Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: UNIPROT:P21817 (RyR1)
 1,154 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Calmodulin (CaM) is a ubiquitous Ca2+-binding protein that regulates the ryanodine receptors (RyRs) by direct binding. CaM inhibits the skeletal muscle ryanodine receptor (RyR1) and cardiac muscle receptor (RyR2) at >1 microm Ca2+ but activates RyR1 and inhibits RyR2 at <1 microm Ca2+. Here we tested whether CaM regulates RyR2 by binding to a highly conserved site identified previously in RyR1. Deletion of RyR2 amino acid residues 3583-3603 resulted in background [35S]CaM binding levels. In single channel measurements, deletion of the putative CaM binding site eliminated CaM inhibition of RyR2 at Ca2+ concentrations below and above 1 microm. Five RyR2 single or double mutants in the CaM binding region (W3587A, L3591D, F3603A, W3587A/L3591D, L3591D/F3603A) eliminated or greatly reduced [35S]CaM binding and inhibition of single channel activities by CaM depending on the Ca2+ concentration. An RyR2 mutant, which assessed the effects of 4 amino acid residues that differ between RyR1 and RyR2 in or flanking the CaM binding domain, bound [35S]CaM and was inhibited by CaM, essentially identical to wild type (WT)-RyR2. Three RyR1 mutants (W3620A, L3624D, F3636A) showed responses to CaM that differed from corresponding mutations in RyR2. The results indicate that CaM regulates RyR1 and RyR2 by binding to a single, highly conserved CaM binding site and that other RyR type-specific sites are likely responsible for the differential functional regulation of RyR1 and RyR2 by CaM.
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PMID:Molecular basis of calmodulin binding to cardiac muscle Ca(2+) release channel (ryanodine receptor). 1270 60

The ryanodine receptor (RyR) is the major calcium (Ca(2+)) release channel in the sarcoplasmic reticulum (SR) of skeletal and cardiac muscle and is required for excitation-contraction (EC) coupling. The 565 kDa RyR protein forms a tetrameric channel that is part of a macromolecular signaling complex that also includes four FK506 binding proteins (FKBPs). The RyR channel complex is localized on specialized regions of the SR, such that the large RyR cytoplasmic domain is closely opposed to the transverse tubule (T-tubule) of the plasma membrane. RyR channel complexes are organized in regular arrays such that neighboring RyRs are in physical contact with each other. We have shown that physical and functional association between RyR1 or RyR2 channels results in coordinated gating behavior termed coupled gating. Coupled gating requires FKBP12 or FKBP12.6 in the RyR1 or RyR2 macromolecular complexes, respectively. FKBPs are known to stabilize single RyR channel function. Coupled gating describes an additional role for FKBPs in the functional coordination of RyR channel complexes that allows clusters of channels to function as "Ca2+ release units" (CRU). In addition, the FKBP-RyR interaction is regulated by PKA phosphorylation. In failing hearts PKA hyperphosphorylation of RyR2 causes depletion of FKBP12.6 from the channel macromolecular complex and may contribute to contractile dysfunction by impairing EC coupling. As FKBPs are potent modulators of RyR channel function, the FKBP-RyR interaction is a focus for determining molecular mechanisms of coupled gating and presents an exciting pharmacologic target for restoration of RyR complex function in diseased states.
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PMID:Immunophilins and coupled gating of ryanodine receptors. 1287 Nov 70

