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Vincenzo Sorrentino - One of the best experts on this subject based on the ideXlab platform.

  • cardiac expression of ryanodine receptor subtype 3 a strategic component in the intracellular ca2 release system of purkinje fibers in large mammalian heart
    Journal of Molecular and Cellular Cardiology, 2017
    Co-Authors: Rebecca E Daniels, Vincenzo Sorrentino, Lawson Miller, Elizabeth W Chia, Masahito Miura, John J Mcguire, Bruno D Stuyvers
    Abstract:

    Abstract Background Three distinct Ca 2 + release channels were identified in dog P-cells: the ryanodine receptor subtype 2 ( RyR2 ) was detected throughout the cell, while the ryanodine receptor subtype 3 ( RYR3 ) and inositol phosphate sensitive Ca 2 + release channel ( InsP3R ) were found in the cell periphery. How each of these channels contributes to the Ca 2 + cycling of P-cells is unclear. Recent modeling of Ca 2 + mobilization in P-cells suggested that Ca 2 + sensitivity of Ca 2 + induced Ca 2 + release ( CICR ) was larger at the P-cell periphery. Our study examined whether this numerically predicted region of Ca 2 + release exists in live P-cells. We compared the regional Ca 2 + dynamics with the arrangement of intracellular Ca 2 + release ( CR ) channels. Methods Gene expression of CR channels was measured by qPCR in Purkinje fibers and myocardium of adult Yucatan pig hearts. We characterized the CR channels protein expression in isolated P-cells by immuno-fluorescence, laser scanning confocal microscopy, and 3D reconstruction. The spontaneous Ca 2 + activity and electrically-evoked Ca 2 + mobilization were imaged by 2D spinning disk confocal microscopy. Functional regions of P-cell were differentiated by the characteristics of local Ca 2 + events. We used the Ca 2 + propagation velocities as indicators of channel Ca 2 + sensitivity. Results RyR2 gene expression was identical in Purkinje fibers and myocardium (6 hearts) while RYR3 and InsP 3 R gene expressions were, respectively, 100 and 16 times larger in the Purkinje fibers. Specific fluorescent immuno-staining of Ca 2 + release channels revealed an intermediate layer of RYR3 expression between a near-membrane InsP3R -region and a central RyR2 -region. We found that cell periphery produced two distinct forms of spontaneous Ca 2 + -transients: (1) large asymmetrical Ca 2 + sparks under the membrane, and (2) typical Ca 2 + -wavelets propagating exclusively around the core of the cell. Larger cell-wide Ca 2 + waves ( CWW s) appeared occasionally traveling in the longitudinal direction through the core of Pcells. Large sparks arose in a micrometric space overlapping the InsP3R expression. The InsP3R antagonists 2-aminoethoxydiphenyl borate (2-APB; 3 μM) and xestospongin C (XeC; 50 μM) dramatically reduced their frequency. The Ca 2 + wavelets propagated in a 5–10 μm thick layered space which matched the intermediate zone of RYR3 expression. The wavelet incidence was unchanged by 2-APB or XeC, but was reduced by 60% in presence of the RYR3 antagonist dantrolene (10 μM). The velocity of wavelets was two times larger (86 ± 16 μm/s; n = 14) compared to CWWs' (46 ± 10 μm/s; n = 11; P 2 + concentration under the membrane which preceded the propagation of Ca 2 + into the interior of the cell. Elevated Ca i propagated at 150 μm/s (147 ± 34 μm/s; n = 5) through the region equivalent to the zone of RYR3 expression. This velocity dropped by 50% (75 ± 24 μm/s; n = 5) in the central region wherein predominant RyR2 expression was detected. Conclusion We identified two layers of distinct Ca 2 + release channels in the periphery of Pcell: an outer layer of InsP 3 Rs under the membrane and an inner layer of RYR3s . The propagation of Ca 2 + events in these layers revealed that Ca 2 + sensitivity of Ca 2 + release was larger in the RYR3 layer compared to that of other sub-cellular regions. We propose that RYR3 expression in P-cells plays a role in the stability of electric function of Purkinje fibers.

  • type 3 ryanodine receptors mediate hypoxia but not neurotransmitter induced calcium release and contraction in pulmonary artery smooth muscle cells
    The Journal of General Physiology, 2005
    Co-Authors: Yunmin Zheng, Vincenzo Sorrentino, Qingsong Wang, Rakesh Rathore, Wanhui Zhang, Joseph E Mazurkiewicz, Harold A Singer, Michael I Kotlikoff, Yongxiao Wang
    Abstract:

