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)

The potential role in Ca2+ release channel function of highly conserved, polar, and small amino acids in predicted transmembrane sequences in the rabbit skeletal muscle ryanodine receptor (RyR1) was investigated through mutagenesis. Acidic amino acids Asp3987, Glu4032, Asp4815, Asp4917, Asp4938, and Asp4969 and amidated residues Asn4034, Asn4037, Asn4574, Asn4805, Asn4806, and Gln4933, and Gly4033 were mutated to Ala, and Ala3988 was mutated to Val. When expressed in HEK-293 cells and challenged with either caffeine or 4-chloro-m-cresol, mutants E4032A, N4806A, D4815A, and D4917A did not respond, indicating that Ca2+ release channel function was impaired. None of these mutants exhibited specific binding of [3H]ryanodine. Mutants N4805A and Q4933A showed a diminished response to both caffeine and 4-chloro-m-cresol, but [3H]ryanodine binding was not altered. Other mutant responses and the responses of mutants E4032D, N4806Q or D, D4815N or E, and D4938N or E were unaltered when compared with RyR1. However, mutants E4032Q, D4917N or E, and Q4933N or E displayed neither caffeine nor 4-chloro-m-cresol response nor [3H]ryanodine binding. Sedimentation assays indicated that the nonfunctional mutants did contain tetrameric complexes, implying that defects in the assembly of a functional channel did not occur with specific mutations in transmembrane sequences. These results support the view that amino acids Glu4032 (M2), Asn4806 (M7), Asp4815 (M7), Asp4917 (M10), and Gln4933 (M10) are involved in channel function and regulation.
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PMID:Functional consequences of mutations of conserved, polar amino acids in transmembrane sequences of the Ca2+ release channel (ryanodine receptor) of rabbit skeletal muscle sarcoplasmic reticulum. 982 55

A rapid assay for high affinity [3H]ryanodine binding to 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid (CHAPS)-solubilized recombinant or native Ca2+ release channel proteins (ryanodine receptor, RyR) was devised. The key to preservation of high affinity [3H]ryanodine binding sites in the presence of increasing concentrations of CHAPS was the addition of phosphatidylcholine. This assay was used to characterize the equilibrium and kinetic properties of [3H]ryanodine binding to recombinant skeletal (RyR1) and cardiac (RyR2) Ca2+ release channels and the effects on binding of physiological modulators including ATP, Ca2+, and Mg2+. Both RyR1 and RyR2 had a single high affinity ryanodine binding site and low affinity sites, but [3H]ryanodine binding to recombinant RyR2 was not sensitive to ATP activation or Ca2+ inactivation and was less sensitive to Mg2+ inhibition. The [3H]ryanodine binding assay was used to estimate the expression level of recombinant RyR2 and RyR1, and to show that RyR2 can be expressed at very high levels in HEK-293 cells. Analysis of the properties of recombinant RyR2 and RyR1 by measurement of intracellular Fura-2 fluorescence revealed that the different properties of RyR2 and RyR1 are retained in the recombinant expressed proteins.
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PMID:Characterization of recombinant rabbit cardiac and skeletal muscle Ca2+ release channels (ryanodine receptors) with a novel [3H]ryanodine binding assay. 983 97

Malignant hyperthermia (MH) and central core disease (CCD) mutations were introduced into full-length rabbit Ca2+ release channel (RYR1) cDNA, which was then expressed transiently in HEK-293 cells. Resting Ca2+ concentrations were higher in HEK-293 cells expressing homotetrameric CCD mutant RyR1 than in cells expressing homotetrameric MH mutant RyR1. Cells expressing homotetrameric CCD or MH mutant RyR1 exhibited lower maximal peak amplitudes of caffeine-induced Ca2+ release than cells expressing wild type RyR1, suggesting that MH and CCD mutants might be "leaky." In cells expressing homotetrameric wild type or mutant RyR1, the amplitude of 10 mM caffeine-induced Ca2+ release was correlated significantly with the amplitude of carbachol- or thapsigargin-induced Ca2+ release, indicating that maximal drug-induced Ca2+ release depends on the size of the endoplasmic reticulum Ca2+ store. The content of endogenous sarco(endo)plasmic reticulum Ca2+-ATPase isoform 2b (SERCA2b), measured by enzyme-linked immunosorbent assay, 45Ca2+ uptake, and confocal microscopy, was increased in HEK-293 cells expressing wild type or mutant RyR1, supporting the view that endoplasmic reticulum Ca2+ storage capacity is increased as a compensatory response to an enhanced Ca2+ leak. When heterotetrameric (1:1) combinations of MH/CCD mutant and wild type RyR1 were expressed together with SERCA1 to enhance Ca2+ reuptake, the amplitude of Ca2+ release in response to low concentrations of caffeine and halothane was higher than that observed in cells expressing wild type RyR1 and SERCA1. In Ca2+-free medium, MH/CCD mutants were more sensitive to caffeine than wild type RyR1, indicating that caffeine hypersensitivity observed with a variety of MH/CCD mutant RyR1 proteins is not dependent on extracellular Ca2+ concentration.
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PMID:Measurement of resting cytosolic Ca2+ concentrations and Ca2+ store size in HEK-293 cells transfected with malignant hyperthermia or central core disease mutant Ca2+ release channels. 987 4

