Ba2+ and Ca2+ ions. In contrast to KcsA (an acid-activated bacterial potassium channel), KirBac1.1 is inhibited by extraliposomal acid (pKa approximately 6). This characterization of KirBac1.1 activity now paves the way for further correlation of structure and function in this model Kir channel.

Function and structure in glycine receptors and some of their relatives.

PubMed

Colquhoun, David; Sivilotti, Lucia G

2004-06-01

In the field of ligand-gated ion channels, recent developments, both in the knowledge of structure and in the measurement of function at the single-channel level, have allowed a sensible start to be made on understanding the relationship between structure and function in these proteins. In this review, the cases of glycine, nicotinic ACh and glutamate receptors are compared and contrasted, and problems such as how binding of agonist causes the channel to open, and why partial agonists are partial, are considered. Some observations, both structural and functional, suggest that more attention needs to be paid to conformational changes that occur before the channel opens. Such changes might account for the interaction found between subunits of the glycine receptor while it is still shut and, perhaps, the agonist-dependent structural changes seen in AMPA receptors. They might also complicate our understanding of the binding-gating problem.

Evolutionary conservation and changes in insect TRP channels.

PubMed

Matsuura, Hironori; Sokabe, Takaaki; Kohno, Keigo; Tominaga, Makoto; Kadowaki, Tatsuhiko

2009-09-10

TRP (Transient Receptor Potential) channels respond to diverse stimuli and thus function as the primary integrators of varied sensory information. They are also activated by various compounds and secondary messengers to mediate cell-cell interactions as well as to detect changes in the local environment. Their physiological roles have been primarily characterized only in mice and fruit flies, and evolutionary studies are limited. To understand the evolution of insect TRP channels and the mechanisms of integrating sensory inputs in insects, we have identified and compared TRP channel genes in Drosophila melanogaster, Bombyx mori, Tribolium castaneum, Apis mellifera, Nasonia vitripennis, and Pediculus humanus genomes as part of genome sequencing efforts. All the insects examined have 2 TRPV, 1 TRPN, 1 TRPM, 3 TRPC, and 1 TRPML subfamily members, demonstrating that these channels have the ancient origins in insects. The common pattern also suggests that the mechanisms for detecting mechanical and visual stimuli and maintaining lysosomal functions may be evolutionarily well conserved in insects. However, a TRPP channel, the most ancient TRP channel, is missing in B. mori, A. mellifera, and N. vitripennis. Although P. humanus and D. melanogaster contain 4 TRPA subfamily members, the other insects have 5 TRPA subfamily members. T. castaneum, A. mellifera, and N. vitripennis contain TRPA5 channels, which have been specifically retained or gained in Coleoptera and Hymenoptera. Furthermore, TRPA1, which functions for thermotaxis in Drosophila, is missing in A. mellifera and N. vitripennis; however, they have other Hymenoptera-specific TRPA channels (AmHsTRPA and NvHsTRPA). NvHsTRPA expressed in HEK293 cells is activated by temperature increase, demonstrating that HsTRPAs function as novel thermal sensors in Hymenoptera. The total number of insect TRP family members is 13-14, approximately half that of mammalian TRP family members. As shown for mammalian TRP channels, this may suggest that single TRP channels are responsible for integrating diverse sensory inputs to maintain the insect sensory systems. The above results demonstrate that there are both evolutionary conservation and changes in insect TRP channels. In particular, the evolutionary processes have been accelerated in the TRPA subfamily, indicating divergence in the mechanisms that insects use to detect environmental temperatures.

The SCN8A encephalopathy mutation p.Ile1327Val displays elevated sensitivity to the anticonvulsant phenytoin.

PubMed

Barker, Bryan S; Ottolini, Matteo; Wagnon, Jacy L; Hollander, Rachel M; Meisler, Miriam H; Patel, Manoj K

2016-09-01

SCN8A encephalopathy (early infantile epileptic encephalopathy; EIEE13) is caused by gain-of-function mutations resulting in hyperactivity of the voltage-gated sodium channel Nav 1.6. The channel is concentrated at the axon initial segment (AIS) and is involved in establishing neuronal excitability. Clinical features of SCN8A encephalopathy include seizure onset between 0 and 18 months of age, intellectual disability, and developmental delay. Seizures are often refractory to treatment with standard antiepileptic drugs, and sudden unexpected death in epilepsy (SUDEP) has been reported in approximately 10% of patients. In a recent study, high doses of phenytoin were effective in four patients with SCN8A encephalopathy. In view of this observation, we have investigated the relationship between the functional effect of the SCN8A mutation p.Ile1327Val and its response to phenytoin. The mutation was introduced into the Scn8a cDNA by site-directed mutagenesis. Channel activity was characterized in transfected ND7/23 cells. The effects of phenytoin (100 μm) on mutant and wild-type (WT) channels were compared. Channel activation parameters were shifted in a hyperpolarizing direction in the mutant channel, whereas inactivation parameters were shifted in a depolarizing direction, increasing Na channel window current. Macroscopic current decay was slowed in I1327V channels, indicating an impairment in the transition from open state to inactivated state. Channel deactivation was also delayed, allowing more channels to remain in the open state. Phenytoin (100 μm) resulted in hyperpolarized activation and inactivation curves as well as greater tonic block and use-dependent block of I1327V mutant channels relative to WT. SCN8A - I1327V is a gain-of-function mutation with altered features that are predicted to increase neuronal excitability and seizure susceptibility. Phenytoin is an effective inhibitor of the mutant channel and may be of use in treating patients with gain-of-function mutations of SCN8A. Wiley Periodicals, Inc. © 2016 International League Against Epilepsy.

The SCN8A encephalopathy mutation p.Ile1327Val displays elevated sensitivity to the anticonvulsant phenytoin

PubMed Central

Barker, Bryan S.; Ottolini, Matteo; Wagnon, Jacy L.; Hollander, Rachel; Meisler, Miriam H.; Patel, Manoj K.

2016-01-01

Objective SCN8A encephalopathy (EIEE13) is caused by gain-of-function mutations resulting in hyperactivity of the voltage-gated sodium channel Nav1.6. The channel is concentrated at the axon initial segment (AIS) and is involved in establishing neuronal excitability. Clinical features of SCN8A encephalopathy include seizure onset between 0–18 months of age, intellectual disability, and developmental delay. Seizures are often refractory to treatment with standard anti-epileptic drugs, and sudden unexpected death in epilepsy (SUDEP) has been reported in approximately 10% of patients. In a recent study, high doses of phenytoin were effective in four patients with SCN8A encephalopathy. In view of this observation, we have investigated the relationship between the functional effect of the SCN8A mutation p.Ile1327Val and its response to phenytoin. Methods The mutation was introduced into the Scn8a cDNA by site-directed mutagenesis. Channel activity was characterized in transfected ND7/23 cells. The effects of phenytoin (100 μM) on mutant and wild type (WT) channels were compared. Results Channel activation parameters were shifted in a hyperpolarizing direction in the mutant channel, while inactivation parameters were shifted in a depolarizing direction, increasing Na channel window current. Macroscopic current decay was slowed in I1327V channels, indicating an impairment in the transition from open state to inactivated state. Channel deactivation was also delayed, allowing more channels to remain in the open state. Phenytoin (100 μM) resulted in hyperpolarized activation and inactivation curves as well as greater tonic block and use dependent block of I1327V mutant channels relative to WT. Significance SCN8A – I1327V is a gain-of-function mutation with altered features that are predicted to increase neuronal excitability and seizure susceptibility. Phenytoin is an effective inhibitor of the mutant channel and may be of use in treating patients with gain-of-function mutations of SCN8A. PMID:27375106

Thermally activated TRP channels: molecular sensors for temperature detection.

PubMed

Castillo, Karen; Diaz-Franulic, Ignacio; Canan, Jonathan; Gonzalez-Nilo, Fernando; Latorre, Ramon

2018-01-24

Temperature sensing is one of the oldest capabilities of living organisms, and is essential for sustaining life, because failure to avoid extreme noxious temperatures can result in tissue damage or death. A subset of members of the transient receptor potential (TRP) ion channel family is finely tuned to detect temperatures ranging from extreme cold to noxious heat, giving rise to thermoTRP channels. Structural and functional experiments have shown that thermoTRP channels are allosteric proteins, containing different domains that sense changes in temperature, among other stimuli, triggering pore opening. Although temperature-dependence is well characterized in thermoTRP channels, the molecular nature of temperature-sensing elements remains unknown. Importantly, thermoTRP channels are involved in pain sensation, related to pathological conditions. Here, we provide an overview of thermoTRP channel activation. We also discuss the structural and functional evidence supporting the existence of an intrinsic temperature sensor in this class of channels, and we explore the basic thermodynamic principles for channel activation. Finally, we give a view of their role in painful pathophysiological conditions.

Deciphering the function of the CNGB1b subunit in olfactory CNG channels.

PubMed

Nache, Vasilica; Wongsamitkul, Nisa; Kusch, Jana; Zimmer, Thomas; Schwede, Frank; Benndorf, Klaus

2016-07-11

Olfactory cyclic nucleotide-gated (CNG) ion channels are key players in the signal transduction cascade of olfactory sensory neurons. The second messengers cAMP and cGMP directly activate these channels, generating a depolarizing receptor potential. Olfactory CNG channels are composed of two CNGA2 subunits and two modulatory subunits, CNGA4, and CNGB1b. So far the exact role of the modulatory subunits for channel activation is not fully understood. By measuring ligand binding and channel activation simultaneously, we show that in functional heterotetrameric channels not only the CNGA2 subunits and the CNGA4 subunit but also the CNGB1b subunit binds cyclic nucleotides and, moreover, also alone translates this signal to open the pore. In addition, we show that the CNGB1b subunit is the most sensitive subunit in a heterotetrameric channel to cyclic nucleotides and that it accelerates deactivation to a similar extent as does the CNGA4 subunit. In conclusion, the CNGB1b subunit participates in ligand-gated activation of olfactory CNG channels and, particularly, contributes to rapid termination of odorant signal in an olfactory sensory neuron.

Effects of the β1 auxiliary subunit on modification of Rat Na{sub v}1.6 sodium channels expressed in HEK293 cells by the pyrethroid insecticides tefluthrin and deltamethrin

DOE Office of Scientific and Technical Information (OSTI.GOV)

He, Bingjun; Soderlund, David M., E-mail: dms6@cornell.edu

We expressed rat Na{sub v}1.6 sodium channels with or without the rat β1 subunit in human embryonic kidney (HEK293) cells and evaluated the effects of the pyrethroid insecticides tefluthrin and deltamethrin on whole-cell sodium currents. In assays with the Na{sub v}1.6 α subunit alone, both pyrethroids prolonged channel inactivation and deactivation and shifted the voltage dependence of channel activation and steady-state inactivation toward hyperpolarization. Maximal shifts in activation were ~ 18 mV for tefluthrin and ~ 24 mV for deltamethrin. These compounds also caused hyperpolarizing shifts of ~ 10–14 mV in the voltage dependence of steady-state inactivation and increased inmore » the fraction of sodium current that was resistant to inactivation. The effects of pyrethroids on the voltage-dependent gating greatly increased the size of sodium window currents compared to unmodified channels; modified channels exhibited increased probability of spontaneous opening at membrane potentials more negative than the normal threshold for channel activation and incomplete channel inactivation. Coexpression of Na{sub v}1.6 with the β1 subunit had no effect on the kinetic behavior of pyrethroid-modified channels but had divergent effects on the voltage-dependent gating of tefluthrin- or deltamethrin-modified channels, increasing the size of tefluthrin-induced window currents but decreasing the size of corresponding deltamethrin-induced currents. Unexpectedly, the β1 subunit did not confer sensitivity to use-dependent channel modification by either tefluthrin or deltamethrin. We conclude from these results that functional reconstitution of channels in vitro requires careful attention to the subunit composition of channel complexes to ensure that channels in vitro are faithful functional and pharmacological models of channels in neurons. - Highlights: • We expressed Na{sub v}1.6 sodium channels with or without β1 subunits in HEK293 cells. • Tefluthrin and deltamethrin shifted channel gating to hyperpolarized potentials. • The β1 subunit had opposite effects on the actions of tefluthrin and deltamethrin. • Auxiliary subunits are required for full reconstitution of channel function. • Channels in HEK293 cells exhibit properties similar to channels in neurons.« less

[Are there imported components for channels and collaterals?].

PubMed

Zhu, Bing

2005-10-01

The medical papyri in ancient Egypt written before 1500 B. C, recorded the metu system which could not be explained by modern anatomic location and was difficultly expounded by biological functions. Most of diseases were treated by dredging the metu, regulating the metu, balancing the metu, removing noxious substances from the metu, restoring the normal functions of the metu. The ancient Egyptian held that the metu forms mutual connecting channel network to carry out flowing of energy and information, with the functions of linking up external and internal organs, which is similar to linking functions of meridians-zang- and fu- organs. In the works of Hippocrates, the father of ancient Greece medicine, 2nd century B. C, also described the linking channels "phleps" on human surface. And in other country with an ancient civilization, India, had a similar channel system. About 15th century B. C, Charaka Samitha of ancient India described human "nadis" channel system. The system carried "rasas" (life liquid) connected with the whole body from the navel. Therefore, it can be held that at the embryonic stage of medicine development, the linking tube similar to channel or vessel was described, but it is interested that met and Chinese "mai" (vessel) have similar pronunciation. And mai in China was created after met.

Direct Interaction between the Voltage Sensors Produces Cooperative Sustained Deactivation in Voltage-gated H+ Channel Dimers*

PubMed Central

Okuda, Hiroko; Yonezawa, Yasushige; Takano, Yu; Okamura, Yasushi; Fujiwara, Yuichiro

2016-01-01

The voltage-gated H+ channel (Hv) is a voltage sensor domain-like protein consisting of four transmembrane segments (S1–S4). The native Hv structure is a homodimer, with the two channel subunits functioning cooperatively. Here we show that the two voltage sensor S4 helices within the dimer directly cooperate via a π-stacking interaction between Trp residues at the middle of each segment. Scanning mutagenesis showed that Trp situated around the original position provides the slow gating kinetics characteristic of the dimer's cooperativity. Analyses of the Trp mutation on the dimeric and monomeric channel backgrounds and analyses with tandem channel constructs suggested that the two Trp residues within the dimer are functionally coupled during Hv deactivation but are less so during activation. Molecular dynamics simulation also showed direct π-stacking of the two Trp residues. These results provide new insight into the cooperative function of voltage-gated channels, where adjacent voltage sensor helices make direct physical contact and work as a single unit according to the gating process. PMID:26755722

Creating Perfused Functional Vascular Channels Using 3D Bio-Printing Technology

PubMed Central

Lee, Vivian K.; Kim, Diana Y.; Ngo, Haygan; Lee, Young; Seo, Lan; Yoo, Seung-Schik; Vincent, Peter A.; Dai, Guohao

2014-01-01

We developed a methodology using 3D bio-printing technology to create a functional in vitro vascular channel with perfused open lumen using only cells and biological matrices. The fabricated vasculature has a tight, confluent endothelium lining, presenting barrier function for both plasma protein and high-molecular weight dextran molecule. The fluidic vascular channel is capable of supporting the viability of tissue up to 5mm in distance at 5 million cells/mL density under the physiological flow condition. In static-cultured vascular channels, active angiogenic sprouting from the vessel surface was observed whereas physiological flow strongly suppressed this process. Gene expression analysis were reported in this study to show the potential of this vessel model in vascular biology research. The methods have great potential in vascularized tissue fabrication using 3D bio-printing technology as the vascular channel is simultaneously created while cells and matrix are printed around the channel in desired 3D patterns. It can also serve as a unique experimental tool for investigating fundamental mechanisms of vascular remodeling with extracellular matrix and maturation process under 3D flow condition. PMID:24965886

Molecular dissection of botulinum neurotoxin reveals interdomain chaperone function.

PubMed

Fischer, Audrey; Montal, Mauricio

2013-12-01

Clostridium botulinum neurotoxin (BoNT) is a multi-domain protein made up of the approximately 100 kDa heavy chain (HC) and the approximately 50 kDa light chain (LC). The HC can be further subdivided into two halves: the N-terminal translocation domain (TD) and the C-terminal Receptor Binding Domain (RBD). We have investigated the minimal requirements for channel activity and LC translocation. We utilize a cellular protection assay and a single channel/single molecule LC translocation assay to characterize in real time the channel and chaperone activities of BoNT/A truncation constructs in Neuro 2A cells. The unstructured, elongated belt region of the TD is demonstrated to be dispensable for channel activity, although may be required for productive LC translocation. We show that the RBD is not necessary for channel activity or LC translocation, however it dictates the pH threshold of channel insertion into the membrane. These findings indicate that each domain functions as a chaperone for the others in addition to their individual functions, working in concert to achieve productive intoxication. Copyright © 2013 Elsevier Ltd. All rights reserved.

Crystal Structure of the Ternary Complex of a NaV C-Terminal Domain, a Fibroblast Growth Factor Homologous Factor, and Calmodulin

DOE Office of Scientific and Technical Information (OSTI.GOV)

Wang, Chaojian; Chung, Ben C.; Yan, Haidun

2012-11-13

Voltage-gated Na{sup +} (Na{sub V}) channels initiate neuronal action potentials. Na{sub V} channels are composed of a transmembrane domain responsible for voltage-dependent Na{sup +} conduction and a cytosolic C-terminal domain (CTD) that regulates channel function through interactions with many auxiliary proteins, including fibroblast growth factor homologous factors (FHFs) and calmodulin (CaM). Most ion channel structural studies have focused on mechanisms of permeation and voltage-dependent gating but less is known about how intracellular domains modulate channel function. Here we report the crystal structure of the ternary complex of a human NaV CTD, an FHF, and Ca{sup 2+}-free CaM at 2.2 {angstrom}.more » Combined with functional experiments based on structural insights, we present a platform for understanding the roles of these auxiliary proteins in NaV channel regulation and the molecular basis of mutations that lead to neuronal and cardiac diseases. Furthermore, we identify a critical interaction that contributes to the specificity of individual NaV CTD isoforms for distinctive FHFs.« less

Roderick MacKinnon - Patents

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CHANNEL PROTEINS, MUTANT PROKARYOTIC CATION CHANNEL PROTEINS, AND USES THEREOF - MacKinnon, Roderick cation channel proteins, and potentially have uses in treating conditions related to the function of SENSOR DOMAINS OF VOLTAGE-DEPENDENT ION CHANNEL PROTEINS AND USES THEREOF - MacKinnon, Roderick; et. al

Pleiotropic function of TRPV4 ion channels in the central nervous system

PubMed Central

Kanju, Patrick; Liedtke, Wolfgang

2016-01-01

TRPV4 ion channels are osmo-mechano-TRP channels with pleiotropic function and expression in many different types of tissues and cells. They have also been found involved in pain and inflammation. Studies have focused on the role of TRPV4 in peripheral sensory neurons, but its expression and function in central nervous glial cells and neurons has also been documented. In this overview, based on the senior author’s lecture at the recent physiology meeting in Dublin, we concisely review evidence of TRPV4 expression and function in the CNS, and how TRPV4 function can be modulated for therapeutic benefit of neuro-psychiatric disorders. Novel TRPV4-inhibitory compounds developed recently in the authors’ lab will also be discussed PMID:27701788

Functional reconstitution of the voltage-regulated sodium channel purified from electroplax of Electrophorus electricus

DOE Office of Scientific and Technical Information (OSTI.GOV)

Rosenberg, R.L.

1985-01-01

The voltage-regulated NA channel is responsible for the depolarization of the excitable cell membrane during the normal action potential. This research has focused on the functional properties of the Na channel, purified from detergent extracts of electroplax membranes of the electric eel, and reconstituted into vesicles of defined phospholipid. These properties were assessed by measuring neurotoxin-modulated ion flux into the reconstituted membrane vesicles and by recording the single-channel currents of the purified channel by the patch-clamp method. The binding of tritiated tetrodotoxin (TTX) was employed as a marker for the purification of the channel. Two high-resolution fractionation steps, based onmore » molecular charge and protein size, were used to obtain a preparation that is 80% homogeneous for a large peptide of 270,000 daltons. Radiotracer /sup 22/Na/sup +/ influx into the vesicles was stimulated by veratridine and by batrachotoxin (BTX) at concentrations of 100 ..mu..M and 5 ..mu..M, respectively. The stimulation by BTX was greater than that by veratridine, and can be as much as 16-fold over control influx levels. The stimulated influx is blocked by TTX with a K/sub i/ of 35 nM, and by local anesthetics in the normal pharmacological range. Large multilamellar vesicles prepared with a freeze-thaw step are suitable for single-channel recording techniques. When excised patches of the reconstituted membranes were voltage-clamped in the absence of activating neurotoxins, voltage-dependent single-channel currents were recorded. These displayed properties similar to those from native membranes of nerve and muscle. These results indicate that the protein purified on the basis of TTX binding is a functional Na channel possessing these functional domains: the ion-selective channel, the voltage sensors controlling activation and inactivation, and the sites of action of TTX, alkaloid neurotoxins, and local anesthetics.« less

The inward rectifier potassium channel Kir2.1 is expressed in mouse neutrophils from bone marrow and liver.

PubMed

Masia, Ricard; Krause, Daniela S; Yellen, Gary

2015-02-01

Neutrophils are phagocytic cells that play a critical role in innate immunity by destroying bacterial pathogens. Channels belonging to the inward rectifier potassium channel subfamily 2 (Kir2 channels) have been described in other phagocytes (monocytes/macrophages and eosinophils) and in hematopoietic precursors of phagocytes. Their physiological function in these cells remains unclear, but some evidence suggests a role in growth factor-dependent proliferation and development. Expression of functional Kir2 channels has not been definitively demonstrated in mammalian neutrophils. Here, we show by RT-PCR that neutrophils from mouse bone marrow and liver express mRNA for the Kir2 subunit Kir2.1 but not for other subunits (Kir2.2, Kir2.3, and Kir2.4). In electrophysiological experiments, resting (unstimulated) neutrophils from mouse bone marrow and liver exhibit a constitutively active, external K(+)-dependent, strong inwardly rectifying current that constitutes the dominant current. The reversal potential is dependent on the external K(+) concentration in a Nernstian fashion, as expected for a K(+)-selective current. The current is not altered by changes in external or internal pH, and it is blocked by Ba(2+), Cs(+), and the Kir2-selective inhibitor ML133. The single-channel conductance is in agreement with previously reported values for Kir2.1 channels. These properties are characteristic of homomeric Kir2.1 channels. Current density in short-term cultures of bone marrow neutrophils is decreased in the absence of growth factors that are important for neutrophil proliferation [granulocyte colony-stimulating factor (G-CSF) and stem cell factor (SCF)]. These results demonstrate that mouse neutrophils express functional Kir2.1 channels and suggest that these channels may be important for neutrophil function, possibly in a growth factor-dependent manner. Copyright © 2015 the American Physiological Society.

The role of transient receptor potential channels in joint diseases.

PubMed

Krupkova, O; Zvick, J; Wuertz-Kozak, K

2017-10-10

Transient receptor potential channels (TRP channels) are cation selective transmembrane receptors with diverse structures, activation mechanisms and physiological functions. TRP channels act as cellular sensors for a plethora of stimuli, including temperature, membrane voltage, oxidative stress, mechanical stimuli, pH and endogenous, as well as, exogenous ligands, thereby illustrating their versatility. As such, TRP channels regulate various functions in both excitable and non-excitable cells, mainly by mediating Ca2+ homeostasis. Dysregulation of TRP channels is implicated in many pathologies, including cardiovascular diseases, muscular dystrophies and hyperalgesia. However, the importance of TRP channel expression, physiological function and regulation in chondrocytes and intervertebral disc (IVD) cells is largely unexplored. Osteoarthritis (OA) and degenerative disc disease (DDD) are chronic age-related disorders that significantly affect the quality of life by causing pain, activity limitation and disability. Furthermore, currently available therapies cannot effectively slow-down or stop progression of these diseases. Both OA and DDD are characterised by reduced tissue cellularity, enhanced inflammatory responses and molecular, structural and mechanical alterations of the extracellular matrix, hence affecting load distribution and reducing joint flexibility. However, knowledge on how chondrocytes and IVD cells sense their microenvironment and respond to its changes is still limited. In this review, we introduced six families of mammalian TRP channels, their mechanisms of activation, as well as, activation-driven cellular consequences. We summarised the current knowledge on TRP channel expression and activity in chondrocytes and IVD cells, as well as, the significance of TRP channels as therapeutic targets for the treatment of OA and DDD.

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

PubMed Central

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

2013-01-01

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

Chloride channels in cancer: Focus on chloride intracellular channel 1 and 4 (CLIC1 AND CLIC4) proteins in tumor development and as novel therapeutic targets.

PubMed

Peretti, Marta; Angelini, Marina; Savalli, Nicoletta; Florio, Tullio; Yuspa, Stuart H; Mazzanti, Michele

2015-10-01

In recent decades, growing scientific evidence supports the role of ion channels in the development of different cancers. Both potassium selective pores and chloride permeabilities are considered the most active channels during tumorigenesis. High rate of proliferation, active migration, and invasiveness into non-neoplastic tissues are specific properties of neoplastic transformation. All these actions require partial or total involvement of chloride channel activity. In this context, this class of membrane proteins could represent valuable therapeutic targets for the treatment of resistant tumors. However, this encouraging premise has not so far produced any valid new channel-targeted antitumoral molecule for cancer treatment. Problematic for drug design targeting ion channels is their vital role in normal cells for essential physiological functions. By targeting these membrane proteins involved in pathological conditions, it is inevitable to cause relevant side effects in healthy organs. In light of this, a new protein family, the chloride intracellular channels (CLICs), could be a promising class of therapeutic targets for its intrinsic individualities: CLIC1 and CLIC4, in particular, not only are overexpressed in specific tumor types or their corresponding stroma but also change localization and function from hydrophilic cytosolic to integral transmembrane proteins as active ionic channels or signal transducers during cell cycle progression in certain cases. These changes in intracellular localization, tissue compartments, and channel function, uniquely associated with malignant transformation, may offer a unique target for cancer therapy, likely able to spare normal cells. This article is part of a special issue itled "Membrane Channels and Transporters in Cancers." Copyright © 2015 Elsevier B.V. All rights reserved.

A two-dimensional analytical modeling for channel potential and threshold voltage of short channel triple material symmetrical gate Stack (TMGS) DG-MOSFET

NASA Astrophysics Data System (ADS)

Tripathi, Shweta

2016-10-01

In the present work, a two-dimensional (2D) analytical framework of triple material symmetrical gate stack (TMGS) DG-MOSFET is presented in order to subdue the short channel effects. A lightly doped channel along with triple material gate having different work functions and symmetrical gate stack structure, showcases substantial betterment in quashing short channel effects to a good extent. The device functioning amends in terms of improved exemption to threshold voltage roll-off, thereby suppressing the short channel effects. The encroachments of respective device arguments on the threshold voltage of the proposed structure are examined in detail. The significant outcomes are compared with the numerical simulation data obtained by using 2D ATLAS™ device simulator to affirm and formalize the proposed device structure.

Gating of human ClC-2 chloride channels and regulation by carboxy-terminal domains

PubMed Central

Garcia-Olivares, Jennie; Alekov, Alexi; Boroumand, Mohammad Reza; Begemann, Birgit; Hidalgo, Patricia; Fahlke, Christoph

2008-01-01

Eukaryotic ClC channels are dimeric proteins with each subunit forming an individual protopore. Single protopores are gated by a fast gate, whereas the slow gate is assumed to control both protopores through a cooperative movement of the two carboxy-terminal domains. We here study the role of the carboxy-terminal domain in modulating fast and slow gating of human ClC-2 channels, a ubiquitously expressed ClC-type chloride channel involved in transepithelial solute transport and in neuronal chloride homeostasis. Partial truncation of the carboxy-terminus abolishes function of ClC-2 by locking the channel in a closed position. However, unlike other isoforms, its complete removal preserves function of ClC-2. ClC-2 channels without the carboxy-terminus exhibit fast and slow gates that activate and deactivate significantly faster than in WT channels. In contrast to the prevalent view, a single carboxy-terminus suffices for normal slow gating, whereas both domains regulate fast gating of individual protopores. Our findings demonstrate that the carboxy-terminus is not strictly required for slow gating and that the cooperative gating resides in other regions of the channel protein. ClC-2 is expressed in neurons and believed to open at negative potentials and increased internal chloride concentrations after intense synaptic activity. We propose that the function of the ClC-2 carboxy-terminus is to slow down the time course of channel activation in order to stabilize neuronal excitability PMID:18801843

Gating of human ClC-2 chloride channels and regulation by carboxy-terminal domains.

PubMed

Garcia-Olivares, Jennie; Alekov, Alexi; Boroumand, Mohammad Reza; Begemann, Birgit; Hidalgo, Patricia; Fahlke, Christoph

2008-11-15

Eukaryotic ClC channels are dimeric proteins with each subunit forming an individual protopore. Single protopores are gated by a fast gate, whereas the slow gate is assumed to control both protopores through a cooperative movement of the two carboxy-terminal domains. We here study the role of the carboxy-terminal domain in modulating fast and slow gating of human ClC-2 channels, a ubiquitously expressed ClC-type chloride channel involved in transepithelial solute transport and in neuronal chloride homeostasis. Partial truncation of the carboxy-terminus abolishes function of ClC-2 by locking the channel in a closed position. However, unlike other isoforms, its complete removal preserves function of ClC-2. ClC-2 channels without the carboxy-terminus exhibit fast and slow gates that activate and deactivate significantly faster than in WT channels. In contrast to the prevalent view, a single carboxy-terminus suffices for normal slow gating, whereas both domains regulate fast gating of individual protopores. Our findings demonstrate that the carboxy-terminus is not strictly required for slow gating and that the cooperative gating resides in other regions of the channel protein. ClC-2 is expressed in neurons and believed to open at negative potentials and increased internal chloride concentrations after intense synaptic activity. We propose that the function of the ClC-2 carboxy-terminus is to slow down the time course of channel activation in order to stabilize neuronal excitability.

A modern ionotropic glutamate receptor with a K(+) selectivity signature sequence.

PubMed

Janovjak, H; Sandoz, G; Isacoff, E Y

2011-01-01

Glutamate is the major excitatory neurotransmitter in the mammalian central nervous system and gates non-selective cation channels. The origins of glutamate receptors are not well understood as they differ structurally and functionally from simple bacterial ligand-gated ion channels. Here we report the discovery of an ionotropic glutamate receptor that combines the typical eukaryotic domain architecture with the 'TXVGYG' signature sequence of the selectivity filter found in K(+) channels. This receptor exhibits functional properties intermediate between bacterial and eukaryotic glutamate-gated ion channels, suggesting a link in the evolution of ionotropic glutamate receptors.

Different roles for the cyclic nucleotide binding domain and amino terminus in assembly and expression of hyperpolarization-activated, cyclic nucleotide-gated channels.

PubMed

Proenza, Catherine; Tran, Neil; Angoli, Damiano; Zahynacz, Kristin; Balcar, Petr; Accili, Eric A

2002-08-16

In mammalian heart and brain, pacemaker currents are produced by hyperpolarization-activated, cyclic nucleotide-gated (HCN) channels, which probably exist as heteromeric assemblies of different subunit isoforms. To investigate the molecular domains that participate in assembly and membrane trafficking of HCN channels, we have used the yeast two-hybrid system, patch clamp electrophysiology, and confocal microscopy. We show here that the N termini of the HCN1 and HCN2 isoforms interacted and were essential for expression of functional homo- or heteromeric channels on the plasma membrane of Chinese hamster ovary cells. We also show that the cyclic nucleotide binding domain (CNBD) of HCN2 was required for the expression of functional homomeric channels. This expression was dependent on a 12-amino acid domain corresponding to the B-helix in the CNBD of the catabolite activator protein. However, co-expression with HCN1 of an HCN2 deletion mutant lacking the CNBD rescued surface immunofluorescence and currents, indicating that a CNBD need not be present in each subunit of a heteromeric HCN channel. Furthermore, neither CNBDs nor other COOH-terminal domains of HCN1 and HCN2 interacted in yeast two-hybrid assays. Thus, interaction between NH(2)-terminal domains is important for HCN subunit assembly, whereas the CNBD is important for functional expression, but its absence from some subunits will still allow for the assembly of functional channels.

Novel screening techniques for ion channel targeting drugs

PubMed Central

Obergrussberger, Alison; Stölzle-Feix, Sonja; Becker, Nadine; Brüggemann, Andrea; Fertig, Niels; Möller, Clemens

2015-01-01

Ion channels are integral membrane proteins that regulate the flux of ions across the cell membrane. They are involved in nearly all physiological processes, and malfunction of ion channels has been linked to many diseases. Until recently, high-throughput screening of ion channels was limited to indirect, e.g. fluorescence-based, readout technologies. In the past years, direct label-free biophysical readout technologies by means of electrophysiology have been developed. Planar patch-clamp electrophysiology provides a direct functional label-free readout of ion channel function in medium to high throughput. Further electrophysiology features, including temperature control and higher-throughput instruments, are continually being developed. Electrophysiological screening in a 384-well format has recently become possible. Advances in chip and microfluidic design, as well as in cell preparation and handling, have allowed challenging cell types to be studied by automated patch clamp. Assays measuring action potentials in stem cell-derived cardiomyocytes, relevant for cardiac safety screening, and neuronal cells, as well as a large number of different ion channels, including fast ligand-gated ion channels, have successfully been established by automated patch clamp. Impedance and multi-electrode array measurements are particularly suitable for studying cardiomyocytes and neuronal cells within their physiological network, and to address more complex physiological questions. This article discusses recent advances in electrophysiological technologies available for screening ion channel function and regulation. PMID:26556400

Novel screening techniques for ion channel targeting drugs.

PubMed

Obergrussberger, Alison; Stölzle-Feix, Sonja; Becker, Nadine; Brüggemann, Andrea; Fertig, Niels; Möller, Clemens

2015-01-01

Ion channels are integral membrane proteins that regulate the flux of ions across the cell membrane. They are involved in nearly all physiological processes, and malfunction of ion channels has been linked to many diseases. Until recently, high-throughput screening of ion channels was limited to indirect, e.g. fluorescence-based, readout technologies. In the past years, direct label-free biophysical readout technologies by means of electrophysiology have been developed. Planar patch-clamp electrophysiology provides a direct functional label-free readout of ion channel function in medium to high throughput. Further electrophysiology features, including temperature control and higher-throughput instruments, are continually being developed. Electrophysiological screening in a 384-well format has recently become possible. Advances in chip and microfluidic design, as well as in cell preparation and handling, have allowed challenging cell types to be studied by automated patch clamp. Assays measuring action potentials in stem cell-derived cardiomyocytes, relevant for cardiac safety screening, and neuronal cells, as well as a large number of different ion channels, including fast ligand-gated ion channels, have successfully been established by automated patch clamp. Impedance and multi-electrode array measurements are particularly suitable for studying cardiomyocytes and neuronal cells within their physiological network, and to address more complex physiological questions. This article discusses recent advances in electrophysiological technologies available for screening ion channel function and regulation.

Physical instrumental vetoes for gravitational-wave burst triggers

NASA Astrophysics Data System (ADS)

Ajith, P.; Hewitson, M.; Smith, J. R.; Grote, H.; Hild, S.; Strain, K. A.

2007-08-01

We present a robust strategy to veto certain classes of instrumental glitches that appear at the output of interferometric gravitational-wave detectors. This veto method is “physical” in the sense that, in order to veto a burst trigger, we make use of our knowledge of the coupling of different detector subsystems to the main detector output. The main idea behind this method is that the noise in an instrumental channel X can be transferred to the detector output (channel H) using the transfer function from X to H, provided the noise coupling is linear and the transfer function is unique. If a nonstationarity in channel H is causally related to one in channel X, the two have to be consistent with the transfer function. We formulate two methods for testing the consistency between the burst triggers in channel X and channel H. One method makes use of the null stream constructed from channel H and the transferred channel X, and the second involves cross correlating the two. We demonstrate the efficiency of the veto by “injecting” instrumental glitches in the hardware of the GEO 600 detector. The veto safety is demonstrated by performing gravitational-wave like hardware injections. We also show an example application of this method using 5 days of data from the fifth science run of GEO 600. The method is found to have very high veto efficiency with a very low accidental veto rate.

