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

A region in the skeletal muscle ryanodine receptor between amino acids 4014 and 4765 was expressed as a trpE fusion protein. Overlay studies revealed that this region bound Ca2+ and ruthenium red, an indicator of Ca(2+)-binding sites. Ca2+ binding was mapped to subregion 13b between amino acids 4246 and 4377, encompassing a predicted high affinity Ca(2+)-binding site, and to subregion 13c between amino acids 4364 and 4529, encompassing two predicted high affinity Ca(2+)-binding sites. Ca2+ binding was then mapped to three shorter sequences, 22(13b1), 36(13c1), and 35(13c2), amino acids long, each encompassing one of the three predicted Ca(2+)-binding sites. Site-directed polyclonal antibodies were raised against these three short sequences and purified on antigen affinity columns. The antibody against sequence 13c2, lying between residues 4478 and 4512, specifically recognized both denatured and native forms of the ryanodine receptor, suggesting that at least part of the 35 amino acid sequence containing the Ca(2+)-binding site is surface-exposed. The affinity purified antibody increased the Ca2+ sensitivity of ryanodine receptor channels incorporated into planar lipid bilayers, resulting in increased open probability and opening time without altering channel conductance. The antibody-activated channel was still modulated by Ca2+, Mg2+, ATP, ryanodine, and ruthenium red. These observations suggest that sequence 13c2 may be involved in Ca(2+)-induced Ca2+ release.
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PMID:Characterization of a Ca2+ binding and regulatory site in the Ca2+ release channel (ryanodine receptor) of rabbit skeletal muscle sarcoplasmic reticulum. 138 18

In this study, the binding of [3H]ryanodine to liver microsomal subfractions was investigated. The specific binding of [3H]ryanodine, as determined both by vacuum filtration and by ultracentrifugation, is to a single class of high-affinity binding sites with a Kd of 10 +/- 2.5 nM and density of 500 +/- 100 and 1200 +/- 200 fmol/mg of protein by the filtration and centrifugation methods respectively. [3H]Ryanodine binding reached equilibrium in about 1 min and 2 min at 36 degrees C and 24 degrees C respectively, and the half-time of dissociation at 37 degrees C was approx. 15 s. The binding of [3H]ryanodine is Ca(2+)-independent: it is slightly stimulated by NaCl, Mg2+, ATP and InsP3 but strongly inhibited by caffeine, diltiazem and sodium dantrolene. Thus the binding of ryanodine to endoplasmic reticulum membranes shares some of the characteristics of its binding to the sarcoplasmic reticulum but also differs from it in several important properties, such as its Ca(2+)-independence, its rapid association and dissociation, and its inhibition by caffeine. The structural similarities between the skeletal muscle and liver binding sites were further explored by employing in vitro DNA amplification techniques, using the known sequence of the skeletal muscle receptor as reference point. The data obtained with this method indicate that the liver does not process mRNA for the skeletal muscle ryanodine receptor.
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PMID:Characterization of high-affinity ryanodine-binding sites of rat liver endoplasmic reticulum. Differences between liver and skeletal muscle. 203 82

We have cloned and sequenced cDNA encoding the Ca2+ release channel (ryanodine receptor) of rabbit cardiac muscle sarcoplasmic reticulum. The cDNA, 16,532 base pairs in length, encodes a protein of 4,969 amino acids with a Mr of 564,711. The deduced amino acid sequence is 66% identical with that of the skeletal muscle ryanodine receptor, but analysis of predicted secondary structures and hydropathy plots suggests that the two isoforms exhibit the same topology in both transmembrane and cytoplasmic domains. A potential ATP binding domain was identified at residues 2619-2652, a potential phosphorylation site at residue 2809, and potential calmodulin binding sites at residues 2775-2807, 2877-2898, and 2998-3016. We suggest that a modulator binding domain in the protein lies between residues 2619 and 3016. Northern blot analysis of mRNA from a variety of tissues demonstrated that the cardiac isoform is expressed in heart and brain, while the skeletal muscle isoform is expressed in both fast- and slow-twitch muscle. No ryanodine receptor mRNA was detected in extracts from smooth muscle or any other non-muscle tissue examined. The two receptors are clearly the products of separate genes, and the gene encoding the cardiac muscle ryanodine receptor was localized to chromosome 1.
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PMID:Molecular cloning of cDNA encoding the Ca2+ release channel (ryanodine receptor) of rabbit cardiac muscle sarcoplasmic reticulum. 238 Jan 70

