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Sample records for channel ryanodine receptor

  1. Modulation of cardiac ryanodine receptor channels by alkaline earth cations.

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    Paula L Diaz-Sylvester

    Full Text Available Cardiac ryanodine receptor (RyR2 function is modulated by Ca(2+ and Mg(2+. To better characterize Ca(2+ and Mg(2+ binding sites involved in RyR2 regulation, the effects of cytosolic and luminal earth alkaline divalent cations (M(2+: Mg(2+, Ca(2+, Sr(2+, Ba(2+ were studied on RyR2 from pig ventricle reconstituted in bilayers. RyR2 were activated by M(2+ binding to high affinity activating sites at the cytosolic channel surface, specific for Ca(2+ or Sr(2+. This activation was interfered by Mg(2+ and Ba(2+ acting at low affinity M(2+-unspecific binding sites. When testing the effects of luminal M(2+ as current carriers, all M(2+ increased maximal RyR2 open probability (compared to Cs(+, suggesting the existence of low affinity activating M(2+-unspecific sites at the luminal surface. Responses to M(2+ vary from channel to channel (heterogeneity. However, with luminal Ba(2+or Mg(2+, RyR2 were less sensitive to cytosolic Ca(2+ and caffeine-mediated activation, openings were shorter and voltage-dependence was more marked (compared to RyR2 with luminal Ca(2+or Sr(2+. Kinetics of RyR2 with mixtures of luminal Ba(2+/Ca(2+ and additive action of luminal plus cytosolic Ba(2+ or Mg(2+ suggest luminal M(2+ differentially act on luminal sites rather than accessing cytosolic sites through the pore. This suggests the presence of additional luminal activating Ca(2+/Sr(2+-specific sites, which stabilize high P(o mode (less voltage-dependent and increase RyR2 sensitivity to cytosolic Ca(2+ activation. In summary, RyR2 luminal and cytosolic surfaces have at least two sets of M(2+ binding sites (specific for Ca(2+ and unspecific for Ca(2+/Mg(2+ that dynamically modulate channel activity and gating status, depending on SR voltage.

  2. Coupling of SK channels, L-type Ca2+ channels, and ryanodine receptors in cardiomyocytes.

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    Zhang, Xiao-Dong; Coulibaly, Zana A; Chen, Wei Chun; Ledford, Hannah A; Lee, Jeong Han; Sirish, Padmini; Dai, Gu; Jian, Zhong; Chuang, Frank; Brust-Mascher, Ingrid; Yamoah, Ebenezer N; Chen-Izu, Ye; Izu, Leighton T; Chiamvimonvat, Nipavan

    2018-03-16

    Small-conductance Ca 2+ -activated K + (SK) channels regulate the excitability of cardiomyocytes by integrating intracellular Ca 2+ and membrane potentials on a beat-to-beat basis. The inextricable interplay between activation of SK channels and Ca 2+ dynamics suggests the pathology of one begets another. Yet, the exact mechanistic underpinning for the activation of cardiac SK channels remains unaddressed. Here, we investigated the intracellular Ca 2+ microdomains necessary for SK channel activation. SK currents coupled with Ca 2+ influx via L-type Ca 2+ channels (LTCCs) continued to be elicited after application of caffeine, ryanodine or thapsigargin to deplete SR Ca 2+ store, suggesting that LTCCs provide the immediate Ca 2+ microdomain for the activation of SK channels in cardiomyocytes. Super-resolution imaging of SK2, Ca v 1.2 Ca 2+ channel, and ryanodine receptor 2 (RyR2) was performed to quantify the nearest neighbor distances (NND) and localized the three molecules within hundreds of nanometers. The distribution of NND between SK2 and RyR2 as well as SK2 and Ca v 1.2 was bimodal, suggesting a spatial relationship between the channels. The activation mechanism revealed by our study paved the way for the understanding of the roles of SK channels on the feedback mechanism to regulate the activities of LTCCs and RyR2 to influence local and global Ca 2+ signaling.

  3. Double-Nanodomain Coupling of Calcium Channels, Ryanodine Receptors, and BK Channels Controls the Generation of Burst Firing.

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    Irie, Tomohiko; Trussell, Laurence O

    2017-11-15

    Action potentials clustered into high-frequency bursts play distinct roles in neural computations. However, little is known about ionic currents that control the duration and probability of these bursts. We found that, in cartwheel inhibitory interneurons of the dorsal cochlear nucleus, the likelihood of bursts and the interval between their spikelets were controlled by Ca 2+ acting across two nanodomains, one between plasma membrane P/Q Ca 2+ channels and endoplasmic reticulum (ER) ryanodine receptors and another between ryanodine receptors and large-conductance, voltage- and Ca 2+ -activated K + (BK) channels. Each spike triggered Ca 2+ -induced Ca 2+ release (CICR) from the ER immediately beneath somatic, but not axonal or dendritic, plasma membrane. Moreover, immunolabeling demonstrated close apposition of ryanodine receptors and BK channels. Double-nanodomain coupling between somatic plasma membrane and hypolemmal ER cisterns provides a unique mechanism for rapid control of action potentials on the millisecond timescale. Copyright © 2017 Elsevier Inc. All rights reserved.

  4. Channel Gating Dependence on Pore Lining Helix Glycine Residues in Skeletal Muscle Ryanodine Receptor.

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    Mei, Yingwu; Xu, Le; Mowrey, David D; Mendez Giraldez, Raul; Wang, Ying; Pasek, Daniel A; Dokholyan, Nikolay V; Meissner, Gerhard

    2015-07-10

    Type 1 ryanodine receptors (RyR1s) release Ca(2+) from the sarcoplasmic reticulum to initiate skeletal muscle contraction. The role of RyR1-G4934 and -G4941 in the pore-lining helix in channel gating and ion permeation was probed by replacing them with amino acid residues of increasing side chain volume. RyR1-G4934A, -G4941A, and -G4941V mutant channels exhibited a caffeine-induced Ca(2+) release response in HEK293 cells and bound the RyR-specific ligand [(3)H]ryanodine. In single channel recordings, significant differences in the number of channel events and mean open and close times were observed between WT and RyR1-G4934A and -G4941A. RyR1-G4934A had reduced K(+) conductance and ion selectivity compared with WT. Mutations further increasing the side chain volume at these positions (G4934V and G4941I) resulted in reduced caffeine-induced Ca(2+) release in HEK293 cells, low [(3)H]ryanodine binding levels, and channels that were not regulated by Ca(2+) and did not conduct Ca(2+) in single channel measurements. Computational predictions of the thermodynamic impact of mutations on protein stability indicated that although the G4934A mutation was tolerated, the G4934V mutation decreased protein stability by introducing clashes with neighboring amino acid residues. In similar fashion, the G4941A mutation did not introduce clashes, whereas the G4941I mutation resulted in intersubunit clashes among the mutated isoleucines. Co-expression of RyR1-WT with RyR1-G4934V or -G4941I partially restored the WT phenotype, which suggested lessening of amino acid clashes in heterotetrameric channel complexes. The results indicate that both glycines are important for RyR1 channel function by providing flexibility and minimizing amino acid clashes. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

  5. SH Oxidation Stimulates Calcium Release Channels (Ryanodine Receptors From Excitable Cells

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    CECILIA HIDALGO

    2000-01-01

    Full Text Available The effects of redox reagents on the activity of the intracellular calcium release channels (ryanodine receptors of skeletal and cardiac muscle, or brain cortex neurons, was examined. In lipid bilayer experiments, oxidizing agents (2,2'-dithiodipyridine or thimerosal modified the calcium dependence of all single channels studied. After controlled oxidation channels became active at sub µM calcium concentrations and were not inhibited by increasing the calcium concentration to 0.5 mM. Subsequent reduction reversed these effects. Channels purified from amphibian skeletal muscle exhibited the same behavior, indicating that the SH groups responsible for modifying the calcium dependence belong to the channel protein. Parallel experiments that measured calcium release through these channels in sarcoplasmic reticulum vesicles showed that following oxidation, the channels were no longer inhibited by sub mM concentrations of Mg2+. It is proposed that channel redox state controls the high affinity sites responsible for calcium activation as well as the low affinity sites involved in Mg2+ inhibition of channel activity. The possible physiological and pathological implications of these results are discussed

  6. Ca2+ and voltage dependence of cardiac ryanodine receptor channel block by sphingosylphosphorylcholine.

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    Yasukochi, Midori; Uehara, Akira; Kobayashi, Sei; Berlin, Joshua R

    2003-03-01

    The effect of sphingosylphosphorylcholine (SPC) on the cytoplasmic Ca(2+) and voltage dependence of channel gating by cardiac ryanodine receptors (RyR) was examined in lipid bilayer experiments. Micromolar concentrations of the lysosphingolipid SPC added to cis solutions rapidly and reversibly decreased the single-channel open probability (P(o)) of reconstituted RyR channels. The SPC-induced decrease in P(o) was marked by an increase in mean closed time and burst-like channel gating. Gating kinetics during intraburst periods were unchanged from those observed in the absence of the sphingolipid, although SPC induced a long-lived closed state that appeared to explain the observed decrease in channel P(o). SPC effects were observed over a broad range of cis [Ca(2+)] but were not competitive with Ca(2+). Interestingly, the sphingolipid-induced, long-lived closed state displayed voltage-dependent kinetics, even though other channel gating kinetics were not sensitive to voltage. Assuming SPC effects represent channel blockade, these results suggest that the blocking rate is independent of voltage whereas the unblocking rate is voltage dependent. Together, these results suggest that SPC binds directly to the cytoplasmic side of the RyR protein in a location in or near the membrane dielectric, but distinct from cytoplasmic Ca(2+) binding sites on the protein.

  7. Intermolecular failure of L-type Ca2+ channel and ryanodine receptor signaling in hypertrophy.

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    Ming Xu

    2007-02-01

    Full Text Available Pressure overload-induced hypertrophy is a key step leading to heart failure. The Ca(2+-induced Ca(2+ release (CICR process that governs cardiac contractility is defective in hypertrophy/heart failure, but the molecular mechanisms remain elusive. To examine the intermolecular aspects of CICR during hypertrophy, we utilized loose-patch confocal imaging to visualize the signaling between a single L-type Ca(2+ channel (LCC and ryanodine receptors (RyRs in aortic stenosis rat models of compensated (CHT and decompensated (DHT hypertrophy. We found that the LCC-RyR intermolecular coupling showed a 49% prolongation in coupling latency, a 47% decrease in chance of hit, and a 72% increase in chance of miss in DHT, demonstrating a state of "intermolecular failure." Unexpectedly, these modifications also occurred robustly in CHT due at least partially to decreased expression of junctophilin, indicating that intermolecular failure occurs prior to cellular manifestations. As a result, cell-wide Ca(2+ release, visualized as "Ca(2+ spikes," became desynchronized, which contrasted sharply with unaltered spike integrals and whole-cell Ca(2+ transients in CHT. These data suggested that, within a certain limit, termed the "stability margin," mild intermolecular failure does not damage the cellular integrity of excitation-contraction coupling. Only when the modification steps beyond the stability margin does global failure occur. The discovery of "hidden" intermolecular failure in CHT has important clinical implications.

  8. Functionally heterogenous ryanodine receptors in avian cerebellum.

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    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.

  9. Ryanodine receptors, a family of intracellular calcium ion channels, are expressed throughout early vertebrate development

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    Wu Houdini HT

    2011-12-01

    Full Text Available Abstract Background Calcium signals ([Ca2+]i direct many aspects of embryo development but their regulation is not well characterised. Ryanodine receptors (RyRs are a family of intracellular Ca2+ release channels that control the flux of Ca2+ from internal stores into the cytosol. RyRs are primarily known for their role in excitation-contraction coupling in adult striated muscle and ryr gene mutations are implicated in several human diseases. Current evidence suggests that RyRs do not have a major role to play prior to organogenesis but regulate tissue differentiation. Findings The sequences of the five zebrafish ryr genes were confirmed, their evolutionary relationship established and the primary sequences compared to other vertebrates, including humans. RyRs are differentially expressed in slow (ryr1a, fast (ryr3 and both types (ryr1b of developing skeletal muscle. There are two ryr2 genes (ryr2a and ryr2b which are expressed exclusively in developing CNS and cardiac tissue, respectively. In addition, ryr3 and ryr2a mRNA is detectable in the initial stages of development, prior to embryonic axis formation. Conclusions Our work reveals that zebrafish ryr genes are differentially expressed throughout the developing embryo from cleavage onwards. The data suggests that RyR-regulated Ca2+ signals are associated with several aspects of embryonic development, from organogenesis through to the differentiation of the musculoskeletal, cardiovascular and nervous system. These studies will facilitate further work to explore the developmental function of RyRs in each of these tissue types.

  10. Association of cardiac myosin binding protein-C with the ryanodine receptor channel: putative retrograde regulation?

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    Stanczyk, Paulina J; Seidel, Monika; White, Judith; Viero, Cedric; George, Christopher H; Zissimopoulos, Spyros; Lai, F Anthony

    2018-06-21

    The cardiac muscle ryanodine receptor-Ca 2+ release channel (RyR2) constitutes the sarcoplasmic reticulum (SR) Ca 2+ efflux mechanism that initiates myocyte contraction, while cardiac myosin binding protein-C (cMyBP-C) mediates regulation of acto-myosin cross-bridge cycling. In this report, we provide the first evidence for the presence of direct interaction between these two proteins, forming a RyR2:cMyBP-C complex. The C-terminus of cMyBP-C binds with the RyR2 N-terminus in mammalian cells and is not mediated by a fibronectin-like domain. Notably, we detected complex formation between both recombinant cMyBP-C and RyR2, as well as with the native proteins in cardiac tissue. Cellular Ca 2+ dynamics in HEK293 cells is altered upon co-expression of cMyBP-C and RyR2, with lowered frequency of RyR2-mediated spontaneous Ca 2+ oscillations, suggesting cMyBP-C exerts a potential inhibitory effect on RyR2-dependent Ca 2+ release. Discovery of a functional RyR2 association with cMyBP-C provides direct evidence for a putative mechanistic link between cytosolic soluble cMyBP-C and SR-mediated Ca 2+ release, via RyR2. Importantly, this interaction may have clinical relevance to the observed cMyBP-C and RyR2 dysfunction in cardiac pathologies, such as hypertrophic cardiomyopathy. © 2018. Published by The Company of Biologists Ltd.

  11. Abnormal interactions of calsequestrin with the ryanodine receptor calcium release channel complex linked to exercise-induced sudden cardiac death

    NARCIS (Netherlands)

    Terentyev, Dmitry; Nori, Alessandra; Santoro, Massimo; Viatchenko-Karpinski, Serge; Kubalova, Zuzana; Gyorke, Inna; Terentyeva, Radmila; Vedamoorthyrao, Srikanth; Blom, Nico A.; Valle, Giorgia; Napolitano, Carlo; Williams, Simon C.; Volpe, Pompeo; Priori, Silvia G.; Gyorke, Sandor

    2006-01-01

    Catecholaminergic polymorphic ventricular tachycardia (CPVT) is a familial arrhythmogenic disorder associated with mutations in the cardiac ryanodine receptor (RyR2) and cardiac calsequestrin (CASQ2) genes. Previous in vitro studies suggested that RyR2 and CASQ2 interact as parts of a multimolecular

  12. Oxygen-coupled Redox Regulation of the Skeletal Muscle Ryanodine Receptor/Ca2+ Release Channel (RyR1)

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    Sun, Qi-An; Wang, Benlian; Miyagi, Masaru; Hess, Douglas T.; Stamler, Jonathan S.

    2013-01-01

    In mammalian skeletal muscle, Ca2+ release from the sarcoplasmic reticulum (SR) through the ryanodine receptor/Ca2+-release channel RyR1 can be enhanced by S-oxidation or S-nitrosylation of separate Cys residues, which are allosterically linked. S-Oxidation of RyR1 is coupled to muscle oxygen tension (pO2) through O2-dependent production of hydrogen peroxide by SR-resident NADPH oxidase 4. In isolated SR (SR vesicles), an average of six to eight Cys thiols/RyR1 monomer are reversibly oxidized at high (21% O2) versus low pO2 (1% O2), but their identity among the 100 Cys residues/RyR1 monomer is unknown. Here we use isotope-coded affinity tag labeling and mass spectrometry (yielding 93% coverage of RyR1 Cys residues) to identify 13 Cys residues subject to pO2-coupled S-oxidation in SR vesicles. Eight additional Cys residues are oxidized at high versus low pO2 only when NADPH levels are supplemented to enhance NADPH oxidase 4 activity. pO2-sensitive Cys residues were largely non-overlapping with those identified previously as hyperreactive by administration of exogenous reagents (three of 21) or as S-nitrosylated. Cys residues subject to pO2-coupled oxidation are distributed widely within the cytoplasmic domain of RyR1 in multiple functional domains implicated in RyR1 activity-regulating interactions with the L-type Ca2+ channel (dihydropyridine receptor) and FK506-binding protein 12 as well as in “hot spot” regions containing sites of mutation implicated in malignant hyperthermia and central core disease. pO2-coupled disulfide formation was identified, whereas neither S-glutathionylated nor sulfenamide-modified Cys residues were observed. Thus, physiological redox regulation of RyR1 by endogenously generated hydrogen peroxide is exerted through dynamic disulfide formation involving multiple Cys residues. PMID:23798702

  13. Involvement of plasma membrane Ca2+ channels, IP3 receptors, and ryanodine receptors in the generation of spontaneous rhythmic contractions of the cricket lateral oviduct.

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    Tamashiro, Hirotake; Yoshino, Masami

    2014-12-01

    In the present study, the isolated cricket (Gryllus bimaculatus) lateral oviduct exhibited spontaneous rhythmic contractions (SRCs) with a frequency of 0.29±0.009 Hz (n=43) and an amplitude of 14.6±1.25 mg (n=29). SRCs completely disappeared following removal of extracellular Ca2+ using a solution containing 5mM EGTA. Application of the non-specific Ca2+ channel blockers Co2+, Ni2+, and Cd2+ also decreased both the frequency and amplitude of SRCs in dose-dependent manners, suggesting that Ca2+ entry through plasma membrane Ca2+ channels is essential for the generation of SRCs. Application of ryanodine (30 μM), which depletes intracellular Ca2+ by locking ryanodine receptor (RyR)-Ca2+ channels in an open state, gradually reduced the frequency and amplitude of SRCs. A RyR antagonist, tetracaine, reduced both the frequency and amplitude of SRCs, whereas a RyR activator, caffeine, increased the frequency of SRCs with a subsequent increase in basal tonus, indicating that RyRs are essential for generating SRCs. To further investigate the involvement of phospholipase C (PLC) and inositol 1,4,5-trisphosphate receptors (IP3Rs) in SRCs, we examined the effect of a PLC inhibitor, U73122, and an IP3R antagonist, 2-aminoethoxydiphenyl borate (2-APB), on SRCs. Separately, U73122 (10 μM) and 2-APB (30-50 μM) both significantly reduced the amplitude of SRCs with little effect on their frequency, further indicating that the PLC/IP3R signaling pathway is fundamental to the modulation of the amplitude of SRCs. A hypotonic-induced increase in the frequency and amplitude of SRCs and a hypertonic-induced decrease in the frequency and amplitude of SRCs indicated that mechanical stretch of the lateral oviduct is involved in the generation of SRCs. The sarcoplasmic reticulum Ca2+-pump ATPase inhibitors thapsigargin and cyclopiazonic acid impaired or suppressed the relaxation phase of SRCs. Taken together, the present results indicate that Ca2+ influx through plasma membrane Ca2

  14. Calcium Occupancy of N-terminal Sites within Calmodulin Induces Inhibition of the Ryanodine Receptor Calcium Release Channel

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    Boschek, Curt B; Jones, Terry E; Squier, Thomas C; Bigelow, Diana J

    2007-08-01

    Calmodulin (CaM) regulates calcium release from intracellular stores in skeletal muscle through its association with the ryanodine receptor (RyR1) calcium release channel, where CaM association enhances channel opening at resting calcium levels and its closing at micromolar calcium levels associated with muscle contraction. A high-affinity CaM-binding sequence (RyRp) has been identified in RyR1, which corresponds to a 30-residue sequence (i.e., K3614 – N3643) located within the central portion of the primary sequence. However, it is currently unclear whether the identified CaM-binding sequence a) senses calcium over the physiological range of calcium-concentrations associated with RyR1 regulation or b) plays a structural role unrelated to the calcium-dependent modulation of RyR1 function. Therefore, we have measured the calcium-dependent activation of the individual domains of CaM in association with RyRp and their relationship to the CaM-dependent regulation of RyR1. These measurements utilize an engineered CaM, permitting the site-specific incorporation of N-(1-pyrene) maleimide at either T34C (PyN-CaM) or T110C (PyC-CaM) in the N- and C-domains, respectively. Consistent with prior measurements, we observe a high-affinity association between both apo- and calcium-activated CaM and RyRp. Upon association with RyRp, fluorescence changes in PyN-CaM or PyC-CaM permit the measurement of the calcium-activation of these individual domains. Fluorescence changes upon calcium-activation of PyC-CaM in association with RyRp are indicative of high-affinity calcium-dependent activation of the C-terminal domain of CaM bound to RyRp at resting calcium levels and the activation of the N-terminal domain at levels of calcium associated cellular activation. In comparison, occupancy of calcium-binding sites in the N-domain of CaM mirrors the calcium-dependence of RyR1 inhibition observed at activating calcium levels, where [Ca]1/2 = 4.3 0.4 μM, suggesting a direct regulation of Ry

  15. Ryanodine receptors, voltage-gated calcium channels and their relationship with protein kinase A in the myocardium

    Czech Academy of Sciences Publication Activity Database

    Petrovič, Miloš; Valeš, Karel; Putnikovič, B.; Djulejič, V.; Mitrovič, D. M.

    2008-01-01

    Roč. 57, č. 2 (2008), s. 141-149 ISSN 0862-8408 R&D Projects: GA ČR(CZ) GA309/07/0271; GA MŠk(CZ) LC554 Institutional research plan: CEZ:AV0Z50110509 Keywords : rynodine receptors * kalcium channels Subject RIV: ED - Physiology Impact factor: 1.653, year: 2008

  16. Insect Ryanodine Receptor: Distinct But Coupled Insecticide Binding Sites for [N-C3H3]Chlorantraniliprole, Flubendiamide, and [3H]Ryanodine

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    Isaacs, André K.; Qi, Suzhen; Sarpong, Richmond; Casida, John E.

    2012-01-01

    Radiolabeled anthranilic diamide insecticide [N-C3H3]chlorantraniliprole was synthesized at high specific activity and compared with phthalic diamide insecticide flubendiamide and [3H]ryanodine in radioligand binding studies with house fly muscle membranes to provide the first direct evidence with a native insect ryanodine receptor that the major anthranilic and phthalic diamide insecticides bind at different allosterically coupled sites, i.e. there are three distinct Ca2+-release channel tar...

  17. Aging Effects of Caenorhabditis elegans Ryanodine Receptor Variants Corresponding to Human Myopathic Mutations

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    Katie Nicoll Baines

    2017-05-01

    Full Text Available Delaying the decline in skeletal muscle function will be critical to better maintenance of an active lifestyle in old age. The skeletal muscle ryanodine receptor, the major intracellular membrane channel through which calcium ions pass to elicit muscle contraction, is central to calcium ion balance and is hypothesized to be a significant factor for age-related decline in muscle function. The nematode Caenorhabditis elegans is a key model system for the study of human aging, and strains were generated with modified C. elegans ryanodine receptors corresponding to human myopathic variants linked with malignant hyperthermia and related conditions. The altered response of these strains to pharmacological agents reflected results of human diagnostic tests for individuals with these pathogenic variants. Involvement of nerve cells in the C. elegans responses may relate to rare medical symptoms concerning the central nervous system that have been associated with ryanodine receptor variants. These single amino acid modifications in C. elegans also conferred a reduction in lifespan and an accelerated decline in muscle integrity with age, supporting the significance of ryanodine receptor function for human aging.

  18. Ryanodine receptor 1 and associated pathologies

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    Fauré , Julien; Lunardi , Joël; Monnier , Nicole; Marty , Isabelle

    2014-01-01

    In skeletal muscle a rise in the cytosolic calcium concentration is the first trigger able to initiate the contraction of the sarcomere. Intracellular calcium levels are tightly controlled by channels and pumps, and it is not surprising that many inherited skeletal muscle disorders arise from mutations altering the players regulating calcium ions concentration (Betzenhauser et al., 2010). In this chapter, we will focus on the pathologies linked to the sarcoplasmic reticulum calcium channel-Ry...

  19. True Molecular Scale Visualization of Variable Clustering Properties of Ryanodine Receptors

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    Isuru Jayasinghe

    2018-01-01

    Full Text Available Summary: Signaling nanodomains rely on spatial organization of proteins to allow controlled intracellular signaling. Examples include calcium release sites of cardiomyocytes where ryanodine receptors (RyRs are clustered with their molecular partners. Localization microscopy has been crucial to visualizing these nanodomains but has been limited by brightness of markers, restricting the resolution and quantification of individual proteins clustered within. Harnessing the remarkable localization precision of DNA-PAINT (<10 nm, we visualized punctate labeling within these nanodomains, confirmed as single RyRs. RyR positions within sub-plasmalemmal nanodomains revealed how they are organized randomly into irregular clustering patterns leaving significant gaps occupied by accessory or regulatory proteins. RyR-inhibiting protein junctophilin-2 appeared highly concentrated adjacent to RyR channels. Analyzing these molecular maps showed significant variations in the co-clustering stoichiometry between junctophilin-2 and RyR, even between nearby nanodomains. This constitutes an additional level of complexity in RyR arrangement and regulation of calcium signaling, intrinsically built into the nanodomains. : Jayasinghe et al. resolve the distribution of single ryanodine receptors (RyRs within intracellular signaling domains in cardiac myocytes with DNA-PAINT, a super-resolution microscopy approach. Individual RyRs are resolved within irregular cluster arrays. Quantitative imaging reveals significant variation in the co-clustering stoichiometry between RyRs and the regulatory protein junctophilin-2. Keywords: nanodomains, DNA-PAINT, single-molecule localization microscopy, ryanodine receptor, super-resolution imaging, junctophilin, heart

  20. CaMKII Regulation of Cardiac Ryanodine Receptors and Inositol Triphosphate Receptors

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    Emmanuel eCamors

    2014-05-01

    Full Text Available Ryanodine receptors (RyRs and inositol triphosphate receptors (InsP3Rs are structurally related intracellular calcium release channels that participate in multiple primary or secondary amplified Ca2+ signals, triggering muscle contraction and oscillatory Ca2+ waves, or activating transcription factors. In the heart, RyRs play an indisputable role in the process of excitation-contraction coupling as the main pathway for Ca2+ release from sarcoplasmic reticulum (SR, and a less prominent role in the process of excitation-transcription coupling. Conversely, InsP3Rs are believed to contribute in subtle ways, only, to contraction of the heart, and in more important ways to regulation of transcription factors. Because uncontrolled activity of either RyRs or InsP3Rs may elicit life-threatening arrhythmogenic and/or remodeling Ca2+ signals, regulation of their activity is of paramount importance for normal cardiac function. Due to their structural similarity, many regulatory factors, accessory proteins, and posttranslational processes are equivalent for RyRs and InsP3Rs. Here we discuss regulation of RyRs and InsP3Rs by CaMKII phosphorylation, but touch on other kinases whenever appropriate. CaMKII is emerging as a powerful modulator of RyR and InsP3R activity but interestingly, some of the complexities and controversies surrounding phosphorylation of RyRs also apply to InsP3Rs, and a clear-cut effect of CaMKII on either channel eludes investigators for now. Nevertheless, some effects of CaMKII on global cellular activity, such as SR Ca2+ leak or force-frequency potentiation, appear clear now, and this constrains the limits of the controversies and permits a more tractable approach to elucidate the effects of phosphorylation at the single channel level.

  1. Novel skeletal muscle ryanodine receptor mutation in a large Brazilian family with malignant hyperthermia.

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    McWilliams, S; Nelson, T; Sudo, R T; Zapata-Sudo, G; Batti, M; Sambuughin, N

    2002-07-01

    Malignant hyperthermia (MH) is an autosomal dominant disorder that predisposes susceptible individuals to a potentially life-threatening crisis when exposed to commonly used anesthetics. Mutations in the skeletal muscle calcium release channel, ryanodine receptor (RYR1) are associated with MH in over 50% of affected families. Linkage analysis of the RYR1 gene region at 19q13 was performed in a large Brazilian family and a distinct disease co-segregating haplotype was revealed in the majority of members with diagnosis of MH. Subsequent sequencing of RYR1 mutational hot spots revealed a nucleotide substitution of C to T at position 7062, causing a novel amino acid change from Arg2355 to Cys associated with MH in the family. Haplotype analysis of the RYR1 gene area at 19q13 in the family with multiple MH members is an important tool in identification of genetic cause underlying this disease.

  2. Cardiac ryanodine receptor in metabolic syndrome: is JTV519 (K201 future therapy?

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    Dincer UD

    2012-04-01

    Full Text Available U Deniz DincerDepartment of Pharmacology, Ufuk University School of Medicine. Mevlana Bulvari, Balgat, Ankara, TurkeyAbstract: Metabolic syndrome is characterized by a combination of obesity, hypertension, insulin resistance, dyslipidemia, and impaired glucose tolerance. This multifaceted syndrome is often accompanied by a hyperdynamic circulatory state characterized by increased blood pressure, total blood volume, cardiac output, and metabolic tissue demand. Experimental, epidemiological, and clinical studies have demonstrated that patients with metabolic syndrome have significantly elevated cardiovascular morbidity and mortality rates. One of the main and frequent complications seen in metabolic syndrome is cardiovascular disease. The primary endpoints of cardiometabolic risk are coronary and peripheral arterial disease, myocardial infarction, congestive heart failure, arrhythmia, and stroke. Alterations in expression and/or functioning of several key proteins involved in regulating and maintaining ionic homeostasis can cause cardiac disturbances. One such group of proteins is known as ryanodine receptors (intracellular calcium release channels, which are the major channels through which Ca2+ ions leave the sarcoplasmic reticulum, leading to cardiac muscle contraction. The economic cost of metabolic syndrome and its associated complications has a significant effect on health care budgets. Improvements in body weight, blood lipid profile, and hyperglycemia can reduce cardiometabolic risk. However, constant hyperadrenergic stimulation still contributes to the burden of disease. Normalization of the hyperdynamic circulatory state with conventional therapies is the most reasonable therapeutic strategy to date. JTV519 (K201 is a newly developed 1,4-benzothiazepine drug with antiarrhythmic and cardioprotective properties. It appears to be very effective in not only preventing but also in reversing the characteristic myocardial changes and preventing

  3. Alteration of Airway Reactivity and Reduction of Ryanodine Receptor Expression by Cigarette Smoke in Mice.

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    Donovan, Chantal; Seow, Huei Jiunn; Royce, Simon G; Bourke, Jane E; Vlahos, Ross

    2015-10-01

    Small airways are a major site of airflow limitation in chronic obstructive pulmonary disease (COPD). Despite the detrimental effects of long-term smoking in COPD, the effects of acute cigarette smoke (CS) exposure on small airway reactivity have not been fully elucidated. Balb/C mice were exposed to room air (sham) or CS for 4 days to cause airway inflammation. Changes in small airway lumen area in response to contractile agents were measured in lung slices in situ using phase-contrast microscopy. Separate slices were pharmacologically maintained at constant intracellular Ca(2+) using caffeine/ryanodine before contractile measurements. Gene and protein analysis of contractile signaling pathways were performed on separate lungs. Monophasic contraction to serotonin became biphasic after CS exposure, whereas contraction to methacholine was unaltered. This altered pattern of contraction was normalized by caffeine/ryanodine. Expression of contractile agonist-specific receptors was unaltered; however, all isoforms of the ryanodine receptor were down-regulated. This is the first study to show that acute CS exposure selectively alters small airway contraction to serotonin and down-regulates ryanodine receptors involved in maintaining Ca(2+) oscillations in airway smooth muscle. Understanding the contribution of ryanodine receptors to altered airway reactivity may inform the development of novel treatment strategies for COPD.

  4. Ryanodine Receptor Calcium Leak in Circulating B-Lymphocytes as a Biomarker in Heart Failure.

    Science.gov (United States)

    Kushnir, Alexander; Santulli, Gaetano; Reiken, Steven R; Coromilas, Ellie; Godfrey, Sarah J; Brunjes, Danielle L; Colombo, Paolo C; Yuzefpolskaya, Melana; Sokol, Seth I; Kitsis, Richard N; Marks, Andrew R

    2018-03-28

    Background -Advances in congestive heart failure (CHF) management depend on biomarkers for monitoring disease progression and therapeutic response. During systole, intracellular Ca2 + is released from the sarcoplasmic reticulum (SR) into the cytoplasm through type 2 ryanodine receptor/Ca2 + release channels (RyR2). In CHF, chronically elevated circulating catecholamine levels cause pathologic remodeling of RyR2 resulting in diastolic SR Ca2 + leak, and decreased myocardial contractility. Similarly, skeletal muscle contraction requires SR Ca2 + release through type-1 ryanodine receptors (RyR1), and chronically elevated catecholamine levels in CHF cause RyR1 mediated SR Ca2 + leak, contributing to myopathy and weakness. Circulating B-lymphocytes express RyR1 and catecholamine responsive signaling cascades, making them a potential surrogate for defects in intracellular Ca2 + handling due to leaky RyR channels in CHF. Methods -Whole blood was collected from patients with CHF, CHF status-post left-ventricular assist devices (LVAD), and controls. Blood was also collected from mice with ischemic CHF, ischemic CHF + S107 (a drug that specifically reduces RyR channel Ca2 + leak), and WT controls. Channel macromolecular complex was assessed by immunostaining RyR1 immunoprecipitated from lymphocyte enriched preparations. RyR1 Ca2 + leak was assessed using flow cytometry to measure Ca2 + fluorescence in B-lymphocytes, in the absence and presence of RyR1 agonists that empty RyR1 Ca2 + stores within the endoplasmic reticulum (ER). Results -Circulating B-lymphocytes from humans and mice with CHF exhibited remodeled RyR1 and decreased ER Ca2 + stores, consistent with chronic intracellular Ca2 + leak. This Ca2 + leak correlated with circulating catecholamine levels. The intracellular Ca2 + leak was significantly reduced in mice treated with the Rycal S107. CHF patients treated with LVAD exhibited a heterogeneous response. Conclusions -In CHF, B-lymphocytes exhibit remodeled leaky

  5. One Dimensional Finite Element Method Approach to Study Effect of Ryanodine Receptor and Serca Pump on Calcium Distribution in Oocytes

    Science.gov (United States)

    Naik, Parvaiz Ahmad; Pardasani, Kamal Raj

    2013-11-01

    Oocyte is a female gametocyte or germ cell involved in reproduction. Calcium ions (Ca2+) impact nearly all aspects of cellular life as they play an important role in a variety of cellular functions. Calcium ions contributes to egg activation upon fertilization. Since it is the internal stores which provide most of the calcium signal, much attention has been focused on the intracellular channels. There are mainly two types of calcium channels which release calcium from the internal stores to the cytoplasm in many cell types. These channels are IP3-Receptor and Ryanodine Receptor (RyR). Further it is essential to maintain low cytosolic calcium concentration, the cell engages the Serco/Endoplasmic reticulum Ca2+ ATPases (SERCA) present on the ER or SR membrane for the re-uptake of cytosolic calcium at the expense of ATP hydrolysis. In view of above an attempt has been made to study the effect of the Ryanodine receptor (RyR) and the SERCA pump on the calcium distribution in oocytes. The main aim of this paper is to study the calcium concentration in absence and presence of these parameters. The FEM is used to solve the proposed Mathematical model under appreciate initial and boundary conditions. The program has been developed in MATLAB 7.10 for the entire problem to get numerical results.

  6. FRET-based localization of fluorescent protein insertions within the ryanodine receptor type 1.

    Directory of Open Access Journals (Sweden)

    Shweta A Raina

    Full Text Available Fluorescent protein (FP insertions have often been used to localize primary structure elements in mid-resolution 3D cryo electron microscopic (EM maps of large protein complexes. However, little is known as to the precise spatial relationship between the location of the fused FP and its insertion site within a larger protein. To gain insights into these structural considerations, Förster resonance energy transfer (FRET measurements were used to localize green fluorescent protein (GFP insertions within the ryanodine receptor type 1 (RyR1, a large intracellular Ca(2+ release channel that plays a key role in skeletal muscle excitation contraction coupling. A series of full-length His-tagged GFP-RyR1 fusion constructs were created, expressed in human embryonic kidney (HEK-293T cells and then complexed with Cy3NTA, a His-tag specific FRET acceptor. FRET efficiency values measured from each GFP donor to Cy3NTA bound to each His tag acceptor site were converted into intermolecular distances and the positions of each inserted GFP were then triangulated relative to a previously published X-ray crystal structure of a 559 amino acid RyR1 fragment. We observed that the chromophoric centers of fluorescent proteins inserted into RyR1 can be located as far as 45 Å from their insertion sites and that the fused proteins can also be located in internal cavities within RyR1. These findings should prove useful in interpreting structural results obtained in cryo EM maps using fusions of small fluorescent proteins. More accurate point-to-point distance information may be obtained using complementary orthogonal labeling systems that rely on fluorescent probes that bind directly to amino acid side chains.

  7. A structural model of the pore-forming region of the skeletal muscle ryanodine receptor (RyR1.

    Directory of Open Access Journals (Sweden)

    Srinivas Ramachandran

    2009-04-01

    Full Text Available Ryanodine receptors (RyRs are ion channels that regulate muscle contraction by releasing calcium ions from intracellular stores into the cytoplasm. Mutations in skeletal muscle RyR (RyR1 give rise to congenital diseases such as central core disease. The absence of high-resolution structures of RyR1 has limited our understanding of channel function and disease mechanisms at the molecular level. Here, we report a structural model of the pore-forming region of RyR1. Molecular dynamics simulations show high ion binding to putative pore residues D4899, E4900, D4938, and D4945, which are experimentally known to be critical for channel conductance and selectivity. We also observe preferential localization of Ca(2+ over K(+ in the selectivity filter of RyR1. Simulations of RyR1-D4899Q mutant show a loss of preference to Ca(2+ in the selectivity filter as seen experimentally. Electrophysiological experiments on a central core disease mutant, RyR1-G4898R, show constitutively open channels that conduct K(+ but not Ca(2+. Our simulations with G4898R likewise show a decrease in the preference of Ca(2+ over K(+ in the selectivity filter. Together, the computational and experimental results shed light on ion conductance and selectivity of RyR1 at an atomistic level.

  8. Membrane depolarization increases ryanodine sensitivity to Ca2+ release to the cytosol in L6 skeletal muscle cells: Implications for excitation-contraction coupling.

    Science.gov (United States)

    Pitake, Saumitra; Ochs, Raymond S

    2016-04-01

    The dihydropyridine receptor in the plasma membrane and the ryanodine receptor in the sarcoplasmic reticulum are known to physically interact in the process of excitation-contraction coupling. However, the mechanism for subsequent Ca(2+) release through the ryanodine receptor is unknown. Our lab has previously presented evidence that the dihydropyridine receptor and ryanodine receptor combine as a channel for the entry of Ca(2+) under resting conditions, known as store operated calcium entry. Here, we provide evidence that depolarization during excitation-contraction coupling causes the dihydropyridine receptor to disengage from the ryanodine receptor. The newly freed ryanodine receptor can then transport Ca(2+) from the sarcoplasmic reticulum to the cytosol. Experimentally, this should more greatly expose the ryanodine receptor to exogenous ryanodine. To examine this hypothesis, we titrated L6 skeletal muscle cells with ryanodine in resting and excited (depolarized) states. When L6 muscle cells were depolarized with high potassium or exposed to the dihydropyridine receptor agonist BAYK-8644, known to induce dihydropyridine receptor movement within the membrane, ryanodine sensitivity was enhanced. However, ryanodine sensitivity was unaffected when Ca(2+) was elevated without depolarization by the ryanodine receptor agonist chloromethylcresol, or by increasing Ca(2+) concentration in the media. Ca(2+) entry currents (from the extracellular space) during excitation were strongly inhibited by ryanodine, but Ca(2+) entry currents in the resting state were not. We conclude that excitation releases the ryanodine receptor from occlusion by the dihydropyridine receptor, enabling Ca(2+) release from the ryanodine receptor to the cytosol. © 2015 by the Society for Experimental Biology and Medicine.

  9. Glucose-Dependent Insulin Secretion in Pancreatic β-Cell Islets from Male Rats Requires Ca2+ Release via ROS-Stimulated Ryanodine Receptors.

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    Paola Llanos

    Full Text Available Glucose-stimulated insulin secretion (GSIS from pancreatic β-cells requires an increase in intracellular free Ca2+ concentration ([Ca2+]. Glucose uptake into β-cells promotes Ca2+ influx and reactive oxygen species (ROS generation. In other cell types, Ca2+ and ROS jointly induce Ca2+ release mediated by ryanodine receptor (RyR channels. Therefore, we explored here if RyR-mediated Ca2+ release contributes to GSIS in β-cell islets isolated from male rats. Stimulatory glucose increased islet insulin secretion, and promoted ROS generation in islets and dissociated β-cells. Conventional PCR assays and immunostaining confirmed that β-cells express RyR2, the cardiac RyR isoform. Extended incubation of β-cell islets with inhibitory ryanodine suppressed GSIS; so did the antioxidant N-acetyl cysteine (NAC, which also decreased insulin secretion induced by glucose plus caffeine. Inhibitory ryanodine or NAC did not affect insulin secretion induced by glucose plus carbachol, which engages inositol 1,4,5-trisphosphate receptors. Incubation of islets with H2O2 in basal glucose increased insulin secretion 2-fold. Inhibitory ryanodine significantly decreased H2O2-stimulated insulin secretion and prevented the 4.5-fold increase of cytoplasmic [Ca2+] produced by incubation of dissociated β-cells with H2O2. Addition of stimulatory glucose or H2O2 (in basal glucose to β-cells disaggregated from islets increased RyR2 S-glutathionylation to similar levels, measured by a proximity ligation assay; in contrast, NAC significantly reduced the RyR2 S-glutathionylation increase produced by stimulatory glucose. We propose that RyR2-mediated Ca2+ release, induced by the concomitant increases in [Ca2+] and ROS produced by stimulatory glucose, is an essential step in GSIS.

  10. Expression of Dihydropyridine and Ryanodine Receptors in Type IIA Fibers of Rat Skeletal Muscle

    International Nuclear Information System (INIS)

    Anttila, Katja; Mänttäri, Satu; Järvilehto, Matti

    2007-01-01

    In this study, the fiber type specificity of dihydropyridine receptors (DHPRs) and ryanodine receptors (RyRs) in different rat limb muscles was investigated. Western blot and histochemical analyses provided for the first time evidence that the expression of both receptors correlates to a specific myosin heavy chain (MHC) composition. We observed a significant (p=0.01) correlation between DHP as well as Ry receptor density and the expression of MHC IIa (correlation factor r=0.674 and r=0.645, respectively) in one slow-twitch, postural muscle (m. soleus), one mixed, fast-twitch muscle (m. gastrocnemius) and two fast-twitch muscles (m. rectus femoris, m. extensor digitorum longus). The highest DHP and Ry receptor density was found in the white part of m. rectus femoris (0.058±0.0060 and 0.057±0.0158 ODu, respectively). As expected, the highest relative percentage of MHC IIa was also found in the white part of m. rectus femoris (70.0±7.77%). Furthermore, histochemical experiments revealed that the IIA fibers stained most strongly for the fluorophore-conjugated receptor blockers. Our data clearly suggest that the expression of DHPRs and RyRs follows a fiber type-specific pattern, indicating an important role for these proteins in the maintenance of an effective Ca 2+ cycle in the fast contracting fiber type IIA

  11. Amyloid β production is regulated by β2-adrenergic signaling-mediated post-translational modifications of the ryanodine receptor.

    Science.gov (United States)

    Bussiere, Renaud; Lacampagne, Alain; Reiken, Steven; Liu, Xiaoping; Scheuerman, Valerie; Zalk, Ran; Martin, Cécile; Checler, Frederic; Marks, Andrew R; Chami, Mounia

    2017-06-16

    Alteration of ryanodine receptor (RyR)-mediated calcium (Ca 2+ ) signaling has been reported in Alzheimer disease (AD) models. However, the molecular mechanisms underlying altered RyR-mediated intracellular Ca 2+ release in AD remain to be fully elucidated. We report here that RyR2 undergoes post-translational modifications (phosphorylation, oxidation, and nitrosylation) in SH-SY5Y neuroblastoma cells expressing the β-amyloid precursor protein (βAPP) harboring the familial double Swedish mutations (APPswe). RyR2 macromolecular complex remodeling, characterized by depletion of the regulatory protein calstabin2, resulted in increased cytosolic Ca 2+ levels and mitochondrial oxidative stress. We also report a functional interplay between amyloid β (Aβ), β-adrenergic signaling, and altered Ca 2+ signaling via leaky RyR2 channels. Thus, post-translational modifications of RyR occur downstream of Aβ through a β2-adrenergic signaling cascade that activates PKA. RyR2 remodeling in turn enhances βAPP processing. Importantly, pharmacological stabilization of the binding of calstabin2 to RyR2 channels, which prevents Ca 2+ leakage, or blocking the β2-adrenergic signaling cascade reduced βAPP processing and the production of Aβ in APPswe-expressing SH-SY5Y cells. We conclude that targeting RyR-mediated Ca 2+ leakage may be a therapeutic approach to treat AD. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

  12. RYR1-related rhabdomyolysis: A common but probably underdiagnosed manifestation of skeletal muscle ryanodine receptor dysfunction.

    Science.gov (United States)

    Voermans, N C; Snoeck, M; Jungbluth, H

    2016-10-01

    Mutations in the skeletal muscle ryanodine receptor (RYR1) gene are associated with a wide spectrum of inherited myopathies presenting throughout life. Malignant hyperthermia susceptibility (MHS)-related RYR1 mutations have emerged as a common cause of exertional rhabdomyolysis, accounting for up to 30% of rhabdomyolysis episodes in otherwise healthy individuals. Common triggers are exercise and heat and, less frequently, viral infections, alcohol and drugs. Most subjects are normally strong and have no personal or family history of malignant hyperthermia. Heat intolerance and cold-induced muscle stiffness may be a feature. Recognition of this (probably not uncommon) rhabdomyolysis cause is vital for effective counselling, to identify potentially malignant hyperthermia-susceptible individuals and to adapt training regimes. Studies in various animal models provide insights regarding possible pathophysiological mechanisms and offer therapeutic perspectives. Copyright © 2016. Published by Elsevier Masson SAS.

  13. The Environmental Neurotoxicant PCB 95 Promotes Synaptogenesis via Ryanodine Receptor-Dependent miR132 Upregulation

    Science.gov (United States)

    Lesiak, Adam; Zhu, Mingyan; Chen, Hao; Appleyard, Suzanne M.; Impey, Soren; Wayman, Gary A.

    2014-01-01

    Non–dioxin-like (NDL) polychlorinated biphenyls (PCBs) are widespread environmental contaminants linked to neuropsychological dysfunction in children. NDL PCBs increase spontaneous Ca2+ oscillations in neurons by stabilizing ryanodine receptor (RyR) calcium release channels in the open configuration, which results in CREB-dependent dendritic outgrowth. In this study, we address the question of whether activation of CREB by NDL PCBs also triggers dendritic spine formation. Nanomolar concentrations of PCB 95, a NDL congener with potent RyR activity, significantly increased spine density and the frequency of miniature EPSCs in primary dissociated rat hippocampal cultures coincident with upregulation of miR132. Inhibition of RyR, CREB, or miR132 as well as expression of a mutant p250GAP cDNA construct that is not suppressed by miR132 blocked PCB 95 effects on spines and miniature EPSCs. PCB 95 also induced spine formation via RyR- and miR132-dependent mechanisms in hippocampal slice cultures. These data demonstrate a novel mechanism of PCB developmental neurotoxicity whereby RyR sensitization modulates spine formation and synaptogenesis via CREB-mediated miR132 upregulation, which in turn suppresses the translation of p250GAP, a negative regulator of synaptogenesis. In light of recent evidence implicating miR132 dysregulation in Rett syndrome and schizophrenia, these findings identify NDL PCBs as potential environmental risk factors for neurodevelopmental disorders. PMID:24431430

  14. Selective expression of the type 3 isoform of ryanodine receptor Ca2+ release channel (RyR3) in a subset of slow fibers in diaphragm and cephalic muscles of adult rabbits

    International Nuclear Information System (INIS)

    Conti, Antonio; Reggiani, Carlo; Sorrentino, Vincenzo

    2005-01-01

    The expression pattern of the RyR3 isoform of Ca 2+ release channels was analysed by Western blot in neonatal and adult rabbit skeletal muscles. The results obtained show that the expression of the RyR3 isoform is developmentally regulated. In fact, RyR3 expression was detected in all muscles analysed at 2 and 15 days after birth while, in adult animals, it was restricted to a subset of muscles that includes diaphragm, masseter, pterygoideus, digastricus, and tongue. Interestingly, all of these muscles share a common embryonic origin being derived from the somitomeres or from the cephalic region of the embryo. Immunofluorescence analysis of rabbit skeletal muscle cross-sections showed that RyR3 staining was detected in all fibers of neonatal muscles. In contrast, in those adult muscles expressing RyR3 only a fraction of fibers was labelled. Staining of these muscles with antibodies against fast and slow myosins revealed a close correlation between expression of RyR3 and fibers expressing slow myosin isoform

  15. Rapid selection for resistance to diamide insecticides in Plutella xylostella via specific amino acid polymorphisms in the ryanodine receptor.

    Science.gov (United States)

    Troczka, Bartlomiej J; Williamson, Martin S; Field, Linda M; Davies, T G Emyr

    2017-05-01

    Diamide insecticides, such as flubendiamide and chlorantraniliprole, are a new class of insecticide with a novel mode of action, selectively activating the insect ryanodine receptor (RyR). They are particularly active against lepidopteran pests of cruciferous vegetable crops, including the diamondback moth, Plutella xylostella. However, within a relatively short period following their commercialisation, a comparatively large number of control failures have been reported in the field. In this review we summarise the current body of knowledge regarding the molecular mechanisms of diamide resistance in P. xylostella. Resistant phenotypes collected from different countries can often be linked to specific target-site mutation(s) in the ryanodine receptors' transmembrane domain. Metabolic mechanisms of resistance have also been proposed. Rapid resistance development is probably a consequence of over-reliance on this one class of chemistry for diamondback moth control. Copyright © 2016 The Authors. Published by Elsevier B.V. All rights reserved.

  16. Effects of pO2 on the activation of skeletal muscle ryanodine receptors by NO: a cautionary note.

    Science.gov (United States)

    Cheong, Eunji; Tumbev, Vassil; Stoyanovsky, Detcho; Salama, Guy

    2005-11-01

    Eu et al., reported that O2 dynamically controls the redox state of 6-8 out of 50 thiols per skeletal ryanodine receptor (RyR1) subunit and thereby tunes the response of Ca2+-release channels to authentic nitric oxide (NO) [J.P. Eu, J. Sun, L. Xu, J.S. Stamler, G. Meissner, The skeletal muscle calcium release channel: coupled O2 sensor and NO signaling functions, Cell 102 (2000) 499-509]. A role for O2 was based on the observation that RyR1 can be activated by submicromolar NO at physiological ( approximately 10 mmHg) but not ambient (approximately 150 mmHg) pO2. At ambient pO2, these critical thiols were oxidized but incubation at low pO2 reset the redox state of these thiols, closed RyR1 channels and made these thiols available for nitrosation by low NO concentrations. Eu et al., postulated the existence of a redox/O2sensor that couples channel activity to NO and pO2 and explained that "the nature of the 'redox/O2 sensor' that couples channel activity to intracellular redox chemistry is a mystery". Here, we re-examined the effect of pO2 on RyR1 and find that incubation of RyR1 at low pO2 did not alter channel activity and NO (0.5-50 microM) failed to activate RyR1 despite a wide range of pO2 pre-incubation conditions. We show that low levels of NO do not activate RyR1, do not reverse the inhibition of RyR1 by calmodulin (CaM) even at physiological pO2. Similarly, the pre-incubation of SR vesicles in low pO2 (for 10-80 min) did not inhibit channel activity or sensitization of RyR1 to NO. We discuss the significance of these findings and propose that caution should be taken when considering a role for pO2 and nitrosation by NO as mechanisms that tune RyRs in striated muscles.

  17. KChIP2 regulates the cardiac Ca2+ transient and myocyte contractility by targeting ryanodine receptor activity.

    Directory of Open Access Journals (Sweden)

    Drew M Nassal

    Full Text Available Pathologic electrical remodeling and attenuated cardiac contractility are featured characteristics of heart failure. Coinciding with these remodeling events is a loss of the K+ channel interacting protein, KChIP2. While, KChIP2 enhances the expression and stability of the Kv4 family of potassium channels, leading to a more pronounced transient outward K+ current, Ito,f, the guinea pig myocardium is unique in that Kv4 expression is absent, while KChIP2 expression is preserved, suggesting alternative consequences to KChIP2 loss. Therefore, KChIP2 was acutely silenced in isolated guinea pig myocytes, which led to significant reductions in the Ca2+ transient amplitude and prolongation of the transient duration. This change was reinforced by a decline in sarcomeric shortening. Notably, these results were unexpected when considering previous observations showing enhanced ICa,L and prolonged action potential duration following KChIP2 loss, suggesting a disruption of fundamental Ca2+ handling proteins. Evaluation of SERCA2a, phospholamban, RyR, and sodium calcium exchanger identified no change in protein expression. However, assessment of Ca2+ spark activity showed reduced spark frequency and prolonged Ca2+ decay following KChIP2 loss, suggesting an altered state of RyR activity. These changes were associated with a delocalization of the ryanodine receptor activator, presenilin, away from sarcomeric banding to more diffuse distribution, suggesting that RyR open probability are a target of KChIP2 loss mediated by a dissociation of presenilin. Typically, prolonged action potential duration and enhanced Ca2+ entry would augment cardiac contractility, but here we see KChIP2 fundamentally disrupts Ca2+ release events and compromises myocyte contraction. This novel role targeting presenilin localization and RyR activity reveals a significance for KChIP2 loss that reflects adverse remodeling observed in cardiac disease settings.

  18. Nitric oxide-induced calcium release: activation of type 1 ryanodine receptor by endogenous nitric oxide.

    Science.gov (United States)

    Kakizawa, Sho; Yamazawa, Toshiko; Iino, Masamitsu

    2013-01-01

    Ryanodine receptors (RyRs), located in the sarcoplasmic/endoplasmic reticulum (SR/ER) membrane, are required for intracellular Ca2+ release that is involved in a wide range of cellular functions. In addition to Ca2+-induced Ca2+ release in cardiac cells and voltage-induced Ca2+ release in skeletal muscle cells, we recently identified another mode of intracellular Ca2+ mobilization mediated by RyR, i.e., nitric oxide-induced Ca2+ release (NICR), in cerebellar Purkinje cells. NICR is evoked by neuronal activity, is dependent on S-nitrosylation of type 1 RyR (RyR1) and is involved in the induction of long-term potentiation (LTP) of cerebellar synapses. In this addendum, we examined whether peroxynitrite, which is produced by the reaction of nitric oxide with superoxide, may also have an effect on the Ca2+ release via RyR1 and the cerebellar LTP. We found that scavengers of peroxynitrite have no significant effect either on the Ca2+ release via RyR1 or on the cerebellar LTP. We also found that an application of a high concentration of peroxynitrite does not reproduce neuronal activity-dependent Ca2+ release in Purkinje cells. These results support that NICR is induced by endogenous nitric oxide produced by neuronal activity through S-nitrosylation of RyR1.

  19. Crystal structure of type I ryanodine receptor amino-terminal [beta]-trefoil domain reveals a disease-associated mutation 'hot spot' loop

    Energy Technology Data Exchange (ETDEWEB)

    Amador, Fernando J.; Liu, Shuang; Ishiyama, Noboru; Plevin, Michael J.; Wilson, Aaron; MacLennan, David H.; Ikura, Mitsuhiko; (Toronto)

    2009-12-01

    Muscle contraction and relaxation is regulated by transient elevations of myoplasmic Ca{sup 2+}. Ca{sup 2+} is released from stores in the lumen of the sarco(endo)plasmic reticulum (SER) to initiate formation of the Ca{sup 2+} transient by activation of a class of Ca{sup 2+} release channels referred to as ryanodine receptors (RyRs) and is pumped back into the SER lumen by Ca{sup 2+}-ATPases (SERCAs) to terminate the Ca{sup 2+} transient. Mutations in the type 1 ryanodine receptor gene, RYR1, are associated with 2 skeletal muscle disorders, malignant hyperthermia (MH), and central core disease (CCD). The evaluation of proposed mechanisms by which RyR1 mutations cause MH and CCD is hindered by the lack of high-resolution structural information. Here, we report the crystal structure of the N-terminal 210 residues of RyR1 (RyR{sub NTD}) at 2.5 {angstrom}. The RyR{sub NTD} structure is similar to that of the suppressor domain of type 1 inositol 1,4,5-trisphosphate receptor (IP3Rsup), but lacks most of the long helix-turn-helix segment of the 'arm' domain in IP3Rsup. The N-terminal {beta}-trefoil fold, found in both RyR and IP{sub 3}R, is likely to play a critical role in regulatory mechanisms in this channel family. A disease-associated mutation 'hot spot' loop was identified between strands 8 and 9 in a highly basic region of RyR1. Biophysical studies showed that 3 MH-associated mutations (C36R, R164C, and R178C) do not adversely affect the global stability or fold of RyRNTD, supporting previously described mechanisms whereby mutations perturb protein-protein interactions.

  20. Ryanodine receptor fragmentation and sarcoplasmic reticulum Ca2+ leak after one session of high-intensity interval exercise.

    Science.gov (United States)

    Place, Nicolas; Ivarsson, Niklas; Venckunas, Tomas; Neyroud, Daria; Brazaitis, Marius; Cheng, Arthur J; Ochala, Julien; Kamandulis, Sigitas; Girard, Sebastien; Volungevičius, Gintautas; Paužas, Henrikas; Mekideche, Abdelhafid; Kayser, Bengt; Martinez-Redondo, Vicente; Ruas, Jorge L; Bruton, Joseph; Truffert, Andre; Lanner, Johanna T; Skurvydas, Albertas; Westerblad, Håkan

    2015-12-15

    High-intensity interval training (HIIT) is a time-efficient way of improving physical performance in healthy subjects and in patients with common chronic diseases, but less so in elite endurance athletes. The mechanisms underlying the effectiveness of HIIT are uncertain. Here, recreationally active human subjects performed highly demanding HIIT consisting of 30-s bouts of all-out cycling with 4-min rest in between bouts (≤3 min total exercise time). Skeletal muscle biopsies taken 24 h after the HIIT exercise showed an extensive fragmentation of the sarcoplasmic reticulum (SR) Ca(2+) release channel, the ryanodine receptor type 1 (RyR1). The HIIT exercise also caused a prolonged force depression and triggered major changes in the expression of genes related to endurance exercise. Subsequent experiments on elite endurance athletes performing the same HIIT exercise showed no RyR1 fragmentation or prolonged changes in the expression of endurance-related genes. Finally, mechanistic experiments performed on isolated mouse muscles exposed to HIIT-mimicking stimulation showed reactive oxygen/nitrogen species (ROS)-dependent RyR1 fragmentation, calpain activation, increased SR Ca(2+) leak at rest, and depressed force production due to impaired SR Ca(2+) release upon stimulation. In conclusion, HIIT exercise induces a ROS-dependent RyR1 fragmentation in muscles of recreationally active subjects, and the resulting changes in muscle fiber Ca(2+)-handling trigger muscular adaptations. However, the same HIIT exercise does not cause RyR1 fragmentation in muscles of elite endurance athletes, which may explain why HIIT is less effective in this group.

  1. Ryanodine receptor fragmentation and sarcoplasmic reticulum Ca2+ leak after one session of high-intensity interval exercise

    Science.gov (United States)

    Place, Nicolas; Ivarsson, Niklas; Venckunas, Tomas; Neyroud, Daria; Brazaitis, Marius; Cheng, Arthur J.; Ochala, Julien; Kamandulis, Sigitas; Girard, Sebastien; Volungevičius, Gintautas; Paužas, Henrikas; Mekideche, Abdelhafid; Kayser, Bengt; Martinez-Redondo, Vicente; Bruton, Joseph; Truffert, Andre; Lanner, Johanna T.; Skurvydas, Albertas; Westerblad, Håkan

    2015-01-01

    High-intensity interval training (HIIT) is a time-efficient way of improving physical performance in healthy subjects and in patients with common chronic diseases, but less so in elite endurance athletes. The mechanisms underlying the effectiveness of HIIT are uncertain. Here, recreationally active human subjects performed highly demanding HIIT consisting of 30-s bouts of all-out cycling with 4-min rest in between bouts (≤3 min total exercise time). Skeletal muscle biopsies taken 24 h after the HIIT exercise showed an extensive fragmentation of the sarcoplasmic reticulum (SR) Ca2+ release channel, the ryanodine receptor type 1 (RyR1). The HIIT exercise also caused a prolonged force depression and triggered major changes in the expression of genes related to endurance exercise. Subsequent experiments on elite endurance athletes performing the same HIIT exercise showed no RyR1 fragmentation or prolonged changes in the expression of endurance-related genes. Finally, mechanistic experiments performed on isolated mouse muscles exposed to HIIT-mimicking stimulation showed reactive oxygen/nitrogen species (ROS)-dependent RyR1 fragmentation, calpain activation, increased SR Ca2+ leak at rest, and depressed force production due to impaired SR Ca2+ release upon stimulation. In conclusion, HIIT exercise induces a ROS-dependent RyR1 fragmentation in muscles of recreationally active subjects, and the resulting changes in muscle fiber Ca2+-handling trigger muscular adaptations. However, the same HIIT exercise does not cause RyR1 fragmentation in muscles of elite endurance athletes, which may explain why HIIT is less effective in this group. PMID:26575622

  2. Simulation of the effect of rogue ryanodine receptors on a calcium wave in ventricular myocytes with heart failure

    International Nuclear Information System (INIS)

    Lu, Luyao; Xia, Ling; Ye, Xuesong; Cheng, Heping

    2010-01-01

    Calcium homeostasis is considered to be one of the most important factors for the contraction and relaxation of the heart muscle. However, under some pathological conditions, such as heart failure (HF), calcium homeostasis is disordered, and spontaneous waves may occur. In this study, we developed a mathematical model of formation and propagation of a calcium wave based upon a governing system of diffusion–reaction equations presented by Izu et al (2001 Biophys. J. 80 103–20) and integrated non-clustered or 'rogue' ryanodine receptors (rogue RyRs) into a two-dimensional (2D) model of ventricular myocytes isolated from failing hearts in which sarcoplasmic reticulum (SR) Ca 2+ pools are partially unloaded. The model was then used to simulate the effect of rogue RyRs on initiation and propagation of the calcium wave in ventricular myocytes with HF. Our simulation results show that rogue RyRs can amplify the diastolic SR Ca 2+ leak in the form of Ca 2+ quarks, increase the probability of occurrence of spontaneous Ca 2+ waves even with smaller SR Ca 2+ stores, accelerate Ca 2+ wave propagation, and hence lead to delayed afterdepolarizations (DADs) and cardiac arrhythmia in the diseased heart. This investigation suggests that incorporating rogue RyRs in the Ca 2+ wave model under HF conditions provides a new view of Ca 2+ dynamics that could not be mimicked by adjusting traditional parameters involved in Ca 2+ release units and other ion channels, and contributes to understanding the underlying mechanism of HF

  3. Simulation of the effect of rogue ryanodine receptors on a calcium wave in ventricular myocytes with heart failure

    Science.gov (United States)

    Lu, Luyao; Xia, Ling; Ye, Xuesong; Cheng, Heping

    2010-06-01

    Calcium homeostasis is considered to be one of the most important factors for the contraction and relaxation of the heart muscle. However, under some pathological conditions, such as heart failure (HF), calcium homeostasis is disordered, and spontaneous waves may occur. In this study, we developed a mathematical model of formation and propagation of a calcium wave based upon a governing system of diffusion-reaction equations presented by Izu et al (2001 Biophys. J. 80 103-20) and integrated non-clustered or 'rogue' ryanodine receptors (rogue RyRs) into a two-dimensional (2D) model of ventricular myocytes isolated from failing hearts in which sarcoplasmic reticulum (SR) Ca2+ pools are partially unloaded. The model was then used to simulate the effect of rogue RyRs on initiation and propagation of the calcium wave in ventricular myocytes with HF. Our simulation results show that rogue RyRs can amplify the diastolic SR Ca2+ leak in the form of Ca2+ quarks, increase the probability of occurrence of spontaneous Ca2+ waves even with smaller SR Ca2+ stores, accelerate Ca2+ wave propagation, and hence lead to delayed afterdepolarizations (DADs) and cardiac arrhythmia in the diseased heart. This investigation suggests that incorporating rogue RyRs in the Ca2+ wave model under HF conditions provides a new view of Ca2+ dynamics that could not be mimicked by adjusting traditional parameters involved in Ca2+ release units and other ion channels, and contributes to understanding the underlying mechanism of HF.

  4. Ryanodine receptor gating controls generation of diastolic calcium waves in cardiac myocytes

    Science.gov (United States)

    Petrovič, Pavol; Valent, Ivan; Cocherová, Elena; Pavelková, Jana

    2015-01-01

    The role of cardiac ryanodine receptor (RyR) gating in the initiation and propagation of calcium waves was investigated using a mathematical model comprising a stochastic description of RyR gating and a deterministic description of calcium diffusion and sequestration. We used a one-dimensional array of equidistantly spaced RyR clusters, representing the confocal scanning line, to simulate the formation of calcium sparks. Our model provided an excellent description of the calcium dependence of the frequency of diastolic calcium sparks and of the increased tendency for the production of calcium waves after a decrease in cytosolic calcium buffering. We developed a hypothesis relating changes in the propensity to form calcium waves to changes of RyR gating and tested it by simulation. With a realistic RyR gating model, increased ability of RyR to be activated by Ca2+ strongly increased the propensity for generation of calcium waves at low (0.05–0.1-µM) calcium concentrations but only slightly at high (0.2–0.4-µM) calcium concentrations. Changes in RyR gating altered calcium wave formation by changing the calcium sensitivity of spontaneous calcium spark activation and/or the average number of open RyRs in spontaneous calcium sparks. Gating changes that did not affect RyR activation by Ca2+ had only a weak effect on the propensity to form calcium waves, even if they strongly increased calcium spark frequency. Calcium waves induced by modulating the properties of the RyR activation site could be suppressed by inhibiting the spontaneous opening of the RyR. These data can explain the increased tendency for production of calcium waves under conditions when RyR gating is altered in cardiac diseases. PMID:26009544

  5. A case of late-onset, thymoma-associated myasthenia gravis with ryanodine receptor and titin antibodies and concomitant granulomatous myositis.

    Science.gov (United States)

    Stefanou, M I; Komorowski, L; Kade, S; Bornemann, A; Ziemann, U; Synofzik, M

    2016-09-13

    Myasthenia gravis is an autoimmune neuromuscular disorder, which has only rarely been reported to co-manifest with myositis. The diagnosis of concomitant myositis in patients with myasthenia gravis is clinically challenging, and requires targeted investigations for the differential diagnosis, including EMG, autoantibody assays, muscle biopsy and, importantly, imaging of the mediastinum for thymoma screening. This report presents a case-vignette of a 72-year-old woman with progressive proximal muscle weakness and myalgias, diagnosed with thymoma-associated myasthenia and bioptically verified granulomatous myositis, with positive autoantibody status for ryanodine receptor and titin antibodies. The diagnosis of concurrent myositis and myasthenia gravis, especially in the presence of ryanodine receptor and titin antibodies, should lead neurologists to adopt different treatment strategies compared to those applied in myasthenia or myositis alone. Moreover, further evidence is warranted that titin and, particularly, ryanodine receptor antibodies may co-occur or be pathophysiologically involved in myasthenia-myositis cases.

  6. Characterization of ryanodine receptor and Ca2+-ATPase isoforms in the thermogenic heater organ of blue marlin (Makaira nigricans).

    Science.gov (United States)

    Morrissette, Jeffery M; Franck, Jens P G; Block, Barbara A

    2003-03-01

    A thermogenic organ is found beneath the brain of billfishes (Istiophoridae), swordfish (Xiphiidae) and the butterfly mackerel (Scombridae). The heater organ has been shown to warm the brain and eyes up to 14 degrees C above ambient water temperature. Heater cells are derived from extraocular muscle fibers and express a modified muscle phenotype with an extensive transverse-tubule (T-tubule) network and sarcoplasmic reticulum (SR) enriched in Ca(2+)-ATPase (SERCA) pumps and ryanodine receptors (RyRs). Heater cells have a high mitochondria content but have lost most of the contractile myofilaments. Thermogenesis has been hypothesized to be associated with release and reuptake of Ca(2+). In this study, Ca(2+) fluxes in heater SR vesicles derived from blue marlin (Makaira nigricans) were measured using fura-2 fluorescence. Upon the addition of MgATP, heater SR vesicles rapidly sequestered Ca(2+). Uptake of Ca(2+) was thapsigargin sensitive, and maximum loading ranged between 0.8 micro mol Ca(2+) mg(-1) protein and 1.0 micro mol Ca(2+) mg(-1) protein. Upon the addition of 10 mmol l(-1) caffeine or 350 micro mol l(-1) ryanodine, heater SR vesicles released only a small fraction of the loaded Ca(2+). However, ryanodine could elicit a much larger Ca(2+) release event when the activity of the SERCA pumps was reduced. RNase protection assays revealed that heater tissue expresses an RyR isoform that is also expressed in fish slow-twitch skeletal muscle but is distinct from the RyR expressed in fish fast-twitch skeletal muscle. The heater and slow-twitch muscle RyR isoform has unique physiological properties. In the presence of adenine nucleotides, this RyR remains open even though cytoplasmic Ca(2+) is elevated, a condition that normally closes RyRs. The fast Ca(2+) sequestration by the heater SR, coupled with a physiologically unique RyR, is hypothesized to promote Ca(2+) cycling, ATP turnover and heat generation. A branch of the oculomotor nerve innervates heater organs

  7. Intense resistance exercise induces early and transient increases in ryanodine receptor 1 phosphorylation in human skeletal muscle.

    Directory of Open Access Journals (Sweden)

    Sebastian Gehlert

    Full Text Available BACKGROUND: While ryanodine receptor 1 (RyR1 critically contributes to skeletal muscle contraction abilities by mediating Ca²⁺ion oscillation between sarcoplasmatic and myofibrillar compartments, AMP-activated protein kinase (AMPK senses contraction-induced energetic stress by phosphorylation at Thr¹⁷². Phosphorylation of RyR1 at serine²⁸⁴³ (pRyR1Ser²⁸⁴³ results in leaky RyR1 channels and impaired Ca²⁺homeostasis. Because acute resistance exercise exerts decreased contraction performance in skeletal muscle, preceded by high rates of Ca²⁺-oscillation and energetic stress, intense myofiber contractions may induce increased RyR1 and AMPK phosphorylation. However, no data are available regarding the time-course and magnitude of early RyR1 and AMPK phosphorylation in human myofibers in response to acute resistance exercise. PURPOSE: Determine the effects and early time-course of resistance exercise on pRyR1Ser²⁸⁴³ and pAMPKThr¹⁷² in type I and II myofibers. METHODS: 7 male subjects (age 23±2 years, height: 185±7 cm, weight: 82±5 kg performed 3 sets of 8 repetitions of maximum eccentric knee extensions. Muscle biopsies were taken at rest, 15, 30 and 60 min post exercise. pRyR1Ser²⁸⁴³ and pAMPKThr¹⁷² levels were determined by western blot and semi-quantitative immunohistochemistry techniques. RESULTS: While total RyR1 and total AMPK levels remained unchanged, RyR1 was significantly more abundant in type II than type I myofibers. pRyR1Ser²⁸⁴³ increased 15 min and peaked 30 min (p<0.01 post exercise in both myofiber types. Type I fibers showed relatively higher increases in pRyR1Ser²⁸⁴³ levels than type II myofibers and remained elevated up to 60 min post resistance exercise (p<0.05. pAMPKThr¹⁷² also increased 15 to 30 min post exercise (p<0.01 in type I and II myofibers and in whole skeletal muscle. CONCLUSION: Resistance exercise induces acutely increased pRyR1Ser²⁸⁴³ and

  8. Pre-Slaughter Stress Affects Ryanodine Receptor Protein Gene Expression and the Water-Holding Capacity in Fillets of the Nile Tilapia.

    Directory of Open Access Journals (Sweden)

    Elenice S R Goes

    Full Text Available Current study evaluated the effect of pre-slaughter stress on serum cortisol levels, pH, colorimetry, water-holding capacity (WHC and gene expression of ryanodine receptors (RyR1 and RyR3 in the Nile tilapia. A 3x4 factorial scheme experiment was conducted comprising three densities (100, 200, 400 kg/m³ with four transportation times (60, 120, 180, and 240 minutes.Transportation times alone reduced cortisol levels up to 180 minutes, followed by increased WHC and mRNA expression, RyR1 and RyR3 (200 kg/m³ density. No effect of density x transportation time interacted on the evaluated parameters. Results provided the first evidence that pre-slaughter stress affected ryanodine gene expression receptors and, consequently, the water-holding capacity in tilapia fillets.

  9. [3H]PN200-110 and [3H]ryanodine binding and reconstitution of ion channel activity with skeletal muscle membranes

    International Nuclear Information System (INIS)

    Hamilton, S.L.; Alvarez, R.M.; Fill, M.; Hawkes, M.J.; Brush, K.L.; Schilling, W.P.; Stefani, E.

    1989-01-01

    Skeletal muscle membranes derived either from the tubular (T) network or from the sarcoplasmic reticulum (SR) were characterized with respect to the binding of the dihydropyridine, [ 3 H]PN200-110, and the alkaloid, [ 3 H]ryanodine; polypeptide composition; and ion channel activity. Conditions for optimizing the binding of these radioligands are discussed. A bilayer pulsing technique is described and is used to examine the channels present in these membranes. Fusion of T-tubule membranes into bilayers revealed the presence of chloride channels and dihydropyridine-sensitive calcium channels with three distinct conductances. The dihydropyridine-sensitive channels were further characterized with respect to their voltage dependence. Pulsing experiments indicated that two different populations of dihydropyridine-sensitive channels existed. Fusion of heavy SR vesicles revealed three different ion channels; the putative calcium release channel, a potassium channel, and a chloride channel. Thus, this fractionation procedure provides T-tubules and SR membranes which, with radioligand binding and single channel recording techniques, provide a useful tool to study the characteristics of skeletal muscle ion channels and their possible role in excitation-contraction coupling

  10. 3D Mapping of the SPRY2 domain of ryanodine receptor 1 by single-particle cryo-EM.

    Directory of Open Access Journals (Sweden)

    Alex Perálvarez-Marín

    Full Text Available The type 1 skeletal muscle ryanodine receptor (RyR1 is principally responsible for Ca(2+ release from the sarcoplasmic reticulum and for the subsequent muscle contraction. The RyR1 contains three SPRY domains. SPRY domains are generally known to mediate protein-protein interactions, however the location of the three SPRY domains in the 3D structure of the RyR1 is not known. Combining immunolabeling and single-particle cryo-electron microscopy we have mapped the SPRY2 domain (S1085-V1208 in the 3D structure of RyR1 using three different antibodies against the SPRY2 domain. Two obstacles for the image processing procedure; limited amount of data and signal dilution introduced by the multiple orientations of the antibody bound in the tetrameric RyR1, were overcome by modifying the 3D reconstruction scheme. This approach enabled us to ascertain that the three antibodies bind to the same region, to obtain a 3D reconstruction of RyR1 with the antibody bound, and to map SPRY2 to the periphery of the cytoplasmic domain of RyR1. We report here the first 3D localization of a SPRY2 domain in any known RyR isoform.

  11. miRNAs regulated overexpression of ryanodine receptor is involved in chlorantraniliprole resistance in Plutella xylostella (L.)

    Science.gov (United States)

    Li, Xiuxia; Guo, Lei; Zhou, Xuguo; Gao, Xiwu; Liang, Pei

    2015-01-01

    The amino acid mutations in ryanodine receptor (RyR) and elevated activity of detoxification enzymes have been associated with the diamide insecticide resistance in the diamondback moth, Plutella xylostella (L.). The up-regulation of P. xylostella RyR mRNA (PxRyR) expression has also been reported in field populations of different graphical origin. However, whether the up-regulation of PxRyR is involved in diamide resistance remains unknown. In this paper, 2.28- to 4.14-fold higher expression of PxRyR was detected in five field collected resistant populations, compared to that in a susceptible population. The expression of PxRyR was up-regulated 5.0- and 7.2-fold, respectively, after P. xylostella was treated with LC50 and LC75 of chlorantraniliprole for 12 h. Suppression of PxRyR using RNA interference restored the toxicity of chlorantraniliprole against the fourth instar larvae from the resistant population. More importantly, the expression of PxRyR is regulated by two miRNAs, miR-7a and miR-8519. These findings provide an empirical evidence of the involvement of miRNAs in the regulation of insecticide resistance, and shed light on the novel targets for the sustainable management of this devastating insect pest. PMID:26370154

  12. Functional analysis of a point mutation in the ryanodine receptor of Plutella xylostella (L.) associated with resistance to chlorantraniliprole.

    Science.gov (United States)

    Guo, Lei; Wang, Yi; Zhou, Xuguo; Li, Zhenyu; Liu, Shangzhong; Pei, Liang; Gao, Xiwu

    2014-07-01

    The diamondback moth, Plutella xylostella (L.) has developed extremely high resistance to chlorantraniliprole and other diamide insecticides in the field. A glycine to glutamic acid substitution (G4946E) in the P. xylostella ryanodine receptor (PxRyR) has been found in two resistant populations collected in Thailand and Philippines and was considered associated with the diamide insecticides resistance but no experimental evidence was provided. The present study aimed to clarify the function of the reported mutation in chlorantraniliprole resistance in P. xylostella. We identified the same mutation (G4946E) in PxRyR from four field collected chlorantraniliprole resistant populations of Plutella xylostella in China. Most importantly, we found that the frequency of the G4946E mutation is significantly correlated to the chlorantraniliprole resistance ratios in P. xylostella (R(2)  = 0.82, P = 0.0003). Ligand binding assays showed that the binding affinities of the PxRyR to the chlorantraniliprole in three field resistant populations were 2.41-, 2.54- and 2.60-times lower than that in the susceptible one. For the first time we experimentally proved that the G4946E mutation in PxRyR confers resistance to chlorantraniliprole in Plutella xylostella. These findings pave the way for the complete understanding of the mechanisms of diamide insecticides resistance in insects. © 2013 Society of Chemical Industry.

  13. Novel mutations and mutation combinations of ryanodine receptor in a chlorantraniliprole resistant population of Plutella xylostella (L.)

