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Sample records for ryanodine receptor subtype

  1. Ryanodine receptor subtype 2 encodes Ca2+ oscillations activated by acetylcholine via the M2 muscarinic receptor/cADP-ribose signalling pathway in duodenum myocytes.

    PubMed

    Fritz, Nicolas; Macrez, Nathalie; Mironneau, Jean; Jeyakumar, Loice H; Fleischer, Sidney; Morel, Jean-Luc

    2005-05-15

    In this study, we characterized the signalling pathway activated by acetylcholine that encodes Ca2+ oscillations in rat duodenum myocytes. These oscillations were observed in intact myocytes after removal of external Ca2+, in permeabilized cells after abolition of the membrane potential and in the presence of heparin (an inhibitor of inositol 1,4,5-trisphosphate receptors) but were inhibited by ryanodine, indicating that they are dependent on Ca2+ release from intracellular stores through ryanodine receptors. Ca2+ oscillations were selectively inhibited by methoctramine (a M2 muscarinic receptor antagonist). The M2 muscarinic receptor-activated Ca2+ oscillations were inhibited by 8-bromo cyclic adenosine diphosphoribose and inhibitors of adenosine diphosphoribosyl cyclase (ZnCl2 and anti-CD38 antibody). Stimulation of ADP-ribosyl cyclase activity by acetylcholine was evaluated in permeabilized cells by measuring the production of cyclic guanosine diphosphoribose (a fluorescent compound), which resulted from the cyclization of nicotinamide guanine dinucleotide. As duodenum myocytes expressed the three subtypes of ryanodine receptors, an antisense strategy revealed that the ryanodine receptor subtype 2 alone was required to initiate the Ca2+ oscillations induced by acetylcholine and also by cyclic adenosine diphosphoribose and rapamycin (a compound that induced uncoupling between 12/12.6 kDa FK506-binding proteins and ryanodine receptors). Inhibition of cyclic adenosine diphosphoribose-induced Ca2+ oscillations, after rapamycin treatment, confirmed that both compounds interacted with the ryanodine receptor subtype 2. Our findings show for the first time that the M2 muscarinic receptor activation triggered Ca2+ oscillations in duodenum myocytes by activation of the cyclic adenosine diphosphoribose/FK506-binding protein/ryanodine receptor subtype 2 signalling pathway.

  2. Modulation of calcium signalling by dominant negative splice variant of ryanodine receptor subtype 3 in native smooth muscle cells.

    PubMed

    Dabertrand, Fabrice; Morel, Jean-Luc; Sorrentino, Vincenzo; Mironneau, Jean; Mironneau, Chantal; Macrez, Nathalie

    2006-07-01

    The ryanodine receptor subtype 3 (RYR3) is expressed ubiquitously but its physiological function varies from cell to cell. Here, we investigated the role of a dominant negative RYR3 isoform in Ca2+ signalling in native smooth muscle cells. We used intranuclear injection of antisense oligonucleotides to specifically inhibit endogenous RYR3 isoform expression. In mouse duodenum myocytes expressing RYR2 subtype and both spliced and non-spliced RYR3 isoforms, RYR2 and non-spliced RYR3 were activated by caffeine whereas the spliced RYR3 was not. Only RYR2 was responsible for the Ca2+-induced Ca2+ release mechanism that amplified Ca2+ influx- or inositol 1,4,5-trisphosphate-induced Ca2+ signals. However, the spliced RYR3 negatively regulated RYR2 leading to the decrease of amplitude and upstroke velocity of Ca2+ signals. Immunostaining in injected cells showed that the spliced RYR3 was principally expressed near the plasma membrane whilst the non-spliced isoform was revealed around the nucleus. This study shows for the first time that the short isoform of RYR3 controls Ca2+ release through RYR2 in native smooth muscle cells.

  3. Contribution of ryanodine receptor subtype 3 to ca2+ responses in Ca2+-overloaded cultured rat portal vein myocytes.

    PubMed

    Mironneau, J; Coussin, F; Jeyakumar, L H; Fleischer, S; Mironneau, C; Macrez, N

    2001-04-06

    Using an antisense strategy, we have previously shown that in vascular myocytes, subtypes 1 and 2 of ryanodine receptors (RYRs) are required for Ca(2+) release during Ca(2+) sparks and global Ca(2+) responses, evoked by activation of voltage-gated Ca(2+) channels, whereas RYR subtype 3 (RYR3) has no contribution. Here, we investigated the effects of increased Ca(2+) loading of the sarcoplasmic reticulum (SR) on the RYR-mediated Ca(2+) responses and the role of the RYR3 by injecting antisense oligonucleotides targeting the RYR subtypes. RYR3 expression was demonstrated by immunodetection in both freshly dissociated and cultured rat portal vein myocytes. Confocal Ca(2+) measurements revealed that the number of cells showing spontaneous Ca(2+) sparks was strongly increased by superfusing the vascular myocytes in 10 mm Ca(2+)-containing solution. These Ca(2+) sparks were blocked after inhibition of RYR1 or RYR2 by treatment with antisense oligolucleotides but not after inhibition of RYR3. In contrast, inhibition of RYR3 reduced the global Ca(2+) responses induced by caffeine and phenylephrine, indicating that RYR3 participated together with RYR1 and RYR2 to these Ca(2+) responses in Ca(2+)-overloaded myocytes. Ca(2+) transients evoked by photolysis of caged Ca(2+) with increasing flash intensities were also reduced after inhibition of RYR3 and revealed that the [Ca(2+)](i) sensitivity of RYR3 would be similar to that of RYR1 and RYR2. Our results show that, under conditions of increased SR Ca(2+) loading, the RYR3 becomes activable by caffeine and local increases in [Ca(2+)](i).

  4. Ryanodine receptors in smooth muscle.

    PubMed

    Guerrero-Hernández, Agustín; Gómez-Viquez, Leticia; Guerrero-Serna, Guadalupe; Rueda, Angélica

    2002-07-01

    The sarcoplasmic reticulum (SR) of smooth muscle is endowed with two different types of Ca2+ release channels, i.e. inositol 1,4,5-trisphosphate receptors (IP3Rs) and ryanodine receptors (RyRs). In general, both release channels mobilize Ca2+ from the same internal store in smooth muscle. While the importance of IP3Rs in agonist-induced contraction is well established, the role of RyRs in excitation-contraction coupling of smooth muscle is not clear. The participation of smooth muscle RyRs in the amplification of Ca2+ transients induced by either opening of Ca2+-permeable channels or IP3-triggered Ca2+ release has been studied. The efficacy of both processes to activate RyRs by calcium-induced calcium release (CICR) is highly variable and not widely present in smooth muscle. Although RyRs in smooth muscle generate Ca2+ sparks that are similar to those observed in striated muscles, the contribution of these local Ca2+ events to depolarization-induced global rise in [Ca2+]i is rather limited. Recent data suggest that RyRs are involved in regulating the luminal [Ca2+] of SR and also in smooth muscle relaxation. This review summarizes studies that were carried out mainly in muscle strips or in freshly isolated myocytes, and that were aimed to determine the physiological role of RyRs in smooth muscle.

  5. Ryanodine receptors as leak channels.

    PubMed

    Guerrero-Hernández, Agustín; Ávila, Guillermo; Rueda, Angélica

    2014-09-15

    Ryanodine receptors are Ca(2+) release channels of internal stores. This review focuses on those situations and conditions that transform RyRs from a finely regulated ion channel to an unregulated Ca(2+) leak channel and the pathological consequences of this alteration. In skeletal muscle, mutations in either CaV1.1 channel or RyR1 results in a leaky behavior of the latter. In heart cells, RyR2 functions normally as a Ca(2+) leak channel during diastole within certain limits, the enhancement of this activity leads to arrhythmogenic situations that are tackled with different pharmacological strategies. In smooth muscle, RyRs are involved more in reducing excitability than in stimulating contraction so the leak activity of RyRs in the form of Ca(2+) sparks, locally activates Ca(2+)-dependent potassium channels to reduce excitability. In neurons the enhanced activity of RyRs is associated with the development of different neurodegenerative disorders such as Alzheimer and Huntington diseases. It appears then that the activity of RyRs as leak channels can have both physiological and pathological consequences depending on the cell type and the metabolic condition.

  6. [Ryanodine receptor, calcium leak and arrhythmias].

    PubMed

    Rueda, Angélica; de Alba-Aguayo, David R; Valdivia, Héctor H

    2014-01-01

    The participation of the ionic Ca(2+) release channel/ryanodine receptor in cardiac excitation-contraction coupling is well known since the late '80s, when various seminal papers communicated its purification for the first time and its identity with the "foot" structures located at the terminal cisternae of the sarcoplasmic reticulum. In addition to its main role as the Ca(2+) channel responsible for the transient Ca(2+) increase that activates the contractile machinery of the cardiomyocytes, the ryanodine receptor releases Ca(2+) during the relaxation phase of the cardiac cycle, giving rise to a diastolic Ca(2+) leak. In normal physiological conditions, diastolic Ca(2+) leak regulates the proper level of luminal Ca(2+), but in pathological conditions it participates in the generation of both, acquired and hereditary arrhythmias. Very recently, several groups have focused their efforts into the development of pharmacological tools to control the altered diastolic Ca(2+) leak via ryanodine receptors. In this review, we focus our interest on describing the participation of cardiac ryanodine receptor in the diastolic Ca(2+) leak under physiological or pathological conditions and also on the therapeutic approaches to control its undesired exacerbated activity during diastole. Copyright © 2013 Instituto Nacional de Cardiología Ignacio Chávez. Published by Masson Doyma México S.A. All rights reserved.

  7. Evidence for ryanodine receptors in Schistosoma mansoni.

    PubMed

    Silva, C L; Cunha, V M; Mendonça-Silva, D L; Noël, F

    1998-10-15

    The present study investigated the presence of ryanodine receptors in the trematode Schistosoma mansoni. [3H]Ryanodine specific binding sites were found in the four subcellular fractions of S. mansoni; however, more binding sites were recovered in the heterogeneous fraction P1 and the microsomal fraction P4, as was thapsigargin-sensitive (Ca2+-Mg2+)ATPase activity, marking the sarco/endoplasmic reticulum calcium ATPase (SERCA) pumps. This binding had an equilibrium dissociation constant (Kd) in the nanomolar range, an apparent maximal number of receptors (Bmax) of about 80 fmol/mg of protein, and was modulated by ions (Ca2+, Mg2+) and some pharmacological tools such as caffeine. Ryanodine was able to accelerate the rate of 45Ca2+ release from actively loaded vesicles, and also to induce a transient contraction of the whole worm. We conclude that ryanodine-sensitive Ca2+ release channels are present in S. mansoni, with properties very similar to the ones present in higher animals.

  8. Detection of calcium release via ryanodine receptors.

    PubMed

    Eu, Jerry P; Meissner, Gerhard

    2012-01-01

    The ryanodine receptor ion channels (RyRs) release Ca(2+) from the endo/sarcoplasmic reticulum in a variety of nonvertebrate and vertebrate species including flies, crustaceans, birds, fish, and amphibians. They are most abundant in skeletal and cardiac muscle, where in response to an action potential, the release of Ca(2+) ions from the sarcoplasmic reticulum through the RyRs into the cytoplasm leads to muscle contraction (i.e., excitation-contraction coupling). Here, we describe how to determine their cellular location using isoform-specific antibodies, their protein levels using an in vitro ((3)H)ryanodine-binding assay, and their cellular release of Ca(2+) using RyR-specific channel agonists and inhibitors.

  9. Ryanodine receptor point mutant E4032A reveals an allosteric interaction with ryanodine.

    PubMed

    Fessenden, J D; Chen, L; Wang, Y; Paolini, C; Franzini-Armstrong, C; Allen, P D; Pessah, I N

    2001-02-27

    The ryanodine receptor (RyR) family of proteins constitutes a unique type of calcium channel that mediates Ca(2+) release from endoplasmic reticulum/sarcoplasmic reticulum stores. Ryanodine has been widely used to identify contributions made by the RyR to signaling in both muscle and nonmuscle cells. Ryanodine, through binding to high- and low-affinity sites, has been suggested to block the channel pore based on its ability to induce partial conductance states and irreversible inhibition. We examined the effect of ryanodine on an RyR type 1 (RyR1) point mutant (E4032A) that exhibits a severely compromised phenotype. When expressed in 1B5 (RyR null/dyspedic) myotubes, E4032A is relatively unresponsive to stimulation by cell membrane depolarization or RyR agonists, although the full-length protein is correctly targeted to junctions and interacts with dihydropyridine receptors (DHPRs) inducing their arrangement into tetrads. However, treatment of E4032A-expressing cells with 200-500 microM ryanodine, concentrations that rapidly activate and then inhibit wild-type (wt) RyR1, restores the responsiveness of E4032A-expressing myotubes to depolarization and RyR agonists. Moreover, the restored E4032A channels remain resistant to subsequent exposure to ryanodine. In single-channel studies, E4032A exhibits infrequent (channel-open probability, P(o) < 0.005) and brief (<250 micros) gating events and insensitivity to Ca(2+). Addition of ryanodine restores Ca(2+)-dependent channel activity exhibiting full, 3/4, 1/2, and 1/4 substates. This evidence suggests that, whereas ryanodine does not occlude the RyR pore, it does bind to sites that allosterically induce substantial conformational changes in the RyR. In the case of E4032A, these changes overcome unfavorable energy barriers introduced by the E4032A mutation to restore channel function.

  10. Ryanodine receptor point mutant E4032A reveals an allosteric interaction with ryanodine

    PubMed Central

    Fessenden, James D.; Chen, Lili; Wang, Yaming; Paolini, Cecilia; Franzini-Armstrong, Clara; Allen, Paul D.; Pessah, Isaac N.

    2001-01-01

    The ryanodine receptor (RyR) family of proteins constitutes a unique type of calcium channel that mediates Ca2+ release from endoplasmic reticulum/sarcoplasmic reticulum stores. Ryanodine has been widely used to identify contributions made by the RyR to signaling in both muscle and nonmuscle cells. Ryanodine, through binding to high- and low-affinity sites, has been suggested to block the channel pore based on its ability to induce partial conductance states and irreversible inhibition. We examined the effect of ryanodine on an RyR type 1 (RyR1) point mutant (E4032A) that exhibits a severely compromised phenotype. When expressed in 1B5 (RyR null/dyspedic) myotubes, E4032A is relatively unresponsive to stimulation by cell membrane depolarization or RyR agonists, although the full-length protein is correctly targeted to junctions and interacts with dihydropyridine receptors (DHPRs) inducing their arrangement into tetrads. However, treatment of E4032A-expressing cells with 200–500 μM ryanodine, concentrations that rapidly activate and then inhibit wild-type (wt) RyR1, restores the responsiveness of E4032A-expressing myotubes to depolarization and RyR agonists. Moreover, the restored E4032A channels remain resistant to subsequent exposure to ryanodine. In single-channel studies, E4032A exhibits infrequent (channel-open probability, Po < 0.005) and brief (<250 μs) gating events and insensitivity to Ca2+. Addition of ryanodine restores Ca2+-dependent channel activity exhibiting full, 3/4, 1/2, and 1/4 substates. This evidence suggests that, whereas ryanodine does not occlude the RyR pore, it does bind to sites that allosterically induce substantial conformational changes in the RyR. In the case of E4032A, these changes overcome unfavorable energy barriers introduced by the E4032A mutation to restore channel function. PMID:11226332

  11. Catecholamime Interactions with the Cardiac Ryanodine Receptor

    NASA Astrophysics Data System (ADS)

    Klipp, Robert Carl

    The cardiac ryanodine receptor (RyR2) is a Ca2+ ion channel found in the sarcoplasmic reticulum (SR), an intracellular membranous Ca2+ storage system. It is well known that a destabilization of RyR2 can lead to a Ca2+ flux out of the SR, which results in an overload of intracellular Ca2+; this can also lead to arrhythmias and heart failure. The catecholamines play a large role in the regulation of RyR2; stimulation of the beta-adrenergic receptor on the cell membrane can lead to a hyperphosphorylation of RyR2, making it more leaky to Ca2+. We have previously shown that strong electron donors will inhibit RyR2. It is hypothesized that the catecholamines, sharing a similar structure with other proven inhibitors of RyR2, will also inhibit RyR2. Here we confirm this hypothesis and show for the first time that the catecholamines, isoproterenol and epinephrine, act as strong electron donors and inhibit RyR2 activity at the single channel level. This data suggests that the catecholamines can influence RyR2 activity at two levels. This offers promising insight into the potential development of a new class of drugs to treat heart failure and arrhythmia; ones that can both prevent the hyperphosphorylation of RyR2 by blocking the beta-adrenergic receptor, but can also directly inhibit the release of Ca2+ from RyR2.

  12. Structure of a mammalian ryanodine receptor.

    PubMed

    Zalk, Ran; Clarke, Oliver B; des Georges, Amédée; Grassucci, Robert A; Reiken, Steven; Mancia, Filippo; Hendrickson, Wayne A; Frank, Joachim; Marks, Andrew R

    2015-01-01

    Ryanodine receptors (RyRs) mediate the rapid release of calcium (Ca(2+)) from intracellular stores into the cytosol, which is essential for numerous cellular functions including excitation-contraction coupling in muscle. Lack of sufficient structural detail has impeded understanding of RyR gating and regulation. Here we report the closed-state structure of the 2.3-megadalton complex of the rabbit skeletal muscle type 1 RyR (RyR1), solved by single-particle electron cryomicroscopy at an overall resolution of 4.8 Å. We fitted a polyalanine-level model to all 3,757 ordered residues in each protomer, defining the transmembrane pore in unprecedented detail and placing all cytosolic domains as tertiary folds. The cytosolic assembly is built on an extended α-solenoid scaffold connecting key regulatory domains to the pore. The RyR1 pore architecture places it in the six-transmembrane ion channel superfamily. A unique domain inserted between the second and third transmembrane helices interacts intimately with paired EF-hands originating from the α-solenoid scaffold, suggesting a mechanism for channel gating by Ca(2+).

  13. Structure of a mammalian ryanodine receptor

    PubMed Central

    Zalk, Ran; Clarke, Oliver B.; des Georges, Amédée; Grassucci, Robert A.; Reiken, Steven; Mancia, Filippo; Hendrickson, Wayne A.; Frank, Joachim; Marks, Andrew R.

    2014-01-01

    Ryanodine receptors (RyRs) mediate rapid release of calcium (Ca2+) from intracellular stores into the cytosol, which is essential for numerous cellular functions including excitation-contraction coupling in muscle. Lack of sufficient structural detail has impeded understanding of RyR gating and regulation. Here, we report the closed-state structure of the 2.3 MDa complex of the rabbit skeletal muscle type 1 RyR (RyR1), solved by single-particle cryo-electron microscopy at an overall resolution of 4.8 Å. We fitted a polyalanine-level model to all 3939 ordered residues in each protomer, defining the transmembrane pore in unprecedented detail and placing all cytosolic domains as tertiary folds. The cytosolic assembly is built on an extended α-solenoid scaffold connecting key regulatory domains to the pore. The RyR1 pore architecture places it in the six-transmembrane (6TM) ion channel superfamily. A unique domain inserted between the second and third transmembrane helices interacts intimately with paired EF-hands originating from the α-solenoid scaffold, suggesting a mechanism for channel gating by Ca2+. PMID:25470061

  14. Flux regulation of cardiac ryanodine receptor channels

    PubMed Central

    Liu, Yiwei; Porta, Maura; Qin, Jia; Ramos, Jorge; Nani, Alma; Shannon, Thomas R.

    2010-01-01

    The cardiac type 2 ryanodine receptor (RYR2) is activated by Ca2+-induced Ca2+ release (CICR). The inherent positive feedback of CICR is well controlled in cells, but the nature of this control is debated. Here, we explore how the Ca2+ flux (lumen-to-cytosol) carried by an open RYR2 channel influences its own cytosolic Ca2+ regulatory sites as well as those on a neighboring channel. Both flux-dependent activation and inhibition of single channels were detected when there were super-physiological Ca2+ fluxes (>3 pA). Single-channel results indicate a pore inhibition site distance of 1.2 ± 0.16 nm and that the activation site on an open channel is shielded/protected from its own flux. Our results indicate that the Ca2+ flux mediated by an open RYR2 channel in cells (∼0.5 pA) is too small to substantially regulate (activate or inhibit) the channel carrying it, even though it is sufficient to activate a neighboring RYR2 channel. PMID:20008518

  15. Insights into the gating mechanism of the ryanodine-modified human cardiac Ca2+-release channel (ryanodine receptor 2).

    PubMed

    Mukherjee, Saptarshi; Thomas, N Lowri; Williams, Alan J

    2014-09-01

    Ryanodine receptors (RyRs) are intracellular membrane channels playing key roles in many Ca(2+) signaling pathways and, as such, are emerging novel therapeutic and insecticidal targets. RyRs are so named because they bind the plant alkaloid ryanodine with high affinity and although it is established that ryanodine produces profound changes in all aspects of function, our understanding of the mechanisms underlying altered gating is minimal. We address this issue using detailed single-channel gating analysis, mathematical modeling, and energetic evaluation of state transitions establishing that, with ryanodine bound, the RyR pore adopts an extremely stable open conformation. We demonstrate that stability of this state is influenced by interaction of divalent cations with both activating and inhibitory cytosolic sites and, in the absence of activating Ca(2+), trans-membrane voltage. Comparison of the conformational stability of ryanodine- and Imperatoxin A-modified channels identifies significant differences in the mechanisms of action of these qualitatively similar ligands.

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

    DTIC Science & Technology

    2015-09-01

    Endocrinol Metab. Jul 2003;285(1):E16-24. 6. Wang C, Swerdloff RS, Iranmanesh A, et al. Transdermal testosterone gel improves sexual function, mood...maintains beneficial effects on sexual function and mood, lean and fat mass, and bone mineral density in hypogonadal men. J Clin Endocrinol Metab. May 2004;89... Reproduction . Mar 2004;127(3):359-366. 23. Andersson DC, Marks AR. Fixing ryanodine receptor Ca leak - a novel therapeutic strategy for contractile

  17. Redox regulation of the ryanodine receptor/calcium release channel.

    PubMed

    Zissimopoulos, S; Lai, F A

    2006-11-01

    The RyR (ryanodine receptor)/calcium release channel contains a number of highly reactive thiol groups that endow it with redox sensitivity. In general, oxidizing conditions favour channel opening, while reducing conditions have the opposite effect. Thiol modification affects the channel sensitivity to its principal effectors, Ca2+, Mg2+ and ATP, and alters RyR protein interactions. Here, we give a brief account of the major findings and prevailing views in the field.

  18. Insights into the Gating Mechanism of the Ryanodine-Modified Human Cardiac Ca2+-Release Channel (Ryanodine Receptor 2)

    PubMed Central

    Mukherjee, Saptarshi; Thomas, N. Lowri; Williams, Alan J.

    2014-01-01

    Ryanodine receptors (RyRs) are intracellular membrane channels playing key roles in many Ca2+ signaling pathways and, as such, are emerging novel therapeutic and insecticidal targets. RyRs are so named because they bind the plant alkaloid ryanodine with high affinity and although it is established that ryanodine produces profound changes in all aspects of function, our understanding of the mechanisms underlying altered gating is minimal. We address this issue using detailed single-channel gating analysis, mathematical modeling, and energetic evaluation of state transitions establishing that, with ryanodine bound, the RyR pore adopts an extremely stable open conformation. We demonstrate that stability of this state is influenced by interaction of divalent cations with both activating and inhibitory cytosolic sites and, in the absence of activating Ca2+, trans-membrane voltage. Comparison of the conformational stability of ryanodine- and Imperatoxin A-modified channels identifies significant differences in the mechanisms of action of these qualitatively similar ligands. PMID:25002270

  19. Inhibitory ryanodine prevents ryanodine receptor-mediated Ca²⁺ release without affecting endoplasmic reticulum Ca²⁺ content in primary hippocampal neurons.

    PubMed

    Adasme, Tatiana; Paula-Lima, Andrea; Hidalgo, Cecilia

    2015-02-27

    Ryanodine is a cell permeant plant alkaloid that binds selectively and with high affinity to ryanodine receptor (RyR) Ca(2+) release channels. Sub-micromolar ryanodine concentrations activate RyR channels while micromolar concentrations are inhibitory. Several reports indicate that neuronal synaptic plasticity, learning and memory require RyR-mediated Ca(2+)-release, which is essential for muscle contraction. The use of micromolar (inhibitory) ryanodine represents a common strategy to suppress RyR activity in neuronal cells: however, micromolar ryanodine promotes RyR-mediated Ca(2+) release and endoplasmic reticulum Ca(2+) depletion in muscle cells. Information is lacking in this regard in neuronal cells; hence, we examined here if addition of inhibitory ryanodine elicited Ca(2+) release in primary hippocampal neurons, and if prolonged incubation of primary hippocampal cultures with inhibitory ryanodine affected neuronal ER calcium content. Our results indicate that inhibitory ryanodine does not cause Ca(2+) release from the ER in primary hippocampal neurons, even though ryanodine diffusion should produce initially low intracellular concentrations, within the RyR activation range. Moreover, neurons treated for 1 h with inhibitory ryanodine had comparable Ca(2+) levels as control neurons. These combined findings imply that prolonged incubation with inhibitory ryanodine, which effectively abolishes RyR-mediated Ca(2+) release, preserves ER Ca(2+) levels and thus constitutes a sound strategy to suppress neuronal RyR function. Copyright © 2015 Elsevier Inc. All rights reserved.

  20. Modulation of ryanodine receptor Ca2+ channels (Review).

    PubMed

    Ozawa, Terutaka

    2010-01-01

    Ryanodine-sensitive Ca2+ release channels (ryanodine receptors, RyRs) play a crucial role in the mobilization of Ca2+ from the sarcoplasmic reticulum (SR) during the excitation-contraction coupling of muscle cells. In skeletal muscle, depolarization of transverse tubules activates the RyR, whereas in cardiac muscle, a Ca2+ influx through an L-type Ca2+ channel activates the RyR. The RyR is also activated by caffeine, a low concentration (<10 µM) of ryanodine or cyclic ADP-ribose. RyR activity is inhibited by Mg2+, ruthenium red, or higher concentrations (≥100 µM) of ryanodine. The activity of RyR channels is modulated by phosphorylation and by associated proteins, including calmodulin (CaM), calsequestrin (CSQ) and FK506-binding proteins (FKBPs). In muscle cells, apoCaM (Ca2+-free CaM) activates the RyR channel, and Ca2+ CaM (Ca2+-bound CaM) inhibits the channel. CSQ can bind approximately 40 moles of Ca2+/mole of CSQ in the SR lumen of muscle cells, and interacts functionally with RyR protein. When the RyR is stimulated, Ca2+ released from the lumen is dissociated from the CSQ- Ca2+ complex. A 12-kDa or 12.6-kDa FK506-binding protein (FKBP12 or FKBP12.6, respectively) is associated with RyR protein. When FKBP12 or FKBP12.6 is dissociated from the FKBP-RyR complex, the RyR is modulated (activated). Phosphorylation of the RyR by cAMP-dependent protein kinase (PKA) and Ca2+/calmodulin-dependent protein kinase II modulates the channel. PKA phosphorylation of the RyR on the skeletal and cardiac muscle SR dissociates FKBP12 or FKBP12.6 from the RyR complex. This review deals with the modulation mechanisms of RyR proteins by associated proteins and phosphorylation.

  1. Ryanodine Receptor Allosteric Coupling and the Dynamics of Calcium Sparks

    PubMed Central

    Groff, Jeffrey R.; Smith, Gregory D.

    2008-01-01

    Puffs and sparks are localized intracellular Ca2+ elevations that arise from the cooperative activity of Ca2+-regulated inositol 1,4,5-trisphosphate receptors and ryanodine receptors clustered at Ca2+ release sites on the surface of the endoplasmic reticulum or the sarcoplasmic reticulum. While the synchronous gating of Ca2+-regulated Ca2+ channels can be mediated entirely though the buffered diffusion of intracellular Ca2+, interprotein allosteric interactions also contribute to the dynamics of ryanodine receptor (RyR) gating and Ca2+ sparks. In this article, Markov chain models of Ca2+ release sites are used to investigate how the statistics of Ca2+ spark generation and termination are related to the coupling of RyRs via local [Ca2+] changes and allosteric interactions. Allosteric interactions are included in a manner that promotes the synchronous gating of channels by stabilizing neighboring closed-closed and/or open-open channel pairs. When the strength of Ca2+-mediated channel coupling is systematically varied (e.g., by changing the Ca2+ buffer concentration), simulations that include synchronizing allosteric interactions often exhibit more robust Ca2+ sparks; however, for some Ca2+ coupling strengths the sparks are less robust. We find no evidence that the distribution of spark durations can be used to distinguish between allosteric interactions that stabilize closed channel pairs, open channel pairs, or both in a balanced fashion. On the other hand, the changes in spark duration, interspark interval, and frequency observed when allosteric interactions that stabilize closed channel pairs are gradually removed from simulations are qualitatively different than the changes observed when open or both closed and open channel pairs are stabilized. Thus, our simulations clarify how changes in spark statistics due to pharmacological washout of the accessory proteins mediating allosteric coupling may indicate the type of synchronizing allosteric interactions exhibited

  2. Functional switching of GABAergic synapses by ryanodine receptor activation

    NASA Astrophysics Data System (ADS)

    Sun, Miao-Kun; Nelson, Thomas J.; Alkon, Daniel L.

    2000-10-01

    The role of the ryanodine receptor (RyR) in modifiability of synapses made by the basket interneurons onto the hippocampal CA1 pyramidal cells was examined in rats. Associating single-cell RyR activation with postsynaptic depolarization increased intracellular free Ca2+ concentrations and reversed the basket interneuron-CA1 inhibitory postsynaptic potential into an excitatory postsynaptic potential. This synaptic transformation was accompanied by a shift of the reversal potential from that of chloride toward that of bicarbonate. This inhibitory postsynaptic potential-excitatory postsynaptic potential transformation was prevented by blocking RyR or carbonic anhydrase. Associated postsynaptic depolarization and RyR activation, therefore, changes GABAergic synapses from excitation filters to amplifier and, thereby, shapes information flow through the hippocampal network.

  3. Calsequestrin interacts directly with the cardiac ryanodine receptor luminal domain

    PubMed Central

    Handhle, Ahmed; Ormonde, Chloe E.; Thomas, N. Lowri; Bralesford, Catherine; Williams, Alan J.; Lai, F. Anthony

    2016-01-01

    ABSTRACT Cardiac muscle contraction requires sarcoplasmic reticulum (SR) Ca2+ release mediated by the quaternary complex comprising the ryanodine receptor 2 (RyR2), calsequestrin 2 (CSQ2), junctin (encoded by ASPH) and triadin. Here, we demonstrate that a direct interaction exists between RyR2 and CSQ2. Topologically, CSQ2 binding occurs at the first luminal loop of RyR2. Co-expression of RyR2 and CSQ2 in a human cell line devoid of the other quaternary complex proteins results in altered Ca2+-release dynamics compared to cells expressing RyR2 only. These findings provide a new perspective for understanding the SR luminal Ca2+ sensor and its involvement in cardiac physiology and disease. PMID:27609834

  4. Sphingosylphosphocholine modulates the ryanodine receptor/calcium-release channel of cardiac sarcoplasmic reticulum membranes.

    PubMed Central

    Betto, R; Teresi, A; Turcato, F; Salviati, G; Sabbadini, R A; Krown, K; Glembotski, C C; Kindman, L A; Dettbarn, C; Pereon, Y; Yasui, K; Palade, P T

    1997-01-01

    Sphingosylphosphocholine (SPC) modulates Ca2+ release from isolated cardiac sarcoplasmic reticulum membranes; 50 microM SPC induces the release of 70 80% of the accumulated calcium. SPC release calcium from cardiac sarcoplasmic reticulum through the ryanodine receptor, since the release is inhibited by the ryanodine receptor channel antagonists ryanodine. Ruthenium Red and sphingosine. In intact cardiac myocytes, even in the absence of extracellular calcium. SPC causes a rise in diastolic Ca2+, which is greatly reduced when the sarcoplasmic reticulum is depleted of Ca2+ by prior thapsigargin treatment. SPC action on the ryanodine receptor is Ca(2+)-dependent. SPC shifts to the left the Ca(2+)-dependence of [3H]ryanodine binding, but only at high pCa values, suggesting that SPC might increase the sensitivity to calcium of the Ca(2+)-induced Ca(2+)-release mechanism. At high calcium concentrations (pCa 4.0 or lower), where [3H]ryanodine binding is maximally stimulated, no effect of SPC is observed. We conclude that SPC releases calcium from cardiac sarcoplasmic reticulum membranes by activating the ryanodine receptor and possibly another intracellular Ca(2+)-release channel, the sphingolipid Ca(2+)-release-mediating protein of endoplasmic reticulum (SCaMPER) [Mao, Kim, Almenoff, Rudner, Kearney and Kindman (1996) Proc.Natl.Acad.Sci. U.S.A 93, 1993-1996], which we have identified for the first time in cardiac tissue. PMID:9078280

  5. A Skeletal Muscle Ryanodine Receptor Interaction Domain in Triadin

    PubMed Central

    Wium, Elize; Dulhunty, Angela F.; Beard, Nicole A.

    2012-01-01

    Excitation-contraction coupling in skeletal muscle depends, in part, on a functional interaction between the ligand-gated ryanodine receptor (RyR1) and integral membrane protein Trisk 95, localized to the sarcoplasmic reticulum membrane. Various domains on Trisk 95 can associate with RyR1, yet the domain responsible for regulating RyR1 activity has remained elusive. We explored the hypothesis that a luminal Trisk 95 KEKE motif (residues 200–232), known to promote RyR1 binding, may also form the RyR1 activation domain. Peptides corresponding to Trisk 95 residues 200–232 or 200–231 bound to RyR1 and increased the single channel activity of RyR1 by 1.49±0.11-fold and 1.8±0.15-fold respectively, when added to its luminal side. A similar increase in [3H]ryanodine binding, which reflects open probability of the channels, was also observed. This RyR1 activation is similar to activation induced by full length Trisk 95. Circular dichroism showed that both peptides were intrinsically disordered, suggesting a defined secondary structure is not necessary to mediate RyR1 activation. These data for the first time demonstrate that Trisk 95′s 200–231 region is responsible for RyR1 activation. Furthermore, it shows that no secondary structure is required to achieve this activation, the Trisk 95 residues themselves are critical for the Trisk 95-RyR1 interaction. PMID:22937102

  6. Cyclic ADP-ribose, the ryanodine receptor and Ca2+ release.

    PubMed

    Sitsapesan, R; McGarry, S J; Williams, A J

    1995-11-01

    In a variety of vertebrate and invertebrate tissues the ryanodine-sensitive Ca2+ channel is the pathway for Ca2+ release from intracellular stores. The mechanism for activation of the ryanodine receptor-channel complex appears to depend both on the ryanodine receptor isoform and the cell type. In addition, a complex combination of endogenous intracellular compounds regulates channel gating. In this article, Rebecca Sitsapesan, Stephen McGarry and Alan Williams review the mechanisms involved in cyclic ADP-ribose (cADPR)-induced Ca2+ release and discuss the likelihood that cADPR-activated Ca2+ release is mediated by one of the recognized isoforms of the ryanodine receptor-Ca2+ channel complex.

  7. Ryanodine is a positive modulator of acetylcholine receptor gating in cochlear hair cells.

    PubMed

    Zorrilla de San Martín, Javier; Ballestero, Jimena; Katz, Eleonora; Elgoyhen, A Belén; Fuchs, Paul A

    2007-12-01

    The efferent synaptic specialization of hair cells includes a near-membrane synaptic cistern, whose presence suggests a role for internal calcium stores in cholinergic inhibition. Calcium release channels from internal stores include 'ryanodine receptors', whose participation is usually demonstrated by sensitivity to the eponymous plant alkaloid, ryanodine. However, use of this and other store-active compounds on hair cells could be confounded by the unusual pharmacology of the alpha9alpha10-containing hair cell nicotinic cholinergic receptor (nAChR), which has been shown to be antagonized by a broad spectrum of compounds. Surprisingly, we found that ryanodine, rather than antagonizing, is a positive modulator of the alpha9alpha10 nAChR expressed in Xenopus oocytes, the first such compound to be found. The effect of ryanodine was to increase the apparent affinity and efficacy for acetylcholine (ACh). Correspondingly, ACh-evoked currents through the isolated cholinergic receptors of inner hair cells in excised mouse cochleas were approximately doubled by 200 microM ryanodine, a concentration that inhibits gating of the ryanodine receptor itself. This unusual positive modulation was not unique to the mammalian receptor. The response to ACh of chicken 'short' hair cells likewise was enhanced in the presence of 100 microM ryanodine. This facilitatory effect on current through the AChR could enhance brief ( approximately 1 s) activation of associated calcium-dependent K(+) (SK) channels in both chicken short hair cells and rat outer hair cells. This novel effect of ryanodine provides new opportunities for the design of compounds that potentiate alpha9alpha10-mediated responses and for potential inner ear therapeutics based on this interaction.

  8. Different Involvement of Type 1, 2, and 3 Ryanodine Receptors in Memory Processes

    ERIC Educational Resources Information Center

    Galeotti, Nicoletta; Quattrone, Alessandro; Vivoli, Elisa; Norcini, Monica; Bartolini, Alessandro; Ghelardini, Carla

    2008-01-01

    The administration of the ryanodine receptor (RyR) agonist 4-Cmc (0.003-9 nmol per mouse intracerebroventricularly [i.c.v.]) ameliorated memory functions, whereas the RyR antagonist ryanodine (0.0001-1 nmol per mouse i.c.v.) induced amnesia in the mouse passive avoidance test. The role of the type 1, 2, and 3 RyR isoforms in memory processes was…

  9. Ryanodine is a Positive Modulator of Acetylcholine Receptor Gating in Cochlear Hair Cells

    PubMed Central

    Zorrilla de San Martín, Javier; Ballestero, Jimena; Katz, Eleonora; Elgoyhen, A. Belén

    2007-01-01

    The efferent synaptic specialization of hair cells includes a near-membrane synaptic cistern, whose presence suggests a role for internal calcium stores in cholinergic inhibition. Calcium release channels from internal stores include ‘ryanodine receptors’, whose participation is usually demonstrated by sensitivity to the eponymous plant alkaloid, ryanodine. However, use of this and other store-active compounds on hair cells could be confounded by the unusual pharmacology of the α9α10-containing hair cell nicotinic cholinergic receptor (nAChR), which has been shown to be antagonized by a broad spectrum of compounds. Surprisingly, we found that ryanodine, rather than antagonizing, is a positive modulator of the α9α10 nAChR expressed in Xenopus oocytes, the first such compound to be found. The effect of ryanodine was to increase the apparent affinity and efficacy for acetylcholine (ACh). Correspondingly, ACh-evoked currents through the isolated cholinergic receptors of inner hair cells in excised mouse cochleas were approximately doubled by 200 μM ryanodine, a concentration that inhibits gating of the ryanodine receptor itself. This unusual positive modulation was not unique to the mammalian receptor. The response to ACh of chicken ‘short’ hair cells likewise was enhanced in the presence of 100 μM ryanodine. This facilitatory effect on current through the AChR could enhance brief (∼1 s) activation of associated calcium-dependent K+ (SK) channels in both chicken short hair cells and rat outer hair cells. This novel effect of ryanodine provides new opportunities for the design of compounds that potentiate α9α10-mediated responses and for potential inner ear therapeutics based on this interaction. PMID:17647061

  10. Ryanodine receptor-mediated arrhythmias and sudden cardiac death

    PubMed Central

    Blayney, Lynda M.; Lai, F. Anthony

    2009-01-01

    The cardiac ryanodine receptor-Ca2+ release channel (RyR2) is an essential sarcoplasmic reticulum (SR) transmembrane protein that plays a central role in excitation–contraction coupling (ECC) in cardiomyocytes. Aberrant spontaneous, diastolic Ca2+ leak from the SR due to dysfunctional RyR2 contributes to the formation of delayed after-depolarisations, which are thought to underlie the fatal arrhythmia that occurs in both heart failure (HF) and in catecholaminergic polymorphic ventricular tachycardia (CPVT). CPVT is an inherited disorder associated with mutations in either the RyR2 or a SR luminal protein, calsequestrin. RyR2 shows normal function at rest in CPVT but the RyR2 dysfunction is unmasked by physical exercise or emotional stress, suggesting abnormal RyR2 activation as an underlying mechanism. Several potential mechanisms have been advanced to explain the dysfunctional RyR2 observed in HF and CPVT, including enhanced RyR2 phosphorylation status, altered RyR2 regulation at luminal/cytoplasmic sites and perturbed RyR2 intra/inter-molecular interactions. This review considers RyR2 dysfunction in the context of the structural and functional modulation of the channel, and potential therapeutic strategies to stabilise RyR2 function in cardiac pathology. PMID:19345240

  11. Intracellular Zinc Modulates Cardiac Ryanodine Receptor-mediated Calcium Release*

    PubMed Central

    Woodier, Jason; Rainbow, Richard D.; Stewart, Alan J.; Pitt, Samantha J.

    2015-01-01

    Aberrant Zn2+ homeostasis is a hallmark of certain cardiomyopathies associated with altered contractile force. In this study, we addressed whether Zn2+ modulates cardiac ryanodine receptor gating and Ca2+ dynamics in isolated cardiomyocytes. We reveal that Zn2+ is a high affinity regulator of RyR2 displaying three modes of operation. Picomolar free Zn2+ concentrations potentiate RyR2 responses, but channel activation is still dependent on the presence of cytosolic Ca2+. At concentrations of free Zn2+ >1 nm, Zn2+ is the main activating ligand, and the dependence on Ca2+ is removed. Zn2+ is therefore a higher affinity activator of RyR2 than Ca2+. Millimolar levels of free Zn2+ were found to inhibit channel openings. In cardiomyocytes, consistent with our single channel results, we show that Zn2+ modulates both the frequency and amplitude of Ca2+ waves in a concentration-dependent manner and that physiological levels of Zn2+ elicit Ca2+ release in the absence of activating levels of cytosolic Ca2+. This highlights a new role for intracellular Zn2+ in shaping Ca2+ dynamics in cardiomyocytes through modulation of RyR2 gating. PMID:26041778

  12. Ryanodine receptor-2 upregulation and nicotine-mediated plasticity.

    PubMed

    Ziviani, Elena; Lippi, Giordano; Bano, Daniele; Munarriz, Eliana; Guiducci, Stefania; Zoli, Michele; Young, Kenneth W; Nicotera, Pierluigi

    2011-01-05

    Nicotine, the major psychoactive component of cigarette smoke, modulates neuronal activity to produce Ca2+-dependent changes in gene transcription. However, the downstream targets that underlie the long-term effects of nicotine on neuronal function, and hence behaviour, remain to be elucidated. Here, we demonstrate that nicotine administration to mice upregulates levels of the type 2 ryanodine receptor (RyR2), a Ca2+-release channel present on the endoplasmic reticulum, in a number of brain areas associated with cognition and addiction, notably the cortex and ventral midbrain. Nicotine-mediated RyR2 upregulation was driven by CREB, and caused a long-lasting reinforcement of Ca2+ signalling via the process of Ca2+-induced Ca2+ release. RyR2 upregulation was itself required for long-term phosphorylation of CREB in a positive-feedback signalling loop. We further demonstrate that inhibition of RyR-activation in vivo abolishes sensitization to nicotine-induced habituated locomotion, a well-characterised model for onset of drug dependence. Our findings, therefore, indicate that gene-dependent reprogramming of Ca2+ signalling is involved in nicotine-induced behavioural changes.

  13. Leaky ryanodine receptors contribute to diaphragmatic weakness during mechanical ventilation

    PubMed Central

    Matecki, Stefan; Dridi, Haikel; Jung, Boris; Saint, Nathalie; Reiken, Steven R.; Scheuermann, Valérie; Mrozek, Ségolène; Umanskaya, Alisa; Petrof, Basil J.; Jaber, Samir; Marks, Andrew R.; Lacampagne, Alain

    2016-01-01

    Ventilator-induced diaphragmatic dysfunction (VIDD) refers to the diaphragm muscle weakness that occurs following prolonged controlled mechanical ventilation (MV). The presence of VIDD impedes recovery from respiratory failure. However, the pathophysiological mechanisms accounting for VIDD are still not fully understood. Here, we show in human subjects and a mouse model of VIDD that MV is associated with rapid remodeling of the sarcoplasmic reticulum (SR) Ca2+ release channel/ryanodine receptor (RyR1) in the diaphragm. The RyR1 macromolecular complex was oxidized, S-nitrosylated, Ser-2844 phosphorylated, and depleted of the stabilizing subunit calstabin1, following MV. These posttranslational modifications of RyR1 were mediated by both oxidative stress mediated by MV and stimulation of adrenergic signaling resulting from the anesthesia. We demonstrate in the murine model that such abnormal resting SR Ca2+ leak resulted in reduced contractile function and muscle fiber atrophy for longer duration of MV. Treatment with β-adrenergic antagonists or with S107, a small molecule drug that stabilizes the RyR1–calstabin1 interaction, prevented VIDD. Diaphragmatic dysfunction is common in MV patients and is a major cause of failure to wean patients from ventilator support. This study provides the first evidence to our knowledge of RyR1 alterations as a proximal mechanism underlying VIDD (i.e., loss of function, muscle atrophy) and identifies RyR1 as a potential target for therapeutic intervention. PMID:27457930

  14. Intracellular Zinc Modulates Cardiac Ryanodine Receptor-mediated Calcium Release.

    PubMed

    Woodier, Jason; Rainbow, Richard D; Stewart, Alan J; Pitt, Samantha J

    2015-07-10

    Aberrant Zn(2+) homeostasis is a hallmark of certain cardiomyopathies associated with altered contractile force. In this study, we addressed whether Zn(2+) modulates cardiac ryanodine receptor gating and Ca(2+) dynamics in isolated cardiomyocytes. We reveal that Zn(2+) is a high affinity regulator of RyR2 displaying three modes of operation. Picomolar free Zn(2+) concentrations potentiate RyR2 responses, but channel activation is still dependent on the presence of cytosolic Ca(2+). At concentrations of free Zn(2+) >1 nm, Zn(2+) is the main activating ligand, and the dependence on Ca(2+) is removed. Zn(2+) is therefore a higher affinity activator of RyR2 than Ca(2+). Millimolar levels of free Zn(2+) were found to inhibit channel openings. In cardiomyocytes, consistent with our single channel results, we show that Zn(2+) modulates both the frequency and amplitude of Ca(2+) waves in a concentration-dependent manner and that physiological levels of Zn(2+) elicit Ca(2+) release in the absence of activating levels of cytosolic Ca(2+). This highlights a new role for intracellular Zn(2+) in shaping Ca(2+) dynamics in cardiomyocytes through modulation of RyR2 gating.

  15. Architecture and conformational switch mechanism of the ryanodine receptor.

    PubMed

    Efremov, Rouslan G; Leitner, Alexander; Aebersold, Ruedi; Raunser, Stefan

    2015-01-01

    Muscle contraction is initiated by the release of calcium (Ca(2+)) from the sarcoplasmic reticulum into the cytoplasm of myocytes through ryanodine receptors (RyRs). RyRs are homotetrameric channels with a molecular mass of more than 2.2 megadaltons that are regulated by several factors, including ions, small molecules and proteins. Numerous mutations in RyRs have been associated with human diseases. The molecular mechanism underlying the complex regulation of RyRs is poorly understood. Using electron cryomicroscopy, here we determine the architecture of rabbit RyR1 at a resolution of 6.1 Å. We show that the cytoplasmic moiety of RyR1 contains two large α-solenoid domains and several smaller domains, with folds suggestive of participation in protein-protein interactions. The transmembrane domain represents a chimaera of voltage-gated sodium and pH-activated ion channels. We identify the calcium-binding EF-hand domain and show that it functions as a conformational switch allosterically gating the channel.

  16. Drosophila Ryanodine Receptors Mediate General Anesthesia by Halothane

    PubMed Central

    Gao, Shuying; Sandstrom, David J.; Smith, Harold E.; High, Brigit; Marsh, Jon W.; Nash, Howard A.

    2012-01-01

    Background Although in vitro studies have identified numerous possible targets, the molecules that mediate the in vivo effects of volatile anesthetics remain largely unknown. The mammalian ryanodine receptor (Ryr) is a known halothane target, and we hypothesized that it has a central role in anesthesia. Methods Gene function of the Drosophila Ryr (dRyr) was manipulated in the whole body or in specific tissues using a collection of mutants and transgenes, and responses to halothane were measured with a reactive climbing assay. Cellular responses to halothane were studied using Ca2+ imaging and patch clamp electrophysiology. Results Halothane potency strongly correlates with dRyr gene copy number, and missense mutations in regions known to be functionally important in mammalian Ryrs gene cause dominant hypersensitivity. Tissue-specific manipulation of dRyr shows that expression in neurons and glia, but not muscle, mediates halothane sensitivity. In cultured cells, halothane-induced Ca2+ efflux is strictly dRyr-dependent, suggesting a close interaction between halothane and dRyr. Ca2+ imaging and electrophysiology of Drosophila central neurons reveal halothane-induced Ca2+ flux that is altered in dRyr mutants and correlates with strong hyperpolarization. Conclusions In Drosophila, neurally-expressed dRyr mediates a substantial proportion of halothane's anesthetic effects in vivo, is potently activated by halothane in vitro, and activates an inhibitory conductance. Our results provide support for Ryr as an important mediator of immobilization by volatile anesthetics. PMID:23254148

  17. Functional Coupling of Ca2+ Channels and Ryanodine Receptors in Cardiac Myocytes

    NASA Astrophysics Data System (ADS)

    Sham, James S. K.; Cleemann, Lars; Morad, Martin

    1995-01-01

    In skeletal muscle, dihydropyridine receptors are functionally coupled to ryanodine receptors of the sarcoplasmic reticulum in triadic or diadic junctional complexes. In cardiac muscle direct physical or functional couplings have not been demonstrated. We have tested the hypothesis of functional coupling of L-type Ca2+ channels and ryanodine receptors in rat cardiac myocytes by comparing the efficacies of Ca2+ in triggering Ca2+ release when the ion enters the cell via the Ca2+ channels or the Na^+/Ca2+ exchanger. Ca2+ transported through the Ca2+ channels was 20-160 times more effective than Ca2+ influx via the Na^+/Ca2+ exchanger in gating Ca2+ release from the sarcoplasmic reticulum, suggesting privileged communication between Ca2+ channels and ryanodine receptors. In support of this hypothesis we found that Ca2+ channels were inactivated by Ca2+ release from the sarcoplasmic reticulum, even though the myoplasmic Ca2+ concentrations were buffered with 10 mM EGTA. The data thus suggest privileged cross signaling between the dihydropyridine and ryanodine receptors such that Ca2+ flux through either the Ca2+ channel or the ryanodine receptor alters the gating kinetics of the other channel.

  18. Decreased expression of ryanodine receptors alters calcium-induced calcium release mechanism in mdx duodenal myocytes.

    PubMed

    Morel, Jean-Luc; Rakotoarisoa, Lala; Jeyakumar, Loice H; Fleischer, Sidney; Mironneau, Chantal; Mironneau, Jean

    2004-05-14

    It is generally believed that alterations of calcium homeostasis play a key role in skeletal muscle atrophy and degeneration observed in Duchenne's muscular dystrophy and mdx mice. Mechanical activity is also impaired in gastrointestinal muscles, but the cellular and molecular mechanisms of this pathological state have not yet been investigated. We showed, in mdx duodenal myocytes, that both caffeine- and depolarization-induced calcium responses were inhibited, whereas acetylcholine- and thapsigargin-induced calcium responses were not significantly affected compared with control mice. Calcium-induced calcium release efficiency was impaired in mdx duodenal myocytes depending only on inhibition of ryanodine receptor expression. Duodenal myocytes expressed both type 2 and type 3 ryanodine receptors and were unable to produce calcium sparks. In control and mdx duodenal myocytes, both caffeine- and depolarization-induced calcium responses were dose-dependently and specifically inhibited with the anti-type 2 ryanodine receptor antibody. A strong inhibition of type 2 ryanodine receptor in mdx duodenal myocytes was observed on the mRNA as well as on the protein level. Taken together, our results suggest that inhibition of type 2 ryanodine receptor expression in mdx duodenal myocytes may account for the decreased calcium release from the sarcoplasmic reticulum and reduced mechanical activity.

  19. Muscarinic Stimulation Facilitates Sarcoplasmic Reticulum Ca Release by Modulating Ryanodine Receptor 2 Phosphorylation Through Protein Kinase G and Ca/Calmodulin-Dependent Protein Kinase II.

    PubMed

    Ho, Hsiang-Ting; Belevych, Andriy E; Liu, Bin; Bonilla, Ingrid M; Radwański, Przemysław B; Kubasov, Igor V; Valdivia, Héctor H; Schober, Karsten; Carnes, Cynthia A; Györke, Sándor

    2016-11-01

    Although the effects and the underlying mechanism of sympathetic stimulation on cardiac Ca handling are relatively well established both in health and disease, the modes of action and mechanisms of parasympathetic modulation are poorly defined. Here, we demonstrate that parasympathetic stimulation initiates a novel mode of excitation-contraction coupling that enhances the efficiency of cardiac sarcoplasmic reticulum Ca store utilization. This efficient mode of excitation-contraction coupling involves reciprocal changes in the phosphorylation of ryanodine receptor 2 at Ser-2808 and Ser-2814. Specifically, Ser-2808 phosphorylation was mediated by muscarinic receptor subtype 2 and activation of PKG (protein kinase G), whereas dephosphorylation of Ser-2814 involved activation of muscarinic receptor subtype 3 and decreased reactive oxygen species-dependent activation of CaMKII (Ca/calmodulin-dependent protein kinase II). The overall effect of these changes in phosphorylation of ryanodine receptor 2 is an increase in systolic Ca release at the low sarcoplasmic reticulum Ca content and a paradoxical reduction in aberrant Ca leak. Accordingly, cholinergic stimulation of cardiomyocytes isolated from failing hearts improved Ca cycling efficiency by restoring altered ryanodine receptor 2 phosphorylation balance. © 2016 American Heart Association, Inc.

  20. Cardiomyocyte ryanodine receptor degradation by chaperone-mediated autophagy.

    PubMed

    Pedrozo, Zully; Torrealba, Natalia; Fernández, Carolina; Gatica, Damian; Toro, Barbra; Quiroga, Clara; Rodriguez, Andrea E; Sanchez, Gina; Gillette, Thomas G; Hill, Joseph A; Donoso, Paulina; Lavandero, Sergio

    2013-05-01

    Chaperone-mediated autophagy (CMA) is a selective mechanism for the degradation of soluble cytosolic proteins bearing the sequence KFERQ. These proteins are targeted by chaperones and delivered to lysosomes where they are translocated into the lysosomal lumen and degraded via the lysosome-associated membrane protein type 2A (LAMP-2A). Mutations in LAMP2 that inhibit autophagy result in Danon disease characterized by hypertrophic cardiomyopathy. The ryanodine receptor type 2 (RyR2) plays a key role in cardiomyocyte excitation-contraction and its dysfunction can lead to cardiac failure. Whether RyR2 is degraded by CMA is unknown. To induce CMA, cultured neonatal rat cardiomyocytes were treated with geldanamycin (GA) to promote protein degradation through this pathway. GA increased LAMP-2A levels together with its redistribution and colocalization with Hsc70 in the perinuclear region, changes indicative of CMA activation. The inhibition of lysosomes but not proteasomes prevented the loss of RyR2. The recovery of RyR2 content after incubation with GA by siRNA targeting LAMP-2A suggests that RyR2 is degraded via CMA. In silico analysis also revealed that the RyR2 sequence harbours six KFERQ motifs which are required for the recognition Hsc70 and its degradation via CMA. Our data suggest that presenilins are involved in RyR2 degradation by CMA. These findings are consistent with a model in which oxidative damage of the RyR2 targets it for turnover by presenilins and CMA, which could lead to removal of damaged or leaky RyR2 channels.

  1. Cardiomyocyte ryanodine receptor degradation by chaperone-mediated autophagy

    PubMed Central

    Pedrozo, Zully; Torrealba, Natalia; Fernández, Carolina; Gatica, Damian; Toro, Barbra; Quiroga, Clara; Rodriguez, Andrea E.; Sanchez, Gina; Gillette, Thomas G.; Hill, Joseph A.; Donoso, Paulina; Lavandero, Sergio

    2013-01-01

    Time for primary review: 15 days Aims Chaperone-mediated autophagy (CMA) is a selective mechanism for the degradation of soluble cytosolic proteins bearing the sequence KFERQ. These proteins are targeted by chaperones and delivered to lysosomes where they are translocated into the lysosomal lumen and degraded via the lysosome-associated membrane protein type 2A (LAMP-2A). Mutations in LAMP2 that inhibit autophagy result in Danon disease characterized by hypertrophic cardiomyopathy. The ryanodine receptor type 2 (RyR2) plays a key role in cardiomyocyte excitation–contraction and its dysfunction can lead to cardiac failure. Whether RyR2 is degraded by CMA is unknown. Methods and results To induce CMA, cultured neonatal rat cardiomyocytes were treated with geldanamycin (GA) to promote protein degradation through this pathway. GA increased LAMP-2A levels together with its redistribution and colocalization with Hsc70 in the perinuclear region, changes indicative of CMA activation. The inhibition of lysosomes but not proteasomes prevented the loss of RyR2. The recovery of RyR2 content after incubation with GA by siRNA targeting LAMP-2A suggests that RyR2 is degraded via CMA. In silico analysis also revealed that the RyR2 sequence harbours six KFERQ motifs which are required for the recognition Hsc70 and its degradation via CMA. Our data suggest that presenilins are involved in RyR2 degradation by CMA. Conclusion These findings are consistent with a model in which oxidative damage of the RyR2 targets it for turnover by presenilins and CMA, which could lead to removal of damaged or leaky RyR2 channels. PMID:23404999

  2. Multiple Modes of Ryanodine Receptor 2 Inhibition by Flecainide

    PubMed Central

    Mehra, D.; Imtiaz, M. S.; van Helden, D. F.; Knollmann, B. C.

    2014-01-01

    Catecholaminergic polymorphic ventricular tachycardia (CPVT) causes sudden cardiac death due to mutations in cardiac ryanodine receptors (RyR2), calsequestrin, or calmodulin. Flecainide, a class I antiarrhythmic drug, inhibits Na+ and RyR2 channels and prevents CPVT. The purpose of this study is to identify inhibitory mechanisms of flecainide on RyR2. RyR2 were isolated from sheep heart, incorporated into lipid bilayers, and investigated by single-channel recording under various activating conditions, including the presence of cytoplasmic ATP (2 mM) and a range of cytoplasmic [Ca2+], [Mg2+], pH, and [caffeine]. Flecainide applied to either the cytoplasmic or luminal sides of the membrane inhibited RyR2 by two distinct modes: 1) a fast block consisting of brief substate and closed events with a mean duration of ∼1 ms, and 2) a slow block consisting of closed events with a mean duration of ∼1 second. Both inhibition modes were alleviated by increasing cytoplasmic pH from 7.4 to 9.5 but were unaffected by luminal pH. The slow block was potentiated in RyR2 channels that had relatively low open probability, whereas the fast block was unaffected by RyR2 activation. These results show that these two modes are independent mechanisms for RyR2 inhibition, both having a cytoplasmic site of action. The slow mode is a closed-channel block, whereas the fast mode blocks RyR2 in the open state. At diastolic cytoplasmic [Ca2+] (100 nM), flecainide possesses an additional inhibitory mechanism that reduces RyR2 burst duration. Hence, multiple modes of action underlie RyR2 inhibition by flecainide. PMID:25274603

  3. Structural determinants of skeletal muscle ryanodine receptor gating.

    PubMed

    Ramachandran, Srinivas; Chakraborty, Asima; Xu, Le; Mei, Yingwu; Samsó, Montserrat; Dokholyan, Nikolay V; Meissner, Gerhard

    2013-03-01

    Ryanodine receptor type 1 (RyR1) releases Ca(2+) from intracellular stores upon nerve impulse to trigger skeletal muscle contraction. Effector binding at the cytoplasmic domain tightly controls gating of the pore domain of RyR1 to release Ca(2+). However, the molecular mechanism that links effector binding to channel gating is unknown due to lack of structural data. Here, we used a combination of computational and electrophysiological methods and cryo-EM densities to generate structural models of the open and closed states of RyR1. Using our structural models, we identified an interface between the pore-lining helix (Tyr-4912-Glu-4948) and a linker helix (Val-4830-Val-4841) that lies parallel to the cytoplasmic membrane leaflet. To test the hypothesis that this interface controls RyR1 gating, we designed mutations in the linker helix to stabilize either the open (V4830W and T4840W) or closed (H4832W and G4834W) state and validated them using single channel experiments. To further confirm this interface, we designed mutations in the pore-lining helix to stabilize the closed state (Q4947N, Q4947T, and Q4947S), which we also validated using single channel experiments. The channel conductance and selectivity of the mutations that we designed in the linker and pore-lining helices were indistinguishable from those of WT RyR1, demonstrating our ability to modulate RyR1 gating without affecting ion permeation. Our integrated computational and experimental approach significantly advances the understanding of the structure and function of an unusually large ion channel.

  4. Modulation of Cardiac Ryanodine Receptor Channels by Alkaline Earth Cations

    PubMed Central

    Diaz-Sylvester, Paula L.; Porta, Maura; Copello, Julio A.

    2011-01-01

    Cardiac ryanodine receptor (RyR2) function is modulated by Ca2+ and Mg2+. To better characterize Ca2+ and Mg2+ binding sites involved in RyR2 regulation, the effects of cytosolic and luminal earth alkaline divalent cations (M2+: Mg2+, Ca2+, Sr2+, Ba2+) were studied on RyR2 from pig ventricle reconstituted in bilayers. RyR2 were activated by M2+ binding to high affinity activating sites at the cytosolic channel surface, specific for Ca2+ or Sr2+. This activation was interfered by Mg2+ and Ba2+ acting at low affinity M2+-unspecific binding sites. When testing the effects of luminal M2+ as current carriers, all M2+ increased maximal RyR2 open probability (compared to Cs+), suggesting the existence of low affinity activating M2+-unspecific sites at the luminal surface. Responses to M2+ vary from channel to channel (heterogeneity). However, with luminal Ba2+or Mg2+, RyR2 were less sensitive to cytosolic Ca2+ and caffeine-mediated activation, openings were shorter and voltage-dependence was more marked (compared to RyR2 with luminal Ca2+or Sr2+). Kinetics of RyR2 with mixtures of luminal Ba2+/Ca2+ and additive action of luminal plus cytosolic Ba2+ or Mg2+ suggest luminal M2+ differentially act on luminal sites rather than accessing cytosolic sites through the pore. This suggests the presence of additional luminal activating Ca2+/Sr2+-specific sites, which stabilize high Po mode (less voltage-dependent) and increase RyR2 sensitivity to cytosolic Ca2+ activation. In summary, RyR2 luminal and cytosolic surfaces have at least two sets of M2+ binding sites (specific for Ca2+ and unspecific for Ca2+/Mg2+) that dynamically modulate channel activity and gating status, depending on SR voltage. PMID:22039534

  5. Mapping domains and mutations on the skeletal muscle ryanodine receptor channel.

    PubMed

    Hwang, Jean H; Zorzato, Francesco; Clarke, Nigel F; Treves, Susan

    2012-11-01

    The skeletal muscle ryanodine receptor isoform 1 (RyR1) is a calcium release channel involved in excitation-contraction coupling, the process whereby an action potential is translated to a cytoplasmic Ca(2+) signal that activates muscle contraction. Dominant and recessive mutations in RYR1 cause a range of muscle disorders, including malignant hyperthermia and several forms of congenital myopathies. Many aspects of disease pathogenesis in ryanodinopathies remain uncertain, particularly for those myopathies due to recessive mutations. A thorough understanding of the ryanodine receptor macromolecular complex and its interactions with proteins and small molecular modulators is an essential starting point from which to investigate disease mechanisms.

  6. Reversible block of the calcium release channel/ryanodine receptor by protamine, a heparin antidote.

    PubMed

    Koulen, P; Ehrlich, B E

    2000-07-01

    Channel activity of the calcium release channel from skeletal muscle, ryanodine receptor type 1, was measured in the presence and absence of protamine sulfate on the cytoplasmic side of the channel. Single-channel activity was measured after incorporating channels into planar lipid bilayers. Optimally and suboptimally calcium-activated calcium release channels were inactivated by the application of protamine to the cytoplasmic side of the channel. Recovery of channel activity was not observed while protamine was present. The addition of protamine bound to agarose beads did not change channel activity, implying that the mechanism of action involves an interaction with the ryanodine receptor rather than changes in the bulk calcium concentration of the medium. The block of channel activity by protamine could be reversed either by removal by perfusion with buffer or by the addition of heparin to the cytoplasmic side of the channel. Microinjection of protamine into differentiated C(2)C(12) mouse muscle cells prevented caffeine-induced intracellular calcium release. The results suggest that protamine acts on the ryanodine receptor in a similar but opposite manner from heparin and that protamine can be used as a potent, reversible inhibitor of ryanodine receptor activity.

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

    PubMed Central

    Baines, Katie Nicoll; Ferreira, Célia; Hopkins, Philip M.; Shaw, Marie-Anne; Hope, Ian A.

    2017-01-01

    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. PMID:28325813

  8. Phosphorylation of the purified cardiac ryanodine receptor by exogenous and endogenous protein kinases.

    PubMed Central

    Hohenegger, M; Suko, J

    1993-01-01

    The ryanodine receptor is the main Ca(2+)-release structure in skeletal and cardiac sarcoplasmic reticulum. In both tissues, phosphorylation of the ryanodine receptor has been proposed to be involved in the regulation of Ca2+ release. In the present study, we have examined the ability of the purified cardiac ryanodine receptor to serve as a substrate for phosphorylation by exogenously added catalytic subunit of the cyclic AMP (cAMP)-dependent protein kinase (PK-A), cyclic GMP (cGMP)-dependent protein kinase (PK-G), or calmodulin-dependent protein kinase (PK-CaM). A large amount of phosphate incorporation was observed for PK-CaM (938 +/- 48 pmol of Pi/mg of purified channel protein), whereas the level of phosphorylation was considerably lower with PK-A or PK-G (345 +/- 139 and 96 +/- 6 pmol/mg respectively). In addition, endogenous PK-CaM activity co-migrates with the ryanodine receptor through several steps of purification, suggesting a strong association of the two proteins. This endogenous PK-CaM activity is abolished by a PK-CaM-specific synthetic peptide inhibitor. Endogenous cAMP- and cGMP-dependent phosphorylation was not observed in the purified ryanodine-receptor preparation. Taken together, these observations imply that PK-CaM is the physiologically relevant protein kinase, capable of phosphorylating the channel protein to a minimum stoichiometry of 2 mol of Pi per mol of tetramer. Images Figure 2 Figure 3 Figure 4 PMID:8257417

  9. Levamisole and ryanodine receptors (II): An electrophysiological study in Ascaris suum

    PubMed Central

    Puttachary, Sreekanth; Robertson, Alan P.; Clark, Cheryl L.; Martin, Richard J.

    2010-01-01

    Resistance to antinematodal drugs like levamisole has increased and there is a need to understand what factors affect the responses to these anthelmintics. In our previous study, we examined the role of ryanodine receptors in muscle contraction pathways. Here we have examined interactions of levamisole receptors, ryanodine receptors (RyRs), the excitatory neuropeptide AF2, and coupling to electrophysiological responses. We examined the effects of a brief application of levamisole on Ascaris suum body muscle under current-clamp. The levamisole responses were characterized as an initial primary depolarization, followed by a slow secondary depolarizing response. We examined the effects of AF2 (KHEYLRFamide), 1 μM applied for 2 min. We found that AF2 potentiated the secondary response to levamisole and had no significant effect on the primary depolarization [1]. Further, the reversal potentials observed during the secondary response suggested that more than one ion was involved in producing this potential. AF2 potentiated the secondary response in the presence of 30 μM mecamylamine suggesting the effect was independent of levamisole sensitive acetylcholine receptors. The secondary response, potentiated by AF2, appeared to be dependent on cytoplasmic events triggered by the primary depolarization. Ion-substitution experiments showed that the AF2 potentiated secondary response was dependent on extracellular calcium and chloride suggesting a role for the calcium-activated anion channel. Caffeine mimicked the AF2 secondary response and 0.1 μM ryanodine inhibited it. 1.0 μM ryanodine increased spiking showing that it affected membrane excitability. A model is proposed showing ryanodine receptors mediating effects of AF2 on levamisole responses. PMID:20064567

  10. Ryanodine and inositol triphosphate receptors modulate facilitation and tetanic depression at the frog neuromuscular junction.

    PubMed

    Silveira, Priscila E; Lima, Ricardo F; Guimarães, Jennifer D S; Molgó, Jordi; Naves, Ligia A; Kushmerick, Christopher

    2015-10-01

    Short-term plasticity of synaptic function is an important physiological control of transmitter release. Short-term plasticity can be regulated by intracellular calcium released by ryanodine and inositol triphosphate (IP3) receptors, but the role of these receptors at the neuromuscular junction is understood incompletely. We measured short-term plasticity of evoked endplate potential (EPP) amplitudes from frog neuromuscular junctions treated with ryanodine, 2-aminoethoxydiphenylborane (2-APB), or 1-[6-[[(17β)-3-methoxyestra-1,3,5(10)-trien-17-yl]amino]hexyl]-1H-pyrrole-2,5-dione (U- 73122). Ryanodine decreases paired-pulse facilitation for intervals <20 ms and markedly decreases tetanic depression. Treatment with 2-APB reduces EPP amplitude, increases paired-pulse facilitation for intervals of <20 ms, and significantly reduces tetanic depression. U-73122 decreases EPP amplitude and decreases paired-pulse depression for intervals <20 ms. Ryanodine, IP3 receptors, and phospholipase C modulate short-term plasticity of transmitter release at the neuromuscular junction. These results suggest possible targets for improving the safety factor of neuromuscular transmission during repetitive activity of the neuromuscular junction. © 2015 Wiley Periodicals, Inc.

  11. Nicotinic acid-adenine dinucleotide phosphate activates the skeletal muscle ryanodine receptor.

    PubMed Central

    Hohenegger, Martin; Suko, Josef; Gscheidlinger, Regina; Drobny, Helmut; Zidar, Andreas

    2002-01-01

    Calcium is a universal second messenger. The temporal and spatial information that is encoded in Ca(2+)-transients drives processes as diverse as neurotransmitter secretion, axonal outgrowth, immune responses and muscle contraction. Ca(2+)-release from intracellular Ca(2+) stores can be triggered by diffusible second messengers like Ins P (3), cyclic ADP-ribose or nicotinic acid-adenine dinucleotide phosphate (NAADP). A target has not yet been identified for the latter messenger. In the present study we show that nanomolar concentrations of NAADP trigger Ca(2+)-release from skeletal muscle sarcoplasmic reticulum. This was due to a direct action on the Ca(2+)-release channel/ryanodine receptor type-1, since in single channel recordings, NAADP increased the open probability of the purified channel protein. The effects of NAADP on Ca(2+)-release and open probability of the ryanodine receptor occurred over a similar concentration range (EC(50) approximately 30 nM) and were specific because (i) they were blocked by Ruthenium Red and ryanodine, (ii) the precursor of NAADP, NADP, was ineffective at equimolar concentrations, (iii) NAADP did not affect the conductance and reversal potential of the ryanodine receptor. Finally, we also detected an ADP-ribosyl cyclase activity in the sarcoplasmic reticulum fraction of skeletal muscle. This enzyme was not only capable of synthesizing cyclic GDP-ribose but also NAADP, with an activity of 0.25 nmol/mg/min. Thus, we conclude that NAADP is generated in the vicinity of type 1 ryanodine receptor and leads to activation of this ion channel. PMID:12102654

  12. Amino acid residues 4425-4621 localized on the three-dimensional structure of the skeletal muscle ryanodine receptor.

    PubMed Central

    Benacquista, B L; Sharma, M R; Samsó, M; Zorzato, F; Treves, S; Wagenknecht, T

    2000-01-01

    We have localized a region contained within the sequence of amino acid residues 4425-4621 on the three-dimensional structure of the skeletal muscle ryanodine receptor (RyR). Mouse monoclonal antibodies raised against a peptide comprising these residues have been complexed with ryanodine receptors and imaged in the frozen-hydrated state by cryoelectron microscopy. These images, along with images of antibody-free ryanodine receptor, were used to compute two-dimensional averaged images and three-dimensional reconstructions. Two-dimensional averages of immunocomplexes in which the ryanodine receptor was in the fourfold symmetrical orientation disclosed four symmetrical regions of density located on the edges of the receptor's cytoplasmic assembly that were absent from control averages of receptor without added antibody. Three-dimensional reconstructions revealed the antibody-binding sites to be on the so-called handle domains of the ryanodine receptor's cytoplasmic assembly, near their junction with the transmembrane assembly. This study is the first to demonstrate epitope mapping on the three-dimensional structure of the ryanodine receptor. PMID:10692321

  13. Structural determinants for activation or inhibition of ryanodine receptors by basic residues in the dihydropyridine receptor II-III loop.

    PubMed Central

    Casarotto, M G; Green, D; Pace, S M; Curtis, S M; Dulhunty, A F

    2001-01-01

    The structures of peptide A, and six other 7-20 amino acid peptides corresponding to sequences in the A region (Thr671- Leu690) of the skeletal muscle dihydropyridine receptor II-III loop have been examined, and are correlated with the ability of the peptides to activate or inhibit skeletal ryanodine receptor calcium release channels. The peptides adopted either random coil or nascent helix-like structures, which depended upon the polarity of the terminal residues as well as the presence and ionisation state of two glutamate residues. Enhanced activation of Ca2+ release from sarcoplasmic reticulum, and activation of current flow through single ryanodine receptor channels (at -40 mV), was seen with peptides containing the basic residues 681Arg Lys Arg Arg Lys685, and was strongest when the residues were a part of an alpha-helix. Inhibition of channels (at +40 mV) was also seen with peptides containing the five positively charged residues, but was not enhanced in helical peptides. These results confirm the hypothesis that activation of ryanodine receptor channels by the II-III loop peptides requires both the basic residues and their participation in helical structure, and show for the first time that inhibition requires the basic residues, but is not structure-dependent. These findings imply that activation and inhibition result from peptide binding to separate sites on the ryanodine receptor. PMID:11371447

  14. Ryanodine receptors are involved in nuclear calcium oscillation in primary pancreatic {beta}-cells

    SciTech Connect

    Zheng, Ji; Chen, Zheng; Yin, Wenxuan; Miao, Lin; Zhou, Zhansong; Ji, Guangju

    2012-06-29

    Highlights: Black-Right-Pointing-Pointer We found that RyRs are expressed on the nuclear envelope in single primary pancreatic {beta}-cells and isolated nuclei. Black-Right-Pointing-Pointer We showed that the pattern of glucose-induced Ca{sup 2+} oscillation in the nucleus and cytosol was similar. Black-Right-Pointing-Pointer Our results demonstrate that ryanodine-sensitive Ca{sup 2+} stores exist and have function in the pancreatic {beta}-cell nucleus. -- Abstract: Ryanodine receptors (RyRs) are mainly located on the endoplasmic reticulum (ER) and play an important role in regulating glucose-induced cytosolic Ca{sup 2+} oscillation in pancreatic {beta}-cells. However, subcellular locations and functions of RyRs on other cell organelles such as nuclear envelope are not well understood. In order to investigate the role of RyRs in nuclear Ca{sup 2+} oscillation we designed and conducted experiments in intact primary pancreatic {beta}-cells. Immunocytochemistry was used to examine the expression of RYRs on the nuclear envelope. Confocal microscopy was used to evaluate the function of RYRs on the nuclear envelope. We found that RyRs are expressed on the nuclear envelope in single primary pancreatic {beta}-cells and isolated nuclei. Laser scanning confocal microscopy studies indicated that application of glucose to the cells co-incubated with Ca{sup 2+} indicator Fluo-4 AM and cell-permeable nuclear indicator Hoechst 33342 resulted in nuclear Ca{sup 2+} oscillation. The pattern of glucose-induced Ca{sup 2+} oscillation in the nucleus and cytosol was similar. The reduction of Ca{sup 2+} oscillation amplitude by ryanodine was much greater in the nucleus though both the cytosol and the nucleus Ca{sup 2+} amplitude decreased by ryanodine. Our results suggest that functional ryanodine receptors not only exist in endoplasmic reticulum but are also expressed in nuclear envelope of pancreatic {beta}-cells.

  15. Selenoprotein N is required for ryanodine receptor calcium release channel activity in human and zebrafish muscle.

    PubMed

    Jurynec, Michael J; Xia, Ruohong; Mackrill, John J; Gunther, Derrick; Crawford, Thomas; Flanigan, Kevin M; Abramson, Jonathan J; Howard, Michael T; Grunwald, David Jonah

    2008-08-26

    Mutations affecting the seemingly unrelated gene products, SepN1, a selenoprotein of unknown function, and RyR1, the major component of the ryanodine receptor intracellular calcium release channel, result in an overlapping spectrum of congenital myopathies. To identify the immediate developmental and molecular roles of SepN and RyR in vivo, loss-of-function effects were analyzed in the zebrafish embryo. These studies demonstrate the two proteins are required for the same cellular differentiation events and are needed for normal calcium fluxes in the embryo. SepN is physically associated with RyRs and functions as a modifier of the RyR channel. In the absence of SepN, ryanodine receptors from zebrafish embryos or human diseased muscle have altered biochemical properties and have lost their normal sensitivity to redox conditions, which likely accounts for why mutations affecting either factor lead to similar diseases.

  16. Pale, soft, exudative turkey--The role of ryanodine receptor variation in meat quality.

    PubMed

    Strasburg, G M; Chiang, W

    2009-07-01

    The poultry industry has made significant advances in growth rate, feed efficiency, and breast muscle yield through intensive breeding of turkeys. However, a meat quality problem known as pale, soft, exudative (PSE) meat presents the industry with a major challenge during periods of stress, such as the onset of a prolonged heat wave. The biochemical characteristics of PSE turkey are strikingly similar to those of PSE pork. Abnormally rapid postmortem metabolism, stimulated in part by high concentrations of calcium ions, may be one of the underlying factors associated with the incidence of PSE turkey. This presentation summarizes our studies on the avian ryanodine receptors and suggests that heat stress may alter the expression pattern of splice variants of ryanodine receptors, which, in turn, could affect postmortem calcium homeostasis.

  17. Analysis of human cultured myotubes responses mediated by ryanodine receptor 1.

    PubMed

    Kobayashi, M; Mukaida, K; Migita, T; Hamada, H; Kawamoto, M; Yuge, O

    2011-03-01

    Malignant hyperthermia is a life-threatening condition caused by autosomal dominant mutations in the ryanodine receptor type 1 gene. Identifying patients predisposed to malignant hyperthermia is done through the Ca-induced Ca release test in Japan. We examined the intracellular calcium concentration in human cultured muscle cells and compared the sensitivity of myotubes to ryanodine receptor type 1 activators based on the Ca-induced Ca release rate. We assessed the utility of this method as an identifying test for predisposition to malignant hyperthermia. Muscle specimens were obtained from 34 individuals undergoing the Ca-induced Ca release test. We cultured myotubes from residual material and monitored changes in intracellular calcium concentration after exposure to the ryanodine receptor type 1 activators caffeine, halothane and 4-chloro-m-cresol by measuring fura-2 fluorescence. We determined the half maximal effective concentrations (EC50) for the test compounds in each myotube and calculated cut-off points using receiver operating characteristic curves. Seventeen patients each were classified into the accelerated and non-accelerated groups based on their Ca-induced Ca release rate. The EC50 values for caffeine, halothane and 4-chloro-m-cresol of the accelerated group were significant lower than those of the non-accelerated group (P < 0.001, P < 0.001 and P < 0.001, respectively). The calculated cut-off points of EC50 values for caffeine, halothane and 4-CmC were 3.62 mM, 2.28 mM and 197 microM, respectively. An increased sensitivity to ryanodine receptor type 1 activators was seen in myotubes in the accelerated group. This functional test on human cultured myotubes indicates that the alteration of their intracellular Ca2+ homeostasis may identify the predisposition to malignant hyperthermia.

