scholarly journals Functional Studies of RYR1  Mutations in the Skeletal Muscle Ryanodine Receptor Using Human RYR1  Complementary DNA

2010 ◽  
Vol 112 (6) ◽  
pp. 1350-1354 ◽  
Author(s):  
Keisaku Sato ◽  
Neil Pollock ◽  
Kathryn M. Stowell
Keyword(s):  
Skeletal Muscle ◽  
Malignant Hyperthermia ◽  
Calcium Channel ◽  
Ryanodine Receptor ◽  
Human Skeletal Muscle ◽  
Binding Assays ◽  
Complementary Dna ◽  
Hek 293 ◽  
Hek 293 Cells ◽  
293 Cells

Background Malignant hyperthermia is associated with mutations within the gene encoding the skeletal muscle ryanodine receptor, the calcium channel that releases Ca from sarcoplasmic reticulum stores triggering muscle contraction, and other metabolic activities. More than 200 variants have been identified in the ryanodine receptor, but only some of these have been shown to functionally affect the calcium channel. To implement genetic testing for malignant hyperthermia, variants must be shown to alter the function of the channel. A number of different ex vivo methods can be used to demonstrate functionality, as long as cells from human patients can be obtained and cultured from at least two unrelated families. Because malignant hyperthermia is an uncommon disorder and many variants seem to be private, including the newly identified H4833Y mutation, these approaches are limited. Methods The authors cloned the human skeletal muscle ryanodine receptor complementary DNA and expressed both normal and mutated forms in HEK-293 cells and carried out functional analysis using ryanodine binding assays in the presence of a specific agonist, 4-chloro-m-cresol, and the antagonist Mg. Results Transiently expressed human ryanodine receptor proteins colocalized with an endoplasmic reticulum marker in HEK-293 cells. Ryanodine binding assays confirmed that mutations causing malignant hyperthermia resulted in a hypersensitive channel, while those causing central core disease resulted in a hyposensitive channel. Conclusions The functional assays validate recombinant human skeletal muscle ryanodine receptor for analysis of variants and add an additional mutation (H4833Y) to the repertoire of mutations that can be used for the genetic diagnosis of malignant hyperthermia.

COOH-terminal association of human smooth muscle calcium channel Cav1.2b with Src kinase protein binding domains: effect of nitrotyrosylation

2007 ◽  
Vol 293 (6) ◽  
pp. C1983-C1990 ◽  
Author(s):  
Minho Kang ◽  
Gracious R. Ross ◽  
Hamid I. Akbarali
Keyword(s):  
Smooth Muscle ◽  
Calcium Channel ◽  
Tyrosine Phosphorylation ◽  
Src Kinase ◽  
Sh2 Domain ◽  
Calcium Currents ◽  
Hek 293 ◽  
Hek 293 Cells ◽  
Binding Domains ◽  
293 Cells

The carboxyl terminus of the calcium channel plays an important role in the regulation of calcium entry, signal transduction, and gene expression. Potential protein-protein interaction sites within the COOH terminus of the L-type calcium channel include those for the SH3 and SH2 binding domains of c-Src kinase that regulates calcium currents in smooth muscle. In this study, we examined the binding sites involved in Src kinase-mediated phosphorylation of the human voltage-gated calcium channel (Cav) 1.2b (hCav1.2b) and the effect of nitrotyrosylation. Cotransfection of human embryonic kidney (HEK)-293 cells with hCav1.2b and c-Src resulted in tyrosine phosphorylation of the calcium channel, which was prevented by nitration of tyrosine residues by peroxynitrite. Whole cell calcium currents were reduced by 58 + 5% by the Src kinase inhibitor PP2 and 64 + 6% by peroxynitrite. Nitrotyrosylation prevented Src-mediated regulation of the currents. Glutathione S-transferase fusion protein of the distal COOH terminus of hCav1.2b (1809-2138) bound to SH2 domain of Src following tyrosine phosphorylation, while binding to SH3 required the presence of the proline-rich motif. Site-directed mutation of Y2134 prevented SH2 binding and resulted in reduced phosphorylation of hCav1.2b. Within the distal COOH terminus, single, double, or triple mutations of Y1837, Y1861, and Y2134 were constructed and expressed in HEK-293 cells. The inhibitory effects of PP2 and peroxynitrite on calcium currents were significantly reduced in the double mutant Y1837-2134F. These data demonstrate that the COOH terminus of hCav1.2b contains sites for the SH2 and SH3 binding of Src kinase. Nitrotyrosylation of these sites prevents Src kinase regulation and may be importantly involved in calcium influx regulation during inflammation.