    Science.gov (United States)

    Guo, Lei; Liang, Pei; Zhou, Xuguo; Gao, Xiwu

    2014-01-01

    A previous study documented a glycine to glutamic acid mutation (G4946E) in ryanodine receptor (RyR) was highly correlated to diamide insecticide resistance in field populations of Plutella xylostella (Lepidoptera: Plutellidae). In this study, a field population collected in Yunnan province, China, exhibited a 2128-fold resistance to chlorantraniliprole. Sequence comparison between resistant and susceptible P. xylostella revealed three novel mutations including a glutamic acid to valine substitution (E1338D), a glutamine to leucine substitution (Q4594L) and an isoleucine to methionine substitution (I4790M) in highly conserved regions of RyR. Frequency analysis of all four mutations in this field population showed that the three new mutations showed a high frequency of 100%, while the G4946E had a frequency of 20%. Furthermore, the florescent ligand binding assay revealed that the RyR containing multiple mutations displayed a significantly lower affinity to the chlorantraniliprole. The combined results suggested that the co-existence of different combinations of the four mutations was involved in the chlorantraniliprole resistance. An allele-specific PCR based method was developed for the diagnosis of the four mutations in the field populations of P. xylostella. PMID:25377064

  14. Crystal structure of ryanodine receptor N-terminal domain from Plutella xylostella reveals two potential species-specific insecticide-targeting sites.

    Science.gov (United States)

    Lin, Lianyun; Liu, Chen; Qin, Juan; Wang, Jie; Dong, Shengjie; Chen, Wei; He, Weiyi; Gao, Qingzhi; You, Minsheng; Yuchi, Zhiguang

    2018-01-01

    Ryanodine receptors (RyRs) are large calcium-release channels located in sarcoplasmic reticulum membrane. They play a central role in excitation-contraction coupling of muscle cells. Three commercialized insecticides targeting pest RyRs generate worldwide sales over 2 billion U.S. dollars annually, but the structure of insect RyRs remains elusive, hindering our understanding of the mode of action of RyR-targeting insecticides and the development of insecticide resistance in pests. Here we present the crystal structure of RyR N-terminal domain (NTD) (residue 1-205) at 2.84 Å resolution from the diamondback moth (DBM), Plutella xylostella, a destructive pest devouring cruciferous crops all over the world. Similar to its mammalian homolog, DBM RyR NTD consists of a beta-trefoil folding motif and a flanking alpha helix. Interestingly, two regions in NTD interacting with neighboring domains showed distinguished conformations in DBM relative to mammalian RyRs. Using homology modeling and molecular dynamics simulation, we created a structural model of the N-terminal three domains, showing two unique binding pockets that could be targeted by potential species-specific insecticides. Thermal melt experiment showed that the stability of DBM RyR NTD was higher than mammalian RyRs, probably due to a stable intra-domain disulfide bond observed in the crystal structure. Previously DBM NTD was shown to be one of the two critical regions to interact with insecticide flubendiamide, but isothermal titration calorimetry experiments negated DBM NTD alone as a major binding site for flubendiamide. Copyright © 2017 Elsevier Ltd. All rights reserved.

  15. A model of cardiac ryanodine receptor gating predicts experimental Ca2+-dynamics and Ca2+-triggered arrhythmia in the long QT syndrome

    Science.gov (United States)

    Wilson, Dan; Ermentrout, Bard; Němec, Jan; Salama, Guy

    2017-09-01

    Abnormal Ca2+ handling is well-established as the trigger of cardiac arrhythmia in catecholaminergic polymorphic ventricular tachycardia and digoxin toxicity, but its role remains controversial in Torsade de Pointes (TdP), the arrhythmia associated with the long QT syndrome (LQTS). Recent experimental results show that early afterdepolarizations (EADs) that initiate TdP are caused by spontaneous (non-voltage-triggered) Ca2+ release from Ca2+-overloaded sarcoplasmic reticulum (SR) rather than the activation of the L-type Ca2+-channel window current. In bradycardia and long QT type 2 (LQT2), a second, non-voltage triggered cytosolic Ca2+ elevation increases gradually in amplitude, occurs before overt voltage instability, and then precedes the rise of EADs. Here, we used a modified Shannon-Puglisi-Bers model of rabbit ventricular myocytes to reproduce experimental Ca2+ dynamics in bradycardia and LQT2. Abnormal systolic Ca2+-oscillations and EADs caused by SR Ca2+-release are reproduced in a modified 0-dimensional model, where 3 gates in series control the ryanodine receptor (RyR2) conductance. Two gates control RyR2 activation and inactivation and sense cytosolic Ca2+ while a third gate senses luminal junctional SR Ca2+. The model predicts EADs in bradycardia and low extracellular [K+] and cessation of SR Ca2+-release terminate salvos of EADs. Ca2+-waves, systolic cell-synchronous Ca2+-release, and multifocal diastolic Ca2+ release seen in subcellular Ca2+-mapping experiments are observed in the 2-dimensional version of the model. These results support the role of SR Ca2+-overload, abnormal SR Ca2+-release, and the subsequent activation of the electrogenic Na+/Ca2+-exchanger as the mechanism of TdP. The model offers new insights into the genesis of cardiac arrhythmia and new therapeutic strategies.

  16. Calcium/calmodulin-dependent kinase II and nitric oxide synthase 1-dependent modulation of ryanodine receptors during β-adrenergic stimulation is restricted to the dyadic cleft.

    Science.gov (United States)

    Dries, Eef; Santiago, Demetrio J; Johnson, Daniel M; Gilbert, Guillaume; Holemans, Patricia; Korte, Sanne M; Roderick, H Llewelyn; Sipido, Karin R

    2016-10-15

    The dyadic cleft, where coupled ryanodine receptors (RyRs) reside, is thought to serve as a microdomain for local signalling, as supported by distinct modulation of coupled RyRs dependent on Ca 2+ /calmodulin-dependent kinase II (CaMKII) activation during high-frequency stimulation. Sympathetic stimulation through β-adrenergic receptors activates an integrated signalling cascade, enhancing Ca 2+ cycling and is at least partially mediated through CaMKII. Here we report that CaMKII activation during β-adrenergic signalling is restricted to the dyadic cleft, where it enhances activity of coupled RyRs thereby contributing to the increase in diastolic events. Nitric oxide synthase 1 equally participates in the local modulation of coupled RyRs. In contrast, the increase in the Ca 2+ content of the sarcoplasmic reticulum and related increase in the amplitude of the Ca 2+ transient are primarily protein kinase A-dependent. The present data extend the concept of microdomain signalling in the dyadic cleft and give perspectives for selective modulation of RyR subpopulations and diastolic events. In cardiac myocytes, β-adrenergic stimulation enhances Ca 2+ cycling through an integrated signalling cascade modulating L-type Ca 2+ channels (LTCCs), phospholamban and ryanodine receptors (RyRs). Ca 2+ /calmodulin-dependent kinase II (CaMKII) and nitric oxide synthase 1 (NOS1) are proposed as prime mediators for increasing RyR open probability. We investigate whether this pathway is confined to the high Ca 2+ microdomain of the dyadic cleft and thus to coupled RyRs. Pig ventricular myocytes are studied under whole-cell voltage-clamp and confocal line-scan imaging with Fluo-4 as a [Ca 2+ ] i indicator. Following conditioning depolarizing pulses, spontaneous RyR activity is recorded as Ca 2+ sparks, which are assigned to coupled and non-coupled RyR clusters. Isoproterenol (ISO) (10 nm) increases Ca 2+ spark frequency in both populations of RyRs. However, CaMKII inhibition reduces

  17. Molecular characterization and expression profiling of ryanodine receptor gene in the pink stem borer, Sesamia inferens (Walker).

    Science.gov (United States)

    Wu, Shun-Fan; Zhao, Dan-Dan; Huang, Jing-Mei; Zhao, Si-Qi; Zhou, Li-Qi; Gao, Cong-Fen

    2018-04-01

    The susceptibilities of three field populations of pink stem borer (PSB), Sesamia inferens (walker) to diamide insecticides, chlorantraniliprole and flubendiamide, were evaluated in this study. The results showed that these PSB field populations were still sensitive to the two diamide insecticides after many years of exposure. To further understand PSB and diamide insecticide, the full-length ryanodine receptor (RyR) cDNA (named as SiRyR), the molecular target of diamide insecticides was cloned from PSB and characterized. The SiRyR gene contains an open reading frame of 15,420 nucleotides, encoding 5140 amino acid residues, which shares 77% to 98% sequence identity with RyR homologous of other insects. All hallmarks of RyR proteins are conserved in the SiRyR protein, including the conserved C-terminal domain with the consensus calcium-biding EF-hands (calcium-binding motif), the six transmembrane domains, as well as mannosyltransferase, IP3R and RyR (pfam02815) (MIR) domains. Real-time qPCR analysis revealed that the highest mRNA expression levels of SiRyR were observed in pupa and adults, especially in males. SiRyR was expressed at the highest level in thorax, and the lowest level in wing. The full genetic characterization of SiRyR could provide useful information for future functional expression studies and for discovery of new insecticides with selective insecticidal activity. Copyright © 2018 Elsevier Inc. All rights reserved.

  18. Voltage-dependent modulation of cardiac ryanodine receptors (RyR2 by protamine.

    Directory of Open Access Journals (Sweden)

    Paula L Diaz-Sylvester

    Full Text Available It has been reported that protamine (>10 microg/ml blocks single skeletal RyR1 channels and inhibits RyR1-mediated Ca2+ release from sarcoplasmic reticulum microsomes. We extended these studies to cardiac RyR2 reconstituted into planar lipid bilayers. We found that protamine (0.02-20 microg/ml added to the cytosolic surface of fully activated RyR2 affected channel activity in a voltage-dependent manner. At membrane voltage (V(m; SR lumen-cytosol = 0 mV, protamine induced conductance transitions to several intermediate states (substates as well as full block of RyR2. At V(m>10 mV, the substate with the highest level of conductance was predominant. Increasing V(m from 0 to +80 mV, decreased the number of transitions and residence of the channel in this substate. The drop in current amplitude (full opening to substate had the same magnitude at 0 and +80 mV despite the approximately 3-fold increase in amplitude of the full opening. This is more similar to rectification of channel conductance induced by other polycations than to the action of selective conductance modifiers (ryanoids, imperatoxin. A distinctive effect of protamine (which might be shared with polylysines and histones but not with non-peptidic polycations is the activation of RyR2 in the presence of nanomolar cytosolic Ca2+ and millimolar Mg2+ levels. Our results suggest that RyRs would be subject to dual modulation (activation and block by polycationic domains of neighboring proteins via electrostatic interactions. Understanding these interactions could be important as such anomalies may be associated with the increased RyR2-mediated Ca2+ leak observed in cardiac diseases.

  19. Calcium-Dependent Energetics of Calmodulin Domain Interactions with Regulatory Regions of the Ryanodine Receptor Type 1 (RyR1)

    Science.gov (United States)

    Newman, Rhonda A.; Sorensen, Brenda R.; Kilpatrick, Adina M.; Shea, Madeline A.

    2014-01-01

    Calmodulin (CaM) plays a vital role in calcium homeostasis by allosterically modulating intracellular calcium channels including the homo-tetrameric human Ryanodine Receptor Type 1 (hRyR1). Apo (calcium-free) CaM activates hRyR1 while calcium-saturated CaM inhibits it. Two CaM-binding regions (residues 1975–1999 and 3614–3643) identified in each RyR1 monomer were proposed to allow CaM to bridge adjacent RyR1 subunits. We explored the distinct roles of CaM domains by using fluorescence anisotropy to determine the affinity of CaM1–148 (full-length), CaM1–80 (N-domain) and CaM76–148 (C-domain) for peptides encompassing hRyR1 residues 1975–1999 or 3614–3643. Both CaM1–148 and CaM76–148 associated in a calcium-independent manner with similar affinities for hRyR1(3614–3643)p while CaM1–80 required calcium and bound ~250-fold more weakly. Association of CaM1–148, CaM1–80 and CaM76–148 with hRyR1(1975–1999)p was much less favorable than with hRyR1(3614–3643)p; differences between the two CaM domains were smaller. Equilibrium calcium titrations monitored by steady-state fluorescence demonstrated that both hRyR1 peptides increased the calcium-binding affinity of both CaM domains. These thermodynamic properties support a prior model in which the CaM C-domain associates with RyR1(3614–3643) at low levels of calcium, positioning CaM to rapidly respond to calcium efflux. However, the affinity of the N-domain of CaM for hRyR1(1975–1999)p is insufficient to explain a model in which CaM bridges adjacent RyR1 subunits within the tetramer. This indicates that other protein factors or properties of the tertiary or quaternary structure of hRyR1 contribute to the energetics of CaM-mediated regulation. PMID:25145833

  20. Transient receptor potential channels in essential hypertension

    DEFF Research Database (Denmark)

    Liu, Daoyan; Scholze, Alexandra; Zhu, Zhiming

    2006-01-01

    The role of nonselective cation channels of the transient receptor potential channel (TRPC) family in essential hypertension has not yet been investigated.......The role of nonselective cation channels of the transient receptor potential channel (TRPC) family in essential hypertension has not yet been investigated....

  1. Bell-shaped calcium-response curves of lns(l,4,5)P3- and calcium-gated channels from endoplasmic reticulum of cerebellum

    Science.gov (United States)

    Bezprozvanny, Llya; Watras, James; Ehrlich, Barbara E.

    1991-06-01

    RELEASE of calcium from intracellular stores occurs by two pathways, an inositol 1,4,5-trisphosphate (InsP3)-gated channel1-3 and a calcium-gated channel (ryanodine receptor)4-6. Using specific antibodies, both receptors were found in Purkinje cells of cerebellum7,8. We have now compared the functional properties of the channels corresponding to the two receptors by incorporating endoplasmic reticulum vesicles from canine cerebellum into planar bilayers. InsP3-gated channels were observed most frequently. Another channel type was activated by adenine nucleotides or caffeine, inhibited by ruthenium red, and modified by ryanodine, characteristics of the ryanodine receptor/channel6. The open probability of both channel types displayed a bell-shaped curve for dependence on calcium. For the InsP3-gated channel, the maximum probability of opening occurred at 0.2 µM free calcium, with sharp decreases on either side of the maximum. Maximum activity for the ryanodine receptor/channel was maintained between 1 and 100 µM calcium. Thus, within the physiological range of cytoplasmic calcium, the InsP3-gated channel itself allows positive feed-back and then negative feedback for calcium release, whereas the ryanodine receptor/channel behaves solely as a calcium-activated channel. The existence in the same cell of two channels with different responses to calcium and different ligand sensitivities provides a basis for complex patterns of intracellular calcium regulation.

  2. Geographic spread, genetics and functional characteristics of ryanodine receptor based target-site resistance to diamide insecticides in diamondback moth, Plutella xylostella.

    Science.gov (United States)

    Steinbach, Denise; Gutbrod, Oliver; Lümmen, Peter; Matthiesen, Svend; Schorn, Corinna; Nauen, Ralf

    2015-08-01

    Anthranilic diamides and flubendiamide belong to a new chemical class of insecticides acting as conformation sensitive activators of the insect ryanodine receptor (RyR). These compounds control a diverse range of different herbivorous insects including diamondback moth, Plutella xylostella (Lepidoptera: Plutellidae), a notorious global pest on cruciferous crops, which recently developed resistance due to target-site mutations located in the trans-membrane domain of the Plutella RyR. In the present study we further investigated the genetics and functional implications of a RyR G4946E target-site mutation we recently identified in a Philippine diamondback moth strain (Sudlon). Strain Sudlon is homozygous for the G4946E mutation and has been maintained under laboratory conditions without selection pressure for almost four years, and still exhibit stable resistance ratios of >2000-fold to all commercial diamides. Its F1 progeny resulting from reciprocal crosses with a susceptible strain (BCS-S) revealed no maternal effects and a diamide susceptible phenotype, suggesting an autosomally almost recessive mode of inheritance. Subsequent back-crosses indicate a near monogenic nature of the diamide resistance in strain Sudlon. Radioligand binding studies with Plutella thoracic microsomal membrane preparations provided direct evidence for the dramatic functional implications of the RyR G4946E mutation on both diamide specific binding and its concentration dependent modulation of [(3)H]ryanodine binding. Computational modelling based on a cryo-EM structure of rabbit RyR1 suggests that Plutella G4946E is located in trans-membrane helix S4 close to S4-S5 linker domain supposed to be involved in the modulation of the voltage sensor, and another recently described mutation, I4790M in helix S2 approx. 13 Å opposite of G4946E. Genotyping by pyrosequencing revealed the presence of the RyR G4946E mutation in larvae collected in 2013/14 in regions of ten different countries where

  3. Ryanodine receptors contribute to the induction of nociceptive input-evoked long-term potentiation in the rat spinal cord slice

    Directory of Open Access Journals (Sweden)

    Zhao Zhi-Qi

    2010-01-01

    Full Text Available Abstract Background Our previous study demonstrated that nitric oxide (NO contributes to long-term potentiation (LTP of C-fiber-evoked field potentials by tetanic stimulation of the sciatic nerve in the spinal cord in vivo. Ryanodine receptor (RyR is a downstream target for NO. The present study further explored the role of RyR in synaptic plasticity of the spinal pain pathway. Results By means of field potential recordings in the adult male rat in vivo, we showed that RyR antagonist reduced LTP of C-fiber-evoked responses in the spinal dorsal horn by tetanic stimulation of the sciatic nerve. Using spinal cord slice preparations and field potential recordings from superficial dorsal horn, high frequency stimulation of Lissauer's tract (LT stably induced LTP of field excitatory postsynaptic potentials (fEPSPs. Perfusion of RyR antagonists blocked the induction of LT stimulation-evoked spinal LTP, while Ins(1,4,5P3 receptor (IP3R antagonist had no significant effect on LTP induction. Moreover, activation of RyRs by caffeine without high frequency stimulation induced a long-term potentiation in the presence of bicuculline methiodide and strychnine. Further, in patch-clamp recordings from superficial dorsal horn neurons, activation of RyRs resulted in a large increase in the frequency of miniature EPSCs (mEPSCs. Immunohistochemical study showed that RyRs were expressed in the dorsal root ganglion (DRG neurons. Likewise, calcium imaging in small DRG neurons illustrated that activation of RyRs elevated [Ca2+]i in small DRG neurons. Conclusions These data indicate that activation of presynaptic RyRs play a crucial role in the induction of LTP in the spinal pain pathway, probably through enhancement of transmitter release.

  4. Bifenthrin causes transcriptomic alterations in mTOR and ryanodine receptor-dependent signaling and delayed hyperactivity in developing zebrafish (Danio rerio).

    Science.gov (United States)

    Frank, Daniel F; Miller, Galen W; Harvey, Danielle J; Brander, Susanne M; Geist, Juergen; Connon, Richard E; Lein, Pamela J

    2018-04-18

    Over the last few decades, the pyrethroid insecticide bifenthrin has been increasingly employed for pest control in urban and agricultural areas, putting humans and wildlife at increased risk of exposure. Exposures to nanomolar (nM) concentrations of bifenthrin have recently been reported to alter calcium oscillations in rodent neurons. Neuronal calcium oscillations are influenced by ryanodine receptor (RyR) activity, which modulates calcium-dependent signaling cascades, including the mechanistic target of rapamycin (mTOR) signaling pathway. RyR activity and mTOR signaling play critical roles in regulating neurodevelopmental processes. However, whether environmentally relevant levels of bifenthrin alter RyR or mTOR signaling pathways to influence neurodevelopment has not been addressed. Therefore, our main objectives in this study were to examine the transcriptomic responses of genes involved in RyR and mTOR signaling pathways in zebrafish (Danio rerio) exposed to low (ng/L) concentrations of bifenthrin, and to assess the potential functional consequences by measuring locomotor responses to external stimuli. Wildtype zebrafish were exposed for 1, 3 and 5 days to 1, 10 and 50 ng/L bifenthrin, followed by a 14 d recovery period. Bifenthrin elicited significant concentration-dependent transcriptional responses in the majority of genes examined in both signaling cascades, and at all time points examined during the acute exposure period (1, 3, and 5 days post fertilization; dpf), and at the post recovery assessment time point (19 dpf). Changes in locomotor behavior were not evident during the acute exposure period, but were observed at 19 dpf, with main effects (increased locomotor behavior) detected in fish exposed developmentally to bifenthrin at 1 or 10 ng/L, but not 50 ng/L. These findings illustrate significant influences of developmental exposures to low (ng/L) concentrations of bifenthrin on neurodevelopmental processes in zebrafish. Copyright © 2018

  5. PCB 136 Atropselectively Alters Morphometric and Functional Parameters of Neuronal Connectivity in Cultured Rat Hippocampal Neurons via Ryanodine Receptor-Dependent Mechanisms

    Science.gov (United States)

    Yang, Dongren; Kania-Korwel, Izabela; Ghogha, Atefeh; Chen, Hao; Stamou, Marianna; Bose, Diptiman D.; Pessah, Isaac N.; Lehmler, Hans-Joachim; Lein, Pamela J.

    2014-01-01

    We recently demonstrated that polychlorinated biphenyl (PCB) congeners with multiple ortho chlorine substitutions sensitize ryanodine receptors (RyRs), and this activity promotes Ca2+-dependent dendritic growth in cultured neurons. Many ortho-substituted congeners display axial chirality, and we previously reported that the chiral congener PCB 136 (2,2′,3,3′,6,6′-hexachlorobiphenyl) atropselectively sensitizes RyRs. Here, we test the hypothesis that PCB 136 atropisomers differentially alter dendritic growth and other parameters of neuronal connectivity influenced by RyR activity. (−)-PCB 136, which potently sensitizes RyRs, enhances dendritic growth in primary cultures of rat hippocampal neurons, whereas (+)-PCB 136, which lacks RyR activity, has no effect on dendritic growth. The dendrite-promoting activity of (−)-PCB 136 is observed at concentrations ranging from 0.1 to 100nM and is blocked by pharmacologic RyR antagonism. Neither atropisomer alters axonal growth or cell viability. Quantification of PCB 136 atropisomers in hippocampal cultures indicates that atropselective effects on dendritic growth are not due to differential partitioning of atropisomers into cultured cells. Imaging of hippocampal neurons loaded with Ca2+-sensitive dye demonstrates that (−)-PCB 136 but not (+)-PCB 136 increases the frequency of spontaneous Ca2+ oscillations. Similarly, (−)-PCB 136 but not (+)-PCB 136 increases the activity of hippocampal neurons plated on microelectrode arrays. These data support the hypothesis that atropselective effects on RyR activity translate into atropselective effects of PCB 136 atropisomers on neuronal connectivity, and suggest that the variable atropisomeric enrichment of chiral PCBs observed in the human population may be a significant determinant of individual susceptibility for adverse neurodevelopmental outcomes following PCB exposure. PMID:24385416

  6. 17beta-estradiol rapidly mobilizes intracellular calcium from ryanodine-receptor-gated stores via a PKC-PKA-Erk-dependent pathway in the human eccrine sweat gland cell line NCL-SG3.

    LENUS (Irish Health Repository)

    Muchekehu, Ruth W

    2008-09-01

    We describe a novel rapid non-genomic effect of 17beta-estradiol (E2) on intracellular Ca2+ ([Ca2+]i) signalling in the eccrine sweat gland epithelial cell line NCL-SG3. E2 had no observable effect on basal [Ca2+]i, however exposure of cells to E2 in the presence of the microsomal Ca2+ ATPase pump inhibitor, thapsigargin, produced a secondary, sustained increase in [Ca2+]i compared to thapsigargin treatment alone, where cells responded with a transient single spike-like increase in [Ca2+]i. The E2-induced increase in [Ca2+]i was not dependent on the presence of extracellular calcium and was completely abolished by ryanodine (100 microM). The estrogen receptor antagonist ICI 182,780 (1 microM) prevented the E2-induced effects suggesting a role for the estrogen receptor in the release of [Ca2+]i from ryanodine-receptor-gated stores. The E2-induced effect on [Ca2+]i could also be prevented by the protein kinase C delta (PKCdelta)-specific inhibitor rottlerin (10 microM), the protein kinase A (PKA) inhibitor Rp-adenosine 3\\

  7. [Endoplasmic-mitochondrial Ca(2+)-functional unit: dependence of respiration of secretory cells on activity of ryanodine- and IP3 - sensitive Ca(2+)-channels].

    Science.gov (United States)

    Velykopols'ka, O Iu; Man'ko, B O; Man'ko, V V

    2012-01-01

    Using Clark oxygen electrode, dependence of mitochondrial functions on Ca(2+)-release channels activity of Chironomus plumosus L. larvae salivary glands suspension was investigated. Cells were ATP-permeabilized in order to enable penetration of exogenous oxidative substrates. Activation of plasmalemmal P2X-receptors (as well as P2Y-receptors) per se does not modify the endogenous respiration of salivary gland suspension. That is, Ca(2+)-influx from extracellular medium does not influence functional activity of mitochondria, although they are located along the basal part of the plasma membrane. Activation of RyRs intensifies endogenous respiration and pyruvate-malate-stimulated respiration, but not succinate-stimulated respiration. Neither activation of IP3Rs (via P2Y-receptors activation), nor their inhibition alters endogenous respiration. Nevertheless, IP3Rs inhibition by 2-APB intensifies succinate-stimulated respiration. All abovementioned facts testify that Ca2+, released from stores via channels, alters functional activity of mitochondria, and undoubtedly confirm the existence of endoplasmic-mitochondrial Ca(2+)-functional unit in Ch. plumosus larvae salivary glands secretory cells. In steady state of endoplasmic-mitochondrial Ca(2+)-functional unit the spontaneous activity of IP3Rs is observed; released through IP3Rs, Ca2+ is accumulated in mitochondria via uniporter and modulates oxidative processes. Activation of RyRs induces the transition of endoplasmic-mitochondrial Ca(2+)-functional unit to the active state, which is required to intensify cell respiration and oxidative phosphorylation. As expected, the transition of endoplasmic-mitochondrial Ca(2+)-functional unit to inactivated state (i. e. inhibition of Ca(2+)-release channels at excessive [Ca2+]i) limits the duration of signal transduction, has protective nature and prevents apoptosis.

  8. Compound K induced apoptosis via endoplasmic reticulum Ca2+ release through ryanodine receptor in human lung cancer cells

    Directory of Open Access Journals (Sweden)

    Dong-Hyun Shin

    2018-04-01

    Full Text Available Background: Extended endoplasmic reticulum (ER stress may initiate apoptotic pathways in cancer cells, and ER stress has been reported to possibly increase tumor death in cancer therapy. We previously reported that caspase-8 played an important role in compound K-induced apoptosis via activation of caspase-3 directly or indirectly through Bid cleavage, cytochrome c release, and caspase-9 activation in HL-60 human leukemia cells. The mechanisms leading to apoptosis in A549 and SK-MES-1 human lung cancer cells and the role of ER stress have not yet been understood. Methods: The apoptotic effects of compound K were analyzed using flow cytometry, and the changes in protein levels were determined using Western blot analysis. The intracellular calcium levels were monitored by staining with Fura-2/AM and Fluo-3/AM. Results: Compound K-induced ER stress was confirmed through increased phosphorylation of eIF2α and protein levels of GRP78/BiP, XBP-1S, and IRE1α in human lung cancer cells. Moreover, compound-K led to the accumulation of intracellular calcium and an increase in m-calpain activities that were both significantly inhibited by pretreatment either with BAPTA-AM (an intracellular Ca2+ chelator or dantrolene (an RyR channel antagonist. These results were correlated with the outcome that compound K induced ER stress-related apoptosis through caspase-12, as z-ATAD-fmk (a specific inhibitor of caspase-12 partially ameliorated this effect. Interestingly, 4-PBA (ER stress inhibitor dramatically improved the compound K-induced apoptosis. Conclusion: Cell survival and intracellular Ca2+ homeostasis during ER stress in human lung cancer cells are important factors in the induction of the compound K-induced apoptotic pathway. Keywords: apoptosis, calcium, compound K, ER stress, lung cancer cells

  9. C[sub 10]-O[sub eq]-N-(4-azido-5-[sup 125]iodo salicyloyl)-[beta]-alanyl-[beta] alanyl ryanodine (Az-[beta]AR), a novel photo-affinity ligand for the ryanodine binding site

    Energy Technology Data Exchange (ETDEWEB)

    Bidasee, K.R.; Besch, H.R. Jr.; Kwon, Sangyeol; Emmick, J.T.; Besch, K.T.; Gerzon, Koert; Humerickhouse, R.A. (Indiana Univ., Indianapolis, IN (United States). School of Medicine)

    1994-01-01

    A high affinity, photoactivatable, radio-iodinated ligand for the ryanodine binding site(s) of the sarcoplasmic reticulum calcium-release channel, C[sub 10]-O[sub e]-N-(4-azido-5-[sup 125]iodo salicyloyl)-[beta]-alanyl-[beta]-alanyl ryanodine (Az-[beta]AR), was synthesized at a specific activity of 1400mCi/mmol. (Author).

  10. Separation and formation of ryanodine from dehydroryanodine. Preparation of tritium-labelled ryanodine

    International Nuclear Information System (INIS)

    Sutko, J.L.; Thompson, L.J.; Schlatterer, R.G.

    1986-01-01

    The commercially available preparation of the naturally occurring diterpene ester ryanodine contains several compounds in addition to ryanodine. These compounds were separated and purified using high performance liquid chromatography. The two major components, ryanodine and dehydroryanodine represented 90% of the material present. A method for the efficient reduction of dehydroryanodine to ryanodine was developed and used to produce ryanodine having tritium atoms at positions 19 and 20 and a specific activity of 60.8 Ci/mmole. (author)

  11. The role of transient receptor potential channels in metabolic syndrome

    DEFF Research Database (Denmark)

    Liu, Daoyan; Zhu, Zhiming; Tepel, Martin

    2008-01-01

    Metabolic syndrome is correlated with increased cardiovascular risk and characterized by several factors, including visceral obesity, hypertension, insulin resistance, and dyslipidemia. Several members of a large family of nonselective cation entry channels, e.g., transient receptor potential (TRP...

  12. The putative imidazoline receptor agonist, harmane, promotes intracellular calcium mobilisation in pancreatic beta-cells.

    Science.gov (United States)

    Squires, Paul E; Hills, Claire E; Rogers, Gareth J; Garland, Patrick; Farley, Sophia R; Morgan, Noel G

    2004-10-06

    beta-Carbolines (including harmane and pinoline) stimulate insulin secretion by a mechanism that may involve interaction with imidazoline I(3)-receptors but which also appears to be mediated by actions that are additional to imidazoline receptor agonism. Using the MIN6 beta-cell line, we now show that both the imidazoline I(3)-receptor agonist, efaroxan, and the beta-carboline, harmane, directly elevate cytosolic Ca(2+) and increase insulin secretion but that these responses display different characteristics. In the case of efaroxan, the increase in cytosolic Ca(2+) was readily reversible, whereas, with harmane, the effect persisted beyond removal of the agonist and resulted in the development of a repetitive train of Ca(2+)-oscillations whose frequency, but not amplitude, was concentration-dependent. Initiation of the Ca(2+)-oscillations by harmane was independent of extracellular calcium but was sensitive to both dantrolene and high levels (20 mM) of caffeine, suggesting the involvement of ryanodine receptor-gated Ca(2+)-release. The expression of ryanodine receptor-1 and ryanodine receptor-2 mRNA in MIN6 cells was confirmed using reverse transcription-polymerase chain reaction (RT-PCR) and, since low concentrations of caffeine (1 mM) or thimerosal (10 microM) stimulated increases in [Ca(2+)](i), we conclude that ryanodine receptors are functional in these cells. Furthermore, the increase in insulin secretion induced by harmane was attenuated by dantrolene, consistent with the involvement of ryanodine receptors in mediating this response. By contrast, the smaller insulin secretory response to efaroxan was unaffected by dantrolene. Harmane-evoked changes in cytosolic Ca(2+) were maintained by nifedipine-sensitive Ca(2+)-influx, suggesting the involvement of L-type voltage-gated Ca(2+)-channels. Taken together, these data imply that harmane may interact with ryanodine receptors to generate sustained Ca(2+)-oscillations in pancreatic beta-cells and that this effect

  13. The action of ryanodine on rat fast and slow intact skeletal muscles.

    Science.gov (United States)

    Fryer, M W; Lamb, G D; Neering, I R

    1989-07-01

    1. The action of ryanodine on force development of bundles dissected from rat extensor digitorum longus (EDL) and soleus muscles has been examined. 2. Ryanodine (100-5000 nM) irreversibly depressed twitch and tetanic tension of both muscle types in a dose-related manner. 3. At concentrations above 250 nM, ryanodine induced a slowly developing, dose-dependent contracture which could not be blocked by 5 mM-Co2+. Increasing the stimulation rate or decreasing the oxygenation of the preparation accelerated the rate of contracture development while the total removal of extracellular Ca2+ was required to prevent it. 4. Following the relaxation of the initial contracture (IC) in Ca2+-free solution, a second type of contracture (SC) could be induced by the readdition of Ca2+. This contracture differed from IC in that it was dependent on Ca2+ in the millimolar range and was prevented by 5 mM-Co2+. Both IC and SC were relaxed by perfusion with Ca2+-free, EGTA-containing solution. 5. Subcontracture doses of ryanodine (100 nM) markedly potentiated caffeine contractures of both muscle types. 6. Asymmetric charge movement in EDL fibres was recorded with the Vaseline-gap technique. The amount of charge moved near threshold was virtually unaffected by the presence of 10 microM-ryanodine over the time examined. 7. The results are consistent with the suggestion that ryanodine locks the calcium release channels of the sarcoplasmic reticulum (SR) in an open subconductance state with reduced conductance. It appears that lowering the external calcium concentration might still inactivate the release channels after they have been blocked open by ryanodine, possibly by an effect on the T-tubular voltage sensor.

  14. Zinc as Allosteric Ion Channel Modulator: Ionotropic Receptors as Metalloproteins

    Science.gov (United States)

    Peralta, Francisco Andrés; Huidobro-Toro, Juan Pablo

    2016-01-01

    Zinc is an essential metal to life. This transition metal is a structural component of many proteins and is actively involved in the catalytic activity of cell enzymes. In either case, these zinc-containing proteins are metalloproteins. However, the amino acid residues that serve as ligands for metal coordination are not necessarily the same in structural proteins compared to enzymes. While crystals of structural proteins that bind zinc reveal a higher preference for cysteine sulfhydryls rather than histidine imidazole rings, catalytic enzymes reveal the opposite, i.e., a greater preference for the histidines over cysteines for catalysis, plus the influence of carboxylic acids. Based on this paradigm, we reviewed the putative ligands of zinc in ionotropic receptors, where zinc has been described as an allosteric modulator of channel receptors. Although these receptors do not strictly qualify as metalloproteins since they do not normally bind zinc in structural domains, they do transitorily bind zinc at allosteric sites, modifying transiently the receptor channel’s ion permeability. The present contribution summarizes current information showing that zinc allosteric modulation of receptor channels occurs by the preferential metal coordination to imidazole rings as well as to the sulfhydryl groups of cysteine in addition to the carboxyl group of acid residues, as with enzymes and catalysis. It is remarkable that most channels, either voltage-sensitive or transmitter-gated receptor channels, are susceptible to zinc modulation either as positive or negative regulators. PMID:27384555

  15. P2X receptor channels in endocrine glands

    Czech Academy of Sciences Publication Activity Database

    Stojilkovic, S. S.; Zemková, Hana

    2013-01-01

    Roč. 2, č. 4 (2013), s. 173-180 ISSN 2190-460X R&D Projects: GA ČR(CZ) GBP304/12/G069 Institutional support: RVO:67985823 Keywords : ATP * purinergic P2X receptor channels * pituitary * endocrine glands Subject RIV: ED - Physiology

  16. The role of transient receptor potential channels in joint diseases.

    Science.gov (United States)

    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.

  17. The role of transient receptor potential channels in joint diseases

    Directory of Open Access Journals (Sweden)

    O Krupkova

    2017-10-01

    Full Text Available ransient 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.

  18. Ion channels in EEG: isolating channel dysfunction in NMDA receptor antibody encephalitis.

    Science.gov (United States)

    Symmonds, Mkael; Moran, Catherine H; Leite, M Isabel; Buckley, Camilla; Irani, Sarosh R; Stephan, Klaas Enno; Friston, Karl J; Moran, Rosalyn J

    2018-04-30

    Neurological and psychiatric practice frequently lack diagnostic probes that can assess mechanisms of neuronal communication non-invasively in humans. In N-methyl-d-aspartate (NMDA) receptor antibody encephalitis, functional molecular assays are particularly important given the presence of NMDA antibodies in healthy populations, the multifarious symptomology and the lack of radiological signs. Recent advances in biophysical modelling techniques suggest that inferring cellular-level properties of neural circuits from macroscopic measures of brain activity is possible. Here, we estimated receptor function from EEG in patients with NMDA receptor antibody encephalitis (n = 29) as well as from encephalopathic and neurological patient controls (n = 36). We show that the autoimmune patients exhibit distinct fronto-parietal network changes from which ion channel estimates can be obtained using a microcircuit model. Specifically, a dynamic causal model of EEG data applied to spontaneous brain responses identifies a selective deficit in signalling at NMDA receptors in patients with NMDA receptor antibody encephalitis but not at other ionotropic receptors. Moreover, though these changes are observed across brain regions, these effects predominate at the NMDA receptors of excitatory neurons rather than at inhibitory interneurons. Given that EEG is a ubiquitously available clinical method, our findings suggest a unique re-purposing of EEG data as an assay of brain network dysfunction at the molecular level.

  19. Acid-sensing ion channels and transient-receptor potential ion channels in zebrafish taste buds.

    Science.gov (United States)

    Levanti, M; Randazzo, B; Viña, E; Montalbano, G; Garcia-Suarez, O; Germanà, A; Vega, J A; Abbate, F

    2016-09-01

    Sensory information from the environment is required for life and survival, and it is detected by specialized cells which together make up the sensory system. The fish sensory system includes specialized organs that are able to detect mechanical and chemical stimuli. In particular, taste buds are small organs located on the tongue in terrestrial vertebrates that function in the perception of taste. In fish, taste buds occur on the lips, the flanks, and the caudal (tail) fins of some species and on the barbels of others. In fish taste receptor cells, different classes of ion channels have been detected which, like in mammals, presumably participate in the detection and/or transduction of chemical gustatory signals. However, since some of these ion channels are involved in the detection of additional sensory modalities, it can be hypothesized that taste cells sense stimuli other than those specific for taste. This mini-review summarizes current knowledge on the presence of transient-receptor potential (TRP) and acid-sensing (ASIC) ion channels in the taste buds of teleosts, especially adult zebrafish. Up to now ASIC4, TRPC2, TRPA1, TRPV1 and TRPV4 ion channels have been found in the sensory cells, while ASIC2 was detected in the nerves supplying the taste buds. Copyright © 2016 Elsevier GmbH. All rights reserved.

  20. Transient receptor potential channel superfamily: Role in lower urinary tract function.

    Science.gov (United States)

    Ogawa, Teruyuki; Imamura, Tetsuya; Nakazawa, Masaki; Hiragata, Shiro; Nagai, Takashi; Minagawa, Tomonori; Yokoyama, Hitoshi; Ishikawa, Masakuni; Domen, Takahisa; Ishizuka, Osamu

    2015-11-01

    Lower urinary tract symptoms associated with neurogenic bladder and overactive bladder syndrome are mediated in part by members of the transient receptor potential channel superfamily. The best studied member of this superfamily is the vanilloid receptor. Other transient receptor potential channels, such as the melastatin receptor and the ankyrin receptor, are also active in the pathogenesis of lower urinary tract dysfunction. However, the detailed mechanisms by which the transient receptor potential channels contribute to lower urinary tract symptoms are still not clear, and the therapeutic benefits of modulating transient receptor potential channel activity have not been proved in the clinical setting. In the present review, to better understand the pathophysiology and therapeutic potential for lower urinary tract symptoms, we summarize the presence and role of different members of the transient receptor potential channel superfamily in the lower urinary tract. © 2015 The Japanese Urological Association.

  1. Molecular determinants of ivermectin sensitivity at the glycine receptor chloride channel

    DEFF Research Database (Denmark)

    Lynagh, Timothy; Webb, Timothy I.; Dixon, Christine L.

    2011-01-01

    Ivermectin is an anthelmintic drug that works by activating glutamate-gated chloride channel receptors (GluClRs) in nematode parasites. GluClRs belong to the Cys-loop receptor family that also includes glycine receptor (GlyR) chloride channels. GluClRs and A288G mutant GlyRs are both activated...

  2. Calcium-Release Channels in Paramecium. Genomic Expansion, Differential Positioning and Partial Transcriptional Elimination

    Science.gov (United States)

    Ladenburger, Eva-Maria; Plattner, Helmut

    2011-01-01

    The release of Ca2+ from internal stores is a major source of signal Ca2+ in almost all cell types. The internal Ca2+ pools are activated via two main families of intracellular Ca2+-release channels, the ryanodine and the inositol 1,4,5-trisphosphate (InsP3) receptors. Among multicellular organisms these channel types are ubiquitous, whereas in most unicellular eukaryotes the identification of orthologs is impaired probably due to evolutionary sequence divergence. However, the ciliated protozoan Paramecium allowed us to prognosticate six groups, with a total of 34 genes, encoding proteins with characteristics typical of InsP3 and ryanodine receptors by BLAST search of the Paramecium database. We here report that these Ca2+-release channels may display all or only some of the characteristics of canonical InsP3 and ryanodine receptors. In all cases, prediction methods indicate the presence of six trans-membrane regions in the C-terminal domains, thus corresponding to canonical InsP3 receptors, while a sequence homologous to the InsP3-binding domain is present only in some types. Only two types have been analyzed in detail previously. We now show, by using antibodies and eventually by green fluorescent protein labeling, that the members of all six groups localize to distinct organelles known to participate in vesicle trafficking and, thus, may provide Ca2+ for local membrane-membrane interactions. Whole genome duplication can explain radiation within the six groups. Comparative and evolutionary evaluation suggests derivation from a common ancestor of canonical InsP3 and ryanodine receptors. With one group we could ascertain, to our knowledge for the first time, aberrant splicing in one thoroughly analyzed Paramecium gene. This yields truncated forms and, thus, may indicate a way to pseudogene formation. No comparable analysis is available for any other, free-living or parasitic/pathogenic protozoan. PMID:22102876

  3. Calcium-release channels in paramecium. Genomic expansion, differential positioning and partial transcriptional elimination.

    Directory of Open Access Journals (Sweden)

    Eva-Maria Ladenburger

    Full Text Available The release of Ca²⁺ from internal stores is a major source of signal Ca²⁺ in almost all cell types. The internal Ca²⁺ pools are activated via two main families of intracellular Ca²⁺-release channels, the ryanodine and the inositol 1,4,5-trisphosphate (InsP₃ receptors. Among multicellular organisms these channel types are ubiquitous, whereas in most unicellular eukaryotes the identification of orthologs is impaired probably due to evolutionary sequence divergence. However, the ciliated protozoan Paramecium allowed us to prognosticate six groups, with a total of 34 genes, encoding proteins with characteristics typical of InsP₃ and ryanodine receptors by BLAST search of the Paramecium database. We here report that these Ca²⁺-release channels may display all or only some of the characteristics of canonical InsP₃ and ryanodine receptors. In all cases, prediction methods indicate the presence of six trans-membrane regions in the C-terminal domains, thus corresponding to canonical InsP₃ receptors, while a sequence homologous to the InsP₃-binding domain is present only in some types. Only two types have been analyzed in detail previously. We now show, by using antibodies and eventually by green fluorescent protein labeling, that the members of all six groups localize to distinct organelles known to participate in vesicle trafficking and, thus, may provide Ca²⁺ for local membrane-membrane interactions. Whole genome duplication can explain radiation within the six groups. Comparative and evolutionary evaluation suggests derivation from a common ancestor of canonical InsP₃ and ryanodine receptors. With one group we could ascertain, to our knowledge for the first time, aberrant splicing in one thoroughly analyzed Paramecium gene. This yields truncated forms and, thus, may indicate a way to pseudogene formation. No comparable analysis is available for any other, free-living or parasitic/pathogenic protozoan.

  4. Expression-dependent pharmacology of transient receptor potential vanilloid subtype 1 channels in Xenopus laevis oocytes

    DEFF Research Database (Denmark)

    Rivera-Acevedo, Ricardo E; Pless, Stephan Alexander; Schwarz, Stephan K W

    2013-01-01

    Transient receptor potential vanilloid subfamily member 1 channels are polymodal sensors of noxious stimuli and integral players in thermosensation, inflammation and pain signaling. It has been shown previously that under prolonged stimulation, these channels show dynamic pore dilation, providing...

  5. Mechanics of channel gating of the nicotinic acetylcholine receptor.

    Directory of Open Access Journals (Sweden)

    Xinli Liu

    2008-01-01

    Full Text Available The nicotinic acetylcholine receptor (nAChR is a key molecule involved in the propagation of signals in the central nervous system and peripheral synapses. Although numerous computational and experimental studies have been performed on this receptor, the structural dynamics of the receptor underlying the gating mechanism is still unclear. To address the mechanical fundamentals of nAChR gating, both conventional molecular dynamics (CMD and steered rotation molecular dynamics (SRMD simulations have been conducted on the cryo-electron microscopy (cryo-EM structure of nAChR embedded in a dipalmitoylphosphatidylcholine (DPPC bilayer and water molecules. A 30-ns CMD simulation revealed a collective motion amongst C-loops, M1, and M2 helices. The inward movement of C-loops accompanying the shrinking of acetylcholine (ACh binding pockets induced an inward and upward motion of the outer beta-sheet composed of beta9 and beta10 strands, which in turn causes M1 and M2 to undergo anticlockwise motions around the pore axis. Rotational motion of the entire receptor around the pore axis and twisting motions among extracellular (EC, transmembrane (TM, and intracellular MA domains were also detected by the CMD simulation. Moreover, M2 helices undergo a local twisting motion synthesized by their bending vibration and rotation. The hinge of either twisting motion or bending vibration is located at the middle of M2, possibly the gate of the receptor. A complementary twisting-to-open motion throughout the receptor was detected by a normal mode analysis (NMA. To mimic the pulsive action of ACh binding, nonequilibrium MD simulations were performed by using the SRMD method developed in one of our laboratories. The result confirmed all the motions derived from the CMD simulation and NMA. In addition, the SRMD simulation indicated that the channel may undergo an open-close (O C motion. The present MD simulations explore the structural dynamics of the receptor under its

  6. A ligand channel through the G protein coupled receptor opsin.

    Directory of Open Access Journals (Sweden)

    Peter W Hildebrand

    Full Text Available The G protein coupled receptor rhodopsin contains a pocket within its seven-transmembrane helix (TM structure, which bears the inactivating 11-cis-retinal bound by a protonated Schiff-base to Lys296 in TM7. Light-induced 11-cis-/all-trans-isomerization leads to the Schiff-base deprotonated active Meta II intermediate. With Meta II decay, the Schiff-base bond is hydrolyzed, all-trans-retinal is released from the pocket, and the apoprotein opsin reloaded with new 11-cis-retinal. The crystal structure of opsin in its active Ops* conformation provides the basis for computational modeling of retinal release and uptake. The ligand-free 7TM bundle of opsin opens into the hydrophobic membrane layer through openings A (between TM1 and 7, and B (between TM5 and 6, respectively. Using skeleton search and molecular docking, we find a continuous channel through the protein that connects these two openings and comprises in its central part the retinal binding pocket. The channel traverses the receptor over a distance of ca. 70 A and is between 11.6 and 3.2 A wide. Both openings are lined with aromatic residues, while the central part is highly polar. Four constrictions within the channel are so narrow that they must stretch to allow passage of the retinal beta-ionone-ring. Constrictions are at openings A and B, respectively, and at Trp265 and Lys296 within the retinal pocket. The lysine enforces a 90 degrees elbow-like kink in the channel which limits retinal passage. With a favorable Lys side chain conformation, 11-cis-retinal can take the turn, whereas passage of the all-trans isomer would require more global conformational changes. We discuss possible scenarios for the uptake of 11-cis- and release of all-trans-retinal. If the uptake gate of 11-cis-retinal is assigned to opening B, all-trans is likely to leave through the same gate. The unidirectional passage proposed previously requires uptake of 11-cis-retinal through A and release of photolyzed all

  7. Sequence genomic organization and expression of two channel catfish Ictalurus punctatus Ghrelin receptors

    Science.gov (United States)

    Two ghrelin receptor (GHS-R) genes were isolated from channel catfish tissue and a bacterial artificial chromosome (BAC) library. The two receptors were characterized by determining tissue distribution, ontogeny of receptor mRNA expression, and effects of exogenous homologous ghrelin administration ...

  8. Sigma-1 Receptor Plays a Negative Modulation on N-type Calcium Channel

    Directory of Open Access Journals (Sweden)

    Kang Zhang

    2017-05-01

    Full Text Available The sigma-1 receptor is a 223 amino acids molecular chaperone with a single transmembrane domain. It is resident to eukaryotic mitochondrial-associated endoplasmic reticulum and plasma membranes. By chaperone-mediated interactions with ion channels, G-protein coupled receptors and cell-signaling molecules, the sigma-1 receptor performs broad physiological and pharmacological functions. Despite sigma-1 receptors have been confirmed to regulate various types of ion channels, the relationship between the sigma-1 receptor and N-type Ca2+ channel is still unclear. Considering both sigma-1 receptors and N-type Ca2+ channels are involved in intracellular calcium homeostasis and neurotransmission, we undertake studies to explore the possible interaction between these two proteins. In the experiment, we confirmed the expression of the sigma-1 receptors and the N-type calcium channels in the cholinergic interneurons (ChIs in rat striatum by using single-cell reverse transcription-polymerase chain reaction (scRT-PCR and immunofluorescence staining. N-type Ca2+ currents recorded from ChIs in the brain slice of rat striatum was depressed when sigma-1 receptor agonists (SKF-10047 and Pre-084 were administrated. The inhibition was completely abolished by sigma-1 receptor antagonist (BD-1063. Co-expression of the sigma-1 receptors and the N-type calcium channels in Xenopus oocytes presented a decrease of N-type Ca2+ current amplitude with an increase of sigma-1 receptor expression. SKF-10047 could further depress N-type Ca2+ currents recorded from oocytes. The fluorescence resonance energy transfer (FRET assays and co-immunoprecipitation (Co-IP demonstrated that sigma-1 receptors and N-type Ca2+ channels formed a protein complex when they were co-expressed in HEK-293T (Human Embryonic Kidney -293T cells. Our results revealed that the sigma-1 receptors played a negative modulation on N-type Ca2+ channels. The mechanism for the inhibition of sigma-1 receptors on

  9. Sigma-1 receptor agonists directly inhibit Nav1.2/1.4 channels.

    Directory of Open Access Journals (Sweden)

    Xiao-Fei Gao

    Full Text Available (+-SKF 10047 (N-allyl-normetazocine is a prototypic and specific sigma-1 receptor agonist that has been used extensively to study the function of sigma-1 receptors. (+-SKF 10047 inhibits K(+, Na(+ and Ca2+ channels via sigma-1 receptor activation. We found that (+-SKF 10047 inhibited Na(V1.2 and Na(V1.4 channels independently of sigma-1 receptor activation. (+-SKF 10047 equally inhibited Na(V1.2/1.4 channel currents in HEK293T cells with abundant sigma-1 receptor expression and in COS-7 cells, which barely express sigma-1 receptors. The sigma-1 receptor antagonists BD 1063,BD 1047 and NE-100 did not block the inhibitory effects of (+-SKF-10047. Blocking of the PKA, PKC and G-protein pathways did not affect (+-SKF 10047 inhibition of Na(V1.2 channel currents. The sigma-1 receptor agonists Dextromethorphan (DM and 1,3-di-o-tolyl-guanidine (DTG also inhibited Na(V1.2 currents through a sigma-1 receptor-independent pathway. The (+-SKF 10047 inhibition of Na(V1.2 currents was use- and frequency-dependent. Point mutations demonstrated the importance of Phe(1764 and Tyr(1771 in the IV-segment 6 domain of the Na(V1.2 channel and Phe(1579 in the Na(V1.4 channel for (+-SKF 10047 inhibition. In conclusion, our results suggest that sigma-1 receptor agonists directly inhibit Na(V1.2/1.4 channels and that these interactions should be given special attention for future sigma-1 receptor function studies.

  10. G-protein mediates voltage regulation of agonist binding to muscarinic receptors: effects on receptor-Na+ channel interaction

    International Nuclear Information System (INIS)

    Cohen-Armon, M.; Garty, H.; Sokolovsky, M.

    1988-01-01

    The authors previous experiments in membranes prepared from rat heart and brain led them to suggest that the binding of agonist to the muscarinic receptors and to the Na + channels is a coupled event mediated by guanine nucleotide binding protein(s) [G-protein(s)]. These in vitro findings prompted us to employ synaptoneurosomes from brain stem tissue to examine (i) the binding properties of [ 3 H] acetylcholine at resting potential and under depolarization conditions in the absence and presence of pertussis toxin; (ii) the binding of [ 3 H]batrachotoxin to Na + channel(s) in the presence of the muscarinic agonists; and (iii) muscarinically induced 22 Na + uptake in the presence and absence of tetrodotoxin, which blocks Na + channels. The findings indicate that agonist binding to muscarinic receptors is voltage dependent, that this process is mediated by G-protein(s), and that muscarinic agonists induce opening of Na + channels. The latter process persists even after pertussis toxin treatment, indicating that it is not likely to be mediated by pertussis toxin sensitive G-protein(s). The system with its three interacting components-receptor, G-protein, and Na + channel-is such that at resting potential the muscarinic receptor induces opening of Na + channels; this property may provide a possible physiological mechanism for the depolarization stimulus necessary for autoexcitation or repetitive firing in heart or brain tissues

  11. Increased migration of monocytes in essential hypertension is associated with increased transient receptor potential channel canonical type 3 channels

    DEFF Research Database (Denmark)

    Zhao, Zhigang; Ni, Yinxing; Chen, Jing

    2012-01-01

    Increased transient receptor potential canonical type 3 (TRPC3) channels have been observed in patients with essential hypertension. In the present study we tested the hypothesis that increased monocyte migration is associated with increased TRPC3 expression. Monocyte migration assay was performe...

  12. The human transient receptor potential vanilloid 3 channel is sensitized via the ERK pathway

    Czech Academy of Sciences Publication Activity Database

    Vyklická, Lenka; Boukalová, Štěpána; Mačíková, Lucie; Chvojka, Štěpán; Vlachová, Viktorie

    2017-01-01

    Roč. 292, č. 51 (2017), s. 21083-21091 ISSN 0021-9258 R&D Projects: GA ČR(CZ) GA15-15839S Institutional support: RVO:67985823 Keywords : epidermal growth factor receptor (EGFR) * extracellular-signal-regulated kinase (ERK) * keratinocyte * phosphorylation * transient receptor potential channels * TRP channels Subject RIV: FH - Neurology OBOR OECD: Neurosciences (including psychophysiology Impact factor: 4.125, year: 2016

  13. 5-HT1A receptors modulate small-conductance Ca2+-activated K+ channels

    DEFF Research Database (Denmark)

    Grunnet, Morten; Jespersen, Thomas; Perrier, Jean-François

    2004-01-01

    Small-conductance calcium-activated potassium channels (SK) are responsible for the medium afterhyperpolarisation (mAHP) following action potentials in neurons. Here we tested the ability of serotonin (5-HT) to modulate the activity of SK channels by coexpressing 5-HT1A receptors with different...

  14. The Role of Canonical Transient Receptor Potential Channels in Seizure and Excitotoxicity

    Directory of Open Access Journals (Sweden)

    Fang Zheng

    2014-04-01

    Full Text Available Canonical transient receptor potential (TRPC channels are a family of polymodal cation channels with some degree of Ca2+ permeability. Although initially thought to be channels mediating store-operated Ca2+ influx, TRPC channels can be activated by stimulation of Gq-coupled G-protein coupled receptors, or by an increase in intracellular free Ca2+ concentration. Thus, activation of TRPC channels could be a common downstream event of many signaling pathways that contribute to seizure and excitotoxicity, such as N-methyl-D-aspartate (NMDA receptor-mediated Ca2+ influx, or metabotropic glutamate receptor activation. Recent studies with genetic ablation of various TRPC family members have demonstrated that TRPC channels, in particular heteromeric TRPC1/4 channels and homomeric TRPC5 channels, play a critical role in both pilocarpine-induced acute seizures and neuronal cell death. However, exact underlying mechanisms remain to be fully elucidated, and selective TRPC modulators and antibodies with better specificity are urgently needed for future research.

  15. The purinergic P2X7 ion channel receptor — a ‘repair’ receptor in bone

    DEFF Research Database (Denmark)

    Jørgensen, Niklas Rye

    2018-01-01

    A strong skeleton relies on adaptation to varying physical demands and on maintenance of the bone tissue in order to avoid accumulation of micro-damage. In bone, the purinergic P2X7 ion channel receptor is expressed on both cells of the stromal lineage such as the bone forming osteoblasts...... and the mechano-sensing osteocytes and on cells belonging to the immune-related monocyte–macrophage lineage, the bone resorbing osteoclasts. Recent studies have demonstrated that the receptor plays important roles in the anabolic responses to mechanical loading on bone and, together with the pannexin1 hemi......-channel, in the process of initiating bone remodeling in response to micro-damage. Thus, the receptor is crucial in skeletal mechano-transduction and in the continuous repair process. However, under pathophysiological conditions such as diabetes with high glucose concentrations or glucocorticoid-treatment the receptor...

  16. Dental enamel cells express functional SOCE channels.

    Science.gov (United States)

    Nurbaeva, Meerim K; Eckstein, Miriam; Concepcion, Axel R; Smith, Charles E; Srikanth, Sonal; Paine, Michael L; Gwack, Yousang; Hubbard, Michael J; Feske, Stefan; Lacruz, Rodrigo S

    2015-10-30

    Dental enamel formation requires large quantities of Ca(2+) yet the mechanisms mediating Ca(2+) dynamics in enamel cells are unclear. Store-operated Ca(2+) entry (SOCE) channels are important Ca(2+) influx mechanisms in many cells. SOCE involves release of Ca(2+) from intracellular pools followed by Ca(2+) entry. The best-characterized SOCE channels are the Ca(2+) release-activated Ca(2+) (CRAC) channels. As patients with mutations in the CRAC channel genes STIM1 and ORAI1 show abnormal enamel mineralization, we hypothesized that CRAC channels might be an important Ca(2+) uptake mechanism in enamel cells. Investigating primary murine enamel cells, we found that key components of CRAC channels (ORAI1, ORAI2, ORAI3, STIM1, STIM2) were expressed and most abundant during the maturation stage of enamel development. Furthermore, inositol 1,4,5-trisphosphate receptor (IP3R) but not ryanodine receptor (RyR) expression was high in enamel cells suggesting that IP3Rs are the main ER Ca(2+) release mechanism. Passive depletion of ER Ca(2+) stores with thapsigargin resulted in a significant raise in [Ca(2+)]i consistent with SOCE. In cells pre-treated with the CRAC channel blocker Synta-66 Ca(2+) entry was significantly inhibited. These data demonstrate that enamel cells have SOCE mediated by CRAC channels and implicate them as a mechanism for Ca(2+) uptake in enamel formation.

  17. FMRFamide-gated sodium channel and ASIC channels: a new class of ionotropic receptors for FMRFamide and related peptides.

    Science.gov (United States)

    Lingueglia, Eric; Deval, Emmanuel; Lazdunski, Michel

    2006-05-01

    FMRFamide and related peptides typically exert their action through G-protein coupled receptors. However, two ionotropic receptors for these peptides have recently been identified. They are both members of the epithelial amiloride-sensitive Na+ channel and degenerin (ENaC/DEG) family of ion channels. The invertebrate FMRFamide-gated Na+ channel (FaNaC) is a neuronal Na+-selective channel which is directly gated by micromolar concentrations of FMRFamide and related tetrapeptides. Its response is fast and partially desensitizing, and FaNaC has been proposed to participate in peptidergic neurotransmission. On the other hand, mammalian acid-sensing ion channels (ASICs) are not gated but are directly modulated by FMRFamide and related mammalian peptides like NPFF and NPSF. ASICs are activated by external protons and are therefore extracellular pH sensors. They are expressed both in the central and peripheral nervous system and appear to be involved in many physiological and pathophysiological processes such as hippocampal long-term potentiation and defects in learning and memory, acquired fear-related behavior, retinal function, brain ischemia, pain sensation in ischemia and inflammation, taste perception, hearing functions, and mechanoperception. The potentiation of ASIC activity by endogenous RFamide neuropeptides probably participates in the response to noxious acidosis in sensory and central neurons. Available data also raises the possibility of the existence of still unknown FMRFamide related endogenous peptides acting as direct agonists for ASICs.

  18. Ion channel profile of TRPM8 cold receptors reveals a novel role of TASK-3 potassium channels in thermosensation

    Science.gov (United States)

    Morenilla-Palao, Cruz; Luis, Enoch; Fernández-Peña, Carlos; Quintero, Eva; Weaver, Janelle L.; Bayliss, Douglas A.; Viana, Félix

    2017-01-01

    Summary Animals sense cold ambient temperatures through the activation of peripheral thermoreceptors that express TRPM8, a cold- and menthol-activated ion channel. These receptors can discriminate a very wide range of temperatures from innocuous to noxious. The molecular mechanism responsible for the variable sensitivity of individual cold receptors to temperature is unclear. To address this question, we performed a detailed ion channel expression analysis of cold sensitive neurons, combining BAC transgenesis with a molecular profiling approach in FACS purified TRPM8 neurons. We found that TASK-3 leak potassium channels are highly enriched in a subpopulation of these sensory neurons. The thermal threshold of TRPM8 cold neurons is decreased during TASK-3 blockade and in mice lacking TASK-3 and, most importantly, these mice display hypersensitivity to cold. Our results demonstrate a novel role of TASK-3 channels in thermosensation, showing that a channel-based combinatorial strategy in TRPM8 cold thermoreceptors leads to molecular specialization and functional diversity. PMID:25199828

  19. Molecular dissection of purinergic P2X receptor channels

    Czech Academy of Sciences Publication Activity Database

    Stojilkovic, S. S.; Tomič, M.; He, M. L.; Yan, Z.; Koshimizu, T.; Zemková, Hana

    2005-01-01

    Roč. 1048, - (2005), s. 116-130 ISSN 0077-8923 Institutional research plan: CEZ:AV0Z50110509 Keywords : ATP * purinergic receptors * deactivation Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 1.971, year: 2005

  20. Differential association of GABAB receptors with their effector ion channels in Purkinje cells.

    Science.gov (United States)

    Luján, Rafael; Aguado, Carolina; Ciruela, Francisco; Cózar, Javier; Kleindienst, David; de la Ossa, Luis; Bettler, Bernhard; Wickman, Kevin; Watanabe, Masahiko; Shigemoto, Ryuichi; Fukazawa, Yugo

    2018-04-01

    Metabotropic GABA B receptors mediate slow inhibitory effects presynaptically and postsynaptically through the modulation of different effector signalling pathways. Here, we analysed the distribution of GABA B receptors using highly sensitive SDS-digested freeze-fracture replica labelling in mouse cerebellar Purkinje cells. Immunoreactivity for GABA B1 was observed on presynaptic and, more abundantly, on postsynaptic compartments, showing both scattered and clustered distribution patterns. Quantitative analysis of immunoparticles revealed a somato-dendritic gradient, with the density of immunoparticles increasing 26-fold from somata to dendritic spines. To understand the spatial relationship of GABA B receptors with two key effector ion channels, the G protein-gated inwardly rectifying K + (GIRK/Kir3) channel and the voltage-dependent Ca 2+ channel, biochemical and immunohistochemical approaches were performed. Co-immunoprecipitation analysis demonstrated that GABA B receptors co-assembled with GIRK and Ca V 2.1 channels in the cerebellum. Using double-labelling immunoelectron microscopic techniques, co-clustering between GABA B1 and GIRK2 was detected in dendritic spines, whereas they were mainly segregated in the dendritic shafts. In contrast, co-clustering of GABA B1 and Ca V 2.1 was detected in dendritic shafts but not spines. Presynaptically, although no significant co-clustering of GABA B1 and GIRK2 or Ca V 2.1 channels was detected, inter-cluster distance for GABA B1 and GIRK2 was significantly smaller in the active zone than in the dendritic shafts, and that for GABA B1 and Ca V 2.1 was significantly smaller in the active zone than in the dendritic shafts and spines. Thus, GABA B receptors are associated with GIRK and Ca V 2.1 channels in different subcellular compartments. These data provide a better framework for understanding the different roles played by GABA B receptors and their effector ion channels in the cerebellar network.

  1. Interaction between Ca++-channel antagonists and α2-adrenergic receptors in rabbit ileal cell membrane

    International Nuclear Information System (INIS)

    Homeidan, F.R.; Wicks, J.; Cusolito, S.; El-Sabban, M.E.; Sharp, G.W.G.; Donowitz, M.

    1986-01-01

    An interaction between Ca ++ -channel antagonists and the α 2 -adrenergic receptor on active electrolyte transport was demonstrated in rabbit ileum. Clonidine, an α 2 -agonist, stimulated NaCl absorption apparently by Ca ++ -channel antagonism since it inhibited 45 Ca ++ uptake across the basolateral membrane and decreased total ileal calcium content. This stimulation was inhibited by the Ca ++ -channel antagonists dl- and l-verapamil and cadmium but not by nifedipine. The binding of 3 H-yohimbine, a specific α 2 -adrenergic antagonist, was studied on purified ileal cell membranes using a rapid filtration technique. dl-Verapamil and Cd ++ inhibited the specific binding of 3 H-yohimbine over the same concentration range in which they affected transport. In contrast, nifedipine had no effect on binding, just as it had no effect on clonidine-stimulated NaCl absorption. These data demonstrate that there is an interaction between Ca ++ -channels and α 2 -adrenergic receptors in ileal basolateral membranes. Some Ca ++ -channel antagonists alter α 2 -adrenergic binding to the receptor and α 2 -agonist binding leads to changes in Ca ++ entry. A close spatial relationship between the Ca ++ -channel and the α 2 -receptor could explain the data

  2. Do cysteine residues regulate transient receptor potential canonical type 6 (TRPC6) channel protein expression?

    DEFF Research Database (Denmark)

    Thilo, Florian; Liu, Ying; Krueger, Katharina

    2012-01-01

    The regulation of calcium influx through transient receptor potential canonical type 6 channel is mandatory for the activity of human monocytes. We submit the first evidence that cysteine residues of homocysteine or acetylcysteine affect TRPC6 expression in human monocytes. We observed that patie......The regulation of calcium influx through transient receptor potential canonical type 6 channel is mandatory for the activity of human monocytes. We submit the first evidence that cysteine residues of homocysteine or acetylcysteine affect TRPC6 expression in human monocytes. We observed...... that patients with chronic renal failure had significantly elevated homocysteine levels and TRPC6 mRNA expression levels in monocytes compared to control subjects. We further observed that administration of homocysteine or acetylcysteine significantly increased TRPC6 channel protein expression compared...... to control conditions. We therefore hypothesize that cysteine residues increase TRPC6 channel protein expression in humans....

  3. Autoantibodies to neurotransmitter receptors and ion channels: from neuromuscular to neuropsychiatric disorders

    Directory of Open Access Journals (Sweden)

    Pilar eMartinez-Martinez

    2013-09-01

    Full Text Available Changes of voltage-gated ion channels and ligand-gated receptor channels caused by mutation or autoimmune attack are the cause of so-called channelopathies in the central and peripheral nervous system. We present the pathophysiology of channelopathies of the neuromuscular junction in terms of loss-of-function and gain-of-function principles. Autoantibodies generally have reduced access to the CNS, but in some cases this is enough to cause disease. A review is provided of recent findings implicating autoantibodies against ligand–activated receptor channels and potassium channels in psychiatric and neurological disorders, including schizophrenia and limbic encephalitis. The emergence of channelopathy-related neuropsychiatric disorders has implications for research and practice.

  4. Signaling by purinergic receptors and channels in the pituitary gland

    Czech Academy of Sciences Publication Activity Database

    Stojilkovic, S. S.; He, M. L.; Koshimizu, T.; Balík, A.; Zemková, Hana

    2010-01-01

    Roč. 314, č. 2 (2010), s. 184-191 ISSN 0303-7207 R&D Projects: GA ČR(CZ) GA305/07/0681 Institutional research plan: CEZ:AV0Z50110509 Keywords : purinergic receptors * ATP * anterior pituitary Subject RIV: ED - Physiology Impact factor: 4.119, year: 2010

  5. Channel opening of γ-aminobutyric acid receptor from rat brain: molecular mechanisms of the receptor responses

    International Nuclear Information System (INIS)

    Cash, D.J.; Subbarao, K.

    1987-01-01

    The function of γ-aminobutyric acid (GABA) receptors, which mediate transmembrane chloride flux, can be studied by use of 36 Cl - isotope tracer with membrane from mammalian brain by quench-flow technique, with reaction times that allow resolution of the receptor desensitization rates from the ion flux rates. The rates of chloride exchange into the vesicles in the absence and presence of GABA were characterized with membrane from rat cerebral cortex. Unspecific 36 Cl - influx was completed in three phases of ca. 3% (t/sub 1/2/ = 0.6 s), 56% (t/sub 1/2 = 82 s), and 41% (t/sub 1/2 = 23 min). GABA-mediated, specific chloride exchange occurred with 6.5% of the total vesicular internal volume. The GABA-dependent 36 Cl - influx proceeded in two phases, each progressively slowed by desensitization. The measurements supported the presence of two distinguishable active GABA receptors on the same membrane mediating chloride exchange into the vesicles. The half-response concentrations were similar for both receptors. The two receptors were present in the activity ratio of ca. 4/1, similar to the ratio of low affinity to high-affinity GABA sites found in ligand binding experiments. The desensitization rates have a different dependence on GABA concentration than the channel-opening equilibria. For both receptors, the measurements over a 2000-fold GABA concentration range required a minimal mechanism involving the occupation of both of the two GABA binding sites for significant channel opening; then the receptors were ca. 80% open. Similarly for both receptors, desensitization was mediated by a different pair of binding sites, although desensitization with only one ligand molecule bound could occur at a 20-fold slower rate

  6. Transient receptor potential channel polymorphisms are associated with the somatosensory function in neuropathic pain patients.

    Directory of Open Access Journals (Sweden)

    Andreas Binder

    Full Text Available Transient receptor potential channels are important mediators of thermal and mechanical stimuli and play an important role in neuropathic pain. The contribution of hereditary variants in the genes of transient receptor potential channels to neuropathic pain is unknown. We investigated the frequency of transient receptor potential ankyrin 1, transient receptor potential melastin 8 and transient receptor potential vanilloid 1 single nucleotide polymorphisms and their impact on somatosensory abnormalities in neuropathic pain patients. Within the German Research Network on Neuropathic Pain (Deutscher Forscbungsverbund Neuropathischer Schmerz 371 neuropathic pain patients were phenotypically characterized using standardized quantitative sensory testing. Pyrosequencing was employed to determine a total of eleven single nucleotide polymorphisms in transient receptor potential channel genes of the neuropathic pain patients and a cohort of 253 German healthy volunteers. Associations of quantitative sensory testing parameters and single nucleotide polymorphisms between and within groups and subgroups, based on sensory phenotypes, were analyzed. Single nucleotide polymorphisms frequencies did not differ between both the cohorts. However, in neuropathic pain patients transient receptor potential ankyrin 1 710G>A (rs920829, E179K was associated with the presence of paradoxical heat sensation (p = 0.03, and transient receptor potential vanilloid 1 1911A>G (rs8065080, I585V with cold hypoalgesia (p = 0.0035. Two main subgroups characterized by preserved (1 and impaired (2 sensory function were identified. In subgroup 1 transient receptor potential vanilloid 1 1911A>G led to significantly less heat hyperalgesia, pinprick hyperalgesia and mechanical hypaesthesia (p = 0.006, p = 0.005 and pG (rs222747, M315I to cold hypaesthesia (p = 0.002, but there was absence of associations in subgroup 2. In this study we found no evidence that genetic

  7. P2Y2 and P2Y4 receptors regulate pancreatic Ca²+-activated K+ channels differently

    DEFF Research Database (Denmark)

    Klærke, Susanne Edeling Hede; Amstrup, Jan; Klærke, Dan Arne

    2005-01-01

    Extracellular ATP is an important regulator of transepithelial transport in a number of tissues. In pancreatic ducts, we have shown that ATP modulates epithelial K+ channels via purinergic receptors, most likely the P2Y2 and P2Y4 receptors, but the identity of the involved K+ channels was not cle...

  8. Regulated appearance of NMDA receptor subunits and channel functions during in vitro neuronal differentiation

    NARCIS (Netherlands)

    Jelitai, Márta; Schlett, Katalin; Varju, Patrícia; Eisel, Ulrich; Madarász, Emília

    The schedule of NMDA receptor subunit expression and the appearance of functional NMDA-gated ion channels were investigated during the retinoic acid (RA) induced neuronal differentiation of NE-4C, a p53-deficient mouse neuroectodermal progenitor cell line. NR2A. NR2B, and NR2D subunit transcripts

  9. Dual Gating Mechanism and Function of P2X7 Receptor Channels

    Czech Academy of Sciences Publication Activity Database

    Khadra, A.; Tomic, M.; Yan, Z.; Zemková, Hana; Sherman, A.; Stojilkovic, S. S.

    2013-01-01

    Roč. 104, č. 12 (2013), s. 2612-2621 ISSN 0006-3495 R&D Projects: GA ČR(CZ) GBP304/12/G069 Institutional support: RVO:67985823 Keywords : purinergic P2X7 receptors * ATP-gated channels * BzATP * dilation * Markov -state model Subject RIV: ED - Physiology Impact factor: 3.832, year: 2013

  10. Increased transient receptor potential canonical type 3 channels in vasculature from hypertensive rats

    DEFF Research Database (Denmark)

    Liu, Daoyan; Yang, Dachun; He, Hongbo

    2009-01-01

    We tested the hypothesis that transient receptor potential canonical type 3 (TRPC3) channels are increased in vascular smooth muscle cells and aortic tissue from spontaneously hypertensive rats (SHR) compared with normotensive Wistar Kyoto rats. Expression of TRPC3 was analyzed by immunohistochem...

  11. Transient receptor potential canonical type 3 channels and blood pressure in humans

    DEFF Research Database (Denmark)

    Thilo, Florian; Baumunk, Daniel; Krause, Hans

    2009-01-01

    There is evidence that transient receptor potential canonical type 3 (TRPC3) cation channels are involved in the regulation of blood pressure, but this has not been studied using human renal tissue. We tested the hypothesis that the expression of TRPC3 in human renal tissue is associated with blood...

  12. Purification and reconstitution of the calcium antagonist receptor of the voltage-sensitive calcium channel

    International Nuclear Information System (INIS)

    Curtis, B.M.

    1986-01-01

    Treatment with digitonin solubilized the calcium antagonist receptor as a stable complex with [ 3 H]nitrendipine from rat brain membranes. The solubilized complex retains allosteric coupling to binding sites for diltiazem, verapamil, and inorganic calcium antagonist sites. The calcium antagonist receptor from cardiac sarcolemma and the transverse-tubule membrane of skeletal muscle is also efficiently solubilized with digitonin and the receptor in all three tissues is a large glycoprotein with a sedimentation coefficient of 20 S. The T-tubule calcium antagonist receptor complex was extensively purified by a combination of chromatography on WGA-Sepharose, ion exchange chromatography, and sedimentation on sucrose gradients to yield preparations estimated to be 41% homogeneous by specific activity and 63% homogeneous by SDS gel electrophoresis. Analysis of SDS gels detect three polypeptides termed α(Mr 135,000), β(Mr 50,000), and γ(Mr 32,000) as noncovalently associated subunits of the calcium antagonist receptor. The α and γ subunits are glycosylated polypeptides, and the molecular weight of the core polypeptides are 108,000 and 24,000 respectively. The calcium antagonist receptor was reconstituted into a phospholipid bilayer by adding CHAPS and exogeneous lipid to the purified receptor followed by rapid detergent removal. This procedure resulted in the incorporation of 45% of the calcium antagonist receptor into closed phospholipid vesicles. Data suggests that the α, β, and γ subunits of the T-tubule calcium antagonist receptor are sufficient to form a functional calcium channel

  13. Isolation and characterization of the inositol trisphosphate receptor from smooth muscle

    International Nuclear Information System (INIS)

    Chadwick, C.C.; Saito, A.; Fleischer, S.

    1990-01-01

    The release of Ca 2+ from internal stores is requisite to muscle contraction. In skeletal muscle and heart, the Ca 2+ release channels (ryanodine receptor) of sarcoplasmic reticulum, involved in excitation-contraction coupling, have recently been isolated and characterized. In smooth muscle, inositol 1,4,5-trisphosphate (IP 3 ) is believed to mobilize Ca 2+ from internal stores and thereby modulate contraction. The authors describe the isolation of an IP 3 receptor from smooth muscle. Bovine aorta smooth muscle microsomes were solubilized with 3-[(3-cholamidopropyl) dimethylammonio]-1-propanesulfonate, and the IP 3 receptor was purified by sucrose gradient centrifugation and column chromatography with heparin-agarose and wheat germ agglutinin-agarose. The receptor is an oligomer of a single polypeptide with a M r of 224,000 as determined by SDS/PAGE. Negative-staining electron microscopy reveals that the receptor is a large pinwheel-like structure having surface dimensions of ∼250 x 250 angstrom with fourfold symmetry. The IP 3 receptor from smooth muscle is similar to the ryanodine receptor with regard to its large size and fourfold symmetry, albeit distinct with regard to appearance, protomer size, and ligand binding

  14. Temperature and Voltage Coupling to Channel Opening in Transient Receptor Potential Melastatin 8 (TRPM8)*♦

    Science.gov (United States)

    Raddatz, Natalia; Castillo, Juan P.; Gonzalez, Carlos; Alvarez, Osvaldo; Latorre, Ramon

    2014-01-01

    Expressed in somatosensory neurons of the dorsal root and trigeminal ganglion, the transient receptor potential melastatin 8 (TRPM8) channel is a Ca2+-permeable cation channel activated by cold, voltage, phosphatidylinositol 4,5-bisphosphate, and menthol. Although TRPM8 channel gating has been characterized at the single channel and macroscopic current levels, there is currently no consensus regarding the extent to which temperature and voltage sensors couple to the conduction gate. In this study, we extended the range of voltages where TRPM8-induced ionic currents were measured and made careful measurements of the maximum open probability the channel can attain at different temperatures by means of fluctuation analysis. The first direct measurements of TRPM8 channel temperature-driven conformational rearrangements provided here suggest that temperature alone is able to open the channel and that the opening reaction is voltage-independent. Voltage is a partial activator of TRPM8 channels, because absolute open probability values measured with fully activated voltage sensors are less than 1, and they decrease as temperature rises. By unveiling the fast temperature-dependent deactivation process, we show that TRPM8 channel deactivation is well described by a double exponential time course. The fast and slow deactivation processes are temperature-dependent with enthalpy changes of 27.2 and 30.8 kcal mol−1. The overall Q10 for the closing reaction is about 33. A three-tiered allosteric model containing four voltage sensors and four temperature sensors can account for the complex deactivation kinetics and coupling between voltage and temperature sensor activation and channel opening. PMID:25352597

  15. Post-translational regulation of P2X receptor channels: modulation by phospholipids

    Directory of Open Access Journals (Sweden)

    Louis-Philippe eBernier

    2013-11-01

    Full Text Available P2X receptor channels mediate fast excitatory signaling by ATP and play major roles in sensory transduction, neuro-immune communication and inflammatory response. P2X receptors constitute a gene family of calcium-permeable ATP-gated cation channels therefore the regulation of P2X signaling is critical for both membrane potential and intracellular calcium homeostasis. Phosphoinositides (PIPn are anionic signaling phospholipids that act as functional regulators of many types of ion channels. Direct PIPn binding was demonstrated for several ligand- or voltage-gated ion channels, however no generic motif emerged to accurately predict lipid-protein binding sites. This review presents what is currently known about the modulation of the different P2X subtypes by phospholipids and about critical determinants underlying their sensitivity to PIPn levels in the plasma membrane.All functional mammalian P2X subtypes tested, with the notable exception of P2X5, have been shown to be positively modulated by PIPn, i.e. homomeric P2X1, P2X2, P2X3, P2X4, and P2X7, as well as heteromeric P2X1/5 and P2X2/3 receptors. Based on various results reported on the aforementioned subtypes including mutagenesis of the prototypical PIPn-sensitive P2X4 and PIPn-insensitive P2X5 receptor subtypes, an increasing amount of functional, biochemical and structural evidence converges on the modulatory role of a short polybasic domain located in the proximal C-terminus of P2X subunits. This linear motif, semi-conserved in the P2X family, seems necessary and sufficient for encoding direct modulation of ATP-gated channels by PIPn. Furthermore, the physiological impact of the regulation of ionotropic purinergic responses by phospholipids on pain pathways was recently revealed in the context of native crosstalks between phospholipase C-linked metabotropic receptors and P2X receptor channels in DRG sensory neurons and microglia.