  18. The ryanodine receptor is expressed in human pancreatic acinar cells and contributes to acinar cell injury.

    PubMed

    Lewarchik, Christopher M; Orabi, Abrahim I; Jin, Shunqian; Wang, Dong; Muili, Kamaldeen A; Shah, Ahsan U; Eisses, John F; Malik, Adeel; Bottino, Rita; Jayaraman, Thottala; Husain, Sohail Z

    2014-09-01

    Physiological calcium (Ca(2+)) signals within the pancreatic acinar cell regulate enzyme secretion, whereas aberrant Ca(2+) signals are associated with acinar cell injury. We have previously identified the ryanodine receptor (RyR), a Ca(2+) release channel on the endoplasmic reticulum, as a modulator of these pathological signals. In the present study, we establish that the RyR is expressed in human acinar cells and mediates acinar cell injury. We obtained pancreatic tissue from cadaveric donors and identified isoforms of RyR1 and RyR2 by qPCR. Immunofluorescence staining of the pancreas showed that the RyR is localized to the basal region of the acinar cell. Furthermore, the presence of RyR was confirmed from isolated human acinar cells by tritiated ryanodine binding. To determine whether the RyR is functionally active, mouse or human acinar cells were loaded with the high-affinity Ca(2+) dye (Fluo-4 AM) and stimulated with taurolithocholic acid 3-sulfate (TLCS) (500 μM) or carbachol (1 mM). Ryanodine (100 μM) pretreatment reduced the magnitude of the Ca(2+) signal and the area under the curve. To determine the effect of RyR blockade on injury, human acinar cells were stimulated with pathological stimuli, the bile acid TLCS (500 μM) or the muscarinic agonist carbachol (1 mM) in the presence or absence of the RyR inhibitor ryanodine. Ryanodine (100 μM) caused an 81% and 47% reduction in acinar cell injury, respectively, as measured by lactate dehydrogenase leakage (P < 0.05). Taken together, these data establish that the RyR is expressed in human acinar cells and that it modulates acinar Ca(2+) signals and cell injury.

  19. Up-regulation of ryanodine receptor expression increases the calcium-induced calcium release and spontaneous calcium signals in cerebral arteries from hindlimb unloaded rats.

    PubMed

    Morel, Jean-Luc; Dabertrand, Fabrice; Porte, Yves; Prevot, Anne; Macrez, Nathalie

    2014-08-01

    Microgravity induces a redistribution of blood volume. Consequently, astronauts' body pressure is modified so that the upright blood pressure gradient is abolished, thereby inducing a modification in cerebral blood pressure. This effect is mimicked in the hindlimb unloaded rat model. After a duration of 8 days of unloading, Ca2+ signals activated by depolarization and inositol-1,4,5-trisphosphate intracellular release were increased in cerebral arteries. In the presence of ryanodine and thapsigargin, the depolarization-induced Ca2+ signals remained increased in hindlimb suspended animals, indicating that Ca2+ influx and Ca2+-induced Ca2+ release mechanism were both increased. Spontaneous Ca2+ waves and localized Ca2+ events were also investigated. Increases in both amplitude and frequency of spontaneous Ca2+ waves were measured in hindlimb suspension conditions. After pharmacological segregation of Ca2+ sparks and Ca2+ sparklets, their kinetic parameters were characterized. Hindlimb suspension induced an increase in the frequencies of both Ca2+ localized events, suggesting an increase of excitability. Labeling with bodipy compounds suggested that voltage-dependent Ca2+ channels and ryanodine receptor expressions were increased. Finally, the expression of the ryanodine receptor subtype 1 (RyR1) was increased in hindlimb unloading conditions. Taken together, these results suggest that RyR1 expression and voltage-dependent Ca2+ channels activity are the focal points of the regulation of Ca2+ signals activated by vasoconstriction in rat cerebral arteries with an increase of the voltage-dependent Ca2+ influx.

  20. Dihydropyridine receptors actively control gating of ryanodine receptors in resting mouse skeletal muscle fibres

    PubMed Central

    Robin, Gaëlle; Allard, Bruno

    2012-01-01

    Contraction of skeletal muscle is triggered by the release of Ca2+ from the sarcoplasmic reticulum (SR) in response to depolarization of the muscle membrane. Depolarization is known to elicit a conformational change of the dihydropyridine receptor (DHPR) in the tubular membrane that controls in a time- and voltage-dependent manner the opening of the ryanodine receptor (RyR), the SR Ca2+ release channel. At rest, it is assumed that RyRs are kept in a closed state imposed by the repressive action of DHPRs; however, a direct control of the RyR gating by the DHPR has up to now never been demonstrated in resting adult muscle. In this study, we monitored slow changes in SR Ca2+ content using the Ca2+ indicator fluo-5N loaded in the SR of voltage-clamped mouse muscle fibres. We first show that external Ca2+ removal induced a reversible SR Ca2+ efflux at −80 mV and prevented SR Ca2+ refilling following depolarization-evoked SR Ca2+ depletion. The dihydropyridine compound nifedipine induced similar effects. The rate of SR Ca2+ efflux was also shown to be controlled in a time- and voltage-dependent manner within a membrane potential range more negative than −50 mV. Finally, intracellular addition of ryanodine produced an irreversible SR Ca2+ efflux and kept the SR in a highly depleted state following depolarization-evoked SR Ca2+ depletion. The fact that resting SR Ca2+ efflux is modulated by conformational changes of DHPRs induced by external Ca2+, nifedipine and voltage demonstrates that DHPRs exert an active control on gating of RyRs in resting skeletal muscle. PMID:23006480

  1. Functional characterisation of the R2452W ryanodine receptor variant associated with malignant hyperthermia susceptibility.

    PubMed

    Roesl, Cornelia; Sato, Keisaku; Schiemann, Anja; Pollock, Neil; Stowell, Kathryn M

    2014-09-01

    Malignant hyperthermia (MH) is a pharmacogenetic disorder that manifests in susceptible individuals exposed to volatile anaesthetics. Over 400 variants in the ryanodine receptor 1 (RYR1) have been reported but relatively few have been definitively associated with susceptibility to MH. This is largely due to the technical challenges of demonstrating abnormal Ca(2+) release from the sarcoplasmic reticulum. This study focuses on the R2452W variant and its functional characterisation with the aim of classifying this variant as MH causative. HEK293 cells were transiently transfected with full-length human wildtype or R2452W mutant RYR1 cDNA. In addition, B-lymphoblastoid cells from blood and myoblasts propagated from in vitro contracture tests were extracted from patients positive for the R2452W variant. All cell lines generated were loaded with the ratiometric dye Fura-2 AM, stimulated with the RYR1-specific agonist 4-chloro-m-cresol and Ca(2+) release from the sarcoplasmic/endoplasmic reticulum was monitored by fluorescence emission. All cells expressing the RYR1 R2452W variant show a significantly higher Ca(2+) release in response to the agonist, 4-chloro-m-cresol, compared to cells expressing RYR1 WT. These results indicate that the R2452W variant results in a hypersensitive ryanodine receptor 1 and suggest that the R2452W variant in the ryanodine receptor 1 is likely to be causative of MH.

  2. Sexual Dimorphism in a Reciprocal Interaction of Ryanodine and IP3 Receptors in the Induction of Hyperalgesic Priming.

    PubMed

    Khomula, Eugen V; Ferrari, Luiz F; Araldi, Dionéia; Levine, Jon D

    2017-02-22

    Hyperalgesic priming, a model of pain chronification in the rat, is mediated by ryanodine receptor-dependent calcium release. Although ryanodine induces priming in both sexes, females are 5 orders of magnitude more sensitive, by an estrogen receptor α (EsRα)-dependent mechanism. An inositol 1,4,5-triphosphate (IP3) receptor inhibitor prevented the induction of priming by ryanodine. For IP3 induced priming, females were also more sensitive. IP3-induced priming was prevented by pretreatment with inhibitors of the sarcoendoplasmic reticulum calcium ATPase and ryanodine receptor. Antisense to EsRα prevented the induction of priming by low-dose IP3 in females. The induction of priming by an EsRα agonist was ryanodine receptor-dependent and prevented by the IP3 antagonist. Thus, an EsRα-dependent bidirectional interaction between endoplasmic reticulum IP3 and ryanodine receptor-mediated calcium signaling is present in the induction of hyperalgesic priming, in females. In cultured male DRG neurons, IP3 (100 μm) potentiated depolarization-induced transients produced by extracellular application of high-potassium solution (20 mm, K20), in nociceptors incubated with β-estradiol. This potentiation of depolarization-induced calcium transients was blocked by the IP3 antagonist, and not observed in the absence of IP3 IP3 potentiation was also blocked by ryanodine receptor antagonist. The application of ryanodine (2 nm), instead of IP3, also potentiated K20-induced calcium transients in the presence of β-estradiol, in an IP3 receptor-dependent manner. Our results point to an EsRα-dependent, reciprocal interaction between IP3 and ryanodine receptors that contributes to sex differences in hyperalgesic priming.SIGNIFICANCE STATEMENT The present study demonstrates a mechanism that plays a role in the marked sexual dimorphism observed in a model of the transition to chronic pain, hyperalgesic priming. This mechanism involves a reciprocal interaction between the endoplasmic

  3. Sexual Dimorphism in a Reciprocal Interaction of Ryanodine and IP3 Receptors in the Induction of Hyperalgesic Priming

    PubMed Central

    Ferrari, Luiz F.

    2017-01-01

    Hyperalgesic priming, a model of pain chronification in the rat, is mediated by ryanodine receptor-dependent calcium release. Although ryanodine induces priming in both sexes, females are 5 orders of magnitude more sensitive, by an estrogen receptor α (EsRα)-dependent mechanism. An inositol 1,4,5-triphosphate (IP3) receptor inhibitor prevented the induction of priming by ryanodine. For IP3 induced priming, females were also more sensitive. IP3-induced priming was prevented by pretreatment with inhibitors of the sarcoendoplasmic reticulum calcium ATPase and ryanodine receptor. Antisense to EsRα prevented the induction of priming by low-dose IP3 in females. The induction of priming by an EsRα agonist was ryanodine receptor-dependent and prevented by the IP3 antagonist. Thus, an EsRα-dependent bidirectional interaction between endoplasmic reticulum IP3 and ryanodine receptor-mediated calcium signaling is present in the induction of hyperalgesic priming, in females. In cultured male DRG neurons, IP3 (100 μm) potentiated depolarization-induced transients produced by extracellular application of high-potassium solution (20 mm, K20), in nociceptors incubated with β-estradiol. This potentiation of depolarization-induced calcium transients was blocked by the IP3 antagonist, and not observed in the absence of IP3. IP3 potentiation was also blocked by ryanodine receptor antagonist. The application of ryanodine (2 nm), instead of IP3, also potentiated K20-induced calcium transients in the presence of β-estradiol, in an IP3 receptor-dependent manner. Our results point to an EsRα-dependent, reciprocal interaction between IP3 and ryanodine receptors that contributes to sex differences in hyperalgesic priming. SIGNIFICANCE STATEMENT The present study demonstrates a mechanism that plays a role in the marked sexual dimorphism observed in a model of the transition to chronic pain, hyperalgesic priming. This mechanism involves a reciprocal interaction between the endoplasmic

  4. CCDI: a new ligand that modulates mammalian type 1 ryanodine receptor (RyR1)

    PubMed Central

    Tian, Chengju; Shao, Chun Hong; Padanilam, Christina; Ezell, Edward; Singh, Jaipaul; Kutty, Shelby; Bidasee, Keshore R

    2014-01-01

    Background and Purpose Ryanodine receptors (RyRs) are Ca2+-release channels on the sarco(endo)plasmic reticulum that modulate a wide array of physiological functions. Three RyR isoforms are present in cells: RyR1, RyR2 and RyR3. To date, there are no reports on ligands that modulate RyR in an isoform-selective manner. Such ligands are not only valuable research tools, but could serve as intermediates for development of therapeutics. Experimental approach Pyrrole-2-carboxylic acid and 1,3-dicyclohexylcarbodiimide were allowed to react in carbon tetrachloride for 24 h at low temperatures and pressures. The chemical structures of the two products isolated were elucidated using NMR spectrometry, mass spectrometry and elemental analyses. [3H]-ryanodine binding, lipid bilayer and time-lapsed confocal imaging were used to determine their effects on RyR isoforms. Key results The major product, 2-cyclohexyl-3-cyclohexylimino-2, 3, dihydro–pyrrolo[1,2-c]imidazol-1-one (CCDI) dose-dependently potentiated Ca2+-dependent binding of [3H]-ryanodine to RyR1, with no significant effects on [3H]-ryanodine binding to RyR2 or RyR3. CCDI also reversibly increased the open probability (Po) of RyR1 with minimal effects on RyR2 and RyR3. CCDI induced Ca2+ transients in C2C12 skeletal myotubes, but not in rat ventricular myocytes. This effect was blocked by pretreating cells with ryanodine. The minor product 2-cyclohexyl-pyrrolo[1,2-c]imidazole-1,3-dione had no effect on either [3H]-ryanodine binding or Po of RyR1, RyR2 and RyR3. Conclusions and implications A new ligand that preferentially modulates RyR1 was identified. In addition to being an important research tool, the pharmacophore of this small molecule could serve as a template for the synthesis of other isoform-selective modulators of RyRs. PMID:24819467

  5. Functional studies of RYR1 mutations in the skeletal muscle ryanodine receptor using human RYR1 complementary DNA.

    PubMed

    Sato, Keisaku; Pollock, Neil; Stowell, Kathryn M

    2010-06-01

    Malignant hyperthermia is associated with mutations within the gene encoding the skeletal muscle ryanodine receptor, the calcium channel that releases Ca from sarcoplasmic reticulum stores triggering muscle contraction, and other metabolic activities. More than 200 variants have been identified in the ryanodine receptor, but only some of these have been shown to functionally affect the calcium channel. To implement genetic testing for malignant hyperthermia, variants must be shown to alter the function of the channel. A number of different ex vivo methods can be used to demonstrate functionality, as long as cells from human patients can be obtained and cultured from at least two unrelated families. Because malignant hyperthermia is an uncommon disorder and many variants seem to be private, including the newly identified H4833Y mutation, these approaches are limited. The authors cloned the human skeletal muscle ryanodine receptor complementary DNA and expressed both normal and mutated forms in HEK-293 cells and carried out functional analysis using ryanodine binding assays in the presence of a specific agonist, 4-chloro-m-cresol, and the antagonist Mg. Transiently expressed human ryanodine receptor proteins colocalized with an endoplasmic reticulum marker in HEK-293 cells. Ryanodine binding assays confirmed that mutations causing malignant hyperthermia resulted in a hypersensitive channel, while those causing central core disease resulted in a hyposensitive channel. The functional assays validate recombinant human skeletal muscle ryanodine receptor for analysis of variants and add an additional mutation (H4833Y) to the repertoire of mutations that can be used for the genetic diagnosis of malignant hyperthermia.

  6. Carbonylation induces heterogeneity in cardiac ryanodine receptor function in diabetes mellitus.

    PubMed

    Shao, Chun Hong; Tian, Chengju; Ouyang, Shouqiang; Moore, Caronda J; Alomar, Fadhel; Nemet, Ina; D'Souza, Alicia; Nagai, Ryoji; Kutty, Shelby; Rozanski, George J; Ramanadham, Sasanka; Singh, Jaipaul; Bidasee, Keshore R

    2012-09-01

    Heart failure and arrhythmias occur at 3 to 5 times higher rates among individuals with diabetes mellitus, compared with age-matched, healthy individuals. Studies attribute these defects in part to alterations in the function of cardiac type 2 ryanodine receptors (RyR2s), the principal Ca(2+)-release channels on the internal sarcoplasmic reticulum (SR). To date, mechanisms underlying RyR2 dysregulation in diabetes remain poorly defined. A rat model of type 1 diabetes, in combination with echocardiography, in vivo and ex vivo hemodynamic studies, confocal microscopy, Western blotting, mass spectrometry, site-directed mutagenesis, and [(3)H]ryanodine binding, lipid bilayer, and transfection assays, was used to determine whether post-translational modification by reactive carbonyl species (RCS) represented a contributing cause. After 8 weeks of diabetes, spontaneous Ca(2+) release in ventricular myocytes increased ~5-fold. Evoked Ca(2+) release from the SR was nonuniform (dyssynchronous). Total RyR2 protein levels remained unchanged, but the ability to bind the Ca(2+)-dependent ligand [(3)H]ryanodine was significantly reduced. Western blotting and mass spectrometry revealed RCS adducts on select basic residues. Mutation of residues to delineate the physiochemical impact of carbonylation yielded channels with enhanced or reduced cytoplasmic Ca(2+) responsiveness. The prototype RCS methylglyoxal increased and then decreased the RyR2 open probability. Methylglyoxal also increased spontaneous Ca(2+) release and induced Ca(2+) waves in healthy myocytes. Treatment of diabetic rats with RCS scavengers normalized spontaneous and evoked Ca(2+) release from the SR, reduced carbonylation of RyR2s, and increased binding of [(3)H]ryanodine to RyR2s. From these data, we conclude that post-translational modification by RCS contributes to the heterogeneity in RyR2 activity that is seen in experimental diabetes.

  7. The Ca2+-release channel/ryanodine receptor is localized in junctional and corbular sarcoplasmic reticulum in cardiac muscle

    PubMed Central

    1993-01-01

    The subcellular distribution of the Ca(2+)-release channel/ryanodine receptor in adult rat papillary myofibers has been determined by immunofluorescence and immunoelectron microscopical studies using affinity purified antibodies against the ryanodine receptor. The receptor is confined to the sarcoplasmic reticulum (SR) where it is localized to interior and peripheral junctional SR and the corbular SR, but it is absent from the network SR where the SR-Ca(2+)-ATPase and phospholamban are densely distributed. Immunofluorescence labeling of sheep Purkinje fibers show that the ryanodine receptor is confined to discrete foci while the SR-Ca(2+)-ATPase is distributed in a continuous network-like structure present at the periphery as well as throughout interior regions of these myofibers. Because Purkinje fibers lack T- tubules, these results indicate that the ryanodine receptor is localized not only to the peripheral junctional SR but also to corbular SR densely distributed in interfibrillar spaces of the I-band regions. We have previously identified both corbular SR and junctional SR in cardiac muscle as potential Ca(2+)-storage/Ca(2+)-release sites by demonstrating that the Ca2+ binding protein calsequestrin and calcium are very densely distributed in these two specialized domains of cardiac SR in situ. The results presented here provide strong evidence in support of the hypothesis that corbular SR is indeed a site of Ca(2+)-induced Ca2+ release via the ryanodine receptor during excitation contraction coupling in cardiac muscle. Furthermore, these results indicate that the function of the cardiac Ca(2+)-release channel/ryanodine receptor is not confined to junctional complexes between SR and the sarcolemma. PMID:8381786

  8. Stressed out: the skeletal muscle ryanodine receptor as a target of stress

    PubMed Central

    Bellinger, Andrew M.; Mongillo, Marco; Marks, Andrew R.

    2008-01-01

    Over the past century, understanding the mechanisms underlying muscle fatigue and weakness has been the focus of much investigation. However, the dominant theory in the field, that lactic acidosis causes muscle fatigue, is unlikely to tell the whole story. Recently, dysregulation of sarcoplasmic reticulum (SR) Ca2+ release has been associated with impaired muscle function induced by a wide range of stressors, from dystrophy to heart failure to muscle fatigue. Here, we address current understandings of the altered regulation of SR Ca2+ release during chronic stress, focusing on the role of the SR Ca2+ release channel known as the type 1 ryanodine receptor. PMID:18246195

  9. The role of ryanodine receptor type 3 in a mouse model of Alzheimer disease

    PubMed Central

    Liu, Jie; Supnet, Charlene; Sun, Suya; Zhang, Hua; Good, Levi; Popugaeva, Elena; Bezprozvanny, Ilya

    2014-01-01

    Dysregulated endoplasmic reticulum (ER) calcium (Ca2+) signaling is reported to play an important role in Alzheimer disease (AD) pathogenesis. The role of ER Ca2+ release channels, the ryanodine receptors (RyanRs), has been extensively studied in AD models and RyanR expression and activity are upregulated in the brains of various familial AD (FAD) models. The objective of this study was to utilize a genetic approach to evaluate the importance of RyanR type 3 (RyanR3) in the context of AD pathology. PMID:24476841

  10. Effect of gadolinium on the ryanodine receptor/sarcoplasmic reticulum calcium release channel of skeletal muscle.

    PubMed

    Sárközi, Sándor; Szegedi, Csaba; Lukács, Balázs; Ronjat, Michel; Jóna, István

    2005-01-01

    The effect of gadolinium ions on the sarcoplasmic reticulum (SR) calcium release channel/ryanodine receptor (RyR1) was studied using heavy SR (HSR) vesicles and RyR1 isolated from rabbit fast twitch muscle. In the [(3)H]ryanodine binding assay, 5 microM Gd(3+) increased the K(d) of the [(3)H]ryanodine binding of the vesicles from 33.8 nM to 45.6 nM while B(max), referring to the binding capacity, was not affected significantly. In the presence of 18 nM[(3)H]ryanodine and 100 microM free Ca(2+), Gd(3+) inhibited the binding of the radiolabeled ryanodine with an apparent K(d) value of 14.7 microM and a Hill coefficient of 3.17. In (45)Ca(2+) experiments the time constant of (45)Ca(2+) efflux from HSR vesicles increased from 90.9 (+/- 11.1) ms to 187.7 (+/- 24.9) ms in the presence of 20 microM gadolinium. In single channel experiments gadolinium inhibited the channel activity from both the cytoplasmic (cis) (IC(50) = 5.65 +/- 0.33 microM, n(Hill) = 4.71) and the luminal (trans) side (IC(50) = 5.47 +/- 0.24 microM, n(Hill) = 4.31). The degree of inhibition on the cis side didn't show calcium dependency in the 100 microM to 1 mM Ca(2+) concentration range which indicates no competition with calcium on its regulatory binding sites. When Gd(3+) was applied at the trans side, EGTA was present at the cis side to prevent the binding of Gd(+3) to the cytoplasmic calcium binding regulatory sites of the RyR1 if Gd(3+) accidentally passed through the channel. The inhibition of the channel did not show any voltage dependence, which would be the case if Gd(3+) exerted its effect after getting to the cis side. Our results suggest the presence of inhibitory binding sites for Gd(3+) on both sides of the RyR1 with similar Hill coefficients and IC(50) values.

  11. Disease mutations in the ryanodine receptor N-terminal region couple to a mobile intersubunit interface.

    PubMed

    Kimlicka, Lynn; Lau, Kelvin; Tung, Ching-Chieh; Van Petegem, Filip

    2013-01-01

    Ryanodine receptors are large channels that release Ca(2+) from the endoplasmic and sarcoplasmic reticulum. Hundreds of RyR mutations can cause cardiac and skeletal muscle disorders, yet detailed mechanisms explaining their effects have been lacking. Here we compare pseudo-atomic models and propose that channel opening coincides with widening of a cytoplasmic vestibule formed by the N-terminal region, thus altering an interface targeted by 20 disease mutations. We solve crystal structures of several disease mutants that affect intrasubunit domain-domain interfaces. Mutations affecting intrasubunit ionic pairs alter relative domain orientations, and thus couple to surrounding interfaces. Buried disease mutations cause structural changes that also connect to the intersubunit contact area. These results suggest that the intersubunit contact region between N-terminal domains is a prime target for disease mutations, direct or indirect, and we present a model whereby ryanodine receptors and inositol-1,4,5-trisphosphate receptors are activated by altering domain arrangements in the N-terminal region.

  12. GABAA receptor subtype involvement in addictive behaviour.

    PubMed

    Stephens, D N; King, S L; Lambert, J J; Belelli, D; Duka, T

    2017-01-01

    GABAA receptors form the major class of inhibitory neurotransmitter receptors in the mammalian brain. This review sets out to summarize the evidence that variations in genes encoding GABAA receptor isoforms are associated with aspects of addictive behaviour in humans, while animal models of addictive behaviour also implicate certain subtypes of GABAA receptor. In addition to outlining the evidence for the involvement of specific subtypes in addiction, we summarize the particular contributions of these isoforms in control over the functioning of brain circuits, especially the mesolimbic system, and make a first attempt to bring together evidence from several fields to understanding potential involvement of GABAA receptor subtypes in addictive behaviour. While the weight of the published literature is on alcohol dependency, the underlying principles outlined are relevant across a number of different aspects of addictive behaviour. © 2016 John Wiley & Sons Ltd and International Behavioural and Neural Genetics Society.

  13. Involvement of ryanodine receptors in neurotrophin-induced hippocampal synaptic plasticity and spatial memory formation

    PubMed Central

    Adasme, Tatiana; Haeger, Paola; Paula-Lima, Andrea C.; Espinoza, Italo; Casas-Alarcón, M. Mercedes; Carrasco, M. Angélica; Hidalgo, Cecilia

    2011-01-01

    Ryanodine receptors (RyR) amplify activity-dependent calcium influx via calcium-induced calcium release. Calcium signals trigger postsynaptic pathways in hippocampal neurons that underlie synaptic plasticity, learning, and memory. Recent evidence supports a role of the RyR2 and RyR3 isoforms in these processes. Along with calcium signals, brain-derived neurotrophic factor (BDNF) is a key signaling molecule for hippocampal synaptic plasticity and spatial memory. Upon binding to specific TrkB receptors, BDNF initiates complex signaling pathways that modify synaptic structure and function. Here, we show that BDNF-induced remodeling of hippocampal dendritic spines required functional RyR. Additionally, incubation with BDNF enhanced the expression of RyR2, RyR3, and PKMζ, an atypical protein kinase C isoform with key roles in hippocampal memory consolidation. Consistent with their increased RyR protein content, BDNF-treated neurons generated larger RyR-mediated calcium signals than controls. Selective inhibition of RyR-mediated calcium release with inhibitory ryanodine concentrations prevented the PKMζ, RyR2, and RyR3 protein content enhancement induced by BDNF. Intrahippocampal injection of BDNF or training rats in a spatial memory task enhanced PKMζ, RyR2, RyR3, and BDNF hippocampal protein content, while injection of ryanodine at concentrations that stimulate RyR-mediated calcium release improved spatial memory learning and enhanced memory consolidation. We propose that RyR-generated calcium signals are key features of the complex neuronal plasticity processes induced by BDNF, which include increased expression of RyR2, RyR3, and PKMζ and the spine remodeling required for spatial memory formation. PMID:21282625

  14. Heterogeneous function of ryanodine receptors, but not IP3 receptors, in hamster cremaster muscle feed arteries and arterioles.

    PubMed

    Westcott, Erika B; Jackson, William F

    2011-05-01

    The roles played by ryanodine receptors (RyRs) and inositol 1,4,5-trisphosphate receptors (IP₃Rs) in vascular smooth muscle in the microcirculation remain unclear. Therefore, the function of both RyRs and IP₃Rs in Ca(²+) signals and myogenic tone in hamster cremaster muscle feed arteries and downstream arterioles were assessed using confocal imaging and pressure myography. Feed artery vascular smooth muscle displayed Ca(²+) sparks and Ca(²+) waves, which were inhibited by the RyR antagonists ryanodine (10 μM) or tetracaine (100 μM). Despite the inhibition of sparks and waves, ryanodine or tetracaine increased global intracellular Ca(²+) and constricted the arteries. The blockade of IP₃Rs with xestospongin D (5 μM) or 2-aminoethoxydiphenyl borate (100 μM) or the inhibition of phospholipase C using U-73122 (10 μM) also attenuated Ca(2+) waves without affecting Ca(²+) sparks. Importantly, the IP₃Rs and phospholipase C antagonists decreased global intracellular Ca(2+) and dilated the arteries. In contrast, cremaster arterioles displayed only Ca(²+) waves: Ca(²+) sparks were not observed, and neither ryanodine (10-50 μM) nor tetracaine (100 μM) affected either Ca(²+) signals or arteriolar tone despite the presence of functional RyRs as assessed by responses to the RyR agonist caffeine (10 mM). As in feed arteries, arteriolar Ca(²+) waves were attenuated by xestospongin D (5 μM), 2-aminoethoxydiphenyl borate (100 μM), and U-73122 (10 μM), accompanied by decreased global intracellular Ca(²+) and vasodilation. These findings highlight the contrasting roles played by RyRs and IP₃Rs in Ca(²+) signals and myogenic tone in feed arteries and demonstrate important differences in the function of RyRs between feed arteries and downstream arterioles.

  15. Heterogeneous function of ryanodine receptors, but not IP3 receptors, in hamster cremaster muscle feed arteries and arterioles

    PubMed Central

    Westcott, Erika B.

    2011-01-01

    The roles played by ryanodine receptors (RyRs) and inositol 1,4,5-trisphosphate receptors (IP3Rs) in vascular smooth muscle in the microcirculation remain unclear. Therefore, the function of both RyRs and IP3Rs in Ca2+ signals and myogenic tone in hamster cremaster muscle feed arteries and downstream arterioles were assessed using confocal imaging and pressure myography. Feed artery vascular smooth muscle displayed Ca2+ sparks and Ca2+ waves, which were inhibited by the RyR antagonists ryanodine (10 μM) or tetracaine (100 μM). Despite the inhibition of sparks and waves, ryanodine or tetracaine increased global intracellular Ca2+ and constricted the arteries. The blockade of IP3Rs with xestospongin D (5 μM) or 2-aminoethoxydiphenyl borate (100 μM) or the inhibition of phospholipase C using U-73122 (10 μM) also attenuated Ca2+ waves without affecting Ca2+ sparks. Importantly, the IP3Rs and phospholipase C antagonists decreased global intracellular Ca2+ and dilated the arteries. In contrast, cremaster arterioles displayed only Ca2+ waves: Ca2+ sparks were not observed, and neither ryanodine (10–50 μM) nor tetracaine (100 μM) affected either Ca2+ signals or arteriolar tone despite the presence of functional RyRs as assessed by responses to the RyR agonist caffeine (10 mM). As in feed arteries, arteriolar Ca2+ waves were attenuated by xestospongin D (5 μM), 2-aminoethoxydiphenyl borate (100 μM), and U-73122 (10 μM), accompanied by decreased global intracellular Ca2+ and vasodilation. These findings highlight the contrasting roles played by RyRs and IP3Rs in Ca2+ signals and myogenic tone in feed arteries and demonstrate important differences in the function of RyRs between feed arteries and downstream arterioles. PMID:21357503

  16. Mecp2 Mediates Experience-Dependent Transcriptional Upregulation of Ryanodine Receptor Type-3.

    PubMed

    Torres, Rodrigo F; Hidalgo, Cecilia; Kerr, Bredford

    2017-01-01

    Mecp2 is a DNA methylation reader that plays a critical role in experience-dependent plasticity. Increasing evidence supports a role for epigenetic modifications in activity-induced gene expression. Hence, candidate genes related to such phenomena are of great interest. Ryanodine receptors are intracellular calcium channels that contribute to hippocampal synaptic plasticity, dendritic spine remodeling, and participate in learning and memory processes. Here we exposed mice to the enriched environment (EE) paradigm, which through increased stimulation induces experience dependent-plasticity, to explore a role for methyl-cytosines, and Mecp2 in directing Ryanodine receptor 3 (Ryr3) transcriptional activity. EE induced a hippocampal-specific increase in the methylation of discrete cytosines located at a Ryr3 isoform promoter; chromatin immunoprecipitation experiments revealed that EE increased Mecp2 binding to this Ryr3 isoform promoter. Interestingly, the experimental paradigm induced robust Ryr3 upregulation, accompanied by miR132-dependent suppression of p250GAP, a pathway driving synaptogenesis. In contrast to WT mice, Mecp2-null mice showed diminished levels of Ryr3 and displayed impaired EE-induced Ryr3 upregulation, compromising miR132 dependent suppression of p250GAP and experience-dependent structural plasticity. Based on these results, we propose that Mecp2 acts as a transcriptional activator of Ryr3, contributing to experience-dependent plasticity.

  17. Mecp2 Mediates Experience-Dependent Transcriptional Upregulation of Ryanodine Receptor Type-3

    PubMed Central

    Torres, Rodrigo F.; Hidalgo, Cecilia; Kerr, Bredford

    2017-01-01

    Mecp2 is a DNA methylation reader that plays a critical role in experience-dependent plasticity. Increasing evidence supports a role for epigenetic modifications in activity-induced gene expression. Hence, candidate genes related to such phenomena are of great interest. Ryanodine receptors are intracellular calcium channels that contribute to hippocampal synaptic plasticity, dendritic spine remodeling, and participate in learning and memory processes. Here we exposed mice to the enriched environment (EE) paradigm, which through increased stimulation induces experience dependent-plasticity, to explore a role for methyl-cytosines, and Mecp2 in directing Ryanodine receptor 3 (Ryr3) transcriptional activity. EE induced a hippocampal-specific increase in the methylation of discrete cytosines located at a Ryr3 isoform promoter; chromatin immunoprecipitation experiments revealed that EE increased Mecp2 binding to this Ryr3 isoform promoter. Interestingly, the experimental paradigm induced robust Ryr3 upregulation, accompanied by miR132-dependent suppression of p250GAP, a pathway driving synaptogenesis. In contrast to WT mice, Mecp2-null mice showed diminished levels of Ryr3 and displayed impaired EE-induced Ryr3 upregulation, compromising miR132 dependent suppression of p250GAP and experience-dependent structural plasticity. Based on these results, we propose that Mecp2 acts as a transcriptional activator of Ryr3, contributing to experience-dependent plasticity. PMID:28659760

  18. Methanethiosulfonate ethylammonium block of amine currents through the ryanodine receptor reveals single pore architecture.

    PubMed

    Anyatonwu, Georgia I; Buck, Edmond D; Ehrlich, Barbara E

    2003-11-14

    The homotetrameric structure of the ryanodine-sensitive intracellular calcium (Ca2+) release channel (ryanodine receptor (RyR)) suggests that the four RyR subunits either combine to form a single pore or that each RyR subunit is an independently conducting pathway. Previously we showed that methanethiosulfonate ethylammonium (MTSEA+) covalently modifies the RyR to reduce current amplitudes in a time-dependent and stepwise manner. To ascertain the number of functionally conducting pores in the RyR, two approaches were combined: modification of the receptor by MTSEA+ and the use of different sized current carriers. Previous reports (Tinker, A., and Williams, A. J. (1993) J. Gen. Physiol. 102, 1107-1129) have shown that the organic cations methylamine, dimethylamine, ethylamine, and trimethylamine are permeant through the RyR but with reduced current amplitude depending upon the diameter of the respective amine. Experiments using the thiol reagent MTSEA+ to modify the channel protein showed that the current amplitudes decrease in steps leading to complete block of the channel when cesium (Cs+) is the current carrier. MTSEA+ modification decreased the number of channel substates as the diameter of the current carrier increased. Comparison of the degree of inhibition of MTSEA+-modified currents allows for differentiation between the two models for channel architecture. These results demonstrate that the conduction pathway for the RyR is comprised of a single central pore.

  19. Ryanodine receptors, calcium signaling and regulation of vascular tone in the cerebral parenchymal microcirculation

    PubMed Central

    Dabertrand, Fabrice; Nelson, Mark T.; Brayden, Joseph E.

    2012-01-01

    The cerebral blood supply is delivered by a surface network of pial arteries and arterioles from which arise (parenchymal) arterioles that penetrate into the cortex and terminate in a rich capillary bed. The critical regulation of cerebral blood flow, locally and globally, requires precise vasomotor regulation of the intracerebral microvasculature. This vascular region is anatomically unique as illustrated by the presence of astrocytic processes that envelope almost the entire basolateral surface of parenchymal arterioles. There are, moreover, notable functional differences between pial arteries and parenchymal arterioles. For example, in pial vascular smooth muscle cells (VSMCs), local calcium release events (“calcium sparks”) through ryanodine receptor (RyR) channels in sarcoplasmic reticulum membrane activate large conductance, calcium-sensitive potassium (BK) channels to modulate vascular diameter. In contrast, VSMCs in parenchymal arterioles express functional RyR and BK channels, but under physiological conditions these channels do not oppose pressure-induced vasoconstriction. Here we summarize the roles of ryanodine receptors in the parenchymal microvasculature under physiologic and pathologic conditions, and discuss their importance in the control of cerebral blood flow. PMID:23216877

  20. Ryanodine receptors are expressed in epidermal keratinocytes and associated with keratinocyte differentiation and epidermal permeability barrier homeostasis.

    PubMed

    Denda, Sumiko; Kumamoto, Junichi; Takei, Kentaro; Tsutsumi, Moe; Aoki, Hirofumi; Denda, Mitsuhiro

    2012-01-01

    Ryanodine receptors (RyRs) have an important role as calcium channels in the regulation of intracellular calcium levels in the nervous system and muscle. In the present study, we investigated the expression of RyR in human epidermis. Immunohistochemical studies and reverse transcription-PCR indicated the expression of RyR type 1, 2, and 3 proteins in epidermal keratinocytes. The expression level of each RyR subtype was higher in differentiating keratinocytes than in proliferative cells. We also demonstrated the functional expression of RyR by calcium imaging. In cultured human keratinocytes, application of the RyR agonist 4-chloro-m-cresol (CMC) induced elevation of the intracellular calcium concentration, and co-application of the RyR antagonist 1,1'-diheptyl-4,4'-bipyridinium dibromide (DHBP) blocked the elevation. Application of CMC accelerated keratinocyte differentiation in vitro. On the other hand, topical application of CMC after tape-stripping of hairless mouse skin delayed barrier recovery, whereas application of an RyR antagonist, dantrolene or DHBP, accelerated the barrier recovery. These results suggest that RyR expressed in epidermal keratinocytes is associated with both differentiation of keratinocytes and epidermal barrier homeostasis.

  1. Structure–function relationships of peptides forming the calcin family of ryanodine receptor ligands

    PubMed Central

    Xiao, Liang; Gurrola, Georgina B.; Zhang, Jing; Valdivia, Carmen R.; SanMartin, Mario; Zamudio, Fernando Z.; Zhang, Liming; Possani, Lourival D.

    2016-01-01

    Calcins are a novel family of scorpion peptides that bind with high affinity to ryanodine receptors (RyRs) and increase their activity by inducing subconductance states. Here, we provide a comprehensive analysis of the structure–function relationships of the eight calcins known to date, based on their primary sequence, three-dimensional modeling, and functional effects on skeletal RyRs (RyR1). Primary sequence alignment and evolutionary analysis show high similarity among all calcins (≥78.8% identity). Other common characteristics include an inhibitor cysteine knot (ICK) motif stabilized by three pairs of disulfide bridges and a dipole moment (DM) formed by positively charged residues clustering on one side of the molecule and neutral and negatively charged residues segregating on the opposite side. [3H]Ryanodine binding assays, used as an index of the open probability of RyRs, reveal that all eight calcins activate RyR1 dose-dependently with Kd values spanning approximately three orders of magnitude and in the following rank order: opicalcin1 > opicalcin2 > vejocalcin > hemicalcin > imperacalcin > hadrucalcin > maurocalcin >> urocalcin. All calcins significantly augment the bell-shaped [Ca2+]-[3H]ryanodine binding curve with variable effects on the affinity constants for Ca2+ activation and inactivation. In single channel recordings, calcins induce the appearance of a subconductance state in RyR1 that has a unique fractional value (∼20% to ∼60% of the full conductance state) but bears no relationship to binding affinity, DM, or capacity to stimulate Ca2+ release. Except for urocalcin, all calcins at 100 nM concentration stimulate Ca2+ release and deplete Ca2+ load from skeletal sarcoplasmic reticulum. The natural variation within the calcin family of peptides offers a diversified set of high-affinity ligands with the capacity to modulate RyRs with high dynamic range and potency. PMID:27114612

  2. Characterization of ryanodine receptor type 1 single channel activity using "on-nucleus" patch clamp.

    PubMed

    Wagner, Larry E; Groom, Linda A; Dirksen, Robert T; Yule, David I

    2014-08-01

    In this study, we provide the first description of the biophysical and pharmacological properties of ryanodine receptor type 1 (RyR1) expressed in a native membrane using the on-nucleus configuration of the patch clamp technique. A stable cell line expressing rabbit RyR1 was established (HEK-RyR1) using the FLP-in 293 cell system. In contrast to untransfected cells, RyR1 expression was readily demonstrated by immunoblotting and immunocytochemistry in HEK-RyR1 cells. In addition, the RyR1 agonists 4-CMC and caffeine activated Ca(2+) release that was inhibited by high concentrations of ryanodine. On nucleus patch clamp was performed in nuclei prepared from HEK-RyR1 cells. Raising the [Ca(2+)] in the patch pipette resulted in the appearance of a large conductance cation channel with well resolved kinetics and the absence of prominent subconductance states. Current versus voltage relationships were ohmic and revealed a chord conductance of ∼750pS or 450pS in symmetrical 250mM KCl or CsCl, respectively. The channel activity was markedly enhanced by caffeine and exposure to ryanodine resulted in the appearance of a subconductance state with a conductance ∼40% of the full channel opening with a Po near unity. In total, these properties are entirely consistent with RyR1 channel activity. Exposure of RyR1 channels to cyclic ADP ribose (cADPr), nicotinic acid adenine dinucleotide phosphate (NAADP) or dantrolene did not alter the single channel activity stimulated by Ca(2+), and thus, it is unlikely these molecules directly modulate RyR1 channel activity. In summary, we describe an experimental platform to monitor the single channel properties of RyR channels. We envision that this system will be influential in characterizing disease-associated RyR mutations and the molecular determinants of RyR channel modulation.

  3. Structure-function relationships of peptides forming the calcin family of ryanodine receptor ligands.

    PubMed

    Xiao, Liang; Gurrola, Georgina B; Zhang, Jing; Valdivia, Carmen R; SanMartin, Mario; Zamudio, Fernando Z; Zhang, Liming; Possani, Lourival D; Valdivia, Héctor H

    2016-05-01

    Calcins are a novel family of scorpion peptides that bind with high affinity to ryanodine receptors (RyRs) and increase their activity by inducing subconductance states. Here, we provide a comprehensive analysis of the structure-function relationships of the eight calcins known to date, based on their primary sequence, three-dimensional modeling, and functional effects on skeletal RyRs (RyR1). Primary sequence alignment and evolutionary analysis show high similarity among all calcins (≥78.8% identity). Other common characteristics include an inhibitor cysteine knot (ICK) motif stabilized by three pairs of disulfide bridges and a dipole moment (DM) formed by positively charged residues clustering on one side of the molecule and neutral and negatively charged residues segregating on the opposite side. [(3)H]Ryanodine binding assays, used as an index of the open probability of RyRs, reveal that all eight calcins activate RyR1 dose-dependently with Kd values spanning approximately three orders of magnitude and in the following rank order: opicalcin1 > opicalcin2 > vejocalcin > hemicalcin > imperacalcin > hadrucalcin > maurocalcin > urocalcin. All calcins significantly augment the bell-shaped [Ca(2+)]-[(3)H]ryanodine binding curve with variable effects on the affinity constants for Ca(2+) activation and inactivation. In single channel recordings, calcins induce the appearance of a subconductance state in RyR1 that has a unique fractional value (∼20% to ∼60% of the full conductance state) but bears no relationship to binding affinity, DM, or capacity to stimulate Ca(2+) release. Except for urocalcin, all calcins at 100 nM concentration stimulate Ca(2+) release and deplete Ca(2+) load from skeletal sarcoplasmic reticulum. The natural variation within the calcin family of peptides offers a diversified set of high-affinity ligands with the capacity to modulate RyRs with high dynamic range and potency.

  4. The mitochondrial ryanodine receptor in rat heart: a pharmaco-kinetic profile.

    PubMed

    Altschafl, Beth A; Beutner, Gisela; Sharma, Virendra K; Sheu, Shey-Shing; Valdivia, Héctor H

    2007-07-01

    A protein discovered within inner mitochondrial membranes (IMM), designated as the mitochondrial ryanodine receptor (mRyR), has been recognized recently as a modulator of Ca(2+) fluxes in mitochondria. The present study provides fundamental pharmacological and electrophysiological properties of this mRyR. Rat cardiac IMM fused to lipid bilayers revealed the presence of a mitochondrial channel with gating characteristics similar to those of classical sarcoplasmic reticulum RyR (SR-RyR), but a variety of other mitochondrial channels obstructed clean recordings. Mitochondrial vesicles were thus solubilized and subjected to sucrose sedimentation to obtain mRyR-enriched fractions. Reconstitution of sucrose-purified fractions into lipid bilayers yielded Cs(+)-conducting, Ca(2+)-sensitive, large conductance (500-800 pS) channels with signature properties of SR-RyRs. Cytosolic Ca(2+) increased the bursting frequency and mean open time of the channel. Micromolar concentrations of ryanodine induced the appearance of subconductance states or inhibited channel activity altogether, while Imperatoxin A (IpTx(a)), a specific activator of RyRs, reversibly induced the appearance of distinct subconductance states. Remarkably, the cardiac mRyR displayed a Ca(2+) dependence of [(3)H]ryanodine binding curve similar to skeletal RyR (RyR1), not cardiac RyR (RyR2). Overall, the mRyR displayed elemental attributes that are present in single channel lipid bilayer recordings of SR-RyRs, although some exquisite differences were also noted. These results therefore provide the first direct evidence that a unique RyR occurs in mitochondrial membranes.

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

    PubMed

    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.

  6. Direct association of the reticulon protein RTN1A with the ryanodine receptor 2 in neurons

    PubMed Central

    Kaya, Levent; Meissner, Barbara; Riedl, Maria Christine; Muik, Martin; Schwarzer, Christoph; Ferraguti, Francesco; Sarg, Bettina; Lindner, Herbert; Schweigreiter, Rüdiger; Knaus, Hans-Günther; Romanin, Christoph; Bandtlow, Christine E.

    2013-01-01

    RTN1A is a reticulon protein with predominant localization in the endoplasmic reticulum (ER). It was previously shown that RTN1A is expressed in neurons of the mammalian central nervous system but functional information remains sparse. To elucidate the neuronal function of RTN1A, we chose to focus our investigation on identifying possible novel binding partners specifically interacting with the unique N-terminus of RTN1A. Using a nonbiased approach involving GST pull-downs and MS analysis, we identified the intracellular calcium release channel ryanodine receptor 2 (RyR2) as a direct binding partner of RTN1A. The RyR2 binding site was localized to a highly conserved 150-amino acid residue region. RTN1A displays high preference for RyR2 binding in vitro and in vivo and both proteins colocalize in hippocampal neurons and Purkinje cells. Moreover, we demonstrate the precise subcellular localization of RTN1A in Purkinje cells and show that RTN1A inhibits RyR channels in [3H]ryanodine binding studies on brain synaptosomes. In a functional assay, RTN1A significantly reduced RyR2-mediated Ca2 + oscillations. Thus, RTN1A and RyR2 might act as functional partners in the regulation of cytosolic Ca2 + dynamics the in neurons. PMID:23454728

  7. Structural and functional characterization of ryanodine receptor-natrin toxin interaction.

    PubMed

    Zhou, Qiang; Wang, Qiong-Ling; Meng, Xing; Shu, Yuyan; Jiang, Tao; Wagenknecht, Terence; Yin, Chang-Cheng; Sui, Sen-Fang; Liu, Zheng

    2008-11-01

    Cysteine-rich secretory proteins (CRISPs) are widely distributed, and notably occur in the mammalian reproductive tract and in the salivary glands of venomous reptiles. Most CRISPs can inhibit ion channels, such as the cyclic nucleotide-gated ion channel, potassium channel, and calcium channel. Natrin is a CRISP that has been purified from snake venom. Its targets include the calcium-activated potassium channel, the voltage-gated potassium channel, and the calcium release channel/ryanodine receptor (RyR). Immunoprecipitation experiments showed that natrin binds specifically to type 1 RyR (RyR1) from skeletal muscle. Natrin was found to inhibit both the binding of ryanodine to RyR1, and the calcium-channel activity of RyR1. Cryo-electron microscopy and single-particle image reconstruction analysis revealed that natrin binds to the clamp domains of RyR1. Docking of the crystal structure of natrin into our cryo-electron microscopy density map of the RyR1 + natrin complex suggests that natrin inhibits RyR1 by stabilizing a domain-domain interaction, and that the cysteine-rich domain of natrin is crucial for binding. These findings help reveal how natrin toxin inhibits the RyR calcium release channel, and they allow us to posit a generalized mechanism that governs the interaction between CRISPs and ion channels.

  8. Dendritic differentiation of cerebellar Purkinje cells is promoted by ryanodine receptors expressed by Purkinje and granule cells.

    PubMed

    Ohashi, Ryo; Sakata, Shin-ichi; Naito, Asami; Hirashima, Naohide; Tanaka, Masahiko

    2014-04-01

    Cerebellar Purkinje cells have the most elaborate dendritic trees among neurons in the brain. We examined the roles of ryanodine receptor (RyR), an intracellular Ca(2+) release channel, in the dendrite formation of Purkinje cells using cerebellar cell cultures. In the cerebellum, Purkinje cells express RyR1 and RyR2, whereas granule cells express RyR2. When ryanodine (10 µM), a blocker of RyR, was added to the culture medium, the elongation and branching of Purkinje cell dendrites were markedly inhibited. When we transferred small interfering RNA (siRNA) against RyR1 into Purkinje cells using single-cell electroporation, dendritic branching but not elongation of the electroporated Purkinje cells was inhibited. On the other hand, transfection of RyR2 siRNA into granule cells also inhibited dendritic branching of Purkinje cells. Furthermore, ryanodine reduced the levels of brain-derived neurotrophic factor (BDNF) in the culture medium. The ryanodine-induced inhibition of dendritic differentiation was partially rescued when BDNF was exogenously added to the culture medium in addition to ryanodine. Overall, these results suggest that RyRs expressed by both Purkinje and granule cells play important roles in promoting the dendritic differentiation of Purkinje cells and that RyR2 expressed by granule cells is involved in the secretion of BDNF from granule cells.

  9. A rare genetic variant of the ryanodine receptor in a suspected malignant hyperthermia susceptible patient.

    PubMed

    MacKay, Emily Jane; Wilkerson, Carlos; Kraeva, Natalia; Rosenberg, Henry; Kennedy, Tara

    2016-09-01

    Malignant hyperthermia (MH) remains a diagnostic challenge. This case report describes the anesthetic management of a suspected intraoperative MH episode and the subsequent, genetic sequence analysis of 3 genes associated with MH. The results of the molecular genetic testing revealed heterozygosity for a rare variant, c.12553G>A (p.Ala4185Thr), in the RYR1 gene encoding the ryanodine receptor. Although the RYR1 gene has previously been implicated in the pathogenesis of MH, (1) this particular variant has only been reported in one other case of MH; (2) the role for diagnostic genetic testing in the diagnosis of MH will be examined. Copyright © 2016 Elsevier Inc. All rights reserved.

  10. Is ryanodine receptor phosphorylation key to the fight or flight response and heart failure?

    PubMed

    Eschenhagen, Thomas

    2010-12-01

    In situations of stress the heart beats faster and stronger. According to Marks and colleagues, this response is, to a large extent, the consequence of facilitated Ca²+ release from intracellular Ca²+ stores via ryanodine receptor 2 (RyR2), thought to be due to catecholamine-induced increases in RyR2 phosphorylation at serine 2808 (S2808). If catecholamine stimulation is sustained (for example, as occurs in heart failure), RyR2 becomes hyperphosphorylated and "leaky," leading to arrhythmias and other pathology. This "leaky RyR2 hypothesis" is highly controversial. In this issue of the JCI, Marks and colleagues report on two new mouse lines with mutations in S2808 that provide strong evidence supporting their theory. Moreover, the experiments revealed an influence of redox modifications of RyR2 that may account for some discrepancies in the field.

  11. Nitric oxide-induced calcium release via ryanodine receptors regulates neuronal function.

    PubMed

    Kakizawa, Sho; Yamazawa, Toshiko; Chen, Yili; Ito, Akihiro; Murayama, Takashi; Oyamada, Hideto; Kurebayashi, Nagomi; Sato, Osamu; Watanabe, Masahiko; Mori, Nozomu; Oguchi, Katsuji; Sakurai, Takashi; Takeshima, Hiroshi; Saito, Nobuhito; Iino, Masamitsu

    2012-01-18

    Mobilization of intracellular Ca(2+) stores regulates a multitude of cellular functions, but the role of intracellular Ca(2+) release via the ryanodine receptor (RyR) in the brain remains incompletely understood. We found that nitric oxide (NO) directly activates RyRs, which induce Ca(2+) release from intracellular stores of central neurons, and thereby promote prolonged Ca(2+) signalling in the brain. Reversible S-nitrosylation of type 1 RyR (RyR1) triggers this Ca(2+) release. NO-induced Ca(2+) release (NICR) is evoked by type 1 NO synthase-dependent NO production during neural firing, and is essential for cerebellar synaptic plasticity. NO production has also been implicated in pathological conditions including ischaemic brain injury, and our results suggest that NICR is involved in NO-induced neuronal cell death. These findings suggest that NICR via RyR1 plays a regulatory role in the physiological and pathophysiological functions of the brain.

  12. Novel targets for treating heart and muscle disease: stabilizing ryanodine receptors and preventing intracellular calcium leak.

    PubMed

    Lehnart, Stephan E

    2007-04-01

    Ryanodine receptors (RyRs) function as intracellular Ca(2+) release channels on the endoplasmic and sarcoplasmic reticulum membranes. In striated muscles, Ca(2+) release through RyRs controls muscle excitation-contraction coupling. RyR channel function is regulated by a cytoplasmic scaffold domain that forms a macromolecular signaling complex including calstabin (formerly known as FK506-binding protein), calmodulin, phosphodiesterase, kinase and phosphatase proteins. An increasing number of genetic and acquired diseases has been associated with intracellular Ca(2+) leak. In heart failure, for instance, the RyR complex becomes altered, resulting in chronic channel dysfunction and chronic sarcoplasmic reticulum Ca(2+) leak. Recently, the efficacy of novel Ca(2+) release channel-stabilizing drugs has been demonstrated in cardiac and skeletal muscle disease models.

  13. Frontrunners of T cell activation: Initial, localized Ca2+ signals mediated by NAADP and the type 1 ryanodine receptor.

    PubMed

    Wolf, Insa M A; Diercks, Björn-Philipp; Gattkowski, Ellen; Czarniak, Frederik; Kempski, Jan; Werner, René; Schetelig, Daniel; Mittrücker, Hans-Willi; Schumacher, Valéa; von Osten, Manuel; Lodygin, Dimitri; Flügel, Alexander; Fliegert, Ralf; Guse, Andreas H

    2015-10-13

    The activation of T cells is the fundamental on switch for the adaptive immune system. Ca(2+) signaling is essential for T cell activation and starts as initial, short-lived, localized Ca(2+) signals. The second messenger nicotinic acid adenine dinucleotide phosphate (NAADP) forms rapidly upon T cell activation and stimulates early Ca(2+) signaling. We developed a high-resolution imaging technique using multiple fluorescent Ca(2+) indicator dyes to characterize these early signaling events and investigate the channels involved in NAADP-dependent Ca(2+) signals. In the first seconds of activation of either primary murine T cells or human Jurkat cells with beads coated with an antibody against CD3, we detected Ca(2+) signals with diameters close to the limit of detection and that were close to the activation site at the plasma membrane. In Jurkat cells in which the ryanodine receptor (RyR) was knocked down or in primary T cells from RyR1(-/-) mice, either these early Ca(2+) signals were not detected or the number of signals was markedly reduced. Local Ca(2+) signals observed within 20 ms upon microinjection of Jurkat cells with NAADP were also sensitive to RyR knockdown. In contrast, TRPM2 (transient receptor potential channel, subtype melastatin 2), a potential NAADP target channel, was not required for the formation of initial Ca(2+) signals in primary T cells. Thus, through our high-resolution imaging method, we characterized early Ca(2+) release events in T cells and obtained evidence for the involvement of RyR and NAADP in such signals.

  14. Calcineurin upregulates local Ca(2+) signaling through ryanodine receptor-1 in airway smooth muscle cells.

    PubMed

    Savoia, Carlo P; Liu, Qing-Hua; Zheng, Yun-Min; Yadav, Vishal; Zhang, Zhen; Wu, Ling-Gang; Wang, Yong-Xiao

    2014-11-15

    Local Ca(2+) signals (Ca(2+) sparks) play an important role in multiple cellular functions in airway smooth muscle cells (ASMCs). Protein kinase Cϵ is known to downregulate ASMC Ca(2+) sparks and contraction; however, no complementary phosphatase has been shown to produce opposite effects. Here, we for the first time report that treatment with a specific calcineurin (CaN) autoinhibitory peptide (CAIP) to block CaN activity decreases, whereas application of nickel to activate CaN increases, Ca(2+) sparks in both the presence and absence of extracellular Ca(2+). Treatment with xestospogin-C to eliminate functional inositol 1,4,5-trisphosphate receptors does not prevent CAIP from inhibiting local Ca(2+) signaling. However, high ryanodine treatment almost completely blocks spark formation and prevents the nickel-mediated increase in sparks. Unlike CAIP, the protein phosphatase 2A inhibitor endothall has no effect. Local Ca(2+) signaling is lower in CaN catalytic subunit Aα gene knockout (CaN-Aα(-/-)) mouse ASMCs. The effects of CAIP and nickel are completely lost in CaN-Aα(-/-) ASMCs. Neither CAIP nor nickel produces an effect on Ca(2+) sparks in type 1 ryanodine receptor heterozygous knockout (RyR1(-/+)) mouse ASMCs. However, their effects are not altered in RyR2(-/+) or RyR3(-/-) mouse ASMCs. CaN inhibition decreases methacholine-induced contraction in isolated RyR1(+/+) but not RyR1(-/+) mouse tracheal rings. Supportively, muscarinic contractile responses are also reduced in CaN-Aα(-/+) mouse tracheal rings. Taken together, these results provide novel evidence that CaN regulates ASMC Ca(2+) sparks specifically through RyR1, which plays an important role in the control of Ca(2+) signaling and contraction in ASMCs.

  15. Calcineurin upregulates local Ca2+ signaling through ryanodine receptor-1 in airway smooth muscle cells

    PubMed Central

    Savoia, Carlo P.; Liu, Qing-Hua; Zheng, Yun-Min; Yadav, Vishal; Zhang, Zhen; Wu, Ling-Gang

    2014-01-01

    Local Ca2+ signals (Ca2+ sparks) play an important role in multiple cellular functions in airway smooth muscle cells (ASMCs). Protein kinase Cϵ is known to downregulate ASMC Ca2+ sparks and contraction; however, no complementary phosphatase has been shown to produce opposite effects. Here, we for the first time report that treatment with a specific calcineurin (CaN) autoinhibitory peptide (CAIP) to block CaN activity decreases, whereas application of nickel to activate CaN increases, Ca2+ sparks in both the presence and absence of extracellular Ca2+. Treatment with xestospogin-C to eliminate functional inositol 1,4,5-trisphosphate receptors does not prevent CAIP from inhibiting local Ca2+ signaling. However, high ryanodine treatment almost completely blocks spark formation and prevents the nickel-mediated increase in sparks. Unlike CAIP, the protein phosphatase 2A inhibitor endothall has no effect. Local Ca2+ signaling is lower in CaN catalytic subunit Aα gene knockout (CaN-Aα−/−) mouse ASMCs. The effects of CAIP and nickel are completely lost in CaN-Aα−/− ASMCs. Neither CAIP nor nickel produces an effect on Ca2+ sparks in type 1 ryanodine receptor heterozygous knockout (RyR1−/+) mouse ASMCs. However, their effects are not altered in RyR2−/+ or RyR3−/− mouse ASMCs. CaN inhibition decreases methacholine-induced contraction in isolated RyR1+/+ but not RyR1−/+ mouse tracheal rings. Supportively, muscarinic contractile responses are also reduced in CaN-Aα−/+ mouse tracheal rings. Taken together, these results provide novel evidence that CaN regulates ASMC Ca2+ sparks specifically through RyR1, which plays an important role in the control of Ca2+ signaling and contraction in ASMCs. PMID:25239916

  16. Mutational analysis of putative calcium binding motifs within the skeletal ryanodine receptor isoform, RyR1.

    PubMed

    Fessenden, James D; Feng, Wei; Pessah, Isaac N; Allen, P D

    2004-12-17

    The functional relevance of putative Ca(2+) binding motifs previously identified with Ca(2+) overlay binding analysis within the skeletal muscle ryanodine receptor isoform (RyR1) was examined using mutational analysis. EF hands between amino acid positions 4081 and 4092 (EF1) and 4116 and 4127 (EF2) were scrambled singly or in combination within the full-length rabbit RyR1 cDNA. These cDNAs were expressed in 1B5 RyR-deficient myotubes and channel function assessed using Ca(2+)-imaging techniques, [(3)H]ryanodine binding measurements, and single channel experiments. In intact myotubes, these mutations did not affect functional responses to either depolarization or RyR agonists (caffeine, 4-chloro-m-cresol) compared with wtRyR1. However, in [(3)H]ryanodine binding measurements, both Ca(2+) activation and inhibition of the EF1 mutant was significantly altered compared with wtRyR1. No high affinity [(3)H]ryanodine binding was observed in membranes expressing the EF2 mutation, although in single channel measurements, the EF2-disrupted channel could be activated by micromolar Ca(2+) concentrations. In addition, micromolar levels of ryanodine placed these channels into the classical half-conductance state, thus indicating that occupancy of high affinity ryanodine binding sites is not required for ryanodine-induced subconductance states in RyR1. Disruption of three additional putative RyR1 calcium binding motifs located between amino acid positions 4254 and 4265 (EF3), 4407 and 4418 (EF4), or 4490 and 4502 (EF5) either singly or in combination (EF3-5) did not affect functional responses in 1B5 myotubes except that the EC(50) for caffeine activation for the EF3 construct was significantly increased compared with wtRyR1. However, in [(3)H]ryanodine binding experiments, the Ca(2+)-dependent activation and inactivation of mutated RyRs containing EF3, EF4, or EF5 was unaffected when compared with wtRyR1.

  17. Antibody probe study of Ca2+ channel regulation by interdomain interaction within the ryanodine receptor.

    PubMed Central

    Kobayashi, Shigeki; Yamamoto, Takeshi; Parness, Jerome; Ikemoto, Noriaki

    2004-01-01

    N-terminal and central domains of ryanodine receptor 1 (RyR1), where many reported malignant hyperthermia (MH) mutations are localized, represent putative channel regulatory domains. Recent domain peptide (DP) probe studies led us to the hypothesis that these domains interact to stabilize the closed state of channel (zipping), while weakening of domain-domain interactions (unzipping) by mutation de-stabilizes the channel, making it leaky to Ca2+ or sensitive to the agonists of RyR1. As shown previously, DP1 (N-terminal domain peptide) and DP4 (central domain peptide) produced MH-like channel activation/sensitization effects, presumably by peptide binding to sites critical to stabilizing domain-domain interactions and resultant loss of conformational constraints. Here we report that polyclonal anti-DP1 and anti-DP4 antibodies also produce MH-like channel activation and sensitization effects as evidenced by about 4-fold enhancement of high affinity [3H]ryanodine binding to RyR1 and by a significant left-shift of the concentration-dependence of activation of sarcoplasmic reticulum Ca2+ release by polylysine. Fluorescence quenching experiments demonstrate that the accessibility of a DP4-directed, conformationally sensitive fluorescence probe linked to the RyR1 N-terminal domain is increased in the presence of domain-specific antibodies, consistent with the view that these antibodies produce unzipping of interacting domains that are of hindered accessibility to the surrounding aqueous environment. Our results suggest that domain-specific antibody binding induces a conformational change resulting in channel activation, and are consistent with the hypothesis that interacting N-terminal and central domains are intimately involved in the regulation of RyR1 channel function. PMID:15027895

  18. Dynamic regulation of ryanodine receptor type 1 (RyR1) channel activity by Homer 1.

    PubMed

    Feng, Wei; Tu, Jiancheng; Pouliquin, Pierre; Cabrales, Elaine; Shen, Xiaohua; Dulhunty, Angela; Worley, Paul F; Allen, Paul D; Pessah, Isaac N

    2008-03-01

    Homer, a family of scaffolding proteins originally identified in neurons, is also expressed in skeletal muscle. Previous studies showed that splice variants of Homer 1 (H1) amplify the gain of the ryanodine receptor type 1 (RyR1) channel complex. Using [3H]ryanodine ([3H]Ry) to probe the conformational state of RyR1, the actions of long- and short-forms of H1 are examined singly and in combination. At < or =200 nM, H1 long-forms (H1b or H1c possessing coiled-coil (CC) domains) and short-forms (H1a or H1EVH1 lacking CC domains) enhance specific [3H]Ry binding to RyR1. However, at a concentration > 200 nM, either H1 form completely inhibited [3H]Ry binding. Importantly, the combinations of H1c+H1EVH1, or H1b+H1a acted in an additive manner to enhance or inhibit [3H]Ry-binding activity. H1a and H1c individually or in combination produced the same dynamic pattern in regulating purified RyR1 channels reconstituted in planar lipid bilayers. In combination, their net action on RyR1 channels depends on total concentrations of H1. These data provide a mechanism by which constitutively and transiently expressed H1 forms can tightly regulate RyR1 channel activity in response to changing levels of expression and degradation of H1 proteins.

  19. Functional calcium release channel formed by the carboxyl-terminal portion of ryanodine receptor.

    PubMed Central

    Bhat, M B; Zhao, J; Takeshima, H; Ma, J

    1997-01-01

    The ryanodine receptor (RyR) is one of the key proteins involved in excitation-contraction (E-C) coupling in skeletal muscle, where it functions as a Ca2+ release channel in the sarcoplasmic reticulum (SR) membrane. RyR consists of a single polypeptide of approximately 560 kDa normally arranged in a homotetrameric structure, which contains a carboxyl (C)-terminal transmembrane domain and a large amino (N)-terminal cytoplasmic domain. To test whether the carboxyl-terminal portion of RyR is sufficient to form a Ca2+ release channel, we expressed the full-length (RyR-wt) and C-terminal (RyR-C, approximately 130 kDa) RyR proteins in a Chinese hamster ovary (CHO) cell line, and measured their Ca2+ release channel functions in planar lipid bilayer membranes. The single-channel properties of RyR-wt were found to be similar to those of RyR from skeletal muscle SR. The RyR-C protein forms a cation-selective channel that shares some of the channel properties with RyR-wt, including activation by cytoplasmic Ca2+ and regulation by ryanodine. Unlike RyR-wt, which exhibits a linear current-voltage relationship and inactivates at millimolar Ca2+, the channels formed by RyR-C display significant inward rectification and fail to close at high cytoplasmic Ca2+. Our results show that the C-terminal portion of RyR contains structures sufficient to form a functional Ca2+ release channel, but the N-terminal portion of RyR also affects the ion-conduction and calcium-dependent regulation of the Ca2+ release channel. Images FIGURE 1 FIGURE 2 FIGURE 3 FIGURE 4 PMID:9284301

  20. Characterization of hadrucalcin, a peptide from Hadrurus gertschi scorpion venom with pharmacological activity on ryanodine receptors

    PubMed Central

    Schwartz, Elisabeth F; Capes, E Michelle; Diego-García, Elia; Zamudio, Fernando Z; Fuentes, Oscar; Possani, Lourival D; Valdivia, Héctor H

    2009-01-01

    Background and purpose: Members of the calcin family, presently including imperatoxin A, maurocalcin, opicalcins and hemicalcin, are basic, 33-mer peptide activators of ryanodine receptors (RyRs), the calcium channels of the sarcoplasmic reticulum (SR) that provide the majority of calcium for muscle contraction. Here we describe hadrucalcin, a novel member of this family. Experimental approach: Hadrucalcin was isolated from the venom of Hadrurus gertschi. Amino acid sequence and mass were determined by Edman degradation and mass spectrometry respectively. A cDNA library was constructed to generate clones for DNA sequence determination. Biological activity of native toxin was confirmed with [3H]ryanodine binding, by using SR vesicles from cardiac and skeletal muscle, and with single skeletal (RyR1) and cardiac (RyR2) channels reconstituted in lipid bilayers. Hadrucalcin was applied to intact ventricular myocytes to investigate effects on calcium transients. The secondary structure of hadrucalcin was computer-modelled by using atomic coordinates from maurocalcin, a structurally similar peptide. Key results: Hadrucalcin is distinguished from previously described congeners by two additional amino acids in its primary sequence and the lack of prominent amphipathicity. Hadrucalcin activated RyRs with high affinity (EC50= 37 nmol·L−1), induced a long-lasting subconductance state on RyR1 and RyR2, and rapidly (lag time ∼2 s) penetrated ventricular cardiomyocytes, eliciting discharge of internal calcium stores and spontaneous contractions. Conclusions and implications: Hadrucalcin is a cell-permeant, powerful activator of RyRs, which has translational potential for targeted delivery of drugs to RyR as novel therapeutic intervention in arrhythmogenic disease. PMID:19389159

  1. Identification of angiotensin II receptor subtypes

    SciTech Connect

    Chiu, A.T.; Herblin, W.F.; McCall, D.E.; Ardecky, R.J.; Carini, D.J.; Duncia, J.V.; Pease, L.J.; Wong, P.C.; Wexler, R.R.; Johnson, A.L.; )

    1989-11-30

    We have demonstrated the existence of two distinct subtypes of the angiotensin II receptor in the rat adrenal gland using radioligand binding and tissue section autoradiography. The identification of the subtypes was made possible by the discovery of two structurally dissimilar, nonpeptide compounds, DuP 753 and EXP655, that show reciprocal selectivity for the two subtypes. In the rat adrenal cortex, DuP 753 inhibited 80% of the total AII binding with an IC50 value on the sensitive sites of 2 x 10(-8) M, while EXP655 displaced only 20%. In the rat adrenal medulla, EXP655 gave 90% inhibition of AII binding with an IC50 value of 3.0 x 10(-8) M, while DuP 753 was essentially inactive. The combination of the two compounds completely inhibited AII binding in both tissues.

  2. Intracellular Ca2+ release through ryanodine receptors contributes to AMPA receptor-mediated mitochondrial dysfunction and ER stress in oligodendrocytes

    PubMed Central

    Ruiz, A; Matute, C; Alberdi, E

    2010-01-01

    Overactivation of ionotropic glutamate receptors in oligodendrocytes induces cytosolic Ca2+ overload and excitotoxic death, a process that contributes to demyelination and multiple sclerosis. Excitotoxic insults cause well-characterized mitochondrial alterations and endoplasmic reticulum (ER) dysfunction, which is not fully understood. In this study, we analyzed the contribution of ER-Ca2+ release through ryanodine receptors (RyRs) and inositol triphosphate receptors (IP3Rs) to excitotoxicity in oligodendrocytes in vitro. First, we observed that oligodendrocytes express all previously characterized RyRs and IP3Rs. Blockade of Ca2+-induced Ca2+ release by TMB-8 following α-amino-3-hydroxyl-5-methyl-4-isoxazole-propionate (AMPA) receptor-mediated insults attenuated both oligodendrocyte death and cytosolic Ca2+ overload. In turn, RyR inhibition by ryanodine reduced as well the Ca2+ overload whereas IP3R inhibition was ineffective. Furthermore, AMPA-triggered mitochondrial membrane depolarization, oxidative stress and activation of caspase-3, which in all instances was diminished by RyR inhibition. In addition, we observed that AMPA induced an ER stress response as revealed by α subunit of the eukaryotic initiation factor 2α phosphorylation, overexpression of GRP chaperones and RyR-dependent cleavage of caspase-12. Finally, attenuating ER stress with salubrinal protected oligodendrocytes from AMPA excitotoxicity. Together, these results show that Ca2+ release through RyRs contributes to cytosolic Ca2+ overload, mitochondrial dysfunction, ER stress and cell death following AMPA receptor-mediated excitotoxicity in oligodendrocytes. PMID:21364659

  3. Clinical features and ryanodine receptor type 1 gene mutation analysis in a Chinese family with central core disease.

    PubMed

    Chang, Xingzhi; Jin, Yiwen; Zhao, Haijuan; Huang, Qionghui; Wang, Jingmin; Yuan, Yun; Han, Ying; Qin, Jiong

    2013-03-01

    Central core disease is a rare inherited neuromuscular disorder caused by mutations in ryanodine receptor type 1 gene. The clinical phenotype of the disease is highly variable. We report a Chinese pedigree with central core disease confirmed by the gene sequencing. All 3 patients in the family presented with mild proximal limb weakness. The serum level of creatine kinase was normal, and electromyography suggested myogenic changes. The histologic analysis of muscle biopsy showed identical central core lesions in almost all of the muscle fibers in the index case. Exon 90-106 in the C-terminal domain of the ryanodine receptor type 1 gene was amplified using polymerase chain reaction. One heterozygous missense mutation G14678A (Arg4893Gln) in exon 102 was identified in all 3 patients. This is the first report of a familial case of central core disease confirmed by molecular study in mainland China.

  4. Seventh Symposium on Subtypes of Musccarinic Receptors.

    DTIC Science & Technology

    1997-01-01

    grants GM07270, HL07312, GM07750 and NS07332. References 1. N.M. NATHANSON, The Muscarinic Receptors, J.A. Brown (ed), 419-454, The Humana Press...R.A., MALENKAR.C. and KAUTERJ.A. Physiol.Revs. 70 514-565 (1990). 6. NORTHR.A. Muscarinic receptor subtypes, J.H.Brown (ed), 341-373, Humana , Clifton...Giannella Dipartimento di Scienze Chimiche, and *Istituto di Farmacologia, Sezione di Anatomia Umana, Universita di Camerino, 62032 Camerino, Italy

  5. Functional Characterization of C-terminal Ryanodine Receptor 1 Variants Associated with Central Core Disease or Malignant Hyperthermia.

    PubMed

    Parker, Remai; Schiemann, Anja H; Langton, Elaine; Bulger, Terasa; Pollock, Neil; Bjorksten, Andrew; Gillies, Robyn; Hutchinson, David; Roxburgh, Richard; Stowell, Kathryn M

    2017-01-01

    Central core disease and malignant hyperthermia are human disorders of skeletal muscle resulting from aberrant Ca2+ handling. Most malignant hyperthermia and central core disease cases are associated with amino acid changes in the type 1 ryanodine receptor (RyR1), the skeletal muscle Ca2+-release channel. Malignant hyperthermia exhibits a gain-of-function phenotype, and central core disease results from loss of channel function. For a variant to be classified as pathogenic, functional studies must demonstrate a correlation with the pathophysiology of malignant hyperthermia or central core disease. We assessed the pathogenicity of four C-terminal variants of the ryanodine receptor using functional analysis. The variants were identified in families affected by either malignant hyperthermia or central core disease. Four variants were introduced separately into human cDNA encoding the skeletal muscle ryanodine receptor. Following transient expression in HEK-293T cells, functional studies were carried out using calcium release assays in response to an agonist. Two previously characterized variants and wild-type skeletal muscle ryanodine receptor were used as controls. The p.Met4640Ile variant associated with central core disease showed no difference in calcium release compared to wild-type. The p.Val4849Ile variant associated with malignant hyperthermia was more sensitive to agonist than wild-type but did not reach statistical significance and two variants (p.Phe4857Ser and p.Asp4918Asn) associated with central core disease were completely inactive. The p.Val4849Ile variant should be considered a risk factor for malignant hyperthermia, while the p.Phe4857Ser and p.Asp4918Asn variants should be classified as pathogenic for central core disease.

  6. Minding the Calcium Store: Ryanodine Receptor Activation as a Convergent Mechanism of PCB Toxicity

    PubMed Central

    Pessah, Isaac N.; Cherednichenko, Gennady; Lein, Pamela J.

    2009-01-01

    Chronic low level polychlorinated biphenyls (PCB) exposures remain a significant public health concern since results from epidemiological studies indicate PCB burden is associated with immune system dysfunction, cardiovascular disease, and impairment of the developing nervous system. Of these various adverse health effects, developmental neurotoxicity has emerged as a particularly vulnerable endpoint in PCB toxicity. Arguably the most pervasive biological effects of PCBs could be mediated by their ability to alter the spatial and temporal fidelity of Ca2+ signals through one or more receptor mediated processes. This review will focus on our current knowledge of the structure and function of ryanodine receptors (RyRs) in muscle and nerve cells and how PCBs and related non-coplanar structures alter these functions. The molecular and cellular mechanisms by which non-coplanar PCBs and related structures alter local and global Ca2+ signaling properties and the possible short and long-term consequences of these perturbations on neurodevelopment and neurodegeneration are reviewed. PMID:19931307

  7. Phosphorylation of the ryanodine receptor mediates the cardiac fight or flight response in mice.

    PubMed

    Shan, Jian; Kushnir, Alexander; Betzenhauser, Matthew J; Reiken, Steven; Li, Jingdong; Lehnart, Stephan E; Lindegger, Nicolas; Mongillo, Marco; Mohler, Peter J; Marks, Andrew R

    2010-12-01

    During the classic "fight-or-flight" stress response, sympathetic nervous system activation leads to catecholamine release, which increases heart rate and contractility, resulting in enhanced cardiac output. Catecholamines bind to β-adrenergic receptors, causing cAMP generation and activation of PKA, which phosphorylates multiple targets in cardiac muscle, including the cardiac ryanodine receptor/calcium release channel (RyR2) required for muscle contraction. PKA phosphorylation of RyR2 enhances channel activity by sensitizing the channel to cytosolic calcium (Ca²+). Here, we found that mice harboring RyR2 channels that cannot be PKA phosphorylated (referred to herein as RyR2-S2808A+/+ mice) exhibited blunted heart rate and cardiac contractile responses to catecholamines (isoproterenol). The isoproterenol-induced enhancement of ventricular myocyte Ca²+ transients and fractional shortening (contraction) and the spontaneous beating rate of sinoatrial nodal cells were all blunted in RyR2-S2808A+/+ mice. The blunted cardiac response to catecholamines in RyR2-S2808A+/+ mice resulted in impaired exercise capacity. RyR2-S2808A+/+ mice were protected against chronic catecholaminergic-induced cardiac dysfunction. These studies identify what we believe to be new roles for PKA phosphorylation of RyR2 in both the heart rate and contractile responses to acute catecholaminergic stimulation.

  8. Effect of ruthenium red, a ryanodine receptor antagonist in experimental diabetes induced vascular endothelial dysfunction and associated dementia in rats.

    PubMed

    Jain, Swati; Sharma, Bhupesh

    2016-10-01

    Diabetes mellitus is considered as a main risk factor for vascular dementia. In the past, we have reported the induction of vascular dementia by experimental diabetes. This study investigates the efficacy of a ruthenium red, a ryanodine receptor antagonist and pioglitazone in the pharmacological interdiction of pancreatectomy diabetes (PaD) induced vascular endothelial dysfunction and subsequent vascular dementia in rats. Attentional set shifting and Morris water-maze test were used for assessment of learning and memory. Vascular endothelial function, blood brain barrier permeability, serum glucose, serum nitrite/nitrate, oxidative stress (viz. aortic superoxide anion, brain thiobarbituric acid reactive species and brain glutathione), brain calcium and inflammation (myeloperoxidase) were also estimated. PaD rats have shown impairment of endothelial function, blood brain barrier permeability, learning and memory along with an increase in brain inflammation, oxidative stress and calcium. Administration of ruthenium red and pioglitazone has significantly attenuated PaD induced impairment of learning, memory, blood brain barrier permeability, endothelial function and biochemical parameters. It may be concluded that ruthenium red, a ryanodine receptor antagonist and pioglitazone, a PPAR-γ agonist may be considered as potent pharmacological agent for the management of PaD induced endothelial dysfunction and subsequent vascular dementia. Ryanodine receptor may be explored further for their possible benefits in vascular dementia.

  9. Is there something fishy about the regulation of the ryanodine receptor in the fish heart?

    PubMed

    Shiels, Holly A; Sitsapesan, Rebecca

    2015-12-01

    What is the topic of this review? Excitation-contraction coupling in fish hearts is maintained over a range of temperatures that would be cardioplegic to most mammals. Here, we review what is known about the fish cardiac ryanodine receptor, and consider how it may be regulated in a different manner from the mammalian cardiac isoforms of this channel. What advances does it highlight? We highlight how a better understanding of the basic gating and conducting properties of fish cardiac ryanodine receptors could provide considerable insight into mechanisms underlying sarcoplasmic reticulum calcium release in fish hearts and the role of the sarcoplasmic reticulum in the evolution of the heart. The fish cardiac sarcoplasmic reticulum (SR) holds large quantities of Ca(2+), but calcium-induced calcium release (CICR) is weak in these myocytes, and contraction and relaxation are largely determined by transsarcolemmal Ca(2+) flux. Many fish species live in a cold and seasonally variable thermal habitat, which could provide challenges to regulation of excitation-contraction coupling. Here, we focus on the cardiac SR Ca(2+)-release channel (RyR2) in fish and ask whether it may be regulated in a different manner from the mammalian RyR2. We review data indicating that fish cardiac RyR are present at lower density, are more spatially separated within the SR membrane and are less responsive to cytosolic Ca(2+) than mammalian RyR2 channels. All of these features would contribute to the weak CICR evident from functional studies. We also consider how CICR can be enhanced in fish myocytes following β-adrenergic stimulation and application of low levels of caffeine, and how acute and chronic temperature change may affect the gating properties of fish RyR2s. It is clear that a lack of insight into the fundamental gating and conductance properties of fish RyR2 channels is hindering our understanding of the role of the SR in fish cardiac excitation-contraction coupling. We conclude by

  10. Disease Mutations in the Ryanodine Receptor Central Region: Crystal Structures of a Phosphorylation Hot Spot Domain

    SciTech Connect

    Yuchi, Zhiguang; Lau, Kelvin; Van Petegem, Filip

    2015-02-09

    Ryanodine Receptors (RyRs) are huge Ca{sup 2+} release channels in the endoplasmic reticulum membrane and form targets for phosphorylation and disease mutations. We present crystal structures of a domain in three RyR isoforms, containing the Ser2843 (RyR1) and Ser2808/Ser2814 (RyR2) phosphorylation sites. The RyR1 domain is the target for 11 disease mutations. Several of these are clustered near the phosphorylation sites, suggesting that phosphorylation and disease mutations may affect the same interface. The L2867G mutation causes a drastic thermal destabilization and aggregation at room temperature. Crystal structures for other disease mutants show that they affect surface properties and intradomain salt bridges. In vitro phosphorylation experiments show that up to five residues in one long loop of RyR2 can be phosphorylated by PKA or CaMKII. Docking into cryo-electron microscopy maps suggests a putative location in the clamp region, implying that mutations and phosphorylation may affect the allosteric motions within this area.