scholarly journals HEK-293 cells possess a carbachol- and thapsigargin-sensitive intracellular Ca2+ store that is responsive to stop-flow medium changes and insensitive to caffeine and ryanodine

1999 ◽  
Vol 343 (1) ◽  
pp. 39-44 ◽  
Author(s):  
Jiefei TONG ◽  
Guo Guang DU ◽  
S. R. Wayne CHEN ◽  
David H. MACLENNAN
Keyword(s):  
Functional Analysis ◽  
Ryanodine Receptor ◽  
Functional Expression ◽  
Hek 293 ◽  
Hek 293 Cells ◽  
Endogenous Expression ◽  
293 Cells ◽  
Medium Change ◽  
Flow Medium ◽  
Stop Flow

Because HEK-293 cells are widely used for the functional expression of channels, exchangers and transporters involved in Ca2+ homoeostasis, the properties of intracellular Ca2+ stores and the methods used for measuring intracellular Ca2+ release in HEK-293 cells were evaluated. Ca2+ imaging was used to show caffeine-, carbachol- and thapsigargin-induced Ca2+ release in HEK-293 cells transfected with ryanodine receptor (RyR) cDNA, but only carbachol- and thapsigargin-induced Ca2+ release in untransfected HEK-293 cells. Intracellular Ca2+ release in untransfected HEK-293 cells was also observed if medium changes were performed by aspirating and replacing fresh medium (stop-flow), but not if medium changes were performed by a continuous over-flow procedure. Stop-flow medium-change-induced Ca2+ release in HEK-293 cells was independent of caffeine and ryanodine, demonstrating that it did not occur through RyR channels. Consistent with these observations was the observation that the level of expression of endogenous RyR proteins was below the limits of detection by Western blotting or [3H]ryanodine binding. Thus the level of endogenous expression of RyR is so low in HEK-293 cells as to provide a negligible background in relation to functional analysis of recombinant RyR molecules. These results are inconsistent with those of Querfurth et al. [Querfurth, Haughey, Greenway, Yacono, Golan and Geiger (1998) Biochem. J. 334, 79-86], who reported higher levels of endogenous RyR expression in untransfected HEK-293 cells.

scholarly journals Pore properties and ionic block of the rabbit epithelial calcium channel expressed in HEK 293 cells

2001 ◽  
Vol 530 (2) ◽  
pp. 183-191 ◽  
Author(s):  
Rudi Vennekens ◽  
Jean Prenen ◽  
Joost G. J. Hoenderop ◽  
René J. M. Bindels ◽  
Guy Droogmans ◽  
...  
Keyword(s):  
Calcium Channel ◽  
Hek 293 ◽  
Hek 293 Cells ◽  
293 Cells ◽  
Pore Properties

scholarly journals Exome Sequencing Reveals Novel Rare Variants in the Ryanodine Receptor and Calcium Channel Genes in Malignant Hyperthermia Families

2013 ◽  
Vol 119 (5) ◽  
pp. 1054-1065 ◽  
Author(s):  
Jerry H. Kim ◽  
Gail P. Jarvik ◽  
Brian L. Browning ◽  
Ramakrishnan Rajagopalan ◽  
Adam S. Gordon ◽  
...  
Keyword(s):  
Malignant Hyperthermia ◽  
Calcium Channel ◽  
Exome Sequencing ◽  
Allele Frequency ◽  
Ryanodine Receptor ◽  
Sanger Sequencing ◽  
Population Sample ◽  
Family Members ◽  
Genetic Diagnosis ◽  
Complementary Dna