  16. Transient receptor potential (TRP) channels as drug targets for diseases of the digestive system

    Science.gov (United States)

    Holzer, Peter

    2011-01-01

    Approximately 20 of the 30 mammalian transient receptor potential (TRP) channel subunits are expressed by specific neurons and cells within the alimentary canal. They subserve important roles in taste, chemesthesis, mechanosensation, pain and hyperalgesia and contribute to the regulation of gastrointestinal motility, absorptive and secretory processes, blood flow, and mucosal homeostasis. In a cellular perspective, TRP channels operate either as primary detectors of chemical and physical stimuli, as secondary transducers of ionotropic or metabotropic receptors, or as ion transport channels. The polymodal sensory function of TRPA1, TRPM5, TRPM8, TRPP2, TRPV1, TRPV3 and TRPV4 enables the digestive system to survey its physical and chemical environment, which is relevant to all processes of digestion. TRPV5 and TRPV6 as well as TRPM6 and TRPM7 contribute to the absorption of Ca2+ and Mg2+, respectively. TRPM7 participates in intestinal pacemaker activity, and TRPC4 transduces muscarinic acetylcholine receptor activation to smooth muscle contraction. Changes in TRP channel expression or function are associated with a variety of diseases/disorders of the digestive system, notably gastro-esophageal reflux disease, inflammatory bowel disease, pain and hyperalgesia in heartburn, functional dyspepsia and irritable bowel syndrome, cholera, hypomagnesemia with secondary hypocalcemia, infantile hypertrophic pyloric stenosis, esophageal, gastrointestinal and pancreatic cancer, and polycystic liver disease. These implications identify TRP channels as promising drug targets for the management of a number of gastrointestinal pathologies. As a result, major efforts are put into the development of selective TRP channel agonists and antagonists and the assessment of their therapeutic potential. PMID:21420431

  17. Single Channel Analysis of Isoflurane and Ethanol Enhancement of Taurine-Activated Glycine Receptors.

    Science.gov (United States)

    Kirson, Dean; Todorovic, Jelena; Mihic, S John

    2018-01-01

    The amino acid taurine is an endogenous ligand acting on glycine receptors (GlyRs), which is released by astrocytes in many brain regions, such as the nucleus accumbens and prefrontal cortex. Taurine is a partial agonist with an efficacy significantly lower than that of glycine. Allosteric modulators such as ethanol and isoflurane produce leftward shifts of glycine concentration-response curves but have no effects at saturating glycine concentrations. In contrast, in whole-cell electrophysiology studies these modulators increase the effects of saturating taurine concentrations. A number of possible mechanisms may explain these enhancing effects, including modulator effects on conductance, channel open times, or channel closed times. We used outside-out patch-clamp single channel electrophysiology to investigate the mechanism of action of 200 mM ethanol and 0.55 mM isoflurane in enhancing the effects of a saturating concentration of taurine. Neither modulator enhanced taurine-mediated conductance. Isoflurane increased the probability of channel opening. Isoflurane also increased the lifetimes of the two shortest open dwell times while both agents decreased the likelihood of occurrence of the longest-lived intracluster channel-closing events. The mechanism of enhancement of GlyR functioning by these modulators is dependent on the efficacy of the agonist activating the receptor and the concentration of agonist tested. Copyright © 2017 by The American Society for Pharmacology and Experimental Therapeutics.

  18. Characterizing ligand-gated ion channel receptors with genetically encoded Ca2++ sensors.

    Directory of Open Access Journals (Sweden)

    John G Yamauchi

    2011-01-01

    Full Text Available We present a cell based system and experimental approach to characterize agonist and antagonist selectivity for ligand-gated ion channels (LGIC by developing sensor cells stably expressing a Ca(2+ permeable LGIC and a genetically encoded Förster (or fluorescence resonance energy transfer (FRET-based calcium sensor. In particular, we describe separate lines with human α7 and human α4β2 nicotinic acetylcholine receptors, mouse 5-HT(3A serotonin receptors and a chimera of human α7/mouse 5-HT(3A receptors. Complete concentration-response curves for agonists and Schild plots of antagonists were generated from these sensors and the results validate known pharmacology of the receptors tested. Concentration-response relations can be generated from either the initial rate or maximal amplitudes of FRET-signal. Although assaying at a medium throughput level, this pharmacological fluorescence detection technique employs a clonal line for stability and has versatility for screening laboratory generated congeners as agonists or antagonists on multiple subtypes of ligand-gated ion channels. The clonal sensor lines are also compatible with in vivo usage to measure indirectly receptor activation by endogenous neurotransmitters.

  19. Muscle Dysfunction in Androgen Deprivation: Role of Ryanodine Receptor

    Science.gov (United States)

    2016-11-01

    reversible pharmacological treatment is a key therapeutic goal in prostate cancer patients. This life prolonging treatment is accompanied by the adverse... reversible pharmacological treatment, is a key therapeutic goal of androgen deprivation therapies (ADT) used in patients with androgen-dependent...gel improves sexual function, mood, muscle strength, and body composition parameters in hypogonadal men. J Clin Endocrinol Metab. Aug 2000;85(8):2839

  20. Functional transient receptor potential vanilloid 1 and transient receptor potential vanilloid 4 channels along different segments of the renal vasculature

    DEFF Research Database (Denmark)

    Chen, L; Kaßmann, M; Sendeski, M

    2015-01-01

    with functional TRPV1 having a narrow, discrete distribution in the resistance vasculature and TRPV4 having more universal, widespread distribution along different vascular segments. We suggest that TRPV1/4 channels are potent therapeutic targets for site-specific vasodilation in the kidney.......AIM: Transient receptor potential vanilloid 1 (TRPV1) and vanilloid 4 (TRPV4) cation channels have been recently identified to promote endothelium-dependent relaxation of mouse mesenteric arteries. However, the role of TRPV1 and TRPV4 in the renal vasculature is largely unknown. We hypothesized...... that TRPV1/4 plays a role in endothelium-dependent vasodilation of renal blood vessels. METHODS: We studied the distribution of functional TRPV1/4 along different segments of the renal vasculature. Mesenteric arteries were studied as control vessels. RESULTS: The TRPV1 agonist capsaicin relaxed mouse...

  1. Channel opening of gamma-aminobutyric acid receptor from rat brain: molecular mechanisms of the receptor responses.

    Science.gov (United States)

    Cash, D J; Subbarao, K

    1987-12-01

    The function of gamma-aminobutyric acid (GABA) receptors, which mediate transmembrane chloride flux, can be studied by use of 36Cl- isotope tracer with membrane from mammalian brain by quench-flow technique, with reaction times that allow resolution of the receptor desensitization rates from the ion flux rates. The rates of chloride exchange into the vesicles in the absence and presence of GABA were characterized with membrane from rat cerebral cortex. Unspecific 36Cl- influx was completed in three phases of ca. 3% (t 1/2 = 0.6 s), 56% (t 1/2 = 82 s), and 41% (t 1/2 = 23 min). GABA-mediated, specific chloride exchange occurred with 6.5% of the total vesicular internal volume. The GABA-dependent 36Cl- influx proceeded in two phases, each progressively slowed by desensitization. The measurements supported the presence of two distinguishable active GABA receptors on the same membrane mediating chloride exchange into the vesicles with initial first-order rate constants of 9.5 s-1 and 2.3 s-1 and desensitizing with first-order rate constants of 21 s-1 and 1.4 s-1, respectively, at saturation. The half-response concentrations were similar for both receptors, 150 microM and 114 microM GABA for desensitization and 105 microM and 82 microM for chloride exchange, for the faster and slower desensitizing receptors, respectively. The two receptors were present in the activity ratio of ca. 4/1, similar to the ratio of "low-affinity" to "high-affinity" GABA sites found in ligand binding experiments. The desensitization rates have a different dependence on GABA concentration than the channel-opening equilibria.(ABSTRACT TRUNCATED AT 250 WORDS)

  2. Allosteric regulation of the P2X4 receptor channel pore dilation

    Czech Academy of Sciences Publication Activity Database

    Zemková, Hana; Khadra, A.; Rokic, Milos Boro; Tvrdoňová, Vendula; Sherman, A.; Stojilkovic, S. S.

    2015-01-01

    Roč. 467, č. 4 (2015), s. 713-726 ISSN 0031-6768 R&D Projects: GA ČR(CZ) GBP304/12/G069; GA MŠk(CZ) ED1.1.00/02.0109 Institutional support: RVO:67985823 Keywords : ATP * purinergic receptor channel * ivermectin * pore dilation * Markov state model Subject RIV: ED - Physiology Impact factor: 3.654, year: 2015

  3. Deciphering the regulation of P2X4 receptor channel gating by ivermectin using Markov models

    Czech Academy of Sciences Publication Activity Database

    Mackay, L.; Zemková, Hana; Stojilkovic, S. S.; Sherman, A.; Khadra, A.

    2017-01-01

    Roč. 13, č. 7 (2017), č. článku e1005643. ISSN 1553-734X R&D Projects: GA ČR(CZ) GBP304/12/G069 Institutional support: RVO:67985823 Keywords : Markov models * ion channel gating * sensory receptors * cation s Subject RIV: ED - Physiology OBOR OECD: Physiology (including cytology) Impact factor: 4.542, year: 2016

  4. Intracellular postsynaptic cannabinoid receptors link thyrotropin-releasing hormone receptors to TRPC-like channels in thalamic paraventricular nucleus neurons.

    Science.gov (United States)

    Zhang, L; Kolaj, M; Renaud, L P

    2015-12-17

    postsynaptic currents, suggesting presynaptic CB receptors are not involved in this situation. Collectively, the data imply that activation of TRH receptors in these midline thalamic neurons engages novel signaling pathways that include postsynaptic intracellular CB1 and CB2 receptors in the activation of TRPC4/5-like channels. Copyright © 2015 IBRO. Published by Elsevier Ltd. All rights reserved.

  5. Distribution and expression of non-neuronal transient receptor potential (TRPV) ion channels in rosacea.

    Science.gov (United States)

    Sulk, Mathias; Seeliger, Stephan; Aubert, Jerome; Schwab, Verena D; Cevikbas, Ferda; Rivier, Michel; Nowak, Pawel; Voegel, Johannes J; Buddenkotte, Jörg; Steinhoff, Martin

    2012-04-01

    Rosacea is a frequent chronic inflammatory skin disease of unknown etiology. Because early rosacea reveals all characteristics of neurogenic inflammation, a central role of sensory nerves in its pathophysiology has been discussed. Neuroinflammatory mediators and their receptors involved in rosacea are poorly defined. Good candidates may be transient receptor potential (TRP) ion channels of vanilloid type (TRPV), which can be activated by many trigger factors of rosacea. Interestingly, TRPV2, TRPV3, and TRPV4 are expressed by both neuronal and non-neuronal cells. Here, we analyzed the expression and distribution of TRPV receptors in the various subtypes of rosacea on non-neuronal cells using immunohistochemistry, morphometry, double immunoflourescence, and quantitative real-time PCR (qRT-PCR) as compared with healthy skin and lupus erythematosus. Our results show that dermal immunolabeling of TRPV2 and TRPV3 and gene expression of TRPV1 is significantly increased in erythematotelangiectatic rosacea (ETR). Papulopustular rosacea (PPR) displayed an enhanced immunoreactivity for TRPV2, TRPV4, and also of TRPV2 gene expression. In phymatous rosacea (PhR)-affected skin, dermal immunostaining of TRPV3 and TRPV4 and gene expression of TRPV1 and TRPV3 was enhanced, whereas epidermal TRPV2 staining was decreased. Thus, dysregulation of TRPV channels also expressed by non-neuronal cells may be critically involved in the initiation and/or development of rosacea. TRP ion channels may be targets for the treatment of rosacea.

  6. Blockade of P2X7 receptors or pannexin-1 channels similarly attenuates postischemic damage.

    Science.gov (United States)

    Cisneros-Mejorado, Abraham; Gottlieb, Miroslav; Cavaliere, Fabio; Magnus, Tim; Koch-Nolte, Friederich; Scemes, Eliana; Pérez-Samartín, Alberto; Matute, Carlos

    2015-05-01

    The role of P2X7 receptors and pannexin-1 channels in ischemic damage remains controversial. Here, we analyzed their contribution to postanoxic depolarization after ischemia in cultured neurons and in brain slices. We observed that pharmacological blockade of P2X7 receptors or pannexin-1 channels delayed the onset of postanoxic currents and reduced their slope, and that simultaneous inhibition did not further enhance the effects of blocking either one. These results were confirmed in acute cortical slices from P2X7 and pannexin-1 knockout mice. Oxygen-glucose deprivation in cortical organotypic cultures caused neuronal death that was reduced with P2X7 and pannexin-1 blockers as well as in organotypic cultures derived from mice lacking P2X7 and pannexin 1. Subsequently, we used transient middle cerebral artery occlusion to monitor the neuroprotective effect of those drugs in vivo. We found that P2X7 and pannexin-1 antagonists, and their ablation in knockout mice, substantially attenuated the motor symptoms and reduced the infarct volume to ~50% of that in vehicle-treated or wild-type animals. These results show that P2X7 receptors and pannexin-1 channels are major mediators of postanoxic depolarization in neurons and of brain damage after ischemia, and that they operate in the same deleterious signaling cascade leading to neuronal and tissue demise.

  7. Micromolar-Affinity Benzodiazepine Receptors Regulate Voltage-Sensitive Calcium Channels in Nerve Terminal Preparations

    Science.gov (United States)

    Taft, William C.; Delorenzo, Robert J.

    1984-05-01

    Benzodiazepines in micromolar concentrations significantly inhibit depolarization-sensitive Ca2+ uptake in intact nerve-terminal preparations. Benzodiazepine inhibition of Ca2+ uptake is concentration dependent and stereospecific. Micromolar-affinity benzodiazepine receptors have been identified and characterized in brain membrane and shown to be distinct from nanomolar-affinity benzodiazepine receptors. Evidence is presented that micromolar, and not nanomolar, benzodiazepine binding sites mediate benzodiazepine inhibition of Ca2+ uptake. Irreversible binding to micromolar benzodiazepine binding sites also irreversibly blocked depolarization-dependent Ca2+ uptake in synaptosomes, indicating that these compounds may represent a useful marker for identifying the molecular components of Ca2+ channels in brain. Characterization of benzodiazepine inhibition of Ca2+ uptake demonstrates that these drugs function as Ca2+ channel antagonists, because benzodiazepines effectively blocked voltage-sensitive Ca2+ uptake inhibited by Mn2+, Co2+, verapamil, nitrendipine, and nimodipine. These results indicate that micromolar benzodiazepine binding sites regulate voltage-sensitive Ca2+ channels in brain membrane and suggest that some of the neuronal stabilizing effects of micromolar benzodiazepine receptors may be mediated by the regulation of Ca2+ conductance.

  8. The Challenge of Interpreting Glutamate-Receptor Ion-Channel Structures.

    Science.gov (United States)

    Mayer, Mark L

    2017-11-21

    Ion channels activated by glutamate mediate excitatory synaptic transmission in the central nervous system. Similar to other ligand-gated ion channels, their gating cycle begins with transitions from a ligand-free closed state to glutamate-bound active and desensitized states. In an attempt to reveal the molecular mechanisms underlying gating, numerous structures for glutamate receptors have been solved in complexes with agonists, antagonists, allosteric modulators, and auxiliary proteins. The embarrassingly rich library of structures emerging from this work reveals very dynamic molecules with a more complex conformational spectrum than anticipated from functional studies. Unanticipated conformations solved for complexes with competitive antagonists and a lack of understanding of the structural basis for ion channel subconductance states further highlight challenges that have yet to be addressed. Published by Elsevier Inc.

  9. Properties of the intracellular transient receptor potential (TRP) channel in yeast, Yvc1.

    Science.gov (United States)

    Chang, Yiming; Schlenstedt, Gabriel; Flockerzi, Veit; Beck, Andreas

    2010-05-17

    Transient receptor potential (TRP) channels are found among mammals, flies, worms, ciliates, Chlamydomonas, and yeast but are absent in plants. These channels are believed to be tetramers of proteins containing six transmembrane domains (TMs). Their primary structures are diverse with sequence similarities only in some short amino acid sequence motifs mainly within sequences covering TM5, TM6, and adjacent domains. In the yeast genome, there is one gene encoding a TRP-like sequence. This protein forms an ion channel in the vacuolar membrane and is therefore called Yvc1 for yeast vacuolar conductance 1. In the following we summarize its prominent features. Copyright 2009 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

  10. Canonical Transient Receptor Potential Channels and Their Link with Cardio/Cerebro-Vascular Diseases.

    Science.gov (United States)

    Xiao, Xiong; Liu, Hui-Xia; Shen, Kuo; Cao, Wei; Li, Xiao-Qiang

    2017-09-01

    The canonical transient receptor potential channels (TRPCs) constitute a series of nonselective cation channels with variable degrees of Ca 2+ selectivity. TRPCs consist of seven mammalian members, TRPC1, TRPC2, TRPC3, TRPC4, TRPC5, TRPC6, and TRPC7, which are further divided into four subtypes, TRPC1, TRPC2, TRPC4/5, and TRPC3/6/7. These channels take charge of various essential cell functions such as contraction, relaxation, proliferation, and dysfunction. This review, organized into seven main sections, will provide an overview of current knowledge about the underlying pathogenesis of TRPCs in cardio/cerebrovascular diseases, including hypertension, pulmonary arterial hypertension, cardiac hypertrophy, atherosclerosis, arrhythmia, and cerebrovascular ischemia reperfusion injury. Collectively, TRPCs could become a group of drug targets with important physiological functions for the therapy of human cardio/cerebro-vascular diseases.

  11. A thermodynamic framework for understanding temperature sensing by transient receptor potential (TRP) channels.

    Science.gov (United States)

    Clapham, David E; Miller, Christopher

    2011-12-06

    The exceptionally high temperature sensitivity of certain transient receptor potential (TRP) family ion channels is the molecular basis of hot and cold sensation in sensory neurons. The laws of thermodynamics dictate that opening of these specialized TRP channels must involve an unusually large conformational standard-state enthalpy, ΔH(o): positive ΔH(o) for heat-activated and negative ΔH(o) for cold-activated TRPs. However, the molecular source of such high-enthalpy changes has eluded neurobiologists and biophysicists. Here we offer a general, unifying mechanism for both hot and cold activation that recalls long-appreciated principles of protein folding. We suggest that TRP channel gating is accompanied by large changes in molar heat capacity, ΔC(P). This postulate, along with the laws of thermodynamics and independent of mechanistic detail, leads to the conclusion that hot- and cold-sensing TRPs operate by identical conformational changes.

  12. Comparison of the calcium release channel of cardiac and skeletal muscle sarcoplasmic reticulum by target inactivation analysis

    International Nuclear Information System (INIS)

    McGrew, S.G.; Inui, Makoto; Chadwick, C.C.; Boucek, R.J. Jr.; Jung, C.Y.; Fleischer, S.

    1989-01-01

    The calcium release channel of sarcoplasmic reticulum which triggers muscle contraction in excitation-contraction coupling has recently been isolated. The channel has been found to be morphologically identical with the feet structures of the junctional face membrane of terminal cisternae and consists of an oligomer of a unique high molecular weight polypeptide. In this study, the authors compare the target size of the calcium release channel from heart and skeletal muscle using target inactivation analysis. The target molecular weights of the calcium release channel estimated by measuring ryanodine binding after irradiation are similar for heart (139,000) and skeletal muscle (143,000) and are smaller than the monomeric unit (estimated to be about 360,000). The target size, estimated by measuring polypeptide remaining after irradiation, was essentially the same for heart and skeletal muscle, 1,061,000 and 1,070,000, respectively, indicating an oligomeric association of protomers. Thus, the calcium release channel of both cardiac and skeletal muscle reacts uniquely with regard to target inactivation analysis in that (1) the size by ryanodine binding is smaller than the monomeric unit and (2) a single hit leads to destruction of more than one polypeptide, by measuring polypeptide remaining. The target inactivation analysis studies indicate that heart and skeletal muscle receptors are structurally very similar

  13. Review article: transient receptor potential channels as possible therapeutic targets in irritable bowel syndrome.

    Science.gov (United States)

    Beckers, A B; Weerts, Z Z R M; Helyes, Z; Masclee, A A M; Keszthelyi, D

    2017-11-01

    Abdominal pain in irritable bowel syndrome (IBS) remains challenging to treat effectively. Researchers have attempted to elucidate visceral nociceptive processes in order to guide treatment development. Transient receptor potential (TRP) channels have been implied in the generation (TRPV1, TRPV4, TRPA1) and inhibition (TRPM8) of visceral pain signals. Pathological changes in their functioning have been demonstrated in inflammatory conditions, and appear to be present in IBS as well. To provide a comprehensive review of the current literature on TRP channels involved in visceral nociception. In particular, we emphasise the clinical implications of these nociceptors in the treatment of IBS. Evidence to support this review was obtained from an electronic database search via PubMed using the search terms "visceral nociception," "visceral hypersensitivity," "irritable bowel syndrome" and "transient receptor potential channels." After screening the abstracts the articles deemed relevant were cross-referenced for additional manuscripts. Recent studies have resulted in significant advances in our understanding of TRP channel mediated visceral nociception. The diversity of TRP channel sensitization pathways is increasingly recognised. Endogenous TRP agonists, including poly-unsaturated fatty acid metabolites and hydrogen sulphide, have been implied in augmented visceral pain generation in IBS. New potential targets for treatment development have been identified (TRPA1 and TRPV4,) and alternative means of affecting TRP channel signalling (partial antagonists, downstream targeting and RNA-based therapy) are currently being explored. The improved understanding of mechanisms involved in visceral nociception provides a solid basis for the development of new treatment strategies for abdominal pain in IBS. © 2017 John Wiley & Sons Ltd.

  14. Regulated appearance of NMDA receptor subunits and channel functions during in vitro neuronal differentiation.

    Science.gov (United States)

    Jelitai, Márta; Schlett, Katalin; Varju, Patrícia; Eisel, Ulrich; Madarász, Emília

    2002-04-01

    The schedule of NMDA receptor subunit expression and the appearance of functional NMDA-gated ion channels were investigated during the retinoic acid (RA) induced neuronal differentiation of NE-4C, a p53-deficient mouse neuroectodermal progenitor cell line. NR2A, NR2B, and NR2D subunit transcripts were present in both nondifferentiated and neuronally differentiated cultures, while NR2C subunits were expressed only transiently, during the early period of neural differentiation. Several splice variants of NR1 were detected in noninduced progenitors and in RA-induced cells, except the N1 exon containing transcripts that appeared after the fourth day of induction, when neuronal processes were already formed. NR1 and NR2A subunit proteins were detected both in nondifferentiated progenitor cells and in neurons, while the mature form of NR2B subunit protein appeared only at the time of neuronal process elongation. Despite the early presence of NR1 and NR2A subunits, NMDA-evoked responses could be detected in NE-4C neurons only after the sixth day of induction, coinciding in time with the expression of the mature NR2B subunit. The formation of functional NMDA receptors also coincided with the appearance of synapsin I and synaptophysin. The lag period between the production of the subunits and the onset of channel function suggests that subunits capable of channel formation cannot form functional NMDA receptors until a certain stage of neuronal commitment. Thus, the in vitro neurogenesis by NE-4C cells provides a suitable tool to investigate some inherent regulatory processes involved in the initial maturation of NMDA receptor complexes. Copyright 2002 Wiley Periodicals, Inc.

  15. Citral sensing by Transient [corrected] receptor potential channels in dorsal root ganglion neurons.

    Directory of Open Access Journals (Sweden)

    Stephanie C Stotz

    2008-05-01

    Full Text Available Transient receptor potential (TRP ion channels mediate key aspects of taste, smell, pain, temperature sensation, and pheromone detection. To deepen our understanding of TRP channel physiology, we require more diverse pharmacological tools. Citral, a bioactive component of lemongrass, is commonly used as a taste enhancer, as an odorant in perfumes, and as an insect repellent. Here we report that citral activates TRP channels found in sensory neurons (TRPV1 and TRPV3, TRPM8, and TRPA1, and produces long-lasting inhibition of TRPV1-3 and TRPM8, while transiently blocking TRPV4 and TRPA1. Sustained citral inhibition is independent of internal calcium concentration, but is state-dependent, developing only after TRP channel opening. Citral's actions as a partial agonist are not due to cysteine modification of the channels nor are they a consequence of citral's stereoisoforms. The isolated aldehyde and alcohol cis and trans enantiomers (neral, nerol, geranial, and geraniol each reproduce citral's actions. In juvenile rat dorsal root ganglion neurons, prolonged citral inhibition of native TRPV1 channels enabled the separation of TRPV2 and TRPV3 currents. We find that TRPV2 and TRPV3 channels are present in a high proportion of these neurons (94% respond to 2-aminoethyldiphenyl borate, consistent with our immunolabeling experiments and previous in situ hybridization studies. The TRPV1 activation requires residues in transmembrane segments two through four of the voltage-sensor domain, a region previously implicated in capsaicin activation of TRPV1 and analogous menthol activation of TRPM8. Citral's broad spectrum and prolonged sensory inhibition may prove more useful than capsaicin for allodynia, itch, or other types of pain involving superficial sensory nerves and skin.

  16. Citral Sensing by TRANSient Receptor Potential Channels in Dorsal Root Ganglion Neurons

    Science.gov (United States)

    Stotz, Stephanie C.; Vriens, Joris; Martyn, Derek; Clardy, Jon; Clapham, David E.

    2008-01-01

    Transient receptor potential (TRP) ion channels mediate key aspects of taste, smell, pain, temperature sensation, and pheromone detection. To deepen our understanding of TRP channel physiology, we require more diverse pharmacological tools. Citral, a bioactive component of lemongrass, is commonly used as a taste enhancer, as an odorant in perfumes, and as an insect repellent. Here we report that citral activates TRP channels found in sensory neurons (TRPV1 and TRPV3, TRPM8, and TRPA1), and produces long-lasting inhibition of TRPV1–3 and TRPM8, while transiently blocking TRPV4 and TRPA1. Sustained citral inhibition is independent of internal calcium concentration, but is state-dependent, developing only after TRP channel opening. Citral's actions as a partial agonist are not due to cysteine modification of the channels nor are they a consequence of citral's stereoisoforms. The isolated aldehyde and alcohol cis and trans enantiomers (neral, nerol, geranial, and geraniol) each reproduce citral's actions. In juvenile rat dorsal root ganglion neurons, prolonged citral inhibition of native TRPV1 channels enabled the separation of TRPV2 and TRPV3 currents. We find that TRPV2 and TRPV3 channels are present in a high proportion of these neurons (94% respond to 2-aminoethyldiphenyl borate), consistent with our immunolabeling experiments and previous in situ hybridization studies. The TRPV1 activation requires residues in transmembrane segments two through four of the voltage-sensor domain, a region previously implicated in capsaicin activation of TRPV1 and analogous menthol activation of TRPM8. Citral's broad spectrum and prolonged sensory inhibition may prove more useful than capsaicin for allodynia, itch, or other types of pain involving superficial sensory nerves and skin. PMID:18461159

  17. Citral sensing by Transient [corrected] receptor potential channels in dorsal root ganglion neurons.

    Science.gov (United States)

    Stotz, Stephanie C; Vriens, Joris; Martyn, Derek; Clardy, Jon; Clapham, David E

    2008-05-07

    Transient receptor potential (TRP) ion channels mediate key aspects of taste, smell, pain, temperature sensation, and pheromone detection. To deepen our understanding of TRP channel physiology, we require more diverse pharmacological tools. Citral, a bioactive component of lemongrass, is commonly used as a taste enhancer, as an odorant in perfumes, and as an insect repellent. Here we report that citral activates TRP channels found in sensory neurons (TRPV1 and TRPV3, TRPM8, and TRPA1), and produces long-lasting inhibition of TRPV1-3 and TRPM8, while transiently blocking TRPV4 and TRPA1. Sustained citral inhibition is independent of internal calcium concentration, but is state-dependent, developing only after TRP channel opening. Citral's actions as a partial agonist are not due to cysteine modification of the channels nor are they a consequence of citral's stereoisoforms. The isolated aldehyde and alcohol cis and trans enantiomers (neral, nerol, geranial, and geraniol) each reproduce citral's actions. In juvenile rat dorsal root ganglion neurons, prolonged citral inhibition of native TRPV1 channels enabled the separation of TRPV2 and TRPV3 currents. We find that TRPV2 and TRPV3 channels are present in a high proportion of these neurons (94% respond to 2-aminoethyldiphenyl borate), consistent with our immunolabeling experiments and previous in situ hybridization studies. The TRPV1 activation requires residues in transmembrane segments two through four of the voltage-sensor domain, a region previously implicated in capsaicin activation of TRPV1 and analogous menthol activation of TRPM8. Citral's broad spectrum and prolonged sensory inhibition may prove more useful than capsaicin for allodynia, itch, or other types of pain involving superficial sensory nerves and skin.

  18. Spontaneous calcium transients in human neural progenitor cells mediated by transient receptor potential channels.

    Science.gov (United States)

    Morgan, Peter J; Hübner, Rayk; Rolfs, Arndt; Frech, Moritz J

    2013-09-15

    Calcium signals affect many developmental processes, including proliferation, migration, survival, and apoptosis, processes that are of particular importance in stem cells intended for cell replacement therapies. The mechanisms underlying Ca(2+) signals, therefore, have a role in determining how stem cells respond to their environment, and how these responses might be controlled in vitro. In this study, we examined the spontaneous Ca(2+) activity in human neural progenitor cells during proliferation and differentiation. Pharmacological characterization indicates that in proliferating cells, most activity is the result of transient receptor potential (TRP) channels that are sensitive to Gd(3+) and La(3+), with the more subtype selective antagonist Ruthenium red also reducing activity, suggesting the involvement of transient receptor potential vanilloid (TRPV) channels. In differentiating cells, Gd(3+) and La(3+)-sensitive TRP channels also appear to underlie the spontaneous activity; however, no sub-type-specific antagonists had any effect. Protein levels of TRPV2 and TRPV3 decreased in differentiated cells, which is demonstrated by western blot. Thus, it appears that TRP channels represent the main route of Ca(2+) entry in human neural progenitor cells (hNPCs), but the responsible channel types are subject to substitution under differentiating conditions. The level of spontaneous activity could be increased and decreased by lowering and raising the extracellular K(+) concentration. Proliferating cells in low K(+) slowed the cell cycle, with a disproportionate increased percentage of cells in G1 phase and a reduction in S phase. Taken together, these results suggest a link between external K(+) concentration, spontaneous Ca(2+) transients, and cell cycle distribution, which is able to influence the fate of stem and progenitor cells.

  19. Deciphering the regulation of P2X4 receptor channel gating by ivermectin using Markov models.

    Directory of Open Access Journals (Sweden)

    Laurent Mackay

    2017-07-01

    Full Text Available The P2X4 receptor (P2X4R is a member of a family of purinergic channels activated by extracellular ATP through three orthosteric binding sites and allosterically regulated by ivermectin (IVM, a broad-spectrum antiparasitic agent. Treatment with IVM increases the efficacy of ATP to activate P2X4R, slows both receptor desensitization during sustained ATP application and receptor deactivation after ATP washout, and makes the receptor pore permeable to NMDG+, a large organic cation. Previously, we developed a Markov model based on the presence of one IVM binding site, which described some effects of IVM on rat P2X4R. Here we present two novel models, both with three IVM binding sites. The simpler one-layer model can reproduce many of the observed time series of evoked currents, but does not capture well the short time scales of activation, desensitization, and deactivation. A more complex two-layer model can reproduce the transient changes in desensitization observed upon IVM application, the significant increase in ATP-induced current amplitudes at low IVM concentrations, and the modest increase in the unitary conductance. In addition, the two-layer model suggests that this receptor can exist in a deeply inactivated state, not responsive to ATP, and that its desensitization rate can be altered by each of the three IVM binding sites. In summary, this study provides a detailed analysis of P2X4R kinetics and elucidates the orthosteric and allosteric mechanisms regulating its channel gating.

  20. Segregation of lipids near acetylcholine-receptor channels imaged by cryo-EM

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    Nigel Unwin

    2017-07-01

    Full Text Available Rapid communication at the chemical synapse depends on the action of ion channels residing in the postsynaptic membrane. The channels open transiently upon the binding of a neurotransmitter released from the presynaptic nerve terminal, eliciting an electrical response. Membrane lipids also play a vital but poorly understood role in this process of synaptic transmission. The present study examines the lipid distribution around nicotinic acetylcholine (ACh receptors in tubular vesicles made from postsynaptic membranes of the Torpedo ray, taking advantage of the recent advances in cryo-EM. A segregated distribution of lipid molecules is found in the outer leaflet of the bilayer. Apparent cholesterol-rich patches are located in specific annular regions next to the transmembrane helices and also in a more extended `microdomain' between the apposed δ subunits of neighbouring receptors. The particular lipid distribution can be interpreted straightforwardly in relation to the gating movements revealed by an earlier time-resolved cryo-EM study, in which the membranes were exposed briefly to ACh. The results suggest that in addition to stabilizing the protein, cholesterol may play a mechanical role by conferring local rigidity to the membrane so that there is productive coupling between the extracellular and membrane domains, leading to opening of the channel.

  1. Polymodal Transient Receptor Potential Vanilloid (TRPV Ion Channels in Chondrogenic Cells

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    Csilla Szűcs Somogyi

    2015-08-01

    Full Text Available Mature and developing chondrocytes exist in a microenvironment where mechanical load, changes of temperature, osmolarity and acidic pH may influence cellular metabolism. Polymodal Transient Receptor Potential Vanilloid (TRPV receptors are environmental sensors mediating responses through activation of linked intracellular signalling pathways. In chondrogenic high density cultures established from limb buds of chicken and mouse embryos, we identified TRPV1, TRPV2, TRPV3, TRPV4 and TRPV6 mRNA expression with RT-PCR. In both cultures, a switch in the expression pattern of TRPVs was observed during cartilage formation. The inhibition of TRPVs with the non-selective calcium channel blocker ruthenium red diminished chondrogenesis and caused significant inhibition of proliferation. Incubating cell cultures at 41 °C elevated the expression of TRPV1, and increased cartilage matrix production. When chondrogenic cells were exposed to mechanical load at the time of their differentiation into matrix producing chondrocytes, we detected increased mRNA levels of TRPV3. Our results demonstrate that developing chondrocytes express a full palette of TRPV channels and the switch in the expression pattern suggests differentiation stage-dependent roles of TRPVs during cartilage formation. As TRPV1 and TRPV3 expression was altered by thermal and mechanical stimuli, respectively, these are candidate channels that contribute to the transduction of environmental stimuli in chondrogenic cells.

  2. A polycystin-type transient receptor potential (Trp channel that is activated by ATP

    Directory of Open Access Journals (Sweden)

    David Traynor

    2017-02-01

    Full Text Available ATP and ADP are ancient extra-cellular signalling molecules that in Dictyostelium amoebae cause rapid, transient increases in cytosolic calcium due to an influx through the plasma membrane. This response is independent of hetero-trimeric G-proteins, the putative IP3 receptor IplA and all P2X channels. We show, unexpectedly, that it is abolished in mutants of the polycystin-type transient receptor potential channel, TrpP. Responses to the chemoattractants cyclic-AMP and folic acid are unaffected in TrpP mutants. We report that the DIF morphogens, cyclic-di-GMP, GABA, glutamate and adenosine all induce strong cytoplasmic calcium responses, likewise independently of TrpP. Thus, TrpP is dedicated to purinergic signalling. ATP treatment causes cell blebbing within seconds but this does not require TrpP, implicating a separate purinergic receptor. We could detect no effect of ATP on chemotaxis and TrpP mutants grow, chemotax and develop almost normally in standard conditions. No gating ligand is known for the human homologue of TrpP, polycystin-2, which causes polycystic kidney disease. Our results now show that TrpP mediates purinergic signalling in Dictyostelium and is directly or indirectly gated by ATP.

  3. Tuning the allosteric regulation of artificial muscarinic and dopaminergic ligand-gated potassium channels by protein engineering of G protein-coupled receptors

    Science.gov (United States)

    Moreau, Christophe J.; Revilloud, Jean; Caro, Lydia N.; Dupuis, Julien P.; Trouchet, Amandine; Estrada-Mondragón, Argel; Nieścierowicz, Katarzyna; Sapay, Nicolas; Crouzy, Serge; Vivaudou, Michel

    2017-01-01

    Ligand-gated ion channels enable intercellular transmission of action potential through synapses by transducing biochemical messengers into electrical signal. We designed artificial ligand-gated ion channels by coupling G protein-coupled receptors to the Kir6.2 potassium channel. These artificial channels called ion channel-coupled receptors offer complementary properties to natural channels by extending the repertoire of ligands to those recognized by the fused receptors, by generating more sustained signals and by conferring potassium selectivity. The first artificial channels based on the muscarinic M2 and the dopaminergic D2L receptors were opened and closed by acetylcholine and dopamine, respectively. We find here that this opposite regulation of the gating is linked to the length of the receptor C-termini, and that C-terminus engineering can precisely control the extent and direction of ligand gating. These findings establish the design rules to produce customized ligand-gated channels for synthetic biology applications. PMID:28145461

  4. Transient Receptor Potential Canonical (TRPC)/Orai1-dependent Store-operated Ca2+ Channels

    Science.gov (United States)

    Sabourin, Jessica; Bartoli, Fiona; Antigny, Fabrice; Gomez, Ana Maria; Benitah, Jean-Pierre

    2016-01-01

    Store-operated Ca2+ entry (SOCE) has emerged as an important mechanism in cardiac pathology. However, the signals that up-regulate SOCE in the heart remain unexplored. Clinical trials have emphasized the beneficial role of mineralocorticoid receptor (MR) signaling blockade in heart failure and associated arrhythmias. Accumulated evidence suggests that the mineralocorticoid hormone aldosterone, through activation of its receptor, MR, might be a key regulator of Ca2+ influx in cardiomyocytes. We thus assessed whether and how SOCE involving transient receptor potential canonical (TRPC) and Orai1 channels are regulated by aldosterone/MR in neonatal rat ventricular cardiomyocytes. Molecular screening using qRT-PCR and Western blotting demonstrated that aldosterone treatment for 24 h specifically increased the mRNA and/or protein levels of Orai1, TRPC1, -C4, -C5, and stromal interaction molecule 1 through MR activation. These effects were correlated with a specific enhancement of SOCE activities sensitive to store-operated channel inhibitors (SKF-96365 and BTP2) and to a potent Orai1 blocker (S66) and were prevented by TRPC1, -C4, and Orai1 dominant negative mutants or TRPC5 siRNA. A mechanistic approach showed that up-regulation of serum- and glucocorticoid-regulated kinase 1 mRNA expression by aldosterone is involved in enhanced SOCE. Functionally, 24-h aldosterone-enhanced SOCE is associated with increased diastolic [Ca2+]i, which is blunted by store-operated channel inhibitors. Our study provides the first evidence that aldosterone promotes TRPC1-, -C4-, -C5-, and Orai1-mediated SOCE in cardiomyocytes through an MR and serum- and glucocorticoid-regulated kinase 1 pathway. PMID:27129253

  5. A combined role of calcium channel blockers and angiotensin receptor blockers in stroke prevention

    Directory of Open Access Journals (Sweden)

    Ji-Guang Wang

    2009-07-01

    Full Text Available Ji-Guang WangCentre for Epidemiological Studies and Clinical Trials, Ruijin Hospital, Shanghai Jiaotong University School of Medicine, Shanghai, ChinaAbstract: Stroke is a leading cause of death and disability worldwide. The importance of lowering blood pressure for reducing the risk of stroke is well established. However, not all the benefits of antihypertensive treatments in stroke can be accounted for by reductions in BP and there may be differences between antihypertensive classes as to which provides optimal protection. Dihydropyridine calcium channel blockers, such as amlodipine, and angiotensin receptor blockers, such as valsartan, represent the two antihypertensive drug classes with the strongest supportive data for the prevention of stroke. Therefore, when combination therapy is required, a combination of these two antihypertensive classes represents a logical approach.Keywords: stroke, angiotensin, calcium channel, cerebrovascular, hypertension, blood pressure

  6. Molecular mechanisms of Cys-loop ion channel receptor modulation by ivermectin

    DEFF Research Database (Denmark)

    Lynagh, Timothy; Lynch, Joseph W.

    2012-01-01

    Ivermectin is an anthelmintic drug that works by inhibiting neuronal activity and muscular contractility in arthropods and nematodes. It works by activating glutamate-gated chloride channels (GluClRs) at nanomolar concentrations. These receptors, found exclusively in invertebrates, belong to the ...... to the neurotransmitter binding site, thus suggesting a mechanism by which ivermectin potentiates neurotransmitter-gated currents. Together, this information provides new insights into the mechanisms of action of this important drug.......) to its site. Several lines of evidence suggest that ivermectin opens the channel pore via a structural change distinct from that induced by the neurotransmitter agonist. Conformational changes occurring at locations distant from the pore can be probed using voltage-clamp fluorometry (VCF), a technique...

  7. Effects of β-estradiol on cold-sensitive receptor channel TRPM8 in ovariectomized rats.

    Science.gov (United States)

    Kubo, Takuro; Tsuji, Shunichiro; Amano, Tsukuru; Yoshino, Fumi; Niwa, Yoko; Kasahara, Kyoko; Yoshida, Saori; Mukaisho, Ken-Ichi; Sugihara, Hiroyuki; Tanaka, Sachiko; Kimura, Fuminori; Takahashi, Kentaro; Murakami, Takashi

    2017-10-30

    Transient receptor potential cation channel subfamily M member 8 (TRPM8) is associated with sensitivity to cold sensation in mammals. A previous study demonstrated that TRPM8 was overexpressed in the skin of ovariectomized (OVX) rats due to the loss of estrogen. In the present study, we investigated whether estrogen replacement restricts overexpression of the TRPM8 channel in the skin of OVX rats. We divided 15 Sprague Dawley rats into three groups: a non-operated group (NON-OPE), an ovariectomy group (OVX), and a group subjected to estrogen replacement during 4 weeks beginning 7 days after ovariectomy (OVX + E2). Five weeks later, TRPM8 channel mRNA and protein in lumbar skin were quantified by real-time RT-PCR, protein ELISA, and immunohistochemistry. The OVX + E2 group exhibited a trend for decreased expression of the TRPM8 channel in the lumbar skin in comparison with the OVX group, whereas ELISA data and immunohistochemistry data and immunohistochemistry graphs relating to TRPM8 protein did not show any obvious differences between the OVX group and the OVX + E2 group. Estrogen replacement may restrict the overexpression of TRPM8 in the dermis of OVX rats.

  8. Increased transient receptor potential vanilloid type 1 (TRPV1) channel expression in hypertrophic heart

    DEFF Research Database (Denmark)

    Thilo, Florian; Liu, Ying; Schulz, Nico

    2010-01-01

    The aim of this study was to compare the expression of transient receptor potential vanilloid type 1 (TRPV1) channels in hypertrophic hearts from transgenic mice showing overexpression of the catalytic subunit alpha of protein phosphatase 2A alpha (PP2Ac alpha) with wild-type mice and with TRPV1-...... alpha transgenic mice compared to wild-type mice and TRPV1-/- mice (8.6±1.3mg/g; 5.4±0.3mg/g; and 5.4±0.4mg/g; respectively; p...

  9. Pulsatile atheroprone shear stress affects the expression of transient receptor potential channels in human endothelial cells

    DEFF Research Database (Denmark)

    Thilo, Florian; Vorderwülbecke, Bernd J; Marki, Alex

    2012-01-01

    in comparison with endothelial cells grown under static conditions. There was a significant association between the expression of TRPC6 and tumor necrosis factor-α mRNA in human vascular tissue. No-flow and atheroprone flow conditions are equally characterized by an increase in the expression of tumor necrosis......The goal of the study was to assess whether pulsatile atheroprone shear stress modulates the expression of transient receptor potential (TRP) channels, TRPC3, TRPC6, TRPM7, and TRPV1 mRNA, in human umbilical vascular endothelial cells. Exposure of cultured vascular endothelial cells to defined...

  10. Do TRPC channels support working memory? Comparing modulations of TRPC channels and working memory through G-protein coupled receptors and neuromodulators.

    Science.gov (United States)

    Reboreda, Antonio; Theissen, Frederik M; Valero-Aracama, Maria J; Arboit, Alberto; Corbu, Mihaela A; Yoshida, Motoharu

    2018-03-01

    Working memory is a crucial ability we use in daily life. However, the cellular mechanisms supporting working memory still remain largely unclear. A key component of working memory is persistent neural firing which is believed to serve short-term (hundreds of milliseconds up to tens of seconds) maintenance of necessary information. In this review, we will focus on the role of transient receptor potential canonical (TRPC) channels as a mechanism underlying persistent firing. Many years of in vitro work have been suggesting a crucial role of TRPC channels in working memory and temporal association tasks. If TRPC channels are indeed a central mechanism for working memory, manipulations which impair or facilitate working memory should have a similar effect on TRPC channel modulation. However, modulations of working memory and TRPC channels were never systematically compared, and it remains unanswered whether TRPC channels indeed contribute to working memory in vivo or not. In this article, we review the effects of G-protein coupled receptors (GPCR) and neuromodulators, including acetylcholine, noradrenalin, serotonin and dopamine, on working memory and TRPC channels. Based on comparisons, we argue that GPCR and downstream signaling pathways that activate TRPC, generally support working memory, while those that suppress TRPC channels impair it. However, depending on the channel types, areas, and systems tested, this is not the case in all studies. Further work to clarify involvement of specific TRPC channels in working memory tasks and how they are affected by neuromodulators is still necessary in the future. Copyright © 2018 Elsevier B.V. All rights reserved.

  11. Evolution of vertebrate transient receptor potential vanilloid 3 channels: opposite temperature sensitivity between mammals and western clawed frogs.

    Directory of Open Access Journals (Sweden)

    Shigeru Saito

    2011-04-01

    Full Text Available Transient Receptor Potential (TRP channels serve as temperature receptors in a wide variety of animals and must have played crucial roles in thermal adaptation. The TRP vanilloid (TRPV subfamily contains several temperature receptors with different temperature sensitivities. The TRPV3 channel is known to be highly expressed in skin, where it is activated by warm temperatures and serves as a sensor to detect ambient temperatures near the body temperature of homeothermic animals such as mammals. Here we performed comprehensive comparative analyses of the TRPV subfamily in order to understand the evolutionary process; we identified novel TRPV genes and also characterized the evolutionary flexibility of TRPV3 during vertebrate evolution. We cloned the TRPV3 channel from the western clawed frog Xenopus tropicalis to understand the functional evolution of the TRPV3 channel. The amino acid sequences of the N- and C-terminal regions of the TRPV3 channel were highly diversified from those of other terrestrial vertebrate TRPV3 channels, although central portions were well conserved. In a heterologous expression system, several mammalian TRPV3 agonists did not activate the TRPV3 channel of the western clawed frog. Moreover, the frog TRPV3 channel did not respond to heat stimuli, instead it was activated by cold temperatures. Temperature thresholds for activation were about 16 °C, slightly below the lower temperature limit for the western clawed frog. Given that the TRPV3 channel is expressed in skin, its likely role is to detect noxious cold temperatures. Thus, the western clawed frog and mammals acquired opposite temperature sensitivity of the TRPV3 channel in order to detect environmental temperatures suitable for their respective species, indicating that temperature receptors can dynamically change properties to adapt to different thermal environments during evolution.

  12. The transient receptor potential, TRP4, cation channel is a novel member of the family of calmodulin binding proteins.

    OpenAIRE

    Trost, C; Bergs, C; Himmerkus, N; Flockerzi, V

    2001-01-01

    The mammalian gene products, transient receptor potential (trp)1 to trp7, are related to the Drosophila TRP and TRP-like ion channels, and are candidate proteins underlying agonist-activated Ca(2+)-permeable ion channels. Recently, the TRP4 protein has been shown to be part of native store-operated Ca(2+)-permeable channels. These channels, most likely, are composed of other proteins in addition to TRP4. In the present paper we report the direct interaction of TRP4 and calmodulin (CaM) by: (1...

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

    Science.gov (United States)

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

    2016-06-01

    Inositol 1,4,5-trisphosphate receptors (IP3 Rs) are a family of ubiquitously expressed intracellular Ca(2+) release channels. Regulation of channel activity by Ca(2+) , nucleotides, phosphorylation, protein binding partners and other cellular factors is thought to play a major role in defining the specific spatiotemporal characteristics of intracellular Ca(2+) signals. These properties are, in turn, believed pivotal for the selective and specific physiological activation of Ca(2+) -dependent effectors. IP3 Rs are also substrates for the intracellular cysteine proteases, calpain and caspase. Cleavage of the IP3 R has been proposed to play a role in apoptotic cell death by uncoupling regions important for IP3 binding from the channel domain, leaving an unregulated leaky Ca(2+) pore. Contrary to this hypothesis, we demonstrate following proteolysis that N- and C-termini of IP3 R1 remain associated, presumably through non-covalent interactions. Further, we show that complementary fragments of IP3 R1 assemble into tetrameric structures and retain their ability to be regulated robustly by IP3 . While peptide continuity is clearly not necessary for IP3 -gating of the channel, we propose that cleavage of the IP3 R peptide chain may alter other important regulatory events to modulate channel activity. In this scenario, stimulation of the cleaved IP3 R may support distinct spatiotemporal Ca(2+) signals and activation of specific effectors. Notably, in many adaptive physiological events, the non-apoptotic activities of caspase and calpain are demonstrated to be important, but the substrates of the proteases are poorly defined. We speculate that proteolytic fragmentation may represent a novel form of IP3 R regulation, which plays a role in varied adaptive physiological processes. © 2015 The Authors. The Journal of Physiology © 2015 The Physiological Society.

  14. TRPV1 channels and the progesterone receptor Sig-1R interact to regulate pain.

    Science.gov (United States)

    Ortíz-Rentería, Miguel; Juárez-Contreras, Rebeca; González-Ramírez, Ricardo; Islas, León D; Sierra-Ramírez, Félix; Llorente, Itzel; Simon, Sidney A; Hiriart, Marcia; Rosenbaum, Tamara; Morales-Lázaro, Sara L

    2018-02-13

    The Transient Receptor Potential Vanilloid 1 (TRPV1) ion channel is expressed in nociceptors where, when activated by chemical or thermal stimuli, it functions as an important transducer of painful and itch-related stimuli. Although the interaction of TRPV1 with proteins that regulate its function has been previously explored, their modulation by chaperones has not been elucidated, as is the case for other mammalian TRP channels. Here we show that TRPV1 physically interacts with the Sigma 1 Receptor (Sig-1R), a chaperone that binds progesterone, an antagonist of Sig-1R and an important neurosteroid associated to the modulation of pain. Antagonism of Sig-1R by progesterone results in the down-regulation of TRPV1 expression in the plasma membrane of sensory neurons and, consequently, a decrease in capsaicin-induced nociceptive responses. This is observed both in males treated with a synthetic antagonist of Sig-1R and in pregnant females where progesterone levels are elevated. This constitutes a previously undescribed mechanism by which TRPV1-dependent nociception and pain can be regulated.

  15. Pore helix domain is critical to camphor sensitivity of transient receptor potential vanilloid 1 channel.

    Science.gov (United States)

    Marsakova, Lenka; Touska, Filip; Krusek, Jan; Vlachova, Viktorie

    2012-04-01

    The recent discovery that camphor activates and strongly desensitizes the capsaicin-sensitive and noxious heat-sensitive channel transient receptor potential vanilloid subfamily member 1 (TRPV1) has provided new insights and opened up new research paths toward understanding why this naturally occurring monoterpene is widely used in human medicine for its local counter-irritant, antipruritic, and anesthetic properties. However, the molecular basis for camphor sensitivity remains mostly unknown. The authors attempt to explore the nature of the activation pathways evoked by camphor and narrow down a putative interaction site at TRPV1. The authors transiently expressed wild-type or specifically mutated recombinant TRPV1 channels in human embryonic kidney cells HEK293T and recorded cation currents with the whole cell, patch clamp technique. To monitor changes in the spatial distribution of phosphatidylinositol 4,5-bisphosphate, they used fluorescence resonance energy transfer measurements from cells transfected with the fluorescent protein-tagged pleckstrin homology domains of phospholipase C. The results revealed that camphor modulates TRPV1 channel through the outer pore helix domain by affecting its overall gating equilibrium. In addition, camphor, which generally is known to decrease the fluidity of cell plasma membranes, may also regulate the activity of TRPV1 by inducing changes in the spatial distribution of phosphatidylinositol-4,5-bisphosphate on the inner leaflet of the plasma membrane. The findings of this study provide novel insights into the structural basis for the modulation of TRPV1 channel by camphor and may provide an explanation for the mechanism by which camphor modulates thermal sensation in vivo.

  16. Transient receptor potential channel ankyrin-1 is not a cold sensor for autonomic thermoregulation in rodents.

    Science.gov (United States)

    de Oliveira, Cristiane; Garami, Andras; Lehto, Sonya G; Pakai, Eszter; Tekus, Valeria; Pohoczky, Krisztina; Youngblood, Beth D; Wang, Weiya; Kort, Michael E; Kym, Philip R; Pinter, Erika; Gavva, Narender R; Romanovsky, Andrej A

    2014-03-26

    The rodent transient receptor potential ankyrin-1 (TRPA1) channel has been hypothesized to serve as a temperature sensor for thermoregulation in the cold. We tested this hypothesis by using deletion of the Trpa1 gene in mice and pharmacological blockade of the TRPA1 channel in rats. In both Trpa1(-/-) and Trpa1(+/+) mice, severe cold exposure (8°C) resulted in decreases of skin and deep body temperatures to ∼8°C and 13°C, respectively, both temperatures being below the reported 17°C threshold temperature for TRPA1 activation. Under these conditions, Trpa1(-/-) mice had the same dynamics of body temperature as Trpa1(+/+) mice and showed no weakness in the tail skin vasoconstriction response or thermogenic response to cold. In rats, the effects of pharmacological blockade were studied by using two chemically unrelated TRPA1 antagonists: the highly potent and selective compound A967079, which had been characterized earlier, and the relatively new compound 43 ((4R)-1,2,3,4-tetrahydro-4-[3-(3-methoxypropoxy)phenyl]-2-thioxo-5H-indeno[1,2-d]pyrimidin-5-one), which we further characterized in the present study and found to be highly potent (IC50 against cold of ∼8 nm) and selective. Intragastric administration of either antagonist at 30 mg/kg before severe (3°C) cold exposure did not affect the thermoregulatory responses (deep body and tail skin temperatures) of rats, even though plasma concentrations of both antagonists well exceeded their IC50 value at the end of the experiment. In the same experimental setup, blocking the melastatin-8 (TRPM8) channel with AMG2850 (30 mg/kg) attenuated cold-defense mechanisms and led to hypothermia. We conclude that TRPA1 channels do not drive autonomic thermoregulatory responses to cold in rodents.

  17. Coarse architecture of the transient receptor potential vanilloid 1 (TRPV1) ion channel determined by fluorescence resonance energy transfer.

    Science.gov (United States)

    De-la-Rosa, Víctor; Rangel-Yescas, Gisela E; Ladrón-de-Guevara, Ernesto; Rosenbaum, Tamara; Islas, León D

    2013-10-11

    The transient receptor potential vanilloid 1 ion channel is responsible for the perception of high temperatures and low extracellular pH, and it is also involved in the response to some pungent compounds. Importantly, it is also associated with the perception of pain and noxious stimuli. Here, we attempt to discern the molecular organization and location of the N and C termini of the transient receptor potential vanilloid 1 ion channel by measuring FRET between genetically attached enhanced yellow and cyan fluorescent protein to the N or C terminus of the channel protein, expressed in transfected HEK 293 cells or Xenopus laevis oocytes. The static measurements of the domain organization were mapped into an available cryo-electron microscopy density of the channel with good agreement. These measurements also provide novel insights into the organization of terminal domains and their proximity to the plasma membrane.

  18. Activation of the Ca2+-sensing receptors increases currents through inward rectifier K+ channels via activation of phosphatidylinositol 4-kinase

    OpenAIRE

    Liu, Chung-Hung; Chang, Hsueh-Kai; Lee, Sue-Ping; Shieh, Ru-Chi

    2016-01-01

    Inward rectifier K+ channels are important for maintaining normal electrical function in many cell types. The proper function of these channels requires the presence of membrane phosphoinositide 4,5-bisphosphate (PIP2). Stimulation of the Ca2+-sensing receptor CaR, a pleiotropic G protein-coupled receptor, activates both Gq/11, which decreases PIP2, and phosphatidylinositol 4-kinase (PI-4-K), which, conversely, increases PIP2. How membrane PIP2 levels are regulated by CaR activation and wheth...

  19. The Proteoglycan Syndecan 4 Regulates Transient Receptor Potential Canonical 6 Channels via RhoA/ROCK Signaling

    DEFF Research Database (Denmark)

    Liu, Ying; Echtermeyer, Frank; Thilo, Florian

    2012-01-01

    OBJECTIVE: Syndecan 4 (Sdc4) modulates signal transduction and regulates activity of protein channels. Sdc4 is essential for the regulation of cellular permeability. We hypothesized that Sdc4 may regulate transient receptor potential canonical 6 (TRPC6) channels, a determinant of glomerular perme...... permeability, in a RhoA/ROCK-dependent manner. METHODS AND RESULTS: Sdc4 knockout (Sdc4(-/-)) mice showed increased glomerular filtration rate and ameliorated albuminuria under baseline conditions and after bovine serum albumin overload (each P...

  20. Negative modulation of NMDA receptor channel function by DREAM/calsenilin/KChIP3 provides neuroprotection?

    Science.gov (United States)

    Wang, KeWei; Wang, Yun

    2012-01-01

    N-methyl-D-aspartate receptors (NMDARs) are glutamate-gated ion channels highly permeable to calcium and essential to excitatory neurotransmission. The NMDARs have attracted much attention because of their role in synaptic plasticity and excitotoxicity. Evidence has recently accumulated that NMDARs are negatively regulated by intracellular calcium binding proteins. The calcium-dependent suppression of NMDAR function serves as a feedback mechanism capable of regulating subsequent Ca2+ entry into the postsynaptic cell, and may offer an alternative approach to treating NMDAR-mediated excitotoxic injury. This short review summarizes the recent progress made in understanding the negative modulation of NMDAR function by DREAM/calsenilin/KChIP3, a neuronal calcium sensor (NCS) protein. PMID:22518099

  1. Marine snail venoms: use and trends in receptor and channel neuropharmacology.

    Science.gov (United States)

    Favreau, Philippe; Stöcklin, Reto

    2009-10-01

    Venoms are rich mixtures of mainly peptides and proteins evolved by nature to catch and digest preys or for protection against predators. They represent extensive sources of potent and selective bioactive compounds that can lead to original active ingredients, for use as drugs, as pharmacological tools in research and for the biotechnology industry. Among the most fascinating venomous animals, marine snails offer a unique set of pharmacologically active components, targeting a wide diversity of receptors and ion channels. Recent advances still continue to demonstrate their huge neuropharmacological potential. In the quest for interesting pharmacological profiles, researchers face a vast number of venom components to investigate within time and technological constraints. A brief perspective on marine snail venom's complexity and features is given followed by the different discovery strategies and pharmacological approaches, exemplified with some recent developments. These advances will hopefully help further uncovering new pharmacologically important venom molecules.

  2. NMDA receptor channels: labeling of MK-801 with iodine-125 and fluorine-18

    International Nuclear Information System (INIS)

    Wieland, D.M.; Kilbourn, M.R.; Yang, D.J.; Laborde, E.; Gildersleeve, D.L.; Van Dort, M.E.; Pirat, J.-L.; Ciliax, B.J.; Young, A.B.

    1988-01-01

    Methods for labeling the glutamate channel blocking agent MK-801 with iodine-125 ( 125 I) and fluorine-18 ( 18 F) are described. Radioiodine was incorporated in the 1- or 3-positions of the aromatic ring of (±)MK-801 by solid-state halogen exchange techniques. Attachment of the [ 18 F]fluoromethyl group to the bridgehead methyl position was achieved by reaction of [ 18 F]fluoride with the triflamide alcohol or the novel cyclic sulfamate recently reported by Merck chemists. Radiochemical yields of (±)13-[ 18 F]-fluoromethyl-MK-801 were >72%, EOB; radiochemical purity > 99%. In competitive binding studies using rat brain homogenates, (±)3-bromo-MK-801 showed greater affinity than (±)MK-801 for the glutamate-linked channel. The experimental log P (2.1 ± 0.1) of MK-801 is optimal for transit of the blood-brain barrier. These preliminary findings support further testing of [ 123 I]iodo-MK-801 and [ 18 F]fluoromethyl-MK-801 as possible agents for in vivo mapping of the glutamate receptor complex. (author)

  3. Nereistoxin and cartap neurotoxicity attributable to direct block of the insect nicotinic receptor/channel.

    Science.gov (United States)

    Lee, Seog-Jong; Tomizawa, Motohiro; Casida, John E

    2003-04-23

    Nereistoxin (NTX) (4-dimethylamino-1,2-dithiolane) is the naturally occurring prototype for cartap [the bis(thiocarbamate) derivative of the NTX dithiol], which is generally regarded as a proinsecticide reverting to NTX. The aim of this study is to define the target site(s) for dithiolanes and dithiol esters. The affinity of [(3)H]NTX was not suitable for binding assays with honeybee (Apis mellifera) head membranes. However, NTX and cartap are equally potent, direct-acting, and competitive displacers of [(3)H]thienylcyclohexylpiperidine binding at the noncompetitive blocker (NCB) site of the Apis nicotinic acetylcholine receptor (nAChR)/channel. NTX also binds at the Apis [(3)H]imidacloprid agonist site, but cartap does not. As candidate metabolic pathways, sequential N-desmethylation and S-oxidation of NTX progressively reduce its potency at the NCB site and toxicity to houseflies. A P450 inhibitor reduces the toxicity of NTX and enhances it with cartap. Surprisingly, cartap is not just a pro-NTX but instead directly induces inhibitory neurotoxicity by blocking the nAChR/channel, whereas NTX may have dual NCB and agonist targets.

  4. Anions mediate ligand binding in Adineta vaga glutamate receptor ion channels.

    Science.gov (United States)

    Lomash, Suvendu; Chittori, Sagar; Brown, Patrick; Mayer, Mark L

    2013-03-05

    AvGluR1, a glutamate receptor ion channel from the primitive eukaryote Adineta vaga, is activated by alanine, cysteine, methionine, and phenylalanine, which produce lectin-sensitive desensitizing responses like those to glutamate, aspartate, and serine. AvGluR1 LBD crystal structures reveal an unusual scheme for binding dissimilar ligands that may be utilized by distantly related odorant/chemosensory receptors. Arginine residues in domain 2 coordinate the γ-carboxyl group of glutamate, whereas in the alanine, methionine, and serine complexes a chloride ion acts as a surrogate ligand, replacing the γ-carboxyl group. Removal of Cl(-) lowers affinity for these ligands but not for glutamate or aspartate nor for phenylalanine, which occludes the anion binding site and binds with low affinity. AvGluR1 LBD crystal structures and sedimentation analysis also provide insights into the evolutionary link between prokaryotic and eukaryotic iGluRs and reveal features unique to both classes, emphasizing the need for additional structure-based studies on iGluR-ligand interactions. Copyright © 2013 Elsevier Ltd. All rights reserved.

  5. Activation of the chemosensing transient receptor potential channel A1 (TRPA1) by alkylating agents.

    Science.gov (United States)

    Stenger, Bernhard; Zehfuss, Franziska; Mückter, Harald; Schmidt, Annette; Balszuweit, Frank; Schäfer, Eva; Büch, Thomas; Gudermann, Thomas; Thiermann, Horst; Steinritz, Dirk

    2015-09-01

    The transient receptor potential ankyrin 1 (TRPA1) cation channel is expressed in different tissues including skin, lung and neuronal tissue. Recent reports identified TRPA1 as a sensor for noxious substances, implicating a functional role in the molecular toxicology. TRPA1 is activated by various potentially harmful electrophilic substances. The chemical warfare agent sulfur mustard (SM) is a highly reactive alkylating agent that binds to numerous biological targets. Although SM is known for almost 200 years, detailed knowledge about the pathophysiology resulting from exposure is lacking. A specific therapy is not available. In this study, we investigated whether the alkylating agent 2-chloroethyl-ethylsulfide (CEES, a model substance for SM-promoted effects) and SM are able to activate TRPA1 channels. CEES induced a marked increase in the intracellular calcium concentration ([Ca(2+)]i) in TRPA1-expressing but not in TRPA1-negative cells. The TRP-channel blocker AP18 diminished the CEES-induced calcium influx. HEK293 cells permanently expressing TRPA1 were more sensitive toward cytotoxic effects of CEES compared with wild-type cells. At low CEES concentrations, CEES-induced cytotoxicity was prevented by AP18. Proof-of-concept experiments using SM resulted in a pronounced increase in [Ca(2+)]i in HEK293-A1-E cells. Human A549 lung epithelial cells, which express TRPA1 endogenously, reacted with a transient calcium influx in response to CEES exposure. The CEES-dependent calcium response was diminished by AP18. In summary, our results demonstrate that alkylating agents are able to activate TRPA1. Inhibition of TRPA1 counteracted cellular toxicity and could thus represent a feasible approach to mitigate SM-induced cell damage.

  6. Expression and distribution of transient receptor potential (TRP) channels in bladder epithelium.

    Science.gov (United States)

    Yu, Weiqun; Hill, Warren G; Apodaca, Gerard; Zeidel, Mark L

    2011-01-01

    The urothelium is proposed to be a sensory tissue that responds to mechanical stress by undergoing dynamic membrane trafficking and neurotransmitter release; however, the molecular basis of this function is poorly understood. Transient receptor potential (TRP) channels are ideal candidates to fulfill such a role as they can sense changes in temperature, osmolarity, and mechanical stimuli, and several are reported to be expressed in the bladder epithelium. However, their complete expression profile is unknown and their cellular localization is largely undefined. We analyzed expression of all 33 TRP family members in mouse bladder and urothelium by RT-PCR and found 22 specifically expressed in the urothelium. Of the latter, 10 were chosen for closer investigation based on their known mechanosensory or membrane trafficking functions in other cell types. Western blots confirmed urothelial expression of TRPC1, TRPC4, TRPV1, TRPV2, TRPV4, TRPM4, TRPM7, TRPML1, and polycystins 1 and 2 (PKD1 and PKD2) proteins. We further defined the cellular and subcellular localization of all 10 TRP channels. TRPV2 and TRPM4 were prominently localized to the umbrella cell apical membrane, while TRPC4 and TRPV4 were identified on their abluminal surfaces. TRPC1, TRPM7, and TRPML1 were localized to the cytoplasm, while PKD1 and PKD2 were expressed on the apical and basolateral membranes of umbrella cells as well as in the cytoplasm. The cellular location of TRPV1 in the bladder has been debated, but colocalization with neuronal marker calcitonin gene-related peptide indicated clearly that it is present on afferent neurons that extend into the urothelium, but may not be expressed by the urothelium itself. These findings are consistent with the hypothesis that the urothelium acts as a sentinel and by expressing multiple TRP channels it is likely it can detect and presumably respond to a diversity of external stimuli and suggest that it plays an important role in urothelial signal

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

    Science.gov (United States)

    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.

  8. First direct electron microscopic visualization of a tight spatial coupling between GABAA-receptors and voltage-sensitive calcium channels

    DEFF Research Database (Denmark)

    Hansen, Gert Helge; Belhage, B; Schousboe, A

    1992-01-01

    Using cerebellar granule neurons in culture it was demonstrated that exposure of the cells to the GABAA receptor agonist 4,5,6,7-tetrahydroisoxazolo[5,4-c]pyridin-3-ol (THIP) leads to an increase in the number of voltage-gated calcium channels as revealed by quantitative preembedding indirect imm...

  9. Localization and Molecular Determinants of the Hanatoxin Receptors on the Voltage-Sensing Domains of a K+ Channel

    Science.gov (United States)

    Li-Smerin, Yingying; Swartz, Kenton J.

    2000-01-01

    Hanatoxin inhibits voltage-gated K+ channels by modifying the energetics of activation. We studied the molecular determinants and physical location of the Hanatoxin receptors on the drk1 voltage-gated K+ channel. First, we made multiple substitutions at three previously identified positions in the COOH terminus of S3 to examine whether these residues interact intimately with the toxin. We also examined a region encompassing S1–S3 using alanine-scanning mutagenesis to identify additional determinants of the toxin receptors. Finally, guided by the structure of the KcsA K+ channel, we explored whether the toxin interacts with the peripheral extracellular surface of the pore domain in the drk1 K+ channel. Our results argue for an intimate interaction between the toxin and the COOH terminus of S3 and suggest that the Hanatoxin receptors are confined within the voltage-sensing domains of the channel, at least 20–25 Å away from the central pore axis. PMID:10828242

  10. Channel catfish (Ictalurus punctatus) leukocytes express estrogen receptor isoforms ERα and ERβ2 and are functionally modulated by estrogens.

    Science.gov (United States)

    Iwanowicz, Luke R; Stafford, James L; Patiño, Reynaldo; Bengten, Eva; Miller, Norman W; Blazer, Vicki S

    2014-09-01

    Estrogens are recognized as modulators of immune responses in mammals and teleosts. While it is known that the effects of estrogens are mediated via leukocyte-specific estrogen receptors (ERs) in humans and mice, leucocyte-specific estrogen receptor expression and the effects of estrogens on this cell population is less explored and poorly understood in teleosts. Here in, we verify that channel catfish (Ictalurus punctaus) leukocytes express ERα and ERβ2. Transcripts of these isoforms were detected in tissue-associated leukocyte populations by PCR, but ERβ2 was rarely detected in PBLs. Expression of these receptors was temporally regulated in PBLs following polyclonal activation by concanavalin A, lipopolysaccharide or alloantigen based on evaluation by quantitative and end-point PCR. Examination of long-term leukocyte cell lines demonstrated that these receptors are differentially expressed depending on leukocyte lineage and phenotype. Expression of ERs was also temporally dynamic in some leukocyte lineages and may reflect stage of cell maturity. Estrogens affect the responsiveness of channel catfish peripheral blood leukocytes (PBLs) to mitogens in vitro. Similarly, bactericidal activity and phorbol 12-myristate 13-acetate induced respiratory burst was modulated by 17β-estradiol. These actions were blocked by the pure ER antagonist ICI 182780 indicating that response is, in part, mediated via ERα. In summary, estrogen receptors are expressed in channel catfish leukocytes and participate in the regulation of the immune response. This is the first time leukocyte lineage expression has been reported in teleost cell lines. Published by Elsevier Ltd.

  11. Phytochemicals from Ruta graveolens Activate TAS2R Bitter Taste Receptors and TRP Channels Involved in Gustation and Nociception

    Directory of Open Access Journals (Sweden)

    Giuseppe Mancuso

    2015-10-01

    Full Text Available Ruta graveolens (rue is a spontaneous plant in the Mediterranean area with a strong aroma and a very intense bitter taste, used in gastronomy and in folk medicine. From the leaves, stems and fruits of rue, we isolated rutin, rutamarin, three furanocoumarins, two quinolinic alkaloids, a dicoumarin and two long chain ketones. Bitter taste and chemesthetic properties have been evaluated by in vitro assays with twenty receptors of the TAS2R family and four TRP ion channels involved in gustation and nociception. Among the alkaloids, skimmianine was active as a specific agonist of T2R14, whereas kokusaginin did not activate any of the tested receptors. The furanocoumarins activates TAS2R10, 14, and 49 with different degrees of selectivity, as well as the TRPA1 somatosensory ion channel. Rutamarin is an agonist of TRPM5 and TRPV1 and a strong antagonist of TRPM8 ion channels.

  12. Phytochemicals from Ruta graveolens Activate TAS2R Bitter Taste Receptors and TRP Channels Involved in Gustation and Nociception.

    Science.gov (United States)

    Mancuso, Giuseppe; Borgonovo, Gigliola; Scaglioni, Leonardo; Bassoli, Angela

    2015-10-16

    Ruta graveolens (rue) is a spontaneous plant in the Mediterranean area with a strong aroma and a very intense bitter taste, used in gastronomy and in folk medicine. From the leaves, stems and fruits of rue, we isolated rutin, rutamarin, three furanocoumarins, two quinolinic alkaloids, a dicoumarin and two long chain ketones. Bitter taste and chemesthetic properties have been evaluated by in vitro assays with twenty receptors of the TAS2R family and four TRP ion channels involved in gustation and nociception. Among the alkaloids, skimmianine was active as a specific agonist of T2R14, whereas kokusaginin did not activate any of the tested receptors. The furanocoumarins activates TAS2R10, 14, and 49 with different degrees of selectivity, as well as the TRPA1 somatosensory ion channel. Rutamarin is an agonist of TRPM5 and TRPV1 and a strong antagonist of TRPM8 ion channels.

  13. Localization of transient receptor potential ion channels in primary and motile cilia of the female murine reproductive organs

    DEFF Research Database (Denmark)

    Teilmann, Stefan C.; Byskov, Anne Grete; Pedersen, Per Amstrup

    2005-01-01

    We have examined the subcellular localization of transient receptor potential (TRP) ion channels and the potential sensory role of cilia in murine female reproductive organs using confocal laser scanning microscopy analysis on ovary and oviduct tissue sections as well as on primary cultures...... of follicular granulosa cells. We show that the Ca2+ permeable cation channel, polycystin-2, as well as polycystin-1, a receptor that forms a functional protein complex with polycystin 2, distinctively localize to primary cilia emerging from granulosa cells of antral follicles in vivo and in vitro. Both...... polycystins are localized to motile oviduct cilia and this localization is greatly increased upon ovulatory gonadotropic stimulation. Further, the Ca2+ permeable cation channel, TRP vaniloid 4 (TRPV4), localizes to a sub-population of motile cilia on the epithelial cells of the ampulla and isthmus with high...

  14. Receptor model for the molecular basis of tissue selectivity of 1,4-dihydropyridine calcium channel drugs

    Science.gov (United States)

    Langs, David A.; Strong, Phyllis D.; Triggle, David J.

    1990-09-01

    Our analysis of the solid state conformations of nifedipine [dimethyl 1,4-dihydro-2,6-dimethyl-4-(2-nitrophenyl)-3,5-pyridinecarboxylate] and its 1,4-dihydropyridine (1,4-DHP) analogues produced a cartoon description of the important interactions between these drugs and their voltage-dependent calcium channel receptor. In the present study a molecular-level detailed model of the 1,4-DHP receptor binding site has been built from the published amino acid sequence of the 215-1 subunit of the voltage-dependent calcium channel isolated from rabbit skeletal muscle transverse tubule membranes. The voltage-sensing component of the channel described in this work differs from others reported for the homologous sodium channel in that it incorporates a water structure and a staggered, rather than eclipsed, hydrogen bonded S4 helix conformation. The major recognition surfaces of the receptor lie in helical grooves on the S4 or voltagesensing α-helix that is positioned in the center of the bundle of transmembrane helices that define each of the four calcium channel domains. Multiple binding clefts defined by Arg-X-X-Arg-P-X-X-S `reading frames' exist on the S4 strand. The tissue selectivity of nifedipine and its analogues may arise, in part, from conservative changes in the amino acid residues at the P and S positions of the reading frame that define the ester-binding regions of receptors from different tissues. The crystal structures of two tissue-selective nifedipine analogues, nimodipine [isopropyl (2-methoxyethyl) 1,4-dihydro-2,6- dimethyl-4-(3-nitrophenyl)-3,5-pyridinecarboxylate] and nitrendipine [ethyl methyl 1,4-dihydro-2,6-dimethyl-4-(3-nitrophenyl)-3,5-pyridinecarboxylate] are reported. Nimodipine was observed to have an unusual ester side chain conformation that enhances the fit to the proposed ester-sensing region of the receptor.