    In this study we examined the expression of RyR subtypes and the role of RyRs in neurotransmitter- and hypoxia-induced Ca2+ release and contraction in pulmonary artery smooth muscle cells (PASMCs). Under perforated patch clamp conditions, maximal activation of RyRs with caffeine or inositol triphosphate receptors (IP3Rs) with noradrenaline induced equivalent increases in [Ca2+]i and Ca2+-activated Cl− currents in freshly isolated rat PASMCs. Following maximal IP3-induced Ca2+ release, neither caffeine nor chloro-m-cresol induced a response, whereas prior application of caffeine or chloro-m-cresol blocked IP3-induced Ca2+ release. In cultured human PASMCs, which lack functional expression of RyRs, caffeine failed to affect ATP-induced increases in [Ca2+]i in the presence and absence of extracellular Ca2+. The RyR antagonists ruthenium red, ryanodine, tetracaine, and dantrolene greatly inhibited submaximal noradrenaline– and hypoxia-induced Ca2+ release and contraction in freshly isolated rat PASMCs, but did not affect ATP-induced Ca2+ release in cultured human PASMCs. Real-time quantitative RT-PCR and immunofluorescence staining indicated similar expression of all three RyR subtypes (RyR1, RyR2, and RYR3) in freshly isolated rat PASMCs. In freshly isolated PASMCs from RYR3 knockout (RYR3−/−) mice, hypoxia-induced, but not submaximal noradrenaline–induced, Ca2+ release and contraction were significantly reduced. Ruthenium red and tetracaine can further inhibit hypoxic increase in [Ca2+]i in RYR3−/− mouse PASMCs. Collectively, our data suggest that (a) RyRs play an important role in submaximal noradrenaline– and hypoxia-induced Ca2+ release and contraction; (b) all three subtype RyRs are expressed; and (c) RYR3 gene knockout significantly inhibits hypoxia-, but not submaximal noradrenaline–induced Ca2+ and contractile responses in PASMCs.

  • RyR1 and RYR3 isoforms provide distinct intracellular Ca2+ signals in HEK 293 cells.
    Journal of cell science, 2002
    Co-Authors: Daniela Rossi, Ilenia Simeoni, Paul D. Allen, Marcella Micheli, Martin Bootman, Peter Lipp, Vincenzo Sorrentino
    Abstract:

    Ryanodine receptors (RyRs) are expressed on the endoplasmic reticulum of many cells, where they form intracellular Ca2+-release channels that participate in the generation of intracellular Ca2+ signals. Here we report studies on the intracellular localisation and functional properties of transfected RyR1 or RYR3 channels in HEK 293 cells. Immunofluorescence studies indicated that both RyR1 and RYR3 did not form clusters but were homogeneously distributed throughout the endoplasmic reticulum. Ca2+ release experiments showed that transfected RyR1 and RYR3 channels responded to caffeine, although with different sensitivity, generating a global release of Ca2+ from the entire endoplasmic reticulum. However, video imaging and confocal microscopy analysis revealed that, in RYR3-expressing cells, local spontaneous Ca2+ release events were observed. No such spontaneous activity was observed in RyR1-expressing cells or in control cells. Interestingly, the spontaneous release events observed in RYR3-expressing cells were restricted to one or two regions of the endoplasmic reticulum, suggesting the formation of a further subcellular organisation of RYR3 in Ca2+ release units. These results demonstrate that different RyR isoforms can engage in the generation of distinct intracellular Ca2+ signals in HEK 293 cells.

  • regulation of calcium sparks and spontaneous transient outward currents by RYR3 in arterial vascular smooth muscle cells
    Circulation Research, 2001
    Co-Authors: Matthias Lohn, Vincenzo Sorrentino, Michael Furstenau, Friedrich C Luft, Hermann Haller, Wolfgang Jessner, Maren Wellner, Maik Gollasch
    Abstract:

    Intracellular Ca 2+ levels control both contraction and relaxation in vascular smooth muscle cells (VSMCs). Ca 2+ -dependent relaxation is mediated by discretely localized Ca 2+ release events through ryanodine receptor (RyR) channels in the sarcoplasmic reticulum (SR). These local increases in Ca 2+ concentration, termed sparks, stimulate nearby Ca 2+ -activated K + (BK) channels causing BK currents (spontaneous transient outward currents or STOCs). STOCs are hyperpolarizing currents that oppose vasoconstriction. Several RyR isoforms are coexpressed in VSMCs; however, their role in Ca 2+ spark generation is unknown. To provide molecular information on RyR cluster function and assembly, we examined Ca 2+ sparks and STOCs in RYR3-deficient freshly isolated myocytes of resistance-sized cerebral arteries from knockout mice and compared them to Ca 2+ sparks in cells from wild-type mice. We used RT-PCR to identify RyR1, RyR2, and RYR3 mRNA in cerebral arteries. Ca 2+ sparks in RYR3-deficient cells were similar in peak amplitude (measured as F/F 0 ), width at half-maximal amplitude, and duration compared with wild-type cell Ca 2+ sparks. However, the frequency of STOCs (between −60 mV and −20 mV) was significantly higher in RYR3-deficient cells than in wild-type cells. Ca 2+ sparks and STOCs in both RYR3-deficient and wild-type cells were inhibited by ryanodine (10 μmol/L), external Ca 2+ removal, and depletion of SR Ca 2+ stores by caffeine (1 mmol/L). Isolated, pressurized cerebral arteries of RYR3-deficient mice developed reduced myogenic tone. Our results suggest that RYR3 is part of the SR Ca 2+ spark release unit and plays a specific molecular role in the regulation of STOCs frequency in mouse cerebral artery VSMCs after decreased arterial tone.

