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1
Dabertrand, Fabrice, Nicolas Fritz, Jean Mironneau, Nathalie Macrez, and Jean-Luc Morel. "Role of RYR3 splice variants in calcium signaling in mouse nonpregnant and pregnant myometrium." American Journal of Physiology-Cell Physiology 293, no. 3 (September 2007): C848—C854. http://dx.doi.org/10.1152/ajpcell.00069.2007.
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Alternative splicing of ryanodine receptor subtype 3 (RYR3) may generate a short isoform (RYR3S) without channel function and a functional full-length isoform (RYR3L). The RYR3S isoform has been shown to negatively regulate the native RYR2 subtype in smooth muscle cells as well as the RYR3L isoform when both isoforms were coexpressed in HEK-293 cells. Mouse myometrium expresses only the RYR3 subtype, but the role of RYR3 isoforms obtained by alternative splicing and their activation by cADP-ribose during pregnancy have never been investigated. Here, we show that both RYR3S and RYR3L isoforms are differentially expressed in nonpregnant and pregnant mouse myometrium. The use of antisense oligonucleotides directed against each isoform indicated that only RYR3L was activated by caffeine and cADP-ribose in nonpregnant myometrium. These RYR3L-mediated Ca2+ releases were negatively regulated by RYR3S expression. At the end of pregnancy, the relative expression of RYR3L versus RYR3S and its ability to respond to cADP-ribose were increased. Therefore, our results suggest that physiological regulation of RYR3 alternative splicing may play an essential role at the end of pregnancy.
2
Zheng, Yun-Min, Qing-Song Wang, Rakesh Rathore, Wan-Hui Zhang, Joseph E. Mazurkiewicz, Vincenzo Sorrentino, Harold A. Singer, Michael I. Kotlikoff, and Yong-Xiao Wang. "Type-3 Ryanodine Receptors Mediate Hypoxia-, but Not Neurotransmitter-induced Calcium Release and Contraction in Pulmonary Artery Smooth Muscle Cells." Journal of General Physiology 125, no. 4 (March 28, 2005): 427–40. http://dx.doi.org/10.1085/jgp.200409232.
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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.
3
Protasi, Feliciano, Alexander Shtifman, Fred J. Julian, and Paul D. Allen. "All three ryanodine receptor isoforms generate rapid cooling responses in muscle cells." American Journal of Physiology-Cell Physiology 286, no. 3 (March 2004): C662—C670. http://dx.doi.org/10.1152/ajpcell.00081.2003.
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The rapid cooling (RC) response in muscle is an increase in cytoplasmic Ca2+concentration ([Ca2+]i) that is probably caused by Ca2+release from the sarcoplasmic reticulum (SR). However, the molecular bases of this response have not been completely elucidated. Three different isoforms of the SR Ca2+release channels, or ryanodine receptors (RyRs), have been isolated (RyR1, RyR2, and RyR3). In the current investigation, the RC response was studied in RyR-null muscle cells (1B5) before and after transduction with HSV-1 virions containing the cDNAs encoding for RyR1, RyR2, or RyR3. Cells were loaded with fluo 4-AM to monitor changes in [Ca2+]iand perfused with either cold (∼0°C), room temperature (RT), or RT buffer containing 40 mM caffeine. Control cells showed no significant response to cold or caffeine, whereas robust Ca2+transients were recorded in response to both RC and caffeine in transduced cells expressing any one of the three RyR isoforms. Our data demonstrate directly that RyRs are responsible for the RC response and that all three isoforms respond in a similar manner. Ca2+release from RyRs is likely caused by a RC-induced conformational change of the channel from the closed to the open state.
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Giannini, G., A. Conti, S. Mammarella, M. Scrobogna, and V. Sorrentino. "The ryanodine receptor/calcium channel genes are widely and differentially expressed in murine brain and peripheral tissues." Journal of Cell Biology 128, no. 5 (March 1, 1995): 893–904. http://dx.doi.org/10.1083/jcb.128.5.893.
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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.
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Tian, Chengju, Caronda J. Moore, Puttappa Dodmane, Chun Hong Shao, Debra J. Romberger, Myron L. Toews, and Keshore R. Bidasee. "Dust from hog confinement facilities impairs Ca2+ mobilization from sarco(endo)plasmic reticulum by inhibiting ryanodine receptors." Journal of Applied Physiology 114, no. 5 (March 1, 2013): 665–74. http://dx.doi.org/10.1152/japplphysiol.00661.2012.
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Individuals working in commercial hog confinement facilities have elevated incidences of headaches, depression, nausea, skeletal muscle weakness, fatigue, gastrointestinal disorders, and cardiovascular diseases, and the molecular mechanisms for these nonrespiratory ailments remain incompletely undefined. A common element underlying these diverse pathophysiologies is perturbation of intracellular Ca2+ homeostasis. This study assessed whether the dust generated inside hog confinement facilities contains compounds that alter Ca2+ mobilization via ryanodine receptors (RyRs), key intracellular channels responsible for mobilizing Ca2+ from internal stores to elicit an array of physiologic functions. Hog barn dust (HBD) was extracted with phosphate-buffered saline, sterile-filtered (0.22 μm), and size-separated using Sephadex G-100 resin. Fractions (F) 1 through 9 (Mw >10,000 Da) had no measurable effects on RyR isoforms. However, F10 through F17, which contained compounds of Mw ≤2,000 Da, modulated the [3H]ryanodine binding to RyR1, RyR2, and RyR3 in a biphasic (Gaussian) manner. The Ki values for F13, the most potent fraction, were 3.8 ± 0.2 μg/ml for RyR1, 0.2 ± 0.01 μg/ml and 19.1 ± 2.8 μg/ml for RyR2 (two binding sites), and 44.9 ± 2.8 μg/ml and 501.6 ± 9.2 μg/ml for RyR3 (two binding sites). In lipid bilayer assays, F13 dose-dependently decreased the open probabilities of RyR1, RyR2, and RyR3. Pretreating differentiated mouse skeletal myotubes (C2C12 cells) with F13 blunted the amplitudes of ryanodine- and K+-induced Ca2+ transients. Because RyRs are present in many cell types, impairment in Ca2+ mobilization from internal stores via these channels is a possible mechanism by which HBD may trigger these seemingly unrelated pathophysiologies.
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OTTINI, Laura, Giovanna MARZIALI, Antonio CONTI, Alexandra CHARLESWORTH, and Vincenzo SORRENTINO. "α and β isoforms of ryanodine receptor from chicken skeletal muscle are the homologues of mammalian RyR1 and RyR3." Biochemical Journal 315, no. 1 (April 1, 1996): 207–16. http://dx.doi.org/10.1042/bj3150207.
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To define the relationship between the two ryanodine receptor (RyR) isoforms present in chicken skeletal muscle, we cloned two groups of cDNAs encoding the chicken homologues of mammalian RyR1 and RyR3. Equivalent amounts of the two chicken isoform mRNAs were detected in thigh and pectoral skeletal muscles. RyR1 and RyR3 mRNAs were co-expressed in testis and cerebellum whereas RyR3 mRNA was expressed also in cerebrum and heart. The full-length sequence of the chicken RyR3 cDNA was established. The RyR3 receptor from chicken had the same general structure as mammalian and amphibian RyRs. The 15089 nt cDNA encoded a 4869-amino-acid-long protein with a molecular mass of 552445. The predicted amino acid sequence of the chicken RyR3 showed 86.9% identity to mammalian RyR3 and 85.6% to frog RyR3. Antibodies specific for chicken RyR1 and RyR3 recognized two different proteins with an apparent molecular mass of about 500 kDa. The two proteins differ slightly in their apparent molecular mass on SDS/PAGE: the protein recognized by antibodies against RyR3 had a higher mobility than the protein recognized by the antiserum against RyR1. Antibodies against RyR1 detected a protein already present in chicken skeletal muscle from 12-day-old embryos and older, while antibodies against RyR3 isoform detected a protein in muscle from only 18-day-old embryos and older. The expression patterns of RyR1 and RyR3 superimpose with those previously reported for the α and the β isoforms respectively. We conclude that α and β isoforms present in chicken skeletal muscle are the homologues of mammalian RyR1 and RyR3.
7
Perez, Claudio F., José R. López, and Paul D. Allen. "Expression levels of RyR1 and RyR3 control resting free Ca2+ in skeletal muscle." American Journal of Physiology-Cell Physiology 288, no. 3 (March 2005): C640—C649. http://dx.doi.org/10.1152/ajpcell.00407.2004.
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To better understand the role of the transient expression of ryanodine receptor (RyR) type 3 (RyR3) on Ca2+ homeostasis during the development of skeletal muscle, we have analyzed the effect of expression levels of RyR3 and RyR1 on the overall physiology of cultured myotubes and muscle fibers. Dyspedic myotubes were infected with RyR1 or RyR3 containing virions at 0.2, 0.4, 1.0, and 4.0 moieties of infection (MOI), and analysis of their pattern of expression, caffeine sensitivity, and resting free Ca2+ concentration ([Ca2+]r) was performed. Although increased MOI resulted in increased expression of each receptor isoform, it did not significantly affect the immunopattern of RyRs or the expression levels of calsequestrin, triadin, or FKBP-12. Interestingly, myotubes expressing RyR3 always had significantly higher [Ca2+]r and lower caffeine EC50 than did cells expressing RyR1. Although some of the increased sensitivity of RyR3 to caffeine could be attributed to the higher [Ca2+]r in RyR3-expressing cells, studies of [3H]ryanodine binding demonstrated intrinsic differences in caffeine sensitivity between RyR1 and RyR3. Tibialis anterior (TA) muscle fibers at different stages of postnatal development exhibited a transient increase in [Ca2+]r coordinately with their level of RyR3 expression. Similarly, adult soleus fibers, which also express RyR3, had higher [Ca2+]r than did adult TA fibers, which exclusively express RyR1. These data show that in skeletal muscle, RyR3 increases [Ca2+]r more than RyR1 does at any expression level. These data suggest that the coexpression of RyR1 and RyR3 at different levels may constitute a novel mechanism by which to regulate [Ca2+]r in skeletal muscle.