Central core disease is a rare, nonprogressive myopathy that is characterized by hypotonia and proximal muscle weakness. In a large Mexican kindred with an unusually severe and highly penetrant form of the disorder, DNA sequencing identified an I4898T mutation in the C-terminal transmembrane/luminal region of the RyR1 protein that constitutes the skeletal muscle ryanodine receptor. All previously reported RYR1 mutations are located either in the cytoplasmic N terminus or in a central cytoplasmic region of the 5,038-aa protein. The I4898T mutation was introduced into a rabbit RYR1 cDNA and expressed in HEK-293 cells. The response of the mutant RyR1 Ca2+ channel to the agonists halothane and caffeine in a Ca2+ photometry assay was completely abolished. Coexpression of normal and mutant RYR1 cDNAs in a 1:1 ratio, however, produced RyR1 channels with normal halothane and caffeine sensitivities, but maximal levels of Ca2+ release were reduced by 67%. [3H]Ryanodine binding indicated that the heterozygous channel is activated by Ca2+ concentrations 4-fold lower than normal. Single-cell analysis of cotransfected cells showed a significantly increased resting cytoplasmic Ca2+ level and a significantly reduced luminal Ca2+ level. These data are indicative of a leaky channel, possibly caused by a reduction in the Ca2+ concentration required for channel activation. Comparison with two other coexpressed mutant/normal channels suggests that the I4898T mutation produces one of the most abnormal RyR1 channels yet investigated, and this level of abnormality is reflected in the severe and penetrant phenotype of affected central core disease individuals.
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PMID:A mutation in the transmembrane/luminal domain of the ryanodine receptor is associated with abnormal Ca2+ release channel function and severe central core disease. 1009 41

Central core disease (CCD) and nemaline myopathy (NM) are congenital myopathies for which differential diagnosis is often based on the presence either of cores or rods. Missense mutations in the skeletal muscle ryanodine receptor gene (RYR1) have been identified in some families with CCD. Mutations in the alpha-tropomyosin and alpha-actin genes have been associated with most dominant forms of NM. Analysis of the RYR1 cDNA in a French family identified a novel Y4796C mutation that lies in the C-terminal channel-forming domain of the RyR1 protein. This mutation was linked not only to a severe and penetrant form of CCD, but also to the presence of rods in the muscle fibres and to the malignant hyperthermia susceptibility (MHS) phenotype. The Y4796C mutation was introduced into a rabbit RYR1 cDNA and expressed in HEK-293 cells. Expression of the mutant RYR1 cDNA produced channels with increased caffeine sensitivity and a significantly reduced maximal level of Ca(2+) release. Single-cell Ca(2+) analysis showed that the resting cytoplasmic level was increased by 60% in cells expressing the mutant channel. These data support the view that the rate of Ca(2+) leakage is increased in the mutant channel. The resulting chronic elevation in myoplasmic concentration is likely to be responsible for the severe expression of the disease. Haplotyping analysis indicated that the mutation arose as a neomutation in the proband. This first report of a neomutation in the RYR1 gene has strong implications for genetic linkage studies of MHS or CCD, two diseases characterized by a genetic heterogeneity.
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PMID:An autosomal dominant congenital myopathy with cores and rods is associated with a neomutation in the RYR1 gene encoding the skeletal muscle ryanodine receptor. 1106 19

We investigated the interaction of the 12 kDa 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 (20 microM) 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 IP33R1 by limited proteolysis. We obtained a 45 kDa 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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PMID:Characterization and mapping of the 12 kDa FK506-binding protein (FKBP12)-binding site on different isoforms of the ryanodine receptor and of the inositol 1,4,5-trisphosphate receptor. 1117 Nov 21

A highly conserved amino acid sequence, GVRAGGGIGD(4831), which may form part of the Ca(2+) release channel pore in RyR2, was subjected to Ala scanning or Ala to Val mutagenesis; function was then measured by expression in HEK-293 cells, followed by Ca(2+) photometry, high affinity [(3)H]ryanodine binding, and single-channel recording. All mutants except I4829A and I4829T (corresponding to the I4897T central core disease mutant in RyR1) displayed caffeine-induced Ca(2+) release in HEK-293 cells; only mutants G4826A, I4829V, and G4830A retained high affinity [(3)H]ryanodine binding; and single-channel function was found for all mutants tested, except for G4822A and A4825V. EC(50) values for caffeine-induced Ca(2+) release were increased for G4822A, R4824A, G4826A, G4828A, and D4831A; decreased for V4823A; and unchanged for A4825V, G4827A, I4829V, and G4830A. Ryanodine (10 microm), which did not stimulate Ca(2+) release in wild type (wt), did so in Ala mutants in amino acids 4823-4827. It inhibited the caffeine response in wt and most mutants, but enhanced the amplitude of caffeine-induced Ca(2+) release in mutant G4828A. It also restored caffeine-induced Ca(2+) release in mutants I4829A and I4829T. In single-channel recordings, mutants I4829V and G4830A retained normal conductance, whereas all others had decreased unitary channel conductances ranging from 27 to 540 picosiemens. Single-channel modulation was retained in G4826A, I4829V, and G4830A, but was lost in other mutants. In contrast to wt and G4826A, I4829V, and G4830A, in which divalent metals were preferentially conducted, mutants with loss of modulation had no selectivity of divalent cations over a monovalent cation. Analysis of Gly(4822) to Asp(4831) mutants in RyR2 supports the view that this highly conserved sequence constitutes part of the ion-conducting pore of the Ca(2+) release channel and plays a key role in ryanodine and caffeine binding and activation.
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PMID:Functional characterization of mutants in the predicted pore region of the rabbit cardiac muscle Ca(2+) release channel (ryanodine receptor isoform 2). 1142 30