TRP channels in the digestive system

PubMed Central

Holzer, Peter

2011-01-01

Several of the 28 mammalian transient receptor potential (TRP) channel subunits are expressed throughout the alimentary canal where they play important roles in taste, chemo- and mechanosensation, thermoregulation, pain and hyperalgesia, mucosal function and homeostasis, control of motility by neurons, interstitial cells of Cajal and muscle cells, and vascular function. While the implications of some TRP channels, notably TRPA1, TRPC4, TRPM5, TRPM6, TRPM7, TRPV1, TRPV4, and TRPV6, have been investigated in much detail, the understanding of other TRP channels in their relevance to digestive function lags behind. The polymodal chemo- and mechanosensory function of TRPA1, TRPM5, TRPV1 and TRPV4 is particularly relevant to the alimentary canal whose digestive and absorptive function depends on the surveillance and integration of many chemical and physical stimuli. TRPV5 and TRPV6 as well as TRPM6 and TRPM7 appear to be essential for the absorption of Ca2+ and Mg2+, respectively, while TRPM7 appears to contribute to the pacemaker activity of the interstitial cells of Cajal, and TRPC4 transduces smooth muscle contraction evoked by muscarinic acetylcholine receptor activation. The implication of some TRP channels in pathological processes has raised enormous interest in exploiting them as a therapeutic target. This is particularly true for TRPV1, TRPV4 and TRPA1, which may be targeted for the treatment of several conditions of chronic abdominal pain. Consequently, blockers of these TRP channels have been developed, and their clinical usefulness has yet to be established. PMID:20932260

Contact resistance extraction methods for short- and long-channel carbon nanotube field-effect transistors

NASA Astrophysics Data System (ADS)

Pacheco-Sanchez, Anibal; Claus, Martin; Mothes, Sven; Schröter, Michael

2016-11-01

Three different methods for the extraction of the contact resistance based on both the well-known transfer length method (TLM) and two variants of the Y-function method have been applied to simulation and experimental data of short- and long-channel CNTFETs. While for TLM special CNT test structures are mandatory, standard electrical device characteristics are sufficient for the Y-function methods. The methods have been applied to CNTFETs with low and high channel resistance. It turned out that the standard Y-function method fails to deliver the correct contact resistance in case of a relatively high channel resistance compared to the contact resistances. A physics-based validation is also given for the application of these methods based on applying traditional Si MOSFET theory to quasi-ballistic CNTFETs.

Marine Toxins Targeting Ion Channels

PubMed Central

Arias, Hugo R.

2006-01-01

This introductory minireview points out the importance of ion channels for cell communication. The basic concepts on the structure and function of ion channels triggered by membrane voltage changes, the so-called voltage-gated ion channels (VGICs), as well as those activated by neurotransmitters, the so-called ligand-gated ion channel (LGICs), are introduced. Among the most important VGIC superfamiles, we can name the voltage-gated Na+ (NaV), Ca2+ (CaV), and K+ (KV) channels. Among the most important LGIC super families, we can include the Cys-loop or nicotinicoid, the glutamate-activated (GluR), and the ATP-activated (P2XnR) receptor superfamilies. Ion channels are transmembrane proteins that allow the passage of different ions in a specific or unspecific manner. For instance, the activation of NaV, CaV, or KV channels opens a pore that is specific for Na+, Ca2+, or K+, respectively. On the other hand, the activation of certain LGICs such as nicotinic acetylcholine receptors, GluRs, and P2XnRs allows the passage of cations (e.g., Na+, K+, and/or Ca2+), whereas the activation of other LGICs such as type A γ-butyric acid and glycine receptors allows the passage of anions (e.g., Cl− and/or HCO3−). In this regard, the activation of NaV and CaV as well as ligand-gated cation channels produce membrane depolarization, which finally leads to stimulatory effects in the cell, whereas the activation of KV as well as ligand-gated anion channels induce membrane hyperpolarization that finally leads to inhibitory effects in the cell. The importance of these ion channel superfamilies is emphasized by considering their physiological functions throughout the body as well as their pathophysiological implicance in several neuronal diseases. In this regard, natural molecules, and especially marine toxins, can be potentially used as modulators (e.g., inhibitors or prolongers) of ion channel functions to treat or to alleviate a specific ion channel-linked disease (e.g., channelopaties).

Functional link between muscarinic receptors and large-conductance Ca2+-activated K+ channels in freshly-isolated human detrusor smooth muscle cells

PubMed Central

Parajuli, Shankar P.; Hristov, Kiril L.; Cheng, Qiuping; Malysz, John; Rovner, Eric S.; Petkov, Georgi V.

2014-01-01

Activation of muscarinic acetylcholine receptors (mAChRs) constitutes the primary mechanism for enhancing excitability and contractility of human detrusor smooth muscle (DSM). Since the large conductance Ca2+-activated K+ (KCa1.1) channels are key regulators of human DSM function, we investigated whether mAChR activation increases human DSM excitability by inhibiting KCa1.1 channels. We used the mAChR agonist, carbachol, to determine the changes in KCa1.1 channel activity upon mAChR activation in freshly-isolated human DSM cells obtained from open bladder surgeries using the perforated whole cell and single KCa1.1 channel patch-clamp recordings. Human DSM cells were collected from 29 patients (23 males and 6 females, average age of 65.9±1.5 years). Carbachol inhibited the amplitude and frequency of KCa1.1 channel-mediated spontaneous transient outward currents and spontaneous transient hyperpolarizations, which are triggered by the release of Ca2+ from ryanodine receptors. Carbachol also caused membrane potential depolarization, which was not observed in the presence of iberiotoxin, a KCa1.1 channel inhibitor, indicating the critical role of the KCa1.1 channels. The potential direct carbachol effects on KCa1.1channels were examined under conditions of removing the major cellular Ca2+ sources for KCa1.1 channel activation with pharmacological inhibitors (thapsigargin, ryanodine, and nifedipine). In the presence of these inhibitors, carbachol did not affect the single KCa1.1 channel open probability and mean KCa1.1 channel conductance (cell-attached configuration) or depolarization-induced whole cell steady-state KCa1.1 currents. The data support the concept that mAChR activation triggers indirect functional KCa1.1 channel inhibition mediated by intracellular Ca2+, thus increasing the excitability in human DSM cells. PMID:24867682

Functional K(ATP) channels in the rat retinal microvasculature: topographical distribution, redox regulation, spermine modulation and diabetic alteration.

PubMed

Ishizaki, Eisuke; Fukumoto, Masanori; Puro, Donald G

2009-05-15

The essential task of the circulatory system is to match blood flow to local metabolic demand. However, much remains to be learned about this process. To better understand how local perfusion is regulated, we focused on the functional organization of the retinal microvasculature, which is particularly well adapted for the local control of perfusion. Here, we assessed the distribution and regulation of functional K(ATP) channels whose activation mediates the hyperpolarization induced by adenosine. Using microvascular complexes freshly isolated from the rat retina, we found a topographical heterogeneity in the distribution of functional K(ATP) channels; capillaries generate most of the K(ATP) current. The initiation of K(ATP)-induced responses in the capillaries supports the concept that the regulation of retinal perfusion is highly decentralized. Additional study revealed that microvascular K(ATP) channels are redox sensitive, with oxidants increasing their activity. Furthermore, the oxidant-mediated activation of these channels is driven by the polyamine spermine, whose catabolism produces oxidants. In addition, our observation that spermine-dependent oxidation occurs predominately in the capillaries accounts for why they generate most of the K(ATP) current detected in retinal microvascular complexes. Here, we also analysed retinal microvessels of streptozotocin-injected rats. We found that soon after the onset of diabetes, an increase in spermine-dependent oxidation at proximal microvascular sites boosts their K(ATP) current and thereby virtually eliminates the topographical heterogeneity of functional K(ATP) channels. We conclude that spermine-dependent oxidation is a previously unrecognized mechanism by which this polyamine modulates ion channels; in addition to a physiological role, spermine-dependent oxidation may also contribute to microvascular dysfunction in the diabetic retina.

Cholesterol up-regulates neuronal G protein-gated inwardly rectifying potassium (GIRK) channel activity in the hippocampus.

PubMed

Bukiya, Anna N; Durdagi, Serdar; Noskov, Sergei; Rosenhouse-Dantsker, Avia

2017-04-14

Hypercholesterolemia is a well known risk factor for the development of neurodegenerative disease. However, the underlying mechanisms are mostly unknown. In recent years, it has become increasingly evident that cholesterol-driven effects on physiology and pathophysiology derive from its ability to alter the function of a variety of membrane proteins including ion channels. Yet, the effect of cholesterol on G protein-gated inwardly rectifying potassium (GIRK) channels expressed in the brain is unknown. GIRK channels mediate the actions of inhibitory brain neurotransmitters. As a result, loss of GIRK function can enhance neuron excitability, whereas gain of GIRK function can reduce neuronal activity. Here we show that in rats on a high-cholesterol diet, cholesterol levels in hippocampal neurons are increased. We also demonstrate that cholesterol plays a critical role in modulating neuronal GIRK currents. Specifically, cholesterol enrichment of rat hippocampal neurons resulted in enhanced channel activity. In accordance, elevated currents upon cholesterol enrichment were also observed in Xenopus oocytes expressing GIRK2 channels, the primary GIRK subunit expressed in the brain. Furthermore, using planar lipid bilayers, we show that although cholesterol did not affect the unitary conductance of GIRK2, it significantly enhanced the frequency of channel openings. Last, combining computational and functional approaches, we identified two putative cholesterol-binding sites in the transmembrane domain of GIRK2. These findings establish that cholesterol plays a critical role in modulating GIRK activity in the brain. Because up-regulation of GIRK function can reduce neuronal activity, our findings may lead to novel approaches for prevention and therapy of cholesterol-driven neurodegenerative disease. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

Chloride channel function is linked to epithelium-dependent airway relaxation.

PubMed

Fortner, C N; Lorenz, J N; Paul, R J

2001-02-01

We previously reported that substance P (SP) and ATP evoke transient, epithelium-dependent relaxation of mouse tracheal smooth muscle. Since both SP and ATP are known to evoke transepithelial Cl- secretion across epithelial monolayers, we tested the hypothesis that epithelium-dependent relaxation of mouse trachea depends on Cl- channel function. In perfused mouse tracheas, the responses to SP and ATP were both inhibited by the Cl- channel inhibitors diphenylamine-2-carboxylate and 5-nitro-2-(3-phenylpropylamino)benzoate. Relaxation to ATP or SP was unaffected by 4,4'-dinitrostilbene-2,2'-disulfonic acid (DNDS), and relaxation to SP was unaffected by either DIDS or DNDS. Replacing Cl- in the buffer solutions with the impermeable anion gluconate on both sides of the trachea inhibited relaxation to SP or ATP. In contrast, increasing the gradient for Cl- secretion using Cl- free medium only in the tracheal lumen enhanced the relaxation to SP or ATP. We conclude that Cl- channel function is linked to receptor-mediated, epithelium-dependent relaxation. The finding that relaxation to SP was not blocked by DIDS suggested the involvement of a DIDS-insensitive Cl- channel, potentially the cystic fibrosis transmembrane conductance regulator (CFTR) Cl- channel. To test this hypothesis, we evaluated tracheas from CFTR-deficient mice and found that the peak relaxation to SP or ATP was not significantly different from those responses in wild-type littermates. This suggests that a DIDS-insensitive Cl- channel other than CFTR is active in the SP response. This work introduces a possible role for Cl- pathways in the modulation of airway smooth muscle function and may have implications for fundamental studies of airway function as well as therapeutic approaches to pulmonary disease.

The Caenorhabditis elegans Iodotyrosine Deiodinase Ortholog SUP-18 Functions through a Conserved Channel SC-Box to Regulate the Muscle Two-Pore Domain Potassium Channel SUP-9

PubMed Central

de la Cruz, Ignacio Perez; Ma, Long; Horvitz, H. Robert

2014-01-01

Loss-of-function mutations in the Caenorhabditis elegans gene sup-18 suppress the defects in muscle contraction conferred by a gain-of-function mutation in SUP-10, a presumptive regulatory subunit of the SUP-9 two-pore domain K+ channel associated with muscle membranes. We cloned sup-18 and found that it encodes the C. elegans ortholog of mammalian iodotyrosine deiodinase (IYD), an NADH oxidase/flavin reductase that functions in iodine recycling and is important for the biosynthesis of thyroid hormones that regulate metabolism. The FMN-binding site of mammalian IYD is conserved in SUP-18, which appears to require catalytic activity to function. Genetic analyses suggest that SUP-10 can function with SUP-18 to activate SUP-9 through a pathway that is independent of the presumptive SUP-9 regulatory subunit UNC-93. We identified a novel evolutionarily conserved serine-cysteine-rich region in the C-terminal cytoplasmic domain of SUP-9 required for its specific activation by SUP-10 and SUP-18 but not by UNC-93. Since two-pore domain K+ channels regulate the resting membrane potentials of numerous cell types, we suggest that the SUP-18 IYD regulates the activity of the SUP-9 channel using NADH as a coenzyme and thus couples the metabolic state of muscle cells to muscle membrane excitability. PMID:24586202

Mechanosensitive Piezo Channels in the Gastrointestinal Tract

PubMed Central

Alcaino, C.; Farrugia, G.; Beyder, A.

2017-01-01

Sensation of mechanical forces is critical for normal function of the gastrointestinal (GI) tract and abnormalities in mechanosensation are linked to GI pathologies. In the GI tract there are several mechanosensitive cell types—epithelial enterochromaffin cells, intrinsic and extrinsic enteric neurons, smooth muscle cells and interstitial cells of Cajal. These cells use mechanosensitive ion channels that respond to mechanical forces by altering transmembrane ionic currents in a process called mechanoelectrical coupling. Several mechanosensitive ionic conductances have been identified in the mechano-sensory GI cells, ranging from mechanosensitive voltage-gated sodium and calcium channels to the mechanogated ion channels, such as the two-pore domain potassium channels K2P (TREK-1) and nonselective cation channels from the transient receptor potential family. The recently discovered Piezo channels are increasingly recognized as significant contributors to cellular mechanosensitivity. Piezo1 and Piezo2 are nonselective cationic ion channels that are directly activated by mechanical forces and have well-defined biophysical and pharmacologic properties. The role of Piezo channels in the GI epithelium is currently under investigation and their role in the smooth muscle syncytium and enteric neurons is still not known. In this review, we outline the current state of knowledge on mechanosensitive ion channels in the GI tract, with a focus on the known and potential functions of the Piezo channels. PMID:28728818

Distribution and function of voltage-gated sodium channels in the nervous system.

PubMed

Wang, Jun; Ou, Shao-Wu; Wang, Yun-Jie

2017-11-02

Voltage-gated sodium channels (VGSCs) are the basic ion channels for neuronal excitability, which are crucial for the resting potential and the generation and propagation of action potentials in neurons. To date, at least nine distinct sodium channel isoforms have been detected in the nervous system. Recent studies have identified that voltage-gated sodium channels not only play an essential role in the normal electrophysiological activities of neurons but also have a close relationship with neurological diseases. In this study, the latest research findings regarding the structure, type, distribution, and function of VGSCs in the nervous system and their relationship to neurological diseases, such as epilepsy, neuropathic pain, brain tumors, neural trauma, and multiple sclerosis, are reviewed in detail.

A complex valued radial basis function network for equalization of fast time varying channels.

PubMed

Gan, Q; Saratchandran, P; Sundararajan, N; Subramanian, K R

1999-01-01

This paper presents a complex valued radial basis function (RBF) network for equalization of fast time varying channels. A new method for calculating the centers of the RBF network is given. The method allows fixing the number of RBF centers even as the equalizer order is increased so that a good performance is obtained by a high-order RBF equalizer with small number of centers. Simulations are performed on time varying channels using a Rayleigh fading channel model to compare the performance of our RBF with an adaptive maximum-likelihood sequence estimator (MLSE) consisting of a channel estimator and a MLSE implemented by the Viterbi algorithm. The results show that the RBF equalizer produces superior performance with less computational complexity.

8-channel prototype of SALT readout ASIC for Upstream Tracker in the upgraded LHCb experiment

NASA Astrophysics Data System (ADS)

Abellan Beteta, C.; Bugiel, S.; Dasgupta, R.; Firlej, M.; Fiutowski, T.; Idzik, M.; Kane, C.; Moron, J.; Swientek, K.; Wang, J.

2017-02-01

SALT is a new 128-channel readout ASIC for silicon strip detectors in the upgraded Upstream Tracker of the LHCb experiment. It will extract and digitise analogue signals from the sensor, perform digital processing and transmit serial output data. SALT is designed in CMOS 130 nm process and uses a novel architecture comprising of an analogue front-end and an ultra-low power (<0.5 mW) fast (40 MSps) sampling 6-bit ADC in each channel. An 8-channel prototype (SALT8), comprising all important functionalities was designed, fabricated and tested. A full 128-channel version was also submitted. The design and test results of the SALT8 prototype are presented showing its full functionality.

Impact Response Characteristics of Polymeric Materials

DTIC Science & Technology

1981-11-01

amplitude-frequency domain. In the language of signal communications an input signal given by some time dependence FAt) is introduced into a " channel ...fixed and not altered by the signal. The channel can be charac- terized by its own function H(t), called the transfer function. This concept can be...rcpresented schematically as follows: Input Signal - [ Channel ] -- Output Signal At) H(t) G(t) In our case the input signal is the impact event, the output

Caenorhabditis elegans TRPV Channels Function in a Modality-Specific Pathway to Regulate Response to Aberrant Sensory Signaling

PubMed Central

Ezak , Meredith J.; Hong , Elizabeth; Chaparro-Garcia , Angela; Ferkey , Denise M.

2010-01-01

Olfaction and some forms of taste (including bitter) are mediated by G protein-coupled signal transduction pathways. Olfactory and gustatory ligands bind to chemosensory G protein-coupled receptors (GPCRs) in specialized sensory cells to activate intracellular signal transduction cascades. G protein-coupled receptor kinases (GRKs) are negative regulators of signaling that specifically phosphorylate activated GPCRs to terminate signaling. Although loss of GRK function usually results in enhanced cellular signaling, Caenorhabditis elegans lacking GRK-2 function are not hypersensitive to chemosensory stimuli. Instead, grk-2 mutant animals do not chemotax toward attractive olfactory stimuli or avoid aversive tastes and smells. We show here that loss-of-function mutations in the transient receptor potential vanilloid (TRPV) channels OSM-9 and OCR-2 selectively restore grk-2 behavioral avoidance of bitter tastants, revealing modality-specific mechanisms for TRPV channel function in the regulation of C. elegans chemosensation. Additionally, a single amino acid point mutation in OCR-2 that disrupts TRPV channel-mediated gene expression, but does not decrease channel function in chemosensory primary signal transduction, also restores grk-2 bitter taste avoidance. Thus, loss of GRK-2 function may lead to changes in gene expression, via OSM-9/OCR-2, to selectively alter the levels of signaling components that transduce or regulate bitter taste responses. Our results suggest a novel mechanism and multiple modality-specific pathways that sensory cells employ in response to aberrant signal transduction. PMID:20176974

Nonlinear model for an optical read-only-memory disk readout channel based on an edge-spread function.

PubMed

Kobayashi, Seiji

2002-05-10

A point-spread function (PSF) is commonly used as a model of an optical disk readout channel. However, the model given by the PSF does not contain the quadratic distortion generated by the photo-detection process. We introduce a model for calculating an approximation of the quadratic component of a signal. We show that this model can be further simplified when a read-only-memory (ROM) disk is assumed. We introduce an edge-spread function by which a simple nonlinear model of an optical ROM disk readout channel is created.

Gompertz type dechanneling functions for protons in <1 0 0>, <1 1 0> and <1 1 1> Si crystal channels

NASA Astrophysics Data System (ADS)

Petrović, S.; Erić, M.; Kokkoris, M.; Nešković, N.

2007-03-01

In this work the energy dependences of the Gompertz type sigmoidal dechanneling function parameters for protons in <1 0 0>, <1 1 0> and <1 1 1> Si crystal channels is investigated theoretically. The proton energy range considered is between 1 and 10 MeV. The original dechanneling functions are generated using a realistic Monte Carlo computer simulation code. We show that the Gompertz type dechanneling function, having two parameters, lc and k, representing the dechanneling range and rate, respectively, approximate accurately the original dechanneling function. It is also shown that the energy dependences of parameters lc and k can be approximated by a linear function and a sum of two exponential functions, respectively. The results obtained can be used for accurate reproduction of experimental proton channeling spectra recorded in the backscattering geometry.

A linkage analysis toolkit for studying allosteric networks in ion channels

PubMed Central

2013-01-01

A thermodynamic approach to studying allosterically regulated ion channels such as the large-conductance voltage- and Ca2+-dependent (BK) channel is presented, drawing from principles originally introduced to describe linkage phenomena in hemoglobin. In this paper, linkage between a principal channel component and secondary elements is derived from a four-state thermodynamic cycle. One set of parallel legs in the cycle describes the “work function,” or the free energy required to activate the principal component. The second are “lever operations” activating linked elements. The experimental embodiment of this linkage cycle is a plot of work function versus secondary force, whose asymptotes are a function of the parameters (displacements and interaction energies) of an allosteric network. Two essential work functions play a role in evaluating data from voltage-clamp experiments. The first is the conductance Hill energy WH[g], which is a “local” work function for pore activation, and is defined as kT times the Hill transform of the conductance (G-V) curve. The second is the electrical capacitance energy WC[q], representing “global” gating charge displacement, and is equal to the product of total gating charge per channel times the first moment (VM) of normalized capacitance (slope of Q-V curve). Plots of WH[g] and WC[q] versus voltage and Ca2+ potential can be used to measure thermodynamic parameters in a model-independent fashion of the core gating constituents (pore, voltage-sensor, and Ca2+-binding domain) of BK channel. The method is easily generalized for use in studying other allosterically regulated ion channels. The feasibility of performing linkage analysis from patch-clamp data were explored by simulating gating and ionic currents of a 17-particle model BK channel in response to a slow voltage ramp, which yielded interaction energies deviating from their given values in the range of 1.3 to 7.2%. PMID:23250867

The Tension-sensitive Ion Transport Activity of MSL8 is Critical for its Function in Pollen Hydration and Germination.

PubMed

Hamilton, Eric S; Haswell, Elizabeth S

2017-07-01

All cells respond to osmotic challenges, including those imposed during normal growth and development. Mechanosensitive (MS) ion channels provide a conserved mechanism for regulating osmotic forces by conducting ions in response to increased membrane tension. We previously demonstrated that the MS ion channel MscS-Like 8 (MSL8) is required for pollen to survive multiple osmotic challenges that occur during the normal process of fertilization, and that it can inhibit pollen germination. However, it remained unclear whether these physiological functions required ion flux through a mechanically gated channel provided by MSL8. We introduced two point mutations into the predicted pore-lining domain of MSL8 that disrupted normal channel function in different ways. The Ile711Ser mutation increased the tension threshold of the MSL8 channel while leaving conductance unchanged, and the Phe720Leu mutation severely disrupted the MSL8 channel. Both of these mutations impaired the ability of MSL8 to preserve pollen viability during hydration and to maintain the integrity of the pollen tube when expressed at endogenous levels. When overexpressed in an msl8-4 null background, MSL8I711S could partially rescue loss-of-function phenotypes, while MSL8F720L could not. When overexpressed in the wild-type Ler background, MSL8I711S suppressed pollen germination, similar to wild-type MSL8. In contrast, MSL8F720L failed to suppress pollen germination and increased pollen bursting, thereby phenocopying the msl8-4 mutant. Thus, an intact MSL8 channel is required for normal pollen function during hydration and germination. These data establish MSL8 as the first plant MS channel to fulfill previously established criteria for assignment as a mechanotransducer. © The Author 2017. Published by Oxford University Press on behalf of Japanese Society of Plant Physiologists. All rights reserved. For permissions, please email: journals.permissions@oup.com.

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

PubMed

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

2015-07-01

Kcnt1 encoded sodium-activated potassium channels (Slack channels) are highly expressed throughout the brain where they modulate the firing patterns and general excitability of many types of neurons. Increasing evidence suggests that Slack channels may be important for higher brain functions such as cognition and normal intellectual development. In particular, recent findings have shown that human Slack mutations produce very severe intellectual disability and that Slack channels interact directly with the Fragile X mental retardation protein (FMRP), a protein that when missing or mutated results in Fragile X syndrome (FXS), the most common form of inherited intellectual disability and autism in humans. We have now analyzed a recently developed Kcnt1 null mouse model in several behavioral tasks to assess which aspects of memory and learning are dependent on Slack. We demonstrate that Slack deficiency results in mildly altered general locomotor activity, but normal working memory, reference memory, as well as cerebellar control of motor functions. In contrast, we find that Slack channels are required for cognitive flexibility, including reversal learning processes and the ability to adapt quickly to unfamiliar situations and environments. Our data reveal that hippocampal-dependent spatial learning capabilities require the proper function of Slack channels. © 2015 Bausch et al.; Published by Cold Spring Harbor Laboratory Press.

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

PubMed Central

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

2015-01-01

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

Unfolding of a Temperature-Sensitive Domain Controls Voltage-Gated Channel Activation.

PubMed

Arrigoni, Cristina; Rohaim, Ahmed; Shaya, David; Findeisen, Felix; Stein, Richard A; Nurva, Shailika Reddy; Mishra, Smriti; Mchaourab, Hassane S; Minor, Daniel L

2016-02-25

Voltage-gated ion channels (VGICs) are outfitted with diverse cytoplasmic domains that impact function. To examine how such elements may affect VGIC behavior, we addressed how the bacterial voltage-gated sodium channel (BacNa(V)) C-terminal cytoplasmic domain (CTD) affects function. Our studies show that the BacNa(V) CTD exerts a profound influence on gating through a temperature-dependent unfolding transition in a discrete cytoplasmic domain, the neck domain, proximal to the pore. Structural and functional studies establish that the BacNa(V) CTD comprises a bi-partite four-helix bundle that bears an unusual hydrophilic core whose integrity is central to the unfolding mechanism and that couples directly to the channel activation gate. Together, our findings define a general principle for how the widespread four-helix bundle cytoplasmic domain architecture can control VGIC responses, uncover a mechanism underlying the diverse BacNa(V) voltage dependencies, and demonstrate that a discrete domain can encode the temperature-dependent response of a channel. Copyright © 2016 Elsevier Inc. All rights reserved.

Unfolding of a temperature-sensitive domain controls voltage-gated channel activation

PubMed Central

Arrigoni, Cristina; Rohaim, Ahmed; Shaya, David; Findeisen, Felix; Stein, Richard A.; Nurva, Shailika Reddy; Mishra, Smriti; Mchaourab, Hassane S.; Minor, Daniel L.

2016-01-01

Voltage-gated ion channels (VGICs) are outfitted with diverse cytoplasmic domains that impact function. To examine how such elements may affect VGIC behavior, we addressed how the bacterial voltage-gated sodium channel (BacNaV) C-terminal cytoplasmic domain (CTD) affects function. Our studies show that the BacNaV CTD exerts a profound influence on gating through a temperature-dependent unfolding transition in a discrete cytoplasmic domain, the neck domain, proximal to the pore. Structural and functional studies establish that the BacNaV CTD comprises a bi-partite four-helix bundle that bears an unusual hydrophilic core whose integrity is central to the unfolding mechanism and that couples directly to the channel activation gate. Together, our findings define a general principle for how the widespread four-helix bundle cytoplasmic domain architecture can control VGIC responses, uncover a mechanism underlying the diverse BacNaV voltage dependencies, and demonstrate that a discrete domain can encode the temperature dependent response of a channel. PMID:26919429

Structural analyses of Ca2+/CaM interaction with NaV channel C-termini reveal mechanisms of calcium-dependent regulation

PubMed Central

Wang, Chaojian; Chung, Ben C.; Yan, Haidun; Wang, Hong-Gang; Lee, Seok-Yong; Pitt, Geoffrey S.

2014-01-01

Ca2+ regulates voltage-gated Na+ (NaV) channels and perturbed Ca2+ regulation of NaV function is associated with epilepsy syndromes, autism, and cardiac arrhythmias. Understanding the disease mechanisms, however, has been hindered by a lack of structural information and competing models for how Ca2+ affects NaV channel function. Here, we report the crystal structures of two ternary complexes of a human NaV cytosolic C-terminal domain (CTD), a fibroblast growth factor homologous factor, and Ca2+/calmodulin (Ca2+/CaM). These structures rule out direct binding of Ca2+ to the NaV CTD, and uncover new contacts between CaM and the NaV CTD. Probing these new contacts with biochemical and functional experiments allows us to propose a mechanism by which Ca2+ could regulate NaV channels. Further, our model provides hints towards understanding the molecular basis of the neurologic disorders and cardiac arrhythmias caused by NaV channel mutations. PMID:25232683

Engineering biosynthetic excitable tissues from unexcitable cells for electrophysiological and cell therapy studies

PubMed Central

Kirkton, Robert D.; Bursac, Nenad

2012-01-01

Patch-clamp recordings in single-cell expression systems have been traditionally used to study the function of ion channels. However, this experimental setting does not enable assessment of tissue-level function such as action potential (AP) conduction. Here we introduce a biosynthetic system that permits studies of both channel activity in single cells and electrical conduction in multicellular networks. We convert unexcitable somatic cells into an autonomous source of electrically excitable and conducting cells by stably expressing only three membrane channels. The specific roles that these expressed channels have on AP shape and conduction are revealed by different pharmacological and pacing protocols. Furthermore, we demonstrate that biosynthetic excitable cells and tissues can repair large conduction defects within primary 2- and 3-dimensional cardiac cell cultures. This approach enables novel studies of ion channel function in a reproducible tissue-level setting and may stimulate the development of new cell-based therapies for excitable tissue repair. PMID:21556054

Connexin 43 and ATP-sensitive potassium channels crosstalk: a missing link in hypoxia/ischemia stress.

PubMed

Ahmad Waza, Ajaz; Ahmad Bhat, Shabir; Ul Hussain, Mahboob; Ganai, Bashir A

2018-02-01

Connexin 43 (Cx43) is a gap junction protein expressed in various tissues and organs of vertebrates. Besides functioning as a gap junction, Cx43 also regulates diverse cellular processes like cell growth and differentiation, cell migration, cell survival, etc. Cx43 is critical for normal cardiac functioning and is therefore abundantly expressed in cardiomyocytes. On the other hand, ATP-sensitive potassium (K ATP ) channels are metabolic sensors converting metabolic changes into electrical activity. These channels are important in maintaining the neurotransmitter release, smooth muscle relaxation, cardiac action potential repolarization, normal physiology of cellular repolarization, insulin secretion and immune function. Cx43 and K ATP channels are part of the same signaling pathway, regulating cell survival during stress conditions and ischemia/hypoxia preconditioning. However, the underlying molecular mechanism for their combined role in ischemia/hypoxia preconditioning is largely unknown. The current review focuses on understanding the molecular mechanism responsible for the coordinated role of Cx43 and K ATP channel protein in protecting cardiomyocytes against ischemia/hypoxia stress.

[Functional electrical stimulation based on a working pattern influences function of lower extremity in subjects with early stroke and effects on diffusion tensor imaging: a randomized controlled trial].

PubMed

Chen, Danfeng; Yan, Tiebin; Li, Guandong; Li, Fangming; Liang, Qitang

2014-10-14

To explore the possible mechanisms for improving lower extremity motor function in patients with early stroke through combining magnetic resonance diffusion tensor imaging (DTI) technology and functional electrical stimulation (FES) based on human walking patterns. From August 2012 to September 2013, a total of 48 eligible patients were stratified according to age, gender, disease course, Brunnstrom staging and types of stroke. And the Minimize software was used to divided them randomly into four-channel FES group (n = 18), dual-channel FES group (n = 15) and comfort stimulation group (n = 15). For all three groups, general medication and standard rehabilitation were provided. Based on normal walking pattern design of FES treatment, four-channel FES groups received the stimulations of quadriceps, hamstring, anterior tibialis and medial gastrocnemius. For the dual-channel FES group, the stimulations of tibialis anterior, peroneus longus and peroneus brevis muscles were applied. In comfort electrical stimulation group, the electrode positions were identical to the stimulation group, but there was no current output during stimulation. Before and after 3-week treatment, three groups received weekly rehabilitation evaluations of Fugl-Meyer assessment (FMA), posture assessment of stroke scale (PASS), Brunel balance assessment (BBA), Berg balance scale (BBS) and modified Barthel index (MBI). Before and after treatment, DTI examination was performed for some patients. Among three groups, general patient profiles and pre-treatment evaluations showed no significant difference. For intra-group comparisons versus pre-treatment, at week 1, 2 and 3, the scores of PASS, BBA, BBS, FMA and MBI had statistically significant differences (P < 0.05); At week 3 post-treatment, when four-channel and double-channel FES groups were compared versus pre-treatment, the scores of ipsilateral FA had statistically significant differences (P < 0.05). At week 1 post-treatment, MBI had statistically significant difference among 3 groups (P = 0.037). As compared with placebo, four-channel group had statistically significant difference [(52 ± 12) vs (38 ± 18), P < 0.05]; At week 2 post-treatment, the scores of PASS and MBI were (29 ± 3, 73 ± 13) in four-channel FES group versus (24 ± 8, 60 ± 17) in dual-channel FES group. And the scores of PASS, BBA, BBS, FMA and MBI were (9 ± 3, 8.3 ± 2.4, 37 ± 7, 22 ± 5, 73 ± 13) in four-channel FES group versus (21 ± 7, 6.2 ± 3.1, 24 ± 16, 15 ± 8, 47 ± 20) in comfort electrical stimulation group. When dual-channel FES and comfort stimulation groups were compared, MBI had significant statistical difference [(60 ± 17) vs (47 ± 20), P < 0.05]. At week 3 post-treatment, four-channel and dual-channel FES groups were compared, there was also statistical significance in FMA [(25 ± 5) vs (20 ± 7), P = 0.055]. The scores of PASS, BBS, FMA and MBI were (31 ± 3, 43 ± 8, 25 ± 5, 81 ± 13) in four-channel FES group versus (25 ± 8, 29 ± 17, 17 ± 9, 54 ± 25) in comfort stimulation group respectively. When dual-channel FES and comfort stimulation groups were compared, the scores of MBI were (71 ± 15) and (54 ± 25) respectively. And the difference was statistically significant (P < 0.05). At week 3 post-treatment, the scores of FA significantly increased [four-channel FES group (0.321 ± 0.172) vs comfort stimulation group (0.217 ± 0.135) (P = 0.020)]. When dual-channel FES group (0.333 ± 0.164) and comfort stimulation group (0.217 ± 0.135) (P = 0.049) were compared, the differences were statistically significant. DTI showed that four-channel FES group increased significantly, but contralateral fiber bundle was not obvious. And the improvements of dual-channel FES and comfort stimulation groups were insignificant. Compared with traditional dual-channel FES, functional electrical stimulation based on human walking patterns is more efficacious. And it helps to restore brain structure and function and promote motor function recovery in patients with early stroke.

Channel processes and watershed function

Treesearch

Tom Lisle

1999-01-01

Purpose of this presentation is to put channel monitoring in context of channel processes and dispel the myth of the learned sage walking up the stream channel observing changes in the channel and extrapolating how these changes came about without looking at the rest of the watershed. The message I want to convey is it is not only O.K.to peek at the rest of the...