Abstract Background: About half of malignant hyperthermia (MH) cases are associated with skeletal muscle ryanodine receptor 1 (RYR1) and calcium channel, voltage-dependent, L type, α1S subunit (CACNA1S) gene mutations, leaving many with an unknown cause. The authors chose to apply a sequencing approach to uncover causal variants in unknown cases. Sequencing the exome, the protein-coding region of the genome, has power at low sample sizes and identified the cause of over a dozen Mendelian disorders. Methods: The authors considered four families with multiple MH cases lacking mutations in RYR1 and CACNA1S by Sanger sequencing of complementary DNA. Exome sequencing in two affecteds per family, chosen for maximum genetic distance, were compared. Variants were ranked by allele frequency, protein change, and measures of conservation among mammals to assess likelihood of causation. Finally, putative pathogenic mutations were genotyped in other family members to verify cosegregation with MH. Results: Exome sequencing revealed one rare RYR1 nonsynonymous variant in each of three families (Asp1056His, Val2627Met, Val4234Leu), and one CACNA1S variant (Thr1009Lys) in the fourth family. These were not seen in variant databases or in our control population sample of 5,379 exomes. Follow-up sequencing in other family members verified cosegregation of alleles with MH. Conclusions: The authors found that using both exome sequencing and allele frequency data from large sequencing efforts may aid genetic diagnosis of MH. In a sample selected by the authors, this technique was more sensitive for variant detection in known genes than Sanger sequencing of complementary DNA, and allows for the possibility of novel gene discovery.

Treatment of Normal Skeletal Muscle with FK506 or Rapamycin Results in Halothane-induced Muscle Contracture

1998 ◽  
Vol 89 (3) ◽  
pp. 693-698. ◽  
Author(s):  
Richard L. Brooksbank ◽  
Margaret E. Badenhorts ◽  
Hyam Isaacs ◽  
Nerina Savage
Keyword(s):  
Carbon Dioxide ◽  
Skeletal Muscle ◽  
Malignant Hyperthermia ◽  
Ryanodine Receptor ◽  
Human Skeletal Muscle ◽  
Receptor Type ◽  
Muscle Strip ◽  
Healthy Human ◽  
Healthy Persons

Background FKBP12 is a protein that is closely associated with the ryanodine receptor type 1 of skeletal muscle and modulates Ca2+ release by the channel. The immunosuppressants FK506 and rapamycin both bind to FKBP12 and in turn dissociate the protein from the ryanodine receptor. By treating healthy human skeletal muscle strips with FK506 or rapamycin and then subjecting the strips to the caffeine-halothane contracture test, this study determined that FK506 and rapamycin alter the sensitivity of the muscle strip to halothane, caffeine, or both. Methods Skeletal muscle strips from 10 healthy persons were incubated in Krebs medium equilibrated with a 95% oxygen and 5% carbon dioxide mixture, which contained either 12 microM FK506 (n = 8) or 12 microM rapamycin (n = 6), for 15 min at 37 degrees C. The strips were subjected to the caffeine-halothane contracture test for malignant hyperthermia according to the European Malignant Hyperthermia Group protocol. Results Treatment of normal skeletal muscle strips with FK506 and rapamycin resulted in halothane-induced contractures of 0.44+/-0.16 g and 0.6+/-0.49 g, respectively, at 2% halothane. Conclusions The results obtained show that pre-exposure of healthy skeletal muscle strips to either FK506 or rapamycin is sufficient to give rise to halothane-induced contractures. This is most likely caused by destabilization of Ca2+ release by the ryanodine receptor as a result of the dissociation of FKBP12. This finding suggests that a mutation in FKBP12 or changes in its capacity to bind to the ryanodine receptor could alter the halothane sensitivity of the skeletal muscle ryanodine receptor and thereby predispose the person to malignant hyperthermia.

scholarly journals Increased sensitivity of the ryanodine receptor to halothane-induced oligomerization in malignant hyperthermia-susceptible human skeletal muscle

2004 ◽  
Vol 96 (1) ◽  
pp. 11-18 ◽  
Author(s):  
Louise Glover ◽  
James J. A. Heffron ◽  
Kay Ohlendieck
Keyword(s):  
Skeletal Muscle ◽  
Sarcoplasmic Reticulum ◽  
Malignant Hyperthermia ◽  
Ryanodine Receptor ◽  
Human Skeletal Muscle ◽  
Dihydropyridine Receptor ◽  
Threshold Concentration ◽  
Release Channel ◽  
Functional Dynamics ◽  
Increased Sensitivity