Mutations G2370A, G2372A, G2373A, G2375A, Y3937A, S3938A, G3939A and K3940A were made in two potential ATP-binding motifs (amino acids 2370-2375 and 3937-3940) in the Ca(2+)-release channel of skeletal-muscle sarcoplasmic reticulum (ryanodine receptor or RyR1). Activation of [(3)H]ryanodine binding by Ca(2+), caffeine and ATP (adenosine 5'-[beta,gamma-methylene]triphosphate, AMP-PCP) was used as an assay for channel opening, since ryanodine binds only to open channels. Caffeine-sensitivity of channel opening was also assayed by caffeine-induced Ca(2+) release in HEK-293 cells expressing wild-type and mutant channels. Equilibrium [(3)H]ryanodine-binding properties and EC(50) values for Ca(2+) activation of high-affinity [(3)H]ryanodine binding were similar between wild-type RyR1 and mutants. In the presence of 1 mM AMP-PCP, Ca(2+)-activation curves were shifted to higher affinity and maximal binding was increased to a similar extent for wild-type RyR1 and mutants. ATP sensitivity of channel opening was also similar for wild-type and mutants. These observations apparently rule out sequences 2370-2375 and 3937-3940 as ATP-binding motifs. Caffeine or 4-chloro-m-cresol sensitivity, however, was decreased in mutants G2370A, G2373A and G2375A, whereas the other mutants retained normal sensitivity. Amino acids 2370-2375 lie within a sequence (amino acids 2163-2458) in which some eight RyR1 mutations have been associated with malignant hyperthermia and shown to be hypersensitive to caffeine and 4-chloro-m-cresol activation. By contrast, mutants G2370A, G2373A and G2375A are hyposensitive to caffeine and 4-chloro-m-cresol. Thus amino acids 2163-2458 form a regulatory domain (malignant hyperthermia regulatory domain 2) that regulates caffeine and 4-chloro-m-cresol sensitivity of RyR1.
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PMID:Mutations to Gly2370, Gly2373 or Gly2375 in malignant hyperthermia domain 2 decrease caffeine and cresol sensitivity of the rabbit skeletal-muscle Ca2+-release channel (ryanodine receptor isoform 1). 1169 96

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.
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PMID:RyR1 and RyR3 isoforms provide distinct intracellular Ca2+ signals in HEK 293 cells. 1204 20

To define the topology of the skeletal muscle ryanodine receptor (RyR1), enhanced GFP (EGFP) was fused in-frame to the C terminus of RyR1, replacing a series of C-terminal deletions that started near the beginning or the end of predicted transmembrane helices M1-M10. The constructs were expressed in HEK-293 (human embryonic kidney cell line 293) or mouse embryonic fibroblast (MEF) cells, and confocal microscopy of intact and saponin-permeabilized cells was used to determine the subcellular location of the truncated fusion proteins. The fusion protein truncated after M3 exhibited uniform cytoplasmic fluorescence, which was lost after permeabilization, indicating that proposed M', M", M1, M2, and M3 sequences are not membrane-associated. The fusion protein truncated at the end of the M4-M5 loop and containing M4 was membrane-associated. All longer truncated fusion proteins were also associated with intracellular membranes. Mapping by protease digestion and extraction of isolated microsomes demonstrated that EGFP positioned after either M5, the N-terminal half of M7 (M7a), or M8 was located in the lumen, and that EGFP positioned after either M4, M6, the C-terminal half of M7 (M7b), or M10 was located in the cytoplasm. These results indicate that RyR1 contains eight transmembrane helices, organized as four hairpin loops. The first hairpin is likely to be made up of M4a-M4b. However, it could be made up from M3-M4, which might form a hairpin loop even though M3 alone is not membrane-associated. The other three hairpin loops are formed from M5-M6, M7a-M7b, and M8-M10. M9 is not a transmembrane helix, but it might form a selectivity filter between M8 and M10.
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PMID:Topology of the Ca2+ release channel of skeletal muscle sarcoplasmic reticulum (RyR1). 1248 42


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