  11. Single-Particle Cryo-EM of the Ryanodine Receptor Channel in an Aqueous Environment

    PubMed Central

    Baker, Mariah R.; Fan, Guizhen

    2015-01-01

    Ryanodine receptors (RyRs) are tetrameric ligand-gated Ca2+ release channels that are responsible for the increase of cytosolic Ca2+ concentration leading to muscle contraction. Our current understanding of RyR channel gating and regulation is greatly limited due to the lack of a high-resolution structure of the channel protein. The enormous size and unwieldy shape of Ca2+ release channels make X-ray or NMR methods difficult to apply for high-resolution structural analysis of the full-length functional channel. Single-particle electron cryo-microscopy (cryo-EM) is one of the only effective techniques for the study of such a large integral membrane protein and its molecular interactions. Despite recent developments in cryo-EM technologies and break-through single-particle cryo-EM studies of ion channels, cryospecimen preparation, particularly the presence of detergent in the buffer, remains the main impediment to obtaining atomic-resolution structures of ion channels and a multitude of other integral membrane protein complexes. In this review we will discuss properties of several detergents that have been successfully utilized in cryo-EM studies of ion channels and the emergence of the detergent alternative amphipol to stabilize ion channels for structure-function characterization. Future structural studies of challenging specimen like ion channels are likely to be facilitated by cryo-EM amenable detergents or alternative surfactants. PMID:26913144

  12. Single-particle cryo-EM of the ryanodine receptor channel in an aqueous environment

    PubMed Central

    Baker, Mariah R.; Fan, Guizhen; Serysheva, Irina I.

    2015-01-01

    Ryanodine receptors (RyRs) are tetrameric ligand-gated Ca2+ release channels that are responsible for the increase of cytosolic Ca2+ concentration leading to muscle contraction. Our current understanding of RyR channel gating and regulation is greatly limited due to the lack of a high-resolution structure of the channel protein. The enormous size and unwieldy shape of Ca2+ release channels make X-ray or NMR methods difficult to apply for high-resolution structural analysis of the full-length functional channel. Single-particle electron cryo-microscopy (cryo-EM) is one of the only effective techniques for the study of such a large integral membrane protein and its molecular interactions. Despite recent developments in cryo-EM technologies and break-through single-particle cryo-EM studies of ion channels, cryospecimen preparation, particularly the presence of detergent in the buffer, remains the main impediment to obtaining atomic-resolution structures of ion channels and a multitude of other integral membrane protein complexes. In this review we will discuss properties of several detergents that have been successfully utilized in cryo-EM studies of ion channels and the emergence of the detergent alternative amphipol to stabilize ion channels for structure-function characterization. Future structural studies of challenging specimen like ion channels are likely to be facilitated by cryo-EM amenable detergents or alternative surfactants. PMID:25844145

  13. Single Ryanodine Receptor Channel Basis of Caffeine's Action on Ca2+ Sparks

    PubMed Central

    Porta, Maura; Zima, Aleksey V.; Nani, Alma; Diaz-Sylvester, Paula L.; Copello, Julio A.; Ramos-Franco, Josefina; Blatter, Lothar A.; Fill, Michael

    2011-01-01

    Caffeine (1, 3, 7-trimethylxanthine) is a widely used pharmacological agonist of the cardiac ryanodine receptor (RyR2) Ca2+ release channel. It is also a well-known stimulant that can produce adverse side effects, including arrhythmias. Here, the action of caffeine on single RyR2 channels in bilayers and Ca2+ sparks in permeabilized ventricular cardiomyocytes is defined. Single RyR2 caffeine activation depended on the free Ca2+ level on both sides of the channel. Cytosolic Ca2+ enhanced RyR2 caffeine affinity, whereas luminal Ca2+ essentially scaled maximal caffeine activation. Caffeine activated single RyR2 channels in diastolic quasi-cell-like solutions (cytosolic MgATP, pCa 7) with an EC50 of 9.0 ± 0.4 mM. Low-dose caffeine (0.15 mM) increased Ca2+ spark frequency ∼75% and single RyR2 opening frequency ∼150%. This implies that not all spontaneous RyR2 openings during diastole are associated with Ca2+ sparks. Assuming that only the longest openings evoke sparks, our data suggest that a spark may result only when a spontaneous single RyR2 opening lasts >6 ms. PMID:21320437

  14. Expression and localization of ryanodine receptors in the frog semicircular canal.

    PubMed

    Perin, Paola; Botta, Laura; Tritto, Simona; Laforenza, Umberto

    2012-01-01

    Several experiments suggest an important role for store-released Ca²⁺ in hair cell organs: drugs targeting IP₃ and ryanodine (RyRs) receptors affect release from hair cells, and stores are thought to be involved in vesicle recycling at ribbon synapses. In this work we investigated the semicircular canal distribution of RyRs by immunofluorescence, using slice preparations of the sensory epithelium (to distinguish cell types) and flat mounts of the simpler nonsensory regions. RyRs were present in hair cells, mostly in supranuclear spots, but not in supporting cells; as regards nonsensory regions, they were also localized in dark cells and cells from the ductus. No labeling was found in nerve terminals, although nerve branches could be observed in proximity to hair cell RyR spots. The differential expression of RyR isoforms was studied by RT-PCR and immunoblotting, showing the presence of RyRα in both ampulla and canal arm and RyRβ in the ampulla only.

  15. Structural mapping of divergent regions in the type 1 ryanodine receptor using fluorescence resonance energy transfer.

    PubMed

    Mahalingam, Mohana; Girgenrath, Tanya; Svensson, Bengt; Thomas, David D; Cornea, Razvan L; Fessenden, James D

    2014-09-02

    Ryanodine receptors (RyRs) release Ca(2+) to initiate striated muscle contraction. Three highly divergent regions (DRs) in the RyR protein sequence (DR1, DR2, and DR3) may confer isoform-specific functional properties to the RyRs. We used cell-based fluorescence resonance energy transfer (FRET) measurements to localize these DRs to the cryoelectron microscopic (cryo-EM) map of the skeletal muscle RyR isoform (RyR1). FRET donors were targeted to RyR1 using five different FKBP12.6 variants labeled with Alexa Fluor 488. FRET was then measured to the FRET acceptors, Cy3NTA or Cy5NTA, targeted to decahistidine tags introduced within the DRs. DR2 and DR3 were localized to separate positions within the "clamp" region of the RyR1 cryo-EM map, which is presumed to interface with Cav1.1. DR1 was localized to the "handle" region, near the regulatory calmodulin-binding site on the RyR. These localizations provide insights into the roles of DRs in RyR allosteric regulation during excitation contraction coupling. Copyright © 2014 Elsevier Ltd. All rights reserved.

  16. Structural mapping of divergent regions in the type 1 ryanodine receptor using fluorescence resonance energy transfer

    PubMed Central

    Mahalingam, Mohana; Girgenrath, Tanya; Svensson, Bengt; Thomas, David D.; Cornea, Razvan L.; Fessenden, James D.

    2014-01-01

    Summary Ryanodine receptors (RyR) release Ca2+ to initiate striated muscle contraction. Three highly divergent regions in the RyR protein sequence (DR1, DR2, DR3) are proposed to confer isoform-specific functional properties to the RyRs. We used cell-based fluorescence resonance energy transfer (FRET) measurements to localize these DRs to the cryo-electron microscopic (EM) map of the skeletal muscle RyR isoform (RyR1). FRET donors were targeted to RyR1 using five different FKBP12.6 variants labeled with Alexa Fluor 488. FRET was then measured to Cy3NTA or Cy5NTA, FRET acceptors targeted to decahistidine tags introduced within the DRs. DR2 and DR3 were localized to separate positions within the “clamp” region of the RyR1 cryo-EM map, which is presumed to interface with Cav1.1. DR1 was localized to the “handle” region, near the regulatory calmodulin binding site on the RyR. These localizations provide new insights into the roles of DRs in RyR allosteric regulation during excitation-contraction coupling. PMID:25132084

  17. Reinterpreting the Anomalous Mole Fraction Effect: The Ryanodine Receptor Case Study

    PubMed Central

    Gillespie, Dirk; Giri, Janhavi; Fill, Michael

    2009-01-01

    Abstract The origin of the anomalous mole fraction effect (AMFE) in calcium channels is explored with a model of the ryanodine receptor. This model predicted and experiments verified new AMFEs in the cardiac isoform. In mole fraction experiments, conductance is measured in mixtures of ion species X and Y as their relative amounts (mole fractions) vary. This curve can have a minimum (an AMFE). The traditional interpretation of the AMFE is that multiple interacting ions move through the pore in a single file. Mole fraction curves without minima (no AMFEs) are generally interpreted as X displacing Y from the pore in a proportion larger than its bath mole fraction (preferential selectivity). We find that the AMFE is also caused by preferential selectivity of X over Y, if X and Y have similar conductances. This is a prediction applicable to any channel and provides a fundamentally different explanation of the AMFE that does not require single filing or multiple occupancy: preferential selectivity causes the resistances to current flow in the baths, channel vestibules, and selectivity filter to change differently with mole fraction, and produce the AMFE. PMID:19843453

  18. Response to caffeine and ryanodine receptor isoforms in mouse skeletal muscles.

    PubMed

    Rossi, R; Bottinelli, R; Sorrentino, V; Reggiani, C

    2001-08-01

    The response to caffeine was studied in mouse muscles [diaphragm, soleus, and extensor digitorum longus (EDL)] with different ryanodine receptor isoform (RyR1, RyR3) composition and in single permeabilized muscle fibers dissected from diaphragm of wild-type (WT) and RyR3-deficient (RyR3-/-) mice at 1, 15, 30, and 60 postnatal days (PND). The caffeine response decreased during development, and, in adult mice, was greater in diaphragm, lower in EDL, and intermediate in soleus. This suggests a direct relation between response to caffeine and RyR3 expression. The lack of RyR3 reduced caffeine response in young, but not in adult mice, and did not abolish the age-dependent variation and the intermuscle differences. In diaphragm single fibers, the response to caffeine increased during development and was reduced in fibers lacking RyR3 both at 15 and 60 PND. A population of fibers highly responsive to caffeine was present in adult WT and disappeared in RyR3-/-. The results confirm the contribution of RyR3 to calcium release for contractile response and clarify the contribution of RyR3 to developmental changes and intermuscle differences.

  19. Hypernitrosylated ryanodine receptor/calcium release channels are leaky in dystrophic muscle

    PubMed Central

    Bellinger, Andrew M.; Reiken, Steven; Carlson, Christian; Mongillo, Marco; Liu, Xiaoping; Rothman, Lisa; Matecki, Stefan; Lacampagne, Alain; Marks, Andrew R.

    2010-01-01

    Duchenne muscular dystrophy (DMD) is characterized by progressive muscle weakness and early death resulting from dystrophin deficiency. Loss of dystrophin results in disruption of a large dystrophin glycoprotein complex (DGC) leading to pathologic calcium (Ca2+)-dependent signals that damage muscle cells 1–5. We have identified a structural and functional defect in the sarcoplasmic reticulum (SR) Ca2+ release channel/ryanodine receptor (RyR1) in the mdx mouse model of muscular dystrophy that may contribute to altered Ca2+ homeostasis in dystrophic muscles. RyR1 isolated from mdx skeletal muscle exhibited an age-dependent increase in S-nitrosylation coincident with dystrophic changes in the muscle. RyR1 S-nitrosylation depleted the channel complex of FKBP12 (or “calstabin1” for calcium channel stabilizing binding protein) resulting in “leaky” channels. Preventing calstabin1 depletion from RyR1 using S107, a compound that binds to the RyR1 channel and enhances the binding affinity of calstabin1 to the nitrosylated channel, inhibited SR Ca2+ leak, reduced biochemical and histologic evidence of muscle damage, improved muscle function and increased exercise performance in mdx mice. Thus, SR Ca2+ leak via RyR1 due to S-nitrosylation of the channel and calstabin1 depletion likely contributes to muscle weakness in muscular dystrophy and preventing the RyR1-mediated SR Ca2+ leak may provide a novel therapeutic approach. PMID:19198614

  20. Cardiac Ryanodine Receptor (Ryr2)-mediated Calcium Signals Specifically Promote Glucose Oxidation via Pyruvate Dehydrogenase.

    PubMed

    Bround, Michael J; Wambolt, Rich; Cen, Haoning; Asghari, Parisa; Albu, Razvan F; Han, Jun; McAfee, Donald; Pourrier, Marc; Scott, Nichollas E; Bohunek, Lubos; Kulpa, Jerzy E; Chen, S R Wayne; Fedida, David; Brownsey, Roger W; Borchers, Christoph H; Foster, Leonard J; Mayor, Thibault; Moore, Edwin D W; Allard, Michael F; Johnson, James D

    2016-11-04

    Cardiac ryanodine receptor (Ryr2) Ca(2+) release channels and cellular metabolism are both disrupted in heart disease. Recently, we demonstrated that total loss of Ryr2 leads to cardiomyocyte contractile dysfunction, arrhythmia, and reduced heart rate. Acute total Ryr2 ablation also impaired metabolism, but it was not clear whether this was a cause or consequence of heart failure. Previous in vitro studies revealed that Ca(2+) flux into the mitochondria helps pace oxidative metabolism, but there is limited in vivo evidence supporting this concept. Here, we studied heart-specific, inducible Ryr2 haploinsufficient (cRyr2Δ50) mice with a stable 50% reduction in Ryr2 protein. This manipulation decreased the amplitude and frequency of cytosolic and mitochondrial Ca(2+) signals in isolated cardiomyocytes, without changes in cardiomyocyte contraction. Remarkably, in the context of well preserved contractile function in perfused hearts, we observed decreased glucose oxidation, but not fat oxidation, with increased glycolysis. cRyr2Δ50 hearts exhibited hyperphosphorylation and inhibition of pyruvate dehydrogenase, the key Ca(2+)-sensitive gatekeeper to glucose oxidation. Metabolomic, proteomic, and transcriptomic analyses revealed additional functional networks associated with altered metabolism in this model. These results demonstrate that Ryr2 controls mitochondrial Ca(2+) dynamics and plays a specific, critical role in promoting glucose oxidation in cardiomyocytes. Our findings indicate that partial RYR2 loss is sufficient to cause metabolic abnormalities seen in heart disease.

  1. Structure of the rabbit ryanodine receptor RyR1 at near-atomic resolution.

    PubMed

    Yan, Zhen; Bai, Xiao-chen; Yan, Chuangye; Wu, Jianping; Li, Zhangqiang; Xie, Tian; Peng, Wei; Yin, Chang-cheng; Li, Xueming; Scheres, Sjors H W; Shi, Yigong; Yan, Nieng

    2015-01-01

    The ryanodine receptors (RyRs) are high-conductance intracellular Ca(2+) channels that play a pivotal role in the excitation-contraction coupling of skeletal and cardiac muscles. RyRs are the largest known ion channels, with a homotetrameric organization and approximately 5,000 residues in each protomer. Here we report the structure of the rabbit RyR1 in complex with its modulator FKBP12 at an overall resolution of 3.8 Å, determined by single-particle electron cryomicroscopy. Three previously uncharacterized domains, named central, handle and helical domains, display the armadillo repeat fold. These domains, together with the amino-terminal domain, constitute a network of superhelical scaffold for binding and propagation of conformational changes. The channel domain exhibits the voltage-gated ion channel superfamily fold with distinct features. A negative-charge-enriched hairpin loop connecting S5 and the pore helix is positioned above the entrance to the selectivity-filter vestibule. The four elongated S6 segments form a right-handed helical bundle that closes the pore at the cytoplasmic border of the membrane. Allosteric regulation of the pore by the cytoplasmic domains is mediated through extensive interactions between the central domains and the channel domain. These structural features explain high ion conductance by RyRs and the long-range allosteric regulation of channel activities.

  2. Ryanodine receptor/calcium release channel PKA phosphorylation: a critical mediator of heart failure progression.

    PubMed

    Wehrens, Xander H T; Lehnart, Stephan E; Reiken, Steven; Vest, John A; Wronska, Anetta; Marks, Andrew R

    2006-01-17

    Defective regulation of the cardiac ryanodine receptor (RyR2)/calcium release channel, required for excitation-contraction coupling in the heart, has been linked to cardiac arrhythmias and heart failure. For example, diastolic calcium "leak" via RyR2 channels in the sarcoplasmic reticulum has been identified as an important factor contributing to impaired contractility in heart failure and ventricular arrhythmias that cause sudden cardiac death. In patients with heart failure, chronic activation of the "fight or flight" stress response leads to protein kinase A (PKA) hyperphosphorylation of RyR2 at Ser-2808. PKA phosphorylation of RyR2 Ser-2808 reduces the binding affinity of the channel-stabilizing subunit calstabin2, resulting in leaky RyR2 channels. We developed RyR2-S2808A mice to determine whether Ser-2808 is the functional PKA phosphorylation site on RyR2. Furthermore, mice in which the RyR2 channel cannot be PKA phosphorylated were relatively protected against the development of heart failure after myocardial infarction. Taken together, these data show that PKA phosphorylation of Ser-2808 on the RyR2 channel appears to be a critical mediator of progressive cardiac dysfunction after myocardial infarction.

  3. Gene profiling of embryonic skeletal muscle lacking type I ryanodine receptor Ca(2+) release channel.

    PubMed

    Filipova, Dilyana; Walter, Anna M; Gaspar, John A; Brunn, Anna; Linde, Nina F; Ardestani, Mostafa A; Deckert, Martina; Hescheler, Jürgen; Pfitzer, Gabriele; Sachinidis, Agapios; Papadopoulos, Symeon

    2016-02-01

    In mature skeletal muscle, the intracellular Ca(2+) concentration rises dramatically upon membrane depolarization, constituting the link between excitation and contraction. This process requires Ca(2+) release from the sarcoplasmic reticulum via the type 1 ryanodine receptor (RYR1). However, RYR1's potential roles in muscle development remain obscure. We used an established RyR1- null mouse model, dyspedic, to investigate the effects of the absence of a functional RYR1 and, consequently, the lack of RyR1-mediated Ca(2+) signaling, during embryogenesis. Homozygous dyspedic mice die after birth and display small limbs and abnormal skeletal muscle organization. Skeletal muscles from front and hind limbs of dyspedic fetuses (day E18.5) were subjected to microarray analyses, revealing 318 differentially expressed genes. We observed altered expression of multiple transcription factors and members of key signaling pathways. Differential regulation was also observed for genes encoding contractile as well as muscle-specific structural proteins. Additional qRT-PCR analysis revealed altered mRNA levels of the canonical muscle regulatory factors Six1, Six4, Pax7, MyoD, MyoG and MRF4 in mutant muscle, which is in line with the severe developmental retardation seen in dyspedic muscle histology analyses. Taken together, these findings suggest an important non-contractile role of RyR1 or RYR1-mediated Ca(2+) signaling during muscle organ development.

  4. Green tea catechins are potent sensitizers of ryanodine receptor type 1 (RyR1).

    PubMed

    Feng, Wei; Cherednichenko, Gennady; Ward, Chris W; Padilla, Isela T; Cabrales, Elaine; Lopez, José R; Eltit, José M; Allen, Paul D; Pessah, Isaac N

    2010-08-15

    Catechins, polyphenols extracted from green tea leaves, have a broad range of biological activities although the specific molecular mechanisms responsible are not known. At the high experimental concentrations typically used polyphenols bind to membrane phospholipid and also are easily auto-oxidized to generate superoxide anion and semiquinones, and can adduct to protein thiols. We report that the type 1 ryanodine receptor (RyR1) is a molecular target that responds to nanomolar (-)-epigallocatechin-3-gallate (EGCG) and (-)-epicatechin-3-gallate (ECG). Single channel analyses demonstrate EGCG (5-10nM) increases channel open probability (Po) twofold, by lengthening open dwell time. The degree of channel activation is concentration-dependent and is rapidly and fully reversible. Four related catechins, EGCG, ECG, EGC ((-)-epigallocatechin) and EC ((-)-epicatechin) showed a rank order of activity toward RyR1 (EGCG>ECG>EGC>EC). EGCG and ECG enhance the sensitivity of RyR1 to activation by < or =100microM cytoplasmic Ca(2+) without altering inhibitory potency by >100microM Ca(2+). EGCG as high as 10microM in the extracellular medium potentiated Ca(2+) transient amplitudes evoked by electrical stimuli applied to intact myotubes and adult FDB fibers, without eliciting spontaneous Ca(2+) release or slowing Ca(2+) transient recovery. The results identify RyR1 as a sensitive target for the major tea catechins EGCG and ECG, and this interaction is likely to contribute to their observed biological activities.

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

    PubMed

    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.

  6. Hypernitrosylated ryanodine receptor calcium release channels are leaky in dystrophic muscle.

    PubMed

    Bellinger, Andrew M; Reiken, Steven; Carlson, Christian; Mongillo, Marco; Liu, Xiaoping; Rothman, Lisa; Matecki, Stefan; Lacampagne, Alain; Marks, Andrew R

    2009-03-01

    Duchenne muscular dystrophy is characterized by progressive muscle weakness and early death resulting from dystrophin deficiency. Loss of dystrophin results in disruption of a large dystrophin glycoprotein complex, leading to pathological calcium (Ca2+)-dependent signals that damage muscle cells. We have identified a structural and functional defect in the ryanodine receptor (RyR1), a sarcoplasmic reticulum Ca2+ release channel, in the mdx mouse model of muscular dystrophy that contributes to altered Ca2+ homeostasis in dystrophic muscles. RyR1 isolated from mdx skeletal muscle showed an age-dependent increase in S-nitrosylation coincident with dystrophic changes in the muscle. RyR1 S-nitrosylation depleted the channel complex of FKBP12 (also known as calstabin-1, for calcium channel stabilizing binding protein), resulting in 'leaky' channels. Preventing calstabin-1 depletion from RyR1 with S107, a compound that binds the RyR1 channel and enhances the binding affinity of calstabin-1 to the nitrosylated channel, inhibited sarcoplasmic reticulum Ca2+ leak, reduced biochemical and histological evidence of muscle damage, improved muscle function and increased exercise performance in mdx mice. On the basis of these findings, we propose that sarcoplasmic reticulum Ca2+ leak via RyR1 due to S-nitrosylation of the channel and calstabin-1 depletion contributes to muscle weakness in muscular dystrophy, and that preventing the RyR1-mediated sarcoplasmic reticulum Ca2+ leak may provide a new therapeutic approach.

  7. Ryanodine Receptor 1 Polymorphism Is Not Associated with Aneurysmal Subarachnoid Hemorrhage or its Clinical Sequelae.

    PubMed

    Hendrix, Philipp; Foreman, Paul M; Harrigan, Mark R; Fisher, Winfield S; Vyas, Nilesh A; Lipsky, Robert H; Lin, Minkuan; Walters, Beverly C; Tubbs, R Shane; Shoja, Mohammadali M; Pittet, Jean-Francois; Mathru, Mali; Griessenauer, Christoph J

    2017-04-01

    The pathophysiologic mechanisms underlying cerebral vasospasm after aneurysmal subarachnoid hemorrhage (aSAH) remain poorly understand. Ryanodine receptors (RYR) are intracellular calcium channels involved in the regulation of vascular smooth muscle cells and cerebrovascular tone and diameter. Previous work reported an association between an RYR polymorphism and cerebral vasospasm. Here, we sought to assess the impact of that RYR polymorphism on aSAH and its clinical sequelae. Blood samples from all patients enrolled in the CARAS (Cerebral Aneurysm Renin Angiotensin System) study were used for genetic evaluation. The RYR1 single nucleotide polymorphism (SNP) rs35364374 was detected using 5'exonuclease (Taqman) genotyping assays. Associations between the RYR1 polymorphism and aSAH and its clinical sequelae were analyzed. Samples from 149 patients with aSAH and 50 controls were available for analysis. Multivariable regression analysis did not show an association of RYR1 SNP rs35364374 with aSAH. Moreover, there was no association of RYR1 SNP rs35364374 with clinical vasospasm, delayed cerebral ischemia, functional outcome at discharge, or functional outcome at last follow-up. Contrary to a previous report, the RYR1 SNP rs35364374 was not associated with aSAH or its clinical sequelae. Copyright © 2017 Elsevier Inc. All rights reserved.

  8. Single ryanodine receptor channel basis of caffeine's action on Ca2+ sparks.

    PubMed

    Porta, Maura; Zima, Aleksey V; Nani, Alma; Diaz-Sylvester, Paula L; Copello, Julio A; Ramos-Franco, Josefina; Blatter, Lothar A; Fill, Michael

    2011-02-16

    Caffeine (1, 3, 7-trimethylxanthine) is a widely used pharmacological agonist of the cardiac ryanodine receptor (RyR2) Ca(2+) release channel. It is also a well-known stimulant that can produce adverse side effects, including arrhythmias. Here, the action of caffeine on single RyR2 channels in bilayers and Ca(2+) sparks in permeabilized ventricular cardiomyocytes is defined. Single RyR2 caffeine activation depended on the free Ca(2+) level on both sides of the channel. Cytosolic Ca(2+) enhanced RyR2 caffeine affinity, whereas luminal Ca(2+) essentially scaled maximal caffeine activation. Caffeine activated single RyR2 channels in diastolic quasi-cell-like solutions (cytosolic MgATP, pCa 7) with an EC(50) of 9.0 ± 0.4 mM. Low-dose caffeine (0.15 mM) increased Ca(2+) spark frequency ∼75% and single RyR2 opening frequency ∼150%. This implies that not all spontaneous RyR2 openings during diastole are associated with Ca(2+) sparks. Assuming that only the longest openings evoke sparks, our data suggest that a spark may result only when a spontaneous single RyR2 opening lasts >6 ms. Copyright © 2011 Biophysical Society. Published by Elsevier Inc. All rights reserved.

  9. A cosmid and yeast artificial chromosome contig containing the complete ryanodine receptor (RYR1) gene

    SciTech Connect

    Rouquier, S.; Giorgi, D.; Trask, B.; Bergmann, A.; De Jong, P. ); Phillips, M.S.; MacLennan, D.H. )

    1993-08-01

    The ryanodine receptor (RYR1) gene is responsible for some forms of malignant hyperthermia and has been localized to 19q13.1. Central core disease is a genetic myopathy that is genetically linked to RYR1. The authors have identified an overlapping set of cosmid and YAC clones that spans more than 800 kb and includes the RYR1 gene ([approximately]205 kb). Cosmids from this region were identified by screening three chromosome 19 cosmid libraries (11-fold coverage) with six subclones representing the entire RYR1 cDNA. Genomic sequences from positive cosmids were then used as probes to identify additional cosmids. A minimally overlapping set of 23 cosmids was assembled into two contigs on the basis of restriction fragment analysis and hybridization data. Three YAC clones were isolated by screening a human YAC library with selected cosmid inserts. Overlaps among these YACs and the cosmid contigs were determined by hybridizing YAC Alu-PCR products to cosmid DNAs. The YACs bridged the gap between the cosmid contigs and extended the contig on both sides. Fluorescence in situ hybridization experiments positioned the RYR1 contig between GPI, MAG, and D19S191 on the proximal side and D19S190, CYP2A, CYP2F, SNRPA, BCKDHA, and other markers on the distal side. The 800-kb contig of cloned reagents will facilitate the detailed characterization of the RYR1 gene and other loci that may be closely related to central core disease. 62 refs., 3 figs., 3 tabs.

  10. The deletion of exon 3 in the cardiac ryanodine receptor is rescued by β strand switching.

    PubMed

    Lobo, Paolo A; Kimlicka, Lynn; Tung, Ching-Chieh; Van Petegem, Filip

    2011-06-08

    Mutations in the cardiac Ryanodine Receptor (RYR2) are linked to triggered arrhythmias. Removal of exon 3 results in a severe form of catecholaminergic polymorphic ventricular tachycardia (CPVT). This exon encodes secondary structure elements that are crucial for folding of the N-terminal domain (NTD), raising the question of why the deletion is neither lethal nor confers a loss of function. We determined the 2.3 Å crystal structure of the NTD lacking exon 3. The removal causes a structural rescue whereby a flexible loop inserts itself into the β trefoil domain and increases thermal stability. The exon 3 deletion is not tolerated in the corresponding RYR1 domain. The rescue shows a novel mechanism by which RYR2 channels can adjust their Ca²⁺ release properties through altering the structure of the NTD. Despite the rescue, the deletion affects interfaces with other RYR2 domains. We propose that relative movement of the NTD is allosterically coupled to the pore region.

  11. Imaging Single Cardiac Ryanodine Receptor Ca2+ Fluxes in Lipid Bilayers

    PubMed Central

    Peng, S.; Publicover, N. G.; Kargacin, G. J.; Duan, D.; Airey, J. A.; Sutko, John L.

    2004-01-01

    In this and an accompanying report we describe two steps, single-channel imaging and channel immobilization, necessary for using optical imaging to analyze the function of ryanodine receptor (RyR) channels reconstituted in lipid bilayers. An optical bilayer system capable of laser scanning confocal imaging of fluo-3 fluorescence due to Ca2+ flux through single RyR2 channels and simultaneous recording of single channel currents was developed. A voltage command protocol was devised in which the amplitude, time course, shape, and hence the quantity of Ca2+ flux through a single RyR2 channel is controlled solely by the voltage imposed across the bilayer. Using this system, the voltage command protocol, and concentrations of Ca2+ (25–50 mM) that result in saturating RyR2 Ca2+ currents, proportional fluo-3 fluorescence was recorded simultaneously with Ca2+ currents having amplitudes of 0.25–14 pA. Ca2+ sparks, similar to those obtained with conventional microscope-based laser scanning confocal systems, were imaged in mouse ventricular cardiomyocytes using the optical bilayer system. The utility of the optical bilayer for systematic investigation of how cellular factors extrinsic to the RyR2 channel, such as Ca2+ buffers and diffusion, alter fluo-3 fluorescent responses to RyR2 Ca2+ currents, and for addressing other current research questions is discussed. PMID:14695257

  12. Charged surface area of maurocalcine determines its interaction with the skeletal ryanodine receptor.

    PubMed

    Lukács, Balázs; Sztretye, Mónika; Almássy, János; Sárközi, Sándor; Dienes, Beatrix; Mabrouk, Kamel; Simut, Cecilia; Szabó, László; Szentesi, Péter; De Waard, Michel; Ronjat, Michel; Jóna, István; Csernoch, László

    2008-10-01

    The 33 amino acid scorpion toxin maurocalcine (MCa) has been shown to modify the gating of the skeletal-type ryanodine receptor (RyR1). Here we explored the effects of MCa and its mutants ([Ala(8)]MCa, [Ala(19)]MCa, [Ala(20)]MCa, [Ala(22)]MCa, [Ala(23)]MCa, and [Ala(24)]MCa) on RyR1 incorporated into artificial lipid bilayers and on elementary calcium release events (ECRE) in rat and frog skeletal muscle fibers. The peptides induced long-lasting subconductance states (LLSS) on RyR1 that lasted for several seconds. However, their average length and frequency were decreased if the mutation was placed farther away in the 3D structure from the critical (24)Arg residue. The effect was strongly dependent on the direction of the current through the channel. If the direction was similar to that followed by calcium during release, the peptides were 8- to 10-fold less effective. In fibers long-lasting calcium release events were observed after the addition of the peptides. The average length of these events correlated well with the duration of LLSS. These data suggest that the effect of the peptide is governed by the large charged surface formed by residues Lys(20), Lys(22), Arg(23), Arg(24), and Lys(8). Our observations also indicate that the results from bilayer experiments mimic the in situ effects of MCa on RyR1.

  13. Structure of the rabbit ryanodine receptor RyR1 at near-atomic resolution

    PubMed Central

    Wu, Jianping; Li, Zhangqiang; Xie, Tian; Peng, Wei; Yin, Changcheng; Li, Xueming; Scheres, Sjors H.W.; Shi, Yigong; Yan, Nieng

    2014-01-01

    The ryanodine receptors (RyRs) are high-conductance intracellular Ca2+ channels that play a pivotal role in the excitation-contraction coupling of skeletal and cardiac muscles. RyRs are the largest known ion channels, with a homotetrameric organization and approximately 5000 residues in each protomer. Here we report the structure of the rabbit RyR1 in complex with its modulator FKBP12 at an overall resolution of 3.8 Å, determined by single-particle electron cryo-microscopy. Three previously uncharacterized domains, named Central, Handle, and Helical domains, display the armadillo repeat fold. These domains, together with the amino-terminal domain, constitute a network of superhelical scaffold for binding and propagation of conformational changes. The channel domain exhibits the voltage-gated ion channel superfamily fold with distinct features. A negative charge-enriched hairpin loop connecting S5 and the pore helix is positioned above the entrance to the selectivity filter vestibule. The four elongated S6 segments form a right-handed helical bundle that closes the pore at the cytoplasmic border of the membrane. Allosteric regulation of the pore by the cytoplasmic domains is mediated through extensive interactions between the Central domains and the channel domain. These structural features explain high ion conductance by RyRs and the long-range allosteric regulation of channel activities. PMID:25517095

  14. DIDS modifies the conductance, gating, and inactivation mechanisms of the cardiac ryanodine receptor.

    PubMed Central

    Hill, Adam Parker; Sitsapesan, Rebecca

    2002-01-01

    The effects of the covalent modifier of amino groups, 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS) on the single-channel properties of purified sheep cardiac ryanodine receptors (RyR) incorporated into planar phospholipid bilayers were investigated. DIDS increased single-channel conductance and open probability (P(o)) and induced unique modifications to the voltage-dependence of gating. The effects of DIDS on conduction and gating were irreversible within the time scale of the experiments, and both effects were dependent on the permeant ion. DIDS induced a greater increase in conductance with Ca(2+) (20%) compared with K(+) (8%) as the permeant ion. After modification by DIDS, all channels could be rapidly inactivated in a voltage-dependent manner. The open probability of the DIDS-modified channel decreased with increasing positive or negative transmembrane potentials; however, inactivation was only observed at negative potentials. Our results demonstrate that inactivation of RyR channels is dependent on the ligand activating the channel, and this will have consequences for the control and termination of sarcoplasmic reticulum Ca(2+) release in cardiac cells. PMID:12023226

  15. Divergent functional properties of ryanodine receptor types 1 and 3 expressed in a myogenic cell line.

    PubMed Central

    Fessenden, J D; Wang, Y; Moore, R A; Chen, S R; Allen, P D; Pessah, I N

    2000-01-01

    Of the three known ryanodine receptor (RyR) isoforms expressed in muscle, RyR1 and RyR2 have well-defined roles in contraction. However, studies on mammalian RyR3 have been difficult because of low expression levels relative to RyR1 or RyR2. Using the herpes simplex virus 1 (HSV-1) helper-free amplicon system, we expressed either RyR1 or RyR3 in 1B5 RyR-deficient myotubes. Western blot analysis revealed that RyR1- or RyR3-transduced cells expressed the appropriate RyR isoform of the correct molecular mass. Although RyR1 channels exhibited the expected unitary conductance for Cs(+) in bilayer lipid membranes, 74 of 88 RyR3 channels exhibited pronounced subconductance behavior. Western blot analysis with an FKBP12/12.6-selective antibody reveals that differences in gating behavior exhibited by RyR1 and RyR3 may be, in part, the result of lower affinity of RyR3 for FKBP12. In calcium imaging studies, RyR1 restored skeletal-type excitation-contraction coupling, whereas RyR3 did not. Although RyR3-expressing myotubes were more sensitive to caffeine than those expressing RyR1, they were much less sensitive to 4-chloro-m-cresol (CMC). In RyR1-expressing cells, regenerative calcium oscillations were observed in response to caffeine and CMC but were never seen in RyR3-expressing 1B5 cells. In [(3)H]ryanodine binding studies, only RyR1 exhibited sensitivity to CMC, but both RyR isoforms responded to caffeine. These functional differences between RyR1 and RyR3 expressed in a mammalian muscle context may reflect differences in association with accessory proteins, especially FKBP12, as well as structural differences in modulator binding sites. PMID:11053126

  16. Divergent functional properties of ryanodine receptor types 1 and 3 expressed in a myogenic cell line.

    PubMed

    Fessenden, J D; Wang, Y; Moore, R A; Chen, S R; Allen, P D; Pessah, I N

    2000-11-01

    Of the three known ryanodine receptor (RyR) isoforms expressed in muscle, RyR1 and RyR2 have well-defined roles in contraction. However, studies on mammalian RyR3 have been difficult because of low expression levels relative to RyR1 or RyR2. Using the herpes simplex virus 1 (HSV-1) helper-free amplicon system, we expressed either RyR1 or RyR3 in 1B5 RyR-deficient myotubes. Western blot analysis revealed that RyR1- or RyR3-transduced cells expressed the appropriate RyR isoform of the correct molecular mass. Although RyR1 channels exhibited the expected unitary conductance for Cs(+) in bilayer lipid membranes, 74 of 88 RyR3 channels exhibited pronounced subconductance behavior. Western blot analysis with an FKBP12/12.6-selective antibody reveals that differences in gating behavior exhibited by RyR1 and RyR3 may be, in part, the result of lower affinity of RyR3 for FKBP12. In calcium imaging studies, RyR1 restored skeletal-type excitation-contraction coupling, whereas RyR3 did not. Although RyR3-expressing myotubes were more sensitive to caffeine than those expressing RyR1, they were much less sensitive to 4-chloro-m-cresol (CMC). In RyR1-expressing cells, regenerative calcium oscillations were observed in response to caffeine and CMC but were never seen in RyR3-expressing 1B5 cells. In [(3)H]ryanodine binding studies, only RyR1 exhibited sensitivity to CMC, but both RyR isoforms responded to caffeine. These functional differences between RyR1 and RyR3 expressed in a mammalian muscle context may reflect differences in association with accessory proteins, especially FKBP12, as well as structural differences in modulator binding sites.

  17. Two regions of the ryanodine receptor calcium channel are involved in Ca(2+)-dependent inactivation.

    PubMed

    Gomez, Angela C; Yamaguchi, Naohiro

    2014-03-04

    Skeletal (RyR1) and cardiac muscle (RyR2) isoforms of ryanodine receptor calcium channels are inhibited by millimollar Ca(2+), but the affinity of RyR2 for inhibitory Ca(2+) is ~10 times lower than that of RyR1. Previous studies demonstrated that the C-terminal quarter of RyR has critical domain(s) for Ca(2+) inactivation. To obtain further insights into the molecular basis of regulation of RyRs by Ca(2+), we constructed and expressed 18 RyR1-RyR2 chimeras in HEK293 cells and determined the Ca(2+) activation and inactivation affinities of these channels using the [(3)H]ryanodine binding assay. Replacing two distinct regions of RyR1 with corresponding RyR2 sequences reduced the affinity for Ca(2+) inactivation. The first region (RyR2 amino acids 4020-4250) contains two EF-hand Ca(2+) binding motifs (EF1, amino acids 4036-4047; EF2, amino acids 4071-4082), and the second region includes the putative second transmembrane segment (S2). A RyR1-backbone chimera containing only EF2 from RyR2 had a modest (not significant) change in Ca(2+) inactivation, whereas another chimera channel carrying only EF1 from RyR2 had a significantly reduced level of Ca(2+) inactivation. The results suggest that EF1 is a more critical determinant for RyR inactivation by Ca(2+). In addition, activities of the chimera carrying RyR2 EF-hands were suppressed at 10-100 μM Ca(2+), and the suppression was relieved by 1 mM Mg(2+). The same effects have been observed with wild-type RyR2. A mutant RyR1 carrying both regions replaced with RyR2 sequences (amino acids 4020-4250 and 4560-4618) showed a Ca(2+) inactivation affinity comparable to that of RyR2, indicating that these regions are sufficient to confer RyR2-type Ca(2+)-dependent inactivation on RyR1.

  18. Evidence for novel caffeine and Ca2+ binding sites on the lobster skeletal ryanodine receptor

    PubMed Central

    Zhang, Jin Jun; Williams, Alan J; Sitsapesan, Rebecca

    1999-01-01

    The effects of Ca2+, ATP and caffeine on the gating of lobster skeletal muscle ryanodine receptors (RyR) was investigated after reconstitution of the channels into planar phospholipid bilayers and by using [3H]-ryanodine binding studies. The single channel studies reveal that the EC50 (60 μM) for activation of the lobster skeletal RyR by Ca2+ as the sole ligand is higher than for any other isoform of RyR studied. Inactivation of the channel by Ca2+ (EC50=1 mM) occurs at concentrations slightly higher than those required to inactivate mammalian skeletal RyR (RyR1) but lower than those required to inactivate mammalian cardiac RyR (RyR2). Lifetime analysis demonstrates that cytosolic Ca2+, as the sole activating ligand, cannot fully open the lobster skeletal RyR (maximum Po approximately 0.2). The mechanism for the increase in open probability (Po) is an increase in both the frequency and the duration of the open events. ATP is a very effective activator of the lobster RyR and can almost fully open the channel in the presence of activating cytosolic [Ca2+]. In the presence of 700 μM Ca2+, 1 mM ATP increased Po to approximately 0.8. Caffeine, often used as a tool to identify the presence of RyR channels, is relatively ineffective and cannot increase Po above the level that can be attained with Ca2+ alone. The results reveal that caffeine increases Po by a different mechanism to that of cytosolic Ca2+ demonstrating that the mechanism for channel activation by caffeine is not ‘sensitization' to cytosolic Ca2+. By studying the mechanisms involved in the activation of the lobster RyR we have demonstrated that the channel responds in a unique manner to Ca2+ and to caffeine. The results strongly indicate that these ligand binding sites on the channel are different to those on mammalian isoforms of RyR. PMID:10193789

  19. Role of malignant hyperthermia domain in the regulation of Ca2+ release channel (ryanodine receptor) of skeletal muscle sarcoplasmic reticulum.

    PubMed

    Zorzato, F; Menegazzi, P; Treves, S; Ronjat, M

    1996-09-13

    A fusion protein encompassing Gly341 of the skeletal muscle ryanodine receptor was used to raise monoclonal antibodies; epitope mapping demonstrates that monoclonal antibody 419 (mAb419) reacts with a sequence a few residues upstream from Gly341. The mAb419 was then used to probe ryanodine receptor (RYR) functions. Our results show that upon incubation of triads vesicles with mAb419 the Ca2+-induced Ca2+ release rate at pCa 8 was increased. Equilibrium evaluation of [3H]ryanodine binding at different [Ca2+] indicates that mAb419 shifted the half-maximal [Ca2+] for stimulation of ryanodine binding to lower value (0.1 versus 1.2 microM). Such functional effects may be due to a direct action of the Ab on the Ca2+ binding domain of the RYR or to the perturbation by the Ab of the intramolecular interaction between the immunopositive region and regulatory domain of the RYR. The latter hypothesis was tested directly using the optical biosensor BIAcore (Pharmacia Biotech Inc.): we show that the immunopositive RYR polypeptide is able to interact with the native RYR complex. Ligand overlays with immunopositive digoxigenin-RYR fusion protein indicate that such an interaction might occur with a calmodulin binding domain (defined by residues 3010-3225) and with a polypeptide defined by residues 799-1172. In conclusion our results suggest that the stimulation by the mAb419 of the RYR channel activity is due to the perturbation of an intramolecular interaction between the immunopositive polypeptide and a Ca2+ regulatory site probably corresponding to a calmodulin binding domain.

  20. Nicotinic acid receptor subtypes and their ligands.

    PubMed

    Soudijn, Willem; van Wijngaarden, Ineke; Ijzerman, Adriaan P

    2007-05-01

    Half a century ago, nicotinic acid (niacin) was introduced into the clinic as the first orally available drug to treat high cholesterol levels and to improve the balance between (V)low density lipoproteins (LDL) and high density lipoproteins (HDL). Remarkably, its putative mechanism of action has only been recently elucidated, particularly because of the cloning of a G protein-coupled receptor (HM74A or GPR109A). This receptor responds to both nicotinic acid and the ketone body beta-hydroxybutyrate, the latter thought to be the more probable endogenous ligand for HM74A. In this review, we will discuss the pharmacology and medicinal chemistry of this receptor subtype and a related one (HM74 or GPR109B). Although still in its infancy, the ligand repertoire is developing, and a number of compound classes have now been described, among which are both full and partial agonists. Antagonists, however, are still lacking, thus compromising thorough pharmacological studies. Mutagenesis experiments have provided clues regarding the ligand binding site; in particular, an arginine residue in transmembrane domain 3 of the receptor seems to recognize the acidic moiety present in nicotinic acid and related substances. HM74A has also been linked to one of the major side effects of nicotinic acid, that is, flushing, since this receptor subtype also occurs in skin immune cells. It is not known yet whether HM74 is also present on these cells. Since nicotinic acid is one of the few available medicines that raise HDL ("good cholesterol") levels, HM74A and HM74 appear promising targets for future pharmacotherapy. (c) 2006 Wiley Periodicals, Inc.

  1. Pharmacologic specificity of alpha-2 adrenergic receptor subtypes

    SciTech Connect

    Petrash, A.; Bylund, D.

    1986-03-01

    The authors have defined alpha-2 adrenergic receptor subtypes in human and rat tissues using prazosin as a subtype selective drug. Prazosin has a lower affinity (250 nM) at alpha-2A receptor and a higher affinity (5 nM) at alpha-2B receptors. In order to determine if other adrenergic drugs are selective for one or the other subtypes, the authors performed (/sup 3/H)yohimbine inhibition experiments with various adrenergic drugs in tissues containing alpha-2A, alpha-2B or both subtypes. Oxymetazoline, WB4101 and yohimbine were found to be 80-, 20- and 10-fold more potent at alpha-2A receptors than at alpha-2B receptors. Phentolamine, adazoxan, (+)- and (-)-mianserin, clonidine, (+)-butaclamol, (-)- and (+)-norepinephrine, epinephrine, dopamine and thioridazine were found to have equal affinities for the two subtypes. These results further validate the subdivision of alpha-2 adrenergic receptors into alpha-2A and alpha-2B subtypes.

  2. Excitation–contraction uncoupling by a human central core disease mutation in the ryanodine receptor

    PubMed Central

    Avila, Guillermo; O'Brien, Jennifer J.; Dirksen, Robert T.

    2001-01-01

    Central core disease (CCD) is a human congenital myopathy characterized by fetal hypotonia and proximal muscle weakness that is linked to mutations in the gene encoding the type-1 ryanodine receptor (RyR1). CCD is thought to arise from Ca2+-induced damage stemming from mutant RyR1 proteins forming “leaky” sarcoplasmic reticulum (SR) Ca2+ release channels. A novel mutation in the C-terminal region of RyR1 (I4898T) accounts for an unusually severe and highly penetrant form of CCD in humans [Lynch, P. J., Tong, J., Lehane, M., Mallet, A., Giblin, L., Heffron, J. J., Vaughan, P., Zafra, G., MacLennan, D. H. & McCarthy, T. V. (1999) Proc. Natl. Acad. Sci. USA 96, 4164–4169]. We expressed in skeletal myotubes derived from RyR1-knockout (dyspedic) mice the analogous mutation engineered into a rabbit RyR1 cDNA (I4897T). Here we show that homozygous expression of I4897T in dyspedic myotubes results in a complete uncoupling of sarcolemmal excitation from voltage-gated SR Ca2+ release without significantly altering resting cytosolic Ca2+ levels, SR Ca2+ content, or RyR1-mediated enhancement of dihydropyridine receptor (DHPR) channel activity. Coexpression of both I4897T and wild-type RyR1 resulted in a 60% reduction in voltage-gated SR Ca2+ release, again without altering resting cytosolic Ca2+ levels, SR Ca2+ content, or DHPR channel activity. These findings indicate that muscle weakness suffered by individuals possessing the I4898T mutation involves a functional uncoupling of sarcolemmal excitation from SR Ca2+ release, rather than the expression of overactive or leaky SR Ca2+ release channels. PMID:11274444

  3. Nebivolol suppresses cardiac ryanodine receptor-mediated spontaneous Ca2+ release and catecholaminergic polymorphic ventricular tachycardia.

    PubMed

    Tan, Zhen; Xiao, Zhichao; Wei, Jinhong; Zhang, Jingqun; Zhou, Qiang; Smith, Chris D; Nani, Alma; Wu, Guogen; Song, Long-Sheng; Back, Thomas G; Fill, Michael; Chen, S R Wayne

    2016-11-15

    β-Blockers are a standard treatment for heart failure and cardiac arrhythmias. There are ∼30 commonly used β-blockers, representing a diverse class of drugs with different receptor affinities and pleiotropic properties. We reported that among 14 β-blockers tested previously, only carvedilol effectively suppressed cardiac ryanodine receptor (RyR2)-mediated spontaneous Ca(2+) waves during store Ca(2+) overload, also known as store overload-induced Ca(2+) release (SOICR). Given the critical role of SOICR in arrhythmogenesis, it is of importance to determine whether there are other β-blockers that suppress SOICR. Here, we assessed the effect of other commonly used β-blockers on RyR2-mediated SOICR in HEK293 cells, using single-cell Ca(2+) imaging. Of the 13 β-blockers tested, only nebivolol, a β-1-selective β-blocker with nitric oxide synthase (NOS)-stimulating action, effectively suppressed SOICR. The NOS inhibitor (N-nitro-l-arginine methyl ester) had no effect on nebivolol's SOICR inhibition, and the NOS activator (histamine or prostaglandin E2) alone did not inhibit SOICR. Hence, nebivolol's SOICR inhibition was independent of NOS stimulation. Like carvedilol, nebivolol reduced the opening of single RyR2 channels and suppressed spontaneous Ca(2+) waves in intact hearts and catecholaminergic polymorphic ventricular tachycardia (CPVT) in the mice harboring a RyR2 mutation (R4496C). Interestingly, a non-β-blocking nebivolol enantiomer, (l)-nebivolol, also suppressed SOICR and CPVT without lowering heart rate. These data indicate that nebivolol, like carvedilol, possesses a RyR2-targeted action that suppresses SOICR and SOICR-evoked VTs. Thus, nebivolol represents a promising agent for Ca(2+)-triggered arrhythmias. © 2016 The Author(s); published by Portland Press Limited on behalf of the Biochemical Society.

  4. Structural and functional conservation of key domains in InsP[subscript 3] and ryanodine receptors

    SciTech Connect

    Seo, Min-Duk; Velamakanni, Saroj; Ishiyama, Noboru; Stathopulos, Peter B.; Rossi, Ana M.; Khan, Samir A.; Dale, Philippa; Li, Congmin; Ames, James B.; Ikura, Mitsuhiko; Taylor, Colin W.

    2012-07-11

    Inositol-1,4,5-trisphosphate receptors (InsP{sub 3}Rs) and ryanodine receptors (RyRs) are tetrameric intracellular Ca{sup 2+} channels. In each of these receptor families, the pore, which is formed by carboxy-terminal transmembrane domains, is regulated by signals that are detected by large cytosolic structures. InsP{sub 3}R gating is initiated by InsP{sub 3} binding to the InsP{sub 3}-binding core (IBC, residues 224-604 of InsP{sub 3}R1) and it requires the suppressor domain (SD, residues 1-223 of InsP{sub 3}R1). Here we present structures of the amino-terminal region (NT, residues 1-604) of rat InsP{sub 3}R1 with (3.6 {angstrom}) and without (3.0 {angstrom}) InsP{sub 3} bound. The arrangement of the three NT domains, SD, IBC-{beta} and IBC-{alpha}, identifies two discrete interfaces ({alpha} and {beta}) between the IBC and SD. Similar interfaces occur between equivalent domains (A, B and C) in RyR1 (ref. 9). The orientations of the three domains when docked into a tetrameric structure of InsP{sub 3}R and of the ABC domains docked into RyR are remarkably similar. The importance of the {alpha}-interface for activation of InsP{sub 3}R and RyR is confirmed by mutagenesis and, for RyR, by disease-causing mutations. Binding of InsP{sub 3} causes partial closure of the clam-like IBC, disrupting the {beta}-interface and pulling the SD towards the IBC. This reorients an exposed SD loop ('hotspot' (HS) loop) that is essential for InsP{sub 3}R activation. The loop is conserved in RyR and includes mutations that are associated with malignant hyperthermia and central core disease. The HS loop interacts with an adjacent NT, suggesting that activation re-arranges inter-subunit interactions. The A domain of RyR functionally replaced the SD in full-length InsP{sub 3}R, and an InsP{sub 3}R in which its C-terminal transmembrane region was replaced by that from RyR1 was gated by InsP{sub 3} and blocked by ryanodine. Activation mechanisms are conserved between InsP{sub 3}R and Ry

  5. Subconductance states in single-channel activity of skeletal muscle ryanodine receptors after removal of FKBP12.

    PubMed Central

    Ahern, G P; Junankar, P R; Dulhunty, A F

    1997-01-01

    FKBP12 was removed from ryanodine receptors (RyRs) by incubation of rabbit skeletal muscle terminal cisternae membranes with rapamycin. The extent of FKBP12 removal was estimated by immunostaining Western blots of terminal cisternae proteins. Single FKBP12-depleted RyR channels, incorporated into planar lipid bilayers, were modulated by Ca2+, ATP, ryanodine, and ruthenium red in the cis chamber and opened frequently to the normal maximum conductance of approximately 230 pS and to substate levels of approximately 0.25, approximately 0.5, and approximately 0.75 of the maximum conductance. Substate activity was rarely seen in native RyRs. Ryanodine did not after the number of conductance levels in FKBP12-depleted channels, but, at a membrane potential of +40 mV, reduced both the maximum and the substate conductances by approximately 50%. FKBP12-stripped channels were activated by a 10-fold-lower [Ca2+] and inhibited by a 10-fold-higher [Ca2+], than RyRs from control-incubated and native terminal cisternae vesicles. The open probability (Po) of these FKBP12-deficient channels was greater than that of control channels at 0.1 microM and 1 mM cis Ca2+ but no different at 10 microM cis Ca2+, where channels showed maximal Ca2+ activation. The approximately 0.25 substate was less sensitive than the maximum conductance to inhibition by Ca2+ and was the dominant level in channels inhibited by 1 mM cis Ca2+. The results show that FKBP12 coordinates the gating of channel activity in control and ryanodine-modified RyRs. Images FIGURE 1 PMID:8994600

  6. Ca2+ entry via AMPA-type glutamate receptors triggers Ca2+-induced Ca2+ release from ryanodine receptors in rat spiral ganglion neurons.

    PubMed

    Morton-Jones, Rachel T; Cannell, Mark B; Housley, Gary D

    2008-04-01

    Ryanodine receptor (RyR)-gated Ca2+ stores have recently been identified in cochlear spiral ganglion neurons (SGN) and likely contribute to Ca2+ signalling associated with auditory neurotransmission. Here, we identify an ionotropic glutamate receptor signal transduction pathway which invokes RyR-gated Ca2+ stores in SGN via Ca2+-induced Ca2+ release (CICR). Ca2+ levels were recorded in SGN in situ within rat cochlear slices (postnatal day 0-17) using the Ca2+ indicator fluo-4. RyR-gated Ca2+ stores were confirmed by caffeine-induced increases in intracellular Ca2+ which were blocked by ryanodine (100 microM) and were independent of external Ca2+. Glutamate evoked comparable increases in intracellular Ca2+, but required the presence of external Ca2+. Ca2+ influx via the glutamate receptor was found to elicit CICR via RyR-gated Ca2+ stores, as shown by the inhibition of the response by prior depletion of the Ca2+ stores with caffeine, the SERCA inhibitor thapsigargin, or ryanodine. The glutamate analogue AMPA (alpha-Amino-3-hydroxy-5-methylisoxazole-4-propionic acid) elicited Ca2+ responses that could be inhibited by caffeine. Glutamate- and AMPA-mediated Ca2+ responses were eliminated with the AMPA/Kainate receptor antagonist DNQX (6,7-dinitroquinoxaline-2,3-dione). These data demonstrate functional coupling between somatic AMPA-type glutamate receptors and intracellular Ca(2+) stores via RyR-dependent CICR in primary auditory neurons.

  7. Enhanced binding of calmodulin to the ryanodine receptor corrects contractile dysfunction in failing hearts.

    PubMed

    Hino, Akihiro; Yano, Masafumi; Kato, Takayoshi; Fukuda, Masakazu; Suetomi, Takeshi; Ono, Makoto; Murakami, Wakako; Susa, Takehisa; Okuda, Shinichi; Doi, Masahiro; Kobayashi, Shigeki; Yamamoto, Takeshi; Koseki, Noritaka; Kyushiki, Hiroyuki; Ikemoto, Noriaki; Matsuzaki, Masunori

    2012-12-01

    The channel function of the cardiac ryanodine receptor (RyR2) is modulated by calmodulin (CaM). However, the involvement of CaM in aberrant Ca(2+) release in diseased hearts remains unclear. Here, we investigated the pathogenic role of defective CaM binding to the RyR2 in the channel dysfunction associated with heart failure. The involvement of CaM in aberrant Ca(2+) release was assessed in normal and pacing-induced failing canine hearts. The apparent affinity of CaM for RyR2 was considerably lower in failing sarcoplasmic reticulum (SR) compared with normal SR. Thus, the amount of CaM bound to RyR2 was markedly decreased in failing myocytes. Expression of the CaM isoform Gly-Ser-His-CaM (GSH-CaM), which has much higher binding affinity than wild-type CaM for RyR1, restored normal CaM binding to RyR2 in both SR and myocytes of failing hearts. The Ca(2+) spark frequency (SpF) was markedly higher and the SR Ca(2+) content was lower in failing myocytes compared with normal myocytes. The incorporation of GSH-CaM into the failing myocytes corrected the aberrant SpF and SR Ca(2+) content to normal levels. Reduced CaM binding to RyR2 seems to play a critical role in the pathogenesis of aberrant Ca(2+) release in failing hearts. Correction of the reduced CaM binding to RyR2 stabilizes the RyR2 channel function and thereby restores normal Ca(2+) handling and contractile function to failing hearts.

  8. RNAi mediated knockdown of the ryanodine receptor gene decreases chlorantraniliprole susceptibility in Sogatella furcifera.

    PubMed

    Yang, Yao; Wan, Pin-Jun; Hu, Xing-Xing; Li, Guo-Qing

    2014-01-01

    The diamide insecticides activate ryanodine receptors (RyRs) to release and deplete intracellular calcium stores from the sarcoplasmic reticulum of muscles and the endoplasmic reticulum of many types of cells. They rapidly interrupt feeding of the target pest and eventually kill the pest due to starvation. However, information about the structure and function of insect RyRs is still limited. In this study, we isolated a 15,985bp full-length cDNA (named SfRyR) from Sogatella furcifera, a serious rice planthopper pest throughout Asia. SfRyR encodes a 5140-amino acid protein, which shares 78-97% sequence identities with other insect homologues, and less than 50% identities with Homo sapiens RyR1-3. All hallmarks of the RyR proteins are conserved in SfRyR. In the N-terminus, SfRyR has a MIR domain, two RIH domains, three SPRY domains, four copies of RyR repeated domain and a RIH-associated domain. In the C-terminus, SfRyR possesses two consensus calcium ion-binding EF-hand motifs, and six transmembrane helices. Temporal and spatial expression analysis showed that SfRyR was widely found in all development stages including egg, first through fifth instar nymphs, macropterous adult females and males. On day 2 fifth-instar nymphs, SfRyR was ubiquitously expressed in the head, thorax and abdomen. Dietary ingestion of dsSfRyR1 and dsSfRyR2 significantly reduced the mRNA level of SfRyR in the treated nymphs by 77.9% and 81.8% respectively, and greatly decreased chlorantraniliprole-induced mortality. Thus, our results suggested that SfRyR gene encoded a functional RyR that mediates chlorantraniliprole toxicity to S. furcifera.

  9. Structural determinants of 4-chloro-m-cresol required for activation of ryanodine receptor type 1.

    PubMed

    Jacobson, Alan R; Moe, Scott T; Allen, P D; Fessenden, James D

    2006-07-01

    4-Chloro-m-cresol (4-CmC) is a clinically relevant activator of the intracellular Ca2+ release channel, the ryanodine receptor isoform 1 (RyR1). In this study, the chemical moieties on the 4-CmC molecule required for its activation of RyR1 were determined using structure-activity relationship analysis with a set of commercially available 4-CmC analogs. Separate compounds each lacking one of the three functional groups of 4-CmC (1-hydroxyl, 3-methyl, or 4-chloro) were poor activators of RyR1. Substitution of different chemical groups for the 1-hydroxyl of 4-CmC resulted in compounds that were poor activators of RyR1, suggesting that the hydroxyl group is preferred at this position. Substitution of hydrophobic groups at the 3-position enhanced bioactivity of the compound relative to 4-CmC, whereas substitution with hydrophilic groups abolished bioactivity. Likewise, 4-CmC analogs with hydrophobic groups substituted into the 4-position enhanced bioactivity, whereas hydrophilic or charged groups diminished bioactivity. 4-CmC analogs containing a single hydrophobic group at either the 3- or 4-position as well as 3,5-disubstituted or 3,4,5-trisubstituted phenols were also effective activators of RyR1. These results indicate that the 1-hydroxyl group of 4-CmC is required for activation of RyR1 and that hydrophobic groups at the 3,4- and 5-positions are preferred. These findings suggest that the 4-CmC binding site on RyR1 most likely consists of a hydrophilic region to interact with the 1-hydroxyl as well as a hydrophobic region(s) to interact with chemical groups at the 3- and/or 4-positions of 4-CmC.

  10. FRET-based trilateration of probes bound within functional ryanodine receptors.

    PubMed

    Svensson, Bengt; Oda, Tetsuro; Nitu, Florentin R; Yang, Yi; Cornea, Iustin; Chen-Izu, Ye; Fessenden, James D; Bers, Donald M; Thomas, David D; Cornea, Razvan L

    2014-11-04

    To locate the biosensor peptide DPc10 bound to ryanodine receptor (RyR) Ca(2+) channels, we developed an approach that combines fluorescence resonance energy transfer (FRET), simulated-annealing, cryo-electron microscopy, and crystallographic data. DPc10 is identical to the 2460-2495 segment within the cardiac muscle RyR isoform (RyR2) central domain. DPc10 binding to RyR2 results in a pathologically elevated Ca(2+) leak by destabilizing key interactions between the RyR2 N-terminal and central domains (unzipping). To localize the DPc10 binding site within RyR2, we measured FRET between five single-cysteine variants of the FK506-binding protein (FKBP) labeled with a donor probe, and DPc10 labeled with an acceptor probe (A-DPc10). Effective donor positions were calculated from simulated-annealing constrained by both the RyR cryo-EM map and the FKBP atomic structure docked to the RyR. FRET to A-DPc10 was measured in permeabilized cardiomyocytes via confocal microscopy, converted to distances, and used to trilaterate the acceptor locus within RyR. Additional FRET measurements between donor-labeled calmodulin and A-DPc10 were used to constrain the trilaterations. Results locate the DPc10 probe within RyR domain 3, ?35 Å from the previously docked N-terminal domain crystal structure. This multiscale approach may be useful in mapping other RyR sites of mechanistic interest within FRET range of FKBP.

  11. FRET-Based Localization of Fluorescent Protein Insertions Within the Ryanodine Receptor Type 1

    PubMed Central

    Raina, Shweta A.; Tsai, Jeffrey; Samsó, Montserrat; Fessenden, James D.

    2012-01-01

    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 Ca2+ 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. PMID:22719904

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

    PubMed

    Raina, Shweta A; Tsai, Jeffrey; Samsó, Montserrat; Fessenden, James D

    2012-01-01

    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.

  13. FRET-Based Trilateration of Probes Bound within Functional Ryanodine Receptors

    PubMed Central

    Svensson, Bengt; Oda, Tetsuro; Nitu, Florentin R.; Yang, Yi; Cornea, Iustin; Chen-Izu, Ye; Fessenden, James D.; Bers, Donald M.; Thomas, David D.; Cornea, Razvan L.

    2014-01-01

    To locate the biosensor peptide DPc10 bound to ryanodine receptor (RyR) Ca2+ channels, we developed an approach that combines fluorescence resonance energy transfer (FRET), simulated-annealing, cryo-electron microscopy, and crystallographic data. DPc10 is identical to the 2460–2495 segment within the cardiac muscle RyR isoform (RyR2) central domain. DPc10 binding to RyR2 results in a pathologically elevated Ca2+ leak by destabilizing key interactions between the RyR2 N-terminal and central domains (unzipping). To localize the DPc10 binding site within RyR2, we measured FRET between five single-cysteine variants of the FK506-binding protein (FKBP) labeled with a donor probe, and DPc10 labeled with an acceptor probe (A-DPc10). Effective donor positions were calculated from simulated-annealing constrained by both the RyR cryo-EM map and the FKBP atomic structure docked to the RyR. FRET to A-DPc10 was measured in permeabilized cardiomyocytes via confocal microscopy, converted to distances, and used to trilaterate the acceptor locus within RyR. Additional FRET measurements between donor-labeled calmodulin and A-DPc10 were used to constrain the trilaterations. Results locate the DPc10 probe within RyR domain 3, ∼35 Å from the previously docked N-terminal domain crystal structure. This multiscale approach may be useful in mapping other RyR sites of mechanistic interest within FRET range of FKBP. PMID:25418089

  14. GREEN TEA CATECHINS ARE POTENT SENSITIZERS OF RYANODINE RECEPTOR TYPE 1 (RYR1)

    PubMed Central

    Feng, Wei; Cherednichenko, Gennady; Ward, Chris W.; Padilla, Isela T.; Cabrales, Elaine; Lopez, José R.; Eltit, José M.; Allen, Paul D.; Pessah, Isaac N.

    2010-01-01

    Catechins, polyphenols extracted from green tea leaves, have a broad range of biological activities although the specific molecular mechanisms responsible are not known. At the high experimental concentrations typically used polyphenols bind to membrane phospholipid and also are easily auto-oxidized to generate superoxide anion and semiquinones, and can adduct to protein thiols. We report that the type 1 ryanodine receptor (RyR1) is a molecular target that responds to nanomolar (−)-epigallocatechin-3-gallate (EGCG) and (−)-epicatechin-3-gallate (ECG). Single channel analyses demonstrate EGCG (5-10nM) increases channel open probability (Po) 2-fold, by lengthening open dwell time. The degree of channel activation is concentration dependent and is rapidly and fully reversible. Four related catechins, EGCG, ECG, EGC ((−)-epigallocatechin) and EC ((−)-epicatechin) showed a rank order of activity toward RyR1 (EGCG>ECG>>EGC>>>EC). EGCG and ECG enhance the sensitivity of RyR1 to activation by ≤100μM cytoplasmic Ca2+ without altering inhibitory potency by >100μM Ca2+. EGCG as high as 10μM in the extracellular medium potentiated Ca2+ transient amplitudes evoked by electrical stimuli applied to intact myotubes and adult FDB fibers, without eliciting spontaneous Ca2+ release or slowing Ca2+ transient recovery. The results identify RyR1 as a sensitive target for the major tea catechins EGCG and ECG, and this interaction is likely to contribute to their observed biological activities. PMID:20471964

  15. Ryanodine receptor cluster fragmentation and redistribution in persistent atrial fibrillation enhance calcium release

    PubMed Central

    Macquaide, Niall; Tuan, Hoang-Trong Minh; Hotta, Jun-ichi; Sempels, Wouter; Lenaerts, Ilse; Holemans, Patricia; Hofkens, Johan; Jafri, M. Saleet; Willems, Rik; Sipido, Karin R.

    2015-01-01

    Aims In atrial fibrillation (AF), abnormalities in Ca2+ release contribute to arrhythmia generation and contractile dysfunction. We explore whether ryanodine receptor (RyR) cluster ultrastructure is altered and is associated with functional abnormalities in AF. Methods and results Using high-resolution confocal microscopy (STED), we examined RyR cluster morphology in fixed atrial myocytes from sheep with persistent AF (N = 6) and control (Ctrl; N = 6) animals. RyR clusters on average contained 15 contiguous RyRs; this did not differ between AF and Ctrl. However, the distance between clusters was significantly reduced in AF (288 ± 12 vs. 376 ± 17 nm). When RyR clusters were grouped into Ca2+ release units (CRUs), i.e. clusters separated by <150 nm, CRUs in AF had more clusters (3.43 ± 0.10 vs. 2.95 ± 0.02 in Ctrl), which were more dispersed. Furthermore, in AF cells, more RyR clusters were found between Z lines. In parallel experiments, Ca2+ sparks were monitored in live permeabilized myocytes. In AF, myocytes had >50% higher spark frequency with increased spark time to peak (TTP) and duration, and a higher incidence of macrosparks. A computational model of the CRU was used to simulate the morphological alterations observed in AF cells. Increasing cluster fragmentation to the level observed in AF cells caused the observed changes, i.e. higher spark frequency, increased TTP and duration; RyR clusters dispersed between Z-lines increased the occurrence of macrosparks. Conclusion In persistent AF, ultrastructural reorganization of RyR clusters within CRUs is associated with overactive Ca2+ release, increasing the likelihood of propagating Ca2+ release. PMID:26490742

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

    PubMed Central

    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

  17. A mechanism for sudden infant death syndrome (SIDS): Stress-induced leak via ryanodine receptors

    PubMed Central

    Tester, David J.; Dura, Miroslav; Carturan, Elisa; Reiken, Steven; Wronska, Anetta; Marks, Andrew R.; Ackerman, Michael J.

    2012-01-01

    BACKGROUND Sudden infant death syndrome (SIDS) is the leading cause of postneonatal mortality in the United States. Mutations in the RyR2-encoded cardiac ryanodine receptor cause the highly lethal catecholaminergic polymorphic ventricular tachycardia (CPVT1) in the young. OBJECTIVE The purpose of this study was to determine the spectrum and prevalence of RyR2 mutations in a large cohort of SIDS cases. METHODS Using polymerase chain reaction, denaturing high performance liquid chromatography, and direct DNA sequencing, a targeted mutational analysis of RyR2 was performed on genomic DNA isolated from frozen necropsy tissue on 134 unrelated cases of SIDS (57 females, 77 males; 83 white, 50 black, 1 Hispanic; average age = 2.7 months). RyR2 mutations were engineered by site-directed mutagenesis, heterologously expressed in HEK293 cells, and functionally characterized using single-channel recordings in planar lipid bilayers. RESULTS Overall, two distinct and novel RyR2 mutations were identified in two cases of SIDS. A 6-month-old black female hosted an R2267H missense mutation, and a 4-week-old white female infant harbored a S4565R mutation. Both nonconservative amino acid substitutions were absent in 400 reference alleles, involved conserved residues, and were localized to key functionally significant domains. Under conditions that simulate stress [Protein Kinase A (PKA) phosphorylation] during diastole (low activating [Ca2+]), SIDS-associated RyR2 mutant channels displayed a significant gain-of-function phenotype consistent with the functional effect of previously characterized CPVT-associated RyR2 mutations. CONCLUSIONS Here we report a novel pathogenic mechanism for SIDS, whereby SIDS-linked RyR2 mutations alter the response of the channels to sympathetic nervous system stimulation such that during stress the channels become “leaky” and thus potentially trigger fatal cardiac arrhythmias. PMID:17556193

  18. A new cytoplasmic interaction between junctin and ryanodine receptor Ca2+ release channels.

    PubMed

    Li, Linwei; Mirza, Shamaruh; Richardson, Spencer J; Gallant, Esther M; Thekkedam, Chris; Pace, Suzy M; Zorzato, Francesco; Liu, Dan; Beard, Nicole A; Dulhunty, Angela F

    2015-03-01

    Junctin, a non-catalytic splice variant encoded by the aspartate-β-hydroxylase (Asph) gene, is inserted into the membrane of the sarcoplasmic reticulum (SR) Ca(2+) store where it modifies Ca(2+) signalling in the heart and skeletal muscle through its regulation of ryanodine receptor (RyR) Ca(2+) release channels. Junctin is required for normal muscle function as its knockout leads to abnormal Ca(2+) signalling, muscle dysfunction and cardiac arrhythmia. However, the nature of the molecular interaction between junctin and RyRs is largely unknown and was assumed to occur only in the SR lumen. We find that there is substantial binding of RyRs to full junctin, and the junctin luminal and, unexpectedly, cytoplasmic domains. Binding of these different junctin domains had distinct effects on RyR1 and RyR2 activity: full junctin in the luminal solution increased RyR channel activity by ∼threefold, the C-terminal luminal interaction inhibited RyR channel activity by ∼50%, and the N-terminal cytoplasmic binding produced an ∼fivefold increase in RyR activity. The cytoplasmic interaction between junctin and RyR is required for luminal binding to replicate the influence of full junctin on RyR1 and RyR2 activity. The C-terminal domain of junctin binds to residues including the S1-S2 linker of RyR1 and N-terminal domain of junctin binds between RyR1 residues 1078 and 2156.

  19. Investigations of the contribution of a putative glycine hinge to ryanodine receptor channel gating.

    PubMed

    Euden, Joanne; Mason, Sammy A; Viero, Cedric; Thomas, N Lowri; Williams, Alan J

    2013-06-07

    Ryanodine receptor channels (RyR) are key components of striated muscle excitation-contraction coupling, and alterations in their function underlie both inherited and acquired disease. A full understanding of the disease process will require a detailed knowledge of the mechanisms and structures involved in RyR function. Unfortunately, high-resolution structural data, such as exist for K(+)-selective channels, are not available for RyR. In the absence of these data, we have used modeling to identify similarities in the structural elements of K(+) channel pore-forming regions and postulated equivalent regions of RyR. This has identified a sequence of residues in the cytosolic cavity-lining transmembrane helix of RyR (G(4864)LIIDA(4869) in RyR2) analogous to the glycine hinge motif present in many K(+) channels. Gating in these K(+) channels can be disrupted by substitution of residues for the hinge glycine. We investigated the involvement of glycine 4864 in RyR2 gating by monitoring properties of recombinant human RyR2 channels in which this glycine is replaced by residues that alter gating in K(+) channels. Our data demonstrate that introducing alanine at position 4864 produces no significant change in RyR2 function. In contrast, function is altered when glycine 4864 is replaced by either valine or proline, the former preventing channel opening and the latter modifying both ion translocation and gating. Our studies reveal novel information on the structural basis of RyR gating, identifying both similarities with, and differences from, K(+) channels. Glycine 4864 is not absolutely required for channel gating, but some flexibility at this point in the cavity-lining transmembrane helix is necessary for normal RyR function.

  20. Calmodulin regulation and identification of calmodulin binding region of type-3 ryanodine receptor calcium release channel.

    PubMed

    Yamaguchi, Naohiro; Xu, Le; Pasek, Daniel A; Evans, Kelly E; Chen, S R Wayne; Meissner, Gerhard

    2005-11-15

    Ryanodine receptors (RyRs) are a family of intracellular Ca(2+) channels that are regulated by calmodulin (CaM). At low Ca(2+) concentrations (<1 microM), CaM activates RyR1 and RyR3 and inhibits RyR2. At elevated Ca(2+) concentrations (>1 microM), CaM inhibits all three RyR isoforms. Here we report that the regulation of recombinant RyR3 by CaM is sensitive to redox regulation. RyR3 in the presence of reduced glutathione binds CaM with 10-15-fold higher affinity, at low and high Ca(2+) concentrations, compared to in the presence of oxidized glutathione. However, compared to RyR1 assayed at low Ca(2+) concentrations under both reducing and oxidizing conditions, CaM binds RyR3 with reduced affinity but activates RyR3 to a greater extent. Under reducing conditions, RyR1 and RyR3 activities are inhibited with a similar affinity at [Ca(2+)] > 1 microM. Mutagenesis studies demonstrate that RyR3 contains a single conserved CaM binding site. Corresponding amino acid substitutions in the CaM binding site differentially affect CaM binding and CaM regulation of RyR3 and those of the two other isoforms. The results support the suggestion that other isoform dependent regions have a major role in the regulation of RyRs by CaM [Yamaguchi et al. (2004) J. Biol. Chem. 279, 36433-36439].

  1. ATP interacts with the CPVT mutation-associated central domain of the cardiac ryanodine receptor.

    PubMed

    Blayney, Lynda; Beck, Konrad; MacDonald, Ewan; D'Cruz, Leon; Nomikos, Michail; Griffiths, Julia; Thanassoulas, Angelos; Nounesis, George; Lai, F Anthony

    2013-10-01

    This study was designed to determine whether the cardiac ryanodine receptor (RyR2) central domain, a region associated with catecholamine polymorphic ventricular tachycardia (CPVT) mutations, interacts with the RyR2 regulators, ATP and the FK506-binding protein 12.6 (FKBP12.6). Wild-type (WT) RyR2 central domain constructs (G(2236)to G(2491)) and those containing the CPVT mutations P2328S and N2386I, were expressed as recombinant proteins. Folding and stability of the proteins were examined by circular dichroism (CD) spectroscopy and guanidine hydrochloride chemical denaturation. The far-UV CD spectra showed a soluble stably-folded protein with WT and mutant proteins exhibiting a similar secondary structure. Chemical denaturation analysis also confirmed a stable protein for both WT and mutant constructs with similar two-state unfolding. ATP and caffeine binding was measured by fluorescence spectroscopy. Both ATP and caffeine bound with an EC50 of ~200-400μM, and the affinity was the same for WT and mutant constructs. Sequence alignment with other ATP binding proteins indicated the RyR2 central domain contains the signature of an ATP binding pocket. Interaction of the central domain with FKBP12.6 was tested by glutaraldehyde cross-linking and no association was found. The RyR2 central domain, expressed as a 'correctly' folded recombinant protein, bound ATP in accord with bioinformatics evidence of conserved ATP binding sequence motifs. An interaction with FKBP12.6 was not evident. CPVT mutations did not disrupt the secondary structure nor binding to ATP. Part of the RyR2 central domain CPVT mutation cluster, can be expressed independently with retention of ATP binding. Copyright © 2013 Elsevier B.V. All rights reserved.

  2. Differential expression of the cardiac ryanodine receptor in normal and arrhythmogenic right ventricular cardiomyopathy canine hearts.

    PubMed

    Meurs, Kathryn M; Lacombe, Veronique A; Dryburgh, Keith; Fox, Philip R; Reiser, Peter R; Kittleson, Mark D

    2006-08-01

    Arrhythmogenic right ventricular cardiomyopathy (ARVC) is a form of cardiomyopathy characterized by ventricular tachyarrhythmias and a fibrofatty infiltrate that is believed to preferentially affect the right ventricle. Mutations in the cardiac ryanodine receptor (RyR2) gene have been identified in some human families with a unique form of ARVC, ARVC2. Although the RyR2 has significant importance in excitation-contraction coupling across the ventricles, mutations in the gene encoding for it appear to have the greatest impact on the right ventricle in ARVC2. Using a canine model (boxer), the RyR2 protein and message RNA in the right ventricle, left ventricle and interventricular septum from normal dogs and dogs with ARVC were investigated by immunoblotting and real time PCR. The cardiac RyR2 message and protein expression were differentially expressed across the cardiac walls in the normal heart, with the lowest concentration expressed in the right ventricle (P < 0.05). The message and protein expression of the RyR2 were reduced in all chambers in the canine model of ARVC. We propose that the increased susceptibility of the right ventricle to ARVC may be associated with the lower baseline protein concentration of RyR2 in the normal right ventricle compared to the left ventricle and interventricular septum and that all three areas are equally affected in this canine model of ARVC. Using this naturally occurring model of canine ARVC, we may have provided new insights into the pathogenesis of this cardiomyopathy.

  3. Blockage of the Ryanodine Receptor via Azumolene Does Not Prevent Mechanical Ventilation-Induced Diaphragm Atrophy.

    PubMed

    Talbert, Erin E; Smuder, Ashley J; Kwon, Oh Sung; Sollanek, Kurt J; Wiggs, Michael P; Powers, Scott K

    2016-01-01

    Mechanical ventilation (MV) is a life-saving intervention for patients in respiratory failure. However, prolonged MV causes the rapid development of diaphragm muscle atrophy, and diaphragmatic weakness may contribute to difficult weaning from MV. Therefore, developing a therapeutic countermeasure to protect against MV-induced diaphragmatic atrophy is important. MV-induced diaphragm atrophy is due, at least in part, to increased production of reactive oxygen species (ROS) from diaphragm mitochondria and the activation of key muscle proteases (i.e., calpain and caspase-3). In this regard, leakage of calcium through the ryanodine receptor (RyR1) in diaphragm muscle fibers during MV could result in increased mitochondrial ROS emission, protease activation, and diaphragm atrophy. Therefore, these experiments tested the hypothesis that a pharmacological blockade of the RyR1 in diaphragm fibers with azumolene (AZ) would prevent MV-induced increases in mitochondrial ROS production, protease activation, and diaphragmatic atrophy. Adult female Sprague-Dawley rats underwent 12 hours of full-support MV while receiving either AZ or vehicle. At the end of the experiment, mitochondrial ROS emission, protease activation, and fiber cross-sectional area were determined in diaphragm muscle fibers. Decreases in muscle force production following MV indicate that the diaphragm took up a sufficient quantity of AZ to block calcium release through the RyR1. However, our findings reveal that AZ treatment did not prevent the MV-induced increase in mitochondrial ROS emission or protease activation in the diaphragm. Importantly, AZ treatment did not prevent MV-induced diaphragm fiber atrophy. Thus, pharmacological inhibition of the RyR1 in diaphragm muscle fibers is not sufficient to prevent MV-induced diaphragm atrophy.