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Vanterpool, Conwin K., Elaine A. Vanterpool, William J. Pearce, and John N. Buchholz. "Advancing age alters the expression of the ryanodine receptor 3 isoform in adult rat superior cervical ganglia." Journal of Applied Physiology 101, no. 2 (August 2006): 392–400. http://dx.doi.org/10.1152/japplphysiol.00167.2006.
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Sympathetic nerves arising from the superior cervical ganglion (SCG) protect the cerebrovasculature during periods of acute hypertension and may play a role in homeostasis of target organs. The functions of these nerves depend on calcium release triggered by activation of ryanodine receptor (RyR) channels. The function of RyR channels is in part dependent on genetic expression and regulation by numerous protein modulators such as neuronal nitric oxide synthase (nNOS) neurons also found in the SCG. We have shown that release of calcium in SCG cells is altered during late maturation and advancing age. However, the underlying molecular mechanisms that may in part account for these data are elusive. Therefore we used molecular techniques to test the hypothesis that advancing age alters the pattern of genetic expression and/or protein levels of RyRs and their modulation by nNOS in the SCG in F344 rats aged 6, 12, and 24 mo. Surprisingly, ryr1 expression was undetectable in all age groups and ryr2 and ryr3 are the predominantly transcribed isoforms in the adult rat SCG. mRNA and protein levels for RyR2 isoform did not change with advancing age. However, ryr3 mRNA levels increased from 6 to 12 mo and declined from 12 to 24 mo. Similarly, RyR3 receptor protein levels also increased from 6 to 12 mo and declined from 12 to 24 mo. Because nNOS and the phosphorylation of the RyRs have been shown to modulate the function of RyRs, total phosphorylation and nNOS protein levels were analyzed in all age groups. Phosphorylation levels of the RyRs were similar in all age groups. However, nNOS protein levels increased from 6 to 12 mo followed by decline from 12 to 24 mo. These data suggest that advancing age selectively impacts the genetic expression and protein levels of RyR3 as well as modulatory nNOS protein levels. In addition, these data may part provide some insight into the possible changes in the function of RyRs that may occur with the normal aging process.
9
Rossi, Daniela, Ilenia Simeoni, Marcella Micheli, Martin Bootman, Peter Lipp, Paul D. Allen, and Vincenzo Sorrentino. "RyR1 and RyR3 isoforms provide distinct intracellular Ca2+signals in HEK 293 cells." Journal of Cell Science 115, no. 12 (June 15, 2002): 2497–504. http://dx.doi.org/10.1242/jcs.115.12.2497.
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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.
10
CONTI, Antonio, L. GORZA, and Vincenzo SORRENTINO. "Differential distribution of ryanodine receptor type 3 (RyR3) gene product in mammalian skeletal muscles." Biochemical Journal 316, no. 1 (May 15, 1996): 19–23. http://dx.doi.org/10.1042/bj3160019.
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Activation of intracellular Ca2+-release channels/ryanodine receptors (RyRs) is a fundamental step in the regulation of muscle contraction. In mammalian skeletal muscle, Ca2+-release channels containing the type 1 isoform of RyR (RyR1) open to release Ca2+ from the sarcoplasmic reticulum (SR) upon stimulation by the voltage-activated dihydropyridine receptor on the T-tubule/plasma membrane. In addition to RyR1, low levels of the mRNA of the RyR3 isoform have been recently detected in mammalian skeletal muscles. Here we report data on the distribution of the RyR3 gene product in mammalian skeletal muscles. Western-blot analysis of SR of individual muscles indicated that, at variance with the even distribution of the RyR1 isoform, the RyR3 content varies among different muscles, with relatively higher amounts being detected in diaphragm and soleus, and lower levels in abdominal muscles and tibialis anterior. In these muscles RyR3 was localized in the terminal cisternae of the SR. No detectable levels of RyR3 were observed in the extensor digitorum longus. Preferential high content of RyR3 in the diaphragm muscle was observed in several mammalian species. In situ hybridization analysis demonstrated that RyR3 transcripts are not restricted to a specific subset of skeletal-muscle fibres. Differential utilization of the RyR3 isoform in skeletal muscle may be relevant to the modulation of Ca2+ release with respect to specific muscle-contraction properties.
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Flucher, Bernhard E., Antonio Conti, Hiroshi Takeshima, and Vincenzo Sorrentino. "Type 3 and Type 1 Ryanodine Receptors Are Localized in Triads of the Same Mammalian Skeletal Muscle Fibers." Journal of Cell Biology 146, no. 3 (August 9, 1999): 621–30. http://dx.doi.org/10.1083/jcb.146.3.621.
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The type 3 ryanodine receptor (RyR3) is a ubiquitous calcium release channel that has recently been found in mammalian skeletal muscles. However, in contrast to the skeletal muscle isoform (RyR1), neither the subcellular distribution nor the physiological role of RyR3 are known. Here, we used isoform-specific antibodies to localize RyR3 in muscles of normal and RyR knockout mice. In normal hind limb and diaphragm muscles of young mice, RyR3 was expressed in all fibers where it was codistributed with RyR1 and with the skeletal muscle dihydropyridine receptor. This distribution pattern indicates that RyR3 is localized in the triadic junctions between the transverse tubules and the sarcoplasmic reticulum. During development, RyR3 expression declined rapidly in some fibers whereas other fibers maintained expression of RyR3 into adulthood. Comparing the distribution of RyR3-containing fibers with that of known fiber types did not show a direct correlation. Targeted deletion of the RyR1 or RyR3 gene resulted in the expected loss of the targeted isoform, but had no adverse effects on the expression and localization of the respective other RyR isoform. The localization of RyR3 in skeletal muscle triads, together with RyR1, is consistent with an accessory function of RyR3 in skeletal muscle excitation–contraction coupling.
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Vaithianathan, Thirumalini, Damodaran Narayanan, Maria T. Asuncion-Chin, Loice H. Jeyakumar, Jianxi Liu, Sidney Fleischer, Jonathan H. Jaggar, and Alejandro M. Dopico. "Subtype identification and functional characterization of ryanodine receptors in rat cerebral artery myocytes." American Journal of Physiology-Cell Physiology 299, no. 2 (August 2010): C264—C278. http://dx.doi.org/10.1152/ajpcell.00318.2009.
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Ryanodine receptors (RyRs) regulate contractility in resistance-size cerebral artery smooth muscle, yet their molecular identity, subcellular location, and phenotype in this tissue remain unknown. Following rat resistance-size cerebral artery myocyte sarcoplasmic reticulum (SR) purification and incorporation into POPE-POPS-POPC (5:3:2; wt/wt) bilayers, unitary conductances of 110 ± 8, 334 ± 15, and 441 ± 27 pS in symmetric 300 mM Cs+ were usually detected. The most frequent (34/40 bilayers) conductance (334 pS) decreased to ≤100 pS when Cs+ was replaced with Ca2+. The predominant conductance displayed 66 bursts/min with at least three open and three closed states. The steady-state activity (NPo)-voltage curve was bell shaped, with NPo drastically decreasing when voltage was switched from −30 to −40 mV. NPo increased when intracellular calcium (Ca2+i) was raised within 0.1–100 μM to abruptly diminish with higher Ca2+i. Thus maximal activity occurred within the Ca2+i range found in rat cerebral artery myocytes under physiological conditions. NPo was reduced by ruthenium red (80 μM), increased monotonically by caffeine (0.1–5 mM) or ryanodine (0.05–5 μM), and unaffected by heparin (2 mg/ml). This phenotype resembles that of cardiac RyR and recombinant RyR2. RT-PCR detected RyR1, RyR2, and RyR3 transcripts in cerebral artery myocytes. However, real-time PCR indicated that RyR2 was 4 and 1.5 times more abundant than RyR1 and RyR3, respectively. Consistently, Western blotting showed that the RyR2 product was very abundant. Immunofluorescence showed that each RyR isoform distributed differentially among subcellular compartments. In particular, RyR2 was drastically stronger in the subplasmalemma than in other compartments, underscoring the predominance of RyR2 in a compartment where SR is abundant. Consistently, RyR from SR-enriched membranes displayed pharmacological specificity typical of RyR2, being activated by digoxin (1 μM), resistant to dantrolene (100 μM), and shifted to a subconductance by neomycin (100 nM). Therefore, RyR2 is the predominant molecular and functional RyR that is expressed in the SR membrane of rat resistance-size cerebral artery myocytes.
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Morrissette, Jeffery, Le Xu, Alexandra Nelson, Gerhard Meissner, and Barbara A. Block. "Characterization of RyR1-slow, a ryanodine receptor specific to slow-twitch skeletal muscle." American Journal of Physiology-Regulatory, Integrative and Comparative Physiology 279, no. 5 (November 1, 2000): R1889—R1898. http://dx.doi.org/10.1152/ajpregu.2000.279.5.r1889.
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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 [3H]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 [3H]ryanodine when compared with RyR1-fast. In single channel recordings, RyR1-slow and RyR1-fast had similar slope conductances. However, the maximum open probability (Po) of RyR1-slow was threefold less than the maximum Po 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 Po of all the RyR channels and displayed less inhibition at millimolar Ca2+. The addition of 5 mM Mg-ATP or 2.5 mM β,γ-methyleneadenosine 5′-triphosphate (AMP-PCP) to the channels increased the Po and [3H]ryanodine binding of both RyR1s but also caused a shift in the Ca2+ dependency curve of RyR1-slow such that Ca2+-dependent inactivation was attenuated. [3H]ryanodine binding data also showed that Mg2+-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 Ca2+ is regulated in these muscle types.
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Yang, Xiao-Ru, Mo-Jun Lin, Kay-Pong Yip, Loice H. Jeyakumar, Sidney Fleischer, George P. H. Leung, and James S. K. Sham. "Multiple ryanodine receptor subtypes and heterogeneous ryanodine receptor-gated Ca2+ stores in pulmonary arterial smooth muscle cells." American Journal of Physiology-Lung Cellular and Molecular Physiology 289, no. 2 (August 2005): L338—L348. http://dx.doi.org/10.1152/ajplung.00328.2004.
