Skeletal Muscle Ryanodine Receptor Mutations Associated with Malignant Hyperthermia Showed Enhanced Intensity and Sensitivity to Triggering Drugs when Expressed in Human Embryonic Kidney Cells

2013 ◽  
Vol 119 (1) ◽  
pp. 111-118 ◽  
Author(s):  
Keisaku Sato ◽  
Cornelia Roesl ◽  
Neil Pollock ◽  
Kathryn M. Stowell
Keyword(s):  
Skeletal Muscle ◽  
Malignant Hyperthermia ◽  
Calcium Channel ◽  
Ryanodine Receptor ◽  
Ex Vivo ◽  
Simian Virus 40 ◽  
T Antigen ◽  
Wild Type ◽  
Human Embryonic Kidney ◽  
Higher Sensitivity

Abstract Background: Mutations within the gene encoding the skeletal muscle calcium channel ryanodine receptor can result in malignant hyperthermia. Although it is important to characterize the functional effects of candidate mutations to establish a genetic test for diagnosis, ex vivo methods are limited because of the low incidence of the disorder and sample unavailability. More than 250 candidate mutations have been identified, but only a few mutations have been functionally characterized. Methods: The human skeletal muscle ryanodine receptor complementary DNA was cloned with or without a disease-related variant. Wild-type and mutant calcium channel proteins were transiently expressed in human embryonic kidney-293 cells expressing the large T-antigen of simian virus 40, and functional analysis was carried out using calcium imaging with fura-2 AM. Six human malignant hyperthermia-related mutants such as R44C, R163C, R401C, R533C, R533H, and H4833Y were analyzed. Cells were stimulated with a specific ryanodine receptor agonist 4-chloro-m-cresol, and intracellular calcium mobility was analyzed to determine the functional aspects of mutant channels. Results: Mutant proteins that contained a variant linked to malignant hyperthermia showed higher sensitivity to the agonist. Compared with the wild type (EC50 = 453.2 µm, n = 18), all six mutants showed a lower EC50 (21.2–170.4 µm, n = 12–23), indicating susceptibility against triggering agents. Conclusions: These six mutations cause functional abnormality of the calcium channel, leading to higher sensitivity to a specific agonist, and therefore could be considered potentially causative of malignant hyperthermia reactions.

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.

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)

2001 ◽  
Vol 360 (1) ◽  
pp. 97-105 ◽  
Author(s):  
Guo Guang DU ◽  
Hideto OYAMADA ◽  
Vijay K. KHANNA ◽  
David H. MacLENNAN
Keyword(s):  
Skeletal Muscle ◽  
Amino Acids ◽  
Malignant Hyperthermia ◽  
Ryanodine Receptor ◽  
Atp Binding ◽  
Regulatory Domain ◽  
Wild Type ◽  
Release Channel ◽  
Binding Motifs ◽  
Channel Opening

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 Ca2+-release channel of skeletal-muscle sarcoplasmic reticulum (ryanodine receptor or RyR1). Activation of [3H]ryanodine binding by Ca2+, caffeine and ATP (adenosine 5′-[β,γ-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 Ca2+ release in HEK-293 cells expressing wild-type and mutant channels. Equilibrium [3H]ryanodine-binding properties and EC50 values for Ca2+ activation of high-affinity [3H]ryanodine binding were similar between wild-type RyR1 and mutants. In the presence of 1mM AMP-PCP, Ca2+-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.

scholarly journals Malignant hyperthermia-associated mutations in the S2-S3 cytoplasmic loop of type 1 ryanodine receptor calcium channel impair calcium-dependent inactivation

2016 ◽  
Vol 311 (5) ◽  
pp. C749-C757 ◽  
Author(s):  
Angela C. Gomez ◽  
Timothy W. Holford ◽  
Naohiro Yamaguchi
Keyword(s):  
Skeletal Muscle ◽  
Malignant Hyperthermia ◽  
Ryanodine Receptor ◽  
Wild Type ◽  
Cytoplasmic Loop ◽  
Muscle Diseases ◽  
Calcium Dependent ◽  
Transmembrane Segments ◽  
Channel Regulation ◽  
Dependent Inactivation