Torque-Summing Brushless Motor

NASA Technical Reports Server (NTRS)

Vaidya, J. G.

1986-01-01

Torque channels function cooperatively but electrically independent for reliability. Brushless, electronically-commutated dc motor sums electromagnetic torques on four channels and applies them to single shaft. Motor operates with any combination of channels and continues if one or more of channels fail electrically. Motor employs single stator and rotor and mechanically simple; however, each of channels electrically isolated from other so that failure of one does not adversely affect others.

Transient Receptor Potential Mucolipin 1 (TRPML1) and Two-pore Channels Are Functionally Independent Organellar Ion Channels*

PubMed Central

Yamaguchi, Soichiro; Jha, Archana; Li, Qin; Soyombo, Abigail A.; Dickinson, George D.; Churamani, Dev; Brailoiu, Eugen; Patel, Sandip; Muallem, Shmuel

2011-01-01

NAADP is a potent second messenger that mobilizes Ca2+ from acidic organelles such as endosomes and lysosomes. The molecular basis for Ca2+ release by NAADP, however, is uncertain. TRP mucolipins (TRPMLs) and two-pore channels (TPCs) are Ca2+-permeable ion channels present within the endolysosomal system. Both have been proposed as targets for NAADP. In the present study, we probed possible physical and functional association of these ion channels. Exogenously expressed TRPML1 showed near complete colocalization with TPC2 and partial colocalization with TPC1. TRPML3 overlap with TPC2 was more modest. TRPML1 and to some extent TRPML3 co-immunoprecipitated with TPC2 but less so with TPC1. Current recording, however, showed that TPC1 and TPC2 did not affect the activity of wild-type TRPML1 or constitutively active TRPML1(V432P). N-terminally truncated TPC2 (TPC2delN), which is targeted to the plasma membrane, also failed to affect TRPML1 and TRPML1(V432P) channel function or TRPML1(V432P)-mediated Ca2+ influx. Whereas overexpression of TPCs enhanced NAADP-mediated Ca2+ signals, overexpression of TRPML1 did not, and the dominant negative TRPML1(D471K) was without affect on endogenous NAADP-mediated Ca2+ signals. Furthermore, the single channel properties of NAADP-activated TPC2delN were not affected by TRPML1. Finally, NAADP-evoked Ca2+ oscillations in pancreatic acinar cells were identical in wild-type and TRPML1−/− cells. We conclude that although TRPML1 and TPCs are present in the same complex, they function as two independent organellar ion channels and that TPCs, not TRPMLs, are the targets for NAADP. PMID:21540176

KV7 channels contribute to paracrine, but not metabolic or ischemic, regulation of coronary vascular reactivity in swine

PubMed Central

Goodwill, Adam G.; Fu, Lijuan; Noblet, Jillian N.; Casalini, Eli D.; Berwick, Zachary C.; Kassab, Ghassan S.; Tune, Johnathan D.

2016-01-01

Hydrogen peroxide (H2O2) and voltage-dependent K+ (KV) channels play key roles in regulating coronary blood flow in response to metabolic, ischemic, and paracrine stimuli. The KV channels responsible have not been identified, but KV7 channels are possible candidates. Existing data regarding KV7 channel function in the coronary circulation (limited to ex vivo assessments) are mixed. Thus we examined the hypothesis that KV7 channels are present in cells of the coronary vascular wall and regulate vasodilation in swine. We performed a variety of molecular, biochemical, and functional (in vivo and ex vivo) studies. Coronary arteries expressed KCNQ genes (quantitative PCR) and KV7.4 protein (Western blot). Immunostaining demonstrated KV7.4 expression in conduit and resistance vessels, perhaps most prominently in the endothelial and adventitial layers. Flupirtine, a KV7 opener, relaxed coronary artery rings, and this was attenuated by linopirdine, a KV7 blocker. Endothelial denudation inhibited the flupirtine-induced and linopirdine-sensitive relaxation of coronary artery rings. Moreover, linopirdine diminished bradykinin-induced endothelial-dependent relaxation of coronary artery rings. There was no effect of intracoronary flupirtine or linopirdine on coronary blood flow at the resting heart rate in vivo. Linopirdine had no effect on coronary vasodilation in vivo elicited by ischemia, H2O2, or tachycardia. However, bradykinin increased coronary blood flow in vivo, and this was attenuated by linopirdine. These data indicate that KV7 channels are expressed in some coronary cell type(s) and influence endothelial function. Other physiological functions of coronary vascular KV7 channels remain unclear, but they do appear to contribute to endothelium-dependent responses to paracrine stimuli. PMID:26825518

KV7 channels contribute to paracrine, but not metabolic or ischemic, regulation of coronary vascular reactivity in swine.

PubMed

Goodwill, Adam G; Fu, Lijuan; Noblet, Jillian N; Casalini, Eli D; Sassoon, Daniel; Berwick, Zachary C; Kassab, Ghassan S; Tune, Johnathan D; Dick, Gregory M

2016-03-15

Hydrogen peroxide (H2O2) and voltage-dependent K(+) (KV) channels play key roles in regulating coronary blood flow in response to metabolic, ischemic, and paracrine stimuli. The KV channels responsible have not been identified, but KV7 channels are possible candidates. Existing data regarding KV7 channel function in the coronary circulation (limited to ex vivo assessments) are mixed. Thus we examined the hypothesis that KV7 channels are present in cells of the coronary vascular wall and regulate vasodilation in swine. We performed a variety of molecular, biochemical, and functional (in vivo and ex vivo) studies. Coronary arteries expressed KCNQ genes (quantitative PCR) and KV7.4 protein (Western blot). Immunostaining demonstrated KV7.4 expression in conduit and resistance vessels, perhaps most prominently in the endothelial and adventitial layers. Flupirtine, a KV7 opener, relaxed coronary artery rings, and this was attenuated by linopirdine, a KV7 blocker. Endothelial denudation inhibited the flupirtine-induced and linopirdine-sensitive relaxation of coronary artery rings. Moreover, linopirdine diminished bradykinin-induced endothelial-dependent relaxation of coronary artery rings. There was no effect of intracoronary flupirtine or linopirdine on coronary blood flow at the resting heart rate in vivo. Linopirdine had no effect on coronary vasodilation in vivo elicited by ischemia, H2O2, or tachycardia. However, bradykinin increased coronary blood flow in vivo, and this was attenuated by linopirdine. These data indicate that KV7 channels are expressed in some coronary cell type(s) and influence endothelial function. Other physiological functions of coronary vascular KV7 channels remain unclear, but they do appear to contribute to endothelium-dependent responses to paracrine stimuli. Copyright © 2016 the American Physiological Society.

On the motion of substance in a channel of a network and human migration

NASA Astrophysics Data System (ADS)

Vitanov, Nikolay K.; Vitanov, Kaloyan N.

2018-01-01

We model the motion of a substance in a channel of a network that consists of chain of (i) nodes of the network and (ii) edges that connect the nodes and form the way for motion of the substance. The nodes of the channel can have different ;leakage;, i.e., some amount of the substance can leave the channel at a node and the rate of leaving can be different for the different nodes of the channel. The nodes close to the end of the channel for some (design or other) reason may be more ;attractive; for the substance in comparison to the nodes around the incoming node of the channel. We discuss channels containing infinite or finite number of nodes. The main outcome of the model is the distribution of the substance along the nodes. Two regimes of functioning of the channels are studied: stationary regime and non-stationary regime. The distribution of the substance along the nodes of the channel for the case of stationary regime is a distribution with a very long tail that contains as particular case the Waring distribution (for channel with infinite number of nodes) or the truncated Waring distribution (for channel with finite number of nodes). In the non-stationary regime of functioning of the channel one observes an exponential increase or exponential decrease of the amount of substance in the nodes. However the asymptotic distribution of the substance among the nodes of the channel in this regime remains stationary. The studied model is applied to the case of migration of humans through a migration channel consisting of chain of countries. In this case the model accounts for the number of migrants entering the channel through the first country of the channel; permeability of the borders between the countries; possible large attractiveness of some countries of the channel; possibility for migrants to obtain permission to reside in a country of the channel. The main outcome of the model is the distribution of migrants along the countries of the channel. We discuss the conditions for concentration of migrants in selected country of the channel. Finally two scenarios of changes of conditions of the functioning of the channel are discussed. It is shown that from the point of view of decreasing of the number of migrants in the countries of the channel it is more effective to concentrate efforts on preventing the entrance of migrants in the first country of the channel when compared to concentration of efforts on decrease of permeability of the borders between the countries of the channel.

The heart and potassium: a banana republic.

PubMed

Khan, Ehsan; Spiers, Christine; Khan, Maria

2013-03-01

The importance of potassium in maintaining stable cardiac function is a clinically understood phenomenon. Physiologically the importance of potassium in cardiac function is described by the large number of different kinds of potassium ions channels found in the heart compared to channels and membrane transport mechanisms for other ions such as sodium and calcium. Potassium is important in physiological homeostatic control of cardiac function, but is also of relevance to the diseased state, as potassium-related effects may stabilize or destabilize cardiac function. This article aims to provide a detailed understanding of potassium-mediated cardiac function. This will help the clinical practitioner evaluate how modulation of potassium ion channels by disease and pharmacological manipulation affect the cardiac patient, thus aiding in decision making when faced with clinical problems related to potassium.

Molecular Biology of Insect Sodium Channels and Pyrethroid Resistance

PubMed Central

Dong, Ke; Du, Yuzhe; Rinkevich, Frank; Nomura, Yoshiko; Xu, Peng; Wang, Lingxin; Silver, Kristopher; Zhorov, Boris S.

2015-01-01

Voltage-gated sodium channels are essential for the initiation and propagation of the action potential in neurons and other excitable cells. Because of their critical roles in electrical signaling, sodium channels are targets of a variety of naturally occurring and synthetic neurotoxins, including several classes of insecticides. This review is intended to provide an update on the molecular biology of insect sodium channels and the molecular mechanism of pyrethroid resistance. Although mammalian and insect sodium channels share fundamental topological and functional properties, most insect species carry only one sodium channel gene, compared to multiple sodium channel genes found in each mammalian species. Recent studies showed that two posttranscriptional mechanisms, alternative splicing and RNA editing, are involved in generating functional diversity of sodium channels in insects. More than 50 sodium channel mutations have been identified to be responsible for or associated with knockdown resistance (kdr) to pyrethroids in various arthropod pests and disease vectors. Elucidation of molecular mechanism of kdr led to the identification of dual receptor sites of pyrethroids on insect sodium channels. Most of the kdr mutations appear to be located within or close to the two receptor sites. The accumulating knowledge of insect sodium channels and their interactions with insecticides provides a foundation for understanding the neurophysiology of sodium channels in vivo and the development of new and safer insecticides for effective control of arthropod pests and human disease vectors. PMID:24704279

Functional expression of purinergic P2 receptors and transient receptor potential channels by the human urothelium.

PubMed

Shabir, Saqib; Cross, William; Kirkwood, Lisa A; Pearson, Joanna F; Appleby, Peter A; Walker, Dawn; Eardley, Ian; Southgate, Jennifer

2013-08-01

In addition to its role as a physical barrier, the urothelium is considered to play an active role in mechanosensation. A key mechanism is the release of transient mediators that activate purinergic P2 receptors and transient receptor potential (TRP) channels to effect changes in intracellular Ca²⁺. Despite the implied importance of these receptors and channels in urothelial tissue homeostasis and dysfunctional bladder disease, little is known about their functional expression by the human urothelium. To evaluate the expression and function of P2X and P2Y receptors and TRP channels, the human ureter and bladder were used to separate urothelial and stromal tissues for RNA isolation and cell culture. RT-PCR using stringently designed primer sets was used to establish which P2 and TRP species were expressed at the transcript level, and selective agonists/antagonists were used to confirm functional expression by monitoring changes in intracellular Ca²⁺ and in a scratch repair assay. The results confirmed the functional expression of P2Y₄ receptors and excluded nonexpressed receptors/channels (P2X₁, P2X₃, P2X₆, P2Y₆, P2Y₁₁, TRPV5, and TRPM8), while a dearth of specific agonists confounded the functional validation of expressed P2X₂, P2X₄, P2Y₁, P2Y₂, TRPV2, TRPV3, TRPV6 and TRPM7 receptors/channels. Although a conventional response was elicited in control stromal-derived cells, the urothelial cell response to well-characterized TRPV1 and TRPV4 agonists/antagonists revealed unexpected anomalies. In addition, agonists that invoked an increase in intracellular Ca²⁺ promoted urothelial scratch repair, presumably through the release of ATP. The study raises important questions about the ligand selectivity of receptor/channel targets expressed by the urothelium. These pathways are important in urothelial tissue homeostasis, and this opens the possibility of selective drug targeting.

Functional expression of purinergic P2 receptors and transient receptor potential channels by the human urothelium

PubMed Central

Shabir, Saqib; Cross, William; Kirkwood, Lisa A.; Pearson, Joanna F.; Appleby, Peter A.; Walker, Dawn; Eardley, Ian

2013-01-01

In addition to its role as a physical barrier, the urothelium is considered to play an active role in mechanosensation. A key mechanism is the release of transient mediators that activate purinergic P2 receptors and transient receptor potential (TRP) channels to effect changes in intracellular Ca2+. Despite the implied importance of these receptors and channels in urothelial tissue homeostasis and dysfunctional bladder disease, little is known about their functional expression by the human urothelium. To evaluate the expression and function of P2X and P2Y receptors and TRP channels, the human ureter and bladder were used to separate urothelial and stromal tissues for RNA isolation and cell culture. RT-PCR using stringently designed primer sets was used to establish which P2 and TRP species were expressed at the transcript level, and selective agonists/antagonists were used to confirm functional expression by monitoring changes in intracellular Ca2+ and in a scratch repair assay. The results confirmed the functional expression of P2Y4 receptors and excluded nonexpressed receptors/channels (P2X1, P2X3, P2X6, P2Y6, P2Y11, TRPV5, and TRPM8), while a dearth of specific agonists confounded the functional validation of expressed P2X2, P2X4, P2Y1, P2Y2, TRPV2, TRPV3, TRPV6 and TRPM7 receptors/channels. Although a conventional response was elicited in control stromal-derived cells, the urothelial cell response to well-characterized TRPV1 and TRPV4 agonists/antagonists revealed unexpected anomalies. In addition, agonists that invoked an increase in intracellular Ca2+ promoted urothelial scratch repair, presumably through the release of ATP. The study raises important questions about the ligand selectivity of receptor/channel targets expressed by the urothelium. These pathways are important in urothelial tissue homeostasis, and this opens the possibility of selective drug targeting. PMID:23720349

[Cognitive Function and Calcium. Structures and functions of Ca2+-permeable channels].

PubMed

Kaneko, Shuji

2015-02-01

Calcium is essential for living organisms where the increase in intracellular Ca2+ concentration functions as a second messenger for many cellular processes including synaptic transmission and neural plasticity. The cytosolic concentration of Ca2+ is finely controlled by many Ca2+-permeable ion channels and transporters. The comprehensive view of their expression, function, and regulation will advance our understanding of neural and cognitive functions of Ca2+, which leads to the future drug discovery.

Down-regulation of T-type Cav3.2 channels by hyperpolarization-activated cyclic nucleotide-gated channel 1 (HCN1): Evidence of a signaling complex

PubMed Central

Fan, Jing; Gandini, Maria A.; Zhang, Fang-Xiong; Chen, Lina; Souza, Ivana A.; Zamponi, Gerald W.

2017-01-01

ABSTRACT Formation of complexes between ion channels is important for signal processing in the brain. Here we investigate the biochemical and biophysical interactions between HCN1 channels and Cav3.2 T-type channels. We found that HCN1 co-immunoprecipitated with Cav3.2 from lysates of either mouse brain or tsA-201 cells, with the HCN1 N-terminus associating with the Cav3.2 N-terminus. Cav3.2 channel activity appeared to be functionally regulated by HCN1. The expression of HCN1 induced a decrease in Cav3.2 Ba2+ influx (IBa2+) along with altered channel kinetics and a depolarizing shift in activation gating. However, a reciprocal regulation of HCN1 by Cav3.2 was not observed. This study highlights a regulatory role of HCN1 on Cav3.2 voltage-dependent properties, which are expected to affect physiologic functions such as synaptic transmission and cellular excitability. PMID:28467171

Investigation of Corner Effect and Identification of Tunneling Regimes in L-Shaped Tunnel Field-Effect-Transistor.

PubMed

Najam, Faraz; Yu, Yun Seop

2018-09-01

Corner-effect existing in L-shaped tunnel field-effect-transistor (LTFET) was investigated using numerical simulations and band diagram analysis. It was found that the corner-effect is caused by the convergence of electric field in the sharp source corner present in an LTFET, thereby increasing the electric field in the sharp source corner region. It was found that in the corner-effect region tunneling starts early, as a function of applied bias, as compared to the rest of the channel not affected by corner-effect. Further, different tunneling regimes as a function of applied bias were identified in the LTFET including source to channel and channel to channel tunneling regimes. Presence of different tunneling regimes in LTFET was analytically justified with a set of equations developed to model source to channel, and channel to channel tunneling currents. Drain-current-gate-voltage (Ids-Vgs) characteristics obtained from the equations is in reasonable qualitative agreement with numerical simulation.

Ras-Association Domain of Sorting Nexin 27 Is Critical for Regulating Expression of GIRK Potassium Channels

PubMed Central

Bodhinathan, Karthik; Taura, Jaume J.; Taylor, Natalie M.; Nettleton, Margaret Y.; Ciruela, Francisco; Slesinger, Paul A.

2013-01-01

G protein-gated inwardly rectifying potassium (GIRK) channels play an important role in regulating neuronal excitability. Sorting nexin 27b (SNX27b), which reduces surface expression of GIRK channels through a PDZ domain interaction, contains a putative Ras-association (RA) domain with unknown function. Deleting the RA domain in SNX27b (SNX27b-ΔRA) prevents the down-regulation of GIRK2c/GIRK3 channels. Similarly, a point mutation (K305A) in the RA domain disrupts regulation of GIRK2c/GIRK3 channels and reduces H-Ras binding in vitro. Finally, the dominant-negative H-Ras (S17N) occludes the SNX27b-dependent decrease in surface expression of GIRK2c/GIRK3 channels. Thus, the presence of a functional RA domain and the interaction with Ras-like G proteins comprise a novel mechanism for modulating SNX27b control of GIRK channel surface expression and cellular excitability. PMID:23536889

Multi-channel orbicularis oculi stimulation to restore eye-blink function in facial paralysis.

PubMed

Somia, N N; Zonnevijlle, E D; Stremel, R W; Maldonado, C; Gossman, M D; Barker, J H

2001-01-01

Facial paralysis due to facial nerve injury results in the loss of function of the muscles of the hemiface. The most serious complication in extreme cases is the loss of vision. In this study, we compared the effectiveness of single- and multiple-channel electrical stimulation to restore a complete and cosmetically acceptable eye blink. We established bilateral orbicularis oculi muscle (OOM) paralysis in eight dogs; the OOM of one side was directly stimulated using single-channel electrical stimulation and the opposite side was stimulated using multi-channel electrical stimulation. The changes in the palpebral fissure and complete palpebral closure were measured. The difference in current intensities between the multi-channel and single-channel simulation groups was significant, while only multi-channel stimulation produced complete eyelid closure. The latest electronic stimulation circuitry with high-quality implantable electrodes will make it possible to regulate precisely OOM contractions and thus generate complete and cosmetically acceptable eye-blink motion in patients with facial paralysis. Copyright 2001 Wiley-Liss, Inc.

Functional interactions at the interface between voltage-sensing and pore domains in the Shaker K(v) channel.

PubMed

Soler-Llavina, Gilberto J; Chang, Tsg-Hui; Swartz, Kenton J

2006-11-22

Voltage-activated potassium (K(v)) channels contain a central pore domain that is partially surrounded by four voltage-sensing domains. Recent X-ray structures suggest that the two domains lack extensive protein-protein contacts within presumed transmembrane regions, but whether this is the case for functional channels embedded in lipid membranes remains to be tested. We investigated domain interactions in the Shaker K(v) channel by systematically mutating the pore domain and assessing tolerance by examining channel maturation, S4 gating charge movement, and channel opening. When mapped onto the X-ray structure of the K(v)1.2 channel the large number of permissive mutations support the notion of relatively independent domains, consistent with crystallographic studies. Inspection of the maps also identifies portions of the interface where residues are sensitive to mutation, an external cluster where mutations hinder voltage sensor activation, and an internal cluster where domain interactions between S4 and S5 helices from adjacent subunits appear crucial for the concerted opening transition.

MOLE 2.0: advanced approach for analysis of biomacromolecular channels

PubMed Central

2013-01-01

Background Channels and pores in biomacromolecules (proteins, nucleic acids and their complexes) play significant biological roles, e.g., in molecular recognition and enzyme substrate specificity. Results We present an advanced software tool entitled MOLE 2.0, which has been designed to analyze molecular channels and pores. Benchmark tests against other available software tools showed that MOLE 2.0 is by comparison quicker, more robust and more versatile. As a new feature, MOLE 2.0 estimates physicochemical properties of the identified channels, i.e., hydropathy, hydrophobicity, polarity, charge, and mutability. We also assessed the variability in physicochemical properties of eighty X-ray structures of two members of the cytochrome P450 superfamily. Conclusion Estimated physicochemical properties of the identified channels in the selected biomacromolecules corresponded well with the known functions of the respective channels. Thus, the predicted physicochemical properties may provide useful information about the potential functions of identified channels. The MOLE 2.0 software is available at http://mole.chemi.muni.cz. PMID:23953065

Asparagine 175 of connexin32 is a critical residue for docking and forming functional heterotypic gap junction channels with connexin26.

PubMed

Nakagawa, So; Gong, Xiang-Qun; Maeda, Shoji; Dong, Yuhua; Misumi, Yuko; Tsukihara, Tomitake; Bai, Donglin

2011-06-03

The gap junction channel is formed by proper docking of two hemichannels. Depending on the connexin(s) in the hemichannels, homotypic and heterotypic gap junction channels can be formed. Previous studies suggest that the extracellular loop 2 (E2) is an important molecular domain for heterotypic compatibility. Based on the crystal structure of the Cx26 gap junction channel and homology models of heterotypic channels, we analyzed docking selectivity for several hemichannel pairs and found that the hydrogen bonds between E2 domains are conserved in a group of heterotypically compatible hemichannels, including Cx26 and Cx32 hemichannels. According to our model analysis, Cx32N175Y mutant destroys three hydrogen bonds in the E2-E2 interactions due to steric hindrance at the heterotypic docking interface, which makes it unlikely to dock with the Cx26 hemichannel properly. Our experimental data showed that Cx26-red fluorescent protein (RFP) and Cx32-GFP were able to traffic to cell-cell interfaces forming gap junction plaques and functional channels in transfected HeLa/N2A cells. However, Cx32N175Y-GFP exhibited mostly intracellular distribution and was occasionally observed in cell-cell junctions. Double patch clamp analysis demonstrated that Cx32N175Y did not form functional homotypic channels, and dye uptake assay indicated that Cx32N175Y could form hemichannels on the cell surface similar to wild-type Cx32. When Cx32N175Y-GFP- and Cx26-RFP-transfected cells were co-cultured, no colocalization was found at the cell-cell junctions between Cx32N175Y-GFP- and Cx26-RFP-expressing cells; also, no functional Cx32N175Y-GFP/Cx26-RFP heterotypic channels were identified. Both our modeling and experimental data suggest that Asn(175) of Cx32 is a critical residue for heterotypic docking and functional gap junction channel formation between the Cx32 and Cx26 hemichannels.

Language Planning, Channel Management, and ESP.

ERIC Educational Resources Information Center

Kennedy, Chris

Channel management, a concept developed in marketing to refer to the process by which a product is moved from production to consumption, uses a channel of distribution operating at several levels, each responsible for one or more of the activities of moving the product forward to the consumer. The function of channel management is to select the…

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

ERIC Educational Resources Information Center

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

2015-01-01

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

Molecular mechanisms of subtype-specific inhibition of neuronal T-type calcium channels by ascorbate.

PubMed

Nelson, Michael T; Joksovic, Pavle M; Su, Peihan; Kang, Ho-Won; Van Deusen, Amy; Baumgart, Joel P; David, Laurence S; Snutch, Terrance P; Barrett, Paula Q; Lee, Jung-Ha; Zorumski, Charles F; Perez-Reyes, Edward; Todorovic, Slobodan M

2007-11-14

T-type Ca2+ channels (T-channels) are involved in the control of neuronal excitability and their gating can be modulated by a variety of redox agents. Ascorbate is an endogenous redox agent that can function as both an anti- and pro-oxidant. Here, we show that ascorbate selectively inhibits native Ca(v)3.2 T-channels in peripheral and central neurons, as well as recombinant Ca(v)3.2 channels heterologously expressed in human embryonic kidney 293 cells, by initiating the metal-catalyzed oxidation of a specific, metal-binding histidine residue in domain 1 of the channel. Our biophysical experiments indicate that ascorbate reduces the availability of Ca(v)3.2 channels over a wide range of membrane potentials, and inhibits Ca(v)3.2-dependent low-threshold-Ca2+ spikes as well as burst-firing in reticular thalamic neurons at physiologically relevant concentrations. This study represents the first mechanistic demonstration of ion channel modulation by ascorbate, and suggests that ascorbate may function as an endogenous modulator of neuronal excitability.

Voltage-dependent K+ channel gating and voltage sensor toxin sensitivity depend on the mechanical state of the lipid membrane.

PubMed

Schmidt, Daniel; MacKinnon, Roderick

2008-12-09

Voltage-dependent K(+) (Kv) channels underlie action potentials through gating conformational changes that are driven by membrane voltage. In this study of the paddle chimera Kv channel, we demonstrate that the rate of channel opening, the voltage dependence of the open probability, and the maximum achievable open probability depend on the lipid membrane environment. The activity of the voltage sensor toxin VsTx1, which interferes with voltage-dependent gating by partitioning into the membrane and binding to the channel, also depends on the membrane. Membrane environmental factors that influence channel function are divisible into two general categories: lipid compositional and mechanical state. The mechanical state can have a surprisingly large effect on the function of a voltage-dependent K(+) channel, including its pharmacological interaction with voltage sensor toxins. The dependence of VSTx1 activity on the mechanical state of the membrane leads us to hypothesize that voltage sensor toxins exert their effect by perturbing the interaction forces that exist between the channel and the membrane.

Voltage-dependent K+ channel gating and voltage sensor toxin sensitivity depend on the mechanical state of the lipid membrane

PubMed Central

Schmidt, Daniel; MacKinnon, Roderick

2008-01-01

Voltage-dependent K+ (Kv) channels underlie action potentials through gating conformational changes that are driven by membrane voltage. In this study of the paddle chimera Kv channel, we demonstrate that the rate of channel opening, the voltage dependence of the open probability, and the maximum achievable open probability depend on the lipid membrane environment. The activity of the voltage sensor toxin VsTx1, which interferes with voltage-dependent gating by partitioning into the membrane and binding to the channel, also depends on the membrane. Membrane environmental factors that influence channel function are divisible into two general categories: lipid compositional and mechanical state. The mechanical state can have a surprisingly large effect on the function of a voltage-dependent K+ channel, including its pharmacological interaction with voltage sensor toxins. The dependence of VSTx1 activity on the mechanical state of the membrane leads us to hypothesize that voltage sensor toxins exert their effect by perturbing the interaction forces that exist between the channel and the membrane. PMID:19050073

Ca2+-dependent phospholipid scrambling by a reconstituted TMEM16 ion channel.

PubMed

Malvezzi, Mattia; Chalat, Madhavan; Janjusevic, Radmila; Picollo, Alessandra; Terashima, Hiroyuki; Menon, Anant K; Accardi, Alessio

2013-01-01

Phospholipid (PL) scramblases disrupt the lipid asymmetry of the plasma membrane, externalizing phosphatidylserine to trigger blood coagulation and mark apoptotic cells. Recently, members of the TMEM16 family of Ca(2+)-gated channels have been shown to be involved in Ca(2+)-dependent scrambling. It is however controversial whether they are scramblases or channels regulating scrambling. Here we show that purified afTMEM16, from Aspergillus fumigatus, is a dual-function protein: it is a Ca(2+)-gated channel, with characteristics of other TMEM16 homologues, and a Ca(2+)-dependent scramblase, with the expected properties of mammalian PL scramblases. Remarkably, we find that a single Ca(2+) site regulates separate transmembrane pathways for ions and lipids. Two other purified TMEM16-channel homologues do not mediate scrambling, suggesting that the family diverged into channels and channel/scramblases. We propose that the spatial separation of the ion and lipid pathways underlies the evolutionary divergence of the TMEM16 family, and that other homologues, such as TMEM16F, might also be dual-function channel/scramblases.

Structural basis for the mutual antagonism of cAMP and TRIP8b in regulating HCN channel function

PubMed Central

Saponaro, Andrea; Pauleta, Sofia R.; Cantini, Francesca; Matzapetakis, Manolis; Hammann, Christian; Donadoni, Chiara; Hu, Lei; Thiel, Gerhard; Banci, Lucia; Santoro, Bina; Moroni, Anna

2014-01-01

cAMP signaling in the brain mediates several higher order neural processes. Hyperpolarization-activated cyclic nucleotide-gated (HCN) channels directly bind cAMP through their cytoplasmic cyclic nucleotide binding domain (CNBD), thus playing a unique role in brain function. Neuronal HCN channels are also regulated by tetratricopeptide repeat-containing Rab8b interacting protein (TRIP8b), an auxiliary subunit that antagonizes the effects of cAMP by interacting with the channel CNBD. To unravel the molecular mechanisms underlying the dual regulation of HCN channel activity by cAMP/TRIP8b, we determined the NMR solution structure of the HCN2 channel CNBD in the cAMP-free form and mapped on it the TRIP8b interaction site. We reconstruct here the full conformational changes induced by cAMP binding to the HCN channel CNBD. Our results show that TRIP8b does not compete with cAMP for the same binding region; rather, it exerts its inhibitory action through an allosteric mechanism, preventing the cAMP-induced conformational changes in the HCN channel CNBD. PMID:25197093

PERTURBATION OF VOLTAGE-SENSITIVE Ca2+ CHANNEL FUNCTION BY VOLATILE ORGANIC SOLVENTS.

EPA Science Inventory

The mechanisms underlying the acute neurophysiological and behavioral effects of volatile organic compounds (VOCs) remain to be elucidated. However, the function of neuronal ion channels is perturbed by VOCs. The present study examined effects of toluene (TOL), trichloroethylene ...

Voltage gated sodium channels as drug discovery targets

PubMed Central

Bagal, Sharan K; Marron, Brian E; Owen, Robert M; Storer, R Ian; Swain, Nigel A

2015-01-01

Voltage-gated sodium (NaV) channels are a family of transmembrane ion channel proteins. They function by forming a gated, water-filled pore to help establish and control cell membrane potential via control of the flow of ions between the intracellular and the extracellular environments. Blockade of NaVs has been successfully accomplished in the clinic to enable control of pathological firing patterns that occur in a diverse range of conditions such as chronic pain, epilepsy, and cardiac arrhythmias. First generation sodium channel modulator drugs, despite low inherent subtype selectivity, preferentially act on over-excited cells which reduces undesirable side effects in the clinic. However, the limited therapeutic indices observed with the first generation demanded a new generation of sodium channel inhibitors. The structure, function and the state of the art in sodium channel modulator drug discovery are discussed in this chapter. PMID:26646477

The voltage-gated proton channel: a riddle, wrapped in a mystery, inside an enigma

PubMed Central

DeCoursey, Thomas E.

2016-01-01

The main properties of voltage gated proton channels are described, along with what is known about how the channel protein structure accomplishes these functions. Just as protons are unique among ions, proton channels are unique among ion channels. Their four transmembrane helices sense voltage, the pH gradient, and conduct protons exclusively. Selectivity is achieved by the unique ability of H3O+ to protonate an Asp-Arg salt bridge. Pathognomonic sensitivity of gating to the pH gradient ensures channel opening only when acid extrusion will result, which is crucial to most biological functions. An exception occurs in dinoflagellates in which H+ influx through HV1 triggers the bioluminescent flash. Pharmacological interventions that promise to ameliorate cancer, asthma, brain damage in ischemic stroke, Alzheimer’s disease, autoimmune diseases, and numerous other conditions, await future progress. PMID:25964989

Structure of the CLC-1 chloride channel from Homo sapiens.

PubMed

Park, Eunyong; MacKinnon, Roderick

2018-05-29

CLC channels mediate passive Cl - conduction, while CLC transporters mediate active Cl - transport coupled to H + transport in the opposite direction. The distinction between CLC-0/1/2 channels and CLC transporters seems undetectable by amino acid sequence. To understand why they are different functionally we determined the structure of the human CLC-1 channel. Its 'glutamate gate' residue, known to mediate proton transfer in CLC transporters, adopts a location in the structure that appears to preclude it from its transport function. Furthermore, smaller side chains produce a wider pore near the intracellular surface, potentially reducing a kinetic barrier for Cl - conduction. When the corresponding residues are mutated in a transporter, it is converted to a channel. Finally, Cl - at key sites in the pore appear to interact with reduced affinity compared to transporters. Thus, subtle differences in glutamate gate conformation, internal pore diameter and Cl - affinity distinguish CLC channels and transporters. © 2018, Park & MacKinnon.

Voltage-gated sodium channels as targets for pyrethroid insecticides.

PubMed

Field, Linda M; Emyr Davies, T G; O'Reilly, Andrias O; Williamson, Martin S; Wallace, B A

2017-10-01

The pyrethroid insecticides are a very successful group of compounds that have been used extensively for the control of arthropod pests of agricultural crops and vectors of animal and human disease. Unfortunately, this has led to the development of resistance to the compounds in many species. The mode of action of pyrethroids is known to be via interactions with the voltage-gated sodium channel. Understanding how binding to the channel is affected by amino acid substitutions that give rise to resistance has helped to elucidate the mode of action of the compounds and the molecular basis of their selectivity for insects vs mammals and between insects and other arthropods. Modelling of the channel/pyrethroid interactions, coupled with the ability to express mutant channels in oocytes and study function, has led to knowledge of both how the channels function and potentially how to design novel insecticides with greater species selectivity.

Direct modulation of the outer mitochondrial membrane channel, voltage-dependent anion channel 1 (VDAC1) by cannabidiol: a novel mechanism for cannabinoid-induced cell death

PubMed Central

Rimmerman, N; Ben-Hail, D; Porat, Z; Juknat, A; Kozela, E; Daniels, M P; Connelly, P S; Leishman, E; Bradshaw, H B; Shoshan-Barmatz, V; Vogel, Z

2013-01-01

Cannabidiol (CBD) is a non-psychoactive plant cannabinoid that inhibits cell proliferation and induces cell death of cancer cells and activated immune cells. It is not an agonist of the classical CB1/CB2 cannabinoid receptors and the mechanism by which it functions is unknown. Here, we studied the effects of CBD on various mitochondrial functions in BV-2 microglial cells. Our findings indicate that CBD treatment leads to a biphasic increase in intracellular calcium levels and to changes in mitochondrial function and morphology leading to cell death. Density gradient fractionation analysis by mass spectrometry and western blotting showed colocalization of CBD with protein markers of mitochondria. Single-channel recordings of the outer-mitochondrial membrane protein, the voltage-dependent anion channel 1 (VDAC1) functioning in cell energy, metabolic homeostasis and apoptosis revealed that CBD markedly decreases channel conductance. Finally, using microscale thermophoresis, we showed a direct interaction between purified fluorescently labeled VDAC1 and CBD. Thus, VDAC1 seems to serve as a novel mitochondrial target for CBD. The inhibition of VDAC1 by CBD may be responsible for the immunosuppressive and anticancer effects of CBD. PMID:24309936

Solution structure and function of the "tandem inactivation domain" of the neuronal A-type potassium channel Kv1.4.