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Ryanodine receptors (RyRs) of pulmonary arterial smooth muscle cells (PASMCs) play important roles in major physiological processes such as hypoxic pulmonary vasoconstriction and perinatal pulmonary vasodilatation. Recent studies show that three subtypes of RyRs are coexpressed and RyR-gated Ca2+ stores are distributed heterogeneously in systemic vascular myocytes. However, the molecular identity and subcellular distribution of RyRs have not been examined in PASMCs. In this study we detected mRNA and proteins of all three subtypes in rat intralobar PASMCs using RT-PCR and Western blot. Quantitative real-time RT-PCR showed that RyR2 mRNA was most abundant, ∼15–20 times more than the other two subtypes. Confocal fluorescence microscopy revealed that RyRs labeled with BODIPY TR-X ryanodine were localized in the peripheral and perinuclear regions and were colocalized with sarcoplasmic reticulum labeled with Fluo-5N. Immunostaining showed that the subsarcolemmal regions exhibited clear signals of RyR1 and RyR2, whereas the perinuclear compartments contained mainly RyR1 and RyR3. Ca2+ sparks were recorded in both regions, and their activities were enhanced by a subthreshold concentration of caffeine or by endothelin-1, indicating functional RyR-gated Ca2+ stores. Moreover, 18% of the perinuclear sparks were prolonged [full duration/half-maximum (FDHM) = 193.3 ± 22.6 ms] with noninactivating kinetics, in sharp contrast to the typical fast inactivating Ca2+ sparks (FDHM = 44.6 ± 3.2 ms) recorded in the same PASMCs. In conclusion, multiple RyR subtypes are expressed differentially in peripheral and perinuclear RyR-gated Ca2+ stores; the molecular complexity and spatial heterogeneity of RyRs may facilitate specific Ca2+ regulation of cellular functions in PASMCs.
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Meissner, Gerhard. "The structural basis of ryanodine receptor ion channel function." Journal of General Physiology 149, no. 12 (November 9, 2017): 1065–89. http://dx.doi.org/10.1085/jgp.201711878.
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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.
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Bultynck, Geert, Daniela Rossi, Geert Callewaert, Ludwig Missiaen, Vincenzo Sorrentino, Jan B. Parys, and Humbert De Smedt. "The Conserved Sites for the FK506-binding Proteins in Ryanodine Receptors and Inositol 1,4,5-Trisphosphate Receptors Are Structurally and Functionally Different." Journal of Biological Chemistry 276, no. 50 (October 11, 2001): 47715–24. http://dx.doi.org/10.1074/jbc.m106573200.
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We compared the interaction of the FK506-binding protein (FKBP) with the type 3 ryanodine receptor (RyR3) and with the type 1 and type 3 inositol 1,4,5-trisphosphate receptor (IP3R1 and IP3R3), using a quantitative GST-FKBP12 and GST-FKBP12.6 affinity assay. We first characterized and mapped the interaction of the FKBPs with the RyR3. GST-FKBP12 as well as GST-FKBP12.6 were able to bind ∼30% of the solubilized RyR3. The interaction was completely abolished by FK506, strengthened by the addition of Mg2+, and weakened in the absence of Ca2+but was not affected by the addition of cyclic ADP-ribose. By using proteolytic mapping and site-directed mutagenesis, we pinpointed Val2322, located in the central modulatory domain of the RyR3, as a critical residue for the interaction of RyR3 with FKBPs. Substitution of Val2322for leucine (as in IP3R1) or isoleucine (as in RyR2) decreased the binding efficiency and shifted the selectivity to FKBP12.6; substitution of Val2322for aspartate completely abolished the FKBP interaction. Importantly, the occurrence of the valylprolyl residue as α-helix breaker was an important determinant of FKBP binding. This secondary structure is conserved among the different RyR isoforms but not in the IP3R isoforms. A chimeric RyR3/IP3R1, containing the core of the FKBP12-binding site of IP3R1 in the RyR3 context, retained this secondary structure and was able to interact with FKBPs. In contrast, IP3Rs did not interact with the FKBP isoforms. This indicates that the primary sequence in combination with the local structural environment plays an important role in targeting the FKBPs to the intracellular Ca2+-release channels. Structural differences in the FKBP-binding site of RyRs and IP3Rs may contribute to the occurrence of a stable interaction between RyR isoforms and FKBPs and to the absence of such interaction with IP3Rs.
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Rousseau, Eric, and Sonia Proteau. "Functional properties of the native type 3 ryanodine receptor Ca2+-release channel from canine diaphragm." Canadian Journal of Physiology and Pharmacology 79, no. 4 (April 1, 2001): 310–19. http://dx.doi.org/10.1139/y00-127.
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mRNA and protein analyses have previously shown that the diaphragm expresses two ryanodine receptor isoforms: RyR1 and RyR3.RyR1 is the main Ca2+-releasing pathway in this muscle type. We now report the conducting, gating, and immunological properties of the native and purified forms of the less abundant RyR3 channel. The conductance of this native Ca2+-release channel was 330 pS in 50 mM/250 mM trans/cis CsCH3SO3. It was activated by Ca2+ concentrations of 1-1000 µM, and did not inactivate at mM concentrations of Ca2+. Both isoforms were purified by either a sucrose density gradient or immunoprecipitation as > 450 kDa proteins on SDS-PAGE. Western blot analysis confirmed the presence of RyR1 and RyR3, which displayed conductances of 740 ± 30 and 800 ± 25 pS, respectively, in 250 mM KCl. We thus provide evidence that one form of the diaphragm SR Ca2+-release channels may be classified as RyR3, with gating properties different from those of the well-characterized RyR1 and RyR2 isoforms.Key words: diaphragm, calcium channel, ryanodine receptors, skeletal muscles, excitation-contraction coupling.
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Ji, Guangju, Morris E. Feldman, Kai Su Greene, Vincenzo Sorrentino, Hong-Bo Xin, and Michael I. Kotlikoff. "RYR2 Proteins Contribute to the Formation of Ca2+ Sparks in Smooth Muscle." Journal of General Physiology 123, no. 4 (March 15, 2004): 377–86. http://dx.doi.org/10.1085/jgp.200308999.
Full textAbstract:
Calcium release through ryanodine receptors (RYR) activates calcium-dependent membrane conductances and plays an important role in excitation-contraction coupling in smooth muscle. The specific RYR isoforms associated with this release in smooth muscle, and the role of RYR-associated proteins such as FK506 binding proteins (FKBPs), has not been clearly established, however. FKBP12.6 proteins interact with RYR2 Ca2+ release channels and the absence of these proteins predictably alters the amplitude and kinetics of RYR2 unitary Ca2+ release events (Ca2+ sparks). To evaluate the role of specific RYR2 and FBKP12.6 proteins in Ca2+ release processes in smooth muscle, we compared spontaneous transient outward currents (STOCs), Ca2+ sparks, Ca2+-induced Ca2+ release, and Ca2+ waves in smooth muscle cells freshly isolated from wild-type, FKBP12.6−/−, and RYR3−/− mouse bladders. Consistent with a role of FKBP12.6 and RYR2 proteins in spontaneous Ca2+ sparks, we show that the frequency, amplitude, and kinetics of spontaneous, transient outward currents (STOCs) and spontaneous Ca2+ sparks are altered in FKBP12.6 deficient myocytes relative to wild-type and RYR3 null cells, which were not significantly different from each other. Ca2+ -induced Ca2+ release was similarly augmented in FKBP12.6−/−, but not in RYR3 null cells relative to wild-type. Finally, Ca2+ wave speed evoked by CICR was not different in RYR3 cells relative to control, indicating that these proteins are not necessary for normal Ca2+ wave propagation. The effect of FKBP12.6 deletion on the frequency, amplitude, and kinetics of spontaneous and evoked Ca2+ sparks in smooth muscle, and the finding of normal Ca2+ sparks and CICR in RYR3 null mice, indicate that Ca2+ release through RYR2 molecules contributes to the formation of spontaneous and evoked Ca2+ sparks, and associated STOCs, in smooth muscle.
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Murayama, Takashi, and Yasuo Ogawa. "RyR1 exhibits lower gain of CICR activity than RyR3 in the SR: evidence for selective stabilization of RyR1 channel." American Journal of Physiology-Cell Physiology 287, no. 1 (July 2004): C36—C45. http://dx.doi.org/10.1152/ajpcell.00395.2003.
Full textAbstract:
We showed that frog α-ryanodine receptor (α-RyR) had a lower gain of Ca2+-induced Ca2+ release (CICR) activity than β-RyR in sarcoplasmic reticulum (SR) vesicles, indicating selective “stabilization” of the former isoform (Murayama T and Ogawa Y. J Biol Chem 276: 2953–2960, 2001). To know whether this is also the case with mammalian RyR1, we determined [3H]ryanodine binding of RyR1 and RyR3 in bovine diaphragm SR vesicles. The value of [3H]ryanodine binding (B) was normalized by the number of maximal binding sites (Bmax), whereby the specific activity of each isoform was expressed. This B/Bmax expression demonstrated that ryanodine binding of individual channels for RyR1 was <15% that for RyR3. Responses to Ca2+, Mg2+, adenine nucleotides, and caffeine were not substantially different between in situ and purified isoforms. These results suggest that the gain of CICR activity of RyR1 is markedly lower than that of RyR3 in mammalian skeletal muscle, indicating selective stabilization of RyR1 as is true of frog α-RyR. The stabilization was partly eliminated by FK506 and partly by solubilization of the vesicles with CHAPS, each of which was additive to the other. In contrast, high salt, which greatly enhances [3H]ryanodine binding, caused only a minor effect on the stabilization of RyR1. None of the T-tubule components, coexisting RyR3, or calmodulin was the cause. The CHAPS-sensitive intra- and intermolecular interactions that are common between mammalian and frog skeletal muscles and the isoform-specific inhibition by FKBP12, which is characteristic of mammals, are likely to be the underlying mechanisms.