Channel activities of skeletal muscle ryanodine receptor (RyR1) are activated by micromolar Ca2+ and inactivated by higher (∼1 mM) Ca2+. To gain insight into a mechanism underlying Ca2+-dependent inactivation of RyR1 and its relationship with skeletal muscle diseases, we constructed nine recombinant RyR1 mutants carrying malignant hyperthermia or centronuclear myopathy-associated mutations and determined RyR1 channel activities by [3H]ryanodine binding assay. These mutations are localized in or near the RyR1 domains which are responsible for Ca2+-dependent inactivation of RyR1. Four RyR1 mutations (F4732D, G4733E, R4736W, and R4736Q) in the cytoplasmic loop between the S2 and S3 transmembrane segments (S2–S3 loop) greatly reduced Ca2+-dependent channel inactivation. Activities of these mutant channels were suppressed at 10–100 μM Ca2+, and the suppressions were relieved by 1 mM Mg2+. The Ca2+- and Mg2+-dependent regulation of S2–S3 loop RyR1 mutants are similar to those of the cardiac isoform of RyR (RyR2) rather than wild-type RyR1. Two mutations (T4825I and H4832Y) in the S4–S5 cytoplasmic loop increased Ca2+ affinities for channel activation and decreased Ca2+ affinities for inactivation, but impairment of Ca2+-dependent inactivation was not as prominent as those of S2–S3 loop mutants. Three mutations (T4082M, S4113L, and N4120Y) in the EF-hand domain showed essentially the same Ca2+-dependent channel regulation as that of wild-type RyR1. The results suggest that nine RyR1 mutants associated with skeletal muscle diseases were differently regulated by Ca2+ and Mg2+. Four malignant hyperthermia-associated RyR1 mutations in the S2–S3 loop conferred RyR2-type Ca2+- and Mg2+-dependent channel regulation.

E1A induces phosphorylation of the retinoblastoma protein independently of direct physical association between the E1A and retinoblastoma products

1991 ◽  
Vol 11 (8) ◽  
pp. 4253-4265
Author(s):  
H G Wang ◽  
G Draetta ◽  
E Moran
Keyword(s):  
Cell Cycle ◽  
Simian Virus 40 ◽  
Simian Virus ◽  
Binding Activity ◽  
T Antigen ◽  
Physical Interaction ◽  
Resting Cells ◽  
Wild Type ◽  
Quiescent Cells ◽  
Kinase Expression

We have studied the initial effects of adenovirus E1A expression on the retinoblastoma (RB) gene product in normal quiescent cells. Although binding of the E1A products to pRB could, in theory, make pRB phosphorylation unnecessary for cell cycle progression, we have found that the 12S wild-type E1A product is capable of inducing phosphorylation of pRB in normal quiescent cells. The induction of pRB phosphorylation correlates with E1A-mediated induction of p34cdc2 expression and kinase activity, consistent with the possibility that p34cdc2 is a pRB kinase. Expression of simian virus 40 T antigen induces similar effects. Induction of pRB phosphorylation is independent of the pRB binding activity of the E1A products; E1A domain 2 mutants do not bind detectable levels of pRB but remain competent to induce pRB phosphorylation and to activate cdc2 protein kinase expression and activity. Although the kinetics of induction are slower, domain 2 mutants induce wild-type levels of pRB phosphorylation and host cell DNA synthesis and yet fail to induce cell proliferation. These results imply that direct physical interaction between the RB and E1A products does not play a required role in the early stages of E1A-mediated cell cycle induction and that pRB phosphorylation is not, of itself, sufficient to allow quiescent cells to divide. These results suggest that the E1A products do not need to bind pRB in order to stimulate resting cells to enter the cell cycle. Indeed, a more important role of the RB binding activity of the E1A products may be to prevent dividing cells from returning to G0.