  15. Novel transcripts of the estrogen receptor α gene in channel catfish

    Science.gov (United States)

    Patino, Reynaldo; Xia, Zhenfang; Gale, William L.; Wu, Chunfa; Maule, Alec G.; Chang, Xiaotian

    2000-01-01

    Complementary DNA libraries from liver and ovary of an immature female channel catfish were screened with a homologous ERα cDNA probe. The hepatic library yielded two new channel catfish ER cDNAs that encode N-terminal ERα variants of different sizes. Relative to the catfish ERα (medium size; 581 residues) previously reported, these new cDNAs encode Long-ERα (36 residues longer) and Short-ERα (389 residues shorter). The 5′-end of Long-ERα cDNA is identical to that of Medium-ERα but has an additional 503-bp segment with an upstream, in-frame translation-start codon. Recombinant Long-ERα binds estrogen with high affinity (Kd = 3.4 nM), similar to that previously reported for Medium-ERα but lower than reported for catfish ERβ. Short-ERα cDNA encodes a protein that lacks most of the receptor protein and does not bind estrogen. Northern hybridization confirmed the existence of multiple hepatic ERα RNAs that include the size range of the ERα cDNAs obtained from the libraries as well as additional sizes. Using primers for RT-PCR that target locations internal to the protein-coding sequence, we also established the presence of several ERα cDNA variants with in-frame insertions in the ligand-binding and DNA-binding domains and in-frame or out-of-frame deletions in the ligand-binding domain. These internal variants showed patterns of expression that differed between the ovary and liver. Further, the ovarian library yielded a full-length, ERα antisense cDNA containing a poly(A) signal and tail. A limited survey of histological preparations from juvenile catfish by in situ hybridization using directionally synthesized cRNA probes also suggested the expression of ERα antisense RNA in a tissue-specific manner. In conclusion, channel catfish seemingly have three broad classes of ERα mRNA variants: those encoding N-terminal truncated variants, those encoding internal variants (including C-terminal truncated variants), and antisense mRNA. The sense variants may

  16. Mutation of I696 and W697 in the TRP box of vanilloid receptor subtype I modulates allosteric channel activation.

    Science.gov (United States)

    Gregorio-Teruel, Lucia; Valente, Pierluigi; González-Ros, José Manuel; Fernández-Ballester, Gregorio; Ferrer-Montiel, Antonio

    2014-03-01

    The transient receptor potential vanilloid receptor subtype I (TRPV1) channel acts as a polymodal sensory receptor gated by chemical and physical stimuli. Like other TRP channels, TRPV1 contains in its C terminus a short, conserved domain called the TRP box, which is necessary for channel gating. Substitution of two TRP box residues-I696 and W697-with Ala markedly affects TRPV1's response to all activating stimuli, which indicates that these two residues play a crucial role in channel gating. We systematically replaced I696 and W697 with 18 native l-amino acids (excluding cysteine) and evaluated the effect on voltage- and capsaicin-dependent gating. Mutation of I696 decreased channel activation by either voltage or capsaicin; furthermore, gating was only observed with substitution of hydrophobic amino acids. Substitution of W697 with any of the 18 amino acids abolished gating in response to depolarization alone, shifting the threshold to unreachable voltages, but not capsaicin-mediated gating. Moreover, vanilloid-activated responses of W697X mutants showed voltage-dependent gating along with a strong voltage-independent component. Analysis of the data using an allosteric model of activation indicates that mutation of I696 and W697 primarily affects the allosteric coupling constants of the ligand and voltage sensors to the channel pore. Together, our findings substantiate the notion that inter- and/or intrasubunit interactions at the level of the TRP box are critical for efficient coupling of stimulus sensing and gate opening. Perturbation of these interactions markedly reduces the efficacy and potency of the activating stimuli. Furthermore, our results identify these interactions as potential sites for pharmacological intervention.

  17. Receptor channel TRPC6 orchestrate the activation of human hepatic stellate cell under hypoxia condition

    International Nuclear Information System (INIS)

    Iyer, Soumya C; Kannan, Anbarasu; Gopal, Ashidha; Devaraj, Niranjali; Halagowder, Devaraj

    2015-01-01

    Hepatic stellate cells (HSCs), a specialized stromal cytotype have a great impact on the biological behaviors of liver diseases. Despite this fact, the underlying mechanism that regulates HSC still remains poorly understood. The aim of the present study was to understand the role of TRPC6 signaling in regulating the molecular mechanism of HSCs in response to hypoxia. In the present study we showed that under hypoxia condition, the upregulated Hypoxia Inducible Factor 1α (HIF1α) increases NICD activation, which in turn induces the expression of transient receptor potential channel 6 (TRPC6) in HSC line lx-2. TRPC6 causes a sustained elevation of intracellular calcium which is coupled with the activation of the calcineurin-nuclear factor of activated T-cell (NFAT) pathway which activates the synthesis of extracellular matrix proteins. TRPC6 also activates SMAD2/3 dependent TGF-β signaling in facilitating upregulated expression of αSMA and collagen. As activated HSCs may be a suitable target for HCC therapy and targeting these cells rather than the HCC cells may result in a greater response. Collectively, our studies indicate for the first time the detailed mechanism of activation of HSC through TRPC6 signaling and thus being a promising therapeutic target. - Highlights: • HIF1α increases NICD, induces TRPC6 in lx2 cells. • TRPC6 a novel regulator in the activation of HSC. • HSCs as target for HCC therapy

  18. Receptor channel TRPC6 orchestrate the activation of human hepatic stellate cell under hypoxia condition

    Energy Technology Data Exchange (ETDEWEB)

    Iyer, Soumya C, E-mail: chidambaram.soumya@gmail.com [Unit of Biochemistry, Department of Zoology, School of Life Sciences, University of Madras, Guindy Campus, Chennai 600025, Tamilnadu (India); Kannan, Anbarasu [Department of Biochemistry, University of Madras, Guindy Campus, Chennai 600025, Tamilnadu (India); Gopal, Ashidha [Unit of Biochemistry, Department of Zoology, School of Life Sciences, University of Madras, Guindy Campus, Chennai 600025, Tamilnadu (India); Devaraj, Niranjali [Department of Biochemistry, University of Madras, Guindy Campus, Chennai 600025, Tamilnadu (India); Halagowder, Devaraj [Unit of Biochemistry, Department of Zoology, School of Life Sciences, University of Madras, Guindy Campus, Chennai 600025, Tamilnadu (India)

    2015-08-01

    Hepatic stellate cells (HSCs), a specialized stromal cytotype have a great impact on the biological behaviors of liver diseases. Despite this fact, the underlying mechanism that regulates HSC still remains poorly understood. The aim of the present study was to understand the role of TRPC6 signaling in regulating the molecular mechanism of HSCs in response to hypoxia. In the present study we showed that under hypoxia condition, the upregulated Hypoxia Inducible Factor 1α (HIF1α) increases NICD activation, which in turn induces the expression of transient receptor potential channel 6 (TRPC6) in HSC line lx-2. TRPC6 causes a sustained elevation of intracellular calcium which is coupled with the activation of the calcineurin-nuclear factor of activated T-cell (NFAT) pathway which activates the synthesis of extracellular matrix proteins. TRPC6 also activates SMAD2/3 dependent TGF-β signaling in facilitating upregulated expression of αSMA and collagen. As activated HSCs may be a suitable target for HCC therapy and targeting these cells rather than the HCC cells may result in a greater response. Collectively, our studies indicate for the first time the detailed mechanism of activation of HSC through TRPC6 signaling and thus being a promising therapeutic target. - Highlights: • HIF1α increases NICD, induces TRPC6 in lx2 cells. • TRPC6 a novel regulator in the activation of HSC. • HSCs as target for HCC therapy.

  19. Discovery of functional monoclonal antibodies targeting G-protein-coupled receptors and ion channels.

    Science.gov (United States)

    Wilkinson, Trevor C I

    2016-06-15

    The development of recombinant antibody therapeutics is a significant area of growth in the pharmaceutical industry with almost 50 approved monoclonal antibodies on the market in the US and Europe. Despite this growth, however, certain classes of important molecular targets have remained intractable to therapeutic antibodies due to complexity of the target molecules. These complex target molecules include G-protein-coupled receptors and ion channels which represent a large potential target class for therapeutic intervention with monoclonal antibodies. Although these targets have typically been addressed by small molecule approaches, the exquisite specificity of antibodies provides a significant opportunity to provide selective modulation of these target proteins. Given this opportunity, substantial effort has been applied to address the technical challenges of targeting these complex membrane proteins with monoclonal antibodies. In this review recent progress made in the strategies for discovery of functional monoclonal antibodies for these challenging membrane protein targets is addressed. © 2016 The Author(s). published by Portland Press Limited on behalf of the Biochemical Society.

  20. Transient receptor potential A1 channel contributes to activation of the muscle reflex.

    Science.gov (United States)

    Koba, Satoshi; Hayes, Shawn G; Sinoway, Lawrence I

    2011-01-01

    This study was undertaken to elucidate the role played by transient receptor potential A1 channels (TRPA1) in activating the muscle reflex, a sympathoexcitatory drive originating in contracting muscle. First, we tested the hypothesis that stimulation of the TRPA1 located on muscle afferents reflexly increases sympathetic nerve activity. In decerebrate rats, allyl isothiocyanate, a TRPA1 agonist, was injected intra-arterially into the hindlimb muscle circulation. This led to a 33% increase in renal sympathetic nerve activity (RSNA). The effect of allyl isothiocyanate was a reflex because the response was prevented by sectioning the sciatic nerve. Second, we tested the hypothesis that blockade of TRPA1 reduces RSNA response to contraction. Thirty-second continuous static contraction of the hindlimb muscles, induced by electrical stimulation of the peripheral cut ends of L(4) and L(5) ventral roots, increased RSNA and blood pressure. The integrated RSNA during contraction was reduced by HC-030031, a TRPA1 antagonist, injected intra-arterially (163 ± 24 vs. 95 ± 21 arbitrary units, before vs. after HC-030031, P reflex. Increases in RSNA in response to injection into the muscle circulation of arachidonic acid, bradykinin, and diprotonated phosphate, which are metabolic by-products of contraction and stimulants of muscle afferents during contraction, were reduced by HC-030031. These observations suggest that the TRPA1 located on muscle afferents is part of the muscle reflex and further support the notion that arachidonic acid metabolites, bradykinin, and diprotonated phosphate are candidates for endogenous agonists of TRPA1.

  1. Histamine facilitates GABAergic transmission in the rat entorhinal cortex: Roles of H1 and H2 receptors, Na+ -permeable cation channels, and inward rectifier K+ channels.

    Science.gov (United States)

    Cilz, Nicholas I; Lei, Saobo

    2017-05-01

    In the brain, histamine (HA) serves as a neuromodulator and a neurotransmitter released from the tuberomammillary nucleus (TMN). HA is involved in wakefulness, thermoregulation, energy homeostasis, nociception, and learning and memory. The medial entorhinal cortex (MEC) receives inputs from the TMN and expresses HA receptors (H 1 , H 2 , and H 3 ). We investigated the effects of HA on GABAergic transmission in the MEC and found that HA significantly increased the frequency of spontaneous inhibitory postsynaptic currents (sIPSCs) with an EC 50 of 1.3 µM, but failed to significantly alter sIPSC amplitude. HA-induced increases in sIPSC frequency were sensitive to tetrodotoxin (TTX), required extracellular Ca 2+ , and persisted when GDP-β-S, a G-protein inactivator, was applied postsynaptically via the recording pipettes, indicating that HA increased GABA release by facilitating the excitability of GABAergic interneurons in the MEC. Recordings from local MEC interneurons revealed that HA significantly increased their excitability as determined by membrane depolarization, generation of an inward current at -65 mV, and augmentation of action potential firing frequency. Both H 1 and H 2 receptors were involved in HA-induced increases in sIPSCs and interneuron excitability. Immunohistochemical staining showed that both H 1 and H 2 receptors are expressed on GABAergic interneurons in the MEC. HA-induced depolarization of interneurons involved a mixed ionic mechanism including activation of a Na + -permeable cation channel and inhibition of a cesium-sensitive inward rectifier K + channel, although HA also inhibited the delayed rectifier K + channels. Our results may provide a cellular mechanism, at least partially, to explain the roles of HA in the brain. © 2017 Wiley Periodicals, Inc. © 2017 Wiley Periodicals, Inc.

  2. Maitotoxin Is a Potential Selective Activator of the Endogenous Transient Receptor Potential Canonical Type 1 Channel in Xenopus laevis Oocytes

    Directory of Open Access Journals (Sweden)

    Pedro L. Flores

    2017-06-01

    Full Text Available Maitotoxin (MTX is the most potent marine toxin known to date. It is responsible for a particular human intoxication syndrome called ciguatera fish poisoning (CFP. Several reports indicate that MTX is an activator of non-selective cation channels (NSCC in different cell types. The molecular identity of these channels is still an unresolved topic, and it has been proposed that the transient receptor potential (TRP channels are involved in this effect. In Xenopus laevis oocytes, MTX at picomolar (pM concentrations induces the activation of NSCC with functional and pharmacological properties that resemble the activity of TRP channels. The purpose of this study was to characterize the molecular identity of the TRP channel involved in the MTX response, using the small interference RNA (siRNA approach and the two-electrode voltage-clamp technique (TEVC. The injection of a specifically designed siRNA to silence the transient receptor potential canonical type 1 (TRPC1 protein expression abolished the MTX response. MTX had no effect on oocytes, even at doses 20-fold higher compared to cells without injection. Total mRNA and protein levels of TRPC1 were notably diminished. The TRPC4 siRNA did not change the MTX effect, even though it was important to note that the protein level was reduced by the silencing of TRPC4. Our results suggest that MTX could be a selective activator of TRPC1 channels in X. laevis oocytes and a useful pharmacological tool for further studies on these TRP channels.

  3. Beta-Estradiol Regulates Voltage-Gated Calcium Channels and Estrogen Receptors in Telocytes from Human Myometrium

    Directory of Open Access Journals (Sweden)

    Adela Banciu

    2018-05-01

    Full Text Available Voltage-gated calcium channels and estrogen receptors are essential players in uterine physiology, and their association with different calcium signaling pathways contributes to healthy and pathological conditions of the uterine myometrium. Among the properties of the various cell subtypes present in human uterine myometrium, there is increasing evidence that calcium oscillations in telocytes (TCs contribute to contractile activity and pregnancy. Our study aimed to evaluate the effects of beta-estradiol on voltage-gated calcium channels and estrogen receptors in TCs from human uterine myometrium and to understand their role in pregnancy. For this purpose, we employed patch-clamp recordings, ratiometric Fura-2-based calcium imaging analysis, and qRT-PCR techniques for the analysis of cultured human myometrial TCs derived from pregnant and non-pregnant uterine samples. In human myometrial TCs from both non-pregnant and pregnant uterus, we evidenced by qRT-PCR the presence of genes encoding for voltage-gated calcium channels (Cav3.1, Ca3.2, Cav3.3, Cav2.1, estrogen receptors (ESR1, ESR2, GPR30, and nuclear receptor coactivator 3 (NCOA3. Pregnancy significantly upregulated Cav3.1 and downregulated Cav3.2, Cav3.3, ESR1, ESR2, and NCOA3, compared to the non-pregnant condition. Beta-estradiol treatment (24 h, 10, 100, 1000 nM downregulated Cav3.2, Cav3.3, Cav1.2, ESR1, ESR2, GRP30, and NCOA3 in TCs from human pregnant uterine myometrium. We also confirmed the functional expression of voltage-gated calcium channels by patch-clamp recordings and calcium imaging analysis of TCs from pregnant human myometrium by perfusing with BAY K8644, which induced calcium influx through these channels. Additionally, we demonstrated that beta-estradiol (1000 nM antagonized the effect of BAY K8644 (2.5 or 5 µM in the same preparations. In conclusion, we evidenced the presence of voltage-gated calcium channels and estrogen receptors in TCs from non-pregnant and pregnant

  4. Beta-Estradiol Regulates Voltage-Gated Calcium Channels and Estrogen Receptors in Telocytes from Human Myometrium.

    Science.gov (United States)

    Banciu, Adela; Banciu, Daniel Dumitru; Mustaciosu, Cosmin Catalin; Radu, Mihai; Cretoiu, Dragos; Xiao, Junjie; Cretoiu, Sanda Maria; Suciu, Nicolae; Radu, Beatrice Mihaela

    2018-05-09

    Voltage-gated calcium channels and estrogen receptors are essential players in uterine physiology, and their association with different calcium signaling pathways contributes to healthy and pathological conditions of the uterine myometrium. Among the properties of the various cell subtypes present in human uterine myometrium, there is increasing evidence that calcium oscillations in telocytes (TCs) contribute to contractile activity and pregnancy. Our study aimed to evaluate the effects of beta-estradiol on voltage-gated calcium channels and estrogen receptors in TCs from human uterine myometrium and to understand their role in pregnancy. For this purpose, we employed patch-clamp recordings, ratiometric Fura-2-based calcium imaging analysis, and qRT-PCR techniques for the analysis of cultured human myometrial TCs derived from pregnant and non-pregnant uterine samples. In human myometrial TCs from both non-pregnant and pregnant uterus, we evidenced by qRT-PCR the presence of genes encoding for voltage-gated calcium channels (Cav3.1, Ca3.2, Cav3.3, Cav2.1), estrogen receptors (ESR1, ESR2, GPR30), and nuclear receptor coactivator 3 (NCOA3). Pregnancy significantly upregulated Cav3.1 and downregulated Cav3.2, Cav3.3, ESR1, ESR2, and NCOA3, compared to the non-pregnant condition. Beta-estradiol treatment (24 h, 10, 100, 1000 nM) downregulated Cav3.2, Cav3.3, Cav1.2, ESR1, ESR2, GRP30, and NCOA3 in TCs from human pregnant uterine myometrium. We also confirmed the functional expression of voltage-gated calcium channels by patch-clamp recordings and calcium imaging analysis of TCs from pregnant human myometrium by perfusing with BAY K8644, which induced calcium influx through these channels. Additionally, we demonstrated that beta-estradiol (1000 nM) antagonized the effect of BAY K8644 (2.5 or 5 µM) in the same preparations. In conclusion, we evidenced the presence of voltage-gated calcium channels and estrogen receptors in TCs from non-pregnant and pregnant human uterine

  5. Oxidizing reagent copper-o-phenanthroline is an open channel blocker of the vanilloid receptor TRPV1

    Czech Academy of Sciences Publication Activity Database

    Toušová, Karolina; Sušánková, Klára; Teisinger, Jan; Vyklický st., Ladislav; Vlachová, Viktorie

    2004-01-01

    Roč. 47, č. 2 (2004), s. 273-285 ISSN 0028-3908 R&D Projects: GA ČR GA305/03/0802; GA ČR GA309/02/1479; GA MŠk LN00B122 Institutional research plan: CEZ:AV0Z5011922 Keywords : vanilloid receptor * TRP channels * capsaicin Subject RIV: ED - Physiology Impact factor: 3.734, year: 2004

  6. Agmatine suppresses peripheral sympathetic tone by inhibiting N-type Ca(2+) channel activity via imidazoline I2 receptor activation.

    Science.gov (United States)

    Kim, Young-Hwan; Jeong, Ji-Hyun; Ahn, Duck-Sun; Chung, Seungsoo

    2016-08-26

    Agmatine, a putative endogenous ligand of imidazoline receptors, suppresses cardiovascular function by inhibiting peripheral sympathetic tone. However, the molecular identity of imidazoline receptor subtypes and its cellular mechanism underlying the agmatine-induced sympathetic suppression remains unknown. Meanwhile, N-type Ca(2+) channels are important for the regulation of NA release in the peripheral sympathetic nervous system. Therefore, it is possible that agmatine suppresses NA release in peripheral sympathetic nerve terminals by inhibiting Ca(2+) influx through N-type Ca(2+) channels. We tested this hypothesis by investigating agmatine effect on electrical field stimulation (EFS)-evoked contraction and NA release in endothelium-denuded rat superior mesenteric arterial strips. We also investigated the effect of agmatine on the N-type Ca(2+) current in superior cervical ganglion (SCG) neurons in rats. Our study demonstrates that agmatine suppresses peripheral sympathetic outflow via the imidazoline I2 receptor in rat mesenteric arteries. In addition, the agmatine-induced suppression of peripheral vascular sympathetic tone is mediated by modulating voltage-dependent N-type Ca(2+) channels in sympathetic nerve terminals. These results suggest a potential cellular mechanism for the agmatine-induced suppression of peripheral sympathetic tone. Furthermore, they provide basic and theoretical information regarding the development of new agents to treat hypertension. Copyright © 2016 Elsevier Inc. All rights reserved.

  7. The Pyrexia transient receptor potential channel mediates circadian clock synchronization to low temperature cycles in Drosophila melanogaster.

    Science.gov (United States)

    Wolfgang, Werner; Simoni, Alekos; Gentile, Carla; Stanewsky, Ralf

    2013-10-07

    Circadian clocks are endogenous approximately 24 h oscillators that temporally regulate many physiological and behavioural processes. In order to be beneficial for the organism, these clocks must be synchronized with the environmental cycles on a daily basis. Both light : dark and the concomitant daily temperature cycles (TCs) function as Zeitgeber ('time giver') and efficiently entrain circadian clocks. The temperature receptors mediating this synchronization have not been identified. Transient receptor potential (TRP) channels function as thermo-receptors in animals, and here we show that the Pyrexia (Pyx) TRP channel mediates temperature synchronization in Drosophila melanogaster. Pyx is expressed in peripheral sensory organs (chordotonal organs), which previously have been implicated in temperature synchronization. Flies deficient for Pyx function fail to synchronize their behaviour to TCs in the lower range (16-20°C), and this deficit can be partially rescued by introducing a wild-type copy of the pyx gene. Synchronization to higher TCs is not affected, demonstrating a specific role for Pyx at lower temperatures. In addition, pyx mutants speed up their clock after being exposed to TCs. Our results identify the first TRP channel involved in temperature synchronization of circadian clocks.

  8. Dynamics of receptor-operated Ca(2+) currents through TRPC channels controlled via the PI(4,5)P2-PLC signaling pathway.

    Science.gov (United States)

    Mori, Masayuki X; Itsuki, Kyohei; Hase, Hideharu; Sawamura, Seishiro; Kurokawa, Tatsuki; Mori, Yasuo; Inoue, Ryuji

    2015-01-01

    Transient receptor potential canonical (TRPC) channels are Ca(2+)-permeable, nonselective cation channels that carry receptor-operated Ca(2+) currents (ROCs) triggered by receptor-induced, phospholipase C (PLC)-catalyzed hydrolysis of phosphatidylinositol 4,5-bisphosphate [PI(4,5)P2]. Within the vasculature, TRPC channel ROCs contribute to smooth muscle cell depolarization, vasoconstriction, and vascular remodeling. However, TRPC channel ROCs exhibit a variable response to receptor-stimulation, and the regulatory mechanisms governing TRPC channel activity remain obscure. The variability of ROCs may be explained by their complex regulation by PI(4,5)P2 and its metabolites, which differentially affect TRPC channel activity. To resolve the complex regulation of ROCs, the use of voltage-sensing phosphoinositide phosphatases and model simulation have helped to reveal the time-dependent contribution of PI(4,5)P2 and the possible role of PI(4,5)P2 in the regulation of ROCs. These approaches may provide unprecedented insight into the dynamics of PI(4,5)P2 regulation of TRPC channels and the fundamental mechanisms underlying transmembrane ion flow. Within that context, we summarize the regulation of TRPC channels and their coupling to receptor-mediated signaling, as well as the application of voltage-sensing phosphoinositide phosphatases to this research. We also discuss the controversial bidirectional effects of PI(4,5)P2 using a model simulation that could explain the complicated effects of PI(4,5)P2 on different ROCs.

  9. Structural domains required for channel function of the mouse transient receptor potential protein homologue TRP1beta.

    Science.gov (United States)

    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.

  10. Revealing dynamically-organized receptor ion channel clusters in live cells by a correlated electric recording and super-resolution single-molecule imaging approach.

    Science.gov (United States)

    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.

  11. Voltage dependent anion channel-1 regulates death receptor mediated apoptosis by enabling cleavage of caspase-8

    International Nuclear Information System (INIS)

    Chacko, Alex D; Liberante, Fabio; Paul, Ian; Longley, Daniel B; Fennell, Dean A

    2010-01-01

    Activation of the extrinsic apoptosis pathway by tumour necrosis factor related apoptosis inducing ligand (TRAIL) is a novel therapeutic strategy for treating cancer that is currently under clinical evaluation. Identification of molecular biomarkers of resistance is likely to play an important role in predicting clinical anti tumour activity. The involvement of the mitochondrial type 1 voltage dependent anion channel (VDAC1) in regulating apoptosis has been highly debated. To date, a functional role in regulating the extrinsic apoptosis pathway has not been formally excluded. We carried out stable and transient RNAi knockdowns of VDAC1 in non-small cell lung cancer cells, and stimulated the extrinsic apoptotic pathway principally by incubating cells with the death ligand TRAIL. We used in-vitro apoptotic and cell viability assays, as well as western blot for markers of apoptosis, to demonstrate that TRAIL-induced toxicity is VDAC1 dependant. Confocal microscopy and mitochondrial fractionation were used to determine the importance of mitochondria for caspase-8 activation. Here we show that either stable or transient knockdown of VDAC1 is sufficient to antagonize TRAIL mediated apoptosis in non-small cell lung cancer (NSCLC) cells. Specifically, VDAC1 is required for processing of procaspase-8 to its fully active p18 form at the mitochondria. Loss of VDAC1 does not alter mitochondrial sensitivity to exogenous caspase-8-cleaved BID induced mitochondrial depolarization, even though VDAC1 expression is essential for TRAIL dependent activation of the intrinsic apoptosis pathway. Furthermore, expression of exogenous VDAC1 restores the apoptotic response to TRAIL in cells in which endogenous VDAC1 has been selectively silenced. Expression of VDAC1 is required for full processing and activation of caspase-8 and supports a role for mitochondria in regulating apoptosis signaling via the death receptor pathway

  12. Voltage-Dependent Inhibition of Glycine Receptor Channels by Niflumic Acid

    Directory of Open Access Journals (Sweden)

    Galyna Maleeva

    2017-05-01

    Full Text Available Niflumic acid (NFA is a member of the fenamate class of nonsteroidal anti-inflammatory drugs. This compound and its derivatives are used worldwide clinically for the relief of chronic and acute pain. NFA is also a commonly used blocker of voltage-gated chloride channels. Here we present evidence that NFA is an efficient blocker of chloride-permeable glycine receptors (GlyRs with subunit heterogeneity of action. Using the whole-cell configuration of patch-clamp recordings and molecular modeling, we analyzed the action of NFA on homomeric α1ΔIns, α2B, α3L, and heteromeric α1β and α2β GlyRs expressed in CHO cells. NFA inhibited glycine-induced currents in a voltage-dependent manner and its blocking potency in α2 and α3 GlyRs was higher than that in α1 GlyR. The Woodhull analysis suggests that NFA blocks α1 and α2 GlyRs at the fractional electrical distances of 0.16 and 0.65 from the external membrane surface, respectively. Thus, NFA binding site in α1 GlyR is closer to the external part of the membrane, while in α2 GlyR it is significantly deeper in the pore. Mutation G254A at the cytoplasmic part of the α1 GlyR pore-lining TM2 helix (level 2′ increased the NFA blocking potency, while incorporation of the β subunit did not have a significant effect. The Hill plot analysis suggests that α1 and α2 GlyRs are preferably blocked by two and one NFA molecules, respectively. Molecular modeling using Monte Carlo energy minimizations provides the structural rationale for the experimental data and proposes more than one interaction site along the pore where NFA can suppress the ion permeation.

  13. Regulation of 1, 4, 5-triphosphate receptor channel gating dynamics by mutant presenilin in Alzheimer's disease cells

    Science.gov (United States)

    Wei, Fang; Li, Xiang; Cai, Meichun; Liu, Yanping; Jung, Peter; Shuai, Jianwei

    2017-06-01

    In neurons of patients with Alzheimer's disease, the intracellular Ca2+ concentration is increased by its release from the endoplasmic reticulum via the inositol 1, 4, 5-triphosphate receptor (IP3R). In this paper, we discuss the IP3R gating dynamics in familial Alzheimer's disease (FAD) cells induced with presenilin mutation PS1. By fitting the parameters of an IP3R channel model to experimental data of the open probability, the mean open time and the mean closed time of IP3R channels, in control cells and FAD mutant cells, we suggest that the interaction of presenilin mutation PS1 with IP3R channels leads the decrease in the unbinding rates of IP3 and the activating Ca2+ from IP3Rs. As a result, the increased affinities of IP3 and activating Ca2+ for IP3R channels induce the increase in the Ca2+ signal in FAD mutant cells. Specifically, the PS1 mutation decreases the IP3 dissociation rate of IP3R channels significantly in FAD mutant cells. Our results suggest possible novel targets for FAD therapeutic intervention.

  14. Distribution profiles of transient receptor potential melastatin- and vanilloid-related channels in rat spermatogenic cells and sperm.

    Science.gov (United States)

    Li, Shilin; Wang, Xinghuan; Ye, Haixia; Gao, Weicheng; Pu, Xiaoyong; Yang, Zhonghua

    2010-03-01

    In the present study, we aimed to investigate the expression and distribution of transient receptor potential melastatin (TRPM)- and vanilloid (TRPV)- related channels in rat spermatogenic cells and spermatozoa. Spermatogenic cells and spermatozoa were obtained from male Sprague-Dawley rats. Reverse transcription polymerase chain reaction (RT-PCR) were used to detect the expression of all TRPM and TRPV channel members with specific primers. Western blot analysis was applied for detecting the expression of TRPM and TRPV channel proteins. Immunohistochemistry staining for TRPM4, TRPM7 and TRPV5 was also performed in rat testis. The mRNAs of TRPM3, TRPM4, TRPM7 and TRPV5 were detected in the spermatogenic cells and spermatozoa in rat. Western blot analysis verified the expression of TRPM4, TRPM7 and TRPV5 in the rat spermatogenic cells and spermatozoa. Immunocytochemistry staining for TRPM and TRPV channel families indicated that TRPM4 and TRPM7 proteins were highly expressed in different stages of spermatogenic cells and spermatozoa, while TRPV5 protein was lowly expressed in these cells. Our results demonstrate that mRNAs or proteins for TRPM3, TRPM4, TRPM7 and TRPV5 exist in rat spermatogenic cells and spermatozoa. These data presented here may assist in elucidating the possible physiological function of TRPM and TRPV channels in spermatogenic cells and spermatozoa.

  15. Canonical transient receptor potential channel 2 (TRPC2): old name-new games. Importance in regulating of rat thyroid cell physiology.

    Science.gov (United States)

    Törnquist, Kid; Sukumaran, Pramod; Kemppainen, Kati; Löf, Christoffer; Viitanen, Tero

    2014-11-01

    In addition to the TSH-cyclic AMP signalling pathway, calcium signalling is of crucial importance in thyroid cells. Although the importance of calcium signalling has been thoroughly investigated for several decades, the nature of the calcium channels involved in signalling is unknown. In a recent series of investigations using the well-studied rat thyroid FRTL-5 cell line, we showed that these cells exclusively express the transient receptor potential canonical 2 (TRPC2) channel. Our results suggested that the TRPC2 channel is of significant importance in regulating thyroid cell function. These investigations were the first to show that thyroid cells express a member of the TRPC family of ion channels. In this review, we will describe the importance of the TRPC2 channel in regulating TSH receptor expression, thyroglobulin maturation, intracellular calcium and iodide homeostasis and that the channel also regulates thyroid cell proliferation.

  16. Gating at the mouth of the acetylcholine receptor channel: energetic consequences of mutations in the alphaM2-cap.

    Directory of Open Access Journals (Sweden)

    Pallavi A Bafna

    2008-06-01

    Full Text Available Gating of nicotinic acetylcholine receptors from a C(losed to an O(pen conformation is the initial event in the postsynaptic signaling cascade at the vertebrate nerve-muscle junction. Studies of receptor structure and function show that many residues in this large, five-subunit membrane protein contribute to the energy difference between C and O. Of special interest are amino acids located at the two transmitter binding sites and in the narrow region of the channel, where CO gating motions generate a lowhigh change in the affinity for agonists and in the ionic conductance, respectively. We have measured the energy changes and relative timing of gating movements for residues that lie between these two locations, in the C-terminus of the pore-lining M2 helix of the alpha subunit ('alphaM2-cap'. This region contains a binding site for non-competitive inhibitors and a charged ring that influences the conductance of the open pore. alphaM2-cap mutations have large effects on gating but much smaller effects on agonist binding, channel conductance, channel block and desensitization. Three alphaM2-cap residues (alphaI260, alphaP265 and alphaS268 appear to move at the outset of channel-opening, about at the same time as those at the transmitter binding site. The results suggest that the alphaM2-cap changes its secondary structure to link gating motions in the extracellular domain with those in the channel that regulate ionic conductance.

  17. Role of aryl hydrocarbon receptor nuclear translocator in KATP channel-mediated insulin secretion in INS-1 insulinoma cells

    International Nuclear Information System (INIS)

    Kim, Ji-Seon; Zheng Haifeng; Kim, Sung Joon; Park, Jong-Wan; Park, Kyong Soo; Ho, Won-Kyung; Chun, Yang-Sook

    2009-01-01

    Aryl hydrocarbon receptor nuclear translocator (ARNT) has been known to participate in cellular responses to xenobiotic and hypoxic stresses, as a common partner of aryl hydrocarbon receptor and hypoxia inducible factor-1/2α. Recently, it was reported that ARNT is essential for adequate insulin secretion in response to glucose input and that its expression is downregulated in the pancreatic islets of diabetic patients. In the present study, the authors addressed the mechanism by which ARNT regulates insulin secretion in the INS-1 insulinoma cell line. In ARNT knock-down cells, basal insulin release was elevated, but insulin secretion was not further stimulated by a high-glucose challenge. Electrophysiological analyses revealed that glucose-dependent membrane depolarization was impaired in these cells. Furthermore, K ATP channel activity and expression were reduced. Of two K ATP channel subunits, Kir6.2 was found to be positively regulated by ARNT at the mRNA and protein levels. Based on these results, the authors suggest that ARNT expresses K ATP channel and by so doing regulates glucose-dependent insulin secretion.

  18. Role of Transient Receptor Potential Ankyrin 1 Ion Channel and Somatostatin sst4 Receptor in the Antinociceptive and Anti-inflammatory Effects of Sodium Polysulfide and Dimethyl Trisulfide

    Directory of Open Access Journals (Sweden)

    István Z. Bátai

    2018-02-01

    Full Text Available Transient receptor potential ankyrin 1 (TRPA1 non-selective ligand-gated cation channels are mostly expressed in primary sensory neurons. Polysulfides (POLYs are Janus-faced substances interacting with numerous target proteins and associated with both protective and detrimental processes. Activation of TRPA1 in sensory neurons, consequent somatostatin (SOM liberation and action on sst4 receptors have recently emerged as mediators of the antinociceptive effect of organic trisulfide dimethyl trisulfide (DMTS. In the frame of the present study, we set out to compare the participation of this mechanism in antinociceptive and anti-inflammatory effects of inorganic sodium POLY and DMTS in carrageenan-evoked hind-paw inflammation. Inflammation of murine hind paws was induced by intraplantar injection of carrageenan (3% in 30 µL saline. Animals were treated intraperitoneally with POLY (17 µmol/kg or DMTS (250 µmol/kg or their respective vehicles 30 min prior paw challenge and six times afterward every 60 min. Mechanical pain threshold and swelling of the paws were measured by dynamic plantar aesthesiometry and plethysmometry at 2, 4, and 6 h after initiation of inflammation. Myeloperoxidase (MPO activity in the hind paws were detected 6 h after challenge by luminescent imaging. Mice genetically lacking TRPA1 ion channels, sst4 receptors and their wild-type counterparts were used to examine the participation of these proteins in POLY and DMTS effects. POLY counteracted carrageenan-evoked mechanical hyperalgesia in a TRPA1 and sst4 receptor-dependent manner. POLY did not influence paw swelling and MPO activity. DMTS ameliorated all examined inflammatory parameters. Mitigation of mechanical hyperalgesia and paw swelling by DMTS were mediated through sst4 receptors. These effects were present in TRPA1 knockout animals, too. DMTS inhibited MPO activity with no participation of the sensory neuron–SOM axis. While antinociceptive effects of

  19. Mechanism of action of the insecticides, lindane and fipronil, on glycine receptor chloride channels.

    Science.gov (United States)

    Islam, Robiul; Lynch, Joseph W

    2012-04-01

    Docking studies predict that the insecticides, lindane and fipronil, block GABA(A) receptors by binding to 6' pore-lining residues. However, this has never been tested at any Cys-loop receptor. The neurotoxic effects of these insecticides are also thought to be mediated by GABA(A) receptors, although a recent morphological study suggested glycine receptors mediated fipronil toxicity in zebrafish. Here we investigated whether human α1, α1β, α2 and α3 glycine receptors were sufficiently sensitive to block by either compound as to represent possible neurotoxicity targets. We also investigated the mechanisms by which lindane and fipronil inhibit α1 glycine receptors. Glycine receptors were recombinantly expressed in HEK293 cells and insecticide effects were studied using patch-clamp electrophysiology. Both compounds completely inhibited all tested glycine receptor subtypes with IC(50) values ranging from 0.2-2 µM, similar to their potencies at vertebrate GABA(A) receptors. Consistent with molecular docking predictions, both lindane and fipronil interacted with 6' threonine residues via hydrophobic interactions and hydrogen bonds. In contrast with predictions, we found no evidence for lindane interacting at the 2' level. We present evidence for fipronil binding in a non-blocking mode in the anaesthetic binding pocket, and for lindane as an excellent pharmacological tool for identifying the presence of β subunits in αβ heteromeric glycine receptors. This study implicates glycine receptors as novel vertebrate toxicity targets for fipronil and lindane. Furthermore, lindane interacted with pore-lining 6' threonine residues, whereas fipronil may have both pore and non-pore binding sites. © 2011 The Authors. British Journal of Pharmacology © 2011 The British Pharmacological Society.

  20. Novel role for the transient potential receptor melastatin 4 channel in guinea pig detrusor smooth muscle physiology

    Science.gov (United States)

    Smith, Amy C.; Hristov, Kiril L.; Cheng, Qiuping; Xin, Wenkuan; Parajuli, Shankar P.; Earley, Scott; Malysz, John

    2013-01-01

    Members of the transient receptor potential (TRP) channel superfamily, including the Ca2+-activated monovalent cation-selective TRP melastatin 4 (TRPM4) channel, have been recently identified in the urinary bladder. However, their expression and function at the level of detrusor smooth muscle (DSM) remain largely unexplored. In this study, for the first time we investigated the role of TRPM4 channels in guinea pig DSM excitation-contraction coupling using a multidisciplinary approach encompassing protein detection, electrophysiology, live-cell Ca2+ imaging, DSM contractility, and 9-phenanthrol, a recently characterized selective inhibitor of the TRPM4 channel. Western blot and immunocytochemistry experiments demonstrated the expression of the TRPM4 channel in whole DSM tissue and freshly isolated DSM cells with specific localization on the plasma membrane. Perforated whole cell patch-clamp recordings and real-time Ca2+ imaging experiments with fura 2-AM, both using freshly isolated DSM cells, revealed that 9-phenanthrol (30 μM) significantly reduced the cation current and decreased intracellular Ca2+ levels. 9-Phenanthrol (0.1–30 μM) significantly inhibited spontaneous, 0.1 μM carbachol-induced, 20 mM KCl-induced, and nerve-evoked contractions in guinea pig DSM-isolated strips with IC50 values of 1–7 μM and 70–80% maximum inhibition. 9-Phenanthrol also reduced nerve-evoked contraction amplitude induced by continuous repetitive electrical field stimulation of 10-Hz frequency and shifted the frequency-response curve (0.5–50 Hz) relative to the control. Collectively, our data demonstrate the novel finding that TRPM4 channels are expressed in guinea pig DSM and reveal their critical role in the regulation of guinea pig DSM excitation-contraction coupling. PMID:23302778

  1. Transient receptor potential ankyrin 1 channel localized to non-neuronal airway cells promotes non-neurogenic inflammation

    DEFF Research Database (Denmark)

    Nassini, Romina; Pedretti, Pamela; Moretto, Nadia

    2012-01-01

    The transient receptor potential ankyrin 1 (TRPA1) channel, localized to airway sensory nerves, has been proposed to mediate airway inflammation evoked by allergen and cigarette smoke (CS) in rodents, via a neurogenic mechanism. However the limited clinical evidence for the role of neurogenic...... inflammation in asthma or chronic obstructive pulmonary disease raises an alternative possibility that airway inflammation is promoted by non-neuronal TRPA1.By using Real-Time PCR and calcium imaging, we found that cultured human airway cells, including fibroblasts, epithelial and smooth muscle cells express...... functional TRPA1 channels. By using immunohistochemistry, TRPA1 staining was observed in airway epithelial and smooth muscle cells in sections taken from human airways and lung, and from airways and lung of wild-type, but not TRPA1-deficient mice. In cultured human airway epithelial and smooth muscle cells...

  2. A glycine residue essential for high ivermectin sensitivity in Cys-loop ion channel receptors

    DEFF Research Database (Denmark)

    Lynagh, Timothy; Lynch, Joseph W.

    2010-01-01

    Ivermectin exerts its anthelmintic effect by activating nematode Cys-loop glutamate-gated receptors. Here we show that a glycine residue at a specific transmembrane domain location is essential for high ivermectin sensitivity in both glycine- and glutamate-gated Cys-loop receptors. We also show...

  3. Functional coupling between heterologously expressed dopamine D(2) receptors and KCNQ channels

    DEFF Research Database (Denmark)

    Ljungstrom, Trine; Grunnet, Morten; Jensen, Bo Skaaning

    2003-01-01

    protein of the G(alphai/o) subtype. Cells of the human neuroblastoma line SH-SY5Y were co-transfected transiently with KCNQ4 and D(2L) receptors. Stimulation of D(2L) receptors increased the KCNQ4 current ( n=6) as determined in whole-cell patch-clamp recordings. The specificity of the dopaminergic...

  4. [Application of Brownian dynamics to the description of transmembrane ion flow as exemplified by the chloride channel of glycine receptor].

    Science.gov (United States)

    Boronovskiĭ, S E; Nartsissov, Ia R

    2009-01-01

    Using the Brownian dynamics of the movement of hydrated ion in a viscous water solution, a mathematical model has been built, which describes the transport of charged particles through a single protein pore in a lipid membrane. The dependences of transmembrane ion currents on ion concentrations in solution have been obtained. It was shown that, if the geometry of a membrane pore is identical to that of the inner part of the glycine receptor channel and there is no ion selectivity, then the values of both chloride and sodium currents are not greater than 0.5 pA at the physiological concentrations of these ions. If local charge heterogeneity caused by charged amino acid residues of transmembrane protein segments is included into the model calculations, the chloride current increases to about 3.7 pA, which exceeds more than seven times the value for sodium ions under the conditions of the complex channel geometry in the range of physiological concentrations of ions in the solution. The model takes changes in the density of charge distribution both inside the channel and near the protein surface into account. The alteration of pore geometry can be also considered as a parameter at the researcher's option. Thus, the model appears as an effective tool for the description of transmembrane currents for other types of membrane channels.

  5. The LILI Motif of M3-S2 Linkers Is a Component of the NMDA Receptor Channel Gate

    Czech Academy of Sciences Publication Activity Database

    Ladislav, Marek; Černý, Jiří; Krůšek, Jan; Balík, Aleš; Vyklický ml., Ladislav

    2018-01-01

    Roč. 11, Apr 6 (2018), č. článku 113. ISSN 1662-5099 R&D Projects: GA ČR(CZ) GA17-02300S; GA TA ČR(CZ) TE01020028; GA MZd(CZ) NV15-29370A; GA MŠk(CZ) LQ1604; GA MŠk(CZ) ED1.1.00/02.0109 Institutional support: RVO:67985823 Keywords : glutamate receptor gating * electrophysiology * spontaneous activity * channel open probability * protein block alphabet * molecular modeling Subject RIV: FH - Neurology OBOR OECD: Neurosciences (including psychophysiology Impact factor: 5.076, year: 2016

  6. Block of GABA(A) receptor ion channel by penicillin: electrophysiological and modeling insights toward the mechanism.

    Science.gov (United States)

    Rossokhin, Alexey V; Sharonova, Irina N; Bukanova, Julia V; Kolbaev, Sergey N; Skrebitsky, Vladimir G

    2014-11-01

    GABA(A) receptors (GABA(A)R) mainly mediate fast inhibitory neurotransmission in the central nervous system. Different classes of modulators target GABA(A)R properties. Penicillin G (PNG) belongs to the class of noncompetitive antagonists blocking the open GABA(A)R and is a prototype of β-lactam antibiotics. In this study, we combined electrophysiological and modeling approaches to investigate the peculiarities of PNG blockade of GABA-activated currents recorded from isolated rat Purkinje cells and to predict the PNG binding site. Whole-cell patch-сlamp recording and fast application system was used in the electrophysiological experiments. PNG block developed after channel activation and increased with membrane depolarization suggesting that the ligand binds within the open channel pore. PNG blocked stationary component of GABA-activated currents in a concentration-dependent manner with IC50 value of 1.12mM at -70mV. The termination of GABA and PNG co-application was followed by a transient tail current. Protection of the tail current from bicuculline block and dependence of its kinetic parameters on agonist affinity suggest that PNG acts as a sequential open channel blocker that prevents agonist dissociation while the channel remains blocked. We built the GABA(A)R models based on nAChR and GLIC structures and performed an unbiased systematic search of the PNG binding site. Monte-Carlo energy minimization was used to find the lowest energy binding modes. We have shown that PNG binds close to the intracellular vestibule. In both models the maximum contribution to the energy of ligand-receptor interactions revealed residues located on the level of 2', 6' and 9' rings formed by a bundle of M2 transmembrane segments, indicating that these residues most likely participate in PNG binding. The predicted structural models support the described mechanism of PNG block. Copyright © 2014 Elsevier Inc. All rights reserved.

  7. Plant-derived cannabinoids modulate the activity of transient receptor potential channels of ankyrin type-1 and melastatin type-8.

    Science.gov (United States)

    De Petrocellis, Luciano; Vellani, Vittorio; Schiano-Moriello, Aniello; Marini, Pietro; Magherini, Pier Cosimo; Orlando, Pierangelo; Di Marzo, Vincenzo

    2008-06-01

    The plant cannabinoids (phytocannabinoids), cannabidiol (CBD), and Delta(9)-tetrahydrocannabinol (THC) were previously shown to activate transient receptor potential channels of both vanilloid type 1 (TRPV1) and ankyrin type 1 (TRPA1), respectively. Furthermore, the endocannabinoid anandamide is known to activate TRPV1 and was recently found to antagonize the menthol- and icilin-sensitive transient receptor potential channels of melastatin type 8 (TRPM8). In this study, we investigated the effects of six phytocannabinoids [i.e., CBD, THC, CBD acid, THC acid, cannabichromene (CBC), and cannabigerol (CBG)] on TRPA1- and TRPM8-mediated increase in intracellular Ca2+ in either HEK-293 cells overexpressing the two channels or rat dorsal root ganglia (DRG) sensory neurons. All of the compounds tested induced TRPA1-mediated Ca2+ elevation in HEK-293 cells with efficacy comparable with that of mustard oil isothiocyanates (MO), the most potent being CBC (EC(50) = 60 nM) and the least potent being CBG and CBD acid (EC(50) = 3.4-12.0 microM). CBC also activated MO-sensitive DRG neurons, although with lower potency (EC(50) = 34.3 microM). Furthermore, although none of the compounds tested activated TRPM8-mediated Ca2+ elevation in HEK-293 cells, they all, with the exception of CBC, antagonized this response when it was induced by either menthol or icilin. CBD, CBG, THC, and THC acid were equipotent (IC(50) = 70-160 nM), whereas CBD acid was the least potent compound (IC(50) = 0.9-1.6 microM). CBG inhibited Ca2+ elevation also in icilin-sensitive DRG neurons with potency (IC(50) = 4.5 microM) similar to that of anandamide (IC(50) = 10 microM). Our findings suggest that phytocannabinoids and cannabis extracts exert some of their pharmacological actions also by interacting with TRPA1 and TRPM8 channels, with potential implications for the treatment of pain and cancer.

  8. Activation of the Ca2+-sensing receptors increases currents through inward rectifier K+ channels via activation of phosphatidylinositol 4-kinase.

    Science.gov (United States)

    Liu, Chung-Hung; Chang, Hsueh-Kai; Lee, Sue-Ping; Shieh, Ru-Chi

    2016-11-01

    Inward rectifier K + channels are important for maintaining normal electrical function in many cell types. The proper function of these channels requires the presence of membrane phosphoinositide 4,5-bisphosphate (PIP 2 ). Stimulation of the Ca 2+ -sensing receptor CaR, a pleiotropic G protein-coupled receptor, activates both G q/11 , which decreases PIP 2 , and phosphatidylinositol 4-kinase (PI-4-K), which, conversely, increases PIP 2 . How membrane PIP 2 levels are regulated by CaR activation and whether these changes modulate inward rectifier K + are unknown. In this study, we found that activation of CaR by the allosteric agonist, NPSR568, increased inward rectifier K + current (I K1 ) in guinea pig ventricular myocytes and currents mediated by Kir2.1 channels exogenously expressed in HEK293T cells with a similar sensitivity. Moreover, using the fluorescent PIP 2 reporter tubby-R332H-cYFP to monitor PIP 2 levels, we found that CaR activation in HEK293T cells increased membrane PIP 2 concentrations. Pharmacological studies showed that both phospholipase C (PLC) and PI-4-K are activated by CaR stimulation with the latter played a dominant role in regulating membrane PIP 2 and, thus, Kir currents. These results provide the first direct evidence that CaR activation upregulates currents through inward rectifier K + channels by accelerating PIP 2 synthesis. The regulation of I K1 plays a critical role in the stability of the electrical properties of many excitable cells, including cardiac myocytes and neurons. Further, synthetic allosteric modulators that increase CaR activity have been used to treat hyperparathyroidism, and negative CaR modulators are of potential importance in the treatment of osteoporosis. Thus, our results provide further insight into the roles played by CaR in the cardiovascular system and are potentially valuable for heart disease treatment and drug safety.

  9. Transcranial Random Noise Stimulation-induced plasticity is NMDA-receptor independent but sodium-channel blocker and benzodiazepines sensitive

    Directory of Open Access Journals (Sweden)

    Leila eChaieb

    2015-04-01

    Full Text Available Background: Application of transcranial random noise stimulation (tRNS between 0.1 and 640 Hz of the primary motor cortex (M1 for 10 minutes induces a persistent excitability increase lasting for at least 60 minutes. However, the mechanism of tRNS-induced cortical excitability alterations is not yet fully understood. Objective: The main aim of this study was to get first efficacy data with regard to the possible neuronal effect of tRNS. Methods: Single-pulse transcranial magnetic stimulation (TMS was used to measure levels of cortical excitability before and after combined application of tRNS at an intensity of 1mA for 10mins stimulation duration and a pharmacological agent (or sham on 8 healthy male participants. Results: The sodium channel blocker carbamazepine showed a tendency towards inhibiting MEPs 5-60 mins poststimulation. The GABAA agonist lorazepam suppressed tRNS-induced cortical excitability increases at 0-20 and 60 min time points. The partial NMDA receptor agonist D-cycloserine, the NMDA receptor antagonist dextromethorphan and the D2/D3 receptor agonist ropinirole had no significant effects on the excitability increases seen with tRNS.Conclusions: In contrast to transcranial direct current stimulation (tDCS, aftereffects of tRNS are seem to be not NMDA receptor dependent and can be suppressed by benzodiazepines suggesting that tDCS and tRNS depend upon different mechanisms.

  10. Voltage-dependent neuromodulation of Na+ channels by D1-like dopamine receptors in rat hippocampal neurons.

    Science.gov (United States)

    Cantrell, A R; Scheuer, T; Catterall, W A

    1999-07-01

    Activation of D1-like dopamine (DA) receptors reduces peak Na+ current in acutely isolated hippocampal neurons through phosphorylation of the alpha subunit of the Na+ channel by cAMP-dependent protein kinase (PKA). Here we report that neuromodulation of Na+ currents by DA receptors via PKA is voltage-dependent in the range of -110 to -70 mV and is also sensitive to concurrent activation of protein kinase C (PKC). Depolarization enhanced the ability of D1-like DA receptors to reduce peak Na+ currents via the PKA pathway. Similar voltage-dependent modulation was observed when PKA was activated directly with the membrane-permeant PKA activator DCl-cBIMPS (cBIMPS; 20 microM), indicating that the membrane potential dependence occurs downstream of PKA. PKA activation caused only a small (-2.9 mV) shift in the voltage dependence of steady-state inactivation and had no effect on slow inactivation or on the rates of entry into the fast or slow inactivated states, suggesting that another mechanism is responsible for coupling of membrane potential changes to PKA modulation. Activation of PKC with a low concentration of the membrane-permeant diacylglycerol analog oleylacetyl glycerol also potentiated modulation by SKF 81297 or cBIMPS, and these effects were most striking at hyperpolarized membrane potentials where PKA modulation was not stimulated by membrane depolarization. Thus, activation of D1-like DA receptors causes a strong reduction in Na+ current via the PKA pathway, but it is effective primarily when it is combined with depolarization or activation of PKC. The convergence of these three distinct signaling modalities on the Na+ channel provides an intriguing mechanism for integration of information from multiple signaling pathways in the hippocampus and CNS.

  11. Increased rhythmicity in hypertensive arterial smooth muscle is linked to transient receptor potential canonical channels

    DEFF Research Database (Denmark)

    Chen, Xiaoping; Yang, Dachun; Ma, Shuangtao

    2010-01-01

    Vasomotion describes oscillations of arterial vascular tone due to synchronized changes of intracellular calcium concentrations. Since increased calcium influx into vascular smooth muscle cells from spontaneously hypertensive rats (SHR) has been associated with variances of transient receptor pot...

  12. Schedule of NMDA receptor subunit expression and functional channel formation in the course of in vitro-induced neurogenesis.

    Science.gov (United States)

    Varju, P; Schlett, K; Eisel, U; Madarász, E

    2001-06-01

    NE-7C2 neuroectodermal cells derived from forebrain vesicles of p53-deficient mouse embryos (E9) produce neurons and astrocytes in vitro if induced by all-trans retinoic acid. The reproducible morphological stages of neurogenesis were correlated with the expression of various NMDA receptor subunits. RT-PCR studies revealed that GluRepsilon1 and GluRepsilon4 subunit mRNAs were transcribed by both non-induced and neuronally differentiated cells. GluRepsilon3 subunit mRNAs were not synthesized by NE-7C2 cells and increased numbers of messages from the GluRepsilon2 gene were detected only after neural network formation. The presence of the GluRzeta1 protein was detected throughout neural induction, whereas retinoic acid-induced neuron formation elevated the amount of exon 21 (C1)- and exon 22 (C2)-containing GluRzeta1 mRNAs and resulted in the appearance of exon 5 (N1)-containing transcripts. NMDA-elicited Ca(2+)-signals were detected only in cells displaying neuronal morphology, but preceding the appearance of synapsin-I immunoreactivity. Our findings demonstrated that, in spite of the presence of subunits necessary for channel formation, functional channels were formed by NE-7C2 cells no sooner than the time of neurite maturation. The data show that the cell line provides a suitable model to analyse the mechanisms involved in NMDA receptor gene expression before the appearance of synaptic communication.

  13. Sildenafil prevents the up-regulation of transient receptor potential canonical channels in the development of cardiomyocyte hypertrophy

    International Nuclear Information System (INIS)

    Kiso, Hironori; Ohba, Takayoshi; Iino, Kenji; Sato, Kazuhiro; Terata, Yutaka; Murakami, Manabu; Ono, Kyoichi; Watanabe, Hiroyuki; Ito, Hiroshi

    2013-01-01

    Highlights: •Transient receptor potential canonical (TRPC1, 3 and 6) are up-regulated by ET-1. •Sildenafil inhibited hypertrophic responses (BNP, Ca entry, NFAT activation). •Sildenafil suppressed TRPC1, 3 and 6 expression. -- Abstract: Background: Transient receptor potential canonical (TRPCs) channels are up-regulated in the development of cardiac hypertrophy. Sildenafil inhibits TRPC6 activation and expression, leading to the prevention of cardiac hypertrophy. However, the effects of sildenafil on the expression of other TRPCs remain unknown. We hypothesized that in addition to its effects of TRPC6, sildenafil blocks the up-regulation of other TRPC channels to suppress cardiomyocyte hypertrophy. Methods and results: In cultured neonatal rat cardiomyocytes, a 48 h treatment with 10 nM endothelin (ET)-1 induced hypertrophic responses characterized by nuclear factor of activated T cells activation and enhancement of brain natriuretic peptide expression and cell surface area. Co-treatment with sildenafil (1 μM, 48 h) inhibited these ET-1-induced hypertrophic responses. Although ET-1 enhanced the gene expression of TRPCs, sildenafil inhibited the enhanced gene expression of TRPC1, C3 and C6. Moreover, co-treatment with sildenafil abolished the augmentation of SOCE in the hypertrophied cardiomyocytes. Conclusions: These results suggest that sildenafil inhibits cardiomyocyte hypertrophy by suppressing the up-regulation of TRPC expression

  14. P2X receptor-ion channels in the inflammatory response in adipose tissue and pancreas-potential triggers in onset of type 2 diabetes?

    DEFF Research Database (Denmark)

    Novak, Ivana; Solini, Anna

    2018-01-01

    -cell and adipose tissue. In the former, P2Y and possibly some P2X receptors-ion channels regulate insulin secretion, but it is still debated whether excessive ATP can via P2X receptors impair β-cell function directly or whether cell damage is due to an excessive systemic release of cytokines. In human adipocytes......, the P2X7 receptor promotes the release of inflammatory cytokines, at least in part via inflammasome activation, likely contributing to systemic insulin resistance. This receptor-inflammasome system is also strongly activated in macrophages infiltrating both pancreas and adipose tissue, mediating...

  15. Distribution of voltage-dependent and intracellular Ca2+ channels in submucosal neurons from rat distal colon.

    Science.gov (United States)

    Rehn, Matthias; Bader, Sandra; Bell, Anna; Diener, Martin

    2013-09-01

    We recently observed a bradykinin-induced increase in the cytosolic Ca2+ concentration in submucosal neurons of rat colon, an increase inhibited by blockers of voltage-dependent Ca2+ (Ca(v)) channels. As the types of Ca(v) channels used by this part of the enteric nervous system are unknown, the expression of various Ca(v) subunits has been investigated in whole-mount submucosal preparations by immunohistochemistry. Submucosal neurons, identified by a neuronal marker (microtubule-associated protein 2), are immunoreactive for Ca(v)1.2, Ca(v)1.3 and Ca(v)2.2, expression being confirmed by reverse transcription plus the polymerase chain reaction. These data agree with previous observations that the inhibition of L- and N-type Ca2+ currents strongly inhibits the response to bradykinin. However, whole-cell patch-clamp experiments have revealed that bradykinin does not enhance Ca2+ inward currents under voltage-clamp conditions. Consequently, bradykinin does not directly interact with Ca(v) channels. Instead, the kinin-induced Ca2+ influx is caused indirectly by the membrane depolarization evoked by this peptide. As intracellular Ca2+ channels on Ca(2+)-storing organelles can also contribute to Ca2+ signaling, their expression has been investigated by imaging experiments and immunohistochemistry. Inositol 1,4,5-trisphosphate (IP3) receptors (IP3R) have been functionally demonstrated in submucosal neurons loaded with the Ca(2+)-sensitive fluorescent dye, fura-2. Histamine, a typical agonist coupled to the phospholipase C pathway, induces an increase in the fura-2 signal ratio, which is suppressed by 2-aminophenylborate, a blocker of IP3 receptors. The expression of IP3R1 has been confirmed by immunohistochemistry. In contrast, ryanodine, tested over a wide concentration range, evokes no increase in the cytosolic Ca2+ concentration nor is there immunohistochemical evidence for the expression of ryanodine receptors in these neurons. Thus, rat submucosal neurons are equipped

  16. Dynamics of receptor-operated Ca2+ Currents Through TRPC Channels Controlled via the PI(4,5P2-PLC Signaling Pathway

    Directory of Open Access Journals (Sweden)

    Masayuki X Mori

    2015-02-01

    Full Text Available Transient receptor potential canonical (TRPC channels are Ca2+-permeable, nonselective cation channels that carry receptor-operated Ca2+ currents (ROCs triggered by receptor-induced, phospholipase C (PLC-catalyzed hydrolysis of phosphatidylinositol 4,5-bisphosphate (PI(4,5P2. Within the vasculature, TRPC channel ROCs contribute to smooth muscle cell depolarization, vasoconstriction and vascular remodeling. However, TRPC channel ROCs exhibit a variable response to receptor-stimulation, and the regulatory mechanisms governing TRPC channel activity remain obscure. The variability of ROCs may be explained by their complex regulation by PI(4,5P2 and its metabolites, which differentially affect TRPC channel activity. To resolve the complex regulation of ROCs, the use of voltage-sensing phosphoinositide phosphatases and model simulation have helped to reveal the time-dependent contribution of PI(4,5P2 and the possible role of PI(4,5P2 in the regulation of ROCs. These approaches may provide unprecedented insight into the dynamics of PI(4,5P2 regulation of TRPC channels and the fundamental mechanisms underlying transmembrane ion flow. Within that context, we summarize the regulation of TRPC channels and their coupling to receptor-mediated signaling, as well as the application of voltage-sensing phosphoinositide phosphatases to this research. We also discuss the controversial bidirectional effects of PI(4,5P2 using a model simulation that could explain the complicated effects of PI(4,5P2 on different ROCs.

  17. The stress protein heat shock cognate 70 (Hsc70) inhibits the Transient Receptor Potential Vanilloid type 1 (TRPV1) channel.

    Science.gov (United States)

    Iftinca, Mircea; Flynn, Robyn; Basso, Lilian; Melo, Helvira; Aboushousha, Reem; Taylor, Lauren; Altier, Christophe

    2016-01-01

    Specialized cellular defense mechanisms prevent damage from chemical, biological, and physical hazards. The heat shock proteins have been recognized as key chaperones that maintain cell survival against a variety of exogenous and endogenous stress signals including noxious temperature. However, the role of heat shock proteins in nociception remains poorly understood. We carried out an expression analysis of the constitutively expressed 70 kDa heat-shock cognate protein, a member of the stress-induced HSP70 family in lumbar dorsal root ganglia from a mouse model of Complete Freund's Adjuvant-induced chronic inflammatory pain. We used immunolabeling of dorsal root ganglion neurons, behavioral analysis and patch clamp electrophysiology in both dorsal root ganglion neurons and HEK cells transfected with Hsc70 and Transient Receptor Potential Channels to examine their functional interaction in heat shock stress condition. We report an increase in protein levels of Hsc70 in mouse dorsal root ganglia, 3 days post Complete Freund's Adjuvant injection in the hind paw. Immunostaining of Hsc70 was observed in most of the dorsal root ganglion neurons, including the small size nociceptors immunoreactive to the TRPV1 channel. Standard whole-cell patch-clamp technique was used to record Transient Receptor Potential Vanilloid type 1 current after exposure to heat shock. We found that capsaicin-evoked currents are inhibited by heat shock in dorsal root ganglion neurons and transfected HEK cells expressing Hsc70 and TRPV1. Blocking Hsc70 with matrine or spergualin compounds prevented heat shock-induced inhibition of the channel. We also found that, in contrast to TRPV1, both the cold sensor channels TRPA1 and TRPM8 were unresponsive to heat shock stress. Finally, we show that inhibition of TRPV1 depends on the ATPase activity of Hsc70 and involves the rho-associated protein kinase. Our work identified Hsc70 and its ATPase activity as a central cofactor of TRPV1 channel function

  18. Cyclophosphamide-induced cystitis reduces ASIC channel but enhances TRPV1 receptor function in rat bladder sensory neurons.

    Science.gov (United States)

    Dang, Khoa; Bielefeldt, Klaus; Gebhart, G F

    2013-07-01

    Using patch-clamp techniques, we studied the plasticity of acid-sensing ion channels (ASIC) and transient receptor potential V1 (TRPV1) channel function in dorsal root ganglia (DRG) neurons retrogradely labeled from the bladder. Saline (control) or cyclophosphamide (CYP) was given intraperitoneally on days 1, 3, and 5. On day 6, lumbosacral (LS, L6-S2) or thoracolumbar (TL, T13-L2) DRG were removed and dissociated. Bladders and bladder DRG neurons from CYP-treated rats showed signs of inflammation (greater myeloperoxidase activity; lower intramuscular wall pH) and increased size (whole cell capacitance), respectively, compared with controls. Most bladder neurons (>90%) responded to protons and capsaicin. Protons produced multiphasic currents with distinct kinetics, whereas capsaicin always triggered a sustained response. The TRPV1 receptor antagonist A-425619 abolished capsaicin-triggered currents and raised the threshold of heat-activated currents. Prolonged exposure to an acidic environment (pH range: 7.2 to 6.6) inhibited proton-evoked currents, potentiated the capsaicin-evoked current, and reduced the threshold of heat-activated currents in LS and TL bladder neurons. CYP treatment reduced density but not kinetics of all current components triggered by pH 5. In contrast, CYP-treatment was associated with an increased current density in response to capsaicin in LS and TL bladder neurons. Correspondingly, heat triggered current at a significantly lower temperature in bladder neurons from CYP-treated rats compared with controls. These results reveal that cystitis differentially affects TRPV1- and ASIC-mediated currents in both bladder sensory pathways. Acidification of the bladder wall during inflammation may contribute to changes in nociceptive transmission mediated through the TRPV1 receptor, suggesting a role for TRPV1 in hypersensitivity associated with cystitis.

  19. Investigation of the transient receptor potential vanilloid 1 (TRPV1) ion channel

    OpenAIRE

    Winter Zoltán

    2013-01-01

    The aims of this research were to determine sensory modalities that may be lost after the RTX treatment of newborn or adult mice, to dissect potential side-effect(s) of molecular neurosurgery, to gather information about the structure and function of the channel by investigating the effects of M2+ on the TRPV1 and by collecting the literature data on the functionally important point mutations of the channel for prospective in silico modeling. The findings of the research work can be summa...

  20. TRPM5, a taste-signaling transient receptor potential ion-channel, is a ubiquitous signaling component in chemosensory cells

    Directory of Open Access Journals (Sweden)

    Hofmann Thomas

    2007-07-01

    Full Text Available Abstract Background A growing number of TRP channels have been identified as key players in the sensation of smell, temperature, mechanical forces and taste. TRPM5 is known to be abundantly expressed in taste receptor cells where it participates in sweet, amino acid and bitter perception. A role of TRPM5 in other sensory systems, however, has not been studied so far. Results Here, we systematically investigated the expression of TRPM5 in rat and mouse tissues. Apart from taste buds, where we found TRPM5 to be predominantly localized on the basolateral surface of taste receptor cells, TRPM5 immunoreactivity was seen in other chemosensory organs – the main olfactory epithelium and the vomeronasal organ. Most strikingly, we found solitary TRPM5-enriched epithelial cells in all parts of the respiratory and gastrointestinal tract. Based on their tissue distribution, the low cell density, morphological features and co-immunostaining with different epithelial markers, we identified these cells as brush cells (also known as tuft, fibrillovesicular, multivesicular or caveolated cells. In terms of morphological characteristics, brush cells resemble taste receptor cells, while their origin and biological role are still under intensive debate. Conclusion We consider TRPM5 to be an intrinsic signaling component of mammalian chemosensory organs, and provide evidence for brush cells being an important cellular correlate in the periphery.

  1. An improved ivermectin-activated chloride channel receptor for inhibiting electrical activity in defined neuronal populations

    DEFF Research Database (Denmark)

    Lynagh, Timothy Peter; Lynch, Joseph W

    2010-01-01

    The ability to silence the electrical activity of defined neuronal populations in vivo is dramatically advancing our understanding of brain function. This technology may eventually be useful clinically for treating a variety of neuropathological disorders caused by excessive neuronal activity...... conductance, homomeric expression, and human origin may render the F207A/A288G alpha1 glycine receptor an improved silencing receptor for neuroscientific and clinical purposes. As all known highly ivermectin-sensitive GluClRs contain an endogenous glycine residue at the corresponding location, this residue...

  2. Pore Helix Domain Is Critical to Camphor Sensitivity of Transient Receptor Potential Vanilloid 1 Channel

    Czech Academy of Sciences Publication Activity Database

    Maršáková, Lenka; Touška, Filip; Krůšek, Jan; Vlachová, Viktorie

    2012-01-01

    Roč. 116, č. 4 (2012), s. 903-917 ISSN 0003-3022 R&D Projects: GA ČR(CZ) GA305/09/0081; GA ČR(CZ) GA202/09/0806; GA ČR(CZ) GD305/08/H037; GA MŠk(CZ) 1M0517; GA MŠk(CZ) LC554; GA ČR(CZ) GBP304/12/G069 Grant - others:Univerzita Karlova(CZ) 25409 Institutional research plan: CEZ:AV0Z50110509 Keywords : vanilloid receptor * camphor * transient receptor potential Subject RIV: ED - Physiology Impact factor: 5.163, year: 2012

  3. Binding of ArgTX-636 in the NMDA receptor ion channel

    DEFF Research Database (Denmark)

    Poulsen, Mette H; Andersen, Jacob; Christensen, Rune

    2015-01-01

    of NMDAR activity and have therapeutic potential for treatment of a variety of brain diseases or as pharmacological tools for studies of the neurobiological role of NMDARs. We have performed a kinetic analysis of the blocking mechanism of the prototypical polyamine toxin NMDAR ion channel blocker...

  4. The Acetylcholine Receptor and Its Ionic Channel as Targets for Drugs and Toxins

    Science.gov (United States)

    1981-12-10

    mlecular target that can have any number of different binding sites and is able to generate levels of energy barriers which &re a direct function of the...Albuquerque, E.X. XEatrachotoxi.-A 20- a -benzoate: A new radioactive ligand for voltage- sensitive sodium channels. Cell. Mol. Neurobio .. 1: 19-40

  5. Single nucleotide polymorphisms and genotypes of transient receptor potential ion channel and acetylcholine receptor genes from isolated B lymphocytes in myalgic encephalomyelitis/chronic fatigue syndrome patients.

    Science.gov (United States)

    Marshall-Gradisnik, Sonya; Johnston, Samantha; Chacko, Anu; Nguyen, Thao; Smith, Peter; Staines, Donald

    2016-12-01

    Objective The pathomechanism of chronic fatigue syndrome/myalgic encephalomyelitis (CFS/ME) is unknown; however, a small subgroup of patients has shown muscarinic antibody positivity and reduced symptom presentation following anti-CD20 intervention. Given the important roles of calcium (Ca 2+ ) and acetylcholine (ACh) signalling in B cell activation and potential antibody development, we aimed to identify relevant single nucleotide polymorphisms (SNPs) and genotypes in isolated B cells from CFS/ME patients. Methods A total of 11 CFS/ME patients (aged 31.82 ± 5.50 years) and 11 non-fatigued controls (aged 33.91 ± 5.06 years) were included. Flow cytometric protocols were used to determine B cell purity, followed by SNP and genotype analysis for 21 mammalian TRP ion channel genes and nine mammalian ACh receptor genes. SNP association and genotyping analysis were performed using ANOVA and PLINK analysis software. Results Seventy-eight SNPs were identified in nicotinic and muscarinic acetylcholine receptor genes in the CFS/ME group, of which 35 were in mAChM3. The remaining SNPs were identified in nAChR delta (n = 12), nAChR alpha 9 (n = 5), TRPV2 (n = 7), TRPM3 (n = 4), TRPM4 (n = 1) mAChRM3 2 (n = 2), and mAChRM5 (n = 3) genes. Nine genotypes were identified from SNPs in TRPM3 (n = 1), TRPC6 (n = 1), mAChRM3 (n = 2), nAChR alpha 4 (n = 1), and nAChR beta 1 (n = 4) genes, and were located in introns and 3' untranslated regions. Odds ratios for these specific genotypes ranged between 7.11 and 26.67 for CFS/ME compared with the non-fatigued control group. Conclusion This preliminary investigation identified a number of SNPs and genotypes in genes encoding TRP ion channels and AChRs from B cells in patients with CFS/ME. These may be involved in B cell functional changes, and suggest a role for Ca 2+ dysregulation in AChR and TRP ion channel signalling in the pathomechanism of CFS/ME.

  6. Computing characterizations of drugs for ion channels and receptors using Markov models

    CERN Document Server

    Tveito, Aslak

    2016-01-01

    Flow of ions through voltage gated channels can be represented theoretically using stochastic differential equations where the gating mechanism is represented by a Markov model. The flow through a channel can be manipulated using various drugs, and the effect of a given drug can be reflected by changing the Markov model. These lecture notes provide an accessible introduction to the mathematical methods needed to deal with these models. They emphasize the use of numerical methods and provide sufficient details for the reader to implement the models and thereby study the effect of various drugs. Examples in the text include stochastic calcium release from internal storage systems in cells, as well as stochastic models of the transmembrane potential. Well known Markov models are studied and a systematic approach to including the effect of mutations is presented. Lastly, the book shows how to derive the optimal properties of a theoretical model of a drug for a given mutation defined in terms of a Markov model.

  7. Transient receptor potential channels encode volatile chemicals sensed by rat trigeminal ganglion neurons.

    Directory of Open Access Journals (Sweden)

    Matthias Lübbert

    Full Text Available Primary sensory afferents of the dorsal root and trigeminal ganglia constantly transmit sensory information depicting the individual's physical and chemical environment to higher brain regions. Beyond the typical trigeminal stimuli (e.g. irritants, environmental stimuli comprise a plethora of volatile chemicals with olfactory components (odorants. In spite of a complete loss of their sense of smell, anosmic patients may retain the ability to roughly discriminate between different volatile compounds. While the detailed mechanisms remain elusive, sensory structures belonging to the trigeminal system seem to be responsible for this phenomenon. In order to gain a better understanding of the mechanisms underlying the activation of the trigeminal system by volatile chemicals, we investigated odorant-induced membrane potential changes in cultured rat trigeminal neurons induced by the odorants vanillin, heliotropyl acetone, helional, and geraniol. We observed the dose-dependent depolarization of trigeminal neurons upon application of these substances occurring in a stimulus-specific manner and could show that distinct neuronal populations respond to different odorants. Using specific antagonists, we found evidence that TRPA1, TRPM8, and/or TRPV1 contribute to the activation. In order to further test this hypothesis, we used recombinantly expressed rat and human variants of these channels to investigate whether they are indeed activated by the odorants tested. We additionally found that the odorants dose-dependently inhibit two-pore potassium channels TASK1 and TASK3 heterologously expressed In Xenopus laevis oocytes. We suggest that the capability of various odorants to activate different TRP channels and to inhibit potassium channels causes neuronal depolarization and activation of distinct subpopulations of trigeminal sensory neurons, forming the basis for a specific representation of volatile chemicals in the trigeminal ganglia.

  8. Specific profiles of ion channels and ionotropic receptors define adipose- and bone marrow derived stromal cells.

    Czech Academy of Sciences Publication Activity Database

    Forostyak, Oksana; Butenko, Olena; Anděrová, Miroslava; Forostyak, Serhiy; Syková, Eva; Verkhratsky, A.; Dayanithi, Govindan

    2016-01-01

    Roč. 16, č. 3 (2016), s. 622-634 ISSN 1873-5061 R&D Projects: GA ČR(CZ) GA14-34077S; GA ČR(CZ) GAP304/11/2373; GA ČR(CZ) GBP304/12/G069 Institutional support: RVO:68378041 Keywords : adipose derived stromal cells * bone marrow stromal cell * Ca(2+) signaling * Ion channels Subject RIV: FH - Neurology Impact factor: 3.494, year: 2016

  9. Modulation of firing and synaptic transmission of serotonergic neurons by intrinsic G protein-coupled receptors and ion channels

    Directory of Open Access Journals (Sweden)

    Takashi eMaejima

    2013-05-01

    Full Text Available Serotonergic neurons project to virtually all regions of the CNS and are consequently involved in many critical physiological functions such as mood, sexual behavior, feeding, sleep/wake cycle, memory, cognition, blood pressure regulation, breathing and reproductive success. Therefore serotonin release and serotonergic neuronal activity have to be precisely controlled and modulated by interacting brain circuits to adapt to specific emotional and environmental states. We will review the current knowledge about G protein-coupled receptors and ion channels involved in the regulation of serotonergic system, how their regulation is modulating the intrinsic activity of serotonergic neurons and its transmitter release and will discuss the latest methods for controlling the modulation of serotonin release and intracellular signaling in serotonergic neurons in vitro and in vivo.

  10. The Transient Receptor Potential (TRP) Channel Family in Colletotrichum graminicola: A Molecular and Physiological Analysis.

    Science.gov (United States)

    Lange, Mario; Weihmann, Fabian; Schliebner, Ivo; Horbach, Ralf; Deising, Holger B; Wirsel, Stefan G R; Peiter, Edgar

    2016-01-01

    Calcium (Ca2+) is a universal second messenger in all higher organisms and centrally involved in the launch of responses to environmental stimuli. Ca2+ signals in the cytosol are initiated by the activation of Ca2+ channels in the plasma membrane and/or in endomembranes. Yeast (Saccharomyces cerevisiae) contains a Ca2+-permeable channel of the TRP family, TRPY1, which is localized in the vacuolar membrane and contributes to cytosolic free Ca2+ ([Ca2+]cyt) elevations, for example in response to osmotic upshock. A TRPY1 homologue in the rice blast fungus is known to be important for growth and pathogenicity. To determine the role of the TRP channel family in the maize pathogen Colletotrichum graminicola, proteins homologous to TRPY1 were searched. This identified not one, but four genes in the C. graminicola genome, which had putative orthologs in other fungi, and which we named CgTRPF1 through 4. The topology of the CgTRPF proteins resembled that of TRPY1, albeit with a variable number of transmembrane (TM) domains additional to the six-TM-domain core and a diverse arrangement of putatively Ca2+-binding acidic motifs. All CgTRPF genes were expressed in axenic culture and throughout the infection of maize. Like TRPY1, all TRPF proteins of C. graminicola were localized intracellularly, albeit three of them were found not in large vacuoles, but co-localized in vesicular structures. Deletion strains for the CgTRPF genes were not altered in processes thought to involve Ca2+ release from internal stores, i.e. spore germination, the utilization of complex carbon sources, and the generation of tip-focussed [Ca2+]cyt spikes. Heterologous expression of CgTRPF1 through 4 in a tryp1Δ yeast mutant revealed that none of the channels mediated the release of Ca2+ in response to osmotic upshock. Accordingly, aequorin-based [Ca2+]cyt measurements of C. graminicola showed that in this fungus, osmotic upshock-triggered [Ca2+]cyt elevations were generated entirely by influx of Ca2

  11. The Transient Receptor Potential (TRP Channel Family in Colletotrichum graminicola: A Molecular and Physiological Analysis.