  • functional properties of the ryanodine receptor type 3 RYR3 ca2 release channel
    The EMBO Journal, 1998
    Co-Authors: Alois Sonnleitner, Antonio Conti, Federica Bertocchini, Hansgeorg Schindler, Vincenzo Sorrentino
    Abstract:

    Single-channel analysis of sarcoplasmic reticulum vesicles prepared from diaphragm muscle, which contains both RyR1 and RYR3 isoforms, revealed the presence of two functionally distinct ryanodine receptor calcium release channels. In addition to channels with properties typical of RyR1 channels, a second population of ryanodine-sensitive channels with properties distinct from those of RyR1 channels was observed. The novel channels displayed close-to-zero open-probability at nanomolar Ca2+ concentrations in the presence of 1 mM ATP, but were shifted to the open conformation by increasing Ca2+ to micromolar levels and were not inhibited at higher Ca2+ concentrations. These novel channels were sensitive to the stimulatory effects of cyclic adenosine 5'-diphosphoribose (cADPR). Detection of this second population of RyR channels in lipid bilayers was always associated with the presence of the RYR3 isoform in muscle preparations used for single-channel measurements and was abrogated by the knockout of the RYR3 gene in mice. Based on the above, we associated the novel population of channels with the RYR3 isoform of Ca2+ release channels. The functional properties of the RYR3 channels are in agreement with a potential qualitative contribution of this channel to Ca2+ release in skeletal muscle and in other tissues.

Andrew R. Marks - One of the best experts on this subject based on the ideXlab platform.

  • essential roles of intracellular calcium release channels in muscle brain metabolism and aging
    Current Molecular Pharmacology, 2015
    Co-Authors: Gaetano Santulli, Andrew R. Marks
    Abstract:

    Abstract Calcium (Ca(2+)) release from intracellular stores controls numerous cellular processes, including cardiac and skeletal muscle contraction, synaptic transmission and metabolism. The ryanodine receptors (RyRs: RyR1, RyR2, RYR3) and inositol 1,4,5-trisphosphate receptors (IP3Rs: IP3R1, IP3R2, IP3R3) are the major Ca(2+) release channels (CRCs) on the endo/sarcoplasmic reticulum (ER/SR). RyRs and IP3Rs comprise macromolecular signaling complexes that include modulatory proteins which regulate channel activity in response to extracellular signals resulting in intracellular Ca(2+) release. Here we focus on the roles of CRCs in heart, skeletal muscle, brain, metabolism, and aging.

  • enhancing calstabin binding to ryanodine receptors improves cardiac and skeletal muscle function in heart failure
    Proceedings of the National Academy of Sciences of the United States of America, 2005
    Co-Authors: Xander H T Wehrens, Steven Reiken, Stephan E Lehnart, Roel Van Der Nagel, Raymond Morales, Zhenzhuang Cheng, Shixiang Deng, Leon J De Windt, Donald W. Landry, Andrew R. Marks
    Abstract:

    Abstract Abnormalities in intracellular calcium release and reuptake are responsible for decreased contractility in heart failure (HF). We have previously shown that cardiac ryanodine receptors (RyRs) are protein kinase A-hyperphosphorylated and depleted of the regulatory subunit calstabin-2 in HF. Moreover, similar alterations in skeletal muscle RyR have been linked to increased fatigability in HF. To determine whether restoration of calstabin binding to RyR may ameliorate cardiac and skeletal muscle dysfunction in HF, we treated WT and calstabin-2-/- mice subjected to myocardial infarction (MI) with JTV519. JTV519, a 1,4-benzothiazepine, is a member of a class of drugs known as calcium channel stabilizers, previously shown to increase calstabin binding to RyR. Echocardiography at 21 days after MI demonstrated a significant increase in ejection fraction in WT mice treated with JTV519 (45.8 ± 5.1%) compared with placebo (31.1 ± 3.1%; P < 0.05). Coimmunoprecipitation experiments revealed increased amounts of calstabin-2 bound to the RyR2 channel in JTV519-treated WT mice. However, JTV519 did not show any of these beneficial effects in calstabin-2-/- mice with MI. Additionally, JTV519 improved skeletal muscle fatigue in WT and calstabin-2-/- mice with HF by increasing the binding of calstabin-1 to RyR1. The observation that treatment with JTV519 improved cardiac function in WT but not calstabin-2-/- mice indicates that calstabin-2 binding to RyR2 is required for the beneficial effects in failing hearts. We conclude that JTV519 may provide a specific way to treat the cardiac and skeletal muscle myopathy in HF by increasing calstabin binding to RyR. calcium FKBP12.6 myocardial infarction contractility

  • intracellular calcium release and cardiac disease
    Annual Review of Physiology, 2005
    Co-Authors: Xander H T Wehrens, Stephan E Lehnart, Andrew R. Marks
    Abstract:

    ▪ Abstract Intracellular calcium release channels are present on sarcoplasmic and endoplasmic reticuli (SR, ER) of all cell types. There are two classes of these channels: ryanodine receptors (RyR) and inositol 1,4,5-trisphosphate receptors (IP3R). RyRs are required for excitation-contraction (EC) coupling in striated (cardiac and skeletal) muscles. RyRs are made up of macromolecular signaling complexes that contain large cytoplasmic domains, which serve as scaffolds for proteins that regulate the function of the channel. These regulatory proteins include calstabin1/calstabin2 (FKBP12/FKBP12.6), a 12/12.6 kDa subunit that stabilizes the closed state of the channel and prevents aberrant calcium leak from the SR. Kinases and phosphatases are targeted to RyR2 channels and modulate RyR2 function in response to extracellular signals. In the classic fight or flight stress response, phosphorylation of RyR channels by protein kinase A reduces the affinity for calstabin and activates the channels leading to increa...