DNA sequence requirements for replication of polyomavirus DNA in vivo and in vitro

1987 ◽  
Vol 7 (10) ◽  
pp. 3694-3704
Author(s):  
C Prives ◽  
Y Murakami ◽  
F G Kern ◽  
W Folk ◽  
C Basilico ◽  
...  
Keyword(s):  
Dna Replication ◽  
Simian Virus 40 ◽  
T Antigen ◽  
Early Gene ◽  
Wild Type ◽  
The Core ◽  
Cell Extracts ◽  
Sequence Requirements

Cell extracts of FM3A mouse cells replicate polyomavirus (Py) DNA in the presence of immunoaffinity-purified Py large T antigen, deoxynucleoside triphosphates, ATP, and an ATP-generating system. This system was used to examine the effects of mutations within or adjacent to the Py core origin (ori) region in vitro. The analysis of plasmid DNAs containing deletions within the early-gene side of the Py core ori indicated that sequences between nucleotides 41 and 57 define the early boundary of Py DNA replication in vitro. This is consistent with previously published studies on the early-region sequence requirements for Py replication in vivo. Deleting portions of the T-antigen high-affinity binding sites A and B (between nucleotides 57 and 146) on the early-gene side of the core ori led to increased levels of replication in vitro and to normal levels of replication in vivo. Point mutations within the core ori region that abolish Py DNA replication in vivo also reduced replication in vitro. A mutant with a reversed orientation of the Py core ori region replicated in vitro, but to a lesser extent that wild-type Py DNA. Plasmids with deletions on the late-gene side of the core ori, within the enhancer region, that either greatly reduced or virtually abolished Py DNA replication in vivo replicated to levels similar to those of wild-type Py DNA plasmids in vitro. Thus, as has been observed with simian virus 40, DNA sequences needed for Py replication in vivo are different from and more stringent than those required in vitro.

scholarly journals Assembly of T-Antigen Double Hexamers on the Simian Virus 40 Core Origin Requires Only a Subset of the Available Binding Sites

1998 ◽  
Vol 18 (5) ◽  
pp. 2677-2687 ◽  
Author(s):  
Woo S. Joo ◽  
Henry Y. Kim ◽  
John D. Purviance ◽  
K. R. Sreekumar ◽  
Peter A. Bullock
Keyword(s):  
Structural Changes ◽  
Simian Virus 40 ◽  
Simian Virus ◽  
T Antigen ◽  
Wild Type ◽  
The Core ◽  
Structural Distortions ◽  
In The Wild ◽  
Sv40 Dna ◽  
Initial Assembly

ABSTRACT Initiation of simian virus 40 (SV40) DNA replication is dependent upon the assembly of two T-antigen (T-ag) hexamers on the SV40 core origin. To further define the oligomerization mechanism, the pentanucleotide requirements for T-ag assembly were investigated. Here, we demonstrate that individual pentanucleotides support hexamer formation, while particular pairs of pentanucleotides suffice for the assembly of T-ag double hexamers. Related studies demonstrate that T-ag double hexamers formed on “active pairs” of pentanucleotides catalyze a set of previously described structural distortions within the core origin. For the four-pentanucleotide-containing wild-type SV40 core origin, footprinting experiments indicate that T-ag double hexamers prefer to bind to pentanucleotides 1 and 3. Collectively, these experiments demonstrate that only two of the four pentanucleotides in the core origin are necessary for T-ag assembly and the induction of structural changes in the core origin. Since all four pentanucleotides in the wild-type origin are necessary for extensive DNA unwinding, we concluded that the second pair of pentanucleotides is required at a step subsequent to the initial assembly process.

scholarly journals Differential ability of a T-antigen transport-defective mutant of simian virus 40 to transform primary and established rodent cells.

1985 ◽  
Vol 5 (5) ◽  
pp. 1043-1050 ◽  
Author(s):  
R E Lanford ◽  
C Wong ◽  
J S Butel
Keyword(s):  
Cell Lines ◽  
Mouse Embryo ◽  
Simian Virus 40 ◽  
Simian Virus ◽  
T Antigen ◽  
Wild Type ◽  
Mouse Embryo Fibroblasts ◽  
Established Cell Lines ◽  
Established Cell ◽  
Embryo Fibroblasts