  4. Blockage of the Ryanodine Receptor via Azumolene Does Not Prevent Mechanical Ventilation-Induced Diaphragm Atrophy

    PubMed Central

    Talbert, Erin E.; Smuder, Ashley J.; Kwon, Oh Sung; Sollanek, Kurt J.; Wiggs, Michael P.; Powers, Scott K.

    2016-01-01

    Mechanical ventilation (MV) is a life-saving intervention for patients in respiratory failure. However, prolonged MV causes the rapid development of diaphragm muscle atrophy, and diaphragmatic weakness may contribute to difficult weaning from MV. Therefore, developing a therapeutic countermeasure to protect against MV-induced diaphragmatic atrophy is important. MV-induced diaphragm atrophy is due, at least in part, to increased production of reactive oxygen species (ROS) from diaphragm mitochondria and the activation of key muscle proteases (i.e., calpain and caspase-3). In this regard, leakage of calcium through the ryanodine receptor (RyR1) in diaphragm muscle fibers during MV could result in increased mitochondrial ROS emission, protease activation, and diaphragm atrophy. Therefore, these experiments tested the hypothesis that a pharmacological blockade of the RyR1 in diaphragm fibers with azumolene (AZ) would prevent MV-induced increases in mitochondrial ROS production, protease activation, and diaphragmatic atrophy. Adult female Sprague-Dawley rats underwent 12 hours of full-support MV while receiving either AZ or vehicle. At the end of the experiment, mitochondrial ROS emission, protease activation, and fiber cross-sectional area were determined in diaphragm muscle fibers. Decreases in muscle force production following MV indicate that the diaphragm took up a sufficient quantity of AZ to block calcium release through the RyR1. However, our findings reveal that AZ treatment did not prevent the MV-induced increase in mitochondrial ROS emission or protease activation in the diaphragm. Importantly, AZ treatment did not prevent MV-induced diaphragm fiber atrophy. Thus, pharmacological inhibition of the RyR1 in diaphragm muscle fibers is not sufficient to prevent MV-induced diaphragm atrophy. PMID:26849371

  5. Cardiac calcium signalling pathologies associated with defective calmodulin regulation of type 2 ryanodine receptor

    PubMed Central

    Arnáiz-Cot, Juan José; Damon, Brooke James; Zhang, Xiao-Hua; Cleemann, Lars; Yamaguchi, Naohiro; Meissner, Gerhard; Morad, Martin

    2013-01-01

    Cardiac ryanodine receptor (RyR2) is a homotetramer of 560 kDa polypeptides regulated by calmodulin (CaM), which decreases its open probability at diastolic and systolic Ca2+ concentrations. Point mutations in the CaM-binding domain of RyR2 (W3587A/L3591D/F3603A, RyR2ADA) in mice result in severe cardiac hypertrophy, poor left ventricle contraction and death by postnatal day 16, suggesting that CaM inhibition of RyR2 is required for normal cardiac function. Here, we report on Ca2+ signalling properties of enzymatically isolated, Fluo-4 dialysed whole cell clamped cardiac myocytes from 10–15-day-old wild-type (WT) and homozygous Ryr2ADA/ADA mice. Spontaneously occurring Ca2+ spark frequency, measured at −80 mV, was 14-fold lower in mutant compared to WT myocytes. ICa, though significantly smaller in mutant myocytes, triggered Ca2+ transients that were of comparable size to those of WT myocytes, but with slower activation and decay kinetics. Caffeine-triggered Ca2+ transients were about three times larger in mutant myocytes, generating three- to four-fold bigger Na+-Ca2+ exchanger NCX currents (INCX). Mutant myocytes often exhibited Ca2+ transients of variable size and duration that were accompanied by similarly alternating and slowly activating INCX. The data suggest that RyR2ADA mutation produces significant reduction in ICa density and ICa-triggered Ca2+ release gain, longer but infrequently occurring Ca2+ sparks, larger sarcoplasmic reticulum Ca2+ loads, and spontaneous Ca2+ releases accompanied by activation of large and potentially arrhythmogenic inward INCX. PMID:23836685

  6. A new cytoplasmic interaction between junctin and ryanodine receptor Ca2+ release channels

    PubMed Central

    Li, Linwei; Mirza, Shamaruh; Richardson, Spencer J.; Gallant, Esther M.; Thekkedam, Chris; Pace, Suzy M.; Zorzato, Francesco; Liu, Dan; Beard, Nicole A.; Dulhunty, Angela F.

    2015-01-01

    ABSTRACT Junctin, a non-catalytic splice variant encoded by the aspartate-β-hydroxylase (Asph) gene, is inserted into the membrane of the sarcoplasmic reticulum (SR) Ca2+ store where it modifies Ca2+ signalling in the heart and skeletal muscle through its regulation of ryanodine receptor (RyR) Ca2+ release channels. Junctin is required for normal muscle function as its knockout leads to abnormal Ca2+ signalling, muscle dysfunction and cardiac arrhythmia. However, the nature of the molecular interaction between junctin and RyRs is largely unknown and was assumed to occur only in the SR lumen. We find that there is substantial binding of RyRs to full junctin, and the junctin luminal and, unexpectedly, cytoplasmic domains. Binding of these different junctin domains had distinct effects on RyR1 and RyR2 activity: full junctin in the luminal solution increased RyR channel activity by ∼threefold, the C-terminal luminal interaction inhibited RyR channel activity by ∼50%, and the N-terminal cytoplasmic binding produced an ∼fivefold increase in RyR activity. The cytoplasmic interaction between junctin and RyR is required for luminal binding to replicate the influence of full junctin on RyR1 and RyR2 activity. The C-terminal domain of junctin binds to residues including the S1–S2 linker of RyR1 and N-terminal domain of junctin binds between RyR1 residues 1078 and 2156. PMID:25609705

  7. Redox modification of ryanodine receptors contributes to sarcoplasmic reticulum Ca2+ leak in chronic heart failure.

    PubMed

    Terentyev, Dmitry; Györke, Inna; Belevych, Andriy E; Terentyeva, Radmila; Sridhar, Arun; Nishijima, Yoshinori; de Blanco, Esperanza Carcache; Khanna, Savita; Sen, Chandan K; Cardounel, Arturo J; Carnes, Cynthia A; Györke, Sandor

    2008-12-05

    Abnormal cardiac ryanodine receptor (RyR2) function is recognized as an important factor in the pathogenesis of heart failure (HF). However, the specific molecular causes underlying RyR2 defects in HF remain poorly understood. In the present study, we used a canine model of chronic HF to test the hypothesis that the HF-related alterations in RyR2 function are caused by posttranslational modification by reactive oxygen species generated in the failing heart. Experimental approaches included imaging of cytosolic ([Ca(2+)](c)) and sarcoplasmic reticulum (SR) luminal Ca(2+) ([Ca(2+)]SR) in isolated intact and permeabilized ventricular myocytes and single RyR2 channel recording using the planar lipid bilayer technique. The ratio of reduced to oxidized glutathione, as well as the level of free thiols on RyR2 decreased markedly in failing versus control hearts consistent with increased oxidative stress in HF. RyR2-mediated SR Ca(2+) leak was significantly enhanced in permeabilized myocytes, resulting in reduced [Ca(2+)](SR) in HF compared to control cells. Both SR Ca(2+) leak and [Ca(2+)](SR) were partially normalized by treating HF myocytes with reducing agents. Conversely, oxidizing agents accelerated SR Ca(2+) leak and decreased [Ca(2+)](SR) in cells from normal hearts. Moreover, exposure to antioxidants significantly improved intracellular Ca(2+)-handling parameters in intact HF myocytes. Single RyR2 channel activity was significantly higher in HF versus control because of increased sensitivity to activation by luminal Ca(2+) and was partially normalized by reducing agents through restoring luminal Ca(2+) sensitivity oxidation of control RyR2s enhanced their luminal Ca(2+) sensitivity, thus reproducing the HF phenotype. These findings suggest that redox modification contributes to abnormal function of RyR2s in HF, presenting a potential therapeutic target for treating HF.

  8. The role of ryanodine receptor type 3 in a mouse model of Alzheimer disease.

    PubMed

    Liu, Jie; Supnet, Charlene; Sun, Suya; Zhang, Hua; Good, Levi; Popugaeva, Elena; Bezprozvanny, Ilya

    2014-01-01

    Dysregulated endoplasmic reticulum (ER) calcium (Ca(2+)) signaling is reported to play an important role in Alzheimer disease (AD) pathogenesis. The role of ER Ca(2+) release channels, the ryanodine receptors (RyanRs), has been extensivelys tudied in AD models and RyanR expression and activity are upregulated in the brains of various familial AD (FAD) models.The objective of this study was to utilize a genetic approach to evaluate the importance of RyanR type 3 (RyanR3) in the context of AD pathology.The expression of RyanR3 was also elevated in hippocampus of APPPS1 mice (Thy1-APPKM670/671NL, Thy1-PS1L166P).In young (≤ 3 mo) APPPS1 mice, the deletion of RyanR3 increased hippocampal neuronal network excitability and accelerated AD pathology, leading to mushroom spine loss and increased amyloid accumulation. In contrast, deletion of RyanR3 in older APPPS1 mice (≥ 6 mo) rescued network excitability and mushroom spine loss, reduced amyloid plaque load and reduced spontaneous seizure occurrence.Our data suggests a dual role for RyanR3 in AD pathology. In young AD neurons, RyanR3 protects AD neurons from synaptic and network dysfunction. In older AD neurons, increased RyanR3 activity contributes to pathology. These results imply that blockade of RyanR3 may be beneficial for those in the later stages of the disease, but RyanR activators may be beneficial when used prior to disease onset or in its initial stages. Caffeine is an activator of RyanRs and our results may help to explain a complex epidemiological connection between coffee consumption in mid-life and risk of AD development in old age.

  9. Ryanodine receptor 2 contributes to hemorrhagic shock-induced bi-phasic vascular reactivity in rats

    PubMed Central

    Zhou, Rong; Ding, Xiao-li; Liu, Liang-ming

    2014-01-01

    Aim: Ryanodine receptor 2 (RyR2) is a critical component of intracellular Ca2+ signaling in vascular smooth muscle cells (VSMCs). The aim of this study was to investigate the role of RyR2 in abnormal vascular reactivity after hemorrhagic shock in rats. Methods: SD rats were hemorrhaged and maintained mean arterial pressure (MAP) at 40 mmHg for 30 min or 2 h, and then superior mesenteric arteries (SMA) rings were prepared to measure the vascular reactivity. In other experiments, SMA rings of normal rats and rat VSMCs were exposed to a hypoxic medium for 10 min or 3 h. SMA rings of normal rats and VSMCs were transfected with siRNA against RyR2. Intracellular Ca2+ release in VSMCs was assessed using Fura-2/AM. Results: The vascular reactivity of the SMA rings from hemorrhagic rats was significantly increased in the early stage (30 min), but decreased in the late stage (2 h) of hemorrhagic shock. Similar results were observed in the SMA rings exposed to hypoxia for 10 min or 3 h. The enhanced vascular reactivity of the SMA rings exposed to hypoxia for 10 min was partly attenuated by transfection with RyR2 siRNA, whereas the blunted vascular reactivity of the SMA rings exposed to hypoxia for 3 h was partly restored by transfection with RyR2 siRNA. Treatment with the RyR agonist caffeine (1 mmol/L) significantly increased Ca2+ release in VSMCs exposed to hypoxia for 10 min or 3 h, which was partially antagonized by transfection with RyR2 siRNA. Conclusion: RyR2-mediated Ca2+ release contributes to the development of bi-phasic vascular reactivity induced by hemorrhagic shock or hypoxia. PMID:25263335

  10. What role does modulation of the ryanodine receptor play in cardiac inotropy and arrhythmogenesis?

    PubMed

    Eisner, D A; Kashimura, T; O'Neill, S C; Venetucci, L A; Trafford, A W

    2009-04-01

    In this article we review the role of the Ryanodine Receptor (RyR) in cardiac inotropy and arrhythmogenesis. Most of the calcium that activates cardiac contraction comes from the sarcoplasmic reticulum (SR) from where it is released through the RyR. The amplitude of the systolic Ca transient depends steeply on the SR Ca content and it is therefore important that SR content be regulated. This regulation occurs via changes of SR Ca content affecting systolic Ca and thence sarcolemmal Ca fluxes. In the steady state, the cardiac myocyte must be in Ca flux balance on each beat and this has implications for understanding even simple inotropic manoeuvres. The main part of the review considers the effects of modulating the RyR on systolic Ca. Potentiation of RyR opening produces an increase of the amplitude of the Ca transient but this effect disappears within a few beats because the increased sarcolemmal efflux of Ca decreases SR Ca content. We conclude that it is therefore unlikely that potentiation of the RyR by phosphorylation plays a dominant role in the actions of positive inotropic agents such as beta-adrenergic stimulation. Some cardiac arrhythmias result from release of Ca from the SR in the form of waves. This is best known to occur when the SR is overloaded with calcium. Mutations in the RyR also produce cardiac arrhythmias attributed to Ca waves due to leaky RyRs and a similar leak has been suggested to contribute to arrhythmias in heart failure. We show that, due to compensatory changes of SR Ca content, simply making the RyR leaky does not produce Ca waves in the steady state and that SR Ca content is critical in determining whether Ca waves occur.

  11. In vitro Modeling of Ryanodine Receptor 2 Dysfunction Using Human Induced Pluripotent Stem Cells

    PubMed Central

    Fatima, Azra; Xu, Guoxing; Shao, Kaifeng; Papadopoulos, Symeon; Lehmann, Martin; Arnáiz-Cot, Juan J.; Rosa, Angelo O.; Nguemo, Filomain; Matzkies, Matthias; Dittmann, Sven; Stone, Susannah L.; Linke, Matthias; Zechner, Ulrich; Beyer, Vera; Hennies, Hans Christian; Rosenkranz, Stephan; Klauke, Baerbel; Parwani, Abdul S.; Haverkamp, Wilhelm; Pfitzer, Gabriele; Farr, Martin; Cleemann, Lars; Morad, Martin; Milting, Hendrik; Hescheler, Juergen; Šaric, Tomo

    2011-01-01

    Background/Aims: Induced pluripotent stem (iPS) cells generated from accessible adult cells of patients with genetic diseases open unprecedented opportunities for exploring the pathophysiology of human diseases in vitro. Catecholaminergic polymorphic ventricular tachycardia type 1 (CPVT1) is an inherited cardiac disorder that is caused by mutations in the cardiac ryanodine receptor type 2 gene (RYR2) and is characterized by stress-induced ventricular arrhythmia that can lead to sudden cardiac death in young individuals. The aim of this study was to generate iPS cells from a patient with CPVT1 and determine whether iPS cell-derived cardiomyocytes carrying patient specific RYR2 mutation recapitulate the disease phenotype in vitro. Methods: iPS cells were derived from dermal fibroblasts of healthy donors and a patient with CPVT1 carrying the novel heterozygous autosomal dominant mutation p.F2483I in the RYR2. Functional properties of iPS cell derived-cardiomyocytes were analyzed by using whole-cell current and voltage clamp and calcium imaging techniques. Results: Patch-clamp recordings revealed arrhythmias and delayed afterdepolarizations (DADs) after catecholaminergic stimulation of CPVT1-iPS cell-derived cardiomyocytes. Calcium imaging studies showed that, compared to healthy cardiomyocytes, CPVT1-cardiomyocytes exhibit higher amplitudes and longer durations of spontaneous Ca2+ release events at basal state. In addition, in CPVT1-cardiomyocytes the Ca2+-induced Ca2+-release events continued after repolarization and were abolished by increasing the cytosolic cAMP levels with forskolin. Conclusion: This study demonstrates the suitability of iPS cells in modeling RYR2-related cardiac disorders in vitro and opens new opportunities for investigating the disease mechanism in vitro, developing new drugs, predicting their toxicity, and optimizing current treatment strategies. PMID:22178870

  12. Ryanodine Receptor Activation Induces Long-Term Plasticity of Spine Calcium Dynamics

    PubMed Central

    Pannasch, Ulrike; Rückl, Martin; Rüdiger, Sten; Schmitz, Dietmar

    2015-01-01

    A key feature of signalling in dendritic spines is the synapse-specific transduction of short electrical signals into biochemical responses. Ca2+ is a major upstream effector in this transduction cascade, serving both as a depolarising electrical charge carrier at the membrane and an intracellular second messenger. Upon action potential firing, the majority of spines are subject to global back-propagating action potential (bAP) Ca2+ transients. These transients translate neuronal suprathreshold activation into intracellular biochemical events. Using a combination of electrophysiology, two-photon Ca2+ imaging, and modelling, we demonstrate that bAPs are electrochemically coupled to Ca2+ release from intracellular stores via ryanodine receptors (RyRs). We describe a new function mediated by spine RyRs: the activity-dependent long-term enhancement of the bAP-Ca2+ transient. Spines regulate bAP Ca2+ influx independent of each other, as bAP-Ca2+ transient enhancement is compartmentalized and independent of the dendritic Ca2+ transient. Furthermore, this functional state change depends exclusively on bAPs travelling antidromically into dendrites and spines. Induction, but not expression, of bAP-Ca2+ transient enhancement is a spine-specific function of the RyR. We demonstrate that RyRs can form specific Ca2+ signalling nanodomains within single spines. Functionally, RyR mediated Ca2+ release in these nanodomains induces a new form of Ca2+ transient plasticity that constitutes a spine specific storage mechanism of neuronal suprathreshold activity patterns. PMID:26098891

  13. Ryanodine receptor type 2 is required for the development of pressure overload-induced cardiac hypertrophy.

    PubMed

    Zou, Yunzeng; Liang, Yanyan; Gong, Hui; Zhou, Ning; Ma, Hong; Guan, Aili; Sun, Aijun; Wang, Ping; Niu, Yuhong; Jiang, Hong; Takano, Hiroyuki; Toko, Haruhiro; Yao, Atsushi; Takeshima, Hiroshi; Akazawa, Hiroshi; Shiojima, Ichiro; Wang, Yuqi; Komuro, Issei; Ge, Junbo

    2011-12-01

    Ryanodine receptor type 2 (RyR-2) mediates Ca(2+) release from sarcoplasmic reticulum and contributes to myocardial contractile function. However, the role of RyR-2 in the development of cardiac hypertrophy is not completely understood. Here, mice with or without reduction of RyR-2 gene (RyR-2(+/-) and wild-type, respectively) were analyzed. At baseline, there was no difference in morphology of cardiomyocyte and heart and cardiac contractility between RyR-2(+/-) and wild-type mice, although Ca(2+) release from sarcoplasmic reticulum was impaired in isolated RyR-2(+/-) cardiomyocytes. During a 3-week period of pressure overload, which was induced by constriction of transverse aorta, isolated RyR-2(+/-) cardiomyocytes displayed more reduction of Ca(2+) transient amplitude, rate of an increase in intracellular Ca(2+) concentration during systole, and percentile of fractional shortening, and hearts of RyR-2(+/-) mice displayed less compensated hypertrophy, fibrosis, and contractility; more apoptosis with less autophagy of cardiomyocytes; and similar decrease of angiogenesis as compared with wild-type ones. Moreover, constriction of transverse aorta-induced increases in the activation of calcineurin, extracellular signal-regulated protein kinases, and protein kinase B/Akt but not that of Ca(2+)/calmodulin-dependent protein kinase II, and its downstream targets in the heart of wild-type mice were abolished in the RyR-2(+/-) one, suggesting that RyR-2 is a regulator of calcineurin, extracellular signal-regulated protein kinases, and Akt but not of calmodulin-dependent protein kinase II activation during pressure overload. Taken together, our data indicate that RyR-2 contributes to the development of cardiac hypertrophy and adaptation of cardiac function during pressure overload through regulation of the sarcoplasmic reticulum Ca(2+) release; activation of calcineurin, extracellular signal-regulated protein kinases, and Akt; and cardiomyocyte survival.

  14. Peptide fragments of the dihydropyridine receptor can modulate cardiac ryanodine receptor channel activity and sarcoplasmic reticulum Ca2+ release.

    PubMed Central

    Dulhunty, Angela F; Curtis, Suzanne M; Cengia, Louise; Sakowska, Magdalena; Casarotto, Marco G

    2004-01-01

    We show that peptide fragments of the dihydropyridine receptor II-III loop alter cardiac RyR (ryanodine receptor) channel activity in a cytoplasmic Ca2+-dependent manner. The peptides were AC (Thr-793-Ala-812 of the cardiac dihydropyridine receptor), AS (Thr-671-Leu-690 of the skeletal dihydropyridine receptor), and a modified AS peptide [AS(D-R18)], with an extended helical structure. The peptides added to the cytoplasmic side of channels in lipid bilayers at > or = 10 nM activated channels when the cytoplasmic [Ca2+] was 100 nM, but either inhibited or did not affect channel activity when the cytoplasmic [Ca2+] was 10 or 100 microM. Both activation and inhibition were independent of bilayer potential. Activation by AS, but not by AC or AS(D-R18), was reduced at peptide concentrations >1 mM in a voltage-dependent manner (at +40 mV). In control experiments, channels were not activated by the scrambled AS sequence (ASS) or skeletal II-III loop peptide (NB). Resting Ca2+ release from cardiac sarcoplasmic reticulum was not altered by peptide AC, but Ca2+-induced Ca2+ release was depressed. Resting and Ca2+-induced Ca2+ release were enhanced by both the native and modified AS peptides. NMR revealed (i) that the structure of peptide AS(D-R18) is not influenced by [Ca2+] and (ii) that peptide AC adopts a helical structure, particularly in the region containing positively charged residues. This is the first report of specific functional interactions between dihydropyridine receptor A region peptides and cardiac RyR ion channels in lipid bilayers. PMID:14678014

  15. Acetylcholine nicotinic receptor subtypes in chromaffin cells.

    PubMed

    Criado, Manuel

    2017-08-08

    In the adrenal gland, acetylcholine released on stimulation of the sympathetic splanchnic nerve activates neuronal-type nicotinic receptors (nAChRs) in chromaffin cells and triggers catecholamine secretion. At least two subtypes of nAChRs have been described in bovine chromaffin cells. The main subtype, a heteromeric assembly of α3, β4 and perhaps α5 subunits, is involved in the activation step of the catecholamine secretion process and is not blocked by the snake toxin α-bungarotoxin. The other is α-bungarotoxin-sensitive, and its functional role has not yet been well defined. The α7 subunit conforms the homomeric structure of this subtype. All nAChR subunits share the same molecular organization and structural data at atomic resolution level are now available for some homomeric and heteromeric ensembles. The α3, β4 and α5 subunits are clustered in genomes of different species, with the transcription factor Sp1 playing a co-ordinating role in the transcriptional regulation of these three subunits. The transcription factor Egr-1 controls the differential expression of α7 nAChR in adrenergic chromaffin cells, as happens with the enzyme phenylethanolamine N-methyl transferase. For unknown reasons, whole cell currents observed in bovine chromaffin cells clearly differ of the ones observed when different combinations of subunit RNAs are injected in oocytes. In addition to the typical nicotinic ligands, a variety of unrelated substances with clinical relevance can target nAChRs in chromaffin cells and, therefore, affect catecholamine secretion. They can act as agonists, antagonists or allosteric modulators.

  16. Characterization of RyR1-slow, a ryanodine receptor specific to slow-twitch skeletal muscle.

    PubMed

    Morrissette, J; Xu, L; Nelson, A; Meissner, G; Block, B A

    2000-11-01

    Two distinct skeletal muscle ryanodine receptors (RyR1s) are expressed in a fiber type-specific manner in fish skeletal muscle (11). In this study, we compare [(3)H]ryanodine binding and single channel activity of RyR1-slow from fish slow-twitch skeletal muscle with RyR1-fast and RyR3 isolated from fast-twitch skeletal muscle. Scatchard plots indicate that RyR1-slow has a lower affinity for [(3)H]ryanodine when compared with RyR1-fast. In single channel recordings, RyR1-slow and RyR1-fast had similar slope conductances. However, the maximum open probability (P(o)) of RyR1-slow was threefold less than the maximum P(o) of RyR1-fast. Single channel studies also revealed the presence of two populations of RyRs in tuna fast-twitch muscle (RyR1-fast and RyR3). RyR3 had the highest P(o) of all the RyR channels and displayed less inhibition at millimolar Ca(2+). The addition of 5 mM Mg-ATP or 2.5 mM beta, gamma-methyleneadenosine 5'-triphosphate (AMP-PCP) to the channels increased the P(o) and [(3)H]ryanodine binding of both RyR1s but also caused a shift in the Ca(2+) dependency curve of RyR1-slow such that Ca(2+)-dependent inactivation was attenuated. [(3)H]ryanodine binding data also showed that Mg(2+)-dependent inhibition of RyR1-slow was reduced in the presence of AMP-PCP. These results indicate differences in the physiological properties of RyRs in fish slow- and fast-twitch skeletal muscle, which may contribute to differences in the way intracellular Ca(2+) is regulated in these muscle types.

  17. Arachidonic acid activates release of calcium ions from reticulum via ryanodine receptor channels in C2C12 skeletal myotubes.

    PubMed

    Muslikhov, E R; Sukhanova, I F; Avdonin, P V

    2014-05-01

    Arachidonic acid causes an increase in free cytoplasmic calcium concentration ([Ca2+]i) in differentiated skeletal multinucleated myotubes C2C12 and does not induce calcium response in C2C12 myoblasts. The same reaction of myotubes to arachidonic acid is observed in Ca2+-free medium. This indicates that arachidonic acid induces release of calcium ions from intracellular stores. The blocker of ryanodine receptor channels of sarcoplasmic reticulum dantrolene (20 µM) inhibits this effect by 68.7 ± 6.3% (p < 0.001). The inhibitor of two-pore calcium channels of endolysosomal vesicles trans-NED19 (10 µM) decreases the response to arachidonic acid by 35.8 ± 5.4% (p < 0.05). The phospholipase C inhibitor U73122 (10 µM) has no effect. These data indicate the involvement of ryanodine receptor calcium channels of sarcoplasmic reticulum in [Ca2+]i elevation in skeletal myotubes caused by arachidonic acid and possible participation of two-pore calcium channels from endolysosomal vesicles in this process.

  18. Calmodulin and immunophilin are required as functional partners of a ryanodine receptor in ascidian oocytes at fertilization.

    PubMed

    Albrieux, M; Moutin, M J; Grunwald, D; Villaz, M

    2000-09-01

    Fertilization of oocytes incites numerous changes relying on Ca(2+) signaling. In inseminated ascidian eggs, an increase in the egg surface membrane, monitored by a change in electrical capacitance, is recorded at the onset of meiosis resumption. This membrane addition to the cell surface is controlled by calcium release through a ryanodine receptor (RyR), sensitive to cyclic ADP-ribose. Using confocal microscopy analysis of ascidian oocytes immunostained with anti-RyR antibody, we show here that this calcium channel is asymmetrically located in the vegetal cortical zone. Interestingly, the increase in cell capacitance occurring at fertilization is correlated with a fluorescent signal, imaged by the marker of vesicle trafficking FM 1-43, located close to the RyR region. Two putative partners of RyR, namely an FKBP-like protein and a calmodulin, are identified in these oocyte extracts by detection of enzyme activity and PCR amplification. Both are necessary to sustain ryanodine receptor activity in these oocytes since the membrane insertion triggered by fertilization is inhibited by the FKBP ligand rapamycin and by a calmodulin antagonist peptide. These findings suggest that exocytosis in ascidian eggs is triggered at fertilization by a functional Ca(2+) release unit operating as a complex of several proteins, including a calmodulin and an immunophilin, around the intracellular calcium channel itself. Copyright 2000 Academic Press.

  19. Hierarchical clustering of ryanodine receptors enables emergence of a calcium clock in sinoatrial node cells

    PubMed Central

    Maltseva, Larissa A.; Juhaszova, Magdalena; Sollott, Steven J.; Lakatta, Edward G.; Maltsev, Victor A.

    2014-01-01

    The sinoatrial node, whose cells (sinoatrial node cells [SANCs]) generate rhythmic action potentials, is the primary pacemaker of the heart. During diastole, calcium released from the sarcoplasmic reticulum (SR) via ryanodine receptors (RyRs) interacts with membrane currents to control the rate of the heartbeat. This “calcium clock” takes the form of stochastic, partially periodic, localized calcium release (LCR) events that propagate, wave-like, for limited distances. The detailed mechanisms controlling the calcium clock are not understood. We constructed a computational model of SANCs, including three-dimensional diffusion and buffering of calcium in the cytosol and SR; explicit, stochastic gating of individual RyRs and L-type calcium channels; and a full complement of voltage- and calcium-dependent membrane currents. We did not include an anatomical submembrane space or inactivation of RyRs, the two heuristic components that have been used in prior models but are not observed experimentally. When RyRs were distributed in discrete clusters separated by >1 µm, only isolated sparks were produced in this model and LCR events did not form. However, immunofluorescent staining of SANCs for RyR revealed the presence of bridging RyR groups between large clusters, forming an irregular network. Incorporation of this architecture into the model led to the generation of propagating LCR events. Partial periodicity emerged from the interaction of LCR events, as observed experimentally. This calcium clock becomes entrained with membrane currents to accelerate the beating rate, which therefore was controlled by the activity of the SERCA pump, RyR sensitivity, and L-type current amplitude, all of which are targets of β-adrenergic–mediated phosphorylation. Unexpectedly, simulations revealed the existence of a pathological mode at high RyR sensitivity to calcium, in which the calcium clock loses synchronization with the membrane, resulting in a paradoxical decrease in

  20. Coupled gating of skeletal muscle ryanodine receptors is modulated by Ca2+, Mg2+, and ATP

    PubMed Central

    Porta, Maura; Diaz-Sylvester, Paula L.; Neumann, Jake T.; Escobar, Ariel L.; Fleischer, Sidney

    2012-01-01

    Coupled gating (synchronous openings and closures) of groups of skeletal muscle ryanodine receptors (RyR1), which mimics RyR1-mediated Ca2+ release underlying Ca2+ sparks, was first described by Marx et al. (Marx SO, Ondrias K, Marks AR. Science 281: 818–821, 1998). The nature of the RyR1-RyR1 interactions for coupled gating still needs to be characterized. Consequently, we defined planar lipid bilayer conditions where ∼25% of multichannel reconstitutions contain mixtures of coupled and independently gating RyR1. In ∼10% of the cases, all RyRs (2–10 channels; most frequently 3–4) gated in coupled fashion, allowing for quantification. Our results indicated that coupling required cytosolic solutions containing ATP/Mg2+ and high (50 mM) luminal Ca2+ (Calum) or Sr2+ solutions. Bursts of coupled activity (events) started and ended abruptly, with all channels activating/deactivating within ∼300 μs. Coupled RyR1 were heterogeneous, where highly active RyR1 (“drivers”) seemed open during the entire coupled event (Po = 1), while other RyR1s (“followers”) displayed abundant flickering and smaller amplitude. Drivers mean open time increased with cytosolic Ca2+ (Cacyt) or caffeine, whereas followers flicker frequency was Cacyt independent and more sensitive to inhibition by cytosolic Mg2+. Coupled events were insensitive to varying lumen-to-cytosol Ca2+ fluxes from ∼1 to 8 pA, which does not corroborate coupling of neighboring RyR1 by local Ca2+-induced Ca2+ release. However, coupling requires specific Calum sites, as it was lost when Calum was replaced by luminal Ba2+ or Mg2+. In summary, coupled events reveal complex interactions among heterogeneous RyR1, differentially modulated by cytosolic ATP/Mg2+, Cacyt, and Calum, which under cell-like ionic conditions may parallel synchronous RyR1 gating during Ca2+ sparks. PMID:22785120

  1. Unbalanced upregulation of ryanodine receptor 2 plays a particular role in early development of daunorubicin cardiomyopathy

    PubMed Central

    Kucerova, Dana; Doka, Gabriel; Kruzliak, Peter; Turcekova, Katarina; Kmecova, Jana; Brnoliakova, Zuzana; Kyselovic, Jan; Kirchhefer, Uwe; Müller, Frank U; Krenek, Peter; Boknik, Peter; Klimas, Jan

    2015-01-01

    Calcium release channel on the sarcoplasmic reticulum of cardiomyocytes (ryanodine receptor type 2, RyR2) plays a critical role in the regulation of calcium and was identified as a crucial factor for development of chronic anthracycline cardiomyopathy. Its early stages are less well described although these determine the later development. Hence, we tested the effect of repeated, short-term anthracycline (daunorubicin) administration on cardiac performance, cardiomyocyte function and accompanied changes in calcium regulating proteins expression. Ten-twelve weeks old male Wistar rats were administered with 6 doses of daunorubicin (DAU, 3 mg/kg, i.p., every 48 h), controls (CON) received vehicle. Left ventricular function (left ventricular pressure, LVP; rate of pressure development, +dP/dt and decline, -dP/dt) was measured using left ventricular catheterization under tribromethanol anaesthesia (15 ml/kg b.w.). Cell shortening was measured in enzymatically isolated cardiomyocytes. The expressions of RyR2 and associated intracellular calcium regulating proteins, cytoskeletal proteins (alpha-actinin, alpha-tubul in) as well as oxidative stress regulating enzymes (gp91phox, MnSOD) were detected in ventricular tissue samples using immunoblotting. mRNA expressions of cardiac damage markers (Nppa and Nppb, atrial and brain natriuretic peptides; Myh6, Myh7 and Myh7b, myosin heavy chain alpha and beta) were detected using RT-PCR. Thiobarbituric acid reactive substances concentration was measured to estimate oxidative stress. DAU rats exhibited significantly depressed left ventricular features (LVP by 14%, +dP/dt by 36% and -dP/dt by 30%; for all P<0.05), in line with concomitant increase in Nppa and Nppb gene expressions (3.23- and 2.18-fold, for both P<0.05), and a 4.34-fold increase in Myh7 (P<0.05). Controversially, we observed increased cell shortening of isolated cardiac cells by 31% (p<0.05). DAU administration was associated with a twofold upregulation of RyR2 (P<0

  2. Expression of ryanodine receptor RyR3 produces Ca2+ sparks in dyspedic myotubes

    PubMed Central

    Ward, Christopher W; Schneider, Martin F; Castillo, Daniel; Protasi, Feliciano; Wang, Yaming; Wayne Chen, S R; Allen, Paul D

    2000-01-01

    Discrete, localized elevations of myoplasmic [Ca2+], Ca2+‘sparks’, were readily detected using the fluorescent Ca2+ indicator fluo-3 and laser scanning confocal microscopy in ‘dyspedic’ 1B5 myotubes, i.e. myotubes which do not express ryanodine receptors (RyRs), transduced with virions containing cDNA for RyR type 3 that were saponin permeabilized to allow dye entry. Ca2+ sparks were never observed in non-transduced RyR null myotubes.The spatial locations of sparks observed in permeabilized myotubes roughly corresponded to regions of RyR protein expression in the same myotube as detected after subsequent fixation and antibody staining.Permeabilized RyR3-transduced myotubes exhibited similar punctate peripheral RyR3 protein immunohistochemical patterns as myotubes fixed before permeabilization indicating that permeabilization did not affect the structural organization of the triad.Ca2+ sparks, recorded in line scan mode, in permeabilized myotubes expressing RyR3 exhibited mean amplitudes (change in fluorescence/mean fluorescence, ΔF/F: 1.20 ± 0.04) and temporal rise times (10-90 %; 6.31 ± 0.12 ms) similar to those of sparks recorded in permeabilized frog skeletal muscle fibres (0.98 ± 0.01; 6.11 ± 0.07, respectively) using the same confocal system. Spatial extent and temporal duration of the Ca2+ sparks were ≈40 % larger in the RyR3-expressing myotube cultures than in frog fibres.Ca2+ sparks recorded in line scan mode often occurred repetitively at the same spatial location in RyR3-expressing myotubes. Such repetitive events were highly reproducible in amplitude and spatio-temporal properties, as previously observed for repetitive mode sparks in frog skeletal muscle.Ca2+ sparks recorded in xy mode were frequently compressed in the y (slower scan) direction compared to the x direction. This asymmetry was reproduced assuming spatially symmetric events having the time course of Ca2+ sparks recorded in line scan (xt) mode.These expression studies

  3. Mechanism of calsequestrin regulation of single cardiac ryanodine receptor in normal and pathological conditions.

    PubMed

    Chen, Haiyan; Valle, Giorgia; Furlan, Sandra; Nani, Alma; Gyorke, Sandor; Fill, Michael; Volpe, Pompeo

    2013-08-01

    Release of Ca(2+) from the sarcoplasmic reticulum (SR) drives contractile function of cardiac myocytes. Luminal Ca(2+) regulation of SR Ca(2+) release is fundamental not only in physiology but also in physiopathology because abnormal luminal Ca(2+) regulation is known to lead to arrhythmias, catecholaminergic polymorphic ventricular tachycardia (CPVT), and/or sudden cardiac arrest, as inferred from animal model studies. Luminal Ca(2+) regulates ryanodine receptor (RyR)2-mediated SR Ca(2+) release through mechanisms localized inside the SR; one of these involves luminal Ca(2+) interacting with calsequestrin (CASQ), triadin, and/or junctin to regulate RyR2 function. CASQ2-RyR2 regulation was examined at the single RyR2 channel level. Single RyR2s were incorporated into planar lipid bilayers by the fusion of native SR vesicles isolated from either wild-type (WT), CASQ2 knockout (KO), or R33Q-CASQ2 knock-in (KI) mice. KO and KI mice have CPVT-like phenotypes. We show that CASQ2(WT) action on RyR2 function (either activation or inhibition) was strongly influenced by the presence of cytosolic MgATP. Function of the reconstituted CASQ2(WT)-RyR2 complex was unaffected by changes in luminal free [Ca(2+)] (from 0.1 to 1 mM). The inhibition exerted by CASQ2(WT) association with the RyR2 determined a reduction in cytosolic Ca(2+) activation sensitivity. RyR2s from KO mice were significantly more sensitive to cytosolic Ca(2+) activation and had significantly longer mean open times than RyR2s from WT mice. Sensitivity of RyR2s from KI mice was in between that of RyR2 channels from KO and WT mice. Enhanced cytosolic RyR2 Ca(2+) sensitivity and longer RyR2 open times likely explain the CPVT-like phenotype of both KO and KI mice.

  4. Ryanodine receptor point mutations confer diamide insecticide resistance in tomato leafminer, Tuta absoluta (Lepidoptera: Gelechiidae).

    PubMed

    Roditakis, Emmanouil; Steinbach, Denise; Moritz, Gerald; Vasakis, Emmanouil; Stavrakaki, Marianna; Ilias, Aris; García-Vidal, Lidia; Martínez-Aguirre, María Del Rosario; Bielza, Pablo; Morou, Evangelia; Silva, Jefferson E; Silva, Wellington M; Siqueira, Ηerbert A A; Iqbal, Sofia; Troczka, Bartlomiej J; Williamson, Martin S; Bass, Chris; Tsagkarakou, Anastasia; Vontas, John; Nauen, Ralf

    2017-01-01

    Insect ryanodine receptors (RyR) are the molecular target-site for the recently introduced diamide insecticides. Diamides are particularly active on Lepidoptera pests, including tomato leafminer, Tuta absoluta (Lepidoptera: Gelechiidae). High levels of diamide resistance were recently described in some European populations of T. absoluta, however, the mechanisms of resistance remained unknown. In this study the molecular basis of diamide resistance was investigated in a diamide resistant strain from Italy (IT-GELA-SD4), and additional resistant field populations collected in Greece, Spain and Brazil. The genetics of resistance was investigated by reciprocally crossing strain IT-GELA-SD4 with a susceptible strain and revealed an autosomal incompletely recessive mode of inheritance. To investigate the possible role of target-site mutations as known from diamondback moth (Plutella xylostella), we sequenced respective domains of the RyR gene of T. absoluta. Genotyping of individuals of IT-GELA-SD4 and field-collected strains showing different levels of diamide resistance revealed the presence of G4903E and I4746M RyR target-site mutations. These amino acid substitutions correspond to those recently described for diamide resistant diamondback moth, i.e. G4946E and I4790M. We also detected two novel mutations, G4903V and I4746T, in some of the resistant T. absoluta strains. Radioligand binding studies with thoracic membrane preparations of the IT-GELA-SD4 strain provided functional evidence that these mutations alter the affinity of the RyR to diamides. In combination with previous work on P. xylostella our study highlights the importance of position G4903 (G4946 in P. xylostella) of the insect RyR in defining sensitivity to diamides. The discovery of diamide resistance mutations in T. absoluta populations of diverse geographic origin has serious implications for the efficacy of diamides under applied conditions. The implementation of appropriate resistance

  5. Aldolase potentiates DIDS activation of the ryanodine receptor in rabbit skeletal sarcoplasmic reticulum

    PubMed Central

    Seo, In-Ra; Moh, Sang Hyun; Lee, Eun Hui; Meissner, Gerhard; Kim, Do Han

    2006-01-01

    DIDS (4,4′-di-isothiocyanostilbene-2,2′-disulfonate), an anion channel blocker, triggers Ca2+ release from skeletal muscle SR (sarcoplasmic reticulum). The present study characterized the effects of DIDS on rabbit skeletal single Ca2+-release channel/RyR1 (ryanodine receptor type 1) incorporated into a planar lipid bilayer. When junctional SR vesicles were used for channel incorporation (native RyR1), DIDS increased the mean Po (open probability) of RyR1 without affecting unitary conductance when Cs+ was used as the charge carrier. Lifetime analysis of single RyR1 activities showed that 10 μM DIDS induced reversible long-lived open events (Po=0.451±0.038) in the presence of 10 μM Ca2+, due mainly to a new third component for both open and closed time constants. However, when purified RyR1 was examined in the same condition, 10 μM DIDS became considerably less potent (Po=0.206±0.025), although the caffeine response was similar between native and purified RyR1. Hence we postulated that a DIDS-binding protein, essential for the DIDS sensitivity of RyR1, was lost during RyR1 purification. DIDS-affinity column chromatography of solubilized junctional SR, and MALDI–TOF (matrix-assisted laser-desorption ionization–time-of-flight) MS analysis of the affinity-column-associated proteins, identified four major DIDS-binding proteins in the SR fraction. Among them, aldolase was the only protein that greatly potentiated DIDS sensitivity. The association between RyR1 and aldolase was further confirmed by co-immunoprecipitation and aldolase-affinity batch-column chromatography. Taken together, we conclude that aldolase is physically associated with RyR1 and could confer a considerable potentiation of the DIDS effect on RyR1. PMID:16817780

  6. Hierarchical clustering of ryanodine receptors enables emergence of a calcium clock in sinoatrial node cells.

    PubMed

    Stern, Michael D; Maltseva, Larissa A; Juhaszova, Magdalena; Sollott, Steven J; Lakatta, Edward G; Maltsev, Victor A

    2014-05-01

    The sinoatrial node, whose cells (sinoatrial node cells [SANCs]) generate rhythmic action potentials, is the primary pacemaker of the heart. During diastole, calcium released from the sarcoplasmic reticulum (SR) via ryanodine receptors (RyRs) interacts with membrane currents to control the rate of the heartbeat. This "calcium clock" takes the form of stochastic, partially periodic, localized calcium release (LCR) events that propagate, wave-like, for limited distances. The detailed mechanisms controlling the calcium clock are not understood. We constructed a computational model of SANCs, including three-dimensional diffusion and buffering of calcium in the cytosol and SR; explicit, stochastic gating of individual RyRs and L-type calcium channels; and a full complement of voltage- and calcium-dependent membrane currents. We did not include an anatomical submembrane space or inactivation of RyRs, the two heuristic components that have been used in prior models but are not observed experimentally. When RyRs were distributed in discrete clusters separated by >1 µm, only isolated sparks were produced in this model and LCR events did not form. However, immunofluorescent staining of SANCs for RyR revealed the presence of bridging RyR groups between large clusters, forming an irregular network. Incorporation of this architecture into the model led to the generation of propagating LCR events. Partial periodicity emerged from the interaction of LCR events, as observed experimentally. This calcium clock becomes entrained with membrane currents to accelerate the beating rate, which therefore was controlled by the activity of the SERCA pump, RyR sensitivity, and L-type current amplitude, all of which are targets of β-adrenergic-mediated phosphorylation. Unexpectedly, simulations revealed the existence of a pathological mode at high RyR sensitivity to calcium, in which the calcium clock loses synchronization with the membrane, resulting in a paradoxical decrease in beating

  7. Ryanodine produces a low frequency stimulation-induced NMDA receptor-independent long-term potentiation in the rat dentate gyrus in vitro.

    PubMed Central

    Wang, Y; Wu, J; Rowan, M J; Anwyl, R

    1996-01-01

    1. The induction of long-term potentiation (LTP) was investigated in the rat dentate gyrus in the presence of ryanodine, an agent which is known to selectively bind to the ryanodine receptor (RyR) Ca2+ channels which regulate Ca2+ release from intracellular Ca2+ stores. 2. In control media, high frequency stimulation (HFS) induced LTP, and prolonged low frequency stimulation (LFS) induced long-term depression (LTD), of field excitatory postsynaptic potentials (EPSPs) and patch clamped excitatory postsynaptic currents (EPSCs). 3. In the presence of ryanodine, at a threshold concentration of about 1 microM, HFS-induced LTP was inhibited, whereas LFS (5 Hz, 900 pulses) now induced LTP. 4. The N-methyl-D-aspartate receptor (NMDAR) antagonist D-2-amino-phosphonopentanoate (D-AP5), at both 50 and 200 microM, did not prevent the induction of LTP by 5 Hz LFS in the presence of ryanodine. This demonstrates the NMDAR independence of LTP induction in the presence of ryanodine. Furthermore, D AP5 reversed the block of HFS-induced LTP by ryanodine. 5. The induction of LTP by 5 Hz LFS in the presence of ryanodine was blocked by lowering extracellular Ca2+, or by rapidly buffering intracellular Ca2+ to very low levels with BAPTA. 6. The induction of LTP by 5 Hz LFS was inhibited by the L-type voltage-gated Ca2+ channel blocker nifedipine, and also by Ni2+ a commonly used T type voltage-gated Ca2+ channel blocker. 7. The 5 Hz LFS-induced LTP in the presence of ryanodine was inhibited by the metabotropic glutamate receptor (mGluR) antagonist (+)-alpha-methyl 4-carboxyphenylglycine (MCPG). 8. The 5 Hz LFS-induced LTP in the presence of ryanodine was blocked by Ruthenium Red, an agent known to block RyR channel opening, and also by thapsigargin, an agent known to block-ATP-dependent Ca2+ uptake into endoplasmic reticulum. 9. The results of the present studies emphasize the importance of intracellular Ca2+ stores in the induction of LTP. PMID:8887781

  8. A variably spliced region in the type 1 ryanodine receptor may participate in an inter-domain interaction.

    PubMed

    Kimura, Takashi; Pace, Suzy M; Wei, Lan; Beard, Nicole A; Dirksen, Robert T; Dulhunty, Angela F

    2007-01-01

    The aim of the present study was to examine residues that are variably spliced in the juvenile and adult isoforms of the skeletal-muscle RyR1 (type 1 ryanodine receptor). The juvenile ASI(-) splice variant is less active than the adult ASI(+) variant and is overexpressed in patients with DM (myotonic dystrophy) [Kimura, Nakamori, Lueck, Pouliquin, Aoike, Fujimura, Dirksen, Takahashi, Dulhunty and Sakoda (2005) Hum. Mol. Genet. 14, 2189-2200]. In the present study, we explore the ASI region using synthetic peptides corresponding to rabbit RyR1 residues Thr3471-Gly3500 either containing [PASI(+)] or lacking [PASI(-)] the ASI residues. Both peptides increased [3H]ryanodine binding to rabbit RyR1s, increased Ca2+ release from sarcoplasmic reti-culum vesicles and increased single RyR1 channel activity. The peptide PASI(-) was more active in each case than PASI(+). [3H]Ryanodine binding to recombinant ASI(+)RyR1 or ASI(-)-RyR1 was enhanced more by PASI(-) than PASI(+), with the greatest increase seen when PASI(-) was added to ASI(-)RyR1. The activation of the RyR channels is consistent with the hypo-thesis that the peptides interrupt an inhibitory inter-domain inter-action and that PASI(-) is more effective at interrupting this interaction than PASI(+). We therefore suggest that the ASI(-) sequence interacts more tightly than the ASI(+) sequence with its binding partner, so that the ASI(-)RyR1 is more strongly inhibited (less active) than the ASI(+)RyR1. Thus the affinity of the binding partners in this inter-domain interaction may deter-mine the activities of the mature and juvenile isoforms of RyR1 and the stronger inhibition in the juvenile isoform may contribute to the myopathy in DM.

  9. Chloroform extract of hog barn dust modulates skeletal muscle ryanodine receptor calcium-release channel (RyR1)

    PubMed Central

    Tian, Chengju; Shao, Chun Hong; Fenster, Danielle S.; Mixan, Mark; Romberger, Debra J.; Toews, Myron L.

    2010-01-01

    Skeletal muscle weakness is a reported ailment in individuals working in commercial hog confinement facilities. To date, specific mechanisms responsible for this symptom remain undefined. The purpose of this study was to assess whether hog barn dust (HBD) contains components that are capable of binding to and modulating the activity of type 1 ryanodine receptor Ca2+-release channel (RyR1), a key regulator of skeletal muscle function. HBD collected from confinement facilities in Nebraska were extracted with chloroform, filtered, and rotary evaporated to dryness. Residues were resuspended in hexane-chloroform (20:1) and precipitates, referred to as HBDorg, were air-dried and studied further. In competition assays, HBDorg dose-dependently displaced [3H]ryanodine from binding sites on RyR1 with an IC50 of 1.5 ± 0.1 μg/ml (Ki = 0.4 ± 0.0 μg/ml). In single-channel assays using RyR1 reconstituted into a lipid bilayer, HBDorg exhibited three distinct dose-dependent effects: first it increased the open probability of RyR1 by increasing its gating frequency and dwell time in the open state, then it induced a state of reduced conductance (55% of maximum) that was more likely to occur and persist at positive holding potentials, and finally it irreversibly closed RyR1. In differentiated C2C12 myotubes, addition of HBD triggered a rise in intracellular Ca2+ that was blocked by pretreatment with ryanodine. Since persistent activation and/or closure of RyR1 results in skeletal muscle weakness, these new data suggest that HBD is responsible, at least in part, for the muscle ailment reported by hog confinement workers. PMID:20576841

  10. Chloroform extract of hog barn dust modulates skeletal muscle ryanodine receptor calcium-release channel (RyR1).

    PubMed

    Tian, Chengju; Shao, Chun Hong; Fenster, Danielle S; Mixan, Mark; Romberger, Debra J; Toews, Myron L; Bidasee, Keshore R

    2010-09-01

    Skeletal muscle weakness is a reported ailment in individuals working in commercial hog confinement facilities. To date, specific mechanisms responsible for this symptom remain undefined. The purpose of this study was to assess whether hog barn dust (HBD) contains components that are capable of binding to and modulating the activity of type 1 ryanodine receptor Ca2+-release channel (RyR1), a key regulator of skeletal muscle function. HBD collected from confinement facilities in Nebraska were extracted with chloroform, filtered, and rotary evaporated to dryness. Residues were resuspended in hexane-chloroform (20:1) and precipitates, referred to as HBDorg, were air-dried and studied further. In competition assays, HBDorg dose-dependently displaced [3H]ryanodine from binding sites on RyR1 with an IC50 of 1.5±0.1 microg/ml (Ki=0.4±0.0 microg/ml). In single-channel assays using RyR1 reconstituted into a lipid bilayer, HBDorg exhibited three distinct dose-dependent effects: first it increased the open probability of RyR1 by increasing its gating frequency and dwell time in the open state, then it induced a state of reduced conductance (55% of maximum) that was more likely to occur and persist at positive holding potentials, and finally it irreversibly closed RyR1. In differentiated C2C12 myotubes, addition of HBD triggered a rise in intracellular Ca2+ that was blocked by pretreatment with ryanodine. Since persistent activation and/or closure of RyR1 results in skeletal muscle weakness, these new data suggest that HBD is responsible, at least in part, for the muscle ailment reported by hog confinement workers.

  11. The ryanodine receptor/calcium channel genes are widely and differentially expressed in murine brain and peripheral tissues

    PubMed Central

    1995-01-01

    Ryanodine receptors (RyRs) are intracellular calcium release channels that participate in controlling cytosolic calcium levels. At variance with the probably ubiquitous inositol 1,4,5-trisphosphate-operated calcium channels (1,4,5-trisphosphate receptors), RyRs have been mainly regarded as the calcium release channels controlling skeletal and cardiac muscle contraction. Increasing evidence has recently suggested that RyRs may be more widely expressed, but this has never been extensively examined. Therefore, we cloned three cDNAs corresponding to murine RyR homologues to carry a comprehensive analysis of their expression in murine tissues. Here, we report that the three genes are expressed in almost all tissues analyzed, where tissue-specific patterns of expression were observed. In the uterus and vas deferens, expression of RyR3 was localized to the smooth muscle component of these organs. In the testis, expression of RyR1 and RyR3 was detected in germ cells. RyR mRNAs were also detected in in vitro-cultured cell lines. RyR1, RyR2, and RyR3 mRNA were detected in the cerebrum and in the cerebellum. In situ analysis revealed a cell type-specific pattern of expression in the different regions of the central nervous system. The differential expression of the three ryanodine receptor genes in the central nervous system was also confirmed using specific antibodies against the respective proteins. This widespread pattern of expression suggests that RyRs may participate in the regulation of intracellular calcium homeostasis in a range of cells wider than previously recognized. PMID:7876312

  12. P2 receptor subtypes in the cardiovascular system.

    PubMed Central

    Kunapuli, S P; Daniel, J L

    1998-01-01

    Extracellular nucleotides have been implicated in a number of physiological functions. Nucleotides act on cell-surface receptors known as P2 receptors, of which several subtypes have been cloned. Both ATP and ADP are stored in platelets and are released upon platelet activation. Furthermore, nucleotides are also released from damaged or broken cells. Thus during vascular injury nucleotides play an important role in haemostasis through activation of platelets, modulation of vascular tone, recruitment of neutrophils and monocytes to the site of injury, and facilitation of adhesion of leucocytes to the endothelium. Nucleotides also moderate these functions by generating nitric oxide and prostaglandin I2 through activation of endothelial cells, and by activating different receptor subtypes on vascular smooth muscle cells. In the heart, P2 receptors regulate contractility through modulation of L-type Ca2+ channels, although the molecular mechanisms involved are still under investigation. Classical pharmacological studies have identified several P2 receptor subtypes in the cardiovascular system. Molecular pharmacological studies have clarified the nature of some of these receptors, but have complicated the picture with others. In platelets, the classical P2T receptor has now been resolved into three P2 receptor subtypes: the P2Y1, P2X1 and P2TAC receptors (the last of these, which is coupled to the inhibition of adenylate cyclase, is yet to be cloned). In peripheral blood leucocytes, endothelial cells, vascular smooth muscle cells and cardiomyocytes, the effects of classical P2X, P2Y and P2U receptors have been found to be mediated by more than one P2 receptor subtype. However, the exact functions of these multiple receptor subtypes remain to be understood, as P2-receptor-selective agonists and antagonists are still under development. PMID:9841859

  13. Clinical Significance of Ryanodine Receptor 1 Gene (RYR1) Variants: Proceedings of the 2013 MHAUS Scientific Conference

    PubMed Central

    Riazi, Sheila; Kraeva, Natalia; Muldoon, Sheila M.; Dowling, James; Ho, Clara; Petre, Maria-Alexandra; Parness, Jerome; Dirksen, Robert T.; Rosenberg, Henry

    2014-01-01

    The Malignant Hyperthermia Association of the United States (MHAUS) and the Department of Anesthesia at the University of Toronto sponsored a Scientific Conference on November 1–2, 2013 in Toronto, Canada. The multidisciplinary group of experts, including clinicians, geneticists and physiologists involved in research related to malignant hyperthermia (MH), shared new insights into the pathophysiology of type-1 ryanodine receptor gene (RYR1)-linked diseases, as well as the relationship between MH and “awake MH” conditions, such as exertional rhabdomyolysis (ER) and exertional heat illness (EHI). In addition, the molecular genetics of MH, and clinical issues related to the diagnosis and management of RYR1-linked disorders, were presented. The conference also honored Dr. David H. MacLennan for his contributions to our understanding of the genetics, pathogenesis and treatment of MH and other RYR1-related myopathies. This report represents a summary of the proceedings of this conference. PMID:25189431

  14. Heterogeneity of muscarinic receptor subtypes in cerebral blood vessels

    SciTech Connect

    Garcia-Villalon, A.L.; Krause, D.N.; Ehlert, F.J.; Duckles, S.P. )

    1991-07-01

    The identity and distribution of muscarinic cholinergic receptor subtypes and associated signal transduction mechanisms was characterized for the cerebral circulation using correlated functional and biochemical investigations. Subtypes were distinguished by the relative affinities of a panel of muscarinic antagonists, pirenzepine, AF-DX 116 (11-2-((2-(diethylaminomethyl)- 1-piperidinyl)acetyl)-5,11-dihydro-6H- pyrido(2,3-b)(1,4)benzodiazepine-6-one), hexahydrosiladifenidol, methoctramine, 4-diphenylacetoxy-N-methylpiperidine methobromide, dicyclomine, para-fluoro-hexahydrosiladifenidol and atropine. Muscarinic receptors characterized by inhibition of (3H)quinuclidinylbenzilate binding in membranes of bovine pial arteries were of the M2 subtype. In contrast pharmacological analysis of (3H)-quinuclidinylbenzilate binding in bovine intracerebral microvessels suggests the presence of an M4 subtype. Receptors mediating endothelium-dependent vasodilation in rabbit pial arteries were of the M3 subtype, whereas muscarinic receptors stimulating endothelium-independent phosphoinositide hydrolysis in bovine pial arteries were of the M1 subtype. These findings suggest that characteristics of muscarinic receptors in cerebral blood vessels vary depending on the type of vessel, cellular location and function mediated.

  15. Three-dimensional structure of ryanodine receptor isoform three in two conformational states as visualized by cryo-electron microscopy.

    PubMed

    Sharma, M R; Jeyakumar, L H; Fleischer, S; Wagenknecht, T

    2000-03-31

    Using cryo-electron microscopy and single particle image processing techniques, we present the first three-dimensional reconstructions of isoform 3 of the ryanodine receptor/calcium release channel (RyR3). Reconstructions were carried out on images obtained from a purified, detergent-solubilized receptor for two different buffer conditions, which were expected to favor open and closed functional states of the channel. As for the heart (RyR2) and skeletal muscle (RyR1) receptor isoforms, RyR3 is a homotetrameric complex comprising two main components, a multidomain cytoplasmic assembly and a smaller ( approximately 20% of the total mass) transmembrane region. Although the isoforms show structural similarities, consistent with the approximately 70% overall sequence identity of the isoforms, detailed comparisons of RyR3 with RyR1 showed one region of highly significant difference between them. This difference indicated additional mass present in RyR1, and it likely corresponds to a region of the RyR1 sequence (residues 1303-1406, known as diversity region 2) that is absent from RyR3. The reconstructions of RyR3 determined under "open" and "closed" conditions were similar to each other in overall architecture. A difference map computed between the two reconstructions reveals subtle changes in conformation at several widely dispersed locations in the receptor, the most prominent of which is a approximately 4 degrees rotation of the transmembrane region with respect to the cytoplasmic assembly.

  16. Myometrial angiotensin II receptor subtypes change during ovine pregnancy.

    PubMed Central

    Cox, B E; Ipson, M A; Shaul, P W; Kamm, K E; Rosenfeld, C R

    1993-01-01

    Although regulation of angiotensin II receptor (AT) binding in vascular and uterine smooth muscle is similar in nonpregnant animals, studies suggest it may differ during pregnancy. We, therefore, examined binding characteristics of myometrial AT receptors in nulliparous (n = 7), pregnant (n = 24, 110-139 d of gestation), and postpartum (n = 21, 5 to > or = 130 d) sheep and compared this to vascular receptor binding. We also determined if changes in myometrial binding reflect alterations in receptor subtype. By using plasma membrane preparations from myometrium and medial layer of abdominal aorta, we determined receptor density and affinity employing radioligand binding; myometrial AT receptor subtypes were assessed by inhibiting [125I]-ANG II binding with subtype-specific antagonists. Compared to nulliparous ewes, myometrial AT receptor density fell approximately 90% during pregnancy (1,486 +/- 167 vs. 130 +/- 16 fmol/mg protein) and returned to nulliparous values > or = 4 wk postpartum; vascular binding was unchanged. Nulliparous myometrium expressed predominantly AT2 receptors (AT1/AT2 congruent to 15%/85%), whereas AT1 receptors predominated during pregnancy (AT1/AT2 congruent to 80%/20%). By 5 d postpartum AT1/AT2 congruent to 40%/60%, and > 4 wk postpartum AT2 receptors again predominated (AT1/AT2 congruent to 15%/85%). In studies of ANG II-induced force generation, myometrium from pregnant ewes (n = 10) demonstrated dose-dependent increases in force (P < 0.001), which were inhibited with an AT1 receptor antagonist. Postpartum myometrial responses were less at doses > or = 10(-9) M (P < 0.05) and unaffected by AT2 receptor antagonists. Vascular and myometrial AT receptor binding are differentially regulated during ovine pregnancy, the latter primarily reflecting decreases in AT2 receptor expression. This is the first description of reversible changes in AT receptor subtype in adult mammals. PMID:8227339

  17. Function and expression of ryanodine receptors and inositol 1,4,5-trisphosphate receptors in smooth muscle cells of murine feed arteries and arterioles.

    PubMed

    Westcott, Erika B; Goodwin, Erica L; Segal, Steven S; Jackson, William F

    2012-04-15

    We tested the hypothesis that vasomotor control is differentially regulated between feed arteries and downstream arterioles from the cremaster muscle of C57BL/6 mice. In isolated pressurized arteries, confocal Ca(2+) imaging of smooth muscle cells (SMCs) revealed Ca(2+) sparks and Ca(2+) waves. Ryanodine receptor (RyR) antagonists (ryanodine and tetracaine) inhibited both sparks and waves but increased global Ca(2+) and myogenic tone. In arterioles, SMCs exhibited only Ca(2+) waves that were insensitive to ryanodine or tetracaine. Pharmacological interventions indicated that RyRs are functionally coupled to large-conductance, Ca(2+)-activated K(+) channels (BK(Ca)) in SMCs of arteries, whereas BK(Ca) appear functionally coupled to voltage-gated Ca2+ channels in SMCs of arterioles. Inositol 1,4,5-trisphosphate receptor (IP3R) antagonists (xestospongin D or 2-aminoethoxydiphenyl borate) or a phospholipase C inhibitor (U73122) attenuated Ca(2+) waves, global Ca(2+) and myogenic tone in arteries and arterioles but had no effect on arterial sparks. Real-time PCR of isolated SMCs revealed RyR2 as the most abundant isoform transcript; arteries expressed twice the RyR2 but only 65% the RyR3 of arterioles and neither vessel expressed RyR1. Immunofluorescent localisation of RyR protein indicated bright, clustered staining of arterial SMCs in contrast to diffuse staining in arteriolar SMCs. Expression of IP(3)R transcripts and protein immunofluorescence were similar in SMCs of both vessels with IP(3)R1>IP(3)R2>IP(3)R3. Despite similar expression of IP(3)Rs and dependence of Ca(2+) waves on IP(3)Rs, these data illustrate pronounced regional heterogeneity in function and expression of RyRs between SMCs of the same vascular resistance network. We conclude that vasomotor control is differentially regulated in feed arteries vs. downstream arterioles.

  18. Function and expression of ryanodine receptors and inositol 1,4,5-trisphosphate receptors in smooth muscle cells of murine feed arteries and arterioles

    PubMed Central

    Westcott, Erika B; Goodwin, Erica L; Segal, Steven S; Jackson, William F

    2012-01-01

    We tested the hypothesis that vasomotor control is differentially regulated between feed arteries and downstream arterioles from the cremaster muscle of C57BL/6 mice. In isolated pressurized arteries, confocal Ca2+ imaging of smooth muscle cells (SMCs) revealed Ca2+ sparks and Ca2+ waves. Ryanodine receptor (RyR) antagonists (ryanodine and tetracaine) inhibited both sparks and waves but increased global Ca2+ and myogenic tone. In arterioles, SMCs exhibited only Ca2+ waves that were insensitive to ryanodine or tetracaine. Pharmacological interventions indicated that RyRs are functionally coupled to large-conductance, Ca2+-activated K+ channels (BKCa) in SMCs of arteries, whereas BKCa appear functionally coupled to voltage-gated Ca2+ channels in SMCs of arterioles. Inositol 1,4,5-trisphosphate receptor (IP3R) antagonists (xestospongin D or 2-aminoethoxydiphenyl borate) or a phospholipase C inhibitor (U73122) attenuated Ca2+ waves, global Ca2+ and myogenic tone in arteries and arterioles but had no effect on arterial sparks. Real-time PCR of isolated SMCs revealed RyR2 as the most abundant isoform transcript; arteries expressed twice the RyR2 but only 65% the RyR3 of arterioles and neither vessel expressed RyR1. Immunofluorescent localisation of RyR protein indicated bright, clustered staining of arterial SMCs in contrast to diffuse staining in arteriolar SMCs. Expression of IP3R transcripts and protein immunofluorescence were similar in SMCs of both vessels with IP3R1>>IP3R2>IP3R3. Despite similar expression of IP3Rs and dependence of Ca2+ waves on IP3Rs, these data illustrate pronounced regional heterogeneity in function and expression of RyRs between SMCs of the same vascular resistance network. We conclude that vasomotor control is differentially regulated in feed arteries vs. downstream arterioles. PMID:22331418

  19. Roles of the NH2-terminal domains of cardiac ryanodine receptor in Ca2+ release activation and termination.

    PubMed

    Liu, Yingjie; Sun, Bo; Xiao, Zhichao; Wang, Ruiwu; Guo, Wenting; Zhang, Joe Z; Mi, Tao; Wang, Yundi; Jones, Peter P; Van Petegem, Filip; Chen, S R Wayne

    2015-03-20

    The NH2-terminal region (residues 1-543) of the cardiac ryanodine receptor (RyR2) harbors a large number of mutations associated with cardiac arrhythmias and cardiomyopathies. Functional studies have revealed that the NH2-terminal region is involved in the activation and termination of Ca(2+) release. The three-dimensional structure of the NH2-terminal region has recently been solved. It is composed of three domains (A, B, and C). However, the roles of these individual domains in Ca(2+) release activation and termination are largely unknown. To understand the functional significance of each of these NH2-terminal domains, we systematically deleted these domains and assessed their impact on caffeine- or Ca(2+)-induced Ca(2+) release and store overload-induced Ca(2+) release (SOICR) in HEK293 cells. We found that all deletion mutants were capable of forming caffeine- and ryanodine-sensitive functional channels, indicating that the NH2-terminal region is not essential for channel gating. Ca(2+) release measurements revealed that deleting domain A markedly reduced the threshold for SOICR termination but had no effect on caffeine or Ca(2+) activation or the threshold for SOICR activation, whereas deleting domain B substantially enhanced caffeine and Ca(2+) activation and lowered the threshold for SOICR activation and termination. Conversely, deleting domain C suppressed caffeine activation, abolished Ca(2+) activation and SOICR, and diminished protein expression. These results suggest that domain A is involved in channel termination, domain B is involved in channel suppression, and domain C is critical for channel activation and expression. Our data shed new insights into the structure-function relationship of the NH2-terminal domains of RyR2 and the action of NH2-terminal disease mutations.

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

    PubMed

    Goes, Elenice S R; Lara, Jorge A F; Gasparino, Eliane; Del Vesco, Ana P; Goes, Marcio D; Alexandre Filho, Luiz; Ribeiro, Ricardo P

    2015-01-01

    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.

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

    PubMed Central

    Lara, Jorge A. F.; Gasparino, Eliane; Del Vesco, Ana P.; Goes, Marcio D.; Alexandre Filho, Luiz

    2015-01-01

    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. PMID:26053858

  2. Role of the dysfunctional ryanodine receptor - Na(+)-Ca(2+)exchanger axis in progression of cardiovascular diseases: What we can learn from pharmacological studies?

    PubMed

    Acsai, Károly; Ördög, Balázs; Varró, András; Nánási, Péter P

    2016-05-15

    Abnormal Ca(2+)homeostasis is often associated with chronic cardiovascular diseases, such as hypertension, heart failure or cardiac arrhythmias, and typically contributes to the basic ethiology of the disease. Pharmacological targeting of cardiac Ca(2+)handling has great therapeutic potential offering invaluable options for the prevention, slowing down the progression or suppression of the harmful outcomes like life threatening cardiac arrhythmias. In this review we outline the existing knowledge on the involvement of malfunction of the ryanodine receptor and the Na(+)-Ca(2+)exchanger in disturbances of Ca(2+)homeostasis and discuss important proof of concept pharmacological studies targeting these mechanisms in context of hypertension, heart failure, atrial fibrillation and ventricular arrhythmias. We emphasize the promising results of preclinical studies underpinning the potential benefits of the therapeutic strategies based on ryanodine receptor or Na(+)-Ca(2+)exchanger inhibition. Copyright © 2016 Elsevier B.V. All rights reserved.

  3. Identifying the receptor subtype selectivity of retinoid X and retinoic acid receptors via quantum mechanics.

    PubMed

    Tsuji, Motonori; Shudo, Koichi; Kagechika, Hiroyuki

    2017-03-01

    Understanding and identifying the receptor subtype selectivity of a ligand is an important issue in the field of drug discovery. Using a combination of classical molecular mechanics and quantum mechanical calculations, this report assesses the receptor subtype selectivity for the human retinoid X receptor (hRXR) and retinoic acid receptor (hRAR) ligand-binding domains (LBDs) complexed with retinoid ligands. The calculated energies show good correlation with the experimentally reported binding affinities. The technique proposed here is a promising method as it reveals the origin of the receptor subtype selectivity of selective ligands.

  4. The foot structure from the type 1 ryanodine receptor is required for functional coupling to store-operated channels.

    PubMed

    Sampieri, Alicia; Diaz-Muñoz, Mauricio; Antaramian, Anaid; Vaca, Luis

    2005-07-01

    In the present study we have explored structural determinants of the functional interaction between skeletal muscle ryanodine receptor (RyR1) and transient receptor potential channel 1 (TRPC1) channels expressed in Chinese hamster ovary cells. We have illustrated a functional interaction between TRPC1 channels and RyR1 for the regulation of store-operated calcium entry (SOCE) initiated after releasing calcium from a caffeine-sensitive intracellular calcium pool. RNA interference experiments directed to reduce the amount of TRPC1 protein indicate that RyR1 associates to at least two different types of store-operated channels (SOCs), one dependent and one independent of TRPC1. In contrast, bradykinin-induced SOCE is completely dependent on the presence of TRPC1 protein, as we have previously illustrated. Removing the foot structure from RyR1 results in normal caffeine-induced release of calcium from internal stores but abolishes the activation of SOCE, indicating that this structure is require for functional coupling to SOCs. The footless RyR1 protein shows a different cellular localization when compared with wild type RyR1. The later protein shows a higher percentage of colocalization with FM-464, a marker of plasma membrane. The implications of the foot structure for the functional and physical coupling to TRPC and SOCs is discussed.

  5. Mediation of Autophagic Cell Death by Type 3 Ryanodine Receptor (RyR3) in Adult Hippocampal Neural Stem Cells.

    PubMed

    Chung, Kyung Min; Jeong, Eun-Ji; Park, Hyunhee; An, Hyun-Kyu; Yu, Seong-Woon

    2016-01-01

    Cytoplasmic Ca(2+) actively engages in diverse intracellular processes from protein synthesis, folding and trafficking to cell survival and death. Dysregulation of intracellular Ca(2+) levels is observed in various neuropathological states including Alzheimer's and Parkinson's diseases. Ryanodine receptors (RyRs) and inositol 1,4,5-triphosphate receptors (IP3Rs), the main Ca(2+) release channels located in endoplasmic reticulum (ER) membranes, are known to direct various cellular events such as autophagy and apoptosis. Here we investigated the intracellular Ca(2+)-mediated regulation of survival and death of adult hippocampal neural stem (HCN) cells utilizing an insulin withdrawal model of autophagic cell death (ACD). Despite comparable expression levels of RyR and IP3R transcripts in HCN cells at normal state, the expression levels of RyRs-especially RyR3-were markedly upregulated upon insulin withdrawal. While treatment with the RyR agonist caffeine significantly promoted the autophagic death of insulin-deficient HCN cells, treatment with its inhibitor dantrolene prevented the induction of autophagy following insulin withdrawal. Furthermore, CRISPR/Cas9-mediated knockout of the RyR3 gene abolished ACD of HCN cells. This study delineates a distinct, RyR3-mediated ER Ca(2+) regulation of autophagy and programmed cell death in neural stem cells. Our findings provide novel insights into the critical, yet understudied mechanisms underlying the regulatory function of ER Ca(2+) in neural stem cell biology.

  6. Stable expression and functional characterisation of the diamondback moth ryanodine receptor G4946E variant conferring resistance to diamide insecticides

    PubMed Central

    Troczka, Bartlomiej J.; Williams, Alan J.; Williamson, Martin S.; Field, Linda M.; Lüemmen, Peter; Davies, T.G. Emyr

    2015-01-01

    Diamides, such as flubendiamide and chlorantraniliprole, belong to a new chemical class of insecticides that act as conformation-sensitive activators of insect ryanodine receptors (RyRs). Both compounds are registered for use against lepidopteran species such as the diamondback moth, Plutella xylostella, a notorious global pest of cruciferous crops. Recently acquired resistance to diamide insecticides in this species is thought to be due to a target-site mutation conferring an amino acid substitution (G4946E), located within the trans-membrane domain of the RyR, though the exact role of this mutation has not yet been fully determined. To address this we have cloned a full-length cDNA encoding the P. xylostella RyR and established clonal Sf9 cell lines stably expressing either the wildtype RyR or the G4946E variant, in order to test the sensitivity to flubendiamide and chlorantraniliprole on the recombinant receptor. We report that the efficacy of both diamides was dramatically reduced in clonal Sf9 cells stably expressing the G4946E modified RyR, providing clear functional evidence that the G4946E RyR mutation impairs diamide insecticide binding. PMID:26424584

  7. Stable expression and functional characterisation of the diamondback moth ryanodine receptor G4946E variant conferring resistance to diamide insecticides.

    PubMed

    Troczka, Bartlomiej J; Williams, Alan J; Williamson, Martin S; Field, Linda M; Lüemmen, Peter; Davies, T G Emyr

    2015-10-01

    Diamides, such as flubendiamide and chlorantraniliprole, belong to a new chemical class of insecticides that act as conformation-sensitive activators of insect ryanodine receptors (RyRs). Both compounds are registered for use against lepidopteran species such as the diamondback moth, Plutella xylostella, a notorious global pest of cruciferous crops. Recently acquired resistance to diamide insecticides in this species is thought to be due to a target-site mutation conferring an amino acid substitution (G4946E), located within the trans-membrane domain of the RyR, though the exact role of this mutation has not yet been fully determined. To address this we have cloned a full-length cDNA encoding the P. xylostella RyR and established clonal Sf9 cell lines stably expressing either the wildtype RyR or the G4946E variant, in order to test the sensitivity to flubendiamide and chlorantraniliprole on the recombinant receptor. We report that the efficacy of both diamides was dramatically reduced in clonal Sf9 cells stably expressing the G4946E modified RyR, providing clear functional evidence that the G4946E RyR mutation impairs diamide insecticide binding.

  8. Mediation of Autophagic Cell Death by Type 3 Ryanodine Receptor (RyR3) in Adult Hippocampal Neural Stem Cells

    PubMed Central

    Chung, Kyung Min; Jeong, Eun-Ji; Park, Hyunhee; An, Hyun-Kyu; Yu, Seong-Woon

    2016-01-01

    Cytoplasmic Ca2+ actively engages in diverse intracellular processes from protein synthesis, folding and trafficking to cell survival and death. Dysregulation of intracellular Ca2+ levels is observed in various neuropathological states including Alzheimer’s and Parkinson’s diseases. Ryanodine receptors (RyRs) and inositol 1,4,5-triphosphate receptors (IP3Rs), the main Ca2+ release channels located in endoplasmic reticulum (ER) membranes, are known to direct various cellular events such as autophagy and apoptosis. Here we investigated the intracellular Ca2+-mediated regulation of survival and death of adult hippocampal neural stem (HCN) cells utilizing an insulin withdrawal model of autophagic cell death (ACD). Despite comparable expression levels of RyR and IP3R transcripts in HCN cells at normal state, the expression levels of RyRs—especially RyR3—were markedly upregulated upon insulin withdrawal. While treatment with the RyR agonist caffeine significantly promoted the autophagic death of insulin-deficient HCN cells, treatment with its inhibitor dantrolene prevented the induction of autophagy following insulin withdrawal. Furthermore, CRISPR/Cas9-mediated knockout of the RyR3 gene abolished ACD of HCN cells. This study delineates a distinct, RyR3-mediated ER Ca2+ regulation of autophagy and programmed cell death in neural stem cells. Our findings provide novel insights into the critical, yet understudied mechanisms underlying the regulatory function of ER Ca2+ in neural stem cell biology. PMID:27199668

  9. Pregnant rat myometrial cells show heterogeneous ryanodine- and caffeine-sensitive calcium stores.

    PubMed

    Martin, C; Hyvelin, J M; Chapman, K E; Marthan, R; Ashley, R H; Savineau, J P

    1999-08-01

    Intracellular Ca(2+) release channels such as ryanodine receptors play crucial roles in the Ca(2+)-mediated signaling that triggers excitation-contraction coupling in muscles. Although the existence and the role of these channels are well characterized in skeletal and cardiac muscles, their existence in smooth muscles, and more particularly in the myometrium, is very controversial. We have now clearly demonstrated the expression of ryanodine receptor Ca(2+) release channels in rat myometrial smooth muscle, and for the first time, intracellular Ca(2+) concentration experiments with indo 1 on single myometrial cells have revealed the existence of a functional ryanodine- and caffeine-sensitive Ca(2+) release mechanism in 30% of rat myometrial cells. RT-PCR and RNase protection assay on whole myometrial smooth muscle demonstrate the existence of all three ryr mRNAs in the myometrium: ryr3 mRNA is the predominant subtype, with much lower levels of expression for ryr1 and ryr2 mRNAs, suggesting that the ryanodine Ca(2+) release mechanism in rat myometrium is largely encoded by ryr3. Moreover, using intracellular Ca(2+) concentration measurements and RNase protection assays, we have demonstrated that the expression, the percentage of cells responding to ryanodine, and the function of these channels are not modified during pregnancy.

  10. Electron-conformational transformations govern the temperature dependence of the cardiac ryanodine receptor gating

    NASA Astrophysics Data System (ADS)

    Moskvin, A. S.; Iaparov, B. I.; Ryvkin, A. M.; Solovyova, O. E.; Markhasin, V. S.

    2015-07-01

    Temperature influences many aspects of cardiac excitation-contraction coupling, in particular, hypothermia increases the open probability ( P open) of cardiac sarcoplasmic reticulum (SR) Ca2+-release channels (ryanodine-sensitive RyR channels) rising the SR Ca2+ load in mammalian myocytes. However, to the best of our knowledge, no theoretical models are available for that effect. Traditional Markov chain models do not provide a reasonable molecular mechanistic insight on the origin of the temperature effects. Here in the paper we address a simple physically clear electron-conformational model to describe the RyR gating and argue that a synergetic effect of external thermal fluctuation forces (Gaussian-Markovian noise) and internal friction via the temperature stimulation/suppression of the open-close RyR tunneling probability can be considered as a main contributor to temperature effects on the RyR gating. Results of the computer modeling allowed us to successfully reproduce all the temperature effects observed for an isolated RyR gating in vitro under reducing the temperature: increase in P open and mean open time without any significant effect on mean closed

  11. Activation of single cardiac and skeletal ryanodine receptor channels by flash photolysis of caged Ca2+.

    PubMed Central

    Györke, S; Vélez, P; Suárez-Isla, B; Fill, M

    1994-01-01

    Single ryanodine-sensitive sarcoplasmic reticulum (SR) Ca2+ release channels isolated from rabbit skeletal and canine cardiac muscle were reconstituted in planar lipid bilayers. Single channel activity was measured in simple solutions (no ATP or Mg2+) with 250 mM symmetrical Cs+ as charge carrier. A laser flash was used to photolyze caged-Ca2+ (DM-nitrophen) in a small volume directly in front of the bilayer. The free [Ca2+] in this small volume and in the bulk solution was monitored with Ca2+ electrodes. This setup allowed fast, calibrated free [Ca2+] stimuli to be applied repetitively to single SR Ca2+ release channels. A standard photolytically induced free [Ca2+] step (pCa 7-->6) was applied to both the cardiac and skeletal release channels. The rate of channel activation was determined by fitting a single exponential to ensemble currents generated from at least 50 single channel sweeps. The time constants of activation were 1.43 +/- 0.65 ms (mean +/- SD; n = 5) and 1.28 +/- 0.61 ms (n = 5) for cardiac and skeletal channels, respectively. This study presents a method for defining the fast Ca2+ regulation kinetics of single SR Ca2+ release channels and shows that the activation rate of skeletal SR Ca2+ release channels is consistent with a role for CICR in skeletal muscle excitation-contraction coupling. PMID:8075325

  12. Functional and biochemical properties of ryanodine receptor type 1 channels from heterozygous R163C malignant hyperthermia-susceptible mice.