PubMed

Wissmann, Ralph; Bildl, Wolfgang; Oliver, Dominik; Beyermann, Michael; Kalbitzer, Hans-Robert; Bentrop, Detlef; Fakler, Bernd

2003-05-02

Cumulative inactivation of voltage-gated (Kv) K(+) channels shapes the presynaptic action potential and determines timing and strength of synaptic transmission. Kv1.4 channels exhibit rapid "ball-and-chain"-type inactivation gating. Different from all other Kvalpha subunits, Kv1.4 harbors two inactivation domains at its N terminus. Here we report the solution structure and function of this "tandem inactivation domain" using NMR spectroscopy and patch clamp recordings. Inactivation domain 1 (ID1, residues 1-38) consists of a flexible N terminus anchored at a 5-turn helix, whereas ID2 (residues 40-50) is a 2.5-turn helix made up of small hydrophobic amino acids. Functional analysis suggests that only ID1 may work as a pore-occluding ball domain, whereas ID2 most likely acts as a "docking domain" that attaches ID1 to the cytoplasmic face of the channel. Deletion of ID2 slows inactivation considerably and largely impairs cumulative inactivation. Together, the concerted action of ID1 and ID2 may promote rapid inactivation of Kv1.4 that is crucial for the channel function in short term plasticity.

The TRPM6 Kinase Domain Determines the Mg·ATP Sensitivity of TRPM7/M6 Heteromeric Ion Channels*

PubMed Central

Zhang, Zheng; Yu, Haijie; Huang, Junhao; Faouzi, Malika; Schmitz, Carsten; Penner, Reinhold; Fleig, Andrea

2014-01-01

The transient receptor potential melastatin member 7 (TRPM7) and member 6 (TRPM6) are divalent cation channel kinases essential for magnesium (Mg2+) homeostasis in vertebrates. It remains unclear how TRPM6 affects divalent cation transport and whether this involves functional homomeric TRPM6 plasma membrane channels or heteromeric channel assemblies with TRPM7. We show that homomeric TRPM6 is highly sensitive to intracellular free Mg2+ and therefore unlikely to be active at physiological levels of [Mg2+]i. Co-expression of TRPM7 and TRPM6 produces heteromeric TRPM7/M6 channels with altered pharmacology and sensitivity to intracellular Mg·ATP compared with homomeric TRPM7. Strikingly, the activity of heteromeric TRPM7/M6 channels is independent of intracellular Mg·ATP concentrations, essentially uncoupling channel activity from cellular energy status. Disruption of TRPM6 kinase phosphorylation activity re-introduces Mg·ATP sensitivity to the heteromeric channel similar to that of TRPM7. Thus, TRPM6 modulates the functionality of TRPM7, and the TRPM6 kinase plays a critical role in tuning the phenotype of the TRPM7·M6 channel complex. PMID:24385424

The TRPM6 kinase domain determines the Mg·ATP sensitivity of TRPM7/M6 heteromeric ion channels.

PubMed

Zhang, Zheng; Yu, Haijie; Huang, Junhao; Faouzi, Malika; Schmitz, Carsten; Penner, Reinhold; Fleig, Andrea

2014-02-21

The transient receptor potential melastatin member 7 (TRPM7) and member 6 (TRPM6) are divalent cation channel kinases essential for magnesium (Mg(2+)) homeostasis in vertebrates. It remains unclear how TRPM6 affects divalent cation transport and whether this involves functional homomeric TRPM6 plasma membrane channels or heteromeric channel assemblies with TRPM7. We show that homomeric TRPM6 is highly sensitive to intracellular free Mg(2+) and therefore unlikely to be active at physiological levels of [Mg(2+)]i. Co-expression of TRPM7 and TRPM6 produces heteromeric TRPM7/M6 channels with altered pharmacology and sensitivity to intracellular Mg·ATP compared with homomeric TRPM7. Strikingly, the activity of heteromeric TRPM7/M6 channels is independent of intracellular Mg·ATP concentrations, essentially uncoupling channel activity from cellular energy status. Disruption of TRPM6 kinase phosphorylation activity re-introduces Mg·ATP sensitivity to the heteromeric channel similar to that of TRPM7. Thus, TRPM6 modulates the functionality of TRPM7, and the TRPM6 kinase plays a critical role in tuning the phenotype of the TRPM7·M6 channel complex.

Role of voltage-gated K(+) channels in regulating Ca(2+) entry in rat cortical astrocytes.

PubMed

Wu, King-Chuen; Kuo, Chang-Shin; Chao, Chia-Chia; Huang, Chieh-Chen; Tu, Yuan-Kun; Chan, Paul; Leung, Yuk-Man

2015-03-01

Astrocytes have multiple functions such as provision of nourishment and mechanical support to the nervous system, helping to clear extracellular metabolites of neurons and modulating synaptic transmission by releasing gliotransmitters. In excitable cells, voltage-gated K(+) (Kv) channels serve to repolarize during action potentials. Astrocytes are considered non-excitable cells since they are not able to generate action potentials. There is an abundant expression of various Kv channels in astrocytes but the functions of these Kv channels remain unclear. We examined whether these astrocyte Kv channels regulate astrocyte "excitability" in the form of cytosolic Ca(2+) signaling. Electrophysiological examination revealed that neonatal rat cortical astrocytes possessed both delayed rectifier type and A-type Kv channels. Pharmacological blockade of both delayed rectifier Kv channels by TEA and A-type Kv channels by quinidine significantly suppressed store-operated Ca(2+) influx; however, TEA alone or quinidine alone did not suffice to cause such suppression. TEA and quinidine together dramatically enhanced current injection-triggered membrane potential overshoot (depolarization); either drug alone caused much smaller enhancements. Taken together, the results suggest both delayed rectifier and A-type Kv channels regulate astrocyte Ca(2+) signaling via controlling membrane potential.

Molecular and functional characterization of Anopheles gambiae inward rectifier potassium (Kir1) channels: a novel role in egg production.

PubMed

Raphemot, Rene; Estévez-Lao, Tania Y; Rouhier, Matthew F; Piermarini, Peter M; Denton, Jerod S; Hillyer, Julián F

2014-08-01

Inward rectifier potassium (Kir) channels play essential roles in regulating diverse physiological processes. Although Kir channels are encoded in mosquito genomes, their functions remain largely unknown. In this study, we identified the members of the Anopheles gambiae Kir gene family and began to investigate their function. Notably, we sequenced the A. gambiae Kir1 (AgKir1) gene and showed that it encodes all the canonical features of a Kir channel: an ion pore that is composed of a pore helix and a selectivity filter, two transmembrane domains that flank the ion pore, and the so-called G-loop. Heterologous expression of AgKir1 in Xenopus oocytes revealed that this gene encodes a functional, barium-sensitive Kir channel. Quantitative RT-PCR experiments then showed that relative AgKir1 mRNA levels are highest in the pupal stage, and that AgKir1 mRNA is enriched in the adult ovaries. Gene silencing of AgKir1 by RNA interference did not affect the survival of female mosquitoes following a blood meal, but decreased their egg output. These data provide evidence for a new role of Kir channels in mosquito fecundity, and further validates them as promising molecular targets for the development of a new class of mosquitocides to be used in vector control. Copyright © 2014 Elsevier Ltd. All rights reserved.

Structure-function of proteins interacting with the α1 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 (α1) associated with four additional polypeptide chains β, α2, δ, and γ, 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 α1-subunit, including calmodulin and multiple members of the small and large GTPase family. Some of these proteins only interact with a subset of α1-subunits and during specific stages of biogenesis. More strikingly, most of the α1-subunit interacting proteins, such as the β-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 α1-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

Differentiation dependent expression of TRPA1 and TRPM8 channels in IMR-32 human neuroblastoma cells.

PubMed

Louhivuori, Lauri M; Bart, Genevieve; Larsson, Kim P; Louhivuori, Verna; Näsman, Johnny; Nordström, Tommy; Koivisto, Ari-Pekka; Akerman, Karl E O

2009-10-01

TRPA1 and TRPM8 are transient receptor potential (TRP) channels involved in sensory perception. TRPA1 is a non-selective calcium permeable channel activated by irritants and proalgesic agents. TRPM8 reacts to chemical cooling agents such as menthol. The human neuroblastoma cell line IMR-32 undergoes a remarkable differentiation in response to treatment with 5-bromo-2-deoxyuridine. The cells acquire a neuronal morphology with increased expression of N-type voltage gated calcium channels and neurotransmitters. Here we show using RT-PCR, that mRNA for TRPA1 and TRPM8 are strongly upregulated in differentiating IMR-32 cells. Using whole cell patch clamp recordings, we demonstrate that activators of these channels, wasabi, allyl-isothiocyanate (AITC) and menthol activate membrane currents in differentiated cells. Calcium imaging experiments demonstrated that AITC mediated elevation of intracellular calcium levels were attenuated by ruthenium red, spermine, and HC-030031 as well as by siRNA directed against the channel. This indicates that the detected mRNA level correlate with the presence of functional channels of both types in the membrane of differentiated cells. Although the differentiated IMR-32 cells responded to cooling many of the cells showing this response did not respond to TRPA1/TRPM8 channel activators (60% and 90% for AITC and menthol respectively). Conversely many of the cells responding to these activators did not respond to cooling (30%). This suggests that these channels have also other functions than cold perception in these cells. Furthermore, our results suggest that IMR-32 cells have sensory characteristics and can be used to study native TRPA1 and TRPM8 channel function as well as developmental expression. Copyright 2009 Wiley-Liss, Inc.

Involvement of Parkin in the ubiquitin proteasome system-mediated degradation of N-type voltage-gated Ca2+ channels.

PubMed

Grimaldo, Lizbeth; Sandoval, Alejandro; Garza-López, Edgar; Felix, Ricardo

2017-01-01

N-type calcium (CaV2.2) channels are widely expressed in the brain and the peripheral nervous system, where they play important roles in the regulation of transmitter release. Although CaV2.2 channel expression levels are precisely regulated, presently little is known regarding the molecules that mediate its synthesis and degradation. Previously, by using a combination of biochemical and functional analyses, we showed that the complex formed by the light chain 1 of the microtubule-associated protein 1B (LC1-MAP1B) and the ubiquitin-proteasome system (UPS) E2 enzyme UBE2L3, may interact with the CaV2.2 channels promoting ubiquitin-mediated degradation. The present report aims to gain further insights into the possible mechanism of degradation of the neuronal CaV2.2 channel by the UPS. First, we identified the enzymes UBE3A and Parkin, members of the UPS E3 ubiquitin ligase family, as novel CaV2.2 channel binding partners, although evidence to support a direct protein-protein interaction is not yet available. Immunoprecipitation assays confirmed the interaction between UBE3A and Parkin with CaV2.2 channels heterologously expressed in HEK-293 cells and in neural tissues. Parkin, but not UBE3A, overexpression led to a reduced CaV2.2 protein level and decreased current density. Electrophysiological recordings performed in the presence of MG132 prevented the actions of Parkin suggesting enhanced channel proteasomal degradation. Together these results unveil a novel functional coupling between Parkin and the CaV2.2 channels and provide a novel insight into the basic mechanisms of CaV channels protein quality control and functional expression.

Physiology and Evolution of Voltage-Gated Calcium Channels in Early Diverging Animal Phyla: Cnidaria, Placozoa, Porifera and Ctenophora

PubMed Central

Senatore, Adriano; Raiss, Hamad; Le, Phuong

2016-01-01

Voltage-gated calcium (Cav) channels serve dual roles in the cell, where they can both depolarize the membrane potential for electrical excitability, and activate transient cytoplasmic Ca2+ signals. In animals, Cav channels play crucial roles including driving muscle contraction (excitation-contraction coupling), gene expression (excitation-transcription coupling), pre-synaptic and neuroendocrine exocytosis (excitation-secretion coupling), regulation of flagellar/ciliary beating, and regulation of cellular excitability, either directly or through modulation of other Ca2+-sensitive ion channels. In recent years, genome sequencing has provided significant insights into the molecular evolution of Cav channels. Furthermore, expanded gene datasets have permitted improved inference of the species phylogeny at the base of Metazoa, providing clearer insights into the evolution of complex animal traits which involve Cav channels, including the nervous system. For the various types of metazoan Cav channels, key properties that determine their cellular contribution include: Ion selectivity, pore gating, and, importantly, cytoplasmic protein-protein interactions that direct sub-cellular localization and functional complexing. It is unclear when these defining features, many of which are essential for nervous system function, evolved. In this review, we highlight some experimental observations that implicate Cav channels in the physiology and behavior of the most early-diverging animals from the phyla Cnidaria, Placozoa, Porifera, and Ctenophora. Given our limited understanding of the molecular biology of Cav channels in these basal animal lineages, we infer insights from better-studied vertebrate and invertebrate animals. We also highlight some apparently conserved cellular functions of Cav channels, which might have emerged very early on during metazoan evolution, or perhaps predated it. PMID:27867359

NS309 decreases rat detrusor smooth muscle membrane potential and phasic contractions by activating SK3 channels

PubMed Central

Parajuli, Shankar P; Hristov, Kiril L; Soder, Rupal P; Kellett, Whitney F; Petkov, Georgi V

2013-01-01

Background and Purpose Overactive bladder (OAB) is often associated with abnormally increased detrusor smooth muscle (DSM) contractions. We used NS309, a selective and potent opener of the small or intermediate conductance Ca2+-activated K+ (SK or IK, respectively) channels, to evaluate how SK/IK channel activation modulates DSM function. Experimental Approach We employed single-cell RT-PCR, immunocytochemistry, whole cell patch-clamp in freshly isolated rat DSM cells and isometric tension recordings of isolated DSM strips to explore how the pharmacological activation of SK/IK channels with NS309 modulates DSM function. Key Results We detected SK3 but not SK1, SK2 or IK channels expression at both mRNA and protein levels by RT-PCR and immunocytochemistry in DSM single cells. NS309 (10 μM) significantly increased the whole cell SK currents and hyperpolarized DSM cell resting membrane potential. The NS309 hyperpolarizing effect was blocked by apamin, a selective SK channel inhibitor. NS309 inhibited the spontaneous phasic contraction amplitude, force, frequency, duration and tone of isolated DSM strips in a concentration-dependent manner. The inhibitory effect of NS309 on spontaneous phasic contractions was blocked by apamin but not by TRAM-34, indicating no functional role of the IK channels in rat DSM. NS309 also significantly inhibited the pharmacologically and electrical field stimulation-induced DSM contractions. Conclusions and Implications Our data reveal that SK3 channel is the main SK/IK subtype in rat DSM. Pharmacological activation of SK3 channels with NS309 decreases rat DSM cell excitability and contractility, suggesting that SK3 channels might be potential therapeutic targets to control OAB associated with detrusor overactivity. PMID:23145946

Mechanosensitive Ca²⁺-permeable channels in human leukemic cells: pharmacological and molecular evidence for TRPV2.

PubMed

Pottosin, Igor; Delgado-Enciso, Iván; Bonales-Alatorre, Edgar; Nieto-Pescador, María G; Moreno-Galindo, Eloy G; Dobrovinskaya, Oxana

2015-01-01

Mechanosensitive channels are present in almost every living cell, yet the evidence for their functional presence in T lymphocytes is absent. In this study, by means of the patch-clamp technique in attached and inside-out modes, we have characterized cationic channels, rapidly activated by membrane stretch in Jurkat T lymphoblasts. The half-activation was achieved at a negative pressure of ~50mm Hg. In attached mode, single channel currents displayed an inward rectification and the unitary conductance of ~40 pS at zero command voltage. In excised inside-out patches the rectification was transformed to an outward one. Mechanosensitive channels weakly discriminated between mono- and divalent cations (PCa/PNa~1) and were equally permeable for Ca²⁺ and Mg²⁺. Pharmacological analysis showed that the mechanosensitive channels were potently blocked by amiloride (1mM) and Gd³⁺ (10 μM) in a voltage-dependent manner. They were also almost completely blocked by ruthenium red (1 μM) and SKF 96365 (250 μM), inhibitors of transient receptor potential vanilloid 2 (TRPV2) channels. At the same time, the channels were insensitive to 2-aminoethoxydiphenyl borate (2-APB, 100 μM) or N-(p-amylcinnamoyl)anthranilic acid (ACA, 50 μM), antagonists of transient receptor potential canonical (TRPC) or transient receptor potential melastatin (TRPM) channels, respectively. Human TRPV2 siRNA virtually abolished the stretch-activated current. TRPV2 are channels with multifaceted functions and regulatory mechanisms, with potentially important roles in the lymphocyte Ca²⁺ signaling. Implications of their regulation by mechanical stress are discussed in the context of lymphoid cells functions. Copyright © 2014 Elsevier B.V. All rights reserved.

[Molecular and functional diversity of ATP-sensitive K+ channels: the pathophysiological roles and potential drug targets].

PubMed

Nakaya, Haruaki; Miki, Takashi; Seino, Susumu; Yamada, Katsuya; Inagaki, Nobuya; Suzuki, Masashi; Sato, Toshiaki; Yamada, Mitsuhiko; Matsushita, Kenji; Kurachi, Yoshihisa; Arita, Makoto

2003-09-01

ATP-sensitive K(+) (K(ATP)) channels comprise the pore-forming subunit (Kir6.1 or Kir6.2) and the regulatory subunit sulfonylurea receptors (SUR1 or SUR2). K(ATP) channels with different combinations of these subunits are present in various tissues and regulate cellular functions. From the analysis of mouse models with targeted deletion of the gene encoding the pore-forming subunit Kir6.1 or Kir6.2, functional roles of K(ATP) channels in various organs have been clarified. Kir6.1(-/-) mice showed sudden death associated with ST elevation and atrioventricular block in ECG, a phenotype resembling Prinzmetal angina in humans. Kir6.2(-/-) mice were more susceptible to generalized seizure during hypoxia than wild-type (WT) mice, suggesting that neuronal K(ATP) channels, probably composed of Kir6.2 and SUR1, play a crucial role for the protection of the brain against lethal damage due to seizure. In Kir6.2(-/-) mice lacking the sarcolemmal K(ATP) channel activity in cardiac cells, ischemic preconditioning failed to reduce the infarct size, suggesting that sarcolemmal K(ATP) channels play an important role in cardioprotection against ischemia/reperfusion injuries in the heart. Mitochondrial K(ATP) channels have been also proposed to play a crucial role in cardioprotection, although the molecular identity of the channel has not been established. Nicorandil and minoxidil, K(+) channel openers activating mitochondrial K(ATP) channels, decreased the mitochondrial membrane potential, thereby preventing the Ca(2+) overload in the mitochondria of guinea-pig ventricular cells. SURs are the receptors for K(+) channel openers and the activating effects on sarcolemmal K(ATP) channels in cardiovascular tissues could be modulated by the interaction of nucleotides. Due to the molecular diversity of the accessory and pore subunits of K(ATP) channels, there would be considerable differences in the tissue selectivity of K(ATP) channel-acting drugs. Studies of Kir6.1 and Kir6.2 knockout mice indicate that K(ATP) channels are involved in the mechanisms of the protection against metabolic stress. Further clarification of physiological as well as pathophysiological roles of K(ATP) channels may lead to a new therapeutic strategy to improve the quality of life.

Dominant-negative suppression of big brain ion channel activity by mutation of a conserved glutamate in the first transmembrane domain.

PubMed

Yool, Andrea J

2007-01-01

The neurogenic protein Drosophila big brain (BIB), which is involved in the process of neuroblast determination, and the water channel aquaporin-1 (AQP1) are among a subset of the major intrinsic protein (MIP) channels that have been found to show gated monovalent cation channel activity. A glutamate residue in the first transmembrane (M1) domain is conserved throughout the MIP family. Mutation of this residue to asparagine in BIB (E71N) knocks out ion channel activity, and when coexpressed with BIB wild-type as shown here generates a dominant-negative effect on ion channel function, measured in the Xenopus oocyte expression system using two-electrode voltage clamp. cRNAs for wild-type and mutant BIB or AQP1 channels were injected individually or as mixtures. The magnitude of the BIB ionic conductance response was greatly reduced by coexpression of the mutant E71N subunit, suggesting a dominant-negative mechanism of action. The analogous mutation in AQP1 (E17N) did not impair ion channel activation by cGMP, but did knock out water channel function, although not via a dominant-negative effect. This contrast in sensitivity between BIB and AQP1 to mutation of the M1 glutamate suggests the possibility of interesting structural differences in the molecular basis of the ion permeation between these two classes of channels. The dominant-negative construct of BIB could be a tool for testing a role for BIB ion channels during nervous system development in Drosophila. The neurogenic protein Drosophila big brain (BIB), which is involved in the process of neuroblast determination, and the water channel aquaporin-1 (AQP1) are among a subset of the major intrinsic protein (MIP) channels that have been found to show gated monovalent cation channel activity. A glutamate residue in the first transmembrane (M1) domain is conserved throughout the MIP family. Mutation of this residue to asparagine in BIB (E71N) knocks out ion channel activity, and when coexpressed with BIB wild-type as shown here generates a dominant-negative effect on ion channel function, measured in the Xenopus oocyte expression system using two-electrode voltage clamp. cRNAs for wild-type and mutant BIB or AQP1 channels were injected individually or as mixtures. The magnitude of the BIB ionic conductance response was greatly reduced by coexpression of the mutant E71N subunit, suggesting a dominant-negative mechanism of action. The analogous mutation in AQP1 (E17N) did not impair ion channel activation by cGMP, but did knock out water channel function, although not via a dominant-negative effect. This contrast in sensitivity between BIB and AQP1 to mutation of the M1 glutamate suggests the possibility of interesting structural differences in the molecular basis of the ion permeation between these two classes of channels. The dominant-negative construct of BIB could be a tool for testing a role for BIB ion channels during nervous system development in Drosophila.

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

PubMed Central

Shipston, Michael J.

2014-01-01

Mechanisms that control surface expression and/or activity of large conductance calcium-activated potassium (BK) channels are important determinants of their (patho)physiological function. Indeed, BK channel dysfunction is associated with major human disorders ranging from epilepsy to hypertension and obesity. S-acylation (S-palmitoylation) represents a major reversible, post-translational modification controlling the properties and function of many proteins including ion channels. Recent evidence reveals that both pore-forming and regulatory subunits of BK channels are S-acylated and control channel trafficking and regulation by AGC-family protein kinases. The pore-forming α-subunit is S-acylated at two distinct sites within the N- and C-terminus, each site being regulated by different palmitoyl acyl transferases (zDHHCs) and acyl thioesterases (APTs). S-acylation of the N-terminus controls channel trafficking and surface expression whereas S-acylation of the C-terminal domain determines regulation of channel activity by AGC-family protein kinases. S-acylation of the regulatory β4-subunit controls ER exit and surface expression of BK channels but does not affect ion channel kinetics at the plasma membrane. Furthermore, a significant number of previously identified BK-channel interacting proteins have been shown, or are predicted to be, S-acylated. Thus, the BK channel multi-molecular signaling complex may be dynamically regulated by this fundamental post-translational modification and thus S-acylation likely represents an important determinant of BK channel physiology in health and disease. PMID:25140154

The stretch-dependent potassium channel TREK-1 and its function in murine myometrium

PubMed Central

Monaghan, Kevin; Baker, Salah A; Dwyer, Laura; Hatton, William C; Sik Park, Kyung; Sanders, Kenton M; Koh, Sang Don

2011-01-01

Smooth muscle of the uterus stays remarkably quiescent during normal pregnancy to allow sufficient time for development of the fetus. At present the mechanisms leading to uterine quiescence during pregnancy and how the suppression of activity is relieved at term are poorly understood. Myometrial excitability is governed by ion channels, and a major hypothesis regarding the regulation of contractility during pregnancy has been that expression of certain channels is regulated by hormonal influences. We have explored the expression and function of stretch-dependent K+ (SDK) channels, which are likely to be due to TREK channels, in murine myometrial tissues and myocytes using PCR, Western blots, patch clamp, intracellular microelectrode and isometric force measurements. TREK-1 is more highly expressed than TREK-2 in myometrium, and there was no detectable expression of TRAAK. Expression of TREK-1 transcripts and protein was regulated during pregnancy and delivery. SDK channels were activated in response to negative pressure applied to patches. SDK channels were insensitive to a broad-spectrum of K+ channel blockers, including tetraethylammonium and 4-aminopyridine, and insensitive to intracellular Ca2+. SDK channels were activated by stretch and arachidonic acid and inhibited by reagents that block TREK-1 channels, l-methionine and/or methioninol. Our data suggest that uterine excitability and contractility during pregnancy is regulated by the expression of SDK/TREK-1 channels. Up-regulation of these channels stabilizes membrane potential and controls contraction during pregnancy and down-regulation of these channels induces the onset of delivery. PMID:21224218

An ion channel library for drug discovery and safety screening on automated platforms.

PubMed

Wible, Barbara A; Kuryshev, Yuri A; Smith, Stephen S; Liu, Zhiqi; Brown, Arthur M

2008-12-01

Ion channels represent the third largest class of targets in drug discovery after G-protein coupled receptors and kinases. In spite of this ranking, ion channels continue to be under exploited as drug targets compared with the other two groups for several reasons. First, with 400 ion channel genes and an even greater number of functional channels due to mixing and matching of individual subunits, a systematic collection of ion channel-expressing cell lines for drug discovery and safety screening has not been available. Second, the lack of high-throughput functional assays for ion channels has limited their use as drug targets. Now that automated electrophysiology has come of age and provided the technology to assay ion channels at medium to high throughput, we have addressed the need for a library of ion channel cell lines by constructing the Ion Channel Panel (ChanTest Corp., Cleveland, OH). From 400 ion channel genes, a collection of 82 of the most relevant human ion channels for drug discovery, safety, and human disease has been assembled.Each channel has been stably overexpressed in human embryonic kidney 293 or Chinese hamster ovary cells. Cell lines have been selected and validated on automated electrophysiology systems to facilitate cost-effective screening for safe and selective compounds at earlier stages in the drug development process. The screening and validation processes as well as the relative advantages of different screening platforms are discussed.

Bupivacaine inhibits large conductance, voltage- and Ca2+- activated K+ channels in human umbilical artery smooth muscle cells

PubMed Central

Martín, Pedro; Enrique, Nicolás; Palomo, Ana R. Roldán; Rebolledo, Alejandro; Milesi, Veronica

2012-01-01

Bupivacaine is a local anesthetic compound belonging to the amino amide group. Its anesthetic effect is commonly related to its inhibitory effect on voltage-gated sodium channels. However, several studies have shown that this drug can also inhibit voltage-operated K+ channels by a different blocking mechanism. This could explain the observed contractile effects of bupivacaine on blood vessels. Up to now, there were no previous reports in the literature about bupivacaine effects on large conductance voltage- and Ca2+-activated K+ channels (BKCa). Using the patch-clamp technique, it is shown that bupivacaine inhibits single-channel and whole-cell K+ currents carried by BKCa channels in smooth muscle cells isolated from human umbilical artery (HUA). At the single-channel level bupivacaine produced, in a concentration- and voltage-dependent manner (IC50 324 µM at +80 mV), a reduction of single-channel current amplitude and induced a flickery mode of the open channel state. Bupivacaine (300 µM) can also block whole-cell K+ currents (~45% blockage) in which, under our working conditions, BKCa is the main component. This study presents a new inhibitory effect of bupivacaine on an ion channel involved in different cell functions. Hence, the inhibitory effect of bupivacaine on BKCa channel activity could affect different physiological functions where these channels are involved. Since bupivacaine is commonly used during labor and delivery, its effects on umbilical arteries, where this channel is highly expressed, should be taken into account. PMID:22688134

Inherited disorders of voltage-gated sodium channels

PubMed Central

George, Alfred L.

2005-01-01

A variety of inherited human disorders affecting skeletal muscle contraction, heart rhythm, and nervous system function have been traced to mutations in genes encoding voltage-gated sodium channels. Clinical severity among these conditions ranges from mild or even latent disease to life-threatening or incapacitating conditions. The sodium channelopathies were among the first recognized ion channel diseases and continue to attract widespread clinical and scientific interest. An expanding knowledge base has substantially advanced our understanding of structure-function and genotype-phenotype relationships for voltage-gated sodium channels and provided new insights into the pathophysiological basis for common diseases such as cardiac arrhythmias and epilepsy. PMID:16075039

Connexins and Pannexins in Vascular Function and Disease.

PubMed

Molica, Filippo; Figueroa, Xavier F; Kwak, Brenda R; Isakson, Brant E; Gibbins, Jonathan M

2018-06-05

Connexins (Cxs) and pannexins (Panxs) are ubiquitous membrane channel forming proteins that are critically involved in many aspects of vascular physiology and pathology. The permeation of ions and small metabolites through Panx channels, Cx hemichannels and gap junction channels confers a crucial role to these proteins in intercellular communication and in maintaining tissue homeostasis. This review provides an overview of current knowledge with respect to the pathophysiological role of these channels in large arteries, the microcirculation, veins, the lymphatic system and platelet function. The essential nature of these membrane proteins in vascular homeostasis is further emphasized by the pathologies that are linked to mutations and polymorphisms in Cx and Panx genes.

CFTR and lung homeostasis

PubMed Central

Matalon, Sadis

2014-01-01

CFTR is a cAMP-activated chloride and bicarbonate channel that is critical for lung homeostasis. Decreases in CFTR expression have dire consequences in cystic fibrosis (CF) and have been suggested to be a component of the lung pathology in chronic obstructive pulmonary disease. Decreases or loss of channel function often lead to mucus stasis, chronic bacterial infections, and the accompanying chronic inflammatory responses that promote progressive lung destruction, and, eventually in CF, lung failure. Here we discuss CFTR's functional role airway surface liquid hydration and pH, in regulation of other channels such as the epithelial sodium channel, and in regulating inflammatory responses in the lung. PMID:25381027

Zinc-dependent multi-conductance channel activity in mitochondria isolated from ischemic brain.

PubMed

Bonanni, Laura; Chachar, Mushtaque; Jover-Mengual, Teresa; Li, Hongmei; Jones, Adrienne; Yokota, Hidenori; Ofengeim, Dimitry; Flannery, Richard J; Miyawaki, Takahiro; Cho, Chang-Hoon; Polster, Brian M; Pypaert, Marc; Hardwick, J Marie; Sensi, Stefano L; Zukin, R Suzanne; Jonas, Elizabeth A

2006-06-21

Transient global ischemia is a neuronal insult that induces delayed cell death. A hallmark event in the early post-ischemic period is enhanced permeability of mitochondrial membranes. The precise mechanisms by which mitochondrial function is disrupted are, as yet, unclear. Here we show that global ischemia promotes alterations in mitochondrial membrane contact points, a rise in intramitochondrial Zn2+, and activation of large, multi-conductance channels in mitochondrial outer membranes by 1 h after insult. Mitochondrial channel activity was associated with enhanced protease activity and proteolytic cleavage of BCL-xL to generate its pro-death counterpart, deltaN-BCL-xL. The findings implicate deltaN-BCL-xL in large, multi-conductance channel activity. Consistent with this, large channel activity was mimicked by introduction of recombinant deltaN-BCL-xL to control mitochondria and blocked by introduction of a functional BCL-xL antibody to post-ischemic mitochondria via the patch pipette. Channel activity was also inhibited by nicotinamide adenine dinucleotide, indicative of a role for the voltage-dependent anion channel (VDAC) of the outer mitochondrial membrane. In vivo administration of the membrane-impermeant Zn2+ chelator CaEDTA before ischemia or in vitro application of the membrane-permeant Zn2+ chelator tetrakis-(2-pyridylmethyl) ethylenediamine attenuated channel activity, suggesting a requirement for Zn2+. These findings reveal a novel mechanism by which ischemic insults disrupt the functional integrity of the outer mitochondrial membrane and implicate deltaN-BCL-xL and VDAC in the large, Zn2+-dependent mitochondrial channels observed in post-ischemic hippocampal mitochondria.

Zinc-Dependent Multi-Conductance Channel Activity in Mitochondria Isolated from Ischemic Brain

PubMed Central

Bonanni, Laura; Chachar, Mushtaque; Jover-Mengual, Teresa; Li, Hongmei; Jones, Adrienne; Yokota, Hidenori; Ofengeim, Dimitry; Flannery, Richard J.; Miyawaki, Takahiro; Cho, Chang-Hoon; Polster, Brian M.; Pypaert, Marc; Hardwick, J. Marie; Sensi, Stefano L.; Zukin, R. Suzanne; Jonas, Elizabeth A.

2015-01-01

Transient global ischemia is a neuronal insult that induces delayed cell death. A hallmark event in the early post-ischemic period is enhanced permeability of mitochondrial membranes. The precise mechanisms by which mitochondrial function is disrupted are, as yet, unclear.Here we show that global ischemia promotes alterations in mitochondrial membrane contact points, a rise in intramitochondrial Zn2+, and activation of large, multi-conductance channels in mitochondrial outer membranes by 1 h after insult. Mitochondrial channel activity was associated with enhanced protease activity and proteolytic cleavage of BCL-xL to generate its pro-death counterpart, ΔN-BCL-xL. The findings implicate ΔN-BCL-xL in large, multi-conductance channel activity. Consistent with this, large channel activity was mimicked by introduction of recombinant ΔN-BCL-xL to control mitochondria and blocked by introduction of a functional BCL-xL antibody to post-ischemic mitochondria via the patch pipette. Channel activity was also inhibited by nicotinamide adenine dinucleotide, indicative of a role for the voltage-dependent anion channel (VDAC) of the outer mitochondrial membrane. In vivo administration of the membrane-impermeant Zn2+ chelator CaEDTA before ischemia or in vitro application of the membrane-permeant Zn2+ chelator tetrakis-(2-pyridylmethyl) ethylenediamine attenuated channel activity, suggesting a requirement for Zn2+. These findings reveal a novel mechanism by which ischemic insults disrupt the functional integrity of the outer mitochondrial membrane and implicate ΔNBCL-xL and VDAC in the large, Zn2+-dependent mitochondrial channels observed in post-ischemic hippocampal mitochondria. PMID:16793892

Transcriptomic analysis of the ion channelome of human platelets and megakaryocytic cell lines.

PubMed

Wright, Joy R; Amisten, Stefan; Goodall, Alison H; Mahaut-Smith, Martyn P

2016-08-01

Ion channels have crucial roles in all cell types and represent important therapeutic targets. Approximately 20 ion channels have been reported in human platelets; however, no systematic study has been undertaken to define the platelet channelome. These membrane proteins need only be expressed at low copy number to influence function and may not be detected using proteomic or transcriptomic microarray approaches. In our recent work, quantitative real-time PCR (qPCR) provided key evidence that Kv1.3 is responsible for the voltage-dependent K+ conductance of platelets and megakaryocytes. The present study has expanded this approach to assess relative expression of 402 ion channels and channel regulatory genes in human platelets and three megakaryoblastic/erythroleukaemic cell lines. mRNA levels in platelets are low compared to other blood cells, therefore an improved method of isolating platelets was developed. This used a cocktail of inhibitors to prevent formation of leukocyte-platelet aggregates, and a combination of positive and negative immunomagnetic cell separation, followed by rapid extraction of mRNA. Expression of 34 channel-related transcripts was quantified in platelets, including 24 with unknown roles in platelet function, but that were detected at levels comparable to ion channels with established roles in haemostasis or thrombosis. Trace expression of a further 50 ion channel genes was also detected. More extensive channelomes were detected in MEG-01, CHRF-288-11 and HEL cells (195, 185 and 197 transcripts, respectively), but lacked several channels observed in the platelet. These "channelome" datasets provide an important resource for further studies of ion channel function in the platelet and megakaryocyte.

The preference of tryptophan for membrane interfaces: insights from N-methylation of tryptophans in gramicidin channels.