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Van Petegem, Filip. "The Ryanodine Receptor: Arrhythmias and Muscle Disorders at High Resolution." Acta Crystallographica Section A Foundations and Advances 70, a1 (August 5, 2014): C798. http://dx.doi.org/10.1107/s2053273314092018.
Full textAbstract:
Calcium ions play crucial roles in our bodies, acting as second messengers in multiple signaling pathways. The resting calcium levels in the cytosol are very low, but can increase rapidly and transiently by influx from the extracellular space, or by release from intracellular stores. The Endoplasmic and Sarcoplasmic Reticulum (ER/SR) form major intracellular calcium stores. The `Ryanodine Receptor' (RyR) is a protein that dictates calcium release from the ER/SR. It forms a huge ion channel with a molecular weight exceeding 2 MegaDalton. RyRs are expressed in multiple cell types, but are particularly abundant in cardiac and skeletal muscle, where they are directly involved in excitation-contraction coupling. Three RyR isoforms exist in mammalian species (RyR1, RyR2, RyR3). Mutations in RyR1 and RyR2 have been linked to a number of devastating genetic disorders, including stress-induced cardiac arrhythmias (CPVT), malignant hyperthermia, and central core disease. In the past 5 years we have been solving crystal structures of several RyR domains. Cryo-electron microscopy structures have helped us to build pseudo-atomic models, allowing us to locate these domains in full-length RyRs. Over 80 disease mutations are scattered throughout the structures. The bulk of these affect domain-domain interactions. By comparing the RyR in the open and closed state, we find that some of these interactions are labile: they are disrupted during channel opening. Many disease mutations weaken these interactions, leading to facilitated channel opening, resulting in premature or prolonged release of calcium. In addition, many other disease mutations affect the same labile interactions allosterically. Stabilizing the closed-state domain-domain interactions may therefore be of therapeutic value.
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BULTYNCK, Geert, Patrick DE SMET, Daniela ROSSI, Geert CALLEWAERT, Ludwig MISSIAEN, Vincenzo SORRENTINO, Humbert DE SMEDT, and Jan B. PARYS. "Characterization and mapping of the 12kDa FK506-binding protein (FKBP12)-binding site on different isoforms of the ryanodine receptor and of the inositol 1,4,5-trisphosphate receptor." Biochemical Journal 354, no. 2 (February 22, 2001): 413–22. http://dx.doi.org/10.1042/bj3540413.
Full textAbstract:
We investigated the interaction of the 12kDa FK506-binding protein (FKBP12) with two ryanodine-receptor isoforms (RyR1 and RyR3) and with two myo-inositol 1,4,5-trisphosphate (IP3) receptor isoforms (IP3R1 and IP3R3). Using glutathione S-transferase (GST)-FKBP12 affinity chromatography, we could efficiently extract RyR1 (42±7% of the solubilized RyR1) from terminal cisternae of skeletal muscle as well as RyR3 (32±4% of the solubilized RyR3) from RyR3-overexpressing HEK-293 cells. These interactions were completely abolished by FK506 (20µM) but were largely unaffected by RyR-channel modulators. In contrast, neither IP3R1 nor IP3R3 from various sources, including rabbit cerebellum, A7r5 smooth-muscle cells and IP3R-overexpressing Sf9 insect cells from Spodoptera frugiperda, were retained on the GST-FKBP12 matrix. Moreover, immunoprecipitation experiments indicated a high-affinity interaction of FKBP12 with RyR1 but not with IP3R1. In order to determine the FKBP12-binding site, we fragmented both RyR1 and IP3R1 by limited proteolysis. We obtained a 45kDa fragment of RyR1 that bound to the GST-FKBP12 matrix, indicating that it retained all requirements for FKBP12 binding. This fragment was identified by its interaction with antibody m34C and must therefore contain its epitope (amino acids 2756–2803). However, no fragment of IP3R1 was retained on the column. These molecular data are in agreement with the lack of correlation between FKBP12 and IP3R1 expression in various cell types. The observation that FKBP12 did not affect IP3-induced Ca2+ release but reduced caffeine-induced Ca2+ release also indicated that mature IP3R1 and IP3R3, in contrast to RyR1 and RyR3, did not display a specific, high-affinity interaction with FKBP12.
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Jo, Michiko, Andrea N. Trujillo, Ying Yang, and Jerome W. Breslin. "Evidence of functional ryanodine receptors in rat mesenteric collecting lymphatic vessels." American Journal of Physiology-Heart and Circulatory Physiology 317, no. 3 (September 1, 2019): H561—H574. http://dx.doi.org/10.1152/ajpheart.00564.2018.
Full textAbstract:
In the current study, the potential contributions of ryanodine receptors (RyRs) to intrinsic pumping and responsiveness to substance P (SP) were investigated in isolated rat mesenteric collecting lymphatic vessels. Responses to SP were characterized in lymphatic vessels in the absence or presence of pretreatment with nifedipine to block L-type Ca2+ channels, caffeine to block normal release and uptake of Ca2+ from the sarcoplasmic reticulum, ryanodine to block all RyR isoforms, or dantrolene to more selectively block RyR1 and RyR3. RyR expression and localization in lymphatics was also assessed by quantitative PCR and immunofluorescence confocal microscopy. The results show that SP normally elicits a significant increase in contraction frequency and a decrease in end-diastolic diameter. In the presence of nifedipine, phasic contractions stop, yet subsequent SP treatment still elicits a strong tonic contraction. Caffeine treatment gradually relaxes lymphatics, causing a loss of phasic contractions, and prevents subsequent SP-induced tonic contraction. Ryanodine also gradually diminishes phasic contractions but without causing vessel relaxation and significantly inhibits the SP-induced tonic contraction. Dantrolene treatment did not significantly impair lymphatic contractions nor the response to SP. The mRNA for all RyR isoforms is detectable in isolated lymphatics. RyR2 and RyR3 proteins are found predominantly in the collecting lymphatic smooth muscle layer. Collectively, the data suggest that SP-induced tonic contraction requires both extracellular Ca2+ plus Ca2+ release from internal stores and that RyRs play a role in the normal contractions and responsiveness to SP of rat mesenteric collecting lymphatics. NEW & NOTEWORTHY The mechanisms that govern contractions of lymphatic vessels remain unclear. Tonic contraction of lymphatic vessels caused by substance P was blocked by caffeine, which prevents normal uptake and release of Ca2+ from internal stores, but not nifedipine, which blocks L-type channel-mediated Ca2+ entry. Ryanodine, which also disrupts normal sarcoplasmic reticulum Ca2+ release and reuptake, significantly inhibited substance P-induced tonic contraction. Ryanodine receptors 2 and 3 were detected within the smooth muscle layer of collecting lymphatic vessels.
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Matsuki, Katsuhito, Daiki Kato, Masashi Takemoto, Yoshiaki Suzuki, Hisao Yamamura, Susumu Ohya, Hiroshi Takeshima, and Yuji Imaizumi. "Negative regulation of cellular Ca2+ mobilization by ryanodine receptor type 3 in mouse mesenteric artery smooth muscle." American Journal of Physiology-Cell Physiology 315, no. 1 (July 1, 2018): C1—C9. http://dx.doi.org/10.1152/ajpcell.00006.2018.
Full textAbstract:
Physiological functions of type 3 ryanodine receptors (RyR3) in smooth muscle (SM) tissues are not well understood, in spite of their wide expression. However, the short isoform of RyR3 is known to be a dominant-negative variant (DN-RyR3), which may negatively regulate functions of both RyR2 and full-length (FL) RyR3 by forming hetero-tetramers. Here, functional roles of RyR3 in the regulation of Ca2+ signaling in mesenteric artery SM cells (MASMCs) were examined using RyR3 homozygous knockout mice (RyR3−/−). Quantitative PCR analyses suggested that the predominant RyR3 subtype in MASMs from wild-type mice (RyR3+/+) was DN-RyR3. In single MASMCs freshly isolated from RyR3−/−, the EC50 of caffeine to induce Ca2+ release was lower than that in RyR3+/+ myocytes. The amplitude and frequency of Ca2+ sparks and spontaneous transient outward currents in MASMCs from RyR3−/− were all larger than those from RyR3+/+. Importantly, mRNA and functional expressions of voltage-dependent Ca2+ channel and large-conductance Ca2+-activated K+ (BK) channel in MASMCs from RyR3−/− were identical to those from RyR3+/+. However, in the presence of BK channel inhibitor, paxilline, the pressure rises induced by BayK8644 in MA vascular beds of RyR3−/− were significantly larger than in those of RyR3+/+. This indicates that the negative feedback effects of BK channel activity on intracellular Ca2+ signaling was enhanced in RyR3−/−. Thus, RyR3, and, in fact, mainly DN-RyR3, via a complex with RyR2 suppresses Ca2+ release and indirectly regulated membrane potential by reducing BK channel activity in MASMCs and presumably can affect the regulation of intrinsic vascular tone.
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Marks, A. R. "Intracellular calcium-release channels: regulators of cell life and death." American Journal of Physiology-Heart and Circulatory Physiology 272, no. 2 (February 1, 1997): H597—H605. http://dx.doi.org/10.1152/ajpheart.1997.272.2.h597.