Four ryanodine receptor type 1 and 2 chimeras (R4, R9, R10, and R16) and their respective wild-type ryanodine receptors (type 1 and 2; wtRyR1 and wtRyR2) were expressed in dyspedic 1B5 to identify possible negative regulatory modules of the Ca2+ release channel that are under the influence of the dihydropyridine receptor (DHPR). Responses of intact 1B5 myotubes expressing each construct to caffeine in the absence or presence of either La3+ and Cd2+ or the organic DHPR blocker nifedipine were determined by imaging single 1B5 myotubes loaded with fluo 4. The presence of La3+ and Cd2+ or nifedipine in the external medium at concentrations known to block Ca2+ entry through the DHPRs significantly decreased the caffeine EC50 of wtRyR1 (2.80 +/- 0.12 to 0.83 +/- 0.09 mM; P < 0.05). On the other hand, DHPR blockade did not significantly alter the caffeine EC50 values of wtRyR2, chimeras R10 and R16, whereas the caffeine EC50 values of chimeras R4 and R9 were significantly increased (1.27 +/- 0.05 to 2.60 +/- 0.16 mM, and 1.15 +/- 0.03 to 2.11 +/- 0.32 mM, respectively; P < 0.05). Despite the fact that all the chimeras form fully functional Ca2+ release channels in situ, sarcoplasmic reticulum (SR) containing R4, R10, and R16 did not possess high-affinity binding of [3H]ryanodine regardless of Ca2+ concentration. These results suggest the presence of an interaction between RyR1 and the DHPR, which is not present in RyR2, that contributes negative control of SR Ca2+ release induced by direct agonists such as caffeine. Although we were unable to define the negative module using RyR1-RyR2 chimeras, they further demonstrated that the RyR is very sensitive to long-range allosterism.
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PMID:Conformational coupling of DHPR and RyR1 in skeletal myotubes is influenced by long-range allosterism: evidence for a negative regulatory module. 1367 3

Phosphorylation of the skeletal muscle (RyR1) and cardiac muscle (RyR2) ryanodine receptors has been reported to modulate channel activity. Abnormally high phosphorylation levels (hyperphosphorylation) at Ser-2843 in RyR1 and Ser-2809 in RyR2 and dissociation of FK506-binding proteins from the receptors have been implicated as one of the causes of altered calcium homeostasis observed during human heart failure. Using site-directed mutagenesis, we prepared recombinant RyR1 and RyR2 mutant receptors mimicking constitutively phosphorylated and dephosphorylated channels carrying a Ser/Asp (RyR1-S2843D and RyR2-S2809D) and Ser/Ala (RyR1-S2843A and RyR2-S2809A) substitution, respectively. Following transient expression in human embryonic kidney 293 cells, the effects of Ca2+, Mg2+, and ATP on channel function were determined using single channel and [3H]ryanodine binding measurements. In both assays, neither the skeletal nor cardiac mutants showed significant differences compared with wild type. Similarly essentially identical caffeine responses were observed in Ca2+ imaging measurements. Co-immunoprecipitation and Western blot analysis showed comparable binding of FK506-binding proteins to wild type and mutant receptors. Finally metabolic labeling experiments showed that the cardiac ryanodine receptor was phosphorylated at additional sites. Taken together, the results did not support the view that phosphorylation of a single site (RyR1-Ser-2843 and RyR2-Ser-2809) substantially changes RyR1 and RyR2 channel function.
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PMID:Characterization of recombinant skeletal muscle (Ser-2843) and cardiac muscle (Ser-2809) ryanodine receptor phosphorylation mutants. 1453 76

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+]i and perfused with either cold ( approximately 0 degrees 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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PMID:All three ryanodine receptor isoforms generate rapid cooling responses in muscle cells. 1459 7

Specific interactions between adjacent ryanodine receptor (RyR) molecules to form ordered two-dimensional arrays in the membrane have been demonstrated using electron microscopy both in situ, in tissues and cells, and in vitro, with the purified protein. RyR interoligomeric association has also been inferred from observations of simultaneous channel gating during multi-RyR channel recordings in lipid bilayers. In this study, we report experiments designed to identify the region(s) of the RyR molecule, participating in this reciprocal interaction. Using epitope-specific antibodies, we identified a RyR tryptic fragment that specifically bound the intact immobilized RyR. Three overlapping RyR fragments encompassing this epitope, expressed using an in vitro mammalian expression system, were immunoprecipitated by RyR. To refine the binding regions, smaller RyR fragments were expressed as glutathione S-transferase (GST) fusion proteins, and their binding to RyR was monitored using a "sandwich" enzyme-linked immunosorbent assay. Three GST-RyR fusion proteins demonstrated specific binding, dependent upon ionic strength. Binding was greatest at 50-150 mm NaCl for two GST-RyR constructs, and a third GST-RyR construct demonstrated maximum binding between 150 and 450 mm NaCl. The binding at high NaCl concentration suggested involvement of a hydrophobic interaction. In silico analysis of secondary structure showed evidence of coil regions in two of these RyR fragment sequences, which might explain these data. In GST pull-down assays, these same three fragments captured RyR2, and two of them retained RyR1. These results identify a region at the center of the linear RyR (residues 2540-3207 of human RyR2) which is able to bind to the RyR oligomer. This region may constitute a specific subdomain participating in RyR-RyR interaction.
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PMID:Ryanodine receptor oligomeric interaction: identification of a putative binding region. 1472