PubMed

Sun, Haiyan; Greathouse, Denise V; Andersen, Olaf S; Koeppe, Roger E

2008-08-08

To better understand the structural and functional roles of tryptophan at the membrane/water interface in membrane proteins, we examined the structural and functional consequences of Trp --> 1-methyl-tryptophan substitutions in membrane-spanning gramicidin A channels. Gramicidin A channels are miniproteins that are anchored to the interface by four Trps near the C terminus of each subunit in a membrane-spanning dimer. We masked the hydrogen bonding ability of individual or multiple Trps by 1-methylation of the indole ring and examined the structural and functional changes using circular dichroism spectroscopy, size exclusion chromatography, solid state (2)H NMR spectroscopy, and single channel analysis. N-Methylation causes distinct changes in the subunit conformational preference, channel-forming propensity, single channel conductance and lifetime, and average indole ring orientations within the membrane-spanning channels. The extent of the local ring dynamic wobble does not increase, and may decrease slightly, when the indole NH is replaced by the non-hydrogen-bonding and more bulky and hydrophobic N-CH(3) group. The changes in conformational preference, which are associated with a shift in the distribution of the aromatic residues across the bilayer, are similar to those observed previously with Trp --> Phe substitutions. We conclude that indole N-H hydrogen bonding is of major importance for the folding of gramicidin channels. The changes in ion permeability, however, are quite different for Trp --> Phe and Trp --> 1-methyl-tryptophan substitutions, indicating that the indole dipole moment and perhaps also ring size and are important for ion permeation through these channels.

The Split Personality of Glutamate Transporters: A Chloride Channel and a Transporter.

PubMed

Cater, Rosemary J; Ryan, Renae M; Vandenberg, Robert J

2016-03-01

Transporters and ion channels are conventionally categorised into distinct classes of membrane proteins. However, some membrane proteins have a split personality and can function as both transporters and ion channels. The excitatory amino acid transporters (EAATs) in particular, function as both glutamate transporters and chloride (Cl(-)) channels. The EAATs couple the transport of glutamate to the co-transport of three Na(+) ions and one H(+) ion into the cell, and the counter-transport of one K(+) ion out of the cell. The EAAT Cl(-) channel is activated by the binding of glutamate and Na(+), but is thermodynamically uncoupled from glutamate transport and involves molecular determinants distinct from those responsible for glutamate transport. Several crystal structures of an EAAT archaeal homologue, GltPh, at different stages of the transport cycle, alongside numerous functional studies and molecular dynamics simulations, have provided extensive insights into the mechanism of substrate transport via these transporters. However, the molecular determinants involved in Cl(-) permeation, and the mechanism by which this channel is activated are not entirely understood. Here we will discuss what is currently known about the molecular determinants involved in EAAT-mediated Cl(-) permeation and the mechanisms that underlie their split personality.

Centrifugal sedimentation for selectively packing channels with silica microbeads in three-dimensional micro/nanofluidic devices.

PubMed

Gong, Maojun; Bohn, Paul W; Sweedler, Jonathan V

2009-03-01

Incorporation of nanofluidic elements into microfluidic channels is one approach for adding filtration and partition functionality to planar microfluidic devices, as well as providing enhanced biomolecular separations. Here we introduce a strategy to pack microfluidic channels with silica nanoparticles and microbeads, thereby indirectly producing functional nanostructures; the method allows selected channels to be packed, here demonstrated so that a separation channel is packed while keeping an injection channel unpacked. A nanocapillary array membrane is integrated between two patterned microfluidic channels that cross each other in vertically separated layers. The membrane serves both as a frit for bead packing and as a fluid communication conduit between microfluidic channels. Centrifugal force-assisted sedimentation is then used to selectively pack the microfluidic channels using an aqueous silica bead suspension loaded into the appropriate inlet reservoirs. This packing approach may be used to simultaneously pack multiple channels with silica microbeads having different sizes and surface properties. The chip design and packing method introduced here are suitable for packing silica particles in sizes ranging from nanometers to micrometers and allow rapid (approximately 10 min) packing with high quality. The liquid/analyte transport characteristics of these packed micro/nanofluidic devices have potential utility in a wide range of applications, including electroosmotic pumping, liquid chromatographic separations, and electrochromatography.

Mechanisms of pyrethroid insecticide-induced stimulation of calcium influx in neocortical neurons

EPA Science Inventory

Pyrethroid insecticides bind to voltage-gated sodium channels (VGSCs) and modify their gating kinetics, thereby disrupting neuronal function. Pyrethroids have also been reported to alter the function of other channel types, including activation of voltage-gated Ca2+ calcium chann...

Physical and functional interaction between integrins and hERG potassium channels.

PubMed

Arcangeli, A; Becchetti, A; Cherubini, A; Crociani, O; Defilippi, P; Guasti, L; Hofmann, G; Pillozzi, S; Olivotto, M; Wanke, E

2004-11-01

Integrins are adhesion receptors capable of transmitting intracellular signals that regulate many different cellular functions. Among integrin-mediated signals, the activation of ion channels can be included. We demonstrated that a long-lasting activation of hERG (human ether-a-go-go-related gene) potassium channels occurs in both human neuroblastoma and leukaemia cells after the activation of the beta1 integrin subunit. This activation is apparently a determining factor inducing neurite extension and osteoclastic differentiation in both the cell types. More recently, we provided evidences that beta1 integrins and hERG channels co-precipitate in both the cell types. Preliminary results suggest that a macromolecular signalling complex indeed occurs between integrins and the hERG1 protein and that hERG channel activity can modulate integrin downstream signalling.

Performance of mixed RF/FSO systems in exponentiated Weibull distributed channels

NASA Astrophysics Data System (ADS)

Zhao, Jing; Zhao, Shang-Hong; Zhao, Wei-Hu; Liu, Yun; Li, Xuan

2017-12-01

This paper presented the performances of asymmetric mixed radio frequency (RF)/free-space optical (FSO) system with the amplify-and-forward relaying scheme. The RF channel undergoes Nakagami- m channel, and the Exponentiated Weibull distribution is adopted for the FSO component. The mathematical formulas for cumulative distribution function (CDF), probability density function (PDF) and moment generating function (MGF) of equivalent signal-to-noise ratio (SNR) are achieved. According to the end-to-end statistical characteristics, the new analytical expressions of outage probability are obtained. Under various modulation techniques, we derive the average bit-error-rate (BER) based on the Meijer's G function. The evaluation and simulation are provided for the system performance, and the aperture average effect is discussed as well.

Ion Channel Genes and Epilepsy: Functional Alteration, Pathogenic Potential, and Mechanism of Epilepsy.

PubMed

Wei, Feng; Yan, Li-Min; Su, Tao; He, Na; Lin, Zhi-Jian; Wang, Jie; Shi, Yi-Wu; Yi, Yong-Hong; Liao, Wei-Ping

2017-08-01

Ion channels are crucial in the generation and modulation of excitability in the nervous system and have been implicated in human epilepsy. Forty-one epilepsy-associated ion channel genes and their mutations are systematically reviewed. In this paper, we analyzed the genotypes, functional alterations (funotypes), and phenotypes of these mutations. Eleven genes featured loss-of-function mutations and six had gain-of-function mutations. Nine genes displayed diversified funotypes, among which a distinct funotype-phenotype correlation was found in SCN1A. These data suggest that the funotype is an essential consideration in evaluating the pathogenicity of mutations and a distinct funotype or funotype-phenotype correlation helps to define the pathogenic potential of a gene.

Cholesterol regulates the endoplasmic reticulum exit of the major membrane protein P0 required for peripheral myelin compaction.

PubMed

Saher, Gesine; Quintes, Susanne; Möbius, Wiebke; Wehr, Michael C; Krämer-Albers, Eva-Maria; Brügger, Britta; Nave, Klaus-Armin

2009-05-13

Rapid impulse conduction requires electrical insulation of axons by myelin, a cholesterol-rich extension of the glial cell membrane with a characteristic composition of proteins and lipids. Mutations in several myelin protein genes cause endoplasmic reticulum (ER) retention and disease, presumably attributable to failure of misfolded proteins to pass the ER quality control. Because many myelin proteins partition into cholesterol-rich membrane rafts, their interaction with cholesterol could potentially be part of the ER quality control system. Here, we provide in vitro and in vivo evidence that the major peripheral myelin protein P0 requires cholesterol for exiting the ER and reaching the myelin compartment. Cholesterol dependency of P0 trafficking in heterologous cells is mediated by a cholesterol recognition/interaction amino acid consensus (CRAC) motif. Mutant mice lacking cholesterol biosynthesis in Schwann cells suffer from severe hypomyelination with numerous uncompacted myelin stretches. This demonstrates that high-level cholesterol coordinates P0 export with myelin membrane synthesis, which is required for the correct stoichiometry of myelin components and for myelin compaction.

The life and contribution of Dr. Ronald Gitelman: a pioneer of modern chiropractic science.

PubMed

Vernon, Howard

2013-03-01

The life and contribution to chiropractic science of Dr. Ronald Gitelman is reviewed. Sources for this article included review of the notes prepared by Dr. Joseph Keating in his "biography" of the Canadian Memorial Chiropractic College (CMCC); review of the important articles published by Dr. Gitelman; review of the important projects undertaken by him along with various colleagues; notes from reminiscences obtained from many of these colleagues and discussions with his family. Dr. Gitelman's academic career spanned from 1963 to the late 1980's. During that time, he made foundational contributions to the development of chiropractic science including: developing the Archives (1974), the first collection of scientific articles supporting chiropractic science (which was subsequently published as the Chiropractic Archives Research Collection (CRAC)); delivering one of the few chiropractic papers at the seminal NINCDS conference (1975) and, developing the collaboration between CMCC and Dr. Kirkaldy-Willis at the University of Saskatoon (1976). He practiced in Toronto from 1961 to 2007. Dr. Gitelman was a pioneer in the development of chiropractic science. He died on October 7, 2012.

PopAffiliator: online calculator for individual affiliation to a major population group based on 17 autosomal short tandem repeat genotype profile.

PubMed

Pereira, Luísa; Alshamali, Farida; Andreassen, Rune; Ballard, Ruth; Chantratita, Wasun; Cho, Nam Soo; Coudray, Clotilde; Dugoujon, Jean-Michel; Espinoza, Marta; González-Andrade, Fabricio; Hadi, Sibte; Immel, Uta-Dorothee; Marian, Catalin; Gonzalez-Martin, Antonio; Mertens, Gerhard; Parson, Walther; Perone, Carlos; Prieto, Lourdes; Takeshita, Haruo; Rangel Villalobos, Héctor; Zeng, Zhaoshu; Zhivotovsky, Lev; Camacho, Rui; Fonseca, Nuno A

2011-09-01

Because of their sensitivity and high level of discrimination, short tandem repeat (STR) maker systems are currently the method of choice in routine forensic casework and data banking, usually in multiplexes up to 15-17 loci. Constraints related to sample amount and quality, frequently encountered in forensic casework, will not allow to change this picture in the near future, notwithstanding the technological developments. In this study, we present a free online calculator named PopAffiliator ( http://cracs.fc.up.pt/popaffiliator ) for individual population affiliation in the three main population groups, Eurasian, East Asian and sub-Saharan African, based on genotype profiles for the common set of STRs used in forensics. This calculator performs affiliation based on a model constructed using machine learning techniques. The model was constructed using a data set of approximately fifteen thousand individuals collected for this work. The accuracy of individual population affiliation is approximately 86%, showing that the common set of STRs routinely used in forensics provide a considerable amount of information for population assignment, in addition to being excellent for individual identification.

Dystrophin Is Required for the Normal Function of the Cardio-Protective KATP Channel in Cardiomyocytes

PubMed Central

Graciotti, Laura; Becker, Jodi; Granata, Anna Luisa; Procopio, Antonio Domenico; Tessarollo, Lino; Fulgenzi, Gianluca

2011-01-01

Duchenne and Becker muscular dystrophy patients often develop a cardiomyopathy for which the pathogenesis is still unknown. We have employed the murine animal model of Duchenne muscular dystrophy (mdx), which develops a cardiomyopathy that includes some characteristics of the human disease, to study the molecular basis of this pathology. Here we show that the mdx mouse heart has defects consistent with alteration in compounds that regulate energy homeostasis including a marked decrease in creatine-phosphate (PC). In addition, the mdx heart is more susceptible to anoxia than controls. Since the cardio-protective ATP sensitive potassium channel (KATP) complex and PC have been shown to interact we investigated whether deficits in PC levels correlate with other molecular events including KATP ion channel complex presence, its functionality and interaction with dystrophin. We found that this channel complex is present in the dystrophic cardiac cell membrane but its ability to sense a drop in the intracellular ATP concentration and consequently open is compromised by the absence of dystrophin. We further demonstrate that the creatine kinase muscle isoform (CKm) is displaced from the plasma membrane of the mdx cardiac cells. Considering that CKm is a determinant of KATP channel complex function we hypothesize that dystrophin acts as a scaffolding protein organizing the KATP channel complex and the enzymes necessary for its correct functioning. Therefore, the lack of proper functioning of the cardio-protective KATP system in the mdx cardiomyocytes may be part of the mechanism contributing to development of cardiac disease in dystrophic patients. PMID:22066028

The regulation of transient receptor potential canonical 4 (TRPC4) channel by phosphodiesterase 5 inhibitor via the cyclic guanosine 3'5'-monophosphate.

PubMed

Wie, Jinhong; Jeong, SeungJoo; Kwak, Misun; Myeong, Jongyun; Chae, MeeRee; Park, Jong Kwan; Lee, Sung Won; So, Insuk

2017-06-01

The transient receptor potential (TRP) protein superfamily consists of a diverse group of cation channels that bear structural similarities to the fruit fly Drosophila TRP. The TRP superfamily is distinct from other groups of ion channels in displaying a large diversity in ion selectivity, modes of activation, and physiological functions. Classical TRP (transient receptor potential canonical (TRPC)) channels are activated by stimulation of Gq-PLC-coupled receptors and modulated by phosphorylation. The cyclic guanosine monophosphate (cGMP)-PKG pathway is involved in the regulation of TRPC3 and TRPC6 channels. Phosphodiesterase (PDE) 5 inhibitor induced muscle relaxation in corporal smooth muscle cells and was used to treat erectile dysfunction by inhibiting cGMP degradation. Here, we report the functional relationship between TRPC4 and cGMP. In human embryonic kidney (HEK) 293 cells overexpressing TRPC4, cGMP selectively activated TRPC4 channels and increased cytosolic calcium level through TRPC4 channel. We investigated phosphorylation sites in TRPC4 channels and identified S688 as an important phosphorylation site for the cGMP-PKG pathway. Cyclic GMP also activated TRPC4-like current with doubly rectifying current-voltage relationship in prostate smooth muscle cell lines. Taken together, these results show that TRPC4 is phosphorylated by the cGMP-PKG pathway and might be an important target for modulating prostate function by PDE5 inhibitors.

Electrophysiological channel interactions using focused multipolar stimulation for cochlear implants

NASA Astrophysics Data System (ADS)

George, Shefin S.; Shivdasani, Mohit N.; Wise, Andrew K.; Shepherd, Robert K.; Fallon, James B.

2015-12-01

Objective. Speech intelligibility with existing multichannel cochlear implants (CIs) is thought to be limited by poor spatial selectivity and interactions between CI channels caused by overlapping activation with monopolar (MP) stimulation. Our previous studies have shown that focused multipolar (FMP) and tripolar (TP) stimulation produce more restricted neural activation in the inferior colliculus (IC), compared to MP stimulation. Approach. This study explored interactions in the IC produced by simultaneous stimulation of two CI channels. We recorded multi-unit neural activity in the IC of anaesthetized cats with normal and severely degenerated spiral ganglion neuron populations in response to FMP, TP and MP stimulation from a 14 channel CI. Stimuli were applied to a ‘fixed’ CI channel, chosen toward the middle of the cochlear electrode array, and the effects of simultaneously stimulating a more apical ‘test’ CI channel were measured as a function of spatial separation between the two stimulation channels and stimulus level of the fixed channel. Channel interactions were quantified by changes in neural responses and IC threshold (i.e., threshold shift) elicited by simultaneous stimulation of two CI channels, compared to stimulation of the test channel alone. Main results. Channel interactions were significantly lower for FMP and TP than for MP stimulation (p < 0.001), whereas no significant difference was observed between FMP and TP stimulation. With MP stimulation, threshold shifts increased with decreased inter-electrode spacing and increased stimulus levels of the fixed channel. For FMP and TP stimulation, channel interactions were found to be similar for different inter-electrode spacing and stimulus levels of the fixed channel. Significance. The present study demonstrates how the degree of channel interactions in a CI can be controlled using stimulation configurations such as FMP and TP; such knowledge is essential in enhancing CI function in complex acoustic environments.

Distribution, expression and functional effects of small conductance Ca-activated potassium (SK) channels in rat myometrium.

PubMed

Noble, Karen; Floyd, Rachel; Shmygol, Andre; Shmygol, Anatoly; Mobasheri, A; Wray, Susan

2010-01-01

Calcium-activated potassium channels are important in a variety of smooth muscles, contributing to excitability and contractility. In the myometrium previous work has focussed on the large conductance channels (BK), and the role of small conductance channels (SK) has received scant attention, despite the finding that over-expression of an SK channel isoform (SK3) results in uterine dysfunction and delayed parturition. This study therefore characterises the expression of the three SK channel isoforms (SK1-3) in rat myometrium throughout pregnancy and investigates their effect on cytosolic [Ca] and force and compares this with that of BK channels. Consistent expression of all SK isoform transcripts and clear immunostaining of SK1-3 was found. Inhibition of SK1-3 channels (apamin, scyllatoxin) significantly inhibited outward current, caused membrane depolarisation and elicited action potentials in previously quiescent cells. Apamin or scyllatoxin increased the amplitude of [Ca] and force in spontaneously contracting myometrial strips throughout gestation. The functional effect of SK inhibition was larger than that of BK channel inhibition. Thus we show for the first time that SK1-3 channels are expressed and translated throughout pregnancy and contribute to outward current, regulate membrane potential and hence Ca signals in pregnant rat myometrium. They contribute more to quiescence that BK channels. 2009 Elsevier Ltd. All rights reserved.

Physical and functional interactions between a glioma cation channel and integrin-β1 require α-actinin

PubMed Central

Rooj, Arun K.; Liu, Zhiyong; McNicholas, Carmel M.

2015-01-01

Major plasma membrane components of the tumor cell, ion channels, and integrins play crucial roles in metastasis. Glioma cells express an amiloride-sensitive nonselective cation channel composed of acid-sensing ion channel (ASIC)-1 and epithelial Na+ channel (ENaC) α- and γ-subunits. Inhibition of this channel is associated with reduced cell migration and proliferation. Using the ASIC-1 subunit as a reporter for the channel complex, we found a physical and functional interaction between this channel and integrin-β1. Short hairpin RNA knockdown of integrin-β1 attenuated the amiloride-sensitive current, which was due to loss of surface expression of ASIC-1. In contrast, upregulation of membrane expression of integrin-β1 increased the surface expression of ASIC-1. The link between the amiloride-sensitive channel and integrin-β1 was mediated by α-actinin. Downregulation of α-actinin-1 or -4 attenuated the amiloride-sensitive current. Mutation of the putative binding site for α-actinin on the COOH terminus of ASIC-1 reduced the membrane localization of ASIC-1 and also resulted in attenuation of the amiloride-sensitive current. Our data suggest a novel interaction between the amiloride-sensitive glioma cation channel and integrin-β1, mediated by α-actinin. This interaction may form a mechanism by which channel activity can regulate glioma cell proliferation and migration. PMID:26108662

Mechanosensitive Piezo Channels in the Gastrointestinal Tract.

PubMed

Alcaino, C; Farrugia, G; Beyder, A

2017-01-01

Sensation of mechanical forces is critical for normal function of the gastrointestinal (GI) tract and abnormalities in mechanosensation are linked to GI pathologies. In the GI tract there are several mechanosensitive cell types-epithelial enterochromaffin cells, intrinsic and extrinsic enteric neurons, smooth muscle cells and interstitial cells of Cajal. These cells use mechanosensitive ion channels that respond to mechanical forces by altering transmembrane ionic currents in a process called mechanoelectrical coupling. Several mechanosensitive ionic conductances have been identified in the mechanosensory GI cells, ranging from mechanosensitive voltage-gated sodium and calcium channels to the mechanogated ion channels, such as the two-pore domain potassium channels K2P (TREK-1) and nonselective cation channels from the transient receptor potential family. The recently discovered Piezo channels are increasingly recognized as significant contributors to cellular mechanosensitivity. Piezo1 and Piezo2 are nonselective cationic ion channels that are directly activated by mechanical forces and have well-defined biophysical and pharmacologic properties. The role of Piezo channels in the GI epithelium is currently under investigation and their role in the smooth muscle syncytium and enteric neurons is still not known. In this review, we outline the current state of knowledge on mechanosensitive ion channels in the GI tract, with a focus on the known and potential functions of the Piezo channels. Copyright © 2017 Elsevier Inc. All rights reserved.

Voltage-gated proton channel in a dinoflagellate

PubMed Central

Smith, Susan M. E.; Morgan, Deri; Musset, Boris; Cherny, Vladimir V.; Place, Allen R.; Hastings, J. Woodland; DeCoursey, Thomas E.

2011-01-01

Fogel and Hastings first hypothesized the existence of voltage-gated proton channels in 1972 in bioluminescent dinoflagellates, where they were thought to trigger the flash by activating luciferase. Proton channel genes were subsequently identified in human, mouse, and Ciona intestinalis, but their existence in dinoflagellates remained unconfirmed. We identified a candidate proton channel gene from a Karlodinium veneficum cDNA library based on homology with known proton channel genes. K. veneficum is a predatory, nonbioluminescent dinoflagellate that produces toxins responsible for fish kills worldwide. Patch clamp studies on the heterologously expressed gene confirm that it codes for a genuine voltage-gated proton channel, kHV1: it is proton-specific and activated by depolarization, its gH–V relationship shifts with changes in external or internal pH, and mutation of the selectivity filter (which we identify as Asp51) results in loss of proton-specific conduction. Indirect evidence suggests that kHV1 is monomeric, unlike other proton channels. Furthermore, kHV1 differs from all known proton channels in activating well negative to the Nernst potential for protons, EH. This unique voltage dependence makes the dinoflagellate proton channel ideally suited to mediate the proton influx postulated to trigger bioluminescence. In contrast to vertebrate proton channels, whose main function is acid extrusion, we propose that proton channels in dinoflagellates have fundamentally different functions of signaling and excitability. PMID:22006335

New perspectives in cyclic nucleotide-mediated functions in the CNS: the emerging role of cyclic nucleotide-gated (CNG) channels.

PubMed

Podda, Maria Vittoria; Grassi, Claudio

2014-07-01

Cyclic nucleotides play fundamental roles in the central nervous system (CNS) under both physiological and pathological conditions. The impact of cAMP and cGMP signaling on neuronal and glial cell functions has been thoroughly characterized. Most of their effects have been related to cyclic nucleotide-dependent protein kinase activity. However, cyclic nucleotide-gated (CNG) channels, first described as key mediators of sensory transduction in retinal and olfactory receptors, have been receiving increasing attention as possible targets of cyclic nucleotides in the CNS. In the last 15 years, consistent evidence has emerged for their expression in neurons and astrocytes of the rodent brain. Far less is known, however, about the functional role of CNG channels in these cells, although several of their features, such as Ca(2+) permeability and prolonged activation in the presence of cyclic nucleotides, make them ideal candidates for mediators of physiological functions in the CNS. Here, we review literature suggesting the involvement of CNG channels in a number of CNS cellular functions (e.g., regulation of membrane potential, neuronal excitability, and neurotransmitter release) as well as in more complex phenomena, like brain plasticity, adult neurogenesis, and pain sensitivity. The emerging picture is that functional and dysfunctional cyclic nucleotide signaling in the CNS has to be reconsidered including CNG channels among possible targets. However, concerted efforts and multidisciplinary approaches are still needed to get more in-depth knowledge in this field.

Identifying cochlear implant channels with poor electrode-neuron interfaces: electrically evoked auditory brain stem responses measured with the partial tripolar configuration.

PubMed

Bierer, Julie Arenberg; Faulkner, Kathleen F; Tremblay, Kelly L

2011-01-01

The goal of this study was to compare cochlear implant behavioral measures and electrically evoked auditory brain stem responses (EABRs) obtained with a spatially focused electrode configuration. It has been shown previously that channels with high thresholds, when measured with the tripolar configuration, exhibit relatively broad psychophysical tuning curves. The elevated threshold and degraded spatial/spectral selectivity of such channels are consistent with a poor electrode-neuron interface, defined as suboptimal electrode placement or reduced nerve survival. However, the psychophysical methods required to obtain these data are time intensive and may not be practical during a clinical mapping session, especially for young children. Here, we have extended the previous investigation to determine whether a physiological approach could provide a similar assessment of channel functionality. We hypothesized that, in accordance with the perceptual measures, higher EABR thresholds would correlate with steeper EABR amplitude growth functions, reflecting a degraded electrode-neuron interface. Data were collected from six cochlear implant listeners implanted with the HiRes 90k cochlear implant (Advanced Bionics). Single-channel thresholds and most comfortable listening levels were obtained for stimuli that varied in presumed electrical field size by using the partial tripolar configuration, for which a fraction of current (σ) from a center active electrode returns through two neighboring electrodes and the remainder through a distant indifferent electrode. EABRs were obtained in each subject for the two channels having the highest and lowest tripolar (σ = 1 or 0.9) behavioral threshold. Evoked potentials were measured with both the monopolar (σ = 0) and a more focused partial tripolar (σ ≥ 0.50) configuration. Consistent with previous studies, EABR thresholds were highly and positively correlated with behavioral thresholds obtained with both the monopolar and partial tripolar configurations. The Wave V amplitude growth functions with increasing stimulus level showed the predicted effect of shallower growth for the partial tripolar than for the monopolar configuration, but this was observed only for the low-threshold channels. In contrast, high-threshold channels showed the opposite effect; steeper growth functions were seen for the partial tripolar configuration. These results suggest that behavioral thresholds or EABRs measured with a restricted stimulus can be used to identify potentially impaired cochlear implant channels. Channels having high thresholds and steep growth functions would likely not activate the appropriate spatially restricted region of the cochlea, leading to suboptimal perception. As a clinical tool, quick identification of impaired channels could lead to patient-specific mapping strategies and result in improved speech and music perception.

Site-3 sea anemone toxins: molecular probes of gating mechanisms in voltage-dependent sodium channels.

PubMed

Smith, Jaime J; Blumenthal, Kenneth M

2007-02-01

Sea anemone toxins, whose biological function is the capture of marine prey, are invaluable tools for studying the structure and function of mammalian voltage-gated sodium channels. Their high degree of specificity and selectivity have allowed for detailed analysis of inactivation gating and assignment of molecular entities responsible for this process. Because of their ability to discriminate among channel isoforms, and their high degree of structural conservation, these toxins could serve as important lead compounds for future pharmaceutical design.

EVALUATING MOLECULAR SITES OF ACTION FOR TOLUENE USING AN IN VIVO MODEL.

EPA Science Inventory

In vitro studies have demonstrated that toluene disrupts the function of several ion channels localized in the brain, including the NMDA-glutamate receptor. This has led to the hypothesis that effects on ion channel function may contribute to toluene neurotoxicity, CNS depres...

Functional Electrical Therapy Systems: Neuroprostheses for Life-Like Reach and Grasp

DTIC Science & Technology

2001-10-25

communication channel by means of an infrared link. Keywords – Neuroprosthesis, therapy, reaching, grasping I. INTRODUCTION Functional electrical...present technology and the next generation. Proc 2nd Int Symp IFESS, Burnaby, pp 139-140, 1997. [7] Ocadiz G, Mayagoitia RE. Programmable eight channel

Reconstitution of Homomeric GluA2flop Receptors in Supported Lipid Membranes

PubMed Central

Baranovic, Jelena; Ramanujan, Chandra S.; Kasai, Nahoko; Midgett, Charles R.; Madden, Dean R.; Torimitsu, Keiichi; Ryan, John F.

2013-01-01

AMPA receptors (AMPARs) are glutamate-gated ion channels ubiquitous in the vertebrate central nervous system, where they mediate fast excitatory neurotransmission and act as molecular determinants of memory formation and learning. Together with detailed analyses of individual AMPAR domains, structural studies of full-length AMPARs by electron microscopy and x-ray crystallography have provided important insights into channel assembly and function. However, the correlation between the structure and functional states of the channel remains ambiguous particularly because these functional states can be assessed only with the receptor bound within an intact lipid bilayer. To provide a basis for investigating AMPAR structure in a membrane environment, we developed an optimized reconstitution protocol using a receptor whose structure has previously been characterized by electron microscopy. Single-channel recordings of reconstituted homomeric GluA2flop receptors recapitulate key electrophysiological parameters of the channels expressed in native cellular membranes. Atomic force microscopy studies of the reconstituted samples provide high-resolution images of membrane-embedded full-length AMPARs at densities comparable to those in postsynaptic membranes. The data demonstrate the effect of protein density on conformational flexibility and dimensions of the receptors and provide the first structural characterization of functional membrane-embedded AMPARs, thus laying the foundation for correlated structure-function analyses of the predominant mediators of excitatory synaptic signals in the brain. PMID:23382380

Expression, purification and functional reconstitution of slack sodium-activated potassium channels.

PubMed

Yan, Yangyang; Yang, Youshan; Bian, Shumin; Sigworth, Fred J

2012-11-01

The slack (slo2.2) gene codes for a potassium-channel α-subunit of the 6TM voltage-gated channel family. Expression of slack results in Na(+)-activated potassium channel activity in various cell types. We describe the purification and reconstitution of Slack protein and show that the Slack α-subunit alone is sufficient for potassium channel activity activated by sodium ions as assayed in planar bilayer membranes and in membrane vesicles.

Molecular biology of insect sodium channels and pyrethroid resistance.

PubMed

Dong, Ke; Du, Yuzhe; Rinkevich, Frank; Nomura, Yoshiko; Xu, Peng; Wang, Lingxin; Silver, Kristopher; Zhorov, Boris S

2014-07-01

Voltage-gated sodium channels are essential for the initiation and propagation of the action potential in neurons and other excitable cells. Because of their critical roles in electrical signaling, sodium channels are targets of a variety of naturally occurring and synthetic neurotoxins, including several classes of insecticides. This review is intended to provide an update on the molecular biology of insect sodium channels and the molecular mechanism of pyrethroid resistance. Although mammalian and insect sodium channels share fundamental topological and functional properties, most insect species carry only one sodium channel gene, compared to multiple sodium channel genes found in each mammalian species. Recent studies showed that two posttranscriptional mechanisms, alternative splicing and RNA editing, are involved in generating functional diversity of sodium channels in insects. More than 50 sodium channel mutations have been identified to be responsible for or associated with knockdown resistance (kdr) to pyrethroids in various arthropod pests and disease vectors. Elucidation of molecular mechanism of kdr led to the identification of dual receptor sites of pyrethroids on insect sodium channels. Many of the kdr mutations appear to be located within or close to the two receptor sites. The accumulating knowledge of insect sodium channels and their interactions with insecticides provides a foundation for understanding the neurophysiology of sodium channels in vivo and the development of new and safer insecticides for effective control of arthropod pests and human disease vectors. Copyright © 2014 Elsevier Ltd. All rights reserved.

In vivo and in vitro functional characterization of Andersen's syndrome mutations.

PubMed

Bendahhou, Saïd; Fournier, Emmanuel; Sternberg, Damien; Bassez, Guillaume; Furby, Alain; Sereni, Carole; Donaldson, Matthew R; Larroque, Marie-Madeleine; Fontaine, Bertrand; Barhanin, Jacques

2005-06-15

The inward rectifier K(+) channel Kir2.1 carries all Andersen's syndrome mutations identified to date. Patients exhibit symptoms of periodic paralysis, cardiac dysrhythmia and multiple dysmorphic features. Here, we report the clinical manifestations found in three families with Andersen's syndrome. Molecular genetics analysis identified two novel missense mutations in the KCNJ2 gene leading to amino acid changes C154F and T309I of the Kir2.1 open reading frame. Patch clamp experiments showed that the two mutations produced a loss of channel function. When co-expressed with Kir2.1 wild-type (WT) channels, both mutations exerted a dominant-negative effect leading to a loss of the inward rectifying K(+) current. Confocal microscopy imaging in HEK293 cells is consistent with a co-assembly of the EGFP-fused mutant proteins with WT channels and proper traffick to the plasma membrane to produce silent channels alone or as hetero-tetramers with WT. Functional expression in C2C12 muscle cell line of newly as well as previously reported Andersen's syndrome mutations confirmed that these mutations act through a dominant-negative effect by altering channel gating or trafficking. Finally, in vivo electromyographic evaluation showed a decrease in muscle excitability in Andersen's syndrome patients. We hypothesize that Andersen's syndrome-associated mutations and hypokalaemic periodic paralysis-associated calcium channel mutations may lead to muscle membrane hypoexcitability via a common mechanism.

In vivo and in vitro functional characterization of Andersen's syndrome mutations

PubMed Central

Bendahhou, Saïd; Fournier, Emmanuel; Sternberg, Damien; Bassez, Guillaume; Furby, Alain; Sereni, Carole; Donaldson, Matthew R; Larroque, Marie-Madeleine; Fontaine, Bertrand; Barhanin, Jacques

2005-01-01

The inward rectifier K+ channel Kir2.1 carries all Andersen's syndrome mutations identified to date. Patients exhibit symptoms of periodic paralysis, cardiac dysrhythmia and multiple dysmorphic features. Here, we report the clinical manifestations found in three families with Andersen's syndrome. Molecular genetics analysis identified two novel missense mutations in the KCNJ2 gene leading to amino acid changes C154F and T309I of the Kir2.1 open reading frame. Patch clamp experiments showed that the two mutations produced a loss of channel function. When co-expressed with Kir2.1 wild-type (WT) channels, both mutations exerted a dominant-negative effect leading to a loss of the inward rectifying K+ current. Confocal microscopy imaging in HEK293 cells is consistent with a co-assembly of the EGFP-fused mutant proteins with WT channels and proper traffick to the plasma membrane to produce silent channels alone or as hetero-tetramers with WT. Functional expression in C2C12 muscle cell line of newly as well as previously reported Andersen's syndrome mutations confirmed that these mutations act through a dominant-negative effect by altering channel gating or trafficking. Finally, in vivo electromyographic evaluation showed a decrease in muscle excitability in Andersen's syndrome patients. We hypothesize that Andersen's syndrome-associated mutations and hypokalaemic periodic paralysis-associated calcium channel mutations may lead to muscle membrane hypoexcitability via a common mechanism. PMID:15831539

Dopamine negatively modulates the NCA ion channels in C. elegans

PubMed Central

Topalidou, Irini; Pereira, Laura

2017-01-01

The NALCN/NCA ion channel is a cation channel related to voltage-gated sodium and calcium channels. NALCN has been reported to be a sodium leak channel with a conserved role in establishing neuronal resting membrane potential, but its precise cellular role and regulation are unclear. The Caenorhabditis elegans orthologs of NALCN, NCA-1 and NCA-2, act in premotor interneurons to regulate motor circuit activity that sustains locomotion. Recently we found that NCA-1 and NCA-2 are activated by a signal transduction pathway acting downstream of the heterotrimeric G protein Gq and the small GTPase Rho. Through a forward genetic screen, here we identify the GPCR kinase GRK-2 as a new player affecting signaling through the Gq-Rho-NCA pathway. Using structure-function analysis, we find that the GPCR phosphorylation and membrane association domains of GRK-2 are required for its function. Genetic epistasis experiments suggest that GRK-2 acts on the D2-like dopamine receptor DOP-3 to inhibit Go signaling and positively modulate NCA-1 and NCA-2 activity. Through cell-specific rescuing experiments, we find that GRK-2 and DOP-3 act in premotor interneurons to modulate NCA channel function. Finally, we demonstrate that dopamine, through DOP-3, negatively regulates NCA activity. Thus, this study identifies a pathway by which dopamine modulates the activity of the NCA channels. PMID:28968387

Dopamine negatively modulates the NCA ion channels in C. elegans.