Full textAbstract:
Intracellular Ca2+-release channels on the sarcoplasmic reticulum of striated muscle [ryanodine receptors (RyRs)] and on the endoplasmic reticulum of almost all types of cells [inositol 1,4,5-trisphosphate receptors (IP3Rs)] comprise a unique family of molecules that are structurally and functionally distinct from all other known ion channels. These channels play crucial roles in Ca2+-mediated signaling that triggers excitation-contraction coupling, T-lymphocyte activation, fertilization, and many other cellular functions. Three forms of RyR have been identified: RyR1, expressed predominantly in skeletal muscle; RyR2, expressed predominantly in cardiac muscle; and RyR3, expressed in specialized muscles and nonmuscle tissues including the brain. RyR channels are tetramers composed of four subunits each with a molecular mass of approximately 560,000 Da. The tetrameric structures of RyR1 and RyR2 are stabilized by a channel-associated protein known as the FK506 binding protein (FKBP). FKBP is the cytosolic receptor for the immunosuppressant drugs FK506 and rapamycin that inhibit the prolyl isomerase activity of FKBP and can dissociate FKBP from RyRs. Rapamycin and FK506 increase the sensitivity of RyRs to agonists such as caffeine and could be a cause of cardiac dysfunction associated with high-dose immunosuppressant therapy by promoting leakage of Ca2+ from the sarcoplasmic reticulum. The role of prolyl isomerase activity of FKBP in regulating RyR function remains uncertain, and several models have been proposed that could explain how the channel is modulated by its association with FKBP. Three forms of IP3Rs (types 1, 2 and 3) have been characterized by cDNA cloning. Most cells have at least one form of IP3R, and many express all three types. Like RyRs, the IP3R channels are tetramers composed of four subunits (approximately 300,000 Da each). IP3R1 function is regulated by at least two major cellular signaling pathways: the second messenger IP3 activates the channel, and phosphorylation by nonreceptor protein tyrosine kinases (e.g., Fyn) increase its open probability. During end-stage human heart failure, RyR2 mRNA and protein are downregulated, whereas IP3R1 is upregulated, suggesting that altered Ca2+-release channel levels may contribute to defects in Ca2+ homeostasis. Cells that are deficient in IP3R1 exhibit defective T cell-receptor signaling and thus cannot be activated by T cell-receptor stimulation. IP3R1-deficient cells are also resistant to induced apoptosis. Thus RyRs and IP3Rs play critical roles in fundamental and diverse signaling phenomena that include excitation-contraction coupling, T-cell activation, and programmed cell death.
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AWAD, Suad S., Heather K. LAMB, Joanna M. MORGAN, William DUNLOP, and James I. GILLESPIE. "Differential expression of ryanodine receptor RyR2 mRNA in the non-pregnant and pregnant human myometrium." Biochemical Journal 322, no. 3 (March 15, 1997): 777–83. http://dx.doi.org/10.1042/bj3220777.
Full textAbstract:
We describe here the expression of the ryanodine receptor isoforms RyR2 and RyR3 in human non-pregnant and pregnant (non-labouring) myometrium, and in isolated cultured myometrial cells. The mRNA encoding the RyR3 isoform was found in both non-pregnant and pregnant myometrial tissue samples; however, the mRNA for RyR2 was found only in pregnant samples. It can be speculated that the appearance of this additional isoform in the pregnant myometrium may increase the ability of this tissue to contract at term. Control of expression of the RyR2 gene may therefore be another example of an up-regulated signalling system in pregnancy. Although the mRNA for RyR3 was expressed in cultured myometrial cells, the mRNA for RyR2 could not be detected. Thus cultured myometrial cells appear to be similar to the non-pregnant myometrium. The cytokine transforming growth factor β (TGF-β) has been reported to alter RyR mRNA expression in many cell types. After treatment with TGF-β, both RyR2 and RyR3 mRNAs could be detected in cultured myometrial cells. These observations support the idea that the expression of the RyR2 isoform is up-regulated both in pregnancy and in TGF-β-treated cultured myometrial cells. Using measurements of 45Ca2+ release, we have further demonstrated that cultured human myometrial cells show a significant augmentation of both the Ca2+-induced Ca2+ release (CICR) mechanism and ryanodine-induced Ca2+ release after treatment with TGF-β. Additionally, caffeine was able to induce Ca2+ release and sensitize the CICR mechanism to ryanodine. Thus we suggest that the appearance of RyR2 mRNA leads to the expression of this receptor/channel protein with identifiable pharmacological characteristics. These results are discussed in the context of the potential role of gene activation in the process of maturation of the human myometrium during pregnancy.
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Clancy, J. S., H. Takeshima, S. L. Hamilton, and M. B. Reid. "Contractile function is unaltered in diaphragm from mice lacking calcium release channel isoform 3." American Journal of Physiology-Regulatory, Integrative and Comparative Physiology 277, no. 4 (October 1, 1999): R1205—R1209. http://dx.doi.org/10.1152/ajpregu.1999.277.4.r1205.
Full textAbstract:
Skeletal muscle expresses at least two isoforms of the calcium release channel in the sarcoplasmic reticulum (RyR1 and RyR3). Whereas the function of RyR1 is well defined, the physiological significance of RyR3 is unclear. Some authors have suggested that RyR3 participates in excitation-contraction coupling and that RyR3 may specifically confer resistance to fatigue. To test this hypothesis, we measured contractile function of diaphragm strips from adult RyR3-deficient mice (exon 2-targeted mutation) and their heterozygous and wild-type littermates. In unfatigued diaphragm, there were no differences in isometric contractile properties (twitch characteristics, force-frequency relationships, maximal force) among the three groups. Our fatigue protocol (30 Hz, 0.25 duty cycle, 37°C) depressed force to 25% of the initial force; however, lack of RyR3 did not accelerate the decline in force production. The force-frequency relationship was shifted to higher frequencies and was depressed in fatigued diaphragm; lack of RyR3 did not exaggerate these changes. We therefore provide evidence that RyR3 deficiency does not alter contractile function of adult muscle before, during, or after fatigue.
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Martin, Cécile, Jean-Marc Hyvelin, Karen E. Chapman, Roger Marthan, Richard H. Ashley, and Jean-Pierre Savineau. "Pregnant rat myometrial cells show heterogeneous ryanodine- and caffeine-sensitive calcium stores." American Journal of Physiology-Cell Physiology 277, no. 2 (August 1, 1999): C243—C252. http://dx.doi.org/10.1152/ajpcell.1999.277.2.c243.
Full textAbstract:
Intracellular Ca2+ release channels such as ryanodine receptors play crucial roles in the Ca2+-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 Ca2+ release channels in rat myometrial smooth muscle, and for the first time, intracellular Ca2+ concentration experiments with indo 1 on single myometrial cells have revealed the existence of a functional ryanodine- and caffeine-sensitive Ca2+ 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 Ca2+ release mechanism in rat myometrium is largely encoded by ryr3. Moreover, using intracellular Ca2+ 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.
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Perni, Stefano, Kurt C. Marsden, Matias Escobar, Stephen Hollingworth, Stephen M. Baylor, and Clara Franzini-Armstrong. "Structural and functional properties of ryanodine receptor type 3 in zebrafish tail muscle." Journal of General Physiology 145, no. 3 (February 9, 2015): 173–84. http://dx.doi.org/10.1085/jgp.201411303.
Full textAbstract:
The ryanodine receptor (RyR)1 isoform of the sarcoplasmic reticulum (SR) Ca2+ release channel is an essential component of all skeletal muscle fibers. RyR1s are detectable as “junctional feet” (JF) in the gap between the SR and the plasmalemma or T-tubules, and they are required for excitation–contraction (EC) coupling and differentiation. A second isoform, RyR3, does not sustain EC coupling and differentiation in the absence of RyR1 and is expressed at highly variable levels. Anatomically, RyR3 expression correlates with the presence of parajunctional feet (PJF), which are located on the sides of the SR junctional cisternae in an arrangement found only in fibers expressing RyR3. In frog muscle fibers, the presence of RyR3 and PJF correlates with the occurrence of Ca2+ sparks, which are elementary SR Ca2+ release events of the EC coupling machinery. Here, we explored the structural and functional roles of RyR3 by injecting zebrafish (Danio rerio) one-cell stage embryos with a morpholino designed to specifically silence RyR3 expression. In zebrafish larvae at 72 h postfertilization, fast-twitch fibers from wild-type (WT) tail muscles had abundant PJF. Silencing resulted in a drop of the PJF/JF ratio, from 0.79 in WT fibers to 0.03 in the morphants. The frequency with which Ca2+ sparks were detected dropped correspondingly, from 0.083 to 0.001 sarcomere−1 s−1. The few Ca2+ sparks detected in morphant fibers were smaller in amplitude, duration, and spatial extent compared with those in WT fibers. Despite the almost complete disappearance of PJF and Ca2+ sparks in morphant fibers, these fibers looked structurally normal and the swimming behavior of the larvae was not affected. This paper provides important evidence that RyR3 is the main constituent of the PJF and is the main contributor to the SR Ca2+ flux underlying Ca2+ sparks detected in fully differentiated frog and fish fibers.
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Rossi, R., R. Bottinelli, V. Sorrentino, and C. Reggiani. "Response to caffeine and ryanodine receptor isoforms in mouse skeletal muscles." American Journal of Physiology-Cell Physiology 281, no. 2 (August 1, 2001): C585—C594. http://dx.doi.org/10.1152/ajpcell.2001.281.2.c585.
Full textAbstract:
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.
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Murayama, Takashi, Toshiharu Oba, Shigeki Kobayashi, Noriaki Ikemoto, and Yasuo Ogawa. "Postulated role of interdomain interactions within the type 1 ryanodine receptor in the low gain of Ca2+-induced Ca2+ release activity of mammalian skeletal muscle sarcoplasmic reticulum." American Journal of Physiology-Cell Physiology 288, no. 6 (June 2005): C1222—C1230. http://dx.doi.org/10.1152/ajpcell.00415.2004.
Full textAbstract:
Ryanodine receptor (RyR) type 1 (RyR1) exhibits a markedly lower gain of Ca2+-induced Ca2+ release (CICR) activity than RyR type 3 (RyR3) in the sarcoplasmic reticulum (SR) of mammalian skeletal muscle (selective stabilization of the RyR1 channel), and this reduction in the gain is largely eliminated using 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid (CHAPS). We have investigated whether the hypothesized interdomain interactions within RyR1 are involved in the selective stabilization of the channel using [3H]ryanodine binding, single-channel recordings, and Ca2+ release from the SR vesicles. Like CHAPS, domain peptide 4 (DP4, a synthetic peptide corresponding to the Leu2442-Pro2477 region of RyR1), which seems to destabilize the interdomain interactions, markedly stimulated RyR1 but not RyR3. Their activating effects were saturable and nonadditive. Dantrolene, a potent inhibitor of RyR1 used to treat malignant hyperthermia, reversed the effects of DP4 or CHAPS in an identical manner. These findings indicate that RyR1 is activated by DP4 and CHAPS through a common mechanism that is probably mediated by the interdomain interactions. DP4 greatly increased [3H]ryanodine binding to RyR1 with only minor alterations in the sensitivity to endogenous CICR modulators (Ca2+, Mg2+, and adenine nucleotide). However, DP4 sensitized RyR1 four- to six-fold to caffeine in the caffeine-induced Ca2+ release. Thus the gain of CICR activity critically determines the magnitude and threshold of Ca2+ release by drugs such as caffeine. These findings suggest that the low CICR gain of RyR1 is important in normal Ca2+ handling in skeletal muscle and that perturbation of this state may result in muscle diseases such as malignant hyperthermia.