    PubMed

    Feng, Wei; Barrientos, Genaro C; Cherednichenko, Gennady; Yang, Tianzhong; Padilla, Isela T; Truong, Kim; Allen, Paul D; Lopez, José R; Pessah, Isaac N

    2011-03-01

    Mutations in ryanodine receptor type 1 (RyR1) confer malignant hyperthermia susceptibility. How inherent impairments in Ca(2+) channel regulation affect skeletal muscle function in myotubes and adult fibers under basal (nontriggering) conditions are not understood. Myotubes, adult flexor digitorum brevis (FDB) fibers, and sarcoplasmic reticulum skeletal membranes were isolated from heterozygous knockin R163C and wild-type (WT) mice. Compared with WT myotubules, R163C myotubes have reduced Ca(2+) transient amplitudes in response to electrical field pulses; however, R163C FDB fibers do not differ in their responses to electrical stimuli, despite heightened cellular cytoplasmic resting Ca(2+) ([Ca(2+)](rest)) and sensitivity to halothane. Immunoblotting of membranes from each genotype shows similar expression of RyR1, FK506 binding protein 12 kDa, and Ca(2+)-ATPase, but RyR1 (2844)Ser phosphorylation in R163C muscle is 31% higher than that of WT muscle (p < 0.001). RyR1 channels reconstituted in planar lipid bilayers reveal ∼65% of R163C channels exhibit ≥2-fold greater open probability (P(o)) than WT, with prolonged mean open dwell times and shortened closed dwell times. [(3)H]Ryanodine (Ry) binding and single-channel analyses show that R163C-RyR1 has altered regulation compared with WT: 1) 3-fold higher sensitivity to Ca(2+) activation; 2) 2-fold greater [(3)H]Ry receptor occupancy; 3) comparatively higher channel activity, even in reducing glutathione buffer; 4) enhanced RyR1 activity both at 25 and 37°C; and 5) elevated cytoplasmic [Ca(2+)](rest). R163C channels are inherently more active than WT channels, a functional impairment that cannot be reversed by dephosphorylation with protein phosphatase. Dysregulated R163C channels produce a more overt phenotype in myotubes than in adult fibers in the absence of triggering agents, suggesting tighter negative regulation of R163C-RyR1 within the Ca(2+) release unit of adult fibers.

  13. Acromegaly: molecular expression of somatostatin receptor subtypes and treatment outcome.

    PubMed

    Bronstein, Marcello D

    2006-01-01

    About a third of acromegalic patients are resistant to the currently commercially available somatostatin analogs (SA) octreotide and lanreotide. Such resistance is related to an overall reduction of somatostatin receptor (SSTR) density or to a differentiated expression of SSTR subtypes. There are five known SSTR subtypes. SSTR2 and SSTR5 are usually expressed in GH-secreting pituitary tumors, and both octreotide and lanreotide bind preferentially to SSTR2 and, to a lesser extent, to SSTR5. SA inhibitory effects on GH secretion and tumor cell proliferation can occur together or be dissociated events, depending on the tumor expression of SSTR subtypes involved in each mechanism. The development of specific somatostatin subtypes analogs, mainly for SSTR5, of a SSTR2-SSTR5 bispecific compound, and of a "universal" analog with high affinity to SSTR1, 2, 3, and 5 showed preliminary, albeit promising results for the treatment of resistant somatotropic adenomas.

  14. Knockout of the BK β4-subunit promotes a functional coupling of BK channels and ryanodine receptors that mediate a fAHP-induced increase in excitability

    PubMed Central

    Wang, Bin; Bugay, Vladislav; Ling, Ling; Chuang, Hui-Hsui; Jaffe, David B.

    2016-01-01

    BK channels are large-conductance calcium- and voltage-activated potassium channels with diverse properties. Knockout of the accessory BK β4-subunit in hippocampus dentate gyrus granule neurons causes BK channels to change properties from slow-gated type II channels to fast-gated type I channels that sharpen the action potential, increase the fast afterhyperpolarization (fAHP) amplitude, and increase spike frequency. Here we studied the calcium channels that contribute to fast-gated BK channel activation and increased excitability of β4 knockout neurons. By using pharmacological blockers during current-clamp recording, we find that BK channel activation during the fAHP is dependent on ryanodine receptor activation. In contrast, L-type calcium channel blocker (nifedipine) affects the BK channel-dependent repolarization phase of the action potential but has no effect on the fAHP. Reducing BK channel activation during the repolarization phase with nifedipine, or during the fAHP with ryanodine, indicated that it is the BK-mediated increase of the fAHP that confers proexcitatory effects. The proexcitatory role of the fAHP was corroborated using dynamic current clamp. Increase or decrease of the fAHP amplitude during spiking revealed an inverse relationship between fAHP amplitude and interspike interval. Finally, we show that the seizure-prone ryanodine receptor gain-of-function (R2474S) knockin mice have an unaltered repolarization phase but larger fAHP and increased AP frequency compared with their control littermates. In summary, these results indicate that an important role of the β4-subunit is to reduce ryanodine receptor-BK channel functional coupling during the fAHP component of the action potential, thereby decreasing excitability of dentate gyrus neurons. PMID:27146987

  15. Beyond classical benzodiazepines: Novel therapeutic potential of GABAA receptor subtypes

    PubMed Central

    Rudolph, Uwe; Knoflach, Frédéric

    2012-01-01

    GABAA receptors are a family of ligand-gated ion channels which are essential for the regulation of central nervous system function. Benzodiazepines – which target GABAA receptors containing the α1, α2, α3, or α5 subunits non-selectively – have been in clinical use for decades and are still among the most widely prescribed drugs for the treatment of insomnia and anxiety disorders. However, their use is limited by side effects and the risk of drug dependence. In the past decade, the identification of separable key functions of GABAA receptor subtypes suggests that receptor subtype-selective compounds could overcome the limitations of classical benzodiazepines and, furthermore, might be valuable for novel indications, such as analgesia, depression, schizophrenia, cognitive enhancement and stroke. PMID:21799515

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

    PubMed Central

    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

  17. Genetic and Biochemical Approaches for In Vivo and In Vitro Assessment of Protein Oligomerization: The Ryanodine Receptor Case Study

    PubMed Central

    Stanczyk, Paulina J.; Lai, F. Anthony; Zissimopoulos, Spyros

    2016-01-01

    Oligomerization is often a structural requirement for proteins to accomplish their specific cellular function. For instance, tetramerization of the ryanodine receptor (RyR) is necessary for the formation of a functional Ca2+ release channel pore. Here, we describe detailed protocols for the assessment of protein self-association, including yeast two-hybrid (Y2H), co-immunoprecipitation (co-IP) and chemical cross-linking assays. In the Y2H system, protein self-interaction is detected by β-galactosidase assay in yeast co-expressing GAL4 bait and target fusions of the test protein. Protein self-interaction is further assessed by co-IP using HA- and cMyc-tagged fusions of the test protein co-expressed in mammalian HEK293 cells. The precise stoichiometry of the protein homo-oligomer is examined by cross-linking and SDS-PAGE analysis following expression in HEK293 cells. Using these different but complementary techniques, we have consistently observed the self-association of the RyR N-terminal domain and demonstrated its intrinsic ability to form tetramers. These methods can be applied to protein-protein interaction and homo-oligomerization studies of other mammalian integral membrane proteins. PMID:27500320

  18. Fluorescence probe study of Ca2+-dependent interactions of calmodulin with calmodulin-binding peptides of the ryanodine receptor.

    PubMed

    Gangopadhyay, Jaya Pal; Grabarek, Zenon; Ikemoto, Noriaki

    2004-10-22

    We have used a highly environment-sensitive fluorescent probe 6-bromoacetyl-2-dimethylaminonaphthalene (badan) to study the interaction between calmodulin (CaM) and a CaM-binding peptide of the ryanodine receptor (CaMBP) and its sub-fragments F1 and F4. Badan was attached to the Thr34Cys mutant of CaM (CaM-badan). Ca(2+) increase in a physiological range of Ca(2+) (0.1-2 microM) produced about 40 times increase in the badan fluorescence. Upon binding to CaMBP, the badan fluorescence of apo-CaM showed a small increase at a slow rate; whereas that of Ca-CaM showed a large decrease at a very fast rate. Upon binding of CaM to the badan-labeled CaMBP, the badan fluorescence showed a small and slow increase at low Ca(2+), and a large and fast increase at high Ca(2+). Thus, the badan probe attached to CaM Cys(34) can be used to monitor conformational changes occurring not only in CaM, but also those in the CaM-CaMBP interface. Based on our results we propose that both the interaction interface and the global conformation of the CaM-CaMBP complex are altered by calcium.

  19. Crystal structures of ryanodine receptor SPRY1 and tandem-repeat domains reveal a critical FKBP12 binding determinant

    PubMed Central

    Yuchi, Zhiguang; Yuen, Siobhan M. Wong King; Lau, Kelvin; Underhill, Ainsley Q.; Cornea, Razvan L.; Fessenden, James D.; Van Petegem, Filip

    2015-01-01

    Ryanodine receptors (RyRs) form calcium release channels located in the membranes of the sarcoplasmic and endoplasmic reticulum. RyRs play a major role in excitation-contraction coupling and other Ca2+-dependent signalling events, and consist of several globular domains that together form a large assembly. Here we describe the crystal structures of the SPRY1 and tandem-repeat domains at 1.2–1.5 Å resolution, which reveal several structural elements not detected in recent cryo-EM reconstructions of RyRs. The cryo-EM studies disagree on the position of SPRY domains, which had been proposed based on homology modelling. Computational docking of the crystal structures, combined with FRET studies, show that the SPRY1 domain is located next to FK506-binding protein (FKBP). Molecular dynamics flexible fitting and mutagenesis experiments suggest a hydrophobic cluster within SPRY1 that is crucial for FKBP binding. A RyR1 disease mutation, N760D, appears to directly impact FKBP binding through interfering with SPRY1 folding. PMID:26245150

  20. Ryanodine receptors selectively contribute to the formation of taste-evoked calcium signals in mouse taste cells

    PubMed Central

    Rebello, Michelle R.; Medler, Kathryn F.

    2010-01-01

    The peripheral taste system uses multiple signaling pathways to transduce a stimulus into an output signal that activates afferent neurons. All of these signaling pathways depend on transient increases in intracellular calcium but the current understanding of these calcium signals is not well-developed. Using molecular and physiological techniques, this study establishes that ryanodine receptors (RyRs), specifically isoform 1, are expressed in taste cells and that their physiological function differs among cell types employing different signaling pathways. RyR1 contributes to some taste-evoked signals that rely on calcium release from internal stores but can also supplement the calcium signal that is initiated by opening VGCCs. In taste cells expressing both signaling pathways, RyR1 contributes to the depolarization-induced calcium signal but not to the calcium signal that depends on calcium release from stores. These data suggest that RyR1 is an important regulator of calcium signaling and that its physiological role in taste cells is dictated by the nature of the calcium signaling mechanisms expressed. PMID:20955474

  1. Crystal structures of ryanodine receptor SPRY1 and tandem-repeat domains reveal a critical FKBP12 binding determinant.

    PubMed

    Yuchi, Zhiguang; Yuen, Siobhan M Wong King; Lau, Kelvin; Underhill, Ainsley Q; Cornea, Razvan L; Fessenden, James D; Van Petegem, Filip

    2015-08-06

    Ryanodine receptors (RyRs) form calcium release channels located in the membranes of the sarcoplasmic and endoplasmic reticulum. RyRs play a major role in excitation-contraction coupling and other Ca(2+)-dependent signalling events, and consist of several globular domains that together form a large assembly. Here we describe the crystal structures of the SPRY1 and tandem-repeat domains at 1.2-1.5 Å resolution, which reveal several structural elements not detected in recent cryo-EM reconstructions of RyRs. The cryo-EM studies disagree on the position of SPRY domains, which had been proposed based on homology modelling. Computational docking of the crystal structures, combined with FRET studies, show that the SPRY1 domain is located next to FK506-binding protein (FKBP). Molecular dynamics flexible fitting and mutagenesis experiments suggest a hydrophobic cluster within SPRY1 that is crucial for FKBP binding. A RyR1 disease mutation, N760D, appears to directly impact FKBP binding through interfering with SPRY1 folding.

  2. HIV-1 Tat Activates Neuronal Ryanodine Receptors with Rapid Induction of the Unfolded Protein Response and Mitochondrial Hyperpolarization

    PubMed Central

    Norman, John P.; Perry, Seth W.; Reynolds, Holly M.; Kiebala, Michelle; De Mesy Bentley, Karen L.; Trejo, Margarita; Volsky, David J.; Maggirwar, Sanjay B.; Dewhurst, Stephen; Masliah, Eliezer; Gelbard, Harris A.

    2008-01-01

    Neurologic disease caused by human immunodeficiency virus type 1 (HIV-1) is ultimately refractory to highly active antiretroviral therapy (HAART) because of failure of complete virus eradication in the central nervous system (CNS), and disruption of normal neural signaling events by virally induced chronic neuroinflammation. We have previously reported that HIV-1 Tat can induce mitochondrial hyperpolarization in cortical neurons, thus compromising the ability of the neuron to buffer calcium and sustain energy production for normal synaptic communication. In this report, we demonstrate that Tat induces rapid loss of ER calcium mediated by the ryanodine receptor (RyR), followed by the unfolded protein response (UPR) and pathologic dilatation of the ER in cortical neurons in vitro. RyR antagonism attenuated both Tat-mediated mitochondrial hyperpolarization and UPR induction. Delivery of Tat to murine CNS in vivo also leads to long-lasting pathologic ER dilatation and mitochondrial morphologic abnormalities. Finally, we performed ultrastructural studies that demonstrated mitochondria with abnormal morphology and dilated endoplasmic reticulum (ER) in brain tissue of patients with HIV-1 inflammation and neurodegeneration. Collectively, these data suggest that abnormal RyR signaling mediates the neuronal UPR with failure of mitochondrial energy metabolism, and is a critical locus for the neuropathogenesis of HIV-1 in the CNS. PMID:19009018

  3. Modal gating transitions in cardiac ryanodine receptors during increases of Ca2+ concentration produced by photolysis of caged Ca2+.

    PubMed

    Zahradníková, A; Dura, M; Györke, S

    1999-08-01

    Channel adaptation is a basic property of the sarcoplasmic reticulum Ca2+-release channels/ryanodine receptors (RyRs). It allows channel activity to decay during sustained increases in the concentration of activating Ca2+. Despite the potential physiological importance of this self-confining process, its molecular mechanism is not well understood. To define the mechanism of adaptation we studied the dynamics of cardiac Ca2+-release channel (RyR) gating using the planar lipid bilayer technique in combination with photolysis of caged Ca2+ (DM-nitrophen). Channels activated by rapid and sustained increases in Ca2+ concentration (from 0.1 to 0.5 micromol/l) displayed three distinct gating modes, manifested as current records with frequent and long openings (H-mode), with rare and short openings (L-mode), and with no openings (I-mode). H-mode channel activity occurred primarily at early times while L- and I-modes predominated at late times after the rapid Ca2+ concentration increase. The decrease in probability of H-mode, mirrored by an increase in the probability of the I-mode, proceeded with a time constant similar to that observed for spontaneous decay in channel activity (i.e., adaptation) in ensemble average records. These results indicate that RyR adaptation transpires by a shift of channel gating from a high open probability mode to low open probability and inactivated modes of the channel.

  4. Oxygen-coupled redox regulation of the skeletal muscle ryanodine receptor-Ca2+ release channel by NADPH oxidase 4.

    PubMed

    Sun, Qi-An; Hess, Douglas T; Nogueira, Leonardo; Yong, Sandro; Bowles, Dawn E; Eu, Jerry; Laurita, Kenneth R; Meissner, Gerhard; Stamler, Jonathan S

    2011-09-20

    Physiological sensing of O(2) tension (partial O(2) pressure, pO(2)) plays an important role in some mammalian cellular systems, but striated muscle generally is not considered to be among them. Here we describe a molecular mechanism in skeletal muscle that acutely couples changes in pO(2) to altered calcium release through the ryanodine receptor-Ca(2+)-release channel (RyR1). Reactive oxygen species are generated in proportion to pO(2) by NADPH oxidase 4 (Nox4) in the sarcoplasmic reticulum, and the consequent oxidation of a small set of RyR1 cysteine thiols results in increased RyR1 activity and Ca(2+) release in isolated sarcoplasmic reticulum and in cultured myofibers and enhanced contractility of intact muscle. Thus, Nox4 is an O(2) sensor in skeletal muscle, and O(2)-coupled hydrogen peroxide production by Nox4 governs the redox state of regulatory RyR1 thiols and thereby governs muscle performance. These findings reveal a molecular mechanism for O(2)-based signaling by an NADPH oxidase and demonstrate a physiological role for oxidative modification of RyR1.

  5. Antiarrhythmic potential of drugs targeting the cardiac ryanodine receptor Ca2+ release channel: case study of dantrolene.

    PubMed

    Acsai, Karoly; Nagy, Norbert; Marton, Zoltan; Oravecz, Kinga; Varro, Andras

    2015-01-01

    Driven by the limitations of the traditional antiarrhythmic pharmacology, current antiarrhythmic research is trying to identify new avenues for the development of specific and safe antiarrhythmic drugs. One of the most promising approaches in this field is the amelioration of the abnormal events in cellular Ca(2+) handling originating from the dysfunction of ryanodine receptor Ca(2+) release complex (RyR), which is an inevitable key factor in the pathology of myocardial dysfunction, remodeling and arrhythmogenesis. Accordingly, both in experimental and clinical situations, inhibition of abnormal activity of RyR, regardless of being the primary cause or a consequence during the pathogenesis appears to exert beneficial effect on disease outcome, including a marked antiarrhythmic defense. Considerable amount of our knowledge in this field originates from studies using dantrolene, a human drug with RyR stabilizing effect. Our review summarizes the cardiovascular pharmacology of dantrolene and the results of its use in experimental models of cardiac diseases, which emphasize a promising perspective for the possible antiarrhythmic application of RyR inhibition in the future.

  6. King-Denborough syndrome with and without mutations in the skeletal muscle ryanodine receptor (RYR1) gene.

    PubMed

    Dowling, James J; Lillis, Suzanne; Amburgey, Kimberley; Zhou, Haiyan; Al-Sarraj, Safa; Buk, Stefan J A; Wraige, Elizabeth; Chow, Gabby; Abbs, Stephen; Leber, Steven; Lachlan, Katherine; Baralle, Diana; Taylor, Alexandra; Sewry, Caroline; Muntoni, Francesco; Jungbluth, Heinz

    2011-06-01

    King-Denborough syndrome (KDS), first described in 1973, is a rare condition characterised by the triad of dysmorphic features, myopathy, and malignant hyperthermia susceptibility (MHS). Autosomal dominant inheritance with variable expressivity has been reported in several cases. Mutations in the skeletal muscle ryanodine receptor (RYR1) gene have been implicated in a wide range of myopathies such as central core disease (CCD), the malignant hyperthermia (MH) susceptibility trait and one isolated patient with KDS. Here we report clinical, pathologic and genetic features of four unrelated patients with KDS. Patients had a relatively uniform clinical presentation but muscle biopsy findings were highly variable. Heterozygous missense mutations in RYR1 were uncovered in three out of four families, of which one mutation was novel and two have previously been reported in MH. Further RyR1 protein expression studies performed in two families showed marked reduction of the RyR1 protein, indicating the presence of allelic RYR1 mutations not detectable on routine sequencing and potentially explaining marked intrafamilial variability. Our findings support the hypothesis that RYR1 mutations are associated with King-Denborough syndrome but that further genetic heterogeneity is likely.

  7. Is ryanodine receptor a calcium or magnesium channel? Roles of K+ and Mg2+ during Ca2+ release.

    PubMed

    Gillespie, Dirk; Chen, Haiyan; Fill, Michael

    2012-06-01

    The ryanodine receptor (RyR) is a poorly selective channel that mediates Ca(2+) release from intracellular Ca(2+) stores. How RyR's selectivity between the physiological cations K(+), Mg(2+), and Ca(2+) affects single-channel Ca(2+) current amplitude is examined using a recent model of RyR permeation. It is found that K(+) provides the vast majority of the countercurrent (through RyR itself) that is needed to prevent the sarcoplasmic reticulum (SR) membrane potential from changing and stopping Ca(2+) release. Moreover, intra-pore competition between Ca(2+) and Mg(2+) defines single RyR Ca(2+) current amplitude. Since both [Mg(2+)] and [Ca(2+)](SR) can change during pathophysiological conditions, the RyR unitary Ca(2+) current amplitude during Ca(2+) release may change significantly due to this Ca(2+)/Mg(2+) competition. Compared to the classic action of Mg(2+) on RyR open probability, these Ca(2+) current amplitude changes have as large or larger effects on overall RyR Ca(2+) mobilization. A new aspect of RyR divalent versus monovalent selectivity is also identified where this kind of selectivity decreases as divalent concentration increases.

  8. Neuronal regeneration in C. elegans requires subcellular calcium release by ryanodine receptor channels and can be enhanced by optogenetic stimulation.

    PubMed

    Sun, Lin; Shay, James; McLoed, Melissa; Roodhouse, Kevin; Chung, Samuel H; Clark, Christopher M; Pirri, Jennifer K; Alkema, Mark J; Gabel, Christopher V

    2014-11-26

    Regulated calcium signals play conserved instructive roles in neuronal repair, but how localized calcium stores are differentially mobilized, or might be directly manipulated, to stimulate regeneration within native contexts is poorly understood. We find here that localized calcium release from the endoplasmic reticulum via ryanodine receptor (RyR) channels is critical in stimulating initial regeneration following traumatic cellular damage in vivo. Using laser axotomy of single neurons in Caenorhabditis elegans, we find that mutation of unc-68/RyR greatly impedes both outgrowth and guidance of the regenerating neuron. Performing extended in vivo calcium imaging, we measure subcellular calcium signals within the immediate vicinity of the regenerating axon end that are sustained for hours following axotomy and completely eliminated within unc-68/RyR mutants. Finally, using a novel optogenetic approach to periodically photo-stimulate the axotomized neuron, we can enhance its regeneration. The enhanced outgrowth depends on both amplitude and temporal pattern of excitation and can be blocked by disruption of UNC-68/RyR. This demonstrates the exciting potential of emerging optogenetic technology to beneficially manipulate cell physiology in the context of neuronal regeneration and indicates a link to the underlying cellular calcium signal. Taken as a whole, our findings define a specific localized calcium signal mediated by RyR channel activity that stimulates regenerative outgrowth, which may be dynamically manipulated for beneficial neurotherapeutic effects.

  9. A novel late-onset axial myopathy associated with mutations in the skeletal muscle ryanodine receptor (RYR1) gene.

    PubMed

    Løseth, Sissel; Voermans, Nicol C; Torbergsen, Torberg; Lillis, Sue; Jonsrud, Christoffer; Lindal, Sigurd; Kamsteeg, Erik-Jan; Lammens, Martin; Broman, Marcus; Dekomien, Gabriele; Maddison, Paul; Muntoni, Francesco; Sewry, Caroline; Radunovic, Aleksandar; de Visser, Marianne; Straub, Volker; van Engelen, Baziel; Jungbluth, Heinz

    2013-06-01

    Mutations in the skeletal muscle ryanodine receptor (RYR1) gene are a common cause of inherited neuromuscular disorders and have been associated with a wide clinical spectrum, ranging from various congenital myopathies to the malignant hyperthermia susceptibility (MHS) trait without any associated weakness. RYR1-related myopathies are usually of early-childhood onset. Here we present 11 patients from 8 families with a late-onset axial myopathy associated with RYR1 variants. Patients presented between the third and seventh decade of life to neuromuscular centres in Norway, the Netherlands and the United Kingdom with predominant axial muscle involvement, comprising variable degrees of lumbar hyperlordosis, scapular winging and/or camptocormia. Marked myalgia was commonly associated. Serum creatine kinase levels were normal or moderately elevated. Muscle imaging showed consistent involvement of the lower paravertebral muscles and the posterior thigh. Muscle biopsy findings were often discrete, featuring variability in fibre size, increased internal nuclei and unevenness of oxidative enzyme staining, but only rarely overt cores. RYR1 sequencing revealed heterozygous missense variants, either previously associated with the MHS trait or localizing to known MHS mutational hotspots. These findings indicate that MHS-related RYR1 mutations may present later in life with prominent axial weakness but not always typical histopathological features. We propose a combined effect of RyR1 dysfunction, aging and particular vulnerability of axial muscle groups as a possible pathogenic mechanism. RYR1 is a candidate for cases with "idiopathic" camptocormia or bent spine syndrome (BSS).

  10. Ryanodine receptor genes of the rice stem borer, Chilo suppressalis: Molecular cloning, alternative splicing and expression profiling.

    PubMed

    Peng, Y C; Sheng, C W; Casida, John E; Zhao, C Q; Han, Z J

    2017-01-01

    The ryanodine receptor (RyR) of the calcium release channel is the main target of anthranilic and phthalic diamide insecticides which have high selective insecticidal activity relative to mammalian toxicity. In this study, the full-length cDNA of Chilo suppressalis RyR (CsRyR) was isolated and characterized. The CsRyR mRNA has an open reading frame (ORF) of 15,387bp nucleotides, which encodes 5128 amino acids with GenBank ID: KR088972. Comparison of protein sequences showed that CsRyR shared high identities with other insects of 77-96% and lower identity to mammals and nematodes with only 42-45%. One alternative splicing site (KENLG) unique to Lepidoptera was found and two exclusive exons of CsRyR (I /II) were revealed. Spatial and temporal expression of CsRyR mRNA was at the highest relative level in 3rd instar larvae and head (including brain and muscle), and at the lowest expression level in egg and fat body. The expression levels of whole body CsRyR mRNA were increased remarkably after injection of 4th instar larvae with chlorantraniliprole at 0.004 to 0.4μg/g. This structural and functional information on CsRyR provides the basis for further understanding the selective action of chlorantraniliprole and possibly other diamide insecticides.

  11. Cardiac ryanodine receptor: Selectivity for alkaline earth metal cations points to the EF-hand nature of luminal binding sites.

    PubMed

    Gaburjakova, Jana; Gaburjakova, Marta

    2016-06-01

    A growing body of evidence suggests that the regulation of cardiac ryanodine receptor (RYR2) by luminal Ca(2+) is mediated by luminal binding sites located on the RYR2 channel itself and/or its auxiliary protein, calsequestrin. The localization and structure of RYR2-resident binding sites are not known because of the lack of a high-resolution structure of RYR2 luminal regions. To obtain the first structural insight, we probed the RYR2 luminal face stripped of calsequestrin by alkaline earth metal divalents (M(2+): Mg(2+), Ca(2+), Sr(2+) or Ba(2+)). We show that the RYR2 response to caffeine at the single-channel level is significantly modified by the nature of luminal M(2+). Moreover, we performed competition experiments by varying the concentration of luminal M(2+) (Mg(2+), Sr(2+) or Ba(2+)) from 8 mM to 53 mM and investigated its ability to compete with 1mM luminal Ca(2+). We demonstrate that all tested M(2+) bind to exactly the same RYR2 luminal binding sites. Their affinities decrease in the order: Ca(2+)>Sr(2+)>Mg(2+)~Ba(2+), showing a strong correlation with the M(2+) affinity of the EF-hand motif. This indicates that the RYR2 luminal binding regions and the EF-hand motif likely share some structural similarities because the structure ties directly to the function.

  12. Caffeine-induced Release of Intracellular Ca2+ from Chinese Hamster Ovary Cells Expressing Skeletal Muscle Ryanodine Receptor

    PubMed Central

    Bhat, Manjunatha B.; Zhao, Jiying; Zang, Weijin; Balke, C. William; Takeshima, Hiroshi; Wier, W. Gil; Ma, Jianjie

    1997-01-01

    The ryanodine receptor (RyR)/Ca2+ release channel is an essential component of excitation–contraction coupling in striated muscle cells. To study the function and regulation of the Ca2+ release channel, we tested the effect of caffeine on the full-length and carboxyl-terminal portion of skeletal muscle RyR expressed in a Chinese hamster ovary (CHO) cell line. Caffeine induced openings of the full length RyR channels in a concentration-dependent manner, but it had no effect on the carboxyl-terminal RyR channels. CHO cells expressing the carboxyl-terminal RyR proteins displayed spontaneous changes of intracellular [Ca2+]. Unlike the native RyR channels in muscle cells, which display localized Ca2+ release events (i.e., “Ca2+ sparks” in cardiac muscle and “local release events” in skeletal muscle), CHO cells expressing the full length RyR proteins did not exhibit detectable spontaneous or caffeine-induced local Ca2+ release events. Our data suggest that the binding site for caffeine is likely to reside within the amino-terminal portion of RyR, and the localized Ca2+ release events observed in muscle cells may involve gating of a group of Ca2+ release channels and/or interaction of RyR with muscle-specific proteins. PMID:9382901

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

    PubMed

    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.

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

    PubMed Central

    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

  15. Role of CaMKIIdelta phosphorylation of the cardiac ryanodine receptor in the force frequency relationship and heart failure.

    PubMed

    Kushnir, Alexander; Shan, Jian; Betzenhauser, Matthew J; Reiken, Steven; Marks, Andrew R

    2010-06-01

    The force frequency relationship (FFR), first described by Bowditch 139 years ago as the observation that myocardial contractility increases proportionally with increasing heart rate, is an important mediator of enhanced cardiac output during exercise. Individuals with heart failure have defective positive FFR that impairs their cardiac function in response to stress, and the degree of positive FFR deficiency correlates with heart failure progression. We have identified a mechanism for FFR involving heart rate dependent phosphorylation of the major cardiac sarcoplasmic reticulum calcium release channel/ryanodine receptor (RyR2), at Ser2814, by calcium/calmodulin-dependent serine/threonine kinase-delta (CaMKIIdelta). Mice engineered with an RyR2-S2814A mutation have RyR2 channels that cannot be phosphorylated by CaMKIIdelta, and exhibit a blunted positive FFR. Ex vivo hearts from RyR2-S2814A mice also have blunted positive FFR, and cardiomyocytes isolated from the RyR2-S2814A mice exhibit impaired rate-dependent enhancement of cytosolic calcium levels and fractional shortening. The cardiac RyR2 macromolecular complexes isolated from murine and human failing hearts have reduced CaMKIIdelta levels. These data indicate that CaMKIIdelta phosphorylation of RyR2 plays an important role in mediating positive FFR in the heart, and that defective regulation of RyR2 by CaMKIIdelta-mediated phosphorylation is associated with the loss of positive FFR in failing hearts.

  16. Ryanodine receptors blockade reduces Amyloid-beta load and memory impairments in Tg2576 mouse model of Alzheimer disease

    PubMed Central

    Oulès, Bénédicte; Del Prete, Dolores; Greco, Barbara; Zhang, Xuexin; Lauritzen, Inger; Sevalle, Jean; Moreno, Sebastien; Paterlini-Bréchot, Patrizia; Trebak, Mohamed; Checler, Frédéric; Benfenati, Fabio; Chami, Mounia

    2012-01-01

    In Alzheimer disease (AD), the perturbation of the endoplasmic reticulum (ER) calcium (Ca2+) homeostasis has been linked to presenilins (PS), the catalytic core in γ-secretase complexes cleaving the amyloid precursor protein (APP) thereby generating amyloid-β (Aβ) peptides. Here we investigate whether APP contributes to ER Ca2+ homeostasis and whether ER Ca2+ could in turn influence Aβ production. We show that overexpression of wild-type human APP (APP695), or APP harboring the Swedish double mutation (APPswe) triggers increased Ryanodine receptors (RyR) expression and enhances RyR-mediated ER Ca2+ release in SH-SY5Y neuroblastoma cells and in APPswe-expressing (Tg2576) mice. Interestingly, dantrolene-induced lowering of RyR-mediated Ca2+ release leads to the reduction of both intracellular and extracellular Aβ load in neuroblastoma cells as well as in primary cultured neurons derived from Tg2576 mice. This Aβ reduction can be accounted for by decreased Thr-668-dependent APP phosphorylation and β- and γ-secretases activities. Importantly, dantrolene diminishes Aβ load, reduces Aβ-related histological lesions and slows down learning and memory deficits in Tg2576 mice. Overall, our data document a key role of RyR in Aβ production and learning and memory performances, and delineate RyR-mediated control of Ca2+ homeostasis as a physiological paradigm that could be targeted for innovative therapeutic approaches. PMID:22915123

  17. The H29D Mutation Does Not Enhance Cytosolic Ca2+ Activation of the Cardiac Ryanodine Receptor.

    PubMed

    Xiao, Zhichao; Guo, Wenting; Yuen, Siobhan M Wong King; Wang, Ruiwu; Zhang, Lin; Van Petegem, Filip; Chen, S R Wayne

    2015-01-01

    The N-terminal domain of the cardiac ryanodine receptor (RyR2) harbors a large number of naturally occurring mutations that are associated with stress-induced ventricular tachyarrhythmia and sudden death. Nearly all these disease-associated N-terminal mutations are located at domain interfaces or buried within domains. Mutations at these locations would alter domain-domain interactions or the stability/folding of domains. Recently, a novel RyR2 mutation H29D associated with ventricular arrhythmia at rest was found to enhance the activation of single RyR2 channels by diastolic levels of cytosolic Ca2+. Unlike other N-terminal disease-associated mutations, the H29D mutation is located on the surface of the N-terminal domain. It is unclear how this surface-exposed H29D mutation that does not appear to interact with other parts of the RyR2 structure could alter the intrinsic properties of the channel. Here we carried out detailed functional characterization of the RyR2-H29D mutant at the molecular and cellular levels. We found that the H29D mutation has no effect on the basal level or the Ca2+ dependent activation of [3H]ryanodine binding to RyR2, the cytosolic Ca2+ activation of single RyR2 channels, or the cytosolic Ca2+- or caffeine-induced Ca2+ release in HEK293 cells. In addition, the H29D mutation does not alter the propensity for spontaneous Ca2+ release or the thresholds for Ca2+ release activation or termination. Furthermore, the H29D mutation does not have significant impact on the thermal stability of the N-terminal region (residues 1-547) of RyR2. Collectively, our data show that the H29D mutation exerts little or no effect on the function of RyR2 or on the folding stability of the N-terminal region. Thus, our results provide no evidence that the H29D mutation enhances the cytosolic Ca2+ activation of RyR2.

  18. The H29D Mutation Does Not Enhance Cytosolic Ca2+ Activation of the Cardiac Ryanodine Receptor

    PubMed Central

    Xiao, Zhichao; Guo, Wenting; Yuen, Siobhan M. Wong King; Wang, Ruiwu; Zhang, Lin; Van Petegem, Filip; Chen, S. R. Wayne

    2015-01-01

    The N-terminal domain of the cardiac ryanodine receptor (RyR2) harbors a large number of naturally occurring mutations that are associated with stress-induced ventricular tachyarrhythmia and sudden death. Nearly all these disease-associated N-terminal mutations are located at domain interfaces or buried within domains. Mutations at these locations would alter domain-domain interactions or the stability/folding of domains. Recently, a novel RyR2 mutation H29D associated with ventricular arrhythmia at rest was found to enhance the activation of single RyR2 channels by diastolic levels of cytosolic Ca2+. Unlike other N-terminal disease-associated mutations, the H29D mutation is located on the surface of the N-terminal domain. It is unclear how this surface-exposed H29D mutation that does not appear to interact with other parts of the RyR2 structure could alter the intrinsic properties of the channel. Here we carried out detailed functional characterization of the RyR2-H29D mutant at the molecular and cellular levels. We found that the H29D mutation has no effect on the basal level or the Ca2+ dependent activation of [3H]ryanodine binding to RyR2, the cytosolic Ca2+ activation of single RyR2 channels, or the cytosolic Ca2+- or caffeine-induced Ca2+ release in HEK293 cells. In addition, the H29D mutation does not alter the propensity for spontaneous Ca2+ release or the thresholds for Ca2+ release activation or termination. Furthermore, the H29D mutation does not have significant impact on the thermal stability of the N-terminal region (residues 1–547) of RyR2. Collectively, our data show that the H29D mutation exerts little or no effect on the function of RyR2 or on the folding stability of the N-terminal region. Thus, our results provide no evidence that the H29D mutation enhances the cytosolic Ca2+ activation of RyR2. PMID:26405799

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

    PubMed Central

    Llanos, Paola; Contreras-Ferrat, Ariel; Barrientos, Genaro; Valencia, Marco; Mears, David; Hidalgo, Cecilia

    2015-01-01

    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. PMID:26046640

  20. Prognostic Value of Somatostatin Receptor Subtypes in Pancreatic Neuroendocrine Tumors.

    PubMed

    Song, Ki Byung; Kim, Song Cheol; Kim, Ji Hun; Seo, Dong-Wan; Hong, Seung-Mo; Park, Kwang-Min; Hwang, Dae Wook; Lee, Jae Hoon; Lee, Young-Joo

    2016-02-01

    Studies on the expression of somatostatin receptor (SSTR) subtypes in pancreatic neuroendocrine tumors (PNETs) are rare. The aim of this study was to determine the expression of the SSTR subtypes via immunohistochemistry analyses and assess the correlation between SSTR subtype expression and prognosis. We examined 199 patients with PNET who underwent surgical resection between January 1995 and December 2010 at the Asan Medical Center. For all cases, medical records, including demographic data, clinical symptoms, radiological findings, postoperative treatment outcomes, and expression of SSTR subtypes, were carefully reviewed. In total, 162 (81.4%) PNETs expressed more than 1 SSTR subtype. Functioning PNET expressed significantly more SSTR subtypes, compared to nonfunctioning PNET. The SSTR2(+) and SSTR5(+) groups had better prognosis than the SSTR2(-) (P = 0.009) and SSTR5(-) groups (P = 0.03), respectively. In the grade 2 PNET of 2010 World Health Organization classification, the SSTR(+) group had better prognosis than SSTR(-) group. The expression of SSTR 2 and 5 were related with good prognosis of PNET. In World Health Organization grade 2 PNET, the SSTR(+) group had better prognosis than SSTR(-) group. The SSTR expression(+) by immunohistochemistry might be related with good prognosis of the patients with surgically resected PNET.

  1. Interdomain Interactions within Ryanodine Receptors Regulate Ca2+ Spark Frequency in Skeletal Muscle

    PubMed Central

    Shtifman, Alexander; Ward, Christopher W.; Yamamoto, Takeshi; Wang, Jianli; Olbinski, Beth; Valdivia, Hector H.; Ikemoto, Noriaki; Schneider, Martin F.

    2002-01-01

    DP4 is a 36-residue synthetic peptide that corresponds to the Leu2442-Pro2477 region of RyR1 that contains the reported malignant hyperthermia (MH) mutation site. It has been proposed that DP4 disrupts the normal interdomain interactions that stabilize the closed state of the Ca2+ release channel (Yamamoto, T., R. El-Hayek, and N. Ikemoto. 2000. J. Biol. Chem. 275:11618–11625). We have investigated the effects of DP4 on local SR Ca2+ release events (Ca2+ sparks) in saponin-permeabilized frog skeletal muscle fibers using laser scanning confocal microscopy (line-scan mode, 2 ms/line), as well as the effects of DP4 on frog SR vesicles and frog single RyR Ca2+ release channels reconstituted in planar lipid bilayers. DP4 caused a significant increase in Ca2+ spark frequency in muscle fibers. However, the mean values of the amplitude, rise time, spatial half width, and temporal half duration of the Ca2+ sparks, as well as the distribution of these parameters, remained essentially unchanged in the presence of DP4. Thus, DP4 increased the opening rate, but not the open time of the RyR Ca2+ release channel(s) generating the sparks. DP4 also increased [3H]ryanodine binding to SR vesicles isolated from frog and mammalian skeletal muscle, and increased the open probability of frog RyR Ca2+ release channels reconstituted in bilayers, without changing the amplitude of the current through those channels. However, unlike in Ca2+ spark experiments, DP4 produced a pronounced increase in the open time of channels in bilayers. The same peptide with an Arg17 to Cys17 replacement (DP4mut), which corresponds to the Arg2458-to-Cys2458 mutation in MH, did not produce a significant effect on RyR activation in muscle fibers, bilayers, or SR vesicles. Mg2+ dependence experiments conducted with permeabilized muscle fibers indicate that DP4 preferentially binds to partially Mg2+-free RyR(s), thus promoting channel opening and production of Ca2+ sparks. PMID:11773235

  2. Subtype Differences in Pre-Coupling of Muscarinic Acetylcholine Receptors

    PubMed Central

    Jakubík, Jan; Janíčková, Helena; Randáková, Alena; El-Fakahany, Esam E.; Doležal, Vladimír

    2011-01-01

    Based on the kinetics of interaction between a receptor and G-protein, a myriad of possibilities may result. Two extreme cases are represented by: 1/Collision coupling, where an agonist binds to the free receptor and then the agonist-receptor complex “collides” with the free G-protein. 2/Pre-coupling, where stable receptor/G-protein complexes exist in the absence of agonist. Pre-coupling plays an important role in the kinetics of signal transduction. Odd-numbered muscarinic acetylcholine receptors preferentially couple to Gq/11, while even-numbered receptors prefer coupling to Gi/o. We analyzed the coupling status of the various subtypes of muscarinic receptors with preferential and non-preferential G-proteins. The magnitude of receptor-G-protein coupling was determined by the proportion of receptors existing in the agonist high-affinity binding conformation. Antibodies directed against the C-terminus of the α-subunits of the individual G-proteins were used to interfere with receptor-G-protein coupling. Effects of mutations and expression level on receptor-G-protein coupling were also investigated. Tested agonists displayed biphasic competition curves with the antagonist [3H]-N-methylscopolamine. Antibodies directed against the C-terminus of the α-subunits of the preferential G-protein decreased the proportion of high-affinity sites, and mutations at the receptor-G-protein interface abolished agonist high-affinity binding. In contrast, mutations that prevent receptor activation had no effect. Expression level of preferential G-proteins had no effect on pre-coupling to non-preferential G-proteins. Our data show that all subtypes of muscarinic receptors pre-couple with their preferential classes of G-proteins, but only M1 and M3 receptors also pre-couple with non-preferential Gi/o G-proteins. Pre-coupling is not dependent on agonist efficacy nor on receptor activation. The ultimate mode of coupling is therefore dictated by a combination of the receptor subtype

  3. Multiple Estrogen Receptor Subtypes Influence Ingestive Behavior in Female Rodents

    PubMed Central

    Santollo, Jessica; Daniels, Derek

    2015-01-01

    Postmenopausal women are at an increased risk of obesity and cardiovascular-related diseases. This is attributable, at least in part, to loss of the ovarian hormone estradiol, which inhibits food and fluid intake in humans and laboratory animal models. Although the hypophagic and anti-dipsogenic effects of estradiol have been well documented for decades, the precise mechanisms underlying these effects are not fully understood. An obvious step toward addressing this open question is identifying which estrogen receptor subtypes are involved and what intracellular processes are involved. This question, however, is complicated not only by the variety of estrogen receptor subtypes that exist, but also because many subtypes have multiple locations of action (i.e. in the nucleus or in the plasma membrane). This review will highlight our current understanding of the roles specific estrogen receptor subtypes play in mediating estradiol’s anorexigenic and anti-dipsogenic effects along with highlighting the many open questions that remain. This review will also describe recent work being performed by our laboratory aimed at answering these open questions. PMID:26037634

  4. Multiple estrogen receptor subtypes influence ingestive behavior in female rodents.

    PubMed

    Santollo, Jessica; Daniels, Derek

    2015-12-01

    Postmenopausal women are at an increased risk of obesity and cardiovascular-related diseases. This is attributable, at least in part, to loss of the ovarian hormone estradiol, which inhibits food and fluid intake in humans and laboratory animal models. Although the hypophagic and anti-dipsogenic effects of estradiol have been well documented for decades, the precise mechanisms underlying these effects are not fully understood. An obvious step toward addressing this open question is identifying which estrogen receptor subtypes are involved and what intracellular processes are involved. This question, however, is complicated not only by the variety of estrogen receptor subtypes that exist, but also because many subtypes have multiple locations of action (i.e. in the nucleus or in the plasma membrane). This review will highlight our current understanding of the roles that specific estrogen receptor subtypes play in mediating estradiol's anorexigenic and anti-dipsogenic effects along with highlighting the many open questions that remain. This review will also describe recent work being performed by our laboratory aimed at answering these open questions.

  5. Isolation of rat genomic clones encoding subtypes of the alpha 2-adrenergic receptor. Identification of a unique receptor subtype.

    PubMed

    Lanier, S M; Downing, S; Duzic, E; Homcy, C J

    1991-06-05

    alpha 2-Adrenergic receptors (alpha 2-AR) exist as subtypes that are expressed in a tissue-specific manner and differ in 1) their ligand recognition properties, 2) their extent of receptor protein glycosylation, and possible 3) their mechanism of signal transduction. Genomic or cDNA clones encoding three receptor subtypes have been characterized; however, both functional and radioligand binding studies in rodents suggest the existence of a fourth receptor subtype. To isolate the rat genes encoding receptor subtypes we screened a rat genomic library with an oligonucleotide probe encompassing the third membrane span of the human C-4 alpha 2-AR. Two intronless rat genes were isolated that encode distinct receptor subtypes (RG10, RG20). RG10 and RG20 encode proteins of 458 and 450 amino acids, respectively, that are 56% homologous and possess the structural features expected of this class of membrane-bound receptors. RG10 identifies a mRNA species of approximately 2500 nucleotides that is found primarily in brain, whereas RG20 identifies a larger mRNA species (approximately 4000 nucleotides) that is found in several tissues including brain, kidney, and salivary gland. RG10 is 88% homologous to the human C-4 alpha 2-AR and exhibits similar binding properties ( [3H]rauwolscine KD = 0.7 +/- 0.3 nM) as determined following transient expression of the receptor in COS-1 cells. RG20 exhibits ligand binding properties distinct from the three receptor subtypes identified by molecular cloning. Saturation binding studies indicate an affinity constant of 15 +/- 1.2 nM for the alpha 2-AR antagonist [3H]rauwolscine, a value 6-20 times higher than that observed for the three cloned receptor subtypes. In competition binding studies the potency order of competing ligands for RG20 is phentolamine greater than idazoxan greater than yohimbine greater than rauwolscine greater than prazosin. Of the three previously cloned alpha 2-AR, RG20 is most closely related to the human C-10 alpha 2-AR

  6. Ca2+ release in muscle fibers expressing R4892W and G4896V type 1 ryanodine receptor disease mutants.

    PubMed

    Lefebvre, Romain; Legrand, Claude; Groom, Linda; Dirksen, Robert T; Jacquemond, Vincent

    2013-01-01

    The large and rapidly increasing number of potentially pathological mutants in the type 1 ryanodine receptor (RyR1) prompts the need to characterize their effects on voltage-activated sarcoplasmic reticulum (SR) Ca(2+) release in skeletal muscle. Here we evaluated the function of the R4892W and G4896V RyR1 mutants, both associated with central core disease (CCD) in humans, in myotubes and in adult muscle fibers. For both mutants expressed in RyR1-null (dyspedic) myotubes, voltage-gated Ca(2+) release was absent following homotypic expression and only partially restored following heterotypic expression with wild-type (WT) RyR1. In muscle fibers from adult WT mice, both mutants were expressed in restricted regions of the fibers with a pattern consistent with triadic localization. Voltage-clamp-activated confocal Ca(2+) signals showed that fiber regions endowed with G4896V-RyR1s exhibited an ∼30% reduction in the peak rate of SR Ca(2+) release, with no significant change in SR Ca(2+) content. Immunostaining revealed no associated change in the expression of either α1S subunit (Cav1.1) of the dihydropyridine receptor (DHPR) or type 1 sarco(endo)plasmic reticulum Ca(2+) ATPase (SERCA1), indicating that the reduced Ca(2+) release resulted from defective RyR1 function. Interestingly, in spite of robust localized junctional expression, the R4892W mutant did not affect SR Ca(2+) release in adult muscle fibers, consistent with a low functional penetrance of this particular CCD-associated mutant.

  7. Characterization of the Molecular Architecture of Human Caveolin-3 and Interaction with the Skeletal Muscle Ryanodine Receptor*

    PubMed Central

    Whiteley, Gareth; Collins, Richard F.; Kitmitto, Ashraf

    2012-01-01

    Caveolin-3 (cav-3), an integral membrane protein, is a building block of caveolae as well as a regulator of a number of physiological processes by facilitating the formation of multiprotein signaling complexes. We report that the expression of cav-3 in insect (Sf9) cells induces caveola formation, comparable in size with those observed in native tissue. We have also purified the recombinant cav-3 determining that it forms an oligomer of ∼220 kDa. We present the first three-dimensional structure for cav-3 (using transmission electron microscopy and single particle analysis methods) and show that nine cav-3 monomers assemble to form a complex that is toroidal in shape, ∼16.5 nm in diameter and ∼ 5.5 nm in height. Labeling experiments and reconstitution of the purified cav-3 into liposomes have allowed a proposal for the orientation of the protein with respect to the membrane. We have identified multiple caveolin-binding motifs within the ryanodine receptor (RyR1) sequence employing a bioinformatic analysis. We have then shown experimentally that there is a direct interaction between recombinant cav-3 nonamers and purified RyR1 homotetramers that would imply that at least one of the predicted cav-3-binding sites is exposed within the fully assembled RyR1 structure. The cav-3 three-dimensional model provides new insights as to how a cav-3 oligomer can bind multiple partners in close proximity to form signaling complexes. Furthermore, a direct interaction with RyR1 suggests a possible role for cav-3 as a modifier of muscle excitation-contraction coupling and/or for localization of the receptor to regions of the sarcoplasmic reticulum. PMID:23071107

  8. Ryanodine Receptor Type 1 Gene Variants in the Malignant Hyperthermia-Susceptible Population of the United States

    PubMed Central

    Brandom, Barbara W.; Bina, Saiid; Wong, Cynthia A.; Wallace, Tarina; Visoiu, Mihaela; Isackson, Paul J.; Vladutiu, Georgirene D.; Sambuughin, Nyamkhishig; Muldoon, Sheila M.

    2013-01-01

    Background Mutations in the ryanodine receptor type 1 gene (RYR1) that encodes the skeletal muscle-specific intracellular calcium (Ca2+) release channel are a cause of malignant hyperthermia (MH). In this study we examined RYR1 mutations in a large number of North American MH-susceptible (MHS) subjects without prior genetic diagnosis. Methods RYR1 was examined in 120 unrelated MHS subjects from the United States in a tiered manner. The alpha-1 subunit of the dihydropyridine receptor gene (CACNA1S) was screened for four variants in subjects in whom no abnormality was found in 100 or more exons of RYR1. Results Ten known causative MH mutations were found in 26 subjects. Variants of uncertain significance in RYR1 were found in 36 subjects, 16 of which are novel. Novel variants in both RYR1 and CACNA1S were found in the one subject who died of MH. Two RYR1 variants were found in 4 subjects. Variants of uncertain significance were found outside and inside the hotspots of RYR1. Maximal contractures in the caffeine-halothane contracture test were greater in those who had a known MH mutation or variant of uncertain significance in RYR1 than in those who did not. Conclusions The identification of novel RYR1 variants and previously observed RYR1 variants of uncertain significance in independent MHS families is necessary for demonstrating the significance of these variants for MH susceptibility and supports the need for functional studies of these variants. Continued reporting of the clinical phenotypes of MH is necessary for interpretation of genetic findings, especially because the pathogenicity of most of these genetic variants associated with MHS remains to be elucidated. PMID:23558838

  9. Levetiracetam Inhibits Both Ryanodine and IP3 Receptor Activated Calcium Induced Calcium Release in Hippocampal Neurons in Culture

    PubMed Central

    Nagarkatti, Nisha; Deshpande, Laxmikant S.; DeLorenzo, Robert J.

    2010-01-01

    Epilepsy affects approximately 1% of the population worldwide, and there is a pressing need to develop new anti-epileptic drugs (AEDs) and understand their mechanisms of action. Levetiracetam (LEV) is a novel AED and despite its increasingly widespread clinical use, its mechanism of action is as yet undetermined. Intracellular calcium ([Ca2+]i) regulation by both inositol 1,4,5-triphosphate receptors (IP3R) and ryanodine receptors (RyR) has been implicated in epileptogenesis and the maintenance of epilepsy. To this end, we investigated the effect of LEV on RyR and IP3R activated calcium-induced calcium release (CICR) in hippocampal neuronal cultures. RyR-mediated CICR was stimulated using the well-characterized RyR activator, caffeine. Caffeine (10mM) caused a significant increase in [Ca2+]i in hippocampal neurons. Treatment with LEV (33μM) prior to stimulation of RyR-mediated CICR by caffeine led to a 61% decrease in the caffeine induced peak height of [Ca2+]i when compared to the control. Bradykinin stimulates IP3R-activated CICR—to test the effect of LEV on IP3R-mediated CICR, bradykinin (1μM) was used to stimulate cells pre-treated with LEV (100μM). The data showed that LEV caused a 74% decrease in IP3R-mediated CICR compared to the control. In previous studies we have shown that altered Ca2+ homeostatic mechanisms play a role in seizure activity and the development of spontaneous recurrent epileptiform discharges (SREDs). Elevations in [Ca2+]i mediated by CICR systems have been associated with neurotoxicity, changes in neuronal plasticity, and the development of AE. Thus, the ability of LEV to modulate the two major CICR systems demonstrates an important molecular effect of this agent on a major second messenger system in neurons. PMID:18406528

  10. Structural basis of kainate subtype glutamate receptor desensitization

    PubMed Central

    Meyerson, Joel R.; Chittori, Sagar; Merk, Alan; Rao, Prashant; Han, Tae Hee; Serpe, Mihaela; Mayer, Mark L.; Subramaniam, Sriram

    2016-01-01

    Glutamate receptors are ligand gated tetrameric ion channels that mediate synaptic transmission in the central nervous system. They are instrumental in vertebrate cognition and their dysfunction underlies diverse diseases1,2. In both the resting and desensitized states of AMPA and kainate subtype glutamate receptors the ion channels are closed while the ligand binding domain, which is physically coupled to the channel, adopts dramatically different conformations3–6. Without an atomic model for the desensitized state, it is not possible to address a central question in receptor gating: how the resting and desensitized receptor states both display closed ion channels, even though they have major differences in quaternary structure of the ligand binding domain. By determining the cryo-EM structure of the kainate receptor GluK2 subtype in its desensitized state at 3.8 Å resolution, we show that desensitization is characterized by establishment of a ring-like structure in the ligand binding domain layer of the receptor. Formation of this “desensitization ring” is mediated by staggered helix contacts between adjacent subunits, which leads to a pseudo four-fold symmetric arrangement of the ligand binding domains, illustrating subtle changes in symmetry that are at the heart of the gating mechanism. Disruption of the desensitization ring is likely the key switch that enables restoration of the receptor to its resting state, thereby completing the gating cycle. PMID:27580033

  11. Structure-activity relationship for noncoplanar polychlorinated biphenyl congeners toward the ryanodine receptor-Ca2+ channel complex type 1 (RyR1).

    PubMed

    Pessah, Isaac N; Hansen, Larry G; Albertson, Timothy E; Garner, C Edwin; Ta, Tram Anh; Do, Zung; Kim, Kyung Ho; Wong, Patty W

    2006-01-01

    Ryanodine receptor isoforms are expressed in both excitable and nonexcitable tissues where they form microsomal Ca2+ release channels broadly involved in shaping cellular signaling. In this report, we provide a detailed structure-activity relationship (SAR) for polychlorinated biphenyl (PCB) congeners and metabolites necessary for enhancing ryanodine receptor type 1 (RyR1) activity using [3H]ryanodine ([3H]Ry) binding analysis. The 2,3,6-Cl PCB configuration is most important for optimal recognition by the RyR1 complex and/or critical for sensitizing its activation. Para substitution(s) diminishes the activity with para-chloro having a higher potency than the corresponding para-hydroxy derivative. The addition of a more bulky para-methyl-sulfonyl group eliminates the activity toward RyR1, supporting the importance of the para positions in binding RyR1. The requirement for an intact major T cell immunophilin FKBP12-RyR1 complex was observed with each of 12 active PCB congeners indicating a common mechanism requiring an immunophilin-regulated Ca2+ release channel. An excellent correlation between the relative potencies for doubling [3H]Ry binding and the corresponding initial rates of PCB-induced Ca2+ efflux indicates that [3H]Ry binding analysis provides a measure of dysregulation of microsomal Ca2+ transport. The SAR for activating RyR1 is consistent with those previously reported in several in vivo and in vitro studies, suggesting that a common mechanism may contribute to the toxicity of noncoplanar PCBs. A practical application of the receptor-based screen developed here with RyR1 is that it provides a quantitative SAR that may be useful in predicting biological activity and risk of mixtures containing noncoplanar PCB congeners that have low or a lack of aryl hydrocarbon receptor activity.

  12. Characterization of muscarinic receptor subtypes in human tissues

    SciTech Connect

    Giraldo, E.; Martos, F.; Gomez, A.; Garcia, A.; Vigano, M.A.; Ladinsky, H.; Sanchez de La Cuesta, F.

    1988-01-01

    The affinities of selective, pirenzepine and AF-DX 116, and classical, N-methylscopolamine and atropine, muscarinic cholinergic receptor antagonists were investigated in displacement binding experiments with (/sup 3/H)Pirenzepine and (/sup 3/H)N-methylscopolamine in membranes from human autoptic tissues (forebrain, cerebellum, atria, ventricle and submaxillary salivary glands). Affinity estimates of N-methylscopolamine and atropine indicated a non-selective profile. Pirenzepine showed differentiation between the M/sub 1/ neuronal receptor of the forebrain and the receptors in other tissues while AF-DX 116 clearly discriminated between muscarinic receptors of heart and glands. The results in human tissues confirm the previously described selectivity profiles of pirenzepine and AF-DX 116 in rat tissues. These findings thus reveal the presence also in man of three distinct muscarinic receptor subtypes: the neuronal M/sub 1/, the cardiac M/sub 2/ and the glandular M/sub 3/.

  13. Pharmacophore development for antagonists at α1 adrenergic receptor subtypes

    NASA Astrophysics Data System (ADS)

    Bremner, J. B.; Coban, B.; Griffith, R.

    1996-12-01

    Many receptors, including α1 adrenergic receptors, have a range of subtypes. This offers possibilities for the development of highly selective antagonists with potentially fewer detrimental effects. Antagonists developed for α1A receptors, for example, would have potential in the treatment of benign prostatic hyperplasia. As part of the molecular design process, structural features necessary for the selective affinity for α1A and α1B adrenergic receptors have been investigated. The molecular modelling software (particularly the Apex module) of Molecular Simulations, Inc. was used to develop pharmacophore models for these two subtypes. Low-energy conformations of a set of known antagonists were used as input, together with a classification of the receptor affinity data. The biophores proposed by the program were evaluated and pharmacophores were proposed. The pharmacophore models were validated by testing the fit of known antagonists, not included in the training set. The critical structural feature for selectivity between the α1A and α1B adrenergic receptor sites is the distance between the basic nitrogen atom and the centre of an aromatic ring system. This will be exploited in the design and synthesis of structurally new selective antagonists for these sites.

  14. Muscarinic receptor subtype selectivity of novel heterocyclic QNB analogues

    SciTech Connect

    Baumgold, J.; Cohen, V.I.; Paek, R.; Reba, R.C. )

    1991-01-01

    In an effort at synthesizing centrally-active subtype-selective antimuscarinic agents, the authors derivatized QNB (quinuclidinyl benzilate), a potent muscarinic antagonist, by replacing one of the phenyl groups with less lipophilic heterocyclic moieties. The displacement of ({sup 3}H)-N-methyl scopolamine binding by these novel compounds to membranes from cells expressing ml - m4 receptor subtypes was determined. Most of the novel 4-bromo-QNB analogues were potent and slightly selective for ml receptors. The 2-thienyl derivative was the most potent, exhibiting a 2-fold greater potency than BrQNB at ml receptors, and a 4-fold greater potency than BrQNB at ml receptors, and a 4-fold greater potency at m2 receptors. This compound was also considerably less lipophilic than BrQNB as determined from its retention time on C18 reverse phase HPLC. This compound may therefore be useful both for pharmacological studies and as a candidate for a radioiodinated SPECT imaging agent for ml muscarinic receptors in human brain.

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

    NASA Astrophysics Data System (ADS)

    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.

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

    PubMed

    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.

  17. Type 1 and type 3 ryanodine receptors generate different Ca(2+) release event activity in both intact and permeabilized myotubes.

    PubMed Central

    Ward, C W; Protasi, F; Castillo, D; Wang, Y; Chen, S R; Pessah, I N; Allen, P D; Schneider, M F

    2001-01-01

    In this investigation we use a "dyspedic" myogenic cell line, which does not express any ryanodine receptor (RyR) isoform, to examine the local Ca(2+) release behavior of RyR3 and RyR1 in a homologous cellular system. Expression of RyR3 restored caffeine-sensitive, global Ca(2+) release and causes the appearance of relatively frequent, spontaneous, spatially localized elevations of [Ca(2+)], as well as occasional spontaneous, propagating Ca(2+) release, in both intact and saponin-permeabilized myotubes. Intact myotubes expressing RyR3 did not, however, respond to K(+) depolarization. Expression of RyR1 restored depolarization-induced global Ca(2+) release in intact myotubes and caffeine-induced global release in both intact and permeabilized myotubes. Both intact and permeabilized RyR1-expressing myotubes exhibited relatively infrequent spontaneous Ca(2+) release events. In intact myotubes, the frequency of occurrence and properties of these RyR1-induced events were not altered by partial K(+) depolarization or by application of nifedipine, suggesting that these RyR1 events are independent of the voltage sensor. The events seen in RyR1-expressing myotubes were spatially more extensive than those seen in RyR3-expressing myotubes; however, when analysis was limited to spatially restricted "Ca(2+) spark"-like events, events in RyR3-expressing myotubes were larger in amplitude and duration compared with those in RyR1. Thus, in this skeletal muscle context, differences exist in the spatiotemporal properties and frequency of occurrence of spontaneous release events generated by RyR1 and RyR3. These differences underscore functional differences between the Ca(2+) release behavior of RyR1 and RyR3 in this homologous expression system. PMID:11720987

  18. N-terminal and central segments of the type 1 ryanodine receptor mediate its interaction with FK506-binding proteins.

    PubMed

    Girgenrath, Tanya; Mahalingam, Mohana; Svensson, Bengt; Nitu, Florentin R; Cornea, Razvan L; Fessenden, James D

    2013-05-31

    We used site-directed labeling of the type 1 ryanodine receptor (RyR1) and fluorescence resonance energy transfer (FRET) measurements to map RyR1 sequence elements forming the binding site of the 12-kDa binding protein for the immunosuppressant drug, FK506. This protein, FKBP12, promotes the RyR1 closed state, thereby inhibiting Ca(2+) leakage in resting muscle. Although FKBP12 function is well established, its binding determinants within the RyR1 protein sequence remain unresolved. To identify these sequence determinants using FRET, we created five single-Cys FKBP variants labeled with Alexa Fluor 488 (denoted D-FKBP) and then targeted these D-FKBPs to full-length RyR1 constructs containing decahistidine (His10) "tags" placed within N-terminal (amino acid residues 76-619) or central (residues 2157-2777) regions of RyR1. The FRET acceptor Cy3NTA bound specifically and saturably to these His tags, allowing distance analysis of FRET measured from each D-FKBP variant to Cy3NTA bound to each His tag. Results indicate that D-FKBP binds proximal to both N-terminal and central domains of RyR1, thus suggesting that the FKBP binding site is composed of determinants from both regions. These findings further imply that the RyR1 N-terminal and central domains are proximal to one another, a core premise of the domain-switch hypothesis of RyR function. We observed FRET from GFP fused at position 620 within the N-terminal domain to central domain His-tagged sites, thus further supporting this hypothesis. Taken together, these results support the conclusion that N-terminal and central domain elements are closely apposed near the FKBP binding site within the RyR1 three-dimensional structure.

  19. N-terminal and Central Segments of the Type 1 Ryanodine Receptor Mediate Its Interaction with FK506-binding Proteins*

    PubMed Central

    Girgenrath, Tanya; Mahalingam, Mohana; Svensson, Bengt; Nitu, Florentin R.; Cornea, Razvan L.; Fessenden, James D.

    2013-01-01

    We used site-directed labeling of the type 1 ryanodine receptor (RyR1) and fluorescence resonance energy transfer (FRET) measurements to map RyR1 sequence elements forming the binding site of the 12-kDa binding protein for the immunosuppressant drug, FK506. This protein, FKBP12, promotes the RyR1 closed state, thereby inhibiting Ca2+ leakage in resting muscle. Although FKBP12 function is well established, its binding determinants within the RyR1 protein sequence remain unresolved. To identify these sequence determinants using FRET, we created five single-Cys FKBP variants labeled with Alexa Fluor 488 (denoted D-FKBP) and then targeted these D-FKBPs to full-length RyR1 constructs containing decahistidine (His10) “tags” placed within N-terminal (amino acid residues 76–619) or central (residues 2157–2777) regions of RyR1. The FRET acceptor Cy3NTA bound specifically and saturably to these His tags, allowing distance analysis of FRET measured from each D-FKBP variant to Cy3NTA bound to each His tag. Results indicate that D-FKBP binds proximal to both N-terminal and central domains of RyR1, thus suggesting that the FKBP binding site is composed of determinants from both regions. These findings further imply that the RyR1 N-terminal and central domains are proximal to one another, a core premise of the domain-switch hypothesis of RyR function. We observed FRET from GFP fused at position 620 within the N-terminal domain to central domain His-tagged sites, thus further supporting this hypothesis. Taken together, these results support the conclusion that N-terminal and central domain elements are closely apposed near the FKBP binding site within the RyR1 three-dimensional structure. PMID:23585572

  20. High prevalence of rare ryanodine receptor type 1 variants in patients suffering from aneurysmatic subarachnoid hemorrhage: A pilot study.

    PubMed

    Coburger, Jan; Kapapa, Thomas; Wirtz, Cristian Rainer; Jurkat-Rott, Karin; Klingler, Werner

    2017-07-24

    Subarachnoid hemorrhage (SAH) remains a challenging neurosurgical disease. The ryanodine receptor type 1 Ca2+ channel (RyR1) plays a crucial role in vasoconstriction and hemostasis. Mutations of the encoding gene, RYR1, are known to cause susceptibility to malignant hyperthermia (MH). Recently, a RYR1 mutation was found to be associated with abnormal bleeding times. Therefore, an assessment of the RYR1 gene might be of high relevance in patients with aneurysmatic SAH. In the presented pilot study, we screened 10 patients suffering from SAH for RYR1 variants and, for the first time in SAH, performed an assessment of pathogenicity of these variants using protein prediction software. Four of the patients showed a RYR1 variant. For three of the variants, p.Glu79Lys, p.Arg885C, p.Glu2635 Val, all three programs predicted pathogenicity. Their prevalence in the general population is very low i.e. under 0.005%. For the fourth variant, p.Pro4501Leu (RS73933023), the results of the prediction programs were discrepant and the prevalence in the general population was high, i.e. almost 0.5%, which is too frequent to be associated with the rare SAH phenotype. Clinical evaluation revealed that no differences concerning neurological outcome, presence of vasospasm, ischemic deficits and mean hospital stay between patients with and without variants were found. However, in our series SAH patients have an increased frequency of rare RYR1 variants. Hence, potentially contributing to the pathogenesis of SAH. Further data is needed to confirm this preliminary result. Copyright © 2017 Elsevier Ltd. All rights reserved.

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

    PubMed

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

    2010-05-26

    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.

  2. Exome sequencing reveals novel rare variants in the ryanodine receptor and calcium channel genes in malignant hyperthermia families.

    PubMed

    Kim, Jerry H; Jarvik, Gail P; Browning, Brian L; Rajagopalan, Ramakrishnan; Gordon, Adam S; Rieder, Mark J; Robertson, Peggy D; Nickerson, Deborah A; Fisher, Nickla A; Hopkins, Philip M

    2013-11-01

    About half of malignant hyperthermia (MH) cases are associated with skeletal muscle ryanodine receptor 1 (RYR1) and calcium channel, voltage-dependent, L type, α1S subunit (CACNA1S) gene mutations, leaving many with an unknown cause. The authors chose to apply a sequencing approach to uncover causal variants in unknown cases. Sequencing the exome, the protein-coding region of the genome, has power at low sample sizes and identified the cause of over a dozen Mendelian disorders. The authors considered four families with multiple MH cases lacking mutations in RYR1 and CACNA1S by Sanger sequencing of complementary DNA. Exome sequencing in two affecteds per family, chosen for maximum genetic distance, were compared. Variants were ranked by allele frequency, protein change, and measures of conservation among mammals to assess likelihood of causation. Finally, putative pathogenic mutations were genotyped in other family members to verify cosegregation with MH. Exome sequencing revealed one rare RYR1 nonsynonymous variant in each of three families (Asp1056His, Val2627Met, Val4234Leu), and one CACNA1S variant (Thr1009Lys) in the fourth family. These were not seen in variant databases or in our control population sample of 5,379 exomes. Follow-up sequencing in other family members verified cosegregation of alleles with MH. The authors found that using both exome sequencing and allele frequency data from large sequencing efforts may aid genetic diagnosis of MH. In a sample selected by the authors, this technique was more sensitive for variant detection in known genes than Sanger sequencing of complementary DNA, and allows for the possibility of novel gene discovery.

  3. Exome Sequencing Reveals Novel Rare Variants in the Ryanodine Receptor and Calcium Channel Genes in Malignant Hyperthermia Families

    PubMed Central

    Kim, Jerry H.; Jarvik, Gail P.; Browning, Brian L.; Rajagopalan, Ramakrishnan; Gordon, Adam S.; Rieder, Mark J.; Robertson, Peggy D.; Nickerson, Deborah A.; Fisher, Nickla A.; Hopkins, Philip M.

    2014-01-01

    Background About half of malignant hyperthermia (MH) cases are associated with skeletal muscle ryanodine receptor 1 (RYR1) and calcium channel, voltage-dependent, L type, α1S subunit (CACNA1S) gene mutations, leaving many with an unknown cause. We chose to apply a sequencing approach to uncover causal variants in unknown cases. Sequencing the exome, the protein-coding region of the genome, has power at low sample sizes and identified the cause of over a dozen Mendelian disorders. Methods We considered four families with multiple MH cases but in whom no mutations in RYR1 and CACNA1S had been identified by Sanger sequencing of complementary DNA. Exome sequencing of two affecteds per family, chosen for maximum genetic distance, were compared. Variants were ranked by allele frequency, protein change, and measures of conservation among mammals to assess likelihood of causation. Finally, putative pathogenic mutations were genotyped in other family members to verify cosegregation with MH. Results Exome sequencing revealed 1 rare RYR1 nonsynonymous variant in each of 3 families (Asp1056His, Val2627Met, Val4234Leu), and 1 CACNA1S variant (Thr1009Lys) in a 4th family. These were not seen in variant databases or in our control population sample of 5379 exomes. Follow-up sequencing in other family members verified cosegregation of alleles with MH. Conclusions Using both exome sequencing and allele frequency data from large sequencing efforts may aid genetic diagnosis of MH. In our sample, it was more sensitive for variant detection in known genes than Sanger sequencing of complementary DNA, and allows for the possibility of novel gene discovery. PMID:24013571

  4. Identification of novel ryanodine receptor 1 (RyR1) protein interaction with calcium homeostasis endoplasmic reticulum protein (CHERP).

    PubMed

    Ryan, Timothy; Sharma, Parveen; Ignatchenko, Alex; MacLennan, David H; Kislinger, Thomas; Gramolini, Anthony O

    2011-05-13

    The ryanodine receptor type 1 (RyR1) is a homotetrameric Ca(2+) release channel located in the sarcoplasmic reticulum of skeletal muscle where it plays a role in the initiation of skeletal muscle contraction. A soluble, 6×-histidine affinity-tagged cytosolic fragment of RyR1 (amino acids 1-4243) was expressed in HEK-293 cells, and metal affinity chromatography under native conditions was used to purify the peptide together with interacting proteins. When analyzed by gel-free liquid chromatography mass spectrometry (LC-MS), 703 proteins were identified under all conditions. This group of proteins was filtered to identify putative RyR interacting proteins by removing those proteins found in only 1 RyR purification and proteins for which average spectral counts were enriched by less than 4-fold over control values. This resulted in 49 potential RyR1 interacting proteins, and 4 were selected for additional interaction studies: calcium homeostasis endoplasmic reticulum protein (CHERP), endoplasmic reticulum-Golgi intermediate compartment 53-kDa protein (LMAN1), T-complex protein, and phosphorylase kinase. Western blotting showed that only CHERP co-purified with affinity-tagged RyR1 and was eluted with imidazole. Immunofluorescence showed that endogenous CHERP co-localizes with endogenous RyR1 in the sarcoplasmic reticulum of rat soleus muscle. A combination of overexpression of RyR1 in HEK-293 cells with siRNA-mediated suppression of CHERP showed that CHERP affects Ca(2+) release from the ER via RyR1. Thus, we propose that CHERP is an RyR1 interacting protein that may be involved in the regulation of excitation-contraction coupling.

  5. Regulation of the skeletal muscle ryanodine receptor/Ca2+-release channel RyR1 by S-palmitoylation.

    PubMed

    Chaube, Ruchi; Hess, Douglas T; Wang, Ya-Juan; Plummer, Bradley; Sun, Qi-An; Laurita, Kennneth; Stamler, Jonathan S

    2014-03-21

    The ryanodine receptor/Ca(2+)-release channels (RyRs) of skeletal and cardiac muscle are essential for Ca(2+) release from the sarcoplasmic reticulum that mediates excitation-contraction coupling. It has been shown that RyR activity is regulated by dynamic post-translational modifications of Cys residues, in particular S-nitrosylation and S-oxidation. Here we show that the predominant form of RyR in skeletal muscle, RyR1, is subject to Cys-directed modification by S-palmitoylation. S-Palmitoylation targets 18 Cys within the N-terminal, cytoplasmic region of RyR1, which are clustered in multiple functional domains including those implicated in the activity-governing protein-protein interactions of RyR1 with the L-type Ca(2+) channel CaV1.1, calmodulin, and the FK506-binding protein FKBP12, as well as in "hot spot" regions containing sites of mutations implicated in malignant hyperthermia and central core disease. Eight of these Cys have been identified previously as subject to physiological S-nitrosylation or S-oxidation. Diminishing S-palmitoylation directly suppresses RyR1 activity as well as stimulus-coupled Ca(2+) release through RyR1. These findings demonstrate functional regulation of RyR1 by a previously unreported post-translational modification and indicate the potential for extensive Cys-based signaling cross-talk. In addition, we identify the sarco/endoplasmic reticular Ca(2+)-ATPase 1A and the α1S subunit of the L-type Ca(2+) channel CaV1.1 as S-palmitoylated proteins, indicating that S-palmitoylation may regulate all principal governors of Ca(2+) flux in skeletal muscle that mediates excitation-contraction coupling.

  6. Characterization of the Ryanodine Receptor Gene With a Unique 3'-UTR and Alternative Splice Site From the Oriental Fruit Moth.

    PubMed

    Sun, L N; Zhang, H J; Quan, L F; Yan, W T; Yue, Q; Li, Y Y; Qiu, G S

    2016-01-01

    The ryanodine receptor (RyR), the largest calcium channel protein, has been studied because of its key roles in calcium signaling in cells. Insect RyRs are molecular targets for novel diamide insecticides. The target has been focused widely because of the diamides with high activity against lepidopterous pests and safety for nontarget organisms. To study our understanding of effects of diamides on RyR, we cloned the RyR gene from the oriental fruit moth, Grapholita molesta, which is the most serious pest of stone and pome tree fruits throughout the world, to investigate the modulation of diamide insecticides on RyR mRNA expression in G. molesta (GmRyR). The full-length cDNAs of GmRyR contain a unique 3'-UTR with 625 bp and an open reading frame of 15,402 bp with a predicted protein consisting of 5,133 amino acids. GmRyR possessed a high level of overall amino acid homology with insect and vertebrate isoforms, with 77-92% and 45-47% identity, respectively. Furthermore, five alternative splice sites were identified in GmRyR. Diagnostic PCR showed that the inclusion frequency of one optional exon (f) differed between developmental stages, a finding only found in GmRyR. The lowest expression level of GmRyR mRNA was in larvae, the highest was in male pupae, and the relative expression level in male pupae was 25.67 times higher than that of in larvae. The expression level of GmRyR in the male pupae was 8.70 times higher than in female pupae, and that in male adults was 5.70 times higher than female adults. © The Author 2016. Published by Oxford University Press on behalf of the Entomological Society of America.

  7. Cell Type Specific Spatial and Functional Coupling Between Mammalian Brain Kv2.1 K+ Channels and Ryanodine Receptors

    PubMed Central

    Mandikian, Danielle; Bocksteins, Elke; Parajuli, Laxmi Kumar; Bishop, Hannah I.; Cerda, Oscar; Shigemoto, Ryuichi; Trimmer, James S.

    2014-01-01

    The Kv2.1 voltage-gated K+ channel is widely expressed throughout mammalian brain where it contributes to dynamic activity-dependent regulation of intrinsic neuronal excitability. Here we show that somatic plasma membrane Kv2.1 clusters are juxtaposed to clusters of intracellular ryanodine receptor (RyR) Ca2+-release channels in mouse brain neurons, most prominently in medium spiny neurons (MSNs) of the striatum. Electron microscopy-immunogold labeling shows that in MSNs, plasma membrane Kv2.1 clusters are adjacent to subsurface cisternae, placing Kv2.1 in close proximity to sites of RyR-mediated Ca2+ release. Immunofluorescence labeling in transgenic mice expressing GFP in specific MSN populations reveals the most prominent juxtaposed Kv2.1-RyR clusters in indirect pathway MSNs. Kv2.1 in both direct and indirect pathway MSNs exhibits markedly lower levels of labeling with phosphospecific antibodies directed against the S453, S563, and S603 phosphorylation site compared to levels observed in neocortical neurons, although labeling for Kv2.1 phosphorylation at S563 was significantly lower in indirect pathway MSNs compared to those in the direct pathway. Finally, acute stimulation of RyRs in heterologous cells causes a rapid hyperpolarizing shift in the voltage-dependence of activation of Kv2.1, typical of Ca2+/calcineurin-dependent Kv2.1 dephosphorylation. Together, these studies reveal that striatal MSNs are distinct in their expression of clustered Kv2.1 at plasma membrane sites juxtaposed to intracellular RyRs, as well as in Kv2.1 phosphorylation state. Differences in Kv2.1 expression and phosphorylation between MSNs in direct and indirect pathways provide a cell- and circuit-specific mechanism for coupling intracellular Ca2+ release to phosphorylation-dependent regulation of Kv2.1 to dynamically impact intrinsic excitability. PMID:24962901

  8. Ion-pulling simulations provide insights into the mechanisms of channel opening of the skeletal muscle ryanodine receptor.

    PubMed

    Mowrey, David D; Xu, Le; Mei, Yingwu; Pasek, Daniel A; Meissner, Gerhard; Dokholyan, Nikolay V

    2017-08-04

    The type 1 ryanodine receptor (RyR1) mediates Ca(2+) release from the sarcoplasmic reticulum to initiate skeletal muscle contraction and is associated with muscle diseases, malignant hyperthermia, and central core disease. To better understand RyR1 channel function, we investigated the molecular mechanisms of channel gating and ion permeation. An adequate model of channel gating requires accurate, high-resolution models of both open and closed states of the channel. To this end, we generated an open-channel RyR1 model using molecular simulations to pull Ca(2+) through the pore constriction site of a closed-channel RyR1 structure determined at 3.8-Å resolution. Importantly, we find that our open-channel model is consistent with the RyR1 and cardiac RyR (RyR2) open-channel structures reported while this paper was in preparation. Both our model and the published structures show similar rotation of the upper portion of the pore-lining S6 helix away from the 4-fold channel axis and twisting of Ile-4937 at the channel constriction site out of the channel pore. These motions result in a minimum open-channel pore radius of ∼3 Å formed by Gln-4933, rather than Ile-4937 in the closed-channel structure. We also present functional support for our model by mutations around the closed- and open-channel constriction sites (Gln-4933 and Ile-4937). Our results indicate that use of ion-pulling simulations produces a RyR1 open-channel model, which can provide insights into the mechanisms of channel opening complementing those from the structural data. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

  9. Postpacing abnormal repolarization in catecholaminergic polymorphic ventricular tachycardia associated with a mutation in the cardiac ryanodine receptor gene.