PubMed

Topalidou, Irini; Cooper, Kirsten; Pereira, Laura; Ailion, Michael

2017-10-01

The NALCN/NCA ion channel is a cation channel related to voltage-gated sodium and calcium channels. NALCN has been reported to be a sodium leak channel with a conserved role in establishing neuronal resting membrane potential, but its precise cellular role and regulation are unclear. The Caenorhabditis elegans orthologs of NALCN, NCA-1 and NCA-2, act in premotor interneurons to regulate motor circuit activity that sustains locomotion. Recently we found that NCA-1 and NCA-2 are activated by a signal transduction pathway acting downstream of the heterotrimeric G protein Gq and the small GTPase Rho. Through a forward genetic screen, here we identify the GPCR kinase GRK-2 as a new player affecting signaling through the Gq-Rho-NCA pathway. Using structure-function analysis, we find that the GPCR phosphorylation and membrane association domains of GRK-2 are required for its function. Genetic epistasis experiments suggest that GRK-2 acts on the D2-like dopamine receptor DOP-3 to inhibit Go signaling and positively modulate NCA-1 and NCA-2 activity. Through cell-specific rescuing experiments, we find that GRK-2 and DOP-3 act in premotor interneurons to modulate NCA channel function. Finally, we demonstrate that dopamine, through DOP-3, negatively regulates NCA activity. Thus, this study identifies a pathway by which dopamine modulates the activity of the NCA channels.

Modulatory mechanisms and multiple functions of somatodendritic A-type K+ channel auxiliary subunits

PubMed Central

Jerng, Henry H.; Pfaffinger, Paul J.

2014-01-01

Auxiliary subunits are non-conducting, modulatory components of the multi-protein ion channel complexes that underlie normal neuronal signaling. They interact with the pore-forming α-subunits to modulate surface distribution, ion conductance, and channel gating properties. For the somatodendritic subthreshold A-type potassium (ISA) channel based on Kv4 α-subunits, two types of auxiliary subunits have been extensively studied: Kv channel-interacting proteins (KChIPs) and dipeptidyl peptidase-like proteins (DPLPs). KChIPs are cytoplasmic calcium-binding proteins that interact with intracellular portions of the Kv4 subunits, whereas DPLPs are type II transmembrane proteins that associate with the Kv4 channel core. Both KChIPs and DPLPs genes contain multiple start sites that are used by various neuronal populations to drive the differential expression of functionally distinct N-terminal variants. In turn, these N-terminal variants generate tremendous functional diversity across the nervous system. Here, we focus our review on (1) the molecular mechanism underlying the unique properties of different N-terminal variants, (2) the shaping of native ISA properties by the concerted actions of KChIPs and DPLP variants, and (3) the surprising ways that KChIPs and DPLPs coordinate the activity of multiple channels to fine-tune neuronal excitability. Unlocking the unique contributions of different auxiliary subunit N-terminal variants may provide an important opportunity to develop novel targeted therapeutics to treat numerous neurological disorders. PMID:24723849

Modulatory mechanisms and multiple functions of somatodendritic A-type K (+) channel auxiliary subunits.

PubMed

Jerng, Henry H; Pfaffinger, Paul J

2014-01-01

Auxiliary subunits are non-conducting, modulatory components of the multi-protein ion channel complexes that underlie normal neuronal signaling. They interact with the pore-forming α-subunits to modulate surface distribution, ion conductance, and channel gating properties. For the somatodendritic subthreshold A-type potassium (ISA) channel based on Kv4 α-subunits, two types of auxiliary subunits have been extensively studied: Kv channel-interacting proteins (KChIPs) and dipeptidyl peptidase-like proteins (DPLPs). KChIPs are cytoplasmic calcium-binding proteins that interact with intracellular portions of the Kv4 subunits, whereas DPLPs are type II transmembrane proteins that associate with the Kv4 channel core. Both KChIPs and DPLPs genes contain multiple start sites that are used by various neuronal populations to drive the differential expression of functionally distinct N-terminal variants. In turn, these N-terminal variants generate tremendous functional diversity across the nervous system. Here, we focus our review on (1) the molecular mechanism underlying the unique properties of different N-terminal variants, (2) the shaping of native ISA properties by the concerted actions of KChIPs and DPLP variants, and (3) the surprising ways that KChIPs and DPLPs coordinate the activity of multiple channels to fine-tune neuronal excitability. Unlocking the unique contributions of different auxiliary subunit N-terminal variants may provide an important opportunity to develop novel targeted therapeutics to treat numerous neurological disorders.

Log Distribution, Persistence, and Geomorphic Function in Streams and Rivers, in the Northeastern U.S.

NASA Astrophysics Data System (ADS)

St Pierre, L.; Burchsted, D.; Warren, D.

2015-12-01

Large wood provides critical ecosystem services such as fish habitat, temperature regulation and bank stabilization. In the northeastern U.S., the distribution of large wood is documented; however, there is little understanding of the movement, longevity and geomorphic function. This research examines the hypothesis that tree species control the persistence and geomorphic function of instream wood in the Appalachian region of the northeastern U.S. To do this, we assessed size, location, and species of logs in New Hampshire rivers, including locations in the White Mountain National Forest (WMNF) where these data were collected ten years ago. We expanded the previous dataset to include assessment of geomorphic function, including creation of diversion channels, pool formation, and sediment storage, among others. We also added new sites in the WMNF and sites on a large rural river in southwestern NH to increase the range of geomorphic variables to now include: confined and unconfined channels; 1st to 4th order streams; low to high gradient; meandering, multithreaded, and straight channels; and land use such as historic logging, modern agriculture, and post-agricultural abandonment. At each study site, we located all large logs (>10cm diameter, > 1m length) and log jams (>3 accumulated logs that provide a geomorphic function) along 100m-700m reaches. We marked each identified log with a numbered tag and recorded species, diameter, length, orientation, GPS location, tag number, and photographs. We assessed function and accumulation, decay, stability, and source classes for each log. Along each reach we measured riparian forest composition and structure and channel width. Preliminary analysis suggests that tree species significantly affects the function of logs: yellow birch and American sycamore are highly represented. Additionally, geomorphic setting also plays a primary role, where unconfined reaches have large logs that provide important functions; those functions are rarely contributed by logs in confined channels. Land use limit the ability of logs to provide habitat for vegetation recruitment, notable in rivers adjacent to agricultural areas that maintain a straight channel; invasive vegetation dominate the banks and there is little to no recruitment of native vegetation.

Ecological Functions of Off-Channel Habitats of the Willamette River, Oregon, Database and Documentation (1997-2001)

EPA Science Inventory

The database from the Ecological Functions of Off-Channel Habitats of the Willamette River, Oregon project (OCH Project) contains data collected from 1997 through 2001 from multiple research areas of the project, and project documents such as the OCH Research Plan, Quality Assura...

Seeking structural specificity: direct modulation of pentameric ligand-gated ion channels by alcohols and general anesthetics.

PubMed

Howard, Rebecca J; Trudell, James R; Harris, R Adron

2014-01-01

Alcohols and other anesthetic agents dramatically alter neurologic function in a wide range of organisms, yet their molecular sites of action remain poorly characterized. Pentameric ligand-gated ion channels, long implicated in important direct effects of alcohol and anesthetic binding, have recently been illuminated in renewed detail thanks to the determination of atomic-resolution structures of several family members from lower organisms. These structures provide valuable models for understanding and developing anesthetic agents and for allosteric modulation in general. This review surveys progress in this field from function to structure and back again, outlining early evidence for relevant modulation of pentameric ligand-gated ion channels and the development of early structural models for ion channel function and modulation. We highlight insights and challenges provided by recent crystal structures and resulting simulations, as well as opportunities for translation of these newly detailed models back to behavior and therapy.

Structural domains required for channel function of the mouse transient receptor potential protein homologue TRP1beta.

PubMed

Engelke, Michael; Friedrich, Olaf; Budde, Petra; Schäfer, Christina; Niemann, Ursula; Zitt, Christof; Jüngling, Eberhard; Rocks, Oliver; Lückhoff, Andreas; Frey, Jürgen

2002-07-17

Transient receptor potential proteins (TRP) are supposed to participate in the formation of store-operated Ca(2+) influx channels by co-assembly. However, little is known which domains facilitate the interaction of subunits. Contribution of the N-terminal coiled-coil domain and ankyrin-like repeats and the putative pore region of the mouse TRP1beta (mTRP1beta) variant to the formation of functional cation channels were analyzed following overexpression in HEK293 (human embryonic kidney) cells. MTRP1beta expressing cells exhibited enhanced Ca(2+) influx and enhanced whole-cell membrane currents compared to mTRP1beta deletion mutants. Using a yeast two-hybrid assay only the coiled-coil domain facilitated homodimerization of the N-terminus. These results suggest that the N-terminus of mTRP1beta is required for structural organization thus forming functional channels.

Structural and Functional Similarities of Calcium Homeostasis Modulator 1 (CALHM1) Ion Channel with Connexins, Pannexins, and Innexins*

PubMed Central

Siebert, Adam P.; Ma, Zhongming; Grevet, Jeremy D.; Demuro, Angelo; Parker, Ian; Foskett, J. Kevin

2013-01-01

CALHM1 (calcium homeostasis modulator 1) forms a plasma membrane ion channel that mediates neuronal excitability in response to changes in extracellular Ca2+ concentration. Six human CALHM homologs exist with no homology to other proteins, although CALHM1 is conserved across >20 species. Here we demonstrate that CALHM1 shares functional and quaternary and secondary structural similarities with connexins and evolutionarily distinct innexins and their vertebrate pannexin homologs. A CALHM1 channel is a hexamer, comprised of six monomers, each of which possesses four transmembrane domains, cytoplasmic amino and carboxyl termini, an amino-terminal helix, and conserved extracellular cysteines. The estimated pore diameter of the CALHM1 channel is ∼14 Å, enabling permeation of large charged molecules. Thus, CALHMs, connexins, and pannexins and innexins are structurally related protein families with shared and distinct functional properties. PMID:23300080

Seeking Structural Specificity: Direct Modulation of Pentameric Ligand-Gated Ion Channels by Alcohols and General Anesthetics

PubMed Central

Trudell, James R.; Harris, R. Adron

2014-01-01

Alcohols and other anesthetic agents dramatically alter neurologic function in a wide range of organisms, yet their molecular sites of action remain poorly characterized. Pentameric ligand-gated ion channels, long implicated in important direct effects of alcohol and anesthetic binding, have recently been illuminated in renewed detail thanks to the determination of atomic-resolution structures of several family members from lower organisms. These structures provide valuable models for understanding and developing anesthetic agents and for allosteric modulation in general. This review surveys progress in this field from function to structure and back again, outlining early evidence for relevant modulation of pentameric ligand-gated ion channels and the development of early structural models for ion channel function and modulation. We highlight insights and challenges provided by recent crystal structures and resulting simulations, as well as opportunities for translation of these newly detailed models back to behavior and therapy. PMID:24515646

The Promise of Microelectrode Array Approaches for Toxicity Testing: Examples with Neuroactive Chemicals

EPA Science Inventory

While high-throughput patch clamping formats provide rapid characterization of chemical effects on ion channel function and kinetics, the limitations of such systems often include the need for channel by channel characterization, requirements for transfected, rather than primary ...

Presynaptic excitability.

PubMed

Jackson, M B

1995-01-01

Based on functional characterizations with electrophysiological techniques, the channels in nerve terminals appear to be as diverse as channels in nerve cell bodies (Table I). While most presynaptic Ca2+ channels superficially resemble either N-type or L-type channels, variations in detail have necessitated the use of subscripts and other notations to indicate a nerve terminal-specific subtype (e.g., Wang et al., 1993). Variations such as these pose a serious obstacle to the identification of presynaptic channels based solely on the effects of channel blockers on synaptic transmission. Pharmacological sensitivity alone is not likely to help in determining functional properties. Crucial details, such as voltage sensitivity and inactivation, require direct examination. It goes without saying that every nerve terminal membrane contains Ca2+ channels as an entry pathway so that Ca2+ can trigger secretion. However, there appears to be no general specification of channel type, other than the exclusion of T-type Ca2+ channels. T-type Ca2+ channels are defined functionally by strong inactivation and low threshold. Some presynaptic Ca2+ channels inactivate (posterior pituitary and Xenopus nerve terminals), and others have a somewhat reduced voltage threshold (retinal bipolar neurons and squid giant synapse). Perhaps it is just a matter of time before a nerve terminal Ca2+ channel is found with both of these properties. The high threshold and strong inactivation of T-type Ca2+ channels are thought to be adaptations for oscillations and the regulation of bursting activity in nerve cell bodies. The nerve terminals thus far examined have no endogenous electrical activity, but rather are driven by the cell body. On functional grounds, it is then reasonable to anticipate finding T-type Ca2+ channels in nerve terminals that can generate electrical activity on their own. The rarity of such behavior in nerve terminals may be associated with the rarity of presynaptic T-type Ca2+ channels. In four of the five preparations reviewed in this chapter--motor nerve, squid giant synapse, ciliary ganglion, and retina bipolar neurons--evidence was presented that supports a location for Ca2+ channels that is very close to active zones of secretion. All of these synapses secrete from clear vesicles, and the speed and specificity of transduction provided by proximity may be a common feature of these rapid synapses. In contrast, the posterior pituitary secretion apparatus may be triggered by higher-affinity Ca2+ receptors and lower concentrations of Ca2+ (Lindau et al., 1992). This would correspond with the slower performance of peptidergic secretion, but because of the large stimuli needed to evoke release from neurosecretosomes, the possibility remains that the threshold for secretion is higher than that reported. While the role of Ca2+ as a trigger of secretion dictates a requirement for voltage-activated Ca2+ channels as universal components of the presynaptic membrane, the presence of other channels is more difficult to predict. Depolarizations caused by voltage-activated Na+ channels activate the presynaptic Ca2+ channels, but whether this depolarization requires Na+ channels in the presynaptic membrane itself may depend on the electrotonic length of the nerve terminal. Variations in density between motor nerve terminals may reflect species differences in geometry. The high Na+ channel density in the posterior pituitary reflects the great electrotonic length of this terminal arbor. Whether Na+ channels are abundant or not in a presynaptic membrane, K+ channels provide the most robust mechanism for limiting depolarization-induced Ca2+ entry. K+ channel blockers enhance transmission at most synapses. In general, K+ channels are abundant in nerve terminals, although their apparent lower priority compared to Ca2+ channels in the eyes of many investigators leaves us with fewer detailed investigations in some preparations. Most nerve terminals have more than

Ageing causes cytoplasmic retention of MaxiK channels in rat corporal smooth muscle cells

PubMed Central

Davies, KP; Stanevsky, Y; Moses, T; Chang, JS; Chance, MR; Melman, A

2007-01-01

The MaxiK channel plays a critical role in the regulation of corporal smooth muscle tone and thereby erectile function. Given that ageing results in a decline in erectile function, we determined changes in the expression of MaxiK, which might impact erectile function. Quantitative-polymerase chain reaction demonstrated that although there is no significant change in transcription of the α- and β-subunits that comprise the MaxiK channel, there are significant changes in the expression of transcripts encoding different splice variants. One transcript, SV1, is 13-fold increased in expression in the ageing rat corpora. SV1 has previously been reported to trap other isoforms of the MaxiK channel in the cytoplasm. Correlating with increased expression of SV1, we observed in older rats there is approximately a 13-fold decrease in MaxiK protein in the corpora cell membrane and a greater proportion is retained in the cytoplasm (approximately threefold). These experiments demonstrate that ageing of the corpora is accompanied by changes in alternative splicing and cellular localization of the MaxiK channel. PMID:17287835

Ageing causes cytoplasmic retention of MaxiK channels in rat corporal smooth muscle cells.

PubMed

Davies, K P; Stanevsky, Y; Tar, M T; Moses, T; Chang, J S; Chance, M R; Melman, A

2007-01-01

The MaxiK channel plays a critical role in the regulation of corporal smooth muscle tone and thereby erectile function. Given that ageing results in a decline in erectile function, we determined changes in the expression of MaxiK, which might impact erectile function. Quantitative-polymerase chain reaction demonstrated that although there is no significant change in transcription of the alpha- and beta-subunits that comprise the MaxiK channel, there are significant changes in the expression of transcripts encoding different splice variants. One transcript, SV1, is 13-fold increased in expression in the ageing rat corpora. SV1 has previously been reported to trap other isoforms of the MaxiK channel in the cytoplasm. Correlating with increased expression of SV1, we observed in older rats there is approximately a 13-fold decrease in MaxiK protein in the corpora cell membrane and a greater proportion is retained in the cytoplasm (approximately threefold). These experiments demonstrate that ageing of the corpora is accompanied by changes in alternative splicing and cellular localization of the MaxiK channel.

Accurate analytical modeling of junctionless DG-MOSFET by green's function approach

NASA Astrophysics Data System (ADS)

Nandi, Ashutosh; Pandey, Nilesh

2017-11-01

An accurate analytical model of Junctionless double gate MOSFET (JL-DG-MOSFET) in the subthreshold regime of operation is developed in this work using green's function approach. The approach considers 2-D mixed boundary conditions and multi-zone techniques to provide an exact analytical solution to 2-D Poisson's equation. The Fourier coefficients are calculated correctly to derive the potential equations that are further used to model the channel current and subthreshold slope of the device. The threshold voltage roll-off is computed from parallel shifts of Ids-Vgs curves between the long channel and short-channel devices. It is observed that the green's function approach of solving 2-D Poisson's equation in both oxide and silicon region can accurately predict channel potential, subthreshold current (Isub), threshold voltage (Vt) roll-off and subthreshold slope (SS) of both long & short channel devices designed with different doping concentrations and higher as well as lower tsi/tox ratio. All the analytical model results are verified through comparisons with TCAD Sentaurus simulation results. It is observed that the model matches quite well with TCAD device simulations.

Ion channels in inflammation.

PubMed

Eisenhut, Michael; Wallace, Helen

2011-04-01

Most physical illness in vertebrates involves inflammation. Inflammation causes disease by fluid shifts across cell membranes and cell layers, changes in muscle function and generation of pain. These disease processes can be explained by changes in numbers or function of ion channels. Changes in ion channels have been detected in diarrhoeal illnesses, pyelonephritis, allergy, acute lung injury and systemic inflammatory response syndromes involving septic shock. The key role played by changes in ion transport is directly evident in inflammation-induced pain. Expression or function of all major categories of ion channels like sodium, chloride, calcium, potassium, transient receptor potential, purinergic receptor and acid-sensing ion channels can be influenced by cyto- and chemokines, prostaglandins, leukotrienes, histamine, ATP, reactive oxygen species and protons released in inflammation. Key pathways in this interaction are cyclic nucleotide, phosphoinositide and mitogen-activated protein kinase-mediated signalling, direct modification by reactive oxygen species like nitric oxide, ATP or protons and disruption of the cytoskeleton. Therapeutic interventions to modulate the adverse and overlapping effects of the numerous different inflammatory mediators on each ion transport system need to target adversely affected ion transport systems directly and locally.

A functional Kv1.2-hERG chimaeric channel expressed in Pichia pastoris

PubMed Central

Dhillon, Mandeep S.; Cockcroft, Christopher J.; Munsey, Tim; Smith, Kathrine J.; Powell, Andrew J.; Carter, Paul; Wrighton, David C.; Rong, Hong-lin; Yusaf, Shahnaz P.; Sivaprasadarao, Asipu

2014-01-01

Members of the six-transmembrane segment family of ion channels share a common structural design. However, there are sequence differences between the members that confer distinct biophysical properties on individual channels. Currently, we do not have 3D structures for all members of the family to help explain the molecular basis for the differences in their biophysical properties and pharmacology. This is due to low-level expression of many members in native or heterologous systems. One exception is rat Kv1.2 which has been overexpressed in Pichia pastoris and crystallised. Here, we tested chimaeras of rat Kv1.2 with the hERG channel for function in Xenopus oocytes and for overexpression in Pichia. Chimaera containing the S1–S6 transmembrane region of HERG showed functional and pharmacological properties similar to hERG and could be overexpressed and purified from Pichia. Our results demonstrate that rat Kv1.2 could serve as a surrogate to express difficult-to-overexpress members of the six-transmembrane segment channel family. PMID:24569544

Computational models of O-LM cells are recruited by low or high theta frequency inputs depending on h-channel distributions

PubMed Central

Sekulić, Vladislav; Skinner, Frances K

2017-01-01

Although biophysical details of inhibitory neurons are becoming known, it is challenging to map these details onto function. Oriens-lacunosum/moleculare (O-LM) cells are inhibitory cells in the hippocampus that gate information flow, firing while phase-locked to theta rhythms. We build on our existing computational model database of O-LM cells to link model with function. We place our models in high-conductance states and modulate inhibitory inputs at a wide range of frequencies. We find preferred spiking recruitment of models at high (4–9 Hz) or low (2–5 Hz) theta depending on, respectively, the presence or absence of h-channels on their dendrites. This also depends on slow delayed-rectifier potassium channels, and preferred theta ranges shift when h-channels are potentiated by cyclic AMP. Our results suggest that O-LM cells can be differentially recruited by frequency-modulated inputs depending on specific channel types and distributions. This work exposes a strategy for understanding how biophysical characteristics contribute to function. DOI: http://dx.doi.org/10.7554/eLife.22962.001 PMID:28318488

Analysis of the performance of a wireless optical multi-input to multi-output communication system.

PubMed

Bushuev, Denis; Arnon, Shlomi

2006-07-01

We investigate robust optical wireless communication in a highly scattering propagation medium using multielement optical detector arrays. The communication setup consists of synchronized multiple transmitters that send information to a receiver array and an atmospheric propagation channel. The mathematical model that best describes this scenario is multi-input to multi-output communication through stochastic slow changing channels. In this model, signals from m transmitters are received by n receiver-detectors. The channel transfer function matrix is G, and its size is n x m. G(i,j) is the transfer function from transmitter i to detector j, and m > or = n. We adopt a quasi-stationary approach in which the channel time variation has a negligible effect on communication performance over a burst. The G matrix is calculated on the basis of the optical transfer function of the atmospheric channel (composed of aerosol and turbulence elements) and the receiver's optics. In this work we derive a performance model using environmental data, such as documented turbulence and aerosol models and noise statistics. We also present the results of simulations conducted for the proposed detection algorithm.

Cyclic GMP-gated CNG channels function in Sema3A-induced growth cone repulsion.

PubMed

Togashi, Kazunobu; von Schimmelmann, Melanie J; Nishiyama, Makoto; Lim, Chae-Seok; Yoshida, Norihiro; Yun, Bokyoung; Molday, Robert S; Goshima, Yoshio; Hong, Kyonsoo

2008-06-12

Cyclic nucleotide-gated channels (CNGCs) transduce external signals required for sensory processes, e.g., photoreception, olfaction, and taste. Nerve growth cone guidance by diffusible attractive and repulsive molecules is regulated by differential growth cone Ca2+ signaling. However, the Ca2+-conducting ion channels that transduce guidance molecule signals are largely unknown. We show that rod-type CNGC-like channels function in the repulsion of cultured Xenopus spinal neuron growth cones by Sema3A, which triggers the production of the cGMP that activates the Xenopus CNGA1 (xCNGA1) subunit-containing channels in interneurons. Downregulation of xCNGA1 or overexpression of a mutant xCNGA1 incapable of binding cGMP abolished CNG currents and converted growth cone repulsion to attraction in response to Sema3A. We also show that Ca2+ entry through xCNGCs is required to mediate the repulsive Sema3A signal. These studies extend our knowledge of the function of CNGCs by demonstrating their requirement for signal transduction in growth cone guidance.

Proton and non-proton activation of ASIC channels

PubMed Central

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

2017-01-01

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

Divergent biophysical properties, gating mechanisms, and possible functions of the two skeletal muscle Ca(V)1.1 calcium channel splice variants.

PubMed

Tuluc, Petronel; Flucher, Bernhard E

2011-12-01

Voltage-gated calcium channels are multi-subunit protein complexes that specifically allow calcium ions to enter the cell in response to membrane depolarization. But, for many years it seemed that the skeletal muscle calcium channel Ca(V)1.1 is the exception. The classical splice variant Ca(V)1.1a activates slowly, has a very small current amplitude and poor voltage sensitivity. In fact adult muscle fibers work perfectly well even in the absence of calcium influx. Recently a new splice variant of the skeletal muscle calcium channel Ca(V)1.1e has been characterized. The lack of the 19 amino acid exon 29 in this splice variant results in a rapidly activating calcium channel with high current amplitude and good voltage sensitivity. Ca(V)1.1e is the dominant channel in embryonic muscle, where the expression of this high calcium-conducting Ca(V)1.1 isoform readily explains developmental processes depending on L-type calcium currents. Moreover, the availability of these two structurally similar but functionally distinct channel variants facilitates the analysis of the molecular mechanisms underlying the unique current properties of the classical Ca(V)1.1a channel.

Mechanism underlying selective regulation of G protein-gated inwardly rectifying potassium channels by the psychostimulant-sensitive sorting nexin 27

PubMed Central

Balana, Bartosz; Maslennikov, Innokentiy; Kwiatkowski, Witek; Stern, Kalyn M.; Bahima, Laia; Choe, Senyon; Slesinger, Paul A.

2011-01-01

G protein-gated inwardly rectifying potassium (GIRK) channels are important gatekeepers of neuronal excitability. The surface expression of neuronal GIRK channels is regulated by the psychostimulant-sensitive sorting nexin 27 (SNX27) protein through a class I (-X-Ser/Thr-X-Φ, where X is any residue and Φ is a hydrophobic amino acid) PDZ-binding interaction. The G protein-insensitive inward rectifier channel (IRK1) contains the same class I PDZ-binding motif but associates with a different synaptic PDZ protein, postsynaptic density protein 95 (PSD95). The mechanism by which SNX27 and PSD95 discriminate these channels was previously unclear. Using high-resolution structures coupled with biochemical and functional analyses, we identified key amino acids upstream of the channel's canonical PDZ-binding motif that associate electrostatically with a unique structural pocket in the SNX27-PDZ domain. Changing specific charged residues in the channel's carboxyl terminus or in the PDZ domain converts the selective association and functional regulation by SNX27. Elucidation of this unique interaction site between ion channels and PDZ-containing proteins could provide a therapeutic target for treating brain diseases. PMID:21422294

Organic toxins as tools to understand ion channel mechanisms and structure.

PubMed

Morales-Lázaro, Sara Luz; Hernández-García, Enrique; Serrano-Flores, Barbara; Rosenbaum, Tamara

2015-01-01

Ion channels constitute a varied class of membrane proteins with pivotal roles in cellular physiology and that are fundamental for neuronal signaling, hormone secretion and muscle contractility. Hence, it is not unanticipated that toxins from diverse organisms have evolved to modulate the activity of ion channels. For instance, animals such as cone snails, scorpions, spiders and snakes use toxins to immobilize and capture their prey by affecting ion channel function. This is a beautiful example of an evolutionary process that has led to the development of an injection apparatus from predators and to the existence of toxins with high affinity and specificity for a given target. Toxins have been used in the field of ion channel biophysics for several decades to gain insight into the gating mechanisms and the structure of ion channels. Through the use of these peptides, much has been learned about the ion conduction pathways, voltage-sensing mechanisms, pore sizes, kinetics, inactivation processes, etc. This review examines an assortment of toxins that have been used to study different ion channels and describes some key findings about the structure-function relationships in these proteins through the details of the toxin-ion channel interactions.

Hydraulic droplet coarsening in open-channel capillaries

NASA Astrophysics Data System (ADS)

Warren, Patrick B.

2016-11-01

Over a range of liquid-solid contact angles, an open-channel capillary with curved or angled sides can show a maximum in the Laplace pressure as a function of the filling state. Examples include double-angle wedges, grooves scored into flat surfaces, steps on surfaces, and the groove between touching parallel cylinders. The liquid in such a channel exhibits a beading instability if the channel is filled beyond the Laplace pressure maximum. The subsequent droplet coarsening takes place by hydraulic transport through the connecting liquid columns that remain in the groove. A mean-field scaling argument predicts the characteristic droplet radius R ˜t1 /7 , as a function of time t . This is confirmed by one-dimensional simulations of the coarsening kinetics. Some remarks are also made on the spreading kinetics of an isolated drop deposited in such a channel.

Reconstruction of an infrared band of meteorological satellite imagery with abductive networks

NASA Technical Reports Server (NTRS)

Singer, Harvey A.; Cockayne, John E.; Versteegen, Peter L.

1995-01-01

As the current fleet of meteorological satellites age, the accuracy of the imagery sensed on a spectral channel of the image scanning system is continually and progressively degraded by noise. In time, that data may even become unusable. We describe a novel approach to the reconstruction of the noisy satellite imagery according to empirical functional relationships that tie the spectral channels together. Abductive networks are applied to automatically learn the empirical functional relationships between the data sensed on the other spectral channels to calculate the data that should have been sensed on the corrupted channel. Using imagery unaffected by noise, it is demonstrated that abductive networks correctly predict the noise-free observed data.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Karch, Andreas; Sato, Yoshiki

In this paper we discuss geodesic Witten diagrams in generic holographic conformal field theories with boundary or defect. Boundary CFTs allow two different de-compositions of two-point functions into conformal blocks: boundary channel and ambient channel. Building on earlier work, we derive a holographic dual of the boundary channel decomposition in terms of bulk-to-bulk propagators on lower dimensional AdS slices. In the situation in which we can treat the boundary or defect as a perturbation around pure AdS spacetime, we obtain the leading corrections to the two-point function both in boundary and ambient channel in terms of geodesic Witten diagrams whichmore » exactly reproduce the decomposition into corresponding conformal blocks on the field theory side.« less

Possible involvement of transient receptor potential ankyrin 1 in Ca2+ signaling via T-type Ca2+ channel in mouse sensory neurons.

PubMed

Nishizawa, Yuki; Takahashi, Kenji; Oguma, Naoko; Tominaga, Makoto; Ohta, Toshio

2018-05-01

T-type Ca 2+ channels and TRPA1 are expressed in sensory neurons and both are associated with pain transmission, but their functional interaction is unclear. Here we demonstrate that pharmacological evidence of the functional relation between T-type Ca 2+ channels and TRPA1 in mouse sensory neurons. Low concentration of KCl at 15 mM (15K) evoked increases of intracellular Ca 2+ concentration ([Ca 2+ ] i ), which were suppressed by selective T-type Ca 2+ channel blockers. RT-PCR showed that mouse sensory neurons expressed all subtypes of T-type Ca 2+ channel. The magnitude of 15K-induced [Ca 2+ ] i increase was significantly larger in neurons sensitive to allylisothiocyanate (AITC, a TRPA1 agonist) than in those insensitive to it, and in TRPA1 -/- mouse sensory neurons. TRPA1 blockers diminished the [Ca 2+ ] i responses to 15K in neurons sensitive to AITC, but failed to inhibit 40 mM KCl-induced [Ca 2+ ] i increases even in AITC-sensitive neurons. TRPV1 blockers did not inhibit the 15K-induced [Ca 2+ ] i increase regardless of the sensitivity to capsaicin. [Ca 2+ ] i responses to TRPA1 agonist were enhanced by co-application with 15K. These pharmacological data suggest the possibility of functional interaction between T-type Ca 2+ channels and TRPA1 in sensory neurons. Since TRPA1 channel is activated by intracellular Ca 2+ , we hypothesize that Ca 2+ entered via T-type Ca 2+ channel activation may further stimulate TRPA1, resulting in an enhancement of nociceptive signaling. Thus, T-type Ca 2+ channel may be a potential target for TRPA1-related pain. © 2017 Wiley Periodicals, Inc.

Upregulation of voltage-gated Ca2+ channels in mouse astrocytes infected with Theiler's murine encephalomyelitis virus (TMEV).

PubMed

Rubio, N; Almanza, A; Mercado, F; Arévalo, M-Á; Garcia-Segura, L M; Vega, R; Soto, E

2013-09-05

Theiler's murine encephalomyelitis virus (TMEV) induces demyelination in susceptible strains of mice through a CD4(+) Th1 T cell-mediated immunopathological process. TMEV infection produces a syndrome in mice that resembles multiple sclerosis. In this work, we focused on the increased expression of the genes encoding voltage-gated Ca(2+) channel subunits in SJL/J mouse astrocytes infected in culture with a BeAn strain of TMEV. Affymetrix DNA murine genome U74v2 DNA microarray hybridized with cRNA from mock- and TMEV-infected astrocytes revealed the upregulation of four sequences encoding Ca(2+)-binding and Ca(2+) channel subunit proteins. The DNA hybridization results were further validated using conventional RT-PCR and quantitative RT-PCR, demonstrating the increased expression of mRNA encoding channel subunit proteins. Western blotting also showed the increased synthesis of L- and N-type channel subunit specific proteins after infection. The reduced expression and the functional upregulation of functional voltage-gated Ca(2+) channels in mock- and TMEV-infected cells, respectively, was demonstrated using voltage clamp experiments. TMEV infection in mouse astrocytes induced a Ca(2+) current with a density proportional to the amount of viral particles used for infection. The use of Ca(2+) channel blockers, nimodipine and ω-conotoxin-GVIA, showed that both functional L- and N-type Ca(2+) channels were upregulated in infected astrocytes. The upregulation of Ca(2+) channels in astrocytes after TMEV infection provides insight into the molecular processes and potential role of astrocyte Ca(2+) dysregulation in the pathophysiology of encephalomyelitis and is important for the development of novel therapeutic strategies leading to prevention of neurodegeneration. Copyright © 2013 IBRO. Published by Elsevier Ltd. All rights reserved.

Molecular basis of potassium channels in pancreatic duct epithelial cells

PubMed Central

Hayashi, Mikio; Novak, Ivana

2013-01-01

Potassium channels regulate excitability, epithelial ion transport, proliferation, and apoptosis. In pancreatic ducts, K+ channels hyperpolarize the membrane potential and provide the driving force for anion secretion. This review focuses on the molecular candidates of functional K+ channels in pancreatic duct cells, including KCNN4 (KCa3.1), KCNMA1 (KCa1.1), KCNQ1 (Kv7.1), KCNH2 (Kv11.1), KCNH5 (Kv10.2), KCNT1 (KCa4.1), KCNT2 (KCa4.2), and KCNK5 (K2P5.1). We will give an overview of K+ channels with respect to their electrophysiological and pharmacological characteristics and regulation, which we know from other cell types, preferably in epithelia, and, where known, their identification and functions in pancreatic ducts and in adenocarcinoma cells. We conclude by pointing out some outstanding questions and future directions in pancreatic K+ channel research with respect to the physiology of secretion and pancreatic pathologies, including pancreatitis, cystic fibrosis, and cancer, in which the dysregulation or altered expression of K+ channels may be of importance. PMID:23962792

Evidence of K+ channel function in epithelial cell migration, proliferation, and repair

PubMed Central

Girault, Alban

2013-01-01

Efficient repair of epithelial tissue, which is frequently exposed to insults, is necessary to maintain its functional integrity. It is therefore necessary to better understand the biological and molecular determinants of tissue regeneration and to develop new strategies to promote epithelial repair. Interestingly, a growing body of evidence indicates that many members of the large and widely expressed family of K+ channels are involved in regulation of cell migration and proliferation, key processes of epithelial repair. First, we briefly summarize the complex mechanisms, including cell migration, proliferation, and differentiation, engaged after epithelial injury. We then present evidence implicating K+ channels in the regulation of these key repair processes. We also describe the mechanisms whereby K+ channels may control epithelial repair processes. In particular, changes in membrane potential, K+ concentration, cell volume, intracellular Ca2+, and signaling pathways following modulation of K+ channel activity, as well as physical interaction of K+ channels with the cytoskeleton or integrins are presented. Finally, we discuss the challenges to efficient, specific, and safe targeting of K+ channels for therapeutic applications to improve epithelial repair in vivo. PMID:24196531

Functional significance of M-type potassium channels in nociceptive cutaneous sensory endings

PubMed Central

Passmore, Gayle M.; Reilly, Joanne M.; Thakur, Matthew; Keasberry, Vanessa N.; Marsh, Stephen J.; Dickenson, Anthony H.; Brown, David A.