    Directory of Open Access Journals (Sweden)

    Mario Lange

    Full Text Available Calcium (Ca2+ is a universal second messenger in all higher organisms and centrally involved in the launch of responses to environmental stimuli. Ca2+ signals in the cytosol are initiated by the activation of Ca2+ channels in the plasma membrane and/or in endomembranes. Yeast (Saccharomyces cerevisiae contains a Ca2+-permeable channel of the TRP family, TRPY1, which is localized in the vacuolar membrane and contributes to cytosolic free Ca2+ ([Ca2+]cyt elevations, for example in response to osmotic upshock. A TRPY1 homologue in the rice blast fungus is known to be important for growth and pathogenicity. To determine the role of the TRP channel family in the maize pathogen Colletotrichum graminicola, proteins homologous to TRPY1 were searched. This identified not one, but four genes in the C. graminicola genome, which had putative orthologs in other fungi, and which we named CgTRPF1 through 4. The topology of the CgTRPF proteins resembled that of TRPY1, albeit with a variable number of transmembrane (TM domains additional to the six-TM-domain core and a diverse arrangement of putatively Ca2+-binding acidic motifs. All CgTRPF genes were expressed in axenic culture and throughout the infection of maize. Like TRPY1, all TRPF proteins of C. graminicola were localized intracellularly, albeit three of them were found not in large vacuoles, but co-localized in vesicular structures. Deletion strains for the CgTRPF genes were not altered in processes thought to involve Ca2+ release from internal stores, i.e. spore germination, the utilization of complex carbon sources, and the generation of tip-focussed [Ca2+]cyt spikes. Heterologous expression of CgTRPF1 through 4 in a tryp1Δ yeast mutant revealed that none of the channels mediated the release of Ca2+ in response to osmotic upshock. Accordingly, aequorin-based [Ca2+]cyt measurements of C. graminicola showed that in this fungus, osmotic upshock-triggered [Ca2+]cyt elevations were generated entirely by

  12. Neurotransmitter Receptors and Their Ionic Channels as Targets for Drugs and Toxins

    Science.gov (United States)

    1985-01-06

    over a period of three months, the glands and venom sacs were removed, frozen in liquid nitrogen then lyophilized. This honey bee wolf (Philanthus...uses a paralytic venom to immobilize honey bee workers and steal their honey . Water extractU oF lvophilized venom sacs and glands injected into honey bee ...specifically with any of these regulatory proteins and to under- stand mechanisms by which these receptors regulate the selective transloca- tion of

  13. Dopamine suppresses neuronal activity of Helisoma B5 neurons via a D2-like receptor, activating PLC and K channels.

    Science.gov (United States)

    Zhong, L R; Artinian, L; Rehder, V

    2013-01-03

    Dopamine (DA) plays fundamental roles as a neurotransmitter and neuromodulator in the central nervous system. How DA modulates the electrical excitability of individual neurons to elicit various behaviors is of great interest in many systems. The buccal ganglion of the freshwater pond snail Helisoma trivolvis contains the neuronal circuitry for feeding and DA is known to modulate the feeding motor program in Helisoma. The buccal neuron B5 participates in the control of gut contractile activity and is surrounded by dopaminergic processes, which are expected to release DA. In order to study whether DA modulates the electrical activity of individual B5 neurons, we performed experiments on physically isolated B5 neurons in culture and on B5 neurons within the buccal ganglion in situ. We report that DA application elicited a strong hyperpolarization in both conditions and turned the electrical activity from a spontaneously firing state to an electrically silent state. Using the cell culture system, we demonstrated that the strong hyperpolarization was inhibited by the D2 receptor antagonist sulpiride and the phospholipase C (PLC) inhibitor U73122, indicating that DA affected the membrane potential of B5 neurons through the activation of a D2-like receptor and PLC. Further studies revealed that the DA-induced hyperpolarization was inhibited by the K channel blockers 4-aminopyridine and tetraethylammonium, suggesting that K channels might serve as the ultimate target of DA signaling. Through its modulatory effect on the electrical activity of B5 neurons, the release of DA in vivo may contribute to a neuronal output that results in a variable feeding motor program. Copyright © 2012 IBRO. Published by Elsevier Ltd. All rights reserved.

  14. Iron mediates N-methyl-D-aspartate receptor-dependent stimulation of calcium-induced pathways and hippocampal synaptic plasticity.

    Science.gov (United States)

    Muñoz, Pablo; Humeres, Alexis; Elgueta, Claudio; Kirkwood, Alfredo; Hidalgo, Cecilia; Núñez, Marco T

    2011-04-15

    Iron deficiency hinders hippocampus-dependent learning processes and impairs cognitive performance, but current knowledge on the molecular mechanisms underlying the unique role of iron in neuronal function is sparse. Here, we investigated the participation of iron on calcium signal generation and ERK1/2 stimulation induced by the glutamate agonist N-methyl-D-aspartate (NMDA), and the effects of iron addition/chelation on hippocampal basal synaptic transmission and long-term potentiation (LTP). Addition of NMDA to primary hippocampal cultures elicited persistent calcium signals that required functional NMDA receptors and were independent of calcium influx through L-type calcium channels or α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptors; NMDA also promoted ERK1/2 phosphorylation and nuclear translocation. Iron chelation with desferrioxamine or inhibition of ryanodine receptor (RyR)-mediated calcium release with ryanodine-reduced calcium signal duration and prevented NMDA-induced ERK1/2 activation. Iron addition to hippocampal neurons readily increased the intracellular labile iron pool and stimulated reactive oxygen species production; the antioxidant N-acetylcysteine or the hydroxyl radical trapper MCI-186 prevented these responses. Iron addition to primary hippocampal cultures kept in calcium-free medium elicited calcium signals and stimulated ERK1/2 phosphorylation; RyR inhibition abolished these effects. Iron chelation decreased basal synaptic transmission in hippocampal slices, inhibited iron-induced synaptic stimulation, and impaired sustained LTP in hippocampal CA1 neurons induced by strong stimulation. In contrast, iron addition facilitated sustained LTP induction after suboptimal tetanic stimulation. Together, these results suggest that hippocampal neurons require iron to generate RyR-mediated calcium signals after NMDA receptor stimulation, which in turn promotes ERK1/2 activation, an essential step of sustained LTP.

  15. In vivo binding and autoradiographic imaging of (+)-3-[125I]Iodo-MK-801 to the NMDA receptor-channel complex in rat brain

    International Nuclear Information System (INIS)

    Gibson, R.E.; Burns, H.D.; Thorpe, H.H.; Waisi Eng; Ransom, R.; Solomon, H.

    1992-01-01

    Radioiodinated (+)-3-Iodo-MK-801 is a high affinity radioligand for the N-methyl-D-aspartate (NMDA) receptor-channel complex. We have demonstrated in vivo localization in the CNS of rat which is stereoselective and blocked by coinjection of unlabeled MK-801. Autoradiography indicates localization in vivo which is in concordance with in vitro autoradiographic studies. These results indicate that radioiodinated (+)-3-Iodo-MK-801 is a useful probe for in vitro and in vivo autoradiographic studies and suggest that radioligands for the NMDA receptor may be developed which will provide in vivo images of receptor distribution in man. (author)

  16. RNA-Sequencing Analyses Demonstrate the Involvement of Canonical Transient Receptor Potential Channels in Rat Tooth Germ Development

    Directory of Open Access Journals (Sweden)

    Jun Yang

    2017-06-01

    Full Text Available Tooth development depends on multiple molecular interactions between the dental epithelium and mesenchyme, which are derived from ectodermal and ectomesenchymal cells, respectively. We report on a systematic RNA sequencing analysis of transcriptional expression levels from the bud to hard tissue formation stages of rat tooth germ development. We found that GNAO1, ENO1, EFNB1, CALM1, SIAH2, ATP6V0A1, KDELR2, GTPBP1, POLR2C, SORT1, and members of the canonical transient receptor potential (TRPC channel family are involved in tooth germ development. Furthermore, Cell Counting Kit 8 (CCK8 and Transwell migration assays were performed to explore the effects of these differentially expressed genes (DEGs on the proliferation and migration of dental pulp stem cells. Immunostaining revealed that TRPC channels are expressed at varying levels during odontogenesis. The identified genes represent novel candidates that are likely to be vital for rat tooth germ development. Together, the results provide a valuable resource to elucidate the gene regulatory mechanisms underlying mammalian tooth germ development.

  17. Transient Receptor Potential Canonical 3 (TRPC3) Channels Are Required for Hypothalamic Glucose Detection and Energy Homeostasis.

    Science.gov (United States)

    Chrétien, Chloé; Fenech, Claire; Liénard, Fabienne; Grall, Sylvie; Chevalier, Charlène; Chaudy, Sylvie; Brenachot, Xavier; Berges, Raymond; Louche, Katie; Stark, Romana; Nédélec, Emmanuelle; Laderrière, Amélie; Andrews, Zane B; Benani, Alexandre; Flockerzi, Veit; Gascuel, Jean; Hartmann, Jana; Moro, Cédric; Birnbaumer, Lutz; Leloup, Corinne; Pénicaud, Luc; Fioramonti, Xavier

    2017-02-01

    The mediobasal hypothalamus (MBH) contains neurons capable of directly detecting metabolic signals such as glucose to control energy homeostasis. Among them, glucose-excited (GE) neurons increase their electrical activity when glucose rises. In view of previous work, we hypothesized that transient receptor potential canonical type 3 (TRPC3) channels are involved in hypothalamic glucose detection and the control of energy homeostasis. To investigate the role of TRPC3, we used constitutive and conditional TRPC3-deficient mouse models. Hypothalamic glucose detection was studied in vivo by measuring food intake and insulin secretion in response to increased brain glucose level. The role of TRPC3 in GE neuron response to glucose was studied by using in vitro calcium imaging on freshly dissociated MBH neurons. We found that whole-body and MBH TRPC3-deficient mice have increased body weight and food intake. The anorectic effect of intracerebroventricular glucose and the insulin secretory response to intracarotid glucose injection are blunted in TRPC3-deficient mice. TRPC3 loss of function or pharmacological inhibition blunts calcium responses to glucose in MBH neurons in vitro. Together, the results demonstrate that TRPC3 channels are required for the response to glucose of MBH GE neurons and the central effect of glucose on insulin secretion and food intake. © 2017 by the American Diabetes Association.

  18. Molecular Docking Analysis of Ginger Active Compound on Transient Receptor Potential Cation Channel Subfamily V Member 1 (TRPV1

    Directory of Open Access Journals (Sweden)

    Fifteen Aprila Fajrin

    2018-02-01

    Full Text Available Ginger had been reported to ameliorate painful diabetic neuropathy (PDN in an animal model. Gingerol and shogaol were active compounds of ginger that potentially act on transient receptor potential cation channel subfamily V member 1 (TRPV1, a key receptor in PDN. This study aims to predict the binding of gingerol and shogaol to TRPV1 using an in silico model. The ligands of the docking study were 3 chemical compounds of each gingerol and shogaol, i.e. 6-shogaol, 8-shogaol, 10-shogaol, 6-gingerol, 8 gingerol and 10-gingerol. Capsaicin, a TRPV1 agonist, was used as a native ligand. The TRPV1 structure was taken from Protein Data Bank (ID 3J9J. The docking analysis was performed using Autodock Vina. The result showed that among the ginger active compounds, 6-shogaol had the strongest binding energy (-7.10 kcal/mol to TRPV1. The 6-shogaol lacked the potential hydrogen bond to Ile265 of TRPV1 protein, which capsacin had. However, it's binding energy towards TRPV1 was not significantly different compared to capsaicin. Therefore, 6-shogaol had potential to be developed as a treatment for PDN.

  19. Intercellular odontoblast communication via ATP mediated by pannexin-1 channel and phospholipase C-coupled receptor activation.

    Directory of Open Access Journals (Sweden)

    Masaki eSato

    2015-11-01

    Full Text Available Extracellular ATP released via pannexin-1 channels, in response to the activation of mechanosensitive-TRP channels during odontoblast mechanical stimulation, mediates intercellular communication among odontoblasts in dental pulp slice preparation dissected form rat incisor. Recently, odontoblast cell lines, such as mouse odontoblast lineage cells, have been widely used to investigate physiological/pathological cellular functions. To clarify whether the odontoblast cell lines also communicate with each other by diffusible chemical substance(s, we investigated the chemical intercellular communication among cells from mouse odontoblast cell lines following mechanical stimulation. A single cell was stimulated using a glass pipette filled with standard extracellular solution. We measured intracellular free Ca2+ concentration ([Ca2+]i by fura-2 in stimulated cells, as well as in cells located nearby. Direct mechanical stimulation to a single odontoblast increased [Ca2+]i, which showed sensitivity to capsazepine. In addition, we observed increases in [Ca2+]i not only in the mechanically stimulated odontoblast, but also in nearby odontoblasts. We could observe mechanical stimulation-induced increase in [Ca2+]i in a stimulated human embryo kidney (HEK 293 cell, but not in nearby HEK293 cells. The increase in [Ca2+]i in nearby odontoblasts, but not in the stimulated odontoblast, was inhibited by adenosine triphosphate (ATP release channel (pannexin-1 inhibitor in a concentration- and spatial-dependent manner. Moreover, in the presence of phospholipase C (PLC inhibitor, the increase in [Ca2+]i in nearby odontoblasts, following mechanical stimulation of a single odontoblast, was abolished. We could record some inward currents evoked from odontoblasts near the stimulated odontoblast, but the currents were observed in only 4.8% of the recorded odontoblasts. The results of this study showed that ATP is released via pannexin-1, from a mechanically stimulated

  20. Molecular Basis of Ion Channels and Receptors Involved in Nerve Excitation, Synaptic Transmission and Muscle Contraction

    Science.gov (United States)

    1993-12-20

    R\\I) (CHARLES A SANDE[RS. Past Chairman {I IElNE 1. KAPLAN . General ,unsel (c-\\ itimol RODNEY W NI(HOI.S. Chief Fiveut,,, Officer [k.x fliui...MUSCARINIC ACETYLCHOLINE RECEPTOR 221 𔃼 -7 acca cr Y. x a ý . a; > -, -~ 2x 1 Ft. 4• ,1; S4" វ - ɜ:4+ 222 ANNALS NEW YORK ACADEMY OF SCIENCES with other...Physiol. 263: C267-C286. 22. KAPLAN , J, H. 1993. Molecular biology of carrier proteins. Cell 72: 13-18. 23. SOEJIMA, M. & A. NOMA. 1984. Mode of

  1. MacMARCKS interacts with the metabotropic glutamate receptor type 7 and modulates G protein-mediated constitutive inhibition of calcium channels

    Czech Academy of Sciences Publication Activity Database

    Bertaso, F.; Lill, Y.; Airas, J.M.; Espeut, J.; Blahoš, Jaroslav; Bockaert, J.; Fagni, L.; Betz, H.; Far, O.E.

    2006-01-01

    Roč. 99, - (2006), s. 288-298 ISSN 0022-3042 R&D Projects: GA ČR GA204/05/0920 Institutional research plan: CEZ:AV0Z50390512 Keywords : calmodulin * metabotropic glutamate receptor * calcium channel Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 4.260, year: 2006

  2. Chemo-nociceptive signalling from the colon is enhanced by mild colitis and blocked by inhibition of transient receptor potential ankyrin 1 channels

    DEFF Research Database (Denmark)

    Mitrovic, Martina; Shahbazian, Anaid; Bock, Elisabeth

    2010-01-01

    Transient receptor potential ankyrin 1 (TRPA1) channels are expressed by primary afferent neurones and activated by irritant chemicals including allyl isothiocyanate (AITC). Here we investigated whether intracolonic AITC causes afferent input to the spinal cord and whether this response is modifi...

  3. Cloning and expression of ligand-gated ion-channel receptor L2 in central nervous system

    International Nuclear Information System (INIS)

    Houtani, Takeshi; Munemoto, Yumi; Kase, Masahiko; Sakuma, Satoru; Tsutsumi, Toshiyuki; Sugimoto, Tetsuo

    2005-01-01

    An orphan receptor of ligand-gated ion-channel type (L2, also termed ZAC according to the presence of zinc ion for channel activation) was identified by computer-assisted search programs on human genome database. The L2 protein shares partial homology with serotonin receptors 5HT3A and 5HT3B. We have cloned L2 cDNA derived from human caudate nucleus and characterized the exon-intron structure as follows: (1) The L2 protein has four transmembrane regions (M1-M4) and a long cytoplasmic loop between M3 and M4. (2) The sequence is conserved in species including chimpanzee, dog, cow, and opossum. (3) Nine exons form its protein-coding region and especially exon 5 corresponds to a disulfide bond region on the amino-terminal side. Our analysis using multiple tissue cDNA panels revealed that at least two splicing variants of L2 mRNA are present. The cDNA PCR amplification study revealed that L2 mRNA is expressed in tissues including brain, pancreas, liver, lung, heart, kidney, and skeletal muscle while 5HT3A mRNA could be detected in brain, heart, placenta, lung, kidney, pancreas, and skeletal muscle, and 5HT3B mRNA in brain, kidney, and skeletal muscle, suggesting different significance in tissue expression of these receptors. Regional expression of L2 mRNA and protein was examined in brain. The RT-PCR studies confirmed L2 mRNA expression in hippocampus, striatum, amygdala, and thalamus in adult brain. The L2 protein was immunolocalized by using antipeptide antibodies. Immunostained tissue sections revealed that L2-like immunoreactivity was dominantly expressed in the hippocampal CA3 pyramidal cells and in the polymorphic layer of the dentate gyrus. We analyzed the expression of L2 protein in HEK293 cells using GFP fusion protein reporter system. Western blots revealed that L2 protein confers sugar chains on the extracellular side. In transfected HEK293 cells, cellular membranes and intracellular puncta were densely labeled with GFP, suggesting selective dispatch to the

  4. ABA signaling in guard cells entails a dynamic protein-protein interaction relay from the PYL-RCAR family receptors to ion channels.

    Science.gov (United States)

    Lee, Sung Chul; Lim, Chae Woo; Lan, Wenzhi; He, Kai; Luan, Sheng

    2013-03-01

    Plant hormone abscisic acid (ABA) serves as an integrator of environmental stresses such as drought to trigger stomatal closure by regulating specific ion channels in guard cells. We previously reported that SLAC1, an outward anion channel required for stomatal closure, was regulated via reversible protein phosphorylation events involving ABA signaling components, including protein phosphatase 2C members and a SnRK2-type kinase (OST1). In this study, we reconstituted the ABA signaling pathway as a protein-protein interaction relay from the PYL/RCAR-type receptors, to the PP2C-SnRK2 phosphatase-kinase pairs, to the ion channel SLAC1. The ABA receptors interacted with and inhibited PP2C phosphatase activity against the SnRK2-type kinase, releasing active SnRK2 kinase to phosphorylate, and activate the SLAC1 channel, leading to reduced guard cell turgor and stomatal closure. Both yeast two-hybrid and bimolecular fluorescence complementation assays were used to verify the interactions among the components in the pathway. These biochemical assays demonstrated activity modifications of phosphatases and kinases by their interaction partners. The SLAC1 channel activity was used as an endpoint readout for the strength of the signaling pathway, depending on the presence of different combinations of signaling components. Further study using transgenic plants overexpressing one of the ABA receptors demonstrated that changing the relative level of interacting partners would change ABA sensitivity.

  5. Functional modulation of cerebral gamma-aminobutyric acidA receptor/benzodiazepine receptor/chloride ion channel complex with ethyl beta-carboline-3-carboxylate: Presence of independent binding site for ethyl beta-carboline-3-carboxylate

    Energy Technology Data Exchange (ETDEWEB)

    Taguchi, J.; Kuriyama, K. (Kyoto Prefectural Univ. of Medicine (Japan))

    1990-05-01

    Effect of ethyl beta-carboline-3-carboxylate (beta-CCE) on the function of gamma-aminobutyric acid (GABA)A receptor/benzodiazepine receptor/chloride ion channel complex was studied. Beta-CCE noncompetitively and competitively inhibited (3H)flunitrazepam binding to benzodiazepine receptor, but not (3H)muscimol binding to GABAA receptor as well as t-(3H)butylbicycloorthobenzoate (( 3H) TBOB) binding to chloride ion channel, in particulate fraction of the mouse brain. Ro15-1788 also inhibited competitively (3H) flunitrazepam binding. On the other hand, the binding of beta-(3H)CCE was inhibited noncompetitively and competitively by clonazepam and competitively by Ro15-1788. In agreement with these results, benzodiazepines-stimulated (3H)muscimol binding was antagonized by beta-CCE and Ro15-1788. Gel column chromatography for the solubilized fraction from cerebral particulate fraction by 0.2% sodium deoxycholate (DOC-Na) in the presence of 1 M KCl indicated that beta-(3H)CCE binding site was eluted in the same fraction (molecular weight, 250,000) as the binding sites for (3H)flunitrazepam, (3H)muscimol and (3H)TBOB. GABA-stimulated 36Cl- influx into membrane vesicles prepared from the bovine cerebral cortex was stimulated and attenuated by flunitrazepam and beta-CCE, respectively. These effects of flunitrazepam and beta-CCE on the GABA-stimulated 36Cl- influx were antagonized by Ro15-1788. The present results suggest that the binding site for beta-CCE, which resides on GABAA receptor/benzodiazepine receptor/chloride ion channel complex, may be different from that for benzodiazepine. Possible roles of beta-CCE binding site in the allosteric inhibitions on benzodiazepine binding site as well as on the functional coupling between chloride ion channel and GABAA receptor are also suggested.

  6. Cerebrovascular endothelin-1 hyper-reactivity is associated with transient receptor potential canonical channels 1 and 6 activation and delayed cerebral hypoperfusion after forebrain ischaemia in rats

    DEFF Research Database (Denmark)

    Johansson, S E; Andersen, X E D R; Hansen, R H

    2015-01-01

    . METHODS: Experimental forebrain ischaemia was induced in Wistar male rats by a two-vessel occlusion model, and the cerebral blood flow was measured by magnetic resonance imaging two days after reperfusion. In vitro vasoreactivity studies, immunofluorescence and quantitative PCR were performed on cerebral...... in the vascular smooth muscle cells was enhanced and correlated with decreased cerebral blood flow two days after forebrain ischaemia. Furthermore, under conditions when voltage-dependent calcium channels were inhibited, endothelin-1-induced cerebrovascular contraction was enhanced and this enhancement...... was presumably mediated by Ca(2+) influx via upregulated transient receptor potential canonical channels 1 and 6. CONCLUSIONS: Our data demonstrates that endothelin-1-mediated influx of extracellular Ca(2+) activates transient receptor potential canonical channels 1 and 6 in cerebral vascular smooth muscle cells...

  7. Molecular pharmacology of cell receptors for cardiac glycosides, opiates, ACTH and ion channel modulators

    Energy Technology Data Exchange (ETDEWEB)

    Hnatowich, M R

    1986-01-01

    The influence of light and oxygen on molecular interactions between the artificial food dye, erythrosine (ERY), and (/sup 3/H)ouabain ((/sup 3/H)OUA) binding sites on (Na/sup +/ + K/sup +/)-ATPase in rat brain and guinea pig heart was investigated. Putative endogenous digitalis-like factors (DLF's) were studied in four in vitro assays for cardiac glycosides. (/sup 3/H)Etorphine binding was characterized in rat brain homogenates, depleted of opioids, from animals acutely and chronically treated with morphine and naloxone, and either unstressed or cold-restraint-stressed. Binding sites for the ion channel modulators (/sup 3/H)verapamil ((/sup 3/H)VER) and (/sup 3/H) phencyclidine ((/sup 3/H)PCP) were characterized in rat brain.

  8. Influence of ER leak on resting cytoplasmic Ca2+ and receptor-mediated Ca2+ signalling in human macrophage.

    Science.gov (United States)

    Layhadi, Janice A; Fountain, Samuel J

    2017-06-03

    Mechanisms controlling endoplasmic reticulum (ER) Ca 2+ homeostasis are important regulators of resting cytoplasmic Ca 2+ concentration ([Ca 2+ ] cyto ) and receptor-mediated Ca 2+ signalling. Here we investigate channels responsible for ER Ca 2+ leak in THP-1 macrophage and human primary macrophage. In the absence of extracellular Ca 2+ we employ ionomycin action at the plasma membrane to stimulate ER Ca 2+ leak. Under these conditions ionomycin elevates [Ca 2+ ] cyto revealing a Ca 2+ leak response which is abolished by thapsigargin. IP 3 receptors (Xestospongin C, 2-APB), ryanodine receptors (dantrolene), and translocon (anisomycin) inhibition facilitated ER Ca 2+ leak in model macrophage, with translocon inhibition also reducing resting [Ca 2+ ] cyto . In primary macrophage, translocon inhibition blocks Ca 2+ leak but does not influence resting [Ca 2+ ] cyto . We identify a role for translocon-mediated ER Ca 2+ leak in receptor-mediated Ca 2+ signalling in both model and primary human macrophage, whereby the Ca 2+ response to ADP (P2Y receptor agonist) is augmented following anisomycin treatment. In conclusion, we demonstrate a role of ER Ca 2+ leak via the translocon in controlling resting cytoplasmic Ca 2+ in model macrophage and receptor-mediated Ca 2+ signalling in model macrophage and primary macrophage. Copyright © 2017 Elsevier Inc. All rights reserved.

  9. Signal-dependent hydrolysis of phosphatidylinositol 4,5-bisphosphate without activation of phospholipase C: implications on gating of Drosophila TRPL (transient receptor potential-like) channel.

    Science.gov (United States)

    Lev, Shaya; Katz, Ben; Tzarfaty, Vered; Minke, Baruch

    2012-01-06

    In Drosophila, a phospholipase C (PLC)-mediated signaling cascade, couples photo-excitation of rhodopsin to the opening of the transient receptor potential (TRP) and TRP-like (TRPL) channels. A lipid product of PLC, diacylglycerol (DAG), and its metabolites, polyunsaturated fatty acids (PUFAs) may function as second messengers of channel activation. However, how can one separate between the increase in putative second messengers, change in pH, and phosphatidylinositol 4,5-bisphosphate (PI(4,5)P(2)) depletion when exploring the TRPL gating mechanism? To answer this question we co-expressed the TRPL channels together with the muscarinic (M1) receptor, enabling the openings of TRPL channels via G-protein activation of PLC. To dissect PLC activation of TRPL into its molecular components, we used a powerful method that reduced plasma membrane-associated PI(4,5)P(2) in HEK cells within seconds without activating PLC. Upon the addition of a dimerizing drug, PI(4,5)P(2) was selectively hydrolyzed in the cell membrane without producing DAG, inositol trisphosphate, or calcium signals. We show that PI(4,5)P(2) is not an inhibitor of TRPL channel activation. PI(4,5)P(2) hydrolysis combined with either acidification or application of DAG analogs failed to activate the channels, whereas PUFA did activate the channels. Moreover, a reduction in PI(4,5)P(2) levels or inhibition of DAG lipase during PLC activity suppressed the PLC-activated TRPL current. This suggests that PI(4,5)P(2) is a crucial substrate for PLC-mediated activation of the channels, whereas PUFA may function as the channel activator. Together, this study defines a narrow range of possible mechanisms for TRPL gating.

  10. Covalent modification of mutant rat P2X2 receptors with a thiol-reactive fluorophore allows channel activation by zinc or acidic pH without ATP.

    Directory of Open Access Journals (Sweden)

    Shlomo S Dellal

    Full Text Available Rat P2X2 receptors open at an undetectably low rate in the absence of ATP. Furthermore, two allosteric modulators, zinc and acidic pH, cannot by themselves open these channels. We describe here the properties of a mutant receptor, K69C, before and after treatment with the thiol-reactive fluorophore Alexa Fluor 546 C(5-maleimide (AM546. Xenopus oocytes expressing unmodified K69C were not activated under basal conditions nor by 1,000 µM ATP. AM546 treatment caused a small increase in the inward holding current which persisted on washout and control experiments demonstrated this current was due to ATP independent opening of the channels. Following AM546 treatment, zinc (100 µM or acidic external solution (pH 6.5 elicited inward currents when applied without any exogenous ATP. In the double mutant K69C/H319K, zinc elicited much larger inward currents, while acidic pH generated outward currents. Suramin, which is an antagonist of wild type receptors, behaved as an agonist at AM546-treated K69C receptors. Several other cysteine-reactive fluorophores tested on K69C did not cause these changes. These modified receptors show promise as a tool for studying the mechanisms of P2X receptor activation.

  11. A possible participation of transient receptor potential vanilloid type 1 channels in the antidepressant effect of fluoxetine.

    Science.gov (United States)

    Manna, Shyamshree S S; Umathe, Sudhir N

    2012-06-15

    The present study investigated the influence of transient receptor vanilloid type 1 (TRPV1) channel agonist (capsaicin) and antagonist (capsazepine) either alone or in combination with traditional antidepressant drug, fluoxetine; or a serotonin hydroxylase inhibitor, para-chlorophenylalanine; or a glutamate N-methyl-D-aspartate (NMDA) receptor agonist, NMDA on the forced swim test and tail suspension test using male Swiss mice. Results revealed that intracerebroventricular injections of capsaicin (200 and 300 μg/mouse) and capsazepine (100 and 200 μg/mouse) reduced the immobility time, exhibiting antidepressant-like activity that was comparable to the effects of fluoxetine (2.5-10 μg/mouse) in both the tests. However, in the presence of inactive dose (10 μg/mouse) of capsazepine, capsaicin (300 μg/mouse) had no influence on the indices of both tests, signifying that the effects are TRPV1-mediated. Further, the antidepressant-like effects of both the TRPV1 ligands were neutralized in mice-pretreated with NMDA (0.1 μg/mouse), suggestive of the fact that decreased glutamatergic transmission might contribute to the antidepressant-like activity. In addition, co-administration of sub-threshold dose of capsazepine (10 μg/mouse) and fluoxetine (1.75 μg/mouse) produced a synergistic effect in both the tests. In contrast, inactive doses of capsaicin (10 and 100 μg/mouse) partially abolished the antidepressant effect of fluoxetine (10 μg/mouse), while its effect was potentiated by active dose of capsaicin (200 μg/mouse). Moreover, pretreatment of mice with para-chlorophenylalanine (300 mg/kg/day × 3 days, i.p.) attenuated the effects of capsaicin and capsazepine, demonstrating a probable interplay between serotonin and TRPV1, at least in parts. Thus, our data indicate a possible role of TRPV1 in depressive-like symptoms. Copyright © 2012 Elsevier B.V. All rights reserved.

  12. Excitatory effect of the A2A adenosine receptor agonist CGS-21680 on spontaneous and K+-evoked acetylcholine release at the mouse neuromuscular junction.

    Science.gov (United States)

    Palma, A G; Muchnik, S; Losavio, A S

    2011-01-13

    The mechanism of action of the A2A adenosine receptor agonist 2-p-(2-carboxyethyl) phenethylamino-5'-N-ethylcarboxamidoadenosine hydrochloride (CGS-21680) in the facilitation of spontaneous (isotonic and hypertonic condition) and K+-evoked acetylcholine (ACh) release was investigated in the mouse diaphragm muscles. At isotonic condition, the CGS-21680-induced excitatory effect on miniature end-plate potential (MEPP) frequency was not modified in the presence of CdCl2 and in a medium free of Ca2+ (0Ca2+-EGTA), but it was abolished after buffering the rise of intracellular Ca2+ with 1,2-bis-(2-aminophenoxy)-ethane-N,N,N',N'-tetraacetic acid tetra(acetoxy-methyl) (BAPTA-AM) and when the Ca2+-ATPase inhibitor thapsigargin was used to deplete intracellular Ca2+ stores. CGS-21680 did not have a direct effect on the Ca2+-independent neurotransmitter-releasing machinery, since the modulatory effect on the hypertonic response was also occluded by BAPTA-AM and thapsigargin. CGS-21680 facilitation on K+-evoked ACh release was not altered by the P/Q-type voltage-dependent calcium channel (VDCC) blocker ω-Agatoxin IVA, but it was completely prevented by both, the L-type VDCC blocker nitrendipine (which is known to immobilize their gating charges), or thapsigargin, suggesting that the effects of CGS-21680 on L-type VDCC and thapsigargin-sensitive internal stores are associated. We found that the VDCC pore blocker Cd2+ (2 mM Ca2+ or 0Ca2+-EGTA) failed to affect the CGS-21680 effect in high K+ whereas nitrendipine in 0Ca2+-EGTA+Cd2+ occluded its action. The blockade of Ca2+ release from endoplasmic reticulum with ryanodine antagonized the facilitating effect of CGS-21680 in control and high K+ concentration. It is concluded that, at the mouse neuromuscular junction, activation of A2A receptors facilitates spontaneous and K+-evoked ACh release by an external Ca2+-independent mechanism but that involves mobilization of Ca2+ from internal stores: during spontaneous ACh release

  13. Angiotensin-2-mediated Ca2+ signaling in the retinal pigment epithelium: role of angiotensin-receptor-associated-protein and TRPV2 channel.

    Directory of Open Access Journals (Sweden)

    Rene Barro-Soria

    Full Text Available Angiotensin II (AngII receptor (ATR is involved in pathologic local events such as neovascularisation and inflammation including in the brain and retina. The retinal pigment epithelium (RPE expresses ATR in its AT1R form, angiotensin-receptor-associated protein (Atrap, and transient-receptor-potential channel-V2 (TRPV2. AT1R and Atrap co-localize to the basolateral membrane of the RPE, as shown by immunostaining. Stimulation of porcine RPE (pRPE cells by AngII results in biphasic increases in intracellular free Ca(2+inhibited by losartan. Xestospongin C (xest C and U-73122, blockers of IP3R and PLC respectively, reduced AngII-evoked Ca(2+response. RPE cells from Atrap(-/- mice showed smaller AngII-evoked Ca(2+peak (by 22% and loss of sustained Ca(2+elevation compared to wild-type. The TRPV channel activator cannabidiol (CBD at 15 µM stimulates intracellular Ca(2+-rise suggesting that porcine RPE cells express TRPV2 channels. Further evidence supporting the functional expression of TRPV2 channels comes from experiments in which 100 µM SKF96365 (a TRPV channel inhibitor reduced the cannabidiol-induced Ca(2+-rise. Application of SKF96365 or reduction of TRPV2 expression by siRNA reduced the sustained phase of AngII-mediated Ca(2+transients by 53%. Thus systemic AngII, an effector of the local renin-angiotensin system stimulates biphasic Ca(2+transients in the RPE by releasing Ca(2+from cytosolic IP3-dependent stores and activating ATR/Atrap and TRPV2 channels to generate a sustained Ca(2+elevation.

  14. Transient receptor potential cation channel, subfamily C, member 5 (TRPC5) is a cold-transducer in the peripheral nervous system.

    Science.gov (United States)

    Zimmermann, Katharina; Lennerz, Jochen K; Hein, Alexander; Link, Andrea S; Kaczmarek, J Stefan; Delling, Markus; Uysal, Serdar; Pfeifer, John D; Riccio, Antonio; Clapham, David E

    2011-11-01

    Detection and adaptation to cold temperature is crucial to survival. Cold sensing in the innocuous range of cold (>10-15 °C) in the mammalian peripheral nervous system is thought to rely primarily on transient receptor potential (TRP) ion channels, most notably the menthol receptor, TRPM8. Here we report that TRP cation channel, subfamily C member 5 (TRPC5), but not TRPC1/TRPC5 heteromeric channels, are highly cold sensitive in the temperature range 37-25 °C. We found that TRPC5 is present in mouse and human sensory neurons of dorsal root ganglia, a substantial number of peripheral nerves including intraepithelial endings, and in the dorsal lamina of the spinal cord that receives sensory input from the skin, consistent with a potential TRPC5 function as an innocuous cold transducer in nociceptive and thermosensory nerve endings. Although deletion of TRPC5 in 129S1/SvImJ mice resulted in no temperature-sensitive behavioral changes, TRPM8 and/or other menthol-sensitive channels appear to underpin a much larger component of noxious cold sensing after TRPC5 deletion and a shift in mechanosensitive C-fiber subtypes. These findings demonstrate that highly cold-sensitive TRPC5 channels are a molecular component for detection and regional adaptation to cold temperatures in the peripheral nervous system that is distinct from noxious cold sensing.

  15. The Allergen Der p3 from House Dust Mite Stimulates Store-Operated Ca2+ Channels and Mast Cell Migration through PAR4 Receptors.

    Science.gov (United States)

    Lin, Yu-Ping; Nelson, Charmaine; Kramer, Holger; Parekh, Anant B

    2018-04-19

    The house dust mite is the principal source of perennial aeroallergens in man. How these allergens activate innate and adaptive immunity is unclear, and therefore, there are no therapies targeting mite allergens. Here, we show that house dust mite extract activates store-operated Ca 2+ channels, a common signaling module in numerous cell types in the lung. Activation of channel pore-forming Orai1 subunits by mite extract requires gating by STIM1 proteins. Although mite extract stimulates both protease-activated receptor type 2 (PAR2) and PAR4 receptors, Ca 2+ influx is more tightly coupled to the PAR4 pathway. We identify a major role for the serine protease allergen Der p3 in stimulating Orai1 channels and show that a therapy involving sub-maximal inhibition of both Der p3 and Orai1 channels suppresses mast cell activation to house dust mite. Our results reveal Der p3 as an important aeroallergen that activates Ca 2+ channels and suggest a therapeutic strategy for treating mite-induced asthma. Copyright © 2018 Elsevier Inc. All rights reserved.

  16. Glycolytic metabolite methylglyoxal inhibits cold and menthol activation of the transient receptor potential melastatin type 8 channel.

    Science.gov (United States)

    Ciobanu, A C; Selescu, T; Gasler, I; Soltuzu, L; Babes, A

    2016-03-01

    Methylglyoxal (MG) is a reactive dicarbonyl compound involved in protein modifications linked to diabetes mellitus. The plasma level of MG is elevated in diabetic patients, particularly those with painful diabetic neuropathy. Diabetic neuropathy is often associated with spontaneous pain and altered thermal perception. This study assesses effects of MG on TRPM8, an ion channel involved in innocuous cold sensing and cold allodynia and also in cold-mediated analgesia. Acute treatment with MG inhibited the activation of recombinant rat and human transient receptor potential melastatin type 8 (TRPM8) by cold and chemical agonists. A similar effect was observed when native TRPM8 was investigated in cultured rat dorsal root ganglion (DRG) neurons. DRG neurons treated with MG for 16-24 hr displayed a significant reduction in the fraction of cold- and menthol-sensitive neurons, most likely expressing TRPM8. The fraction of allyl isothiocyanate-sensitive neurons was also reduced, and the coexpression among different neuronal populations was affected. The same prolonged exposure to MG significantly reduced the expression of TRPM8 at the mRNA level. Overall, our data provide evidence for decreased activity and expression level of TRPM8 in the presence of MG, which may be linked to some of the alterations in pain and temperature sensing reported by diabetic patients. © 2015 Wiley Periodicals, Inc. © 2015 Wiley Periodicals, Inc.

  17. Competitive inhibition of the nondepolarizing muscle relaxant rocuronium on nicotinic acetylcholine receptor channels in the rat superior cervical ganglia.

    Science.gov (United States)

    Zhang, Chengmi; Wang, Zhenmeng; Zhang, Jinmin; Qiu, Haibo; Sun, Yuming; Yang, Liqun; Wu, Feixiang; Zheng, Jijian; Yu, Weifeng

    2014-05-01

    A number of case reports now indicate that rocuronium can induce a number of serious side effects. We hypothesized that these side effects might be mediated by the inhibition of nicotinic acetylcholine receptors (nAChRs) at superior cervical ganglion (SCG) neurons. Conventional patch clamp recordings were used to study the effects of rocuronium on nAChR currents from enzymatically dissociated rat SCG neurons. We found that ACh induced a peak transient inward current in rat SCG neurons. Additionally, rocuronium suppressed the peak ACh-evoked currents in rat SCG neurons in a concentration-dependent and competitive manner, and it increased the extent of desensitization of nAChRs. The inhibitory rate of rocuronium on nAChR currents did not change significantly at membrane potentials between -70 and -20 mV, suggesting that this inhibition was voltage independent. Lastly, rocuronium preapplication enhanced its inhibitory effect, indicating that this drug might prefer to act on the closed state of nAChR channels. In conclusion, rocuronium, at clinically relevant concentrations, directly inhibits nAChRs at the SCG by interacting with both opened and closed states. This inhibition is competitive, dose dependent, and voltage independent. Blockade of synaptic transmission in the sympathetic ganglia by rocuronium might have potentially inhibitory effects on the cardiovascular system.

  18. Autoantibody-induced internalization of CNS AQP4 water channel and EAAT2 glutamate transporter requires astrocytic Fc receptor.

    Science.gov (United States)

    Hinson, Shannon R; Clift, Ian C; Luo, Ningling; Kryzer, Thomas J; Lennon, Vanda A

    2017-05-23

    Aquaporin-4 (AQP4) water channel-specific IgG distinguishes neuromyelitis optica (NMO) from multiple sclerosis and causes characteristic immunopathology in which central nervous system (CNS) demyelination is secondary. Early events initiating the pathophysiological outcomes of IgG binding to astrocytic AQP4 are poorly understood. CNS lesions reflect events documented in vitro following IgG interaction with AQP4: AQP4 internalization, attenuated glutamate uptake, intramyelinic edema, interleukin-6 release, complement activation, inflammatory cell recruitment, and demyelination. Here, we demonstrate that AQP4 internalization requires AQP4-bound IgG to engage an astrocytic Fcγ receptor (FcγR). IgG-lacking Fc redistributes AQP4 within the plasma membrane and induces interleukin-6 release. However, AQP4 endocytosis requires an activating FcγR's gamma subunit and involves astrocytic membrane loss of an inhibitory FcγR, CD32B. Interaction of the IgG-AQP4 complex with FcγRs triggers coendocytosis of the excitatory amino acid transporter 2 (EAAT2). Requirement of FcγR engagement for internalization of two astrocytic membrane proteins critical to CNS homeostasis identifies a complement-independent, upstream target for potential early therapeutic intervention in NMO.

  19. Design and synthesis of enantiomerically enriched, radiolabeled MK-801 analogs as potential radiotracers for imaging and autoradiographic studies of the NMDA receptor-ion channel complex

    International Nuclear Information System (INIS)

    Eng, W.S.; Burns, H.D.; Gibson, R.E.; Ransom, R.W.; Thorpe, H.; Fioravanit, C.; Britcher, S.F.; Magill, C.A.; Solomon, H.F.; Dannals, R.F.; Wilson, A.A.; Ravert, H.T.; Wagner, H.N.

    1989-01-01

    MK-801 is a potent, non-competitive antagonist for the N-methyl-D-asspartate (NMDA) receptor-ion channel complex. This complex is though to be involved in nerve cell damage in stroke patients when excess calcium is released through the activated channel. A thorough understanding of drug interactions with the NMDA receptor complex could lead to improved therapy for reducing hypoxic-ischemic neuronal injuries in stroke patients. Based on the results of extensive structure-activity studies, the authors have developed several enantiomerically enriched, radiolabeled analogs of MK-801, including: 3-1231-MK-801 for Single Photon Emission Computed tomography (SPECT); 3-1251-MK-801 for in-vivo and in-vitro autoradiography; 8-11C-MeO-MK-801 for Positron Emission Tomography (PET). Details of the synthesis of these radiotracers and their application to both in-vitro and in-vivo studies are described

  20. Modulation of voltage-gated Ca2+ channels by G protein-coupled receptors in celiac-mesenteric ganglion neurons of septic rats.

    Directory of Open Access Journals (Sweden)

    Mohamed Farrag

    Full Text Available Septic shock, the most severe complication associated with sepsis, is manifested by tissue hypoperfusion due, in part, to cardiovascular and autonomic dysfunction. In many cases, the splanchnic circulation becomes vasoplegic. The celiac-superior mesenteric ganglion (CSMG sympathetic neurons provide the main autonomic input to these vessels. We used the cecal ligation puncture (CLP model, which closely mimics the hemodynamic and metabolic disturbances observed in septic patients, to examine the properties and modulation of Ca2+ channels by G protein-coupled receptors in acutely dissociated rat CSMG neurons. Voltage-clamp studies 48 hr post-sepsis revealed that the Ca2+ current density in CMSG neurons from septic rats was significantly lower than those isolated from sham control rats. This reduction coincided with a significant increase in membrane surface area and a negligible increase in Ca2+ current amplitude. Possible explanations for these findings include either cell swelling or neurite outgrowth enhancement of CSMG neurons from septic rats. Additionally, a significant rightward shift of the concentration-response relationship for the norepinephrine (NE-mediated Ca2+ current inhibition was observed in CSMG neurons from septic rats. Testing for the presence of opioid receptor subtypes in CSMG neurons, showed that mu opioid receptors were present in ~70% of CSMG, while NOP opioid receptors were found in all CSMG neurons tested. The pharmacological profile for both opioid receptor subtypes was not significantly affected by sepsis. Further, the Ca2+ current modulation by propionate, an agonist for the free fatty acid receptors GPR41 and GPR43, was not altered by sepsis. Overall, our findings suggest that CSMG function is affected by sepsis via changes in cell size and α2-adrenergic receptor-mediated Ca2+ channel modulation.

  1. P2Y receptor-mediated transient relaxation of rat longitudinal ileum preparations involves phospholipase C activation, intracellular Ca(2+) release and SK channel activation.

    Science.gov (United States)

    Mader, Felix; Krause, Ludwig; Tokay, Tursonjan; Hakenberg, Oliver W; Köhling, Rüdiger; Kirschstein, Timo

    2016-05-01

    Purinergic signaling plays a major role in the enteric nervous system, where it governs gut motility through a number of P2X and P2Y receptors. The aim of this study was to investigate the P2Y receptor-mediated motility in rat longitudinal ileum preparations. Ileum smooth muscle strips were prepared from rats, and fixed in an organ bath. Isometric contraction and relaxation responses of the muscle strips were measured with force transducers. Drugs were applied by adding of stock solutions to the organ bath to yield the individual final concentrations. Application of the non-hydrolyzable P2 receptor agonists α,β-Me-ATP or 2-Me-S-ADP (10, 100 μmol/L) dose-dependently elicited a transient relaxation response followed by a sustained contraction. The relaxation response was largely blocked by SK channel blockers apamin (500 nmol/L) and UCL1684 (10 μmol/L), PLC inhibitor U73122 (100 μmol/L), IP3 receptor blocker 2-APB (100 μmol/L) or sarcoendoplasmic Ca(2+) ATPase inhibitor thapsigargin (1 μmol/L), but not affected by atropine, NO synthase blocker L-NAME or tetrodotoxin. Furthermore, α,β-Me-ATP-induced relaxation was suppressed by P2Y1 receptor antagonist MRS2179 (50 μmol/L) or P2Y13 receptor antagonist MRS2211 (100 μmol/L), and was abolished by co-application of the two antagonists, whereas 2-Me-S-ADP-induced relaxation was abolished by P2Y6 receptor antagonist MRS2578 (50 μmol/L). In addition, P2Y1 receptor antagonist MRS2500 (1 μmol/L) not only abolished α,β-Me-ATP-induced relaxation, but also suppressed 2-Me-S-ADP-induced relaxation. P2Y receptor agonist-induced transient relaxation of rat ileum smooth muscle strips is mediated predominantly by P2Y1 receptor, but also by P2Y6 and P2Y13 receptors, and involves PLC, IP3, Ca(2+) release and SK channel activation, but is independent of acetylcholine and NO release.

  2. Activation of Mechanosensitive Transient Receptor Potential/Piezo Channels in Odontoblasts Generates Action Potentials in Cocultured Isolectin B4-negative Medium-sized Trigeminal Ganglion Neurons.

    Science.gov (United States)

    Sato, Masaki; Ogura, Kazuhiro; Kimura, Maki; Nishi, Koichi; Ando, Masayuki; Tazaki, Masakazu; Shibukawa, Yoshiyuki

    2018-04-27

    Various stimuli to the dentin surface elicit dentinal pain by inducing dentinal fluid movement causing cellular deformation in odontoblasts. Although odontoblasts detect deformation by the activation of mechanosensitive ionic channels, it is still unclear whether odontoblasts are capable of establishing neurotransmission with myelinated A delta (Aδ) neurons. Additionally, it is still unclear whether these neurons evoke action potentials by neurotransmitters from odontoblasts to mediate sensory transduction in dentin. Thus, we investigated evoked inward currents and evoked action potentials form trigeminal ganglion (TG) neurons after odontoblast mechanical stimulation. We used patch clamp recordings to identify electrophysiological properties and record evoked responses in TG neurons. We classified TG cells into small-sized and medium-sized neurons. In both types of neurons, we observed voltage-dependent inward currents. The currents from medium-sized neurons showed fast inactivation kinetics. When mechanical stimuli were applied to odontoblasts, evoked inward currents were recorded from medium-sized neurons. Antagonists for the ionotropic adenosine triphosphate receptor (P2X 3 ), transient receptor potential channel subfamilies, and Piezo1 channel significantly inhibited these inward currents. Mechanical stimulation to odontoblasts also generated action potentials in the isolectin B 4 -negative medium-sized neurons. Action potentials in these isolectin B 4 -negative medium-sized neurons showed a short duration. Overall, electrophysiological properties of neurons indicate that the TG neurons with recorded evoked responses after odontoblast mechanical stimulation were myelinated Aδ neurons. Odontoblasts established neurotransmission with myelinated Aδ neurons via P2X 3 receptor activation. The results also indicated that mechanosensitive TRP/Piezo1 channels were functionally expressed in odontoblasts. The activation of P2X 3 receptors induced an action potential

  3. Development of therapeutic antibodies to G protein-coupled receptors and ion channels: Opportunities, challenges and their therapeutic potential in respiratory diseases.

    Science.gov (United States)

    Douthwaite, Julie A; Finch, Donna K; Mustelin, Tomas; Wilkinson, Trevor C I

    2017-01-01

    The development of recombinant antibody therapeutics continues to be a significant area of growth in the pharmaceutical industry with almost 50 approved monoclonal antibodies on the market in the US and Europe. Therapeutic drug targets such as soluble cytokines, growth factors and single transmembrane spanning receptors have been successfully targeted by recombinant monoclonal antibodies and the development of new product candidates continues. Despite this growth, however, certain classes of important disease targets have remained intractable to therapeutic antibodies due to the complexity of the target molecules. These complex target molecules include G protein-coupled receptors and ion channels which represent a large target class for therapeutic intervention with monoclonal antibodies. Although these targets have typically been addressed by small molecule approaches, the exquisite specificity of antibodies provides a significant opportunity to provide selective modulation of these important regulators of cell function. Given this opportunity, a significant effort has been applied to address the challenges of targeting these complex molecules and a number of targets are linked to the pathophysiology of respiratory diseases. In this review, we provide a summary of the importance of GPCRs and ion channels involved in respiratory disease and discuss advantages offered by antibodies as therapeutics at these targets. We highlight some recent GPCRs and ion channels linked to respiratory disease mechanisms and describe in detail recent progress made in the strategies for discovery of functional antibodies against challenging membrane protein targets such as GPCRs and ion channels. Copyright © 2016 Elsevier Inc. All rights reserved.

  4. MS-377, a selective sigma receptor ligand, indirectly blocks the action of PCP in the N-methyl-D-aspartate receptor ion-channel complex in primary cultured rat neuronal cells.

    Science.gov (United States)

    Karasawa, Jun-ichi; Yamamoto, Hideko; Yamamoto, Toshifumi; Sagi, Naoki; Horikomi, Kazutoshi; Sora, Ichiro

    2002-02-22

    MS-377 ((R)-(+)-1-(4-chlorophenyl)-3-[4-(2-methoxyethyl)piperazin-1-yl]methyl-2-pyrrolidinone L-tartrate) is a antipsychotic agent that binds to sigma-1 receptor. MS-377 showed anti-dopaminergic and anti-serotonergic activities and antagonistic action against phencyclidine (PCP)-induced behaviors in an animal model. These anti-psychotic activities of MS-377 are attributable to association with sigma-1 receptor. However, the mechanism by which the sigma-1 receptor ligands exact those numerous effects remains to be elucidated. In the present study, we evaluated the effect of MS-377 on N-methyl-D-aspartate (NMDA) receptor ion-channel complex in primary cultured rat neuronal cells. First, we examined the effect of MS-377 on NMDA-induced Ca2+ influx with fura-2/ AM loaded cells. MS-377 showed no effects on the basal Ca2+ concentration and NMDA-induced Ca2+ influx by itself PCP and SKF-10047 reduced the NMDA-induced increase in intracellular Ca2+ concentration. Pre-incubation of 1 microM MS-377 was found to significantly block the reduction by PCP or SKF-10047 of the NMDA-induced Ca2+ influx. Second, the effect of MS-377 on [3H]MK-801 intact cell binding was examined. PCP, haloperidol and (+)-pentazocine inhibited [3H]MK-801 binding, although MS-377 showed no effect by itself Pre-treatment of MS-377 markedly reversed the inhibition of [3H]MK-801 binding by PCP in a dose-dependent manner. These effects of MS-377 may depend on its affinity for the sigma-1 receptor, because MS-377 is a selective sigma-1 receptor ligand without any affinity for NMDA receptor ion-channel complex. These observations suggest that the MS-377 indirectly modulated the NMDA receptor ion-channel complex, and the anti-psychotic activities of MS-377, in part, are attributable to such on action via sigma-1 receptor.

  5. Calcium channel blockers, more than diuretics, enhance vascular protective effects of angiotensin receptor blockers in salt-loaded hypertensive rats.

    Directory of Open Access Journals (Sweden)

    Eiichiro Yamamoto

    Full Text Available The combination therapy of an angiotensin receptor blocker (ARB with a calcium channel blocker (CCB or with a diuretic is favorably recommended for the treatment of hypertension. However, the difference between these two combination therapies is unclear. The present work was undertaken to examine the possible difference between the two combination therapies in vascular protection. Salt-loaded stroke-prone spontaneously hypertensive rats (SHRSP were divided into 6 groups, and they were orally administered (1 vehicle, (2 olmesartan, an ARB, (3 azelnidipine, a CCB, (4 hydrochlorothiazide, a diuretic, (5 olmesartan combined with azelnidipine, or (6 olmesartan combined with hydrochlorothiazide. Olmesartan combined with either azelnidipine or hydrochlorothiazide ameliorated vascular endothelial dysfunction and remodeling in SHRSP more than did monotherapy with either agent. However, despite a comparable blood pressure lowering effect between the two treatments, azelnidipine enhanced the amelioration of vascular endothelial dysfunction and remodeling by olmesartan to a greater extent than did hydrochlorothiazide in salt-loaded SHRSP. The increased enhancement by azelnidipine of olmesartan-induced vascular protection than by hydrochlorothiazide was associated with a greater amelioration of vascular nicotinamide adenine dinucleotide phosphate (NADPH oxidase activation, superoxide, mitogen-activated protein kinase activation, and with a greater activation of the Akt/endothelial nitric oxide synthase (eNOS pathway. These results provided the first evidence that a CCB potentiates the vascular protective effects of an ARB in salt-sensitive hypertension, compared with a diuretic, and provided a novel rationale explaining the benefit of the combination therapy with an ARB and a CCB.

  6. Sigma-1 receptor agonist increases axon outgrowth of hippocampal neurons via voltage-gated calcium ions channels.

    Science.gov (United States)

    Li, Dong; Zhang, Shu-Zhuo; Yao, Yu-Hong; Xiang, Yun; Ma, Xiao-Yun; Wei, Xiao-Li; Yan, Hai-Tao; Liu, Xiao-Yan

    2017-12-01

    Sigma-1 receptors (Sig-1Rs) are unique endoplasmic reticulum proteins that have been implicated in both neurodegenerative and ischemic diseases, such as Alzheimer's disease and stroke. Accumulating evidence has suggested that Sig-1R plays a role in neuroprotection and axon outgrowth. The underlying mechanisms of Sig-1R-mediated neuroprotection have been well elucidated. However, the mechanisms underlying the effects of Sig-1R on axon outgrowth are not fully understood. To clarify this issue, we utilized immunofluorescence to compare the axon lengths of cultured naïve hippocampal neurons before and after the application of the Sig-1R agonist, SA4503. Then, electrophysiology and immunofluorescence were used to examine voltage-gated calcium ion channel (VGCCs) currents in the cell membranes and growth cones. We found that Sig-1R activation dramatically enhanced the axonal length of the naïve hippocampal neurons. Application of the Sig-1R antagonist NE100 and gene knockdown techniques both demonstrated the effects of Sig-1R. The growth-promoting effect of SA4503 was accompanied by the inhibition of voltage-gated Ca 2+ influx and was recapitulated by incubating the neurons with the L-type, N-type, and P/Q-type VGCC blockers, nimodipine, MVIIA and ω-agatoxin IVA, respectively. This effect was unrelated to glial cells. The application of SA4503 transformed the growth cone morphologies from complicated to simple, which favored axon outgrowth. Sig-1R activation can enhance axon outgrowth and may have a substantial influence on neurogenesis and neurodegenerative diseases. © 2017 John Wiley & Sons Ltd.

  7. Transient receptor potential melastatin subfamily member 2 cation channel regulates detrimental immune cell invasion in ischemic stroke.

    Science.gov (United States)

    Gelderblom, Mathias; Melzer, Nico; Schattling, Benjamin; Göb, Eva; Hicking, Gordon; Arunachalam, Priyadharshini; Bittner, Stefan; Ufer, Friederike; Herrmann, Alexander M; Bernreuther, Christian; Glatzel, Markus; Gerloff, Christian; Kleinschnitz, Christoph; Meuth, Sven G; Friese, Manuel A; Magnus, Tim

    2014-11-01

    Brain injury during stroke results in oxidative stress and the release of factors that include extracellular Ca(2+), hydrogen peroxide, adenosine diphosphate ribose, and nicotinic acid adenine dinucleotide phosphate. These alterations of the extracellular milieu change the activity of transient receptor potential melastatin subfamily member 2 (TRPM2), a nonselective cation channel expressed in the central nervous system and the immune system. Our goal was to evaluate the contribution of TRPM2 to the tissue damage after stroke. In accordance with current quality guidelines, we independently characterized Trpm2 in a murine ischemic stroke model in 2 different laboratories. Gene deficiency of Trpm2 resulted in significantly improved neurological outcome and decreased infarct size. Besides an already known moderate neuroprotective effect of Trpm2 deficiency in vitro, ischemic brain invasion by neutrophils and macrophages was particularly reduced in Trpm2-deficient mice. Bone marrow chimeric mice revealed that Trpm2 deficiency in the peripheral immune system is responsible for the protective phenotype. Furthermore, experiments with mixed bone marrow chimeras demonstrated that Trpm2 is essential for the migration of neutrophils and, to a lesser extent, also of macrophages into ischemic hemispheres. Notably, the pharmacological TRPM2 inhibitor, N-(p-amylcinnamoyl)anthranilic acid, was equally protective in the stroke model. Although a neuroprotective effect of TRPM2 in vitro is well known, we can show for the first time that the detrimental role of TRPM2 in stroke primarily depends on its role in activating peripheral immune cells. Targeting TRPM2 systemically represents a promising therapeutic approach for ischemic stroke. © 2014 American Heart Association, Inc.

  8. TPC2 is a novel NAADP-sensitive Ca2+ release channel, operating as a dual sensor of luminal pH and Ca2+.

    Science.gov (United States)

    Pitt, Samantha J; Funnell, Tim M; Sitsapesan, Mano; Venturi, Elisa; Rietdorf, Katja; Ruas, Margarida; Ganesan, A; Gosain, Rajendra; Churchill, Grant C; Zhu, Michael X; Parrington, John; Galione, Antony; Sitsapesan, Rebecca

    2010-11-05

    Nicotinic acid adenine dinucleotide phosphate (NAADP) is a molecule capable of initiating the release of intracellular Ca(2+) required for many essential cellular processes. Recent evidence links two-pore channels (TPCs) with NAADP-induced release of Ca(2+) from lysosome-like acidic organelles; however, there has been no direct demonstration that TPCs can act as NAADP-sensitive Ca(2+) release channels. Controversial evidence also proposes ryanodine receptors as the primary target of NAADP. We show that TPC2, the major lysosomal targeted isoform, is a cation channel with selectivity for Ca(2+) that will enable it to act as a Ca(2+) release channel in the cellular environment. NAADP opens TPC2 channels in a concentration-dependent manner, binding to high affinity activation and low affinity inhibition sites. At the core of this process is the luminal environment of the channel. The sensitivity of TPC2 to NAADP is steeply dependent on the luminal [Ca(2+)] allowing extremely low levels of NAADP to open the channel. In parallel, luminal pH controls NAADP affinity for TPC2 by switching from reversible activation of TPC2 at low pH to irreversible activation at neutral pH. Further evidence earmarking TPCs as the likely pathway for NAADP-induced intracellular Ca(2+) release is obtained from the use of Ned-19, the selective blocker of cellular NAADP-induced Ca(2+) release. Ned-19 antagonizes NAADP-activation of TPC2 in a non-competitive manner at 1 μM but potentiates NAADP activation at nanomolar concentrations. This single-channel study provides a long awaited molecular basis for the peculiar mechanistic features of NAADP signaling and a framework for understanding how NAADP can mediate key physiological events.

  9. Different effects of pentobarbital on two γ-aminobutyrate receptors from rat brain: channel opening, desensitization, and an additional conformational change

    International Nuclear Information System (INIS)

    Cash, D.J.; Subbarao, K.

    1988-01-01

    The effect of pentobarbital on the responses of the γ-aminobutyric acid (GABA) receptor from rat brain was studied in quantitative measurements of GABA-mediated chloride-exchange rates (reflecting channel-opening equilibrium) and receptor desensitization rates by using 36 Cl - tracer ion with negative membrane vesicles. Pentobarbital effected the two phases of 36 Cl - influx in different ways, supporting previous evidence that these are mediated by two different receptors. Both the chloride-exchange rate and the desensitization rate of the faster desensitizing receptor were increased by pentobarbital at concentrations above 20 μM by an allosteric effect shifting the response curve to lower GABA concentrations. A similar enhancement of the responses of the slower desensitizing receptor occurred up to 200 μM pentobarbital. Two pentobarbital effector sites were involved in the allosteric mechanism. Above 500 μM pentobarbital, both the initial chloride-exchange rate and the desensitization rate of the slower desensitizing receptor were decreased. This inhibition, which was immediate, occurred with saturating as well as low GABA concentrations and therefore was not attributed to decreased GABA binding but to inhibitory sites for pentobarbital, different from the allosteric activating sites and the GABA binding sites. The chloride ion exchange activity was seen to recover with time, at concentrations above 1000 μM pentobarbital, in a process with a very steep dependence on pentobarbital concentration. This reactivation was attributed to the conversion of an initial form of the receptor to a final form that was less inhibited by pentobarbital. Comparisons of the effects of pentobarbital on desensitization and on high-affinity ligand binding measurements suggest that increased GABA binding at equilibrium reflects an increased conversion to the desensitized state

  10. Length and amino acid sequence of peptides substituted for the 5-HT3A receptor M3M4 loop may affect channel expression and desensitization.

    Directory of Open Access Journals (Sweden)

    Nicole K McKinnon

    Full Text Available 5-HT3A receptors are pentameric neurotransmitter-gated ion channels in the Cys-loop receptor family. Each subunit contains an extracellular domain, four transmembrane segments (M1, M2, M3, M4 and a 115 residue intracellular loop between M3 and M4. In contrast, the M3M4 loop in prokaryotic homologues is <15 residues. To investigate the limits of M3M4 loop length and composition on channel function we replaced the 5-HT3A M3M4 loop with two to seven alanine residues (5-HT3A-A(n = 2-7. Mutants were expressed in Xenopus laevis oocytes and characterized using two electrode voltage clamp recording. All mutants were functional. The 5-HT EC(50's were at most 5-fold greater than wild-type (WT. The desensitization rate differed significantly among the mutants. Desensitization rates for 5-HT3A-A(2, 5-HT3A-A(4, 5-HT3A-A(6, and 5-HT3A-A(7 were similar to WT. In contrast, 5-HT3A-A(3 and 5-HT3A-A(5 had desensitization rates at least an order of magnitude faster than WT. The one Ala loop construct, 5-HT3A-A(1, entered a non-functional state from which it did not recover after the first 5-HT application. These results suggest that the large M3M4 loop of eukaryotic Cys-loop channels is not required for receptor assembly or function. However, loop length and amino acid composition can effect channel expression and desensitization. We infer that the cytoplasmic ends of the M3 and M4 segments may undergo conformational changes during channel gating and desensitization and/or the loop may influence the position and mobility of these segments as they undergo gating-induced conformational changes. Altering structure or conformational mobility of the cytoplasmic ends of M3 and M4 may be the basis by which phosphorylation or protein binding to the cytoplasmic loop alters channel function.

  11. Development and utilization of a fluorescence-based receptor-binding assay for the site 5 voltage-sensitive sodium channel ligands brevetoxin and ciguatoxin.

    Science.gov (United States)

    McCall, Jennifer R; Jacocks, Henry M; Niven, Susan C; Poli, Mark A; Baden, Daniel G; Bourdelais, Andrea J

    2014-01-01

    Brevetoxins are a family of ladder-frame polyether toxins produced during blooms of the marine dinoflagellate Karenia brevis. Consumption of fish exposed to K. brevis blooms can lead to the development of neurotoxic shellfish poisoning. The toxic effects of brevetoxins are due to activation of voltage-sensitive sodium channels (VSSCs) in cell membranes. Binding of toxins has historically been measured using a radioligand competition assay that is fraught with difficulty. In this study, we developed a novel fluorescence-based binding assay for the brevetoxin receptor. Several fluorophores were conjugated to polyether brevetoxin-2 and used as the labeled ligand. Brevetoxin analogs were able to compete for binding with the fluorescent ligands. This assay was qualified against the standard radioligand receptor assay for the brevetoxin receptor. Furthermore, the fluorescence-based assay was used to determine relative concentrations of toxins in raw extracts of K. brevis culture, and to determine ciguatoxin affinity to site 5 of VSSCs. The fluorescence-based assay was quicker, safer, and far less expensive. As such, this assay can be used to replace the current radioligand assay and will be a vital tool for future experiments examining the binding affinity of various ligands for site 5 on sodium channels.

  12. Activation of the ζ receptor 1 suppresses NMDA responses in rat retinal ganglion cells.

    Science.gov (United States)

    Zhang, X-J; Liu, L-L; Jiang, S-X; Zhong, Y-M; Yang, X-L

    2011-03-17

    The sigma receptor 1 (σR1) has been shown to modulate the activity of several voltage- and ligand-gated channels. Using patch-clamp techniques in rat retinal slice preparations, we demonstrated that activation of σR1 by SKF10047 (SKF) or PRE-084 suppressed N-methyl-D-aspartate (NMDA) receptor-mediated current responses from both ON and OFF type ganglion cells (GCs), dose-dependently, and the effect could be blocked by the σR1 antagonist BD1047 or the σR antagonist haloperidol. The suppression by SKF of NMDA currents was abolished with pre-incubation of the G protein inhibitor GDP-β-S or the Gi/o activator mastoparan. We further explored the intracellular signaling pathway responsible for the SKF-induced suppression of NMDA responses. Application of either cAMP/the PKA inhibitor Rp-cAMP or cGMP/the PKG inhibitor KT5823 did not change the SKF-induced effect, suggesting the involvement of neither cAMP/PKA nor cGMP/PKG pathway. In contrast, suppression of NMDA responses by SKF was abolished by internal infusion of the phosphatidylinostiol-specific phospholipase C (PLC) inhibitor U73122, but not by the phosphatidylcholine-PLC inhibitor D609. SKF-induced suppression of NMDA responses was dependent on intracellular Ca2+ concentration ([Ca2+]i), as evidenced by the fact that the effect was abolished when [Ca2+]i was buffered with 10 mM BAPTA. The SKF effect was blocked by xestospongin-C/heparin, IP3 receptor antagonists, but unchanged by ryanodine/caffeine, ryanodine receptor modulators. Furthermore, application of protein kinase C inhibitors Bis IV and Gö6976 eliminated the SKF effect. These results suggest that the suppression of NMDA responses of rat retinal GCs caused by the activation of σR1 may be mediated by a distinct [Ca2+]i-dependent PLC-PKC pathway. This effect of SKF could help ameliorate malfunction of GCs caused by excessive stimulation of NMDA receptors under pathological conditions. Copyright © 2011 IBRO. Published by Elsevier Ltd. All rights

  13. Calcium-dependent expression of transient receptor potential canonical type 3 channels in patients with chronic kidney disease

    DEFF Research Database (Denmark)

    Liu, Ying; Krueger, Katharina; Hovsepian, Anahit

    2011-01-01

    patients with chronic kidney disease and 19 age- and sex-matched healthy control subjects. TRPC3 channels were identified by immunoblotting using specific antibodies and TRPC3 protein was further confirmed by mass spectrometry. We observed a significant increase of TRPC3 channel protein expression...

  14. Potential role of melastatin-related transient receptor potential cation channel subfamily M gene expression in the pathogenesis of urinary bladder cancer.

    Science.gov (United States)

    Ceylan, Gülay Güleç; Önalan, Ebru Etem; Kuloğlu, Tuncay; Aydoğ, Gülten; Keleş, İbrahim; Tonyali, Şenol; Ceylan, Cavit

    2016-12-01

    Urinary bladder cancer is one of the most common malignancies of the urinary tract. Ion channels and calcium homeostasis are involved in almost all basic cellular mechanisms. The transient receptor potential cation channel subfamily M (TRPM) takes its name from the melastatin protein, which is classified as potential tumor suppressor. To the best of our knowledge, there have been no previous studies in the literature investigating the role of these ion channels in bladder cancer. The present study aimed to determine whether bladder cancer is associated with mRNA expression levels of TRPM ion channel genes, and whether there is the potential to conduct further studies to establish novel treatment modalities. The present study included a total of 47 subjects, of whom 40 were bladder cancer patients and 7 were controls. Following the histopathological evaluation for bladder carcinoma, the mRNA and protein expression of TRPM were examined by reverse transcription-quantitative polymerase chain reaction (RT-qPCR) and immunohistochemistry in tumor and normal tissues, in order to determine whether there is a difference in the expression of these channels in tumor and normal tissues. Immunoreactivity for TRPM2, TRPM4, TRPM7 and TRPM8 was observed in epithelial bladder cells in the two groups. RT-qPCR revealed a significant increase in TRPM7 expression in bladder cancer tissue compared to the controls (healthy bladder tissue), whereas no differences in TRPM2 or TRPM4 expression levels were observed. There were significant reductions in the expression levels of TRPM5 and TRPM8 in bladder cancer tissues. In the present study, the effects of TRP ion channels on the formation of bladder cancer was investigated. This study is instructive for TRPM2, TRPM4, TRPM5, TRPM7 and TRPM8 and their therapeutic role in bladder cancer. The results support the fact that these gens can be novel targets and can also be tested for during the treatment of bladder cancer.

  15. Pharmacovigilance database search discloses ClC-K channels as a novel target of the AT1 receptor blockers valsartan and olmesartan.

    Science.gov (United States)

    Imbrici, Paola; Tricarico, Domenico; Mangiatordi, Giuseppe Felice; Nicolotti, Orazio; Lograno, Marcello Diego; Conte, Diana; Liantonio, Antonella

    2017-07-01

    Human ClC-K chloride channels are highly attractive targets for drug discovery as they have a variety of important physiological functions and are associated with genetic disorders. These channels are crucial in the kidney as they control chloride reabsorption and water diuresis. In addition, loss-of-function mutations of CLCNKB and BSND genes cause Bartter's syndrome (BS), whereas CLCNKA and CLCNKB gain-of-function polymorphisms predispose to a rare form of salt sensitive hypertension. Both disorders lack a personalized therapy that is in most cases only symptomatic. The aim of this study was to identify novel ClC-K ligands from drugs already on the market, by exploiting the pharmacological side activity of drug molecules available from the FDA Adverse Effects Reporting System database. We searched for drugs having a Bartter-like syndrome as a reported side effect, with the assumption that BS could be causatively related to the block of ClC-K channels. The ability of the selected BS-causing drugs to bind and block ClC-K channels was then validated through an integrated experimental and computational approach based on patch clamp electrophysiology in HEK293 cells and molecular docking simulations. Valsartan and olmesartan were able to block ClC-Ka channels and the molecular requirements for effective inhibition of these channels have been identified. These results suggest additional mechanisms of action for these sartans further to their primary AT 1 receptor antagonism and propose these compounds as leads for designing new potent ClC-K ligands. © 2017 The British Pharmacological Society.

  16. Characterization of P2Y receptors mediating ATP induced relaxation in guinea pig airway smooth muscle: involvement of prostaglandins and K+ channels.

    Science.gov (United States)

    Montaño, Luis M; Cruz-Valderrama, José E; Figueroa, Alejandra; Flores-Soto, Edgar; García-Hernández, Luz M; Carbajal, Verónica; Segura, Patricia; Méndez, Carmen; Díaz, Verónica; Barajas-López, Carlos

    2011-10-01

    In airway smooth muscle (ASM), adenosine 5'-triphosphate (ATP) induces a relaxation associated with prostaglandin production. We explored the role of K(+) currents (I (K)) in this relaxation. ATP relaxed the ASM, and this effect was abolished by indomethacin. Removal of airway epithelium slightly diminished the ATP-induced relaxation at lower concentration without modifying the responses to ATP at higher concentrations. ATPγS and UTP induced a concentration-dependent relaxation similar to ATP; α,β-methylene-ATP was inactive from 1 to 100 μM. Suramin or reactive blue 2 (RB2), P2Y receptor antagonists, did not modify the relaxation, but their combination significantly reduced this effect of ATP. The relaxation was also inhibited by N-ethylmaleimide (NEM; which uncouples G proteins). In myocytes, the ATP-induced I (K) increment was not modified by suramin or RB2 but the combination of both drugs abolished it. This increment in the I (K) was also completely nullified by NEM and SQ 22,536. 4-Amynopyridine or iberiotoxin diminished the ATP-induced I (K) increment, and the combination of both substances diminished ATP-induced relaxation. The presence of P2Y(2) and P2Y(4) receptors in smooth muscle was corroborated by Western blot and confocal images. In conclusion, ATP: (1) produces relaxation by inducing the production of bronchodilator prostaglandins in airway smooth muscle, most likely by acting on P2Y(4) and P2Y(2) receptors; (2) induces I (K) increment through activation of the delayed rectifier K(+) channels and the high-conductance Ca(2+)-dependent K(+) channels, therefore both channels are implicated in the ATP-induced relaxation; and (3) this I (K) increment is mediated by prostaglandin production which in turns increase cAMP signaling pathway.

  17. Prostanoid-dependent bladder pain caused by proteinase-activated receptor-2 activation in mice: Involvement of TRPV1 and T-type Ca2+ channels

    Directory of Open Access Journals (Sweden)

    Maho Tsubota

    2018-01-01

    Full Text Available We studied the pronociceptive role of proteinase-activated receptor-2 (PAR2 in mouse bladder. In female mice, intravesical infusion of the PAR2-activating peptide, SLIGRL-amide (SL, caused delayed mechanical hypersensitivity in the lower abdomen, namely ‘referred hyperalgesia’, 6–24 h after the administration. The PAR2-triggered referred hyperalgesia was prevented by indomethacin or a selective TRPV1 blocker, and restored by a T-type Ca2+ channel blocker. In human urothelial T24 cells, SL caused delayed prostaglandin E2 production and COX-2 upregulation. Our data suggest that luminal PAR2 stimulation in the bladder causes prostanoid-dependent referred hyperalgesia in mice, which involves the activation of TRPV1 and T-type Ca2+ channels.

  18. The metabolic impact of β-hydroxybutyrate on neurotransmission: Reduced glycolysis mediates changes in calcium responses and KATP channel receptor sensitivity

    DEFF Research Database (Denmark)

    Lund, Trine Meldgaard; Ploug, K.B.; Iversen, Anne

    2015-01-01

    -hydroxybutyrate might change neuronal function as there is a known coupling between metabolism and neurotransmission. The purpose of this study was to shed light on the effects of the ketone body β-hydroxybutyrate on glycolysis and neurotransmission in cultured murine glutamatergic neurons. Previous studies have shown...... an effect of β-hydroxybutyrate on glucose metabolism, and the present study further specified this by showing attenuation of glycolysis when β-hydroxybutyrate was present in these neurons. In addition, the NMDA receptor-induced calcium responses in the neurons were diminished in the presence of β...... to a combination of glucose and R-β-hydroxybutyrate in cultured neurons. Using the latter combination, glycolysis was diminished, NMDA receptor-induced calcium responses were lower, and the KATP channel blocker glibenclamide caused a higher transmitter release....

  19. A simple and highly selective 2,2-diferrocenylpropane-based multi-channel ion pair receptor for Pb(2+) and HSO4(-).

    Science.gov (United States)

    Wan, Qian; Zhuo, Ji-Bin; Wang, Xiao-Xue; Lin, Cai-Xia; Yuan, Yao-Feng

    2015-03-28

    A structurally simple, 2,2-diferrocenylpropane-based ion pair receptor 1 was synthesized and characterized by (1)H NMR, (13)C NMR, HRMS, elemental analyses, and single-crystal X-ray diffraction. The ion pair receptor 1 showed excellent selectivity and sensitivity towards Pb(2+) with multi-channel responses: a fluorescence enhancement (more than 42-fold), a notable color change from yellow to red, redox anodic shift (ΔE1/2 = 151 mV), while HSO4(-) promoted fluorescence enhancement when Pb(2+) or Zn(2+) was bonded to the cation binding-site. (1)H NMR titration and density functional theory were performed to reveal the sensing mechanism based on photo-induced electron transfer (PET).

  20. Modulation of the transient receptor potential vanilloid channel TRPV4 by 4alpha-phorbol esters: a structure-activity study

    DEFF Research Database (Denmark)

    Klausen, Thomas Kjaer; Pagani, Alberto; Minassi, Alberto

    2009-01-01

    The mechanism of activation of the transient receptor potential vanilloid 4 (TRPV4) channel by 4alpha-phorbol esters was investigated by combining information from chemical modification of 4alpha-phorbol-didecanoate (4alpha-PDD, 2a), site-directed mutagenesis, Ca(2+) imaging, and electrophysiology....... Binding of 4alpha-phorbol esters occurs in a loop in the TM3-TM4 domain of TRPV4 that is analogous to the capsaicin binding site of TRPV1, and the ester decoration of ring C and the A,B ring junction are critical for activity. The lipophilic ester groups on ring C serve mainly as a steering element...

  1. Contribution of the putative inner-pore region to the gating of the Transient Receptor Potential Vanilloid Subtype 1 Channel (TRPV1)

    Czech Academy of Sciences Publication Activity Database

    Sušánková, Klára; Ettrich, Rüdiger; Vyklický st., Ladislav; Teisinger, Jan; Vlachová, Viktorie

    2007-01-01

    Roč. 27, č. 28 (2007), s. 7578-7585 ISSN 0270-6474 R&D Projects: GA ČR(CZ) GA305/06/0319; GA ČR(CZ) GA303/07/0915; GA MŠk(CZ) 1M0517; GA MŠk(CZ) LC554; GA MŠk LC06010 Institutional research plan: CEZ:AV0Z50110509; CEZ:AV0Z60870520 Keywords : capsaicin * vanilloid receptor * TRP channels Subject RIV: ED - Physiology Impact factor: 7.490, year: 2007

  2. On the ligand binding profile and desensitization of plant ionotropic glutamate receptor (iGluR)-like channels functioning in MAMP-triggered Ca2+ influx

    DEFF Research Database (Denmark)

    Kwaaitaal, Mark Adrianus Cornelis J; Maintz, Jens; Cavdar, Meltem

    2012-01-01

    in the putative agonist binding profile and potential mode of desensitization of MAMP-activated plant iGluRs. Based on results from pharmacological inhibition and desensitization experiments, we propose that plant iGluR complexes responsible for the MAMP-triggered Ca ( 2+) signature have a binding profile...... that combines the specificities of mammalian NMDA-and non-NMDA types of iGluRs, possibly reflecting the evolutionary history of plant and animal iGluRs. We further hypothesize that, analogous to the mammalian NMDA-NR1 receptor, desensitization of plant iGluR-like channels might involve binding of the ubiquitous...

  3. The metabolic impact of β-hydroxybutyrate on neurotransmission: Reduced glycolysis mediates changes in calcium responses and KATP channel receptor sensitivity.