Microsomal sarcoplasmic reticulum (SR) fractions from lobster skeletal muscle were found to bind [3H]-ryanodine. [3H]-ryanodine binding was enhanced by AMP, Ca2+ and caffeine, and significantly diminished by ATP, Ba2+ and Sr2+. Furthermore, dantrolene and ruthenium red, two classical inhibitors of Ca2+ release from the SR, blocked [3H]-ryanodine binding. Similarly, tetracaine, known to block the charge movement associated with excitation-contraction coupling in vertebrate muscle, inhibited the binding of the alkaloid. Our lobster SR preparation exhibited a single high-affinity ryanodine binding site (Kd = 6.6 nM, Bmax = 10 pmol/mg protein). Since SDS-PAGE of the SR proteins revealed a major band c. 565 kDa which comigrated with the putative ryanodine receptor from both rat and chicken skeletal muscle, we concluded that lobster skeletal muscle is equipped with the 565 kDa ryanodine receptor. Finally, incorporation of the SR microsomal fraction from lobster into planar bilayer membranes revealed the presence of a ryanodine-sensitive Ca2+ channel activity (160 pS in symmetrical 200 mM CsCl solutions). We concluded that both the crustacean and vertebrate skeletal muscle ryanodine receptor share the relevant properties such as molecular weight and affinity for ryanodine and inositol 1,4,5 triphosphate. However, there are important differences between the two receptors including differential effects of the alkaloid on the Ca2+ release channel and modulation of the receptor by nucleotides.
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PMID:Properties of the ryanodine receptor present in the sarcoplasmic reticulum from lobster skeletal muscle. 751 63

In earlier studies (Chen, S. R. W., Zhang, L., and MacLennan, D. H. (1992) J. Biol. Chem. 267, 23318-23326), an amino acid sequence, designated 13c2, lying between amino acid residues 4478 and 4512 in the skeletal muscle ryanodine receptor was shown, through the use of a polyclonal antibody, to be involved in Ca(2+)-induced Ca2+ release. In the present study, an immobilized synthetic peptide, PEPEPEPEPE, corresponding to part of the predicted high affinity Ca2+ binding site between residues 4489 and 4499, was used to purify specific antibodies from an anti-13c2 rabbit antiserum. The effect of this affinity-purified, anti-peptide (anti-13cp1) antibody on Ca2+ release channel function was then characterized using single channel recordings across planar lipid bilayers. The anti-peptide antibody inhibited Ca(2+)- or caffeine-activated channel activities without closing the channel but did not diminish ATP-activated channel activity. The addition of ATP reversed the inhibition of the Ca(2+)- or caffeine-activated channel by the antibody, and the antibody-bound, ATP-activated channel was further modulated by Mg2+, ryanodine, and ruthenium red. The major epitopes in the anti-13c2 antibody, previously shown to activate the Ca2+ release channel by increasing the Ca2+ sensitivity of the channel, did not lie in the PE repeat. These results suggest that the PE repeat sequence forms a site involved in the Ca2+ activation pathway.
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PMID:Antibodies as probes for Ca2+ activation sites in the Ca2+ release channel (ryanodine receptor) of rabbit skeletal muscle sarcoplasmic reticulum. 768 61