2012-01-01

M-channels carry slowly activating potassium currents that regulate excitability in a variety of central and peripheral neurons. Functional M-channels and their Kv7 channel correlates are expressed throughout the somatosensory nervous system where they may play an important role in controlling sensory nerve activity. Here we show that Kv7.2 immunoreactivity is expressed in the peripheral terminals of nociceptive primary afferents. Electrophysiological recordings from single afferents in vitro showed that block of M-channels by 3 μM XE991 sensitized Aδ- but not C-fibers to noxious heat stimulation and induced spontaneous, ongoing activity at 32°C in many Aδ-fibers. These observations were extended in vivo: intraplantar injection of XE991 selectively enhanced the response of deep dorsal horn (DH) neurons to peripheral mid-range mechanical and higher range thermal stimuli, consistent with a selective effect on Aδ-fiber peripheral terminals. These results demonstrate an important physiological role of M-channels in controlling nociceptive Aδ-fiber responses and provide a rationale for the nocifensive behaviors that arise following intraplantar injection of the M-channel blocker XE991. PMID:22593734

Spinocerebellar ataxia type 6 knockin mice develop a progressive neuronal dysfunction with age-dependent accumulation of mutant CaV2.1 channels

PubMed Central

Watase, Kei; Barrett, Curtis F.; Miyazaki, Taisuke; Ishiguro, Taro; Ishikawa, Kinya; Hu, Yuanxin; Unno, Toshinori; Sun, Yaling; Kasai, Sayumi; Watanabe, Masahiko; Gomez, Christopher M.; Mizusawa, Hidehiro; Tsien, Richard W.; Zoghbi, Huda Y.

2008-01-01

Spinocerebellar ataxia type 6 (SCA6) is a neurodegenerative disorder caused by CAG repeat expansions within the voltage-gated calcium (CaV) 2.1 channel gene. It remains controversial whether the mutation exerts neurotoxicity by changing the function of CaV2.1 channel or through a gain-of-function mechanism associated with accumulation of the expanded polyglutamine protein. We generated three strains of knockin (KI) mice carrying normal, expanded, or hyperexpanded CAG repeat tracts in the Cacna1a locus. The mice expressing hyperexpanded polyglutamine (Sca684Q) developed progressive motor impairment and aggregation of mutant CaV2.1 channels. Electrophysiological analysis of cerebellar Purkinje cells revealed similar Ca2+ channel current density among the three KI models. Neither voltage sensitivity of activation nor inactivation was altered in the Sca684Q neurons, suggesting that expanded CAG repeat per se does not affect the intrinsic electrophysiological properties of the channels. The pathogenesis of SCA6 is apparently linked to an age-dependent process accompanied by accumulation of mutant CaV2.1 channels. PMID:18687887

Modulation of low-voltage-activated T-type Ca²⁺ channels.

PubMed

Zhang, Yuan; Jiang, Xinghong; Snutch, Terrance P; Tao, Jin

2013-07-01

Low-voltage-activated T-type Ca²⁺ channels contribute to a wide variety of physiological functions, most predominantly in the nervous, cardiovascular and endocrine systems. Studies have documented the roles of T-type channels in sleep, neuropathic pain, absence epilepsy, cell proliferation and cardiovascular function. Importantly, novel aspects of the modulation of T-type channels have been identified over the last few years, providing new insights into their physiological and pathophysiological roles. Although there is substantial literature regarding modulation of native T-type channels, the underlying molecular mechanisms have only recently begun to be addressed. This review focuses on recent evidence that the Ca(v)3 subunits of T-type channels, Ca(v)3.1, Ca(v)3.2 and Ca(v)3.3, are differentially modulated by a multitude of endogenous ligands including anandamide, monocyte chemoattractant protein-1, endostatin, and redox and oxidizing agents. The review also provides an overview of recent knowledge gained concerning downstream pathways involving G-protein-coupled receptors. This article is part of a Special Issue entitled: Calcium channels. Copyright © 2012 Elsevier B.V. All rights reserved.

Calculation of the electron wave function in a graded-channel double-heterojunction modulation-doped field-effect transistor

NASA Technical Reports Server (NTRS)

Mui, D. S. L.; Patil, M. B.; Morkoc, H.

1989-01-01

Three double-heterojunction modulation-doped field-effect transistor structures with different channel composition are investigated theoretically. All of these transistors have an In(x)Ga(1-x)As channel sandwiched between two doped Al(0.3)Ga(0.7)As barriers with undoped spacer layers. In one of the structures, x varies from 0 from either heterojunction to 0.15 at the center of the channel quadratically; in the other two, constant values of x of 0 and 0.15 are used. The Poisson and Schroedinger equations are solved self-consistently for the electron wave function in all three cases. The results showed that the two-dimensional electron gas (2DEG) concentration in the channel of the quadratically graded structure is higher than the x = 0 one and slightly lower than the x = 0.15 one, and the mean distance of the 2DEG is closer to the center of the channel for this transistor than the other two. These two effects have important implications on the electron mobility in the channel.

Isolated pores dissected from human two-pore channel 2 are functional

PubMed Central

Penny, Christopher J.; Rahman, Taufiq; Sula, Altin; Miles, Andrew J.; Wallace, B. A.; Patel, Sandip

2016-01-01

Multi-domain voltage-gated ion channels appear to have evolved through sequential rounds of intragenic duplication from a primordial one-domain precursor. Whereas modularity within one-domain symmetrical channels is established, little is known about the roles of individual regions within more complex asymmetrical channels where the domains have undergone substantial divergence. Here we isolated and characterised both of the divergent pore regions from human TPC2, a two-domain channel that holds a key intermediate position in the evolution of voltage-gated ion channels. In HeLa cells, each pore localised to the ER and caused Ca2+ depletion, whereas an ER-targeted pore mutated at a residue that inactivates full-length TPC2 did not. Additionally, one of the pores expressed at high levels in E. coli. When purified, it formed a stable, folded tetramer. Liposomes reconstituted with the pore supported Ca2+ and Na+ uptake that was inhibited by known blockers of full-length channels. Computational modelling of the pore corroborated cationic permeability and drug interaction. Therefore, despite divergence, both pores are constitutively active in the absence of their partners and retain several properties of the wild-type pore. Such symmetrical ‘pore-only’ proteins derived from divergent channel domains may therefore provide tractable tools for probing the functional architecture of complex ion channels. PMID:27941820

Isolated pores dissected from human two-pore channel 2 are functional.

PubMed

Penny, Christopher J; Rahman, Taufiq; Sula, Altin; Miles, Andrew J; Wallace, B A; Patel, Sandip

2016-12-12

Multi-domain voltage-gated ion channels appear to have evolved through sequential rounds of intragenic duplication from a primordial one-domain precursor. Whereas modularity within one-domain symmetrical channels is established, little is known about the roles of individual regions within more complex asymmetrical channels where the domains have undergone substantial divergence. Here we isolated and characterised both of the divergent pore regions from human TPC2, a two-domain channel that holds a key intermediate position in the evolution of voltage-gated ion channels. In HeLa cells, each pore localised to the ER and caused Ca 2+ depletion, whereas an ER-targeted pore mutated at a residue that inactivates full-length TPC2 did not. Additionally, one of the pores expressed at high levels in E. coli. When purified, it formed a stable, folded tetramer. Liposomes reconstituted with the pore supported Ca 2+ and Na + uptake that was inhibited by known blockers of full-length channels. Computational modelling of the pore corroborated cationic permeability and drug interaction. Therefore, despite divergence, both pores are constitutively active in the absence of their partners and retain several properties of the wild-type pore. Such symmetrical 'pore-only' proteins derived from divergent channel domains may therefore provide tractable tools for probing the functional architecture of complex ion channels.

Mitochondrial Ion Channels/Transporters as Sensors and Regulators of Cellular Redox Signaling

PubMed Central

Ryu, Shin-Young; Jhun, Bong Sook; Hurst, Stephen

2014-01-01

Abstract Significance: Mitochondrial ion channels/transporters and the electron transport chain (ETC) serve as key sensors and regulators for cellular redox signaling, the production of reactive oxygen species (ROS) and nitrogen species (RNS) in mitochondria, and balancing cell survival and death. Although the functional and pharmacological characteristics of mitochondrial ion transport mechanisms have been extensively studied for several decades, the majority of the molecular identities that are responsible for these channels/transporters have remained a mystery until very recently. Recent Advances: Recent breakthrough studies uncovered the molecular identities of the diverse array of major mitochondrial ion channels/transporters, including the mitochondrial Ca2+ uniporter pore, mitochondrial permeability transition pore, and mitochondrial ATP-sensitive K+ channel. This new information enables us to form detailed molecular and functional characterizations of mitochondrial ion channels/transporters and their roles in mitochondrial redox signaling. Critical Issues: Redox-mediated post-translational modifications of mitochondrial ion channels/transporters and ETC serve as key mechanisms for the spatiotemporal control of mitochondrial ROS/RNS generation. Future Directions: Identification of detailed molecular mechanisms for redox-mediated regulation of mitochondrial ion channels will enable us to find novel therapeutic targets for many diseases that are associated with cellular redox signaling and mitochondrial ion channels/transporters. Antioxid. Redox Signal. 21, 987–1006. PMID:24180309

Estimating discharge in rivers using remotely sensed hydraulic information

USGS Publications Warehouse

Bjerklie, D.M.; Moller, D.; Smith, L.C.; Dingman, S.L.

2005-01-01

A methodology to estimate in-bank river discharge exclusively from remotely sensed hydraulic data is developed. Water-surface width and maximum channel width measured from 26 aerial and digital orthophotos of 17 single channel rivers and 41 SAR images of three braided rivers were coupled with channel slope data obtained from topographic maps to estimate the discharge. The standard error of the discharge estimates were within a factor of 1.5-2 (50-100%) of the observed, with the mean estimate accuracy within 10%. This level of accuracy was achieved using calibration functions developed from observed discharge. The calibration functions use reach specific geomorphic variables, the maximum channel width and the channel slope, to predict a correction factor. The calibration functions are related to channel type. Surface velocity and width information, obtained from a single C-band image obtained by the Jet Propulsion Laboratory's (JPL's) AirSAR was also used to estimate discharge for a reach of the Missouri River. Without using a calibration function, the estimate accuracy was +72% of the observed discharge, which is within the expected range of uncertainty for the method. However, using the observed velocity to calibrate the initial estimate improved the estimate accuracy to within +10% of the observed. Remotely sensed discharge estimates with accuracies reported in this paper could be useful for regional or continental scale hydrologic studies, or in regions where ground-based data is lacking. ?? 2004 Elsevier B.V. All rights reserved.

TPC2 polymorphisms associated with a hair pigmentation phenotype in humans result in gain of channel function by independent mechanisms.

PubMed

Chao, Yu-Kai; Schludi, Verena; Chen, Cheng-Chang; Butz, Elisabeth; Nguyen, O N Phuong; Müller, Martin; Krüger, Jens; Kammerbauer, Claudia; Ben-Johny, Manu; Vollmar, Angelika M; Berking, Carola; Biel, Martin; Wahl-Schott, Christian A; Grimm, Christian

2017-10-10

Two-pore channels (TPCs) are endolysosomal cation channels. Two members exist in humans, TPC1 and TPC2. Functional roles associated with the ubiquitously expressed TPCs include VEGF-induced neoangiogenesis, LDL-cholesterol trafficking and degradation, physical endurance under fasting conditions, autophagy regulation, the acrosome reaction in sperm, cancer cell migration, and intracellular trafficking of pathogens such as Ebola virus or bacterial toxins (e.g., cholera toxin). In a genome-wide association study for variants associated with human pigmentation characteristics two coding variants of TPC2, rs35264875 (encoding M484L) and rs3829241 (encoding G734E), have been found to be associated with a shift from brown to blond hair color. In two recent follow-up studies a role for TPC2 in pigmentation has been further confirmed. However, these human polymorphic variants have not been functionally characterized until now. The development of endolysosomal patch-clamp techniques has made it possible to investigate directly ion channel activities and characteristics in isolated endolysosomal organelles. We applied this technique here to scrutinize channel characteristics of the polymorphic TPC2 variants in direct comparison with WT. We found that both polymorphisms lead to a gain of channel function by independent mechanisms. We next conducted a clinical study with more than 100 blond- and brown/black-haired individuals. We performed a genotype/phenotype analysis and subsequently isolated fibroblasts from WT and polymorphic variant carriers for endolysosomal patch-clamp experimentation to confirm key in vitro findings.

Epithelial Sodium and Acid-Sensing Ion Channels

NASA Astrophysics Data System (ADS)

Kellenberger, Stephan

The epithelial Na+ channel (ENaC) and acid-sensing ion channels (ASICs) are non-voltage-gated Na+ channels that form their own subfamilies within the ENaC/degenerin ion channel family. ASICs are sensors of extracellular pH, and ENaC, whose main function is trans-epithelial Na+ transport, can sense extra- and intra-cellular Na+. In aldosterone-responsive epithelial cells of the kidney, ENaC plays a critical role in the control of sodium balance, blood volume and blood pressure. In airway epithelia, ENaC has a distinct role in controlling fluid reabsorption at the air-liquid interface, thereby determining the rate of mucociliary transport. In taste receptor cells of the tongue, ENaC is involved in salt taste sensation. ASICs have emerged as key sensors for extracellular protons in central and peripheral neurons. Although not all of their physiological and pathological functions are firmly established yet, there is good evidence for a role of ASICs in the brain in learning, expression of fear, and in neurodegeneration after ischaemic stroke. In sensory neurons, ASICs are involved in nociception and mechanosensation. ENaC and ASIC subunits share substantial sequence homology and the conservation of several functional domains. This chapter summarises our current understanding of the physiological functions and of the mechanisms of ion permeation, gating and regulation of ENaC and ASICs.

Modeling channel interference in an orbital angular momentum-multiplexed laser link

NASA Astrophysics Data System (ADS)

Anguita, Jaime A.; Neifeld, Mark A.; Vasic, Bane V.

2009-08-01

We study the effects of optical turbulence on the energy crosstalk among constituent orbital angular momentum (OAM) states in a vortex-based multi-channel laser communication link and determine channel interference in terms of turbulence strength and OAM state separation. We characterize the channel interference as a function of C2n and transmit OAM state, and propose probability models to predict the random fluctuations in the received signals for such architecture. Simulations indicate that turbulence-induced channel interference is mutually correlated across receive channels.

HCN1 channels in cerebellar Purkinje cells promote late stages of learning and constrain synaptic inhibition

PubMed Central

Rinaldi, Arianna; Defterali, Cagla; Mialot, Antoine; Garden, Derek L F; Beraneck, Mathieu; Nolan, Matthew F

2013-01-01

Neural computations rely on ion channels that modify neuronal responses to synaptic inputs. While single cell recordings suggest diverse and neurone type-specific computational functions for HCN1 channels, their behavioural roles in any single neurone type are not clear. Using a battery of behavioural assays, including analysis of motor learning in vestibulo-ocular reflex and rotarod tests, we find that deletion of HCN1 channels from cerebellar Purkinje cells selectively impairs late stages of motor learning. Because deletion of HCN1 modifies only a subset of behaviours involving Purkinje cells, we asked whether the channel also has functional specificity at a cellular level. We find that HCN1 channels in cerebellar Purkinje cells reduce the duration of inhibitory synaptic responses but, in the absence of membrane hyperpolarization, do not affect responses to excitatory inputs. Our results indicate that manipulation of subthreshold computation in a single neurone type causes specific modifications to behaviour. PMID:24000178

Using iPSC Models to Probe Regulation of Cardiac Ion Channel Function.

PubMed

Bruyneel, Arne A N; McKeithan, Wesley L; Feyen, Dries A M; Mercola, Mark

2018-05-25

Cardiovascular disease is the leading contributor to mortality and morbidity. Many deaths of heart failure patients can be attributed to sudden cardiac death due primarily to ventricular arrhythmia. Currently, most anti-arrhythmics modulate ion channel conductivity or β-adrenergic signaling, but these drugs have limited efficacy for some indications, and can potentially be proarrhythmic. Recent studies have shown that mutations in proteins other than cardiac ion channels may confer susceptibility to congenital as well as acquired arrhythmias. Additionally, ion channels themselves are subject to regulation at the levels of channel expression, trafficking and post-translational modification; thus, research into the regulation of ion channels may elucidate disease mechanisms and potential therapeutic targets for future drug development. This review summarizes the current knowledge of the molecular mechanisms of arrhythmia susceptibility and discusses technological advances such as induced pluripotent stem cell-derived cardiomyocytes, gene editing, functional genomics, and physiological screening platforms that provide a new paradigm for discovery of new therapeutic targets to treat congenital and acquired diseases of the heart rhythm.

Ion-binding properties of the ClC chloride selectivity filter

PubMed Central

Lobet, Séverine; Dutzler, Raimund

2006-01-01

The ClC channels are members of a large protein family of chloride (Cl−) channels and secondary active Cl− transporters. Despite their diverse functions, the transmembrane architecture within the family is conserved. Here we present a crystallographic study on the ion-binding properties of the ClC selectivity filter in the close homolog from Escherichia coli (EcClC). The ClC selectivity filter contains three ion-binding sites that bridge the extra- and intracellular solutions. The sites bind Cl− ions with mM affinity. Despite their close proximity within the filter, the three sites can be occupied simultaneously. The ion-binding properties are found conserved from the bacterial transporter EcClC to the human Cl− channel ClC-1, suggesting a close functional link between ion permeation in the channels and active transport in the transporters. In resemblance to K+ channels, ions permeate the ClC channel in a single file, with mutual repulsion between the ions fostering rapid conduction. PMID:16341087

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

PubMed

Nielsen, Jens Jung; Kristensen, Michael; Hellsten, Ylva; Bangsbo, Jens; Juel, Carsten

2003-02-01

The present study investigated the localization of ATP-sensitive K+ (KATP) channels in human skeletal muscle and the functional importance of these channels for human muscle K+ distribution at rest and during muscle activity. Membrane fractionation based on the giant vesicle technique or the sucrose-gradient technique in combination with Western blotting demonstrated that the KATP channels are mainly located in the sarcolemma. This localization was confirmed by immunohistochemical measurements. With the microdialysis technique, it was demonstrated that local application of the KATP channel inhibitor glibenclamide reduced (P < 0.05) interstitial K+ at rest from approximately 4.5 to 4.0 mM, whereas the concentration in the control leg remained constant. Glibenclamide had no effect on the interstitial K+ accumulation during knee-extensor exercise at a power output of 60 W. In contrast to in vitro conditions, the present study demonstrated that under in vivo conditions the KATP channels are active at rest and contribute to the accumulation of interstitial K+.

Structural model of the open–closed–inactivated cycle of prokaryotic voltage-gated sodium channels

PubMed Central

Bagnéris, Claire; Naylor, Claire E.; McCusker, Emily C.

2015-01-01

In excitable cells, the initiation of the action potential results from the opening of voltage-gated sodium channels. These channels undergo a series of conformational changes between open, closed, and inactivated states. Many models have been proposed for the structural transitions that result in these different functional states. Here, we compare the crystal structures of prokaryotic sodium channels captured in the different conformational forms and use them as the basis for examining molecular models for the activation, slow inactivation, and recovery processes. We compare structural similarities and differences in the pore domains, specifically in the transmembrane helices, the constrictions within the pore cavity, the activation gate at the cytoplasmic end of the last transmembrane helix, the C-terminal domain, and the selectivity filter. We discuss the observed differences in the context of previous models for opening, closing, and inactivation, and present a new structure-based model for the functional transitions. Our proposed prokaryotic channel activation mechanism is then compared with the activation transition in eukaryotic sodium channels. PMID:25512599

Evolutionarily Conserved, Multitasking TRP Channels: Lessons from Worms and Flies

PubMed Central

Venkatachalam, Kartik; Luo, Junjie; Montell, Craig

2015-01-01

The Transient Receptor Potential (TRP) channel family is comprised of a large group of cation-permeable channels, which display an extraordinary diversity of roles in sensory signaling. TRPs allow animals to detect chemicals, mechanical force, light, and changes in temperature. Consequently, these channels control a plethora of animal behaviors. Moreover, their functions are not limited to the classical senses, as they are cellular sensors, which are critical for ionic homeostasis and metabolism. Two genetically tractable invertebrate model organisms, Caenorhabditis elegans and Drosophila melanogaster, have led the way in revealing a wide array of sensory roles and behaviors that depend on TRP channels. Two overriding themes have emerged from these studies. First, TRPs are multitasking proteins, and second, many functions and modes of activation of these channels are evolutionarily conserved, including some that were formerly thought to be unique to invertebrates, such as phototransduction. Thus, worms and flies offer the potential to decipher roles for mammalian TRPs, which would otherwise not be suspected. PMID:24961975

Rhynchophylline from Uncaria rhynchophylla functionally turns delayed rectifiers into A-Type K+ channels.

PubMed

Chou, Chun-Hsiao; Gong, Chi-Li; Chao, Chia-Chia; Lin, Chia-Huei; Kwan, Chiu-Yin; Hsieh, Ching-Liang; Leung, Yuk-Man

2009-05-22

Rhynchophylline (1), a neuroprotective agent isolated from the traditional Chinese medicinal herb Uncaria rhynchophylla, was shown to affect voltage-gated K(+) (Kv) channel slow inactivation in mouse neuroblastoma N2A cells. Extracellular 1 (30 microM) accelerated the slow decay of Kv currents and shifted the steady-state inactivation curve to the left. Intracellular dialysis of 1 did not accelerate the slow current decay, suggesting that this compound acts extracellularly. In addition, the percent blockage of Kv currents by this substance was independent of the degree of depolarization and the intracellular K(+) concentration. Therefore, 1 did not appear to directly block the outer channel pore, with the results obtained suggesting that it drastically accelerated Kv channel slow inactivation. Interestingly, 1 also shifted the activation curve to the left. This alkaloid also strongly accelerated slow inactivation and caused a left shift of the activation curve of Kv1.2 channels heterologously expressed in HEK293 cells. Thus, this compound functionally turned delayed rectifiers into A-type K(+) channels.

The hitchhiker’s guide to the voltage-gated sodium channel galaxy

PubMed Central

2016-01-01

Eukaryotic voltage-gated sodium (Nav) channels contribute to the rising phase of action potentials and served as an early muse for biophysicists laying the foundation for our current understanding of electrical signaling. Given their central role in electrical excitability, it is not surprising that (a) inherited mutations in genes encoding for Nav channels and their accessory subunits have been linked to excitability disorders in brain, muscle, and heart; and (b) Nav channels are targeted by various drugs and naturally occurring toxins. Although the overall architecture and behavior of these channels are likely to be similar to the more well-studied voltage-gated potassium channels, eukaryotic Nav channels lack structural and functional symmetry, a notable difference that has implications for gating and selectivity. Activation of voltage-sensing modules of the first three domains in Nav channels is sufficient to open the channel pore, whereas movement of the domain IV voltage sensor is correlated with inactivation. Also, structure–function studies of eukaryotic Nav channels show that a set of amino acids in the selectivity filter, referred to as DEKA locus, is essential for Na+ selectivity. Structures of prokaryotic Nav channels have also shed new light on mechanisms of drug block. These structures exhibit lateral fenestrations that are large enough to allow drugs or lipophilic molecules to gain access into the inner vestibule, suggesting that this might be the passage for drug entry into a closed channel. In this Review, we will synthesize our current understanding of Nav channel gating mechanisms, ion selectivity and permeation, and modulation by therapeutics and toxins in light of the new structures of the prokaryotic Nav channels that, for the time being, serve as structural models of their eukaryotic counterparts. PMID:26712848

Molecular Targets for Antiepileptic Drug Development

PubMed Central

Meldrum, Brian S.; Rogawski, Michael A.

2007-01-01

Summary This review considers how recent advances in the physiology of ion channels and other potential molecular targets, in conjunction with new information on the genetics of idiopathic epilepsies, can be applied to the search for improved antiepileptic drugs (AEDs). Marketed AEDs predominantly target voltage-gated cation channels (the α subunits of voltage-gated Na+ channels and also T-type voltage-gated Ca2+ channels) or influence GABA-mediated inhibition. Recently, α2–δ voltage-gated Ca2+ channel subunits and the SV2A synaptic vesicle protein have been recognized as likely targets. Genetic studies of familial idiopathic epilepsies have identified numerous genes associated with diverse epilepsy syndromes, including genes encoding Na+ channels and GABAA receptors, which are known AED targets. A strategy based on genes associated with epilepsy in animal models and humans suggests other potential AED targets, including various voltage-gated Ca2+ channel subunits and auxiliary proteins, A- or M-type voltage-gated K+ channels, and ionotropic glutamate receptors. Recent progress in ion channel research brought about by molecular cloning of the channel subunit proteins and studies in epilepsy models suggest additional targets, including G-protein-coupled receptors, such as GABAB and metabotropic glutamate receptors; hyperpolarization-activated cyclic nucleotide-gated cation (HCN) channel subunits, responsible for hyperpolarization-activated current Ih; connexins, which make up gap junctions; and neurotransmitter transporters, particularly plasma membrane and vesicular transporters for GABA and glutamate. New information from the structural characterization of ion channels, along with better understanding of ion channel function, may allow for more selective targeting. For example, Na+ channels underlying persistent Na+ currents or GABAA receptor isoforms responsible for tonic (extrasynaptic) currents represent attractive targets. The growing understanding of the pathophysiology of epilepsy and the structural and functional characterization of the molecular targets provide many opportunities to create improved epilepsy therapies. PMID:17199015

ClC-K chloride channels: emerging pathophysiology of Bartter syndrome type 3.

PubMed

Andrini, Olga; Keck, Mathilde; Briones, Rodolfo; Lourdel, Stéphane; Vargas-Poussou, Rosa; Teulon, Jacques

2015-06-15

The mutations in the CLCNKB gene encoding the ClC-Kb chloride channel are responsible for Bartter syndrome type 3, one of the four variants of Bartter syndrome in the genetically based nomenclature. All forms of Bartter syndrome are characterized by hypokalemia, metabolic alkalosis, and secondary hyperaldosteronism, but Bartter syndrome type 3 has the most heterogeneous presentation, extending from severe to very mild. A relatively large number of CLCNKB mutations have been reported, including gene deletions and nonsense or missense mutations. However, only 20 CLCNKB mutations have been functionally analyzed, due to technical difficulties regarding ClC-Kb functional expression in heterologous systems. This review provides an overview of recent progress in the functional consequences of CLCNKB mutations on ClC-Kb chloride channel activity. It has been observed that 1) all ClC-Kb mutants have an impaired expression at the membrane; and 2) a minority of the mutants combines reduced membrane expression with altered pH-dependent channel gating. Although further investigation is needed to fully characterize disease pathogenesis, Bartter syndrome type 3 probably belongs to the large family of conformational diseases, in which the mutations destabilize channel structure, inducing ClC-Kb retention in the endoplasmic reticulum and accelerated channel degradation. Copyright © 2015 the American Physiological Society.

An extensive investigation of work function modulated trapezoidal recessed channel MOSFET

NASA Astrophysics Data System (ADS)

Lenka, Annada Shankar; Mishra, Sikha; Mishra, Satyaranjan; Bhanja, Urmila; Mishra, Guru Prasad

2017-11-01

The concept of silicon on insulator (SOI) and grooved gate help to lessen the short channel effects (SCEs). Again the work function modulation along the metal gate gives a better drain current due to the uniform electric field along the channel. So all these concepts are combined and used in the proposed MOSFET structure for more improved performance. In this work, trapezoidal recessed channel silicon on insulator (TRC-SOI) MOSFET and work function modulated trapezoidal recessed channel silicon on insulator (WFM-TRC-SOI) MOSFET are compared with DC and RF parameters and later linearity of both the devices is tested. An analytical model is formulated by using a 2-D Poisson's equation and develops a compact equation for threshold voltage using minimum surface potential. In this work we analyze the effect of negative junction depth and the corner angle on various device parameters such as minimum surface potential, sub-threshold slope (SS), drain induced barrier lowering (DIBL) and threshold voltage. The analysis interprets that the switching performance of WFM-TRC-SOI MOSFET surpasses TRC-SOI MOSFET in terms of high Ion/Ioff ratio and also the proposed structure can minimize the short channel effects (SCEs) in RF application. The validity of proposed model has been verified with simulation result performed on Sentaurus TCAD device simulator.

Genotype–phenotype correlations in neonatal epilepsies caused by mutations in the voltage sensor of Kv7.2 potassium channel subunits

PubMed Central

Miceli, Francesco; Soldovieri, Maria Virginia; Ambrosino, Paolo; Barrese, Vincenzo; Migliore, Michele; Cilio, Maria Roberta; Taglialatela, Maurizio

2013-01-01

Mutations in the KV7.2 gene encoding for voltage-dependent K+ channel subunits cause neonatal epilepsies with wide phenotypic heterogeneity. Two mutations affecting the same positively charged residue in the S4 domain of KV7.2 have been found in children affected with benign familial neonatal seizures (R213W mutation) or with neonatal epileptic encephalopathy with severe pharmacoresistant seizures and neurocognitive delay, suppression-burst pattern at EEG, and distinct neuroradiological features (R213Q mutation). To examine the molecular basis for this strikingly different phenotype, we studied the functional characteristics of mutant channels by using electrophysiological techniques, computational modeling, and homology modeling. Functional studies revealed that, in homomeric or heteromeric configuration with KV7.2 and/or KV7.3 subunits, both mutations markedly destabilized the open state, causing a dramatic decrease in channel voltage sensitivity. These functional changes were (i) more pronounced for channels incorporating R213Q- than R213W-carrying KV7.2 subunits; (ii) proportional to the number of mutant subunits incorporated; and (iii) fully restored by the neuronal Kv7 activator retigabine. Homology modeling confirmed a critical role for the R213 residue in stabilizing the activated voltage sensor configuration. Modeling experiments in CA1 hippocampal pyramidal cells revealed that both mutations increased cell firing frequency, with the R213Q mutation prompting more dramatic functional changes compared with the R213W mutation. These results suggest that the clinical disease severity may be related to the extent of the mutation-induced functional K+ channel impairment, and set the preclinical basis for the potential use of Kv7 openers as a targeted anticonvulsant therapy to improve developmental outcome in neonates with KV7.2 encephalopathy. PMID:23440208

Near-Channel Versus Watershed Controls on Sediment Rating Curves

NASA Astrophysics Data System (ADS)

Vaughan, Angus A.; Belmont, Patrick; Hawkins, Charles P.; Wilcock, Peter

2017-10-01

Predicting riverine suspended sediment flux is a fundamental problem in geomorphology, with important implications for water quality, land and water resource management, and aquatic ecosystem health. To advance understanding, we evaluated environmental and landscape factors that influence sediment rating curves (SRCs). We generated SRCs with recent total suspended solids (TSSs) and discharge data from 45 gages on 36 rivers throughout the state of Minnesota, USA. Watersheds range from 32 to 14,600 km2 and represent distinct settings regarding topography, land cover, and geologic history. Rivers exhibited three distinct SRC shapes: simple power functions, threshold power functions, and peaked or negative-slope functions. We computed SRC exponents and coefficients (describing the steepness of the relation and the TSS concentration at median flows, respectively). In addition to quantifying watershed topography, climate/hydrology, geology, soil type, and land cover, we used lidar topography to characterize the near-channel environment upstream of gages. We used random forest models to analyze relations between SRC parameters and attributes of the watershed and the near-channel environment. The models correctly classify 78% of SRC shapes and explain 37%-60% of variance in SRC parameters. We find that SRC steepness (exponent) is strongly related to near-channel morphological characteristics including near-channel relief, channel gradient, and presence of lakes along the local channel network, but not to land use. In contrast, land use influences TSS concentrations at moderate and low flow. These findings suggest that the near-channel environment controls changes in TSS as flows increase, whereas land use drives median and low flow TSS conditions.

Focus on Kir7.1: physiology and channelopathy

PubMed Central

Kumar, Mohit; Pattnaik, Bikash R

2014-01-01

Genetic studies have linked alterations in Kir7.1 channel to diverse pathologies. We summarize functional relevance of Kir7.1 channel in retinal pigment epithelium (RPE), regulation of channel function by various cytoplasmic metabolites, and mutations that cause channelopathies. At the apical membrane of RPE, K+ channels contribute to subretinal K+ homeostasis and support Na+/K+ pump and Na+-K+-2Cl− cotransporter function by providing a pathway for K+ secretion. Electrophysiological studies have established that barium- and cesium-sensitive inwardly rectifying K+ (Kir) channels make up a major component of the RPE apical membrane K+ conductance. Native human RPE expresses transcripts for Kir1.1, Kir2.1, Kir2.2, Kir3.1, Kir3.4, Kir4.2, and Kir6.1, albeit at levels at least 50-fold lower than Kir7.1. Kir7.1 is structurally similar to other Kir channels, consisting of 2 trans-membrane domains, a pore-forming loop that contains the selectivity filter, and 2 cytoplasmic polar tails. Within the cytoplasmic structure, clusters of amino acid sequences form regulatory domains that interact with cellular metabolites and control the opening and closing of the channel. Recent evidence indicated that intrinsic sequence motifs present in Kir7.1 control surface expression. Mutant Kir7.1 channels are associated with inherited eye pathologies such as Snowflake Vitreoretinal Degeneration (SVD) and Lebers Congenital Amaurosis (LCA16). Based on the current evidence, mutations implicated in channelopathies have the potential to be used for genetic testing to diagnose blindness due to Kir7.1. PMID:25558901

Revealing dynamically-organized receptor ion channel clusters in live cells by a correlated electric recording and super-resolution single-molecule imaging approach.

PubMed

Yadav, Rajeev; Lu, H Peter

2018-03-28

The N-methyl-d-aspartate (NMDA) receptor ion-channel is activated by the binding of ligands, along with the application of action potential, important for synaptic transmission and memory functions. Despite substantial knowledge of the structure and function, the gating mechanism of the NMDA receptor ion channel for electric on-off signals is still a topic of debate. We investigate the NMDA receptor partition distribution and the associated channel's open-close electric signal trajectories using a combined approach of correlating single-molecule fluorescence photo-bleaching, single-molecule super-resolution imaging, and single-channel electric patch-clamp recording. Identifying the compositions of NMDA receptors, their spatial organization and distributions over live cell membranes, we observe that NMDA receptors are organized inhomogeneously: nearly half of the receptor proteins are individually dispersed; whereas others exist in heterogeneous clusters of around 50 nm in size as well as co-localized within the diffraction limited imaging area. We demonstrate that inhomogeneous interactions and partitions of the NMDA receptors can be a cause of the heterogeneous gating mechanism of NMDA receptors in living cells. Furthermore, comparing the imaging results with the ion-channel electric current recording, we propose that the clustered NMDA receptors may be responsible for the variation in the current amplitude observed in the on-off two-state ion-channel electric signal trajectories. Our findings shed new light on the fundamental structure-function mechanism of NMDA receptors and present a conceptual advancement of the ion-channel mechanism in living cells.