  • immunophilins and coupled gating of ryanodine receptors
    Current Topics in Medicinal Chemistry, 2003
    Co-Authors: Stephan E Lehnart, Fannie Huang, Steven O Marx, Andrew R. Marks
    Abstract:

    The ryanodine receptor (RyR) is the major calcium (Ca2+) release channel in the sarcoplasmic reticulum (SR) of skeletal and cardiac muscle and is required for excitation-contraction (EC) coupling. The 565 kDa RyR protein forms a tetrameric channel that is part of a macromolecular signaling complex that also includes four FK506 binding proteins (FKBPs). The RyR channel complex is localized on specialized regions of the SR, such that the large RyR cytoplasmic domain is closely opposed to the transverse tubule (T-tubule) of the plasma membrane. RyR channel complexes are organized in regular arrays such that neighboring RyRs are in physical contact with each other. We have shown that physical and functional association between RyR1 or RyR2 channels results in coordinated gating behavior termed coupled gating. Coupled gating requires FKBP12 or FKBP12.6 in the RyR1 or RyR2 macromolecular complexes, respectively. FKBPs are known to stabilize single RyR channel function. Coupled gating describes an additional role for FKBPs in the functional coordination of RyR channel complexes that allows clusters of channels to function as “Ca2+ release units” (CRU). In addition, the FKBP-RyR interaction is regulated by PKA phosphorylation. In failing hearts PKA hyperphosphorylation of RyR2 causes depletion of FKBP12.6 from the channel macromolecular complex and may contribute to contractile dysfunction by impairing EC coupling. As FKBPs are potent modulators of RyR channel function, the FKBP-RyR interaction is a focus for determining molecular mechanisms of coupled gating and presents an exciting pharmacologic target for restoration of RyR complex function in diseased states.

  • involvement of the cardiac ryanodine receptor calcium release channel in catecholaminergic polymorphic ventricular tachycardia
    Journal of Cellular Physiology, 2002
    Co-Authors: Andrew R. Marks, Silvia G Priori, Mirella Memmi, Kimmo Kontula, Paivi Laitinen
    Abstract:

    The cardiac ryanodine receptor (RyR2), the major calcium release channel on the sarcoplasmic reticulum (SR) in cardiomyocytes, has recently been shown to be involved in at least two forms of sudden cardiac death (SCD): (1) Catecholaminergic polymorphic ventricular tachycardia (CPVT) or familial polymorphic VT (FPVT); and (2) Arrhythmogenic right ventricular dysplasia type 2 (ARVD2). Eleven RyR2 missense mutations have been linked to these diseases. All eleven RyR2 mutations cluster into 3 regions of RyR2 that are homologous to the three malignant hyperthermia (MH)/central core disease (CCD) mutation regions of the skeletal muscle ryanodine receptor/calcium release channel RyR1. MH/CCD RyR1 mutations have been shown to alter calcium-induced calcium release. Sympathetic nervous system stimulation leads to phosphorylation of RyR2 by protein kinase A (PKA). PKA phosphorylation of RyR2 activates the channel. In conditions associated with high rates of SCD such as heart failure RyR2 is PKA hyperphosphorylated resulting in “leaky” channels. SR calcium leak during diastole can generate “delayed after depolarizations” that can trigger fatal cardiac arrhythmias (e.g., VT). We propose that RyR2 mutations linked to genetic forms of catecholaminergic-induced SCD may alter the regulation of the channel resulting in increased SR calcium leak during sympathetic stimulation. J. Cell. Physiol. 190: 1–6, 2002. © 2002 Wiley-Liss, Inc.

Takashi Murayama - One of the best experts on this subject based on the ideXlab platform.

  • regulatory mechanisms of ryanodine receptor ca 2 release channel revealed by recent advancements in structural studies
    Journal of Muscle Research and Cell Motility, 2020
    Co-Authors: Haruo Ogawa, Nagomi Kurebayashi, Toshiko Yamazawa, Takashi Murayama
    Abstract:

    Ryanodine receptors (RyRs) are huge homotetrameric Ca(2+) release channels localized to the sarcoplasmic reticulum. RyRs are responsible for the release of Ca(2+) from the SR during excitation-contraction coupling in striated muscle cells. Recent revolutionary advancements in cryo-electron microscopy have provided a number of near-atomic structures of RyRs, which have enabled us to better understand the architecture of RyRs. Thus, we are now in a new era understanding the gating, regulatory and disease-causing mechanisms of RyRs. Here we review recent advances in the elucidation of the structures of RyRs, especially RyR1 in skeletal muscle, and their mechanisms of regulation by small molecules, associated proteins and disease-causing mutations.

  • a tryptophan residue in the caffeine binding site of the ryanodine receptor regulates ca 2 sensitivity
    Communications biology, 2018
    Co-Authors: Takashi Murayama, Nagomi Kurebayashi, Haruo Ogawa, Seiko Ohno, Minoru Horie, Takashi Sakurai
    Abstract:

    Ryanodine receptors (RyRs) are Ca2+ release channels in the sarcoplasmic reticulum of skeletal and cardiac muscles and are essential for muscle contraction. Mutations in genes encoding RyRs cause various muscle and arrhythmogenic heart diseases. Although RyR channels are activated by Ca2+, the actual mechanism of Ca2+ binding remains largely unknown. Here, we report the molecular basis of Ca2+ binding to RyRs for channel activation and discuss its implications in disease states. RyR1 and RyR2 carrying mutations in putative Ca2+ and caffeine-binding sites were functionally analysed. The results were interpreted with respect to recent near-atomic resolution RyR1 structures in various ligand states. We demonstrate that a tryptophan residue in the caffeine-binding site controls the structure of the Ca2+-binding site to regulate the Ca2+ sensitivity. Our results reveal the initial step of RyR channel activation by Ca2+ and explain the molecular mechanism of Ca2+ sensitization by caffeine and disease-causing mutations.