We have tested the periodate-oxidized ATP analogue 2',3'-dialdehyde adenosine triphosphate (oATP) as a ligand for the skeletal muscle ryanodine receptor/Ca(2+)-release channel. Ca2+ efflux from passively loaded heavy sarcoplasmic reticulum vesicles of skeletal muscle is biphasic. oATP stimulates the initial phase of Ca2+ release in a concentration-dependent manner (EC50 160 microM), and the efflux proceeds with a half-time in the range 100-200 ms. This oATP-modulated initial rapid Ca2+ release was specifically inhibited by millimolar concentrations of Mg2+ and micromolar concentrations of Ruthenium Red, indicating that the effect of oATP was mediated via the ryanodine receptor. The purified Ca(2+)-release channel was incorporated into planar lipid bilayers, and single-channel recordings were carried out to verify a direct interaction of oATP with the ryanodine receptor. Addition of oATP to the cytoplasmic side activated the channel with an EC50 of 76 microM, which is roughly 30-fold higher than the apparent affinity of ATP. The oATP-induced increase in the open probability of the ryanodine receptor displays a steep concentration-response curve with a Hill coefficient of approximately 2, which suggests a co-operativity of the ATP binding sites in the tetrameric protein. oATP binds to the ryanodine receptor in a quasi-irreversible manner via Schiff base formation between the aldehyde groups of oATP and amino groups in the nucleotide binding pocket. This allows for the covalent specific incorporation of [alpha-32P]oATP by borhydride reduction. A typical adenine nucleotide binding site cannot be identified in the primary sequence of the ryanodine receptor. Our results demonstrate that oATP can be used to probe the structure and function of the nucleotide binding pocket of the ryanodine receptor and presumably of other ATP-regulated ion channels.
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PMID:Activation and labelling of the purified skeletal muscle ryanodine receptor by an oxidized ATP analogue. 775 53

The ryanodine receptor is a channel for Ca2+ release from intracellular stores. By PCR analysis, we identified two alternatively spliced regions in mRNA of the mouse skeletal muscle ryanodine receptor (sRyR). The splice variants were characterized by the presence or absence of 15 bp (ASI) and 18 bp (ASII) exons. The exclusion of these exons results in the absence of the regions corresponding to Ala3481-Gln3485 and Val3865-Asn3870, respectively, of rabbit sRyR; these amino acid sequences exist in the modulatory region, where sites for phosphorylation and binding of Ca2+, calmodulin and ATP are postulated to be. We also detected sRyR in brain and heart as well as in skeletal muscle, and the splicing patterns were found to be tissue-specific. Only the ASII-lacking isoform was detected in heart, whereas in other tissues the ASII-containing isoform was predominant. The splicing patterns were also found to change during development. In skeletal muscle, the ASI-containing isoform increased gradually from embryo to adult. The ASII-lacking isoform abruptly increased upon birth, but the ASII-containing isoform increased steadily afterwards. In cerebrum, the ratio of the ASII-containing isoform to the ASII-lacking one increased abruptly during embryonic days 14 and 18. These findings suggest that the alternative splicing of ASI and ASII, by affecting the modulatory region, generates functionally different sRyR isoforms in a tissue-specific and developmentally regulated manner.
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PMID:Tissue-specific and developmentally regulated alternative splicing in mouse skeletal muscle ryanodine receptor mRNA. 783 48

Ryanodine receptors are key molecules in excitation-contraction coupling of skeletal muscle. They form the pore of the calcium release channel, which is regulated by Ca and ATP. Multiple proton titration sites are involved in controlling the different open states of the channel, as indicated by the following: i) the channel had a biphasic response to changes in proton concentrations around neutral pH; ii) the activities of the channel were inhibited by acidic pHs in a highly cooperative manner; and iii) the channel exhibited pronounced hysteresis to changes in pH. Four distinct conductance states can be identified in the single ryanodine-activated calcium release channel. The distribution of the multiple conductance states depends on the level of [Ca], ATP, and pH in the recording solution. The data are consistent with the multimeric structure of the skeletal muscle ryanodine receptor.
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PMID:Highly cooperative and hysteretic response of the skeletal muscle ryanodine receptor to changes in proton concentrations. 794 77