It is known that the two types of FK506-binding proteins FKBP12 and FKBP12.6 are tightly associated with the skeletal (RyR1) and cardiac ryanodine receptors (RyR2), respectively, and their interactions are important for channel functions of the RyR. In the case of cardiac muscle, three amino acid residues (Gln-31, Asn-32, and Phe-59) of FKBP12.6 could be essential for the selective binding to RyR2 (Xin, H. B., Rogers, K., Qi, Y., Kanematsu, T., and Fleischer, S. (1999) J. Biol. Chem. 274, 15315-15319). In this study to identify amino acid residues of FKBP12 that are important for the selective binding to RyR1, we mutated 9 amino acid residues of FKBP12 that differ from the counterparts of FKBP12.6 (Q3E, R18A, E31Q, D32N, M49R, R57A, W59F, H94A, and K105A), and we examined binding properties of these mutants to RyR1 by in vitro binding assay by using glutathione S-transferase-fused proteins of the mutants and Triton X-100-solubilized, FKBP12-depleted rabbit skeletal sarcoplasmic reticulum vesicles. Among the nine mutants tested, only Q3E and R18A lost their selective binding ability to RyR1. Furthermore, co-immunoprecipitation of RyR1 with 33 various mutants for the 9 positions produced by introducing different size, charge, and hydrophobicity revealed that an integration of the hydrogen bonds by the irreplaceable Gln-3 and the hydrophobic interactions by the residues Arg-18 and Met-49 could be a possible mechanism for the binding of FKBP12 to RyR1. Therefore, these results suggest that the N-terminal regions of FKBP12 (Gln-3 and Arg-18) and Met-49 are essential and unique for binding of FKBP12 to RyR1 in skeletal muscle.
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PMID:N-terminal region of FKBP12 is essential for binding to the skeletal ryanodine receptor. 1503 87

The cellular and molecular processes underlying the regulation of ryanodine receptor (RyR) Ca(2+) release in smooth muscle cells (SMCs) are incompletely understood. Here we show that FKBP12.6 proteins are expressed in pulmonary artery (PA) smooth muscle and associated with type-2 RyRs (RyR2), but not RyR1, RyR3, or IP(3) receptors (IP(3)Rs) in PA sarcoplasmic reticulum. Application of FK506, which binds to FKBPs and dissociates these proteins from RyRs, induced an increase in [Ca(2+)](i) and Ca(2+)-activated Cl(-) and K(+) currents in freshly isolated PASMCs, whereas cyclosporin, an agent known to inhibit calcineurin but not to interact with FKBPs, failed to induce an increase in [Ca(2+)](i). FK506-induced [Ca(2+)](i) increase was completely blocked by the RyR antagonist ruthenium red and ryanodine, but not the IP(3)R antagonist heparin. Hypoxic Ca(2+) response and hypoxic vasoconstriction were significantly enhanced in FKBP12.6 knockout mouse PASMCs. FK506 or rapamycin pretreatment also enhanced hypoxic increase [Ca(2+)](i), but did not alter caffeine-induced Ca(2+) release (SR Ca(2+) content) in PASMCs. Norepinephrine-induced Ca(2+) release and force generation were also markedly enhanced in PASMCs from FKBP12.6 null mice. These findings suggest that FKBP12.6 plays an important role in hypoxia- and neurotransmitter-induced Ca(2+) and contractile responses by regulating the activity of RyRs in PASMCs.
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PMID:Role of FKBP12.6 in hypoxia- and norepinephrine-induced Ca2+ release and contraction in pulmonary artery myocytes. 1503 51