  • putative roles of type 3 ryanodine receptor isoforms RYR3
    Trends in Cardiovascular Medicine, 2000
    Co-Authors: Yasuo Ogawa, Nagomi Kurebayashi, Takashi Murayama
    Abstract:

    Abstract Ca 2 + -release from the sarcoplasmic or endoplasmic reticulum, the intracellular Ca 2 + store, is mediated by the ryanodine receptor (RyR) and/or the inositol trisphosphate receptor (IP3R). While IP3R is a ligand(IP3)-operated channel, RyR can be gated by a ligand (Ca 2 + ) and/or mechanical coupling with the voltage sensor. There are three genetically distinct isoforms among RyR in mammals: RyR1–3. RyR1, the primary isoform in the skeletal muscle, can be gated by direct or indirect coupling with the conformation change of the α1S subunit of dihydropyridine receptor (DHPR) on the T-tubules (transversely invaginated sarcolemma) upon depolarization of skeletal muscles or by the increased cytoplasmic Ca 2 + (Ca 2 + -induced Ca 2 + release, CICR). RyR2, the primary isoform in the cardiac ventricular muscle (and, in a lesser amount, the brain), can be gated by Ca 2 + which flows in through DHPR, especially the α1C subunit on depolarization. RYR3 is distributed ubiquitously in various tissues and may be coexpressed with RyR1 and RyR2. RYR3 is considered to be similar to RyR2 in the respect that it can be activated by Ca 2 + , in view of the lack of available evidence to show the activation by the α1S subunit. Therefore, it is anticipated that RYR3 might take part through CICR in Ca 2 + signaling in smooth muscle and other non-muscle cells. To address the possible involvement of the CICR mechanism in the Ca 2 + signal transduction, it is critical to assess the effect of Mg 2 + on the CICR activity and the cytoplasmic concentration of Mg 2 + . In this brief review, our discussion focuses on the effects of Ca 2 + and Mg 2 + on the activity of RYR3.

  • further characterization of the type 3 ryanodine receptor RYR3 purified from rabbit diaphragm
    Journal of Biological Chemistry, 1999
    Co-Authors: Takashi Murayama, Toshiharu Oba, Eisaku Katayama, Hideto Oyamada, Katsuji Oguchi, Masakazu A R Kobayashi, Kazuyuki Otsuka, Yasuo Ogawa
    Abstract:

    We characterized type 3 ryanodine receptor (RYR3) purified from rabbit diaphragm by immunoaffinity chromatography using a specific antibody. The purified receptor was free from 12-kDa FK506-binding protein, although it retained the ability to bind 12-kDa FK506-binding protein. Negatively stained images of RYR3 show a characteristic rectangular structure that was indistinguishable from RyR1. The location of the D2 segment, which exists uniquely in the RyR1 isoform, was determined as the region around domain 9 close to the corner of the square-shaped assembly, with use of D2-directed antibody as a probe. The RYR3 homotetramer had a single class of high affinity [3H]ryanodine-binding sites with a stoichiometry of 1 mol/mol. In planar lipid bilayers, RYR3 displayed cation channel activity that was modulated by several ligands including Ca2+, Mg2+, caffeine, and ATP, which is consistent with [3H]ryanodine binding activity. RYR3 showed a slightly larger unit conductance and a longer mean open time than RyR1. Whereas RyR1 showed two classes of channel activity with distinct open probabilities (P o), RYR3 displayed a homogeneous and steeply Ca2+-dependent activity withP o ∼1. RYR3 was more steeply affected in the channel activity by sulfhydryl-oxidizing and -reducing reagents than RyR1, suggesting that the channel activity of RYR3 may be transformed more precipitously by the redox state. This is also a likely explanation for the difference in the Ca2+ dependence of RYR3 between [3H]ryanodine binding and channel activity.

  • characterization of type 3 ryanodine receptor RYR3 of sarcoplasmic reticulum from rabbit skeletal muscles
    Journal of Biological Chemistry, 1997
    Co-Authors: Takashi Murayama, Yasuo Ogawa
    Abstract:

    Abstract We investigated type 3 isoform (RYR3) of ryanodine receptor in rabbit skeletal muscles using an antibody specific for RYR3. By Western blot analysis and by immunoprecipitation, a single polypeptide for RYR3 was detected in sarcoplasmic reticulum vesicles from rabbit diaphragm but not in those from back muscle. The molecular mass was slightly smaller than that of RyR1, the major isoform in skeletal muscles. Each of RyR1 and RYR3 formed a homotetramer in rabbit diaphragm. RYR3 had a single class of [3H]ryanodine binding sites of high affinity (K D = 1.6 nm). From theB max of the binding, the content of RYR3 was estimated to be only 0.6% of RyR1 in rabbit diaphragm. [3H]Ryanodine binding to RYR3 was biphasically dependent on Ca2+, as is true of RyR1, and was stimulated further by adenine nucleotide, caffeine, or high salt concentration. Procaine and ruthenium red inhibited the binding. RYR3 was more resistant to Mg2+ inhibition than RyR1. Interestingly, RYR3 showed about a 7-fold lower Ca2+ sensitivity for activation than RyR1. Comparison with the counterparts in bullfrog skeletal muscles indicates that the Ca2+ sensitivities of RYR3 homologs are similar to each other, whereas those of RyR1 homologs are species-specific.