The transforming potential and oncogenicity of a simian virus 40 (SV40) mutant affecting T-antigen (T-ag), SV40(cT)-3, was examined in an effort to dissect T-ag functions in transformation. SV40(cT)-3 has a point mutation at nucleotide 4434 that abolishes the transport of T-ag to the nucleus but does not affect its association with the cell surface. Transfection-transformation assays were performed with primary cells and established cell lines of mouse and rat origin. The efficiency of transformation for established cell lines by SV40(cT)-3 was comparable to that of wild-type SV40, indicating that transformation of established cell lines can occur in the absence of detectable amounts of nuclear T-ag. Transformation of primary mouse embryo fibroblasts by SV40(cT)-3 was markedly influenced by culture conditions; the relative transforming frequency was dramatically reduced in assays involving focus formation in low serum concentrations or anchorage-independent growth. Immunofluorescence tests revealed that the transformed mouse embryo fibroblasts partially transport the mutant cT-ag to the cell nucleus. Transformed cell lines induced by SV40(cT)-3 did not differ in growth properties from wild-type transformants. SV40(cT)-3 was completely defective for the transformation of primary baby rat kidney cells, a primary cell type unable to transport the mutant T-ag to the nucleus. The intracellular localization of cellular protein p53 was found to mimic T-ag distribution in all the transformants analyzed. The mutant virus was weakly oncogenic in vivo: the induction of tumors in newborn hamsters by SV40(cT)-3 was reduced in incidence and delayed in appearance in comparison to wild-type SV40. These observations suggest that cellular transformation is regulated by both nuclear and surface-associated forms of SV40 T-ag.

Separation of simian virus 40 large-T-antigen-transforming and origin-binding functions from the ability to block differentiation

1988 ◽  
Vol 8 (3) ◽  
pp. 1380-1384 ◽  
Author(s):  
V Cherington ◽  
M Brown ◽  
E Paucha ◽  
J St Louis ◽  
B M Spiegelman ◽  
...  
Keyword(s):  
Dehydrogenase Activity ◽  
Adipocyte Differentiation ◽  
Simian Virus 40 ◽  
Simian Virus ◽  
Lipid Binding ◽  
T Antigen ◽  
Large T Antigen ◽  
Wild Type ◽  
Glycerophosphate Dehydrogenase ◽  
Triglyceride Accumulation

Wild-type simian virus 40 large T antigen is very effective at blocking adipocyte differentiation in 3T3-F442A cells as assayed by triglyceride accumulation, induction of glycerophosphate dehydrogenase activity, and expression of mRNAs for glycerophosphate dehydrogenase, the adipocyte serine protease adipsin, and the putative lipid-binding protein adipocyte P2. Point mutants defective for either origin-specific DNA binding or transformation blocked differentiation as completely as wild type.

Caffeine sensitivity of native RyR channels from normal and malignant hyperthermic pigs: effects of a DHPR II–III loop peptide

2004 ◽  
Vol 286 (4) ◽  
pp. C821-C830 ◽  
Author(s):  
Esther M. Gallant ◽  
James Hart ◽  
Kevin Eager ◽  
Suzanne Curtis ◽  
Angela F. Dulhunty
Keyword(s):  
Skeletal Muscle ◽  
Sarcoplasmic Reticulum ◽  
Malignant Hyperthermia ◽  
Lipid Bilayers ◽  
Ryanodine Receptor ◽  
High Affinity ◽  
Enhanced Sensitivity ◽  
Skeletal Muscle Contraction ◽  
Activation Mechanisms ◽  
Standard Tests

Enhanced sensitivity to caffeine is part of the standard tests for susceptibility to malignant hyperthermia (MH) in humans and pigs. The caffeine sensitivity of skeletal muscle contraction and Ca2+ release from the sarcoplasmic reticulum is enhanced, but surprisingly, the caffeine sensitivity of purified porcine ryanodine receptor Ca2+-release channels (RyRs) is not affected by the MH mutation (Arg615Cys). In contrast, we show here that native malignant hyperthermic pig RyRs (incorporated into lipid bilayers with RyR-associated lipids and proteins) were activated by caffeine at 100- to 1,000-fold lower concentrations than native normal pig RyRs. In addition, the results show that the mutant ryanodine receptor channels were less sensitive to high-affinity activation by a peptide (CS) that corresponds to a part of the II–III loop of the skeletal dihydropyridine receptor (DHPR). Furthermore, subactivating concentrations of peptide CS enhanced the response of normal pig and rabbit RyRs to caffeine. In contrast, the caffeine sensitivity of MH RyRs was not enhanced by the peptide. These novel results showed that in MH-susceptible pig muscles 1) the caffeine sensitivity of native RyRs was enhanced, 2) the sensitivity of RyRs to a skeletal II–III loop peptide was depressed, and 3) an interaction between the caffeine and peptide CS activation mechanisms seen in normal RyRs was lost.

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