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KISELYOV, Kirill, Dong Min SHIN, Nikolay SHCHEYNIKOV, Tomohiro KUROSAKI, and Shmuel MUALLEM. "Regulation of Ca2+-release-activated Ca2+ current (Icrac) by ryanodine receptors in inositol 1,4,5-trisphosphate-receptor-deficient DT40 cells." Biochemical Journal 360, no. 1 (November 8, 2001): 17–22. http://dx.doi.org/10.1042/bj3600017.
Full textAbstract:
Persistence of capacitative Ca2+ influx in inositol 1,4,5-trisphosphate (IP3) receptor (IP3R)-deficient DT40 cells (DT40IP3R-/−) raises the question of whether gating of Ca2+-release activated Ca2+ current (Icrac) by conformational coupling to Ca2+-release channels is a general mechanism of gating of these channels. In the present work we examined the properties and mechanism of activation of Icrac Ca2+ current in wild-type and DT40IP3R-/− cells. In both cell types passive depletion of internal Ca2+ stores by infusion of EGTA activated a Ca2+ current with similar characteristics and time course. The current was highly Ca2+-selective and showed strong inward rectification, all typical of Icrac. The activator of ryanodine receptor (RyR), cADP-ribose (cADPR), facilitated activation of Icrac, and the inhibitors of the RyRs, 8-N-cADPR, ryanodine and Ruthenium Red, all inhibited Icrac activation in DT40IP3R-/− cells, even after complete depletion of intracellular Ca2+ stores by ionomycin. Wild-type and DT40IP3R-/− cells express RyR isoforms 1 and 3. RyR levels were adapted in DT40IP3R-/− cells to a lower RyR3/RyR1 ratio than in wild-type cells. These results suggest that IP3Rs and RyRs can efficiently gate Icrac in DT40 cells and explain the persistence of Icrac gating by internal stores in the absence of IP3Rs.
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Lifshitz, Lawrence M., Jeffrey D. Carmichael, F. Anthony Lai, Vincenzo Sorrentino, Karl Bellvé, Kevin E. Fogarty, and Ronghua ZhuGe. "Spatial organization of RYRs and BK channels underlying the activation of STOCs by Ca2+ sparks in airway myocytes." Journal of General Physiology 138, no. 2 (July 11, 2011): 195–209. http://dx.doi.org/10.1085/jgp.201110626.
Full textAbstract:
Short-lived, localized Ca2+ events mediate Ca2+ signaling with high efficiency and great fidelity largely as a result of the close proximity between Ca2+-permeable ion channels and their molecular targets. However, in most cases, direct evidence of the spatial relationship between these two types of molecules is lacking, and, thus, mechanistic understanding of local Ca2+ signaling is incomplete. In this study, we use an integrated approach to tackling this issue on a prototypical local Ca2+ signaling system composed of Ca2+ sparks resulting from the opening of ryanodine receptors (RYRs) and spontaneous transient outward currents (STOCs) caused by the opening of Ca2+-activated K+ (BK) channels in airway smooth muscle. Biophysical analyses of STOCs and Ca2+ sparks acquired at 333 Hz demonstrate that these two events are associated closely in time, and approximately eight RYRs open to give rise to a Ca2+ spark, which activates ∼15 BK channels to generate a STOC at 0 mV. Dual immunocytochemistry and 3-D deconvolution at high spatial resolution reveal that both RYRs and BK channels form clusters and RYR1 and RYR2 (but not RYR3) localize near the membrane. Using the spatial relationship between RYRs and BK channels, the spatial-temporal profile of [Ca2+] resulting from Ca2+ sparks, and the kinetic model of BK channels, we estimate that an average Ca2+ spark caused by the opening of a cluster of RYR1 or RYR2 acts on BK channels from two to three clusters that are randomly distributed within an ∼600-nm radius of RYRs. With this spatial organization of RYRs and BK channels, we are able to model BK channel currents with the same salient features as those observed in STOCs across a range of physiological membrane potentials. Thus, this study provides a mechanistic understanding of the activation of STOCs by Ca2+ sparks using explicit knowledge of the spatial relationship between RYRs (the Ca2+ source) and BK channels (the Ca2+ target).
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Eckhardt, Jan, Christoph Bachmann, Marijana Sekulic-Jablanovic, Volker Enzmann, Ki Ho Park, Jianjie Ma, Hiroshi Takeshima, Francesco Zorzato, and Susan Treves. "Extraocular muscle function is impaired in ryr3−/− mice." Journal of General Physiology 151, no. 7 (May 13, 2019): 929–43. http://dx.doi.org/10.1085/jgp.201912333.
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Calcium is an ubiquitous second messenger mediating numerous physiological processes, including muscle contraction and neuronal excitability. Ca2+ is stored in the ER/SR and is released into the cytoplasm via the opening of intracellular inositol trisphosphate receptor and ryanodine receptor calcium channels. Whereas in skeletal muscle, isoform 1 of the RYR is the main channel mediating calcium release from the SR leading to muscle contraction, the function of ubiquitously expressed ryanodine receptor 3 (RYR3) is far from clear; it is not known whether RYR3 plays a role in excitation–contraction coupling. We recently reported that human extraocular muscles express high levels of RYR3, suggesting that such muscles may be useful to study the function of this isoform of the Ca2+ channel. In the present investigation, we characterize the visual function of ryr3−/− mice. We observe that ablation of RYR3 affects both mechanical properties and calcium homeostasis in extraocular muscles. These changes significantly impact vision. Our results reveal for the first time an important role for RYR3 in extraocular muscle function.
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Tsai, Shu-Huei, Emily Yun-Chia Chang, Yi-Cheng Chang, Siow-Wey Hee, Yun-Chih Tsai, Tien-Jyun Chang, and Lee-Ming Chuang. "Knockdown of RyR3 Enhances Adiponectin Expression Through an atf3-Dependent Pathway." Endocrinology 154, no. 3 (March 1, 2013): 1117–29. http://dx.doi.org/10.1210/en.2012-1515.
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Abstract Adiponectin is an important adipose-specific protein, which possesses insulin (INS)-sensitizing, antiinflammatory, and antiatherosclerotic functions. However, its regulation remains largely unknown. In this study, we identified that ryanodine receptor (RyR)3 plays an important role in the regulation of adiponectin expression. RyR3 was expressed in 3T3-L1 preadipocytes, and its level was decreased upon adipogenesis. Silencing of RyR3 expression in 3T3-L1 preadipocytes resulted in up-regulated adiponectin promoter activity, enhanced adiponectin mRNA expression, and more adiponectin protein secreted into the medium. An inverse relation between RyR3 and adiponectin mRNA levels was also observed in adipose tissues of db/db mice. In addition, knockdown of RyR3 with small interfering RNA (siRNA) in db/db mice and high-fat diet-fed obese mice increased serum adiponectin level, improved INS sensitivity, and lowered fasting glucose levels. These effects were in parallel with decreased mitochondrial Ca2+, increased mitochondrial mass, and reduced activating transcription factor 3 (atf3) expression. Overexpression of atf3 in 3T3-L1 preadipocytes blocked the effect of RyR3 silencing on adiponectin expression, indicating that an atf3-dependent pathway mediates the effect downstream of RyR3 silencing. Our data suggest that RyR3 may be a new therapeutic target for improving INS sensitivity and related metabolic disorders.
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Chun, Lois G., Christopher W. Ward, and Martin F. Schneider. "Ca2+ sparks are initiated by Ca2+ entry in embryonic mouse skeletal muscle and decrease in frequency postnatally." American Journal of Physiology-Cell Physiology 285, no. 3 (September 2003): C686—C697. http://dx.doi.org/10.1152/ajpcell.00072.2003.
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“Spontaneous” Ca2+ sparks and ryanodine receptor type 3 (RyR3) expression are readily detected in embryonic mammalian skeletal muscle but not in adult mammalian muscle, which rarely exhibits Ca2+ sparks and expresses predominantly RyR1. We have used confocal fluorescence imaging and systematic sampling of enzymatically dissociated single striated muscle fibers containing the Ca2+ indicator dye fluo 4 to show that the frequency of spontaneous Ca2+ sparks decreases dramatically from embryonic day 18 (E18) to postnatal day 14 (P14) in mouse diaphragm and from P1 to P14 in mouse extensor digitorum longus fibers. In contrast, the relative levels of RyR3 to RyR1 protein remained constant in diaphragm muscles from E18 to P14, indicating that changes in relative levels of RyR isoform expression did not cause the decline in Ca2+ spark frequency. E18 diaphragm fibers were used to investigate possible mechanisms underlying spark initiation in embryonic fibers. Spark frequency increased or decreased, respectively, when E18 diaphragm fibers were exposed to 8 or 0 mM Ca2+ in the extracellular Ringer solution, with no change in either the average resting fiber fluo 4 fluorescence or the average properties of the sparks. Either CoCl2 (5 mM) or nifedipine (30 μM) markedly decreased spark frequency in E18 diaphragm fibers. These results indicate that Ca2+ sparks may be triggered by locally elevated [Ca2+] due to Ca2+ influx via dihydropyridine receptor L-type Ca2+ channels in embryonic mammalian skeletal muscle.
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Mattei, M. G., G. Giannini, F. Moscatelli, and V. Sorrentino. "Chromosomal Localization of Murine Ryanodine Receptor Genes RYR1, RYR2, and RYR3 by in Situ Hybridization." Genomics 22, no. 1 (July 1994): 202–4. http://dx.doi.org/10.1006/geno.1994.1362.