Yasuo Ogawa - One of the best experts on this subject based on the ideXlab platform.

  • mg2 activates the ryanodine receptor type 2 ryr2 at intermediate ca2 concentrations
    American Journal of Physiology-cell Physiology, 2007
    Co-Authors: Akihito Chugun, Hiroshi Takeshima, Osamu Sato, Yasuo Ogawa
    Abstract:

    To clarify whether activity of the ryanodine receptor type 2 (RyR2) is reduced in the sarcoplasmic reticulum (SR) of cardiac muscle, as is the case with the ryanodine receptor type 1 (RyR1), Ca2+-d...

  • putative roles of type 3 ryanodine receptor isoforms RYR3
    Trends in Cardiovascular Medicine, 2000
    Co-Authors: Yasuo Ogawa, Nagomi Kurebayashi, Takashi Murayama
    Abstract:

    Abstract Ca 2 + -release from the sarcoplasmic or endoplasmic reticulum, the intracellular Ca 2 + store, is mediated by the ryanodine receptor (RyR) and/or the inositol trisphosphate receptor (IP3R). While IP3R is a ligand(IP3)-operated channel, RyR can be gated by a ligand (Ca 2 + ) and/or mechanical coupling with the voltage sensor. There are three genetically distinct isoforms among RyR in mammals: RyR1–3. RyR1, the primary isoform in the skeletal muscle, can be gated by direct or indirect coupling with the conformation change of the α1S subunit of dihydropyridine receptor (DHPR) on the T-tubules (transversely invaginated sarcolemma) upon depolarization of skeletal muscles or by the increased cytoplasmic Ca 2 + (Ca 2 + -induced Ca 2 + release, CICR). RyR2, the primary isoform in the cardiac ventricular muscle (and, in a lesser amount, the brain), can be gated by Ca 2 + which flows in through DHPR, especially the α1C subunit on depolarization. RYR3 is distributed ubiquitously in various tissues and may be coexpressed with RyR1 and RyR2. RYR3 is considered to be similar to RyR2 in the respect that it can be activated by Ca 2 + , in view of the lack of available evidence to show the activation by the α1S subunit. Therefore, it is anticipated that RYR3 might take part through CICR in Ca 2 + signaling in smooth muscle and other non-muscle cells. To address the possible involvement of the CICR mechanism in the Ca 2 + signal transduction, it is critical to assess the effect of Mg 2 + on the CICR activity and the cytoplasmic concentration of Mg 2 + . In this brief review, our discussion focuses on the effects of Ca 2 + and Mg 2 + on the activity of RYR3.

  • further characterization of the type 3 ryanodine receptor RYR3 purified from rabbit diaphragm
    Journal of Biological Chemistry, 1999
    Co-Authors: Takashi Murayama, Toshiharu Oba, Eisaku Katayama, Hideto Oyamada, Katsuji Oguchi, Masakazu A R Kobayashi, Kazuyuki Otsuka, Yasuo Ogawa
    Abstract:

    We characterized type 3 ryanodine receptor (RYR3) purified from rabbit diaphragm by immunoaffinity chromatography using a specific antibody. The purified receptor was free from 12-kDa FK506-binding protein, although it retained the ability to bind 12-kDa FK506-binding protein. Negatively stained images of RYR3 show a characteristic rectangular structure that was indistinguishable from RyR1. The location of the D2 segment, which exists uniquely in the RyR1 isoform, was determined as the region around domain 9 close to the corner of the square-shaped assembly, with use of D2-directed antibody as a probe. The RYR3 homotetramer had a single class of high affinity [3H]ryanodine-binding sites with a stoichiometry of 1 mol/mol. In planar lipid bilayers, RYR3 displayed cation channel activity that was modulated by several ligands including Ca2+, Mg2+, caffeine, and ATP, which is consistent with [3H]ryanodine binding activity. RYR3 showed a slightly larger unit conductance and a longer mean open time than RyR1. Whereas RyR1 showed two classes of channel activity with distinct open probabilities (P o), RYR3 displayed a homogeneous and steeply Ca2+-dependent activity withP o ∼1. RYR3 was more steeply affected in the channel activity by sulfhydryl-oxidizing and -reducing reagents than RyR1, suggesting that the channel activity of RYR3 may be transformed more precipitously by the redox state. This is also a likely explanation for the difference in the Ca2+ dependence of RYR3 between [3H]ryanodine binding and channel activity.

  • characterization of type 3 ryanodine receptor RYR3 of sarcoplasmic reticulum from rabbit skeletal muscles
    Journal of Biological Chemistry, 1997
    Co-Authors: Takashi Murayama, Yasuo Ogawa
    Abstract:

    Abstract We investigated type 3 isoform (RYR3) of ryanodine receptor in rabbit skeletal muscles using an antibody specific for RYR3. By Western blot analysis and by immunoprecipitation, a single polypeptide for RYR3 was detected in sarcoplasmic reticulum vesicles from rabbit diaphragm but not in those from back muscle. The molecular mass was slightly smaller than that of RyR1, the major isoform in skeletal muscles. Each of RyR1 and RYR3 formed a homotetramer in rabbit diaphragm. RYR3 had a single class of [3H]ryanodine binding sites of high affinity (K D = 1.6 nm). From theB max of the binding, the content of RYR3 was estimated to be only 0.6% of RyR1 in rabbit diaphragm. [3H]Ryanodine binding to RYR3 was biphasically dependent on Ca2+, as is true of RyR1, and was stimulated further by adenine nucleotide, caffeine, or high salt concentration. Procaine and ruthenium red inhibited the binding. RYR3 was more resistant to Mg2+ inhibition than RyR1. Interestingly, RYR3 showed about a 7-fold lower Ca2+ sensitivity for activation than RyR1. Comparison with the counterparts in bullfrog skeletal muscles indicates that the Ca2+ sensitivities of RYR3 homologs are similar to each other, whereas those of RyR1 homologs are species-specific.