    PubMed

    Nof, Eyal; Belhassen, Bernard; Arad, Michael; Bhuiyan, Zahurul A; Antzelevitch, Charles; Rosso, Raphael; Fogelman, Rami; Luria, David; El-Ani, Dalia; Mannens, Marcel M A M; Viskin, Sami; Eldar, Michael; Wilde, Arthur A M; Glikson, Michael

    2011-10-01

    Catecholaminergic polymorphic ventricular tachycardia (CPVT) is an arrhythmogenic disease for which electrophysiological studies (EPS) have shown to be of limited value. This study presents a CPVT family in which marked postpacing repolarization abnormalities during EPS were the only consistent phenotypic manifestation of ryanodine receptor (RyR2) mutation carriers. The study was prompted by the observation of transient marked QT prolongation preceding initiation of ventricular fibrillation during atrial fibrillation in a boy with a family history of sudden cardiac death (SCD). Family members underwent exercise and pharmacologic electrocardiographic testing with epinephrine, adenosine, and flecainide. Noninvasive clinical test results were normal in 10 patients evaluated, except for both epinephrine- and exercise-induced ventricular arrhythmias in 1. EPS included bursts of ventricular pacing and programmed ventricular extrastimulation reproducing short-long sequences. Genetic screening involved direct sequencing of genes involved in long QT syndrome as well as RyR2. Six patients demonstrated a marked increase in QT interval only in the first beat after cessation of ventricular pacing and/or extrastimulation. All 6 patients were found to have a heterozygous missense mutation (M4109R) in RyR2. Two of them, presenting with aborted SCD, also had a second missense mutation (I406T- RyR2). Four family members without RyR2 mutations did not display prominent postpacing QT changes. M4109R- RyR2 is associated with a high incidence of SCD. The contribution of I406T to the clinical phenotype is unclear. In contrast to exercise testing, marked postpacing repolarization changes in a single beat accurately predicted carriers of M4109R- RyR2 in this family. Copyright © 2011 Heart Rhythm Society. Published by Elsevier Inc. All rights reserved.

  10. Ryanodine receptor blockade reduces amyloid-β load and memory impairments in Tg2576 mouse model of Alzheimer disease.

    PubMed

    Oulès, Bénédicte; Del Prete, Dolores; Greco, Barbara; Zhang, Xuexin; Lauritzen, Inger; Sevalle, Jean; Moreno, Sebastien; Paterlini-Bréchot, Patrizia; Trebak, Mohamed; Checler, Frédéric; Benfenati, Fabio; Chami, Mounia

    2012-08-22

    In Alzheimer disease (AD), the perturbation of the endoplasmic reticulum (ER) calcium (Ca²⁺) homeostasis has been linked to presenilins, the catalytic core in γ-secretase complexes cleaving the amyloid precursor protein (APP), thereby generating amyloid-β (Aβ) peptides. Here we investigate whether APP contributes to ER Ca²⁺ homeostasis and whether ER Ca²⁺ could in turn influence Aβ production. We show that overexpression of wild-type human APP (APP(695)), or APP harboring the Swedish double mutation (APP(swe)) triggers increased ryanodine receptor (RyR) expression and enhances RyR-mediated ER Ca²⁺ release in SH-SY5Y neuroblastoma cells and in APP(swe)-expressing (Tg2576) mice. Interestingly, dantrolene-induced lowering of RyR-mediated Ca²⁺ release leads to the reduction of both intracellular and extracellular Aβ load in neuroblastoma cells as well as in primary cultured neurons derived from Tg2576 mice. This Aβ reduction can be accounted for by decreased Thr-668-dependent APP phosphorylation and β- and γ-secretases activities. Importantly, dantrolene diminishes Aβ load, reduces Aβ-related histological lesions, and slows down learning and memory deficits in Tg2576 mice. Overall, our data document a key role of RyR in Aβ production and learning and memory performances, and delineate RyR-mediated control of Ca²⁺ homeostasis as a physiological paradigm that could be targeted for innovative therapeutic approaches.

  11. Restraint effects on stress-related hormones and blood natural killer cell cytotoxicity in pigs with a mutated ryanodine receptor.

    PubMed

    Ciepielewski, Z M; Stojek, W; Glac, W; Wrona, D

    2013-05-01

    A mutation in the ryanodine receptor gene (RYR1) of the calcium release channel is responsible for increased stress susceptibility in pigs. In the present study, the relation of a mutation in RYR1 with the neuroendocrine (stress-related hormone) response and the immune defense represented by natural killer cell cytotoxicity (NKCC) during a 4-h restraint and recovery phase in 60 male pigs was investigated. Blood samples were collected from pigs previously divided into RYR1 genotypes (nn, Nn, NN), based on PCR amplification and restriction analyses. The blood samples collected during the restraint and recovery phases of the experiment were used to determine NKCC ((51)Cr-release assay), large granular lymphocyte number (hematologic method), and plasma concentrations of prolactin (PRL), GH, ACTH, and cortisol (COR) (by specific RIA). The greatest degree of NKCC response (P < 0.05) to restraint stress relative to controls was observed for the stress-susceptible homozygote group (nn). Measures of stress-related hormones were positively correlated with NKCC during the entire experimental period (P < 0.001 for all investigated hormones) in the nn group. Immunostimulatory effects in the early (0-60 min) phase of restraint were associated with increased hormone responses, especially PRL and GH. In the late (180-240 min) phase of stress and the recovery phase (480 min), a decrease in immune response was accompanied by an elevated COR response in all RYR1 genotypes. Moreover, divergent responses of both PRL (greatest in nn, P < 0.001) and GH (greatest in NN, P < 0.001) to the 4-h restraint were observed. Our results suggest that stress-susceptible RYR1-mutated homozygotes develop a greater level of immune defense, including cytotoxic activity of NK cells, and accompanied by more pronounced stress-induced changes in neuroendocrine response than stress-resistant heterozygous (Nn) and homozygous (NN) pigs.

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

    PubMed Central

    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

  13. Skeletal muscle ryanodine receptor mutations associated with malignant hyperthermia showed enhanced intensity and sensitivity to triggering drugs when expressed in human embryonic kidney cells.

    PubMed

    Sato, Keisaku; Roesl, Cornelia; Pollock, Neil; Stowell, Kathryn M

    2013-07-01

    Mutations within the gene encoding the skeletal muscle calcium channel ryanodine receptor can result in malignant hyperthermia. Although it is important to characterize the functional effects of candidate mutations to establish a genetic test for diagnosis, ex vivo methods are limited because of the low incidence of the disorder and sample unavailability. More than 250 candidate mutations have been identified, but only a few mutations have been functionally characterized. The human skeletal muscle ryanodine receptor complementary DNA was cloned with or without a disease-related variant. Wild-type and mutant calcium channel proteins were transiently expressed in human embryonic kidney-293 cells expressing the large T-antigen of simian virus 40, and functional analysis was carried out using calcium imaging with fura-2 AM. Six human malignant hyperthermia-related mutants such as R44C, R163C, R401C, R533C, R533H, and H4833Y were analyzed. Cells were stimulated with a specific ryanodine receptor agonist 4-chloro-m-cresol, and intracellular calcium mobility was analyzed to determine the functional aspects of mutant channels. Mutant proteins that contained a variant linked to malignant hyperthermia showed higher sensitivity to the agonist. Compared with the wild type (EC50=453.2 µM, n=18), all six mutants showed a lower EC50 (21.2-170.4 µM, n=12-23), indicating susceptibility against triggering agents. These six mutations cause functional abnormality of the calcium channel, leading to higher sensitivity to a specific agonist, and therefore could be considered potentially causative of malignant hyperthermia reactions.

  14. Integrin ligands mobilize Ca2+ from ryanodine receptor-gated stores and lysosome-related acidic organelles in pulmonary arterial smooth muscle cells.

    PubMed

    Umesh, Anita; Thompson, Michael A; Chini, Eduardo N; Yip, Kay-Pong; Sham, James S K

    2006-11-10

    Extracellular matrix (ECM) protein receptors, or integrins, participate in vascular remodeling and the systemic myogenic response. Synthetic ligands and ECM fragments regulate the vascular smooth muscle cell contractile state by altering intracellular Ca2+ levels ([Ca2+]i). Information on the Ca2+ effect of integrins in vascular smooth muscle cells is limited, but nonexistent in pulmonary arterial smooth muscle cells (PASMCs). We therefore characterized integrin expression in endothelium-denuded pulmonary arteries, and explored [Ca2+]i mobilization pathways induced by soluble ligands in rat PASMCs. Reverse transcriptase-PCR showed mRNA expression of integrins alpha1, alpha2, alpha3, alpha4, alpha5, alpha7, alpha8, alpha(v), beta1, beta3, and beta4, and immunoblots of alpha5, alpha(v), beta1, and beta3 confirmed protein expression. Exposure of PASMCs to integrin-binding peptides (0.5 mM) containing the arginine-glycine-aspartate (RGD) motif elicited [Ca2+]i responses with an order of potency of GRGDNP > GRGDSP > GRGDTP = cyclo-RGD. Pharmacological analysis revealed that the GRGDSP-induced Ca2+ response was unrelated to Ca2+ influx and the inositol triphosphate receptor-gated Ca2+ store, but partially blocked by ryanodine or inhibition of lysosome-related acidic organelles with bafilomycin A1. Simultaneous inhibition of both pathways was necessary to abolish the response. GRGDSP treatment increased cyclic ADP-ribose, the endogenous activator of ryanodine receptors, by 70%. GRGDSP also rapidly reduced Lysotracker Red accumulation, confirming direct modulation of acidic organelles. These data are the first demonstration of integrin-mediated Ca2+ regulation in PASMCs. The presence of an array of integrins, and activation of ryanodine-sensitive Ca2+ stores and lysosome-like organelles by GRGDSP suggest important roles for integrin-dependent Ca2+ signaling in regulating PASMC function.

  15. Participation of inositol trisphosphate and ryanodine receptors in Bufo arenarum oocyte activation.

    PubMed

    Ajmat, M T; Bonilla, F; Zelarayán, L; Bühler, M I

    2011-05-01

    Calcium is considered the most important second messenger at fertilization. Transient release from intracellular stores is modulated through both agonist-gated channels, IP₃Rs and RyRs, which can be found individually or together depending on the oocyte species. Using the four commonly used compounds (thimerosal, caffeine, heparin and ruthenium red), we investigated the existence and interdependence of both IP₃Rs and RyRs in mature Bufo arenarum oocytes. We found that caffeine, a well known specific RyRs agonist, was able to trigger oocyte activation in a dose-dependent manner. Microinjection of 10 mM caffeine showed 100% of oocytes exhibiting characteristic morphological criteria of egg activation. Ruthenium red, the specific RyR blocker, was able to inhibit oocyte activation induced either by sperm or caffeine. Our present findings provide the first reported evidence of the existence of RyR in frogs. We further explored the relationship between IP₃Rs and RyRs in B. arenarum oocytes by exposing them to the agonists of one class after injecting a blocker of the other class of receptor. We found that thimerosal overcame the inhibitory effect of RyR on oocyte activation, indicating that IP₃Rs function as independent receptors. In contrast, previous injection of heparin delayed caffeine-induced calcium release, revealing a relative dependence of RyRs on functional IP₃Rs, probably through a CICR mechanism. Both receptors play a role in Ca²+ release mechanisms although their relative contribution to the activation process is unclear.

  16. The EF-hand Ca2+ Binding Domain Is Not Required for Cytosolic Ca2+ Activation of the Cardiac Ryanodine Receptor.

    PubMed

    Guo, Wenting; Sun, Bo; Xiao, Zhichao; Liu, Yingjie; Wang, Yundi; Zhang, Lin; Wang, Ruiwu; Chen, S R Wayne

    2016-01-29

    Activation of the cardiac ryanodine receptor (RyR2) by elevating cytosolic Ca(2+) is a central step in the process of Ca(2+)-induced Ca(2+) release, but the molecular basis of RyR2 activation by cytosolic Ca(2+) is poorly defined. It has been proposed recently that the putative Ca(2+) binding domain encompassing a pair of EF-hand motifs (EF1 and EF2) in the skeletal muscle ryanodine receptor (RyR1) functions as a Ca(2+) sensor that regulates the gating of RyR1. Although the role of the EF-hand domain in RyR1 function has been studied extensively, little is known about the functional significance of the corresponding EF-hand domain in RyR2. Here we investigate the effect of mutations in the EF-hand motifs on the Ca(2+) activation of RyR2. We found that mutations in the EF-hand motifs or deletion of the entire EF-hand domain did not affect the Ca(2+)-dependent activation of [(3)H]ryanodine binding or the cytosolic Ca(2+) activation of RyR2. On the other hand, deletion of the EF-hand domain markedly suppressed the luminal Ca(2+) activation of RyR2 and spontaneous Ca(2+) release in HEK293 cells during store Ca(2+) overload or store overload-induced Ca(2+) release (SOICR). Furthermore, mutations in the EF2 motif, but not EF1 motif, of RyR2 raised the threshold for SOICR termination, whereas deletion of the EF-hand domain of RyR2 increased both the activation and termination thresholds for SOICR. These results indicate that, although the EF-hand domain is not required for RyR2 activation by cytosolic Ca(2+), it plays an important role in luminal Ca(2+) activation and SOICR.

  17. Low dose acute alcohol effects on GABAA receptor subtypes

    PubMed Central

    Wallner, Martin; Hanchar, H. Jacob; Olsen, Richard W.

    2010-01-01

    GABAA receptors (GABAARs) are the main inhibitory neurotransmitter receptors and have long been implicated in mediating at least part of the acute actions of ethanol. For example, ethanol and GABAergic drugs including barbiturates and benzodiazepines share many pharmacological properties. Besides the prototypical synaptic GABAAR subtypes, nonsynaptic GABAARs have recently emerged as important regulators of neuronal excitability. While high doses (≥100 mM) of ethanol have been reported to enhance activity of most GABAAR subtypes, most abundant synaptic GABAARs are essentially insensitive to ethanol concentrations that occur during social ethanol consumption (<30 mM). However, extrasynaptic δ and β3 subunit-containing GABAARs, associated in the brain with α4or α6 subunits, are sensitive to low millimolar ethanol concentrations, as produced by drinking half a glass of wine. Additionally, we found that a mutation in the cerebellar α6 subunit (α6R100Q), initially reported in rats selectively bred for increased alcohol sensitivity, is sufficient to produce increased alcohol-induced motor impairment and further increases of alcohol sensitivity in recombinant α6β3δ receptors. Furthermore, the behavioral alcohol antagonist Ro15-4513 blocks the low dose alcohol enhancement on α4/6/β3δ receptors, without reducing GABA-induced currents. In binding assays α4β3δ GABAARs bind [3H] Ro15-4513 with high affinity, and this binding is inhibited, in an apparently competitive fashion, by low ethanol concentrations, as well as analogs of Ro15-4513 that are active to antagonize ethanol or Ro15-4513’s block of ethanol. We conclude that most low to moderate dose alcohol effects are mediated by alcohol actions on alcohol/Ro15-4513 binding sites on GABAAR subtypes. PMID:16814864

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

    PubMed

    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

  19. Muscarinic receptor subtypes in human and rat colon smooth muscle.

    PubMed

    Gómez, A; Martos, F; Bellido, I; Marquez, E; Garcia, A J; Pavia, J; Sanchez de la Cuesta, F

    1992-06-09

    Muscarinic receptor subtypes in human and rat colon smooth muscle homogenates were characterized with [3H]N-methylscopolamine ([3H]NMS) by ligand binding studies. [3H]NMS saturation experiments show the existence of a homogeneous population of non-interacting binding sites with similar affinity (KD values of 1.38 +/- 0.20 nM in human colon smooth muscle and 1.48 +/- 0.47 nM in rat colon smooth muscle) and with Hill slopes close to unity in both samples of tissue. However, a significant (P less than 0.01) increase in muscarinic receptor density (Bmax) is found in human colon (29.9 +/- 2.9 fmol/mg protein) compared with rat colon (17.2 +/- 1.5 fmol/mg protein). Inhibition of [3H]NMS binding by non-labelled compounds shows the following order in human colon: atropine greater than AF-DX 116 greater than pirenzepine. Whereas in rat colon the rank order obtained is atropine greater than pirenzepine greater than AF-DX 116. Atropine and pirenzepine bind to a homogeneous population of binding sites, although pirenzepine shows higher affinity to bind to the sites present in rat colon (Ki = 1.08 +/- 0.08 microM) than those in human colon (Ki = 1.74 +/- 0.02 microM) (P less than 0.05). Similarly, IC50 values obtained in AF-DX 116 competition experiments were significantly different (P less than 0.01) in human colon (IC50 = 1.69 +/- 0.37 microM) than in rat colon (IC50 = 3.78 +/- 0.75 microM). Unlike atropine and pirenzepine, the inhibition of [3H]NMS binding by AF-DX 116 did not yield a simple mass-action binding curve (nH less than 1, P less than 0.01) suggesting the presence of more than one subtype of muscarinic receptor in both species. Computer analysis of these curves with a two binding site model suggests the presence of two populations of receptor. The apparent Ki1 value for the high affinity binding site is 0.49 +/- 0.07 microM for human colon smooth muscle and 0.33 +/- 0.05 microM for rat colon smooth muscle. The apparent Ki2 for the low affinity binding site is 8

  20. The Role of Parvalbumin, Sarcoplasmatic Reticulum Calcium Pump Rate, Rates of Cross-Bridge Dynamics, and Ryanodine Receptor Calcium Current on Peripheral Muscle Fatigue: A Simulation Study

    PubMed Central

    Neumann, Verena

    2016-01-01

    A biophysical model of the excitation-contraction pathway, which has previously been validated for slow-twitch and fast-twitch skeletal muscles, is employed to investigate key biophysical processes leading to peripheral muscle fatigue. Special emphasis hereby is on investigating how the model's original parameter sets can be interpolated such that realistic behaviour with respect to contraction time and fatigue progression can be obtained for a continuous distribution of the model's parameters across the muscle units, as found for the functional properties of muscles. The parameters are divided into 5 groups describing (i) the sarcoplasmatic reticulum calcium pump rate, (ii) the cross-bridge dynamics rates, (iii) the ryanodine receptor calcium current, (iv) the rates of binding of magnesium and calcium ions to parvalbumin and corresponding dissociations, and (v) the remaining processes. The simulations reveal that the first two parameter groups are sensitive to contraction time but not fatigue, the third parameter group affects both considered properties, and the fourth parameter group is only sensitive to fatigue progression. Hence, within the scope of the underlying model, further experimental studies should investigate parvalbumin dynamics and the ryanodine receptor calcium current to enhance the understanding of peripheral muscle fatigue. PMID:27980606

  1. The Role of Parvalbumin, Sarcoplasmatic Reticulum Calcium Pump Rate, Rates of Cross-Bridge Dynamics, and Ryanodine Receptor Calcium Current on Peripheral Muscle Fatigue: A Simulation Study.

    PubMed

    Röhrle, Oliver; Neumann, Verena; Heidlauf, Thomas

    2016-01-01

    A biophysical model of the excitation-contraction pathway, which has previously been validated for slow-twitch and fast-twitch skeletal muscles, is employed to investigate key biophysical processes leading to peripheral muscle fatigue. Special emphasis hereby is on investigating how the model's original parameter sets can be interpolated such that realistic behaviour with respect to contraction time and fatigue progression can be obtained for a continuous distribution of the model's parameters across the muscle units, as found for the functional properties of muscles. The parameters are divided into 5 groups describing (i) the sarcoplasmatic reticulum calcium pump rate, (ii) the cross-bridge dynamics rates, (iii) the ryanodine receptor calcium current, (iv) the rates of binding of magnesium and calcium ions to parvalbumin and corresponding dissociations, and (v) the remaining processes. The simulations reveal that the first two parameter groups are sensitive to contraction time but not fatigue, the third parameter group affects both considered properties, and the fourth parameter group is only sensitive to fatigue progression. Hence, within the scope of the underlying model, further experimental studies should investigate parvalbumin dynamics and the ryanodine receptor calcium current to enhance the understanding of peripheral muscle fatigue.

  2. Direct detection of calmodulin tuning by ryanodine receptor channel targets using a Ca2+-sensitive acrylodan-labeled calmodulin.

    PubMed

    Fruen, Bradley R; Balog, Edward M; Schafer, Janet; Nitu, Florentin R; Thomas, David D; Cornea, Razvan L

    2005-01-11

    Calmodulin (CaM) activates the skeletal muscle ryanodine receptor (RyR1) at nanomolar Ca(2+) concentrations but inhibits it at micromolar Ca(2+) concentrations, indicating that binding of Ca(2+) to CaM may provide a molecular switch for modulating RyR1 channel activity. To directly examine the Ca(2+) sensitivity of RyR1-complexed CaM, we used an environment-sensitive acrylodan adduct of CaM. The resulting (ACR)CaM probe displayed high-affinity binding to, and Ca(2+)-dependent regulation of, RyR1 similar to that of unlabeled wild-type (WT) CaM. Upon addition of Ca(2+), (ACR)CaM exhibited a substantial (>50%) decrease in fluorescence (K(Ca) = 2.7 +/- 0.8 microM). A peptide derived from the RyR1 CaM binding domain (RyR1(3614)(-)(43)) caused an even more pronounced Ca(2+)-dependent fluorescence decrease, and a >or=10-fold leftward shift in its K(Ca) (0.2 +/- 0.1 microM). In the presence of intact RyR1 channels in SR vesicles, (ACR)CaM fluorescence spectra were distinct from those in the presence of RyR1(3614)(-)(43), although a Ca(2+)-dependent decrease in fluorescence was still observed. The K(Ca) for (ACR)CaM fluorescence in the presence of SR (0.8 +/- 0.4 microM) was greater than in the presence of RyR1(3614)(-)(43) but was consistent with functional determinations showing the conversion of (ACR)CaM from channel activator (apoCaM) to inhibitor (Ca(2+)CaM) at Ca(2+) concentrations between 0.3 and 1 microM. These results indicate that binding to RyR1 targets evokes significant changes in the CaM structure and Ca(2+) sensitivity (i.e., CaM tuning). However, changes resulting from binding of CaM to the full-length, tetrameric channels are clearly distinct from changes caused by the RyR1-derived peptide. We suggest that the Ca(2+) sensitivity of CaM when in complex with full-length channels may be tuned to respond to physiologically relevant changes in Ca(2+).

  3. Identification of a key determinant of ryanodine receptor type 1 required for activation by 4-chloro-m-cresol.

    PubMed

    Fessenden, James D; Perez, Claudio F; Goth, Sam; Pessah, Isaac N; Allen, Paul D

    2003-08-01

    4-Chloro-m-cresol (4-CmC) is a potent and specific activator of the intracellular Ca2+ release channel, the ryanodine receptor (RyR). We have previously shown that RyR1 expressed in dyspedic 1B5 myotubes is activated by 4-CmC, whereas RyR3 is not (Fessenden, J. D., Wang, Y., Moore, R. A., Chen, S. R. W., Allen, P. D., and Pessah, I. N. (2000) Biophys. J. 79, 2509-2525). To identify region(s) on RyR1 that are responsible for mediating activation by 4-CmC, we expressed RyR1-RyR3 chimeric proteins in dyspedic 1B5 myotubes and then measured 4-CmC-induced increases in intracellular Ca2+. Substitution of the C-terminal third of RyR1 into RyR3 imparted 4-CmC sensitivity to the resulting chimera, thus suggesting that determinants required for activation by 4-CmC are located in this region. We subdivided the C-terminal third of RyR1 into smaller segments and identified two overlapping regions of RyR1 (amino acids 3769-4180 and 4007-4382) that each imparted 4-CmC sensitivity to RyR3. Substitution of the 173 amino acids of RyR1 common to these two chimeras (amino acids 4007-4180) also weakly restored 4-CmC sensitivity in the resulting chimera. To confirm these findings, we created a complementary set of chimeras containing RyR3 substitutions in RyR1. Substitution of the RyR3 C terminus into RyR1 disrupted 4-CmC sensitivity in the resulting chimera. In addition, substitution of the corresponding RyR3 sequence into positions 4007-4180 of RyR1 disrupted 4-CmC sensitivity. Taken together, these results suggest that essential determinants required for activation of RyR1 by 4-CmC reside within a 173-amino acid region between residues 4007 and 4180.

  4. Site-Specific Labeling of the Type 1 Ryanodine Receptor Using Biarsenical Fluorophores Targeted to Engineered Tetracysteine Motifs

    PubMed Central

    Fessenden, James D.; Mahalingam, Mohana

    2013-01-01

    The type 1 ryanodine receptor (RyR1) is an intracellular Ca2+ release channel that mediates skeletal muscle excitation contraction coupling. While the overall shape of RyR1 has been elucidated using cryo electron microscopic reconstructions, fine structural details remain elusive. To better understand the structure of RyR1, we have previously used a cell-based fluorescence resonance energy transfer (FRET) method using a fused green fluorescent protein (GFP) donor and a fluorescent acceptor, Cy3NTA that binds specifically to short poly-histidine ‘tags’ engineered into RyR1. However, the need to permeabilize cells to allow Cy3NTA entry as well as the noncovalent binding of Cy3NTA to the His tag limits future applications of this technique for studying conformational changes of the RyR. To overcome these problems, we used a dodecapeptide sequence containing a tetracysteine (Tc) motif to target the biarsenical fluorophores, FlAsH and ReAsH to RyR1. These compounds freely cross intact cell membranes where they then bind covalently to the tetracysteine motif. First, we used this system to conduct FRET measurements in intact cells by fusing a yellow fluorescent protein (YFP) FRET donor to the N-terminus of RyR1 and then targeting the FRET acceptor, ReAsH to an adjacent Tc tag. Moderate energy transfer (∼33%) was observed whereas ReAsH incubation of a YFPRyR1 fusion protein lacking the Tc tag resulted in no detectable FRET. We also developed a FRET-based system that did not require RyR fluorescent protein fusions by labeling N-terminal Tc-tagged RyR1 with FlAsH, a FRET donor and then targeting the FRET acceptor Cy3NTA to an adjacent decahistidine (His10) tag. A high degree of energy transfer (∼66%) indicated proper binding of both compounds to these unique recognition sequences in RyR1. Thus, these two systems should provide unprecedented flexibility in future FRET-based structural determinations of RyR1. PMID:23724080

  5. Site-specific labeling of the type 1 ryanodine receptor using biarsenical fluorophores targeted to engineered tetracysteine motifs.

    PubMed

    Fessenden, James D; Mahalingam, Mohana

    2013-01-01

    The type 1 ryanodine receptor (RyR1) is an intracellular Ca(2+) release channel that mediates skeletal muscle excitation contraction coupling. While the overall shape of RyR1 has been elucidated using cryo electron microscopic reconstructions, fine structural details remain elusive. To better understand the structure of RyR1, we have previously used a cell-based fluorescence resonance energy transfer (FRET) method using a fused green fluorescent protein (GFP) donor and a fluorescent acceptor, Cy3NTA that binds specifically to short poly-histidine 'tags' engineered into RyR1. However, the need to permeabilize cells to allow Cy3NTA entry as well as the noncovalent binding of Cy3NTA to the His tag limits future applications of this technique for studying conformational changes of the RyR. To overcome these problems, we used a dodecapeptide sequence containing a tetracysteine (Tc) motif to target the biarsenical fluorophores, FlAsH and ReAsH to RyR1. These compounds freely cross intact cell membranes where they then bind covalently to the tetracysteine motif. First, we used this system to conduct FRET measurements in intact cells by fusing a yellow fluorescent protein (YFP) FRET donor to the N-terminus of RyR1 and then targeting the FRET acceptor, ReAsH to an adjacent Tc tag. Moderate energy transfer (∼33%) was observed whereas ReAsH incubation of a YFPRyR1 fusion protein lacking the Tc tag resulted in no detectable FRET. We also developed a FRET-based system that did not require RyR fluorescent protein fusions by labeling N-terminal Tc-tagged RyR1 with FlAsH, a FRET donor and then targeting the FRET acceptor Cy3NTA to an adjacent decahistidine (His10) tag. A high degree of energy transfer (∼66%) indicated proper binding of both compounds to these unique recognition sequences in RyR1. Thus, these two systems should provide unprecedented flexibility in future FRET-based structural determinations of RyR1.

  6. Ryanodine receptor sensitivity governs the stability and synchrony of local calcium release during cardiac excitation-contraction coupling.

    PubMed

    Wescott, Andrew P; Jafri, M Saleet; Lederer, W J; Williams, George S B

    2016-03-01

    Calcium-induced calcium release is the principal mechanism that triggers the cell-wide [Ca(2+)]i transient that activates muscle contraction during cardiac excitation-contraction coupling (ECC). Here, we characterize this process in mouse cardiac myocytes with a novel mathematical action potential (AP) model that incorporates realistic stochastic gating of voltage-dependent L-type calcium (Ca(2+)) channels (LCCs) and sarcoplasmic reticulum (SR) Ca(2+) release channels (the ryanodine receptors, RyR2s). Depolarization of the sarcolemma during an AP stochastically activates the LCCs elevating subspace [Ca(2+)] within each of the cell's 20,000 independent calcium release units (CRUs) to trigger local RyR2 opening and initiate Ca(2+) sparks, the fundamental unit of triggered Ca(2+) release. Synchronization of Ca(2+) sparks during systole depends on the nearly uniform cellular activation of LCCs and the likelihood of local LCC openings triggering local Ca(2+) sparks (ECC fidelity). The detailed design and true SR Ca(2+) pump/leak balance displayed by our model permits investigation of ECC fidelity and Ca(2+) spark fidelity, the balance between visible (Ca(2+) spark) and invisible (Ca(2+) quark/sub-spark) SR Ca(2+) release events. Excess SR Ca(2+) leak is examined as a disease mechanism in the context of "catecholaminergic polymorphic ventricular tachycardia (CPVT)", a Ca(2+)-dependent arrhythmia. We find that that RyR2s (and therefore Ca(2+) sparks) are relatively insensitive to LCC openings across a wide range of membrane potentials; and that key differences exist between Ca(2+) sparks evoked during quiescence, diastole, and systole. The enhanced RyR2 [Ca(2+)]i sensitivity during CPVT leads to increased Ca(2+) spark fidelity resulting in asynchronous systolic Ca(2+) spark activity. It also produces increased diastolic SR Ca(2+) leak with some prolonged Ca(2+) sparks that at times become "metastable" and fail to efficiently terminate. There is a huge margin of safety for

  7. Site-specific modification of calmodulin Ca²(+) affinity tunes the skeletal muscle ryanodine receptor activation profile.

    PubMed

    Jiang, Jie; Zhou, Yubin; Zou, Jin; Chen, Yanyi; Patel, Priya; Yang, Jenny J; Balog, Edward M

    2010-11-15

    The skeletal muscle isoform of the ryanodine receptor Ca²(+)-release channel (RyR1) is regulated by Ca²(+) and CaM (calmodulin). CaM shifts the biphasic Ca²(+)-dependence of RyR1 activation leftward, effectively increasing channel opening at low Ca²(+) and decreasing channel opening at high Ca²(+). The conversion of CaM from a RyR1 activator into an inhibitor is due to the binding of Ca²(+) to CaM; however, which of CaM's four Ca²(+)-binding sites serves as the switch for this conversion is unclear. We engineered a series of mutant CaMs designed to individually increase the Ca²(+) affinity of each of CaM's EF-hands by increasing the number of acidic residues in Ca²(+)-chelating positions. Domain-specific Ca²(+) affinities of each CaM variant were determined by equilibrium fluorescence titration. Mutations in sites I (T26D) or II (N60D) in CaM's N-terminal domain had little effect on CaM Ca²(+) affinity and regulation of RyR1. However, the site III mutation N97D increased the Ca²(+)-binding affinity of CaM's C-terminal domain and caused CaM to inhibit RyR1 at a lower Ca²(+) concentration than wild-type CaM. Conversely, the site IV mutation Q135D decreased the Ca²(+)-binding affinity of CaM's C-terminal domain and caused CaM to inhibit RyR1 at higher Ca²(+) concentrations. These results support the hypothesis that Ca²(+) binding to CaM's C-terminal acts as the switch converting CaM from a RyR1 activator into a channel inhibitor. These results indicate further that targeting CaM's Ca²(+) affinity may be a valid strategy to tune the activation profile of CaM-regulated ion channels.

  8. Designing calcium release channel inhibitors with enhanced electron donor properties: stabilizing the closed state of ryanodine receptor type 1.

    PubMed

    Ye, Yanping; Yaeger, Daniel; Owen, Laura J; Escobedo, Jorge O; Wang, Jialu; Singer, Jeffrey D; Strongin, Robert M; Abramson, Jonathan J

    2012-01-01

    New drugs with enhanced electron donor properties that target the ryanodine receptor from skeletal muscle sarcoplasmic reticulum (RyR1) are shown to be potent inhibitors of single-channel activity. In this article, we synthesize derivatives of the channel activator 4-chloro-3-methyl phenol (4-CmC) and the 1,4-benzothiazepine channel inhibitor 4-[-3{1-(4-benzyl) piperidinyl}propionyl]-7-methoxy-2,3,4,5-tetrahydro-1,4-benzothiazepine (K201, JTV519) with enhanced electron donor properties. Instead of activating channel activity (~100 μM), the 4-methoxy analog of 4-CmC [4-methoxy-3-methyl phenol (4-MmC)] inhibits channel activity at submicromolar concentrations (IC(50) = 0.34 ± 0.08 μM). Increasing the electron donor characteristics of K201 by synthesizing its dioxole congener results in an approximately 16 times more potent RyR1 inhibitor (IC(50) = 0.24 ± 0.05 μM) compared with K201 (IC(50) = 3.98 ± 0.79 μM). Inhibition is not caused by an increased closed time of the channel but seems to be caused by an open state block of RyR1. These alterations to chemical structure do not influence the ability of these drugs to affect Ca(2+)-dependent ATPase activity of sarcoplasmic/endoplasmic reticulum Ca(2+)-ATPase type 1. Moreover, the FKBP12 protein, which stabilizes RyR1 in a closed configuration, is shown to be a strong electron donor. It seems as if FKBP12, K201, its dioxole derivative, and 4-MmC inhibit RyR1 channel activity by virtue of their electron donor characteristics. These results embody strong evidence that designing new drugs to target RyR1 with enhanced electron donor characteristics results in more potent channel inhibitors. This is a novel approach to the design of new, more potent drugs with the aim of functionally modifying RyR1 single-channel activity.

  9. Role of Cys³⁶⁰² in the function and regulation of the cardiac ryanodine receptor.

    PubMed

    Mi, Tao; Xiao, Zhichao; Guo, Wenting; Tang, Yijun; Hiess, Florian; Xiao, Jianmin; Wang, Yundi; Zhang, Joe Z; Zhang, Lin; Wang, Ruiwu; Jones, Peter P; Chen, S R Wayne

    2015-04-01

    The cardiac Ca²⁺ release channel [ryanodine receptor type 2 (RyR2)] is modulated by thiol reactive agents, but the molecular basis of RyR2 modulation by thiol reagents is poorly understood. Cys³⁶³⁵ in the skeletal muscle RyR1 is one of the most hyper-reactive thiols and is important for the redox and calmodulin (CaM) regulation of the RyR1 channel. However, little is known about the role of the corresponding cysteine residue in RyR2 (Cys³⁶⁰²) in the function and regulation of the RyR2 channel. In the present study, we assessed the impact of mutating Cys³⁶⁰² (C³⁶⁰²A) on store overload-induced Ca²⁺ release (SOICR) and the regulation of RyR2 by thiol reagents and CaM. We found that the C³⁶⁰²A mutation suppressed SOICR by raising the activation threshold and delayed the termination of Ca²⁺ release by reducing the termination threshold. As a result, C³⁶⁰²A markedly increased the fractional Ca²⁺ release. Furthermore, the C³⁶⁰²A mutation diminished the inhibitory effect of N-ethylmaleimide on Ca²⁺ release, but it had no effect on the stimulatory action of 4,4'-dithiodipyridine (DTDP) on Ca²⁺ release. In addition, Cys³⁶⁰² mutations (C³⁶⁰²A or C³⁶⁰²R) did not abolish the effect of CaM on Ca²⁺-release termination. Therefore, RyR2-Cys³⁶⁰² is a major site mediating the action of thiol alkylating agent N-ethylmaleimide, but not the action of the oxidant DTDP. Our data also indicate that residue Cys³⁶⁰² plays an important role in the activation and termination of Ca²⁺ release, but it is not essential for CaM regulation of RyR2.

  10. Total internal reflectance fluorescence imaging of genetically engineered ryanodine receptor-targeted Ca(2+) probes in rat ventricular myocytes.

    PubMed

    Pahlavan, Sara; Morad, Marin

    2017-09-01

    The details of cardiac Ca(2+) signaling within the dyadic junction remain unclear because of limitations in rapid spatial imaging techniques, and availability of Ca(2+) probes localized to dyadic junctions. To critically monitor ryanodine receptors' (RyR2) Ca(2+) nano-domains, we combined the use of genetically engineered RyR2-targeted pericam probes, (FKBP-YCaMP, Kd=150nM, or FKBP-GCaMP6, Kd=240nM) with rapid total internal reflectance fluorescence (TIRF) microscopy (resolution, ∼80nm). The punctate z-line patterns of FKBP,(2)-targeted probes overlapped those of RyR2 antibodies and sharply contrasted to the images of probes targeted to sarcoplasmic reticulum (SERCA2a/PLB), or cytosolic Fluo-4 images. FKBP-YCaMP signals were too small (∼20%) and too slow (2-3s) to detect Ca(2+) sparks, but the probe was effective in marking where Fluo-4 Ca(2+) sparks developed. FKBP-GCaMP6, on the other hand, produced rapidly decaying Ca(2+) signals that: a) had faster kinetics and activated synchronous with ICa(3) but were of variable size at different z-lines and b) were accompanied by spatially confined spontaneous Ca(2+) sparks, originating from a subset of eager sites. The frequency of spontaneously occurring sparks was lower in FKBP-GCaMP6 infected myocytes as compared to Fluo-4 dialyzed myocytes, but isoproterenol enhanced their frequency more effectively than in Fluo-4 dialyzed cells. Nevertheless, isoproterenol failed to dissociate FKBP-GCaMP6 from the z-lines. The data suggests that FKBP-GCaMP6 binds predominantly to junctional RyR2s and has sufficient on-rate efficiency as to monitor the released Ca(2+) in individual dyadic clefts, and supports the idea that β-adrenergic agonists may modulate the stabilizing effects of native FKBP on RyR2. Copyright © 2017 Elsevier Ltd. All rights reserved.

  11. Camkii-Dependent Phosphorylation of Cardiac Ryanodine Receptors Regulates Cell Death In Cardiac Ischemia/Reperfusion Injury

    PubMed Central

    Di Carlo, Mariano N.; Said, Matilde; Ling, Haiyun; Valverde, Carlos A.; De Giusti, Verónica; Sommese, Leandro; Palomeque, Julieta; Aiello, Alejandro E.; Skapura, Darlene G.; Rinaldi, Gustavo; Respress, Jonathan L.; Brown, Joan Heller; Wehrens, Xander H.T.; Salas, Margarita A.; Mattiazzi, Alicia

    2014-01-01

    Ca2+-Calmodulin kinase II (CaMKII) activation is deleterious in cardiac ischemia/reperfusion (I/R). Moreover, inhibition of CaMKII-dependent phosphorylations at the sarcoplasmic reticulum (SR) prevents CaMKII-induced I/R damage. However, the downstream targets of CaMKII at the SR level, responsible for this detrimental effect, remain unclear. In the present study we aimed to dissect the role of the two main substrates of CaMKII at the SR level, phospholamban (PLN) and ryanodine receptors (RyR2), in CaMKII-dependent I/R injury. In mouse hearts subjected to global I/R (45/120 min), phosphorylation of the primary CaMKII sites, S2814 on cardiac RyR2 and of T17 on PLN, significantly increased at the onset of reperfusion whereas PKA-dependent phosphorylation of RyR2 and PLN did not change. Similar results were obtained in vivo, in mice subjected to regional myocardial I/R (1/24 hrs). Knock-in mice with an inactivated serine 2814 phosphorylation site on RyR2 (S2814A), significantly improved post-ischemic mechanical recovery, reduced infarct size and decreased apoptosis. Conversely, knock-in mice, in which CaMKII site of RyR2 is constitutively activated (S2814D), significantly increased infarct size and exacerbated apoptosis. In S2814A and S2814D mice subjected to regional myocardial ischemia, infarct size was also decreased and increased respectively. Transgenic mice with double-mutant non-phosphorylatable PLN (S16A/T17A) in the PLN knockout background (PLNDM) also showed significantly increased post-ischemic cardiac damage. This effect cannot be attributed to PKA-dependent PLN phosphorylation and was not due to the enhanced L-type Ca2+ current, present in these mice. Our results reveal a major role for the phosphorylation of S2814 site on RyR2 in CaMKII-dependent I/R cardiac damage. In contrast, they showed that CaMKII-dependent increase in PLN phosphorylation during reperfusion opposes rather than contributes to I/R damage. PMID:24949568

  12. The random-coil 'C' fragment of the dihydropyridine receptor II-III loop can activate or inhibit native skeletal ryanodine receptors.

    PubMed Central

    Haarmann, Claudia S; Green, Daniel; Casarotto, Marco G; Laver, Derek R; Dulhunty, Angela F

    2003-01-01

    The actions of peptide C, corresponding to (724)Glu-Pro(760) of the II-III loop of the skeletal dihydropyridine receptor, on ryanodine receptor (RyR) channels incorporated into lipid bilayers with the native sarcoplasmic reticulum membrane show that the peptide is a high-affinity activator of native skeletal RyRs at cytoplasmic concentrations of 100 nM-10 microM. In addition, we found that peptide C inhibits RyRs in a voltage-independent manner when added for longer times or at higher concentrations (up to 150 microM). Peptide C had a random-coil structure indicating that it briefly assumes a variety of structures, some of which might activate and others which might inhibit RyRs. The results suggest that RyR activation and inhibition by peptide C arise from independent stochastic processes. A rate constant of 7.5 x 10(5) s(-1).M(-1) was obtained for activation and a lower estimate for the rate constant for inhibition of 5.9 x 10(3) s(-1).M(-1). The combined actions of peptide C and peptide A (II-III loop sequence (671)Thr-Leu(690)) showed that peptide C prevented activation but not blockage of RyRs by peptide A. We suggest that the effects of peptide C indicate functional interactions between a part of the dihydropyridine receptor and the RyR. These interactions could reflect either dynamic changes that occur during excitation-contraction coupling or interactions between the proteins at rest. PMID:12620094

  13. Myoplasmic resting Ca2+ regulation by ryanodine receptors is under the control of a novel Ca2+-binding region of the receptor

    PubMed Central

    Chen, Yanyi; Xue, Shenghui; Zou, Juan; Lopez, Jose R.; Yang, Jenny J.; Perez, Claudio F.

    2014-01-01

    Passive SR (sarcoplasmic reticulum) Ca2+ leak through the RyR (ryanodine receptor) plays a critical role in the mechanisms that regulate [Ca2+]rest (intracellular resting myoplasmic free Ca2+ concentration) in muscle. This process appears to be isoform-specific as expression of either RyR1 or RyR3 confers on myotubes different [Ca2+]rest. Using chimaeric RyR3–RyR1 receptors expressed in dyspedic myotubes, we show that isoform-dependent regulation of [Ca2+]rest is primarily defined by a small region of the receptor encompassing amino acids 3770–4007 of RyR1 (amino acids 3620–3859 of RyR3) named as the CLR (Ca2+ leak regulatory) region. [Ca2+]rest regulation by the CLR region was associated with alteration of RyRs’ Ca2+-activation profile and changes in SR Ca2+-leak rates. Biochemical analysis using Tb3+-binding assays and intrinsic tryptophan fluorescence spectroscopy of purified CLR domains revealed that this determinant of RyRs holds a novel Ca2+-binding domain with conformational properties that are distinctive to each isoform. Our data suggest that the CLR region provides channels with unique functional properties that modulate the rate of passive SR Ca2+ leak and confer on RyR1 and RyR3 distinctive [Ca2+]rest regulatory properties. The identification of a new Ca2+-binding domain of RyRs with a key modulatory role in [Ca2+]rest regulation provides new insights into Ca2+-mediated regulation of RyRs. PMID:24635445

  14. Defining breast cancer intrinsic subtypes by quantitative receptor expression.

    PubMed

    Cheang, Maggie C U; Martin, Miguel; Nielsen, Torsten O; Prat, Aleix; Voduc, David; Rodriguez-Lescure, Alvaro; Ruiz, Amparo; Chia, Stephen; Shepherd, Lois; Ruiz-Borrego, Manuel; Calvo, Lourdes; Alba, Emilio; Carrasco, Eva; Caballero, Rosalia; Tu, Dongsheng; Pritchard, Kathleen I; Levine, Mark N; Bramwell, Vivien H; Parker, Joel; Bernard, Philip S; Ellis, Matthew J; Perou, Charles M; Di Leo, Angelo; Carey, Lisa A

    2015-05-01

    To determine intrinsic breast cancer subtypes represented within categories defined by quantitative hormone receptor (HR) and HER2 expression. We merged 1,557 cases from three randomized phase III trials into a single data set. These breast tumors were centrally reviewed in each trial for quantitative ER, PR, and HER2 expression by immunohistochemistry (IHC) stain and by reverse transcription-quantitative polymerase chain reaction (RT-qPCR), with intrinsic subtyping by research-based PAM50 RT-qPCR assay. Among 283 HER2-negative tumors with <1% HR expression by IHC, 207 (73%) were basal-like; other subtypes, particularly HER2-enriched (48, 17%), were present. Among the 1,298 HER2-negative tumors, borderline HR (1%-9% staining) was uncommon (n = 39), and these tumors were heterogeneous: 17 (44%) luminal A/B, 12 (31%) HER2-enriched, and only 7 (18%) basal-like. Including them in the definition of triple-negative breast cancer significantly diminished enrichment for basal-like cancer (p < .05). Among 106 HER2-positive tumors with <1% HR expression by IHC, the HER2-enriched subtype was the most frequent (87, 82%), whereas among 127 HER2-positive tumors with strong HR (>10%) expression, only 69 (54%) were HER2-enriched and 55 (43%) were luminal (39 luminal B, 16 luminal A). Quantitative HR expression by RT-qPCR gave similar results. Regardless of methodology, basal-like cases seldom expressed ER/ESR1 or PR/PGR and were associated with the lowest expression level of HER2/ERBB2 relative to other subtypes. Significant discordance remains between clinical assay-defined subsets and intrinsic subtype. For identifying basal-like breast cancer, the optimal HR IHC cut point was <1%, matching the American Society of Clinical Oncology and College of American Pathologists guidelines. Tumors with borderline HR staining are molecularly diverse and may require additional assays to clarify underlying biology. ©AlphaMed Press.

  15. Phytoestrogens from Psoralea corylifolia reveal estrogen receptor-subtype selectivity.

    PubMed

    Xin, D; Wang, H; Yang, J; Su, Y-F; Fan, G-W; Wang, Y-F; Zhu, Y; Gao, X-M

    2010-02-01

    The seed of Psoralea corylifolia L. (PCL), a well-known traditional Chinese medicine, has been applied as a tonic or an aphrodisiac agent and commonly used as a remedy for bone fracture, osteomalacia and osteoporosis in China. In our study, the estrogen receptor subtype-selective activities of the extracts and compounds derived from PCL were analyzed using the HeLa cell assay. The different fractions including petroleum ether, CH(2)Cl(2) and EtOAc fractions of the EtOH extract of PCL showed significant activity in activating either ERalpha or ERbeta whereas the n-BuOH fraction showed no estrogenic activity. Further chromatographic purification of the active fractions yielded seven compounds including the two coumarins isopsoralen and psoralen, the four flavonoids isobavachalcone, bavachin, corylifol A and neobavaisoflavone, and the meroterpene phenol, bakuchiol. In reporter gene assay, the two coumarins (10(-8)-10(-5)M) acted as ERalpha-selective agonists while the other compounds (10(-9)-10(-6)M) activated both ERalpha and ERbeta. The estrogenic activities of all compounds could be completely suppressed by the pure estrogen antagonist, ICI 182,780, suggesting that the compounds exert their activities through ER. Only psoralen and isopsoralen as ERalpha agonists promoted MCF-7 cell proliferation significantly. Although all the compounds have estrogenic activity, they may exert different biological effects. In conclusion, both ER subtype-selective and nonselective activities in compounds derived from PCL suggested that PCL could be a new source for selective estrogen-receptor modulators.

  16. An Extended Structure-Activity Relationship of Nondioxin-Like PCBs Evaluates and Supports Modeling Predictions and Identifies Picomolar Potency of PCB 202 Towards Ryanodine Receptors.

    PubMed

    Holland, Erika B; Feng, Wei; Zheng, Jing; Dong, Yao; Li, Xueshu; Lehmler, Hans-Joachim; Pessah, Isaac N

    2017-01-01

    Nondioxin-like polychlorinated biphenyls (NDL PCBs) activate ryanodine-sensitive Ca(2+ )channels (RyRs) and this activation has been associated with neurotoxicity in exposed animals. RyR-active congeners follow a distinct structure-activity relationship and a quantitative structure-activity relationship (QSAR) predicts that a large number of PCBs likely activate the receptor, which requires validation. Additionally, previous structural based conclusions have been established using receptor ligand binding assays but the impact of varying PCB structures on ion channel gating behavior is not understood. We used [(3)H]Ryanodine ([(3)H]Ry) binding to assess the RyR-activity of 14 previously untested PCB congeners evaluating the predictability of the QSAR. Congeners determined to display widely varying potency were then assayed with single channel voltage clamp analysis to assess direct influences on channel gating kinetics. The RyR-activity of individual PCBs assessed in in vitro assays followed the general pattern predicted by the QSAR but binding and lipid bilayer experiments demonstrated higher potency than predicted. Of the 49 congeners tested to date, tetra-ortho PCB 202 was found to be the most potent RyR-active congener increasing channel open probability at 200 pM. Shifting meta-substitutions to the para-position resulted in a > 100-fold reduction in potency as seen with PCB 197. Non-ortho PCB 11 was found to lack activity at the receptor supporting a minimum mono-ortho substitution for PCB RyR activity. These findings expand and support previous SAR assessments; where out of the 49 congeners tested to date 42 activate the receptor demonstrating that the RyR is a sensitive and common target of PCBs. © The Author 2016. Published by Oxford University Press on behalf of the Society of Toxicology. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.

  17. The ryanodine receptor pore blocker neomycin also inhibits channel activity via a previously undescribed high-affinity Ca(2+) binding site.

    PubMed

    Laver, Derek R; Hamada, Tomoyo; Fessenden, James D; Ikemoto, Noriaki

    2007-12-01

    In this study, we present evidence for the mechanism of neomycin inhibition of skeletal ryanodine receptors (RyRs). In single-channel recordings, neomycin produced monophasic inhibition of RyR open probability and biphasic inhibition of [(3)H]ryanodine binding. The half-maximal inhibitory concentration (IC(50)) for channel blockade by neomycin was dependent on membrane potential and cytoplasmic [Ca(2+)], suggesting that neomycin acts both as a pore plug and as a competitive antagonist at a cytoplasmic Ca(2+) binding site that causes allosteric inhibition. This novel Ca(2+)/neomycin binding site had a neomycin affinity of 100 nM: and a Ca(2+) affinity of 35 nM,: which is 30-fold higher than that of the well-described cytoplasmic Ca(2+) activation site. Therefore, a new high-affinity class of Ca(2+) binding site(s) on the RyR exists that mediates neomycin inhibition. Neomycin plugging of the channel pore induced brief (1-2 ms) conductance substates at 30% of the fully open conductance, whereas allosteric inhibition caused complete channel closure with durations that depended on the neomycin concentration. We quantitatively account for these results using a dual inhibition model for neomycin that incorporates voltage-dependent pore plugging and Ca(2+)-dependent allosteric inhibition.

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

    PubMed

    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.

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

    PubMed Central

    Pitake, Saumitra

    2015-01-01

    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 Ca2+ 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 Ca2+ 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 Ca2+ 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 Ca2+ was elevated without depolarization by the ryanodine receptor agonist chloromethylcresol, or by increasing Ca2+ concentration in the media. Ca2+ entry currents (from the extracellular space) during excitation were strongly inhibited by ryanodine, but Ca2+ entry currents in the resting state were not. We conclude that excitation releases the ryanodine receptor from occlusion by the dihydropyridine receptor, enabling Ca2+ release from the ryanodine receptor to the cytosol. PMID:26643865

  20. Muscarinic M3 receptor subtype gene expression in the human heart.

    PubMed

    Hellgren, I; Mustafa, A; Riazi, M; Suliman, I; Sylvén, C; Adem, A

    2000-01-20

    The heart is an important target organ for cholinergic function. In this study, muscarinic receptor subtype(s) in the human heart were determined using reverse transcription-polymerase chain reaction. Our results demonstrated muscarinic receptor M2 and M3 subtype RNA in left/right atria/ventricles of donor hearts. Receptor autoradiography analysis using selective muscarinic ligands indicated an absence of M1 receptor subtype in the human heart. The level of muscarinic receptor binding in atria was two to three times greater than in ventricles. Our results suggest that muscarinic receptors in the human heart are of the M2 and M3 subtypes. This is the first report of M3 receptors in the human myocardium.

  1. FKBP12 modulation of the binding of the skeletal ryanodine receptor onto the II-III loop of the dihydropyridine receptor.

    PubMed Central

    O'Reilly, Fiona M; Robert, Mylène; Jona, Istvan; Szegedi, Csaba; Albrieux, Mireille; Geib, Sandrine; De Waard, Michel; Villaz, Michel; Ronjat, Michel

    2002-01-01

    In skeletal muscle, excitation-contraction coupling involves a functional interaction between the ryanodine receptor (RyR) and the dihydropyridine receptor (DHPR). The domain corresponding to Thr(671)-Leu(690) of the II-III loop of the skeletal DHPR alpha(1)-subunit is able to regulate RyR properties and calcium release from sarcoplasmic reticulum, whereas the domain corresponding to Glu(724)-Pro(760) antagonizes this effect. Two peptides, covering these sequences (peptide A(Sk) and C(Sk), respectively) were immobilized on polystyrene beads. We demonstrate that peptide A(Sk) binds to the skeletal isoform of RyR (RyR1) whereas peptide C(Sk) does not. Using surface plasmon resonance detection, we show that 1) domain Thr(671)-Leu(690) is the only sequence of the II-III loop binding with RyR1 and 2) the interaction of peptide A(Sk) with RyR1 is not modulated by Ca(2+) (pCa 9-2) nor by Mg(2+) (up to 10 mM). In contrast, this interaction is strongly potentiated by the immunophilin FKBP12 (EC(50) = 10 nM) and inhibited by both rapamycin (IC(50) = 5 nM) and FK506. Peptide A(Sk) induces a 300% increase of the opening probability of the RyR1 incorporated in lipid bilayer. Removal of FKBP12 from RyR1 completely abolishes this effect of domain A(Sk) on RyR1 channel behavior. These results demonstrate a direct interaction of the RyR1 with the discrete domain of skeletal DHPR alpha(1)-subunit corresponding to Thr(671)-Leu(690) and show that the association of FKBP12 with RyR1 specifically modulates this interaction. PMID:11751303

  2. Identification of two H3-histamine receptor subtypes

    SciTech Connect

    West, R.E. Jr.; Zweig, A.; Shih, N.Y.; Siegel, M.I.; Egan, R.W.; Clark, M.A. )

    1990-11-01

    The H3-histamine receptor provides feedback inhibition of histamine synthesis and release as well as inhibition of other neurotransmitter release. We have characterized this receptor by radioligand binding studies with the H3 agonist N alpha-(3H)methylhistamine ((3H)NAMHA). The results of (3H)NAMHA saturation binding and NAMHA inhibition of (3H)NAMHA binding were consistent with an apparently single class of receptors (KD = 0.37 nM, Bmax = 73 fmol/mg of protein) and competition assays with other agonists and the antagonists impromidine and dimaprit disclosed only a single class of sites. In contrast, inhibition of (3H)NAMHA binding by the specific high affinity H3 antagonist thioperamide revealed two classes of sites (KiA = 5 nM, BmaxA = 30 fmol/mg of protein; KiB = 68 nM, BmaxB = 48 fmol/mg of protein). Burimamide, another antagonist that, like thioperamide, contains a thiourea group, likewise discriminated between two classes of sites. In addition to differences between some antagonist potencies for the two receptors, there is a differential guanine nucleotide sensitivity of the two. The affinity of the H3A receptor for (3H) NAMHA was reduced less than 2-fold, whereas (3H)NAMHA binding to the H3B receptor was undetectable in the presence of guanosine 5'-O-(3-thiotriphosphate). The distinction between H3A and H3B receptor subtypes, the former a high affinity and the latter a low affinity thioperamide site, draws support from published in vitro data.

  3. Localization of nigrostriatal dopamine receptor subtypes and adenylate cyclase

    SciTech Connect

    Filloux, F.; Dawson, T.M.; Wamsley, J.K.

    1988-04-01

    Quantitative autoradiography using (/sup 3/H)-SCH 23390, (/sup 3/H)-sulpiride and (/sup 3/H)-forskolin was used to assess the effects of single and combined neurotoxin lesions of the nigrostriatal pathway in the rat brain on dopamine (DA) receptor subtypes and adenylate cyclase (AC), respectively. Ibotenic acid (IA) lesions of the caudate-putamen (CPu) resulted in near total loss of both (/sup 3/H)-SCH 23390 and of (/sup 3/H)-forskolin binding in the ipsilateral CPu and substantia nigra reticulata (SNR). (/sup 3/H)-sulpiride binding in the CPu was only partially removed by this same lesion, and nigral (/sup 3/H)-sulpiride binding was virtually unchanged. 6-Hydroxydopamine (6-OHDA) and IA lesions of the substantia nigra compacta (SNC) did not affect (/sup 3/H)-SCH 23390 or (/sup 3/H)-forskolin binding, but largely removed (/sup 3/H)-sulpiride binding in the SNC. A 6-OHDA lesion of the nigrostriatal pathway followed by an ipsilateral IA injection of the CPu failed to further reduce (/sup 3/H)-sulpiride binding in the CPu. These results demonstrate that postsynaptic DA receptors in the CPu are of both the D1 and D2 variety; however, a portion of D2 receptors in the CPu may be presynaptic on afferent nerve terminals to this structure. D1 receptors in the SNR are presynaptic on striatonigral terminals, whereas the D2 receptors of the SNC are autoreceptors on nigral DA neurons. The existence of presynaptic D2 receptors on nigrostriatal DA-ergic terminals could not be confirmed by this study. Co-localization of D1 receptors and AC occurs in both the CPu and SNR.

  4. Genotypic Prediction of Co-receptor Tropism of HIV-1 Subtypes A and C

    PubMed Central

    Riemenschneider, Mona; Cashin, Kieran Y.; Budeus, Bettina; Sierra, Saleta; Shirvani-Dastgerdi, Elham; Bayanolhagh, Saeed; Kaiser, Rolf; Gorry, Paul R.; Heider, Dominik

    2016-01-01

    Antiretroviral treatment of Human Immunodeficiency Virus type-1 (HIV-1) infections with CCR5-antagonists requires the co-receptor usage prediction of viral strains. Currently available tools are mostly designed based on subtype B strains and thus are in general not applicable to non-B subtypes. However, HIV-1 infections caused by subtype B only account for approximately 11% of infections worldwide. We evaluated the performance of several sequence-based algorithms for co-receptor usage prediction employed on subtype A V3 sequences including circulating recombinant forms (CRFs) and subtype C strains. We further analysed sequence profiles of gp120 regions of subtype A, B and C to explore functional relationships to entry phenotypes. Our analyses clearly demonstrate that state-of-the-art algorithms are not useful for predicting co-receptor tropism of subtype A and its CRFs. Sequence profile analysis of gp120 revealed molecular variability in subtype A viruses. Especially, the V2 loop region could be associated with co-receptor tropism, which might indicate a unique pattern that determines co-receptor tropism in subtype A strains compared to subtype B and C strains. Thus, our study demonstrates that there is a need for the development of novel algorithms facilitating tropism prediction of HIV-1 subtype A to improve effective antiretroviral treatment in patients. PMID:27126912

  5. Genotypic Prediction of Co-receptor Tropism of HIV-1 Subtypes A and C.

    PubMed

    Riemenschneider, Mona; Cashin, Kieran Y; Budeus, Bettina; Sierra, Saleta; Shirvani-Dastgerdi, Elham; Bayanolhagh, Saeed; Kaiser, Rolf; Gorry, Paul R; Heider, Dominik

    2016-04-29

    Antiretroviral treatment of Human Immunodeficiency Virus type-1 (HIV-1) infections with CCR5-antagonists requires the co-receptor usage prediction of viral strains. Currently available tools are mostly designed based on subtype B strains and thus are in general not applicable to non-B subtypes. However, HIV-1 infections caused by subtype B only account for approximately 11% of infections worldwide. We evaluated the performance of several sequence-based algorithms for co-receptor usage prediction employed on subtype A V3 sequences including circulating recombinant forms (CRFs) and subtype C strains. We further analysed sequence profiles of gp120 regions of subtype A, B and C to explore functional relationships to entry phenotypes. Our analyses clearly demonstrate that state-of-the-art algorithms are not useful for predicting co-receptor tropism of subtype A and its CRFs. Sequence profile analysis of gp120 revealed molecular variability in subtype A viruses. Especially, the V2 loop region could be associated with co-receptor tropism, which might indicate a unique pattern that determines co-receptor tropism in subtype A strains compared to subtype B and C strains. Thus, our study demonstrates that there is a need for the development of novel algorithms facilitating tropism prediction of HIV-1 subtype A to improve effective antiretroviral treatment in patients.

  6. Molecular and cellular analysis of human histamine receptor subtypes

    PubMed Central

    Seifert, Roland; Strasser, Andrea; Schneider, Erich H.; Neumann, Detlef; Dove, Stefan; Buschauer, Armin

    2013-01-01

    The human histamine receptors hH1R and hH2R constitute important drug targets, and hH3R and hH4R have substantial potential in this area. Considering the species-specificity of pharmacology of HxR orthologs, it is important to analyze hHxRs. Here,we summarize current knowledge of hHxRs endogenously expressed in human cells and hHxRs recombinantly expressed in mammalian and insect cells. We present the advantages and disadvantages of the various systems. We also discuss problems associated with the use of hHxR antibodies, an issue of general relevance for G-protein-coupled receptors (GPCRs). There is much greater overlap in activity of ‘selective’ ligands for other hHxRs than the cognate receptor subtype than generally appreciated. Studies with native and recombinant systems support the concept of ligand-specific receptor conformations, encompassing agonists and antagonists. It is emerging that for characterization of hHxR ligands, one cannot rely on a single test system and a single parameter. Rather, multiple systems and parameters have to be studied. Although such studies are time-consuming and expensive, ultimately, they will increase drug safety and efficacy. PMID:23254267

  7. Adrenergic receptor subtypes in the cerebral circulation of newborn piglets

    SciTech Connect

    Wagerle, L.C.; Delivoria-Papadopoulos, M.

    1987-06-01

    The purpose of this study was to identify the ..cap alpha..-adrenergic receptor subtype mediating cerebral vasoconstriction during sympathetic nerve stimulation in the newborn piglet. The effect of ..cap alpha../sub 1/- and ..cap alpha../sub 2/-antagonists prazosin and yohimbine on the cerebrovascular response to unilateral electrical stimulation (15 Hz, 15 V) of the superior cervical sympathetic trunk was studied in 25 newborn piglets. Regional cerebral blood flow was measured with tracer microspheres. Sympathetic stimulation decreased blood flow to the ipsilateral cerebrum hippocampus, choroid plexus, and masseter muscle. ..cap alpha../sub 1/-Adrenergic receptor blockade with prazosin inhibited the sympathetic vasoconstriction in the cerebrum, hippocampus, and masseter muscle and abolished it in the choroid plexus. ..cap alpha../sub s/-Adrenergic receptor blockade with yohimbine had no effect. Following the higher dose of yohimbine, however, blood flow to all brain regions was increased by approximately two-fold, possibly due to enhanced cerebral metabolism. These data demonstrate that vascular ..cap alpha../sub 1/-adrenergic receptors mediate vasoconstriction to neuroadrenergic stimulation in cerebral resistance vessels in the newborn piglet.

  8. [Subtype-specific clinically important effects of alpha 2-adrenergic receptors].

    PubMed

    Shishkina, G T; Dygalo, N N

    2002-01-01

    A- B- and C-subtypes of alpha 2-adrenoreceptors present in all mammals are involved in responses to currently existing subtype-nonselective ligands of these receptors widely used in medicine. Each of the subtypes has its own specific distribution in tissue and cells, onthogenetic pattern, specific regulation of activity and expression, and, as result, specific physiological functions. The latter suggests opportunities of using the subtype-specific for correction of the functions depending on this receptor. The article reviews the role of individual subtypes of alpha 2-adrenoreceptors in regulation of neurochemical transmission of cardiovascular system, psychoemotional state and development of psychic disorders, and also male sexual behaviour.

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

    SciTech Connect

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

    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.

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

    SciTech Connect

    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

  11. Calcium occupancy of N-terminal sites within calmodulin induces inhibition of the ryanodine receptor calcium release channel.

    PubMed

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

    2007-09-18

    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 presently unclear whether the identified CaM-binding sequence in association with CaM (a) senses calcium over the physiological range of calcium concentrations associated with RyR1 regulation or alternatively, (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 of both apo-CaM and calcium-activated CaM with RyRp. Upon association with RyRp, fluorescence changes in PyN-CaM or PyC-CaM permit the measurement of the calcium-dependent 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 at resting calcium levels; at calcium levels associated with muscle contraction, activation of the N-terminal domain occurs with concomitant increases in the fluorescence intensity of PyC-CaM that is associated with structural changes within the CaM-binding sequence of RyR1. Occupancy of calcium-binding sites in the N

  12. Malignant hyperthermia and the clinical significance of type-1 ryanodine receptor gene (RYR1) variants: proceedings of the 2013 MHAUS Scientific Conference.

    PubMed

    Riazi, Sheila; Kraeva, Natalia; Muldoon, Sheila M; Dowling, James; Ho, Clara; Petre, Maria-Alexandra; Parness, Jerome; Dirksen, Robert T; Rosenberg, Henry

    2014-11-01

    The Malignant Hyperthermia Association of the United States and the Department of Anesthesia at the University of Toronto sponsored a Scientific Conference on November 1-2, 2013 in Toronto, ON, Canada. The multidisciplinary group of experts, including clinicians, geneticists, and physiologists involved in research related to malignant hyperthermia (MH), shared new insights into the pathophysiology of diseases linked to the type-1 ryanodine receptor gene (RYR1) as well as the relationship between MH and "awake MH" conditions, such as exertional rhabdomyolysis and exertional heat illness. In addition, the molecular genetics of MH and clinical issues related to the diagnosis and management of disorders linked to RYR1 were presented. The conference also honoured Dr. David H. MacLennan for his contributions to our understanding of the genetics, pathogenesis, and treatment of MH and other RYR1-related myopathies. This report represents a summary of the proceedings of this conference.

  13. Growing vascular endothelial cells express somatostatin subtype 2 receptors

    PubMed Central

    Watson, J C; Balster, D A; Gebhardt, B M; O'Dorisio, T M; O'Dorisio, M S; Espenan, G D; Drouant, G J; Woltering, E A

    2001-01-01

    We hypothesized that non-proliferating (quiescent) human vascular endothelial cells would not express somatostatin receptor subtype 2 (sst 2) and that this receptor would be expressed when the endothelial cells begin to grow. To test this hypothesis, placental veins were harvested from 6 human placentas and 2 mm vein disks were cultured in 0.3% fibrin gels. Morphometric analysis confirmed that 50–75% of cultured vein disks developed radial capillary growth within 15 days. Sst 2 gene expression was determined by reverse transcription-polymerase chain reaction (RT-PCR) analysis of the RNA from veins before culture and from tissue-matched vein disks that exhibited an angiogenic response. The sst 2 gene was expressed in the proliferating angiogenic sprouts of human vascular endothelium. The presence of sst 2 receptors on proliferating angiogenic vessels was confirmed by immunohistochemical staining and in vivo scintigraphy. These results suggest that sst 2 may be a unique target for antiangiogenic therapy with sst 2 preferring somatostatin analogues conjugated to radioisotopes or cytotoxic agents. © 2001 Cancer Research Campaign http://www.bjcancer.com PMID:11461088

  14. Hooked on benzodiazepines: GABAA receptor subtypes and addiction

    PubMed Central

    Tan, Kelly R.; Rudolph, Uwe; Lüscher, Christian

    2011-01-01

    Benzodiazepines are widely used clinically to treat anxiety and insomnia. They also induce muscle relaxation, control epileptic seizures, and can provoke amnesia. Moreover, benzodiazepines are often abused after chronic clinical treatment but also for recreational purposes. Within weeks, tolerance to the pharmacological effects can develop, in addition to dependence and even addiction in vulnerable individuals. Here, we review recent observations from animal models regarding the cellular and molecular basis that may underlie the addictive properties of benzodiazepines. These data reveal how benzodiazepines, acting through specific GABAA receptor subtypes, activate midbrain dopamine neurons and how this may hijack the mesolimbic reward system. Such findings have important implications for the future design of benzodiazepines with reduced or even absent addiction liability. PMID:21353710

  15. Differential regulation of SK and BK channels by Ca2+ signals from Ca2+ channels and ryanodine receptors in guinea-pig urinary bladder myocytes

    PubMed Central

    Herrera, Gerald M; Nelson, Mark T

    2002-01-01

    Small-conductance (SK) and large-conductance (BK) Ca2+-activated K+ channels are key regulators of excitability in urinary bladder smooth muscle (UBSM) of guinea-pig. The overall goal of this study was to define how SK and BK channels respond to Ca2+ signals from voltage-dependent Ca2+ channels (VDCCs) in the surface membrane and from ryanodine-sensitive Ca2+ release channels or ryanodine receptors (RyRs) in the sarcoplasmic reticulum (SR) membrane. To characterize the role of SK channels in UBSM, the effects of the SK channel blocker apamin on phasic contractions were examined. Apamin caused a dose-dependent increase in the amplitude of phasic contractions over a broad concentration range (10−10 to 10−6m). To determine the effects of Ca2+ signals from VDCCs and RyRs to SK and BK channels, whole cell membrane current was measured in isolated myocytes bathed in physiological solutions. Depolarization (-70 to +10 mV for 100 ms) of isolated myocytes caused an inward Ca2+ current (ICa), followed by an outward current. The outward current was reduced in a dose-dependent manner by apamin (10−10 to 10−6m), and designated ISK. ISK had a mean amplitude of 53.8 ± 6.1 pA or ∼1.4 pA pF−1 at +10 mV. The amplitude of ISK correlated with the peak ICa. Blocking ICa abolished ISK. In contrast, ISK was insensitive to the RyR blocker ryanodine (10 μM). These data indicate that Ca2+ signals from VDCCs, but not from RyRs, activate SK channels. BK channel currents (IBK) were isolated from other currents by using the BK channel blockers tetraethylammonium ions (TEA+; 1 mm) or iberiotoxin (200 nm). Voltage steps evoked transient and steady-state IBK components. Transient BK currents have previously been shown to result from BK channel activation by local Ca2+ release through RyRs (‘Ca2+ sparks’). Transient BK currents were inhibited by ryanodine (10 μM), as expected, and had a mean amplitude of 152.6 pA at +10 mV. The mean number of transient BK currents during a

  16. Hypocretin (orexin) receptor subtypes differentially enhance acetylcholine release and activate g protein subtypes in rat pontine reticular formation.

    PubMed

    Bernard, René; Lydic, Ralph; Baghdoyan, Helen A

    2006-04-01

    The hypothalamic peptides hypocretin-1 (orexin A) and -2 (orexin B) promote wakefulness by mechanisms that are not well understood. Defects in hypocretinergic neurotransmission underlie the human sleep disorder narcolepsy. Hypocretins alter cell excitability via two receptor subtypes, hypocretin receptor subtype 1 (hcrt-r1) and hypocretin receptor subtype 2 (hcrt-r2). This study aimed to identify G protein subtypes activated by hypocretin in rat pontine reticular nucleus oral part (PnO) and the hypocretin receptor subtype modulating acetylcholine (ACh) release in the PnO. G protein activation was quantified using in vitro [(35)S]guanylyl-5'-O-(gamma-thio)triphosphate autoradiography. ACh release was measured using in vivo microdialysis and high-performance liquid chromatography. Hypocretin-1-stimulated G protein activation was significantly decreased by pertussis toxin, demonstrating that some hypocretin receptors in rat PnO activate inhibitory G proteins. Hypocretin-1-stimulated ACh release was not blocked by pertussis toxin, supporting the conclusion that the hypocretin receptors modulating ACh release in rat PnO activate stimulatory G proteins. Hypocretin-1 and -2 each caused a concentration-dependent increase in ACh release with similar potencies, indicating that hcrt-r2 modulates ACh release in PnO. Hypocretin-1 caused a significantly greater increase in ACh release than hypocretin-2, suggesting a role for hcrt-r1 in the modulation of PnO ACh release. Taken together, these data provide the first evidence that hypocretin receptors in rat PnO signal via inhibitory and stimulatory G proteins and that ACh release in rat PnO is modulated by hcrt-r2 and hcrt-r1. One mechanism by which hypocretin promotes arousal may be to increase ACh release in the pontine reticular formation.

  17. Role of amino-terminal half of the S4-S5 linker in type 1 ryanodine receptor (RyR1) channel gating.

    PubMed

    Murayama, Takashi; Kurebayashi, Nagomi; Oba, Toshiharu; Oyamada, Hideto; Oguchi, Katsuji; Sakurai, Takashi; Ogawa, Yasuo

    2011-10-14

    The type 1 ryanodine receptor (RyR1) is a Ca(2+) release channel found in the sarcoplasmic reticulum of skeletal muscle and plays a pivotal role in excitation-contraction coupling. The RyR1 channel is activated by a conformational change of the dihydropyridine receptor upon depolarization of the transverse tubule, or by Ca(2+) itself, i.e. Ca(2+)-induced Ca(2+) release (CICR). The molecular events transmitting such signals to the ion gate of the channel are unknown. The S4-S5 linker, a cytosolic loop connecting the S4 and S5 transmembrane segments in six-transmembrane type channels, forms an α-helical structure and mediates signal transmission in a wide variety of channels. To address the role of the S4-S5 linker in RyR1 channel gating, we performed alanine substitution scan of N-terminal half of the putative S4-S5 linker (Thr(4825)-Ser(4829)) that exhibits high helix probability. The mutant RyR1 was expressed in HEK cells, and CICR activity was investigated by caffeine-induced Ca(2+) release, single-channel current recordings, and [(3)H]ryanodine binding. Four mutants (T4825A, I4826A, S4828A, and S4829A) had reduced CICR activity without changing Ca(2+) sensitivity, whereas the L4827A mutant formed a constitutive active channel. T4825I, a disease-associated mutation for malignant hyperthermia, exhibited enhanced CICR activity. An α-helical wheel representation of the N-terminal S4-S5 linker provides a rational explanation to the observed activities of the mutants. These results suggest that N-terminal half of the S4-S5 linker may form an α-helical structure and play an important role in RyR1 channel gating.

  18. Molecular Characterization, mRNA Expression and Alternative Splicing of Ryanodine Receptor Gene in the Brown Citrus Aphid, Toxoptera citricida (Kirkaldy)

    PubMed Central

    Wang, Ke-Yi; Jiang, Xuan-Zhao; Yuan, Guo-Rui; Shang, Feng; Wang, Jin-Jun

    2015-01-01

    Ryanodine receptors (RyRs) play a critical role in regulating the release of intracellular calcium, which enables them to be effectively targeted by the two novel classes of insecticides, phthalic acid diamides and anthranilic diamides. However, less information is available about this target site in insects, although the sequence and structure information of target molecules are essential for designing new control agents of high selectivity and efficiency, as well as low non-target toxicity. Here, we provided sufficient information about the coding sequence and molecular structures of RyR in T. citricida (TciRyR), an economically important pest. The full-length TciRyR cDNA was characterized with an open reading frame of 15,306 nucleotides, encoding 5101 amino acid residues. TciRyR was predicted to embrace all the hallmarks of ryanodine receptor, typically as the conserved C-terminal domain with consensus calcium-biding EF-hands (calcium-binding motif) and six transmembrane domains, as well as a large N-terminal domain. qPCR analysis revealed that the highest mRNA expression levels of TciRyR were observed in the adults, especially in the heads. Alternative splicing in TciRyR was evidenced by an alternatively spliced exon, resulting from intron retention, which was different from the case of RyR in Myzus persicae characterized with no alternative splicing events. Diagnostic PCR analysis indicated that the splicing of this exon was not only regulated in a body-specific manner but also in a stage-dependent manner. Taken together, these results provide useful information for new insecticide design and further insights into the molecular basis of insecticide action. PMID:26154764

  19. Characterization of muscarinic cholinergic receptor subtypes in human peripheral lung

    SciTech Connect

    Bloom, J.W.; Halonen, M.; Yamamura, H.I.

    1988-02-01

    The authors have characterized the muscarinic cholinergic receptor subtypes in human peripheral lung membranes using the selective muscarinic antagonist (/sup 3/H)pirenzepine ((/sup 3/H)PZ) and the classical muscarinic antagonist (/sup 3/H)(-)-quinuclidinyl benzilate. High-affinity binding with pharmacologic specificity was demonstrated for both radioligands. The high affinity Kd for (/sup 3/H)PZ binding determined from saturation isotherms was 5.6 nM, and the Kd for (/sup 3/H)(-)-quinuclidinyl benzilate binding was 14.3 pM. Approximately 62% of the total muscarinic binding sites in human peripheral lung bind (/sup 3/H)PZ with high affinity. There was no significant effect of the guanine nucleotide, guanyl-5'-yl imidodiphosphate, on the inhibition of (/sup 3/H)(-)-quinyclidinyl benzilate binding by the muscarinic agonist carbachol in peripheral lung membranes. If the muscarinic receptor with high affinity for PZ has an important role in bronchoconstriction, its characterization could result in the development of more selective bronchodilators.

  20. Adenosine receptors and diabetes: Focus on the A(2B) adenosine receptor subtype.

    PubMed

    Merighi, Stefania; Borea, Pier Andrea; Gessi, Stefania

    2015-09-01

    Over the last two decades, diabetes mellitus has become one of the most challenging health problems worldwide. Diabetes mellitus, classified as type I and II, is a pathology concerning blood glucose level in the body. The nucleoside adenosine has long been known to affect insulin secretion, glucose homeostasis and lipid metabolism, through activation of four G protein coupled adenosine receptors (ARs), named A1, A2A, A2B and A3. Currently, the novel promising subtype to develop new drugs for diabetes treatment is the A2BAR subtype. The use of selective agonists and antagonists for A2BAR subtype in various diabetic animal models allowed us to identify several effects of A2BAR signaling in cell metabolism. In particular, the focus of this review is to summarize the studies on purinergic signaling associated with diabetes through A2BARs modulation.

  1. Sound Waves Induce Neural Differentiation of Human Bone Marrow-Derived Mesenchymal Stem Cells via Ryanodine Receptor-Induced Calcium Release and Pyk2 Activation.

    PubMed

    Choi, Yura; Park, Jeong-Eun; Jeong, Jong Seob; Park, Jung-Keug; Kim, Jongpil; Jeon, Songhee

    2016-10-01

    Mesenchymal stem cells (MSCs) have shown considerable promise as an adaptable cell source for use in tissue engineering and other therapeutic applications. The aims of this study were to develop methods to test the hypothesis that human MSCs could be differentiated using sound wave stimulation alone and to find the underlying mechanism. Human bone marrow (hBM)-MSCs were stimulated with sound waves (1 kHz, 81 dB) for 7 days and the expression of neural markers were analyzed. Sound waves induced neural differentiation of hBM-MSC at 1 kHz and 81 dB but not at 1 kHz and 100 dB. To determine the signaling pathways involved in the neural differentiation of hBM-MSCs by sound wave stimulation, we examined the Pyk2 and CREB phosphorylation. Sound wave induced an increase in the phosphorylation of Pyk2 and CREB at 45 min and 90 min, respectively, in hBM-MSCs. To find out the upstream activator of Pyk2, we examined the intracellular calcium source that was released by sound wave stimulation. When we used ryanodine as a ryanodine receptor antagonist, sound wave-induced calcium release was suppressed. Moreover, pre-treatment with a Pyk2 inhibitor, PF431396, prevented the phosphorylation of Pyk2 and suppressed sound wave-induced neural differentiation in hBM-MSCs. These results suggest that specific sound wave stimulation could be used as a neural differentiation inducer of hBM-MSCs.

  2. Role of Mg(2+) in Ca(2+)-induced Ca(2+) release through ryanodine receptors of frog skeletal muscle: modulations by adenine nucleotides and caffeine.

    PubMed

    Murayama, T; Kurebayashi, N; Ogawa, Y

    2000-04-01

    Mg(2+) serves as a competitive antagonist against Ca(2+) in the high-affinity Ca(2+) activation site (A-site) and as an agonist of Ca(2+) in the low-affinity Ca(2+) inactivation site (I-site) of the ryanodine receptor (RyR), which mediates Ca(2+)-induced Ca(2+) release (CICR). This paper presents the quantitative determination of the affinities for Ca(2+) and Mg(2+) of A- and I-sites of RyR in frog skeletal muscles by measuring [(3)H]ryanodine binding to purified alpha- and beta-RyRs and CICR activity in skinned fibers. There was only a minor difference in affinity at most between alpha- and beta-RyRs. The A-site favored Ca(2+) 20- to 30-fold over Mg(2+), whereas the I-site was nonselective between the two cations. The RyR in situ showed fivefold higher affinities for Ca(2+) and Mg(2+) of both sites than the purified alpha- and beta-RyRs with unchanged cation selectivity. Adenine nucleotides, whose stimulating effect was found to be indistinguishable between free and complexed forms, did not alter the affinities for cations in either site, except for the increased maximum activity of RyR. Caffeine increased not only the affinity of the A-site for Ca(2+) alone, but also the maximum activity of RyR with otherwise minor changes. The results presented here suggest that the rate of CICR in frog skeletal muscles appears to be too low to explain the physiological Ca(2+) release, even though Mg(2+) inhibition disappears.