    Science.gov (United States)

    Lund, Trine M; Ploug, Kenneth B; Iversen, Anne; Jensen, Anders A; Jansen-Olesen, Inger

    2015-03-01

    Glucose is the main energy substrate for neurons, and ketone bodies are known to be alternative substrates. However, the capacity of ketone bodies to support different neuronal functions is still unknown. Thus, a change in energy substrate from glucose alone to a combination of glucose and β-hydroxybutyrate might change neuronal function as there is a known coupling between metabolism and neurotransmission. The purpose of this study was to shed light on the effects of the ketone body β-hydroxybutyrate on glycolysis and neurotransmission in cultured murine glutamatergic neurons. Previous studies have shown an effect of β-hydroxybutyrate on glucose metabolism, and the present study further specified this by showing attenuation of glycolysis when β-hydroxybutyrate was present in these neurons. In addition, the NMDA receptor-induced calcium responses in the neurons were diminished in the presence of β-hydroxybutyrate, whereas a direct effect of the ketone body on transmitter release was absent. However, the presence of β-hydroxybutyrate augmented transmitter release induced by the KATP channel blocker glibenclamide, thus giving an indirect indication of the involvement of KATP channels in the effects of ketone bodies on transmitter release. Energy metabolism and neurotransmission are linked and involve ATP-sensitive potassium (KATP ) channels. However, it is still unclear how and to what degree available energy substrate affects this link. We investigated the effect of changing energy substrate from only glucose to a combination of glucose and R-β-hydroxybutyrate in cultured neurons. Using the latter combination, glycolysis was diminished, NMDA receptor-induced calcium responses were lower, and the KATP channel blocker glibenclamide caused a higher transmitter release. © 2014 International Society for Neurochemistry.

  4. Modulation of CaV1.2 calcium channel by neuropeptide W regulates vascular myogenic tone via G protein-coupled receptor 7.

    Science.gov (United States)

    Ji, Li; Zhu, Huayuan; Chen, Hong; Fan, Wenyong; Chen, Junjie; Chen, Jing; Zhu, Guoqing; Wang, Juejin

    2015-12-01

    Neuropeptide W (NPW), an endogenous ligand for the G protein-coupled receptor 7 (GPR7), was first found to make important roles in central nerve system. In periphery, NPW was also present and regulated intracellular calcium homeostasis by L-type calcium channels. This study was designed to discover the effects of NPW-GPR7 on the function of CaV1.2 calcium channels in the vascular smooth muscle cells (VSMCs) and vasotone of arterial vessels. By whole-cell patch clamp, we studied the effects of NPW-23, the active form of NPW, on the CaV1.2 channels in the heterologously transfected human embryonic kidney 293 cells and VSMCs isolated from rat. Living system was used to explore the physiological function of NPW-23 in arterial myogenic tone. To investigate the pathological relevance, NPW mRNA level of mesenteric arteries was measured in the hypertensive and normotensive rats. NPW's receptor GPR7 was coexpressed with CaV1.2 channels in arterial smooth muscle. NPW-23 increased the ICa,L in transfected human embryonic kidney 293 cells and VSMCs via GPR7, which could be abrogated by phospholipase C (PLC)/protein kinase C (PKC) inhibitors, not protein kinase A or protein kinase G inhibitor. After NPW-23 application, the expression of pan phospho-PKC was increased; moreover, intracellular diacylglycerol level, the second messenger catalyzed by PLC, was increased 1.5-2-fold. Application with NPW-23 increased pressure-induced vasotone of the rat mesenteric arteries. Importantly, the expression of NPW was decreased in the hypertensive rats. NPW-23 regulates ICa,L via GPR7, which is mediated by PLC/PKC signaling, and such a mechanism plays a role in modulating vascular myogenic tone, which may involve in the development of vascular hypertension.

  5. Intragastric Dai-Kenchu-To, a Japanese herbal medicine, stimulates colonic motility via transient receptor potential cation channel subfamily V member 1 in dogs.

    Science.gov (United States)

    Kikuchi, Daisuke; Shibata, Chikashi; Imoto, Hirofumi; Naitoh, Takeshi; Miura, Koh; Unno, Michiaki

    2013-08-01

    Japanese herbal medicine, also known as Kampo, is used for various diseases in Japan. One of those medicines, Dai-Kenchu-To (DKT), is considered clinically effective for adhesive bowel obstruction and chronic constipation. Although scientific evidence of DKT to improve adhesive bowel obstruction was shown in several previous reports, mechanism of DKT to improve constipation remains unknown. Our aim was to study the effect of intragastric DKT on colonic motility and defecation, and the involvement of various receptors in DKT-induced colonic contractions. Five beagle dogs were instructed with serosal strain-gauge force transducers to measure circular muscle activity at the proximal, middle, and distal colon. Dogs are suitable for a present study to administer the drugs repeatedly to the same individual and look at its effect on colonic motility. We studied the effects of DKT (2.5 or 5 g) administered into the stomach on colonic motility. Muscarinic receptor antagonist atropine, nicotinic receptor antagonist hexamthonium, or 5-hydroxytryptamine-3 receptor antagonist ondansetron was injected intravenously 10 min before DKT administration. Capsazepine, an antagonist to transient receptor potential cation channel subfamily V member 1 (TRPV1), was administered into the stomach 5 min before DKT administration. Intragastric DKT (2.5 or 5 g) induced colonic contractions within 10 min after administration but did not induce defecation. Pretreatment with atropine, hexamthonium, ondansetron, or capsazepine inhibited DKT-induced colonic contractions. These results indicate that orally administered DKT stimulates colonic motility via TRPV1, muscarinic, nicotinic, and 5-hydroxytryptamine-3 receptors, thereby providing scientific support for the efficacy of oral DKT in chronic constipation.

  6. (/sup 3/H)MK-801 labels a site on the N-methyl-D-aspartate receptor channel complex in rat brain membranes

    Energy Technology Data Exchange (ETDEWEB)

    Wong, E H; Knight, A R; Woodruff, G N

    1988-01-01

    The potent noncompetitive N-methyl-D-aspartate (NMDA) receptor antagonist (/sup 3/H)MK-801 bound with nanomolar affinity to rat brain membranes in a reversible, saturable, and stereospecific manner. The affinity of (/sup 3/H)MK-801 was considerably higher in 5 mM Tris-HCl (pH 7.4) than in previous studies using Krebs-Henseleit buffer. (/sup 3/H)MK-801 labels a homogeneous population of sites in rat cerebral cortical membranes with KD of 6.3 nM and Bmax of 2.37 pmol/mg of protein. This binding was unevenly distributed among brain regions, with hippocampus greater than cortex greater than olfactory bulb = striatum greater than medulla-pons, and the cerebellum failing to show significant binding. Detailed pharmacological characterization indicated (/sup 3/H)MK-801 binding to a site which was competitively and potently inhibited by known noncompetitive NMDA receptor antagonists, such as phencyclidine, thienylcyclohexylpiperidine (TCP), ketamine, N-allylnormetazocine (SKF 10,047), cyclazocine, and etoxadrol, a specificity similar to sites labelled by (/sup 3/H)TCP. These sites were distinct from the high-affinity sites labelled by the sigma receptor ligand (+)-(/sup 3/H)SKF 10,047. (/sup 3/H)MK-801 binding was allosterically modulated by the endogenous NMDA receptor antagonist Mg2+ and by other active divalent cations. These data suggest that (/sup 3/H)MK-801 labels a high-affinity site on the NMDA receptor channel complex, distinct from the NMDA recognition site, which is responsible for the blocking action of MK-801 and other noncompetitive NMDA receptor antagonists.

  7. Molecular analysis of the sea anemone toxin Av3 reveals selectivity to insects and demonstrates the heterogeneity of receptor site-3 on voltage-gated Na+ channels.

    Science.gov (United States)

    Moran, Yehu; Kahn, Roy; Cohen, Lior; Gur, Maya; Karbat, Izhar; Gordon, Dalia; Gurevitz, Michael

    2007-08-15

    Av3 is a short peptide toxin from the sea anemone Anemonia viridis shown to be active on crustaceans and inactive on mammals. It inhibits inactivation of Na(v)s (voltage-gated Na+ channels) like the structurally dissimilar scorpion alpha-toxins and type I sea anemone toxins that bind to receptor site-3. To examine the potency and mode of interaction of Av3 with insect Na(v)s, we established a system for its expression, mutagenized it throughout, and analysed it in toxicity, binding and electrophysiological assays. The recombinant Av3 was found to be highly toxic to blowfly larvae (ED50=2.65+/-0.46 pmol/100 mg), to compete well with the site-3 toxin LqhalphaIT (from the scorpion Leiurus quinquestriatus) on binding to cockroach neuronal membranes (K(i)=21.4+/-7.1 nM), and to inhibit the inactivation of Drosophila melanogaster channel, DmNa(v)1, but not that of mammalian Na(v)s expressed in Xenopus oocytes. Moreover, like other site-3 toxins, the activity of Av3 was synergically enhanced by ligands of receptor site-4 (e.g. scorpion beta-toxins). The bioactive surface of Av3 was found to consist mainly of aromatic residues and did not resemble any of the bioactive surfaces of other site-3 toxins. These analyses have portrayed a toxin that might interact with receptor site-3 in a different fashion compared with other ligands of this site. This assumption was corroborated by a D1701R mutation in DmNa(v)1, which has been shown to abolish the activity of all other site-3 ligands, except Av3. All in all, the present study provides further evidence for the heterogeneity of receptor site-3, and raises Av3 as a unique model for design of selective anti-insect compounds.

  8. Roselle Polyphenols Exert Potent Negative Inotropic Effects via Modulation of Intracellular Calcium Regulatory Channels in Isolated Rat Heart.

    Science.gov (United States)

    Lim, Yi-Cheng; Budin, Siti Balkis; Othman, Faizah; Latip, Jalifah; Zainalabidin, Satirah

    2017-07-01

    Roselle (Hibiscus sabdariffa Linn.) calyces have demonstrated propitious cardioprotective effects in animal and clinical studies; however, little is known about its action on cardiac mechanical function. This study was undertaken to investigate direct action of roselle polyphenols (RP) on cardiac function in Langendorff-perfused rat hearts. We utilized RP extract which consists of 12 flavonoids and seven phenolic acids (as shown by HPLC profiling) and has a safe concentration range between 125 and 500 μg/ml in this study. Direct perfusion of RP in concentration-dependent manner lowered systolic function of the heart as shown by lowered LVDP and dP/dt max , suggesting a negative inotropic effect. RP also reduced heart rate (negative chronotropic action) while simultaneously increasing maximal velocity of relaxation (positive lusitropic action). Conversely, RP perfusion increased coronary pressure, an indicator for improvement in coronary blood flow. Inotropic responses elicited by pharmacological agonists for L-type Ca 2+ channel [(±)-Bay K 8644], ryanodine receptor (4-chloro-m-cresol), β-adrenergic receptor (isoproterenol) and SERCA blocker (thapsigargin) were all abolished by RP. In conclusion, RP elicits negative inotropic, negative chronotropic and positive lusitropic responses by possibly modulating calcium entry, release and reuptake in the heart. Our findings have shown the potential use of RP as a therapeutic agent to treat conditions like arrhythmia.

  9. Expression of the transient receptor potential channels TRPV1, TRPA1 and TRPM8 in mouse trigeminal primary afferent neurons innervating the dura

    Science.gov (United States)

    2012-01-01

    Background Migraine and other headache disorders affect a large percentage of the population and cause debilitating pain. Activation and sensitization of the trigeminal primary afferent neurons innervating the dura and cerebral vessels is a crucial step in the “headache circuit”. Many dural afferent neurons respond to algesic and inflammatory agents. Given the clear role of the transient receptor potential (TRP) family of channels in both sensing chemical stimulants and mediating inflammatory pain, we investigated the expression of TRP channels in dural afferent neurons. Methods We used two fluorescent tracers to retrogradely label dural afferent neurons in adult mice and quantified the abundance of peptidergic and non-peptidergic neuron populations using calcitonin gene-related peptide immunoreactivity (CGRP-ir) and isolectin B4 (IB4) binding as markers, respectively. Using immunohistochemistry, we compared the expression of TRPV1 and TRPA1 channels in dural afferent neurons with the expression in total trigeminal ganglion (TG) neurons. To examine the distribution of TRPM8 channels, we labeled dural afferent neurons in mice expressing farnesylated enhanced green fluorescent protein (EGFPf) from a TRPM8 locus. We used nearest-neighbor measurement to predict the spatial association between dural afferent neurons and neurons expressing TRPA1 or TRPM8 channels in the TG. Results and conclusions We report that the size of dural afferent neurons is significantly larger than that of total TG neurons and facial skin afferents. Approximately 40% of dural afferent neurons exhibit IB4 binding. Surprisingly, the percentage of dural afferent neurons containing CGRP-ir is significantly lower than those of total TG neurons and facial skin afferents. Both TRPV1 and TRPA1 channels are expressed in dural afferent neurons. Furthermore, nearest-neighbor measurement indicates that TRPA1-expressing neurons are clustered around a subset of dural afferent neurons. Interestingly, TRPM

  10. Three C-terminal residues from the sulphonylurea receptor contribute to the functional coupling between the KATP channel subunits SUR2A and Kir6.2

    Science.gov (United States)

    Dupuis, Julien P; Revilloud, Jean; Moreau, Christophe J; Vivaudou, Michel

    2008-01-01

    Cardiac ATP-sensitive potassium (KATP) channels are metabolic sensors formed by the association of the inward rectifier potassium channel Kir6.2 and the sulphonylurea receptor SUR2A. SUR2A adjusts channel gating as a function of intracellular ATP and ADP and is the target of pharmaceutical openers and blockers which, respectively, up- and down-regulate Kir6.2. In an effort to understand how effector binding to SUR2A translates into Kir6.2 gating modulation, we examined the role of a 65-residue SUR2A fragment linking transmembrane domain TMD2 and nucleotide-binding domain NBD2 that has been shown to interact with Kir6.2. This fragment of SUR2A was replaced by the equivalent residues of its close homologue, the multidrug resistance protein MRP1. The chimeric construct was expressed in Xenopus oocytes and characterized using the patch-clamp technique. We found that activation by MgADP and synthetic openers was greatly attenuated although apparent affinities were unchanged. Further chimeragenetic and mutagenetic studies showed that mutation of three residues, E1305, I1310 and L1313 (rat numbering), was sufficient to confer this defective phenotype. The same mutations had no effects on channel block by the sulphonylurea glibenclamide or by ATP, suggesting a role for these residues in activatory – but not inhibitory – transduction processes. These results indicate that, within the KATP channel complex, the proximal C-terminal of SUR2A is a critical link between ligand binding to SUR2A and Kir6.2 up-regulation. PMID:18450778

  11. Phosphorylation of Ser1928 mediates the enhanced activity of the L-type Ca2+ channel Cav1.2 by the β2-adrenergic receptor in neurons.

    Science.gov (United States)

    Qian, Hai; Patriarchi, Tommaso; Price, Jennifer L; Matt, Lucas; Lee, Boram; Nieves-Cintrón, Madeline; Buonarati, Olivia R; Chowdhury, Dhrubajyoti; Nanou, Evanthia; Nystoriak, Matthew A; Catterall, William A; Poomvanicha, Montatip; Hofmann, Franz; Navedo, Manuel F; Hell, Johannes W

    2017-01-24

    The L-type Ca 2+ channel Ca v 1.2 controls multiple functions throughout the body including heart rate and neuronal excitability. It is a key mediator of fight-or-flight stress responses triggered by a signaling pathway involving β-adrenergic receptors (βARs), cyclic adenosine monophosphate (cAMP), and protein kinase A (PKA). PKA readily phosphorylates Ser 1928 in Ca v 1.2 in vitro and in vivo, including in rodents and humans. However, S1928A knock-in (KI) mice have normal PKA-mediated L-type channel regulation in the heart, indicating that Ser 1928 is not required for regulation of cardiac Ca v 1.2 by PKA in this tissue. We report that augmentation of L-type currents by PKA in neurons was absent in S1928A KI mice. Furthermore, S1928A KI mice failed to induce long-term potentiation in response to prolonged theta-tetanus (PTT-LTP), a form of synaptic plasticity that requires Ca v 1.2 and enhancement of its activity by the β 2 -adrenergic receptor (β 2 AR)-cAMP-PKA cascade. Thus, there is an unexpected dichotomy in the control of Ca v 1.2 by PKA in cardiomyocytes and hippocampal neurons. Copyright © 2017, American Association for the Advancement of Science.

  12. The Transient Receptor Potential Melastatin 7 Channel Regulates Pancreatic Cancer Cell Invasion through the Hsp90α/uPA/MMP2 pathway

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    Pierre Rybarczyk

    2017-04-01

    Full Text Available Pancreatic ductal adenocarcinoma (PDAC is an aggressive malignancy with a very poor prognosis. There is an urgent need to better understand the molecular mechanisms that regulate PDAC cell aggressiveness. The transient receptor potential melastatin 7 (TRPM7 is a nonselective cationic channel that mainly conducts Ca2+ and Mg2+. TRPM7 is overexpressed in numerous malignancies including PDAC. In the present study, we used the PANC-1 and MIA PaCa-2 cell lines to specifically assess the role of TRPM7 in cell invasion and matrix metalloproteinase secretion. We show that TRPM7 regulates Mg2+ homeostasis and constitutive cation entry in both PDAC cell lines. Moreover, cell invasion is strongly reduced by TRPM7 silencing without affecting the cell viability. Conditioned media were further studied, by gel zymography, to detect matrix metalloproteinase (MMP secretion in PDAC cells. Our results show that MMP-2, urokinase plasminogen activator (uPA, and heat-shock protein 90α (Hsp90α secretions are significantly decreased in TRPM7-deficient PDAC cells. Moreover, TRPM7 expression in human PDAC lymph node metastasis is correlated to the channel expression in primary tumor. Taken together, our results show that TRPM7 is involved in PDAC cell invasion through regulation of Hsp90α/uPA/MMP-2 proteolytic axis, confirming that this channel could be a promising biomarker and possibly a target for PDAC metastasis therapy.

  13. Tris-hydroxymethyl-aminomethane enhances capsaicin-induced intracellular Ca2+ influx through transient receptor potential V1 (TRPV1 channels

    Directory of Open Access Journals (Sweden)

    Satoshi Murakami

    2016-02-01

    Full Text Available Non-selective transient receptor potential vanilloid (TRPV cation channels are activated by various insults, including exposure to heat, acidity, and the compound capsaicin, resulting in sensations of pain in the skin, visceral organs, and oral cavity. Recently, TRPV1 activation was also demonstrated in response to basic pH elicited by ammonia and intracellular alkalization. Tris-hydroxymethyl aminomethane (THAM is widely used as an alkalizing agent; however, the effects of THAM on TRPV1 channels have not been defined. In this study, we characterized the effects of THAM-induced TRPV1 channel activation in baby hamster kidney cells expressing human TRPV1 (hTRPV1 and the Ca2+-sensitive fluorescent sensor GCaMP2 by real-time confocal microscopy. Notably, both capsaicin (1 μM and pH 6.5 buffer elicited steep increases in the intracellular Ca2+ concentration ([Ca2+]i, while treatment with THAM (pH 8.5 alone had no effect. However, treatment with THAM (pH 8.5 following capsaicin application elicited a profound, long-lasting increase in [Ca2+]i that was completely inhibited by the TRPV1 antagonist capsazepine. Taken together, these results suggest that hTRPV1 pre-activation is required to provoke enhanced, THAM-induced [Ca2+]i increases, which could be a mechanism underlying pain induced by basic pH.

  14. Structural Analysis and Deletion Mutagenesis Define Regions of QUIVER/SLEEPLESS that Are Responsible for Interactions with Shaker-Type Potassium Channels and Nicotinic Acetylcholine Receptors.

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    Meilin Wu

    Full Text Available Ly6 proteins are endogenous prototoxins found in most animals. They show striking structural and functional parallels to snake α-neurotoxins, including regulation of ion channels and cholinergic signaling. However, the structural contributions of Ly6 proteins to regulation of effector molecules is poorly understood. This question is particularly relevant to the Ly6 protein QUIVER/SLEEPLESS (QVR/SSS, which has previously been shown to suppress excitability and synaptic transmission by upregulating potassium (K channels and downregulating nicotinic acetylcholine receptors (nAChRs in wake-promoting neurons to facilitate sleep in Drosophila. Using deletion mutagenesis, co-immunoprecipitations, ion flux assays, surface labeling and confocal microscopy, we demonstrate that only loop 2 is required for many of the previously described properties of SSS in transfected cells, including interactions with K channels and nAChRs. Collectively our data suggest that QVR/SSS, and by extension perhaps other Ly6 proteins, target effector molecules using limited protein motifs. Mapping these motifs may be useful in rational design of drugs that mimic or suppress Ly6-effector interactions to modulate nervous system function.

  15. Structural Analysis and Deletion Mutagenesis Define Regions of QUIVER/SLEEPLESS that Are Responsible for Interactions with Shaker-Type Potassium Channels and Nicotinic Acetylcholine Receptors.

    Science.gov (United States)

    Wu, Meilin; Liu, Clifford Z; Joiner, William J

    2016-01-01

    Ly6 proteins are endogenous prototoxins found in most animals. They show striking structural and functional parallels to snake α-neurotoxins, including regulation of ion channels and cholinergic signaling. However, the structural contributions of Ly6 proteins to regulation of effector molecules is poorly understood. This question is particularly relevant to the Ly6 protein QUIVER/SLEEPLESS (QVR/SSS), which has previously been shown to suppress excitability and synaptic transmission by upregulating potassium (K) channels and downregulating nicotinic acetylcholine receptors (nAChRs) in wake-promoting neurons to facilitate sleep in Drosophila. Using deletion mutagenesis, co-immunoprecipitations, ion flux assays, surface labeling and confocal microscopy, we demonstrate that only loop 2 is required for many of the previously described properties of SSS in transfected cells, including interactions with K channels and nAChRs. Collectively our data suggest that QVR/SSS, and by extension perhaps other Ly6 proteins, target effector molecules using limited protein motifs. Mapping these motifs may be useful in rational design of drugs that mimic or suppress Ly6-effector interactions to modulate nervous system function.

  16. Adrenomedullin increases the short-circuit current in the rat prostate: Receptors, chloride channels, the effects of cAMP and calcium ions and implications on fluid secretion.

    Science.gov (United States)

    Liao, S B; Cheung, K H; Cheung, M P L; Wong, P F; O, W S; Tang, F

    2014-05-01

    In this study, we have investigated the effects of adrenomedullin on chloride and fluid secretion in the rat prostate. The presence of adrenomedullin (ADM) in rat prostate was confirmed using immunostaining, and the molecular species was determined using gel filtration chromatography coupled with an enzyme-linked assay for ADM. The effects of ADM on fluid secretion were studied by short-circuit current technique in a whole mount preparation of the prostate in an Ussing chamber. The results indicated that the ADM level was higher in the ventral than the dorso-lateral prostate and the major molecular species was the active peptide. ADM increased the short-circuit current through both the cAMP- and calcium-activated chloride channels in the ventral lobe, but only through the calcium-activated channels in the dorso-lateral lobe. These stimulatory effects were blocked by the calcitonin gene-related peptide (CGRP) receptor antagonist, hCGRP8-37. We conclude that ADM may regulate prostatic fluid secretion through the chloride channels, which may affect the composition of the seminal plasma bathing the spermatozoa and hence fertility. © 2014 American Society of Andrology and European Academy of Andrology.

  17. The quaternary lidocaine derivative, QX-314, exerts biphasic effects on transient receptor potential vanilloid subtype 1 channels in vitro

    DEFF Research Database (Denmark)

    Rivera-Acevedo, Ricardo E; Pless, Stephan Alexander; Ahern, Christopher A

    2011-01-01

    concentrations (less than 1 mM), QX-314 potently inhibited capsaicin-evoked TRPV1 currents with an IC₅₀ of 8.0 ± 0.6 μM. CONCLUSIONS: The results of this study show that the quaternary lidocaine derivative QX-314 exerts biphasic effects on TRPV1 channels, inhibiting capsaicin-evoked TRPV1 currents at lower...... channels. METHODS: The authors conducted an in vitro laboratory study in which they expressed TRPV1 and TRPV4 channels in Xenopus laevis oocytes and recorded cation currents with the two-electrode voltage clamp method. They used confocal microscopy for Ca²⁺ imaging in TRPV1 transient transfected tsA201...

  18. Pharmacological Targeting Of Neuronal Kv7.2/3 Channels: A Focus On Chemotypes And Receptor Sites.

    Science.gov (United States)

    Miceli, Francesco; Soldovieri, Maria Virginia; Ambrosino, Paolo; Manocchio, Laura; Medoro, Alessandro; Mosca, Ilaria; Taglialatela, Maurizio

    2017-10-12

    The Kv7 (KCNQ) subfamily of voltage-gated potassium channels consists of 5 members (Kv7.1-5) each showing a characteristic tissue distribution and physiological roles. Given their functional heterogeneity, Kv7 channels represent important pharmacological targets for development of new drugs for neuronal, cardiac and metabolic diseases. In the present manuscript, we focus on describing the pharmacological relevance and the potential therapeutic applications of drugs acting on neuronally-expressed Kv7.2/3 channels, placing particular emphasis on the different modulator chemotypes, and highlighting their pharmacodynamic and, whenever possible, pharmacokinetic peculiarities. The present work is based on an in-depth search of the currently available scientific literature, and on our own experience and knowledge in the field of neuronal Kv7 channel pharmacology. Space limitations impeded to describe the full pharmacological potential of Kv7 channels; thus, we have chosen to focus on neuronal channels composed of Kv7.2 and Kv7.3 subunits, and to mainly concentrate on their involvement in epilepsy. An astonishing heterogeneity in the molecular scaffolds exploitable to develop Kv7.2/3 modulators is evident, with important structural/functional peculiarities of distinct compound classes. In the present work we have attempted to show the current status and growing potential of the Kv7 pharmacology field. We anticipate a bright future for the field, and we express our hopes that the efforts herein reviewed will result in an improved treatment of hyperexcitability (or any other) diseases. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.

  19. Over-Expression of Dopamine D2 Receptor and Inwardly Rectifying Potassium Channel Genes in Drug-Naive Schizophrenic Peripheral Blood Lymphocytes as Potential Diagnostic Markers

    Directory of Open Access Journals (Sweden)

    Ágnes Zvara

    2005-01-01

    Full Text Available Schizophrenia is one of the most common neuropsychiatric disorders affecting nearly 1% of the human population. Current diagnosis of schizophrenia is based on complex clinical symptoms. The use of easily detectable peripheral molecular markers could substantially help the diagnosis of psychiatric disorders. Recent studies showed that peripheral blood lymphocytes (PBL express subtypes of D1 and D2 subclasses of dopamine receptors. Recently, dopamine receptor D3 (DRD3 was found to be over-expressed in schizophrenic PBL and proposed to be a diagnostic and follow-up marker for schizophrenia. In this study we screened PBL of 13 drug-naive/drug-free schizophrenic patients to identify additional markers of schizophrenia. One of the benefits of our study is the use of blood samples of non-medicated, drug-naive patients. This excludes the possibility that changes detected in gene expression levels might be attributed to the medication rather than to the disorder itself. Among others, genes for dopamine receptor D2 (DRD2 and the inwardly rectifying potassium channel (Kir2.3 were found to be over-expressed in microarray analysis. Increased mRNA levels were confirmed by quantitative real-time PCR (QRT-PCR using the SybrGreen method and dual labeled TaqMan probes. The use of both molecular markers allows a more rapid and precise prediction of schizophrenia and might help find the optimal medication for schizophrenic patients.

  20. Decreased expression of transient receptor potential channels in cerebral vascular tissue from patients after hypertensive intracerebral hemorrhage

    DEFF Research Database (Denmark)

    Thilo, Florian; Suess, Olaf; Liu, Ying

    2011-01-01

    , TRPC5, TRPC6, TRPM4, TRPM6, and TRPM7 channels were detected in cerebral vascular tissue by quantitative real-time RT-PCR. Control cerebral vascular tissue was obtained from normotensive patients who underwent neurosurgical operation because of brain tumor. To examine a possible relation between...

  1. Involvement of transient receptor potential A1 channel in algesic and analgesic actions of the organic compound limonene.

    Science.gov (United States)

    Kaimoto, T; Hatakeyama, Y; Takahashi, K; Imagawa, T; Tominaga, M; Ohta, T

    2016-08-01

    TRPA1 is a Ca-permeable nonselective cation channel expressed in sensory neurons and acts as a nocisensor. Recent reports show that some monoterpenes, a group of naturally occurring organic compounds, modulate TRP channel activity. Here, we report that limonene, being contained in citrus fruits and mushrooms, shows a unique bimodal action on TRPA1 channel. We examine the effects of limonene on sensory neurons from wild-type, TRPV1- and TRPA1-gene-deficient mice and on heterologously expressed channels in vitro. Molecular determinants were identified with using mutated channels. Cellular excitability is monitored with ratiometric Ca imaging. Nociceptive and analgesic actions of limonene are also examined in vivo. In wild-type mouse sensory neurons, limonene increased the intracellular Ca(2+) concentration ([Ca(2+) ]i ), which was inhibited by selective inhibitors of TRPA1 but not TRPV1. Limonene-responsive neurons highly corresponded to TRPA1 agonist-sensitive ones. Limonene failed to stimulate sensory neurons from the TRPA1 (-/-) mouse. Heterologously expressed mouse TRPA1 was activated by limonene. Intraplantar injection of limonene elicited acute pain, which was significantly less in TRPA1 (-/-) mice. Systemic administration of limonene reduced nociceptive behaviours evoked by H2 O2 . In both heterologously and endogenously expressed TRPA1, a low concentration of limonene significantly inhibited H2 O2 -induced TRPA1 activation. TRPA1 activation by limonene was abolished in H2 O2 -insensitive cysteine-mutated channels. Topically applied limonene stimulates TRPA1, resulting in elicitation of acute pain, but its systemic application inhibits nociception induced by oxidative stress. Because limonene is a safe compound, it may be utilized for pain control due to its inhibition of TRPA1 channels. What does this study add: Limonene, a monoterpene in essential oils of various plants, has been known for its antitumor and anti-inflammatory properties. However, molecular

  2. Schedule of NMDA receptor subunit expression and functional channel formation in the course of in vitro-induced neurogenesis

    NARCIS (Netherlands)

    Varju, P; Schlett, K; Eisel, U; Madarasz, E

    NE-7C2 neuroectodermal cells derived from forebrain vesicles of p53-deficient mouse embryos (E9) produce neurons and astrocytes in vitro if induced by all-trans retinoic acid. The reproducible morphological stages of neurogenesis were correlated with the expression of various NMDA receptor subunits.

  3. Serotonin 2C receptor activates a distinct population of arcuate pro-opiomelanocortin neurons via TRPC channels

    Science.gov (United States)

    Serotonin 2C receptors (5-HT2CRs) expressed by pro-opiomelanocortin (POMC) neurons of hypothalamic arcuate nucleus regulate food intake, energy homeostasis ,and glucose metabolism. However, the cellular mechanisms underlying the effects of 5-HT to regulate POMC neuronal activity via 5-HT2CRs have no...

  4. Opposing Roles of Calcium and Intracellular ATP on Gating of the Purinergic P2X2 Receptor Channel

    Directory of Open Access Journals (Sweden)

    Milos B. Rokic

    2018-04-01

    Full Text Available P2X2 receptors (P2X2R exhibit a slow desensitization during the initial ATP application and a progressive, calcium-dependent increase in rates of desensitization during repetitive stimulation. This pattern is observed in whole-cell recordings from cells expressing recombinant and native P2X2R. However, desensitization is not observed in perforated-patched cells and in two-electrode voltage clamped oocytes. Addition of ATP, but not ATPγS or GTP, in the pipette solution also abolishes progressive desensitization, whereas intracellular injection of apyrase facilitates receptor desensitization. Experiments with injection of alkaline phosphatase or addition of staurosporine and ATP in the intracellular solution suggest a role for a phosphorylation-dephosphorylation in receptor desensitization. Mutation of residues that are potential phosphorylation sites identified a critical role of the S363 residue in the intracellular ATP action. These findings indicate that intracellular calcium and ATP have opposing effects on P2X2R gating: calcium allosterically facilitates receptor desensitization and ATP covalently prevents the action of calcium. Single cell measurements further revealed that intracellular calcium stays elevated after washout in P2X2R-expressing cells and the blockade of mitochondrial sodium/calcium exchanger lowers calcium concentrations during washout periods to basal levels, suggesting a role of mitochondria in this process. Therefore, the metabolic state of the cell can influence P2X2R gating.

  5. receptores

    Directory of Open Access Journals (Sweden)

    Salete Regina Daronco Benetti

    2006-01-01

    Full Text Available Se trata de un estudio etnográfico, que tuvo lo objetivo de interpretar el sistema de conocimiento y del significado atribuidos a la sangre referente a la transfusión sanguínea por los donadores y receptores de un banco de sangre. Para la colecta de las informaciones se observaron los participantes y la entrevista etnográfica se realizó el análisis de dominio, taxonómicos y temáticos. Los dominios culturales fueron: la sangre es vida: fuente de vida y alimento valioso; creencias religiosas: fuentes simbólicas de apoyos; donación sanguínea: un gesto colaborador que exige cuidarse, gratifica y trae felicidad; donación sanguínea: fuente simbólica de inseguridad; estar enfermo es una condición para realizar transfusión sanguínea; transfusión sanguínea: esperanza de vida; Creencias populares: transfusión sanguínea como riesgo para la salud; donadores de sangre: personas benditas; donar y recibir sangre: como significado de felicidad. Temática: “líquido precioso que origina, sostiene, modifica la vida, provoca miedo e inseguridad”.

  6. Upregulated Expression of Transient Receptor Potential Cation Channel Subfamily V Receptors in Mucosae of Patients with Oral Squamous Cell Carcinoma and Patients with a History of Alcohol Consumption or Smoking.

    Science.gov (United States)

    Sakakibara, Akiko; Sakakibara, Shunsuke; Kusumoto, Junya; Takeda, Daisuke; Hasegawa, Takumi; Akashi, Masaya; Minamikawa, Tsutomu; Hashikawa, Kazunobu; Terashi, Hiroto; Komori, Takahide

    2017-01-01

    Transient receptor potential cation channel (subfamily V, members 1-4) (TRPV1-4) are expressed in skin and neurons and activated by external stimuli in normal mucosae of all oral cavity sites. The oral cavity is exposed to various stimuli, including temperature, mechanical stimuli, chemical substances, and changes in pH, and, notably, the risk factors for oncogenic transformation in oral squamous epithelium are the same as the external stimuli received by TRPV1-4 receptors. Hence, we examined the relationship between oral squamous cell carcinoma (SCC) and TRPV1-4 expression. Oral SCC patients (n = 37) who underwent surgical resection were included in this study. We investigated the expression of TRPV1-4 by immunohistochemical staining and quantification of TRPV1-4 mRNA in human oral mucosa. In addition, we compared the TRPV1-4 levels in mucosa from patients with SCC to those in normal oral mucosa. The receptors were expressed in oral mucosa at all sites (tongue, buccal mucosa, gingiva, and oral floor) and the expression was stronger in epithelia from patients with SCC than in normal epithelia. Furthermore, alcohol consumption and tobacco use were strongly associated with the occurrence of oral cancer and were found to have a remarkable influence on TRPV1-4 receptor expression in normal oral mucosa. In particular, patients with a history of alcohol consumption demonstrated significantly higher expression levels. Various external stimuli may influence the behavior of cancer cells. Overexpression of TRPV1-4 is likely to be a factor in enhanced sensitivity to external stimuli. These findings could contribute to the establishment of novel strategies for cancer therapy or prevention.

  7. Roles of purinergic P2X receptors as pacemaking channels and modulators of calcium-mobilizing pathway in pituitary gonadotrophs

    Czech Academy of Sciences Publication Activity Database

    Zemková, Hana; Balík, Aleš; Jiang, Y.; Kretschmannová, K.; Stojilkovic, S. S.

    2006-01-01

    Roč. 20, č. 6 (2006), s. 1423-1436 ISSN 0888-8809 R&D Projects: GA AV ČR(CZ) IAA5011408; GA AV ČR(CZ) IAA5011103; GA MŠk(CZ) LC554 Institutional research plan: CEZ:AV0Z50110509 Keywords : purinergic receptors * electrical activity * pituitary Subject RIV: FH - Neurology Impact factor: 4.967, year: 2006

  8. Cyclic ADP ribose-dependent Ca2+ release by group I metabotropic glutamate receptors in acutely dissociated rat hippocampal neurons.

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    Jong-Woo Sohn

    Full Text Available Group I metabotropic glutamate receptors (group I mGluRs; mGluR1 and mGluR5 exert diverse effects on neuronal and synaptic functions, many of which are regulated by intracellular Ca(2+. In this study, we characterized the cellular mechanisms underlying Ca(2+ mobilization induced by (RS-3,5-dihydroxyphenylglycine (DHPG; a specific group I mGluR agonist in the somata of acutely dissociated rat hippocampal neurons using microfluorometry. We found that DHPG activates mGluR5 to mobilize intracellular Ca(2+ from ryanodine-sensitive stores via cyclic adenosine diphosphate ribose (cADPR, while the PLC/IP(3 signaling pathway was not involved in Ca(2+ mobilization. The application of glutamate, which depolarized the membrane potential by 28.5±4.9 mV (n = 4, led to transient Ca(2+ mobilization by mGluR5 and Ca(2+ influx through L-type Ca(2+ channels. We found no evidence that mGluR5-mediated Ca(2+ release and Ca(2+ influx through L-type Ca(2+ channels interact to generate supralinear Ca(2+ transients. Our study provides novel insights into the mechanisms of intracellular Ca(2+ mobilization by mGluR5 in the somata of hippocampal neurons.

  9. Essential role of NMDA receptor channel ε4 subunit (GluN2D in the effects of phencyclidine, but not methamphetamine.

    Directory of Open Access Journals (Sweden)

    Yoko Hagino

    Full Text Available Phencyclidine (PCP, a noncompetitive N-methyl-D-aspartate (NMDA receptor antagonist, increases locomotor activity in rodents and causes schizophrenia-like symptoms in humans. Although activation of the dopamine (DA pathway is hypothesized to mediate these effects of PCP, the precise mechanisms by which PCP induces its effects remain to be elucidated. The present study investigated the effect of PCP on extracellular levels of DA (DA(ex in the striatum and prefrontal cortex (PFC using in vivo microdialysis in mice lacking the NMDA receptor channel ε1 or ε4 subunit (GluRε1 [GluN2A] or GluRε4 [GluN2D] and locomotor activity. PCP significantly increased DA(ex in wildtype and GluRε1 knockout mice, but not in GluRε4 knockout mice, in the striatum and PFC. Acute and repeated administration of PCP did not increase locomotor activity in GluRε4 knockout mice. The present results suggest that PCP enhances dopaminergic transmission and increases locomotor activity by acting at GluRε4.

  10. Cryo-EM structure of the cytoplasmic domain of murine transient receptor potential cation channel subfamily C member 6 (TRPC6).

    Science.gov (United States)

    Azumaya, Caleigh M; Sierra-Valdez, Francisco; Cordero-Morales, Julio F; Nakagawa, Terunaga

    2018-05-11

    The kidney maintains the internal milieu by regulating the retention and excretion of proteins, ions, and small molecules. The glomerular podocyte forms the slit diaphragm of the ultrafiltration filter, whose damage leads to progressive kidney failure and focal segmental glomerulosclerosis (FSGS). The canonical transient receptor potential 6 (TRPC6) ion channel is expressed in the podocyte and mutations in its cytoplasmic domain cause FSGS in humans. In vitro evaluation of disease-causing mutations in TRPC6 has revealed that these genetic alterations result in abnormal ion channel gating. However, the mechanism whereby the cytoplasmic domain modulates TRPC6 function is largely unknown. Here we report a cryoEM structure of the cytoplasmic domain of murine TRPC6 at 3.8Å resolution. The cytoplasmic fold of TRPC6 is characterized by an inverted dome-like chamber pierced by four radial horizontal helices that converge into a vertical coiled-coil at the central axis. Unlike in other TRP channels, TRPC6 displays a unique domain swap that occurs at the junction of the horizontal helices and coiled-coil. Multiple FSGS mutations converge at the buried interface between the vertical coiled-coil and the ankyrin repeats, which form the dome, suggesting these regions are critical for allosteric gating modulation. This functionally critical interface is a potential target for drug design. Importantly, dysfunction in other family members leads to learning deficits (TRPC1/4/5) and ataxia (TRPC3). Our data provide a structural framework for the mechanistic investigation of the TRPC family. Published under license by The American Society for Biochemistry and Molecular Biology, Inc.

  11. Activation of CRH receptor type 1 expressed on glutamatergic neurons increases excitability of CA1 pyramidal neurons by the modulation of voltage-gated ion channels

    Directory of Open Access Journals (Sweden)

    Stephan eKratzer

    2013-07-01

    Full Text Available Corticotropin-releasing hormone (CRH plays an important role in a substantial number of patients with stress-related mental disorders, such as anxiety disorders and depression. CRH has been shown to increase neuronal excitability in the hippocampus, but the underlying mechanisms are poorly understood. The effects of CRH on neuronal excitability were investigated in acute hippocampal brain slices. Population spikes (PS and field excitatory postsynaptic potentials (fEPSP were evoked by stimulating Schaffer-collaterals and recorded simultaneously from the somatic and dendritic region of CA1 pyramidal neurons. CRH was found to increase PS amplitudes (mean  Standard error of the mean; 231.8  31.2% of control; n=10 while neither affecting fEPSPs (104.3 ± 4.2%; n=10 nor long-term potentiation (LTP. However, when Schaffer-collaterals were excited via action potentials (APs generated by stimulation of CA3 pyramidal neurons, CRH increased fEPSP amplitudes (119.8 ± 3.6%; n=8 and the magnitude of LTP in the CA1 region. Experiments in slices from transgenic mice revealed that the effect on PS amplitude is mediated exclusively by CRH receptor 1 (CRHR1 expressed on glutamatergic neurons. The effects of CRH on PS were dependent on phosphatase-2B, L- and T-type calcium channels and voltage-gated potassium channels but independent on intracellular Ca2+-elevation. In patch-clamp experiments, CRH increased the frequency and decay times of APs and decreased currents through A-type and delayed-rectifier potassium channels. These results suggest that CRH does not affect synaptic transmission per se, but modulates voltage-gated ion currents important for the generation of APs and hence elevates by this route overall neuronal activity.

  12. Tarantula huwentoxin-IV inhibits neuronal sodium channels by binding to receptor site 4 and trapping the domain ii voltage sensor in the closed configuration.

    Science.gov (United States)

    Xiao, Yucheng; Bingham, Jon-Paul; Zhu, Weiguo; Moczydlowski, Edward; Liang, Songping; Cummins, Theodore R

    2008-10-03

    Peptide toxins with high affinity, divergent pharmacological functions, and isoform-specific selectivity are powerful tools for investigating the structure-function relationships of voltage-gated sodium channels (VGSCs). Although a number of interesting inhibitors have been reported from tarantula venoms, little is known about the mechanism for their interaction with VGSCs. We show that huwentoxin-IV (HWTX-IV), a 35-residue peptide from tarantula Ornithoctonus huwena venom, preferentially inhibits neuronal VGSC subtypes rNav1.2, rNav1.3, and hNav1.7 compared with muscle subtypes rNav1.4 and hNav1.5. Of the five VGSCs examined, hNav1.7 was most sensitive to HWTX-IV (IC(50) approximately 26 nM). Following application of 1 microm HWTX-IV, hNav1.7 currents could only be elicited with extreme depolarizations (>+100 mV). Recovery of hNav1.7 channels from HWTX-IV inhibition could be induced by extreme depolarizations or moderate depolarizations lasting several minutes. Site-directed mutagenesis analysis indicated that the toxin docked at neurotoxin receptor site 4 located at the extracellular S3-S4 linker of domain II. Mutations E818Q and D816N in hNav1.7 decreased toxin affinity for hNav1.7 by approximately 300-fold, whereas the reverse mutations in rNav1.4 (N655D/Q657E) and the corresponding mutations in hNav1.5 (R812D/S814E) greatly increased the sensitivity of the muscle VGSCs to HWTX-IV. Our data identify a novel mechanism for sodium channel inhibition by tarantula toxins involving binding to neurotoxin receptor site 4. In contrast to scorpion beta-toxins that trap the IIS4 voltage sensor in an outward configuration, we propose that HWTX-IV traps the voltage sensor of domain II in the inward, closed configuration.

  13. Transient Receptor Potential channels: What's happening? Reflections in the wake of the 2009 TRP Meeting, Karolinska Institutet, Stockholm.

    Science.gov (United States)

    Goswami, Chandan; Islam, Md Shahidul

    2010-01-01

    More than 150 participants from 25 countries gathered in Stockholm during 25(th) to 27(th) Sept 2009 to attend the meeting "TRP channels: from sensory signaling to human disease" and enjoyed an international, intensive and vibrant meeting. This meeting shed lights on the recent advances made in this field of research in different sectors of biology, and identified directions for future research and the areas where TRP channels could be used as potential targets for prevention and treatment of human diseases. The participants of this meeting shared their recent largely unpublished data, state-of-the-art techniques and their critical views which would push research in this field forward in the new decade. Another major outcome of this meeting was the realization that extensive work remains to be done to develop the necessary tools and enhance the quality of research in this area so that the prevailing controversies can be resolved. In this report we summarize the latest scientific excitements, some critical issues, as well as some future directions for research that were addressed and discussed in this meeting.

  14. Novel role of cold/menthol-sensitive transient receptor potential melastatine family member 8 (TRPM8) in the activation of store-operated channels in LNCaP human prostate cancer epithelial cells.

    NARCIS (Netherlands)

    Thebault, S.C.; Lemonnier, L.; Bidaux, G.; Flourakis, M.; Bavencoffe, A.; Gordienko, D.; Roudbaraki, M.; Delcourt, P.; Panchin, Y.; Shuba, Y.; Skryma, R.; Prevarskaya, N.

    2005-01-01

    Recent cloning of a cold/menthol-sensitive TRPM8 channel (transient receptor potential melastatine family member 8) from rodent sensory neurons has provided the molecular basis for the cold sensation. Surprisingly, the human orthologue of rodent TRPM8 also appears to be strongly expressed in the

  15. Ca 2+ signaling by plant Arabidopsis thaliana Pep peptides depends on AtPepR1, a receptor with guanylyl cyclase activity, and cGMP-activated Ca 2+ channels

    KAUST Repository

    Qia, Zhi

    2010-11-18

    A family of peptide signaling molecules (AtPeps) and their plasma membrane receptor AtPepR1 are known to act in pathogendefense signaling cascades in plants. Little is currently known about the molecular mechanisms that link these signaling peptides and their receptor, a leucine-rich repeat receptor-like kinase, to downstream pathogen-defense responses. We identify some cellular activities of these molecules that provide the context for a model for their action in signaling cascades. AtPeps activate plasma membrane inwardly conducting Ca 2+ permeable channels in mesophyll cells, resulting in cytosolic Ca 2+ elevation. This activity is dependent on their receptor as well as a cyclic nucleotide-gated channel (CNGC2). We also show that the leucine-rich repeat receptor- like kinase receptor AtPepR1 has guanylyl cyclase activity, generating cGMP from GTP, and that cGMP can activate CNGC2- dependent cytosolic Ca 2+ elevation. AtPep-dependent expression of pathogen-defense genes (PDF1.2, MPK3, and WRKY33) is mediated by the Ca 2+ signaling pathway associated with AtPep peptides and their receptor. The work presented here indicates that extracellular AtPeps, which can act as danger-associated molecular patterns, signal by interaction with their receptor, AtPepR1, a plasma membrane protein that can generate cGMP. Downstream from AtPep and AtPepR1 in a signaling cascade, the cGMP-activated channel CNGC2 is involved in AtPep- and AtPepR1-dependent inward Ca 2+ conductance and resulting cytosolic Ca 2+ elevation. The signaling cascade initiated by AtPeps leads to expression of pathogen- defense genes in a Ca 2+-dependent manner.

  16. Nifedipine, a calcium channel blocker, inhibits advanced glycation end product (AGE)-elicited mesangial cell damage by suppressing AGE receptor (RAGE) expression via peroxisome proliferator-activated receptor-gamma activation

    International Nuclear Information System (INIS)

    Matsui, Takanori; Yamagishi, Sho-ichi; Takeuchi, Masayoshi; Ueda, Seiji; Fukami, Kei; Okuda, Seiya

    2009-01-01

    The interaction between advanced glycation end products (AGE) and their receptor RAGE mediates the progressive alteration in renal architecture and loss of renal function in diabetic nephropathy. Oxidative stress generation and inflammation also play a central role in diabetic nephropathy. This study investigated whether and how nifedipine, a calcium channel blocker (CCB), blocked the AGE-elicited mesangial cell damage in vitro. Nifedipine, but not amlodipine, a control CCB, down-regulated RAGE mRNA levels and subsequently reduced reactive oxygen species (ROS) generation in AGE-exposed mesangial cells. AGE increased mRNA levels of vascular cell adhesion molecule-1 (VCAM-1) and induced monocyte chemoattractant protein-1 (MCP-1) production in mesangial cells, both of which were prevented by the treatment with nifedipine, but not amlodipine. The beneficial effects of nifedipine on AGE-exposed mesangial cells were blocked by the simultaneous treatment of GW9662, an inhibitor of peroxisome proliferator-activated receptor-γ (PPAR-γ). Although nifedipine did not affect expression levels of PPAR-γ, it increased the PPAR-γ transcriptional activity in mesangial cells. Our present study provides a unique beneficial aspect of nifedipine on diabetic nephropathy; it could work as an anti-inflammatory agent against AGE by suppressing RAGE expression in cultured mesangial cells via PPAR-γ activation.

  17. Oral treatment with essential oil of Hyptis spicigera Lam. (Lamiaceae) reduces acute pain and inflammation in mice: Potential interactions with transient receptor potential (TRP) ion channels.

    Science.gov (United States)

    Simões, Róli Rodrigues; Coelho, Igor Dos Santos; Junqueira, Stella Célio; Pigatto, Glauce Regina; Salvador, Marcos José; Santos, Adair Roberto Soares; de Faria, Felipe Meira

    2017-03-22

    The genus Hyptis comprehends almost 400 species widespread in tropical and temperate regions of America. The use of Hyptis spicigera Lam. (Lamiaceae) is reported in traditional medicine due to its gastroprotective, anti-inflammatory and analgesic properties. The rationale of this study was to investigate the potential use of the essential oil of H. spicigera (EOHs) as analgesic. The antinociceptive effect of EOHs was verified analyzing acute nocifensive behavior of mice induced by chemical noxious stimuli [i.e., formalin and transient receptor potential (TRP) channels agonists]. We also verified the effects of EOHs on locomotor activity and motor performance in mice. Finally, we investigate the involvement of central afferent C-fibers with EOHs analgesic effect. EOHs presented antinociceptive effect at 300 and 1000mg/kg on formalin-induced pain behavior model, presenting 50% and 72% of inhibition during the first phase (ED 50 =292mg/kg), and 85% and 100% during de second phase (ED 50 =205mg/kg), respectively. Temperature of the hind paw was reduced by EOHs treatment in a dose-dependent manner; oedema was diminished only by EOHs 1000mg/kg. EOHs does not impaired locomotor activity or motor performance. For mice injected with capsaicin, a TRPV1 activator, EOHs (1000mg/kg, ED 50 =660mg/kg) showed decreased (63%) nociceptive behavior. When injected with cinnamaldehyde (TRPA1 activator), mice treated with EOHs showed 23%, 43% and 66% inhibition on nociceptive behavior (100, 300 and 1000mg/kg, respectively; ED 50 402mg/kg). When mice were injected with menthol (TRPM8 activator), EOHs showed 29%, 59% and 98% inhibition of nociceptive behavior (100, 300 and 1000mg/kg, respectively; with ED 50 =198mg/kg. Finally, when desensitized mice were injected with menthol, EOHs (300mg/kg) does not show antinociceptive effect. This study demonstrated the efficacy of EOHs on experimental models of nociception. We have found the involvement of TRP channels V1, A1 and M8 with EOHs

  18. Regulation of basal and reserve cardiac pacemaker function by interactions of cAMP mediated PKA-dependent Ca2+ cycling with surface membrane channels

    Science.gov (United States)

    Vinogradova, Tatiana M.; Lakatta, Edward G.

    2009-01-01

    Decades of intensive research of primary cardiac pacemaker, the sinoatrial node, have established potential roles of specific membrane channels in the generation of the diastolic depolarization, the major mechanism allowing sinoatrial node cells generate spontaneous beating. During the last three decades, multiple studies made either in the isolated sinoatrial node or sinoatrial node cells have demonstrated a pivotal role of Ca2+ and, specifically Ca2+-release from sarcoplasmic reticulum, for spontaneous beating of cardiac pacemaker. Recently, spontaneous, rhythmic local subsarcolemmal Ca2+ releases from ryanodine receptors during late half of the diastolic depolarization have been implicated as a vital factor in the generation of sinoatrial node cells spontaneous firing. Local Ca2+ releases are driven by a unique combination of high basal cAMP production by adenylyl cyclases, high basal cAMP degradation by phosphodiesterases and a high level of cAMP-mediated PKA-dependent phosphorylation. These local Ca2+ releases activate an inward Na+-Ca2+ exchange current which accelerates the terminal diastolic depolarization rate and, thus, controls the spontaneous pacemaker firing. Both the basal primary pacemaker beating rate and its modulation via β-adrenergic receptor stimulation appear to be critically dependent upon intact RyR function and local subsarcolemmal sarcoplasmic reticulum generated Ca2+ releases. This review aspires to integrate the traditional viewpoint that has emphasized the supremacy of the ensemble of surface membrane ion channels in spontaneous firing of the primary cardiac pacemaker, and these novel perspectives of cAMP-mediated PKA-dependent Ca2+ cycling in regulation of the heart pacemaker clock, both in the basal state and during β-adrenergic receptor stimulation. PMID:19573534

  19. Microparticle-mediated transfer of the viral receptors CAR and CD46, and the CFTR channel in a CHO cell model confers new functions to target cells.

    Directory of Open Access Journals (Sweden)

    Gaëlle Gonzalez

    Full Text Available Cell microparticles (MPs released in the extracellular milieu can embark plasma membrane and intracellular components which are specific of their cellular origin, and transfer them to target cells. The MP-mediated, cell-to-cell transfer of three human membrane glycoproteins of different degrees of complexity was investigated in the present study, using a CHO cell model system. We first tested the delivery of CAR and CD46, two monospanins which act as adenovirus receptors, to target CHO cells. CHO cells lack CAR and CD46, high affinity receptors for human adenovirus serotype 5 (HAdV5, and serotype 35 (HAdV35, respectively. We found that MPs derived from CHO cells (MP-donor cells constitutively expressing CAR (MP-CAR or CD46 (MP-CD46 were able to transfer CAR and CD46 to target CHO cells, and conferred selective permissiveness to HAdV5 and HAdV35. In addition, target CHO cells incubated with MP-CD46 acquired the CD46-associated function in complement regulation. We also explored the MP-mediated delivery of a dodecaspanin membrane glycoprotein, the CFTR to target CHO cells. CFTR functions as a chloride channel in human cells and is implicated in the genetic disease cystic fibrosis. Target CHO cells incubated with MPs produced by CHO cells constitutively expressing GFP-tagged CFTR (MP-GFP-CFTR were found to gain a new cellular function, the chloride channel activity associated to CFTR. Time-course analysis of the appearance of GFP-CFTR in target cells suggested that MPs could achieve the delivery of CFTR to target cells via two mechanisms: the transfer of mature, membrane-inserted CFTR glycoprotein, and the transfer of CFTR-encoding mRNA. These results confirmed that cell-derived MPs represent a new class of promising therapeutic vehicles for the delivery of bioactive macromolecules, proteins or mRNAs, the latter exerting the desired therapeutic effect in target cells via de novo synthesis of their encoded proteins.

  20. Subtype-selective regulation of IP(3) receptors by thimerosal via cysteine residues within the IP(3)-binding core and suppressor domain.

    Science.gov (United States)

    Khan, Samir A; Rossi, Ana M; Riley, Andrew M; Potter, Barry V L; Taylor, Colin W

    2013-04-15

    IP(3)R (IP(3) [inositol 1,4,5-trisphosphate] receptors) and ryanodine receptors are the most widely expressed intracellular Ca(2+) channels and both are regulated by thiol reagents. In DT40 cells stably expressing single subtypes of mammalian IP(3)R, low concentrations of thimerosal (also known as thiomersal), which oxidizes thiols to form a thiomercurylethyl complex, increased the sensitivity of IP(3)-evoked Ca(2+) release via IP(3)R1 and IP(3)R2, but inhibited IP(3)R3. Activation of IP(3)R is initiated by IP(3) binding to the IBC (IP(3)-binding core; residues 224-604) and proceeds via re-arrangement of an interface between the IBC and SD (suppressor domain; residues 1-223). Thimerosal (100 μM) stimulated IP(3) binding to the isolated NT (N-terminal; residues 1-604) of IP(3)R1 and IP(3)R2, but not to that of IP(3)R3. Binding of a competitive antagonist (heparin) or partial agonist (dimeric-IP(3)) to NT1 was unaffected by thiomersal, suggesting that the effect of thimerosal is specifically related to IP(3)R activation. IP(3) binding to NT1 in which all cysteine residues were replaced by alanine was insensitive to thimerosal, so too were NT1 in which cysteine residues were replaced in either the SD or IBC. This demonstrates that thimerosal interacts directly with cysteine in both the SD and IBC. Chimaeric proteins in which the SD of the IP(3)R was replaced by the structurally related A domain of a ryanodine receptor were functional, but thimerosal inhibited both IP(3) binding to the chimaeric NT and IP(3)-evoked Ca(2+) release from the chimaeric IP(3)R. This is the first systematic analysis of the effects of a thiol reagent on each IP(3)R subtype. We conclude that thimerosal selectively sensitizes IP(3)R1 and IP(3)R2 to IP(3) by modifying cysteine residues within both the SD and IBC and thereby stabilizing an active conformation of the receptor.

  1. Subtype-selective regulation of IP3 receptors by thimerosal via cysteine residues within the IP3-binding core and suppressor domain

    Science.gov (United States)

    Khan, Samir A.; Rossi, Ana M.; Riley, Andrew M.; Potter, Barry V. L.; Taylor, Colin W.

    2013-01-01

    IP3R (IP3 [inositol 1,4,5-trisphosphate] receptors) and ryanodine receptors are the most widely expressed intracellular Ca2+ channels and both are regulated by thiol reagents. In DT40 cells stably expressing single subtypes of mammalian IP3R, low concentrations of thimerosal (also known as thiomersal), which oxidizes thiols to form a thiomercurylethyl complex, increased the sensitivity of IP3-evoked Ca2+ release via IP3R1 and IP3R2, but inhibited IP3R3. Activation of IP3R is initiated by IP3 binding to the IBC (IP3-binding core; residues 224–604) and proceeds via re-arrangement of an interface between the IBC and SD (suppressor domain; residues 1–223). Thimerosal (100 μM) stimulated IP3 binding to the isolated NT (N-terminal; residues 1–604) of IP3R1 and IP3R2, but not to that of IP3R3. Binding of a competitive antagonist (heparin) or partial agonist (dimeric-IP3) to NT1 was unaffected by thiomersal, suggesting that the effect of thimerosal is specifically related to IP3R activation. IP3 binding to NT1 in which all cysteine residues were replaced by alanine was insensitive to thimerosal, so too were NT1 in which cysteine residues were replaced in either the SD or IBC. This demonstrates that thimerosal interacts directly with cysteine in both the SD and IBC. Chimaeric proteins in which the SD of the IP3R was replaced by the structurally related A domain of a ryanodine receptor were functional, but thimerosal inhibited both IP3 binding to the chimaeric NT and IP3-evoked Ca2+ release from the chimaeric IP3R. This is the first systematic analysis of the effects of a thiol reagent on each IP3R subtype. We conclude that thimerosal selectively sensitizes IP3R1 and IP3R2 to IP3 by modifying cysteine residues within both the SD and IBC and thereby stabilizing an active conformation of the receptor. PMID:23282150

  2. Stimulation of Oxytocin Receptor during Early Reperfusion Period Protects the Heart against Ischemia/Reperfusion Injury: the Role of Mitochondrial ATPSensitive Potassium Channel, Nitric Oxide, and Prostaglandins

    Directory of Open Access Journals (Sweden)

    Alireza Imani

    2015-10-01

    Full Text Available Postconditioning is a simple and safe strategy for cardioprotection and infarct size limitation. Ourprevious study showed that oxytocin (OT exerts postconditioning effect on ischemic/reperfused isolated ratheart. The aim of this study was to investigate the involvement of OT receptor, mitochondrial ATP-sensitivepotassium channel (mKATP, nitric oxide (NO and cyclooxygenase (COX pathways in OTpostconditioning. Isolated rat hearts were divided into10 groups and underwent 30 min of regional ischemiafollowed by 120 min of reperfusion (n =6. In I/R (ischemia/reperfusion group, ischemia and reperfusionwere induced without any treatment. In OT group, oxytocin was perfused 5 min prior to beginning ofreperfusion for 25 min. In groups 3-6, atosiban (oxytocin receptor blocker, L-NAME (N-Nitro-L-ArginineMethyl Ester, non-specific nitric oxide synthase inhibitor, 5-HD (5-hydroxydecanoate, mKATP inhibitorand indomethacin (cyclooxygenase inhibitor were infused prior to oxytocin administration. In others, thementioned inhibitors were perfused prior to ischemia without oxytocin infusion. Infarct size, ventricularhemodynamic, coronary effluent, malondialdehyde (MDA and lactate dehydrogenase (LDH were measuredat the end of reperfusion. OT perfusion significantly reduced infarct size, MDA and LDH in comparison withIR group. Atosiban, 5HD, L-NAME and indomethacin abolished the postconditioning effect of OT. Perfusionof the inhibitors alone prior to ischemia had no effect on infarct size, hemodynamic parameters, coronaryeffluent and biochemical markers as compared with I/R group. In conclusion, this study indicates thatpostconditioning effects of OT are mediated by activation of mKATP and production of NO andProstaglandins (PGs.

  3. Interaction of xenobiotics with estrogen receptors α and β and a putative plasma sex hormone-binding globulin from channel catfish (Ictalurus punctatus)

    Science.gov (United States)

    Gale, William L.; Patino, Reynaldo; Maule, Alec G.

    2004-01-01

    Estrogens are important regulators of physiological functions. Although environmental contaminants (xenoestrogens) which interfere with estrogen signaling are of increasing concern, there is only limited information about their ability to interact with estrogen-binding proteins (SHBG) or receptors (ER). Recombinant ER?? and ?? were obtained after transient transfection of COS-7 cells with channel catfish ER cDNA. Plasma from adult female channel catfish was the source of SHBG. Tritiated estradiol ( 3H-E2) was used in standard radioligand-binding assays to characterize the binding properties of channel catfish SHBG (ccfSHBG) and to estimate the inhibition constants for various estrogenic compounds. Binding of 3H-E2 to ccfSHBG was saturable and of high affinity with a Kd (??SE) of 1.9??0.14nM and a Bmax of 14.3??2.4pmol/mg protein (n=3 assays). Additionally, ccfSHBG displayed binding specificity for androgens and estrogens. Endosulfan, 4-nonylphenol, and 4-octylphenol displaced 3H-E2 binding to ccfSHBG albeit only at very high concentrations, whereas dieldrin and atrazine showed little displacement activity even at the highest concentrations used. The synthetic estrogen ethynylestradiol had higher affinity than E2 for ccfSHBG. This finding differs from results with human and rainbow trout SHBG. The alkylphenolic compounds (4-octylphenol and 4-nonylphenol) displayed some ability to displace 3H-E2 binding from ER?? and ?? at high concentrations, but dieldrin and atrazine had little binding activity for both ER subtypes and endosulfan for ER??. The xenobiotics tested generally showed equivalent or greater affinity for ER?? than ER??, whereas natural estrogens had much greater affinity for ER?? than ER??. These observations suggest that results of studies using fish tissue ER extracts must be interpreted with caution, since both ER subtypes may be present, and that the binding of xenoestrogens to SHBG must be taken into account for proper assessment of endocrine

  4. Salmon lice (Lepeophtheirus salmonis) showing varying emamectin benzoate susceptibilities differ in neuronal acetylcholine receptor and GABA-gated chloride channel mRNA expression.

    Science.gov (United States)

    Carmichael, Stephen N; Bron, James E; Taggart, John B; Ireland, Jacqueline H; Bekaert, Michaël; Burgess, Stewart Tg; Skuce, Philip J; Nisbet, Alasdair J; Gharbi, Karim; Sturm, Armin

    2013-06-18

    Caligid copepods, also called sea lice, are fish ectoparasites, some species of which cause significant problems in the mariculture of salmon, where the annual cost of infection is in excess of €300 million globally. At present, caligid control on farms is mainly achieved using medicinal treatments. However, the continued use of a restricted number of medicine actives potentially favours the development of drug resistance. Here, we report transcriptional changes in a laboratory strain of the caligid Lepeophtheirus salmonis (Krøyer, 1837) that is moderately (~7-fold) resistant to the avermectin compound emamectin benzoate (EMB), a component of the anti-salmon louse agent SLICE® (Merck Animal Health). Suppression subtractive hybridisation (SSH) was used to enrich transcripts differentially expressed between EMB-resistant (PT) and drug-susceptible (S) laboratory strains of L. salmonis. SSH libraries were subjected to 454 sequencing. Further L. salmonis transcript sequences were available as expressed sequence tags (EST) from GenBank. Contiguous sequences were generated from both SSH and EST sequences and annotated. Transcriptional responses in PT and S salmon lice were investigated using custom 15 K oligonucleotide microarrays designed using the above sequence resources. In the absence of EMB exposure, 359 targets differed in transcript abundance between the two strains, these genes being enriched for functions such as calcium ion binding, chitin metabolism and muscle structure. γ-aminobutyric acid (GABA)-gated chloride channel (GABA-Cl) and neuronal acetylcholine receptor (nAChR) subunits showed significantly lower transcript levels in PT lice compared to S lice. Using RT-qPCR, the decrease in mRNA levels was estimated at ~1.4-fold for GABA-Cl and ~2.8-fold for nAChR. Salmon lice from the PT strain showed few transcriptional responses following acute exposure (1 or 3 h) to 200 μg L-1 of EMB, a drug concentration tolerated by PT lice, but toxic for S lice

  5. Ghrelin upregulates the phosphorylation of the GluN2B subunit of the NMDA receptor by activating GHSR1a and Fyn in the rat hippocampus.

    Science.gov (United States)

    Berrout, Liza; Isokawa, Masako

    2018-01-01

    Ghrelin and its receptor GHSR1a have been shown to exert numerous physiological functions in the brain, in addition to the well-established orexigenic role in the hypothalamus. Earlier work indicated that ghrelin stimulated the phosphorylation of the GluN1 subunit of the NMDA receptor (NMDAR) and enhanced synaptic transmission in the hippocampus. In the present study, we report that the exogenous application of ghrelin increased GluN2B phosphorylation. This increase was independent of GluN2B subunit activity or NMDAR channel activity. However, it depended on the activation of GHSR1a and Fyn as it was blocked by D-Lys3-GHRP-6 and PP2, respectively. Inhibitors for G-protein-regulated second messengers, such as Rp-cAMP, H89, TBB, ryanodine, and thapsigargin, unexpectedly enhanced GluN2B phosphorylation, suggesting that cAMP, PKA, casein kinase II, and cytosolic calcium signaling may oppose to the effect of ghrelin on the phosphorylation of GluN2B. Our findings suggest that 1) GluN2B is likely a molecular target of ghrelin and GHSR1a-driven signaling cascades, and 2) the ghrelin-mediated phosphorylation of GluN2B depends on Fyn activation under complex negative regulation by other second messengers. Copyright © 2017 Elsevier B.V. All rights reserved.

  6. Downregulation of transient receptor potential M6 channels as a cause of hypermagnesiuric hypomagnesemia in obese type 2 diabetic rats.

    Science.gov (United States)

    Takayanagi, Kaori; Shimizu, Taisuke; Tayama, Yosuke; Ikari, Akira; Anzai, Naohiko; Iwashita, Takatsugu; Asakura, Juko; Hayashi, Keitaro; Mitarai, Tetsuya; Hasegawa, Hajime

    2015-06-15

    We assessed the expression profile of Mg(2+)-transporting molecules in obese diabetic rats as a cause of hypermagnesiuric hypomagnesemia, which is involved in the development of insulin resistance, hypertension, and coronary diseases. Kidneys were obtained from male Otsuka Long-Evans Tokushima fatty (OLETF) and Long-Evans Tokushima Otsuka (LETO) obese diabetic rats at the ages of 16, 24, and 34 wk. Expression profiles were studied by real-time PCR and immunohistochemistry together with measurements of urine Mg(2+) excretion. Urine Mg(2+) excretion was increased in 24-wk-old OLETF rats and hypomagnesemia was apparent in 34-wk-old OLETF rats but not in LETO rats (urine Mg(2+) excretion: 0.16 ± 0.01 μg·min(-1)·g body wt(-1) in 24-wk-old LETO rats and 0.28 ± 0.01 μg·min(-1)·g body wt(-1) in 24-wk-old OLETF rats). Gene expression of transient receptor potential (TRP)M6 was downregulated (85.5 ± 5.6% in 34-wk-old LETO rats and 63.0 ± 3.5% in 34-wk-old OLETF rats) concomitant with Na(+)-Cl(-) cotransporter downregulation, whereas the expression of claudin-16 in tight junctions of the thick ascending limb of Henle was not different. The results of the semiquantitative analysis of immunohistochemistry were consistent with these findings (TRPM6: 0.49 ± 0.04% in 16-wk-old LETO rats, 0.10 ± 0.01% in 16-wk-old OLETF rats, 0.52 ± 0.03% in 24-wk-old LETO rats, 0.10 ± 0.01% in 24-wk-old OLETF rats, 0.48 ± 0.02% in 34-wk-old LETO rats, and 0.12 ± 0.02% in 34-wk-old OLETF rats). Gene expression of fibrosis-related proinflammatory cytokines as well as histological changes showed that the hypermagnesiuria-related molecular changes and tubulointerstitial nephropathy developed independently. TRPM6, located principally in distal convoluted tubules, appears to be a susceptible molecule that causes hypermagnesiuric hypomagnesemia as a tubulointerstitial nephropathy-independent altered tubular function in diabetic nephropathy. Copyright © 2015 the American Physiological

  7. The Drosophila nicotinic acetylcholine receptor subunits Dα5 and Dα7 form functional homomeric and heteromeric ion channels

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    Lansdell Stuart J

    2012-06-01

    Full Text Available Abstract Background Nicotinic acetylcholine receptors (nAChRs play an important role as excitatory neurotransmitters in vertebrate and invertebrate species. In insects, nAChRs are the site of action of commercially important insecticides and, as a consequence, there is considerable interest in examining their functional properties. However, problems have been encountered in the successful functional expression of insect nAChRs, although a number of strategies have been developed in an attempt to overcome such difficulties. Ten nAChR subunits have been identified in the model insect Drosophila melanogaster (Dα1-Dα7 and Dβ1-Dβ3 and a similar number have been identified in other insect species. The focus of the present study is the Dα5, Dα6 and Dα7 subunits, which are distinguished by their sequence similarity to one another and also by their close similarity to the vertebrate α7 nAChR subunit. Results A full-length cDNA clone encoding the Drosophila nAChR Dα5 subunit has been isolated and the properties of Dα5-, Dα6- and Dα7-containing nAChRs examined in a variety of cell expression systems. We have demonstrated the functional expression, as homomeric nAChRs, of the Dα5 and Dα7 subunits in Xenopus oocytes by their co-expression with the molecular chaperone RIC-3. Also, using a similar approach, we have demonstrated the functional expression of a heteromeric ‘triplet’ nAChR (Dα5 + Dα6 + Dα7 with substantially higher apparent affinity for acetylcholine than is seen with other subunit combinations. In addition, specific cell-surface binding of [125I]-α-bungarotoxin was detected in both Drosophila and mammalian cell lines when Dα5 was co-expressed with Dα6 and RIC-3. In contrast, co-expression of additional subunits (including Dα7 with Dα5 and Dα6 prevented specific binding of [125I]-α-bungarotoxin in cell lines, suggesting that co-assembly with other nAChR subunits can block maturation of correctly folded nAChRs in

  8. Possible involvement of the transient receptor potential vanilloid type 1 channel in postoperative adhesive obstruction and its prevention by a kampo (traditional Japanese) medicine, daikenchuto.

    Science.gov (United States)

    Tokita, Yohei; Yamamoto, Masahiro; Satoh, Kazuko; Nishiyama, Mitsue; Iizuka, Seiichi; Imamura, Sachiko; Kase, Yoshio

    2011-01-01

    This study focused on the localization of transient receptor potential vanilloid type 1 (TRPV1) in the intestines in postoperative adhesion model rats and investigated the underlying mechanism for the anti-adhesion action of daikenchuto (DKT), especially in relation to TRPV1. Postoperative intestinal adhesion was induced by sprinkling talc in the small intestine. The expression of TRPV1 mRNA was examined by in situ hybridization and real-time RT-PCR. The effects of DKT and its major ingredient, hydroxy sanshool, with or without ruthenium red, a TRP-channel antagonist, on talc-induced intestinal adhesions were evaluated. The level of TRPV1 mRNA was higher in the adhesion regions of talc-treated rats than in normal small intestine of sham-operated rats. Localization of TRPV1 mRNA expression was identified in the submucosal plexus of both sham-operated and talc-treated rats; and in talc-treated rats, it was observed also in the myenteric plexus and regions of adhesion. Capsaicin, DKT, and hydroxy sanshool significantly prevented formation of intestinal adhesions. The effects of DKT and hydroxy sanshool were abrogated by subcutaneous injection of ruthenium red. These results suggest that pharmacological modulation of TRPV1 might be a possible therapeutic option in postoperative intestinal adhesion, which might be relevant to the prevention of postoperative adhesive obstruction by DKT.

  9. Presynaptic muscarinic receptors, calcium channels, and protein kinase C modulate the functional disconnection of weak inputs at polyinnervated neonatal neuromuscular synapses.

    Science.gov (United States)

    Santafe, M M; Garcia, N; Lanuza, M A; Tomàs, M; Besalduch, N; Tomàs, J

    2009-04-01

    We studied the relation among calcium inflows, voltage-dependent calcium channels (VDCC), presynaptic muscarinic acetylcholine receptors (mAChRs), and protein kinase C (PKC) activity in the modulation of synapse elimination. We used intracellular recording to determine the synaptic efficacy in dually innervated endplates of the levator auris longus muscle of newborn rats during axonal competition in the postnatal synaptic elimination period. In these dual junctions, the weak nerve terminal was potentiated by partially reducing calcium entry (P/Q-, N-, or L-type VDCC-specific block or 500 muM magnesium ions), M1- or M4-type selective mAChR block, or PKC block. Moreover, reducing calcium entry or blocking PKC or mAChRs results in unmasking functionally silent nerve endings that now recover neurotransmitter release. Our results show interactions between these molecules and indicate that there is a release inhibition mechanism based on an mAChR-PKC-VDCC intracellular cascade. When it is fully active in certain weak motor axons, it can depress ACh release and even disconnect synapses. We suggest that this mechanism plays a central role in the elimination of redundant neonatal synapses, because functional axonal withdrawal can indeed be reversed by mAChRs, VDCCs, or PKC block.

  10. Nuclear receptor CAR specifically activates the two-pore K+ channel Kcnk1 gene in male mouse livers, which attenuates phenobarbital-induced hepatic hyperplasia.

    Science.gov (United States)

    Saito, Kosuke; Moore, Rick; Negishi, Masahiko

    2013-03-01

    KCNK1, a member of the family of two-pore K(+) ion channels, is specifically induced in the livers of male mice after phenobarbital treatment. Here, we have determined the molecular mechanism of this male-specific activation of the Kcnk1 gene and characterized KCNK1 as a phenobarbital-inducible antihyperplasia factor. Upon activation by phenobarbital, nuclear receptor CAR binds the 97-bp response element (-2441/-2345) within the Kcnk1 promoter. This binding is observed in the livers of male mice, but not in the livers of female mice and requires the pituitary gland, because hypophysectomy abrogates it. Hyperplasia further progressed in the livers of Kcnk1 ( -/- ) male mice compared with those of Kcnk1 ( +/+ ) males after phenobarbital treatment. Thus, KCNK1 suppresses phenobarbital-induced hyperplasia. These results indicate that phenobarbital treatment induces KCNK1 to elicit a male-specific and growth-suppressing signal. Thus, KCNK1 and Kcnk1 ( -/- ) mice provide an experimental tool for further investigation into the molecular mechanism of CAR-mediated promotion of the development of hepatocellular carcinoma in mice.

  11. Transient receptor potential ion channel Trpm7 regulates exocrine pancreatic epithelial proliferation by Mg2+-sensitive Socs3a signaling in development and cancer

    Directory of Open Access Journals (Sweden)

    Nelson S. Yee

    2011-03-01

    Genetic analysis of pancreatic development has provided new insights into the mechanisms underlying the formation of exocrine pancreatic neoplasia. Zebrafish sweetbread (swd mutants develop hypoplastic acini and dysmorphic ducts in the exocrine pancreas, with impeded progression of cell division cycle and of epithelial growth. Positional cloning and allelic complementation have revealed that the swd mutations affect the transient receptor potential melastatin-subfamily member 7 (trpm7 gene, which encodes a divalent cation-permeable channel with kinase activity. Supplementary Mg2+ partially rescued the exocrine pancreatic defects of the trpm7 mutants by improving cell-cycle progression and growth and repressing the suppressor of cytokine signaling 3a (socs3a gene. The role of Socs3a in Trpm7-mediated signaling is supported by the findings that socs3a mRNA level is elevated in the trpm7 mutants, and antisense inhibition of socs3a expression improved their exocrine pancreatic growth. TRPM7 is generally overexpressed in human pancreatic adenocarcinoma. TRPM7-deficient cells are impaired in proliferation and arrested in the G0-G1 phases of the cell division cycle. Supplementary Mg2+ rescued the proliferative defect of the TRPM7-deficient cells. Results of this study indicate that Trpm7 regulates exocrine pancreatic development via the Mg2+-sensitive Socs3a pathway, and suggest that aberrant TRPM7-mediated signaling contributes to pancreatic carcinogenesis.

  12. NMDA receptors in mouse anterior piriform cortex initialize early odor preference learning and L-type calcium channels engage for long-term memory.

    Science.gov (United States)

    Mukherjee, Bandhan; Yuan, Qi

    2016-10-14

    The interactions of L-type calcium channels (LTCCs) and NMDA receptors (NMDARs) in memories are poorly understood. Here we investigated the specific roles of anterior piriform cortex (aPC) LTCCs and NMDARs in early odor preference memory in mice. Using calcium imaging in aPC slices, LTCC activation was shown to be dependent on NMDAR activation. Either D-APV (NMDAR antagonist) or nifedipine (LTCC antagonist) reduced somatic calcium transients in pyramidal cells evoked by lateral olfactory tract stimulation. However, nifedipine did not further reduce calcium in the presence of D-APV. In mice that underwent early odor preference training, blocking NMDARs in the aPC prevented short-term (3 hr) and long-term (24 hr) odor preference memory, and both memories were rescued when BayK-8644 (LTCC agonist) was co-infused. However, activating LTCCs in the absence of NMDARs resulted in loss of discrimination between the conditioned odor and a similar odor mixture at 3 hr. Elevated synaptic AMPAR expression at 3 hr was prevented by D-APV infusion but restored when LTCCs were directly activated, mirroring the behavioral outcomes. Blocking LTCCs prevented 24 hr memory and spared 3 hr memory. These results suggest that NMDARs mediate stimulus-specific encoding of odor memory while LTCCs mediate intracellular signaling leading to long-term memory.