Mutations in the skeletal muscle RyR1 isoform of the ryanodine receptor (RyR) Ca2+-release channel confer susceptibility to malignant hyperthermia, which may be triggered by inhalational anesthetics such as halothane. Using immunoblotting, we show here that the ryanodine receptor, calmodulin, junctin, calsequestrin, sarcalumenin, calreticulin, annexin-VI, sarco(endo)plasmic reticulum Ca2+-ATPase, and the dihydropyridine receptor exhibit no major changes in their expression level between normal human skeletal muscle and biopsies from individuals susceptible to malignant hyperthermia. In contrast, protein gel-shift studies with halothane-treated sarcoplasmic reticulum vesicles from normal and susceptible specimens showed a clear difference. Although the α2-dihydropyridine receptor and calsequestrin were not affected, clustering of the Ca2+-ATPase was induced at comparable halothane concentrations. In the concentration range of 0.014–0.35 mM halothane, anesthetic-induced oligomerization of the RyR1 complex was observed at a lower threshold concentration in the sarcoplasmic reticulum from patients with malignant hyperthermia. Thus the previously described decreased Ca2+-loading ability of the sarcoplasmic reticulum from susceptible muscle fibers is probably not due to a modified expression of Ca2+-handling elements, but more likely a feature of altered quaternary receptor structure or modified functional dynamics within the Ca2+-regulatory apparatus. Possibly increased RyR1 complex formation, in conjunction with decreased Ca2+ uptake, is of central importance to the development of a metabolic crisis in malignant hyperthermia.

scholarly journals HSP70 Binds to the Fast-twitch Skeletal Muscle Sarco(endo)plasmic Reticulum Ca2+-ATPase (SERCA1a) and Prevents Thermal Inactivation

2004 ◽  
Vol 279 (50) ◽  
pp. 52382-52389 ◽  
Author(s):  
A. Russell Tupling ◽  
Anthony O. Gramolini ◽  
Todd A. Duhamel ◽  
Hiroya Kondo ◽  
Michio Asahi ◽  
...  
Keyword(s):  
Skeletal Muscle ◽  
Thermal Inactivation ◽  
Fluorescein Isothiocyanate ◽  
Nucleotide Binding ◽  
Binding Domain ◽  
Nucleotide Binding Domain ◽  
Hek 293 ◽  
Hek 293 Cells ◽  
293 Cells ◽  
Fast Twitch

This study examined whether HSP70 could bind to and protect against thermal inactivation of SERCA1a, the SERCA isoform expressed in adult fast-twitch skeletal muscle. Sarcoplasmic reticulum vesicles prepared from rat gastrocnemius muscle were incubated with purified HSP70 at both 37 and 41 °C for either 30, 60, or 120 min. Maximal SERCA1a activity (μmol/g protein/min) in the absence of HSP70 was reduced progressively with time, with greater reductions occurring at 41 °C compared with 37 °C. HSP70 protected against thermal inactivation of SERCA1a activity at 37 °C but not at 41 °C and only at 30 and 60 min but not at 120 min. HSP70 also protected against reductions in binding capacity for fluorescein isothiocyanate, a fluorescent probe that binds to Lys515in the nucleotide binding domain of SERCA, at 30 and 60 min but not at 120 min, an effect that was independent of temperature. HEK-293 cells were co-transfected with cDNAs encoding rabbit SERCA1a and human HSP-EYFP and subjected to 40 °C for 1 h. Immunohistochemistry revealed nearly complete co-localization of SERCA1a with HSP70 under these conditions. Co-immunoprecipitation showed physical interaction between HSP70 and SERCA1a under all thermal conditions bothin vitroand in HEK-293 cells. Modeling showed that the fluorescein isothiocyanate-binding site of intact SERCA1a in the E2 form lies in its close proximity to a potential interaction site between SERCA1a and HSP70. These results indicate that HSP70 can bind to SERCA1a and, depending on the severity of heat stress, protect SERCA1a function by stabilizing the nucleotide binding domain.

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