  3. 3,5-Di-t-butyl catechol is a potent human ryanodine receptor 1 activator, not suitable for the diagnosis of malignant hyperthermia susceptibility.

    PubMed

    Lacava, Caterina; Michalek-Sauberer, Andrea; Kraft, Birgit; Sgaragli, Giampietro; Sipos, Elisabeth; Höller, Carmen; Kress, Hans Georg; Fusi, Fabio; Weigl, Lukas G

    2012-07-01

    3,5-Di-t-butyl catechol (DTCAT) releases Ca(2+) from rat skeletal muscle sarcoplasmic reticulum (SR) vesicles. Hence, it is a candidate for use as a substitute for halothane or caffeine in the in vitro contracture test for the diagnosis of susceptibility to malignant hyperthermia (MH). To characterize the effect of DTCAT at cell level, Ca(2+) release experiments were performed on cultured, human skeletal muscle myotubes using the fluorescent Ca(2+) indicator fura2-AM. DTCAT was also assayed in the in vitro contracture test on human skeletal muscle bundles obtained from individuals diagnosed susceptible (MHS), normal (MHN) or equivocal for halothane (MHEH) and compared to the standard test substances caffeine and halothane. DTCAT increased, in a concentration-dependent manner and with a higher efficacy as compared to caffeine, the free, intracellular Ca(2+) levels of cultured MHN and MHS skeletal muscle myotubes. This effect was similar in both types of myotubes and involved the release of Ca(2+) from SR stores as well as Ca(2+)-influx from the extracellular space. Inhibition of ryanodine receptors either with ryanodine or with ruthenium red markedly reduced DTCAT-induced increase in intracellular Ca(2+) concentration while abolishing that induced by caffeine. In MHN skeletal muscle bundles, DTCAT induced contractures with an EC(50) value of 160 ± 91 μM. However, the sensitivity of MHS or MHEH muscles to DTCAT was similar to that of MHN muscles. In conclusion, DTCAT is not suitable for the diagnosis of MH susceptibility due to its failure to discriminate between MHN and MHS muscles. Copyright © 2012 Elsevier Ltd. All rights reserved.

  4. Divergent Activity Profiles of Type 1 Ryanodine Receptor Channels Carrying Malignant Hyperthermia and Central Core Disease Mutations in the Amino-Terminal Region.

    PubMed

    Murayama, Takashi; Kurebayashi, Nagomi; Yamazawa, Toshiko; Oyamada, Hideto; Suzuki, Junji; Kanemaru, Kazunori; Oguchi, Katsuji; Iino, Masamitsu; Sakurai, Takashi

    2015-01-01

    The type 1 ryanodine receptor (RyR1) is a Ca2+ release channel in the sarcoplasmic reticulum of skeletal muscle and is mutated in several diseases, including malignant hyperthermia (MH) and central core disease (CCD). Most MH and CCD mutations cause accelerated Ca2+ release, resulting in abnormal Ca2+ homeostasis in skeletal muscle. However, how specific mutations affect the channel to produce different phenotypes is not well understood. In this study, we have investigated 11 mutations at 7 different positions in the amino (N)-terminal region of RyR1 (9 MH and 2 MH/CCD mutations) using a heterologous expression system in HEK293 cells. In live-cell Ca2+ imaging at room temperature (~25 °C), cells expressing mutant channels exhibited alterations in Ca2+ homeostasis, i.e., an enhanced sensitivity to caffeine, a depletion of Ca2+ in the ER and an increase in resting cytoplasmic Ca2+. RyR1 channel activity was quantitatively evaluated by [3H]ryanodine binding and three parameters (sensitivity to activating Ca2+, sensitivity to inactivating Ca2+ and attainable maximum activity, i.e., gain) were obtained by fitting analysis. The mutations increased the gain and the sensitivity to activating Ca2+ in a site-specific manner. The gain was consistently higher in both MH and MH/CCD mutations. Sensitivity to activating Ca2+ was markedly enhanced in MH/CCD mutations. The channel activity estimated from the three parameters provides a reasonable explanation to the pathological phenotype assessed by Ca2+ homeostasis. These properties were also observed at higher temperatures (~37 °C). Our data suggest that divergent activity profiles may cause varied disease phenotypes by specific mutations. This approach should be useful for diagnosis and treatment of diseases with mutations in RyR1.

  5. Blockade of Cocaine or σ Receptor Agonist Self Administration by Subtype-Selective σ Receptor Antagonists.

    PubMed

    Katz, Jonathan L; Hiranita, Takato; Kopajtic, Theresa A; Rice, Kenner C; Mesangeau, Christophe; Narayanan, Sanju; Abdelazeem, Ahmed H; McCurdy, Christopher R

    2016-07-01

    The identification of sigma receptor (σR) subtypes has been based on radioligand binding and, despite progress with σ1R cellular function, less is known about σR subtype functions in vivo. Recent findings that cocaine self administration experience will trigger σR agonist self administration was used in this study to assess the in vivo receptor subtype specificity of the agonists (+)-pentazocine, PRE-084 [2-(4-morpholinethyl) 1-phenylcyclohexanecarboxylate hydrochloride], and 1,3-di-o-tolylguanidine (DTG) and several novel putative σR antagonists. Radioligand binding studies determined in vitro σR selectivity of the novel compounds, which were subsequently studied for self administration and antagonism of cocaine, (+)-pentazocine, PRE-084, or DTG self administration. Across the dose ranges studied, none of the novel compounds were self administered, nor did they alter cocaine self administration. All compounds blocked DTG self administration, with a subset also blocking (+)-pentazocine and PRE-084 self administration. The most selective of the compounds in binding σ1Rs blocked cocaine self administration when combined with a dopamine transport inhibitor, either methylphenidate or nomifensine. These drug combinations did not decrease rates of responding maintained by food reinforcement. In contrast, the most selective of the compounds in binding σ2Rs had no effect on cocaine self administration in combination with either dopamine transport inhibitor. Thus, these results identify subtype-specific in vivo antagonists, and the utility of σR agonist substitution for cocaine self administration as an assay capable of distinguishing σR subtype selectivity in vivo. These results further suggest that effectiveness of dual σR antagonism and dopamine transport inhibition in blocking cocaine self administration is specific for σ1Rs and further support this dual targeting approach to development of cocaine antagonists. U.S. Government work not protected by U

  6. Blockade of Cocaine or σ Receptor Agonist Self Administration by Subtype-Selective σ Receptor Antagonists

    PubMed Central

    Hiranita, Takato; Kopajtic, Theresa A.; Rice, Kenner C.; Mesangeau, Christophe; Narayanan, Sanju; Abdelazeem, Ahmed H.; McCurdy, Christopher R.

    2016-01-01

    The identification of sigma receptor (σR) subtypes has been based on radioligand binding and, despite progress with σ1R cellular function, less is known about σR subtype functions in vivo. Recent findings that cocaine self administration experience will trigger σR agonist self administration was used in this study to assess the in vivo receptor subtype specificity of the agonists (+)-pentazocine, PRE-084 [2-(4-morpholinethyl) 1-phenylcyclohexanecarboxylate hydrochloride], and 1,3-di-o-tolylguanidine (DTG) and several novel putative σR antagonists. Radioligand binding studies determined in vitro σR selectivity of the novel compounds, which were subsequently studied for self administration and antagonism of cocaine, (+)-pentazocine, PRE-084, or DTG self administration. Across the dose ranges studied, none of the novel compounds were self administered, nor did they alter cocaine self administration. All compounds blocked DTG self administration, with a subset also blocking (+)-pentazocine and PRE-084 self administration. The most selective of the compounds in binding σ1Rs blocked cocaine self administration when combined with a dopamine transport inhibitor, either methylphenidate or nomifensine. These drug combinations did not decrease rates of responding maintained by food reinforcement. In contrast, the most selective of the compounds in binding σ2Rs had no effect on cocaine self administration in combination with either dopamine transport inhibitor. Thus, these results identify subtype-specific in vivo antagonists, and the utility of σR agonist substitution for cocaine self administration as an assay capable of distinguishing σR subtype selectivity in vivo. These results further suggest that effectiveness of dual σR antagonism and dopamine transport inhibition in blocking cocaine self administration is specific for σ1Rs and further support this dual targeting approach to development of cocaine antagonists. PMID:27189970

  7. Angiotensin II AT2 receptor subtype: an uprising frontier in cardiovascular disease?

    PubMed

    Volpe, Massimo; Musumeci, Beatrice; De Paolis, Paola; Savoia, Carmine; Morganti, Alberto

    2003-08-01

    The renin-angiotensin system (RAS) plays a pivotal role in the regulation of fluid, electrolyte balance and blood pressure, and is a modulator of cellular growth and proliferation. Biological actions of RAS are linked to the binding of the effector molecule, angiotensin II (AngII), to specific membrane receptors, mostly the AT1 subtype and, to a lesser extent, other subtypes. Following the identification and characterization of the AT2 subtype receptor, it has been proposed that a complex interaction between AngII and its receptors may play an important role in the effects of RAS. In this paper current information on AngII subtype receptors--their structure, regulation and intracellular signalling--are reviewed, with a particular emphasis on the potential relevance for cardiovascular pathophysiology. In addition, we discuss modulation of expression of the AT2 receptor and its interaction with the AT1 receptor subtype, as well as the potential effects of this receptor on blood pressure regulation. A better understanding of the integrated effects of the AngII subtype receptors may help to elucidate the function of the RAS, as well as their participation in the mechanisms of cardiovascular disease and attendant therapeutic implications.

  8. Isoform-Specific Interactions Between Halothane and the Ryanodine Receptor Ca2+-Release Channel: Implications for Malignant Hyperthermia and the Protein Theory of Anaesthetic Action

    NASA Astrophysics Data System (ADS)

    Frömming, G. R.; Ohlendieck, K.

    General anaesthetics exhibit a relatively close relationship between their pharmacological potency and their lipid solubility and may thus act by non-specific perturbation of biomembranes. However, more recent data on anaesthetic action suggests that inhalational drugs such as halothane bind directly to hydrophobic protein domains, thereby modulating important receptor functions. In support of this protein theory of anaesthetic action our native gel analysis presented here shows that halothane induces oligomerization of the skeletal muscle ryanodine receptor (RyR) 1 Ca2+-release channel, but not its cardiac RyR-2 isoform. Thus, inhalational anaesthetics are not only able to influence protein-protein interactions directly but also appear to differentiate between protein isoforms and/or configurations. This suggests that distinct peptide binding sites exist for these pharmacological agents. In addition, similar mutations in the RyR-2 isoform, which would trigger an episode of malignant hyperthermia in skeletal muscle fibres via abnormal RyR-1 isoforms, would probably not induce an increase in cardiac Ca2+-release upon administration of halothane.

  9. Classification of Dopamine Receptor Genes in Vertebrates: Nine Subtypes in Osteichthyes.

    PubMed

    Yamamoto, Kei; Fontaine, Romain; Pasqualini, Catherine; Vernier, Philippe

    2015-01-01

    Dopamine neurotransmission regulates various brain functions, and its regulatory roles are mediated by two families of G protein-coupled receptors: the D1 and D2 receptor families. In mammals, the D1 family comprises two receptor subtypes (D1 and D5), while the D2 family comprises three receptor subtypes (D2, D3 and D4). Phylogenetic analyses of dopamine receptor genes strongly suggest that the common ancestor of Osteichthyes (bony jawed vertebrates) possessed four subtypes in the D1 family and five subtypes in the D2 family. Mammals have secondarily lost almost half of the ancestral dopamine receptor genes, whereas nonmammalian species kept many of them. Although the mammalian situation is an exception among Osteichthyes, the current classification and characterization of dopamine receptors are based on mammalian features, which have led to confusion in the identification of dopamine receptor subtypes in nonmammalian species. Here we begin by reviewing the history of the discovery of dopamine receptors in vertebrates. The recent genome sequencing of coelacanth, gar and elephant shark led to the proposal of a refined scenario of evolution of dopamine receptor genes. We also discuss a current problem of nomenclature of dopamine receptors. Following the official nomenclature of mammalian dopamine receptors from D1 to D5, we propose to name newly identified receptor subtypes from D6 to D9 in order to facilitate the use of an identical name for orthologous genes among different species. To promote a nomenclature change which allows distinguishing the two dopamine receptor families, a nomenclature consortium is needed. This comparative perspective is crucial to correctly interpret data obtained in animal studies on dopamine-related brain disorders, and more fundamentally, to understand the characteristics of dopamine neurotransmission in vertebrates.

  10. Oxygen-coupled redox regulation of the skeletal muscle ryanodine receptor-Ca2+ release channel by NADPH oxidase 4

    PubMed Central

    Sun, Qi-An; Hess, Douglas T.; Nogueira, Leonardo; Yong, Sandro; Bowles, Dawn E.; Eu, Jerry; Laurita, Kenneth R.; Meissner, Gerhard; Stamler, Jonathan S.

    2011-01-01

    Physiological sensing of O2 tension (partial O2 pressure, pO2) plays an important role in some mammalian cellular systems, but striated muscle generally is not considered to be among them. Here we describe a molecular mechanism in skeletal muscle that acutely couples changes in pO2 to altered calcium release through the ryanodine receptor–Ca2+-release channel (RyR1). Reactive oxygen species are generated in proportion to pO2 by NADPH oxidase 4 (Nox4) in the sarcoplasmic reticulum, and the consequent oxidation of a small set of RyR1 cysteine thiols results in increased RyR1 activity and Ca2+ release in isolated sarcoplasmic reticulum and in cultured myofibers and enhanced contractility of intact muscle. Thus, Nox4 is an O2 sensor in skeletal muscle, and O2-coupled hydrogen peroxide production by Nox4 governs the redox state of regulatory RyR1 thiols and thereby governs muscle performance. These findings reveal a molecular mechanism for O2-based signaling by an NADPH oxidase and demonstrate a physiological role for oxidative modification of RyR1. PMID:21896730

  11. Ca2+ Current and Charge Movements in Skeletal Myotubes Promoted by the β-Subunit of the Dihydropyridine Receptor in the Absence of Ryanodine Receptor Type 1

    PubMed Central

    Ahern, Chris A.; Sheridan, David. C.; Cheng, Weijun; Mortenson, Lindsay; Nataraj, Priya; Allen, Paul; Waard, Michel De; Coronado, Roberto

    2003-01-01

    The β-subunit of the dihydropyridine receptor (DHPR) enhances the Ca2+ channel and voltage-sensing functions of the DHPR. In skeletal myotubes, there is additional modulation of DHPR functions imposed by the presence of ryanodine receptor type-1 (RyR1). Here, we examined the participation of the β-subunit in the expression of L-type Ca2+ current and charge movements in RyR1 knock-out (KO), β1 KO, and double β1/RyR1 KO myotubes generated by mating heterozygous β1 KO and RyR1 KO mice. Primary myotube cultures of each genotype were transfected with various β-isoforms and then whole-cell voltage-clamped for measurements of Ca2+ and gating currents. Overexpression of the endogenous skeletal β1a isoform resulted in a low-density Ca2+ current either in RyR1 KO (36 ± 9 pS/pF) or in β1/RyR1 KO (34 ± 7 pS/pF) myotubes. However, the heterologous β2a variant with a double cysteine motif in the N-terminus (C3, C4), recovered a Ca2+ current that was entirely wild-type in density in RyR1 KO (195 ± 16 pS/pF) and was significantly enhanced in double β1/RyR1 KO (115 ± 18 pS/pF) myotubes. Other variants tested from the four β gene families (β1a, β1b, β1c, β3, and β4) were unable to enhance Ca2+ current expression in RyR1 KO myotubes. In contrast, intramembrane charge movements in β2a-expressing β1a/RyR1 KO myotubes were significantly lower than in β1a-expressing β1a/RyR1 KO myotubes, and the same tendency was observed in the RyR1 KO myotube. Thus, β2a had a preferential ability to recover Ca2+ current, whereas β1a had a preferential ability to rescue charge movements. Elimination of the double cysteine motif (β2a C3,4S) eliminated the RyR1-independent Ca2+ current expression. Furthermore, Ca2+ current enhancement was observed with a β2a variant lacking the double cysteine motif and fused to the surface membrane glycoprotein CD8. Thus, tethering the β2a variant to the myotube surface activated the DHPR Ca2+ current and bypassed the requirement for RyR1

  12. Subtype-selective regulation of IP(3) receptors by thimerosal via cysteine residues within the IP(3)-binding core and suppressor domain.

    PubMed

    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.

  13. Is there life in the horny layer? Dihydropyridine and ryanodine receptors in the skin of female and male chickens (Gallus domesticus).

    PubMed

    Peltonen, Liisa M; Mänttäri, Satu

    2008-05-01

    Previous findings in pigeons and chickens show that Ca(2+) may be accumulated inside the cornified skin cells and that Ca(2+) microenvironments with a lower- or higher-than-blood concentration may exist in the skin. It has been suggested that the skin may function as a secretory pathway or a reservoir for Ca(2+) recycling. To test this hypothesis, we studied the dermis and epidermis of female and male chickens in vivo to find out whether cellular mechanisms exist for the accumulation, recycling or secretion of Ca(2+). For calcium influx and intracellular Ca(2+) release, respectively, the density of dihydropyridine receptors (DHPRs) and ryanodine receptors (RyRs) was examined, using high-affinity (-)-enantiomers of dihydropyridine and ryanodine labelled with fluorophores. To investigate Ca(2+) utilization in the skin, the systemic and local activity of the enzyme alkaline phosphatase (ALP) and the concentration of ionic Ca(2+) were measured in plasma and in cutaneous extracellular fluid, collected by suction blister technique. We found that both DHPRs and RyRs were present in all skin layers from dermis to horny layer. However, receptor densities were highest in the surface layers. With a basic calcium-rich diet, receptor densities were higher in males, particularly in the dermis and mid-epidermis. After a reduction in the nutritional Ca(2+) input, receptor densities in males decreased to the same level as in females, in which the receptor densities were not affected by the amount of Ca(2+) in the diet or that resulting from coming out of lay. The extracellular concentration of ionic Ca(2+) per se was not found to affect the density of DHPRs and RyRs in the skin. Spatially, RyRs seem to be located in the periphery of the sebokeratinocyte. ALP activity was shown to be lower in the extracellular fluid than in the plasma in both sexes. However, activity in both extracellular domains increased significantly in females that had come out of lay. This was probably

  14. TARP subtypes differentially and dose-dependently control synaptic AMPA receptor gating.

    PubMed

    Milstein, Aaron D; Zhou, Wei; Karimzadegan, Siavash; Bredt, David S; Nicoll, Roger A

    2007-09-20

    A family of transmembrane AMPA receptor regulatory proteins (TARPs) profoundly affects the trafficking and gating of AMPA receptors (AMPARs). Although TARP subtypes are differentially expressed throughout the CNS, it is unclear whether this imparts functional diversity to AMPARs in distinct neuronal populations. Here, we examine the effects of each TARP subtype on the kinetics of AMPAR gating in heterologous cells and in neurons. We report a striking heterogeneity in the effects of TARP subtypes on AMPAR deactivation and desensitization, which we demonstrate controls the time course of synaptic transmission. In addition, we find that some TARP subtypes dramatically slow AMPAR activation kinetics. Synaptic AMPAR kinetics also depend on TARP expression level, suggesting a variable TARP/AMPAR stoichiometry. Analysis of quantal synaptic transmission in a TARP gamma-4 knockout (KO) mouse corroborates our expression data and demonstrates that TARP subtype-specific gating of AMPARs contributes to the kinetics of native AMPARs at central synapses.

  15. [11C]Carfentanil Binds Preferentially to μ-Opioid Receptor Subtype 1 Compared to Subtype 2.

    PubMed

    Eriksson, Olof; Antoni, Gunnar

    2015-01-01

    The positron emission tomography (PET) ligand [(11)C]carfentanil is a selective agonist for μ-opioid receptors and has been used for studying μ-opioid receptors in the human brain. However, it is unknown if [(11)C]carfentanil binding differentiates between subtype receptors μ1 and μ2. In this study, we investigated whether μ1 and μ2 can be studied separately through receptor subtype-selective inhibition of [(11)C]carfentanil by pharmacologic intervention. [(11)C]Carfentanil binding characteristics on rat brain sections were assessed either alone or in the presence of the μ-receptor inhibitor cyprodime or the μ1-specific inhibitor naloxonazine. [(11)C]Carfentanil binding in the living rat brain was similarly studied by small animal PET/computed tomography during baseline conditions or following displacement by cyprodime or naloxonazine. Autoradiography binding studies on rat brain sections demonstrated that [(11)C]carfentanil has higher affinity and binding potential for μ1 than for μ2. [(11)C]Carfentanil binding to μ2 in vivo could not be detected following specific blocking of μ1, as predicted from the low binding potential for μ2 as measured in vitro. [(11)C]Carfentanil binding is preferential for μ1 compared to μ2 in vitro and in vivo. Clinical studies employing [(11)C]carfentanil are therefore likely biased to measure μ1 rather than μ2.

  16. [(11)C]Carfentanil Binds Preferentially to μ-Opioid Receptor Subtype 1 Compared to Subtype 2.

    PubMed

    Eriksson, Olof; Antoni, Gunnar

    2015-09-01

    The positron emission tomography (PET) ligand [(11)C]carfentanil is a selective agonist for μ-opioid receptors and has been used for studying μ-opioid receptors in the human brain. However, it is unknown if [(11)C]carfentanil binding differentiates between subtype receptors μ1 and μ2. In this study, we investigated whether μ1 and μ2 can be studied separately through receptor subtype-selective inhibition of [(11)C]carfentanil by pharmacologic intervention. [(11)C]Carfentanil binding characteristics on rat brain sections were assessed either alone or in the presence of the μ-receptor inhibitor cyprodime or the μ1-specific inhibitor naloxonazine. [(11)C]Carfentanil binding in the living rat brain was similarly studied by small animal PET/computed tomography during baseline conditions or following displacement by cyprodime or naloxonazine. Autoradiography binding studies on rat brain sections demonstrated that [(11)C]carfentanil has higher affinity and binding potential for μ1 than for μ2. [(11)C]Carfentanil binding to μ2 in vivo could not be detected following specific blocking of μ1, as predicted from the low binding potential for μ2 as measured in vitro. [(11)C]Carfentanil binding is preferential for μ1 compared to μ2 in vitro and in vivo. Clinical studies employing [(11)C]carfentanil are therefore likely biased to measure μ1 rather than μ2.

  17. Multiple actions of phi-LITX-Lw1a on ryanodine receptors reveal a functional link between scorpion DDH and ICK toxins.

    PubMed

    Smith, Jennifer J; Vetter, Irina; Lewis, Richard J; Peigneur, Steve; Tytgat, Jan; Lam, Alexander; Gallant, Esther M; Beard, Nicole A; Alewood, Paul F; Dulhunty, Angela F

    2013-05-28

    We recently reported the isolation of a scorpion toxin named U1-liotoxin-Lw1a (U1-LITX-Lw1a) that adopts an unusual 3D fold termed the disulfide-directed hairpin (DDH) motif, which is the proposed evolutionary structural precursor of the three-disulfide-containing inhibitor cystine knot (ICK) motif found widely in animals and plants. Here we reveal that U1-LITX-Lw1a targets and activates the mammalian ryanodine receptor intracellular calcium release channel (RyR) with high (fM) potency and provides a functional link between DDH and ICK scorpion toxins. Moreover, U1-LITX-Lw1a, now described as ϕ-liotoxin-Lw1a (ϕ-LITX-Lw1a), has a similar mode of action on RyRs as scorpion calcines, although with significantly greater potency, inducing full channel openings at lower (fM) toxin concentrations whereas at higher pM concentrations increasing the frequency and duration of channel openings to a submaximal state. In addition, we show that the C-terminal residue of ϕ-LITX-Lw1a is crucial for the increase in full receptor openings but not for the increase in receptor subconductance opening, thereby supporting the two-binding-site hypothesis of scorpion toxins on RyRs. ϕ-LITX-Lw1a has potential both as a pharmacological tool and as a lead molecule for the treatment of human diseases that involve RyRs, such as malignant hyperthermia and polymorphic ventricular tachycardia.

  18. The cysteine-rich secretory protein domain of Tpx-1 is related to ion channel toxins and regulates ryanodine receptor Ca2+ signaling.

    PubMed

    Gibbs, Gerard M; Scanlon, Martin J; Swarbrick, James; Curtis, Suzanne; Gallant, Esther; Dulhunty, Angela F; O'Bryan, Moira K

    2006-02-17

    The cysteine-rich secretory proteins (Crisp) are predominantly found in the mammalian male reproductive tract as well as in the venom of reptiles. Crisps are two domain proteins with a structurally similar yet evolutionary diverse N-terminal domain and a characteristic cysteine-rich C-terminal domain, which we refer to as the Crisp domain. We presented the NMR solution structure of the Crisp domain of mouse Tpx-1, and we showed that it contains two subdomains, one of which has a similar fold to the ion channel regulators BgK and ShK. Furthermore, we have demonstrated for the first time that the ion channel regulatory activity of Crisp proteins is attributed to the Crisp domain. Specifically, the Tpx-1 Crisp domain inhibited cardiac ryanodine receptor (RyR) 2 with an IC(50) between 0.5 and 1.0 microM and activated the skeletal RyR1 with an AC(50) between 1 and 10 microM when added to the cytoplasmic domain of the receptor. This activity was nonvoltage-dependent and weakly voltage-dependent, respectively. Furthermore, the Tpx-1 Crisp domain activated both RyR forms at negative bilayer potentials and showed no effect at positive bilayer potentials when added to the luminal domain of the receptor. These data show that the Tpx-1 Crisp domain on its own can regulate ion channel activity and provide compelling evidence for a role for Tpx-1 in the regulation of Ca(2+) fluxes observed during sperm capacitation.

  19. Multiple actions of φ-LITX-Lw1a on ryanodine receptors reveal a functional link between scorpion DDH and ICK toxins

    PubMed Central

    Smith, Jennifer J.; Vetter, Irina; Lewis, Richard J.; Peigneur, Steve; Tytgat, Jan; Lam, Alexander; Gallant, Esther M.; Beard, Nicole A.; Alewood, Paul F.; Dulhunty, Angela F.

    2013-01-01

    We recently reported the isolation of a scorpion toxin named U1-liotoxin-Lw1a (U1-LITX-Lw1a) that adopts an unusual 3D fold termed the disulfide-directed hairpin (DDH) motif, which is the proposed evolutionary structural precursor of the three-disulfide-containing inhibitor cystine knot (ICK) motif found widely in animals and plants. Here we reveal that U1-LITX-Lw1a targets and activates the mammalian ryanodine receptor intracellular calcium release channel (RyR) with high (fM) potency and provides a functional link between DDH and ICK scorpion toxins. Moreover, U1-LITX-Lw1a, now described as φ-liotoxin-Lw1a (φ-LITX-Lw1a), has a similar mode of action on RyRs as scorpion calcines, although with significantly greater potency, inducing full channel openings at lower (fM) toxin concentrations whereas at higher pM concentrations increasing the frequency and duration of channel openings to a submaximal state. In addition, we show that the C-terminal residue of φ-LITX-Lw1a is crucial for the increase in full receptor openings but not for the increase in receptor subconductance opening, thereby supporting the two-binding-site hypothesis of scorpion toxins on RyRs. φ-LITX-Lw1a has potential both as a pharmacological tool and as a lead molecule for the treatment of human diseases that involve RyRs, such as malignant hyperthermia and polymorphic ventricular tachycardia. PMID:23671114

  20. Comprehensive behavioral phenotyping of ryanodine receptor type 3 (RyR3) knockout mice: decreased social contact duration in two social interaction tests.

    PubMed

    Matsuo, Naoki; Tanda, Koichi; Nakanishi, Kazuo; Yamasaki, Nobuyuki; Toyama, Keiko; Takao, Keizo; Takeshima, Hiroshi; Miyakawa, Tsuyoshi

    2009-01-01

    Dynamic regulation of the intracellular Ca2+ concentration is crucial for various neuronal functions such as synaptic transmission and plasticity, and gene expression. Ryanodine receptors (RyRs) are a family of intracellular calcium release channels that mediate calcium-induced calcium release from the endoplasmic reticulum. Among the three RyR isoforms, RyR3 is preferentially expressed in the brain especially in the hippocampus and striatum. To investigate the behavioral effects of RyR3 deficiency, we subjected RyR3 knockout (RyR3-/-) mice to a battery of behavioral tests. RyR3-/- mice exhibited significantly decreased social contact duration in two different social interaction tests, where two mice can freely move and make contacts with each other. They also exhibited hyperactivity and mildly impaired prepulse inhibition and latent inhibition while they did not show significant abnormalities in motor function and working and reference memory tests. These results indicate that RyR3 has an important role in locomotor activity and social behavior.

  1. Detection of a novel mutation in the ryanodine receptor gene in an Irish malignant hyperthermia pedigree: correlation of the IVCT response with the affected and unaffected haplotypes.

    PubMed Central

    Keating, K E; Giblin, L; Lynch, P J; Quane, K A; Lehane, M; Heffron, J J; McCarthy, T V

    1997-01-01

    Defects in the ryanodine receptor (RYR1) gene are associated with malignant hyperthermia (MH), an autosomal dominant disorder of skeletal muscle and one of the main causes of death resulting from anaesthesia. Susceptibility to MH (MHS) is determined by the level of tension generated in an in vitro muscle contracture test (IVCT) in response to caffeine and halothane. To date, mutation screening of the RYR1 gene in MH families has led to the identification of eight mutations. We describe here the identification of a novel mutation, Arg552Trp, in the RYR1 gene, which is clearly linked to the MHS phenotype in a large, well characterised Irish pedigree. Considering that the RYR1 protein functions as a tetramer, correlation of the IVCT with the affected and unaffected haplotypes was performed on the pedigree to investigate if the normal RYR1 allele in affected subjects contributes to the variation in the IVCT. The results show that the normal RYR1 allele is unlikely to play a role in IVCT variation. Images PMID:9138151

  2. Resistance to diamide insecticides in diamondback moth, Plutella xylostella (Lepidoptera: Plutellidae) is associated with a mutation in the membrane-spanning domain of the ryanodine receptor.

    PubMed

    Troczka, Bartek; Zimmer, Christoph T; Elias, Jan; Schorn, Corinna; Bass, Chris; Davies, T G Emyr; Field, Linda M; Williamson, Martin S; Slater, Russell; Nauen, Ralf

    2012-11-01

    Diamide insecticides such as chlorantraniliprole and flubendiamide are a new class of insecticide that selectively target insect ryanodine receptors (RyR), a distinct class of homo-tetrameric calcium release channels which play a pivotal role in calcium homeostasis in numerous cell types. Resistance to these insecticides has recently been reported in the diamondback moth, Plutella xylostella (Lepidoptera: Plutellidae), a global lepidopteran pest of cruciferous crops. In the present study a region of the gene encoding the proposed diamide binding site of the RyR from P. xylostella collected from the Philippines and Thailand and found to be over 200-fold resistant to both chlorantraniliprole and flubendiamide compared to susceptible strains, were amplified by RT-PCR and sequenced. Comparison of the sequence with those from several susceptible reference strains revealed non-synonymous mutations in each of the resistant strains that in both cases lead to a glycine to glutamic acid substitution (G4946E) in the protein. The independent evolution of the same amino acid substitution within a highly conserved region of the proposed diamide binding site in two geographically separated resistant strains of P. xylostella strongly suggests a causal association with diamide resistance. Furthermore we designed a pyrosequencing-based diagnostic assay for resistance monitoring purposes that can be used to detect the G4946E mutation in field-collected samples of diamondback moth. The implications of the reported findings for resistance management strategies are discussed.

  3. Localization of a novel ryanodine receptor gene (RYR3) to human chromosome 15q14-q15 by in situ hybridization

    SciTech Connect

    Sorrentino, V. Istituto Scientifico H. San Raffaele, Milan ); Giannini, G. ); Malzac, P.; Mattei, M.G. )

    1993-10-01

    Ryanodine receptors (RyRs) are intracellular Ca[sup 2+] release channels responsible for the release of Ca[sup 2+] from intracellular stores following transduction of many different extracellular stimuli. The genes of the two RyRs, originally described in the sarcoplasmic reticulum of skeletal (RYR1) or cardiac (RYR2) muscles, have been cloned and mapped on chromosome 19q13.1 and chromosome 1, respectively, in humans. The RYR1 gene has been shown to be tightly linked to the locus for malignant hyperthermia susceptibility (MHS). A single-point mutation in RYR1 has been identified as the possible cause of MHS in swine and of at least some forms of MHS in human. Another MHS locus has been mapped on human chromosome 17q11-q24. We have recently cloned a third RyR (RYR3) from mink, which appears to be widely expressed. A RyR with 94% homology to mink cDNA has also been isolated from rabbit brain.

  4. Multi-minicore disease and atypical periodic paralysis associated with novel mutations in the skeletal muscle ryanodine receptor (RYR1) gene.

    PubMed

    Zhou, Haiyan; Lillis, Suzanne; Loy, Ryan E; Ghassemi, Farshid; Rose, Michael R; Norwood, Fiona; Mills, Kerry; Al-Sarraj, Safa; Lane, Russell J M; Feng, Lucy; Matthews, Emma; Sewry, Caroline A; Abbs, Stephen; Buk, Stefan; Hanna, Michael; Treves, Susan; Dirksen, Robert T; Meissner, Gerhard; Muntoni, Francesco; Jungbluth, Heinz

    2010-03-01

    The skeletal muscle ryanodine receptor plays a crucial role in excitation-contraction (EC) coupling and is implicated in various congenital myopathies. The periodic paralyses are a heterogeneous, dominantly inherited group of conditions mainly associated with mutations in the SCN4A and the CACNA1S genes. The interaction between RyR1 and DHPR proteins underlies depolarization-induced Ca(2+) release during EC coupling in skeletal muscle. We report a 35-year-old woman presenting with signs and symptoms of a congenital myopathy at birth and repeated episodes of generalized, atypical normokalaemic paralysis in her late teens. Genetic studies of this patient revealed three heterozygous RYR1 substitutions (p.Arg2241X, p.Asp708Asn and p.Arg2939Lys) associated with marked reduction of the RyR1 protein and abnormal DHPR distribution. We conclude that RYR1 mutations may give rise to both myopathies and atypical periodic paralysis, and RYR1 mutations may underlie other unresolved cases of periodic paralysis with unusual features.

  5. Nitric Oxide-induced Activation of the Type 1 Ryanodine Receptor Is Critical for Epileptic Seizure-induced Neuronal Cell Death.

    PubMed

    Mikami, Yoshinori; Kanemaru, Kazunori; Okubo, Yohei; Nakaune, Takuya; Suzuki, Junji; Shibata, Kazuki; Sugiyama, Hiroki; Koyama, Ryuta; Murayama, Takashi; Ito, Akihiro; Yamazawa, Toshiko; Ikegaya, Yuji; Sakurai, Takashi; Saito, Nobuhito; Kakizawa, Sho; Iino, Masamitsu

    2016-09-01

    Status epilepticus (SE) is a life-threatening emergency that can cause neurodegeneration with debilitating neurological disorders. However, the mechanism by which convulsive SE results in neurodegeneration is not fully understood. It has been shown that epileptic seizures produce markedly increased levels of nitric oxide (NO) in the brain, and that NO induces Ca(2+) release from the endoplasmic reticulum via the type 1 ryanodine receptor (RyR1), which occurs through S-nitrosylation of the intracellular Ca(2+) release channel. Here, we show that through genetic silencing of NO-induced activation of the RyR1 intracellular Ca(2+) release channel, neurons were rescued from seizure-dependent cell death. Furthermore, dantrolene, an inhibitor of RyR1, was protective against neurodegeneration caused by SE. These results demonstrate that NO-induced Ca(2+) release via RyR is involved in SE-induced neurodegeneration, and provide a rationale for the use of RyR1 inhibitors for the prevention of brain damage following SE.

  6. Imperatoxin A, a Cell-Penetrating Peptide from Scorpion Venom, as a Probe of Ca2+-Release Channels/Ryanodine Receptors

    PubMed Central

    Gurrola, Georgina B.; Capes, E. Michelle; Zamudio, Fernando Z.; Possani, Lourival D.; Valdivia, Héctor H.

    2010-01-01

    Scorpion venoms are rich in ion channel-modifying peptides, which have proven to be invaluable probes of ion channel structure-function relationship. We previously isolated imperatoxin A (IpTxa), a 3.7 kDa peptide activator of Ca2+-release channels/ryanodine receptors (RyRs) [1,2,3] and founding member of the calcin family of scorpion peptides. IpTxa folds into a compact, mostly hydrophobic molecule with a cluster of positively-charged, basic residues polarized on one side of the molecule that possibly interacts with the phospholipids of cell membranes. To investigate whether IpTxa permeates external cellular membranes and targets RyRs in vivo, we perfused IpTxa on intact cardiomyocytes while recording field-stimulated intracellular Ca2+ transients. To further investigate the cell-penetrating capabilities of the toxin, we prepared thiolated, fluorescent derivatives of IpTxa. Biological activity and spectroscopic properties indicate that these derivatives retain high affinity for RyRs and are only 5- to 10-fold less active than native IpTxa. Our results demonstrate that IpTxa is capable of crossing cell membranes to alter the release of Ca2+ in vivo, and has the capacity to carry a large, membrane-impermeable cargo across the plasma membrane, a finding with exciting implications for novel drug delivery. PMID:20668646

  7. Malignant hyperthermia-associated mutations in the S2-S3 cytoplasmic loop of type 1 ryanodine receptor calcium channel impair calcium-dependent inactivation.

    PubMed

    Gomez, Angela C; Holford, Timothy W; Yamaguchi, Naohiro

    2016-11-01

    Channel activities of skeletal muscle ryanodine receptor (RyR1) are activated by micromolar Ca(2+) and inactivated by higher (∼1 mM) Ca(2+) To gain insight into a mechanism underlying Ca(2+)-dependent inactivation of RyR1 and its relationship with skeletal muscle diseases, we constructed nine recombinant RyR1 mutants carrying malignant hyperthermia or centronuclear myopathy-associated mutations and determined RyR1 channel activities by [(3)H]ryanodine binding assay. These mutations are localized in or near the RyR1 domains which are responsible for Ca(2+)-dependent inactivation of RyR1. Four RyR1 mutations (F4732D, G4733E, R4736W, and R4736Q) in the cytoplasmic loop between the S2 and S3 transmembrane segments (S2-S3 loop) greatly reduced Ca(2+)-dependent channel inactivation. Activities of these mutant channels were suppressed at 10-100 μM Ca(2+), and the suppressions were relieved by 1 mM Mg(2+) The Ca(2+)- and Mg(2+)-dependent regulation of S2-S3 loop RyR1 mutants are similar to those of the cardiac isoform of RyR (RyR2) rather than wild-type RyR1. Two mutations (T4825I and H4832Y) in the S4-S5 cytoplasmic loop increased Ca(2+) affinities for channel activation and decreased Ca(2+) affinities for inactivation, but impairment of Ca(2+)-dependent inactivation was not as prominent as those of S2-S3 loop mutants. Three mutations (T4082M, S4113L, and N4120Y) in the EF-hand domain showed essentially the same Ca(2+)-dependent channel regulation as that of wild-type RyR1. The results suggest that nine RyR1 mutants associated with skeletal muscle diseases were differently regulated by Ca(2+) and Mg(2+) Four malignant hyperthermia-associated RyR1 mutations in the S2-S3 loop conferred RyR2-type Ca(2+)- and Mg(2+)-dependent channel regulation. Copyright © 2016 the American Physiological Society.

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

    PubMed

    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

  9. Analysis of the entire ryanodine receptor type 1 and alpha 1 subunit of the dihydropyridine receptor (CACNA1S) coding regions for variants associated with malignant hyperthermia in Australian families.

    PubMed

    Gillies, R L; Bjorksten, A R; Du Sart, D; Hockey, B M

    2015-03-01

    Defects in the genes coding for the skeletal muscle ryanodine receptor (RYR1) and alpha 1 subunit of the dihydropyridine receptor (CACNA1S) have been identified as causative for malignant hyperthermia (MH). Sixty-two MH susceptible individuals presenting to the same diagnostic centre had copy deoxyribonucleic acid, derived from muscle ribonucleic acid, sequenced to identify variants with the potential to be responsible for the MH phenotype in both RYR1 and CACNA1S. These genetic findings were combined with clinical episode details and in vitro contracture test results to improve our understanding of the Australian MH cohort. Twelve novel variants were identified in RYR1 and six in CACNA1S. Known RYR1 causative mutations were identified in six persons and novel variants in RYR1 and CACNA1S in a further 17 persons. Trends indicated higher mutation identification in those with more definitive clinical episodes and stronger in vitro contracture test responses.

  10. Differential alterations in muscarinic receptor subtypes in Alzheimer's disease: implications for cholinergic-based therapies.

    PubMed

    Flynn, D D; Ferrari-DiLeo, G; Levey, A I; Mash, D C

    1995-01-01

    Molecular subtypes of muscarinic receptors (m1-m5) are novel targets for cholinergic replacement therapies in Alzheimer's disease (AD). However, knowledge concerning the relative distribution, abundance and functional status of these receptors in human brain and AD is incomplete. Recent data from our laboratory have demonstrated a defect in the ability of the M1 receptor subtype to form a high affinity agonist-receptor-G protein complex in AD frontal cortex. This defect is manifested by decreased M1 receptor-stimulated GTPgammaS binding and GTPase activity and by a loss in receptor-stimulated phospholipase C activity. Normal levels of G proteins suggest that the aberrant receptor-G protein interaction may result from an altered form of the m1 receptor in AD. The combined use of radioligand binding and receptor-domain specific antibodies has permitted the re-examination of the status of muscarinic receptor subtypes in the human brain. In AD, normal levels of m1 receptor [3H]-pirenzepine binding contrasted with diminished m1 immunoreactivity, further suggesting that there is an altered form of the m1 receptor in the disease. Reduced m2 immunoreactivity was consistent with decreased numbers of m2 binding sites. Increased levels of m4 receptors were observed in both binding and immunoreactivity measurements. These findings suggest one possible explanation for the relative ineffectiveness of cholinergic replacement therapies used to date and suggest potential new directions for development of effective therapeutic strategies for AD.

  11. Autoradiographic visualization of muscarinic receptor subtypes in human and guinea pig lung

    SciTech Connect

    Mak, J.C.; Barnes, P.J. )

    1990-06-01

    Muscarinic receptor subtypes have been localized in human and guinea pig lung sections by an autoradiographic technique, using (3H)(-)quinuclidinyl benzilate (( 3H)QNB) and selective muscarinic antagonists. (3H)QNB was incubated with tissue sections for 90 min at 25 degrees C, and nonspecific binding was determined by incubating adjacent serial sections in the presence of 1 microM atropine. Binding to lung sections had the characterization expected for muscarinic receptors. Autoradiography revealed that muscarinic receptors were widely distributed in human lung, with dense labeling over submucosal glands and airway ganglia, and moderate labeling over nerves in intrapulmonary bronchi and of airway smooth muscle of large and small airways. In addition, alveolar walls were uniformly labeled. In guinea pig lung, labeling of airway smooth muscle was similar, but in contrast to human airways, epithelium was labeled but alveolar walls were not. The muscarinic receptors of human airway smooth muscle from large to small airways were entirely of the M3-subtype, whereas in guinea pig airway smooth muscle, the majority were the M3-subtype with a very small population of the M2-subtype present. In human bronchial submucosal glands, M1- and M3-subtypes appeared to coexist in the proportions of 36 and 64%, respectively. In human alveolar walls the muscarinic receptors were entirely of the M1-subtype, which is absent from the guinea pig lung. No M2-receptors were demonstrated in human lung. The localization of M1-receptors was confirmed by direct labeling with (3H)pirenzepine. With the exception of the alveolar walls in human lung, the localization of muscarinic receptor subtypes on structures in the lung is consistent with known functional studies.

  12. GABA A/Bz receptor subtypes as targets for selective drugs.

    PubMed

    Da Settimo, F; Taliani, S; Trincavelli, M L; Montali, M; Martini, C

    2007-01-01

    The gamma-aminobutyric acid type A (GABA(A)) receptors are the major inhibitory neuronal receptors in the mammalian brain. Their activation by GABA opens the intrinsic ion channel, enabling chloride flux into the cell with subsequent hyperpolarization. Several GABA(A) receptor subunit isoforms have been cloned, the major isoform containing alpha, beta, and gamma subunits, and a regional heterogeneity associated with distinct physiological effects has been suggested. As a variety of allosteric ligands can modulate GABA-gated conductance changes through binding to distinct sites, the development of subtype-selective ligands may lead to the selective treatment of GABA system-associated pathology. In particular, the best characterized binding site is the benzodiazepine site (BzR), localized at the alpha/gamma subunit interface, in which the alpha subunit is the main determinant of BzR ligand action selectivity. The alpha1-containing BzR have been proposed to be responsible for the sedative action; the alpha2 and/or the alpha3 subtypes have been suggested to mediate the anxiolytic activity and the myorelaxation effects, and the alpha5 subtype has been associated with cognition processes. The discovery of alpha-selective subtype ligands may help in the specific treatment of anxiety, sleep disorders, convulsions and memory deficits with fewer side effects. Selectivity may be achieved by two approaches: selective affinity or selective efficacy. Selective affinity needs a compound to bind with a higher affinity to one receptor subtype compared with another, whereas subtype-selective efficacy relies on a compound binding to all subtypes, but having different efficacies at various subtypes. The status of BzR ligands, subdivided on the basis of their main chemical structural features, is reviewed in relation to structure-activity relationships which determine their affinity or efficacy selectivity for a certain BzR subtype.

  13. Expression and function of ryanodine receptor related pathways in PCB tolerant Atlantic killifish (Fundulus heteroclitus) from New Bedford Harbor, MA, USA.

    PubMed

    Fritsch, Erika B; Stegeman, John J; Goldstone, Jared V; Nacci, Diane E; Champlin, Denise; Jayaraman, Saro; Connon, Richard E; Pessah, Isaac N

    2015-02-01

    Atlantic killifish (Fundulus heteroclitus) thrive in New Bedford Harbor (NBH), MA, highly contaminated with polychlorinated biphenyls (PCBs). Resident killifish have evolved tolerance to dioxin-like (DL) PCBs, whose toxic effects through the aryl hydrocarbon receptor (AhR) are well studied. In NBH, non-dioxin like PCBs (NDL PCBs), which lack activity toward the AhR, vastly exceed levels of DL congeners yet how killifish counter NDL toxic effects has not been explored. In mammals and fish, NDL PCBs are potent activators of ryanodine receptors (RyR), Ca(2+) release channels necessary for a vast array of physiological processes. In the current study we compared the expression and function of RyR related pathways in NBH killifish with killifish from the reference site at Scorton Creek (SC, MA). Relative to the SC fish, adults from NBH displayed increased levels of skeletal muscle RyR1 protein, and increased levels of FK506-binding protein 12 kDa (FKBP12) an accessory protein essential for NDL PCB-triggered changes in RyR channel function. In accordance with increased RyR1 levels, NBH killifish displayed increased maximal ligand binding, increased maximal response to Ca(2+) activation and increased maximal response to activation by the NDL PCB congener PCB 95. Compared to SC, NBH embryos and larvae had increased levels of mtor and ryr2 transcripts at multiple stages of development, and generations, while levels of serca2 were decreased at 9 days post-fertilization in the F1 and F2 generations. These findings suggest that there are compensatory and heritable changes in RyR mediated Ca(2+) signaling proteins or potential signaling partners in NBH killifish.

  14. Juxtaglomerular cell CaSR stimulation decreases renin release via activation of the PLC/IP(3) pathway and the ryanodine receptor.

    PubMed

    Ortiz-Capisano, M Cecilia; Reddy, Mahendranath; Mendez, Mariela; Garvin, Jeffrey L; Beierwaltes, William H

    2013-02-01

    The calcium-sensing receptor (CaSR) is a G-coupled protein expressed in renal juxtaglomerular (JG) cells. Its activation stimulates calcium-mediated decreases in cAMP content and inhibits renin release. The postreceptor pathway for the CaSR in JG cells is unknown. In parathyroids, CaSR acts through G(q) and/or G(i). Activation of G(q) stimulates phospholipase C (PLC), and inositol 1,4,5-trisphosphate (IP(3)), releasing calcium from intracellular stores. G(i) stimulation inhibits cAMP formation. In afferent arterioles, the ryanodine receptor (RyR) enhances release of stored calcium. We hypothesized JG cell CaSR activation inhibits renin via the PLC/IP(3) and also RyR activation, increasing intracellular calcium, suppressing cAMP formation, and inhibiting renin release. Renin release from primary cultures of isolated mouse JG cells (n = 10) was measured. The CaSR agonist cinacalcet decreased renin release 56 ± 7% of control (P < 0.001), while the PLC inhibitor U73122 reversed cinacalcet inhibition of renin (104 ± 11% of control). The IP(3) inhibitor 2-APB also reversed inhibition of renin from 56 ± 6 to 104 ± 11% of control (P < 0.001). JG cells were positively labeled for RyR, and blocking RyR reversed CaSR-mediated inhibition of renin from 61 ± 8 to 118 ± 22% of control (P < 0.01). Combining inhibition of IP(3) and RyR was not additive. G(i) inhibition with pertussis toxin plus cinacalcet did not reverse renin inhibition (65 ± 12 to 41 ± 8% of control, P < 0.001). We conclude stimulating JG cell CaSR activates G(q), initiating the PLC/IP(3) pathway, activating RyR, increasing intracellular calcium, and resulting in calcium-mediated renin inhibition.

  15. Expression and function of ryanodine receptor related pathways in PCB tolerant Atlantic killifish (Fundulus heteroclitus) from New Bedford Harbor, MA, USA

    PubMed Central

    Fritsch, Erika B.; Stegeman, John J.; Goldstone, Jared V.; Nacci, Diane E.; Champlin, Denise; Jayaraman, Saro; Connon, Richard E.; Pessah, Isaac N.

    2015-01-01

    Atlantic killifish (Fundulus heteroclitus) thrive in New Bedford Harbor (NBH), MA, highly contaminated with polychlorinated biphenyls (PCBs). Resident killifish have evolved tolerance to dioxin-like (DL) PCBs, whose toxic effects through the aryl hydrocarbon receptor (AhR) are well studied. In NBH, non-dioxin like PCBs (NDL PCBs), which lack activity toward the AhR, vastly exceed levels of DL congeners yet how killifish counter NDL toxic effects has not been explored. In mammals and fish, NDL PCBs are potent activators of ryanodine receptors (RyR), Ca2+ release channels necessary for a vast array of physiological processes. In the current study we compared the expression and function of RyR related pathways in NBH killifish with killifish from the reference site at Scorton Creek (SC, MA). Relative to the SC fish, adults from NBH displayed increased levels of skeletal muscle RyR1 protein, and increased levels of FK506-binding protein 12 kDa (FKBP12), an accessory protein essential for NDL PCB-triggered changes in RyR channel function. In accordance with increased RyR1 levels, NBH killifish displayed increased maximal ligand binding, increased maximal response to Ca2+ activation and increased maximal response to activation by the NDL PCB congener PCB 95. Compared to SC, NBH embryos and larvae had increased levels of mtor and ryr2 transcripts at multiple stages of development, and generations, while levels of serca2 were decreased at 9 days post-fertilization in the F1 and F2 generations. These findings suggest that there are compensatory and heritable changes in RyR mediated Ca2+ signaling proteins or potential signaling partners in NBH killifish. PMID:25546006

  16. Angiotensin receptor subtype mediated physiologies and behaviors: New discoveries and clinical targets

    PubMed Central

    Wright, John W.; Yamamoto, Brent J.; Harding, Joseph W.

    2008-01-01

    The renin–angiotensin system (RAS) mediates several classic physiologies including body water and electrolyte homeostasis, blood pressure, cyclicity of reproductive hormones and sexual behaviors, and the regulation of pituitary gland hormones. These functions appear to be mediated by the angiotensin II (AngII)/AT1 receptor subtype system. More recently, the angiotensin IV (AngIV)/AT4 receptor subtype system has been implicated in cognitive processing, cerebroprotection, local blood flow, stress, anxiety and depression. There is accumulating evidence to suggest an inhibitory influence by AngII acting at the AT1 subtype, and a facilitory role by AngIV acting at the AT4 subtype, on neuronal firing rate, long-term potentiation, associative and spatial learning, and memory. This review initially describes the biochemical pathways that permit synthesis and degradation of active angiotensin peptides and three receptor subtypes (AT1, AT2 and AT4) thus far characterized. There is vigorous debate concerning the identity of the most recently discovered receptor subtype, AT4. Descriptions of classic and novel physiologies and behaviors controlled by the RAS are presented. This review concludes with a consideration of the emerging therapeutic applications suggested by these newly discovered functions of the RAS. PMID:18160199

  17. Positive somatostatin receptor scintigraphy correlates with the presence of somatostatin receptor subtype 2.

    PubMed Central

    John, M; Meyerhof, W; Richter, D; Waser, B; Schaer, J C; Scherübl, H; Boese-Landgraf, J; Neuhaus, P; Ziske, C; Mölling, K; Riecken, E O; Reubi, J C; Wiedenmann, B

    1996-01-01

    Somatostatin receptor scintigraphy (SRS) is positive in approximately 75% of all patients with neuroendocrine gastroenteropancreatic tumours. This study aimed to identify specific somatostatin receptor (sstr) subtypes, which are responsible for the in vivo binding of the widely used somatostatin analogue, octreotide in human neuroendocrine gastroenteropancreatic tumours. Twelve patients underwent SRS with radiolabelled octreotide. After surgical resection, tumour tissues were analysed in vitro for somatostatin and octreotide binding sites by autoradiography. In addition, for the first time, sstr subtype mRNA expression was examined by semiquantitative reverse transcription polymerase chain reaction (RT-PCR). Tumour tissues from all SRS positive patients were positive by autoradiography. Semiquantitative RT-PCR revealed most prominently sstr2 expression in scintigraphically positive tumours. Two SRS negative tumours contained in vitro octreotide binding sites as well as high levels of sstr1 and sstr2 mRNAs. Positive SRS is mainly due to sstr2. sstr1, 3, 4, and probably 5 are less important for in vivo octreotide binding. False negative scintigraphic results seem to be influenced by factors independent of the expression of specific sstr. Images Figure 4 PMID:8566856

  18. Decreased inward rectifying K+ current and increased ryanodine receptor sensitivity synergistically contribute to sustained focal arrhythmia in the intact rabbit heart

    PubMed Central

    Myles, Rachel C; Wang, Lianguo; Bers, Donald M; Ripplinger, Crystal M

    2015-01-01

    Heart failure (HF) results in dramatic electrophysiological remodelling, including increased sensitivity of ryanodine receptors (RyRs) and decreased inward rectifying K+ current (IK1), which predisposes HF myocytes to delayed afterdepolarizations and triggered activity. Therefore, we sought to determine the role of increased RyR sensitivity and decreased IK1 in contributing to focal arrhythmia in the intact non-failing heart. Optical mapping of transmembrane potential and intracellular Ca2+ was performed in Langendorff-perfused rabbit hearts (n = 15). Local β-adrenergic receptor stimulation with noradrenaline (norepinephrine; NA, 50 μl, 250 μm) was applied to elicit focal activity (premature ventricular complexes (PVCs) or ventricular tachycardia (VT ≥ 3 beats)). NA was administered under control conditions (CTL) and following pretreatment with 50 μm BaCl2 to reduce IK1, or 200 μm caffeine (Caff) to sensitize RyRs, both alone and in combination. Local NA injection resulted in Ca2+-driven PVCs arising from the injection site in all hearts studied. No increase in NA-mediated PVCs was observed following pretreatment with either BaCl2 or Caff alone (CTL: 1.1 ± 0.7, BaCl2: 1.0 ± 0.7, Caff: 1.3 ± 0.8 PVCs/injection, P not significant). However, pretreatment with the combination of BaCl2 + Caff resulted in a significant increase in PVCs (2.3 ± 2.8 PVCs/injection, P < 0.05 vs. CTL, BaCl2, Caff). Additionally, pretreatment with BaCl2 + Caff led to sustained monomorphic VT arising from the NA application site in all hearts studied, which lasted up to 6 min following a single NA injection. VT was never observed under any other condition suggesting synergism between increased RyR sensitivity and decreased IK1 in contributing to focal activity. These findings may have important implications for the understanding and prevention of focal arrhythmia in HF. Key points Heart failure leads to dramatic electrophysiological remodelling as a result of

  19. Muscarinic receptor-stimulated phosphatidylinositol turnover in the rat corpus striatum: role of muscarinic receptor subtypes and regulation

    SciTech Connect

    Monsma, F.J.

    1987-01-01

    The coupling between the M1 and M2 muscarinic receptor subtypes and phosphatidylinositol (Pl) hydrolysis has been examined in the corpus striatum and cerebral cortex of the rat brain. Receptor binding by the various muscarinic ligands was assessed using a preparation of intact brain cell aggregates, under similar conditions as the assay of Pl hydrolysis. In striatal cell aggregates, (/sup 3/H)-quinuclidinyl benzilate ((/sup 3/H)-QNB) bound to a single class of muscarinic sites with high affinity, inhibition of (/sup 3/H)-QNB binding by muscarinic receptor ligands which exhibit selectivity for subtypes of the muscarinic receptor revealed the presence of both the M1 and M2 subtypes in approximately equal numbers.

  20. Caffeine-mediated inhibition of calcium release channel inositol 1,4,5-trisphosphate receptor subtype 3 blocks glioblastoma invasion and extends survival.

    PubMed

    Kang, Sang Soo; Han, Kyung-Seok; Ku, Bo Mi; Lee, Yeon Kyung; Hong, Jinpyo; Shin, Hye Young; Almonte, Antoine G; Woo, Dong Ho; Brat, Daniel J; Hwang, Eun Mi; Yoo, Seung Hyun; Chung, Chun Kee; Park, Sung-Hye; Paek, Sun Ha; Roh, Eun Joo; Lee, Sung Joong; Park, Jae-Yong; Traynelis, Stephen F; Lee, C Justin

    2010-02-01

    Calcium signaling is important in many signaling processes in cancer cell proliferation and motility including in deadly glioblastomas of the brain that aggressively invade neighboring tissue. We hypothesized that disturbing Ca(2+) signaling pathways might decrease the invasive behavior of giloblastoma, extending survival. Evaluating a panel of small-molecule modulators of Ca(2+) signaling, we identified caffeine as an inhibitor of glioblastoma cell motility. Caffeine, which is known to activate ryanodine receptors, paradoxically inhibits Ca(2+) increase by inositol 1,4,5-trisphospate receptor subtype 3 (IP(3)R3), the expression of which is increased in glioblastoma cells. Consequently, by inhibiting IP(3)R3-mediated Ca(2+) release, caffeine inhibited migration of glioblastoma cells in various in vitro assays. Consistent with these effects, caffeine greatly increased mean survival in a mouse xenograft model of glioblastoma. These findings suggest IP(3)R3 as a novel therapeutic target and identify caffeine as a possible adjunct therapy to slow invasive growth of glioblastoma.

  1. Cloning of two adenosine receptor subtypes from mouse bone marrow-derived mast cells.

    PubMed

    Marquardt, D L; Walker, L L; Heinemann, S

    1994-05-01

    Adenosine potentiates the stimulated release of mast cell mediators. Pharmacologic studies suggest the presence of two adenosine receptors, one positively coupled to adenylate cyclase and the other coupled to phospholipase C activation. To identify mast cell adenosine receptor subtypes, cDNAs for the A1 and A2a adenosine receptors were obtained by screening a mouse brain cDNA library with the use of PCR-derived probes. Mouse bone marrow-derived mast cell cDNA libraries were constructed and screened with the use of A1 and A2a cDNA probes, which revealed the presence of A2a, but not A1, receptor clones. A putative A2b receptor was identified by using low stringency mast cell library screening. Northern blotting of mast cell poly(A)+ RNA with the use of receptor subtype probes labeled single mRNA bands of 2.4 kb and 1.8 kb for the A2a and A2b receptors, respectively. In situ cells. An A2a receptor-specific agonist failed to enhance mast cell mediator release, which suggests that the secretory process is modulated through the A2b and/or another receptor subtype. By using RNase protection assays, we found that mast cells that had been cultured in the presence of N-ethylcarboxamidoadenosine for 24 h exhibited a decrease in both A2a and A2b receptor RNA levels. Cells that had been cultured for 1 to 2 days in the presence of dexamethasone demonstrated increased amounts of A2a receptor mRNA, but no identifiable change in A2b receptor mRNA. Mast cells possess at least two adenosine receptor subtypes that may be differentially regulated.

  2. Prognosis of metastatic breast cancer subtypes: the hormone receptor/HER2-positive subtype is associated with the most favorable outcome.

    PubMed

    Lobbezoo, Dorien J A; van Kampen, Roel J W; Voogd, Adri C; Dercksen, M Wouter; van den Berkmortel, Franchette; Smilde, Tineke J; van de Wouw, Agnes J; Peters, Frank P J; van Riel, Johanna M G H; Peters, Natascha A J B; de Boer, Maaike; Borm, George F; Tjan-Heijnen, Vivianne C G

    2013-10-01

    Contrary to the situation in early breast cancer, little is known about the prognostic relevance of the hormone receptor (HR) and human epidermal growth factor receptor 2 (HER2) in metastatic breast cancer. The objectives of this study were to present survival estimates and to determine the prognostic impact of breast cancer subtypes based on HR and HER2 status in a recent cohort of metastatic breast cancer patients, which is representative of current clinical practice. Patients diagnosed with metastatic breast cancer between 2007 and 2009 were included. Information regarding patient and tumor characteristics and treatment was collected. Patients were categorized in four subtypes based on the HR and HER2 status of the primary tumor: HR positive (+)/HER2 negative (-), HR+/HER2+, HR-/HER2+ and triple negative (TN). Survival was estimated using the Kaplan-Meier method. Cox proportional hazards model was used to determine the prognostic impact of breast cancer subtype, adjusted for possible confounders. Median follow-up was 21.8 months for the 815 metastatic breast cancer patients included; 66 % of patients had the HR+/HER2- subtype, 8 % the HR-/HER2+ subtype, 15 % the TN subtype and 11 % the HR+/HER2+ subtype. The longest survival was observed for the HR+/HER2+ subtype (median 34.4 months), compared to 24.8 months for the HR+/HER2- subtype, 19.8 months for the HR-/HER2+ subtype and 8.8 months for the TN subtype (P < 0.0001). In the multivariate analysis, subtype was an independent prognostic factor, as were initial site of metastases and metastatic-free interval. The HR+/HER2+ subtype was associated with the longest survival after diagnosis of distant metastases.

  3. Overlap of dopaminergic, adrenergic, and serotoninergic receptors and complementarity of their subtypes in primate prefrontal cortex

    SciTech Connect

    Goldman-Rakic, P.S.; Lidow, M.S.; Gallager, D.W. )

    1990-07-01

    Quantitative in vitro autoradiography was used to determine and compare the areal and laminar distribution of the major dopaminergic, adrenergic, and serotonergic neurotransmitter receptors in 4 cytoarchitectonic regions of the prefrontal cortex in adult rhesus monkeys. The selective ligands, 3H-SCH-23390, 3H-raclopride, 3H-prazosin, and 3H-clonidine were used to label the D1 and D2 dopamine receptor subtypes and the alpha 1- and alpha 2-adrenergic receptors, respectively, while 125I-iodopindolol was used to detect beta-adrenergic receptors. The radioligands, 3H-5-hydroxytryptamine and 3H-ketanserin labeled, respectively, the 5-HT1 and 5-HT2 receptors. Densitometry was performed on all cortical layers and sublayers for each of the 7 ligands to allow quantitative as well as qualitative comparison among them in each cytoarchitectonic area. Although each monoamine receptor was distributed in a distinctive laminar-specific pattern that was remarkably similar from area to area, there was considerable overlap among the dopaminergic, adrenergic, and serotoninergic receptors, while subtypes of the same receptor class tended to have complementary laminar profiles and different concentrations. Thus, the D1 dopamine, the alpha 1- and alpha 2-adrenergic, and the 5-HT1 receptors were present in highest relative concentration in superficial layers I, II, and IIIa (the S group). In contrast, the beta 1- and beta 2-adrenergic subtypes and the 5-HT2 receptor had their highest concentrations in the intermediate layers, IIIb and IV (the I group), while the D2 receptor was distinguished by relatively high concentrations in the deep layer V compared to all other layers (the D class). Thus, clear laminar differences were observed in the D1 vs D2 dopaminergic, the alpha- vs beta-adrenergic, and the 5-HT1 vs 5-HT2 serotoninergic receptor subtypes in all 4 areas examined.

  4. Contribution of impaired myofibril and ryanodine receptor function to prolonged low-frequency force depression after in situ stimulation in rat skeletal muscle.

    PubMed

    Watanabe, Daiki; Kanzaki, Keita; Kuratani, Mai; Matsunaga, Satoshi; Yanaka, Noriyuki; Wada, Masanobu

    2015-06-01

    The aim of this study was to examine whether prolonged low-frequency force depression (PLFFD) that occurs in situ is the result of decreased myofibrillar Ca(2+) sensitivity and/or reduced sarcoplasmic reticulum (SR) Ca(2+) release. Intact rat gastrocnemius muscles were electrically stimulated via the sciatic nerve until force was reduced to ~50% of the initial and dissected 30 min following the cessation of stimulation. Skinned fibre and whole muscle analyses were performed in the superficial region composed exclusively of type IIB fibres. Fatiguing stimulation significantly reduced the ratio of force at low frequency to that at high frequency to 65% in skinned fibres (1 vs. 50 Hz) and 73% in whole muscles (20 vs. 100 Hz). In order to evaluate changes in myofibrillar Ca(2+) sensitivity and ryanodine receptor caffeine sensitivity, skinned fibres were activated in Ca(2+)- and caffeine-containing solutions, respectively. Skinned fibres from fatigued muscles displayed decreased caffeine sensitivity together with increased myofibrillar Ca(2+) sensitivity. Treatment with 2,2'-dithiodipyridine and reduced glutathione induced a smaller increase in myofibrillar Ca(2+)sensitivity in fatigued than in rested fibres. In fatigued muscles, S-glutathionylation of troponin I was increased and submaximal SR Ca(2+) release, induced by 4-chloro-m-cresol, was decreased. These findings suggest that in the early stage of PLFFD that occurs in fast-twitch muscles of exercising animals and humans, S-glutathionylation of troponin I may attenuate PLFFD by increasing myofibrillar Ca(2+) sensitivity and that under such a circumstance, PLFFD may be ascribable to failure of SR Ca(2+) release.

  5. The I4895T Mutation in the Type 1 Ryanodine Receptor Induces Fiber-Type Specific Alterations in Skeletal Muscle that Mimic Premature Aging

    PubMed Central

    Boncompagni, Simona; Loy, Ryan E.; Dirksen, Robert T.; Franzini-Armstrong, Clara

    2010-01-01

    SUMMARY The I4898T (IT) mutation in type 1 ryanodine receptor (RyR1), the Ca2+ release channel of the sarcoplasmic reticulum (SR) is linked to a form of central core disease (CCD) in humans and results in a non leaky channel and excitation-contraction uncoupling. We characterized age- and fiber type-dependent alterations in muscle ultrastructure, as well as the magnitude and spatiotemporal properties of evoked Ca2+ release in heterozygous Ryr1I4895T/WT (IT/+) knock-in mice on a mixed genetic background. The results indicate a classical but mild CCD phenotype that includes muscle weakness and the presence of mitochondrial-deficient areas in type I fibers. Electrically-evoked Ca2+ release is significantly reduced in single FDB fibers from young and old IT/+ mice. Structural changes are strongly fiber type-specific, affecting type I and IIB/IIX fibers in very distinct ways, and sparing type IIA fibers. Ultrastructural alterations in our IT/+ mice are also present in wild type, but at a lower frequency and older ages, suggesting that the disease mutation on the mixed background promotes an acceleration of normal age-dependent changes. The observed functional and structural alterations and their similarity to age-associated changes are entirely consistent with the known properties of the mutated channel, which result in reduced calcium release as is also observed in normal aging muscle. In strong contrast to these observations, a subset of patients with the analogous human heterozygous mutation and IT/+ mice on an inbred 129S2/SvPasCrl background exhibit a more severe disease phenotype, which is not directly consistent with the mutated channel properties. PMID:20961389

  6. Polydatin protects cardiac function against burn injury by inhibiting sarcoplasmic reticulum Ca2+ leak by reducing oxidative modification of ryanodine receptors.

    PubMed

    Jiang, Xin; Liu, Wenjuan; Deng, Jianxin; Lan, Liqin; Xue, Xiang; Zhang, Cuicui; Cai, Gaorui; Luo, Xinping; Liu, Jie

    2013-07-01

    Our recent studies demonstrate that burn trauma induces leaky sarcoplasmic reticulum (SR) in heart due to excessively active ryanodine receptor (RyR) function. SR Ca(2+) leak causes partial depletion of SR Ca(2+) content and disturbances in intracellular Ca(2+) homeostasis, resulting in the pathogenesis of burn-generated cardiac dysfunction. This study investigated the role of polydatin, a resveratrol glucoside, in preventing SR leak and its therapeutic effect against burn-generated cardiac dysfunction. We found that polydatin treatment improved cardiac function impaired by burn injury of 30% of total body surface area. Parallel to the alterations in cardiac function, polydatin significantly increased the defective systolic Ca(2+) transient and contractility in burn-traumatized cardiomyocytes. Burn injury increased the occurrence of Ca(2+) sparks. The enhancement of Ca(2+) spark-mediated SR leak caused partial depletion of SR Ca(2+) content in burn-traumatized cardiomyocytes. Furthermore, we found that the content of free thiols (the number of reduced cysteines) in RyR2 in cardiomyocytes determined by the monobromobimane fluorescence of RyR2 was decreased markedly in burn-traumatized hearts. Polydatin treatment decreased intracellular reactive oxygen species levels and restored the amount of free thiols in RyR2 in burns. Concomitantly, polydatin corrected Ca(2+) spark-mediated SR leak and restored SR Ca(2+) load. The systolic Ca(2+) transient and cellular contractility were significantly increased by polydatin treatment. Taken together, the present findings provide the first evidence demonstrating that polydatin prevents enhanced Ca(2+) spark-mediated SR leak by reducing oxidative stress in RyR2 in burn-traumatized heart, leading to protection of cardiac function against burn injury. Copyright © 2013 Elsevier Inc. All rights reserved.

  7. Reduced aerobic capacity causes leaky ryanodine receptors that trigger arrhythmia in a rat strain artificially selected and bred for low aerobic running capacity

    PubMed Central

    Høydal, MA; Stølen, TO; Johnsen, AB; Alvez, M; Catalucci, D; Condorelli, G; Koch, LG; Britton, SL; Smith, GL; Wisløff, U

    2014-01-01

    Aim Rats selectively bred for inborn Low Capacity of Running (LCR) display a series of poor health indices where as rats selected for High Capacity of Running (HCR) display a healthy profile. We hypothesized that selection of low aerobic capacity over generations leads to a phenotype with increased diastolic Ca2+ leak that trigger arrhythmia. Methods We used rats selected for HCR (N=10) or LCR (N=10) to determine the effect of inborn aerobic capacity on Ca2+ leak and susceptibility of ventricular arrhythmia. We studied isolated FURA2/AM loaded cardiomyocytes to detect Ca2+-handling and function on an inverted epi-fluorescence microscope. To determine arrhythmogenicity we did a final experiment with electrical burst pacing in Langendorff perfused hearts. Results Ca2+-handling was impaired by reduced Ca2+ amplitude, prolonged time to 50% Ca2+ decay, and reduced sarcoplasmic reticulum (SR) Ca2+-content. Impaired Ca2+ removal was influenced by reduced SR Ca2+ ATP-ase 2a (SERCA2a) function and increased sodium/Ca2+-exchanger (NCX) in LCR rats. Diastolic Ca2 leak was 87% higher in LCR rats. The leak was reduced by CaMKII inhibition. Expression levels of phosphorylated theorine-286 CaMKII levels and increased RyR2 phosphorylation at the Serine-2814 site mechanistically support our findings of increased leak in LCR. LCR rats had significantly higher incidence of ventricular fibrillation. Conclusion Selection of inborn low aerobic capacity over generations leads to a phenotype with increased risk of ventricular fibrillation. Increased phosphorylation of CaMKII at serine-2814 at the cardiac ryanodine receptor appears as an important mechanism of impaired Ca2+ handling and diastolic Ca2+ leak that results in increased susceptibility to ventricular fibrillation. PMID:24444142

  8. Leaky ryanodine receptors delay the activation of store overload-induced Ca2+ release, a mechanism underlying malignant hyperthermia-like events in dystrophic muscle.

    PubMed

    Cully, Tanya R; Launikonis, Bradley S

    2016-04-15

    The mouse model of Duchenne muscular dystrophy, the mdx mouse, displays changes in Ca(2+)homeostasis that may lead to the pathology of the muscle. Here we examine the activation of store overload-induced Ca(2+)release (SOICR) in mdx muscle. The activation of SOICR is associated with the depolymerization of the sarcoplasmic reticulum (SR) Ca(2+)buffer calsequestrin and the reduction of SR Ca(2+)buffering power (BSR). The role of SOICR in healthy and dystrophic muscle is unclear. Using skinned fibers we show that lowering the Mg(2+)concentration can activate discrete Ca(2+)release events that did not necessarily lead to activation of SOICR. However, SOICR waves could propagate into these fiber segments. The average delay to activation of SOICR in mdx fibers was longer than in wild-type (WT) fibers. In the lowered Ca(2+)-buffered environment following large SOICR events, brief waves in mdx fibers displayed a low amplitude and propagation rate, in contrast to WT fibers that showed a range of amplitudes correlated with wave propagation rate. The distinct properties of SOICR in mdx fibers were consistent with a ryanodine receptor (RyR) that was leakier to Ca(2+)than in WT. The consequence of delayed SOICR and leaky RyRs is prolonged high BSRand a reduction in free Ca(2+)concentration inside the SR as total SR calcium drops. We present a hypothesis that SOICR activation is required in healthy muscle and that this mechanism works suboptimally in mdx fibers to fail to limit the activation of store-operated Ca(2+)entry. Copyright © 2016 the American Physiological Society.

  9. Ryanodine Receptor Phosphorylation by CaMKII Promotes Spontaneous Ca2+ Release Events in a Rodent Model of Early Stage Diabetes: the Arrhythmogenic Substrate

    PubMed Central

    Sommese, Leandro; Valverde, Carlos A; Blanco, Paula; Castro, María Cecilia; Rueda, Omar Velez; Kaetzel, Marcia; Dedman, John; Anderson, Mark E.; Mattiazzi, Alicia; Palomeque, Julieta

    2016-01-01

    Background Heart failure and arrhythmias occur more frequently in patients with type 2 diabetes (T2DM) than in the general population. T2DM is preceded by a prediabetic condition marked by elevated reactive oxygen species (ROS) and subclinical cardiovascular defects. Although multifunctional Ca2+ calmodulin-dependent protein kinase II (CaMKII) is ROS-activated and CaMKII hyperactivity promotes cardiac diseases, a link between prediabetes and CaMKII in the heart is unprecedented. Objectives to prove the hypothesis that increased ROS and CaMKII activity contribute to heart failure and arrhythmogenic mechanisms in early stage diabetes. Methods-Results Echocardiography, electrocardiography, biochemical and intracellular Ca2+ (Ca2+i) determinations were performed in fructose-rich diet -induced impaired glucose tolerance, a prediabetes model, in rodents. Fructose-rich diet rats showed decreased contractility and hypertrophy associated with increased CaMKII activity, ROS production, oxidized CaMKII and enhanced CaMKII-dependent ryanodine receptor (RyR2) phosphorylation compared to rats fed with control diet. Isolated cardiomyocytes from fructose-rich diet showed increased spontaneous Ca2+i release events associated with spontaneous contractions, which were prevented by KN-93, a CaMKII inhibitor, or addition of Tempol, a ROS scavenger, to the diet. Moreover, fructose-rich diet myocytes showed increased diastolic Ca2+ during the burst of spontaneous Ca2+i release events. Micetreated with Tempol or with sarcoplasmic reticulum-targeted CaMKII-inhibition by transgenic expression of the CaMKII inhibitory peptide AIP, were protected from fructose-rich diet-induced spontaneous Ca2+i release events, spontaneous contractions and arrhythmogenes is in vivo, despite ROS increases. Conclusions RyR2 phosphorylation by ROS-activated CaMKII, contributes to impaired glucose tolerance-induced arrhythmogenic mechanisms, suggesting that CaMKII inhibition could prevent prediabetic

  10. Stabilization of the skeletal muscle ryanodine receptor ion channel-FKBP12 complex by the 1,4-benzothiazepine derivative S107.

    PubMed

    Mei, Yingwu; Xu, Le; Kramer, Henning F; Tomberlin, Ginger H; Townsend, Claire; Meissner, Gerhard

    2013-01-01

    Activation of the skeletal muscle ryanodine receptor (RyR1) complex results in the rapid release of Ca(2+) from the sarcoplasmic reticulum and muscle contraction. Dissociation of the small FK506 binding protein 12 subunit (FKBP12) increases RyR1 activity and impairs muscle function. The 1,4-benzothiazepine derivative JTV519, and the more specific derivative S107 (2,3,4,5,-tetrahydro-7-methoxy-4-methyl-1,4-benzothiazepine), are thought to improve skeletal muscle function by stabilizing the RyR1-FKBP12 complex. Here, we report a high degree of nonspecific and specific low affinity [(3)H]S107 binding to SR vesicles. SR vesicles enriched in RyR1 bound ∼48 [(3)H]S107 per RyR1 tetramer with EC(50) ∼52 µM and Hillslope ∼2. The effects of S107 and FKBP12 on RyR1 were examined under conditions that altered the redox state of RyR1. S107 increased FKBP12 binding to RyR1 in SR vesicles in the presence of reduced glutathione and the NO-donor NOC12, with no effect in the presence of oxidized glutathione. Addition of 0.15 µM FKBP12 to SR vesicles prevented FKBP12 dissociation; however, in the presence of oxidized glutathione and NOC12, FKBP12 dissociation was observed in skeletal muscle homogenates that contained 0.43 µM myoplasmic FKBP12 and was attenuated by S107. In single channel measurements with FKBP12-depleted RyR1s, in the absence and presence of NOC12, S107 augmented the FKBP12-mediated decrease in channel activity. The data suggest that S107 can reverse the harmful effects of redox active species on SR Ca(2+) release in skeletal muscle by binding to RyR1 low affinity sites.

  11. Overexpression of ryanodine receptor type 1 enhances mitochondrial fragmentation and Ca2+-induced ATP production in cardiac H9c2 myoblasts.

    PubMed

    O-Uchi, Jin; Jhun, Bong Sook; Hurst, Stephen; Bisetto, Sara; Gross, Polina; Chen, Ming; Kettlewell, Sarah; Park, Jongsun; Oyamada, Hideto; Smith, Godfrey L; Murayama, Takashi; Sheu, Shey-Shing

    2013-12-01

    Ca(+) influx to mitochondria is an important trigger for both mitochondrial dynamics and ATP generation in various cell types, including cardiac cells. Mitochondrial Ca(2+) influx is mainly mediated by the mitochondrial Ca(2+) uniporter (MCU). Growing evidence also indicates that mitochondrial Ca(2+) influx mechanisms are regulated not solely by MCU but also by multiple channels/transporters. We have previously reported that skeletal muscle-type ryanodine receptor (RyR) type 1 (RyR1), which expressed at the mitochondrial inner membrane, serves as an additional Ca(2+) uptake pathway in cardiomyocytes. However, it is still unclear which mitochondrial Ca(2+) influx mechanism is the dominant regulator of mitochondrial morphology/dynamics and energetics in cardiomyocytes. To investigate the role of mitochondrial RyR1 in the regulation of mitochondrial morphology/function in cardiac cells, RyR1 was transiently or stably overexpressed in cardiac H9c2 myoblasts. We found that overexpressed RyR1 was partially localized in mitochondria as observed using both immunoblots of mitochondrial fractionation and confocal microscopy, whereas RyR2, the main RyR isoform in the cardiac sarcoplasmic reticulum, did not show any expression at mitochondria. Interestingly, overexpression of RyR1 but not MCU or RyR2 resulted in mitochondrial fragmentation. These fragmented mitochondria showed bigger and sustained mitochondrial Ca(2+) transients compared with basal tubular mitochondria. In addition, RyR1-overexpressing cells had a higher mitochondrial ATP concentration under basal conditions and showed more ATP production in response to cytosolic Ca(2+) elevation compared with nontransfected cells as observed by a matrix-targeted ATP biosensor. These results indicate that RyR1 possesses a mitochondrial targeting/retention signal and modulates mitochondrial morphology and Ca(2+)-induced ATP production in cardiac H9c2 myoblasts.