  13. Ligand induced change of β2 adrenergic receptor from active to inactive conformation and its implication for the closed/open state of the water channel: insight from molecular dynamics simulation, free energy calculation and Markov state model analysis.

    Science.gov (United States)

    Bai, Qifeng; Pérez-Sánchez, Horacio; Zhang, Yang; Shao, Yonghua; Shi, Danfeng; Liu, Huanxiang; Yao, Xiaojun

    2014-08-14

    The reported crystal structures of β2 adrenergic receptor (β2AR) reveal that the open and closed states of the water channel are correlated with the inactive and active conformations of β2AR. However, more details about the process by which the water channel states are affected by the active to inactive conformational change of β2AR remain illusive. In this work, molecular dynamics simulations are performed to study the dynamical inactive and active conformational change of β2AR induced by inverse agonist ICI 118,551. Markov state model analysis and free energy calculation are employed to explore the open and close states of the water channel. The simulation results show that inverse agonist ICI 118,551 can induce water channel opening during the conformational transition of β2AR. Markov state model (MSM) analysis proves that the energy contour can be divided into seven states. States S1, S2 and S5, which represent the active conformation of β2AR, show that the water channel is in the closed state, while states S4 and S6, which correspond to the intermediate state conformation of β2AR, indicate the water channel opens gradually. State S7, which represents the inactive structure of β2AR, corresponds to the full open state of the water channel. The opening mechanism of the water channel is involved in the ligand-induced conformational change of β2AR. These results can provide useful information for understanding the opening mechanism of the water channel and will be useful for the rational design of potent inverse agonists of β2AR.

  14. Comparative effect of angiotensin II type I receptor blockers and calcium channel blockers on laboratory parameters in hypertensive patients with type 2 diabetes

    Directory of Open Access Journals (Sweden)

    Nishida Yayoi

    2012-05-01

    Full Text Available Abstract Background Both angiotensin II type I receptor blockers (ARBs and calcium channel blockers (CCBs are widely used antihypertensive drugs. Many clinical studies have demonstrated and compared the organ-protection effects and adverse events of these drugs. However, few large-scale studies have focused on the effect of these drugs as monotherapy on laboratory parameters. We evaluated and compared the effects of ARB and CCB monotherapy on clinical laboratory parameters in patients with concomitant hypertension and type 2 diabetes mellitus. Methods We used data from the Clinical Data Warehouse of Nihon University School of Medicine obtained between Nov 1, 2004 and July 31, 2011, to identify cohorts of new ARB users (n = 601 and propensity-score matched new CCB users (n = 601, with concomitant mild to moderate hypertension and type 2 diabetes mellitus. We used a multivariate-adjusted regression model to adjust for differences between ARB and CCB users, and compared laboratory parameters including serum levels of triglyceride (TG, total cholesterol (TC, non-fasting blood glucose, hemoglobin A1c (HbA1c, sodium, potassium, creatinine, alanine aminotransferase (ALT, aspartate aminotransferase (AST, gamma-glutamyltransferase (GGT, hemoglobin and hematocrit, and white blood cell (WBC, red blood cell (RBC and platelet (PLT counts up to 12 months after the start of ARB or CCB monotherapy. Results We found a significant reduction of serum TC, HbA1c, hemoglobin and hematocrit and RBC count and a significant increase of serum potassium in ARB users, and a reduction of serum TC and hemoglobin in CCB users, from the baseline period to the exposure period. The reductions of RBC count, hemoglobin and hematocrit in ARB users were significantly greater than those in CCB users. The increase of serum potassium in ARB users was significantly greater than that in CCB users. Conclusions Our study suggested that hematological adverse effects and

  15. Angiotensin Converting-Enzyme Inhibitors, Angiotensin Receptor Blockers, and Calcium Channel Blockers Are Associated with Prolonged Vascular Access Patency in Uremic Patients Undergoing Hemodialysis.

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    Fu-An Chen

    Full Text Available Vascular access failure is a huge burden for patients undergoing hemodialysis. Many efforts have been made to maintain vascular access patency, including pharmacotherapy. Angiotensin converting enzyme inhibitor (ACE-I, angiotensin receptor blocker (ARB, and calcium channel blocker (CCB are known for their antihypertensive and cardio-protective effects, however, their effects on long-term vascular access patency are still inconclusive.We retrospectively enrolled patients commencing maintenance hemodialysis between January 1, 2000, and December 31, 2006 by using National Health Insurance Research Database in Taiwan. Primary patency was defined as the date of first arteriovenous fistula (AVF or arteriovenous graft (AVG creation to the time of access thrombosis or any intervention aimed to maintain or re-establish vascular access patency. Cox proportional hazards models were used to adjust the influences of patient characteristics, co-morbidities and medications.Total 42244 patients were enrolled in this study, 37771 (89.4% used AVF, 4473 (10.6% used AVG as their first long term dialysis access. ACE-I, ARB, and CCB use were all associated with prolonged primary patency of AVF [hazard ratio (HR 0.586, 95% confidence interval (CI 0.557-0.616 for ACE-I use; HR 0.532, CI 0.508-0.556 for ARB use; HR 0.485, CI 0.470-0.501 for CCB use] and AVG (HR 0.557, CI 0.482-0.643 for ACE-I use, HR 0.536, CI 0.467-0.614 for ARB use, HR 0.482, CI 0.442-0.526 for CCB use.In our analysis, ACE-I, ARB, and CCB were strongly associated with prolonged primary patency of both AVF and AVG. Further prospective randomized studies are still warranted to prove the causality.

  16. Calcium channel blockers, angiotensin receptor blockers, and angiotensin-converting enzyme inhibitors: Effectiveness in combination with diuretics or β-blockers for treating hypertension

    Directory of Open Access Journals (Sweden)

    John D Bisognano

    2007-11-01

    Full Text Available John D Bisognano1, Trent McLaughlin2, Craig S Roberts3, Simon SK Tang31Internal Medicine Department, Cardiology Division, the University of Rochester Medical Center, Rochester, NY, USA; 2NDC Health, Phoenix, Arizona, USA; 3Pfizer Inc, New York, NY, USAAbstract: This retrospective database analysis compared the effectiveness of dihydropyridine calcium channel blockers (DHPs, angiotensin-converting enzyme (ACE inhibitors, and angiotensin receptor blockers (ARBs added to diuretics or β-blockers. Adults with hypertension treated with diuretic or β-blocker monotherapy between 1998 and 2001 were identified from a large US electronic medical records database of primary care practices. Patients were required to have a baseline blood pressure (BP ≥140/90 mmHg (≥130/80 mmHg for diabetes mellitus and recorded BP measurements within 6 months before and 1–12 months following index date. Patients were matched 1:1:1 by propensity score to correct for differences in baseline characteristics. 1875 patients met study criteria and 660 (220 in each cohort were matched based on propensity scores. Matched cohorts had no significant differences in baseline characteristics. Mean changes in systolic/diastolic BP were –17.5/–8.8, –15.7/–6.3, and –13.0/–8.0 mmHg with DHPs, ACE inhibitors, and ARBs, respectively. Joint National Committee on the Prevention, Detection, Evaluation, and Treatment of High BP 6/7 goal attainment for each regimen was 47.3%, 40.0%, and 32.2%, respectively. DHPs, ACE inhibitors, and ARBs improved BP when added to patients’ β-blocker or diuretic therapy. The greatest benefits were observed with DHPs, followed by ACE inhibitors, then ARBs.Keywords: hypertension, amlodipine besylate, lisinopril, valsartan, Joint National Committee (JNC 6 and 7

  17. Pannexin channels mediate the acquisition of myogenic commitment in C2C12 reserve cells promoted by P2 receptor activation

    Science.gov (United States)

    Riquelme, Manuel A.; Cea, Luis A.; Vega, José L.; Puebla, Carlos; Vargas, Aníbal A.; Shoji, Kenji F.; Subiabre, Mario; Sáez, Juan C.

    2015-01-01

    The acquisition of myoblast commitment to the myogenic linage requires rises in intracellular free Ca2+ concentration ([Ca2+]i). Putative cell membrane pathways involved in these [Ca2+]i increments are P2 receptors (P2Rs) as well as connexin (Cx) and/or pannexin (Panx) hemichannels and channels (Cx HChs and Panx Chs), respectively, which are known to permeate Ca2+. Reserve cells (RCs) are uncommitted myoblasts obtained from differentiated C2C12 cell cultures, which acquire commitment upon replating. Regarding these cells, we found that extracellular ATP increases the [Ca2+]i via P2Rs. Moreover, ATP increases the plasma membrane permeability to small molecules and a non-selective membrane current, both of which were inhibited by Cx HCh/Panx1Ch blockers. However, RCs exposed to divalent cation-free saline solution, which is known to activate Cx HChs (but not Panx Chs), did not enhance membrane permeability, thus ruling out the possible involvement of Cx HChs. Moreover, ATP-induced membrane permeability was inhibited with blockers of P2Rs that activate Panx Chs. In addition, exogenous ATP induced the expression of myogenic commitment and increased MyoD levels, which was prevented by the inhibition of P2Rs or knockdown of Panx1 Chs. Similarly, increases in MyoD levels induced by ATP released by RCs were inhibited by Panx Ch/Cx HCh blockers. Myogenic commitment acquisition thus requires a feed-forward mechanism mediated by extracellular ATP, P2Rs, and Panx Chs. PMID:26000275

  18. Alterations in expression of Cat-315 epitope of perineuronal nets during normal ageing, and its modulation by an open-channel NMDA receptor blocker, memantine.

    Science.gov (United States)

    Yamada, Jun; Ohgomori, Tomohiro; Jinno, Shozo

    2017-06-15

    The perineuronal net (PNN), a specialized aggregate of the extracellular matrix, is involved in neuroprotection against oxidative stress, which is now recognized as a major contributor to age-related decline in brain functions. In this study, we investigated the age-related molecular changes of PNNs using monoclonal antibody Cat-315, which recognizes human natural killer-1 (HNK-1) glycan on aggrecan-based PNNs. Western blot analysis showed that the expression levels of Cat-315 epitope in the hippocampus were higher in middle-aged (MA, 12-month-old) mice than in young adult (YA, 2-month-old) mice. Although there were no differences in the expression levels of Cat-315 epitope between old age (OA, 20-month-old) and MA mice, Cat-315 immunoreactivity was also detected in astrocytes of OA mice. To focus on Cat-315 epitope in PNNs, we used YA and MA mice in the following experiments. Optical disector analysis showed that there were no differences in the numbers of Cat-315-positive (Cat-315 + ) PNNs between YA and MA mice. Fluorescence intensity analysis indicated that Cat-315 immunoreactivity in PNNs increased with age in the dorsal hippocampus, which is mainly involved in cognitive functions. Administration of an open-channel blocker of NMDA receptor, memantine, reduced the expression levels of Cat-315 epitope in the hippocampus. Furthermore, the numbers of glutamatergic and GABAergic terminals colocalized with Cat-315 epitope around parvalbumin-positive neurons were decreased by memantine. These findings provide novel insight into the involvement of PNNs in normal brain ageing, and suggest that memantine may counteract the age-related alterations in expression levels of Cat-315 epitope via regulation of its subcellular localization. © 2017 Wiley Periodicals, Inc.

  19. Clinical presentation of anti-N-methyl-d-aspartate receptor and anti-voltage-gated potassium channel complex antibodies in children: A series of 24 cases.

    Science.gov (United States)

    Konuskan, Bahadir; Yildirim, Mirac; Topaloglu, Haluk; Erol, Ilknur; Oztoprak, Ulkuhan; Tan, Huseyin; Gocmen, Rahsan; Anlar, Banu

    2018-01-01

    The symptomatology and paraclinical findings of antibody-mediated encephalitis, a relatively novel disorder, are still being characterized in adults and children. A high index of suspicion is needed in order to identify these cases among children presenting with various neurological symptoms. The aim of this study is to examine the clinical, demographic and laboratory findings and outcome of children with anti-NMDAR and anti-VGKC encephalitis for any typical or distinctive features. Cases diagnosed with anti-N-Methyl d-aspartate receptor (NMDAR) and anti-voltage gated potassium channel (VGKC) antibody-mediated encephalopathy in four major child neurology centers are described. In four years, 16 children with NMDAR and 8 children with VGKC antibody-associated disease were identified in the participating centers. The most frequent initial manifestation consisted of generalized seizures and cognitive symptoms in both groups. Movement abnormalities were frequent in anti-NMDAR patients and autonomic symptoms, in anti-VGKC patients. Cerebrospinal fluid (CSF) protein, cell count and IgG index were normal in 9/15 anti-NMDAR and 5/8 anti-VGKC patients tested. EEG and MRI findings were usually nonspecific and non-contributory. The rate and time of recovery was not related to age, sex, acute or subacute onset, antibody type, MRI, EEG or CSF results. Treatment within 3 months of onset was associated with normal neurological outcome. Our results suggest anti-NMDAR and VGKC encephalopathies mostly present with non-focal neurological symptoms longer than 3 weeks. In contrast with adult cases, routine CSF testing, MRI and EEG did not contribute to the diagnosis in this series. Copyright © 2017 European Paediatric Neurology Society. Published by Elsevier Ltd. All rights reserved.

  20. [Effects of transient receptor potential melastatin 8 cation channels on inflammatory reaction induced by cold temperatures in human airway epithelial cells].

    Science.gov (United States)

    Li, Min-chao; Perelman, Juliy M; Kolosov, Victor P; Zhou, Xiang-dong

    2011-10-01

    To explore the role of transient receptor potential melastatin 8 cation channels (TRPM8) in cold-induced production of inflammatory factors in airway epithelial cells and related signal transduction mechanism. The 16HBE human airway epithelial cells were stimulated with cold temperature (18°C). In intervention experiments, cells were pretreated with TRPM8 channel antagonist BCTC, protein kinase C (PKC) specific inhibitor calphostin C and transfected with TRPM8 shRNA or control shRNA respectively, and thereafter cold stimulation was applied. Cells were divided into 6 groups: a control group (incubated at 37°C), a cold stimulation group, a cold stimulation + BCTC group, a cold stimulation + TRPM8 shRNA group, a cold stimulation + control shRNA group, a cold stimulation + calphostin C group. Western blot was performed to show the extent of knockdown in TRPM8 protein expression in the TRPM8 shRNA transfected cells. Dynamics of relative concentration of intracellular Ca(2+) in the former 5 groups were measured by calcium imaging techniques. Images were taken at one frame per 10 seconds. The levels of interleukin (IL)-6, IL-8, tumor necrosis factor (TNF)-α mRNA and protein were detected by real-time PCR and ELISA respectively. The highest relative concentration of intracellular calcium in cold stimulation group (2.36 ± 0.24) was higher than that of control group (1.01 ± 0.02) (t = 12.52, P cold stimulation group (t = 6.69 and 9.12, all P cold stimulation group[0.66 ± 0.16, 0.77 ± 0.15, 0.73 ± 0.09 and (92 ± 13) ng/L, (125 ± 22) ng/L, (88 ± 12) ng/L ] were significantly higher than those in control group [0.37 ± 0.08, 0.32 ± 0.07, 0.48 ± 0.10 and (52 ± 8) ng/L, (50 ± 9) ng/L, (61 ± 8) ng/L] (t = 3.20 - 6.26, all P cold stimulation + BCTC group [0.42 ± 0.09, 0.52 ± 0.13, 0.52 ± 0.12 and (72 ± 8) ng/L, (92 ± 14) ng/L, (68 ± 11) ng/L], cold stimulation + TRPM8 shRNA group [0.41 ± 0.10, 0.49 ± 0.08, 0.50 ± 0.08 and (60 ± 12) ng/L, (89 ± 14) ng

  1. Neuropharmacology of purinergic receptors in human submucous plexus: Involvement of P2X₁, P2X₂, P2X₃ channels, P2Y and A₃ metabotropic receptors in neurotransmission.

    Science.gov (United States)

    Liñán-Rico, A; Wunderlich, J E; Enneking, J T; Tso, D R; Grants, I; Williams, K C; Otey, A; Michel, K; Schemann, M; Needleman, B; Harzman, A; Christofi, F L

    2015-08-01

    The role of purinergic signaling in human ENS is not well understood. We sought to further characterize the neuropharmacology of purinergic receptors in human ENS and test the hypothesis that endogenous purines are critical regulators of neurotransmission. LSCM-Fluo-4/(Ca(2+))-imaging of postsynaptic Ca(2+) transients (PSCaTs) was used as a reporter of synaptic transmission evoked by fiber tract electrical stimulation in human SMP surgical preparations. Pharmacological analysis of purinergic signaling was done in 1,556 neurons (identified by HuC/D-immunoreactivity) in 235 ganglia from 107 patients; P2XR-immunoreactivity was evaluated in 19 patients. Real-time MSORT (Di-8-ANEPPS) imaging tested effects of adenosine on fast excitatory synaptic potentials (fEPSPs). Synaptic transmission is sensitive to pharmacological manipulations that alter accumulation of extracellular purines: Apyrase blocks PSCaTs in a majority of neurons. An ecto-NTPDase-inhibitor 6-N,N-diethyl-D-β,γ-dibromomethyleneATP or adenosine deaminase augments PSCaTs. Blockade of reuptake/deamination of eADO inhibits PSCaTs. Adenosine inhibits fEPSPs and PSCaTs (IC50 = 25 µM), sensitive to MRS1220-antagonism (A3AR). A P2Y agonist ADPβS inhibits PSCaTs (IC50 = 111 nM) in neurons without stimulatory ADPbS responses (EC50 = 960 nM). ATP or a P2X1,2,2/3 (α,β-MeATP) agonist evokes fast, slow, biphasic Ca(2+) transients or Ca(2+) oscillations (ATP,EC50 = 400 mM). PSCaTs are sensitive to P2X1 antagonist NF279. Low (20 nM) or high (5 µM) concentrations of P2X antagonist TNP-ATP block PSCaTs in different neurons; proportions of neurons with P2XR-immunoreactivity follow the order P2X2 > P2X1 > P2X3; P2X1 + P2X2 and P2X3 + P2X2 are co-localized. RT-PCR identified mRNA-transcripts for P2X1-7, P2Y1,2,12-14R. Purines are critical regulators of neurotransmission in human ENS. Purinergic signaling involves P2X1, P2X2, P2X3 channels, P2X1 + P2X2 co-localization and inhibitory P2Y or A3 receptors. These are

  2. Volume Regulated Channels

    DEFF Research Database (Denmark)

    Klausen, Thomas Kjær

    of volume perturbations evolution have developed system of channels and transporters to tightly control volume homeostasis. In the past decades evidence has been mounting, that the importance of these volume regulated channels and transporters are not restricted to the defense of cellular volume...... but are also essential for a number of physiological processes such as proliferation, controlled cell death, migration and endocrinology. The thesis have been focusing on two Channels, namely the swelling activated Cl- channel (ICl, swell) and the transient receptor potential Vanilloid (TRPV4) channel. I: Cl......- serves a multitude of functions in the mammalian cell, regulating the membrane potential (Em), cell volume, protein activity and the driving force for facilitated transporters giving Cl- and Cl- channels a major potential of regulating cellular function. These functions include control of the cell cycle...

  3. A Close Association of RyRs with Highly Dense Clusters of Ca2+-activated Cl− Channels Underlies the Activation of STICs by Ca2+ Sparks in Mouse Airway Smooth Muscle

    Science.gov (United States)

    Bao, Rongfeng; Lifshitz, Lawrence M.; Tuft, Richard A.; Bellvé, Karl; Fogarty, Kevin E.; ZhuGe, Ronghua

    2008-01-01

    Ca2+ sparks are highly localized, transient releases of Ca2+ from sarcoplasmic reticulum through ryanodine receptors (RyRs). In smooth muscle, Ca2+ sparks trigger spontaneous transient outward currents (STOCs) by opening nearby clusters of large-conductance Ca2+-activated K+ channels, and also gate Ca2+-activated Cl− (Cl(Ca)) channels to induce spontaneous transient inward currents (STICs). While the molecular mechanisms underlying the activation of STOCs by Ca2+ sparks is well understood, little information is available on how Ca2+ sparks activate STICs. In the present study, we investigated the spatial organization of RyRs and Cl(Ca) channels in spark sites in airway myocytes from mouse. Ca2+ sparks and STICs were simultaneously recorded, respectively, with high-speed, widefield digital microscopy and whole-cell patch-clamp. An image-based approach was applied to measure the Ca2+ current underlying a Ca2+ spark (ICa(spark)), with an appropriate correction for endogenous fixed Ca2+ buffer, which was characterized by flash photolysis of NPEGTA. We found that ICa(spark) rises to a peak in 9 ms and decays with a single exponential with a time constant of 12 ms, suggesting that Ca2+ sparks result from the nonsimultaneous opening and closure of multiple RyRs. The onset of the STIC lags the onset of the ICa(spark) by less than 3 ms, and its rising phase matches the duration of the ICa(spark). We further determined that Cl(Ca) channels on average are exposed to a [Ca2+] of 2.4 μM or greater during Ca2+ sparks. The area of the plasma membrane reaching this level is <600 nm in radius, as revealed by the spatiotemporal profile of [Ca2+] produced by a reaction-diffusion simulation with measured ICa(spark). Finally we estimated that the number of Cl(Ca) channels localized in Ca2+ spark sites could account for all the Cl(Ca) channels in the entire cell. Taken together these results lead us to propose a model in which RyRs and Cl(Ca) channels in Ca2+ spark sites localize

  4. A close association of RyRs with highly dense clusters of Ca2+-activated Cl- channels underlies the activation of STICs by Ca2+ sparks in mouse airway smooth muscle.

    Science.gov (United States)

    Bao, Rongfeng; Lifshitz, Lawrence M; Tuft, Richard A; Bellvé, Karl; Fogarty, Kevin E; ZhuGe, Ronghua

    2008-07-01

    Ca(2+) sparks are highly localized, transient releases of Ca(2+) from sarcoplasmic reticulum through ryanodine receptors (RyRs). In smooth muscle, Ca(2+) sparks trigger spontaneous transient outward currents (STOCs) by opening nearby clusters of large-conductance Ca(2+)-activated K(+) channels, and also gate Ca(2+)-activated Cl(-) (Cl((Ca))) channels to induce spontaneous transient inward currents (STICs). While the molecular mechanisms underlying the activation of STOCs by Ca(2+) sparks is well understood, little information is available on how Ca(2+) sparks activate STICs. In the present study, we investigated the spatial organization of RyRs and Cl((Ca)) channels in spark sites in airway myocytes from mouse. Ca(2+) sparks and STICs were simultaneously recorded, respectively, with high-speed, widefield digital microscopy and whole-cell patch-clamp. An image-based approach was applied to measure the Ca(2+) current underlying a Ca(2+) spark (I(Ca(spark))), with an appropriate correction for endogenous fixed Ca(2+) buffer, which was characterized by flash photolysis of NPEGTA. We found that I(Ca(spark)) rises to a peak in 9 ms and decays with a single exponential with a time constant of 12 ms, suggesting that Ca(2+) sparks result from the nonsimultaneous opening and closure of multiple RyRs. The onset of the STIC lags the onset of the I(Ca(spark)) by less than 3 ms, and its rising phase matches the duration of the I(Ca(spark)). We further determined that Cl((Ca)) channels on average are exposed to a [Ca(2+)] of 2.4 microM or greater during Ca(2+) sparks. The area of the plasma membrane reaching this level is <600 nm in radius, as revealed by the spatiotemporal profile of [Ca(2+)] produced by a reaction-diffusion simulation with measured I(Ca(spark)). Finally we estimated that the number of Cl((Ca)) channels localized in Ca(2+) spark sites could account for all the Cl((Ca)) channels in the entire cell. Taken together these results lead us to propose a model in which

  5. Transcriptional Repression and Protein Degradation of the Ca2+-Activated K+ Channel KCa1.1 by Androgen Receptor Inhibition in Human Breast Cancer Cells

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    Anowara Khatun

    2018-04-01

    Full Text Available The large-conductance Ca2+-activated K+ channel KCa1.1 plays an important role in the promotion of breast cancer cell proliferation and metastasis. The androgen receptor (AR is proposed as a therapeutic target for AR-positive advanced triple-negative breast cancer. We herein investigated the effects of a treatment with antiandrogens on the functional activity, activation kinetics, transcriptional expression, and protein degradation of KCa1.1 in human breast cancer MDA-MB-453 cells using real-time PCR, Western blotting, voltage-sensitive dye imaging, and whole-cell patch clamp recording. A treatment with the antiandrogen bicalutamide or enzalutamide for 48 h significantly suppressed (1 depolarization responses induced by paxilline (PAX, a specific KCa1.1 blocker and (2 PAX-sensitive outward currents induced by the depolarizing voltage step. The expression levels of KCa1.1 transcripts and proteins were significantly decreased in MDA-MB-453 cells, and the protein degradation of KCa1.1 mainly contributed to reductions in KCa1.1 activity. Among the eight regulatory β and γ subunits, LRRC26 alone was expressed at high levels in MDA-MB-453 cells and primary and metastatic breast cancer tissues, whereas no significant changes were observed in the expression levels of LRRC26 and activation kinetics of PAX-sensitive outward currents in MDA-MB-453 cells by the treatment with antiandrogens. The treatment with antiandrogens up-regulated the expression of the ubiquitin E3 ligases, FBW7, MDM2, and MDM4 in MDA-MB-453 cells, and the protein degradation of KCa1.1 was significantly inhibited by the respective siRNA-mediated blockade of FBW7 and MDM2. Based on these results, we concluded that KCa1.1 is an androgen-responsive gene in AR-positive breast cancer cells, and its down-regulation through enhancements in its protein degradation by FBW7 and/or MDM2 may contribute, at least in part, to the antiproliferative and antimetastatic effects of antiandrogens in

  6. Selectivity and evolutionary divergence of metabotropic glutamate receptors for endogenous ligands and G proteins coupled to phospholipase C or TRP channels.

    Science.gov (United States)

    Kang, Hye Jin; Menlove, Kit; Ma, Jianpeng; Wilkins, Angela; Lichtarge, Olivier; Wensel, Theodore G

    2014-10-24

    To define the upstream and downstream signaling specificities of metabotropic glutamate receptors (mGluR), we have examined the ability of representative mGluR of group I, II, and III to be activated by endogenous amino acids and catalyze activation of G proteins coupled to phospholipase C (PLC), or activation of G(i/o) proteins coupled to the ion channel TRPC4β. Fluorescence-based assays have allowed us to observe interactions not previously reported or clearly identified. We have found that the specificity for endogenous amino acids is remarkably stringent. Even at millimolar levels, structurally similar compounds do not elicit significant activation. As reported previously, the clear exception is L-serine-O-phosphate (L-SOP), which strongly activates group III mGluR, especially mGluR4,-6,-8 but not group I or II mGluR. Whereas L-SOP cannot activate mGluR1 or mGluR2, it acts as a weak antagonist for mGluR1 and a potent antagonist for mGluR2, suggesting that co-recognition of L-glutamate and L-SOP arose early in evolution, and was followed later by divergence of group I and group II mGluR versus group III in l-SOP responses. mGluR7 has low affinity and efficacy for activation by both L-glutamate and L-SOP. Molecular docking studies suggested that residue 74 corresponding to lysine in mGluR4 and asparagine in mGluR7 might play a key role, and, indeed, mutagenesis experiments demonstrated that mutating this residue to lysine in mGluR7 enhances the potency of L-SOP. Experiments with pertussis toxin and dominant-negative Gα(i/o) proteins revealed that mGluR1 couples strongly to TRPC4β through Gα(i/o), in addition to coupling to PLC through Gα(q/11). © 2014 by The American Society for Biochemistry and Molecular Biology, Inc.

  7. Selectivity and Evolutionary Divergence of Metabotropic Glutamate Receptors for Endogenous Ligands and G Proteins Coupled to Phospholipase C or TRP Channels*

    Science.gov (United States)

    Kang, Hye Jin; Menlove, Kit; Ma, Jianpeng; Wilkins, Angela; Lichtarge, Olivier; Wensel, Theodore G.

    2014-01-01

    To define the upstream and downstream signaling specificities of metabotropic glutamate receptors (mGluR), we have examined the ability of representative mGluR of group I, II, and III to be activated by endogenous amino acids and catalyze activation of G proteins coupled to phospholipase C (PLC), or activation of Gi/o proteins coupled to the ion channel TRPC4β. Fluorescence-based assays have allowed us to observe interactions not previously reported or clearly identified. We have found that the specificity for endogenous amino acids is remarkably stringent. Even at millimolar levels, structurally similar compounds do not elicit significant activation. As reported previously, the clear exception is l-serine-O-phosphate (l-SOP), which strongly activates group III mGluR, especially mGluR4,-6,-8 but not group I or II mGluR. Whereas l-SOP cannot activate mGluR1 or mGluR2, it acts as a weak antagonist for mGluR1 and a potent antagonist for mGluR2, suggesting that co-recognition of l-glutamate and l-SOP arose early in evolution, and was followed later by divergence of group I and group II mGluR versus group III in l-SOP responses. mGluR7 has low affinity and efficacy for activation by both l-glutamate and l-SOP. Molecular docking studies suggested that residue 74 corresponding to lysine in mGluR4 and asparagine in mGluR7 might play a key role, and, indeed, mutagenesis experiments demonstrated that mutating this residue to lysine in mGluR7 enhances the potency of l-SOP. Experiments with pertussis toxin and dominant-negative Gαi/o proteins revealed that mGluR1 couples strongly to TRPC4β through Gαi/o, in addition to coupling to PLC through Gαq/11. PMID:25193666

  8. Distinct Mechanism of Cysteine Oxidation-Dependent Activation and Cold Sensitization of Human Transient Receptor Potential Ankyrin 1 Channel by High and Low Oxaliplatin

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    Takahito Miyake

    2017-11-01

    Full Text Available Oxaliplatin, a third-generation platinum-based chemotherapeutic agent, displays unique acute peripheral neuropathy triggered or enhanced by cold, and accumulating evidence suggests that transient receptor potential ankyrin 1 (TRPA1 is responsible. TRPA1 is activated by oxaliplatin via a glutathione-sensitive mechanism. However, oxaliplatin interrupts hydroxylation of a proline residue located in the N-terminal region of TRPA1 via inhibition of prolyl hydroxylase (PHD, which causes sensitization of TRPA1 to reactive oxygen species (ROS. Furthermore, PHD inhibition endows cold-insensitive human TRPA1 (hTRPA1 with ROS-dependent cold sensitivity. Since cysteine oxidation and proline hydroxylation regulate its activity, their association with oxaliplatin-induced TRPA1 activation and acquirement of cold sensitivity were investigated in the present study. A high concentration of oxaliplatin (1 mM induced outward-rectifier whole-cell currents and increased the intracellular Ca2+ concentration in hTRPA1-expressing HEK293 cells, but did not increase the probability of hTRPA1 channel opening in the inside-out configuration. Oxaliplatin also induced the rapid generation of hydrogen peroxide, and the resultant Ca2+ influx was prevented in the presence of glutathione and in cysteine-mutated hTRPA1 (Cys641Ser-expressing cells, whereas proline-mutated hTRPA1 (Pro394Ala-expressing cells showed similar whole-cell currents and Ca2+ influx. By contrast, a lower concentration of oxaliplatin (100 μM did not increase the intracellular Ca2+ concentration but did confer cold sensitivity on hTRPA1-expressing cells, and this was inhibited by PHD2 co-overexpression. Cold sensitivity was abolished by the mitochondria-targeting ROS scavenger mitoTEMPO and was minimal in cysteine-mutated hTRPA1 (Cys641Ser or Cys665Ser-expressing cells. Thus, high oxaliplatin evokes ROS-mediated cysteine oxidation-dependent hTRPA1 activation independent of PHD activity, while a lower

  9. Activation of Phosphatidylinositol-Linked Dopamine Receptors Induces a Facilitation of Glutamate-Mediated Synaptic Transmission in the Lateral Entorhinal Cortex.

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    Iulia Glovaci

    Full Text Available The lateral entorhinal cortex receives strong inputs from midbrain dopamine neurons that can modulate its sensory and mnemonic function. We have previously demonstrated that 1 µM dopamine facilitates synaptic transmission in layer II entorhinal cortex cells via activation of D1-like receptors, increased cAMP-PKA activity, and a resulting enhancement of AMPA-receptor mediated currents. The present study assessed the contribution of phosphatidylinositol (PI-linked D1 receptors to the dopaminergic facilitation of transmission in layer II of the rat entorhinal cortex, and the involvement of phospholipase C activity and release of calcium from internal stores. Whole-cell patch-clamp recordings of glutamate-mediated evoked excitatory postsynaptic currents were obtained from pyramidal and fan cells. Activation of D1-like receptors using SKF38393, SKF83959, or 1 µM dopamine induced a reversible facilitation of EPSCs which was abolished by loading cells with either the phospholipase C inhibitor U-73122 or the Ca2+ chelator BAPTA. Neither the L-type voltage-gated Ca2+ channel blocker nifedipine, nor the L/N-type channel blocker cilnidipine, blocked the facilitation of synaptic currents. However, the facilitation was blocked by blocking Ca2+ release from internal stores via inositol 1,4,5-trisphosphate (InsP3 receptors or ryanodine receptors. Follow-up studies demonstrated that inhibiting CaMKII activity with KN-93 failed to block the facilitation, but that application of the protein kinase C inhibitor PKC(19-36 completely blocked the dopamine-induced facilitation. Overall, in addition to our previous report indicating a role for the cAMP-PKA pathway in dopamine-induced facilitation of synaptic transmission, we demonstrate here that the dopaminergic facilitation of synaptic responses in layer II entorhinal neurons also relies on a signaling cascade dependent on PI-linked D1 receptors, PLC, release of Ca2+ from internal stores, and PKC activation which is

  10. Neuropharmacology of Purinergic Receptors in Human Submucous Plexus: Involvement of P2X1, P2X2, P2X3 Channels, P2Y and A3 Metabotropic Receptors in Neurotransmission

    Science.gov (United States)

    Liñán-Rico, A.; Wunderlich, JE.; Enneking, JT.; Tso, DR.; Grants, I.; Williams, KC.; Otey, A.; Michel, K.; Schemann, M.; Needleman, B.; Harzman, A.; Christofi, FL.

    2015-01-01

    P2X1, P2X2, P2X3 channels, P2X1+P2X2 co-localization and inhibitory P2Y or A3 receptors. These are potential novel therapeutic targets for neurogastroenterology. PMID:25724083

  11. Follicle-stimulating hormone receptor-mediated uptake of 45Ca2+ by proteoliposomes and cultured rat sertoli cells: Evidence for involvement of voltage-activated and voltage-independent calcium channels

    International Nuclear Information System (INIS)

    Grasso, P.; Reichert, L.E. Jr.

    1989-01-01

    We have previously reported incorporation into liposomes of Triton X-100-solubilized FSH receptor-G-protein complexes derived from purified bovine calf testis membranes. In the present study we have used this model system to show that FSH induces flux of 45Ca2+ into such proteoliposomes in a hormone-specific concentration-dependent manner. FSH, inactivated by boiling, had no stimulatory effect on 45Ca2+ flux, nor did isolated alpha- or beta-subunits of FSH. Addition of GTP (or its analogs 5'-guanylylimidodiphosphate and guanosine-5'-O-[3-thiotriphosphate]) or sodium fluoride (in the presence or absence of GTP or its analogs) failed to induce 45Ca2+ flux into proteoliposomes, suggesting that the uptake of 45Ca2+ was receptor, and not G-protein, related. Voltage-independent (ruthenium red and gadolinium chloride) and voltage-activated (methyoxyverapamil and nifedipine) calcium channel-blocking agents reduced FSH-stimulated 45Ca2+ flux into proteoliposomes to control levels. FSH also induced uptake of 45Ca2+ by cultured rat Sertoli cells. Ruthenium red and gadolinium chloride had no effect on basal levels of 45Ca2+ uptake or estradiol secretion by cultured rat Sertoli cells, nor did methoxyverapamil or nifedipine. All four calcium channel blockers, however, were able to reduce FSH-induced 45Ca2+ uptake to basal levels and FSH-stimulated conversion of androstenedione to estradiol by up to 50%, indicating an involvement of Ca2+ in FSH-stimulated steroidogenesis. Our results suggest that the well documented changes in intracellular calcium levels consequent to FSH binding may be due, at least in part, to an influx of calcium through FSH receptor-regulated calcium channels

  12. Synergistic activation of vascular TRPC6 channel by receptor and mechanical stimulation via phospholipase C/diacylglycerol and phospholipase A2/¿-hydroxylase/20-HETE pathways

    DEFF Research Database (Denmark)

    Inoue, Ryuji; Jensen, Lars Jørn; Jian, Zhong

    2009-01-01

    ). Single TRPC6 channel activity evoked by carbachol was also enhanced by a negative pressure added in the patch pipette. Mechanical potentiation of carbachol- or OAG-induced I(TRPC6) was abolished by small interfering RNA knockdown of cytosolic phospholipase A(2) or pharmacological inhibition of omega...... or Arg8 vasopressin was greatly enhanced by mechanical stimuli via 20-HETE production. Furthermore, myogenic response of pressurized mesenteric artery was significantly enhanced by weak receptor stimulation dependently on 20-HETE production. These results collectively suggest that simultaneous operation...

  13. Up-regulated Ectonucleotidases in Fas-Associated Death Domain Protein- and Receptor-Interacting Protein Kinase 1-Deficient Jurkat Leukemia Cells Counteract Extracellular ATP/AMP Accumulation via Pannexin-1 Channels during Chemotherapeutic Drug-Induced Apoptosis.

    Science.gov (United States)

    Boyd-Tressler, Andrea M; Lane, Graham S; Dubyak, George R

    2017-07-01

    Pannexin-1 (Panx1) channels mediate the efflux of ATP and AMP from cancer cells in response to induction of extrinsic apoptosis by death receptors or intrinsic apoptosis by chemotherapeutic agents. We previously described the accumulation of extracellular ATP /AMP during chemotherapy-induced apoptosis in Jurkat human leukemia cells. In this study, we compared how different signaling pathways determine extracellular nucleotide pools in control Jurkat cells versus Jurkat lines that lack the Fas-associated death domain (FADD) or receptor-interacting protein kinase 1 (RIP1) cell death regulatory proteins. Tumor necrosis factor- α induced extrinsic apoptosis in control Jurkat cells and necroptosis in FADD-deficient cells; treatment of both lines with chemotherapeutic drugs elicited similar intrinsic apoptosis. Robust extracellular ATP/AMP accumulation was observed in the FADD-deficient cells during necroptosis, but not during apoptotic activation of Panx1 channels. Accumulation of extracellular ATP/AMP was similarly absent in RIP1-deficient Jurkat cells during apoptotic responses to chemotherapeutic agents. Apoptotic activation triggered equivalent proteolytic gating of Panx1 channels in all three Jurkat cell lines. The differences in extracellular ATP/AMP accumulation correlated with cell-line-specific expression of ectonucleotidases that metabolized the released ATP/AMP. CD73 mRNA, and α β -methylene-ADP-inhibitable ecto-AMPase activity were elevated in the FADD-deficient cells. In contrast, the RIP1-deficient cells were defined by increased expression of tartrate-sensitive prostatic acid phosphatase as a broadly acting ectonucleotidase. Thus, extracellular nucleotide accumulation during regulated tumor cell death involves interplay between ATP/AMP efflux pathways and different cell-autonomous ectonucleotidases. Differential expression of particular ectonucleotidases in tumor cell variants will determine whether chemotherapy-induced activation of Panx1 channels

  14. Adrenomedullin increased the short-circuit current in the pig oviduct through chloride channels via the CGRP receptor: mediation by cAMP and calcium ions but not by nitric oxide.

    Science.gov (United States)

    Liao, S B; Cheung, K H; Cheung, M P L; To, Y T; O, W S; Tang, F

    2013-10-01

    The oviduct serves as a site for the fertilization of the ovum and the transport of the conceptus down to the uterus for implantation. In this study, we investigated the presence of adrenomedullin (ADM) and its receptor component proteins in the pig oviduct. The effect of ADM on oviductal secretion, the specific receptor, and the mechanisms involved were also investigated. The presence of ADM and its receptor component proteins in the pig oviduct were confirmed using immunostaining. Short-circuit current (I(sc)) technique was employed to study chloride ion secretion in the oviductal epithelium. ADM increased I(sc) through cAMP- and calcium-activated chloride channels, and this effect could be inhibited by the CGRP receptor antagonist, hCGRP8-37. In contrast, the nitric oxide synthase inhibitor, L-NG-nitroarginine methyl ester (L-NAME), could not block the effect of ADM on I(sc). In summary, ADM may increase oviductal fluid secretion via chloride secretion independent of the nitric oxide pathway for the transport of sperm and the conceptus.

  15. Brain-derived neurotrophic factor (BDNF) induces sustained intracellular Ca2+ elevation through the up-regulation of surface transient receptor potential 3 (TRPC3) channels in rodent microglia.

    Science.gov (United States)

    Mizoguchi, Yoshito; Kato, Takahiro A; Seki, Yoshihiro; Ohgidani, Masahiro; Sagata, Noriaki; Horikawa, Hideki; Yamauchi, Yusuke; Sato-Kasai, Mina; Hayakawa, Kohei; Inoue, Ryuji; Kanba, Shigenobu; Monji, Akira

    2014-06-27

    Microglia are immune cells that release factors, including proinflammatory cytokines, nitric oxide (NO), and neurotrophins, following activation after disturbance in the brain. Elevation of intracellular Ca(2+) concentration ([Ca(2+)]i) is important for microglial functions such as the release of cytokines and NO from activated microglia. There is increasing evidence suggesting that pathophysiology of neuropsychiatric disorders is related to the inflammatory responses mediated by microglia. Brain-derived neurotrophic factor (BDNF) is a neurotrophin well known for its roles in the activation of microglia as well as in pathophysiology and/or treatment of neuropsychiatric disorders. In this study, we sought to examine the underlying mechanism of BDNF-induced sustained increase in [Ca(2+)]i in rodent microglial cells. We observed that canonical transient receptor potential 3 (TRPC3) channels contribute to the maintenance of BDNF-induced sustained intracellular Ca(2+) elevation. Immunocytochemical technique and flow cytometry also revealed that BDNF rapidly up-regulated the surface expression of TRPC3 channels in rodent microglial cells. In addition, pretreatment with BDNF suppressed the production of NO induced by tumor necrosis factor α (TNFα), which was prevented by co-adiministration of a selective TRPC3 inhibitor. These suggest that BDNF induces sustained intracellular Ca(2+) elevation through the up-regulation of surface TRPC3 channels and TRPC3 channels could be important for the BDNF-induced suppression of the NO production in activated microglia. We show that TRPC3 channels could also play important roles in microglial functions, which might be important for the regulation of inflammatory responses and may also be involved in the pathophysiology and/or the treatment of neuropsychiatric disorders. © 2014 by The American Society for Biochemistry and Molecular Biology, Inc.

  16. Current and calcium responses to local activation of axonal NMDA receptors in developing cerebellar molecular layer interneurons.

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    Bénédicte Rossi

    Full Text Available In developing cerebellar molecular layer interneurons (MLIs, NMDA increases spontaneous GABA release. This effect had been attributed to either direct activation of presynaptic NMDA receptors (preNMDARs or an indirect pathway involving activation of somato-dendritic NMDARs followed by passive spread of somatic depolarization along the axon and activation of axonal voltage dependent Ca(2+ channels (VDCCs. Using Ca(2+ imaging and electrophysiology, we searched for preNMDARs by uncaging NMDAR agonists either broadly throughout the whole field or locally at specific axonal locations. Releasing either NMDA or glutamate in the presence of NBQX using short laser pulses elicited current transients that were highly sensitive to the location of the spot and restricted to a small number of varicosities. The signal was abolished in the presence of high Mg(2+ or by the addition of APV. Similar paradigms yielded restricted Ca(2+ transients in interneurons loaded with a Ca(2+ indicator. We found that the synaptic effects of NMDA were not inhibited by blocking VDCCs but were impaired in the presence of the ryanodine receptor antagonist dantrolene. Furthermore, in voltage clamped cells, bath applied NMDA triggers Ca(2+ elevations and induces neurotransmitter release in the axonal compartment. Our results suggest the existence of preNMDARs in developing MLIs and propose their involvement in the NMDA-evoked increase in GABA release by triggering a Ca(2+-induced Ca(2+ release process mediated by presynaptic Ca(2+ stores. Such a mechanism is likely to exert a crucial role in various forms of Ca(2+-mediated synaptic plasticity.

  17. TRP channels: an overview

    DEFF Research Database (Denmark)

    Pedersen, Stine Falsig; Owsianik, Grzegorz; Nilius, Bernd

    2005-01-01

    The TRP ("transient receptor potential") family of ion channels now comprises more than 30 cation channels, most of which are permeable for Ca2+, and some also for Mg2+. On the basis of sequence homology, the TRP family can be divided in seven main subfamilies: the TRPC ('Canonical') family......, the TRPV ('Vanilloid') family, the TRPM ('Melastatin') family, the TRPP ('Polycystin') family, the TRPML ('Mucolipin') family, the TRPA ('Ankyrin') family, and the TRPN ('NOMPC') family. The cloning and characterization of members of this cation channel family has exploded during recent years, leading...... to a plethora of data on the roles of TRPs in a variety of tissues and species, including mammals, insects, and yeast. The present review summarizes the most pertinent recent evidence regarding the structural and functional properties of TRP channels, focusing on the regulation and physiology of mammalian TRPs....

  18. Multiple P2Y receptors couple to calcium-dependent, chloride channels in smooth muscle cells of the rat pulmonary artery

    Directory of Open Access Journals (Sweden)

    Gurney Alison M

    2005-10-01

    Full Text Available Abstract Background Uridine 5'-triphosphate (UTP and uridine 5'-diphosphate (UDP act via P2Y receptors to evoke contraction of rat pulmonary arteries, whilst adenosine 5'-triphosphate (ATP acts via P2X and P2Y receptors. Pharmacological characterisation of these receptors in intact arteries is complicated by release and extracellular metabolism of nucleotides, so the aim of this study was to characterise the P2Y receptors under conditions that minimise these problems. Methods The perforated-patch clamp technique was used to record the Ca2+-dependent, Cl- current (ICl,Ca activated by P2Y receptor agonists in acutely dissociated smooth muscle cells of rat small (SPA and large (LPA intrapulmonary arteries, held at -50 mV. Contractions to ATP were measured in isolated muscle rings. Data were compared by Student's t test or one way ANOVA. Results ATP, UTP and UDP (10-4M evoked oscillating, inward currents (peak = 13–727 pA in 71–93% of cells. The first current was usually the largest and in the SPA the response to ATP was significantly greater than those to UTP or UDP (P -1 and changed little during agonist application. The non-selective P2 receptor antagonist suramin (10-4M abolished currents evoked by ATP in SPA (n = 4 and LPA (n = 4, but pyridoxalphosphate-6-azophenyl-2',4'-disulphonic acid (PPADS (10-4M, also a non-selective P2 antagonist, had no effect (n = 4, 5 respectively. Currents elicited by UTP (n = 37 or UDP (n = 14 were unaffected by either antagonist. Contractions of SPA evoked by ATP were partially inhibited by PPADS (n = 4 and abolished by suramin (n = 5. Both antagonists abolished the contractions in LPA. Conclusion At least two P2Y subtypes couple to ICl,Ca in smooth muscle cells of rat SPA and LPA, with no apparent regional variation in their distribution. The suramin-sensitive, PPADS-resistant site activated by ATP most resembles the P2Y11 receptor. However, the suramin- and PPADS-insensitive receptor activated by UTP and UDP

  19. Synthesis and evaluation of 1-[l brace]1-[5-(2'-[[sup 18]F]fluoroethyl)-2-thienyl]cyclohexyl[r brace]piperidine as a potential in vivo radioligand for the NMDA receptor-channel complex. [N-methyl-D-aspartate receptor

    Energy Technology Data Exchange (ETDEWEB)

    Orita, Kazuhiro; Sasaki, Shigeki; Maeda, Minoru [Kyushu Univ., Fukuoka (Japan). Faculty of Pharmaceutical Sciences; Hashimoto, Atsushi; Nishikawa, Toru [National Inst. of Neuroscience, Tokyo (Japan); Yugami, Tomoko; Umezu, Kohei [Mitsubishi Kasei Corp., Yokohama (Japan). Research Center

    1993-10-01

    1-[l brace]1-[5-(2'-[[sup 18]F]Fluoroethyl)-2-thienyl]cyclohexyl[r brace]piperidine ([sup 18]FE-TCP) was prepared a fluorine-substituted analogue of the potent NMDA receptor channel blocker, 1-[1-(2-thienyl)cyclohexyl]piperidine (TCP), by the mesylate displacement with [[sup 18]F]fluoride ion with isolated radiochemical yields of 6-12%, and the synthesis time including a two step HPLC purification was 120 min. The regional distribution in rat brain after i.v. injection of [sup 18]FE-TCP was heterogeneous and similar to the known distribution of phencyclidine recognition sites, with hippocampus-cerebellum, striatum-cerebellum and cerebral cortex-cerebellum concentration ratios of 2.08, 1.7 and 1.54, respectively, 15 min post-injection. Furthermore, this localized regional cerebral distribution was blocked by co-injection with the unlabelled FE-TCP or pretreatment with cis-2-hydroxymethyl-r-1-(N-piperidyl)-1-(2-thienyl)cyclohexane, with the greatest reductions seen in the hippocampus followed by the striatum and cerebral cortex. However, relatively low receptor binding affinity and high non-specific binding due to its high lipophilicity suggest that [sup 18]FE-TCP may not be a suitable radioligand for in vivo PET investigations of the NMDA receptor-channel complex. (Author).

  20. Conserved Residues within the Putative S4–S5 Region Serve Distinct Functions among Thermosensitive Vanilloid Transient Receptor Potential (TRPV) Channels

    Czech Academy of Sciences Publication Activity Database

    Boukalová, Štěpána; Maršáková, Lenka; Teisinger, Jan; Vlachová, Viktorie

    2010-01-01

    Roč. 285, č. 53 (2010), s. 41455-41462 ISSN 0021-9258 R&D Projects: GA ČR GA305/09/0081; GA ČR GAP301/10/1159; GA AV ČR(CZ) IAA600110701; GA MŠk(CZ) 1M0517; GA MŠk(CZ) LC554 Grant - others:Univerzita Karlova(CZ) 26110 Institutional research plan: CEZ:AV0Z50110509 Keywords : vanilloid receptor * voltage sensor * transient receptor potential Subject RIV: FH - Neurology Impact factor: 5.328, year: 2010

  1. Cav1.2 channels mediate persistent chronic stress-induced behavioral deficits that are associated with prefrontal cortex activation of the p25/Cdk5-glucocorticoid receptor pathway

    Directory of Open Access Journals (Sweden)

    Charlotte C. Bavley

    2017-12-01

    Full Text Available Chronic stress is known to precipitate and exacerbate neuropsychiatric symptoms, and exposure to stress is particularly pathological in individuals with certain genetic predispositions. Recent genome wide association studies have identified single nucleotide polymorphisms (SNPs in the gene CACNA1C, which codes for the Cav1.2 subunit of the L-type calcium channel (LTCC, as a common risk variant for multiple neuropsychiatric conditions. Cav1.2 channels mediate experience-dependent changes in gene expression and long-term synaptic plasticity through activation of downstream calcium signaling pathways. Previous studies have found an association between stress and altered Cav1.2 expression in the brain, however the contribution of Cav1.2 channels to chronic stress-induced behaviors, and the precise Cav1.2 signaling mechanisms activated are currently unknown. Here we report that chronic stress leads to a delayed increase in Cav1.2 expression selectively within the prefrontal cortex (PFC, but not in other stress-sensitive brain regions such as the hippocampus or amygdala. Further, we demonstrate that while Cav1.2 heterozygous (Cav1.2+/− mice show chronic stress-induced depressive-like behavior, anxiety-like behavior, and deficits in working memory 1–2 days following stress, they are resilient to the effects of chronic stress when tested 5–7 days later. Lastly, molecular studies find a delayed upregulation of the p25/Cdk5-glucocorticoid receptor (GR pathway in the PFC when examined 8 days post-stress that is absent in Cav1.2+/− mice. Our findings reveal a novel Cav1.2-mediated molecular mechanism associated with the persistent behavioral effects of chronic stress and provide new insight into potential Cav1.2 channel mechanisms that may contribute to CACNA1C-linked neuropsychiatric phenotypes.

  2. Synthesis of 3-[18F]-fluoromethyl-TCP1, a potential tool for PET study of the NMDA receptor channel complex

    International Nuclear Information System (INIS)

    Ponchant, M.; Crouzel, C.

    1992-01-01

    In an attempt to visualize the NMDA glutamatergic receptors and after checking the biological activity of the cold 3-fluoromethyl-TCP, 3-[ 18 F]-fluoro-methyl-TCP was synthesized by a nucleophilic substitution of 3-bromomethyl-TCP with [ 18 F - ]. (author)

  3. Additive effects of cilnidipine, an L-/N-type calcium channel blocker, and an angiotensin II receptor blocker on reducing cardiorenal damage in Otsuka Long-Evans Tokushima Fatty rats with type 2 diabetes mellitus

    Directory of Open Access Journals (Sweden)

    Mori Y

    2014-06-01

    Full Text Available Yutaka Mori,1,2 Shizuka Aritomi,3 Kazumi Niinuma,3 Tarou Nakamura,3 Kenichi Matsuura,1 Junichi Yokoyama,1 Kazunori Utsunomiya1 1Division of Diabetes and Endocrinology, Department of Internal Medicine, The Jikei University School of Medicine, Minato-ku, Japan; 2Department of Clinical Research, National Hospital Organization, Utsunomiya National Hospital, Utsunomiya, Japan; 3Research Center, Ajinomoto Pharmaceuticals Co, Ltd, Kanagawa, Japan Abstract: Cilnidipine (Cil, which is an L-/N-type calcium channel blocker (CCB, has been known to provide renal protection by decreasing the activity of the sympathetic nervous system (SNS and the renin–angiotensin system. In this study, we compared the effects of the combination of Cil and amlodipine (Aml, which is an L-type CCB, with an angiotensin (Ang II receptor blocker on diabetic cardiorenal damage in spontaneously type 2 diabetic rats. Seventeen-week-old Otsuka Long-Evans Tokushima Fatty rats were randomly assigned to receive Cil, Aml, valsartan (Val, Cil + Val, Aml + Val, or a vehicle (eight rats per group for 22 weeks. Antihypertensive potencies were nearly equal among the CCB monotherapy groups and the combination therapy groups. The lowering of blood pressure by either treatment did not significantly affect the glycemic variables. However, exacerbations of renal and heart failure were significantly suppressed in rats administered Cil or Val, and additional suppression was observed in those administered Cil + Val. Although Val increased the renin–Ang system, Aml + Val treatment resulted in additional increases in these parameters, while Cil + Val did not show such effects. Furthermore, Cil increased the ratio of Ang-(1–7 to Ang-I, despite the fact that Val and Aml + Val decreased the Ang-(1–7 levels. These actions of Cil + Val might be due to their synergistic inhibitory effect on the activity of the SNS, and on aldosterone secretion through N-type calcium channel antagonism and Ang II

  4. Brain Damage from Soman-Induced Seizures Is Greatly Exacerbated by Dimethyl sulfoxide (DMSO): Modest Neuroprotection by 2-Aminoethyl diphenylborinate (2- APB), a Transient Receptor Potential Channel Inhibitor and Inositol 1,4,5-triphosphate Receptor Antagonist

    Science.gov (United States)

    2008-03-04

    stereotypy, and wet-dog shakes. Overt motor convulsions were characterized by rhythmic clonic jerks of head and forepaws, rearing, salivation and Straub...Dale LB, Bhattacharya M, Anborgh PH , Murdoch B, Bhatia M, Nakanishi S, Ferguson SS. G protein-coupled receptor kinase-mediated desensitization of

  5. Distribution Profile of Inositol 1,4,5-Trisphosphate Receptor/Ca2+ Channels in α and β Cells of Pancreas: Dominant Localization in Secretory Granules and Common Error in Identification of Secretory Granule Membranes.

    Science.gov (United States)

    Hur, Yong Suk; Yoo, Seung Hyun

    2015-01-01

    The α and β cells of pancreatic islet release important hormones in response to intracellular Ca increases that result from Ca releases through the inositol 1,4,5-trisphoshate receptor (IP3R)/Ca channels. Yet no systematic studies on distribution of IP3R/Ca channels have been done, prompting us to investigate the distribution of all 3 IP3R isoforms. Immunogold electron microscopy was performed to determine the presence and the relative concentrations of all 3 IP3R isoforms in 2 major organelles secretory granules (SGs) and the endoplasmic reticulum of α and β cells of rat pancreas. All 3 IP3R isoforms were present in SG membranes of both cells, and the IP3R concentrations in SGs were ∼2-fold higher than those in the endoplasmic reticulum. Moreover, large halos shown in the electron microscope images of insulin-containing SGs of β cells were gap spaces that resulted from separation of granule membranes from the surrounding cytoplasm. These results strongly suggest the important roles of SGs in IP3-induced, Ca-dependent regulatory secretory pathway in pancreas. Moreover, the accurate location of SG membranes of β cells was further confirmed by the location of another integral membrane protein synaptotagmin V and of membrane phospholipid PI(4,5)P2.

  6. Altered Elementary Calcium Release Events and Enhanced Calcium Release by Thymol in Rat Skeletal Muscle

    OpenAIRE

    Szentesi, Péter; Szappanos, Henrietta; Szegedi, Csaba; Gönczi, Monika; Jona, István; Cseri, Julianna; Kovács, László; Csernoch, László

    2004-01-01

    The effects of thymol on steps of excitation-contraction coupling were studied on fast-twitch muscles of rodents. Thymol was found to increase the depolarization-induced release of calcium from the sarcoplasmic reticulum, which could not be attributed to a decreased calcium-dependent inactivation of calcium release channels/ryanodine receptors or altered intramembrane charge movement, but rather to a more efficient coupling of depolarization to channel opening. Thymol increased ryanodine bind...

  7. TRP Channels in Human Prostate

    Directory of Open Access Journals (Sweden)

    Carl Van Haute

    2010-01-01

    Full Text Available This review gives an overview of morphological and functional characteristics in the human prostate. It will focus on the current knowledge about transient receptor potential (TRP channels expressed in the human prostate, and their putative role in normal physiology and prostate carcinogenesis. Controversial data regarding the expression pattern and the potential impact of TRP channels in prostate function, and their involvement in prostate cancer and other prostate diseases, will be discussed.

  8. Functional modifications of acid-sensing ion channels by ligand-gated chloride channels.

    Directory of Open Access Journals (Sweden)

    Xuanmao Chen

    Full Text Available Together, acid-sensing ion channels (ASICs and epithelial sodium channels (ENaC constitute the majority of voltage-independent sodium channels in mammals. ENaC is regulated by a chloride channel, the cystic fibrosis transmembrane conductance regulator (CFTR. Here we show that ASICs were reversibly inhibited by activation of GABA(A receptors in murine hippocampal neurons. This inhibition of ASICs required opening of the chloride channels but occurred with both outward and inward GABA(A receptor-mediated currents. Moreover, activation of the GABA(A receptors modified the pharmacological features and kinetic properties of the ASIC currents, including the time course of activation, desensitization and deactivation. Modification of ASICs by open GABA(A receptors was also observed in both nucleated patches and outside-out patches excised from hippocampal neurons. Interestingly, ASICs and GABA(A receptors interacted to regulate synaptic plasticity in CA1 hippocampal slices. The activation of glycine receptors, which are similar to GABA(A receptors, also modified ASICs in spinal neurons. We conclude that GABA(A receptors and glycine receptors modify ASICs in neurons through mechanisms that require the opening of chloride channels.

  9. Post-Translational Modifications of TRP Channels

    Directory of Open Access Journals (Sweden)

    Olaf Voolstra

    2014-04-01

    Full Text Available Transient receptor potential (TRP channels constitute an ancient family of cation channels that have been found in many eukaryotic organisms from yeast to human. TRP channels exert a multitude of physiological functions ranging from Ca2+ homeostasis in the kidney to pain reception and vision. These channels are activated by a wide range of stimuli and undergo covalent post-translational modifications that affect and modulate their subcellular targeting, their biophysical properties, or channel gating. These modifications include N-linked glycosylation, protein phosphorylation, and covalent attachment of chemicals that reversibly bind to specific cysteine residues. The latter modification represents an unusual activation mechanism of ligand-gated ion channels that is in contrast to the lock-and-key paradigm of receptor activation by its agonists. In this review, we summarize the post-translational modifications identified on TRP channels and, when available, explain their physiological role.

  10. Differential Effects of TRPA and TRPV Channels on Behaviors of

    Directory of Open Access Journals (Sweden)

    Jennifer Thies

    2016-01-01

    Full Text Available 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.

  11. The analgesic effect of trans-resveratrol is regulated by calcium channels in the hippocampus of mice.

    Science.gov (United States)

    Wang, Weijie; Yu, Yingcong; Li, Jing; Wang, Lin; Li, Zhi; Zhang, Chong; Zhen, Linlin; Ding, Lianshu; Wang, Gang; Sun, Xiaoyang; Xu, Ying

    2017-08-01

    Resveratrol has been widely studied in terms of it's potential to slow the progression of many diseases. But little is known about the mechanism of action in neuropathic pain. Neuropathic pain is the main type of chronic pain associated with tissue injury. Calcium channels and calcium/caffeine-sensitive pools are associated with analgesic pathway involving neuropathic pain. Our previous study suggested that the antinociceptive effect of resveratrol was involved in Ca 2+ /calmodulin-dependent signaling in the spinal cord of mice. The aim of this study was to explore the involvement of Ca 2+ in analgesic effects of trans-resveratrol in neuropathic pain and signal pathway in hippocampus. Hot plate test was used to assess antinociceptive response when mice were treated with trans-resveratrol alone or in combination with Mk 801, nimodipine, CaCl 2 , ryanodine or EGTA. The effects of trans-resveratrol and the combination on Ca 2+ /calmodulin-dependent protein kinase II (CaMKII) and BDNF (brain-derived neurotrophic factor) expression in hippocampus were also investigated. The results showed that trans-resveratrol increased paw withdraw latency in the hot plate test. The effect of resveratrol was enhanced by Mk 801 and nimodipine. Central administration of Ca 2+ , however, abolished the antinociceptive effects of resveratrol. In contrast, centrally administered EGTA or ryanodine improved trans-resveratrol induced antinociception. There was a significant increase in p-CaMKII and BDNF expression in the hippocampus when resveratrol were combined with Mk 801, nimodipine, ryanodine and EGTA. Administration of CaCl 2 blocked changes in p-CaMKII and BDNF levels in the hippocampus. These findings suggest that trans-resveratrol exerts the effects of antinociception through regulation of calcium channels and calcium/caffeine-sensitive pools.

  12. Ion channeling

    International Nuclear Information System (INIS)

    Erramli, H.; Blondiaux, G.

    1994-01-01

    Channeling phenomenon was predicted, many years ago, by stark. The first channeling experiments were performed in 1963 by Davies and his coworkers. Parallely Robinson and Oen have investigated this process by simulating trajectories of ions in monocrystals. This technique has been combined with many methods like Rutherford Backscattering Spectrometry (R.B.S.), Particles Induced X-rays Emission (P.I.X.E) and online Nuclear Reaction (N.R.A.) to localize trace elements in the crystal or to determine crystalline quality. To use channeling for material characterization we need data about the stopping power of the incident particle in the channeled direction. The ratios of channeled to random stopping powers of silicon for irradiation in the direction have been investigated and compared to the available theoretical results. We describe few applications of ion channeling in the field of materials characterization. Special attention is given to ion channeling combined with Charged Particle Activation Analysis (C.P.A.A.) for studying the behaviour of oxygen atoms in Czochralski silicon lattices under the influence of internal gettering and in different gaseous atmospheres. Association between ion channeling and C.P.A.A was also utilised for studying the influence of the growing conditions on concentration and position of carbon atoms at trace levels in the MOVPE Ga sub (1-x) Al sub x lattice. 6 figs., 1 tab., 32 refs. (author)

  13. Soluble adenylyl cyclase in vascular endothelium: gene expression control of epithelial sodium channel-α, Na+/K+-ATPase-α/β, and mineralocorticoid receptor.

    Science.gov (United States)

    Schmitz, Boris; Nedele, Johanna; Guske, Katrin; Maase, Martina; Lenders, Malte; Schelleckes, Michael; Kusche-Vihrog, Kristina; Brand, Stefan-Martin; Brand, Eva

    2014-04-01

    The Ca(2+)- and bicarbonate-activated soluble adenylyl cyclase (sAC) has been identified recently as an important mediator of aldosterone signaling in the kidney. Nuclear sAC has been reported to stimulate cAMP response element-binding protein 1 phosphorylation via protein kinase A, suggesting an alternative cAMP pathway in the nucleus. In this study, we analyzed the sAC as a potential modulator of endothelial stiffness in the vascular endothelium. We determined the contribution of sAC to cAMP response element-mediated transcriptional activation in vascular endothelial cells and kidney collecting duct cells. Inhibition of sAC by the specific inhibitor KH7 significantly reduced cAMP response element-mediated promoter activity and affected cAMP response element-binding protein 1 phosphorylation. Furthermore, KH7 and anti-sAC small interfering RNA significantly decreased mRNA and protein levels of epithelial sodium channel-α and Na(+)/K(+)-ATPase-α. Using atomic force microscopy, a nano-technique that measures stiffness and deformability of living cells, we detected significant endothelial cell softening after sAC inhibition. Our results suggest that the sAC is a regulator of gene expression involved in aldosterone signaling and an important regulator of endothelial stiffness. Additional studies are warranted to investigate the protective action of sAC inhibitors in humans for potential clinical use.

  14. α/sub i/-3 cDNA encodes the α subunit of G/sub k/, the stimulatory G protein of receptor-regulated K+ channels

    International Nuclear Information System (INIS)

    Codina, J.; Olate, J.; Abramowitz, J.; Mattera, R.; Cook, R.G.; Birnbaumer, L.

    1988-01-01

    cDNA cloning has identified the presence in the human genome of three genes encoding α subunits of pertussis toxin substrates, generically called G/sub i/. They are named α/sub i/-1, α/sub i/-2 and α/sub i/-3. However, none of these genes has been functionally identified with any of the α subunits of several possible G proteins, including pertussis toxin-sensitive G/sub p/'s, stimulatory to phospholipase C or A 2 , G/sub i/, inhibitory to adenylyl cyclase, or G/sub k/, stimulatory to a type of K + channels. The authors now report the nucleotide sequence and the complete predicted amino acid sequence of human liver α/sub i/-3 and the partial amino acid sequence of proteolytic fragments of the α subunit of human erythrocyte G/sub k/. The amino acid sequence of the proteolytic fragment is uniquely encoded by the cDNA of α/sub i/-3, thus identifying it as α/sub k/. The probable identity of α/sub i/-1 with α/sub p/ and possible roles for α/sub i/-2, as well as additional roles for α/sub i/-1 and α/sub i/-3 (α/sub k/) are discussed

  15. Channel box

    International Nuclear Information System (INIS)

    Tanabe, Akira.

    1993-01-01

    In a channel box of a BWR type reactor, protruding pads are disposed in axial position on the lateral side of a channel box opposing to a control rod and facing the outer side portion of the control rod in a reactor core loaded state. In the initial loading stage of fuel assemblies, channel fasteners and spacer pads are abutted against each other in the upper portion between the channel boxes sandwiching the control rod therebetween. Further, in the lower portion, a gap as a channel for the movement of the control rod is ensured by the support of fuel support metals. If the channel box is bent toward the control rod along with reactor operation, the pads are abutted against each other to always ensure the gap through which the control rod can move easily. Further, when the pads are brought into contact with each other, the bending deformation of the channel box is corrected by urging to each other. Thus, the control rod can always be moved smoothly to attain reactor safety operation. (N.H.)

  16. Surface channeling

    International Nuclear Information System (INIS)

    Sizmann, R.; Varelas, C.

    1976-01-01

    There is experimental evidence that swift light ions incident at small angles towards single crystalline surfaces can lose an appreciable fraction of their kinetic energy during reflection. It is shown that these projectiles penetrate into the bulk surface region of the crystal. They can travel as channeled particles along long paths through the solid (surface channeling). The angular distribution and the depth history of the re-emerged projectiles are investigated by computer simulations. A considerable fraction of the penetrating projectiles re-emerges from the crystal with constant transverse energy if the angle of incidence is smaller than the critical angle for axial channeling. Analytical formulae are derived based on a diffusion model for surface channeling. A comparison with experimental data exhibits the relevance of the analytical solutions. (Auth.)

  17. Spark Channels

    Energy Technology Data Exchange (ETDEWEB)

    Haydon, S. C. [Department of Physics, University of New England, Armidale, NSW (Australia)

    1968-04-15

    A brief summary is given of the principal methods used for initiating spark channels and the various highly time-resolved techniques developed recently for studies with nanosecond resolution. The importance of the percentage overvoltage in determining the early history and subsequent development of the various phases of the growth of the spark channel is discussed. An account is then given of the recent photographic, oscillographic and spectroscopic investigations of spark channels initiated by co-axial cable discharges of spark gaps at low [{approx} 1%] overvoltages. The phenomena observed in the development of the immediate post-breakdown phase, the diffuse glow structure, the growth of the luminous filament and the final formation of the spark channel in hydrogen are described. A brief account is also given of the salient features emerging from corresponding studies of highly overvolted spark gaps in which the spark channel develops from single avalanche conditions. The essential differences between the two types of channel formation are summarized and possible explanations of the general features are indicated. (author)

  18. Pannexin-1 channels in epilepsy.

    Science.gov (United States)

    Aquilino, Mark S; Whyte-Fagundes, Paige; Zoidl, Georg; Carlen, Peter L

    2017-09-05

    Pannexin-1 (Panx1) expression is raised in several animal seizure models and in resected human epileptic brain tissue, suggesting relevance to epilepsy. Multiple factors that are characteristic of seizures are thought to regulate Panx1 channel opening, including elevated levels of extracellular K + . Panx1, when open, 1) releases ATP, glutamate, and other metabolites into the extracellular medium, and 2) may depolarize the membrane due to a channel reversal potential around 0mV. Resultant ATP release from stimulated Panx1 can activate purinergic receptors, including P2X7 receptors. Glutamate and other signaling molecules released by Panx1 opening may have both excitatory and inhibitory actions on seizure generation. This review examines the critical and complex roles of Panx1 channels in epilepsy, which could provide a basis for future therapeutics. Copyright © 2017 Elsevier B.V. All rights reserved.

  19. Functional inactivation of hypocretin 1 receptors in the medial prefrontal cortex affects the pyramidal neuron activity and gamma oscillations: An in vivo multiple-channel single-unit recording study.

    Science.gov (United States)

    He, C; Chen, Q-H; Ye, J-N; Li, C; Yang, L; Zhang, J; Xia, J-X; Hu, Z-A

    2015-06-25

    The hypocretin signaling is thought to play a critical role in maintaining wakefulness via stimulating the subcortical arousal pathways. Although the cortical areas, including the medial prefrontal cortex (mPFC), receive dense hypocretinergic fibers and express its receptors, it remains unclear whether the hypocretins can directly regulate the neural activity of the mPFC in vivo. In the present study, using multiple-channel single-unit recording study, we found that infusion of the SB-334867, a blocker for the Hcrtr1, beside the recording sites within the mPFC substantially exerted an inhibitory effect on the putative pyramidal neuron (PPN) activity in naturally behaving rats. In addition, functional blockade of the Hcrtr1 also selectively reduced the power of the gamma oscillations. The PPN activity and the power of the neural oscillations were not affected after microinjection of the TCS-OX2-29, a blocker for the Hcrtr2, within the mPFC. Together, these data indicate that endogenous hypocretins acting on the Hcrtr1 are required for the normal neural activity in the mPFC in vivo, and thus might directly contribute cortical arousal and mPFC-dependent cognitive processes. Copyright © 2015 IBRO. Published by Elsevier Ltd. All rights reserved.

  20. The structurally novel Ca2+ channel blocker Ro 40-5967, which binds to the [3H] desmethoxyverapamil receptor, is devoid of the negative inotropic effects of verapamil in normal and failing rat hearts

    International Nuclear Information System (INIS)

    Clozel, J.P.; Veniant, M.; Osterrieder, W.

    1990-01-01

    Ro 40-5967 is a structurally novel Ca 2+ channel blocker that binds to the verapamil-type receptor of cardiac membranes but that has been shown in isolated guinea-pig hearts to be about ten times less potent a negative inotropic agent than verapamil. The goals of the present study were to confirm these findings in vitro in isolated perfused rat hearts as well as in vivo in conscious rats and to compare Ro 40-5967 to verapamil. The effects of Ro 40-5967 and verapamil were tested not only in normal rats, but also in rats with heart failure induced by chronic myocardial infarction. In isolated Langendorff hearts (without heart failure), no decrease of contractility was observed with Ro 40-5967 up to complete AV block. In contrast, verapamil decreased contractility with an IC50 of 100 nM. In isolated, electrically stimulated rat papillary muscles, the IC50 values for the decrease of contractile force were 15,000 and 440 nM for Ro 40-5967 and verapamil, respectively. In vivo, Ro 40-5967 did not decrease left ventricular contractility (as assessed by changes of dP/dt max +) in rats without and with heart failure. In contrast, verapamil was markedly negative inotropic in both conditions

  1. Cell surface expression of channel catfish leukocyte immune-type receptors (IpLITRs) and recruitment of both Src homology 2 domain-containing protein tyrosine phosphatase (SHP)-1 and SHP-2.

    Science.gov (United States)

    Montgomery, Benjamin C S; Mewes, Jacqueline; Davidson, Chelsea; Burshtyn, Deborah N; Stafford, James L

    2009-04-01

    Channel catfish leukocyte immune-type receptors (IpLITRs) are immunoglobulin superfamily (IgSF) members believed to play a role in the control and coordination of cellular immune responses in teleost. Putative stimulatory and inhibitory IpLITRs are co-expressed by different types of catfish immune cells (e.g. NK cells, T cells, B cells, and macrophages) but their signaling potential has not been determined. Following cationic polymer-mediated transfections into human cell lines we examined the surface expression, tyrosine phosphorylation, and phosphatase recruitment potential of two types of putative inhibitory IpLITRs using 'chimeric' expression constructs and an epitope-tagged 'native' IpLITR. We also cloned and expressed the teleost Src homology 2 domain-containing protein tyrosine phosphatases (SHP)-1 and SHP-2 and examined their expression in adult tissues and developing zebrafish embryos. Co-immunoprecipitation experiments support the inhibitory signaling potential of distinct IpLITR-types that bound both SHP-1 and SHP-2 following the phosphorylation of tyrosine residues within their cytoplasmic tail (CYT) regions. Phosphatase recruitment by IpLITRs represents an important first step in understanding their influence on immune cell effector functions and suggests that certain inhibitory signaling pathways are conserved among vertebrates.

  2. β-Adrenergic receptor stimulation inhibits proarrhythmic alternans in postinfarction border zone cardiomyocytes: a computational analysis.

    Science.gov (United States)

    Tomek, Jakub; Rodriguez, Blanca; Bub, Gil; Heijman, Jordi

    2017-08-01

    The border zone (BZ) of the viable myocardium adjacent to an infarct undergoes extensive autonomic and electrical remodeling and is prone to repolarization alternans-induced cardiac arrhythmias. BZ remodeling processes may promote or inhibit Ca 2+ and/or repolarization alternans and may differentially affect ventricular arrhythmogenesis. Here, we used a detailed computational model of the canine ventricular cardiomyocyte to study the determinants of alternans in the BZ and their regulation by β-adrenergic receptor (β-AR) stimulation. The BZ model developed Ca 2+ transient alternans at slower pacing cycle lengths than the control model, suggesting that the BZ may promote spatially heterogeneous alternans formation in an infarcted heart. β-AR stimulation abolished alternans. By evaluating all combinations of downstream β-AR stimulation targets, we identified both direct (via ryanodine receptor channels) and indirect [via sarcoplasmic reticulum (SR) Ca 2+ load] modulation of SR Ca 2+ release as critical determinants of Ca 2+ transient alternans. These findings were confirmed in a human ventricular cardiomyocyte model. Cell-to-cell coupling indirectly modulated the likelihood of alternans by affecting the action potential upstroke, reducing the trigger for SR Ca 2+ release in one-dimensional strand simulations. However, β-AR stimulation inhibited alternans in both single and multicellular simulations. Taken together, these data highlight a potential antiarrhythmic role of sympathetic hyperinnervation in the BZ by reducing the likelihood of alternans and provide new insights into the underlying mechanisms controlling Ca 2+ transient and repolarization alternans. NEW & NOTEWORTHY We integrated, for the first time, postmyocardial infarction electrical and autonomic remodeling in a detailed, validated computer model of β-adrenergic stimulation in ventricular cardiomyocytes. Here, we show that β-adrenergic stimulation inhibits alternans and provide novel insights

  3. MARKETING CHANNELS

    Directory of Open Access Journals (Sweden)

    Ljiljana Stošić Mihajlović

    2014-07-01

    Full Text Available Marketing channel is a set of entities and institutions, completion of distribution and marketing activities, attend the efficient and effective networking of producers and consumers. Marketing channels include the total flows of goods, money and information taking place between the institutions in the system of marketing, establishing a connection between them. The functions of the exchange, the physical supply and service activities, inherent in the system of marketing and trade. They represent paths which products and services are moving after the production, which will ultimately end up buying and eating by the user.

  4. BDE-47 and 6-OH-BDE-47 modulate calcium homeostasis in primary fetal human neural progenitor cells via ryanodine receptor-independent mechanisms

    NARCIS (Netherlands)

    Gassmann, Kathrin; Schreiber, Timm; Dingemans, Milou M L; Krause, Guido; Roderigo, Claudia; Giersiefer, Susanne; Schuwald, Janette; Moors, Michaela; Unfried, Klaus; Bergman, Åke; Westerink, Remco H S; Rose, Christine R.; Fritsche, Ellen

    2014-01-01

    Polybrominated diphenyl ethers (PBDEs) are bioaccumulating flame retardants found in rising concentrations in human tissue. Epidemiological and animal studies have raised concern for their potential to induce developmental neurotoxicity (DNT). Considering the essential role of calcium homeostasis in

  5. A meta-analysis of the effect of angiotensin receptor blockers and calcium channel blockers on blood pressure, glycemia and the HOMA-IR index in non-diabetic patients.