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Huddleston, A. Tara, Wei Tang, Hiroshi Takeshima, Susan L. Hamilton, and Eric Klann. "Superoxide-Induced Potentiation in the Hippocampus Requires Activation of Ryanodine Receptor Type 3 and ERK." Journal of Neurophysiology 99, no. 3 (March 2008): 1565–71. http://dx.doi.org/10.1152/jn.00659.2007.
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Reactive oxygen species (ROS) are required for the induction of long-term potentiation (LTP) and behave as signaling molecules via redox modifications of target proteins. In particular, superoxide is necessary for induction of LTP, and application of superoxide to hippocampal slices is sufficient to induce LTP in area CA1. Although a rise in postsynaptic intracellular calcium is necessary for LTP induction, superoxide-induced potentiation does not require calcium flux through N-methyl-d-aspartate (NMDA) receptors. Ryanodine receptors (RyRs) mediate calcium-induced calcium release from intracellular stores and have been shown to modulate LTP. In this study, we investigated the highly redox-sensitive RyRs and L-type calcium channels as calcium sources that might mediate superoxide-induced potentiation. In agreement with previous studies of skeletal and cardiac muscle, we found that superoxide enhances activation of RyRs in the mouse hippocampus. We identified a functional coupling between L-type voltage-gated calcium channels and RyRs and identified RyR3, a subtype enriched in area CA1, as the specific isoform required for superoxide-induced potentiation. Superoxide also enhanced the phosphorylation of extracellular signal-regulated kinase (ERK) in area CA1, and ERK was necessary for superoxide-induced potentiation. Thus superoxide-induced potentiation requires the redox targeting of RyR3 and the subsequent activation of ERK.
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Iacomino, Nicola, Letizia Scandiffio, Fabio Conforti, Erika Salvi, Maria Cristina Tarasco, Federica Bortone, Stefania Marcuzzo, et al. "Muscle and Muscle-like Autoantigen Expression in Myasthenia Gravis Thymus: Possible Molecular Hint for Autosensitization." Biomedicines 11, no. 3 (February 28, 2023): 732. http://dx.doi.org/10.3390/biomedicines11030732.
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The thymus is widely recognized as an immunological niche where autoimmunity against the acetylcholine receptor (AChR) develops in myasthenia gravis (MG) patients, who mostly present thymic hyperplasia and thymoma. Thymoma-associated MG is frequently characterized by autoantibodies to the muscular ryanodine receptor 1 (RYR1) and titin (TTN), along with anti-AChR antibodies. By real-time PCR, we analyzed muscle—CHRNA1, RYR1, and TTN—and muscle-like—NEFM, RYR3 and HSP60—autoantigen gene expression in MG thymuses with hyperplasia and thymoma, normal thymuses and non-MG thymomas, to check for molecular changes potentially leading to an altered antigen presentation and autoreactivity. We found that CHRNA1 (AChR-α subunit) and AIRE (autoimmune regulator) genes were expressed at lower levels in hyperplastic and thymoma MG compared to the control thymuses, and that the RYR1 and TTN levels were decreased in MG versus the non-MG thymomas. Genes encoding autoantigens that share epitopes with AChR-α (NEFM and HSP60), RYR1 (neuronal RYR3), and TTN (NEFM) were up-regulated in thymomas versus hyperplastic and control thymuses, with distinct molecular patterns across the thymoma histotypes that could be relevant for autoimmunity development. Our findings support the idea that altered muscle autoantigen expression, related with hyperplastic and neoplastic changes, may favor autosensitization in the MG thymus, and that molecular mimicry involving tumor-related muscle-like proteins may be a mechanism that makes thymoma prone to developing MG.
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Thorne, George D., and Richard J. Paul. "Effects of organ culture on arterial gene expression and hypoxic relaxation: role of the ryanodine receptor." American Journal of Physiology-Cell Physiology 284, no. 4 (April 1, 2003): C999—C1005. http://dx.doi.org/10.1152/ajpcell.00158.2002.
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Organ culture specifically inhibits vasorelaxation to acute hypoxia and preferentially decreases specific voltage-dependent K+channel expression over other K+ and Ca2+channel subtypes. To isolate further potential oxygen-sensing mechanisms correlated with altered gene expression, we performed differential display analysis on RNA isolated from control and cultured coronary arterial rings. We hypothesize that organ culture results in altered gene expression important for vascular smooth muscle contractility important to the mechanism of hypoxia-induced relaxation. Our results indicate a milieu of changes suggesting both up- and downregulation of several genes. The altered expression pattern of two positive clones was verified by Northern analysis. Subsequent screening of a porcine cDNA library indicated homology to the ryanodine receptor (RyR). RT-PCR using specific primers to the three subtypes of RyR shows an upregulation of RyR2 and RyR3 after organ culture. Additionally, the caffeine- and/or ryanodine-sensitive intracellular Ca2+store was significantly more responsive to caffeine activation after organ culture. Our data indicate that organ culture increases expression of specific RyR subtypes and inhibits hypoxic vasorelaxation. Importantly, ryanodine blunted hypoxic relaxation in control coronary arteries, suggesting that upregulated RyR might play a novel role in altered intracellular Ca2+ handling during hypoxia.
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Dahan, Diana, Thomas Ducret, Jean-François Quignard, Roger Marthan, Jean-Pierre Savineau, and Eric Estève. "Implication of the ryanodine receptor in TRPV4-induced calcium response in pulmonary arterial smooth muscle cells from normoxic and chronically hypoxic rats." American Journal of Physiology-Lung Cellular and Molecular Physiology 303, no. 9 (November 1, 2012): L824—L833. http://dx.doi.org/10.1152/ajplung.00244.2011.
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There is a growing body of evidence indicating that transient receptor potential (TRP) channels are implicated in calcium signaling and various cellular functions in the pulmonary vasculature. The aim of this study was to investigate the expression, functional role, and coupling to reticulum calcium channels of the type 4 vanilloid TRP subfamily (TRPV4) in the pulmonary artery from both normoxic (Nx) and chronically hypoxic (CH) rats. Activation of TRPV4 with the specific agonist 4α-phorbol-12,13-didecanoate (4α-PDD, 5 μM) increased the intracellular calcium concentration ([Ca2+]i). This effect was significantly reduced by a high concentration of ryanodine (100 μM) or chronic caffeine (5 mM) that blocked ryanodine receptor (RyR) but was insensitive to xestospongin C (10 μM), an inositol trisphosphate receptor antagonist. Inhibition of RyR1 and RyR3 only with 10 μM of dantrolene did not attenuate the 4α-PDD-induced [Ca2+]i increase. Western blotting experiments revealed the expression of TRPV4 and RyR2 with an increase in both receptors in pulmonary arteries from CH rats vs. Nx rats. Accordingly, the 4α-PDD-activated current, measured with patch-clamp technique, was increased in pulmonary artery smooth muscle cells (PASMC) from CH rats vs. Nx rats. 4α-PDD increased isometric tension in artery rings, and this response was also potentiated under chronic hypoxia conditions. 4α-PDD-induced calcium response, current, and contraction were all inhibited by the selective TRPV4 blocker HC-067047. Collectively, our findings provide evidence of the interplay between TRPV4 and RyR2 in the Ca2+ release mechanism and contraction in PASMC. This study provides new insights onto the complex calcium signaling in PASMC and point out the importance of the TRPV4-RyR2 signaling pathway under hypoxic conditions that may lead to pulmonary hypertension.
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Frank, Daniel F., Galen W. Miller, Richard E. Connon, Juergen Geist, and Pamela J. Lein. "Transcriptomic profiling of mTOR and ryanodine receptor signaling molecules in developing zebrafish in the absence and presence of PCB 95." PeerJ 5 (November 29, 2017): e4106. http://dx.doi.org/10.7717/peerj.4106.
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The mechanistic target of rapamycin (mTOR) and ryanodine receptor (RyR) signaling pathways regulate fundamental processes of neurodevelopment, and genetic mutations within these pathways have been linked to neurodevelopmental disorders. While previous studies have established that these signaling molecules are expressed in developing zebrafish, a detailed characterization of the ontogenetic profile of these signaling molecules is lacking. Thus, we evaluated the spatiotemporal expression of key transcripts in mTOR and RyR signaling pathways in wildtype zebrafish at 24, 72 and 120 hours post fertilization (hpf). We further determined whether transcriptional profiles of a subset of genes in both pathways were altered by exposure to PCB 95 (2,2′,3,5′,6-pentachlorobiphenyl), a pervasive environmental contaminant known to cause developmental neurotoxicity in mammalian systems via RyR-dependent mechanisms. Quantitative PCR revealed that transcription generally increased across development. Genes in the signaling pathway upstream of the mTORC1 complex, and the RyR-paralogs, ryr2a and ryr3, were robustly upregulated, and in situ hybridization of ryr3 coincided with a transcriptional shift from muscle to neuronal tissue after 24 hpf. Static waterborne exposure to PCB 95 beginning at 6 hpf significantly altered transcription of genes in both pathways. These changes were concentration- and time-dependent, and included downregulation of rptor, a member of the mTORC1 complex, at both 72 and 120 hpf, and increased transcript levels of the RyR paralog ryr2b and downstream target of RyR signaling, Wingless-type 2ba (wnt2ba) at 72 hpf. The detailed transcriptomic profiling of key genes within these two signaling pathways provides a baseline for identifying other environmental factors that modify normal spatiotemporal expression patterns of mTOR and RyR signaling pathways in the developing zebrafish, as illustrated here for PCB 95.
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Hoyer, Joachim, Meike Kuehn, Christiane Degenhardt, Norbert Runkel, Martin Paul, MC Benjamin Franklin, and Ralf Koehler. "Expression of Ryanodine Receptor 3 and Trp Channels in Endothelium of Human Mesenteric Artery: A Single-Cell Rt-Pcr and Patch-Clamp Analysis in Situ ." Hypertension 36, suppl_1 (October 2000): 719. http://dx.doi.org/10.1161/hyp.36.suppl_1.719-d.