  12. Calcium-dependent energetics of calmodulin domain interactions with regulatory regions of the Ryanodine Receptor Type 1 (RyR1).

    PubMed

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

    2014-01-01

    Calmodulin (CaM) allosterically regulates the homo-tetrameric human Ryanodine Receptor Type 1 (hRyR1): apo CaM activates the channel, while (Ca(2+))4-CaM inhibits it. CaM-binding RyR1 residues 1975-1999 and 3614-3643 were proposed to allow CaM to bridge adjacent RyR1 subunits. Fluorescence anisotropy titrations monitored the binding of CaM and its domains to peptides encompassing hRyR(11975-1999) or hRyR1(3614-3643). Both CaM and its C-domain associated in a calcium-independent manner with hRyR1(3614-3643) while N-domain required calcium and bound ~250-fold more weakly. Association with hRyR1(11975-1999) was weak. Both hRyR1 peptides increased the calcium-binding affinity of both CaM domains, while maintaining differences between them. These energetics support the CaM C-domain association with hRyR1(3614-3643) at low calcium, positioning CaM to respond to calcium efflux. However, the CaM N-domain affinity for hRyR(11975-1999) alone was insufficient to support CaM bridging adjacent RyR1 subunits. Other proteins or elements of the hRyR1 structure must contribute to the energetics of CaM-mediated regulation.

  13. Bidirectional coupling between ryanodine receptors and Ca2+ release-activated Ca2+ (CRAC) channel machinery sustains store-operated Ca2+ entry in human T lymphocytes.

    PubMed

    Thakur, Pratima; Dadsetan, Sepehr; Fomina, Alla F

    2012-10-26

    The expression and functional significance of ryanodine receptors (RyR) were investigated in resting and activated primary human T cells. RyR1, RyR2, and RyR3 transcripts were detected in human T cells. RyR1/2 transcript levels increased, whereas those of RyR3 decreased after T cell activation. RyR1/2 protein immunoreactivity was detected in activated but not in resting T cells. The RyR agonist caffeine evoked Ca(2+) release from the intracellular store in activated T cells but not in resting T cells, indicating that RyR are functionally up-regulated in activated T cells compared with resting T cells. In the presence of store-operated Ca(2+) entry (SOCE) via plasmalemmal Ca(2+) release-activated Ca(2+) (CRAC) channels, RyR blockers reduced the Ca(2+) leak from the endoplasmic reticulum (ER) and the magnitude of SOCE, suggesting that a positive feedback relationship exists between RyR and CRAC channels. Overexpression of fluorescently tagged RyR2 and stromal interaction molecule 1 (STIM1), an ER Ca(2+) sensor gating CRAC channels, in HEK293 cells revealed that RyR are co-localized with STIM1 in the puncta formed after store depletion. These data indicate that in primary human T cells, the RyR are coupled to CRAC channel machinery such that SOCE activates RyR via a Ca(2+)-induced Ca(2+) release mechanism, which in turn reduces the Ca(2+) concentration within the ER lumen in the vicinity of STIM1, thus facilitating SOCE by reducing store-dependent CRAC channel inactivation. Treatment with RyR blockers suppressed activated T cell expansion, demonstrating the functional importance of RyR in T cells.

  14. Ca(2+)-activated ion currents triggered by ryanodine receptor-mediated Ca(2+) release control firing of inhibitory neurons in the prepositus hypoglossi nucleus.

    PubMed

    Saito, Yasuhiko; Yanagawa, Yuchio

    2013-01-01

    Spontaneous miniature outward currents (SMOCs) are known to exist in smooth muscles and peripheral neurons, and evidence for the presence of SMOCs in central neurons has been accumulating. SMOCs in central neurons are induced through Ca(2+)-activated K(+) (K(Ca)) channels, which are activated through Ca(2+)-induced Ca(2+) release from the endoplasmic reticulum via ryanodine receptors (RyRs). Previously, we found that some neurons in the prepositus hypoglossi nucleus (PHN) showed spontaneous outward currents (SOCs). In the present study, we used whole cell recordings in slice preparations of the rat brain stem to investigate the following: 1) the ionic mechanisms of SOCs, 2) the types of neurons exhibiting frequent SOCs, and 3) the effect of Ca(2+)-activated conductance on neuronal firing. Pharmacological analyses revealed that SOCs were induced via the activation of small-conductance-type K(Ca) (SK) channels and RyRs, indicating that SOCs correspond to SMOCs. An analysis of the voltage responses to current pulses of the fluorescence-expressing inhibitory neurons of transgenic rats revealed that inhibitory neurons frequently exhibited SOCs. Abolition of SOCs via blockade of SK channels enhanced the frequency of spontaneous firing of inhibitory PHN neurons. However, abolition of SOCs via blockade of RyRs reduced the firing frequency and hyperpolarized the membrane potential. Similar reductions in firing frequency and hyperpolarization were also observed when Ca(2+)-activated nonselective cation (CAN) channels were blocked. These results suggest that, in inhibitory neurons in the PHN, Ca(2+) release via RyRs activates SK and CAN channels, and these channels regulate spontaneous firing in a complementary manner.

  15. Ryanodine receptor phosphorylation by CaMKII promotes spontaneous Ca(2+) release events in a rodent model of early stage diabetes: The arrhythmogenic substrate.

    PubMed

    Sommese, Leandro; Valverde, Carlos A; Blanco, Paula; Castro, María Cecilia; Rueda, Omar Velez; Kaetzel, Marcia; Dedman, John; Anderson, Mark E; Mattiazzi, Alicia; Palomeque, Julieta

    2016-01-01

    Heart failure and arrhythmias occur more frequently in patients with type 2 diabetes (T2DM) than in the general population. T2DM is preceded by a prediabetic condition marked by elevated reactive oxygen species (ROS) and subclinical cardiovascular defects. Although multifunctional Ca2+ calmodulin-dependent protein kinase II (CaMKII) is ROS-activated and CaMKII hyperactivity promotes cardiac diseases, a link between prediabetes and CaMKII in the heart is unprecedented. To prove the hypothesis that increased ROS and CaMKII activity contribute to heart failure and arrhythmogenic mechanisms in early stage diabetes. Echocardiography, electrocardiography, biochemical and intracellular Ca2+ (Ca2+i) determinations were performed in fructose-rich diet-induced impaired glucose tolerance, a prediabetes model, in rodents. Fructose-rich diet rats showed decreased contractility and hypertrophy associated with increased CaMKII activity, ROS production, oxidized CaMKII and enhanced CaMKII-dependent ryanodine receptor (RyR2) phosphorylation compared to rats fed with control diet. Isolated cardiomyocytes from fructose-rich diet showed increased spontaneous Ca2+i release events associated with spontaneous contractions, which were prevented by KN-93, a CaMKII inhibitor, or addition of Tempol, a ROS scavenger, to the diet. Moreover, fructose-rich diet myocytes showed increased diastolic Ca2+ during the burst of spontaneous Ca2+i release events. Mice treated with Tempol or with sarcoplasmic reticulum-targeted CaMKII-inhibition by transgenic expression of the CaMKII inhibitory peptide AIP, were protected from fructose-rich diet-induced spontaneous Ca2+i release events, spontaneous contractions and arrhythmogenesis in vivo, despite ROS increases. RyR2 phosphorylation by ROS-activated CaMKII, contributes to impaired glucose tolerance-induced arrhythmogenic mechanisms, suggesting that CaMKII inhibition could prevent prediabetic cardiovascular complications and/or evolution. Copyright

  16. AB283. SPR-10 Down-regulation of ryanodine receptor gene expression in murine urinary bladder smooth muscle following partial bladder outlet obstruction

    PubMed Central

    Boopathi, Ettickan; Javed, Elham; Addya, Shankar; Fortina, Paolo; Zderic, Stephen; Wein, Alan; Chacko, Samuel

    2016-01-01

    Objective Urinary bladder smooth muscle (UBSM) displays spontaneous action potentials and this potential is related to the phasic nature of spontaneous contractions in this tissue. The amplitude of a phasic contraction depends on the increase in Ca2+ entry caused by membrane depolarization. Ryanodine receptors (RyRs) in UBSM decreases the force production by decreasing the frequency of phasic contractions through interactions with large-conductance Ca2+-activated K+ (BK) and small-conductance Ca2+-activated K+ (SK) channels. Microarray and network analysis were employed to determine the changes in mRNA in 14-day obstructed murine bladders. We found that obstruction significantly down-regulated the RyRs in bladder smooth muscle (BSM). Methods Male C57Bl/6 mice were surgically obstructed and kept for 14 days. Sham-operated mice served as a control. Bladders were excised; urothelium scraped off with a scalpel, and the serosa was removed. BSM obtained from PBOO and sham control animals were used for microarray and western blotting Results Pathway-based analysis of these gene signatures showed significant number of under-expressed genes in obstructed bladder and they were mapped to proteins involved in calcium signaling. We focused our work on RyR protein expression in BSM. There was a four-fold reduction of RyR3 in BSM in 14-day obstructed groups as shown by microarray and immunoblotting compared to that of sham-operated animals. Conclusions These results confirm that the RyR gene expression is down-regulated in obstructed murine bladder smooth muscle. Funding Source(s) None

  17. CRISPR/Cas9 mediated G4946E substitution in the ryanodine receptor of Spodoptera exigua confers high levels of resistance to diamide insecticides.

    PubMed

    Zuo, Yayun; Wang, Hui; Xu, Yanjun; Huang, Jianlei; Wu, Shuwen; Wu, Yidong; Yang, Yihua

    2017-10-01

    Diamide insecticides selectively activate insect ryanodine receptors (RyRs), inducing uncontrolled release of calcium ions, and causing muscle contraction, paralysis and eventually death. The RyR(G4946E) substitution associated with diamide resistance has been identified in three lepidopteran pests, Plutella xylostella, Tuta absoluta and Chilo suppressalis. Recently, the T. absoluta RyR(G4946V) mutation was knocked into the model insect Drosophila melanogaster by CRISPR/Cas9 mediated genome editing and provided in vivo functional confirmation for its role in diamide resistance. In the present study, we successfully introduced the RyR(G4946E) mutation with CRISPR/Cas9 technology into a lepidopteran pest of global importance, Spodoptera exigua. The genome-edited strain (named 4946E) homozygous for the SeRyR(G4946E) mutation exhibited 223-, 336- and >1000-fold resistance to chlorantraniliprole, cyantraniliprole and flubendiamide, respectively when compared to the wild type strain (WHS) of S. exigua. Reciprocal crossing experiments revealed that the target-site resistance in strain 4946E underlies an autosomal and almost recessive mode of inheritance for anthranilic diamides, whereas it was completely recessive for flubendiamide. Our results not only provided in vivo functional validation of the RyR(G4946E) mutation in conferring high levels of resistance to diamide insecticides for the first time in a controlled genetic background of a lepidopteran pest, but also revealed slight differences on the level of resistance between anthranilic diamides (chlorantraniliprole and cyantraniliprole) and flubendiamide conferred by the SeRyR(G4946E) mutation. Copyright © 2017 Elsevier Ltd. All rights reserved.

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

    PubMed Central

    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

  19. PCB 136 atropselectively alters morphometric and functional parameters of neuronal connectivity in cultured rat hippocampal neurons via ryanodine receptor-dependent mechanisms.

    PubMed

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

    2014-04-01

    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 100 nM 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.

  20. Conformation-dependent stability of junctophilin 1 (JP1) and ryanodine receptor type 1 (RyR1) channel complex is mediated by their hyper-reactive thiols.

    PubMed

    Phimister, Andrew J; Lango, Jozsef; Lee, Eun Hui; Ernst-Russell, Michael A; Takeshima, Hiroshi; Ma, Jianjie; Allen, Paul D; Pessah, Isaac N

    2007-03-23

    Junctophilin 1 (JP1), a 72-kDa protein localized at the skeletal muscle triad, is essential for stabilizing the close apposition of T-tubule and sarcoplasmic reticulum membranes to form junctions. In this study we report that rapid and selective labeling of hyper-reactive thiols found in both JP1 and ryanodine receptor type 1 (RyR1) with 7-diethylamino-3-(4'-maleimidylphenyl)-4-methylcoumarin, a fluorescent thiol-reactive probe, proceeded 12-fold faster under conditions that minimize RyR1 gating (e.g. 10 mM Mg2+) compared with conditions that promote high channel activity (e.g. 100 microM Ca2+, 10 mM caffeine, 5 mM ATP). The reactivity of these thiol groups was very sensitive to oxidation by naphthoquinone, H2O2, NO, or O2, all known modulators of the RyR1 channel complex. Using preparative SDS-PAGE, in-gel tryptic digestion, high pressure liquid chromatography, and mass spectrometry-based peptide sequencing, we identified 7-diethylamino-3-(4'-maleimidylphenyl)-4-methylcoumarin-thioether adducts on three cysteine residues of JP1 (101, 402, and 627); the remaining five cysteines of JP1 were unlabeled. Co-immunoprecipitation experiments demonstrated a physical interaction between JP1 and RyR1 that, like thiol reactivity, was sensitive to RyR1 conformation and chemical status of the hyper-reactive cysteines of JP1 and RyR1. These findings support a model in which JP1 interacts with the RyR1 channel complex in a conformationally sensitive manner and may contribute integral redox-sensing properties through reactive sulfhydryl chemistry.

  1. Spinal Cord Injury Leads to Hyperoxidation and Nitrosylation of Skeletal Muscle Ryanodine Receptor-1 Associated with Upregulation of Nicotinamide Adenine Dinucleotide Phosphate Oxidase 4.

    PubMed

    Liu, Xin-Hua; Harlow, Lauren; Graham, Zachary A; Bauman, William A; Cardozo, Christopher

    2017-02-27

    Spinal cord injury (SCI) results in marked atrophy and dysfunction of skeletal muscle. There are currently no effective treatments for SCI-induced muscle atrophy or the dysfunction of the remaining muscle tissue. Nicotinamide adenine dinucleotide phosphate (NADPH) oxidase-4 (Nox4) produces reactive oxygen species (ROS) in sarcoplasmic reticulum (SR) and has been identified as an important O2 sensor in skeletal muscle. Ryanodine receptors (RyRs) are calcium (Ca(2+)) channels that are responsible for Ca(2+) release from SR. In skeletal muscle, type1 RyR (RyR1) is predominantly functional. RyR1 is regulated by multiple proteins, including calstabin1, which assures that they close appropriately once contraction has ceased. RyR1 function is also regulated by oxidation and redox-dependent cysteine nitrosylation. Excessive oxidation/nitrosylation of RyR1 is associated with dissociation of calstabin1 and reduced muscle force generation. However, whether Nox4 levels in skeletal muscle are elevated or whether RyR1 is oxidized or nitrosylated after SCI has not been determined. In this study, we examined Nox4 expression, oxidation/nitrolysation status, and association of calstabin1 with RyR1 in skeletal muscle derived from rats that were subjected to T4 complete transection (SCI), and observed elevated expression of Nox4 messenger RNA and protein in muscle after SCI associated with enhanced binding of Nox4 to RyR1, increased oxidation and nitrosylation of RyR1, and dissociation of calstabin1 from RyR1 in SCI rat muscle. Our data suggest that RyR1 dysfunction resulting from excessive oxidation/nitrosylation may contribute to reduced specific force after SCI and suggest that Nox4 may be the source of ROS responsible for increased oxidation and nitrosylation of RyR1.

  2. Recombinant Atrial Natriuretic Peptide Prevents Aberrant Ca2+ Leakage through the Ryanodine Receptor by Suppressing Mitochondrial Reactive Oxygen Species Production Induced by Isoproterenol in Failing Cardiomyocytes

    PubMed Central

    Susa, Takehisa; Nanno, Takuma; Ishiguchi, Hironori; Myoren, Takeki; Nishimura, Shigehiko; Kato, Takayoshi; Hino, Akihiro; Oda, Tetsuro; Okuda, Shinichi; Yamamoto, Takeshi; Yano, Masafumi

    2016-01-01

    Catecholamines induce intracellular reactive oxygen species (ROS), thus enhancing diastolic Ca2+ leakage through the ryanodine receptor during heart failure (HF). However, little is known regarding the effect of atrial natriuretic peptide (ANP) on ROS generation and Ca2+ handling in failing cardiomyocytes. The aim of the present study was to clarify the mechanism by which an exogenous ANP exerts cardioprotective effects during HF. Cardiomyocytes were isolated from the left ventricles of a canine tachycardia-induced HF model and sham-operated vehicle controls. The degree of mitochondrial oxidized DNA was evaluated by double immunohistochemical (IHC) staining using an anti-VDAC antibody for the mitochondria and an anti-8-hydroxy-2′-deoxyguanosine antibody for oxidized DNA. The effect of ANP on ROS was investigated using 2,7-dichlorofluorescin diacetate, diastolic Ca2+ sparks assessed by confocal microscopy using Fluo 4-AM, and the survival rate of myocytes after 48 h. The double IHC study revealed that isoproterenol (ISO) markedly increased oxidized DNA in the mitochondria in HF and that the ISO-induced DNA damage was markedly inhibited by the co-presence of ANP. ROS production and Ca2+ spark frequency (CaSF) were increased in HF compared to normal controls, and were further increased in the presence of ISO. Notably, ANP significantly suppressed both ISO-induced ROS and CaSF without changing sarcoplasmic reticulum Ca2+ content in HF (p<0.01, respectively). The survival rate after 48 h in HF was significantly decreased in the presence of ISO compared with baseline (p<0.01), whereas it was significantly improved by the co-presence of ANP (p<0.01). Together, our results suggest that ANP strongly suppresses ISO-induced mitochondrial ROS generation, which might correct aberrant diastolic Ca2+ sparks, eventually contributing to the improvement of cardiomyocyte survival in HF. PMID:27657534

  3. Ryanodine receptor-mediated Ca2+ release underlies iron-induced mitochondrial fission and stimulates mitochondrial Ca2+ uptake in primary hippocampal neurons

    PubMed Central

    SanMartín, Carol D.; Paula-Lima, Andrea C.; García, Alejandra; Barattini, Pablo; Hartel, Steffen; Núñez, Marco T.; Hidalgo, Cecilia

    2014-01-01

    Mounting evidence indicates that iron accumulation impairs brain function. We have reported previously that addition of sub-lethal concentrations of iron to primary hippocampal neurons produces Ca2+ signals and promotes cytoplasmic generation of reactive oxygen species. These Ca2+ signals, which emerge within seconds after iron addition, arise mostly from Ca2+ release through the redox-sensitive ryanodine receptor (RyR) channels present in the endoplasmic reticulum. We have reported also that addition of synaptotoxic amyloid-β oligomers to primary hippocampal neurons stimulates RyR-mediated Ca2+ release, generating long-lasting Ca2+ signals that activate Ca2+-sensitive cellular effectors and promote the disruption of the mitochondrial network. Here, we describe that 24 h incubation of primary hippocampal neurons with iron enhanced agonist-induced RyR-mediated Ca2+ release and promoted mitochondrial network fragmentation in 43% of neurons, a response significantly prevented by RyR inhibition and by the antioxidant agent N-acetyl-L-cysteine. Stimulation of RyR-mediated Ca2+ release by a RyR agonist promoted mitochondrial Ca2+ uptake in control neurons and in iron-treated neurons that displayed non-fragmented mitochondria, but not in neurons with fragmented mitochondria. Yet, the global cytoplasmic Ca2+ increase induced by the Ca2+ ionophore ionomycin prompted significant mitochondrial Ca2+ uptake in neurons with fragmented mitochondria, indicating that fragmentation did not prevent mitochondrial Ca2+ uptake but presumably decreased the functional coupling between RyR-mediated Ca2+ release and the mitochondrial Ca2+ uniporter. Taken together, our results indicate that stimulation of redox-sensitive RyR-mediated Ca2+ release by iron causes significant neuronal mitochondrial fragmentation, which presumably contributes to the impairment of neuronal function produced by iron accumulation. PMID:24653672

  4. The effects of ryanodine receptor (RYR1) mutation on natural killer cell cytotoxicity, plasma cytokines and stress hormones during acute intermittent exercise in pigs.

    PubMed

    Ciepielewski, Z M; Stojek, W; Borman, A; Myślińska, D; Pałczyńska, P; Kamyczek, M

    2016-04-01

    Stress susceptibility has been mapped to a single recessive gene, the ryanodine receptor 1 (RYR1) gene or halothane (Hal) gene. Homozygous (Hal(nn)), mutated pigs are sensitive to halothane and susceptible to Porcine Stress Syndrome (PSS). Previous studies have shown that stress-susceptible RYR1 gene mutated homozygotes in response to restraint stress showed an increase in natural killer cell cytotoxicity (NKCC) accompanied by more pronounced stress-related hormone and anti-inflammatory cytokine changes. In order to determine the relationship of a RYR1 gene mutation with NKCC, plasma cytokines and stress-related hormones following a different stress model - exercise - 36 male pigs (representing different genotypes according to RYR1 gene mutation: NN, homozygous dominant; Nn, heterozygous; nn, homozygous recessive) were submitted to an intermittent treadmill walking. During the entire experiment the greatest level of NKCC and the greatest concentrations of interleukin (IL-) 6, IL-10, IL-12, interferon (IFN-)γ and tumor necrosis factor-α and stress-related hormones (adrenaline, prolactin, beta-endorphin) were observed in nn pigs, and the greatest concentration of IL-1 and growth hormone in NN pigs. Immunostimulatory effects of intermittent exercise on NKCC in nn pigs were concomitant with increases in IL-2, IL-12 and IFN-γ, the potent NKCC activators. Our findings suggest that stress-susceptible pigs RYR1 gene mutated pigs develop a greater level of NKCC and cytokine production in response to exercise stress. These results suggest that the heterogeneity of immunological and neuroendocrine response to exercise stress in pigs could be influenced by RYR1 gene mutation.

  5. Modulation of the adaptive response to stress by brain activation of selective somatostatin receptor subtypes

    PubMed Central

    Stengel, Andreas; Rivier, Jean; Taché, Yvette

    2013-01-01

    Somatostatin-14 was discovered in 1973 in the hypothalamus as a peptide inhibiting growth hormone release. Somatostatin interacts with five receptor subtypes (sst1–5) which are widely distributed in the brain with a distinct, but overlapping, expression pattern. During the last few years, the development of highly selective peptide agonists and antagonists provided new insight to characterize the role of somatostatin receptor subtypes in the pleiotropic actions of somatostatin. Recent evidence in rodents indicates that the activation of selective somatostatin receptor subtypes in the brain blunts stress-CRF related ACTH release (sst2/5), sympathetic-adrenal activaton (sst5), stimulation of colonic motility (sst1), delayed gastric emptying (sst5), suppression of food intake (sst2) and the anxiogenic-like (sst2) response. These findings suggest that brain somatostatin signaling pathways may play an important role in dampening CRF-mediated endocrine, sympathetic, behavioral and visceral responses to stress. PMID:23287111

  6. Differential subcellular distribution of rat brain dopamine receptors and subtype-specific redistribution induced by cocaine

    PubMed Central

    Voulalas, Pamela J.; Schetz, John; Undieh, Ashiwel S.

    2011-01-01

    We investigated the subcellular distribution of dopamine D1, D2 and D5 receptor subtypes in rat frontal cortex, and examined whether psychostimulant-induced elevation of synaptic dopamine could alter the receptor distribution. Differential detergent solubilization and density gradient centrifugation were used to separate various subcellular fractions, followed by semi-quantitative determination of the relative abundance of specific receptor proteins in each fraction. D1 receptors were predominantly localized to detergent-resistant membranes, and a portion of these receptors also floated on sucrose gradients. These properties are characteristic of proteins found in lipid rafts and caveolae. D2 receptors exhibited variable distribution between cytoplasmic, detergent-soluble and detergent-resistant membrane fractions, yet were not present in buoyant membranes. Most D5 receptor immunoreactivity was distributed into the cytoplasmic fraction, failing to sediment at forces up to 300,000g, while the remainder was localized to detergent-soluble membranes in cortex. D5 receptors were undetectable in detergent-resistant fractions or raft-like subdomains. Following daily cocaine administration for seven days, a significant portion of D1 receptors translocated from detergent-resistant membranes to detergent-soluble membranes and the cytoplasmic fraction. The distributions of D5 and D2 receptor subtypes were not significantly altered by cocaine treatment. These data imply that D5 receptors are predominantly cytoplasmic, D2 receptors are diffusely distributed within the cell, whereas D1 receptors are mostly localized to lipid rafts within the rat frontal cortex. Dopamine receptor subtype localization is susceptible to modulation by pharmacological manipulations that elevate synaptic dopamine, however the functional implications of such drug-induced receptor warrant further investigation. PMID:21236347

  7. Pirenzepine binding to membrane-bound, solubilized and purified muscarinic receptor subtypes

    SciTech Connect

    Baumgold, J.

    1986-05-01

    Muscarinic receptors were purified to near-homogeneity from bovine cortex, an area rich in the putative M1 subtype, and from bovine pons/medulla, an area rich in the putative M2 subtype. In both cases, the receptors were solubilized in digitonin and purified over an affinity column. Both the cortical and pons/medulla preparations yielded receptor proteins of 70,000 daltons. Pirenzepine binding was deduced from its competition with /sup 3/H-N-methyl scopolamine. The binding of pirenzepine to membrane-bound receptors from cortex was best described by a two site model, with approximately half the sites having a Ki of 6.4 x 10/sup -9/ M and the remaining sites having a Ki of 3.5 x 10/sup -7/ M. Membrane-bound receptors from pons/medulla bound pirenzepine according to a one-site model with a Ki of 1.1 x 10/sup -7/ M. After solubilization the two-site binding of cortical receptors became a one-site binding, Ki = 1.1 x 10/sup -7/M. This value was still five-fold lower than that of soluble receptors from pons/medulla. After purification however the affinity of pirenzepine for the pons/medulla receptor increased so that the two putative subtypes bound pirenzepine with approximately the same affinity. These findings suggest that the different pirenzepine binding characteristics used to define muscarinic receptor subtypes are not inherent in the receptor protein itself but may be due to coupling factors associated with the receptor.

  8. Selectivity of dobutamine for adrenergic receptor subtypes: in vitro analysis by radioligand binding.

    PubMed

    Williams, R S; Bishop, T

    1981-06-01

    The cardiovascular responses elicited by dobutamine are distinctly different from those produced by other adrenergic or dopaminergic agonists. To test the hypothesis that dobutamine could have differential affinities for adrenergic receptor subtypes, and that such subtype selectivity could be related to its relatively unique pharmacologic properties, we assessed the ability of dobutamine to displace adrenergic radioligands from membrane receptors in a number of tissues of previously characterized adrenergic receptor subtype. For beta adrenergic receptors identified by (-) [(3)H]dihydroalprenolol (DHA), dobutamine had significantly greater affinity for the beta(1) subtype (K(D) = 2.5 muM in rat heart and 2.6 muM in turkey erythrocyte) than for the beta(2) subtype (K(D) = 14.8 muM in frog heart and 25.4 muM in rat lung) (P < 0.001). For alpha adrenergic receptors, dobutamine had markedly greater affinity for the alpha(1)-subtype identified by [(3)H]prazosin (K(D) = 0.09 muM in rat heart and 0.14 muM in rabbit uterus) than for the alpha(2)-subtype identified by [(3)H]dihydroergocryptine (DHE) (K(D) = 9.3 muM in human platelet) or by [(3)H]yohimbine (K(D) = 5.7 muM in rabbit uterus) (P < 0.001). Like other beta(1)-agonists, in the absence of guanine nucleotide, dobutamine competition curves for DHA binding in rat heart demonstrated two classes of binding sites, with one site of significantly higher affinity (K(D) = 0.5 muM, P = 0.008) than the single class of binding sites (K(D) = 5.2 muM) identified in the presence of guanine nucleotide. However, unlike beta(2)- or alpha(2)-agonists, dobutamine displacement of DHA binding in rat lung or of DHE binding in human platelets demonstrated only a single class of binding sites, and guanine nucleotide had only minimal effects. We conclude that dobutamine is selective for beta(1) as opposed to beta(2), and for alpha(1) as opposed to alpha(2) adrenergic receptors. Furthermore, guanine nucleotide effects on dobutamine binding

  9. Estrogen receptor subtype- and promoter-specific modulation of aryl hydrocarbon receptor-dependent transcription.

    PubMed

    Wihlén, Björn; Ahmed, Shaimaa; Inzunza, José; Matthews, Jason

    2009-06-01

    In this study, we examined the role of estrogen receptors (ER) in aryl hydrocarbon receptor (AHR)-dependent transactivation. Chromatin immunoprecipitation assays showed that AHR agonists differentially induced recruitment of ERalpha to the AHR target genes CYP1A1 and CYP1B1. Cotreatment with 17beta-estradiol significantly increased beta-naphthoflavone (BNF)- and 2,3,7,8-tetrachlorodibenzo-p-dioxin-induced recruitment of ERalpha to CYP1A1, whereas 3,3'-diindolylmethane induced promoter occupancy of ERalpha at CYP1A1 that was unaffected by cotreatment with 17beta-estradiol. Cyclical recruitment of AHR and ERalpha to CYP1A1 was only observed in cells treated with BNF. Stable and subtype-specific knockdown of ERalpha or ERbeta using shRNA showed that suppression of ERalpha significantly reduced, whereas knockdown of ERbeta significantly enhanced, AHR agonist-induced Cyp1a1 expression in HC11 mouse mammary epithelial cells. AHR agonist-induced Cyp1b1 expression was reduced by ERbeta knockdown but unaffected by ERalpha knockdown. The siRNA-mediated knockdown of ERalpha in MCF-7 human breast cancer cells did not affect 2,3,7,8-tetrachlorodibenzo-p-dioxin-dependent regulation of CYP1A1 and CYP1B1 mRNA expression. In agreement with our in vitro findings in the HC11 cells, ERalpha knockout mice exhibit reduced BNF-dependent induction of Cyp1a1 mRNA. These results establish ligand- and promoter-specific influences on the cyclical recruitment patterns for AHR and show ER species-, subtype-, and promoter-specific modulation of AHR-dependent transcription.

  10. Binding and functional properties of hexocyclium and sila-hexocyclium derivatives to muscarinic receptor subtypes.

    PubMed Central

    Waelbroeck, M.; Camus, J.; Tastenoy, M.; Feifel, R.; Mutschler, E.; Tacke, R.; Strohmann, C.; Rafeiner, K.; Rodrigues de Miranda, J. F.; Lambrecht, G.

    1994-01-01

    1. We have compared the binding properties of several hexocyclium and sila-hexocyclium derivatives to muscarinic M1 receptors (in rat brain, human neuroblastoma (NB-OK 1) cells and calf superior cervical ganglia), rat heart M2 receptors, rat pancreas M3 receptors and M4 receptors in rat striatum, with their functional antimuscarinic properties in rabbit vas deferens (M1/M4-like), guinea-pig atria (M2), and guinea-pig ileum (M3) muscarinic receptors. 2. Sila-substitution (C/Si exchange) of hexocyclium (-->sila-hexocyclium) and demethyl-hexocyclium (-->demethyl-sila-hexocyclium) did not significantly affect their affinities for muscarinic receptors. By contrast, sila-substitution of o-methoxy-hexocyclium increased its affinity 2 to 3 fold for all the muscarinic receptor subtypes studied. 3. The p-fluoro- and p-chloro-derivatives of sila-hexocyclium had lower affinities than the parent compound at the four receptor subtypes, in binding and pharmacological studies. 4. In binding studies, o-methoxy-sila-hexocyclium (M1 = M4 > or = M3 > or = M2) had a much lower affinity than sila-hexocyclium for the four receptor subtypes, and discriminated the receptor subtypes more poorly than sila-hexocyclium (M1 = M3 > M4 > M2). This is in marked contrast with the very clear selectivity of o-methoxy-sila-hexocyclium for the prejunctional M1/M4-like heteroreceptors in rabbit vas deferens. 5. The tertiary amines demethyl-hexocyclium, demethyl-sila-hexocyclium and demethyl-o-methoxy-sila-hexocyclium had 10 to 30 fold lower affinities than the corresponding quaternary ammonium derivatives. PMID:8075869

  11. Expression of purinergic P2X receptor subtypes 1, 2, 3 and 7 in equine laminitis.

    PubMed

    Zamboulis, Danae E; Senior, Mark; Clegg, Peter D; Milner, Peter I

    2013-11-01

    Tissue sensitisation and chronic pain have been described in chronic-active laminitis in the horse, making treatment of such cases difficult. Purinergic P2X receptors are linked to chronic pain and inflammation. The aim of this study was to examine the expression of purinergic P2X receptor subtypes 1, 2, 3 and 7 in the hoof, palmar digital vessels and nerve, dorsal root ganglia and spinal cord in horses with chronic-active laminitis (n=5) compared to non-laminitic horses (n=5). Immunohistochemical analysis was performed on tissue sections using antibodies against P2X receptor subtypes 1-3 and 7. In horses with laminitis, there was a reduction in the thickness of the tunica media layer of the palmar digital vein as a proportion of the whole vessel diameter (0.48±0.05) compared to the non-laminitic group (0.57±0.04; P=0.02). P2X receptor subtype 3 was expressed in the smooth muscle layer (tunica media) of the palmar digital artery of horses with laminitis, but was absent in horses without laminitis. There was strong expression of P2X receptor subtype 7 in the proliferating, partially keratinised, epidermal cells of the secondary epidermal lamellae in the hooves of horses with laminitis, but no immunopositivity in horses without laminitis.

  12. Cockroach GABAB receptor subtypes: molecular characterization, pharmacological properties and tissue distribution.

    PubMed

    Blankenburg, S; Balfanz, S; Hayashi, Y; Shigenobu, S; Miura, T; Baumann, O; Baumann, A; Blenau, W

    2015-01-01

    γ-aminobutyric acid (GABA) is the predominant inhibitory neurotransmitter in the central nervous system (CNS). Its effects are mediated by either ionotropic GABAA receptors or metabotropic GABAB receptors. GABAB receptors regulate, via Gi/o G-proteins, ion channels, and adenylyl cyclases. In humans, GABAB receptor subtypes are involved in the etiology of neurologic and psychiatric disorders. In arthropods, however, these members of the G-protein-coupled receptor family are only inadequately characterized. Interestingly, physiological data have revealed important functions of GABAB receptors in the American cockroach, Periplaneta americana. We have cloned cDNAs coding for putative GABAB receptor subtypes 1 and 2 of P. americana (PeaGB1 and PeaGB2). When both receptor proteins are co-expressed in mammalian cells, activation of the receptor heteromer with GABA leads to a dose-dependent decrease in cAMP production. The pharmacological profile differs from that of mammalian and Drosophila GABAB receptors. Western blot analyses with polyclonal antibodies have revealed the expression of PeaGB1 and PeaGB2 in the CNS of the American cockroach. In addition to the widespread distribution in the brain, PeaGB1 is expressed in salivary glands and male accessory glands. Notably, PeaGB1-like immunoreactivity has been detected in the GABAergic salivary neuron 2, suggesting that GABAB receptors act as autoreceptors in this neuron.

  13. Oxygen-coupled redox regulation of the skeletal muscle ryanodine receptor/Ca2+ release channel (RyR1): sites and nature of oxidative modification.

    PubMed

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

    2013-08-09

    In mammalian skeletal muscle, Ca(2+) release from the sarcoplasmic reticulum (SR) through the ryanodine receptor/Ca(2+)-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 Ca(2+) 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.

  14. Effects of absolute configuration of IQNP on muscarinic receptor subtype selectivity in vitro and in vivo

    SciTech Connect

    McPherson, D.W.; Lambert, C.R.; Knapp, F.F.

    1994-05-01

    IQNP, a high affinity muscarinic ligand with high cerebral uptake and long retention, contains two chiral centers in addition to vinyl iodide sterochemistry. The various diastereomers, in which the 3-quinuclidinyl moiety has the R configuration, have been prepared and evaluated in vitro and in vivo. These data show that muscarinic receptor subtype selectivity is dramatically affected by the configuration of the acetate center and vinyl iodide. In vitro studies show that E-(R,R)-IQNP is 100 times more selective for ml than m2 subtype as compared to E-(R,S), which was confirmed by in vivo results. In contrast, in vivo, Z-(R,R) has high uptake in m2 rich tissues (heart and cerebellum). In vitro studies are being performed on the Z isomers. Blocking studies with subtype-specific ligands confirm these data which illustrate the importance of molecular configuration on receptor subtype selectivity. These combined studies demonstrate that these isomers of IQNP are good candidates for future studies of receptor subtypes.

  15. Evidence that human Fc gamma receptor IIA (CD32) subtypes are not receptors for oxidized LDL.

    PubMed

    Morganelli, P M; Groveman, D S; Pfeiffer, J R

    1997-11-01

    Several lines of evidence suggest that clearance of oxidized LDL (oxLDL) immune complexes by macrophage IgG Fc receptors (Fc gamma Rs) plays a role in atherogenesis. Ox-LDL may also be cleared directly by Fc gamma Rs, as shown for murine Fc gamma RII-B2. In humans, the homologous Fc gamma R is Fc gamma RIIA (CD32), which is abundantly expressed on monocytes and macrophages and shares 60% sequence identity with murine Fc gamma RII-B2. As murine Fc gamma RII-B2 and human Fc gamma RIIA also share similar IgG ligand-binding properties, the purpose of this study was to test the hypothesis that human CD32 is a receptor for oxLDL. For these studies we used transfected Chinese hamster ovary (CHO) cells, monocytes, and cell lines that functionally express either of two Fc gamma RIIA subtypes (R131 or H131) and assayed binding or degradation of several preparations of oxLDL. The integrity of all oxLDL preparations was checked by studying their ability to react with CHO cells expressing human type I scavenger receptors and by other characteristics of lipoprotein oxidation. Although we showed that each preparation of oxLDL could recognize class A or class B scavenger receptors, we did not detect any differences in the binding or degradation of any type of oxLDL preparation among control versus CHO cell transfectants. Using monocytes that express Fc gamma RIIA and CD36, we showed that the binding of oxLDL was inhibited by antibodies to CD36, but not by Fc gamma RIIA antibodies. Thus, the data do not support the hypothesis that human Fc gamma RIIA is by itself a receptor for oxLDL. We conclude that human CD32 can mediate uptake of lipoprotein immune complexes, but does not mediate uptake of oxLDL in the absence of anti-oxLDL antibodies. OxLDL may interact with human mononuclear phagocytes directly via other types of receptors, such as class A and class B scavenger receptors or CD68.

  16. Identification of Receptor Ligands and Receptor Subtypes Using Antagonists in a Capillary Electrophoresis Single-Cell Biosensor Separation System

    NASA Astrophysics Data System (ADS)

    Fishman, Harvey A.; Orwar, Owe; Scheller, Richard H.; Zare, Richard N.

    1995-08-01

    A capillary electrophoresis system with single-cell biosensors as a detector has been used to separate and identify ligands in complex biological samples. The power of this procedure was significantly increased by introducing antagonists that inhibited the cellular response from selected ligand-receptor interactions. The single-cell biosensor was based on the ligand-receptor binding and G-protein-mediated signal transduction pathways in PC12 and NG108-15 cell lines. Receptor activation was measured as increases in cytosolic free calcium ion concentration by using fluorescence microscopy with the intracellular calcium ion indicator fluo-3 acetoxymethyl ester. Specifically, a mixture of bradykinin (BK) and acetylcholine (ACh) was fractionated and the components were identified by inhibiting the cellular response with icatibant (HOE 140), a selective antagonist to the BK B_2 receptor subtype (B_2BK), and atropine, an antagonist to muscarinic ACh receptor subtypes. Structurally related forms of BK were also identified based on inhibiting B_2BK receptors. Applications of this technique include identification of endogenous BK in a lysate of human hepatocellular carcinoma cells (Hep G2) and screening for bioactivity of BK degradation products in human blood plasma. The data demonstrate that the use of antagonists with a single-cell biosensor separation system aids identification of separated components and receptor subtypes.

  17. Cloning of a novel G protein-coupled receptor, SLT, a subtype of the melanin-concentrating hormone receptor.

    PubMed

    Mori, M; Harada, M; Terao, Y; Sugo, T; Watanabe, T; Shimomura, Y; Abe, M; Shintani, Y; Onda, H; Nishimura, O; Fujino, M

    2001-05-25

    A DNA fragment encoding an amino acid sequence possessing common features to the G protein-coupled receptor (GPCR) superfamily was found in the human genomic sequence, and from this information, the full-length cDNA of a novel GPCR, designated SLT, was cloned from the human hippocampus cDNA library. SLT showed the highest homology to the melanin-concentrating hormone (MCH) receptor, SLC-1 (31.5% identity), and to a lesser extent, to the somatostatin (SST) receptor subtypes. MCH exhibited agonistic behavior when applied to the SLT-expressing CHO cells at subnanomolar doses whereas more than 200 known peptides, including SST and cortistatin, did not. These results indicated that MCH is the cognate ligand of the SLT receptor and that this newly cloned GPCR is the second subtype of the MCH receptor. Quantitative polymerase chain reaction analysis of the SLT gene expression in human tissues showed that the SLT receptor is expressed mainly in brain areas including the cerebral cortex, amygdala, hippocampus, and corpus callosum, as well as in a limited number of peripheral tissues. The distribution of the SLT nearly overlapped that of SLC-1, suggesting that some of the neural functions of MCH may be mediated by both of these receptor subtypes.

  18. LTD expression is independent of glutamate receptor subtype.

    PubMed

    Granger, Adam J; Nicoll, Roger A

    2014-01-01

    Long-term depression (LTD) is a form of synaptic plasticity that plays a major role in the activity-dependent reshaping of synaptic transmission. LTD is expressed as a decrease in synaptic AMPA receptor number, though the exact mechanism remains controversial. Several lines of evidence have suggested necessary roles for both the GluA1 and GluA2 subunits, and specifically certain interactions with their cytoplasmic tails. However, it is unclear if either GluA1 or GluA2 are absolutely required for LTD. We tested this hypothesis using constitutive knock-outs and single-cell molecular replacement of AMPA receptor subunits in mouse hippocampus. We found that neither GluA1 or GluA2 are required for normal expression of LTD, and indeed a normal decrease in synaptic transmission was observed in cells in which all endogenous AMPA receptors have been replaced by kainate receptors. Thus, LTD does not require removal of specific AMPA receptor subunits, but likely involves a more general modification of the synapse and its ability to anchor a broad range of receptor proteins.

  19. Oestrogen and progesterone receptor expression in subtypes of canine mammary tumours in intact and ovariectomised dogs.

    PubMed

    Mainenti, M; Rasotto, R; Carnier, P; Zappulli, V

    2014-10-01

    The objective of this study was to investigate as a potential prognostic indicator the relationship between histological subtype of canine mammary tumours (CMTs) and oestrogen-α (ORα) and progesterone (PR) receptor expression. Using immunohistochemistry, receptor expression in neoplastic epithelial cells was assessed in 12 different subtypes in 113 CMTs (34 benign, 79 malignant) and 101 surrounding normal tissues. Sixty-eight and 45 CMTs were from intact and ovariectomised bitches, respectively. Histological subtype strongly influenced ORα/PR expression: simple and complex adenomas as well as simple tubular carcinomas exhibited the greatest expression, whereas immunohistochemical labelling for these receptors was weakest in carcinoma and malignant myoepitheliomas, as well as in solid/anaplastic carcinomas and comedocarcinomas. Receptor expression was generally higher in benign relative to malignant neoplasms, and in the latter it was significantly lower in ovariectomised vs. intact bitches. Lymphatic invasion, mitotic index, nodule diameter, and tumour grade were significantly associated with ORα/PR expression. Although not found to be an independent prognostic indicator, tumours from dogs with <10% cells with ORα/PR expression had a poorer prognosis. Lymphatic invasion, the state of the margins of excision, and mitotic index were found to be independent prognostic indicators. Overall, the results suggest that differences in histological subtype and whether or not a bitch has been ovariectomised should be considered when evaluating the significance of ORα and PR expression in CMTs. Copyright © 2014 Elsevier Ltd. All rights reserved.

  20. A Molecular and Chemical Perspective in Defining Melatonin Receptor Subtype Selectivity

    PubMed Central

    Chan, King Hang; Wong, Yung Hou

    2013-01-01

    Melatonin is primarily synthesized and secreted by the pineal gland during darkness in a normal diurnal cycle. In addition to its intrinsic antioxidant property, the neurohormone has renowned regulatory roles in the control of circadian rhythm and exerts its physiological actions primarily by interacting with the G protein-coupled MT1 and MT2 transmembrane receptors. The two melatonin receptor subtypes display identical ligand binding characteristics and mediate a myriad of signaling pathways, including adenylyl cyclase inhibition, phospholipase C stimulation and the regulation of other effector molecules. Both MT1 and MT2 receptors are widely expressed in the central nervous system as well as many peripheral tissues, but each receptor subtype can be linked to specific functional responses at the target tissue. Given the broad therapeutic implications of melatonin receptors in chronobiology, immunomodulation, endocrine regulation, reproductive functions and cancer development, drug discovery and development programs have been directed at identifying chemical molecules that bind to the two melatonin receptor subtypes. However, all of the melatoninergics in the market act on both subtypes of melatonin receptors without significant selectivity. To facilitate the design and development of novel therapeutic agents, it is necessary to understand the intrinsic differences between MT1 and MT2 that determine ligand binding, functional efficacy, and signaling specificity. This review summarizes our current knowledge in differentiating MT1 and MT2 receptors and their signaling capacities. The use of homology modeling in the mapping of the ligand-binding pocket will be described. Identification of conserved and distinct residues will be tremendously useful in the design of highly selective ligands. PMID:24018885

  1. Role of the M3 Muscarinic Acetylcholine Receptor Subtype in Murine Ophthalmic Arteries After Endothelial Removal

    PubMed Central

    Gericke, Adrian; Steege, Andreas; Manicam, Caroline; Böhmer, Tobias; Wess, Jürgen; Pfeiffer, Norbert

    2014-01-01

    Purpose. We tested the hypothesis that the M3 muscarinic acetylcholine receptor subtype mediates cholinergic responses in murine ophthalmic arteries after endothelial removal. Methods. Muscarinic receptor gene expression was determined in ophthalmic arteries with intact and with removed endothelium using real-time PCR. To examine the role of the M3 receptor in mediating vascular responses, ophthalmic arteries from M3 receptor-deficient mice (M3R−/−) and respective wild-type controls were studied in vitro. Functional studies were performed in nonpreconstricted arteries with either intact or removed endothelium using video microscopy. Results. In endothelium-intact ophthalmic arteries, mRNA for all five muscarinic receptor subtypes was detected, but M3 receptor mRNA was most abundant. In endothelium-removed ophthalmic arteries, M1, M2, and M3 receptors displayed similar mRNA expression levels, which were higher than those for M4 and M5 receptors. In functional studies, acetylcholine evoked vasoconstriction in endothelium-removed arteries from wild-type mice that was virtually abolished after incubation with the muscarinic receptor blocker atropine, indicative of the involvement of muscarinic receptors. In concentration-response experiments, acetylcholine and carbachol concentration-dependently constricted endothelium-removed ophthalmic arteries from wild-type mice, but produced only negligible responses in arteries from M3R−/− mice. In contrast, acetylcholine concentration-dependently dilated ophthalmic arteries with intact endothelium from wild-type mice, but not from M3R−/− mice. Responses to the nitric oxide donor nitroprusside and to KCl did not differ between ophthalmic arteries from wild-type and M3R−/− mice, neither in endothelium-intact nor in endothelium-removed arteries. Conclusions. These findings provide evidence that in murine ophthalmic arteries the muscarinic M3 receptor subtype mediates cholinergic endothelium-dependent vasodilation

  2. Cloning and expression of a cDNA for mouse prostaglandin E receptor EP2 subtype.

    PubMed

    Honda, A; Sugimoto, Y; Namba, T; Watabe, A; Irie, A; Negishi, M; Narumiya, S; Ichikawa, A

    1993-04-15

    A functional cDNA clone encoding mouse EP2 subtype of prostaglandin (PG) E receptor was isolated from a mouse cDNA library by cross-hybridization with the mouse EP3 subtype PGE receptor cDNA. The mouse EP2 receptor consists of 513 amino acid residues with putative seven-transmembrane domains. In contrast to EP3 receptor, this receptor possesses long third intracellular loop and carboxyl-terminal tail. [3H] PGE2 specifically bound to the membrane of mammalian COS cells transfected with the cDNA. The binding to the membrane was displaced with unlabeled PG in the order of PGE2 = PGE1 > iloprost > or = PGF2 alpha > or = PGD2. The binding was also inhibited by misoprostol, an EP2 and EP3 agonist, but not by sulprostone, an EP1 and EP3 agonist, and SC-19220, an EP1 antagonist. PGE2 markedly increased cAMP level in COS cells transfected with the cDNA. These results suggest that this receptor is EP2 subtype. Northern blot analysis demonstrated that the EP2 mRNA is widely expressed in various tissues, the abundant expression being observed in ileum, thymus, and mastocytoma P-815 cells.

  3. GABA(A) receptor subtype-selectivity of novel bicuculline derivatives.

    PubMed

    Ramerstorfer, Joachim; Foppa, Verena; Thiery, Hanna; Hermange, Philippe; Janody, Simon; Berger, Michael L; Dodd, Robert H; Sieghart, Werner

    2015-01-01

    GABA(A) receptors are the major inhibitory neurotransmitter receptors in the central nervous system and are targets of clinically important drugs modulating GABA induced ion flux by interacting with distinct allosteric binding sites. ROD 185 is a previously investigated structural analogue of the GABA site antagonist bicuculline, and a positive allosteric modulator acting via the benzodiazepine binding site. Here, we investigated 13 newly synthesized structural analogues of ROD 185 for their interaction with rat GABA(A) receptors. Using [(3)H]flunitrazepam binding assays, we identified four compounds exhibiting a higher affinity for the benzodiazepine binding site than ROD 185. Two electrode voltage clamp electrophysiology at recombinant GABA(A) receptors indicated that most of these compounds positively modulated GABA-induced currents at these receptors. Additionally, these experiments revealed that this compound class not only interacts with the benzodiazepine binding site at αβγ receptors but also with a novel, so far unidentified binding site present in αβ receptors. Compounds with a high affinity for the benzodiazepine binding site stimulated GABA-induced currents stronger at αβγ than at αβ receptors and preferred α3β3γ2 receptors. Compounds showing equal or smaller effects at αβγ compared to αβ receptors differentially interacted with various αβ or αβγ receptor subtypes. Surprisingly, five of these compounds interacting with αβ receptors showed a strong stimulation at α6β3γ2 receptors. The absence of any direct effects at GABA(A) receptors, as well as their potential selectivity for receptor subtypes not being addressed by benzodiazepines, make this compound class to a starting point for the development of drugs with a possible clinical importance.

  4. Long-term effects of aripiprazole exposure on monoaminergic and glutamatergic receptor subtypes: comparison with cariprazine.

    PubMed

    Choi, Yong Kee; Adham, Nika; Kiss, Béla; Gyertyán, István; Tarazi, Frank I

    2017-01-06

    This study examined the chronic effects of aripiprazole and cariprazine on serotonin (5-HT1A and 5-HT2A) and glutamate (NMDA and AMPA) receptor subtypes. In addition, the effects of aripiprazole on D2 and D3 receptors were tested and compared with previously reported cariprazine data. Rats received vehicle, aripiprazole (2, 5, or 15 mg/kg), or cariprazine (0.06, 0.2, or 0.6 mg/kg) for 28 days. Receptor levels were quantified using autoradiographic assays on brain sections from the medial prefrontal cortex (MPC), dorsolateral frontal cortex (DFC), nucleus accumbens (NAc), caudate-putamen medial (CPu-M), caudate-putamen lateral (CPu-L), hippocampal CA1 (HIPP-CA1) and CA3 (HIPP-CA3) regions, and the entorhinal cortex (EC). Similar to previous findings with cariprazine, aripiprazole upregulated D2 receptor levels in various regions; D3 receptor changes were less than those reported with cariprazine. All aripiprazole doses and higher cariprazine doses increased 5-HT1A receptors in the MPC and DFC. Higher aripiprazole and all cariprazine doses increased 5-HT1A receptors in HIPP-CA1 and HIPP-CA3. Aripiprazole decreased 5-HT2A receptors in the MPC, DFC, HIPP-CA1, and HIPP-CA3 regions. Both compounds decreased NMDA receptors and increased AMPA receptors in select brain regions. Long-term administration of aripiprazole and cariprazine had similar effects on 5-HT1A, NMDA, and AMPA receptors. However, cariprazine more profoundly increased D3 receptors while aripiprazole selectively reduced 5-HT2A receptors. These results suggest that the unique actions of cariprazine on dopamine D3 receptors, combined with its effects on serotonin and glutamate receptor subtypes, may confer the clinical benefits, safety, and tolerability of this novel compound in schizophrenia and bipolar mania.

  5. Receptor subtypes involved in the presynaptic and postsynaptic actions of dopamine on striatal interneurons.

    PubMed

    Centonze, Diego; Grande, Cristina; Usiello, Alessandro; Gubellini, Paolo; Erbs, Eric; Martin, Ana B; Pisani, Antonio; Tognazzi, Nadia; Bernardi, Giorgio; Moratalla, Rosario; Borrelli, Emiliana; Calabresi, Paolo

    2003-07-16

    By stimulating distinct receptor subtypes, dopamine (DA) exerts presynaptic and postsynaptic actions on both large aspiny (LA) cholinergic and fast-spiking (FS) parvalbumin-positive interneurons of the striatum. Lack of receptor- and isoform-specific pharmacological agents, however, has hampered the progress toward a detailed identification of the specific DA receptors involved in these actions. To overcome this issue, in the present study we used four different mutant mice in which the expression of specific DA receptors was ablated. In D1 receptor null mice, D1R-/-, DA dose-dependently depolarized both LA and FS interneurons. Interestingly, SCH 233390 (10 microm), a D1-like (D1 and D5) receptor antagonist, but not l-sulpiride (3-10 microm), a D2-like (D2, D3, D4) receptor blocker, prevented this effect, implying D5 receptors in this action. Accordingly, immunohistochemical analyses in both wild-type and D1R-/- mice confirmed the expression of D5 receptors in both cholinergic and parvalbumin-positive interneurons of the striatum. In mice lacking D2 receptors, D2R-/-, the DA-dependent inhibition of GABA transmission was lost in both interneuron populations. Both isoforms of D2 receptor, D2L and D2S, were very likely involved in this inhibitory action, as revealed by the electrophysiological analysis of the effect of the DA D2-like receptor agonist quinpirole in two distinct mutants lacking D2L receptors and expressing variable contents of D2S receptors. The identification of the receptor subtypes involved in the actions of DA on different populations of striatal cells is essential to understand the circuitry of the basal ganglia and to develop pharmacological strategies able to interfere selectively with specific neuronal functions.

  6. Muscarinic and dopaminergic receptor subtypes on striatal cholinergic interneurons

    SciTech Connect

    Dawson, V.L.; Dawson, T.M.; Wamsley, J.K. )

    1990-12-01

    Unilateral stereotaxic injection of small amounts of the cholinotoxin, AF64A, caused minimal nonselective tissue damage and resulted in a significant loss of the presynaptic cholinergic markers (3H)hemicholinium-3 (45% reduction) and choline acetyltransferase (27% reduction). No significant change from control was observed in tyrosine hydroxylase or tryptophan hydroxylase activity; presynaptic neuronal markers for dopamine- and serotonin-containing neurons, respectively. The AF64A lesion resulted in a significant reduction of dopamine D2 receptors as evidenced by a decrease in (3H)sulpiride binding (42% reduction) and decrease of muscarinic non-M1 receptors as shown by a reduction in (3H)QNB binding in the presence of 100 nM pirenzepine (36% reduction). Saturation studies revealed that the change in (3H)sulpiride and (3H)QNB binding was due to a change in Bmax not Kd. Intrastriatal injection of AF64A failed to alter dopamine D1 or muscarinic M1 receptors labeled with (3H)SCH23390 and (3H)pirenzepine, respectively. In addition, no change in (3H)forskolin-labeled adenylate cyclase was observed. These results demonstrate that a subpopulation of muscarinic receptors (non-M1) are presynaptic on cholinergic interneurons (hence, autoreceptors), and a subpopulation of dopamine D2 receptors are postsynaptic on cholinergic interneurons. Furthermore, dopamine D1, muscarinic M1 and (3H)forskolin-labeled adenylate cyclase are not localized to striatal cholinergic interneurons.

  7. Molecular Characteristics, mRNA Expression, and Alternative Splicing of a Ryanodine Receptor Gene in the Oriental Fruit Fly, Bactrocera dorsalis (Hendel)

    PubMed Central

    Yuan, Guo-Rui; Shi, Wen-Zhi; Yang, Wen-Jia; Jiang, Xuan-Zhao; Dou, Wei; Wang, Jin-Jun

    2014-01-01

    Ryanodine receptors (RyRs) are a distinct class of ligand-gated channels controlling the release of calcium from intracellular stores. The emergence of diamide insecticides, which selectively target insect RyRs, has promoted the study of insect RyRs. In the present study, the full-length RyR cDNA (BdRyR) was cloned and characterized from the oriental fruit fly, Bactrocera dorsalis (Hendel), a serious pest of fruits and vegetables throughout East Asia and the Pacific Rim. The cDNA of BdRyR contains a 15,420-bp open reading frame encoding 5,140 amino acids with a predicted molecular weight of 582.4 kDa and an isoelectric point of 5.38. BdRyR shows a high level of amino acid sequence identity (78 to 97%) to other insect RyR isoforms. All common structural features of the RyRs are present in the BdRyR, including a well-conserved C-terminal domain containing consensus calcium-binding EF-hands and six transmembrane domains, and a large N-terminal domain. Quantitative real-time PCR analyses revealed that BdRyR was expressed at the lowest and highest levels in egg and adult, respectively, and that the BdRyR expression levels in the third instar larva, pupa and adult were 166.99-, 157.56- and 808.56-fold higher, respectively, than that in the egg. Among different adult body parts, the highest expression level was observed in the thorax compared with the head and abdomen. In addition, four alternative splice sites were identified in the BdRyR gene, with the first, ASI, being located in the central part of the predicted second spore lysis A/RyR domain. Diagnostic PCR analyses revealed that alternative splice variants were generated not only in a tissue-specific manner but also in a developmentally regulated manner. These results lay the foundation for further understanding the structural and functional properties of BdRyR, and the molecular mechanisms for target site resistance in B. dorsalis. PMID:24740254

  8. Effects of CaMKII-Mediated Phosphorylation of Ryanodine Receptor Type 2 on Islet Calcium Handling, Insulin Secretion, and Glucose Tolerance

    PubMed Central

    Dixit, Sayali S.; Wang, Tiannan; Manzano, Eiffel John Q.; Yoo, Shin; Lee, Jeongkyung; Chiang, David Y.; Ryan, Nicole; Respress, Jonathan L.; Yechoor, Vijay K.; Wehrens, Xander H. T.

    2013-01-01

    Altered insulin secretion contributes to the pathogenesis of type 2 diabetes. This alteration is correlated with altered intracellular Ca2+-handling in pancreatic β cells. Insulin secretion is triggered by elevation in cytoplasmic Ca2+ concentration ([Ca2+]cyt) of β cells. This elevation in [Ca2+]cyt leads to activation of Ca2+/calmodulin-dependent protein kinase II (CAMKII), which, in turn, controls multiple aspects of insulin secretion. CaMKII is known to phosphorylate ryanodine receptor 2 (RyR2), an intracellular Ca2+-release channel implicated in Ca2+-dependent steps of insulin secretion. Our data show that RyR2 is CaMKII phosphorylated in a pancreatic β-cell line in a glucose-sensitive manner. However, it is not clear whether any change in CaMKII-mediated phosphorylation underlies abnormal RyR2 function in β cells and whether such a change contributes to alterations in insulin secretion. Therefore, knock-in mice with a mutation in RyR2 that mimics its constitutive CaMKII phosphorylation, RyR2-S2814D, were studied. This mutation led to a gain-of-function defect in RyR2 indicated by increased basal RyR2-mediated Ca2+ leak in islets of these mice. This chronic in vivo defect in RyR2 resulted in basal hyperinsulinemia. In addition, S2814D mice also developed glucose intolerance, impaired glucose-stimulated insulin secretion and lowered [Ca2+]cyt transients, which are hallmarks of pre-diabetes. The glucose-sensitive Ca2+ pool in islets from S2814D mice was also reduced. These observations were supported by immunohistochemical analyses of islets in diabetic human and mouse pancreata that revealed significantly enhanced CaMKII phosphorylation of RyR2 in type 2 diabetes. Together, these studies implicate that the chronic gain-of-function defect in RyR2 due to CaMKII hyperphosphorylation is a novel mechanism that contributes to pathogenesis of type 2 diabetes. PMID:23516528

  9. Amino acid residues Gln4020 and Lys4021 of the ryanodine receptor type 1 are required for activation by 4-chloro-m-cresol.

    PubMed

    Fessenden, James D; Feng, Wei; Pessah, Isaac N; Allen, Paul D

    2006-07-28

    The ryanodine receptor type 1 (RyR1) and type 2 (RyR2), but not type 3 (RyR3), are efficiently activated by 4-chloro-m-cresol (4-CmC). We previously showed that a 173-amino acid segment of RyR1 (residues 4007-4180) is required for channel activation by 4-CmC (Fessenden, J. D., Perez, C. F., Goth, S., Pessah, I. N., and Allen, P. D. (2003) J. Biol. Chem. 278, 28727-28735). In the present study, we used site-directed mutagenesis to identify individual amino acid(s) within this region that mediate 4-CmC activation. In RyR1, substitution of 11 amino acids conserved between RyR1 and RyR2, but divergent in RyR3, with their RyR3 counterparts reduced 4-CmC sensitivity to the same degree as substitution of the entire 173-amino acid segment. Further analysis of various RyR1 mutants containing successively smaller numbers of these mutations identified 2 amino acid residues (Gln(4020) and Lys(4021)) that, when mutated to their RyR3 counterparts (Leu(3873) and Gln(3874)), abolished 4-CmC activation of RyR1. Mutation of either of these residues alone did not abolish 4-CmC sensitivity, although Q4020L partially reduced 4-CmC-induced Ca(2+) transients. In addition, mutation of the corresponding residues in RyR3 to their RyR1 counterparts (L3873Q/Q3874K) imparted 4-CmC sensitivity to RyR3. Recordings of single RyR1 channels indicated that 4-CmC applied to either the luminal or cytoplasmic side activated the channel with equal potency. Secondary structure modeling in the vicinity of the Gln(4020)-Lys(4021) dipeptide suggests that the region contains a surface-exposed region adjacent to a hydrophobic segment, indicating that both hydrophilic and hydrophobic regions of RyR1 are necessary for 4-CmC binding to the channel and/or to translate allosteric 4-CmC binding into channel activation.

  10. Förster Resonance Energy Transfer Measurements of Ryanodine Receptor Type 1 Structure Using a Novel Site-Specific Labeling Method

    PubMed Central

    Fessenden, James D.

    2009-01-01

    Background While the static structure of the intracellular Ca2+ release channel, the ryanodine receptor type 1 (RyR1) has been determined using cryo electron microscopy, relatively little is known concerning changes in RyR1 structure that accompany channel gating. Förster resonance energy transfer (FRET) methods can resolve small changes in protein structure although FRET measurements of RyR1 are hampered by an inability to site-specifically label the protein with fluorescent probes. Methodology/Principal Findings A novel site-specific labeling method is presented that targets a FRET acceptor, Cy3NTA to 10-residue histidine (His) tags engineered into RyR1. Cy3NTA, comprised of the fluorescent dye Cy3, coupled to two Ni2+/nitrilotriacetic acid moieties, was synthesized and functionally tested for binding to His-tagged green fluorescent protein (GFP). GFP fluorescence emission and Cy3NTA absorbance spectra overlapped significantly, indicating that FRET could occur (Förster distance = 6.3 nm). Cy3NTA bound to His10-tagged GFP, quenching its fluorescence by 88%. GFP was then fused to the N-terminus of RyR1 and His10 tags were placed either at the N-terminus of the fused GFP or between GFP and RyR1. Cy3NTA reduced fluorescence of these fusion proteins by 75% and this quenching could be reversed by photobleaching Cy3, thus confirming GFP-RyR1 quenching via FRET. A His10 tag was then placed at amino acid position 1861 and FRET was measured from GFP located at either the N-terminus or at position 618 to Cy3NTA bound to this His tag. While minimal FRET was detected between GFP at position 1 and Cy3NTA at position 1861, 53% energy transfer was detected from GFP at position 618 to Cy3NTA at position 1861, thus indicating that these sites are in close proximity to each other. Conclusions/Significance These findings illustrate the potential of this site-specific labeling system for use in future FRET-based experiments to elucidate novel aspects of RyR1 structure. PMID

  11. Förster resonance energy transfer measurements of ryanodine receptor type 1 structure using a novel site-specific labeling method.

    PubMed

    Fessenden, James D

    2009-10-12

    While the static structure of the intracellular Ca(2+) release channel, the ryanodine receptor type 1 (RyR1) has been determined using cryo electron microscopy, relatively little is known concerning changes in RyR1 structure that accompany channel gating. Förster resonance energy transfer (FRET) methods can resolve small changes in protein structure although FRET measurements of RyR1 are hampered by an inability to site-specifically label the protein with fluorescent probes. A novel site-specific labeling method is presented that targets a FRET acceptor, Cy3NTA to 10-residue histidine (His) tags engineered into RyR1. Cy3NTA, comprised of the fluorescent dye Cy3, coupled to two Ni(2+)/nitrilotriacetic acid moieties, was synthesized and functionally tested for binding to His-tagged green fluorescent protein (GFP). GFP fluorescence emission and Cy3NTA absorbance spectra overlapped significantly, indicating that FRET could occur (Förster distance = 6.3 nm). Cy3NTA bound to His(10)-tagged GFP, quenching its fluorescence by 88%. GFP was then fused to the N-terminus of RyR1 and His(10) tags were placed either at the N-terminus of the fused GFP or between GFP and RyR1. Cy3NTA reduced fluorescence of these fusion proteins by 75% and this quenching could be reversed by photobleaching Cy3, thus confirming GFP-RyR1 quenching via FRET. A His(10) tag was then placed at amino acid position 1861 and FRET was measured from GFP located at either the N-terminus or at position 618 to Cy3NTA bound to this His tag. While minimal FRET was detected between GFP at position 1 and Cy3NTA at position 1861, 53% energy transfer was detected from GFP at position 618 to Cy3NTA at position 1861, thus indicating that these sites are in close proximity to each other. These findings illustrate the potential of this site-specific labeling system for use in future FRET-based experiments to elucidate novel aspects of RyR1 structure.

  12. Malignant hyperthermia susceptibility arising from altered resting coupling between the skeletal muscle L-type Ca2+ channel and the type 1 ryanodine receptor

    PubMed Central

    Eltit, Jose Miguel; Bannister, Roger A.; Moua, Ong; Altamirano, Francisco; Hopkins, Philip M.; Pessah, Isaac N.; Molinski, Tadeusz F.; López, Jose R.; Beam, Kurt G.; Allen, Paul D.

    2012-01-01

    Malignant hyperthermia (MH) susceptibility is a dominantly inherited disorder in which volatile anesthetics trigger aberrant Ca2+ release in skeletal muscle and a potentially fatal rise in perioperative body temperature. Mutations causing MH susceptibility have been identified in two proteins critical for excitation–contraction (EC) coupling, the type 1 ryanodine receptor (RyR1) and CaV1.1, the principal subunit of the L-type Ca2+ channel. All of the mutations that have been characterized previously augment EC coupling and/or increase the rate of L-type Ca2+ entry. The CaV1.1 mutation R174W associated with MH susceptibility occurs at the innermost basic residue of the IS4 voltage-sensing helix, a residue conserved among all CaV channels [Carpenter D, et al. (2009) BMC Med Genet 10:104–115.]. To define the functional consequences of this mutation, we expressed it in dysgenic (CaV1.1 null) myotubes. Unlike previously described MH-linked mutations in CaV1.1, R174W ablated the L-type current and had no effect on EC coupling. Nonetheless, R174W increased sensitivity of Ca2+ release to caffeine (used for MH diagnostic in vitro testing) and to volatile anesthetics. Moreover, in CaV1.1 R174W-expressing myotubes, resting myoplasmic Ca2+ levels were elevated, and sarcoplasmic reticulum (SR) stores were partially depleted, compared with myotubes expressing wild-type CaV1.1. Our results indicate that CaV1.1 functions not only to activate RyR1 during EC coupling, but also to suppress resting RyR1-mediated Ca2+ leak from the SR, and that perturbation of CaV1.1 negative regulation of RyR1 leak identifies a unique mechanism that can sensitize muscle cells to MH triggers. PMID:22547813

  13. Modulation of sarcoplasmic reticulum Ca2+ release in skeletal muscle expressing ryanodine receptor impaired in regulation by calmodulin and S100A1.

    PubMed

    Yamaguchi, Naohiro; Prosser, Benjamin L; Ghassemi, Farshid; Xu, Le; Pasek, Daniel A; Eu, Jerry P; Hernández-Ochoa, Erick O; Cannon, Brian R; Wilder, Paul T; Lovering, Richard M; Weber, David; Melzer, Werner; Schneider, Martin F; Meissner, Gerhard

    2011-05-01

    In vitro, calmodulin (CaM) and S100A1 activate the skeletal muscle ryanodine receptor ion channel (RyR1) at submicromolar Ca(2+) concentrations, whereas at micromolar Ca(2+) concentrations, CaM inhibits RyR1. One amino acid substitution (RyR1-L3625D) has previously been demonstrated to impair CaM binding and regulation of RyR1. Here we show that the RyR1-L3625D substitution also abolishes S100A1 binding. To determine the physiological relevance of these findings, mutant mice were generated with the RyR1-L3625D substitution in exon 74, which encodes the CaM and S100A1 binding domain of RyR1. Homozygous mutant mice (Ryr1(D/D)) were viable and appeared normal. However, single RyR1 channel recordings from Ryr1(D/D) mice exhibited impaired activation by CaM and S100A1 and impaired CaCaM inhibition. Isolated flexor digitorum brevis muscle fibers from Ryr1(D/D) mice had depressed Ca(2+) transients when stimulated by a single action potential. However, during repetitive stimulation, the mutant fibers demonstrated greater relative summation of the Ca(2+) transients. Consistently, in vivo stimulation of tibialis anterior muscles in Ryr1(D/D) mice demonstrated reduced twitch force in response to a single action potential, but greater summation of force during high-frequency stimulation. During repetitive stimulation, Ryr1(D/D) fibers exhibited slowed inactivation of sarcoplasmic reticulum Ca(2+) release flux, consistent with increased summation of the Ca(2+) transient and contractile force. Peak Ca(2+) release flux was suppressed at all voltages in voltage-clamped Ryr1(D/D) fibers. The results suggest that the RyR1-L3625D mutation removes both an early activating effect of S100A1 and CaM and delayed suppressing effect of CaCaM on RyR1 Ca(2+) release, providing new insights into CaM and S100A1 regulation of skeletal muscle excitation-contraction coupling.

  14. Redox modification of ryanodine receptors by mitochondria-derived reactive oxygen species contributes to aberrant Ca2+ handling in ageing rabbit hearts.

    PubMed

    Cooper, Leroy L; Li, Weiyan; Lu, Yichun; Centracchio, Jason; Terentyeva, Radmila; Koren, Gideon; Terentyev, Dmitry

    2013-12-01

    Ageing is associated with a blunted response to sympathetic stimulation and an increased risk of arrhythmia and sudden cardiac death. Aberrant calcium (Ca(2+)) handling is an important contributor to the electrical and contractile dysfunction associated with ageing. Yet, the specific molecular mechanisms underlying abnormal Ca(2+) handling in ageing heart remain poorly understood. In this study, we used ventricular myocytes isolated from young (5-9 months) and old (4-6 years) rabbit hearts to test the hypothesis that changes in Ca(2+) homeostasis are caused by post-translational modification of ryanodine receptors (RyRs) by mitochondria-derived reactive oxygen species (ROS) generated in the ageing heart. Changes in parameters of Ca(2+) handling were determined by measuring cytosolic and intra-sarcoplasmic reticulum (SR) Ca(2+) dynamics in intact and permeabilized ventricular myocytes using confocal microscopy. We also measured age-related changes in ROS production and mitochondria membrane potential using a ROS-sensitive dye and a mitochondrial voltage-sensitive fluorescent indicator, respectively. In permeablized myocytes, ageing did not change SERCA activity and spark frequency but decreased spark amplitude and SR Ca(2+) load suggesting increased RyR activity. Treatment with the antioxidant dithiothreitol reduced RyR-mediated SR Ca(2+) leak in permeabilized myocytes from old rabbit hearts to the level comparable to young. Moreover, myocytes from old rabbits had more depolarized mitochondria membrane potential and increased rate of ROS production. Under β-adrenergic stimulation, Ca(2+) transient amplitude, SR Ca(2+) load, and latency of pro-arrhythmic spontaneous Ca(2+) waves (SCWs) were decreased while RyR-mediated SR Ca(2+) leak was increased in cardiomyocytes from old rabbits. Additionally, with β-adrenergic stimulation, scavenging of mitochondrial ROS in myocytes from old rabbit hearts restored redox status of RyRs, which reduced SR Ca(2+) leak, ablated most

  15. Molecular characteristics, mRNA expression, and alternative splicing of a ryanodine receptor gene in the oriental fruit fly, Bactrocera dorsalis (Hendel).

    PubMed

    Yuan, Guo-Rui; Shi, Wen-Zhi; Yang, Wen-Jia; Jiang, Xuan-Zhao; Dou, Wei; Wang, Jin-Jun

    2014-01-01

    Ryanodine receptors (RyRs) are a distinct class of ligand-gated channels controlling the release of calcium from intracellular stores. The emergence of diamide insecticides, which selectively target insect RyRs, has promoted the study of insect RyRs. In the present study, the full-length RyR cDNA (BdRyR) was cloned and characterized from the oriental fruit fly, Bactrocera dorsalis (Hendel), a serious pest of fruits and vegetables throughout East Asia and the Pacific Rim. The cDNA of BdRyR contains a 15,420-bp open reading frame encoding 5,140 amino acids with a predicted molecular weight of 582.4 kDa and an isoelectric point of 5.38. BdRyR shows a high level of amino acid sequence identity (78 to 97%) to other insect RyR isoforms. All common structural features of the RyRs are present in the BdRyR, including a well-conserved C-terminal domain containing consensus calcium-binding EF-hands and six transmembrane domains, and a large N-terminal domain. Quantitative real-time PCR analyses revealed that BdRyR was expressed at the lowest and highest levels in egg and adult, respectively, and that the BdRyR expression levels in the third instar larva, pupa and adult were 166.99-, 157.56- and 808.56-fold higher, respectively, than that in the egg. Among different adult body parts, the highest expression level was observed in the thorax compared with the head and abdomen. In addition, four alternative splice sites were identified in the BdRyR gene, with the first, ASI, being located in the central part of the predicted second spore lysis A/RyR domain. Diagnostic PCR analyses revealed that alternative splice variants were generated not only in a tissue-specific manner but also in a developmentally regulated manner. These results lay the foundation for further understanding the structural and functional properties of BdRyR, and the molecular mechanisms for target site resistance in B. dorsalis.

  16. The Arrhythmogenic Calmodulin p.Phe142Leu Mutation Impairs C-domain Ca2+ Binding but Not Calmodulin-dependent Inhibition of the Cardiac Ryanodine Receptor.

    PubMed

    Søndergaard, Mads Toft; Liu, Yingjie; Larsen, Kamilla Taunsig; Nani, Alma; Tian, Xixi; Holt, Christian; Wang, Ruiwu; Wimmer, Reinhard; Van Petegem, Filip; Fill, Michael; Chen, S R Wayne; Overgaard, Michael Toft

    2017-01-27

    A number of point mutations in the intracellular Ca(2+)-sensing protein calmodulin (CaM) are arrhythmogenic, yet their underlying mechanisms are not clear. These mutations generally decrease Ca(2+) binding to CaM and impair inhibition of CaM-regulated Ca(2+) channels like the cardiac Ca(2+) release channel (ryanodine receptor, RyR2), and it appears that attenuated CaM Ca(2+) binding correlates with impaired CaM-dependent RyR2 inhibition. Here, we investigated the RyR2 inhibitory action of the CaM p.Phe142Leu mutation (F142L; numbered including the start-Met), which markedly reduces CaM Ca(2+) binding. Surprisingly, CaM-F142L had little to no aberrant effect on RyR2-mediated store overload-induced Ca(2+) release in HEK293 cells compared with CaM-WT. Furthermore, CaM-F142L enhanced CaM-dependent RyR2 inhibition at the single channel level compared with CaM-WT. This is in stark contrast to the actions of arrhythmogenic CaM mutations N54I, D96V, N98S, and D130G, which all diminish CaM-dependent RyR2 inhibition. Thermodynamic analysis showed that apoCaM-F142L converts an endothermal interaction between CaM and the CaM-binding domain (CaMBD) of RyR2 into an exothermal one. Moreover, NMR spectra revealed that the CaM-F142L-CaMBD interaction is structurally different from that of CaM-WT at low Ca(2+) These data indicate a distinct interaction between CaM-F142L and the RyR2 CaMBD, which may explain the stronger CaM-dependent RyR2 inhibition by CaM-F142L, despite its reduced Ca(2+) binding. Collectively, these results add to our understanding of CaM-dependent regulation of RyR2 as well as the mechanistic effects of arrhythmogenic CaM mutations. The unique properties of the CaM-F142L mutation may provide novel clues on how to suppress excessive RyR2 Ca(2+) release by manipulating the CaM-RyR2 interaction. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

  17. The Arrhythmogenic Calmodulin p.Phe142Leu Mutation Impairs C-domain Ca2+ Binding but Not Calmodulin-dependent Inhibition of the Cardiac Ryanodine Receptor*

    PubMed Central

    Liu, Yingjie; Larsen, Kamilla Taunsig; Nani, Alma; Tian, Xixi; Holt, Christian; Wang, Ruiwu; Fill, Michael

    2017-01-01

    A number of point mutations in the intracellular Ca2+-sensing protein calmodulin (CaM) are arrhythmogenic, yet their underlying mechanisms are not clear. These mutations generally decrease Ca2+ binding to CaM and impair inhibition of CaM-regulated Ca2+ channels like the cardiac Ca2+ release channel (ryanodine receptor, RyR2), and it appears that attenuated CaM Ca2+ binding correlates with impaired CaM-dependent RyR2 inhibition. Here, we investigated the RyR2 inhibitory action of the CaM p.Phe142Leu mutation (F142L; numbered including the start-Met), which markedly reduces CaM Ca2+ binding. Surprisingly, CaM-F142L had little to no aberrant effect on RyR2-mediated store overload-induced Ca2+ release in HEK293 cells compared with CaM-WT. Furthermore, CaM-F142L enhanced CaM-dependent RyR2 inhibition at the single channel level compared with CaM-WT. This is in stark contrast to the actions of arrhythmogenic CaM mutations N54I, D96V, N98S, and D130G, which all diminish CaM-dependent RyR2 inhibition. Thermodynamic analysis showed that apoCaM-F142L converts an endothermal interaction between CaM and the CaM-binding domain (CaMBD) of RyR2 into an exothermal one. Moreover, NMR spectra revealed that the CaM-F142L-CaMBD interaction is structurally different from that of CaM-WT at low Ca2+. These data indicate a distinct interaction between CaM-F142L and the RyR2 CaMBD, which may explain the stronger CaM-dependent RyR2 inhibition by CaM-F142L, despite its reduced Ca2+ binding. Collectively, these results add to our understanding of CaM-dependent regulation of RyR2 as well as the mechanistic effects of arrhythmogenic CaM mutations. The unique properties of the CaM-F142L mutation may provide novel clues on how to suppress excessive RyR2 Ca2+ release by manipulating the CaM-RyR2 interaction. PMID:27927985

  18. Rank-based genome-wide analysis reveals the association of Ryanodine receptor-2 gene variants with childhood asthma among human populations

    PubMed Central

    2013-01-01

    Background The standard approach to determine unique or shared genetic factors across populations is to identify risk alleles in one population and investigate replication in others. However, since populations differ in DNA sequence information, allele frequencies, effect sizes, and linkage disequilibrium patterns, SNP association using a uniform stringent threshold on p values may not be reproducible across populations. Here, we developed rank-based methods to investigate shared or population-specific loci and pathways for childhood asthma across individuals of diverse ancestry. We performed genome-wide association studies on 859,790 SNPs genotyped in 527 affected offspring trios of European, African, and Hispanic ancestry using publically available asthma database in the Genotypes and Phenotypes database. Results Rank-based analyses showed that there are shared genetic factors for asthma across populations, more at the gene and pathway levels than at the SNP level. Although the top 1,000 SNPs were not shared, 11 genes (RYR2, PDE4D, CSMD1, CDH13, ROBO2, RBFOX1, PTPRD, NPAS3, PDE1C, SEMA5A, and CTNNA2) mapped by these SNPs were shared across populations. Ryanodine receptor 2 (RYR2, a statin response-related gene) showed the strongest associa