  • Properties of RYR3 ryanodine receptor isoform in mammalian brain.
    The Journal of biological chemistry, 1996
    Co-Authors: Takashi Murayama, Yasuo Ogawa
    Abstract:

    Abstract Although the RNA for the third isoform (RYR3) of ryanodine receptor (RyR), a Ca release channel, is detected in specific regions of mammalian brain, little is known about the protein. We investigated RYR3 in rabbit brain, using an antibody raised against the synthetic peptide corresponding to amino acid sequence 4375-4387 of rabbit RYR3, the homologue of bullfrog β-RyR. The antibody which reacted with bullfrog β-RyR, but not with the other isoforms, Ryr1 or Ryr2, specifically precipitated a single polypeptide from rabbit brain microsomes having a size similar to β-RyR. Sucrose gradient ultracentrifugation revealed that RYR3 forms a homotetramer, as true of the other isoforms. Being consistent with the distribution of its RNA, RYR3 was abundantly expressed in hippocampus, corpus striatum, and diencephalon. RYR3 demonstrated Ca-dependent [3H]ryanodine binding, and caffeine increased its Ca sensitivity. The Ca sensitivity of RYR3 was also enhanced in a medium containing 1 M NaCl, as observed with β-RyR. [3H]Ryanodine binding gave an estimate of RYR3 which would be only 2% or less of total RyR in rabbit brain. These results confirm the expression of functional RYR3 in mammalian brain which is similar to nonmammalian β-RyR.

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  • the structural basis of ryanodine receptor ion channel function
    The Journal of General Physiology, 2017
    Co-Authors: Gerhard Meissner
    Abstract:

    Large-conductance Ca2+ release channels known as ryanodine receptors (RyRs) mediate the release of Ca2+ from an intracellular membrane compartment, the endo/sarcoplasmic reticulum. There are three mammalian RyR isoforms: RyR1 is present in skeletal muscle; RyR2 is in heart muscle; and RYR3 is expressed at low levels in many tissues including brain, smooth muscle, and slow-twitch skeletal muscle. RyRs form large protein complexes comprising four 560-kD RyR subunits, four ∼12-kD FK506-binding proteins, and various accessory proteins including calmodulin, protein kinases, and protein phosphatases. RyRs share ∼70% sequence identity, with the greatest sequence similarity in the C-terminal region that forms the transmembrane, ion-conducting domain comprising ∼500 amino acids. The remaining ∼4,500 amino acids form the large regulatory cytoplasmic "foot" structure. Experimental evidence for Ca2+, ATP, phosphorylation, and redox-sensitive sites in the cytoplasmic structure have been described. Exogenous effectors include the two Ca2+ releasing agents caffeine and ryanodine. Recent work describing the near atomic structures of mammalian skeletal and cardiac muscle RyRs provides a structural basis for the regulation of the RyRs by their multiple effectors.

  • two ef hand motifs in ryanodine receptor calcium release channels contribute to isoform specific regulation by calmodulin
    Cell Calcium, 2017
    Co-Authors: Angela C Gomez, Gerhard Meissner, Daniel A Pasek, Naohiro Yamaguchi
    Abstract:

    The mammalian ryanodine receptor Ca2+ release channel (RyR) has a single conserved high affinity calmodulin (CaM) binding domain. However, the skeletal muscle RyR1 is activated and cardiac muscle RyR2 is inhibited by CaM at submicromolar Ca2+. This suggests isoform-specific domains are involved in RyR regulation by CaM. To gain insight into the differential regulation of cardiac and skeletal muscle RyRs by CaM, RyR1/RyR2 chimeras and mutants were expressed in HEK293 cells, and their single channel activities were measured using a lipid bilayer method. All RyR1/RyR2 chimeras and mutants were inhibited by CaM at 2μM Ca2+, consistent with CaM inhibition of RyR1 and RyR2 at micromolar Ca2+ concentrations. An RyR1/RyR2 chimera with RyR1 N-terminal amino acid residues (aa) 1-3725 and RyR2 C-terminal aa 3692-4968 were inhibited by CaM at <1μM Ca2+ similar to RyR2. In contrast, RyR1/RyR2 chimera with RyR1 aa 1-4301 and RyR2 4254-4968 was activated at <1μM Ca2+ similar to RyR1. Replacement of RyR1 aa 3726-4298 with corresponding residues from RyR2 conferred CaM inhibition at <1μM Ca2+, which suggests RyR1 aa 3726-4298 are required for activation by CaM. Characterization of additional RyR1/RyR2 chimeras and mutants in two predicted Ca2+ binding motifs in RyR1 aa 4081-4092 (EF1) and aa 4116-4127 (EF2) suggests that both EF-hand motifs and additional sequences in the large N-terminal regions are required for isoform-specific RyR1 and RyR2 regulation by CaM at submicromolar Ca2+ concentrations.