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P146 Ca 2+ mobilization plays an important role in endothelial function by stimulating Ca 2+ -dependent synthesis of vasodilating factors. In addition to InsP 3 - mediated Ca 2+ store depletion, Ca 2+ release from ryanodine-sensitive pools and Ca 2+ -influx through cation channels of the TRP-gene family have been suggested to be involved in endothelial Ca 2+ signaling. In cultured endothelial cells (EC) the function and expression of ryanodine-receptors (RyR) and TRP channels might differ substantially from those in native endothelium of human blood vessels. We, therefore, characterized expression and function of RyR and TRP channels in EC of small human mesenteric artery (MA) by use of single-cell RT-PCR and patch-clamp techniques in situ. MA were isolated from colon specimens of patients subjected to hemicolectomy. For single-cell RT-PCR and PC experiments single human mesenteric artery EC (HMAEC) were harvested directly from the luminal vessel with the patch pipette. Expression of the RyR subtype 3, but not of the RyR1 and RyR2 subtpyes was detected in 25% of HMAEC samples. Correspondingly, in PC experiments in HMAEC, application of caffeine (0.5 mM) induced Ca 2+ -release from ryanodine-sensitive stores and subsequently activation of Ca 2+ -activated K + channels leading to a sustained endothelial hyperpolarization from a resting potential of 28 ± 3 mV to -46 ± 4 mV. Single HMAEC expressed the TRP subtypes, TRP1 and TRP3, but not TRP 4 and 6. The TRP1 was the predominantly expressed TRP subtype as expression was detected in 16% of HMAEC. TRP3 expression was detected in only 3% of HMAEC. In single-channel and whole-cell patch clamp experiments in HMAEC, InsP 3 -mediated Ca 2+ -store depletion in response to bradykinin (100 nM) activated TRP related nonselective cation currents leading to Ca 2+ entry. In conclusion, Ca 2+ release from ryanodine-sensitive stores mediated by RyR3 and Ca 2+ entry through TRP1 might represent important components of endothelial Ca 2+ signaling and thereby of endothelial function in intact human blood vessels.
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Savoia, Carlo P., Qing-Hua Liu, Yun-Min Zheng, Vishal Yadav, Zhen Zhang, Ling-Gang Wu, and Yong-Xiao Wang. "Calcineurin upregulates local Ca2+ signaling through ryanodine receptor-1 in airway smooth muscle cells." American Journal of Physiology-Lung Cellular and Molecular Physiology 307, no. 10 (November 15, 2014): L781—L790. http://dx.doi.org/10.1152/ajplung.00149.2014.
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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.
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Ooashi, Noriko, Akira Futatsugi, Fumie Yoshihara, Katsuhiko Mikoshiba, and Hiroyuki Kamiguchi. "Cell adhesion molecules regulate Ca2+-mediated steering of growth cones via cyclic AMP and ryanodine receptor type 3." Journal of Cell Biology 170, no. 7 (September 19, 2005): 1159–67. http://dx.doi.org/10.1083/jcb.200503157.
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Axonal growth cones migrate along the correct paths during development, not only directed by guidance cues but also contacted by local environment via cell adhesion molecules (CAMs). Asymmetric Ca2+ elevations in the growth cone cytosol induce both attractive and repulsive turning in response to the guidance cues (Zheng, J.Q. 2000. Nature. 403:89–93; Henley, J.R., K.H. Huang, D. Wang, and M.M. Poo. 2004. Neuron. 44:909–916). Here, we show that CAMs regulate the activity of ryanodine receptor type 3 (RyR3) via cAMP and protein kinase A in dorsal root ganglion neurons. The activated RyR3 mediates Ca2+-induced Ca2+ release (CICR) into the cytosol, leading to attractive turning of the growth cone. In contrast, the growth cone exhibits repulsion when Ca2+ signals are not accompanied by RyR3-mediated CICR. We also propose that the source of Ca2+ influx, rather than its amplitude or the baseline Ca2+ level, is the primary determinant of the turning direction. In this way, axon-guiding and CAM-derived signals are integrated by RyR3, which serves as a key regulator of growth cone navigation.
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MOUTON, Jérôme, Isabelle MARTY, Michel VILLAZ, Anne FELTZ, and Yves MAULET. "Molecular interaction of dihydropyridine receptors with type-1 ryanodine receptors in rat brain." Biochemical Journal 354, no. 3 (March 8, 2001): 597–603. http://dx.doi.org/10.1042/bj3540597.
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In striated muscles, Ca2+ release from internal stores through ryanodine receptor (RyR) channels is triggered by functional coupling to voltage-activated Ca2+ channels known as dihydropyridine receptors (DHPRs) located in the plasma membrane. In skeletal muscle, this occurs by a direct conformational link between the tissue-specific DHPR (Cav1.1) and RyR1, whereas in the heart the signal is carried from the cardiac-type DHPR (Cav1.2) to RyR2 by calcium ions acting as an activator. Subtypes of both channels are expressed in the central nervous system, but their functions and mechanisms of coupling are still poorly understood. We show here that complexes immunoprecipitated from solubilized rat brain membranes with antibodies against DHPR of the Cav1.2 or Cav1.3 subtypes contain RyR. Only type-1 RyR is co-precipitated, although the major brain isoform is RyR2. This suggests that, in neurons, DHPRs could communicate with RyRs by way of a strong molecular interaction and, more generally, that the physical link between DHPR and RyR shown to exist in skeletal muscle can be extended to other tissues.
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Torres, Rodrigo F., and Bredford Kerr. "Spatial Learning Is Associated with Antagonist Outcomes for DNA Methylation and DNA Hydroxymethylation in the Transcriptional Regulation of the Ryanodine Receptor 3." Neural Plasticity 2021 (August 11, 2021): 1–8. http://dx.doi.org/10.1155/2021/9930962.
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Increasing attention has been drawn to the role that intracellular calcium stores play in neuronal function. Ryr3 is an intracellular calcium channel that contributes to hippocampal long-term potentiation, dendritic spine function, and higher cognitive processes. Interestingly, stimuli that increase neuronal activity upregulate the transcriptional activity of Ryr3 and augment DNA methylation in its proximal promoter. However, if these observations are valid for complex behavioral tasks such as learning and memory remains being evaluated. Relative expression analysis revealed that spatial learning increased the hippocampal levels of Ryr3, whereas mice trained using a visible platform that resulted in no spatial association showed reduced expression. Interestingly, we also observed that specific DNA modifications accompanied these opposite transcriptional changes. Increased DNA methylation was observed in hippocampal samples from spatially trained mice, and increased DNA hydroxymethylation was found in samples from mice trained using a visible platform. Both DNA modifications were not altered in control regions, suggesting that these changes are not generalized, but rather specific modifications associated with this calcium channel’s transcriptional regulation. Our two experimental groups underwent the same physical task differing only in the spatial learning component, highlighting the tight relationship between DNA modifications and transcriptional activity in a relevant context such as behavioral training. Our results complement previous observations and suggest that DNA modifications are a reliable signal for the transcriptional activity of Ryr3 and can be useful to understand how conditions such as aging and neuropathological diseases determine altered Ryr3 expression.
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Yang, Dongmei, Zui Pan, Hiroshi Takeshima, Caihong Wu, Ramakrishnan Y. Nagaraj, Jianjie Ma, and Heping Cheng. "RyR3 Amplifies RyR1-mediated Ca2+-induced Ca2+Release in Neonatal Mammalian Skeletal Muscle." Journal of Biological Chemistry 276, no. 43 (August 10, 2001): 40210–14. http://dx.doi.org/10.1074/jbc.m106944200.
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Vega-Vásquez, Ignacio, Pedro Lobos, Jorge Toledo, Tatiana Adasme, Andrea Paula-Lima, and Cecilia Hidalgo. "Hippocampal dendritic spines express the RyR3 but not the RyR2 ryanodine receptor isoform." Biochemical and Biophysical Research Communications 633 (December 2022): 96–103. http://dx.doi.org/10.1016/j.bbrc.2022.10.024.
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Haseeb, Mohsin, and Paul D. Thompson. "The effect of statins on RyR and RyR-associated disease." Journal of Applied Physiology 131, no. 2 (August 1, 2021): 661–71. http://dx.doi.org/10.1152/japplphysiol.01003.2020.
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We sought to review the effects of statins on the ryanodine receptor (RyR) and on RyR-associated diseases, with an emphasis on catecholaminergic polymorphic ventricular tachycardia (CPVT). Statins can affect skeletal muscle and produce statin-associated muscle symptoms (SAMS) but have no adverse effects on cardiac muscle. These contrasting effects may be due to differences in how statins affect the skeletal (RyR1) and cardiac (RyR2) RyR. We searched PubMed to identify English language articles reporting the pathophysiology of the RyR, the effect of statins on RyR function, and on RyR-associated genetic diseases. We selected 150 articles for abstract review, 96 of which provided sufficient information to be included and were reviewed in detail. Fifteen articles highlighted the interaction of statins with the RyR. Nine identified the interaction of statins with RyR1, six addressed the interaction of statins with RyR2, 13 suggested that statins reduce ventricular arrhythmias (VA), and seven suggested that statins increase the risk of malignant hyperthermia (MH). In general, statins increase RyR1 and decrease RyR2 activity. We identified no articles examining the effect of statins on CPVT, a condition often caused by defects in RyR2. Statins appear to increase the risk of MH and decrease the risk of ventricular arrhythmia. The effect of statins on CPVT has not been directly examined, but statins’ reduction in RyR2 function and their apparent reduction in VA suggest that they may be beneficial in this condition.
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Perez, Claudio F., Andrew Voss, Isaac N. Pessah, and Paul D. Allen. "RyR1/RyR3 Chimeras Reveal that Multiple Domains of RyR1 Are Involved in Skeletal-Type E-C Coupling." Biophysical Journal 84, no. 4 (April 2003): 2655–63. http://dx.doi.org/10.1016/s0006-3495(03)75071-1.
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