    Science.gov (United States)

    Yang, Yue; Wei, Ri-bao; Xing, Yue; Tang, Lu; Zheng, Xiao-yong; Wang, Zi-cheng; Gao, Yu-wei; Li, Min-xia; Chen, Xiang-mei

    2013-12-01

    This study compared the efficacy of angiotensin receptor blockers (ARBs) and calcium channel blockers (CCBs) in the effect of insulin resistance (IR) as assessed using the homeostasis model assessment of insulin resistance (HOMA-IR) in non-diabetic patients. The MEDLINE, EMBASE, and Cochrane Library databases were searched to identify studies published before December 2012 that investigated the use of ARBs and CCBs to determine the effect on the HOMA-IR index in non-diabetics. Parameters on IR and blood pressure were collected. Review Manager 5.2 and Stata 12.0 were used to perform the meta-analysis. Fixed and random effects models were applied to various aspects of the meta-analysis, which assessed the therapeutic effects of the two types of drug using the HOMA-IR index in non-diabetic patients. The meta-analysis included five clinical trials. Patient comparisons before and after treatment with ARBs and CCBs revealed that ARBs reduced the HOMA-IR index (weighted mean difference (WMD) -0.65, 95% confidence interval (CI) -0.93 to -0.38) and fasting plasma insulin (FPI) (WMD -2.01, 95% CI -3.27 to -0.74) significantly more than CCBs. No significant differences in the therapeutic effects of these two types of drug on blood pressure were observed. Given that there are no significant differences in the therapeutic effects of ARBs and CCBs on blood pressure, as ARBs are superior to CCBs in their effect on the HOMA-IR index in non-diabetics, they might be a better choice in hypertension patients without diabetes. © 2013.

  6. Estradiol up-regulates L-type Ca2+ channels via membrane-bound estrogen receptor/phosphoinositide-3-kinase/Akt/cAMP response element-binding protein signaling pathway.

    Science.gov (United States)

    Yang, Xiaoyan; Mao, Xiaofang; Xu, Gao; Xing, Shasha; Chattopadhyay, Ansuman; Jin, Si; Salama, Guy

    2018-05-01

    In long QT syndrome type 2, women are more prone than men to the lethal arrhythmia torsades de pointes. We previously reported that 17β-estradiol (E2) up-regulates L-type Ca 2+ channels and current (I Ca,L ) (∼30%) in rabbit ventricular myocytes by a classic genomic mechanism mediated by estrogen receptor-α (ERα). In long QT syndrome type 2 (I Kr blockade or bradycardia), the higher Ca 2+ influx via I Ca,L causes Ca 2+ overload, spontaneous sarcoplasmic reticulum Ca 2+ release, and reactivation of I Ca,L that triggers early afterdepolarizations and torsades de pointes. The purpose of this study was to investigate the molecular mechanisms whereby E2 up-regulates I Ca,L , which are poorly understood. H9C2 and rat myocytes were incubated with E2 ± ER antagonist, or inhibitors of downstream transcription factors, for 24 hours, followed by western blots of Cav1.2α1C and voltage-clamp measurements of I Ca,L . Incubation of H9C2 cells with E2 (10-100 nM) increased I Ca,L density and Cav1.2α1C expression, which were suppressed by the ER antagonist ICI182,780 (1 μM). Enhanced I Ca,L and Cav1.2α1C expression by E2 was suppressed by inhibitors of phosphoinositide-3-kinase (Pi3K) (30 μM LY294002; P L via plasma membrane ER and by activating Pi3K, Akt, and CREB signaling. The promoter regions of the CACNA1C gene (human-rabbit-rat) contain adjacent/overlapping binding sites for p-CREB and ERα, which suggests a synergistic regulation by these pathways. Copyright © 2018 Heart Rhythm Society. Published by Elsevier Inc. All rights reserved.

  7. GABA receptor imaging

    Energy Technology Data Exchange (ETDEWEB)

    Lee, Jong Doo [Yonsei University College of Medicine, Seoul (Korea, Republic of)

    2007-04-15

    GABA is primary an inhibitory neurotransmitter that is localized in inhibitory interneurons. GABA is released from presynaptic terminals and functions by binding to GABA receptors. There are two types of GABA receptors, GABA{sub A}-receptor that allows chloride to pass through a ligand gated ion channel and GABA{sub B}-receptor that uses G-proteins for signaling. The GABA{sub A}-receptor has a GABA binding site as well as a benzodiazepine binding sites, which modulate GABA{sub A}-receptor function. Benzodiazepine GABAA receptor imaging can be accomplished by radiolabeling derivates that activates benzodiazepine binding sites. There has been much research on flumazenil (FMZ) labeled with {sup 11}C-FMZ, a benzodiazepine derivate that is a selective, reversible antagonist to GABAA receptors. Recently, {sup 18}F-fluoroflumazenil (FFMZ) has been developed to overcome {sup 11}C's short half-life. {sup 18}F-FFMZ shows high selective affinity and good pharmacodynamics, and is a promising PET agent with better central benzodiazepine receptor imaging capabilities. In an epileptic focus, because the GABA/benzodiazepine receptor amount is decreased, using '1{sup 1}C-FMZ PET instead of {sup 18}F-FDG, PET, restrict the foci better and may also help find lesions better than high resolution MR. GABA{sub A} receptors are widely distributed in the cerebral cortex, and can be used as an viable neuronal marker. Therefore it can be used as a neuronal cell viability marker in cerebral ischemia. Also, GABA-receptors decrease in areas where neuronal plasticity develops, therefore, GABA imaging can be used to evaluate plasticity. Besides these usages, GABA receptors are related with psychological diseases, especially depression and schizophrenia as well as cerebral palsy, a motor-related disorder, so further in-depth studies are needed for these areas.

  8. GABA receptor imaging

    International Nuclear Information System (INIS)

    Lee, Jong Doo

    2007-01-01

    GABA is primary an inhibitory neurotransmitter that is localized in inhibitory interneurons. GABA is released from presynaptic terminals and functions by binding to GABA receptors. There are two types of GABA receptors, GABA A -receptor that allows chloride to pass through a ligand gated ion channel and GABA B -receptor that uses G-proteins for signaling. The GABA A -receptor has a GABA binding site as well as a benzodiazepine binding sites, which modulate GABA A -receptor function. Benzodiazepine GABAA receptor imaging can be accomplished by radiolabeling derivates that activates benzodiazepine binding sites. There has been much research on flumazenil (FMZ) labeled with 11 C-FMZ, a benzodiazepine derivate that is a selective, reversible antagonist to GABAA receptors. Recently, 18 F-fluoroflumazenil (FFMZ) has been developed to overcome 11 C's short half-life. 18 F-FFMZ shows high selective affinity and good pharmacodynamics, and is a promising PET agent with better central benzodiazepine receptor imaging capabilities. In an epileptic focus, because the GABA/benzodiazepine receptor amount is decreased, using '1 1 C-FMZ PET instead of 18 F-FDG, PET, restrict the foci better and may also help find lesions better than high resolution MR. GABA A receptors are widely distributed in the cerebral cortex, and can be used as an viable neuronal marker. Therefore it can be used as a neuronal cell viability marker in cerebral ischemia. Also, GABA-receptors decrease in areas where neuronal plasticity develops, therefore, GABA imaging can be used to evaluate plasticity. Besides these usages, GABA receptors are related with psychological diseases, especially depression and schizophrenia as well as cerebral palsy, a motor-related disorder, so further in-depth studies are needed for these areas

  9. Channel Power in Multi-Channel Environments

    NARCIS (Netherlands)

    M.G. Dekimpe (Marnik); B. Skiera (Bernd)

    2004-01-01

    textabstractIn the literature, little attention has been paid to instances where companies add an Internet channel to their direct channel portfolio. However, actively managing multiple sales channels requires knowing the customers’ channel preferences and the resulting channel power. Two key

  10. Insulin receptors

    International Nuclear Information System (INIS)

    Kahn, C.R.; Harrison, L.C.

    1988-01-01

    This book contains the proceedings on insulin receptors. Part A: Methods for the study of structure and function. Topics covered include: Method for purification and labeling of insulin receptors, the insulin receptor kinase, and insulin receptors on special tissues

  11. 3D structure of muscle dihydropyridine receptor

    Directory of Open Access Journals (Sweden)

    Montserrat Samsó

    2015-01-01

    Full Text Available Excitation contraction coupling, the rapid and massive Ca2+ release under control of an action potential that triggers muscle contraction, takes places at specialized regions of the cell called triad junctions. There, a highly ordered supramolecular complex between the dihydropyridine receptor (DHPR and the ryanodine receptor (RyR1 mediates the quasi‐instantaneous conversion from T‐tubule depolarization into Ca2+ release from the sarcoplasmic reticulum (SR. The DHPR has several key modules required for EC coupling: the voltage sensors and II‐III loop in the alpha1s subunit, and the beta subunit. To gain insight into their molecular organization, this review examines the most updated 3D structure of the DHPR as obtained by transmission electron microscopy and image reconstruction. Although structure determination of a heteromeric membrane protein such as the DHPR is challenging, novel technical advances in protein expression and 3D labeling facilitated this task. The 3D structure of the DHPR complex consists of a main body with five irregular corners around its perimeter encompassing the transmembrane alpha 1s subunit besides the intracellular beta subunit, an extended extracellular alpha 2 subunit, and a bulky intracellular II‐III loop. The structural definition attained at 19 Å resolution enabled docking of the atomic coordinates of structural homologs of the alpha1s and beta subunits. These structural features, together with their relative location with respect to the RyR1, are discussed in the context of the functional data.

  12. Channelling and electromagnetic radiation of channelling particles

    International Nuclear Information System (INIS)

    Kalashnikov, N.

    1983-01-01

    A brief description is presented of the channelling of charged particles between atoms in the crystal lattice. The specificities are discussed of the transverse motion of channelling particles as are the origin and properties of quasi-characteristic radiation of channelling particles which accompany transfers from one band of permissible energies of the transverse motion of channelling particles to the other. (B.S.)

  13. Direct proton conductance through the TRPV1 pore and multimerization of TRPV channel subunits

    OpenAIRE

    Hellwig, Nicole Barbara

    2010-01-01

    TRPV1-induced intracellular acidification: The vanilloid receptor-related transient receptor potential channels (TRPV) belong to the superfamily of hexahelical cation channels and are integral components of thermosensation, pain perception and Ca2+-reabsorption in kidney and intestine. The vanilloid receptor (TRPV1), a poorly selective cation channel, is expressed in dorsal root ganglion (DRG) neurons and is regulated by diverse stimuli including capsaicin, endovanilloids and heat. Since a...

  14. Ion channels: molecular targets of neuroactive insecticides.

    Science.gov (United States)

    Raymond-Delpech, Valérie; Matsuda, Kazuhiko; Sattelle, Benedict M; Rauh, James J; Sattelle, David B

    2005-11-01

    Many of the insecticides in current use act on molecular targets in the insect nervous system. Recently, our understanding of these targets has improved as a result of the complete sequencing of an insect genome, i.e., Drosophila melanogaster. Here we examine the recent work, drawing on genetics, genomics and physiology, which has provided evidence that specific receptors and ion channels are targeted by distinct chemical classes of insect control agents. The examples discussed include, sodium channels (pyrethroids, p,p'-dichlorodiphenyl-trichloroethane (DDT), dihydropyrazoles and oxadiazines); nicotinic acetylcholine receptors (cartap, spinosad, imidacloprid and related nitromethylenes/nitroguanidines); gamma-aminobutyric acid (GABA) receptors (cyclodienes, gamma-BHC and fipronil) and L-glutamate receptors (avermectins). Finally, we have examined the molecular basis of resistance to these molecules, which in some cases involves mutations in the molecular target, and we also consider the future impact of molecular genetic technologies in our understanding of the actions of neuroactive insecticides.

  15. Channel Modeling

    Science.gov (United States)

    Schmitz, Arne; Schinnenburg, Marc; Gross, James; Aguiar, Ana

    For any communication system the Signal-to-Interference-plus-Noise-Ratio of the link is a fundamental metric. Recall (cf. Chapter 9) that the SINR is defined as the ratio between the received power of the signal of interest and the sum of all "disturbing" power sources (i.e. interference and noise). From information theory it is known that a higher SINR increases the maximum possible error-free transmission rate (referred to as Shannon capacity [417] of any communication system and vice versa). Conversely, the higher the SINR, the lower will be the bit error rate in practical systems. While one aspect of the SINR is the sum of all distracting power sources, another issue is the received power. This depends on the transmitted power, the used antennas, possibly on signal processing techniques and ultimately on the channel gain between transmitter and receiver.

  16. Channeling experiment

    International Nuclear Information System (INIS)

    Abelin, H.; Birgersson, L.; Widen, H.; Aagren, T.; Moreno, L.; Neretnieks, I.

    1990-07-01

    Channeling of water flow and tracer transport in real fractures in a granite body at Stripa have been investigated experimentally. The experimental site was located 360 m below the ground level. Two kinds of experiments were performed. In the single hole experiments, 20 cm diameter holes were drilled about 2.5 m into the rock in the plane of the fracture. Specially designed packers were used to inject water into the fracture in 5 cm intervals all along the fracture trace in the hole. The variation of the injection flowrates along the fracture were used to determine the transmissivity variations in the fracture plane. Detailed photographs were taken from inside the hole and the visual fracture aperture was compared with the injection flowrates in the same locations. Geostatistical methods were used to evaluate the results. Five holes were measured in great detail. In addition 7 holes were drilled and scanned by simpler packer systems. A double hole experiment was performed where two parallel holes were drilled in the same fracture plane at nearly 2 m distance. Pressure pulse tests were made between the holes in both directions. Tracers were injected in 5 locations in one hole and monitored for in many locations in the other hole. The single hole experiment and the double hole experiment show that most of the fracture planes are tight but that there are open sections which form connected channels over distances of at least 2 meters. It was also found in the double hole experiment that the investigated fracture was intersected by at least one fracture between the two holes which diverted a large amount of the injected tracers to several distant locations at the tunnel wall. (authours)

  17. ASIC3 channels in multimodal sensory perception.

    Science.gov (United States)

    Li, Wei-Guang; Xu, Tian-Le

    2011-01-19

    Acid-sensing ion channels (ASICs), which are members of the sodium-selective cation channels belonging to the epithelial sodium channel/degenerin (ENaC/DEG) family, act as membrane-bound receptors for extracellular protons as well as nonproton ligands. At least five ASIC subunits have been identified in mammalian neurons, which form both homotrimeric and heterotrimeric channels. The highly proton sensitive ASIC3 channels are predominantly distributed in peripheral sensory neurons, correlating with their roles in multimodal sensory perception, including nociception, mechanosensation, and chemosensation. Different from other ASIC subunit composing ion channels, ASIC3 channels can mediate a sustained window current in response to mild extracellular acidosis (pH 7.3-6.7), which often occurs accompanied by many sensory stimuli. Furthermore, recent evidence indicates that the sustained component of ASIC3 currents can be enhanced by nonproton ligands including the endogenous metabolite agmatine. In this review, we first summarize the growing body of evidence for the involvement of ASIC3 channels in multimodal sensory perception and then discuss the potential mechanisms underlying ASIC3 activation and mediation of sensory perception, with a special emphasis on its role in nociception. We conclude that ASIC3 activation and modulation by diverse sensory stimuli represent a new avenue for understanding the role of ASIC3 channels in sensory perception. Furthermore, the emerging implications of ASIC3 channels in multiple sensory dysfunctions including nociception allow the development of new pharmacotherapy.

  18. Mechanosensitive Piezo Channels in the Gastrointestinal Tract.

    Science.gov (United States)

    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.

  19. Ca 2+ signaling by plant Arabidopsis thaliana Pep peptides depends on AtPepR1, a receptor with guanylyl cyclase activity, and cGMP-activated Ca 2+ channels

    KAUST Repository

    Qia, Zhi; Verma, Rajeev K.; Gehring, Christoph A; Yamaguchi, Yube; Zhao, Yichen; Ryan, Clarence A.; Berkowitz, Gerald A.

    2010-01-01

    receptor- like kinase receptor AtPepR1 has guanylyl cyclase activity, generating cGMP from GTP, and that cGMP can activate CNGC2- dependent cytosolic Ca 2+ elevation. AtPep-dependent expression of pathogen-defense genes (PDF1.2, MPK3, and WRKY33

  20. Lessons from crystal structures of kainate receptors

    DEFF Research Database (Denmark)

    Møllerud, Stine; Frydenvang, Karla Andrea; Pickering, Darryl S

    2017-01-01

    Kainate receptors belong to the family of ionotropic glutamate receptors. These receptors assemble from five subunits (GluK1-5) into tetrameric ion channels. Kainate receptors are located at both pre- and postsynaptic membranes in the central nervous system where they contribute to excitatory...... synaptic transmission and modulate network excitability by regulating neurotransmitter release. Dysfunction of kainate receptors has been implicated in several neurological disorders such as epilepsy, schizophrenia and depression. Here we provide a review on the current understanding of kainate receptor...

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

    Science.gov (United States)

    Borbiro, Istvan; Badheka, Doreen; Rohacs, Tibor

    2015-01-01

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

  2. Function and expression of the epithelial Ca(2+) channel family: comparison of mammalian ECaC1 and 2.

    NARCIS (Netherlands)

    Hoenderop, J.G.J.; Vennekens, R.; Müller, D.G.; Prenen, J.; Droogmans, G.; Bindels, R.J.M.; Nilius, B.

    2001-01-01

    1. The epithelial Ca(2+) channel (ECaC) family represents a unique group of Ca(2+)-selective channels that share limited homology to the ligand-gated capsaicin receptors, the osmolarity-sensitive channel OTRPC4, as well as the transient receptor potential family. Southern blot analysis demonstrated

  3. Somatostatin receptors

    DEFF Research Database (Denmark)

    Møller, Lars Neisig; Stidsen, Carsten Enggaard; Hartmann, Bolette

    2003-01-01

    functional units, receptors co-operate. The total receptor apparatus of individual cell types is composed of different-ligand receptors (e.g. SRIF and non-SRIF receptors) and co-expressed receptor subtypes (e.g. sst(2) and sst(5) receptors) in characteristic proportions. In other words, levels of individual......-peptides, receptor agonists and antagonists. Relatively long half lives, as compared to those of the endogenous ligands, have been paramount from the outset. Motivated by theoretical puzzles or the shortcomings of present-day diagnostics and therapy, investigators have also aimed to produce subtype...

  4. Epithelial calcium channels: from identification to function and regulation.

    NARCIS (Netherlands)

    Hoenderop, J.G.J.; Nilius, B.; Bindels, R.J.M.

    2003-01-01

    The epithelial calcium channels TRPV5 and TRPV6 have been studied extensively in the epithelial tissues controlling Ca(2+) homeostasis and exhibit a range of distinctive properties that distinguish them from other transient receptor potential (TRP) channels. These two novel members of the

  5. Structural Studies of Nicotinic Acetylcholine Receptors

    DEFF Research Database (Denmark)

    Shahsavar, Azadeh; Gajhede, Michael; Kastrup, Jette

    2016-01-01

    Nicotinic acetylcholine receptors (nAChRs) are members of the pentameric ligand-gated ion channel superfamily that play important roles in control of neurotransmitter release in the central and peripheral nervous system. These receptors are important therapeutic targets for development of drugs...

  6. Citizens and service channels: channel choice and channel management implications

    NARCIS (Netherlands)

    Pieterson, Willem Jan

    2010-01-01

    The arrival of electronic channels in the 1990s has had a huge impact on governmental service delivery. The new channels have led to many new opportunities to improve public service delivery, not only in terms of citizen satisfaction, but also in cost reduction for governmental agencies. However,

  7. Ion Channels and Zinc: Mechanisms of Neurotoxicity and Neurodegeneration

    Directory of Open Access Journals (Sweden)

    Deborah R. Morris

    2012-01-01

    Full Text Available Ionotropic glutamate receptors, such as NMDA, AMPA and kainate receptors, are ligand-gated ion channels that mediate much of the excitatory neurotransmission in the brain. Not only do these receptors bind glutamate, but they are also regulated by and facilitate the postsynaptic uptake of the trace metal zinc. This paper discusses the role of the excitotoxic influx and accumulation of zinc, the mechanisms responsible for its cytotoxicity, and a number of disorders of the central nervous system that have been linked to these neuronal ion channels and zinc toxicity including ischemic brain injury, traumatic brain injury, and epilepsy.

  8. Atropisomers of 2,2',3,3',6,6'-hexachlorobiphenyl (PCB 136) exhibit stereoselective effects on activation of nuclear receptors in vitro.

    Science.gov (United States)

    Pěnčíková, Kateřina; Brenerová, Petra; Svržková, Lucie; Hrubá, Eva; Pálková, Lenka; Vondráček, Jan; Lehmler, Hans-Joachim; Machala, Miroslav

    2017-11-09

    PCB 136 is an environmentally relevant chiral PCB congener, which has been found in vivo to be present in form of rotational isomers (atropisomers). Its atropselective biotransformation or neurotoxic effects linked with sensitization of ryanodine receptor suggest that it might interact also with other intracellular receptors in a stereospecific manner. However, possible atropselective effects of PCB 136 on nuclear receptor transactivation remain unknown. Therefore, in this study, atropselective effects of PCB 136 on nuclear receptors controlling endocrine signaling and/or expression of xenobiotic and steroid hormone catabolism were investigated. PCB136 atropisomers were found to exert differential effects on estrogen receptor (ER) activation; (+)-PCB 136 was estrogenic, while (-)-PCB 136 was antiestrogenic. In contrast, inhibition of androgen receptor (AR) activity was not stereospecific. Both PCB136 stereoisomers induced the constitutive androgen receptor (CAR)-dependent gene expression; however, no significant stereospecificity of PCB 136 atropisomers was observed. PCB136 was a partial inducer of the pregnane X receptor (PXR)-dependent gene expression. Here, (-)-PCB 136 was a significantly more potent inducer of PXR activity than (+)-PCB 136. Taken together, the present results indicate that at least two nuclear receptors participating in endocrine regulation or metabolism, ER and PXR, could be regulated in an atropselective manner by chiral PCB 136. The enantioselective enrichment of PCB atropisomers in animal and human tissues may thus have significant consequences for endocrine-disrupting effects of chiral ortho-substituted PCB congeners.

  9. TAURINE REGULATION OF VOLTAGE-GATED CHANNELS IN RETINAL NEURONS

    Science.gov (United States)

    Rowan, Matthew JM; Bulley, Simon; Purpura, Lauren; Ripps, Harris; Shen, Wen

    2017-01-01

    Taurine activates not only Cl−-permeable ionotropic receptors, but also receptors that mediate metabotropic responses. The metabotropic property of taurine was revealed in electrophysiological recordings obtained after fully blocking Cl−-permeable receptors with an inhibitory “cocktail” consisting of picrotoxin, SR95531, and strychnine. We found that taurine’s metabotropic effects regulate voltage-gated channels in retinal neurons. After applying the inhibitory cocktail, taurine enhanced delayed outward rectifier K+ channels preferentially in Off-bipolar cells, and the effect was completely blocked by the specific PKC inhibitor, GF109203X. Additionally, taurine also acted through a metabotropic pathway to suppress both L- and N-type Ca2+ channels in retinal neurons, which were insensitive to the potent GABAB receptor inhibitor, CGP55845. This study reinforces our previous finding that taurine in physiological concentrations produces a multiplicity of metabotropic effects that precisely govern the integration of signals being transmitted from the retina to the brain. PMID:23392926

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

    Science.gov (United States)

    Kaczmarek, Leonard K.

    2013-01-01

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

  11. Cardiac actions of phencyclidine in isolated guinea pig and rat heart: possible involvement of slow channels

    International Nuclear Information System (INIS)

    Temma, K.; Akera, T.; Ng, Y.C.

    1985-01-01

    The mechanisms responsible for the positive inotropic effect of phencyclidine were studied in isolated preparations of guinea pig and rat heart. In electrically paced left atrial muscle preparations, phencyclidine increased the force of contraction; rat heart muscle preparations were more sensitive than guinea pig heart muscle preparations. The positive inotropic effect of phencyclidine was not significantly reduced by a combination of phentolamine and nadolol; however, the effect was competitively blocked by verapamil in the presence of phentolamine and nadolol. Inhibition of the outward K+ current by tetraethylammonium chloride also produced a positive inotropic effect; however, the effect of tetraethylammonium was reduced by phentolamine and nadolol, and was almost insensitive to verapamil. The inotropic effect of phencyclidine was associated with a marked prolongation of the action potential duration and a decrease in maximal upstroke velocity of the action potential, with no change in the resting membrane potential. The specific [ 3 H]phencyclidine binding observed with membrane preparations from guinea pig ventricular muscle was saturable with a single class of high-affinity binding site. This binding was inhibited by verapamil, diltiazem, or nitrendipine, but not by ryanodine or tetrodotoxin. These results suggest that the positive inotropic effect of phencyclidine results from enhanced Ca 2+ influx via slow channels, either by stimulation of the channels or secondary to inhibition of outward K + currents

  12. USACE Navigation Channels 2012

    Data.gov (United States)

    California Natural Resource Agency — This dataset represents both San Francisco and Los Angeles District navigation channel lines. All San Francisco District channel lines were digitized from CAD files...

  13. Calcium channel blocker overdose

    Science.gov (United States)

    ... this page: //medlineplus.gov/ency/article/002580.htm Calcium-channel blocker overdose To use the sharing features on this page, please enable JavaScript. Calcium-channel blockers are a type of medicine used ...

  14. Quantum Channels With Memory

    International Nuclear Information System (INIS)

    Rybar, T.

    2012-01-01

    Quantum memory channels represent a very general, yet simple and comprehensible model for causal processes. As such they have attracted considerable research interest, mostly aimed on their transfer capabilities and structure properties. Most notably it was shown that memory channels can be implemented via physically naturally motivated collision models. We also define the concept of repeatable channels and show that only unital channels can be implemented repeat ably with pure memory channels. In the special case of qubit channels we also show that every unital qubit channel has a repeatable implementation. We also briefly explore the possibilities of stroboscopical simulation of channels and show that all random unitary channels can be stroboscopically simulated. Particularly in qubit case, all indivisible qubit channels are also random unitary, hence for qubit all indivisible channels can be stroboscopically simulated. Memory channels also naturally capture the framework of correlated experiments. We develop methods to gather and interpret data obtained in such setting and in detail examine the two qubit case. We also show that for control unitary interactions the measured data will never contradict a simple unitary evolution. Thus no memory effects can be spotted then. (author)

  15. Eight channel fast scalar

    Energy Technology Data Exchange (ETDEWEB)

    Waddoup, W D; Stubbs, R J [Durham Univ. (UK)

    1977-11-01

    An eight channel 64-bit scaler has been constructed with a static CMOS memory. Scaling frequencies are independently variable, at each channel, as are the number of bits/channel. The scaler, when used in conjunction with a multichannel charge to time converter results in a very flexible, gated multichannel ADC.

  16. KV7 potassium channels

    DEFF Research Database (Denmark)

    Stott, Jennifer B; Jepps, Thomas Andrew; Greenwood, Iain A

    2014-01-01

    Potassium channels are key regulators of smooth muscle tone, with increases in activity resulting in hyperpolarisation of the cell membrane, which acts to oppose vasoconstriction. Several potassium channels exist within smooth muscle, but the KV7 family of voltage-gated potassium channels have been...

  17. Receptor studies in biological psychiatry

    International Nuclear Information System (INIS)

    Fujiwara, Yutaka

    1992-01-01

    Recent advances in the pharmacological treatment of endogenous psychosis have led to the development of biological studies in psychiatry. Studies on neurotransmitter receptors were reviewed in order to apply positron-emission tomograph (PET) for biological psychiatry. The dopamine (DA) hypothesis for schizophrenia was advanced on the basis of the observed effects of neuroleptics and methamphetamine, and DA(D 2 ) receptor supersensitivity measured by PET and receptor binding in the schizophrenic brain. The clinical potencies of neuroleptics for schizophrenia were correlated with their abilities to inhibit the D 2 receptor, and not other receptors. The σ receptor was expected to be a site of antipsychotic action. However, the potency of drugs action on it was not correlated with clinical efficacy. Haloperidol binds with high affinity to the σ receptor, which may mediate acute dystonia, an extrapyramidal side effect of neuroleptics. Behavioral and neurochemical changes induced by methamphetamine treatment were studied as an animal model of schizophrenia, and both a decrease of D 2 receptor density and an increase of DA release were detected. The monoamine hypothesis for manic-depressive psychosis was advanced on the basis of the effect of reserpine, monoamine oxidase inhibitor and antidepressants. 3 H-clonidine binding sites were increased in platelet membranes of depressive patients, 3 H-imipramine binding sites were decreased. The GABA A receptor is the target site for the action of anxiolytics and antiepileptics such as benzodiazepines and barbiturates. Recent developments in molecular biology techniques have revealed the structure of receptor proteins, which are classified into two receptor families, the G-protein coupled type (D 2 ) and the ion-channel type (GABA A ). (J.P.N.)

  18. Neuroactive Steroids: Receptor Interactions and Responses

    Directory of Open Access Journals (Sweden)

    Kald Beshir Tuem

    2017-08-01

    Full Text Available Neuroactive steroids (NASs are naturally occurring steroids, which are synthesized centrally as de novo from cholesterol and are classified as pregnane, androstane, and sulfated neurosteroids (NSs. NASs modulate many processes via interacting with gamma-aminobutyric acid (GABA, N-methyl-d-aspartate, serotonin, voltage-gated calcium channels, voltage-dependent anion channels, α-adrenoreceptors, X-receptors of the liver, transient receptor potential channels, microtubule-associated protein 2, neurotrophin nerve growth factor, and σ1 receptors. Among these, NSs (especially allopregnanolone have high potency and extensive GABA-A receptors and hence demonstrate anticonvulsant, anesthetic, central cytoprotectant, and baroreflex inhibitory effects. NSs are also involved in mood and learning via serotonin and anti-nociceptive activity via T-type voltage-gated Ca2+ channels. Moreover, they are modulators of mitochondrial function, synaptic plasticity, or regulators of apoptosis, which have a role in neuroprotective via voltage-dependent anion channels receptors. For proper functioning, NASs need to be in their normal level, whereas excess and deficiency may lead to abnormalities. When they are below the normal, NSs could have a part in development of depression, neuro-inflammation, multiple sclerosis, experimental autoimmune encephalitis, epilepsy, and schizophrenia. On the other hand, stress and attention deficit disorder could occur during excessive level. Overall, NASs are very important molecules with major neuropsychiatric activity.

  19. Analysis of odorant receptor protein function in the yellow fever mosquito, aedes aegypti

    Science.gov (United States)

    Odorant receptors (ORs) in insects are ligand-gated ion channels comprised of two subunits: a variable receptor and an obligatory co-receptor (Orco). This protein receptor complex of unknown stoichiometry interacts with an odor molecule leading to changes in permeability of the sensory dendrite, th...

  20. Receptor assay

    Energy Technology Data Exchange (ETDEWEB)

    Kato, K; Ibayashi, H [Kyushu Univ., Fukuoka (Japan). Faculty of Medicine

    1975-05-01

    This paper summarized present status and problems of analysis of hormone receptor and a few considerations on clinical significance of receptor abnormalities. It was pointed that in future clinical field quantitative and qualitative analysis of receptor did not remain only in the etiological discussion, but that it was an epoch-making field of investigation which contained the possiblity of artificial change of sensitivity of living body on drugs and the development connected directly with treatment of various diseases.

  1. Fine Channel Networks

    Science.gov (United States)

    1997-01-01

    A color image of fine channel networks on Mars; north toward top. The scene shows heavily cratered highlands dissected by dendritic open channel networks that dissect steep slopes of impact crater walls. This image is a composite of Viking high-resolution images in black and white and low-resolution images in color. The image extends from latitude 9 degrees S. to 5 degrees S. and from longitude 312 degrees to 320 degrees; Mercator projection. The dendritic pattern of the fine channels and their location on steep slopes leads to the interpretation that these are runoff channels. The restriction of these types of channels to ancient highland rocks suggests that these channels are old and date from a time on Mars when conditions existed for precipitation to actively erode rocks. After the channels reach a low plain, they appear to end. Termination may have resulted from burial by younger deposits or perhaps the flows percolated into the surface materials and continued underground.

  2. TRP channel proteins and signal transduction.

    Science.gov (United States)

    Minke, Baruch; Cook, Boaz

    2002-04-01

    TRP channel proteins constitute a large and diverse family of proteins that are expressed in many tissues and cell types. This family was designated TRP because of a spontaneously occurring Drosophila mutant lacking TRP that responded to a continuous light with a transient receptor potential (hence TRP). In addition to responses to light, TRPs mediate responses to nerve growth factor, pheromones, olfaction, mechanical, chemical, temperature, pH, osmolarity, vasorelaxation of blood vessels, and metabolic stress. Furthermore, mutations in several members of TRP-related channel proteins are responsible for several diseases, such as several tumors and neurodegenerative disorders. TRP-related channel proteins are found in a variety of organisms, tissues, and cell types, including nonexcitable, smooth muscle, and neuronal cells. The large functional diversity of TRPs is also reflected in their diverse permeability to ions, although, in general, they are classified as nonselective cationic channels. The molecular domains that are conserved in all members of the TRP family constitute parts of the transmembrane domains and in most members also the ankyrin-like repeats at the NH2 terminal of the protein and a "TRP domain" at the COOH terminal, which is a highly conserved 25-amino acid stretch with still unknown function. All of the above features suggest that members of the TRP family are "special assignment" channels, which are recruited to diverse signaling pathways. The channels' roles and characteristics such as gating mechanism, regulation, and permeability are determined by evolution according to the specific functional requirements.

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

    Science.gov (United States)

    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.

  4. Ion channels in plants.

    Science.gov (United States)

    Hedrich, Rainer

    2012-10-01

    Since the first recordings of single potassium channel activities in the plasma membrane of guard cells more than 25 years ago, patch-clamp studies discovered a variety of ion channels in all cell types and plant species under inspection. Their properties differed in a cell type- and cell membrane-dependent manner. Guard cells, for which the existence of plant potassium channels was initially documented, advanced to a versatile model system for studying plant ion channel structure, function, and physiology. Interestingly, one of the first identified potassium-channel genes encoding the Shaker-type channel KAT1 was shown to be highly expressed in guard cells. KAT1-type channels from Arabidopsis thaliana and its homologs from other species were found to encode the K(+)-selective inward rectifiers that had already been recorded in early patch-clamp studies with guard cells. Within the genome era, additional Arabidopsis Shaker-type channels appeared. All nine members of the Arabidopsis Shaker family are localized at the plasma membrane, where they either operate as inward rectifiers, outward rectifiers, weak voltage-dependent channels, or electrically silent, but modulatory subunits. The vacuole membrane, in contrast, harbors a set of two-pore K(+) channels. Just very recently, two plant anion channel families of the SLAC/SLAH and ALMT/QUAC type were identified. SLAC1/SLAH3 and QUAC1 are expressed in guard cells and mediate Slow- and Rapid-type anion currents, respectively, that are involved in volume and turgor regulation. Anion channels in guard cells and other plant cells are key targets within often complex signaling networks. Here, the present knowledge is reviewed for the plant ion channel biology. Special emphasis is drawn to the molecular mechanisms of channel regulation, in the context of model systems and in the light of evolution.

  5. The number and distribution of AMPA receptor channels containing fast kinetic GluA3 and GluA4 subunits at auditory nerve synapses depend on the target cells.

    Science.gov (United States)

    Rubio, María E; Matsui, Ko; Fukazawa, Yugo; Kamasawa, Naomi; Harada, Harumi; Itakura, Makoto; Molnár, Elek; Abe, Manabu; Sakimura, Kenji; Shigemoto, Ryuichi

    2017-11-01

    The neurotransmitter receptor subtype, number, density, and distribution relative to the location of transmitter release sites are key determinants of signal transmission. AMPA-type ionotropic glutamate receptors (AMPARs) containing GluA3 and GluA4 subunits are prominently expressed in subsets of neurons capable of firing action potentials at high frequencies, such as auditory relay neurons. The auditory nerve (AN) forms glutamatergic synapses on two types of relay neurons, bushy cells (BCs) and fusiform cells (FCs) of the cochlear nucleus. AN-BC and AN-FC synapses have distinct kinetics; thus, we investigated whether the number, density, and localization of GluA3 and GluA4 subunits in these synapses are differentially organized using quantitative freeze-fracture replica immunogold labeling. We identify a positive correlation between the number of AMPARs and the size of AN-BC and AN-FC synapses. Both types of AN synapses have similar numbers of AMPARs; however, the AN-BC have a higher density of AMPARs than AN-FC synapses, because the AN-BC synapses are smaller. A higher number and density of GluA3 subunits are observed at AN-BC synapses, whereas a higher number and density of GluA4 subunits are observed at AN-FC synapses. The intrasynaptic distribution of immunogold labeling revealed that AMPAR subunits, particularly GluA3, are concentrated at the center of the AN-BC synapses. The central distribution of AMPARs is absent in GluA3-knockout mice, and gold particles are evenly distributed along the postsynaptic density. GluA4 gold labeling was homogenously distributed along both synapse types. Thus, GluA3 and GluA4 subunits are distributed at AN synapses in a target-cell-dependent manner.

  6. Chloride channels as tools for developing selective insecticides.

    Science.gov (United States)

    Bloomquist, Jeffrey R

    2003-12-01

    Ligand-gated chloride channels underlie inhibition in excitable membranes and are proven target sites for insecticides. The gamma-aminobutyric acid (GABA(1)) receptor/chloride ionophore complex is the primary site of action for a number of currently used insecticides, such as lindane, endosulfan, and fipronil. These compounds act as antagonists by stabilizing nonconducting conformations of the chloride channel. Blockage of the GABA-gated chloride channel reduces neuronal inhibition, which leads to hyperexcitation of the central nervous system, convulsions, and death. We recently investigated the mode of action of the silphinenes, plant-derived natural compounds that structurally resemble picrotoxinin. These materials antagonize the action of GABA on insect neurons and block GABA-mediated chloride uptake into mouse brain synaptoneurosomes in a noncompetitive manner. In mammals, avermectins have a blocking action on the GABA-gated chloride channel consistent with a coarse tremor, whereas at longer times and higher concentrations, activation of the channel suppresses neuronal activity. Invertebrates display ataxia, paralysis, and death as the predominant signs of poisoning, with a glutamate-gated chloride channel playing a major role. Additional target sites for the avermectins or other chloride channel-directed compounds might include receptors gated by histamine, serotonin, or acetylcholine.The voltage-sensitive chloride channels form another large gene family of chloride channels. Voltage-dependent chloride channels are involved in a number of physiological processes including: maintenance of electrical excitability, chloride ion secretion and resorption, intravesicular acidification, and cell volume regulation. A subset of these channels is affected by convulsants and insecticides in mammals, although the role they play in acute lethality in insects is unclear. Given the wide range of functions that they mediate, these channels are also potential targets for

  7. Compound Wiretap Channels

    Directory of Open Access Journals (Sweden)

    Shlomo Shamai (Shitz

    2009-01-01

    Full Text Available This paper considers the compound wiretap channel, which generalizes Wyner's wiretap model to allow the channels to the (legitimate receiver and to the eavesdropper to take a number of possible states. No matter which states occur, the transmitter guarantees that the receiver decodes its message and that the eavesdropper is kept in full ignorance about the message. The compound wiretap channel can also be viewed as a multicast channel with multiple eavesdroppers, in which the transmitter sends information to all receivers and keeps the information secret from all eavesdroppers. For the discrete memoryless channel, lower and upper bounds on the secrecy capacity are derived. The secrecy capacity is established for the degraded channel and the semideterministic channel with one receiver. The parallel Gaussian channel is further studied. The secrecy capacity and the secrecy degree of freedom (s.d.o.f. are derived for the degraded case with one receiver. Schemes to achieve the s.d.o.f. for the case with two receivers and two eavesdroppers are constructed to demonstrate the necessity of a prefix channel in encoder design. Finally, the multi-antenna (i.e., MIMO compound wiretap channel is studied. The secrecy capacity is established for the degraded case and an achievable s.d.o.f. is given for the general case.

  8. Compound Wiretap Channels

    Directory of Open Access Journals (Sweden)

    Kramer Gerhard

    2009-01-01

    Full Text Available Abstract This paper considers the compound wiretap channel, which generalizes Wyner's wiretap model to allow the channels to the (legitimate receiver and to the eavesdropper to take a number of possible states. No matter which states occur, the transmitter guarantees that the receiver decodes its message and that the eavesdropper is kept in full ignorance about the message. The compound wiretap channel can also be viewed as a multicast channel with multiple eavesdroppers, in which the transmitter sends information to all receivers and keeps the information secret from all eavesdroppers. For the discrete memoryless channel, lower and upper bounds on the secrecy capacity are derived. The secrecy capacity is established for the degraded channel and the semideterministic channel with one receiver. The parallel Gaussian channel is further studied. The secrecy capacity and the secrecy degree of freedom ( are derived for the degraded case with one receiver. Schemes to achieve the for the case with two receivers and two eavesdroppers are constructed to demonstrate the necessity of a prefix channel in encoder design. Finally, the multi-antenna (i.e., MIMO compound wiretap channel is studied. The secrecy capacity is established for the degraded case and an achievable is given for the general case.

  9. ATP Release Channels

    Directory of Open Access Journals (Sweden)

    Akiyuki Taruno

    2018-03-01

    Full Text Available Adenosine triphosphate (ATP has been well established as an important extracellular ligand of autocrine signaling, intercellular communication, and neurotransmission with numerous physiological and pathophysiological roles. In addition to the classical exocytosis, non-vesicular mechanisms of cellular ATP release have been demonstrated in many cell types. Although large and negatively charged ATP molecules cannot diffuse across the lipid bilayer of the plasma membrane, conductive ATP release from the cytosol into the extracellular space is possible through ATP-permeable channels. Such channels must possess two minimum qualifications for ATP permeation: anion permeability and a large ion-conducting pore. Currently, five groups of channels are acknowledged as ATP-release channels: connexin hemichannels, pannexin 1, calcium homeostasis modulator 1 (CALHM1, volume-regulated anion channels (VRACs, also known as volume-sensitive outwardly rectifying (VSOR anion channels, and maxi-anion channels (MACs. Recently, major breakthroughs have been made in the field by molecular identification of CALHM1 as the action potential-dependent ATP-release channel in taste bud cells, LRRC8s as components of VRACs, and SLCO2A1 as a core subunit of MACs. Here, the function and physiological roles of these five groups of ATP-release channels are summarized, along with a discussion on the future implications of understanding these channels.

  10. Involvement of direct inhibition of NMDA receptors in the effects of sigma-receptor ligands on glutamate neurotoxicity in vitro.

    Science.gov (United States)

    Nishikawa, H; Hashino, A; Kume, T; Katsuki, H; Kaneko, S; Akaike, A

    2000-09-15

    This study was performed to examine the roles of the N-methyl-D-aspartate (NMDA) receptor/phencyclidine (PCP) channel complex in the protective effects of sigma-receptor ligands against glutamate neurotoxicity in cultured cortical neurons derived from fetal rats. A 1-h exposure of cultures to glutamate caused a marked loss of viability, as determined by Trypan blue exclusion. This acute neurotoxicity of glutamate was prevented by NMDA receptor antagonists. Expression of sigma(1) receptor mRNA in cortical cultures was confirmed by reverse transcription polymerase chain reaction (RT-PCR). sigma Receptor ligands with affinity for NMDA receptor channels including the PCP site, such as (+)-N-allylnormetazocine ((+)-SKF10,047), haloperidol, and R(-)-N-(3-phenyl-1-propyl)-1-phenyl-2-aminopropane ((-)-PPAP), prevented glutamate neurotoxicity in a concentration-dependent manner. In contrast, other sigma-receptor ligands without affinity for NMDA receptors, such as carbetapentane and R(+)-3-(3-hydroxyphenyl)-N-propylpiperidine ((+)-3-PPP), did not show neuroprotective effects. Putative endogenous sigma receptor ligands such as pregnenolone, progesterone, and dehydroepiandrosterone did not affect glutamate neurotoxicity. The protective effects of (+)-SKF10,047, haloperidol, and (-)-PPAP were not affected by the sigma(1) receptor antagonist rimcazole. These results suggested that a direct interaction with NMDA receptors but not with sigma receptors plays a crucial role in the neuroprotective effects of sigma receptor ligands with affinity for NMDA receptors.

  11. Pharmacological Conversion of a Cardiac Inward Rectifier into an Outward Rectifier Potassium Channel.

    Science.gov (United States)

    Moreno-Galindo, Eloy G; Sanchez-Chapula, Jose A; Tristani-Firouzi, Martin; Navarro-Polanco, Ricardo A

    2016-09-01

    Potassium (K(+)) channels are crucial for determining the shape, duration, and frequency of action-potential firing in excitable cells. Broadly speaking, K(+) channels can be classified based on whether their macroscopic current outwardly or inwardly rectifies, whereby rectification refers to a change in conductance with voltage. Outwardly rectifying K(+) channels conduct greater current at depolarized membrane potentials, whereas inward rectifier channels conduct greater current at hyperpolarized membrane potentials. Under most circumstances, outward currents through inwardly rectifying K(+) channels are reduced at more depolarized potentials. However, the acetylcholine-gated K(+) channel (KACh) conducts current that inwardly rectifies when activated by some ligands (such as acetylcholine), and yet conducts current that outwardly rectifies when activated by other ligands (for example, pilocarpine and choline). The perplexing and paradoxical behavior of KACh channels is due to the intrinsic voltage sensitivity of the receptor that activates KACh channels, the M2 muscarinic receptor (M2R). Emerging evidence reveals that the affinity of M2R for distinct ligands varies in a voltage-dependent and ligand-specific manner. These intrinsic receptor properties determine whether current conducted by KACh channels inwardly or outwardly rectifies. This review summarizes the most recent concepts regarding the intrinsic voltage sensitivity of muscarinic receptors and the consequences of this intriguing behavior on cardiac physiology and pharmacology of KACh channels. Copyright © 2016 by The American Society for Pharmacology and Experimental Therapeutics.

  12. Role of TRP Channels in Dinoflagellate Mechanotransduction.

    Science.gov (United States)

    Lindström, J B; Pierce, N T; Latz, M I

    2017-10-01

    Transient receptor potential (TRP) ion channels are common components of mechanosensing pathways, mainly described in mammals and other multicellular organisms. To gain insight into the evolutionary origins of eukaryotic mechanosensory proteins, we investigated the involvement of TRP channels in mechanosensing in a unicellular eukaryotic protist, the dinoflagellate Lingulodinium polyedra. BLASTP analysis of the protein sequences predicted from the L. polyedra transcriptome revealed six sequences with high similarity to human TRPM2, TRPM8, TRPML2, TRPP1, and TRPP2; and characteristic TRP domains were identified in all sequences. In a phylogenetic tree including all mammalian TRP subfamilies and TRP channel sequences from unicellular and multicellular organisms, the L. polyedra sequences grouped with the TRPM, TPPML, and TRPP clades. In pharmacological experiments, we used the intrinsic bioluminescence of L. polyedra as a reporter of mechanoresponsivity. Capsaicin and RN1734, agonists of mammalian TRPV, and arachidonic acid, an agonist of mammalian TRPV, TRPA, TRPM, and Drosophila TRP, all stimulated bioluminescence in L. polyedra. Mechanical stimulation of bioluminescence, but not capsaicin-stimulated bioluminescence, was inhibited by gadolinium (Gd 3+ ), a general inhibitor of mechanosensitive ion channels, and the phospholipase C (PLC) inhibitor U73122. These pharmacological results are consistent with the involvement of TRP-like channels in mechanosensing by L. polyedra. The TRP channels do not appear to be mechanoreceptors but rather are components of the mechanotransduction signaling pathway and may be activated via a PLC-dependent mechanism. The presence and function of TRP channels in a dinoflagellate emphasize the evolutionary conservation of both the channel structures and their functions.

  13. Determination of Disulfide Bond Connectivity of Cysteine-rich Peptide IpTx{sub a}

    Energy Technology Data Exchange (ETDEWEB)

    Lee, Chul Won; Kim, Jim Il [Chonnam National Univ., Gwangju (Korea, Republic of); Sato, Kazuki [Fukuoka Women' s Univ., Fukuoka (Japan)

    2013-06-15

    Cysteine-rich peptides stabilized by intramolecular disulfide bonds have often been isolated from venoms of microbes, animals and plants. These peptides typically have much higher stability and improved biopharmaceutical properties compared to their linear counterparts. Therefore the correct disulfide bond formation of small proteins and peptides has been extensively studied for a better understanding of their folding mechanism and achieving efficient generation of the naturally occurring biologically active product. Imperatoxin A (IpTx{sub a}), a peptide toxin containing 6 cysteine residues, was isolated from the venom of scorpion Pandinus imperator, selectively binds the ryanodine receptors and activates Ca{sup 2+} release from sarcoplasmic reticulum (SR). IpTx{sub a} increases the binding of ryanodine to ryanodine receptors (RyRs) and encourages reconstituted single channel to induce subconductance states.

  14. Skeletal muscle excitation-contraction coupling: who are the dancing partners?

    Science.gov (United States)

    Rebbeck, Robyn T; Karunasekara, Yamuna; Board, Philip G; Beard, Nicole A; Casarotto, Marco G; Dulhunty, Angela F

    2014-03-01

    There is an overwhelming body of work supporting the idea that excitation-contraction coupling in skeletal muscle depends on a physical interaction between the skeletal muscle isoform of the dihydropyridine receptor L-type Ca(2+) channel and the skeletal isoform of the ryanodine receptor Ca(2+) release channel. A general assumption is that this physical interaction is between "critical" residues that have been identified in the II-III loop of the dihydropyridine receptor alpha subunit and the ryanodine receptor. However, despite extensive searches, the complementary "critical" residues in the ryanodine receptor have not been identified. This raises the possibility that the coupling proceeds either through other subunits of the dihydropyridine receptor and/or other co-proteins within the large RyR1 protein complex. There have been some remarkable advances in recent years in identifying proteins in the RyR complex that impact on the coupling process, and these are considered in this review. A major candidate for a role in the coupling mechanism is the beta subunit of the dihydropyridine receptor, because specific residues in both the beta subunit and ryanodine receptor have been identified that facilitate an interaction between the two proteins and these also impact on excitation-contraction coupling. This role of beta subunit remains to be fully investigated as well as the degree to which it may complement any other direct or indirect voltage-dependent coupling interactions between the DHPR alpha II-III loop and the ryanodine receptor. Copyright © 2014. Published by Elsevier Ltd.

  15. Imaging the PCP site of the NMDA ion channel

    Energy Technology Data Exchange (ETDEWEB)

    Waterhouse, Rikki N. E-mail: rnw7@columbia.edu

    2003-11-01

    The N-methyl-D-aspartate (NMDA) ion channel plays a role in neuroprotection, neurodegeneration, long-term potentiation, memory, and cognition. It is implicated in the pathophysiology of several neurological and neuropsychiatric disorders including Parkinson's Disease, Huntington's Chorea, schizophrenia, alcoholism and stroke. The development of effective radiotracers for the study of NMDA receptors is critical for our understanding of their function, and their modulation by endogenousr substances or therapeutic drugs. Since the NMDA/PCP receptor lies within the channel, it is a unique target and is theoretically accessible only when the channel is in the active and 'open' state, but not when it is in the inactive or 'closed' state. The physical location of the NMDA/PCP receptor not only makes it an important imaging target but also complicates the development of suitable PET and SPECT radiotracers for this site. An intimate understanding of the biochemical, pharmacological, physiological and behavioral processes associated with the NMDA ion channel is essential to develop improved imaging agents. This review outlines progress made towards the development of radiolabeled agents for PCP sites of the NMDA ion channel. In addition, the animal and pharmacological models used for in vitro and in vivo assessment of NMDA receptor targeted agents are discussed.

  16. Imaging the PCP site of the NMDA ion channel

    International Nuclear Information System (INIS)

    Waterhouse, Rikki N.

    2003-01-01

    The N-methyl-D-aspartate (NMDA) ion channel plays a role in neuroprotection, neurodegeneration, long-term potentiation, memory, and cognition. It is implicated in the pathophysiology of several neurological and neuropsychiatric disorders including Parkinson's Disease, Huntington's Chorea, schizophrenia, alcoholism and stroke. The development of effective radiotracers for the study of NMDA receptors is critical for our understanding of their function, and their modulation by endogenousr substances or therapeutic drugs. Since the NMDA/PCP receptor lies within the channel, it is a unique target and is theoretically accessible only when the channel is in the active and 'open' state, but not when it is in the inactive or 'closed' state. The physical location of the NMDA/PCP receptor not only makes it an important imaging target but also complicates the development of suitable PET and SPECT radiotracers for this site. An intimate understanding of the biochemical, pharmacological, physiological and behavioral processes associated with the NMDA ion channel is essential to develop improved imaging agents. This review outlines progress made towards the development of radiolabeled agents for PCP sites of the NMDA ion channel. In addition, the animal and pharmacological models used for in vitro and in vivo assessment of NMDA receptor targeted agents are discussed

  17. Receptor binding studies of the living heart

    International Nuclear Information System (INIS)

    Syrota, A.

    1988-01-01

    Receptors form a class of intrinsic membrane proteins (or glycoproteins) defined by the high affinity and specificity with which they bind ligands. Many receptors are associated directly or indirectly with membrane ion channels that open or close after a conformational change of the receptor induced by the binding of the neurotransmitter. Changes in number and/or affinity of cardiac neurotransmitter receptors have been associated with myocardial ischemia and infarction, congestive heart failure, and cardiomyopathy as well as diabetes or thyroid-induced heart muscle disease. These alterations of cardiac receptors have been demonstrated in vitro on membrane homogenates from samples collected mainly during surgery or postmortem. The disadvantage of these in vitro binding techniques is that receptors lose their natural environment and their relationships with the other components of the tissue

  18. HIPPI and Fibre Channel

    International Nuclear Information System (INIS)

    Tolmie, D.E.

    1992-01-01

    The High-Performance Parallel Interface (HIPPI) and Fibre Channel are near-gigabit per second data communications interfaces being developed in ANSI standards Task Group X3T9.3. HIPPI is the current interface of choice in the high-end and supercomputer arena, and Fibre Channel is a follow-on effort. HIPPI came from a local area network background, and Fibre Channel came from a mainframe to peripheral interface background

  19. Nuclear reactor coolant channels

    International Nuclear Information System (INIS)

    Macbeth, R.V.

    1978-01-01

    Reference is made to coolant channels for pressurised water and boiling water reactors and the arrangement described aims to improve heat transfer between the fuel rods and the coolant. Baffle means extending axially within the channel are provided and disposed relative to the fuel rods so as to restrict flow oscillations occurring within the coolant from being propagated transversely to the axis of the channel. (UK)

  20. New Channels, New Possibilities

    DEFF Research Database (Denmark)

    Pieterson, Willem; Ebbers, Wolfgang; Østergaard Madsen, Christian

    2017-01-01

    In this contribution we discuss the characteristics of what we call the fourth generation of public sector service channels: social robots. Based on a review of relevant literature we discuss their characteristics and place into multi-channel models of service delivery. We argue that social robots......-channel models of service delivery. This is especially relevant given the current lack of evaluations of such models, the broad range of channels available, and their different stages of deployment at governments around the world. Nevertheless, social robots offer an potentially very relevant addition...

  1. Calcium Channel Blockers

    Science.gov (United States)

    ... Certain calcium channel blockers interact with grapefruit products. Kaplan NM, et al. Treatment of hypertension: Drug therapy. In: Kaplan's Clinical Hypertension. 11th ed. Philadelphia, Pa.: Wolters Kluwer ...

  2. A channel profile analyser

    International Nuclear Information System (INIS)

    Gobbur, S.G.

    1983-01-01

    It is well understood that due to the wide band noise present in a nuclear analog-to-digital converter, events at the boundaries of adjacent channels are shared. It is a difficult and laborious process to exactly find out the shape of the channels at the boundaries. A simple scheme has been developed for the direct display of channel shape of any type of ADC on a cathode ray oscilliscope display. This has been accomplished by sequentially incrementing the reference voltage of a precision pulse generator by a fraction of a channel and storing ADC data in alternative memory locations of a multichannel pulse height analyser. Alternative channels are needed due to the sharing at the boundaries of channels. In the flat region of the profile alternate memory locations are channels with zero counts and channels with the full scale counts. At the boundaries all memory locations will have counts. The shape of this is a direct display of the channel boundaries. (orig.)

  3. Role of TRP channels in the cardiovascular system.

    Science.gov (United States)

    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.

  4. Effect of membrane hyperpolarization induced by a K+ channel opener on histamine-induced Ca2+ mobilization in rabbit arterial smooth muscle.

    Science.gov (United States)

    Watanabe, Y; Suzuki, A; Suzuki, H; Itoh, T

    1996-03-01

    1. The role of membrane hyperpolarization on agonist-induced contraction was investigated in intact and alpha-toxin-skinned smooth muscles of rabbit mesenteric artery by use of the ATP-sensitive K+ channel opener, (-)-(3S,4R)-4-(N-acetyl-N-hydroxyamino)-6-cyano-3,4-dihydro-2,2- dimethyl-2H-1-benzopyran-3-ol (Y-26763), and either histamine (Hist) or noradrenaline (NA). 2. Hist (3 microM) and NA (10 microM) both produced a phasic, followed by a tonic increase in intracellular Ca2+ concentration ([Ca2+]i) and force. Y-26763 (10 microM) potently inhibited the NA-induced phasic and tonic increase in [Ca2+]i and force. In contrast, Y-26763 attenuated the Hist-induced phasic increase in [Ca2+]i and force but had almost no effect on the tonic response. However, ryanodine-treatment of muscles in order to inhibit the function of intracellular Ca2+ storage sites altered the action of Y-26763 which now attenuated the Hist-induced tonic increase in [Ca2+]i and force in a concentration-dependent manner (at concentrations > 1 microM). Glibenclamide (10 microM) attenuated the inhibitory action of Y-26763. 3. Hist (3 microM) depolarized the smooth muscle cells to the same extent as NA (10 microM). In the absence of either agonist, Y-26763 (over 30 nM) hyperpolarized the membrane and glibenclamide inhibited this hyperpolarization. Y-26763 (10 microM) almost abolished the NA-induced membrane depolarization, but only slightly attenuated the Hist-induced membrane depolarization in which the delta (delta) value (the difference before and after application of Hist) was not modified by any concentration of Y-26763. In ryanodine-treated smooth muscle cells, Y-26763 hyperpolarized the membrane and potently inhibited the membrane depolarization induced by Hist. 4. In ryanodine-treated muscle, Y-26763 had no measurable effect on the Hist-induced [Ca2+]i-force relationship. Y-26763 also had no apparent effect on the myofilament Ca(2+)-sensitivity in the presence of Hist in alpha

  5. Selectivity of Odorant Receptors in Insects

    Science.gov (United States)

    2012-07-13

    Luetje, C. W., and Robertson, H. M. (2007). A honey bee odorant receptor for the queen substance 9-oxo-2-decenoic acid. Proc. Natl. Acad. Sci. U.S.A...since they might be exposed to a greater number of pharmacolog- ically active compounds than other conventional ligand-gated ion channels and G- protein ...2008). Drosophila odorant receptors are novel seven transmembrane domain proteins that can signal independently of heterotrimeric G proteins

  6. Dynamic mobility of functional GABAA receptors at inhibitory synapses.

    Science.gov (United States)

    Thomas, Philip; Mortensen, Martin; Hosie, Alastair M; Smart, Trevor G

    2005-07-01

    Importing functional GABAA receptors into synapses is fundamental for establishing and maintaining inhibitory transmission and for controlling neuronal excitability. By introducing a binding site for an irreversible inhibitor into the GABAA receptor alpha1 subunit channel lining region that can be accessed only when the receptor is activated, we have determined the dynamics of receptor mobility between synaptic and extrasynaptic locations in hippocampal pyramidal neurons. We demonstrate that the cell surface GABAA receptor population shows no fast recovery after irreversible inhibition. In contrast, after selective inhibition, the synaptic receptor population rapidly recovers by the import of new functional entities within minutes. The trafficking pathways that promote rapid importation of synaptic receptors do not involve insertion from intracellular pools, but reflect receptor diffusion within the plane of the membrane. This process offers the synapse a rapid mechanism to replenish functional GABAA receptors at inhibitory synapses and a means to control synaptic efficacy.

  7. Structural and Molecular Modeling Features of P2X Receptors

    Directory of Open Access Journals (Sweden)

    Luiz Anastacio Alves

    2014-03-01

    Full Text Available Currently, adenosine 5'-triphosphate (ATP is recognized as the extracellular messenger that acts through P2 receptors. P2 receptors are divided into two subtypes: P2Y metabotropic receptors and P2X ionotropic receptors, both of which are found in virtually all mammalian cell types studied. Due to the difficulty in studying membrane protein structures by X-ray crystallography or NMR techniques, there is little information about these structures available in the literature. Two structures of the P2X4 receptor in truncated form have been solved by crystallography. Molecular modeling has proven to be an excellent tool for studying ionotropic receptors. Recently, modeling studies carried out on P2X receptors have advanced our knowledge of the P2X receptor structure-function relationships. This review presents a brief history of ion channel structural studies and shows how modeling approaches can be used to address relevant questions about P2X receptors.

  8. G-protein-coupled inward rectifier potassium channels involved in corticostriatal presynaptic modulation.

    Science.gov (United States)

    Meneses, David; Mateos, Verónica; Islas, Gustavo; Barral, Jaime

    2015-09-01

    Presynaptic modulation has been associated mainly with calcium channels but recent data suggests that inward rectifier potassium channels (K(IR)) also play a role. In this work we set to characterize the role of presynaptic K(IR) channels in corticostriatal synaptic transmission. We elicited synaptic potentials in striatum by stimulating cortical areas and then determined the synaptic responses of corticostriatal synapsis by using paired pulse ratio (PPR) in the presence and absence of several potassium channel blockers. Unspecific potassium channels blockers Ba(2+) and Cs(+) reduced the PPR, suggesting that these channels are presynaptically located. Further pharmacological characterization showed that application of tertiapin-Q, a specific K(IR)3 channel family blocker, also induced a reduction of PPR, suggesting that K(IR)3 channels are present at corticostriatal terminals. In contrast, exposure to Lq2, a specific K(IR)1.1 inward rectifier potassium channel, did not induce any change in PPR suggesting the absence of these channels in the presynaptic corticostriatal terminals. Our results indicate that K(IR)3 channels are functionally expressed at the corticostriatal synapses, since blockage of these channels result in PPR decrease. Our results also help to explain how synaptic activity may become sensitive to extracellular signals mediated by G-protein coupled receptors. A vast repertoire of receptors may influence neurotransmitter release in an indirect manner through regulation of K(IR)3 channels. © 2015 Wiley Periodicals, Inc.

  9. A linearization of quantum channels

    Science.gov (United States)

    Crowder, Tanner

    2015-06-01

    Because the quantum channels form a compact, convex set, we can express any quantum channel as a convex combination of extremal channels. We give a Euclidean representation for the channels whose inverses are also valid channels; these are a subset of the extreme points. They form a compact, connected Lie group, and we calculate its Lie algebra. Lastly, we calculate a maximal torus for the group and provide a constructive approach to decomposing any invertible channel into a product of elementary channels.

  10. Omni channel fashion shopping

    NARCIS (Netherlands)

    Kemperman, A.D.A.M.; van Delft, L.; Borgers, A.W.J.; Pantano, E.

    2015-01-01

    This chapter gives insight into consumers' online and offline fashion shopping behavior, consumers' omni-channel usage during the shopping process, and consumer fashion shopper segments. Based on a literature review, omni-channel shopping behavior during the shopping process was operationalized.

  11. Channel electron multipliers

    International Nuclear Information System (INIS)

    Seidman, A.; Avrahami, Z.; Sheinfux, B.; Grinberg, J.

    1976-01-01

    A channel electron multiplier is described having a tubular wall coated with a secondary-electron emitting material and including an electric field for accelerating the electrons, the electric field comprising a plurality of low-resistive conductive rings each alternating with a high-resistive insulating ring. The thickness of the low-resistive rings is many times larger than that of the high-resistive rings, being in the order of tens of microns for the low-resistive rings and at least one order of magnitude lower for the high-resistive rings; and the diameter of the channel tubular walls is also many times larger than the thickness of the high-resistive rings. Both single-channel and multiple-channel electron multipliers are described. A very important advantage, particularly in making multiple-channel multipliers, is the simplicity of the procedure that may be used in constructing such multipliers. Other operational advantages are described

  12. Cardiac potassium channel subtypes

    DEFF Research Database (Denmark)

    Schmitt, Nicole; Grunnet, Morten; Olesen, Søren-Peter

    2014-01-01

    About 10 distinct potassium channels in the heart are involved in shaping the action potential. Some of the K(+) channels are primarily responsible for early repolarization, whereas others drive late repolarization and still others are open throughout the cardiac cycle. Three main K(+) channels...... drive the late repolarization of the ventricle with some redundancy, and in atria this repolarization reserve is supplemented by the fairly atrial-specific KV1.5, Kir3, KCa, and K2P channels. The role of the latter two subtypes in atria is currently being clarified, and several findings indicate...... that they could constitute targets for new pharmacological treatment of atrial fibrillation. The interplay between the different K(+) channel subtypes in both atria and ventricle is dynamic, and a significant up- and downregulation occurs in disease states such as atrial fibrillation or heart failure...

  13. Cl- channels in apoptosis

    DEFF Research Database (Denmark)

    Wanitchakool, Podchanart; Ousingsawat, Jiraporn; Sirianant, Lalida

    2016-01-01

    A remarkable feature of apoptosis is the initial massive cell shrinkage, which requires opening of ion channels to allow release of K(+), Cl(-), and organic osmolytes to drive osmotic water movement and cell shrinkage. This article focuses on the role of the Cl(-) channels LRRC8, TMEM16/anoctamin......, and cystic fibrosis transmembrane conductance regulator (CFTR) in cellular apoptosis. LRRC8A-E has been identified as a volume-regulated anion channel expressed in many cell types. It was shown to be required for regulatory and apoptotic volume decrease (RVD, AVD) in cultured cell lines. Its presence also......(-) channels or as regulators of other apoptotic Cl(-) channels, such as LRRC8. CFTR has been known for its proapoptotic effects for some time, and this effect may be based on glutathione release from the cell and increase in cytosolic reactive oxygen species (ROS). Although we find that CFTR is activated...

  14. CHANNEL ESTIMATION TECHNIQUE

    DEFF Research Database (Denmark)

    2015-01-01

    A method includes determining a sequence of first coefficient estimates of a communication channel based on a sequence of pilots arranged according to a known pilot pattern and based on a receive signal, wherein the receive signal is based on the sequence of pilots transmitted over the communicat......A method includes determining a sequence of first coefficient estimates of a communication channel based on a sequence of pilots arranged according to a known pilot pattern and based on a receive signal, wherein the receive signal is based on the sequence of pilots transmitted over...... the communication channel. The method further includes determining a sequence of second coefficient estimates of the communication channel based on a decomposition of the first coefficient estimates in a dictionary matrix and a sparse vector of the second coefficient estimates, the dictionary matrix including...... filter characteristics of at least one known transceiver filter arranged in the communication channel....

  15. Voltage-gated sodium channels in taste bud cells.

    Science.gov (United States)

    Gao, Na; Lu, Min; Echeverri, Fernando; Laita, Bianca; Kalabat, Dalia; Williams, Mark E; Hevezi, Peter; Zlotnik, Albert; Moyer, Bryan D

    2009-03-12

    Taste bud cells transmit information regarding the contents of food from taste receptors embedded in apical microvilli to gustatory nerve fibers innervating basolateral membranes. In particular, taste cells depolarize, activate voltage-gated sodium channels, and fire action potentials in response to tastants. Initial cell depolarization is attributable to sodium influx through TRPM5 in sweet, bitter, and umami cells and an undetermined cation influx through an ion channel in sour cells expressing PKD2L1, a candidate sour taste receptor. The molecular identity of the voltage-gated sodium channels that sense depolarizing signals and subsequently initiate action potentials coding taste information to gustatory nerve fibers is unknown. We describe the molecular and histological expression profiles of cation channels involved in electrical signal transmission from apical to basolateral membrane domains. TRPM5 was positioned immediately beneath tight junctions to receive calcium signals originating from sweet, bitter, and umami receptor activation, while PKD2L1 was positioned at the taste pore. Using mouse taste bud and lingual epithelial cells collected by laser capture microdissection, SCN2A, SCN3A, and SCN9A voltage-gated sodium channel transcripts were expressed in taste tissue. SCN2A, SCN3A, and SCN9A were expressed beneath tight junctions in subsets of taste cells. SCN3A and SCN9A were expressed in TRPM5 cells, while SCN2A was expressed in TRPM5 and PKD2L1 cells. HCN4, a gene previously implicated in sour taste, was expressed in PKD2L1 cells and localized to cell processes beneath the taste pore. SCN2A, SCN3A and SCN9A voltage-gated sodium channels are positioned to sense initial depolarizing signals stemming from taste receptor activation and initiate taste cell action potentials. SCN2A, SCN3A and SCN9A gene products likely account for the tetrodotoxin-sensitive sodium currents in taste receptor cells.

  16. Amplified Cold Transduction in Native Nociceptors by M-Channel Inhibition

    Czech Academy of Sciences Publication Activity Database

    Vetter, I.; Hein, A.; Sattler, S.; Hessler, S.; Touška, Filip; Bressan, E.; Parra, A.; Hager, U.; Leffler, A.; Boukalová, Štěpána; Nissen, M.; Lewis, R. J.; Belmonte, C.; Alzheimer, Ch.; Huth, T.; Vlachová, Viktorie; Reeh, P. W.; Zimmermann, K.

    2013-01-01

    Roč. 33, č. 42 (2013), s. 16627-16641 ISSN 0270-6474 Institutional support: RVO:67985823 Keywords : nociceptor * channels of HEK293T * receptor TRPM8 Subject RIV: ED - Physiology Impact factor: 6.747, year: 2013

  17. The Vasopressin Type-2 Receptor and Prostaglandin Receptors EP2 and EP4 can Increase Aquaporin-2 Plasma Membrane Targeting Through a cAMP Independent Pathway

    DEFF Research Database (Denmark)

    Olesen, Emma Tina Bisgaard; Moeller, Hanne Bjerregaard; Assentoft, Mette

    2016-01-01

    Apical membrane targeting of the collecting duct water channel aquaporin-2 (AQP2) is essential for body water balance. As this event is regulated by Gs coupled 7-transmembrane receptors such as the vasopressin type 2 receptor (V2R) and the prostanoid receptors EP2 and EP4, it is believed to be c...

  18. Characterization of astrocytic and neuronal benzodiazepine receptors

    Energy Technology Data Exchange (ETDEWEB)

    Bender, A.S.

    1988-01-01

    Primary cultures of astrocytes and neurons express benzodiazepine receptors. Neuronal benzodiazepine receptors were of high-affinity, K{sub D} values were 7.5-43 nM and the densities of receptors (B{sub max}) were 924-4131 fmol/mg protein. Astrocytes posses a high-affinity benzodiazepine receptor, K{sub D} values were 6.6-13 nM. The B{sub max} values were 6,033-12,000 fmol/mg protein. The pharmacological profile of the neuronal benzodiazepine receptor was that of the central-type benzodiazepine receptor, where clonazepam has a high-affinity and Ro 5-4864 (4{prime}-chlorodiazepam) has a low-affinity. Whereas astrocytic benzoidazepine receptor was characteristic of the so called peripheral-type benzodiazepine receptors, which shows a high-affinity towards Ro 5-4863, and a low-affinity towards clonazepam. The astrocytic benzodiazepine receptors was functionally correlated with voltage dependent calcium channels, since dihydropyridines and benzodiazepines interacted with ({sup 3}H) diazepam and ({sup 3}H) nitrendipine receptors with the same rank order of potency, showing a statistically significant correlation. No such correlation was observed in neurons.

  19. Sodium Channel Mutations and Pyrethroid Resistance in Aedes aegypti

    Directory of Open Access Journals (Sweden)

    Yuzhe Du

    2016-10-01

    Full Text Available Pyrethroid insecticides are widely used to control insect pests and human disease vectors. Voltage-gated sodium channels are the primary targets of pyrethroid insecticides. Mutations in the sodium channel have been shown to be responsible for pyrethroid resistance, known as knockdown resistance (kdr, in various insects including mosquitoes. In Aedes aegypti mosquitoes, the principal urban vectors of dengue, zika, and yellow fever viruses, multiple single nucleotide polymorphisms in the sodium channel gene have been found in pyrethroid-resistant populations and some of them have been functionally confirmed to be responsible for kdr in an in vitro expression system, Xenopus oocytes. This mini-review aims to provide an update on the identification and functional characterization of pyrethroid resistance-associated sodium channel mutations from Aedes aegypti. The collection of kdr mutations not only helped us develop molecular markers for resistance monitoring, but also provided valuable information for computational molecular modeling of pyrethroid receptor sites on the sodium channel.

  20. Interaction of H2S with Calcium Permeable Channels and Transporters

    Directory of Open Access Journals (Sweden)

    Weihua Zhang

    2015-01-01

    Full Text Available A growing amount of evidence has suggested that hydrogen sulfide (H2S, as a gasotransmitter, is involved in intensive physiological and pathological processes. More and more research groups have found that H2S mediates diverse cellular biological functions related to regulating intracellular calcium concentration. These groups have demonstrated the reciprocal interaction between H2S and calcium ion channels and transporters, such as L-type calcium channels (LTCC, T-type calcium channels (TTCC, sodium/calcium exchangers (NCX, transient receptor potential (TRP channels, β-adrenergic receptors, and N-methyl-D-aspartate receptors (NMDAR in different cells. However, the understanding of the molecular targets and mechanisms is incomplete. Recently, some research groups demonstrated that H2S modulates the activity of calcium ion channels through protein S-sulfhydration and polysulfide reactions. In this review, we elucidate that H2S controls intracellular calcium homeostasis and the underlying mechanisms.

  1. Beltless Translocation Domain of Botulinum Neurotoxin A Embodies a Minimum Ion-conductive Channel*

    OpenAIRE

    Fischer, Audrey; Sambashivan, Shilpa; Brunger, Axel T.; Montal, Mauricio

    2011-01-01

    Botulinum neurotoxin, the causative agent of the paralytic disease botulism, is an endopeptidase composed of a catalytic domain (or light chain (LC)) and a heavy chain (HC) encompassing the translocation domain (TD) and receptor-binding domain. Upon receptor-mediated endocytosis, the LC and TD are proposed to undergo conformational changes in the acidic endocytic environment resulting in the formation of an LC protein-conducting TD channel. The mechanism of channel formation and the conformat...

  2. TRP channel functions in the gastrointestinal tract.

    Science.gov (United States)

    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.

  3. Coherifying quantum channels

    Science.gov (United States)

    Korzekwa, Kamil; Czachórski, Stanisław; Puchała, Zbigniew; Życzkowski, Karol

    2018-04-01

    Is it always possible to explain random stochastic transitions between states of a finite-dimensional system as arising from the deterministic quantum evolution of the system? If not, then what is the minimal amount of randomness required by quantum theory to explain a given stochastic process? Here, we address this problem by studying possible coherifications of a quantum channel Φ, i.e., we look for channels {{{Φ }}}{ \\mathcal C } that induce the same classical transitions T, but are ‘more coherent’. To quantify the coherence of a channel Φ we measure the coherence of the corresponding Jamiołkowski state J Φ. We show that the classical transition matrix T can be coherified to reversible unitary dynamics if and only if T is unistochastic. Otherwise the Jamiołkowski state {J}{{Φ }}{ \\mathcal C } of the optimally coherified channel is mixed, and the dynamics must necessarily be irreversible. To assess the extent to which an optimal process {{{Φ }}}{ \\mathcal C } is indeterministic we find explicit bounds on the entropy and purity of {J}{{Φ }}{ \\mathcal C }, and relate the latter to the unitarity of {{{Φ }}}{ \\mathcal C }. We also find optimal coherifications for several classes of channels, including all one-qubit channels. Finally, we provide a non-optimal coherification procedure that works for an arbitrary channel Φ and reduces its rank (the minimal number of required Kraus operators) from {d}2 to d.

  4. CANDU channel flow verification

    International Nuclear Information System (INIS)

    Mazalu, N.; Negut, Gh.

    1997-01-01

    The purpose of this evaluation was to obtain accurate information on each channel flow that enables us to assess precisely the level of reactor thermal power and, for reasons of safety, to establish which channel is boiling. In order to assess the channel flow parameters, computer simulations were done with the NUCIRC code and the results were checked by measurements. The complete channel flow measurements were made in the zero power cold condition. In hot conditions there were made flow measurements using the Shut Down System 1 (SDS 1) flow devices from 0.1 % F.P. up to 100 % F.P. The NUCIRC prediction for CANDU channel flows and the measurements by Ultrasonic Flow Meter at zero power cold conditions and SDS 1 flow channel measurements at different reactor power levels showed an acceptable agreement. The 100 % F.P. average errors for channel flow of R, shows that suitable NUCIRC flow assessment can be made. So, it can be done a fair prediction of the reactor power distribution. NUCIRC can predict accurately the onset of boiling and helps to warn at the possible power instabilities at high powers or it can detect the flow blockages. The thermal hydraulic analyst has in NUCIRC a suitable tool to do accurate predictions for the thermal hydraulic parameters for different steady state power levels which subsequently leads to an optimal CANDU reactor operation. (authors)

  5. Sub-cellular distribution and translocation of TRP channels.

    Science.gov (United States)

    Toro, Carlos A; Arias, Luis A; Brauchi, Sebastian

    2011-01-01

    Cellular electrical activity is the result of a highly complex processes that involve the activation of ion channel proteins. Ion channels make pores on cell membranes that rapidly transit between conductive and non-conductive states, allowing different ions to flow down their electrochemical gradients across cell membranes. In the case of neuronal cells, ion channel activity orchestrates action potentials traveling through axons, enabling electrical communication between cells in distant parts of the body. Somatic sensation -our ability to feel touch, temperature and noxious stimuli- require ion channels able to sense and respond to our peripheral environment. Sensory integration involves the summing of various environmental cues and their conversion into electrical signals. Members of the Transient Receptor Potential (TRP) family of ion channels have emerged as important mediators of both cellular sensing and sensory integration. The regulation of the spatial and temporal distribution of membrane receptors is recognized as an important mechanism for controlling the magnitude of the cellular response and the time scale on which cellular signaling occurs. Several studies have shown that this mechanism is also used by TRP channels to modulate cellular response and ultimately fulfill their physiological function as sensors. However, the inner-working of this mode of control for TRP channels remains poorly understood. The question of whether TRPs intrinsically regulate their own vesicular trafficking or weather the dynamic regulation of TRP channel residence on the cell surface is caused by extrinsic changes in the rates of vesicle insertion or retrieval remain open. This review will examine the evidence that sub-cellular redistribution of TRP channels plays an important role in regulating their activity and explore the mechanisms that control the trafficking of vesicles containing TRP channels.

  6. The meth brain: methamphetamines alter brain functions via NMDA receptors

    Czech Academy of Sciences Publication Activity Database

    Proft, Juliane; Weiss, Norbert

    2015-01-01

    Roč. 34, č. 1 (2015), s. 1-3 ISSN 0231-5882 R&D Projects: GA ČR GA15-13556S Institutional support: RVO:61388963 Keywords : ion channel * methamphetamine * piriform cortex * NMDA receptor * AMPA receptor Subject RIV: CE - Biochemistry Impact factor: 0.892, year: 2015

  7. Reconfigurable virtual electrowetting channels.

    Science.gov (United States)

    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