  • ca2 sparks operated by membrane depolarization require isoform 3 ryanodine receptor channels in skeletal muscle
    Proceedings of the National Academy of Sciences of the United States of America, 2007
    Co-Authors: Sandrine Pouvreau, Gerhard Meissner, Leandro Royer, Gustavo Brum, Eduardo Rios, Jingsong Zhou
    Abstract:

    Stimuli are translated to intracellular calcium signals via opening of inositol trisphosphate receptor and ryanodine receptor (RyR) channels of the sarcoplasmic reticulum or endoplasmic reticulum. In cardiac and skeletal muscle of amphibians the stimulus is depolarization of the transverse tubular membrane, transduced by voltage sensors at tubular–sarcoplasmic reticulum junctions, and the unit signal is the Ca2+ spark, caused by concerted opening of multiple RyR channels. Mammalian muscles instead lose postnatally the ability to produce sparks, and they also lose RYR3, an isoform abundant in spark-producing skeletal muscles. What does it take for cells to respond to membrane depolarization with Ca2+ sparks? To answer this question we made skeletal muscles of adult mice expressing exogenous RYR3, demonstrated as immunoreactivity at triad junctions. These muscles showed abundant sparks upon depolarization. Sparks produced thusly were found to amplify the response to depolarization in a manner characteristic of Ca2+-induced Ca2+ release processes. The amplification was particularly effective in responses to brief depolarizations, as in action potentials. We also induced expression of exogenous RyR1 or yellow fluorescent protein-tagged RyR1 in muscles of adult mice. In these, tag fluorescence was present at triad junctions. RyR1-transfected muscle lacked voltage-operated sparks. Therefore, the voltage-operated sparks phenotype is specific to the RYR3 isoform. Because RYR3 does not contact voltage sensors, their opening was probably activated by Ca2+, secondarily to Ca2+ release through junctional RyR1. Physiologically voltage-controlled Ca2+ sparks thus require a voltage sensor, a master junctional RyR1 channel that provides trigger Ca2+, and a slave parajunctional RYR3 cohort.

  • ryanodine receptors in muscarinic receptor mediated bronchoconstriction
    Journal of Biological Chemistry, 2005
    Co-Authors: Wanglei Du, Jonathan A Stiber, Paul B Rosenberg, Gerhard Meissner, Jerry P Eu
    Abstract:

    Ryanodine receptors (RyRs), intracellular calcium release channels essential for skeletal and cardiac muscle contraction, are also expressed in various types of smooth muscle cells. In particular, recent studies have suggested that in airway smooth muscle cells (ASMCs) provoked by spasmogens, stored calcium release by the cardiac isoform of RyR (RyR2) contributes to the calcium response that leads to airway constriction (bronchoconstriction). Here we report that mouse ASMCs also express the skeletal muscle and brain isoforms of RyRs (RyR1 and RYR3, respectively). In these cells, RyR1 is localized to the periphery near the cell membrane, whereas RYR3 is more centrally localized. Moreover, RyR1 and/or RYR3 in mouse airway smooth muscle also appear to mediate bronchoconstriction caused by the muscarinic receptor agonist carbachol. Inhibiting all RyR isoforms with > or = 200 microM ryanodine attenuated the graded carbachol-induced contractile responses of mouse bronchial rings and calcium responses of ASMCs throughout the range of carbachol used (50 nM to > or = 3 microM). In contrast, inhibiting only RyR1 and RYR3 with 25 microM dantrolene attenuated these responses caused by high (>500 nM) but not by low concentrations of carbachol. These data suggest that, as the stimulation of muscarinic receptor in the airway smooth muscle increases, RyR1 and/or RYR3 also mediate the calcium response and thus bronchoconstriction. Our findings provide new insights into the complex calcium signaling in ASMCs and suggest that RyRs are potential therapeutic targets in bronchospastic disorders such as asthma.

  • characterization of recombinant skeletal muscle ser 2843 and cardiac muscle ser 2809 ryanodine receptor phosphorylation mutants
    Journal of Biological Chemistry, 2003
    Co-Authors: Mirko Stange, Naohiro Yamaguchi, David Balshaw, Gerhard Meissner
    Abstract:

    Phosphorylation of the skeletal muscle (RyR1) and cardiac muscle (RyR2) ryanodine receptors has been reported to modulate channel activity. Abnormally high phosphorylation levels (hyperphosphorylation) at Ser-2843 in RyR1 and Ser-2809 in RyR2 and dissociation of FK506-binding proteins from the receptors have been implicated as one of the causes of altered calcium homeostasis observed during human heart failure. Using site-directed mutagenesis, we prepared recombinant RyR1 and RyR2 mutant receptors mimicking constitutively phosphorylated and dephosphorylated channels carrying a Ser/Asp (RyR1-S2843D and RyR2-S2809D) and Ser/Ala (RyR1-S2843A and RyR2-S2809A) substitution, respectively. Following transient expression in human embryonic kidney 293 cells, the effects of Ca2+, Mg2+, and ATP on channel function were determined using single channel and [3H]ryanodine binding measurements. In both assays, neither the skeletal nor cardiac mutants showed significant differences compared with wild type. Similarly essentially identical caffeine responses were observed in Ca2+ imaging measurements. Co-immunoprecipitation and Western blot analysis showed comparable binding of FK506-binding proteins to wild type and mutant receptors. Finally metabolic labeling experiments showed that the cardiac ryanodine receptor was phosphorylated at additional sites. Taken together, the results did not support the view that phosphorylation of a single site (RyR1-Ser-2843 and RyR2-Ser-2809) substantially changes RyR1 and RyR2 channel function.