Ryanodine receptors/Ca2+ release channels play an important role in regulating the intracellular free calcium concentrations in both muscle and nonmuscle cells. Ryanodine, a neutral plant alkaloid, specifically binds to and modulates these Ca2+ release channels. In the work described here, we characterize the interaction of a tritium-labeled, photoactivable derivative of ryanodine (3H-labeled 10-O-[3-(4-azidobenzamido)propionyl]ryanodine ([3H]ABRy)) with the ryanodine receptor of skeletal, cardiac, and brain membranes. Scatchard analysis demonstrates that this ligand binds to a single class of high affinity sites in skeletal muscle triads. Furthermore, competition binding assays of [3H]ryanodine with skeletal, cardiac, and brain membranes in the presence of increasing concentrations of unlabeled ABRy illustrate that this azido derivative of ryanodine is able to specifically displace [3H]ryanodine from its binding site(s). Analysis of the effects of Ca2+, ATP, and KCl on [3H]ABRy binding in triad membranes shows a similar modulation of binding to that seen in these membranes with [3H]ryanodine. Photoaffinity labeling of triads with [3H]ABRy resulted in specific and covalent incorporation of [3H]ABRy into a 565-kDa protein that was shown to be the skeletal muscle ryanodine receptor. Digestion of the labeled ryanodine receptor revealed a [3H]ABRy-labeled 76-kDa tryptic fragment that was identified with an antibody directed against the COOH-terminal of the receptor. These results demonstrate that the 76-kDa COOH-terminal tryptic fragment contains the high affinity binding site for ryanodine.
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PMID:Photoaffinity labeling of the ryanodine receptor/Ca2+ release channel with an azido derivative of ryanodine. 817 31

The photoaffinity analog of ATP, 3'-O-(4-benzoyl)benzoyl-adenosine 5'-triphosphate (Bz2ATP) was used to covalently label and to identify the ATP binding site of the skeletal muscle ryanodine receptor. Like ATP, Bz2ATP stimulates up to fivefold the binding of ryanodine to its receptor. Photoactivation by ultraviolet light of the benzophenone group in the [alpha-32P]Bz2ATP results in covalent binding of [alpha-32P]Bz2ATP to the 450-kDa polypeptide, the ryanodine receptor's subunit. An apparent molar stiochiometry of Bz2ATP to the tetrameric ryanodine receptor complex of 1.146 +/- 0.087 (n = 2) was estimated. The covalent binding of [alpha-32P]Bz2ATP was inhibited by ATP and analogous compounds in the order: ATP = AdoPP[CH2]P = ADP = Ado = cAMP > AMP > ITP = GTP. Similar specificity was obtained for the stimulation of ryanodine binding by these nucleotides. ATP increased the ryanodine binding affinity by about sixfold. The polycationic dye ruthenium red, known as an inhibitor of Ca2+ release and ryanodine binding, inhibited the labeling of the ryanodine receptor by [alpha-32P]Bz2ATP. Tryptic digestion of the ryanodine receptor revealed a [alpha-32P]Bz2ATP-labeled 76-kDa tryptic fragment. Digestion of either the [alpha-32P]Bz2ATP-labeled 450-kDa or the 76-kDa polypeptides with S. aureus resulted in the appearance of four labeled fragments of 39, 33, 27 and 13 kDa, where the 39-kDa fragment is the precursor of the 27-kDa and 13-kDa fragments. The results suggest that the regulation of Ca2+ release by ATP involves an ATP binding site(s) located on the 27-kDa and 13-kDa fragments of the ryanodine receptor protein.
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PMID:Characterization and photoaffinity labeling of the ATP binding site of the ryanodine receptor from skeletal muscle. 838 21


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