Closed-channel culture system for efficient and reproducible differentiation of human pluripotent stem cells into islet cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Hirano, Kunio; Konagaya, Shuhei; Turner, Alexander

Human pluripotent stem cells (hPSCs) are thought to be a promising cell-source solution for regenerative medicine due to their indefinite proliferative potential and ability to differentiate to functional somatic cells. However, issues remain with regard to achieving reproducible differentiation of cells with the required functionality for realizing human transplantation therapies and with regard to reducing the potential for bacterial or fungal contamination. To meet these needs, we have developed a closed-channel culture device and corresponding control system. Uniformly-sized spheroidal hPSCs aggregates were formed inside wells within a closed-channel and maintained continuously throughout the culture process. Functional islet-like endocrine cell aggregatesmore » were reproducibly induced following a 30-day differentiation protocol. Our system shows an easily scalable, novel method for inducing PSC differentiation with both purity and functionality. - Highlights: • A simple, closed-channel-based, semi-automatic culture system is proposed. • Uniform cell aggregate formation and culture is realized in microwell structure. • Functional islet cells are successfully induced following 30-plus-day protocol. • System requires no daily medium replacement and reduces contamination risk.« less

Glycine Potentiates AMPA Receptor Function through Metabotropic Activation of GluN2A-Containing NMDA Receptors

PubMed Central

Li, Li-Jun; Hu, Rong; Lujan, Brendan; Chen, Juan; Zhang, Jian-Jian; Nakano, Yasuko; Cui, Tian-Yuan; Liao, Ming-Xia; Chen, Jin-Cao; Man, Heng-Ye; Feng, Hua; Wan, Qi

2016-01-01

NMDA receptors are Ca2+-permeable ion channels. The activation of NMDA receptors requires agonist glutamate and co-agonist glycine. Recent evidence indicates that NMDA receptor also has metabotropic function. Here we report that in cultured mouse hippocampal neurons, glycine increases AMPA receptor-mediated currents independent of the channel activity of NMDA receptors and the activation of glycine receptors. The potentiation of AMPA receptor function by glycine is antagonized by the inhibition of ERK1/2. In the hippocampal neurons and in the HEK293 cells transfected with different combinations of NMDA receptors, glycine preferentially acts on GluN2A-containing NMDA receptors (GluN2ARs), but not GluN2B-containing NMDA receptors (GluN2BRs), to enhance ERK1/2 phosphorylation independent of the channel activity of GluN2ARs. Without requiring the channel activity of GluN2ARs, glycine increases AMPA receptor-mediated currents through GluN2ARs. Thus, these results reveal a metabotropic function of GluN2ARs in mediating glycine-induced potentiation of AMPA receptor function via ERK1/2 activation. PMID:27807405

DOE Office of Scientific and Technical Information (OSTI.GOV)

Vesely, P.; Michel, N.; Toivanen, J.

For over four decades, the Skyrme functional within various parametrizations has been used to calculate nuclear properties. In the last few years there was a number of attempts to improve its performance and introduce generalized forms. In particular, the most general phenomenological quasi-local energy density functional, which contains all combinations of density, spin-density, and their derivatives up to the sixth order (N{sup 3}LO), was proposed in reference [1]. Since in the phenomenological functional approaches the particle-particle (pp) interaction channel is treated independently from the particle-hole (ph) channel, there remains a question of what pairing interaction is suitable to use withinmore » the N{sup 3}LO energy functional. In our study, we use the separable, finite-range, translationally invariant form given in [2], which we generalize to the arbitrary angular momentum channel. We discuss the application of this pairing interaction within the N{sup 3}LO energy functional.« less

Preliminary Studies of Acute Cadmium Administration Effects on the Calcium-Activated Potassium (SKCa and BKCa) Channels and Na+/K+-ATPase Activity in Isolated Aortic Rings of Rats.

PubMed

Vassallo, Dalton V; Almenara, Camila C P; Broseghini-Filho, Gilson Brás; Teixeira, Ariane Calazans; da Silva, David Chaves F; Angeli, Jhuli K; Padilha, Alessandra S

2018-06-01

Cadmium is an environmental pollutant closely linked with cardiovascular diseases that seems to involve endothelium dysfunction and reduced nitric oxide (NO) bioavailability. Knowing that NO causes dilatation through the activation of potassium channels and Na + /K + -ATPase, we aimed to determine whether acute cadmium administration (10 μM) alters the participation of K + channels, voltage-activated calcium channel, and Na + /K + -ATPase activity in vascular function of isolated aortic rings of rats. Cadmium did not modify the acetylcholine-induced relaxation. After L-NAME addition, the relaxation induced by acetylcholine was abolished in presence or absence of cadmium, suggesting that acutely, this metal did not change NO release. However, tetraethylammonium (a nonselective K + channels blocker) reduced acetylcholine-induced relaxation but this effect was lower in the preparations with cadmium, suggesting a decrease of K + channels function in acetylcholine response after cadmium incubation. Apamin (a selective blocker of small Ca 2+ -activated K + channels-SK Ca ), iberiotoxin (a selective blocker of large-conductance Ca 2+ -activated K + channels-BK Ca ), and verapamil (a blocker of calcium channel) reduced the endothelium-dependent relaxation only in the absence of cadmium. Finally, cadmium decreases Na + /K + -ATPase activity. Our results provide evidence that the cadmium acute incubation unaffected the calcium-activated potassium channels (SK Ca and BK Ca ) and voltage-calcium channels on the acetylcholine vasodilatation. In addition, acute cadmium incubation seems to reduce the Na + /K + -ATPase activity.

Isolation and Characterization of a High Affinity Peptide Inhibitor of ClC-2 Chloride Channels*

PubMed Central

Thompson, Christopher H.; Olivetti, Pedro R.; Fuller, Matthew D.; Freeman, Cody S.; McMaster, Denis; French, Robert J.; Pohl, Jan; Kubanek, Julia; McCarty, Nael A.

2009-01-01

The ClC protein family includes voltage-gated chloride channels and chloride/proton exchangers. In eukaryotes, ClC proteins regulate membrane potential of excitable cells, contribute to epithelial transport, and aid in lysosomal acidification. Although structure/function studies of ClC proteins have been aided greatly by the available crystal structures of a bacterial ClC chloride/proton exchanger, the availability of useful pharmacological tools, such as peptide toxin inhibitors, has lagged far behind that of their cation channel counterparts. Here we report the isolation, from Leiurus quinquestriatus hebraeus venom, of a peptide toxin inhibitor of the ClC-2 chloride channel. This toxin, GaTx2, inhibits ClC-2 channels with a voltage-dependent apparent KD of ∼20 pm, making it the highest affinity inhibitor of any chloride channel. GaTx2 slows ClC-2 activation by increasing the latency to first opening by nearly 8-fold but is unable to inhibit open channels, suggesting that this toxin inhibits channel activation gating. Finally, GaTx2 specifically inhibits ClC-2 channels, showing no inhibitory effect on a battery of other major classes of chloride channels and voltage-gated potassium channels. GaTx2 is the first peptide toxin inhibitor of any ClC protein. The high affinity and specificity displayed by this toxin will make it a very powerful pharmacological tool to probe ClC-2 structure/function. PMID:19574231

The L‐type Ca2+ channel facilitates abnormal metabolic activity in the cTnI‐G203S mouse model of hypertrophic cardiomyopathy

PubMed Central

Viola, Helena; Johnstone, Victoria; Cserne Szappanos, Henrietta; Richman, Tara; Tsoutsman, Tatiana; Filipovska, Aleksandra; Semsarian, Christopher

2016-01-01

Key points Genetic mutations in cardiac troponin I (cTnI) are associated with development of hypertrophic cardiomyopathy characterized by myocyte remodelling, disorganization of cytoskeletal proteins and altered energy metabolism.The L‐type Ca2+ channel is the main route for calcium influx and is crucial to cardiac excitation and contraction. The channel also regulates mitochondrial function in the heart by a functional communication between the channel and mitochondria via the cytoskeletal network.We find that L‐type Ca2+ channel kinetics are altered in cTnI‐G203S cardiac myocytes and that activation of the channel causes a significantly greater increase in mitochondrial membrane potential and metabolic activity in cTnI‐G203S cardiac myocytes.These responses occur as a result of impaired communication between the L‐type Ca2+ channel and cytoskeletal protein F‐actin, involving decreased movement of actin–myosin and block of the mitochondrial voltage‐dependent anion channel, resulting in a ‘hypermetabolic’ mitochondrial state.We propose that L‐type Ca2+ channel antagonists, such as diltiazem, might be effective in reducing the cardiomyopathy by normalizing mitochondrial metabolic activity. Abstract Genetic mutations in cardiac troponin I (cTnI) account for 5% of families with hypertrophic cardiomyopathy. Hypertrophic cardiomyopathy is associated with disorganization of cytoskeletal proteins and altered energy metabolism. The L‐type Ca2+ channel (ICa‐L) plays an important role in regulating mitochondrial function. This involves a functional communication between the channel and mitochondria via the cytoskeletal network. We investigate the role of ICa‐L in regulating mitochondrial function in 25‐ to 30‐week‐old cardiomyopathic mice expressing the human disease‐causing mutation Gly203Ser in cTnI (cTnI‐G203S). The inactivation rate of ICa‐L is significantly faster in cTnI‐G203S myocytes [cTnI‐G203S: τ1 = 40.68 ± 3.22, n = 10 vs. wild‐type (wt): τ1 = 59.05 ± 6.40, n = 6, P < 0.05]. Activation of ICa‐L caused a greater increase in mitochondrial membrane potential (Ψm, 29.19 ± 1.85%, n = 15 vs. wt: 18.84 ± 2.01%, n = 10, P < 0.05) and metabolic activity (24.40 ± 6.46%, n = 8 vs. wt: 9.98 ± 1.57%, n = 9, P < 0.05). The responses occurred because of impaired communication between ICa‐L and F‐actin, involving lack of dynamic movement of actin–myosin and block of the mitochondrial voltage‐dependent anion channel. Similar responses were observed in precardiomyopathic mice. ICa‐L antagonists nisoldipine and diltiazem decreased Ψm to basal levels. We conclude that the Gly203Ser mutation leads to impaired functional communication between ICa‐L and mitochondria, resulting in a ‘hypermetabolic’ state. This might contribute to development of cTnI‐G203S cardiomyopathy because the response is present in young precardiomyopathic mice. ICa‐L antagonists might be effective in reducing the cardiomyopathy by altering mitochondrial function. PMID:27062056

Disruption of ATP-sensitive potassium channel function in skeletal muscles promotes production and secretion of musclin

DOE Office of Scientific and Technical Information (OSTI.GOV)

Sierra, Ana, E-mail: ana-sierra@uiowa.edu; Subbotina, Ekaterina, E-mail: ekaterina-subbotina@uiowa.edu; Zhu, Zhiyong, E-mail: zhiyong-zhu@uiowa.edu

Sarcolemmal ATP-sensitive potassium (K{sub ATP}) channels control skeletal muscle energy use through their ability to adjust membrane excitability and related cell functions in accordance with cellular metabolic status. Mice with disrupted skeletal muscle K{sub ATP} channels exhibit reduced adipocyte size and increased fatty acid release into the circulation. As yet, the molecular mechanisms underlying this link between skeletal muscle K{sub ATP} channel function and adipose mobilization have not been established. Here, we demonstrate that skeletal muscle-specific disruption of K{sub ATP} channel function in transgenic (TG) mice promotes production and secretion of musclin. Musclin is a myokine with high homology tomore » atrial natriuretic peptide (ANP) that enhances ANP signaling by competing for elimination. Augmented musclin production in TG mice is driven by a molecular cascade resulting in enhanced acetylation and nuclear exclusion of the transcription factor forkhead box O1 (FOXO1) – an inhibitor of transcription of the musclin encoding gene. Musclin production/secretion in TG is paired with increased mobilization of fatty acids and a clear trend toward increased circulating ANP, an activator of lipolysis. These data establish K{sub ATP} channel-dependent musclin production as a potential mechanistic link coupling “local” skeletal muscle energy consumption with mobilization of bodily resources from fat. Understanding such mechanisms is an important step toward designing interventions to manage metabolic disorders including those related to excess body fat and associated co-morbidities. - Highlights: • ATP-sensitive K{sup +} channels regulate musclin production by skeletal muscles. • Lipolytic ANP signaling is promoted by augmented skeletal muscle musclin production. • Skeletal muscle musclin transcription is promoted by a CaMKII/HDAC/FOXO1 pathway. • Musclin links adipose mobilization to energy use in K{sub ATP} channel deficient skeletal muscle.« less

A New Splice Variant of Large Conductance Ca2+-activated K+ (BK) Channel α Subunit Alters Human Chondrocyte Function.

PubMed

Suzuki, Yoshiaki; Ohya, Susumu; Yamamura, Hisao; Giles, Wayne R; Imaizumi, Yuji

2016-11-11

Large conductance Ca 2+ -activated K + (BK) channels play essential roles in both excitable and non-excitable cells. For example, in chondrocytes, agonist-induced Ca 2+ release from intracellular store activates BK channels, and this hyperpolarizes these cells, augments Ca 2+ entry, and forms a positive feed-back mechanism for Ca 2+ signaling and stimulation-secretion coupling. In the present study, functional roles of a newly identified splice variant in the BK channel α subunit (BKαΔe2) were examined in a human chondrocyte cell line, OUMS-27, and in a HEK293 expression system. Although BKαΔe2 lacks exon2, which codes the intracellular S0-S1 linker (Glu-127-Leu-180), significant expression was detected in several tissues from humans and mice. Molecular image analyses revealed that BKαΔe2 channels are not expressed on plasma membrane but can traffic to the plasma membrane after forming hetero-tetramer units with wild-type BKα (BKαWT). Single-channel current analyses demonstrated that BKα hetero-tetramers containing one, two, or three BKαΔe2 subunits are functional. These hetero-tetramers have a smaller single channel conductance and exhibit lower trafficking efficiency than BKαWT homo-tetramers in a stoichiometry-dependent manner. Site-directed mutagenesis of residues in exon2 identified Helix2 and the linker to S1 (Trp-158-Leu-180, particularly Arg-178) as an essential segment for channel function including voltage dependence and trafficking. BKαΔe2 knockdown in OUMS-27 chondrocytes increased BK current density and augmented the responsiveness to histamine assayed as cyclooxygenase-2 gene expression. These findings provide significant new evidence that BKαΔe2 can modulate cellular responses to physiological stimuli in human chondrocyte and contribute under pathophysiological conditions, such as osteoarthritis. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

A-1048400 is a novel, orally active, state-dependent neuronal calcium channel blocker that produces dose-dependent antinociception without altering hemodynamic function in rats.

PubMed

Scott, Victoria E; Vortherms, Timothy A; Niforatos, Wende; Swensen, Andrew M; Neelands, Torben; Milicic, Ivan; Banfor, Patricia N; King, Andrew; Zhong, Chengmin; Simler, Gricelda; Zhan, Cenchen; Bratcher, Natalie; Boyce-Rustay, Janel M; Zhu, Chang Z; Bhatia, Pramila; Doherty, George; Mack, Helmut; Stewart, Andrew O; Jarvis, Michael F

2012-02-01

Blockade of voltage-gated Ca²⁺ channels on sensory nerves attenuates neurotransmitter release and membrane hyperexcitability associated with chronic pain states. Identification of small molecule Ca²⁺ channel blockers that produce significant antinociception in the absence of deleterious hemodynamic effects has been challenging. In this report, two novel structurally related compounds, A-686085 and A-1048400, were identified that potently block N-type (IC₅₀=0.8 μM and 1.4 μM, respectively) and T-type (IC₅₀=4.6 μM and 1.2 μM, respectively) Ca²⁺ channels in FLIPR based Ca²⁺ flux assays. A-686085 also potently blocked L-type Ca²⁺ channels (EC₅₀=0.6 μM), however, A-1048400 was much less active in blocking this channel (EC₅₀=28 μM). Both compounds dose-dependently reversed tactile allodynia in a model of capsaicin-induced secondary hypersensitivity with similar potencies (EC₅₀=300-365 ng/ml). However, A-686085 produced dose-related decreases in mean arterial pressure at antinociceptive plasma concentrations in the rat, while A-1048400 did not significantly alter hemodynamic function at supra-efficacious plasma concentrations. Electrophysiological studies demonstrated that A-1048400 blocks native N- and T-type Ca²⁺ currents in rat dorsal root ganglion neurons (IC₅₀=3.0 μM and 1.6 μM, respectively) in a voltage-dependent fashion. In other experimental pain models, A-1048400 dose-dependently attenuated nociceptive, neuropathic and inflammatory pain at doses that did not alter psychomotor or hemodynamic function. The identification of A-1048400 provides further evidence that voltage-dependent inhibition of neuronal Ca²⁺ channels coupled with pharmacological selectivity vs. L-type Ca²⁺ channels can provide robust antinociception in the absence of deleterious effects on hemodynamic or psychomotor function. Copyright © 2011 Elsevier Inc. All rights reserved.

Functional validation of Ca2+-binding residues from the crystal structure of the BK ion channel.

PubMed

Kshatri, Aravind S; Gonzalez-Hernandez, Alberto J; Giraldez, Teresa

2018-04-01

BK channels are dually regulated by voltage and Ca 2+ , providing a cellular mechanism to couple electrical and chemical signalling. Intracellular Ca 2+ concentration is sensed by a large cytoplasmic region in the channel known as "gating ring", which is formed by four tandems of regulator of conductance for K + (RCK1 and RCK2) domains. The recent crystal structure of the full-length BK channel from Aplysia californica has provided new information about the residues involved in Ca 2+ coordination at the high-affinity binding sites located in the RCK1 and RCK2 domains, as well as their cooperativity. Some of these residues have not been previously studied in the human BK channel. In this work we have investigated, through site directed mutagenesis and electrophysiology, the effects of these residues on channel activation by voltage and Ca 2+ . Our results demonstrate that the side chains of two non-conserved residues proposed to coordinate Ca 2+ in the A. californica structure (G523 and E591) have no apparent functional role in the human BK Ca 2+ sensing mechanism. Consistent with the crystal structure, our data indicate that in the human channel the conserved residue R514 participates in Ca 2+ coordination in the RCK1 binding site. Additionally, this study provides functional evidence indicating that R514 also interacts with residues E902 and Y904 connected to the Ca 2+ binding site in RCK2. Interestingly, it has been proposed that this interaction may constitute a structural correlate underlying the cooperative interactions between the two high-affinity Ca 2+ binding sites regulating the Ca 2+ dependent gating of the BK channel. This article is part of a Special Issue entitled: Beyond the Structure-Function Horizon of Membrane Proteins edited by Ute Hellmich, Rupak Doshi and Benjamin McIlwain. Copyright © 2017 Elsevier B.V. All rights reserved.

A complicated complex: Ion channels, voltage sensing, cell membranes and peptide inhibitors.

PubMed

Zhang, Alan H; Sharma, Gagan; Undheim, Eivind A B; Jia, Xinying; Mobli, Mehdi

2018-04-21

Voltage-gated ion channels (VGICs) are specialised ion channels that have a voltage dependent mode of action, where ion conduction, or gating, is controlled by a voltage-sensing mechanism. VGICs are critical for electrical signalling and are therefore important pharmacological targets. Among these, voltage-gated sodium channels (Na V s) have attracted particular attention as potential analgesic targets. Na V s, however, comprise several structurally similar subtypes with unique localisations and distinct functions, ranging from amplification of action potentials in nociception (e.g. Na V 1.7) to controlling electrical signalling in cardiac function (Na V 1.5). Understanding the structural basis of Na V function is therefore of great significance, both to our knowledge of electrical signalling and in development of subtype and state selective drugs. An important tool in this pursuit has been the use of peptides from animal venoms as selective Na V modulators. In this review, we look at peptides, particularly from spider venoms, that inhibit Na V s by binding to the voltage sensing domain (VSD) of this channel, known as gating modifier toxins (GMT). In the first part of the review, we look at the structural determinants of voltage sensing in VGICs, the gating cycle and the conformational changes that accompany VSD movement. Next, the modulation of the analgesic target Na V 1.7 by GMTs is reviewed to develop bioinformatic tools that, based on sequence information alone, can identify toxins that are likely to inhibit this channel. The same approach is also used to define VSD sequences, other than that from Na V 1.7, which are likely to be sensitive to this class of toxins. The final section of the review focuses on the important role of the cellular membrane in channel modulation and also how the lipid composition affects measurements of peptide-channel interactions both in binding kinetics measurements in solution and in cell-based functional assays. Copyright © 2018 Elsevier B.V. All rights reserved.

Effects of Volatile Aromatic Anesthetics on Voltage-Gated Na+ Channels Expressed in Xenopus Oocytes

PubMed Central

Horishita, Takafumi; Eger, Edmond I; Harris, R. Adron

2008-01-01

Background Many inhaled anesthetics inhibit voltage-gated sodium channels at clinically relevant concentrations, and suppression of neurotransmitter release by these agents results, at least partly, from decreased presynaptic sodium channel activity. Volatile aromatic anesthetics can inhibit N-methyl-D-aspartate (NMDA) receptor function and enhance γ-amino butyric acid A (GABAA) receptor function, but these effects depend strongly on the chemical properties of the aromatic ompounds. The present study tested whether diverse aromatic anesthetics consistently inhibit sodium channel function. Methods We studied the effect of eight aromatic anesthetics on Nav1.2 sodium channels with β1 subunits, using whole-cell, two-electrode voltage-clamp techniques in Xenopus oocytes. Results All aromatic anesthetics inhibited INa (sodium currents) at a holding potential which produce half-maximal current (V1/2) (partial depolarization); inhibition was modest with 1,3,5-trifluorobenzene (8 ± 2%), pentafluorobenzene (13 ± 2%), and hexafluorobenzene (13 ± 2%), but greater with benzene (37 ± 2%), fluorobenzene (39 ± 2%), 1,2-difluorobenzene (48 ± 2%), 1,4-difluorobenzene (31 ± 3%), and 1,2,4-trifluorobenzene (33 ± 1%). Such dichotomous effects were noted by others for NMDA and GABAA receptors. Parallel, but much smaller inhibition, was found for INa at a holding potential which produced near maximal current (−90 mV) (VH-90), and hexafluorobenzene caused small (6 ± 1%) potentiation of this current. These changes in sodium channel function were correlated with effectiveness for inhibiting NMDA receptors, with lipid solubility of the compounds, with molecular volume, and with cation-π interactions. Conclusion Aromatic compounds vary in their actions on the kinetics of sodium channel gating and this may underlie their variable inhibition. The range of inhibition produced by MAC concentrations of inhaled anesthetics indicates that sodium channel inhibition may underlie the action of some of these anesthetics but not others. PMID:18931215

Aberrant Splicing Induced by Dysregulated Rbfox2 Produces Enhanced Function of CaV1.2 Calcium Channel and Vascular Myogenic Tone in Hypertension.

PubMed

Zhou, Yingying; Fan, Jia; Zhu, Huayuan; Ji, Li; Fan, Wenyong; Kapoor, Isha; Wang, Yue; Wang, Yuan; Zhu, Guoqing; Wang, Juejin

2017-12-01

Calcium influx from activated voltage-gated calcium channel Ca V 1.2 in vascular smooth muscle cells is indispensable for maintaining myogenic tone and blood pressure. The function of Ca V 1.2 channel can be optimized by alternative splicing, one of post-transcriptional modification mechanisms. The splicing factor Rbfox2 is known to regulate the Ca V 1.2 pre-mRNA alternative splicing events during neuronal development. However, Rbfox2's roles in modulating the key function of vascular Ca V 1.2 channel and in the pathogenesis of hypertension remain elusive. Here, we report that the proportion of Ca V 1.2 channels with alternative exon 9* is increased by 10.3%, whereas that with alternative exon 33 is decreased by 10.5% in hypertensive arteries. Surprisingly, the expression level of Rbfox2 is increased ≈3-folds, presumably because of the upregulation of a dominant-negative isoform of Rbfox2. In vascular smooth muscle cells, we find that knockdown of Rbfox2 dynamically increases alternative exon 9*, whereas decreases exon 33 inclusion of Ca V 1.2 channels. By patch-clamp studies, we show that diminished Rbfox2-induced alternative splicing shifts the steady-state activation and inactivation curves of vascular Ca V 1.2 calcium channel to hyperpolarization, which makes the window current potential to more negative. Moreover, siRNA-mediated knockdown of Rbfox2 increases the pressure-induced vascular myogenic tone of rat mesenteric artery. Taken together, our data indicate that Rbfox2 modulates the functions of vascular Ca V 1.2 calcium channel by dynamically regulating the expressions of alternative exons 9* and 33, which in turn affects the vascular myogenic tone. Therefore, our work suggests a key role for Rbfox2 in hypertension, which provides a rational basis for designing antihypertensive therapies. © 2017 American Heart Association, Inc.

Site-Directed Spin Labeling Reveals Pentameric Ligand-Gated Ion Channel Gating Motions

PubMed Central

Dellisanti, Cosma D.; Ghosh, Borna; Hanson, Susan M.; Raspanti, James M.; Grant, Valerie A.; Diarra, Gaoussou M.; Schuh, Abby M.; Satyshur, Kenneth; Klug, Candice S.; Czajkowski, Cynthia

2013-01-01

Pentameric ligand-gated ion channels (pLGICs) are neurotransmitter-activated receptors that mediate fast synaptic transmission. In pLGICs, binding of agonist to the extracellular domain triggers a structural rearrangement that leads to the opening of an ion-conducting pore in the transmembrane domain and, in the continued presence of neurotransmitter, the channels desensitize (close). The flexible loops in each subunit that connect the extracellular binding domain (loops 2, 7, and 9) to the transmembrane channel domain (M2–M3 loop) are essential for coupling ligand binding to channel gating. Comparing the crystal structures of two bacterial pLGIC homologues, ELIC and the proton-activated GLIC, suggests channel gating is associated with rearrangements in these loops, but whether these motions accurately predict the motions in functional lipid-embedded pLGICs is unknown. Here, using site-directed spin labeling (SDSL) electron paramagnetic resonance (EPR) spectroscopy and functional GLIC channels reconstituted into liposomes, we examined if, and how far, the loops at the ECD/TMD gating interface move during proton-dependent gating transitions from the resting to desensitized state. Loop 9 moves ∼9 Å inward toward the channel lumen in response to proton-induced desensitization. Loop 9 motions were not observed when GLIC was in detergent micelles, suggesting detergent solubilization traps the protein in a nonactivatable state and lipids are required for functional gating transitions. Proton-induced desensitization immobilizes loop 2 with little change in position. Proton-induced motion of the M2–M3 loop was not observed, suggesting its conformation is nearly identical in closed and desensitized states. Our experimentally derived distance measurements of spin-labeled GLIC suggest ELIC is not a good model for the functional resting state of GLIC, and that the crystal structure of GLIC does not correspond to a desensitized state. These findings advance our understanding of the molecular mechanisms underlying pLGIC gating. PMID:24260024

Channel width dependence of electrical characteristics of a-Si:H TFTs under bending stresses

NASA Astrophysics Data System (ADS)

Oh, Hyungon; Cho, Kyoungah; Kim, Sangsig

2017-04-01

In this study, we investigate the electrical characteristics of bendable a-Si:H thin-film transistors (TFTs) with various channel widths as a function of bending stress. Compared with a narrower channel TFT, a wider channel TFT exhibits a stable performance even at a bending strain of 1.3%. Our stress and strain distribution analysis reveals an inverse relationship between the channel width and the channel stress. As the channel width widens from 8 to 50 μm, the stress experienced by the middle channel region decreases from 545 to 277 MPa. Moreover, a 50 μm-channel-width TFT operates stably even after a 15 000 bending cycle while the 8 μm-channel-width TFT fails to operate after a 2000 bending cycle.

Functional studies of RYR1 mutations in the skeletal muscle ryanodine receptor using human RYR1 complementary DNA.

PubMed

Sato, Keisaku; Pollock, Neil; Stowell, Kathryn M

2010-06-01

Malignant hyperthermia is associated with mutations within the gene encoding the skeletal muscle ryanodine receptor, the calcium channel that releases Ca from sarcoplasmic reticulum stores triggering muscle contraction, and other metabolic activities. More than 200 variants have been identified in the ryanodine receptor, but only some of these have been shown to functionally affect the calcium channel. To implement genetic testing for malignant hyperthermia, variants must be shown to alter the function of the channel. A number of different ex vivo methods can be used to demonstrate functionality, as long as cells from human patients can be obtained and cultured from at least two unrelated families. Because malignant hyperthermia is an uncommon disorder and many variants seem to be private, including the newly identified H4833Y mutation, these approaches are limited. The authors cloned the human skeletal muscle ryanodine receptor complementary DNA and expressed both normal and mutated forms in HEK-293 cells and carried out functional analysis using ryanodine binding assays in the presence of a specific agonist, 4-chloro-m-cresol, and the antagonist Mg. Transiently expressed human ryanodine receptor proteins colocalized with an endoplasmic reticulum marker in HEK-293 cells. Ryanodine binding assays confirmed that mutations causing malignant hyperthermia resulted in a hypersensitive channel, while those causing central core disease resulted in a hyposensitive channel. The functional assays validate recombinant human skeletal muscle ryanodine receptor for analysis of variants and add an additional mutation (H4833Y) to the repertoire of mutations that can be used for the genetic diagnosis of malignant hyperthermia.

Voltage-dependent ion channels in the mouse RPE: comparison with Norrie disease mice.

PubMed

Wollmann, Guido; Lenzner, Steffen; Berger, Wolfgang; Rosenthal, Rita; Karl, Mike O; Strauss, Olaf

2006-03-01

We studied electrophysiological properties of cultured retinal pigment epithelial (RPE) cells from mouse and a mouse model for Norrie disease. Wild-type RPE cells revealed the expression of ion channels known from other species: delayed-rectifier K(+) channels composed of Kv1.3 subunits, inward rectifier K(+) channels, Ca(V)1.3 L-type Ca(2+) channels and outwardly rectifying Cl(-) channels. Expression pattern and the ion channel characteristics current density, blocker sensitivity, kinetics and voltage-dependence were compared in cells from wild-type and Norrie mice. Although no significant differences were observed, our study provides a base for future studies on ion channel function and dysfunction in transgenic mouse models.

Molecular Basis of Cardiac Delayed Rectifier Potassium Channel Function and Pharmacology.

PubMed

Wu, Wei; Sanguinetti, Michael C

2016-06-01

Human cardiomyocytes express 3 distinct types of delayed rectifier potassium channels. Human ether-a-go-go-related gene (hERG) channels conduct the rapidly activating current IKr; KCNQ1/KCNE1 channels conduct the slowly activating current IKs; and Kv1.5 channels conduct an ultrarapid activating current IKur. Here the authors provide a general overview of the mechanistic and structural basis of ion selectivity, gating, and pharmacology of the 3 types of cardiac delayed rectifier potassium ion channels. Most blockers bind to S6 residues that line the central cavity of the channel, whereas activators interact with the channel at 4 symmetric binding sites outside the cavity. Copyright © 2016 Elsevier Inc. All rights reserved.

[Voltage-gated potassium channels and human neurological diseases].

PubMed

Jin, Hong-Wei; Wang, Xiao-Liang

2002-01-01

Voltage-gated potassium channels (Kv) is the largest, most complex in potassium channel superfamily. It can be divided into Kv alpha subunit and auxiliary two groups. The roles of some Kv channels types, e.g. rapidly inactivating (A-Type channel) and muscarine sensitive channels (M-type channel) are beginning to be understood. They are prominent in nervous system, acting in delicate and accurate ways to control or modify many physiological and pathological functions including membrane excitability, neurotransmitter release, cell proliferation or degeneration, signal transduction in neuronal network. Many human neurological disease pathogenesis are found to be related to mutant of Kv-channels subunit or subtype, such as, learning and memory impairing, ataxia, epilepsy, deafness, etc.

A nanoscale bio-inspired light-harvesting system developed from self-assembled alkyl-functionalized metallochlorin nano-aggregates

NASA Astrophysics Data System (ADS)

Ocakoglu, Kasim; Joya, Khurram S.; Harputlu, Ersan; Tarnowska, Anna; Gryko, Daniel T.

2014-07-01

Self-assembled supramolecular organization of nano-structured biomimetic light-harvesting modules inside solid-state nano-templates can be exploited to develop excellent light-harvesting materials for artificial photosynthetic devices. We present here a hybrid light-harvesting system mimicking the chlorosomal structures of the natural photosynthetic system using synthetic zinc chlorin units (ZnChl-C6, ZnChl-C12 and ZnChl-C18) that are self-aggregated inside the anodic aluminum oxide (AAO) nano-channel membranes. AAO nano-templates were modified with a TiO2 matrix and functionalized with long hydrophobic chains to facilitate the formation of supramolecular Zn-chlorin aggregates. The transparent Zn-chlorin nano-aggregates inside the alkyl-TiO2 modified AAO nano-channels have a diameter of ~120 nm in a 60 μm length channel. UV-Vis studies and fluorescence emission spectra further confirm the formation of the supramolecular ZnChl aggregates from monomer molecules inside the alkyl-functionalized nano-channels. Our results prove that the novel and unique method can be used to produce efficient and stable light-harvesting assemblies for effective solar energy capture through transparent and stable nano-channel ceramic materials modified with bio-mimetic molecular self-assembled nano-aggregates.Self-assembled supramolecular organization of nano-structured biomimetic light-harvesting modules inside solid-state nano-templates can be exploited to develop excellent light-harvesting materials for artificial photosynthetic devices. We present here a hybrid light-harvesting system mimicking the chlorosomal structures of the natural photosynthetic system using synthetic zinc chlorin units (ZnChl-C6, ZnChl-C12 and ZnChl-C18) that are self-aggregated inside the anodic aluminum oxide (AAO) nano-channel membranes. AAO nano-templates were modified with a TiO2 matrix and functionalized with long hydrophobic chains to facilitate the formation of supramolecular Zn-chlorin aggregates. The transparent Zn-chlorin nano-aggregates inside the alkyl-TiO2 modified AAO nano-channels have a diameter of ~120 nm in a 60 μm length channel. UV-Vis studies and fluorescence emission spectra further confirm the formation of the supramolecular ZnChl aggregates from monomer molecules inside the alkyl-functionalized nano-channels. Our results prove that the novel and unique method can be used to produce efficient and stable light-harvesting assemblies for effective solar energy capture through transparent and stable nano-channel ceramic materials modified with bio-mimetic molecular self-assembled nano-aggregates. Electronic supplementary information (ESI) available. See DOI: 10.1039/c4nr01661k

The relation of turbulence to diffusion in open-channel flows

USGS Publications Warehouse

Keefer, Thomas N.

1971-01-01

The exponent in the power-law equation describing the decay of scalar quantities downstream of a jet is a linear function of the shear velocity of the channel. The length of the core region of a jet is a power-law function of the jet strength with the exponent depending on boundary roughness.

Acid-sensing ion channels: trafficking and synaptic function.

PubMed

Zha, Xiang-ming

2013-01-02

Extracellular acidification occurs in the brain with elevated neural activity, increased metabolism, and neuronal injury. This reduction in pH can have profound effects on brain function because pH regulates essentially every single biochemical reaction. Therefore, it is not surprising to see that Nature evolves a family of proteins, the acid-sensing ion channels (ASICs), to sense extracellular pH reduction. ASICs are proton-gated cation channels that are mainly expressed in the nervous system. In recent years, a growing body of literature has shown that acidosis, through activating ASICs, contributes to multiple diseases, including ischemia, multiple sclerosis, and seizures. In addition, ASICs play a key role in fear and anxiety related psychiatric disorders. Several recent reviews have summarized the importance and therapeutic potential of ASICs in neurological diseases, as well as the structure-function relationship of ASICs. However, there is little focused coverage on either the basic biology of ASICs or their contribution to neural plasticity. This review will center on these topics, with an emphasis on the synaptic role of ASICs and molecular mechanisms regulating the spatial distribution and function of these ion channels.

Pacific Northwest tide channel utilization by fish as an ecosystem service

EPA Science Inventory

Background/Question/Methods: Saltwater marsh tide channels are considered to be important in the ecology of estuarine fish serving both as a refuge and as a provider of enhanced food resources. However, this presumed function of tide channels in Pacific Northwest estuaries has ...