The ultrastructural localization of ryanodine receptors (RyR) in sinus endothelial cells of the rat spleen was examined by confocal laser scanning and electron microscopy by using isoform-specific antibodies to each of the RyR isoforms. Immunofluorescence microscopy of tissue cryosections revealed RyR3 to be localized, with a strand-like form, in the superficial layer and within the cytoplasm of endothelial cells. Antibodies to RyR1 and RyR2 did not react indicating RyR3 was the predominant isoform. RyR3 was observed over the cortical layer of actin filaments in the apical part and beneath stress fibers in the basal part of the endothelial cells. The distribution of Ca2+-storing tubulovesicular-structures within endothelial cells was established by tissue sections treated with osmium ferricyanide selectively to stain the sarcoplasmic reticulum and transverse tubules in muscle cells; electron microscopy revealed densely stained tubulovesicular structures located throughout the sinus endothelial cells and interconnected at various sites. These structures closely apposed the plasma membrane at the apical, lateral, and basal surfaces of the cells and occasionally ran closely parallel to the plasma membrane and near to the mitochondria. Immunogold electron microscopy revealed RyR in the membranes of the nucleus, tubulovesicular structures, and subplasmalemmal cisternae. In the subplasmalemmal cisternae at the apical, lateral, and basal surfaces, RyR was detected on the membranes near to the plasma membrane. Labeling was also present on the membranes of tubulovesicular structures near to caveolae and on the cristae of the mitochondria. Thus, RyR probably participates in Ca2+ signal transduction and/or mechanosignal transduction in sinus endothelial cells.
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PMID:Localization of ryanodine receptor 3 in the sinus endothelial cells of the rat spleen. 1520 40

Calmodulin (CaM) inhibits the skeletal muscle ryanodine receptor-1 (RyR1) and cardiac muscle RyR2 at micromolar Ca(2+) but activates RyR1 and inhibits RyR2 at submicromolar Ca(2+) by binding to a single, highly conserved CaM-binding site. To identify regions responsible for the differential regulation of RyR1 and RyR2 by CaM, we generated chimeras encompassing and flanking the CaM-binding domain. We found that the exchange of the N- and C-terminal flanking regions differentially affected RyR1 and RyR2. A RyR1/RyR2 chimera with an N-terminal flanking RyR2 substitution (RyR2 amino acid (aa) 3537-3579) was activated by CaM in single channel measurements at both submicromolar and micromolar Ca(2+). A RyR2/RyR1 chimera with a C-terminal flanking the 86-amino acid RyR1 substitution (RyR1 aa 3640-3725) bound (35)S-CaM but was not inhibited by CaM at submicromolar Ca(2+). In this region, five non-conserved amino acid residues (RyR1 aa 3680 and 3682-3685 and RyR2 aa 3647 and 3649-3652) differentially affect RyR helical probability. Substitution of the five amino acid residues in RyR1 with those of RyR2 showed responses to CaM comparable with wild type RyR1. In contrast, substitution of the five amino acid residues in RyR2 with those of RyR1 showed loss of CaM inhibition, whereas substitution of the five RyR2 sequence residues in the RyR2 chimera containing the RyR1 calmodulin-binding domain and C-flanking sequence restored wild type RyR2 inhibition by CaM at submicromolar Ca(2+). The results suggest that different regions are involved in CaM modulation of RyR1 and RyR2. They further suggest that five non-conserved amino acids in the C-terminal region flanking the CaM-binding domain have a key role in CaM inhibition of RyR2.
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PMID:Different regions in skeletal and cardiac muscle ryanodine receptors are involved in transducing the functional effects of calmodulin. 1521 35


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